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During normal pulp tissue healing, inflammatory cytokines, such as tumor necrosis factor-alpha (TNF-α) or interleukins, act in the initial 48 hours (inflammatory phase) and play important roles not only as chemo-attractants of inflammatory cells and stem/progenitor cells but also in inducing a cascade of reactions toward tissue regeneration or reparative dentin formation or both. Previous reports have shown that inflammatory cytokines regulate the differentiation capacity of dental pulp stem/progenitor cells (DPCs), but none has interrogated the impact of these cytokines on the stem cell phenotype of stem/progenitor cells. This study investigated the effects of a short-term treatment with TNF-α on the stem cell phenotype and differentiation ability of human DPCs.    Methods  An  in vivo  mouse model of pulp exposure was performed for analysis of expression of the mesenchymal stem cell marker CD146 in DPCs during the initial stage of inflammatory response. For  in vitro  studies, human DPCs were isolated and incubated with TNF-α for 2 days and passaged to eliminate TNF-α completely. Analysis of stem cell phenotype was performed by quantification of cells positive for mesenchymal stem cell markers SSEA-4 (stage-specific embryonic antigen 4) and CD146 by flow cytometry as well as by quantitative analysis of telomerase activity and mRNA levels of  OCT-4  and  NANOG . Cell migration, colony-forming ability, and differentiation toward odontogenesis and adipogenesis were also investigated.    Results  The pulp exposure model revealed a strong staining for CD146 during the initial inflammatory response, at 2 days after pulp exposure.  In vitro  experiments demonstrated that a short-term (2-day) treatment of TNF-α increased by twofold the percentage of SSEA-4 +  cells. Accordingly, STRO-1, CD146, and SSEA-4 protein levels as well as  OCT-4  and  NANOG  mRNA levels were also significantly upregulated upon TNF-α treatment. A short-term TNF-α treatment also enhanced DPC function, including the ability to form cell colonies, to migrate, and to differentiate into odontogenic and adipogenic lineages.    Conclusions  A short-term treatment with TNF-α enhanced the stem cell phenotype, migration, and differentiation ability of DPCs.

A short-term treatment with tumor necrosis factor-alpha enhances stem cell phenotype of human dental pulp cells