Journal of International Dental and Medical Research ISSN 1309-100X Bovine Hydroxyapatite Gelatin Chitosan Scaffolds

http://www.ektodermaldisplazi.com/journal.htm Anita Yuliati, and et al

The Profile of Crosslinked Bovine Hydroxyapatite Gelatin Chitosan Scaffolds

with 0.25% Glutaraldehyde
Anita Yuliati1*, Nadia Kartikasari2, Elly Munadziroh1, Devi Rianti1
1. Department of Dental Material, Faculty of Dental Medicine, Universitas Airlangga, Surabaya, Indonesia.
2. Departement of Periodontic, Faculty of Dental Medicine, Universitas Airlangga, Surabaya, Indonesia.

Abstract

Bone defect is still a major problem in dentistry. In oral cavity, trauma, congenital disorder,
periodontitis, and tumor can damage bones. A biomimetic scaffold made of bovine hydroxyapatite
(BHA), gelatin (GEL), and chitosan (K) (BHA-GEL-K) is on a development in recent researches.
This research tends to develop BHA-GEL-K scaffolds combined with 0.25% glutaraldehyde as
cross linking agent.
Scaffolds’ profiles were examined with FTIR, porosity size by using SEM, and compressive
strength by using autograph. The adhesion of bone narrow mesenchymal stem cell (BM-MSC) to
crosslinked BHA-GEL-K scaffolds with 0.25% glutaraldehyde were examined by using SEM.
BHA, GEL, K and glutaraldehyde components are bonded to each other. These scaffolds have
121.21 μm of porosity and 912.86 + 83.81 kPa of compressive strength value. These scaffolds
have good adhesion to BM-MSC. The adhesion of scaffolds increases since day 1 to 7.
The profile of BHA-GEL-K scaffolds that crosslinked to 0.25% glutaraldehyde may fulfills a
requirement of bone tissue engineering.
Experimental article (J Int Dent Med Res 2017; 10: (1), pp. 151-155)
Keywords: Scaffolds, bovine-hydroxyapatite, gelatin, chitosan, crosslink.
Received date: 09 February 2017 Accept date: 01 March 2017

Introduction role as a temporary extracellular matrix for cell

adhesion medium. The adhered cells on
Bone defect is still a main problem in scaffolds are shown to proliferate, differentiate,
dentistry. In oral cavity, trauma, congenital and form new tissue with the assist of molecular
disorder, periodontitis, and tumor can damage signal.2
bones. Bones are capable to self-regenerate. In In bone regeneration, it needs some
some cases, damages and bone resorptions are criteria in scaffolds making such as (a)
too fatal that lead to self-regenerate reduction. biocompatible, so that cells are able to grow,
This case might cause bone unification to adhere to scaffolds’ surface, and proliferate; (b)
hamper (nonunion) and the support of osteoconductive and osteoinductive materials; (c)
surrounding tissues to reduce.1 capable of handling a bearing load; and (e)
The most used treatment for bone defect having porosity structure and porosity size at
is bonegraft. In recent years, the method is rarely >100 μm to facilitate cell penetration, tissues’
used due to some its disadvantages. growth and vascularization.4
Researchers are starting to use tissue- Biomimetic scaffolds that were made of
engineering method.2 Tissue engineering is an hydroxyapatite (HA), gelatin (GEL), and chitosan
approaching method, which tends to repair or (K) (HA-GEL-K) is now on a development.5-8
replace the damaged body parts by using cells, Hydroxyapatite (Ca10(PO4)6(OH)2) has a
scaffolds, and molecular signals.3 Scaffolds take biocompatible, bioactive, and osteoconductive
structure.4 HA (hydroxyapatite) could be
collected from synthetic and natural materials
*Corresponding author:
and it could be found from cow bone (bovine
Anita Yuliati
Department of Dental Material, hydroxyapatite/BHA). BHA is a non-toxic material
Faculty of Dental Medicine, Universitas Airlangga, that has similarities with HA in humans.9 Gelatin
Jl. Mayjen Prof.Dr. Moestopo 47, Surabaya, Indonesia.
E-mail: [email protected]
is a derivation of collagen from organic structure
of bone. Gelatin has a biodegradable,

