An in vitro coculture approach to study the interplay between dental pulp cells and Streptococcus mutans.

AIM: To develop a new coculture system that allows exposure of dental pulp cells (DPCs) to Streptococcus mutans and dentine matrix proteins (eDMP) to study cellular interactions in dentine caries. METHODOLOGY: Dental pulp cells and S. mutans were cocultured with or without eDMP for 72 h. Cell proliferation and viability were assessed by cell counting and MTT assays, while bacterial growth and viability were determined by CFU and LIVE/DEAD staining. Glucose catabolism and lactate excretion were measured photometrically as metabolic indicators. To evaluate the inflammatory response, the release of cytokines and growth factors (IL-6, IL-8, TGF-ß1, VEGF) was determined by ELISA. Non-parametric statistical analyses were performed to compare all groups and time points (Mann-Whitney U test or Kruskal-Wallis test; α = .05). RESULTS: While eDMP and especially S. mutans reduced the number and viability of DPCs (p ≤ .0462), neither DPCs nor eDMP affected the growth and viability of S. mutans during coculture (p > .0546). The growth of S. mutans followed a common curve, but the death phase was not reached within 72 h. S. mutans consumed medium glucose in only 30 h, whereas in the absence of S. mutans, cells were able to catabolize glucose throughout 72 h, resulting in the corresponding amount of l-lactate. No change in medium pH was observed. S. mutans induced IL-6 production in DPCs (p ≤ .0011), whereas eDMP had no discernible effect (p > .7509). No significant changes in IL-8 were observed (p > .198). TGF-ß1, available from eDMP supplementation, was reduced by DPCs over time. VEGF, on the other hand, was increased in all groups during coculture. CONCLUSIONS: The results show that the coculture of DPCs and S. mutans is possible without functional impairment. The bacterially induced stimulation of proinflammatory and regenerative cytokines provides a basis for future investigations and the elucidation of molecular biological relationships in pulp defence against caries.

A critical analysis of clinical research methods to study regenerative endodontics.

Regenerative endodontic treatment such as revitalization provides a treatment option for immature teeth with pulp necrosis. The main difference to the alternative procedure, the apical plug, is the induction of a blood clot inside the canal as a scaffold for healing and new tissue formation. Due to the biology-based and minimally-invasive nature of the treatment, revitalization has raised considerable interest in recent years. Whereas the procedure is fairly new and recommendations from endodontic societies have been in place only for a few years, the treatment protocol has evolved over the past two decades. Evidence has been created, not only from laboratory and animal work, but also from clinical studies including case reports, cohort studies and eventually prospective randomized controlled clinical trials, systematic reviews and meta-analyses. However, the research methods and clinical studies with subsequent reports oftentimes present with methodical limitations, which makes it difficult to objectively assess the value of this treatment modality. Several open questions remain, including the need for a more differentiated indication of revitalization after different traumatic injuries, the long-term prognosis of treated teeth and the true benefits for the patient. Therefore, this review aims to identify and reflect on such limitations, scrutinizing study design, diagnostic tools, procedural details and outcome parameters. A core outcome set is also proposed in this context, which can be considered in future clinical investigations. These considerations may lead to a more detailed and stringent planning and execution of future studies in order to create high-quality evidence for the treatment modality of revitalization and thus provide more robust data, create a larger body of knowledge for clinicians and further specify current recommendations.