多孔纳米纤维微球联合BMP-2/SDF-1双控释系统诱导年轻恒牙根尖再发育

Trauma or dental caries arrests root development of immature permanent teeth, resulting in difficulty of their long-term preservation. The traditional clinical treatment for these teeth is apexification, which is not able to achieve ideal apex regeneration, making these teeth susceptible to fracture. Therefore, the injectable microspheres and controlled release was combined and adopted by our group to realize apex regeneration of immature permanent teeth. Nanofibrous microspheres and BMP-2 controlled release were developed to effectively promote odontogenic differention of stem cells of apical papilla (SCAP). These injectable microspheres could be applied to repair irregularly shaped pulp cavity by promoting development of dentin-like tissue. However, their capability of loading stem cells and degradation rate in vivo is still needing to be optimized. In addition, the role of microspheres and BMP-2 controlled release in SCAP regulation is largely unknown. Based on previous study, porous nanofibrous microspheres will be developed to provide a three-dimensional physicochemical and biological microenvironment for stem cell migration and proliferation. Meanwhile, sequential delivery of BMP-2/SDF-1 will be proposed and optimized to facilitate SCAP and endothelial cell recruitment to the apical pulp cavity by SDF-1 and subsequent in situ odontogenic differentiation of SCAP by BMP-2, facilitating apex regeneration of immature permanent teeth. Moreover, mechanism of new microspheres and dual delivery of BMP-2/SDF-1 in SCAP regulation will be explored. The accomplishment of this project will provide a reasonable rationale and feasible method for apex regeneration of immature permanent teeth.

外伤或龋坏等原因会导致年轻恒牙牙根发育中断，难以长期保存。临床常用的根尖诱导成形术不易达到其理想的根尖发育，远期根折几率较大。故我们试图联合可注射微球与控释技术实现其根尖再发育。前期构建的纳米纤维微球可直接注射入不规则的髓腔系统，并与BMP-2控释技术结合有效促进根尖牙乳头干细胞的成牙分化。但这种微球在容纳干细胞能力及体内降解性方面还有待优化，其与控释技术联合调控干细胞成牙分化的具体机制仍然不明。本研究拟在前期基础上发展多孔且具有交联结构的纳米纤维微球，为干细胞迁移分化提供更有利微环境；并构建BMP-2/SDF-1时序双控释系统，先吸引根尖牙乳头干细胞及血管内皮细胞迁移入根尖髓腔再促进成牙分化，实现根尖牙本质再发育；进而探讨此微球及控释系统影响干细胞迁移及成牙分化的分子机制，为临床上年轻恒牙根尖再发育提供理论依据和技术支持。

外伤或龋坏等原因会引起牙髓感染或坏死，导致年轻恒牙牙根发育中断，难以长期保存。临床常用的根尖诱导成形术治疗周期长且不易达到理想的根尖发育，远期根折几率较大。因此我们试图将可注射纳米纤维微球支架与BMP-2/SDF-1双控释技术相结合促进其根尖再发育。我们首先构建了可注射的纳米纤维微球，通过SEM、DNA含量测试、HE和免疫组化染色证实其具有良好的组织相容性和促根尖牙乳头干细胞（SCAP）成牙分化的能力。其次，通过Western blot和流式细胞分析证实SCAP表面有SDF-1的受体CXCR4, Transwell和CCK-8检测证实100ng/ml 的SDF-1既不影响SCAP的增殖又能显著促进SCAP的迁移; 通过RT-PCR和矿化实验证实100ng/ml的BMP-2可以有效促进了SCAP的分化。进一步我们成功构建了双缓释微球，通过调整PLGA微球大小和孔隙率实现了BMP-2/SDF-1的有序释放，体内HE和Masson结果证实这种双缓释系统促进了类牙本质再生。最后，RT-PCR和Western blot结果证实BMP-2 和SDF-1协同性的促进了SCAP的成牙分化。通过siRNA下调CXCR4的表达，我们发现.SDF-1/CXCR4轴参与了BMP-2介导的Smad1/5 和 Erk1/2的激活，表明SDF-1/CXCR4轴可能参与了BMP-2诱导的SCAP的成牙分化。本课题构建出了具有良好生物相容性和生物可退化性的可注射型支架材料，并建立完善的BMP-2/SDF-1双因子控释系统，促进年轻恒牙的根尖再发育，进一步研究了SDF-1/BMP-2双控释系统对 SCAP 迁移及成牙分化能力影响的具体机制，为临床上年轻恒牙的长期保存提供理论依据和技术支持。