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Effect of Leukocyte-Platelet Rich Fibrin (L-PRF) on Tissue Regeneration and Proliferation of Human Gingival Fibroblast Cells Cultured Using a Modified Method

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Tissue Engineering and Regenerative Medicine Aims and scope

Abstract

Background:

An in vitro study on rapid culturing method of human gingival fibroblast cells (HGFCs) was established to investigate the potential use of the leukocyte-platelet rich fibrin (L-PRF) in tissue engineering technology, different medical fields, including periodontology and implantology.

Methods:

Eight biopsies were obtained from eight different donors and a modified culturing technique was developed to obtain HGFCs. The modified 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide MTT assay was used to compare the cell viability when the modified culturing method was used in comparison to the standard method. Blood samples were collected from the same patients and L-PRF was isolated using a standard protocol. The releases of platelet-derived growth factor-AA and transforming growth factor-beta1 at various time intervals were observed using enzyme-linked immunosorbent assay (ELISA) kit. The proliferative effect of L-PRF on HGFCs was assessed by the cell counting kit—8 assay.

Results:

A simple and rapid modified method for in vitro HGFC culture yielded a cellular monolayer within three to nine days after cell culture. L-PRF with three-dimensional polymer fibers released growth factors that peaked during the first three hours and continued to produce up to 10 days. The L-PRF presented a dose-dependent effect on HGFCs proliferation where HGFCs proliferation increased with an increase in L-PRF concentration.

Conclusion:

The modified technique for the culture of HGFCs might be useful for the development of future experimental and clinical studies, besides L-PRF has great therapeutic potential in oral surgery fields.

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References

  1. Reid CB, Cloos J, Snow GB, Braakhuis BJ. A simple and reliable technique for culturing of human oral keratinocytes and fibroblasts. Acta Otolaryngol. 1997;117:628–33.

    Article  CAS  Google Scholar 

  2. Mak K, Manji A, Gallant-Behm C, Wiebe C, Hart DA, Larjava H, et al. Scarless healing of oral mucosa is characterized by faster resolution of inflammation and control of myofibroblast action compared to skin wounds in the red Duroc pig model. J Dermatol Sci. 2009;56:168–80.

    Article  CAS  Google Scholar 

  3. Winning TA, Townsend GC. Oral mucosal embryology and histology. Clin Dermatol. 2000;18:499–511.

    Article  CAS  Google Scholar 

  4. Wanichpakorn S, Kedjarune-Laggat U. Primary cell culture from human oral tissue: gingival keratinocytes, gingival fibroblasts and periodontal ligament fibroblasts. Songklanakarin J Sci Technol. 2010;32:327–31.

    CAS  Google Scholar 

  5. Kedjarune U, Pongprerachok S, Arpornmaeklong P, Ungkusonmongkhon K. Culturing primary human gingival epithelial cells: comparison of two isolation techniques. J Craniomaxillofac. 2001;29:224–31.

    Article  CAS  Google Scholar 

  6. Klingbeil MF, Herson MR, Cristo EB, dos Santos Pinto D Jr, Yoshito D, Mathor MB. Comparison of two cellular harvesting methods for primary human oral culture of keratinocytes. Cell Tissue Bank. 2009;10:197–204.

    Article  Google Scholar 

  7. Lauer G, Otten JE, von Specht BU, Schilli W. Cultured gingival epithelium: a possible suitable material for pre-prosthetic surgery. J Craniomaxillofac Surg. 1991;19:21–6.

    Article  CAS  Google Scholar 

  8. Malekzadeh R, Hollinger JO, Buck D, Adams DF, McAllister BS. Isolation of human osteoblast-like cells and in vitro amplification for tissue engineering. J Periodontol. 1998;69:1256–62.

    Article  CAS  Google Scholar 

  9. Salkin LM, Freedman AL, Stein MD, Bassiouny MA. A longitudinal study of untreated mucogingival defects. J Periodontol. 1987;58:164–6.

