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. 2019 Feb 28;20(5):1053.
doi: 10.3390/ijms20051053.

Platelet-Rich Fibrin Extract: A Promising Fetal Bovine Serum Alternative in Explant Cultures of Human Periosteal Sheets for Regenerative Therapy

Tomoyuki Kawase  1 , Masaki Nagata  2 , Kazuhiro Okuda  3 , Takashi Ushiki  4 , Yoko Fujimoto  5 , Mari Watanabe  6 , Akira Ito  7 , Koh Nakata  8
Affiliations

Platelet-Rich Fibrin Extract: A Promising Fetal Bovine Serum Alternative in Explant Cultures of Human Periosteal Sheets for Regenerative Therapy

Tomoyuki Kawase et al. Int J Mol Sci. .

Abstract

In 2004, we developed autologous periosteal sheets for the treatment of periodontal bone defects. This regenerative therapy has successfully regenerated periodontal bone and augmented alveolar ridge for implant placement. However, the necessity for 6-week culture is a limitation. Here, we examined the applicability of a human platelet-rich fibrin extract (PRFext) as an alternative to fetal bovine serum (FBS) for the explant culture of periosteal sheets in a novel culture medium (MSC-PCM) originally developed for maintaining mesenchymal stem cells. Small periosteum tissue segments were expanded in MSC-PCM + 2% PRFext for 4 weeks, and the resulting periosteal sheets were compared with those prepared by the conventional method using Medium199 + 10% FBS for their growth rate, cell multilayer formation, alkaline phosphatase (ALP) activity, and surface antigen expression (CD73, CD90, and CD105). Periosteal sheets grew faster in the novel culture medium than in the conventional medium. However, assessment of cell shape and ALP activity revealed that the periosteal cells growing in the novel medium were relatively immature. These findings suggest that the novel culture medium featuring PRFext offers advantages by shortening the culture period and excluding possible risks associated with xeno-factors without negatively altering the activity of periosteal sheets.

Keywords: bone grafting material; differentiation; growth; periosteal sheet; platelet-rich fibrin.

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Conflict of interest statement

A.I. who is an employee of Kohjin Bio, Co., Ltd. was involved in the collection, analyses and interpretation of data. Author T.K., M.N., K.O., T.U., Y.F., M.W. and K.N. state that there are no conflicts of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results.

Figures

Figure 1
Figure 1
Effects of different culture media on the onset of periosteal cell outgrowth. The data obtained from periosteum samples derived from four independent donors are shown. X-axis: types of culture media. Statistical analysis was performed by Kruskal–Wallis one-way analysis of variance, followed by Steel–Dwass multiple comparison test. No significant difference was observed between the groups. N = 3, 4, 5, 6, or 9 replicates.
Figure 2
Figure 2
Photomicrographs of periosteal cells in the central and peripheral regions of periosteal sheets cultured in different culture media. (A) Medium199 + 10% fetal bovine serum (FBS), (B) MSC-PCM + 4% FBS, (C) MSC-PCN + 2% platelet-rich fibrin extract (PRFext). Bar = 50 µm. PTS: periosteum tissue segment.
Figure 3
Figure 3
Effects of different culture media on the growth of periosteal sheets. The data obtained from periosteum samples derived from four independent donors are shown. X-axis: time periods (weeks) of explant culture. N = 2 (6 weeks), 3 (6 weeks), 4, 5, or 7 replicates. Statistical analysis was performed by Kruskal–Wallis one-way analysis of variance, followed by Steel–Dwass multiple comparison test. * p < 0.05 as compared with the control group (Medium199 + 10% FBS) at same time points. ** p < 0.05 as compared with the other experimental group (MSC-PCM + 4% FBS) at same time points.
Figure 4
Figure 4
Effects of different culture media on the alkaline phosphatase (ALP) activity and size of periosteal sheets. Fixed individual periosteal sheets were first stained for ALP activity (positive: dark blue-purple) and subsequently treated with Safranin-O (Saf.-O) for the evaluation of their sizes. We used 60 mm culture dishes.
Figure 5
Figure 5
Effects of different culture media on the thickness of periosteal sheets. In von Kossa staining, calcium deposits were stained black. These data are representative of five independent experiments. hematoxylin and eosin (HE) staining. Bar = 200 µm.
Figure 6
Figure 6
Effects of different culture media on the expression of platelet-derived growth factor-B (PDGF-B), transforming growth factor beta 1 (TGFβ1), and collagen type I in the central region of periosteal sheets (outgrowth area). Immunohistochemical staining with visualization using 3′-diaminobenzidine (DAB) (positive: dark brown). These data are representative of five independent experiments. Bar = 50 µm.
Figure 7
Figure 7
Effects of different culture media on the expression of surface antigens. Only periosteal sheets cultured with MSC-PCM + 2% PRFext were compared with the control sheets (Sample 21). X-axis: type of surface antigens. Statistical analysis was performed using the Mann–Whitney rank-sum test and no significant difference was observed between the two groups. N = 3 or 4 replicates.
Figure 8
Figure 8
Phases in the process of periosteal sheet cultures.
Figure 9
Figure 9
Graphic summary of preparation of platelet-rich fibrin (PRF) extract.
See this image and copyright information in PMC

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