We have previously reported on the use of Bay K8644-release strategies in combination with perfusion–compression bioreactor systems for up regulating bone formation in three-dimensional PLLA scaffolds. Here we report on the analysis of Bay activity following its release from our PLLA scaffolds over the culture period imposed in our tissue engineering protocol using UV spectroscopy in combination with whole cell patch clamping techniques. Bay was released continually from scaffolds within the physiological range required for agonist activity (1–10μM). Patch clamping allowed for the effects of Bay released from scaffolds to be monitored directly with respect to osteoblast electrophysiology. A characteristic shift in the current–voltage (I–V) relationship of L-type VOCC currents was observed in rat osteoblast sarcoma (ROS) cells patched in a solution with Bay released from scaffolds following 14 and 28 days incubation, with statistically significant differences observed in peak currents compared to non-Bay controls. An increase in the magnitude of the peak inward currents was also noted. The electrophysiological response of osteoblasts in the presence of Bay released from scaffolds demonstrates that the released Bay is stable and maintains its bioactivity following culture of up to 28 days.