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We have developed a novel multicomponent nano-hydroxyapatite-poly(D,L-lactide-co-glycolide)-collagen biomaterial (nHAP-PLGA-collagen) with mechanical properties similar to human cancellous bone. To demonstrate the bone forming capacity of nHAP-PLGA-collagen prior to in vivo experiments, nHAP-PLGA-collagen films and 3D porous scaffolds were seeded with human mesenchymal stem cells (hMSCs) to characterize cell proliferation and osteogenic differentiation. Over 21 days hMSCs seeded on 2D nHAP-PLGA-collagen films proliferate, form nodules, deposit mineral and express high alkaline phosphatase activity (ALP) indicating commitment of hMSCs towards osteogenic lineage. When seeded in 3D scaffolds, hMSCs migrate throughout the connected porous network of the nHAP-PLGA-collagen scaffold and proliferate to fill the scaffold voids. Over 35 days, cells express ALP, osteocalcin and deposit minerals with kinetics similar to osteogenesis in vivo. Adipogenic or chondrogenic differentiation is not detected in 3D constructs, indicating that in an osteogenic environment the presence of bone ECM specific molecules in nHAP-PLGA-collagen scaffolds support homogeneous bone tissue development. This ability of nHAP-PLGA-collagen matrices to provide biochemical stimulation to support osteogenesis from stem cells along with its high mechanical strength suggests that nHAP-PLGA-collagen is a suitable biomaterial for bone regeneration. This platform technology of covalently attaching ECM proteins and molecules with synthetic and natural polymers to adjust material properties and biochemical signaling has a potential for a wider range of applications in tissue engineering and regenerative medicine.