2.1. Wear-Corrosion Tests
The interaction of physiological fluids with the bearing surfaces of hip implants is of great importance in the research of artificial joint lubrication, although this study has been so far little explored.
The effect of sliding of the alumina ball on the HCCoCr alloys is clearly shown in the drastic change in the open circuit potential. As an example, Figure 1
shows the change in the open circuit potential when the HCCoCr surfaces in PBS supplemented with 3 g/L HA (PBS-HA) are subjected to wear. The open circuit potential without wear was around −0.25 V versus Ag/AgCl, decreasing sharply when the alumina ball (pin) started the circular movement under 5 N load at 120 rpm. At this moment, the open circuit potential decreased until achieving values of about −0.55 V vs. Ag/AgCl, i.e., about 300 mV, and remained constant until the end of the test. The reduction in the potential value towards more negative values indicates that the HCCoCr surface becomes electrochemically active. This variation is due to the breakdown of the passive film under sliding, promoting the release of metallic ions and particles.
panels a and b show the coefficient of friction (COF) for HCCoCr/alumina pair in PBS and PBS supplemented with 3 g/L HA (PBS-HA) during anodic potentiodynamic polarization and the anodic polarization curves drawn at 10 mV/min of HCCoCr in PBS and PBS containing 3 g/L HA under wear conditions (between point 1 and 2 in Figure 2
a), respectively. The anodic polarization curve of HCCoCr in PBS-HA without wear has been also added in Figure 2
b for comparative reasons. It can be seen that under sliding at the corrosion potential (before point 1 in Figure 2
a), the COF was significantly higher in PBS than in PBS-HA. This result agrees with the hypothesis that the hyaluronic acid has a known lubricant role in the joint, acting as a shock absorber [25
] and thus facilitating smooth joint movement by reducing friction between both surfaces. At the next stage (from point 1 to 2, in Figure 2
a), the difference between both COF (in PBS and PBS-HA) remained, but higher fluctuations were detected. The fluctuations could be related to the continuous formation of hard particulate matter that enhances friction between both counterparts and decreases the friction when ejected from the track to surrounding areas where it accumulates (Figure 3
). The load applied on the CoCr surfaces while sliding activates mechano-chemical reactions, causing not only the detachment of the passive film [26
] but also bulk material resulting in an increase of COF. The hyaluronic acid in PBS maintains the lubricant effect during most of the wear corrosion tests.
As a consequence of mechanically assisted corrosion, the passive film on the HCCoCr surface was rapidly broken in both media, PBS and PBS-HA, producing an increase of approximately 3 orders of magnitude in current (Figure 2
b) with respect to the anodic polarization curve without wear. Corrosion progresses on the wear track drawn by the sliding of alumina ball on the HCCoCr disks (Figure 3
). Having in mind the wide passive region seen in the anodic polarization curve drawn without wear (Figure 2
), the potential applied could be employed in forming rapidly the new oxide film. However, the sliding rate is quick enough to avoid the repassivation and formation of new protective chromium oxides. The constant value of the current density around 1 mA (three orders of magnitude higher than without wear) indicates that under these experimental conditions (5 N load and sliding rate of 120 rpm), the passive film is destroyed and remains in an active state until the end of the test.
shows the secondary electron (SE) images of the tracks of HCCoCr in PBS-HA after wear corrosion tests, at the corrosion potential (PBS-HA) and under anodic potentiodynamic polarization (PBS-HA+POL). In both cases (a) and (b), debris is accumulated in the immediate vicinity of the wear tracks, but the surface inside the track is especially altered when anodic potentiodynamic potential is applied. Figure 4
shows, as an example, the semiquantitative analysis taken by EDS of the three areas of interest in the HCCoCr alloy immersed in PBS-HA after wear corrosion under polarization (PBS-HA-POL): away from the track (spectrum 1), immediate vicinity (spectrum 2), and inside the track (spectrum 3). The most important feature found is the high % C content accumulated in the vicinity of the track. It means that the debris is mainly composed of C and O, the greatest proportion probably coming from the hyaluronic acid.
The morphology and chemical characterization of the wear particles detached during wear corrosion tests revealed some interesting results. Figure 5
shows, as an example, the secondary electron image of wear particles collected from the tribocorrosion test in PBS containing 3 g/L HA and the semiquantitative analysis of some particles, identified from 1 to 6 and marked in blue color.
