Collagen and hyaluronic acid interpenetrating polymer networks for tissue engineering

I

LI

Collagen and Hyaluronic Acid Interpenetrating Polymer Networks for Tissue Engineering

8f NMASSACHU

7

Or TE

NNOby

SET CHN

13

BRARIES

I

Mark D.

Brigham

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.

TS INSTITUTE

2LOGY

2008

RiES

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.

A'¢ / ""'

Author

Department of Electrical Engineering and Computer Science

/ ,n /7 1 August 21, 2007

Certified by_

Ali Khademhosseini Assistant Professo f Medicine and Health Sciences and Technology

Thesis Supervisor

J/ l. I IL

-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 collagen-MeHA IPN hydrogels represent a powerful new material for tissue engineering applications.

Thesis Supervisor: Ali Khademhosseini

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 which affect cell attachment, differentiation, viability, and

proliferation[34, 35]. 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

Hydrogen-bonded helical domain

)rnm / I0-ii I

.t

*--I

r

\\ I** .. '/ -8 nm / • \\ I e 0 ...---__ ___I -__ MICR( - - FIBRIL cross--linked cross-linked )-FIBRIL .0!• UNDULATED FIBER

0

1 - 500 diamei TISSUE

Figure 1: Collagen structure and fiber formation. [20]

--Hyaluronan

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

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 370C.

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 cm2 _{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% CO2, 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.

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 FITC-collagen and standard stock FITC-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

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 370C 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 -200C 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

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

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% CO2, 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% CO2, 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

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 370_{C 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 370C. Aliquots B and C were cooled to 40C. Aliquot A and B were then mixed, by

pipetting, with NIH-3T3s at a concentration of 2x106 cells/ml. For Aliquot C, a solution of

NIH-3T3 cells was cooled to 40C 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 2x106

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

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.

L.Ut-u. 1.OE-04 0.0E+00 3.0E-03 - w/4.1mg/mi 1.0E-03 8.OE-04 6.OE-04 4.OE-04 2.OE-04 0.OE+00 2.5E-03 2.0E-03 1.5E-03 1.0E-03 5.0E-04 O.OE+O0 0.0% 0.5% 1.0% 1.5% 2.0% 0.0% 0.5% 1.0% 1.5% 2.0% 0.0% 1.0% 2.0%

Strain C Strain D 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. IOU 140 * 120 , 100 80 S60 40 2 o 20 0 A -2fl ~ 80*wI4.1 glm colage T

T

2.5wt% HA 5wt% HA 7wt%/o HA rrn A 2 i iw/4.1 mg/mLcollagen • MeHA only Nw/4.1 mg/mLcollagen EMeHA only

-R

aw

TT

-- T-m ,,,,. 1F1-MU

-IOU -140 C- 120 2 100 -0 E 80 60 60

-E 40

20 0

T

I

* MeHA only Ow/ 4.1 mg/mL collagen *+ --- 1-r-2.5 wt% HA 5 wt% HA 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 *.

,4 •#"1

2.0t A 5wt A

*~

II

14U 120 a. S100

80

0 _E 0 0 > 60 40 E 0 20 0 E MeHA only Uw/ 4.1 mg/mL collagen 2.5% HA 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 *.

-T

**rr

45

1

*

40 ' tV 35 Q. ~ ~ 30 :; '8 25 I _MeHAonly :I ~ 20 .w/4.1mg/mLcollagen 'ii

=

15 ~ Q. E ₁₀ 0 0 5 0

A

2.5% HA 5%HA 7%HA 1.GOE-04 1.40E-04 1.20E-04 IiQ. 1.00E-04 ! _8.00E-OS III III f _G.OOE-OS

..

III 4.00E-OS 2.00E-OS O.OOE+OO B 0.0% -w/4.1 mg/ml collagen -MeHAonly 0.5% 1.0% 1.5% 2.0% Strain 9.00E-04 8.00E-04 7.00E-04 Ii G.OOE-04 Q. ! S.OOE-04 III 4.00E-04 III f ~ 3.00E-04 2.00E-04 1.00E-04 O.OOE+OO C 0.0% -w/4.1 mg/ml collagen -MeHAonly 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.

ou 50 40 0

E 30

a20 o E30_{20 2} E 0 10 0-*· 2.5 wt% HA 5 wt% HA 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 *.

S T * N MeHA only Uw/ 4.1 mg/mL collagen .. 2.0t A 5wt A 7wH •

,-7-3.2 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 370C, 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

FITC-collagen 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, FITC-collagen 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.

E

v

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. A I I I I R

-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

9-12A. 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

_C

E

F

_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.

I OU 160 .o 140 120 =a 100 80 * 60 E o 40 20

I

SHA only *w/4.1 mg/mL c* collagen *5 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.

*bn L

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

2500 2000 -E E 1500

-U

1000 500 - 0-T

I

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).

I~ _ _

collagen w/ irgacure collagen w/out TCPS irgacure

----

I

I

collagen w/irgacure collagen w/out TCPS irgacure

3.6 NIH-3T3 culture on collagen hydrogels with and without Irgacure washing

To further investigate cell attachment and proliferation on collagen with Irgacure,

NIH-3T3 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/mm2 at 10.5 h and

grew to an average density of 3500 cells/mm2 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 3T3s and were imaged at 4, 8, 12, 24 and 31 h to monitor cell attachment.

NIH-3T3 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/mm2.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

B

C

E

D

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.

- 4.1 mg/ml collagen, no

irgacure

- 4.1 mg/ml collagen w/

irgacure

0 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.

7000 6000 5000 E E 4000 3000

2000

1000 0 -1 on0

--- collagen, no irgacure 1800 1600 1400 1200 1000 800 600 400 200 0 -w/ irgacure and w/ irgacure no I" I I - I 10 20 30

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.

--- collagen washing - - collagen washing -200 ___ _ i I I I Ir

·I-1

A

B

C

E

D

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

-** I I Wu /o 80% -70% -, 60% -:M 50% -40% -S30% 20% 10% -0%

-TI,

a 37 d C MeHA 37 de C

F

NIH-3T3

* 4 degC MeHA, 37 deg C

NIH-3T3

04 deg C MeHA, 4 deg C

NIH-3T3

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.

Tr

_ _~~____1__1____1____~

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 370C.

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.

A

C

E

Figure 22: Photobleaching of FITC-MeHA solution

A) First image of FITC-MeHA photobleaching at time 30 sec.

B) Image of FITC-MeHA after 5 min exposure to 490 nm light. C) Image of FITC-MeHA at 10 min exposure to 490 nm light.

D) Image of FITC-MeHA it 15 min exposure to 490 nm light.

E) Plot of grayscale fluorescent intensities of major diagonal of images A-D.

-AA --3,uu 250 LM eq 200 g 150 5 100 50 0 1- 30 sec --- 5 min - - - - 10 min -- - 15 min 0 200 400 600 800 1000 Pixel # on Diagonal ----~

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 40C 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

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