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Title: [Semaphorin 3A-stimulated bone marrow mesenchymal stem cells sheets promotes osteogenesis of type 2 diabetic rat].
Author: Qiao Q, Song YL, Li FL.
Journal: Zhonghua Kou Qiang Yi Xue Za Zhi; 2018 May 09; 53(5):333-338. PubMed ID: 29972992.
Abstract:
Objective: To evaluate the effect of semaphorin 3A (Sema3A) pre-treated bone marrow mesenchymal stem cells (BMSC) sheets on new bone formation in type 2 diabetes mellitus rats. Methods: Type 2 diabetes mellitus (T2DM) were induced by injection of streptozotocin, and the BMSC were isolated, controlled, identified and induced into cell sheets. Fifteen T2DM rats were randomly divided into control, sheets and Sema3A-sheets group and the calvarial critical size defect (CSD) model of rats were established. The defect zone of rats from control group were implanted with bone powder. The defect zone of rats from sheets group were implanted with bone powder and BMSC sheets. The defect zone of rats from Sema3A-sheets group were implanted with bone powder and BMSC sheets pretreated with 1.0 mg/L Sema3A. After 8 weeks, the bone samples were harvested and analyzed by micro-CT scanning, HE staining for the evaluation of new bone formation, and the immunohistochemical analysis for the expression of osteogenesis-related proteins including type Ⅰ collagen (COL- Ⅰ ), bone morphogenetic protein-2 (BMP-2), and osteocalcin (OCN). Results: The BMSC were isolated and cultured, and oil red O and Alizarin red S staining proved the multi-potential differentiation. Eight weeks after the establishment of calvarial CSD model, Sema3A-sheet group showed the most abundant new bone formation (0.516±0.070), with increased bone volume fraction, namely bone volume/tissue volume (BV/TV) compared with sheets group (0.319±0.050) and control group (0.224±0.037) (P<0.05), and the sheets group showed increased BV/TV compared with control group (P<0.05). While trabecular thickness (Tb.Th) control group showed no difference in three groups (P>0.05). HE staining also confirmed that Sema3A-sheets group showed the most new bone formation. Sheet group (0.174±0.051) compared showed difference with control group (0.099±0.033) (P< 0.05), and Sema3A-sheet group (0.421±0.069) showed increased bone formation compared with sheet group and control group (P<0.05). Immunohistochemistry showed that BMSC sheet increased the expression of osteogenesis-related proteins including COL-Ⅰ, BMP-2 and OCN, while Sema3A pretreatment showed more obvious increase of the expression of COL-Ⅰ and OCN. Conclusions: The combined implantation of bone powder and Sema3A stimulated BMSC sheets significantly increased bone regeneration in vivo. Therefore, Sema3A pre-treated BMSC sheets transplantation provides a new strategy for restoring bone defect in T2DM. 目的： 评价信号素3A刺激的骨髓间充质干细胞（bone marrow mesenchymal stem cell，BMSC）膜片对2型糖尿病大鼠新骨形成的影响。 方法： 通过注射链脲佐菌素构建2型糖尿病大鼠模型，分离、培养BMSC并鉴定，诱导成膜；使用随机数字表法将15只2型糖尿病大鼠随机分为对照组、膜片组、信号素3A膜片组（每组5只），构建大鼠颅骨极限骨缺损模型，对照组植入骨粉，膜片组植入BMSC细胞膜片和骨粉，信号素3A膜片组植入用1.0 mg/L信号素3A刺激的BMSC细胞膜片和骨粉，8周后取材分别行显微CT扫描及HE染色，分析新骨形成情况，免疫组化观察Ⅰ型胶原、骨形态发生蛋白2和骨钙蛋白的表达。 结果： 分离培养的2型糖尿病大鼠BMSC经茜素红、油红O染色鉴定具有多向分化潜能。构建大鼠颅骨极限骨缺损模型8周后，显微CT扫描可见，信号素3A膜片组新骨形成最多，骨体积分数（bone volume/tissue volume，BV/TV）（0.516±0.070）显著大于膜片组（0.319±0.050）和对照组（0.224±0.037）（P<0.05），膜片组BV/TV显著大于对照组（P<0.05）；而3组骨小梁厚度差异无统计学意义（P>0.05）。HE染色分析显示，信号素3A膜片组新骨形成比值（0.421±0.069）显著大于膜片组（0.174±0.051）和对照组（0.099±0.033）（P<0.05），膜片组新骨形成比值显著大于对照组（P<0.05）。免疫组化染色观察可见，膜片组Ⅰ型胶原、骨形态发生蛋白2、骨钙蛋白阳性表达多于对照组，而信号素3A膜片组Ⅰ型胶原、骨钙蛋白阳性表达多于膜片组和对照组。 结论： 联合植入信号素3A刺激的BMSC细胞膜片和骨粉，可显著增加2型糖尿病大鼠的骨再生能力。.

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