Modification of Bone Grafts for Orthopaedic Procedures

描述(申请人提供)：植骨是目前填补大量骨缺损的黄金标准。然而，一旦骨移植被耐甲氧西林金黄色葡萄球菌(MRSA)感染，由于缺乏抗菌药物生物利用度的血管和灌流，感染很难治疗。植骨感染不仅导致微生物栖息，还会引发先天免疫炎性细胞因子风暴。炎症旨在击退病原体，但过度的炎症会破坏骨骼，就像埃博拉病毒感染通过一系列细胞因子风暴摧毁宿主一样。美国卫生与公众服务部(HHS)正在努力将感染降至最低，这是最重要的质量和安全指标之一。因此，迫切需要开发一种新的策略来预防或减少骨移植相关的感染。我们的建议制定了一种双膦酸盐偶联纳米微泡(BNB)的治疗应用，该泡泡可以负载万古霉素和炎性激酶抑制剂，用于治疗MRSA骨移植感染。在技术上，我们对BNB的载药量、释放动力学、骨附着和杀菌作用进行了优化。从机制的角度来看，微阵列、蛋白激酶阵列和确认性RT-PCR显示，MRSA在MRSA感染的成骨细胞中诱导了主要由pERK1/2介导的炎性-破骨细胞因子风暴。OSX-CRE；DNMEK1成骨细胞ERK1/2磷酸化缺陷小鼠或AZD 6244(临床级别的MEK1-ERK1/2磷酸化抑制剂)治疗在体内和体外均表现出对细菌毒素或MRSA的先天炎症反应和破骨细胞生成的抑制。我们假设了一个中心假设，即万古霉素+AZD6244负载的BNB减少了术后骨感染和MRSA感染后炎性移植物的吸收。目标1(抗微生物技术)：确定载药BNB移植物在体外预防感染和抗MRSA感染的天然免疫炎症反应的有效性；目的2(机制)：确定pERK1/2在MRSA诱导的细胞因子风暴和随后的体内外炎症相关骨丢失中的作用；以及目标3(临床前翻译)：在感染的骨移植动物模型中建立一种新的预防和治疗MRSA感染和BNB炎症的方案。我们将使用临床级别的pERK1/2抑制剂和OSX-CRE；在体外对MRSA感染的人和小鼠成骨细胞中RANKL、MCSF、肿瘤坏死因子、IL-1b和IL-6mRNA的表达进行一系列的机制实验。我们将进行临床相关的小鼠股骨移植实验，通过分析放射学、显微CT、组织学和生物力学测试数据，确定万古霉素+AZD-BNB是否可以预防或减少MRSA感染骨栅后的感染和过度炎症。所提出的技术和治疗概念可以扩展到预防或治疗其他类型的骨或生物植入物感染和高影响的细菌菌株。

DESCRIPTION (provided by applicant): Bone grafting is currently the gold standard for filling in massive skeletal defects. However, once a bone graft is infected by methicillin-resistant S. aureus (MRSA) a common pathogen, infection is very difficult to treat due to the lack of vascularity and perfusion for antimicrobial bioavailability. Bone graft infection results in not ony microbial habitation, but also triggers innate immune inflammatory cytokine storms. Inflammation is meant to repel pathogens but excessive inflammation destroys bone just as the Ebola viral infection through a series of cytokine storms destroys its host. The U.S. Department of Health and Human Services (HHS) is pushing to minimize infection, one of the most important 'Quality and Safety' indicators. Therefore, there is a desperate need for developing a new strategy to prevent or reduce bone graft-associated infections. Our proposal formulates a therapeutic application of bisphosphonate-conjugated nano-micro-size-bubbles (BNB) that can load vancomycin and inflammatory kinase inhibitors for the treatment of MRSA bone graft infections. Technically, we optimized drug loading, release-kinetics, bone attachment, and the bactericidal effect of BNB. From a mechanism perspective, microarray, protein kinase array, and confirmatory RT-PCR showed MRSA- induction of inflammatory-osteoclastogenic cytokine storms that were primarily mediated by pERK1/2 in MRSA-infected osteoblasts. Osx-Cre;DN MEK1 mice with defective ERK1/2 phosphorylation in osteoblasts or AZD 6244 (a clinical grade MEK1-ERK1/2 phosphorylation inhibitor) treatments exhibited suppressed innate inflammatory response and osteoclastogenesis in response to bacterial toxins or MRSA in vivo and in vitro. We posit a central hypothesis that vancomycin+AZD6244-loaded BNB reduce post-operative bone infections and inflammatory graft resorption following MRSA infection. Three Specific Aims are Aim 1 (Anti-Microbial Technology): To establish the efficacy of drug-loaded-BNB grafts to prevent infection and innate immune inflammatory reaction against MRSA infection in vitro; Aim 2 (Mechanism): To determine a role of pERK1/2 in MRSA-induced cytokine storms by osteoblasts and subsequent inflammation-associated bone loss in vitro and in vivo; and Aim 3 (Pre-Clinical Translation): To establish a new preventive & therapeutic regimen concurrently treating MRSA infection and inflammation with BNB in an animal model of infected bone grafting. We will conduct a series of mechanistic experiments using a clinical-grade pERK1/2 inhibitor and Osx-Cre;DN MEK1 mice with defective ERK1/2 phosphorylation regarding mRNA expression of RANKL, MCSF, TNF, IL-1b, IL-6 in MRSA-infected human and murine osteoblasts in vitro. We will conduct clinically relevant intercalary femoral bone grafting experiments in mice and determine whether vancomycin + AZD-BNB prevents or reduces infection and excessive inflammation following MRSA-infected bone grating by analyzing radiographs, microCT, histology, and biomechanical testing data. The proposed technique and therapeutic concepts may be extended to prevent or treat other types of bone or bio-implant infections and bacterial strains with high impact.