Bone alkaline phosphatase and osteocalcin expression of rat's Gingival mesenchymal stem cells cultured in platelet-rich fibrin for bone remodeling (in vitro study)

ABSTRACT

Objective: The aim of this study was to analyze the osteogenic differentiation of rat GMSCs cultured in PRF for bone remodeling. Materials and Methods: GMSCs were isolated from the lower gingival tissue of four healthy, 250 g, 1-month old, male rats (Rattus norvegicus) cut into small fragments, cultured for 2 weeks, and subsequently passaged every 4–5 days. GMSCs isolated in passage 3 were characterized by CD34, CD45, CD44, CD73, CD90, and CD105 using fluorescein isothiocyanate immunocytochemistry (ICC) examination. GMSCs in passage 3–5 cultured in five M24 plates (N = 108; n = 6/group) for 7, 14, and 21 days with three different mediums as follows: Control (−) group: α-Modified Eagle Medium; Control (+) group: High-dose glucose Dulbecco's Modified Eagle's Medium (DMEM-HG) + osteogenic medium; and treatment group: DMEM-HG + osteogenic medium + PRF. GMSCs were osteogenic differentiation cultured in vitro in three different mediums by bone alkaline phosphatase (BALP) and osteocalcin (OSC) marker using ICC monoclonal antibody. Statistical Analysis Used: The one-way analysis of variance was performed (P < 0.05) based on Shapiro–Wilk and Levene's tests (P > 0.05). Results: GMSCs were shown to present + CD44, +CD73, +CD90, +CD105 and − CD34, − and CD45 expression as MSCs markers. The treatment group showed the highest BALP expression (16.00 ± 1.732) on day 7, while OSC expression (13.67 ± 2.309) on day 21 showed the statistically significant difference between groups (P < 0.05). Conclusion: GMSCs cultured in PRF demonstrated potential osteogenic differentiation ability capable of accelerating in vitro bone remodeling by enhancing BALP and OSC expression.

Key words:

Bone alkaline phosphatase - gingival mesenchymal stem cells - osteocalcin - osteogenic differentiation - platelet-rich fibrin

