The MC3T3-E1 cell line, a clonal pre-osteoblastic cell line derived from newborn mouse calvaria, was grown in α-minimum essential medium (α-MEM; ICN Pharmaceuticals, Inc.) supplemented with 10% fetal bovine serum (FBS; Thermo Fisher Scientific), 50 µg/ml ascorbate 2-phosphate, 10 mM β-glycerophosphate, and 40 mM HEPES (pH 7.4), as described (24). Mouse NIH3T3 fibroblasts were purchased from Riken (Tsukuba) and maintained in Dulbecco's modified Eagle's medium (DMEM; Thermo Fisher Scientific) supplemented with FBS. All cells were cultured at 37°C in a humidified atmosphere containing 5% CO₂ and 95% air.

Male Wistar rats, aged 8–10 weeks and weighing 400–450 g, were obtained from Clea Japan Inc., Tokyo, Japan). The rats were housed individually in a barrier facility for laboratory animals with a 12 h light-dark cycle and allowed food and water ad libitum. All surgical procedures were performed under general anesthesia with sevoflurane (Mylan; 4% for the induction and 3% for the maintenance), with local anaesthesia provided by 2% lidocaine (250 µg/kg) if necessary. Rats were sacrificed by intraperitoneal injection of over dose (120 mg/kg) of sodium pentobarbital (Kyoritsu) under general anaesthesia with sevoflurane.

All animal experiments were approved by the animal ethics committee of Ohu University (Koriyama, Japan) and done in the Animal Facility where animals were cared by the Animal Care Staff according to compliance by the ARRIVE guidelines (no. 2017-14). Number of rats used for preparation of PRF were as follows: One donor rat for a set of in vitro experiment and one donor rat for two recipient rats to treat defect. One in vivo experiment used eight rats as the recipient, which were divided into two groups: One group was used as PRF-grafted group and the other one was as the control. Thus, we minimized number of rats and used total 23 rats including repetition.

PRF was prepared as described (7,25,26), with slight modifications. Briefly, rats were anaesthetized with sevoflurane. Whole blood (6 ml) was collected from the apex of the heart using a 23-gauge needle and BD Vacutainer^® Blood Collection Tubes (Becton-Dickinson) without anti-coagulants. Immediately after collection, the blood was centrifuged at 890 × g at room temperature for 13 min. The intermediate layer was defined as PRF to be subjected to in vitro and in vivo experiments (27).

We used preosteoblastic cell line MC3T3-E1 cells, which was well established model for osteoblastic differentiation, to analyze the effects of PRF on osteoblastic differentiation according to Ogino et al (27) with slight modifications. MC3T3-E1 cells that reached 70% confluence in 6-well culture plates were induced to undergo osteoblastic differentiation by the addition of 50 µg/ml ascorbate 2-phosphate and 10 mM β-glycerophosphate, as described (24), with the culture medium renewed every 2–3 days. PRF (21 µg/cm²) was placed at the center of a cell monolayer sheet and the treatment was started at the same time.

Total RNA was extracted from samples with acid guanidinium thiocyanate-phenol-chloroform (AGPC) and reverse-transcribed to cDNA with a High-Capacity cDNA Reverse Transcription Kit (Thermo Fisher Scientific). The resulting cDNA was PCR amplified using a GoTaq^® Real-Time qPCR Kit (Bio-Rad) with specific primers (Table I) in a Thermal Cycler Dice Real Time System (TP-870; Takara). Results were normalized relative to Actb (β-actin) mRNA expression levels in the same samples.

The TCF-4 binding motif was cloned by PCR and inserted into the pGL3-OT vector (Addgene #16558). Following transfection of this vector into MC3T3-E1 cells using Xfect Transfection Reagent (Takara), the cells were stimulated with PRF (0.2 g/well; 21 µg/cm²) in 6-well plates for 24 h. TCF-4 binding activity was evaluated using Dual-Luciferase Reporter Assay System (Promega). Cells were co-transfected with the pGL4.75 [hRluc (Renilla reniformis)/CMV] vector to control for transfection efficiency.

Mineralized matrix in culture plates was stained with AR-S as described (24). Briefly, cells were fixed in 70% ethanol for 1 h at room temperature and stained with 40 mM AR-S at pH 4.2 for 10 min at room temperature. After washing with deionized water and Ca²⁺- and Mg²⁺-free phosphate buffered saline [PBS(−)], AR-S-positivity was quantified using Molecular Imager (Bio-Rad).

Cells were washed twice with PBS(−) and sonicated in ice-cold 0.1 M Tris-HCl (pH 7.2) containing 0.1% Triton X-100. ALP activity was determined using a Lab Assay ALP kit (Wako), according to the Bessey-Lowry method.

Proteins in cell lysates (20 µg), prepared in RIPA buffer, and those in conditioned medium (CM, 10 µg), which were concentrated by acetone precipitation, were separated by sodium dodecyl sulfate (SDS)-10% polyacrylamide gel electrophoresis (PAGE) and transferred onto polyvinylidene difluoride (PVDF) membranes. The membranes were sequentially incubated with primary antibody, biotinylated anti-rabbit IgG or anti-mouse IgG (Jackson ImmunoResearch Laboratories) as secondary antibody, and horseradish peroxidase (HRP)-conjugated streptavidin (Bio-Rad). Signals were detected using Luminate^™ Forte Western HRP Substrate (Merck Millipore). The primary antibodies used in this study included anti-RANKL antibody (BioLegend), anti-M-CSF antibody (Abcam), anti-OPG antibodies, and anti-β-actin antibodies (both from GeneTex).

