Naturally Derived Scaffolds For Treatment of Volumetric Muscle Loss

NATURALLY DERIVED SCAFFOLDS FOR TREATMENT
OF VOLUMETRIC MUSCLE LOSS
BY
HANNAH BESSER BAKER
A Dissertation Submitted to the Graduate Faculty of
WAKE FOREST UNIVERSITY SCHOOL OF ARTS AND SCIENCES
in Partial Fulfillment of the Requirements
for the Degree of
DOCTOR OF PHILOSOPHY
Biomedical Engineering
August, 2015
Winston-Salem, NC
Approved By:
George J. Christ, Ph.D., Advisor and Chair
Graça Almeida-Porada, M.D., Ph.D.
Aaron S. Goldstein, Ph.D.
Robert W. Grange, Ph.D.
David L. Kaplan, Ph.D.
Aaron M. Mohs, Ph.D.

DEDICATION
This dissertation is dedicated to my mother, Sheena Besser Baker, whose untiring work
ethic and resilience have been my inspiration and for having instilled in me a stubborn
independence and resourcefulness for which I am truly grateful.
ii
ACKNOWLEDGEMENTS
This work would not have been possible without the guidance and support of many
people over the last five years and for that I am sincerely grateful. I would like to first
acknowledge and thank my advisor Dr. George Christ for his support and mentorship, for
providing me with many amazing opportunities to work first hand on several aspects of
translational research, and for helping me develop as a researcher by holding me to a high
standard.
I would also like to thank my committee members: Dr. Aaron Mohs, Dr. Graça
Almeida-Porada, Dr. Aaron Goldstein, Dr. Robert Grange, and Dr. David Kaplan for their
input on this work and support over the last couple years. I would especially like to
acknowledge Dr. Mohs for serving as my interim advisor and Dr. Koudy Williams for
taking over as principal investigator on ongoing animal protocols in the months between
Dr. Christ’s relocation to UVa and my defense. Further I would also like to acknowledge
and thank WFIRM for providing me with the opportunity to be a NIBIB T32 fellow and
for the support during my fellowship from Dr. Christ, Dr. Anthony Atala, and Joan
Schanck. I would also like to thank Dr. JP McQuilling for his comradery during our time
spent together as fellows.
Many people at WFIRM played crucial roles in completing this research. I would
like to thank the administrators who have helped make everything run smoothly, especially
Joanne Gray and Donna Tucker. A special thanks is owed to the WFIRM technicians and
especially Cathy Mathis and Cindy Zimmerman whose expertise and support in histology
and immunohistochemistry has helped tremendously. I would also like to thank the surgery
iii
core, the animal care staff, and especially Cindy Miller, Tammy Cockerham, and Cara
Clouse for assistance, guidance, and advice over the years on many animal protocols.
I owe my past and present lab family at WFIRM and UVa many thanks for
supporting me in work and life: Drs. Ben Corona, Catherine Ward, Masood Machingal,
and David Burmeister, Ashley Starr, Christine Koval, Dr. Mevan Siriwardane, Dr. Juliana
Passipieri, Manasi Madhavkar, Chris Bergman, Benjamin Rowe, Mona Zarif-Pour, and Dr.
John Scott, and collaborators and friends in the Marini lab: Kristina Stumpf and Lysette
Mutkus. A special thanks to Dr. John Scott for providing many weeks of assistance on the
rat LD project. I would also like to specially acknowledge Manasi Vadhavkar who
provided countless hours of assistance in cell culture and animal work and for teaching me
many lab techniques. Another very special thanks is owed to Chris Bergman, who assisted
in several facets of every project in this dissertation and especially for having endured
hundreds of hours of organ bath testing for the rat LD project, for which I am extremely
appreciative.
I am exceptionally thankful to have conducted these studies start to finish alongside
Dr. Juliana Passipieri. I have learned so much from Juliana who has a remarkable skill set
and knowledge base. Together we have been an effective and efficient team. I am going to
miss working with Juliana tremendously.
I am very grateful for my friends and extended lab family in the UVa Christ Lab,
Danny Lovell, Ellen Mintz, and Dr. Juliana Passipieri, for their assistance and support
throughout the last several months as I was traveling between Winston-Salem,
Charlottesville, and Baltimore. A very special thank you to Danny and the outstanding UVa
undergrads for conquering an unfathomable amount of histology for the rat LD project.
iv
My time in graduate school would not have been nearly as enjoyable without the
support of my family and the friendships I have made. I would like to acknowledge my
family members for their love and support throughout my grad school years: my mother
Sheena Baker, my father Steve Baker, my step-mother Cheryl Baker, and my brothers
and sisters: Mark Nater, Kristan Nater, Sarah and my brother in law Mike Burns, Amber
and my brother in law Jimmar Wilson, and Rosemary Baker.
I would also like to recognize the great friendships and endless support of fellow
graduate students which have made this time so wonderfully fun and deserve
acknowledgement: Anthony and Jess Santago, Kiks Kowalczewski, JP and Alyssa
McQuilling, Nick Vavalle and Heather Wallin, Tanner Hill, Jill and David Hobson, Jenni
and Chris Jordan, Aleks and Steph Skardal, Megan Johnston, Chris and Sarah Jo Bergman,
Roche De Guzman, Dipen Vyas, Matthew Brovold and Samantha Caldwell, Ashley
Wagoner, Daniel Deegan, Shaida Moghaddassi, Emmy McKee, Sean and Jess Murphy,
Peter Alexander and Amie Severino, Leah and Chris Stuart, Cale and Stephanie
Fahrenholtz, Brittany Eldridge, Jessica Swanner, Hesh Devarasetty, Sam Pendergraft,
Colin Fennelly, Stephen Rego and Amritha Kidiyoor, Kadie Vanderman, Saloomeh
Mokhtari, Elie Zakhem, and Susan Zhao.
I have been incredibly lucky to have great roommates and amazing friends in Emma
Moran and Doug Evans, Liz Graham and Kevin Gurysh, and Danny Lovell who have made
my time in Winston so much fun. I also owe several friends outside of Winston a big thank
you for their continuous support academically and in life over the years: Allison
Hulchanski, Matt Bailey, Jessica Marino, David Axe, Eric Weissmann, Shekhar Gadkaree,
Dan Mendelson, and Monica Lentz.
v
I would like to give a special shout out to Daniel Stovall and Shreya Raghavan for
being such great and supportive friends and helping me generously throughout the process
of applying for post graduate opportunities and beyond and to Jes Bolduc for being such a
great listener throughout this crazy time of life.
Finally, I want to specially acknowledge Abritee Dhal and Mike Kelen. Abritee has
truly kept me sane throughout our friendship in grad school and Mike has been wonderfully
understanding, patient, and helpful over the last two years. Abritee and Mike have been so
selflessly supportive especially during these last incredibly stressful and crazy couple of
years and I thank them both dearly for it.
vi
TABLE OF CONTENTS
List of Figures and Tables………….…………………………..……………………….viii
List of Abbreviations……...…………………………………...………………………..xvi
Abstract…...……………………………………………………………………………..xix
CHAPTER
I. Introduction……...……………………………………………..………………….1
II. Cell and Growth Factor Loaded Keratin Hydrogels for Treatment of Volumetric
Muscle Loss in a Mouse Model……………………………………………….....60
III. Keratin Hydrogel Enhances In Vivo Skeletal Muscle Function in a Rat Model of
Volumetric Muscle Loss………………………………………………………....99
IV. Investigation of Tissue Engineered Muscle Repair Constructs for Use in Cleft Lip
Secondary Revision Procedures in a Rat Model………………………………..134
V. Summary, Conclusions, and Future Directions…………………………….......241
APPENDIX
1. Implantation of In Vitro Tissue Engineered Muscle Repair Constructs and Bladder
Acellular Matrix Partially Restore In Vivo Skeletal Muscle Function in a Rat Model
of Volumetric Muscle Loss Injury……………….………………...……...……269
2. Ethical Considerations in Tissue Engineering Research: Case Studies in
Translation……………………………………………………………………..308
4. Growth Factor Release from Keratin Hydrogel Formulation Used in Mouse LD
and Rat TA Models (Unpublished Data from KeraNetics, LLC)…..................351
Curriculum Vitae……..…………………………………………………………..……353
vii
LIST OF FIGURES AND TABLES
CHAPTER I: Introduction
Figure 1. Skeletal Muscle Form and Histology of Immature and Mature Myofibers........6
Figure 2. Skeletal Muscle Injury and Repair in VML…………………………………....8
Table I. Summary of Key Growth Factors and Trophic Factors involved in Skeletal
Muscle Repair……………………………………………………………………………12
Figure 3. Platform of Technologies for TE Skeletal Muscle…………………………....26
Figure 4. Keratin Structure.………………………………………………………….…..28
Figure 5. Growth Factor Release from 20% Keratin Gels………………………………30
Figure 6. TEMR Technology……………………………………………………………33
Figure 7. Data from Study Using TEMR in Mouse LD (from Machingal, 2011 used with
permission)……………………………………………………………………………….35
Figure 8. Muscle Regeneration in a Mouse LD VML model (Corona, 2012 used with
permission)……………………………………………………………………………….36
Figure 9. Data from TEMR Study in Rat TA (from Corona, 2014 used with
permission)……………………………………………………………………………….39
CHAPTER II: Cell and Growth Factor Loaded Keratin Hydrogels for Treatment of
Volumetric Muscle Loss in a Mouse Model
Table I. List of Groups, Components, and Number of Animals (n)……….....................68
Figure 1. Surgical Flow of VML injury and Keratin Implant…………...........................69
viii
Table II. Summary of Latissimus Dorsi Muscle Physical and Functional Measurement
Data……………………………………………………………………………………....72
Figure 2. Muscle Function Analysis at 2 Months Post-Surgery…………………...……74
Figure 3. Selected Force-Frequency Curves with Fitted Dose-Response Curves……....76
Figure 4. Representative Tissue Gross Morphology…………………………..…….…..77
Figure 5. Mouse LD Cell and Tissue Morphology and Functional Protein Expression in
Uninjured, NR, and BAM Treated Tissues…………………………………………...….79
KN, KN+I, KN+b, and KN+I+b Treated Tissues…………………..……………..……..81
KN+M, KN+M+I, KN+M+b, and KN+M+I+b Treated Tissues…………………..……83
CHAPTER III: Keratin Hydrogel Enhances In Vivo Skeletal Muscle Function in a Rat
Model of Volumetric Muscle Loss
Table I. Design of Experimental Groups Submitted to VML Injury…..........................103
Figure 1. Creation of VML in Rat TA and Injection of Keratin Hydrogel………….....106
Figure 2. Contribution of Keratin Hydrogel Formulation to Muscle Regeneration at 4 and
8 Weeks…………………………………………………………………………………110
Table II. In Vivo Functional Analysis of TA Muscle at 8 Weeks Post-Injury…...….…113
Figure 3. Keratin Hydrogel Promotes Continuous Functional Recovery over Time…..115
Table III. In Vivo Functional Analysis of TA Muscle at 12 Weeks Post-Injury……....117
Figure 4. Gross Morphology and Histological Analysis of TA after Keratin Based
Hydrogel Treatment…………………………………………………………………….119
ix
CHAPTER IV: Investigation of Tissue Engineered Muscle Repair Constructs for Use in
Cleft Lip Secondary Revision Procedures in a Rat Model
Table I. Treatment Groups……………………………………………………………..140
Figure 1. Surgical Flow of Rat LD VML Injury and repair with TEMR…………..….140
Table II. Summary of Latissimus Dorsi Muscle Physical and Functional Measurement
Data at 2 Months Post-Injury…………………………….……………………………..144
Table III. Summary of Latissimus Dorsi Muscle Physical and Functional Measurement
Data at 4 Months Post-Injury………………………………………………….……..…146
Table IV. Summary of Latissimus Dorsi Muscle Physical and Functional Measurement
Data at 6 Months Post-Injury……………………………………………….………..…148
Figure 2. Force-Frequency Curve with Dose-Response Curves for Males at 2mos…...150
Figure 3. Comparison of Po across Treatment Groups and Timepoints in Males…...…151
Figure 4. Comparison of Pt across Treatment Groups and Timepoints in Males………151
Figure 5. Comparison of P30 Hz across Treatment Groups and Timepoints in Males…..152
Figure 6. Comparison of Po across Treatment Groups and Timepoints in Females..….152
Figure 7. Comparison of Pt across Treatment Groups and Timepoints in Females……153
Figure 8. Comparison of P30 Hz across Treatment Groups and Timepoints in Females..153
Table V. Body Weight at Implant and Explant, Defect Weight & Size, and LD Weight
and Lo for Males and Females………………………………………………………….154
Figure 9. Macroscopic Comparison of Selected Representative Tissues across Treatment
Groups for Males at 2mos………………………………………………………………157
Figure 10. Macroscopic Comparison of Selected Representative Tissues across
Treatment Groups for Males at 4mos……………………………..……………………158
x
Treatment Groups for Males at 6mos……………………………………..……………159
Treatment Groups for Females at 2mos………………………………………...………160
Table VI. Summary of Macroscopic Findings in Males…………………………….…163
Table VII. Summary of Macroscopic Findings in Females………………………...….164
Figure 15. Hematoxylin and Eosin and Masson’s Trichrome Staining of Tissues from
Selected Representative Tissues: 2mos Male TEMR…………………………………..168
Selected Representative Tissues: 2mos Male BAM……………………..……………..170
Selected Representative Tissues: 2mos Male NR…………………..…………………..172
Selected Representative Tissues: 2mos Female TEMR……………………….………..174
Selected Representative Tissues: 2mos Female BAM……………………...………..…176
Selected Representative Tissues: 2mos Female NR………………………..………..…178
xi
Selected Representative Tissues: 4mos Male TEMR……………………………..……180
Selected Representative Tissues: 4mos Male BAM…………………..………..………182
Selected Representative Tissues: 4mos Male NR……………..…………………..……184
Selected Representative Tissues: 4mos Female TEMR……………………….…..……186
Selected Representative Tissues: 4mos Female BAM…………………………….....…188
Selected Representative Tissues: 4mos Female NR…………………………..……..…190
Selected Representative Tissues: 6mos Male BAM………………………..…………..194
Selected Representative Tissues: 6mos Male NR………………………………………196
Selected Representative Tissues: 6mos Female TEMR………….……………………..198
xii
Selected Representative Tissues: 6mos Male TEMR……………………………..……202
Table VIII. Summary of Key Histological Findings for TEMR and BAM Treated
Tissues in Males…………………...……………………………………………………204
Table IX. Summary of Key Histological Findings for TEMR and BAM Treated Tissues
in Females……………………………………………….……………………………...206
Supplemental Figure 1. Macroscopic Images of all 2mos Male TEMR Tissues..……215
Supplemental Figure 2. Macroscopic Images of all 2mos Male BAM Tissues………216
Supplemental Figure 3. Macroscopic Images of all 2mos Male NR Tissues…....……217
Supplemental Figure 5. Macroscopic Images of all 4mos Male BAM Tissues....……219
Supplemental Figure 6. Macroscopic Images of all 4mos Male NR Tissues..………..220
Supplemental Figure 8. Macroscopic Images of all 6mos Male BAM Tissues............222
Supplemental Figure 9. Macroscopic Images of all 6mos Male NR Tissues..…….….223
Supplemental Figure 10. Macroscopic Images of all 2mos Female TEMR Tissues….224
Supplemental Figure 11. Macroscopic Images of all 2mos Female BAM Tissues…...225
Supplemental Figure 12. Macroscopic Images of all 2mos Female NR Tissues….….226
Supplemental Figure 15. Macroscopic Images of all 4mos Female NR Tissues……..229
xiii
Supplemental Figure 18. Macroscopic Images of all 6mos Female NR Tissues……..232
CHAPTER V: Summary, Conclusions, and Future Directions
Table I. Experimental Groups for Optimization of Keratin Hydrogel Cellularity……..253
Table II. Experimental Groups for In Vivo Keratin Hydrogel Coupled Growth Factor
Dose Response Experiment………………………………………………………….....256
Table III. Experimental Groups for Mechanical Testing of Test Articles in Series with
Rat LD Muscle…......…………………………………………………………………...258
Table IV. Experimental Groups for Study of Laminate and Multi-Seeded TEMR……260
APPENDIX 1: Implantation of In Vitro Tissue Engineered Muscle Repair Constructs and
Bladder Acellular Matrix Partially Restore In Vivo Skeletal Muscle Function in a Rat Model
of Volumetric Muscle Loss Injury
Table I. TA muscle in vivo functional capacity 12 weeks post-injury……………..…293
Table II. Summary of BAM scaffold and TEMR construct in vivo performance…….294
Figure 1 TA muscle VML injury repair with BAM scaffold and TEMR constructs…295
Figure 2. In vivo isometric torque of the anterior crural muscles before and after TA
muscle synergist ablation and VML…………………………………………....296
Figure 3. TEMR improves functional recovery of rat TA muscle with VML injury…297
Figure 4. TA muscle gross morphology 12 weeks after VML and treatment…….......298
Figure 5. TA muscle tissue morphology following transplantation of BAM and TEMR
constructs……………………………………………………………….………299
xiv
Figure 6. TA muscle fiber generation and inflammation following transplantation of
BAM and TEMR constructs……………………………………………………300
APPENDIX 3. Growth Factor Release from Keratin Hydrogel Formulation Used in
Mouse LD and Rat TA Models (Unpublished Data from KeraNetics, LLC)
Figure 1. Release of IGF-1 and bFGF from Blends of KOS:KTN……………...........351
xv
LIST OF ABBREVIATIONS
AA: Antibiotic Antimycotic
ADSC: Adipose Derived Stem Cell
BAM: Bladder Acellular Matrix
BMMSC: Bone Marrow Mesenchymal Stem Cell
BMP: Bone Morphogenic Protein
DMEM: Dulbecco’s Modified Eagle Medium
ECM: Extracellular Matrix
EDS: Glutamic Acid Aspartic Acid Serine
FBS: Fetal Bovine Serum
FGF: Fibroblast Growth Factor (bFGF, FGF-2: Basic Fibroblast Growth Factor)
GAG: Glycosaminoglycan
GIF: Gamma Interferon
GMP: Good Manufacturing Process
HGF: Hepatocyte Growth Factor
HS: Horse Serum
HSPG: Heparan Sulfate Proteoglycans
IGF: Insulin-like Growth factor
IL: Interleukin
IND: Investigational New Drug
KAP: Keratin Associated Protein
KIF: Keratin Intermediate Filament
xvi
KOS: Keratose
KTN: Kerateine
LD: Latissimus Dorsi
LDV: Leucine Aspartic Acid Valine
LIF: Leukemia Inhibitory Factor
M1: Type 1 Macrophage
M2: Type 2 Macrophage
MHC: Myosin Heavy Chain
MMP: Matrix Metalloproteinase
MPC: Muscle Progenitor Cell
NGF: Nerve Growth Factor
NR: No Repair
OCT: Optimum Cutting Temperature
Po: Maximum Isometric Contraction Force
PAA: Peracetic Acid
PBS: Phosphate Buffered Saline
PCSA: Physiological Cross Sectional Area
PFA: Paraformaldehyde
QSR: Quality Service Regulation
TA: Tibialis Anterior
TE: Tissue Engineered
TGA: Thioglycolic Acid
TEMR: Tissue Engineered Muscle Repair
xvii
TGF: Transforming Growth Factor
TNF-α: Tissue Necrosis Factor alpha
VEGF: Vascular Endothelial Growth Factor
VML: Volumetric Muscle Loss
xviii
ABSTRACT
Volumetric muscle loss (VML) is defined as the irrecoverable loss of function in
skeletal muscle as a result of tissue loss. Herein, cell and growth factor loaded keratin
hydrogels and Tissue Engineered Muscle Repair (TEMR) constructs consisting of cell
seeded, cyclic stretched bladder acellular matrix (BAM), were investigated in VML
injuries in which the overriding hypothesis was that these technologies would improve
functional recovery and tissue morphology in otherwise irrecoverable injuries.
In the presented studies, investigation of keratin hydrogels in mouse latissimus
dorsi (LD) and rat tibialis anterior (TA) injuries determined that keratin combined with
insulin like growth factor-1, IGF-1, and basic fibroblast growth factor, bFGF, (KN+I+b)
resulted in the highest recovered contraction force. In the mouse, improvement was
significantly greater than that of all groups besides keratin alone and keratin combined with
muscle progenitor cells and IGF-1. In the rat, these findings were different from no repair
and BAM, but not from keratin alone or KN+b. Histological findings in both studies
indicated keratin combined with both growth factors, KN+I+b, resulted in improved tissue
quality with evidence of new muscle tissue formation.
In the investigation of TEMR in a novel model of VML with bio-relevance to
secondary revision of cleft lip, constructs were introduced into a surgically created defect
in the rat LD which was ~4 times larger than our previously largest model. To determine
the effectiveness of TEMR in the new model, in vitro functional analysis as well as
morphological and histological analysis were conducted on whole excised muscles at 2, 4,
6 months after implant. Results indicated that while the greatest improvement in function
was seen in TEMR treated tissue at 2 months, the findings were not significantly different
xix
from BAM treated and untreated tissues and no difference was seen at later timepoints.
Interestingly, morphological and histological findings indicated that TEMR resulted in
improved vascularity and attenuated inflammation compared with BAM treated tissues.
Investigation of these technologies indicated their capacity to improve muscle
function and form and contributed to the overall goal of our work to develop a platform of
TE skeletal muscle therapies capable of treating a diversity of VML presentations.
xx
CHAPTER I
Introduction
Hannah Besser Baker
Department of Biomedical Engineering
Wake Forest University School of Medicine, Winston-Salem, NC 27157
Affiliations: Virginia Tech-Wake Forest School of Biomedical Engineering and Sciences,
Winston-Salem, NC 27157; Wake Forest Institute for Regenerative Medicine,
Winston-Salem, NC 27101
Note: This chapter represents the majority of a manuscript in preparation for submission
as a review of tissue engineering approaches to treating Volumetric Muscle Loss injuries.
This chapter was composed by Hannah Besser Baker with editorial guidance from
George J. Christ, Ph.D.
1
Clinical Significance
Extremity and craniofacial traumatic injuries are common among civilians and
active military personnel alike. In a report detailing the resources devoted to injuries on the
battlefield, extremity trauma ranked as the most common injury, having the longest
inpatient period, and accounting for 65% of the total disability costs calculated (1). High
energy explosions are responsible for as much as 81% of war conflict extremity injuries (1,
2). In civilians traumatic injuries are typically a result of gunshot wounds and motor
vehicle accidents, while on the battlefield, explosions are the major cause. In addition to
gunshot wounds, civilians may face volumetric muscle loss as a result of several other
indications. Some include the removal of cancerous tissue, congenital defects such as cleft
lip or palate, and muscle-neurological indications such as Bell’s palsy and Moebius
Syndrome (3, 4). Among the most prevalent of non-traumatic VML indications is cleft lip
which occurs alone or with cleft palate in 10.5/10,000 live births in the United States (5).
VML varies greatly in presentation with a wide range of complexity and severity,
especially when several tissues are involved. For example, a blast injury to the extremities,
head, or neck may result in burns, bone fracture, and can cause a variety of muscular
injuries that can lead to loss of muscle function and amputation of the limbs (6, 7).
Standard Treatment
When a significant portion of muscle has been lost, surgical management using
muscle flap transfer is the standard treatment (7-10). As a result of the various conditions
surrounding volumetric muscle loss, the native structure of the muscle, including the shape,
the vascularity, and the innervation, must be considered when choosing a repair strategy;
2
not just one type of graft is advisable for all situations. For example, in reconstruction of a
large craniofacial muscle defect the latissimus dorsi myocutaneous flap may be preferred
for its greater size. However, in the case of facial reanimation the neurovascular transfer of
a gracilis free flap may be preferred due to its particular innervation. Moreover, in the case
of lower extremity trauma, the flap which most adequately fills the space and has the most
similar vasculature is chosen (3, 9, 11).
While microsurgical technique has made major strides in reducing the donor site
morbidity and increasing the aesthetic quality of muscle flap grafts for traumatic VML and
orofacial defects, such setbacks remain present. Flap transfers have high risk of
complication and, due to the complexity of the procedure and lengthy hospitalization time,
are also costly (12, 13). For muscular defects such as cleft lip, primary surgical revision
involves repositioning of the skin and muscular tissue of the nose and nasal floor, however
this treatment suffers from graft tissue deficiency, scar formation, and stigmatic secondary
deformities (5, 14, 15). In addition, revision of secondary cleft lip/palate deformities often
requires an additional muscular graft and thus further donor site morbidity (15).
Research Considerations
The limitations of conventional treatments for VML have spurred tissue
engineering research endeavors for alternative therapies. It is important to note that unlike
minor injuries such as contusions and over exertion injuries and severe trauma injuries such
as compartment syndrome and ischemia-reperfusion injuries, VML injuries to skeletal
muscle pose an exceptional challenge due to the physical loss of muscle and the
surrounding matrix. Significant loss of tissue coupled with the absence of proper
3
scaffolding for regeneration results in injuries that cannot properly heal through the natural
regenerative process (16). Without intervention, the remodeling phase of the healing
process results in mostly non-functional scar tissue (17-21).
Skeletal Muscle Background
Skeletal Muscle Structure and Function
Comprising almost half of the human body’s mass, skeletal muscle enables
coordinated motion to the body through attachments to the skeleton (19). Muscle consists
of bundles of muscle fibers of which there are three types: type I which are slow twitch
fibers that do not fatigue easily, type IIA fibers that are fast twitch fibers that are resistant
to fatigue, and type IIX which are fast twitch and not resistant to fatigue. These fibers exist
as groups of bundled muscle fibers surrounded by connective tissue and interwoven with
the nerves and blood vessels that are vital to proper muscle function. The connective tissue
layers extend beyond the length of the muscle tissue to form tendons. A single muscle is
covered tendon to tendon with an outer protective sheath of connective tissue, the
epimysium, which holds together several bundles of muscle fibers called fascicles. Each
fascicle is surrounded by a connective tissue layer called perimysium. The individual fibers
within fascicles are multinucleated muscle cells, myocytes, which are each surrounded by
a plasma membrane, several layers of extracellular matrix which are referred to as the
sarcolemma, as well as an additional outer connective tissue layer called endomysium.
Below the sarcolemma are three additional layers of extracellular matrix proteins which
make up the basal lamina. Between the basal lamina and the sarcolemma there exists a
4
quiescent population of satellite cells that will regenerate tissue after injury (Figure 1) (19,
22).
Contained within the sarcolemma are the cellular organelles including nuclei,
which reside just below the sarcolemma in mature muscle fibers, the Golgi apparatus,
mitochondria, and the sarcoplasmic reticulum. Each myocyte contains several myofibrils
which are bundles of contractile proteins, myofilaments. Myofilaments consist of thick
filaments, made mostly of the protein myosin, and thin filaments, made mostly of the
protein actin. Actin and myosin overlap to form the basic contractile unit of muscle, the
sarcomere.
The sarcoplasmic reticulum consists of branches that surround each myofibril and
form terminal cisterna that store calcium for muscle contraction. At the midpoint of the
overlap between actin and myosin filaments, the terminal cisternae abut invaginations
called transverse tubules that travel from the surface of the sarcolemma deep into the
muscle cells along the sarcoplasmic reticulum (23).
The motor nerves which drive muscle function have axons that branch many times
into terminal axons that each interact with only one myocyte at the motor end plate of the
neuromuscular junction. A group of myocytes innervated by the same nerve-cell axon are
called a motor unit. An action potential propagated down a motor nerve will cause release
of acetylcholine at the presynaptic axon into the synaptic cleft. The acetylcholine binds
receptors on an adjacent post synaptic region of the sarcolemma which causes
depolarization of the sarcolemma and t-tubules.
5
Figure 1. Skeletal Muscle Form and Histology of Immature and Mature
Myofibers. Muscle exists as bundled bundles of fibers surrounded by connective
tissue and interwoven with nerves and blood vessels. The epimysium is a protective
sheath surrounding the whole muscle. The perimysium surrounds bundles of fibers
called fascicles. The endomysium surrounds individual fibers consisting of Myocytes
and satellite cells. Upon injury satellite cells are activated to become myoblasts which
differentiate into immature myotubes which become mature fibers of which the nuclei
are present at the periphery of fibers. (Figure from Huard, 2002 used with permission)
6
Activated voltage gated dihydropyridine receptors on the t-tubule will induce a
conformational change in colocalized ryanodine receptors on the sarcoplasmic reticulum
which causes a transient release of calcium from the terminal cisternae into the cytoplasm.
The released calcium ions interact with the protein troponin-C on actin filaments resulting
in a conformational change that allows the actin to bind myosin and contraction of the
sarcomere to occur. The contraction ends when acetylcholinesterases break down
acetylcholine, calcium is recycled to the sarcoplasmic reticulum, and troponin conforms to
prevent actin-myosin interaction (23).
Muscle can contract in three ways: eccentric, isometric, and concentric. In eccentric
contraction the muscle contracts with less force than the applied load and lengthens. During
isometric contraction, the muscle contracts with a force equal to the load and there is no
change in length. In concentric contraction the muscle contracts with a force greater than
the load and shortens (23).
Skeletal Muscle Injury Pathology
Skeletal muscle trauma is variable in pathology as well as severity, however the
manifestation of distinct trauma injuries are similar at the cellular level. The major
processes of skeletal muscle damage and healing have been well documented (17-20, 24).
These phases overlap with one another, but can be distinguished as destruction, repair, and
remodeling (Figure 2) (17).
7
Figure 2. Skeletal Muscle Injury and Repair in VML. Injury resulted in
disruption of the basal lamina (a). Invading macrophages clear the debris (b).
Activated satellite cells migrate to the injury site and differentiate into myoblasts
while fibroblasts begin to create scar tissue (c). The myoblasts fuse to form
myotubes at the ends of the injured fibers (d). In VML the fibroblast production of
scar tissue exceeds the regenerative capacity of muscle and a scar tissue gap is
formed within the defect (e). The resulting scar formation will cause denervation
for previously innervated tissue (f). (Figure from Turner, 2012 used with
permission)
8
Destruction of Damaged Tissue: Necrosis and Phagocytosis
Mechanical disruption of the sarcolemma and basal lamina results in an influx of
extracellular calcium activating calcium-dependent proteases, such as calpains, which
dismantle myofibers and the cytoskeleton. This process is localized to the injured fibers
through the formation of a contraction band-hematoma in the void. Further destruction on
either side of the contraction band arises as circulating inflammatory cells are recruited and
activated by cytokines secreted by the injured tissue. Additional inflammatory cells are
also released and activated from within the muscle and disrupted blood vessels. The first
inflammatory cells to congregate around the damaged fibers are neutrophils, present
between two and 24 hours after injury. These are followed by an initial wave of phagocytic
macrophages which peaks around 48 hours. Thereafter a second wave of macrophages
arrives, peaking around four days and remaining for as long as several weeks. The early
arriving cells begin to degrade the necrotized tissue and release cytokines and growth
factors which amplify the recruitment of inflammatory cells and promote the release of
satellite cells from nearby intact basal lamina. An abundance of research supports the
theory of subpopulations of macrophages playing distinct roles in muscle repair (25-29).
Early arriving macrophages (type 1 macrophages) are associated with the degradation of
necrotized tissue through phagocytosis and with the release of pro-inflammatory factors.
Later arriving macrophages (type 2 macrophages) are considered to be responsible for
regeneration of tissue by inhibiting degradation through the secretion of anti-inflammatory
proteins and promoting the fusion of satellite cells to myofibers.
9
Repair of the Injured Myofibers
Satellite cells are primarily responsible for repairing injured muscle (30). These
cells are either physically released upon disruption of the basal lamina or migrate from the
healthy tissue to damaged areas. Growth factors, chemokines, and cytokines secreted by
muscle and inflammatory cells and released from stores within the extracellular matrix
control the activation, proliferation, and differentiation of the satellite cells. Upon
activation, cells will rapidly proliferate. Proliferating cells divide into both an
undifferentiated cell population to repopulate the store of quiescent satellite cells as well
as muscle precursor cells. The precursor cells differentiate into myoblasts and fuse to intact
fibers or to one another to form multinucleated myotubes which will also fuse to uninjured
fibers. Finally growing myotubes will mature into myofibers in which the nuclei move
from a central to a peripheral location (31, 32).
Remodeling of the Tissue
Newly formed myofibers will begin to fill the injury site, eventually contacting the
contraction band. By this time the contraction band has been reorganized by invading
fibroblasts. Initially, the inflammatory cells break down the hematoma which is replaced
by a network blood derived fibrin and fibronectin. The cross-linked network provides a site
for invading fibroblasts to adhere and subsequently secrete extracellular matrix proteins.
With time the contraction band is able to provide the injury site with the necessary
mechanical integrity to withstand contractions, but this reorganization makes it difficult for
the muscle fiber stumps on either side to penetrate and reunite. If the damage to the muscle
is too severe, as in volumetric muscle loss, the fibroblast ECM of the band will have created
10
an impenetrable scar. The muscle fiber stumps will attempt to reunite by branching and
even traveling outside of the basal lamina in an effort to push through the scar tissue, but
ultimately will form a musculotendinous junction with the scar tissue. In cases of less
severe damage in which the basal lamina remains intact, the regenerated myofibers will
extend from either side of the injury until they reunite. Muscle that regenerates in this
fashion is morphologically and histologically indistinguishable from undamaged tissue
(31, 32).
Chemical and Physical Cues Guiding Regeneration
After muscle tissue has been damaged, there are intricate interactions between cells
and the extracellular matrix and the milieu of secreted factors which play large roles in the
overall regenerative process. These central regenerative mechanisms are important
considerations in development of a tissue engineered treatment for VML.
Secreted Factors
Substantial effort has been put into isolating the factors that regulate the major
processes of muscle injury repair and regeneration. Several authors have thoroughly
reviewed the source and mechanisms of a wide variety of these factors, only the key factors
will be summarized here (17-21, 24, 33). A table has been included for reference (Table I).
11
Factor Source Role References
Allen, 1995;
Activation of satellite cells,
Bischoff, 1997
Myocytes, recruitment of MPCs to
HGF Hayashi, 2004;
MPCs injury site, inhibition of
Tatsumi, 1998;
differentiation
Tatsumi, 2004
Allen, 1989;
Activation and
Myocytes, Bischoff, 1997
proliferation of satellite
macrophages, Charge, 2004;
FGF cells, inhibition of
endothelial Florini, 1996
differentiation, potential
cells, platelets Sheehan, 1994;
influence on IGF-1
Thompson, 1989
Promotes differentiation,
Allen, 1989;
MPCs, in presence of mitogen
IGF-1 Florini, 1996
macrophages such as FGF promotes
Hayashi, 2004
proliferation
Allen, 1989;
Inhibits myoblast Bischoff, 1997
Macrophages,
proliferation and Charge, 2004;
vascular
TGF-β1 differentiation, promotes Florini, 1989
endothelial
differentiation of MPCs Kirk, 2000;
cells, platelets
into myofibroblasts Li, 2002; Li, 2004
Massague, 1986
Promote myoblast Arnold, 2007;
TNF-α and Type 1
proliferation, impair Charge, 2004
IL-1β macrophages
differentiation Ciciliot, 2010
Promote proliferation of Charge, 2004;
LIF Myocytes
MPCs Wang, 2008
Promote transition from
Differentiating
IL-4 M1 to M2 phenotype in Arnold, 2007
myoblasts
macrophages
Induce M2 phenotype,
Type 2 recruitment of myoblasts, Arnold, 2007;
IL-10
macrophages promotion of Deng, 2012
differentiation
Table I. Summary of Key Growth Factors and Trophic Factors involved in
Skeletal Muscle Repair.
12
HGF and FGF: Activators of Satellite Cells and Proliferation, Inhibitors of
Differentiation
Hepatocyte growth factor, HGF, is secreted by uninjured skeletal muscle cells and
muscle progenitor cells and is stored in the extracellular matrix. It has been shown to be
the major cytokine responsible for the activation of quiescent satellite cells both in vitro
