hydrogel paper final

Optimization of Gelatin and Microbial Transglutaminase Hydrogels for Cardiac Tissue Engineering

Andre&Lai1,&Nicolaus&Jakowec2,&Kevin&Chin3&and&Megan&L.&McCain3&

1"Diamond"Bar"High"School,"Diamond"Bar,"USA;"2Loyola"High"School,"Los"Angeles,"USA"

3Department"of"Biomedical"Engineering,"Viterbi"School"of"Engineering,"University"of"Southern"California,"Los"Angeles,"CA"90089"

Abstract Tissue engineering has the potential to restore proper cardiovascular function to hearts damaged by cardiovascular diseases and malformations. In the field of tissue engineering, defining the structural parameters and chemical composition of hydrogel scaffolds used for cell culture remains a crucial area of study. Because tissue scaffolds will be surgically implanted onto a patient’s damaged organ, such as the heart, hydrogels must be fabricated in such a way as to withstand physiological conditions while permitting the growth of cultured cells. For this study, we optimized two key components of a hydrogel scaffold for cardiac tissue engineering: gelatin, a derivative of collagen, and microbial transglutaminase (mTg), an enzyme crosslinking reagent that makes the gelatin thermostable. We tested the absorbency of hydrogels fabricated with various combinations of the two substances by comparing their weights before and after overnight immersion in phosphate buffered saline solution (PBS) or water in an incubator (37°C) or at room temperature (21°C). Hydrogels with mTg crosslinker were more durable, as they showed less mass change than hydrogels with no mTg crosslinker, illustrating the enzyme’s essential role in optimal hydrogel fabrication. A stable hydrogel scaffold could then be used for cell culture. In summary, we found optimal concentrations of gelatin and mTg for developing biomimetic hydrogel scaffolds for cardiac tissue engineering. Keywords: Cardiomyocytes, Hydrogel, Microbial Transglutaminase Introduction Heart disease accounts for 600,000 deaths each year in the United States and costs an annual sum of over $100 billion in health care services [1]. Heart disease carries a colossal toll on the nation’s health and will accelerate its damage if a solution to cardiac ailments is not found soon. The heart, while a vital organ, lacks sufficient regenerative capacity and cannot correct tissue injury or malfunction. Thus, in the precarious scenario of heart failure, we are forced to rely on therapeutic strategies to repair the wounded heart and restore optimal physiological function to the cardiac tissue. A promising solution to cardiac disorders involves replacing the diseased tissue of the heart with new, healthier cells. Chong et. al. (2014) demonstrated that human embryonic-stem-cell-derived cardiomyocytes (hESC-CMs) can be injected into the hearts of non-human primates to produce significant remuscularization [2]. However, this method of straight injection