• REFERENCES

• 1 Barzilay R, Melamed E, Offen D. Introducing transcription factors to multipotent mesenchymal stem cells: Making transdifferentiation possible. Stem Cells 2009; 27: 2509-15
• 2 Egusa H, Sonoyama W, Nishimura M, Atsuta I, Akiyama K. Stem cells in dentistry – part I: Stem cell sources. J Prosthodont Res 2012; 56: 151-65
• 3 Ponnaiyan D, Jegadeesan V. Comparison of phenotype and differentiation marker gene expression profiles in human dental pulp and bone marrow mesenchymal stem cells. Eur J Dent 2014; 8: 307-13
• 4 Augello A, Kurth TB, deBari C. Mesenchymal stem cells: A perspective from in vitro cultures to in vivo migration and niches. Eur Cells Mater 2010; 20: 121-33
• 5 Law S, Chaudhuri S. Mesenchymal stem cell and regenerative medicine: Regeneration versus immunomodulatory challenges. Am J Stem Cells 2013; 2: 22-38
• 6 Gao F, Chiu SM, Motan DA, Zhang Z, Chen L, Ji HL. et al. Mesenchymal stem cells and immunomodulation: Current status and future prospects. Cell Death Dis 2016; 7: e2062
• 7 Miran S, Mitsiadis TA, Pagella P. Innovative dental stem cell-based research approaches: The future of dentistry. Stem Cells Int 2016; 2016: 7231038
• 8 Diomede F, Rajan TS, Gatta V, D'Aurora M, Merciaro I, Marchisio M. et al. Stemness maintenance properties in human oral stem cells after long-term passage. Stem Cells Int 2017; 2017: 5651287
• 9 Egusa H, Sonoyama W, Nishimura M, Atsuta I, Akiyama K. Stem cells in dentistry – Part II: Clinical applications. J Prosthodont Res 2012; 20: 229-48
• 10 Niibe K, Suehiro F, Oshima M, Nishimura M, Kuboki T, Egusa H. et al. Challenges for stem cell-based “regenerative prosthodontics”. J Prosthodont Res 2017; 61: 3-5
• 11 Baek HS, Lee HS, Kim BJ, Chung IK, Kim CH, Jin SM. et al. Effect of platelet-rich fibrin on repair of defect in the articular disc in rabbit temporomandibular joint by platelet-rich fibrin. Tissue Eng Regen Med 2011; 8: 530-5
• 12 Kazemi D, Fakhrjou A, Dizaji VM, Alishahi MK. Effect of autologous platelet rich fibrin on the healing of experimental articular cartilage defects of the knee in an animal model. Biomed Res Int 2014; 2014: 486436
• 13 Kamadjaja MJ, Salim S, Rantam FA. Osteogenic potential differentiation of human amnion mesenchymal stem cell with chitosan- carbonate apatite. Bali Med J 2016; 5: 71-7
• 14 Zeidán-Chuliá F, Noda M. “Opening” the mesenchymal stem cell tool box. Eur J Dent 2009; 3: 240-9
• 15 Granéli C, Thorfve A, Ruetschi U, Brisby H, Thomsen P, Lindahl A. et al. Novel markers of osteogenic and adipogenic differentiation of human bone marrow stromal cells identified using a quantitative proteomics approach. Stem Cell Res 2014; 12: 153-65
• 16 Nugraha AP, Narmada IB, Ernawati DS, Dinaryanti A, Hendrianto E, Ihsan IS. et al. Osteogenic potential of gingival stromal progenitor cells cultured in platelet rich fibrin is predicted by core-binding factor subunit-α1/Sox9 expression ratio (in vitro) [version 1; referees: 1 approved, 2 approved with reservations]. F1000Research 2018; 7: 1134
• 17 Rantam FA, Nugraha AP, Narmada IB, Ernawati DS, Widodo ADW, Lestari P. et al. Gingival mesenchymal stem cells from wistar rat's gingiva (Rattus Novergicus) - Isolation and characterization (In Vitro Study). J Int Med Res 2018; 11: 694-9
• 18 Jin SH, Lee JE, Yun JH, Kim I, Ko Y, Park JB. et al. Isolation and characterization of human mesenchymal stem cells from gingival connective tissue. J Periodontal Res 2015; 50: 461-7
• 19 Borie E, Oliví DG, Orsi IA, Garlet K, Weber B, Beltrán V. et al. Platelet-rich fibrin application in dentistry: A literature review. Int J Clin Exp Med 2015; 8: 7922-9
• 20 Rahmawati D, Roestamadji RI, Yuliati A, Bramantoro T. Osteogenic ability of combined hematopoetic stem cell, hydroxyapatite graft and platelet rich fibrin on rats (Rattus novergicus). J Krishna Instit Med Sci Univ 2017; 6: 88-95
• 21 Ekizer A, Yalvac ME, Uysal T, Sonmez MF, Sahin F. Bone marrow mesenchymal stem cells enhance bone formation in orthodontically expanded maxillae in rats. Angle Orthod 2015; 85: 394-9
• 22 Kuo YC, Au HK, Hsu JL, Wang HF, Lee CJ, Peng SW. et al. IGF-1R promotes symmetric self-renewal and migration of alkaline phosphatase+germ stem cells through HIF-2α-OCT4/CXCR4 loop under hypoxia. Stem Cell Reports 2018; 10: 524-37
• 23 Kobayashi E, Flückiger L, Fujioka-Kobayashi M, Sawada K, Sculean A, Schaller B. et al. Comparative release of growth factors from PRP, PRF, and advanced-PRF. Clin Oral Investig 2016; 20: 2353-60
• 24 Li Q, Reed DA, Min L, Gopinathan G, Li S, Dangaria SJ. et al. Lyophilized platelet-rich fibrin (PRF) promotes craniofacial bone regeneration through runx2. Int J Mol Sci 2014; 15: 8509-25
• 25 Cortese A, Pantaleo G, Borri A, Caggiano M, Amato M. Platelet-rich fibrin (PRF) in implant dentistry in combination with new bone regenerative technique in elderly patients. Int J Surg Case Rep 2016; 28: 52-6
• 26 Yang S, En L, Yanwei G, Mingguo W. Platelet-rich fibrin promotes osteogenic differentiation of bone marrow mesenchymal stem cells through canonical Wnt/β-catenin signaling pathway. Int J Oral Maxillofac Surg 2015; 44: e310
• 27 Loebel C, Czekanska EM, Bruderer M, Salzmann G, Alini M, Stoddart MJ. et al. In vitro osteogenic potential of human mesenchymal stem cells is predicted by runx2/Sox9 ratio. Tissue Eng Part A 2015; 21: 115-23
• 28 Duan X, Lin Z, Lin X, Wang Z, Wu Y, Ji M. et al. Study of platelet-rich fibrin combined with rat periodontal ligament stem cells in periodontal tissue regeneration. J Cell Mol Med 2018; 22: 1047-55
• 29 Dohan Ehrenfest DM, Diss A, Odin G, Doglioli P, Hippolyte MP, Charrier JB. et al. In vitro effects of choukroun's PRF (platelet-rich fibrin) on human gingival fibroblasts, dermal prekeratinocytes, preadipocytes, and maxillofacial osteoblasts in primary cultures. Oral Surg Oral Med Oral Pathol Oral Radiol Endod 2009; 108: 341-52
• 30 He L, Lin Y, Hu X, Zhang Y, Wu H. A comparative study of platelet-rich fibrin (PRF) and platelet-rich plasma (PRP) on the effect of proliferation and differentiation of rat osteoblasts in vitro . Oral Surg Oral Med Oral Pathol Oral Radiol Endod 2009; 108: 707-13
• 31 Huang FM, Yang SF, Zhao JH, Chang YC. Platelet-rich fibrin increases proliferation and differentiation of human dental pulp cells. J Endod 2010; 36: 1628-32
• 32 Dohan Ehrenfest DM, Doglioli P, dePeppo GM, Del Corso M, Charrier JB. Choukroun's platelet-rich fibrin (PRF) stimulates in vitro proliferation and differentiation of human oral bone mesenchymal stem cell in a dose-dependent way. Arch Oral Biol 2010; 55: 185-94
• 33 Chang IC, Tsai CH, Chang YC. Platelet-rich fibrin modulates the expression of extracellular signal-regulated protein kinase and osteoprotegerin in human osteoblasts. J Biomed Mater Res A 2010; 95: 327-32
• 34 Paldanius PM. The Role of Osteocalcin in Human Bone Metabolism and Glucose Homeostasis. Academic Disertation 2017 https://www.helda.helsinki.fi/bitstream/handle/10138/173087/THEROLEO.pdf?sequence=1 . [Last accessed on 2018 Jun 01]
• 35 Nakamura A, Dohi Y, Akahane M, Ohgushi H, Nakajima H, Funaoka H. et al. Osteocalcin secretion as an early marker of in vitro osteogenic differentiation of rat mesenchymal stem cells. Tissue Eng Part C Methods 2009; 15: 169-80
• 36 Li Q, Pan S, Dangaria SJ, Gopinathan G, Kolokythas A, Chu S. et al. Platelet-rich fibrin promotes periodontal regeneration and enhances alveolar bone augmentation. Biomed Res Int 2013; 2013: 638043