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.

Dentine matrix proteins: isolation and effects on human pulp cells.

AIM: To establish a simplified and efficient protocol for the isolation and concentration of matrix proteins from human dentine, and to assess the effects of extracted dentine matrix proteins (eDMP) on the behaviour of human pulp cells. METHODOLOGY: Matrix proteins were isolated from human dentine, purified, concentrated and characterized with protein and enzyme-linked immunosorbent assays (ELISA). Culture media were supplemented with eDMP in different concentrations, referred to as eDMP 1-10 000, to assess viability and proliferation of human pulp cells by DNA and MTT assays; apoptotic events were quantified by flow cytometry. Chemotactic effects of eDMP were assessed in a modified Boyden chamber assay. Expression levels of odontoblastic marker genes in pulp cells cultured with eDMPs were determined by real-time quantitative PCR, and the ability to induce mineralization was demonstrated by alizarin red staining. Nonparametric statistical analysis was performed to pairwise compare different groups at all time-points (Mann-Whitney U-test, α = 0.05). RESULTS: High concentrations of eDMP exhibited significant antiproliferative effects (P ≤ 0.023) after 5 (eDMP 1000) and 7 days (eDMP 500) without affecting cell viability. Apoptosis was barely influenced (P ≥ 0.089). eDMP exerted a concentration-dependent chemotactic stimulus on dental pulp cells with statistical significance already at low dosage (P = 0.006 at eDMP 10). Changes in gene expression indicated a differentiation into odontoblast-like cells, which was corroborated by findings of mineral nodule formation. CONCLUSIONS: A novel, effective and time-saving protocol for isolation and concentration of dentine matrix proteins is presented. As eDMP stimulates chemotaxis, differentiation and mineralization without affecting viability, endogenous dentine matrix proteins might be valuable for approaches to regenerate or engineer dental pulp.

A training model for revitalization procedures.

AIM: To develop an inexpensive simulation model for training of revitalization procedures. METHODOLOGY: A replica of an immature maxillary central incisor was equipped with a mock blood reservoir at the root apex and embedded in a plaster model. Mock blood consisted of water supplemented with red pigments and fibrinogen, whilst thrombin was inserted into the root canal to allow for clot formation. RESULTS: A true-to-life training model for revitalization procedures was established, where the induction of bleeding and formation of a blood clot can be mimicked. The model can be fastened to a patient mannequin and thus closely simulate the clinical setting. CONCLUSIONS: The newly developed model can be used for training purposes, both for dental students and for practitioners, to perform the treatment steps of a revitalization procedure.

Ultrasonic activation of irrigants increases growth factor release from human dentine.

OBJECTIVES: Bioactive proteins are sequestered in human dentine and play a decisive role in dental pulp regeneration and repair. They can be released and exposed on the dentine surface by acids, but also chelators, such as ethylenediaminetetraacetic acid (EDTA). The objectives of this study were (i) to evaluate whether ultrasonic activation of irrigants in the root canal will promote growth factor release from dentine and (ii) to collect bioactive proteins in a physiological solution. MATERIALS AND METHODS: Human dentine disks underwent irrigation with and without ultrasonic activation. The protocols included treatment by either a single or two consecutive steps with 10 % EDTA and phosphate-buffered saline (PBS), where each sample was treated three times. To mimic clinical conditions, selected irrigation regimens were applied to root canals of extracted human teeth after preparation. Amounts of transforming growth factor ß1 (TGF-ß1) in solution were quantified using enzyme-linked immunosorbent assays. Nonparametric statistical analysis was performed to compare different groups as well as repetitions within a group (Mann-Whitney U test, α = 0.05). Additionally, morphological changes of dentine surfaces were visualized by scanning electron microscopy (SEM). RESULTS: TGF-ß1 was not detectable after irrigation of dentine with PBS, neither with nor without ultrasonic activation. Irrigation with EDTA released TGF-ß1, and ultrasonic activation of EDTA enhanced this effect. However, preceding EDTA conditioning enabled the release of bioactive proteins into PBS solution. Similar results were observed in dentine disks and root canals. Visualization of dentine surfaces after different treatment revealed superficial erosion after ultrasonic activation irrespective of the irrigant solution, but different degrees of exposure of organic substance. CONCLUSIONS: Ultrasonic activation enhances growth factor release from human dentine. Bioactive proteins can be isolated in physiological solvents and may act as autologous supplements for regenerative endodontic treatment or pulp tissue engineering. CLINICAL RELEVANCE: Autologous growth factors from human dentine can advance treatment strategies in dental pulp tissue engineering.

EDTA conditioning of dentine promotes adhesion, migration and differentiation of dental pulp stem cells.

AIM: To evaluate the effect of dentine conditioning on migration, adhesion and differentiation of dental pulp stem cells. METHODOLOGY: Dentine discs prepared from extracted human molars were pre-treated with EDTA (10%), NaOCl (5.25%) or H2 O. Migration of dental pulp stem cells towards pre-treated dentine after 24 and 48 h was assessed in a modified Boyden chamber assay. Cell adhesion was evaluated indirectly by measuring cell viability. Expression of mineralization-associated genes (COL1A1, ALP, BSP, DSPP, RUNX2) in cells cultured on pre-treated dentine for 7 days was determined by RT-qPCR. Nonparametric statistical analysis was performed for cell migration and cell viability data to compare different groups and time-points (Mann-Whitney U-test, α = 0.05). RESULTS: Treatment of dentine with H2 O or EDTA allowed for cell attachment, which was prohibited by NaOCl with statistical significance (P = 0.000). Furthermore, EDTA conditioning induced cell migration towards dentine. The expression of mineralization-associated genes was increased in dental pulp cells cultured on dentine after EDTA conditioning compared to H2 O-pre-treated dentine discs. CONCLUSIONS: EDTA conditioning of dentine promoted the adhesion, migration and differentiation of dental pulp stem cells towards or onto dentine. A pre-treatment with EDTA as the final step of an irrigation protocol for regenerative endodontic procedures has the potential to act favourably on new tissue formation within the root canal.

