Shen S, Sun S J, Ge S H
Department of Periodontology, School and Hospital of Stomatology, Cheeloo College of Medicine, Shandong University & Shandong Key Laboratory of Oral Tissue Regeneration & Shandong Engineering Laboratory for Dental Materials and Oral Tissue Regeneration, Jinan 250012, China.
Department of Prosthodontics, School and Hospital of Stomatology, Cheeloo College of Medicine, Shandong University & Shandong Key Laboratory of Oral Tissue Regeneration & Shandong Engineering Laboratory for Dental Materials and Oral Tissue Regeneration, Jinan 250012, China.
Zhonghua Kou Qiang Yi Xue Za Zhi. 2021 Mar 9;56(3):268-275. doi: 10.3760/cma.j.cn112144-20200611-00334.
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. 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. After 10 d of culture, Wnt3a with concentrations of 20-500 μg/L significantly promoted the proliferation (<0.01) and the migration (<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 (<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] (<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 (<0.01). Wnt3a might promote the proliferation, migration and osteogenic differentiation of PDLSC and the alveolar bone regeneration.
探讨Wnt3a对牙周膜干细胞（PDLSC）增殖、迁移及成骨分化的影响，明确Wnt3a在小鼠实验性牙周炎牙槽骨再生中的作用。实验分别用5种不同浓度（0、20、100、200、500μg/L）的Wnt3a刺激PDLSC。采用细胞计数法检测细胞增殖，用Transwell法评估细胞迁移，通过实时定量PCR（RT-qPCR）检测成骨相关基因Ⅰ型胶原（Col-Ⅰ）、 runt相关转录因子2（Runx2）的表达。将聚乳酸-乙醇酸共聚物（PLGA）-Wnt3a-透明质酸（HA）水凝胶局部注射到实验性牙周炎小鼠的龈沟内。水凝胶注射1、2、4和8周后，获取上颌牙槽骨样本。采用显微CT、苏木精-伊红（HE）染色及碱性磷酸酶（ALP）、Runx2、骨钙素（OCN）等成骨相关标志物的免疫组化染色评估牙槽骨再生情况。培养10 d后，浓度为20 - 500μg/L的Wnt3a显著促进PDLSC的增殖（<0.01）和迁移（<0.01）。培养21 d后，5个浓度组Col-Ⅰ mRNA表达水平分别为0.96±0.27、1.90±0.47、2.18±0.24、2.32±0.15和1.99±0.43，Runx2 mRNA表达水平分别为1.08±0.15、3.19±0.17、6.19±0.28、9.19±0.41和5.55±±0.06。与阴性对照组相比，二者表达均有显著统计学差异（<0.05）。在1、2、4和8周时，与牙周炎组[（710.3±10.2）、（614.0±16.4）、（564.3±12.5）、（502.3±6.8）μm]相比，Wnt3a水凝胶组从牙骨质-釉质界到牙槽嵴顶的距离[（497.3±18.2）、（455.7±12.5）、（401.0±8.5）、（362.3±15.5）μm]更小（<0.01），牙周炎症更轻。免疫组化结果显示，与牙周炎组相比，Wnt3a水凝胶组骨相关蛋白ALP（0.72±0.01）、Runx2（0.77±0.03）和OCN（0.72±0.07）的表达水平升高（<0.01）。Wnt3a可能促进PDLSC的增殖、迁移及成骨分化和牙槽骨再生。
