1 EFFECTS OF LIVER EXTRACELLULAR MATRIX GEL STIFFNESS ON PRIMARY HEPATOCYTE FUNCTION BY DANIEL B. DEEGAN A Dissertation Submitted to the Graduate Faculty of WAKE FOREST UNIVESITY GRADUATE SCHOOL OF ARTS AND SCIENCES in Partial Fulfillment of the Requirements for the Degree of DOCTOR OF PHILOSOPHY Molecular Medicine and Translational Sciences December 2015 Winston-Salem, North Carolina Copyright Daniel B. Deegan 2015 (If Applicable) Approved By: Thomas D. Shupe, Ph.D., Advisor Emmanuel C. Opara, Ph.D., Chair Graça D. Almeida-Porada, M.D., Ph.D. Michael C. Seeds, Ph.D. Shay Soker, Ph.D.
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1
EFFECTS OF LIVER EXTRACELLULAR MATRIX GEL STIFFNESS ON
PRIMARY HEPATOCYTE FUNCTION
BY
DANIEL B. DEEGAN
A Dissertation Submitted to the Graduate Faculty of
WAKE FOREST UNIVESITY GRADUATE SCHOOL OF ARTS AND SCIENCES
in Partial Fulfillment of the Requirements
for the Degree of
DOCTOR OF PHILOSOPHY
Molecular Medicine and Translational Sciences
December 2015
Winston-Salem, North Carolina
Copyright Daniel B. Deegan 2015 (If Applicable)
Approved By:
Thomas D. Shupe, Ph.D., Advisor
Emmanuel C. Opara, Ph.D., Chair
Graça D. Almeida-Porada, M.D., Ph.D.
Michael C. Seeds, Ph.D.
Shay Soker, Ph.D.
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DEDICATION AND ACKNOWLEDGEMENTS
A special thanks to my PI and advisor Dr. Tom Shupe for his guidance, patience,
and selflessness in mentoring me and helping me to complete the graduate school
process. Thanks to my undergraduate mentor at Virginia Tech, Dr. John McDowell, for
sparking my interest in laboratory research. Thanks to my previous mentors Dr. Tamer
Aboushwareb and Dr. Bryon Petersen for their assistance throughout graduate school.
Thanks to my labmate Cindy Zimmerman for her help and support on technical work of
my project. Thanks to my committee members, Dr. Emmanuel Opara, Dr. Graça
Almeida-Porada, Dr. Michael Seeds, and Dr. Shay Soker for their guidance on my thesis
project. Thanks to Dr. Aleksander Skardal for his support and expertise in hydrogel
formation. Thanks to Dr. Bridget Brosnihan for her counsel and advisement during
difficult times. Thanks to the Molecular Medicine and Translational Sciences program
and the Wake Forest Institute for Regenerative Medicine. Finally, thank you to my family
and friends for keeping me sane throughout the struggles of graduate school.
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TABLE OF CONTENTS
List of Figures and Tables………………………………………………………………5-6
in the liver. Without corrections in matrix deposition, cirrhosis leads to complications and
total loss of liver function.
Fibrosis is not only the result of increased matrix synthesis but additionally from
reduced matrix degradation. During normal liver repair, HSCs maintain an equilibrium
between production of MMPs and tissue inhibitor of metalloproteinases (TIMPs) (57).
However, during fibrosis, TIMPs, especially TIMP1 and TIMP2, are secreted at a greater
rate and block any action of MMPs (49). This inhibition, along with increases in matrix
production, worsens liver health and function. To treat liver disease, therapies need to
both revert HSCs and other ECM secreting cells to quiescent states and block the release
of TIMPs. Through these actions, balance and normal liver function can be restored.
6. Stiffness in Tissue Microenvironments
Increased collagen deposition and crosslinking during fibrosis cause changes in
the mechanical properties of the ECM. Mechanical properties of tissue
microenvironments contribute to the overall function and health of the organ systems
they comprise. These forces include shear stress induced by laminar flow in the
vasculature, tension from attachment to adjacent cells, and compression or bending of
tissue in response to external stimuli (129). One of the physical factors with the greatest
impact on cell behavior is stiffness. Stiffness is the mechanical property that represents
rigidity or the resistance to deformity following an applied force (130, 131). Variations in
stiffness are found in both the cells and extracellular matrix (ECM) that constitute tissue.
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6.1 ECM Stiffness
ECM stiffness varies with tissue or organ type. Brain and fat are two of the softest
tissue types, while bone has the most rigid matrix composition and is several-fold stiffer
(132). The mechanical properties of ECM directly correlate to tissue function. Physical
characteristics of the matrix are determined by its composition of structural molecules,
the tension generated by cell attachment and migration, and exogenous physiological
forces such as blood flow.(133, 134) Stiffness also depends on location within the tissue
or organ. Gradients of bone and skeletal muscle stiffness can be found in the body. ECM
composition in the liver changes from the periportal to the pericentral regions of lobules
resulting in changes in cell phenotype, even among cells of the same lineage (22, 135).
Because of differences in the molecular arrangement throughout these liver
microstructures, one can infer that variations in stiffness also exist. A clinical study
supported this concept by measuring stiffness of liver sections in separate locations.
Results showed differing measurements between lobes of same liver and among smaller
areas within each lobe (136). Organ and tissue physiology show that the slightest
differences in mechanical properties can alter function of adult cells.
Studies have additionally shown the abilities of stiffness to direct stem cells
towards specific cell lineages. In a previous study, mesenchymal stem cells (MSCs) were
differentiated on varying substrate stiffnesses into three different cell lineages;
neurogenic, myogenic, and osteogenic cells (137). A separate group’s analysis revealed
that effects of growth factors on MSCs also changed with substrate stiffness. TGF-β
differentiated MSCs into smooth muscle cells (SMCs) at a low stiffness while
upregulating expression of chondrogenic markers at a higher stiffness level (138). By
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simply changing this mechanical property, cell development and growth was directed to
mimic cells of a certain tissue type.
Cells not only change function but also migrate according to stiffness gradients.
In previous assays, MSCs under cell culture conditions traveled to areas of higher
substrate stiffness. The hypothesis stated that the cells migrate toward an injury-related
stiffness or a more scar-like site in order to aid in repair (139, 140). This phenomenon
called durotaxis has been observed in several cell types and is thought to be related to
mechanisms of development and wound healing (141). These studies show that physical
cues are just as important as chemical stimuli in determining cell arrangement, activation,
and phenotype.
6.2 Cellular Stiffness
Cell stiffness is directly correlated to the mechanical properties of the substrate to which
it is attached. This material could be the underlying ECM, a tissue culture biomaterial, or
a neighboring cell. Cell rigidity also depends on the cell lineage type, the cell’s maturity,
and the functional state or health of the cell (142). Cell stiffness ranges from softer
epithelial cells to stiffer smooth muscle cells and rigid osteocytes. Stem cells and
progenitor cells adapt their physical structures as they mature and differentiate (143,
144).
Certain disease and repair conditions can direct mechanical properties. Cells adapt
to dynamic physical environments and can change their activation states accordingly.
During the development of atherosclerosis, endothelial cells increase in stiffness which,
along with plaque build-up, limits flow of oxygen-rich blood (145, 146). Valvular
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interstitial cells activate and become fibroblastic in diseased valves. They return to
quiescent states following repair and remodeling (147). In cancer, cells with decreased
stiffness have the highest metastatic potential (148). This concept holds true in ovarian
cancer cells where stiffness can be used as a biomarker for invasiveness (142).
Mechanical properties regulate cell-cell and cell-matrix interactions. Stiffness of
cells and the ECM can have independent or cumulative effects that cause systemic
imbalances and disease. Cells and matrix can also interact to balance physical conditions
and maintain normal tissue homeostasis and function. Determining methods to achieve a
healthy stiffness state in tissue will lead to effective treatments to correct disorders or
diseases in patients.
7. Liver ECM Stiffness during Developmental or Repair States
Stiffness is not a static property in liver tissue but fluctuates with organ
development, disease, and repair. In early liver development, a low stiffness environment
maintains progenitor cells in their undifferentiated form in the endoderm. As ECM
structures are created, cytokine and environmental cues eventually initiate cell migration
and differentiation of these cells into mature parenchymal liver cells (149, 150). These
observations are supported by in vitro studies which have shown the ability of low
stiffness substrates to maintain expression of stem cell markers in hepatic progenitor cells
(151).
Normal liver repair or regeneration is also initiated by elements of stiffness.
Matrix-producing hepatic stellate cells and endothelial cells are activated by damage or
increases in stiffness and migrate to these areas for repair (59). In these situations, MMPs
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are required to degrade proteolytic-resistant collagens. These MMPs break-up or remove
damaged or excess ECM to allow formation of new matrix structures (37, 152). Physical
properties of the ECM as well as cell signaling enable new cells to repopulate these
repaired sites to restore normal tissue function (58).
In the instance of tissue resection such as a partial hepatectomy, the lost ECM
mass is fully regenerated de novo with the help of the activated matrix-producing cells.
The provisional matrix produced during early stages of repair or regeneration contains
uncrosslinked collagen I (58). In progenitor cell-mediated regeneration, this softer
composition initially maintains the hepatoblast phenotype of cells until ECM structures
are fully formed with the addition of collagen IV and the crosslinking of collagen I (153).
Regeneration initiated by proliferating hepatocytes occurs simultaneously to matrix
production by HSCs. However, physical cues provided by the ECM contribute to control
of the initiation and cessation of cell propagation (31, 35, 154).
