ISOLATION AND CHARACTERIZATION OF HUMAN GINGIVAL FIBROBLASTS

• Author / Creator

  Diar-Bakirly, Samira

• Background: Gingival fibroblasts (GFs) that exhibit characteristics similar to adult stem cells are also known as gingival mesenchymal stem cells (GMSCs). These cells can be isolated using either tissue explants or enzymatic digestion, however it is unknown whether the isolation method influences the GMSCs potential for differentiation. In addition, no specific mesenchymal stem cell (MSC) marker has been reported to identify and distinguish GMSCs from GFs. Recently, the cell surface molecule known as cluster of differentiation (CD) 146 has been identified as a potential MSC cell surface marker.
  Objective: To investigate the differentiation potential of GMSCs based on CD146 expression.
  Hypothesis: CD146 is the MSC surface marker that identifies GMSCs among a population of GFs regardless of the isolation method employed.
  Materials and methods: GFs were isolated by two techniques: tissue explants or enzymatic digestion. GFs were cultured and expanded in a medium containing fibroblast growth factor 2 (FGF-2), and magnetically sorted according to CD146 expression. Four experimental groups were collected: CD146low and CD146high from explant-derived isolation and CD146low and CD146high from digestion-derived isolation. Each group of cells was expanded and then tested for stem cell markers using flow cytometry before it was subjected to osteogenic and chondrogenic differentiation. Multilineage differentiation outcome was tested after 21 days using histology, immunofluorescence, real-time quantitative PCR (qPCR), and glycosaminoglycan (GAG) to DNA ratio (GAG/DNA) assays.
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  Results: As confirmation of osteogenic differentiation, alizarin red staining was positive for all groups with no significant difference between osteogenic gene expressions. The absence of Safranin O staining accompanied by low GAG production negates chondrogenic differentiation. This lack of chondrogenesis was further confirmed by immunofluorescence assay, which indicated no deposition of collagen type II in the extracellular matrix (ECM) of GFs aggregates.
  Conclusion: CD146 is not the specific stem cell surface marker to identify and enrich the GMSC population. Its expression did not enhance the osteogenic potential differentiation potential of sorted GFs regardless of the implemented isolation method

• Subjects / Keywords

  • Gingival fibroblasts
  • Dental stem cells
  • Osteogenic and chondrogenic differentiation

• Graduation date

  Fall 2019

• Type of Item

  Thesis

• Degree

  Master of Science

• DOI

  https://doi.org/10.7939/r3-0q68-8j24

• License

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