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Dental calculi can cause gingival bleeding and periodontitis, yet the mechanism underlying the formation of such mineral build-ups, and in particular the role of the local microenvironment, are unclear. Here we show that the formation of dental calculi involves bacteria in local mature biofilms converting the DNA in neutrophil extracellular traps (NETs) from being degradable by the enzyme DNase I to being degradation resistant, promoting the nucleation and growth of apatite. DNase I inhibited NET-induced mineralization in vitro and ex vivo, yet plasma DNases were ineffective at inhibiting ectopic mineralization in the oral cavity in rodents. The topical application of the DNA-intercalating agent chloroquine in rodents fed with a dental calculogenic diet reverted NET DNA to its degradable form, inhibiting the formation of calculi. Our findings may motivate therapeutic strategies for the reduction of the prevalence of the deposition of bacteria-driven calculi in the oral cavity., (© 2024. The Author(s), under exclusive licence to Springer Nature Limited.)

During the Early Bronze Age, populations of the western Eurasian steppe expanded across an immense area of northern Eurasia. Combined archaeological and genetic evidence supports widespread Early Bronze Age population movements out of the Pontic-Caspian steppe that resulted in gene flow across vast distances, linking populations of Yamnaya pastoralists in Scandinavia with pastoral populations (known as the Afanasievo) far to the east in the Altai Mountains 1,2 and Mongolia 3 . Although some models hold that this expansion was the outcome of a newly mobile pastoral economy characterized by horse traction, bulk wagon transport 4-6 and regular dietary dependence on meat and milk 5 , hard evidence for these economic features has not been found. Here we draw on proteomic analysis of dental calculus from individuals from the western Eurasian steppe to demonstrate a major transition in dairying at the start of the Bronze Age. The rapid onset of ubiquitous dairying at a point in time when steppe populations are known to have begun dispersing offers critical insight into a key catalyst of steppe mobility. The identification of horse milk proteins also indicates horse domestication by the Early Bronze Age, which provides support for its role in steppe dispersals. Our results point to a potential epicentre for horse domestication in the Pontic-Caspian steppe by the third millennium BC, and offer strong support for the notion that the novel exploitation of secondary animal products was a key driver of the expansions of Eurasian steppe pastoralists by the Early Bronze Age., (© 2021. The Author(s).)

Consuming the milk of other species is a unique adaptation of Homo sapiens, with implications for health, birth spacing and evolution. Key questions nonetheless remain regarding the origins of dairying and its relationship to the genetically-determined ability to drink milk into adulthood through lactase persistence (LP). As a major centre of LP diversity, Africa is of significant interest to the evolution of dairying. Here we report proteomic evidence for milk consumption in ancient Africa. Using liquid chromatography tandem mass spectrometry (LC-MS/MS) we identify dairy proteins in human dental calculus from northeastern Africa, directly demonstrating milk consumption at least six millennia ago. Our findings indicate that pastoralist groups were drinking milk as soon as herding spread into eastern Africa, at a time when the genetic adaptation for milk digestion was absent or rare. Our study links LP status in specific ancient individuals with direct evidence for their consumption of dairy products.

The cleaning and physicochemical properties on tooth root biointerfaces are pivotal for periodontal healing. Herein, this work investigated the impact of multi-treatment on the physicochemical features of tooth root surfaces and the responsive behavior of human gingival fibroblasts (hGFs). It was found that the combination of various mechanical treatments significantly affects the topographical pattern and size as well as wettability on tooth root surfaces. Furthermore, biological experiments revealed that hGF behaviors (i.e., cell adhesion, shape, spreading, arrangement, and viability) were regulated by the topography and wettability of tooth root surfaces. Also, there was no significant difference in the protein expression of NLRP3 inflammasome and IL-1β in hGFs among tooth root surfaces under various treatments. This study provides new insights to efficiently remove the dental calculus and to understand the interaction between the tooth root interface and cell, which could guide the clinical operation and thereby is more conducive to periodontal recovery., Competing Interests: Declaration of Competing Interest The authors declare no conflict of interests, (Copyright © 2019 Elsevier B.V. All rights reserved.)