Imbalances in these reparative processes can cause increases in stiffness and liver
dysfunction. Liver stiffness is an important parameter in the prognosis of liver diseases
including cirrhosis, hepatitis, and hepatocellular carcinoma (HCC) (155, 156). During a
fibrotic state, HSCs undergo uncontrolled activation and deposit excess amounts of
laminin and collagen, especially collagen IV (43, 59). Fibrosis also occurs because of a
loss of MMP production and increase in collagen crosslinking by lysyl oxidases (LOX)
(157). All these factors contribute to an increase in stiffness and lead to a loss of
hepatocyte phenotype and cell death (151). The liver is typically able to repair itself if the
causative stresses are eliminated. However, prolonged fibrosis can cause worsening
levels of stiffness and cellular impairment which eventually lead to irreversible cirrhosis
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and liver failure (43, 130, 151). Elevated liver stiffness is also a precursor for the
development of HCC (158). It can predict both the appearance and metastatic potential of
tumors. Increased stiffness correlates with increased proliferation of cancer cells and
greater resistance to chemotherapeutic agents (151).
Novel research and treatments for liver disease attempt to address the mechanisms
that cause changes in tissue stiffness. Therapeutic strategies include inhibition of
activated stellate cells to limit ECM accumulation and developing agents to block LOX
to block collagen crosslinking. Groups are also investigating methods to better control the
release and action of a variety of MMPs to enable better manipulation of matrix
degradation to soften fibrotic tissue. Studies have shown that once cells are returned to a
normal stiffness environment, full function and health can be restored. Obtaining better
control over ECM stiffness levels will lead to greater management of liver disease.
8. Effects of Stiffness on Mechanotransduction and Focal Adhesion Signaling
8.1 Cell Adhesion Molecules
Cells sense mechanical properties of their microenvironment through cell
attachment initiated by integrins, syndecans, or other glycoproteins (159). Integrins are
the most common cell adhesion molecules (CAMs) and are composed of both an alpha
and beta subunit (160). These structural components determine the integrin’s specificity
to certain ECM molecules including collagens, fibronectin, laminin, or vitronectin (161).
Integrin engagement is vital for survival and normal phenotype of all epithelial cell types
(162). These transmembrane structures initiate signal transduction by formation of focal
adhesions that connect the ECM with the cytoskeleton. This mechanical force-induced
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signaling allows cells to sense and interact with the surrounding substrate and affects cell
proliferation, motility, and function (163, 164).
Other adhesion molecules also have roles in regulating cell behavior. Syndecans
are transmembrane proteins that are linked to GAG chains of heparan sulfate and
chondroitin sulfate (165). Syndecans, with the aid of these GAGs, bind and concentrate
important growth factors, such as the liver HGF and EGF (166, 167). Although not as
prevalent as integrins, these proteoglycans also form anchoring junctions and can bind to
collagens and fibronectin of the ECM to support cell to matrix attachment. In addition,
syndecans can act alongside certain integrins to promote cell-cell adhesion (159).
Cadherins are a large family of glycoproteins that are the main effectors of cell-
cell binding. They provide connections between actin filaments of various cells to help
determine morphology and motility (168). Several classes of these calcium dependent
structures exist. Expression of E-cadherin (epithelial-cadherin) is specifically vital to
epithelial cell viability and polarity. E-cadherin forms homophilic cell-cell junctions
between hepatocytes within the liver and is critical to tissue formation (168, 169). Loss of
E-cadherin in vivo has been correlated with loss of cell phenotype and the development of
metastases in patients (170).
Tight junctions are another type of intercellular complex important to epithelial
cell adhesion, cell polarity, and function (171, 172). These structures prevent leaking of
material between cells and allow for direct diffusion and active transport of molecules
and ions between cells (173). Claudins, especially claudin-1, are required for structural
maintenance of tight junctions (174). Although not an integral structural component, the
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tight junction protein occludin is a central protein involved in cell polarity and directional
migration. Occludin enables organization of actin filaments in response to stimuli that
lead to formation of cell protrusions and cell movement (175).
8.2 Mechanotransduction and Cytoskeletal Regulation
Cell adhesion molecules are fundamental for sensing the physical characteristics
of adjacent cells and the ECM or underlying cell culture material. Although all CAMs are
important to normal cellular functions, integrins are the molecules that are essential for
detecting substrate stiffness (176). Bound integrins develop small focal adhesion
complexes and stimulate assembly of more mature focal adhesions through recruitment
of kinases and adaptor proteins to enable cellular mechanotransduction (177, 178).
Cytoplasmic proteins serve as mediators to amplify or modify the signals generated from
integrin attachment. Signals are passed bidirectionally through either inside-out or
outside-in activation to affect cell phenotype and create changes in the surrounding ECM
microenvironment (179, 180).
During cell attachment, several different focal adhesion molecules are recruited to
interact at the cytoplasmic domains of the integrins. One of the first proteins to bind to
the integrin tail regions is talin. Talin is a key component of focal adhesions that
functionally activates integrins and determines their affinity for specific ligands (181).
Talin recruits the adaptor proteins paxillin and vinculin to focal adhesion complexes to
regulate the actin cytoskeleton (182). These linkages generate strain between integrins
and cytoskeletal structures to allow cells to sense substrate mechanical properties, which
in turn direct morphology and motility (183). Vinculin localizes to focal adhesion sites as
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well as cadherin junctions. It directly connects talin to actin filaments within the cell.
Presence of vinculin is required for cell attachment, cell spreading, and filopodia
formation (184-186). Paxillin is another scaffold component that joins with talin to
control focal adhesion stability and turnover. It also interacts with different intracellular
kinases to regulate assembly of focal adhesion complexes and organization of the
cytoskeleton (183).
Focal adhesion kinase (FAK) is one of the main kinases that co-localizes with
integrins and affects their activation (179, 183). FAK has an integral role in
mechanotransduction. In previous studies utilizing FAK negative fibroblasts, mechanical
stiffness did not initiate normal cellular responses, and cell migration tendencies were
blocked (187). To initiate responses, FAK interacts with Src tyrosine kinase as well as
adaptor proteins and dozens of other focal adhesion signaling molecules (164). FAK
contains C-terminal and N-terminal domains that specify activity and transduction of
signals. The C-terminal domain allows FAK to migrate, attach, and disrupt focal
adhesion sites, while the N-terminus binds and regulates localization of ligands such as
paxillin (188, 189). Integrin attachment activates FAK by inducing kinase clustering and
autophosphorylation. This action subsequently allows Src to bind, activate, and further
phosphorylate FAK at various amino acid residues (164, 190).
Depending on the site targeted, phosphorylation of FAK affects a wide variety of
proteins and intracellular pathways to execute different and sometimes opposing
functions. FAK is important in cell viability and stimulates phosphoinositide 3-kinase
(PI3K) mediated upregulation of protein kinase B (AKT) (191). FAK also supports
survival by serving as a scaffold for phosphorylation of Crk-associated substrate (CAS)
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which activates c-Jun N-terminal kinases (JNK), mitogen-activated protein kinase 1
(MAPK1), and mitogen-activated protein kinase 3 (MAPK3) (192). Previous studies have
shown the ability to prevent anoikis, or detachment related apoptosis, in epithelial cells
through FAK expression (193, 194).
In addition to influencing viability and proliferation, activated FAK in
combination with Src can phosphorylate talin and lead to the disassembly of focal
adhesion complexes (195, 196). Under unique conditions, FAK has also been observed to
strengthen integrin attachment and enhance focal adhesion formation (197, 198).
Differences in results of these studies could be explained by the ability of FAK to
stimulate at least four separate pathways of actin assembly and cell motility (199). PI3K
and CAS pathways are not only FAK targets implicated in cell survival, but also
stimulate cell spreading and migration (164, 200). Both PI3K and CAS serve as
regulators of the downstream Ras homolog family member (Rho) GTPase, Ras-related
C3 botulinum toxin substrate (Rac) (192, 201). FAK also regulates function of the
adaptor protein growth factor receptor-bound 7 (GRB7) as well as neural Wiskott-
Aldrich syndrome protein (N-WASP) (202). N-WASP controls actin polymerization and
remodeling through the actin-related protein (Arp) 2/3 complex and another type of Rho
GTPase, cell division control protein 42 (CDC42) (164, 199). Studies have also shown
that FAK can actually suppress a third type of Rho GTPase, Ras homolog family member
A (RhoA), to promote focal adhesion turnover (203).
FAK regulates Rho GTPases that are vital to focal adhesion assembly and
disassembly, actin organization, and ultimately cell motility. The three main Rho
GTPases (Rac1, CDC42, and RhoA) work in concert with each other to initiate cell
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migration (204). However, cells seeded on varying ECM molecules or stiffnesses can
produce activation profiles of the Rac1 and CDC42 that differ from RhoA in timing or
expression levels. In research on fibroblasts seeded on fibronectin, Rac1 and CDC42
were stimulated at early time points post-seeding, while RhoA was activated much later
in vitro (199). These previous studies show that FAK activation occurs at various
phosphorylation sites or affects different pathways to control the two sets of Rho
GTPases (205).
Rac1 and CDC42 stimulate actin polymerization and extension of cellular
processes for movement. Rac1 is directly involved in formation of lamellipodia at the
leading edge of polarized cells (206, 207). Besides activation by FAK, paxillin can bind
and stimulate Rac1 activity. CDC42 binds and activates n-WASP to create thin
projections called filopodia past the frontal cellular boundary (183). Both GTPases and
FAK are required for durotaxis and other forms of directional migration controlled by
feedback signaling mechanisms (208). Lower substrate stiffness levels have been shown
to support turnover of focal adhesion sites, easier alteration of cell morphology, and
simpler cell detachment for motility (199).
RhoA can act in opposition to cell movement by increasing the assembly and
formation of mature focal adhesions and contractile actin stress fibers through stimulation
of Rho-associated protein kinase (ROCK). Increases in RhoA correlate with increases in
stiffness and create greater cytoskeletal organization, actin polarization, and stabilization
and anchorage of cell structures (209). However, overexpression of RhoA can actually
lead to build-up of stress fibers and EMT which promotes the development of cancer
(210, 211). In contrast to formation of stable adhesions, RhoA is also essential to
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generate traction force for migration on substrates. Cells can initiate stronger traction
forces on stiffer or inflexible surfaces (209). The tradeoff is that greater stiffness in
substrates creates more stable focal adhesions that make cell detachment more difficult
(199). Therefore, an intermediate, substrate stiffness is optimal for traction and speed for
cell motility.
