Research J. Pharm. and Tech.

15(8): August 2022

ISSN 0974-3618 (Print) www.rjptonline.org

0974-360X (Online)

RESEARCH ARTICLE

The Viability of Collagen Peptide from Osphronemus goramy Fish Scale

Extract on Human Gingival Fibroblast
Noer Ulfah1, Samuel Rehuel Santoso2, Lambang Bargowo1, Shafira Kurnia1,
Chiquita Prahasanti3
1
Department of Periodontology, Faculty of Dental Medicine Universitas Airlangga,
Jl. Prof. Dr. Moestopo No. 47 Surabaya – Indonesia.
2
Periodontology Specialist Program Student, Faculty of Dental Medicine Universitas Airlangga,
Jl. Prof. Dr. Moestopo No. 47 Surabaya – Indonesia.
3
Professor of Department of Periodontology, Faculty of Dental Medicine Universitas Airlangga,
Jl. Prof. Dr. Moestopo No. 47 Surabaya – Indonesia.
*Corresponding Author E-mail: chiquita-p-s@fkg.unair.ac.id

ABSTRACT:
Background: Damage to the periodontium tissue requires a regenerative treatment to increase the dimensions of
the lost bone. This research was conducted to find another regenerative material with the use of type 1 collagen
peptides derived from gourami fish scales. This study was conducted to test the viability of gourami scales
collagen peptides on Human Gingival Fibroblast cells for 24 hours. Purpose: To determine the viability of
Human Gingival Fibroblasts (HGF) after the administration of collagen peptides of gourami fish scales.
Method: HGF was taken from healthy gingiva and planted in 96 well plates. The type 1 collagen peptides of
gourami fish scales with concentrations of 0.32 mg / ml, 0.16 mg / ml, 0.04 mg / ml, 0.02 mg / ml and 0.01 mg /
ml were inserted into each well and incubated for 24 hours. MTT Assay was performed to see the viability of
fibroblast cells. Results: There was an increase in the viability value from a concentration of 0.32 mg/ml to 0.01
mg/ml. The concentration of 0.01 mg/ml showed the highest viability. Conclusion: The collagen peptide is a
potential substance for tissue engineering. The concentration of 0.01 mg/ml collagen peptides shows the highest
HGF viability.

KEYWORDS: Regenerative, Collagen peptides, Viability, Human gingival fibroblast, Tissue engineering.

INTRODUCTION: The ideal scaffold requirements include osteoconductive,

Recently, the development of biomaterial research for
regeneration purposes has increased. This biomaterial
research has the concept that the substrate is not only
focused on damaged tissue structures but also integrates
with surrounding tissue and stimulates regeneration 1–4.
Until now, there is a lot of research on natural and
synthetic graft materials for tissue engineering.
However, the material which has the best
biocompatibility and bioactivity is not yet founded 5. The
biomaterial innovation for tissue engineering design is
focused on the physicochemical suitable scaffolds for
cell attachment and proliferation, and differentiation for
the formation of specific organ tissues6,7.
Received on 17.04.2021 Modified on 21.11.2021
Accepted on 04.04.2022 © RJPT All right reserved
Research J. Pharm. and Tech. 2022; 15(8):3497-3501.
DOI: 10.52711/0974-360X.2022.00586
osteoinductive, osteogenic, biodegradable, good
microstructure, and proper mechanical properties. Also,
the most important requirement is the ability to stimulate
cell adhesion and to maintain tissue function. Polymer
materials that have been developed in tissue engineering
include polyglycolic acid (PGA), polylactic acid (PLA),
polylactic-co-glicolic acid (PLGA), chitosan, and
collagen8–11. These polymers have been considered a
material that has many functional advantages because of
its high biocompatibility, biodegradable, and low
toxicity12–15.
The polymer that has the potential for tissue engineering
is collagen, which mass is ranging at 25-35% mass per
total protein weight in vertebrates. Collagen is a major
components of the extracellular matrix which found in
blood vessel, tendon, ligament, skin, cartilage,
intervertebral disc, and bone16,17. Collagen contains RGD

