Quantifying the Race for the Surface via IV-MLSM

摘要 植入物相关感染是肌肉骨骼组织工程的祸根。总计超过150万 每年进行髋关节和全膝关节置换手术，骨感染主要由 金黄色葡萄球菌仍然是与肌肉骨骼相关的最严重和最具破坏性的风险之一 植入物。几十年来，人们一直认为，在生物环境中添加外来物质 为细菌附着、定植和顽固生物膜的形成提供了避风港。在此基础上 Dogma，建立了“表面竞赛”的概念来解释 植入物定植的宿主细胞和细菌。为了让这场比赛偏向主办方，各种 抗菌生物材料、表面涂层、药物和免疫疗法已经进行了测试。虽然很多人都有 根据体外研究结果和动物模型的初步结果，没有一种药物被证实有效 在临床试验中。虽然对于缺乏临床翻译有几种解释，但一个被广泛接受的 缺点是过度依赖化验(例如静态生物膜、菌落形成单位(CFU)、最低 抑制浓度(MIC)和横断面结果(例如静态放射学和显微镜检查)， 不能如实评估体内感染过程。因此，这个节目的科学前提是真实的 肌肉骨骼植入物表面浮游细菌生长的活体时间定量 对这些细菌的天然宿主反应，对于评估新的预防和治疗方法至关重要 显著抑制定居和生物膜形成的干预措施。为此，我们开创了 活体多光子激光扫描显微镜(IV-MLSM)在种植体相关小鼠模型中的应用 骨髓炎。我们对红色荧光S的增殖和表面覆盖率进行了初步研究。 金黄色与股骨内金属植入物上绿色荧光宿主细胞的表面覆盖率 在3小时内展示对地面的比赛是非常有活力和完整的。在本节目的目标1中， 我们将证实这些发现，并正式建立表面竞赛的实时动力学 标准护理不锈钢植入物，以及标准护理非肠道抗生素的疗效 金黄色葡萄球菌的甲氧西林敏感和耐药菌株。我们还将评估天蓝色的金黄色葡萄球菌 Ly6GCre/ROSAtdTomato/CSF1R-EGFP小鼠对种植体表面定植和细菌清除的量化 (蓝色)由中性粒细胞(橙色)和巨噬细胞(绿色)在体内。在目标2中，我们将测试假设 先前描述的抗菌植入物的功效(“烧成”氮化硅(Si3N4)和3D打印的钛) 是因为在争夺表面的过程中，它们有能力偏爱宿主细胞，而不是细菌的定植。在 完成这一高风险-高回报计划，我们将有新的体内结果指标来阐明 控制表面竞争的分子和细胞机制，以及经验阈值 评估肌肉骨骼组织工程中抗菌干预的效果。

Abstract Implant-associated infections are the bane of musculoskeletal tissue engineering. With over 1.5 million total hip and total knee replacement procedures performed each year, bone infection primarily caused by Staphylococcus aureus remains among the most severe and devastating risks associated with musculoskeletal implants. It has been understood for decades that the addition of a foreign material to a biological environment provides a haven for bacterial attachment, colonization and recalcitrant biofilm formation. Based on this dogma, the concept of the “race for the surface” has been established to explains the competition between host cells and bacteria for implant colonization. To bias this competition in favor of the host, various antimicrobial biomaterials, surface coatings, drugs and immunotherapies have been tested. While many have shown promise based on in vitro findings and preliminary results in animal models, none have proven efficacy in clinical trials. While there are several explanations for the lack of clinical translation, a broadly accepted shortcoming has been the over reliance on assays (e.g. static biofilm, colony formation units (CFU), minimum inhibitory concentration (MIC)), and cross-sectional outcomes (e.g. static radiology and microscopy), which cannot faithfully assess the in vivo infection process. Thus, the Scientific Premise of this program is that real time in vivo quantification of planktonic bacterial growth on the surface of musculoskeletal implants, and the innate host response to these bacteria, is critical for the evaluation of novel prophylactic and therapeutic interventions that significantly inhibit colonization and biofilm formation. To this end, we have pioneered the use of intravital multiphoton laser scanning microscopy (IV-MLSM) with a murine model of implant-associated osteomyelitis. Our preliminary studies quantifying the proliferation and surface coverage of red fluorescent S. aureus, versus surface coverage of green fluorescent host cells on a metal implant within the femur demonstrate that the race for the surface is very dynamic and complete within 3hrs. In Aim 1 of this program, we will confirm these findings, and formally establish the real time kinetics of the race of the surface on standard of care stainless steel implants, and the efficacy of standard of care parenteral antibiotics against methicillin sensitive and resistant strains of S. aureus. We will also assess cerulean S. aureus in Ly6GCre/ROSAtdTomato/Csf1r-EGFP mice to quantify implant surface colonization, and clearance of bacteria (blue) by neutrophils (orange) and macrophages (green) in vivo. In Aim 2, we will test the hypothesis that the efficacy of previously described antimicrobial implants (“as fired” silicon nitride (Si3N4) and 3D-printed titanium) is due to their ability to favor host cells over bacterial colonization during the race for the surface. At the completion of this high risk-high reward program, we will have new in vivo outcome metrics to elucidate the molecular and cellular mechanisms that govern the race for the surface, and empirical threshold values to assess the efficacy of antimicrobial interventions for musculoskeletal tissue engineering.