In addition to FAK’s effects on Rho GTPase expression, integrin-linked kinase
(ILK) can act in parallel to influence cell morphology and motility (212). ILK recruits
PINCH1 to focal adhesions to regulate assembly and disassembly during cell migration.
ILK also complexes with Engulfment and Cell Motility 2 (ELMO2) and RhoG for cell
polarization (213). Similar to FAK, ILK can stimulate PI3K-induced activation of Rac1
to control cytoskeletal rearrangement and formation of cellular processes (214, 215).
As discussed, mechanical sensing and signaling involves a complex array of focal
adhesion sites, intracellular kinases, and Rho GTPases. These various molecules act in
conjunction to affect the actin cytoskeleton and form cellular protrusions. The outside-in
signaling and resultant inside-out adjustments applied by the cell can affect morphology,
motility, and ultimately the overall function of the cell. Understanding the mechanisms
and actions of important effectors like FAK, ILK, and Rho GTPases in specific cell types
under defined culture conditions will allow researchers to better model disease and more
precisely manipulate substrates for use in testing or therapies.
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CHAPTER 2
Decellularization and the Incorporation of Liver Extracellular
Matrix in Cell Culture Substrates
Daniel B. Deegan
Content
1. ECM Isolation through Decellularization Methods .………………..……61
3. Results and Advantages of ECM Use in Cell Culture……..………….…67
4. Altering Stiffness in Cell Culture Substrates……………………………..69
4.1 Methods for Manipulating Hydrogel Stiffness ……….…….………..69
4.2 Measuring Stiffness……………………………………………..…….73
5. Conclusions and Potential Applications…………………………………..77
61
In order to optimize cell culture conditions, substrates must contain the proper
milieu of structural components and growth factors. These molecules also must be
present in the correct ratios to grow and maintain specific cell types. The method of
decellularization solves this problem by removing native cells from tissue while
preserving the natural ECM arrangement. The byproduct is a non-immunogenic scaffold
that can be directly seeded or indirectly incorporated into tissue culture substrates and
cell therapies. In turn, these substrates can be utilized to test the effects of certain
substrate properties on cell signaling and function. Our own assays used the versatile
nature of liver ECM hydrogels in order to study the effects of mechanical stiffness on
primary hepatocyte function.
1. ECM Isolation through Decellularization Methods
A wide variety of approaches were developed to decellularize tissue or whole
organs for ECM isolation and eventual use in cell culture. Methods are dependent upon
the tissue and animal type being processed. Variations exist in the mechanical or
chemical procedures used. Protocols utilize different physical forces, compounds, and
reagents for cellular removal. Incubations in solutions and subsequent washing steps vary
in duration. These diverse procedures for decellularization have experienced a wide range
of success and failure.
Each decellularization technique possesses a set of advantages and disadvantages.
One of the first methods developed in this field ruptured cells through utilization of
distilled water and salt solutions in combination with mechanical force (1). Use of
hypertonic and hypotonic solutions to lyse cells does minimum harm to the underlying
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ECM structure but on the other hand, does not remove smaller cellular components from
the tissue (2-8). Addition of physical disruption and agitation of tissue promotes full
clearance, however, it can also lead to damage of the intact architecture and unintended
removal of ECM constituents (9).
Another early decellularization technique implemented the use of freezing and
thawing to eliminate cells from tissue constructs (10-14). This simple method does not
require creation of solutions or the involvement of physical force. Repeated freeze thaw
effectively kills and lyses cells but can ultimately degrade certain proteins of the ECM,
especially growth factors which exhibit limited half-lives (15). Because of the sensitivity
of specific molecules and the unknown extent of ECM damage due to this process, freeze
thaw cycles should be kept to a minimum.
Newer methods incorporated varying concentrations of enzymatic reagents and
detergents into decellularization protocols. Enzymes including trypsin and DNase are
often used to treat scaffolds for cellular removal (16-24). These compounds are highly
successful in liberating cells from the ECM and break down immunogenic cellular
components such as nucleic acids (16). During natural processes in the body, apoptotic
cells trigger caspase-activated DNases which degrade DNA into 180 base pair fragments.
These nucleic acids are further degraded by DNase II of macrophages to avoid initiation
of innate immune reactions (25). For these reasons, important criteria established for
decellularization include degrading any residual DNA fragments to below 200 base pairs
(bp) in length and reducing DNA levels to below 50 ng of DNA per mg of dried tissue
(9). These actions are vital in avoiding immune responses when these substrates are
implanted into in vivo systems. Despite abilities to reduce immunoreactivity, lengthy
63
enzymatic treatments can also lead to destruction of ECM proteins and
glycosaminoglycans (GAGs) (26). Difficulties also exist in subsequent removal of
enzymes from the scaffold. Remnant enzymes could be harmful to animal models or
human patients and induce their own immune reactions.
An alternative option to enzymes for elimination of cellular material is detergents.
Previous publications have reported advantages of detergents over enzymatic methods in
maintenance of mechanical and elastic properties of the ECM (27). Detergents vary in
structure and strength, which both dictate concentrations applied and length of time
utilized. The two main types of detergents used in decellularization are ionic and non-
ionic detergents. Ionic detergents contain a charged head group and hydrophobic tail
group. This structure allows for rapid and effective disruption of the cellular membrane
and full removal of all cellular components from tissue (16, 24, 28-31). However,
because of its strength, this compound can easily denature or remove ECM molecules
such as GAGs and basement membrane proteins if not limited in concentration or
duration of use (32, 33). In comparison, non-ionic detergents are gentle on the underlying
matrix. Their hydrophilic groups are uncharged but are still able to disrupt lipid bonds
(28, 34-38). However, their mild nature means greater detergent concentrations and
longer washes are required for full cellular removal. Non-ionic detergents are often
combined with other reagents to ensure complete and timely decellularization (7, 39, 40).
The decellularization method designed by our research group and chosen for use
in this thesis project involves use of both ionic and non-ionic detergents. The
decellularization protocol has been optimized for use with livers of rat models. The
process takes advantage of the rat’s intact vasculature in order to perfuse solutions
64
Fig.1) Rat Liver Decellularization, adapted from Shupe et al (1)
A) PBS washing removes blood from the liver. B) Triton-X 100 detergent solubilizes lipid
membranes, while C) SDS detergent removes remnant cellular material.
throughout the organ. This approach enables solutions to infiltrate all areas of the tissue
for maximal contact with cells. The impact is faster and more efficient decellularization
as compared to simply immersing tissue surfaces. These developed methods allow
preservation of structural and biologically active components vital for effective growth
and maintenance of cells. For whole organ engineering, perfusion also allows for later
use of the vasculature during reseeding of cells. Through this established protocol,
decellularized liver tissue or substrates supplemented with the isolated ECM improve
liver cell viability and long term function in the cell culture assays of this thesis work.
65
2. Substrate Forms
ECM isolated from the decellularization process can be utilized in several
different configurations for many different applications. The intended objective
determines how this material must be used.
2.1 Decellularized Tissue Constructs
One of the main uses of decellularized organs and vascular constructs is for tissue
engineering. With a large disparity between donor organs and patients requiring
transplantations, the goal of organ engineering is to narrow this gap by supplying lab
grown tissues, preferably with patients’ own cells to avoid an immune response. To
engineer complex organs, one of the primary objectives is to create a fully cellularized
and patent vasculature. This task is made easier by maintaining an intact vessel basement
membrane.
Several groups have successfully generated re-endothelialized and functional
vascular grafts through seeding of decellularized scaffolds (41-46). Similar strides have
been made at a reduced scale in the recellularization of small animal organs or small
tissue samples (39, 47-49). Decellularization not only preserves the ECM composition
but also the structural architecture of the tissue. This remnant microarchitecture allows
cells to attach, align, and self-organize into proper functional units within both
parenchymal and vascular regions.
Challenges remain in whole organ engineering. Consistency of decellularized
tissue samples and the ability to control the physical and biochemical properties of the
decellularized matrix remain a problem. The exact physical and biological toll various
66
detergents take on ECM during decellularization is not fully controllable or known.
Certain key growth factors or smaller microstructures may need to be replaced or
reconstructed in order to produce an optimally functional cell scaffold. Microscopic
structures are easily damaged by decellularization reagents, including glomeruli of the
kidney, as well as bile canaliculi and sinusoids of the liver. Future studies should focus
on modifying and improving methods for preserving these delicate but important
structures. Research is still needed to optimize exact concentrations of reagents, duration
of individual steps, and storage methods for decellularized tissue. Studies also need to
gain a better understanding of complex interactions within tissue-specific ECM,
including the binding and biological activity of growth factors. New knowledge will
build upon current advances and lead the field closer to achieving functional tissue
products.
2.2 ECM Hydrogels
Hydrogels provide a versatile platform to isolate and test individual physical and
chemical properties. The flexibility of gel substrates allows for alteration of properties
such as stiffness, growth factor content, and ECM composition. These substrates can be
applied to everything from drug testing to a variety of novel therapies and treatments.
Supplementing these gels with organ-specific ECM improves attachment, growth, and
phenotype of cells similar to levels exhibited in fully functional tissue.
One of the uses of ECM hydrogels is to reproduce conditions found in vivo in order to
model certain disease or repair states. Three-dimensional cell culture in gels effectively
model tumors and metastatic phenotypes for cancer research (50, 51). High stiffness gels
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can simulate diseases like liver cirrhosis, while lower stiffness mimics a developmental
or regenerative environment (52, 53). An upregulation of cellular production of specific
growth factors and matrix proteins also occurs during disease or regeneration (54-57).