produced poor integration of cells into the cardiac tissue, resulting in uncontrolled cell death and cell migration away from the site of injection. Therefore, we must look for an alternative method that allows controlled cell integration. Cellular scaffolds offer such a controlled method; cells can be organized into functional tissue that can be surgically grafted onto the heart. Through tissue engineering, cells can be constructed in hydrogels and grafted into a patient’s heart using cellular scaffolds, restoring proper cardiovascular function and prolonging the patient’s life. This emerging field has the potential for effective tissue repair of a patient’s degrading vital organs and even tissue enhancement for improved physiological function. When engineering tissues in vitro, proper scaffolds for culturing cells are necessary to accurately mimic the physiological environment of humans. Scaffolds allow us to replicate the mechanical and biological influences a cell receives within the human body and creates an environment that promotes cells and tissues to grow and behave physiologically [3,4]. Traditional scaffolds consist of glass and tissue culture plastic. However both of these types of scaffolds are stiff and thus not physiological. This has led to the use of Polydimethylsiloxane (PDMS) and Polyacrylamide gels. However, PDMS scaffolds also have a stiffness that contrasts physiological conditions, hindering the growth and proliferation of cells on such surfaces. On the other hand, Polyacrylamide Gels, while characterized by a stiffness that is tunable within physiological range, are toxic and thus do not provide a suitable environment for long term culture of cells. These undesirable qualities have led to the development of more biocompatible hydrogels for scaffold use. Current hydrogel scaffolds include Alginate hydrogels. Unfortunately, these hydrogels are not native to the heart and thus require additional fabrication steps for fibronectin adhesion [5]. For this reason, an alternative type of hydrogel must be utilized. One potential scaffold that is both physically stable and biocompatible is microbial transglutaminase (mTg)-crosslinked gelatin hydrogels. Gelatin is naturally derived from collagen, a protein commonly found within the extracellular matrix. As a result, gelatin is innately non-toxic and highly suitable for cell adhesion. Furthermore, the stiffness of gelatin can be adjusted depending on the amount of gelatin and mTg crosslinker used, adding an extra advantage for gelatin hydrogels. The objective of our project was to determine the optimal concentration of gelatin and mTg to create a hydrogel scaffold that is biomimetic without compromise in physical stability. One disadvantage of gelatin is that it melts at physiological temperatures. Therefore, mTg crosslinker is used to stabilize the gelatin. We hypothesized that the proper concentration of mTg and gelatin would form an ideal hydrogel that could sustain long-term cardiomyocyte growth in vitro. In our experiment, we tested various combinations of different concentrations of gelatin and mTg hydrogels to determine the optimal mixture of gelatin and mTg that would produce the most stable scaffold. The different hydrogels were then placed in one of four conditions: PBS/37°, PBS/Room Temperature, Water/37°, Water/Room Temperature to test how temperature and osmolarity can affect the hydrogel scaffold. This allowed us to obtain percent mass change data influenced by concentration of gelatin, concentration of mTg, solution placed in, and temperature stored in.

Methods Fabrication of mTg-Crosslinked Gelatin Hydrogels 10 mL solutions of 5%, 10%, and 15% w/v gelatin from porcine skin (175 Bloom, Type A, Sigma--Aldrich, St. Louis, MO) were prepared with 0%, 2%, 5%, 10%, and 15% microbial transglutaminase (mTg, Ajinomoto, Fort Lee, NJ), with Millipore water or PBS as the medium for a total of two sets of 15 solutions. 15-mL tubes filled with 10 mL of Millipore water were placed in a water bath at 65°C to dissolve the gelatin and prevent solidification. The microbial Transglutaminase was then added to the 65°C gelatin solution. Next, disposable pipettes were used to transfer the mTg-crosslinked gelatin hydrogel into 60mm petri dishes to be cured overnight at room temperature (Figure 1).

Figure 1. Example of a hydrogel consisting of 15% Gelatin and 10% mTg in a 60mm

petri dish. Initial Mass Measurements and Separation into Different Conditions After the gels were cured overnight, industrial razor blades (VWR, Surgical Carbon Steel) were used to cut the gels into 4 individual hydrogels of approximately 21mm x 21mm x 10mm. Each individual gel was weighed in grams using an electronic scale (Mettler Toledo). Following mass measurements, the gels were placed in 16mm petri dishes. Half of the petri dishes were filled with 5mL of 1x PBS solution, while the other half were filled with 5mL of Millipore water. The 60 gels, 30 submersed in water and 30 submersed in PBS, were then separated into two equal groups to be left overnight in one of the following two conditions: room temperature or 37°C (Figure 2).

Figure 2. Set of 60 Hydrogels resting in 4 different conditions.

Final Mass Measurements of Hydrogels After the 60 hydrogels were left overnight, the gels were weighed again to observe any mass changes. The gels were gently dried with a Kim Wipe to remove any excess liquid and then placed on the scale to be weighed. Optimizing the Hydrogel and Statistical Analysis The data was then compiled using Microsoft Excel (Microsoft Excel for Mac 2011, Version 14.4.2) and graphed to show percent mass change. Percent mass change was calculated using the following formula:

