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**SCHREYER HONORS COLLEGE
**

DEPARTMENT OF BIOENGINEERING

THE NOVEL DESIGN OF A BIOREACTOR FOR IN VITRO PROLIFERATION AND

DIFFERENTIATION OF HUMAN MESENCHYMAL STEM CELLS

JOSHUA D. SALVI

Spring 2009

A thesis

submitted in partial fulfillment

of the requirements

for a baccalaureate degree

in Bioengineering

with honors in Bioengineering

Reviewed and approved* by the following:

Henry J. Donahue

Baker Professor of Cellular and Molecular Physiology

Thesis Co-Supervisor

Peter J. Butler

Associate Professor of Bioengineering

Thesis Co-Supervisor

William O. Hancock

Associate Professor of Bioengineering

Honors Adviser

* Signatures are on file in the Schreyer Honors College.

We approve the thesis of Joshua D. Salvi:

Date of Signature

_____________________________________ ______________

Henry J. Donahue

Baker Professor of Cellular and Molecular Physiology

Thesis Co-Supervisor

_____________________________________ ______________

Peter J. Butler

Associate Professor of Bioengineering

Thesis Co-Supervisor

_____________________________________ ______________

William O. Hancock

Associate Professor of Bioengineering

Honors Adviser

9-6939-2728

ABSTRACT

Continued use of autografts and allografts for bone-tissue therapy has serious

implications, including the decreased strength of such grafts in vivo over time [2]. A

novel solution to this problem is the use of tissue-engineered implants from the patient’s

bone marrow. Numerous techniques have already been used to build either two- or three-

dimensional scaffolds. Additionally, previous studies have demonstrated that oscillating

fluid flow-induced shear stresses will aid in the chemotransport among osteoblastic

lineages. In an exploratory series of studies, two-dimensional nanoscale substrates have

been analyzed with their flat counterparts under both static and flow conditions.

Furthermore, these protocols have been simulated through finite element analysis in

COMSOL by solving for various stresses encountered by cells under oscillating fluid

flow. Expanding these two-dimensional substrata to three-dimensional scaffolds,

progenitor cells, including human mesenchymal stem cells (hMSCs) and human bone

marrow stromal cells (hBMSCs), have been proliferated while maintaining their

differentiation potential into the osteoblastic lineage in vitro. These studies culminated in

the design of a three-dimensional bioreactor in which finite element analysis was used to

optimize the stress distribution and perfusion throughout the volume of a scaffold. The

purpose of these experiments was to explore the field of tissue engineering as it applies to

the musculoskeletal sciences. This reductionist approach to tissue engineering resulted in

the proposal of a new in silico method and an analysis of key parameters for successful in

vitro stem cell tissue culture.

TABLE OF CONTENTS

ABSTRACT .................................................................................................................................... 3

INTRODUCTION ......................................................................................................................... 6

Background .............................................................................................................................. 6

Bone Grafts ....................................................................................................................... 6

Skeletal Tissue Engineering Approaches .......................................................................... 7

Progenitor Cell Lines ...................................................................................................... 12

Finite Element Analysis ................................................................................................... 15

Substrates and Scaffolds.................................................................................................. 16

Bioreactors ...................................................................................................................... 18

Literature Review .................................................................................................................. 21

Two-Dimensional Substrata ............................................................................................ 21

Three-Dimensional Scaffolds .......................................................................................... 25

Bioreactor Designs .......................................................................................................... 30

Finite Element Analyses .................................................................................................. 34

Literature Review Summary ............................................................................................ 36

Proposal ................................................................................................................................. 39

Two-Dimensional Substrata ............................................................................................ 39

Three-Dimensional Scaffolds .......................................................................................... 39

Biophysical Stimuli .......................................................................................................... 40

Bioreactor Design ........................................................................................................... 40

Finite Element Analyses .................................................................................................. 41

Thesis Statement and Hypotheses ................................................................................... 43

MATERIALS AND METHODS ................................................................................................ 44

Nanoscale Substrate Fabrication ............................................................................................ 44

Salt Leaching and 3D Scaffolds ............................................................................................. 45

Cell Harvesting ...................................................................................................................... 46

Cell Culture ............................................................................................................................ 47

Oscillating Fluid Flow ........................................................................................................... 48

Fluorescent Markers .............................................................................................................. 49

Alkaline Phosphatase Assay .................................................................................................. 51

Substrate Characterization ..................................................................................................... 52

Simulation of the 2D Microenvironment ............................................................................... 53

Bioreactor Design .................................................................................................................. 55

Statistics ................................................................................................................................. 56

RESULTS ..................................................................................................................................... 57

2D Substrate Characterization ............................................................................................... 57

3D Scaffold Characterization ................................................................................................. 60

Mechanosensitivity of Stem Cells on Substrates ................................................................... 63

FACS Analysis ...................................................................................................................... 66

AP Activity ............................................................................................................................ 71

Finite Element Analysis of Cell Confluence ......................................................................... 74

Finite Element Analysis of Cell Height ................................................................................. 78

FEA of Cells Cultured on Various Nanotopographies .......................................................... 85

3D Bioreactor Design by the Finite Element Method ........................................................... 89

DISCUSSION ............................................................................................................................... 96

Two-Dimensional Substrata Characterization ....................................................................... 96

Findings from AFM Imaging ........................................................................................... 96

Drawbacks of Cell Height Estimates .............................................................................. 97

Three-Dimensional Scaffold Characterization ...................................................................... 98

Findings from SEM Imaging ........................................................................................... 98

Drawbacks of Porosity Calculations ............................................................................... 99

Stem Cell Growth on 2D Substrates .................................................................................... 101

Mechanosensitivity of Stem Cells .................................................................................. 101

Proliferation and Differentiation Potentials ................................................................. 102

Alkaline Phosphatase Activity ....................................................................................... 103

Stem Cell Growth on 3D Scaffolds ..................................................................................... 105

Alkaline Phosphatase Activity ....................................................................................... 105

Comparison with 2D Substrata ..................................................................................... 106

Finite Element Analysis ....................................................................................................... 107

Discussions of Cell Confluence and Hydrophobicity .................................................... 107

Benefits over Empirical Data Collection ...................................................................... 109

Benefits of Oscillating Fluid Flow ................................................................................ 109

Summary of FEM as a Tool in Cell Culture .................................................................. 110

Bioreactor Design ................................................................................................................ 111

Satisfaction of Design Criteria and Specifications ....................................................... 111

Comparison of Two Models .......................................................................................... 113

Analysis of Design ......................................................................................................... 114

CONCLUSIONS ........................................................................................................................ 116

Methods of Biomaterial Characterization ............................................................................ 116

Ability of Substrates and Scaffolds to Regulate Stem Cell Activity ................................... 118

Finite Element Method as a Novel Tool in Tissue Engineering .......................................... 120

Summary of Bioreactor Properties Key for Success ............................................................ 122

Closing Remarks .................................................................................................................. 124

REFERENCES ........................................................................................................................... 127

APPENDICES ............................................................................................................................ 132

Appendix A: Acknowledgments .......................................................................................... 132

Appendix B: Health Insurance Portability and Accountability Act (HIPAA) ..................... 133

Appendix C: Funding Sources ............................................................................................. 134

Appendix D: Supplemental Sketches .................................................................................. 135

Appendix E: Summary of Common Tissue Engineering Growth Factors ........................... 137

Appendix F: COMSOL Model Reports ............................................................................... 138

Appendix G: Academic Vita ................................................................................................ 208

INTRODUCTION

Background:

Bone Grafts:

Ten years ago, there were 650,000 reported cases of bone allograft transplantation

[9]. Bone transplantation is key in rebuilding diseased tissues. These include the hips,

shoulders, knees, and spine. Grafts are also key in the repair of bone loss from fractures

or cancers [10]. Allografts are tissue transplantations between individuals of the same

species. In this case, bone is grafted from one human being to another. Autografts, on the

other hand, are grafts of tissue from one location on an individual to another location of

that same individual. Autografts are far less common than allografts, due mainly to the

constraints of individual patients. For example, osteoporotic bone is virtually useless in

autograft transplantation, and allografts are thus necessary. Xenografts, methods rarely

used in orthopedic surgery, involve tissue grafts from one species to another. Bone

allografts are the most common due to their osteoconductive properties and the inclusion

of osteogenic factors that induce bone tissue growth [9]. These factors include bone tissue

progenitors (such as mesenchymal stem cells), osteoblasts, and osteocytes.

Allografts are harvested either from a living donor or cadaveric sources within 24

hours of death [9]. Key components of this harvesting include the maintenance of cell

viability and prevention of allograft infection. This is not to say that current methods are

perfect, but they have been successful nonetheless. Risk of HIV infection is very low, at a

rate of 1 in 150,471. If lymph nodes are tested prior to transplantation, this rate has been

reduced to as low as 1 in 1.67 million [9]. There have also been cases of HCV

transmission, though these were rare. Though current sterilization methods are not

6

perfect, grafts rarely lead to disease transmission. Nonetheless, a viable alternative

without such transmission, similar to the functionality of autografts, would be preferred.

Other factors affect allograft performance. With continued use in vivo, allografts

lead to mechanical failure of bone. Using human models, it has recently been

demonstrated that a decreased bone mineral density, degradation of material properties

(such as elasticity), and increased numbers of micro-fissures resulted with time. These

data were correlated to the 60% failure rate of allografts within 10 years after

transplantation [2].

These structural anomalies are a function of age. The donor must be 45 or

younger in order to decrease failure rates. Furthermore, additional tissue processing for

sterility (e.g. freeze-thaw cycles) increase failure rates [9]. These data can be correlated to

a trade-off between preventing transmission of disease and preventing mechanical failure.

Thus, current bone graft methods are limited by the lack of donors, possible

disease transmission, and known mechanical failure with time. A tissue engineering

method incorporating autograft principles but assuring the osteogenic potential would act

as one viable solution.

Skeletal Tissue Engineering Approaches:

When considering the potential for tissue regeneration by tissue engineering, a

unique example can be found in nature. Amphibian limb models are classic in that they

demonstrate that a limb can be naturally regenerated after injury. The unique factor in

these regeneration models is the initial development of a regeneration blastema prior to

continued development of the limb. After inducing injury in a number of amphibians,

7

ectopic blastemas followed by ectopic limbs were witnessed within 60 days post-wound.

Furthermore, if a skin graft was added to the wound location, amphibians demonstrated a

73% rate of limb regeneration [11]. One model is illustrated in Figure 1.

Figure 1. The Stepwise Model for Limb Regeneration [11].

Shown in Figure 1 is a stepwise model for limb regeneration in amphibians. After

the injury, the key factors in limb regeneration (as opposed to skin regeneration alone or

blastema regression) include continued proliferation as signals from nerves and

fibroblasts are prevalent. The three pathways include wound healing (as seen in humans),

bump formation, and complete limb regeneration. Note that dedifferentiation to

progenitor cell lines was required in order to form the regeneration blastema. These

phenomena in limb regeneration remain mysterious by mechanism, but the key factor of

utilizing one’s own cells to regenerate tissue becomes the focal point of skeletal tissue

engineering [11].

In response to the phenomena, orthopedists have developed a number of methods

in skeletal tissue engineering, specifically to combat current issues with bone grafts [9].

8

Among the many strategies in skeletal tissue engineering, there are three basic

approaches to tissue engineering [12]:

1. In situ Regeneration of Skeletal Tissue

2. Isolation and Culture of Cells, Followed by Reimplantation

3. Combination of Cells and Scaffolds to Implant a Bone-Like or Cartilage-Like

Construct

The in situ regeneration is similar to that encountered in amphibian limb models,

where a graft or scaffold will stimulate the growth of skeletal tissue. The second method,

however, involves in vitro culture of progenitor cells, typically harvested from human

bone marrow. These aggregates are then injected into the patient without an

accompanying scaffold. Finally, the third and most popular approach uses a complete

three-dimensional model of bone or cartilage. However, the cells within the scaffold must

have reached maturity prior to implantation [12, 13].

Considering two-dimensional and three-dimensional biomaterials specifically,

there are numerous biomaterial factors that can influence cell-surface interactions. Within

seconds to minutes, cellular fluid adhesion is affected by surface wettability, and protein

adhesion can be affected by local pH, ionic composition, and temperature. Cell

attachment is then influenced by van der Waal’s forces. Finally, hours later, cell adhesion

and spreading are affected by matrix proteins and cytoskeleton proteins [12]. Two key

factors in cell-surface interactions include surface topography and surface chemistry.

Surface chemistry is quantified through wettability, zeta potential, and elemental

composition (i.e. ESCA). However, the focus of the following studies is surface

topography. Osteoblastic cells significantly respond to surface morphology, and surface

9

roughness studies result in controversial conclusions. Lithographic methods and

numerous microscale substrates resulted in conflicting results, and further data are

required in order to obtain meaningful conclusions [12-14].

Similar methods and progenitor cells from bone marrow have been involved in

myocardial regeneration. These approaches have also taken advantage of scaffold

structures similar to chondrocyte repair. Though the final product may vary greatly, it

was demonstrated that the same progenitor cell lines can be used for numerous

applications [15].

The coupling of these approaches can be found in the development of bioreactors

for skeletal tissue engineering. Prior to developing a bioreactor, multiple cell lines,

substrates, and growth factors must be analyzed for potential differentiation and

proliferation in vitro. Furthermore, the bioreactor design is not the end result. Bone tissue

constructs must then be analyzed through implantation studies prior to the development

of a final product [16]. These approaches are summarized in Figure 2.

Figure 2. Outline of the Tissue Engineering Process [16].

10

When designing a bioreactor, one must consider how nutrients and growth factors

will be supplied to cells in culture. Furthermore, perfusion seeding of scaffolds may be

required if a three-dimensional scaffold is involved. An analysis of intercellular and

intracellular interaction of cells cultured in these scaffolds is also required. Nonetheless,

the bioreactor system must account for multiple scaffold types, cell types, and scaffold

dimensions. These reactors have included tissue culture flasks, agitated vessels, packed

beds, fluidized beds, and membrane bioreactors [9, 12, 13, 16-18]. A systems view of

bioreactor designs can be found in Figure 3.

Figure 3. A Systems View of the Ideal Bioreactor in Tissue Engineering. Note the number

of factors that must be considered in the design of these systems [16].

11

Progenitor Cell Lines:

Stem cells are unique in that they can continue to proliferate for long periods of

times, are unspecialized, and can differentiate into multiple lineages. Human embryonic

stem cells (hESCs) are harvested from a blastocyst initiated by in vitro fertilization [19].

The focus of this study, however, is on adult stem cells. Specifically, human

mesenchymal stem cells (hMSCs) are harvested from umbilical cord blood, bone

marrow, or fat in somatic tissue. There are two types of adult stem cells. Hematopoietic

stem cells give rise to erythrocytes, leukocytes, and platelets in the blood. However,

human bone marrow stromal cells (hBMSCs) are non-hematopoietic, meaning that they

do not give rise to blood cells. Instead, stromal cells differentiate into osteoblasts,

chondrocytes, adipocytes, and other connective tissue. Plasticity is the phenomenon by

which stromal cells harvested from one tissue type can differentiate into the lineage of

another phenotype. For example, bone marrow stromal cells can differentiate into bone,

cardiac muscle, and skeletal muscle lineages [20]. The differentiation pathways

illustrating such transdifferentiation are illustrated in Figure 4.

12

Figure 4. Comparison of hematopoietic (Top) and stromal (Bottom) stem cell

differentiation [20].

Considering the construction of filaments in the extracellular matrix, the common

pattern witnessed is that of branching morphogenesis. Compare this to the branching of a

tree, where continued growth leads to a fairly complex matrix of numerous branches.

This morphogenesis becomes key in the differentiation of progenitor cells, whereby the

intercellular space becomes complex in a three-dimensional matrix. In skeletal tissue

engineering, mimicking this branching morphogenesis in vitro through the creation of a

three-dimensional scaffold on which progenitor cells are cultured is key.

In bone, the branching of tissue by the dynamic nature of osteoblastic growth and

osteoclastic decay leads to a lighter and more structurally sound system. In fact, such

properties also lead to improved diffusion across the mesenchyme [21, 22]. However,

another strong consideration is the method by which differentiation occurs.

13

Initially, pluripotent stem cells can differentiate to the myoblast, fibroblast,

chondrocyte, adipocyte, or osteoblastic lineages. Initially, however, these cells must

commit as a progenitor cell that can then differentiate to all but the myoblastic and

fibroblastic lineages. After the addition of osteogenic media with synthetic

gluococorticoid, dexamethasone, and bone morphogenic protein (BMP-2) enhances

differentiation potential to the osteoblastic lineage. These osteoblasts can then further

mature to bone lining cells or osteocytes [23]. The aforementioned differentiation

pathways are illustrated in Figure 5.

The major issue with tissue engineering, however, has been that continued

expansion of progenitor cells in vitro significantly reduces their differentiation potential.

Tissue engineering techniques thus attempt to mimic the in vivo environment of bone in

order to maximize this differentiation [21, 24, 25]. This technique typically requires some

combination of growth factors, biomaterials, and biophysical signals witnessed in vivo.

Figure 5. The Multiple Differentiation Pathways for Osteoblasts [22].

14

Finite Element Analysis:

When considering the biomechanics of bone, orthopedists and biomedical

engineers often turn to structural analyses by the finite element method (or finite element

analysis, FEA). FEA allows for the discretization of complex geometries, granting much

greater flexibility in the study of mechanics than finite volume methods (FVM). The key

difference between FEA and FVM is the analysis by nodes or volumes, respectively.

Typically, finite element analyses require the use of computer technology for discrete

approximations [26].

FEA is useful in solving complex differential equations by first denoting a

number of finite elements or nodes and then approximating between them. First, consider

any differential equation. For our purposes, let this be a second-order differential

equation in one dimension. This equation can be generalized to a decomposition of

I × u = ¡, where L is some linear operator. Using dot products, we can then state the

function I(:) = (I:) · : - 2(¡ · :), and I(u) is the minimum of I × u = ¡. The next

phase then uses an arbitrary variable in order to determine that (Iu) · : = ¡ · :. We can

then solve for this equation instead of solving for I × u = ¡. This equation is known as

the Galerkin form, and it now requires that L only be a stationary point as opposed to a

linear operator. This method for solving equations by nodal approximations becomes key

when solving for complex geometries [26, 27].

As mentioned previously, finite element analysis requires the use of

computational methods. In this particular study, COMSOL Multiphysics (formerly

FEMLAB) has been used. COMSOL is unique in that it can not only analyze complex

geometries by finite element analysis, but it can also couple multiple physics modules

15

into a single geometry. The program was initially developed by Germund Dahlquist at the

Royal Institute of Technology in Stockholm, Sweden [28]. This created the ability to

couple structure stress-strain analyses with the fluid mechanics of a geometry as

implemented by steady-state Navier-Stokes equations [29].

A sample application of finite element analyses to studies of skeletal tissue

engineering is that of bone remodeling. In the morphogenesis of skeletal tissue, bone

undergoes changes of mechanical properties and changes of relaxed lengths. These are

typically studied independently, but FEMLAB (the predecessor to COMSOL) allowed

the use of multiphysics to study them simultaneously. By using microspin velocity as the

rate by which bone remodeling occurs, and noting both the current state and relaxed state

of each element, constitutive equations could then be coupled. Finally, these models were

able to find the anisotropic elasticity with respect to time. Though not described in detail

here, this simulation exemplifies an excellent application of the finite element method to

studies of skeletal tissue remodeling [30].

Substrates and Scaffolds:

As mentioned previously, the key issue in the continued expansion of progenitor

cells in vitro is mimicking the in vivo environment. Typically, cells are often cultured on

flat surfaces, such as plasma-cleaned glass, polystyrene, or quartz slides. Not

surprisingly, cells cultured on these substrates lose their differentiation potential with

continued expansion.

To combat this issue, biomedical engineers have fabricated a number of two-

dimensional substrates that mimic the in vivo environment. The first method, the one

16

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17

Though demineralization improves allografts and autografts after implantation,

mineralization becomes key when analyzing three-dimensional scaffolds. As mentioned

previously, these scaffolds provide a matrix upon which progenitor cells can grow into

mature bone. This construct is then re-implanted into the patient from whom bone

marrow is derived. Electro-spun nanofibers, hydrogels, carbon nanotubes, and a

multitude of other materials and approaches have been used in the development of

scaffolds. However, it has been demonstrated recently that mineralization by calcium and

phosphorous-containing materials provide additional support to the scaffold and induce

further growth of osteoblasts [33]. These properties thus become key in the development

of scaffolds.

Bioreactors:

The bioreactor provides a sterile environment for in vitro cell culture. The goal of

the design is to mimic in vivo conditions, thus improving proliferation and differentiation

of mesenchymal stem cells. Skeletal tissue engineering bioreactors include those with

fluid flow or mechanical stretching [34-37]. These provide dynamic mechanisms for the

differentiation into numerous lineages.

In a myriad of past studies, static cultures were involved. Though static cultures

have been simple to implement, these cultures result in non-homogeneous cell

distributions. Additionally, extracellular matrix proteins are not deposited uniformly,

adversely affecting the biomechanical properties of cells cultured in three-dimensional

scaffolds. This phenomenon is expected, since cells in vivo are grown under mechanical

18

loads by interstitial fluid flow [34]. As a result, implementation of biophysical signals

will be integral in this study.

Spinner flask bioreactors, on the other hand, use a magnetic stir bar to thoroughly

mix the media. Cells cultured in these bioreactors demonstrated increased proliferation

and uniformity of distribution. However, the spinner flask bioreactor was not the optimal

device for in vitro osteogenesis due to decreased cell adhesion under turbulent flow

patterns [34].

Originally developed by NASA, the rotating wall vessel (RWV) bioreactor

implements two concentric cylinders, with the outer cylinder rotating. Scaffolds then

float in the annular space after centrifugal forces and gravitational forces are balanced.

Therefore, the culture conditions are microgravity-like. This bioreactor system is

excellent for inducing chondrogenesis, so it is widely involved in cartilage tissue

engineering. However, the rotating wall vessel bioreactor does not significantly induce

osteogenesis. One factor discovered by NASA, though, was that turbulence in bioreactors

is unfavorable. Though turbulence increases mixing in bioreactors, such fluid flow

properties significantly reduce cell activity. Thus, this bioreactor functions through

laminar flow profiles [34, 38].

Flow perfusion bioreactors are models very similar to the one developed in this

study. These devices perfuse media through scaffolds with a pump mechanism and can be

used for uniform seeding of cells in three-dimensional scaffolds. Additionally, flow

perfusion bioreactors improve mass transport throughout the interior of the scaffold,

unlike the spinner flask and rotating wall vessel models. Furthermore, fluid shear stress

has been demonstrated to induce osteogenesis in vitro. Studies comparing static and

19

perfused scaffolds in a flow perfusion bioreactor found that calcium deposition

significantly improved in these devices. Since interstitial fluid flow is a key component of

in vivo bone growth, this bioreactor acts as a significant improvement over the

aforementioned models [16, 34, 39].

Finally, continued mechanical loading of cells is believed to significantly improve

mass transports of nutrients throughout the interstitial space of the scaffold. For example,

bioreactors have induced mechanical loading by oscillating fluid flow, substrate bending,

longitudinal stretching, and compressive loading [34, 40].

20

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22

Upon using the polymer demixing method, it was found that various

weight/weight ratios would result in very different topographical features. When poly(L-

lactic acid)/polystyrene demixing was performed, increased ratios of polystyrene would

result in a topography more similar to nanopits rather than nanoislands (Figure 8). After

demixing, poly(L-lactic acid) would segregate to the upper surface of the polymer film,

mimicking similar culture dishes. After analysis by XPS and contact angle (wettability),

it was found that the PLLA/PS 70/30 w/w resulted in the most in vivo like conditions [1].

When human fetal osteoblastic cells (hFOB) were cultured on each of these

surfaces, both cell morphology and adhesion were measured. Additionally, these

substrata were compared with both flat poly(L-lactic acid) and polystyrene surfaces. In

doing so, it was thus determined that cell area was significantly greater (p < 0.01) on both

the poly(L-lactic acid)/polystyrene 70/30 and 90/10 w/w substrata when compared with

the flat controls. Furthermore, cell adhesion was also significantly greater (p < 0.01) on

these same substrata when compared with the flat controls. These demixed films

correspond to nanoislands as opposed to the nanopits illustrated in Figure 9.b. These data

demonstrate that osteoblastic cells will preferentially grow and adhere to various surfaces

based upon surface topography alone. This conclusion corresponds well with tissue

engineering concepts described previously in the background [1].

Nanoscale surface characteristics were further characterized by integrin

expression and osteopontin regulation in hFOBs cultured on nanoscale substrata [47]. It

was discovered that surface topography and wettability influence osteoblastic

extracellular matrix protein expression [47]. Furthermore, additional studies confirmed

that nanoscale surface topography positively influences cell morphology and adhesion

23

when other biomaterials were used [1, 45, 47-52]. The key issue, however, was that cell

activity and both proliferation and differentiation potentials were not measured in these

experiments. Though adhesion may be increase significantly, proliferation and the

maintenance of differentiation potential in vitro are the true keys to skeletal tissue

engineering. Without such data acquisition, it cannot yet be concluded that these substrata

are in fact beneficial to osteoblastic differentiation of human bone marrow stromal cells

and other mesenchymal progenitors.

However, other studies have demonstrated that inducing biophysical signals via

oscillating fluid flow may increase proliferation and possibly differentiation potential.

The data in support of this theory simply used flat substrates as opposed to the

aforementioned nanoscale topographies. It was demonstrated that calcium signaling and

upregulation of the MAP kinases ERK 1/2 result from oscillating fluid flow-induced

shear stresses [8, 39, 53-56]. Again, polymer demixed films were not involved in these

experiments. It should also be noted that a similar study analyzed the elastic moduli of

hFOBs cultured on numerous nanoscale substrata using contact-mode atomic force

microcopy (AFM). It was concluded that cells cultured on 11-38 nm nanoislands

displayed significantly greater elastic moduli when compared with those on flat

polystyrene or plasma-cleaned glass surfaces [57]. Intuition would then beg the

hypothesis that some combination of induced biophysical signals and surface topography

would result in increased apparent shear stresses due to the increased elastic moduli of

cells cultured on these substrata. Thus, such a combination may in fact provide a more

versatile and adept method for maintenance of stem cell differentiation potential with

continued expansion in vitro.

24

Three-Dimensional Scaffolds:

Though it has been previously demonstrated that two-dimensional nanoscale

topographies increase focal adhesion and expression of extracellular matrix proteins, it

has been widely disputed that three-dimensional scaffolds provide a more adept

microenvironment due to increased surface area and a better matrix for bone growth in

skeletal tissue engineering. Numerous studies have analyzed such scaffolds, but none of

the scaffolds to be mentioned involve a nanotopographic substrate as a comparative

measure along with induced biophysical signals to promote progenitor cell activity [4].

Figure 10. Structures of PEG4600DM (A), PEG526MMA-nLA-fluvastatin (B), acrylated-

PEG3400-RGDS (C), and methacrylated heparin (D) used for hydrogel fabrication [4].

As mentioned previously, bone morphogenic protein-2 (BMP2) becomes

necessary in the commitment of progenitor cells to the osteoblastic lineages (Figure 5).

Recent studies in multiple laboratories have used PEG hydrogels to create a scaffold via

25

copolymerization (Figure 10). Note that the structures of these polymers have a high

molecular weight and a high density of ester functional groups. These become key in the

interaction with BMP2. The PEG and heparin copolymers noncovalently bonded to the

BMP2, resulting in a more adept method for controlling a sustained level of osteogenic

growth factors within the scaffold [4]. As a result, these data were correlated to increased

differentiation potential of progenitor cells into the osteoblastic lineage in vitro on these

three-dimensional hydrogels.

However, hydrogels can be expensive to fabricate and maintain, and other simpler

scaffolds have also been thoroughly explored. Another example was the development of

collagen and collagen-hydroxyapatite scaffolds. The key concept in the development of

these three-dimensional structures was the utilization of biomaterials inherent in the

extracellular matrix of bone. Scanning electron microscopy images and pore sizes of

these two scaffolds are depicted in Figure 11.

