Scanning electron microscopy (–) confirmed that a modest temperature oxidation treatment could be used to introduce nanoscale structural features to the Ti surfaces. In this study, the oxidation temperature (i.e., 740°C) and gaseous environment ( i.e., synthetic air) were fixed while the duration of the process was varied. The surfaces of the starting PT samples were relatively smooth on the microscale (CLM Sa = 0.43 ± 0.02 µm), although surface pits, presumably resulting from the PT acid pickling process, were detected (). After 45 minutes of controlled oxidation (NMPT45), a low density of nanoscale protuberances was observed to have formed on the specimen surfaces (), with protuberance sizes ranging from about 40 to 200 nm in diameter () and about 10 to 150 nm in height. After 90 minutes of modification (NMPT90), the entire surface was homogeneously covered with a relatively high density of nanoscale structures (), which ranged in size from about 40 to 360 nm in diameter () and about 60 to 350 nm in height. Following 180 minutes of modification (NMPT180), the nanostructures coalesced into coarser structures () that spanned about 500 to 1000 nm in diameter and about 80 to 500 nm in height. The mass increase of the oxidized samples was also monitored by TG analyses and correlated to changes in surface topography. Indeed, by coupling weight gain measurements to the resulting surface topography, TG analyses may be used to monitor the time required for the generation of a high surface density of nanoscale structures on titanium implants of various geometries.
Figure 2 NM-treatment of (a) PT surfaces via oxidation in flowing synthetic air (21% O2, 79% N2) at 740°C for times of: (b) 45 minutes; (c) 90 minutes; (d) 180 minutes. The modification process introduced: (b) nanoscale protuberances with low surface coverage (more ...)
SEM images of the surfaced of (a) PT, (b) NMPT, (c) SLA, and (d) NMSLA samples used for further surface characterization and for cell experiments. The NM treatment consisted of oxidation in flowing synthetic air for 90 min at 740°C.
The NM treatment was also applied to SLA substrates that possessed a greater degree of microscale roughness (CLM Sa = 3.29 ± 0.18 µm) than for the PT specimens. NMSLA samples were generated using the same oxidation conditions as for the NMPT90 samples (i.e., 740°C, 90 min, synthetic flowing air). At low magnifications (), SEM analyses revealed a similar microscale topography for the SLA and NMSLA samples. However, at intermediate and higher magnifications (), NMSLA surfaces were observed to possess a relatively high and uniform density of nanoscale structures.
Figure 3 SEM images of starting SLA samples (a, c, e), and of NMSLA samples (b, d, f) generated via oxidation in flowing synthetic air at 740°C for 90 minutes. These images indicate that the NM process yielded a relatively high density of nanoscale structures (more ...)
After verifying that a NM treatment (740°C, 90 min., synthetic flowing air) could be used to introduce a relatively high density of nanoscale structural features to Ti surfaces that were relatively smooth or rough at the microscale, this treatment was applied to Ti specimens for further surface characterization and for use in cell experiments. Cell interactions with four types of specimens were examined: PT (), NMPT (), SLA () and NMSLA (). The microscale and nanoscale topography of these samples was measured quantitatively using CLM and AFM, respectively (). As expected, the mean values of microscale (CLM-derived) roughness and peak-to-valley height obtained for the PT and NMPT specimens were lower than for the SLA and NMSLA samples. Additionally, the average values of the microscale (CLM-derived) roughness of the nano-modified samples, NMPT and NMSLA, were slightly lower than for the respective controls. The mean nanoscale (AFM-derived) roughness of the NMPT specimens was apparently higher than for the PT controls (), although little statistical difference in the mean nanoscale roughness could be discerned between the SLA and NMSLA specimens. However, the NMPT and NMSLA surfaces shared noticeably higher (and similar) mean values of nanoscale peak-to-valley height relative to the PT and SLA surfaces. The combined CLM and AFM analyses were consistent with the presence of a relatively high density of nanoscale features on the NMPT and NMSLA specimens with little or no statistical change in the microscale topography.
Mean ± one standard deviation (SD) values of roughness (Sa) and peak-to-valley height (Sz) of the different titanium surfaces examined using atomic force microscopy (AFM) and confocal laser microscopy (CLM).
Water contact angle measurements indicated that all of the samples exhibited relatively hydrophobic behavior (). The contact angles measured for the SLA and NMSLA samples were significantly larger than for the PT and NMPT samples (), which was consistent with the enhanced mean values of microscale roughness (CLM-derived Sa values) and microscale peak-to-valley height (CLM-derived Sz values) for the SLA and NMSLA samples ().
Figure 5 Surface characterization data of the NM-treated samples, which were oxidized in flowing synthetic air for 90 min at 740°C, and their controls. (a–d) Optical images of water contact angles on PT, SLA, NMPT, and NMSLA surfaces. The contact (more ...)
Mean values of NMPT/PT and NMSLA/SLA O and Ti concentration ratios ± one standard deviation (SD) as determined by x-ray photoelectron spectroscopy (XPS).
General surveys of the surface chemistry of the different specimens by XPS analyses revealed the presence of appreciable oxygen and titanium. Within statistical error, the concentrations of oxygen and titanium on the PT and NMPT surfaces, and of oxygen and titanium on the SLA and NMSLA surfaces, were similar (). However, a detectable change in the phase content on the Ti surfaces after the NM treatment was revealed by XRD and TEM analyses (). XRD analyses of the surfaces of the PT and SLA samples yielded major diffraction peaks for α-Ti (ICDD 01-089-3073) and did not yield detectable diffraction peaks for crystalline oxides of titanium (). The SLA samples also exhibited additional diffraction peaks of modest intensity that were attributed to titanium hydride (TiH2, ICDD 04-008-1386). Both NMPT and NMSLA specimens exhibited relatively intense diffraction peaks for the rutile polymorph of TiO2 (ICDD 01-071-6411). The α-Ti diffraction peaks in the NM-treated samples also appeared to shift to lower two-theta values. TEM analysis of an ion-milled cross-section of the NMPT sample () revealed the presence of a compact and conformal oxide layer on the Ti surface. The average thickness of this oxide layer, generated within 90 min at 740°C in air, was about 1.2 µm. Selected area electron diffraction (SAED) analysis () of this oxide scale yielded a diffraction pattern that was consistent with the presence of only the rutile polymorph of TiO2 (as had also been revealed by the XRD analyses of NM-treated specimens).
Mean values of water contact angle ± one standard deviation (SD).
Osteoblasts were sensitive to the surface modifications. The number of MG63 osteoblast cells, as deduced from DNA measurements (), and the alkaline phosphatase specific activity () for the NMPT, SLA, and NMSLA samples were statistically lower than for the PT specimens. This reduction in cell content and ALP activity paralleled an increase in mean nanoscale roughness (NMPT vs. PT) and the microscale roughness (SLA and NMSLA vs. PT). While the levels of osteocalcin, osteoprotegerin, and vascular endothelial growth factor () measured for the PT and NMPT samples were not noticeably different, statistically-significant increases in the levels of these markers were observed for the SLA specimens, which paralleled the increase in microscale roughness for the SLA specimens relative to the PT and NMPT samples (). Further statistically significant increases in the osteocalcin, osteoprotegerin, and VEGF levels over the SLA specimens was observed for the NMSLA specimens.
Figure 6 Effects of nanoscale surface features and microscale surface roughness on osteoblast differentiation. MG63 cells were plated on PT, NMPT, SLA, and NMSLA surfaces and grown to confluence. The NM treatment consisted of oxidation in flowing synthetic air (more ...)