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Collagen XVIII is a basement membrane (BM) component, whereas MMP-20 (enamelysin) is a matrix metalloproteinase predominantly expressed in teeth. Since MMP-20 was found to degrade collagen XVIII, we studied the co-expression of these proteins in dental tissues. Collagen XVIII surrounded the developing tooth during early and late bell stages and was also present in developing enamel. Western blotting indicated that developing enamel contains collagen XVIII N-terminal fragments of the frizzled variant. Enamelysin was co-localized with collagen XVIII in the developing enamel matrix and stratum intermedium. Electron microscope analysis showed that total mineral, calcium and phosphorus contents of enamel were slightly increased in collagen XVIII null mice but the analysis revealed no visible defects in the enamel or dentin structures. In odontogenic tumors MMP-20 and collagen XVIII were co-localized in the enamel-like tumor matrix. Our results show that collagen XVIII is present in developing teeth, but its absence seems not to be critical for the development of the teeth.

Matrix metalloproteinases (MMPs) and their specific tissue inhibitors (TIMPs) participate into extracellular matrix degradation in physiological and pathological conditions. We hypothesized that MMP expression in pulp tissue changes in response to caries attack and investigated the gene expression profiles of MMPs and TIMPs in pulp tissue of sound and carious teeth with cDNA microarray. cDNA microarray demonstrated an extremely high MMP-13 (collagenase-3) mRNA expression in pooled pulp samples of sound and carious teeth, with less pronounced expression of MMP-16 (MT3-MMP) and TIMP-1. Real-time quantitative polymerase chain reaction of individual pulp samples revealed a wide range of the MMP-13 expression level between pulp samples with possible downregulation of MMP-13 expression during caries progression. Western blot and immunohistochemical staining confirmed the presence of MMP-13 with no observable differences between sound and carious teeth pulp tissues. The results reveal that MMP-13 is expressed and synthesized in pulp tissue, an interesting feature considering the very limited expression of MMP-13 in normal adult tissues. Further studies with a larger sample size are needed to clarify the changes in MMP-13 expression during caries progression.

Objective: We sought to assess the efficacy of chlorhexidine (CHX) and calcium hydroxide, Ca(OH)(2), against Enterococcus faecalis in vitro., Study Design: The effect of CHX (0.2% and 2% in gel or solution) and Ca(OH)(2) (alone or with 0.2% CHX gel) was evaluated by using the agar diffusion test and an in vitro human root inoculation method, to measure zone of inhibition or bacterial growth with optical density analysis, respectively. For optical density analysis, samples from infected root canals were collected after 7 days of medication and were cultured for 24 hours in brain-heart infusion to detect viable bacteria., Results: In the agar diffusion test, CHX was effective against E faecalis in a concentration-dependent fashion, but Ca(OH)(2) alone had no effect. In the root canal inoculation test, CHX was significantly more effective against E faecalis than Ca(OH)(2) was (P < .05), but there were no significant differences between the modes of medication or concentrations of CHX., Conclusions: CHX is effective against E faecalis in vitro. Further in vivo studies are needed to confirm the value of CHX in clinical treatment.

Placement of orifice plugs has been suggested to augment the seal of conventional root canal fillings. This study assessed in vivo the efficacy of white mineral trioxide aggregate (MTA) plugs in preventing periapical inflammation subsequent to coronal inoculation of root-filled teeth. The two-rooted mandibular premolars of six beagle dogs were conventionally prepared and filled with gutta-percha and sealer. A white MTA orifice plug was placed into one canal in each tooth. Pulp chambers were inoculated with plaque except for 12 teeth (negative control), and restored. Radiographs were taken at regular intervals. At 10 months, dogs were killed and jaw blocks processed for histology. None of the roots revealed radiographic or histologic evidence of severe inflammation. Mild inflammation was observed in 17% and 39% of the roots with and without an orifice plug, respectively (McNemar, p > 0.05). Without development of severe inflammation, the seal augmentation efficacy of MTA orifice plugs could not be determined.

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.