    Article  CAS  Google Scholar 

  10. Hürzele MB, Weng D. Functional and esthetic outcome enhancement of periodontal surgery by application of plastic surgery principles. Int J Periodontics Restorative Dent. 1999;19:36–43.

    Google Scholar 

  11. Newman MG, Takei HH, Klokkevold PR, Carranza FA, editors. St Louis Missouri: Saunders Elsevier; 2006. p. 1023–5.

  12. Anfossi G, Trovati M, Mularoni E, Massucco P, Calcamuggi G, Emanuelli G. Influence of propranolol on platelet aggregation and thromboxane B2 production from platelet-rich plasma and whole blood. Prostaglandins Leukot Essent Fatty Acids. 1989;36:1–7.

    Article  CAS  Google Scholar 

  13. Fijnheer R, Pietersz RN, de Korte D, Gouwerok CW, Dekker WJ, Reesink HW, Platelet activation during preparation of platelet concentrates: a comparison of the platelet-rich plasma and the buffy coat methods. Transfusion. 1990;30:634–8.

    Article  CAS  Google Scholar 

  14. Marx RE. Platelet-rich plasma: evidence to support its use. J Oral Maxillofac Surg. 2004;62:489–96.

    Article  Google Scholar 

  15. Lekovic V, Milinkovic I, Aleksic Z, Jankovic S, Stankovic P, Kenney EB, et al. Platelet-rich fibrin and bovine porous bone mineral versus platelet-rich fibrin in the treatment of intrabony periodontal defects. J Periodontal Res. 2012;47:409–17.

    Article  CAS  Google Scholar 

  16. Shivashankar VY, Johns DA, Vidyanath S, Sam G. Combination of platelet rich fibrin, hydroxyapatite and PRF membrane in the management of large inflammatory periapical lesion. J Conserv Dent. 2013;16:261–4.

    Article  CAS  Google Scholar 

  17. Medina-Porqueres I, Alvarez-Juarez P. The efficacy of platelet-rich plasma injection in the management of hip osteoarthritis: a systematic review protocol. Musculoskelet Care. 2016;14:121–5.

    Article  Google Scholar 

  18. Mudalal M, Zhou Y. Biological additives and platelet concentrates for tissue engineering on regenerative dentistry basic Science and concise review. Asian J Pharm. 2017;11:255–63.

    CAS  Google Scholar 

  19. Choukroun J, Adda F, Schoeffler C, Vervelle A. Une opportunité en paro-implantologie: le PRF. Implantodontie. 2001;42:e62.

    Google Scholar 

  20. Salamanna F, Veronesi F, Maglio M, Della Bella E, Sartori M, Fini M. New and emerging strategies in platelet-rich plasma application in musculoskeletal regenerative procedures: general overview on still open questions and outlook. Biomed Res Int. 2015;2015:846045.

    Article  Google Scholar 

  21. Chang YC, Zhao JH. Effects of platelet-rich fibrin on human periodontal ligament fibroblasts and application for periodontal infrabony defects. Aust Dent J. 2011;56:365–71.

    Article  Google Scholar 

  22. Qin J, Wang L, Sun Y, Sun X, Wen C, Shahmoradi M, et al. Concentrated growth factor increases Schwann cell proliferation and neurotrophic factor secretion and promotes functional nerve recovery in vivo. Int J Mol Med. 2016;37:493–500.

  23. Saczko J, Dominiak M, Kulbacka J, Chwiłkowska A, Krawczykowska H. A simple and established method of tissue culture of human gingival fibroblasts for gingival augmentation. Folia Histochem Cytobiol. 2008;46(1):117–9.

    Article  Google Scholar 

  24. Hassell T, Stanek EJ III. Evidence that healthy human gingiva contains functionally heterogeneous fibroblast subpopulations. Arch Oral Biol. 1983;28:617–25.

    Article  CAS  Google Scholar 

  25. Mosmann T. Rapid colorimetric assay for cellular growth and survival: application to proliferation and cytotoxicity assays. J Immunol Methods. 1983;65:55–63.