and in vivo (34-37). HGF is untethered from the extracellular matrix upon mechanical
stretch or local injury and acts in a paracrine-autocrine manner by binding to the c-met
receptors on nearby satellite cells (34, 35). Studies have demonstrated the presence of the
activated form of HGF at early time points after injury surrounding myocytes and myotubes
in vivo (37, 38). Additionally, these investigations further demonstrated the established
effects of HGF as well as its chemotactic effects on nearby satellite cells (24, 38). When
added to in vitro cultures of myocytes in differentiation media, increased dosage of HGF
resulted in increased cell proliferation whereas the formation of myotubes decreased (24,
34-37, 39, 40).
Several studies have shown that a handful of cytokines in the Fibroblast Growth
Factor family, FGFs, have also been implicated in satellite cell activation and proliferation
as well as inhibition of MPC differentiation (24, 38, 41-43). Also similar to HGF, FGFs
act directly on satellite cells via FGF receptors and are stored in the basement membrane
and connective tissue surrounding myofibers through attachment to proteoglycans (44).
Unlike HGF, several cell types and blood borne platelets present in the muscle injury
environment have been found to produce FGFs including macrophages and vascular
endothelial cells (38, 43). In experiments in which in vitro cultures of muscle derived cells
13
were treated with a combination of growth factors, it was demonstrated that FGFs may
influence the activity of IGFs; however this mechanism remains unclear (43, 45).
IGF
Insulin-like growth factors I and II, IGF-I and IGF-II, are well known regulators of
regeneration in many tissues (24). IGFs are produced by myocytes as well as invading
macrophages and are present in the active form during the early and middle time points of
regeneration (37). Both IGFs have been shown to promote the contradictory activities of
proliferation and differentiation of muscle precursor cells; however, the mechanism
surrounding the differential activity is debated (37, 45). It has been suggested that in the
presence of a mitogen such as FGF or HGF, IGF-1 will also act as a mitogen and in the
absence of a mitogen IGF-1 will promote differentiation (43).
TGF-β1
Transforming growth factor beta 1, TGF-β1, is a key negative regulator during
regeneration. TGF-β1 is secreted by invading immune cells, endothelial cells, and platelets
(38, 46). It is well documented as acting to inhibit myoblast proliferation and differentiation
(24, 38, 43, 47-49). Alternatively, TGF-β gradients have been shown to recruit adjacent
activated satellite cells (38). While TGF- β is known to inhibit the differentiation of satellite
cells into myoblasts, it has been demonstrated to cause the differentiation of activated
muscle precursor cells into myofibroblasts (38, 43, 46, 50). Therefore, it is no coincidence
that studies have shown an increased expression of TGF- β1 to be associated with increased
extracellular matrix production and scar formation (17).
14
Additional Relevant Trophic Factors
Several other secreted factors isolated from regenerating skeletal muscle have been
proven to play a role in repair and remodeling events. Macrophages contribute greatly to
these processes and have been deemed essential for proper regeneration to take place (26,
27). Early arriving inflammatory or type I macrophages in culture with satellite cells
promote proliferation and inhibit differentiation (18, 24, 27). Type I macrophages secrete
the inflammatory cytokine tumor necrosis factor alpha, TNF-α, and interlukin-1β, IL-1β,
which promote myoblast proliferation and impair differentiation (51). Leukemia inhibitory
factor, LIF, of the IL-6 family of cytokines is secreted by muscle cells and has been shown
to increase proliferation of MPCs in culture (24, 51). Differentiating myoblasts secrete IL-
4 which in culture with type I macrophages, pro-inflammatory M1 population of
macrophages, alters their phenotype to that of a later arriving, anti-inflammatory or type II
macrophage, M2 population (27). Anti-inflammatory factors secreted by the M2 phenotype
include interlukin-10 and TGF-β which are respectively associated with inducing the M2
phenotype and with regulating the recruitment and differentiation of myoblasts (27, 52).
The Extracellular Matrix
The extracellular matrix (ECM) of skeletal muscle consisting of the basal lamina
and the sarcolemma physically and chemically guides the activation, proliferation, and
differentiation of satellite cells released after an injury (53). These properties are derived
from the proportions and arrangement of constituent proteins and polysaccharides. The
ECM is made up of collagens I and IV, laminins, fibronectin, and the glycosaminoglycans
(GAGs): heparan sulfate, chondroitin sulfate, and hyaluronic acid (54). The strength and
15
structure of the sarcolemma or stromal matrix results from a network collagen I fibers
decorated with hyaluronan and GAGs. The basement membrane is primarily made up of
the non-fibrillar collagen IV interwoven with fibronectin and laminin (33).
Cell integrin attachment sites on collagen I promote myoblast cell binding and
collagens I and IV as well as laminin promote myoblast proliferation. Components of the
stromal and basement matrix: Fibronectin and hyaluronan, are known to promote the
differentiation of myoblasts to myocytes (55). Heparan sulfate proteoglycans, HSPG, play
a very important role in the regulation of growth factors. Specifically, the growth factors
HGF and FGF which play roles in satellite cell activation and proliferation are stored in an
inactive state while tethered to HSPGs in the ECM (39, 40).
It has been shown that the ECM provides physical cues which can guide the differentiation
of stem cells according to stiffness (56, 57). In addition, the specific ultrastructural
properties of the remaining intact basal lamina guide alignment of the fusing myocytes and
new myofibers (31).
Mechanical Cues
Together, the ECM and the secreted factors enable the satellite cells to repair an
injury. A key mechanism enabling this collaboration is the effect of mechanical stiffness
on regenerating muscle tissue. Myocytes and especially fibroblasts sense and react to the
stiffness of their environment through mechanoreceptors (40, 57, 58). For muscle cells and
fibroblasts mechanical sensing directly involves ECM components. As the surrounding
intact muscle fibers contract and pull on the ECM surrounding the damaged tissue, the
filaments extending from the muscle cells and fibroblasts which are attached to the ECM
16
are physically pulled (40). For fibroblasts this results in increased secretion of scar tissue
proteins, while for muscle cells this promotes alignment and fusion (33, 50). In addition,
injury and further stretch on the ECM result in the release of growth factors bound to GAGs
which regulate the activation, proliferation, and differentiation of myoblasts (36, 39). This
is thought to begin with the secretion of matrix metalloproteinases, MMPs, by muscle cells
which stimulates nitric oxide synthesis. Nitric oxide mediates the release of HGF and IGF
from the ECM, two growth factors responsible for the proliferation and differentiation of
myoblasts (38-40).
Tissue Engineering Approaches to Volumetric Muscle Loss
Animal Models
To study treatments aimed to restore structure and function in VML, animal models
which adequately recapitulate the injury pathology are necessary. To mimic the human
condition, a proper animal model of volumetric muscle loss injury should entail the
physical removal of muscle tissue which results in a permanent loss of function. Further,
investigators must employ physiologically relevant assays of muscle form and function to
indicate that irrecoverable damage has been achieved. Several animal models exist which
have given insight into the regenerative process of skeletal muscle injuries, without causing
the permanent damage seen in VML. These models have however, successfully mimicked
the short term damage common to sports injuries and more importantly have captured the
general process of regeneration associated with all muscle injures (24). Methods used to
mimic and study sports related muscle damage have included rodent eccentric contraction
injuries and thermal injuries which produce large functional deficits at early time points,
17
but tend to regain almost all function within four weeks (59-64). Other models of muscle
injury have entailed the use myotoxins which disrupt membranes, cause cell lysis, and
inhibit neuro-transduction. The application of these toxins enabled the study of muscle
damage and repair mechanisms, but similarly to the sports injury models the toxin induced
injures fully recovered within three weeks or less (24, 47, 65, 66). While appropriate for
investigating repair and regeneration, these models resulted in full functional recovery
within weeks after the injury and are therefore not comparable to a VML injury. Several
groups have indeed developed animal models of VML in which there is permanent tissue
and functional loss. Surgical resection of tissue from muscles is one approach which results
in a functional deficit proportional to the amount of tissue removed. Groups have created
defects in this manner primarily in rodent and dog hindlimbs and abdominal walls (67-74).
Beyond the use of histological analysis, whole muscle force production capacity is a widely
employed physiologic measurement for studying skeletal muscle conditions. Whole
muscle peak tetanic force may be evaluated through nerve stimulation in vivo or electrical
or chemical stimulation in vitro through the use of a force transducer attached to the active
moment arm in vivo (the foot in the case of the hindlimb) or attached to either end of an
isolated muscle in vitro. When used with proper controls, such devices have enabled
researchers to quantitatively assess the extent of damage and recovery present for a given
VML model and investigated therapy (59, 61, 63, 64, 67, 74-76). Physiologically relevant
analyses such as functional testing serve to corroborate and give merit to histological
evidence. Realistic models of skeletal muscle injury coupled with appropriate assays will
serve to minimize translational distance and will provide an honest approximation of the
clinical application of a given technology.
18
Growth Factors
As previously described, many growth factors and cytokines influence the
regeneration of skeletal muscle after injury. These factors, usually secreted by cells
localized to the injury site, may be introduced artificially as human recombinant proteins.
Direct injection of IGF-1, basic-FGF (bFGF or FGF-2), and to a small extent nerve growth
factor, NGF, into a lacerated mouse hindlimb have enhanced the number and diameter of
newly formed muscle fibers and improved the ex vivo whole muscle force generation (77,
78). The exact role of the FGFs, such as bFGF, in vivo is debated as bFGF as well as FGFs
-4, and -6 have been shown to improve healing in some studies and to have no effect in
others (24, 79-82). Several groups have investigated the impact of LIF on regenerating
muscle in vivo in models of induced muscular dystrophy as well as traumatic injuries (24,
83, 84). In a rodent crush injury model, injections of LIF to the injury site increased the
size but not the number of regenerating myofibers (83). Perhaps a more feasible approach
to muscle regeneration using growth factors is the idea of limiting fibrosis at the wound
site (19). In preventing excessive scar tissue formation, the approach is to inhibit the
activity of TGF-β. TGF- β3, gamma interferon or GIF, and the molecule decorin have been
shown successful in inhibiting scar tissue formation in vivo (14, 77, 85). In a rodent
laceration model, injections of decorin alone improved muscle contraction force over
injections of IGF and decorin and IGF together; however IGF combined with decorin gave
the greatest new myofiber number and diameter (77). In a similar study, GIF also improved
force generation recovery and decreased scar tissue formation (85). The use of growth
factors for treatment of VML suffers due to the complex physical, spatial, and temporal
regulation and from promiscuity of growth factors among cells present in the injury site.
19
Nonetheless, the use of growth factors for treatment of VML has shown positive results,
with perhaps the most promising endeavors surrounding the use of antifibrotic factors.
Cellular Therapies
As it is well established that satellite cells are the major players in the natural repair
process, several researchers have investigated injectable muscle derived and purified
satellite cell therapies for skeletal muscle injuries (86-88). However, other investigators
have demonstrated that non-muscle stem cells are capable of differentiating into muscle
progenitor cells and promoting regeneration in skeletal muscle injuries (89-94). In such
studies, investigators have undertaken the use of primary muscle cells as well as adipose
derived stem cells (ADSCs) and bone marrow mesenchymal stem cells (BMMSCs) among
others for treatment of cryo-injury, laceration, crush injury, and loss of function due to
nerve denervation-reinnervation and cardiotoxin injection. In a study in which primary
myoblasts were injected into severely cryo-damaged mouse soleus muscles, the cell treated
muscles had significantly greater twitch and tetany tensions ex vivo (87). Similarly in a
rabbit tibialis anterior denervation-reinnervation model, primary isolated myoblasts
enabled improved in situ max contraction force at two months post injury, but at 14 months
there was no difference between treated and non-treated muscle contraction force (88).
Labeled ADSCs injected into a cardiotoxin damaged tibialis anterior muscles in rabbits
were found to be present in regenerating tissues fifteen days later and resulted in improved
muscle contraction force at two months post injection when compared with untreated
animals with damaged tissues. However, use of the freshly isolated ADSCs resulted in
adipose tissue development along the injection site (89). In a laceration injury in the rat
20
soleus, an injection of isolated ADSCs and matrigel resulted in improved ex vivo tetanic
contraction force relative to matrigel alone at two weeks post injury; however at four weeks
the contraction forces for the treatments were not different (92). Bone marrow
mesenchymal stem cells, have been investigated in a variety of regenerative medicine
applications, including their role in skeletal muscle injury repair (4). In crush injury model
in the rat soleus, direct injection of BMMSCs resulted in increased twitch and tetanic in
situ contraction force relative to untreated injured muscles (91, 93). Cell therapy shows
promise for the injuries described in these studies such as laceration cryo-damage, however
such cell therapies have not been investigated with success in larger VML injury models
in which the muscle tissue is no longer present. Further, direct cell transplant therapies
suffer from issues surrounding viability after transplant (95, 96)
Naturally Derived Scaffolds
As muscle progenitor cells along with other key cell types have been identified as
necessary components in skeletal muscle healing, the use of a variety of scaffolds alone to
attract cells to the injury site has been investigated. Several groups have researched the use
of decellularized extracellular matrices for use in treating skeletal muscle injuries. One
such study found that repair of a rat tendon injury using the BAM increased the presence
of multipotent progenitor cells when compared with control tissue, showcasing the ability
to act as a cellular chemoattractant (97). It has also been shown that after decellularization
such ECM scaffolds retain the protein structure, growth factors, and glycosaminoglycans
known to be involved with guiding muscle repair (98-101). Further, acellular ECM
scaffolds have also been shown to promote a favorable immune response as was the case
21
when BAM and an acellular body wall matrix were used to repair a body wall defect in rats
and the majority of invading macrophage populations were the favorable type two variety
at several early and later time points after implant (25). This finding also correlated to a
greater remodeling of the implant.
Several other studies have demonstrated varied success of various acellular ECM
scaffolds in repairing an abdominal wall defects. In studies using small intestine submucosa
and acellular skeletal muscle matrix in animal models of abdominal wall defects there was
evidence of replacement of the scaffolds with host tissue and the presence of functional
skeletal muscle within the scaffold (68, 102, 103). However such findings are contradicted
by others such as one case in which a diaphragm acellular matrix implant in an abdominal
wall defect in rabbits led to fibrosis and did not enable muscle formation within the graft
site (70). Similarly varied results have been found using acellular matrices in extremity
muscles. In studies using acellular ECM in hindlimb defects in rats and dogs, results
showed evidence of moderate cell infiltration within the scaffolds but without functional
improvement (69, 73). Another study evidenced the opposite phenomena: using acellular
skeletal muscle matrix in a rat hindlimb defect resulted in fibrotic tissue formation with a
lack of muscle cell presence within the scaffold, however there was functional
improvement when compared with no treatment. The author has attributed this to
“functional fibrosis,” in which the fibrotic tissue bears weight and prevents overload injury
to the surrounding muscle (104). In a study using porcine BAM to treat defects in mouse
and human quadriceps there was similar improvement in function with only minimal
evidence of muscle formation within the implants (105).
22
Other naturally derived scaffolds such as silk fibroin, keratin, alginate, and chitosan
have been investigated in several applications including skeletal muscle regeneration due
to their capacity to be manipulated into various structures and to act as drug delivery
vehicles (106-108). In one study silk fibroin-chitosan blend scaffold was found to
outperform an acellular matrix when used for abdominal wall repair in guinea pigs. The
blended scaffold better integrated with host tissue and retained a greater tensile strength
when compared with human acellular dermal matrix (109).
Other successful use of tunable natural scaffolds has included the use of hydrogels
as growth factor carriers. In one case alginate beads loaded with IGF-1 and or VEGF were
implanted in a limb ischemia model in rodents and resulted in increased muscle fiber
regeneration and angiogenesis respectfully (and increased angiogenesis and fiber
regeneration when used together) (110). It is apparent that naturally derived scaffolds alone
and coupled with growth factors show promise in treating skeletal muscle injuries. While
ECM scaffolds stand to promote cellular infiltration at the injury site and improve muscle
function, these scaffolds have a varied capacity for regenerating muscle tissue within the
scaffold and in the worst cases resulted in non-functional, fibrotic tissue. On the other hand,
synthesized natural materials such as alginate, silk, and keratin offer the potential to deliver
drugs or growth factors to the injury site to further improve recruitment and infiltration of
cells and new tissue formation, but these have not yet shown efficacy in VML models.
23
Combination Therapies: Scaffolds, Cells, and Preconditioning
More complex strategies to treat VML injuries have involved the use of a
combinations of scaffolds, cells, growth factors, and preconditioning. As with the use of
naturally derived scaffolds alone, investigators have tested a variety of ECM as well as
other materials in conjunction with different cell types in several skeletal muscle injury
models (111, 112). Several authors have used natural scaffolds seeded with primary
isolations of skeletal muscle cells with success (67, 74, 76, 113-116). In a notable study
using a photo-cross linking hyaluronan based hydrogel loaded with freshly isolated satellite
cells, muscle progenitor cells, or freshly isolated muscle fibers in a mouse tibialis anterior
defect, the gel and satellite cell combination was found to enable a much greater repair of
muscle tissue and functional recovery compared with use of the gel alone or the gels with
MPCs or fibers (113). Fibrin constructs, as gels and microthreads, loaded with MPCs in
rodent defects were also shown to improve neo tissue formation, reduce collagen
deposition, and improve functional recovery compared with non-treated injuries (114,
115).
Other investigators have utilized growth factors either directly or through genetic
modification of cells in combination with scaffolds and MPCs for treatment of skeletal
muscle injury. Alginate loaded with MPCs, IGF-1, and vascular endothelial growth factor
(VEGF) improved functional recovery, cell engraftment, and minimized scar formation in
a combined ischemia and toxin injury in mice (116). However, another study using alginate
loaded with either MPCs or MPCs made to over express FGF-2 in a crush injury in rats
found that while FGF-2 over expressing cells in alginate evidenced modulation of
24
apoptosis and proliferation of injured tissue, neither treatment resulted in significant
improvements to muscle function compared with no treatment (117).
In addition to the use of growth factors in combination with cells and natural
scaffolds, another strategy to improve the success of tissue engineered constructs for
skeletal muscle entails preconditioning scaffolds using mechanical or electrical stimuli.
Use of bioreactors to provide these cues to cell seeded scaffolds in vitro aims to enhance
the differentiation and alignment of cells on the construct to improve the ability of the
scaffold to restore function in vivo (118). Use of cyclic stretch alone or coupled with
electrical preconditioning in vitro has been found to promote contractile protein synthesis,
to increase myotube formation, diameter, and alignment as well as to improve the
excitability and force production of the cells on the scaffold (119-122). Further, when
preconditioned allogeneic cell seeded BAM scaffolds were used in a mouse model of VML
injury, functional recovery was greater than that enabled by static cultured cell seeded
scaffolds (67, 74). While combination therapies have demonstrated the ability to promote
the restoration of form and function in skeletal muscle injury models, it is worth noting that
the regulatory pathway for these complex technologies remains an obstacle for
investigators (123).
Keratin Hydrogels and Tissue Engineered Muscle Repair Constructs
Further Development of a Platform of Technologies for VML
Tissue engineering approaches to treatment of VML are diverse and unlimited
when one considers the possibilities enabled by combination therapies. To match the
variety and complexity of skeletal muscle injuries and pathologies, a multitude of treatment
25
options are necessary. In establishing a platform of such treatments for VML presentations,
cell and growth factor loaded keratin hydrogels and cell seeded and preconditioned BAM
are to be investigated as is described in this proposal (Figure 3). To gain a better
understanding of these technologies and their potential for use in treating VML, necessary
background and foundational research shall be reviewed.
Figure 3. Platform of Technologies for TE Skeletal Muscle. All technologies are
cell delivery capable while silk and keratin can be altered to achieve a desired
degradation rate and can be modified to deliver drugs. The geometry of each therapy
facilitates its use. TEMR for muscles with sheet like architecture, Silk for solid
geometry injuries, and keratin hydrogels for complex geometries. In this proposal,
studies to investigate keratin hydrogels and TEMR for VML are outlined
26
Keratin Structure and Isolation
In human hair, trichocytic keratins are intermediate filaments which make up the
cortex and cuticle of hair fibers. They can be classified into three types: alpha, beta, and
gamma keratins of which the alpha and gamma components have been investigated (Figure
4a). Beta keratins make up the cuticle of the hair fiber and are difficult to isolate and have
structural properties which have not yet been found useful (124). Within the cortex, alpha
keratins assemble together into fibrous structures referred to as keratin intermediate
filaments (KIF) which make up 50-60% of the material and provide structure (Figure 4b).
Gamma keratins provide a supporting matrix and are referred to as keratin associated
proteins (KAPs). Gamma keratins make up 20-30% of the cortex. Alpha keratins exist as
heterodimers of type I (acidic) and type II (neutral-basic) keratins which further assemble
into 10nm intermediate filaments. Type I and type II keratins have a central alpha-helical
structure and a non-helical end domain but differ in amino acid composition and molecular
weight. Type I keratins have a higher molecular weight (40-60kDa) and a lower sulfur
content while type II keratins have a lower molecular weight (10-25kDa) and a higher
sulfur content (125-128).
27
Figure 4. Keratin Structure. Keratin intermediate filaments make up the bulk of hair
fibers (A). Acidic type I and basic type II keratins form helical dimers which
polymerize to form tetramers that make up protofibrils that further polymerize to form
intermediate fibers (B). Oxidation extraction of keratin converts cysteine bonds to
sulfonic acid while reductive extraction breaks the bonds but does not alter the
cysteine groups allowing the bonds to reform (C). The resulting proteins are referred
to as keratose and kerateine respectively and have distinct properties. (Keratin hair
illustration from hairkeratins.com, secondary to tertiary structure image from Sigma,
keratose and kerateine image from manuscript under review used with permission
from KeraNetics, LLC (Saul et al)).
28
Cortical keratins can be extracted by first chemically disrupting disulfide crosslinks
holding the fibers together followed by the use of denaturing solvents to solubilize the
proteins. The alpha and gamma fractions may then be purified using filtration and dialysis
(124). Reducing agents used for extraction will result in non-cross linked cysteine residues
for which the resulting keratins are referred to as kerateines (KTN). When oxidizing agents
are used in the extraction process the cysteine disulfide bonds are broken and converted to
sulfonic acid groups and the resulting extraction is referred to as keratose (KOS) (129,
130). Differences in the cysteine residue chemistry between KOS and KTN result in
different physical behaviors. Unpublished experiments carried out by collaborators using
keratin gels have demonstrated that KTN gels undergo a longer degradation time, have an
order of magnitude lower ratio of elastic to viscous moduli, and have a slower release of
loaded growth factors when compared with KOS, due to the reformation of disulfide bonds
in the free cysteine groups (Figure 4c)*. Specifically, KTN will form disulfide cross links
which result in a slower degradation rate and longer release profile of growth factors as
well as resulting in a stiffer less easily flowing gel (Figure 5)*. As such, the two materials
can be combined in specific ratios to achieve a desired degradation and drug or growth
factor release period.
*Denotes unpublished data used with permission of collaborators at KeraNetics, LLC.
29
Figure 5. Growth Factor Release from 20% Keratin Gels. 20% keratose gels (A)
showed a quicker release of growth factors than 20% kerateine gels (B). The release of
growth factors from both gels was first order, dependent on the degradation of the gel
(A, B).
30
Keratin Hydrogels for Use in Regenerative Medicines
Keratins have several useful properties for use in regenerative medicine
applications. Extracted alpha keratins have been found to contain the cell recognition
amino acid sequences leucine-aspartic acid-valine (LDV) and glutamic acid-aspartic acid-
serine (EDS) (131, 132). Keratins are capable of adhering and activating platelets which
led investigators to believe keratins have receptors for the β1 and β3 integrin present on
platelets (133). Keratins have also been indicated in the promotion of a favorable immune
response. In an in vitro study in which a monocytic cell line was seeded onto keratin
macrophages exhibited the type 2 anti-inflammatory, pro-remodeling phenotype.
Macrophages were found to secrete more anti-inflammatory cytokines, and a decreased
amount of inflammatory cytokines (134). The favorable interactions of keratin biomaterials
with cells in addition to the capacity to be manipulated into a variety of constructs and the
ability to act as a drug delivery vehicle has led many investigators to utilize keratin in a
variety of regenerative medicine applications (126).
Keratin hydrogels in particular have been successful in several applications,
including bone and nerve repair, treatment of cardiac infarctions, and use in promoting
hemostasis (124, 133, 135-140). Encouraged by these successes, keratin hydrogels with
and without the addition of cells and growth factors will be investigated in a rodent model
of VML for use as a skeletal muscle injury treatment as explained in this proposal.
31
Development of a Tissue Engineered Muscle Repair Construct
As reviewed previously, combination products involving the use of ECM scaffolds
and cells have great potential for skeletal muscle tissue engineering. One such scaffold,
which will be referred to as a Tissue Engineered Muscle Repair (TEMR) construct consists
of a porcine derived bladder acellular matrix (BAM) scaffold seeded with primary muscle
progenitor cells and subjected to cyclic stretch preconditioning (Figure 6) (67). Initial
studies carried out by Moon et al sought to determine the effect of cyclic stretch
preconditioning on a bioengineered muscle scaffold which in this study was a human
skeletal muscle cell seeded BAM scaffold. In this work, it was determined that use of a
stretch regimen for one week in vitro resulted in enhanced tissue organization and cellular
alignment. Further, at one week after a subcutaneous implant onto the LD of athymic mice,
retrieved constructs stained positive the muscle markers α-actin and myosin heavy chain,
MHC (122). With the framework in place: a stretch protocol and an ECM scaffold material
to pursue, TEMR development was underway.
32
Figure 6. TEMR Technology. Primary isolated myoblasts (A) are seeded onto BAM
scaffolds (B). The cell seeded scaffolds are subjected to cyclic stretch in a bioreactor
(C) which increases the number and size of multinucleated myofibers (D). The
scaffolds are then ready to implant (E).
33
In the first major study utilizing TEMR, BAM scaffolds alone were compared with
the cell seeded, preconditioned, TEMR, constructs for their ability to restore function to a
VML injury in the mouse LD (67). Two months after implant into the defect, explanted
TEMR scaffold treated muscles subjected to electrical stimuli had a maximal contraction
of ~72% of the contralateral control muscles, while the BAM and no repair, NR, groups
were at ~46% and ~45% (Figure 7d). Immunohistochemistry indicated the presence of new
muscle tissue at the interface and within the scaffold as well as the presence of
neurovascular bundles (Figure 7e, 7a-c). In a follow up study investigating the effects of
the addition of MPCs during the week of bioreactor preconditioning also using the mouse
LD defect model, TEMR constructs with and without the addition of MPCs gave similarly
favorable functional and morphologic outcomes. TEMR treated tissues examined at the
interface of the implant and the native tissue stained positive for the force transmission
proteins desmin and myosin the excitation-contraction coupling proteins ryanodine
receptor 1 and junctiphilin 1 (Figure 8) (74).
34
Figure 7. Data from Study Using TEMR in Mouse LD (from Machingal, 2011
used with permission). Neurovascular bundles could be seen within the site of the
TEMR implant (A) with positive staining for vasculature (von Willebrand’s factor, B)
and neurofilaments (NF-200, C). The formation of new muscle tissue within the
implant region was further evidenced by positive staining for desmin filaments (E).
Maximum isometric muscle contraction forces are shown at two months post-surgery
(D) for native tissue (blue), NR (red), TEMR treated (green) and BAM treated (purple)
samples.
35
Figure 8. Muscle Regeneration in a Mouse LD VML model (Corona, 2012 used
with permission). All histology shown was conducted on TEMR treated LD muscles.
Images were taken at the interface (A-E) and within the scaffold region (F-J). Muscles
were stained with Masson’s trichrome (A, F), for the functional proteins desmin (B,G)
and myosin (C, H) and the excitation-contraction coupling proteins ryanodine recptor-
1 (D, I) and junctophilin-1 (E, J).
36
The TEMR scaffold (one seeding, original protocol) was later employed in a VML
model in the rat TA (76). Interestingly, in this system the TEMR treatment gave two
distinct results referred to as low and high responders. While the high responders gave a
functional recovery significantly greater than that enabled by BAM or no repair, the low
responders resulted in a loss of function (Figure 9i). Similar to previous results from
Machingal et al, high responders yielded tissues with evidence of new muscle formation
and less of an inflammatory response when compared with NR, BAM, and TEMR low
responders (Figure 9ii-iii). Taken together, the results of these TEMR studies indicate the
benefits of this technology in VML models while highlighting some potential pitfalls. For
example, the resulting subset of TEMR low responders in the study carried out by Corona
et al in the rat model should be a forethought to planning future studies with this treatment.
One theory is that the cellular density exposed to the injury site was compromised when
applying the TEMR technology in the larger VML injury model.
Research using TEMR constructs has showcased the ability of the constructs to
integrate with the host tissue and enable functional recovery in an otherwise irrecoverable
defect in two VML models with distinct geometries. These successes have inspired its use
for VML indications outside of trauma. As covered earlier there are many VML
presentations which include traumatic injury, but also acquired diseases which affect the
musculature and congenital birth defects involving muscle such as cleft lip and cleft palate.
In the study proposed herein, TEMR constructs will be investigated for application in a
VML model which has bio-relevance to a cleft lip secondary revision.
37
Figure 9. Data from TEMR Study in Rat TA (from Corona, 2014 used with
permission). Maximum contraction force relative to control muscles is shown for NR,
BAM, and negative and positive TEMR groups at 4 weeks in top row, 8 weeks in the
middle row, and for 12 weeks in the bottom row (i). A Masson’s trichrome stain was
conducted (ii) on longitudinal sections (A-E) and cross sections (F-J) for control
muscles (A, F), NR (B, G), BAM (C, H), TEMR high responders (D, I) and TEMR
low responders (E, J). Muscle samples were stained for myosin (MF-20, A-C) and
macrophages (CD-68, D-F) (iii). BAM samples are shown in A and D, TEMR high
responders in B and E, and TEMR low responders in C and F.
38
Summary
The repair and regeneration of skeletal muscle after an injury is a complex process
involving multiple cell types, factors, and physiologic cues (17-20, 24). VML indications
are characterized by their ability to overwhelm the native capacity of skeletal muscle to
repair and remodel tissue after injury (7). TE therapies offer a means of enabling repair and
regeneration for VML (19, 21, 86, 141-143). When examining potential treatments for
skeletal muscle repair or remodeling in animal models, it is of key importance that the
chosen models accurately mimic the clinical indication for the investigated treatment. In
the case of VML injury, this means induced muscle injuries must be irrecoverable without
intervention (7). For treatment of muscle injury, investigators have examined the
effectiveness of cell therapies, naturally derived scaffolds, growth factors and
combinations of the three as well with accompanying bioreactor preconditioning with a
range of results (141-143). Among the most exciting interventions were combination
therapies utilizing natural scaffolds (67, 74, 76, 113, 116, 117, 144). In developing a
platform of technologies capable of treating the vast presentation of VML, two exceptional
combination therapies using natural scaffolds will be further investigated: cell and growth
factor loaded keratin hydrogels and a cell seeded and preconditioned ECM scaffold
(TEMR).
39
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59
CHAPTER II
Cell and Growth Factor Loaded Keratin Hydrogels for Treatment of Volumetric Muscle
Loss in a Mouse Model
Hannah B. Bakera,b; Juliana A. Passipieria,c; Mevan Siriwardanea; Mary D. Ellenburgd;
Justin M. Saule; Luke Burnettd; Seth Tomblynd; George J. Christa,b,c,f
Affiliations:
a
Wake Forest Institute for Regenerative Medicine, Wake Forest University, Winston-
Salem, NC 27157, USA

