Cartilage tissue engineering with heterogeneous and clonal mesenchymal stem cell populations: Multi-scale analysis of maturation, stability, and response to environmental stressors

Osteoarthritis is a disease of high incidence with significant clinical impact. Unfortunately, joint arthroplasty remains the gold standard treatment as there has been limited success in long term cartilage repair with biological treatments. While advances have been made in cartilage tissue engineering, resulting in the in vitro development of a mechanically viable tissue, much of this progress has been restricted to chondrocyte-based engineered tissues, and these cells are limited in their availability. Mesenchymal stem cells are one possible alternative cell source for cartilage repair strategies; however, they have yet to produce a mechanically stable tissue comparable to chondrocytes cultured identically. Thus, the objective of this dissertation was to use a multi-scale approach to better characterize, between these two cell types, where differences in matrix production and construct mechanics arise, the time scales during which chondrocytes and MSCs diverge in their production of a mechanically stable tissue, and the environmental factors that may be impacting MSC health. Furthermore, we assessed if there are clonal subpopulations with a greater propensity for chondrogenic differentiation. Through assessment of regional mechanical properties of cell-laden constructs, we found that MSCs are in fact capable of producing mechanically functional matrix equivalent to that produced by chondrocytes. However, due to nutritional stress, the health and viability of these cells is severely impacted in regions of constructs that are nutrient deprived. By modulating nutrient (glucose) and metabolic (oxygen) concentrations in the growth media, we found that glucose concentration had a greater impact on cell health than low oxygen tension. However, with increased culture time, regardless of nutrient provision, MSC-based constructs underwent mechanical failure (with loss of GAG content) , suggesting innate instability of this stem cell population. Probing subpopulations of heterogeneous MSC isolates for chondrogenic potential revealed that both inter- and intra- colony heterogeneity exists, with a small fraction of colony subpopulations showing greater chondrogenic potential. Collectively, this work highlights potential pitfalls that are encountered when developing a stem cell based cartilage in vitro, which may further be exacerbated in vivo, but also provides future directions that may result in a clinically successful stem cell based cartilage replacement.