Lecture 12

Biopolymeric Materials: Polysaccharide

 Natural Resilin

Resilin in the wing hinge of a

dragonfly Tendon under white and UV light Sound-producing tymbal of a cicada

 An energy-storing, insect structural protein

 Found in specialized compartments of insect bodies
 Functions where efficient energy storage and repetitive locomotions are required

Weis-Togh, T., J. Exp. Biol. 1960, 37, 889; Elvin et al. Nature 2005, 437, 999. Li, L. and Kiick, K. L Polymer Science: A Comprehensive Reference, Vol 9.
2012.
 Recombinantly Synthesized Resilin-Like Polypeptides
 Resilin gene identification in 2001 (D. melanogaster CG15290)

 First recombinant resilin reported in 2005 (Rec1-resilin first exon of CG15920, in E. coli)

Rec1-resilin Dros 16
 First exon of CG15920 gene  (GGRPSDSYGAPGGGN)16
 D. melanogaster

Elvin et al. Nature 2005, 437, 999.

Kim et al. Protein Expression Purif. 2007, 52, 230.
 Recombinantly Synthesized RLPs
An16 Resilin-ChBD
 (AQTPSSQYGAP)16  Full native resilin protein of CG15920 gene
 an Anopheles gambiae homologue

Qin et al. Biomacromolecules. 2009, 10, 3227.

Lyons et al. Biomacromolecules. 2009, 10, 3009.

GB1-resilin based polyprotein RLP-ELP-CLP

 Titin-mimicking, passive elasticity of  Combining elasticity and strength
muscles

Bracalello et al. Biomacromolecules. 2011, 12, 2957.

Lv et al. Nature. 2010, 465, 69.
 Recombinantly Synthesized RLPs
Rec1-resilin
 UCST and LCST, dual-phase behavior
 pH sensitive photophysical properties
Dutta et al. Angewandte Chemie. 2011, 50, 4428.
Simulated resilin-sequences
 both sequences (natural, half charge, zero
charge, non-polar)
 polarity improves mechanical properties
Kappiyoor et al. Soft Matter. 2011, 7, 11006.
5
 Excellent Mechanical Properties of Resilin
 Unique Properties:
 Good fatigue resistance
 High resilience  Useful high-frequency responsiveness
 Efficient energy storage  Useful water solubility
 Low stiffness, soft

Natural Natural Vocal

elastin resilin
ELPs RLPs
fold
Non-polar 95% 66% N/A N/A N/A
Acidic N/A N/A
0.6% 16% N/A
residues
Modulus(Eint) 1.1MPa 0.6-0.9MPa 0.2-1.0MPa 25-40kPa 33kPa
Extensibility 90% 200% 90-500% 320% 30%-200%
94-100% High
Resilience 90% 93% 70-80%
(>95%?)

Resilin-like polypeptides as a new class of bio-elastomer in the engineering of mechanically active tissues
such as heart, blood vessels and vocal folds.
Weis-Fogh. J. Mol. Biol., 1961, 648; Li. et al. Polymer Chemistry, 2010, 1, 1160; Kutty, J. K. et al., Tissue Engineering: Part B, 2009, 15, 249. 6
Engineering an RLP-based Bio-elastomer

 Can resilin sequence be modified and retain useful elastomeric properties?

Vocal Fold Anatomy

Epithelium

Lamina Propria
(LP)

Vocalis Muscle

Courtesy of Drs W. Montgomery and J. Kobler

• LP: layered structure, extremely soft and pliable

• Fibroblasts in LP maintain the ECM
• Sustain high impact collisions up to 30% strain at 75-1000 Hz

Titze, I. R. J. Acoust. Soc. Am. 1989, 85, 901-906 .

