P. J. Lumley

Ultrastructural Localisation of TGF-β Exposure in Dentine by Chemical Treatment

Transforming growth factor-β (TGF-β) sequestered in dentine matrix has an important role in dental tissue repair after injury and its exposure at sites of injury may stimulate tertiary dentinogenesis. This study aimed to investigate the expression of TGF-β isoforms in mature human dentine matrix and the ability of chemical treatments to expose TGF-β on the cut surface of dentine using gold immunolabelling and subsequent scanning electron microscopy examination. TGF-β1 was the only isoform that could be detected in human dentine and the nature of the chemical treatment of the tissue influenced its detection. EDTA treatment provided good exposure of TGF-β1 on the dentine surface, whilst citric acid and sodium hypochlorite treatments revealed lesser amounts of this isoform. Only minimal staining for TGF-β1 was observed in samples treated with phosphate-buffered saline. TGF-β2 and -β3 could not be detected in the specimens with any of the treatments. This study suggests that TGF-β1 is the only TGF-β isoform expressed by human odontoblasts to be sequestered in dentine implying that differences in isoform–extracellular matrix interactions may exist. Information on chemical treatment of tissue specimens for immunostaining may provide a useful basis for selection of tissue preparation techniques for clinical restorative treatment procedures to facilitate TGF-β mediated reparative processes at sites of dental injury.

Tooth slice organ culture for cytotoxicity assessment of dental materials

The purpose of this work was to develop a tooth slice organ culture method to assess the response of the cells of the dental pulp to commonly used dental materials and products. Wistar rat tooth slices were grown in culture for two and ten days in the presence of dental materials. After culture, the tooth tissues were processed and the responses of the pulpal cells were analysed histomorphometrically. Cytotoxic cell destruction was observed following the direct application of test materials to tooth slices (n = 298) after 10 days in culture (MANOVA, P = 0.0001), whilst the restoration of prepared deep dentine cavities (n = 30), with test products, did not result in a significant amount of pulpal injury (MANOVA, P = 0.287). In rank order of causing pulpal injury, the test materials from the most to the least cell destructive, was; Salicylic acid. Calcium hydroxide, Kalzinol zinc oxide eugenol, high-mercury Amalgam, Prime & Bond, Dycal, Barium sulphate, Hypocal, Scotchbond, Calasept, Life and One-step. Tooth slice organ culture, provided a cytotoxicity screening method for dental materials, bearing a closer physiological resemblance to the clinical situation than cell culture screening methods. Tooth slice culturing may have the potential to replace some types of in vivo animal experimentation, as there is a clear need to reduce this form of testing.

Molecular characterization of young and mature odontoblasts

UNLABELLED The odontoblast is the secretory cell responsible for primary, secondary and tertiary reactionary dentinogenesis. We provide evidence that the changes in secretory activity of odontoblasts reflect differential transcriptional control and that common regulatory processes may exist between dentine and bone. INTRODUCTION Based on the hypothesis that differential dentine secretion (primary and secondary dentinogenesis) is associated with changes in the transcriptional control within the cell, we have investigated the transcriptome of odontoblasts at young and mature stages and subsequently used this information to identify key regulatory intracellular pathways involved in this process. MATERIALS AND METHODS We used microarray analysis to compare the transcriptome of early stage (primary dentinogenesis) and late stage (secondary dentinogenesis) odontoblasts from 30 month old bovine teeth. Secondarily, we used post-array sqRT-PCR to confirm the differential expression of 23 genes in both populations of odontoblasts. Finally, immunohistochemistry was performed on bovine and murine tissues with antibodies to DMP1 and anti-phospho p38 proteins. RESULTS DMP-1 and osteocalcin gene expression were up-regulated in the mature odontoblasts, whereas collagen I, DSPP, TGF-beta1 and TGF-beta1R gene expression were down-regulated. Microarray analysis highlighted 574 differentially regulated genes (fold change>2 - p<0.05). This study supports further existing similarities between pulp cells and bone cells. Using post-array Sq-RT-PCR we characterized transcript levels of genes involved in the p38 MAP kinase pathway (PTPRR, NTRKK2, MAPK13, MAP2K6, MKK3). Differential p38 gene activation was confirmed by immunohistochemistry for p38 protein in murine teeth. Finally, immunohistochemistry for DMP1 indicated that odontoblasts involved in primary and secondary dentinogenesis may coexist in the same tooth. CONCLUSION As established in bone cells, the transcriptome of the odontoblast was shown here to evolve with their stage and functional maturity. Identification of the involved signalling pathways, as highlighted for p38, will enable the deciphering of physiology and pathology of mineralised tissue formation.

