Bone repair in elderly individuals is challenging due to slower regeneration rates. To enhance stem cell response and tissue repair, biomimetic substrates with physical and chemical stimuli are investigated. Here, we optimized osteoblast patterns on polydimethylsiloxane (PDMS) substrates and deposited a nano layer of hydroxyapatite (nHA) using a sputtering technique to mimic the bone extracellular matrix (ECM) and improve differentiation. Substrate morphology, tensile strength, and biocompatibility were assessed. Differentiation and proliferation were evaluated using various staining techniques and real-time PCR. The results showed effective printing of cell patterns on substrates, with no alteration in surface topography following nHA deposition. All substrates exhibited good cytocompatibility. The combination of nHA as a chemical stimulus and cell patterns as a physical stimulus increased stem cell differentiation significantly, as evidenced by enhanced alkaline phosphatase (ALP) activity on nHA-coated substrates (97%). Real-time PCR analysis revealed higher expression of osteoblast-specific genes (RNX2 and OSC) on nHA-coated patterned substrates. The simultaneous physical and chemical changes in the cell environment promoted increased differentiation and proliferation during each step of stem cell differentiation. In conclusion, these substrates hold promise as a viable method for bone regeneration, providing valuable insights for tissue engineering application.