Qiao Q, Song Y L, Li F L
Department of Stomatology, Shanxi Medical University, Taiyuan 030012, China.
Department of Implantation, School of Stomatology, The Fourth Military Medical University & State Key Laboratory of Military Stomatology & National Clinical Research Center for Oral Diseases & Shaanxi Engineering Research Center for Dental Materials and Advanced Manufacture, Xi'an 710032, China.
Zhonghua Kou Qiang Yi Xue Za Zhi. 2018 May 9;53(5):333-338. doi: 10.3760/cma.j.issn.1002-0098.2018.05.009.
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. 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). 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) (<0.05), and the sheets group showed increased BV/TV compared with control group (<0.05). While trabecular thickness (Tb.Th) control group showed no difference in three groups (>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) (< 0.05), and Sema3A-sheet group (0.421±0.069) showed increased bone formation compared with sheet group and control group (<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. The combined implantation of bone powder and Sema3A stimulated BMSC sheets significantly increased bone regeneration . Therefore, Sema3A pre-treated BMSC sheets transplantation provides a new strategy for restoring bone defect in T2DM.
评估信号素3A（Sema3A）预处理的骨髓间充质干细胞（BMSC）片对2型糖尿病大鼠新骨形成的影响。通过注射链脲佐菌素诱导建立2型糖尿病（T2DM）大鼠模型，分离、培养、鉴定并诱导BMSC形成细胞片。将15只T2DM大鼠随机分为对照组、细胞片组和Sema3A - 细胞片组，建立大鼠颅骨临界尺寸缺损（CSD）模型。对照组大鼠的缺损区植入骨粉；细胞片组大鼠的缺损区植入骨粉和BMSC片；Sema3A - 细胞片组大鼠的缺损区植入骨粉和用1.0 mg/L Sema3A预处理的BMSC片。8周后，采集骨样本，通过显微CT扫描、HE染色评估新骨形成情况，并进行免疫组织化学分析以检测成骨相关蛋白Ⅰ型胶原（COL - Ⅰ）、骨形态发生蛋白 - 2（BMP - 2）和骨钙素（OCN）的表达。分离培养BMSC，油红O和茜素红S染色证明其具有多向分化能力。颅骨CSD模型建立8周后，Sema3A - 细胞片组新骨形成最为丰富（0.516±0.070），与细胞片组（0.319±0.050）和对照组（0.224±0.037）相比，骨体积分数即骨体积/组织体积（BV/TV）增加（<0.05），细胞片组的BV/TV较对照组增加（<0.05）。而三组间骨小梁厚度（Tb.Th）对照组无差异（>0.05）。HE染色也证实Sema3A - 细胞片组新骨形成最多。细胞片组（0.174±0.051）与对照组（0.099±0.033）相比有差异（<0.05），Sema3A - 细胞片组（0.421±0.069）与细胞片组和对照组相比骨形成增加（<0.05）。免疫组织化学显示BMSC片增加了包括COL - Ⅰ、BMP - 2和OCN在内的成骨相关蛋白的表达，而Sema3A预处理使COL - Ⅰ和OCN的表达增加更明显。骨粉与Sema3A联合植入刺激BMSC片显著增加骨再生。因此，Sema3A预处理的BMSC片移植为修复T2DM大鼠骨缺损提供了一种新策略。
