Collagen and Hyaluronic Acid Interpenetrating Polymer Networks
for Tissue Engineering
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Mark D.Brigham
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S.B. Computer Science, M.I.T., 2006
Submitted to the Department of Electrical Engineering and Computer Science
in Partial Fulfillment of the Requirements for the Degree of
Master of Engineering in Electrical Engineering and Computer Science
at the Massachusetts Institute of Technology
August 2007
Copyright 2007 Mark D. Brigham. All rights reserved.
The author hereby grants to M.I.T. permission to reproduce and
to distribute publicly paper and electronic copies of this thesis document in whole and in
part in any medium now known or hereafter created.
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Author
Department of Electrical Engineering and Computer Science
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August 21, 2007
Certified by_
Assistant Professo
Ali Khademhosseini
f Medicine and Health Sciences and Technology
Thesis Supervisor
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Certified by
Utkan Demirci
structor (Assistant Professor TBA) of
Medicine and.Health Se-ieces and Technology
The 9 C/Supervisor
Accepted by
-- ---Arthur C. Smith
Professor of Electrical Engineering
Chairman, Department Committee on Graduate Theses
ARCHIVE b
1
Acknowledgments
There are many people to whom I owe a great deal of gratitude for their help in this work,
both inside and outside the lab. Thanks to all the members of Khademhosseini Lab for their hard
work and assistance. Thanks to Alex Bick for his excellent work and determination. Thanks to
Amel Bendali, without whose help Team Lab Coat never would have existed and this project
would not have been nearly as successful. Thanks to Professor Utkan Demirci for his support and
advice. A million thanks to Professor Ali Khademhosseini for his undying passion for our work
and his friendship and mentoring. Most of all I am eternally grateful to my parents, Mike and
Jean, my brother, Matt, and my fianc6/elebear, Joy, for their love and faith.
Collagen and Hyaluronic Acid Interpenetrating Polymer Networks for Tissue Engineering
by
Mark D. Brigham
Submitted to the
Department of Electrical Engineering and Computer Science
August 27, 2007
In Partial Fulfillment of the Requirements for the Degree of
Master of Engineering in Electrical Engineering and Computer Science
ABSTRACT
Interpenetrating Polymer Networks (IPNs) represent a strategy for combining the properties of
several polymeric materials into a single network. In this thesis, collagen and methacrylated
hyaluronic acid are combined in IPNs to produce a range of new biocompatible. The fabrication
method allows for control of compressive strength of the IPN hydrogels. The materials are
confirmed to be homogeneous at microscopic scales with fluorescent techniques. The IPNs are
used for cell encapsulation and have the potential to be used for surface cell culture. The
mechanical properties can be adjusted to match those of cardiac tissue. Thus, when combined
with the properties of biocompatibility, viable cell encapsulation, and cell culture, the collagenMeHA IPN hydrogels represent a powerful new material for tissue engineering applications.
Thesis Supervisor: Ali Khademhosseini
Title: Assistant Professor of Medicine and Health Sciences and Technology
1 Introduction
Since the emergence of the field of tissue engineering, may attempts have been made
towards the generation of cardiac tissue engineered organs. Biodegradable scaffolds made from
either natural[l, 2] or synthetic[3-5] materials are seen as having great potential value in this
area. The scaffolds function as a 3D structure on which the cardiac tissue may be induced to
grow. By mimicking the in vivo conditions of the tissue, while delivering nutrients, oxygen, and
other soluble factors to the tissue constructs[4-7], tissue engineers hope to provide the ideal
environment for producing cardiac organ constructs in vitro. The inclusion
of the
microenvironmental factors observed in vivo is a major avenue used to enhance the function of
these engineered organs. For example, by stimulating the tissues with pulsatile electric fields,
cardiac structures were formed with improved functionality[7].
Additionally, mechanical
stimulation, like that seen in cardiac contraction, has been shown to enhance cardiac tissue
formation[8]. Although the fabrication of functional myocardial constructs has been reported[7],
their differentiation levels have not yet achieved those of adult tissues and there are no reports on
tissue engineering terminally differentiated cardiac tissues. Thus, it is desirable to formulate
alternative approaches and materials to more precisely control the organization of cellular tissue
engineering and to advance the biomimetic properties of scaffolding materials.
Hydrogels are biologically or artificially derived polymers cross-linked in solution to
produce polymer network gels with very high water content, 95% to 99%. Cell laden hydrogels
have attracted great interest as scaffolding materials for tissue engineering because of their high
water content, biocompatibility and mechanical properties, which resemble those of natural
tissues[9, 10]. Hydrogels have been used for tissue engineering of bone[l 1-13], cartilage[14-16],
and other tissues[17, 18]. By adding cells to a hydrogel precursor prior to the gelling process,
cells can be distributed homogeneously throughout the gel. Biodegradable hydrogels are
particularly attractive because of the potential for the tissue to replace the initial scaffold as the
cells proliferate, generate extracellular matrix components, and the tissue structure develops.
Differentiated cardiac tissues have been engineered by casting neonatal rat cardiac myocytes into
collagen gels and subsequently subjecting them to cyclic mechanical stretch[19].
Hydrogels from natural sources can be derived from polymers such as collagen,
hyaluronic acid (HA), fibrin, alginate, agarose or chitosan[9]. The specific properties of natural
polymers depends heavily on their origin and composition. Several natural polymers used as
hydrogels, such as collagen and HA, are components of the mammalian ECM, the advantages of
which include low toxicity and biocompatibility.
Collagen and other mammalian derived protein-based polymers are effective matrices for
cellular growth, as they contain many cell signaling domains present in the in vivo ECM.
Collagen gels can be naturally created without chemical modifications, simply by neutralizing a
collagen suspension and providing a sufficiently warm environment, 19-37°C[20], Fig 1.
However, these gels are mechanically weak. Various methods have been developed such as
chemical crosslinking[21, 22], crosslinking with UV or temperature[21, 23], or in mixture with
other polymeric agents[21, 24], though none have shown sufficient cell viability during
fabrication. Collagen degradation is mediated naturally by proteins such as collagenase.
The most abundant heteropolysaccharides in the body are the glycosaminoglycans
(GAGs). GAGs are located primarily on the surface of cells or in the ECM. HA is a GAG which
is particularly prevalent during wound healing and in joints.
Covalently crosslinked HA
hydrogels can be formed by multiple chemical modification means[25-28], most commonly via
the methacrylation of HA macromers to produce UV crosslinkable polymer solutions[29], Fig 2.
Methacrylated hyaluronic acid (MeHA) hydrogels are biodegradable by an enzyme called
hyaluronidase[27]. HA is particularly appealing for tissue engineering as it is naturally present
in great abundance in a variety of tissues[30-32].
Previously, HA scaffolds have been used for
tissue engineering of various tissues[2, 27, 33].
To use hydrogels in various tissue engineering applications, it is desirable to control their
mechanical
properties
proliferation[34, 35].
which
affect
cell
attachment,
differentiation,
viability,
and
Mechanically, hydrogels are remarkably similar to biological tissues.
Generally, hydrogels exhibit excellent elastic characteristics and when loaded to deformations of
20% or less, they typically rebound remarkably well.
To control the mechanical properties of hydrogels a number of parameters such as the
density and chemistry of the crosslinks, as well as the concentration, chemistry and molecular
weight of the precursors can be modified. In general, brain tissues exhibit elasticity between 0.1
kPa and 1 kPa, muscle tissue ~10 kPa (between 0.1 to >40kPa), and collagenous bone
approximately 100 kPa. Furthermore, the human heart has an elasticity of -31 kPa[36]. It has
been demonstrated that by merely seeding stem cells onto substrates of varying mechanical
properties, the stem cells will differentiate into the tissue precursor most similar to the
underlying substrate[34]. Therefore there is a need for generating hydrogels that can mimic the
mechanical, biological and physical properties of native tissues. However, despite significant
progress, many current approaches to fabricate hydrogels do not result in the synthesis of
constructs with desired mechanical and chemical properties. Limitations with generating
mechanically robust hydrogels that can withstand the in vivo environment include the need for
low overall concentration of material, the requirement for degradation and the need for
cytocompatibility.
Interpenetrating polymer networks (IPNs) are a powerful method of modifying hydrogel
properties. An IPN is a mixture of two or more crosslinked networks that are mixed together at
the molecular level. When only one polymer of the IPN is crosslinked and the other is left in its
linear form, the system is referred to as a semi-IPN. Conversely, when both types of polymer are
crosslinked the system is called a full-IPN. IPNs help to improve the mechanical strength and
resiliency of the overall polymer and many studies have demonstrated that the mechanical
properties of IPNs are significantly greater than their individual components[37-42]. It is seen
that the increase in strength, failure stress and stiffness of IPN hydrogels can be achieved while
retaining elasticity. When a shear stress is applied to an IPN hydrogel, physical entanglements
formed among polymer chains store the energy and therefore enhance the tensile strength.
