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Characteristics and properties of modified gelatin cross-linked with saline for tissue engineering applications

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Characteristics and properties of modified gelatin cross-linked with saline for tissue engineering applications

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Content CHARACTERISTICS AND PROPERTIES OF MODIFIED GELATIN
CROSS-LINKED WITH SILANE FOR TISSUE ENGINEERING
APPLICATIONS
Copyright 2004
by
BoSun Kwon
A Thesis Presented to the
FACULTY OF THE GRADUATE SCHOOL
UNIVERSITY OF SOUTHERN CALIFORNIA
In Partial Fulfillment of the
Requirements for the Degree
MASTER OF SCIENCE
(BIOMEDICAL ENGINEERING)
May 2004
BoSun Kwon
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UMI Number: 1422415
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ACKNOWLEDGEMENTS
I would like to express my gratitude for the support and knowledge devoted to me
by Professor Bo Han and Marcel E. Nimni. I would also like to express my
appreciation to Dr. Tzung K. Hsiai and Dr. Michael C.K. Khoo to help me as
committee members. Special thanks to my colleague, Jun Oh, Jaewoo Choi and
Sungwoo Kim. And, I appreciate to my roommate, Junkwan Lee. Finally, the author
would like to thank Seongln Yun, sincerely.
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TABLE OF CONTENTS
ACKNOWLEDGEMENTS.................... ii
LIST OF TABLES.................................................................................................. v
LIST OF FIGURES................................................................................................. vi
ABSTRACT.............................................................................................................. vii
CHAPTER
1. INTRODUCTION............................................................................................ 1
1.1. Natural Polymer.............................................................................. 1
1.1.1. Collagen as a natural Polymer........................................................ 1
1.1.2. Gelatin as a natural Polymer................................................. 2
1.2. Synthetic Polymer................................................................................... 3
1.2.1. Alternative of synthetic polymer...................................................... 3
1.2.2. Structure and reaction mechanism of silane................................... 4
2. MATERIALS AND METHODS................ 6
2.1. Preparation of samples through silanation............................................ 6
2.2. After curing and drying............................................................................ 6
2.3. Formation of modified gelatin................................................................. 7
2.3.1. Preparation of sponge...................................................................... 7
2.3.2. Preparation of film............................................................................ 7
2.3.3. Preparation of powder...................................................................... 7
2.4. Characterization.......................... 8
2.4.1. DSC and TGA................................................................................... 8
2.4.2. Swelling measurement (EWC)......................................................... 8
2.4.3. Mechanical test................................................................................ 9
2.4.4. Cell proliferation and cytotoxicity test............................................. 9
2.4.5. Biodegradation test .................................................. 9
2.4.6. FT-IR analysis............................ 10
3. RESULTS......................... -.......... 11
3.1. Thermal analysis............................................ 11
3.1.1. DSC measurement......................................... 11
3.1.2. TGA messurement .................................................................... 14
3.2. Physical analysis................................. 15
3.2.1. Swelling test.................. 16
3.2.2. Structure image of sponge with spectroscopy............................... 18
iii
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3.3. Mechanical analysis. ............................................................................ 19
3.4. Biological analysis................................................ 22
3.4.1. Cell attachment and proliferation.......................... 22
3.4.2. Biodegradation test..................................................................... 23
3.5. FT-IR analysis.......................................................................................... 26
4. DISCUSSION AND CONCLUTION............................................................. 29
BIBLIOGRAPHY............................... 33
iv
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LIST OF TABLES
TABLE
3.1 The data of thermal properties...................................................................... 11
3.2 The data from the mechanical test............................................................... 20
3.3 FTIR frequencies of native and modified gelatin........................................... 30
v
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LIST OF FIGURES
FIGURE