Exogenous growth factors can be bound to active sites in ECM supplemented gels, or
ratios of specific structural molecules can be altered. This capacity for modifications
allows accurate modeling of ECM microenvironments for many tissue types and
physiological scenarios. Allows us to explore effects of composition
2.3 Species Differences
For this thesis project, decellularized rat liver tissue was integrated into HA gels
for preliminary primary rat hepatocyte studies and subsequently, primary human
hepatocyte culture. ECM components including collagens, polysaccharides, and
fibronectin are highly ubiquitous in mammalian tissue (58, 59). Molecular structures and
cell binding sites of these proteins are highly conserved between rats and humans and
interact with common cellular factors (60-62). GAGs and associated growth factors also
contain conserved structures and functions (58, 63, 64). In our own assays, we have
observed human growth factors including HGF and EGF stimulating rat cells and vice
versa. Because of their structural similarities, species differences between rat and human
liver ECM proved to be inconsequential in our assays.
3. Results and Advantages of ECM Use in Cell Culture
Traditional cell culture methods use synthetic materials or incorporate single
protein coatings like collagen into substrates for cell attachment and growth. With the
development of decellularization, a fuller representation of organ-specific ECM
68
components could be included. Several studies have shown the advantages of culturing
different cell types on natural, tissue-specific ECM substrates compared to purely
synthetic substrates. ECM from different tissue types can direct stem cells towards
various cell lineages (65-70). In liver culture systems, specific ECM cues can promote
the differentiation of hepatic progenitor cells into fully mature hepatocytes or
cholangiocytes (44, 71-74).
To develop effective and successful treatments, long term viability and phenotype
maintenance needs to be supported. Heparin and HA hydrogels are favorable substrate
bases because these molecules are naturally found in the body, do not induce immune
reactions upon implantation, and have the ability to bind and present growth factors (75,
76). We hypothesize addition of tissue-specific ECM would allow cells to function at
higher levels for use in cell based therapies. Previous studies have shown how tissue
specific ECM improves long term cell function in vitro. Both liver progenitor cells and
adult hepatocytes seeded onto isolated liver ECM maintained viability and function over
longer periods of time than cells grown on tissue culture plastic or collagen alone (44, 77-
79). Decellularized matrix has supported growth and phenotype of cells of several
different tissue types for a variety of uses (80-82). By recreating a natural
microenvironment in culture, cells are able to function and interact as they would within
normal, healthy tissue of the body.
With the addition of diverse species of GAGs present in natural ECM, greater
control over growth factor binding and release could be achieved to aid the efficiency of
cell therapies. Improvements in phenotypic maintenance were observed in cells
encapsulated with a combination of several structural molecules and growth factors,
69
especially hepatocyte growth factor (HGF) and epidermal growth factor (EGF) with
hepatocytes (83, 84). New data on the cellular effects of various substrate compositions
and properties will enable better design of cell therapies. Combining innovative methods
and knowledge will optimize their effectiveness in combating or correcting diseases in
patients.
4. Altering Stiffness in Cell Culture Substrates
4.1 Methods for Manipulating Hydrogel Stiffness
As mentioned in the previous chapter, physical properties are as important as the
presence of specific ECM components for differentiation or maintenance of various cell
types during seeding of intact tissue. Mechanical properties are modified by a wide
variety of strategies and techniques in cell culture. Previous groups have used increased
concentrations of proteins or synthetic compounds such as acrylamide to bolster substrate
stiffness (85-87). However, these alterations can influence cells in ways beyond stiffness,
and tend to complicate assay results. To isolate the property of stiffness during
experiments, the best techniques avoid addition of biologically active molecules.
One of the simplest and most common methods for modifying stiffness is through
substrate crosslinking. Crosslinking allows precise manipulation with limited
confounding effects on attachment sites or biological activity of the substrate. Stiffness
alterations can be made by varying the crosslinker type, the structure and arm length of
the crosslinker, the crosslinker concentration, or the duration of the crosslinker treatment
(88). The choice for crosslinker type is contingent on which molecules are available in
the substrate. These reagents chemically react with several different moieties including;
70
thiols, amines, carboxyls, hydroxyls, and carbonyls (89). Crosslinkers can also be
photoreactive and are activated by exposure to ultraviolet light (90, 91).
A crosslinker is either classified as homofunctional or heterofunctional, reacting
and covalently binding to one or more classes of functional groups. Homofunctional
crosslinkers act at rates and strengths dependent on the concentration of the crosslinker,
as well as the density of reactive groups in the substrate’s constituents. Greater
concentrations of the reagent cause a faster and higher degree of crosslinking. A larger
quantity of active sites in the substrate also translates to quicker bonding and stiffer
materials (92). Multiple homofunctional crosslinker types can be used in procedures to
gradually stiffen materials during activities such as bioprinting (93). Unlike
homofunctional reagents, a single heterofunctional linker type can be utilized in multiple
step reactions to produce viable substrates. Instead of combining all substrate components
at once, the crosslinker is allowed to react with one molecule before additional
compounds are added. This process provides greater control over specificity of
interactions and spacing of bonds for more exact material construction (93, 94).
Properties of the crosslinker are not only dependent on its chemical specificity but
also vary according to the length and number of structural arms it contains. A longer
distance between crosslinks creates a looser assembly of compounds and a softer
substrate. Therefore, to increase stiffness, one must use a crosslinker that is shorter in
length (92, 95). The typical reagent is bifunctional and holds two binding sites. However,
crosslinkers have been engineered to contain as many as eight arms. Greater amount of
branching leads to more reactive ends and a greater degree of binding. The geometry of
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multi-armed crosslinkers is also more compact which generates tighter packed and stiffer
structures (96).
For this thesis project, our group chose a polyethylene glycol (PEG) based
compound for crosslinking applications during formation of liver ECM hydrogels. PEG
based chemicals are nontoxic and do not influence cell function (97). They also do not
initiate immune reactions upon implantation in vivo (98, 99). Because PEG is water-
soluble, clumping of the compound is avoided, and crosslinking is evenly spaced within
the polymer (100). PEG creates hydrophilic substrates which have been shown to
promote better cell adhesion, proliferation, spreading, and function than hydrophobic
materials (101, 102). The combination of these characteristics makes PEG an ideal
crosslinker for use in primary hepatocyte culture and development of cell treatments.
Liver ECM gels were created by mixing solubilized liver matrix with hyaluronic
acid and denatured collagen solutions. These HA gel components were specifically
modified to contain reactive thiols for crosslinking (103). Acrylate is a type of molecule
often attached to a PEG spacer to form a crosslinker that contains reactive double bonds
which bind to sulfhydryl groups. Our assays tested several geometric variations of PEG
acrylate in order to solidify the liver hydrogels at various stiffnesses.
Polyethylene glycol diacrylate (PEGDA) is one of the most commonly used
crosslinkers. It has a two-arm linear structure that produces a softer gel type (104). This
substrate stiffness was used as a baseline for our assays. PEG acrylate variations
containing four or eight arms also exist (105). More acrylate arms allow for a greater
number of reactive ends to bind thiol groups. In our own assays, PEGDA, four-arm PEG
72
acrylate, and eight-arm PEG acrylate were added to HA-ECM gel constituents at various
concentrations. Results showed that eight-arm PEG acrylate at the highest concentration
of 6% weight by volume produced the highest stiffness.
Although most combinations of crosslinker geometry and concentration produced
distinctively different stiffness levels, a saturation point of each crosslinker was also
determined. Higher concentrations of crosslinker past this limit did not significantly
affect stiffness, regardless of additional reaction time (Figure 3). This upper limit is
reached when all thiol groups are utilized. To ensure excess crosslinker is not retained in
the gel, no concentration above the observed saturation threshold was chosen for
subsequent assays. Furthermore, gels were thoroughly washed with PBS to clear out
excess crosslinker.
Fig.2) PEG Crosslinker Structures, adapted from Zhu et al (119) Greater PEG branching or arm number creates a tighter molecular network and increases
substrate stiffness.
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4.2 Measuring Stiffness
Several methods and instrumentation were developed to measure the property of
stiffness. For centuries, the simple practice of palpation was used to diagnose patients
(106). Clinicians now utilize a non-invasive method called elastography to image and
quantitate stiffness of intact organs. This technique employs the use of ultrasound or
magnetic resonance imaging (MRI).
Several variations of ultrasound elastography evolved over time. Early forms
involved application of a manual force to the exterior of patients which was then
measured internally by ultrasound to calculate organ stiffness (107). Accuracy improved
through use of mechanical pulse generators and more advanced sonography systems.
During transient elastography, an automated device creates shear waves that penetrate
through the skin into organs of the abdominal cavity such as the liver (108, 109). The
0
200
400
600
800
1000
1200
1400
1% 2% 4%
G' (P
a)
Crosslinker Concentration % (w/v)
Crosslinked Gel Stiffness
Fig.3) PEGDA Crosslinking Saturation- HA-ECM gels were crosslinked with 1%,
2, or 4% w/v PEGDA (3.4 kDa). Only the 1% and 2% PEGDA gels were
significantly different (*p<.05, Student’s t test, n=4)
74
velocity of the waves is measured by ultrasound as they pass through the tissue of
interest. This data allows clinicians to calculate the stiffness of a large representative area
of the organ for diagnosis (110). Instead of acting on the surface of the skin, newer
adaptations employ concentrated sonography waves to generate acoustic radiation forces
at the site of measurement within the tissue (111-113). These processes improve the
distance pulses can be directed within the body. Acoustic radiation forces also increase
the precision of deep tissue ultrasound measurements through their ability to travel across
fluid build-up in the peritoneal cavity which normally impedes transient elastography
(110, 112).
