# Raman Spectroscopic Investigation of Osteoclastic Activity under the Influence of Bisphosphonate
## 4 Discussion  
In this study, we explored the spectroscopic fingerprints of osteoclast differentiation and activity in presence of RANKL and bisphosphonates.  
Results from microscopic investigations indicated that remodeling have happened on five of the eight different substrates, while only four of the eight samples showed signs of osteocl differentiation by TRAP (Figure 2) and by the presence of the Raman fingerprint band at about 1375 cm\({}^{-1}\) (Figure 8).  
The limited remodeling observed on the dentine and octacalcium phosphate controls can be associated with the action of the macrophage RAW-264.7 cells [34]. In vivo, this specific remodeling action is usually associated with bone tissue inflammation [35]. It has been previously suggested that macrophages can in particular degrade the organic bone matrix [34], which would explain why the octacalcium phosphate controls did not show clear signs of remodeling.  
Bone remodeling caused by macrophages can also be a result of various foreign body reactions [36], even if the in vitro simplified culture environment does not reflect the complexity of a real in vivo situation [37].  
The remarkable difference between the Raman spectra of Figures 8b and 8d constitutes a spectroscopic clue for cellular differentiation: when the spectra are dominated by the relatively strong amides signals from the substrate (dentine), spectroscopic differences between the four substrates are limited to the intensity of the signal. This suggests that RANKL and bi-phosphonate do not affect the substrates directly. Clear differences rise when the octacalcium phosphate substrates are exposed to RANKL: new bands appear at various Raman shifts, in particular an intense and sharp band at about 1375 cm\({}^{-1}\).  
As the Raman signals in these regions for octacalcium phosphates substrates are generated only by the cells on their surface, we can label these intense bands as spectroscopic fingerprints for cellular differentiation into osteoclasts.  
Various biomolecules would feature bands at similar Raman shifts, but between them, thymine and thymidine are the most consistent. Thanks to their double-ring structure [38], all purines have a relatively strong Raman intensity. Nevertheless, the signal rising at  
Figure 13: PCA analysis for the amide region of the octacalcium phosphate samples, PC1 related to the bandwidth of the 960 cm\({}^{-1}\) phosphate vibration and PC2 related to the intensity of the phenylalanine band at about 1004 cm\({}^{-1}\).  
about 1375 cm\({}^{-1}\) is a specific marker for thymine, thymidine and their complexes [39; 40], associated with the 8 CH\({}_{3}\) vibrational mode [41]. Between all the possible complexes, thymidine phosphorylase is associated with the production and proliferation of osteoclasts in in vitro models [42]. Previous authors suggested that thymidine phosphorylase induces the production of significant numbers of osteoclasts [43], and its relationship with RANKL has already been explored in the previous literature [44]: when osteoclasts resorb bone matrix, thymidine phosphorylase is released [45].