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.

项目摘要 /摘要 每年大约有1500万个骨折，下颌骨可维持大部分骨折 颅面骨骼。重要的是，愈合受损的患病率很重要，并且仍然是未得到的 临床需求。了解直接断裂愈合的机制对于发展至关重要 改进的疗法。下颌骨通过内软骨骨化的过程愈合，其中软骨 中间形式，后来被骨骼代替。最近的工作揭示了一种新的内软骨模型 软骨中间体转变成成骨细胞的骨细胞骨细胞形成形成的成骨细胞 与入侵脉管系统相邻的区域的新骨。软骨细胞的基础机制 尚未探索转变的，但是我的初步数据以及先前发表的工作， 表明规范Wnt信号传导可能是软骨细胞转分化的中心介体。为了这 项目我旨在了解内侧骨修复过程中规范Wnt信号传导的作用，然后测试 强烈激活Wnt信号的新型水溶性分子的治疗作用。中央 该项目的假设是，规范的Wnt信号传导通过诱导来调节软骨细胞转变 成骨程序和通过施用Wnt-Surrogate激活WNT途径 通过增加软骨细胞向成骨细胞的转化率来加速下颌骨骨折的愈合。 在我的第一个目标中，为了确定内软骨断裂修复过程中的典型Wnt信号传导的作用，我将使用 转基因小鼠菌株有条件地抑制或激活包含软骨细胞中的经典Wnt信号传导 断裂愈伤组织。我将通过测量来评估Wnt信号传导对软骨细胞跨分化的影响 软骨对骨转化的速率。 Wnt信号对细胞重新编程的影响将是 通过测量软骨基因和成骨基因的表达水平和模式来确定 使用QPCR，原位杂交，免疫组织化学和立体学的软骨细胞。 在第二个目标中，我将测试一种新型的替代wnt配体的治疗作用，以促进断裂修复。 加西亚实验室（斯坦福）为我们提供了强烈激活Wnt的Wnt-Surogate 体外信号传导。为了确定Wnt-Surogate的成骨作用，将评估软骨外植体 基质矿化和碱性磷酸酶活性在体外。此外，下颌骨骨折模型将是 评估骨矿物质密度和愈合速率。实验（DOE）方法的设计将是 用于优化Wnt-Surogate给药的剂量和时机，将应用于进一步的体内 分析Wnt-Surogate对生物力学强度和骨形成速率的影响。一起 这项研究将提供有关规范Wnt信号在软骨细胞中的作用的不幸信息 Wnt-Surogate作为裂缝愈合的新方法的转分化和临床前证据。