1.1 The structure of gelatin................................................................................. 3
1.2 Reaction mechanism of silane and gelatin................................................. 4
3.1 DSC curves of native and modified gelatins................................................ 14
3.2 TGA curves of native gelatin and modified gelatin...................................... 15
3.3 EWC curves according to time function....................................................... 17
3.4 Spectroscopy image of sponge of native and modified gelatin in
aqueous conditions....................................................................................... 18
3.5 External view of native and modified gelatin films....................................... 19
3.6 Tensile properties of native gelatin and modified gelatins.......................... 21
3.7 Image of Spectroscopy for cell growth......................................................... 22
3.8 Cell growth in plain gelatin and modified gelatin......................................... 23
3.9 Biodegradation test with sircol assay in a pepsin........................................ 25
3.10 FT-IR spectra................................................................................................. 28
4.1 Cell Grow pattern for cell proliferation after 10 day..................................... 31
4.2 Surface structure from optical spectroscopy................................................ 32
vi
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ABSTRACT
With the radical development in tissue engineering and biomaterials, the chemically
and biologically modified gelatin with proposed scaffolds is in need of the repair,
reconstruction and regeneration of human living organs and tissues. With the
respect of this compelling need, the objective of present work is to establish the
stable cross-linking conditions and to create optimized materials on silane modified
gelatin. Silane as a crosslinker organizes an interpenetrating polymer network on
gelatin. Samples that were building up strong hydrogen bonds and siloxane bond
were formed as hydrogel, film and sponge through diverse experimental conditions;
pH, the ratio G/S and after-curing. In result, film and sponge of modified gelatin
showed more physical resistance to maintain their shapes. In cyto-biocompatibility
test, this cross-linked gelatin with silane validated biocompatible property and
evidence as suitable medium for promoting cell adhesion and proliferation. In
addition, the stiff curve of tensile properties of gelatin-modified film (thickness:
10-130 //m) obtained from mechanical test demonstrated strong interconnected
bond, as maintaining the elongation at break of native gelatin. The data from DSC,
TGA, and FT-IR confirmed the degree of silane-mediated cross-linking.
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1. Introduction
1.1. Natural Polymer
Natural polymers provide the products which could exclude toxicity and
biocompatibility problems. They are available substances on which cells can be
adhered and proliferated readily. On the other hand, rapid dissolution in aqueous
and atmospheric environments limits practical applications in tissue engineering and
lacks proper mechanical and physical properties.[15,22,25] For compelling need
able to overcome these problems, many studies were attempted to improve
characteristics of natural polymer like collagen, gelatin, etc, as using cross-linker.[7]
1.1.1. Collagen as a natural polymer
Collagen, one of the abundant proteins makes up 30% of all proteins in
human body, and is actively processed as biomaterial in many practical
applications; transplantation of organs and tissue, surgical construction, use of
mechanical devices, or supplementation of metabolic products.[16] Collagen
molecules are responsible for a structural scaffold to other tissue and assembled
functionally as bonds, cartilage, skin and tendon. In addition, the physiochemical
and biological properties of collagen[3] such as low inflammatory and cytotocixity
response[3] , haemostatic properties[12] and promotion of cellular growth[5] have
provided great advantages in tissue engineering application. However, its’
1
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instability in vivo and in vitro limits its application and problems in reaction control
generated when modifying collagen introduce denatured form of collagen, gelatin.
1.1.2. Gelatin as a natural polymer
Recently, the use of gelatin as a polymer has been reported in the various
fields: pharmaceutical products[6], drug delivery release systems[4,6,9,23,] wound
closure care[3], framework for physiological organ and tissue[8], etc. The great
interest in gelatin was originated in the fact that this natural polymer permits the
biocompatible properties and provides highly reactive functional sites like hydroxyl
group and amide group which can be reacted with coupling agents. Also, this is
the optimal material able to be manipulated as hydrogel[9,11], film[24,26] and
sponge[8] for the diverse application purposes in tissue engineering. On the other
hand, the intrinsic hydrophobic property of these polymers restricts their application
as cell colonizing materials.[28] [Fig. 1-1] The structure of gelatin as a natural
material is unstable and dissolved at room temperature and in water solution as
