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 植入物相关的感染是肌肉骨骼组织工程的祸根。总共超过150万 每年进行的髋关节和总膝盖置换手术，主要由 金黄色葡萄球菌仍然是与肌肉骨骼有关的最严重和毁灭性的风险之一 植入物。几十年来，人们已经了解到在生物环境中添加异物 为细菌附着，定殖和顽固生物膜形成提供了避风港。基于此 教条，已经建立了“表面种族”的概念来解释 宿主细胞和植入物定植的细菌。为了偏向于主持人，各种各样的竞争 已经测试了抗菌生物材料，表面涂料，药物和免疫疗法。虽然有很多 基于体外发现和动物模型的初步结果显示出希望 在临床试验中。尽管缺乏临床翻译有几种解释，但已被广泛接受 缺点是对测定的过度缓解（例如静态生物膜，菌落形成单元（CFU），最低 抑制浓度（MIC）和横截面结果（例如静态放射学和显微镜），它们 不能忠实地评估体内感染过程。那个程序的科学前提是真实 时间在体内数量的浮游细菌在肌肉骨骼的表面上生长，而 先天宿主对这些细菌的反应对于评估新型预防和治疗至关重要 显着抑制定殖和生物膜形成的干预措施。为此，我们开创了 使用与植入物相关的鼠模型的浸润式多光子激光扫描显微镜（IV-MLSM） 骨髓炎。我们的初步研究量化了红色荧光S的增殖和表面覆盖范围。 金黄色的金黄色 证明表面的竞赛非常动态，并且在3小时内完成。在该计划的AIM 1中， 我们将确认这些发现，并正式建立表面种族种族的实时动力学 护理标准不锈钢即将 金黄色葡萄球菌的甲氧西林敏感和抗性菌株。我们还将评估Cerulean S.金黄色葡萄球菌 LY6GCRE/ROSATDTOMATO/CSF1R-EGFP小鼠以量化植入物表面定植，并清除细菌 （蓝色）由中性粒细胞（橙色）和巨噬细胞（绿色）在体内。在AIM 2中，我们将检验以下假设 先前描述的抗微生物的有效性（“如火如荼”氮化硅（SI3N4）和3D打印的钛） 是由于它们在竞赛中促使宿主细胞比细菌定植的能力。在 完成此高风险高奖励计划的完成，我们将拥有新的体内结果指标来阐明 控制表面种族的分子和细胞机制，以及经验阈值 评估肌肉骨骼组织工程抗菌干预措施的效率。