Introduction: In autopsy cases staged for sporadic Parkinson's disease (PD), the neuropathology is characterized by a preclinical phase that targets the enteric nervous system of the gastrointestinal tract (GIT). Therefore, the ENS might be a source of potential (presymptomatic) PD biomarkers., Methods: In this clinically based study, we examined the alpha-synuclein (αSyn) concentration in an easily accessible protein storage medium of the GIT, dental calculus, in 21/50 patients with PD and 28/50 age- and gender-matched controls using ELISA., Results: αSyn was detectable in dental calculus and the median concentration in the control patients was 8.6 pg/mg calculus (interquartile range 2.6-13.1 pg/mg). αSyn concentrations were significantly influenced by blood contamination and samples with a hemoglobin concentration of > 4000 ng/mL were excluded. There was no significant difference of αSyn concentrations in the dental calculus of PD patients (5.76 pg/mg, interquartile range 2.91-9.74 pg/mg) compared to those in controls (p = 0.40)., Conclusion: The total αSyn concentration in dental calculus is not a suitable biomarker for sporadic PD. Disease-related variants such as oligomeric or phosphorylated αSyn in calculus might prove to be more specific.

Background: Chlorhexidine gluconate (CHG) has been proven to be effective in preventing and controlling biofilm formation. At the same time, an increase in calculus formation is known as one of considerable side effects. The purpose of this study was to investigate whether mineral deposition preceding a calculus formation would occur at an early stage after the use of CHG using an in vitro saliva-related biofilm model., Methods: Biofilms were developed on the MBEC™ device in brain heart infusion (BHI) broth containing 0.5% sucrose at 37 °C for 3 days under anaerobic conditions. Biofilms were periodically exposed to 1 min applications of 0.12% CHG every 12 h and incubated for up to 2 days in BHI containing a calcifying solution. Calcium and phosphate in the biofilm were measured using atomic absorption spectrophotometry and a phosphate assay kit, respectively. Morphological structure was observed using a scanning electron microscope (SEM), and chemical composition was analyzed with an electron probe microanalyzer (EPMA)., Results: The concentrations of Ca and Pi following a single exposure to CHG increased significantly compared with the control. Repeatedly exposing biofilms to CHG dose-dependently increased Ca deposition, and the amount of Ca was five times as much as that of the control. Pi levels in CHG-treated biofilms were significantly higher than those from the control group (p < 0.05); however, the influence of the number of exposures was limited. Analyses using an SEM and EPMA showed many clusters containing calcium and phosphate complexes in CHG-treated biofilms. Upon composition analysis of the clusters, calcium was detected at a greater concentration than phosphate., Conclusions: Findings suggested that CHG may promote mineral uptake into the biofilm soon after its use. It is necessary to disrupt the biofilm prior to the start of a CHG mouthwash in order to reduce the side effects associated with this procedure. The management of patients is also important.

Background/Purpose. Chronic periodontitis is an inflammatory disease of gums that causes loss of supporting structures of teeth, that is, gingiva, periodontal ligament, cementum, and alveolar bone. Levels of various cytokines in the serum, gingival tissues, and gingival crevicular fluid in patients with chronic periodontitis have been studied, but limited data are available on the level of cytokines in saliva. Therefore, a study was designed to determine levels of salivary IL-6 and IL-17 in patients with calculus associated chronic periodontitis. Materials and Methods. It was a comparative, cross-sectional study that is comprised of 41 healthy controls and 41 calculus associated chronic periodontitis patients (CP patients). According to the degree of attachment loss, CP patients were subcategorized as mild (CAL 1-2 mm), moderate (CAL 3-4 mm), and severe (CAL > 5 mm) forms of periodontitis. Salivary levels of IL-6 and IL-17 were determined using enzyme-linked immunosorbent assay (ELISA) technique. Data was analyzed using SPSS 20.0. Results. Between healthy controls and CP patients (moderate and severe disease), a statistically significant difference was observed in the concentrations of IL-6 and IL-17. In CP patients, the highest mean ± SD of salivary IL-6 and IL-17 was observed in severe CP, followed by moderate and mild CP. Regarding level of IL-6, a statistically significant difference was observed between mild and severe disease and between moderate and severe subcategories of CP patients. Similarly, statistically significant difference was observed in the level of IL-17 between mild and moderate, mild and severe disease, and moderate and severe disease. Conclusion. The levels of salivary IL-6 and IL-17 were increased significantly in calculus associated CP patients as compared to healthy controls and these levels increased with the progression of CP. Clinical Significance. Salivary levels of IL-6 and IL-17 may help in the subcategorization of CP.

