Ascorbic Acid, Inflammatory Cytokines (IL-1β/TNF-α/IFN-γ), or Their Combination's Effect on Stemness, Proliferation, and Differentiation of Gingival Mesenchymal Stem/Progenitor Cells

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.


Introduction
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. Subsequently, an inflammatory reaction is mounted, with the release of inflammatory cytokines, most prominently tumor necrosis factor alpha (TNFα), interleukin (IL) 1 beta (IL-1β), IL-4, IL-6, and interferon gamma (IFN-γ) [1]. 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.
Ascorbic acid (AA) is one of the pivotal biomolecule, with decisive effects on wound healing and collagen biosynthesis [2]. Investigations on adult [3,4], embryonic [5], and induced pluripotent [6] stem/progenitor cells outlined the beneficial effects of AA on increasing cellular proliferation, impeding apoptosis, and triggering pluripotency markers' expression. Furthermore, 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].
Gingival mesenchymal stem/progenitor cells (G-MSCs) possess notable periodontal reparative and regenerative potentials [9,10], with inflammation-resistance properties [11] and immunomodulation abilities in their local microenvironment [12]. The latest investigations outlined an individual G-MSCs-TLRs' expression profile [13] and defined two TLR-generated immunomodulatory phenotypes in G-MSCs challenged by TLR-agonists [14]. G-MSCs obtained from inflamed gingival tissues demonstrated a differentiation/regenerative aptitude comparable to G-MSCs from uninflamed tissues [15]. Furthermore, recent studies outlined positive short-term stimulatory effects of controlled inflammatory microenvironments on the G-MSCs' pluripotency, proliferation, and differentiation attributes [16][17][18]. Short-term inflammatory stimuli in isolation or combined with retinol supplementation boosted the stemness, proliferative, and differentiation capabilities of G-MSCs [19]. Recently, AA was shown to possess the ability to similarly enhance G-MSCs' proliferative aptitude and pluripotency marker expression [20]. The current study's aim is to investigate for the first time the AA and controlled inflammatory impacts, isolated or combined, on pluripotency, proliferation, Wnt/β-catenin pathway activation, and differentiation of G-MSCs.

Isolation/Characterization of G-MSCs.
Human G-MSCs were obtained from healthy gingival collars (n = 5) at the Christian-Albrechts University of Kiel. The patient was taken as the experimental unit, and cells were not pooled. The present study was approved by the Ethics Committee of Christian-Albrechts University of Kiel (IRB D513/17). Cells' isolation, culturing in basic medium and immuno-magnetic cell sorting, employing anti-STRO-1 antibody (BioLegend, San Diego, CA, USA) and anti-IgM MicroBeads (Miltenyi-Biotec, Bergisch Gladbach, Germany), was done as formerly described [19,21] [24] to test the cells metabolic activity. No phenol-red serum-free medium (RPMI 1640, PAN-Biotech, Aidenbach, Germany) and 0.5 mg/ml MTT-labelling reagent was added to the G-MSCs cultures and left for 4 hours, followed by 1 ml of the Solubilization solution (37°C, 5% CO 2 , overnight). The spectrophotometrical absorbance was recorded at 570 nm wavelength (MultiskanGO Microplate Spectrophotometer, Thermo Fisher). Metabolic activity 2 Stem Cells International was calculated using standard curves. The assays were conducted in duplicate and averaged. Second passage 1.63/cm 2 G-MSCs were cultivated in 10cm-diameter dishes for the CFUs assay. On the 14 th day, cell cultures were fixed using 100% methanol (ice-cold, for 10 min) and stained with 0.1% crystal violet for 10 min. CFUs were evaluated by two independent examiners, using phasecontrast inverted microscopy. Aggregations of ≥50 cells were counted as a colony.
2.6. G-MSCs' mRNA Expression. To test for the pluripotency gene expressions (Nanog, octamer-binding-transcriptionfactor 4A (Oct4A) as well as sex-determining-region-Y-box 2 (Sox2)), mRNA extraction was done in the four experimental groups outlined above at 24, 72, and 120 hours, using the RNeasy kit (Qiagen, Hilden, Germany). Complementary DNA (cDNA) was synthesized from RNA (1 μg/μl) via reverse transcription (QuantiTect reverse transcription kit, Qiagen) in a volume of 20 μl reaction mixture (4 pmol of each primer, 10 μl of the LightCycler Probes Master mixture (Roche) and 5 μl specimen cDNA). Real-time polymerase chain reaction (rt-PCR; LightCycler 96 Real-Time PCR System, Roche Molecular Biochemicals, Indianapolis, Indiana, USA) was conducted. Nineteen possible reference genes were preexamined to decide on the most suitable reference gene in the G-MSCs, which would not be regulated by the experiment (NormFinder). Except for PGK1, all were regulated. Hence, PGK1 (housekeeping gene) was determined to be employed as a reference gene ( Table 1). The relative quantification of the examined genes was conducted employing the 2 -ΔΔCt method and assays done in triplicate and averaged. Gene expressions of each of the tested genes were normalized to PGK1.

