The DPSCs were isolated from healthy (n = 3) and expanded dental pulps for the (I) characterization of the mesenchymal phenotype (immunophenotyping, proliferation curve and folding time, cell viability, senescence, colony formation, electron microscopy (II) expression profile of collagen type I / Col I, MMPs / TIMPs / RECK by immunofluorescence (IF) in the undifferentiated DPSCs, (III) characterization of biomarkers of osteogenesis after induction (ATP-4, FRA-1, SOST, ORP150, Connexin 43 and E11 / gp38 gene expression, RUNX2, BSP, ATF-4, And formation of nodules of mineralization by red alizarin); (IV) gene and protein expression profile of MMPs / TIMPs / RECK after induction of in vitro differentiation for 35 days; (V) protein expression profile of MMP-2 and MMP-14 after induction of in vitro differentiation by 35 days by western blot; (VI) evaluation of the interaction of DPSCs seeded on collagen and collagen / chitosan blends and osteogenesis after induction of differentiation in vitro by 35 days by scanning electron microscopy; (VII) evaluation of bone ectopic formation of DPSCs seeded on collagen / chitosan blends, pre-differentiated for 7 days in vitro and implanted in subcutaneous nude mice for 42 days by histological analysis; And (VIII) immunohistochemical evaluation of TIMPs during ectopic bone formation in vivo. DPSCs showed positivity for cell surface markers characteristic of MSCs, high proliferative rate, cellular metabolism appropriate to their cellular status, low density clonogenic potential, cytoplasm containing elongated mitochondria and rough endoplasmic reticulum as well as defined nuclei and capacity for adipogenic differentiation, But with some differences. Col I, some MMPs, all TIMPs and RECKs were expressed in the undifferentiated DPSCs. During the induction of osteogenesis, we verified that there was peak alkaline phosphatase activity in 14 days as well as the beginning of formation of mineralization nodules between 7-21 days. On the other hand, the levels of transcripts for the genes related to the involvement with the osteoblastic lineage (RUNX2), and in the osteoblast / osteocyte transition were quite different between the DPSCs of different patients, potentially reflecting different stages of differentiation or composition of stem cells since we worked With heterogenous populations of DPSCs. In the same way, there was a variation in the MMPs / Inhibitors gene expression as a function of the DPSC evaluated. Overall, most MMPs / Inhibitors were upregulated during induction of osteoblastic differentiation relative to undifferentiated DPSCs and MMPs -9, -19 and -25 were not expressed. There was a relationship between the profile of the osteoblastic differentiation markers and the expression of the MMPs / Inhibitors, where we verified 3 different profiles. Protein levels of MMPs -2 and -14 were higher than those of DPSCs from 28 to 35 days, periods related to the mineralization phase. The histological analysis of the ectopic bone formation revealed, in the experimental groups, the blende associated with the pre-differentiated DPSCs can be degraded and remodeled in a time-dependent manner and is able to form an osteoid matrix of 28 to 42 days. By immunohistochemical analysis, TIMPs were widely expressed by mesenchymal cells. Thus, we suggest that MMPs / TIMPs / RECK may play important roles in maintaining the undifferentiated state of DPSCs as well as acting at different stages of osteoblastic differentiation and in vitro mineralization. In addition, we suggest that the blends seeded with DPSCs may stimulate ectopic bone formation, therefore, being osteogenic and that TIMPs may actively participate in the tissue remodeling processes involved.
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