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To clarify the terminology and its application in the context of materials placed in contact with tissues in living systems.Review the nature of both the biological and chemical reactions occurring in such contexts with a view to a classification of types of behaviour and thus types of material.A range of types of behaviour was identified with clear diagnostic characteristics. Materials could thus be classified accordingly, with the possibility of more than one type of behaviour being exhibited. 'Bioactivity' was concluded to depend solely on the modulation of a natural biological process, but excluding pharmaceutical action.Clarity in the characterisation and labelling of materials and their behaviours in biological systems will benefit the interpretation of the literature, the design of experiments, and future research directions.

Objective Acids, such as those used in adhesive dentistry, have been shown to solubilize bioactive molecules from dentin. These dentin matrix components (DMC) may promote cell proliferation and differentiation, and ultimately contribute to dentin regeneration. The objective of this study was to evaluate the potential for varying concentrations of DMC extracted from human dentin by phosphoric acid of a range of pHs to stimulate proliferation and mineralization of two different cultured pulp cell populations. Methods DMC were solubilized from powdered human dentin (7 days – 4 °C) by phosphoric acid of pH 1, 3, and 5 and also, EDTA. Extracts were dialyzed for 7 days against distilled water and lyophilized. Undifferentiated mouse dental pulp cells (OD-21) and cells of the odontoblast-like cell line (MDPC-23) were seeded in six-well plates (1 × 10 5 ) and cultured for 24 h in DMEM (Dulbecco's modified Eagle's medium) containing 10% (v/v) FBS (fetal bovine serum). The cells were washed with serum-free medium and then treated with different concentrations of DMC (0.01, 0.1, 1.0 and 10.0 μg/ml) daily in serum free medium for 7 days. After 3, 5 (MDPC-23 only), and 7 days of treatment, cell proliferation was measured using 10 vol% Alamar blue solution, which was added to each well for 1 h. Cell numbers were first measured by cell counting (Trypan blue; n = 5) and Alamar blue fluorescence to validate the assay, which was then used for the subsequent assessments of proliferation. Mineralization was assessed by Alizarin Red S assay after 12 days exposure to DMC ( n = 5). Controls were media-only (DMEM) and dexamethasone (DEX; positive control). Results were analysed by ANOVA/Tukey's ( p ≤ 0.05). Results There was a linear correlation between cell counts and Alamar blue fluorescence ( R 2 > 0.96 for both cell types) , verifying the validity of the Alamar blue assay for these cell types. In general, there was a dose-dependent trend for enhanced cell proliferation with higher concentration of DMC for both cell lines, especially at 10.0 μg/ml. DEX exposure resulted in significantly higher mineralization, but did not affect cell proliferation. DMC exposure demonstrated significantly greater mineralization than media-only control for 10 μg/ml for all extracts, and at lower concentrations for EDTA and pH 5 extracts. Significance Human dentin matrix components solubilized by acids at pH levels found in commercial dentin adhesives enhanced cell proliferation and mineralization of mouse and rat undifferentiated dental pulp cells when presented in adequate concentration.

Introduction The development of regenerative endodontic therapies offers exciting opportunities for future improvements in treatment outcomes. Methods Advances in our understanding of regenerative events at the molecular and cellular levels are helping to underpin development of these therapies, although the various strategies differ in the translational challenges they pose. The identification of a variety of bioactive molecules, including growth factors, cytokines, chemokines, and matrix molecules, sequestered within dentin and dental pulp provides the opportunity to present key signaling molecules promoting reparative and regenerative events after injury. Results and Conclusions The protection of the biological activity of these molecules by mineral in dentin before their release allows a continuing supply of these molecules, while avoiding the short half-life and the non-human origin of exogenous molecules. The ready release of these bioactive molecules by the various tissue preparation agents, medicaments, and materials commonly used in endodontics highlights the opportunities for translational regenerative strategies exploiting these molecules with little change to existing clinical practice.

The transcription factor Nuclear Factor I-C (NFIC) has been implicated in the regulation of tooth root development, where it may be anticipated to impact on the behavior of stem cells from the apical papilla (SCAPs) and root odontoblast activity. We hypothesized that NFIC may provide an important target for promoting dentin/root regeneration. In the present study, the effects of NFIC on the proliferation and differentiation of SCAPs were investigated. Over-expression of NFIC increased cell proliferation, mineralization nodule formation and alkaline phosphatase (ALP) activity in SCAPs. Furthermore, NFIC up-regulated the mRNA levels of odontogenic-related markers, ALP, osteocalcin and collagen type I as well as dentin sialoprotein protein levels. In contrast, knockdown of NFIC by si-RNA inhibited the mineralization capacity of SCAPs and down-regulated the expression of odontogenic-related markers. In conclusion, the results indicated that upregulation of NFIC activity in SCAPs may promote osteo/odontoblastic differentiation of SCAPs.