Maximum elongation at break were observed for different type of IPNs such as sequential-IPNs
composed of polyurethane (PU) and polymethylmethacrylate (PMMA)[39], semi-IPN hydrogels
based on bacterial poly(3-hydroxybutyrate) and net-PEG[40], protein/synthetic polymer hybrid
IPNs of poly (N-isopropylacrylamide) (PNIPAAm) with Bombyx mori silk fibroin[43],
composite hydrogels of one or more ethylenically-unsaturated monomers, and a multiolefinic
crosslinking agent[44] as well as a number of other systems[38, 41, 45]. Thus, IPN formation
produces materials that are stronger than the hydrogel copolymers of similar water content. IPNs
have been used for a number of biological applications ranging from tissue engineering and drug
delivery to synthesis of sutures. For example, they have been used to study stem cells[46] and
control cell behavior[47]. However, few studies have aimed to use the ability of IPNs to enhance
mechanical properties of hydrogels in tissue engineering, in particular with respect to
overcoming the weak mechanical properties of materials such as collagen. By creating IPNs of
MeHA and collagen, we hope to produce materials with controllable robust mechanical
properties while maintaining the cell adhesive properties of collagen. The use of collagen and
MeHA is particularly appealing since both components of the IPNs are biologically compatible,
biodegradable and have shown significant promise in tissue engineering.
COLLAGEN STRUCTURE
MOLECULE
cross-linked
end domain
helical domain
Hydrogen-bonded
----
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-8 nm
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/ •
I0-
ii
I
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\\
I e 0
...---__
r
___I
.0!•
MICR( )-FIBRIL
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'/
cross--linked
cross-linked
FIBRIL
UNDULATED
FIBER
0
1 - 500
diamei
TISSUE
Figure 1: Collagen structure and fiber formation. [20]
Hyaluronan
"7s·a"
Ha
HA-MA
Figure 2: Methacrylated hyaluronic acid synthesis and photocrosslinking
2 Materials and Methods
2.1 Materials
Tissue culture medium and serum for NIH-3T3 fibroblasts was purchased from Gibco
Invitrogen Corporation. Tissue culture medium, supplements, and primary human umbilical vein
endothelial cells (HUVECs) were purchased from Cambrex Biosciences. Cambrex Biosciences
is now a part of Lonza Corporation.
2.2 Microscopy
All microscopic imaging was performed with a Nikon Eclipse TE2000-U inverted
microscope with a mercury-arc lamp. Images were captured by a SPOT RTKE camera from
Diagnostic Instruments, Inc. All blue excitation (green emission) was performed at 495 nm. All
green excitation (red emission) was performed at 590 nm.
2.3 UV exposure
All UV exposures were performed using an EXFO OmniCure Series 2000 at 200
mW/cm 2 and 365 nm wavelength.
2.4 Methacrylated hyaluronic acid synthesis
Methacrylated hyaluronic acid (MeHA) was synthesized by the addition of methacrylic
anhydride (Sigma-Aldrich) to a solution of I wt % HA (Lifecore Hyaluronan Division) in
deionized water. The final methacrylic anhydride concentration was 1 wt %. The molecular
weight of HA macromers was 60 kDa. The pH was adjusted to and maintained between 8 and 9
via the addition of IM NaOH (Sigma-Aldrich) and reacted on ice for 24 h. This synthesis was
previously described [29] and is shown in Fig 2. For purification, the solution was dialyzed in
100 fold volume of deionized water for 48 h using cellulose dialysis tubing (Spectrum
Laboratories, Inc., 7-8 kDa cutoff). The final macromer product was obtained by lyophilization
for 72 h. Once dried, the MeHA was dissolved at a concentration of 10 wt % in IX phosphate
buffered saline (PBS) overnight at 370 C.
2.5 PDMS mold fabrication
Molds for controlling hydrogel size and shape were fabricated from polydimethylsiloxane
(PDMS) (Dow Coming Corp.). Sheets of PDMS 1, 2, and 3 mm thick were formed by mixing
silicone elastomer and curing agent in a 6:1 ratio and pouring the mixture into Petri dishes. The
dishes were placed in a 70°C oven, leveled, and allowed to cure for 2 h. The PDMS sheets were
then peeled from the dishes, cut into 2 cm 2 sections and perforated by a 5 mm (for 1 mm thick
sheets) or 8 mm (for 2 and 3 mm thick sheets) biopsy punch (Sklar Corp.). The surfaces of the
PDMS molds were made hydrophilic by plasma treatment for 7 min (Model #: PDC-001,
Harrick Plasma). The PDMS molds were then bonded to plasma treated glass slides (Fisher
Scientific).
2.6 Cell culture
All cells were passaged and utilized under sterile tissue culture hoods and stored in a 95%
air/5% CO 2 , 100% humid 37oC incubator. NIH-3T3 cells were cultured in 10% fetal bovine
serum (FBS), 0.2% Penicillin-Streptomycin in Dulbecco's modified eagle medium (DMEM).
Confluent dishes of NIH-3T3 cells were fed every 24 h and passaged every 2-3 days in a 1:4
subculture ratio. HUVECs were cultured in EGM-2 medium from Lonza, fed every 24 h and
passaged every 4-5 days in a 1:4 culture ratio. HUVECs were utilized between passage 4 and 7.
2.7 Collagen preconcentration
Collagen solutions in 0.02M acetic acid (BD Biosciences, Trevigen Corp) with
concentrations in the range of 5.0-10.59 mg/ml were frozen in liquid nitrogen and lyophilized for
24 h. Once freeze dried, the collagen was redissolved in 0.02M acetic acid (Fluka) at a
concentration of 15.7 mg/ml.
2.8 IPN fabrication
During the experimental design process, several iterations of the IPN fabrication method
were used in attempts to improve the uniformity of the gels. Below are the complete fabrication
processes for all iterations. Example volumes for the mixtures described are provided in Table 1.
Set #
Name
1
4.1 mg/ml collagen
2
2.5 wt % MeHA + 4.1 mg/ml collagen
3
5.0 wt %MeHA + 4.1 mg/ml collagen
4
7.0 wt % MeHA + 4.1 mg/ml collagen
5
2.5 wt %MeHA
6
5.0 wt % MeHA
7
7.0 wt % MeHA
Set #
1
2
3
4
5
6
7
10 wt %stock MeHA
0
175
350
490
175
350
490
10X PBS
70
52.5
35
21
0
0
0
1X PBS
0
0
0
0
525
350
210
1M NaOH
4.2
4.2
4.2
4.2
0
0
0
15.7 mg/ml collagen stock
183
183
183
183
0
0
0
deionized water
443
285
128
1.8
0
0
0
irgacure (33 wt %in methanol)
13.3
13.3
13.3
13.3
13.3
13.3
13.3
Table 1: Sample Components of IPN prepolymers.
A) Set numbers and final concentrations.
B) Solution components in IPN fabrication. All values have unit of gl.
2.8.1 Iteration 1
Collagen-HA IPNs were fabricated from 15.7 mg/ml stock collagen solution and the 10
wt % MeHA prepolymer synthesized previously. The collagen solution was neutralized with
10X phosphate buffered saline (PBS, Invitrogen) and IM NaOH. The collagen solution was then
diluted with deionized water to a concentration such that, when mixed with a corresponding
amount of MeHA, the final collagen solution was 4.1 mg/ml. MeHA was stored at 37°C prior to
IPN fabrication. After preparation of the collagen solution, MeHA prepolymer was pipetted into
the collagen solution to produce the desired concentration of MeHA. MeHA solutions without
collagen were also prepared by diluting the 10 wt % MeHA. Overall, the following combinations
were produced: 4.1 mg/ml collagen with 2.5 wt % MeHA, 4.1 mg/ml collagen with 5.0 wt %
MeHA, 4.1 mg/ml collagen with 7.0 wt % MeHA, 2.5 wt % MeHA without collagen, 5.0 wt %
MeHA without collagen, 7.0 wt % MeHA without collagen, and 4.1 mg/ml collagen without
MeHA. Finally, 1.5 wt % photoinitiator solution (33 wt % Irgacure (Ciba) dissolved in methanol
(Sigma-Aldrich)) was added to each prepolymer mixture.
After all prepolymer components had been combined, the solutions were mixed with a
vortexer for 15 seconds, pipetted into PDMS molds, and irradiated with UV light for 180 sec.
The gels were then placed in a 95% air/5% CO2, 100% humid 37°C incubator for 2 h before
being removed from the PDMS molds for analysis or experimentation.
2.8.2 Iteration 2
The second iteration of IPN fabrication was identical to the first with the exceptions that
the 10 wt % MeHA prepolymer solution was stored at 40C after dissolving overnight at 370C and
the solutions were mixed for 30 sec by hand in lieu of vortexing.