Background: Odontogenic keratocyst (KC) differs from other epithelial odontogenic cysts in regard to increased epithelial proliferation and a strong tendency to recur. Laminin-5 (Ln-5) is an epithelial anchoring filament component, which after modulation by certain matrix metalloproteinases (MMPs), like MMP-2 and MMP-13, induces epithelial cell migration., Methods: Using in situ hybridization and immunohistochemistry, we studied the Ln-5 gamma-2 chain expression related to the expression of MMP-2, -8, and -13 in different odontogenic cysts, including radicular cysts (RC; n = 11), follicular cysts (FC; n = 11), and odontogenic keratocysts (KC; n = 16)., Results: Ln-5 mRNA was present in all cysts examined, while less than half of KCs and RCs (33 and 40%, respectively) demonstrated MMP-2 mRNA. MMP-13 mRNA was present in all KC samples. Ln-5 protein was located as a continuous ribbon in BM zone of all KCs, and MMP-2 and MMP-13 immunoreactivities colocated significantly with Ln-5 in that area. MMP-8 was expressed by stromal macrophages and epithelial goblet cells, but never located in BM zone., Conclusions: Our results indicate that the colocalization of Ln-5 with MMP-2 or MMP-13, but not with MMP-8, in BM zone of KCs, may be related to special characteristics of KC.

Objective: The aim of this in vitro study was to assess the substantive antimicrobial activity of different medicaments in human root dentin., Study Design: Canals of 98 roots were enlarged to standard size and medicated for 7 days with the following: (1) 2% chlorhexidine (CHX) gel, (2) 0.2% CHX gel, (3) 2% CHX solution, (4) Ca(OH)(2), (5) Ca(OH)(2)+ 0.2% CHX gel, (6) 2% CHX solution + a 25% CHX-containing controlled-release device, (7) saline, and (8) gel vehicle. After medication, canals were inoculated with Enterococcus faecalis for 21 days. Dentin samples were collected with Gates-Glidden burs into brain heart infusion broth, and bacterial growth was assessed with spectrophotometric analysis of optical density after 72 hours of incubation., Results: Mean optical densities were significantly lower for groups with 2% CHX (1, 3, and 6) when compared with those of the controls (P < .05, analysis of variance with the Tukey test). Other groups did not differ significantly from the controls., Conclusions: Canal dressing for 1 week with 2% CHX may provide residual antimicrobial activity against E faecalis.

The favourable response of exposed pulp tissue against a variety of materials used for pulp capping in experimental conditions, as observed by hard tissue (reparative dentine) formation, demonstrates an intrinsic capacity of pulp tissue for healing. However, in the clinical situation, in which a pulpal exposure is usually accompanied by a long-term external irritation with the subsequent long-term inflammatory response to that irritation, the outcome of pulp capping procedures is not as predictable. While some of the factors related to the defensive reactions and healing after pulp exposure and capping procedures are well understood, the mechanisms and importance of others remain less well-known. Understanding the mechanisms regulating the spread of inflammation and necrosis in pulp tissue, and the factors regulating healing after closure of the wound, would facilitate the development of new and better treatment procedures with more predictable outcomes. In this review, some of the aspects considered to be important in pulpal wound healing are discussed.

Background: Radiation therapy for head and neck tumour patients may lead to decreased salivary flow, oral mucosal lesions and increased caries experience. Salivary matrix metalloproteinases (MMPs) may participate in the pathogenesis of mucosal lesions and dentinal caries. The aims of this study were: (i) to assay the presence, molecular forms and proteolytic activity of MMP-8 (collagenase-2) and MMP-9 (gelatinase B) in the whole saliva of head and neck cancer patients having radiation therapy; (ii) to see whether salivary pH affects the activity of MMP-8 and MMP-9; and (iii) to find out the possible connection between MMP-8 and MMP-9 with the eruption of oral mucosal lesions during radiation therapy., Methods: The whole saliva samples of 39 head and neck cancer patients having radiation therapy were collected before, during and after radiation therapy, and saliva flow rate, pH, buffer capacity, Streptococcus mutans, Lactobacillus and Candida albicans were measured. Any oral mucosal lesions were examined during each visit. The levels of MMP-8 were measured by immunofluorometric assay (IFMA) and the presence of different MMP-8 forms was analysed using Western immunoblotting. The presence and molecular forms of MMP-9 were analyzed by gelatin zymography. MMP-9 capture activity assay was used to determine the APMA-activated MMP-9 activity (total) and the endogenously active MMP-9 (free activity)., Results: Salivary flow rate, buffer capacity and pH decreased, and the levels of Lactobacilli increased significantly, during the first half of the radiation therapy. The endogenously activated salivary MMP-9 correlated with low salivary pH (P = 0.013). No connection was found between the oral mucosal lesions and salivary MMP-8 or MMP-9., Conclusions: In this study, salivary MMP-8 or MMP-9 did not correlate with the presence of radiation induced oral mucosal lesions, but the activation of MMP-9 may be dependent on pH.

