Collagen Chitosan Scaffold Composition Variation with Addition of Lauric Acid

Plasticized for Skin Tissue Engineering on Burn Cases

Ewing Dian Setyadi1, Prihartini Widiyanti2,3, Djoni Izak Rudyarjo4

1
Bachelor of Biomedical Engineering Study Program, Faculty of Science and

Technology, Universitas Airlangga, Indonesia

2
Biomedical Engineering Study Program, Faculty of Science and Technology,

Universitas Airlangga, Indonesia

3
Institute of Tropical Disease, Universitas Airlangga, Indonesia
4
Physics Study Program, Faculty of Science and Technology, Universitas

Airlangga, Indonesia

ABSTRACT

The prevalence of burns in the world is more than 800 cases per one million people

each year and this is the second highest cause of death due to trauma after traffic

accident. Many studies are turning to skin subtitute methods of tissue engineering.

The purpose of this study is to determine the composition of the collagen, chitosan,

and lauric acid scaffold, as well as knowing the results of the characterization of the

scaffold. The synthesis of chitosan collagen lauric acid scaffold as a skin tissue

engineered by using freeze dried method. Results from making manufacture of

collagen chitosan lauric acid scaffold, was characterized mechanically, chemically

and biologically by FTIR, SEM, cytotoxicity, biodegradation, tensile strength. The

results of FTIR showed physical interaction indicated by decrease in absorption

wave number in the amide I when adding chitosan. Interactions between collagen-

chitosan with lauric acid plasticizer are indicated by the wave number of ester groups

C = O where the C = O indicates carboxylic acid groups. From the morphology test,

the result obtained is that pore diameter size ranges from 94.11 to 140.1 μm for

samples A, B, C, D, which are the range of normal pore size scaffold 63-150 μm,

while sample E has value below the standard which is about 37.87 to 47.36 µm.

From oxytotoxicity assay, the result obtained is the percentage value of living cells

between 20.11 to 21.51%. This value is below 50% the standard value of living cells.

Incompatibility is made possible because of human error mainly the replication of

washing process over the standard. Degradation testing obtained a value of 19.44%

-40% by weight which is degraded during the 7 days of observation. Tensile test

obtained a range of values of 0.192 MPa - 3.53 MPa. Only sample A (3.53 MPa) and

B (1.935 MPa) meets the standard values of skin tissue scaffold that is 1-24 MPa.

Based on the results of the characteristics of this study, composite chitosan collagen

scaffold lauric acid has a potential candidate for skin tissue engineering for skin

burns cases.

Keywords: scaffold, collagen, chitosan, lauric acid, skin tissue engineering

INTRODUCTION

Burns are a common cause of trauma and can result in relatively high amount

of morbidity and mortality compared to other causes of injury. The incidence of burns

in the world amounts to more than 800 cases per one million people each year and

is the second highest cause of death due to trauma after traffic accidents

(Hettiaratchy and Dziewulski, 2004). The burn will cause damage to various organs,
including the skin. On degree III burns, very deep wounds occur which damage

organs beneath the skin such as muscles, nerves, bones. There is no process of

spontaneous healing in which the proliferation of the epithelial layer (reepithelization)

on the edge of the wound does not occur (Moenadjat, 2001). Thus, a healing method

for handling cases of third-degree burns is required.

In recent decades, several methods of skin substitution such as xenografts,

allografts, and autografts have been used for wound healing. However, because the

ability of a substance or antigen to induce an immune response (antigenicity) is high

or there is limited donor tissue, skin replacement methods mentioned above cannot

be used widely (Boyce, 2011 and Schul, 2005). Therefore, there have been more

research conducted to approach tissue engineering. One important factor

in engineering skin tissue is the construction of scaffold. In a three-dimensional

scaffold, there is an extracellular matrix that serves as a template which is needed

for host infiltration and physical support to guide differentiation and proliferation of

