Enhanced Osteogenic Differentiation by cAMP-loaded Layer-by-layer Self-assembly on Gelatin-Based Hydroxyapatite

Objectives: To investigate osteogenic differentiation of cyclic adenosine monophosphate (cAMP) encapsulated stem cells from apical papilla (SCAPs) by layer-by-layer (LBL) self-assembly on gelatin based hydroxyapatite (HA) scaffold.

Methods: Gelatin gel were alternately soaked in solution of CaCl₂ and Na₂HPO₄ to prepare the Gel-HA scaffold. The physico-chemical characteristics of Gel-HA were analyzed by scanning electron microscopy (SEM) and energy dispersive X-ray analysis (EDX). SCAPs were encapsulated with cAMP using LBL self-assembled gelatin and alginate polyelectrolytes (LBL-cAMP-SCAPs). SCAPs loaded LBL self-assembled without cAMP was used as negative control (LBL-SCAPs). The cellular morphology of LBL-cAMP-SCAPs and LBL-SCAPs on Gel-HA was analyzed by SEM for 21 days. Cell proliferation and viability was assessed by Cell Counting Kit-8 and live/dead staining, respectively after 1 and 7 days. The osteogenic differentiation of SCAPs was evaluated by alkaline phosphatase activity (ALP) and the expression of osteogenic related genes after 21 days.
Results: SEM revealed that interconnected pore architecture with HA crystals embedded within the gelatin surface. EDX confirmed the prominent element on the surface of Gel-HA was Calcium and Phosphorous. SEM revealed that both LBL-cAMP-SCAPs and LBL-SCAPs on Gel-HA exhibited spheroid morphology after 1 day, and filopodial structure after 7 days. LBL-cAMP-SCAPs formed extra cellular matrix on the surface of Gel-HA after 21 days. No significant difference in cell proliferation and viability was found between LBL-cAMP-SCAPs and LBL-SCAPs. LBL-cAMP-SCAPs seeded on the Gel-HA showed higher level of ALP activity than LBL-SCAPs.
Conclusions: cAMP encapsulated by LBL self-assembly enhanced osteogenic differentiation of SCAPs on gelatin based hydroxyapatite scaffold.