Biochemical and biomechanical gradients for directed bone marrow stromal cell differentiation toward tendon and bone

Substrates with mechanical property gradients and various extracellular matrix ligand loadings were evaluated for their ability to direct bone marrow stromal cell differentiation along osteogenic and tenogenic lineages. After verifying reproducible mechanical compliance characteristics of commercial hydrogel gradient substrates, substrates were functionalized with whole length fibronectin or collagen, both of which are found in skeletal structures and are relevant to cell-matrix signalling. Bone marrow stromal cells were seeded onto the substrates in growth media and cultured first to examine cell attachment and morphology, indicating higher levels of attachment on collagen substrates after 1 h, and increased spreading and organization trends after 24 h. Differentiation studies showed an increase in osteoblast differentiation on fibronectin substrates while collagen substrates lacked osteogenic differentiation. Osteogenic differentiation decreased on substrates of lower stiffness and lower ligand density. Molecular investigations revealed an increase in relevant signalling molecules for osteoblasts that were consistent with differentiation studies, but detected the presence of tenoblast markers on collagen substrates within a narrow range of stiffness. Our results indicate that mechanovariant substrates do hold promise as a culture platform for directed differentiation to tendon and bone by altering gene level expression of relevant signalling molecules. This study aids in understanding the molecular mechanisms that drive differentiation from substrate based cues, and could aid the design of therapeutic biomaterials at the transition from tendon to bone.