pointed out above.[4,7,14,] In order to make up for this structural deficiency of
gelatin, chemical cross-linking procedures giving rise to the formation of structural
stability have been considered.
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v v v li ,1 )4 - c
i
O f,
t t >->
» .* /
v -
r
8~— *
r
" " 1
[Figure 1-1] The structure of gelatin. A typical structure is -Ala-Gly-Pr-Gly-Glu-4Hyp-Gly-Pro-. From
www.isbu.ac.uk
1.2. Synthetic Polymer
New need of biomaterials have made novel synthetic polymer which has
applicable biocompatible scaffold. Even if various synthetic polymers provide
excellent mechanical and physical properties, cytotoxicity restricts their applications
in vivo. For example, the use of formaldehyde and glutaldehyde as a cross-linker
can induce the toxicity, if the residual of cross linkers is not removed completely. [6]
Also, these lack biological information as impregnated into macro-biomolecules. In
order to overcome these adverse aspects and find alternative, various cross-linking
agents such as genioin[6], chitosan, sugar[23], propunoicdirydrazide[29], etc have
been studied.
3
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1.2.1. Alternative of synthetic polymer
In this respect, introducing silane coupling agents as cross-linker and
developing the properties of modified materials with this material has been focus on
this study. In addition to its good physical properties and inert chemical function.
Biocompatibility of silicone material has long been proved as medical device and
transplantation.[26]
1.2.2. Structure and reaction mechanism of silane
RSiCQCH,)*
D.
RSi(OHi«
D.
2RSi(OCH,)«
3CHs OH
2'HaO
H O -S i- O -S i- O -S i- C H
OH Ah OH
OH ^
a ist
R
I
H O - S i - O - S i - O - S i - O H
\ A ,
■tSSSSfi—
H O -Si-O — S j-O -S i-O H
[Figure 1-2] Reaction Mechanism of Silane and Gelatin .A: Hydrolysis, B: Condensation,
C: Mixing Gelatin and Silane D: Hydrogen bonding, E: Expected structure [4]
The alkoxy silane has two properties based on functional groups; organic
functional and inorganic functional group.[4, 8] [Fig. 1-2] The general formula is
4
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RnS!X(4-n )
The inorganic alkoxy functional group ‘X’ [ex: methoxy, ethoxy, etc] provides the
linkage with inorganic substrates and react with the hydroxyl group. In the reaction
of silane, hydrolysis occurs in the alkoxy group. This produces silanol and proceeds
self-condensation. The tendency toward self-condensation can be controlled by
using distilled water and by selecting carefully pH range, 4-6. Finally, each silicone
of organosilane is oriented toward hydroxyl sites on collagen peptide based upon
hydrogen bond as linkage with the substrate (gelatin). During curing, a covalent
bond is formed with substrates and liberates the water. On the other hand, ‘R’ is the
non-hydrolyzable organic functional group [ex: vinyl, chloro, amino, etc] which
provides the organic compatibility and produces a covalent bond with organic
material. Possessing organic groups in silane coupling agents seems to provide
sufficient medium with gelatin to cell and living tissue. However, residual silane
materials and unexpected byproducts can conduct side effects like toxicity. In order
to remove the presence of this thermal treatment and dialysis has to be executed.
The purpose of the present study shows characteristics and properties of modified
gelatin and finds the optimized material before this new synthetic polymer applies to
tissue or medical therapeutics.
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2. Materials and Methods
2.1. Preparation of samples through Silanation
The silanes were used z-6040(3-methacryloxypropyltrimethoxysilane), z-
6040(3-glycidoxypropyltrimethoxysilane), and z-6094 (aminoethylaminopropylric
methoxysilane) from Dow Corning. The pure gelatin was purchased from Aldrich-
SIGMA(from calf bone, -225 Bloom). All materials were immersed in adjusted water
to pH 4.5, 5.0 or 5.5 with 1N HCI and 1N NaCl. First, silane which has
concentration, 5%, 10%, and 20 % (v/v) was sufficiently stirred in the pH adjusted-
water in order to complete hydrolysis and self-condensation at ambient condition for
around 1 hour. Gelatin solution of 5% or 10% (w/v) was dissolved in adjusted water
at 601 for 3 min. Gelatin was subsequently modified with silane with the
concentration ratio of 1:1, 1:2, and 1:4. The mixture was mechanically stirred in
order to hydrolyze and crosslink two compounds at 60-65° C for 10min. In the
procedure of preparation, the change of pH was detected.
2.2. After curing and drying
The resulting solutions were treated by three methods for curing and drying;
(1); at over 100 °C for 10min, (2); at 60-65 ° C for 10min and (3); at ambient
condition for 24 hours. At the end of the reaction, each solution was dialyzed for 72
hours against water to get rid of untreated chemicals. These were maintained in
clean and sterilized room before applying them to various shapes.
6
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2.3. Formation of modified-gelatin
2.3.1. Preparation of sponge
Each sample was poured into 15ml polystyrene tube to 8ml. The gel-typed
samples were frozen for forming the sponge in the freezer-dryer for 1 day. After the
reaction was completed, the tubes were removed and sponges were lyophilized out.