(final mass - initial mass) / initial mass * 100 Then, the experiment described in the Fabrication of Microbial Transglutaminase- Crosslinked Gelatin Hydrogels was then repeated twice for a total of 3 times. Once the experiments were repeated 3 times over, we took the average of the data sets and calculated standard deviation to format error bars. T-tests were then performed using an online program found at http://www.graphpad.com/quickcalcs/ttest1.cfm to determine statistical significance between different mTg concentration values within the same gelatin concentration. Preparing Glass Coverslips Glass coverslips (22mm x 22mm) were covered along the edges with low-adhesive tape (3M, St. Paul, MN). Small strips of tape were cut and placed on each side of the coverslip. This created an outer border of tape that was slightly raised, leaving an inner area of glass. Next, the coverslips were immersed in 0.1 M NaOH for 5 min, followed by 0.5% APTES in 95% ethanol for 5 min and 0.5% glutaraldehyde for 30 min. The coverslips were then rinsed in Millipore water 3 times and placed in a 65°C incubator for 20 min to allow them to dry. Hydrogel solutions of 5%, 10%, and 15% w/v gelatin from porcine skin (175 Bloom, Type A, Sigma--Aldrich, St. Louis, MO) with 2% and 10% microbial transglutaminase (mTg, Ajinomoto, Fort Lee, NJ) were then created with Millipore water as the medium. The solution was then quickly pipetted onto the taped activated coverslips (Figure 3A). Polydimethylsiloxane (PDMS, Sylgard 184, Dow Corning, Midland, MI) stamps with 10 µm x 10 µm line features were then inverted onto the hydrogel solution so that the stamp was resting on the taped edges of the coverslip. The gelatin on the coverslip with the stamp was then left to cure overnight. After the gelatin was cured overnight, the coverslip and stamp were immersed in Millipore water to facilitate the removal of the stamp from the gelatin and coverslip. Once the stamp was removed, the pieces of tape on the edge of the coverslip were carefully peeled off (Figure 3B & C). We used an industrial razor blade (VWR, Surgical Carbon Steel) to assist this process by cutting off any excess gel that was stuck on the tape. Once the tape was peeled off, the coverslip was placed in a 6 well cell culture plate (Costar, Corning, NY) and rinsed once with PBS. The coverslip was then re-immersed in

PBS and then sterilized in a UV-ozone cleaner for one minute. After, they were stored at 4°C until cell seeding. Cell Culture A graduate student cultured and seeded cardiomyocyte cells on the coverslips we prepared according to previously established protocols. Briefly, Cardiac myocytes were isolated from two-day old neonatal rat hearts and seeded onto the micromolded gelatin substrates. Cells were then cultured for four days in a 5% CO2, 37°C incubator to allow the cells to form tissue. Immunostaining and Image Analysis The engineered cardiac tissues on the gelatin hydrogels were fixed using 4% paraformaldehyde (Electron Microscopy Sciences, Hatfield, PA) and 0.5% Triton-X (Sigma--Aldrich, St. Louis, MO). They were then stained to visualize their nuclei and cytoskeleton according to previously established protocols. The coverslips were stained with the primary antibody, mouse alpha actinin, which stains sarcomeres, and let to rest for 1 -2 hours. The coverslips were then rinsed in PBS and incubated with DAPI to stain nuclei, Phalloidin-488 to stain actin filaments, and anti-mouse-546 for 1-2 hours. Following this procedure, the coverslips were mounted onto glass slides, preserved with ProLong Gold Antifade, and sealed with nail polish. The slides were then stored in a -20°C freezer until we were ready to image them. The slides were imaged under a Nikon Eclipse Ti light microscope (Melville, New York) with both 20x and 60x objective.

