Objective. The dentin pH at different sites following placement of calcium hydroxide paste using 2 different methods was evaluated.Study design. One hundred twenty Extracted teeth were instrumented and randomly divided into 6 different groups. Then they were dressed with calcium hydroxide by 2 different methods using paper points or Lentulo spiral, according to their respective groups, and stored. After the experimental period of time, half of the samples were cut transversally into slices and the other half split longitudinally and rinsed with distilled water. The dentin pH of cervical, middle, and apical thirds was measured from the root canal wall surface and I mm away from the root canal lumen in the inner dentin. The results were compared and statistically analyzed.Results. The highest pH values were obtained on the root canal walls when the calcium hydroxide was placed with Lentulo spiral filler (P>.05). There was a significant reduction in pH values in the inner dentin. When the cross sections of teeth were compared at 7 days, the Lentulo group was statistically different only in the apical third.Conclusions. Placement of the calcium hydroxide paste with a Lentulo spiral with subsequent compaction with the blunt end of a sterile paper point obtained a higher pH value on the canal walls and in the inner dentin than paper points only.

Aim To compare the tuned-aperture computed tomography system of imaging to conventional D-speed film for their ability to identify root canals in extracted human molars.Methodology Thirteen maxillary and six mandibular human molars were mounted in acrylic blocks to simulate clinical conditions by surrounding the teeth with a radiodense structure. The teeth were then imaged with conventional D-speed film using a standard paralleling technique, and with a modified orthopantomograph OP100 machine using a Schick no. 2 size CCD sensor as the image receptor. The source images were registered and TACT slices were generated using TACT Workbench(TM) Software. Three observers were asked to identify the number of canals in the conventional film group and the TACT image group using specific criteria. Ground truth was established by cross-sectioning the teeth at the coronal, middle, and apical thirds of the roots and directly visualizing the root canal morphology.Results TACT imaging detected 36% of 4th canals in maxillary molars and 80% of third canals in mandibular molars. Conventional film detected 0% of fourth canals in maxillary molars and 0% of third canals in mandibular molars. The differences in canal detection between the two techniques were statistically significant (Wilcoxon matched pair sign rank test, P = 0.001).Conclusions In this study, the TACT system of digital imaging was superior to conventional film in the detection of root canals in human molars and may be useful for the detection of root canals that will probably be missed upon conventional X-ray examination.

This study compared the ability of the TACT system of imaging and conventional D-speed film to detect simulated resorptive defects in cadaver teeth and jaws. Mandibular human jaw blocks were prepared and then split through the teeth in a mesial to distal direction so that the mandibular halves could be opened and reassembled when necessary. One half of each of 42 teeth was extracted, and areas on the tooth and corresponding bone identified to be studied. The jaws were opened and reassembled after no defects or after defects of different sizes (0.25, 0.5, 1.0, 2.0 mm) were placed in the tooth and the corresponding bone at predetermined evaluation areas. The teeth were imaged after each stage with conventional D-speed film using a standard paralleling technique, and with a modified orthopantomograph OP100 machine using a Schick #2 size CCD sensor as the image receptor. The source images were registered and TACT slices were generated using TACT Workbench Software. Three observers were asked to identify the presence of resorptive defects in the conventional film group and the TACT image group using specific criteria. TACT imaging was statistically superior to conventional radiographs in detecting defects that were present (Wilcoxon matched pairs signed ranks test P=0.003). This was true for all sizes of defects examined. There was no difference between the two modalities when a defect was not present.

Due to their peripheral location in the dental pulp and their cellular extension into dentin, odontoblasts are the first pulpal cells to encounter dental pathogens. The association of odontoblasts with immunoglobulins and dendritic cells during microbial invasion of dentin implies that these cells may possess a role in the innate and adaptive pulpal immune responses, however this has not been examined. A pivotal step in the innate immune response is the detection of foreign antigen and the recruitment of immune effector cells to the area. IL-8 is a potent chemotactic cytokine that plays an important role in the inflammatory response. The purpose of this study was to determine if odontoblasts are capable of expressing the pro-inflammatory chemokine IL-8. Human odontoblasts from intact, noncarious third molars were maintained in culture and exposed to Escherichia coli lipopolysaccharide (LPS) (serotype 055:B5) on day 4 for 8-10 h in a humidified 5% CO2 incubator. Control and experimental samples were assayed by reverse transcription-polymerase chain reaction (RT-PCR) and Western blot for the production of IL-8 mRNA and protein. Analysis of the PCR products revealed that cells of the odontoblast layer maintained in this culture model constitutively expressed low levels of IL-8, which were increased in response to E. coli LPS exposure. Western blotting confirmed that the mRNA was translated into protein. These results imply that odontoblasts are capable of producing of pro-inflammatory mediators, thereby actively participating in the recruitment of neutrophils in response to bacterial by-products.

