Evaluation of Keratinocytes and T-Lymphocytes Following Augmentation of Keratinized Gingiva

Objectives: While several studies focused on the in vitro and in vivo behavior of oral fibroblasts following gingival augmentation, data on oral keratinocytes (OK) is scarce. Due to previous difficulties in isolating and characterizing different OK sub-populations, a thorough analysis of the cellular behavior following their contact with various oral dermal substitutes is lacking. The aim of the present study was to analyze the behavior of different populations of OK and inflammatory cells, responsible for re-constructing and maintaining oral epithelial tissue architecture, following the augmentation of the keratinized oral mucosa using a 3D-collagen matrix.
Methods: Different groups of OK and T-lymphocytes were isolated from biopsies harvested before the surgical procedure as well as after 7 and 14 days after the augmentation procedure. The cells were characterized for different stem and differentiation markers and in vitro parameters such as cell viability, cell size and colony-forming efficiency. The methods included magnetic separation of different cell populations, histological staining of oral biopsies, immunofluorescence and flow cytometry. For OK progenitor cells in vitro behaviour, results from five independent experiments are shown as means ± SD. For each independent experiment the same number of cells was used. Statistical analysis was performed using ANOVA. Statistical significance was accepted at p<0.05.
Results: At each analyzed time point the majority of cells that repopulate the matrix are actively proliferating/progenitor OK with the phenotype integrin alfa6beta4+CD71+. These cells display in vitro characteristics similar to progenitor cells analyzed before the matrix placement. T-lymphocytes expressed CD8 and CD69 markers, while CD25 was absent.
Conclusions: The study shows that at two weeks after the collagen membrane placement, the healing process seems to be macroscopically complete, with no abnormal immune response, but the oral epithelial architecture is still forming, with the majority of OK being transit amplifying cells.