Figure 3 (A) Gelatin Hydrogel Scaffold on an Activated coverslip (22x22mm) w/ taped edges. (10% gelatin, 10% mTg) (B) Cross-section view of patterned hydrogel scaffold on a glass coverslip (22x22mm). (10% gelatin, 10% mTg) (C) Top view of hydrogel scaffold. (10% gelatin, 10% mTg)

A B

C

Results Optimal Hydrogel Concentrations An optimal hydrogel scaffold recapitulates the mechanical properties of cardiac tissue and is also stable under physiological conditions, including changes in temperature and osmolarity. The aim of this paper was to find the corresponding concentrations of gelatin and mTg to create a hydrogel that would fit this definition. Three different concentrations of gelatin were tested with five different concentrations of mTg to test a total of 15 different solutions in four various conditions: PBS/37°, PBS/Room Temperature, Water/37°, Water/Room. Percent mass change data was then recorded for each gel after the gels were let to rest overnight in their respective conditions. Statistics were then performed after the experiment was repeated three times over. The data allowed us to determine which hydrogel scaffold was most ideal in terms of minimal percent mass change. Hydrogel mixtures involving 0% mTg crosslinker melted completely under physiological temperatures and swelled heavily under room temperature, above 30% mass change in most instances. The hydrogels consisting of 2% mTg showed promising and ideal results when placed in PBS or water at room temperature, but had significant mass change when placed at physiological temperatures. Likewise, the hydrogels consisting of 5% mTg had very similar results and, while stable under room temperature, had significant mass change at 37°C. In contrast, the hydrogels with 10% mTg showed minimal percent mass change when compared to those with only 2% or 5% mTg crosslinker, while the hydrogel with 15% had similar results to that of 10% mTg, and are not statistically significant based on calculated p-values (Supplementary Tables 1-8). Average data, percent mass change data, and statistical analysis involving standard deviation and t-test show that the most optimal hydrogel combination in terms of minimal percent mass change is 10% gelatin / 10% mTg and 10% gelatin / 15% mTg. In our tests, the 10% gelatin / 10% mTg hydrogel had a percent mass change of -5% ± 4% while the 10% gelatin / 15% mTg hydrogel had a percent mass change of -4% ± 4%, both under physiological conditions (Figure 4). The use of PBS as a medium as opposed to using water produced no significant advantage in terms of percent mass change. Similar trends and patterns can be seen between the hydrogel data of the two mediums.

Figure 4. Average percent mass change results of hydrogels placed in different conditions.

Creating a Homogeneous Solution A common issue when fabricating the hydrogels was the lack of a completely homogeneous solution and the presence of air bubbles. This issue was more pronounced within the hydrogels that had more mTg crosslinker and may have an uncertain effect on cell culture. Higher concentrations of gelatin caused some of the gelatin to precipitate and rest at the bottom of the test tube. As a result, additional mixing with the vortex machine was required. However, performing additional mixing produced an excess of air bubbles. While large bubbles were siphoned out with droppers, many miniscule air bubbles remained encapsulated within the gel after the solution was mixed using the vortex machine. Preparing the gelatin and mTg separately as two different solutions, and then mixing the solutions together at the same time into a larger tube resolved this issue. This procedure helped create a completely homogeneous solution with minimal air bubble formation within the hydrogel. Scaffold Performance Once we established the physical properties of the hydrogel scaffolds, we then proceeded to culture cardiac myocytes onto our scaffolds to determine their potential for tissue engineering. Gelatin hydrogels scaffold were micromolded on a glass coverslip to allow the formation of tissue. They were then seeded with cardiomyocytes and left to grow for three nights. Shortly after, the tissue was fixed, stained for sarcomeres and imaged under an inverted light microscope. Image analysis revealed that the cardiac tissue engineered onto the scaffolds with 10% mTg crosslinker had a more visible linear pattern than those engineered onto scaffolds with only 2% mTg crosslinker (Figure 5 & 6). This result is consistent with the percent mass change data in that the linear patterns of the scaffolds with only 2% mTg probably melted in the incubator, while the scaffold with the 10% mTg kept the linear structure. Overall, the scaffold with 10% mTg crosslinker proved to be more stable and biomimetic.

Figure 5. Left: Stained cardiomyocytes on 5% gelatin, 2% mTg hydrogel scaffold.

Right: Stained cardiomyocytes on 5% gelatin, 10% mTg hydrogel scaffold.

Figure 6. Left: Stained cardiomyocytes on 10% gelatin, 2% mTg hydrogel scaffold.

