| Cartilage is formed through the process of chondrogenesis, in which mesenchymal stem cells (MSCs) differentiate to become chondrocytes. Chondrocytes then synthesize all of the components of the intricate extracellular matrix (ECM) of which cartilage is comprised. Articular cartilage has a very poor reparative ability and extracellular matrix degradation leads to the eventual destruction of the tissue. Tissue engineering presents a possible avenue for its repair and regeneration. Because hydrogels are mimetic of the native, 3-dimensional, water-swollen environment in which chondrocytes exist, they have been the focus of much research in terms of cartilage tissue engineering, but success has been limited. This thesis describes the design and characterization of poly(ethylene diacrylate) (PEGDA) and poly(ethylene) (PEG)-based semi-interpenetrating networks that facilitated the formation of cartilage by human MSCs. Alterations in the molecular weight of the PEGDA crosslinker and the ratio of PEGDA:PEG, resulted in substantial changes in extracellular matrix elaboration. Proteoglycan content was significantly increased through lowering the PEGDA:PEG ratio at the lowest PEGDA molecular weight used (6 kDa). Furthermore, the profile of proteoglycan deposition within the scaffold was substantially altered with lower PEGDA:PEG ratio and with higher PEGDA molecular weight. With higher PEGDA molecular weight, toluidine blue staining revealed intercellular/interterritorial deposition of proteoglycan, as compared with pericellular deposition exhibited by constructs fabricated at PEGDA:PEG of 2:1 with PEGDA (6 kDa). Collagen content, however, was lower in constructs fabricated with higher PEGDA molecular weight at a PEGDA:PEG ratio of 1:2. The effects of culture conditions on subsequent matrix development in the optimized polymer hydrogel were also examined. Initial experiments defined the optimal cell seeding density that produced maximum matrix deposition throughout the construct at 25 x106 cells/ml. Pretreatment of monolayer cells with defined chondrogenic medium retarded matrix elaboration, while withdrawal of either TGF-beta1, or dexamethasone (DEX) lowered collagen content per cell at all timepoints. These findings are informative for in vitro tissue engineering of cartilage, but may also aid in vivo implantation strategies for hydrogels that contain MSCs rather than preformed cartilage. |
