Histology of human teeth: Standard and specific staining methods revisited.

OBJECTIVE: Histological techniques have long been an integral part of dental research. Especially the processing of complex tissues poses specific challenges, however, literature offers only few technical references. Objectives of this study were therefore to optimize histological staining methods and compile detailed protocols for preparation and staining of dental tissues. METHODS: Human teeth were collected and fixed with 4 % formaldehyde solution after extraction. Subsequently, teeth were decalcified in 17 % EDTA or Morse's solution over a period of 28 days. The extent of decalcification was determined by weight loss and radiography. After sectioning, histological staining methods were optimized for their use on teeth. These included hematoxylin-eosin, Masson trichrome, Masson-Goldner trichrome and May-Gruenwald-Giemsa staining. Nerve fibres were visualized by luxol fast blue staining and Bodian silver staining. In addition, specific methods like TRAP, modified Brown and Brenn as well as picrosirius red staining with light polarization or fluorescence were applied and optimized. RESULTS: Preparation of an artificial access to the pulp chamber was essential to ensure prompt penetration of the chemicals. Decalcification with Morse's solution took at least two weeks but was more efficient than 17 % ETDA, where thorough demineralization was achieved only after three weeks. The staining methods exhibited differences not only regarding their ability to display specific structures of interest, but also in terms of reproducibility. CONCLUSION: High-quality histology of teeth can only be achieved after optimal tissue preparation and accurate staining. A complementary use of staining techniques is necessary to answer specific research questions.

Penetration depth of irrigants into root dentine after sonic, ultrasonic and photoacoustic activation.

AIM: To compare penetration depths of endodontic irrigants into the dentinal tubules of extracted teeth when using several activation methods. METHODOLOGY: The root canals of 90 extracted human teeth were prepared to size 40, .06 taper. The straight and round-shaped root canals were distributed randomly into six groups, and final irrigation was performed with EDTA and sodium hypochlorite as follows: (I) manual dynamic activation, (II) Ultrasonic, (III) Sonic, (IV) PIPS (photon-induced photoacoustic streaming, (V) SWEEPS (shock-wave enhanced emission photoacoustic streaming) and (0) control without final irrigation or activation. Subsequently, methylene blue was inserted into the canals and activated according to the groups (I-V). Teeth were sectioned horizontally, imaged under a light microscope, and dye penetration depths were measured in six sections per tooth and 24 points on a virtual clock-face per section. Data were analysed statistically by nonparametric tests for whole teeth and separately for coronal, middle and apical thirds. RESULTS: Penetration of dye into the dentinal tubules was lowest for the controls. Median penetration depths amounted to 700-900 µm for groups I-V with differences in the apical thirds between group I and the other test groups. Minimum penetration depths were significantly greater for PIPS in the apical thirds (P ≤ 0.046). CONCLUSIONS: Greater penetration depths occurred in the apical thirds for ultrasonic, sonic and laser-induced activation compared to manual dynamic activation. PIPS was associated with deeper penetration of irrigants. The novel SWEEPS mode did not increase irrigant penetration.

Interactive effects of LPS and dentine matrix proteins on human dental pulp stem cells.

AIM: To investigate the combinatorial effects of lipopolysaccharide (LPS) and extracted dentine matrix proteins (eDMP) on regenerative and inflammatory responses in human dental pulp stem cells (DPSCs). METHODOLOGY: Culture media were supplemented with several concentrations of LPS, eDMP and combinations of both. Cell viability was assessed over 1 week by MTT assay; cell survival was evaluated after 24 h and 7 days by flow cytometry. The expression of mineralization-associated marker genes was determined by real-time quantitative polymerase chain reaction (RT-qPCR). To analyse the inflammatory response, secretion of interleukin 6 (IL-6) was quantified in the initial and the late phase of cell culture by enzyme-linked immunosorbent assay (ELISA). Data were treated nonparametrically and Mann-Whitney U-tests were performed to compare all experimental groups (α = 0.05). RESULTS: Whereas LPS had no impact on viability, eDMP led to a concentration-dependent decrease, which was significant after 7 days (P ≤ 0.024). A moderate decline of cell survival induced by LPS was detected after 48 h (P ≤ 0.026), whereas eDMP was able to reverse this effect. eDMP alone caused increased expression of tested marker genes, LPS had no regulatory effect. Combined eDMP and LPS induced an upregulation of collagen type I and osteocalcin, whereas expression levels of dentine matrix acidic phosphoprotein and dentine sialophosphoprotein were similar to the control. IL-6-secretion was increased by LPS over time. eDMP markedly elevated initial production of IL-6 (P ≤ 0.002), but suppressed LPS-induced cytokine production in the later phase. CONCLUSIONS: Lipopolysaccharide did not affect cell viability but interfered with odontoblast-like cell differentiation of DPSCs. Proteins from the dentine matrix may have a protective effect, attenuate the detrimental impact of LPS and thus play an important role during pulp repair.