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.

Isolation of Endogenous TGF-ß1 from Root Canals for Pulp Tissue Engineering: A Translational Study.

Cell homing for dental pulp tissue engineering has been advocated as a feasible approach to regenerate dental pulp in a clinical setting. In order to develop a translational protocol for clinical application, we wanted to determine the effects of disinfectants on the availability of growth factors from the root canal, the amount that can be obtained in this context, and whether they can be processed for use in tissue engineering procedures. The extraction of growth factors should also be confirmed in a clinical setting. Root canals were prepared in 36 extracted mature teeth, and the amount of TGF-ß1 in solution was quantified after different irrigation protocols (sodium hypochlorite, chlorhexidine) and after intracanal medication (calcium hydroxide). Centrifugal filters with a cut-off of 10,000 Da and 3000 Da were used for efficient concentration, and volumes and amounts of retained TGF-ß1 were measured at different time points. During conventional endodontic treatment, ethylenediaminotetraacetic acid (EDTA) solution was collected after ultrasonic activation from the root canals of mature teeth of 38 patients, and growth factor content was quantified via enzyme-linked immunosorbent assay (ELISA). Irrigation with sodium hypochlorite reduced TGF-ß1 release into EDTA. This effect was partially reversed by canal enlargement after the use of sodium hypochlorite and by subsequent use of calcium hydroxide. A few minutes of centrifugation with a cut-off of 10,000 Da reduced the initial volume of the irrigant by 90% and led to a continuous increase in concentration to the same extent. Furthermore, TGF-ß1 was obtained from root canals of mature teeth during endodontic treatment in quantities that have been shown to elicit desirable cellular responses in a subsequent clinical application. A mixture with a suitable scaffold material and injection into the root canal has the potential to promote dental pulp regeneration.