Transcriptome Analysis Reveals Modulation of Human Stem Cells from the Apical Papilla by Species Associated with Dental Root Canal Infection

Abstract

Interaction of oral bacteria with stem cells from the apical papilla (SCAP) can negatively affect the success of regenerative endodontic treatment (RET). Through RNA-seq transcriptomic analysis, we studied the effect of the oral bacteria Fusobacterium nucleatum and Enterococcus faecalis, as well as their supernatants enriched by bacterial metabolites, on the osteo- and dentinogenic potential of SCAPs in vitro. We performed bulk RNA-seq, on the basis of which differential expression analysis (DEG) and gene ontology enrichment analysis (GO) were performed. DEG analysis showed that E. faecalis supernatant had the greatest effect on SCAPs, whereas F. nucleatum supernatant had the least effect (Tanimoto coefficient = 0.05). GO term enrichment analysis indicated that F. nucleatum upregulates the immune and inflammatory response of SCAPs, and E. faecalis suppresses cell proliferation and cell division processes. SCAP transcriptome profiles showed that under the influence of E. faecalis the upregulation of VEGFA, Runx2, and TBX3 genes occurred, which may negatively affect the SCAP’s osteo- and odontogenic differentiation. F. nucleatum downregulates the expression of WDR5 and TBX2 and upregulates the expression of TBX3 and NFIL3 in SCAPs, the upregulation of which may be detrimental for SCAPs’ differentiation potential. In conclusion, the present study shows that in vitro, F. nucleatum, E. faecalis, and their metabolites are capable of up- or downregulating the expression of genes that are necessary for dentinogenic and osteogenic processes to varying degrees, which eventually may result in unsuccessful RET outcomes. Transposition to the clinical context merits some reservations, which should be approached with caution.

Microbiological assessment of success and failure in pulp revitalization: a randomized clinical trial using calcium hydroxide and chlorhexidine gluconate in traumatized immature necrotic teeth

“…Although the treatment protocol we used is standardized and we applied passive ultrasonic irrigation [ 50 ], these efforts might not be enough to eliminate the bacterial biofilm. Furthermore, low concentrations of irrigation solutions have been proposed due to concerns about the potential cytotoxicity of high concentrations of NaOCl solutions on stem cells of the apical papilla [ 51 ]: the higher concentrations of NaOCl used, the more cytotoxic effects on survival and differentiation of the stem cells [ 52 , 53 ]. In fact, it has been found that even if bacterial levels are reduced when using 6% NaOCl concentration, a total elimination of the microorganisms might not be guaranteed [ 47 ].…”

“…Therefore, microbial correlations with host phenotype might jeopardize the treatment outcomes of PR [ 76 ]. In the presence of bacterial species, the immune and inflammatory response of SCAPs is upregulated, but cell division and proliferation are downregulated [ 51 ]. Data obtained from animal models confirm that bacteria present even as a low-grade infection have negative effects on bone healing [ 77 , 78 ].…”

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.…”