Osphronemus goramy scales-derived type 1 Collagen induces RUNX2 and Osteocalcin expression: An in vivo Study

 

Reza Dony Hendrawan1, Chiquita Prahasanti2, Okkinardo Arief1,

I Komang Evan Wijaksana2, Lambang Bargowo2, Irma Josefina Savitri2, Wibi Riawan3

1Postgraduate Student of Periodontology Specialist Programme, Faculty of Dental Medicine,

Universitas Airlangga, Surabaya.

2Lecturer of Departement of Periodontology, Faculty of Dental Medicine, Universitas Airlangga, Surabaya.

3Department of Clinical Pathology, Faculty of Medicine, Universitas Brawijaya, Malang.

*Corresponding Author E-mail: chiquita-p-s@fkg.unair.ac.id

 

ABSTRACT:

Introduction: Alveolar bone defects need bone augmentation therapies by subtituting with bone material. Gourami (Osphronemus goramy) fish scale comprises type 1 collagen and it has been used as scaffolding material in bone tissue engineering. As alternative bone graft material, the scales have a big potency to promote osteogenesis in periodontal bone defect when autografts are not feasible. This study will analise Runx2 and Osteocalcin expression in wistar rat alveolar bone induced by type 1 collagen derived from gourami fish scale. Methods: 32 male Wistar rats were divided into four groups; control group—7 days (C7), treatment group—7 days (P7), control group—14 days (C14), and treatment group—14 days (P14). The left mandibular incisivus was extracted and the tooth socket was treated with 10mg collagen. The rats were euthanized (at day 7th and 14th) and immunohistochemistry was performed using monoclonal antibodies anti-RUNX2 and anti-osteocalcin. Results: After seven days and 14days, the expression of RUNX2 and osteocalcin in the treatment group increased significantly (p<0.05) compared with the control group. Conclusion: Type 1 collagen from gourami (Osphronemus goramy) fish scales increases RUNX2 and osteocalcin expression as a bone growth marker.

 

KEYWORDS: Gourami (Osphronemus goramy) fish scales, Type I collagen, RUNX2, Osteocalcin, Bone graft.

 

 


INTRODUCTION: 

Periodontal disease causes periodontal tissue destruction, and in such cases, one of the treatments is bone augmentation, a regenerative therapy used to repair the periodontal tissue defect, especially, the alveolar bone1,2. Socket preservation is one aspect of bone augmentation therapy and is performed by placing the bone graft in the tooth socket and covering the socket, with or without a barrier membrane. The purpose of this therapy is to preserve alveolar bone volume after tooth extraction3.

 

In bone augmentation therapy, the bone graft acts as a scaffold that provides space for cells and initiates osteoblast migration and differentiation4. A collagen scaffold is used in tissue engineering for bone regeneration because this material has good biocompatibility, high porosity, and low antigenicity5,6. There are various sources of collagen scaffolds. Fish scales is one of the alternative sources that has tremendous potency but the majority of the scales will be ended as an organic waste5,6,7. Gourami fish scales (Osphronemus goramy) contain collagen type 18 and induce minimal immunological and inflammatory responses. Moreover, it is relatively easy to extract collagen from this material5,8.

 

One indicator of alveolar bone regeneration is enhancement of RUNX2 and osteocalcin expression. In osteogenesis, RUNX2 is a transcription factor that is responsible for chondrocyte hypertrophy, endochondral bone formation, and vascular invasion of cartilage. RUNX2 also controls pluripotent mesenchymal cell differentiation into osteoblasts9. Osteocalcin is an extracellular matrix protein that is produced by osteoblasts in the bone remodeling phase10. This material plays an important role in connecting the mineral and organic matrix, acts as an indicator for osteoblast activity, and is a signaling molecule for bone remodeling11. This study aims to observe the increase in RUNX2 and osteocalcin expression in the alveolar bone of the Wistar rat (Rattus norvegicus strain wistar) treated by gourami fish scale type 1 collagen. Furthermore, this experiment will be a precursor experiment for developing this material.