 Modular Design of Resilin-like Polypeptides

GGGGDQK[(GGRPSDSFGAPGGGNGGRPSDSFGAPGGGNGGRPSDSFGAPGGGNGGK)2GGGRGDSPG]2GGPQ
GIWGQGGRGGCKAAKRPKAAKDKQTKGEDLGDPMASMTGGQQMG

GGRPSDSFGAPGGGN putative consensus motif from D. melanogaster

Lysines available for cross-linking
Modules added for biological functions:
 RGDSPG derived from fibronectin for cell adhesion via α5β1 intergrin
 GPQGIWGQ, MMP sequence from α(I) collagen from human for scaffold degradation
 CKAAKRPKAAKDKQTK, Heparin-binding domain for heparin binding and controlled delivery
and release of growth factors
Hersel, et al. Biomaterials, 2003, 24, 4385; Nagase, et al. Biopolymers, 1996, 40, 399; Liu, et al. Proc. Natl. Acad. Sci. 1997, 94, 1739; Yamaguchi et al, Biomacromolecules, 2005, 6, 1921.
Mechanically and Biologically Tunable RLPs

Independent control of biological domains decoupled from the mechanical properties

 Cross-linked RLPs Form Elastic Hydrogels

10000

1000
M odulus (Pa)

100

10 G'
G''
 Cross-over in less than 2 mins, fast gelation
1

allows in vivo injection

0.1
0 5 10 15
Time (min)
50 60
 Hydrogel formation complete after 10 min ,
Time sweep for in situ cross-linking of RLP12 50-fold difference between G’ and G’’
Li. et al., Biomacromolecules, 2011, 12, 2302; Lim et al. Biomacromolecules, 2008, 9, 222; Nie et al. Acta Biomaterialia, 2008, 5, 865.
 Tunable Elastic Shear Moduli of RLPs
-NH2:-OH
10wt% 15wt% 20wt% 25wt%
/[RLP12]
1:0.5 600±100 Pa
1:1 1275±120 Pa 1600±200 Pa 2800±500 Pa 4450±430 Pa
1:2 1850±320 Pa 4600±225 Pa 9300±630 Pa
1:4 6850±220 Pa
1:5 10600±165 Pa

10000

 Moduli encompass those reported for vocal

fold
M odulus (P a)

1000
 The solid-like behavior of these RLPs-
G' 1:0.5
G'
G'
1:1
1:2
based hydrogels is expected as the RLPs
G' 1:4

100
G' 1:5 are covalently cross-linked.
0.1 1 10 100
Angular frequency (rad/s)

Li et al., Biomacromolecules, 2011, 12, 2302-2310; Chan et al., J. Acoust. Soc. Am., 2004, 115, 3161-3170.
 Cyclic Stress-strain Characterizations
Bausch & Lomb, NY
Dr. Jeffery Linhardt

15000
30000
1:4

3 repeats 1:2
10000 30% strain 1:2 20000
Stress (Pa)

Stress(P a)
1:1
5000 10000
30%
60%
100%
0
0 0 20 40 60 80 100
0 10 20 30
Strain (%) Strain (%)

Various cross-linking ratios for 20wt% 1:2 cross-linking ratio for 20wt% RLPs
RLPs up to 30%, 60% and 100% strain
Li, L. et al., Biomacromolecules, 2011, 2302; Kutty, J. K. et al., Tissue Engineering: Part B, 2009, 15, 249.
Tensile testing: stress-strain cycles/hysteresis/resilience
20
ELP-based hydrogels
RLP
15 RLP+RLP-RGD
RLP-RGD+RLP-MMP
Stress (kPa)

10

0
0 5 10 15 20 25 30
Strain (%)
Wu et al., Biomacromolecules, 2008, 9, 1787

 Low energy loss, high resilience and negligible hysteresis for RLP-
based hydrogels compared with ELP-based hydrogels.

Li et al., Soft Matter, 2013, 9, 665; Li et al., Front. Chem. 2014, 2, 21.
 Dynamic Mechanical Testing: stress relaxation of RLPs
125
RLP
Elastin-like polypeptides
100 100% (VPGVG)n
Stress (kPa)

75 60%

50
30%

25
15%

0
0 3 6 9 12
Time (min) Wu et al., Biomacromolecules, 2008, 9, 1787

 Negligible stress-relaxation was observed indicating RLPs are elastomeric.

 RLP hydrogels behave almost like an ideal elastic solid, Immediate chain response to dynamic
mechanical loading
Li et al., Front. Chem. 2014, 2, 21.
 Attachment of hMSCs in 2D cell culture
100% RLP 100% RLP-RDG

50% RLP 100% RLP-RGD

50% RLP-
RGD hMSCs display
different
morphologies on
various RLP-based
hydrogels
 Encapsulation of hMSCs in 3D culture
Day 21 Z View
XY View

 Fast gelation enabled the homogeneous

distribution of cells.

