A sleep bruxism detection system based on sensors in a splint - pilot clinical data.

It is difficult in a dental setting to accurately diagnose sleep bruxism and to objectively assess the severity, frequency or natural history of the condition in an individual patient. Yet this information is essential for the management of sleep bruxism and to plan appropriate dental treatment. The objective of this study was to clinically test a device that could be used to record bruxism events in a home environment. Pressure sensors were developed for use under the surface of an occlusal splint, and circuitry was designed to facilitate the recording and wireless transmission of the pressure sensor signal to a computer. Controlled mandibular movements were carried out in vivo to simulate bruxism and non-bruxism patterns. These patterns of force application were graphically presented to two examiners who were asked to identify the type of activity represented by the force curves. Examiners were largely able to distinguish bruxism from non-bruxism activity; the sensitivity ranged from 80% to 100% and the specificity from 75% to 100%. Using sensors in an occlusal splint, it is possible to recognise the typical tooth contact patterns seen in sleep bruxism. Such a device may be useful for monitoring sleep bruxism over an extended period at home.

Assessment of occlusion after placement of stainless steel crowns in children - a pilot study.

Many stainless steel crowns (SSCs) disrupt the occlusion in children, but stabilisation appears to occur within a short period post-placement. The extent and mechanism of these short-term occlusal changes in children are unknown. This study sought to determine whether placement of a SSC changes the maximum intercuspation position (MIP) in children, whether the MIP returns to normal within 4 weeks and whether local anaesthesia had an effect on the child's ability to achieve MIP. The T-Scan(®) III was used for the measurement of occlusal contacts. Reliability and reproducibility of the system was determined using a calibration exercise where MIP recordings were taken of eleven children not undergoing any dental treatment. For the main study, the percentage of total occlusal force on each tooth was recorded in 20 children preoperatively, after local anaesthesia, after SSC placement and 4 weeks postoperatively. There was no significant difference in MIP (P = 0·435) preoperatively and post-administration of local anaesthesia. There was a significant difference between the preoperative force on a tooth and the reading after crown placement (P = 0·0013, Wilcoxon test). By 4 weeks, there was no significant difference overall between post-SSC placement and the preoperative value for the tooth (P = 0·3). Administration of local anaesthesia did not affect the ability of a child to attain MIP. Maximum intercuspation position was disturbed by the placement of a SSC in seven of 20 cases. When MIP was disturbed, in most cases, it returned to preoperative status within 4 weeks of crown placement.

Maxillary anterior tooth dimensions and proportions in an Irish young adult population.

This study was undertaken in a young Irish population to determine the dimensions and ratios of the six maxillary anterior teeth. One hundred and nine Irish subjects (age 18-25 inclusive) had irreversible hydrocolloid impressions made of their maxillary dentition poured in type V stone. Clinical crown dimensions were measured with a digital calliper. The stone casts were digitally photographed in a standardised manner enabling calculation of various ratios between the maxillary anterior teeth. Sexual dimorphism existed for various tooth dimensions; most notably canine teeth were in the region of 0·8 mm longer and 0·6 mm wider in males. Central and lateral incisors were found to be 0·5 mm wider in males. It is, therefore, recommended that dimensional tooth guidelines should be given for each of the sexes and not on a population basis. With regard to tooth proportion ratios, no significant differences were found between genders or the left and right sides for any of the measurements or ratios measured. The digitally recorded tooth proportions were similar for both sexes, and the Golden Proportion guidelines could only be applied to the lateral incisor/central incisor widths (0·618). Identified width proportions for the canine/central incisor were 0·58 and for canine/lateral incisor 0·89.

The effect of two root canal sealers on the retentive strength of glass fibre endodontic posts.

This study was undertaken to examine the effect of eugenol-containing and non-eugenol-containing root canal sealers on the retention strength of glass fibre endodontic posts (ParaPost Fibre White) luted with a resin cement (ParaPost cement). We also examined the mode of failure that occurred visually by using scanning electron microscopy. Seventy-two single rooted, recently extracted, premolar teeth were root canal treated and randomly divided into two groups. Group 1 was obturated with gutta percha and a calcium hydroxide-based sealer (Sealapex, Kerr). Group 2 was obturated with gutta percha and a eugenol-based sealer (Tubli-Seal Kerr). The teeth were stored for 1 week in distilled water at 37 degrees C and then prepared for 9 mm posts with a 1.40-mm drill. The matching glass fibre post was luted with a resin cement following the manufacturer's instructions. The samples were stored for 1 week and thermocycled. The posts were removed from the root canals using a calibrated testing machine in tensile mode. The mean dislodging force for group 1 was 190.46 N and for group 2 was 183.8 N, with standard deviations of 54.9 and 56.0 N respectively. The t-test indicated no significant difference between the two groups. Failure of the posts occurred mainly within the resin layer. This study showed that under experimental conditions there was no statistically significant difference between Sealapex sealer and Tubli-Seal sealer on the retention of glass fibre posts using a resin cement.