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(Arg-Gly-Asp) and non-RGD which can bind to surface solution to clean off the blood, then placed in a transport
cells related to integrins to facilitate migration, adhesion, medium.
proliferation, and cell differentiation18. Collagen has
been widely used in tissue engineering because of its The gingival tissue was washed 3 times using 10%
good biocompatibility, biodegradable, low antigenicity Phosphate Buffer Saline (PBS) containing penicillin and
properties, and has a good attachment to cells19,20. The streptomycin antibiotics to avoid the possibility of
usage of certain molecules in the scaffold is effective in bacterial contamination. The tissue was cut into 1mm 3
increasing stem cells growth and also triggering and added with collagenase for 30 minutes at 37°C. The
osteogenic differentiation because of its ability to tissue was centrifuged for 6 minutes.
provide signals for osteogenesis stimulator5,21,22.
The obtained cells were cultured with growth medium
In this study the collagen peptide of gourami fish scale and incubated at 37°C with 5% CO2 incubator for 3 days
obtained via enzymatic hydrolysis process. The purpose and observed every day until confluent. The cultured
of this study is to investigate the viability of human mediums were changed every 3 days until the cells were
gingival fibroblast exposed to collagen peptide derived enough to be passaged.
from gourami fish.
The Control Group and Experimental Group
MATERIAL AND METHODS: Division:
This is a laboratory experimental research with Post-Test The groups were divided into a control group and
Only control group design. This research was held in the experimental groups. The experimental groups were
Stem Cell Research and Development Center of divided into 5 groups. The collagen peptide solution was
Airlangga University and the Biochemistry laboratory of used for the experimental groups. Collagen peptide
Brawijaya University. The ethical clearance for this solution was immersed in α-MEM for 24 hours before
research with registeration number been divided into 5 groups.
193/HRECC.FODM/VII/2018 was given by the
Bioethics Committee of Faculty of Dental Medicine, The 5 experimental groups were: the group I contained
Universitas Airlangga, Surabaya. The patients have 0.32mg/ml collagen peptide solution, group II contained
given their consent for this research. 0.16mg/ml, group III contained 0.04mg/ml, group IV
contained 0.02mg/ml, group V contained 0.01mg/ml.
The preparation of gourami fish scale peptide
collagen: After 24 hours of incubation at 37°C and 5% CO 2, the
The gourami fish scales were washed using NaCl and culture medium and the collagen peptide solution were
dried. The scales were soaked in 1M NaOH solution at removed. The culture cells were washed using 5% PBS
4°C for 24 hours to remove non-collagen protein, the and were given MTT reagent. The microplates were
NaOH solution is replaced every 8 hours. The scales incubated again for 4 hours at 37°C and 5% CO 2. After
were washed to neutralize the pH, then immersed in the incubation, Dimethyl sulfoxide (DMSO) solutions
isobutanol to remove the fat. EDTA 1 N as a chelating were added. ELISA Reader was used to analyze the
agent was used for the decalcification process with the result.
addition of acetic acid (acid solubility collagen) 0.5 M.
0.1gr of pepsin enzyme was added and agitated with an MTT assay:
ultrasonic device at 4°C for 18 hours and divided into The Fibroblast cells were transferred to a 96-well
small tubes.0.5 M NaCl was added then centrifuged at a microplate. The extracted collagen peptide then added.
speed of 4000 rpm and then washed again. Later, 25μL MTT reagent was inserted in each
microplate incubated for 4 hours. The plates were stored
The lyophilization process was done using a freeze-dried
at 95% humidity with 37° C and 5% CO2. Cell viability
method to remove water content at -76°C and ambient
was determined after 24 hours later using the standard
temperature of 23.6°C for 12 hours until. The fish scales
colorimetric MTT assay to observe viable cells. The 96-
collagen was combined with 5 mg of pepsin enzyme and
well microplate was absorbed by the ELISA reader at a
1.25ml of sodium phosphate buffer solution (0.1 M pH
wavelength of 595nm.
7.5). The mixture then placed on the orbital shaker at
55°C and 50rpm for 120 minutes. Last, the water bath
Statistical Analysis:
was done at 75°C for 10 minutes.
The Shapiro-Wilk test were used for normal distribution
The Fibroblast Cells Management: test, while Levene’s test were used for homogeneity.
Gingival tissue was harvested from free gingiva of first ANOVA were used for statistical analysis with the
premolar tooth which was extracted for orthodontic significance value was set at 0.05.
treatment. The gingival tissue was washed with a saline
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RESULTS: The process of making collagen peptides requires a

