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FAM20C (family with sequence similarity 20-member C), a kinase that phosphorylates secretory proteins, plays essential roles in various biological processes. In humans, mutations in FAM20C gene cause Raine syndrome, an autosomal recessive hereditary disease manifesting a broad spectrum of developmental defects including skeletal and craniofacial deformities. Our previous studies revealed that inactivation of Fam20c in mice led to hypophosphatemic rickets and that high phosphate (hPi) diet significantly improved the development of the skeleton in Fam20c-deficient mice. In this study, we evaluated the effects of hPi diet on the formation of dentin in Fam20c-deficient mice, using plain x-ray radiography, micro–computed tomography (μCT), histology, and immunohistochemistry. Plain x-ray radiography and μCT analyses showed that the hPi diet improved the dentin volume fraction and dentin mineral density of the Fam20c-deficient mice. Histology analyses further demonstrated that the hPi diet dramatically improved the integrity of the mandibular first molars and prevented pulp infection and dental abscesses in Fam20c-deficient mice. Our results support that the hPi diet significantly increased the formation and mineralization of dentin in Fam20c-deficient mice, implying that hypophosphatemia is a significant contributor to the dentin defects in Fam20c-deficient subjects.

This study was designed to assess the possible protective effect of alpha-lipoic acid (ALA) on apical periodontitis (AP)-induced cardiac injury. Wistar albino rats were randomized into four groups: control; ALA; AP; and ALA + AP. Rats of the control and ALA groups were not endodontically treated, but saline and ALA (100 mg kg-1 ) were administered. In rats of the AP and ALA + AP groups, the pulp chambers of mandibular first molar teeth were exposed and left open for 30 d to induce AP. Saline and ALA (100 mg kg-1 ) were administered intraperitoneally every 24 h during the experiment. At the end of the experiment, the rats were killed. Establishment of AP was verified by radiographic and histopathological evaluation. Serum alkaline phosphatase (ALP), lactate dehydrogenase (LDH), creatine kinase (CK), and superoxide dismutase (SOD) activities were determined using an automated biochemical analyzer, and the structural cardiac injury was assessed pathologically. Serum ALP, LDH, and CK activities were elevated, and SOD activities were decreased, in the AP group. The changed enzyme activities were significantly normalized by treatment with ALA. We conclude that ALA administration alleviated the AP-induced heart injury and improved cardiac structure and function, and therefore this agent may be of potential therapeutic value in protecting cardiac tissue from systemic injury caused by AP.

In the well-vascularized dental pulp vascular endothelial growth factor A (VEGF-A) is expressed. Vascular endothelial growth factor A is a member of the VEGF family, which includes VEGFs-B, -C, and -D. The latter three have not been investigated in the pulp. Vascular endothelial growth factors C and D are the only ligands for vascular endothelial growth factor receptor (VEGFR)-3, which is usually expressed in lymphatic endothelium. They can also activate VEGFR-2, the main angiogenic receptor. We aimed to study VEGFs signalling in human dental pulp at the gene level and to identify the cellular source for protein expression using immunolabelling. All VEGFs (-A, -B, -C, and -D) were expressed in the pulp and may exert both autocrine and paracrine effects in blood vessels and immune cells found to be equipped with VEGFRs-2 and -3. Lymphatic vessel endothelial hyaluronan receptor-positive macrophages, known to be involved in angiogenesis, were found in the pulp, whereas lymphatic vessels were not detected. Twenty-six of 84 VEGF signalling genes, including VEGFR-3, were expressed at a significantly higher level in the pulp than in the control periodontal ligament. In conclusion, the normal human pulp represents a tissue with relatively high VEGF signalling involving both immune responses and vascular activity.

Tooth agenesis is a common craniofacial congenital malformation in humans, but little is known about the mechanisms of root resorption in this condition. The purpose of this study was to investigate the mechanisms of root resorption in primary molars without successors. An animal model without permanent tooth germs was established by surgery in beagles. The times of onset of primary molar root resorption, with and without successors, were compared. The distribution of immune cells, odontoclasts, and their activating factors were determined by histochemistry and immunohistochemistry. Root resorption of primary mandibular molars without successors began later than physiological resorption. In primary molars without permanent germs, odontoclasts and immune cells were present mainly in the apical pulp at the start of root resorption, whereas in control teeth receptor activator of nuclear factor-κB ligand (RANKL)-positive cells were found mainly in the region of the periodontal ligament. CD14(+) and CD3(+) cells were found in both the pulp and the periodontal ligament region. These results suggest that the dental pulp of primary molars, as well as immune cells, may play an important role in root resorption in primary molars without permanent tooth germs.

Occlusion is commenced by contact of a tooth with an opposing tooth and is the mechanical force working against the periodontal ligament (PDL). However, the influences of occlusion during root development remain uncertain. By extracting the unerupted counterpart molars of rats, we established a non-occlusal model that directly examined the effects of the absence of occlusion in developing molars using micro-computed tomography (μ-CT) and histological procedures. The μ-CT data for experimental molars confirmed no attrition and hypogenesis of the alveolar bone. Root lengths in experimental groups increased more than in control groups. Histological findings of experimental molars showed a wide crown pulp, a long and narrow root, immature Sharpey's fibers, and hypogenesis of cementum. Proliferating cells localized in Hertwig's epithelial root sheath (HERS), the apical pulp, and the PDL of experimental teeth. Furthermore, cell-proliferative activity in experimental roots exceeded that in normal roots. These data indicate that cell proliferation is decreased by occlusion during root formation. Thus, occlusion is one factor that regulates root elongation.

Changes in fluid perfusion through tubules may affect the sensitivity of exposed and restored dentine. The rate of perfusion is dependent on the structure and composition of dentine, particularly at the surface. This work analyzed the effect of treatment with an Erbium, Chromium-doped: Yttrium Scandium Gallium Garnet (Er,Cr:YSGG) laser on dentine perfusion. Extracted molars were sectioned above the mid-coronal portion, and below the cemento-enamel junction, to create crown segments. The pulp was extirpated and the dentine treated with the Er,Cr:YSGG laser, a diamond bur or sandpaper. Each specimen was mounted, with the pulp chamber oriented upwards, on a petri-dish cover in order to permit a tube filled with water to be connected to the pulp. Movement of the water meniscus over 24 h provided a measurement of the volume of water that filtered across the dentine. The dishes contained water to provide a moist environment in the relevant specimens, or were left dry to provide dry conditions. Specimens were perfused for 24 h with water pressures ranging from 20 to 60 cm. The results (in μl mm(-2) d(-1) ) showed a significant difference in the perfusion rate between treatments. The difference between perfusion in wet and dry conditions was highly significant. Laser and bur treatment of dentine significantly affected perfusion, which was higher in the former than in the latter. Therefore, laser-treated dentine may be more sensitive than bur-cut dentine.