b
VT-WFU School of Biomedical Engineering and Sciences, Winston-Salem, NC 27157
c
Biomedical Engineering Department, University of Virginia, Charlottesville, VA, 22908,
USA
d
KeraNetics, LLC, Suite 168, 391 Technology Way, Winston-Salem, NC 27101
e
Department of Chemical and Paper Engineering, Miami University, Oxford, OH 45056,
USA
f
Orthopedics Department, University of Virginia, Charlottesville, VA, 22908, USA
Note: This manuscript was prepared by Hannah B. Baker and has not yet been submitted.
She (primarily) and Dr. Juliana A. Passipieri (secondarily) were responsible for the
majority of the work described.
60
Introduction
Skeletal muscle injuries and disorders which result in permanent loss of function
have been defined as Volumetric Muscle Loss injuries by Grogan et al (1). Such injuries
are common among active military personnel wherein musculoskeletal trauma makes up
60-70% of all injuries (2). In addition to traumatic injuries, VML can also arise as a result
of excision of cancerous tissue, congenital birth defects such as cleft lip and cleft palate,
and as a result of acquired diseases such as Bell’s palsy (3-6) . Presently, the gold standard
of treatment for VML is an autologous or cadaveric muscle graft. While these interventions
have become increasingly successful with advances in microsurgery, flap transfer
procedures still suffer from donor-host compatibility in the case of cadaveric tissue and
from donor site morbidity when using autologous tissues (7-9). Current efforts in tissue
engineering research seek to develop of a variety of therapies which overcome these issues
and are capable of meeting the needs of the wide variety of VML presentations.
Researchers have investigated several biomaterial scaffolds including synthetic and
biologically derived materials for use in skeletal muscle indications. Biologic materials are
of particular interest in tissue engineering applications due to their availability and
favorable structural and chemical characteristics (10-12). Investigators have applied a
variety of biologic materials alone or in concert with cells, drugs, or factors in several
different animal models of muscle injury (13, 14). Among these approaches, extracellular
matrix (ECM) derived materials have shown great promise for use in tissue engineering
applications and a number of ECM constructs have been FDA approved for several
indications including soft tissue repair, rotator cuff reinforcement, and superficial wound
healing (10). Prior work in our group has focused on the use of such an ECM scaffold
61
coupled with cells and physiologic preconditioning in rodent models of VML. In studies
using this approach, we observed a significant improvement in functional recovery (15-
17). In building a platform of technologies capable of treating a wide variety of VML
indications, our group has begun to explore other biologic scaffold materials including
keratin hydrogels, the subject of this study.
Keratin biomaterials have a host of properties favorable for regenerative medicine
applications and pertinent to skeletal muscle repair. Isolated keratin proteins allow cell
attachment via known cell recognition sequences (18, 19) and reductively isolated keratins
(kerateines) have been found to promote platelet adhesion and coagulation (20). Keratin
hydrogels are capable of achieving sustained release of drugs and growth factors (21-23).
Furthermore, keratin biomaterials have been shown to induce differentiation of monocytes
into the favorable pro-regeneration type-2 population of macrophage (24). As it would
follow, these properties of keratin biomaterials have afforded positive results in a wide
range of regenerative medicine applications including burn healing, bone regeneration, and
nerve regeneration and have demonstrated potential for use in skeletal muscle repair
applications.
Trichocytic keratins which make up human hair are classified as alpha, beta, and
gamma wherein the alpha fraction makes up the majority of the hair fibers, the beta fraction
is present in the cortex of the fibers, and the globular gamma fraction provides the
supporting matrix around filaments (12). The keratins can be solubilized through disruption
of the disulfide bonds to open the cortex and denature the fibers into constitutive proteins
through either an oxidative or reductive process (25, 26). Oxidative extraction yields
keratins with altered cysteine residues that can no longer cross link with other keratins and
62
are referred to as keratose. Reductive isolation results in keratin referred to as kerateine in
which the cysteine group disulfide bonds are broken but the cysteine residues are unaltered
and able to reform disulfide bonds. As a result keratose has a faster degradation rate than
kerateine and the two can be combined to achieve a desired degradation rate.
In the current study, several properties of keratin hydrogels were utilized in
developing and employing a construct to promote muscle repair. Keratin hydrogels made
up of the kerateine and keratose were combined in a manner to achieve degradation within
8 weeks and sustained release of growth factors over this time. In addition, keratin
hydrogels served as a delivery vehicle for autologous skeletal muscle progenitor cells
(MPCs) to the site of injury. Insulin-like growth factor-1 (IGF-1) and basic fibroblast
growth factor (bFGF) were chosen for investigation based upon their known effects on the
skeletal muscle cell population. IGF-1 has been demonstrated to promote differentiation of
satellite cells and fusion of myoblasts while studies using bFGF indicated it promotes
satellite cell activation and proliferation (27-31).
The overriding hypothesis driving this study is that keratin hydrogels will promote
regeneration of skeletal muscle in VML by encouraging a favorable environment for tissue
repair and regeneration. It is further hypothesized that the addition of cells and growth
factors known to influence the skeletal muscle cell population will complement and
enhance the regenerative capacity of keratin alone. To investigate this hypothesis, keratin
hydrogels with or without the addition of a cellular component, growth factors, or a
combination will be examined in an established model of VML for their ability to enable
restoration of function and native morphology.
63
Methods
Keratin Extraction
Keratins were extracted using a patented process in a quality system
regulation/good manufacturing process, QSR/GMP, facility at KeraNetics, LLC. Briefly,
a cold solution of 0.5M thioglycolic acid (TGA) in sodium hydroxide (reductive
extraction for kerateine) or a 2% peracetic acid (PAA) solution (oxidative extraction for
keratose) were added to chopped human hair (chemicals were purchased from Sigma
Aldrich, St. Louis, MO; hair was purchased from World Response Group, Huntsville,
AL). For reductive extraction, the reducing solution was added to hair in a mixing tank
for 12 hours at 37 °C followed by two washes in 100mM Tris base (Sigma) and water.
The solution was then centrifuged, filtered, dialyzed against a 100kDa molecular weight
cutoff cellulose membrane, and neutralized to pH 7.4. For oxidative extraction with PAA,
the hair was handled in a similar manner. Both extractions were followed by acid
precipitation of the alpha fraction, centrifugation, re-dissolution in buffer, and dialyzed in
water against a 30 kDa nominal low molecular weight cutoff filter. The resulting
solutions were lyophilized, ground, and sterilized by 2Mrad gamma irradiation.
BAM Preparation
Briefly, porcine derived bladder was washed and trimmed to obtain the lamina
propria, which was placed in 0.05% trypsin (Hyclone, Logan, UT) for 1 h at 37 °C. The
bladder was then transferred to Dulbecco’s modified Eagle’s Medium, DMEM, solution
supplemented with 10% FBS and 1% antibiotic antimycotic, AA, (Hyclone) and kept
overnight at 4 °C. The preparation was then washed in a solution containing 1% triton-X
64
(Sigma) and 0.1% ammonium hydroxide (Fischer Scientific, Pittsburgh, PA) in de-ionized
water for 4 days at 4 °C. Finally, the bladder was then washed in deionized water for 3
days at 4 °C. The decellularized scaffold was further dissected to obtain a scaffold of 0.2–
0.4mm thickness. The scaffolds were then cut and draped onto custom made silicone molds
with a 4.5cm2 working area.
Cell Culture
Mouse MPCs were isolated from the tibialis anterior and soleus muscles of 4-8
week old C57/Bl6 mice purchased from Charles River Laboratories. Muscles were
sterilized in iodine and washed with PBS before transfer to a 0.2% w/v collagenase
(Worthington Biochemical, Lakewood, NJ) low glucose DMEM (Hyclone) solution where
they were finely minced and then allowed to further digest at 37 °C for 2 hours. The cell
slurry was plated on matrigel (BD Biosciences, San Jose, CA) coated tissue culture plastic
in myogenic media consisting of high glucose DMEM supplemented with 20% fetal bovine
FBS, 10% horse serum (HS), 1% chick embryo extract, and 1% AA (Hyclone). Three days
after plating the media was changed to seeding media consisting of low glucose DMEM
supplemented with 15% FBS and 1% AA (Hyclone). At 70-80% confluence cells were
passaged.
KOS:KTN Optimization and Cell Capacity
Prior to investigating the hydrogel in the mouse model, the ratio of KOS to KTN
which enabled a sustained release of both growth factors was determined via ELISA
analysis of solution surrounding growth factor loaded hydrogels (data shown in appendix:
65
3). The gels were prepared with selected ratios of KOS to KTN in which varied
concentrations (w/v) of keratin powder to sterile PBS were also examined. The results
indicated that a 70:30 blend of 15% KOS and 7% KTN resulted in the sustained release of
both IGF-1 and bFGF over the course of a month (later time points not examined).
Based on rheology data collected in an ongoing study (data not shown), the elastic
modulus of hydrogel which allows incorporation of cells and may still be extruded through
a surgical syringe is ~100Pa. The maximum number of cells which could be incorporated
into the optimized blend and still yield a hydrogel which was flowable in nature and
capable of being extruded through a surgical syringe was 1.7x106 MPCs/ml in the KOS
fraction. It may be assumed that the addition of this concentration of cells resulted in a
hydrogel which did not have an elastic modulus significantly greater than 100Pa. As such
it is worth noting that the concentration of cells used in this study was limited by the
viscosity of the hydrogel.
Hydrogel Preparation
For the growth factor containing groups, IGF-1 and/or bFGF (Peprotech, Rocky
Hill, NJ) were diluted in sterile water to yield a final concentration of 100 µg/mL of each
growth factor. Growth factor solutions were added to KTN powders to make a 7% w/v
hydrogel. KOS was added to either PBS or a serum free media cell suspension of
~1.65x106/mL passage 2 mouse MPCs to form a 15% w/v hydrogel. The KTN and KOS
hydrogels were then combined at a 70:30 KOS:KTN ratio by passing the gels between
coupled syringes.
66
Mouse Latissimus Dorsi VML Model and Hydrogel Implant
Eight week old female C57/BL6 mice were purchased from Charles River
Laboratories. The VML model was created by resecting ~50% of the area of the LD
muscle, ~16±3 mg, as was done in previous studies (15, 17). After creation of the defect
animals were put into one of the following treatment groups: no repair (NR), BAM, keratin
alone, and keratin with a combination of cells, IGF-1, and bFGF (groups listed in Table I,
surgery shown in Figure 1). For the BAM treatment, scaffolds were folded and secured
with 6-0 vicryl (Ethicon, Sommerville, NJ) at the proximal and distal ends of the defect.
For the NR and keratin groups, after creation of the defect, the fascia and mammary fat pad
were closed with 6-0 vicryl. For treatment with keratin hydrogels, 0.1-0.2ml of the
hydrogel was injected into the fascial pocket over the injury site. In all groups the skin was
then sutured with 5-0 prolene (Ethicon) and animals were allowed to recover. At 8 weeks
post implant, animals were sacrificed and the experimental and contralateral LD muscles
were explanted for functional and histological analysis.
67
TABLE I. TREATMENT GROUP COMPONENTS
Treatment Group Number of Components
(abbreviation) animals (n) Keratin MPCs IGF-1 bFGF
No Repair
9
(NR)
Bladder Acellular Matrix
8
(BAM)
Keratin
(KN)
6 +
Keratin+IGF-1
(KN+I)
8 + +
Keratin+bFGF
(KN+b)
8 + +
Keratin+IGF1+bFGF
(KN+I+b)
8 + + +
Keratin+MPC
(KN+M)
8 + +
Keratin+MPC+IGF-1
(KN+M+I)
8 + + +
Keratin+MPC+bFGF
(KN+M+ b)
9 + + +
Keratin+MPC+IGF-1+bFGF
(KN+M+I+b)
8 + + +
Table I. List of Groups, Components, and Number of Animals (n)
68
Figure 1. Surgical flow of VML Injury and Keratin Implant. Muscle is isolated
in (A) with proposed defect region outlined; defect is created and outlined in (B).
Fascia is partially closed and keratin hydrogel is injected into the fascial pocket
(C). Fascia and skin are then closed and the animal is allowed to recover.
Muscle Function Analysis
The entire LD muscle was isolated from the thoracolumbar fascia to the humeral
tendon from mice while under anesthesia and the tendon and facial ends were tied with 5-
0 silk suture (Ethicon) and transferred to ice cold Krebs-Ringer buffer solution (Sigma;
composition: pH 7.4; concentration in mM: 121.0 NaCl, 5.0 KCl, 0.5 MgCl2, 1.8 CaCl2,
24.0 NaHCO3, 0.4NaH2PO4, and 5.5 glucose). Muscles were transferred to individual
chambers of a DMT 750 tissue organ bath system (DMT, Ann Arbor, MI) filled with
Krebs-Ringer buffer at 35 °C bubbled with 95% O2 and 5% CO2. The muscles were
positioned between custom-made platinum electrodes with the proximal tendon attached
to a force transducer and the distal tendon to a fixed support. Direct muscle electrical
stimulation (0.2ms pulse at 30 V) was applied across the LD muscle using a Grass S88
stimulator (Grass, Warwick, RI). Real time display and recording of all force
measurements were performed on a PC with Power Lab/8sp (AD Instruments, Colorado
Springs, CO). Once the LD muscles were mounted in the organ bath, the muscles were
69
allowed to equilibrate for 5 min prior to determining optimal physiological muscle length
(Lo) via a series of twitch contractions. Force as a function of stimulation frequency (1–
250Hz) was measured at 37 °C during isometric contractions (750ms trains of 0.2ms
pulses), with 2 min between contractions. The maximal force of contraction, Po, was
normalized to an approximate physiological cross-sectional area (PCSA), which was
calculated using the following equation, where muscle density is 1.06 g/cm3:
𝑃𝑃𝑃𝑃𝑃𝑃𝑃𝑃 = (𝑤𝑤𝑤𝑤𝑤𝑤 𝑤𝑤𝑤𝑤𝑤𝑤𝑤𝑤ℎ𝑡𝑡 (𝑔𝑔)/ [𝑚𝑚𝑚𝑚𝑚𝑚𝑚𝑚𝑚𝑚𝑚𝑚 𝑑𝑑𝑑𝑑𝑑𝑑𝑑𝑑𝑑𝑑𝑑𝑑𝑑𝑑 (𝑔𝑔/𝑐𝑐𝑐𝑐3) 𝑥𝑥 𝑚𝑚𝑚𝑚𝑚𝑚𝑚𝑚𝑚𝑚𝑚𝑚 𝑙𝑙𝑙𝑙𝑙𝑙𝑙𝑙𝑙𝑙ℎ, 𝐿𝐿𝑜𝑜 (𝑐𝑐𝑐𝑐)]
Equation 1. Physiological Cross Sectional Area (PCSA) Calculation
Additionally the force-frequency curves were fit to the following dose response equation:
𝑓𝑓(𝑥𝑥) = 𝑚𝑚𝑚𝑚𝑚𝑚 − (𝑚𝑚𝑚𝑚𝑚𝑚 − min)/[1 + (𝑥𝑥 ⁄ 𝐸𝐸𝐸𝐸 50 )−𝑛𝑛 ]
Equation 2. Dose Response Formula
where x is the stimulation frequency in Hz, min is the lowest observed force generated,
equivalent to the twitch force or Pt, max is the largest observed force or Po, EC50 is the
stimulation frequency which yields half the amplitude of the maximum observed force (Po-
Pt), and n is the slope of the linear portion of the force-frequency curve.
Immunohistochemistry and Imaging
Hematoxylin and eosin and Masson’s trichrome stains were conducted by standard
techniques to determine the basic morphology of cells in and around the implant and to
observe any inflammatory response. Immunohistochemical staining was performed using
antibody to detect myosin (MF-20, 1:10) acquired from Developmental Studies Hybridoma
Bank, Iowa City, IA. Biotinylated anti-mouse IgG (MKB-2225, 1:250, Vector Laboratories
Inc.) secondary antibody was used to detect primary antibodies. The sections were next
70
treated with Avidin Biotin Complex Reagent (PK-7100, Vector Laboratories Inc.) and then
visualized using a NovaRED substrate kit (SK-4800, Vector Laboratories Inc.). Finally,
the sections were counterstained using Gill’s Hematoxylin (GHS280, Sigma-Aldrich).
Tissue sections without primary antibody were used as negative controls. Images were
captured and digitized (DM4000B Leica Upright Microscope).
Statistical Analysis
Functional data were statistically analyzed using one way ANOVA. When
significance was found (α set to 0.05) a post-hoc multiple comparison test was used to
compare group means (α set to 0.05) using a Fisher Least Significant Difference (LSD)
significance test. Statistical analysis was conducted using GraphPad Prism 6.0 for
Windows La Jolla, California.
Results
In Vitro Functional Assessment
In vitro force measurement was conducted as described at 8 weeks after implant on
whole LD operated and contralateral control muscles harvested from the ten treatment
groups referenced in Table I. Measured physical and functional parameters for each group
and parameter statistics are listed in Table II, charts of Po and specific Po are shown in
Figure 2.
71
72
Table II. Summary of Latissimus Dorsi Physical and Functional Measurement
Data. Values are mean ± standard error of the mean. Muscle mass was measured
after completion of functional testing. Lo is the optimal muscle length associated
with peak twitch response (Pt). PCSA was determined using the measured Lo and
wet weight using Equation 1. Peak twitch (Pt), contraction force at 80hz (P80), and
maximal isometric contraction force (Po) were measured by soliciting contractions
via direct electric stimuli (0.2ms, 30V) in the organ bath setup. Measured Po was
normalized to PCSA to determine specific force (Specific Po). The stimulation
frequency which yielded 50% of the absolute maximum amplitude (EC50) and the
slope of the linear portion of the force-frequency curves shown in Fig. 3 (n
coefficient) were determined by fitting the curves to function described by
Equation 2. Superscript denotations indicate statistically different group means (p
< 0.05) from: aUninjured, bNR, cBAM, dKN, eKN+IGF-1, fKN+ bFGF, gKN+IGF-
h
1+bFGF, KN+MPC, iKN+MPC+IGF-1, jKN+MPC+bFGF, kKN+MPC+IGF-
1+bFGF, and lall other groups.
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74
Figure 2. Muscle Function Analysis at 2 Months Post-Surgery. Maximum isometric contraction force as a result of electrical
field stimulation (1-250hz, 0.2ms, 30V) (A) normalized to PCSA (B). For both measurements Uninjured n=46, NR n=9, BAM
n=8, KN n=6, KN+I n=8, KN+b n=8, KN+I+b n=8, KN+M n=8, KN+M+I n=8, KN+M+b n=9, KN+M+I+b n=8. Acellular
groups are colored green, cellularized are colored blue. Significant differences are denoted as follows: * p<0.05, ** p<0.01, ***
p<0.001, † different from all other groups p<0.001, # different from all other groups p<0.0001
The average created defect mass was 16.0±3 mg with no significant difference
between treatment groups. The injury created in this study resulted in a 58% reduction in
maximum isometric contraction force (Po), a 55% reduction in peak twitch force (Pt), and
a 64% reduction in measured submaximal tetanic contraction at 80hz (P80 Hz) in the No
Repair (NR) group relative to uninjured control muscles.
Among the treatment groups keratin+IGF-1+bFGF enabled the greatest recovery
of Po, Pt, P80 Hz, and had the highest specific Po. The Po values measured for KN+I+b treated
muscles were significantly greater than all treatment groups’ values excluding KN and
KN+M+I. Measured Pt values were also significantly greater than KN+b, KN+M, and
KN+M+I+b and specific Po values for KN+I+b were significantly greater than BAM and
KN+b values. In addition KN+M+I treatments also resulted in specific Po values which
were significantly higher than BAM and KN+b values.
Analysis of dose response curves fit to force-frequency data (selected curves shown
in Figure 3) indicated that EC50 values for all treatments were increased relative to control
values while curve slope values were not different between any groups (Table II). EC50 for
the KN+I+b group had the largest shift relative to uninjured values and was significantly
different from EC50 values for NR, BAM, KN+b, KN+M, and KN+M+I.
Muscle masses of BAM treated muscles were significantly greater than those of
any other group including uninjured tissues, equating to 176% of control values. KN+b
treated muscles were also significantly heavier than control tissues with values measuring
140% of uninjured tissue values. Similarly, BAM treated tissues had the longest optimal
muscle Lo of the treatment groups and this measurement was not significantly different
from uninjured muscles.
75
Figure 3. Selected Force-Frequency Curves with Fitted Dose-Response
Curves. Two months post-surgery contractions elicited via electrical stimuli from
1-250Hz is shown for Uninjured, KN+I+b, BAM, and NR muscles. Data has been
fit with dose response curves according to Equation 2. Curve fitting was used to
determine EC50 and Hill Slope values shown in Table II.
Morphology
Upon completion of functional analysis and physical measurements, experimental
and control tissues were placed next to one another in tissue culture plastic for
morphological comparison. Representative tissue samples from each treatment group are
shown in Figure 4. Among treatment groups, it was observed that BAM and KN+b treated
muscles were consistently larger in size and carried more fatty-connective tissue than the
other treatment groups’ tissue. While uninjured tissues had a distinct organized vasculature
compared to that seen in experimental muscles, a notable difference in vasculature
structures among the groups was not observable at the gross morphology level.
76
Figure 4. Representative Tissue Gross Morphology. Images shown were taken
following functional assessment carried out at 2 months post-surgery.
Representative samples from each group are shown.
77
Histology
All tissues were subjected to histological processing as described. Depicted in
Figures 5-7 are images of uninjured and experimental tissue sections stained with
hematoxylin and eosin, Masson’s Trichrome, and for the contractile protein myosin. Tissue
sections were examined at the interface between injured and uninjured tissue for the
presence of adipose and collagen deposition, and for evidence of reorganization and
regeneration of muscle tissue.
In NR tissues adipose and collagen deposition are present along the injury site with
mononuclear cells evenly distributed throughout the defect site. A small extent of new
muscle formation was evidenced with short and thin fibers dispersed along the region
between native tissue and the observed band of adipose and scar tissue. In BAM treated
muscles the injury-native tissue border resembles that seen in NR tissue with a much
greater volume of collagen present consistent with the presence of non-remodeled BAM
scaffold.
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79
Figure 5. Mouse LD Cell and Tissue Morphology and Functional Protein
Expression in Uninjured, NR, and BAM Treated Tissues. Treatment groups
presented: uninjured control (A, D, G), NR (B, E, H), BAM (C, F, I). Cell and tissue
morphology was examined through hematoxylin and eosin staining (A-C) wherein
nuclei are stained blue-purple and cytoplasm and cellular proteins are stained pink
and Masson’s trichrome staining (D-F) in which tissue stains red, collagen stains
blue, and nuclei stain black. The presence of new muscle tissue formation within
the defect and treatment site was evaluated by staining for myosin (MF-20) shown
in brown (G-I). (*) denotes adipose tissue deposits; (#) denotes collagen
deposition/scar tissue; (‡) denotes native tissue; and new muscle formation is
indicated with a black arrow.
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81
Figure 6. Mouse LD Morphology and Functional Protein Expression in KN,
KN+I, KN+b, and KN+I+b. Treatment groups presented: KN (A, E, I), KN+I (B,
F, J), and KN+b (C, G, K), and KN+I+b (D, H, L). Morphology was examined
through hematoxylin and eosin staining (A-D) wherein nuclei are stained blue-
purple and cytoplasm and cellular proteins are stained pink and Masson’s trichrome
staining (E-H) in which tissue stains red, collagen stains blue, and nuclei stain
black. The presence of new muscle tissue formation within the defect and treatment
site was evaluated by staining for myosin (MF-20) shown in brown (I-L). (*)
denotes adipose tissue deposits; (#) denotes collagen deposition/scar tissue; (‡)
denotes native tissue; and new muscle formation is indicated with a black arrow.
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83
Figure 7. Mouse LD Morphology and Functional Protein Expression in
KN+M, KN+M+I, KN+M+b, and KN+M+I+b. Treatment groups presented:
KN+M (A, E, I), KN+M+I (B, F, J), KN+M+b (C, G, K), and KN+M+I+b (D, H,
L). Morphology was examined through hematoxylin and eosin staining (A-D)
wherein nuclei are stained blue-purple and cytoplasm and cellular proteins are
stained pink and Masson’s trichrome staining (E-H) in which tissue stains red,
collagen stains blue, and nuclei stain black. The presence of new muscle tissue
formation within the defect and treatment site was evaluated by staining for myosin
(MF-20) shown in brown (I-L). (*) denotes adipose tissue deposits; (#) denotes
collagen deposition/scar tissue; (‡) denotes native tissue; and new muscle
formation is indicated with a black arrow.
84
Cell morphology among the keratin treatment groups was varied with regard to the
presence of scar tissue, adipose and mononuclear cells, and neo-tissue formation.
Compared with BAM and NR groups, KN treated tissue showed much less collagen
presence, but a greater presence of adipose tissue and mononuclear cells which had formed
granulomas at the injury site. At the border of the defect site thin muscle fibers travelling
along the axis of the native tissue are evident with more continuous fibers present than in
the BAM and NR groups.
Unlike KN alone, KN+M tissue showed a large degree of scar tissue formation with
some adipose tissue deposition, a more disperse mononuclear cell presence, and a moderate
layer of organized but interrupted thin new muscle fibers. KN+I and KN+M+I+b had injury
borders that had larger layers of adipose tissue present with little to no scar tissue formation
and newly formed thin muscle fibers which traveled along the same axis as the native tissue
but did not infiltrate into the region of adipose tissue deposition- similar to that seen in
KN+M.
KN+I+b tissues had similar cell morphology to KN+M+I+b and KN+I tissues with
exception that muscle fibers could be observed up to and within the layer of adipose tissue
present. KN+b and KN+M+b tissues were similar in appearance to KN+M with a greater
presence of scar tissue as well as adipose deposition. Notably, KN+b tissues were observed
to have a greater extent of new muscle tissue formation, however the neo-tissues appeared
as rafts of muscle scattered and separated within the extensive fat and scar tissue present,
which also appeared to be interrupting the native tissue adjacent to the injury site.
85
Lastly, KN+M+I sections resembled the bFGF groups in that there was a large
presence of both fat and scar tissue, however in the KN+M+I tissues the deposition was
not as extensive or invasive as in the case of KN+b. In addition, the new muscle tissue
formation was more similar to that seen in KN+I+b in that it was visible within the scar
and fat deposition, but as an extension from the native tissue and not in rafts as in KN+b
tissues.
Discussion
In previous studies our group has developed two distinct models of VML injury in
rodents in which to examine the effectiveness of a BAM scaffold loaded with MPCs and
subjected to cyclic stretch preconditioning (15-17). With these established models it is
possible to evaluate additional technologies such as keratin hydrogels as potential
treatments for VML injuries. Keratin was a viable candidate for a muscle repair application
due to its known capacity to mitigate inflammation, to influence the coagulation cascade
and neurogenesis, and to act as a cell and drug delivery vehicle (18, 20, 23, 24, 32, 33).
Several studies have illustrated the importance of a cellular component in tissue
engineering technologies aimed to promote skeletal muscle repair and regeneration in
VML injuries (15-17, 34-40). Of these studies, those conducted by our group which
employed the same VML model used in the present study indicated that a cellular
component in addition to the scaffold used did not just improve function, but was necessary
for any functional improvement to occur (15, 17). Investigators have also shown that
growth factors alone or coupled with cell and scaffold based therapies can improve muscle
recovery after injury in vivo (37, 41, 42). These studies as well as in vitro investigations of
86
the effect of growth factors showcase the important role of IGF-1 and bFGF within
regenerating skeletal muscle (29-31). The culmination of these findings resulted in the
hypothesis for this study which is that keratin hydrogels will improve muscle repair and
regeneration in a VML injury model and that the regenerative capacity will be
complemented through the addition of a combination of MPCs, IGF-1, and bFGF. The
novelty of this work lies therein: this is the first known application of keratin hydrogels,
let alone the combination of keratin, MPCs, and growth factors, in an in vivo VML model.
The hypothesis of this study was addressed through use of an established injury
model and physiological and histochemical assays. The major goal of this work was to
determine the potential of keratin for use in such a model and to utilize the carrier capacity
of keratin hydrogels to further glean insight into the effectiveness of keratin in combination
with cells and growth factors. Furthermore, this study would allow for the study of the in
vivo interactions between keratin, cells, and growth factors in an active wound environment
and resulting effects on the repair of skeletal muscle.
Use of in vitro functional assessment indicated that the combination of keratin, IGF-
1, and bFGF resulted in the greatest recovery after injury according to metrics surrounding
contraction force (Po, Pt, P80 Hz, and Specific Po). Interestingly and unexpectedly, this
treatment yielded the greatest recovery of absolute and specific contraction force in the
absence of a cellular component. It is worth noting, however, that the functional results
observed for these measurements for KN+I+b treated muscles were not significantly
different from keratin alone or keratin coupled with cells and IGF-1. Further, the addition
of cells to the same combination keratin loaded with MPCs, IGF-1, and bFGF, KN+M+I+b,
resulted in a significantly lower Pt and Po than KN+I+b. This raises the question of how
87
the addition of cells influenced recovery. If the groups with cells are grouped together and
the same is done for the groups without cells, there is no significant difference between the
Po of the two groups (data not shown). In a previous study from our group in which cells
were coupled to an ECM scaffold, chemical and physical differentiation cues resulted in a
greater functional improvement at 2 months post-implant compared with the addition of
cells in the absence of differentiation cues (17). Similarly in the current study, cells were
combined with keratin and not cultured in a differentiation medium or subjected to
physiologic preconditioning which may account for why the addition of cells did not
impact functional recovery. Moreover and perhaps a more relevant comparison is the
concentration of cells applied in each model. In the previous studies utilizing this VML
model, implanted scaffolds carried ~6x106 MPCs (1x106 MPC/cm2 per side on a 1x3cm
construct) compared with ~1.2-2.3x105 MPCs in the injected volume of hydrogel used for
the cell groups in this study which constitutes a 26-50 fold difference.
Further investigation using cell morphology analysis gives insight into how the
cells and growth factors may have impacted the resulting functional outcomes. While the
cell morphology observed among the keratin treatment groups showed variation,
characteristic elements differed between groups which experienced a greater recovery of
function and those which did not. For example KN+I+b tissues showed evidence of new
tissue formation which extended from the native tissue well into the site where muscle was
removed which was unlike the poorly recovered groups such as BAM, KN+M, KN+I, and
KN+M+b where new muscle tissue was visible only in a thin layer along the border of the
injury, and was small in area relative to the fat and scar tissue layers present. In addition,
KN+I+b, KN+M+I, and KN treated tissues showed a thinner, more moderate level of
88
adipose and scar tissue deposition in comparison with lower performing groups such as
KN+b, KN+M+b, KN+M, and KN+M+I+b which had extensive fat and/or scar tissue
formation. For KN+b tissues, the scar and fat tissue appeared to separate new tissue
formation into smaller rafts of tissue and to disrupt surrounding native tissue which may
explain why the KN+b group had a lower P0 and Pt than NR muscles (Table II).
In the literature, there are several examples of studies in which bFGF and IGF-1
enhanced skeletal muscle repair in vivo; however, these growth factors have also been
shown to lead to increased connective and scar tissue formation representative of the
observations in this study (37, 41, 43). IGF-1 acts on a variety of cell types, promoting
proliferation in fibroblasts, myoblasts, and chondrocytes and differentiation also in
myoblasts and in other cell types including adipocytes (27, 44). In one study, sustained
release of IGF-1 from microspheres in a rat model resulted in adipose tissue formation
believed to be a result of IGF-1 promoted differentiation of infiltrating cells into mature
adipocytes (45). Thus, the increased level of lipid containing cells observed in the IGF-1
groups is potentially a result of IGF-1 mediated differentiation of invading cells with an
immature phenotype. Likewise, bFGF is secreted by and promotes proliferation in a
variety of cell types including fibroblasts (27). As such, a possible mechanism behind the
increased fibrosis seen in bFGF groups is the increased proliferation of invading fibroblasts
resulting in a greater ECM deposition and scar tissue formation. Based on the results of the
individual growth factors it would be difficult to predict the favorable results seen in this
study, however similar success has been seen in other studies. For example, in an in vitro
study combining IGF-1 and bFGF in culture of muscle derived cells, cell proliferation was
greater than use of either of the growth factors alone (31) and when applied to an in vivo
89
muscle laceration injury, IGF-1 and bFGF resulted in significantly improved twitch force
(42).
The addition of cells for treatment of skeletal muscle injury has been shown to
mitigate the inflammatory process which studies have indicated to be a result of interaction
with invading macrophages (46). In the present study, the concentration of cells may not
have been sufficient to result in a favorable attenuation of inflammation: herein cellularized
groups did not promote a greater functional recovery than the acellular groups and in nearly
all samples examined did not appear to cause a decrease in connective and fibrotic tissue
generation. In previous studies wherein the addition of cells led to decreased inflammation
and promotion of functional recovery, a greater number of cells was introduced by the
therapy (1-6x106+ in cited studies versus 1.2-2.3x105 MPCs used in this study)(15-17, 40).
As such, for the number of cells introduced in this study, the histological and functional
evidence suggests that this volume of cells may have been capable of recruiting
macrophages and fibroblasts to promote an inflammatory response, but may not have been
sufficient in promoting an anti-inflammatory progression of the immune cell population
necessary for promotion of muscle regeneration.
This study sought to investigate keratin hydrogels in combination with cells and
growth factors implicated in regulation of skeletal muscle growth and development. In
doing so, a previously developed murine model of VML was utilized to test the
combination of keratin hydrogels loaded with combinations of MPCs, IGF-1, and bFGF.
Functional analysis indicated that the combination of keratin and both growth factors
resulted the greatest recovery which was notably not significantly greater than that seen in
the application of keratin alone or keratin combined with MPCs and IGF-1. Histological
90
findings indicated that improved recovery was associated with a greater presence of neo-
tissue formation and a decreased lipid and fibrotic tissue formation. Further, the functional
and histological data indicated that the cell concentration used in this study was likely not
sufficient to result in improved functional recovery or a decrease in fibrosis and connective
tissue deposition. Further efforts to increase cell concentration while maintaining the
desired physical properties of the keratin hydrogel would be an insightful follow up to this
study. The results of this study further showcased the utility of keratin hydrogels in tissue
engineering and served to illustrate its potential as a treatment for VML injuries.
91
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CHAPTER III
Keratin Hydrogel Enhances In Vivo Skeletal Muscle Function in a Rat Model of
Volumetric Muscle Loss
Juliana A. Passipieria,b; Hannah B. Bakera,c; Mevan Siriwardanea; Mary D. Ellenburgd;
Justin M. Saule; Luke Burnettd; Seth Tomblynd; George J. Christa,b,c,f
Affiliations:
a
Wake Forest Institute for Regenerative Medicine, Wake Forest University, Winston-
Salem, NC 27157, USA

