Wear Particle Disease and NF-kappa B Signaling

描述(申请人提供)：全关节置换术(TJR)是一种非常成功的外科手术；然而，轴承表面的磨损限制了患者的长期生存。磨损颗粒会刺激慢性炎症，延迟骨整合，并导致假体周围骨溶解和种植体松动。颗粒诱导的炎症导致促炎因子的释放；这些事件主要由转录因子核因子kappaB(核因子kappaB)介导，核因子kappaB是促炎基因激活的关键信号分子。这项资助的目的是利用体外和体内模型调节核因子？B的表达，以减轻颗粒诱导的炎症和骨溶解。该研究提案将为人类假体周围骨溶解的治疗提供实质性的策略。具体目的1：证明当小鼠或人巨噬细胞体外暴露于有/无粘附性内毒素的聚乙烯颗粒时，核因子B诱骗寡核苷酸(ODN)能抑制促炎基因和蛋白的表达。具体目的#2：证明当细胞体外暴露于具有/不具有粘附性内毒素的临床相关聚乙烯颗粒时，核因子？B诱骗ODN不会对小鼠或人的成骨细胞产生重大不良影响。具体目标#3：利用我们验证的小鼠股骨植入模型，证明局部注射核因子？B诱骗ODN可以抑制与持续输注聚乙烯颗粒相关的种植体周围炎症和骨溶解。具体目标#4：利用我们的小鼠模型，证明局部传递核因子？B诱骗ODN可以抑制外源性报告巨噬细胞向颗粒输注区域的系统性迁移。小鼠和人的巨噬细胞和骨祖细胞(OPC)将与临床相关的聚乙烯颗粒内毒素一起培养。核因子？B诱骗ODN、杂乱诱骗ODN或什么都不添加到培养物中。检测细胞存活率和增殖能力；从培养上清液(ELISA法和Western印迹法)和细胞(RT-PCR法)分别在蛋白和基因水平检测肿瘤坏死因子、白介素1、白介素6、单核细胞趋化蛋白1和RANKL。培养的OPC将检测碱性磷酸酶和骨钙素的表达，骨基质的形成将使用Von Kossa染色进行定量。使用我们的小鼠股骨植入模型持续注入聚乙烯颗粒将导致系统性报告巨噬细胞向颗粒注入部位转运(使用生物发光和免疫荧光显微镜)，增加局部骨转换(使用microPET扫描)和骨溶解(使用定量microCT)。这些影响将通过本地递送的核因子？B诱骗ODN来缓解。TJR将继续产生聚乙烯碎片，即使是产生更小、潜在更具生物活性的颗粒的较新聚乙烯也是如此。这项拟议的研究具有重要的创新性和临床意义；它将提出潜在的局部药理学策略，以减轻磨损颗粒引起的慢性炎症反应和假体周围的骨溶解。 公共卫生相关性：全关节置换术(TJR)是治疗终末期关节炎的一种非常成功的外科手术，然而TJR的寿命受到轴承表面磨损的限制。磨损颗粒会刺激慢性炎症反应，导致植入物周围局部骨质破坏。这项拨款申请测试了一种新的翻译策略，通过干扰细胞内控制炎症介质产生的主要信号分子(转录因子NFkB)来减轻颗粒相关的骨破坏。这一策略很有可能延长人类TJR的寿命。

DESCRIPTION (provided by applicant): Total joint replacement (TJR) is a highly successful surgical procedure; however the long-term survivorship is limited by wear of the bearing surfaces. Wear particles stimulate chronic inflammation that delays osseointegration, and leads to peri-prosthetic osteolysis and implant loosening. Particle-induced inflammation results in the release of pro-inflammatory factors; these events are mediated primarily by the transcription factor Nuclear Factor kappa B (NF?B), a critical signaling molecule in the activation of pro- inflammatory genes. The purpose of this grant is to modulate NF?B expression in order to mitigate particle- induced inflammation and osteolysis using in vitro and in vivo models. The research proposal will yield substantive strategies for treatment of periprosthetic osteolysis in humans. Specific Aim #1: To demonstrate that an NF?B decoy oligodeoxynucleotide (ODN) will abrogate the expression of pro-inflammatory genes and proteins when murine or human macrophages are exposed to clinically relevant polyethylene particles with/without adherent endotoxin in vitro. Specific Aim #2: To demonstrate that an NF?B decoy ODN will have no major adverse effects on murine or human osteoprogenitors when the cells are exposed to clinically relevant polyethylene particles with/without adherent endotoxin in vitro. Specific Aim #3: To demonstrate that local delivery of the NF?B decoy ODN inhibits peri-implant inflammation and osteolysis associated with continuous infusion of polyethylene particles using our validated murine femoral implant model. Specific Aim #4: To demonstrate that local delivery of the NF?B decoy ODN inhibits systemic migration of exogenous, reporter macrophages to the area of particle infusion using our murine model. Murine and human macrophages and osteoprogenitor cells (OPCs) will be cultured with clinically relevant polyethylene particles endotoxin. NF?B decoy ODN, scrambled decoy ODN or nothing will be added to the cultures. Cellular viability and proliferation will be assessed; TNF, IL-1, IL-6, MCP-1 and RANKL will be assayed at the protein and gene levels from the supernatants (ELISA and Western blot) and cells (RT- PCR) respectively. OPC cultures will be assayed for alkaline phosphatase and osteocalcin expression, and bone matrix formation will be quantitated using Von Kossa staining. Continuous infusion of polyethylene particles using our murine femoral implant model will result in systemic reporter macrophage trafficking to the site of particle infusion (using bioluminescence and immunofluorescence microscopy), increased local bone turnover (using microPET scans) and osteolysis (using quantitative microCT). These effects will be mitigated by the local delivery of NF?B decoy ODN. Polyethylene debris will continue to be generated from TJRs, even with newer polyethylenes that produce smaller, potentially more biologically active particles. The proposed research is important, innovative and clinically relevant; it will suggest potential local pharmacological strategies to mitigate the chronic inflammatory reaction to wear particles and periprosthetic osteolysis. PUBLIC HEALTH RELEVANCE: Total joint replacement (TJR) is a highly successful surgical procedure for end-stage arthritis, however the longevity of TJRs is limited by wear of the bearing surfaces. Wear particles stimulate a chronic inflammatory reaction that leads to local bone destruction around the implant. This grant application tests a novel translational strategy to mitigate particle-associated bone destruction by interfering with the primary signaling molecule (the transcription factor NFkB) within cells that controls the production of inflammatory mediators. This strategy has a high likelihood of extending the lifetime of TJRs in humans.