Thymoquinone (TQ), the key active component of Nigella sativa (NS), demonstrates very promising biomedical anti-inflammatory, antioxidant, antimicrobial and anticancer properties. Several investigations have inspected the modulative activities of TQ on different stem/progenitor cell types, but its possible role in the regulation of gingival mesenchymal stem/progenitor cells (G-MSCs) has not yet been characterized. For the first time, this study investigates the effects of TQ on G-MSCs’ stemness and Toll-like receptor expression profiles. G-MSCs (n = 5) were isolated, sorted via anti-STRO-1 antibodies and then disseminated on cell culture dishes to create colony-forming units (CFUs), and their stem/progenitor cell attributes were characterized. TQ stimulation of the G-MSCs was performed, followed by an examination of the expression of pluripotency-related factors using RT-PCR and the expression profiles of TLRs 1–10 using flowcytometry, and they were compared to a non-stimulated control group. The G-MSCs presented all the predefined stem/progenitor cells’ features. The TQ-activated G-MSCs displayed significantly higher expressions of TLR3 and NANOG with a significantly reduced expression of TLR1 (p < 0.05, Wilcoxon signed-rank test). TQ-mediated stimulation preserves G-MSCs’ pluripotency and facilitates a cellular shift into an immunocompetent-differentiating phenotype through increased TLR3 expression. This characteristic modulation might impact the potential therapeutic applications of G-MSCs.

The present study explores for the first time the effect of hyperbaric oxygen (HBO) on gingival mesenchymal stem cells’ (G-MSCs) gene expression profile, intracellular pathway activation, pluripotency, and differentiation potential under an experimental inflammatory setup. G-MSCs were isolated from five healthy individuals (n = 5) and characterized. Single (24 h) or double (72 h) HBO stimulation (100% O2, 3 bar, 90 min) was performed under experimental inflammatory [IL-1β (1 ng/mL)/TNF-α (10 ng/mL)/IFN-γ (100 ng/mL)] and non-inflammatory micro-environment. Next Generation Sequencing and KEGG pathway enrichment analysis, G-MSCs’ pluripotency gene expression, Wnt-/β-catenin pathway activation, proliferation, colony formation, and differentiation were investigated. G-MSCs demonstrated all mesenchymal stem/progenitor cells’ characteristics. The beneficial effect of a single HBO stimulation was evident, with anti-inflammatory effects and induction of differentiation (TLL1, ID3, BHLHE40), proliferation/cell survival (BMF, ID3, TXNIP, PDK4, ABL2), migration (ABL2) and osteogenic differentiation (p < 0.05). A second HBO stimulation at 72 h had a detrimental effect, significantly increasing the inflammation-induced cellular stress and ROS accumulation through HMOX1, BHLHE40, and ARL4C amplification and pathway enrichment (p < 0.05). Results outline a positive short-term single HBO anti-inflammatory, regenerative, and differentiation stimulatory effect on G-MSCs. A second (72 h) stimulation is detrimental to the same properties. The current results could open new perspectives in the clinical application of short-termed HBO induction in G-MSCs-mediated periodontal reparative/regenerative mechanisms.