b
Biomedical Engineering Department, University of Virginia, Charlottesville, VA, 22908,
USA
c
VT-WFU School of Biomedical Engineering and Sciences, Winston-Salem, NC 27157;
d
KeraNetics, LLC, Suite 168, 391 Technology Way, Winston-Salem, NC 27101;
e
Department of Chemical and Paper Engineering, Miami University, Oxford, OH 45056,
USA;
f
Orthopedics Department, University of Virginia, Charlottesville, VA, 22908, USA
Note: This manuscript was prepared by Dr. Juliana A. Passipieri and has not yet been
submitted. She (primarily) and Hannah B. Baker (secondarily) were responsible for the
majority of the work described.
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Introduction
The natural progression of muscle injury involves a series of interdependent and
overlapping phases that include muscle degradation/inflammation, regeneration, and
remodeling (1). The muscle repair process is detailed reviewed in (2).
However, when the extent of the injury goes beyond the intrinsic regenerative
mechanism, the irrecoverable loss of tissue is referred to as volumetric muscle loss (VML)
(3). It can be the result of numerous congenital and/or acquired diseases, as well as
traumatic accidents, as those experienced by the military population during battlefield
injuries.
Currently, the survival rate of battlefield injuries is the highest in history (4) due to
significant improvements in medical training, tactical warfare, and advances in personal
armor. Consequently, there are an increasing number of wounded warriors with serious but
survivable injuries facing permanent disability and disfigurement. It is paramount that
every one of them has maximal potential for functional recovery.
Present therapeutic options are very limited and highly associated to local morbidity
and mortality (5). As a result, focus has turned to tissue-engineering technologies to
develop more effective treatments for large muscle injuries. A popular approach is the
implantation of growth factor-releasing biological scaffolds, which can alter the
macrophage phenotype response from scar formation to nascent muscle remodeling (6).
Acellular extracellular matrix (ECM) is an effective biological scaffold used to optimize
muscle repair, specifically when it is combined with a cellular component (e.g. muscle
progenitor cells – MPC) (7-10). However, every injury is unique and it is necessary to
explore a combination of treatments to efficiently and effectively restore muscle function.
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Keratin-based biomaterials are at the forefront of tissue engineering applications
due to their tissue compatibility, degradability (11), non-antigenic, and non-immunogenic
properties (12). Medical and biotechnology applications of these biomaterials have been
presented in several studies showing their in vitro compatibility with different cell types
(13-15), and their contributions to bone, cardiac muscle and peripheral nerve regeneration
(16-20) as well as wound healing in vivo (21, 22).
Keratin, an intermediate filament protein that constitutes hair, wool, nails and other
epidermal appendageal structures (23), is processed via oxidation, creating a non-
covalently cross-link form known as keratose (KOS) or reduction, creating a disulfide
cross-linked form known as kerateine (KTN). As the mammalian metabolism is incapable
of synthetizing the enzyme responsible for keratin breakdown, the degradation rate of
keratin based-biomaterials is easily manipulated through the combination of different ratios
of the cross-linked and non cross-linked forms.
The easy manipulation of the degradation rate enables the controlled release of
physiologically relevant molecules (i.e. growth factor, antibiotics), potentially increasing
the clinical relevance of keratin-based biomaterials. Growth factors are essential in the
complex network of regulatory pathways responsible for triggering and maintaining
through cell proliferation, differentiation and growth, being intrinsically released from the
extracellular matrix surrounding injured tissue (24, 25) and by inflammatory cells during
the initial phase of tissue healing (26, 27).
Basic fibroblast growth factor (bFGF or FGF-2) is known to directly stimulate
muscle regeneration by activating satellite cell proliferation, enhancing satellite cell
recruitment to the injury site, and, due to its strong angiogenic effect (28, 29), by improving
101
overall blood perfusion at the injury site. Likewise, Insulin-like Growth Factor 1 (IGF-1)
also plays an important role in muscle maintenance and repair. IGF-1 influences muscle
metabolism, via muscle cell differentiation and proliferation (30) and it stimulates satellite
cell proliferation in rat skeletal muscle (31).
In this context, to investigate a novel skeletal muscle tissue engineering strategy,
we hypothesized whether keratin hydrogel would support muscle regeneration as well as
provide a suitable delivery platform for growth factors and ex-vivo grown cells. To address
this issue, we used a rat Tibialis Anterior (TA) VML injury model to evaluate the effects
of keratin hydrogel formulations with IGF-1 and/or bFGF and/or MPC on muscle
regeneration.
Methods
Keratin hydrogel preparation
Keratins were extracted using a proprietary patent-pending process in a Quality
Systems Regulated facility at KeraNetics, LLC in a manner similar to those previously
published (32, 33). The lyophilized powders were weighed to produce a 15% keratose
(KOS) and 7% kerateine (KTN) weight to volume hydrogel. The KOS was hydrated with
either DPBS, if the gel did not contain cells, or was hydrated with cell culture media
(without fetal bovine serum) if the gel contained MPCs.
The KTN gel was hydrated with water, when growth factors were impregnated into
the gels. Individual gels are given approximately 30 minutes to spontaneously form
hydrogels at 37°C. The gels are then mixed together placing 70% KOS gel and 30% KTN
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gel into one mixture. The concentration of growth factor used was 100µg/ml and the cell
concentration was 1.15 x 106 MPC/ml in the final product.
Treatment groups
Treatment groups and group sizes are listed in Table I and are as follows: Keratin
(n=18), Keratin + IGF (n=18), Keratin + bFGF (n=18), Keratin + IGF + bFGF (n=18),
Keratin + MPC (n=8), Keratin + MPC + IGF (n=8), Keratin + MPC + bFGF (n=8), Keratin
+ MPC + IGF + bFGF (n=8). Two negative control groups were included: no repair group
(NR, n=17), and BAM group (n=17) (Bladder Acellular Matrix, n=17), performed as
previously described (8).
Table I: Design of experimental groups submitted to VML injury model.
MPC
Group IGF-1 bFGF
Group Keratin 1.15x106 BAM
size (n) 100µg/mL 100µg/mL
cells/mL
No repair 17 - - - - -
BAM 17 - - - - +
Keratin 18 + - - - -
KN + I 18 + + - - -
KN+ b 18 + - + - -
KN + I + b 18 + + + - -
KN + M 8 + - - + -
KN + M + I 8 + + - + -
KN + M + b 8 + - + + -
KN + M + I + b 8 + + + + -
KN: keratin; I: IGF-1, insulin-like growth factor 1;b: bFGF, basic fibroblast growth
factor; MPC: muscle progenitor cell; BAM: bladder acellular matrix.
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Muscle Progenitor Cell (MPC) isolation and culture
MPCs were isolated from the soleus and TA muscles of 4-6 weeks old male Lewis
rats (Charles River Laboratories) as previously described (8-10). Briefly, after sterilization
in iodine and consecutive washes in sterile PBS, muscles were minced into small pieces by
hand, and incubated in 0.2% collagenase (Worthington Biochemicals) solution prepared in
low glucose Dulbecco’s modified Eagle’s medium (DMEM; Hyclone) for 2 hours at 37
°C. Muscle tissue fragments were plated onto tissue culture dishes coated with Matrigel
(1:50 dilution, BD Biosciences) in myogenic medium containing DMEM high glucose
supplemented with 20% fetal bovine serum (FBS), 10% horse serum, 1% chicken embryo
extract, and 1% antibiotic/antimycotic (Hyclone). Cells were passaged at 70%–80%
confluence, cultured in low-glucose DMEM supplemented with 15% FBS and 1%
antibiotic/antimycotic, and impregnated in the keratin gel at second passage.
Animal care
This study was conducted in compliance with the Animal Welfare Act, the
Implementing Animal Welfare Regulations, and in accordance with the principles of the
Guide for the Care and Use of Laboratory Animals. The Wake Forest University Health
Sciences School of Medicine Animal Care and Use Committee approved all animal
procedures. Adult female Lewis rats (Charles River Laboratories) weighting 203.4 + 0.8 g,
at 11-13 weeks of age, were individually housed in a vivarium accredited by the American
Association for the Accreditation of Laboratory Animal Care, and provided with food and
water ad libitum.
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Surgical Procedures
Surgical creation of volumetric muscle loss (VML) injury was performed in the TA
muscle as previously reported (8, 34). A longitudinal incision was made on the lateral
aspect of the left lower leg, followed by blunt separation of the skin from the underlying
fascia. Similarly, the fascia covering the anterior crural muscles was separated using blunt
dissection. The proximal and distal tendons of the Extensor Hallicus Longus (EHL) and
Extensor Digitorum Longus (EDL) muscles were then isolated and ablated. TA muscle
corresponds to 0.17% of the total body weight, as previously determined (8, 34). VML
injury was created by excising approximately 20% of the TA muscle weight at the middle
third of the muscle (Figure 1A).
105
Figure 1. Creation of VML in Rat TA and Injection of Keratin Hydrogel. Rat
hind limb is shown with anterior side facing forward. Anterior crural compartment
muscles are exposed by opening the skin and fascia and the EDL and EHL are
ablated tendon to tendon (not shown). The longitudinal middle third, ~1cm, is
notched 0.5cm across the width of the TA and ~0.25cm depth of muscle is removed
from the rectangular region (A). The surrounding fascia is closed, leaving one gap
for a needle to be inserted and ~200µl of hydrogel is injected into the fascia-muscle
pocket (B). The remaining fascial gap is closed, the skin is closed and the animal
is allowed to recover.
106
The BAM scaffold was sutured directly over the defect as previously described (8).
The fascia was closed with 6-0 vicryl sutures, and approximately 200µl of the hydrogel
were injected using a 19G needle over the muscle defect through the sutured fascia (Figure
1B).
The skin closure was performed with 5-0 prolene using interrupted sutures, and
skin glue was applied between the skin sutures to help prevent the incision from opening.
Ketoprofen (0.03 mg/kg; subcutaneously) was administered every twenty-four hours for
three days.
In vivo Functional Analysis
At 4, 8 and 12 weeks after surgery, rats were anesthetized (1.5 – 2.5% isoflurane)
and the left hind limb was aseptically prepared. The rat was placed supine on a heated
platform and the left knee was bent to a 90-degree angle and was secured using a stabilizing
rod. The left foot was taped to a footplate attached to the shaft of an Aurora Scientific
305C-LR-FP servomotor, which was controlled using a computer. Sterilized percutaneous
needle electrodes were inserted through the skin for stimulation of the left common
peroneal nerve. Electrical stimulus was applied using an Aurora Scientific stimulator with
a constant current SIU (Model 701C). Stimulation voltage and needle electrode placement
were optimized with a series of twitch contractions at 1 Hz. Contractile function of the
anterior crural muscles was assessed by measuring peak isometric tetanic torque derived
from the maximal response to a range of stimulation frequencies (10 - 200 Hz). After
functional testing, animals were allowed to recover on the heated platform and returned to
107
the vivarium. For terminal time points, animals were euthanized via CO2 inhalation and
the TA muscle harvested.
Gross morphology analysis of TA muscle
After animal euthanasia, skin on the hind limbs was removed and the fascia layer
was gently detached and removed. Once exposed, injured TA muscle and its contralateral
uninjured control were imaged, and then harvested. The weights of the samples were
measured, followed by preparation for histological analysis.
Histology and immunohistochemistry
TA muscles from all experimental groups were fixed in 4% paraformaldehyde
overnight at 4 °C. Five-micrometer-thick serial sections were cut from the paraffin
embedded blocks and submitted to Masson’s trichrome staining and Gill’s Hematoxylin-
Eosin staining following standard procedures. Histological analysis was performed in
longitudinal sections of the muscle at approximately 50 to 150 microns deep into the wound
bed. Images were captured and digitized (DM4000B Leica Upright Microscope; Leica
Microsystems) at varying magnifications.
Statistics
Numeric data are presented as Mean + SEM. Functional data were analyzed using
one-way or two-way ANOVA and Fisher’s PLSD post-hoc test using Prism (GraphPad
Software Inc, La Jolla, CA). Statistical significance was achieved at an alpha < 0.05.
108
Results
Creation of volumetric muscle loss (VML) injury in the rat tibialis anterior
None of the animals died during the surgical procedure and no post-implantation mortality
was recorded. The weight of the muscle excised from the animals in each treatment group
was similar, reflecting the creation of comparable VML injuries (NR: 73.3 ± 0.6 mg; BAM:
72.9 ± 0.7 mg; KN: 73.9 ± 0.6 mg; KN+I: 72.7 ± 1.1 mg; KN+b: 72.4 ± 0.9 mg; KN+I+b:
70.4 ± 1.0 mg; KN+M: 67.8 ± 0.5 mg; KN+M+I: 73.9 ± 1.4 mg; KN+M+b: 68.1 ± 0.83
mg; KN+M+I+b: 72.4 ± 0.98 mg).
In vivo Functional analysis after VML injury
Animal weights were similar among the groups at the time of baseline functional
analysis, surgery, and functional analysis at 4, 8 and 12 weeks post- surgery (Figure 2 A).
Additionally, the similarity in body weight reflects the lack of adverse reaction to the
various treatments. All data collected were normalized to body weight, in order to control
the effect of normal growth on peak isometric torque production over the course of the
study.
Mean values for the baseline (pre-injury) maximal isometric torque in response to
peroneal nerve stimulation are shown in Figure 2B for all animals and treatment groups.
The results demonstrate that the initial strength prior to VML injury is remarkably
consistent, and statistically indistinguishable, among animals in all treatment groups.
109
110
Figure 2. Contribution of Keratin Hydrogel Formulation to Muscle
Regeneration at 4 and 8 Weeks. A) Body weights of study animals reveal normal
healthy weight gain in all groups. B) Baseline contraction force resulting from
peroneal nerve stimulation and measured with a footplate force transducer. Peak
isometric torque measured at C) 4 weeks. D) 8 weeks. No significant advantage
resulted from the inclusion of cells in the injected hydrogel treatment Individual
responses are normalized to the respective group mean of the initial maximum pre-
injury isometric torque response and their body weight. Data are presented as Mean
+ SEM. *-Significantly different at the p<0.05 level using Fisher’s PLSD test; after
performing a Two-Way ANOVA for matched samples. Group sample sizes are
listed in parentheses.
111
For the first phase of the study, seven to eight animals were randomized into 10
different treatments. The mean isometric torque values normalized to pre-injury baseline
force and body weight at 4 and 8 weeks after surgery are shown in Figure 2C and Figure
2D, respectively. A two-way ANOVA was used to compare functional outcomes among
the different treatment groups over time. Statistical analysis revealed that most treatment
groups showed improvement relative to the negative control BAM group, but overall, no
significant advantage resulted from the inclusion of cells in the injected hydrogel treatment.
Moreover, as illustrated, at 4 weeks (Figure 2C) and 8 weeks post-VML injury (Figure 2D
and Table II), the groups treated with only keratin or keratin containing growth factor(s)
actually showed significant improvement over the groups that had cells embedded within
the keratin hydrogel.
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Table II. In Vivo Functional Analysis of TA Muscle at 8 Weeks Post-Injury. Values are mean ± SEM. Values denoted with
the same letter ate not significantly different (p>0.05), while values without a like letter denotation are significantly different
113
(p<0.05). # Deficit calculated from unoperated animals. BAM: bladder acellular matrix; b: basic fibroblast growth factor;
I: insulin like growth factor type 1; M: muscle progenitor cells; K: keratin; TA: tibialis anterior; wt: weight.
Animals were euthanized at 8 weeks after surgery and an independent second phase
of the study was conducted. Nine to ten animals were randomly assigned to one of 6
treatment groups (i.e., the 4 groups containing cells were eliminated), and the studies were
extended to a 12-week time point to ensure that the extent of functional recovery was more
completely evaluated for these groups (as indicated in (8)).
Placement/implantation of keratin hydrogels (with the exception of KN+I) at the
site of VML injury in the TA was associated with significant and sustained increases in
functional recovery, relative to No Repair (NR) and/or implantation of BAM alone, at all
time points post-VML injury (Figure 3). Moreover, at 12 weeks post-VML injury, the peak
isometric torque response to peroneal nerve stimulation in all keratin treatment groups
(again, except for KN+I) was significantly greater than that observed for both the NR and
BAM treatment groups (Figure 3C and Table III).
Analysis of TA muscle wet weight showed significant reduction of muscle mass in
all experimental groups relative to their contralateral control. However, after 12 weeks, the
TA wet weight of the samples in the KN+b and KN+I+b groups were improved compared
to the other experimental groups (Figure 3D).
114
115
Figure 3. Keratin Hydrogel Promotes Continuous Functional Recovery over
Time. Peak isometric torque production for the treatment groups that combined
formulations of Keratin and growth factors at 4 weeks (A), 8 weeks (B) and 12
weeks (C) are presented. Implantation of all keratin-based treatment groups, except
keratin + IGF, resulted in significant and sustained functional recovery relative to
BAM and/or NR. Wet weight measurements of the injured TA muscle and their
contralateral control are shown in (D). Individual responses are normalized to the
respective group mean of the initial maximum pre-injury isometric torque response
and their body weight. Data is presented as Mean + SEM. *-Significantly different
at the p<0.05 level using Fisher’s PLSD test; after performing a Two-Way ANOVA
for matched samples. Group sample sizes are listed in parentheses.
116
Table III. In Vivo Functional Analysis of TA Muscle at 12 Weeks Post-Injury. Values are mean ± SEM. Values denoted
117
with the same letter ate not significantly different (p>0.05), while values without a like letter denotation are significantly different
(p<0.05). # Deficit calculated from unoperated animals. BAM: bladder acellular matrix; b: basic fibroblast growth factor; I:
insulin like growth factor type 1; K: keratin; TA: tibialis anterior; wt: weight.
Gross Morphology and histological Analysis of TA muscle
Macroscopically, the implants were well tolerated by the recipient animals, with no
episodes infection or seroma. Significant gross morphological modifications were evident
at 12 weeks after the surgical procedure (Figure 4 i-vi). The wound bed was nearly
indistinguishable in the KN (Figure 4 iv), KN+b (Figure 4 v), and KN+I+b (Figure 4 vi)
groups, where signs of regeneration were evident by the presence of viable tissue at the
injury site close to the native muscle, reduced fibrosis and inflammatory infiltrate (Figure
4 I-X).
Corroborating the functional data, the KN+I group showed signs of atrophy at the
injury site (Figure 4 iii), especially when compared to the other keratin treated groups.
Nevertheless, new tissue formation was still observed at the injury site (Figure 4 M-P).
The BAM implant was well integrated with the surrounding wound bed (Figure 4
ii) and filled the void created by the surgical excision of the muscle. However,
histologically, the lack of newly formed tissue as well as increased collagen matrix
formation and inflammatory cell infiltration were evident and corroborated with
diminished functional recovery (Figure 4 E-H).
Different from every group, the wound bed present in the NR group created was
very prominent (Figure 4 i) and the lack of muscle or fibrosis formation at the initial
150mm inside the wound bed confirmed the insufficient regeneration (Figure 4 A-D).
118
119
Figure 4. Gross Morphology and Histological Analysis of TA after Keratin
Based Hydrogel Treatment. Distinguishable differences in the gross appearance
of each group were evident 12 weeks after VML injury. Injury site was apparent in
the NR (i), and, in less extent, in the KN + I group (iv). New tissue formation
completed most of the wound bed in the KN (iii), KN + b (v), and KN + I + b (vi).
BAM implant was incorporated into the wound bed in the BAM group (ii).
Hematoxylin- Eosin (red/pink = cell cytoplasm; violet = nuclei) and Mason’s
Thrichrome (red = tissue; blue = collagen; black = nuclei) staining of longitudinal
section from NR (A-D), BAM (E-H), KN (I-L), KN + I (M-P), KN + b (Q-T) and
KN + I + b (U-X) groups. Arrows denote regions with new muscle fiber formation.
Stars denote regions with native muscle.
120
Discussion
Skeletal muscle has a robust capacity for regeneration after injury, based on the
proliferation and differentiation of resident quiescent satellite cells (35). However, there
are no commercially available products to treat irreversible muscle injuries that result from
traumatic accidents, or acquired and/or congenital diseases.
Our group recently showed the efficacy of TEMR (tissue engineered muscle repair)
construct implantation to restore function in a VML injury model in the TA muscle. The
TEMR constructs were created by seeding muscle progenitor cells (MPCs) onto a bladder
acellular matrix (BAM) and subjecting it to cyclic mechanical preconditioning (10%
stretch) in a bioreactor prior to implantation. In fact, the presence of MPCs in the construct
played a crucial role in the regeneration process as shown by the greater functional recovery
on the TEMR group compared to the group implanted with unseeded BAM constructs (8).
Despite the encouraging outcome from the TEMR technology studies, a
combination of different tissue engineered approaches are necessary to treat the ample
variety of traumatic injuries and diseases that compromise soft tissue.
Keratin hydrogel is a malleable biological scaffold, with future clinical applications
promoting minimally invasive surgical procedures. Additionally, its material properties
(e.g. degradation rate) can be easily tailored to promote sustainable release of biological
compounds (20, 33) that closely influence tissue regeneration (e.g. growth factors).
IGF-1 and bFGF are locally produced molecules that play crucial roles in the
complex signaling cascade that stimulates recruitment, proliferation and differentiation of
satellite cells (36, 37). The incorporation of these growth factors into keratin hydrogel
allows for their controlled release over time, avoiding loss of biological activity due to
121
diffusion and/or enzymatic inactivation (38).
Herein, keratin hydrogels were a 70% KOS/30% KTN combination, as used in
previous studies (32, 33), and contained a combination of IGF-1 and/or bFGF (Table 1).
Functional recovery of the TA muscle after VML injury for all treatment groups is
represented in Figure 3. The maximal isometric torque in the NR and BAM groups
corroborates with data previously published (8, 34), indicating precision in the
reproduction of the TA injury model. Hydrogel implantation mediated restoration of
significant functional capacity that continued to improve over the course of 12 weeks, when
the functional recovery observed by keratin alone was indistinguishable from other
effective keratin hydrogels containing growth factors (Keratin + bFGF and Keratin + IGF
+ bFGF).
These observations highlight the efficacy of the keratin biomaterial as a potentially
important platform for treatment of VML injury. To put these observations into
perspective, it should be noted that the synergists (EDL and EHL) are removed at the time
of the surgical VML injury, resulting in a permanent ≈20% functional deficit (see (8) for
details). This means that after removing approximately 20% of the TA muscle, the total
functional defect is ≈50%, of which only 30% is recoverable. Thus, the maximal functional
recovery possible is 0.8 of the pre-injury baseline maximal isometric torque response. Both
NR and BAM treatment groups exhibited maximal torque responses of ≤0.6, while the
keratin hydrogel treatments (Keratin alone, Keratin + bFGF and Keratin+ bFGF + IGF)
exhibited values of ≥0.7, ranging as high as 0.74. This corresponds to ≈90% functional
recovery. These are robust and reproducible functional recoveries on a substantial number
of animals.
122
Qualitative morphological and histological analysis corroborates with functional
data. The TA muscle treated with keratin-based hydrogels had distinguishable morphology
from the untreated group (NR) (Figure 4), and evidence of new muscle fiber formation and
diminished fibrosis related to the BAM treated group (Figure 5).
Interestingly, keratin-only implantation promoted morphological and functional
improvement even in the absence of growth factor (IGF-1 and bFGF). It is possible that
during muscle regeneration, keratin biomaterial modulates cell proliferation, cell adhesion,
and gene expression in a similar manner as seen in nerve repair (16, 32, 39, 40). However,
the data from this study does not provide sufficient basis to formulate an explanation for
the molecular mechanics behind the functional and morphological improvement in the
experimental groups. Further investigation regarding the influence of the keratin-based
hydrogel over skeletal muscle regeneration is warranted.
The contribution of controlled time-released growth factors to muscle healing have
been extensively discussed (reviewed in (41)). However, throughout the literature, there is
clear evidence that the addition of cells component to tissue-engineered scaffolds is also
crucial to enhance functional recovery in a VML injury model (8-10, 42-44). Synthetic and
biological hydrogels have been used as a cell delivery system in a variety of injury models
in vivo (45-55). Through hydrogel implantation, myoblasts formed new muscle fibers in a
skeletal muscle injury in one study (45), while bone marrow mesenchymal stem cells were
shown to improve cardiac remodeling after myocardial infarction in another (54). This
study has shown that addition of MPCs (at the concentration used herein) used to the
keratin formulation did not significantly improve muscle regeneration.
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Based on our experience with the keratin hydrogel, the number of MPC implanted
was consistent with the hydrogel’s limitations regarding viscosity. The number of MPCs
is remarkably smaller compared to the number of cells used in the above-mentioned
studies. Our previous work has revealed that implantation of too few myoblasts in this
VML injury setting may actually be associated with diminished functional regeneration
(8). The data are consistent with the supposition that before hydrogels can be used in
muscle repair, further optimization of keratin hydrogel properties are required to
accommodate the delivery of a higher cell yield.
Conclusion
We have shown for the first time that keratin hydrogel, successfully used for bone
and nerve regeneration, promotes significant functional improvement of severely damaged
skeletal muscle. The recovery observed after keratin implantation is achieved through a
minimally invasive transplantation procedure. This procedure has apparent equivalence to
the efficacy of materials used for cell delivery in the same animal model, but with the
advantage of not requiring a cellular component, a xenogeneic scaffold (BAM), or
bioreactor preconditioning.
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CHAPTER IV
Investigation of Tissue Engineered Muscle Repair Constructs for Use in Cleft Lip
Secondary Revision Procedures in a Rat Model
Hannah Besser Baker
Note: This chapter was composed by Hannah Besser Baker
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Introduction
Volumetric Muscle Loss (VML) has been defined by Grogan et al as the
irrecoverable loss of function (1). This definition came about through the study of traumatic
injuries observed in active military personnel wherein musculoskeletal traumas make up
as much as 70% of reported injuries (2). It is important to consider that in addition to
traumatic injuries, several other skeletal muscle pathologies and indications are associated
with irrecoverable loss of function. Such conditions include acquired diseases of the
musculature i.e. Bell’s palsy, surgical resection of tissue concurrent with cancer treatment,
and congenital birth defects such as cleft lip and cleft palate (CL/CP) (3, 4). Among these
presentations, cleft lip and cleft palate (CL/CP) are extremely common- occurring in 1 in
1,000 live births the United States (5). Current strategies for repair of CL/CP largely
surround the use of neighboring tissues to act as flaps to bridge tissue voids (6-8).
Advancements in surgical repair strategy have continued to improve patient outcomes after
CL/CP revision; however, secondary revision is typically required (9-12). Present standard
procedures for correction of the most frequent secondary lip and nasal deformities utilize
regional tissue flaps. These procedures can be complicated by tissue deficiency as well as
the quality of the available tissue which can be impaired as a result of the primary repair
procedure (9, 10, 13, 14). Tissue engineered grafts offer an alternative to tissue flaps and
the often associated need to implement tissue expansion devices or regimens to provide
sufficient tissue area for use in secondary revisions (8, 10, 12, 13).
The crucial concern in secondary as well as primary revision of CL/CP is
establishment of continuity and function in the orbicularis oris muscle (OOM) (8, 9, 11,
15, 16). As such, tissue engineering strategies should provide additional graft material
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which facilitates favorable OOM form and function. Numerous investigators have shown
the utility of decellularized extracellular matrix based technologies in a variety of tissue
engineering applications including promotion of skeletal muscle regeneration in animal
models of VML (17-27). Among these studies, our group has illustrated the capacity of a
porcine bladder acellular matrix (BAM) seeded with autologous muscle cells and subjected
to axial stretch preconditioning to confer recovery of muscle function and intrinsic
morphology in two distinct rodent models of VML (18, 25, 28).
To investigate an established technology for use as a graft in a (CL/CP) secondary
revision procedure in an in vivo study, an appropriate animal model would accommodate
a graft size and surgical application akin to revision procedures with local tissue flaps.
Investigators have indicated the use of rat latissimus dorsi (LD) for studying muscle repair
and VML and have highlighted similarities in architecture between rat LD and human
craniofacial muscles such as the OOM (21, 29). We have recently developed a model in
the rat LD with a defect area larger in size than used in our previous studies. The new
model allows surgical incorporation of a test article by methods which have also been
applied when using autologous tissue flaps in CL/CP revision.
We hypothesize that Tissue Engineered Muscle Repair (TEMR) constructs
developed in previous studies will enable return of both function and form by promotion
of favorable integration with the host tissue and development of new muscle within a VML
model with bio-relevance to cleft lip secondary revision. Moreover we hypothesize that the
addition of a cellular component and stretch preconditioning will improve the recovery in
the TEMR repaired muscles relative to those treated with BAM alone.
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To evaluate the proposed hypotheses, the newly developed VML model was
employed to investigate both TEMR and BAM constructs in both male and female rats. To
capture the full potential of muscle repair and to investigate potential long term outcomes
this study examined functional and morphological recovery at 2, 4 and 6 months after
injury.
Methods
BAM Preparation
BAM scaffolds were prepared as previously described (25, 28). Briefly, the bladder
was washed and trimmed to obtain the lamina propria, which was placed in 0.05% trypsin
(Hyclone, Logan, UT) for 1 hour at 37 °C. The bladder was then transferred to Dulbecco’s
modified Eagle’s Medium, DMEM, solution supplemented with 10% FBS and 1%
antibiotic antimycotic, AA, (Hyclone) and kept overnight at 4 °C. The preparation was then
washed in a solution containing 1% triton-X (Sigma) and 0.1% ammonium hydroxide
(Fischer Scientific, Pittsburgh, PA) in de-ionized water for 4 days at 4 °C. Finally, the
bladder was then washed in deionized water for 3 days at 4 °C. The decellularized scaffold
was further dissected to obtain a scaffold of 0.2–0.4mm thickness. The scaffolds were then
cut and draped onto custom made silicone molds with a 5.4cm2 working area.
Cell Isolation and Culture
Rat MPCs were isolated from the tibialis anterior and soleus muscles of 4 week old
(male or female depending on the sex of the recipient rats) Lewis Rats purchased from
Charles River Laboratories. Muscles were sterilized in iodine and washed with PBS before
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transfer to a 0.2% w/v type 1 collagenase (Worthington Biochemical, Lakewood, NJ) low
glucose DMEM solution (Hyclone, Logan, UT) where they were finely minced and then
allowed to further digest at 37 °C for 2 hours. The cell slurry was plated on collagen coated
dishes for 24 hours (as a fibroblast reduction measure) in myogenic media consisting of
high glucose DMEM supplemented with 20% fetal bovine serum (FBS) (Hyclone), 10%
horse serum (HS), 1% chick embryo extract, and 1% antibiotic/antimitotic (AA) (Hyclone).
The following day, the slurry was moved to matrigel (BD) coated plates in myogenic
media. Three days after plating the cells on matrigel plates, the media was changed to
seeding media consisting of low glucose DMEM supplemented with 15% FBS and 1% AA
(Hyclone). At 70-80% confluence cells were passaged. At passage 2 cells were seeded onto
one side of BAM scaffolds at 1x106 cells/cm2, 5.4x106 MPCs/side. 24 hours later passage
2 cells were seeded onto the other side of the BAM scaffold at 1x106cells/cm2. Three days
after the first side was seeded, the scaffolds were switched to differentiation media
consisting of F12 DMEM, 2% HS, 1%AA (Hyclone). After 3 days scaffolds were given
fresh differentiation media.
Mechanical Stretch Preconditioning
After a total of 10 days of static culture on the scaffolds, scaffolds were transferred
to a bioreactor for stretch preconditioning which has been previously described (25). In
seeding media within the bioreactor, scaffolds were mounted onto the two adjacent ledges
with the long side of the scaffold spanning the gap between the ledges. The gap length was
adjusted to ~3cm. Scaffolds were then clamped to the ledges, and the bioreactor was
flooded with seeding media. A proprietary software was used to interact with a Phidget
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controller for operating the stepper motor of the bioreactor (Phidgets Inc., Calgary, CAN).
Using the software-phidget-motor interface, the scaffolds were stretched and relaxed 10%
of the initial gap length 3 times each minute for the first 5 minutes of each hour for 5 days.
Rat LD VML Model and Implantation of TEMR
12 week old Lewis rats were purchased from Charles River Laboratories. Both male
and female animals were used equally across all groups and time points (Table I). The
VML defect was created by making an incision along the spine, separating the fascia, and
removing a 1.7cm wide and 2.5cm long rectangular section of tissue, ~2mm from the lateral
edge of the muscle and with the lower lateral corner in line with the last palpable rib. For
the NR group, the defect was left alone. For the BAM and TEMR treatment groups, one
BAM or TEMR construct was secured below the defect window with 6-0 vicryl suture by
making ten equally spaced knots. Another construct was then secured above the window
in a similar fashion (Figure 1). The fascia was then sutured closed with 6-0 vicryl and the
skin was closed with 4-0 prolene. The animals were then allowed to recover.
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Table I. Treatment Groups
Figure 1. Surgical Flow of Rat LD VML Injury and repair with
TEMR. The LD muscle is exposed and defect is outlined (A), the
defect is created (B), and TEMR constructs are sutured below and
above the created defect (C).
140
Muscle Function Analysis
The entire LD muscle was isolated from the thoracolumbar fascia to the humeral
tendon from rats while under anesthesia and the tendon and facial ends were tied with 0-0
silk suture (Ethicon) and transferred to ice cold Krebs-Ringer buffer solution (Sigma;
composition: pH 7.4; concentration in mM: 121.0 NaCl, 5.0 KCl, 0.5 MgCl2, 1.8 CaCl2,
24.0 NaHCO3, 0.4 NaH2PO4, and 5.5 glucose). Muscles were transferred to individual
chambers of a DMT 750 tissue organ bath system (DMT, Ann Arbor, MI) filled with
Krebs-Ringer buffer at 25 °C bubbled with 95% O2 and 5% CO2. The muscles were
positioned between custom-made platinum electrodes with the proximal tendon attached
to a force transducer and the distal tendon to a fixed support. Direct muscle electrical
stimulation (0.2ms pulse at 30 V) was applied across the LD muscle using a Grass S88
stimulator (Grass, Warwick, RI). Real time display and recording of all force
measurements were performed on a PC with Power Lab/8sp (AD Instruments, Colorado
Springs, CO). Once the LD muscles were mounted in the organ bath, the muscles were
allowed to equilibrate for 5 min prior to determining optimal physiological muscle length
(Lo) via a series of twitch contractions. Force as a function of stimulation frequency (1–
150Hz) was measured at 25 °C during isometric contractions (750ms trains of 0.2ms
pulses), with 2 min between contractions. The maximal force of isometric contraction, Po,
was normalized to an approximate physiological cross-sectional area (PCSA), which was
calculated using the following equation, where muscle density is 1.06 g/cm3:
𝑃𝑃𝑃𝑃𝑃𝑃𝑃𝑃 = (𝑤𝑤𝑤𝑤𝑤𝑤 𝑤𝑤𝑤𝑤𝑤𝑤𝑤𝑤ℎ𝑡𝑡 (𝑔𝑔)/ [𝑚𝑚𝑚𝑚𝑚𝑚𝑚𝑚𝑚𝑚𝑚𝑚 𝑑𝑑𝑑𝑑𝑑𝑑𝑑𝑑𝑑𝑑𝑑𝑑𝑑𝑑 (𝑔𝑔/𝑐𝑐𝑐𝑐3) 𝑥𝑥 𝑚𝑚𝑚𝑚𝑚𝑚𝑚𝑚𝑚𝑚𝑚𝑚 𝑙𝑙𝑙𝑙𝑙𝑙𝑙𝑙𝑙𝑙ℎ, 𝐿𝐿𝑜𝑜 (𝑐𝑐𝑐𝑐)]
Equation 1: Physiological Cross Sectional Area (PCSA) Calculation.
141
Additionally the force-frequency curves were fit to the following dose response equation:
𝑓𝑓(𝑥𝑥) = 𝑚𝑚𝑚𝑚𝑚𝑚 − (𝑚𝑚𝑚𝑚𝑚𝑚 − min)/[1 + (𝑥𝑥 ⁄ 𝐸𝐸𝐸𝐸 50 )−𝑛𝑛 ]
Equation 2: Dose Response Equation.
where x is the stimulation frequency in Hz, min is the lowest observed force generated,
equivalent to the twitch force or Pt, max is the largest observed force or Po, EC50 is the
stimulation frequency which yields half the amplitude of the maximum observed force (Po-
Pt), and n is the slope of the linear portion of the force-frequency curve.
Immunohistochemistry and Imaging
No explanted LDs were specifically dedicated for the use of immunohistochemical
analysis, all histology and immunohistochemistry were performed on muscles that had
been through the organ bath analysis. When preparing the tissue, the rat LD, the muscle
was cut in half transversely in the middle of the defect site such that the samples were a
more manageable size for frozen and paraffin sectioning. Hematoxylin and eosin and
Masson’s trichrome stains were conducted by standard techniques to determine the basic
morphology of cells in and around the implant and to observe any inflammatory response.
Images were captured and digitized using a DM4000B Leica Upright Microscope.
Statistical Analysis
Data values are shown as mean ± SEM unless otherwise noted. Data were
statistically analyzed using one way ANOVA. When significance was found (α set to 0.05)
a post-hoc multiple comparison test was used to compare group means (α set to 0.05) using
142
a Fisher Least Significant Difference (LSD) significance test. Statistical analysis was
conducted using GraphPad Prism 6.0 for Windows La Jolla, California.
Results
In vitro force measurement was conducted as described at 2, 4, and 6 months after
implant on whole LD operated and contralateral control muscles harvested from rats in the
three treatment groups at each time point Table I. Measured physical and functional
parameters for each group and parameter statistics are listed in Tables II-IV. EC50 and Hill
slope were calculated by fitting the dose-response function in Equation 2 to force-
frequency curves as shown in Figure 2 for 2 months post-injury male groups. Comparison
of Po, Pt, and P30 Hz is shown for all groups at each time point for males and females (Figures
3-8).
143
144
Table II. Summary of Latissimus Dorsi Muscle Physical and Functional
Measurement Data at 2 Months Post-Injury. Values are mean ± standard error
of the mean. Muscle weight was measured after completion of functional testing.
Lo is the optimal muscle length associated with peak twitch response (Pt). PCSA
was determined using the measured Lo and wet weight using Equation 1. Peak
twitch (Pt), contraction force at 30hz (P30), and maximal isometric contraction force
(Po) were measured by soliciting contractions via direct electric stimuli (0.2ms,
30V) in the organ bath setup. Measured Po was normalized to PCSA to determine
specific force (Specific Po). The stimulation frequency which yielded 50% of the
absolute maximum amplitude (EC50) and the slope of the linear portion of the force-
frequency curves as shown in Figure 2 for selected data sets (n coefficient) were
determined by fitting the curves to function described by Equation 2. Significant
differences (p < 0.05) between TEMR, BAM, and NR treated muscles were
determined by ANOVA and further evaluated using a Fisher Least Significant

