Articular cartilage provides a bearing surface for transmitting forces across joints. The poor ability of cartilage to self-repair has motivated efforts to engineer replacement tissues, and mesenchymal stem cells (MSCs), which can undergo chondrogenesis, have emerged as a promising cell source. To date however, the functional properties of MSCbased constructs remain lower than those of the native tissue and of chondrocyte-based constructs cultured identically. Therefore, the overall objective of this thesis is to better understand the transcriptional and functional limitations underlying chondrogenic differentiation and enhance MSC chondrogenesis.

Toward this end, established tissue engineering strategies from the chondrocyte literature were applied. Specifically, the effects of cell seeding density, media formulation and mechanical stimulation were examined with respect to functional growth. Transient application of TGF-β3 improved the compressive properties of MSC-laden constructs, but only when constructs were formed at a higher seeding density. Long-term dynamic compression initiated 3 days after MSC encapsulation impaired functional properties;​ in contrast, dynamic compression initiated after 3 weeks of chondrogenic pre-culture improved mechanical function. While these strategies enhanced functional chondrogenesis, the compressive properties achieved were ~50% of native tissue levels and did not reach chondrocyte levels. To understand the basis of this difference, microarray analysis was carried out to compare these two cells types and a set of molecular factors were identified as mis-expressed during MSC chondrogenesis.

Although work up to this point focused on optimizing compressive properties, the tensile properties of articular cartilage are also critical to its functional role. In this work, we characterized the tensile properties of MSC-based constructs and demonstrated functional parity with chondrocyte-based constructs. To further enhance these properties, a novel sliding contact bioreactor was developed to better replicate physiologic joint loading conditions. Long-term application of loading to MSC-laden constructs improved not only tensile properties, but instilled biochemical inhomogeneity, reminiscent of native articular cartilage.

Overall, the work outlined in this thesis represents a significant advancement in engineering cartilage replacements as well as in understanding MSC chondrogenesis. Using a multi-faceted approach, we explored potential routes toward overcoming limitations in chondrogenesis and demonstrated that MSCs are responsive to their chemical and mechanical environment.