The role of Australian rural radiographers in radiographic interpretation, communication and disclosure of their radiographic opinion with a specific focus on plain film radiography was examined in a two phase, exploratory interpretive study. Data were collected using questionnaires and interviews and analysed thematically. This reports one of the key themes identified in the thematic data analysis. ‘Disclosure of Radiographic Opinion to Patients’ comprises the three interrelated sub-themes Acting Ethically, Selective Disclosure and Filtered Truth. It is wholly concerned with the ways in which rural radiographers choose to disclose their radiographic opinion to patients. Without a clear picture of where they stand medico-legally, rural radiographers draw on experience and a strong ethical framework as the basis for these complex decisions. Rural radiographers frame their disclosures to patients in a manner that is governed by the diagnostic, therapeutic and emotional impact the information disclosed may have on the patient. Disclosure to patients was found to be selective, often diagnostically vague and ethically filtered.

The effects of cryopreservation on mesenchymal stem cell (MSC) phenotype are not well documented; however this process is of increasing importance for regenerative therapies. This study examined the effect of cryopreservation (10% dimethyl-sulfoxide) on the morphology, viability, gene-expression and relative proportion of MSC surface-markers on cells derived from rat adipose, bone marrow and dental pulp. Cryopreservation significantly reduced the number of viable cells in bone marrow and dental pulp cell populations but had no observable effect on adipose cells. Flow cytometry analysis demonstrated significant increases in the relative expression of MSC surface-markers, CD90 and CD29/CD90 following cryopreservation. sqRT-PCR analysis of MSC gene-expression demonstrated increases in pluripotent markers for adipose and dental pulp, together with significant tissue-specific increases in CD44, CD73–CD105 following cryopreservation. Cells isolated from different tissue sources did not respond equally to cryopreservation with adipose tissue representing a more robust source of MSCs.

The traditional concept of replacing diseased tooth/pulp tissues by inert materials (restoration) is being challenged by recent advances in pulp biology leading to regenerative strategies aiming at the generation of new vital tissue. New tissue formation in the pulp chamber can be observed after adequate infection control and the formation of a blood clot. However, differentiation of true odontoblasts is still more speculative, and the approach is largely limited to immature teeth with open apices. A more systematic approach may be provided by the adoption of the tissue engineering concepts of using matrices, suitable (stem) cells, and signaling molecules to direct tissue events. With these tools, pulplike constructs have already been generated in experimental animals. However, a number of challenges still remain for clinical translation of pulp regeneration (eg, the cell source [resident vs nonresident stem cells, the latter associated with cell-free approaches], mechanisms of odontoblast differentiation, the pulp environment, the role of infection and inflammation, dentin pretreatment to release fossilized signaling molecules from dentin, and the provision of suitable matrices). Transition as a process, defined by moving from one form of “normal” to another, is based not only on the progress of science but also on achieving change to established treatment concepts in daily practice. However, it is clear that the significant recent achievements in pulp biology are providing an exciting platform from which clinical translation of dental pulp regeneration can advance.

From the Unit of Oral Biology, School of Dentistry, University of Birmingham, St Chads Queensway, Birmingham, United Kingdom. This paper is based on a presentation from the International Association for Dental Research (IADR) Pulp Biology and Regeneration Group Satellite Meeting, which was held March 24–26, 2013 in San Francisco, California. Address requests for reprints to Dr Anthony J. Smith, Unit of Oral Biology, School of Dentistry, University of Birmingham, St Chads Queensway, Birmingham B4 6NN, UK. E-mail address: a.j.smith@bham.ac.uk 0099-2399/$ see front matter Copyright a 2014 American Association of Endodontists. http://dx.doi.org/10.1016/j.joen.2014.01.037

Dental tissue infection and disease result in acute and chronic activation of the innate immune response, which is mediated by molecular and cellular signaling. Different cell types within the dentin-pulp complex are able to detect invading bacteria at all stages of the infection. Indeed, at relatively early disease stages, odontoblasts will respond to bacterial components, and as the disease progresses, core pulpal cells including fibroblasts, stems cells, endothelial cells, and immune cells will become involved. Pattern recognition receptors, such as Toll-like receptors expressed on these cell types, are responsible for detecting bacterial components, and their ligand binding leads to the activation of the nuclear factor-kappa B and p38 mitogen-activated protein (MAP) kinase intracellular signaling cascades. Subsequent nuclear translocation of the transcription factor subunits from these pathways will lead to proinflammatory mediator expression, including increases in cytokines and chemokines, which trigger host cellular defense mechanisms. The complex molecular signaling will result in the recruitment of immune system cells targeted at combating the invading microbes; however, the trafficking and antibacterial activity of these cells can lead to collateral tissue damage. Recent evidence suggests that if inflammation is resolved relatively low levels of proinflammatory mediators may promote tissue repair, whereas if chronic inflammation ensues repair mechanisms become inhibited. Thus, the effects of mediators are temporal context dependent. Although containment and removal of the infection are keys to enable dental tissue repair, it is feasible that the development of anti-inflammatory and immunomodulatory approaches, based on molecular, epigenetic, and photobiomodulatory technologies, may also be beneficial for future endodontic treatments.