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., (© 2017 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

Aim: Matrix-Gla Protein (MGP) is one of the major Gla-containing protein associated with calcification process. It also has a high affinity for Ca 2+ and hydroxyapatite. In this study we aimed to evaluate the MGP rs4236 [A/G] gene polymorphism in association with subgingival dental calculus. Also a possible relationship between MGP gene polymorphism and serum and GCF levels of MGP were examined., Material and Methods: MGP rs4236 [A/G] gene polymorphism was investigated in 110 patients with or without subgingival dental calculus, using polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) techniques. Additionally, serum and GCF levels of MGP of the patients were compared according to subgingival dental calculus., Results: Comparison of patients with and without subgingival dental calculus showed no statistically significant difference in MGP rs4236 [A/G] gene polymorphism (p=0.368). MGP concentrations in GCF of patients with subgingival dental calculus were statistically higher than those without subgingival dental calculus (p=0.032). However, a significant association was not observed between the genotypes of AA, AG and GG of the MGP rs4236 gene and the serum and GCF concentrations of MGP in subjects., Conclusion: In this study, it was found that MGP rs4236 [A/G] gene polymorphism was not to be associated with subgingival dental calculus. Also, that GCF MGP levels were detected higher in patients with subgingival dental calculus than those without subgingival dental calculus independently of polymorphism, may be the effect of adaptive mechanism to inhibit calculus formation., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

Milk is a major food of global economic importance, and its consumption is regarded as a classic example of gene-culture evolution. Humans have exploited animal milk as a food resource for at least 8500 years, but the origins, spread, and scale of dairying remain poorly understood. Indirect lines of evidence, such as lipid isotopic ratios of pottery residues, faunal mortality profiles, and lactase persistence allele frequencies, provide a partial picture of this process; however, in order to understand how, where, and when humans consumed milk products, it is necessary to link evidence of consumption directly to individuals and their dairy livestock. Here we report the first direct evidence of milk consumption, the whey protein β-lactoglobulin (BLG), preserved in human dental calculus from the Bronze Age (ca. 3000 BCE) to the present day. Using protein tandem mass spectrometry, we demonstrate that BLG is a species-specific biomarker of dairy consumption, and we identify individuals consuming cattle, sheep, and goat milk products in the archaeological record. We then apply this method to human dental calculus from Greenland's medieval Norse colonies, and report a decline of this biomarker leading up to the abandonment of the Norse Greenland colonies in the 15(th) century CE.

Background: Oral health and dental maintenance have become part of the standard of care for pediatric liver transplant recipients. These individuals tend to suffer particularly from dental problems, such as gingival enlargement, gingivitis, poor oral hygiene, dental hypoplasia, and caries. Saliva composition influences oral hygiene and disease states. We investigated saliva composition and its association with the oral health of young recipients of liver transplants., Methods: In 70 patients, 36 liver transplant recipients (ages 2-23 years) and 34 healthy controls (ages 4-21 years), we measured the following variables: (a) oral hygiene, (b) gingival inflammation, (c) caries status, (d) dental calculus formation, (e) oral mucosal pH, and (f) salivary protein composition., Results: Lower mean decayed, missing, and filled teeth index (P=0.0038), higher mean gingival index (P=0.0001), and higher mean calculus score (P=0.003) were found in the transplanted study group compared with the control. The mean mucosal pH for seven intraoral sites was higher in the transplant group (P=0.0006). The median salivary albumin concentration was significantly lower in the transplant group (P=0.01), as was the median salivary albumin/total protein ratio (P=0.0002)., Conclusions: In post-liver transplant pediatric recipients, low incidence of caries, together with high incidence of dental calculus, could be attributed to elevated oral mucosal pH. Salivary albumin and immunoglobulin A levels were relatively low in these patients. Clinicians should pay particular attention to the oral health and dental care of liver transplanted children.

