Metallic biomaterials possess the combination of properties required for load-bearing applications. However, despite the good bulk characteristics, there are still an undesirable number of implants failures. In order to overcome such concerns, improvements could be achieved by designing biomaterials where the bulk and the surface are independently tailored with regenerative capabilities. Therefore, the objective of this study is to improve the osteointegration of Ti-surfaces by adding organic and biomolecules. We hypothesize that the hydroxyl groups presented in TiO2 surface will interact with the functional groups of the organic molecules that will enhance the DMP[1] peptide interaction, improve cell adhesion and differentiation, leading to an implant success.
Titanium cp discs were polished until Ra≈150 nm then cleaned with deionized water. The samples were etched and hydroxylated in Piranha solution. TiO2 deposition was performed by spin coating technique and then annealed at 830°C. The samples were divided in five groups. The control group consists in Ti substrate coated with TiO2, and the other four TiO2 surface were functionalized with 3-mercaptopropionic acid (MPA), 3-4 aminophenyl propionic acid (APPA), 3- aminopropyltrimetoxysilane (APTMS) and polyethylene glycol (PEG). Peptide pA (ESQES) and peptide pB (QESQSEQDS) were diluted in the ratio 1:4, respectively, in order to have a concentration of 1mg/mL. White Light Interferometry (WLI) was used to determine average surface roughness. To determine the chemical states of the elements X-ray Photoelectron Spectroscopy (XPS) analysis was carried out.
Surface measurements demonstrated that the titanium surface modified with TiO2 presented low roughness than the biofunctionalized titanium (Figure 1). Surface roughness between 0.5 and 1.5 µm, as the presented ones, has shown improved cell adhesion[2]]. The XPS analysis revealed an increase in the intensity of C 1s for all samples, indicating the success in the functionalization process (Figue 2a). Different contributions of oxygen can be observed in the high resolution spectra of O 1s from TiO2 (Figure 2). The first three peaks at lower energy are typical for the metal oxide bonds in TiO2, the other one located in 531.2 eV is assigned to the Ti-OH bonds[3]. It indicates that the surface contains hydroxyl groups acting as anchoring points for the formation of monolayers. APTMS attachment is preferential by silane groups because Si-OH reacts fast by condensation forming Si-O-Si groups. This covalent bond stabilizes the monolayer and also allows the binding with peptides by NH2 anchoring group[4]. For MPA the results indicate that this molecule bind with TiO2 surface by COOH groups (Figure 3b). PEG has several hydroxyl groups available for derivatization which cross linked with each other forming multilayer upon the TiO2 surface, as shown by the increase of C 1s and O 1s in PEG survey followed by a decrease in Ti 2p intensity. In order to test the surface properties, human mesenchymal stem cells were cultured on the modified substrates and tested for their cellular adhesive properties and their differentiation potential.
These findings suggest that the bio-functionalization of the Ti based substrates with organic and biomolecules could possibly open the door for designing better implants and their use in regenerative medicine.
FAPESP (2014/27015-0; 2014/01713-3; 2014/20471-0); CEPID (2013/07296-2); Brodie Endowment fund
References:
[1] P. Silva-Bermudez, & S. E. Rodil, Surf. Coat. Technol. 233, 147–158 (2013).
[2] K. Balani et al. Biosurfaces: A Materials Science and Engineering Perspective. John Wiley & Sons Inc.: New Jersey, 2015.
[3] J. F. Moulder et al. Handbook of X-ray Photoelectron Spectroscopy. Perkin-Elmer Corporation: Minnesota, 1992.
[4] S.P. Pujari, et al. Angew. Chem. Int. Ed. 53, 6322–6356 (2014).