Human Tooth Slices Research Articles

Interaction of doped magnesium, zinc and fluoride ions on hydroxyapatite crystals grown on etched human enamel

ObjectivesTo explore the interaction of added magnesium, zinc, and fluoride ions on calcium phosphate crystal organization, morphology, and crystallinephases in enamel remineralization. MethodsDemineralized human tooth slices were immersed in a remineralization solution at pH = 7.3. Mg2+, Zn2+, and F− ions were added at different concentrations. The tooth specimens were stored at 37 °C for 16 h for the formation of a remineralization layer. Crystals formed on the enamel surface were analyzed with FE-SEM for morphology and organization. The elemental composition and mineral phase of these crystals were analyzed with EDS and XRD, respectively. ResultsIncreasing F− concentration affected the size and shape of the hydroxyapatite crystals while Mg2+ did not directly affect the morphology of the hydroxyapatite crystals. In the presence of combined F− with Mg2+, small needle-like crystals with a greater density were formed. Although high Zn2+ concentration tended to inhibit crystal formation, dense needle-like crystals formed homogenous layers after combination of F− with low concentration of Zn2+. The elemental analysis indicated that the Ca/P ratio without F− and Mg2+ was similar to OCP phase, while the Ca/P ratio was close to HA in the presence of F− and Mg2+. ConclusionThere is a synergistic effect of magnesium, zinc and fluoride ions on hydroxyapatite crystallization in enamel remineralization.

Biomimetic chitosan against bioinspired nanohydroxyapatite for repairing enamel surfaces

In this study, chitosan was employed as a novel biomimetic mineralization model to repair damaged enamel to compare its performance with that of bioinspired zinc-doped nanohydroxyapatite. Fifty human premolar tooth slices were prepared, and artificial caries lesions were induced to produce demineralized enamel surfaces. The etched slices were randomly divided into two groups: a chitosan-hydrogel-treated group and a zinc-doped nanohydroxyapatite-treated group. In vitro assessment using energy-dispersive X-ray analysis, X-ray diffraction analysis and scanning electron microscopy was conducted at the baseline, demineralization and remineralization stages. Baseline results were matched with those for normal enamel; a marked reduction in the calcium (Ca)/phosphorus (P) ratio to 1·12 and the lack of the characteristic hydroxyapatite diffraction peaks were detected for demineralized enamel. The remineralization stage revealed evident recovery of the mineral contents (the calcium/phosphorus ratio was 1·61 for the chitosan-treated group and 1·58 for the bioinspired-nanohydroxyapatite-treated one), with apparent distinctive X-ray diffraction patterns of hydroxyapatite in both groups. Scanning electron microscopic analysis showed the absence of etched enamel porosity, with the formation of a newly formed rod-like apatite layer, similar to natural enamel, which extended over the treated enamel surfaces of both groups. Chitosan hydrogel is recommended as a biomimetic mineralization smart system for repairing demineralized carious enamel.

Decellularized human periodontal ligament for periodontium regeneration

Regenerating the periodontal ligament (PDL) is a crucial factor for periodontal tissue regeneration in the presence of traumatized and periodontally damaged teeth. Various methods have been applied for periodontal regeneration, including tissue substitutes, bioactive materials, and synthetic scaffolds. However, all of these treatments have had limited success in structural and functional periodontal tissue regeneration. To achieve the goal of complete periodontal regeneration, many studies have evaluated the effectiveness of decellularized scaffolds fabricated via tissue engineering. The aim of this study was to fabricate a decellularized periodontal scaffold of human tooth slices and determine its regeneration potential. We evaluated two different protocols applied to tooth slices obtained from human healthy third molars. The extracellular matrix scaffold decellularized using sodium dodecyl sulfate and Triton X-100, which are effective in removing nuclear components, was demonstrated to preserve an intact structure and composition. Furthermore, the decellularized scaffold could support repopulation of PDL stem cells near the cementum and expressed cementum and periodontal-ligament-related genes. These results show that decellularized PDL scaffolds of human teeth are capable of inducing the proliferation and differentiation of mesenchymal stem cells, thus having regeneration potential for use in future periodontal regenerative tissue engineering.