Figure 11. Collagen (A) and Collagen-Hydroxyapatite (ColHA; B) scaffolds. ColHA

frozen at -30°C (C) and -80°C (D), along with porosities (E).

26

Note that most pores were on the order of 50 to 200 microns, near the range of

marrow stromal cells (approximately 20-40 microns). After culturing osteoblastic cells on

these scaffolds, it was discovered that the collagen-hydroxyapatite scaffolds promoted

osteoconduction both in vitro and in vivo [5]. However, these scaffolds must be stored at

-30°C or -80°C, significantly decreasing their versatility in tissue engineering. The

concept of utilizing physiologic biomaterials will be applied in this study, but a scaffold

with more versatile properties would act as an improvement to the aforementioned

scaffolds.

A novel method for development used peptide-amphiphile (PA) molecules to

create a self-assembled network of nanofibers. The concept of electrospun nanofibers has

been popular recently, but these scaffolds tend to have very little three-dimensional

depth; in fact, it is argued by this investigator that these fiber networks resemble more of

a two-dimensional sheet rather than a three-dimensional scaffold. However, cells cultured

on these nanofiber networks displayed a significantly greater level of mesenchymal

progenitor cell proliferation when compared with two-dimensional tissue culture plates.

Furthermore alkaline phosphatase activity was also significantly increased on these

networks when compared with the two-dimensional plates. Again, note that these plates

were basic tissue culture substrates, and the networks were not compared with other

scaffolds nor with multiple surface topographies. Finally, osteocalcin was significantly

increased on these networks [58-64]. Osteocalcin is a key hormone in the development of

the extracellular matrix in skeletal tissue engineering. Though the nanofiber networks

provide a significant improvement over tissue culture plates, it must be noted again that

27

these networks are not truly three-dimensional scaffolds when compared with the other

aforementioned scaffolds.

Some studies delve into comparisons between scaffolds, but these comparisons

typically only involve commercially-available scaffolds. For example, one study

compared a β-tricalcium phosphate ceramic with an open-celled poly(lactic acid) foam.

Poly(lactic acid) has been approved by the Food and Drug Administration for therapeutic

use due to its biodegradability. Typical quantification methods in these studies analyzed

alkaline phosphatase, osteopontin, and osteocalcin through assays [65]. However, all of

these were in static culture, and there was only comparison between commercially-

available three-dimensional scaffolds. None of the studies compared these scaffolds with

unapproved biomaterials or two-dimensional substrata. This study will attempt to

compare multiple scaffolds and substrata.

Many tissue engineers are now beginning to grasp the concept that cell seeding is

enhanced through the application of a bioreactor. One example is the use of poly(DL-

lactic-co-glycolic acid) porous scaffolds to enhance osteoblastic proliferation in vitro.

Cell seeding was significantly enhanced by the utilization of a spinner flask bioreactor

[3]. As mentioned previously, however, these bioreactors do not provide optimal fluid

flow patterns. Poly(DL-lactic-co-glycolic acid) scaffolds also demonstrate yet another

opportunity to use porous polymers in a three-dimensional scaffold as opposed to

physiologic biomaterials such as collagen or collagen-hydroxyapatite. Furthermore, the

polymer used in this method was also approved by the FDA, so a similar biomaterial

would thus be preferred in the development of the scaffold in this particular study.

28

Putting biomaterial properties aside, a key component in the fabrication of

optimal three-dimensional scaffolds is the mesh size preferred by osteoblastic cells and

their progenitors. Using a titanium fiber mesh, it was determined that mesh size and

porosity does, in fact, affect cell responses to growth on the scaffolds. Under both static

and flow conditions, alkaline phosphatase activity was significantly altered between 20

micron and 40 micron average pore sizes. Furthermore, mineralization and osteopontin

secretion were also significantly affected by mesh sizes in the corresponding scaffolds.

Figure 12.A depicts the titanium mesh after four days of culture. By day 8 in culture

(Figure 12.B), mesenchymal stem cell concentration began to increase. Finally, day 16

(Figure 12.C) of culture demonstrates a confluence of cells and mineralization within the

construct [66].

Figure 12. Titanium/MSC constructs after static culture.

The three-dimensional scaffolds described here demonstrate that numerous

methods were used analyzing various biomaterials and porosities. However, studies have

29

yet to compare many types of scaffolds with their two-dimensional counterparts while

inducing biophysical signals in vitro.

Bioreactor Designs:

One of the most common methods for seeding cells in three-dimensional scaffolds

is by the use of flow perfusion bioreactors. Each of these reactors are key for initial

scaffold seeding, but a bioreactor with oscillating fluid flow has yet to be devised.

Detailed below are a few of these bioreactor designs and their respective qualities.

A flow perfusion bioreactor typically involves some flow chamber with

unidirectional flow from a pump that will perfuse media with cells through the path of

least resistance. An example of this bioreactor is detailed in Figure 13 below [3].

Figure 13. Schematic of a standard flow perfusion bioreactor. Note that no flow occurs

around the outer perimeter.

The bioreactor detailed above simply involved the basic design of the reactor

system and peristaltic flow loop [3]. However, the seeding efficiency was not analyzed,

begging the question as to whether or not the aforementioned system was truly

advantageous over other similar bioreactor systems. Does the flow and seeding in a

perfusion bioreactor provide uniform seeding density? Does the modification of scaffold

30

porosity affect the perfusion or shear stresses throughout the scaffold while perfusing

media through the scaffold? These questions have not yet been answered by Bancroft in

his preliminary design studies.

Years prior to the design of the bioreactor by Bancroft, many tissue engineers

developed small flow chambers for two-dimensional substrata. In essence, these were

also similar in that they perfused media through the system. Furthermore, the study also

analyzed continuous unidirectional flow over bone marrow stromal cells in culture. Note

that this flow is not oscillatory as occurs in vivo, but the use of continuous flow in vitro

still provides some mechanism for improvement over static culture. Many flow loops

have a tendency to simply used peristaltic pumps in a unidirectional fashion, but

oscillatory fluid flow was rarely used in these studies [3, 67].

Using highly porous collagen microspheres for long term in vitro culture of bone

marrow cells in an arguably two-dimensional bioreactor system, biophysical stimuli were

maintained in the bioreactor system under unidirectional peristaltic flow. In this particular

case, “long term” flow referred to culture of murine bone marrow cells over a four month

period. The bioreactor system provided a significantly beneficial environment over

traditional static culture in flasks. The authors of the study pointed out, however, that

maintenance of biophysical and biochemical stimuli was not as successful as would be

preferred. Future bioreactors should incorporate such maintenance, and other possibilities

also include an improvement of culture media or the growth factors involved in the

experiments [67].

Additional studies then analyzed the seeding and differentiation of human

mesenchymal stem cells (hMSCs) in unidirectional peristaltic flow systems with a

31

chamber similar to that constructed in the previous study. The goal was to analyze the

effects of flow rates and then the capability for differentiation with time in vitro [7].

The scaffolds analyzed in these studies were PET matrices, which were in fact

two-dimensional meshes as opposed to three-dimensional scaffolds. Most perfusion

bioreactor systems, aside from being peristaltic unidirectional as opposed to continuous

oscillatory flow, have typically involved near-two-dimensional matrices instead of three-

dimensional alternatives. After setting up the flow loop, it was determined that there

exists an inverse relationship between flow rate and seeding density. This is to say that

lower flow rates result in much greater seeding densities. However, cells did not begin to

proliferate significantly until after the first four weeks had passed, as depicted in Figure

14 below [7].

Figure 14. Cell density seeded on three PET scaffolds with time in a peristaltic flow

chamber [7].

The bioreactor studies with PET meshes demonstrated that faster seeding kinetics

resulted in perfusion systems as opposed to static culture. Furthermore, differentiation

32

into the osteoblastic lineage was significantly enhanced in these two-dimensional

systems. Please note that nanoscale topographies, oscillating fluid flow-induced

biophysical signals, and primary donor cells were not used in these experiments. Such a

lack of analysis leaves a large gap open for study when compared with numerous studies

[7, 66, 68-78]. As a result, we must consider alternative bioreactor systems.

Nonetheless, this perfusion bioreactor system was quite successful in its attempt

to culture cells to confluence and induce differentiation into the osteoblastic lineage. An

example of the PET mesh after culture in the perfusion bioreactor is detailed in Figure 15

below. After 40 days of culture, a dense cellular mass was witnessed under scanning

electron microscopy. It can thus be determined that bioreactor systems provide numerous

advantages over classic static culture in flasks [7].

Figure 15. SEM image of PET scaffold with hMSCs cultured for 40 days [7].

As can be seen, numerous flow perfusion bioreactor systems have been

developed. However, these systems involved unidirectional flow as opposed to the

oscillatory flow occurring in vivo. Additionally, many bioreactors tend to implement two-

dimensional scaffolds as opposed to true three-dimensional constructs. By maintaining

33

biophysical and biochemical signals in vitro, a novel bioreactor design would better

mimic the in vivo environment through oscillatory flow and three-dimensional constructs.

Finite Element Analyses:

Mentioned before, finite element analyses are integral in the estimation of

physical properties in more complex geometries. In tissue engineering, these analyses are

more common in the simulation of numerous bioreactor systems, specifically spinning

flask bioreactors and perfusion flow bioreactors. However, fluid flow over cells cultured

on nanoscale topographies has yet to be analyzed through finite element methods.

Regarding the simulation of flow perfusion bioreactors, the most common method

used is the Lattice-Boltzmann method, where physical three-dimensional space is broken

into a number of nodes. The Lattice-Boltzmann method is very similar to the method

described previously. The important factor to consider, however, is that fluid flow

simulations with the Lattice-Boltzmann method simplify the Navier-Stokes equations to a

second-order set of equations and assume that all fluids are Newtonian fluids. As a result,

the calculations with these methods are very rough estimates of the reality at hand [79].

The methods used in literature were successful in the simulations of velocity

fields through bioreactors, and shear stresses were coupled with velocities at the solid-

fluid interface. In coupling empirical data regarding scaffold properties and then

analyzing flow fields, the flow field properties existing within the flow perfusion

bioreactors could thus be estimated. An example simulation is detailed in Figure 16

below. The upper image depicts the transverse velocity field through a three-dimensional

scaffold in mm/s. The simulation image on the bottom of Figure 16 displays the same

34

three-dimensional scaffold, but with a top view [79]. Note, again, that the velocity

gradients can then be correlated to shear stresses.

Figure 16. Velocity fields in a flow perfusion bioreactor as estimated by the LB method.

The upper image depicts transverse flow through a scaffold from a side view, and the

bottom image depicts the same flow from a top view [79].

Finite element analyses become key in the design of an optimal bioreactor system.

Many tissue engineers have failed to perform these simulations prior to bioreactor design,

much unlike the approach taken in this study. Furthermore, simulations have been limited

to bioreactors in the literature. One possibility would be to examine the biophysical

signals experienced by cells on multiple nanoscale substrata.

35

Literature Review Summary:

The table below is meant to compare many of the current techniques used in bone-tissue engineering. Notice that a single study

does not use both 2D and 3D substrates while coupling these with biophysical signals. Additionally, the use of computer simulations

(finite element analysis) is unique to this study. The proposal listed on this table will be described in the next section.

Author Biomaterial 2D/3D Cell Line

Desired

Lineage

Biophysical

Signals

Quantification Novel Aspects

Wang, ‘99

[67]

Bovine Collagen

Scaffolds

2D

Murine

Bone

Marrow

(C57BL/6J)

Expansion No

CFU-GM

Assay,

Hemacytometry

Porous

Microspheres

Bancroft,

‘03

[3]

poly(DL-lactic-co-

glycolic acid) Scaffolds

3D

Rat Marrow

Stromal

Osteogenic No

Hemacytometry,

PCR

3D Bioreactor

Holtorf, ‘05

[66]

Titanium Fiber Mesh 3D hBMSC Osteogenic Yes

ALP,

Osteopontin,

Ca

2+

Deposition

Mesh Variability,

Constant Porosity

Lim, ‘05

[1], [45]

poly(L-lactic

acid)/polystyrene

Demixed Substrata

2D hFOB

Adhesion

and

Expansion

No

Adhesion, ALP,

SIMS,

Hemacytometry

Nanoscale

Substrata

Porter, ‘05

[79]

N/A 3D N/A N/A N/A

Lattice-

Boltzman

Computational

Modeling

36

Trojani, ‘05

[6]

(Si-HPMC)-based Hydrogel 3D Osteosarcoma Osteogenic No

ALP,

Mineralization,

RT-PCR

(TGFβ,

Interleukin,

etc.)

Injectable Hydrogel,

pH Maintenance

Zhao, ‘05

[7]

poly(ethylene terepthalate)

Mesh

3D hMSC Osteogenic No Cell Density Perfusion Bioreactor

Hee, ‘06

[65]

poly(lactic acid) Foam, Beta

TCP Scaffolds

3D

Human

Dermal

Fibroblasts

Osteogenic No

Scaffold Dry

Weight, ALP,

Histology,

Osteopontin

Comparison of

Commercially

Available Scaffolds

Hosseinkhani,

‘06

[61]

Peptide-Amphiphile

Nanofibers

“3D” hMSC Osteogenic No

ALP,

Osteocalcin

“Three-Dimensional”

Nanofibers

Riddle, ‘06

[55]

Glass Slides 2D hMSC Expansion Yes

Intracellular

Calcium

Application of

Mechanical Loading

Benoit, ‘07

[4]

poly(ethylene glycol)

Hydrogels

3D hMSC Osteogenic No BMP2, ALP

Bidirectional

Interaction between

Cells and Hydrogel

Hansen, ‘07

[57]

polystyrene/polybromostyrene

Demixed Substrata

2D MC3T3-E1 Expansion No

Cellular

Modulus

Cell Stiffness

correlated with

Nanoscale Substrata

Lim, ‘07

[50]

poly(L-lactic

acid)/polystyrene

Demixed Substrata

2D hFOB

Adhesion

and

Expansion

No

FAK, pY397,

Integrin

Adhesion on

Nanoscale Substrata

Riddle, ‘07

[56]

Glass Slides 2D hBMSC Expansion Yes

ATP, Western

Blot,

Intracellular

Calcium,

Calcineurin

Fluid Flow-Induced

Proliferation

37

38

Dawson, ‘08

[5]

Type I Collagen Scaffolds 3D hBMSC

Osteogenic,

Chondrogenic

No

Histology,

micro-CT,

ALP

Microchannels

Promote Osteogenesis

PROPOSED

1. PLLA/PS Demixed

Substrata

2. PS/PBrS Demixed

Substrata

3. Calcium Phosphate

Scaffolds

4. PLLA Salt Leached

Scaffolds

5. Simulated Bioreactor

2D

3D

hMSC

hBMSC

Osteogenic

Yes

(Empirical

and

Simulated)

AFM, SEM,

ALP, Flow

Cytometry,

Intracellular

Calcium,

Finite Element

Analysis

1. Comparison of

2D and 3D

Protocols

2. Design of a 3D

Perfusion

Bioreactor with

Biophysical

Signals

3. Simulations of

Oscillating

Fluid Flow

Proposal:

Two-Dimensional Substrata:

This study will involve the fabrication and analysis of two-dimensional nanoscale

substrates with variations in surface topography. Specifically, the goal will be to analyze

the responses of progenitor cell lines on randomly distributed nanoislands as opposed to

nanopits or uniform topographies as fabricated by photolithographic methods.

To accomplish this polymer demixing will be used with the following immiscible

polymers:

1. Poly(L-lactic acid)/Polystyrene (PLLA/PS) 70/30 w/w

2. Polystyrene/Polybromostyrene (PS/PBrS) 60/40 w/w

Finally, the surface chemistry will be maintained among these substrates. These

will then be compared with flat controls of the same chemistry. In this case, either

poly(L-lactic acid) or polystyrene will be analyzed as the negative control.

Three-Dimensional Scaffolds:

Multiple scaffold variations will then be analyzed and compared with the results

of two-dimensional substrata. The scaffolds will be compared by relative porosities, and

progenitor cell activity will be compared both between the scaffolds and the

aforementioned two-dimensional substrata.

The scaffolds to be analyzed in this study include the following:

1. FDA-Approved BD© Calcium Phosphate Scaffold

2. BoneMedik© Calcium Phosphate Coral Scaffold

39

3. Salt-Leached Poly(L-lactic acid) polymer scaffolds (150-710 micron NaCl

crystals)

It should be noted that the wide range of salt crystals will be further divided into

relative sizes to obtain hypothetical variations in porosities. These scaffolds will vary

greatly in surface chemistry, surface topography, and relative porosities. For this reason,

a direct comparison or causal effects between scaffold features will be difficult to

ascertain. Nonetheless, these three-dimensional scaffolds will later be used in a fabricated

bioreactor.

Biophysical Stimuli:

Bone in vivo undergoes biophysical stimuli by oscillating fluid flow through the

insterstitial space. Thus, an important factor will be the addition of these stimuli to the

tissue engineering protocol.

Using oscillating fluid flow, shear stresses will be induced within the physiologic

range. Specifically, stresses will be induced at 5, 10, and 20 dyne/cm

2

. This flow will be

coupled with the two dimensional substrata only due to the lack of a current bioreactor

system for such flow. However, simulations of the bioreactor system being developed

will determine flow rates necessary to induce such shear stresses throughout the volume

of each scaffold.

Bioreactor Design:

A bioreactor will not be fabricated in this study, but a design will be created for

potential fabrication at a later date. Such a design involves certain criteria to be met. The

40

bioreactor system will be used for long-term culture of human bone marrow stromal cells

for differentiation into the osteoblastic lineage in vitro. All of the prior studies eventually

lead up to the design of such a bioreactor, so these criteria cannot be taken lightly. The

design criteria and specifications have been tabulated below:

Design Criteria Design Specifications

The bioreactor must maintain

physiologic shear stresses as witnessed in

vivo.

Maintain a uniform shear stress throughout

the volume of the scaffold at 5, 10, and 20

dynes/cm

2

.

Standard scaffolds must fit within the

bioreactor volume.

Ensure the bioreactor has a variable

diameter between 2 and 10 mm.

The bioreactor must withstand

oscillating fluid flow conditions.

Determine a symmetrical geometry capable

of withstanding 1 Hz oscillations.

Flow must be uniform throughout the

scaffold volume.

Simulate the flow profiles throughout the

volume of the scaffold to ensure all are

greater than zero.

The bioreactor must work for all

scaffolds.

After determining the relative porosities of

each scaffold, repeat the above analyses

with each porosity value.

Finite Element Analyses:

COMSOL Multiphysics will be used extensively in the design of the

aforementioned bioreactor. The above specifications must be met, and finite element

analyses will determine the optimal geometry, flow rates, and scaffold properties key in

the meeting of these specifications before a final design is reached.

Additionally, this study will simulate fluid flow of media over cells cultured on

the nanoscale substrata described previously. The goal will be to analyze the biophysical

stimuli experienced by cells under oscillating fluid flow. It will be interesting to

41

determine where turbulence may result and what these flow patterns look like. In essence,

these simulations will be used solely for exploratory purposes.

42

Thesis Statement and Hypotheses:

Thesis Statement:

Thiough a numbei of expeiimental piotocols, skeletal tissue engineeiing methous

will be mouifieu anu maue optimal (i.e., incieaseu giowth anu uiffeientiation) foi

maintenance of stem cell piolifeiation anu uiffeientiation in vitio by mimicking

the in vivo extiacellulai milieu of bone anu inuuceu biophysical signals.

Hypothesis 1:

Nanoscale substiates select foi subpopulations of piogenitoi cells thiough

uiffeientiation into the osteoblastic lineage, as influenceu by suiface

chaiacteiistics, incluuing chemistiy anu topogiaphy

Hypothesis 2:

Hypothesis 3:

A bioieactoi uesigneu with finite element methous will satisfy all the ciiteiia

neeueu foi long‐teim cultuie on thiee‐uimensional scaffolus with inuuceu

biophysical signals.

The same piogenitoi cell lines cultuieu on thiee‐uimensional calcium phosphate

scaffolus will uisplay significantly maintaineu uiffeientiation potential with

continueu expansion in vitro compaieu with two‐uimensional substiata anu flat

contiols.

43

MATERIALS AND METHODS

Nanoscale Substrate Fabrication:

Two methods for polymer demixing have been demonstrated to result in the same

surface chemistry and similar surface topography characteristics. Polybromostyrene

(PBrS), with a molecular weight of 65x10

3

amu, and polystyrene (PS), with a molecular

weight of 289x10

3

amu, blend solutions were involved in one method. Another blend

solution included polystyrene (PS, M

W

= 289x10

3

) and poly(L-lactic acid) (PLLA). In

order to assure nanoisland topography, PS/PBrS was blended in 60/40 w/w, and

PLLA/PS blended in 70/30 w/w. The blended, immiscible polymers were then dissolved

in chloroform in concentrations of 0.5%, 1.0%, 2.0%, and 3.0%. The varying

concentrations would hypothetically increase the scale of the nanoislands as

concentration was increased.

Once blended, the polymer solutions were then dispensed onto quartz slides or

glass cover slips depending upon the particular protocol. Quartz slides were used for flow

experiments, and cover slips were involved in numerous assays, including alkaline

phosphatase assays. These were then spin casted at 4000 rpm for 30 seconds. In doing so,

the volatile chloroform rapidly evaporated, allowing segregation of the two blended

polymers. The films were sealed and allowed to dry for 24 hours.

For the PS/PBrS polymer films, an annealing process was required to assure

uniform surface chemistry. The spin-cast films were heated to the glass transition

temperature (T

g

) of PS but below the T

g

of PBrS. Film concentrations of 0.5% and 1.0%

were annealed for 1 hour, and spin-cast films of 2.0% and 3.0% were annealed for 2

44

hours. The annealing process allowed the PS composition of the films to segregate to the

air-film interface.

After the fabrication process was complete, substrates were sealed and allowed to

cool for 24 hours if annealing was performed. Annealing was not required for PLLA/PS

spin-cast films. In preparation for cell culture, all substrates were exposed to UV

radiation for 60 minutes prior to seeding.

Salt Leaching and 3D Scaffolds:

Poly(L-lactic acid) was initially dissolved in chloroform with NaCl crystals of

various diameters. The diameters were divided into three categories: 150-300 μm, 300-

500 μm, and 500-710 μm. The mixture was poured into a Petri dish such that the liquid

was approximately 3-5 mm deep. It was then sealed and allowed to dry for 48 hours.

The solid PLLA/NaCl scaffold was then leached three times with ddH

2

O in order

to dissolve and thus remove all NaCl crystals from the PLLA scaffold interface.

Hypothetically, crystals of increased diameters would result in pore sizes of increased

diameters. These diameters would then be correlated to an increase in porosity between

the scaffolds. The salt-leached PLLA scaffolds were then cut into cylindrical scaffolds

approximately 3-5 mm thick and 6 mm in diameter.

The additional three-dimensional scaffolds included a coral scaffold as

manufactured by BoneMedik© and a calcium phosphate scaffold as manufactured by

Becton-Dickson©. These scaffolds were then cut into cylinders that were 4 mm thick and

6 mm in diameter to remain uniform with the salt-leached PLLA scaffolds.

45

To prepare the scaffolds for cell culture, all scaffolds were immersed in 100%

EtOH for 24 hours. The ethanol was then aspirated and all scaffolds were exposed to UV

radiation for 60 minutes. This process was repeated three times to ensure complete

sterilization of all scaffolds prior to seeding.

Cell Harvesting:

Human mesenchymal stem cells (hMSCs) were obtained from Cambrex©. These

cells were from a distributer, so a primary donor was preferable.

Another cell line included primary human bone marrow stromal cells (hBMSCs),

another type of progenitor stem cell. To harvest these cells, human bone marrow was first

obtained from a patient at the Milton S. Hershey Medical Center through a protocol

defined by the Internal Review Board (IRB). The marrow was harvested from the rimings

of the femoral head of a 43 year old male patient undergoing hip surgery. Cells were then

washed and separated using ficoll gradient (1.077 g/ml). Cells located at the interphase of

this gradient were collected. They were then plated at a density of 2x10

5

cells/cm

2

in

growth medium composed of low glucose Dulbecco’s Modified Eagle Medium

(DMEM), 10% fetal bovine serum (FBS), 1% Penn/Strep, and 1% L-glutamine. After

four days of incubation, non-adherent cells were removed and adherent cells maintained

in growth medium with media changes every two to three days.

46

Cell Culture:

hMSCs and hBMSCs were maintained in low glucose DMEM, 10% FBS, 1%

FBS, 1% P/S, and 1% L-glutamine until required for experimental protocols, with media

changes every 2-3 days. When cells reached a confluence of 70-90%, they were then

trypsinized and passed to prevent the natural differentiation that occurs when progenitor

cells reach confluence. The hMSCs were used up to a passage of 7-8, and the hBMSCs

were viable up to a passage of 4-5. When ready for data acquisition, cells were then

seeded onto the corresponding substrates or scaffolds with either growth or osteogenic

media used. Osteogenic differentiation media consisted of low glucose DMEM, 50 µg/ml

ascorbic acid phosphate, 10 nM dexamethasone, and 10 nM β-glycerol phosphate.

Cells were then seeded onto two dimensional nanoscale substrates at a density of

4x10

3

cells/cm

2

. For three dimensional scaffolds, cells were seeded at 5x10

6

cells/cm

3

.

This seeding would occur after sterilization of the appropriate substrates or scaffolds.

Scaffold seeding was performed via the vacuum filtration method. Substrate seeding was

performed with a pipette.

Cells were then allowed to expand, proliferate, and differentiate for a period

defined by the particular protocol. Media changes took place every 2-3 days, and cells

were incubated at 37°C up to 12-14 days.

47

Oscillating Fluid Flow:

After hMSCs were cultured on various nanoscale substrata, they were then

cleaned with PBS three times and loaded onto a vacuum flow chamber (Figure 17). Clear

flow media was perfused through the chamber, and all were attached to the pneumatic

pump depicted in Figure 17..

Figure 17. Pneumatic pump (A) and flow chamber (B).

The pneumatic pump induced oscillating fluid flow. This flow rate could then be

correlated to shear stresses acting upon the substrata by the following equation: ¡

w

=

µ

60

wH

2

, where τ

w

is the shear stress at the wall, μ is the dynamic viscosity of the flow

media, Q is the amplitude of the sinusoidal flow rate, W is the width of the flow chamber,

and H is the height of the flow chamber. Since the dynamic viscosity, width of the

chamber, and height of the chamber were constant, the shear stress at the wall could then

48

be controlled by adjusting the amplitude of the sinusoidal flow rate. This is assuming

Poiseuille, laminar flow rates in a Newtonian fluid. In doing so, the pneumatic pump was

then used to induce shear stresses by oscillating fluid flow at 5, 10, and 20 dyne/cm

2

.

The fluid flow protocol called for the flow chamber to be coupled with a

fluorescent microscope calibrated for Fura-2 fluorescent markers. The first 180 seconds

were under static conditions, followed by 180 seconds of fluid flow. The entire period of

360 seconds was then recorded temporally by absolute changes in fluorescence.

Fluorescent Markers:

For short-term oscillating fluid flow experiments, Fura Red fluorescent dyes were

loaded for 30 minutes prior to data collection. Fura 2 was not involved due to the fact that

it would adsorb to the polystyrene or poly(L-lactic acid) substrata. The fluorescent

marker acted as a measurement of intracellular calcium concentrations ([Ca

2+

]

i

). After the

absolute fluorescence was recorded for 360 seconds, the data was outputted to a PC. Cells

were manually selected, and each experiment included 15-40 cells within the data range.

Data processing included an initial average (μ) of absolute fluorescence during the

static period (0-180 s). During the flow period, the peak fluorescence was then

determined. If the peak fluorescence surpassed the average plus four times the standard

deviation of static fluorescence (μ+4σ), the cell was considered to have responded. This

same analysis was performed for each manually selected cell within each short-term

oscillating fluid flow experiment to determine the mechanosensitivity of cells as

determined by [Ca

2+

]

i

response.

