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系统相关的最严重和最具破坏性的风险之一， 植入物.几十年来，人们已经认识到，向生物环境中添加外来物质 为细菌附着、定殖和寄生生物膜形成提供了避风港。基于此 教条，“表面竞赛”的概念已经建立，以解释之间的竞争 用于植入定殖的宿主细胞和细菌。为了使这场比赛偏向东道主， 已经测试了抗微生物生物材料、表面涂层、药物和免疫疗法。虽然许多人 基于体外研究结果和动物模型的初步结果， 在临床试验阶段虽然对缺乏临床翻译有几种解释，但广泛接受的是 缺点是过度依赖于测定（例如静态生物膜、菌落形成单位（CFU）、最小菌落形成单位（CFU））。 抑制浓度（MIC））和横断面结果（例如静态放射学和显微镜检查）， 不能真实地评估体内感染过程。因此，这个程序的科学假设是真实的， 肌肉骨骼植入物表面上的细菌生长的时间体内定量，以及 先天宿主对这些细菌的反应，对于评价新的预防和治疗药物是至关重要的。 这些干预措施显著抑制定殖和生物膜形成。为此，我们开创了 活体多光子激光扫描显微镜（IV-MLSM）在种植体相关性肿瘤小鼠模型中的应用 骨髓炎我们的初步研究定量的增殖和表面覆盖的红色荧光S。 金黄色葡萄球菌，相对于股骨内金属植入物上绿色荧光宿主细胞的表面覆盖 证明了表面的比赛是非常动态的，并在3小时内完成。在该方案的目标1中， 我们将证实这些发现，并正式建立表面竞赛的真实的时间动力学。 标准护理不锈钢植入物，以及标准护理肠外抗生素对 甲氧西林敏感和耐药菌株。金黄色。我们还将评估天蓝色S。金黄色 Ly6GCre/ROSAtdTomato/Csf1r-EGFP小鼠用于定量植入物表面定殖和细菌清除 （蓝色）被体内嗜中性粒细胞（橙子）和巨噬细胞（绿色）吞噬。在目标2中，我们将检验以下假设： 先前描述的抗微生物植入物（"烧制"氮化硅（Si3N4）和3D打印钛）的功效 是由于它们在表面竞争过程中有利于宿主细胞而不是细菌定植的能力。在 完成这个高风险-高回报计划后，我们将有新的体内结果指标来阐明 分子和细胞的机制，管理比赛的表面，和经验阈值， 评估抗菌干预对肌肉骨骼组织工程的有效性。

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.