釉基质蛋白纳米微球缓释系统协同根尖区干细胞群促进根尖再生的作用机制研究

The interraction of epithelium and mesenchymal play crital role in teeth development.The micro environment of the apical epithelial signals disappeared after the completion development of permanent teeth, no longer interaction with apical stem cell group,therefore periapical surgery is difficult to form a similar physiological apical closure.Previous researches of scholars and us have shown EMPs are synthesized and secreted by ameloblasts from epithelium, and play a crucial role during the root apex development in epithelial root sheath. Due to its origin and stability, current researches on its effects in dental pulp regeneration are rare. This research is based on the previous developed nano fiber microspheres to built an enamel matrix protein delivery system by double emulsion chemical cross linking and thermally induced phase separation, observe its effects on the dental pulp stem cell, apical papilla stem cells, periodontal ligament stem cells and bone marrow mesenchymal stem cells , investigate the regulatory network regulated by RunX2 , test its effects on the regeneration of periapical tissue in ectopic and in situ at the same time, find the optimal portfolio of stem cell and epithelial signals, so as to lay the theoretical and material basis for further elucidating the mechanisms of promoting apical repair and exploring the influence of environment on the dental apical pulp regeneration, provide new ideas for clinical vital pulp conservation and studying biological composite materials for periapical diseases.

牙齿的发育矿化是上皮与间充质相互作用的结果。恒牙发育完成后，根尖微环境的上皮信号消失，不再与根尖区干细胞群相互作用，故而根尖周手术后根尖区难以形成生理性的根尖愈合。本课题组拟在前期分离纯化的釉基质蛋白和成功构建的纳米微球的基础上，构建与釉基质蛋白功能单位高度相似的纳米纤维微球-釉基质蛋白缓释体系。试图通过重建牙根发育的微环境，重启上皮-间充质相互作用，观察缓释的釉基质蛋白这一重要上皮信号与根尖干细胞群协同作用下的细胞生物学影响，探讨RUNX2为核心的调控网络对其牙向/骨向分化的影响机制，在体内异位和原位观察各种干细胞对根尖组织的再生效果，筛选根尖再生的上皮信号-根尖干细胞群最优组合策略，为进一步研究临床牙髓病、根尖病的生物治疗及根尖周组织的再生修复提供新思路。

背景：牙髓干细胞的牙本质分化有助于牙髓-牙本质再生。牙釉质基质衍生物(EMD)被认为是牙形成过程中诱导干细胞向成牙本质细胞分化的重要上皮信号，已广泛应用于临床牙周组织再生。本研究旨在探讨EMD对DPSCs增殖、牙本质分化和牙髓牙本质再生的作用及其机制。.研究内容:体外部分：采用CCK-8法检测细胞增殖;然后通过碱性磷酸酶活性及染色、茜素红染色、实时荧光定量PCR、western blot检测EMD对DPSCs成牙分化的作用及MAPK信号通路参与情况。体内部分：通过扫描电子显微镜明确VitroGel 3D-RGD的生物相容性后，将水凝胶混合物注射到裸鼠皮下，取材后进行HE染色、Masson三色染色、Von Kossa染色和DSPP、OCN 、CD31免疫组化染色。.结果：EMD不影响DPSCs的增殖，但能促进DPSCs在体内外的牙源性分化。阻断MAPK通路抑制EMD对DPSCs分化的促进作用。此外，VitroGel 3D-RGD可为DPSCs的增殖、分化和再生提供有利的支持作用。.结论：EMD可以在体外促进DPSCs的成牙本质方向分化，但不影响其增殖；EMD可以在体内促进DPSCs的成牙本质向分化、血管形成以及矿化硬组织再生；EMD通过MAPK信号通路调控DPSCs的成牙本质方向分化。