According to the results of reading OD (Optical chemical or enzymatic hydrolysis process under
Density), the average results of the obtained research controlled conditions24,25. One of the best hydrolysis
result are shown in Table 5.1 processes is the enzymatic method because its
advantages, such as specificity, hydrolysis control, and
Table 5.1 Absorbance results in the treatment group collagen less waste; for those reasons, this method is more widely
peptide extract for 24 hours.
Treatment N Mean
used. Nevertheless, extraction methods may has
Cells Control 4 0.634 ±0.056 favorably and unfavorably that affect collagen
Concentration 0.32 mg/ml 4 0.832 ±0.020 characteristics, such as: thermal stability, molar mass,
Concentration 0.16 mg/ml 4 0.848 ±0.167 water-retaining capacity, and gel-forming capacity.
Concentration 0.04 mg/ml 4 0.878 ±0.050 Enzymatic hydrolysis process is simple, efficient, and
Concentration 0.02 mg/ml 4 0.882 ±0.040
involves mild alkaline conditions that do not denature
Concentration 0.01 mg/ml 4 0.907 ±0.065
proteins19,26.
Based on the results of the study it can be seen that the
average value of the absorbance of the cell control group The enzyme used in the hydrolysis process is a protease
for 24hours was 0.634. The lowest absorbance of the which is also called peptidase. Peptidase is an enzyme of
collagen peptide group at a concentration of 0.32mg/ml a hydrolase group and acts by breaking protein bonds
was 0.832 and the highest at a concentration of 0.01 into peptides27,28. In this study, the peptidase enzyme
mg/ml was 0.907. used is the pepsin enzyme. Enzymatic hydrolysis of
collagen produces biologically active peptides with
The results of the viability of peptide collagen to human antioxygenic and antimicrobial capacities29.
gingival fibroblasts are shown in table 5.2. The highest
viability of the collagen peptide group at the The absorbance value determines cell viability and is
concentration of 0.01ml/mg (150.11%) and the lowest influenced by several factors, i.e the type of solvent, pH,
percentage at the concentration of 0.32mg/ml (136.35%). temperature, high electrolyte concentration, and the
presence of disruptor substances. If the value of
Tabel 5.2 Viability results in the treatment group gourami scale
collagen extract for 24 hours. absorbance produced higher than the control, it shows a
Concentration Percentage of Living Cells (%) high ability of cell proliferation. But if the proliferation
0.32 mg/ml 136.35% ±0.164 rate is too high it will result in cell death30–32.
0.16 mg/ml 139.20% ±0.437
0.04 mg/ml 144.75% ±0.108 Based on the results, the treatment groups show a
0.02 mg/ml 145.40% ±0.235
0.01 mg/ml 150.12% ± 0.083 significantly higher absorbance value than the control
group. The results showed that there was a gradual
Statistical Analysis: increase in the value of viability in the treatment of
The measurement results are tabulated according to each collagen peptides from the highest concentration (0.32
group sample. Statistical tests were carried out using mg/ml) to the lowest concentration (0.01mg/ml). The
ANOVA One Way at the significance level of Sig. <0.05 research data shows that there is a significant difference
and significant differences were obtained, then continued in the whole group of collagen peptide solution
by performing multiple comparisons using post hoc test treatments to cell control.
Tukey HSD which concluded that all groups had
significant differences except the concentration of 0.16 The results showed there was a gradual increase in the
mg/ml and 0.32mg/ml. value of viability in the treatment of collagen peptide
solutions from the highest concentration (0.32mg/ml) to
DISCUSSION: the lowest concentration (0.01mg/ml). The research data
A natural origin collagen biomaterial from marine began showed that there was a significant difference in the