 These cell-gel constructs were able to maintain

mechanical integrity and cell viability up to 3 weeks
20wt% polypeptide
50% RLP + 50% RLP-RGD

Li et al., Soft Matter, 2013, 9, 665-673.

 In vivo subcutaneous RLP hydrogel injection
Soluble RLP injection: 0.5wt%, 1wt%, 2.5wt% and 5wt% didn’t induce any inflammatory response

20x Muscle
Minimal
inflammatory
Dermis cells, tissues
Soluble RLP appear
injection histologically
Day 5 & 21 normal
Epidermis

RLP hydrogel in vivo injection: 10wt% mild inflammatory response

20x RLP hydrogel

Mild
inflammatory
response in
deep dermis,
accumulation
of neutrophils

Li et al., in preparation
 Carbohydrate and Polysaccharides
 Polysaccharide structure:
- Chain of sugars: CxHyOz
- Bonded by glycosidic bonds

 Function:
- Energy storage (e.g., starch and cellulose)
Glucose monomers
Humans have enzymes to digest
- In connective tissue, polysaccharides influence hydrophilicity
Glycosaminoglycans (GAGs)
- Cell surface receptors: polysaccharides on the surface of cells where they act
as “road signs” allowing molecules to distinguish one cell from another
 Glycosaminoglycans (GAGs)
 10% by weight of the ECM but fill most of space
 Unbranched polysaccharide chains
 Disaccharide subunits
 Amino sugar, highly charged, hydrophilic
 Swelling enables matrix to withstand compressive forces
 4 groups
 Hyaluronan (HA)
 chondroitin sulfate, dermatan sulfate
 heparan sulfate, heparin
 keratan sulfate
Structures and properties of common polysaccharides
 Carbohydrates: glucose, repeating unit

Glucose
 Stable six member ring
 Boat Conformation is possible
 Chair Conformation is more stable
 At equilibrium about 36% exists in the alpha
form and 64% exists in the beta form.
 The open chain form of D-glucose in water
is a very small percentage (0.02%)
Carbohydrates: Disaccharides
Carbohydrates: Polysaccharides
 Natural Polymers - Polysaccharides

Biomaterial applications for polysaccharides

 Glycosaminoglycans: Tissue engineering of load bearing tissue. GAGs are
mixed with collagen in degradable scaffolds to regenerate viable tissue.
 Heparin: Anti-coagulant for blood, angiogenesis

 Chitin: Unique properties to prevent infection and promote homeostasis

 Dextran: Injected to “tighten” ligament

 Hyaluronic Acid (HA) and alginate (AA): hydrogel tissue scaffold

GAG chains occupy large amounts of space and form hydrated gels
 Unbranched polysaccharide chains composed of repeating disaccharide units
 Acidic Polysaccharides
Hyaluronic Acid (HA) Alginate (AA)
β(1-4) linked 2-acetamide-2-deoxy-
D-glucose and β(1-3) linked D- (1-4) linked -D-mannuronic acid and
glucuronic acid -D-guluronic acid

CH3

OH O C
COO
OH HN
O O
HO O
HO O O
O O HO
O HO HO
NH OOC
C HO
O
CH3

n
• HA is known to be biocompatible, • AA is non-tissue reactive*
non-antigenic, non-inflammatory, • AA can be ionically crosslinked with
and generally non-tissue reactive Ca2+ to form a hydrogel
• Clinically used for viscosurgery • AA has been extensively researched
and viscosupplementation for tissue engineering applications
• Non-Newtonian polymer • Newtonian polymer
Hyaluronic Acid

 A linear non-sulfated glycosaminoglycan (GAG)

