Bone defects resulting from trauma, disease, or congenital abnormalities present formidable clinical challenges, necessitating advanced regenerative strategies. In this study, a novel bone tissue engineering approach utilizing the osteoinductive properties of collagen/hydroxyapatite (HA) hydrogels and the structural support provided by 3D-printed polylactic acid (PLA) scaffolds was investigated. Specifically, MG63 osteoblast-like cells were encapsulated within collagen/HA hydrogels formulated at an optimized 5:5 ratio and subsequently loaded into PLA lattices. Cell viability, osteogenic differentiation, and mineralization, assessed through live/dead assays, alkaline phosphatase (ALP) activity, osteogenic gene expression analysis, alizarin red S (ARS) staining, field-emission scanning electron microscopy (FE-SEM), and micro-computed tomography (micro-CT) analyses were conducted in vitro. The results demonstrated that the 5:5 collagen/HA hydrogel supported significantly enhanced cell proliferation compared to other tested ratios and the collagen control group. Under bone morphogenetic protein 2 (BMP-2)-induced osteogenic conditions, the composite hydrogel exhibited markedly higher ALP activity and upregulated key osteogenic markers, including ALP and Osterix, indicating robust early differentiation. ARS staining and FE-SEM imaging revealed accelerated and more uniform mineral deposition in the collagen/HA group. These findings were corroborated by 3D micro-CT analysis, which showed near-complete mineralization of the scaffold interior by Day 30. These findings suggest that integrating HA into collagen hydrogels improves the biological environment for osteoblast proliferation and differentiation while promoting nucleation and mineralized extracellular matrix growth. The innovative strategy of encapsulating cells within the hydrogel before scaffold loading maximizes direct cell-material interactions, thereby facilitating more efficient osteogenic signaling compared to traditional composite scaffold fabrication methods. This composite scaffold design demonstrates strong potential for accelerating bone regeneration and improving clinical outcomes in bone defect repair.