Guiding chondrogenesis through controlled growth factor presentation with polymer microspheres in high density stem cell system

Due to the prevalence of osteoarthritis in the United States and worldwide, there is a critical and growing need for replacement cartilage tissues. Motivated by this need, tissue engineering strategies combine principles of biology and engineering to generate living tissues for the repair and replacement of damaged or diseased cartilage. This thesis aimed to develop systems to direct stem cell chondrogenesis through investigation of the hypothesis that the chondrogenic potential of stem cells in high density culture can be guided through controlling growth factor presentation via incorporated TGF-beta1 loaded biopolymer microspheres. The chondrogenic differentiation of human mesenchymal stem cells (hMSC) in aggregates incorporated with TGF-beta1 releasing poly(lactide-co-glycolide) (PLGA) microspheres was regulated through varying the amounts of incorporated microspheres, and glycosaminoglycan (GAG) production after 3 weeks was comparable to that of traditional cell aggregates cultured in growth factor-containing medium. A system of genipin-crosslinked microspheres was also developed, enabling sustained TGF-beta1 delivery via cell-mediated microsphere degradation at rates controlled by the level of hydrogel crosslinking. These gelatin microspheres were shown to regulate GAG production in hMSC aggregates from 3 different donors at levels dependent on the amount of incorporated microspheres, growth factor concentration, and microsphere degradation rates. This gelatin microsphere incorporated hMSC system was scaled up for the production of self-assembling microsphere incorporated cell sheets more appropriately sized for the treatment of clinical cartilage defects. The inclusion of fast- or slow-degrading microspheres with or without loaded TGF-beta1 significantly increased sheet thickness and compressive equilibrium modulus, and enabled more uniform matrix deposition by comparison to cell-only sheets. Sheets incorporated with fast-degrading microspheres containing TGF-beta1 produced significantly more GAG and GAG per DNA than all other groups tested and stained more intensely for type II collagen. These findings demonstrated improved cartilage formation in microsphere-incorporated cell sheets, and described a tailorable system for the chondrogenic induction of hMSCs. Overall, these high density stem cell systems incorporated with TGF-beta1 releasing microspheres enable chondrogenic differentiation at controllable levels without requiring culture in growth factor containing medium. This technology could enable earlier implantation and facilitate cartilage tissue formation within the body, enhancing the potential for construct integration and offering promise for cartilage tissue engineering strategies.