2.8.3 Iteration 3
The third iteration was identical to the second with the exception that, after the
prepolymer components were combined, the solutions were placed in a Labquake shaker
rotisserie (Barnstead Thermolyne) for 24 h at 4°C before pipetting into PDMS molds.
2.8.4 Iteration 4
The fourth iteration was identical to the second with the exception that, after the
prepolymer components were combined, the solutions were mechanically stirred at -60 rpm for
24 h at 4°C before pipetting into PDMS molds.
2.8.5 Iteration 5
The fifth iteration was identical to the fourth with the exception that the photoinitiator
was added 5 min prior to the end of the 24 h stirring period instead of being combined with the
other prepolymer components prior to stirring.
2.9 Compressive strain testing of collagen-MeHA IPNs
For each of iterations 1-5, the IPN hydrogels were removed from the 2 mm by 8 mm
PDMS molds and placed in IX PBS. The IPNs were then allowed to swell to equilibrium in PBS
for 24 h at 370 C. The swollen hydrogels were removed from the PBS and placed on glass slides.
Stress versus strain curves were generated for each hydrogel (n=5) using an Instron 5542
mechanical tester. The gels were compressed at a rate of 20% per min until the sample fractured
or had been compressed to 20% of its original thickness. Using the small strain (<10%) model,
the compressive modulus (Young's modulus) of the IPNs was determined by a linear fit to the
initial section of the stress v. strain curves.
2.10 FITC-collagen and MeHA IPN fabrication and imaging
To characterize the mixing of collagen and MeHA, fluorescein isothiocyanate (FITC)
conjugated collagen (Sigma-Aldrich) and HA IPNs were fabricated. Prior to fabrication FITCcollagen and standard stock collagen were mixed in a 1:50 ratio. The final concentrations of
FITC-collagen and HA in the prepolymer solutions were 4.1 mg/ml and 5 wt %, respectively. To
analyze the homogeneity of the mixing process, two different solutions were prepared. In the
18
first solution, the collagen and HA were mixed by pipetting for 1 minute. In the second solution,
the collagen and HA were not mixed, but instead were pipetted into the mold simultaneously.
Additionally, FITC-collagen-only and HA-only gels were prepared as controls. The prepolymer
solutions were placed in 1 mm by 5 mm PDMS molds and exposed to UV light for 180 sec.
After UV treatment, the gels were incubated at 370 C for 2 h. The gels were then microscopically
imaged. The hydrogels were exposed for 2 sec using the 495 nm filter.
2.11 Methacrylated FITC-HA synthesis
To improve IPN homogeneity studies, methacrylated FITC-HA was synthesized from
lyophilized FITC-HA conjugate (Invitrogen, Molecular Probes, MW=200kDa). The lyophilized
FITC-HA was dissolved overnight at 37oC in deionized water to a concentration of 5mg/ml. It
was then further diluted to 100 gpg/ml and tested for fluorescence under 495 nm excitation.
Following the protocol for hyaluronic acid methacrylation, 100 ýtg of FITC-HA was mixed with
100 mg HA and a solution of 1 wt % HA (FITC and non-FITC mixture) and 1 wt % methacrylic
anhydride in deionized water was created. The pH was adjusted to between 8 and 9 by the
addition of IM NaOH. The solution was reacted for 24 h on ice. After completion of the
reaction, the solution was dialyzed against deionized water for 48 h. The purified solution was
lyophilized for 72 h. The dried product was stored at -20 0 C until use. To test for fluorescent
conjugate stability and successful methacrylation, the dried FITC-HA was dissolved in lX PBS
at a 5 wt % concentration and mixed with 1.5 wt % photoinitiator solution. Two sets of solutions
were pipetted into 1mm by 5 mm PDMS molds. One set was immediately imaged under 495 nm
excitation with an exposure time of 1 sec. The second set was photocrosslinked by UV exposure
and then imaged under 495 nm excitation with an exposure time of 1 sec.
To examine the photobleaching effects of fluorescent imaging on the FITC-HA, a time
series of fluorescent images was taken. After being UV irradiated for 180 sec, the FITC-HA
hydrogel was examined with the fluorescent microscope. While being constantly excited with
495 nm light, the camera captured an image, 1 sec exposure, every 30 sec for 15 min.
2.12 Texas Red-X collagen labeling
To improve IPN homogeneity studies, collagen was labeled using Texas Red-X protein
stain (Invitrogen). A collagen solution in 0.02M acetic acid was diluted to 2 mg/ml. A 0.5 ml
sample of 2 mg/ml collagen was mixed with 50 pl of IM sodium bicarbonate and kept on ice. A
vial of Texas Red-X was warmed to 23'C and mixed with 10 pl of dimethyl sulfoxide (DMSO).
The 0.5 ml sample of collagen and sodium bicarbonate was added to the Texas Red-X stain vial.
The solution was stirred for 24 h on ice to allow the Texas Red-X to conjugate with the collagen.
After completion of the staining, the labeled collagen was purified in a separation column filled
with purification resin (Invitrogen) designed for separation of the excess Texas Red-X dye from
proteins with MW> 15kDa. The staining solution, containing the labeled collagen and excess dye,
was poured into the separation column and kept at 4°C. Periodically, a lX PBS elution buffer
was poured into the column to allow the protein and excess dye to traverse the resin and separate.
Every 1 h, the column was examined with a handheld UV lamp to observe the progress of the
fluorescent bands. When the faster moving band, which contained the labeled collagen, reached
the end of the column, it was collected and dialyzed against 0.02M acetic acid for 48 h. After
20
dialysis, the solution was frozen with liquid nitrogen and lyophilized for 72 h. The dried collagen
product was dissolved at a concentration of 1 mg/ml. To test the fluorescent labeling, the Texas
Red-X collagen solution was mixed in a 1:10 ratio with the stock collagen solution. The mixture
was neutralized with 10X PBS and IM NaOH, then pipetted into 2 mm by 8 mm PDMS molds.
The solutions in the molds were treated with UV for 180 sec and then incubated at 37oC for 2
hours to allow the collagen to gel. Following incubation, the gels were imaged by excitation with
590 nm light with an exposure time of 1 sec.
2.13 HUVEC culture on collagen-MeHA IPNs
Collagen-MeHA IPN hydrogels were fabricated according to the protocol described
above in IPN fabrication iteration 4. The hydrogels, 100 •l each, were fabricated in 3 mm by 8
mm PDMS molds, leaving 100 gtl of empty volume above the hydrogels. A 250,000 cell/ml
solution of HUVECs in in cell culture media was prepared for seeding on top of the IPNs. A 50
•l volume of cell suspension, -12,500 cells, was pipetted onto each hydrogel. The seeded
hydrogels were then placed in a 95% air/5% CO 2, 100% humid 37°C incubator for 2 h to allow
for HUVEC attachment. To observe HUVEC attachment and proliferation, the IPN surfaces
were imaged at 2, 12 and 24 h.
2.14 HUVEC culture on collagen hydrogels
To test the effect of Irgacure on the attachment and proliferation of HUVECs, 4.1 mg/ml
collagen hydrogels were prepared. In one set of gels, the standard 1.5 wt % Irgacure was
included. In the other set, the Irgacure was excluded. The solutions were pipetted into 3 mm by 8
mm PDMS molds and irradiated with UV for 180 sec. The hydrogels were then placed in a 95%
air/5% CO 2, 100% humid 37°C incubator for 2 h to allow the collagen to gel. After 2 h in the
incubator, 50 tl of 250,000 cell/ml HUVEC suspension was pipetted onto each hydrogel. The
HUVECs were allowed to attach for I h before the solution was aspirated, washed with IX PBS
and a 50 tl volume of HUVEC culture media was added on top of each hydrogel. The hydrogel
surfaces were imaged 24 h after seeding.
2.15 NIH-3T3 culture on collagen hydrogels with and without Irgacure washing
Two separate experiments were conducted to analyze the effect of Irgacure on the
attachment and proliferation of NIH-3T3s on collagen hydrogels. In both experiments, 4.1
mg/mL solutions of lyophilized and non-lyophilized collagen, both with and without 1.5 wt %
Irgacure, were mixed on ice and neutralized with 10X PBS and IM NaOH. The solutions were
pipetted, in 100 pl volumes, into 3 mm by 8 mm PDMS molds and allowed to gel for 2 h at
370C.
At this point in the first experiment, 50 jl of 250,000 cell/ml NIH-3T3 solution was
pipetted onto each hydrogel. The hydrogels were microscopically imaged at 2, 4, 8, 10.5 and 24
h. Just prior to the 24 h imaging, the media was aspirated and replaced with fresh culture media,
to remove unattached cells.
At the same point in the second experiment, the hydrogels were removed from the PDMS
molds and placed in Petri dishes with NIH-3T3 culture media. To provide physical agitation, the
Petri dishes were placed on a Reliable Scientific tilting stage in a 37°C room for 36 h. The
22
collagen gels were then removed from the Petri dishes, placed in smaller wells, and covered with
200 pl of 250,000 cell/ml NIH-3T3 solution. The hydrogels were microscopically imaged at 4, 8,
12, 24, and 31 h.