Less invasive excavation methods have been suggested for deep caries lesions. We tested the effects of stepwise vs. direct complete excavation, 1 yr after the procedure had been carried out, in 314 adults (from six centres) who had received treatment of a tooth with deep caries. The teeth had caries lesions involving 75% or more of the dentin and were centrally randomized to stepwise or direct complete excavation. Stepwise excavation resulted in fewer pulp exposures compared with direct complete excavation [difference: 11.4%, 95% confidence interval (CI) (1.2; 21.3)]. At 1 yr of follow-up, there was a statistically significantly higher success rate with stepwise excavation, with success being defined as an unexposed pulp with sustained pulp vitality without apical radiolucency [difference: 11.7%, 95% CI (0.5; 22.5)]. In a subsequent nested trial, 58 patients with exposed pulps were randomized to direct capping or partial pulpotomy. We found no significant difference in pulp vitality without apical radiolucency between the two capping procedures after more than 1 yr [31.8% and 34.5%; difference: 2.7%, 95% CI (-22.7; 26.6)]. In conclusion, stepwise excavation decreases the risk of pulp exposure compared with direct complete excavation. In view of the poor prognosis of vital pulp treatment, a stepwise excavation approach for managing deep caries lesions is recommended.

– The dissolving effects of seven endodontic solutions on unprepared root canal walls of young intact human premolars were evaluated with the scanning electron microscope. After incubation at 37°C in distilled water or isotonic saline for 10 min the predentinal surfaces showed no changes as compared with untreated controls. The demineralizers Decal® and Largal Ultra® had little effect on the organic tissues but caused some decalcification where mineralized dentin was exposed. Nelex® caused concentration-dependent coagulation of the residual pulp tissue, making typical ring structures on the surfaces. Sodium hypochlorite at 2.5% and 5.0% dissolved most of the predentin, exposing the globular appearance of the mineralizing front. Salvizol® produced small globules on otherwise intact surfaces. Dissolution of both the organic and inorganic tissue of the root canal wall would require the combined use of two of the solutions studied.

Transforming growth factor-beta1 (TGF-beta1) and nerve growth factor (NGF) have been detected in pulp tissues after injury and are implicated in the differentiation of odontoblast-like cells and in pulp tissue repair. We examined TGF-beta1-mediated regulation of NGF and investigated its signaling pathways in human dental pulp cells. Analyses by reverse transcription-polymerase chain reaction (RT-PCR) and enzyme-linked immunosorbent assay (ELISA) revealed that TGF-beta1 (1 ng ml(-1)) induced NGF mRNA and protein expression through the phosphorylation of p38 mitogen-activated protein kinase (MAPK) and c-Jun N-terminal kinase (JNK). Blockade of the p38 MAPK and JNK pathways with the respective upstream inhibitors (SB203580 and SP600125) abolished the TGF-beta1-mediated induction of NGF. In addition, SB225002, a G-protein-coupled receptor antagonist, and staurosporine, a serine-threonine kinase inhibitor, partially inhibited TGF-beta1-mediated induction of NGF. Phospho-p38 MAPK was suppressed by SB225002, whereas phospho-JNK was inhibited by staurosporine. We conclude that TGF-beta1 up-regulates NGF in human dental pulp cells. This suggests that TGF-beta1 plays a role in NGF regulation during pulp tissue repair. The signal of TGF-beta1 involves the activation of MAPK, especially p38 and JNK. We suggest that crosstalk between TGF-beta1 and G-protein-coupled receptor signaling also participates in the inductive mechanism.

Microorganisms remaining beneath restorations can cause secondary caries and pulp damage. Because of this, antimicrobial treatment could be useful. The aim of this study was to evaluate the antibacterial effect of the HealOzone device on Streptococcus mutans and to compare it with the already proven activity of two dentin-bonding systems. Thirty-five human molars were divided into 5 groups. Cavities were then cut into the teeth (n = 28 cavities per group). After sterilization, the teeth were left in broth cultures of 10(6) colony-forming units (CFU) ml(-1) of S. mutans at 36 degrees C for 48 h. The appropriate treatment followed (group A, control; group B, Clearfil SE Bond; group C, Clearfil Protect Bond; group D, 40 s of treatment with ozone; and group E, 80 s of treatment with ozone), and the cavities were then filled with composite resin. After 72 h, the restorations were removed, dentin chips were collected with an excavator, and the total number of microorganisms was determined. All treatments significantly reduced the number of S. mutans present compared with the control group. The antimicrobial effect of both bonding systems and treatment with 80 s of ozone was significantly higher than the 40 s ozone treatment. In conclusion, HealOzone and the bonding systems show striking antimicrobial effects against S. mutans.

Primary cells in culture have a limited capacity to divide and soon reach a non-proliferative state. This cellular senescence limits the investigation of cells derived from human pulp concerning cellular pathways, gene regulation, mechanisms of dentin formation, or responses to material exposure. To overcome this problem, primary human pulp-derived cells were established and transfected with a plasmid containing coding sequences of Simian Virus 40 (SV40) large T-antigen. This resulted in the establishment of several cell clones showing an extension of life span. Expression of T-antigen transcripts and protein was verified by reverse transcription-polymerase chain reaction (RT-PCR) and immunohistochemistry. Primary human pulp cells were cultured until senescence (i.e. up to passage 7) and transfected cells could be cultured to passage 18 after transfection, when a cellular crisis with massive cell death occurred. One clone escaped from crisis and has been maintained in culture for 55 wk. Experiments were performed to characterize transfected cells in comparison to primary cells. Cell morphology and proliferation were analyzed, and expression of cell-specific gene transcripts and proteins (including collagen types I and III, alkaline phosphatase, bone sialoprotein, osteocalcin, and dentin sialophosphoprotein and dentin matrix protein I) was detected by RT-PCR and immunohistochemistry. Transfection of human pulp-derived cells resulted in an immortalized cell line retaining many of the phenotypic characteristics observed in primary cells.

The purpose of this study was to investigate pulp cell responses during hypoxia and reoxygenation. Pulp tissues obtained from beagle dogs were cultured. In the control group, pulp cells were incubated in normoxic conditions (20% O2) for 1-4 d. In the hypoxia group, pulp cells were incubated under hypoxic conditions (2% O2) for 1-4 d. In the reoxygenation group, pulp cells were first incubated under hypoxic conditions for 24 h, and were then incubated in normoxic conditions (20% O2) for one to three additional days. Cell viability, MTT (3- (4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) reduction assay, cellular proliferation, and alkaline phosphatase (ALPase) activity were determined. Expression of heat shock protein 70 (HSP70) and vascular endothelial growth factor (VEGF) was analysed by Western blotting. Hypoxia inducible factor-1alpha (HIF-1alpha) in pulp cells was analysed by reverse transcriptase polymerase chain reaction (RT-PCR). The cell growth rate and ALPase activity were significantly higher in the hypoxia group than in the control group. After reoxygenation, cellular proliferation and ALPase activity decreased to the level of the control group while HSP70 expression increased. Hypoxia inducible factor-1alpha expression was detected in pulp cells, and VEGF expression (which is regulated by HIF-1alpha) increased under hypoxic conditions. These results suggest that dynamic responses to hypoxia and reoxygenation occur in pulp cells.

