LepR阳性干细胞在种植体骨结合中的作用及机制研究

Stem cells homing and differentiation are fairly essential for dental implant osseointegration. However, there are few reports on the specific roles and mechanisms of stem cells in the process of osseointegration. LepR+ cells are newly discovered skeletal stem cells in long bone, which are the major source of bone cells in bone marrow and play a key role in injury repair. Our previous study indicated that LepR+ cells could accumulate and go through osteogenic differentiation in the tooth extraction socket healing process. Thus, we hypothesize that LepR+ cells play an important role in implant osseointegration. Here, we will use LepR-Cre;tdTomato;Col2.3-GFP transgenic fluorescent reporter mice to demonstrate that LepR can be used as a reliable in vivo marker in jaw bone using lineage tracing assay as well as in vitro cell culture and identification. By establishing implant placement mouse model, along with tissue clearing technique and two-photon microscopy 3D imaging, we aim to investigate the role of LepR+ stem cells in osseointegration. We will further perform parabiosis mouse model and RNA-Seq technology to dissect the source and molecular regulation mechanisms of LepR+ stem cells during osseointegration. Our results will reveal the role of stem cells in osseointegration and provide an insight for dental treatment strategy of stem cells based osseointegration enhancement.

干细胞的归巢和分化是实现种植体骨结合的必经环节。然而，关于干细胞在骨结合过程中的具体作用和机制鲜有报道。LepR+干细胞是新发现的长骨骨骼干细胞，是骨髓中骨细胞的主要来源，并在损伤修复中发挥关键作用。申请人前期发现LepR+细胞在颌骨拔牙创愈合中大量聚集，并向成骨细胞分化。因而假设LepR+干细胞可能在种植体骨结合中具有重要作用。本课题拟利用LepR-Cre;tdTomato;Col2.3-GFP荧光报告小鼠，通过干细胞谱系追踪实验及体外细胞培养鉴定等手段验证LepR可以作为颌骨干细胞的可靠体内标志；通过建立种植体植入模型，结合组织透明化和双光子3D成像技术探讨LepR+干细胞在骨结合中的作用；通过构建异体共生小鼠模型和RNA-Seq技术明确骨结合过程中LepR+干细胞的来源及分子调控机制。研究结果将揭示骨结合过程中干细胞的作用，为基于干细胞的调控机制寻找促进骨结合的临床策略提供依据。

干细胞的募集和分化是骨再生的必经环节。目前长骨中已经发现了多种干细胞，但颌骨中干细胞的种类和功能尚不明确。鉴于长骨与颌骨在发育起源、成骨方式和干细胞体外增殖分化特性等方面有诸多不同，颌骨干细胞的种类和成骨特性可能与长骨干细胞存在差异。瘦素受体(leptin receptor,LepR)是近年来发现的小鼠长骨骨髓中重要的成体干细胞特异性标记物，LepR阳性(LepR+)细胞是长骨中成骨细胞和脂肪细胞的主要来源，而LepR+细胞在颌骨损伤愈合及种植体骨结合中的作用尚不清楚。本课题主要通过构建LepR-Cre;tdTomato;Col2.3-GFP 荧光小鼠、LepR-Cre;iDTR 基因敲除小鼠及LepR-Cre;Pth1rfl/fl条件性基因敲除小鼠，建立小鼠拔牙窝愈合模型及种植体骨结合模型，利用干细胞谱系追踪技术、免疫荧光染色、荧光双标染色、MicroCT、组织透明化等多种实验方法，证实颌骨中的LepR+细胞在生理状态下是静止的，其数量随着年龄增长而增加，并逐渐分化为成骨细胞；在拔牙后LepR+细胞被激活，并且在拔牙窝愈合及种植体骨结合过程中生成了大部分成骨细胞。同时，LepR+细胞的消融影响了拔牙窝的正常愈合，进一步验证了LepR+细胞在颌骨骨再生中的重要作用。进一步的机制研究表明，体内PTH注射可以促进颌骨LepR+细胞增殖和成骨分化；在LepR+细胞中条件性敲除pth1r基因会影响拔牙窝的正常愈合，说明颌骨LepR+细胞受PTH/PTH1R信号调控。