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）：总关节置换（TJR）是一项非常成功的手术程序；但是，长期的生存受到轴承表面的磨损的限制。磨损颗粒会刺激慢性炎症，从而延迟骨整合，并导致螺旋周期骨溶解和植入物松动。粒子诱导的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kappa b（NF？b）介导的，这是促炎性基因激活中的关键信号分子。该赠款的目的是调节NF？b表达，以便使用体外和体内模型来减轻颗粒诱导的炎症和骨溶解。该研究建议将产生实质性的策略，以治疗人体内的骨溶解。具体目的＃1：证明NF？B诱饵寡脱氧核苷酸（ODN）将消除当鼠或人类巨噬细胞暴露于临床上相关的聚乙烯颗粒的情况下，促炎性基因和蛋白质的表达，具有/没有依从性内毒素的临床相关的聚乙烯颗粒。具体目的＃2：证明NF？b诱饵ODN不会在细胞暴露于具有/没有粘附性内毒素的临床相关的聚乙烯颗粒上时，对鼠或人骨基因生成剂不会产生重大不利影响。特定目的＃3：证明NF？b诱饵ODN的局部递送抑制了使用我们经过验证的汞股植入物模型连续输注聚乙烯颗粒相关的植入植物周围炎症和骨溶解。具体目的4：证明NF？B诱饵ODN的局部递送抑制了使用我们的鼠模型将外源性记者巨噬细胞的全身迁移到粒子输注的区域。 鼠类和人类巨噬细胞和骨基源细胞（OPC）将用临床相关的聚乙烯颗粒内毒素培养。 nf？b诱骗的odn，炒诱饵ODN，或者什么也不会添加到文化中。将评估细胞活力和增殖； TNF，IL-1，IL-6，MCP-1和RANKL将分别在上清液（ELISA和Western blot）和细胞（RT-PCR）的蛋白质和基因水平上测定。 OPC培养物将用于碱性磷酸酶和骨钙素表达，并使用von Kossa染色来定量骨基质形成。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）和骨化（使用定量微观）。 NF？b诱饵ODN的局部输送将减轻这些影响。即使使用较新的聚乙烯产生较小，潜在的生物学活性颗粒，也将继续从TJR产生聚乙烯碎片。拟议的研究是重要的，创新的且在临床上相关的。这将提出潜在的局部药理策略，以减轻慢性炎症反应，以使颗粒和人假体骨溶解剂。 公共卫生相关性：总关节置换（TJR）是终末期关节炎的非常成功的外科手术，但是TJR的寿命受轴承表面的磨损限制。磨损颗粒刺激慢性炎症反应，从而导致植入物周围的局部骨骼破坏。该赠款应用测试了一种新型的翻译策略，可通过干扰控制炎症介质产生的细胞中的主要信号分子（转录因子NFKB）来减轻与粒子相关的骨破坏。这种策略很有可能延长人类TJR的寿命。