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ORIGINAL RESEARCH
Year : 2021  |  Volume : 12  |  Issue : 2  |  Page : 73-78

Expression of ALP and TGF-β in Osteoblast Cell Cultures after Administering Collagen Peptide Derived from Gouramy (Osphronemus goramy) Fish Scales


Department of Periodontology, Faculty of Dental Medicine, Airlangga University, Indonesia

Date of Submission02-Oct-2020
Date of Decision01-Dec-2020
Date of Acceptance28-May-2021
Date of Web Publication26-Jul-2021

Correspondence Address:
Chiquita Prahasanti
Department of Periodontology, Faculty of Dental Medicine, Airlangga University, Jl. Prof. Dr Moestopo 47, Surabaya
Indonesia
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/denthyp.denthyp_153_20

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  Abstract 


Introduction: Collagen peptide extracted from gouramy fish scale has noncytotoxic effects and good viability on osteoblast cells. Various characteristic tests also show that it could form as scaffold with the potential to be a biomaterial graft used in tissue engineering. Studies in this field are vital considering the fact that graft is highly essential for the development and success of regeneration therapy. The aim of the study was to examine the expression of alkaline phosphatase (ALP) and transforming growth factor-β (TGF-β) in osteoblast cell cultures after administering collagen peptide derived from gouramy fish scale. Material and Methods: Osteoblast cells were put into 60 well plates divided into two groups. The first group was to analyze the expression of ALP, while the second group was for TGF-β. Then, each of the group was divided into five other groups consisting of cell control and culture of osteoblast cell having collagen peptide derived from gouramy fish scale with four different concentrations. The collagen peptide was extracted through enzymatic method. Then, the immunocytochemistry assay was used to detect the expression of ALP and TGF-β in the osteoblast cell cultures derived from calvaria mice after administering collagen peptide from the fish scale. The statistical methods used include Multivariate Analysisof Variance (MANOVA) analysis with a significance value of 0.000 (P < 0.05) and Tukey Honest Significance Test (HSD). Results: The statistical results showed that the collagen peptide derived from gouramy fish scales with various concentrations produced significantly different expressions of ALP and TGF-β. Conclusion: The expression of ALP and TGF-β in osteoblast cell cultures increased after administering the collagen peptide derived from the gouramy fish scales.

Keywords: Alkaline phosphatase, Fisheries, collagen peptide gouramy scales, oral health, transforming growth factor-β


How to cite this article:
Tionardus M, Dwija Putra IG, Ulfah N, Krismariono A, Setiawatie EM, Prahasanti C. Expression of ALP and TGF-β in Osteoblast Cell Cultures after Administering Collagen Peptide Derived from Gouramy (Osphronemus goramy) Fish Scales. Dent Hypotheses 2021;12:73-8

How to cite this URL:
Tionardus M, Dwija Putra IG, Ulfah N, Krismariono A, Setiawatie EM, Prahasanti C. Expression of ALP and TGF-β in Osteoblast Cell Cultures after Administering Collagen Peptide Derived from Gouramy (Osphronemus goramy) Fish Scales. Dent Hypotheses [serial online] 2021 [cited 2021 Sep 25];12:73-8. Available from: http://www.dentalhypotheses.com/text.asp?2021/12/2/73/322518



Key Messages: The expression of ALP and TGF-ß in osteoblast cell cultures increased after administering the collagen peptide derived from the gouramy fish scales.


  Introduction Top


The balance between inflammatory mediators and their counterregulatory molecules may be fundamental for determining the outcome of the immune pathology of periodontal disease characterized by inflammation and bone loss. Through some biomarker, there is correlation between periodontal disease and systemic diseases. Transforming growth factor-β (TGF-β) and vascular endothelial growth factor are involved in the inflammation and regulation of immune responses, especially in rheumatic disease.[1] Soluble urokinase-type plasminogen activator receptor in patients with coronary heart disease and periodontitis is higher than healthy ones.[2]

Treatment modalities from nonsurgical to surgical are proposed to correct the defect caused by periodontal disease such as periodontitis. The first step in therapy is aimed at guiding behavior change by motivating the patient to undertake successful removal of supragingival dental biofilm and risk factor control. Nutraceutical agent, when combined with scaling and root planing, was demonstrated to be effective in reducing periodontal parameters and controlling the levels of inflammatory mediators and pain in patients with periodontitis.[3]