Vascular endothelial growth factor (VEGF), a potent pro-angiogenic factor, might regulate the neovascularization observed in the pulp of teeth with deep caries. The purpose of this in vitro study was to evaluate the effect of bacterial lipopolysaccharides (LPS) on VEGF expression in dental pulp cells. Mouse odontoblast-like cells (MDPC-23) or undifferentiated pulp cells (OD-21) were exposed to 0-20 microg ml-1Escherichia coli LPS or 0-80 microg ml-1Prevotella intermedia LPS. As controls, mouse macrophages or gingival fibroblasts were exposed to LPS, since these cells are known to secrete VEGF. The VEGF expression was evaluated by reverse transcriptase polymerase chain reaction or enzyme-linked immunosorbent assay. The baseline expression levels of VEGF protein were higher in MDPC-23 and OD-21 than in fibroblasts or macrophages. Vascular endothelial growth factor protein expression was upregulated in MDPC-23 and macrophages exposed to E. coli LPS, but not in OD-21 cells or fibroblasts. Higher concentrations of P. intermedia LPS were required to induce VEGF expression in MDPC-23 cells. Treatment with LPS did not affect VEGF expression at the mRNA level in any of the cells evaluated. These results demonstrate that bacterial LPS upregulates VEGF expression in odontoblast-like cells and macrophages, and suggest that the regulation of VEGF expression occurs primarily at a post-transcriptional level.

Previous studies have demonstrated that (at least) matrix metalloproteinase (MMP)-2, -8, -9, -14 and -20 are expressed by human odontoblasts. Here, we analysed the expression of 19 MMPs and their specific tissue inhibitors (TIMP)-1, -2 and -3) -1, -2 and -3 in mature human odontoblasts and pulp tissue. Since MMP-20 is almost exclusively expressed by the dentin-pulp complex cells, we further analysed the effect of transforming growth factor (TGF)-beta1 and bone morphogenetic protein (BMPs)-2 on its expression. Matrix metalloproteinase-9 served as a positive control for growth factor responsiveness. It was found that MMP-1, -2, -9, -10, -11, -13, -14, -15, -16, -17, -19, -20 and -23, in addition to TIMP-1, -2 and -3 were expressed by both odontoblasts and pulp tissue. Neither MMP-3 nor MMP-12 were expressed in odontoblasts or pulp tissue, and MMP-7, -8, -24 and -25 were expressed only in the odontoblasts; MMP-2, -10, -11, -14 and -20 were expressed more abundantly by odontoblasts, whereas pulp tissue expressed more MMP-13 and MMP-17. Transforming growth factor-beta1 (1 ng ml(-1)) and BMP-2 (100 ng ml(-1)) did not markedly affect MMP-20 mRNA expression. In contrast, TGF-beta1 alone and with BMP-2 significantly upregulated MMP-9 mRNA by 2.4-fold and by 2.6-fold, respectively, in odontoblasts, while in pulp tissue no effects could be detected. The wide-scale expression of MMPs and TIMPs by mature human odontoblasts and pulp tissue suggests that they may participate in dentin matrix organization prior to mineralization, and that growth factors may further control dentin matrix modeling by differentially regulating individual MMPs.

The role of matrix metalloproteinases (MMPs) in the breakdown of pulp tissue of teeth with severe caries has not yet been directly elucidated. This study was to determine the levels of selected MMPs and the overall gelatinolytic activity in clinically healthy and inflamed human dental pulps of 29 healthy subjects, aged 10-19 yr. Seventeen pulps were collected from subjects diagnosed with symptomatic pulpitis, and 18 control pulps were obtained from 12 subjects following premolar extraction for orthodontic reasons. The levels of MMP-1, MMP-2, MMP-3 and MMP-9 were determined with enzyme-linked immunosorbent assay. Densitometric analysis of gelatin zymograms was used to assay gelatinolytic activity in pulp supernatants. The MMP-1 levels were below the detection limit for both groups. Levels of MMP-2 and MMP-3 were significantly lower in symptomatic vs. clinically healthy pulps. In contrast, levels of MMP-9 in inflamed pulps were significantly higher than those recorded in clinically normal pulps. The overall gelatinolytic activity was elevated in inflamed pulps compared with healthy counterparts. Further, the gelatinolytic activity was positively correlated with MMP-9 levels. The data obtained suggest a key role of MMP-9 in the breakdown of inflamed human dental pulp tissue.

Proteolytic processing and degradation of enamel matrix proteins appears to be an essential feature of dental enamel formation. The source and character of proteolytic activity in the enamel matrix of developing teeth changes as enamel formation progresses. Two proteinases have been isolated from the extracellular enamel matrix of developing teeth: enamelysin (MMP-20), a matrix metalloproteinase, and kallikrein-4 (KLK4), a serine proteinase. Here, we ask if the expression of MMP-20 and KLK4 correlate with the stage-associated changes in the digestion of enamel proteins. Using in situ hybridization, we localized MMP-20 and KLK4 mRNA in mouse maxillary first molars on postnatal days 1, 2, 3, 5, 6, 7, 9, 11, and 14. Enamelysin signal was first detected in preameloblasts, ameloblasts, and odontoblasts on day 2, but not in ameloblasts covering the enamel-free zone. Enamelysin signal declined in ameloblasts on day 6 but persisted in the dental pulp. In contrast, KLK4 transcripts were first observed on day 3 in pulp and on day 6 in ameloblasts covering the enamel-free zone. the KLK4 signal was present in maturation-stage ameloblasts on days 9, 11, and 14. The expression patterns of MMP-20 and KLK4 by ameloblasts in mouse molars are stage-specific and complementary.

The aim of this study was to investigate the presence of substance P (SP), neurokinin A (NKA) and calcitonin gene-related peptide (CGRP) in the gingival crevicular fluid of teeth diagnosed with pain of pulpal origin compared with clinically healthy teeth, and to detect any changes in the levels of these neuropeptides in gingival crevicular fluid after removal of the pulp from the painful teeth. Gingival crevicular fluid was collected at baseline from one interproximal site at a painful and a non-painful contralateral tooth from 54 adult patients. Sampling was repeated after 1 wk in a subset of 21 subjects. Samples were analysed for SP, NKA, and CGRP using radioimmunoassay. The mean levels of SP and NKA were significantly higher in gingival crevicular fluid from painful teeth compared with non-painful teeth. The level of SP in the GCF of painful teeth fell significantly 1 wk after pulpectomy. In contralateral teeth, there were no significant differences in the levels of SP and NKA after 1 wk. It is concluded that SP and NKA are present in significantly greater amounts in the GCF of painful teeth compared with healthy teeth.

