Regenerative Enhancement of Aged Chondrocytes via Cytoskeletal Modulation

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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.