Hedgehog信号通路在磨损颗粒诱导假体周围骨溶解中的作用及机制

Although much effort has been made to improve the efficacy of total joint arthroplasty, aseptic loosening (AL) secondary to peri-prosthetic osteolysis remains the leading cause of prosthesis failure and revision surgery. Wear particles due to prosthesis wear accumulated at the bone-prosthesis interface are thought to play a central role in the initiation and development of aseptic loosening. Although the precise mechanism remains unclear, there is growing evidence to suggest that wear particle-induced chronic inflammation and osteolysis play a critical role in AL disease. Hedgehog (Hh) signaling is essential for proper embryonic development and functions in distinct tissues during adult life. It regulates many aspects of cell proliferation, differentiation, function, apoptosis and death. The pathway is activated when one of three Hh ligands, Indian, Sonic, or Desert Hh (Ihh, Shh, and Dhh) binds to the membrane protein Patched. Upon ligand-binding, Patched’s inhibition of smoothened is relieved leading to activation of Hh signaling cascade. Signal transduction leads to activation of Gli transcription factors and expression of Hh targets genes. However, the role of Hh signal playing in the regulation of AL is still unknown. We have demonstrated that the protein level of Ihh was significantly increased in wear particle-induced osteoblast cells. Meanwhile, the nuclear accumulation of Gli1 was markedly increased as determined by western blot analysis. In addition, we also founded that Ihh highly expressed in the osteolytic tissue induced by titanium particles, and the presence of exogenous Ihh promotes titanium particle-induced osteolysis in a murine calvaria model. Accordingly, we hypothesize that Hh signaling pathway was activated in the area of bone-prosthesis interface during aseptic loosening, subsequently impeded the proliferation and differentiation of osteoblast, and then makes osteolysis more serious. This may provide a new explanation for AL disease. In the current study, we decided to investigate the important role of Hh signaling pathway in chronic inflammation and osteolysis of AL process. Furthermore, we also investigate the role of Hh in osteoblast cell proliferation, differentiation and apoptosis. In order to clarify the mechanism of Hh in particle-induced bone resorption, we investigate the effects of Hh in the regulation of osteoclast differentiation stimulated with the receptor activator of nuclear factor kappa B ligand, macrophage colony-stimulating factor and polyethylene particles or titanium particles. Meanwhile, we also investigate the mechanism of Hh signal pathway playing in the interaction between osteoblast and osteoclast in AL scenario. In this study, we performed a detailed analysis of how Hh signal affects chronic inflammation and osteolysis in calvaria of mice with particle stimulated. The purpose of this study was to clarify the relationship between Hh signaling and particle-induced osteolysis, and provide a promising therapeutic target for prevention and treatment of AL.

无菌性松动（AL）是制约人工假体使用寿命的关键因素，至今仍未获得根本性解决。AL确切机制尚未阐明，多认为与磨损颗粒引起的慢性炎症和骨溶解有关。已知Hedgehog（Hh）是调控细胞生长、发育和分化的关键通路。项目组预研结果证实磨损颗粒作用后的成骨细胞（OB）Hh通路被活化，且Hh通路活化关键因子Ihh在骨溶解局部高表达。据此，我们推测磨损颗粒通过活化Hh通路，调控OB增殖与分化，导致骨形成减少，加剧AL的发生。为了验证该假说，本项目从分子、细胞、动物、AL患者标本等层面研究以下内容：① 明确Hh关键因子在AL患者样本中的表达、分布特点及其与骨溶解的相关性；② 探讨Hh对OB增殖、凋亡和分化的影响及机制；③ 探讨Hh对破骨细胞活化的影响；④ 明晰Hh对OB和破骨细胞交互作用的影响；⑤ 明确靶向干预Hh对磨损颗粒诱导骨溶解的作用。本课题将从新的视觉阐明AL发生的机制，并为AL的防治提供新靶点。

假体周围骨溶解（PPO）是一种因植入假体磨损产生颗粒导致的骨质破坏疾病，使植入假体产生无菌性松动，最终导致关节假体失效。其主要病理特征是磨损颗粒导致的假体/骨界面发生异常的骨重建，导致破骨细胞过度活化，成骨细胞功能受损。磨损颗粒条件下成骨细胞骨形成能力下降，破骨细胞过度骨吸收是导致PPO骨溶解的主要原因。Hedgehog信号通路在成骨细胞与破骨细胞的生理过程均起重要的调节作用；因此Hedgehog信号通路可能在磨损颗粒诱导的假体周围骨溶解中发挥关键性作用。我们的研究结果发现：在钛颗粒干预条件下的成骨细胞分化过程中Hedgehog信号通路下游效应转录因子GLI1被显著下调；糖原合成酶激酶3β（GSK-3β）的抑制剂TWS119通过调控GSK-3β的活性，上调转录因子GLI1水平促进成骨细胞分化；TWS119通过上调Hedgehog信号通路增强体内成骨能力，改善骨溶解进程；此外， Hedgehog信号通路同样参与破骨细胞的活化，GLI1通过正向调节DNMTs参与破骨细胞形成；GLI1特异性抑制剂GANT58在体外抑制核因子κ B受体活化因子配体RANKL诱导的破骨细胞分化。上述结果首次表明，Hedgehog信号通路参与PPO钛颗粒条件下的成骨细胞以及破骨细胞的活化。Hedgehog信号通路有可能成为颗粒导致植入假体松动治疗的潜在靶点。