Background: This study aimed to determine whether dental calculus formation is really higher among patients with chronic kidney disease undergoing hemodialysis than among controls. Furthermore, the study evaluated correlations between dental calculus formation and dental plaque, variables that are related to renal disease and/or saliva composition., Methods: The Renal Group was composed of 30 patients undergoing hemodialysis, whereas the Healthy Group had 30 clinically healthy patients. Stimulated whole saliva and parotid saliva were collected. Salivary flow rate and calcium and phosphate concentrations were determined. In the Renal Group the saliva collection was carried out before and after a hemodialysis session. Patients from both groups received intraoral exams, oral hygiene instructions, and dental scaling. Three months later, the dental calculus was measured by the Volpe-Manhold method to determine the rate of dental calculus formation., Results: The Renal Group presented a higher rate of dental calculus formation (p < 0.01). Correlation was observed between rate of dental calculus formation and whole saliva flow rate in the Renal Group after a hemodialysis session (r = 0.44, p < 0.05). The presence of dental calculus was associated with phosphate concentration in whole saliva from the Renal Group (p < 0.05)., Conclusion: In conclusion, patients undergoing hemodialysis presented accelerated dental calculus formation, probably due to salivary variables.

Background and Objective: High mobility group chromosomal protein B1 (HMGB1) and N2 (HMGN2), two members of high mobility group (HMG) family, play important role in inflammation. The purpose of this study was to investigate the expression of HMGB1 and HMGN2 in periodontistis., Materials and Methods: The expression of HMGB1 and HMGN2 mRNA in gingival tissues and gingival crevicular fluid (GCF) in chronic periodontitis (CP), generalised aggressive periodontitis (G-AgP) patients and healthy subjects was detected by real-time PCR. The protein level of HMGB1 and HMGN2 in peri-implant crevicular fluid (PICF), peri-implant crevicular fluid of peri-implantitis (PI-PICF) and normal patients was determined by Western blotting. Furthermore, IL-1β, IL-6, IL-8, TNF-α and HMGB1 levels in GCF, PI-PICF and healthy-PICF samples from different groups were determined by ELISA., Results: HMGN2 expression was increased in inflamed gingival tissues and GCF from CP and G-ApG groups compared to control group. HMGB1 expression was the highest in the gingival tissues and GCF from CP patients and was accompanied by increased concentrations of IL-1β, IL-6, IL-8 proinflammaory cytokines., Conclusion: To our knowledge, this is the first study reporting that the expression of HMGB1 and HMGN2 was increased in the gingival tissues and GCF in CP and G-AgP and the PICF in PICF. Our data suggest that HMGB1 may be a potential target for the therapy of periodontitis and PI., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

Background: A large amount of calculus may hamper the efficacy of daily oral hygiene and thereby accelerate plaque formation. Salivary concentrations of orthophosphate and pyrophosphate are important in preventing calculus formation. Activity of orthophosphate, pyrophosphate, and pyrophosphatase was studied in whole saliva in calculus-forming groups and plaque-forming groups., Methods: The material for this study consists of 60 healthy individuals (age range: 15 to 30 years; mean age: 22 years). Depending on calculus index score, individuals were divided into four groups, each of 15 patients: Group 1, calculus index score 0 to 0.6; Group 2, calculus index score 0.7 to 1.8; Group 3, calculus index score 1.9 to 3; and Group 4, plaque group where index varied from 0 to 3. The saliva was collected and biochemically analyzed for concentration of orthophosphate, pyrophosphate, and pyrophosphatase., Results: The mean values of orthophosphate in Groups 1, 2, 3, and 4 were 0.2559, 1.3639, 1.7311, and 0.1868 mM, respectively. The mean values of pyrophosphate in Groups 1, 2, 3, and 4 were 0.3258, 0.1091, 0.0314, and 0.3860 mM, respectively. The mean values of pyrophosphatase in Groups 1, 2, 3, and 4 were 10.7937, 15.4249, 27.2900, and 7.5427 units/ml, respectively., Conclusions: A holistic approach toward the control of periodontal disease should include antiplaque and anticalculus agents. The results are conclusive that the components orthophosphate, pyrophosphate, and pyrophosphatase present in saliva have a very significant role to play in formation and inhibition of calculus. This study reinforces the idea of using pyrophosphate and newer bisphosphonates as potential anticalculus agents.