Introduction Lipopolysaccharide (LPS) has been implicated in mesenchymal stem cell differentiation processes. Wnt5a, one of the “non-canonical” Wnt family members, is important in signaling stem cell differentiation and in the inflammatory responses of immune cells. Here we studied whether LPS can regulate the expression of Wnt5a in human dental pulp stem cells (hDPSCs) and investigated the intracellular signaling pathways activated by LPS. Methods Wnt5a mRNA and protein expression changes in hDPSCs were investigated by real-time polymerase chain reaction analysis and enzyme-linked immunosorbent assay. In addition, real-time polymerase chain reaction, enzyme-linked immunosorbent assay, and luciferase activity assays were used to determine whether toll-like receptor 4 (TLR4), myeloid differentiating factor 88 (MyD88), nuclear factor kappa B (NF-kB), or the phosphatidylinositol 3-OH kinase (PI3K)/AKT pathways are involved in LPS-induced Wnt5a expression. The activation of PI3K and AKT in hDPSCs was measured by Western blot analysis. Results Wnt5a mRNA and protein expression was rapidly increased in response to LPS in a time- and dose-dependent manner. LPS-induced Wnt5a expression was effectively attenuated by administration of a TLR4 neutralizing antibody, MyD88 inhibitory peptide, PI3-kinase inhibitors (LY294002 and wortmannin), an AKT inhibitor, or NF-κB inhibitor (pyrrolidine dithiocarbamate), IκBa phosphorylation inhibitor (Bay 117082), or IκB protease inhibitor (L-1-tosylamido-2-phenylethyl chloromethyl ketone). Treatment of hDPSCs with LPS activated PI3-kinase (p85) and AKT signaling in a time-dependent manner. Moreover, LPS-mediated increases in κB-luciferase activity were diminished by the overexpression of dominant negative mutants of TLR4, MyD88, p85, AKT, and IκBa. Conclusions These results demonstrated that LPS-induced Wnt5a expression was mediated through the TLR4/MyD88/PI3-kinase/AKT pathway, which then initiated NF-κB activation in hDPSCs.

Background and aimsCommon non-coding variations within the TCF7L2 locus have a strong influence on type 2 diabetes (T2D) susceptibility through uncharacterised mechanisms. An islet-specific functional polymorphism has been identified, although this does not explain the association between genotype and gene expression in other cell types. This study sought to identify these other functional TCF7L2 variants.Methods and resultsAlternative splicing and gene expression from TCF7L2 was examined from peripheral blood mononuclear cells from 100 healthy Caucasians using two T2D-associated SNPs, rs7903146 and rs12255372. Electrophoretic mobility shift assays and luciferase reporter assays were performed with these SNPs and those in strong LD to determine potential SNP functionality.Individuals homozygous for rs7903146 and rs12255372 T2D risk alleles (TT/TT) expressed 2.6-fold greater levels of TCF7L2 mRNA compared to individuals homozygous for the non-risk alleles (CC/GG, p = 0.006), although differentially spliced TCF7L2 transcripts did not differ by T2D risk-associated genotype. From SNPs identified to be in strong LD with the T2D-associated SNPs, rs7903146 and rs12255372, five (rs4132670, rs4506565, rs7903146, rs7901695, rs17747324) demonstrated allele-specific binding in electrophoretic mobility shift assays (EMSA). In luciferase reporter assays, rs4132670 exhibited 1.3-fold higher levels of enhancer activity in the Huh7 cell line (p = 3.8 × 10−5) and 2-fold higher levels in a WiDr colon carcinoma cell line (p = 0.008).ConclusionsThese results suggest that rs4132670, located in a region of chromatin accessibility, is a non-tissue-specific candidate functional SNP that has the potential to play a role in TCF7L2 gene expression and T2D risk.

Biological solutions for the repair and regeneration of the dental tissues offer significant potential for improved clinical treatment outcomes. Translation of dental tissue-engineering approaches to the clinic will make considerable contributions to these outcomes in the future, but exploiting the natural regenerative potential of dentin-pulp to enhance wound-healing responses offers solutions for maintaining pulp vitality now. Strategies to harness the natural regenerative potential of the pulp must be based on a sound biological understanding of the cellular and molecular events taking place, and require careful consideration of the interplay of infection, inflammation, and regeneration.

Objective Dentine sialophosphoprotein (DSPP) was initially thought to be unique for dentine formation during tooth development, whilst recent reports have shown a much broader expression pattern such as in bone, periodontium and inner ear. Our goal was to explore its expression and potential impact during early nasal cartilage formation in comparison with tooth development. Study design We investigated DSPP expression in the nasal cartilage by immunohistochemistry and in situ hybridisation. We also cloned a 719 bp partial DSPP cDNA from nasal cartilage and analysed its homology to the published mouse DSPP cDNA sequence. In addition, quantitative RT-PCR was undertaken to compare the expression pattern of DSPP in nasal cartilage and tooth germs during embryonic development. Results The expression of DSPP in mouse nasal chondrocytes was detected using in situ hybridisation and immunohistochemical staining. The quantitative RT-PCR data showed that expression levels of DSPP in nasal cartilage are similar to that of tooth: low at E18, and increased during development with the peak level at P3. Furthermore, DSPP levels in nasal cartilage are lower than tooth but higher than bone. Conclusion DSPP is expressed in nasal cartilage, and a similar temporal expression pattern in cartilage and tooth indicates the potential importance of DSPP during development.

