# Raman Spectroscopic Investigation of Osteoclastic Activity under the Influence of Bisphosphonate
## 3 Results
The control sample of Figure 5a shows a limited increase in surface roughness when compared with the sample exposed to bisphosphonates in Figure 5b. The surface roughness further increases moving to the sample treated with RANKL, in Figure 5c, and with RANKL and bisphosphonates, in Figure 5d. The small, distributed porosities visible in Figure 5b are still partially observable on the control sample, but completely disappear due to the osteocl remodeling in presence of RANKL. As previously observed for dentine, the action of bisphosphonates decreases the amount of remodeling occurring in presence of RANKL, as clearly observable by comparing the roughness of Figure 4c,d. Figure 6 shows the diffraction patterns obtained on the dentine and octacalcium phosphate substrate as a function of the experimental conditions. It can be observed that the four spectra of Figure 6a, related to dentine, are similar to each other and feature the bands conventionally associated with hydroxyapatite crystals [22], the more intense being the band related to the \(\langle 211\rangle\) orientation, followed by the \(\langle 002\rangle\) and then the \(\langle 004\rangle\). Between the four spectra, the main differences appear to be the relative intensities of these three bands. For the octacalcium phosphate substrates of Figure 6b, another set of bands is clearly visible, resulting in a slightly different XRD pattern when compared with Figure 6a. Bands at 31, 34, 46 and 52\({}^{\circ}\) and related to tri-calcium phosphate [23; 24] show a maximum intensity for the sample tested with RANKL, indicating that the hydroxyapatite phase was completely dissolved by the osteocl activity. Interestingly, the further addiction of bisphosphonate reduces the intensity of the bands, but only by about 10%. Figure 7 shows the main spectroscopic information that could be extracted from the X-ray diffraction patterns of Figure 6, in particular, concerning the preferential orientation of the hydroxyapatite crystals and the phase ratio between hydroxyapatite and tri-calcium phosphate. On both the dentine (Figure 7a) and phosphate (Figure 7b) substrates, the ratio between the intensity of the \(\langle 004\rangle\) and \(\langle 211\rangle\) orientation peaks appears to be mainly influenced by the presence of RANKL, growing from \(\sim\)0.4 to \(\sim\)0.5 or even to \(\sim\)0.6 for dentine  
Figure 5: Images of the surface of the octacalcium phosphate substrates after in vitro testing. (**a**) octalcium phosphate, (**b**) octalcium phosphate with bisphosphonate, (**c**) octacalcium phosphate with RANKL and (**d**) octacalcium phosphate with both bisphosphonate and RANKL. and from \(\sim\)0.3 to \(\sim\)0.5 for octacalcium phosphate. No statistically significant difference could be observed in samples containing bisphosphonate. These results suggest that the osteoclastic activity is strongly promoted by RANKL and manifests with a preferential erosion of the \(\langle 211\rangle\)-oriented crystals when compared with the \(\langle 004\rangle\)-oriented crystals. Further evidence for the role of RANKL in enhancing the erosion rate of hydroxyapatite can be deducted from Figure 7c: the relative intensity of the diffraction bands related to octacalcium phosphate increase by 5-fold in the presence of RANKL. Bisphosphonate also plays a statistically relevant role, reducing the dissolution activity of osteoclasts by about 20%, when in presence of RANKL. Figure 6: X-ray diffraction patterns for the two different materials, at different conditions. (**a**) Dentine and (**b**) octacalcium phosphate. Peaks related to hydroxyapatite are marked in (**a**), while only the peaks related to octacalcium phosphate are marked in (**b**). Figure 7: Spectroscopic information extracted from the X-ray diffraction patterns of Figure 7: I\({}_{004}\)/I\({}_{211}\) intensity ratio for (**a**) dentine and (**b**) octacalcium phosphate and (**c**) I\({}_{\text{TCP}}\)/I\({}_{\text{HA}}\) ratio for octacalcium phosphate. * indicate \(p<0.05\).