Objective: We studied the effects of food components on the in vitro formation of calcium phosphate precipitates., Materials and Methods: The effects of food components, such as starch, soybean flour, fish meal, rapeseed oil, and coconut oil, on calcium phosphate precipitation were studied using a pH drop method., Results: Although the addition of starch had no effect on the rate of precipitation of amorphous calcium phosphate (ACP), it increased both the rate of transformation of ACP to hydroxyapatite (HAP) and the induction time (i.e. time for the initiation of transformation of ACP to HAP to occur); this was irrespective of the heat treatment of the starch. Amylopectin (insoluble constituent of starch) was effective in increasing the rate of HAP transformation, but amylose (soluble constituent of starch) was not. Oil specimen obtained from rapeseed (400 microl ml(-1)) increased the entire reaction of calcium phosphate precipitation, but that from coconut did not. Protein food, such as soybean flour and fish meal, decreased the rate of transformation of ACP to HAP and increased the induction time, while they had no effect on the rate of ACP precipitation., Conclusion: These results suggest that carbohydrate and oil (both are staple diets for the humans) enhance oral calcification (dental calculus formation or re-mineralization of tooth enamel), while side dishes of protein food would decrease it.

The aims of this study were to evaluate (i): whether vitamin C in chewing gum, alone or in combination with carbamide, influences calculus formation, and (ii) whether carbamide affects the release, stability and uptake of vitamin C in a chewing gum. In two test series (Series I and II), 30 subjects, all calculus formers, participated. They were instructed to chew on five (Series I) or 10 (Series II) pieces of gum per day for a period of 3 months. The chewing gums were: vitamin C (60 mg, Series I), non-vitamin C (Series I) and vitamin C + carbamide (30 mg + 30 mg, Series II). In both series, no gum was used as a negative control. Calculus formation was scored on three lingual sites on the six anterior mandibular teeth according to the Volpe-Manhold index. The effect on plaque and gingivitis was also determined. A significant reduction in the total calculus score was observed after the use of vitamin C (33%) and vitamin C + carbamide (12%) gums compared with no gum use; this reduction was most pronounced in the heavy calculus formers. A reduced amount of visible plaque was also observed after use of vitamin C and non-vitamin C gum, but only the vitamin C gum reduced the number of bleeding sites (37%). In a separate study, the release, stability and uptake of vitamin C were evaluated using the iodine titration method in both saliva and urine after exposure to the following gums: vitamin C + carbamide (30 mg + 30 mg) and vitamin C (30 mg). There was no indication that carbamide affected the release, stability or uptake of vitamin C when used in a chewing gum.

