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.

局灶性关节软骨缺损（FACD）见于相当一部分人群，患病率为60 在老年人中。骨关节炎（OA）是一种经常由FACD引发的退行性疾病，影响着2700万美国人， 其中约60%的男性和约70%的65岁以上女性。OA也是导致残疾的主要原因 老年人，目前没有治疗方法可用于完全软骨再生和关节功能的恢复。 自体软骨细胞植入（ACI）是一种先进的再生治疗FACD，减少或 延迟OA启动。然而，ACI不推荐用于老年患者，主要是因为 增殖能力和功能的软骨细胞收获自老年患者，谁因此不能 获得足够的功能性软骨细胞用于透明软骨再生。衰老的机制和 软骨细胞中由于自然老化而伴随的生物活性损失是未知的。有趣的是， 软骨细胞显示刚性细胞骨架的形成增加，这表明细胞骨架可能是 与软骨细胞老化密切相关在另一种人工制品中也观察到类似的现象。 软骨细胞衰老是由体外大量扩增引起的，称为“去分化”， 通过软骨细胞表型的丧失，以及微丝的破坏，已经报道部分逆转了 去分化状态因此，我们假设，一个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 环境将逆转老化的软骨细胞回到稳定状态， 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与细胞骨架结构之间的关系。 和软骨细胞的状态（包括年轻和老年，健康和患病），以及关键蛋白的表达谱。 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充分描述软骨细胞， 在健康或疾病老化期间的状态，这在以前没有报道过，但也揭示了生物学的 软骨细胞老化在目标2中，我们将测试不同细胞因子干扰剂的有效性和安全性 以及对老化软骨细胞增殖能力和表型的治疗方案。重建 细胞骨架将在三维培养条件下进行，以适应三维培养的重要要求。 环境中维持软骨细胞表型。我们的目标是找出最好的细胞分裂素干扰 在逆转衰老状态中的试剂和条件。最佳的软骨形成能力 再生的软骨细胞将通过皮下植入细胞、包封的 在软骨支持性水凝胶支架内，植入SCID小鼠。成功的结果将导致生产 的生物活性软骨细胞，使老年人的细胞为基础的再生治疗， 健康的老龄化，除了获得深入了解软骨老化的潜在机制。