49

hBMSCs cultured for either 7 or 12 days in both growth medium and osteogenic

differentiation media were harvested and then tagged with SSEA-4, CD73 (SH3/4),

CD90 (Thy-1), or CD105 (SH2, Endoglin) primary antibodies. hBMSCs without

antibodies served as negative controls. We chose to examine these markers because

SSEA-4, an embryonic antigen previously believed to be specifically expressed by

human embryonic stem cells has been shown to identify stem cells that are osteogenic in

vivo [80]; CD73 and CD105 react with bone progenitor cells, but not with osteoblasts or

osteocytes; and expression of CD90 in hBMSC is correlated with osteogenic potential.

These cells were then washed three times with PBS and suspended for

fluorescence-activated cell sorting (FACS) analysis. This analysis first uses flow

cytometry to sort cells by size and complexity. After sorting, the fluorescence of cells

was then measured and correlated with each of the antibodies. Each experiment analyzed

10,000 counts, which resulted in 7,000-9,500 cells per experiment on average. Controls

were used to determine the fluorescence of nonresponding cells, and a threshold was

determined for each experimental protocol in order to determine the percentage of cells

responding to the corresponding antibodies.

50

Alkaline Phosphatase Assay:

Alkaline phosphatase is a hydrolase enzyme key in the dephosphorylation of

numerous molecules in the intracellular environment of cells. The alkaline phosphatase

(ALP) assay is key in measuring the activity of this enzyme, which can then be correlated

to the overall activity of cells.

An alkaline phosphatase assay kit was involved in this protocol. Two previously

prepared standards with 0.9% NaCl and blanks were first created to develop a standard

calibration curve for optical density and absorbance. Human mesenchymal stem cells

cultured for 12 days on two dimensional substrata and three dimensional scaffolds were

then lysed. The cell lysate was added to 65 mM phenolphthalein monophosphate in 7.8 M

2-amino-2-methyl-1-propanol with a pH of 10.5. It should be noted that ALP requires an

alkaline environment for optimal activity, as indicated by its name.

To this mixture, 0.1 M phosphate buffer with a pH of 11.2 was added. The

mixture was diluted progressively in order to create a curve for each of the cell lysates.

The phosphate buffer acts as a color stabilizer when measuring the alkaline phosphatase

activity for the cells cultured on each of the substrates. These assays were then automated

in order to measure absorbance relative to blank samples and calibration curves. The

absorbance is directly correlated to cell ALP activity.

51

Substrate Characterization:

After fabrication of the two dimensional nanoscale substrata, the samples were

dried and prepared for atomic force microscopy (AFM). AFM was key in the analysis of

the nanoscale topography of these nanoislands. Specifically, the method allowed for

characterization of the scales and random distribution of the rough surfaces of these

nanoscale substrata. All AFM was performed under dry conditions.

The three dimensional scaffolds were not characterized utilizing AFM. Instead,

scanning electron microscopy (SEM) was used to characterize the three dimensional

characteristics and pore sizes among each of the scaffolds. SEM required that all

scaffolds were dried and coated with gold (Au) prior to analysis. Scanning electron

microscopy images were then outputted to ImageJ in order to determine the relative

porosities between the scaffold types. The software considered the relative white-to-black

balance along each of the upper surfaces of the scaffolds. This balance was then

correlated with the relative pore sizes along the scaffold. Porosity is normally defined by

the following equation: O =

v

¡

v

T

, where V

v

is the volume of the void space, V

T

is the total

volume of the bulk material, and the porosity is the ratio between the two. Since SEM

simply characterizes a two-dimensional image of a three-dimensional material, porosity

is redefined by the following equation: e =

A

¡

A

T

=

N

B

P

N

T

P

, where A

v

is the area of the void

space, A

T

is the total area of the surface, and estimated porosity is the ratio between these

two. In terms of imaging, A

v

is redefined by N

B

P

, which is the total number of black

pixels after the image undergoes a threshold algorithm, and A

T

is redefined by N

1

P

, which

is the total number of pixels in the image. This definition of porosity acts as a very rough

estimate and should only be considered as a relative porosity measure used to compare

52

scaffolds. The absolute value is, in essence, inaccurate due to the assumption that these

pores remain continuous throughout the bulk of the material, which is not true in reality.

Nevertheless, these relative values play key roles in the comparison between scaffolds.

Wettability was previously determined in order to compare the relative

hydrophobicities among different substrata. The measure of hydrophobicity on the

surfaces of biomaterials plays a key role in cell adhesion and morphology. Decreased

hydrophobicity, or increased hydrophilicity, leads to better adhesion and a wider spread

of cells when analyzing their morphologies or histologies. Again, these data were

previously determined, and the measure of wettability simply ensured uniform surface

chemistry among substrata.

Simulation of the 2D Microenvironment:

COMSOL Multiphysics was used to simulate the two-dimensional

microenvironment of cells cultured on nanoscale topographies. Included in the

comparison were different levels of cell confluence, differing elastic moduli of cells

cultured on numerous substrata, and an analysis of the flow patterns over a cell cultured

on a hydrophobic substrate.

When comparing the cells cultured on multiple substrata, data from a contact-

mode atomic force microscopy (AFM) study by Joshua Hansen were used as inputs for

the moduli of cells on these surfaces. Cells cultured on plasma-cleaned glass had an

average modulus of 7000 Pa, those on flat polystyrene had a modulus of approximately

4000 Pa, cells on 11 nm nanoislands (PS/PBrS 40/60 w/w) displayed an elastic modulus

of 9000 Pa, and those cultured on 38 nm nanoislands had an approximate modulus of

53

12000 Pa. Incompressible Navier-Stokes fluid flow was coupled with a stress-strain

analysis to determine the apparent shear stresses induced on the surfaces of these cells.

When analyzing oscillatory fluid flow, one must ensure that the flow remains

laminar if assumptions and equations are to hold. The maximum Reynolds number was

16.302 using the follow ng c i riteria:

• Inlet Pressure: P

ct

=

ìnI

u.Sk × cos(æt) + P

utm

• Outlet Pres - .Sk + P sure: P

outIct

= u × cos(æt)

utm

• Constants: æ = 2n roJ¡s; P

utm

= 1S1 kPo; k = 4uuu N¡m

3

The incompressible Navier-Stokes equations and the assumptions following

include:

•

ðu

t

µ

ð

+ µ

•

uvu = v|-µI + µ(vu +(vu)

1

)] +F

vu = û

• µ|vu +(vu)

1

] = û

These equations for fluid flow analyses can then be coupled with a stress-strain

analysis to determine shear stresses throughout the two-dimensional microenvironment.

In the analysis of shear stresses, the normalized von Mises stress was used:

o

¡M

=

1

√2

¹

(o

x

- o

y

)

2

+ (o

y

- o

z

)

2

+ (o

z

- o

x

)

2

+ 6¡

xy

2

+ 6¡

zx

2

+ 6¡

yz

2

All of these data were then analyzed to determine flow patterns, values of shear

stresses at multiple locations, and the absolute flow rates in various geometries.

Note: COMSOL model reports detailing the inputs and outputs from the

simulations can be found in Appendix F.

54

Bioreactor Design:

In the design of the bioreactor, COMSOL Multiphysics was also used to create

various geometries and alter the flow rates. Porosity values previously determined were

used as inputs, with a permeability of zero, when simulating the insertion of a scaffold in

each bioreactor geometry. Furthermore, the previous equations and assumptions as

involved in the two-dimensional microenvironment were also used in the three-

dimensional analysis of various bioreactors. However, to conserve computing power, the

three-dimensional geometry was simplified to a cylindrical coordinate system with axial

symmetry. In turn, this allowed the three-dimensional system to be analyzed through two-

dimensional simulations.

To satisfy the aforementioned design criteria for a bioreactor, multiple geometries

were first used as inputs in the bioreactor. Specifically, two geometries, one with a

confluence with the scaffold and another with a gap, were used. The next variable altered

was the porosity of the scaffold from the relative values determined with SEM and

ImageJ. The simulation then displayed various flow profiles through the bioreactor and

the corresponding scaffold. Additionally, the incompressible Navier-Stokes equations

were coupled with a stress-strain analysis of the scaffold, and the von Mises stresses

throughout the volume of the scaffold were determined.

Design criteria were met if the bioreactor and corresponding scaffold displayed

uniform flow profiles and consistent von Mises stresses throughout the scaffold volume.

This criterion should remain true for all relative porosity values, indicating that the same

bioreactor could hypothetically be used under multiple conditions.

55

Once the geometry was determined, multiple flow rates were used in order to

determine the flow rate or pressure value necessary to ensure von Mises stress of 5, 10,

and 20 dyne/cm

2

throughout the volume of the scaffold. Analysis was simply performed

through an iterative design process until the optimal flow rates were determined.

Again, the final geometry was the key component of this analysis. No physical

bioreactor was fabricated in this study. The purpose was simply to optimize the system

and demonstrate that FEA can be used as an excellent design tool in bioreactor

fabrication for tissue engineering.

Note: COMSOL model reports detailing the inputs and outputs from the

simulations can be found in Appendix F.

Statistics:

All numerical data were analyzed with one-way analysis of variance (ANOVA).

Significance was then determined with the Tukey Post Hoc Test. These methods assumed

a normal distribution of data, independent samples of data, equal variances of the

populations, and Simple Random Samples (SRS). Furthermore, values were considered

significant if p<0.05 and very significant if p<0.01.

56

RESULTS

2D Substrate Characterization:

Polystyrene/Polybromostyrene (PS/PBrS 40/60 w/w) demixed substrata were

dried and subsequently characterized with atomic force microscopy (AFM). AFM images

of these surfaces are depicted in Figure 18 below.

Figure 18. AFM Images of PS/PBrS 40/60 w/w polymer demixed substrata.

The figure above depicts PS/PBrS 40/60 w/w demixed substrata from

concentrations of 0.5%, 1.0%, and 2.0% after dissolution in chloroform. The 0.5%

PS/PBrS 40/60 w/w resulted in nanoislands of approximately 11 nm in height, 1.0% in

nanoislands of approximately 38 nm height, and 2.0% in nanoislands of approximately 85

nm in height. Note that the diameters of these nanoislands are random, and the

distribution of the surface topography is also random. Compare these substrata with those

resulting from UV photolithography, which results in uniform distribution of nanoscale

or microscale topographies. It should be noted that the average diameters of the 11 nm

nanoislands are 0.5 to 0.9 μm, 38 nm nanoislands with diameters of 0.7 to 1.0 μm, and 85

nm nanoislands with diameters from as small as 0.5 μm on average to as great as 1.7 μm

57

on average. This variation could thus correspond to a difference in overall surface area

between the nanoscale topographies.

Poly(L-lactic acid)/Polystyrene (PLLA/PS 70/30 w/w) demixed substrata were

also fabricated, dried, and analyzed with AFM. The AFM images from these analyses are

depicted in Figure 19 below. Titles listed above each figure are average island heights.

Figure 19. AFM Images of PLLA/PS 70/30 w/w polymer demixed substrata.

The image above depicts the substrata fabricated from 0.5%, 1.0%, 2.0%, and

3.0% PLLA/PS 70/30 w/w polymer demixing. Note that the corresponding nanoisland

heights were on the same order as the PS/PBrS 40/60 w/w demixed films; however, the

topographical characterization was much different than the other polymers. The

nanoscale for 0.5% films was approximately 12 nm in height, 1.0% films were around 21

nm in height, 2.0% films were approximately 45 nm, and the 3.0% demixed substrata

were around 80 nm in height. These substrata were even less uniform than their PS/PBrS

counterparts. The diameters also significantly changed from the 12-21 nm nanoislands to

the 45-80 nm nanoislands. Diameters for the 12-21 nm nanoislands were approximately

0.1-0.3 μm, and the diameters for the 45-80 nm nanoislands were approximately 0.5-1.0

58

μm. As before, this variation could hypothetically correspond to a variation in the surface

area among the different nanoscale substrata.

59

3D Scaffold Characterization:

Each of the three

imensional scaffolds was

The BD© scaffold is

d

analyzed with scanning

electron microscopy (SEM).

The SEM images for these

scaffolds are shown in Figure

20.

shown in Figure 20.A and 20.B

at different scales. Note that

the pore sizes may be very

large in this particular scaffold,

but the continuous porosity of

the BD© calcium phosphate

scaffold is very low. The

BoneMedik© coral scaffold is

shown in Figure 20.C and 20.D

at different scales. Note that

although the pore sizes appear

much smaller, the depth of

pores appear to be much

greater in these coral scaffolds.

60

Figure 20. SEM Images of scaffolds(A/B-BD©; C/D-Coral; E/F-PLLA(150 μm);

G/H-PLLA(300 μm); I/J-PLLA(500 μm).

Furthermore, there is much greater uniformity of pores in the coral scaffold as opposed to

its calcium phosphate counterpart. The salt-leached PLLA scaffolds are shown in Figures

20.(E-J). Figures 20.E and 20.F depict the scaffolds with leached NaCl crystals ranging in

diameter from 150 to 300 μm at two different scales, 20.G and 20.H depict those from

leached salt crystals of 300 to 500 μm, and 20.I and 20.J depict PLLA scaffolds whose

leached crystals ranged in diameter from 500 to 710 μm. Note that the pore sizes

increased with the size of the NaCl crystals, but not by much. Furthermore, it appeared

that “pits” were actually leached from the PLLA polymer as opposed to true pores. It

would thus be hypothesized that proper cell seeding would not be possible in such

scaffolds when compared with the coral or calcium phosphate scaffolds.

Using these SEM images, the relative porosities were then determined with

ageJ

Figure 21. Estimated porosities calculated in ImageJ from SEM images of 3D scaffolds. (*p<0.05 w/ PLLA(300-500);

**p<0.01 w/ PLLA(300-500); ***p<0.001 w/ PLLA(300-500); +++p<0.001 w/ PLLA(500-710) N=10)

Im through the protocol in Materials and Methods – Substrate Characterization.

Figure 21 shows the results from these porosity calculations.

61

The “gel” as shown in this graph was another scaffold fabricated from ground

calcium phosphate, fibrin, and thrombin. The process was similar to the formation of a

blood clot in vivo. However, these scaffolds were not properly analyzed due to a

breakdown of the fibrin/thrombin composite upon drying.

Note that the salt-leached PLLA scaffolds with 300-500 μm diameters displayed

porosities significantly greater than the BD© calcium phosphate scaffolds and the

BoneMedik© coral scaffolds. Furthermore, the PLLA scaffolds with NaCl crystals

ranging from 500-710 μm diameters displayed significantly (p<0.001) greater porosities

than all other scaffolds, including the salt-leached PLLA scaffolds with 150-300 μm

diameter crystals. These data demonstrate a significant variation among estimated

porosities between the num

scaffolds shown previously have demonstrated that the coral and calcium phosphate

scaffolds actually have true, continuous pores when compared with the PLLA scaffolds.

Thus, it could be argued that these porosity estimates are not true indicators of cell

performance on each of the three dimensional scaffolds. Instead, these data will be

involved in the simulations of multiple scaffolds in each of the bioreactor geometries

with finite element analysis.

erous scaffolds. However, note that the morphology of the

62

Mechanosensitivity of Stem Cells on Substrates:

Human mesenchymal stem cells were cultured in Dulbecco’s Modified Eagle

Medium (DMEM) with 10% fetal bovine serum (FBS), 1% penicillin-streptomycin, and

1% L-glutamine. Randomly distributed nanoisland textures with varying heights (12, 21,

5, and 80 nm) were fabricated using poly(L-lactic acid)/polystyrene (70/30 w/w)

emixing techniques in concentrations of 0.5%, 1%, 2%, and 3% w/w. The solutions

ere spin-casted on quartz slides at 4000 rpm for 30 s, completing substrate fabrication.

lat PLLA surfaces were also created using this process.

Oscillating fluid flow has been demonstrated to induce an increase in intracellular

alcium ([Ca

2+

]

i

). Increased [Ca

2+

]

i

activates MAP kinases ERK1/2, resulting in increased

or mechanosensitivity. hMSCs

h of the nanotopographies.

4

d

w

F

c

cellular proliferation. Thus, [Ca

2+

]

i

acts as a marker f

cultured on the nanotopographical substrates were placed on a vacuum-sealed oscillating

fluid flow chamber. Shear stresses of 5, 10, and 20 dyne/cm

2

were induced by this

oscillating fluid flow. Fura Red AM stain was used to measure [Ca

2+

]

i

over time within

the cells through fluorescence microscopy. The initial 180s were static, and the final 180s

included oscillating fluid flow-induced shear stresses.

Figure 22 depicts the percentage of cells responding under static and fluid flow

conditions at each of the shear stresses after culture on eac

63

Figure 22. % Cell Response on various nanotopographies under different shear stresses

after 180 s. (*p<0.05 w/ Flat; N=3)

At 5 dyne/cm

2

, the cells cultured on the 12 nm nanoislands displayed significantly

greater [Ca

2+

]

i

response compared with the flat control. Furthermore, shear stresses

differences were noted b of 10 dyne/cm

2

and 20

yne/cm

2

.

Figure 23. Absolute increase in fluorescence under fluid flow. (*p<0.05 w/ Flat;

#p<0.05 w/ 12 nm; ##p<0.01 w/ 12 nm; N=3)

continued to increase with corresponding shear stresses. Due to this increase, no

etween substrates under conditions

d

Depicted in Figure 23 are the absolute cellular response results, measured as a

change in fluorescence from the static baseline.

64

Cells on 12 nm nanoisland topographies demonstrated significantly greater

mechanosensitivity than cells on 45 nm and 80 nm nanoislands at 5 dyne/cm

2

.

Additionally, significant differences occurred between the 45-80 nm nanoislands and the

12 nm nanoislands along with the flat PLLA surfaces at 10 dyne/cm

2

.

Finally, Figure 24 depicts the percent increase in fluorescence from a baseline set

to 1

er fluid flow. (*p<0.05 w/ Flat; #p<0.05

w/ 12 nm; N=3)

There were no significant differences in percent increase among the substrates

under shear stresses of 5 dyne/cm

2

and 20 dyne/cm

2

. However, the cells cultured on 80

nm nanoislands under oscillating fluid flow-induced shear stresses of 10 dyne/cm

2

displayed a significantly lower percent increase when compared with both the flat PLLA

00% for each of the shear stress conditions.

Figure 24. Percent increase in fluorescence und

control and the cells cultured on 12 nm nanoislands.

65

FACS Analysis:

Multiple stem cell markers were used to determine whether substrate nanoscale

topographies affect stem cell differentiation behavior in vitro by using fluorescence-

PS/PBrS 40/60 w/w demixed films at varying spin-casting concentrations

displayed randomly distributed nanoisland textures with varying island heights (11, 38,

and 85 nm). After annealing, PS segments segregate to the film surface, and hBMSCs

responses to nanotopographic scale could be assessed under the same surface chemistry

of polystyrene. hBMSCs cultured on 11 nm substrates for 7 days displayed a significantly

lower SSEA-4, CD73, and CD105 positive cell percentage relative to flat control and

larger nanoisland surfaces (Figure 26). After 12 days of culture, cells cultured in

osteogenic differentiation media resulted in lower positive percentages relative to cells in

growth media, regardless of textured or flat surfaces (Figure 27). Cell response to CD90

after 7 days of culture displayed similar trends but did not demonstrate significant results.

Figure 25 depicts a sample output from FACS analysis.

activated cell sorting (FACS) analysis.

66

SSEA-4 (Osteogenic) SSEA-4 (Growth)

Control

11 nm

38 nm

85 nm

Flat

Control

11 nm

38 nm

85 nm

Flat

Figure 25. FACS Output for SSEA‐4 fluorescent markers.

The curves in Figure 25 display the fluorescence of a total of 10,000 counts.

level, and the percentage of counts beyond the

esho

Figures 26/27. hBMSC response to SSEA-4, CD73, CD90, and CD 105 in growth and osteogenic media after and 12

days of cultur m; N=3)

Greater fluorescence would be shifted to the right. In each experiment, the fluorescent

counts were compared with the same control. A threshold was set for each experiment

based upon the control’s fluorescence

thr ld (minus the artifacts in the control) allowed for a comparison in the percentage

of cells responding to each marker utilizing a FACS analysis. These data are displayed in

Figures 26 and 27 below.

7

e. (*p<0.05 w/ Flat; **p<0.01 w/ Flat; ##p<0.01 w/ 85 n

67

The above data depict the responses of hBMSCs cultured on 11 nm, 38 nm, and

85 nm nanoislands. hBMSCs cultured on flat polystyrene surfaces were used as a control.

Cells were cultured for either 7 or 12 days in growth or osteogenic differentiation media

and then tagged with either SSEA-4, CD73, CD90, or CD105 antibodies. Outputs similar

to the data shown previously were collected for each of these experiments, and the

percentage of cells responding was thus calculated.

First, note the general differences between all cells cultured in growth versus

osteogenic media on both Day 7 and Day 12. The general trend was a decrease in percent

response to antibodies, or an increase in differentiation, from growth to osteogenic media.

Such a decrease was an expected result, and it demonstrated success in data collection.

Next, look at the data for cells cultured for 7 days in growth media. There were no

significant differences among the substrates for SSEA-4, CD90, or CD105 antibodies.

However, cells cultured on the 11 nm substrata displayed significantly lower antibody

response to CD73 when compared with both the flat control and those cultured on 85 nm

nanoislands. Though this was significant, the response was still greater than 80%. Thus,

this unexpected result in significance was attributed more to significance by statistics

than by biological means since most cells remained undifferentiated.

Analyzing data from Day 7 in osteogenic differentiation media yields much

different results. First, note that significant differentiation of cells tagged with CD90

antibody diminished any significance among the substrata. However, cells cultured on 11

nm nanoislands displayed significantly lower responses to SSEA-4, CD73, and CD105

antibodies when compared with both the flat control and 85 nm nanoislands. This

decrease in response is correlated to an increased osteogenic potential and thus

68

differentiation of cells cultured on the 11 nm nanoislands. Again, this agrees with the data

found for mechanosensitivity of stem cells cultured on 12 nm nanoislands as shown in

Figure 22. Thus, we have an increased osteogenic potential and increased

mechanosensitivity of hMSCs on 11-12 nm nanoislands, and these data are significant

when compared with other scales of topographies and flat controls. Again, surface

chemistry remained consistent among the substrata.

Focusing now on the cells cultured for 12 days in growth media, significant

results have also occurred. First, note that the overall cell responses to different

antibodies in growth media after 12 days were generally lower than those cultured for 7

days. Such differences could have been due to some natural differentiation occurring as

s cultured on 11 nm

hBMSCs reached confluence. However, stem cells cultured on 38 nm nanoislands for 12

days in growth media displayed a significantly greater response to the CD90 antibody

when compared with the flat control. Furthermore, progenitor cells cultured on the 11 nm

nanoislands for 12 days in growth media also displayed a significantly greater fluorescent

response to SSEA-4 antibody when compared with the flat control. These increases could

be attributed a significantly increased rate of progenitor cell proliferation on these

substrata. However, this hypothesis conflicts with the response of cell

nanoislands for 7 days in growth media and tagged with CD73.

Finally, progenitor cells cultured in osteogenic differentiation media for 12 days

demonstrated a generally lower response to all antibodies when compared with those

cultured in growth media. However, cells cultured on 11 nm nanoislands and tagged with

CD105 antibody displayed significantly decreased fluorescent response compared with

the flat control. These data are again correlated to an increased osteogenic potential, and

69

these data are once again coupled with the increased mechanosensitivity of stem cells on

12 nm nanoisland topographies. All other cells tagged with SSEA-4, CD73, and CD90

antibodies displayed such a low response that no significant differences were noted

among the substrata.

70

AP Activity:

Human mesenchymal stem cells (hMSCs) were cultured on the BD© calcium

phosphate scaffolds, BoneMedik© coral scaffolds, flat polystyrene surfaces, and 11 nm

nanoislands topographies fabri PS/PBrS 40/60 w/w polymer demixing. The

rogenitor cells were cultured in osteogenic differentiation media for 12 days. Cells were

med for each of the scaffolds

and substrates (N=6). The AP activity was normalized by the number of proteins in the

lysate, resulting in units of Sigma Units (arbitrary units) per protein.

Depicted in Figure 28 below is the AP activity for cells cultured on flat

polystyrene and 11 nm nanoislands.

Figure 28. AP Activity for cells cultured on flat PS and 11 nm nanoislands. (***p<0.001

w/ 11 nm; N=6)

As expected, hMSCs cultured on the 11 nm nanoisland substrata displayed

significantly greater AP activity when compared with the flat substrate. These data are

further coupled with the data stating that progenitor cells cultured on 11-12 nm

cated from

p

lysed, and an alkaline phosphatase activity assay was perfor

***

71

72

splay significantly increased mechanosensitivity, osteogenic potential, and

Figure 29. AP Activity for cells cultured on BD© calcium phosphate scaffolds and

BoneMedik© coral scaffolds. (**p<0.01 w/ coral; N=6)

The progenitor cells cultured on the BoneMedik© coral scaffolds displayed

significantly greater alkaline phosphatase activity when compared with the BD© calcium

BD© scaffold is currently undergoi l, but the BoneMedik© scaffold is

ot. However, the analysis of the morphology of these scaffolds demonstrated previously

nanoislands di

now AP activity when compared with flat controls of the same surface chemistry (i.e.

PLLA or PS).

Depicted in Figure 29 below is the AP activity for each of the three dimensional

scaffolds in this experiment.

**

phosphate scaffolds. In one sense, such a result is unexpected, due to the fact that the

ng FDA approva

n

that cell seeding may be more efficient on the coral scaffold than on the calcium

phosphate scaffold due to increased continuity of more continuous pores on the coral

72

scaffold. Furthermore, the relative porosity of the BoneMedik© scaffold was also greater

than the estimated porosity of the BD© scaffold.

73

Finite Element Analysis of Cell Confluence:

Numerous studies thus far have involved oscillatory fluid flow to induce shear

tresses on the surfaces of progenitor cells. However, the microscale and macroscale

element analysis (FEA) was

therefore employed to simulate fluid flow over these cells. In doing so, the data can then

predict both flow patterns and possibly cell responses to such flow. Cell biologists

typically do not employ such methods, making this particular study novel in its bio-

computational aspects.

The initial simulation in COMSOL Multiphysics attempted to analyze the effects

of cell confluence on fluid flow patterns. As opposed to oscillatory fluid flow,

unidirectional flow of media was employed. The pressure difference, as opposed to being

sinusoidal in nature, was now a constant at ∆P = µgI, where ∆P is the pressure

difference across the bioreactor, ρ is the density of the fluid in kg/m

3

(in this case, water

was used with ρ = 1000 kg/m

3

), g is the acceleration due to gravity (g = 9.81 m/s

2

), and L

is the length of the bioreactor being simulated in meters. Figure 30 below depicts the

macroscopic view of flow over cells cultured on less hydrophilic surfaces with varying

levels of confluence. Note that there were very little differences in flow patterns within

the bioreactor.

s

patterns of flow have yet to be analyzed in detail. Finite

74

75

Analysis of cell confluence; macroscopic view. Incompressible Navier-

Stokes with a Newtonian fluid was assumed.

Although there were minor disturbances in the flow profiles, most notably with

50% and 100% confluence at the upper boundary of the bioreactor, significant

differences in flow patterns were not noted. Thus, it was recommended that a

Figure 30. FEA

75

microscopic view of flow over cells be analyzed due to the inconclusive results from this

preliminary study.

Figure 31 below depicts the microscopic view of flow over cells with various

confluence. Note the velocity field contour lines in addition to the color surface.

Figure 31. FEA of cell confluence; microscopic view. (A) 50% confluence with cell at

entrance to flow field; (B) 100% confluence with cell at entrance to flow field; (C) 100%

confluence with high hydrophobicity; (D) 50% confluence without cell at entrance.