2.16 Volumetric swelling analysis of collagen-MeHA IPNs
Collagen-MeHA IPN hydrogels were made in 2 mm by 8 mm PDMS molds according to
the protocol given in IPN fabrication iteration 4 above. After the 2 h incubation to allow for
collagen gelling, the hydrogels were removed from the PDMS molds and placed well plates
containing lX PBS. The well plates were then stored at 370C for 24 h so that the IPNs could
swell to equilibrium volume. The gels were then removed from the PBS and excess liquid was
blotted away with Kimwipes. The gels were weighed and placed in Petri dishes. The gels were
lyophilized for 24 h and weighed again. The volumetric swelling ratio, swollen mass divided by
dried mass, was then calculated for each gel.
2.17 Scanning electron microscopy of collagen-MeHA IPNs
To aid in understanding the structure of the networks, collagen-HA IPNs were examined
using scanning electron microscopy (SEM). Collagen-MeHA IPN hydrogels were made in 2 mm
by 8 mm PDMS molds according to the protocol given in IPN fabrication iteration 4 above.
After the 2 h incubation to allow for collagen gelling, the hydrogels were removed from the
PDMS molds and placed well plates containing lX PBS. The well plates were then stored at
370C for 24 h so that the IPNs could swell to equilibrium volume. The hydrogels were then
frozen with liquid nitrogen and lyophilized for 24 h. The dried IPNs were mounted on SEM
pucks with conductive tape and coated with -6 nm of gold-palladium using a Quorumtech
SC7640 sputter coater. The gold-palladium coated networks were then placed in a Jeol JSM6060
scanning electron microscope and imaged at 100x - 5000x magnification. The acceleration
voltage was 5kV and the spot size was set to 50. Images were captured in scan 4, super high
quality mode. Surface and cross sectional views were imaged for each IPN combination.
2.18 NIH-3T3 encapsulation in MeHA hydrogels
A 5 wt % solution of MeHA was prepared and split into three aliquots. Aliquot A was
warmed to 370 C. Aliquots B and C were cooled to 40 C. Aliquot A and B were then mixed, by
pipetting, with NIH-3T3s at a concentration of 2x10 6 cells/ml. For Aliquot C, a solution of NIH3T3 cells was cooled to 40 C at a rate of lVC per min using a Nalgene Cryo lPC Freezing
Container. After cooling, the cells were mixed with Aliquot C at a concentration of 2x10 6
cells/ml. Aliquots A, B, and C were then pipetted into 1 mm by 5 mm PDMS molds and
irradiated with UV light for 180 sec. A set of hydrogels from each aliquot was immediately
incubated in a solution containing calcein and ethidium homodimer. After a 10 min incubation
period, the hydrogels were removed from the solution, wash with IX PBS and imaged under a
fluorescent microscope. At 495 nm excitation, live cells fluoresced green due to the metabolism
of calcein. At 590 nm excitation, dead cells fluoresced red due to the binding of homodimer to
DNA. The remaining hydrogels were placed in NIH-3T3 culture media and placed in a 95%
air/5% CO2, 100% humid 37°C incubator for 24 h and then stained for live/dead assaying by the
same procedure described above.
2.20 Statistical analysis
Data sets were analyzed using the Student's T-test. Any reports of statistically significant
differences were supported by > 95% confidence levels.
3 Results
3.1 Collagen-MeHA IPN fabrication and mechanical testing
The protocol used in iteration #1 for collagen-MeHA IPN fabrication produced hydrogels
with large variations in their compressive moduli. Hydrogels prepared without collagen resulted
in average Instron compressive moduli of 2.6 +/- 1.4 kPa, 33.0 +/- 16.4 kPa, and 75.2 +/- 15.0
kPa for 2.5 wt%, 5.0 wt%, and 7.0 wt% MeHA, respectively. Hydrogels prepared with 4.1
mg/ml collagen resulted in average Instron compressive moduli of 5.3 +/- 3.2 kPa, 33.3 +/- 10.4
kPa, and 69.2 +/- 71.2 kPa for 2.5 wt%, 5.0 wt%, and 7.0 wt% MeHA, respectively. The
compressive moduli and standard deviations are summarized in Fig 3A. In this set of
compressive tests and all hereafter, 4.1 mg/ml collagen only hydrogels fell below the noise
threshold of the Instron 5542. Stress versus strain plots of the compressions, Fig 3B-D, showed
an increase in linearity and diminishing effects of noise with increasing MeHA concentration.
Two exceptions were stress versus strain curves with sub-linear order growth, even in the 2%
strain range, Fig 3C, and saw-toothed stress versus strain curves, Fig 3D. The stress versus strain
plots for each MeHA concentration displayed a great deal of overlap, as expected from the high
standard deviations of the compressive moduli measurements.
Several observations were made during the fabrication process. When the 23°C 10 wt%
MeHA was added to the collagen prepolymer solutions, a cloudy, flaky precipitate began to form
in the solution. The formation of the precipitate added to the already high viscosity of the 10
wt% MeHA solution. Additionally, vortexing the more viscous solutions, such as 7 wt% MeHA
with and without collagen, had little agitative effect and, in fact, appeared to decrease the
uniformity of the prepolymer mixtures.
The protocol used in iteration #2, 4°C MeHA and 30 sec thorough hand mixing, produced
IPN hydrogels with statistically significant differences between 2.5 wt% HA with and without
collagen and between 7.0 wt% HA with and without collagen. Hydrogels prepared without
collagen resulted in average Instron compressive moduli of 1.7 +/- 0.7 kPa, 27.2 +/- 22.0 kPa,
and 51.9 +/- 13.4 kPa for 2.5 wt%, 5.0 wt%, and 7.0 wt% MeHA, respectively. Hydrogels
prepared with 4.1 mg/ml collagen resulted in average Instron compressive moduli of 9.9 +/- 6.2
kPa, 73.3 +/- 56.3 kPa, and 108.2 +/- 38.6 kPa for 2.5 wt%, 5.0 wt%, and 7.0 wt% MeHA,
respectively. The compressive moduli and standard deviations are summarized in Fig 4. The
cloudy precipitate observed in iteration #1 was not seen during fabrication of IPNs using the
iteration #2 protocol.
The protocol used in iteration #3, 4"C MeHA and 24 h Labquake shaker rotisserie
mixing, produced IPN hydrogels with statistically significant differences between 2.5 wt% HA
with and without collagen and between 5.0 wt% MeHA with and without collagen. However, the
variation in the compressive moduli measured for 7.0 wt% MeHA with collagen was nearly 90%
of the average value. To summarize, as shown graphically in Fig 5, hydrogels prepared without
collagen resulted in average Instron compressive moduli of 2.0 +/- 0.7 kPa, 17.8 +/- 6.6 kPa, and
63.1 +/- 21.0 kPa for 2.5 wt%, 5.0 wt%, and 7.0 wt% MeHA, respectively. Hydrogels prepared
with 4.1 mg/ml collagen resulted in average Instron compressive moduli of 9.9 +/- 2.7 kPa, 31.7
+/- 8.7 kPa, and 62.6 +/- 55.6 kPa for 2.5 wt%, 5.0 wt%, and 7.0 wt% MeHA, respectively.
Although the cloudy precipitate seen in iteration #1 did not form using the iteration #3 protocol,
it was clear that the Labquake rotisserie was unacceptable for mixing the more viscous,
concentrated prepolymers. Specifically, solutions of 5.0 wt% MeHA and lower could be seen
flowing back and forth inside the microtubes while turning on the rotisserie, while the 7.0 wt%
MeHA solutions remained in one side of the tubes and had no observable flow.
The protocol used in iteration #4, 4°C MeHA and stirring at -60 rpm for 24 h, produced
collagen-MeHA IPN hydrogels with statistically significant differences between 5.0 wt% MeHA
with and without collagen and between 7.0 wt% MeHA with and without collagen. Additionally,
2.5 wt% MeHA with collagen had consistent, measurable compressive moduli, while 2.5 wt%
MeHA was too soft and fell below the sensitivity of the Instron 5542. In summary, shown in Fig
6A, hydrogels prepared without collagen resulted in average compressive moduli of 3.00 +/- 0.65
kPa and 10.26 +/- 2.17 kPa for 5.0 wt% and 7.0 wt% MeHA, respectively. Hydrogels with 4.1
mg/ml collagen had compressive moduli of 0.17 +/- 0.08 kPa, 5.50 +/- 1.15 kPa, and 33.48 +/5.3 kPa for 2.5 wt%, 5.0 wt%, and 7.0 wt% MeHA, respectively. As can be seen in Fig 6B-C,
though the compressive moduli may be lower in this iteration, the overlap between hydrogels
with and without collagen is much smaller. Both 5.0 wt % and 7.0 wt % had statistically
significant differences with greater than 99% confidence levels.