Introduction Lipopolysaccharide (LPS) has been shown to regulate the function of odontoblasts. However, the molecular mechanisms of the effect of LPS on odontoblasts are poorly understood. Decorin (DCN), one of the major matrix proteoglycans, is known to affect the mineralization of teeth. In this study, we investigated whether LPS can regulate the expression of DCN in odontoblasts and determined the intracellular signaling pathways triggered by LPS. Methods The DCN messenger RNA and protein expression changes in mouse odontoblast-lineage cells (OLCs) were detected by real-time polymerase chain reaction (PCR) analysis and enzyme-linked immunosorbent assay (ELISA). Whether TLR4, myeloid differentiating factor 88 (MyD88), nuclear factor-kappa B (NF-κB), or mitogen-activated protein kinase (MAPK) pathways were involved in the LPS-induced DCN expression was determined by examined real-time PCR, ELISA, and luciferase activity assay. The activation of extracellular signal-regulated kinase (ERK), p38, and JNK in OLCs was measured by Western blot analysis. Results We found that the mouse OLCs expressed DCN. DCN messenger RNA was rapidly induced by LPS in a time- and dose-dependent manner. Pretreatment with a MyD88 inhibitory peptide, a TLR4 antibody, or a specific inhibitor for NF-κB or I Kappa B alpha (IκBα) significantly inhibited LPS-induced DCN expression. Moreover, the LPS-mediated increase in κB-luciferase activity in OLCs was suppressed by the overexpression of dominant negative mutants of TLR4, MyD88, and IκBα but not by a dominant negative mutant of TLR2. In addition, LPS stimulation activated the ERK, p38, and JNK MAPK pathways. The pretreatment of OLCs with specific inhibitors of the ERK, p38, and JNK MAPK pathways markedly offset the LPS-induced up-regulation of DCN expression. Conclusions Our results show that LPS stimulation can up-regulate the gene expression of DCN via the TLR4, MyD88, NF-κB, and MAPK pathways in odontoblast cells.

National and international standards detail in vitro methodologies for assessing toothpaste abrasivity using human enamel and dentine coupled with profilometric or radioisotope analysis. This profilometry-based study investigates the influence of brushing simulator design and choice of standard abrasive on enamel/dentine wear. Four simulators were evaluated including the flat-bed and V8 Grabenstetter designs described in the present BS EN ISO 11609: 1998 standard, together with the two abrasives prescribed therein. Vertical reciprocating-action and rotary-action simulators were also assessed. Mean abrasion depths for enamel and dentine specimen groups using the rotary-action simulator with either abrasive were ca. 0.9 μm and 4–5 μm respectively, significantly higher vs. comparators ( p = 0.0001; CV ∼ 0.2). Mean abrasion depths determined using the flat-bed and V8 simulators were ≤0.1 μm (CV, 0.2–0.8) for enamel and ≤0.7 μm (CV, 0.2–0.6) for dentine. The vertical reciprocating-action simulator elicited intermediate levels of wear, but high design-driven variance (CV, 0.6–0.7). Mean enamel abrasion depths were independent of abrasive ( p ≥ 0.534), however, mean dentine abrasion depths were significantly different for the vertical reciprocating and rotary-action simulators ( p = 0.0026 and 0.0014 respectively). This study demonstrates the importance of simulator design in the assessment of dentifrice abrasivity, which is key to delivering the desired sensitivity and discriminatory power. Various limitations in design of the simulators studied highlight opportunities for improvements in simulator design.

Histone deacetylase inhibitors (HDACis) alter the homeostatic balance between 2 groups of cellular enzymes, histone deacetylases (HDACs) and histone acetyltransferases (HATs), increasing transcription and influencing cell behavior. This study investigated the potential of 2 HDACis, valproic acid (VPA) and trichostatin A (TSA), to promote reparative processes in pulp cells as assayed by viability, cell cycle, and mineralization analyses.VPA (0.125-5 mmol/L) and TSA (12.5-400 nmol/L) were applied to a pulp-derived cell population and compared with unsupplemented controls. Cell proliferation and viability were evaluated by trypan blue staining and cell counting, whereas cell cycle and apoptosis were analyzed by immunocytochemical staining with antibodies for p53, phosphorylated p53, Bcl-2 homologous antagonist/killer (BAK), caspase-3 and p21(WAF1/CIP), and DNA staining with Hoechst 33342. For mineralization analysis, cultures were stained with Alizarin red and quantified spectrophotometrically. Relative gene expression levels of mineralization associated markers were analyzed using reverse-transcriptase polymerase chain reaction. One-way analysis of variance and Tukey post hoc tests were applied to the data (P.05).VPA and TSA reduced cell proliferation dose dependently with no significant effect on cell viability except at 400 nmol/L TSA. The transcription factor p21(WAF1/CIP) was significantly increased at the highest concentration of TSA but not VPA. Significant increases (P.05) in the apoptosis marker protein active caspase-3 and cell cycle alterations were only evident at the maximum concentrations of TSA/VPA, whereas HDACi-induced mineralization per cell was stimulated dose dependently with a significant increase in the expression of the dentinogenic-associated transcript, dentine matrix protein-1.These results indicate that HDACis are capable of epigenetically modulating pulp cell behavior, signifying their therapeutic potential for augmenting biomaterials, and stimulating regenerative responses in the damaged pulp.

