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Despite the innate regenerative capabilities of bone tissue, self-repair is impaired when an injury exceeds the critical size threshold because of trauma, congenital, or pathological conditions. Autologous transplantation is the gold standard to reconstruct large bone defects. However, this method has drawbacks such as limited amount of graftable material, limited accessibility, and donor site morbidity. For these reasons, alternative regenerative medicine and tissue engineering approaches are being developed, including implantable scaffolds.The use of in vitro-made scaffolds containing biomaterials that mimic the functional characteristics and composition of extracellular bone matrix has been favored in 3D vs 2D in vitro culture systems. Adult stem cells such as mesenchymal stem cells (MSCs), that give rise to bone building cells, have been used in combination with various biomaterials. The development of an implantable scaffold with or without cells requires extensive in vitro validation and optimization prior to its testing in vivo. Thus, the primary aim of this thesis was to develop a 3D model for the optimization of MSC differentiation. Further aims were to utilize this 3D model to evaluate the MSC response to a novel osteogenic biomaterial. To achieve these objectives human bone marrow MSCs (BMSCs) were utilized in various hydrogels in combination with chemical differentiation factors or biomaterials. Moreover, the osteogenic capability of the tested biomaterials and their induced inflammatory/angiogenic responses were investigated, and the culture conditions were optimized for clinical application. In this thesis, a comparison between 2D and 3D (hydrogels) in vitro culture models was developed with the purpose of studying osteogenic differentiation in BSMCs. Testing various hydrogels revealedt he superiority of type I collagen hydrogels for the osteogenic 3D in vitro culture system. Further, cell culture conditions were improved for the expansion and differentiation of BMSCs to fulfill clinically approved standards according to Good Manufacturing Practice (GMP) conditions. Comparisons between fetal bovine serum (FBS) and human platelet lysate (PLT) showed superior cellular differentiation in FBS, while PLT enhanced cell proliferation. Based on the developed 3D model, the osteogenic properties of a novel nanoporous silica calcium phosphate (nSCP) material were investigated. The results indicated that nSCP was osteoinductive, involving different pathways compared with the traditional chemical differentiation protocols or other tested osteogenic biomaterials. Finally, the inflammatory and angiogenic responses from human BMSCs and an immortalized monocyte cell line (THP-1) exposed to nSCP in the established 3D model were assessed.The results indicated limited inflammatory effect of nSCP, while inducing the secretion of pro-angiogenic cytokines. The bioactivity of these released factors was confirmed in an assay using human endothelial cells.Taken together, this thesis presents a 3D in vitro model for studying osteogenic differentiation in MSCs, which can be utilized to evaluate, validate, and optimize biomaterial candidates for bone regeneration applications.