Objectives: To determine the suitability of a dense collagen gel as a vehicle for sustained delivery of plasmid DNA in calvarial osteoblast culture.
Methods: Fetal rat calvarial osteoblasts were grown in DMEM supplemented with 10% fetal calf serum. On the day prior to transfection, cells were seeded at a density of 50,000 cells/well into 48-well plates. Plasmid DNA, encoding Transforming growth factor-beta 3 (Tgf-beta 3), combined with Geneporter (Gene Therapy systems, Inc.) was mixed with either collagen gel (32 mg/ml, NeuColl, Inc.) or media. Collagen with DNA (1.0 ug/ml) or media with DNA (0.25 ug/ml) was added to the wells then supernatants were collected at various time points up to 14 days. DNA release from the gel was measured by spectrophotometry and Tgf-beta 3 production was quantified by ELISA.
Results: DNA release from collagen indicated gradual release of DNA over time, up to 17 days. Plasmid DNA released from the collagen gel was able to transfect and generate Tgf-beta 3 protein in calvarial osteoblasts. Tgf-beta 3 production was observed up to 14 days in both experimental groups. The most efficient transfection was achieved with DNA in media at early time points. However, cells exposed to collagen with DNA demonstrated elevated Tgf-beta 3 production levels as late as 14 days.
Conclusions: Collagen gel can provide sustained release of viable plasmid DNA which can result in prolonged osteoblast transfection and elevated growth factor production. This suggests that use of collagen gel as a vehicle may provide a strategy to mediate localized and controlled gene delivery in vivo.
(Supported by a grant from NIH, NIAMS RO35RO3AR46382)