<p>This study investigated the in vitro biological behaviour of human mesenchymal stem cells grown on acrylate-based substrates functionalized with biomolecules that play a significant role in the extracellular matrix, specifically gelatin (derived from collagen) and heparin. Synthetic supports were synthesised by free radical polymerization in the form of flat films, prepared by solvent casting, and microspheres, produced via an oil-in-water emulsion, with gelatin or heparin grafted onto theirs surface. The viability and proliferation of primary cells obtained from bone marrow aspirates were analysed in 2D monolayer environments, and in 3D environments using microspheres. In addition, the gene expression of osteogenic and chondrogenic markers was studied in cells expanded in basal medium before reaching confluence in both 2D and 3D environments, to assess the influence of the grafted biomolecules and dimensionality on the early commitment of MSCs. Significant changes were induced by the interaction of cells with the grafted biomolecules, which suggested the commitment of the human bone marrow mesenchymal stem cells (hBMMSCs) to specific lineages and loss of multipotency. The culture was continued in basal or osteogenic medium for up to 21&#xa0;days in 2D environments after confluence, revealing significant differences between the functionalised supports and either the non-functionalised substrates and standard polystyrene culture wells (tissue culture plastic, TCP). These findings highlight the potential of this approach to advance bone tissue engineering applications and contribute to the development of innovative and effective bone regeneration strategies.</p>