ODONTOBLASTS DERIVED FROM DOG ENDOMETRIAL STEM CELLS ENCAPSULATED IN FIBRIN GEL ASSOCIATED WITH BMP-2 FOR DENTIN REGENERATION

Dental hard tissues are known to be extremely important to be safeguarded, since their regeneration does not occur: in particular, enamel is a tissue without cells, and dentin regeneration often appears disorganized. The most advanced tissue engineering techniques, applied to regenerative treatment for dental tissue, include stem cell cultures that can be differentiated into target tissues on a three- dimensional network to provide a scaffold, which plays a crucial role in tissue engineering. Endometrial stem cells (EnSCs) isolated from canine endometrium have been identified as a source of mesenchymal stem cells (MSCs) characterized by a selfrenewal capability. Furthermore, growth factors play an essential role in cell proliferation and differentiation: bone morphologic protein-2 (BMP-2), which belongs to the transforming growth factor β (TGF- β) family, is fundamental in the growth and regeneration of skeletal tissues and has shown odontogenic and osteogenic properties, both in vitro and in vivo. Besides, BMP-2 stimulates the differentiation of dental pulp stem cells (DPSCs) into odontoblasts. This study aimed to solve dental pathologies utilizing tissue engineering methodologies. The study employed an in vitro model to analyze the proliferation and odontogenic differentiation of canine endometrial stem cells (C-EnSCs) isolated from the biopsy of the uterine endometrium. The dentin regeneration potential of odontoblast-like cells (OD) derived from CEnSCs was evaluated in rodent models. C-EnSCs were isolated using alizarin red staining in order to detect the differentiation into odontoblasts and the expression of two odontoblastic markers, the dentin sialophosphoprotein (DSPP) and dentin matrix protein-1 (DMP1) genes by qRT-PCR. Then, C-EnSCs were characterized via flow cytometry, using the conjugated antibodies to CD90, CD105, CD34, and CD45. These cells were encapsulated within fibrin gel supplemented with signaling molecules to establish proper conditions for cellular proliferation and differentiation. OD cells were combined with BMP-2 to stimulate dentin formation in vivo. The experimental model, employed for assessing the regenerative efficacy of cells and biomaterials, involved the preparation of the left maxillary first molar in twenty male Wistar rats, for direct pulp capping. The animals were divided into four cohorts: group 1 served as the control without treatment, group 2 received fibrin alone, group 3 received fibrin with ODs (fibrin/ODs), and group 4 received fibrin with ODs and BMP-2 (fibrin/ODs/BMP-2). SEM analysis Journal of Biological Research 2024; volume 97:(s1) assessed the organization of C-EnSCs attached and spread in fibrin hydrogel, appeared as a 3D open porous and interconnected porosity. Morphological examinations revealed the differentiation of C-EnSCs into ODs. Additionally, histomorphometric and histomorphological analysis of treated teeth, using hematoxylin-eosin staining, demonstrated that fibrin gel combined with BMP-2 at a concentration of 100 ng/mL provided an optimal microenvironment for dentin tissue regeneration in rodents. In conclusion, these results showed the potential utility of OD cells derived from CEnSCs encapsulated in fibrin gel supplemented with BMP-2 for direct pulp capping and dentin regeneration.