Multilineage
Potential of Stimulated G-MSCs. G-MSCs were prestimulated for five days in the experimental groups designated above, followed by osteogenic (21 days), adipogenic (21 days), or chondrogenic (35 days) differentiation. mRNA expression of alkaline phosphatase (ALP) and Runtrelated transcription factor 2 (Runx2), as well as the qualitative and quantitative Alizarin red staining, was conducted to assess the G-MSCs' osteogenic aptitude. For the quantification of the Alizarin red staining, 200 μl 10% acetic acid was supplemented into the osteogenically induced G-MSCs cultures for 30 minutes on a shaker, followed by detachment of the cellular monolayer, and transferred into a 1.5 ml tubes (Eppendorf) and vortexing for 30 seconds. Following 10 minutes 85°C heating and ice-cooling, the mixture was centrifuged at 20,000 rpm (15 minutes). 200 μl supernatant was transported to a new 1.5-ml tube and the pH neutralized with 10% ammonium hydroxide. Spectrophotometrical absorbance of 50 μl of the sample and Alizarin red standards were recorded at OD405 (Thermo Fisher), and relative Alizarin red quantities were determined [25].
mRNA expression for lipoprotein lipase (LPL) and proliferator-activated receptor gamma (PPAR-γ) and quantitative and qualitative Oil-Red-O evaluation were assessed as evidence for the adipogenic differentiation aptitude. For Oil-Red-O quantification, isopropanol (1 ml/well) was added, and the cultures were incubated for 15 minutes on a shaker, and 100 μl of the resultant mixture's spectrophotometrical absorbance was measured at OD540 (Thermo Fisher), and finally, the relative Oil-Red-O quantities were determined [26].
To assess chondrogenic differentiation, Aggrecan (ACAN) mRNA expression and Alcian blue/nuclear-fastred staining were evaluated. For quantification of Alcian blue and nuclear-fast-red staining, automated digital image quantification of the chondrogenic differentiation was performed as previously described [27]. All primers used in the realtime PCR were supplied by Roche (Table 1).

Statistical
Analysis. The normality of the data was evaluated, using the Shapiro-Wilk-test. Differences in intracellular total β-catenin, CFUs, MTT, mRNA expression, and quantitative adipogenic and osteogenic differentiation between the experimental groups were analyzed employing the Friedmantest. Differences in MTT and mRNA expression at different time points and pairwise comparisons were conducted using the Wilcoxon-signed-rank-test (SPSS 11.5, IBM, Chicago, IL, USA). The significance level was set at p = 0:05.

