Gli1+间充质祖细胞在牙种植体周骨改建中的作用和机制研究

Osseointegration is the basis of dental implant restoration, while a long period of peri-implant tissue healing becomes one of major drawbacks of this treatment. Progenitors with high expression of Gli1 in the alveolar bone have the ability to rehabilitate bone injury, but the mechanism of osteogenesis regulation in the implant osseointegration remains to be elucidated. Previous experiments showed that Gli1+ cells were induced to proliferate after implantation, then differentiate to bone tissue spontaneously， transforming into osteocytes in peri-implant tissue. Accordingly, the applicant firstly proposed that Gli1+ cells are significant progenitors involved in and regulating alveolar tissue repair and bone remodeling. Regulating the biomechanical load after implant implantation will promote the proliferation and differentiation of Gli1+ cells and shrunken the osseointegration duration. Based on the in vivo and in vitro experiments, we established alveolar bone injury healing and dental implant models. Combining cell lineage tracing, this project aimed mainly to investigate: ① Gli1+ cells in persistent and instant expression in alveolar bone remodeling; ② in vitro and in vivo functional verification of Gli1+ cells in alveolar bone healing; ③ the mechanisms of Gli1+ cells involved peri-implant bone remodeling after functional occlusal loading. The project will reveal important cellular sources of peri-implant bone remodeling and provide theoretical basis for the positive regulation mechanism of osseointegration.

种植体骨结合的形成是牙种植修复的核心基础，然而种植体周组织愈合时间较长成为该治疗手段的一大弊端。祖细胞具有骨损伤修复的能力，但有关其在种植体骨结合中成骨调节的具体机制尚未阐明。前期实验发现：种植体植入后诱发Gli1+细胞增殖并向成骨方向分化，最终形成种植体周骨细胞。据此，申请人首次提出“Gli1+细胞是参与并调控种植体周组织修复和骨改建的重要祖细胞，合适的种植体负载力可促进细胞增殖分化，缩短骨结合时间”。本项目将在前期基础上，通过建立牙槽骨损伤愈合、牙种植修复模型，采用细胞谱系示踪模型重点探讨：①Gli1+细胞在牙槽骨改建过程的持续性与即时性表达；②Gli1+细胞在牙槽骨愈合中促成骨作用的功能性验证；③牙种植体即刻负重对Gli1+细胞在牙槽骨改建中的影响及机制研究。本课题有望揭示种植体周骨组织修复的重要细胞来源，为种植体骨结合正向调节机制提供重要理论依据。

种植体骨结合的形成是牙种植修复的核心基础，种植体周骨组织修复的重要细胞来源，为种植体骨结合正向调节机制尚不明确。本课题通过构建Gli1-CreERT2;Tdtomato，Axin2-CreERT2;Ai14，Gli1-CreERT2;Ai14，Axin2-CreERT2; Ai14;Gli1-LacZ及Gli1-CreERT2; β-cateninflox/flox ; Ai14小鼠模型和小鼠鼻窦底提升细胞示踪模型，阐明Gli1+间充质干细胞在种植体周骨改建的增殖、骨向分化作用，将谱系示踪技术及LacZ染色技术结合，阐明Gli1+间充质干细胞中与Wnt信号通路在蛋白水平的共定位及共表达情况，采用RNAscope技术进一步检测Wnt信号通路关键基因β-catenin的在基因层面的共表达情况，在体内水平确定两者空间表达关系；通过在Gli1+间充质干细胞中特异性敲除Wnt信号，检测骨缝骨改建及Gli1+细胞的激活状态，进一步阐明种植体周及骨缝Gli1+干细胞在骨改建的调控作用。