Regenerative Enhancement of Aged Chondrocytes via Cytoskeletal Modulation

通过细胞骨架调节增强老化软骨细胞的再生

• 批准号: 9372731
• 负责人: ROCKY S TUAN
• 金额: $ 23.33万
• 依托单位: UNIVERSITY OF PITTSBURGH AT PITTSBURGH
• 依托单位国家: 美国
• 财政年份: 2017
• 资助国家: 美国
• 起止时间: 2017-09-15 至 2019-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9372731
• 关键词: Affect; Age-Years; Aging; Aging-Related Process; Alternative Therapies; American; Animals; Architecture; Autologous; Back; Biological; Biology; Cartilage; Cell Aging; Cell Proliferation; Cell Transplants; Cells; Chondrocytes; Chondrogenesis; Cytoskeletal Modeling; Cytoskeleton; Data; Defect; Degenerative Disorder; Degenerative polyarthritis; Dimensions; Disease; Effectiveness; Elderly; Encapsulated; Environment; Future; Gelatin; Generations; Goals; Harvest; Human; Hyaline Cartilage; Hyaluronic Acid; Hydrogels; Impairment; In Vitro; Individual; Intermediate Filaments; Joints; Knee; Laboratories; Length; Life; Life Expectancy; Light; Longevity; Methods; Microfilaments; Microtubules; Mitochondria; Molecular Profiling; Mus; Natural regeneration; Operative Surgical Procedures; Outcome; Outcome Study; Pathway interactions; Patients; Phenotype; Population; Prevalence; Procedures; Production; Prosthesis; Publishing; Quality of life; Reagent; Rejuvenation; Replacement Arthroplasty; Reporting; Research; Resources; Role; SCID Mice; Safety; Sampling; Stress; Structure; Testing; Tissues; Treatment Protocols; Woman; age related; aged; aging population; arthropathies; articular cartilage; base; cartilage development; cartilage regeneration; clinical application; disability; experience; healthy aging; human disease; implantation; in vivo; joint function; joint injury; men; mobility enhancement; older patient; regenerative; regenerative therapy; restoration; scaffold; senescence; subcutaneous; telomere; three dimensional cell culture

Focal articular cartilage defects (FACD) are seen in a significant fraction of the population, with 60% prevalence in the aged. Osteoarthritis (OA), a degenerative disease often initiated by FACD, affects 27 million Americans, including ~60% of men and ~70% of women over 65 years of age. OA is also the dominant cause of disability in the aged, and no therapy is currently available for fully cartilage regeneration and restoration of joint function. Autologous chondrocyte implantation (ACI) is an advanced regenerative treatment for FACD, reducing or delaying OA initiation. However, ACI is not recommended for old patients, primarily because of the impaired proliferation capacity and function of chondrocytes harvested from aged patients, who are therefore unable to receive sufficient, functional chondrocytes for hyaline cartilage regeneration. The mechanism of senescence and accompanying bioactivity loss in chondrocytes due to natural aging is not known. Interestingly, senescent chondrocytes display increased formation of a rigid cytoskeleton, suggesting that the cytoskeleton may be critically involved in chondrocyte aging. A similar phenomenon is also observed in another type of artefactual chondrocyte senescence caused by extensive in vitro expansion, termed “dedifferentiation” and characterized by loss of chondrocytic phenotype, and disruption of microfilaments has been reported to partially reverse the dedifferentiation status. Therefore, we hypothesize that a highly structured cytoskeleton accompanies the chondrocyte aging process, and a re-organization of the cytoskeleton in three dimensional (3D) environment will reverse aging chondrocytes back to a stable state with reparative potential comparable to that of young chondrocytes. In Aim 1 we will first analyze the relationship between cytoskeletal organization and the state of chondrocytes (including young and old, healthy and diseased), and profile expression of key molecules involved in chondrogenesis, cell proliferation, as well as cytoskeletal dynamics during aging process. Results from these studies will not only allow us to develop a set of criteria to fully delineate chondrocyte cell state during healthy or diseased aging, which has not been reported before, but also shed light on the biology of chondrocyte aging. In Aim 2, we will test the effectiveness and safety of different cytoskeleton-disrupting agents and treatment regimens on proliferation capacity and phenotype of aging chondrocytes. The re-establishment of cytoskeleton will be conducted under 3D culture conditions to accommodate the important requirement of 3D environment in maintaining chondrocyte phenotype. The goal is to identify the best cytoskeleton-disrupting agent(s) and conditions in reversing the senescent state. The cartilage formation capacity of optimally rejuvenated chondrocytes will be further tested in vivo by subcutaneous implantation of the cells, encapsulated within a chondrosupportive hydrogel scaffold, into SCID mice. Successful outcomes will lead to the production of biologically active chondrocytes to enable cell-based regenerative therapy for the aged towards functional and healthy aging, in addition to gaining in-depth understanding of the underlying mechanism of aging in cartilage.

很大一部分人群患有局灶性关节软骨缺损 (FACD)，患病率为 60% 在老年人中。骨关节炎 (OA) 是一种通常由 FACD 引发的退行性疾病，影响着 2700 万美国人， 包括 65 岁以上的约 60% 的男性和约 70% 的女性。 OA 也是导致残疾的主要原因 对于老年人来说，目前还没有可以使软骨完全再生和恢复关节功能的治疗方法。 自体软骨细胞植入 (ACI) 是一种先进的 FACD 再生治疗方法，可减少或 延迟 OA 启动。然而，不建议老年患者进行 ACI，主要是因为老年患者的功能受损。 从老年患者身上采集的软骨细胞的增殖能力和功能，因此无法 获得足够的功能性软骨细胞以进行透明软骨再生。衰老的机制与 由于自然老化导致软骨细胞生物活性丧失的情况尚不清楚。有趣的是，衰老 软骨细胞显示出刚性细胞骨架的形成增加，表明细胞骨架可能是 与软骨细胞老化密切相关。在另一种类型的人工制品中也观察到类似的现象 由广泛的体外扩增引起的软骨细胞衰老，称为“去分化”，其特征是 据报道，通过软骨细胞表型的丧失和微丝的破坏可以部分逆转 去分化状态。因此，我们假设高度结构化的细胞骨架伴随着 软骨细胞老化过程以及三维 (3D) 细胞骨架的重组 环境会将老化的软骨细胞逆转到具有可比修复潜力的稳定状态 到年轻的软骨细胞。在目标1中，我们将首先分析细胞骨架组织之间的关系 软骨细胞的状态（包括年轻和年老，健康和患病），以及关键的表达谱 参与软骨形成、细胞增殖以及衰老过程中细胞骨架动力学的分子。 这些研究的结果不仅使我们能够制定一套标准来完全描述软骨细胞 健康或患病衰老过程中的状态，这以前从未有过报道，但也揭示了 软骨细胞老化。在目标2中，我们将测试不同细胞骨架破坏剂的有效性和安全性 以及对衰老软骨细胞增殖能力和表型的治疗方案。的重建 细胞骨架将在3D培养条件下进行，以满足3D的重要要求 维持软骨细胞表型的环境。目标是确定最佳的细胞骨架破坏剂 逆转衰老状态的药物和条件。最佳软骨形成能力 再生软骨细胞将通过皮下植入细胞进行进一步的体内测试，封装 在软骨支持水凝胶支架内，进入 SCID 小鼠。成功的结果将导致生产 具有生物活性的软骨细胞，使老年人能够进行基于细胞的再生治疗，实现功能性和功能性 健康衰老，除了深入了解软骨衰老的潜在机制。