Abstract
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.
Highlights
Periodontitis is an inflammatory disorder of tooth-investing and tooth-supporting tissues, branded by a gradual damage of alveolar bone, periodontal attachment, and eventually gingiva, associated with bacterial dysbiosis
Characteristic colony-forming units (CFUs) were demonstrated by the gingival mesenchymal stem/progenitor cells (G-MSCs) (Figure 1(b)), which were negative for CD14, CD34, weakly positive for CD45, and highly positive for CD73, CD90, and CD105 surface markers expression (Figure 1(c))
Proliferation, migration, homing, differentiation, and maturation [28]. These healing stages primarily take place in an initially inflamed periodontal microenvironment, with inflammatory cytokines orchestrating the course of the inflammatory periodontal disease progression [29], possible healing/regeneration [30], as well as periodontal stem/progenitor cells’ attributes [18, 19]
Summary
Periodontitis is an inflammatory disorder of tooth-investing and tooth-supporting tissues, branded by a gradual damage of alveolar bone, periodontal attachment, and eventually gingiva, associated with bacterial dysbiosis. The commencement of this multifaceted disease process commonly entails challenging of the periodontal immune-inflammatory system through virulent microbial biofilms. Duration and intensity of the resultant host reaction govern the personalized course and outcome of the inflammatory process, as well as affect the outcome of any subsequent periodontal reparative/regenerative approach. In additional to its host modulatory effects in periodontal disease [2], it was suggested that AA supplementation could positively affect the outcome of periodontal reparative/regenerative therapies [7, 8]
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