Lipopolysaccharide (LPS), a cell wall component of Gram negative anaerobic bacteria, has been implicated in the pathogenesis of periapical disease resulting from infected root canals. Calcium hydroxide [Ca(OH)2] has been shown to be an effective medicament in such infections, reducing the microbial titre within the canal. It has been proposed that the therapeutic effect of Ca(OH)2 may also be the result of direct inactivation of LPS. The aim of this study was to investigate whether the toxic potential of an Escherichia coli LPS could be reduced or eliminated by Ca(OH)2. Four concentrations of E. coli LPS ranging from 1-1000 ng/ml sterile water were incubated in duplicate either with 25 mg Ca(OH)2 or sterile water alone. Controls consisted of Ca(OH)2 without LPS or sterile water only. Monocytes were collected from peripheral blood by centrifuging through a gradient and plated to a specific density. Adherent monocytes were incubated for 4 days at 37 degrees C with 5% CO2 in M199 medium with 10% autologous serum. The different LPS solutions were added to the wells on day 5. After 4 h the supernatants were collected and quantitatively assayed for TNF-alpha using a commercial ELISA kit. Statistical analysis was performed with ANOVA. Results indicated that Ca(OH)2 is able to eliminate the ability of an E. coli LPS to stimulate TNF-alpha production in peripheral blood monocytes (P < 0.0001).

SummaryInhibiting antibodies in patients with hemophilia A pose a significant therapeutic dilemma in the treatment of bleeding episodes. The genetic factors which predispose hemophiliacs to inhibitors and the optimal method for inhibitor suppression remain obscure. Hence, an animal model of the human FVIII inhibitor response is of potential value. Sprague-Dawley rats immunized with human recombinant FVIII (rFVIII) subsequently developed abnormal coagulation parameters coincident with the development of an immune response to the human protein. The epitopes for the resultant rat anti-rFVIII antibodies were mapped using a random fragment expression library constructed from the FVIII cDNA. Antigenic regions located within the Al, First and Second Acidic and B domains were mapped. Rat immunoglobulins reactive with the individual epitopes were immunoaffinity purified and assayed for inhibitory activity. Several of the epitopes mapped using the rat antibodies were similar to regions previously shown to be antigenic for human inhibitors. By contrast, no epitopes were mapped to the A2 domain with the techniques used. This may be due to the possible presence of conformational epitopes in this area which cannot undergo fragmentation and still retain antigenicity or the presence of relatively low concentrations of antibodies to this region. The rat model shares some similarity with both the auto- and alloimmune human response to FVIII and therefore may be a valuable model for studies on the induction and suppression of the inhibitor response.

Hemophilia A is a clotting disorder that is due to reduced or absent coagulation factor VIII (FVIII) activity. In approximately 25% of people with severe hemophilia A, standard treatment with intravenous plasma-derived or recombinant FVIII (rFVIII) induces anti-FVIII antibodies that inhibit FVIII activity (inhibitors). We describe the development of a rat model to study the formation of inhibitors. Immunization of rats with human rFVIII in adjuvant induced an anti-human rFVIII antibody response characteristic of an anti-FVIII inhibitor response in hemophilia A patients. The rats exhibited a rapid, polyclonal secondary antibody response to human rFVIII. These antibodies were reactive against epitopes located in the heavy and light chains. All the rFVIII-immunized rats developed antibodies against the FVIII C2 domain, a region of major reactivity in hemophilia A patients with inhibitors. Furthermore, competition ELISAs demonstrated that rat and human anti-FVIII antibodies recognized identical or overlapping epitopes of the FVIII molecule. The rat anti-FVIII antibodies also functioned as human FVIII inhibitors with titers ranging from 120 to 2048 Bethesda Units (B.U.). We propose that this rat model may be useful to investigate immune responses to FVIII and may lead to better therapies for FVIII inhibitors.

A simple, reliable technique for establishing cultures of human pulpal fibroblasts is presented. The technique relies on a sequential digestion of minced pulpal tissue with collagenase and trypsin and produces confluent cultures in 7 to 10 d from four to five pulps.

Bone adapts to physical deformation in vivo, yet the mechanism of the adaptive process remains unknown. One reason for this perplexity has been the difficulty in examining the effects of a well-defined deformation regimen on individual bone cells. With the utilization of novel, flexible-bottomed cell culture plates, one can study the effects of cyclic strain on the morphologic and biochemical adaptations of individual osteoblasts in vitro. Avian, calvarial osteoblast-like cells, from passes 2-5, responded to cyclic strain, by increasing their rates of DNA synthesis and cell division during the first 72 h after initiation of a continuous deformation regimen comprised of 3 cycles per min of 0-24% elongation. In addition, within hours after initiation of the deformation regimen, cells oriented 90 degrees to the applied strain field at the periphery of the culture plate in the region of maximum strain and elongation.

The effects of applied cyclic tensional deformation and relaxation on cultured bovine aortic endothelial cells were examined. Endothelial cells from passages 3 to 9 were seeded in flexible-bottomed plates and allowed to attach for 24 hours. Endothelial cells in the experimental group (n = 6 wells per time point) were placed in a vacuum-operated stress-providing instrument that exerted an average elongation of 10% at maximum downward deflection of the culture plate bottom. The stretched endothelial cells were subjected to repeating cycles of 10 seconds elongation and 10 seconds relaxation from days 1 through 7 in culture. Endothelial cells in the control group (n = 6 wells per time point) were subjected to similar incubation conditions as the experimental group but without tensional deformation. Tritiated thymidine was added to cells 24 hours before harvesting. On days 0, 1, 3, 5, and 7 cells were counted and analyzed for trichloroacetic acid-precipitable tritiated thymidine incorporation. The results showed that 3 cycles/min mechanical stretching stimulated deoxyribonucleic acid synthesis and endothelial cell division. We conclude that cyclic tensional deformation may stimulate endothelial cell proliferation. It is possible that naturally occurring cyclic mechanical deformation in vivo, such as the repetitive stretching and relaxation of aortic tissue by the heart, may invoke a particular pattern of synthesis and division in endothelial cells.