The statistical results of the effect of the corrosive medium and polarization applied in the wear corrosion tests on the chemical composition of the wear-detached particles collected appear in Table 1
. In this table, three condition numbers assigned to 1, the PBS corrosive medium, 2, the PBS-HA corrosive medium without applying polarization, and 3, the PBS-HA corrosive medium applying polarization (PBS-HA-POL), have been considered. Mean, standard deviation, minimum and maximum value, C25 and C75, and median are shown.
It can be seen that the particles are mainly composed of Co, Cr, Mo, P, C and O, with some traces of Al in some isolated particles. The Kruskal–Wallis test indicated that there are significant differences in the levels of Cr, Co, Mo, O and C, comparing the different conditions, i.e., depending on the composition of corrosive medium (PBS-condition 1 or PBS-HA-condition 2) and the application of polarization in wear corrosion tests (PBS-HA-POL, condition 3, and PBS-HA, condition 2). However, no significant differences in P and Al levels were obtained.
The results of the post hoc Mann–Whitney test used to determine which pairs differed among them are shown in Table 2
. Cr levels are significantly higher in condition number 3 than 2 (p
= 0.021). Co and Mo levels are significantly higher in condition number 3 than 1 and 2 (p
= 0.002 and p
= 0.001, p
= 0.025 and p
= 0.002, respectively). O levels are significantly lower in condition number 3 than 1 and 2 (p
= 0.002 in both cases). C levels are significantly lower in condition number 3 than 2 (p
In summary, the statistical analysis confirmed that factors such as “composition of the corrosive medium” and “polarization applied” have an influence on the dependent variable chemical composition of the particles that is discussed immediately below.
The main significant effect of the addition of hyaluronic acid in the PBS to the wear particles detached is observed in the increase of the C content in the chemical composition of the particles. In both media (PBS, condition 1, and PBS-HA, condition 2), particles are mainly composed of Cr and O, followed by P and some Co. This chemical composition can be directly linked to the detachment of the native passive film during the wear corrosion test.
It has been proven by XPS (data not shown) that the immersion of the HCCoCr surfaces in PBS-HA causes a decrease in the Co species in the passive film and the enrichment in chromium oxide where phosphorus is included. It has been reported in literature that phosphate is adsorbed upon freshly exposed metal at the same time that ions are released into the solution until the passive layer is formed, whose composition varies significantly depending upon the environment [27
]. Lewis et al. established that the corrosion, especially when associated with mechanical wear, is controlled by phosphate anions that absorb or react with the Co and Cr dissolution products. This promotes the formation of a mixed composition of phosphates, hydroxides, and oxides originating from the bulk metal.
This means that most of the particles collected after the wear corrosion tests in PBS and PBS-HA come from the native passive film (whose thickness is about 5–7 nm) and are mainly composed of chromium oxide and phosphate.
With respect to applying polarization during the wear corrosion tests in PBS-HA (condition 3), this factor has an important effect on the chemical composition of the wear particles detached. In this condition, particles are mainly composed of Cr and Co, followed by O, P, and Mo. The main significant effect of the polarization is the significant enrichment in Co, Cr, and Mo in the chemical composition of the detached particles. In this case, the wear particles produced under anodic polarization increased the Co/Cr ratio (with a value of 0.9 in comparison with a value of 0.3 found in PBS-HA without polarization). As wear particles obtained without polarization, these particles also contained P, although in a low proportion (Table 1
). It has been reported in the literature [16
] that the potential applied on the HCCoCr induces a change in the chemical composition of the passive film. Díaz et al. established that the increase in polarization (from 0.5 to 0.7 V) induced the preferential dissolution of cobalt whereas chromium was concentrated in the surface oxide film [16
]. The passive film grown at a potential of 0.5 V vs. Ag/AgCl (into the passive region of the anodic polarization curve) consisted predominantly of Cr2
. However, the oxidation at a potential of 0.7 V vs. Ag/AgCl caused the appearance of Cr (VI) in the passive film but Co was not increased. In the case of wear corrosion under anodic potentiodynamic polarization, the continuous sliding of the alumina ball on the HCCoCr surface did not allow the regeneration of the oxide film. Instead, an active state stimulated by polarization was induced on the surface where bulk material was directly exposed and detached to the electrolyte. Considering this situation, the results reveal that the anodic polarization on CoCr surfaces under wear-corrosion processes accelerated and induced the release of larger metallic particles with higher Co content coming from the base material.