Right: Stained cardiomyocytes on 10% gelatin, 10% mTg hydrogel scaffold. Discussion The goal of this experiment was to obtain the optimal concentrations of gelatin and mTg in the composition of a hydrogel that would deter gel degradation and limit water adsorption so that it could be used as a scaffold for tissue engineering. In other words, our objective was to design a hydrogel that would best retain its structure under physiological conditions. To design such a gel, a variety of combinations of gelatin and mTg were tested. We found that a hydrogel with a composition of 10% gelatin and 15% mTg showed the lowest mass change under the physiologically relevant temperature of 37C, while a hydrogel with a composition of 15% gelatin and 15% mTg achieved similar results. Overall, hydrogels composed of 15% mTg had significantly better mass consistency than gels of lower concentrations (2%, 5%, 10%) of mTg, reinforcing the knowledge that higher concentrations of mTg correspond with greater degrees of cross-linking due to the enzyme’s catalytic role in network-crosslinking.

The absence of mTg crosslinker produced hydrogels of highly absorbent character in comparison with hydrogels of 2%, 5%, 10%, and 15% mTg. Hydrogels of 0% mTg adsorbed up to 70% of their dry weight in water at 21°C, while hydrogels of as little as 2% mTg adsorbed a maximum of 15%. Furthermore, all hydrogels with 0% mTg crosslinker composition completely melted in the incubator at 37°C; no other gel with mTg crosslinker melted. Such a striking disparity between gels of no mTg crosslinker and gels with as little as 2% mTg crosslinker highlights the cross-linking enzyme as a crucial component when designing hydrogels for scaffolding tissue. Surprisingly, no significant difference in results was observed between hydrogels of the two tested mediums, PBS and water. The structures of both gels are acutely similar and undergo matching degrees of crosslinking probably because the solution PBS is water-based and thus fulfills the same polar interactions that characterize chemical crosslinking. Since the swelling of hydrogels is influenced by the structure of the hydrogel network and since both gels had water molecules perform the crosslinking reactions, hydrogels of PBS and water displayed similar levels of adsorption. After testing the hydrogels against physiological conditions, we wanted to test how the hydrogel would perform as a tissue engineering scaffold by micromolding substrates and seeding them with cardiac myocytes. For scaffold performance 10% mTg hydrogels maintained their linear stamp patterns and served as a proper template for cardiac tissue. In contrast, the linear stamp patterns of 2% mTg melted under physiological temperature and did not facilitate the growth of cardiomyocytes into properly aligned cardiac tissue. The role of tissue engineering in the field of biomedical engineering has unlimited applications where cell regeneration and tissue replacement are highly driven pursuits. Since heart disease is rapidly escalating as the leading cause of morbidity, we must look for alternative solutions this complicated issue. Through tissue engineering hearts scarred by myocardial infarction can be restored with healthy cardiomyocytes transplanted via hydrogel scaffolds. However, tissue grafting will not be restricted to cardiac tissue. Patients who suffer from neurodegenerative disorders, such as Alzheimer’s and Parkinson’s disease (PD), experience neural cell loss and tissue degradation in the brain. Moreover, stroke and traumatic brain injury (TBI) patients experience debilitating destruction of neural tissue and the formation of brain cavities. Considering the poor regenerative capability of neural tissue, tissue engineering is left as the only means to replace these lost cells. Stem cells, while feasible and effective at significant substitution of tissue, are currently an inaccurate tool for a target as precarious as the human brain. Integrating stem cells with scaffolds, such as gelatin hydrogels, and grafting the scaffolded tissue directly onto the site of degeneration could reduce the possibility of cell migration and cell death. Furthermore, the architecture of a hydrogel can be designed so as to reflect that of the brain by using techniques such as micromolding; in this way, neurons seeded onto hydrogels can grow in complexes that mirror those of the brain, promoting functional integration of the cultured cells [6]. Furthermore, a scaffold with an elastic modulus

