Immunohistochemical localization of osteopontin in human pulp stones

The organic matrix component of human pulp stones was investigated by immunohistochemistry. Two pulp stones were extracted from the upper molar teeth of two patients suffering from irreversible pulpitis. Both were formed in the center of the pulp cavity and located apart from the dentin walls. After demineralization, serial sections of the stones were prepared and subjected to immunohistochemical procedures using specific antibodies to type I collagen and noncollagenous proteins (osteopontin, osteonectin, and osteocalcin), which are reported to be involved in calcified matrix formation. Type I collagen was localized evenly in the stones, indicating that it is a major matrix component of pulp stones. Strong immunostaining of osteopontin appeared in the peripheral area of the stones, whereas osteonectin and osteocalcin were not detected. We previously reported that dental pulp cells produced osteopontin in vitro. Osteopontin has been commonly found in other pathological calcification, such as urinary stones, atherosclerotic plaques, and dental calculus. Taken together, the present findings suggest that osteopontin produced by dental pulp cells is possibly associated with calcification of the pulp stone matrix.     

Anticalculus effect of a triclosan mouthwash containing phytate: a double-blind, randomized, three-period crossover trial

Background and Objective:  Dental calculus occurs as a consequence of supersaturation of saliva with respect to calcium phosphates. This mineralization of dental plaque can be delayed by the presence of crystallization inhibitors, such as pyrophosphate or bisphosphonates. Phytate inhibits brushite and hydroxyapatite crystallization and has the potential to prevent dental calculi formation. The aim of the present study was to examine the effects of phytate and zinc, administered in a mouthwash solution, to prevent the formation of dental calculus.
Material and Methods:  Healthy dental plaque-forming volunteers ( n  = 25) took part in a randomized, double-blind, three-period crossover clinical study to assess the efficacy of a phytate-containing mouthwash in relation to control and placebo effects. Subjects rinsed their mouths for 1 min, twice each day, with 20 mL of the test solution, without ingestion. Mouthwash efficacy was assessed through quantification of the amounts of calcium, phosphorus and magnesium present in the residues obtained by dental cleaning, performed by a single trained examiner.
Results:  A good correlation was found among total calcium, magnesium and phosphorus in calcified dental plaque residues, indicating that any of these variables is adequate for evaluating the reduction of plaque crystallization as calcium phosphate. A statistically significant decrease in total calcium, magnesium and phosphorus was found in the phytate-treatment period compared with control and placebo periods, demonstrating the efficacy of the proposed treatment in reducing dental calculus formation.
Conclusion:  The high efficacy of phytate in reducing dental calculus formation suggests that this substance may be an effective treatment for preventing the development of calculus deposits.     

Dental calculus: the calcified biofilm and its role in disease development

Dental calculus represents the first fossilized record of bacterial communities as a testimony of evolutionary biology. The development of dental calculus is a dynamic process that starts with a nonmineralized biofilm which eventually calcifies. Nonmineralized dental biofilm entraps particles from the oral cavity, including large amounts of oral bacteria, human proteins, viruses and food remnants, and preserves their DNA. The process of mineralization involves metabolic activities of the bacterial colonies and strengthens the attachment of nonmineralized biofilms to the tooth surface. From a clinical point of view, dental calculus always harbors a living, nonmineralized biofilm, jeopardizing the integrity of the dento‐gingival or implanto‐mucosal unit. This narrative review presents a brief historical overview of dental calculus formation and its clinical relevance in modern periodontal practice.     

Extracellular matrix mineralization in periodontal tissues: Noncollagenous matrix proteins,enzymes, and relationship to hypophosphatasia and X‐linked hypophosphatemia