    Article  CAS  Google Scholar 

  26. Miron R, Choukroun J, Ghanaati S. Controversies related to scientific report describing g-forces from studies on platelet-rich fibrin: necessity for standardization of relative centrifugal force values. Int J Growth Factors Stem Cells Dent. 2018;1:80–6.

    Article  Google Scholar 

  27. Kobayashi M, Kawase T, Horimizu M, Okuda K, Wolff LF, Yoshie H. A proposed protocol for the standardized preparation of PRF membranes for clinical use. Biologicals. 2012;40:323–9.

    Article  CAS  Google Scholar 

  28. Dohan Ehrenfest DM, Doglioli P, de Peppo 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.

    Article  CAS  Google Scholar 

  29. Pini Prato GP, Rotundo R, Mognoni C, Soranzo C. Tissue engineering technology for gingival augmentation procedures: a case report. Int J Periodontics Restorative Dent. 2000;20:552–9.

    CAS  PubMed  Google Scholar 

  30. Castillo TN, Pouliot MA, Kim HJ, Dragoo JL. Comparison of growth factor and platelet concentration from commercial platelet-rich plasma separation systems. Am J Sprots Med. 2011;39:266–71.

    Article  Google Scholar 

  31. Mudalal M, Sun X, Li X, Zhou Y. The evaluation of leukocyte-platelet rich fibrin as an antiinflammatory autologous biological additive: a novel in vitro study. Saudi Med J. 2019;40:657–68.

    Article  Google Scholar 

  32. Boora P, Rathee M, Bhoria M. Effect of platelet rich fibrin (PRF) on peri-implant soft tissue and crestal bone in one-stage implant placement: a randomized controlled trial. J Clin Diagn Res. 2015;9:ZC18–21.

    PubMed  Google Scholar 

  33. Wang Z, Mudalal M, Sun Y, Liu Y, Wang J, Wang Y, et al. The effects of leukocyte-platelet rich fibrin (L-PRF) on suppression of the expressions of the pro-inflammatory cytokines, and proliferation of Schwann cell, and neurotrophic factors. Sci Rep. 2020;10:2421.

    Article  CAS  Google Scholar 

  34. Morales-Ruiz M, Fulton D, Sowa G, Languino LR, Fujio Y, Walsh K, et al. Vascular endothelial growth factor–stimulated actin reorganization and migration of endothelial cells is regulated via the serine/threonine kinase Akt. Circ Res. 2000;86:892–6.

    Article  CAS  Google Scholar 

  35. Chan CM, Chang HH, Wang VC, Huang CL, Hung CF. Inhibitory effects of resveratrol on PDGF-BB-induced retinal pigment epithelial cell migration via PDGFRβ, PI3K/Akt and MAPK pathways. PLoS One. 2013;8:e56819.

    Article  CAS  Google Scholar 

  36. Maucksch C, McGregor A, Yang M, Gordon RJ, Connor B. IGF-I redirects doublecortin-positive cell migration in the normal adult rat brain. Neuroscience. 2013;241:106–15.

    Google Scholar 

  37. Tohidnezhad M, Bayer A, Rasuo B, Hock JVP, Kweider N, Fragoulis A, et al. Platelet-released growth factors modulate the secretion of cytokines in synoviocytes under inflammatory joint disease. Mediators Inflamm. 2017;2017:1046438.

    Article  Google Scholar 

Download references

Acknowledgements

1This research article was supported by the department of implantology, Stomatology Hospital of Jilin university. 2Jilin Province of Science and Technology for Development (serial number 20160101138JC).

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Correspondence to Xiaolin Sun or Yanmin Zhou.

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Authors have no conflict of interests, and the work was not supported or funded by any companies.

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The study protocol was approved by the institutional review board of School and Hospital of Stomatology at Jilin University (IRB no. 3).

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Mudalal, M., Wang, Z., Mustafa, S. et al. Effect of Leukocyte-Platelet Rich Fibrin (L-PRF) on Tissue Regeneration and Proliferation of Human Gingival Fibroblast Cells Cultured Using a Modified Method. Tissue Eng Regen Med 18, 895–904 (2021). https://doi.org/10.1007/s13770-021-00360-1

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