mimicking natural brain ECM will likely improve survival rate of implanted neural cells. Cheng et. al. (2013) demonstrated that neural stem cells transplanted via peptide hydrogels onto mouse brain lesions improved the survival rate of cells and supplanted the surgery-induced lesions of the brain [7]. In addition to their role in tissue replacement, engineered tissues provide a method of testing drug toxicity and promise to uplift the pharmaceutical industry from its rut of drug failure. Cells and tissues engineering on biomimetic scaffolds such as hydrogels, in contrast to animal models and laboratory tests, are mechanically tunable and chemically functionalized for cell attachment and long-term culture; various conditions can be situated to test the drug’s efficacy across many parameters, a feat not fully accomplished in animal and petri dish settings. The so-called “organs-on-chips” are a desirable alternative, or at least a complement to, the current in vivo and in-vitro drug testing methods [8]. Our results contribute to novel methods of tissue replacement and cell modeling. While the techniques described aimed towards designing a hydrogel scaffold suitable for cell cultures, many challenges lay ahead: what scaffold architectures best facilitate cardiac tissue formation, how can we transplant tissue scaffolds without activating the body’s immune system, to what extent can we improve the physiological function of organs through tissue engineering? With such questions answered, we will begin to unfold the true potential of tissue engineering and revolutionize the way in which damaged tissue is repaired and broken hearts are healed. Acknowledgements We would personally like to thank: Dr. Megan L McCain for allowing us to be a part of her incredible lab, giving us unparalleled lab experience, as well as for mentoring us throughout the summer; Dr. Cocozza and Mrs. Sabogal for accepting us into this unique program and providing us this exciting opportunity to participate in a research laboratory; Kevin Chin for mentoring us within our lab; all our labmates, Jasper Hsu, Davi Leite, Nethika Ariyasinghe, Archana Bettadapur, Gio Suh, and Clara Hua; and lastly Windsong Trust for funding and supporting this exclusive summer program. Reference Citations [1] “Heart Disease Facts.” cdc.gov. Centers for Disease Control and Prevention,

2014. Web. 10 Jul. 2014. [2] Chong James J. H., Xiulan Yang, Creighton W. Don, et al. Human embryonic-

stem-cell-derived cardiomyocytes regenerate non-human primate hearts. Nature 2014;510:273-279.

[3] Lee HyeongJin, GeunHyung Kim. Enhanced cellular activities of

polycaprolactone/alginate-based cell-laden hierarchical scaffolds for hard tissue engineering applications. Journal of Colloid and Interface Science 2014;430:315-325.

[4] Yung C.W., L.Q. Wu, J.A. Tullman, G.F. Payne, W.E. Bentley, T.A. Barbari. Transglutaminase crosslinked gelatin as a tissue engineering scaffold. Journal of Biomedical Materials Research Part A 2007;1039-1046.

[5] McCain Megan L., Ashutosh Agarwal, Haley W. Nesmith, Alexander P. Nesmith,

Kevin Kit Parker. Micromolded ggelatin hydrogels for extended culture of engineered cardiac tissues. Biomaterials 2014;30:1-10.

[6] Aurand Emily R., Jennifer Wagner, Craig Lanning, Kimberly B. Bjugstad.

Building Biocompatible Hydrogels for Tissue Engineering of the Spinal Cord. Journal of Functional Biomaterials 2012;3:839-963.

[7] Cheng Tzu-Yun, Ming-Hong Chen, Wen-Han Chang, Ming-Yuan Huang, Tzu-

Wei Wang. Neural stem cells encapsulated in a functionalized self-assembling peptide hydrogel for brain tissue engineering. Biomaterials 2013;34:2005-2016.

[8] Capulli A. K., K. Tian, N. Mehandru, A. Bukhta, S. F. Choudhury, M. Suchyta,

K.K. Parker. Approaching the in vitro clinical trial: engineering organs on chips. Royal Society of Chemistry 2014;DOI:10.1039/c4lc00276h.

