Development of Cell-laden Hydrogel Composites for Osteochondral Tissue Engineering
Mikos, Antonios G.
Doctor of Philosophy
Articular cartilage is a flexible connective tissue that enables the frictionless and painless articulation of bones in synovial joints throughout the body. Given its avascular nature, articular cartilage tissue inherently exhibits a compromised endogenous capacity for regeneration upon damage. Defects caused by disease or trauma often lead to chronic pain and osteoarthritis as current clinical treatments are still unable to achieve long-term repair. Hence, tissue engineers are developing innovative technologies to provide strategies for successful cartilage repair and regeneration. This thesis focuses on the development and evaluation of cell-based osteochondral tissue engineering solutions based on injectable and biodegradable polymer biomaterials for bone and cartilage repair. First, we developed and characterized oligo(poly(ethylene glycol) fumarate) (OPF)-based hydrogels for osteochondral tissue engineering applications. We investigated the main effects of five main hydrogel fabrication parameters (the poly(ethylene glycol) molecular weight (PEG MW), the crosslinker-to-OPF carbon-carbon double bond ratio (DBR), the crosslinker type, the crosslinking density of encapsulated gelatin microparticles, and the incubation medium composition) as well as their interaction effects on the swelling behavior and degradation of OPF hydrogel composites. We found that increasing the PEG MW increased the mean swelling ratio and decreased the mean mass remaining %, while changing the crosslinker type from methylene bisacrylamide (MB) to PEG diacrylate yielded the opposite effect. Additionally, we found that the swelling of hydrogels fabricated with higher PEG MW or with MB were more sensitive to increases in DBR. From these results, we showed that the swelling and degradation properties of OPF-based hydrogels can be precisely tuned through the modulation of these five fabrication parameters. The second part of this thesis investigated the potential of bilayered OPF hydrogel composites encapsulating chondrogenically and osteogenically pre- differentiated MSCs in a spatially controlled fashion for osteochondral tissue repair. We demonstrated that MSCs that underwent 7 days (CG7), but not 14 days (CG14), of chondrogenic pre-differentiation most closely resembled the phenotype of native hyaline cartilage when combined with osteogenically pre-differentiated (OS) cells in a bilayered OPF hydrogel. We found that the respective chondrogenic and osteogenic phenotypes of encapsulated MSCs were maintained for up to 28 days in vitro without the need for external growth factors. When taken in vivo, the delivery of CG7 cells, as opposed to CG14 cells, in combination with OS cells via a bilayered OPF hydrogel composite stimulated morphologically superior cartilage repair. Indeed, the present work showed that cartilage regeneration in osteochondral defects can be enhanced by MSCs that are chondrogenically and osteogenically pre-differentiated prior to implantation. Longer chondrogenic pre-differentiation periods, however, resulted in diminished cartilage repair. The final section of this thesis investigated the use of poly(L-lysine) (PLL), previously shown to up-regulate condensation during cartilage development in vitro, as an early chondrogenic stimulant of MSCs encapsulated in OPF hydrogels. We showed that PLL incorporation resulted in early enhancements of type II collagen and aggrecan gene expression as well as increased type II/type I collagen expression ratios when compared to blank controls. We also demonstrated that PLL enhanced N-cadherin gene expression of encapsulated MSCs under certain conditions, suggesting that PLL also likely induced pre-cartilaginous condensation.