They were maintained in a desiccator until use.
2.3.2. Preparation of film
After curing the solution, the solutions were cooled rapidly with ice for 20
min, in order to form gel. Gel-typed samples which had high viscosity were
pressed with same pressure in the plastic sheet and maintained in the refrigerator at
4°C for 2 hours. After they were sufficiently dried in the ambient condition, the
sheet of each film was manipulated to suitable shape for mechanical, thermal, and
FT-IR analyses.
2.3.3. Preparation of powder
5 g of film of modified gelatin was promptly frozen with nitrogen oxide and
grinded with an impactor in the freezer-mill (spex). After 5 min, they was sieved with
using sieve with opening micrometer (<75 //m) and maintained at 35 °C until use.
7
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2.4. Characterization
2.4.1. DSC and TGA analysis
The analysis of thermal properties in cross-linked gelatin with silane were
conducted by using TA Instruments DSC 2920 (differential scanning calorimeter)
and TGA2050 (thermo-gravimetric analysis). For DSC, Preweighted films were
hermetically sealed in aluminum TA pans and scanned at the heating rate of
10 °C/min under inert gas (N2) flow (65ml/min). The reference was an empty pan.
The glass transition temperature (Tg ) and melting temperature (Tm ) of each film was
measured in temperature range from 20 to 300 °C. For TGA, sponges of samples
were prepared and put in aluminum pan and scanned at heat rate of 20 °C/min
under inert gas (N2 ) flow (65ml/min).
2.4.2. Swelling test (Equilibrium water contents)
The preweighted film samples, W0, were dipped in distilled water adjusted
at pH 7.0 in order to measure equilibrium water contents (EWC). After water on
the surface of film due to water transport was removed, the increased weight of
swollen film, Ws, was recorded at various time intervals. [7, 9] The degree of
swollenness can be calculated by curves of water uptake as a function of time
according to following equation.
EWC(%)=((M4 - W0 )/ Ws ) X 100
8
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2.4.3. Mechanical test
Mechanical properties were measured using of MTS 858 Mini Bionix with
force transducer (Interface Company) at rate of loading 5mm/sec, tendon moment
arm of 35mm and ulcer angle of 30 degrees. Type os loading was pull-out and
fixation type was 8 x 95 mm screw. Measurement was at room temperature with
film of each sample, 10mm wide, 60mm long, and thickness of each sample.[26]
Three replicates was tested for each film and average values of elongation at break,
and tensile strength, and modulus were determined. [20]
2.4.4. Cell proliferation and Cytotoxicity Test
Fifty milliliters of plain gelatin or silanated gelatin were pipetted to the bottom
of 24-well plate and air was dried for 24 hours. Before plating cells, wells were
washed with PBS twice. 5x104 human fibroblast was plated in a 24-well plate with
the supplement of DMEM medium (Cellgro) and 10% fetal bovine serum. Cells were
incubated at 37 °C with 5% CO2 incubator. Medium was changed every 2 days.
Cells were counted 78-hours later.
2.4.5. Biodegradation test
Degradation of silanated gelatin was performed using sircol collagen assay
(Sircol Dye Reagent, Alkali reagent, biocolor Ltd.). In degradation of test film of native
gelatin was used as a control [7], Each film was immersed in 1ml pepsin and
9
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incubated at 37 ° C for the desired duration. 100 pi of each solution was taken into
microcentrifuge tube adjusted with 100 pi of 0.5 M acetic acid. After 1ml of Sircol
dye reagent was added into this tube, they were mixed in the shaker for 30 minutes.
During this time period, the dye reagent bound specifically to collagen and collagen-
dye complex was precipitated. Micro centrifuge was used at 6000 rpm for 5minutes
in order to pack the collagen-dye complex at the bottom of the tubes.
Subsequently, the unbound dye solution was removed and 1ml of alkali reagent was
added to each tube in order to solve the collagen-dye complex. After 5min, the
concentration of collagen-dye complex was examined in OD5 5 0 using multi-well plate
reader {Kinetic Macroplate reader, UV Max) for measuring the biodegradation property
in each sample.
2.4.6. FT-IR analysis of modified gelatin
Fourier transform infrared (FT-IR) spectra were acquired on Thermo Nicolet,
370 FT-IR, Avastar in order to confirm the cross-linking between gelatin and silane.
To obtain the data, the film with thickness (< 40 pm ) of each sample was used.
The resolution was ranged from 400 to 4000 cm'1 .
10
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3. Results
3.1. Thermal Properties
3.1.1. DSC measurement
DSC was used to access the thermal behavior and structural change in the
native and cross-liked gelatin [23]. The coupling agent, silane, allowed totally different
physical properties to modified gelatin.[Table3-1] Also, figure 3-1 shows the results
of thermal behavior according to G/S ratios, pH and after curing.
Type of
sample
Condition of
Reaction
Tm
VC]
Tg
VC]
H
(mJ/mg)
Gelatin 5%(w/v) at pH 5.0 70.75 222.02 312.3
Sponge
After curing (1) 118.19 235.65 106.7
(g/s=1:2
After curing (2) 60.63 216.27 50.67
at pH 5.0) After curing (3) 75.80 220.57 63.38
g/s=1:1 at pH 5.0 112.81 212.82 238.09