a
Difference post-hoc analysis. denotes significant difference from both other
treatment groups
145
146
Table III. Summary of Latissimus Dorsi Muscle Physical and Functional
frequency curves as shown in Figure 2 for selected data sets (n coefficient) were

a
treatment groups
147
148
Table IV. Summary of Latissimus Dorsi Muscle Physical and Functional
frequency curves shown in Figure 2 for selected data sets (n coefficient) were

a
treatment groups
149
Figure 2. Force-Frequency Curve with Dose-Response Curves for Males at
2mos. Two months post-surgery contractions elicited via electrical stimuli from 1-
150Hz is shown for male groups. Data has been fit with dose response curves
according to Equation 2. Curve fitting was used to determine EC50 and Hill Slope
values shown in Tables II. Data points shown are group mean ± SEM for each
stimulation frequency.
150
Figure 3. Comparison of Po across Treatment Groups and Timepoints in
Males. Maximal isometric contraction force, Po, is shown for each group at 2, 4,
and 6 months post-injury. Forces were measured by eliciting contractions via direct
electric stimuli (0.2ms, 30V) from 1-150Hz in the organ bath setup.
Figure 4. Comparison of Po across Treatment Groups and Timepoints in
Females. Maximal isometric contraction force, Po, is shown for each group at 2, 4,
and 6 months post-injury. Forces were measured by eliciting contractions via direct
electric stimuli (0.2ms, 30V) from 1-150Hz in the organ bath setup.
151
Figure 5. Comparison of Pt across Treatment Groups and Timepoints in
Males. Peak twitch force, Pt, is shown for each group at 2, 4, and 6 months post-
injury. Twitches were measured by eliciting contractions via direct electric stimuli
(0.2ms, 30V) at 1Hz in the organ bath setup.
Figure 6. Comparison of Pt across Treatment Groups and Timepoints in
Females. Peak twitch force, Pt, is shown for each group at 2, 4, and 6 months post-
injury. Twitches were measured by eliciting contractions via direct electric stimuli
(0.2ms, 30V) at 1Hz in the organ bath setup.
152
Figure 7. Comparison of P30 Hz across Treatment Groups and Timepoints in
Males. Submaximal contraction force at 30Hz, P30 Hz, is shown for each group at
2, 4, and 6 months post-injury. Forces were measured by eliciting contractions via
direct electric stimuli (0.2ms, 30V) at 30 Hz in the organ bath setup.
Figure 8. Comparison of P30 Hz across Treatment Groups and Timepoints in
Females. Submaximal contraction force at 30Hz, P30 Hz, is shown for each group at
2, 4, and 6 months post-injury. Forces were measured by eliciting contractions via
direct electric stimuli (0.2ms, 30V) at 30 Hz in the organ bath setup.
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Animal and Model Specifications
Male rats weighed 308 ± 2g and female rats weighed 195 ± 1g at the time of
implant. The average created defect weighed 377 ± 4mg and measured 4.34 ± 0.02cm2 in
males and 267 ± 3mg and 4.28 ± 0.02 cm2 in females with no significant difference between
treatment groups. Control LD weight and length at the time of explant was not significantly
different between time points in either male or female rats (2.23 ± 0.02g and 6.93 ± 0.06cm
in males and 1.21 ± 0.01g and 6.49 ± 0.06cm in females) while body weight was
significantly different at each time point for both sexes (Table V). The injury created in
this study represented 17% of measured total weight of the contralateral control LD at the
time of explant for males and 22% for females.
Table V. Body Weight at Implant and Explant, Defect Weight & Size, and LD
Weight and Lo for Males and Females. Body weights at implant and explant,
defect size and weight, and control LD weight and optimal length, lo, at explant are
shown for males and females for each of the study time points. Statistical
differences were determined by ANOVA p < 0.05. *denotes difference from all
other timepoints (for males and females separately) p < 0.0001.
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In Vitro Functional Assessment
For males and females respectively injury resulted in an 80% and 73% reduction in
maximum isometric contraction force (Po), an 86% and 77% reduction in peak twitch force
(Pt), and an 83% and 75% reduction in measured submaximal contraction at 30Hz (P30 Hz)
in the No Repair (NR) group relative to uninjured control muscles evaluated at two months.
Two Months Post-Injury
In both males and females TEMR treated muscles had the highest Po, Pt, P30 Hz, and
specific Po though these findings were not significantly different from those of BAM and
NR treated muscles. Weight and length measurements were not significantly different
among the treatment groups in males while female BAM treated muscles were significantly
heavier than TEMR or NR treated tissues. Analysis of dose response curves fit to force-
frequency data (Example shown in Figure 2) indicated that EC50 values and hill slope
coefficients were not different among treatment groups.
Four Months Post-Injury
At four month post injury there were no differences in Po, Pt, P30 Hz or specific Po
between treatments in either males or females. Female BAM treated muscles remained the
heaviest, but were not significantly heavier than TEMR or NR treated muscles. Similar to
the two month time point there were no significant differences among weight and length
between treatment groups for either sex. Determined EC50 values were significantly higher
for male NR muscles and significantly lower for female NR muscles compared with the
155
other treatment groups. Hill slope coefficients were not significantly different between
treatment groups for either sex.
Six Months Post-Injury
As observed at the previous Po, Pt, P30 Hz and specific Po were not different among
treatment groups for either sex. Female BAM treated muscles were still the heaviest, but
not different. Male NR muscles weighed the least but were longest (ns for either parameter)
which contributed to a significantly lower PCSA compared with TEMR and BAM treated
muscles. EC50 and hill slope coefficients were not different among male experimental
tissues while female TEMR data had a significant forward shift in EC50 without a
significant difference in hill slope compared with BAM and NR groups.
Morphology
Macroscopically, there were visible similarities in tissue morphology within
treatment groups. To showcase patterns within treatment groups, time points, and for males
and females, images of all samples are shown in Supplemental Figures 1-18 and three
selected representative samples from each group are shown for each time point for males
and females in Figures 9-14. A summary of findings surrounding tissue density within the
implant region, vascular structure presence, adipose or scar tissue presence, and apparent
blood clots or inflammation is shown in Tables VI-VII for BAM and TEMR treated tissues.
156
Treatment Groups for Males at 2mos. Representative TEMR treated tissues are
shown in A-C, BAM treated samples in D-F, and NR samples are shown in G-I.
Samples are shown with the experimental muscle on the left and the uninjured
contralateral control muscle on the right.
157
Treatment Groups for Females at 2mos. Representative TEMR treated tissues
are shown in A-C, BAM treated samples in D-F, and NR samples are shown in G-
I. Samples are shown with the experimental muscle on the left and the uninjured
158
159
160
161
162
Table VI. Summary of Macroscopic Findings in Males. BAM and TEMR
treated tissues were evaluated for tissue quality. Parameters of interest which were
evaluated include connective tissue density, and presence of blood vessels, scar
tissue, and apparent blood clots or inflammation within the connective tissue layers.
The assessment used a scale from (–): little to no evidence, to (+++): extensive
presence. Reported findings for males were relative to male BAM and TEMR
treated tissues.
163
Table VII. Summary of Macroscopic Findings in Females. BAM and TEMR
treated tissues were evaluated for tissue quality. Parameters of interest which were
evaluated include connective tissue density, and presence of blood vessels, scar
tissue, and apparent blood clots or inflammation within the connective tissue layers.
The assessment used a scale from (–): little to no evidence, to (+++): extensive
presence. Reported findings for females were relative to female BAM and TEMR
treated tissues. *Note BAM scaffold remnants in one sample.
164
TEMR treated tissues
Tissues which were treated with TEMR differed between males and females but
were similar for given time points for each sex and resulted in the greatest tissue quality
relative to NR and BAM treated tissues. In males TEMR treated tissues had tissue present
within the defect site which was more opaque at each subsequent time point. Vascular
structures were present at 2 months and to a greater extent at 4 and 6 months. For males
the TEMR treated tissue did not appear to be inflamed and there was no evident scar tissue
or adipose tissue deposition near or within the implant region. For females the tissue quality
was visibility decreased in comparison with male TEMR treated tissues with regard to
opacity, vascularity, and inflammation. Tissue within defects for females treated with
TEMR was visibly less dense than in males at all timepoints. In females blood clots could
be found in samples at each time point and scar tissue could be seen at 6 months. Female
tissues treated with TEMR, like males, did have vascular structures present within the
implant region at each time point but to a lesser extent than was seen in males.
BAM treated tissues
Similar to the morphology seen in TEMR treated tissues, BAM treated muscles’
morphology differed between males and females but similarities were visible within time
points for each sex and resulted in tissue quality which overall surpassed that seen in NR
tissues. For males at 2 months, BAM treated defects were filled in with connective tissue
to a lesser extent than what was seen in TEMR treated muscles at each time point. The
tissue layer tore easily leaving holes visible in samples (Figure 11E-F, Figure 13F). Within
the implant region, some dark clots were visible at 2 and 4 months, but these findings were
165
not present at the 6 month time point. At 6 months BAM treated tissues were more opaque
than at previous time points, tissues were free of the previously seen clots. Within the BAM
treated tissues, unlike TEMR treated tissues, a small extent of adipose and scar tissue
deposition was apparent at the 6 month time point. BAM treated tissues had vascular
structures present within the region of the defect at 2 months with increasing presence at 4
and 6 months, however the vascularity was visibly less than that seen in TEMR treated
tissues for each time point. In females treated with BAM, a tissue layer formed that was
thinner and more frail than that seen in female TEMR tissues for each time point, resulting
in gaps and tears not seen in TEMR treated tissues. The tissue layer in females had blood
clots present, similar to those seen in male BAM treated muscles, at each time point
including 6 months. Unique to female BAM treated tissues was the presence of remaining
BAM scaffold at 2 months such as that seen in Figure 10F. A small extent of vascular
structures were present at each time point within the implant region though, like that seen
in male BAM tissues, to a lesser extent than was seen in TEMR treated tissues.
NR tissues
At all three time points and in both sexes, tissues in which there was no repair (NR
tissues) had a distinct macroscopic appearance (Figures 9-14 G-I). In males, NR muscles
had thin translucent connective tissue layers at 2 months which tore and resulted in holes
due to handling in some samples. The connective tissue layer appeared thicker at 4 months
and thickest at 6 months, but the layer remained translucent at both subsequent time points.
Little to no vascular structures were visible within the connective tissue layers at the 2 and
4 month time points, while at 6 months a greater, though still modest, extent of vascular
166
structures could be seen. Female NR tissues were very similar in appearance to male NR
tissues, unlike the differences seen between the sexes in TEMR and BAM treated tissues.
In female NR tissues, a very thin, translucent, and delicate connective tissue layer was
visible at 2 and 4 months and appeared slightly more substantial at 6 months. The tissue
layer which formed was the least dense of the treatment groups and tore as a result of
handling at all timepoints for females. There was no apparent inflammation or scar or
adipose tissue deposition in the female NR tissues. Vascular structures were not visible
within defect regions for any female NR tissue at any time point.
Histology
As described, following functional testing explanted tissues were fixed and
processed for histological analysis. Hematoxylin and eosin staining and Masson’s
trichrome staining enabled tissue level visualization of the selected representative samples
(Figures 9-14) for each treatment group with additional images of Masson’s trichrome
staining within the implant region, when feasible, for TEMR and BAM treated tissues
(note: samples for macroscopic comparison were also used for histological
comparison)(Figures 15-32). Samples were evaluated for overall presence and apparent
density of the connective tissue layer at the border of and within the created defect and for
presence and location of adipose and scar tissue deposition, neurovascular structures, and
mononuclear cell infiltrate. A summary of findings for TEMR and BAM groups can be
found in Tables VIII-IX.
167
168
Figure 15. Hematoxylin and Eosin and Masson’s Trichrome Staining of
Tissues from Selected Representative Tissues: 2mos Male TEMR. Following
functional testing explanted tissues were fixed and processed for histological
analysis. Hematoxylin and eosin staining (A-C) and Masson’s trichrome staining
(D-F) enabled tissue level visualization of the native tissue-defect and implant
border for the selected representative samples from each treatment group.
Additional images of Masson’s trichrome staining within the implant region (G-I)
are shown for TEMR and BAM treated tissues for samples wherein the implant
region was both intact and distinct from the native tissue interface. Samples were
evaluated for overall presence and apparent density of the connective tissue layer
at the border of native tissue and the defect and within the created defect and for
presence and location of adipose and scar tissue deposition, neurovascular
structures (denoted with white arrows), and mononuclear cell infiltrates (denoted
with black arrows).
169
170
Tissues from Selected Representative Tissues: 2mos Male BAM. Following
with black arrows).
171
172
Tissues from Selected Representative Tissues: 2mos Male NR. Following
border for the selected representative samples from each treatment group. Samples
were evaluated for overall presence and apparent density of the connective tissue
layer at the border of native tissue and the defect and within the created defect and
for presence and location of adipose and scar tissue deposition, neurovascular
with black arrows).
173
174
Tissues from Selected Representative Tissues: 2mos Female TEMR. Following
with black arrows).
175
176
Tissues from Selected Representative Tissues: 2mos Female BAM. Following
with black arrows).
177
178
Tissues from Selected Representative Tissues: 2mos Female NR. Following
with black arrows).
179
180
with black arrows).
181
182
with black arrows).
183
184
with black arrows).
185
186
with black arrows).
187
188
with black arrows).
189
190
with black arrows).
191
192
with black arrows).
193
194
with black arrows).
195
196
with black arrows).
197
198
with black arrows).
199
200
with black arrows).
201
202
with black arrows).
203
Table VIII. Summary of Key Histological Findings for TEMR and BAM
Treated Tissues in Males. BAM and TEMR tissues sections from representative
samples were evaluated for apparent connective tissue density, mononuclear cell
infiltrates, and presence of neurovascular structures at the interface between native
tissue and the defect-implant region and within the implant region. The assessment
used a scale from (–): little to no evidence, to (+++): extensive presence. Findings
for males were relative to male BAM and TEMR treated tissues.
204
Table IX. Summary of Key Histological Findings for TEMR and BAM
Treated Tissues in Females. BAM and TEMR tissues sections from representative
samples were evaluated for apparent connective tissue density, mononuclear cell
infiltrates, and presence of neurovascular structures at the interface between native
tissue and the defect-implant region and within the implant region. The assessment
used a scale from (–): little to no evidence, to (+++): extensive presence. Findings
for females were relative to female BAM and TEMR treated tissues.
205
TEMR treated tissues
Males and females treated with TEMR had histological presentations with notable
similarities which highlighted the characteristics of repair associated with TEMR treatment
(Figures 15, 21, and 27 and 18, 24, and 30). These similarities included vascular structure
presence and little to no inflammation at all timepoints. Comparison also highlighted a
divergence in observed tissue quality between males and females (also observed in
comparing males and females in BAM and NR treatment groups).
At 2 months male TEMR treated muscles showed a dense, collagen rich tissue
bridge which spanned the defect created in the native tissue. Vascular structures were
present to a great extent in all evaluated samples at the borders and well within the
implanted constructs. A moderate mononuclear cell presence was observed in one sample
(Figure 15A) while other samples did not show signs of inflammation. At 4 months, the
collagen rich tissue bridge remained intact with vascular structures present throughout and
with some visible adipose tissue deposition within the tissue bridge. Mononuclear cells
were not observed in any of the samples at this timepoint. At 6 months, tissues appeared
similar to those observed at the 4 month time point: the connective tissue layer was collagen
dense and free of mononuclear cells infiltration in all samples. Two samples at this time
point presented with well-defined vascular structures near the implant-native tissue border
which were larger in diameter than vascular structures observed at previous time points for
TEMR (Figure 27A-B).
Female tissues treated with TEMR had thin connective tissue layers present at 2
months post-surgery which appeared to be more collagenous closer to the border of the
defect with increasing adipose presence further from the border and within the implant. In
206
two samples moderate mononuclear cell infiltrates could be observed with a coagulations
of cells visible in one sample (Figure 18A-B). Vascular structures were visible at the border
of the implant and native tissue with decreasing presence further from the native tissue-
implant border. At 4 months female TEMR treated tissues showed a dense collagenous
tissue ‘cap’ beginning to form at the edge of the native tissue. Within the observed collagen
cap, vascular structures were seen. Beyond the collagenous cap tissue layer, the connective
tissue was observed to have a greater extent of adipose tissue deposition and fewer vascular
structures. There was no evidence of mononuclear cell infiltration or inflammation in any
sample at 4 months. The overall tissue density and quality at 6 months was similar to that
seen at 4 months. There was however, an increased presence of vascular cells within the
adipose rich region of the tissue bridge (within the area occupied by the implant).
BAM treated tissues
Males and females treated with BAM had similarities in tissue morphology with
regard to tissue density and inflammation. Comparison of males and females also
exemplified the differences seen in overall tissue quality between the sexes that had also
been seen in TEMR treated tissues (Figures 16, 22, and 28 and 19, 25, and 31).
Males treated with BAM at 2 months had a dense collagenous layer formed within
the implant region. Mononuclear cells could be seen within the layer in all samples with a
visible mass of cells present in one sample (Figure 16C). New blood vessels could be seen
within the tissue layer, with a higher concentration of structures closer to the implant-
muscle interface. At 4 months, the tissue density appeared to be decreased further away
from the native tissue and further into the implant region. Male BAM samples were
207
observed to have little to no mononuclear cell presence surrounding the edge of the implant
at 4 months compared with 2 months. At 4 months, issues had more frequent vascular
structures present at the border than within the implant. At 6 months the tissue density
appeared unchanged. Mononuclear cell presence had decreased as well and vascularity also
appeared to be less prevalent beyond the tissue-implant border than that observed at 4
months.
Females treated with BAM had thin collagen rich tissue layers within the implant
site at 2 months. There was visible inflammation in all samples with congregations of
mononuclear cells observed in each sample. Vascular structures could be seen within the
connective tissue layer in the vicinity of the inflammatory regions and along the edges of
the native tissue-implant interface. At 4 months, tissue density within the implant region
appeared have decreased and had a higher presence of adipose cells. There was also no
visible inflammation at this time point. At 4 months in BAM treated females, there were
few visible vascular structures along the interface or within the implant site. Histological
findings were similar at 6 months with more collagen present along the border of the
implant region which included a greater extent of vascular structures than was seen at 4
months and little to no visible mononuclear cells present.
NR tissues
Among treatment groups, male and female NR tissues were the most similar in
overall appearance and tissue quality (Figures 17, 23, and 29 and 20, 26, and 32). Males
developed a thin adipose heavy tissue layer within the region of the defect at 2 months.
Within the connective tissue layer there was little to no evidence of new vascular structure
208
formation nor was there evidence of inflammation. At 4 months the connective layer was
more collagen heavy forming a cap along the native tissue edge with no change in
vascularity or inflammation. The tissue cap seen at 4 months could also been seen at 6
months and appeared to be thicker. At 6 months there was also no inflammation present
nor extensive new vascular structure compared with the prior timepoints. In females, at 2
months there was a thin connective tissue layer rich with adipose cells. There was little to
no vascular structures or inflammation present compared with other treatment groups in
females. At 4 months, the thin connective tissue layer remained adipose rich and a cap of
tissue surrounding the native tissue could be seen as was observed in male NR tissues.
Observed vascularity remained minimal at 4 months. The tissue layer surrounding the
native tissue was more well defined and collagen heavy at 6 months. As was seen in
previous timepoints for NR female tissues, there was little to no evidence of vascular
formation within the implant region as well as no visible signs of inflammation.
Discussion
Tissue engineering strategies for CL/CP revision have largely surrounded cartilage
and bone repair for CP applications (30-37). Among CL/CP TE research there have been
few endeavors focused on soft tissue repair (21, 38-41). As it would follow, there also
existed a lack of animal models approximating human craniofacial muscles for
investigation of treatment for CL/CP (21, 42-46). As such, the first major goal of the
present research was to develop an injury model in the rat latissimus dorsi approximating
secondary revision of CL. Secondly, the intent of this study was to test the capacity of a
previously established technology to promote muscle healing and regeneration in the newly
209
developed model. The therapy of interest, TEMR, has shown promising results in models
of VML carried out in the mouse latissimus dorsi and the rat tibialis anterior. Results
indicated improved recovery of muscle cell and tissue morphology and corresponding
significantly improved contraction forces (18, 25, 28). As preservation of the OOM is a
major necessity of CL/CP revision, the TEMR technology was well suited for investigation
as a TE alternative graft material (8, 16). Herein, we hypothesized that TEMR would
promote recovery of morphology and function in a newly developed model of VML with
relevance to CL/CP secondary revision.
To test the hypothesis of this study, a new VML model was developed utilizing the
rat latissimus dorsi which would allow the evaluation of TEMR as well as other future
therapies of interest as potential candidates for use in CL/CP revision. In evaluating the
effect of TEMR on muscle repair and regeneration in this model, functional and
morphological outcomes were assessed in comparison with non-repaired (NR) injuries and
BAM alone treated injuries. By investigating the treatments alongside one another in both
males and females and over the course of 6 months, the study enabled the investigation of
the effectiveness of TEMR, it showcased the value of including a cellular component in
VML treatment, and gave insight into recovery timelines in VML.
Use of in vitro contraction force assessment indicated that the model resulted in an
80% reduction in maximal contraction force for NR males compared with control muscles
at 2 months post-surgery and that the contraction force improved to a ~50% reduction by
4 months and remained unchanged at 6 months. In females the same injury size, although
accounting for a greater percentage of the total muscle weight (22% in females versus 17%
in males), resulted in a ~70% reduction in force at 2 months and did not improve until 6
210
months at which time the deficit improved to a 60% reduction of maximal force compared
with control muscles. While the model did result in an injury in which there was
improvement in function over time, the maximal recovery did plateau for males at 4 months
and remained even lower for females at 6 months with 50% and 60% maximal contraction
deficits respectively. The resulting functional deficits in this model are similar to those
created in previously established rodent models of VML and as such this model can
reasonably be considered to result in true VML (18, 25, 28).
Unexpectedly, while functional recovery was greatest for TEMR compared with
NR and BAM at 2 months for males and females the differences in parameters were not
different and at later time points there were no significant differences between treatment
groups for males or females in the assessed contraction parameters. Interestingly the lack
of differences seen functionally did not correlate with the macroscopic and histologic
findings for which there were clear differences between treatment groups. Namely, NR
tissues showed a distinct void where the defect was created while BAM and TEMR treated
tissues had connective tissue layers which for most males and some females spanned the
region of the defect and persisted through the 6 month time point. Furthermore, TEMR
treated tissues in both males and females appeared to be in a distinctly better condition than
either NR or BAM treated tissues. TEMR treated tissues appeared more homogenous
overall macroscopically and had fewer incidences of holes or tears and apparent
inflammation both macroscopically and in histological results. In addition, within the
TEMR tissue’s implant regions there was a greater incidence of vascular structures
compared with BAM tissues in both males and females at all timepoints.
211
The differences between TEMR and BAM treated tissues seen macroscopically and
through histological analysis in spite of differences in functional recovery prompt further
assessment of the impact of the addition of a cellular component. The addition of MPCs
and preconditioning was previously shown to improve both morphology and functional
recovery (18, 25, 28). However, in this study only the former was demonstrated and while
tissue quality within the implant was more vascular and TEMR was apparently less
inflammatory than BAM, there was no evidence of neo-muscle formation as was seen
alongside improved function in those studies (18, 25, 28). In prior work in which TEMR
was employed in a mouse model of VML in the LD muscle a 3x1cm strip carrying 1x106
MPCs/cm2 per side or 6x106 MPCs total was implanted into a defect weighing ~25mg (25,
28). The size of the defect created in the mouse LD model was approximately 11 fold less
than the defect created in the female rat LD in this study, 256mg, and 15 fold less than in
males, 377mg, but in this model only ~3 fold the number of cells was employed: ~17x106
MPCs versus 6x106 MPCs (number of cells implanted in this study based on seeding 1x106
MPCs per side for two scaffolds cut to fit a 4.25cm2 defect). Therefore it may be postulated
that the concentration of cells used in this model was insufficient in promoting functional
recovery for the size of the injury generated based on previous successes. The literature
suggests that implanted muscle progenitor cells may impact regeneration through direct
generation of new muscle or indirectly through paracrine effects on surrounding cells (47-
51). In this study, the histologic findings indicate that while the implanted MPCs were not
capable of resulting in new muscle tissue formation they had paracrine effects on
surrounding tissue which resulted in improved angiogenesis within the implanted
construct. In addition, the cellular component may be attributed to the improved immune
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modulation seen in TEMR treated tissues as previous studies have indicated that
phagocytosis of muscle cellular debris by macrophages promotes transformation from the
pro-inflammatory to anti-inflammatory phenotype (51).
When measuring success in a CL/CP secondary revision procedure, aesthetic
restoration is a key concern for patients and has been indicated as the primary reason for
undergoing revision procedures in most cases (11-14). The findings of this study indicated
the potential of TEMR constructs for use as a graft material in secondary CL/CP revision
procedures capable of promoting improved tissue morphology. Herein, TEMR
demonstrated the ability to bridge a large defect with a connective tissue layer wherein the
tissue was demonstrated both macroscopically and histologically to be well vascularized
and non-inflammatory. However, the concentration of MPCs applied in the study did not
improve functional recovery relative to NR or BAM treatments as we had seen in previous
work (18, 25, 28). These outcomes will help shape future studies wherein the goal will be
to enhance neo-tissue formation within the region of the implant and correspondingly
increase functional improvement while maintaining or improving the macroscopic tissue
quality that was demonstrated in this study. Compelling follow up investigations might
include increasing the number of TEMR constructs implanted into the defect or
incorporating the addition of a second cell seeding during preconditioning. Adding more
layers of TEMR to the implant would serve to increase the number of cells added and could
potentially improve the longevity of the implant to encourage an improved balance of tissue
remodeling to scaffold degradation within the implant region. Use of an additional cell
seeding of undifferentiated MPCs during preconditioning could benefit this model through
213
promotion of new muscle formation within the scaffold region with potential for
accompanied functional improvement as was shown by Corona et al (28).
This study has benefited skeletal muscle TE research through the introduction of a
VML model in which the size poses a novel challenge for existing technologies wherein
success demonstrates a high potential for translation. The hypothesis of this study could be
partially accepted: TEMR was capable of promoting improved tissue quality after VML
albeit in the absence of improved functional recovery and new muscle generation.
Nonetheless, this work has indicated the potential of TEMR for use in CL/CP secondary
revision and has elucidated the benefits conferred through the addition of cells in TE which
future studies involving TEMR should strive to build upon.
214
Supplemental Figure 1. Macroscopic Images of all 2mos Male TEMR Tissues.
Excised whole LD muscles are shown after undergoing functional testing. Experimental
LD muscle is shown on the left and contralateral uninjured control muscle is shown on
the right in each image.
215
Supplemental Figure 2. Macroscopic Images of all 2mos Male BAM Tissues. Excised
whole LD muscles are shown after undergoing functional testing. Experimental LD
muscle is shown on the left and contralateral uninjured control muscle is shown on the
right in each image.
216
Supplemental Figure 3. Macroscopic Images of all 2mos Male NR Tissues. Excised
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219
220
221
222
223
Supplemental Figure 10. Macroscopic Images of all 2mos Female TEMR Tissues.
224
Supplemental Figure 11. Macroscopic Images of all 2mos Female BAM Tissues.
225
Supplemental Figure 12. Macroscopic Images of all 2mos Female NR Tissues.
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229
230
231
232
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CHAPTER V
Summary, Conclusions, and Future Directions
Hannah Besser Baker
Note: This chapter was composed by Hannah Besser Baker
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Introduction
The work presented in this dissertation further demonstrated the potential of natural
biomaterials for use in skeletal muscle TE applications and represents an effort to further
develop a platform of technologies aimed to meet the diverse presentations of VML. The
two materials of interest: human hair derived keratin hydrogels and porcine derived BAM
showcase the utility and diversity of natural scaffolds. Both are benefitted by certain
intrinsic properties known to many natural biomaterials including the capacity to bind cells
through protein sequences, degradation into biocompatible components, and accessibility
with the capacity to be manufactured on a large scale (1-9).
However similar the two materials may be found to be, they are also widely
different physically especially when comparing the states of the materials investigated in
these studies, a hydrogel versus sheet like scaffold which both offer unique benefits.
Keratin hydrogels may act as cell and drug delivery vehicles, capable of achieving
sustained release of a drug of interest over the course of degradation (10-12). The hydrogel
can flow to fill tortuous voids and in doing so can deliver carried materials to locations
other scaffold geometries cannot interact with as efficiently. The unique biochemical
properties of keratin allow it to be isolated in two distinct ways yielding different
degradation rates. The different keratins may be combined in variable ratios to achieve a
desired degradation time, perhaps unique to a specific injury anatomy, pathology, or
patient.
The BAM scaffold has the strength to retain sutures and therefore can transmit
force- an especially important consideration in skeletal muscle tissue engineering (13-20).
Further, while strong, the collagen rich ECM scaffold is also flexible which allows it to
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undergo uniaxial stretch enabling physiological preconditioning of seeded cells. The
resulting benefits are a more aligned and mature cell phenotype found to be directly
correlated with improved functional recovery in VML models (13-16).
In covering the work carried out using these two promising materials, the following
chapter will serve to first summarize and refresh the findings of the literature search in the
first chapter and of the studies presented in the subsequent chapters. Thereafter the findings
from each study will compared to highlight observed patterns and phenomena and to
suggest limitations of these efforts. A section detailing potential future endeavors will
follow and lastly translational considerations will be addressed.
Summary of Chapters
The literature review presented in the first chapter covered skeletal muscle clinical
indications, biology with a focus on regeneration, and current tissue engineering strategies
for treating VML with a focus on natural biomaterials. Review of the research literature
highlighted the importance of animal models and animal model selection in skeletal muscle
research and particularly when studying VML. The research reviewed also shed light on
the complexity of chemical cues involved in repair of muscle and showed examples of
successful use of growth factors in TE applications. Finally the review covered background
research surrounding the two materials used in this dissertation, keratin hydrogels and
TEMR, and emphasized their utility for use in skeletal muscle repair applications.
The following two chapters investigated the novel application of keratin hydrogels
in VML models. In these studies keratin hydrogels consisting of a blend of KOS and KTN
keratins capable of achieving sustained release of IGF-1 and bFGF were examined. The
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growth factors were chosen due to their known effects on skeletal muscle cells: IGF-1 is
associated with promoting satellite cell differentiation and fusion of myoblasts and bFGF
is associated with resulting in satellite cell activation and fusion (21-24). In both studies,
we hypothesized that keratin hydrogels would result in improved recovery of muscle
function and morphology and that the addition of cells and selected growth factors would
further enhance recovery.
In chapter 2, keratin hydrogels were applied in a previously established VML model
in the mouse LD (13, 16). In this model, functional recovery was measured in whole
excised LD muscles at a terminal time point through measurement of contraction force
elicited through electrical stimulation using an a specialized organ bath set-up. Histological
analysis was then conducted on tissues after they have been functionally assessed and
consisted of examining overall tissue morphology via hematoxylin and eosin and Masson’s
trichrome staining and staining for the functional protein myosin. At 8 weeks post-surgery,
tissues treated with keratin in combination with IGF-1 and bFGF in the absence of cells
resulted in the greatest improvement in functional recovery with maximum tetanic
contraction forces significantly greater than all groups besides keratin alone and
KN+M+IGF-1. In addition, histology results indicated that new muscle tissue could be
seen in KN+I+b treated tissue extending from the edge of the native tissue and into the
layer of deposited connective tissue. The results indicated that the number of cells used did
not improve functional recovery or attenuate the immune response more effectively than
acellular keratin groups. These findings showed keratin hydrogels were capable of
enhancing functional and morphological recovery in a mouse skeletal muscle injury model
and encouraged the further exploration of keratin biomaterials to treat VML.
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Chapter 3 presented the application of same blend of keratin hydrogel coupled with
growth factors and cells used in chapter 2 in a second previously established model of VML
in the rat TA. In the rat TA VML model, functional recovery was measured in vivo at 4, 8,
and 12 weeks post-surgery. TA contraction forces were measured via electrical stimulation
of the peroneal nerve and measurement of subsequent dorsiflexion through a foot plate
force transducer. In this method, force measurements could be taken at several timepoints
in the same animal. Histological analysis was then conducted on tissues removed at 12
weeks after surgery. Overall tissue morphology was examined via hematoxylin and eosin
and Masson’s trichrome staining.
At 8 weeks post-surgery tissues treated with keratin in combination with IGF-1 in
the absence of cells resulted in the greatest improvement in functional recovery with
maximum tetanic contraction forces significantly greater than NR. At 12 weeks post-
surgery, tissues treated with KN+I+b were significantly greater than BAM and NR treated
tissues, but were not different from KN or KN+b treated tissues.
In addition, histology results at 12 weeks indicated that greater evidence of new
muscle tissue formation could be seen in all keratin treated tissues. The results at the 8
week time point (at which cellular groups were included in the study) illustrated that the
number of cells used did not improve functional recovery to a greater extent than the
acellular keratin groups. These findings in the rat TA model provided further evidence of
keratin hydrogels enhancing functional and morphological recovery in a muscle injury and
solidified the basis for further exploration of keratin biomaterials for VML treatment.
TEMR was further investigated for use in VML in Chapter 4 which sought to
evaluate TEMR for use in CL/CP secondary revision procedures by implementing the
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technology in a newly developed model. The developed injury model consisted of a
rectangular injury in the rat LD which was roughly 4 times larger in weight than the
previously largest injury model used by our group in the rat TA. The unique surgical model
enabled the TEMR constructs to be applied in two layers: one below and above the created
defect in a fashion applicable to CL/CP revision. The created injury was reported to result
in an 80% deficit relative to uninjured control tissues at 2 months post injury in males and
a 70% deficit in females.
Use of the TEMR construct resulted in the highest contraction parameters at 2
months post implant in both males and females, but these findings were not significant
compared with NR and BAM treated tissues and at 4 and 6 months there were also no
significant differences in function between the groups in either sex. Contrary to the
functional data, the macroscopic images and histological findings indicated that NR tissues
did not regenerate and TEMR treatment resulted in greater tissue quality and vascularity
and better attenuated inflammation compared with BAM treated tissues.
Therefore, it was suggested that the number of cells used in this study was
insufficient in improving functional recovery, but the cells likely played a role in improving
tissue quality through either paracrine effects, interaction with invading cells, or a
combination of both. As such the study merited the further exploration of TEMR for use
in treating VML and indicated its potential as a graft material for use in CL/CP secondary
revision procedures.
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Comparison of Research Findings
Comparing Keratin Hydrogel Application in Two Distinct Rodent Models of VML
Previous work using TEMR showed the technology was capable of significantly
improving functional recovery in two distinct rodent injury models (13, 15, 16). Moving
forward from these successes, the next step in further developing a platform of TE
technologies for VML was to investigate other potential biomaterial scaffolds. Based on
several beneficial characteristics, keratin hydrogels were a strong candidate for
investigation in the existing VML models. Perhaps not surprisingly, keratin hydrogels were
successful in promoting significant improvement in function in both the mouse LD and the
rat TA models.
Key differences in the models make the improvement in function enabled by keratin
in both studies worthy of further consideration. The mouse LD injury resulted in a ~16mg
defect and the rat TA injury resulted in a ~70mg. In the mouse model between and 100-
200µl of hydrogel was injected and similarly in the rat model ~200µl was injected. It could
be argued that for the volume injected in both studies, wound beds were receiving similar
doses of keratin and depending on the group investigated, a similar concentration of growth
factors and/or cells.
It is worth noting, that in both cases the number of cells implanted, 1.2-2.3x105
MPCs, was arguably too low to contribute to functional recovery as much higher
concentrations were used in prior work with success. In a previous study using TEMR in
the rat TA model ~3x106 MPCs were introduced and ~6x106* MPCs were applied in two
previous investigations using TEMR in the mouse LD (* not including additional cell
seeding done during preconditioning for one group in Corona, 2012) (13, 15, 16).
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In spite of the difference in injury size (rat TA defect was ~4x larger than the mouse
LD defect) the dose of keratin given was effective in both studies, though the timeline of
recovery appeared to be different in the two models. In the mouse model, at 8 weeks post
injury the greatest improvement in function was enabled by keratin in combination with
both growth factors which was significantly greater than all treatment groups besides
keratin alone and keratin combined with cells and IGF-1. At 8 weeks in the rat TA model,
only KN+IGF-1 was significantly greater than NR. However, at 12 weeks post-implant in
the rat model KN+I was no longer different from NR and KN+I+b, keratin alone, and
KN+b were all significantly greater than NR (with deficits approximating data seen at 8
weeks in K+I treated tissues), suggesting that keratin enabled recovery in the rat TA model
took place over a longer time course. In looking at the improvement conferred in each
model, the highest improvement in function was enabled by KN+I+b in both groups. In the
mouse model at 8 weeks, KN+I+b had a 32% functional deficit relative to control tissues
compared with NR tissues which had a 58% deficit. At 12 weeks post-surgery in the rat
model, KN+I+b treated tissues had contraction forces equating to a 29% deficit relative to
control forces whereas NR tissues had a 43% deficit. Therefore, the dose of hydrogel
enabled a 26% improvement over no treatment in the mouse model and a 14%
improvement in the rat model. While a greater improvement in the mouse model may be
expected considering the differences in the defect sizes and resulting relative doses of
hydrogel, these reasons alone are likely not strictly responsible. Other potential factors
might include muscle specific and species specific differences in muscle regeneration rate.
In addition, differences in how the injury models altered the muscle biomechanics could
also play a role in how the injuries healed. As keratin hydrogels have demonstrated a
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capacity to improve muscle function in two distinct models, the ongoing investigation of
keratin for treating VML, as well as investigation into the considerations posed here, is
encouraged.
Comparing Keratin Hydrogels and TEMR Applied in Rodent Models of VML
In working towards developing a platform of technologies for treating VML
injuries our group has also developed, and is continuing to develop, several in vivo VML
injury models. The collection of these animal models aims to address a variety of potential
injuries and represents a diversity of wound geometries, muscle anatomies, and relative
sizes. In the set of experiments covered by this dissertation, three different models were
employed in studying two different natural biomaterial scaffolds. Previously, the
application of cell and growth factor loaded keratin hydrogels in two different rodent
models was compared. This discussion will consider the use of cells and how the materials
interfaced with the injured tissues in the studies.
In all three studies, the cell density applied in the various injury models was
discussed and considered to be suboptimal. For example, and as previously stated, in the
mouse LD study of keratin hydrogels the number of cells delivered was 26-50 times lower
than that used in previous studies employing TEMR in the mouse LD. Also previously
mentioned was that in the rat LD model for an injury 11-15 times greater in defect weight
than that in the mouse LD, only 3 times as many cells were applied when comparing
numbers from the previous TEMR studies (using the mouse LD model). While both sets
of studies indicated an insufficient number of cells were used, it should be noted that the
determined discrepancy was much greater between the keratin studies and previous work
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than between the study of TEMR in the rat LD and previous work: 25-50x versus 3-5x
fewer cells respectively. In addition, and potentially more importantly, the cells presented
in the rat LD model were subjected to chemical and physical differentiation cues whereas
the cells delivered in the keratin studies were early passage non-fused muscle derived cells.
The considerable difference in cell number (and arguably as a result of the differences in
cell phenotype) may have caused the differences observed in histology for cellular and
non-cellular groups between the studies. TEMR appeared to improve tissue quality over
BAM whereas cellularized keratin hydrogels did not in comparison with acellular keratin
groups.
Nonetheless, the concentrations of cells used in all three studies were still unable
to enable significant functional improvement whereas growth factor loaded keratin
hydrogels alone were. Which brings to attention the consideration of how the gels
interacted with the surrounding tissue to result in improved function versus application of
BAM scaffolds. As stated repeatedly, it is hypothesized that the cells delivered on the
TEMR scaffold in the rat LD were too few to result in functional improvement relative to
BAM, but there was also no significant difference between BAM, TEMR, and NR. This
suggests that the presence of the scaffolds alone in this study was not capable of providing
a sufficient tissue bridge between non-injured tissues to conduct force transmission and
improve contraction forces over NR, a finding which has been termed functional fibrosis
(25). Interestingly, in the keratin studies BAM treated tissues actually had lower
contraction forces than NR treated tissues. Potentially this is a result of the volume of
scaffold used. Perhaps there was too little scaffold used in the rat LD defect for any
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functional fibrosis to occur and in the mouse LD and rat TA models, in the absence of cells,
the volume of BAM used was actually detrimental to recovery and regeneration of tissue.
While suture retaining ECM scaffolds are intrinsically capable of achieving
functional fibrosis, injectable keratin hydrogels are not. The significant functional
improvement conferred by the hydrogels was not a result of the remodeling of a physical
bridge as occurs in ECM scaffolds, but instead must be attested to the interaction of the gel
with tissue. Based on previous studies, several mechanisms could be possible. Potentially,
the hydrogels promoted a more favorable environment for regeneration by influencing the
conversion of invading monocytes toward an anti-inflammatory phenotype (26). Or,
perhaps the keratin hydrogels recruited invading blood borne BMMSCs which then
differentiated toward muscle and aided in regeneration (5-7, 27-30) The mechanism
surrounding this interaction was not explicitly explored in either keratin study presented,
but certainly deserves further investigation as keratin has shown a great potential for use
in treating VML.
Suggested Future Research
In summarizing and thoughtfully comparing the findings presented in this
dissertation, serval avenues of future work have been brought to attention. Naturally, the
following suggested efforts are not exhaustive; instead the outlined studies strive to address
major limitations and to present studies which would clearly and directly further this area
of research. Several future studies are described briefly below: optimization of keratin
hydrogel cellularity, in vivo examination of dose-response to selected growth factors in a
muscle injury, assessment of mechanical properties of scaffolds alone and in series with
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muscle, and examination of the effect of an additional cell seeding or the use of additional
scaffold layers in the rat LD model.
Optimization of Keratin Hydrogel Cellularity
The major limitation of the keratin studies presented in chapters 2 and 3 was the
cellularity of the constructs applied in the VML models. The blend of keratose and
kerateine employed in the two studies was developed under constraints surrounding
degradation and accompanying growth factor release time. A 70:30 blend of 15% KOS and
7% KTN resulted in sustained release of both IGF-1 and bFGF over the course of 4 weeks
(Appendix 3). However, this blend was not capable of carrying more than 1.7x106 rodent
MPCs in the KOS fraction due to viscosity becoming too high for extrusion of the gel
through a large gauge needle (16-19 gauge surgical needles). Work submitted for
publication showed 12% KOS and 8% KTN gels had elastic moduli measured to be
~100Pa, which was associated with gels that were flowable in nature. Since the gels
described in that study had similar w/v KOS and KTN values to those used in this research
and that all of the formulations were delivered via syringe in an in vivo model it is assumed
here that the blend used in these studies was also roughly ~100Pa and that increasing the
number of cells would cause gels to exceed this target elastic moduli (data not shown from
Tomblyn et al, unpublished results).
In order to overcome the viscosity issue and improve the cellularity, the KOS:KTN
blend would need to be optimized in order to maintain the release profile of IGF-1 and
bFGF and the desired elastic moduli of ~100Pa while increasing the cellularity. Methods
necessary to optimize these parameters include culture and expansion of rodent MPCS,
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testing the elastic moduli of the gels with a rheometer, and conducting ELISA on solutions
surrounded growth factor loaded gels to determine percent release of each factor. To plan
optimizing the w/v of KOS and KTN and ratio of each used, one might begin by examining
lower concentrations of keratin in the KOS blend, which holds the cells, and examining
subsequent increases and decreased in KTN w/v and within each group determining the
maximum cell capacity without exceeding 100Pa as shown in Table I.
The expected outcome from this proposed optimization study would be
identification of a keratin blend which allows a greater cell concentration while
maintaining desirable release rates of IGF-1 and bFGF and striving to maintain an elastic
moduli near 100Pa.
70% KOS 30% KTN 30% KTN 30% KTN

