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.
目的： 探究Wnt3a对人牙周膜干细胞（periodontal ligament stem cell，PDLSC）增殖、迁移和成骨分化的影响，确定Wnt3a对小鼠实验性牙周炎牙槽骨再生的作用。 方法： 分别用不同质量浓度的Wnt3a（0、20、100、200、500 μg/L）刺激PDLSC（计为5组），培养2、4、7或10 d后通过细胞计数检测细胞增殖，通过Transwell实验检测细胞迁移；培养21 d时通过实时荧光定量PCR检测成骨相关基因Ⅰ型胶原、runt 相关转录因子2（runt-related transcription factor 2，Runx2）的表达情况。将Wnt3a蛋白包裹在聚乳酸-羟基乙酸共聚物微球复合透明质酸水凝胶中，注入牙周炎小鼠的龈沟中。在1、2、4和8周取上颌牙槽骨样本，用显微计算机断层扫描、HE染色及骨形成标志物碱性磷酸酶（alkaline phosphatase，ALP）、Runx2和骨钙蛋白免疫组化染色评估牙槽骨再生情况。 结果： 培养10 d后，与0 μg/L Wnt3a组相比，Wnt3a在20~500 μg/L时均可显著促进PDLSC增殖（P<0.01）。培养21 d时，0、20、100、200及500 μg/L组Ⅰ型胶原mRNA的表达量分别为0.96±0.27、1.90±0.47、2.18±0.24、2.32±0.15、1.99±0.43，Runx2 mRNA的表达量分别为1.08±0.15、3.19±0.17、6.19±0.28、9.19±0.41、5.55±0.06，Ⅰ型胶原和Runx2 mRNA的表达与0 μg/L Wnt3a组相比差异均有统计学意义（P<0.05）。在注射水凝胶第1、2、4、8周后，包裹Wnt3a的水凝胶组小鼠上颌第二磨牙颊侧釉质牙骨质界至牙槽嵴顶的距离［分别为（497.3±18.2）、（455.7±12.5）、（401.0±8.5）、（362.3±15.5） μm］与牙周炎组小鼠［分别为（710.3±10.2）、（614.0±16.4）、（564.3±12.5）、（502.3±6.8） μm］相比均显著减少（P<0.01），牙周炎症减轻。注射水凝胶4周后免疫组化结果显示，与牙周炎组相比，Wnt3a水凝胶组成骨相关基因ALP（0.72±0.01）、Runx2（0.77±0.03）及骨钙蛋白（0.72±0.07）的表达水平均显著升高（P<0.01）。 结论： Wnt3a可以促进PDLSC的增殖、迁移和成骨分化以及实验性牙周炎小鼠的牙槽骨再生。.