Spatial and Temporal Role of the Runx3 Transcription Factor in Secondary Fracture Healing

Runx3 转录因子在二次骨折愈合中的时空作用

• 批准号: 10454763
• 负责人: HICHAM M DRISSI
• 金额: --
• 依托单位: VETERANS HEALTH ADMINISTRATION
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-04-01 至 2024-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10454763
• 关键词: Age; Aging; Biomechanics; Blood Vessels; Blood capillaries; Bone Tissue; Bone callus; Cartilage; Cell Differentiation process; Cell Lineage; Cells; Chondrocytes; Clinical; Data; Diabetes Mellitus; Economic Burden; Encapsulated; Evaluation; Event; Femoral Fractures; Flow Cytometry; Fracture; Frequencies; Gelatin; Healthcare Systems; Histologic; Histology; Hydrogels; Image; Impairment; Incidence; Knockout Mice; LoxP-flanked allele; Mediating; Mesenchymal; MicroRNAs; Modality; Modeling; Molecular; Molecular Analysis; Molecular Target; Monitor; Mus; Names; Osteoblasts; Osteocytes; Osteogenesis; Osteoporosis; Osteoporotic; Pathway interactions; Patients; Periosteal Cell; Periosteum; Pharmaceutical Preparations; Population; Process; RNA; RUNX3 gene; Regulator Genes; Regulatory Pathway; Reporter; Reporting; Repression; Roentgen Rays; Role; Site; Smoking; Sorting - Cell Movement; Tamoxifen; Testing; Time; Tissues; Torsion; Transgenic Mice; Translational Research; Vascular blood supply; Veterans; Work; angiogenesis; bone; bone fracture repair; bone healing; bone imaging; bone mass; bone repair; cartilaginous; clinically relevant; comorbidity; conditional knockout; experimental study; healing; ineffective therapies; laser capture microdissection; long bone; lost work time; microCT; multiphoton microscopy; nanofiber; new therapeutic target; novel; novel therapeutic intervention; novel therapeutics; osteogenic; osteoporosis with pathological fracture; pre-clinical; prevent; progenitor; skeletal stem cell; stem cells; success; transcription factor; two photon microscopy

Clinical Dilemma: The frequency of impaired fracture healing is increased with aging as well as in the presence of other patient-related factors such as smoking, osteoporosis, and diabetes. Treatment of fractures in this setting continues to pose a significant economic burden on the US healthcare system due to increases in time lost from work as well as increases in the expenses associated with fracture-associated complications. While various bone anabolic drugs are successful in increasing homeostatic bone mass in osteoporotic patients and decreasing fracture incidence, they have not demonstrated significant success in enhancing fracture repair. Therefore, identifying novel molecular targets to accelerate secondary fracture healing in this very common setting remains of paramount importance. Relevance to the VA: According to the Office of VA Inspector General report in 2010, osteoporotic patients who suffered a single fracture present a higher incidence of subsequent fractures (20-fold increase) than unaffected populations. Impaired or delayed bony union following fracture of long bones prevents or delays a significant percentage of VA patients from resuming their daily activities and returning to work. Ineffective treatment of these fractures maximizes the economic burden on the VA healthcare system. Identifying novel molecular targets to enhance secondary bone repair remains of paramount importance. The objective of this translational research application is to enhance secondary fracture healing by targeting novel regulatory pathways that enhance periosteal cell-induced osteogenesis and angiogenesis during fracture callus formation. Scientific premise: We provide compelling preliminary evidence of the following: 1. Runx3 is expressed in chondrocytes, osteoblasts and osteocytes of C57BL6j murine long bones. 2. Runx3 expression levels are increased in soft cartilaginous calluses and subsequently decreased in bony calluses of murine femoral fractures. 3. Conditional deletion of Runx3 in periosteal cells (cKO) resulted in enhanced secondary bone healing as evidenced by histological, histomorphometric, and molecular analyses. 4. The cellular mechanisms underlying these positive effects on secondary bone healing implicate increased osteogenesis as well as angiogenesis of fractured periosteal cells from Runx3 cKO compared to control mice. 5. Use of multiphoton microscopy demonstrate the feasibility of tracking Prx1+ skeletal progenitor cells during bone repair and longitudinally monitor the bone healing process for lineage tracing experiments. Here we hypothesize that Runx3 is a molecular switch that controls the transition from cartilaginous to bony callus, and its deletion in the chondrogenic cell lineage will accelerate secondary fracture healing. To verify this hypothesis, we propose to first establish the effects of stage-specific repression of Runx3 on secondary fracture healing (Aim 1). We will then determine the mechanisms via which Runx3 controls mesenchymal cell differentiation into the chondro/osteogenic lineages (Aim 2). Finally, we will assess the efficacy of Runx3 inhibition during fracture repair in control C57BL6 mice through controlled and sustained delivery of miRNA encapsulated hydrogel and examine the rate of bone healing and biomechanical strength of healed bone (Aim 3). Impact: Defining the pathways that governthe transition from soft to bony callus will help identify new therapies to accelerate secondary fracture healing. Here, we will establish Runx3 as a novel therapeutic target.

