Canonical Wnt Signaling as a Novel Regulator of Chondrocyte to Osteoblast Transdifferentiation during Endochondral Bone Repair in the Mandible

项目总结/摘要 每年约有1500万例骨折，其中下颌骨骨折占大多数 颅面骨骼的结构重要的是，愈合受损的患病率很高， 临床需要了解直接骨折愈合的机制对于发展 改进的疗法。下颌骨通过软骨内骨化过程愈合，其中软骨 中间形式，后来被骨骼取代。最近的研究揭示了一种新的软骨内分泌模型 骨化，其中软骨中间体的软骨细胞转分化成成骨细胞， 在邻近侵入脉管系统的区域的新骨。软骨细胞的作用机制 转分化还没有被探索，但我的初步数据，沿着以前发表的工作， 表明经典Wnt信号可能是软骨细胞转分化的中心介质。为此 项目I旨在了解经典Wnt信号在软骨内骨修复过程中的作用，然后测试 一种新型水溶性分子的治疗效果，它能强烈激活Wnt信号传导。中央 该项目的假设是经典Wnt信号通过诱导软骨细胞转分化来调节软骨细胞转分化， 通过施用Wnt-替代物成骨程序和Wnt途径的活化 通过增加软骨细胞转化成骨细胞的速率加速下颌骨骨折愈合。 为了确定经典Wnt信号在软骨内骨折修复中的作用，在我的第一个目标中，我将使用 条件性抑制或激活软骨细胞中典型Wnt信号传导的转基因小鼠品系，包括 骨折骨痂我将评估Wnt信号传导对软骨细胞转分化的影响， 软骨向骨转化的速度Wnt信号传导对细胞重编程的影响将是 通过测量成软骨和成骨基因的表达水平和模式来确定， 使用qPCR、原位杂交、免疫组织化学和体视学对软骨细胞进行检测。 在第二个目标中，我将测试一种新的替代Wnt配体促进骨折修复的治疗效果。 Garcia实验室（斯坦福大学）好心地为我们提供了强烈激活Wnt的Wnt-替代物 体外信号传导。为了确定Wnt替代物的成骨作用，将评估软骨外植体的成骨作用。 体外基质矿化和碱性磷酸酶活性。此外，下颌骨骨折模型将 评估骨矿物质密度和愈合率。实验设计（DOE）方法将被 用于优化Wnt-替代品给药的剂量和时间，这将应用于进一步的体内研究。 分析Wnt-替代物对生物力学强度和骨形成率的影响。两者合计 本研究将提供有关经典Wnt信号在软骨细胞中作用的重要信息， 转分化和临床前证据表明Wnt-替代物是骨折愈合的一种新方法。

PROJECT SUMMARY / ABSTRACT There are approximately 15 million bone fractures annually and the mandible sustains the majority of fractures of the craniofacial skeleton. Importantly, prevalence of impaired healing is significant and remains an unmet clinical need. Understanding the mechanisms that direct fracture healing is imperative to the development of improved therapies. The mandible heals through the process of endochondral ossification, in which a cartilage intermediate forms and is later replaced by bone. Recent work has revealed a new model of endochondral ossification in which chondrocytes of the cartilage intermediate transdifferentiate into osteoblasts that form the new bone at a region adjacent to the invading vasculature. The mechanisms underlying chondrocyte transdifferentiation have not been explored, but my preliminary data, along with previously published work, indicate that canonical Wnt signaling may be a central mediator of chondrocyte transdifferentiation. For this project I aim to understand the role of canonical Wnt signaling during endochondral bone repair, and then test the therapeutic effect of a novel, water-soluble molecule that strongly activates Wnt signaling. The central hypothesis for this project is that canonical Wnt signaling regulates chondrocyte transdifferentiation by inducing the osteogenic program and that activation of the Wnt pathway through administration of Wnt-Surrogate accelerates mandible fracture healing by increasing the rate of conversion of chondrocytes to osteoblasts. To determine the role of canonical Wnt signaling during endochondral fracture repair, in my first Aim, I will use transgenic mouse strains to conditionally inhibit or activate canonical Wnt signaling in chondrocytes comprising the fracture callus. I will assess the effects of Wnt signaling on chondrocyte transdifferentiation by measuring the rate of cartilage to bone conversion. The effect of Wnt signaling on cellular re-programming will be determined by measuring the expression levels and patters of chondrogenic and osteogenic genes in chondrocytes using qPCR, in situ hybridization, immunohistochemistry, and stereology. In the second Aim, I will test the therapeutic effect of a novel surrogate Wnt ligand to promote fracture repair. The Garcia Laboratory (Stanford) has kindly provided us with the Wnt-Surrogate that strongly activates Wnt signaling in vitro. To determine the osteogenic effects of Wnt-surrogate, cartilage explants will be assessed for matrix mineralization and alkaline phosphatase activity in vitro. Additionally, mandible fracture models will be assessed for bone mineral density and rate of healing. Design of Experiments (DOE) methodologies will be used to optimize the dose and timing of Wnt-Surrogate administration, which will be applied to further in vivo analysis of the effect of Wnt-Surrogate on biomechanical strength and rate of bone formation. Taken together this study will provide improtant information regarding the role of canonical Wnt signaling in chondrocyte transdifferentiation and pre-clinical evidence for Wnt-Surrogate as a novel approach to fracture healing.