As a mineralised connective tissue, dentine is well adapted to its functional role as a major structural component of the tooth. Although similar in composition to bone, dentine matrix is not remodelled physiologically and traditionally, has been regarded as a rather inert tissue. Nevertheless, dentine–pulp demonstrates strong regenerative potential which allows it to respond to disease and traumatic injury. Such responses are strongly influenced by cell–matrix interactions and modified by disease processes, including infection and inflammation. The identification of many bioactive molecules bound within dentine matrix has allowed their potential involvement in regenerative and other tissue responses to be better understood and new opportunities to be recognised for novel clinical therapies.

Angiogenesis is key to both the development and regeneration of the dentin-pulp complex. Objective We hypothesized that proangiogenic signaling molecules sequestered in dentin matrix can be solubilised to induce angiogenic events. Methods Matrix components were extracted from powdered sound human dentin with EDTA and their dose-dependent (0.0001-5 mg/mL) effects examined in endothelial cells in an in vitro angiogenic tube formation assay, proliferation assay, and transcriptional regulation of the VEGF and VEGF-R2 genes. Results Lower concentrations of dentin matrix components were found to show proangiogenic activity, whereas higher concentrations suppressed angiogenic activity. Conclusion This study highlights that the release of dentin matrix components after dental injury can contribute to the angiogenic events that support pulp regeneration.

Dental pulp tissue engineering is an emerging field that can potentially have a major impact on oral health. However, the source of morphogens required for stem cell differentiation into odontoblasts and the scaffold characteristics that are more conducive to odontoblastic differentiation are still unclear. This study investigated the effect of dentin and scaffold porogen on the differentiation of human dental pulp stem cells (DPSCs) into odontoblasts.Poly-L-lactic acid (PLLA) scaffolds were prepared in pulp chambers of extracted human third molars using salt crystals or gelatin spheres as porogen. DPSCs seeded in tooth slice/scaffolds or control scaffolds (without tooth slice) were either cultured in vitro or implanted subcutaneously in immunodefficient mice.DPSCs seeded in tooth slice/scaffolds but not in control scaffolds expressed putative odontoblastic markers (DMP-1, DSPP, and MEPE) in vitro and in vivo. DPSCs seeded in tooth/slice scaffolds presented lower proliferation rates than in control scaffolds between 7 and 21 days (p0.05). DPSCs seeded in tooth slice/scaffolds and transplanted into mice generated a tissue with morphological characteristics similar to those of human dental pulps. Scaffolds generated with gelatin or salt porogen resulted in similar DPSC proliferation. The porogen type had a relatively modest impact on the expression of the markers of odontoblastic differentiation.Collectively, this work shows that dentin-related morphogens are important for the differentiation of DPSC into odontoblasts and for the engineering of dental pulp-like tissues and suggest that environmental cues influence DPSC behavior and differentiation potential.

Objectives Dental tissue disease and trauma provides an excellent model for the interaction between tissue defence and regenerative processes and has application to many of the body's other tissues. Following dental tissue infection, characterised by caries, the molecular and cellular mediators of the immune/inflammatory processes clearly impact on the dental tissues’ natural regenerative responses. This review of the literature was performed to better understand how these two processes interact and identify whether cross-talk may provide novel areas for future research and subsequent translation into clinical application. Data and sources A review of the literature was performed using the PubMed database resource and this was followed by extensive hand searching using reference lists from relevant articles. Conclusions Frequently, the dental tissue inflammatory and regenerative processes are seen as both distinct and antagonistic and subsequently have often been studied in isolation; however, both direct and indirect data are now emerging which indicate significant inter-relationship. Whilst the ensuing inflammatory process will result in dental tissue breakdown and molecular signalling which may impede regeneration, low grade inflammation, potentially induced by mechanical trauma and tissue necrosis, may promote regenerative mechanisms, including angiogenic and stem cell processes. Notably, the locally derived growth factors, neuropeptides, cytokines and chemokines, released from the host dentine matrix and by resident pulpal cells, immune cells, neurons and/or dying cells, will modulate defence and repair processes within the tissue.

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.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.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.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 change2 - p0.05). 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.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.

Variation of sea-surface and water pressure above the sea bed induces temporal variation of submarine groundwater discharge over time scales ranging from seconds to years. Hydrodynamic theory and measurements suggest that wave-induced exchange between a permeable sediment bed and overlying water column is significant but conventional seepage-meter studies have focussed mainly on tidal dynamics and ignored waves. At Cockburn Sound in Western Australia we measured wave-induced flow reversals through a seepage meter of amplitude 60 cm d - 1 ; however, it was unclear whether the measured flows were real seepage or, partly or wholly, an artifact of wave action on the seepage meter. A numerical model of seepage patterns beneath a vented benthic chamber demonstrated an observer effect introduced by the chamber and not previously identified. Placing a chamber on the sediment bed disturbed the pressure field and changed both the pattern and magnitude of the wave-induced flow. A separate analysis of benthic-chamber movements under the action of shallow surface waves established that micron-scale movements of the chamber at the wave frequency were sufficient to produce apparent seepage amplitudes of O(1–100) cm d−1. We concluded that wave action is a key control on bed seepage and should not be neglected without justification in direct-measurement studies of marine bed discharge. A systematic error during each wave cycle can accumulate to a significant measurement error if the wave cycle error is large or if wave-induced flow is the dominant component of the seepage. In the latter case, the error could potentially be misinterpreted as a steady seepage component.