Three-dimensional culture of dental pulp stem cells in direct contact to tricalcium silicate cements.

OBJECTIVES: Calcium silicate cements are biocompatible dental materials applicable in contact with vital tissue. The novel tricalcium silicate cement Biodentine™ offers properties superior to commonly used mineral trioxide aggregate (MTA). Objective of this study was to evaluate its cytocompatibility and ability to induce differentiation and mineralization in three-dimensional cultures of dental pulp stem cells after direct contact with the material. MATERIALS AND METHODS: Test materials included a new tricalcium silicate (Biodentine™, Septodont, Saint-Maur-des-Fossés, France), MTA (ProRoot® MTA, DENSPLY Tulsa Dental Specialities, Johnson City, TN, USA), glass ionomer (Ketac™ Molar Aplicap™, 3M ESPE, Seefeld, Germany), human dentin disks and polystyrene. Magnetic activated cell sorting for to the surface antigen STRO-1 was performed to gain a fraction enriched with mesenchymal stem cells. Samples were allowed to set and dental pulp stem cells in collagen carriers were placed on top. Scanning electron microscopy of tricalcium silicate cement surfaces with and without cells was conducted. Cell viability was measured for 14 days by MTT assay. Alkaline phosphatase activity was evaluated (days 3, 7, and 14) and expression of mineralization-associated genes (COL1A1, ALP, DSPP, and RUNX2) was quantified by real-time quantitative PCR. Nonparametric statistical analysis for cell viability and alkaline phosphatase data was performed to compare different materials as well as time points (Mann-Whitney U test, α = 0.05). RESULTS: Cell viability was highest on tricalcium silicate cement, followed by MTA. Viability on glass ionomer cement and dentin disks was significantly lower. Alkaline phosphatase activity was lower in cells on new tricalcium silicate cement compared to MTA, whereas expression patterns of marker genes were alike. CONCLUSIONS: Increased cell viability and similar levels of mineralization-associated gene expression in three-dimensional cell cultures on the novel tricalcium silicate cement and mineral trioxide aggregate indicate that the material is cytocompatible and bioactive. CLINICAL RELEVANCE: The tested new tricalcium silicate cement confirms its suitability as an alternative to MTA in vital pulp therapy.

Infection and immunity in broiler chicken breeders vaccinated with a temperature-sensitive mutant of Mycoplasma gallisepticum and impact on performance of offspring.

A comparison of infection and immunity to Mycoplasma gallisepticum (MG) in broiler chicken breeders vaccinated with a temperature-sensitive mutant of MG versus nonvaccinated chickens, and the impact on the performance of their offspring was conducted. Infection and immunity in breeders were assessed by culture and enzyme-linked immunoassay, respectively. However, performance in their offspring was assessed by studying MG infection in embryos, occurrence of infection titers to MG in relation to mortality, and feed conversion in the broilers. Five out of 10 broiler chicken breeder flocks raised on the same multiple-age farm with a long history of mycoplasmosis were vaccinated intraocularly once with a temperature-sensitive MG mutant vaccine (ts-11) at an average age of 7.5 wk; another five breeder flocks were left as unvaccinated controls exposed to field MG. The average recoveries of ts-11 organisms from tracheas and infraorbital sinuses of 41-wk-old vaccinates were 88 and 84%, respectively. No field MG organisms were recovered from vaccinates between 15 and 41 wk of age. The recovery of field MG organisms from tracheas and sinuses of nonvaccinated chickens increased to an average of 100% at 41 wk of age. A significant decrease (P < 0.05) in the average percentage of MG-seroconverted breeders occurred in ts-11-vaccinated flocks in comparison with nonvaccinated, MG-infected flocks at 15, 20, 23, 29, 32, 36, and 41 wk of age. The average infection prevalence by MG in the vitelline membrane of 7-d-old embryos produced by the five unvaccinated breeder flocks peaked at 79% when their respective hatching eggs were collected at 36 wk of breeder's age. Embryos of ts-11-vaccinated flocks had zero prevalence of MG infection at all times between 29 and 57 wk of breeder's age. Seroconversion to MG (average of 17.7%) at 42 d of age was only present in sera of 10 offspring broiler flocks of the nonvaccinated breeders. However, a lack of seroconversion to MG occurred in 10, 42-d-old offspring broiler flocks of the five ts-11-vaccinated breeder flocks. This lack was associated with a lower, better average feed-conversion ratio (2.05) (P < 0.05) and a lower average mortality percentage (5.3%) (P < 0.05) in comparison with those obtained in the offspring of the five unvaccinated, MG-infected breeder flocks. The results indicate that vaccination of broiler chicken breeders with a temperature-sensitive mutant of MG prevented infection by field MG in tracheas and infraorbital sinuses of these breeders and in the vitelline membranes of their embryos. In addition, the broiler offspring of the vaccinated breeders had a better production performance.