Fraction Fraction Fraction Fraction
Control
15 5 7 9
(% KN w/v)
Test set 1
12 5 7 9
(% KN w/v)
Test set 2
10 5 7 9
(% KN w/v)
Test set 3
8 5 7 9
(% KN w/v)
Table I. Experimental Design for Optimizing Keratin Hydrogel Cellularity
In vivo ‘Dose Response’ to IGF-1 and bFGF Loaded Keratin Hydrogels in VML
The concentration of growth factors administered in the studies presented in
chapters 2 and 3 were based on work wherein the goal was to showcase the ability of keratin
hydrogels to deliver both cells and growth factors, but concentrations were not selected
based on previous work or data suggesting optimal concentrations (data from manuscript
253
under review, Tomblyn et al). The chosen concentrations (100ug/mL of hydrogel or in the
dissertation research studies ~10ug/injection in the mouse LD and ~25ug/injection in the
rat TA) were efficacious in combination with VEGF in causing increased vascular
structures and were attributed to promoting viable muscle tissue. Vascular structures could
be seen within the cell loaded gel and cells in the region stained positive for intermediate
filament desmin at 4 weeks after implant into a subcutaneous dorsal pouch in a mouse (data
not shown, summarized from manuscript under review by Tomblyn et al). In the present
studies, IGF-1 and bFGF administered alone in keratin hydrogels did not improve muscle
function or result in new muscle tissue formation, but when combined (in the absence of
cells) the mixture supported significantly improved function in both a mouse LD injury
and a rat TA injury (chapters 2 and 3 respectively). While these results are promising, a
future study might aim to better understand the interaction between the two growth factors
and within a muscle injury. Specifically a study of long term functional ‘dose-response’ to
varied concentrations of the growth factors may shed light on the complex interaction
between growth factors, regenerating tissue, and surrounding and invading cell
populations. Such an endeavor is certainly daunting, but ultimately if growth factors were
deemed necessary through further research more (and much more complex) studies would
need to be undertaken prior to clinical translation.
The methods involved with pursuing this study include: rodent VML surgery and
associated functional testing to examine resulting functional recovery and histology and
immunohistochemistry to examine cell and tissue morphology with specific consideration
given to identification of immune cell congregation and inflammation, new muscle tissue
presence, vascularization, and scar tissue deposition. In order to glean the most information
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from the study, it is suggested that the rat TA model of VML be used in order to measure
functional recovery relatively non-invasively with the option of measuring at multiple time
points prior to termination and so that the tissue tested for function may also be used for
histology (unlike the mouse LD model wherein it is preferable to use different tissues for
function and histology as the organ bath tested tissues tend to be of poorer quality for
histology). To simplify a study examining dose-response to growth factors one might wish
to only investigate 10 fold or 5 fold changes in concentrations perhaps as outlined in Table
II. As there might be effects due to both concentration and ratio of the growth factors used
it will be important to include combinations wherein the growth factors are both the same
concentration and the ratio is the same between test sets (i.e. 10µg/ml IGF-1:10µg/ml bFGF
would be the same ratio as that used in chapters 2 and 3, 1:1; 1µg/ml IGF-1:10µg/ml bFGF
and 10 µg/ml IGF-1:100 µg/ml bFGF would be looking at a 1:10 ratio in both cases).
As the interaction of growth factors and their effects on remodeling tissue is
difficult to predict, it is also difficult to propose expected outcomes from this study. Based
on histologic findings from chapters 2 and 3 is seems likely that increased adipose and scar
tissue deposition will be found in groups with higher IGF-1 and bFGF concentrations
respectively. Further, varied concentrations might result entirely different results as it could
alter the interaction between the growth factors. Previous work has shown that in the
presence of bFGF, IGF-1 will behave like a mitogen instead of promoting differentiation
and fusion in muscle- a relationship which this proposed work may determine to be
concentration dependent (21).
255
IGF-1 bFGF bFGF bFGF
Control Test Set
100 500 100 10
(µg factor/ml gel)
Test set 1
10 500 100 10
(µg factor/ml gel)
Test set 3
500 500 100 10
(µg factor/ml gel)
Table II. Experimental Groups for In Vivo Keratin Hydrogel Coupled
Growth Factor Dose Response Experiment
Investigation of Mechanical Properties of Injured and Treated Tissues
Throughout the discussion of the effectiveness of keratin hydrogels and TEMR
constructs in various models of VML in studies previous to this dissertation and within,
there have been several mentions of mechanical interactions between a scaffold and the
wound environment it is applied to. With ECM based scaffolds such as BAM and TEMR,
the material is flexible, but strong enough to hold sutures which anchor the constructs in
place within a wound bed. However, for keratin hydrogels and other materials which
cannot retain sutures, the physical anchoring of the test article cannot serve to bridge one
end of a muscle injury to the other and potentially enabling transmission of contraction and
forces. Further, bridging the gap or enabling force transmission in muscle injuries has been
suggested to protect the surrounding muscle by shielding it from excess strain which could
result in degeneration of tissue (15, 31, 32). In addition to the immediate physical bridge
offered by certain materials, some may result in improvement in the mechanical integrity
of regenerating tissue or remodeling scaffold by interacting with the surrounding tissue via
256
the addition of a cellular component or deliverable substances such as growth factors or
drugs.
To investigate the impact on mechanical properties as a result of VML and
treatment with various scaffolds, mechanical testing of a variety of different materials
could be conducted in several settings. The test parameters of interest might include elastic
modulus and failure strength. Uniaxial tensile testing could be carried out on scaffolds
before and after cell seeding and preconditioning, on muscle strips in series with constructs
immediately after surgical attachment or after being in in vivo, or on strips of muscle in the
vicinity of the defect. An example of a list of groups for testing in shown in Table III.
Methods used to compare the mechanical properties of the scaffolds alone and after
implantation in vivo include cell culture and cyclic stretch bioreactor assembly and
programming, rodent surgery, and use of a mechanical testing system and interpretation of
the resulting data. It should be noted that as muscle is viscoelastic, a set strain rate should
be employed for all tests. Based on previous studies, the expected outcomes of such a study
would be that the cell seeded constructs have a greater stiffness than in non-cell seeded
constructs more closely resembling muscle strips and that the failure strength in cell seeded
constructs in series with tissue is improved relative to BAM in series with muscle just after
implant and after 4 weeks in vivo (33). Studying the mechanical properties of the scaffolds
alone and in situ would help determine how the materials contribute to functional recovery
and may aid in further development of TE therapies for VML.
257
Table III. Experimental Groups for Mechanical Testing of Test Articles in
Series with Rat LD Muscle.
258
Investigation into Laminate TEMR and Multi-Seeded TEMR in Rat LD Model
Lastly, another potential set of studies which may serve to further the research
presented in this dissertation involves additional exploration of TEMR in the rat LD model.
Specifically, it was discussed that the use of TEMR in the large injury model developed in
the rat LD might benefit from an increased cell number. Two such methods of improving
the cell number delivered to the injury site would be to add additional layers of TEMR
(potentially 4 instead of 2 as used previously) or to increase the cell number on each TEMR
construct. The introduction of additional scaffold layers stands to benefit the current model
as more scaffold material may stand to improve force transduction through functional
fibrosis alone- even without a cellular component. Previous work indicated that the
addition of a cell seeding during preconditioning improves functional recovery (16).
As such, these two strategies could be employed separately and together in the rat
LD model in an effort to improve functional recovery. Potential treatment groups for use
in a 2-6 month study are shown in Table IV. Methods involved with this study include cell
culture, assembly and programming of a cyclic stretch bioreactor, rodent surgery, in vitro
functional testing using the organ bath set-up, and immunohistochemistry.
Expected outcomes would be that the addition of TEMR layers results in a longer
scaffold degradation time, serves to improve functional recovery, and tissue quality
compared with single layered TEMR constructs. Further it would be expected that multi-
seeded TEMR results in improved functional recovery and correlated improved tissue
morphology including new muscle tissue formation.
259
Number of Number of Additional
2 Mos. Groups animals (n) Scaffolds (n) MPCs added
TEMR 6 2 no
Double TEMR 6 4 no
TEMR+MPC 6 2 yes
Double
6 4 yes
TEMR+MPC
Table IV. Experimental Groups for Study of Laminate and Multi-Seeded
TEMR
Clinical Translation Considerations
Keratin hydrogels and TEMR have both been shown to have promise for use as a
VML treatment. Conventions within the FDA regulatory processes and agencies have been
historically centered on drugs, medical devices, and biologics alone, however over the last
two decades those processes have had to evolve, and continue to evolve, to accommodate
TE products in which combinations of cells, dugs, and devices are used (34-37).
On top of being a combination therapy, both products face unique challenges prior
to and after submission of an IND in clinical trials. The scaffold material in the products,
the keratin hydrogel alone and the BAM, will need to be manufactured according to Good
Manufacturing Process (GMP) guidelines under which elimination of batch to batch
variability, which is an innate obstacle for naturally derived scaffolds, will need to be
ensured. For keratin hydrogels, streamlined manufacturing is already in use for other
keratin products via KeraNetics, LLC. Manufacturing with GMP for TEMR will also have
to accommodate cell seeding of scaffolds and the use of the cyclic stretch bioreactor which
poses a unique challenge in itself by adding an enabling technology into the process.
260
As part of the IND application, both products must demonstrate non-toxicity
through animal studies. Though these combinations have not been through the vetting
process yet, many of their components have. The individual constituents to consider are
MPCs, BAM, keratin hydrogel, and the administration of growth factors. Clinical use of
allogeneic multipotent stem cells such as BMMSCs have indicated their safety in several
applications and research studies using further differentiated adult stem cells, like MPCs,
have also demonstrated safety (38-40). Many ECM scaffold based materials have gone
through clinical trials successfully and examples of clinically available products include
patches for wound healing, rotator cuff repair, and hernia repair (9, 41). Similarly, a keratin
hydrogel product has also obtained IND status and was submitted for use in a clinical trial
(42, 43).
The remaining component is the application of growth factors. As previously
mentioned in the discussion of potential future studies, the application of growth factors in
TE research is inherently complex. Natural growth factor production and regulation is
dynamic and intricate and through the work of many investigators there have been great
strides made in understanding these mechanisms and their application in tissue engineering
(21, 23, 44-47). The addition of extrinsic factors can have undesirable effects as was seen
in high dose BMP-2 releasing INFUSE devices applied in the cervical spine which led to
ectopic bone formation in the trachea- which was believed to be a result of too high a
concentration of BMP-2 (48). As such, further studies must be carried out in order to
determine safe and effective doses and release rates of growth factors from keratin
hydrogels prior to an IND submission surrounding their combination.
261
Conclusion
The culmination of the work presented in this dissertation has furthered the overall
goal of developing a platform of technologies capable of treating VML. This dissertation
entailed the first time application of cell and growth factor loaded keratin hydrogels in a
VML model and a novel application of TEMR in a newly developed VML model with
relevance to cleft lip secondary revision procedures. In these studies keratin hydrogels and
TEMR have shown promise for clinical translation for treatment of muscle injuries. As
such, future studies which seek to further this research and bring these therapies closer to
translation are well warranted.
262
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268
APPENDIX 1
Implantation of in vitro tissue engineered muscle repair (TEMR) constructs and bladder
acellular matrices (BAM) partially restore in vivo skeletal muscle function in a rat model
of volumetric muscle loss (VML) injury
Corona, B.T.1,2, Ward, C.L.1,2, Baker, H.B.1, Walters, T.J.2, & Christ, G.J.1
Affiliations:
1
Wake Forest Institute for Regenerative Medicine, Winston Salem, NC
2
United States Army Institute of Surgical Research, Department of Extremity Trauma and
Regenerative Medicine, Fort Sam Houston, TX
Note: this manuscript has been published and is being used with permission
269
Acknowledgements: We would like to thank Ms. Manasi Vadhavkar for her technical
assistance during surgery and functional testing and Mr. Christopher Bergman for his
technical assistance with histological procedures. The opinions or assertions contained
herein are the private views of the authors and are not to be construed as official or
reflecting the views of the Department of Defense or the United States Government. The
authors (btc, clw, and tjw) are employees of the U.S. government and this work was
prepared as part of their official duties. This work was supported in part by the Armed
Forces Institute for Regenerative Medicine (W81XWH-08-2-0032) and by the U.S. Army
Medical Research and Medical Command (W81XWH-09-2-0177).
Corresponding Author:
George J. Christ, Ph.D.
Professor of Regenerative Medicine
Wake Forest Institute for Regenerative Medicine
Wake Forest University Baptist Medical Center
Richard H. Dean Biomedical Research Building, Room 257
391 Technology Way
Ph: 336-713-7296
Fax: 336-713-7290
Email: gchrist@wakehealth.edu
270
Abstract
The frank loss of a large volume of skeletal muscle (i.e., volumetric muscle loss;
VML) can lead to functional debilitation and presents a significant problem to civilian and
military medicine. Current clinical treatment for VML involves the use of free muscle flaps
and physical rehabilitation; however, neither are effective in promoting regeneration of
skeletal muscle to replace the tissue that was lost. Towards this end, skeletal muscle tissue
engineering therapies have recently shown great promise in offering an unprecedented
treatment option for VML. In the current study, we further extend our recent progress
(Machingal et al., 2011, Tissue Eng; Corona et al., 2012, Tissue Eng) in the development
of tissue engineered muscle repair (TEMR) constructs (i.e., muscle derived cells (MDCs)
seeded on a bladder acellular matrix (BAM) preconditioned with uniaxial mechanical
strain) for the treatment of VML. TEMR constructs were implanted into a VML defect in
a tibialis anterior muscle of Lewis rats and observed out to 12 weeks post-injury. The
salient findings of the study were: 1) TEMR constructs exhibited a highly variable capacity
to restore in vivo function of injured TA muscles, wherein TEMR positive responders (n =
6) promoted a ≈61% improvement but negative responders (n = 7) resulted in no
improvement compared to non-repaired controls, 2) TEMR positive and negative
responders exhibited differential immune responses that may underlie these variant
responses, 3) BAM scaffolds (n = 7) without cells promoted an ≈26% functional
improvement compared to uninjured muscles, 4) TEMR positive responders promoted
muscle fiber regeneration within the initial defect area while BAM scaffolds did so only
sparingly. These findings indicate that TEMR constructs can improve the in vivo functional
capacity of the injured musculature at least in part by promoting generation of functional
271
skeletal muscle fibers. In short, the degree of functional recovery observed following
TEMR implantation (BAM + MDCs) was 2.3X-fold greater than that observed following
implantation of BAM alone. As such, this study confirms and extends prior work to further
underscore the utility of including a cellular component in the tissue engineering strategy
for VML injury.
Introduction
Craniofacial and extremity soft tissue trauma resulting in the permanent loss of
skeletal muscle mass is a clinical challenge for both military and civilian medicine. This
type of injury has been termed volumetric muscle loss (VML) and is defined as “the
traumatic or surgical loss of skeletal muscle with resultant functional impairment.”1 The
current standard of care for VML injury involves the use of free muscle transfer (i.e.,
muscle flaps) followed by extensive physical rehabilitation. However, free muscle flaps do
not significantly restore muscle function to the injured tissue, and are often primarily used
for bony coverage. Functional muscle transfer, in which vasculature and innervation are
surgically restored to the graft have been shown to improve strength to an injured muscle
group2 but these procedures require an extraordinary level of surgical expertise that is not
likely available at most treatment centers. In either case, the use of large muscle flaps
presents an added complication of donor site morbidity to the patient.3 Lastly, while there
is little evidence that physical rehabilitation alone is sufficient to significantly restore
strength to muscle with VML injury, physical rehabilitation performed with an energy-
storing ankle-foot orthosis has recently proven to be successful in restoring function to
patients with extremity trauma.4
272
In this scenario, skeletal muscle tissue engineering and regenerative medicine
therapies offer a promising treatment paradigm for severe soft tissue trauma resulting in
the loss of a large volume of musculature. Therapeutic strategies are under development
for the repair of VML to craniofacial and extremity muscles, and similar injury conditions
(e.g., abdominal wall repair).5-15 Serving as a foundation for many of these strategies
biological extracellular matrices (ECMs) derived from the intestine and bladder (porcine),
and skeletal muscle (variety of species) have been studied in in vivo preclinical skeletal
muscle injury models previously.7, 8, 10, 12, 15, 16

Most studies have shown limited
regeneration of muscle fibers after transplantation (e.g.,8, 9, 15, 17). The only clinical report
currently available describes the use of an acellular matrix for the treatment of quadriceps
muscle with VML injury.18 While these initial findings are encouraging for the overall
tissue engineering paradigm, only somewhat minor strength gains were observed with
continued physical therapy.18
As an extension of this approach, the therapeutic benefit of a combined
transplantation of biological scaffold with stem or progenitor cells has also been
characterized thoroughly in preclinical studies in vivo.5, 6, 8-10, 19-21

The rationale for
inclusion of a cellular component to skeletal muscle tissue engineering therapies is based
on the numerous beneficial effects that have been reported. For example, depending on the
cell type used, transplanted cells can improve revascularization, modulate the
inflammatory response, reduce fibrosis, or contribute directly to muscle fiber
regeneration.22-25 In fact, the combination of bone marrow mesenchymal stem cells with
rat muscle extracellular matrix,9, 10 freshly isolated satellite cells with hyaluronan-based
gel,21 and muscle derived progenitor cells with bladder acellular matrix8 have all
273
demonstrated partial restoration of force production following implantation in surgically-
created VML injuries. In all cases, the observed degree of functional muscle repair and
regeneration was superior to the delivery of the respective matrix alone, providing clear
evidence for the therapeutic benefit of including a cellular component to tissue engineering
strategies designed for the treatment of VML.
In this regard, our recent efforts have exploited this latter strategy and focused on
development of an in vitro method to create tissue engineered muscle repair (TEMR)
constructs for the treatment of skeletal muscle tissue following traumatic injury.5, 8, 26 To
date, we have demonstrated that TEMR constructs, which have been created by seeding
Lewis rat muscle derived cells (MDCs) on a bladder acellular matrix (BAM), mediate
restoration of significant functional capacity (60-70% recovery) to VML injured latissimus
dorsi (LD) muscle in nude mice.5, 8 This recovery appears to involve, at least to some
extent, regeneration of a portion of the muscle fibers that were surgically removed. In
support of this supposition, we have observed presumptive formation (regeneration) of new
muscle fibers in the area of the defect where the construct was transplanted, and
furthermore, noted that these regenerating fibers express key proteins required for force
production and transmission, as well as supporting vascular and neural structures.5, 8
Herein, we report our most recent progress in developing TEMR constructs for
eventual clinical use. In the current study, the therapeutic potential of TEMR constructs
was tested in a Lewis rat tibialis anterior (TA) muscle VML model. The use of the model
introduced three important advances relative to our previous studies of the repair of VML
injury: 1) the muscle defect is approximately four-fold greater by weight in the female
Lewis rat TA muscle than the nude mouse LD muscle, 2) the Lewis rat has a competent
274
immune system, and 3) functional analysis is performed in vivo with neural stimulation
nominally requiring regenerating muscle fibers to be innervated to contribute to functional
recovery. The results of this study indicate that TEMR constructs as well as BAM scaffolds
can improve neural-stimulated in vivo functional recovery following VML injury.
However, there was marked variability in the therapeutic response of TEMR constructs
that appears to be related to a differential inflammatory response within the TEMR
construct population. Discussion is provided to highlight underlying mechanisms for these
findings and future directions for the translation of the TEMR technology.
Methods
Experimental Design: This study was designed to perform repeated in vivo functional
measurements with the same animal (leg) over a three month period using a repeated
measures experimental design. Baseline in vivo functional assessments were performed via
neural stimulation before VML injury was surgically created in the TA muscle and then
every month thereafter for three months. After the last in vivo assessment, TA muscles
were harvested for morphological and histological analysis. Because the extensor hallicus
longus (EHL), extensor digitorum longus (EDL), and TA muscle are innervated by the
common peroneal nerve and the EDL and EHL muscles hypertrophy in response to VML
injury in the TA muscle, in all experimental VML groups the EDL and EHL muscles were
ablated. To account for any potential strength changes of the TA muscle due to synergist
ablation and to provide a theoretical baseline for TA muscle recovery, a group of rats served
as a surgical-sham wherein the synergists were ablated but no VML injury was created in
the TA muscle. In total there were 5 experimental groups in this study: 1) An un-operated
275
group that served as a age-matched cage controls (n = 8), a sham/ablation group in which
the TA muscle did not have a VML defect but underwent sham surgery with ablation of
the EDL muscle (n = 6), a VML injured but non-repaired group (n = 4), a VML injured
with BAM repair group (n = 7), and a VML injured TEMR repaired group (n = 13). All
VML injured groups underwent EDL muscle ablation.
Animals: Male Lewis rats aged 3 – 4 weeks used as donors for muscle derived cells
(MDCs), as previously reported.5, 8 VML injury studies were performed with female Lewis
rats aged 12 – 14 weeks at the beginning of the study. All rats were purchased from Harlan
laboratories. All animal procedures were approved by the Wake Forest University IACUC.
Bladder Acellular Matix (BAM) Preparation: BAM scaffolds were prepared from porcine
urinary bladder as previously described.5, 8 Briefly, the bladder was washed and trimmed
to obtain a crude sample of the lamina propria (i.e., some muscle tissue remaining
attached), which was placed in 0.05% Trypsin (Hyclone, Logan, UT) for one hour at 37°C.
The bladder was then transferred to DMEM solution supplemented with 10% FBS and 1%
Antibiotic/Antimycotic and kept overnight at 4˚C. The preparation was then washed in a
solution containing 1% Triton X (Sigma-Aldrich, St Louis, MO) and 0.1% Ammonium
hydroxide (Fisher Scientific, Fairlown, NJ) in de-ionized water for 4 days at 4°C. Finally,
the bladder was then washed in de-ionized water for 3 days at 4°C. The decellularized
scaffold was then further dissected by hand to remove any remaining muscle tissue layers
to obtain a scaffold with of 0.2-0.4 mm thickness. The prepared acellular matrix was then
cut into strips of 3 cm X 2 cm size and placed onto a custom designed seeding chamber
276
made of silicon (McMaster Carr, Cleveland, OH). Scaffolds and silicon seeding chambers
were then individually placed in culture dishes and sterilized by ethylene oxide.
TEMR Construct Creation: TEMR constructs were generated following previously
described cell culture, seeding, and bioreactor preconditioning protocols.5, 8 Briefly, BAM
scaffolds were seeded with MDCs (≈1 million per cm2) on each side, cultured statically for
10 days allowing for proliferation and differentiation, and then preconditioned in a
bioreactor with uniaxial mechanical strain (10% strain, 3 stretches per minute for the first
five minutes of each hour) as described in detail elsewhere.5, 8
Surgical Procedures: Surgical creation of VML injury was performed in the TA muscle as
previously reported.27 Briefly, a longitudinal skin incision was made along the lateral
aspect of the left lower leg. The skin was separated bluntly from the underlying fascia
covering the anterior compartment. The fascia covering the anterior crural muscles was
then separated using blunt dissection. The proximal and distal tendons of the EDL muscle
were then isolated and severed. Next, the EHL muscle located underneath the TA muscle
was isolated and ablated. Care was taken to avoid disruption of the retinaculum holding
the distal tendon of the TA muscle in position. VML injury was created in the TA muscle
by excising approximately 20% of the TA muscle weight at the middle third of the muscle
(Figure 1C).27 A 20% TA muscle defect was approximated using linear regression
determined experimentally (n = 16) to estimate TA muscle mass from rat body weight: TA
wet weight (mg) = 1.553 x Body Weight (g) + 83.084 (R2 = 0.85).
Repair of the TA muscle was accomplished by folding TEMR or BAM constructs
(≈3 x 0.5 cm) in triplicate creating an ≈1 x 0.5 cm construct with three layers (Figure 1A).
277
The transplant therefore approximated the volume of the defect. The thrice-folded
transplant was placed in the defect and the four corners and four margins were sutured (6-
0 Vicryl) to the borders of the defect (Figure 1D). The fascia was closed with 6-0 vicryl
and the skin was closed with 5-0 prolene using interrupted sutures. Skin glue was applied
between the skin sutures to help prevent the incision from opening. Following surgery,
buprenorphine (0.05 mg/kg; sc) was administered every twelve hours for three days.
In vivo Functional Analysis: Torque production of the left anterior crural muscles
(primarily due to the contraction of the TA and EDL muscles) was measured in vivo using
similar methodology as previously described.27-30 After rats were anesthetized (1.5 – 2.5%
isoflurane), the left hindlimb was aseptically prepared. The rat was then placed on a heated
platform. The left knee was clamped and the left foot was secured to a custom-made foot
plate that is attached to the shaft of an Aurora Scientific 305C-LR-FP servomotor, which
in turn was controlled using a PC. Sterilized percutaneous needle electrodes were inserted
through the skin for stimulation of the left common peroneal nerve. Electrical stimulus was
applied using a Grass S88 stimulator with a constant current SIU (Grass Model PSIU6).
Stimulation voltage and needle electrode placement were optimized first with a series of
twitch contractions at 1 hz and then with 5 to 10 isometric contractions (400 ms train of
0.05 - 0.1 ms pulses at 150 Hz). Contractile function of the anterior crural muscles was
assessed by measuring peak isometric tetanic torque derived from the maximal response to
a range of stimulation frequencies (100 - 200 Hz). For real-time analysis of torque and
length changes, voltage outputs were sampled at 4000 Hz, converted to a digital signal
using an A/D board (National Instruments PCI 6221) and recorded using a PC loaded with
278
a custom-made Labview®-based program (provided by the U.S. Army Institute of Surgical
Research).
Morphological Analysis of VML Injured TA Muscle: TA muscles were isolated from the
leg and laid on a platform with the superior aspect of the muscle facing away from the
platform. At the middle of the proximal and distal thirds of the muscle, the width of the
muscle was measured using digital calipers.
Histology and Immunohistochemistry: TA muscles from all experimental groups were
fixed in 10% neutral buffered formalin and stored in 60% ethanol. All samples were
processed (ASP300S, Leica Microsystems, Bannockburn, IL) and then embedded in
paraffin (EG1160, Leica Microsystems, Bannockburn, IL). Seven µm thick serial sections
were cut from the paraffin embedded blocks and Masson’s Trichrome staining and
immunohistochemical staining was performed using standard procedures as previously
described.5 Immunohistochemical staining was performed using antibodies to detect
myosin (MF-20, 1:10, Developmental Studies Hybridoma Bank, Iowa City, IA) and
macrophages (CD68, 1:50, Serotec, Raleigh, NC). Images were captured and digitized
(DM4000B Leica Upright Microscope, Leica Microsystems, Bannockburn, IL) at varying
magnifications.
Statistics: Morphological and functional data were analyzed using one- and two-way
ANOVAs as indicated in the text. Upon finding a significant ANOVA, post-hoc
comparison testing of parameters of interest was performed using Fisher’s LSD. Statistical
279
significance was achieved at an alpha < 0.05. Values presented are mean ± SEM. Unless
otherwise stated, all mean values are expressed as the arithmetic mean.
Notably during the course of the study, TEMR responses demonstrated a markedly
greater variability than either NR or BAM (Figure 3). Because of the relatively large
variability, the sample size for the TEMR group was increased to 13 observations in an
effort to reduce the variability within this group. Ultimately, the coefficient of variation
(CV; standard deviation/mean) for the recovery of peak torque 12 weeks post-injury was
approximately two times greater for TEMR treated (CV = 0.20) than no repair (CV = 0.07)
or BAM (CV = 0.11) groups. In accordance with this greater variance, the recovery of peak
torque (12 wk Post-Injury: Pre) ranged from 0.36 to 0.71, indicating a range of functional
recoveries from essentially zero (no recovery) to complete TA muscle functional recovery,
respectively, across thirteen observations (Figure 3C). In light of this divergent response,
the TEMR group was separated into positive and negative responders based on the
following criteria: A TA muscle transplanted with a TEMR construct that recovered a peak
torque value greater than 1 standard deviation (determined from a combination of BAM
and TEMR observation; mean = 0.57, sd = 0.09) above the mean for the no repair group
was considered a positive responder.
Results
Synergist Muscle Ablation: Repeated in vivo functional testing was performed on each
animal at a monthly interval over a three month time frame to assess the functional capacity
of the TA muscle following VML injury and/or repair. In order to accurately assess the
impact of the VML injury as well as recovery of the TA muscle over time in the various
280
treatment groups, the anterior crural muscle synergists (EDL and EHL muscles) to the TA
muscle were ablated at the time of VML injury. The EDL and EHL muscles comprised a
combined 20.3 ± 1.3% of the anterior crural muscle wet weight (i.e., Uninjured TA, EDL,
& EHL muscle wet weight: 421.0 ± 6.6, 96.7 ± 1.2, 10.9 ± 0.3 mg, respectively), suggesting
the potential for TA muscle overload. Functionally, removal of the EDL and EHL muscles
resulted in a -29.4 ± 7.4% loss of peak torque from 4 to 12 weeks post-ablation when
normalized to pre-injury values (Figure 2). Twelve weeks post-ablation, wet weight was
similar among TA muscles from the ablated (446 ± 35 mg) and contralateral control (440
± 27 mg) legs (p = 0.701), indicating that synergist ablation did not provide a significant
overload to uninjured muscle similar that reported for the posterior compartment.31
Importantly, because synergist ablation resulted in a ≈30% reduction of normalized peak
tetanic torque, a complete recovery of TA muscle function following VML injury is
reflected by the return of ≈70% of pre-injury tetanic torque on that same animal using this
metric.
Creation of VML Injury: VML injury was created by surgically excising ≈20% of muscle
mass from the middle third of the TA muscle. Following surgical excision, animals were
divided into 3 treatment groups, no repair (NR), BAM implantation, and TEMR
implantation groups. Equivalent injuries were created in all treatment groups, as reflected
by the similar weight of muscle removed from the animals in the NR (70.8 ± 6.7 mg), BAM
(63.1 ± 3.1 mg), and TEMR (69.3 ± 1.6 mg) treatment groups). For non-repaired muscles
(surgical defect and synergist ablation), peak tetanic torque was reduced compared to pre-
injury values by 51.5 ± 2.8, 51.4 ± 3.2, and 49.6 ± 3.0% at 4, 8, and 12 weeks post-injury,
281
respectively, indicating that over this time-frame the VML injury did not recover
spontaneously (Figure 3). Twelve weeks post-injury, TA muscle wet weight was ≈17%
less for the VML injured NR group than in contralateral control muscles. Together these
results indicate that the TA muscle surgical defect produces an irrecoverable torque deficit
of ≈20% of the TA muscle functional capacity out to 12 weeks post-injury (with the
remainder of the ≈50% torque deficit attributed to ablation of the EDL and EHL).
Treatment In vivo of VML Injury
TA Muscle Strength Assessment: VML injury was repaired by either transplanting BAM
scaffolds or TEMR constructs to the defect site immediately after the injury. While body
weight was similar among all experimental groups (i.e., no repair, BAM, and TEMR)
during the study, over the 12 weeks post-injury each group gained significant body weight
(Figure 3A; Experimental Group x Time ANOVA p = 0.625, Time ANOVA p < 0.001).
To control for increases in in vivo torque production due to animal growth, peak torque
was normalized to body weight (Nmm/kg body wt).32, 33 There were no differences in pre-
injury (anterior crural muscles intact) torque values among the experimental groups (Figure
3B). As such, the degree of functional recovery observed for no repair, BAM or TEMR
groups was determined by calculating the ratio of peak torque at each post-injury time
relative to each respective pre-injury group mean. The pre-injury group mean was used as
a conservative approach to minimize any undue bias of a single pre-injury measurement
(Figure 3C).
A two-factor ANOVA was used to compare functional outcomes among the
different treatment groups over time. Statistical analysis revealed a significant effect of
282
treatment group (p<0.001), a significant effect of time (p<0.049), but no interaction
between time and treatment group (p=0.591). More specifically, all treatment groups
displayed improved function from 4-12 weeks post-injury. However, post-hoc analysis
revealed that at each time point, BAM significantly improved TA muscle functional
recovery compared to NR or TEMR negative responders (which were not different from
each other), while TEMR positive responders significantly improved functional recovery
to a greater extent than NR, BAM, and TEMR negative responders (Figure 3).
Consistent with the aforementioned observations, comparison of absolute peak
torque values among control (Un-operated and Sham/Synergist Ablation) and treatment
(i.e., no-repair, BAM, and TEMR positive and negative responders) groups at twelve weeks
post-injury revealed that TEMR positive responders had significantly greater peak
isometric torque values compared to non-repaired and BAM-repaired groups (Table 1).
Further, when isometric torque was normalized to TA muscle wet weight (an estimate of
specific torque), BAM treated muscles exhibited a significant deficit compared to control
groups (e.g., vs. Sham, p = 0.0278), while TEMR positive responders were similar to
control values (Table 1). Collectively, these findings indicate that TEMR positive
responders, and to a lesser extent BAM, improves the functional capacity of VML injured
muscle out to 12 weeks post-injury.
Morphological Characterization of Retrieved TA Muscle: VML injury resulted in
significant deformation and gross morphological remodeling of the TA muscle (Figure
4A). In non-repaired VML injured muscle, a longitudinal fissure was commonly observed
283
where the defect was originally created. Both BAM and TEMR positive responder groups,
defined by their functional response, appeared to partially fill this void, attenuating the
atrophic appearance of the muscle. Consistent with this observation, both BAM and TEMR
positive responders partially improved the distal width of the TA muscle (Figure 4B & C).
Functional recovery observed 12 weeks post-injury was significantly and positively
correlated with this improvement in distal TA muscle width (Figure 4D). Similarly, TA
muscle wet weight following VML injury was partially improved by BAM and TEMR
positive responders 12 weeks post-injury (Table 1).
Qualitative Histological Characterization of Retrieved TA Muscle: Twelve weeks post-
injury VML injured non-repaired TA muscle presented with fibrotic scarring at the defect
site with signs of aberrant muscle fiber generation marked by a few small disorganized
muscle fibers (Figure 5B&G). For treated VML injured muscle, the functional outcomes
were generally matched by the qualitative appearance of generated muscle fibers at the site
of the defect: that is, TEMR positive responders and BAM treated TA muscle presented
with signs of muscle fiber regeneration (Figures 5 & 6), marked by the formation of a band
of muscle fibers that regenerated in close proximity to the remaining muscle mass. TEMR
negative responders exhibited little to no signs of muscle regeneration (Figures 5E, J &
6C). In corroboration with the variable functional recovery with TEMR transplantation,
TEMR positive and negative responders appeared to have a differential immune response
– TEMR negative responders were marked with a robust macrophage (CD68 + cells)
presence taking the form of foreign body giant cells (Figure 6F), while TEMR positive
responders were marked also by macrophage presence but in a manner phenotypically
284
similar, albeit apparently greater in magnitude, than BAM treated TA muscles (Figure E &
D). A general summary of the performance of BAM scaffolds and TEMR constructs are
provided in Table 2.
Discussion
Over the past decade a focused research effort has been placed on developing
therapeutic strategies aimed at generating a clinically relevant volume of skeletal muscle
for the purpose of ultimately restoring function to traumatized musculoskeletal tissue.
While a variety of tissue engineering approaches have been successful in promoting partial
regeneration of lost skeletal muscle fibers at the site of VML, ultimately an improvement
in force production (i.e., net torque production) of the injured musculature is the most
meaningful treatment outcome. The primary finding of this study is that transplantation of
the TEMR construct and BAM is associated with significant increases in the magnitude of
TA muscle contraction (i.e., torque) following electrical stimulation of the peroneal nerve
in vivo, for up to twelve weeks post-VML injury in an immune competent Lewis rat model
(Figure 3; Table 1). Importantly, TEMR transplantation resulted in a highly variable
therapeutic response, wherein no (zero) to full recovery of function was observed. Twelve
weeks post-injury, TEMR positive responders (n = 6 of 13 observations; see Methods for
full details) and BAM transplantation promoted a mean recovery of 26 and 61% of the
functional deficit, respectively (Table 1). On the other hand, TEMR negative responders
(n = 7 of 13 observations) did not improve the functional recovery of VML injured muscle.
Our previous findings with TEMR transplantation in a nude mouse LD muscle VML injury
model5, 8 and other studies13, 16 support these findings: that TEMR positive responders and
285
to a lesser extent, BAM transplantation, can improve the functional capacity of skeletal
muscle with VML.
Sheet-based acellular matrix tissue engineering approaches with and without the
inclusion of a stem or progenitor cell source have previously been shown to promote
skeletal muscle fiber regeneration.5, 6, 8-10, 13, 15-17, 19 The current study confirms and extends
our prior work in the murine LD VML injury model5, 8

