Decoupling the role of stiffness from other hydroxyapatite signalling cues in periosteal derived stem cell differentiation

Bone extracellular matrix (ECM) is a natural composite made of collagen and mineral hydroxyapatite (HA). Dynamic cell-ECM interactions play a critical role in regulating cell differentiation and function. Understanding the principal ECM cues promoting osteogenic differentiation would be pivotal for both bone tissue engineering and regenerative medicine. Altering the mineral content generally modifies the stiffness as well as other physicochemical cues provided by composite materials, complicating the cause-effect analysis of resultant cell behaviour. To isolate the contribution of mechanical cues from other HA-derived signals, we developed and characterised composite HA/gelatin scaffolds with different mineral contents along with a set of stiffness-matched HA-free gelatin scaffolds. Samples were seeded with human periosteal derived progenitor cells (PDPCs) and cultured over 7 days, analysing their resultant morphology and gene expression. Our results show that both stiffness and HA contribute to directing PDPC osteogenic differentiation, highlighting the role of stiffness in triggering the expression of osteogenic genes and of HA in accelerating the process, particularly at high concentrations.