As broadly demonstrated for the formation of a functional skeleton, proper mineralization of periodontal alveolar bone and teeth – where calcium phosphate crystals are deposited and grow within an extracellular matrix – is essential for dental function. Mineralization defects in tooth dentin and cementum of the periodontium invariably lead to a weak (soft or brittle) dentition in which teeth become loose and prone to infection and are lost prematurely. Mineralization of the extremities of periodontal ligament fibers (Sharpey's fibers) where they insert into tooth cementum and alveolar bone is also essential for the function of the tooth‐suspensory apparatus in occlusion and mastication. Molecular determinants of mineralization in these tissues include mineral ion concentrations (phosphate and calcium), pyrophosphate, small integrin‐binding ligand N‐linked glycoproteins and matrix vesicles. Amongst the enzymes important in regulating these mineralization determinants, two are discussed at length here, with clinical examples given, namely tissue‐nonspecific alkaline phosphatase and phosphate‐regulating gene with homologies to endopeptidases on the X chromosome. Inactivating mutations in these enzymes in humans and in mouse models lead to the soft bones and teeth characteristic of hypophosphatasia and X‐linked hypophosphatemia, respectively, where the levels of local and systemic circulating mineralization determinants are perturbed. In X‐linked hypophosphatemia, in addition to renal phosphate wasting causing low circulating phosphate levels, phosphorylated mineralization‐regulating small integrin‐binding ligand N‐linked glycoproteins, such as matrix extracellular phosphoglycoprotein and osteopontin, and the phosphorylated peptides proteolytically released from them, such as the acidic serine‐ and aspartate‐rich‐motif peptide, may accumulate locally to impair mineralization in this disease.     

The association of calprotectin level in gingival crevicular fluid with gingival index and the activities of collagenase and aspartate aminotransferase in adult periodontitis patients

BACKGROUND: Calprotectin, a major cytosol protein of leukocytes, exists in plasma and other body fluids of healthy human subjects. Since the calprotectin concentration rises markedly in some inflammatory diseases including rheumatoid arthritis, this protein has been thought to be a marker of inflammatory disease. Recently, we identified calprotectin in human dental calculus and gingival crevicular fluid (GCF), and found that the calprotectin concentration in GCF from patients with periodontitis was significantly higher than that in GCF from healthy subjects. In the present study, the association of GCF calprotectin level with GCF volume, gingival index (GI), and levels of biochemical markers including collagenase and aspartate aminotransferase (AST) in GCF was investigated to clarify the relationship between GCF calprotectin level and periodontal inflammation. METHODS: Ninety GCF samples collected from periodontal pockets with a probing depth of more than 4 mm in 54 patients with adult periodontitis were used for these assays. The GCF volume was measured, and GI in each site was recorded. The calprotectin content in GCF samples was determined by ELISA using a specific antibody. The activity of collagenase or AST was measured by a respective assay kit. RESULTS: The total amount of calprotectin and GCF volume showed a highly significant correlation (r = 0.64, P <0.0001), whereas the calprotectin concentration had no correlation with the GCF volume (r = 0.01, P= 0.924). The mean calprotectin concentration in GCF increased with the degree of GI, and the concentration in individual samples was significantly correlated with the GI score (r = 0.56, P<0.0001). Significant positive correlations were observed in GCF calprotectin versus collagenase (r = 0.57, P <0.0001) and GCF calprotectin versus AST levels (r = 0.40, P <0.005). CONCLUSIONS: From the present results and our previous findings, it is shown that the GCF calprotectin level significantly correlates not only with clinical indicators but also with current biochemical marker levels and that calprotectin may be a useful marker for periodontal inflammation.     

A Large Chondroitin Sulfate Proteoglycan, Versican, in Porcine Predentin

Proteoglycans and their constituent glycosaminoglycan (GAG) have been proposed to be involved in the inhibition of mineralization in unmineralized tissue, predentin. Among the proteoglycans secreted by odontoblasts, we focused on the large chondroitin sulfate proteoglycan, versican, for its large binding capacity for calcium ions. The aims of this study were the determination of the full-length sequence and splicing variants of the porcine versican, and the detection of versican in the porcine predentin. The complete coding sequence of the porcine versican mRNA was cloned to be 11,775 nucleotides long and encode 3,924 amino acids, and four splicing variants, V0, V1, V2 and V3, were characterized in the isolated porcine cartilage cells. The number of potential GAG attachment sites was 15 in the V0 variant, 13 in the V1 variant, 2 in the V2 variant and 0 in the V3 variant. They were deposited in DDBJ. The V1 variant was determined by RT-PCR in the odontoblasts, dental papilla cells, dental follicle cells, periodontal ligament cells, dental pulp cells, and gingival cells of pigs, although a small amount of the V0 valiant was found in the dental papilla cells. The predentin was prepared from developing porcine permanent incisor tooth germs and its soluble proteins were extracted in order to be partially characterized by protein and proteinase profiles. The versican V1 cleavage products were detected in the predentin extract by Western blotting analysis. These results suggested that the versican splice variant V1 implicates both the control of the mineralization and the activities of the predentin metalloproteinases, because it has 13 GAG chains that bind a large amount of calcium.     

Characterization of dental follicle cells in developing mouse molar.

