Release kinetics of VEGF(165 )from a collagen matrix and structural matrix changes in a circulation model

BACKGROUND: Current approaches in bone regeneration combine osteoconductive scaffolds with bioactive cytokines like BMP or VEGF. The idea of our in-vitro trial was to apply VEGF(165 )in gradient concentrations to an equine collagen carrier and to study pharmacological and morphological characteristics of the complex in a circulation model. METHODS: Release kinetics of VEGF(165 )complexed in different quantities in a collagen matrix were determined in a circulation model by quantifying protein concentration with ELISA over a period of 5 days. The structural changes of the collagen matrix were assessed with light microscopy, native scanning electron microscopy (SEM) as well as with immuno-gold-labelling technique in scanning and transmission electron microscopy (TEM). RESULTS: We established a biological half-life for VEGF(165 )of 90 minutes. In a half-logarithmic presentation the VEGF(165 )release showed a linear declining gradient; the release kinetics were not depending on VEGF(165 )concentrations. After 12 hours VEGF release reached a plateau, after 48 hours VEGF(165 )was no longer detectable in the complexes charged with lower doses, but still measurable in the 80 μg sample. At the beginning of the study a smear layer was visible on the surface of the complex. After the wash out of the protein in the first days the natural structure of the collagen appeared and did not change over the test period. CONCLUSIONS: By defining the pharmacological and morphological profile of a cytokine collagen complex in a circulation model our data paves the way for further in-vivo studies where additional biological side effects will have to be considered. VEGF(165 )linked to collagen fibrils shows its improved stability in direct electron microscopic imaging as well as in prolonged release from the matrix. Our in-vitro trial substantiates the position of cytokine collagen complexes as innovative and effective treatment tools in regenerative medicine and and may initiate further clinical research.