Quantifying the Race for the Surface via IV-MLSM

通过 IV-MLSM 量化表面竞赛

• 批准号: 10618393
• 负责人: Edward M. Schwarz
• 金额: $ 16.94万
• 依托单位: UNIVERSITY OF ROCHESTER
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-05-05 至 2024-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10618393
• 关键词: 3D Print; Animal Model; Antibiotics; Antimicrobial Effect; Bacteria; Bacterial Attachment Site; Biocompatible Materials; Biological; Biological Assay; Bone Surface; Cells; Clinical; Clinical Trials; Development; Environment; Evaluation; Failure; Femur; Growth; Hip region structure; Histology; Image; Immune response; Immunotherapy; Implant; In Vitro; Infection; Intervention; Kinetics; Laser Scanning Microscopy; Macrophage; Mesenchymal Stem Cells; Metals; Methicillin; Microbial Biofilms; Microscopy; Minimum Inhibitory Concentration measurement; Modeling; Molecular; Mus; Musculoskeletal; Nafcillin; Operative Surgical Procedures; Oranges; Osseointegration; Osteoblasts; Osteomyelitis; Outcome; Pharmaceutical Preparations; Procedures; Process; Proliferating; Property; Race; Radiology Specialty; Risk; Stainless Steel; Staphylococcus aureus; Surface; Testing; Therapeutic Intervention; Time; Tissue Engineering; Titanium; Transgenic Mice; Vancomycin; antimicrobial; bacterial resistance; biomechanical test; clinical translation; efficacy evaluation; high reward; high risk; host colonization; implant associated infection; implantation; in vivo; interest; knee replacement arthroplasty; methicillin resistant Staphylococcus aureus; mouse model; multi-photon; neutrophil; novel; novel strategies; prevent; programs; prophylactic; prospective; recruit; resistant strain; septic; silicon nitride; standard of care; success; surface coating

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.

抽象的 植入物相关感染是肌肉骨骼组织工程的祸根，总数超过 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 几十年来人们一直认为，向生物环境中添加外来物质。 在此基础上，为细菌附着、定植和顽固的生物膜形成提供了避风港。 教条，“表面竞赛”的概念被建立来解释之间的竞争 为了使这种竞争有利于宿主，有多种方法可以用于植入物定植的宿主细胞和细菌。 抗菌生物材料、表面涂层、药物和免疫疗法已经过测试。 根据体外研究结果和动物模型的初步结果显示出希望，但没有一个被证明有效 虽然对于缺乏临床转化有多种解释，但有一种被广泛接受的解释。 缺点是过度依赖测定（例如静态生物膜、菌落形成单位 (CFU)、最小 抑制（MIC））和横截面结果（例如静态放射学和显微浓缩镜），其中 无法忠实地评估体内感染过程因此，该计划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体内时间定量，以及 宿主对这些细菌的先天反应对于评估新型预防和治疗药物至关重要 为此，我们率先采取了显着抑制定植和生物膜形成的干预措施。 使用活体多光子激光扫描显微镜（IV-MLSM）与植入相关的小鼠模型 我们的初步研究量化了红色荧光链球菌的增殖和表面覆盖。 金黄色葡萄球菌，与股骨内金属植入物上绿色荧光宿主细胞的表面覆盖率 证明表面竞赛非常动态且在 3 小时内完成 在本计划的目标 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 不锈钢植入物的护理标准，以及肠外抗生素护理标准的功效 我们还将评估金黄色葡萄球菌的甲氧西林敏感和耐药菌株。 Ly6GCre/ROSAtdTomato/Csf1r-EGFP 小鼠可量化植入物表面定植和细菌清除 在目标 2 中，我们将检验以下假设： 先前描述的抗菌植入物（“烧制”氮化硅 (Si3N4) 和 3D 打印钛）的功效 这是因为它们在争夺表面的过程中能够有利于宿主细胞而不是细菌定植。 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 控制表面竞争的分子和细胞机制，以及经验阈值 评估抗菌干预措施对肌肉骨骼组织工程的功效。