cells into targeted functional tissues (Humatcher, 2001 and Si-Nae Park,

2002). Scaffold made of collagen has been applied in many applications because it

has useful properties, such as homeostatic effect and low antigenicity for the

characteristic use in tissue engineering applications (O'Brien et al., 2005). However,

the quick nature biodegradation on collagen becomes a problem that may limit the

use of these materials (Ma, L et al., 2003), so the scaffold of mixed collagen and a

biodegradable polymer have been used for the manufacture of the scaffold. One of

such efforts is by mixing collagen with chitosan. Chitosan has a high potential

in tissue engineering applications. One of the effects of chitosan in wound healing or

wound healing is the formation of granulation tissue with angiogenesis, inducing

fibroblasts to release interleukins, which are involved in migration and fibroblast

proliferation (Wang, 2003).

In 2006, Tangsadthakun, et al. made use of scaffold material of collagen and

chitosan with collagen composition variation chitosan (1: 9, 3: 7, 1: 1, 7: 3, 9: 1) for

skin tissue engineering. In the study, the result is unsatisfactory in its mechanical

testing; thus, in this experiment, lauric acid plasticizer was aded which is expected to

improve its mechanical properties. Characteristics of chitosan-collagen scaffold-

lauric acid test are determined by FTIR, SEM test, cytotoxicity test, biodegradation,

and tensile strength tests.

RESEARCH METHOD

Materials

Materials used in this study are collagen from BATAN products, chitosan from

Biochitosan Indonesia products, lauric acid from Shindo, Inc., acetic acid, simulated

body fluid (SBF), and distilled water.

Method

The preparation process for this first sample scaffold was initiated by

dissolving 1 gram of chitosan in 100 ml 0.5M acetic acid (1% (w / v)). Collagen was

dissolved until it became homogeneous with a magnetic stirrer for approximately 6

hours. Then, a chitosan solution was made by dissolving 1 gram of chitosan in 100

ml 0.5M acetic acid (1% (w / v)). Chitosan was dissolved until it became

homogenous with a magnetic stirrer for approximately 3 hours. Afterwards, the

manufacture of lauric acid solution was made by dissolving 1 gram of lauric acid into

100ml of ethanol 96% (1% (w / v)). Lauric acid was dissolved until it became
homogeneous with a magnetic stirrer at a temperature of 44 o C. Next, 5 samples

were created for variations in the composition of collagen: chitosan with a

composition ratio of 1: 9, 3: 7, 1: 1, 7: 3: 9: 1. Then, each sample was dissolved

(in stirrer) with lauric acid solution with a magnetic stirrer until it became

homogeneous. Then, the homogeneous solution was frozen at -40 ° C for 24 hours

and then put in freeze drying for 7-8 hours to produce a porous material.

Fourier Transform Infrared (FTIR) Test

Collagen-chitosan-lauric acid scaffold was crushed and cut small along with

KBr powder in a mortar. Then the sample pieces and KBr powder that was mixed

were inserted into the FTIR testing machine. FTIR analysis was performed using the

Shimadzu FTIR, 4000.

Scanning Electron Microscope (SEM) Test

The morphology of the scaffold collagen-chitosan-lauric acid were analyzed

using SEM (Inspect S50.FEI Corp., Japan) previously cut with a size of 12-25 mm,

and then given gold plating. To stick it, a double-sided tape is required.