In rats, sucrose increases dental caries and impairs odontoblastic function by reducing dentine apposition during primary dentinogenesis. A high-sucrose diet also affects negatively a pulp or dentine function that appears to regulate solute or fluid movement within rat dentinal tissue. In earlier work it was found that carbamyl phosphate could significantly reverse sucrose-induced cariogenesis and also stimulate sucrose-depressed movement of dentinal fluid through a mechanism involving parotid function(s). In the current study, the possibility that carbamyl phosphate could overcome the sucrose-induced reduction in dentine apposition was examined. Weanling rats were fed a high-sucrose diet supplemented or not with carbamyl phosphate for 5 weeks. Dentine apposition was measured planimetrically in sagittal sections of the molars. The effect of carbamyl phosphate was similarly tested in parotidectomized animals. Carbamyl phosphate significantly reduced the deleterious effect of sucrose on dentine apposition by 58% in the first molars. However, the reduction in dentine apposition that followed parotidectomy was not altered by carbamyl phosphate supplementation. The possibility that the beneficial effect of carbamyl phosphate on dentinogenesis involves a parotid function is entertained.

Objectives: The first objective of this study was to verify the presence of and identify the molecular forms of matrix metalloproteinases (MMPs), including collagenases (MMP-1, MMP-8, and MMP-13) and gelatinases (MMP-2 and MMP-9), in the synovial fluid (SF) of mild and severe temporomandibular joint internal derangement (TMJ-ID). Another objective was to evaluate whether the SF MMPs are potential diagnostic markers that reflect the stage of intra-articular inflammation in the TMJ., Study Design: The subjects were 44 patients with mild (n = 16) or severe (n = 28) TMJ-ID; they were classified on the basis of subjective symptoms, clinical and radiographic findings, and surgical observations. The patients were surgically treated, and SF samples were collected immediately before the operation. The collagenase activity of SF samples was analyzed by means of a type I collagen degradation assay. The levels and molecular forms of the SF MMPs as well as the tissue inhibitors of MMPs (TIMP-1 and TIMP-2) were analyzed with Western immunoblotting and gelatin zymography., Results: The SF of both the mild and the severe TMJ-ID patients exhibited free collagenase activity and activity capable of further degrading the (3/4)(alphaA) fragments. Ninety-two-kilodalton proMMP-9 and its 121-kD complex form, as well as 72-kD proMMP-2 were significantly increased in the mild TMJ-ID group (P <.05 in all cases). Both 70- to 80-kD neutrophil type and 45- to 55-kD mesenchymal cell-type MMP-8 (corresponding to the latent and active forms) were observed in mild and severe TMJ-ID SF, but they predominated in mild TMJ-ID. Both MMP-1 and MMP-13 were observed in both groups, and in mild TMJ-ID SF the low-molecular weight forms of MMP-1 indicated activation of the enzyme., Conclusions: The degradation of type I collagen in the TMJ is evidently due to the collective action of many collagenolytic MMPs present in the SF of patients with mild and severe TMJ-ID. The elevated levels of MMP-2, MMP-9, and MMP-8 in the SF of patients with mild TMJ-ID eventually reflect the active phase of TMJ destruction. These observations may have considerable diagnostic and therapeutic significance in the management of TMJ disorders.

Since growth factors have been suggested to regulate dentin collagen formation in response to external irritation, we investigated the effect of TGF-β1 on proα1(I) collagen mRNA expression in cultured mature human odontoblasts and pulpal fibroblasts, as well as cultured human pulp tissue, using quantitative PCR. Cultured gingival fibroblasts (GF) and osteoblasts (OB) served as controls. Also, type I collagen synthesis in cultured odontoblasts and pulp tissue, as well as type III collagen synthesis in odontoblasts, were studied by measuring respective procollagen (PINP and PIIINP) secretion into culture media with radioimmunoassay (RIA). Odontoblasts expressed significantly higher basic level of type I collagen mRNA than pulp tissue or pulp fibroblasts in culture, but markedly lower level than GF and OB cells. TGF-β1 (10 ng/ml) had negligible effects on type I collagen mRNA expression or PINP synthesis in cultured odontoblasts and pulp tissue, and PIIINP synthesis in the odontoblasts. In PF cells, the effect of TGF-β1 depended on culturing conditions; a 6-fold increase in mRNA expression was observed using serum-free medium but no effect was seen in the cells cultured with 10% FBS. In contrast, GF cells serving as controls were not markedly affected by the culture conditions, with 2-3-fold increase in mRNA expression by TGF-β1. These experiments demonstrate that mature human odontoblasts are capable of synthesizing type III collagen protein, and that TGF-β1 has negligible effect on mature human odontoblast and pulp tissue collagen expression.