A bone defect-healing model in calvaria was adapted to the mental foramen bone to assess the effects of PRF on bone regeneration (1,28–31). Briefly, following anesthetisation with 4% sevoflurane, both sides of the mental foramen bone were treated with 100 µg of lidocaine and each side was surgically drilled to make holes 2.0 mm in diameter while avoiding injury to the roots of the teeth. The bone defect was filled with PRF (doses equivalent to 0.3 g/defect area) on one side (PRF-grafted group, n=4) and without PRF on the other side (non-grafted or control group, n=4). Four days after surgery, rats were sacrificed by over dose of pentobarbital subject to histological analysis.

Samples of mandibular tissue were obtained from rats under sodium pentobarbital anaesthesia and fixed in phosphate-buffered 4% paraformaldehyde (pH 7.2) at 4°C for 24 h. The samples were decalcified with 10% EDTA (pH 7.0), which was changed every 2–3 days, at 4°C for 4 weeks. The tissue samples were embedded in paraffin and sectioned. The sections were stained with haematoxylin and eosin (H&E) and then tartrate-resistant acid phosphatase (TRAP) using a commercial TRAP staining kit (Wako), according to the manufacturer's protocol.

OPG was detected by immunohistochemistry, as previously described (32–34). Specimens were blocked with 5% skim milk and incubated sequentially with anti-OPG antibodies (GeneTex), biotinylated anti-rabbit IgG (Jackson ImmunoResearch Laboratories, West Grove, PA, USA), and fluorescein isothiocyanate (FITC)-conjugated streptavidin. Signals were observed with a fluorescence microscope (Axio Observer, Carl Zeiss Microscopy GmbH).

Protein concentration was determined by the Bradford method, using a Bio-Rad protein assay kit, with bovine serum albumin (BSA) as the standard.

Representative results from three independent experiments (unless otherwise noted) were shown and statistical significance between two groups was evaluated by Student's t-tests. Simple regression analysis was used to compare two slopes obtained by the least squares method. A P-value <0.05 was considered statistically significant.

MC3T3-E1 cells were treated with PRF to evaluate OPG production during osteoblastic differentiation. PRF treatment gradually but significantly increased expression of Tnfrsf11b mRNA, encoding OPG, for 12 days (Fig. 1A-C). In contrast, Tnfsf11 mRNA, encoding RANKL, was constitutively expressed but not affected by treatment of cells with PRF. Expression of Csf1 mRNA, encoding M-CSF was transiently lowered in PRF treated cells on day 4, with no significant differences at later times. Western blot analysis showed that PRF strongly upregulated the expression of OPG, but not of RANKL or M-CSF, by these cells (Fig. 1D). PRF, however, did not induce expression of Tnfrsf11b mRNA in non-osteoblastic NIH3T3 fibroblasts (Fig. 1E), although non-osteoblastic cells, such as gingival fibroblasts (35), adipocytes (36), and endothelial cells (37), can produce OPG. PRF also increased the ratio of OPG-encoding mRNA to RANKL-encoding mRNA in these cells (Fig. 2).

Treatment of MC3T3-E1 cells with PRF did not induce formation of nodules resulting from mineral deposition (Fig. 3A). PRF reduced ALP mRNA level on day 4, with low ALP mRNA level maintained through day 8, although ALP mRNA level was higher on day 8 than on day 4 (Fig. 3B). ALP activity was also lower in the presence than in the absence of PRF on day 8 (Fig. 3C), ALP activity in PRF-treated cells was higher on day 8 than on day 4, whereas ALP activity in control cells was higher on day 4 than on day 8, suggesting that PRF regulates bone regeneration by delaying the peak of osteoblast differentiation. The expression of other osteoblastic marker genes, encoding Runx2, BMP2, and BMP4, were not affected by PRF treatment (Fig. 3D-F).

In assessing the effect of PRF on Wnt signaling which plays a role in OPG expression, we found that PRF had no effect on the expression of Wnt3a and Wnt5a (Fig. 4A and B). In addition, PRF did not significantly activate the transcription of β-catenin from a TCF-4 binding motif containing a reporter vector (Fig. 4C). These findings suggested that the Wnt pathway is not involved in the induction of Tnfrsf11b mRNA in MC3T3-E1 cells.

To determine whether PRF mediates OPG induction and osteoclastogenesis in vivo, a bone defect-healing model in calvaria was adapted for use in mental foramen bone. A bone defect area 2.7 mm in diameter treated with a low-level laser was reported filled with calcium (~180 mg/g-tissue) and phosphorus (~115 mg/g-tissue) within 2 weeks (28). Both values were 1.4 times higher than in control, non-laser treated, bone. After an additional 2 weeks, these increases in calcium and phosphorus deposition were 1.3- and 1.2-fold higher than in control, respectively, suggesting that early stage bone repair process, within 2 weeks after surgery, was extremely important. Therefore, we created a small defect (2.0 mm in diameter) and observed early stage regeneration. After 4 days, OPG-positive cells were 2.7-fold more abundant in the PRF-engrafted than in the control region (Fig. 5A). Conversely, PRF grafting decreased TRAP positivity to 41.7% of that observed in the control region (Fig. 5B). Taken together, these findings indicate that an elevation in OPG/RANKL ratio may be critical during early stage osteoblastic differentiation.