Platelet-rich plasma inhibits RANKL-induced osteoclast differentiation through activation of Wnt pathway during bone remodeling

Abstract

Platelet-rich plasma (PRP) is used in the clinic as an autologous blood product to stimulate bone regeneration and chondrogenesis. Numerous studies have demonstrated that PRP affects bone remodeling by accelerating osteoblast formation. With the research perspective focusing on osteoclasts, the present study established a mouse model of mandibular advancement to examine the effect of PRP on osteoclast differentiation induced by modification of the dynamics of the temporomandibular joint (TMJ). The lower incisors of the mice were trimmed by 1 mm and the resultant change in mandibular position during the process of eating induced condylar adaptation to this change. PRP significantly increased the bone mass and decreased osteoclastic activity, in vitro as well as in vivo. Mechanistically, the reduced expression of receptor activator of nuclear factor-κB ligand (RANKL)‑induced differentiation marker genes, including nuclear factor of activated T-cells, cytoplasmic 1, c-fos and tartrate-resistant acid phosphatase, and that of the resorptive activity marker genes such as cathepsin k, carbonic anhydrase 2 and matrix metalloproteinase 9, indicated that PRP suppresses RANKL-induced osteoclast differentiation. A microarray analysis revealed that several genes associated with the Wnt pathway were differentially expressed, which indicated the involvement of this pathway in osteoclast differentiation. Furthermore, the activation of the Wnt pathway was verified by reverse transcription-quantitative polymerase chain reaction and immunoblot analysis of Dickkopf-related protein 1 and β-catenin. The results of the present study indicated that PRP inhibits osteoclast differentiation through activation of the Wnt pathway.

Figure 1

PRP alleviates bone loss in a mouse model of mandibular advancement. (A) Four-week-old mice were sacrificed on day 28 after the first mandibular forward movement, and radiographs of the transverse sections of the mandibular condyles were obtained using a micro-CT scanner. Scale bar, 100 µm. (B) Representative photomicrographs of sections subjected to histochemical staining for total collagen. Scale bar, 100 µm. (C) BV/TV, Tb.N and Tb.Sp of the mandibular condyles were determined by analysis of micro-CT data with Xelis software. No significant difference was observed with respect to trabecular thickness between the control and experimental groups (P>0.05). (D-F) The proportion of red-stained collagenous area within the total area in the entire section, on (D) day 7, (E) day 14 and (F) day 28 in the PRP group was significantly greater than that in the control group. Values are expressed as the mean ± standard deviation (n=5). ^**P<0.01. PRP, platelet-rich plasma; RANKL, receptor activator of nuclear factor-κB ligand; BV/TV, bone volume per total volume; Tb.Sp, trabecular separation; Tb.Th, trabecular thickness; CT, computed tomography; Con, control.

Figure 2

Effects of PRP on bone homeostasis and osteoclastic differentiation. The dissected mandibular condyles of day 7 (A), day 14 (B) and day 28 (C) were fixed, decalcified, embedded and sectioned (eft). The sections were stained for TRAP (top) and subjected to Goldner's trichrome staining (bottom). Scale bar, 100 µm. The OcN/BP and ObN/BP were determined by histomorphometric analysis (right). Values are expressed as the mean ± standard deviation (n=5). ^*P<0.05 and ^**P<0.01. TRAP, tartrate resistant acid phosphatase; OcN, number of osteoclasts; ObN, number of osteoblasts; BP, bone perimeter; PRP, platelet-rich plasma; Con, control.

Figure 3

PRP suppresses the early stage of RANKL-induced osteoclastogenesis but enhances osteoblast differentiation. (A) Bone marrow-derived macrophages were cultured with macrophage colony stimulating factor (20 ng/ml) and RANKL (20 ng/ml) in the presence or absence of 1% PRP for 3 days. The cells were fixed, permeabilized and stained for TRAP. Images were captured under a light microscope (top left) and the number of TRAP-positive osteoclasts (>3 nuclei) per field was determined (top right). Scale bar, 200 µm. Bone marrow stromal cells were stained with Alizarin red and examined under a microscope (magnification, x100; bottom left) and the wells of 12-well plates were observed under image scanner (bottom right). (B and C) Effect of PRP on NFATc1, c-fos, TRAP, Ctsk, CAR2 and MMP9 mRNA expression in osteoclasts. All values are expressed as the mean ± standard deviation (n=3). ^*P<0.05 and ^**P<0.01. PRP, platelet-rich plasma; RANKL, receptor activator of nuclear factor-κB ligand; TRAP, tartrate resistant acid phosphatase; Oc, osteoclasts; NFATc1, nuclear factor of activated T-cells, cytoplasmic 1; Ctsk, cathepsin k; CAR2, carbonic anhydrase 2; MMP9, matrix metalloproteinase 9.

Figure 4

Gene chip analysis. (A) Heat map indicating the hierarchical clustering of differentially expressed genes in the samples treated with PRP in comparison with that in the control groups. Each row represents one gene and each column represents one group. The expression levels are represented by the following colors: Red indicates high relative expression and green signifies low relative expression. (B) Top 9 downregulated pathways based on Kyoto Encyclopedia of Genes and Genomes and Biocarta databases. The enrichment score equals the -Log P-value. PRP, platelet-rich plasma; CAM, cell adhesion molecule; Con, control.

Figure 5

The Wnt signaling pathway is activated during osteoclast differentiation. PRP increased the induction of β-catenin and reduced Dkk1 in RANKL-treated osteoclasts. Bone marrow-derived macrophages were incubated with macrophage colony stimulating factor (20 ng/ml), RANKL (20 ng/ml) and 1% PRP for the indicated durations. (A) The protein expression levels of Dkk1 and β-catenin were determined by western blot analysis. (B and C) Protein bands of Dkk1 and β-catenin were quantified by densitometry, revealing that the expression of Dkk1 was declined while β-catenin was increased compared with that in the control group at days 3 and 5 (P<0.01). (D) PRP treatment for 3 days increased β-catenin mRNA expression, while it decreased Dkk1 mRNA expression in the PRP-treated group during osteoclast differentiation, which was well consistent with the results of the western blot analysis. Values are expressed as the mean ± standard deviation (n=3). ^**P<0.01. PRP, platelet-rich plasma; RANKL, receptor activator of nuclear factor-κB ligand; Dkk1, Dickkopf-related protein 1; Con, control.

Figure 6

Schematic illustration of the WNT signaling pathway activated by PRP. PRP inhibited the expression of Dkk1, which accelerates Wnt ligand binding with the Wnt receptor. PRP upregulated the expression of NFATc1, which suppresses phosphorylation of β-catenin. Cyclin D1 is one of the target genes of β-catenin, and β-catenin and NFATc1 contribute to osteoclast differentiation by combination of RANKL and RANK. PRP, platelet-rich plasma; RANKL, receptor activator of nuclear factor-κB ligand; Dkk1, dickkopf-related protein 1; NFATc1, nuclear factor of activated T-cells, cytoplasmic 1.