 

MATERIAL AND METHODS:

Extraction of collagen:

This experiment had been through ethic commission (Health Research Ethical Clearance, Faculty of Dentistry, Universitas Airlangga) with registration number 663/HRECC.FOOM/X/2019. The freshly collected fish scales were washed and cleaned in water and dried for 12 days. Dried fish scales were soaked in sodium hydroxide solution 1M (Merck, Germany) at a temperature of 4oC for 24hours. Every eight hours, the solution was changed. The scales were also stirred intermittently, and the fish scales were washed by aquadest to neutralize the pH. The fish scales were soaked again in 10% isobutanol (Merck) to remove fat (defatted). For decalcification, 250ml EDTA (Merck) was added to 80g fish scales in eight hours. The fish scales were then washed again by aquadest. 800ml 0,5 M acetat acid (Merck, Germany) and 0,1g pepsin enzyme (Worthington®) was added and stirred by an ultrasonic machine (DAWE) at a frequency of 3040 Hz, temperature of 4oC for 3hours. The material was washed by aquadest and filtered. Sodium chloride 0,5M (Merck, Germany) was added and centrifuged at 4000rpm in a 15ml tube for 10 minutes to precipitate the liquid. Then the material was lyophilized by a freeze dryer (VirTis) machine at -76oC condenser temperature and 23.6oC ambient temperature for 12hours until the water was used up. The extracted collagen was sterilized by ethylene oxide and the material was ready for use.

 

Based on our previous study, the isolated colagen was characterized by using scanning electron microscopy (SEM) analysis, fourier transform infrared spectroscopy (FTIR) analysis, and invitro enzymatic biodegradation analysis12.

 

This research was conducted on 32 healthy male Wistar rats aged three months and weighing 150–300grams. The rats were randomly divided into four observation groups: control—7 days (C7), treatment—7days (P7), control—14days (C14), and treatment—14days (P14). The animals were anesthetized intramuscularly with ketamine (1,04mg/kg) (Ivanes®, Indonesia) before extracting teeth. In all groups, the left incisivus mandibula was extracted. In the treatment group, the socket was filled with 10mg gourami fish scale collagen. Observations were recorded on the seventh and the fourteenth day. The left incisivus mandibula was fixed in 10% buffer formalin to prevent tissue breakdown.

 

Immunohistochemistry protocol:

RUNX2 and osteocalcin expression was observed quantitatively by immunohistochemistry technique. The samples were washed with Phosphate-Buffered Saline (PBS) pH 7.4 one (1) time for 5min followed by blocking endogenous peroxide using 3% H2O2 for 20 minutes. Next, samples were washed using PBS pH 7.4 three times for 5 minutes. Blocking unspecific proteins was conducted with 5% FBS containing 0.25% Triton X-100. Next, Washing using PBS pH 7.4 three times for 5 minutes. The samples were incubated by using rat Runx2 monoclonal antibody (Santa Cruz Biotechnology®, USA) and anti-osteocalcin (Santa Cruz Biotechnology®, USA) 60minutes. Washing with PBS pH 7.4 three times for 5minutes. Incubate using HRP conjugated anti-rat for 40minutes. After being washed, DAB (diamino benzidine) was given and incubated for 10minutes. Washing with PBS pH 7.4 three times for 5 minutes. Wash with dH2O for 5 minutes. Counterstaining using Mayer Hematoxylen was incubated for 10minutes and washed with dH2O. Data were acquired based on manually quantification of color-intensity expressed using monoclonal antibodies anti-RUNX2 and anti-osteocalcin.

 

Statistical analysis:

Statistical analysis was conducted by using SPSS® 27 (IBM®) for windows. The results were analyzed using Levene’s homogenity statistic test, Kolmogorov-Smirnov for normality test and analysis of variance ANOVA test following post hoc Tukey’s tests.

 

RESULTS:

The positive expression of RUNX2 and Osteocalcin showed as brown color in cells (Figure 1). The application of type-1 collagen from gourami scales can enhance the expression of RUNX2 and osteocalcin in 7 days and 14 days (table 1). The highest expression of RUNX2 and osteocalcin were found in 14-days treatment group (P14). However, both RUNX2 and Osteocalcin expression, there was no significant difference between P7 and P14 (p>0.05) (table 2).