g/s=1:2 at pH 5.0 110.90 220.08 244.60
Film
g/s=1:4 at pH 5.0 66.06 218.50 239.22
g/s=1:2 at pH 4.5 78.12 201.27 80.51
g/s=1:2 at pH 5.5 85.26 218.15 38.11
[Table 3-1] The data of thermal properties. The ramp is 10°C/min.
Thermal and physical tendency in gelatin was indicated as a reference in
this figure. The result according to pH change in DSC exhibited that the reaction
between gelatin and silane at pH 5.0 was completed better than that in other pH, 4.5
and 5.5 because Tm , Tg, and H of the former was higher than the others.[Fig2-2
11
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(a)] Through the figure 2-2 (b), the influence of the ratios of gelatin and silane in
the reaction of both was explained. Although the peaks of Tm in the films were
moved to left according to contents of silane in modified gelatin, the values of AH
were largely dissipated at the film with the ratio (G/S=1:2). Also, glass trasition
temperature was appeared at the highest value among of samples with the same
ratio. Figure 3-2 (c) shows DSC curves relative to after-curing in sponge typed
samples. The values of Tm _ Tg and H were 118.19, 235.65°C and 106.7mJ/mg,
respectively. These values were higher than that of samples treated by other after
curing. It explained the reaction in gelatin and silane were completed well and
strong bonds in modified gelatin were interconnected at after-curing (1).
Moreover, the glass transition was observed in figure 3-1 and Table 1-1. Tg of
native gelatin occurred at 222.02 °C . Normally, gelatin dried commercially exhibits
intermolecular micro-crystalline junctions, but heating over this temperature, Tg
induces phase transition. [20] In case of modified gelatin with the ratio(G/S=1/2)
treated at pH 5.0 and after curing (1), the different values of Tm > Tg and H
comparing to others could be suggested basic property like phase transition, melting
temperature, and meting enthalpy was formed to optimized resultant.
Consequently, this modified gelatin was chosen as an optimal material in this
experiment from DSC data. Thus, this modified gelatin reacted with silane shows
stable behavior and has strong cross-linked structure on the thermal change.
12
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H e a t F lo w [mW/mg] 05
a)
o
-2
■ 4
Pure Gelatin
G elatin:Silane=1:2 at pH 4.5
G elatin:Silane=1:2 at pH 5.0
Gelatin:Silane=1:2 at pH 5.5
■ 6
50 100 150 200 250
Temperature [°C ]
o.o -
-0.5 -
- 2.0 -
Pure Gelatin
Ge!atin:Silane=1
Gelatin:Silane=1
Gelatin:Silane=1
-2.5 -
-3.0
50 100 150 200 250
Temperature [°C ]
13
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c)
- 0.2
— After-curing (1)
After-curing (2)
— — — After-curing (3)
-0.4 -
05
£ - 0.6 -
§
- 0 .3 -
5
O
HI
'S
a »
X
50 100 150 200 250
Temperature [°C ]
[Figure 3-1] DSC curves of native and modified gelatins. This was plotted a) according to pH at
the ratio [G/S=1:2], b) according to the ratios at pH=5.0, c) according to after-curing in sponge
at same conditions; the ratio [G/S=1/2] and pH=5.0. The rate of ramp is 10 C/min
3.1.2. TGA Measurement
The figure 3-2 shows the weight loss as increasing the temperature. TGA
curves explained the degree of stability of the materials. The rate of weight loss
according to temperature was decreased in the film conditioned with the ratio
(G/S=1:2) at pH 5.0 after curing (3) explained this modified gelatin was stable in
structural change than native gelatin. In addition to DSC curves, this TGA curves
confirmed the sample chosen from DSC shows stable structure.
14
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1 00
80 -
m
w
o
60 -
JC
0)
'o >
5
—— Pure Gelatin
Gelatin:Silane=1:2
— — Gelatin:Silane=2:1
40 -
300 400 500 600 100 200 0
Temperature [ °C ]
[Figure 3-2] TGA curves of native gelatin and modified gelatin. The samples were treated
at the same pH, 5.0. The rate of ramp is 20°C/min.
3.2. Physical analysis
In the preliminary physical experiment, each sponge and film was boiled
until 90 °C in the distilled water adjusted to pH 5.5. In this condition, only gelatin
was melted, while others had kept their shape continuously. As reported table 3-2,
most of modified gelatin showed unchanged shape but the gelatin was completely
dissolved in extreme pH condition; 1.3 and 13.8, respectively. In these conditions,
the sponge of modified gelatin got just torn like fiber after 10 day. Modified gelatin
with silane proved stable and strong cross-linked structure than that of native gelatin
dissolved completely. In order to confirm this fact, the swelling test was performed
and the image of spectroscopy was referred.
15
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3.2.1. Swelling Test
Figure 3.3 shows the dependence of the swelling behavior of native gelatin
and modified gelatins. They were plotted on the average of three films out of each
sample. In the Fig 3-3-a), the EWC of films containing silane, regardless of ratios
exhibited marginal change in the swelling rate, comparing to native gelatin which
was swelled and reached rapidly to equilibrium after 1day. The film treated at pH
5.0 was appeared at the lower swelling rate comparing to others.[Fig 3-3 a)] In
addition to DSC curves in terms of pH change, different value of EWC in each film
treated at different pH showed the reaction of silane is easily afffeced by pH
