Review of 36,537 patient records for tooth health and longevity of dental restorations.

To develop an automatic system for utilizing electronic dental records, a data mining system to extract the diagnostic and treatment codes from the records for an intermediate file and automatic drawing of Kaplan-Meier-type survival curves was first created. Then this intermediate file was analyzed with SAS software for the scientific determination of Kaplan-Meier survival of tooth/surface-specific healthy time and survival of restorations in each permanent tooth, health center, and age cohort and also combined. All patients born in 1985, 1990 or 1995 in 28 health centers in Finland were analyzed. Patients classified as caries-active were those who had caries in any first permanent molar under the age of 8 years, while resistant patients did not have caries in these teeth before 10 years. In the younger age cohorts, a shortening of survival of caries-free teeth was seen. The shortest caries-free survival was seen in mandibular and maxillary molars in the youngest age cohort. Occlusal surfaces of molars determined their caries onsets and proximal caries occurred equally in molars, incisors and premolars, whereas canines or mandibular incisors did not have caries in these age cohorts. Caries-prone subjects had the shortest survival in all their teeth. The median longevity of all restorations was 11.7 years, with great variation between health centers and teeth. Because of the great variation between individual teeth, the tooth-specific approach seems appropriate in both caries epidemiology and material sciences.

Has dental caries prevalence some connection with caries index values in adults?

DMF index values have been used for 70 years and were originally meant to describe both dental status and treatment need in elementary school children. Since then its application to caries experience and severity determination has expanded. Today, WHO has standardized its use in oral health surveys in describing past and present caries experience in adults and the elderly as well. This expansion to all age cohorts creates some problems if the index is limited to dental caries, which can be easily avoided when individual values are not combined. This has been performed in some of the 7,187 DMF index publications included in PubMed. The high number of scientific articles using this index underlines its leading role in the present epidemiological caries research. On the other hand, WHO uses different determinations for clinical dental caries and missing teeth in ICD-10, the 10th revision of International Classification of Diseases, which does not include dental restorations. Combining the individual parameters of Oral Health Surveys and diseases of ICD-10, and analyzing the mean value of each parameter separately, will give a precise picture of dental health at different ages.

A practice-based study of the variation of diagnostics of dental caries in new and old patients of different ages.

Data mining of digital dental records provides possibilities for analysing the variation between dentists when diagnosing caries. A total of 71,317 male and 82,302 female subjects visited the health centres in Vantaa and Kemi during the 'digital era' (1994-2005). As subjects were classified as 'new patients' at the first examination, all re-examinations of the same subjects thereafter by the same dentist produced 'old patients'. A mean number of decayed surfaces (DS) was counted as a function of the age of the subject during the follow-up. The significance of the difference between old and new patients was determined by the Mann-Whitney test for each age cohort at the cross sections, and for the whole follow-up. Caries was seen to affect new patients more than the old ones in both health centres after the age of 20 years. The mean DS values were the same, reaching about 1 for new and old patients at the age of 15 years. The mean DS had a peak for new patients in both health centres at the age of 25 years and another peak around 45-50 years in Kemi. With a few exceptions there was a significant difference between the DS values of new and old patients at most cross sections and for the whole follow-up time. Evidently dentists examine new patients more carefully than their old patients. After the age of 18 years patients may have changed their dentists because they have finished the free-of-charge treatment period.

Baseline expression and effect of TGF-ß1 on Type I and III collagen mRNA and protein synthesis in human odontoblasts and pulp cellsIn Vitro.

Since growth factors have been suggested to regulate dentin collagen formation in response to external irritation, we investigated the effect of TGF-ß1 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-ß1 (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-ß1 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-ß1. These experiments demonstrate that mature human odontoblasts are capable of synthesizing type III collagen protein, and that TGF-ß1 has negligible effect on mature human odontoblast and pulp tissue collagen expression.

The relation of stimulated salivary flow rate and pH to Lactobacillus and yeast concentrations in saliva.

This study demonstrates that the presence of yeast, but not lactobacillus infection, is related to salivary flow rate. Salivary pH is correlated with the primary infection of both lactobacilli and yeasts. The low output of saliva appears to influence the quantity of lactobacilli more than that of yeasts. The flow rate was lower and the presence of yeasts was higher in females than in males.

Age dependency of stimulated salivary flow rate, pH, and lactobacillus and yeast concentrations.

No significant influence of age on the stimulated salivary flow rate or pH was observed in a cross-sectional study of unmedicated adults. The females had a lower output than did the males in every age group. A tendency toward higher salivary counts of lactobacilli and yeasts with aging was observed.

Changes of phosphomonoesterase activity in human dentine during the formation of the root of the tooth.

The changes of acid and alkaline phosphatase activities were quantitatively analyzed in different layers of dentine in developing and intact states of human teeth. The peripulpal areas revealed the highest activities. In the mineralizing apical area the alkaline phosphatase activity was ten times higher than that in fully-formed teeth.

Evidence that high-sucrose diet reduces dentin formation and disturbs mineralization in rat molars.