The protocol used in iteration #5, 4°C MeHA and stirring at -60 rpm for 24 h with
photoinitiator addition 5 min prior to the end of stirring, produced collagen-MeHA IPN
hydrogels with statistically significant differences between 2.5 wt% MeHA with and without
collagen and between 7.0 wt% MeHA with and without collagen. The data for iteration #5 is
summarized in Fig 7.
iw/4.1
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rrn
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i •
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-
,,,,.
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C
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1.OE-04
-
0.5%
1.0%
Strain
1.5%
2.0%
0.0%
D
1.0%
2.0%
Strain
Figure 3: Instron Mechanical Testing of Iteration #1 Collagen-MeHA IPNs
A) Summary of compressive moduli for hydrogels prepared using IPN fabrication iteration
#1 under 20% thickness per min compression by the Instron 5542. No statistically
significant differences existed between collagen and non-collagen hydrogels for any
MeHA concentration.
B) Stress versus strain curves for 2.5 wt %MeHA hydrogels.
C) Stress versus strain curves for 5.0 wt %MeHA hydrogels.
D) Stress versus strain curves for 7.0 wt %MeHA hydrogels.
,4•#"1
IOU
-
140
2 100
0
E
I
T
C- 120
-
* MeHA only
Ow/ 4.1 mg/mL collagen
80
60
60
-
E 40
20
*+
0
2.0t
A
2.5 wt% HA
5wt
A ---
5 wt% HA
7wH
1-r-
7 wt% HA
Figure 4: Instron Mechanical Testing of Iteration #2 Collagen-MeHA IPNs
Summary of compressive moduli for hydrogels prepared using IPN fabrication iteration #2 under
20% thickness per min compression by the Instron 5542. Statistically significant differences are
denoted by *.
**rr
14U
120
a.
S100
I
80
0E 0
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E
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E MeHA only
Uw/ 4.1 mg/mL collagen
-T
0
20
0
2.5% HA5rAH
2.5% HA
I
5% HA
7% HA
Figure 5: Instron Mechanical Testing of Iteration #3 Collagen-MeHA IPNs
Summary of compressive moduli for hydrogels prepared using IPN fabrication iteration #3 under
20% thickness per min compression by the Instron 5542. Statistically significant differences are
denoted by *.
45
1
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tV 35
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collagen
-MeHAonly
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-w/4.1 mg/ml
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-MeHAonly
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B
0.0%
0.5%
1.0%
1.5%
2.0%
C
Strain
0.0%
0.5%
1.0%
1.5%
2.0%
Strain
Figure 6: Instron Mechanical Testing of Iteration #4 Collagen-MeHA IPNs
A) Summary of compressive moduli for hydrogels prepared using IPN fabrication iteration
#4 under 20% thickness per min compression by the Instron 5542. Statistically significant
differences are denoted by *. 2.5 wt % MeHA without collagen was below the sensitivity
of the Instron 5542 and is not included.
B) Stress versus strain curves for 5.0 wt % MeHA hydrogels.
C) Stress versus strain curves for 7.0 wt % MeHA hydrogels.
32
..
ou
*
50
40
0
MeHA only
Uw/ 4.1 mg/mL collagen
E 30
N
a20
o
E30
20
2
E
0 10
S T
*·
0-
A
2.0t
2.5 wt% HA
A • ,-75wt
5 wt% HA
7wH
7 wt% HA
Figure 7: Instron Mechanical Testing of Iteration #4 Collagen-MeHA IPNs
Summary of compressive moduli for hydrogels prepared using IPN fabrication iteration
#4 under 20% thickness per min compression by the Instron 5542. Statistically significant
differences are denoted by *.
3.2 FITC-collagen and MeHA IPNs
FITC-collagen and MeHA IPN hydrogels were fabricated and imaged as described in
section 2.10. One set of FITC-collagen and MeHA IPNs was mixed by repeated pipetting for 1
min. Another set was left unfixed prior to UV treatment. After UV treatment and 2 h incubation
at 370 C, the fluorescent intensity of the central 400x400 pixel block was compared between the
well mixed and unmixed hydrogels. The well mixed IPNs had an average grayscale intensity of
106.0 +/- 15.7, while the unmixed had an average grayscale intensity of 120.5 +/- 33.9.
Fluorescent images, as well as fluorescent intensity plots, are shown for the well mixed
hydrogels in Fig 8A-C and for the unmixed hydrogels in Fig 8D-F. Both top and side view
images indicated that the well mixed hydrogels have more uniformly distributed FITC-collagen
amd MeHA. To control for any MeHA autofluorescence, images of MeHA without FITCcollagen were also captured and are shown in Fig 8G-I. The average grayscale intensity of the
central 400x400 pixel block for the MeHA control was 10.3 +/-3.2. As a positive control, FITCcollagen without MeHA was also imaged, Fig 8J-L. It was both the brightest and most uniform
of all the hydrogels, with an average grayscale intensity of 145.8 +/- 19.5.
I
A
I
I
I
E
v
R
-
Figure 8: FITC-collagen and MeHA IPNs
A) D) G) J) Top view 10x fluorescent images of well mixed FITC-collagen and MeHA,
unmixed FITC-collagen and MeHA, MeHA only, and FITC-collagen only hydrogels,
respectively.
B) E) H) K) Surface plots of fluorescent intensity for top views, 400x400 pixels, of well
mixed FITC-collagen and MeHA, unmixed FITC-collagen and MeHA, MeHA only, and
FITC-collagen only hydrogels, respectively.
C) F) I) L) Cross-sectional 4x fluorescent images of well mixed FITC-collagen and MeHA,
unmixed FITC-collagen and MeHA, MeHA only, and FITC-collagen only hydrogels,
respectively.
3.3 Scanning electron microscopy of collagen-MeHA IPN hydrogels
Collagen-MeHA IPN hydrogels were examined with a scanning electron microscope to
determine the physical structure of the IPNs. The micrographs highlight the differences between
the different materials. The fibrous structure of 4.1 mg/ml collagen is clearly visible in Fig 912A. For hydrogels containing MeHA, the images show that as MeHA concentration increases,
Fig 9-12 B, D, F and C, E, D, the walls of the networks become thicker. Additionally, a
comparison between IPNs with and without collagen, but the same MeHA concentration, e.g. Fig
9-12 D and E, shows that the IPNs without collagen appear less smooth, flakier than those with
collagen. Collagen fibers can also be seen in the SEM images 2.5 wt% MeHA with collagen. The
collagen fibers are less visible at higher MeHA concentrations.
A
B
D
F
3
Figure 9: 100x Scanning Electron Microscopy
A)-G) show 100X SEM images of 4.1 mg/ml collagen, 2.5 wt% MeHA + collagen, 2.5 wt%
MeHA only, 5 wt% MeHA + collagen, 5 wt% MeHA only, 7 wt% MeHA + collagen, and 7 wt%
MeHA only, respectively.
C
D
E
F
G
Figure 10: 500x Scanning Electron Microscopy
A)-G) show 500X SEM images of 4.1 mg/ml collagen, 2.5 wt% MeHA + collagen, 2.5 wt%
MeHA only, 5 wt% MeHA + collagen, 5 wt% MeHA only, 7 wt% MeHA + collagen, and 7 wt%
MeHA only, respectively.
A
B
I
F
C
E
G
Figure 11: 1000x Scanning Electron Microscopy
A)-G) show 1000X SEM images of 4.1 mg/ml collagen, 2.5 wt% MeHA + collagen, 2.5 wt%
MeHA only, 5 wt% MeHA + collagen, 5 wt% MeHA only, 7 wt% MeHA + collagen, and 7 wt%
MeHA only, respectively.
Figure 12: 2000x Scanning Electron Microscopy
A)-G) show 2000X SEM images of 4.1 mg/ml collagen, 2.5 wt% MeHA + collagen, 2.5 wt%
MeHA only, 5 wt% MeHA + collagen, 5 wt% MeHA only, 7 wt% MeHA + collagen, and 7 wt%
MeHA only, respectively.
3.4 Volumetric swelling of collagen-MeHA IPN hydrogels
The equilibrium volumetric swelling ratios of all collagen-MeHA combinations are
shown in Fig 13. For each concentration of MeHA, a statistically significant decrease in
volumetric swelling ratio was observed when 4.1 mg/ml collagen was added to MeHA only
hydrogels. For MeHA hydrogels both with and without collagen increased MeHA concentration
led to a decrease in swelling. The average volumetric swelling ratio decreased from 141 at 2.5
wt% to 36 at 7.0 wt% for MeHA without collagen and decreased from 72 at 2.5 wt% to 29 at 7.0
wt% for MeHA with 4.1 mg/ml collagen. Overall, 4.1 mg/ml collagen hydrogels without MeHA
had the lowest average volumetric swelling ratio at 21.