to be developed because of its structural similarity to whole treatment group of collagen peptide solution to
mammalian collagen. It has good biocompatibility, ease cell control. The data in the study are in accordance with
of extraction, water-soluble, low production costs, and the theory of loading-dose in drugs, ie the higher the
biodegradable. These unique properties make collagen dose given, the therapeutic effect on the target cell is no
peptide could be used as a promising biomaterial for better than the lower dose33.
tissue engineering. This material has tremendous
potential use for tissue regeneration applications This study showed that the viability of collagen peptides
including bone, cartilage, skin, blood vessel tissue was higher than the control group with the highest
regeneration, and teeth because of its good physical results at the lowest concentration of 0.01mg/ml. The
characteristics and physicochemical properties 13,23. application of collagen peptides derived from fish has
also been reported by several other researchers to have a
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good outcome in the field of tissue regeneration.
Research by Liu and Sun shows that the application of
collagen peptides derived from tilapia scales on rat
mesenchymal stem cells also shows an increase in
osteogenic and endothelial markers34. Yamada, et al. also
reported that an administration of fish collagen peptide
to human osteoblasts with low concentrations increases
the proliferation and differentiation of osteoblasts
observed with increased expression of ALP markers,
osteocalcin, osteopontin, BMP-2, and β3 integrin35. Hu,
et al. reported that an administration of collagen peptides
derived from fish with certain concentrations can
increase osteoblast cell proliferation and suppress
osteoclast cell proliferation28.
Collagen as biomaterial can be used to accelerate the
wound healing process by forming an extracellular
matrix for cells attachment periodontal tissue, especially 13.
fibroblast cell36. Collagen derived from gourami scales
can be considered as an alternative material for tissue 14.
engineering because of its abundant collagen content and
good biocompatibility.
CONCLUSION:
Collagen peptides from gourami fish scales are viable to
16.
Human Gingival Fibroblast cells. The results of this
study indicate that the concentration of collagen peptides
and viability is inversely proportional but still higher
than the control group. The highest viability results are 17.
found at the concentration of 0.01mg/ml which is
150.11% 18.
CONFLICT OF INTEREST:
The authors declare no conflict of interest.
REFERENCES:
1. Sheehy EJ, Cunniffe GM, O’Brien FJ. Collagen-based
biomaterials for tissue regeneration and repair. Pept Proteins as
Biomater Tissue Regen Repair. 2018:127-150. doi:10.1016/B978-
0-08-100803-4.00005-X
2. Shi S, Jiang W, Zhao T, et al. The application of nanomaterials in
controlled drug delivery for bone regeneration. J Biomed Mater
Res - Part A. 2015;103(12):3978-3992. doi:10.1002/jbm.a.35522
3. Hossain A, Roy S, Guin PS. The Importance of Advance
Biomaterials in Modern Technology: A Review. Asian J Res
Chem. 2017;10(4):441-453. doi:10.5958/0974-4150.2017.00073.6
4. Diksha, Sazal P. Role of Stem Cells in treatment of different
Diseases. Res J Pharm Technol. 2018;11(8):3667-3678.
doi:10.5958/0974-360X.2018.00674.1
5. Bilem I, Chevallier P, Plawinski L, Sone ED, Durrieu MC,
Laroche G. RGD and BMP-2 mimetic peptide crosstalk enhances
osteogenic commitment of human bone marrow stem cells. Acta
Biomater. 2016;36:132-142. doi:10.1016/j.actbio.2016.03.032
6. León-Mancilla BH, Araiza-Téllez MA, Flores-Flores JO, Piña-
Barba MC. Physico-chemical characterization of collagen
scaffolds for tissue engineering. J Appl Res Technol.
2016;14(1):77-85. doi:10.1016/j.jart.2016.01.001
7. Indira R, Harihara Priya G, Tamizharuvi T, Jaisankar V. Synthesis
