# SEM and FT-MIR Analysis of Human Demineralized Dentin Matrix: An In Vitro Study
## 4 Discussion
The BMPs induced new bone formation through endochondral (indirect) and intramembranous (direct) ossification, as shown by the histological findings of [PERSON] et al. [30]. In our study, the dentin particles underwent a demineralization process with 12% EDTA. This agent is widely used in endodontics as a chelating agent for the enlargement of the canals and the removal of the smear layer. The longer the EDTA works on dentin, the more evident its effects are on dentin, as demonstrated by the release of phosphorus [38]. The specimens in our study were divided based on the exposure time of EDTA (0 min; 2 min; 5 min; 10 min; 60 min). The effect of the demineralizing agent was demonstrated by the analysis of the weight of the specimens, the FT-MIR analysis, and also SEM. The weight of each specimen was lower after the demineralization process. One of the most used methods to confirm demineralization is SEM [18]. The results of our SEM analysis showed that the longer the EDTA was allowed to work, the less rough, or smoother, the surfaces of the particles became. Furthermore, it was observed that the longer the duration of demineralization, the greater the number of denital tubules exposed on the same surface area of the particles. [PERSON] et al. [12] conducted an in vivo study comparing non-demineralized, partially demineralized, and completely demineralized dentin specimens. Similarly to our study, the SEM analysis showed that the surfaces of the demineralized dentin specimens were smoother while those of the non-demineralized dentin were rougher. Interestingly, the osteoblasts adhered only to the surfaces of demineralized dentin but not to those of non-demineralized dentin. The authors hypothesized that the exposure of collagen fibers following demineralization could promote the adhesion of osteoblasts. The specimens of partially and completely demineralized dentin matrix, above all, showed greater osteogenic power than the non-demineralized ones. Similar SEM results were achieved by [PERSON] et al. [18] performed a SEM analysis of dentin particles, reaching results similar to those of our study. The specimens of demineralized dentin showed greater exposure of the dentinal tubules and smoother surfaces compared to the non-demineralized specimens. Furthermore, the surfaces of the demineralized dentin particles demonstrated better suitability for cell proliferation (human dental pulp stem cells) than the surfaces of the non-demineralized particles. The authors also reported that the surfaces of the demineralized dentin particles were less biocompatible than those of the deproteinized dentin particles. It was suggested that these results were due to the demineralization process, during which a certain amount of the proteins may have been denatured. The authors also assumed that if the release of proteins following the demineralization process exceeds a certain threshold, it could be unfavorable, resulting in lethality for the cells. The FT-MIR analysis was performed to investigate the functional groups on the surfaces of dentin particles [18]. This analysis offered information regarding the degree of demineralization for each specimen. The results of our study showed that the inorganic mineral component (CO\({}_{3}\)\({}^{2-}\) and PO\({}_{4}\)\({}^{3-}\)) content decreased with increasing duration of exposure to 12% EDTA. In contrast, the protein component remained unchanged. The reference spectra values were considered on the basis of findings from previous investigations [19; 20; 21]. Clearly, demineralization is effective but within a certain range. If the dentin is poorly demineralized, it results in a poorly osteoinductive scaffold. Nevertheless, if the demineralization is excessive, the substrate becomes ineffective. Therefore, an appropriate balance must be achieved. The demineralized dentine particles have a porous structure due to the presence of the dentinal tubules. This means that the dentin particles may act as an osteoconductive scaffold, which allows cells proliferation.