 

Table 1: The mean of Runx2 and Osteocalcin expression

Group

n

Mean

Standard Deviation

Runx2

Osteocalcin

Runx2

Osteocalcin

C 7

8

5.79

5.91

1.763

1.770

P 7

8

12.46

12.66

2.126

2.128

C 14

8

4.23

4.25

1.202

1.203

P 14

8

14.11

14.21

2.157

2.160

Notes: control—7 days (C7), treatment—7 days (P7), control—14 days (C14), and treatment—14 days (P14).

 

Table 2. Post Hoc Tukey HSD analysis of Runx2 and Osteocalcin expression

 

C7

P7

C14

P14

C7 (Runx2)

C7 (Osteocalcin)

 

0.000*

0.000*

0.046*

0.035*

0.000*

0.000*

P7 (Runx2)

P7 (Osteocalcin)

0.000*

0.000*

 

0.000*

0.000*

0.60

0.50

C14 (Runx2)

C14 (Osteocalcin)

0.046*

0.035*

0.000*

0.000*

 

0.000*

0.000*

P14 (Runx2)

P14(Osteocalcin)

0.000*

0.000*

0.60

0.50

0.000*

0.000*

 

Notes: control—7 days (C7), treatment—7 days (P7), control—14 days (C14), and treatment—14 days (P14). (*) significant difference (p<0.05)

 

RUNX2 expression in treatment group and control group in 7 days and 14 days:

RUNX2 and osteocalcin are expressed both in 7 days and 14 days after collagen treatment (Figure 1). As shown in table 2, RUNX2 expression was increased in the treatment group and there is a significant difference between the treatment and control groups in seven days and 14 days (p-value = 0.000 for both 7 days and 14 days)

 

Figure 1: Immunohistochemistry of Osteocalcin and RUNX2 expression (yellow arrows) post 7 days and 14 days collagen type-1 treatment. The pictures was taken in magnification 1000x.

 

*

 

*

 
Figure 2: Graphic comparison of RUNX2 expression in treatment group and control group for seven days. C is the control group and P is the treatment group; (*) p-value = 0.000

 

Osteocalcin expression in treatment group and control group in 7 days and 14 days:

Osteocalcin expression increased in the treatment group; there was a significant difference between the treatment and control groups in both 7 days and 14 days (p value = 0.000) (figure 3)

 

*

 

*

 
Figure 3: Graphic comparison of osteocalsin expression in treatment group and control group for 7 and 14 days. C is the control group and P is the treatment group; (*) p-value = 0.000

 

DISCUSSION:

This experiment used gourami fish scale collagen extracted by two methods, acid and enzyme. Acid extraction was performed using acetate acid because it can split the collagen crosslink with non-collagen protein that makes the collagen more soluble during the extraction process. High solubility makes the extraction process easier13.

 

During the process, the function of the enzyme is to control denaturation temperature by controlling the cross-bonding collagen molecule and peptide bonding to preserve the collagen structure. Pepsin enzyme is chosen because, compared to other enzymes, it can preserve the triple helix structure of type 1 collagen. The result is a collagen with better denaturation temperature that is not easily damaged12. Sotelo et al. (2015) have succeeded extracting type 1 collagen from catfish skin and brownbanded bamboo shark with acid and enzyme with better denaturation temperature14.

 

RUNX2, as a gen marker that supports osteoblasts, is a main regulator and transcription factor that controls osteoblast function and differentation15. As a transcription factor, RUNX2 controls the expression of mayor genes matrix protein in bone through direct bonding with osteoblast-specific cis-acting element (OSE). OSE is an osteoblast gen promotor as osteopontin, alkaline phosphatase, type-1 collagen, osteocalcin, and bone sialoprotein. The binding of RUNX2 with OSE will regulate osteogenesis and initiate bone mesenchymal condensation in a growing bone. During the immature stage, RUNX2 will enhance the osteogenesis process16.

 

Osteocalcin, produced by osteoblasts, is the most abundant protein found in bone. This protein is an initiator for hydroxyapatite crystal formation and plays a role in bone mechanical function. Osteocalcin, together with osteopontin, forms a bridge that absorbs the outer energy, thus protecting the bone from fracture17. A study conducted by Vimalraj et al. (2015) showed that RUNX2 can trigger osteocalcin expression with bonding mechanism to cis-acting element as osteocalcin promotor18. The result of the study is the same as the outcome of this experiment, which shows improvement in the RUNX2 expression followed by enhancement of osteocalcin expression.