demonstrating that TEMR
transplantation can result in a low to moderate level of muscle fiber regeneration inside the
transplanted construct within three months after transplantation in an animal model with a
competent immune system. Additionally, in the current study BAM transplantation also
promoted muscle fiber regeneration within the same time frame, similar to that reported
recently in the mouse quadriceps following transplantation of subintestinal submucosa-
ECM material.17. On the other hand, TEMR negative responders exhibited virtually no
evidence of myogenesis in the defect area. This raises the ongoing debate as to whether
inclusion of a cellular source with a matrix is necessary to maximize the therapeutic
response of ECM-based tissue engineering devices. There is currently ample evidence
within the literature to support each approach e.g.,9, 16, 20, 21 While the findings of this study
do not settle this question, they do indicate that the inclusion of a cellular component with
a biological ECM has the potential (i.e., TEMR positive responders) to promote a 61%
functional recovery, a 2.3-fold increase in recovery compared to that elicited by BAM
alone (26% recovery), in this rodent model of VML injury. Clearly, further work is required
to increase the reproducibility of the desired functional outcome, and thus, the eventual
translational value of the TEMR technology.
286
The existing literature suggests that the mechanism of functional recovery after
VML injury mediated by scaffold-based tissue engineering approaches is multifactorial.
Of the VML studies that have reported functional improvements following therapy (e.g.,5,
8, 9, 13
), most have demonstrated a partial regeneration of the volume of muscle tissue loss
– the magnitude of tissue regeneration in these reports and in the current study, however,
are comparable to others that did not measure function. This suggests that functional gains
mediated by delivery of acellular scaffolds with and without and stem or progenitor cell
source across multiple VML models are due not only to the regeneration of muscle fibers,
but also by potentially; 1) improving the functional capacity of the remaining muscle mass
(e.g., functional hypertrophy34), 2) augmenting the efficiency of force transmission across
the defect35-38, or 3) protecting the remaining muscle mass from continued injury related to
mechanical overload that may lead to persistent inflammation and fibrosis39, 40.
Highlighting these possibilities, recently it has been shown that transplantation of
a decellularized muscle ECM, which promoted strictly fibrosis within the defect area, still
resulted in enhanced functional restoration at intermittent time points (e.g., 2 and 4 months
post-injury).41 As an extension of these results, in the current study we observed a
significant atrophy of VML injured muscles that was most severe in the distal third of the
tissue (distal to the defect site)- reminiscent of the effect of laceration on skeletal muscle.42-
44
Interestingly, the atrophic response of the distal portion of the TA muscle was attenuated
with TEMR and BAM transplantation, in a manner directly related to each group’s
functional recovery (Figure 4D). The benefit of mechanical loading on tissue regeneration
has long been appreciated,45 wherein increased and decreased activity can positively and
negatively impact regeneration, respectively. In this context, it is plausible that the
287
transplantation of an acelluar scaffold may improve function by promoting the formation
of a mechanical bridge that is capable of improving force transmission throughout the
remaining muscle.41 This concept appears to parallel the biological response of skeletal
muscle to disruptions in cytoskeletal architecture (e.g., desmin knockout mice46), with
repeated lengthening contraction training39, 40, and clinically following laceration of the
biceps brachii muscle.47 Future studies should address the mechanistic basis for the
functional effects of various tissue engineering strategies following VML injury, as the
mechanisms that drive these distinct aspects of functional recovery will provide important
insight into the development of efficient regenerative strategies.
To continue the development of TEMR technology towards clinical applications, it
is important to identify why this group as a whole presented such a wide range of functional
outcomes in this VML injury model. One possibility is that the additional mechanical
manipulation involved with insertion of the TEMR scaffold in the TA VML injury model
(relative to the previously reported LD model) results in a decline in the retention of, or
damage to, the cellular component at implantation. These disturbances would effectively
diminish the presumptive myogenic5, 8, angiogenic 48, or immunomodulatory 49 effects of
MDCs. This supposition is consistent with the clear delineation in the regeneration of
muscle fibers and macrophage infiltration characterizing the positive and negative TEMR
responders (Figure 6). Moreover, delivery of the “damaged” TEMR construct may have
increased inflammation in a manner similar to that observed upon transplantation of grafted
whole tissue50, and may have therefore interfered with improvements in function mediated
via transplantation of BAM alone.
288
The primary findings of this paper highlight that the transplantation of
decellularized ECM with (TEMR positive responders) and without (BAM) the inclusion
of MDCs can promote significant functional recovery in skeletal muscle with VML injury.
Further, the above findings also highlight the potentially greater regenerative capacity of
therapies that include stem or progenitor cells in addition to a scaffold. Specifically, this
possibility is evidenced by the fact that TEMR positive responders promoted a 2.3-fold
greater functional recovery than did BAM alone. On the other hand, these findings also
point to the additional complexities of cell inclusion, as neither functional recovery nor
muscle fiber regeneration was observed in the TEMR negative responders. The high degree
of variance among TEMR responders appears to be related to pre-surgical handling of the
constructs (e.g., physical manipulation), and therefore, we are confident that further
optimization of this technology (i.e., reduced surgical handling, improved cellular seeding,
layering of scaffold sheets or utilization of more favorable scaffold geometries) will
mitigate these issues. In short, while there is still significant room for functional
improvement, these initial studies provide important guidance for future development of
tissue engineering therapies, and furthermore, document the importance of studying these
technologies in distinct animal models and VML injuries in order to develop the strategies
that will best accommodate the full spectrum of functional and cosmetic deficits that result
from such injuries.
289
Figure Legends
Figure 1. TA muscle VML injury repair with BAM scaffold and TEMR constructs.
A) A 3 x 0.5 cm strip of BAM or TEMR was folded in triplicate prior to transplantation.
B) TEMR constructs were comprised of BAM with an aligned cellular content (MDCs
were seeded at 1 million cells per cm2). Scale bar = 50 µm. C) VML injury was surgically
created by making a ≈ 1 x 0.5 x 0.3 cm defect in the left TA muscle and was D) immediately
treated with BAM or TEMR constructs that were sutured in the fresh wound bed as
depicted and described in the methods.
muscle synergist ablation and VML. A) Representative digitized torque tracings are
presented for uninjured intact anterior crural muscles (TA, EDL, & EHL muscles), and
after ablation of the EDL and EHL muscles and subsequent VML. Note: Following
ablation (4 – 12 weeks), the TA muscle produced ≈70% of net anterior crural muscle
torque. Subsequent VML of the TA muscle (see methods) resulted in a further ≈20%
deficit, resulting in a combined ≈50% deficit compared to pre-injury (Intact) torque. B)
TA muscle peak isometric torque (Nmm/kg body wt) normalized to pre-injury values was
stable from four to twelve weeks post-synergist ablation.
Figure 3. TEMR improves functional recovery of rat TA muscle with VML injury.
A) Group mean rat body weight over the course of 12 weeks. No differences were observed
among groups at each time point (see results). All means at each time point demarked with
different letters are significantly different (p < 0.05). B) To control for the effect of normal
growth over the three month study on peak isometric torque production, torque was
290
normalized to body weight (Nmm/kg body weight). Prior to VML, the average peak
isometric torque for each experimental group was similar. C) Individual responses at each
post-injury time point (4, 8, & 12 weeks) were normalized to their respective pre-injury
group mean. Individual responses and group means at each time point post-injury are
presented. Across all time points, BAM significantly restored torque to a greater extent
than NR or TEMR negative responders (*), while TEMR positive responders improved
torque recovery to a greater extent than all other experimental groups (†). Where
applicable, values are listed as mean ± SEM. Group sample sizes are listed in parentheses
in the 12-week response in panel C.
Figure 4. TA muscle gross morphology 12 weeks after VML and treatment. A) The
gross appearance of VML injured muscle without (NR, no repair) and with (BAM or
TEMR) treatment was clearly distinguishable from uninjured muscle. Note: A longitudinal
fissure in the middle third of the muscle was visible in NR muscles. Implantation of either
BAM or TEMR filled this void. B & C) TA muscles appeared atrophied, with distinctly
smaller widths of the proximal and distal thirds of the muscle. D) The extent of atrophy in
the distal TA width was positively correlated with functional recovery 12 weeks post-
injury. Values are means ± SEM. Means at each time point demarked with different letters
are significantly different (p < 0.05).
291
Figure 5. TA muscle tissue morphology following transplantation of BAM and TEMR
constructs. TA muscles were harvested 12 weeks after VML injury. Longitudinal (A – E)
and cross-sections (F – J) from uninjured (A, F), non-repaired (B, G), BAM-repaired (C,
H), and TEMR positive (D, I) and negative (E, I) responders were stained with Mason’s
Trichrome (Red = Tissue; Blue = Collagen; Black = Nuclei). *denotes the area of the defect
where the constructs were transplanted. Scale bars = 50 µm.
Figure 6. TA muscle fiber generation and inflammation following transplantation of
BAM and TEMR constructs. TA muscles were harvested 12 weeks after VML injury.
Longitudinal sections were probed for myosin expression (MF20; A – C) and cross sections
were probed for macrophage presence (CD68; D – F) in BAM (A, D), TEMR positive (B,
E) and TEMR negative (C, F) responder muscles. *denotes the area of the defect where the
constructs were transplanted. Scale bars = 50 µm.
292
293
294
Figure 1. TA muscle VML injury repair with BAM scaffold and TEMR constructs
295
muscle synergist ablation and VML
296
Figure 3. TEMR improves functional recovery of rat TA muscle with VML injury
297
Figure 4. TA muscle gross morphology 12 weeks after VML and treatment.
298
Figure 5. TA muscle tissue morphology following
transplantation of BAM and TEMR constructs
299
300
Figure 6. TA muscle fiber generation and inflammation
following transplantation of BAM and TEMR constructs

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APPENDIX 2
Ethical Considerations in Tissue Engineering Research: Case Studies in Translation
Hannah B. Bakera, John P. McQuillinga, Nancy M. P. Kingb*
Affiliations:
a
Department of Biomedical Engineering, Virginia Tech-Wake Forest School of
Biomedical Engineering and Sciences; Wake Forest Institute for Regenerative Medicine,
391 Technology Way, Winston-Salem, NC 27101
b
Department of Social Sciences and Health Policy and Wake Forest Institute for
Regenerative Medicine, Wake Forest School of Medicine; Center for Bioethics, Health,
and Society and Graduate Program in Bioethics, Wake Forest University
Medical Center Blvd, Winston-Salem, NC 27157

