机械应力调控种植体周应力靶细胞群对骨整合及骨感知的影响研究

Regeneration of the peri-implant tissue comprises bone remodeling and reconstruction of nerve endings. Mechanical stress not only directly have effect on proliferation and osteo-differentiation of bone marrow mesenchymal stem cells(BMSC), but also directly regulates proliferation and function of Schwann cell and thus may affect the regeneration of the nerve endings. Therefore, we hypothesis that there may exists stress-targeted cell populations in the peri-implant tissue, including Schwann cells and BMSC, which are regulated by mechanical stress in the bone remodelling process and intereaction with each other, therefore affect osseointegration and osseoperception. .This project will study the impact of different forms of mechanical stress on proliferation, adhesion, functional activity of Schwann cell in vitro, and then choose the optimal stress in reference of our early results for proliferation and fuctional activity of both BMSCs and Schwann cells. To study the interaction of BMSC and SC,these two cells will be co-cultured directly. Schwann cells will be transfected with Green fluorescent protein (GFP) and bone marrow mesenchymal stem cells with red fluorescent protein(RFP), flow sorting by the GFP and RFP will follow the direct co-cultured. And then, transfected BMSC and Schwann cells will be three-dimensional cultured in the Collagen gel scaffold and mechanically loaded. Finally, the bone chamber loading model will be used to simulate the implant osseointegration and peri-implant tissue reconstruction process in vivo. The first and second premolars in both sides of madible of beagle will be extracted and implanted of the outer bone chamber immediately. Three months later, Schwann cells transfected with GFP and BMSC transfected with RFP will be implanted in the inner bone chamber and mechanically loaded with controllable stress optimal for cell functional activity and proliferation. Bone remodeling and nerve endings reconstruction will be examined by micro-CT, nerve electrophysiological techniques, real-time PCR, western blot or immunofluorescence. We aimed to study the effects of mechanical stress on osseoperception and osseointegration of dental implants, and to find the optimal range of stress that can stimulate the peri-implant bone remodeling and osteoperception.

种植体周围组织再生包括骨改建及感受机械刺激的神经末梢的重建。机械应力不仅直接作用于BMSC影响骨改建，同时也直接调控神经再生的关键细胞即雪旺细胞的增殖及功能，进而可能影响神经末梢的再生。因此种植体周围可能存在以雪旺细胞和BMSC为主的应力靶细胞群，在骨愈合过程中感受机械应力刺激并相互作用，直接或间接影响种植体骨整合。本研究首先在体外观察不同形式应力对雪旺细胞增殖、粘附、功能活性因子表达的影响，并结合前期实验结果筛选最适应力；荧光标记两种细胞，直接共培养及三维培养雪旺细胞和BMSC，并对其进行应力加载，观察应力对两种细胞共培养后增殖和功能的影响。最后采用双套管骨小室应力加载装置体内加载可控应力，通过显微CT、神经电生理技术及分子生物学技术检测应力对种植体周围骨改建和神经末梢重建的影响。从而了解应力对种植体周围神经末梢的损伤修复和骨再生的作用，寻找能刺激种植体周围骨改建和骨感知的最适应力范围。

种植体周围组织再生包括骨改建及感受机械刺激的神经末梢的重建。机械应力不仅直接作用于间充质干细胞影响骨改建，同时也直接调控神经再生的关键细胞即雪旺细胞的增殖及功能，进而影响神经末梢的再生。因此种植体周围可能存在以雪旺细胞和间充质干细胞为主的应力靶细胞群，在骨愈合过程中感受机械应力刺激并相互作用，直接或间接影响种植体骨整合。本研究在体外观察不同形式的应力如牵张应力，压缩应力及低频脉冲超声应力对脂肪间充质干细胞及雪旺细胞的增殖、粘附、功能活性因子表达的影响，并筛选出最适应力形式；同时以低频脉冲超声为主要的加载应力形式，重点观察雪旺细胞在该应力形式下的增殖、神经营养功能、细胞骨架的变化及其调控机制；进一步共培养雪旺细胞及脂肪干细胞，并对其进行应力加载，观察应力对两种细胞共培养状态中细胞增殖和功能的影响。研究结果表明，机械应力刺激（动态牵张应力，动态压缩应力，低频脉冲超声）通过作用于雪旺细胞可以促进神经损伤后的修复进程，表现在促进雪旺细胞的增殖，维持雪旺细胞的去分化状态及促进其神经营养因子分泌，维持其效应呈现时相依赖性。机械应力刺激（动态牵张应力，动态压缩应力，低频脉冲超声）可以促进间充质干细胞的成骨分化，ERK1/2及p38通路参与了机械应力促进成骨分化的调控作用；机械应力刺激（动态牵张应力，动态压缩应力，低频脉冲超声）作用于雪旺细胞可以促进神经损伤后的修复进程，表现在促进雪旺细胞的增殖，维持雪旺细胞的去分化状态及促进其神经营养因子分泌，其效应呈现时相依赖性；其中低频脉冲超声可以通过自分泌及旁分泌作用显著增加雪旺细胞的神经营养作用，促进其髓鞘形成功能及迁移粘附，从而加快神经的修复进程；间充质干细胞的成骨作用和雪旺细胞的神经修复功能是相互影响的，成骨向分化的间充质干细胞及机械应力共同作用可以强化雪旺细胞的髓鞘形成能力。本研究结果的科学意义在于揭示应力信号在种植体周围骨改建的作用、应力对于神经损伤修复的调控作用，从而在临床上提高种植的成功率，提高患者的感觉功能和心理认知。