Modification of Bone Grafts for Orthopaedic Procedures

描述（申请人提供）：骨移植是目前大量骨骼缺损修复的金标准。然而，一旦骨移植物被耐甲氧西林金黄色葡萄球菌（MRSA）感染，由于缺乏血管和灌注的抗菌生物利用度，感染是非常难以治疗的。植骨感染不仅会导致微生物居住，还会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种双膦酸盐共轭纳米微尺寸气泡（BNB）的治疗应用，它可以装载万古霉素和炎症激酶抑制剂来治疗MRSA骨移植感染。在技术上，我们优化了BNB的载药、释放动力学、骨附着和杀菌效果。从机制角度来看，微阵列、蛋白激酶阵列和验证性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;在成骨细胞或AZD 6244（临床级MEK1-ERK1/2磷酸化抑制剂）治疗中，ERK1/2磷酸化缺陷的DN MEK1小鼠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炎症反应和破骨细胞生成。我们提出一个中心假设，即万古霉素+ azd6244负载BNB可减少MRSA感染后的术后骨感染和炎症性移植物吸收。三个特定目的：目的1（抗微生物技术）：在体外建立载药bnb移植物预防MRSA感染和先天免疫炎症反应的效果；目的2（机制）：确定pERK1/2在体外和体内mrsa诱导的成骨细胞细胞因子风暴和随后的炎症相关骨质流失中的作用；目的3（临床前转化）：在感染植骨动物模型中建立BNB同时治疗MRSA感染和炎症的新防治方案。我们将使用临床级pERK1/2抑制剂和Osx-Cre进行一系列机制实验；ERK1/2磷酸化缺陷的DN MEK1小鼠对mrsa感染的人和鼠成骨细胞中RANKL、MCSF、TNF、IL-1b、IL-6 mRNA表达的影响我们将对小鼠进行临床相关的股骨骨间植骨实验，通过x线片、显微ct、组织学和生物力学测试数据分析，确定万古霉素+ AZD-BNB是否能预防或减少mrsa感染骨光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.