variation. The figure 3-3-b) explains the condition of pH5.0 s optimal environment for
reaction with silane. As the content of silane in the modified gelatin was increased,
the rate of the EWC was decreased. It’s estimated this property is caused by the
property of waterproof of silane, itself and dense interconnected network. The
decreased rate of swelling of modified gelatin containing silane prohibits dissolution
in aqueous and atmospheric environment differently from gelatin dissolved in same
conditions.
16
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■V
>7..
80 -
Gelatin:Silane=1:2 at pH 5.0
Gelatin:Silane=1:2 at pH 5.5
Gelatin:Silane=1:2 at pH 4.5
Pure Gelatin
60 -
O
40 -
20 -
0 1 6 2 3 4 5
Time [ Day ]
b)
£
o
8 0 -
Gelatin:Silane=1
Gelatin :Silane=1
Gelatin:Silane=1
Gelatin :Silane=2
Pure Gelatin
60 -
40 -
.. a?
20 -
0 1 2 3 4 5 6
Time [ Day ]
[Figure 3-3] EWC curves according to time function, a) according to pH, b) according the ratio
the same pH 5.0 with fixed ratio (G/S=1/2)
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3.2.2. Structure of sponge recorded with Spectroscopy
The spectroscopy gave us further evidence of cross-linking like shown in
figure 3-4. Gelatin’s organization was less dense than modified gelatin in the
spectroscopy image. Sponges of cross-linked gelatin with silane show a network of
interconnected fiber shapes. It might be explained this tangled external structure
like twine can provide more stable scaffold as medium for cell adhesion and
proliferation.[12]
[Figure 3-4] Spectroscopy image of sponge of native and modified gelatin in aqueous
conditions, a) pure gelatin, b) gelatin : silane = 1:2 at after cure (3).
18
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3.3. Mechanical Test
Figure 3.5 shows the shape of film in the modified gelatin and native gelatin.
There were no changes in extent of transparency between both. The optimized
gelatin folded was not broken like gelatin like shown in figure 3.5-b). After
modifying the gelatin with silane, the elastic property of native gelatin was maintained
in the modified gelatin.
[Fig 3-5] External view of native and modified gelatin films. Bar indicated 10mm.
These properties were confirmed through the mechanical test. Main tensile
properties; tensile strength and elongation at break, and Young’s modulus were
measured with the film of native gelatin and modified gelatin with the G/S ratio
19
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treated at pH 5.0.[Table 3-2, Figure 3-6] Tensile strength of modified gelatin was
increased 4 times higher than that of native gelatin shown in figure 3-6 (a), while the
elongation values at break between modified gelatin and native gelatin were 16.8
and 14.8 respectively.[Fig3-6 (b)] It demonstrated the elastic modulus of native
gelatin was transferred unchangeably to modified gelatin. In case of Young’s
Modulus which is the yardstick of the degree of rigidity shown in figure 3-6 (c), that
of modified gelatin was around 4.5 times than that of native gelatin. Also, as the
slope and was increased, both moduli demonstrated the modified gelatin was stiffer
than the native gelatin like shown figure 3-6 (d) in which each stress value of one
among of measured samples was plotted according to strain values. This
difference of this modulus was caused by the strong covalent bond of silane linked
with the substrate, gelatin.[20, 26]
Tensile Strength
[MPa]
Elongation at
break
Young’s
Modulus [MPa]
Native Gelatin 18.63±0.37 16.82±6.41 119.91+47.97
Modified Gelatin
(the ratio : g/s=1/2 at
pH 5.0 and after curing (1))
72.62± 10.73 14.8+4.73 534.89+202.74
[Table 3-2] The data from the mechanical test. The rate of loading is 5mm/min. each film size was 3
mm wide and 60 mm long. Data were calculated as means of the values of three films of each sample
20
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Y oung's M odulus [M Pa]
GMn G &&iS la& 42 G M n G£Etiri9lare=12
aoo-,
600-
400 -
200 -
G eia tin G ela tin :S Ia n ep 1 :2
160
140
1 2 0
M DdfiedGel^in with the ratio (gfe=#1)
& pH 5 0 a id r fte r a riix i (3)
• O - FueGeisfin
1 0 0
80
60
40
20
0
0.00 0.05 0.10 0.15 0.20
S tra in [(ItfVl*,]
[Figure 3.6] Tensile properties of native gelatin and modified gelatin with the ratio (g/s=2/1) at pH
5.0 and after curing (3) a) tensile strength, b) elongation at break, c) Young’s modulus, d) stress-
strain curve of one film in each sample, where the stiffer the slope, the more rigidity. The rate of
loading is 5mm/min. each film size was 3 mm wide and 60 mm long. Data were calculated as
means of the values of three films of each sample. Bar indicated the standard deviation.
21
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3.4. Biological analysis
3.4.1 Cytotoxicity and Cell proliferation and attachment
The results of the cell pattern and proliferation on the hydrogel typed
samples are shown in figure 3-7. Cells were attached firmly on the silanated
gelatin and spreaded with fixed pattern.[Fig. 3-7. b and d]. The cells on an image
of the modified gelatin gathered denser than plain gelatin [Fig 3-7 (a) and (c)].
Cells tended to align uniformly on the modified gelatin surface comparing to plain
gelatin where orientation of cells was more random. Figure 3-8 shows that cell
[Figure 3-7] Image of Spectroscopy for cell growth a) pure gelatin at 10x10. b) modified gelatin
