# Micro-Raman Spectroscopy of Dental Implants Subjected to Different Surface Treatments
## 2 Materials and Methods  
To study the presence and the transformation of TiO\({}_{2}\) at the implant surface at different manufacturing stages, experimental Ti-6 Al-4V implants were produced by FMD Medical Devices (Rome, Italy). All the samples were obtained from the same titanium alloy lot and underwent the same manufacturing procedures, like those used for commercial Ti dental implants, excepting from those characterizing the surface. In this study, samples consisted of screw-form implants measuring 5.5 mm in diameter and 14 mm in length. A couple of implants for each surface treatment were examined. The basic common procedures consisted of: 1) machining (milling) of the initial rod in the presence of a lubricant; 2) ultrasonication bath with solvents, and 3) packaging in glass tubes under laminar flow hood and dry heat sterilization at 170 \({}^{\circ}\)C for 3 h.  
The surface treatments are listed in Table 2. Surface treatment procedures consisted of: Al\({}_{2}\)O\({}_{3}\) sandblasting with particles with an average size of 180-200 \(\upmu\)m; etching with a HNO\({}_{3}\) (64.5%) and HF (0.5%) solution for 15 s; direct current anodization at 60 V for 15 s; etching with a HNO\({}_{3}\) (65%) solution for 15 s; low-pressure plasma (LPP) treatment with Ar gas for a period of 20 min at 0.35 mbar with an Atto Plasma Cleaner with integrated PC control (Diener electronic GmbH, Ephausen, Germany). A randomly selected site in the apical and in the collar zone was analyzed by RS for each of the implants tested. Spectra were considered identical when the Raman shifts of the peaks appeared at the same wavenumber, and the relative intensities of the major peaks demonstrated a less than 20% height difference [36]. Micro-Raman measures were conducted at room temperature. A LabRAM Dilor spectrometer (HORIBA, Japan) and an Olympus microscope HS BX40 (Shinjuku Monolith, Japan) were used for measurements. A He-Ne laser with a wavelength of 632.8 nm was employed to produce the excitation radiation. The implants were mounted on a motorized xy stage and tested with a laser spot of about \begin{table}
\begin{tabular}{c c c c} \hline \hline
**Code** & **Surface Treatment** & **Code** & **Surface Treatment** \\ \hline N1 & \begin{tabular}{c} Machining + ultrasonic cleaner + packaging \\ and sterilization \\ \end{tabular} & N1A & N1 + Plasma \\ \hline N2 & \begin{tabular}{c} Machining + Al\({}_{2}\)O\({}_{3}\) blasting + ultrasonic \\ cleaner + packaging and sterilization \\ \end{tabular} & N2A & N2 + Plasma \\ \hline N3 & \begin{tabular}{c} Machining + ultrasonic cleaner + HNO\({}_{3}\)\(\backslash\)HF \\ etching + packaging and sterilization \\ \end{tabular} & N3A & N3 + Plasma \\ \hline N4 & \
\begin{tabular}{c} Machining + Al\({}_{2}\)O\({}_{3}\) blasting + ultrasonic \\ cleaner + HNO\({}_{3}\)\(\backslash\)HF etching + packaging and sterilization \\ \end{tabular} & N4A & N4 +Plasma \\ \hline N5 &
\begin{tabular}{c} Machining + Al\({}_{2}\)O\({}_{3}\) blasting + ultrasonic \\ cleaner + HNO\({}_{3}\)\(\backslash\)HF etching + anodization + HNO\({}_{3}\) etching + packaging and sterilization \\ \end{tabular} & N5A & N5 + Plasma \\ \hline \hline \end{tabular}
\end{table} 
Table 2: List of the surface treatments examined. 2 \(\upmu\)m in diameter and with a 50x objective. In this case the numerical aperture was NA = 0.95 with a focal depth at the minimum confocal hole width equal to 10 microns in transparent materials. A long working distance objective was also used to focus the light on the valleys between the threads of the screw. The spectral resolution was about 1 cm\({}^{-1}\). Neutral filters with different optical density irradiated the samples at different light intensities leading to power density values from \(5\times 10^{3}\) W/cm\({}^{2}\) to \(5\times 10^{5}\) W/cm\({}^{2}\). As a detector, a cooled CCD camera was used, with typical integration times of about 2 min. Attention was paid to the acquisition of background signals in order to perform reliable background subtractions for each sample.