Release kinetics of VEGF165 from a collagen matrix and structural matrix changes in a circulation model

Abstract

BackgroundCurrent approaches in bone regeneration combine osteoconductive scaffolds with bioactive cytokines like BMP or VEGF. The idea of our in-vitro trial was to apply VEGF165 in gradient concentrations to an equine collagen carrier and to study pharmacological and morphological characteristics of the complex in a circulation model.MethodsRelease kinetics of VEGF165 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).ResultsWe established a biological half-life for VEGF165 of 90 minutes. In a half-logarithmic presentation the VEGF165 release showed a linear declining gradient; the release kinetics were not depending on VEGF165 concentrations. After 12 hours VEGF release reached a plateau, after 48 hours VEGF165 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.ConclusionsBy 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. VEGF165 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.

Short term storage stability at room temperature of two different platelet-rich plasma preparations from equine donors and potential impact on growth factor concentrations

“…composites with biomaterials and others) and conditions like application mode including activation and indication of PRP formulations to get a more precise impression of the individual kinetics of growth factors in a PRP setting. Thus, for example Kleinheinz and colleagues found an enhancement of VEGF 165 (vascular endothelial growth factor) half-life from 90 min for free VEGF 165 up to 48 h when embedded into a collagen matrix [20]. Presuming different half-life data of growth factors in dependence on the respective preparation and setting it seems to be essential to estimate cytokine stability over time not only for the main classifications of liquid platelet-rich products (P-PRP, L-PRP) but also for each individual preparation protocol currently in use.…”

Extracellular regulation of VEGF: Isoforms, proteolysis, and vascular patterning

“…Heparin can release and disperse FGF2 and FGF10, enabling activation of cells at a distance and altering gradients (99, 100). Similarly, VEGF release by proteases and heparanases produces diffusible VEGF that can escape subsequent rebinding to the matrix and is resistant to degradation (13, 101). Carriers may also enhance the spatial complexity of the local VEGF gradients (21, 102).…”

Local application of VEGF compensates callus deficiency after acute soft tissue trauma—results using a limb‐shortening distraction procedure in rabbit tibia

“…12,26 The effect of this local application was insufficient for fracture healing in segmental defect models. Recently, Kleinheinz et al 16,27 demonstrated a linear declining gradient in release kinetics of a VEGF-soaked collagen I carrier. On the basis of this in vitro release kinetics of different concentrations of rhVEGF 165 , the calculated half-life, and known concentrations required for semi-maximal biologic activity of rhVEGF 165 (ED 50 ¼ 2-6 ng/ml) for activating endothelial cells, we assessed the optimal concentration of rhVEGF 165 per implant (12 mg).…”