*
corresponding author, nmpking@wakehealth.edu, 336.716.4289
Note: This manuscript has been submitted for publication. Hannah B. Baker, John P.
McQuilling, and Nancy M. P. King co-wrote this manuscript.
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Abstract
Tissue engineering research is a complex process that requires investigators to focus on the
relationship between their research and anticipated gains in both knowledge and treatment
improvements. The ethical considerations arising from tissue engineering research are
similarly complex when addressing the translational progression from bench to bedside,
and investigators in the field of tissue engineering act as moral agents at each step of their
research along the translational pathway, from early benchwork and preclinical studies to
clinical research. This review highlights the ethical considerations and challenges at each
stage of research, by comparing issues surrounding two translational tissue engineering
technologies: the bioartificial pancreas and a tissue engineered skeletal muscle construct.
We present relevant ethical issues and questions to consider at each step along the
translational pathway, from the basic science bench to preclinical research to first-in-
human clinical trials. Topics at the bench level include maintaining data integrity,
appropriate reporting and dissemination of results, and ensuring that studies are designed
to yield results suitable for advancing research. Topics in preclinical research include the
principle of “modest translational distance” and appropriate animal models. Topics in
clinical research include key issues that arise in early-stage clinical trials, including
selection of patient-subjects, disclosure of uncertainty, and defining success. The
comparison of these two technologies and their ethical issues brings to light many
challenges for translational tissue engineering research and provides guidance for
investigators engaged in development of any tissue engineering technology.
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Highlights
• Investigators at all stages of translational research are moral agents
• Every trial should produce results that are able to guide researchers in designing the
next study
• Well-designed translational research has value even when ultimately unsuccessful
• Increased discussion of ethics promotes transparency and knowledge-sharing in
research
Keywords:
human research ethics, animal research ethics, translational research ethics, tissue-
engineered skeletal muscle, bioartificial pancreas
Abbreviations (nonstandard):
TESM = tissue engineered skeletal muscle
BAP = bioartificial pancreas
TM = therapeutic misconception
TD = translational distance
FIH = first-in-human
CRO = contract research organization
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1. Introduction
Tissue engineering research is regenerative medicine research that emphasizes
combining cells, tissues, and various enabling technologies, from scaffolds and capsules to
bioreactors, to develop tissues and other biomaterials capable of replacing or augmenting
physiological and biochemical functions impaired by illness or injury [1, pp. 215-216; 2,
p. 11]. Research to develop so-called “combination products” is complex, both
scientifically and from a regulatory standpoint. The ethical issues that arise in tissue
engineering research are likewise complex and worthy of careful examination [3-5]. It is
often helpful to address research ethics issues in the context of specific research case
studies [6]. This essay enumerates some basic ethical considerations for tissue engineering
research, and examines in detail their application to two representative research case
studies: tissue-engineered skeletal muscle (TESM) constructs and the bioartificial pancreas
(BAP).
It is currently popular, even necessary, to describe research—especially research
involving novel biotechnologies—as “translational.” In addition to being trendy, however,
the term is important, because it helps to illustrate the need for and value of viewing all
health-related research comprehensively relating each line of research to anticipated
knowledge gains and health improvements. Translation should not imply certainty that a
line of research will lead neatly to safe and effective products or treatments. Rather, it
should signal the responsibility of investigators to think ahead along the line of research,
to anticipate the relationship between study design and research ethics, and to consider and
periodically reconsider how to do research that has scientific and societal value, regardless
of the direction taken along the translational pathway from one experiment to the next [7].
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Investigators at all stages of translational research are moral agents, with
profession-specific moral duties that apply to the design and conduct of their research.
These duties can be described generally, but must also be articulated and applied to the
specific context of a given study. This essay therefore first addresses common ethical
considerations in tissue engineering research at the laboratory, preclinical, and human
research stages. There are important ethical considerations common to all research, and
additional considerations that particular research stages have in common. Next, we
consider these ethical issues as they arise in our two case studies, which were chosen for
their differences, with the goal of comparison and contrast. It is our hope that examining
the application and relative importance of the ethical considerations affecting the design
and conduct of studies of TESM and BAP along the translational pathway will help to
model similar thinking for tissue engineering researchers working with different tissue
constructs, and thus make it easier to engage in thoughtful research at all stages from the
bench to the bedside.
1.1. In the laboratory (in vitro and pre-clinical animal research)
Much tissue engineering research is still in preclinical stages. Although ethical
issues are often underaddressed in research until human trials have begun [8], there are
many issues worthy of consideration at the bench. These include: data integrity,
responsible reporting and dissemination of results, and ensuring that every study is
designed and conducted so that it can yield results suitable to decide on the next research
steps [9]. It is important to recognize that, at any stage, the results of well-designed and
properly conducted research might lead not forward, but back or in a different direction
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entirely, to refine and expand knowledge at an earlier stage or to explore and develop newly
identified possibilities [7].
The use of animal models at preclinical stages remains vital to the success of tissue
engineering research. Efforts are underway to minimize the use of animals, but potential
alternatives like computer modeling and body-on-a-chip organoid arrays have significant
limitations and require considerable further development [10]. Researchers therefore must
consider the three Rs of animal research -- Reduce, Refine, and Replace [11]. The choice
of animal models, and their humane and appropriate use, helps to ensure that the research
transition from animals to humans adheres to the principle of “modest translational distance”
described by Jonathan Kimmelman. Translational distance (TD) refers to the number and
size of inferential leaps from animals to humans [7], pp. 117-122] – in other words, it is a
measure of uncertainty. In first-in-human (FIH) and other early-stage research, modest TD
may provide an analytical model for considering the relationship of research design and
ethics – a role that cannot be filled by the concept of clinical equipoise, which applies only
to later-stage research such as trials comparing experimental interventions to standard
treatment. Clinical equipoise justifies asking patient-subjects to risk receiving an unproven
intervention in a clinical trial when there is sufficient evidence that reasonable clinicians
consider both the unproven intervention and currently available standard treatment to be a
reasonable treatment choice under the circumstances [12]. Early-stage research cannot
offer the potential for direct benefit to patient-subjects that may be available in later-stage
research. Instead, modest TD justifies asking patient-subjects in early-stage research to risk
receiving an unproven intervention only when the “inference gap” is small enough to
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predict both that the clinical trial can yield useful results and that it can adequately protect
their safety – not because it is reasonable to anticipate direct benefit.
1.2. Clinical research with patients as subjects
Much has been written about ethical issues in clinical trials [13-15]; this body of
scholarly literature, and the issues it addresses, continues to grow. For tissue engineering,
like other regenerative medicine research, it is most important to address key issues arising
in early-stage clinical trials [16-18]. Those issues include: moving into human subjects;
designing FIH trials; selecting patient-subjects; and informed consent.
In order to move from preclinical to FIH and other early-stage trials, several
questions must be answered in the affirmative:
• Has enough preclinical information been collected so that the only reasonable way
to learn more is to move to humans? This is another way of asking whether modest
TD has been achieved.
• Has enough been done to reduce the risks of harm to humans, and to maximize the
likelihood that the intervention will ultimately show benefit in humans? This
question about the appropriate balancing of the risks of harm and potential benefits
applies at all stages of clinical research, but is particularly important in early-stage
research, because what counts as potential direct benefit to patient-subjects in these
trials is limited and uncertain at best [19].
• Has the point of irreducible uncertainty been reached? In early-stage research, this
question is more applicable than the question “Have the risks of harm been
minimized as far as possible?” because it acknowledges that some risks of harm are
still unknown, and that it is never truly possible to eliminate all uncertainty.
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• Is the amount of irreducible uncertainty small enough that it is fair to subjects to
ask them to become involved in the research? Addressing this evaluative question
is a key component of the researcher’s ethical inquiry. The answer is less important
than the reasons that can be given to support an affirmative answer. Like the more
familiar requirement that risks of harm and potential benefits “must be balanced
and shown to be in a favorable ratio” [15], this reasoning forms the basis of
productive discussions between researchers, their colleagues, and oversight bodies
like funders, study sections, and institutional review boards (IRBs).
Two key considerations that relate the design of clinical trials to research ethics
considerations are scientific validity and social value. A valid study is methodologically
rigorous, designed and powered to provide useful answers to the questions it asks –
including negative answers. A valuable study asks a socially meaningful and/or
scientifically useful question [14]. Value in research is usually defined as progressive
value—that is, whether a study’s results are able to move the line of research directly
forward to the next phase. However, FIH and other early-stage research is also highly
likely to have translational value that is not progressive, but may be reciprocal, iterative,
or collateral [7], pp. 92-94]. Reciprocal value highlights the necessity of and informs new
preclinical work. Iterative value helps to inform the clinical trial itself by providing new
information that can be productively used for in-trial modifications as the trial goes forward.
Collateral value helps one or more different trials, by making new information, experience,
and techniques more broadly available to researchers in related research or related fields.
Researchers who recognize the broad applicability of study data and design translational
research to take advantage of many different types of value can readily plan both to address
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what would otherwise simply be regarded as failures of research progress and to enhance
the value of successful research.
Selecting patient-subjects from whom scientifically useful data can be collected and
who are also able to make well-informed choices about research participation is especially
important in early-stage research. Choosing the population from whom the most may be
learned and who also can most readily be helped to make autonomous choices about
participation may be challenging, as sometimes those are two different populations of
potential patient-subjects [16,20]. Researchers must take care to provide good information,
not only to potential subjects in the consent form and process, but also to the media and in
publications discussing study results. Good information reduces the likelihood that the
therapeutic misconception (TM) will cause potential subjects, their families, oversight
bodies, the media and the public, and even fellow researchers to overestimate the potential
benefits of the research or to underestimate its risks of harm [21,22]. The need for long-
term follow-up, both to monitor the health and function of patient-subjects after the
intervention and to determine the intervention’s success or failure, is often overlooked, not
only in study design and budgeting, but also in disclosure to potential subjects.
2. Case study 1: Ethical considerations in translation of TESM technologies
2.1. Introduction to the technology
In this case study, the TESM consists of a cell-seeded biomaterial construct used to
enable repair and/or remodeling in a muscle loss injury or pathology. Such a technology
aims to replace the current standard of care, which is the use of host or cadaveric muscle
flaps for repair and reconstruction. Using host muscle flaps means creating a new, ideally
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less consequential region of muscle loss in order to repair the initial injury. Using cadaveric
muscle flaps requires an immunosuppressive regimen. In contrast, using TESM, if
perfected, would also require surgical implantation, but would not create an additional
injury or the need for immunosuppression.
In future human studies, the generation of a TESM construct would begin weeks
prior to implant with isolation of muscle cells from a biopsy taken from a patient-subject.
Those cells would then be expanded in culture and eventually seeded onto a biocompatible
scaffold. Next, depending on the particular TESM technology employed, the cellularized
scaffold may be subjected to additional physiological cues to guide further maturation of
the cells on the construct [23,24] Finally, the construct would be sutured into the tissue
void.
Initial proof of concept for a TESM technology must be established in vitro prior
to any animal studies; this alone can take years. This initial effort would be followed by in
vivo studies, which seek to first develop a working animal model of a muscle injury that
the investigator is interested in treating. This in vivo work can be inherently lengthy, as
each study will need to carry on long enough to capture the full potential recovery for the
muscle pathology of interest, which can vary from days for minor injuries, such as sports-
related muscle damage, to months for volumetric muscle loss injuries [23,25]. The
complexity of this research suggests unique ethical considerations at each stage leading up
to clinical translation. This case study explores those issues at both early and pre-clinical
stages and in clinical trials.
2.2. Research ethics in the lab
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As noted previously, for preclinical research early on in a translational project, the
primary ethical considerations should focus on data integrity, both for the value of the
science and for the sake of future beneficiaries of the research. Every trial should produce
results that are able to guide researchers in designing the next study. In animal studies, in
addition, researchers should apply the three Rs to the maximum extent possible.
2.2.1. In vitro
Ethical considerations at the bench include: keeping good records, practicing good
data collection and management, transparency of data-sharing and realistic representation
of study results, and preparing for transition to animal studies.
In aiming to conduct authentic translational research on TESM, even early stage
investigators must be cognizant of the potential beneficiaries of the technology. Work at
the bench and in the culture hood may someday help treat a wounded warrior, a cancer
patient, or someone born with a congenital birth defect. As such, ethical considerations at
this stage are vital. It means that the quirk identified in the protocol but left undocumented
might be the difference between successful translation of work into the clinic and loss of
progress. Details—in cell culture, making up reagents, and handling of constructs—are all
very important, and good documentation is a key requirement. Staying aware of the
potential benefits of responsible laboratory practices and the ramifications of failure to
attend to detail is important in day-to-day research even at the early stages [9].
In complex research like TESM, it may be necessary to involve an unbiased third
party research group such as a contract research organization (CRO) to repeat some of the
research. Doing so at an early stage may make it easier to reduce TD, show proof of
concept for animal studies, and prepare to move toward clinical trials.
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In addition to facilitating direct translational progress, in vitro research can also
have knowledge-generating results with broader applications. In TESM research, culture
of muscle cells on scaffolds can help to inform both the use and development of enabling
technologies such as specialized imaging modalities and the use of muscle cells in other
applications [26,27] – important examples of reciprocal and collateral value.
2.2.2. In vivo
Meeting the goals of the three Rs is challenging in TESM research. With respect
to the first R, “Replace”, there exists no potential alternative, such as a computer model or
a cell line, which can completely replace the use of animals to study TESM technology in
a physiologically meaningful manner. Studying muscle cells seeded onto biomaterial
scaffolds can provide useful information about the interaction between the cells and the
construct, but cell-seeded scaffolds alone cannot recapitulate the necessary physiologic
environment. Thus, for in vivo work, the other two Rs, “Reduce” and “Refine”, inform the
planning of humane animal studies. These two Rs help to ensure that the number of animals
used is minimized, and that animals experience the least amount of pain possible in a study
and are housed and cared for according to established guidelines [28].
Reduction entails both making use of only the best animal model and determining
the optimum number of animals needed to achieve significance in results. For example, in
a muscle injury model it would be unwise to test a large number of TESM interventions in
small groups of animals, as this could create additional “noise” in an experiment, impeding
determination of efficacy for any particular intervention. Refinement entails not only
ensuring adequate analgesia for all animals used, but also ensuring that the use of animals
is parsimonious and targeted. This might include conducting in vitro studies and studies
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with smaller animals (e.g., rodents) to develop techniques for injury production and
application of TESM prior to use in a large animal model (e.g., sheep or pigs).
Because TESM is a complex technology with cellular, biomaterial, and bioreactor
components, all of which must be fully functional for success, the risk profile may be
heightened, as there are several risks of harm associated with each component. Ensuring
modest TD requires demonstrating through pre-clinical evidence that these risks are
manageable. Species to species differences must be ruled out as much as possible when
moving from animals to humans. Ensuring modest TD is particularly difficult in this
context. Although volumetric muscle loss in human patients is not standardizable, creating
a “spontaneous” and unique injury in animal models through surgically induced irregular
trauma or blasts is, difficult to justify in both regulatory and ethical terms. Thus, it is
reasonable to create more uniform and less traumatic defects in animal models, especially
in non-human primates, both for ethical reasons and to establish proof of concept. Even
so, it will be challenging to gather generalizable data because there will always be
irreducible differences in injuries, both across animals and between animals and human
patient-subjects.
Additional considerations when matching animal models of muscle pathologies to
humans include (1) developing a model in which the untreated animals will recover (or not
recover) in the same manner/timeline as the clinical presentation of the particular injury
and (2) matching the muscle fiber type and anatomy in the animal model site to the fiber
type or fiber orientation planned for the clinical application. Studies should also include
both male and female animals of varying ages. Finally, if cells are used, the same cell
source or an equivalent source should be appropriate and planned for human trials.
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For maximal knowledge generation, animal studies of TESM using an injury model
require proper controls. A negative control for TESM animal studies entails use of a no-
repair group in which an injury is created and allowed to heal without surgical intervention.
This is necessary for muscle injury models in order to study the extent to which the
intervention enables repair and regeneration within the injury site. Positive controls -- the
use of an uninjured control group or treatment with autologous and allogeneic flap transfers
– are also necessary in order to begin determining the effectiveness of the TESM strategy
under study relative to the current standard of care. The ultimate goal of this line of research
is to determine whether TESM can replace flap transfer. In order to show whether TESM
can overcome the problems of graft failure associated with cadaveric transplant and the
cosmetic impairment caused by resection of the patient’s own tissue, clinically relevant
animal studies involving TESM need appropriate controls.
Finally, reducing TD also means ensuring data transparency and both internal and
external validity. Internal validity [7, p.119] is a measure of the strength of causal
inferences arising from trial data, and thus of the predictive power of a trial. Ways to
improve internal validity include randomization and limiting variability within and across
arms (even when blinding is not possible). For TESM studies, one option is ensuring the
ability to repeat the work done previously within the same lab group, by maintaining good
lab notes. Turnover of students, post-doctoral fellows, and lab technicians can hinder this
process. External validity is most fundamentally challenged by the inherent divergence
between animal models and human subjects, and can best be addressed through close
correspondence in study designs and goals, increased use of pilot and feasibility studies in
FIH settings, transparency, and minimization of bias [7, pp. 120-121]. This may be
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accomplished by making use of a third party research group such as a CRO to repeat some
of the research in order to prove efficacy in animal studies. Finally, pre-clinical data should
be published, so that scientists can learn from one another and further the field of their
research in general. Failures of transparency increase the likelihood that a technology with
low odds of ultimate success will make it to clinical trials.
2.3. Research ethics in early-stage human trials
Every clinical trial should produce results that are able to guide researchers in
designing the next study. Because the ultimate goal of medical research is to improve
clinical practice, all trials should be designed for both validity and value. Trial design and
conduct must also address protections for research subjects: Risks of harm to subjects
should be clearly identified and minimized, and the potential, or lack thereof, for direct
benefit must be addressed; this includes defining success and failure, and examining what
can be done for patient-subjects if/when the experimental intervention fails. Addressing
these considerations in first-in-human and early-stage TESM research may pose special
challenges.
2.3.1 Patient-subject selection and informed consent
Fair and appropriate subject selection is a key component of scientifically and
ethically sound clinical trial design. Should the first subjects in a TESM construct study be
patients with extensive injuries, or with small defects? What control groups are most
appropriate? Early-stage TESM research in humans will necessarily focus only on repair
of injuries and defects of relatively small volumes, primarily because of the need to develop
or provide a vascular supply. Vascularization is an essential aspect of TE research that
requires further development [29]. Consideration might also be given, however, to
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enrolling patients with more extensive injuries, if proof of concept data could be gathered
prior to standard repair or amputation. Thus, early-stage TESM research might also be
undertaken in patient-subjects with more extensive injuries if there is sufficient time to do
so before standard treatment with cadaveric muscle or flap transfer is attempted. It would
be critical to design and conduct such studies so that (1) standard treatment would remain
available, (2) delaying standard treatment would be minimally disadvantageous to patient-
subjects, and (3) a clear and robust consent process can be undertaken so that patient-
subjects understand the study design and the harm-benefit balance and can make free and
informed decisions about participation. In addition, it might be useful to collect data from
patient-subjects who choose not to have any repair. In early-stage TESM trials where
vascular supply does not pose a barrier, randomization of patient-subjects who would
otherwise choose standard treatment to no-treatment or sham surgery is not ethically
justifiable when the design requires withholding reasonably effective standard treatment
[14,30,31].
A patient who volunteers to take part in a clinical trial should know first and
foremost that he or she is a research partner who, by joining the study, will further scientific
research aimed at advancing the field regardless of the outcome. Potential
patient-subjects must be told that the goal of the study is to find out if TESM technology
can restore function and improve appearance in the area of muscle injury. Outcomes for
patient-subjects are unknown and will vary. Research is not treatment, but rather an
investigation into a possible solution. In TESM research, the need to seek consent to
participation from patients with recent traumatic injuries may make it more difficult to
convey essential information effectively. And even well-informed potential subjects may
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still believe strongly that in spite of the risk of failure, TESM is sure to work for them.
Such patient-subjects may be expressing acceptable optimism, or may be significantly
underestimating the risks of harm, overestimating potential benefits, and mistakenly
viewing research as treatment – that is, some may be affected by the therapeutic
misconception (TM) [21,32-36]. TM can often be minimized by careful discussion and
expectation management with patients who are potential subjects during screening and
interviews prior to enrollment, as well as continuing discussion during trial participation.
A high degree of attention to informed consent for patient-subjects with recent injuries will
be essential in TESM research [37].
In FIH trials of TESM interventions, there are several important “unknowns.”
Injuries created in animals will not be the same as the injuries in patients: anatomical
differences will be present, and the injuries will be of different geometries and complexities.
In general, every injury is uniquely complex. Thus, researchers in FIH TESM trials must
identify (and then explain to potential subjects) what is unpredictable and unknown. This
is true regardless of study design, as it applies to pilot and feasibility studies as much as it
does to trials with more than one arm.
In translating tissue engineering research like TESM to human patient-subjects, the
risks include not only the risks of harm associated with all surgical procedures, such as
infection at the implant site, but also the risk of failure to meet patient expectations. TESM
is a permanent alteration to the patient-subject’s body, which is only reversible by
additional surgery [5]. Potential subjects must be told that if the experimental intervention
were to fail, an additional surgery would be needed to correct the defect, possibly entailing
replacement of the experimental implant with a standard flap transfer, which poses all the
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risks of additional surgery and potentially adds the risk of rejection of the flap. Moreover,
a TESM graft might not be physically rejected but might nonetheless fail to meet the
patient-subject’s expectations about appearance or functional recovery. To mitigate
dissatisfaction with a TESM repair outcome, the patient-subject’s expectations should be
addressed and discussed before enrollment – a process similar to standard consent practice
in plastic and reconstructive surgery.
2.3.2. Success and failure in TESM
Several metrics could be used to assess the degree of success achieved in a TESM
study. For repairs involving load-bearing muscles such as those in the extremities, function
could be analyzed through electromyography or more simply through physical strength
tests administered by a physical therapist. Another important metric is the appearance of
the treated muscle after surgery. Improving the aesthetic quality of muscles in the face and
neck or the extremities can greatly improve self-confidence and quality of life, even if the
muscles are not fully functional. Assessment of personal qualitative metrics should be
conducted through careful interviews with patient-subjects before and after surgery. These
measurements are likely to vary in importance from person to person, and for each person,
some bodily characteristics may be more important than others. Before beginning a clinical
trial, these metrics must be clearly defined and perhaps ranked by patient-subjects before,
during, and after the process of surgery and recovery. For some patient-subjects who may
have experienced trauma associated with their muscle loss injuries, mental health and
wellbeing should be especially carefully evaluated to help determine the impact of TESM
on overall quality of life.
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The above considerations suggest that human clinical trials of a TESM construct
should explicitly interrogate the meaning of success. What counts as success is likely to
depend in large part on the preferences and values of patient-subjects, and thus may vary
considerably between individuals. Success, as defined by the goals and expectations of
patient-subjects, could focus on restoring appearance associated with damaged muscle, or
on recovering muscle function to varying degrees, from simple weight-bearing to returning
to competitive athletics. In this respect, research outcomes in TESM are more “patient-
centered” [60] than is typical in many trials. Researchers should discuss realistic
expectations with potential subjects, learn what patient-subjects seek, and consult with
them both immediately after the intervention and as a matter of long-term follow-up to
evaluate quality of life and assess success. Functional outcomes can be identified and
physiologically relevant outcome measures can be applied, but if, ultimately, patients are
not satisfied with their outcomes, TESM was not successful. Similarly, patients who are
satisfied with a non-functional repair, perhaps due to the restoration of desired appearance,
will deem TESM successful. As success is ultimately crucial in all translational research,
investigators and subjects must talk together about what success means in TESM research
on an individual basis.
Of course, well-designed translational research has value even when ultimately
unsuccessful. Failure in a well-designed FIH TESM study supports investigating potential
reciprocal, iterative, or collateral value. Reciprocal value could be finding out that patient-
subjects with a pre-existing condition react differently from other patient-subjects, leading
to a preclinical TESM study done in animals with that condition. Procedural nuances
necessary for success may present iterative value in FIH trials. Surgical techniques may be
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refined extensively from one patient-subject to the next in a single protocol [38], and many
other in-trial procedural modifications (e.g. special post-operative treatment, a specific
exercise, bed rest protocol, or specialized physical therapy) may also be developed. Finally,
exposing more surgeons and specialists to TESM is by itself a major foreseeable collateral
value. For instance, muscle scaffolds may be discovered to have new, unanticipated
surgical applications. Thus, research that can identify potential reciprocal, iterative, and
collateral value can respond productively to apparent failure, providing hope that the
research may continue and return to additional clinical trials.
3. Case Study 2: Ethical considerations in the bioartificial pancreas (BAP)
3.1. Introduction to the technology
The BAP is in development as an alternative to standard treatments for Type 1
diabetes. Standard treatments include daily insulin injections, continuous infusion insulin
pumps, pancreas transplantation, and clinical islet transplantation. Research into islet cell
microencapsulation technologies began as early as 1980, in order to overcome the need for
immunosuppression that accompanies transplantation of allogeneic cells, tissues, and
organs. The BAP utilizes either insulin-producing β-cells or aggregates of these cells which
are known as islets. These cells are incorporated into either a macro- or microencapsulating
device, and then implanted into the body for the regulation of blood glucose levels. Several
BAP models have been studied; one key feature of current BAP research addresses optimal
implantation sites, which include the peritoneal cavity and the omentum [39,40]. There
are many similarities between the ethical considerations arising in TESM and the BAP
during pre-clinical research and human trials, but the BAP also poses several ethical
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considerations that are meaningfully different from those in TESM. Type 1 diabetes is a
chronic condition, with several alternative treatment options. Because of the multiple
treatment options available for Type 1 diabetes, the performance of the BAP comes under
additional scrutiny—the BAP needs to alleviate the long-term comorbidities of Type 1
diabetes and avoid both hypoglycemic and hyperglycemic events. If the BAP is unable to
improve upon the treatment provided through exogenous insulin injection or the use of an
insulin pump, it will not be a viable treatment alternative. The BAP must also ultimately
prove functional over the long-term. Thus, questions arise about the acceptable balance of
harms and benefits and the meaning of success and failure for this tissue engineering
technology, as they do for TESM, but the answers are likely to be different.
3.2. Research ethics in the lab
The same general concerns about data integrity, transparency, and anticipation of
translation to animal and human studies discussed in Case 1 apply to BAP research.
Animal study of the BAP is farther advanced than is TESM research, but different aspects
of BAP research are at very different stages. As always, it is important to follow good
laboratory practices and maintain a clear and transparent record of all data in order to
identify risks of harm that may be associated with a potential future therapy and to avoid
redundant or repetitive studies. Several additional issues unique to the BAP also arise at
the earliest stages of bench research.
3.2.1. In vitro
Most BAPs utilize whole islets; thus, the technology requires researchers to work
with primary cell lines, which are in limited supply and can only be cultured or stored for
a finite amount of time. This feature has led to an important ethical consideration in the
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laboratory: islets can be maintained in vitro for only a limited time before transplantation,
and there can be significant batch to batch variation between islet isolations. It is thus
essential that the tissue be evaluated as thoroughly as possible in vitro prior to
transplantation, and it is problematic when in vitro evaluation is not sufficient. Some of the
techniques commonly used are insufficient to evaluate both the functionality and the
viability of the islet cells in culture [41]. Utilizing the most appropriate in vitro tests
includes identifying those that are as close to clinical tests as possible, to minimize TD.
For the BAP, this means using in vitro tests that have been shown to strongly correlate with
transplant outcomes, such as tests that measure the metabolic activity or insulin secretion
rates of islets as opposed to more traditional live-dead viability stains [42,41].
The use of more predictive in vitro tests is especially important when considering
the three “R’s” of animal research when moving to animal models: reduction, refinement
and replacement. Ideally, the use of more accurate in vitro tests should reduce the number
of animals or groups needed when research progresses to in vivo studies.
A second issue specific to the BAP also relates to the limited availability of human
cadaveric pancreases. The urgent need for alternative tissue sources affects BAP research
from the earliest stages, because it gives rise to an important ethical issue surrounding
BAPs: the use of non-human tissue in humans. Using xenografts, most often from porcine
tissue, requires a very high level of awareness and vigilance with regard to the risks and
safeguards associated with non-human tissues [43-45]. Of chief concern with porcine cells
is the transfer of zoonotic disease like porcine endogenous retrovirus (PERV) [46]. As a
result of these differences, the risks of harm and potential benefits for the BAP, at every
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stage of research and ultimately in the clinic, are significantly different from those for
TESM.
3.2.2. In vivo
Identification of the appropriate small animal model and the use of proper controls
for the chosen animal model are important considerations in BAP research, in order both
to conform to the three Rs and to reduce TD. As with TESM, it is especially important to
use a small animal model that best represents the clinical disease. In the case of a BAP, it
is important to consider whether it is preferable to utilize a knockout model (where the
disease develops spontaneously) or an induced model for diabetes (where the animal is
given a chemical such as streptozotocin to cause disease). It is also important to consider
the appropriateness of using an immunocompromised or an immunocompetent animal
model. Using the former concentrates attention on factors other than the immunoprotection
afforded by the BAP, thus, for example, facilitating research on vascularization, which is
as essential for success of the BAP as for TESM. However, using immunocompromised
animals may increase the number of animal studies necessary before moving into humans
to test a BAP with an immunoprotective coating.
The most appropriate animal models in BAP research will always include large
animals. Although large animal models are not necessarily required for all TESM, large
animal models are required for islet cell transplantation because diabetic conditions in
rodents are significantly different from those in humans [47]. When using large diabetic
animal models for BAP studies, the reproducibility of the model is a critical factor. It is
also important to consider the clinical relevance of the insulin therapy given to controls.
330
In the case of the BAP, this means identifying the most appropriate and accurate protocol
for insulin therapy, in order to maintain maximal translational relevance to the clinic.
Like TESM research, animal studies of the BAP are complex, often involving
multiple surgical procedures, to induce diabetes and to implant the BAP. Especially in
research involving creation of an omentum pouch to improve vascularization of the BAP
it is essential to determine how best to maintain study protocol integrity while allowing for
the flexibility needed to improve surgical techniques over the course of the study.
Several considerations unique to the BAP also apply to design of animal studies.
Studies of a BAP that utilizes immunoprotective coatings to prevent immune rejection,
must be able to demonstrate that these coatings remain intact over the course of the study.
It is also important to adhere to clinically relevant islet acceptance criteria [48,49] in
constructing and using the BAP in vivo.
Determining the point at which the research is ready for human trials raises several
provocative considerations in BAP research that differ from TESM research. First,
determining the outer limits of the harm-benefit balance in BAP requires special attention
to ethical and design questions in animal studies before translation to human studies. One
issue is the role of immunosuppressants in BAP research. Ideally, the BAP would not
require the use of immunosuppressants, because long-term immunosuppressant use poses
risks that could outweigh the BAP’s benefits. Thus, the most desired endpoint of BAP
research is an encapsulated device that can restore a sufficient degree of pancreatic function
and that has an immunoprotective coating that is effective over the long-term. However,
studying the use of immunosuppressants with the BAP could ultimately facilitate the earlier
clinical availability of devices that could benefit seriously ill patients, for whom the
331
increased risk might be a reasonable choice under the circumstances. Thus, two very
different clinical applications may follow from research designed for different patient
populations with different options and needs.
Along the same lines, because there are different BAPs, there are different harm-
benefit assessments that can lead to different conclusions. Some BAPs carry higher risks
but are viewed as acceptably risky for broad clinical use. BAPs that make use of xenografts
are risk-bearing due to the concerns associated with the use of porcine or other non-human
tissues, in particular zoonotic disease, but the limited availability of human tissue makes
research focused on the use of porcine tissues reasonable under current circumstances.
Thus, research has proceeded using both allografts and xenografts in non-
immunosuppressed animals [40]. In contrast, however, some types of BAP constructs are
now considered unacceptably risky. BAP constructs that connect directly to the circulatory
system are riskier because there is a higher chance of clot formation and rejection.
Additionally, these devices cannot be retrieved or removed as easily as devices that are
simply implanted. These lines of research have been supplanted as more has been learned
about the inherent risks associated with these approaches [40].
Additional factors to consider when determining the point at which a BAP is ready
for human trials include study duration, size, and reproducibility. As with TESM studies,
one method to externally verify results is the use of a CRO. Challenges here include
establishment of the diabetic animals, technology transfer, and the training of CRO staff
[46,50,51].
Finally, when planning in vivo studies it is necessary to consider how they can be
designed to better identify the causes of failure if they are unsuccessful at achieving insulin
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independence. The long history of BAP research includes many examples of research that
has provided important iterative value (e.g. in-trial surgical modifications), reciprocal
value (e.g. research focusing on better vascularization to improve the longevity of the BAP
and research utilizing different islet cell sources), or collateral value (e.g. development of
BAPs intended only for limited patient populations, such as those for whom long-term
immunosuppression does not pose a significant risk), even though the ultimate goal of BAP
research has not yet been reached.
3.3. Research ethics in early-stage clinical trials
As compared to TESM, the BAP has one distinct advantage when moving into
clinical trials, and this is the historical precedent for the use of islet cell transplantation.
For BAP, unlike TESM, there is both a network of islet transplant centers and a set of
published donor selection criteria, as well as established guidelines for involvement in an
islet transplant study [48,49]. In this respect, BAP research benefits from the precedent
and expertise of related biotechnologies -- that is, from the collateral value generated by
prior research. BAP researchers can also make use of existing infrastructure and guidance
to help design and conduct trials that minimize risks of harm, maximize the production of
generalizable knowledge, and fully inform potential subjects.
3.3.1 Patient-subject selection and informed consent
Another difference from TESM appears in the selection of patients as research
subjects. Several populations of diabetic patients who are potential subjects for BAP
research have characteristics that put them at greater risk but also may make them better
candidates for some early-phase designs. One group, mentioned previously, is seriously
ill patients, whose disease course and anticipated longevity might in some circumstances
333
make them suitable candidates for BAP designs requiring immunosuppression. Another
such population is patients who have reduced awareness of hypoglycemia. This is a
dangerous condition [52]; consequently, these individuals may be candidates for early
clinical trials involving either islet transplantation or the BAP, since current insulin
therapies are inadequate for them. For populations like these, early-stage research
involving patient-subjects with diabetes most resembles Phase I oncology research, where
patients are chosen as subjects because there are no adequate standard treatments currently
available to them. Unfortunately, TM can be exacerbated in such trials, as it is tempting to
move from “nothing else works well for you” to “this research is your best hope” even
though direct benefit is very unlikely. As always, a clear and complete consent process is
the best way to avoid TM when patients are enrolled as research subjects under such
circumstances.
As trial designs change and scientific thinking about best subject populations also
changes, it may be worth speculating about how BAP research might be regarded if it were
thought that better data could be gathered from young, treatment-naive patient-subjects
than from patient-subjects for whom nothing else has proven effective. A contemporary
parallel might be found in current research using a new smartphone-based application to
regulate insulin pump delivery based on continuous data readouts [53]. In this Type 1
diabetes intervention, a sensor is used to monitor blood sugar in the patient-subject, and
blood sugar levels are managed through a dual pump to supply insulin (to lower blood
sugar levels) and glucagon (to prevent blood sugar level drops) to achieve normoglycemia.
Some patient-subjects in ongoing trials are teenagers, which is a departure from the
traditional adults-first model. This choice of subject population more closely resembles
334
the most likely patient-subjects in FIH trials of TESM constructs: younger patients with
smaller injuries or defects. Determining which patients are the best subjects in FIH trials,
especially when the potential subject populations are as different as very young, treatment-
naive patient subjects and adult patient-subjects for whom no adequate alternatives exist,
represents an extremely important ethical and design challenge. Which patient-subjects
should be first is an ongoing issue in gene transfer research and many other novel
biotechnologies as well [2,28].
3.3.2. Xenografting and risk in the BAP
One of the most discussed issues involving the BAP, which does not arise in TESM
research, is the use of xenografts. As previously mentioned, the use of porcine islets is one
solution to the shortage of available human donor islets. However, the use of xenografts
dramatically increases the risks associated with the BAP. Of particular concern is the risk
of spreading zoonotic diseases. As a result of this increased risk, there has been a
significant amount of research focused on creating disease-free pigs, with a particular focus
on pigs free of PERV. In response to the increased research into xenografts, the
International Xenotransplantation Association has published a consensus statement [54],
which reviews the key ethical requirements of an international regulatory framework for
clinical trials of xenotransplantation, the recommendations on source pigs, release criteria,
and necessary preclinical data required to justify a clinical trial, strategies to prevent the
transmission of PERV, patient selection, and an outline for informed consent [55-58]. Risk
reduction in this area may in the future change what is regarded as an acceptable risk-
benefit balance in BAP human studies and related trials.
3.3.3. Success, failure, and the BAP
335
For the BAP to become a successful treatment, it has to function better than insulin
therapy with its attendant complications. Yet what that means in terms of defining success
in BAP research may not be as clear-cut as it seems. On the one hand, it could be
reasonable to expect that the BAP should ultimately prove a genuine cure. On the other
hand, what if the BAP transforms what can be described as a progressively fatal disease
into a chronic disease? If partial functional correction is an acceptable goal, then a BAP
might be introduced into the treatment armamentarium as another useful “halfway
technology” that is not a cure. BAPs that meet this goal of partial functional correction
would reduce the need for insulin therapy and thus reduce the occurrence of hypoglycemic
unawareness and stabilize or reduce the comorbidities of Type 1 diabetes.
If the BAP is likely to reduce but not eliminate the need for exogenous insulin, then
the preferences and perspectives of patient-subjects may be highly relevant in determining
what counts as success for the BAP, even though there is no aesthetic component as in
TESM. Because Type 1 diabetes is a chronic and progressive condition that begins to
change most patients’ lives at a relatively young age, potential subjects may be more
knowledgeable and under less decision-making pressure than in TESM research, where
most potential subjects will have recently experienced a wide variety of muscle injuries
and loss. Thus, potential subjects for BAP research may consider the impact of different
research outcomes on their decisions to participate and their ultimate satisfaction from a
very different emotional and psychological vantage point. Moreover, the consequences of
partial success—reducing but not eliminating the need for exogenous insulin and the
effects of the many comorbidities of Type 1 diabetes – are quite different from those
potentially faced by TESM subjects.
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4. Discussion and conclusions
4.1. Strengths and limitations
By examining in some detail two very different examples of tissue engineering
research, we have been able to focus in-depth on some key issues that are not as carefully
reviewed elsewhere. However, a case-based review cannot address all of the many relevant
ethical issues arising in tissue engineering research.
The use of cells and cell lines has been thoroughly examined elsewhere [1,5,16].
So has the problem of access and cost, which is of considerable concern for all novel
biotechnologies [1,2,16]. The overall goals of tissue engineering include not only
developing treatments that are more effective than currently available alternatives, but also
keeping costs low and improving access. The complexity of tissue engineering
interventions represents a challenge to achievement of this goal, but it is nonetheless one
toward which the field strives [59].
4.2. Key lessons learned
Several key ethical issues emerge from these case studies. First, it is essential to
look for iterative value in tissue engineering research. Because tissue engineering research
methods are complex and mixed, including surgery, cells, and a wide range of enabling
technologies in animal and human trials, innovations are almost inevitable. Researchers
should anticipate the likelihood of innovation, and should design and describe protocols
that can not only accommodate innovations but also convert them into study features when
warranted [15,60]. Clear and close communication with research oversight bodies is
therefore essential, both to ensure the proper balance of flexibility and reproducibility in
337
tissue engineering protocols and to amend protocols when amendment is necessary to
capture methodological innovations.
Second, data transparency is critical, even though it is increasingly difficult to
achieve in the current highly competitive funding environment. For tissue engineering
research, it is particularly vital that researchers publish data at all stages, especially data
from early clinical trials, regardless of success or failure. Being transparent about the
research strategy and results of the trial enables scientists at earlier stages of development
to see what is and is not working with the model being used. This will fuel and inspire
future work and advance the field as a whole [61,62]. In addition, making the results of
these trials publicly available and understandable to the lay public will inform those
considering participation in such trials and help to set the stage for understanding the
benefits and limitations not only of trial participation but also of any future treatments and
new research directions that may later emerge from the research.
Third, much work is needed to increase parsimony in animal research while
identifying best animal models and designing effective adjuncts and alternatives to use of
animals. As noted earlier, body-on-a-chip and organ-on-a-chip research shows great
promise in this regard.
Fourth, it may be time to abandon FDA’s traditional phase designations in order to
take better account of novel trial designs and the increasingly nuanced considerations
influencing subject selection. Like Phase I oncology trials and most if not all FIH and other
early-stage clinical trials of novel biotechnologies, early TESM and BAP clinical trials
necessarily enroll patients as subjects, instead of the “healthy volunteers” traditionally
enrolled in Phase I drug trials, and may at times begin with older or younger subjects, and
338
experienced or treatment-naive subjects. Clear justification for a clinical trial’s design and
goals is paramount regardless of phase designation, and thorough consideration of the
implications of enrolling patients as subjects, including but not limited to the consent form
and process, are essential. A related challenge arises when complex tissue engineering
intervention trials cannot effectively or ethically be randomized and blinded. Patients who
are potential subjects may have strong and reasonable preferences for one arm over another,
as their goals and quality of life assessments may differ in ways that significantly affect
their choices about participation. It may be prudent to seek new ways to gather important
information in research that does not culminate in “gold standard” randomized controlled
trials. Much attention is now being given to “patient-centered outcomes” in clinical
research [63,64]. Incorporating patients’ perspectives into research could be well-suited to
some tissue engineering trials, and could increase enrollment.
Methodological challenges will inevitably arise in new trial designs, including not
only statistical design and data credibility but also a potential increase in the likelihood of
TM in investigators, the media, the public, and patient-subjects. These challenges can be
effectively addressed with clear and transparent justification for each step along the
research pathway, strong preclinical data, exemplary communication, and the creativity
that arises from collaborative scholarship along the translational pathway. The result could
yield intervention profiles that are more predictive of outcomes in clinical practice.
Finally, not only is long-term follow-up essential, but in addition, researchers can
play a critical role in facilitating translation to clinical care for these complex tissue
engineering interventions. Clinical use of cell-seeded scaffolds and capsules in TESM and
BAP will continue to pose challenges even after successful research. One often overlooked
339
issue is the infrastructure needed for feasible and efficient tissue engineering treatments.
For instance, hospital personnel would need to be trained in handling cell-scaffold products
and hospitals would need to affiliate with a Good Manufacturing Process facility for
generation of the constructs [65]. Several biomaterial scaffolds have been approved by the
FDA for a variety of applications, including rotator cuff repair, hernia repair, and heart
valve patches [66]. Familiarity with these devices may facilitate acceptance and use of
tissue engineering technologies in the future, as long as researchers and sponsors plan well
in advance for effective clinical translation.
We encourage detailed examination of additional case examples of research using
tissue engineering and other cell-based interventions. Case-based discussion of ethics and
design questions throughout the translational trajectory is the best way we know of to
promote transparency, encourage discussion, and maximize the knowledge gained from
this important research.
340
Acknowledgments
The authors thank Drs. George Christ and Emmanuel Opara for their review of earlier
versions of this paper. Research for this publication was supported in part by the National
Institute of Biomedical Imaging and Bioengineering of the National Institutes of Health
under Award Number T32EB014836. The content is solely the responsibility of the authors
and does not necessarily represent the official views of the National Institutes of Health
(HBB, JPMcQ).
341
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APPENDIX 3
Hannah Besser Baker
Note: The following figure represents unpublished data used with permission from
KeraNetics, LLC, Winston-Salem, NC 27101
351
Figure 1. Release of IGF-1 and bFGF from Blends of KOS:KTN
352
CURRICULUM VITAE
Hannah Besser Baker, B.S.

Wake Forest Institute for Regenerative Medicine • 391 Technology Way
(651) 402-4941 • hbaker@wakehealth.edu • 6/29/2015
Education
WAKE FOREST UNIVERSITY WINSTON-SALEM, NC
Doctor of Philosophy in Biomedical Engineering Anticipated July 13th, 2015
Virginia Tech-Wake Forest School of Biomedical Engineering and Sciences
• Thesis Committee: George Christ, Ph.D. (Chairman), Grace Almeida-Porada, Ph.D., Aaron
Goldstein, Ph.D., Robert Grange, Ph.D., David Kaplan, Ph.D., Aaron Mohs, Ph.D.
• Awards: Wake Forest Institute for Regenerative Medicine NIBIB T32 Fellowship
UNIVERSITY OF ROCHESTER ROCHESTER, NY
Bachelor of Science in Biomedical Engineering May, 2010
Concentration in Cell and Tissue Engineering with Minor in Biology
• Honors: Dean’s List; Bausch and Lomb Scholarship for Outstanding Achievement in
Sciences
• Involvement: Sigma Delta Tau Sorority; Order of Omega Greek Honor Society; UR
Biodiesel Team
Research Experience
WAKE FOREST INSTITUTE FOR REGENERATIVE MEDICINE WINSTON-SALEM, NC
Biomedical Engineering Graduate Student, Christ Lab Fall 2010-Present
• Skeletal muscle research focused on the use of preconditioned naturally derived materials
for repair and regeneration of volumetric muscle loss associated with craniofacial and
extremity injuries of active military personnel and congenital birth defects including cleft
lip and cleft palate
• Projects have included the investigation of cellularized porous aligned silk, cell and growth
factor loaded keratin hydrogels, and cell loaded and bioreactor preconditioned bladder
acellular matrix in rodent injury models of volumetric muscle loss
• Current efforts surround a preclinical study involving the development of a rodent defect
model with clinical relevance to cleft lip and subsequent investigation of tissue engineered
muscle repair therapies in this model for use in cleft lip secondary revision procedures
RADIATION & ONCOLOGY, STRONG MEDICAL CENTER ROCHESTER, NY

Research Assistant to Dr. Walter O’Dell Fall 2009
• Provided programming assistance for research software which aimed to model and
predict brain tumor metastasis from diffusion MRI images in order to optimize radiation
therapy
353
NEW JERSEY INSTITUTE OF TECHNOLOGY NEWARK, NJ
BioMEMs Summer Institute, Research Experience for Undergraduates Summer 2009
• Trained in microfabrication, microfluidics, and soft lithography with emphasis on
biological applications; designed and manufactured a MEMs device which utilized
ultrasound for the examination of osteoporosis
LAKE REGION MANUFACTURING CHASKA, MN
Quality Assurance Intern Summer 2007
• Conducted mechanical tests on medi cal devi ce guidewires and packaging; prepared
validation reports for aging studies; gained experience in product R&D and clean room
protocol
Publications
Benjamin T. Corona, Ph.D., Catherine L. Ward, Ph.D., Hannah B. Baker, Thomas J. Walters,
Ph.D., George J. Christ, Ph.D. Implantation of in vitro tissue engineered muscle repair
(TEMR) constructs and bladder acellular matrices (BAM) partially restore in vivo skeletal
muscle function in a rat model of volumetric muscle loss (VML) injury. Tissue Engineering
Part A 20 (3-4), 705-715. December, 2013
Oral Presentations
• Hannah B. Baker. Growth Factor Loaded Keratin Hydrogels for Treatment of a Sheet-
like VML Injury in Mice. Tissue Engineering and Regenerative Medicine International
Society Americas Annual Conference, Washington, D.C. December 13-16th, 2014.
• Hannah B. Baker. Keratin Hydrogels as a Cell and Growth Factor Delivery Vehicle for
Treatment of Volumetric Muscle Loss. Biomedical Engineering Society Annual Meeting,
San Antonio, TX.
October 22-25th, 2014.
• Hannah B. Baker. Keratin Hydrogels as a Delivery System for the Regenerative

Treatment of Volumetric Muscle Loss. North Carolina Tissue Engineering and
Regenerative Medicine Society Annual Conference, Winston-Salem, NC. October 18th,
2013.
Poster Presentations
• Hannah B. Baker, Catherine L. Ward, Ph.D., Masood Machingal, Ph.D., Benjamin T.
Corona, Ph.D., Jelena Rnjak, Ph.D., David L. Kaplan, Ph.D., George J. Christ, Ph.D.
Porous Aligned Silk Constructs for Treatment of Volumetric Muscle Loss Injury in a Rat
Model. North Carolina Tissue Engineering and Regenerative Medicine Society Annual
Conference. Raleigh, NC. September 10th, 2012.
• Hannah B. Baker, Catherine L. Ward, Ph.D., Benjamin T. Corona, Ph.D., Masood

Machingal, Ph.D., Jelena Rnjak, Ph.D., David L. Kaplan, Ph.D., George J. Christ, Ph.D.
Further development of a tissue engineered muscle repair (TEMR) construct for the
treatment of volumetric muscle loss (VML) injuries. Armed Forces Institute for
Regenerative Medicine All Hands Meeting. St. Pete Beach, FL. February 13-15th, 2012.
354
Skills
• Cell culture: rodent muscle primary cell isolation, aseptic technique, mammalian
cell line maintenance, scaffold seeding, stretch bioreactor sterile assembly and scaffold
loading technique
• Histology: immunohistochemistry, fluorescent and brightfield microscopy, paraffin
and frozen tissue processing and sectioning
• In vivo experimentation: Rodent skeletal muscle surgical defect and implant surgeries
and dissection, rodent post procedural care, in vivo and ex vivo muscle function testing
• Characterization techniques: Scanning electron microscopy, mechanical testing,
calcium imaging
• GMP Training: Standard Operating Procedure and Master Batch Record preparation and
execution
• Applications: Microsoft Office, LabVIEW, GraphPad, Chart, Image J
• Programming Applications: MATLAB, Mathematica
Biomedical Engineering Design Projects

UNIVERSITY OF ROCHESTER ROCHESTER, NY
Biomedical Engineering Senior Design Project: DPN Diagnostics Pedi-Map 2009-2010
• In a team of four students, designed and built diagnostic device prototype to identify and
track peripheral neuropathies in the foot in order to identify regions at risk for ulceration
UR Biodiesel Independent Study 2009-2010

• With a team of students, created a processor to convert waste vegetable oil from campus
dining halls into fuel for campus buses
• Aided in development, day to day processing, training, and guided efforts to ensure
program sustainability
Leadership Experience
WAKE FOREST INSTITUTE FOR REGENERATIVE MEDICINE WINSTON-SALEM, NC
Biomedical Engineering Graduate Student Researcher Fall 2010-Present
• Summer Research Mentor Summer 2012-2014

Served as a mentor to 11 summer scholars and rotating graduate students on guided
projects and facilitated training in histology, cell culture, microscopy, mechanical testing,
and animal care and use procedures
• Invited Speaker at the Shepherd Center of Winston Salem January 16th, 2014
Presented an overview of the history and progress of tissue engineering to a group of
seniors as part of a series offered in collaboration with the Wake Forest Translational
Science Institute Speakers Bureau
• Invited Speaker at Atkins Academic & Technology High School October 25th, 2013
Presented on Biomedical Engineering as an academic pursuit and career path to interested
students as part of a lecture series geared toward young women interested in science and
engineering careers
355
• Healthcare Career Day Tour Presenter October 20th, 2012
• Explained the research of the WFIRM Physiology Lab to 60 touring high school students
and parents interested in learning more about biomedical research and career opportunities
• First Lego League Volunteer, Winston-Salem, NC Fall 2011, 2013

Provided weekly advice and guidance for a team of students as they built and programmed
a robot and challenge course for a competitive robotics exhibition
UNIVERSITY OF ROCHESTER, BIOMEDICAL ENGINEERING ROCHESTER, NY

• Teaching Assistant: Biosystems and Biosignals Spring 2010
UNIVERSITY OF ROCHESTER, CHEMISTRY ROCHESTER, NY

• Teaching Assistant: General Chemistry II 2008-2010
SIGMA DELTA TAU SORORITY ROCHESTER, NY

• President 2009-2010, Treasurer Fall 2008 2007-2010
• Fundraising Chair Spring 2008, Philanthropy Chair, Spring 2008
SIMLEY SENIOR HIGH SCHOOL INVER GROVE HEIGHTS, MN

• Class Valedictorian June, 2006
356

Naturally Derived Scaffolds For Treatment of Volumetric Muscle Loss

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NATURALLY DERIVED SCAFFOLDS FOR TREATMENT

OF VOLUMETRIC MUSCLE LOSS

HANNAH BESSER BAKER

A Dissertation Submitted to the Graduate Faculty of

WAKE FOREST UNIVERSITY SCHOOL OF ARTS AND SCIENCES

in Partial Fulfillment of the Requirements

for the Degree of

George J. Christ, Ph.D., Advisor and Chair

Graça Almeida-Porada, M.D., Ph.D.

Aaron S. Goldstein, Ph.D.

Robert W. Grange, Ph.D.

David L. Kaplan, Ph.D.

Aaron M. Mohs, Ph.D.

independence and resourcefulness for which I am truly grateful.

translational research, and for helping me develop as a researcher by holding me to a high

spent together as fellows.

I am exceptionally thankful to have conducted these studies start to finish alongside

miss working with Juliana tremendously.

throughout the last several months as I was traveling between Winston-Salem,

and my brother in law Jimmar Wilson, and Rosemary Baker.

acknowledgement: Anthony and Jess Santago, Kiks Kowalczewski, JP and Alyssa

Fahrenholtz, Brittany Eldridge, Jessica Swanner, Hesh Devarasetty, Sam Pendergraft,

Mokhtari, Elie Zakhem, and Susan Zhao.

Dan Mendelson, and Monica Lentz.

great listener throughout this crazy time of life.

years and I thank them both dearly for it.

List of Figures and Tables………….…………………………..……………………….viii

Muscle Loss in a Mouse Model……………………………………………….....60

Volumetric Muscle Loss………………………………………………………....99

Secondary Revision Procedures in a Rat Model………………………………..134

V. Summary, Conclusions, and Future Directions…………………………….......241

1. Implantation of In Vitro Tissue Engineered Muscle Repair Constructs and Bladder

of Volumetric Muscle Loss Injury……………….………………...……...……269

2. Ethical Considerations in Tissue Engineering Research: Case Studies in

4. Growth Factor Release from Keratin Hydrogel Formulation Used in Mouse LD

and Rat TA Models (Unpublished Data from KeraNetics, LLC)…..................351

Figure 2. Skeletal Muscle Injury and Repair in VML…………………………………....8

Figure 3. Platform of Technologies for TE Skeletal Muscle…………………………....26

Figure 4. Keratin Structure.………………………………………………………….…..28

Figure 5. Growth Factor Release from 20% Keratin Gels………………………………30

Figure 6. TEMR Technology……………………………………………………………33

Volumetric Muscle Loss in a Mouse Model

Table I. List of Groups, Components, and Number of Animals (n)……….....................68

Figure 1. Surgical Flow of VML injury and Keratin Implant…………...........................69

Figure 2. Muscle Function Analysis at 2 Months Post-Surgery…………………...……74

Figure 3. Selected Force-Frequency Curves with Fitted Dose-Response Curves……....76

Figure 4. Representative Tissue Gross Morphology…………………………..…….…..77

Uninjured, NR, and BAM Treated Tissues…………………………………………...….79

KN, KN+I, KN+b, and KN+I+b Treated Tissues…………………..……………..……..81

KN+M, KN+M+I, KN+M+b, and KN+M+I+b Treated Tissues…………………..……83

Model of Volumetric Muscle Loss

Table I. Design of Experimental Groups Submitted to VML Injury…..........................103

Figure 1. Creation of VML in Rat TA and Injection of Keratin Hydrogel………….....106

Figure 2. Contribution of Keratin Hydrogel Formulation to Muscle Regeneration at 4 and

Table II. In Vivo Functional Analysis of TA Muscle at 8 Weeks Post-Injury…...….…113

Figure 3. Keratin Hydrogel Promotes Continuous Functional Recovery over Time…..115

Table III. In Vivo Functional Analysis of TA Muscle at 12 Weeks Post-Injury……....117

Figure 4. Gross Morphology and Histological Analysis of TA after Keratin Based

Cleft Lip Secondary Revision Procedures in a Rat Model

Table I. Treatment Groups……………………………………………………………..140

Data at 2 Months Post-Injury…………………………….……………………………..144

Data at 4 Months Post-Injury………………………………………………….……..…146

Data at 6 Months Post-Injury……………………………………………….………..…148