临床困境：骨折愈合受损的频率随着年龄的增长而增加 是否存在其他与患者相关的因素，例如吸烟、骨质疏松症和糖尿病。骨折治疗 在这种情况下，由于医疗费用的增加，继续对美国医疗保健系统造成重大经济负担 工作时间损失以及与骨折相关并发症相关的费用增加。 虽然各种骨合成代谢药物成功地增加了骨质疏松症患者的稳态骨量 患者并降低骨折发生率，但他们在增强 骨折修复。因此，识别新的分子靶点来加速骨折的二次愈合 非常常见的环境仍然至关重要。 与 VA 的相关性：根据 VA 监察长办公室 2010 年的报告，骨质疏松症患者 发生过一次骨折的人后续骨折的发生率比 未受影响的人群。长骨骨折后骨愈合受损或延迟可预防或延迟骨折 很大一部分 VA 患者无法恢复日常活动并重返工作岗位。无效 这些骨折的治疗使退伍军人管理局医疗保健系统的经济负担最大化。识别小说 增强二次骨修复的分子靶标仍然至关重要。此举的目的 转化研究应用是通过针对新的监管来增强继发性骨折愈合 在骨折愈伤组织形成过程中增强骨膜细胞诱导的成骨和血管生成的途径。 科学前提：我们提供以下令人信服的初步证据： 1. Runx3在C57BL6j鼠长骨的软骨细胞、成骨细胞和骨细胞中表达。 2. Runx3表达水平在软软骨愈伤组织中增加，随后在骨性组织中减少 小鼠股骨骨折的老茧。 3. 骨膜细胞中 Runx3 的条件性缺失 (cKO) 导致二次骨愈合增强，如下所示： 通过组织学、组织形态计量学和分子分析来证明。 4. 这些对二次骨愈合的积极作用背后的细胞机制表明， 与对照相比，Runx3 cKO 骨折骨膜细胞的成骨和血管生成 老鼠。 5. 使用多光子显微镜证明了追踪 Prx1+ 骨骼祖细胞的可行性 骨修复并纵向监测骨愈合过程以进行谱系追踪实验。 在这里，我们假设 Runx3 是一个分子开关，控制从软骨到 骨愈伤组织，其在软骨形成细胞谱系中的缺失将加速继发性骨折 康复。 为了验证这一假设，我们建议首先确定 Runx3 的阶段特异性抑制对 骨折二次愈合（目标 1）。然后我们将确定 Runx3 控制的机制 间充质细胞分化为软骨/成骨谱系（目标 2）。最后，我们将评估 通过控制和持续的控制，在对照 C57BL6 小鼠骨折修复过程中 Runx3 抑制的功效 递送 miRNA 封装的水凝胶并检查骨愈合速度和生物力学强度 骨头愈合（目标 3）。 影响：定义控制从软愈伤组织到骨愈伤组织转变的途径将有助于确定新的疗法 加速骨折的二次愈合。在这里，我们将把 Runx3 确立为一个新的治疗靶点。