Figures 31.A and 31.B depict unidirectional fluid flow of cells with 50%

confluence and 100% confluence, respectively. In both cases, a cell was located at the

e

the flow patterns, such es of following cells.

hus, it could be argued that the shear stresses along the surfaces of these cells would

ntrance to the flow region. In doing so, this initial bump in the flow significantly altered

that flow was at or near zero along the surfac

T

also be significantly lower.

When increasing hydrophobicity significantly and ignoring entrance region

effects, Figure 31.C depicts the flow along cells in such a bioreactor at the microscopic

76

level. Note that the flow is nonzero along the upper region of the cells, but the space

between cells exhibited flow rates at or near zero. Thus, this unidirectional flow

mulat

rfaces of

such cells are non-uniform in nature.

Finally, Figure 31.D depicts the culture of cells on hydrophilic surfaces at 50%

confluence beyond the entrance region. In this case, the lack of flow initially witnessed in

Figures 31.A and 31.B becomes minimal. Furthermore, some flow occurs over a greater

surface area of the cells, and the shear stresses become more uniform when compared

with those in Figure 31.C. Therefore, these data suggest that cells with a lower

confluence and on hydrophilic surfaces will have more uniform shear stresses under

os

si ion suggests that the shear stresses between cells in extremely hydrophobic

conditions are also at or near zero. Furthermore, the shear stresses along the su

cillating fluid flow than their hydrophobic counterparts at a higher confluence.

77

Finite Element Analysis of Cell Height:

The next study attempted to examine the various effects of culture of cells on

surfaces differing in hydrophobicity under fluid flow conditions, using cell height as the

independent variable. Though the core of this thesis analyzes surface morphology under

constant surface chemistry, such chemistry should still be considered when developing a

Figure 32. Cell with a very high contact angle with respect to the surface under constant,

unidirectional flow.

bioreactor. Considering that cell morphology is controlled by the hydrophobicity of

substrata, it would be predicted that the apparent shear stresses would also vary as surface

chemistry is altered if cell height were to increase with hydrophobicity. First, consider the

extreme case of a cell with a very high contact angle with respect to the culture surface

under unidirectional flow, as shown in Figure 32.

78

This study demonstrates a few important characteristics of such extreme

ypothetically fabricate negative effects

on cell adhesion, distribution of shear stresses, and conservation of energy. In other

words, the assumptions necessary for reproducibility in experimental protocols would

break down if such turbulence were to occur.

As a result, this extreme condition predicts that oscillatory fluid flow provides a

more consistent and beneficial experimental condition than protocols utilizing

unidirectional constant or unidirectional peristaltic fluid flow of media over cells extreme

increases in cell height.

The study then continues by examining oscillatory fluid flow and altering the cell

height among the simulations. The purpose was to determine the general differences

between relative shear stresses induced by oscillating fluid flow on cells cultured on

substrata with varying levels of hydrophobicity. It was hypothesized that cells with lower

height would exhibit greater shear stresses, but those with greater cell height would have

shear stresses concentrated near the center of the cell. This hypothesis stemmed from the

conditions. First, the high cell height may lead to a lack of flow at edge of the cell on

both sides of flow. Such height change leads to a concentration of higher shear stresses at

the upper surface of the cell. It would thus be predicted that cell shear stresses with

increased cell height would be non-uniform in nature. As a result, oscillatory fluid flow

would be more beneficial than unidirectional flow in distributing the shear stresses along

the surface of this cell with increased height.

Furthermore, note that eddies begin to form in the distal region of flow, just

beyond the right side of the cell in the flow field. These eddies can lead to turbulence if

velocity is increased, and such turbulence could h

79

pr nary simulation shown in Figure 32. The results of these simulations are displayed

in Figure 33.

elimi

cell height. Oscillatory fluid flow of media was used, and these data depict the simulation

depicts the von Mises stress distribution in the various simulations.

Figure 33. Shear stresses along the surfaces of cells cultured on substrata with varying

at t = 3.0 seconds. Red arrows depict the velocity field, and the grayscale gradient

80

Note that the flow profiles were similar among the various conditions, except at

the cell surfaces. As cell height was increased, the fluid flow profile was further

disturbed. It is very difficult to quantify the von Mises stresses from these simulation

outputs, but the purpose is to depict and define the various levels of cell height involved

in this protocol. Figure 34 demonstrates the first quantification of these data in terms of

relative shear stresses at the center of each cell in the experiments.

Figure 34. Shear stresses at the cell center over 3.0 seconds, along with the

corresponding averages for each variance in cell height.

First, note the wide variation in von Mises stresses from t = 0.0 s to t = 3.0 s

between cells cultured on various substrata. As can easily be seen, the amplitudes of these

sinusoidal patterns vary greatly, with those for the 7.5 µm-high cells having the greatest

peak amplitudes at nearly 120 dyn/cm

2

. The flat control was generally the lowest in its

How ater shear

stresses would be concentrated at the cell center on cells of greater height was nearly

time-dependent shear stresses at the cell center.

ever, upon averaging these data, the hypothesized trend that gre

81

observed. The flat control expectedly exhibited the lowest average shear stress at the cell

center, followed by the 1.5 µm- and 3 µm-high cells. Additionally, the very 7.5 µm-high

cells exhibited the greatest average shear stress at this location. However, the 5 µm-high

cells did not follow this trend. Such results could have been due to an error in the

simulation or some unpredicted factor in shear stress versus cell height. Nonetheless, all

other substrata followed predicted trends.

Figure 35 depicts the same data for various substrata at the left adhesion point

between the cell and the surface under oscillating fluid flow for 3.0 seconds.

Figure 35. Shear stresses at the leftmost point on the cell over 3.0 seconds, along with

the corresponding averages for each variance in cell height.

es very difficult to analyze these time-

Unlike the previous location, the von Mises stresses at the leftmost point on each

cell from t = 0.0 s to t = 3.0 s was very similar in its sinusoidal pattern among cells

cultured on various substrata. In fact, it becom

dependent data at this location. Nevertheless, one can easily locate very high peaks for

the flat control and 1.5 µm-high cells.

82

Upon averaging these time-dependent shear stresses, one finds a trend very much

unlike that found at the center of the cell. The flat control exhibited the greatest average

shear stress over this period of 3.0 seconds. Furthermore, the trend demonstrated that

shear stress at this adhesion point decreased with increasing cell height. This trend was

hypothesized from the preliminary cell height data, predicting that most shear stresses

would be concentrated away from this point under conditions where height is greatest.

F

Finally, these data were again analyzed at the rightmost point on each cell over a

period of 3.0 seconds for each substrate in Figure 36.

igure 36. Shear stresses at the rightmost point on the cell over 3.0 seconds, along with

the corresponding averages for each variance in cell height.

Considering that oscillatory fluid flow was used, similar trends in data were

displayed at the distal location in the flow field. These data further demonstrate the

benefit of oscillatory fluid flow over unidirectional flow, where such consistency would

not be exhibited as demonstrated previously for the cell with increased height. The trend

83

depicting decreasing shear stress with increasing cell height was once again noted at this

location.

Thus, these data demonstrate the benefits of decreased cell height and the use of

oscillatory fluid flow in the distribution of more uniform shear stresses across the surface

of cells.

84

FEA of Cells Cultured on Various Nanotopographies:

All of the previous simulations lead up to this series of experiments. Finite

lemen

lass, flat polystyrene, and two polystyrene/polybromostyrene (PS/PBrS 40/60

/w) demixed substrates. The nanoislands simulated were 11 nm and 38 nm high

[57], cells cultured on these

surfaces exhibited the following approximate elastic moduli:

• Plasma-Cleaned Glass: E

cell

≈ 7000 Pa

• Flat Polystyrene: E

cell

≈ 4000 Pa

• 11 nm Nanoislands (PS/PBrS 40/60 w/w): E

cell

≈ 9000 Pa

• 38 nm Nanoislands (PS/PBrS 40/60 w/w): E

cell

≈ 12000 Pa

The purpose of these experiments was thus to analyze the differences in apparent

von Mises stresses under the same flow conditions. In order to do so, all cells were

simulated as 3 µm-high cells with consistency among the aforementioned oscillatory

fluid flow conditions. Additionally, the velocity fields were analyzed in order to

determine whether these conditions were upheld. As shown previously, velocity fields

should not be significantly altered when flow occurs over various nanoscale substrata

(Figure 30). Furthermore, hydrophilic surfaces and oscillatory fluid flow have been

demonstrated to exhibit the greatest level of shear stress distribution according to prior

experimentation (Figures 33-36).

Although it would be predicted that these assumptions will hold true, it is still

helpful to verify these assumptions by initially analyzing the velocity fields and von

e t analysis with COMSOL Multiphysics was employed to analyze oscillatory fluid

flow over cells cultured on nanoscale substrates. These substrates included plasma-

cleaned g

w

topographies. According to past analyses of these substrates

85

Mises stress gradients at various time points and on the various substrata. These data

have been depicted in Figure 37.

Figure 37. FEA simulation of oscillatory fluid flow on various substrata at t = 0.5 s, t =

2.3 s, and t = 3.0 s. Red arrows depict the velocity field, and the grayscale gradient

depicts the von Mises stress distribution in the various simulations.

Though the size of the image makes it difficult to completely analyze these

simulations in detail, it is helpful to analyze each of the columns to compare among the

86

four substrata (Figure 37.a-d) at each time point. Notice that there are no significant

differences in the velocity field as demonstrated by the grayscale streamlines and the red

arrows. The middle column, at t = 2.3 seconds, depicts the peak velocity in the sinusoidal

fluid flow simulation. Again, even at the peak flow, there is no significant difference in

velocity field and von Mises stress distribution among the various surfaces. This analysis

allows for a consistent examination of the apparent shear stresses along the cell surface.

In order to analyze the average shear stresses along the cell surface, the von Mises

stress was quantified for each element along the arc length of the cell surface. Each of

these elements was then averaged with the others to find this apparent shear stress at the

cell surface. These average shear stresses at t = 0.5 s, t = 2.3 s, and t = 3.0 s are depicted

in Figure 38 below.

Figure 38. Average shear stresses in the FEA of oscillatory fluid flow over various

substrata at t = 0.5 s, t = 2.3 s, and t = 3.0 s. (*p<0.05; **p<0.01; ***p<0.001)

At t = 0.5 s, there were significant differences in apparent shear stresses among

multiple surfaces. Cells cultured on 38 nm nanoislands displayed significantly greater

shear stresses than both the cells cultured on plasma-cleaned glass and those cells

cultured on flat polystyrene. Furthermore, cells cultured on 11 nm nanoislands were also

87

significantly greater in apparent shear stresses than those cultured on flat polystyrene.

Thus, the apparent shear stresses exhibited under oscillatory fluid flow was always

significantly greater on the various nanoscale topographies than their flat counterparts at

this time point.

At t = 2.3 s, there was no significance for those cells cultured on 11 nm

nanoislands. However, cells cultured on 38 nm nanoisland topographies were once again

red on 38 nm nanoisland substrates were once again significantly

greater in apparent shear stresses than their flat counterparts. Furthermore, those cultured

on 11 nm nanoislands exhibited significantly greater apparent shear stresses than the cells

cultured on flat polystyrene.

The key result of this study was the finding that cells cultured on 11 and 38 nm

nanoisland topographies exhibited significantly greater apparent shear stresses under the

same oscillatory flow conditions than their flat counterparts. This increase in apparent

she of

cells

significantly greater than those cultured on plasma-cleaned glass and flat polystyrene.

These data suggest that the significant increase in apparent shear stresses is upheld under

peak flow conditions.

Finally, at 3.0 s, the significance in data similar to the t = 0.5 s time point

returned. Cells cultu

ar stress could thus be correlated to other findings of increased mechanosensitivity

cultured on these nanoscale substrata when compared with flat controls.

88

3D Bioreactor Design by the Finite Element Method:

As previously described, the ultimate purpose of these preliminary studies is to

design a novel bioreactor capable of maintaining many of the aforementioned optimal

properties. The design of such a two-dimensional bioreactor has been analyzed above, but

roughout the scaffold volume, (2) ensuring the bioreactor diameter

t symmetrical bioreactor geometries, both satisfying the 2 to

the utilization of three-dimensional scaffolds in a bioreactor where oscillating fluid flow

is induced must now be developed with the five design criteria set forth in the project

proposal located in the Introduction.

The bioreactor design must satisfy the design criteria by (1) maintaining a

uniform shear stress th

is between 2 and 10 mm, (3) allowing for 1 Hz oscillations of fluid flow over time

through a symmetrical geometry, (4) ensuring all velocities are greater than zero in the

scaffold volume, and (5) repeating the optimal geometry simulation for each previously

analyzed scaffold by comparing simulations with the relative porosities calculated earlier.

To do so, two differen

10 mm diameter requirement, were first created with COMSOL Multiphysics. The first

geometry suspended the scaffold in the center of the bioreactor, and the second geometry

tightly held the scaffold against the bioreactor walls. First, however, the general geometry

satisfying the Criteria 2 and 3 are depicted in Figure 39.

89

Figure 39. Initial geometry with FEA setup for analysis of scaffolds. This geometry has a

Scaffold

diam

e upper-right image depicts the

volume scale factor. As can be seen, fluid flow will occur more easily throughout the

length of the bioreactor, but shear stresses will be very high where the volume scale

factor is very low (at the two flow ports and the center of the bioreactor). This

symmetrical design was then simplified to a 2D axisymmetrical simulation. Symmetry

significantly decreases required computing power for FEA. The bottom-right image then

eter between 2 and 10 mm, and it is symmetrical for use with 1 Hz oscillations of

fluid flow.

The upper-left image in Figure 39 depicts the general geometry. Note that it is

symmetrical for use with 1 Hz oscillatory fluid flow. Th

90

depicts the area of interest, where the scaffold will be placed. The numerical values in

this design were arbitrary at this point in order to show general geometry and design

setup.

The next phase of the design process then compared the tight and loose scaffold

geometries. The purpose was to determine which geometry would better satisfy Criterion

1, which states that the bioreactor must exhibit uniform shear stresses throughout the

volume of the scaffold. A simulation of the BD© scaffold using the Brinkman model for

porous media in both tight and loose geometries is shown in Figure 40.

Figures 40.A and 40.B compare the stresses between both loose and tight

geometries for the BD© scaffold as shown by the color gradient. Note that the tight

Figure 40. Bioreactor simulations of the BD© scaffold in tight and loose geometries.

91

geometry (Figure 40.B) has a high concentration of shear stress near the axis of

symmetry, with very little von Mises stresses near the outer perimeter of the scaffold. On

the other hand, the loose geometry exhibited von Mises stresses with a more uniform

concentrated near

the center of the scaffold with very little flow near the outer perimeter. Thus, perfusion of

media throughout the scaffolds followed nearly the same pattern between the two

geometries.

Therefore, the loose geometry was considered the better choice due to its

satisfaction of Criterion 1. The shear stresses were more uniformly distributed throughout

the volume of the BD© calcium phosphate scaffold in this particular geometry, providing

a better level of mechanotransduction as shown by previous experiments. Perfusion was

nearly the same between the two bioreactor geometries, so the key factor was simply the

distribution of von Mises stress.

With the optimal bioreactor geometry chosen, the next phase was to ensure that

velocity throughout the volume of the scaffold remained greater than zero for each of the

aforementioned scaffolds. To do so, each scaffold was simulated in the loose geometry

bi e

rc length of the scaffold from the axis of symmetry (r = 0) to the outer edge of the

distribution throughout the volume of the scaffold. The uniformity was far from perfect,

but this novel design demonstrates a benefit to loose-fitting bioreactors with their

scaffolds.

Figures 40.C and 40.D compare the velocity fields between the loose and tight

geometries for the BD© scaffold. In both cases, most of the flow was

oreactor, and the velocity field was quantified. The velocity of each element along th

a

scaffold (r = R) was determined, and these were than averaged along the z-axis of the

92

scaffold. Provided was an average velocity gradient from r = 0 to r = R for each scaffold

under the same conditions. The output of these analyses is depicted in Figure 41 below.

Figure 41. Average velocities from r = 0 to r = R in the loose bioreactor geometry for

each of the scaffolds. These data were then averaged again along the arc length to

produce the graph on the right.

e velocity gradients appear very similar among all the

ensure that the same bioreactor could hypothetically be used for numerous scaffolds

First, note that the prediction of a greater velocity near the center (r = 0) and a

diminishing velocity near the outer perimeter (r = R) was verified according to the graph

on the left in Figure 41. Furthermore, Criterion 4 was satisfied by demonstrating that the

velocity did not drop below zero on average throughout the arc length of each scaffold. It

is very interesting to note that th

scaffolds. Upon averaging these data along the arc length of the scaffold, the graph on the

right in Figure 41 further demonstrates that these velocities do not significantly change as

porosity is significantly altered (Figure 21). Thus, this same bioreactor geometry could be

used for numerous scaffolds with significantly different porosities.

The final criterion, Criterion 5, required that all the scaffolds be analyzed and

their shear stresses (in this case, von Mises stresses) be compared. The purpose is to

93

under the same protocol. Using the same method as in Figure 41, von Mises stresses were

now calculated along the arc length of each scaffold in the loose bioreactor geometry

from r = 0 to r = R. These data are depicted in Figure 42 below.

Fig h

of the previously quantified scaffolds.

ure 42. FEA simulation of von Mises stress in the loose bioreactor geometry for eac

Note once again that all of the scaffolds displayed very similar distribution of

shear stresses along the arc length of the scaffolds from r = 0 to r = R. These data were

then averaged once again, and the graph on the right of Figure 42 depicts the average

shear stresses in each of the scaffolds. As hypothesized, there was no significant

difference in average shear stress among the various scaffolds. However, the patterns

mimicked the porosities of each scaffold (Figure 21). Nonetheless, this lack of

significance demonstrates that the bioreactor could be used for numerous scaffolds with

significantly different porosities under the same protocol. As a result, Criterion 5 was

thus satisfied.

As a result, this novel three-dimensional bioreactor design satisfied all of the

aforementioned criteria. Furthermore, the benefits of fluid flow previously demonstrated

on two-dimensional substrata could now be examined in three-dimensional scaffolds.

94

Thus, the results come full-circle from the simplification of a three-dimensional in vivo

environment to two-dimensional in vitro studies of biophysical signals and then the

expansion of this two-dimensional study into a three-dimensional in vivo bioreactor based

upon such prior data regarding the benefits and analyses of two-dimensional culture in

bioreactors in vitro.

95

DISCUSSION

Two-Dimensional Substrata Characterization:

Findings from AFM Imaging:

Previous studies have successfully involved methods to determine the three-

dimensional topographic characteristics of two-dimensional substrata at an order of

agnitude equivalent to nanometer [1, 48]. The images from atomic force microscopy

racteristics at the nanoscale.

By increasing the concentration of polymer in chloroform solvent, the nanoscale was thus

increased. PS/PBrS 40/60 w/w films were found to elicit nanoislands increasing from 11

nm to 85 nm, while the solute (i.e. polymer) concentration was increased from 0.5% to

2.0%. PLLA/PS 70/30 w/w films elicited similar nanotopographical characteristics,

increasing from 12 nm to 80 nm in nanoisland height, while the solute concentration was

increased from 0.5% to 3.0% in chloroform.

The extracellular milieu of bone in vivo is characterized by nanotopographies of

the same order of magnitude [13]. Though the number varies, it is widely believed that

such nanoislands would be approximately 10-30 nm in height. Thus, it was demonstrated

that these polymer demixing techniques successfully mimicked the nanoscale of in vivo

bone tissue.

Another finding was the contrast with lithographic methods. Typically,

lithography results in an ordered patterning of topographical features [31]. The problem

with such a method is that it does not successfully mimic the in vivo environment, due to

the fact that the extracellular milieu of bone is in fact very disordered. In contrast with

lithographic methods which could already successfully create the same nanoscale as

m

(AFM) demonstrated the presence of three-dimensional cha

96

previously mentioned, this stu e a disordered patterning of

FM, one can easily witness a random

e height varies greatly among the islands, along with the

lity.

rther varied. Though variation is not necessarily problematic, such a

dy wished to provid

nanoislands. When analyzing the images from A

assortment of nanoislands. Th

width of each nanoisland. Furthermore, the nanotopography is randomly dispersed along

the surface of the film. Such random distribution demonstrated success of these polymer

films in improving upon current lithographic methods in mimicking the extracellular

milieu of in vivo bone tissue through both scale and disorder.

Drawbacks of Cell Height Estimates:

A skilled technician in atomic force microscopy understands issues resulting from

the cantilever. Even with a sharp cantilever, AFM images depict rounded surfaces where

surfaces are instead sharp. Thus, it should be noted that the round nature of these islands

may not be accurate when compared with rea

Another issue results from the random character of the nanoscale topographies.

For example, larger (80-85 nm; Figures 18 and 19) topographies demonstrate an

exaggeration of this phenomenon. Though the nanoscale is labeled as 80 nm or 85 nm,

islands actually vary in height to an extreme degree. Furthermore, the width of these

islands is fu

phenomenon must be recognized throughout further discussion of results.

97

Three-Dimensional Scaffold Characterization:

Findings from SEM Imaging:

Scanning electron microscopy (SEM) allowed for microscopic analysis of five

different three-dimensional scaffolds. In doing so, numerous differences between the

various scaffolds were revealed. Firstly, the BD© calcium phosphate scaffolds (Figures

20A and 20B) demonstrated similar textures and topographies when compared with the

BoneMedik© coral scaffolds (Figures 20C and 20D). However, the calcium phosphate

caffolds did not appear to have deep pores, and the scaffold instead illustrated a series of

ding from a seemingly solid calcium phosphate

ts.

could then be compared with

other scaffolds found in previous studies. Collagen and collagen-hydroxyapatite

constructs were illustrated with a rough texture similar to that of the scaffolds found in

the poly(L-lactic acid) constructs [5]. When compared with scaffolds created from

s

three-dimensional topographies exten

construct. The coral scaffolds, on the other hand, demonstrated pores with much deeper

topographies, leading to better perfusion throughout the scaffold. Furthermore, the

calcium phosphate scaffold images depicted much larger topographies when compared

with the smaller pores of the coral scaffolds.

The three poly(L-lactic acid) polymer scaffolds demonstrated a very different

texture than both the coral and calcium phosphate scaffolds. As the NaCl salt crystals

were increased in diameter, polymer “shells” became larger accordingly. However, the

pores in these scaffolds were not true pores; instead, the topographies demonstrated

microscale pits as opposed to true microscale pores. These images became integral in

understand the differences in cell and media perfusion among the construc

Textures depicted from SEM images of the scaffolds

98

nanofibers, the coral and calcium phosphate scaffolds demonstrated the deep pores found

. Such deep pores became useful in the perfusion of media

ading

imation of porosity is the concept that three-

mens

in these constructs [66, 71-73]

le to efficient cell seeding. Thus, these data provided beneficial insights into the

drawbacks of collagen scaffolds through analyses of pore depth and construct texture and

benefits of coral scaffolds by comparison with nanofibrous scaffolds in previous studies.

The estimates of porosity were then calculated from these SEM images utilizing

contrast comparison techniques in ImageJ. As mentioned before, these data demonstrated

increased estimated porosities in the polymer scaffolds. However, such findings must be

met with a skeptical attitude, especially due to the aforementioned lack of pore depth in

such poly(L-lactic acid) constructs.

Drawbacks of Porosity Calculations:

One of the greatest issues with the est

di ional constructs are simplified to two-dimensional images. In doing so, the

representation severely loses its accuracy. A mock scaffold is depicted in Figure 43.

Figure 43. A comparison of the 2D representation of a 3D construct (A) with the actual

3D construct. Notice the loss of depicted pores in these representations.

A

B

99

In Figure 43, notice that the two-dimensional image in Figure 43A depicts only

five pores. ImageJ would then be used to calculate the porosity, estimating that these five

pores are continuous throughout the volume of the construct. Thus, it would be estimated

that the porosity would be approximately 20-25% according to the two-dimensional

representation. However, the reality is much different. The three-dimensional image in

Figure 43B depicts pores lost in the two-dimensional image. As can be imagined, the

actual porosity may thus be much greater than the estimate due to the loss of pores by

two-dimensional simplification. Therefore, estimated porosities are inaccurate and may in

fact be much lower than reality.

100

Stem Cell Growth on 2D Substrates:

Mechanosensitivity of Stem Cells:

As depicted in Figures 22-24, the mechanosensitivity of mesenchymal stem cells

(hMSCs) cultured on PLLA/PS 70/30 w/w demixed films was studied by analyzing the

relative fluorescence of Fura Red stain. When analyzing the percentage of cells

responding in Figure 22, one finds that, at a shear stress of 5 dyne/cm

2

, hMSCs cultured

on 12 nm nanoislands displayed a significantly greater response when compared with

cells cultured on 21 nm nanoislands, 45 nm nanoislands, 80 nm nanoislands, and flat

PLLA controls. At greater stresses (10 and 20 dyne/cm

2

), the responses were too great to

tion that mechanosensitivity

of stem cells cultured on nanoscale topographies is significantly greater than cells

cultured on flat controls while the surface chemistry is held constant.

Figure 24, as opposed to illustrating the percentage of cells responding, showed

the absolute fluorescent response above the average baseline during static conditions. It

was observed that cells cultured on larger (45 nm and 80 nm) nanoislands displayed

significantly lower absolute fluorescent responses than hMSCs cultured on 12 nm

nanoislands. This difference demonstrated a variance among sizes of nanotopographies in

the mechanosensitivity. Thus, it was observed that cells not only preferred nanoscale

topographies, but a preference existed for certain scales (i.e. 12 nm) over others (45 and

80 nm).

Finally, Figure 25 depicted the percent increase in fluorescence above the baseline

during static conditions. Similar to the data in Figure 24, hMSCs were observed to exhibit

some preference among the substrates. These data thus became critical in the

determine any significance. Such data illustrate the observa

101

development of a bioreactor. If cells exhibit preferences in mechanosensitivity at

ear stresses and also among varying nanoscale

these substrates for

slands.

different fluid flow induced sh

topographies, such conditions should be implemented in the bioreactor. As a result, the

bioreactor design used oscillatory fluid flow, along with varying three-dimensional

porosities based upon these two-dimensional data.

Proliferation and Differentiation Potentials:

Utilizing data from fluorescence activated cell sorting (FACS) analysis (Figure

25) led to an analysis of proliferation and differentiation potentials of human bone

marrow stromal cells (hBMSCs) cultured on PS/PBrS 40/60 w/w demixed films.

Specifically, these films displayed nanoscales of 11 nm, 38 nm, and 85 nm. A flat

polystyrene control was also analyzed. hBMSCs were cultured on

either 7 or 12 days, and harvested cells were tagged with SSEA-4, CD73, CD90, or

CD105 primary antibodies with a fluorescent secondary antibody.

After seven days, the cells were harvested from culture in either growth or

osteogenic media. It was observed that cells cultured on 11 nm nanoislands in osteogenic

media displayed significantly lower SSEA-4, CD73, and CD105 reactivity compared

with the flat control and 85 nm nanoisland substrates. This decrease in reactivity was

directly correlated with an increase in differentiation potential of hBMSCs cultured on 11

nm nanoi

After twelve days, the cells were harvested from culture in either growth or

osteogenic media. hBMSCs cultured on 11 nm nanoislands in growth media displayed

significantly greater reactivity to SSEA-4 compared with the flat control. Such reactivity

102

was directly correlated with an increase in proliferation potential for cells cultured on

these substrata. Additionally, hBMSCs cultured on these same 11 nm substrates

displayed significantly lower CD105 reactivity when compared with flat controls. These

data further verified the observation that cell differentiation potential was increased on

these substrata.

Thus, proliferation and differentiation potentials were also found to have

aphies when compared with their flat

parison of the two samples, it was observed that cells cultured on 11 nm

increased on specific nanoisland topogr

counterparts. Combined with previous data, we can now infer that substrate topography

regulates progenitor cell mechanosensitivity, proliferation, and differentiation potentials.

