INVESTIGATING CULTURE CONDITIONS OF IN VITRO CARTILAGE MATURATION

Articular cartilage (AC) provides an interface between bones within joints that serves to minimize friction, absorb and distribute load along the joint, and facilitate movement. Cartilage has poor self-repair abilities following injury, in part due to the limited migration of chondrocytes and the avascular nature of the tissue which impedes the ability of progenitor cells to reach the site of injury. As a result, cartilage defects are at risk of progressing into osteoarthritis (OA), a degenerative disease of the whole joint. OA affects almost 5 million Canadians and can cause pain, severely reduce joint mobility, and negatively impact quality of life. Cartilage tissue engineering is a field that aims to develop strategies to repair cartilage defects with a combination of cells, biomaterials, and/or cues, either biochemical or biophysical in nature, to guide tissue formation. Tissue engineering strategies can include an in vitro maturation phase to create cartilage constructs with sufficient mechanical properties to withstand the cyclic loads present in the joint upon implantation. Identifying optimal culture conditions during this in vitro maturation phase is key for the generation of functional cartilage constructs. Typical tissue engineering strategies use supraphysiological glucose concentrations to ensure there is sufficient glucose in the media for energy production and proteoglycan synthesis between media changes; however, studies have found that these elevated glucose concentrations may elicit catabolic processes in the chondrocytes. We hypothesized that culturing constructs at physiological glucose concentrations in larger media volumes, to prevent glucose depletion, would generate cartilage constructs with superior biochemical properties. To test this hypothesis, primary chondrocytes were cultured in physiological (5 mM) and supraphysiologic (25 mM) glucose concentrations at low (2 mL) and high (11 mL) media volumes. The composition of the tissue and the different metabolic pathways conducted by chondrocytes were then evaluated. Our results indicated that high media volumes generate constructs with significantly higher proteoglycan and collagen content, the two major components of the extracellular matrix. Physiological glucose concentrations had no apparent effect on matrix accumulation; however, histological sections suggest that this culture condition may provide improved cell morphology. The glucose consumption rate was comparable for all four media conditions which suggests that the constructs may have similar matrix synthesis rates. Lactate concentration was significantly higher in low media volumes which may lead to a more acidic environment. The levels of both bioenergetic molecules quantified in the constructs, adenosine triphosphate (ATP) and inorganic polyphosphate (polyP), follow similar trends as the levels of matrix components; however, the relationship between ATP and polyP remains poorly understood. This thesis provides insight into the optimal culture conditions for engineered cartilage by demonstrating that media volume is an important culture parameter for matrix accumulation. Future work is required to understand the mechanisms behind this effect of media volume, to characterize effects of glucose concentration at the cellular level, and to identify key nutrients that will form functional cartilage constructs.