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Irreversible pulpitis is an inflammation of the tooth pulp caused by an opportunity-driven invasion of the pulp space by oral microbiota typically prevalent in the oral cavity. Microbial organisms are extensively recognised to be the fundamental cause of endodontic infections and treatment failures. Previously, bacterial species responsible for these infections were largely recognised using conventional microbial culture techniques, lending credence to the widely held belief that anaerobic Gram-negative bacteria frequently enter the pulp space and trigger endodontic infections. The advent of novel technologies grants the advantage of detecting and studying microbial populations via an amalgamation of the modern “Omics” techniques and meticulous bioinformatics analysis, additionally detecting the metatranscriptome, metaproteome and metabolome along with the metagenome. Amongst these analytical strategies, metagenomic analyses are essentially pragmatic for investigating the oral microbiome. Metagenomics favor not only assessment of microbial composition in diseased conditions, but also contributes to detection of novel, potentially pathogenic species inclusive of non-viable bacteria. The present review describes current knowledge of root canal microbiome, including its composition and functional attributes, the novel strategies available for detection of microbiome as well as challenges associated and provides some crucial pointers for areas of future research.

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Introduction: The standard isolation and expansion of human Dental Pulp Stem Cells (DPSCs) under invitro conditions normally involve the usage of Fetal Bovine Serum (FBS). However, its animal-origin poses possible concerns for clinically relevant procedures. This critical issue compels the use of Xenogeneic-Free (XF) or human-origin alternatives to FBS for culture expansion and differentiation of DPSCs to determine the usefulness for translating into therapeutic clinical applications. Aim: To evaluate the cellular characteristics and expression of pluripotent markers in DPSCs cultured using Serum-Containing Medium (SCM-DPSCs) and Serum-Free Medium (SFM-DPSCs). Materials and Methods: This in-vitro descriptive study was conducted at NITTE (Deemed to be University), Mangaluru, Karnataka, India, from June 2019 to August 2020. DPSCs were isolated from impacted third molars. The culture expanded DPSCs in serum-containing and serum-free media were analysed on their morphology, viability, proliferation rate, Population Doubling Time (PDT), Alkaline Phosphatase (ALP) activity, cell surface markers expression, osteogenic and adipogenic potential, and the relative expression of selected pluripotent genes. Results: The primary culture of DPSCs established in SCM and SFM showed spindle shaped fibroblastic morphology with >80% viability from passage 1 (P1) to P4. A significant (p-value<0.05) difference in the proliferation rates in terms of cell numbers between SCM-DPSCs and SFM-DPSCs was observed (day 6: 3×105 vs 0.8×105; day 9: 5.8×105 vs 1.27×105; day 12: 7.8×105vs 1.56×105, respectively). The average PDT values recorded in SCM- and SFM-DPSCs were 44.33 hours and 58.41 hours, respectively. A slightly higher expression of ALP activity was observed in SCM-DPSCs than in SFM-DPSCs. Flow cytometry analysis showed that both DPSCs were positive for CD29, CD73, CD90, and negative for CD34 and CD45. The expression of OCT4 and NANOG was relatively higher in SCM-DPSCs compared to SFM-DPSCs. Further, SCM-DPSCs showed the higher levels of SOX2 and SSEA4, but did not exhibit any significant differences in their expression levels. Conclusion: The results showed that DPSCs in FBS displayed better growth kinetics and stemness markers expression along with more propensities towards lineage differentiation. SFM can be used to establish and expand DPSCs with characteristics of multipotent stem cells, but needs further research for its optimisation.