Discussion
Reparative/regenerative approaches in periodontology are based principally on recapitulating the chief periodontal developmental events, encompassing stem/progenitor cells' proliferation, migration, homing, differentiation, and finally maturation [28]. Clinically, 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]. Apart from its important roles in periodontal wound healing, tissue regeneration [31], and collagen synthesis of bone, teeth, and gingiva, AA demonstrates potent cellular protective antioxidative properties in response to periodontitis-induced oxidative inflammatory reactions [32,33]. It further possesses immunomodulatory abilities, which could markedly downregulate IL-1α, IL-1β, IL-6, tumor necrosis factor beta (TNF-β), and nitric oxide production in periodontal lesions [34,35]. AA could further induce a pluripotent stage [36] and enhance the reprogramming efficiency [6] in embryonic stem cells.
Comparable to oral wound healing [47], periodontal wound healing undergoes five phases, namely a hemostatic phase, an inflammatory phase, a cellular recruitment and proliferation phase, and finally a tissue remodeling one. Hereby, G-MSCs rely primarily on their stemness in performing theses periodontal tissue reparative and regenerative actions, primarily proliferation and multilineage differentiation potential. The current study examined the effect of the inflammatory cytokines mixture together and in combination with AA on the pluripotency markers' expression, namely Nanog, Oct4A, and Sox2 at 24, 72, and 120 hours. In line with earlier studies, in the present one Nanog, Oct4A, and Sox2 expressions were amplified in the AA- [3,20,48] and inflammation-challenged [19,49] G-MSCs. The AAinduced increase in the pluripotency markers Oct4A and Nanog could be ascribed primarily to the capability of AA, similar to Retinol [19], to activate the ten-eleventranslocation (TET) demethylases, eliminating methylation of the DNA and thereby inciting intracellular epigenetic reprogramming actions, encompassing pluripotency amplification [19,50,51]    Stem Cells International that a combination of AA and inflammation could augment their action on the expression of nuclear markers of pluripotency in the G-MSCs, thereby upregulating their stemness. Cellular proliferation represents a primary requirement of any periodontal regenerative/reparative approach to obtain cellular counts able of executing the ensuing phases of migration/homing and functional tissue differentiation. A fundamental hallmarks of stem/progenitor cells remains to be their ability for colonogenic self-renewal, demonstrated through their CFUs producing potential. Inflammatory stimulation, in line with previous investigations [17], appeared to enhance G-MSCs' metabolic activity at 72 hours. Surprisingly, AA stimulation initially appeared to decrease cellular metabolic activity in the early phase and even diminish the augmenting effects of the inflammatory stimuli in the inflammatory/AA group. The observed attenuation of G-MSCs' metabolic activity, while inducing proliferation could be attributed to the earlier reported property of AA to suppress cellular growth arrest encoding genes, namely growth arrest/-DNA-damage-inducible 45α (Gadd45a) as well as apoptosisinducing genes, namely caspase-1 [35]. It further can be ascribed to the potential of AA to increase ERK1/2 phosphorylation with a concomitant attenuation of the mitogenactivated kinase pathway [52]. In the current study, AA exhibited over 14 days a proliferative-induction potential, which was previously reported in longer-term cultures [48,53], and significantly higher CFUs formation. This property could be primarily attributed to an increase in the AAmediated upregulation of the proliferation-related-Fos-transcriptional-factor [52]. In contrast, the tested inflammatory microenvironment drove the G-MSCs to self-senescence on lengthier stimulation [17]. Short-term inflammatory stimuli  Figure 2: Wnt/β-catenin pathway activation, MTT, and CFUs following ascorbic acid and inflammatory stimulation of G-MSCs: ELISA examination of the Wnt/β-catenin signaling pathway for total intracellular β-catenin following G-MSCs' challenging by ascorbic acid and inflammation (a; box and whisker plots with medians/quartiles). Relative G-MSCs metabolic activity following ascorbic acid and inflammatory stimulation for 24 h and 72 h (b; box and whisker plots with medians/quartiles). CFUs-assay/CFUs' numbers following G-MSCs' stimulation via ascorbic acid and inflammation (c; box and whisker plots with medians/quartiles). Significant differences denoted with asterisks (n = 5, * p < 0:05; * * p < 0:01; Wilcoxon-signed-rank-test). Abbreviations: CFUs: colony-forming units; β-catenin: total β-catenin.  [18,45], which was evident by the reduced total intracellular β-catenin observed in the challenged groups. Yet on excessive stimulation, TNF-α could induce self-senescence of the stem/progenitor cells, especially in the presence of IFN-γ and cultivation at lower cellular densities, as in the CFU experiment. Through altering the IFN-γ-activated, nonapoptotic form of TNF-receptor-superfamily-member-6 (Fas) signaling into a caspase 3-and caspase 8-associated proapoptotic cascade, the G-MSCs' apoptotic pathway could have been activated [54]. AA and short-term controlled inflammation appear to have opposed biphasic impacts on the short-term as well as the long-term G-MSCs' proliferation.

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Notably, the greatest multilineage differentiation capability was evident in the AA group, emphasizing the importance of an inflammation-free environment for a successful multilineage differentiation of G-MSCs boosted by AA.
Combined, current results point at enhanced G-MSCs' characteristics in the presence of AA, which, apart from an initially observed synergistic effect on cellular metabolism as well as Sox2 and Oct4A pluripotency markers expression, were attenuated in the presence of an inflammatory microenvironment. The observed effects appeared to be associated with a Wnt/β-catenin pathway activation. Similar to previous investigations, precise short-termed microenvironmental inflammatory stimuli could enhance early cellular attributes and pluripotency, while an AA-induced boosting of cellular proliferation and differentiation would require an inflammation-free microenvironment. The present results denote that an early short-termed controlled G-MSCs' inflammatory stimulation, followed by a AA stimulation in an inflammation-free microenvironment could provide an interesting scheme for enhancing their cellular attributes in regenerative approaches.

Data Availability
All data used to support the findings of this study are included within the article.

Conflicts of Interest
All authors declare no conflict of interest related to this work.