Human odontoblast cell numbers after dental injury

OBJECTIVES The purpose of this study was to measure the changes in odontoblast cell numbers in response to cavity restoration variables and patient factors, and the effect these factors have on dental repair by tertiary dentinogenesis. The number of vital odontoblasts is a critical factor for pulpal repair following restorative surgery, and yet little information is available on these cell numbers. METHODS Class V non-exposed cavities were prepared in the buccal surface of intact first or second premolar teeth of 27 patients, between 9 and 17 years of age. Following tooth extraction (28-163 days) the area of reactionary dentine and the area of the odontoblasts were measured histomorphometrically. RESULTS Patient factors, as well as cavity preparation and restoration variables, had little effect on the numbers of odontoblasts per pulpal unit area. However, the age of the patient did appear to have an effect on the reactionary dentine secretory capacity of odontoblasts per unit area, and on the relative number of odontoblasts beneath cut dentinal tubules. CONCLUSIONS Odontoblast cell numbers were maintained following the preparation of cavities cut into dentine with a 0.5mm residual dentine thickness. The repair capacity of the pulp-dentine complex would appear to be age dependent, this may explain differences in the success of various restorative treatments between patients.

Ultrasound in dentistry. Part 2—periodontology and endodontics

Ultrasound in the kHz frequency range is used widely in clinical dentistry. The most common uses are in the fields of periodontology and endodontics. The ultrasonic scaler works by the vibratory chipping action of the oscillating tip and is assisted by the presence of cavitational activity in the associated cooling water. When assessing clinical studies it is often difficult to interpret results from different workers due to the lack of standardization of the ultrasonic scaler. Operators should be aware of the oscillatory pattern of different instruments. Endosonics utilizes an ultrasonically oscillating endodontic file to clean and shape the root canal prior to obturation. The cleaning ability of such files is assisted by the occurrence of acoustic microstreaming forces. The endosonic file is prone to constraint when it contacts the canal wall which alters its oscillatory pattern. Clinical techniques should be modified to reduce this problem.

The MAP Kinase Pathway Is Involved in Odontoblast Stimulation via p38 Phosphorylation

INTRODUCTION We have previously shown that the p38 gene is highly expressed in odontoblasts during active primary dentinogenesis, but is drastically down-regulated as cells become quiescent in secondary dentinogenesis. Based on these observations, we hypothesized that p38 expression might be upregulated, and the protein activated by phosphorylation, when odontoblasts are stimulated such as during tertiary reactionary dentinogenesis. METHODS We stimulated immortalized, odontoblast-like MDPC-23 cells, alone or in combination, with heat-inactivated Streptococcus mutans, EDTA-extracted dentine matrix proteins (DMPs), or growth factors, including transforming growth factor (TGF)-beta1, tumor necrosis factor-alpha (TNF-alpha), and adrenomedullin (ADM). We used ELISA to measure the resulting phosphorylation of the p38 protein, as well as its degree of nuclear translocation. RESULTS Our results suggest that the p38-MAPKinase pathway is activated during odontoblast stimulation in tertiary dentinogenesis by both p38 phosphorylation and enhanced nuclear translocation. CONCLUSIONS Data indicate that odontoblast behaviour therefore potentially recapitulates that during active primary dentinogenesis.