In addition to its caries-promoting effect, a high-sucrose diet reduces the apposition of mineralized dentin in young rats. This study was undertaken to test whether it has a similar effect on the width of the as-yet-uncalcified matrix, predentin. Female Wistar rats were weaned at the age of 3 weeks and fed for 7 weeks with either a high-sucrose diet, a non-cariogenic raw potato starch diet, or a non-cariogenic commercial powdered rat food (for reference). The sucrose diet induced the greatest number of caries lesions. Dentin formation was smaller and the predentin zone wider in rats fed a sucrose diet when compared with rats fed the reference diet. In rats fed a starch diet, dentin formation was smaller than in rats fed a reference diet, but no alterations in the areas of caries lesions or predentin width were observed. Widening of the predentin zone in rats fed a high-sucrose diet may reflect changes in odontoblast function, such as reduced matrix synthesis and possibly disturbed mineralization. Dentinal caries progression may thus be modulated by odontoblast function, not as much by matrix formation but rather by mineralization.

Dentin caries recording with Schiff's reagent, fluorescence, and back-scattered electron image.

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.

Localization of estrogen-receptor-related antigen in human odontoblasts.

Estrogen receptors have been demonstrated in many osteogenic cell lines. Recently, we showed that estrogen deficiency induced by ovariectomy caused enhanced dentin formation in adult rats, suggesting that estrogen receptors may be present in dental tissues. Nothing is known about estrogen receptors in human teeth. We used immunohistochemical staining and immuno-blotting to demonstrate the presence of estrogen receptors in human pulp and/or the pulpo-dentinal border. Unerupted human wisdom teeth were surgically removed, frozen in liquid nitrogen, and prepared for immunological studies. Western blot analysis with monoclonal antibodies specific for human estrogen-receptor-related antigens demonstrated an approximately 29-kDa clear double band in the material scraped from the predentin-odontoblast border and in the fluid that emerged into the pulpal chamber, evidently from the odontoblasts. A weaker double band was also present in pulpal tissue samples. By immunohistochemical staining, estrogen-receptor-related antigens were visualized in the predentinal-odontoblast region and in the pulpal blood vessels. Our results suggest the presence of estrogen receptors in human teeth, and thus the previously reported enhancement of the dentin formation in rats after ovariectomy may be mediated via these receptors.

Mineral element analysis of carious and sound rat dentin by electron probe microanalyzer combined with back-scattered electron image.

We recently demonstrated the advantages of back-scattered electron images (COMPO) in the visualization of dentinal caries, and the relationship of the change in the dentin fluorescence pattern in caries lesions. However, the exact nature of these changes is not known. In this paper, the nature of the changes in the areas with reduced mineral content in COMPO images was investigated. We examined the relation of changes in mineral elements and the appearance of soft carious and sound dentin in COMPO images using a scanning electron microscope (SEM) equipped with an electron probe microanalyzer (EPMA). Rat molars with small dentinal caries lesions just under the DEJ were chosen for the study. The Ca, P, Na, Mg, Zn, F, and total contents were determined by EPMA from five different dentin sites, and the Ca/P and Mg/Ca ratios were calculated. Generally, the lowest contents were found in caries lesions and highest in mantle dentin, with the exceptions of Mg and Zn. The Ca/P ratio was lowest in mantle dentin and highest in carious dentin. The results confirm that the change in fluorescence in the dentinal caries lesion is correlated with the very initial changes in mineral content, and that EPMA used in combination with COMPO images is a useful tool for determining small changes in mineral elements in the carious and adjacent areas of dentin.

A novel organ culture method to study the function of human odontoblasts in vitro: gelatinase expression by odontoblasts is differentially regulated by TGF-beta1.

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 a high-sucrose diet on dentin formation and dental caries in hyperinsulinemic rats.

A high-sucrose diet decreases dentin formation and its minerals, but the mechanisms behind the effect are largely unknown. We studied the combined and separate effects of sucrose and insulin on dentin formation and mineral metabolism in growing rats. At weaning, animals were randomized into 4 groups: control/sucrose diets both with and without external insulin (1 U/x 100 g body weight daily). After 4 weeks, we measured areas of dentin formation, numbers and areas of dentinal caries lesions, and serum and urine glucose, insulin, Ca, Na, K, and P. Exogenous insulin increased serum and urine insulin levels and decreased serum glucose level, but did not affect dentin formation or dentinal caries lesion formation or progression. A high-sucrose diet decreased dentin formation independently of insulin. The differences in serum and urine minerals between the groups were minor. The findings confirm that sucrose-diet-induced reduction in dentinogenesis is independent of insulin and loss of minerals in urine.

The localization of matrix metalloproteinase-20 (MMP-20, enamelysin) in mature human teeth.