Since growth factors have been suggested to regulate dentin collagen formation in response to external irritation, we investigated the effect of TGF-beta 1 on pro alpha 1 (I) collagen mRNA expression in cultured mature human odontoblasts and pulpal fibroblasts, as well as cultured human pulp tissue, using quantitative PCR. Cultured gingival fibroblasts (GF) and osteoblasts (OB) served as controls. Also, type I collagen synthesis in cultured odontoblasts and pulp tissue, as well as type III collagen synthesis in odontoblasts, were studied by measuring respective procollagen (PINP and PIIINP) secretion into culture media with radio-immunoassay (RIA). Odontoblasts expressed significantly higher basic level of type I collagen mRNA than pulp tissue or pulp fibroblasts in culture, but markedly lower level than GF and OB cells. TGF-beta 1 (10 ng/ml) had negligible effects on type I collagen mRNA expression or PINP synthesis in cultured odontoblasts and pulp tissue, and PIIINP synthesis in the odontoblasts. In PF cells, the effect of TGF-beta 1 depended on culturing conditions; a 6-fold increase in mRNA expression was observed using serum-free medium but no effect was seen in the cells cultured with 10% FBS. In contrast, GF cells serving as controls were not markedly affected by the culture conditions, with 2-3-fold increase in mRNA expression by TGF-beta 1. These experiments demonstrate that mature human odontoblasts are capable of synthesizing type III collagen protein, and that TGF-beta 1 has negligible effect on mature human odontoblast and pulp tissue collagen expression.

Recent findings show that matrix metalloproteinase-8 (MMP-8) is expressed, in addition to neutrophils, by human chondrocytes, cultured fibroblasts, and endothelial cells. We investigated the expression of MMP-8 in other human mesenchyme-derived cells, odontoblasts, and pulp tissue. Odontoblasts and pulp tissue were collected from extracted human teeth for MMP-8 mRNA analysis with reverse-transcription/polymerase chain-reaction (RT-PCR) and Southern blot. The expression, localization, and secretion of MMP-8 protein were studied with Western blot, immunohistochemistry, and immunofluorometric assay. The effect of TGF-beta1 (10 ng/mL) on the expression, secretion, and concentration of secreted MMP-8 was studied by odontoblast and pulp tissue culture methods (Tjäderhane et al., 1998a). RT-PCR demonstrated MMP-8 mRNA expression in native and cultured odontoblasts and pulp tissue and cultured pulp fibroblasts, with a 522-bp transcript comparable with that of bone marrow cells. The specificity of PCR was confirmed with Southern blot. Western blot with MMP-8-specific antibody detected 65- and 50-kDa proteins in native samples, representing latent and active forms of mesenchymal-type MMP-8, and in the conditioned odontoblast culture media, 50-kDa protein was observed. TGF-beta down-regulated the MMP-8 mRNA and concentration of secreted protein in both cultures. Immunohistochemical staining detected MMP-8 in odontoblasts. These findings indicate that mesenchyme-derived cells of the dentin-pulp complex express, synthesize, and activate MMP-8, which may, in concert with odontoblast-derived gelatinases, participate in organization of dentin organic matrix prior to mineralization.

Odontoblasts cannot be cultured by traditional cell culture methods, thus restricting in vitro studies. Here we present an organ culture method for human odonto-blasts that utilizes the pulp chamber as a culture crucible. Crowns of human third molars were dissected, pulp was gently removed, and the odontoblasts attached to and in the walls of the pulp chambers were cultured in serum-free OPTI-MEM medium, or DMEM/Ham's F12 medium containing 10% serum. Pulp tissues were cultured separately. Cell content and morphology were analyzed by SEM, and the removed pulps were examined by light microscopy. Proteins secreted into the medium with or without TGF-beta1 supplementation were metabolically labeled with [35S]methionine, and the total protein content was assessed by TCA precipitation and SDS-PAGE/fluorography. To assess the role of gelatinolytic enzymes on dentin matrix remodeling, we used enzymography to analyze the effect of TGF-beta1 on gelatinase A and B expression. SEM revealed odontoblasts in pulp chambers after 5 days of culture, with only few or no fibroblasts, and no alterations in the odontoblast cell morphology or differences between the cells cultured in serum-free and serum-containing media. Rarely were any odontoblasts present in pulp tissue. Radiolabeling revealed protein synthesis and secretion until day 6 in both the odontoblast and pulp cultures, with no marked differences between TGF-beta1-treated and control cultures. The level of gelatinase A remained constant up to 7 days, while gelatinase B expression was always low and decreased with time in culture. However, gelatinase B levels were markedly increased upon TGF-beta1 treatment of cells and remained high to day 7. The results suggest that this method provides a novel technique for the study of human odontoblasts in vitro and that odontoblasts can be cultured even in serum-free conditions.