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L
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120
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=a
100
80
*
E
o
c*
*5
60
only
*w/4.1 mg/mL
collagen
40
20
I
I
Collagen only 2.5wt% HA
5wt% HA
7wt% HA
Figure 13: Volumetric Swelling Ratio of Collagen-MeHA IPN Hydrogels
The volumetric swelling ratio was calculated by measuring the swollen mass divided by
the dried mass of each hydrogel. Statistically significant differences are denoted by *
Statistically significant differences existed between the collagen only hydrogels and all other
mixtures.
3.5 HUVEC culture on collagen-MeHA IPNs and collagen hydrogels
The imaging for the experiment described in section 2.13 revealed that no HUVECs had
attached to any of the collagen-MeHA IPN hydrogels. More curiously, no HUVECs attached or
grew on the 4.1 mg/ml collagen only hydrogels. The data is not presented here, though repeated
trials confirmed the findings.
In an attempt to explain the lack of HUVEC attachment and proliferation on the collagen
hydrogels, an experiment was conducted in which collagen gels, some with irgacure and some
without Irgacure were seeded with HUVECs, as described in section 2.14. After 24 h of
attachment time, the culture media was aspirated to remove unattached cells and the collagen
hydrogels were imaged. The results, summarized in Fig 14, indicate that, while the collagen
without Irgacure performed as well as TCPS, the collagen with Irgacure had essentially no
HUVEC attachment or growth.
_
I~
_
2500
T
I
2000 E
E 1500 -
U
1000
500 0-
----
collagen w/irgacure
collagen w/ irgacure
collagen w/out
I
w/out
collagen
irgacure
irgacure
TCPS
I
TCPS
Figure 14: HUVEC culture on collagen with and without Irgacure
Above is a comparison of collagen without Irgacure photoinitiator and with Irgacure, but
no washing. After 24 h of incubation, very few, if any, HUVEC had attached to the collagen with
Irgacure hydrogels. The HUVEC attachment and proliferation on collagen without Irgacure was
comparable to that of tissue culture polystyrene (TCPS).
3.6 NIH-3T3 culture on collagen hydrogels with and without Irgacure washing
To further investigate cell attachment and proliferation on collagen with Irgacure, NIH3T3 cells were seeded onto collagen hydrogels with and without Irgacure and imaged at 2, 4, 8,
10.5 and 24 h to monitor cell attachment progress. As with the HUVEC experiment, NIH-3T3
cells seeded onto collagen with Irgacure failed to culture any cells, Fig 15B,D,F. On the other
hand, collagen without Irgacure had an average NIH-3T3 density of 370 cells/mm 2 at 10.5 h and
grew to an average density of 3500 cells/mm 2 at 24 h. This more favorable NIH-3T3
proliferation pattern can be seen developing in Fig 15A,C,E. It can be noted that some cells
appear in Fig 15D, collagen with Irgacure at 10.5 h after seeding. However, the cells are round
and of high contrast, suggesting they are not attached. The lack of NIH-3T3 cells in Fig 15F
confirms that the unattached cells were aspirated prior to the 24 h imaging. The results are
summarized in Fig 16.
In an extension to the previous experiment, the above procedure was repeated; prior to
seeding NIH-3T3 cells on the collagen surfaces, the hydrogels, both with and without Irgacure,
were washed in media on a tilting stage for 24 h. After washing, the collagen hydrogels were
seeded with NIH-3T3s and were imaged at 4, 8, 12, 24 and 31 h to monitor cell attachment. NIH3T3 cells successfully attached and proliferated on both sets of collagen hydrogels. Fig 17 shows
a plot of NIH-3T3 density as a function of time. Due to the large variation in cell densities,
statistical differences could not be determined. However, at 31 h, collagen with Irgacure had
reached an NIH-3T3 density of 720 cells/mm 2. Microscopic images captured at 4, 12, and 24 h
after seeding are shown for collagen without Irgacure, Fig 18A,C,E, and collagen with Irgacure,
Fig 18B,D,F.
A
A
B
B
C
D
E
F
Figure 15: Microscopic images of collagen hydrogels with and without irgacure
A) C) E) Collagen without irgacure at 4, 10, and 24 h after NIH-3T3 seeding. The culture
medium was aspirated and replaced just prior to the 24 imaging.
B) D) F) Collagen with irgacure at 4, 10 and 24 h after NIH-3T3 seeding. The culture
medium was aspirated and replaced just prior to the 24 imaging.
7000
6000
5000
E
E
4000
-
4.1 mg/ml collagen, no
irgacure
-
4.1 mg/ml collagen w/
irgacure
3000
2000
1000
0
0
-1 on0
Time after NIH-3T3 seeding (h)
Figure 16: NIH-3T3 Attachment vs. Time for unwashed collagen hydrogels
Initially, 50 pl of 250,000 cell/ml NIH-3T3 cell suspension was pipetted onto the
collagen hydrogels. Hydrogel surfaces were imaged at the indicated time points. Only attached
cells, exhibiting morphology typical of NIH-3T3 cells were included in the counting. Unattached
cells were removed by aspiration just prior to the 24 h imaging.
1800 1600 1400 1200 -
---
collagen, no irgacure
---
collagen w/ irgacure and
1000 800 600 400 -
washing
1
-
-
collagen w/ irgacure no
washing
200 -
0 -200
·I- ___
_
I" I
i
I
I
I
Ir
I
-
I
30
20
10
Time after NIH-3T3 seeding (h)
Figure 17: NIH-3T3 Attachment vs. Time for collagen hydrogels with 36 h washing
Initially, 200 pl of 250,000 cell/ml NIH-3T3 cell suspension was pipetted into the wells
containing the collage collagen hydrogels, covering the hydrogels with sell suspension. Hydrogel
surfaces were imaged at the indicated time points. Only attached cells, exhibiting morphology
typical of NIH-3T3 cells were included in the counting. Unattached cells were removed by
aspiration just prior to the 24 h imaging.
A
A
B
B
C
D
E
F
Figure 18: Microscopic images of collagen hydrogels with and without irgacure, including
36 h washing
A) C) E) Collagen without irgacure at 4, 12, and 24 h after NIH-3T3 seeding. The culture
medium was aspirated and replaced just prior to the 24 imaging.
B) D) F) Collagen with irgacure at 4, 12 and 24 h after NIH-3T3 seeding. The culture
medium was aspirated and replaced just prior to the 24 imaging.
3.7 NIH-3T3 encapsulation in MeHA hydrogels
NIH-3T3 cells encapsulated in 5.0 wt% MeHA were assayed with calcein and ethidium
homodimer to examine cell viability immediately after encapsulation and after a 24 h incubation
period. To determine the effect of prepolymer and cell temperature on post-encapsulation cell
viability, the three protocols described in section 2.18 were all followed and separate live/dead
assays were performed on each group. The three groups, 37°C MeHA/37°C NIH-3T3, 4°C
MeHA/37°C NIH-3T3, and 4oC MeHA/4°C NIH-3T3, had cell viabilities, immediately after
encapsulation, of 77 +/- 7%, 81 +/- 3%, and 78 +/- 6%, respectively, Fig 19. There were no
significant differences between the three groups. After 24 h of incubation, cell viability had
decreased in all three groups to 58 +/- 25%, 48 +/-16%, and 54 +/-19%, respectively, Fig 19.
Despite the large variances of the 24 h groups, statistically significant differences existed
between the immediate viability and the 24 h viability for all three protocols. Live/dead images
of 0 and 24 h time points are shown in Fig 20.
_~~____1__1____1____~
_
Wu/o
-**
80% 70% ,
I
Tr
60% -
TI,
F
a
:M 50% -
37
d
C
MeHA
37
de
C
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40% -
* 4 degC MeHA, 37 deg C
NIH-3T3
S30% -
20% -
04 deg C MeHA, 4 deg C
NIH-3T3
10% 0% -
I
-
0 hours
!
24 hours
Time after NIH-3T3 encapsulation
Figure 19: NIH-3T3 viability following encapsulation in MeHA
After being encapsulated in MeHA hydrogels, the encapsulated NIH-3T3 cells were
stained with a live/dead assay to determine the effects of hydrogel fabrication, UV exposure,
and incubation in media for 24 h on cell viability. Statistically significant differences existed
between the 0 and 24 h time points for each of the three fabrication methods. No statistically
significant differences existed between the three fabrication methods at either of the time points.
Figure 20: Live/Dead Imaging of NIH-3T3 cells encapsulated in MeHA
A) Fluorescent image of live (green) and dead (red) cells immediately after encapsulation in
a 5 wt% MeHA hydrogel.
B) Fluorescent image of live and dead cells encapsulated in a 5 wt% MeHA hydrogel, after
24 h incubation at 370 C.
