Liu Guolin, Xu Guoquan, Gao Zhenhua, Liu Zhenhai, Xu Junji, Wang Jinsong, Zhang Chunmei, Wang Songlin
Molecular Laboratory for Gene Therapy and Tooth Regeneration, Beijing Key Laboratory of Tooth Regeneration and Function Reconstruction, Capital Medical University School of Stomatology, Beijing, PR China.
Cells Tissues Organs. 2016;201(1):65-76. doi: 10.1159/000440952. Epub 2015 Nov 17.
The aim of this study was to investigate the effect of demineralized dentin matrix (DDM) on dental pulp stem cells (DPSCs) and the potential of complexes with DPSCs and DDM for mineralized tissue formation. Stem cells derived from the dental pulp of healthy pigs aged 18 months were isolated and cultured. DPSCs were incubated with alpha-minimum essential medium treated with DDM extract at 1 mg/ml (DDM1) or 10 mg/ml (DDM10). The concentrations of 3 growth factors in DDM extract was measured by enzyme-linked immunosorbent assay. Adhesion of DPSCs on DDM and hydroxyapatite-tricalcium phosphate (HA-TCP) surfaces was observed using scanning electron microscopy. Cell proliferation was evaluated with cell counting kit-8 and migration by Transwell migration assays. Odontoblastic differentiation was assessed by alkaline phosphatase (ALP) and alizarin red staining, ALP activity and real-time polymerase chain reaction analysis of markers of ALP, runt-related transcription factor 2, type I collagen, dentin matrix acidic phosphoprotein-1, osteonectin and dentin sialophosphoprotein (DSPP). Finally, DPSCs were combined with DDM and placed subcutaneously in nude mice for 12 weeks; DPSCs combined with HA-TCP and DDM alone served as controls. DDM could promote DPSC adhesion, migration and odontoblastic differentiation. Mineralized tissue formation was observed with the DPSC and DDM combination and the DPSC and HA-TCP combination. The mineralized tissue of the DPSC + DDM combination stained positive for DSPP, similar to the dentin tissue. These results indicate that DDM induces DPSC odontoblastic differentiation, suggesting applications for dentin regeneration.
本研究旨在探讨脱矿牙本质基质（DDM）对牙髓干细胞（DPSCs）的影响，以及DPSCs与DDM复合物形成矿化组织的潜力。分离并培养了来自18月龄健康猪牙髓的干细胞。将DPSCs与用1mg/ml（DDM1）或10mg/ml（DDM10）的DDM提取物处理的α-最低必需培养基孵育。通过酶联免疫吸附测定法测量DDM提取物中3种生长因子的浓度。使用扫描电子显微镜观察DPSCs在DDM和羟基磷灰石-磷酸三钙（HA-TCP）表面的粘附情况。用细胞计数试剂盒-8评估细胞增殖，并通过Transwell迁移试验评估迁移情况。通过碱性磷酸酶（ALP）和茜素红染色、ALP活性以及对ALP、 runt相关转录因子2、I型胶原、牙本质基质酸性磷酸蛋白-1、骨连接蛋白和牙本质涎磷蛋白（DSPP）标志物的实时聚合酶链反应分析来评估成牙本质细胞分化。最后，将DPSCs与DDM组合并皮下植入裸鼠12周；将DPSCs与HA-TCP和单独的DDM组合作为对照。DDM可促进DPSC的粘附、迁移和成牙本质细胞分化。在DPSC与DDM组合以及DPSC与HA-TCP组合中均观察到矿化组织形成。DPSC + DDM组合的矿化组织对DSPP染色呈阳性，类似于牙本质组织。这些结果表明DDM可诱导DPSC成牙本质细胞分化，提示其在牙本质再生中的应用前景。