Dental follicle has been implicated as the origin of alveolar bone, cementum and periodontal ligament, but there is no direct evidence of their cellular lineage. The present pilot study was designed to characterize the phenotype of cultured cells obtained from the dental follicle of neonatal mouse molars. Developing mandibular molars from 6-day-old CD-1 mice were subjected to 1% trypsin in Hank's balanced salt solution. After trypsinization, the dental follicle was enucleated from the tooth germ and separated from the associated epithelial root sheath. Pure dental follicle tissue was cultured in alpha-minimal essential medium containing 10% fetal bovine serum and antibiotics. The nature of the cultured follicle cells was determined in situ by immunocytochemical staining for type I and III collagen, fibronectin, and alkaline phosphatase expression. Earlier phenotypic markers for mineralization such as bone sialoprotein and osteopontin were also examined by in situ hybridization of matched molar tissues. The extracellular matrix proteins (such as type I collagen and fibronectin) were moderately expressed cytochemically. However, type III collagen was strongly stained. Gene expression of bone sialoprotein and osteopontin was detected in sections of mouse molars of similar age. The ALPase activity showed moderate to strong intensity in these primary cultured cells and responded to 1,25(OH)2 vitamin D3 treatment. Cytokeratin stains were not noted in these cells. In conclusion, the 6-day-old dental follicle cells exhibit partial characteristics of a mineralized tissue-forming phenotype even though the expression of osteopontin, type I collagen and fibronectin was low at this stage.     

Dentonin, a MEPE fragment, initiates pulp-healing response to injury

Phosphorylated extracellular matrix proteins, including matrix extracellular phosphoprotein (MEPE), are involved in the formation and mineralization of dental tissues. In this study, we evaluated the potential of Dentonin, a synthetic peptide derived from MEPE, to promote the formation of reparative dentin. Agarose beads, either soaked with Dentonin or unloaded, were implanted into the pulps of rat molars, and examined 8, 15, and 30 days after treatment. At day 8, Dentonin promoted the proliferation of pulp cells, as visualized by PCNA-labeling. RP59-positive osteoblast progenitors were located around the Dentonin-soaked beads. PCNA- and RP59-labeling were decreased at day 15, while osteopontin, weakly labeled at day 8, was increased at 15 days, but dentin sialoprotein was undetectable at any time. At 8 days, precocious reparative dentin formation occurred in pulps containing Dentonin-soaked beads, with formation slowing after 15 days. These results suggest that Dentonin affects primarily the initial cascade of events leading to pulp healing.     

Glycosaminoglycan-like components in oral calculus, parotid saliva, and dental plaque

Abstract— A comparative investigation was performed on glycosaminoglycan-like fractions obtained from calculus, parotid saliva, and dental plaque. The calculus (sub-gingival and supragingival) was obtained from extracted teeth. Human parotid saliva was collected with an acrylic cup and the dental plaque was gathered from different individuals. The samples were hydrolyzed with papain and the glycosaminoglycan-like compounds were precipitated with the conventional methods. The analyses were made from the material precipitated with cetylpyridinium chloride (CPC). The results showed that the CPC-precipitate of the plaque material contained less uronic acids, sulphate. hexoses. sialic acid, and proteins than did calculus and saliva. The results revealed further that the CPC-precipitate derived from calculus contained phosphate and some calcium. Both saliva and plaque were devoid of these components. The plaque sample lacked the fractions existing in pherograms of both calculus preparation and preparation of parotid saliva (alcian'blue positive). A part of the fractions in the pherograms of calculus and saliva could be stained with lissamine green.     

Compatibility of Type IV Dental Stones With Polysulfide Impression Materials

PURPOSE: In the American National Standards Institute/American Dental Association specification no. 19, compatibility of impression materials with dental stones is assessed by the presence of a 20-microns-wide line reproduced on an unmodified calcium sulfate dihydrate cast. In actual dental practice, modified type IV dental stones are used, although little is known of their compatibility with polysulfide impression materials. This study evaluated the compatibility of 6 polysulfide impression materials and 11 modified type IV dental stones. MATERIALS AND METHODS: A line 20 microns wide was etched on four glass dies. Four samples of each combination of impression material and dental stone were prepared according to the manufacturer's directions with an additional 3 minutes for the final setting time. Compatibility was determined by the presence of the reproduced line on the dental stones, as observed under low angle 10 x magnification by four rater groups. RESULTS: The line was reproduced on all of the impression specimens, and the examiners recorded 66 positive identifications of the line on the stone casts out of a possible 1,056 ratings for a total of 6.25% of the specimens. Out of a possible 66 impression-stone combinations, only 18 reproduced the 20-microns line. The combinations reproducing the lines most frequently (75%) were Neoplex with Blue Die Stone (Columbus Dental, St Louis, MO) and Coeflex with Indic Die Stone (Coe Lab Inc, Chicago, IL). CONCLUSIONS: The study showed that many combinations of polysulfide impression materials and modified type IV dental stones did not reproduce the 20-microns line; therefore, not every polysulfide is compatible with every type IV dental stone.     