Magnetic Resonance Elastography (MRE) incorporates these same mechanisms to
propagate shear waves. However, the velocity of these waves is subsequently measured
by MRI. Unlike ultrasound technology, a three-dimensional image of the entire tissue is
produced alongside a map of various stiffness values (114, 115). This tool provides a
more complete analysis of the organ being tested. MRE has proven to be a reliable tool
for detection of fibrosis in diseased liver (116). It has also successfully evaluated
stiffnesses in organs such as the brain that were unattainable by ultrasound methods
(117). Further refinement is needed to optimize the use of MRE in other tissue or organ
types (114).
In a laboratory setting, a rheometer is one of the most popular instruments used to
measure stiffness for samples prepared ex vivo. Unlike non-invasive clinical methods,
Rheometry is used to analyze biopsies or excised tissue outside of a living organism. This
technique is also commonly used in calculating stiffness of hydrogels, biomaterials, or
synthetic substrates used in cell culture. Because samples are isolated from irrelevant
75
tissues, fluids, or other confounding factors, rheometry increases accuracy and precision
of measurements compared to ultrasound and MRE (118). Engineers developed several
variations of rheometers based upon the material being tested and the type of data
required for analyses.
Rotational or shear rheometry is often used to measure stiffness in viscoelastic
soft tissue, hydrogels, or polymers (119, 120). These methods were applied in our own
assays to determine stiffness in liver ECM hydrogels and decellularized liver tissue.
During testing with these rheometers, a sample is stabilized on a fixed platform or plate.
An oscillating geometry in the form of a cone, plate, or cylinder is subsequently lowered
until it comes into contact with the sample (118). From the various forces applied, an
elastic modulus can be measured and calculated. The elastic modulus, or resistance to
deformity, can be expressed by three types of calculations; Young’s modulus, bulk
modulus and shear modulus. Young’s modulus is the ratio of tensile stress to tensile
strain or the force required to compress an object (121). The bulk modulus is the amount
of force needed to uniformly deform a material or the ratio of volumetric stress to
volumetric strain (122). The shear modulus describes how a sample responds to opposing
forces parallel to its surface and is calculated by dividing shear stress by shear strain
(123).
In the stiffness tests we performed, the Discovery Series HR-2 Rheometer (TA
Instruments, New Castle, DE) was used with a parallel plate geometry. Oscillation stress
sweeps incrementally increased shear stress on the samples up to 10.0 Pa and were run at
a constant oscillatory frequency and axial force. Both the shear storage modulus (G’) and
shear loss modulus (G’’) were measured and recorded. The storage modulus describes the
76
elastic properties or stored energy, while the loss modulus describes the viscous
characteristics or energy lost as heat (124). The storage modulus was the main stiffness
value utilized for reporting and comparison of hydrogels in our assays.
Shear rheometry provides an overall representation of stiffness in materials by
assessing a substantial portion of the substrate. For rheology measurements of smaller
microstructures or individual cells, atomic force microscopy (AFM) is required. An
atomic force microscope contains a high resolution nano-indenter or tip and detection
probe that scans indented samples to evaluate microscopic areas for stiffness (125-128).
Instead of measuring the combined effects of cell and ECM stiffness, AFM allows
separate analysis of components. Singular structures and cells in a sample can easily be
distinguished and measured to assess conditions such as cancer (129). Multiple readings
of a larger homogenous substrate can also be obtained and averaged to calculate more
wide-ranging stiffness (130, 131).
Despite AFM’s exceptional resolution, disadvantages also exist. AFM performed
on a heterogeneous sample can give an incomplete or inaccurate representation of the
material tested if too few readings are acquired or only a partial area is covered (132).
Problems can also arise depending on the indenter type used. Improper tips cause artifacts
to develop in samples (127, 131). Indenters can also adhere to softer materials and cause
miscalculations of stiffness (133).
Every analysis of stiffness presents unique objectives and obstacles. Clinicians
require a quick and noninvasive strategy, while a laboratory setting affords greater time
and control over samples. By effectively evaluating the overall stiffness of cell culture
77
substrates, shear rheometry demonstrated the greatest ability to study altered stiffness in
our liver ECM gels.
5. Conclusions and Potential Applications
In previous studies, hepatocytes cultured on different substrate stiffnesses have
been reported to have generalized effects. Low stiffness is associated with a growth
arrested and differentiated adult phenotype. High stiffness is associated with increased
viability and cell proliferation but also dedifferentiation and possible initiation of EMT
(53). However, the exact stiffness ranges defined in these studies and substrate
compositions vary greatly. Stiffness levels are also often at non-physiologically
achievable levels.
The assays we conducted utilized liver specific ECM isolated by decellularization
to form gels so that substrate components were similar to what composes normal liver
tissue. We also designed our study to focus on a narrow physiological relevant range of
stiffness. The adjustable system was created to culture primary human hepatocytes to
determine optimal conditions for cell function and distinguish specific mechanisms for
stiffness-induced changes in cell mechanics and morphology. We hypothesized that the
substrate stiffness closest to the actual stiffness of native liver tissue promotes the highest
long term function.
This knowledge can enable advancement in the creation of substrates for liver cell
therapies to treat disease in patients. This technology was developed to easily manipulate
and test effects of mechanical properties on cells. Future studies can use these abilities to
simulate the effects of disease states in a variety of organ systems and determine disease
78
mechanisms in order to form possible treatment strategies. Increased dimensions of
complexity in architecture and cell types could also be added to the system. These ECM-
containing hydrogels could eventually be used for tissue engineering applications.
Because of its semi-fluidic properties, cells encapsulated in gels can be injected into
numerous sites in vivo (134-138). Gels can also be “printed” into various patterns for
tissue construction. Bioprinting is a new technology that rapidly deposits substrates into a
predetermined form. The process controls substrate shape, composition, and physical
properties like stiffness. After gels are printed into constructs, materials can be fully
cross-linked depending upon the desired tissue type. Bioprinted tissues currently in
development include everything from skin patches and vessels to complex organs like the
liver (80, 139-142). With the use of ECM hydrogels and new emerging technologies, the
possibilities for new therapeutic products and treatments are endless.
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CHAPTER 3
Stiffness of Hyaluronic Acid Gels Containing Liver
Extracellular Matrix Supports Human Hepatocyte Function
and Alters Cell Morphology
Daniel B. Deegan
This chapter includes and expands upon the results of the published manuscript,
“Stiffness of Hyaluronic Acid Gels Containing Liver Extracellular Matrix Supports
Human Hepatocyte Function and Alters Cell Morphology.”
Manuscript Reference:
Deegan D.B., Zimmerman C., Skardal A, Atala A., Shupe T., Stiffness of Hyaluronic Acid Gels
Containing Liver Extracellular Matrix Supports Hepatocyte Function and Alters Cell Morphology. Journal
of the Mechanical Behavior of Biomedical Materials. 2015; pp. 87-103
90
Abstract
Tissue engineering and cell based liver therapies have utilized primary hepatocytes with
limited success due to the failure of hepatocytes to maintain their phenotype in vitro. In
order to overcome this challenge, hyaluronic acid (HA) cell culture substrates were
formulated to closely mimic the composition and stiffness of the normal liver cellular
microenvironment. The stiffness of the substrate was modulated by adjusting HA
hydrogel crosslinking. Additionally, the repertoire of bioactive molecules within the HA
substrate was bolstered by supplementation with normal liver extracellular matrix
(ECM). Primary human hepatocyte viability and phenotype were determined over a
narrow physiologically relevant range of substrate stiffnesses from 600 to 4600 Pa in
both the presence and absence of liver ECM. Cell attachment, viability, and organization
of the actin cytoskeleton improved with increased stiffness up to 4600 Pa. These
differences were not evident in earlier time points or substrates containing only HA.
However, gene expression for the hepatocyte markers hepatocyte nuclear factor 4 alpha
(HNF4α) and albumin significantly decreased on the 4600 Pa stiffness at day 7 indicating
that cells may not have maintained their phenotype long-term at this stiffness. Function,
as measured by albumin secretion, varied with both stiffness and time in culture, peaking
at day 7 at the 1200 Pa stiffness, slightly below the stiffness of normal liver ECM at 3000
Pa. Overall, gel stiffness affected primary human hepatocyte cell adhesion, functional
marker expression, and morphological characteristics dependent on both the presence of
liver ECM in gel substrates and time in culture.
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1. Introduction
Rising incidence and cost of liver disease as well as a limited supply of
transplantable organs have increased demand for new liver treatments. Experimental
treatments currently being developed include liver cell transplant on biological scaffolds
(1-5). However, several limitations intrinsic to primary hepatocytes have slowed
development of these technologies. Challenges remain in the maintenance of viability and
cell function of primary hepatocytes outside of the normal liver microenvironment (5).
Therefore, developing substrates that support hepatocyte viability and function is critical
to maximize the efficacy of primary hepatocytes in tissue engineering and cell therapies
for liver disease.
Previous studies have identified several advantages of using tissue-specific ECM
instead of generic matrix formulations such as Matrigel for supporting primary cells (6).
Cells on natural liver ECM demonstrated increased ability to proliferate and maintain
function over extended time periods (7). Hepatocytes cultured on intact or solubilized
organ specific matrix also maintained normal cell morphologies as compared to cells
grown on tissue culture plastic or collagen (3, 8, 9). The underlying mechanisms for this
improved phenotypic stability are still unknown but likely relate to inclusion of several
components of the natural liver microenvironment, including cell and growth factor
binding sites.
For this study, decellularization of normal liver was used to isolate acellular ECM
for incorporation into hyaluronic acid (HA) hydrogels. The decellularization process
involved perfusion of the organ with detergents to remove native cellular material, while
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preserving structural proteins and glycosaminoglycans (GAGs) (10). Removal of native
cells leaves a non-immunogenic ECM scaffold that can be seeded with hepatocytes that
remain viable for several weeks (11).