52
3.8 FITC-MeHA Hydrogels and photobleaching
The methacrylation and photopolymerization process described in section 2.11 was
successful in creating FITC-MeHA hydrogels. After synthesis and mixing of the prepolymer
solution, one set of FITC-MeHA in PDMS molds was imaged with 495 nm excitation. As seen in
Fig 21A, with the 1 sec exposure time necessary to image the post-UV FITC-MeHA hydrogels,
the prepolymer, without UV exposure, fluoresced strongly enough to saturate the image field
completely. After a 60 second UV exposure time, reduced from 180 sec to limit photobleaching,
the FITC-MeHA had the texture and appearance of typical 5.0 wt% MeHA hydrogels. The gels
were imaged under 495 nm excitation, fluorescing significantly less than the pre-UV solution,
but strongly enough to capture a clear green image of the FITC-MeHA with a 1 sec exposure
time, Fig 21B. Background fluorescence imaging is shown in Fig 21C.
Sample images and fluorescent profiles of the time-lapse characterization of FITC-MeHA
solution photobleaching are shown in Fig 22. The center of the solution had already been
exposed to UV, explaining the darker area in the center of Fig 22A. Photobleaching in the center
of the image occurred rapidly during the first 5 min, but slowed as the center approached
background fluorescence levels, Fig 22E. Fluorescent profiles plotted in Fig 22E were the
intensity values for the pixels of the main diagonals from Fig 22A-D.
A
B
C
Figure 21: FITC-MeHA Fluorescent Images
A) Green fluorescent emission image of FITC-MeHA prepolymer solution prior to UV
exposure. Most of image pixels are saturated at a grayscale value of 255.
B) Green fluorescent emission image of FITC-MeHA hydrogel after 60 sec UV exposure.
Some flakes of FITC-HA are noticeable in the image.
C) Green fluorescent emission image of background showing little to no fluorescence.
A
C
~
----AA --
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250
LM
eq 200
130 sec
g
150
------- 5 min
- - - - 10 min
5
100
--
- 15 min
50
0
0
200
400
600
800
1000
Pixel # on Diagonal
E
Figure 22: Photobleaching of FITC-MeHA solution
A)
B)
C)
D)
E)
First image of FITC-MeHA photobleaching at time 30 sec.
Image of FITC-MeHA after 5 min exposure to 490 nm light.
Image of FITC-MeHA at 10 min exposure to 490 nm light.
Image of FITC-MeHA it 15 min exposure to 490 nm light.
Plot of grayscale fluorescent intensities of major diagonal of images A-D.
3.10 Texas Red-X collagen
Collagen was successfully stained with Texas Red-X protein stain and imaged in
solution, where red fluorescence was observed with 590 nm excitation Fig 23A. The solution
gave no emission under 495 nm exposure, Fig 23B, and showed little photobleaching from
exposure to the blue wavelength, Fig 23C. Texas Red-X collagen hydrogels were successfully
fabricated by mixing the Texas Red-X collagen solution with stock collagen.
A
C
Figure 23: Texas Red-X collagen fluorescent images
A) Texas Red-X collagen solution emitting red fluorescence under 590 nm excitation.
B) Fluorescent green emission image to check for green autofluorescence and test the
photobleaching of the Texas Red-X collagen solution
C) Texas Red-X collagen solution emitting red fluorescence under 590 nm excitation after
exposure to 495 nm light. The image was taken to determine the effect of 495 nm
exposure on the red spectrum emission of the Texas Red-X stain.
4 Discussion
4.1 Collagen-MeHA IPN fabrication, uniformity, and compressive modulus
A primary goal of this project is to create collagen-MeHA IPN hydrogels with
controllable properties. Even in the first experiments with the materials, it was clear that
mixtures could be formed, but it was also clear that create homogeneous mixtures with uniform
and predictable properties was less than trivial. The iterations of the IPN fabrication process
were undertaken to refine the protocol and to produce IPNs with predictable mechanical
properties specifically compressive modulus. Table 2 shows the coefficient of variation for each
of the IPN mixtures within each iteration.
Initially, there was concern that the more viscous solutions would not mix well. The
collagen prepolymer solution had to be kept at 40 C once it had been neutralized. However,
iteration #1 included the use of 37°C 10 wt% MeHA so as to take advantage of the decreased
viscosity at 37°C. The effect was counterproductive due to the formation of the flaky white
precipitate, which was consistent with the formation of collagen[20]. It appeared that the
addition of the 37°C MeHA quickly raised the temperature of the collagen, causing it to gel
inside of the solution. The effect was more pronounced in the higher MeHA concentration
solutions, which led to poor mixing and, later, to large variations in hydrogel compressive
moduli. Because the collagen formed prior to UV treatment, it is unlikely that true IPNs were
actually formed. Another downside of the protocol used in iteration #1 was the use of the
vortexer, as it appeared to cause the two solutions to separate, rather than mix, and may have
contributed to the poor uniformity of the hydrogels.
Iteration #1 also provided opportunities to examine several anomalous stress versus strain
curves seen in Fig 3C,D. Compression of one 7 wt% MeHA hydrogel without collagen resulted
in a saw-tooth shape, Fig 3C. This was caused by bubbles trapped between the Instron 5542 plate
and the hydrogel. As each bubble bursts, a rapid drop in stress is seen in the stress versus strain
curve. The problem may be remedied by compressing the gel to burst the trapped bubbles, then
relaxing the compression back to the initial height before beginning the compression experiment.
The other anomalous stress versus strain curve was the hydrogel with sub-linear order growth
seen Fig 3D. This curve can potentially be explained by microfractures in the hydrogel. Though
such a test was not performed, the microfracture hypothesis could be confirmed by relaxing the
compression and examining the plot for hysteresis.
Iteration #2 provided little improvement over the protocol used in iteration #1. Keeping
the MeHA at 4°C instead of 37'C may have prevented the collagen in the IPNs from forming
prematurely, but also made the solution more difficult to mix due to increased viscosity. The
mixing via pipette was clearly not enough to result in more homogeneous mixtures. As seen in
Table 2, the coefficient of variation decreased, in comparison to iteration #1, for the 7.0 wt%
MeHA with collagen and 2.5 wt% MeHA without collagen solutions. However, the coefficient
of variation increased for all other solutions in comparison to iteration #1. Similarly, the average
coefficient of variation for iteration #2, 0.54, showed no improvement over the average
coefficient of variation for iteration #3, 0.52. Despite these shortcomings in reducing statistical
variance, IPN fabrication was more successful overall, as statistically significant differences
were seen between average compressive moduli of hydrogels with and without collagen for 2.5
and 7.0 wt% MeHA hydrogels. This is most likely due to the elimination of premature collagen
gelling; it is possible that the MeHA network was able to form during the UV crosslinking to
provide an initial structure and assure that the collagen remained distributed throughout the
network, prohibiting the collagen from condensing into thicker membranes, as was seen in
iteration #1.
The statistical variations observed in iteration #2 were still unacceptably large and, thus,
further attempts were made to improve hydrogel uniformity via better mixing. Iteration #3
attempted to utilize the Labquake rotisserie shaker achieve improved mixing with vibration and
inversion. Watching the mixing, it was clear that the rotisserie was successfully combining the
lower viscosity mixtures, which was confirmed in the compressive testing; all the 2.5 and 5.0
wt% solutions with and without collagen all had coefficients of variation of 37% or below.
Unfortunately, it was equally clear that the Labquake had little effect on the most viscous of the
solutions, 7.0 wt% MeHA with collagen; it remained in the bottom of the tube, unagitated and
not flowing at all as it was overturned. Again, the observation was confirmed with a coefficient
of variation in compressive modulus of 89%. During the preparation, it was more difficult to
predict the uniformity of the 7.0 wt% MeHA without collagen. While on the rotisserie, it was not
churning as easily as the 2.5 and 5.0 wt% solutions, though it showed a small amount of flow
while being inverted. In the end, the 7.0 wt% MeHA without collagen was well mixed and fairly
uniform as it resulted in a coefficient of variation of 33%. Although iteration #3 produced
statistically significant differences in compressive modulus between the 2.5 wt% and 5.0 wt%
MeHA with and without collagen, the nearly 90% variation in 7.0 wt% MeHA with collagen was
still unacceptably large, leading to the changes in fabrication seen in iteration #4.
The protocol used in iteration #4 varied from iteration #3 only in its use of mixing with
magnetic stir bars at -60 rpm for 24 h in lieu of the Labquake rotisserie shaker. This change had
dramatic effects on the values and variation of the average compressive moduli for the collagen60
MeHA IPNs. Statistically significant differences between hydrogels with and without occurred
for all MeHA concentrations, Fig 6. This provides evidence that combining the collagen and
MeHA into IPNs results in improvements in at least one mechanical characteristic when
compared to each of the hydrogels individually.
Another noticeable effect of the 24h stirring was an overall reduction in compressive
modulus from previous iterations. The most likely cause of this is the precipitation of the
photoinitiator out of solution, resulting in a lower photoinitiator concentration during UV
treatment, which is known to decrease stiffness[48]. Despite these decreases, the average
compressive modulus of the 7.0 wt% MeHA with collagen, 33.5 kPa, falls very close to the
target compressive modulus given for cardiac tissue in section 1, 31 kPa. The ability to
encapsulate cells inside the 7.0 wt% MeHA with collagen and to culture cells on the surface of
the IPNs would make the material a potentially valuable tool for cardiac tissue engineering.