The effect of increasing dietary sucrose concentration on dentin formation and dentinal caries progression was studied. Weanling Wistar rats received 15, 30 or 43 g/100 g sucrose in a diet; for reference, another group was fed a nonpurified diet. At the onset, tetracycline was injected to mark the dentin formed during the experiment. After 6 wk, lower molars were sectioned sagittally; the areas and thicknesses of the dentin formation during the experiment and dentinal caries lesions were quantified separately in the first and second molars. Feeding the 43% sucrose diet resulted in a significantly lower dentin formation than in other diet groups (P < 0.05). The differences obtained from the area measurements were supported by thickness measurements. In the first molar, the 43% sucrose diet resulted in a significantly greater area of dentinal caries than in the other sucrose groups. The number and severity of caries lesions clearly increased as the concentration of sucrose in the diet increased (r = 0.5, P < 0.05 and r = 0.6, P < 0.05, respectively). This study suggests that the increase in the concentration of sucrose in the diet reduces dentin formation and increases the area of dentinal caries as well as the number and severity of caries lesions; the critical sucrose concentration appears to be between 30 and 43 g/100 g.

The effect of a low dose of glucocorticoids on dentin formation and progression of dental caries was studied in young rats. The animals were inoculated in the mouth with Streptococcus sobrinus and fed ad libitum a high-sucrose, a high-starch, or a reference diet. The cortisone or placebo pellet was implanted subcutaneously in the back of the necks of the animals. After a 6-week experimental period the areas of dentin formation and dentinal caries were quantified planimetrically. Schiff's staining was used to classify caries. Cortisone reduced the dentin formation both in high-starch and reference diets, but it did not further affect the reducing effect of the high-sucrose diet on dentin formation. Cortisone did not affect the number or severity of dentinal carious lesions.

A xylitol-induced reduction in dentin formation has been observed in molars of post-weanling rats, but the effect in older animals with a slower rate of secondary dentinogenesis has not been investigated. We examined the effects of different concentrations of dietary xylitol on dentinogenesis in molars of young adult rats. The control group received a commercial basal diet, while for the other 3 groups, the same diet was supplemented with 5, 10 and 20% of xylitol. After 8 weeks, dentin thickness beneath the main fissures in the mandibular 1st and 2nd molars was measured. The final body weights did not differ between the groups. Dentin formation in the 1st molars of the 10 and 20% xylitol groups was significantly lower as compared to the controls, whereas no difference was found between these 2 groups. In conclusion, dietary xylitol reduces dentin formation in young adult rats in spite of the slow rate of dentinogenesis.

The effects of dietary calcium deficiency and subsequent replenishment of the diet with calcium alone or with xylitol were studied. Thirty 3-week-old Wistar rats were labelled with an i.p. tetracycline injection. Twenty rats were fed a diet with 0.026% calcium (Ca-deficient); 10 received a 0.5% Ca diet (controls). After 3 weeks the tetracycline labelling was repeated. Replenishment of the diet was introduced for Ca-deficient rats, and 10 received additional 5% xylitol in the diet. After 4 weeks the labelling was repeated and the animals were decapitated. Dentine formation was measured by the tetracycline stripes in the lower first and second molars. Calcium deficiency during the first 3 weeks reduced dentine formation. In the control and xylitol groups, a much smaller amount of dentine was formed during the recovery period. With Ca alone, dentine formation was faster than in the controls or Ca-xylitol group and did not differ from the Ca-deficient period. These results indicate that in rat molars the odontoblasts can accelerate the rate of dentine formation when calcium is restored to the diet, at least during primary dentinogenesis. This effect was not seen when 5% xylitol was added to the replenishment diet.

