Effect of inflammation on mesenchymal stem cell from periodontal tissues

Author(s)

Mayra Laino Albiero

• Document type: Doctoral Thesis
• Press: Piracicaba, SP.
• Institution: Universidade Estadual de Campinas (UNICAMP). Faculdade de Odontologia de Piracicaba
• Defense date: 2017-02-17

Examining board members

Karina Gonzales Silverio; Bruna Rabelo Amorim; Bruno Bueno Silva; Thaisângela Rodrigues; Francisco Humberto Nociti Junior

• Advisor: Karina Gonzales Silverio

Abstract

The present study evaluated the effect of the inflammatory process on mesenchymal stem cell properties from the periodontal ligament (PDLSCs) and on the expression of the mesenchymal cell surface markers in the periodontal tissues. Initially PDLSCs purified with CD105 marker (hPDL-CD105+) were exposed to Porphyromonas gingivalis total protein extract (PgPE), and cells metabolism, inflammatory activity, proliferative capacity and osteogenic differentiation were evaluated. Subsequently, it was analyzed the expression of the mesenchymal cell surface markers after the induction of periodontal disease in vivo, and confirmed in vitro. For this, hPDL-CD105+ were exposed to different concentrations of PgPE and evaluated for cellular metabolism (MTT assay) to determine the non-cytotoxic PgPE concentration. Apoptosis (Annexin V and 7-AAD markers) was assessed by flow cytometry technique. Proinflammatory activity was investigated by 1) gene expression of interleukin 1-beta (IL-1?), interleukin 6 (IL-6) and tumor necrosis factor alpha (TNF-?) by RT-qPCR, 2) presence of IL-6 with immunofluorescence assay 3) qPCR array of IL6/STAT3 signaling pathway. To evaluate osteogenic differentiation capacity, mineral nodules formation was assessed with alizarin red staining (AR) and gene expression for Runt-related transcription factor 2 (RUNX2), alkaline phosphatase (ALP) and osteocalcin (OCN) by RT-qPCR. Experimental periodontal disease by ligature was induced in Wistar rats for 7 days, after this period one group with disease induction only was euthanized (n=8) and one group was received scaling and root planning after removal of the ligature (n=8), and together with the control group (n=8), were euthanized at day 14. From the periodontal tissues collected from rats, the expression of mesenchymal cell surface markers (STRO-1, CD105, CD166, CD146) was evaluated by immunohistochemistry and RT-qPCR gene expression. The same cell surface markers were evaluated by flow cytometry in hPDL-CD105+ exposed to PgPE. Results showed that doses lower than 3 ?g/ml were non-cytotoxic, and were not able to induce apoptosis. hPDL-CD105+ showed increased expression of proinflammatory cytokines exposed to 2ug/ml PgPE, and this was confirmed by an increased expression of 28 inflammatory genes related to the IL6/STAT3 pathway. In addition, osteogenic potential was maintained in the presence of PgPE, with significant deposition of mineral nodules and expression of the osteogenic genes. In rat periodontal tissues, inflammatory disease induced an increase in CD105 and CD166 surface markers gene expression and this result was confirmed in situ at protein level. hPDL-CD105+ stimulated with PgPE showed an increase in STRO-1 stem cell marker. And this increase is positively correlated to increased IL-6 gene expression in hPDL-CD105+. These data suggest that there was a clear modulation of the cell surface markers related to the mesenchymal stem cell phenotype in vitro and in vivo, and that the presence of PgPE did not affect cell viability and osteogenic differentiation capacity of hPDL-CD105+, preserving its biological properties (AU)

• FAPESP's process: 14/01827-9 - Influence of Porphyromonas gingivalis exposure on the mesenchymal stem cell phenotype of cells isolated from human periodontal ligament
• Grantee: Mayra Laino Albiero
• Support Opportunities: Scholarships in Brazil - Doctorate