and Characterization of Novel Bio-Elastomers for Tissue
Engineering. Asian J Res Chem. 2013;6(9):814-818.
https://ajrconline.org/HTMLPaper.aspx?Journal=Asian Journal of
3500
Research in Chemistry; PID=2013-6-9-4.
8. Neto AS, Ferreira JMF. Synthetic and marine-derived porous
scaffolds for bone tissue engineering. Materials (Basel).
2018;11(9). doi:10.3390/ma11091702
9. Nikolova MP, Chavali MS. Recent advances in biomaterials for
3D scaffolds: A review. Bioact Mater. 2019;4(October):271-292.
doi:10.1016/j.bioactmat.2019.10.005
10. Nugraha AP, Rezkita F, Puspitaningrum MS, et al. Gingival
Mesenchymal Stem Cells and Chitosan Scaffold to Accelerate
Alveolar Bone Remodelling in Periodontitis: A Narrative Review.
Res J Pharm Technol. 2020;13(5):2502-2506. doi:10.5958/0974-
360X.2020.00446.1
11. Aswin K, Jothishwar S, Nayagam VC, Priya G. Scaffolds for
Biomolecule Delivery and Controlled Release–A Review. Res J
Pharm Technol. 2018;11(10):4719-4730. doi:10.5958/0974-
360X.2018.00861.2
12. Cvrček L, Horáková M. Chapter 14 - Plasma Modified Polymeric
Materials for Implant Applications. In: Thomas S, Mozetič M,
Cvelbar U, Špatenka P, K.M. PBT-N-TPT for PM, eds. Elsevier;
2019:367-407. doi:https://doi.org/10.1016/B978-0-12-813152-
7.00014-7
Lim YS, Ok YJ, Hwang SY, Kwak JY, Yoon S. Marine collagen
as a promising biomaterial for biomedical applications. Mar
Drugs. 2019;17(8). doi:10.3390/md17080467
Radhika G, Sreelakhsmi Divya P, Prashanty Reddy G, Venkatesh
P, Ravindra Reddy K. An Overview on Regenerative Medicine.
Res J Pharm Technol. 2010;3(3):727-728.
15. Agarwal S, Jhunjhunwala V, Priya G. Fabrication and
Morphological Analysis of Gelatin-Alginate Scaffolds. Res J
Pharm Technol. 2018;11(9):3816-3818. doi:10.5958/0974-
360X.2018.00699.6
McKee TJ, Perlman G, Morris M, Komarova S V. Extracellular
matrix composition of connective tissues: a systematic review and
meta-analysis. Sci Rep. 2019;9(1):1-15. doi:10.1038/s41598-019-
46896-0
Kusindarta DL. The Role of Extracellular Matrix in Tissue
Regeneration. In: Kaoud HWE-HA hay E-S, ed. Rijeka:
IntechOpen; 2018:Ch. 5. doi:10.5772/intechopen.75728
Kruger TE, Miller AH, Wang J. Collagen scaffolds in bone
sialoprotein-mediated bone regeneration. Sci World J.
2013;2013(I). doi:10.1155/2013/812718
19. Meyer M. Processing of collagen based biomaterials and the
resulting materials properties. Biomed Eng Online. 2019;18(1):1-
74. doi:10.1186/s12938-019-0647-0
20. Calabrese G, Gulino R, Giuffrida R, et al. In vivo evaluation of
biocompatibility and chondrogenic potential of a cell-free
collagen-based scaffold. Front Physiol. 2017;8(NOV):1-11.
doi:10.3389/fphys.2017.00984
21. Ko E, Yang K, Shin J, Cho SW. Polydopamine-assisted
osteoinductive peptide immobilization of polymer scaffolds for
enhanced bone regeneration by human adipose-derived stem cells.
Biomacromolecules. 2013;14(9):3202-3213.
doi:10.1021/bm4008343
22. Ridhanya, Rajakumari K. Skin Wound Healing: An update on the
Current knowledge and Concepts. Res J Pharm Technol.
2019;12(3):1448-1452. doi:10.5958/0974-360X.2019.00240.3
23. Zulkifeli NRAN, Zain HHM, Zainol I, Musa NHC. The properties
of Hydrolysed Collagen from Oreochromis mossambicus’s scale
and their effect towards Cell viability. Res J Pharm Technol.
2020;13(12):5855-5860. doi:10.5958/0974-360X.2020.01020.3
24. León-López A, Morales-Peñaloza A, Martínez-Juárez VM,
Vargas-Torres A, Zeugolis DI, Aguirre-Álvarez G. Hydrolyzed
Collagen—Sources and Applications. Molecules.
2019;24(22):4031. doi:10.3390/molecules24224031
25. Hong H, Fan H, Chalamaiah M, Wu J. Preparation of low-
molecular-weight, collagen hydrolysates (peptides): Current
progress, challenges, and future perspectives. Food Chem.
2019;301(July):125222. doi:10.1016/j.foodchem.2019.125222
26. Schmidt MM, Dornelles RCP, Mello RO, et al. Collagen
extraction process. Int Food Res J. 2016;23(3):913-922.
 Research J. Pharm. and Tech. 15(8): August 2022

