The action of stem cells is mediated by their paracrine secretions which comprise the secretory profile. Various approaches can be used to modify the secretory profile of stem cells. Creating a hypoxic environment is one method. The present study aims to demonstrate the influence of CoCl2 in generating hypoxic conditions in a dental pulp stem cell (DPSCs) culture, and the effect of this environment on their secretory profile. DPSCs that were isolated from human permanent teeth were characterized and treated with different concentrations of CoCl2 to assess their viability by an 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay and proliferation by a cell counting kit (CCK)-8 assay. The gene expression level of hypoxia-inducible factor 1-alpha (HIF-1α) was analyzed by quantitative real time polymerase chain reaction (qRT-PCR) to demonstrate a hypoxic environment. Comparative evaluation of the growth factors and cytokines were done by cytometric bead array. Gene expression levels of transcription factors OCT4 and SOX2 were analyzed by qRT-PCR to understand the effect of CoCl2 on stemness in DPSCs. DPSCs were positive for MSC-specific markers. Doses of CoCl2, up to 20 µM, did not negatively affect cell viability; in low doses (5 µM), it promoted cell survival. Treatment with 10 µM of CoCl2 significantly augmented the genetic expression of HIF-1α. Cells treated with 10 µM of CoCl2 showed changes in the levels of growth factors and cytokines produced. It was very evident that CoCl2 also increased the expression of OCT4 and SOX2, which is the modulation of stemness of DPSCs. A CoCl2 treatment-induced hypoxic environment modulates the secretory profile of DPSCs.

Autogenous gingival grafts used for root coverage or gingival augmentation procedures often result in donor site morbidity. Living cellular constructs as an exogenous alternative have been proven to be associated with lower morbidity. With the available background information, the present study aims to assess if quercetin-induced living cell constructs, derived from dental pulp stem cells, have the potential to be applied as a tool for soft tissue augmentation. The characterized dental pulp stem cells (positive for CD73, CD90, and negative for CD34, HLA-DR) were expanded in Dulbecco’s Modified Eagle’s medium (DMEM) supplemented with 10 mM quercetin. The handling properties of the quercetin-induced dental pulp stem cell constructs were assessed by visual, and tactile sensation. A microscopic characterization using hematoxylin and eosin staining, and qRT-PCR-based analysis for stemness-associated genes (OCT4, NANOG, SOX2, and cMyc) was also performed. Dental pulp stem cells without quercetin administration were used as the control. Dental pulp stem cell constructs induced by quercetin easily detached from the surface of the plate, whereas there was no formation in the control cells. It was also simple to transfer the induced cellular construct on the flattened surface. Microscopic characterization of the constructs showed cells embedded in a tissue matrix. Quercetin also increased the expression of stemness-related genes. The use of quercetin-induced DPSC living constructs for soft tissue augmentation could provide an alternative to autogenous soft tissue grafts to lower patient morbidity and improve esthetic outcomes.