Amelogenin and enamelin are structural proteins in the enamel matrix of developing teeth. The temporal and spatial patterns of enamelin expression in developing mouse molars have not been characterized, while controversy remains with respect to amelogenin expression by odontoblasts and cementoblasts. Here we report the results of in situ hybridization analyses of amelogenin and enamelin expression in mouse molars from postnatal days 1, 2, 3, 7, 9, 14, and 21. Amelogenin and enamelin mRNA in maxillary first molars was first observed in pre-ameloblasts on the cusp slopes at day 2. The onsets of amelogenin and enamelin expression were approximately synchronous with the initial accumulation of predentin matrix. Both proteins were expressed by ameloblasts throughout the secretory, transition, and early maturation stages. Enamelin expression terminated in maturation stage ameloblasts on day 9, while amelogenin expression is still detected in maturation stage ameloblasts on day 14. No amelogenin expression was observed in day 21 mouse molars. Amelogenin and enamelin RNA messages were restricted to ameloblasts. No expression was observed in pulp, bone, or along the developing root. We conclude that amelogenin and enamelin are enamel-specific and do not directly participate in the formation of dentin or cementum in developing mouse molars.

The neuropeptide substance P (SP) is found within a subpopulation of nociceptive afferent nerve fibres and has been shown to be upregulated in a variety of sites following peripheral inflammation. The aim of this study was to investigate the expression of SP within human teeth, both in health and disease, and to seek a correlation between reported pain history and SP expression within pulpal nerves. Coronal pulps were removed from 62 permanent mandibular molars with a known pain history. Teeth were categorised as intact, moderately or grossly carious. Using indirect immunofluorescence, sections were double-labelled for the general neuronal marker protein gene product 9.5 (PGP 9.5) and for SP. Image analysis was then used to quantify the percentage area of PGP 9.5-labelled tissue which was also labelled for SP. Throughout the pulp, the expression of SP was found to be significantly increased with the progression of caries. Furthermore, SP expression was significantly greater in grossly carious painful specimens than in grossly carious asymptomatic specimens. These data would suggest that the expression of SP within pulpal nerves undergoes dynamic changes following caries, which may have an important clinical significance in terms of inflammation and pain experience.

In vitro investigations of cell-specific metabolism and cell interactions as well as biocompatibility studies are often hampered by the limited lifespan of primary cells originating from target tissues like the oral mucosa, gingiva or pulp. Pulp cells, as do other primary cells, undergo senescence after several passages in vitro. However, senescence can be overcome by transfection of primary cells with oncogenes like the HPV 18 (human papillomavirus 18) E6/E7 oncogene, resulting in immortalized cell lines. The purpose of our study was to establish and preliminary characterize an immortalized bovine dental papilla-derived cell line by transfection with HPV 18 E6/E7 for future use in biocompatibility testing of dental materials. First passage dental papilla-derived cells from molar tooth germs of 6-month-old calves were transfected by electroporation with pUC18 LCR E6/E7 coding for the HPV 18 E6/E7 oncogene. Cells underwent crisis after 5 wk in culture. Distinct cell colonies arose after about 9 wk. Cells were cloned by single cell dilution in passage 15 and 17. Out of three cell clones maintained in culture, two cell clones showed cell death after 28 and 30 wk, respectively. One cell clone overcame a second crisis after 38 wk and was maintained in culture until passage 75. Stable gene expression of HPV 18 E6/E7 oncogenes was verified by polymerase chain reaction (PCR) and immunohistochemistry. Reverse transcription (RT)-PCR revealed that the established cell line (passage 50) expresses procollagen type I, alkaline phosphatase and osteocalcin. This suggests that the immortalization with HPV 18 E6/E7 results in a cell line, which maintained phenotypic characteristics of odontoblast-like cells.

The expression of hyaluronan in human tooth germs was studied by using a biotinylated hyaluronan-binding complex and quantitative digital image analysis. At the cap stage, dental papilla exhibited a moderate staining, while intense reaction was observed in the apical portion of presecretory ameloblasts, stellate reticulum, and in dental basement membrane. When the enamel and dentine matrices started to develop, a strong hyaluronan reaction was evident in the young enamel and the apical portion of secretory ameloblasts. No hyaluronan could be detected in the secretory ameloblasts and enamel matrix of the early (9-wk-old) post-natal stage. It is concluded that hyaluronan may play a transitory role in the early phase of the development of the enamel matrix organization. A very weak signal was observed in the wall of dentin tubules, whereas the rest of the dentine matrix was not stained. The odontoblasts and the pulp were also moderately stained, and these reactions gradually decreased with age, suggesting that hyaluronan may also contribute to the development of dentine matrix and pulp.

Recombinant human BMP-7 (bone morphogenetic protein-7, osteogenic protein-1) is osteogenic, dentinogenic and cementogenic when implanted into the appropriate tissue in vivo. However, most studies characterizing the induction of these tissues have implanted BMP-7 into freshly surgerized, clinically healthy tissues. To determine if BMP-7 is dentinogenic in inflamed dental pulps, we applied BMP-7 to inflamed ferret pulps. A single application of 5 microg of a commercial preparation of lipopolysaccharide (LPS) from Salmonella typhimurium directly to the coronal pulp induced a reversible mixed inflammatory exudate of moderate intensity within 3 d. Treatment with a single application of 2.5, 7.5 or 25 microg recombinant human BMP-7/mg collagen (2 mg total mass/tooth) induced reparative dentinogenesis in controls but not LPS treated dental pulps. These data reveal that a single application of up to 50 microg/tooth of exogenous recombinant BMP-7 is insufficient to induce reparative dentinogenesis in ferret teeth with reversible pulpitis. Given that pulp cells in the inflamed tissues likely retain the capacity to respond to exogenous BMP-7, it is possible that insufficient active recombinant protein is available to induce tissue formation in experimentally inflamed dental pulps.

Non-amalgam filling materials may release substances which have been shown to be toxic in cytotoxicity tests and implantation studies. However, results from systemic toxicity tests do not indicate any unacceptable risk to the patient's general health, but data for non-amalgam dental filling materials are scarce in comparison to amalgam. Although estrogen-like effects of one fissure sealant have been claimed, no conclusions can be drawn at present for the patient from these in vitro data because of the limitation of the test methods and materials used. Some components of composite resins/dentin adhesives and a resin-modified glass ionomer cement were mutagenic mainly in in vitro tests. Due to the limitations of the test systems and the comparatively high concentrations needed to elicit the reactions, no unacceptable risk can yet be derived from those data for the patient. However, a no-touch technique is recommended for the dental personnel. As with amalgam, local reactions of the pulp are not expected with alternative filling materials, if the pulp tissue is not exposed and if bacterial penetration is avoided. The latter requirement is still difficult to fulfill, especially for composite resin systems and related materials in posterior teeth situations. Slight gingival reactions to alternative filling materials and to amalgams are mainly attributed to plaque accumulation. From all these data it can be concluded that, for the time being, it is not possible to rank dental filling materials in respect to their biocompatibility, and it is evident that biocompatibility must be considered to the same extent for both amalgams and commonly used or recommended alternative filling materials.

