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Identification of pericytes in periodontal ligament and characterization as mesenchymal stem cells

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Mércia Jussara da Silva Cunha
Total Authors: 1
Document type: Doctoral Thesis
Press: Piracicaba, SP.
Institution: Universidade Estadual de Campinas (UNICAMP). Faculdade de Odontologia de Piracicaba
Defense date:
Examining board members:
Karina Gonzales Silverio; Bruna Rabelo Amorim; Ruthinéia Diógenes Alves Uchôa Lins; Denise Carleto Andia; Francisco Humberto Nociti Junior
Advisor: Karina Gonzales Silverio

It is not clear in literature that the periodontal ligament cells (PDL), characterized as mesenchymal stem cells (MSC), correspond to the same cell populations identified as pericytes. Moreover, there is no evidence regarding to these cell distribution in healthy periodontal tissues and in tissues damaged by chronic inflammatory periodontal disease (DPIC). In this way, the aims of the present study were: 1) to isolate and to characterize phenotypically populations of pericytes from human periodontal ligament, and 2) to evaluate the distribution of pericytes/MSC in healthy periodontal tissues and in tissues damaged by DPIC. For this purpose, populations of pericytes (CD146+ cells) were purified as from three cell populations of PDL and the positive and negative portions were characterized about: 1) undifferentiated mesenchymal phenotype (expression of markers CD105, CD166, STRO-1, and OCT-4), and about endothelial/hematopoietic phenotype (expression of markers CD34 and CD45) using flow cytometry, 2) expression of OCT4/POU5F1 by immunocytochemistry, and 3) potential to differentiate into osteoblastic phenotype (mineralization assays by Alizarin Red and gene expression of RUNX2, ALP, and OCN by PCRq technique) and adipogenic phenotype (Oil Red O assay and gene expression of PPAR?2 and LPL). In order to determine the distribution pattern of cells CD146+, CD166+, and STRO-1+ on periodontal tissues, it was used an in vivo model of DPIC experimental induction, placing subgingival cotton ligatures on first lower molar of 20 male Wistar rats. The contralateral molar was used as control (healthy). After 3 and 14 days, animals were sacrificed, its jaws were processed and histological sections were analyzed by histometry and immunohistochemistry. Flow cytometry analysis confirmed the efficiency of magnetic separation with 95.14% (± 2.46%) of cells expressing CD146 marker. More than 90% of the PDL-CD146+ cells co-expressed markers CD105 and CD166. Only one of the populations PDL-CD146+ did not express CD166 marker, nevertheless it was the only population to express STRO-1 marker and the transcriptional factor OCT-4/POU5F1. All PDL-CD146+ cell populations exhibited potential of osteoblastic/cementoblastic differentiation with a significant increase on expression of RUNX2, ALP, and OCN (p < 0.05), which did not occur in PDL-CD146- cell populations. When undergoing to adipogenic differentiation, only population PDL-CD146+ CD105+ CD166+ STRO1+ OCT4+ was able to differentiate into adipocyte-like cells, showing a significant increase in the expression of specific genes (LPL e PPAR?2) (p < 0.05). Evaluating the pattern of distribution of cells CD146+, CD166+, and STRO-1+, it is observed that these cells are distributed in the region of ligament and connective tissue, next to cementum and bone tissue. It was also seen on the wall of blood vessels and agglomerates close to it, evidencing that these may be its niches. These findings demonstrate that only cells that express concomitantly the surfaces antigens CD146 and STRO-1 and the transcriptional factor OCT-4/POU5F are able to differentiate into osteoblasts and adipocytes. The PDL-CD146- cells do not have the characteristic of multipotentiality. Immunostained cells to antigens CD146, CD166, and STRO-1 are distributed in PDL and in perivascular space of healthy teeth and teeth with periodontal disease (AU)

FAPESP's process: 14/14472-4 - Identification of pericytes in periodontal ligament and characterization as mesenchymal stem cells
Grantee:Mércia Jussara da Silva Cunha
Support Opportunities: Scholarships in Brazil - Doctorate