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，我们开发了一种新型的小鼠模型，使用连接输注泵的髓内股植入物进行连续颗粒输注，以了解磨损颗粒诱导的骨溶解的基本生物学过程。这种临床相关的模型通过将颗粒连续输送到骨-种植体界面来更接近地模拟假体环境。本R01基金申请的目的是通过体外和体内模型阐明趋化因子导向巨噬细胞（MAC）和骨祖细胞（OPC）向颗粒运输的生物学过程。这项工作将强调颗粒诱导的MAC和OPC趋化和运输的机制，并提出缓解骨溶解的潜在策略和目标。明确目标#1。研究表明，由颗粒激发的MAC诱导的局部MAC和OPC体外趋化部分是由两种特异性C-C趋化因子介导的：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。利用报告基因和生物发光技术（BLI）证明，从远处引入的mac和opc将迁移到持续注入聚乙烯颗粒的股骨中。使用[18F]氟离子正电子发射断层扫描（PET），我们将证明颗粒输注引起局部骨代谢反应增强。具体目标#3。证明细胞在体内由趋化因子MCP-1和MIP-11介导从远程位置到颗粒植入区域的运输。干扰这些趋化因子和随后的细胞运输会减弱异物反应，减少骨溶解，降低骨代谢活性。野生型小鼠mac将在体外暴露于聚乙烯颗粒。野生型（CCR1++, CCR2++）， CCR1-（对MIP-11无反应）和CCR2-（对MCP-1无反应）mac和OPCs的局部迁移将在体外transwell细胞迁移装置中进行定量评估。我们将测试MCP-1和MIP-11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小鼠模型，髓内股内植入物和装载聚乙烯颗粒的输注泵。报告型野生型、CCR1—和CCR2—MACs和OPCs将于术后第6天通过裸鼠尾静脉注射。在术后第5天（第6天注射细胞前1天）、7、8、14、21和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扫描将记录骨溶解。进一步的研究将使用注入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、趋化因子引导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.