Targeting angiogenesis for fracture nonunion treatment under inflammatory diseases

摘要 骨折不愈合是一个重要的临床问题。在美国，大约有160万块骨头 骨折每年都会遇到延长愈合或不愈合的情况。骨折不愈合的治疗通常包括 在实践中，复杂和大量的程序，有时需要多次手术，因此增加了 医疗保健的成本，并导致明显的病人残疾。患有这些临床疾病的主要人群 并发症是患有炎性疾病的患者，例如，老年患者、吸烟者、糖尿病患者或类风湿患者 关节炎（RA）患者，强调慢性全身性炎症在骨折中的潜在有害作用 修复。 该建议的首要假设是慢性炎症导致骨折不愈合 通过Dnmt 3b下调介导的血管生成缺陷，以及OPN和CXCL 12的局部递送恢复 炎症条件下的血管生成和骨折修复。这一假设得到了我们初步的支持。 数据，其中我们显示Dnmt 3b在骨折修复过程中在骨折骨痂中高度表达， MPCs中主要的DNA甲基转移酶（Dnmt）对细胞因子有反应。根据我们的建议，我们提供 炎症信号以NF-κ B依赖性方式抑制Dnmt 3b的证据。一致，小鼠 软骨细胞中的Dnmt 3b功能丧失（LOF）显示受损的血管生成和骨折修复; 软骨细胞中的功能获得（GOF）在体外显示出对炎症的保护作用， 小鼠的骨折修复。机制上，炎症和Dnmt 3b LOF介导的血管生成缺陷 与OPN（骨桥蛋白）和CXCL 12（C-X-C基序趋化因子配体12）的下调一致， 外源性OPN和CXCL 12可以恢复体外血管生成能力。为了进一步研究当地 为了在体内递送OPN和CXCL 12，我们已经开发了负载有OPN的优化的生物材料片， CXCL 12，并显示了强大的血管生成过程和恢复骨折愈合的RA小鼠。 提出了三个主要的具体目标。具体目标1将描述 炎症通过下调Dnmt 3b减少骨折修复过程中的血管生成。具体目标2将 确定OPN和CXCL 12对血管生成的最佳释放动力学。具体目标3将决定 持续OPN和CXCL 12释放对小鼠血管生成和骨折不愈合的治疗作用。的 提出的研究将增强我们对全身性炎症（通过NF-κ B）机制的理解， κB通路）通过Dnmt 3 B影响血管生成过程。这项工作将建立一个重要的治疗 选择，以改善血管生成和骨折愈合。

ABSTRACT Fracture nonunion poses a significant clinical problem. In the United States, approximately 1.6 million bone fractures encounter prolonged healing or nonunion each year. Fracture nonunion treatment usually involves complicated and massive procedures in practice, and sometimes needs multiple surgeries, therefore increases the cost of health care and results in marked patient disability. The major population bearing with these clinical complications are patients with inflammatory conditions, e.g, elder patients, smoking, diabetic or rheumatoid arthritis (RA) patients, highlighting the potential deleterious role of chronic systemic inflammation in fracture r epair. The overarching hypothesis of this proposal is that chronic inflammation results in fracture nonunion through Dnmt3b downregulation mediated angiogenesis defect, and local delivery of OPN and CXCL12 restores angiogenesis and fracture repair under inflammatory conditions. This hypothesis is supported by our preliminary data wherein we show that Dnmt3b is highly expressed in fracture callus during fracture repair and Dnmt3b is the major DNA methyltransferase (Dnmt) responsive to cytokines in MPCs. Relevant to our proposal, we provide evidence that inflammatory signals inhibit Dnmt3b in an NF-κB-dependent manner. Consistently, mice with Dnmt3b loss-of-function (LOF) in chondrocytes display impaired angiogenesis and fracture repair; and Dnmt3b gain-of-function (GOF) in chondrocytes shows protective effect from inflammation in vitro and accelerates fracture repair in mice. Mechanistically, angiogenesis defect mediated by inflammation and Dnmt3b LOF coincide with downregulation of OPN (Osteopontin) and CXCL12 (C-X-C Motif Chemokine Ligand 12) and exogenous OPN and CXCL12 can restore angiogenesis capacity in vitro. To further examine the efficacy of local delivery of OPN and CXCL12 in vivo, we have developed an optimized biomaterial sheet loaded with OPN and CXCL12 and showed a robust angiogenesis process and a restoration of fracture union in RA mice. Three main Specific Aims are proposed. Specific Aim 1 will delineate the mechanism by which inflammation reduces angiogenesis via downregulating Dnmt3b during fracture repair. Specific Aim 2 will determine the optimal release kinetics of OPN and CXCL12 on angiogenesis. Specific Aim 3 will determine the therapeutic effect of sustained OPN and CXCL12 release on angiogenesis and fracture nonunion in mice. The proposed studies will enhance our understanding of mechanisms by which systemic inflammation (via the NF- κB pathway) affects the angiogenic process through Dnmt3b. This work will establish an important therapeutic option to improve the angiogenesis and fracture healing.