at 20x10 (the ratio of silane and geltin (1:2)). c) pure gelatin at 20x10. d) modified gelatin at 20x10
(the ratio of silane and geltin (1:2))
22
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
proliferation w a s significantly enhanced on modified gelatin with ratio [G/S=1/2]
conditioned at pH5.0. However, modified gelatin with the ratio [G/S=1/4] leaded to
cell death. It was supposed that untreated silane materials were remained after
reaction in this sample. Silane as a coupling agent is very sensitive to pH change.
Modified gelatin with silane reacted at pH 5.5 hardly provided the stable medium for
cell growth. Also, these figures proved coincidently gelatin chemically modified with
silane was no toxicity to cells.
o
o
H I
a
£
3
z
m
O
25 -
20 -
15 -
10
Pure Gelatin
Gelatin: Silane:
Gelatin: Silane:
Gelatin: Silane:
1:2 at pH 5.0 and after cure 3)
1:2 at pH 5.5 and after cure 3)
1:4 at pH 5.0 and after cure 3)
2
Days
[Figure 3-8] Cell growth in plain gelatin and modified gelatin that has ratio of gelatin to
silane; 1:2[pH5.0, 5.5] and 1:4. Bar indicated the standard deviation.
23
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3.4.2. Biodegradation Test
The results of biodegradation test based on sircol assay are shown in figure
3-9 for a pepsin. The elements which were degraded in the pepsin, were died by
sircol dye reagent. The values recorded by a plate reader were plotted as a time
function. The biodegradation rate of modified gelatin was reached slowly to the
equilibrium than that of gelatin which degraded in 1 day. After 10 days, the rate of
degradation in native gelatin was 6 times faster than that modified gelatin.
However, the rate of degradation of modified gelatin demonstrated almost the same
values regardless of the ratio of gelatin/silane. As shown in the swelling test, It
was estimated silane interconnected strongly with the substrate, gelatin, interfered
the activity of pepsin. In the view of the degradation rate, modified gelatin is
suitable for the application in which the maintenance of structure and scaffolds is
demanded for a long term in vivo.
24
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550
12
10 -
8 6
2 -
Gelatin:Silane=1:2
.........n .................
Gelatin:Silane=1:4
“ -... T ....- ■ . Gelatin:Silane=2:1
-------- v ------- Gelatin coaded with Glutaldehyde
-------- .
Pure Gelatin
f
T
0
■m "'
~r
50 100 150
Time [ hour ]
200 250
2.5
2.0 -
o
in
in
Q
o
0.5 -
0.0 -
250 0 50 100 150 200
H ire [Hour]
[Figure 3-9] Biodegradation test with sircol assay in a pepsin. Data recorded by plate reader, b)
is enlarged from a). The value from OD550 was plotted as a time function.
25
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3.5. FTIR analysis
FT-IR tests were executed with the
film of each sample (< 40 //m) in order to find
Bond Vibrations Frequency(cm'1 )
evidence and reaction degree of silane-
N=H stretch 3340
modified cross-linking with gelatin. In Fig 3-
O-H stretch 3072
10, FT-IR spectra of pure gelatin and
C-H stretch 2931-2862
modified gelatins with various ratios between
C=0 stretch 1650
gelatin and silane were shown. The FT-IR
N-0 stretch 1540-1556
spectrum of native gelatin (a) was typical of
C-0 stretch 1454
a protein like collagen. The main C-Si stretch 1336 and 1238
characteristic absorbance bands for gelatin C-0 and Si-0 stretch 1180
are N-H, C=0, and N-0 modes, respectably,
C=C stretch 900
at 3320, 1650, and 1540 cm'1 . The line of a)
[Table 3-3] FTIR frequencies of native and
and b) were almost same absorbance modified gelatin.
peaks except for Si-0 bond vibration peak. The peak of (b) explained the gelatin
dominated this sample with ratio [G/S=2/1], In figure 3-10 (c), (d), (e), new additional
peaks were located at 1337 and 1127 cm'1 which indicated C-Si and Si-O. These
peaks proved the stretching vibration of and C-Si and Si-0 which provides rigid
evidence of the cross-linking of gelatin. Comparing c), d), and e) lines, while the
area of C=0, N-H, O-H and N-0 stretch vibration was narrower and lower, Si-0 and
26
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C-Si bond vibration peaks was higher, respectively. Also, this demonstrated the
reaction between gelatin and silane was completed well with hydrogen bond
covalent bond, after bonds of C=0 and N-0 were broken. However, the size of the
C-0 peak at 1100 cm'1 of e) can be never overlooked, because it might be
summarized as a presence of untreated silane functional groups.[9,19,20,27]
27
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A a / V \
[Figure 3.10] FTIR spectra: a) pure gelatin, b) Modified gelatin with the ratio between gelatin and
silane b) 2:1, c) 1:1, d) 1:2, e) 1:4.
28
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4. Discussion and Conclusion
Various fabrication processes for the reaction between gelatin and silane
were reported including self-condensation of silane, sol-gel method and absorption
method [2,20,27,5,19] as a previous step for application to tissue engineering. In
order to find the optimal experiment conditions, change of pH, various ratio [G/S]
and three after-curing were used in the preparation of the solution of gelatin mixed
with silane. Change of pH among of these conditions tranformed largely the
properties of the modified gelatin verified from the DSC data. In figure 3-1 (a), the