 

The comparison score for RUNX2 expression between control group and treatment group both 7 and 14 days showed significant enhancement of RUNX2 expression (figure 2). Expression of osteocalcin is also enhanced significantly in the treatment group compared with the control group in seven days and 14 days—C7 compared with P7 and C14 compared with P14 (figure 3). This shows that the material can trigger RUNX2 and osteocalcin expression as a bone growth marker. The expression of the marker was enhanced because this material contains collagen type 1 and hydroxyapatite as a biocomposite19. During osteogenesis, osteoblasts secrete type 1 collagen and non-collagen protein as osteocalcin, bone sialoprotein, and osteopontin. In osteogenesis, collagen works as a container as well as a location to start mineralization of extracellular matrix6.

 

From the author’s previous experiment, collagen extracted from gourami fish scales completely degraded within seven days. This degradation rate needs to be optimised to cope with an ideal scaffold. As the scaffold is expected to be absorbed by the body, controlled resorption is crucial in order to create space for the growth of new bone tissue12.

 

When placed in a tooth socket, collagen, as extracellular matrix, will be tied by integrin alfa 2 beta 1 2β1). This process will trigger phosphorylation via extracellular related kinase (ERK) pathway as a part of mitogen-activated protein kinase (MAPK) signaling pathway. After that, phosphorilating extracellular related kinase (P-ERK) will accumulate in the nucleus. P-ERK and RUNX2 will tie osteocalcin as the promotor gene that induces the osteoblast. RUNX2 and osteocalcin bonding takes place because osteocalcin has two osteoblastic cis-acting elements (OSE) that are OSEa and OSEb as RUNX2 target gene20. After that, RUNX2 will increase self-transcription and other osteoblast induction markers, including osteocalcin. This is the process for RUNX2 and osteocalcin enhancement in this experiment. As a basic component of bone, collagen is an osteoconductive and biocompatible material, which increases osteoinductive, osteointegration, and mechanical properties in the mineralization process of bone formation. Therefore, it shows potential as a bone graft material21.

 

CONCLUSION:

Gourami fish scale collagen enhances the expression of RUNX2 and osteocalcin as a bone growth marker.

 

CONFLICT OF INTEREST:

The authors have no conflicts of interest regarding this investigation.

 

REFERENCES:

1.     Urban IA, Monje A. Guided Bone Regeneration in Alveolar Bone Reconstruction. Oral and Maxillofacial Surgery Clinics of North America. 2019; 31(2): 331–338. doi:10.1016/j.coms.2019.01.003

2.     Shirai Y. Okuda K. Kubota T. Wolff LF. Yoshie H. The comparative effectiveness of granules or blocks of superporous hydroxyapatite for the treatment of intrabony periodontal defects. Open Journal of Stomatology. 2012; 2(2): 81–87. doi.org/10.4236/ojst.2012.22015

3.     Maiorana C, Poli PP, Deflorian M, Testori T, Mandelli F, Nagursky H, et al. Alveolar socket preservation with demineralised bovine bone mineral and a collagen matrix. J Periodontal Implant Sci. 2017; 47:194–210. doi.org/10.5051/jpis.2017.47.4.194

4.     Prahasanti C, Perdana S. The Roles of Insulin Growth Factors-1 (IGF-1) in Bone Graft to increase Osteogenesis. Research Journal of Pharmacy and Technology. 2022; 15(4): 1737-2. doi.org/10.52711/0974-360X.2022.00291

5.     Kumar B. Bhat A. Kumar K. Lakshmanan P. John A, Silvipriya K. Collagen: Animal Sources and Biomedical Application. J Appl Pharm Sci. 2015; 5:123–127. doi.org/10.7324/JAPS.2015.50322

6.     Ferreira AM. Gentile P. Chiono V. Ciardelli G. Collagen for bone tissue regeneration. Acta Biomater. 2012; 8: 3191–3200. doi.org/10.1016/j.actbio.2012.06.014

7.     Shalaby M. Agwa M. Saeed H. Khedr SM. Morsy O. & El-Demellawy MA. Fish Scale Collagen Preparation, Characterization and Its Application in Wound Healing. Journal of Polymers and the Environment. 2020; 28(1): 166–178. doi.org/10.1007/s10924-019-01594-w