Nerve growth factor signalling in pathology and regeneration of human teeth

Nerve growth factor (NGF) is a key regulator of the development and differentiation of neuronal and non-neuronal cells. In the present study we examined the distribution of NGF and its low and high-affinity receptors, p75NTR and TrkA respectively, in permanent human teeth under normal and pathological conditions. In intact functional teeth, NGF, p75NTR and TrkA are weakly expressed in dental pulp fibroblasts and odontoblasts that are responsible for dentine formation, while the NGF and p75NTR molecules are strongly expressed in nerve fibres innervating the dental pulp. In carious and injured teeth NGF and TrkA expression is upregulated in a selective manner in odontoblasts surrounding the injury sites, indicating a link between NGF signalling and dental tissue repair events. Accordingly, NGF and TrkA expression is strongly upregulated in cultured primary human dental mesenchymal cells during their differentiation into odontoblasts. Targeted release of NGF in cultured human tooth slices induced extensive axonal growth and migration of Schwann cells towards the NGF administration site. These results show that NGF signalling is strongly linked to pathological and regenerative processes in human teeth and suggest a potential role for this neurotrophic molecule in pulp regeneration.

(117) Lipopolysaccharide but not lipoteichoic acid induces inflammation and sensitization of nerve ending in the dental pulp

Painful pulpitis is an inflammation of the dental pulp caused by bacterial proliferation near the pulp after tooth damage or caries. Previous data show that toxins produced by bacteria are particularly important in the induction of inflammation, and the toxin lipopolysaccharide can directly activate sensory neurons. In this study we evaluated the effects of the bacterial toxins lipopolysaccharide (LPS) from E. coli and lipoteichoic acid (LTA) from Bacillus subtilis on neuronal sensitization and cytokine production in an ex vivo model using human tooth slices. Healthy 3rd molars were collected and sectioned to obtain 2-3 slices, 1mm thick, per tooth. Tooth slices were treated with either LPS (1 and 100ug/ml), LTA (1 and 100ug/ml) or control media culture for 3 or 24h. After incubation with LPS or vehicle, calcitonin gene related peptide (CGRP) release from peripheral sensory nerve endings was stimulated with capsaicin (6 mM), and the supernatent was collected, concentrated and CGRP was quantified by ELISA. The release of the cytokines (IFNγ, IL10, IL12p70, IL13, IL1β IL2, IL4, IL6, IL8 and TNFα) in the culture media was quantified with an MSD multiplex assay kit. Although 3h treatment with LPS did not induce changes in CGRP or cytokine release compared to control tooth slices, 24h treatment with LPS (100μg/ml) increased the release of CGRP from sensory nerve endings, pro-inflammatory cytokines, IFNγ, IL1β and TNFα and the anti-inflammatory cytokine IL10 from the dental pulp. In contrast, 24h treatment with LTA (1 and 100 μg/ml) did not affect the level of CGRP or cytokine release compared to control tooth slices. In conclusion, LPS is a more important toxin than LTA in producing neuronal sensitization and inflammation that contribute to painful pulpitis.

Radiopacity of Composite Luting Cements Using a Digital Technique.

The aim of this in vitro study was to evaluate the radiopacity of 20 common dental composite luting materials using a digital technique. A 1-mm-thick specimen of each material with a human tooth slice and aluminium step wedge were tested using digital radiographs under four combinations of exposure and voltage. The radiopacity in pixels was determined using computer software. The equivalent thickness of aluminium for each material was then calculated based on the calibration curve. All tested materials except one had higher radiopacity than dentin (p>α; α = 0.01), and 80% of the materials had radiopacity above enamel value (p>α; α = 0.01). Moreover, 40% of tested materials had radiopacity of three times above the minimal International Organization for Standardization (ISO) values for composite luting cements. At all exposure values, the highest radiopacity was for Solocem and Multilink groups of materials, at three to six times above dentin radiopacity. Only Variolink Veneer showed radiopacity below dentin and enamel. Composite luting materials should have radiopacity above ISO values or greater than the dentin or enamel equivalent. The highest radiopacity values were for the Solocem and Multilink family composite luting cements. Clinicians should choose materials with high radiopacity values, and manufacturers should be aware of the radiopacity values when introducing materials on the market.

