Activation of Osteoblastic Function on Titanium Surface with Titanium-Doped Hydroxyapatite Nanoparticle Coating: An In Vitro Study.

PURPOSE: Titanium-doped hydroxyapatite (TiHA) nanoparticles contain titanium atoms in the hydroxyapatite lattice, which can physicochemically functionalize the titanium surface without modification of the surface topography. This study aimed to evaluate the physicochemical properties of machined or microroughened titanium surfaces coated with TiHA nanoparticles and the functions of osteoblasts cultured on them. MATERIALS AND METHODS: Titanium disks with commercially available surface topography, such as machined or sandblasted, large-grit, and acid-etched (SLA) surfaces, were coated with TiHA. The disks with original or TiHA-coated surfaces were evaluated in topography, wettability, and chemical composition. Osteoblastic cells from rat femurs were cultured on the disks and evaluated in proliferation and differentiation. RESULTS: TiHA coating changed from hydrophobicity to hydrophilicity on both machined and SLA surfaces. Calcium and phosphate atoms were detected all over the surface with TiHA coating regardless of the surface topography. However, the considerable change in the inherent surface topographies was not observed on both types of surfaces after TiHA coating. Osteoblastic proliferative activity at day 4 was increased by TiHA coating on both types of surfaces. TiHA coating did not enhance expressions of bone matrix-related genes such as osteocalcin, osteopontin, bone sialoprotein, alkaline phosphatase, and collagen I. However, depositions of collagen, osteocalcin, and calcium in the culture at days 7 and 20 were increased on both types of surface topographies with TiHA coating. CONCLUSION: TiHA coating enhanced extracellular matrix formation on smooth and microroughened titanium surfaces by increasing osteoblastic proliferative activity without the deterioration of differentiation through hydrophilic and chemical functionalization.

Effects of Ti(IV) substitution on protein adsorption behaviors of calcium hydroxyapatite particles.

The fundamental experiments on the adsorption behaviors of proteins onto photocatalytic Ti(IV)-doped calcium hydroxyapatite (TiHap) particles with varied amounts of Ti(IV) ions doped (called as original particle) were examined comparing to those onto the calcium hydroxyapatite (CaHap) ones. The heat treated TiHaps and CaHap particles at 650°C for 1h were also examined (called as heat treated particle). The Ti/(Ca+Ti) atomic ratio (X(Ti)) of the TiHap particles was varied between 0 and 0.20. Since the surface acidity of the particles was increased by increase in X(Ti) value, the negative zeta potential (zp) of the particles was increased. All the adsorption isotherms of bovine serum albumin (BSA), myoglobin (MGB) and lysozyme (LSZ) from 1×10(-4)mol/dm(3) KCl solution were the pseudo-Langmuirian type. The saturated amounts of adsorbed LSZ (n(S)(LSZ)) values onto the original particles were increased with increase in the negative zp of the particles. However, the saturated amounts of adsorbed BSA (n(S)(BSA)) values were decreased by increase in the negative zp except at X(Ti)=0.05 where n(S)(BSA) value exhibited a maximum. In the case of MGB, the saturated amounts of adsorbed MGB (n(s)(MGB)) values were less dependent on the zp of the particles. These results were explained by changing the electrostatic forces between protein molecules and TiHap particles by doping Ti(IV) ions. On the other hand, n(S)(BSA), n(S)(LSZ) and n(s)(MGB) values onto the heat treated particles were larger than the original particles in each particle system, though no relationship to the X(Ti) value was recognized in each protein system. This result was interpreted to the formation of ß-TCP crystal phase in both the CaHap and TiHap particles by the heat treatment. The Ca(2+) ions produced by dissolution from ß-TCP phase may exert as binders between BSA and surfaces of the heat treated particles. The weak binder effects of Ca(2+) and PO(4)(3-) ions were observed for the adsorptions of LSZ and MGB.

Decomposition of proteins by photocatalytic Ti(IV)-doped calcium hydroxyapatite particles.

The decomposition of protein molecules on the surface of Ti(IV)-doped calcium hydroxyapatite (TiHap) particles with a Ti/(Ca+Ti) atomic ratio among 0-0.20 under UV irradiation of 365 nm in wavelength was disclosed. The acidic bovine serum albumin (BSA), neutral myoglobin (MGB) and basic lysozyme (LSZ) were employed as a model of pathogenic proteins. The photocatalytic activities of TiHap particles were estimated from the decomposition of each protein under 1 mW/cm(2) UV irradiation dispersed in 10 mL quartz tube. The concentrations of each protein in the supernatant after centrifugation during the UV irradiation were determined both by a HPLC and a SDS-Polyacrylamide Gel Electrophoresis (SDS-PAGE) analysis methods. No change in BSA concentration ([BSA]) by UV irradiation was observed for all the unheated original TiHap particles with low photocatalytic activity. The similar results were observed for the systems employed heat treated particles endowed a high photocatalytic activity by heat treatment at 650°C for 1h. These results indicated that the decomposition of BSA molecules is hard to take place. The heme structured MGB molecules are decomposed by UV irradiation irrespective of the presence of TiHap particles. In the case of heat treated particles, MGB molecules were further decomposed by the UV irradiation. The strongest photocatalytic activity was observed for the decomposition systems of LSZ by using heat treated particles. In this system, all the TiHap particles completely decomposed LSZ molecules after started the UV irradiation. It was concluded that the ability of decomposition of proteins is strongly related to the molecular weight and rigidity of proteins molecules. The LSZ molecule with low molecular weight and rigid structure was easily decomposed on the surface of heat treated TiHap particles under UV irradiation.

