- Abstract Human bone marrow-derived mesenchymal stem cells (hMSCs) are promising cell candidates for cartilage regeneration. Building the appropriate microenvironment for cell differentiation in response to exogenous stimuli is a critical step towards the clinical utilization of hMSCs. In this study, the effects of RGD peptide immobilization onto macro-porous alginate scaffolds on TGF-β1-induced hMSC chondrogenesis were evaluated. The results revealed different cell morphology, viability and proliferation extent in the RGD-immobilized vs. un-modified scaffolds. The TGF-β1-induced activation of both Smad-dependent (SMAD2) and Smad-independent (ERK1/2) signaling pathways was stronger and persisted for over 3 weeks in the RGD-immobilized scaffolds, indicating greater accessibility of the cells to the inducer. By contrast, in the un-modified alginate scaffolds, the cells aggregated into compacted clusters resulting in lesser effects of TGF-β1. The efficient and prolonged exposure to the chondrogenic inducer in the RGD-modified scaffolds ensured the appropriate progression of MSC differentiation from the initial phase of cell condensation until the appearance of committed chondrocytes, at 3 weeks of cultivation. Taken together, our results highlight the fundamental importance of the microenvironment design of the scaffold as well as the presentation of the inductive cue for inducing efficient stem-cell controlled differentiation.