Sustained delivery of bioactive cytokine using a dense collagen gel vehicle: Collagen gel delivery of bioactive cytokine

Abstract

The use of cytokines as localized therapeutic agents is limited by the lack of a satisfactory delivery system. The aim of the current investigation was to determine the release kinetics and bioactivity of a simplified cytokine/collagen gel system designed to achieve extended, local delivery of bioactive cytokines at sites of premature cranial suture fusion (craniosynostosis). Cytokine release was determined by ELISA measurements of Tgf-beta3 collected in media. Cytokine bioactivity was determined by measuring the effect of conditioned media, containing released Tgf-beta3, on mink lung epithelial cell proliferation and osteoblast alkaline phosphatase activity. Osteoblast response was evaluated by measuring proliferation of cells cultured on collagen gel containing Tgf-beta3 using an AlamarBlue assay. Gels loaded with 100 and 500 ng of Tgf-beta3 produced a sustained release over 14 days with a pattern of initial large release followed by a gradual reduction in the amount released over the time. The reduced release over time was correlated to the amount initially loaded. Mink lung epithelial cell assay results indicated that Tgf-beta3 released from the collagen gel retained its bioactivity following incorporation into the collagen gel and release into the media. This bioactivity was further illustrated by a decreased alkaline phosphatase activity measured in osteoblasts cultured on the gels loaded with Tgf-beta3. Osteoblast proliferation assays demonstrated that the collagen gel has an inherent inhibitory effect on osteoblast cell number. This collagen gel/cytokine delivery system can retain and release bioactive cytokine over a prolonged period. These results will allow for better optimization of future in vitro and in vivo studies directed at improving the treatment of craniosynostosis.