 Alternating units of D-glucuronic acid and N-acetyl-D-glucosamine
 Vitreous of the eye to the extracellular matrix of cartilage tissue
 Highly negatively charged, large water content, swelling pressure,
resist compressive forces in tissues and joints
 Biocompatible, biodegradable, bioactive, non-immunogenic and
non-thrombogenic
Hyaluronic acid has properties that are desirable for
tissue scaffolds
 6.4ug HA in each mg protein in vocal fold, major
component of VF ECM
 Naturally derived  Extensive H-bonding leads to high viscosity solution
 Non-immunogenic  Loosely coiled conformation structure of HA
 Non-adhesive functions as a shock absorber, acting as a tissue
 Bioactive damper that protects the vocal fold edges from the
oscillatory trauma during phonation
 Osmotic, viscoelastic and space-filling properties
affect the thickness and viscosity of VF
 HA interacts with VFFs and MSCs through various
cell surface receptors e.g., CD44
 Anti-inflammatory mediator (high molecular weight),
inhibit macrophage migration and aggregation
 Covalently Cross-linked HA Hydrogels
 Rapid degradation by hyaluronidase, 3-5 days after injection in vocal fold, unsuitable for tissue
engineering applications

Chemical Modification Necessary!

Burdick et al, 2011, Adv. Healthcare Mater., 23, H41-H56.

HA-based Hydrogels in Tissue Engineering Applications
HA-based Hydrogels in Tissue Engineering Applications

Silk fibroin + HA Sponge Electrospun Nanofibrous HA-based Hydrogel

 3D matrix for cartilage tissue  photo-crosslink to spatially control hydrogel patterns and
engineering tethered biomolecules to guide cell behavior

Foss et al, 2013, Biomacromolecules, 14, 38-47. Wade et al, 2015, Advanced Materials, doi: 10.1002/adma.201404993
Examples of photo-crosslinked HA scaffold structures
RLP+HA Chemically Photo-pattern-able Hydrogel

Photo-chemically cross-linked HA
 Pattern-able surfaces, scaffolds and
hydrogels
 Control substrate modulus
 Guided cell behavior for tissue
engineering

Khetan et al, 2010, Biomaterials, 31, 8228-8234; Gramlich et al, 2013, Biomaterials, 34, 9803-9811

Introducing Photo-triggered Crosslinking Chemistry to RLP

Acrylated RLP

Resilin-like polypeptide
Spatially controlled behavior of stem cells in 3D hydrogels
HA-MA Hydrogels: Blocks, Films, and Microcapsules

contain blue dextran (2 x 106 g/mol) for ease of

visualization (the gels are normally translucent)
Photo-crosslinked HA-MA, HA-MA/AA, and AA-MA hydrogels are stable for more than 4 years!
Repair of stroke infarct by HA hydrogel-encapsulated NPCs
Compression study of AA/HA hydrogel
Stress vs. Strain
40
– – – – – – H A H y d r o g e l
 – – – L V G H y d ro g e l

Compressive Moduli

at low strain
30
 Hydrogel E (Pa)
 6% AA-MA 20
S tre s s (k P a )


3% HA-MA 18
20
8% HA-MA 68

17% HA-MA 130


10


  



 
 
 
0 

0 20 40 60 80 100
S tr a in ( % ) U n iv e r s a l V 2 .6 D T A I n s tr u m e n t s

38
 Midterm Overview
 Biomaterials, Types, Structure Features, Properties (mechanical, biological, biocompatibility), think
about what features/properties are required to design a certain biomaterials for an application.
 Molecular forces in materials, (micro)structures, bulk versus interface
 Mechanical properties, stress-strain, Young’s modulus, stiffness, viscoelasticity, dynamic mechanics
 Mechanical properties on cell behavior and function, soft versus stiff, elastic versus plastic, how to read
rheological data, storage versus loss modulus
 Basic cell biology and extracellular matrix, know their structural features and biological functions.
 Biomaterials versus Cell ECM, think about the similarity and differences regarding designing materials.
 Basic polymer principles, types of polymers, repeating units, structure, calculate Mw, morphology
 Radical and condensation polymerization, give you monomers yield polymer structure, vice versa
 Polypeptide, features of repeating units, properties, thermal and mechanical
Natural Polymers - Proteins
• Strengthening mechanisms
• Covalent crosslinking of protein chains
• CHEMICAL: Decellularizes, anti-infection,
strengthens, makes non-biodegradable, cells
can’t penetrate, can cause foreign body
response (e.g., glutaraldehyde, carbodiimides,
polyepoxy)
• PHYSICAL: Heat treatments or compression to
make physical crosslinks without adding
chemical residues
• ENZYMATIC: Natural, cells can penetrate
• e.g., Transglutaminases (free amine and glutamine)
to get stable structures – clots, skin, hair
44