Evaluation of oral keratinocyte progenitor and T-lymphocite cells response during early healing after augmentation of keratinized gingiva with a 3D collagen matrix - a pilot study

Abstract

Background: The aim of the present study is to analyze the behavior of selected populations of oral keratinocytes and T-lymphocytes, responsible for re-constructing and maintaining the oral epithelial tissue architecture, following augmentation of the keratinized oral mucosa using a 3D-collagen matrix.

Methods: Different groups of oral keratinocytes were isolated from biopsies harvested from 3 patients before the surgical procedure, as well as 7 and 14 days after the augmentation procedure. T-lymphocytes were isolated from peripheral blood at same timepoints. Keratinocytes were characterized for stem and differentiation markers, such as p63, cytokeratin 10 and 14, and in vitro parameters, such as cell viability, cell size and colony-forming efficiency. T-lymphocytes were analyzed for viability and the expression of various cluster of differentiation markers. The methods included magnetic separation of cell populations, immunofluorescence, flow cytometry, and histology of oral biopsies.

Results: Both at 7 and 14 days, the majority of cells that repopulate the matrix were actively proliferating/progenitor oral keratinocytes with the phenotype integrin alfa6beta4 + CD71+. These cells display in vitro characteristics similar to the progenitor cells analyzed before the matrix placement. T-lymphocytes expressed CD8 and CD69 markers, while CD25 was absent.

Conclusion: The study shows that two weeks after the collagen membrane placement, the healing process appeared to be histologically complete, with no abnormal immune response induced by the matrix, however, with a higher than usual content of active proliferating cells, the majority of keratinocytes being characterized as transit amplifying cells.

Keywords: Collagen matrix; Keratinocytes; Oral mucosa; T-lymphocytes.

Fig. 1

Images describing the surgical procedure: a) initial situation with deficit of keratinized gingiva; b) mucosal fenestration with apically positioned flap; c) the collagen matrix sutured in place; d) one week after the surgery; e) ten days after the surgery, immediately after the removal of the sutures; f) two weeks after the surgery

Fig. 2

Cell viability. Graph representing the percentage of α6β4+/CD71+ cells cells at different time points: 0, 7 and 14 days. At each analyzed time point most cells were found viable in all analyzed samples, with rare necrosis (each sample identifies different individuals; n = 5, p <0.05 Student’s t-test)

Fig. 3

Colony forming efficiency. Graph representing the colony forming potential of α6β4+/CD71+ cells at baseline, 7 and 14 days, respectively (number of CFUs). Following magnetic separation, cells were stained with crystal violet; colonies larger than 20 cells were counted individually using an image software (each sample identifies different individuals; n = 5, p <0.05 Student’s t-test)

Fig. 4

Cell size. Graph representing the percentage of α6β4+/CD71+ cells displaying the same size as the α6β4pos CD71pos oral keratinocyte progenitor cells from pristine sites (each sample identifies different individuals; n = 5, p <0.05 Student’s t-test)

Fig. 5

Immunofluorescence. Oral keratinocyte progenitor cells at 14 days expressed different stem- and differentiation markers (nuclei stained with DAPI); original magnification (x40). p63 - a specific oral keratinocyte stem cells marker; CK14 - differentiation marker usually positive for keratinocytes found in the basal layers of the epithelia; CK10 - differentiation marker expressed on cells found in the upper layers of the oral epithelia

Fig. 6

Oral mucosa and collagen matrix. Oral tissue samples before the procedure (a)(HE, x10), one week (b)(HE, x40) and 2 weeks after the procedure (c)(HE, x40). Histological assessment shows that 7 days following surgery, the oral epithelia is still forming, while at 14 days the architecture closely resembles the pristine gingiva (Scale bar - 100μm)