A comparison of the osteogenic potential of adult rat mesenchymal stem cells cultured in 2-D and on 3-D collagen glycosaminoglycan scaffolds.

Adult mesenchymal stem cells (MSCs) have the capability to differentiate along several lineages including those of bone, cartilage, tendon and muscle, thus offering huge potential for the field of tissue engineering. The purpose of this study was to characterise the differentiation capacity of rat MSCs cultured on standard plastic coverslips in 2 dimensions and on a novel collagen glycosaminoglycan scaffold in the presence of a standard combination of osteoinductive factors. Cells were cultured for 3, 7, 14 and 21 days and several markers of osteogenesis were analysed. While the initial response of the cells in 3-D seemed to be faster than cells cultured in 2-D, as evidenced by collagen type I expression, later markers showed that osteogenic differentiation of MSCs took longer in the 3-D environment of the collagen GAG scaffold compared to standard 2-D culture conditions. Furthermore, it was shown that complete scaffold mineralisation could be evoked within a 6 week timeframe. This study further demonstrates the potential use of MSC-seeded collagen GAG scaffolds for bone tissue engineering applications.

Direct mechanical measurement of geodesic structures in rat mesenchymal stem cells.

During numerous biological processes, cell adhesion, cell migration and cell spreading are vital. These basic biological functions are regulated by the interaction of cells with their extracellular environment. To examine the morphology and mechanical changes occurring in mesenchymal stem cells cultured on a mechanically rigid substrate, atomic force microscopy and fluorescence microscopy were employed. Investigations of the cells revealed both linear and geodesic F-actin configurations. No particular cell characteristics or intra-cellular location were implicated in the appearance of the geodesic structures. However, the length of time the cells were cultured on the substrate correlated with the percentage appearance of the geodesic structures. Calculating energy dissipation from cell images acquired by dynamic mode atomic force microscopy, it was observed that the vertices of the geodesic structures had significantly higher energy dissipation compared to the linear F-actin and the glass. This supports work by Lazarides [J. Cell Biol. 68, 202-219 (1976)], who postulated that the vertices of these geodesic structures should have a greater flexibility. Our results also support predictions based on the microfilament tensegrity model. By understanding the basic principles of cell ultrastructure and cell mechanics in relation to different extracellular environments, a better understanding of physiological and pathological process will be elicited.

Matrix metalloproteinase expression by endothelial cells in collagen lattices changes during co-culture with fibroblasts and upon induction of decorin expression.

EA.hy 926 cells, a derivative of human umbilical vein endothelial cells, in the presence of fibroblasts show the phenomena of angiogenesis, express the proteoglycan decorin and escape apoptosis, when they are maintained in collagen lattices, while fibroblast-free cultures do not show these changes. Virus-mediated decorin expression can substitute for the presence of fibroblasts. Since the expression of matrix metalloproteinases (MMPs) is an essential step in the formation of capillaries, several MMPs and tissue inhibitors of metalloproteinases (TIMPs) were investigated. MMP-1, MMP-2, MMP-9, and the cell-associated MMP-14 were augmented on the protein level in the presence of fibroblasts. No effect was seen with respect to MMP-3, TIMP-1, and TIMP-2. Semiquantitative RT-PCRs of endothelial cells in co-culture revealed a 7-, 19-, and 11-fold increase for mRNAs of MMP-1, MMP-2, and MMP-14 after six days, respectively. Virus-mediated decorin expression also was accompanied by an up-regulation of these MMPs. The expression of MMP-1 mRNAs increased 5-fold after 2 days and gradually declined thereafter. In contrast, MMP-2 and MMP-14 showed a 7-fold and a 14-fold increase on day two which returned to basal levels within 24 h, indicating that the expression of MMP-1 is differentially regulated from MMP-2 and MMP-14. In spite of the upregulation of the proteases, an enhanced degradation of decorin was not observed. These results indicate that the expression of decorin is a sufficient signal in EA.hy 926 cells for a finely tuned induction of selected MMPs which are involved in angiogenesis whereas the up-regulation of MMPs does not lead to the degradation of the responsible proteoglycan.

Evaluation of salivary gland acinar and ductal cell-specific promoters in vivo with recombinant adenoviral vectors.