Objective Vascular endothelial growth factor (VEGF) has been implicated in the regulation of dental pulp and dentine repair. Therapeutic ultrasound was shown to be effective for fracture repair. We investigated whether low frequency ultrasound influences the production of VEGF by odontoblast-like cells. Moreover, we examined the direct effects of VEGF on odontoblast-like cell proliferation. Design MDPC-23, an established odontoblast-like cell line, was exposed to increasing intensities of 30 kHz ultrasound using an ultrasonic tip probe. Results After 24 h cell culture, WST-1 analysis of cell viability and number showed a dose-dependent decrease in the number of viable cells with increasing ultrasound power. However, the relative concentration of VEGF as analysed by ELISA and normalised to cell number was significantly increased in the culture supernatants indicating an ultrasound-induced stimulation of odontoblastic VEGF secretion. Analysis of VEGF gene expression by sqRT-PCR revealed the expression of the main VEGF isoforms in the MDPC-23 cells, i.e. VEGF 120 and VEGF 164 as well as to a minor extent VEGF 188 . Low power ultrasound increased gene expression of all VEGF isoforms. Addition of recombinant VEGF to the cell cultures significantly stimulated cell proliferation. Gene expression of the VEGF receptors Flt1/VEGFR1 and KDR/VEGFR2 was detected in the MDPC-23, suggesting the possibility that VEGF may act on the odontoblast-like cells in an autocrine manner. Conclusions Our results indicate that ultrasound promoted VEGF expression and production by odontoblast-like cells and that VEGF may have autocrine effects on these cells. It is proposed that ultrasound may influence odontoblast activity and dentine repair by modulating production of endogenous growth factors in the dentine-pulp complex.

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.

The input of groundwater-borne nutrients to Adelaide's (South Australia) coastal zone is not well known but could contribute to the ongoing decline of seagrass in the area. As a component of the Adelaide Coastal Waters Study (ACWS), the potential for using the radium quartet ( 223 Ra, 224 Ra, 226 Ra and 228 Ra) and 222 Rn to evaluate submarine groundwater discharge (SGD) was evaluated. Potential isotopic signatures for SGD were assessed by sampling groundwater from three regional aquifers potentially contributing SGD to the ACWS area. In addition, intertidal groundwater was sampled at two sand beach sites. In general, the regional groundwaters were enriched in long-lived Ra isotopes ( 226 Ra and 228 Ra) and in 222 Rn relative to intertidal groundwater. Radium activity (but not 222 Rn activity) was positively correlated to salinity in groundwater from one of the regional aquifers and in intertidal groundwater. Radium isotope ratios ( 223 Ra/ 226 Ra, 224 Ra/ 226 Ra and 228 Ra/ 226 Ra) were less variable than individual Ra isotope activities within potential SGD sources. Recirculated seawater (estimated from the intertidal groundwater samples with seawater-like salinities) also had distinctly higher Ra isotope ratios than the regional groundwaters. The activities for all radioisotopes were relatively low in seawater. The activity of the short-lived 223 Ra and 224 Ra were highest at the shoreline and declined exponentially with distance offshore. In contrast, 228 Ra and 226 Ra activities had a weak linear declining trend with distance offshore. Rn-222 activity was at or near background in all seawater samples. The pattern of enrichment in short-lived Ra isotopes and the lack of 222 Rn in seawater suggest that seawater recirculation is the main contributor to SGD in the ACWS area. Preliminary modeling of the offshore flux of 228 Ra and 226 Ra suggest that the SGD flux to the ACWS area ranges between 0.2 and 3 · 10 − 3 m 3 (m of shoreline) − 1 s − 1 .

In this study, the effects of low frequency ultrasound (US) were examined on odontoblasts, the primary cell responsible for dentinogenesis and dentine repair. An established odontoblast-like cell line, MDPC-23, was subjected to 30 kHz ultrasound at three different power settings. US induced a marginal level of cell death (3% to 4%) at lower amplitudes rising to 25% cell death at the highest power tested. The latter was reflected in a 30% decrease in cell attachment after 4 to 24 h of culture, while the number of adherent cells was reduced by approximately 10% to 15% in the lower power groups. Cell replication after 24 h, as measured by BrdU incorporation, showed no significant changes in the US-treated groups. Gene expression analyses demonstrated a moderate dose-dependent increase in the expression of GAPDH (glyseraldehyde-3-phosphate dehydrogenase)-normalised collagen type I, osteopontin (OPN), transforming growth factor-β1 (TGFβ1) and the heat shock protein (hsp) 70. The greatest change was found in the expression of the small hsp 25/27, which showed a two- to six-fold increase following US treatment. No significant effects were observed for alkaline phosphatase (ALP) and core-binding factor A1 (CBFA1/Runx2) expression levels. This is the first report describing US effects on odontoblasts. Further studies are warranted to elucidate US effects on odontoblast function and to evaluate US as a therapeutic application in dentine repair. (E-mail: b.a.scheven@bham.ac.uk )