Many previous studies utilized ECM coated tissue culture plastic for growing
primary hepatocytes. However, this method is only suitable for two-dimensional culture
and is not easily translated into a three-dimensional form for use in cell based therapies
(12, 13). The current study uses a hydrogel that may be formed into three-dimensional
units suitable for transplantation. Preliminary findings indicated that solubilized ECM
combined with hyaluronic acid gel (HA) maintained a normal epithelial morphology and
strong tight junction formation with little evidence of transition to a fibroblast-like
morphology, commonly seen in traditional collagen cultures. Solubilization of the ECM
also prevented hepatocyte apoptosis induced by phagocytosis of cryomilled ECM
powders. Other studies published by our group demonstrated that HA based hydrogels
avoided an immunogenic response measured by human lymphocyte proliferation, while
other gel types such as rat tail collagen 1 induced a low level immune response (11, 14).
Better phenotype maintenance and biocompatibility upon transplantation signaled that
HA gel was the best substrate for incorporating decellularized liver in future assays.
In the development of a liver ECM substrate, the physical characteristic of
stiffness is important to cell physiology and mechanics. The general conclusions of
previous publications indicate that stiff substrates promote hepatocyte spreading,
proliferation, and dedifferentiation; while soft substrates promote maintenance of the
functional hepatocyte phenotype (15, 16). Substrate stiffness also regulates cell
aggregation, growth factor responsiveness, and cell motility with the highest cell
93
migration occurring on intermediate stiffness levels (17, 18). However, several of these
previous studies were done on stiffness ranges well above physiological liver stiffness,
which has been reported anywhere from 1.5 to 8.5 kPa (19-24).
For the current study, the crosslinker concentration was the sole variable for
adjusting gel stiffness. Crosslinking eliminated potential confounding effects found in
more complex systems where concentrations of structural compounds are altered. Gel
stiffnesses were also limited to a narrow, physiologically relevant range of 600-4600 Pa
that bracketed the stiffness of normal liver ECM at 3000 Pa. Assays were conducted to
measure overall hepatocyte function as well as the mechanisms by which substrate
stiffness affected cell phenotype. Specific tests performed in this study were designed to
explore the mechanisms by which substrate stiffness affected substrate/cell adhesion,
primary hepatocyte function, and morphology. Cell junction formation is critical for the
maintenance of epithelial cell phenotype; regulating a broad range of cellular properties
through cell-cell communication, adhesion, and diffusion of water and solutes (25, 26).
Tight junctions act as semi-permeable barriers that establish cell polarity, which is crucial
for the maintenance of bile canaliculi (27, 28). The tight junction protein, claudin-1, is
required for maintenance of the selective barrier properties between adjacent hepatocytes
(29). Occludin is not essential for maintaining tight junction structures, but plays a role in
regulating cytoskeletal organization, cell polarity, and cell migration (30-32).
The effects of substrate stiffness on intracellular signaling were also studied
through measurement of two cytoplasmic kinases: focal adhesion kinase (FAK) and
integrin-linked kinase (ILK). Cells adapt their cytoskeletal structure to specific
microenvironmental conditions through interactions between focal adhesion molecules
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and intracellular kinases. Appropriate expression of these kinases in hepatocytes is
required for normal growth factor responsiveness, prevention of apoptosis, and normal
metabolic function (33-35). Silencing or overexpression of these factors can lead to
cellular dysfunction and, in the case of neoplasia, tumor progression (36). Increased
expression also mediates cytoskeletal reorganization and cell protrusion formation which
drives cell migration (37, 38). In other experimental systems, FAK and ILK expression
have been shown to correlate with cell junction remodeling, specifically through
interactions with occludin. FAK and ILK also act through downstream activation of Ras
homolog family member (Rho) GTPases; cell division control protein 42 (CDC42), Ras-
related C3 botulinum toxin substrate (Rac1), and Ras homolog family member A (RhoA)
(39, 40). CDC42 and Rac are the two GTPases involved in actin cytoskeletal
reorganization for cell motility, including formation of cells extensions called filopodia
and lamellipodia. RhoA expression increases focal adhesion and stress fiber formation
and is correlated with a more contractile morphology (41-44).
By studying the stiffness responsive factors that influence hepatocyte structure
and function, we have gained insights into the mechanisms through which substrate
stiffness affects the viability and phenotypic stability of primary hepatocytes. These
findings have informed the development of a transplantable gel containing liver specific
ECM at an optimal stiffness for therapeutic use in hepatocyte cell therapies.
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2. Materials and Methods
2.1 Liver Decellularization
Intact rat livers were decellularized according to our group’s previously published
methods (10). Ten week old male F344 rats were euthanized by administering a lethal
dose of sodium pentobarbital (100 mg/kg). Livers were then cannulated and perfused by
methods previously diagrammed and reported in our cell isolation protocol (45). In brief,
a euthanized rat was pinned on a dissection board, and a mid-line incision was made
through the peritoneum starting from the diaphragm down to the groin. Visceral organs
were shifted to expose the inferior vena cava (IVC) and liver. The IVC was cannulated
with a 20-gauge catheter and tied in place with sutures. The portal vein was then cut, and
the superior vena cava (SVC) was clamped. Solutions were held in flasks at 37°C in a
water bath (Fisher Scientific, Pittsburgh, PA) and then perfused through the catheter at a
flow rate of 5 mL/minute using tubing and a Masterflex L/S peristaltic pump (Cole-
Parmer Instrument Company, Vernon Hill, IL).
100 mL of PBS was first perfused to clear residual blood. The liver was then
perfused with approximately 300 mL each of 1%, 2% and 3% Triton X-100 solutions in
PBS. A final solution of 0.1% SDS in PBS was used to clear remaining DNA and Triton
detergent. The liver was then fully excised, and overnight PBS washing was used to
remove residual SDS from the matrices. All animal procedures were approved by the
Institutional Animal Care and Use Committee (IACUC) at Wake Forest University.
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2.2 Immunohistochemistry (IHC) on Decellularized Tissue
Following decellularization, we immunostained rat livers to assess cell clearance
and retention of structural components such as collagens and laminin. As reported in our
previous manuscript, decellularization completely removed cellular material from tissue
sections, while collagen and laminin were preserved and represented (Figure 1) (10).
For this study, Alcian blue staining for mucopolysaccharides that comprise GAGs
was performed on frozen decellularized liver sections. Tissue was frozen in OCT blocks
and cut into 5 μm slices that were then fixed in acetone on slides at 4ºC for two minutes.
Sections were then stained according to a previously established protocol (46). In short,
Fig.1) IHC of Decellularized Liver Tissue, adapted from Shupe et al (10)
A) Hematoxylin and eosin staining (H&E) shows full cellular removal. B)
Trichrome staining shows presence of structural collagens. C) Staining for
laminin and D) collagen IV indicate presence of a basement membrane.
97
sections were incubated in 0.5% Alcian blue 8G solution in 0.5% acetic acid for 30
minutes (Sigma-Aldrich, St. Louis, MO). Sections were then washed with PBS and
imaged.
2.3 GAG Function in Decellularized Tissue
To assess the ability of residual GAGs to bind growth factors following
decellularization, 5 mg samples of decellularized liver were incubated at room
temperature for one hour in PBS containing an array of concentrations of recombinant
epidermal growth factor (EGF) and human hepatocyte growth factor (HGF) up to 1000
ng/mL (Peprotech, Rocky Hill, NJ). Following pre-treatment, tissue was washed for 1
hour at 4ºC in four separate 50 mL volumes of PBS to remove unbound growth factor.
We confirmed that following the fourth wash, greater washing of up to ten washes
created no differences in bound growth factor quantity. Following lyophilization,
cryomilling, and solubilization of the tissue matrix in HCl-pepsin buffer solution, we
quantified EGF or HGF levels in each sample by ELISA (RayBiotech Inc., Norcross,
GA). Samples were normalized through measurement of total protein using the Pierce
BCA Protein Assay Kit (Thermo, Rockport, IL).
2.4 Liver ECM Solubilization
Gels with added ECM were created according to previously published protocols
from our group (8). In brief, decellularized liver tissue was frozen at -80ºC and
lyophilized using a SuperModulyo Freeze Dryer (Thermo, Waltham, MA). Samples were
maintained under vacuum at a pressure of 0.1 mbar and temperature of -80ºC for 24
hours. Tissue was then ground in liquid nitrogen using a mortar and pestle until a fine
98
powder was attained. Liver ECM was solubilized by mixing lyophilized decellularized
tissue with 0.1 N HCl and pepsin (Porcine gastric mucosa, 3400 units of protein, Fischer
Scientific, Fair Lawn, NJ). After an incubation of 48 hours at room temperature, the
solution was centrifuged and filtered with a 0.2 µm syringe filter. The pH was adjusted to
7.0 and the total protein concentration to 1.0 mg/mL.
2.5 Solubilized Liver ECM Quantitative Analysis
DNA from solubilized liver ECM solutions was isolated and purified using the
Qiagen DNeasy Kit. DNA amounts were quantified using a set of standards and the
Quant-iT PicoGreen reagent kit (Invitrogen Corp., Carlsbad, CA) that could detect lower
than 1 ng/mL of DNA in solution.
GAG levels were quantified using the Blyscan sGAG Assay kit (Biocolor,
Newtownabbey, UK). 40 μL of solubilized ECM was mixed with 160 μL of 0.2 M
sodium phosphate buffer containing 1 mg/mL of papain and incubated at 65ºC for 3
hours. Samples were then mixed with Blyscan dye, pelleted, and washed. After
dissociation reagent was added, samples and GAG standards were loaded into a 96 well
plate and measured in a SpectraMax M5 Multi-Mode Microplate Reader at an absorbance
of 656 nm (Molecular Devices, Sunnyvale, CA).
Collagen levels were quantified using the Sircol Soluble Collagen Assay kit
(Biocolor, Newtownabbey, UK). 20 μL of solubilized ECM was mixed with 130 μL of
0.5 M acetic acid containing 0.1 mg/mL pepsin and then incubated overnight at 4ºC.
Samples were then mixed with Sircol dye, pelleted, and washed. Dissociation reagent
99
was added, and samples and standards were read in a 96 well plate at an absorbance of
555 nm.