Adenoviral vectors efficiently deliver exogenous genes to salivary glands. There are two general epithelial cell types, with very different functions, in salivary glands--acinar and ductal. To determine if gene expression can be restricted in vivo to either general cell type using a relatively cell/tissue-specific promoter in conjunction with adenovirus-mediated gene transfer, we tested the human amylase and kallikrein promoters. For initial studies the sensitive reporter gene luciferase was used in two adenoviral constructs. The adenovirus AdAMY-luc contains the human salivary gland amylase promoter (-1003 to +2)(AMY1C) and AdKALL-luc contains the human tissue kallikein promoter (-315 to -1)(KLK1). The adenovirus AdKALL-hAQP1 was also used to test a therapeutic gene, human aquaporin-1 (hAQP1), potentially of importance in treating surviving ductal cells in irradiation-damaged glands. Luciferase expression after AdAMY-luc delivery in vivo directly to the parotid, submandibular, and sublingual glands, as well as to the lungs, and intravenously via the femoral vein, was restricted to the three salivary glands and the pancreas. AdKALL-luc delivery via the same routes resulted in a more general distribution of luciferase expression, although greatest luciferase activity was seen in salivary glands and lung. Luciferase activity after AdAMY-luc delivery was proportionally greater (approximately 14-fold) in acinar cells, whereas luciferase activity after AdKALL-luc delivery was proportionally greater (approximately 9-fold) in ductal cells. The expression of hAQP1 after AdKALL-hAQP1 gene transfer was mainly observed in ductal cells in vivo. AdKALL-hAQP1 was as useful as AdCMV-hAQP1 in increasing salivary flow rates of irradiated rats. This study demonstrates that adenoviral vectors containing the relatively cell/tissue-specific AMY1C or KLK1 promoters may be useful for targeting therapeutic gene expression in salivary glands.

PKC and ERK1/2 regulate amylase promoter activity during differentiation of a salivary gland cell line.

The addition of transforming growth factor alpha (TGFalpha) to a human submandibular gland cell line (HSG) cultured on basement membrane extract Matrigel, synergistically activates the acinar cell-specific salivary amylase promoter. Signaling through beta1 integrins and increased phosphorylation of ERK1/2 are involved in the increased promoter activity. Phorbol-12-myristate-13-acetate (PMA) and thapsigargin increase amylase promoter activity, suggesting that phorbol ester and calcium-dependent protein kinase C (PKC) pathways are also involved. The combination of specific inhibitors of PKC and MEK1 inhibits the amylase promoter. Inhibitors of the calcium-dependent PKC isoforms alpha, beta, and gamma decrease the promoter activity; however, PKCbeta is not detectable in HSG cells. TGFalpha alters the cellular localization of PKCalpha but not -gamma, suggesting PKCalpha is involved in TGFalpha upregulation of the amylase promoter. Furthermore, rottlerin, a PKCdelta-specific inhibitor, increases the promoter activity, suggesting PKC isoforms differentially regulate the amylase promoter. In conclusion, beta1-integrin and TGFalpha signaling pathways regulate the amylase promoter activity in HSG cells. In response to Matrigel and TGFalpha, the activation of both PKCalpha and phosphorylation of ERK1/2 results in synergistic activation of the amylase promoter. Published 2000 Wiley-Liss, Inc.

Salivary glands as a model for craniofacial applications of gene transfer.

The potential applications of gene transfer technology to all branches of medicine are increasing. It is quite likely that within the next 10-20 years surgical practice routinely will utilize gene transfer, at least adjunctively. The purpose of this review is to familiarize the oral and maxillofacial surgeon with this technology. Studies performed with salivary glands in animal models are presented as examples of proof of concept.

Adenoviral vector cytotoxicity depends in part on the transgene encoded.

First-generation adenoviral vectors induce G(2)/M arrest and cell death at high multiplicities of infection (m.o.i.'s) in vitro. It is unclear whether this cytotoxicity is entirely adenoviral gene related or influenced in part by the encoded transgene. We examined this question in epithelial cells using seven vectors at relatively low (50) or higher (200) m.o.i.'s. The vectors contained no transgene (+/-promoter), transgenes encoding a cytoplasmic reporter protein (two luciferase constructs; beta-galactosidase), or transgenes encoding a secretory protein (alpha1-antitrypsin; growth hormone). After 24 h with a m.o.i. of 50, vectors encoding cytoplasmic reporter proteins led to greatest cytotoxicity (approximately 35-40% cells in G(2)/M). Vectors without a transgene resulted in lower cytotoxicity (approximately 15%, minus, or 23%, plus promoter, cells in G(2)/M). Vectors encoding secretory proteins led to approximately 22-25% cells in G(2)/M. A similar pattern resulted when cell number was measured. Results were unrelated to the steady-state levels of transgene product. At the higher m.o.i., all vectors caused substantial growth retardation. This is the first demonstration that adenoviral vector-induced cytotoxic effects are in part related to the transgene encoded.