Regenerative periodontal surgery is one of the treatment modalities to correct bone loss caused by periodontitis. The process of bone healing and regeneration is an important part of regenerative periodontal surgery.[4],[5],[6] Usually, bone is formed through three processes: osteogenesis, modelling, and remodelling. These processes are mediated by osteoblasts that work closely with bone-re­sorbing osteoclasts, which together forming a bone multicel­lular unit. Also, the osteoblasts synthesize extracellular matrix of the bones in the process known as osteogenesis, the osteoclasts carve out its shape so as to fit the physical environment in a process known as modelling, and then it adjusts to the demands of the body growth in a process known as remodelling.[4] In addition, the mineralization of osteoblasts is important for bone regeneration, while osteogenesis and mineralization promote bone repair. Then, the migration of osteoblasts to a damaged area always repairs the bone injury by triggering bone regeneration.[7]

The emergence of bioactive materials in recent years resulted in the new progress recorded in the periodontal defect regeneration.[4] These biomaterials possess osteoinductive properties and then recruit and induce precursor cells in order to differentiate into osteogenic cells, thereby forming inductive bone. Regenerative medicine is made up of three components: cells, nutrients, and collagen. The nutrients could be in the form of growth factors and cytokines chemicals, while collagen could serve as scaffold materials.[8],[9] In addition, collagen substrates usually affect some growth characteristics of cells and balance various facets of cell behavior such as the adhesion, proliferation, and differentiation of cells. Then, the use of bioactive natural organic materials such as fish skin is very essential in the formation of scaffold and also used as an alternative source of collagen.[8],[9],[10]

Collagen is a vital source of biomaterials in different aspects, such as injectable collagen solutions, regenerative medicine, tissue engineering, and drug delivery. It also has the capacity of forming a highly organized intricate three-dimensional architecture of woven fibre networks through self-aggregation and cross-linking. These networks have the ability of resisting tensile stress in multiple directions and supporting cell growth.[11] Also, collagen molecules are made up of a combination of three triple helix chains consisting of 1000 glycine, 360 proline, and 300 hydroxyproline. Then, due to its high spatial structure and molecular weight, naturally, pure collagen does not dissolve in water. But in clinical applications, the collagen scaffold used with bone tissue is usually biodegradable. Also, after the biodegradation process, the collagen splits into collagen peptides that interact directly with the cell.[12]

Fish collagen extracted from the scales, skin, and bone have been extensively used in most laboratories worldwide because of its bioactive properties, such as low antigenicity, excellent biocompatibility, cell proliferation potential, and high biodegradability.[13] It also has various advantages such as high degree of safety, low cost, high absorbability, and no religious barrier when compared with collagen extracted from land-based animals. In addition, the collagens derived from fish skin or scale waste are the type I collagen and the most plentiful protein in fish and human body. Moreover, fish collagens are highly applicable and promising biomaterials in the field of medicine.[14]

Previous studies have demonstrated that gouramy scales collagen can be a good alternative source of collagen because it has high viability against osteoblast cells.[15] Gouramy fish (O. goramy), which live in freshwater, are commonly consumed for their meat, while their scales and bone are considered as wastes.[15],[16]

Alkaline phosphatase (ALP) and TGF-β play a role in bone metabolism. ALP is a molecular marker for osteoblast differentiation and bone formation. It is usually secreted to extracellular matrix alongside Ca2+ salt, thereby increasing the concentration of local phosphate and promoting the mineralization of the matrix.[17] Strong cell differentiation activities always result to high ALP secretion, especially on the level of osteoblasts differentiation. Additionally, the measurement of the activity of ALP in osteoblasts culture medium is considered as a simple, accurate, and fast assay method.[18]

Furthermore, TGF-β acts as a chemoattractant for osteoprogenitor cells, thereby recruiting these cells to the site of formation of new bone or remodelling.[19] The three TGF-β isoforms identified in mammals include TGF-β1, TGF-β2, and TGF-β3. These three have similar biological activities; however, TGF-β1 is the major isoform of TGF-β.[20] TGF-β1 has the capacity of recruiting osteoblasts precursors during bone formation and also produces bone matrix by stimulating osteoblasts differentiation.[7]

Fish collagen has been widely researched for food, cosmetics, and medicine. However, use of collagen derived from fish scales for research in the field of biomaterials is still not widely carried out. The aim of this study is to analyze the ability of osteogenic differentiation of osteoblast cell culture after administration of different concentrations of gouramy fish scale collagen peptide by ALP and TGF-β expression.


