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，长期生存率受到关节面磨损的限制。磨损颗粒刺激慢性炎症，延迟骨整合，并导致假体周围骨质溶解和植入物松动。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κ B（NF？B），一种在促炎基因激活中的关键信号分子。该补助金的目的是调节NF？B表达以减轻颗粒诱导的炎症和骨质溶解。该研究提案将产生实质性的战略，治疗假体周围骨溶解在人类。具体目标#1：证明NF？当小鼠或人巨噬细胞体外暴露于有/无粘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？当细胞在体外暴露于具有/不具有粘附内毒素的临床相关的聚乙烯颗粒时，B诱饵ODN将对鼠或人骨祖细胞没有主要的不利影响。具体目标#3：证明NF的局部递送？B诱饵ODN抑制植入物周围炎症和骨质溶解与连续输注聚乙烯颗粒使用我们的验证小鼠股骨植入物模型。具体目标#4：证明NF的局部递送？使用我们的小鼠模型，B诱饵ODN抑制外源性报告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（OPC）将与临床相关聚乙烯颗粒内毒素一起培养。NF？将B诱饵ODN、加扰诱饵ODN或不加任何东西添加到培养物中。将评估细胞活力和增殖;将分别在上清液（ELISA和蛋白质印迹）和细胞（RT-PCR）的蛋白质和基因水平上测定TNF、IL-1、IL-6、MCP-1和RANKL。将测定OPC培养物的碱性磷酸酶和骨钙素表达，并使用Von Kossa染色定量骨基质形成。使用我们的鼠股骨植入物模型连续输注聚乙烯颗粒将导致全身报告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microPET扫描）和骨质溶解（使用定量microCT）。这些影响将减轻当地交付的NF？B诱饵ODN。TJR将继续产生聚乙烯碎片，即使是产生更小、可能更具生物活性颗粒的新型聚乙烯。拟议的研究是重要的，创新的和临床相关的;它将建议潜在的局部药理学策略，以减轻对磨损颗粒和假体周围骨质溶解的慢性炎症反应。 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，但TJR的寿命受到关节面磨损的限制。磨损颗粒刺激慢性炎症反应，导致植入物周围的局部骨破坏。这项资助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。这种策略很有可能延长TJR在人类中的寿命。