27. Jin HX, Xu HP, Li Y, Zhang QW, Xie H. Preparation and

evaluation of peptides with potential antioxidant activity by
microwave assisted enzymatic hydrolysis of collagen from sea
cucumber Acaudina molpadioides obtained from Zhejiang
province in China. Mar Drugs. 2019;17(3).
doi:10.3390/md17030169
28. Hu CH, Yao CH, Chan TM, et al. Effects of different
concentrations of collagenous peptide from fish scales on
osteoblast proliferation and osteoclast resorption. Chin J Physiol.
2016;59(4):191-201. doi:10.4077/CJP.2016.BAE398
29. Gomez-Guillen MC, Gimenez B, Lopez-Caballero ME, Montero
MP. Functional and bioactive properties of collagen and gelatin
from alternative sources: A review. Food Hydrocoll.
2011;25(8):1813-1827. doi:10.1016/j.foodhyd.2011.02.007
30. Bahi M, Jacob C, Khairan K. Efek sitotoksik haarlem oil terhadap
HL- 60 cell line dan steinernema fetiae. J Kedokt Hewan.
2013;10(2):109-114.
31. Mayerhöfer TG, Pipa A V., Popp J. Beer’s Law-Why Integrated
Absorbance Depends Linearly on Concentration. ChemPhysChem.
2019;20(21):2748-2753. doi:10.1002/cphc.201900787
32. Amin MN, Hidayat M, Permatasari N, Choiron MA. The
Differences Color Intensity, pH, Absorbance Value of Frangipani
(Plumeria accuminata Ait) Sap During Storage. Res J Pharm
Technol. 2020;13(12):5871-5875. doi:10.5958/0974-
360X.2020.01023.9
33. McCormack JP, Allan GM, Virani AS. Is bigger better? An
argument for very low starting doses. Cmaj. 2011;183(1):65-69.
doi:10.1503/cmaj.091481
34. Liu C, Sun J. Potential application of hydrolyzed fish collagen for
inducing the multidirectional differentiation of rat bone marrow
mesenchymal stem cells. Biomacromolecules. 2014;15(1):436-
443. doi:10.1021/bm401780v
35. Yamada S, Yoshizawa Y, Kawakubo A, Ikeda T, Yanagiguchi K,
Hayashi Y. Early gene and protein expression associated with
osteoblast differentiation in response to fish collagen peptides
powder. Dent Mater J. 2013;32(2):233-240.
doi:10.4012/dmj.2012-188
36. Mahesh L, Kurtzman GM, Shukla S. Regeneration in Periodontics:
Collagen-A Review of Its Properties and Applications in Dentistry.
Compend Contin Educ Dent. 2015;36(5):358-363.

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