Cytotoxicity Test

Preparation of fibroblast cell culture was performed in a laminar flow. This

MTT test used BHK-21 cell culture in the form of monolayer with Eagle medium and

5% FBS grown in culture bottles roux that was then treated in incubation at 37C for

48 hours. Cell cultures were incubated for 48 hours and were washed with PBS 5

times. Cells with a density of 2x 105 were included in 100 μL medial (medium

Eagle's 86%, penicillin streptomycin 1% Fungizone 100 units / mL), and then

transferred into 96-microwell plate. Each sample was sterilized with UV light for
longer than overnight, dissolving 0.05 grams of the sample in 1 ml of ethanol. 50 μL

of the sample solution was then inserted in a 96-microwell plate , and 24-hour

incubation was carried out at a temperature of 37C. The reaction of MTT of 5 mg / ml

was then diluted in PBS and added to the media as much as 10 μL for

each well, and then incubated for 4 hours in a temperature of 37 ° C. The DMSO

solvent was added to each well of 50 μL and then centrifuged for 5 minutes at 30

rpm. After the centrifuge process, the optical density of the cell could be calculated

by using a tool commonly called Elisa Reader.

Biodegradation Test

1 Liter of distilled water was mixed with the composition of Simulated Body

Fluid (SBF) of K 2 HPO 4 .3H 2 O, CaCl 2 .2H 2 O, NaCl,

NaHCO 3, Na 2 SO 4, KCl, HCl, MgCl 2 .6H 2 O, and (HOCH 2) 3CNH 2. The

temperature of the solution was adjusted to 36.5 ° C and the pH was adjusted to pH

7 with a solution of HCl 1 M. The scaffold was cut measuring 1 cm x 1 cm and put

into plastic bottle, and then added with 3 ml of SBF solution. The bottle was then

sealed. Afterwards, the bottles were stored at 37 ° C for 7 days for observation.

Tensile Strength test

The scaffold was cut into the shape of a standard specimen for tensile

strength test. Then, the thickness of the scaffold was measured with a digital

micrometer. The tip of the membrane attached to the test equipment and towing load

is mounted on the load unit of kgf. The scaffold was then drawn at a certain speed to

drop out. The tools used for the tensile test were in the form of the autograph Imada

HV-500NII.
RESULTS AND DISCUSSION

Fourier Transform Infrared (FTIR) Test

(a) (b)

71.4 71.5

65 65
826,64 1467,65
60 1467,62 60 1383,63
1382,60 502,60 3752,60 502,61
1305,62 1226,58
55 55
1637,55 1226,57 599,56 1637,57 600,57
%T 559,54 %T
50 50 560,53
2851,49 2851,51
45 45 1029,40
2920,45 2920,47
1027,44
40 40
3379,40 3429,39
35.0 35.0
4000.0 3000 2000 1500 1000 450.0 4000.0 3000 2000 1500 1000 450.0
cm-1 cm-1

(c) (d)

(e)
Figure 1. IR spectrum from collagen/chitosan/lauric acid from scaffold with the

corresponding composition of (a)1/9/2, (b)3/7/2, (c)5/5/2, (d)7/3/2 dan (e)9/1/2

-1
Chitosan typical absorption peaks at 3379.4 to 3448.84 cm wave number

which is a hydroxyl group (OH). Aliphatic group (-CH 2 and -CH 3) is located in the
-1.
absorption wave number 2920.37 to 2956.32 cm Absorption peak wave number

1653.05 to 1654.98 cm -1 has a functional group C = O stretching. The absorption

-1
wave number 1543.10 to 1560.46 cm is -NH 2 bending. The presence of

CO stretching group of primary alcohol groups are shown in the absorption wave

number 1458.23 to 1471.74 cm -1.. The absorption from 1140.86 to 1151.5 cm -

1
wave number is -COC- glycosidic connection between chitosan monomer.

Typical absorption peak of collagen is located in the absorption wave number

-1
3421.83 to 3448.84 cm which is a hydroxyl group (OH). In the absorption wave

number 1653.05 to 1654.98 cm -1 Amide I. Amide II are shown at the absorption

wave number 1543.10 to 1560.46 cm -1.

