The field of tissue engineering has continually been described as “cells, signals, and scaffolds.” The current thesis work describes the evaluation of a continuously-graded microsphere-based scaffold technology for the regeneration of the osteochondral interface where cartilage and bone have been damaged due to degenerative conditions, such as osteoarthritis. This scaffold technology used spatiotemporal release of bioactive factors from a biodegradable polymer for simultaneous differentiation of stem cells into bone and cartilage. The work in the current thesis evaluated the scaffold formulation in vitro, in vivo, and addressed the feasibility of adding osteoconductive materials to the first generation design for enhanced bone regeneration. Lastly, bioactive signal delivery and bioactivity was investigated as a consequence of preparatory methods used to construct the bioactive scaffolds. Results from the body of in vitro studies included stimulation of gene expression, increased biochemical production, and tissue synthesis. Specifically, gradient constructs outperformed control constructs in glycosaminoglycan content, produced twice as much collagen, and were capable of facilitating regional tissue synthesis. In vivo studies demonstrated the feasibility and potential efficacy of bioactive factor inclusion at two different osteochondral interfaces. In the New Zealand White rabbit knee, implants with embedded signals gradients were capable of regenerating more bone tissue than negative controls. When used in a smaller defect site, such as the New Zealand White rabbit mandibular condyle, the bioactive scaffolds were beneficial in regenerating thicker layers of cartilage. Moreover, this thesis has bridged the gradient-based microsphere scaffold design from concept to practice, in addition to opening new areas of investigation for further refinement of the technology.