Volume ∙ 10 ∙ Number ∙ 1 ∙ 2017 Page 151

 Journal of International Dental and Medical Research ISSN 1309-100X Bovine Hydroxyapatite Gelatin Chitosan Scaffolds
http://www.ektodermaldisplazi.com/journal.htm Anita Yuliati, and et al

biocompatible, and low antigenicity property. This Compressive Strength Test

material has Arginine-Glycine-Aspartic acid Compressive strength test is performed
sequences that modulate cell adhesion and a by using autograph (Shimadzu Ag-10 TE). The
high number of accessible functional groups that samples are pressed at 10 mm/minute with 100
eases a modification with crosslinking agent.10 kN load cell. The calculation of compressive
Chitosan is a natural polymer material that has a strength is using formula.13
hydrophilic surface that makes adhesion and cell
2
proliferation work well. Chitosan is a Compressive strength (N/mm )= Force (Newton)
2
biocompatible, bioactive, osteoconductive, and Surface area (mm )
biodegradable material. 11
BHA, gelatin, and chitosan combination is Adhesion Test
expected to resemble bones’ natural composition. Scaffolds were soaked on medium DMEM
Researchers used to use BHA-GEL-K with for 1 day. The scaffolds are set into well of M24
70:15:15 (w/w) ratios with a good result; however and medium suspension which contain 2 x 106
the mechanical properties of the scaffolds were BM–MSC are added as well. The plate is
not optimal.8 In this study, BHA-GEL-K scaffolds incubated in 37oC temperature with 5% CO2 for 3
had crosslinked with 0.25% glutaraldehyde to hours. After 3 hours, 500 μl of medium is added
increase its mechanical and biological properties. per well. Afterwards, the plate is incubated for 1
and 7 days. The seeded-BM-MSC-scaffolds are
Materials and methods fixed by using 2% glutaraldehyde in 4oC. Then,
the scaffolds are washed with PBS (3 times for 5
This study uses BHA (particle size at minutes) in 4oC. The PBS is replaced by 1% of
<150 μm that made of cow bone purchased from osmic acid for 1-2 hours in 4oC and is followed by
RSUD Dr. Soetomo, Surabaya), gelatin washing with PBS. The scaffolds are dehydrated
(Rousselot, Ghuangdong, China), chitosan by using alcohol in a level at 30%, 50%, 70%,
(Sigma 93646, USA), deacetylation degree at 90% and 100% (2 times) for 15-20 minutes.
>81%) and 0.25% glutaraldehyde. Afterwards, the solvent is replaced with amyl
acetate absolute, which is a preservative for
BHA-GEL-K Scaffolds Preparation waiting its drying time. Scaffolds are dried by
Acetic acid 2% 9 ml and 0.375 gr gelatin using critical point drying tool (CPD). After dried,
were mixed using magnetic stirrer in 100oC. the seeded cell in scaffold is observed by using
BHA 1.75 gr was mixed with 5 ml distilled water. SEM with 200x of magnification (JEOL, JSM-
The BHA mixture was mixed to gelatin and acetic T1000 Scanning Microscope, Japan).
acid mixture. Chitosan 0.375 gr and 2 ml of 10%
NaOH were added. The mixture was put into a Results
scaffolds’ mold and frozen at -80°C for 24 hours
and then it was freeze-dried for 2 x 24 hours.8 FTIR
The finished scaffolds are soaked in The result of the analysis of crosslinked
0.25% glutaraldehyde for 24 hours. After 24 BHA-GEL-K scaffold with 0.25% glutaraldehyde
hours, the scaffolds are soaked in distillated aqua shows in Figure 1. The results show the peak at
for 24 hours. Then the scaffolds are frozen at - 3274.93cm-1, 2931,98 cm-1, 1636,87 cm-1,
40oC for 24 hours and dry frozen for 2 x 24 1541,33 cm-1, 1086,80 cm-1, 1025,98 cm-1,
hours.12 961,49 cm-1, 627,08 cm-1, 598,62 cm-1, and
FTIR (Fourier Transform Infrared 562,07 cm-1. The component of crosslinked
Spectroscopy) BHA-GEL-K scaffolds with 0.25% glutaraldehyde
FTIR is performed by using FTIR Thermo interacts with each other. It is shown as a
Scientific examination tool, Nicolet, iS10 with formation of amide group (3274 cm-1), C-N (1080
400-4000 cm-1 wavelength. The reading is cm-1), and Ca-O (562,07 cm-1).
performed by using OMNIC program. Porosity
Porosity Test SEM result of crosslinked BHA-GEL-K
Porosity test is performed by using SEM scaffolds with 0.25% glutaraldehyde shows in
(FEI, Inspect-S50). Picture is taken at 100x Figure 2. The smallest porosity size of the
magnification. crosslinked BHA-GEL-K scaffolds with 0.25%