Supplementary Materials Table Set 1 (PBS$at$37°C$w/$water$as$medium)$$

P5Values$for$5%$Gelatin$$%$mTg$ 0$ 2$ 5$ 10$ 15$

0$ x" x" x" x" x"2$ x" x" 0.0171" 0.0002" 0.0001"5$ x" 0.0171" x" 0.0005" 0.0001"10$ x" 0.0002" 0.0005" x" 0.0443"15$ x" 0.0001" 0.0001" 0.0443" x"

P5Values$for$10%$Gelatin$$

%$mTg$ 0$ 2$ 5$ 10$ 15$0$ x" x" x" x" x"2$ x" x" 0.3937" 0.0003" 0.002"5$ x" 0.3937" x" 0.0002" 0.0021"10$ x" 0.0003" 0.0002" x" 0.1751"15$ x" 0.002" 0.0021" 0.1751" x"

P5Values$for$15%$Gelatin$"

%$mTg$ 0$ 2$ 5$ 10$ 15$0$ x" x" x" x" x"2$ x" x" 0.4596" 0.0017" 0.0004"5$ x" 0.4596" x" 0.0068" 0.0013"

10$ x" 0.0017" 0.0068" x" 0.0444"15$ x" 0.0004" 0.0013" 0.0444" x"

*Values in red have a P-Value > 0.05 and are thus not statistically significant

Table Set 2 (PBS$at$21°C$w/$water$as$medium)$

P5Values$for$5%$Gelatin$"%$mTg$ 0$ 2$ 5$ 10$ 15$

0$ x" 0.0003" 0.0005" 0.0024" 0.0456"2$ 0.0003" x" 0.5546" 0.0749" 0.0061"5$ 0.0005" 0.5546" x" 0.0769" 0.007"10$ 0.0024" 0.0749" 0.0769" x" 0.0428"15$ 0.0456" 0.0061" 0.007" 0.0428" x"


%$mTg$ 0$ 2$ 5$ 10$ 15$0$ x" 0.001" 0.0005" 0.0022" 0.0216"2$ 0.001" x" 0.0019" 0.0155" 0.0106"5$ 0.0005" 0.0019" x" 0.0007" 0.0025"10$ 0.0022" 0.0155" 0.0007" x" 0.0479"15$ 0.0216" 0.0106" 0.0025" 0.0479" x"


%$mTg$ 0$ 2$ 5$ 10$ 15$0$ x" 0.0012" 0.0003" 0.0006" 0.0026"2$ 0.0012" x" 0.026" 0.823" 0.2253"5$ 0.0003" 0.026" x" 0.0077" 0.0079"10$ 0.0006" 0.823" 0.0077" x" 0.1191"15$ 0.0026" 0.2253" 0.0079" 0.1191" x"


Table Set 3 (Water$at$37°C$w/$water$as$medium)


0$ x" x" x" x" x"2$ x" x" 0.191" 0.0002" 0.0008"5$ x" 0.191" x" 0.5642" 0.3121"10$ x" 0.0002" 0.5642" x" 0.1126"15$ x" 0.0008" 0.3121" 0.1126" x"






Table Set 4 (Water$at$21°C$w/$water$as$medium)


0$ x" 0.0026" 0.0016" 0.0028" 0.0033"2$ 0.0026" x" 0.0656" 0.9283" 0.3216"5$ 0.0016" 0.0656" x" 0.081" 0.0122"10$ 0.0028" 0.9283" 0.081" x" 0.4"15$ 0.0033" 0.3216" 0.0122" 0.4" x"


%$mTg$ 0$ 2$ 5$ 10$ 15$0$ x" 0.0006" 0.0004" 0.0495" 0.0025"2$ 0.0006" x" 0.0213" 0.4352" 0.0863"5$ 0.0004" 0.0213" x" 0.1795" 0.0141"10$ 0.0495" 0.4352" 0.1795" x" 0.8428"15$ 0.0025" 0.0863" 0.0141" 0.8428" x"


%$mTg$ 0$ 2$ 5$ 10$ 15$0$ x" 0.0015" 0.0007" 0.001" 0.0034"2$ 0.0015" x" 0.0002" 0.0003" 0.2332"5$ 0.0007" 0.0002" x" 0.0121" 0.0121"