Incidence of root face alteration after ultrasonic retrograde cavity preparation

PROBLEM Ultrasonic root end preparation is now a recognized clinical procedure. Advantages claimed include improved access to the surgical site (because of reduced root end bevel), and faster more conservative preparation of the root end cavity. However, cracking of the root face has been reported after preparation. OBJECTIVES The aim of this study was to determine the incidence of root end cracking at varying ultrasonic power levels with a replica technique and scanning electron microscopy. STUDY DESIGN The root canals of 55 single-rooted teeth were prepared to size 40 apically and obturated with laterally condensed gutta perch and sealer. Root ends were resected at 90 degrees 3 mm from the apex. Class 1 cavities (n = 5) were prepared with retro tips in a Neosonic handpiece with different power settings. Time of preparation and load applied were constant. Another experimental group (n = 5) was prepared with a no. 1 rotary bur. A replica technique with addition-cured silicone impression material and epoxy resin was used to prevent drying artefacts. Specimens were viewed under scanning electron microscope for alterations of the root face. RESULTS Results showed no root face cracking across the full range of instrument power settings, although chipping of the retrograde cavity margins was observed. No cracking was noted in bur-prepared teeth.

Dental regeneration and materials—a partnership

Considerable focus on the biocompatibility of dental materials over the last three decades has provided a platform for a wealth of studies on the cellular and molecular responses of the cells of the pulp to injury, both from the disease process and from subsequent restorative intervention. These studies have been fundamental to understanding not only how we can achieve a biocompatible response during restoration of dental disease but also how we can exploit the pulpal cellular responses to achieve wound healing and tissue regeneration in the dentine–pulp complex. This article examines the responses of the pulp to injury and the events leading to tissue regeneration. As new biologically based regenerative therapies emerge for the dental tissues, it is important that these develop in partnership with more traditional approaches using dental materials.

Effect of exposure intensity and post-cure temperature storage on hardness of contemporary photo-activated composites

OBJECTIVES The effect of variation in post-exposure storage temperature (18 vs. 37 degrees C) and light intensity (200 vs. 500mW/cm(2)) on micro-hardness of seven light-activated resin composite materials, cured with a Prismetics Mk II (Dentsply) light activation unit, were studied. METHODS Hardness values at the upper and lower surfaces of 2mm thick disc shaped specimens of seven light-cured resin composite materials (Herculite XRV and Prodigy/Kerr, Z100 and Silux Plus/3M, TPH/Dentsply, Pertac-Hybrid/Espe, and Charisma/Kulzer), which had been stored dry, were determined 24h after irradiation with a Prismetics Mk II (Dentsply) light activation unit. RESULTS Hardness values varied with product, surface, storage temperature, and curing light intensity. In no case did the hardness at the lower surface equal that of the upper surface, and the combination of 500mW/cm(2) intensity and 37 degrees C storage produced the best hardness results at the lower surface. CONCLUSIONS Material composition had a significant influence on surface hardness. Only one of the seven products (TPH) produced a mean hardness values at the lower surface >80% of the maximum mean upper surface hardness obtained for the corresponding product at 500mW/cm(2) intensity/37 degrees C storage temperature when subjected to all four test regimes. Despite optimum post-cure storage conditions, 200mW/cm(2) intensity curing for 40s will not produce acceptable hardness at the lower surface of 2mm increments of the majority of products tested.

The flexural properties of endodontic post materials

OBJECTIVES To measure the flexural strengths and moduli of endodontic post materials and to assess the effect on the calculated flexural properties of varying the diameter/length (D/L) ratio of three-point bend test samples. METHODS Three-point bend testing of samples of 2mm diameter metal and fiber-reinforced composite (FRC) rods was carried out and the mechanical properties calculated at support widths of 16 mm, 32 mm and 64 mm. Weibull analysis was performed on the strength data. RESULTS The flexural strengths of all the FRC post materials exceeded the yield strengths of the gold and stainless steel samples; the flexural strengths of two FRC materials were comparable with the yield strength of titanium. Stainless steel recorded the highest flexural modulus while the titanium and the two carbon fiber materials exhibited similar values just exceeding that of gold. The remaining glass fiber materials were of lower modulus within the range of 41-57 GPa. Weibull modulus values for the FRC materials ranged from 16.77 to 30.09. Decreasing the L/D ratio produced a marked decrease in flexural modulus for all materials. SIGNIFICANCE The flexural strengths of FRC endodontic post materials as new generally exceed the yield strengths of metals from which endodontic posts are made. The high Weibull modulus values suggest good clinical reliability of FRC posts. The flexural modulus values of the tested posts were from 2-6 times (FRC) to 4-10 times (metal) that of dentin. Valid measurement of flexural properties of endodontic post materials requires that test samples have appropriate L/D ratios.