Variability in Capsaicin-stimulated Calcitonin Gene-related Peptide Release from Human Dental Pulp

IntroductionThe unique innervation and anatomic features of dental pulp contribute to the remarkable finding that any physical stimulation of pulpal tissue is painful. Furthermore, when pathological processes such as caries affect teeth and produce inflammation of the pulp, the pain experienced can be quite intense and debilitating. To better understand these underlying neurobiological mechanisms and identify novel analgesic targets for pulpally derived pain, we have developed a powerful ex vivo model using human tooth slices. MethodsNoncarious, freshly extracted teeth were collected and sectioned longitudinally into 1-mm-thick slices containing both dental pulp and the surrounding mineralized tissues. Tooth slices from 36 patients were exposed to 60 μmol/L capsaicin to stimulate the release of calcitonin gene-related peptide (CGRP) from nerve terminals in the pulp. Patient factors were analyzed for their effects on capsaicin-stimulated CGRP release using a mixed model analysis of variance. ResultsApproximately one third of the variability observed in capsaicin-evoked CGRP release was attributable to differences between individuals. In terms of individual factors, there was no effect of anesthesia type, sex, or age on capsaicin-stimulated CGRP release. Using a within-subject study design, a significant effect of capsaicin on CGRP release was observed. ConclusionsCapsaicin-stimulated CGRP release from dental pulp is highly variable between individuals. A within-subject study design improves the variability and maximizes the potential of this powerful translational model to test the efficacy of novel pharmacotherapeutic agents on human peripheral nociceptors.

L-mimosine increases the production of vascular endothelial growth factor in human tooth slice organ culture model.

To assess the pro-angiogenic and pro-inflammatory capacity of the dentine-pulp complex in response to the prolyl hydroxylase inhibitor L-mimosine in a tooth slice organ culture model. Human teeth were sectioned transversely into 600-μm-thick slices and cultured in medium supplemented with serum and antibiotics. Then, pulps were stimulated for 48h with L-mimosine. Pulps were subjected to viability measurements based on formazan formation in MTT assays. In addition, histological evaluation of pulps was performed based on haematoxylin and eosin staining. Culture supernatants were subjected to immunoassays for vascular endothelial growth factor (VEGF) to determine the pro-angiogenic capacity and to immunoassays for interleukin (IL)-6 and IL-8 to assess the pro-inflammatory response. Interleukin-1 served as pro-inflammatory control. Echinomycin was used to inhibit hypoxia-inducible factor-1 (HIF-1) alpha activity. Data were analysed using Student's t-test and Mann-Whitney U test. Pulps within tooth slices remained vital upon L-mimosine stimulation as indicated by formazan formation and histological evaluation. L-mimosine increased VEGF production when normalized to formazan formation in the pulp tissue of the tooth slices (P<0.05). This effect on VEGF was reduced by echinomycin (P<0.01). Changes in normalized IL-6 and IL-8 levels upon treatment with L-mimosine did not reach the level of significance (P>0.05), whilst treatment with IL-1, which served as positive control, increased IL-6 (P<0.05) and IL-8 levels (P<0.05). The prolyl hydroxylase inhibitor L-mimosine increased VEGF production via HIF-1 alpha in the tooth slice organ culture model whilst inducing no prominent increase in IL-6 and IL-8. Pre-clinical studies will reveal if these in vitro effects translate into dental pulp regeneration.

A hydrogel scaffold that maintains viability and supports differentiation of dental pulp stem cells

ObjectivesThe clinical translation of stem cell-based Regenerative Endodontics demands further development of suitable injectable scaffolds. Puramatrix™ is a defined, self-assembling peptide hydrogel which instantaneously polymerizes under normal physiological conditions. Here, we assessed the compatibility of Puramatrix™ with dental pulp stem cell (DPSC) growth and differentiation. MethodsDPSC cells were grown in 0.05–0.25% Puramatrix™. Cell viability was measured colorimetrically using the WST-1 assay. Cell morphology was observed in 3D modeling using confocal microscopy. In addition, we used the human tooth slice model with Puramatrix™ to verify DPSC differentiation into odontoblast-like cells, as measured by expression of DSPP and DMP-1. ResultsDPSC survived and proliferated in Puramatrix™ for at least three weeks in culture. Confocal microscopy revealed that cells seeded in Puramatrix™ presented morphological features of healthy cells, and some cells exhibited cytoplasmic elongations. Notably, after 21 days in tooth slices containing Puramatrix™, DPSC cells expressed DMP-1 and DSPP, putative markers of odontoblastic differentiation. SignificanceCollectively, these data suggest that self-assembling peptide hydrogels might be useful injectable scaffolds for stem cell-based Regenerative Endodontics.

