Concurrent multi-lineage differentiation of mesenchymal stem cells through spatial presentation of growth factors

Severe tendon and ligament injuries are estimated to affect between 300 000 and 400 000 people annually. Surgical repairs of these injuries often have poor long-term clinical outcomes because of resection of the interfacial tissue-the enthesis-and subsequent stress concentration at the attachment site. A healthy enthesis consists of distinct regions of bone, fibrocartilage, and tendon, each with distinct cell types, extracellular matrix components, and architecture, which are important for tissue function. Tissue engineering, which has been proposed as a potential strategy for replacing this tissue, is currently limited by its inability to differentiate multiple lineages of cells from a single stem cell population within a single engineered construct. In this study, we develop a multi-phasic gelatin methacrylate hydrogel construct system for spatial presentation of proteins, which is then validated for multi-lineage differentiation towards the cell types of the bone-tendon enthesis. This study determines growth factor concentrations for differentiation of mesenchymal stem cells towards osteoblasts, chondrocytes/fibrochondrocytes, and tenocytes, which maintain similar differentiation profiles in 3D hydrogel culture as assessed by qPCR and immunofluorescence staining. Finally, it is shown that this method is able to guide heterogeneous and spatially confined changes in mesenchymal stem cell genes and protein expressions with the tendency to result in osteoblast-, fibrochondrocyte-, and tenocyte-like expression profiles. Overall, we demonstrate the utility of the culture technique for engineering other musculoskeletal tissue interfaces and provide a biochemical approach for recapitulating the bone-tendon enthesisin vitro.