Enhancing the angiogenesis and osteogenesis of low elastic modulus Ti-Nb-Zr-Sn alloy surface through grafting growth factors-containing gelatin nanoparticles

Abstract

The study aimed to apply a multiple surface modification for low elastic modulus Ti-24 % Nb-4 % Zr-8 % Sn (Ti2448) alloy, to enhance its biological properties for bone implant applications. We employed a combined sandblasting/acid etching/alkaline etching process to modify the Ti2448 alloy surfaces with mixed nano/submicron/micron-scaled pits and super wettability. We prepared gelatin nanoparticles (GNPs) loaded with growth factors: vascular endothelial growth factor/platelet-derived growth factor (VEGF/PDGF) and/or bone morphogenetic protein-2 (BMP-2). The growth factors-containing GNPs were then sequentially grafted onto the surface-modified Ti2448 alloy in a layered (single layer: BMP-2 or VEGF/PDGF; double layer: inner BMP-2 and outer VEGF/PDGF) or mixed BMP-2/VEGF/PDGF manner. Surface characterizations, including morphology, roughness, surface energy, and cytotoxicity, of Ti2448 alloy were analyzed. For biological response, we investigated the tube formation ability of human umbilical vein endothelial cells (HUVECs) and the cell responses (adhesion, proliferation, and mineralization) of human bone marrow mesenchymal stem cells (hMSCs). The results demonstrated the successful preparation of GNPs with 200∼300 nm particle size and their successful loading with the growth factors using natural crosslinker genipin. The growth factor-containing GNPs were effectively grafted onto the surface-modified hydrophilic Ti2448 alloy without significantly reducing its wettability (water contact angle remained below 40°). The results of the study showed that the GNPs-grafted Ti2448 alloy surfaces, with the GNPs loaded with VEGF/PDGF and/or BMP-2, exhibited enhanced biological properties. The GNPs-grafted surfaces were found to be non-toxic and capable of promoting tube formation of HUVECs, particularly when single VEGF/PDGF layer or double (inner BMP-2/ outer VEGF/PDGF) layer was loaded on GNPs (enhancing the cumulative tube length approximately 1.4 times that of no GNPs-grafted surfaces). Moreover, the GNPs-grafted surfaces enhanced the adhesion-related proteins and extracellular matrix mineralization of hMSCs, particularly with the loading of single VEGF/PDGF or mixed VEGF/PDGF/BMP-2 on GNPs (enhancing αvβ3 integrin patch number > 3.5 times, vinculin patch number > 2 times, and mineralization approximately 1.4 times). Based on these findings, we concluded that the proposed Ti2448 alloy surfaces, when grafted with VEGF/PDGF- and/or BMP-2-containing GNPs, have the potential for bone implant applications. The surface modifications on the Ti2448 alloy may promote angiogenesis in the early stage of bone repair (through VEGF/PDGF) and osteogenesis in the late stage (through BMP-2), thereby accelerating the overall bone healing process.