Construction and function of a recombinant adenovirus encoding a human aquaporin 1-green fluorescent protein fusion product.

Transfer of the human aquaporin 1 (hAQP1) gene provides a novel way to potentially correct the severe salivary hypofunction associated with therapeutic radiation for head and neck cancer. To facilitate the study of individual cells transduced with this gene, we have designed a fusion product of the hAQP1 and jellyfish green fluorescent protein (GFP) cDNAs. An expression plasmid, pACCMVhAQP1GFP, and a recombinant adenovirus, AdhAQP1GFP, encoding this fusion product were constructed. Both the recombinant plasmid and virus directed the expression of the encoded, 55-kDa fusion protein (hAQP1GFP), which was detected in the plasma membranes of several epithelial cell lines (293, SMIE, and A5). hAQP1GFP was functionally active and facilitated fluid movement across a polarized salivary epithelial cell monolayer (approximately 5-fold noninfected controls) in response to an osmotic gradient. In response to a hypotonic challenge, individual epithelial cells expressing the fusion protein exhibited significantly more capacitance (used herein as an indicator of cell swelling) than control cells. Conversely, in response to a hypertonic challenge, individual infected cells shrunk more rapidly (approximately 2- to 3-fold) and to a greater extent than control cells. We conclude that AdhAQP1GFP is a useful experimental tool to identify and study individual cells expressing a water channel transgene.

Identification of CDK4 as a target of c-MYC.

The prototypic oncogene c-MYC encodes a transcription factor that can drive proliferation by promoting cell-cycle reentry. However, the mechanisms through which c-MYC achieves these effects have been unclear. Using serial analysis of gene expression, we have identified the cyclin-dependent kinase 4 (CDK4) gene as a transcriptional target of c-MYC. c-MYC induced a rapid increase in CDK4 mRNA levels through four highly conserved c-MYC binding sites within the CDK4 promoter. Cell-cycle progression is delayed in c-MYC-deficient RAT1 cells, and this delay was associated with a defect in CDK4 induction. Ectopic expression of CDK4 in these cells partially alleviated the growth defect. Thus, CDK4 provides a direct link between the oncogenic effects of c-MYC and cell-cycle regulation.

Genomic integration and gene expression by a modified adenoviral vector.

A replication-deficient recombinant adenovirus encoding luciferase was constructed using 5' and 3' long terminal repeat (LTR) sequences of the Moloney murine leukemia virus. Gene expression was observed in cultured cells in vitro and in submandibular gland, cortex, and caudate nucleus for as long as three months in vivo. The vector integrated randomly into the genome of both dividing and nondividing cells as determined by fluorescence in situ hybridization (FISH) (10-15% of cells in vitro and 5% in rat spleen in vivo), gene walking, Southern hybridization, and polymerase chain reaction (PCR), in the absence of transcomplementing reverse transcriptase or integrase activity. The new vector combines the high titer and versatility of adenoviral vectors with the long-term gene expression and integration of retroviral vectors.

Safety and efficacy of adenovirus-mediated transfer of the human aquaporin-1 cDNA to irradiated parotid glands of non-human primates.

This study evaluated the safety and efficacy of a single administration of a recombinant adenovirus encoding human aquaporin-1 (AdhAQP1) to the parotid glands of adult rhesus monkeys. In anticipation of possible clinical use of this virus to correct irradiation damage to salivary glands, AdhAQP1 was administered (at either 2 x 10(9) or 1 x 10(8) plaque-forming units/gland) intraductally to irradiated glands and to their contralateral nonirradiated glands. Radiation (single dose, 10 Gy) significantly reduced salivary flow in exposed glands. Virus administration resulted in gene transfer to irradiated and nonirradiated glands and was without untoward local (salivary) or systemic (sera chemistry, complete blood count) effects in all animals. However, the effect of AdhAQP1 administration varied and did not result in a consistent positive effect on salivary flow rates for all animals under these experimental conditions. We conclude that a single adenoviral-mediated gene transfer to primate salivary glands is well-tolerated, although its functional utility in enhancing fluid secretion from irradiated parotid glands is inconsistent.

The need to introduce gene therapy to the dental curriculum.

Recombinant DNA technology is finding its way into many aspects of clinical medicine. No application currently is more dramatic than gene therapy. Proofs of principle have already been established for gene therapy targeted to oral tissues, and more are likely to be demonstrated in the near future. The dental curriculum must begin to include the biological basis of DNA-based therapies and other related biomedical science progress.

