Ascorbic Acid, Inflammatory Cytokines (IL-1βTNF-αIFN-γ)‎, or Their Combination’s Effect on Stemness, Proliferation, and Differentiation of Gingival Mesenchymal StemProgenitor Cells

Abstract EN

Objective.

Ascorbic acid (AA) and controlled inflammatory stimuli are postulated to possess the ability to independently exert positive effects on a variety of proliferative, pluripotency, and differentiation attributes of gingival mesenchymal stem/progenitor cells (G-MSCs).

The current study’s objective was to explore and compare for the first time the impact of the major inflammatory cytokines (IL-1β/TNF-α/IFN-γ), AA, or their combination on multipotency/pluripotency, proliferative, and differentiation characteristics of G-MSCs.

Design.

Human G-MSCs (n=5) were isolated and cultured in basic medium (control group), in basic medium with major inflammatory cytokines; 1 ng/ml IL-1β, 10 ng/ml TNF-α, and 100 ng/ml IFN-γ (inflammatory group), in basic medium with 250 μmol/l AA (AA group) and in inflammatory medium supplemented by AA (inflammatory/AA group).

All media were renewed three times per week.

In stimulated G-MSCs intracellular β-catenin at 1 hour, pluripotency gene expression at 1, 3, and 5 days, as well as colony-forming units (CFUs) ability and cellular proliferation over 14 days were examined.

Following a five-days stimulation in the designated groups, multilineage differentiation was assessed via qualitative and quantitative histochemistry as well as mRNA expression.

Results.

β-Catenin significantly decreased intracellularly in all experimental groups (p=0.002, Friedman).

AA group exhibited significantly higher cellular counts on days 3, 6, 7, and 13 (p<0.05) and the highest CFUs at 14 days [median-CFUs (Q25/Q75); 40 (15/50), p=0.043].

Significantly higher Nanog expression was noted in AA group [median gene-copies/PGK1 (Q25/Q75); 0.0006 (0.0002/0.0007), p<0.01, Wilcoxon-signed-rank].

Significant multilineage differentiation abilities, especially into osteogenic and chondrogenic directions, were further evident in the AA group.

Conclusions.

AA stimulation enhances G-MSCs’ stemness, proliferation, and differentiation properties, effects which are associated with a Wnt/β-catenin signaling pathway activation.

Apart from initially boosting cellular metabolism as well as Sox2 and Oct4A pluripotency marker expression, inflammation appeared to attenuate these AA-induced positive effects.

Current results reveal that for AA to exert its beneficial effects on G-MSCs’ cellular attributes, it requires to act in an inflammation-free microenvironment.