Wnt3a promotes osteogenic differentiation of periodontal ligament stem cell and regeneration of alveolar bone in experimental periodontitis

Objective: To explore the effects of Wnt3a on the proliferation, migration and osteogenic differentiation of periodontal ligament stem cell (PDLSC) and to identify the role of Wnt3a in alveolar bone regeneration in mouse experimental periodontitis. Methods: The experiments were conducted by stimulating PDLSC using Wnt3a of 5 different concentrations (0, 20, 100, 200, 500 μg/L) respectively. Cell proliferation was detected by cell-counting assay, cell migration was evaluated by Transwell assay and the expressions of osteogenic related genes collagen Ⅰ (Col-Ⅰ), runt-related transcription factor 2 (Runx2) were examined by real-time quantitative PCR (RT-qPCR). Poly lactic-co-glycolic acid (PLGA)-Wnt3a-hyaluronic acid (HA) hydrogel was injected locally into the gingival sulcus of mice with experimental periodontitis. After 1, 2, 4, and 8 weeks of hydrogel injection, samples of maxillary alveolar bone were obtained. Micro-CT, HE staining and immunohistochemical staining of osteogenesis related markers, such as alkaline phosphatase (ALP), Runx2, osteocalcin (OCN), were used to evaluate alveolar bone regeneration. Results: After 10 d of culture, Wnt3a with concentrations of 20-500 μg/L significantly promoted the proliferation (P<0.01) and the migration (P<0.01) of PDLSC. After 21 d of culture, the expression levels of Col-Ⅰ mRNA were 0.96±0.27, 1.90±0.47, 2.18±0.24, 2.32±0.15 and 1.99±0.43 in 5 concentration groups respectively, and the expression levels of Runx2 mRNA were 1.08±0.15, 3.19±0.17, 6.19±0.28, 9.19±0.41 and 5.55±0.06, respectively. Both expressions had significant statistical differences compared with the negative control group (P<0.05). At 1, 2, 4, and 8 weeks, the Wnt3a hydrogel group had less distance [(497.3±18.2), (455.7±12.5), (401.0±8.5), (362.3±15.5) μm] from the cemento-enamel junction to alveolar bone crest compared with the periodontitis group [(710.3±10.2), (614.0±16.4), (564.3±12.5), (502.3±6.8) μm] (P<0.01) and weaker periodontal inflammation. Immunohistochemical results showed that the expression levels of bone-related proteins of ALP (0.72±0.01), Runx2 (0.77±0.03) and OCN (0.72±0.07) in the Wnt3a hydrogel group were increased compared with the periodontitis group (P<0.01). Conclusions: Wnt3a might promote the proliferation, migration and osteogenic differentiation of PDLSC and the alveolar bone regeneration.