# SEM and FT-MIR Analysis of Human Demineralized Dentin Matrix: An In Vitro Study
## 3 Results
### FT-MIR  
MIR spectra were acquired for each specimen in the 4000-600 cm\({}^{-1}\) spectral range, and NIR in the 10000-4000 cm\({}^{-1}\) range. The main MIR absorption bands considered in this study are shown in Figure 3, which shows the spectrum of the dentin not subjected to demineralization (T0). Based on previous studies [19; 20; 21], the main absorption bands analyzed in this study were: (i) \(\sim\)1649 cm\({}^{-1}\) band corresponding to the protein component of dentin (band A); (ii) \(\sim\)1021 cm\({}^{-1}\) band corresponding to the phosphate ion PO\({}_{4}\)\({}^{3-}\) (band B); (iii) \(\sim\)872 cm\({}^{-1}\) band corresponding to the stretching of the carbonate CO\({}_{3}\)\({}^{2-}\) (band C).  
\begin{table}
\begin{tabular}{c c c c c} \hline \hline
**Specimen** & **T2** & **T5** & **T10** & **T60** \\ \hline D1 & 0.075 g & 0.075 g & 0.075 g & 0.075 g \\ D2 & 0.093 g & 0.081 g & 0.051 g & 0.024 g \\ D3 & 0.061 g & 0.057 g & 0.034 g & 0.014 g \\ \hline \hline \end{tabular}
\end{table}
Table 2: The weights of the specimens (expressed in g) in D1 (triturated and centrifuged dentin), D2 (triturated and demineralized dentin), and D3 (triturated, demineralized, and centrifuged dentin).  
Figure 2: Instruments used in this study. (**a**) SpeedVac Concentrator, Savant SPD111V was used for the elimination of residual water; (**b**) FT-IR Perkin Elmer Spectrum One. Acquisition range 4000–450 cm\({}^{-1}\); (**c**) scanning electron microscope (Supra 40, Zeiss).  
The variations in the MIR spectral profiles of the analyzed specimens (T0, T2, T5, T10, T60) were acquired in the spectral range 4000-600 cm\({}^{-1}\). Then, the spectra were normalized compared to the 1649 cm\({}^{-1}\) band, which corresponded to the protein component of dentin, and which remained constant even after the demineralization treatment. Through this procedure, it was possible to observe the trends of the PO4\({}^{3-}\) and CO3\({}^{2-}\) groups (Figure 4).  
The concentrations of the inorganic components of dentin (PO4\({}^{3-}\) and CO3\({}^{2-}\) groups) decreased with increasing exposure time to the demineralizing agent. The amount of the mineralized component of the dentin was greater in T0, followed by T2, T5, T10. The T60 specimens were subject to the longest time of demineralization and showed the least amount of mineralized component.  
The ratio between band B (1021 cm\({}^{-1}\)), which described the percentage of PO4\({}^{3-}\), and band A (1649 cm\({}^{-1}\)), which corresponded to the protein component, was measured to determine the percentage of mineralization of each specimen (Figure 5a). Band C (872 cm\({}^{-1}\)), which reflected the CO3\({}^{2-}\) percentage, was also compared with band A (1649 cm\({}^{-1}\)), which corresponded to the protein component (Figure 5b).  
Figure 4: Spectra of the analyzed specimens normalized at the level of the 1649 cm\({}^{-1}\) band, which corresponded to the protein component of dentin. Reductions were observed in the band at 1021 cm\({}^{-1}\), which resembled the phosphate group, and at 872 cm\({}^{-1}\), which corresponded to the carbonate group.  
Figure 3: Spectrum acquired through FT-MIR analysis of dentin particles not subjected to demineralization with 12% EDTA.  
At T0, dentin was not treated with 12% EDTA, so it was considered as the control group, assuming 100% mineralization. Next, we calculated the quantity of PO4\({}^{3-}\) and CO3\({}^{2-}\) as a percentage of each specimen (Figure 6).