peak of Tg at pH 4.5, 5.0 and 5.5, respectively, followed by endothermic heat flow
was 220.08, 201.27 and 218.15°C, . Although Tg of the film treated at pH 5.0 was
very close to that treated at pH 5.5, Tm and melting enthalpy of the former was
higher than that of the latter. The difference between each Tg and AH of both could
meant that change of pH affected either the melting temperature or the amount of
enthalpy and was estimated that the sample treated at pH 5.0 was strongly cross-
linked. Also, in Fig 3-1 b) and c), it might be concluded that the curing time as well
as the ratio [G/S] of the modified gelatin influenced Tg, Tm and AH and formed cross-
linked interpenetrating polymer network with improved properties [4], Where, the
film with the ratio [g/s=1/2] reacted at pH 5.0 and curing (3) exhibited good thermal
results than others. Also, TGA curves [Fig. 3-2] in addition to other test supported
this fact. Thus, the sample with these conditions was chosen as the optimized.
29
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The evidence of cross-linking was confirmed with the analysis of FT-IR
spectra. When comparing to Fig. 3-8 a) and b) dominated by gelatin’s properties,
the interchanged network between gelatin and silane was validated from the new
additional peak at 1336 and 1238 cm"1 indicating C-Si and Si-0 bond vibration. As
the contents of silane in modified gelatin was increased, the values of peaks of
stretching vibrations of N=H, O-H, and N-0 were decreased and peaks of stretching
vibrations, Si-0 and C-S were increased as a rigid evidence. However, the peak
1100 cm'1 from C-0 bond vibration might be from the presence of untreated silane
compound and unexpected products generating toxicity. Thus, after completing the
reaction, the course of sterilization was needed in order to remove the toxicity.
Biocompatibility studies were performed coincidently. Although the
physiochemical properties of synthetic polymer showed good results in vitro, it can’t
be verified as a biopolymer with biocompatibility in vivo. The cell growth on the
surface coated with hydrogel of each sample was observed by spectroscopy. After
10 days of incubation, cells grown on optimized G/S substrate reached higher level
than pure gelatin and the control.[Fig 4-1] It was remarked that optimized sample
provided not only non-cytotoxicity, but also scaffolds as a medium for cell
attachment and proliferation. The images of the surface morphology in optimized
sample explained this status.[Fig 4-2] It might be estimated from the morphology of
modified gelatin that lots of hole and valleys in the image of this sample leaded cell
patterning and proliferation. In biodegradation test, the optimized sample was
degraded with slow rate of degradation in a pepsin. Also, EWC from swelling test
30
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demonstrated non-soluble network formation was conducted in the reaction
between silane and gelatin, regardless of contents of silane. Through the controlling
the long active biodegradation ability and maintenance of the shape in aqueous
environment, this material can be applied for drug delivery system, wound closure
care, pharmaceutical products pharmaceutical products and repair of damaged
tissue or organs
*
[Figure 4-1] Cell grow pattern. After 10 day, cell was proliferated in each sample, a) control, b)
native gelatin, c) modified gelatin treated with the ratio (g/s1/2) and at pH 5.0
31
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[Fig 4-2] Surface structure from optical spectroscopy a) control, b) modified gelatin treated with
the ratio [G/S=1/2] and at pH 5.0
The strength of cross-linked interpenetrating network was reported from mechanical
test. The optimized sample maintaining the elongation at break almost same to
gelatin showed the higher tensile strength and Young’s modulus. This property
allows the range of application to extend to various fields in tissue engineering; the
scaffold cultivating growth factor, anchored bio-implant for repair of damaged
neuron, regeneration of cartilage connected with bond.
32
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BIBLIOGRAPHY
[1] Burmania, Jeanine A., Gabriel J. Martinez-Diaz, Weiyuan John Kao, “Synthesis
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Asset Metadata

Creator Kwon, Bosun (author)

Core Title Characteristics and properties of modified gelatin cross-linked with saline for tissue engineering applications

School Graduate School

Degree Master of Science

Degree Program Biomedical Engineering

Publisher University of Southern California (original), University of Southern California. Libraries (digital)

Tag engineering, biomedical,OAI-PMH Harvest

Language English

Contributor Digitized by ProQuest (provenance)

Advisor Han, Bo (committee chair), Nimni, Marcel E. (committee chair), Hsiai, Tzung K. (committee member), Khoo, Michael C.K. (committee member)

Permanent Link (DOI) https://doi.org/10.25549/usctheses-c16-318821

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Access Conditions The author retains rights to his/her dissertation, thesis or other graduate work according to U.S. copyright law. Electronic access is being provided by the USC Libraries in agreement with the au...

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