Previous studies have also verified that focal adhesions also increase on the same

substrata [1]. Combining all of these data allows for the observation that nanoscale

substrata of 10-40 nm height provide an excellent alternative to flat counterparts

commonly used in tissue culture protocols.

Alkaline Phosphatase Activity:

Human mesenchymal stem cells (hMSCs) cultured on 11 nm nanoislands from

PS/PBrS 40/60 w/w demixed films and flat polystyrene substrata were harvested after 12

days of culture in osteogenic media. Alkaline phosphatase assays were performed on

each of the samples (Figure 28).

After com

nanoislands displayed significantly greater alkaline phosphatase (AP) activity than

hMSCs cultured on flat polystyrene controls. Thus, cells have been demonstrated to

display increased intracellular activity based solely upon surface morphology.

103

When these data are compared with previous studies, the data demonstrate a

consistent observation that progenitor cells cultured on 11-12 nm nanoislands display

significantly greater cell alkaline phosphatase activity, mechanosensitivity, proliferation

potential, and differentiation potential. Thus, surface morphology alone can regulate stem

cell activity.

104

Stem Cell Growth on 3D Scaffolds:

Alkaline Phosphatase Activity:

Similar to the aforementioned studies of cells cultured on two-dimensional

substrates, human mesenchymal stem cells (hMSCs) were also studied on three-

dimensional scaffolds. hMSCs were cultured on either BD© calcium phosphate or

samples were then analyzed for alkaline phosphatase (AP) activity, normalized by the

number of total proteins in the sample (Figure 29).

After analysis of these data, it was observed that progenitor cells cultured on the

BoneMedik© coral scaffolds displayed significantly greater AP activity than those cells

cultured on the BD© calcium phosphate scaffolds.

This observation was expected when considering the differences in porosities and

surface morphologies (Figures 20, 21). The coral scaffold was observed to have a greater

porosity value than the calcium phosphate scaffold. Additionally, the morphology of the

coral scaffolds were observed to have deeper and more uniform pores than the BD©

calcium phosphate scaffold samples after examination by scanning electron microscopy.

Thus, it could be argued that increased perfusion and deeper pores allowed for better flow

of osteogenic media throughout the volume of the scaffolds. Such flow, in turn, would

lead to greater alkaline phosphatase activity in cells.

BoneMedik© coral scaffolds for 12 days in osteogenic media prior to harvesting. These

105

Comparison with 2D Substrata:

One will notice that the alkaline phosphatase activities were not directly

mpar

tes ranged from 125-250 sigma units

e.

co ed statistically. No analysis was performed due to major differences in seeding

density and differences in protocols between the two-dimensional and three-dimensional

samples.

However, the number of sigma units per protein was on average greater among

two-dimensional samples (Figure 28) than on three-dimensional samples (Figure 29).

Specifically, averages for two-dimensional substra

per protein, whereas those for three-dimensional scaffolds ranged from 10-70 sigma units

per protein. When comparing the calcium phosphate scaffolds with the two-dimensional

substrata, there was an order of magnitude differenc

These observations should be taken with a grain of salt, due the aforementioned

differences in protocol and lack of statistical analyses between two- and three-

dimensional substrates and scaffolds.

106

Finite Element Analysis:

Discussions of Cell Confluence and Hydrophobicity:

FEM simulations were used in this study in order to analyze fluid flow patterns

inside a cell cultured flow chamber. Firstly, we examined the variability of fluid flow

patterns among various levels of cell confluence. At the macroscopic level, no significant

ls of confluence, 50% confluent or

r stresses. However, we suspect that the lower

tory effects from cell-to-cell

communication. Thus, there may be an optimal cell confluence level that allows both

uniform, positive fluid flow effects and cell-to-cell communication effects.

As regards cell height effects, FEM demonstrated a lack of flow in the distal

region of cells when the cell height is greater. Furthermore, these data suggest that

oscillating fluid flow used in some bioreactor systems [8, 39, 40] would be more

beneficial than constant or peristaltic unidirectional fluid flow found in similar

bioreactors due to a more uniform distribution of shear stresses along cell surfaces [7, 35,

74, 76, 77, 81]. In analyses using oscillating fluid flow conditions, cells with larger

differences were noted. Thus, cell confluence was concluded to have no major effect on

the flow profiles in bioreactors. However, microscopic analyses demonstrated that little

to no flow would occur between highly confluent cells. When cell confluence was

decreased, fluid flow could more easily occur between cells. Furthermore, cells in the

entrance region of flow in the bioreactor would alter the patterns of flow over cells

downstream. It would thus be assumed that lower leve

less, would be optimal in the design of bioreactors utilizing such fluid flow. This lower

confluence allows for a better distribution of fluid flow across cell surfaces and thus a

more even distribution of wall shea

confluence may be a trade-off considering potential stimula

107

heights lead to more concentrated shear stresses near the center of the cell. With

reading), the shear stresses at the

ith a greater

decreasing cell height (relevant to increased cell sp

center of the cells decreased on average and the shear stresses at the left and right of the

cell increased. These relative changes imply a more efficient distribution of shear stresses

with decreasing cell height. Taken together, these data suggest that bioreactors should use

oscillating fluid flow in combination with substrates that stimulate cell spreading for a

better distribution of wall shear stresses in the cell culture system.

Our previous studies demonstrated that cells cultured on various nanoscale

topographies will exhibit different biophysical properties, including varying elastic

moduli, cell adhesion, and morphology [1, 45, 57]. Continuing with finite element

analyses utilizing oscillating fluid flow, data from these studies was implemented to

determine any potential differences in the apparent shear stresses of cells cultured on

these various substrates. It was demonstrated in this study that cells cultured on 11 and 38

nm nanoislands exhibited a significantly greater apparent shear stress than cells on flat

surface. Such shear stress variation was due to an increased elastic modulus present in the

cells cultured on these surfaces. Increased cell stiffness is correlated with an increased

apparent shear stress, and these data support the hypothesis that cells w

elastic modulus will exhibit increased apparent shear stresses under oscillating fluid flow

conditions.

108

Benefits over Empirical Data Collection:

The success of finite element analyses was witnessed in the examination of

factors not easily determined empirically through the demonstration that cells cultured on

nanotopographic substrata exhibited increased apparent shear stresses relative to cells on

flat surfaces. Furthermore, FEM was used in such a way that an experiment could be set

up to empirically validate these results through parallel analyses. Thus, these methods

provide an excellent segue into exploring future routes of study.

Benefits of Oscillating Fluid Flow:

A key element of the data presented was the demonstrated benefit of oscillating

fluid flow as a superior method to its unidirectional counterpart. Flow is more evenly

distributed in the bioreactor system over time, leading to a more even distribution of von

Mises stresses across cell membranes. This distribution allows such systems to be more

predictive due to a better estimate of the flow rates and shear stresses at the walls of a

bioreactor. Thus, it can be concluded that oscillating fluid flow provides greater benefits

than constant or peristaltic unidirectional fluid flow systems.

109

Summary of FEM as a Tool in Cell Culture

actor systems, this same

ethod could provide insight not easily acquired through empirical methods.

FEM was successfully used to analyze flow patterns over cells, as demonstrated

by the data previously presented. FEM provides an excellent tool in the prediction of

experimental protocols and the design of bioreactor systems. Though the bioreactor

system presented here was simulated as a two-dimensional substrate simplified to a single

plane, FEM can easily be expanded to analyze more complex geometries. When

discussing the difference in perfusion of three-dimensional biore

m

110

Bioreactor Design:

Satisfaction of Design Criteria and Specifications:

This particular design specification was accomplished in two manners. First,

uniform shear stresses were witnessed in the loose variation of the bioreactor design, as

depicted in Figure 40.A. The shear stresses throughout this particular design’s volume

were uniform.

Additionally, it was specified that the design must satisfy physiologically correct

shear stresses. The target ranges were 5, 10, and 20 dyne/cm

2

. By altering input

velocities, the shear stresses could be adequately controlled. Average shear stresses were

shown to be 5.36, 10.54, and 21.88 dyne/cm

2

in this particular model. Figure 44 depicts

the changes in shear stress along the r=0 to r=R arc length of the scaffold.

Figure 44. Average shear stresses along the arc length of the loose scaffold model were

successfully maintained at approximately 21.88, 10.54, and 5.36 dyne/cm

2

.

Criterion/Specification 1: The bioreactor must maintain physiologic shear stresses as

witnessed in vivo. It must maintain uniform shear stresses throughout the volume of

the scaffold at 5, 10, and 20 dyne/cm

2

.

111

e

h 3 mm (loose) or 4 mm (tight). In both cases, the

Though the bioreactors simulated in Figure 39 and Figure 40 did not undergo

oscillating fluid flow, the benefits of 1 Hz oscillations were previously determined in the

finite element analyses of oscillating fluid flow over two-dimensional substrata.

However, the symmetrical nature about the r-plane should lead to the capability of

withstanding oscillatory fluid flow without harm to the scaffold.

Flow profiles were depicted in Figure 40 and Figure 41. Figure 40 demonstrated

through a surface map of both tight (Figure 40.D) and loose (Figure 40.C) scaffolds that

the loose bioreactor design led to a more uniform distribution of velocity through the

volume of the scaffold.

Additionally, velocities along the arc length of the loose scaffold was simulated

f

Criterion/Specification 2: Standard scaffolds must fit within the bioreactor volume.

ing somewhere between 2.0 and The bioreactor must have a variable diameter rang

10.0 mm.

The bioreactor is depicted in Figure 39. Though it may be difficult to witness, th

eight of the scaffolds were set to either

bioreactor was modeled such that it could support scaffolds ranging in diameter from 5

mm to 8 mm. This value was well within the range specified previously.

Criterion/Specification 3: The bioreactor must withstand oscillating fluid flow

conditions. A

oscillations.

symmetrical geometry must be determined which can withstand 1 Hz

Criterion/Specification 4: Flow must be uniform throughout the scaffold volume.

Flow profiles must be simulated throughout the volume of the scaffold to ensure that

all are greater than zero.

or different types of scaffolds. In all cases, a parabolic velocity profile was maintained,

112

w ns

a

osity values as

d in shear

st and

Figure 42 depict these differences. It was observed that scaffold porosity changes did not

correlate to significant changes in velocity profiles or shear stress profiles along the arc

length of each scaffold. According to these FEM simulations, the bioreactor should

perform equally well for all porosities examined in this study.

Comparison of Two Models:

el

in city profiles and shear stresses throughout the volumes of

ease in the scaffold’s perfusion ratio compared with that of the

of the scaffold within the bioreactor. This particular model assumed that the scaffold

here velocity was greatest at r=0 and smallest at r=R. Even with no-slip conditio

ssumed, the velocity never dropped below zero.

The loose bioreactor design (Figures 40.A and 40.C) was chosen as the optimal

bioreactor design. This bioreactor was simulated with various por

Criterion

deter

/Specification 5: The bioreactor must work for all scaffolds. After

mining the relative porosities of each scaffold, these values must then be inserted

into iterative simulations of the bioreactor of choice.

etermined from SEM characterization of scaffolds. In doing so, the changes

ress and velocity along the arc length of each scaffold was determined. Figure 41

The tight bioreactor model was far less successful than the loose bioreactor mod

exhibiting uniform velo

scaffolds. This comparison of success was observed in finite element simulations as

depicted in Figure 40. Notice that the loose bioreactor is more capable of exhibiting

uniform shear stresses throughout scaffold volumes. Such stress distribution would thus

be correlated to an incr

tight bioreactor.

The only issue with the loose bioreactor model is the need for secured suspension

113

simply floated in the volume of the bioreactor, which could potentially cause harm to

both the cells and scaffolds alike. To prevent this issue, it would be beneficial to include

so magnitude

sm n

disruption of fluid flow. Empirical data must then be collected after fabrication of the

scaffold with the securing mechanism in place. Such a design is depicted in Figure 45.

igur

success, however, perfusion studies must

me type of securing mechanism with adjustable supports one order of

aller than the bioreactor diameter. This difference in size should prevent issues i

Scaffold

z

r

Supports with

Screws for

Adjustment

Flow

F e 45. Loose scaffold with six tight cables to suspend the scaffold within the volume

of the bioreactor.

Analysis of Design:

This bioreactor design satisfied all of the design criteria and specifications set

forth previously. Additionally, further design components have been prepared in order to

increase the likelihood of success for such a bioreactor.

Before the design can be considered a

be performed whereby a prototype is first manufactured and then set up with a

114

un

sc

un

S

o

4

nidirectiona

caffold with

The n

nidirectiona

pecifically,

f fluid flow

l or oscillato

the bioreact

next phase

l fluid flow

the initial te

and with dif

ory pump. C

tor in order t

would the

on cells cult

est would be

fferent shear

Cells of any

to verify the

en be to a

tured in each

e an alkaline

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y type will t

data from fi

analyze the

h scaffold w

phosphatas

his design sc

then be perfu

inite elemen

effects of

with this part

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chematic is o

fused throug

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oscillatory

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outlined in F

gh the

and

actor.

eriods

Figure

6.

Figur

Fabricat

bioreact

from CA

model.

re 46. Future

tion of

tor

AD

A

t

f

e directions f

Analysis of

perfusion

capabilitie

cells in var

scaffolds w

the newly‐

fabricated

bioreactor

for bioreacto

f

es of

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with

‐

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or design.

Analysis o

long‐term

fluid flow

progenito

cells in th

volume o

various

scaffolds

within thi

bioreacto

of

m

of

or

e

f

is

or.

115

CONCLUSIONS

Methods of Biomaterial Characterization:

Numerous methods can be used to characterize biomaterial properties. Surface

chemistry properties can be analyzed with X-ray photoelectron spectroscopy (XPS), and

tensiometry can be analyzed with contact angle measurements, for example. However,

the key analysis in this study was surface morphology. In order to do so, both atomic

force microscopy (AFM) and scanning electron microscopy (SEM) were involved.

After characterization of two different two-dimensional substrata, it was

discovered that AFM was very successful in predicting surface morphology. The method

not only provided useful data, but it was simple to employ. The significant drawback to

data from AFM was that it will oftentimes depict rough surfaces with rounded edges.

Such an artifact is due to the nature of the cantilever and was not corrected in the

previous data presented in this work.

SEM was used in order to characterize the morphology of three-dimensional

scaffolds. It was very useful in estimating the pore sizes and thus the relative porosities of

numerous scaffolds. However, these relative porosities varied significantly from the true

porosity values, as illustrated previously. Thus, other methods than electron microscopy

would be beneficial to determine the total available void volume relative to the total

scaffold volume (i.e. the porosity).

Therefore, the methods involved in this study were successful in that they

adequately described the morphology of various two-dimensional substrata and three-

dimensional scaffolds. However, these morphologies had their flaws, though the most

significant flaws resulted from estimates of three-dimensional porosity values from the

116

twwo-dimensio onal surfacee images inn the SEM characterization of thhe various tthree-

dimensional s scaffolds.

th

m

It wou

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methods have

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117

A

ch

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in

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Ability of Su

Surfac

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The p

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ifferentiation

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Figure 48

Increase

mechanosens

among 12

nanoscale sub

ubstrates an

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orosity amon

tem cells cul

reviously pr

stem cell r

sitivity, as

these surfa

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(AP) activi

n. The key

ubstrates com

38 nm) nan

have been su

. Summary o

Two

d

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nm

bstrata

Inc

activ

nm

s

d Scaffolds

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ng scaffolds,

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resented data

response for

determined

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ty, increase

was that th

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noscale subst

ummarized in

of human me

sur

Stem C

o‐Dimens

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substrata

to Regulate

, including in

were used a

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hese data ill

h flat surfa

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n Figure 48.

esenchymal s

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Increased st

proliferation

11‐38 nm na

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onstrated the

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Incr

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Activity:

noscale subs

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utility of sp

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118

lative

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ponse,

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m cell

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nal

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anoscale

ta over

noscales

It should also be noted that increased responsiveness has been correlated with

rther studies.

urements, and antibody studies through

fluorescence-activated cell sorting (FACS). These studies would thus parallel those

previously completed for two-dimensional substrata.

previous studies of increased elastic moduli among cells cultured on these specific two-

dimensional nanoscale substrata. This relationship between cell stiffness and biological

response to materials could be explored through fu

The biological response of stem cells cultured on three-dimensional scaffolds was

also briefly analyzed. Unlike the studies of two-dimensional substrata, only alkaline

phosphatase activity was assayed. After analysis, the data illustrated that the

BoneMedik© coral scaffolds displayed significantly greater AP activity compared with

the BD© calcium phosphate scaffold. This variance could have been a result of

differences in porosity as previously discussed. However, the key finding was that not all

scaffolds are created equal. Whether this is a function of surface chemistry, surface,

morphology, or some combination of biomaterial properties is yet to be discovered.

Future studies analyzing the biological response to three-dimensional scaffolds

could include studies of AP activity under oscillating fluid flow conditions, analyses of

mechanosensitivity by intracellular calcium meas

119

F

H

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Finite Eleme

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120

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The applications in Figure 49 have been divided in macroscopic and microscopic

with various geometries and inputs for

tro can be coupled with physical outputs

eterm

utilities. It should be noted, however, that these were simply the uses demonstrated in this

paper. Thus, the applications illustrated in Figure 49 are not all-inclusive.

Another novel application of the finite element method in tissue engineering was

found in the design of bioreactors. FEM provides the ability to predict perfusion, flow

properties, deformation, stresses, and more

various bioreactors. For example, it may be useful to determine whether eddies or

turbulence result in a mixing chamber. FEM can predict this through both velocity

profiles and Reynolds number calculations. The utility of FEM as a design tool in tissue

engineering was illustrated in the bioreactor designed in this paper.

Thus, the finite element method acts as an excellent tool for predictive studies in

tissue engineering. It is recommended that FEM be coupled with future studies as a

preliminary method when designing experiments. In doing so, biological responses to

materials and bioreactors determined in vi

d ined in silico.

121

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122

bioreactors with a loose fitting illustrated better distribution of flow rates and shear

rrelates to

increased seeding efficiencies, increased biomechanical stimuli by fluid flow over

adhered cells, and thus more efficient tissue growth in the scaffold. As a result, the key

parameter for any bioreactor must be its perfusion efficiency.

Finally, laminar flow must be maintained in all bioreactor designs. Turbulence

has been demonstrated to break down focal adhesions and shear cells off surfaces. Such

flow would lead to a degradation of tissue growth efficiency in the scaffold. If the

Reynolds number is kept below 2300 and eddies do not appear in the current, it can be

assumed that laminar flow is maintained in the device. Such a parameter must be

analyzed by the finite element method.

ity of

is me

stresses throughout the volume of the scaffold under consideration.

Future bioreactors must be compared by their relative levels of perfusion

throughout the volume of the scaffold being studied. Increased perfusion co

Bioreactor designs in the future should use FEM as a design tool. The util

th thod in tissue engineering has been adequately demonstrated in this paper through

a preliminary design. The purpose of this study was to demonstrate key parameters for

bioreactors and introduce a new method for bioreactor design. With these goals in mind,

the design was a success.

123

Closing Remarks:

The field of skeletal tissue engineering is interdisciplinary in nature. This study

ion of tissue engineering,

umero

ing laws have not yet been confirmed. With so

any p

t method was introduced as a novel technique for tissue

s were successfully analyzed.

Additionally, various bioreactor geometries were considered. As a result, key properties

required for bioreactor optimization were determined. It was determined that FEM would

be best-suited as a preliminary mechanism to be coupled with tissue engineering studies.

alone involved skills from cell biology, bioengineering, computer science, and

biochemistry. Such a diverse field requires numerous approaches, and a new method has

been introduced here. Furthermore, the changing landscape of tissue engineering is

dependent upon legislation over cell line restrictions, support through funding sources,

and the capability of in vivo studies. Through an explorat

n us abilities and limitations present in the field have hopefully been illuminated.

Taking a reductionist approach to bioreactor design, individual properties have

been analyzed in lieu of the design of an entire system. Both two-dimensional and three-

dimensional biomaterials were characterized. Biological responses on these materials

were then analyzed and compared. The field of biomaterials is yet another discipline to

be added to the list, one whose govern

m ossible biological responses to materials, a myriad of assays and microscopic

techniques was required. In the end, only the cellular response could be analyzed. This

provided an optimal set of materials with specific parameters upon which cells will

proliferate and differentiate preferentially.

The finite elemen

engineering design purposes. Two-dimensional flow profile

124

As a result, a new approach to tissue engineering is proposed. This process is illustrated

Figure 51. An iterative design process for design of “in vitro” bioreactors, including a

As depicted in Figure 51, the process will begin the computational modeling of

in silico to a bioreactor during fabrication. As before,

stem cells will be harvested and cultured in the in vitro bioreactor. After the study is

These will then be modeled by FEM once again for re-optimization, and the process will

This study was thus successful in demonstrating the complete process as depicted

in Figure 51. All of the specific aims set forth in the Introduction have been

in Figure 51.

Preliminary

Modeling (FEM, in

silico)

Computational

Bioreactor

vitro)

Assessment of

or Failure

Bioreactor Success Fabrication (in

preliminary computational modeling step.

some bioreactor or specific properties to be assessed in a bioreactor. The optimal design

as determined will then be applied

Cell Culture in Stem Cell

Bioreactor (in vitro) Harvesting (in vivo)

complete, data will be analyzed to determine successes and/or failures in the design.

continue. Such an approach deviates slightly from the traditional approach to skeletal

tissue engineering.

125

accomplished, and this study has thus been a success. The field of tissue engineering is a

vast terrain waiting to be explored. These pages have only delved into the outer perimeter

of the field, hopefully whetting one’s appetite for further exploration. It is the hope of this

investigator that such exploration will not only be considered but also pursued.

126

REFERENCES

1. Lim, J.Y., et al., Osteoblast adhesion on poly(L-lactic acid)/polystyrene demixed

thin film blends: effect of nanotopography, surface chemistry, and wettability.

Biomacromolecules, 2005. 6(6): p. 3319-27.

2. Wheeler, D.L. and W.F. Enneking, Allograft bone decreases in streng

over time. Clin Orthop Relat Res, 2005(435): p. 36-42.

th in vivo

3. Bancroft, G.N., V.I. Sikavitsas, and A.G. Mikos, Design of a flow perfusion

bioreactor system for bone tissue-engineering applications. Tissue Eng, 2003.

ltifunctional hydrogels that

promote osteogenic hMSC differentiation through stimulation and sequestering of

BMP2. Adv Funct Mater, 2007. 17(13): p. 2085-2093.

5. Dawson, J.I., et al., Development of specific collagen scaffolds to support the

osteogenic and chondrogenic differentiation of human bone marrow stromal cells.

Biomaterials, 2008. 29(21): p. 3105-16.

6. Trojani, C., et al., Three-dimensional culture and differentiation of human

osteogenic cells in an injectable hydroxypropylmethylcellulose hydrogel.

Biomaterials, 2005. 26(27): p. 5509-17.

7. Zhao, F. and T. Ma, Perfusion bioreactor system for human mesenchymal stem

cell tissue engineering: dynamic cell seeding and construct development.

Biotechnol Bioeng, 2005. 91(4): p. 482-93.

8. Donahue, T.L., et al., Mechanosensitivity of bone cells to oscillating fluid flow

induced shear stress may be modulated by chemotransport. J Biomech, 2003.

36(9): p. 1363-71.

9. Felix Bronner, M.F.-C., Antonios Mikos (eds.), Engineering of functional skeletal

tissues. Vol. 1. 2007: London : Springer.

10. MedLine-Plus. Bone Grafts. [cited; Available from:

http://www.nlm.nih.gov/medlineplus/bonegrafts.html

9(3): p. 549-54.

4. Benoit, D.S., S.D. Collins, and K.S. Anseth, Mu

.

11. Endo, T., S.V. Bryant, and D.M. Gardiner, A stepwise model system for limb

regeneration. Dev Biol, 2004. 270(1): p. 135-45.

12. Ulrich Meyer, H.P.W., Bone and cartilage engineering [electronic resource]

2006, Berlin; New York: Springer.

13. Hollinger, J.O., Bone tissue engineering. 2005, Boca Raton: CRC Press. 363.

14. Mao, J.J., Translational approaches in tissue engineering and regenerative

medicine. 2008, Boston, Mass.: Artech House.

15. Nabil Dib, D.A.T., Edward B. Diethrich, editors, Stem cell therapy and tissue

engineering for cardiovascular repair [electronic resource] : from basic research

to clinical applications 2006, New York, NY: Springer.

16. Al-Rubeai, J.C.a.M., Bioreactors for tissue engineeering [electronic resource] :

principles, design and operation. Vol. 1. 2005, London: Springer.

17. Caplan, V.M.G.a.A.I., Orthopedic tissue engineering : basic science and practice

Vol. XV. 2004, New York: Marcel Dekker. 338.

18. Hatton, A.P.H.a.P.V., Biopolymer methods in tissue engineering Methods in

molecular biology. Vol. XIII. c2004, Totowa, N.J: Humana Press.

127

19. Stocum, D.L., Regenerative biology and medicine. 2006, Amsterdam ; Boston:

Elsevier Academic Press

21.

23.

24.

27.

30.

38.

.

20. Health, N.I.o. Stem Cell Basics. Stem Cell Information 2008 [cited 2008 October

12].

Darwin J. Prockop, D.G.P., Bruce A. Bunnell, Mesenchymal stem cells : methods

and protocols Methods in molecular biology. 2008, Totowa, N.J.: Humana

Press,. 192.

22. Davies, J.A., Branching morphogenesis [electronic resource] Molecular biology

intelligence unit. 2006, Georgetown, Tex.: Landes Bioscience.

Lian, J.B. and G.S. Stein, Development of the osteoblast phenotype: molecular

mechanisms mediating osteoblast growth and differentiation. Iowa Orthop J,

1995. 15: p. 118-40.

Bunting, K.D., Hematopoietic stem cell protocols 2ed. Methods in molecular

biology. 2008, Totowa, N.J.: Humana

25. Growth Factor and Signal Transduction Conference (13th : 2004 : Ames, I. Stem

cell biology : development and plasticity in Growth Factor and Signal

Transduction Conference. 2004. Ames, Iowa: New York Academy of Sciences.

26. Tang, H. (1995) Mathematics of the Finite Element Method. Volume,

Strang, G., An Analysis of the Finite Element Method. 1973: Prentice Hall, Inc.

28. Mathematics, S.f.I.a.A. (2005) Obituary: Germund Dahlquist. Volume,

29. AB., C. (2008) COMSOL Multiphysics. Volume,

Teresi, A.D.S.N.S.Q.L., Modeling Bone Remodeling. Proceedings of the

COMSOL Multiphysics User's Conference 2005 Paris, 2005.

31. de Mel, A., et al., Biofunctionalization of Biomaterials for Accelerated in Situ

Endothelialization: A Review. Biomacromolecules, 2008.

32. McMahon, L.A., F.J. O'Brien, and P.J. Prendergast, Biomechanics and

mechanobiology in osteochondral tissues. Regen Med, 2008. 3(5): p. 743-59.

33. Kretlow, J.D. and A.G. Mikos, Review: mineralization of synthetic polymer

scaffolds for bone tissue engineering. Tissue Eng, 2007. 13(5): p. 927-38.

34. Abousleiman, R.I. and V.I. Sikavitsas, Bioreactors for tissues of the

musculoskeletal system. Adv Exp Med Biol, 2006. 585: p. 243-59.

35. Chromiak, J.A., et al., Bioreactor perfusion system for the long-term maintenance

of tissue-engineered skeletal muscle organoids. In Vitro Cell Dev Biol Anim,

1998. 34(9): p. 694-703.

36. Freed, L.E., et al., Tissue engineering of cartilage in space. Proc Natl Acad Sci U

S A, 1997. 94(25): p. 13885-90.

37. Freed, L.E., et al., Microgravity cultivation of cells and tissues. Gravit Space Biol

Bull, 1999. 12(2): p. 57-66.

G. Vunjak-Novakovic, e.a., Microgravity studies of cells and tissues,. Ann N Y

Acad Sci,, 2002. 974: p. 504-17.

