# SEM and FT-MIR Analysis of Human Demineralized Dentin Matrix: An In Vitro Study
## 2 Materials and Methods  
### Experimental Design and Specim Preparation  
For this study, we used extracted teeth with unfavorable prognoses with the consent of patients. The criteria for exclusion were: (i) teeth with carious lesions; (ii) teeth with root canal treatment. After extraction of the specimens, the enamel and root cement were removed with a dental drill (Figure 1a). Next, each tooth was washed with physiological solution, rinsed with an air-spray tool, and stored at \(-20\)\({}^{\circ}\)C. The teeth were placed in a sterile container (Figure 1b) and ground using a Smart Dentin Grinder (KometaBio Inc., Cresskill, NJ, USA) for 3 s (Figure 1c). Then, the dental powder was sieved to distinguish two specimens with different grain sizes: the first with granules of smaller sizes (<300 \(\upmu\)m) (A); and the second with granules of sizes between 300 \(\upmu\)m and 1200 \(\upmu\)m (B). The specimens were collected in sterile containers (Figure 1d,e). In this study, we used only the second specimen (B), while the first was excluded (specimen A).  
Following the instructions of the machine producer, the dentinal powder was immersed in basic alcohol for 10 min in a sterile glass container. The basic alcohol was composed of 0.5 M NaOH and 20% ethanol. Next, the dentinal powder was picked up and washed two times with sterile phosphate-buffered saline solution (3 min for each wash) (Figure 1f). The dentinal powder was washed with a physiological solution and rinsed as much as possible.  
The dentinal powder was randomly divided into five groups (0.075 g of dentin each) based on the time of demineralization (Table 1): T2, 2 min; T5, 5 min; T10, 10 min; T60, 60 min. T0 was not-demineralized and was considered as the control group.  
12 %EDTA was used to demineralize the dentin. Specifically, 1.7 g EDTA disodium salt was dissolved in 10 mL distilled water at 25 \({}^{\circ}\)C. A saturated solution was obtained, from which only the supernatant was taken. Through this process, we obtained EDTA at 12%. The choice of this concentration was made according to previous articles published in the literature [12; 17]. An amount of 1.1 mL EDTA was applied for the established time using a specific tool (Vortex). Once the process was completed, EDTA was removed, and the specimens were stored at \(-20\) \({}^{\circ}\)C. Each specimen was washed two times with a physiologic solution (400 \(\upmu\)L) to stop the demineralizing process. Next, the physiologic solution was aspirated.  
The water had to be removed from the specimens to allow the spectrophotometry analysis. This process was completed by putting the specimens in a centrifuge (SpeedVac Concentrator, Savant SPD111V) (Figure 2a) for 30 min. The specimens were also weighed at different times: (i) ground and centrifuged dentin, before the demineralization process (D1); (ii) ground and demineralized dentin (D2); (iii) ground and centrifuged dentin after demineralization process (D3).  
\begin{table}
\begin{tabular}{c c} \hline \hline
**Nomenclature** & **Demineralization Process** \\ \hline T0 & not-demineralized \\ T2 & 2 min in 12\% EDTA dentin particles \\ T5 & 5 min in 12\% EDTA dentin particles \\ T10 & 10 min in 12\% EDTA dentin particles \\ T60 & 60 min in 12\% EDTA dentin particles \\ \hline \end{tabular}
\end{table}
Table 1: Group nomenclature based on demineralization process.  
Figure 1: Specim preparation: (**a**) Example of the final specimens after enamel and cementum removal with a dental drill; (**b**) specimens in the sterile trituration chamber; (**c**) detail of Smart Dentin Grinder machine (KometaBio Inc., Cresskill, NJ, USA); (**d**) ground dentin with particle size less than 300 \(\upmu\)m; (**e**) ground dentin with particle size between 300 \(\upmu\)m and 1200 \(\upmu\)m; (**f**) dentin particulate immersed in a sterile container with sterile saline solution.