Calcium hydroxide (Ca(OH)(2)) has been used extensively to induce dentine regeneration through formation of dentine bridges at sites of pulp exposure after dental tissue injury, however, the biological processes underpinning these events are unclear. We hypothesise that growth factors and other bio-active molecules, sequestered within dentine matrix, may be released by the action of Ca(OH)(2) and signal gene expression in pulp cells, which mediates the changes in cell behaviour observed during regeneration. Powdered sound, human dentine samples were extracted with either 0.02 m Ca(OH)(2), pH 11.7 or 10% EDTA, pH 7.2 ( a control known extractant of bio-active and other ECM molecules from dentine) over a 14-day period. Extracts were compared for non-collagenous protein (NCP) and glycosaminoglycan (GAG) content using dye binding assays and protein compositions were analysed by 1D-polyacrylamide gel electrophoresis (1D-PAGE) and TGF-beta1 ELISA. The effects of extracts on TGF-beta1, Collagen-1alpha and Nestin gene expression were analysed using semi-quantitative RT-PCR in the dental MDPC-23, OD-21 and fibroblastic Swiss 3T3 cell lines following 24h of exposure. Ca(OH)(2) solubilised NCPs and GAGs from the dentine ECM, although with a lower yield than the EDTA solution and with different kinetics. 1D-PAGE analysis demonstrated some differences in profiles for proteins solubilised from dentine by Ca(OH)(2) and EDTA. Both solutions released TGF-beta1 from the dentine with higher concentrations present in the EDTA (1.395 +/- 0.036 ng/mg) versus the Ca(OH)(2) (0.364 +/- 0.012 ng/mg) extract. Notably, both extracts induced similar gene expression profiles in all cell lines. These data provide a rational explanation for the action of Ca(OH)(2) during pulp capping in which the cellular activities involved in dentine bridge formation may be mediated through release of growth factors and other bio-active molecules from the dentine by Ca(OH)(2).

Many studies of periodic forcing and response in aquifers have focused on describing the induced fluctuations in hydraulic head, without much consideration of the time-dependent flows. Visualization techniques presented in this paper can be applied to obtain a more physically intuitive impression of groundwater motion in aquifers that undergo periodic fluctuations of hydraulic head. The concepts of velocity ellipse and displacement ellipse are introduced as methods for visualizing oscillatory flows associated with individual forcing modes. Cyclical trajectories illustrate the potential complexity of flow paths that can arise due to superposition of modal responses. The full periodic motion that results due to superposition of the mean flow and modal flows is visualized using streaklines. An animated time series of streaklines provides an intuitive impression of the flow and affords insight into apparent dispersion phenomenon that can arise due to periodic fluctuations in both the strength and direction of groundwater flow. Electronic animations are available from the authors.

HIV-1 reverse transcriptase can remove chain terminators from blocked DNA ends through a nucleotide-dependent mechanism. We show that the catalytic efficiency of the removal reaction can vary several hundred-fold in different sequence contexts and is most strongly affected by the nature of the base pair at the 3'-primer terminus and the six base pairs upstream of it. Similar effects of the upstream sequence were observed with primer-templates terminated with 2',3'-dideoxy-AMP, 2',3'-dideoxy-CMP, or 2',3'-dideoxy-GMP. However, the removal of 2',3'-dideoxy-TMP or 3'-azido-2',3'-dideoxy-TMP was much less influenced by upstream primer-template sequence, and the rate of excision of these thymidylate analogues was greater than or equal to that of the other chain-terminating residues in each sequence context tested. These results strongly indicate that the primer terminus and adjacent upstream base pairs interact with reverse transcriptase in a sequence-dependent manner that affects the removal reaction. We conclude that primer-template sequence context is a major factor to consider when evaluating the removal of different chain terminators by HIV-1 reverse transcriptase.

Isolation of sufficient quantities of pure populations of odontoblasts from healthy and diseased teeth will facilitate our understanding of dentinogenesis during development and repair. Here we describe a novel Piezo-power microdissection (PPMD) technique for the isolation of pure populations of odontoblasts and pulpal tissue from formalin-fixed, paraffin-embedded, mature, healthy and carious human teeth. Odontoblasts and pulpal tissue gene expression were subsequently studied in ribonucleic acid isolated from PPMD preparations using a semi-quantitative reverse transcription polymerase chain reaction approach. Data confirmed that the genes for dentine sialophosphoprotein and Nestin are preferentially expressed in odontoblasts, whilst the genes for both collagen-1alpha and collagen-3alpha were expressed preferentially in pulpal tissue, particularly in carious samples. PPMD provides a novel and powerful approach to isolate pure populations of dental tissues and cells from fixed specimens for subsequent downstream molecular analyses.

