Modification of Bone Grafts for Orthopaedic Procedures

描述（由申请人提供）：骨移植目前是填充大块骨骼缺损的金标准。然而，一旦骨移植物被耐甲氧西林S。金黄色葡萄球菌（MRSA）是一种常见的病原体，由于缺乏血管分布和灌注以获得抗微生物生物利用度，感染非常难以治疗。骨移植感染不仅导致微生物的定居，而且还引发先天免疫炎症细胞因子风暴。炎症是为了击退病原体，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破坏宿主一样。美国卫生与公众服务部（HHS）正在努力减少感染，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感染。我们的建议制定了一个治疗应用的双膦酸盐共轭纳米微米尺寸的气泡（BNB），可以加载万古霉素和炎症激酶抑制剂的MRSA骨移植感染的治疗。在技术上，我们优化了BNB的载药量、释放动力学、骨附着和杀菌作用。从机制的角度来看，微阵列，蛋白激酶阵列，和验证性RT-PCR显示MRSA诱导的炎症破骨细胞因子风暴，主要是由pERK 1/2介导的MRSA感染的成骨细胞。Osx-Cre;成骨细胞中ERK 1/2磷酸化缺陷的DN MEK1小鼠或AZD 6244（一种临床级MEK1-ERK 1/2磷酸化抑制剂）治疗在体内和体外对细菌毒素或MRSA的反应中表现出抑制的先天性炎症反应和破骨细胞生成。我们证实了一个中心假设，即万古霉素+AZD 6244负载的BNB减少了MRSA感染后的术后骨感染和炎性移植物吸收。三个具体目标是目标1抗微生物技术：建立载药BNB移植物体外抗MRSA感染和天然免疫炎症反应的效果（机制）：确定pERK 1/2在体外和体内成骨细胞的MRSA诱导的细胞因子风暴和随后的炎症相关骨丢失中的作用;（临床前翻译）：建立一种新的预防和治疗方案，同时治疗感染骨移植动物模型中的MRSA感染和BNB炎症。我们将使用临床级pERK 1/2抑制剂和ERK 1/2磷酸化缺陷的Osx-Cre; DN MEK1小鼠进行一系列关于MRSA感染的人和鼠成骨细胞中RANKL、MCSF、TNF、IL-1b、IL-6 mRNA表达的机制实验。我们将在小鼠中进行临床相关的股骨间植骨实验，并通过分析X线片、microCT、组织学和生物力学测试数据来确定万古霉素+AZD-BNB是否预防或减少MRSA感染骨格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具有高影响的细菌菌株。

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.