  Material and Methods Top


The ethical clearance of this study for the research was approved by the Health Research Ethical Clearance Commission (No.279/HRECC.FODM/X/ 2018). The collagen from gouramy scales was extracted through enzymatic method. The addition of the enzyme pepsin increases its solubility because pepsin is a protein-breaking enzyme. It breaks down telopeptides containing intermolecular cross bonds without affecting the integrity of the triple helix of collagen. In addition, the collagen produced through this extraction process is known as pepsin soluble collagen. Then, pepsin in acetic acid was added to increase the solubility of collagen present in the bones, fins, and skin of the fish to obtain higher yields.[12],[21],[22] The enzymatic hydrolysis was conducted using papain enzyme with the extracted fish scale collagen. The aim was to produce collagen peptides with molecular weight of about 750 to 1300 Da needed for the laboratory test.

The culture of the osteoblast cells was isolated by sequential trypsin digestion of calvaria bone from 2 days old Wistar rat. These osteoblast cells were grown in the medium and incubated in a 5% CO2 incubator at a temperature of 37°C with close observation. Upon growing to 80% confluency, the cell was washed twice with phosphate buffer saline and then 0.2% trypsin-EDTA (Ethylenediaminetetraacetic acid) was added to release cells from the flask. The cell density was measured using a haemocytometer and about 7.5 × 103 cell density in 100 μL media was transferred into a 60-well plate.

The osteoblast cell cultures in the 60-well plate were divided into two groups in order to observe cell biomarkers. The first group, comprising 30 wells, was used to analyze ALP expression, while the second group with another 30 wells was used to analyze TGF-β expression. Then, each biomarker observation group was further divided into five treatment groups with two repetitions, made up of the untreated or cell control group, while the treatment groups were given hydrolyzed collagen peptides at a concentration of 0.0125, 0.05, 0.2, and 0.8 mg/mL. These were 10 well plates in total and the process was repeated three times comprising 24, 48, and 72 hours to create the observation group with a total of 30 well plates. Then, the immunocytochemistry technique was conducted using monoclonal antibodies to detect ALP and TGF-β in osteoblast cell cultures after administering the collagen from fish scales.

The statistical analysis of the readings was conducted using Stastical Package for the Social Sciences (SPSS) and the results of all the quantitative data were expressed as mean ± standard deviation. Also, Multivariate Analysisof Variance (MANOVA) analysis was conducted to test whether there were differences among groups significantly based on several variables. The basis of decision making for this analysis was the significance value smaller than alpha 5%, indicating a significant difference between the biomarker expression based on the time of observation and the concentration of collagen.


  Results Top


The level of expression of the osteogenic markers, ALP and TGF-β, of the osteoblast cells were detected through the immunocytochemistry assay after being cultured for 24, 48, and 72 hours. The results showed a significant increase in the expression of ALP and TGF-β in the cultures of the treatment group at 24, 48, and 72 hours compared with the control. Also, the expression of ALP and TGF-β reached the maximum level at 72 hours of observation. The results of ALP biomarkers in both [Figure 1] and [Figure 2] showed that the highest concentration of collagen peptide in the gouramy scales was at a concentration of 0.8 mg/mL with the control group. However, the most significant time was observed in [Figure 2], where ALP biomarkers were formed at 72 hours of observation. Then, for TGF-β biomarkers, the highest concentration of collagen peptide in the gouramy scales was found at the concentration of 0.8 mg/mL with the control group. Similarly, the most significant time of observation, as shown in Diagram 2 when TGF-β biomarkers were formed, was found at 72 hours or third day of observation.
Figure 1 ALP expression in osteoblast cell culture when varied concentrations of gourami fish scale collagen were administered in observational time. Cell control (A, B), 0.0125 mg/ml (C, D), 0.05 mg/ml (E, F), 0.2 mg/ml (G, H) and 0.8 mg/ml (I, J)

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Figure 2 TGF-β expression in osteoblast cell culture when varied concentrations of gourami fish scale collagen were administered in observational time. Cell control (A, B), 0.0125 mg/ml (C, D), 0.05 mg/ml (E, F), 0.2 mg/ml (G, H) and 0.8 mg/ml (I, J)

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  Discussion Top


This in vitro study showed that the ALP and TGF-β expression increased the expression of ALP and TGF-β in cultures of the treatment group at 24, 48, and 72 hours compared with the control. This is consistent with the statement of Jafary et al.[23] that there was an increase in the expression of ALP and TGF-β at third day of differentiation [Figure 3] and [Figure 4].
Figure 3 Graphical expression of ALP in osteoblast cell culture when varied concentrations of gourami fish scale collagen were administered in observational time