Lauric acid absorption spectrum has the characteristics of an alkyl group (CH)

that appears at wave number 2920.37 to 2956.32 cm -1. Furthermore, lauric acid has
-1.
carbonyl group (C = O) at wave number 1653.05 to 1654.98 cm

The most dominant interaction between the molecules of collagen and

chitosan molecules are physical interactions, as shown by the decrease in

absorption wave number in the amide I during the addition of chitosan

(Tangsadthakun et al., 2006). Meanwhile, according to Fernandes et.al (2011)

compounds OH, C = O, -NH 2 are formed from composite collagen-chitosan derived

from compounds contained in the incorporation of collagen and chitosan. In addition,

the interaction between the collagen-chitosan with lauric acid plasticizer is indicated

by the wave number of ester group C = O in which the C = O shows the carboxylic
acid group. The analysis indicates that the COOH functional group turned into a

group -COOC- (Nirvana, 2012).

This is possible because, in addition to chitosan experiencing cross belt with

collagen, cross belt also occurs in groups C = O and NH 2 groups (rr

Rohindra et.al., 2004). Therefore, the scaffold can be degraded (Darni et al., 2009).

Scanning Electron Microscope (SEM) Test

(A) 500x Zoom (B) 500x Zoom

(C) 250x Zoom (D) 500x Zoom

(E) 1000x Zoom

 Figure 2. Test result of scaffold with composition variation of collagen-

chitosan-lauric acid (A) 1:9:2 (B) 3:7:2 (C) 5:5:2 (D) 7:3:2 dan (E) 9:1:2

In sample A, variations in the composition of the collagen-chitosan-lauric acid

1: 9: 2 has a pore range from 94.11 to 108.23 lm. In B sample, the result of an

increase in collagen composition causes the variation of collagen-chitosan-lauric

acid to become 3: 7: 2. An increase in the size of pores pore diameter range of 102.5

to 112.5 µm is obtained. Similarly, samples C and D with the respective variation of

the composition of the collagen-chitosan-lauric acid 5: 5: 2 and 7: 3: 2, have a pore

range of 120.05 to 131.02 μm and 122.1 to 140, 1 µm.

The smaller the pore size in the sample, the stronger are the material

properties exhibited in tensile strength test, while the greater the larger the pores, the

more fragile the sample will be. This is because that samples with large pore size is

caused by the composition of collagen which is bigger than the composition of the

chitosan, so the amine group on the chitosan is only few and makes is fiber bond

smaller, causing the fragile nature of the scaffold (Tan, 2001 and Arpornmaeklong,

2007).

The pore size of the four samples meets the standard pore size skin

scaffold for tissue engineering applications of 63-150 μm (O'Brien, 2005). However,

in samples E by variation of the composition of collagen-chitosan-lauric acid 9: 1: 2,

the size of the pore diameter of the scaffold has decreased drastically, which is only

37.87 to 47.36 µm. Moreover, when observed using SEM, hardly any pore that was

found. This is because the sample formed is already fragile due to the composition of

collagen that has exceeded the optimal limit so that scaffold formed

is brittle (Krishna, 2011).

Cytotoxicity Test

Figure 3. Relation of the Percentage of Live Cells with Composition Variation

on the Scaffold Collagen:Chitosan:Lauric Acid

The five scaffold samples showed values below the standard of the toxicity of

a sample. This is indicated by the percentage of living cells of the five

samples scaffold which showed a value below 50% ranges from 20.11 to

21.51%. The value does not match what was expected, because the sample is said

to be non-toxic when life cell percentage is above 50% (Meiyanto et

al, 2008). Mismatch in the research results can be caused by several factors,

including cell media that did not match the sample,

pH, incubation temperature, organic composition of the solvent, up to the repeated

washing of PBS (Dias et al, 1998).

When testing cytotoxicity in this study, samples were under repeated PBS

washing. This can make the cells attached to the media go wasted so that when

observed under a light microscope, the microwell plate did not show any cell. That is

the reason why low percentage of live cells was read on Elisa Reader. Besides,
since this study uses materials that are acid such as acetic acid and lauric acid, it is

necessary to do acidity test to determine the degree of acidity of the samples

because the pH that is too acidic can be one of the factors that causes the decrease

of cell viability percentage, and when the pH is too acidic, cells cannot propagate.