2.2. Macrophage Cell Response
Macrophages are a primary immune cell type and the main cellular type involved in inflammatory processes [1
] and in host response [28
], so their biologic host response to wear particles generated from the implanted materials is of great interest.
Macrophage response to wear particles derived from the tribocorrosion assays was evaluated by measuring the effect on cell toxicity and respiratory activity.
Cytotoxicity induced by HCCoCr wear particles was analyzed by measuring LDH activity released from cells (Figure 6
), whose levels increase upon plasma membrane damage, a sign of cell death [29
]. As is shown in Figure 6
, exposure of macrophages cultures to wear particles induced a degree of cytotoxicity that was mainly dependent on the conditions used during wear-corrosion assays and particle concentration. As shown in Figure 6
panel A, particles concentration of 0.5 mg/mL obtained in PBS produced almost 58% cytotoxicity, a percentage that was significantly reduced to almost 12% when wear particles were generated from tribocorrosion tests in the presence of 3 g/L of hyaluronic acid (PBS-HA), an effect that could indicate a protective role of the hyaluronic acid on the metallic surface under wear stress conditions (Table 3
). Concentrations of 1 mg/mL of wear particles from the PBS test produced an increase in the macrophage cytotoxicity to almost 75%, a value elevated in comparison with the cytotoxicity induced by the wear particles obtained in PBS containing 3 g/L of HA, where cytotoxicity reached 14% (data not shown). No additional increase in the cytotoxicity was observed at higher concentrations of wear particles (2 mg/mL) generated in PBS as macrophages cytotoxicity appeared comparable to the one elicited by exposure to lower concentrations of particles (0.5 and 1 mg/mL), where approximately a 64% cytotoxicity was detected (data not shown).
Particles produced in PBS containing 3 g/L of HA at concentrations of 2 mg/mL elicited an increase in the macrophages cytotoxicity that reached almost 46% (Figure 6
, panel B, PBS-HA). Although such an increase was higher than the one produced by particles concentrations of 0.5 and 1 mg/mL, which were 12% and 14%, respectively, it was reduced to 24% when polarization conditions characteristic of damaged tissue were applied (Figure 6
, panel B, PBS-HA+POL). Although the statistical analysis of the data from Figure 6
panel B (Table 3
) gave no significant differences between results analyzed here, the application of anodic polarization to HA aqueous solution seems to have important observable differences on the mean value of the cytotoxicity. This feature could be relevant and, for this reason, verification by other biocompatibility assays is required. With this purpose, the wear particles collected from the tribocorrosion assays of CoCr alloy in PBS-HA without and applying anodic polarization were tested on macrophages cultures by measuring the mitochondrial activity. It is well known that the mitochondrial activity measurement is directly proportional to the number of metabolically active cells in culture [29
] constituting a measure of cell viability and biocompatibility. As it is shown with Figure 7
by white bars, wear particles collected in the PBS-HA produced a gradual and significant reduction in the mitochondrial respiratory response of macrophages. This result seemed to be directly related to the concentration of particles to which macrophages were exposed (Table 4
). Nevertheless, no reduction in the mitochondrial respiratory activity was observed in macrophages exposed to wear particles generated when polarization was applied during wear-corrosion tests. No significant effects in respiratory activity were observed in the range of particles concentrations tested (Figure 7
, black dotted bars, and Table 5
). The results suggest that the polarization conditions in the wear-corrosion assays in PBS containing HA at the approximate concentration found in synovial fluid seem to be beneficial to macrophage viability and biocompatibility.
The dose-dependence effect on mitochondrial respiratory activity by particles detached in PBS-HA could be explained by the chemical composition of wear particles collected from wear-corrosion tests in this solution as a decrease in Co was observed, as well as an enrichment in chromium oxide, a compound with high toxicity [30
] in comparison with the composition of PBS-HA-POL wear particles. The results suggest that polarization conditions applied to an HA aqueous solution at the approximate concentration found in synovial fluid produce changes in material tribocorrosion behavior inducing wear particles that seem to be beneficial to macrophage viability and biocompatibility. Data that could explain the higher biocompatibility of wear particles generated in PBS-HA+POL could be related to the fact that under these conditions, wear processes did not allow the regeneration of the oxide film. This event could determine the creation of an active state where CoCr base material was directly exposed to the electrolyte without enough time to build up the new oxide film that induced the release of metallic particles with higher Co content probably coming from the base material.