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 Journal of International Dental and Medical Research ISSN 1309-100X Bovine Hydroxyapatite Gelatin Chitosan Scaffolds
http://www.ektodermaldisplazi.com/journal.htm Anita Yuliati, and et al

glutaraldehyde is 103.8 μm whilst the biggest

size is 146.0 μm. The average of its pore is
121.21 μm.

Figure 1. FTIR result of crosslinked BHA-GEL-K

scaffolds with 0.25% glutaraldehyde.

Figure 2. SEM result of crosslinked BHA-GEL-K

scaffolds with 0.25% glutaraldehyde (100x c
magnification). Figure 3. SEM results of the crosslinked BHA-
GEL-K scaffold (a), adhesion of BM-MSC on
Compressive Strength scaffold day 1 (b), and day 7 (c).
The lowest compressive strength value of
the crosslinked scaffold is at 780 kPa whilst the Discussion
highest is at 1030 kPa. The average compressive
strength value of the crosslinked scaffold is at The crosslinked scaffold is a
912.86 +83.81 kPa. compounding material to obtain suitable scaffolds
Adhesion that could be applied well on bone tissue
The adhesion test is executed by using regeneration. The function of BHA in the
SEM after 1 and 7 days of incubation. Figure 3 scaffolds roles as main component of
shows the BM-MSC adhesion on crosslinked osteoconductive inorganic matrix; gelatin as main
BHA-GEL-K scaffold with 0.25% glutaraldehyde. organic component that induces cell adhesion;

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 Journal of International Dental and Medical Research ISSN 1309-100X Bovine Hydroxyapatite Gelatin Chitosan Scaffolds
http://www.ektodermaldisplazi.com/journal.htm Anita Yuliati, and et al

and chitosan as osteoconductive organic due to cell division. The doubling time of BM-
component. MSC is 49.9 ± 4.2.19
The component of BHA, gelatin, chitosan
and glutaraldehyde in scaffold interacts to each Conclusions
other. Phosphate and hydroxyl group is an HA
component that bonds with other materials such Crosslinked BHA-GEL-K scaffold with
as gelatin and chitosan. Hydroxyl group in HA 0.25% glutaraldehyde seems to have a suitable
could form hydrogen bonding with hydroxyl group requirement for bone tissue regeneration. BM-
from K. Phosphate group could bond with COO- MSC might adhere and proliferate to the
ion and NH3+ from GEL and K.14 The remaining of crosslinked scaffold. It makes the scaffold as the
HA component that roles in forming the other biomaterial preference for tissue engineering
material bonding are Ca2+. Calcium forms based of bone tissue regeneration.
hydrogen bonding with NH2also interacts with
COO- and COOH on gelatin and chitosan Declaration of Interest
surface.14
One of the must-have characteristics of The authors report no conflict of interest
the scaffold is a good proportional of porosity. and the article is fund by Hibah RKAT 2016 by
The minimum size of pore on scaffold is at 100- Faculty of Dental Medicine, Universitas Airlangga.
150 μm.15 The small size cause limitation of
migrated cell, and interruption of diffused nutrition.
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