Chemokine Directed Cell Trafficking during Continuous Infusion of Wear Particles

描述(申请人提供)：在我们现有的R21资助下，我们开发了一种新的小鼠持续颗粒输注模型，该模型使用连接到输液泵的股骨髓内植入物，以了解磨损颗粒诱导骨溶解所涉及的基本生物学过程。这种与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。这项R01拨款申请的目的是利用体外和体内模型，阐明趋化因子导向的巨噬细胞(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靶点。目的1.证实MACs体外诱导的局部MAC和OPC趋化作用部分由两种特异性C-C趋化因子介导：巨噬细胞趋化蛋白-1(MCP-1)和巨噬细胞炎性蛋白-1α(MIP-11)。具体目标#2.利用报告基因和生物发光(BLI)技术，证明从远程站点引入的Mac和OPC将迁移到股骨，在那里连续注入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)，我们将证明局部骨代谢对颗粒注射的反应增强。具体目的#3.证明体内细胞从远端到颗粒植入区域的转运是由趋化因子MCP-1和MIP-11介导的。干扰这些趋化因子和随后的细胞转运将钝化异物反应，减少骨溶解，并降低骨代谢活性。野生型小鼠Mac在体外将暴露于聚乙烯颗粒。野生型(CCR1++，CCR2++)、CCR1--(对MIP-11不反应)和CCR2-(对MCP-1不反应)Mac和OPC的局部迁移将在Transwell细胞迁移装置中进行定量评估。将测试MCP-1和MIP-11抗体对细胞趋化的影响。体内研究将使用我们的小鼠持续颗粒输注、股骨髓内植入和充填聚乙烯颗粒的输液泵模型。术后第6天经尾静脉注射野生型、CCR1-和CCR2-MACs和OPC。在术后第5天(第6天注射细胞的前一天)、第7天、第8天、第14天、第21天和第28天，用序贯定量BLI和PET扫描观察细胞的运输和代谢活性。显微CT扫描将记录骨质溶解。其他研究将使用注入的趋化因子抗体和趋化因子基因敲除小鼠作为植入受体。这项研究将阐明颗粒诱导、趋化因子导向的细胞转运的重要机制，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.