Angiogenic functionalisation of titanium surfaces using nano-anchored VEGF – an in vitro study

Abstract

The aim of the present study was to test the hypothesis that sandblasted and acid etched titanium surfaces can be functionalised with vascular endothelial growth factor (VEGF) using oligonucleotides for anchorage and slow release. rhVEGF165 molecules were conjugated to strands of 30-mer non-coding DNA oligonucleotides (ODN) and hybridised to complementary ODN anchor strands which had been immobilised to the surface of sandblasted/acid etched (SAE) Ti specimens. Specimens with non-conjugated VEGF adsorbed to ODN anchor strands and to blank SAE surfaces served as controls. Specifi c binding of conjugated VEGF exhibited the highest percentage of immobilised VEGF (71.0 %), whereas non-conjugated VEGF only achieved 53.2 and 30.7 %, respectively. Cumulative release reached 54.0 % of the immobilised growth factor in the group of specifi cally bound VEGF after 4 weeks, whereas non-conjugated VEGF adsorbed to ODN strands released 78.9 % and VEGF adsorbed to SAE Ti surfaces released 97.4 %. Proliferation of human umbilical vein endothelial cells (HUVECs) was signifi cantly increased on the surfaces with specifi cally bound VEGF compared to the control surfaces and SAE Ti surfaces without VEGF. Moreover, the released conjugated VEGF exhibited biological activity by induction of von Willebrand Factor (vWF) in mesenchymal stem cells. It is concluded that the angiogenic functionalisation of SAE titanium surfaces can be achieved by conjugation of VEGF to ODN strands and hybridisation to complementary ODN strands that are anchored to the titanium surface. The angiogenic effect is exerted both through the immobilised and the released portion of the growth factor.

Improved Anchorage of Ti6Al4V Orthopaedic Bone Implants through Oligonucleotide Mediated Immobilization of BMP-2 in Osteoporotic Rats

“…Nevertheless, we did not observe significant effects on both bone-implant contact and pullout force up to 12 weeks after implantation although an extended in vitro release of the growth factor and in vitro bioactivity of the released conjugated VEGF-A could previously be shown [25]. This may be partly explained by recent data of Lui et al demonstrating that intracellular effects of VEGF-A but not the extracellular interaction with membrane-bound receptors are responsible for induction of osteogenic differentiation of mesenchymal stem cells [41].…”

“…After several desorption steps (three times in the ethanolic acetate buffer, twice in sterilized ultrapure water; each for 30 s) to remove merely adsorbed and not entrapped anchor strands the implants were packed under dry argon atmosphere and sterilized by gamma irradiation with a standard dose of 25 kGy [14], [16]. Conjugates of complementary strands of nucleic acid and recombinant human rhBMP-2 (Reliatech, Braunschweig, Germany) or recombinant human rhVEGF-A (VEGF165, Reliatech) respectively were hybridized to the surface modified with anchor strands directly before implantation under sterile conditions [25], [26]. A final washing step was performed to eliminate unbound growth factors.…”

“…Previous in vitro studies using the oligonucleotide-mediated immobilization technology indicated that BMP-2 and VEGF-A could be effectively bound to titanium surfaces. The growth factors were released over an extended time span and induced stimulatory effects on proliferation and osteogenic differentiation of mesenchymal stem cells as well as proliferative effects on endothelial cells in vitro [25], [26]. Biological activity of released oligonucleotide conjugated BMP-2 and VEGF-A was preserved as indicated by the induction of alkaline phosphatase in C2C12 cells or van Willebrand Factor in mesenchymal stem cells in vitro [25], [26].…”

Utilizing DNA for functionalization of biomaterial surfaces

“…The same was true if VEGF instead of BMP-2 was used [77]. In both cases, hybridized growth factors were the most stably bound [77,78,97] (Fig. In both cases, hybridized growth factors were the most stably bound [77,78,97] (Fig.…”

“…As this process is performed in a slightly acidic electrolyte with pH < IEP TiO2 , the oxide surface is positively charged, and the interaction is, thus, driven by a combination of electrostatic interaction of the negatively charged terminal ODN phosphate group with the positively charged oxide and chemisorption [46,73,74]. To achieve constant conditions in the interface substrate/electrolyte during oxide growth, the polarization is performed in the galvanostatic mode with a current density of 10 mAÁcm À2 to a final potential of 7.5-14.5 V Ag/AgCl that is reached within a few seconds [20, 70,[75][76][77][78][79]. For the specified electrolyte conditions, the process results in a thickness growth of~1 nmÁV À1 at the two interfaces metal/oxide and oxide/electrolyte [7].…”