The periodontal ligament and dental pulp connective tissues are foetal-like, and their process of ageing may therefore differ from other tissues of mesenchymal origin. Several studies have already highlighted the lack of age changes in the extracellular matrix (ECM), but more needs to be known about cellular changes. For this study, tissues from the first molar teeth of Wistar rats aged 12 wk and 104 wk were compared by immunolocalisation of cytoskeletal components. Tissues from the first molar were immunostained, employing a panel of 16 monoclonal antibodies against cytokeratins, vimentin, F-actin and tubulin. Within the pulp, labelling for vimentin in both odontoblasts and pulpal fibroblasts and F-actin in the cell processes of odontoblasts was detected at both ages but with marked reduction in labelling in the older tissue. Within the periodontal ligament, vimentin labelling was weaker in the aged tissue, especially nearer the cementum. More significantly, the fibroblasts of the aged periodontal ligament expressed cytokeratin 19. In contrast to reports of little age change to the ECM, the cells of the pulp, and particularly the periodontal ligament, show marked changes to their cytoskeletal components.

This study was designed to investigate in vivo i) the role of TGF-beta as an active component of the dentin matrix during induction of reparative dentinogenesis; and ii) the ability of TGF-beta1 isoform to induce dentinogenic events. Dental pulps of dog molars and canines were mechanically exposed, and the following implants were placed intrapulpally for 42 d: i) Demineralized or native autogenous dentin matrix preincubated with a TGF-beta-neutralizing antibody; ii) Millipore filters soaked with solution containing 100 ng/ml of TGF-beta1 from human platelets; iii) biomatrices and filters soaked in control solutions. After incubation of biomatrices with the TGF-beta-neutralizing antibody, demineralized dentin completely lost its inductive activity, while native dentin was only able to stimulate formation of fibrodentin. Millipore filters soaked with TGF-beta1 were consistently surrounded by a thick zone of tubular matrix lined with high columnar polarized cells. The data provide evidence that pulp cells can express their dentinogenic potential in response to an appropriate surface containing exogenous TGF-beta1, and that the dentinogenic activity of dentin matrix may at least partly be ascribed to TGF-beta molecule(s).

The distribution of nerve fibers with special reference to their positional relationship with blood vessels in the pulp of the rat mandibular incisor was investigated by light and electron microscopic immunohistochemistry using polyclonal anti-substance P antibody. In the center of the pulp, numerous immunoreactive nerve fibers with varicosities were found along larger blood vessels. In the periphery of the pulp, there were two layers of capillary plexus; an odontoblastic and a subodontoblastic. Many nerve fibers, showing varicosities, were located in the subodontoblastic region, and some revealed a close association with pericytes of capillaries of the subodontoblastic plexus. A few nerve fibers entered the odontoblast layer, but no nerve fibers were seen to be associated with the odontoblastic capillary plexus. The results suggest that the local regulation of blood flow takes place not only in larger blood vessels but also in the capillaries of the rat incisor pulp.

Information concerning cell proliferation and differentiation in dental pulp may be important to understand tooth response to exogenous stimuli. Since few data concerning human dental pulp are available, we have investigated the growth fraction and the localization of proliferating cells in pulp tissue of third molars of young adult human males and females, using flow cytometry and immunohistochemistry. Flow cytometric analysis demonstrates a low proliferative activity of pulp tissue that appears to be confined to radicular pulp, as revealed by immunohistochemical detection of proliferating cells. No polyploid or aneuploid cell populations could be identified, and G2-blocked cells, if any, represented a negligible cell population. Odontoblasts, cells of the sub-odontoblastic layer, and cells of coronal pulp were found to be not proliferating under normal conditions. These data provide the basis for future investigations on proliferative activity and regenerative potentiality of human pulp cells in experimental and clinical situations.

The constitutive (hsc 70) and inducible (hsp 70) isoforms of heat shock protein 70 are important members of the superfamily of stress related proteins that protect and promote the recovery of cells from physiological and pathologic stress. The goal of this study was to define the baseline expression of hsc 70 and hsp 70 in disease-free, minimally stressed human dental pulp of the adult 3rd molar. Immunolocalization demonstrated moderate to heavy staining intensity for hsc 70 in both the cytoplasm and nucleus of odontoblasts and fibroblasts comprising the human pulp. Endothelial and smooth muscle cells displayed weak to moderate immunoreactivity for hsc 70 in both the cytoplasm and nucleus. Schwann cells demonstrated only weak nuclear staining for hsc 70. No immunoreactivity for hsp 70 was observed in any cell type in human pulp. Western, northern, and RT-PCR analysis of pulp preparations confirmed the expression of hsc 70 mRNA and protein within components of the pulp. These results demonstrate that cells of the human pulp express, under conditions of minimal stimulation, a key component of the stress response protein superfamily. The expression of hsc 70 under conditions of minimal stress may provide pulp components an advantage in resisting cell injury when stress occurs.

Dentin formation is preferred in the healing response of the pulp to pulp-capping agents during vital pulp therapy. Enhancement of the dentinogenic differentiation of dental pulp cells is thought to accelerate pulp repair. The aim of this study was to evaluate the dentinogenic activity of small molecules (three flavonoids and phenamil) that have been shown previously to induce osteoblast differentiation. Among the flavonoids (quercetin, genistein and baicalin), quercetin induced the highest alkaline phosphatase (ALP) activity of human dental pulp (HDP) cells. Phenamil, an amiloride derivative, elicited higher ALP activity than quercetin. However, increased expression of dentin sialophosphoprotein (DSPP) mRNA and mineral deposition were seen in cultures treated with quercetin compared with phenamil. This would seem to suggest that quercetin is the most dentinogenic agent among the tested chemicals. The increase in ALP activity in the quercetin-treated cells was not affected by ICI 182,780, an estrogen receptor inhibitor, and was partially blocked by PD98059, an extracellular signal-regulated kinase 1/2 (ERK1/2) inhibitor. This suggests that ERK1/2 is activated in the quercetin-induced differentiation of HDP cells without the mediation of estrogen receptors, which are known to be involved in osteoblast differentiation induced by quercetin.

The ability of 2 calcium hydroxide-containing materials to induce initiation of reparative dentinogenesis was tested at sites remote from the dentinogenically-active regions of the pulp periphery. Pieces of the cements, Dycal and Life, were implanted in central parenchymal sites of dog dental pulps for periods of 6, 14 and 42 days, respectively. Similar pieces were placed in peripheral capping sites as controls. The responses were analyzed by light and transmission electron microscopy. Induction of tubular dentin matrix lined with elongated and polarized odontoblast-like cells was only seen at peripheral capping sites. In response to the centrally implanted cements, only atubular hard tissue with lining fibroblast-like cells was deposited.

The use of iontophoresis for facilitated diffusion of vasoactive agents into the dental pulp was investigated in lower incisor teeth of anaesthetized rats. Acetylcholine, carbachol and noradrenaline were iontophoresed with anodal and sodium nitroprusside with cathodal direct current through a superficial dentin exposure. Pulpal blood flow was measured with laser Doppler flowmetry. Current intensities below 100 microA of both polarities, using sodium chloride as a medium, caused no or minor afferent nerve-induced vasodilation, but excited sympathetic fibres of the pulp in a current-dependent manner. The current threshold for facilitated diffusion of acetylcholine was about 20 microA. The vascular responses to the cholinergic and noradrenergic drugs appeared within a minute after the onset of current and they were abolished by systemic administration of atropine and phenoxy benzamine, respectively. Iontophoresis of acetylcholine (40-100 microA for 20-120 s) caused a 3-fold increase of pulpal blood flow which was not dose-dependent; carbachol provoked a high-magnitude, long-lasting vasodilation and so did sodium nitroprusside. Noradrenaline caused a long-lasting vasoconstriction. In denervated rats iontophoresis of carbachol had effects similar to those seen in intact animals. None of the drugs used locally had any effect on systemic blood pressure. The results of this study indicate that iontophoresis can be used for delivery of vasoactive agents from an exposed dentin surface into the pulp in sufficient quantity to elicit drug-specific local vascular responses without causing systemic vascular effects.

