# Raman Spectroscopic Investigation of Osteoclastic Activity under the Influence of Bisphosphonate
## 3 Results
The first two principal components that were identified by the software resulted to be due to the full-width half-maximum (FWHM) of the octacalcium phosphate band located at about 960 cm\({}^{-1}\) and due to the presence of the phenylalanine band at about 1004 cm\({}^{-1}\). It can be observed that, in the presence of osteocl activity, the width of the band reduces, reaching the minimum value for the samples exposed only to RANKL. This is due to the preferential osteocl erosion of the most defective apatite crystals, which exposes underlying material with higher degrees of crystallinity, which takes a longer time to be remodeled by osteoblasts [33]. The band width is the maximum for the samples exposed to bisphosphonates, and both the control and the samples exposed to both RANKL and bisphosphonate have an intermediate behavior, with a larger distribution on the PC1 axis. For PC2, as the remodeling proceeds, higher amounts of collagen tissues are exposed to the Raman probe, resulting in a higher intensity of the 1004 cm\({}^{-1}\) band. It is worth noticing that both the control and the sample exposed to RANKL and bisphosphonates appear to have a preferentially bi-modal scattering distribution on the PC2 axis. Figure 4:0 Raman imaging of the octacalcium phosphate substrate exposed to both RANKL and bisphosphonates. The red color identifies the phosphate groups, blue identifies the amides (I, II and III) and green identifies the band at 1370 cm\({}^{-1}\) associated with differentiated osteoclasts. Figure 11 shows the results obtained on the same spectral region for the octacalcium phosphate substrates. While the width of the phosphate band seems to follow a similar trend for PC1, the distribution is more scattered due to the presence of two different phases which are eroded at different rates but have similar vibration profiles for PO43- around 960 cm\({}^{-1}\). For the phenylalanine band at about 1004 cm\({}^{-1}\), as the only source for this signal is the cells (either undifferentiated RAW-264.7 or osteoclasts) on the surface of the samples, as there is no collagen in the octacalcium phosphate samples, the intensity of the band is scattered, and the contribution of the PC2 is limited to just 4.6%, making the variations in PC1 (crystallinity) the predominant effect. Figure 12 shows the results of the principal component analysis performed on the dentine substrates, in the spectral window for amides. The PC1 component is associated with the intensity of the amide III bands located at about 1250 cm\({}^{-1}\) (8(NH)) and 1280 cm\({}^{-1}\) (\(\alpha\)-helix), amide I bands at about 1615 cm\({}^{-1}\) (C=C) and 1660 cm\({}^{-1}\) (C=O), as well as 8(CH)-specific vibrations, located at about 1460 cm\({}^{-1}\) and 1320 cm\({}^{-1}\). Meanwhile, PC2 is related to the osteoclast differentiation fingerprint band located at about 1375 cm\({}^{-1}\). Due to the presence of amides for both the substrate and the cells, the contribution of PC1 is somehow limited, and the scattering is high in both PCs. In the case of PC2, in particular, the signal produced by the substrate is intense enough to cover most of the band, making the results less reliable. Figure 13 shows the results of the PCA analysis in the amide region of the octacalcium phosphate samples. Unlike the dentine substrates, in octacalcium phosphate, the only contribution for the amides is due to the presence of the osteoclasts. For this reason, the intensity of the PC2 band associated with differentiated cells is higher and the band itself is clearly observable on the spectra. The PC1 results on the intensity of the amide bands suggest that the osteoclasts have grown more on samples containing RANKL when compared with samples containing bisphosphonates and the control. This is in line with the size of the osteoclast cells, which are often referred to as \"giant cells\". PC2 confirms that the same trend is followed for the differentiation.