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

项目摘要/摘要 每年大约有1500万例骨折，大部分骨折发生在下颌骨。 头面部骨骼的特征。重要的是，愈合受损的患病率很高，而且仍未得到满足。 临床需要。了解指导骨折愈合的机制对发展骨折至关重要。 改进治疗方法。下颌骨通过软骨内骨化过程愈合，在软骨内骨化过程中，软骨 中间形式，后来被骨头取代。最近的研究揭示了一种新的软骨内模型 软骨中间的软骨细胞转化为成骨细胞而形成的骨化。 在与侵入的血管系统相邻的区域处新生骨。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，以及之前发表的工作， 提示规范的Wnt信号可能是软骨细胞转分化的中枢调节因子。为了这个 项目I旨在了解规范的Wnt信号在软骨内骨修复中的作用，然后测试 一种新型的、能强烈激活Wnt信号的水溶性分子的治疗效果。中环 该项目的假设是规范的Wnt信号通过诱导软骨细胞转分化来调节软骨细胞的转分化 成骨程序和通过给予WNT替代物激活WNT途径 通过增加软骨细胞向成骨细胞的转化来加速下颌骨骨折的愈合。 为了确定规范的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 有条件地抑制或激活软骨细胞中规范Wnt信号的转基因小鼠品系，包括 骨折的骨痂。我将通过测量来评估Wnt信号在软骨细胞转分化中的作用 软骨到骨的转换率。Wnt信号对细胞重新编程的影响将是 通过测量成软骨和成骨基因的表达水平和模式来确定 软骨细胞定量聚合酶链式反应、原位杂交、免疫组织化学和体视学。 在第二个目的中，我将测试一种新的替代Wnt配体促进骨折修复的治疗效果。 加西亚实验室(斯坦福大学)好心地为我们提供了强烈激活WNT的WNT代理 在体外发出信号。为了确定WNT替代物的成骨效果，将对软骨移植进行评估 体外基质矿化和碱性磷酸酶活力。此外，下颌骨骨折模型将被 评估骨密度和愈合率。实验设计(DOE)方法将是 用于优化WNT替代给药的剂量和时机，将在体内进一步应用 WNT替代物对生物力学强度和成骨率的影响分析加在一起 这项研究将为规范的Wnt信号在软骨细胞中的作用提供重要信息 Wnt替代物作为骨折愈合的一种新方法的转分化和临床前证据。

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.