In rats fed an essential fatty acid deficient (EFAD) diet, either during pregnancy (DN) or for 4 wk postnatally (ND), the cell density in the central part of the pulp increased about two- and threefold, respectively, of that in rats who had received a conventional diet containing sunflower oil. Cells were especially numerous around capillaries. The cell density was also increased twofold in the subodontoblastic layer in the outer part of the pulp, cells being smaller in ND compared with DN. In contrast, the- odonloblasts were reduced in height, and the Hohl cells formed a thin layer in EFAD rats. This emphasizes some aspects of pulp specificity which reacted differently from odontoblasts. We suggest that the function of killer cells which normally destroy cells at the periphery of the pulp may be impaired by the diet, leading to cell accumulation.

This study examined the defensive ability of human dental pulp against H 2 O 2 in healthy and reversible and irreversible pulpitis tissues through determination of catalase activity by spectrophotometric methods. Thirty-five systemically healthy patients were donors of the pulp tissue, and pulp conditions were assessed using clinical and X-ray evaluations. Catalase activity was 1.61 ′ 0.23 U mg - 1 protein in the healthy tissues, 2.99 ′ 0.45 U mg - 1 protein in the reversible pulpitis tissues, and 2.44 ′ 467 mU mg - 1 protein in the irreversible pulpitis tissues. All differences between the groups were statistically significant. These results point to a role for catalase during dental pulp inflammation in humans, and therefore demonstrate an inherent biological defense system against reactive oxidants in human dental pulp.

Extracted teeth from five deceased patients treated by renal transplantation were examined radiographically and histologically, and compared with teeth from healthy persons of approximately the same age. In three of the patients with a renal transplant, the radiographs showed normal pulp chamber sizes, but histologic examination revealed a widened predentin zone, approximately four times greater than in the controls. In two of the patients the radiographs showed marked reduction of the pulp chamber size. The histologic changes of these teeth were mainly an extensive amount of secondary dentin along the pulp chamber walls and the root canals, with a markedly reduced pulp space. One explanation for the difference between the patients was that patients with extensive pulp calcifications were given a higher total amount of corticosteroids than patients with a widened predentin zone.

Class V cavities were prepared in 47 caries-free premolars intended to be extracted for orthodontic reasons. A powdered gold filling material, Goldent, which can be hand condensed, was used for direct fillings. The following test groups were used: (A) cavity preparation, no filling, (B) cavity filled with Goldent only, (C) cavity lined with Tubulitec and filled with Goldent, (D) cavity with Goldent filling, sealed with IRM, and (E) positive and negative controls. The teeth were extracted after 1-200 days and the pulpal response was histologically examined. The results revealed that only in Group B, i.e. when the cavity was filled with Goldent, was the risk of a pulpal reaction obvious, although the material per se (Group D) did not influence the pulpal tissue to any serious degree. Probably the Goldent material cannot provide a marginal seal tight enough to prevent microleakage, allowing microorganisms to give rise to pulp damage and inflammation. In those cases where the cavity had been isolated with Tubulitec liner before the gold restoration was made, the pulp was effectively protected from damage caused by microorganisms.

The amount of secondary dentin in a tooth has been used as one of several parameters in methods for age estimation. Dentin deposition has been measured according to various scoring systems, and the Pearson correlation coefficient with age has been found to be approximately 0.6. In the present study of 1000 teeth, molars excluded, secondary dentin was estimated according to three scoring systems. The teeth were prepared according to the half tooth technique. In addition, the area of the coronal pulp and the widths of the root and pulp chamber were measured in a stereomicroscope at the cemento-enamel junction and at three other defined points along the root. The Pearson correlation coefficient between age and secondary dentin varied in different types of teeth. Of the scoring systems, scores according to Johanson were most strongly correlated with age (r = 0.59 to 0.74). Correlation between age and the coronal pulp area varied from -0.47 to -0.72, and the range between age and ratio between pulp- and tooth width at the cemento-enamel junction was from -0.46 to -0.77. Correlations between age and ratio between sum of pulp widths and the sum of tooth widths for all four such measurements ranged from -0.58 to -0.81. Multiple regression analyses showed that by combining several types of measurements, the correlation with age was increased. A tendency was also observed towards reduced speed of secondary dentin formation in the elderly and in women.

In essence, endodontics as a clinical discipline is concerned with the prevention and treatment of pulpal and periapical infection. In recent research the infective process has been investigated as well as the mechanisms by which the pulp and periodontium deal with microbial insults. With regard to the pulp, findings on the hemodynamics of pulpitis suggest that the inflammatory response in this tissue is much less influenced by the special anatomic environment of the tooth than was previously believed. Pulpal diseases are being underdiagnosed, mostly because of inadequate examination methods. Laser Doppler flowmetry which gives a vascular rather than a nervous response may gain importance in pulpal diagnostics in the future. It is established that apical periodontitis with bone resorption cannot develop in the absence of bacteria in the root canal system. Root canal infection is characterized by a wide variety of combinations of relatively few anaerobic bacteria, and bacterial synergism plays an important role in maintaining the infection. Microbial invasion of an apical granuloma may take place. Non-oral and environmental organisms like Pseudomonas aeruginosa are frequently isolated from treatment-resistant cases. Success of endodontic treatment depends on the reduction or elimination of the infecting bacteria. This may predictably be obtained after a thorough chemo-mechanical instrumentation and disinfection of the root canal with calcium hydroxide. The standardized technique which entails the preparation of a cylindrical apical box with removal of significant amounts of dentin near the root apex predictably gives a clean canal. This technique has provided excellent clinical and radiographic results in well documented follow-up studies.

This study investigated the characteristics of two dental pulp cell populations during the early stages of crown formation in porcine teeth. A transplantation method was developed to reproduce epithelial cell-mesenchymal cell interactions during odontogenesis (tooth development). The technique allowed two types of cells/tissue to be combined in vivo. Populations of cells localized in the cervical loop epithelium region, dental pulp horn, and dental pulp core chambers were isolated and dissociated into single cells. Each population was examined for its gene-expression pattern using both semiquantitative and quantitative reverse transcription-polymerase chain reaction (RT-PCR) analyses, and for its tissue-formation capability by combining the cervical loop epithelial cells with either pulp horn cells or pulp core cells on biodegradable collagen scaffolds that were subsequently examined using histology and immunohistology. Gene-expression patterns showed that pulp horn cells were more mature than pulp core cells. Cervical loop epithelial cells combined with pulp horn cells mainly reconstituted dentin-cementum structures. By contrast, cervical loop epithelial cells combined with pulp core cells reconstituted enamel-dentin structures. These results suggest that mesenchymal cells residing in a specific location of the pulp possess a specific tissue-formation potential when combined with epithelial cells.

