Enzyme-Responsive Assembly of Cysteine-Terminated Heparan Sulfate Proteoglycan Ligands for Osteoinductive Biphasic Scaffold Formation

Traditional osteoinductive strategies that rely on exogenous growth factors often face challenges, such as uncontrolled differentiation and transient efficacy. To overcome these limitations, we engineered a cysteine-terminated heparan sulfate proteoglycan (HSPG) binding peptide that self-assembles into an enzyme-responsive, biphasic scaffold, emulating the native bone extracellular matrix. This scaffold enables spatiotemporally regulated phosphate release and mineralization in response to alkaline phosphatase (ALP) activity, thereby preventing premature calcification. In vitro, the scaffold promotes osteogenic differentiation of human mesenchymal stem cells (hMSCs) by enhancing focal adhesion maturation, increasing actomyosin contractility, and activating YAP-dependent transcription. By integrating ALP-triggered mineralization with HSPG-targeting, this dynamic and biomimetic platform offers a growth-factor-free strategy for controlled osteoinduction. This strategy supports the design of osteoinductive materials that balance the bioactivity and mineralization control.