Investigation Of Mesenchymal Stem Cells For Cartilage Repair
The aim of the thesis project was to identify a growth factor that would enhance the in vitro chondrogenesis of the equine mesenchymal stem cell (MSC) and subsequently to assess the capacity of the growth factor expressing MSC to improve cartilage repair and osteoarthritis (OA) prevention in vivo. Previous efforts on MSC enhanced cartilage repair have focused on implantation of MSCs to articular cartilage defects. This approach is only applicable to acute and focal articular cartilage injury and does not apply to the OA joint. Global joint disease, as in OA, is far more common than focal cartilage injury and no disease modifying therapies of regenerative therapies are currently available. Short term tracking studies of intra-articular injection of autologous MSCs were performed as part of this dissertation. Studies revealed that MSCs efficiently engraft the synovial membrane but not articular cartilage, whether normal or diseased. Differing three-dimensional culture systems were tested for suitability as a system to study in vitro chondrogenesis of MSCs. In chondrogenic media, fibrin alginate culture and pellet culture (500,000 cell pellets) were superior for chondrogenic induction to agarose, alginate alone and 250,000 cells pellets. For in vitro MSC chondrogenesis, supplementation with an isoform of transforming growth factor beta (TGF-[beta]) is required. TGF-[beta]1, -[beta]2 and -[beta]3 have been used and although TGF-[beta]1 is the most frequently reported both in vitro and in vivo, it is not clear which is superior for chondrogenic induction and prevention of hypertrophy. The chondrogenic effect of the 3 isoforms was tested in MSC pellet culture. All 3 isoforms resulted in MSC chondrogenesis, however, TGF-[beta]3 had clear enhancement of prevention of hypertrophy. Growth factor supplementation in vivo is difficult to maintain because of the short halflife of injected or implanted growth factors. Gene therapy techniques to induce growth factor expression by injected or implanted MSCs would allow for continuous growth factor supplementation to the joint. Adenoviral vectors for expression of TGF-[beta]1, -[beta]2 and -[beta]3 were constructed and tested in MSC pellet culture to confirm transgene expression and efficacy for chondrogenic induction in long term 3-dimensional culture. Adenoviral transgene expression of TGF-[beta]3 resulted in chondrogenic induction and reduced progression toward hypertrophy compared to Ad TGF-[beta]1 and -[beta]2. Given the synovial distribution of MSCs injected to the arthritic joint, injection of growth factor enhanced and anti-catabolic MSCs to the middle carpal joint of horses in the osteochondral fragmentation model of OA was tested. This resulted in improved control of joint disease evidenced by improved effusion scores and range of motion in the week following treatment injection, reduced cartilage MMP13 and synovial IL1b expression, reduced synovial fibrosis and a strong trend of increased cartilage glycosaminoglycan content.
Nixon, Alan J
Kotlikoff, Michael I.; Todhunter, Rory James; Jin, Moonsoo
Ph.D. of Veterinary Medicine
Doctor of Philosophy
dissertation or thesis