MMP-20 (enamelysin), the matrix metalloproteinase family member discovered in the enamel organ, has also been detected in odontoblasts during dentin formation. We studied the presence and localization of MMP-20 in mature human teeth in health and disease. In immunohistochemistry, MMP-20-positive staining was observed most intensively in the radicular odontoblastic layer and also in dilated dentinal tubuli of caries lesions. By Western blotting, MMP-20 was detected in odontoblasts and pulp tissue of both sound and carious teeth, in dentinal fluid and dentin of sound teeth, but not in soft carious dentin. We conclude that MMP-20 produced during primary dentinogenesis is incorporated into dentin and may be released during caries progression. The main cellular source of MMP-20 in the dentin-pulp complex is the odontoblasts, which secrete MMP-20 into the dentinal fluid.

Applying modern survival analysis methods to longitudinal dental caries studies.

Before the 1960s, tooth-specific caries risk was reported to be highest at 2 to 4 years after eruption. We studied the tooth-specific caries risk in three contemporary age cohorts in Finland. All together, 4072 boys and girls were followed annually from age 6 to age 18+ years in three age cohorts born in the 1960s, 1970s, and 1980s. We used a survival model and Bayesian inferential methods in the statistical analyses to establish the secular changes during this period. The analysis was based on the caries risk in individual teeth as a function of tooth age instead of summary measures, such as DMFS values. Our first finding was a marked overall decrease of caries. Moreover, analyses of the 1960 and 1970 cohorts revealed that the risk in molar teeth was highest immediately after eruption; in the youngest cohort, however, the risks of individual teeth were so low that no such dependencies on tooth age could be established.

The activation and function of host matrix metalloproteinases in dentin matrix breakdown in caries lesions.

Matrix metalloproteinases (MMPs) are a family of enzymes which, in concert, are capable of degrading collagen. We investigated whether human MMPs could participate in the degradation of dentin organic matrix after demineralization. We performed Western blot analyses using MMP-specific antibodies to identify MMPs in human dental caries lesions. Enzymography and functional activity assays, with 125I-labeled gelatin as substrate or quantitating the degradation of type I collagen, were used to determine the activity of purified and salivary gelatinolytic (MMP-2 and MMP-9) and collagenolytic (MMP-8) enzymes with and without acid-activation in pHs relevant to caries. Respective analyses were done with caries-related bacteria. We performed electron microscope analyses to assess the degradative activity of sterilized salivary host MMPs on demineralized human dentin. Human MMP-2, MMP-8, and MMP-9 were identified in demineralized dentinal lesions. The latent purified forms of these enzymes were activated at low pH (4.5), followed by neutralization, mimicking the conditions during caries progression. Incubation of human saliva at low pH followed by neutralization resulted in a four-fold increase in the gelatinolytic activity. No gelatinolytic or collagenolytic activity was observed in bacterial samples. The activated enzymes in saliva degraded demineralized dentin organic matrix in vitro. These results demonstrate the pH-dependent activation mechanism of MMPs, which may have a distinct role in different physiological and pathological conditions. They further demonstrate that host MMPs, activated by bacterial acids, have a crucial role in the destruction of dentin by caries.

The expression of MMP-8 in human odontoblasts and dental pulp cells is down-regulated by TGF-beta1.

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.

The effects of MMP inhibitors on human salivary MMP activity and caries progression in rats.

Previous studies suggest that salivary and pulp-derived host enzymes, matrix metalloproteinases (MMPs), may be involved in dentin caries pathogenesis. To study the inhibition of acid-activated human salivary MMPs by non-antimicrobial chemically modified tetracyclines (CMTs), we used a functional activity assay with 125I-labeled gelatin as a substrate. To address the role of MMPs in the progression of fissure caries in vivo, we administered the MMP inhibitors CMT-3 and zoledronate to young rats per os for 7 weeks, 5 days a week. Caries lesions were visualized by Schiff reagent in sagittally sectioned mandibular molars. Marked reduction in gelatinolytic activity of human salivary MMPs was observed with CMT-3. CMT-3 and zoledronate, both alone and in combination, also reduced dentin caries progression in the rats. These results suggest that MMPs have an important role in dentin caries pathogenesis, and that MMP inhibitors may prove to be useful in the prevention of caries progression.

Regulation and interactions of MT1-MMP and MMP-20 in human odontoblasts and pulp tissue in vitro.

MT1-MMP is a cell-membrane-bound metalloenzyme that activates other proMMPs such as proMMP-2 and -13. We studied MT1-MMP expression in mature human odontoblasts and pulp tissue, the regulation of MT1-MMP expression by growth factors TGF-beta1 and BMP-2, and the activation of odontoblast-derived MMP-20 by MT1-MMP. MT1-MMP mRNA is expressed by native and cultured mature human odontoblasts and pulp tissue. Western blot analysis of human odontoblasts and pulp tissue detects 65- and 51-kDa pro- and active forms of MT1-MMP, and smaller truncated MT1-MMP forms. BMP-2 down-regulates MT1-MMP expression in odontoblasts and pulp tissue, while TGF-beta1, alone or with BMP-2, decreases MT1-MMP mRNA levels only slightly. We also demonstrate that MT1-MMP is capable of converting proMMP-20 into a form corresponding to the active MMP-20. In conclusion, this study demonstrates the expression and differential regulation of MT1-MMP in human dentin-pulp complex cells, and the activation of MMP-20 by MT1-MMP.