颅颌骨缝牵张成骨中Gli1+间充质干细胞时空效应的透明化观察及调控机制研究

Craniofacial sutures are important sites of maxilla and facial bone growth. Orthopedic therapies aim to treat patients with dentofacial deformities and malocclusion by loading exogenous tensile force on craniofacial sutures. However, the current understanding of the cell sources for new formed bones under mechanical stimulation and the regulatory mechanisms remains to be elucidated. Our previous study identified that Gli1+ cells within the suture mesenchyme as the main MSC population for craniofacial bones, and that the sutures provide the niche for Gli1+ MSCs, which is positively regulated by Wnt signaling pathway. In this study, the applicant first proposed that tensile stress activates the proliferation and osteogenic differentiation of sutural Gli1+ MSCs by regulating Wnt signaling pathway, which promotes the bone remodeling on suture edge. Tissue clearing technique enables visualization of whole tissues in 3-D by turning tissues transparent, and PEGASOS method have technological superiority on clearing hard tissues. By combining PEGASOS tissue clearing method with mechanobiology study methods for the first time, this study is aiming to analyze the spatiotemporal effects of Gli1+ MSCs in sutural distraction osteogenesis process, as well as elucidating the regulatory role of Wnt signaling pathway in tensile stress induced osteogenic differentiation of sutural Gli1+ MSCs. The project will not only provide a richer scientific connotation for optimizing the mechanism of dentofacial orthopedics, but also lay solid foundation for providing the regulatory targets of promoting maxillofacial growth and remodeling in orthopedic treatment.

颅颌骨缝是颅颌面骨骼生长的重要生长区，矫形治疗通过应力刺激颅颌骨缝牵张成骨可有效治疗多种颅颌面发育畸形，但力学刺激引起骨缝成骨的细胞来源及相关调控机制尚未阐明。课题组前期研究发现：Gli1+细胞在颅颌面作为间充质干细胞而存在，骨缝是其特殊的干细胞龛，且Wnt信号通路对Gli1+干细胞具有正向调控作用。据此，申请者首次提出牵张应力可能通过调控Wnt信号从而激活骨缝Gli1+干细胞增殖分化以促进骨改建发生的新观点。由于申请人前期研发的PEGASOS组织透明化技术对研究硬组织独具优势，本课题将首次结合组织透明化技术与力学生物学研究手段，从三维层面观察分析Gli1+干细胞参与骨缝牵张成骨的时空动态过程，初步探讨Wnt信号通路对牵张应力刺激下Gli1+干细胞参与骨缝及周围骨改建的调控机制。该课题将为颅颌面发育畸形的矫形治疗理念提供更丰富的科学内涵，亦将为矫形治疗中颌面部生长改建机制研究奠定基础。

颅颌骨缝是颅颌面骨骼生长的重要生长区，矫形治疗通过应力刺激颅颌骨缝牵张成骨可有效治疗多种颅颌面发育畸形，但力学刺激引起骨缝成骨的细胞来源及相关调控机制尚未阐明。该项目从二维及三维层面观察分析Gli1+SuSCs参与骨缝牵张成骨的动态时空过程，并进一步探索其中调控机制，获得以下重要结果：1、成功构建了小鼠骨缝牵张标准模型，三维追踪明确骨缝牵张后骨改建周期为3～4周；2、成功构建Gli1-CreERT2;Ai14小鼠模型，明确了骨缝牵张刺激Gli1+SuSCs生物学特性变化，呈现其在全颅缝三维空间中的增殖分化特征，发现力学刺激后骨缝干细胞在1～3天迅速增殖、达到增殖高峰，随后进行分化、形成新骨，尽管新骨在牵张2周左右基本完全形成，但此时骨改建尚未完成，随后1～2周时间骨改建继续发生、骨形态改建完成；3、成功构建Gli1-CreERT2;β-cateninflox/flox ; Ai14小鼠模型，结合qPCR等技术，阐明Gli1+SuSCs中特异性Wnt信号敲除对力学刺激骨缝牵张成骨的影响及调控机制，成骨-破骨平衡失调是骨缝牵张后骨改建异常的主要原因。本项目将首次结合组织透明化技术与力学生物学研究手段，从三维层面观察分析体内Gli1+SuSCs参与骨缝牵张成骨的时空动态过程、探索体内干细胞特异性Wnt信号通路对该过程的调控机制，为颅颌面发育畸形的矫形治疗理念提供更丰富的科学内涵，为矫形治疗中颌面部生长改建机制研究奠定基础。