The lymphatic system is important for immune barrier function and for tissue fluid balance. During inflammation, lymphangiogenesis takes place to enhance the transport of filtered fluid, proteins, and immune cells. Dental tissue is frequently exposed to inflammatory insults, but the lymphatic system and its responses to injury have not been investigated in detail using specific lymphatic markers. We aimed to study this system and to establish whether lymphangiogenesis takes place during wound healing. Immunostaining of the lymphatic endothelial hyaluronan receptor-1 (LYVE-1) and vascular endothelial growth factor receptor-3 (VEGFR-3) demonstrated initial lymphatics in the coronal molar pulp, whereas in incisors the initial lymphatics were found only in the apical part. In molars, lymphatic vessels exit the pulp through the apex and lateral canals. In interdental bone, transverse lymphatics were found, raising the possibility that an infection can be spread from the periodontal ligament to a neighbouring tooth. LYVE-1(+) and VEGFR-3(+) immune cells were found in both molar and incisor pulps, and phenotyping of the cells showed that they are of a monocytic lineage. In inflamed pulp these cells were not observed. Macrophages are suggested to contribute directly to the formation of lymphatic vessels after pulp exposure.

— Class II antigen expressing cells are generally associated with the early phase of the immune response. Dendritic cells and macrophages expressing these cell surface antigens have recently been demonstrated and characterized in the dental pulp. The present study was undertaken to determine when the pulp receives its immunologic defense potential by examining the temporal appearance of class II antigen expressing cells in the rat incisor pulp. Pulp tissue specimens obtained at various time periods from a gestational age of 16 days to 14 wk after birth were examined by immunohistochemistry using O x 6 as a primary antibody and the ABC-technique. Comparisons were made with tissue samples from the spleen, intestine, skin and oral mucosa. At birth, all tissues, except for the pulp, presented cells expressing class II antigen with a dendritic appearance in a number and orientation resembling the mature tissue. A complete distribution of these cells was not seen in the dental pulp until 7 weeks following birth. Data show that the dental pulp acquires its ultimate structural arrangement of immune cells later than other tissues.

Neutrophils and monocytes/macrophages (MO), found in oral mucosa and gingival sulcus, phagocytose and kill bacteria using products produced during a respiratory burst. 2-Hydroxyethyl-methacrylate (HEMA) is a major component released from resin glass ionomer and dental adhesives. Hence, in pulp and gingiva, phagocytes can come into contact with unpolymerized HEMA monomers. The aim of this study was to examine the effects of exposure to HEMA on neutrophil and monocyte bactericidal function. Blood collected from five female volunteers was exposed in vitro to HEMA for 2 h and then phagocytosis, respiratory burst, and cellular integrity were measured using flow cytometry. Respiratory burst was quantified by measuring fluorescent rhodamine 123 generated via oxidation of dihydrorhodamine 123. Cellular membrane integrity was evaluated by staining with propidium iodide. The respiratory burst activity of the neutrophils was significantly decreased by exposure to 7.5 and 15 mM HEMA. No significant effect of HEMA was seen on the number of granulocytes or monocytes capable of performing respiratory burst. Furthermore, there was no significant effect of HEMA on the phagocytic activity of the monocytes or the granulocytes. In conclusion, HEMA did not affect the phagocytosis activity of neutrophils; however, the ability of the cells to kill internalized prey was significantly reduced.

Ectopic tooth transplants are known to receive rich innervation of local neurons, but the precise location and structural features of neurites in the pulp and periodontal ligament (PDL) of such transplants are unclear. In this experiment, the molar tooth germs of rat embryos and incisors of young rats were subcutaneously transplanted into the dorsal regions of rats and processed, at various time intervals, for immunohistochemical demonstration of neural elements. Teeth with periodontal tissue elements developed in most of the molar transplants in 6 or 8 wk and received rich innervation, including some autonomic fibres, in the pulp. Nerve elements were also confirmed to be present in the PDL of these transplants, including specialized nerve ending-like structures reminiscent of the periodontal Ruffini endings. Mechanoreceptor-like structures were also induced in the regenerated PDL of similarly transplanted incisors, although the success rate was low. We conclude that rich and highly ordered innervation of the pulp, and occasional development of mechanoreceptors in the regenerated PDL of ectopic dental transplants, imply a high probability of successful induction of teeth with both nociceptive and mechanical sensations in the ectopic tooth and/or tooth germ transplant systems, although differentiation of mechanoreceptor-like nerve endings occurred in only a few rare cases.

Human permanent teeth were examined in the scanning electron microscope after demineralization and exposure to preparative procedures based on hydrogen peroxide, trypsin, and EDTA. These substances removed the inorganic material, the cellular structures, the homogeneous connective tissue ground substance, and interfibrillar matrix. The remaining tissue components comprised a network of distinct collagen fibers whose organization was related to the type of tissue in which these were incorporated. A similar or identical method has not been developed or applied to teeth previously. Dentin and predentin comprised a compact mass of fibers which basically were parallel to the continuously growing interior surface of the predentin, or arranged at an acute angle to this plane. Collagen fibers in the pulp were numerous, but lacked any particular orientation in most areas. Interodontoblastic fibers crossed the odontoblastic zone at a right angle to the pulp chamber wall and mingled with collagen fibers in predentin. When previously published findings of ours are taken into account, it is possible to conclude that other factors than the organization of the collagen fibers are responsible for the stainability of these fibers in predentin and in interglobular dentin with silver methenamine, and that aldehyde groups on collagen fibers in predentin may be actively and directly involved in the mineralization of the dentin.

The aim of this in vitro study was to determine the diffusion of 2-hydroxyethyl methacrylate (HEMA) released from different bonding systems (BS) through dentin. Occlusal cavities with a remaining dentin thickness (RDT) of 0.5 mm (n=90) and 0.25 mm (n=80), respectively, were prepared in dentin discs of non-carious human molars. Artificial pulp chambers were attached to the pulpal side of each dentin disc. Bonding systems were applied with (Clearfil SE Bond, OptiBond FL, OptiBond Solo Plus) or without (AdheSE, Adper Prompt L-Pop, Clearfil SE Bond, OptiBond FL, OptiBond Solo Plus Self Etch, Xeno III) prior phosphoric acid etching. HEMA was detected by gas chromatography/mass spectrometry (n=10 per BS and RDT). The highest mean HEMA concentration was found in the 0.25 mm RDT group treated with OptiBond FL (13.3 microg) and the lowest mean HEMA concentration was detected in the 0.5 mm RDT group treated with AdheSE (0.5 microg). At 0.25 mm RDT the quantities of HEMA recovered in the artificial pulp chambers were significantly higher than at 0.5 mm RDT, except for Clearfil SE Bond. Etching with phosphoric acid increased the detected HEMA quantities compared with self-etch BS. In deep cavity preparations, etching with phosphoric acid should be avoided in favor of the use of self-etch BS.

