Gelatin microspheres crosslinked with genipin for local delivery of growth factors

A main challenge in tissue engineering and regenerative medicine is achieving local and efficient growth factor release to guide cell function. Gelatin is a denatured form of collagen that cells can bind to and degrade through enzymatic action. In this study, gelatin microspheres were used to release bone morphogenetic protein 2 (BMP2). Spherical microparticles with diameters in the range of 2-6 µm were created by an emulsification process and were stabilized by crosslinking with the small molecule genipin. The degree of crosslinking was varied by controlling the incubation time in genipin solution. Loading rate studies, using soy bean trypsin inhibitor as a model protein, showed rapid protein uptake over the first 24 h, followed by a levelling off and then a further increase after approximately 3 days, as the microspheres swelled. Growth factor release studies using microspheres crosslinked to 20%, 50% and 80% of saturation and then loaded with BMP2 showed that higher degrees of crosslinking resulted in higher loading efficiency and slower protein release. After 24 h, the concentration profiles produced by all microsphere formulations were steady and approximately equal. Microspheres incubated with adult human mesenchymal stem cells accumulated preferentially on the cell surface, and degraded over time in culture. BMP2-loaded microspheres caused a three- to eight-fold increase in expression of the bone sialoprotein gene after 14 days in culture, with more crosslinked beads producing a greater effect. These results demonstrate that genipin-crosslinked gelatin microspheres can be used to deliver growth factors locally to cells in order to direct their function.