10$ 0.001" 0.0003" 0.0121" x" 0.0308"15$ 0.0034" 0.2332" 0.0121" 0.0308" x"


Table Set 5 (PBS$at$37°C$w/$PBS$as$medium)


0$ x" x" x" x" x"2$ x" x" 0.0036" 0.0011" 0.0025"5$ x" 0.0036" x" 0.0091" 0.0113"10$ x" 0.0011" 0.0091" x" 0.3326"15$ x" 0.0025" 0.0113" 0.3326" x"

P5Values$for$10%$Gelatin"





Table Set 6 (PBS$at$21°C$w/$PBS$as$medium)$$

P5Values$for$5%$Gelatin"%$mTg$ 0$ 2$ 5$ 10$ 15$

0$ x" 0.0544" 0.0381" 0.1083" 0.1466"2$ 0.0544" x" 0.1254" 0.1603" 0.0251"5$ 0.0381" 0.1254" x" 0.0566" 0.0125"10$ 0.1083" 0.1603" 0.0566" x" 0.4595"15$ 0.1466" 0.0251" 0.0125" 0.4595" x"


%$mTg$ 0$ 2$ 5$ 10$ 15$0$ x" 0.0001" 0.0001" 0.0001" 0.0051"2$ 0.0001" x" 0.0062" 0.0341" 0.1047"5$ 0.0001" 0.0062" x" 0.0256" 0.0254"10$ 0.0001" 0.0341" 0.0256" x" 0.047"15$ 0.0051" 0.1047" 0.0254" 0.047" x"


%$mTg$ 0$ 2$ 5$ 10$ 15$0$ x" 0.0033" 0.0014" 0.0046" 0.0057"2$ 0.0033" x" 0.0012" 0.372" 0.1041"5$ 0.0014" 0.0012" x" 0.2136" 0.0024"10$ 0.0046" 0.372" 0.2136" x" 0.1464"15$ 0.0057" 0.1041" 0.0024" 0.1464" x"


Table Set 7 (Water$at$37°C$w/$PBS$as$medium)$$


0$ x" x" x" x" x"2$ x" x" 0.0375" 0.0008" 0.0003"5$ x" 0.0375" x" 0.0528" 0.0149"10$ x" 0.0008" 0.0528" x" 0.1602"15$ x" 0.0003" 0.0149" 0.1602" x"




%$mTg$ 0$ 2$ 5$ 10$ 15$0$ x" x" x" x" x"2$ x" x" 0.4959" 0.0059" 0.0056"5$ x" 0.4959" x" 0.0086" 0.0072"

10$ x" 0.0059" 0.0086" x" 0.0718"15$ x" 0.0056" 0.0072" 0.0718" x"


Table Set 8 (Water$at$21°C$w/$PBS$as$medium)$$


0$ x" 0.0001" 0.0001" 0.0001" 0.0001"2$ 0.0001" x" 0.0041" 0.0546" 0.0035"5$ 0.0001" 0.0041" x" 0.0027" 0.0007"10$ 0.0001" 0.0546" 0.0027" x" 0.0303"15$ 0.0001" 0.0035" 0.0007" 0.0303" x"


%$mTg$ 0$ 2$ 5$ 10$ 15$0$ x" 0.0032" 0.0014" 0.0012" 0.0244"2$ 0.0032" x" 0.0024" 0.001" 0.0852"5$ 0.0014" 0.0024" x" 0.02" 0.0152"10$ 0.0012" 0.001" 0.02" x" 0.0098"15$ 0.0244" 0.0852" 0.0152" 0.0098" x"


%$mTg$ 0$ 2$ 5$ 10$ 15$0$ x" 0.0011" 0.0004" 0.0008" 0.0074"2$ 0.0011" x" 0.0011" 0.0423" 0.4438"5$ 0.0004" 0.0011" x" 0.0775" 0.0258"10$ 0.0008" 0.0423" 0.0775" x" 0.1012"15$ 0.0074" 0.4438" 0.0258" 0.1012" x"


hydrogel paper final

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