Over the past twenty years, the field of tissue engineering and regenerative medicine (TERM) has been significantly impacted by the emergence of 3D bioprinting technology. This advancement has enabled the precise printing of tissues composed of a single cell type, with remarkable resolution and fidelity. Nevertheless, achieving the desired functionality of tissues has remained a challenge due to the absence of diverse cell populations and variations in microenvironment distribution. Traditional 3D bioprinting methods have struggled to provide an effective approach for incorporating multiple cells and biomaterials in a controlled manner. The use of interchangeable syringe-based systems has often led to issues such as delamination between interfaces, particularly hindering the fabrication of interconnected constructs like cartilage and bone tissue. In this study, we introduce a new approached based on the possibility of compartmentalization of biomaterials and cells, controlling density over a gradient architecture to closely mimic osteochondral defects. By incorporating flow-focusing and passive mixer printhead modules, we achieved rapid and dynamic modulation of fiber diameter and material composition, driving compartmentalization of human bone marrow stromal cells (HBMSCs) into distinct three-dimensional layers with defined density patterns, demonstrating functional responses based on final concentration. Experiments conducted ex vivo and in vivo confirmed the functionality of 3D Bioprinted constructs containing patterned growth factors and cellular components. Consequently, this approach enables the simulation of diverse cellular environments and proliferation pathways within the same construct, a capability not achievable with conventional bioprinting techniques. These findings present new opportunities for fabricating functionally graded materials and physiologically relevant skeletal tissue substitutes, for the support in TERM applications for an ageing population.