Radiopacity evaluation of composite restorative resins and bonding agents using digital and film x-ray systems

The purpose of this in vitro study was to explore the radiopacity of composite resins and bonding materials using film and phosphor plates. Nine composite dental resin specimens and human tooth slices were exposed together with an aluminium stepwedge using dental film and phosphor plates. Eight dentin bonding specimens were prepared and exposed in a similar manner. Their radiopacity on film was assessed using a transmission densitometer, and the radiopacity with phosphor plates was assessed digitally using the system's own software (Digora). Data were analysed using one-way analysis of variance (ANOVA) and post-hoc Tukey tests (P<.05). Film and phosphor plate radiopacity values were compared using simple regression analysis. Excellent linear correlation was found between film and phosphor plates for both composite resins and bonding agents. The composite materials Spectrum Tph and Natural Look exhibited the highest radiopacity with film and with phosphor plates, respectively. All the dentin bonding agents tested exhibited lower radiopacity than dentin. Synergy, Ice, Filtek Silorane, Filtek Z250, Clearfil Majesty Posterior, Herculite Classic, Spectrum Tph, and Natural Look composite materials exhibited greater radiopacity than dentin, and all the dentin bonding agents tested exhibited lower radiopacity than either enamel or dentin.

Long term effect of Low Intensity Pulsed Ultrasound on a human tooth slice organ culture

ObjectiveInvestigate the effect of therapeutic Low Intensity Pulsed Ultrasound (LIPUS) on human dentine–pulp complex in an in vitro model. Design92 premolars were extracted from 23 adolescent orthodontic patients. The premolars were sectioned transversely into 600μm thick slices. The slices were divided into two main groups according to how often the LIPUS was applied (single or daily application), and then subdivided into five subgroups each (5, 10, 15 and 20min and one control group). The tooth slices were cultured at (37°C/5% CO2) in a humidified incubator where medium was changed every 48h. LIPUS was applied using a 3.9cm2 transducer that produces an incident intensity of 30mW/cm2. After five days, tissue was harvested for histomorphometrical analysis and real time PCR to investigate expression of genes of interest (Collagen I, DMP1, DSPP, TGF-β1, RANKL and OPG). ResultsHistomorphometric analyses revealed that odontoblast cell count was higher in the single application groups (5, 10 and 15min, respectively) than in the control and other treatment groups. Predentin thickness was higher in the single application group (10, 5 and 15min) respectively than in the daily application group and the control groups, however they were not significantly different from each other. Real time PCR demonstrated no statistically significant difference between the groups in the expression of Collagen I, DMP1, TGF-β1, DSPP, RANKL and OPG. ConclusionReproducible responses from cultured dentine–pulp complex were observed in groups with single application of LIPUS for 5, 10 and 15min.

Biomimetic Synthesis of Fluorapatite Coating

In the present study, a simple method of regenerating microstructure of human tooth under near-physiological conditions (pH 7.0, 37 °C, 1 atm) was developed. Commercial gelatin was used as matrix materials in this method, which nucleated the formation of fluorapatite (FA) nanocrystals and regulated the growth of nanocrystals. As a result, the resulting thin FA coatings had been prepared on human tooth slices and sintered hydroxyapatite disks, which were in tight contact with the substrates. Besides, the morphologies of FA nanocrystals changed from acicular to hexagonal with the exchange cycle of gel increased. Electron dispersive spectrometer analysis indicated that some sodium and carbonate ions were incorporated into the FA crystal lattices and the calcium to phosphorus ratio was approximate 1.58. The mechanical properties of the resulting FA coating were investigated through nanoindentation system, which showed the similar hardness with dentin. In conclusion, this method demonstrated a potential application to repair tooth damage in dental clinics.