Protein adsorption behaviors onto photocatalytic Ti(IV)-doped calcium hydroxyapatite particles.

The fundamental experiments on the adsorption behaviors of proteins onto photocatalytic Ti(4+)-doped calcium hydroxyapatite (TiHap) particles were examined comparing to those onto the calcium hydroxyapatite (CaHap) and commercially available typical titanium oxide (TiO(2)) photocatalyst (TKP-101). The heat treated TiHap and CaHap particles were also used after treated these particles at 650°C for 1h (abbreviated as TiHap650 and CaHap650, respectively). All the adsorption isotherms of bovine serum albumin (BSA), myoglobin (MGB) and lysozyme (LSZ) from 1×10(-4)mol/dm(3) KCl solution were the Langmuirian type. The saturated amounts of adsorbed BSA (n(s)(BSA)) for the CaHap650 particles was higher than that for CaHap. Similar results were observed for TiHap and TiHap650. The adsorption of LSZ exhibited the same result of BSA, while the saturated amounts of adsorbed LSZ (n(s)(LSZ)) value on the TiHap were much higher than CaHap. However, the saturated amounts of adsorbed MGB (n(s)(MGB)) are almost equal to those for the CaHap and TiHap nevertheless whether these particles were heat treated at 650°C or not. The TKP-101 exhibited extremely small adsorption capacity of all proteins due to its small particle size of ca. 4nm in diameter. The independence of the n(s)(MGB) value on the zeta potential (zp) of the particles was explained by the electrostatical neutrality of MGB molecules. On the other hand, the n(s)(LSZ) values were increased with increase in the negative zp of the particles. This fact was explained by increasing the electrostatic attractive forces between negatively charged particles and positively charged LSZ. However, the n(s)(BSA) values exhibit maxima for the heat treated TiHap650 and CaHap650 particles. This result was interpreted to the formation of ß-TCP crystal phase by the heat treatment. The produced Ca(2+) ions by dissolution from ß-TCP phase may exert as binders between BSA and surfaces of the heat treated particles.

Effects of modification of calcium hydroxyapatites by trivalent metal ions on the protein adsorption behavior.

The effects of modification of calcium hydroxyapatites (Hap; Ca10(PO4)6(OH)2) by trivalent metal ions (Al(III), La(III), and Fe(III)) on protein adsorption behavior were examined using bovine serum albumin (BSA; isoelectric point (iep) = 4.7 and molecular mass (M(s)) = 67,200 Da). The Al(III)-, La(III)-, and Fe(III)-substituted Hap particles were prepared by the coprecipitation method with different atomic ratios, metal/(Ca + metal), abbreviated as X(metal). The particles precipitated at X(metal) = 0 (original-Hap) were rod-like and 10 x 36 nm2 in size. The short, rod-like original-Hap particles were elongated upon adding metal ions up to X(metal) = 0.10, and the extent of the particle growth was in the order of La(III) < Al(III) << Fe(III). The crystallinity of the materials was slightly lowered by increasing X(metal) for all systems. The adsorption isotherms of BSA onto the Al(III)-, La(III)-, and Fe(III)-substituted Hap particles showed the Langmuirian type. The saturated amounts of adsorbed BSA (n(s)(BSA)) values were strongly dependent on X(metal) in each system. The n(s)(BSA) values for the Fe(III)-substituted Hap system were increased with an increase in X(Fe) (X(metal) value of Hap particles substituted with Fe(III)); the n(s)(BSA) value obtained at X(Fe) = 0.10 was 2.7-fold more than that for the original-Hap particle, though those for the La(III) system were decreased to ca. 1/5. On the other hand, the n(s)(BSA) values for the Al(III) system were decreased with substitution of small amounts of Al(III), showing a minimum point at X(Al) = 0.01, but they were increased again at X(Al) over 0.03. Since the concentrations of hetero metal ions dissolved from the particles exhibited extremely low values, the possibility of binder effects of trivalent cations dissolved from the particle surface for adsorbing BSA to trivalent-ion-substituted Hap particles was excluded. The increase of n(s)(BSA) by an increase in X(Fe) was explained by elongation of mean particle length along with the production of surface hydroxo ions, such as Fe(OH)2+ or Fe(OH)2+, to induce the hydrogen bond between the Fe(III)-substituted Hap surface and BSA molecules, though the number of original C sites established by Ca(II) atoms was reduced. In the case of La(III)-substituted Hap particles, the number of original C sites established by Ca(II) atoms was reduced by La(III) substitution but the mean particle length remained almost constant. Furthermore, surface hydroxo La(III) groups were absent. Therefore, the reduction of n(s)(BSA) was explained by both the unaltered mean particle length and their low surface hydrophilicity. The change of n(s)(BSA) values by X(Al) resembled that of the mean particle length. These results implied that both the mean particle length and surface hydrophilicity of Al(III)-, La(III)-, and Fe(III)-substituted Hap particles are determining factors of the adsorption amounts of BSA.