8.     Yamada S, Yamamoto K, Ikeda T, Yanagiguchi K, Hayashi Y. Potency of fish collagen as a scaffold for regenerative medicine. Biomed Res Int. 2014; 1-8. doi.org/10.1155/2014/302932

9.     Pereira R dos S. Menezes JD. Bonardi JP. Griza GL. Okamoto R. Hochuli-Vieira E. Histomorphometric and immunohistochemical assessment of RUNX2 and VEGF of BiogranTM and autogenous bone graft in human maxillary sinus bone augmentation: A prospective and randomized study. Clin Implant Dent Relat Res. 2017; 19: 867–875. doi.org/10.1111/cid.12507

10.   Singh S, Kumar D, Lal AK. Serum osteocalcin as a diagnostic biomarker for primary osteoporosis in women. J Clin Diagnostic Res 2015; 9(8): RC04–RC07. doi.org/10.7860/JCDR/2015/14857.6318

11.   Blair HC, Larrouture QC, Li Y, Lin H, Beer-Stoltz D, Liu L, Tuan RS, Robinson LJ, Schlesinger PH, Nelson DJ. Osteoblast Differentiation and Bone Matrix Formation In Vivo and In Vitro. Tissue Eng Part B Rev. 2017; 23(3): 268-280. doi.org/10.1089/ten.TEB.2016.0454

12.   Tangguh HL. Prahasanti C. Ulfah N, Krismariono A. Characterization of pepsin-soluble collagen extracted from gourami (Osphronemus goramy) scales. Niger J Clin Pract. 2021; 24: 89-92. doi.org/10.4103/njcp.njcp_516_19

13.   Schmidt MM. Dornelles RCP. Mello RO. Kubota EH. Mazutti MA. Kempka AP. et al. Collagen extraction process. Int Food Res J. 2016; 23: 913–922.

14.   Sotelo CG. Comesańa MB. Ariza PR. Ricardo I. Characterization of Collagen from Different Discarded Fish Species of the West Coast of the Iberian Peninsula Characterization of Collagen from Different Discarded Fish Species. J Aquat Food Prod Technol. 2015: 0–12. doi.org/10.1080/10498850.2013.865283

15.   Bruderer M. Richards RG. Alini M. Stoddart MJ. Role and regulation of runx2 in osteogenesis. Eur Cells Mater. 2014; 28: 269–286. doi.org/10.22203/ecm.v028a19

16.   Liu TM, Eng Hin Lee. Transcriptional Regulatory Cascades in Runx2-Dependent. Tissue Eng Part B Revn. 2013; 19: 254–263. doi.org/10.1089/ten.TEB.2012.0527

17.   Zoch ML. Clemens TL. Riddle RC. New insights into the biology of osteocalcin. Bone. 2016; 82: 42–49 doi.org/10.1016/j.bone.2015.05.046

18.   Vimalraj S. Arumugam B. Miranda PJ. Selvamurugan N. Runx2: Structure, function, and phosphorylation in osteoblast differentiation. Int J Biol Macromol. 2015; 78: 202–208. doi.org/10.1016/j.ijbiomac.2015.04.008

19.   Ahmed R. Getachew AT. Cho YJ. Chun BS. Application of bacterial collagenolytic proteases for the extraction of type I collagen from the skin of bigeye tuna (Thunnus obesus). LWT. 2018; 89: 44–51. doi.org/10.1016/j.lwt.2017.10.024

20.   Langenbach F. Handschel J. Effects of dexamethasone , ascorbic acid and β -glycerophosphate on the osteogenic differentiation of stem cells in vitro. Stem Cell Res Ther. 2013; 4(117): 1-7. doi.org/10.1186/scrt328

21.   Zhang D. Wu X. Chen J. Lin K. Bioactive Materials The development of collagen based composite scaffolds for bone regeneration. Bioact Mater. 2018; 3: 129–138. doi.org/10.1016/j.bioactmat.2017.08.004

 

 

 

 

 

Received on 24.01.2023           Modified on 27.06.2023

Accepted on 03.10.2023          © RJPT All right reserved

Research J. Pharm. and Tech 2024; 17(1):137-141.

DOI: 10.52711/0974-360X.2024.00022