# Raman Spectroscopic Investigation of Osteoclastic Activity under the Influence of Bisphosphonate
## 3 Results  
Figure 2 shows the results of the TRAP staining. Figure 2a shows the macroscopic images of representative samples after testing, while Figure 2b,c show cells that were collected from a dentine substrate with Figure 2a and without Figure 2b RANKL. Both the samples with only RANKL and with RANKL and alendronate were affected by the TRAP staining, with a complete coverage of the substrate in the case of RANKL. Staining also affected the octacalcium phosphate control samples with undifferentiated RAW-264.7 cells, which shows a pale pink tint on about 50% of the overall surface. As expected, the samples treated with bisphosphonate do not show any pigmentation, due to its ability to reduce osteoclast activity by decreasing progenitor development and recruitment and by promoting osteoclast apoptosis [17]. Figure 2a,b show the presence of osteoclasts and macrophage RAW-264.7 cells on the sample with RANKL and the control, respectively. Another clear effect of the bone remodeling activity is the change of morphology of the surfaces after in vitro testing. Figure 3 shows laser microscopy scans of the full surfaces by stitching together 30 single images acquired at 5 magnifications, for each sample. On the surface of the control sample (Figure 3a), the machining marks are still clearly visible (almost vertical lines), but the surface appears to be coarse due to the presence of small, uniformly distributed porosities. Adding bisphosphonates to the medium strongly reduces the osteocl activity, as visible in Figure 3b. The machining marks are clear and regular, and no other porosities can be observed on the surface. When the medium contains RANKL, the amount of osteocl activity increases, as is clearly observable in Figure 3c: the surface appears to be coarser, and the machining marks are barely visible. In Figure 3d, where RANKL and bisphosphonates are combined together, the machine marks are visible, but at the same time the osteocl remodeling of the surface is more marked compared with  
Figure 2: TRAP staining results on the different samples, as observed by optical microscopy. Color contrast has been digitally enhanced using ImageJ software: (**a**) macro images of representative samples for the eight sample groups; (**b**) optical microscope of cells differentiated by RANKL; (**c**) optical microscope of cells from a control dentine sample without RANKL. Both (**b**,**c**) show cells collected from samples and deposited on glass substrate for optical analysis. Figure 4 shows higher magnification images of the sample's surfaces after in vitro testing. On three of the four surfaces (with the exception of the sample treated with just bisphosphonates in Figure 4b), there are clear morphological marks of the osteoclastic remodeling activity: the surface appears to be coarse and globular, with porosities of depth up to 10 microns, in particular in the case of the samples treated with RANKL, in Figure 4c. In Figure 4b, on the other hand, in presence of bisphosphonates, only the original marks caused by the sample machining are visible, and are still partially visible in Figure 4d when both bisphosphonates and RANKL are present at the same time. Figure 4: Images of the surface of the dentine substrates after in vitro testing. (**a**) dentine control, (**b**) dentine with bisphosphonate, (**c**) dentine with RANKL, (**d**) dentine with both phosphonate and RANKL. Figure 3: Low-magnification laser microscopy images of the surface of the samples. (**a**) dentine control, (**b**) dentine with bisphosphonate, (**c**) dentine with RANKL, (**d**) dentine with both phosphonate and RANKL, (**e**) octalum phosphate, (**f**) octacium phosphate with bisphosphonate, (**g**) octacalcium phosphate with RANKL and (**h**) octacalcium phosphate with both bisphosphonate and RANKL. Figure 5 shows the higher magnification images acquired on the surface of the octacalcium phosphate samples after in vitro testing. Unlike dentine, octacalcium phosphate substrates were porous and coarse.