39. Riddle, R.C., A.F. Taylor, and H.J. Donahue, Fluid flow assays. Methods Mol

Biol, 2008. 455: p. 335-45.

40. Alford, A.I., C.R. Jacobs, and H.J. Donahue, Oscillating fluid flow regulates gap

junction communication in osteocytic MLO-Y4 cells by an ERK1/2 MAP kinase-

dependent mechanism small star, filled. Bone, 2003. 33(1): p. 64-70.

128

41. Kim, E.J., et al., Modulating human connective tissue progenitor cell behavior on

cellulose acetate scaffolds by surface microtextures. J Biomed Mater Res A,

42. ., et al., Fabrication and evaluation of calcium phosphate cement scaffold

43. al., Stop-flow lithography to generate cell-laden microgel particles.

44. glycol) hydrogel

45. man foetal osteoblastic cell response to polymer-demixed

46. rst Vogel, Harry Heinzelmann and Martha

47. l

sue Eng,

skeletal

ct, 2004. 4(4): p. 396-8.

graphic patterning.

50. teoblast focal adhesion

p. 504-

52. l growth and

53.

otransduction. J Orthop Res, 2008.

human bone marrow stromal cell proliferation. J

55. ase and calcium signaling mediate fluid flow-induced

l cells. J Bone Miner Res, 2007. 22(4): p. 589-600.

2008.

Li, X

with controlled internal channel architecture and complex shape. Proc Inst Mech

Eng [H], 2007. 221(8): p. 951-8.

Panda, P., et

Lab Chip, 2008. 8(7): p. 1056-61.

Subramani, K. and M.A. Birch, Fabrication of poly(ethylene

micropatterns with osteoinductive growth factors and evaluation of the effects on

osteoblast activity and function. Biomed Mater, 2006. 1(3): p. 144-54.

Lim, J.Y., et al., Hu

nanotopographic interfaces. J R Soc Interface, 2005. 2(2): p. 97-108.

Caterina Minelli, I.G., Rolf Eckert, Ho

Liley, Organization of nanoscale objects via polymer demixing. Colloid and

Polymer Science, 2004. 282(11): p. 1274-1278.

Lim, J.Y., et al., Integrin expression and osteopontin regulation in human feta

osteoblastic cells mediated by substratum surface characteristics. Tis

2005. 11(1-2): p. 19-29.

48. Lim, J.Y. and H.J. Donahue, Biomaterial characteristics important to

tissue engineering. J Musculoskelet Neuronal Intera

49. Lim, J.Y. and H.J. Donahue, Cell sensing and response to micro- and

nanostructured surfaces produced by chemical and topo

Tissue Eng, 2007. 13(8): p. 1879-91.

Lim, J.Y., et al., The regulation of integrin-mediated os

and focal adhesion kinase expression by nanoscale topography. Biomaterials,

2007. 28(10): p. 1787-97.

51. Lim, J.Y., et al., Systematic variation in osteoblast adhesion and phenotype with

substratum surface characteristics. J Biomed Mater Res A, 2004. 68(3):

12.

Lim, J.Y., et al., Surface energy effects on osteoblast spatia

mineralization. Biomaterials, 2008. 29(12): p. 1776-84.

Riddle, R.C. and H.J. Donahue, From streaming-potentials to shear stress: 25

years of bone cell mechan

54. Riddle, R.C., K.R. Hippe, and H.J. Donahue, Chemotransport contributes to the

effect of oscillatory fluid flow on

Orthop Res, 2008. 26(7): p. 918-24.

Riddle, R.C., et al., MAP kin

human mesenchymal stem cell proliferation. Am J Physiol Cell Physiol, 2006.

290(3): p. C776-84.

56. Riddle, R.C., et al., ATP release mediates fluid flow-induced proliferation of

human bone marrow stroma

57. Hansen, J.C., et al., Effect of surface nanoscale topography on elastic modulus of

individual osteoblastic cells as determined by atomic force microscopy. J

Biomech, 2007. 40(13): p. 2865-71.

129

58. Hosseinkhani, H., et al., Bone regeneration through controlled release of bone

morphogenetic protein-2 from 3-D tissue engineered nano-scaffold. J Control

Release, 2007. 117(3): p. 380-6.

6-44.

stem cells using self-assembled peptide

61.

27(22): p.

62. ponge self-

63. collagen sponge self-assembled

64.

ter Res A, 2008. 84(2): p. 291-9.

ermal

66.

d in a flow perfusion bioreactor. J Biomed

67.

cad Sci, 1992. 665: p. 274-84.

9-42.

Proc Natl Acad Sci U S

70. r mesh scaffolds

71. . Mikos, Flow perfusion culture induces the

72. nsen, and A.G. Mikos, Modulation of cell differentiation in

59. Hosseinkhani, H., et al., Enhanced angiogenesis through controlled release of

basic fibroblast growth factor from peptide amphiphile for tissue regeneration.

Biomaterials, 2006. 27(34): p. 583

60. Hosseinkhani, H., M. Hosseinkhani, and H. Kobayashi, Proliferation and

differentiation of mesenchymal

amphiphile nanofibers. Biomed Mater, 2006. 1(1): p. 8-15.

Hosseinkhani, H., et al., Osteogenic differentiation of mesenchymal stem cells in

self-assembled peptide-amphiphile nanofibers. Biomaterials, 2006.

4079-86.

Hosseinkhani, H., et al., Ectopic bone formation in collagen s

assembled peptide-amphiphile nanofibers hybrid scaffold in a perfusion culture

bioreactor. Biomaterials, 2006. 27(29): p. 5089-98.

Hosseinkhani, H., et al., Bone regeneration on a

peptide-amphiphile nanofiber hybrid scaffold. Tissue Eng, 2007. 13(1): p. 11-9.

Tian, F., et al., Quantitative analysis of cell adhesion on aligned micro- and

nanofibers. J Biomed Ma

65. Hee, C.K., M.A. Jonikas, and S.B. Nicoll, Influence of three-dimensional scaffold

on the expression of osteogenic differentiation markers by human d

fibroblasts. Biomaterials, 2006. 27(6): p. 875-84.

Holtorf, H.L., et al., Scaffold mesh size affects the osteoblastic differentiation of

seeded marrow stromal cells culture

Mater Res A, 2005. 74(2): p. 171-80.

Wang, T.Y. and J.H. Wu, A continuous perfusion bioreactor for long-term bone

marrow culture. Ann N Y A

68. Alvarez-Barreto, J.F., et al., Flow perfusion improves seeding of tissue

engineering scaffolds with different architectures. Ann Biomed Eng, 2007. 35(3):

p. 42

69. Datta, N., et al., In vitro generated extracellular matrix and fluid shear stress

synergistically enhance 3D osteoblastic differentiation.

A, 2006. 103(8): p. 2488-93.

Gomes, M.E., et al., Influence of the porosity of starch-based fibe

on the proliferation and osteogenic differentiation of bone marrow stromal cells

cultured in a flow perfusion bioreactor. Tissue Eng, 2006. 12(4): p. 801-9.

Holtorf, H.L., J.A. Jansen, and A.G

osteoblastic differentiation of marrow stroma cell-scaffold constructs in the

absence of dexamethasone. J Biomed Mater Res A, 2005. 72(3): p. 326-34.

Holtorf, H.L., J.A. Ja

bone tissue engineering constructs cultured in a bioreactor. Adv Exp Med Biol,

2006. 585: p. 225-41.

73. Holtorf, H.L., et al., Flow perfusion culture of marrow stromal cells seeded on

porous biphasic calcium phosphate ceramics. Ann Biomed Eng, 2005. 33(9): p.

1238-48.

130

74. Li, X., et al., [Development of rotating perfusion bioreactor system and

application for bone tissue engineering]. Sheng Wu Yi Xue Gong Cheng Xue Za

Zhi, 2007. 24(1): p. 66-70.

75. Sikavitsas, V.I., et al., Mineralized matrix deposition by marrow stromal

osteoblasts in 3D perfusion culture increases with increasing fluid shear forces.

Proc Natl Acad Sci U S A, 2003. 100(25): p. 14683-8.

83.

76. Sikavitsas, V.I., et al., Flow perfusion enhances the calcified matrix deposition of

marrow stromal cells in biodegradable nonwoven fiber mesh scaffolds. Ann

Biomed Eng, 2005. 33(1): p. 63-70.

77. Wang, L., et al., Flow perfusion culture of human fetal bone cells in large beta-

tricalcium phosphate scaffold with controlled architecture. J Biomed Mater Res

A, 2008.

78. Wang, Y., et al., Application of perfusion culture system improves in vitro and in

vivo osteogenesis of bone marrow-derived osteoblastic cells in porous ceramic

materials. Tissue Eng, 2003. 9(6): p. 1205-14.

79. Porter, B., et al., 3-D computational modeling of media flow through scaffolds in

a perfusion bioreactor. J Biomech, 2005. 38(3): p. 543-9.

80. Gang, E.J., et al., SSEA-4 identifies mesenchymal stem cells from bone marrow.

Blood, 2007. 109(4): p. 1743-51.

81. Ouyang, A. and S.T. Yang, A two-stage perfusion fibrous bed bioreactor system

for mass production of embryonic stem cells. Expert Opin Biol Ther, 2008. 8(7):

p. 895-909.

82. Notes, C. Bone Structure. Volume,

Vertebrate Embryology, Brown University.

131

APPENDIX A

ACKNOWLEDGMENTS

• Dr. Henry Donahue - I began working with Dr. Donahue in 2007, and I am very grateful

for all he has provided me as I completed this thesis. Previous work in his lab led to the

insights in oscillating fluid flow that became the cornerstone of this thesis. Without Dr.

•

Donahue, this thesis would not be possible.

Dr. Peter Butler – My work with Dr. Butler spanned two summers of the Biomaterials

and Bionanotechnology Summer Institute (BBSI) and two years in the Department of

Bioengineering. He has provided me with training in COMSOL for the finite element

analyses in this thesis, and I cannot thank him enough for all he has done.

Dr. Jung Yul Lim • – I worked with Dr. Lim under the guidance of Dr. Donahue to develop

the nanoscale substrates in this thesis. Training me at first with a high level of guidance

and later without, he was instrumental in the development of my work in cell culture and

polymer science.

• Dianne McDonald – Mrs. McDonald was the acting administrator of the BBSI program,

and I worked very closely with her to develop myself during those two summers.

• Drs. Yue Zhang and Christopher Niyibizi, Jacqueline Yanoso – These two brilliant

investigators and graduate student assisted me in the flow cytometry studies of stem cells

on 2D substrates.

Govey • Dr. Ryan Riddle, Amanda Taylor, Peter – These students, among others in the

d me heavily in my studies.

• Dr. William Hancock

Department of Musculoskeletal Sciences, assiste

– Dr. Hancock provided insights into writing techniques and

presentations necessary for the completion and defense of this thesis.

• Dr. Margaret Slattery – Dr. Slattery was instrumental in g

in the Department of Bioengineering.

uiding me during my education

• Carol Boring – Without Carol, I would never have graduated in time, and this thesis

would not be complete.

• Bioengineering Faculty – All of the bioengineering faculty provided me with the skills

necessary to complete this work. Dr. Keefe Manning provided a strong foundation in

biofluid mechanics, Dr. Andrew Webb provided a background in statistics, Dr. Ryan

Clement granted me training in MATLAB, and all of the other faculty/staff should be

acknowledged equally.

• SURIP, Step-Up, BBSI Students – These students, some of whom will one day be

colleagues, motivated me and kept me sane when experiments failed. They deserve more

credit than these lines deserve.

• Students of Bioengineering – My fellow students in Bioengineering at Penn State assisted

me in statistical analyses and revising this work. They were also integral in providing

positive feedback throughout the years.

• My Family – This is self-explanatory, but my family provided the most support, a level

of support that I find to be a diamond in the rough.

• All other acquaintances – Those that I met along the way, Students with whom I

interacted, Friends I have made – All of them deserve my eternal thanks for both this

thesis and a wonderful college experience.

132

APPENDIX B

HEALTH INSURANCE PORTABILITY AND

ACCOUNTABILITY ACT (HIPAA)

All materials and information contained in this thesis are believed to be accurate and

nd distributed without alteration.

under

ials should consult professional legal counsel.

reliable; however, the Schreyer Honors College, the Department of Bioengineering, and

The Pennsylvania State University assume no responsibility for the use of the materials

and information.

Unless otherwise noted on an individual document, the Schreyer Honors College grants

permission to copy and distribute files, documents, and information for non-commercial

use provided they are copied a

All human subjects and cell lines have been approved by the IRB at Penn State

compliance with HIPAA.

Parties using these mater

Please , 2002. All

materials dated prior to August 14, 2002 should be reviewed in conjunction with the

note: The Final HIPAA Privacy Rule was published in August 14

Final Privacy Rule published in the federal register on August 14, 2002.

133

APPENDIX C

FUNDING SOURCES

hese studies have been funded jointly by the National Science Foundation (NSF) and

the National Institutes of Health (NIH). The grants established the BBSI, and research

• NSF

T

funds from the BBSI grants were used in the completion of these studies

: EEC-0234026

• NIH: AG13087

Additionally, funding has been provided from the following sources for travel and

• Department of Bioengineering

research:

• Schreyer Honors College

• The Pennsylvania State University

• Pennsylvania Tobacco Fund

134

APPENDIX D

SUPPLEMENTAL SKETCHES

Figure A1. Anatomy of the long bone [82].

135

Figure A2. Pathways for stem cell harvesting and differentiation [83].

136

APPENDIX E

COMMON GROWTH FACTORS

Factor Class/Family Known Types/Examples Applications

BMP1, BMP2, BMP3, BMP4,

Bone Morphogenic Proteins

(BMPs)

BMP5, BMP6, BMP7, BMP8a, Formation of bone and cartilage

BMP8b, BMP10, BMP15

Epidermal Growth Factor

(EGF)

EGF, HB-EGF, Epigen

Regulation of cell growth,

proliferation, and differentiation

Erythropoietin (EPO) EPO, Epogen, Betapoietin Control of erythropoeisis

Fibroblast Growth Factor

(FGF)

FGF1-10, FHF1-4

Angiogenesis, wound healing,

embryonic development

Granulocyte-Colony

Stimulating Factor (G-CSF)

Stimulation of bone marrow to

GCSF/CSF3 produce granulocytes and stem

cells

Granulocyte-Macrophage

Colony Stimulating Factor

(GM-CSF)

Same as above Same as above

Growth Differentiation Factor 9

(GDF9)

Development of the primary

GDF9

follicles in the ovary

Hepatocyte Growth Factor

(HGF)

HGF/SF

Regulation of cell growth, cell

motility and morphogenesis

Insulin-Like Growth Factor

(IGF)

IGF1R, IGF2R, IGF-1, IGF-2, Communication with physiologic

IGFP1-6 environment

Myostatin (GDF-8) GDF-8 Limit of muscle tissue growth

Nerve Growth Factor (NGF)

Differentiation and survival of

NGF, Neutrotrophins

target neurons

Platelet-Derived Growth Factor

(PDGF)

PDGFA, PDGFB, PDGFC,

PDGFD, PDGFAB

Cell growth and division,

Angiogenesis

Thrombopoeitin (TPO)

Production of platelets by the

TPO/THPO

bone marrow

Transforming Growth Factor

Alpha (TGF-α)

TGF-α

Epithelial development, Neural

cell proliferation, Upregulated in

cancer

Transforming Growth Factor

Beta (TGF-β)

Proliferation, Cellular

Differentiation, Immunity,

TGF-β

Cancer, Heart Disease, Diabetes,

Marfan Syndrome

Vascular Endothelial Growth

Factor (VEGF)

VEGF-A, VEGF-B, VEGF-C,

VEGF-D, PIGF

Angiogenesis, Vasculogenesis,

Vasodilation, Lymphangiogenesis,

Inflammation, Wound healing,

Cancer

137

APPENDIX F

COMSOL MODEL REPORTS

COMSOL Model Report – Cell Height Study

1 ents

odel Rep

• Table of Contents

• Constants

• Ge

• Postprocessing

•

2. Model Properties

Property Value

. Table of Cont

• Title - COMSOL M ort

• Model Properties

• Geometry

om1

• Solver Settings

Variables

Model name

Author

Company

Department

Reference

URL

Saved date Jun 19, 2008 2:00:43 PM

C 20 reation date Jun 12, 08 10:36:24 AM

COMS OMSOL 3.4.0 OL version C .248

File name: G:\COMSOL\1.mph

138

Application modes and modules :

• Geom1 (2D

o Inco

o Plane Stress (Structural Mechanics Module)

Name Expression Value Description

used in this model

)

mpressible Navier-Stokes

3. Constants

w 2*pi[rad/s] frequency

Patm 0[Pa]

k 40[Pa/m]

4. Geometry

Number of geometries: 1

4.1. Geom1

139

4.1.1. Point mode

140

4.1.2. Boundary mode

141

4.1.3. Subdomain mode

5. Geom1

Space dimensions: 2D

Independent variables: x, y, z

5.1. Mesh

5.1.1. Mesh Statistics

Number of degrees of freedom 3584

Number of mesh points 224

Number of elements 393

Triangular 393

Quadrilateral 0

Number of boundary elements 53

Number of vertex elements 7

Minimum element quality 0.751

Element area ratio 0.027

142

5.2. Application Mode: Incompressible Navier-

)

mpressible Navier-Stokes

ion Mode Properties

Property Value

Stokes (ns

Application mode type: Inco

Application mode name: ns

5.2.1. Applicat

Default element type Lagrange - P

2

P

1

Analysis type Transient

Corner smoothing Off

Frame Fram f) e (re

Weak constraints Off

Constraint type Ideal

143

5.2.2. Variables

Dependent variables: u, v, p, nxw, nyw

Shape functions: shlag(2,'u'), shlag(2,'v'), shlag(1,'p')

Interior boundaries not active

5.2.3. Boundary Settings

Boundary 1 2, 4, 6-7 3

Type Inlet Wall Wall

intype p uv uv

walltype noslip noslip slip

Pressure (p0) Pa 0.5*k*cos(w*t)+Patm 0 0

Boundary 5

Type Outlet

intype uv

walltype noslip

Pressure (p0) Pa -0.5*k*cos(w*t)+Patm

5.2.4. Subdomain Settings

Subdomain 1

Integration order (gporder) 4 4 2

Constraint order (cporder) 2 2 1

5.3. Application Mode: Plane Stress (smps)

tural Mechanics Module)

mode nam s

e Unit Description

Application mode type: Plane Stress (Struc

Application e: smp

5.3.1. Scalar Variables

Name Variable Valu

t_old_ini t_old_ini_sm Initial condition previous time step (contact

with dynamic friction)

ps -1 s

144

5.3.2. Application Mode Properties

Property Value

Default element type Lagrange - Quadratic

Analysis type Static

Large deformation On

Specify eigenvalues using Eigenfrequency

Create frame On

Deform frame Frame (deform)

Frame Frame (ref)

Weak constrai s Off nt

Constraint typ Ideal e

5.3.3. Variables

Dependent variables: u2, v2, p2

functions: shlag(2,'u2'), shlag(2,'v2')

2 6-7 1, 5

Shape

Interior boundaries not active

5.3.4. Boundary Settings

Boundary -4

Follower pressure (P) Pa 0 p 0

loadcond distr_force follower_press distr_force

constrcond free free fixed

5.3.5. Subdomain Settings

The subdomain settings only contain default values.

6. Solver Settings

Solve using a script: off

Analysis type Transient

Auto select solver On

Solver Time dependent

Solution form Automatic

Symmetric Off

Adaption Off

145

6.1. Direct (PARDISO)

Solver type: Linear system

Va

solver

Parameter lue

Preordering algorithm Nested dissection

Row preordering On

Pivoting perturbation 1.0E-8

Relative tolerance 1.0E-6

Factor in error estimate 400.0

Check tolerances On

6.2. Time Stepping

Value Parameter

Times 0:0.1:3

Relative tolerance 0.01

Absolute tolerance 0.0010

Times to store in output Specified times

Time steps taken by solver Free

Manual tuning of step size Off

Initial time step E-6 1

Maximum time step 1.0

Maximum BDF order 5

Singular mass matrix Maybe

Consistent initialization of DAE systems ackward Eu B ler

Error estimation strategy Exclude algebraic

Allow complex numbers Off

6.3. Advanced

Parameter Value

Constraint handling method Elimination

Null-space function Automatic

Assembly block siz 1000 e

Use Hermitian transpose of constraint matrix and in symmetry

n

Off

detectio

Use complex funct al input Off ions with re

Stop if error due to efined operation On und

Store solution on f Off ile

146

Type of scaling Automatic

Manual scaling

Row equilibration On

Manual control of reassembly Off

Load constant On

Constraint constant On

Mass constant On

Damping (mass) consta On nt

Jacobian constant On

Constraint Jacobian constant On

7. Postprocessing

147

8. Variables

ion Unit Expression

8.1. Point

Name Descript

Fxg_smps Point load in global x dir. N 0

Fyg_smps Point load in global y dir. N 0

disp_smps Total displacement m sqrt(real(u2)^2+real(v2)^2)

8.2. Boundary

8.2.1. Boundary 1-5

Name Description Unit Expression

K_x_ns Viscous force per

area, x

component

Pa eta_ns * (2 * nx_ns * ux+ny_ns * (uy+vx))

T_x_ns Total force per

area, x

component

Pa -nx_ns * p+2 * nx_ns * eta_ns * ux+ny_ns *

eta_ns * (uy+vx)

K_y_ns Viscous force per

area, y

component

Pa eta_ns * (nx_ns * (vx+uy)+2 * ny_ns * vy)

T_y_ns Total force per

area, y

component

Pa -ny_ns * p+nx_ns * eta_ns * (vx+uy)+2 *

ny_ns * eta_ns * vy

Fxg_smps Edge load in

global x-dir.

N/m 0

Fyg_smps Edge load in

global y-dir.

N/m 0

disp_smps Total

displacement

m sqrt(real(u2)^2+real(v2)^2)

Tax_smps Surface traction

(force/area) in x

dir.

Pa (F11_smps * Sx_smps+F12_smps *

Sxy_smps) * nx_smps+(F11_smps *

Sxy_smps+F12_smps * Sy_smps) *

ny_smps

Tay_smps Surface traction Pa (F21_smps * Sx_smps+F22_smps *

(force/area) in y

dir.

Sxy_smps) * nx_smps+(F21_smps *

Sxy_smps+F22_smps * Sy_smps) *

ny_smps

148

8.2.2. Boundary 6-7

ription Unit Expression Name Desc

K_x_ns Viscous force per

area, x

Pa eta_ns * (2 * nx_ns * ux+ny_ns * (uy+vx))

component

T_x_ns Total force per Pa -

area, x e

component

nx_ns p+2 * nx_ns * eta_ns * ux+ny_ns *

ns (uy+vx)

*

ta_ *

K_y_ns Viscous force per Pa eta_ns * (nx_ns * (vx+uy)+2 * ny_ns * vy)

area, y

component

T_y_ns Total force pe

area, y

r

component

Pa -ny_ns * p+nx_ns * eta_ns * (vx+uy)+2 *

ny_ns * eta_ns * vy

Fxg_smps P_smps * dvol_deform * Edge load in

global x-dir.

N/m -nx2_smps *

(1+wz_smps) * thickness_smps/dvol

Fyg_smps N/m -ny2_smps * P_smps * dvol_deform * Edge load in

global y-dir. (1+wz_smps) * thickness_smps/dvol

disp_smps

t

Total

displacemen

m sqrt(real(u2)^2+real(v2)^2)

Tax_smps

Sxy_smps+F12_smps * Sy_smps) *

ny_smps

Surface traction

(force/area) in x

dir.

Pa (F11_smps * Sx_smps+F12_smps *

Sxy_smps) * nx_smps+(F11_smps *

Tay_smps

s+(F21_smps *

_smps+F22_smps * Sy_smps) *

_smps

Surface traction

(force/area) in y

dir.

Pa (F21_smps * Sx_smps+F22_smps *

Sxy_smps) * nx_smp

Sxy

ny

8.3. Subdomain

Unit Name Description Expression

U_ns Velocity field m/s sqrt(u^2+v^2)

V_ns Vorticity 1/s vx-uy

divU_ns Divergence of

velocity field

1/s ux+vy

cellRe_ns Cell Reynolds

number

1 rho_ns * U_ns * h/eta_ns

res_u_ns Equation

residual for u

N/m^3 _x_ns-

s * (2 * uxx+uyy+vxy)

rho_ns * (ut+u * ux+v * uy)+px-F

eta_n

res_sc_u_ns Shock

capturing

N/m^3 rho_ns * (ut+u * ux+v * uy)+px-F_x_ns

149

residual for u

res_v_ns Equation

v

N/m^3 rho_ns * (vt+u * vx+v * vy)+py-F_y_ns-

(vxx+uyx+2 * vyy) residual for eta_ns *

res_sc_v_n

ing

r v

3 s Shock

captur

residual fo

N/m^ rho_ns * (vt+u * vx+v * vy)+py-F_y_ns

beta_x_ns Convective

t

k ^

field, x

componen

g/(m 2*s) rho_ns * u

beta_y_ns

t

^ Convective

field, y

componen

kg/(m 2*s) rho_ns * v

Dm_ns Mean

diffusio

coefficient

n

Pa*s eta_ns

da_ns Total time

scale factor

k 3 g/m^ rho_ns

taum_ns GLS time-

scale

m

h)))

^3*s/kg nojac(min(timestep/rho_ns,0.5 *

h/max(rho_ns * U_ns,6 * eta_ns/

tauc_ns GLS

scale

time- m^2/s in(1,rho_ns *

U_ns * h/eta_ns))

nojac(0.5 * U_ns * h * m

Fxg_smps Body load in

global x-dir.

N 2 /m^ 0

Fyg_smps dy load in

global y-dir.

N/m^2 Bo 0

disp_smps Total

displacement

m sqrt(real(u2)^2+real(v2)^2)

sx_smps sx normal

stress global

Pa

smps+Sxy_smps *

F12_smps)+F12_smps * (Sxy_smps *

F11_smps+Sy_smps *

F12_smps))/J_smps

(F11_smps * (Sx_smps *

F11_

sys.

sy_smps (Sx_smps *

y_smps *

2_smps * (Sxy_smps *

ps+Sy_smps *

ps))/J_smps

sy normal

stress global

sys.

Pa (F21_smps *

F21_smps+Sx

ps)+F2 F22_sm

F21_sm

F22_sm

sxy_smps sxy shear

stress global

sys.

Pa (F11_smps * (Sx_smps *

F21_smps+Sxy_smps *

F22_smps)+F12_smps * (Sxy_smps *

F21_smps+Sy_smps *

F22_smps))/J_smps

ex_smps ex norm

strain glob

al

al

1 u2x+0.5 * (u2x^2+v2x^2)

150

sys.

ey_smps ey normal

strain global

sys.

1 v2y+0.5 * (u2y^2+v2y^2)

ez_smps 1 -nu_smps * (ex_smps/((1+nu_smps) *

(1-2 *

ey_smps/((1+nu_smps) * (1-

2 * nu_smps))) * (1+nu_smps) * (1-2 *

nu_smps)/(1-nu_smps)

ez normal

strain

nu_smps))+

exy_smps exy shear

strain gl

sys.

obal

2x+u2x * u2y+v2x * v2y) 1 0.5 * (u2y+v

Sx_smps ond * ((1-nu_smps) *

ex_smps/((1+nu_smps) * (1-2 *

nu_smps))+nu_smps *

s/((1+nu_smps) * (1-2 *

Sx Sec

Piola-

Kirchhoff

global sys.

Pa E_smps

ey_smp

nu_smps))+nu_smps *

ez_smps/((1+nu_smps) * (1-2 *

nu_smps)))

Sy_smps

mps) *

_smps/((1+nu_smps) * (1-2 *

nu_smps))+nu_smps *

_smps/((1+nu_smps) * (1-2 *

nu_smps)))

Sy Second

Piola-

Kirchhoff

global sys.

