Wear Particle Disease and NF-kappa B Signaling

磨损颗粒病和 NF-kappa B 信号传导

• 批准号: 8419372
• 负责人: STUART B GOODMAN
• 金额: $ 35.34万
• 依托单位: STANFORD UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2012
• 资助国家: 美国
• 起止时间: 2012-09-15 至 2017-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8419372
• 关键词: Adverse effects; Alkaline Phosphatase; Applications Grants; Area; Arthritis; Biological Assay; Bioluminescence; Bone Matrix; CCL2 gene; Cell Adhesion Molecules; Cell Culture Techniques; Cell Proliferation; Cells; Chronic; Continuous Infusion; Disease; Dose; Endotoxins; Enzyme-Linked Immunosorbent Assay; Event; Foreign-Body Reaction; Gene Proteins; Genes; Grant; Human; Immunofluorescence Microscopy; Implant; In Vitro; Inflammation; Inflammation Mediators; Inflammatory; Infusion procedures; Interleukin-1; Interleukin-6; Joints; Laboratories; Long Term Survivorship; Longevity; Macrophage Activation; Mediating; Metals; Methods; Minor; Modeling; Mus; NF-kappa B; Operative Surgical Procedures; Osseointegration; Osteocalcin; Osteogenesis; Osteolysis; Outcome; Patients; Polyethylenes; Procedures; Production; Proliferation Marker; Prosthesis; Reaction; Replacement Arthroplasty; Reporter; Research; Research Proposals; Reverse Transcriptase Polymerase Chain Reaction; Scanning; Signal Transduction; Signaling Molecule; Site; Staging; Staining method; Stains; Surface; TNF gene; TNFSF11 gene; Testing; Transactivation; Up-Regulation; Western Blotting; bone; bone loss; bone turnover; chemokine; clinically relevant; cytokine; in vivo Model; innovation; macrophage; migration; novel; osteoprogenitor cell; particle; particle exposure; response; trafficking; transcription factor; treatment strategy

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.

描述（由申请人提供）：全关节置换术（TJR）是一种非常成功的外科手术；然而，长期的寿命受到轴承表面磨损的限制。磨损颗粒刺激慢性炎症，延缓骨整合，并导致假体周围骨溶解和植入物松动。颗粒性炎症导致促炎因子的释放；这些事件主要是由转录因子核因子κ B (NF？B)，促炎基因激活的关键信号分子。这项拨款的目的是调节NF？B的表达，以减轻颗粒诱导的炎症和骨溶解在体外和体内模型。该研究计划将为人类假体周围骨溶解的治疗提供实质性的策略。具体目标#1：证明NF？在体外实验中，当小鼠或人巨噬细胞暴露于具有或不具有黏附内毒素的临床相关聚乙烯颗粒时，B诱饵寡脱氧核苷酸（ODN）将消除促炎基因和蛋白的表达。具体目标#2：证明NF？B诱饵ODN对小鼠或人的骨祖细胞没有重大的不良影响，当细胞在体外暴露于具有或不具有黏附内毒素的临床相关聚乙烯颗粒时。具体目标#3：证明NF的本地交付？B诱饵ODN抑制与持续注入聚乙烯颗粒相关的植入物周围炎症和骨溶解，使用我们验证的小鼠股骨植入物模型。具体目标#4：证明NF的本地交付？在我们的小鼠模型中，B诱饵ODN抑制外源性报告性巨噬细胞向颗粒输注区域的全身迁移。小鼠和人巨噬细胞和骨祖细胞（OPCs）将与临床相关的聚乙烯颗粒内毒素培养。NF吗?B诱饵ODN，打乱诱饵ODN或什么都不添加到培养物中。评估细胞活力和增殖；将分别从上清（ELISA和Western blot）和细胞（RT- PCR）中检测TNF、IL-1、IL-6、MCP-1和RANKL的蛋白和基因水平。OPC培养物将检测碱性磷酸酶和骨钙素的表达，骨基质形成将使用Von Kossa染色进行定量。在我们的小鼠股骨植入物模型中，持续输注聚乙烯颗粒会导致系统性报告性巨噬细胞运输到颗粒输注部位（使用生物发光和免疫荧光显微镜），增加局部骨转换（使用微pet扫描）和骨溶解（使用定量微ct）。这些影响将通过局部递送NF？B诱饵；聚乙烯碎片将继续从TJRs中产生，即使是产生更小、可能更具生物活性颗粒的新型聚乙烯。建议的研究是重要的、创新的和临床相关的；这将建议潜在的局部药理学策略来减轻磨损颗粒和假体周围骨溶解的慢性炎症反应。