Objectives: Many restorative materials are claimed to be successful in preventing bacterial microleakage and minimizing pulp inflammation. However, information regarding the in vivo performance of materials in comparison with each other is limited. The aim of this study was to evaluate and compare the pulp response of nine restorative materials when placed in non-exposed monkey cavities. Methods: 279 standardized non-exposed Class V cavities, were prepared into buccal dentin. Cavities were restored with a number of materials in the following categories: Zinc oxide eugenol (ZnOE), Calcium hydroxide [Ca(OH)2], zinc phosphate (ZP), Resin-modified glass ionomer (RMGI), Composite resin (CR), Bonded amalgam (BA), Gutta-percha (GP), Compomer and Silicate. Pulp tissues were collected and evaluated at short, intermediate and long-term intervals according to ISO guidelines; employing histomorphometric analysis, Spearman's rho and ANOVA statistics. Pulp responses were categorized according to FDI, ISO and ADA standards. Bacteria were detected using McKay stains. Results: Pulp inflammation was found to be correlated to bacterial microleakage around the restoration (p ≤0.0001). The frequency of bacterial microleakage was found to vary between restorative materials (p ≤0.0001). In rank order of preventing bacterial microleakage from best to the worst; RMGI (100%), BA (88%), ZnOE (86%), CR (80%), GP (64%), Ca(OH)2 (58%), compomer (42%), silicate (36%) and ZP (0%). Significance: The most effective restorative materials to prevent bacterial microleakage and pulp injury from inflammatory activity were RMGI, BA, ZnOE and CR restorations.

Hedgehogs are becoming increasingly popular as pets. This article provides a, broad overview of those factors with which practitioners must be familiar to provide guidance to hedgehog owners. The taxonomy, anatomy, and natural history of the common pet species are presented along with commonly observed behaviors. Information on the reproduction and neonatal management is described. Current recommendations for handling, housing, and feeding these animals are also discussed.

Appropriate selection of medical students is a fundamental prerequisite if medical schools are to produce competent and caring doctors. The selection criteria for entry to the medical degree course at the University of Newcastle, New South Wales, are unique in Australia. The purpose of this study was to identify admission criteria that may predict performance in the first postgraduate (intern) year. Performance ratings were obtained from the clinical supervisors of two graduating classes of Newcastle University medical students during their five terms in internship (first postgraduate year). At least one rating was obtained for 93% of interns. A subset analysis of interns with multiple ratings (57%) showed that combining previous study in both humanities and science before medical school entry was predictive of higher intern performance ratings. These interns were rated more favourably than those who had studied science alone. Moreover, students who had earlier studied both humanities and science were twice as likely to complete their medical degree as those who had studied science alone. Age, gender, admission interview results, written psychometric test scores, academic marks, and whether previous tertiary study had been undertaken prior to medical school entry were not predictive of intern performance ratings. Subject spread, including a background in humanities, is important for effective medical practice, at least in the immediate postgraduate period. Perhaps it is time to evaluate the admission criteria by which medical students are selected.

A method is described for computerized measurement of blood pressure (BP) in small animals. As described, the system measures BP in up to four experimental animals at one time by sequential sampling but can be expanded to take data from up to 10. It requires an Apple II computer, and is run from a Grass pen-recording system. Pulse rate and systolic and diastolic BP are measured, and these variables are used to calculate mean arterial pressure (MAP) and the time integral of MAP minute by minute. The data are displayed in real time and stored hourly on floppy disc for further analysis.

Chromatin released by two nucleases under various ionic conditions has been fractionated by chromatography on ECTHAM-cellulose. Mg2+ -soluble chromatin, which according to Gottesfeld and Partington is enriched in transcribed DNA sequences (Gottesfeld, J.M. and Partington, G.A., (1977) Cell 12, 953-962) and produced by DNAase II digestion at intermediate ionic strength, comprises material eluting from ECTHAM-cellulose at 80-100 mM Cl-, pH 6.8-7.0, whereas bulk, Mg2+ -insoluble chromatin comprises more tightly binding material. Free hnRNP particles elute at 30 mM Cl-, pH 6.8. Oligonucleosomes, which according to Dimitriadis and Tata are enriched in transcribed sequences (Dimitriadis, G.J. and Tata, J.R. (1980) Biochem. J. 187, 467-477) and produced by micrococcal nuclease digestion at physiological ionic strength, also elute predominantly at 80-100 mM Cl-, pH 6.8-7.0. When liver nuclei are digested with micrococcal nuclease at low ionic strength, the most rapidly released chromatin is enriched in nascent RNA and hnRNP particles, and binds weakly to ECTHAM-cellulose. More slowly solubilised chromatin, containing fewer hnRNP particles, binds much more strongly to ECTHAM-cellulose. In confirmation of results with mechanically sheared chromatin, the affinity of particular chromatin fractions is not dependent on the size of chromatin particles, rather it reflects the differing composition, and in particular the non-histone protein and hnRNP content, which, we propose, determines the conformation adopted by different chromatin fractions in the cation conditions used for elution from ECTHAM-cellulose.