Different sodium-dependent inorganic phosphate (P(i)) uptake mechanisms play a major role in cellular P(i) homeostasis. The function and detailed distribution patterns of the type III Na(+)-phosphate cotransporter, PiT-2, in different organs during development are still largely unknown. We therefore examined the temporospatial expression patterns of Pit2 during murine odontogenesis. Odontoblasts were always devoid of Pit2 expression, whereas a transient, but strong, expression was detected in young secretory ameloblasts. However, the stratum intermedium and, later on, the papillary layer and cells of the subodontoblastic layer, exhibited high levels of Pit2 mRNA, which increased gradually as the tooth matured. Hormonal treatment or P(i) starvation of tooth germs in vitro did not alter Pit2 levels or patterns of expression, indicating mechanisms of regulation different from those of PiT-1 or other cell types. PiT-2 also functions as a retroviral receptor, and functional membrane-localized protein was confirmed throughout the dental papilla/pulp by demonstrating cellular permissiveness to infection by a gammaretrovirus that uses PiT-2 as a receptor. The distinct pattern of Pit2 expression during odontogenesis suggests that its P(i)-transporter function may be important for homeostasis of dental cells and not specifically for mineralization of the dental extracellular matrices. The expression of viral receptors in enamel-forming cells and the dental pulp may be of pathological significance.

Remodelling of the periodontium after application of mechanical forces constitutes the basis of clinical orthodontics and various immunoregulatory molecules are involved in this process. The aim of this study was to investigate the expression of the cytokines interleukin-1alpha (IL-1alpha) and tumor necrosis factor-alpha (TNF-alpha) in dental tissues during the early phases of orthodontic tooth movement. Eightteen male Wistar rats were used. All maxillary right first molars were moved orthodontically, with a force of 0.5 N, for 3 h, 1 d, and 3 d. The contralateral sides served as untreated controls. Parasagittal sections of the maxillary molars and the surrounding tissues were subjected to immunohistochemical staining for IL-1alpha or TNF-alpha, and were evaluated with light microscopy. IL-1alpha and TNF-alpha were expressed in the bone and periodontal ligament (PDL) along the roots of the orthodontically moved molars and in the gingiva. Increased expression of both cytokines was observed in the aforementioned areas after 1 and 3 d of tooth movement. The pulp tissue exhibited only minor changes in cytokine expression during tooth movement. The results suggest that mechanical stress results in almost immediate inflammatory reactions in various dental tissues.

Recombinant amelogenin gene splice products A+4 and A-4, implanted in the pulp, induce the recruitment, proliferation, and differentiation of reparative cells. Our aim was to investigate the precocious events occurring in the pulp 1 d and 3 d after implantation of agarose beads alone or loaded with A+4 or A-4. Proliferation and cell recruitment towards an odonto/osteogenic phenotype were visualized by detection of the proliferation cell nuclear antigen (PCNA) and RP59. After implantation of beads alone or loaded with A+4, at day 3, pulp cells were moderately immunopositive for osteopontin (OP), whereas labeling was strongly positive upon treatment with A-4. Dentin sialoprotein (DSP) labeling was not detectable. Parallel in vitro studies were carried out on odontoblastic and mesenchymal progenitor cells in order to evaluate the effect of the amelogenin peptides on the expression of a series of marker genes involved in the odontoblastic/osteogenic/chondrogenic differentiation pathways. Altogether, our results suggest that the 'signaling' effects of the amelogenin peptides A+4 and A-4 may differ according to the type of target cells, their stage of differentiation, the time of treatment, and the type of amelogenin peptide (A+4 or A-4).

Recent evidence suggests that the sympathetic nervous system may have a role in modulating neurogenic inflammation and bone remodelling. Neuropeptide Y (NPY) is a well-characterized neuropeptide transmitter in the peripheral sympathetic nervous system. NPY is known to be present in human dental pulp; however, quantitative data on NPY levels in pulpal health and disease in an adult population remain to be determined. The aims of the current study were to assess, quantitatively, NPY levels by radioimmunoassay and confirm the distribution of NPY fibres by immunocytochemistry in carious and non-carious adult human pulp tissue. Our results suggest changes in the levels and distribution of NPY in human dental pulp during the caries process, with significantly higher levels of NPY in carious compared with non-carious adult human teeth. Within the carious samples studied, our finding, that NPY levels were significantly elevated in mild/moderate caries, concurs with the hypothesis that NPY could have a modulatory role in pulpal inflammation and in reparative dentine formation.

Pulpal responses to gallium-aluminum-arsenide (GaAlAs) laser irradiation applied to the tooth remains to be elucidated. This study aimed to evaluate the effect of the GaAlAs laser on odontoblasts using immunohistochemistry for heat-shock protein (HSP)-25, which labels mature and newly differentiated odontoblasts. The mesial surface of the upper right first molar of 8-wk-old Wistar rats was lased at an output power of 0.5-1.5 W for 180 s. The animals were perfusion-fixed at intervals of 6 h to 30 d after irradiation. At 6 h to 7 d, the intensity of HSP-25-immunoreactivity was found to be disturbed in the coronal odontoblast-layer in an energy-dependent manner. At 30 d, tertiary dentin with/without bone-like tissue was formed abundantly in the dental pulp. Statistical analysis revealed that the area occupied by the new hard tissues was significantly wider in 1.5 W-lased specimens than in 0.5 W-lased specimens. An intense HSP-25 immunoreactivity was seen in the odontoblasts underlying the tertiary dentin, whereas immunoreactivity was weak around the bone-like tissue. It was concluded that the GaAlAs laser may induce the formation of tertiary dentin by influencing the secretory activity of odontoblasts. However, higher energies may cause irreversible changes to the pulp, often leading to the formation of an intrapulpal bone-like tissue.

Nitric oxide (NO) is a biological effector molecule involved in a large variety of reactions and, as synthesized by inducible nitric oxide synthase (iNOS), has many important roles in inflammatory conditions. This study aimed to evaluate the anti-inflammatory effects of an iNOS-specific inhibitor, N-(3-(aminomethyl)benzyl)acetamidine (1400W), on experimentally induced rat pulpitis in the upper incisors of 6-wk-old male Wistar rats. 1400W (1 mg kg(-1)), the non-specific NOS inhibitor N(G)-nitro-L-arginine methyl ester (L-NAME, 50 mg kg(-1)), or sterile saline (control) were administered before the application of lipopolysaccharide (LPS). Rats were killed 3, 6, 9, 12, 24, 48, and 72 h after LPS application, and immunocompetent cells were detected immunohistochemically. The numbers of granulocytes infiltrating into the pulp were significantly depressed in the 1400W group compared with the saline and L-NAME groups. The kinetics of the macrophages and Ia(+) cells in the 1400W group were similar to those in the L-NAME group, while the maximum numbers in both groups were significantly reduced compared with those in the saline group. These results suggest that NO may be responsible for the infiltration of immunocompetent cells in the progress of pulpitis, and that 1400W is a promising candidate for controlling pulpal inflammatory reactions.