Pa E_smps * (nu_smps *

ex_smps/((1+nu_smps) * (1-2 *

nu_smps))+(1-nu_s

ey

ez

Sz_smps Sz Se

Piola-

cond

Kirchhoff

global sys.

Pa 0

Sxy_smps Sxy econd

Piola-

Kirchhoff

Pa S

global sys.

E_smps * exy_smps/(1+nu_smps)

wz_smps

tive of

out-of-plane

displacement

1 sqrt(1+2 * Out of plane

deriva

if(1+2 * ez_smps<0,-1,-1+

ez_smps))

K_smps Bulk modulus s)) Pa E_smps/(3 * (1-2 * nu_smp

G_smps

lus

Pa Shear

modu

0.5 * E_smps/(1+nu_smps)

mises_smps von Mises Pa sqrt(sx_smps^2+sy_smps^2-sx_smps *

) stress sy_smps+3 * sxy_smps^2

Ws_smps Strain energy ^2 _smps * J/m 0.5 * thickness_smps * (ex

151

density sx_smps+ey_smps * sy_smps+2 *

exy_smps * sxy_smps)

evol_smps 1 -1+Jel_smps Volumetric strain

F11_smps Deformation 1 1+u2x

gradient 11

comp.

F12_smps Deformation

gradient 12

comp.

1 u2y

F21_smps Deformation 1

gradient 21

comp.

v2x

F22_smps Deformation

nt 22

1 1+v2y

gradie

comp.

F33_smps Deformation 1

gradient 33

comp.

1+wz_smps

detF_smps Determina

deformation

gradient

1 12_smps nt of F33_smps * (F11_smps * F22_smps-F

* F21_smps)

J_smps Volume ratio 1 detF_smps

Jel_smps Elastic volume

ratio

1 J_smps

invF11_smps

comp.

1 ps Inverse of

deformation

gradient 11

F22_smps * F33_smps/detF_sm

invF12_smps Inverse of 1

deformation

gradient 12

comp.

-F12_smps * F33_smps/detF_smps

invF21_smps 1 -F21_smps * F33_smps/detF_smps Inverse of

deformation

gradient 21

comp.

invF22_smps Inverse

1 F11_smps * F33_smps/detF_smps of

deformation

gradient 22

comp.

invF33_smps

tF_smps

Inverse of

deformation

gradient 33

comp.

1 (F11_smps * F22_smps-F21_smps *

F12_smps)/de

sz_smps sz normal stress

s. global sy

Pa 0

tresca_smps s

s2_smps),abs(s2_smps-

Tresca stres Pa max(max(abs(s1_smps-

s3_smps)),abs(s1_smps-s3_smps))

152

COMSOL el Report – C

1. Table of Contents

e - ode eport

• Table of Contents

el

• Geome

• Geom1

olver

stpr

• Variab

2. Model Properties

Mod ell Confluence Study

• Titl COMSOL M l R

• Mod Properties

try

• S Settings

• Po ocessing

les

Property Value

Model name

Author

Company

Department

Reference

URL

Saved date 2008 2:06:39 PM Jun 6,

Creation date un 6, 2008 1:31:37 PM J

COMSOL ver OL .0.248 sion COMS 3.4

File name: G:\COMSOL\10%-Confluency.mph

Application modes and modules used in this model:

• Geom1 (2D)

o Plane Strain (Structural Me

o Incompressible Navier-Stokes

chanics Module)

153

3. Geometry

Number of geometries: 1

3.1. Geom1

154

3.1.1. Point mode

155

3.1.2. Boundary mode

156

3.1.3. Subdomain mode

4. Geom1

Space dimensions: 2D

Independent variables: x, y, z

4.1. Mesh

4.1.1. Mesh Statistics

Number of degrees of freedom 28861

Number of mesh points 1757

Number of elements 3263

Triangular 3263

Quadrilateral 0

Number of boundary elements 249

Number of vertex elements 28

Minimum element quality 0.691

Element area ratio 0

157

4.2. Application Mode: Plane Strain (smpn)

type: Plane Strain (Structural Mechanics Module)

: smpn

Value Unit Description

Application mode

Application mode name

4.2.1. Scalar Variables

Name Variable

t_old_ini t_old_ini_smpn -1 s Initial condition previous time step (contact

with dynamic friction)

4.2.2. Application Mode Properties

Property Value

Default element type ange - Quadratic Lagr

Analysis type tatic S

Large deformation On

Specify eigenvalues using nfrequency Eige

Create frame Off

158

Deform frame Frame (ref)

Frame Frame (ref)

Weak constraints Off

Constraint type Ideal

4.2.3. Variables

Dependent variables: u2, v2, p2

Shape functions: shlag(2,'u2'), shlag(2,'v2')

Interior boundaries not active

4.2.4. Subdomain Settings

Subdomain 1

name Solid domain

4.3. Application Mode: Incompressible Navier-

Stokes (ns)

Application mode type: Incompressible Navier-Stokes

Application mode name: ns

4.3.1. Application Mode Properties

Property Value

Default element type Lagrange - P

2

P

1

Analysis type Stationary

Corner s Off moothing

Frame Frame (ref)

Weak constraints Off

Constraint type Ideal

4.3.2. Variables

u, v,

lag(2,'u lag(2,'v'), shlag(1,'p')

Dependent variables: p, nxw, nyw

Shape functions: sh '), sh

Interior boundaries not active

159

4.3.3. Boundary Settin

1 2-11, 13-28 12

gs

Boundary

Type Inlet Wall Open boundary

Normal inflow velocity (U0in) m/s 0.38095238 1 1

4.3.4. Subdomain Settings

Subdomain 1

Integration order (gporder) 4 4 2

Constraint order (cporder) 2 2 1

5. Solver Settings

Solve using a script: off

is type Analys Static

Auto select solver On

Solver Stationary

Solution form Automatic

Symmetric auto

Adaption Off

5.1. Direct (PARDISO)

Solver type: Linear system solver

Parameter Value

Preordering algorithm issection Nested d

Row preordering On

Pivoting perturbation 1.0E-8

Relative tolerance 1.0E-6

Factor in error estima .0 te 400

Check tolerances On

160

5.2. Stationary

Parameter Value

Linearity utoma A tic

Relative tolerance 1.0E-6

Maximum number of iterations 25

Manual tuning of damping parameters Off

Highly nonlinear problem On

Initial damping factor 1.0

Minimum damping factor 1.0E-4

Restriction for step size upd 10.0 ate

5.3. Advanced

Parameter Value

Constraint handlin d Elimination g metho

Null-space function Automatic

Assembly block siz 1000 e

Use Hermitian tran onstraint matrix and in symmetry Off spose of c

detection

Use complex funct ith real input Off ions w

Stop if error due to undefined operation On

Store solution on file Off

Type of scaling None

Manual scaling

Row equilibration On

Manual control of reasse Off mbly

Load constant On

Constraint constant On

Mass constant On

Damping (mass) consta On nt

Jacobian constant On

Constraint Jacobian con t On stan

161

6. Postprocessing

s

ion Unit Expression

7. Variable

7.1. Point

Name Descript

Fxg_smpn Point load in global x dir. N 0

Fyg_smpn Point load in global y dir. N 0

disp_smpn Total displacement m sqrt(real(u2)^2+real(v2)^2)

7.2. Boundary

Name Description Unit Expression

Fxg_smpn Edge load in

global x-dir.

N/m 0

Fyg_smpn Edge load in

global y-dir.

N/m 0

162

disp_smpn Total

displacement

m sqrt(real(u2)^2+real(v2)^2)

Tax_smpn Surface traction

(force/area) in x

dir.

Pa (F11_smpn * Sx_smpn+F12_smpn *

Sxy_smpn) * nx_smpn+(F11_smpn *

Sxy_smpn+F12_smpn * Sy_smpn) *

ny_smpn

Tay_smpn Surface traction

(force/area) in y

dir.

Pa (F21_smpn * Sx_smpn+F22_smpn *

Sxy_smpn) * nx_smpn+(F21_smpn *

Sxy_smpn+F22_smpn * Sy_smpn) *

ny_smpn

K_x_ns Viscous force per

area, x

component

Pa eta_ns * (2 * nx_ns * ux+ny_ns * (uy+vx))

T_x_ns Total force per

area, x

component

Pa -nx_ns * p+2 * nx_ns * eta_ns * ux+ny_ns *

eta_ns * (uy+vx)

K_y_ns Viscous force per

area, y

component

Pa eta_ns * (nx_ns * (vx+uy)+2 * ny_ns * vy)

T_y_ns Total force per

area, y

component

Pa -ny_ns * p+nx_ns * eta_ns * (vx+uy)+2 *

ny_ns * eta_ns * vy

7.3. Subdomain

iption Unit Expression Name Descr

Fxg_smpn Body load in

bal x-dir.

N/m^2 0

glo

Fyg_smpn Body load in

global y-dir.

N/m^2 0

disp_smpn m sqrt(real(u2)^2+real(v2)^2) Total

displacemen

t

sx_smpn sx normal Pa

stress global

F

F11_smpn+Sxy_smpn *

F12_smpn)+F12_smpn * (Sxy_smpn *

F11_smpn+Sy_smpn *

F12_smpn))/J_smpn

( 11_smpn * (Sx_smpn *

sys.

sy_smpn

l

Pa n * (Sx_smpn *

F21_smpn+Sxy_smpn *

F22_smpn)+F22_smpn * (Sxy_smpn *

F21_smpn+Sy_smpn *

F22_smpn))/J_smpn

sy normal

stress globa

sys.

(F21_smp

sz_smpn sz normal Pa Sz_smpn * F33_smpn^2/J_smpn

163

stre

sys.

ss global

sxy_smpn sxy shear

stress global

sy

P

s.

a

pn *

mpn+Sy_smpn *

(F11_smpn * (Sx_smpn *

F21_smpn+Sxy_smpn *

F22_smpn)+F12_smpn * (Sxy_sm

F21_s

F22_smpn))/J_smpn

ex_smpn

rain global

sys.

ex normal

st

1 u2x+0.5 * (u2x^2+v2x^2)

ey_smpn

global

ey normal

strain

sys.

1 v2y+0.5 * (u2y^2+v2y^2)

exy_smpn

global

1 exy shear

strain

sys.

0.5 * (u2y+v2x+u2x * u2y+v2x * v2y)

Sx_smpn

ex_smpn/((1+nu_smpn) * (1-2 *

nu_smpn))+nu_smpn *

Sx Second

Piola-

Kirchhoff

Pa

global sys.

E_smpn * ((1-nu_smpn) *

ey_smpn/((1+nu_smpn) * (1-2 *

nu_smpn)))

Sy_smpn d

Piola-

Pa E_smpn * (nu_smpn *

ex_smpn/((1+nu_smpn) * (1-2 *

nu_smpn))+(1-nu_smpn) *

ey_smpn/((1+nu_smpn) * (1-2 *

Sy Secon

Kirchhoff

global sys.

nu_smpn)))

Sz_smpn Sz Second

Piola-

Kirchhoff

global sys.

Pa E_smpn * nu_smpn *

_smpn/((1+nu_smpn) * (1-2 *

nu_smpn))+ey_smpn/((1+nu_smpn) * (1-

nu_smpn)))

(ex

2 *

Sxy_smpn

obal sys.

Pa E_smpn * exy_smpn/(1+nu_smpn) Sxy Second

Piola-

Kirchhoff

gl

K_smpn Bulk

modulus

Pa E_smpn/(3 * (1-2 * nu_smpn))

G_smpn Shear

modulus

Pa 0.5 * E_smpn/(1+nu_smpn)

mises_smpn von Mises

stress

Pa sqrt(sx_smpn^2+sy_smpn^2+sz_smpn^2

-sx_smpn * sy_smpn-sy_s

sz_smpn-sx_smpn * sz_smpn+3 *

sxy_smpn^2)

mpn *

Ws_smpn Strain

energy

J/m^2 ex_smpn *

sy_smpn+2 *

0.5 * thickness_smpn * (

sx_smpn+ey_smpn *

164

density exy_smpn * sxy_smpn)

evol_smpn Volumetric 1 -1+Jel_smpn

strain

ez_smpn

global

sys.

1 ez normal

strain

0

F11_smpn Deformation

1

gradient 11

comp.

1+u2x

F12_smpn Deformation

1 u2y

gradient 12

comp.

F21_smpn Deformation

1 v2x

gradient 21

comp.

F22_smpn Deformation

1 1+v2y

gradient 22

comp.

F33_smpn Deformation

comp.

1

gradient 33

1+ez_smpn

detF_smpn

mpn- Determinant

of

deformation

gradient

1 F33_smpn * (F11_smpn * F22_s

F12_smpn * F21_smpn)

J_smpn Volume ratio 1 detF_smpn

Jel_smpn Elastic

volume ratio

1 J_smpn

invF11_smp 1

n

Inverse of

deformation

gradient 11

comp.

F22_smpn * F33_smpn/detF_smpn

invF12_smp

2

1 -F12_smpn * F33_smpn/detF_smpn

n

Inverse of

ation deform

gradient 1

comp.

invF21_smp

_smpn

n

Inverse of

deformation

gradient 21

comp.

1 -F21_smpn * F33_smpn/detF

invF22_smp Inverse of

t 22

1

n deformation

gradien

comp.

F11_smpn * F33_smpn/detF_smpn

165

invF33_smp f

t 33

1 F21_smpn *

tF_smpn n

Inverse o

deformation

gradien

comp.

(F11_smpn * F22_smpn-

F12_smpn)/de

tresca_smpn

Pa max(max(abs(s1_smpn-

s2_smpn),abs(s2_smpn-

n)),abs(s1_smpn-s3_smpn))

Tresca

stress

s3_smp

U_ns Velocity m/s sqrt(u^2+v^2) field

V_ns Vorticity 1/s vx-uy

divU_ns Divergence

of velocity

field

1/s y ux+v

cellRe_ns Cell

Reynolds

number

ns * U_ns * h/eta_ns 1 rho_

res_u_ns Equation

residual for u

N/m^3 * (u * ux+v * uy)+px-F_x_ns-

eta_ns * (2 * uxx+uyy+vxy)

rho_ns

res_sc_u_ns Shock N/m^3 rho_ns * (u * ux+v * uy)+px-F_x_ns

capturing

residual for u

res_v_ns Equation N/m^3 rho_ns * (u * vx+v * vy)+py-F_y_ns-

residual for v eta_ns * (vxx+uyx+2 * vyy)

res_sc_v_ns Shock

or v

N/m^3 y-F_y_ns

capturing

residual f

rho_ns * (u * vx+v * vy)+p

beta_x_ns /(m^2*s Convective

field, x

component

kg

)

rho_ns * u

beta_y_ns Convective

field, y

component

/(m^2*s

)

kg rho_ns * v

Dm_ns Mean Pa*s eta_ns

diffusion

coefficient

da_ns Total

ctor

kg/m^3 rho_ns time

scale fa

taum_ns GLS time-

scale

m^3*s/kg

eta_ns/h))

nojac(0.5 * h/max(rho_ns * U_ns,6 *

tauc_ns m^2/s nojac(0.5 * U_ns * h * min(1,rho_ns *

U_ns * h/eta_ns))

GLS time-

scale

166

COMSOL R ort –

1. Table of Contents

• Title - COMSOL Model Report

• Table of Contents

el

• Consta

• Geom1

• Solver

tpro

• Variabl

2. Model Properties

Model ep Young’s Modulus Study

• Mod Properties

nts

• Geometry

Settings

• Pos cessing

es

Property Value

Model name

Author

Company

Department

Reference

URL

Saved date 008 1:32:46 PM Jul 9, 2

Creation date , 2 6:2 Jun 12 008 10:3 4 AM

COMSOL ver L 3.4.0.248 sion COMSO

File name: G:\COMSOL\11 nm.mph

Application m d modules used in

• Geom1 (2D)

o Plane Stress (Structural Mechanics Module)

odes an this model:

o Incompressible Navier-Stokes

167

3. Constants

Name Expression Value Description

w 2*pi[rad/s] frequency

Patm 0[Pa]

k 40[Pa/m]

4. Geometry

Number of geometries: 1

4.1. Geom1

168

4.1.1. Point mode

169

4.1.2. Boundary mode

170

4.1.3. Subdomain mode

5. Geom1

Space dimensions: 2D

Independent variables: x, y, z

5.1. Mesh

5.1.1. Mesh Statistics

Number of degrees of freedom 2451

Number of mesh points 155

Number of elements 265

Triangular 265

Quadrilateral 0

Number of boundary elements 43

Number of vertex elements 7

Minimum element quality 0.741

Element area ratio 0.077

171

5.2. Application Mode: Incompressible Navier-

)

mpressible Navier-Stokes

ion Mode Properties

Property Value

Stokes (ns

Application mode type: Inco

Application mode name: ns

5.2.1. Applicat

Default element type Lagrange - P

2

P

1

Analysis type Transient

Corner smoothing Off

Frame Fram f) e (re

Weak constraints Off

Constraint type Ideal

172

5.2.2. Variables

Dependent variables: u, v, p, nxw, nyw

Shape functions: shlag(2,'u'), shlag(2,'v'), shlag(1,'p')

Interior boundaries not active

5.2.3. Boundary Settings

Boundary 1 2, 4, 6-7 3

Type Inlet Wall Wall

intype p uv uv

walltype noslip noslip slip

Pressure (p0) Pa 0.5*k*cos(w*t)+Patm 0 0

Boundary 5

Type Outlet

intype uv

walltype noslip

Pressure (p0) Pa -0.5*k*cos(w*t)+Patm

5.2.4. Subdomain Settings

Subdomain 1

Integration order (gporder) 4 4 2

Constraint order (cporder) 2 2 1

5.3. Application Mode: Plane Stress (smps)

tural Mechanics Module)

mode nam s

e Unit Description

Application mode type: Plane Stress (Struc

Application e: smp

5.3.1. Scalar Variables

Name Variable Valu

t_old_ini t_old_ini_sm Initial condition previous time step (contact

with dynamic friction)

ps -1 s

5.3.2. Application Mode Properties

Property Value

173

Default element type Lagrange - Quadratic

Analysis type Static

Large deformation On

Specify eigenvalues using Eigenfrequency

Create frame On

Deform frame Frame (deform)

Frame Frame (ref)

Weak constraints Off

Constraint type Ideal

5.3.3. Variab s

nt variables: u2, v2, p2

r bound e tive

und r ngs

Boundary 2-4 6-7 1, 5

le

Depende

Shape functions: shlag(2,'u2'), shlag(2,'v2')

Interio ari s not ac

5.3.4. Bo a y Setti

Follower pressure (P) Pa 0 p 0

loadcond distr_force follower_press distr_force

constrcond free free fixed

5.3.5. Subdomain Settings

Subdomain 1

Young's modulus (E) Pa 9000

6. Solver Settings

Analysis type Transient

Solve using a script: off

Auto sele On ct solver

Solver Time depend t en

Solution form Automatic

Symmetric Off

Adaption Off

174

175

6.1. Direct (PARDISO)

Solver type: Linear system solver

Parameter Value

Preordering algorithm Nested dissection

Row preordering On

Pivoting perturbation 1.0E-8

Relative tolerance 1.0E-6

Factor in error estimate 400.0

Check tolerances On

6.2. Time Stepping

Parameter Value

Times 0:0.1:3

Relative tolerance 0.01

Absolute tolerance 0.0010

Times to store in output Specified times

Time steps taken by solver Free

Manual tuning of step size Off

Initial time step 1E-6

Maximum time step 1.0

Maximum BDF order 5

Singular mass matrix Maybe

Consistent initialization of DAE systems Backward Euler

Error estimation strategy Exclude algebraic

Allow complex numbers Off

6.3. Advanced

Parameter Value

Constraint handling method Elimination

Null-space function Automatic

Assembly block size 1000

Use Hermitian transpose of constraint matrix and in symmetry

detection

Off

Use complex functions with real input Off

Stop if error due to undefined operation On

Store solution on file Off

176

Type of scaling Automatic

Manual scaling

Row equilibration On

Manual control of reassembly Off

Load constant On

Constraint constant On

Mass constant On

Damping (mass) consta On nt

Jacobian constant On

Constraint Jacobian constant On

7. Postprocessing

177

8. Variables

ion Unit Expression

8.1. Point

Name Descript

Fxg_smps Point load in global x dir. N 0

Fyg_smps Point load in global y dir. N 0

disp_smps Total displacement m sqrt(real(u2)^2+real(v2)^2)

8.2. Boundary

8.2.1. Boundary 1-5

Name Description Unit Expression

K_x_ns Viscous force per

area, x

component

Pa eta_ns * (2 * nx_ns * ux+ny_ns * (uy+vx))

T_x_ns Total force per

area, x

component

Pa -nx_ns * p+2 * nx_ns * eta_ns * ux+ny_ns *

eta_ns * (uy+vx)

K_y_ns Viscous force per

area, y

component

Pa eta_ns * (nx_ns * (vx+uy)+2 * ny_ns * vy)

T_y_ns Total force per

area, y

component

Pa -ny_ns * p+nx_ns * eta_ns * (vx+uy)+2 *

ny_ns * eta_ns * vy

Fxg_smps Edge load in

global x-dir.

N/m 0

Fyg_smps Edge load in

global y-dir.

N/m 0

disp_smps Total

displacement

m sqrt(real(u2)^2+real(v2)^2)

Tax_smps Surface traction

(force/area) in x

dir.

Pa (F11_smps * Sx_smps+F12_smps *

Sxy_smps) * nx_smps+(F11_smps *

Sxy_smps+F12_smps * Sy_smps) *

ny_smps

Tay_smps Surface traction Pa (F21_smps * Sx_smps+F22_smps *

(force/area) in y

dir.

Sxy_smps) * nx_smps+(F21_smps *

Sxy_smps+F22_smps * Sy_smps) *

ny_smps

178

8.2.2. Boundary 6-7

ription Unit Expression Name Desc

K_x_ns Viscous force per

area, x

Pa eta_ns * (2 * nx_ns * ux+ny_ns * (uy+vx))

component

T_x_ns Total force per Pa -

area, x e

component

nx_ns p+2 * nx_ns * eta_ns * ux+ny_ns *

ns (uy+vx)

*

ta_ *

K_y_ns Viscous force per Pa eta_ns * (nx_ns * (vx+uy)+2 * ny_ns * vy)

area, y

component

T_y_ns Total force pe

area, y

r

component

Pa -ny_ns * p+nx_ns * eta_ns * (vx+uy)+2 *

ny_ns * eta_ns * vy

Fxg_smps P_smps * dvol_deform * Edge load in

global x-dir.

N/m -nx2_smps *

(1+wz_smps) * thickness_smps/dvol

Fyg_smps N/m -ny2_smps * P_smps * dvol_deform * Edge load in

global y-dir. (1+wz_smps) * thickness_smps/dvol

disp_smps

t

Total

displacemen

m sqrt(real(u2)^2+real(v2)^2)

Tax_smps

Sxy_smps+F12_smps * Sy_smps) *

ny_smps

Surface traction

(force/area) in x

dir.

Pa (F11_smps * Sx_smps+F12_smps *

Sxy_smps) * nx_smps+(F11_smps *

Tay_smps

s+(F21_smps *

_smps+F22_smps * Sy_smps) *

_smps

Surface traction

(force/area) in y

dir.

Pa (F21_smps * Sx_smps+F22_smps *

Sxy_smps) * nx_smp

Sxy

ny

8.3. Subdomain

Unit Name Description Expression

U_ns Velocity field m/s sqrt(u^2+v^2)

V_ns Vorticity 1/s vx-uy

divU_ns Divergence of

velocity field

1/s ux+vy

cellRe_ns Cell Reynolds

number

1 rho_ns * U_ns * h/eta_ns

res_u_ns Equation

residual for u

N/m^3 _x_ns-

s * (2 * uxx+uyy+vxy)

rho_ns * (ut+u * ux+v * uy)+px-F

eta_n

res_sc_u_ns Shock

capturing

N/m^3 rho_ns * (ut+u * ux+v * uy)+px-F_x_ns

179

residual for u

res_v_ns Equation

v

N/m^3 rho_ns * (vt+u * vx+v * vy)+py-F_y_ns-

(vxx+uyx+2 * vyy) residual for eta_ns *

res_sc_v_n

ing

r v

3 s s Shock

captur

residual fo

N/m^ rho_ns * (vt+u * vx+v * vy)+py-F_y_n

beta_x_ns Convective

t

k

field, x

componen

g/(m^2*s) rho_ns * u

beta_y_ns

t

Convective

field, y

componen

kg/(m^2*s) rho_ns * v

Dm_ns Mean diffusion

coeffic

ient

P a*s eta_ns

da_ns Total

kg/m^3 rho_ns time

scale factor

taum_ns m^3*s/kg nojac(min(timestep/rho_ns,0.5

)))

GLS time-

scale

*

h/max(rho_ns * U_ns,6 * eta_ns/h

tauc_ns

e

m^2/s ns * GLS time-

scal

nojac(0.5 * U_ns * h * min(1,rho_

U_ns * h/eta_ns))

Fxg_smps N/m^2 0 Body load in

global x-dir.

Fyg_smps

bal y-dir.

N/m^2 Body load in

glo

0

disp_smps Total

displacement

m sqrt(real(u2)^2+real(v2)^2)

sx_smps

ess global

sys.

Pa

smps)+F12_smps * (Sxy_smps *

F11_smps+Sy_smps *

F12_smps))/J_smps

sx normal

str

(F11_smps * (Sx_smps *

F11_smps+Sxy_smps *

F12_

sy_smps sy normal Pa (F21_smps * (Sx_smps *

xy_smps *

2_smps * (Sxy_smps *

ps))/J_smps

stress global

sys.

F21_smps+S

F22_smps)+F2

ps+Sy_smps * F21_sm

F22_sm

sxy_smps ps * (Sx_smps *

y_smps *

F21_smps+Sy_smps *

sxy shear stress

global sys.

Pa (F11_sm

F21_smps+Sxy_smps *

F22_smps)+F12_smps * (Sx

F22_smps))/J_smps

ex_smps

s.

1 ex normal strain

global sy

u2x+0.5 * (u2x^2+v2x^2)

ey_smps train 1 v2y+0.5 * (u2y^2+v2y^2) ey normal s

180

global sys.

ez_smps ez normal strain

smps) * (1-2

1 -nu_smps * (ex_smps/((1+nu_smps) * (1-2

* nu_smps))+ey_smps/((1+nu_

* nu_smps))) * (1+nu_smps) * (1-2 *

nu_smps)/(1-nu_smps)

exy_smps 1 0.5 * (u2y+v2x+u2x * u2y+v2x * v2y) exy shear strain

global sys.

Sx_smps Sx Second

Piola-Kirchhoff

global sys.

Pa E_smps * ((1-nu_smps) *

ex_smps/((1+nu_smps) * (1-2 *

nu_smps))+nu_smps *

+nu_smps) * (1-2 *

nu_smps))+nu_smps *

ez_smps/((1+nu_smps) * (1-2 * nu_smps)))

ey_smps/((1

Sy_smps * (nu_smps *

+nu_smps) * (1-2 *

/((1+nu_smps) * (1-2 *

nu_smps))+nu_smps *

mps)))

Sy Second

Piola-Kirchhoff

global sys.

Pa E_smps

ex_smps/((1

nu_smps))+(1-nu_smps) *

ey_smps

ez_smps/((1+nu_smps) * (1-2 * nu_s

Sz_smps Sz ond

off

Pa Sec

Piola-Kirchh

global sys.

0

Sxy_smps Sxy Second

Piola-Kirchhoff

global sys.

smps * exy_smps/(1+nu_smps) Pa E_

wz_smps Out of plane

derivative of

out-of-

displacement

plane

+2 * ez_smps<0,-1,-1+sqrt(1+2 *

ez_smps))

1 if(1

K_smps Bulk modulus ) Pa E_smps/(3 * (1-2 * nu_smps)

G_smps Pa Shear modulus 0.5 * E_smps/(1+nu_smps)

mises_smps ises

stress

Pa

)

von M sqrt(sx_smps^2+sy_smps^2-sx_smps *

sy_smps+3 * sxy_smps^2

Ws_smps Strain energy J/m^2 (ex_smps *

density

0.5 * thickness_smps *

sx_smps+ey_smps * sy_smps+2 *

exy_smps * sxy_smps)

evol_smps Volumetric

1

strain

-1+Jel_smps

F11_smps Deformation 1

gradient 11

comp.