To investigate the effects of a high sucrose diet and xylitol on secondary dentinogenesis and dentinal caries, a part of the sucrose in a high-cariogenic diet was replaced by xylitol. Fifty-four 3-wk-old Wistar rats were labeled with tetracycline and divided into groups. One group received a high sucrose diet (43% sucrose). In two other groups 5% and 20% of the sucrose was replaced by xylitol. A control group received non-cariogenic food. Six weeks later the mandibles were sectioned sagittally. Schiff staining was used to classify the caries, and the areas of dentin formed during the experiment and dentinal caries in first and second molars were measured planimetrically. The high sucrose diet reduced dentin formation of the molars, and this reduction was further increased by xylitol. Caries initiation and dentinal caries progression were significantly reduced by 20% xylitol, whereas only a slight reduction in caries progression was observed with 5% xylitol. Also, a negative correlation between the dentin formation and dentinal caries progression was observed in the high sucrose and 5% xylitol groups. In conclusion, xylitol together with high sucrose reduced dentin formation and dentinal caries progression, and the effect was dose-dependent.

The effects of high-sucrose and high-starch diets on dentine apposition were examined. One group of young Wistar rats (20 rats) received a high-sucrose (43%) diet; for the high-starch group (16 rats), sucrose was replaced with potato flour. The control group (18 rats) received standard laboratory rat food. The onset of dentine formation was marked with tetracycline. After 5 weeks lower molars were sectioned sagittally, and the areas of the dentine apposition and those of the dentinal caries were quantified. Dentine apposition was independent of sex. The high-sucrose diet reduced dentine apposition and induced caries progression. A negative correlation between dentine apposition and caries progression in the molars was found. This study suggests that a high-sucrose diet itself reduces primary dentine apposition.

After being weaned, the Wistar rats (12) were fed on a sucrose diet for five weeks to induce dental caries. Tetracycline was injected intraperitoneally into 7 of them to label the mineralizing dentin front. Five rats without tetracycline injection were used to verify spontaneous fluorescence. The evidently carious area under one prominent fissure from each mandibular molar hemisected sagittally in the midline was photographed under ultraviolet light so that autofluorescence would be revealed. The jaws were then stained with Schiff's reagent and photographed under normal light and again under ultraviolet light. The areas of all the lesions were quantified planimetrically as they appeared on the photographs. The mineral contents of the areas were verified with the back-scattered electron images. The caries lesions revealed with Schiff's reagent also exhibited a change in the color of the dentin fluorescence regardless of the tetracycline labeling. The areas of these lesions followed the shapes of the lesions stained by Schiff's reagent but they were greater. Staining with Schiff's reagent was repeatable after the specimen was washed with ethanol for a few weeks. The loss of minerals was seen in the areas stained with Schiff's reaction but was also related to the change in dentin fluorescence, which seems to be a more sensitive indicator of the caries progression than Schiff's reagent, especially in the early phase of the carious process.

An evaluation of Gram's method of staining of 601 nonvital dental pulps was performed. It gave positive results in 60.1% of the cases, the most frequent finding was gram-positive cocci. Preoperative x-ray status correlated significantly with the staining results but the size of the radiolucency did not. Previously treated teeth gave less positive staining results than other teeth. There was an obvious correlation between presence of bacteria and various complications (p < 0.001), but no differences were noted between the combinations of bacteria. The teeth that were the most resistant to complications seemed to be the lower canines. The staining results did not correlate with sex, systemic diseases, or preoperative symptoms. We believe that Gram's method of staining is a useful, rapid aid in root canal therapy, especially in complicated cases.

To determine the effect of phenytoin (PHT) on dentin apposition and the rate of caries progression in dentin, 40 Wistar rats were treated daily for 35 days with intraperitoneal injections of 5,5-diphenylhydantoin. Twenty-eight controls received saline with pH adjusted to that of PHT. One PHT and one control group were fed a 43% sucrose diet, and the others a non-cariogenic pellet diet. Streptococcus sobrinus was inoculated to induce caries. Schiff staining was used to determine caries. The areas of dentin apposition and dentinal caries lesions were quantified after tetracycline staining. The root lengths were measured. PHT reduced slightly the dentin apposition and activated significantly the progression of the dentinal carious lesions. No difference in caries initiation was found. The high-sucrose diet reduced dentin apposition and increased the rate of progression of existing caries greatly. Our study suggests that both the high-sucrose diet and PHT have an effect on secreting odontoblasts, which can be seen as an alteration in dentin apposition and caries progression rates in dentin.