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

规范 Wnt 信号作为下颌骨软骨内骨修复过程中软骨细胞向成骨细胞转分化的新型调节剂

• 批准号: 9278967
• 负责人: Sarah Anne Wong
• 金额: $ 4.03万
• 依托单位: UNIVERSITY OF CALIFORNIA, SAN FRANCISCO
• 依托单位国家: 美国
• 财政年份: 2016
• 资助国家: 美国
• 起止时间: 2016-07-01 至 2021-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9278967
• 关键词: Alkaline Phosphatase; Apoptosis; Applications Grants; Binding; Biological; Biomechanics; Bone Density; Bone Regeneration; Bone callus; Cartilage; Chondrocytes; Clinical; Clinical Data; Data; Dental; Development; Dose; Esthetics; Fellowship; Fluorochrome; Foundations; Fracture; Fracture Healing; Gene Expression; Genes; Glycogen Synthase Kinase 3; Goals; Growth Factor; Health; Human Cell Line; Immunohistochemistry; Impaired wound healing; In Situ Hybridization; In Vitro; Incubated; Invaded; Label; Laboratories; Letters; Ligands; Mandible; Mandibular Fractures; Measures; Mediator of activation protein; Methodology; Modeling; Mouse Strains; Osteoblasts; Osteogenesis; Pathway interactions; Pattern; Periosteum; Physiologic Ossification; Prevalence; Process; Publishing; Regulation; Research; Role; Scientist; Signal Transduction; Skeleton; Stem cells; Techniques; Testing; Therapeutic; Therapeutic Effect; Training; Transgenic Mice; United States; Universities; WNT Signaling Pathway; Water; Work; base; beta catenin; bone; career; craniofacial; design; experimental study; healing; improved; in vitro activity; in vivo; inhibitor/antagonist; innovation; maxillofacial; microCT; mineralization; novel; novel strategies; osteogenic; osteoprogenitor cell; pre-clinical; programs; receptor; repaired; therapeutic evaluation; transdifferentiation

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.

项目概要/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。重要的是，愈合受损的患病率很高，而且仍然是一个未得到满足的问题 临床需要。了解直接骨折愈合的机制对于发展骨折至关重要 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，后来被骨头取代。最近的工作揭示了一种新的软骨内模型 骨化，其中软骨中间体的软骨细胞转分化为成骨细胞，形成骨 邻近侵入脉管系统的区域出现新骨。软骨细胞的潜在机制 转分化尚未被探索，但我的初步数据以及之前发表的工作， 表明经典 Wnt 信号传导可能是软骨细胞转分化的核心介质。为了这 我的项目旨在了解典型 Wnt 信号在软骨内骨修复过程中的作用，然后进行测试 一种新型水溶性分子强烈激活 Wnt 信号传导的治疗效果。中央 该项目的假设是，经典的 Wnt 信号通过诱导软骨细胞转分化来调节 成骨程序以及通过施用 Wnt-Surrogate 激活 Wnt 通路 通过提高软骨细胞向成骨细胞的转化率来加速下颌骨骨折的愈合。 为了确定典型 Wnt 信号在软骨内骨折修复过程中的作用，在我的第一个目标中，我将使用 转基因小鼠品系有条件地抑制或激活软骨细胞中的经典 Wnt 信号传导，包括 骨折愈伤组织。我将通过测量来评估 Wnt 信号传导对软骨细胞转分化的影响 软骨到骨的转化率。 Wnt 信号传导对细胞重编程的影响 通过测量软骨形成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分析。 在第二个目标中，我将测试一种新型替代 Wnt 配体促进骨折修复的治疗效果。 加西亚实验室（斯坦福）慷慨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 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 用于优化 Wnt-Surrogate 给药的剂量和时间，这将应用于进一步的体内研究 分析 Wnt-Surrogate 对生物力学强度和骨形成率的影响。合在一起 这项研究将提供关于典型 Wnt 信号在软骨细胞中的作用的重要信息 Wnt-Surrogate 作为骨折愈合新方法的转分化和临床前证据。