Directional x-ray dark-field imaging of strongly ordered systems

Recently a novel grating based x-ray imaging approach called directional x-ray dark-field imaging was introduced. Directional x-ray dark-field imaging yields information about the local texture of structures smaller than the pixel size of the imaging system. In this work we extend the theoretical description and data processing schemes for directional dark-field imaging to strongly scattering systems, which could not be described previously. We develop a simple scattering model to account for these recent observations and subsequently demonstrate the model using experimental data. The experimental data includes directional dark-field images of polypropylene fibers and a human tooth slice.

Evaluation of the radiopacity of different root canal sealers

The aims of this study were to compare the radiopacity of 8 root canal sealers relative to gutta-percha and dentin in standard discs and to evaluate the effect of these sealers on the radiopacity of root canal fillings in simulated canals. Radiographs were taken of 1-mm-thick specimens of 8 root canal sealers (Diaket, Endion, MTA, Endofil, Roeko Seal, Sealite, AH26, AH Plus) and gutta-percha, a 1-mm-thick human tooth slice, and aluminum stepwedge.Simulated canals were used to evaluate the effect of the sealer on radiopacity of the root fillings. After enlargement of the simulated canals with ProTaper instrument, root canals were filled with these 8 sealers alone and with single ProTaper gutta-percha cone. Radiographs of all filled simulated canals were taken with an aluminum stepwedge. All the sealers demonstrated greater radiopacity than dentin (0.7940 mm Al) (P < .001). AH Plus showed the highest radiopacity in the standard disc group (Group 1) (8.9881 mm Al) (P < .001), and in the sealer group in simulated canals (Group 2) (9.2100 mm Al) (P < .001). In the sealer plus gutta-percha group in simulated canals (Group 3), Sealite plus gutta-percha showed greater radiopacity (8.4460 mm Al) (P < .001). Whether the opacity of the sealers alone is more or less than 3 mm Al, their radiopacity is increased when they are used in combination with gutta-percha, because of its higher radiopacity. However, when sealers are used in conjunction with gutta-percha, they can affect the radiopacity of the root canal filling according to their type and thickness.

Dental Pulp Tissue Engineering with Stem Cells from Exfoliated Deciduous Teeth

Stem cells from human exfoliated deciduous teeth (SHED) have been isolated and characterized as multipotent cells. However, it is not known whether SHED can generate a dental pulp-like tissue in vivo. The purpose of this study was to evaluate morphologic characteristics of the tissue formed when SHED seeded in biodegradable scaffolds prepared within human tooth slices are transplanted into immunodeficient mice. We observed that the resulting tissue presented architecture and cellularity that closely resemble those of a physiologic dental pulp. Ultrastructural analysis with transmission electron microscopy and immunohistochemistry for dentin sialoprotein suggested that SHED differentiated into odontoblast-like cells in vivo. Notably, SHED also differentiated into endothelial-like cells, as demonstrated by B-galactosidase staining of cells lining the walls of blood-containing vessels in tissues engineered with SHED stably transduced with LacZ. This work suggests that exfoliated deciduous teeth constitute a viable source of stem cells for dental pulp tissue engineering.

In vitro study of the effects of copper ion on osteoclastic resorption in various dental mineralized tissues

To investigate the effects of copper ion on osteoclastic resorption in various dental mineralized tissues. Osteoclasts were separated from long-limb bones of neonatal rabbits, and cultured with de-activated human tooth slices and glass slices. The cells in the experiment group were treated with (1 x 10(-14))-1 x (10(-4)) mol/L copper + 10% (volume fraction) fetal calf serum (FCS) + alpha-MEM, while the cells in control group cells were grown in 10% FCS + alpha-MEM. Osteoclasts on glass slices were stained by TRAP staining. The absorption pits on tooth slices were observed by inverted phase contrast microscope. The resorbing activity was evaluated with the concentration of calcium in the supernatant liquid of osteoclasts. The ratio between the concentration of calcium in the experiment group and control group was termed as the resorption index. The isolated cells were multinuclear and TRAP positive in cytoplasma. Osteoclasts resorbed teeth slices first on the cementum and dentin. Compared with those on bone slices, the lacunae on the dental slices numbered less, with smaller volume and shallower in depth. Microscopy showed that the number and area of absorption pits formed on treated tissues were less than those on control tissues. The content of calcium in the supernatant liquid decreased in the concentrations of 1 x 10(-14) mol/L - 1 x 10(-4) mol/L copper, especially in the group of 1 x 10(-10) mol/L copper at 3rd day (P < 0.05) and 1 x 10(-4) mol/L, 1 x 10(-10) - 1 x 10(-12) mol/L copper at 7th day (P < 0.05). Their resorption index was lower than one. Extracellular copper ion can inhibit osteoclastic resorption on dental slices.