The effect of disinfectants on the properties of dental gypsum, part 2: Surface properties

PURPOSE: This study is part of an ongoing investigation to evaluate the surface properties of dental stones mixed with disinfection solutions, and to determine the effect of adding gum arabic and calcium hydroxide on the same properties. MATERIALS AND METHODS: Aqueous solutions of 2 chemical disinfectants were used in mixing 2 types of dental stones (type III and type V). These dental stones were modified further by adding 1% gum arabic and 0.132% calcium hydroxide to their hemihydrate powders before mixing. Five specimens prepared from each type of dental stone were classified into 7 groups according to the hemihydrate powder modification and mixing liquid/powder ratio. Surface roughness was tested by 2-dimensional profilometery and scanning electron microscopy (SEM). Knoop hardness testing was carried out, and detail reproduction was assessed using ADA specification 25 in addition to SEM and 3-dimensional profilometer studies. RESULTS: Dental stones mixed with chemical disinfectants showed higher average roughness (R(a)) values than those of the controls. However, adding gum arabic and calcium hydroxide to the hemihydrate powders before mixing restored values to the level of the control. The additives seemed to have a role in the improvement of surface hardness. There was no significant difference between the experimental and the control group in the terms of detail reproduction. CONCLUSIONS: Using SEM, 3-dimensional profilometry, and ADA testing methods, we found that the surface roughness of stone casts was adversely affected by using the disinfectant solutions as mixing water substitutes. Gum arabic and calcium hydroxide additives can yield a harder stone surface without compromising other surface properties.     

Isolation and identification of acid glycosaminoglycans in oral calculus

Samples of supra- and subgingival calculus were pooled, decalcified in EDTA and the organic matrix solubilized by autoclaving and proteolytic digestion. Acid glycosaminoglycans precipitated with ethanol and cetyl pyridinium chloride were studied both by chemical and electrophoretic techniques. Chondroitin sulphate, hyaluronic acid and possibly heparan sulphate were shown to be present in the organic phase. It is suggested that chondroitin sulphate may have a role in the mineralization of dental calculus as postulated for other calcified tissues.     

Genetic etiology and dental pulp cell deficiency of hypophosphatasia

Hypophosphatasia is caused by mutations of the tissue-non-specific alkaline phosphatase (TNSALP) gene with deficiency of dentin structure. The aim of this study was to examine whether TNSALP mutation in dental pulp cells contributes to dentin dysplasia in hypophosphatasia. Mutation analysis showed that compound heterozygous mutations of TNSALP were identified in three hypophosphatasia patients, including 3 novel mutation sites. Exfoliated teeth from the patients showed abnormal dentin mineralization and loss of cementum, as assessed by ground sections and scanning electron microscope analysis. Dental pulp cells isolated from one of the patients showed a significantly reduced TNSALP activity and mineralization capacity when compared with those in dental pulp cells from the unaffected individuals. Our results suggested that dentin dysplasia in hypophosphatasia may be associated with the decreased mineralization ability of dental pulp cells.     

氟对牙发育影响作用的研究进展

氟是人体必需的微量元素,在机体和牙的发育过程中有着重要的作用。牙在发育过程中摄入过量的氟,将导致氟斑牙的发生。氟通过对矿化相关的基质、基质蛋白、蛋白酶、相关细胞以及离子跨膜转运等多种途径影响正常的牙矿化模式,造成牙的矿化异常。下面就氟对牙发育、釉基质、基质蛋白酶、成釉细胞、牙本质基质蛋白和蛋白聚糖的影响作用的研究进展作一综述。     

Effect of retinoic acid on osteopontin expression in rat clonal dental pulp cells

We studied the effect of retinoic acid on osteopontin synthesis and the mRNA expression in rat clonal dental pulp cells, RPC-C2A. An immunoprecipitation assay clarified that retinoic acid caused an increase in phosphorylated osteopontin synthesis that was dose-dependent, and marked increases were observed at retinoic acid concentrations of 10(-6) to 10(-5) M (1.7-fold). A Northern blotting analysis revealed a similar pattern of increase in osteopontin mRNA expression of up to 6.2-fold of control levels. Because osteopontin has an important role in the mineralization process, these results suggest that retinoic acid regulates mineralization, which takes place in the pulp cavity, including reparative dentin formation.     