Biodegradation Test

Figure 4. Relation between the Percentage of Biodegradability to Scaffold

Composition Variation of Collagen:Chitosan:Lauric Acid

Sample A underwent the shortest degradation process, while sample D

underwent the longest. It can be said that the more the composition of the chitosan is

in the scaffold, the longer is the time required for degradation or, in other words, the

more collagen the scaffold composition contains, the faster is the degradation

processes that occurs. This can be seen during the day 7 of soaking, where the

sample A was degraded 19.46%, the samples B and C were degraded 22.22% and

34.48%, respectively, while sample D was degraded 37.93%. Sample E was not

done because the biodegradation test sample was in nearly powder form.
 The rate scaffold degradation is influenced by the composition of collagen and

chitosan, where the chitosan composition is more comparable the composition of

collagen. The scaffold degradation time becomes longer because more than a

chitosan composition of collagen causes steric hindrance better than scaffold with

more composition of scaffold. Wherein the steric hindrance between molecules

making positions are increasingly docked together, such ties are getting stronger and

make the chitosan degraded in a longer period of time(Taravel, 1996).From the

results of biodegradation test, the chitosan-collagen scaffold lauric acid degrade a

maximum of 40% during the 7 days of observation. It makes the scaffold contain

potential to be applied to third-degree burns that have a healing process for 21 days.

Tensile Strength Test

Figure 5. Relation between the Value of Tensile Strength towards the Scaffold

Variation Composition

In sample A, the tensile strength obtained was 3,529 MPa. Tensile strength

obtained for samples B, C, D and E in decreasing order were 1.935 MPa, 0.779

MPa, 0.192 MPa and 0 MPa. Of the five samples, only samples A and B meets the
 standard values of tensile strength to be the scaffold of human skin on the abdomen

based on Table 2.3 (Annaid et al, 2011) which is in the range of 1-24 MPa UTS

value. This value is better than the research of Tangsadthakun et al. in 2006which

had the value of tensile strength of only 0.5 to 1 MPa.

That is because the carboxylic groups contained in lauric acid and amine in

the chitosan is bound to the same C atom so that lauric acid is able to increase the

value of the tensile strength of the scaffold. The decrease of tensile strength

in scaffold samples C, D, E because of too many collagen compositions were used,

and the composition of the chitosan is too little to make the amino group of chitosan

chains which serves to strengthen ties on scaffold fibers to decrease. That makes

the decreasing value of the tensile strength of the scaffold (Tan, 2001 and

Arpornmaeklong, 2007).

CONCLUSION

1. The FTIR test results showed that the most dominant interaction between collagen

molecules and the molecules of chitosan is physical interaction. Interactionbetween

collagen-chitosan with lauric acid plasticizer indicated by the wave number of ester

group C = O. In the morphology test, samples A, B, C and D meet scaffold pore size

standard for skin tissue engineering applications of 63-150 μm. In assay MTT

cytotoxicity test, the percentage of living cells of the five samples of scaffold showed

a value below 50% in the percentage of living cells range from 20.11 to 21.51. In the

degradation test,chitosan-collagen-lauric acid scaffold is degraded 19.44% -37.93%

during the 7 days of observation. In the tensile strength test, only samples A and B
 meet the standard value of the tensile strength of 3.53 MPa and 1.935 MPa. The

value is within the standard value range of UTS scaffold for skin tissue of 1-24 MPa.

2. Sample A of collagen chitosan is the best composition among the other variations of

collagen-chitosan scaffold with the addition of lauric acid as a plasticizer (1: 9: 2) as

a means of skin tissue engineering skin for burn cases since it meets 4 of the 5

characterizations, which are FTIR, SEM, degradation, and tensile strength tests.

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