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Figure 4 Graphical expression of TGF-β in osteoblast cell culture when varied concentrations of gourami fish scale collagen were administered in observational time

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Periodontitis, one of the periodontal disease forms, may lead to tooth loss if left untreated. It negatively affects chewing function and aesthetics and impairs quality of life. Periodontitis features may include loss of tissue attachment, bone loss, periodontal pocketing formation, and gingival bleeding. Periodontitis is preventable and treatable in the majority of cases. Early diagnosis and treatment can increase the tooth survival for maintaining quality of life of the patient.[24]

Biomaterial in the form of scaffold plays a vital role in bone tissue engineering, considering the fact that it influences the matrices of tissue formation. Also, selecting the most suitable material in preparing a better scaffold during bone tissue engineering is a key step toward creating a tissue engineered product.[12]

The role of ALP is vital in extracellular matrix formation, while TGF-β plays a major role in repairing of bones.[22] The results of the test conducted on these two markers showed a relationship between concentration and time duration. According to Jafary et al.[23] there was an increased in the expression of ALP at the third day of differentiation, which then decreased within 7 days. This is an indication that the matrix maturation started after 3 days in the treated cells.[23]

ALP is a metalloenzyme produced by epithelial cells in osteoblasts, fibroblasts, polymorphonuclear (PMNs), and macrophages. It acts as an early indicator of both osteoblast differentiation and cellular activities in general. ALP as an osteogenic marker and shows the activity of hard tissue formation and mineralization. The role of this enzyme during bone mineralization is to form an alkaline atmosphere in the osteoid tissue formed, thereby allowing calcium to be easily formed in the tissue. Also, the enzyme increases the concentration of phosphates in the bone, thereby aiding the formation of calcium-phosphate bonds in the form of crystalline hydroxyapatite that then turn into crystals and settle inside the bone. ALP also plays a role during the formation of phosphate from pyrophosphate that helps in bone mineralization. In general, its activity increases in blood plasma when osteoblast activity increases and prolongs simultaneously.[25] In addition, a high level of ALP is an indication of bone turnover and inflammation in the tissues, and there was a slight increase on the second day after stimulation, followed by a steady increase. Also accompanied by an increase in osteoblast differentiation until the 14th day.[25],[26],[27]

During the differentiation and maturation of osteoblasts, ALP is usually the most important biochemical marker. Its activity and localization are both needed for bone development and differentiation.[28] In addition, the significant increase in its expression after being exposed to collagen peptide is the most fundamental biological effect on osteoblast cells. This is because collagen from the fish scales has both physical and chemical stimuli during the proliferation process. Then, the expression of ALP during the mineralization process is an indication of a strong relationship between its increased expression and bone formation, which facilitates its use as an indicator of bone growth.[27]During the osteoblast differentiation, TGF-β increases the production of extracellular matrix proteins in the bone. Kasagij and Chen[29] reported that TGF-βRI and TGF-βRII receptors in osteoblasts from calvaria mice increasingly functioned during osteoblasts differentiation. This shows that osteoblasts are less sensitive to TGF-ß associated with the superfamily of TGF-ß1. Then, after the activation of TGF-β, the differentiation process leading to mature osteoblasts is regulated by bone skeletal protein (BMP), which is also a member of the superfamily of TGF-β.[29]

The TGF-β family is made up of three groups, which are TGF-β1, TGF-β2, and TGF-β3. The TGF-β1 plays important roles in bone tissue regeneration and remodelling as well as bone mass preservation.[30] The osteoblast cells release large quantities of TGF-β1 that play an important role in bone turnover. Osteogenesis and cell migration are also regulated by TGF-β1 through the upregulation of osteogenic and migration-associated genes.[7]

Furthermore, a study by Tachi et al.[31] showed that TGF-β1 has the capacity of increasing the osteoconduction activity of BMP2, where osteoblast maturation is increased by the presence of TGF-β1. Both cytokines BMP2 and TGF-β1 also play important roles in the healing process and differentiation of mesenchymal cells into chondrocytes and osteoblasts.

Based on the results of the immunocytochemical assay, the expression of ALP and TGF-β was obvious in the osteoblast cell culture after administering the collagen derived from the O. goramy fish scales. In conclusion, the expression of ALP and TGF-β is enhanced after the application of O. goramy fish scale collagen in osteoblast culture.

Financial support and sponsorship

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Conflicts of interest

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