# SEM and FT-MIR Analysis of Human Demineralized Dentin Matrix: An In Vitro Study
## 4 Discussion
In addition, the adhesion of these cells could prevent the absorption of DDM particles and start the formation of new bone. For these reasons, in our study, demineralized dentin particles ranging in size from 300 \(\upmu\)m to 1200 \(\upmu\)m were used, excluding the smaller-sized particles (<300 \(\upmu\)m). Many studies have reported different tooth processing methods. Generally, there are four main categories [17]: (i) extraction of non-collagen proteins from dentin; (ii) demineralization; (iii) elimination of the organic matrix (denaturation); (iv) use of tooth particles without modification. Denaturation is a little-used method because it eliminates the proteins in the matrix, including the growth factors responsible for the osteoinductive capacity of the dentin itself. Currently, the most used protocol is demineralization, as demonstrated by a large number of studies in the literature. The most used demineralizing agents are: (i) EDTA [17; 24; 27]; (ii) HNO\({}_{3}\)[12; 27]; and (iii) HCl [28; 29; 30]. Demineralization does not affect the organic component of the dentin or damage the growth factors contained therein. This process increases the osteoinductivity of the dentin particles, since it promotes the release of growth factors [11], favors the adhesion of osteoblasts through the exposure of collagen fibers [12], and reduces dentin's antigenicity. Demineralization is necessary because crystalline hydroxyapatite inhibits the release of growth factors, such as BMPs [11]. It has been observed that the amount of time during which a demineralizing agent acts influences the characteristics of dentin. An excessive demineralization can damage the dentin structure and adversely affect the composition and function of odontogenic factors. On the other hand, a mild demineralization produces a scaffold with poor osteoinductive capabilities [18]. [PERSON] et al. [27] performed an FIB/SEM analysis of the demineralized (HNO\({}_{3}\) 2%) dentin matrix grafted in a rat calvaria bone defect model. This method allowed the 3D reconstruction of the interface between the implanted dentin particles and the surrounding bone. The diameters of the exposed dental tubules averaged 3 \(\upmu\)m. Mesenchymal cells, such as osteoblasts and bone tissue cells, are between 10-20 \(\upmu\)m in size, while osteoclasts are 20-100 \(\upmu\)m. The results of this study showed that osteocytes surrounded the grafted dentin particles, forming a network on their surfaces. In addition, cytoplasmatic extensions of osteocytes were observed in the dental tubules contained within the dentin particles. The authors also hypothesized a possible biological sequence of events that occurs when demineralized dentin particles are grafted into the recipient site. The release of BMPs induces mesenchymal cells to differentiate into osteoblasts, as also shown by the immunohistochemical analysis of [PERSON] et al. [31]. These cells produce a matrix that undergoes mineralization and forms new bone. At this point, the osteoblasts differentiate into osteocytes, which adhere to the surface of the demineralized dentin particles, forming a network on their surface. Cytoplasmic extensions from the network spread into the denital tubules contained within the dentin particles. Afterward, the dentin particles will be reabsorbed and replaced by new bone, as confirmed by [PERSON] et al. [13]. BMPs belong to the large family of TGF-\(\beta\). [PERSON] [32] was the first to describe their biological activity. In the following years, considerable efforts were made to isolate these growth factors and study both their in vitro and in vivo features. The BMPs promote the differentiation of mesenchymal cells into osteoblasts and chondroblasts [33], participating in the development of bone and cartilage [34], rather than the formation of dental hard tissues [35]. [PERSON] et al. [36,37] showed that BMPs obtained from demineralized dentin have osteoinductive properties similar to those derived from bone tissue.