Iteration #4 produced the hydrogels with the lowest average coefficient of variation for all
iterations at 25%.
Finally, in an effort to demonstrate improved mechanical properties as well as to produce
a range of stiffer gels, in addition to those formed in iteration #4, the protocol used in iteration #5
was adopted. In this iteration, the photoinitiator was added only for the final five min of the 24h
stirring, in the hope that the collagen and MeHA would become well mixed during the 24 h
mixing and that the photoinitiator would not precipitate out of solution, resulting in stiffer IPNs
with low variability in their compressive moduli. Although the iteration had an average
coefficient of variation of only 30%, no statistical variation was observed between the 5.0 wt%
MeHA hydrogels with and without collagen. On the other hand, statistically significant
differences were seen for the 2.5 and 7.0 wt% MeHA hydrogels with and without collagen.
61
Additionally, an increase in compressive modulus for each mixture in iteration #5 was seen in
comparison to iteration #4. Thus, the iteration was considered partially successful, though could
still be improved by optimizing the photoinitiator mixing time.
2.5 wt% w/
collagen
5.0 wt% w/
collagen
7.0 wt% w/
collagen
2.5 wt%
MeHA only
5.0 wt%
MeHA only
7.0 wt%
MeHA only
Average
0.590645
0.623523
0.268266
0.460219
0.238837
0.310945
0.767642
0.274564
0.209537
0.18312
1.029442
0.356822
0.887144
0.157986
0.141691
0.5346
0.42406
0.363327
N/A
0.575112
0.497631
0.811539
0.369742
0.217307
0.23091
0.199185
0.259184
0.332656
0.211817
0.450007
0.527075
0.540462
0.41595
0.251373
0.30328
Iter. #
1
2
3
4
5
Table 2: Coefficients of variation for IPN fabrication iterations
4.2 Hydrogel and IPN visualization: FITC-collagen/MeHA IPNs and SEM
The fabrication and fluorescent imaging of the FITC-collagen/MeHA hydrogels, Fig 8,
provided several insights into the new material combination. First, by comparing the images of
the FITC-collagen/MeHA hydrogels, Fig 8A-C, to the images of the MeHA only hydrogels Fig
8G-I, one can see that the FITC-collagen remains in the hydrogels during UV crosslinking and
collagen gelling. This confirms that the hydrogels contain both collagen and MeHA. Further, by
comparing the fluorescent images of the well mixed versus unmixed, Fig 8D-F, FITCcollagen/MeHA hydrogels, it is clear that the two materials do not separate during fabrication
and that phase separation is not inhibiting the IPN formation. This conclusion is further
supported by the coefficients of variation of the fluorescent intensities of the well mixed and
unmixed hydrogels, which were 15% and 28% respectively. Further support for the conclusion
comes from the comparison of the well mixed FITC-collagen/MeHA IPNs to the FITC-collagen
only control hydrogels. The coefficient of variation for the FITC-collagen was 13%, only slightly
below that of the well mixed FITC-collagen/MeHA. From this one can conclude that, at 10X
magnification, collagen is as uniformly distributed in the collagen-MeHA IPNs as it is in
collagen only hydrogels of the same collagen concentration.
Another aid to PIN visualization was the use of SEM to examine the freeze-dried
structure of the gels. The evidence of the collagen-MeHA IPNs was strongest in the images of
the 2.5 wt% MeHA with collagen, as many collagen fibers could be seen. Because of the density
in MeHA networks in the higher MeHA concentrations, the collagen fibers were less readily
visualized. In the future, two approaches could be taken to make the SEM of even greater value.
First, many more 2.5 wt% images could be taken and the number of visible collagen fibers could
be counted or quantified in some other way. Additionally, it would be interesting to hold the
64
MeHA concentration steady and increase the collagen concentration incrementally to observe the
effect on the microstructure and to see the formation of denser collagen networks inside the
MeHA networks.
FITC-MeHA and Texas Red-X hydrogel visualization was explored and could, in the
future, strengthen the results seen for the FITC-collagen/MeHA IPNs described above. The two
fluorescent materials could be combined and used to image both collagen and MeHA distribution
in hydrogels simultaneously. Since the FITC-MeHA and Texas Red-X collagen have different
excitement and excitation ranges, merging of the two emission range images would help to more
precisely visualize the material homogeneity, particularly with the help of high magnification
confocal microscopy.
4.3 Cell culture on collagen and collagen-MeHA IPNs
When the experiment described in Section 2.13 showed that HUVECs would not grow on
the collagen hydrogels, it seemed very likely that the excess irgacure in the hydrogel was
responsible, as all other characteristics of the collagen fabrication and components were identical
to those typically used in collagen hydrogels for scaffolding and cell culture. The suspicion of
Irgacure as the responsible component was confirmed by the collagen with and without Irgacure
experiment described in the first portion of Section 2.15 and summarized in Fig 14. After this
experiment, the study was switched to the attachment and growth of NIH-3T3 cells because of
their high proliferation rate. The collagen with and without Irgacure experiment was repeated
with NIH-3T3 cells. Once again, the results, Fig 15 and Fig 16, confirmed that the Irgacure
collagen gels were completely unsuitable for cell culture. It was clear that the excess Irgacure
would have to be removed after IPN fabrication in order to allow the materials to be sufficiently
cell adhesive. Fortunately, this washing process was successful, as seen in Fig 17, and the NIH3T3 cells could be successfully cultured on the washed collagen hydrogels. The next point of
extension is to perform the washing step on collagen-MeHA IPNs and determine if NIH-3T3s
can be cultured on their surfaces. Experiments are currently underway and initial results show
that NIH-3T3 culture is possible and appears promising. If NIH-3T3s can be cultured on the
IPNs, the experiments will once again turn to HUVEC culture with the eventual goal of
endothelialized microchannels of collagen-MeHA. If the collagen-MeHA is not suitable for cell
culture, the process will be modified to include the adsorption of additional collagen onto the
surface of the hydrogels. A similar adsorption process has been successfully used in our
laboratory previously[49].
4.4 Cell encapsulation in MeHA and collagen-MeHA IPNs
One concern in the fabrication process was the effect that the cold temperature necessary
for collagen mixing may negatively affect the viability of cells encapsulated in the collagenMeHA IPNs. The experiment described in section 2.18 and summarized in Fig 19 demonstrated
that the MeHA temperature had no significant effect on encapsulated NIH-3T3 viability.
Because cells are commonly encapsulated in collagen hydrogels with high viability[50], it is
reasonable to predict that the addition of collagen to the solution will have no negative effect on
encapsulated cell viability. Thus, it is likely that the collagen-MeHA IPNs will be useful as a cell
encapsulation material for tissue engineering constructs. It will be useful to determine whether,
once encapsulated, the cells can be harvested from the IPNs. Collagen only and MeHA only
hydrogels have had cells harvested from them using collagenase[50] and hyaluronidase[48],
respectively.
5 Conclusions and future work
As discussed previously, the ability to control the properties of hydrogels holds great
potential value in the filed of tissue engineering. In this thesis, collagen-MeHA IPNs were
fabricated to have a number of potentially advantageous properties. Through a series of
iterations, a fabrication process was developed that allowed for the creation of uniform,
mechanically robust and controllable IPN hydrogels. It was shown that the inclusion of collagen
into the network, thereby creating the IPN, had a positive effect on the compressive modulus of
the IPNs across a variety of different polymer concentrations. IPNs were created in the target
compressive modulus of cardiac tissue, equal to -31 kPa. Additionally, the microstructure and
homogeneity of these IPNs were examined with various forms of microscopy. Further, the IPNs
were shown to have cell viability of >75% during hydrogel fabrication. Many steps were taken to
harness the cell adhesive properties of collagen in the IPNs. Although full cell cultures have not
yet been demonstrated on the collagen-MeHA IPNs, a serious engineering issue, the negative
effects of photoinitiator on cell adhesion, was overcome for collagen hydrogels. Overall a new
hybrid material was created which can be mechanically tuned, used for cell encapsulation, and
potentially for surface cell culture.
Many
future
investigations
were
described
in
Chapter
4,
including
further
experimentation into cell adhesion on the IPNs and more advanced fluorescent visualizations
using FITC-MeHA, Texas Red-X, and high magnification confocal imaging. On a larger scale,
the materials will be used for the creation of cardiac tissue structures with endothelialized
microvasculature in vitro. Additionally, a third component, self-assembling peptide amphiphiles
will be included into the IPNs to provide addition chemical and cellular sensitivity. Overall, this
newly developed and characterized material could hold great value in the future of tissue
engineering when developed into cardiac tissue structures.
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