1+u2x

F12_smps Deformation

gradient 12

comp.

1 u2y

F21_smps Deformation

gradient 21

comp.

1 v2x

F22_smps Deformation 1 1+v2y

181

gradient 22

comp.

F33_smps Deformation

gradient 33

comp.

1 1+wz_smps

detF_smps

F12_smps * F21_smps)

Determinant of

deformation

gradient

1 F33_smps * (F11_smps * F22_smps-

J_smps 1 Volume ratio detF_smps

Jel_smps Elastic e

ratio

1 volum J_smps

invF11_smps Inverse of

deformation

1

gradient 11

comp.

F22_smps * F33_smps/detF_smps

invF12_smps

deformation

gradient 12

comp.

1 ps Inverse of -F12_smps * F33_smps/detF_sm

invF21_smps 1_smps * F33_smps/detF_smps Inverse of

deformation

gradient 21

comp.

1 -F2

invF22_smps

1 F11_smps * F33_smps/detF_smps Inverse of

deformation

gradient 22

comp.

invF33_smps 1 (F11_smps * F22_smps-F21_smps *

F12_smps)/detF_smps

Inverse of

deformation

gradient 33

comp.

sz_smps sz normal s

global s

tress

ys.

Pa 0

tresca_smps

-s3_smps))

Tresca stress Pa max(max(abs(s1_smps-

s2_smps),abs(s2_smps-

s3_smps)),abs(s1_smps

182

COMSOL Model Report – Bioreactor Design

1. Table of Contents

• Model

• Consta

• Geom2

• Materials/Coefficients Library

• Postpr

• Variab

rtie

Property lue

• Title - COMSOL Model Report

• Table of Contents

Properties

nts

• Geome

• Geom1

try

• Solver Settings

ocessing

les

2. Model Prope

Va

s

Model name

Author

Company

Department

Reference

URL

Saved date Jun 24, 2008 2:22:41 PM

Creation date Jun 9, 2008 1:27:09 PM

COMSOL version COMSOL 3.4.0.248

File name: G:\COMSOL\Bioreactor-2D-BD.mph

183

Application modes and modules used in this model:

• Geom1 (Axial symmetry (2D))

o Axial Symmetry, Stress-Strain (Structural Mechanics Module)

o Incompressible Navier-Stokes (Chemical Engineering Module)

• Geom2 (3D)

3. Constants

Name Expression Value Description

T 273[K] temperature

4. Geometry

Number of geometries: 2

4.1. Geom1

184

4.1.1. Point mode

185

4.1.2. Boundary mode

186

4.1.3. Subdomain mode

187

4.2. Geom2

188

4.2.1. Point mode

189

4.2.2. Edge mode

190

4.2.3. Boundary mode

191

4.2.4. Subdomain mode

5. Geom1

Space dimensions: Axial symmetry (2D)

Independent variables: R, PHI, Z

5.1. Mesh

5.1.1. Mesh Statistics

Number of degrees of freedom 3498

Number of mesh points 230

Number of elements 358

Triangular 358

Quadrilateral 0

Number of boundary elements 127

Number of vertex elements 20

Minimum element quality 0.646

Element area ratio 0.02

192

5.2. Application Mode: Axial Symmetry, Stress-

axi)

ariables

alue Unit Description

Strain (sm

Application mode type: Axial Symmetry, Stress-Strain (Structural Mechanics

Module)

Application mode name: smaxi

5.2.1. Scalar V

Name Variable V

t_old_ini t_old_ini_smaxi -1 s Initial condition previous time step (contact

with dynamic friction)

193

5.2.2. Application Mode Properties

Property Value

Default element type Lagrange - Quadratic

Analysis type Static

Large deformation On

Specify eigenvalues using Eigenfrequency

Create frame Off

Deform frame Frame (ref)

Frame Frame (ref)

Weak constraints Off

Constraint type Ideal

5.2.3. Variables

Dependent variables: uor, w, p

Shape functions: shlag(2,'uor'), shlag(2,'w')

Interior boundaries active

5.2.4. Boundary Settings

25 13

Boundary 4, 6, 8, 12, 14, 16- 1, 3, 5, 7, 9, 11, 2, 15

2

Edge load (force/area) Z-dir. N/m

(Fz)

0 0 0

constrcond free sym fixed

Boundary 10

Edge load (force/area) Z-dir. (Fz

2

) N/m p4

constrcond free

5.2.5. S in Set

, 5

ubdoma tings

Subdomain 1-2 -6 3 4

Young's modulus (E) Pa 1e9[Pa]

(Polyethylene) (Polyethylene)

2.4e9 1e9[Pa]

Density (rho) kg/m

3

930[kg/m^3] 930[kg/m^3]

ylene)

1190[kg/m^3]

(Polyethylene) (Polyeth

Thermal expansion 1/K 150e-6[1/K] 150e-6[1

coeff. (alpha) (Polyethylene)

/K]

(Polyethylene)

70e-6[1/K]

Poisson's ratio (nu) 1 0.33 0.40 0.40

194

5.3. Application Mode: Incompressible Navier-

(chns)

: Inc okes (Chemical Engineering

ation Mod s

e

Stokes

Application mo

Module)

de type ompressible Navier-St

Application mode name: chns

5.3.1. Applic e Propertie

Property Valu

Default element type ge - P

2

P

1

Lagran

Analysis type Stationary

Corner smoothing Off

Weakly compressible flow Off

Turbulence model None

Realizability Off

Non-Newtonian flow Off

Brinkman on by default Off

Two-phase flow Single-phase flow

Swirl velocity Off

Frame Frame (ref)

Weak constraints Off

Constraint type Ideal

5.3.2. Variables

variables: u4, v4, w2, , lo logd2, logw2, phi2, nrw2, nzw2

y S ttings

7, 9,

Dependent p4 gk2,

Shape functions: shlag(2,'u4'), shlag(2,'v4'), shlag(1,'p4')

Interior boundaries not active

5.3.3. Boundar e

Boundary 1, 3, 5, 11, 13 2 15

Type Symmetry b bound oundary Open ary Inlet

Normal inflow velocity (U0 0.03 in) m/s 1 1

Volume per time uni ) t (V0 m

3

/s 0 0 0.016

flowtype velocity volume velocity

Boundary 16-23, 25

195

Type Wall

Normal inflow velocity (U0in) m/s 1

Volume per time unit (V0) m

3

/s 0

flowtype velocity

5.3.4. Subdomain Settings

Subdomain 1-3, 5-6 4

Integration order (gporder) 4 4 2 4 4 2

Constraint order (cporder) 2 2 1 2 2 1

3

Density (rho) kg/m 1040 1040

Dynamic viscosity (eta) Pa·s 0.00078 0.00078

Porosity (epsilonp) 1 1 0.119375

Permeability (k)

2

m 1 0.119375

Flow in porous media (Brinkman equations) 0 1

(brinkmaneqns)

6. Geom2

Space dimensions: 3D

dent variables: x, y

stics

Indepen , z

6.1. Mesh

6.1.1. Mesh Stati

Number of degrees of freedom 3498

Number of mesh points 2010

Number of elements 7709

Tetrahedral 7709

Prism 0

Hexahedral 0

Number of boundary elements 3066

Triangular 3066

Quadrilateral 0

Number of edge elements 544

Number of vertex elements 56

Minimum element quality 0.279

Element volume ratio 0.008

196

7. Materials/Coefficients Library

7.1. PMMA

Parameter Value

Heat capacity at constant pressure (C) 1420[J/(kg*K)]

Young's modulus (E) 3e9[Pa]

Thermal expansion coeff. (alph 70e-6[1/K] a)

Relative permittivity (epsilonr) 3.0

Thermal conductivity (k) 0.19[W/(m*K)]

Poisson's ratio (nu) 0.40

Density (rho) 1190[kg/m^3]

7.2. Polyethylene

Value Parameter

Heat capacity at constant pressure (C) 1900[J/(kg*K)]

Young's modulus (E) 1e9[Pa]

197

Thermal expansion coeff. (alpha) 150e-6[1/K]

Relative permittivity (epsilonr) 2.3

Thermal conductivity (k) 0.38[W/(m*K)]

Density (rho) 930[kg/m^3]

7.3. Ethanol, liquid

Parameter Value

Heat capacity at constant pressure (C) Cp(T[1/K])[J/(kg*K)]

Dynamic viscosity (eta) eta(T[1/K])[Pa*s]

Thermal conductivity (k) k(T[1/K])[W/(m*K)]

Kinematic viscosity (nu0) nu0(T[1/K])[m^2/s]

Density (rho) rho(T[1/K])[kg/m^3]

7.3.1. Functions

Function Expression Derivatives Complex

output

nu0(T) (10^(1.16E-05*T^2-1.54e-

2*T+0.608))/(-1.426e-

3*T^2-0.1167*T+948.62)

diff((10^(1.16E-05*T^2-1.54e-

2*T+0.608))/(-1.426e-3*T^2-

0.1167*T+948.62),T)

false

Cp(T) 20.7*T-3840 diff(20.7*T-3840,T) false

rho(T) -1.426e-3*T^2- diff(-1.426e-3*

0.1167*T+948.62

T^2-

0.1167*T+948.62,T)

false

eta(T) 10^(1.16

2*T+0.6

E-05*T^2-1.54e-

08)

diff(10^(1.16E-05*T^2-1.54e-

2*T+0.608),T)

false

k(T) -1.03e-3*T+0.4848 d 3e-3*T+0.4848,T) false iff(-1.0

8. Solver Settings

Solve using a script: off

Analysis type Static

Auto select solver On

Solver Stationary

Solution form Automatic

Symmetric auto

Adaption Off

198

8.1. Direct (UMFPACK)

solver

Parameter Value

Solver type: Linear system

Pivot threshold 0.1

Memory allocation factor 0.7

8.2. Stationary

Parameter Value

Linearity Automatic

Relative tolerance 1.0E-6

Maximum number of iterations 25

Manual tuning of damping parameters Off

Highly nonlinear problem Off

Initial damping factor 1.0

Minimum damping factor 1.0E-4

Restriction for step size update 10.0

8.3. Ad

Paramete Value

vanced

r

Constraint im handling method El ination

Null-space Automatic function

Assembly b 100 lock size 0

Use Hermitian transpose of constraint matrix and in symmetry detection Off

Use complex functions with real input Off

Stop if error due to undefined operation On

Store solution on file Off

Type of scaling None

Manual scaling

Row equilibration On

Manual control of reassembly Off

Load constant On

Constraint constant On

Mass constant On

Damping (mass) con t On stan

Jacobian constant On

Constraint Jacobian constant On

199

9. Postprocessing

200

10. Variables

10.1. Point

Name Description Unit Expression

FRg_smaxi Point load in global R dir. N 0

FZg_smaxi Point load in global Z dir. N 0

disp_smaxi Total displacement m sqrt(real(uaxi_smaxi)^2+real(w)^2)

uaxi_smaxi R-displacement m uor * R

uaxiR_smaxi R derivative of R-

displacement

1 uorR * R+uor

uaxiZ_smaxi Z derivative of R

displacement

1 uorZ * R

uaxi_t_smaxi R-velocity m/s diff(uaxi_smaxi,t)

201

10.2. Boundary

10.2.1. Boundary 1-9, 11-25

Name Description Unit Expression

FRg_smaxi Edge load in

global R-dir.

N/m^2 0

FZg_smaxi Edge load in

global Z-dir.

N/m^2 0

disp_smaxi Total

displacement

m sqrt(real(uaxi_smaxi)^2+real(w)^2)

uaxi_smaxi R-

displacement

m uor * R

uaxiR_smaxi R derivative of

R-

displacement

1 uorR * R+uor

uaxiZ_smaxi Z derivative of

R displacement

1 uorZ * R

uaxi_t_smaxi R-velocity m/s diff(uaxi_smaxi,t)

TaR_smaxi Surface

traction

(force/area) in

Pa (F11_smaxi * SR_smaxi+F13_smaxi *

SRZ_smaxi) * nR_smaxi+(F11_smaxi *

SRZ_smaxi+F13_smaxi * SZ_smaxi) *

nZ_smaxi R dir.

TaZ_smaxi Surface

traction

e/area) in

.

Pa (F31_smaxi * SR_smaxi+F33_smaxi *

SRZ_smaxi) * nR_smaxi+(F31_smaxi *

SRZ_smaxi+F33_smaxi * SZ_smaxi) *

nZ_smaxi

(forc

Z dir

K_R_chns Pa eta_chns * (2 * nR_chns * u4R+nZ_chns * Viscous force

per area, R

component

(u4Z+v4R))

T_R_chns Total force per Pa -nR

area, R

component

_chn

u4R Z_

s * p4+2 * nR_chns * eta_chns *

chns * eta_chns * (u4Z+v4R) +n

K_Z_chns Viscous force

per area, Z

P

component

a eta_chns * (nR_chns * (v4R+u4Z)+2 *

nZ_chns * v4Z)

T_Z_chns

component

Pa -nZ_chns * p4+nR_chns * eta_chns *

(v4R+u4Z)+2 * nZ_chns * eta_chns * v4Z

Total force per

area, Z

202

10.2.2. Boundary 10

Name Description Unit Expression

FRg_smaxi Edge load in N/m^2

global R-dir.

0

FZg_smaxi Edge load in

global Z-dir.

N/m^2 FZ_smaxi

disp_smaxi Total

displacement

rt(real(uaxi_smaxi)^2+real(w)^2) m sq

uaxi_smaxi R-

displacement

m uor * R

uaxiR_smaxi erivative of +uor R d

R-

displacement

1 uorR * R

uaxiZ_smaxi Z derivative of

R displacement

1 uorZ * R

uaxi_t_smaxi R-velocity m/s maxi,t) diff(uaxi_s

TaR_smaxi

) in

Pa (F11_smaxi * SR_smaxi+F13_smaxi *

smaxi+(F11_smaxi *

Surface

traction

a (force/are

R dir.

SRZ_smaxi) * nR_

SRZ_smaxi+F13_smaxi * SZ_smaxi) *

nZ_smaxi

TaZ_smaxi

a) in

Pa Surface

traction

(force/are

Z dir.

(F31_smaxi * SR_smaxi+F33_smaxi *

SRZ_smaxi) * nR_smaxi+(F31_smaxi *

SRZ_smaxi+F33_smaxi * SZ_smaxi) *

nZ_smaxi

K_R_chns Pa * Viscous force

per area, R

component

eta_chns * (2 * nR_chns * u4R+nZ_chns

(u4Z+v4R))

T_R_chns Total force per

area, R

component

Pa -nR_chns * p4+2 * nR_chns * eta_chns *

u4R+nZ_chns * eta_chns * (u4Z+v4R)

K_Z_chns Viscous force

per area, Z

component

Pa eta_chns * (nR_chns * (v4R+u4Z)+2 *

nZ_chns * v4Z)

T_Z_chns Total force per

area, Z

component

Pa -nZ_chns * p4+nR_chns * eta_chns *

(v4R+u4Z)+2 * nZ_chns * eta_chns * v4Z

203

10.3. Subdomain

U Name Descriptio

n

nit Expression

FRg_smaxi Body load

in global R-

dir.

N/m^3 0

FZg_smaxi Bod

in global Z-

y load

ir.

N ^3

d

/m 0

disp_smaxi

t

m sqrt(real(uaxi_smaxi)^2+real(w)^2) Total

displaceme

n

uaxi_smaxi m uor * R R-

displaceme

nt

uaxiR_smaxi R derivative

eme

1

of R-

displac

nt

uorR * R+uor

uaxiZ_smaxi Z rivative

eme

1 de

of R

displac

nt

uorZ * R

uaxi_t_smaxi R-velocity m/s axi,t) diff(uaxi_sm

sR_smaxi sR normal

stress

global sys.

P

SRZ_smaxi *

F13_smaxi)+F13_smaxi * (SRZ_smaxi *

a (F11_smaxi * (SR_smaxi *

F11_smaxi+

F11_smaxi+SZ_smaxi *

F13_smaxi))/J_smaxi

sZ_smaxi

.

Pa (F31_smaxi * (SR_smaxi *

_smaxi * (SRZ_smaxi *

F31_smaxi+SZ_smaxi *

sZ normal

stress

global sys

F31_smaxi+SRZ_smaxi *

F33_smaxi)+F33

F33_smaxi))/J_smaxi

sPHI_smaxi sPHI

stress

Pa

normal

SPHI_smaxi * F22_smaxi^2/J_smaxi

sRZ_smaxi sRZ shear

stress

Pa (F11_smaxi * (SR_smaxi *

F31_smaxi+SRZ_smaxi *

_smaxi * (SRZ_smaxi *

smaxi *

global sys. F33_smaxi)+F13

F31_smaxi+SZ_

F33_smaxi))/J_smaxi

eR_smaxi eR normal 1 uorR * R+uor+0.5 * ((uorR *

204

strain global

sys.

R+uor)^2+wR^2)

eZ_smaxi

l

rZ * R)^2+wZ^2) eZ normal

strain globa

sys.

1 wZ+0.5 * ((uo

ePHI_smaxi ePHI

normal

strain

1 uor+0.5 * uor^2

eRZ_smaxi eRZ shear

strain global

uorR * R+uor) * uorZ

* R+wR * wZ)

sys.

1 0.5 * (uorZ * R+wR+(

SR_smaxi SR Second

Piola-

Kir

global sys.

chhoff

eR_smaxi/((1+nu_smaxi) * (1-2 *

xi))+nu_smaxi *

axi/((1+nu_smaxi) * (1-2 *

nu_smaxi))+nu_smaxi *

Pa E_smaxi * ((1-nu_smaxi) *

nu_sma

ePHI_sm

eZ_smaxi/((1+nu_smaxi) * (1-2 *

nu_smaxi)))

SZ_smaxi Pa E_smaxi * (nu_smaxi *

eR_smaxi/((1+nu_smaxi) * (1-2 *

xi/((1+nu_smaxi) * (1-2 *

nu_smaxi))+(1-nu_smaxi) *

eZ_smaxi/((1+nu_smaxi) * (1-2 *

nu_smaxi)))

SZ Second

Piola-

Kirchhoff

global sys.

nu_smaxi))+nu_smaxi *

ePHI_sma

SPHI_smaxi SPHI

Second

Piola-

Kirchhoff

Pa E_smaxi * (nu_smaxi *

1-2 *

xi) * (1-2 *

eR_smaxi/((1+nu_smaxi) * (

nu_smaxi))+(1-nu_smaxi) *

ePHI_smaxi/((1+nu_smaxi) * (1-2 *

nu_smaxi))+nu_smaxi *

eZ_smaxi/((1+nu_sma

nu_smaxi)))

SRZ_smaxi SRZ

Piola-

Kirchhoff

sys.

Pa u_smaxi)

Second

global

E_smaxi * eRZ_smaxi/(1+n

K_smaxi Bulk

s

Pa E_smaxi/(3 * (1-2 * nu_smaxi))

modulu

G_smaxi Shear

modulus

) Pa 0.5 * E_smaxi/(1+nu_smaxi

mises_smaxi

stress

Pa x

xi-sPHI_smaxi *

sZ_smaxi+3 *

von Mises sqrt(sR_smaxi^2+sPHI_smaxi^2+sZ_sma

i^2-sR_smaxi * sPHI_sma

sZ_smaxi-sR_smaxi *

sRZ_smaxi^2)

205

Ws_smaxi

y

J/m^3 sR_smaxi+ePHI_smaxi *

sPHI_smaxi+eZ_smaxi * sZ_smaxi+2 *

Strain

energ

density

0.5 * (eR_smaxi *

eRZ_smaxi * sRZ_smaxi)

evol_smaxi Volumetric

1 -1+Jel_smaxi

strain

F11_smaxi Deform

gradient

comp.

ation

11

1 1+uaxiR_smaxi

F13_smaxi Deformation

gradient 13

comp

.

1 uaxiZ_smaxi

F22_smaxi Deformation

gradie

comp.

nt 22

1 1+uor

F31_smaxi Deformation

gradient 31

comp.

1 wR

F33_smaxi Deformation

1

gradient 33

comp.

1+wZ

detF_smaxi Determinant

deformation

gradient

1 xi-

of

F22_smaxi * (F11_smaxi * F33_sma

F13_smaxi * F31_smaxi)

J_smaxi Volume

ratio

1 detF_smaxi

Jel_smaxi Elastic

tio

1

volume ra

J_smaxi

invF11_smaxi Inverse

deformation

gradient 11

.

1 of

comp

F22_smaxi * F33_smaxi/detF_smaxi

invF13_smaxi Inverse

tion

3

of

deforma

gradient 1

comp.

1 -F13_smaxi * F22_smaxi/detF_smaxi

invF22_smaxi Inverse of

n

2

maxi *

deformatio

t 2 gradien

comp.

1 (F11_smaxi * F33_smaxi-F31_s

F13_smaxi)/detF_smaxi

invF31_smaxi

t 31

comp.

Inverse of

deformation

gradien

1 -F31_smaxi * F22_smaxi/detF_smaxi

206

invF33_smaxi Inverse of

tion

33

deforma

gradient

comp.

1 F11_smaxi * F22_smaxi/detF_smaxi

tresca_smaxi Tresca Pa max(max(abs(s1_smaxi-

_smaxi-

s3_smaxi)),abs(s1_smaxi-s3_smaxi))

stress s2_smaxi),abs(s2

U_chns m/s sqrt(u4^2+v4^2) Velocity

field

V_chns Vorticity 1/s u4Z-v4R

divU_chns Divergence 1/s u4R+v4Z+u4/R

of velocity

field

cellRe_chns Cell 1 rho_chns * U_chns * h/eta_chns

Reynolds

number

res_u4_chns Equation Pa R * (rho_chns * (u4 * u4R+v4 * u4Z)+p4R-

ns)+2 * eta_chns * (u4/R-u4R)-

eta_chns * R * (2 * u4RR+u4ZZ+v4RZ)

residual for

u4

F_r_ch

res_sc_u4_ch

idual for

Pa R * (rho_chns * (u4 * u4R+v4 * u4Z)+p4R-

ns

Shock

capturing

res

u4

F_r_chns)+2 * eta_chns * (u4/R-u4R)

res_v4_chns Equation

for

Pa R * (rho_chns * (u4 * v4R+v4 * v4Z)+p4Z-

ta_chns * (R *

(v4RR+u4ZR)+2 * R * v4ZZ+u4Z+v4R)

residual

v4

F_z_chns)-e

res_sc_v4_chns hns * (u4 * v4R+v4 * v4Z)+p4Z-

F_z_chns)

Shock

capturing

residual for

v4

Pa R * (rho_c

beta_R_chns Convective Pa*s R * rho_chns * u4

field, R

component

beta_Z_chns Convective

field, Z

component

Pa*s R * rho_chns * v4

Dm_chns Mean kg/s R * eta_chns

diffusion

coefficient

da_chns Total

ctor

kg/m^2 R * rho_chns time

scale fa

taum_chns GLS time-

scale

m^3*s/k

g eta_chns/h))

nojac(0.5 * h/max(rho_chns * U_chns,6 *

tauc_chns m^2/s nojac(0.5 * U_chns * h * min(1,rho_chns *

U_chns * h/eta_chns))

GLS time-

scale

207

APPE

NDIX G

Schreyer Honors College

A DE

dem ta o alvi

CA MIC VITA

Aca ic Vi f Joshua Damian S

ical Inf Biograph ormation

Name: Joshua Salvi

601 King n Roa

5

Damian

Address:

York,

3

PA

sto

17402

d

E-Mail ID:

jds519

Education

Major: eering, op

Honors: eering

ove sign

iation of H ls

Thesis Advisors: Henry J. Donahue (

er J. B (Uni

Honors Advisor: O. Hancock

Work Experienc

Bioengin

Bioengin

tion in Chemical Engineering

Thesis Title: The N

Different

l De of a Bioreactor for in vitro Proliferation and

uman Mesenchymal Stem Cel

Hershey, PA)

Pet

William

utler versity Park, PA)

e

08/2007-05/2009 a art ering

aduate Researcher

at ul es at University Park

rviso er J.

5/2009 ta e

t o

he D ion;

teer

iso y

Penn St

Undergr

vestig

te Dep ment of Bioengine

In

Supe

or in m

r: Pet

tiple laboratori

Butler

05/2007-0

Penn S

Investiga

c

te Colleg

or and V

of Medicine

lunteer

Resear

Volun

d in the

ed in the

epartment of Orthopaedics and Rehabilitat

Hershey Medical Center

Superv r: Henr J. Donahue

208

01/2006-05/2009 Penn State

SI Leader, T

Assisted students in CHEM 110(12), BIOE 201, PHYS 211, and

MATH

Superv wsky, William Hancock

07/2002-05/2005 Circuit City Stores, Inc.

S t

F hnology repair

visor: Varied

Learning Center

utor, Teaching Assistant

140

isor: Janice Smith, Herbert Lipo

ales Counselor, Customer Service Assistan

ocused on sales in home electronics and tec

Super

Awards and Leadership

Academic Awards: Bioengineering Student M

John W. White Graduate

arshal

Fellowship

Moffitt Scholarship in Engineering

Presidential Volunteer Service Award

America’s Scholar of Promise Award

wards:

eadership:

tary

Evan Pugh Scholar, Junior and Senior Awards

Vaun A.

Other A Eagle Scout

Memberships: Tau Beta Pi

Sigma Xi

Golden Key International Honour Society

Biomedical Engineering Society (BMES)

L

BMES Chapter President

Debate Team Captain

Student Council Secre

Research

Publications: Jung Yul Lim, Joshua D. Salvi, Ryan C. Riddle, Henry J.

ent

s in Cell Culture.

Conferences:

ture on specific

for subpopulations of stem cells

Donahue. Nanotopography regulation of stem cell

mechanosensitivity.

Joshua D. Salvi, Jung Yul Lim, Henry J. Donahue. Finite Elem

Analyses of Fluid Flow Condition

Joshua D. Salvi, Jung Yul Lim, Yue Zhang, Jacqueline Yanoso,

Donahue, Cul Christopher Niyibizi, Henry J.

nanoscale topographies selects

209

210

tial. 55th Orthopaedic Research

Las Vegas, Nevada, USA.

specific nanoscale topographies selects for subpopulations of stem

enic potential. 2008 Biomedical

8, St. Louis, MO,

Jung Yul Lim, Joshua D. Salvi, Ryan C. Riddle, Henry J.

Donahue, Nanoscale substrate topography regulates stem cell

anosensitivity. 54th Orthopaedic Research Society (ORS),

#22, March 2-5, 2008, San Francisco, CA, USA.

y J. Donahue, Substrate

s to fluid flow.

), September 26-29,

with increased osteogenic poten

Society (ORS), February 22-25, 2009,

Joshua D. Salvi, Jung Yul Lim, Henry J. Donahue, Culture on

cells with increased osteog

Engineering Society (BMES) October 2-4, 200

USA.

mech

Joshua D. Salvi, Jung Yul Lim, Henr

nanotopography affects stem cell responsivenes

2007 Biomedical Engineering Society (BMES

2007, Los Angeles, CA, USA.

Extracurricular Involvement

Department: Bioengineering Curriculum Committee

Representative

ommunity Service: Circle K International

PA

ills

Biomedical Engineering Society

Faculty Senate Liaison

C

Shaver’s Creek Volunteer

Boy Scouts of America

High School Tutor in York,

Related Sk

MATLAB

ab Techniques:

Fluorescence Microscopy, Confocal Microscopy

Distillation, Organic Synthesis

Programming: C++/C#

JavaScript, HTML

L Cell Culture, Light Microscopy

Atomic Force Microscopy, Electron Microscopy

Fluorescence-Activated Cell Sorting, Cytometry

AP Assays, Western Blot, PCR

Polymer Demixing

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