Site specific mineral composition and microstructure of human supra-gingival dental calculus

Dental calculus has been implicated in the aetiology of several periodontal conditions. Its prevention and removal are therefore desirable clinical goals. While it is known that calculus is very variable in chemical composition, crystallinity and crystallite size little is known about site specific variability within a dentition and between individuals. With this in mind, a study was undertaken to investigate the comparative site specific nature and composition of human dental supra-gingival dental calculus obtained from 66 male patients visiting for their dental check-up using fluorescent X-ray spectroscopy, X-ray diffractometry and Fourier transform infrared spectroscopy. The supra-gingival dental calculus formed on the lingual surfaces of lower anterior teeth and the buccal surfaces of upper molar teeth were classified into four types based on calcium phosphate phases present. There was significant difference in composition of the crystal phase types between lower and upper teeth (p<0.01). There was no significant difference in crystal size between dental calculus on anterior or molar teeth of all samples. The degree of crystallinity of dental calculus formed on the upper molar teeth was higher than that formed on the lower anterior teeth (p<0.01). The CO(3)(2-) contents in dental calculus formed on the lower anterior teeth were higher than on upper molar teeth (p<0.05) which might explain the difference in crystallinity. Magnesium and Si contents and Ca:P ratio on the other hand showed no significant difference between lower and upper teeth. It was concluded that the crystal phases, crystallinity and CO(3)(2-) contents of human dental supra-gingival dental calculus is related to its location in the mouth.     

改良Mallory‘三色法对牙胚矿化过程的观察

目的：观察牙釉质和牙本质基质矿化过程的动态变化,为阐明牙轴质和牙本质的形成机制提供依据。方法：21和32周自然流产的胎儿2例。分离含有牙胚的上、下颌骨,固定脱钙,25g/L火棉胶、石腊双重包埋,常规组织学切片,用改良Mallor's三爸和HE染色,镜下观察。结果：在牙胚矿化过程中,不同时期、不同矿化层呈现不同的颜色变化。结论：改良Mallor's三色法较HE染色更为清楚的观察到牙胚发育中组织矿化过程的变化。     

Growth-hormone-stimulated dentinogenesis in Lewis dwarf rat molars.

In dentinogenesis, certain growth factors, matrix proteoglycans, and proteins are directly or indirectly dependent on growth hormone. The hypothesis that growth hormone up-regulates the expression of enzymes, sialoproteins, and other extracellular matrix proteins implicated in the formation and mineralization of tooth and bone matrices was tested by the treatment of Lewis dwarf rats with growth hormone over 5 days. The molar teeth were processed for immunohistochemical demonstration of bone-alkaline phosphatase, bone morphogenetic proteins-2 and -4, osteocalcin, osteopontin, bone sialoprotein, and E11 protein. Odontoblasts responded to growth hormone by more cells expressing bone morphogenetic protein, alkaline phosphatase, osteocalcin, and osteopontin. No changes were found in bone sialoprotein or E11 protein expression. Thus, growth hormone may stimulate odontoblasts to express several growth factors and matrix proteins associated with dentin matrix biosynthesis in mature rat molars.     

Calcium and phosphate concentrations and precipitate formation in whole saliva from smokers and non-smokers

Previous reports in the literature indicate that smoking is associated with increased plaque accumulation and/or mineralization. This might be due to an effect of tobacco smoke on properties of saliva. The present study examined the stability of components in fresh and incubated saliva in smokers and non-smokers. Saliva from smokers, collected immediately after smoking a cigarette, did not differ significantly from that of non-smokers in the turbidity developing after incubation (24 h, 37°C), which may reflect aggregation of salivary components by mechanisms analogous to those involved in plaque formation. This finding is in agreement with our previous result (Macgregor. Edgar & Greenwood 1985) which showed that the rate of plaque formation did not differ between smokers and non-smokers. Although an intimation of higher initial calcium concentrations, greater precipitation of calcium and development of more turbidity during incubation was observed in the salivas of smokers than those of non-smokers, the major reason for the greater plaque accumulation and calculus in smokers is probably inadequate oral hygiene.