2.6 ECM Gel Formation
Liver ECM solution was combined at a 1:1 ratio with thiol modified HA gel
components (HyStem Hydrogels, ESI BIO, Alameda, CA). Components include 3,3′-
Dithiobis(propanoic hydrazide) modified HA (Glycosil or HA-DTPH, Mw 168 kDa, Mn
79 kDa, polydispersity index 2.13), chemically modified gelatin-DTPH (Gelin-S, Mw
∼50 kDa), and heparin-DTPH (Heprasil, Mw ∼17-19 kDa). Protocols for synthesis of
these proprietary compounds can be found in the literature (47, 48). HA alone does not
provide sites for attachment during cell seeding. By including gelatin, or denatured
collagen, and supplemented liver ECM, adequate binding sites for cells were supplied. At
a sufficient stiffness, these molecules enable timely and efficient cell adhesion that
resembles what is observed in seeding of tissue scaffolds or protein coated tissue culture
plastic. These additions allow the hydrogels to mimic the composition and physical
properties of a normal tissue microenvironment.
To test the effects of mechanical properties, three different gel stiffnesses were
created. For the two lower stiffnesses, 1% and 2% weight/volume polyethylene glycol
diacrylate (PEGDA/Extralink, 3.4 kDa) were added to other gel components (ESI BIO,
Alameda, CA). The stiffest gel was created with 6% weight/volume 4-arm PEG acrylate
(20 kDa) (Creative PEGWorks, Chapel Hill, NC). As controls, gels containing only
components of the HA gel kit were created without the addition of solubilized ECM.
100
Gel solutions were added to wells of chamber slides or tissue culture plates and
allowed to fully crosslink for two hours. Gels completely filled the growth area of the
wells and polymerized at a thickness of over 1.5 mm to prevent the tissue culture plastic
bottom from contributing to the stiffness of the substrate. For cell culture assays
involving immunocytochemistry (ICC), 125 μL of each gel type was layered per well into
8-well Permanox Plastic Nunc Lab-Tek Chamber Slides to fill an area 80 mm² (Thermo
Scientific, Waltham, MA). For stiffness quantification, 325 µL of gel was added to 24-
well plates to fill an area of 200 mm². For assays measuring function, viability, and gene
expression, 50 µL of each gel type was layered into 96-well plates to fill an area of 32
were subtracted. Sample measurements were standardized by DNA amount (**p>.01,
Student’s t test, n=6)
134
Supplementary Fig.2) RT-qPCR measured expression of A) HNF4α, B) SNAIL, and C) Claudin-1
in primary hepatocytes grown on ECM containing HA gels of varying stiffnesses at 2 days post-
seeding. No significant differences were found. D) SNAIL expression was also measured at day 7
post-seeding. Expression decreased from day 2, but no differences were found between stiffnesses.
Expression was standardized relative to normal liver expression (p>.05, Student’s t test, n=3).
135
Supplementary Fig. 3) A) RT-qPCR measured expression of integrin beta-1 in primary hepatocytes
grown on ECM containing HA gels of varying stiffnesses at 2 days post-seeding (n=3, Student’s t test,
*p<.05). B) Expression was also measured at day 7 post-seeding, averaged between all stiffnesses,
and compared between gels containing only HA and gels supplemented with ECM (n=9, Student’s t
test, *p<.05). All expression values were standardized relative to normal liver expression.
136
Supplementary Fig. 4) Primary rat hepatocytes were seeded for 2 days on 600 Pa HA gels
supplemented with liver ECM that were untreated or pretreated with combined solutions of
EGF and HGF. Calcein AM stained live cells green, or ethidium homodimer stained dead cells
red. Viability staining on A) untreated or B) pretreated ECM gels showed increased cell
viability with growth factor treatment. C) Cell attachment and D) E-cadherin expression also
increased on growth factor pretreated ECM gels (*p<.05, ***p<.001, DNA n=5, RT qPCR n=3)
137
Supplementary Fig. 5) Human HSCs were seeded for 2 days on HA gels supplemented with liver
ECM at mean stiffnesses of 600, 1200, and 4600 Pa. A) HGF levels per well were measured in the
media by ELISA. B) Total GAG production per well was measured by a colorimetric assay (*p<.05,
**p<.01, ***p<.001, HGF n=3, GAG n=5)
138
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CHAPTER 4
Conclusions and Future Work
Daniel B. Deegan
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The field of regenerative medicine uses a variety of tools, technologies, and
advanced models to gain knowledge of disease mechanisms and to develop new therapies
and treatments. Each approach has a set of advantages and disadvantages depending on
the field of research. Three-dimensional liver spheroids, or organoids, recreate entire
organ systems on a miniature scale (1-5). Spheroids can be maintained in a variety of
substrates like hydrogels. In microfluidic devices, organoids are arranged in a circuit and
interact in a way that mimics how organ systems work together (6, 7). Consequently, they
are a great platform for toxicology and drug testing. Because of their longevity and long
term maintenance of function, testing can be carried out in vitro without the need for
complex in vivo models. However, organoids are not always suitable for ECM based
studies. These cell structures form tight junctions which prevent manipulation of ECM
composition and mechanical properties at the core of the organoids. Since cells only
sense ECM composition and mechanical forces in adjacent areas, these cell centers would
be relatively unaffected by substrate alterations.
Decellularized tissue is an excellent biomaterial for tissue engineering. Optimized
decellularization methods preserve ECM components in their native architecture and
maintain physiological proportions in the tissue. This scaffold material enables cells to
home to specific sites of the matrix and either sustain function or, in the cases of stem
cells, differentiate into appropriate cell phenotypes (8-10). However, challenges remain
in producing consistent decellularized products. The success of cell removal or the
remaining composition of the decellularized tissue can vary because of differences
between animals and differences in the size or vascularity of the organ. When designing
studies on small tissue segments, it can be hard to control for structural variances in the
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tissue that may exist even within the same organ. Matrix samples used for cellular assays
that are excised from different regions of the organ, certain areas that are not fully
decellularized, or sections damaged by the perfusion process can create error or deviation
in the results. It is also difficult to precisely manipulate physical and chemical properties
of the intact matrix. Without a complete analysis and mapping of the tissue, methods to
initiate these changes could be unpredictable.
Hydrogel substrates are viable alternatives to intact decellularized tissue
constructs. Although no longer possessing the native microarchitecture, gels enable better
control and manipulation of individual substrate properties. The concentration of specific
molecules in the hydrogels can be controlled and altered. In the case of this thesis study,
ECM from decellularized tissue was solubilized and incorporated into HA gel substrates
to replicate a natural liver microenvironment. Since the matrix of the entire organs was
solubilized in a homogenous solution, there was little variability within the material. All
regions of the ECM and multiple liver samples were combined, stored, and utilized
across multiple assays, allowing for consistent composition of the substrates. ECM
molecules were also quantified to ensure that all assays used the same total concentration
of solubilized material, and there were similar ratios of structural and bioregulatory
molecules. This ability standardized substrate production and resulted in more consistent
results and less experimental error.
This thesis work demonstrates that HA gels supplemented with ECM provided
some of the same functional advantages for seeded cells as fully intact matrix scaffolds.
Use of ECM containing gels in past studies were shown to greatly improve long term
viability and function of culture of cells, as compared to traditional in vitro methods (11,
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12). Our human hepatocyte assays showed increases in viability, adhesion, and cell
junction formation. To determine the mechanism for some of the improvements seen in
primary hepatocyte attachment and viability, expression of integrin β-1 was analyzed in
cells seeded on ECM supplemented gels versus cells on HA only gels. At day 7,
hepatocyte expression increased on gel substrates with the addition of liver matrix.
Although only one integrin class was measured, integrin β-1 may be the most
critical type for maintenance of cell function. Integrin β-1 is the most highly expressed
beta-integrin, dimerizing with more than 10 alpha-integrin subunits (13). These integrin
complexes directly interact with the actin cytoskeleton (14). Integrin β-1 expression
likely correlates with overall integrin expression in the presence of liver ECM and
explains the increases in hepatocyte attachment and viability relative to cells seeded on
simpler substrates. To establish this conclusion, expression of a greater variety of integrin
types could be analyzed. Varying concentrations of solubilized liver matrix could also be
used in substrate formation to determine if concentration dependent changes were
observed. As controls, certain integrins could also be blocked with increasing
concentrations of integrin-specific antibodies or RGD peptides in order to measure
reduction in binding or survival of the hepatocytes. Manipulations of the cells and the
adaptable gel substrate could help determine the most likely mechanisms for differences
observed.
Greater control of the composition of the substrate also correlates with better
control of the chemistry and physical properties of the material. These characteristics can
be adjusted by adding chemically-modified compounds to the substrate preparation.
Thiol-modified HA, heparin, and collagen were used for gel formation in our study. PEG-
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acrylate crosslinker reacted with these moieties to solidify hydrogels and alter stiffness
within a narrow range. Uniform composition of our ECM supplemented gels allowed
creation of sample replicates at equivalent stiffness levels. Different concentrations and
configurations of the crosslinker also generated adjustable degrees of stiffness to
establish separate experimental groups. Crosslinking can vary the property of stiffness
without modifying the concentration of functional elements in the ECM substrates. These
advantages make ECM supplemented gels optimal for use in cell culture assays to
identify the effects of substrate stiffness variations.
