Release kinetics of VEGF165 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 VEGF165 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 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).


RESULTS
We 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 microg 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. 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.
Kleinheinz
Johannes
J
Department of Cranio-Maxillofacial Surgery, Research Unit Vascular Biology of Oral, Structures, University Hospital Muenster, Waldeyerstrasse 30, D-48149 Muenster, Germany. Johannes.Kleinheinz@ukmuenster.de
Jung
Susanne
S
Wermker
Kai
K
Fischer
Carsten
C
Joos
Ulrich
U
D000818:Animals; D001861:Bone Regeneration / Q000502:physiology*; D003094:Collagen / Q000378:metabolism*; D016207:Cytokines / Q000378:metabolism*; D006736:Horses; D006801:Humans; D066298:In Vitro Techniques; D023421:Models, Animal; D042461:Vascular Endothelial Growth Factor A / Q000378:metabolism*
D016207:Cytokines; C467484:VEGFA protein, human; D042461:Vascular Endothelial Growth Factor A; D003094:Collagen