Lyophilized platelet-rich fibrin (PRF) promotes craniofacial bone regeneration through Runx2

Abstract

Freeze-drying is an effective means to control scaffold pore size and preserve its composition. The purpose of the present study was to determine the applicability of lyophilized Platelet-rich fibrin (LPRF) as a scaffold for craniofacial tissue regeneration and to compare its biological effects with commonly used fresh Platelet-rich fibrin (PRF). LPRF caused a 4.8-fold±0.4-fold elevation in Runt-related transcription factor 2 (Runx2) expression in alveolar bone cells, compared to a 3.6-fold±0.2-fold increase when using fresh PRF, and a more than 10-fold rise of alkaline phosphatase levels and mineralization markers. LPRF-induced Runx2 expression only occurred in alveolar bone and not in periodontal or dental follicle cells. LPRF also caused a 1.6-fold increase in osteoblast proliferation (p<0.001) when compared to fresh PRF. When applied in a rat craniofacial defect model for six weeks, LPRF resulted in 97% bony coverage of the defect, compared to 84% for fresh PRF, 64% for fibrin, and 16% without scaffold. Moreover, LPRF thickened the trabecular diameter by 25% when compared to fresh PRF and fibrin, and only LPRF and fresh PRF resulted in the formation of interconnected trabeculae across the defect. Together, these studies support the application of lyophilized PRF as a biomimetic scaffold for craniofacial bone regeneration and mineralized tissue engineering.

Figure 1.

Macro-, micro-, and submicrographic comparison of fresh and lyophilized PRF structure. (A,B) are macrophotographs of fresh and lyophilized Platelet-rich fibrin (PRF) preparations, showing cell-free preparations on the left, while the preparations on the right contained red blood cells; (C–F) are scanning electron micrographs at 300-fold magnification (C,D) and 2000-fold magnification (E,F). (A,C,E) are from fresh PRF and (B,D,F) are from lyophilized PRF. A scale bar in the left-hand panel serves as a reference for the level of magnification in both panels.

Figure 2.

Effects of fresh and lyophilized PRF on proliferation and migration of periodontal progenitor populations. (A–C) illustrate the results of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide colorimetric proliferation assays when dental follicle progenitors (A), periodontal ligament progenitors (B), and alveolar bone osteoblast progenitors (C) were cultured on PRF-related substrates. The three different substrates used in the proliferation study, fresh PRF, lyophilized PRF, and DMEM medium, are distinguished by line patterns (A); (D) Difference in chemotaxis behavior between periodontal progenitors when cultured in PRF-conditioned media and DMEM medium. The three experimental groups are distinguished by shades of gray.

Figure 3.

Effect of fresh and lyophilized PRF, and DMEM medium on mineralization behavior of periodontal progenitor populations. (A–D) are based on results from alkaline phosphatase staining assays and (E–H) are based on alizarin red S mineralization assays. In (A–C,E–G), alkaline phosphatase or alizarin red staining in periodontal progenitor cells cultured for 7, 14, and 21 days were compared. Different co-culture conditions (fresh or lyophilized PRF and DMEM) are distinguished by different bar patterns which are identified in the bar legend above Figure A. The three periodontal progenitor populations compared in this study, dental follicle, periodontal ligament, and alveolar bone are labeled on the x-axis of the graphs in (A–C,E–G); (D,H) illustrate the differences in mineralization behavior between alveolar bone progenitors co-cultured with fresh or lyophilized PRF and DMEM; (D) is a photograph of the alkaline phosphate stained 6-well plate and (H) is a photograph of the alizarin red stained 6-well plate.

Figure 4.

Differences in mineralization-associated gene expression patterns in co-cultures of periodontal progenitors and centrifuged blood derivatives. (A–C) are real-time RT-PCR assays for the osteoblast transcription factor Runx2; and (D–F) are real-time RT-PCR assays for the calcification inhibitor Matrix Gla Protein. Different co-culture conditions (fresh or lyophilized PRF and DMEM) are distinguished by different bar patterns identified in the upper right corner of the figure. The three periodontal progenitor populations compared in this study, dental follicle, periodontal ligament, and alveolar bone are labeled on the x-axis of the graphs in (A–F).

Figure 5.

Comparison between lyophilized and fresh PRF in subcutaneous implants. Subcutaneous implants of fresh (A,C) and lyophilized PRF (B,D) were dissected from nude mice after seven days (A,B) and 14 days (C,D) of implantation. The samples were fixed and processed for paraffin sections and stained with hematoxylin and eosin. Note the loose and porous structure of the lyophilized PRF (B) when compared to fresh PRF (A) after seven days of implantation. The lyophilized PRF scaffold was infiltrated by fibroblast-like cells (D), while the fresh PRF remained intact (C) after 14 days of implantation.

Figure 6.

Analysis of bone regeneration in critical size calvarial defects using fibrin gel, fresh and lyophilized PRF as scaffolds. (A,C,E,G) are three-dimensional cranial microCT images of control (A), fibrin gel (C), fresh PRF (E) and lyophilized PRF (G) after six weeks of implantation. Regenerated bone was visualized in blue; (B,D,F,H) are photomicrographs of decalcified paraffin sections through control (B), fibrin gel (D), fresh PRF (F), and lyophilized PRF (H) six-week implants stained with Mallory’s procedure. Collagen-containing trabecular bone was stained in blue and fibrin-containing tissue in pink; (I,J) illustrate the results from our morphometric analysis of the regenerated tissue covering the calvarial defect; (I) documents the relative calvarial defect area by dividing the original area of the defect with a diameter of 5 mm by the area covered in the control, fibrin gel, fresh and lyophilized PRF treatment groups, and (J) represents the morphometric analysis of bone versus fiber ratio in all four groups. **: p < 0.01, and ***: p < 0.001.