Radiopacity of calcium hydroxide cement compared with human tooth structure

All materials added to teeth should present an adequate radiopacity to allow the detection of secondary caries. Usually, in extensive cavities, base materials like calcium hydroxide cement are used for the purpose of protecting the pulp. In an attempt to improve the efficiency of radiographic detection of this material, this study aimed to determine the radiopacity of three calcium hydroxide cements and to compare the radiopacity of these materials with dentin and enamel. Radiographs were taken of 1-mm thick specimens of three calcium hydroxide cements: Hydro-C, Dycal and Life, an aluminium stepwedge, a lead foil, and one 1-mm thick human tooth slice. Densitometric measurements were obtained after radiographic processing. The radiopacity values of the calcium hydroxide cements, dentin and enamel were expressed in terms of the equivalent thickness of aluminium. The analysis of variance indicated statistically significant difference only for Life, which presented the lowest radiopacity when compared to the other cements. However, all cements and enamel possessed a radiopacity equivalent to 2mm Al, while dentin presented a radiopacity equivalent to 1mm Al. All tested cements presented a similar radiopacity to that of enamel and they meet the ISO 4049 specifications.

In vitro study of a neodynium:yttrium aluminum perovskite laser on human nonexposed pulp after cavity preparation.

The aim of this study was to investigate dental pulp reactions after a neodynium:yttrium aluminum perovskite laser pulse on the dentinal floor of occlusal cavities in an in vitro model. A Lokki dt laser was used at 30 Hz, 5 W, and 160 mJ for 0.5 s. The pulp reactions were analyzed in a previously described human tooth slice cultured model. The following markers were identified by immunohistochemistry: collagens I, III, and IV and HLA-DR-positive cells. After 4 days of culture, under laser pulse, a concentration of type III collagen beneath the odontoblast layer, a higher level of vessels and an accumulation of HLA-DR-positive cells were routinely observed subjacent to the cavity. This laser treatment leads to the first step of rapid pulp repair under culture conditions.

TGF-beta1 induces accumulation of dendritic cells in the odontoblast layer.

TGF-beta1 released from dentin degraded by bacterial or iatrogenic agents is suspected to influence dental pulp response, including the modulation of cell migration. To determine the consequences of TGF-beta1 action on pulp immune cells, we analyzed, by immunohistochemistry, the effect of transdentinally diffusing TGF-beta1 on their localization in a human tooth slice culture model. TGF-beta1 induced an accumulation of HLA-DR-positive cells in both odontoblast and subodontoblast layers of the stimulated zone. Together with HLA-DR, these cells co-expressed Factor XIIIa and CD68, two features of immature antigen-presenting dendritic cells (DC), as well as the TGF-beta1 specific receptor TbetaRII. In contrast, no effect could be detected on the localization of either mature DC-LAMP-positive DC or of T- and B-lymphocytes. Analysis of these data suggests that TGF-beta1 released from dentin degraded by bacterial or iatrogenic agents could be involved in the immune response of the dental pulp resulting from tooth injury.

Effects of Alginate Hydrogels and TGF-β1 on Human Dental Pulp Repair In Vitro

The objective of this study was to investigate the use of alginate hydrogels to present either exogenous or endogenous transforming growth factor (TGF)- g 1 to the dentin-pulp complex to signal reparative processes. Hydrogels were prepared, applied to cultured human tooth slices and the effects on tertiary dentinogenesis examined histologically. Both TGF- g 1-containing and acid-treated alginate hydrogels, but not untreated hydrogels, upregulated dentin matrix secretion and induced odontoblast-like cell differentiation with subsequent secretion of regular tubular dentin matrix on cut pulpal surfaces. It is concluded that TGF- g 1 can signal both induction of odontoblast-like cell differentiation and upregulation of their matrix secretion in the human dentin-pulp complex. Alginate hydrogels provide an appropriate matrix in which dental regeneration can take place and may also be useful for delivery of growth factors, including TGF- g s, to enhance the natural regenerative capacity of the dental pulp.