*3.3. Characterization Methods*

X-ray diffraction (XRD) analyses were carried out on a Bruker AXS D8 advance diffractometer (Bruker, Karlsruhe, Germany) using the Bragg–Brentano configuration. The excitation of the tube was fixed at 30 KV and 20 mA, the slit was fixed at 0.6 mm, and the probe size was around 6 × 12 mm. A position-sensitive detector (LynxEye, Bruker) was used to collect the data between 10◦ and 70◦ (2θ), with a 0.015◦ step size and a 0.4 s/step acquisition speed. With this configuration, and considering the peaks of interest (the (−111) and (111) monoclinic peaks and the (101) tetragonal peak, respectively, located around 28.3◦, 31.5◦, and 30.1◦), 90% of the XRD signal comes from the first 17 μm below the surface. The monoclinic fraction was determined from the integrated intensities of the XRD peaks after subtracting a linear baseline, using Garvie and Nicholson's equation [30].

Raman spectra were collected with a single spectrograph (Horiba Jobin Yvon LabRAM HR800) with a grating of 1800 gr/mm and Ar+ laser excitation at 514.5 nm wavelength. The laser power on the sample was maintained at ~2 mW with a 100× long-working distance objective to avoid excessive laser-induced heating. With the chosen optical configuration, the laser had a lateral resolution of ~1 μm and a penetration depth of 4.2 μm or 15 μm when the confocal pinhole was fixed at 100 μm or 1000 μm, respectively (intended as the depth from which 90% of the signal comes from). These values were determined following the procedure outlined in Pezzotti et al. [31]. On each specimen, three adjacent points (>10 μm apart) were measured. The collected spectra were fitted with Gaussian–Lorentzian functions after subtracting a linear baseline; the integrated intensity values of monoclinic and tetragonal peaks were used to calculate Vm with both the Clarke/Adar and Katagiri equations and compared with the results of the XRD analyses. An example of the fitted spectrum after baseline subtraction is shown in Figure 1.

**Figure 1.** Example of a fitted Raman spectrum of Delta using the procedure followed in this paper. The spectrum was taken on a head specimen in a region with a high monoclinic fraction.

#### **4. Results**

#### *4.1. In Vitro Aging Study*

Figure 2 and Table 1 present the results of Vm measurements by Raman spectroscopy carried out on Delta femoral heads and inserts, both as received and after the aging procedure. The values of Vm were calculated from the integrated intensity of peaks belonging to the monoclinic and tetragonal phase after the fitting procedure described in Section 3. As it can clearly be seen, the values obtained with the through-focus configuration are smaller than those obtained with the confocal one; in the latter case, due to the smaller probe depth, the volume closer to the surface of the sample was analyzed. Hence, this result shows that the monoclinic fraction is higher in the vicinity of sample surfaces. Aged samples reveal a higher monoclinic content (up to a factor of 2 and higher), as expected, and the difference from the pristine state is larger near the sample surface. Moreover, inserts have a higher monoclinic content due to the raw (grinded backside) surface finish.

**Figure 2.** Values of Vm measured by Raman spectroscopy and XRD on both non-aged and in vitro aged Delta heads (**a**) and inserts (**b**). Conf. = confocal pinhole closed down to 100 μm (penetration depth of Raman signal: 4.2 μm); Th. F. = confocal pinhole fully open (1000 μm)—penetration depth of Raman signal: 15 μm.

Comparing values obtained with the Clarke/Adar and Katagiri equations, it is evident that a higher monoclinic content results from the Katagiri equation. This is in line with the findings of Tabares and Anglada [24], who concluded that the Clarke/Adar formula underestimated the monoclinic content for powders. However, a direct comparison between the through-focus Raman results and the XRD results (which have a very similar penetration depth of ~15 μm and 17 μm, respectively) shows that, indeed, it is the

Clarke/Adar equation that provides the best correspondence with the XRD measurements. This is valid on both sample types and for both pristine and aged specimens. This is more evident from Figure 3, where a direct comparison between Vm by XRD and Raman is provided for all samples both in the (a) confocal and (b) through-focus configurations. In Figure 3a, both equations overestimate the Vm by Raman, and this is due to the difference in the volume probed by the two techniques (with confocal Raman, the probe depth is much smaller). For the through-focus case (Figure 3b), where a direct comparison between Raman and XRD is more pertinent due to the very similar penetration depth, the Katagiri equation clearly overestimates (by a factor of 2.5) the monoclinic content, whereas the Clarke/Adar equation provides only a slightly lower Vm than XRD. This latter equation seems thus more suitable for the determination of Vm in the case of aged femoral heads, where the monoclinic content is not constant over the probed depth, keeping in mind that the obtained value is then a weighted average over 15 μm under the surface, as with Vm obtained by XRD.

**Table 1.** Values of Vm measured by Raman spectroscopy and XRD on both non-aged and in vitro aged Delta heads and inserts. Confocal Raman results belong to a depth up to ~4 μm below the surface (pinhole diameter: 100 μm). Through-focus Raman data correspond to a fully opened confocal pinhole (1000 μm) and thus encompass a depth of 15 μm. The penetration depth of XRD is 17 μm.


**Figure 3.** Comparison between all Vm values measured by XRD and Raman spectroscopy and on both non-aged and in vitro aged Delta heads and inserts. (**a**) Confocal = confocal pinhole closed down to 100 μm (penetration depth of Raman signal: 4.2 μm); (**b**) Through Focus = confocal pinhole fully open (1000 μm)—penetration depth of Raman signal: 15 μm. The Through Focus measurement mode (with the Clarke/Adar equation) best reproduces the XRD results due to the similar probed volume (penetration depth of XRD: 17 μm.
