# Raman Spectroscopic Investigation of Osteoclastic Activity under the Influence of Bisphosphonate
## 3 Results
The average Raman spectra collected on the surface of the various samples are presented in Figure 8. For dentine substrates (Figure 8a,b), both the hydroxyapatite region (Figure 8a) and the amide region (Figure 8b) are in line with previous literature references, in particular showing the presence of PO4\({}^{3-}\)[25], proline and hydroxyproline [25], tyrosine [26], HPO4[27] and various amide-related vibrations confirming the presence of a well-developed collagenous matrix [28]. Due to the overlapping between vibration frequencies from cells and collagenous tissue of the substrate, no clear spectroscopic confirmation of the presence of osteoclasts can be obtained from these spectral windows. When compared to dentine, the spectra of the hydroxyapatite region for the octacaccium phosphate samples are simpler, with one main peak between 950 and 1000 cm\({}^{-1}\) and a few, barely observable smaller bands at higher Raman shifts (Figure 8c). The vibration band related to PO4\({}^{3-}\) appears to be more complex, with a clear shoulder at about 1000 cm\({}^{-1}\) that intensifies on the samples treated with RANKL. This intensity variation is caused by the preferential dissolution action of osteoclasts towards hydroxyapatite than towards octacaccium phosphates, as previously observed in Figure 6b. The amides region of Figure 8d features numerous bands that were barely visible or even completely absent in Figure 8b, due to the simultaneous presence of both collagen and osteoclasts. A few distinctive spectroscopic features appear only in the control sample and in the samples treated with RANKL, in particular between 1240 cm\({}^{-1}\) and 1280 cm\({}^{-1}\), between 1350 cm\({}^{-1}\) and 1380 cm\({}^{-1}\) around 1450 cm\({}^{-1}\) and again at about 1630 cm\({}^{-1}\). Some of these features were previously reported in the literature for osteoclasts observed by Raman spectroscopy [29] and represent possible spectroscopic markers for cell differentiation into fully grown osteoclasts [30]. The intense band at about 1370 cm\({}^{-1}\) has been previously associated with the presence of tryptophan [31], which is also known for playing a role in bone remodeling and osteocl differentiation; however, considering the band position  
Figure 8: Average Raman spectra collected on the surface of the samples. (**a**) Dentine hydroxyapatite region, (**b**) dentine amides region, (**c**) octacaccium phosphate hydroxyapatite region and (**d**) octacaccium phosphate amides region. and relative intensity, it is most likely due to the presence of thymidine [32], produced by cell precursors. Figure 9 shows the Raman imaging map acquired on the octacalcium phosphate substrate in presence of both RANKL and bisphosphonates. The blue color, associated with the presence of amides, marks the locations of not differentiated RAW cells. Green, associated with the intensity of the band located at 1370 cm\({}^{-1}\), marks the location of the differentiated osteoclasts. Red marks the presence of the octacalcium phosphate substrate by showing the distribution of the intensity of the band at about 960 cm\({}^{-1}\) due to PO\({}_{4}\)\({}^{3-}\) vibrations. It can be observed that green areas are always overlapping to blue (due to the presence of amides in both differentiated and not differentiated cells), but blue areas cover most of the surface. This result suggests that most of the RAW cells were differentiated but with an uneven coverage of the substrate. Raman imaging results were limited by the low signal-to-noise ratio and by the overlapping between signals originating from different molecular species. Principal component analysis was applied to the imaging after dividing them into 100 subregions. Results for dentine substrates, in the spectral region around 1000 cm\({}^{-1}\), are shown in Figure 10.  
Figure 10 shows the principal component analysis for the dentine samples in the apatite region.