Musculoskeletal tissue injuries remain a significant challenge in orthopaedics research. For example, currently, millions of patients are suffering from cartilage injuries, with associated annual financial costs of more than $100 billion dollars. There are several viable clinical options to address these injuries. In this context, human mesenchymal stem cells (hMSCs) are a promising cell source for cartilage tissue engineering as they are capable of differentiating down the chondrogenic pathway, can be obtained from bone marrow in a minimally invasive manner, and are easily grown in culture. Although differentiation of hMSCs is regulated by soluble molecules, insoluble biochemical signals and mechanical cues, the combinatorial effects of mechanical loading and biomaterial signals are largely unknown. Our central hypothesis is that the use of high-throughput systems (HTSs) under mechanical stimulation can be used to elucidate single and synergistic microenvironmental factors for directing the chondrogenic differentiation of hMSCs, and thereby functional engineered cartilage formation in vitro and in a clinically relevant in vivo model. The information obtained from the HTS will help in the development of macroscale constructs with enhanced chondrogenesis, and the performance of these constructs will be validated in vivo by treating critical-sized articular cartilage defects. These goals will be accomplished by achieving the following specific Aims: (1) to develop three dimensional (3D) combinatorial HTS hydrogel-based microarrays, consisting of different extracellular matrix molecules and growth factors, which can be mechanically deformed to mimic the chondrogenic microenvironment of hMSCs, (2) to evaluate quantitatively the chondrogenic differentiation response of hMSCs in 3D combinatorial HTS microarrays and macroscale constructs selected from these microarrays, and (3) to determine the potential of hMSC-laden constructs with HTS-identified compositions and mechanical stimulation regimes to induce cartilage regeneration in vivo. The orthopedic community would benefit from a better understanding of these chondroinductive microenvironments that will ultimately induce neo-tissue formation and will represent a viable alternative to current clinical therapies. While the ultimate objective of this research is to engineer clinically relevant articular cartilage therapies, this HTS can also be applicable to test regeneration strategies for other tissues.