Following the development and creation of liver ECM supplemented gels, this
thesis evaluated the effects of substrate stiffness on primary hepatocyte function. Results
revealed hepatocyte gene marker expression and the functional output of albumin varied
with stiffness and time in culture. In early time points, higher gel stiffness stimulated
greater metabolic activity and albumin output. However, after metabolic functions
stabilized for 7 days, hepatocytes grown on an intermediate stiffness of 1200 Pa produced
albumin at the greatest rate. This observation was correlated to a significant drop off in
gene expression of hepatic markers by cells on the highest stiffness of 4600 Pa. To test
the hypothesis that the highest 4600 Pa stiffness causes a loss in long term phenotype,
experiments could utilize time points past day 7. These assays would indicate if observed
variances in function and gene expression widen between cells seeded on the different
stiffnesses in the long term. It would also be relevant to examine if markers for EMT like
SNAIL were upregulated by cells on the highest stiffness group.
The balance between EMT and MET is vital in liver development and could
possibly have an integral role in liver repair and regeneration. It is theorized that
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following injury, epithelial cells undergo EMT and travel to the mesenchyme to
proliferate and produce ECM molecules. These cells then differentiate into hepatocytes or
cholangiocytes to repopulate and restore the liver parenchyma (15). Imbalances in this
process can lead to increases in fibroblasts and a progression of disease (16). During liver
disease, many experts theorize that the stiffening ECM microenvironment promotes
additional EMT of hepatocytes and contributes to worsening fibrosis and eventual organ
failure (16). Our work has helped delineate a proper substrate stiffness for long term
maintenance of hepatocyte phenotype as well as a stiffness range where EMT could be
initiated. Future experiments could be designed using the liver ECM gels to further
replicate events of liver repair or liver disease in culture and study the delicate balance
between EMT and MET.
While studying the effects of stiffness on hepatocyte phenotype, assays were
designed to study the mechanisms behind observed changes in attachment, viability, and
morphology. Experiments monitored gene and protein expression of FAK and ILK, two
integrin-localized kinases with vital roles in mechanotransduction and cytoskeletal
remodeling. As previously discussed, hepatocytes on all gel stiffness expressed the FAK
and ILK genes at levels higher than cells of normal liver tissue. However, at day 7 post-
seeding, FAK and ILK gene and protein expression levels were highest in the lower
stiffness levels, with FAK concentrated at the leading edge of epithelial cell layers.
Although FAK and ILK have been shown to have effects on viability, trends we observed
seemed to correlate most closely with morphological changes in the hepatocytes,
specifically with formation of cellular projections.
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Previous studies have shown FAK and ILK expression stimulates pathways
controlling cytoskeletal structures. In addition, overexpression of these kinases has been
observed in migrating cells. With this knowledge, expression of the downstream targets
and actin regulating Rho GTPases were measured to determine if differences in
cytoskeleton activation and regulation were occurring in human hepatocytes seeded on
ECM gels of varying stiffnesses. Cells expressed RhoA, which controls stress fiber
formation and contractility, on stiffer ECM gels and exhibited greater attachment and
assembly of actin fibers. CDC42, which initiates polarization and filopodia formation,
decreased with substrate stiffness. This result demonstrated how FAK, ILK, and CDC42
overexpression correlates with lower substrate stiffness, morphological changes, and
most likely, cell migration.
Further experimentation could help confirm detected morphological and
migratory trends and help strengthen our hypotheses. Determining exact mechanisms and
sites of FAK and ILK phosphorylation induced by substrate stiffness could help explain
the numerous functions of these kinases. Previous studies have shown blocking FAK and
ILK in cell culture assays may not be ideal because deficiencies in FAK and ILK lead to
reductions in viability. However, plasmid transfections could be used to upregulate FAK,
ILK, or CDC42 in primary hepatocytes, as has been successful in previous studies (17).
Site specific mutations could also be used to pinpoint the importance of specific
phosphorylation sites. RhoA could also be upregulated to determine if increased
expression initiates development of a more anchored phenotype with greater actin
expression. Induction of morphological effects similar to what was observed on the
varying gel stiffnesses would support our hypotheses and further implicate FAK, ILK,
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and the Rho GTPases in the changes we observed. Hepatocyte culture experiments and
imaging specifically designed to track and map cell motility and migration throughout
time points past 7 days would also more definitively validate our theories.
In addition to analyzing the expression and localization of FAK, ILK, and the
Rho GTPases, additional targets related to mechanotransduction could be included. Src
tyrosine kinase binds to and phosphorylates various FAK sites. Several adaptor proteins
including PI3K, CAS, GRB7, and N-WASP associate with FAK and act as intermediates
between FAK and the Rho GTPases. Similar to FAK, ILK also stimulates the PI3K
pathway. Unique adaptor proteins of ILK include PINCH1 and ELMO2 which aid in
cellular polarization. Other focal adhesion components important to mechanosensing,
integrin attachment, and actin modification include talin, vinculin, and paxillin. Assays
testing expression and localization of all these proteins would provide a clearer
understanding of the interactions between ECM gel stiffness, substrate sensing, and the
regulation of the hepatocyte cytoskeleton and focal adhesion formation.
ECM composition and substrate stiffness play important roles in hepatocyte
phenotype; however, growth factor presence also represents a factor in determining the
fates of cells. Thesis studies have shown the ability of ECM gel substrates to bind active
growth factor for cellular use (Chapter 3- Figure 2). Future assays like phospho-labeling
will be required to pinpoint exact binding and interaction sites in the gels. Nevertheless,
the presented studies demonstrate the ability to manipulate the growth factor
concentration in the liver cell substrates. With these ECM supplemented gels, future
assays could be designed to measure how stiffness intensifies or reduces growth factor
effects on liver cells.
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Preliminary studies exhibited the ability of HGF to bind to our liver hydrogels and
improve cell junction formation and hepatocyte function on low stiffness gels (Chapter 3-
Supplementary Figure 4). A previous publication showed similar results with HGF and
EGF-treated hepatocytes seeded on Matrigel of varying stiffnesses. Growth factor co-
stimulation improved aggregation and function on the lower stiffness, while the converse
occurred at the higher stiffness level (18). Additional experiments on HGF and EGF
stimulated epithelial cells implicated stiffness in cell polarization and migratory
responses (19-22). Other research has also shown the direct effects of substrate stiffness
on transforming growth factor beta (TGF-β)-induced dedifferentiation of hepatocytes and
possible EMT (23). Studies could be conducted to determine if these same changes occur
on our liver ECM gels. Further data could define the exact stiffness ranges that induce
these responses and the molecular mechanisms behind these behaviors.
Opportunities exist to improve both the complexity and overall function of the
hepatocyte cell culture system we have developed. Other cell types could be individually
seeded on the ECM supplemented substrates to test effects of stiffness on different cells
of the liver, or co-culture of multiple cell types could determine how they interact and
function in varied mechanical environments. Hepatic stellate cells (HSCs) are one of
most important liver cell types involved in ECM production and maintenance. HSCs exist
in either a quiescent or myofibroblast form. In vivo these cells are vital to both hepatocyte
and stem cell mediated regeneration and repair. They secrete and assemble ECM
molecules including fibronectin, collagens, and GAGs (24, 25). During a fibrotic disease
state, hepatic stellate cells deposit excess amounts of collagen, a process worsened by
myofibroblastic activation and proliferation on stiffening ECM. Several fields from
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cancer biology to regenerative medicine also explored their ability to secrete growth
factors. Assays revealed HSCs produce HGF, EGF, TGFα, and multiple insulin-like
growth factor binding proteins within the liver (26-28).
Previous studies have shown the abilities of stiff substrates to stimulate
myofibroblastic differentiation of HSCs (29, 30). Less is known about the function of
quiescent cells in the normal liver state and the exact stiffness range where
myofibroblastic activation occurs. Results on the liver ECM gels determined that hepatic
stellate cell production of GAGs and HGF were time and stiffness dependent. HGF
production was highest in the lowest gel stiffness, while ECM production was greatest on
the highest gel stiffness (Chapter 3- Supplementary Figure 5). More detailed studies
would reveal if these cells could substantially modify the composition or physical
properties of the substrate microenvironment. Co-culture with primary hepatocytes
should improve long term viability of the cells and provide insight into the relationships
between cell types and certain substrate properties. In addition, modeling a fibrotic
stiffness and determining methods of controlling excess ECM production and
maintenance of hepatocyte function could help in the understanding and treatment of
liver diseases like cirrhosis.
Besides increasing diversity of cell types grown on the substrate, increasing the
three dimensionality of this system could improve capabilities to model disease states and
enable advances in tissue engineering. Hepatocytes are arranged in epithelial sheets in the
liver. 2D systems replicate individual cell layers, but not the complex interactions of the
entire tissue. New 3D bioprinting technology could provide a method of creating these
3D structures. This tool enables careful layering of cells and gel substrate. Using
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knowledge gained from this thesis, a bioprinter could alternate printing cell sheets and
ECM gel layers at a set stiffness level to produce a tissue-like system. In this way, in
vitro modeling could better mimic an in vivo environment, while still providing an
adjustable platform to study specific ECM properties and mechanisms.
Despite the advantages of a fully 3D gel system, new challenges and
complications are created that are non-existent in 2D culture. Gel swelling, pore size, and
the flow of cell nutrients through the substrate all become important. Crosslinking affects
stiffness on a gel layer, but could have confounding effects in 3D. Substrate fabrication
and multiple gel properties would need to be monitored, tested, and studied to prevent the
introduction of unwanted variables. Although several areas still need to be addressed, the
abilities and flexibility of ECM gel substrates provide a promising future.
Overall, liver ECM gels improve long term cell function and allow isolation and
testing of single substrate-related properties. This thesis work elucidated specific
mechanisms of mechanotransduction and cytoskeletal regulation and determined stiffness
ranges where hepatocyte function was optimized in vitro. Future advances and
development of this substrate system could further clarify molecular interactions and
produce more accurate disease models. Liver ECM gels could also be utilized in cell
encapsulation technologies or cell therapies to repair liver injuries or disorders. The thesis
established methods to integrate a natural liver ECM microenvironment in gel substrates
at a physiological stiffness range. This new knowledge and innovation has facilitated
numerous possibilities to pursue and can ultimately lead to future advances and practical
therapies or treatments.
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