Osteoarthritis (OA), a debilitating musculoskeletal disease that is characterized by cartilage degeneration, is one of the leading causes of disability in older adults. In 2006, it was estimated to affect 27 million adults in the United States, carrying with it a financial burden of over $89 billion in direct costs each year. The prevalence of cartilage injuries, in combination with the inability of cartilage to self-repair and regenerate, makes cartilage degeneration one of the great challenges facing medical science. The most common treatments for severe cartilage damage aim either to minimize its symptoms or, at the other extreme, replace the problematic joint with artificial implants. Based upon these two limited options, it is clear that an immense need exists for tissue engineered solutions to regenerate, repair and replace damaged cartilage with a mechanically and a biologically functional equivalent. One approach to engineering biological cartilage replacements uses adult mesenchymal stem cells (MSCs), of which adipose tissue is an abundant source. However, after over a decade of active research on ADSC chondrogeneis, our understanding of how both biochemical and mechanical factors influence the differentiation capacity of ADSCs is sparse. This void in the fundamental understanding of ADSC behavior forms the basis for this dissertation.

The first aim in this dissertation strives to achieve a more reliable and accurate method for assessing ADSC chondrogenesis by improving on the existing dimethylmethylene blue (DMMB) assay, which is widely used to determine the sulfated glycosaminoglycan (sGAG) content of biological samples. It is a critical step, without which the proper understanding of ADSC chondrogenesis may not be reached. The second aim looks at how the growth factors TGF-β3 and BMP-6 affect ADSC chondrogenesis, answering the question of whether both growth factors are necessary to induce chondrogenesis. The third aim examines whether ADSCs are responsive to dynamic compressive loading and at what time points they are most responsive. Knowing the degree to which and when ADSCs are mechanosensitive can help pave way for the use of mechanical stimulation to enhance ADSC chondrogenesis.

Results from the first aim showed that the parameters of pH and wavelength were paramount to enhancing assay sensitivity and accuracy for assessing sGAG in tissueengineered samples. The commonly used DMMB protocol (pH3, with readings at only one absorbance wavelength) can significantly overestimate sGAG content and result in incorrect conclusions about sGAG accumulation within samples. To correct this problem, a simple and fast alteration to the assay was found, namely lowering DMMB dye pH to 1.5 and reading the absorbance at two different wavelengths (e.g. 525nm and 595nm), that resulted in more accurate measurements. This finding is important to the field of tissue engineering where the DMMB assay is commonly used.

Results from the second aim demonstrated that there was little added benefit to supplementing chondrogenic media with 50 ng/ml BMP-6 for the differentiation of ADSC-agarose constructs. TGF-β3 (10 ng/ml) alone was sufficient to initiate a chondrogenic response as measured and confirmed by different assays and multiple experiments. Whether the discrepancy between our results and those found in literature was due to inadequate levels of BMP-6 used in this study or the tissue construct format (agarose compared with pellet culture) is yet to be determined.

Results from the third aim showed that ADSCs responded most strongly to dynamic compression at the earliest time points and lost their ability to respond to dynamic compression with increased time in free-swell culture in chondrogenic medium. Interestingly, ADSCs cultured in basal media remained sensitive to dynamic compression throughout culture. The result of decreased mechanosensitivity with time in free-swell culture of ADSCs has not been previously reported in literature and is a novel finding that can improve understanding of the biomechanical behavior of ADSCs, potentially allowing for optimization of mechanical loading as an anabolic stimulus for ADSC chondrogenesis.