Histatin 3-mediated killing of Candida albicans: effect of extracellular salt concentration on binding and internalization.

Human saliva contains histidine-rich proteins, histatins, which have antifungal activity in vitro. The mechanism by which histatins are able to kill Candida albicans may have clinical significance but is currently unknown. Using radiolabeled histatin 3, we show that the protein binds to C. albicans spheroplasts in a manner that is dependent on time and concentration. Binding to the spheroplasts was saturable and could be competed with unlabeled histatin 3. A single histatin 3 binding site with a K(d) = 5.1 microM was detected. Histatin 3 binding resulted in potassium and magnesium efflux, predominantly within the first 30 min of incubation. Studies with fluorescent histatin 3 demonstrate that the protein is internalized by C. albicans and that translocation of histatin inside the cell is closely associated with cell death. Histatin binding, internalization, and cell death are accelerated in low-ionic-strength conditions. Indeed, a low extracellular salt concentration was essential for cell death to occur, even when histatin 3 was already bound to the cell. The interaction of histatin 3 with C. albicans, and subsequent cell death, is inhibited at low temperature. These results demonstrate that the candidacidal activity of histatin 3 is not due exclusively to binding at the cell surface but also involves subsequent interactions with the cell.

Effect of clodronate on macrophage depletion and adenoviral-mediated transgene expression in salivary glands.

Expression of transgenes from adenoviral vectors is short-lived in salivary glands, in part because of immune responses to the virus and/or transgene product. Previous studies demonstrated that depletion of macrophages with multilamellar liposomes containing clodronate (Cl2MBP) increases adenoviral-mediated transgene expression in the liver. This technique was tested in salivary glands. Rats were treated with Cl2MBP-liposomes or control liposomes by femoral vein, intraperitoneal, or carotid artery injections. Thereafter, a recombinant adenovirus, AdCMVluciferase, was instilled intraductally in submandibular glands (SMGs), or delivered to the liver via femoral vein injection. Marked depletion (>94%) of liver macrophages and increased levels of luciferase activity in the liver (45-fold higher than controls) were present in animals receiving Cl2MBP-liposomes. In contrast, the same treatment never depleted more than 41% of SMG macrophages nor increased luciferase activity in SMGs, regardless of the route of administration. In conclusion, while macrophage depletion with Cl2MBP-liposomes is associated with markedly increased adenoviral-mediated transgene expression, this strategy was ineffective for salivary glands.

Management of gingival overgrowth associated with generalized enamel defects in a child.

Gingival overgrowth is usually associated with systemic conditions or treatment (e.g. blood dyscrasias, anti-epileptic or immunosuppressive agents). A child is presented, who had enlarged gingiva associated with a generalized enamel defect (amelogenesis imperfecta (AI), hypoplastic type) and document the periodontal and restorative management of this case.

Paracrine or virus-mediated induction of decorin expression by endothelial cells contributes to tube formation and prevention of apoptosis in collagen lattices.

Resting endothelial cells express the small proteoglycan biglycan, whereas sprouting endothelial cells also synthesize decorin, a related proteoglycan. Here we show that decorin is expressed in endothelial cells in human granulomatous tissue. For in vitro investigations, the human endothelium-derived cell line, EA.hy 926, was cultured for 6 or more days in the presence of 1% fetal calf serum on top of or within floating collagen lattices which were also populated by a small number of rat fibroblasts. Endothelial cells aligned in cord-like structures and developed cavities that were surrounded by human decorin. About 14% and 20% of endothelial cells became apoptotic after 6 and 12 days of co-culture, respectively. In the absence of fibroblasts, however, the extent of apoptosis was about 60% after 12 days, and cord-like structures were not formed nor could decorin production be induced. This was also the case when lattices populated by EA.hy 926 cells were maintained under one of the following conditions: 1) 10% fetal calf serum; 2) fibroblast-conditioned media; 3) exogenous decorin; or 4) treatment with individual growth factors known to be involved in angiogenesis. The mechanism(s) by which fibroblasts induce an angiogenic phenotype in EA.hy 926 cells is (are) not known, but a causal relationship between decorin expression and endothelial cell phenotype was suggested by transducing human decorin cDNA into EA.hy 926 cells using a replication-deficient adenovirus. When the transduced cells were cultured in collagen lattices, there was no requirement of fibroblasts for the formation of capillary-like structures and apoptosis was reduced. Thus, decorin expression seems to be of special importance for the survival of EA.hy 926 cells as well as for cord and tube formation in this angiogenesis model.