Combined Transcriptomic and Protein Array Cytokine Profiling of Human Stem Cells from Dental Apical Papilla Modulated by Oral Bacteria

Abstract

Stem cells from the apical papilla (SCAP) are a promising resource for use in regenerative endodontic treatment (RET) that may be adversely affected by oral bacteria, which in turn can exert an effect on the success of RET. Our work aims to study the cytokine profile of SCAP upon exposure to oral bacteria and their supernatants—Fusobacterium nucleatum and Enterococcus faecalis—as well as to establish their effect on the osteogenic and immunogenic potentials of SCAP. Further, we target the presence of key proteins of the Wnt/β-Catenin, TGF-β, and NF-κB signaling pathways, which play a crucial role in adult osteogenic differentiation of mesenchymal stem cells, using the Western blot (WB) technique. The membrane-based sandwich immunoassay and transcriptomic analysis showed that, under the influence of F. nucleatum (both bacteria and supernatant), the production of pro-inflammatory cytokines IL-6, IL-8, and MCP-1 occurred, which was also confirmed at the mRNA level. Conversely, E. faecalis reduced the secretion of the aforementioned cytokines at both mRNA and protein levels. WB analysis showed that SCAP co-cultivation with E. faecalis led to a decrease in the level of the key proteins of the Wnt/β-Catenin and NF-κB signaling pathways: β-Catenin (p = 0.0068 *), LRP-5 (p = 0.0059 **), and LRP-6 (p = 0.0329 *), as well as NF-kB (p = 0.0034 **) and TRAF6 (p = 0.0285 *). These results suggest that oral bacteria can up- and downregulate the immune and inflammatory responses of SCAP, as well as influence the osteogenic potential of SCAP, which may negatively regulate the success of RET.

Exploring the impact of oral bacteria remnants on stem cells from the Apical papilla: mineralization potential and inflammatory response

“…In our past studies, we have shown how short-term modulation of SCAP by bacteria can affect their viability, immune response, and gene expression for osteogenesis and dentinogenesis ( Razghonova et al., 2022 ; Zymovets et al., 2022 ). However, to assess whether bacterial influence alone, without the presence of osteo/dentinogenic induction medium, can stimulate SCAPs mineralization and thus influence their differentiation potential, a minimum of 21 days of bacterial exposure is required, which is the necessary duration for cell differentiation ( Abuarqoub et al., 2015 ).…”

“…The BDNF, GM-CSF, and VEGF-A, were chosen for their important role in osteo/odontogenic cells differentiation and regeneration ( Saito et al., 2011 ; Kolar et al., 2017 ; Liu et al., 2018 )). The pro-inflammatory markers—IL-8, IL-6, MCP-1, and IL-17A were selected based on the results of previous studies, indicating the upregulation of these markers in SCAPs under bacterial influence ( Razghonova et al., 2022 ; Zymovets et al., 2022 ) as well as IL-1a and IL-1β, as they are involved in innate pro-inflammatory response ( Dinarello, 2018 ).…”

“…Previously we showed that bacteria, which are mostly associated with traumatized infected root canals ( Manoharan et al., 2020 ), mainly Fusobacterium nucleatum and Enterococcus faecalis , can upregulate inflammatory response and negatively regulate the expression of genes that are important for osteo- and dentinogenesis in human SCAPs after 24 hours of co-cultivation ( Razghonova et al., 2022 ; Zymovets et al., 2022 ). Notwithstanding the results indicating the primary response of SCAP to bacterial stimuli after 24 hours, longer exposure time is necessary to see whether bacterial interaction with SCAP can alter their differentiation potential.…”

“…Notwithstanding the results indicating the primary response of SCAP to bacterial stimuli after 24 hours, longer exposure time is necessary to see whether bacterial interaction with SCAP can alter their differentiation potential. However, the co-cultivation of SCAPs with live bacteria for 21 days (the time required to see differentiation of SCAP towards osteoblasts or odontoblasts) ( Abuarqoub et al., 2015 ) cannot be implemented due to the rapid death of cells after more than 24 hours of co-cultivation with bacteria ( Zymovets et al., 2022 ).…”

Stem Cells from the Apical Papilla (SCAPs): Past, Present, Prospects, and Challenges

“…For example, Fusobacterium nucleatum triggers pro-inflammatory chemokine and cytokine generation in SCAPs while Enterococcus faecalis inhibits the same [73]. In addition, E. faecalis, but not F. nucleatum, can downregulate the expression levels of key proteins in Wnt/β-Catenin and NF-κB signaling pathways [76].…”