Chemokine Directed Cell Trafficking during Continuous Infusion of Wear Particles

描述（由申请人提供）：在我们现有的R21资助下，我们开发了一种使用连接到输液泵的髓内股骨植入物进行连续颗粒输注的新型小鼠模型，以了解磨损颗粒诱导骨质溶解的基本生物学过程。该临床相关模型通过将颗粒连续输送到骨-植入物界面，更接近地模拟假体环境。该R 01授权申请的目的是使用体外和体内模型阐明趋化因子指导的巨噬细胞（MAC）和骨祖细胞（OPC）向颗粒运输的生物学过程。这项工作将突出的机制，颗粒诱导MAC和OPC的趋化性和运输，并建议潜在的战略和目标，以减轻骨质溶解。具体目标#1。证明颗粒激发MAC诱导的体外局部MAC和OPC趋化性部分由两种特异性C-C趋化因子介导：巨噬细胞趋化蛋白-1（MCP-1）和巨噬细胞炎症蛋白-1 α（MIP-11）。具体目标#2。使用报告基因和生物发光（BLI）证明从远程部位引入的MAC和OPC将迁移至股骨，其中持续输注的聚乙烯颗粒正在体内输送。使用[18F]氟离子正电子发射断层扫描（PET）扫描，我们将证明局部骨代谢对粒子输注的反应增强。具体目标#3证明细胞从体内远端部位向颗粒植入区域的运输是由趋化因子MCP-1和MIP-11介导的。干扰这些趋化因子和随后的细胞运输将减弱异物反应，减少骨质溶解，并降低骨代谢活性。野生型鼠MAC将在体外暴露于聚乙烯颗粒。将在体外transwell细胞迁移装置中定量评估野生型（CCR 1 ++、CCR 2 ++）、CCR 1--（对MIP-11无反应）和CCR 2--（对MCP-1无反应）MAC和OPC的局部迁移。将测试针对MCP-1和MIP-11的抗体对细胞趋化性的影响。体内研究将使用我们的连续颗粒输注、髓内股骨植入物和装载聚乙烯颗粒的输注泵的小鼠模型。在手术后第6天，通过裸鼠的尾静脉注射报告基因野生型、CCR 1--和CCR 2--MAC和OPC。在术后第5天（第6天注射细胞前一天）、第7天、第8天、第14天、第21天和第28天，使用连续定量BLI和PET扫描跟踪细胞运输和代谢活性。MicroCT扫描将记录骨质溶解。其他研究将使用输注的趋化因子抗体和趋化因子敲除小鼠作为植入受体。这项研究将阐明颗粒诱导的，趋化因子导向的细胞运输的重要机制，并提出新的治疗策略。

DESCRIPTION (provided by applicant): Under our existing R21 grant, we developed a novel murine model of continuous particle infusion using an intramedullary femoral implant connected to an infusion pump in order to understand fundamental biological processes involved in wear particle-induced osteolysis. This clinically relevant model simulates the prosthetic environment more closely by using continuous delivery of particles to the bone-implant interface. The purpose of this R01 grant application is to elucidate the biological processes of chemokine-directed macrophage (MAC) and osteoprogenitor cell (OPC) trafficking to particles, using in vitro and in vivo models. This work will highlight the mechanisms of particle-induced MAC and OPC chemotaxis and trafficking, and suggest potential strategies and targets for mitigation of osteolysis. Specific Aim #1. To demonstrate that local MAC and OPC chemotaxis in vitro induced by particle-challenged MACs is mediated in part by two specific C-C chemokines: Macrophage Chemoattractant Protein-1 (MCP-1) and Macrophage Inflammatory Protein-1 alpha (MIP-11). Specific Aim #2. To demonstrate that MACs and OPCs introduced from a remote site will migrate to the femur in which continuously infused polyethylene particles are being delivered in vivo, using reporter genes and bioluminescence (BLI). Using [18F] fluoride ion Positron Emission Tomography (PET) scanning we will demonstrate a heightened local bone metabolic response to particle infusion. Specific Aim #3. To demonstrate that cell trafficking from remote sites to the particle-implant area in vivo is mediated by the chemokines MCP-1 and MIP-11. Interference with these chemokines and subsequent cell trafficking will blunt the foreign body reaction, decrease osteolysis, and decrease bone metabolic activity. Wild type murine MACs will be exposed to polyethylene particles in vitro. Local migration of wild type (CCR1++, CCR2++), CCR1-- (not responsive to MIP-11), and CCR2-- (not responsive to MCP-1) MACs and OPCs will be assessed quantitatively in a transwell cell migration apparatus in vitro. The effects of antibodies to MCP-1 and MIP-11 on cell chemotaxis will be tested. In vivo studies will use our murine model of continuous particle infusion, intramedullary femoral implant and infusion pump loaded with polyethylene particles. Reporter wild type, CCR1--, and CCR2-- MACs and OPCs will be injected via the tail vein of nude mice at day 6 post-operatively. Cell trafficking and metablic activity will be followed using sequential quantitative BLI and PET scanning at post-operative days 5 (one day before injection of cells on day 6), 7 , 8, 14, 21 and 28. MicroCT scans will document osteolysis. Additional studies will use infused chemokine antibodies, and chemokine knockout mice as the implant recipient. This research will elucidate important mechanisms of particle-induced, chemokine-directed cell trafficking and suggest new treatment strategies.