1. 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

2. 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.

3. 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.

4. 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

5. 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

6. 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.

7. 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.

8. 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.

9. 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

10. 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.

11. [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"

12. 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"
P5Values$for$10%$Gelatin$"
%$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"
P5Values$for$15%$Gelatin$"
%$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"
*Values in red have a P-Value > 0.05 and are thus not statistically significant
Table Set 3 (Water$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" 0.191" 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"

13. P5Values$for$10%$Gelatin$"
%$mTg$ 0$ 2$ 5$ 10$ 15$
0$ x" x" x" x" x"
2$ x" x" 0.4225" 0.0125" 0.0075"
5$ x" 0.4225" x" 0.0001" 0.0018"
10$ x" 0.0125" 0.0001" x" 0.0682"
15$ x" 0.0075" 0.0018" 0.0682" x"
P5Values$for$15%$Gelatin$"
%$mTg$ 0$ 2$ 5$ 10$ 15$
0$ x" x" x" x" x"
2$ x" x" 0.2982" 0.0008" 0.0003"
5$ x" 0.2982" x" 0.0008" 0.0003"
10$ x" 0.0008" 0.0008" x" 0.0156"
15$ x" 0.0003" 0.0003" 0.0156" x"
*Values in red have a P-Value > 0.05 and are thus not statistically significant
Table Set 4 (Water$at$21°C$w/$water$as$medium)
P5Values$for$5%$Gelatin$"
%$mTg$ 0$ 2$ 5$ 10$ 15$
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"
P5Values$for$10%$Gelatin$"
%$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"
P5Values$for$15%$Gelatin$"
%$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"

14. 10$ 0.001" 0.0003" 0.0121" x" 0.0308"
15$ 0.0034" 0.2332" 0.0121" 0.0308" x"
*Values in red have a P-Value > 0.05 and are thus not statistically significant
Table Set 5 (PBS$at$37°C$w/$PBS$as$medium)
P5Values$for$5%$Gelatin$"
%$mTg$ 0$ 2$ 5$ 10$ 15$
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"
%$mTg$ 0$ 2$ 5$ 10$ 15$
0$ x" x" x" x" x"
2$ x" x" 0.4539" 0.01" 0.0035"
5$ x" 0.4539" x" 0.019" 0.0043"
10$ x" 0.01" 0.019" x" 0.0167"
15$ x" 0.0035" 0.0043" 0.0167" x"
P5Values$for$15%$Gelatin"
%$mTg$ 0$ 2$ 5$ 10$ 15$
0$ x" x" x" x" x"
2$ x" x" 0.8319" 0.0073" 0.0091"
5$ x" 0.8319" x" 0.0022" 0.0072"
10$ x" 0.0073" 0.0022" x" 0.1153"
15$ x" 0.0091" 0.0072" 0.1153" x"
*Values in red have a P-Value > 0.05 and are thus not statistically significant
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"

15. P5Values$for$10%$Gelatin"
%$mTg$ 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"
P5Values$for$15%$Gelatin"
%$mTg$ 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"
*Values in red have a P-Value > 0.05 and are thus not statistically significant
Table Set 7 (Water$at$37°C$w/$PBS$as$medium)$
$
P5Values$for$5%$Gelatin"
%$mTg$ 0$ 2$ 5$ 10$ 15$
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"
P5Values$for$10%$Gelatin"
%$mTg$ 0$ 2$ 5$ 10$ 15$
0$ x" x" x" x" x"
2$ x" x" 0.308" 0.0011" 0.005"
5$ x" 0.308" x" 0.0153" 0.0067"
10$ x" 0.0011" 0.0153" x" 0.0141"
15$ x" 0.005" 0.0067" 0.0141" x"
P5Values$for$15%$Gelatin"
%$mTg$ 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"

16. 10$ x" 0.0059" 0.0086" x" 0.0718"
15$ x" 0.0056" 0.0072" 0.0718" x"
*Values in red have a P-Value > 0.05 and are thus not statistically significant
Table Set 8 (Water$at$21°C$w/$PBS$as$medium)$
$
P5Values$for$5%$Gelatin"
%$mTg$ 0$ 2$ 5$ 10$ 15$
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"
P5Values$for$10%$Gelatin"
%$mTg$ 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"
P5Values$for$15%$Gelatin"
%$mTg$ 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"
*Values in red have a P-Value > 0.05 and are thus not statistically significant