The fabrication of double-layered chitosan/gelatin/genipin nanosphere coating for sequential and controlled release of therapeutic proteins

Bone regeneration is a complicated process and includes a number of distinct and sequential stages of coordinated cellular actions under the regulation of multiple growth factors. Therefore, bone grafting materials in which growth factors can be incorporated and released in a programmed order in line with the bone tissue healing process may lead to desirable clinical outcomes. In the present study, a double-layered chitosan/gelatin/genipin (d-CSG/G) nanosphere coating is developed by using layer-by-layer electrophoretic deposition and genipin crosslinking. The surface morphology, physicochemical and mechanical properties of the coatings are explored. Cytochrome C is used as a therapeutic model protein and is successfully loaded on the inner and outer layers of the coating. The protein release can be controlled by the loading position, genipin concentration and thickness of the outer layer. Furthermore, the cell response to the coatings was evaluated. Real-time polymerase chain reactions, immunofluorescence staining and extracellular matrix mineralization assay confirmed that the functions of the loaded growth factor are fully preserved after fabrication. Overall, the d-CSG/G nanosphere coating could be a promising growth factor delivery system to promote bone tissue regeneration.