Dental calculus is composed of inorganic components and organic matrix. Brushite, dicalcium phosphate dihydrate, octacalcium phosphate, hydroxyapatite, and whitlockite form the mineral part of dental calculus. Salivary proteins selectively adsorb on the tooth surface to form an acquired pellicle. It is followed by the adherence of various oral micro-organisms. Fimbriae, flagella, and some other surface proteins are essential for microbial adherence. Microbial co-aggregation and co-adhesion enable some micro-organisms, which are incapable of adhering, to adhere to the pellicle-coated tooth surface. Once organisms attach to the tooth surface, new genes could be expressed so that mature dental plaque can form and biofilm bacteria assume increased resistance to antimicrobial agents. Supersaturation of saliva and plaque fluid with respect to calcium phosphates is the driving force for plaque mineralization. Both salivary flow rate and plaque pH appear to influence the saturation degree of calcium phosphates. Acidic phospholipids and specific proteolipids present in cell membranes play a key role in microbial mineralization. The roles of crystal growth inhibitors, promoters, and organic acids in calculus formation are discussed. Application of biofilm culture systems in plaque mineralization is concisely reviewed. Anti-calculus agents used--centering on triclosan plus polyvinyl methyl ether/maleic acid copolymer, pyrophosphate plus polyvinyl methyl ether/maleic acid copolymer, and zinc ion-in commercial dentifrices are also discussed in this paper.

We present here the first study of the local environment of zinc ions in biological calcium phosphates. It was suggested from in vitro studies that zinc inhibits the formation of hydroxyapatite and promotes the formation of more soluble phases, like tricalcium phosphate. Several mechanisms of zinc - calcium phosphate interaction were proposed, yielding either to the adsorption or to the incorporation of zinc ions into the phosphate structure. The results obtained here show that, under in vivo conditions, the zinc atoms are fully incorporated into the crystalline structure of the calcium phosphates.

Several individual species of dental plaque bacteria have the ability to initiate the precipitation of calcium phosphate minerals in vitro; other plaque components have been shown to inhibit mineralisation. We have examined subjects' overall plaque mineralisation promoter and inhibitor properties, and have attempted to correlate them with supragingival calculus development over 6 months. Three-day-old plaque was collected from 22 adult subjects at the start and end of the study. To detect promoter activity, the plaque was placed in a suspension of brushite, the liquid phase of which was supersaturated with respect to hydroxyapatite. The extent of mineralisation was determined by the rise in phosphate concentration over 4 days. To detect inhibitor activity, plaque was placed in a similar suspension, which also contained hydroxyapatite. Promoter activity was compared with that hydroxyapatite, and inhibitor activity was compared with polyaspartate. The subjects' teeth were scaled at the start of the study, and calculus deposition was measured at the end using the Volpe Manhold method. Most plaque samples showed some promoter or inhibitor activity, or both, but no significant correlation existed between these activities and a subject's development of calculus. A significant inverse correlation existed between plaque mineralisation promoter activity and its inhibitor activity at the start of the study. Our results suggest that the nucleating and mineralisation inhibitory properties of young plaque will probably not be a useful target for a practical preventive methodology for supragingival calculus.

To non mucin proteins of human saliva belong: cystatins, statherin, histatins and acidic proline-rich protein. These saliva proteins influence hard and soft tissues by forming a pellicle layer on oral mucosa and enamel, by taking part in removing bacteria or initiating of bacterial colonization. Most of them are able to inhibit the formation of dental calculus and control the calcium phosphate homeostasis.

This paper discusses the different types of calcium phosphate in calculus and the role of plaque pH in controlling calcium phosphate precipitation (calculus) or dissolution (caries). There is great site-specificity of deposition of supragingival but not subgingival calculus, with highest amounts of the former being present on the lingual aspects of the mandibular anterior teeth. This may be because the plaque there is most alkaline as that region has the highest salivary film velocity and the lowest salivary sugar concentration during consumption of sugar-containing food and drinks. The importance of calculus removal for good gingival health is emphasised.

Four interlocking lines of research carried out during the Directorship of Dr TW Cutress in the Dental Research Unit were: plaque urea metabolism, which led to the study of plaque pH responses and their control; development of plaque-like biofilm model systems; plaque mineralisation to calculus; and plaque demineralisation of tooth tissue in caries. New modes of regulation of oral bacterial urea metabolism and its role in the mouth were discovered, especially a role as a pH-rise factor and in mineralisation processes. The development of microcosm plaques, consortia of major plaque species, and of the multi-plaque artificial mouth with the ability to measure pH continuously, has substantiated the theory that plaque thickness and fluid flow are important in determining plaque pH. For the first time, formation of large pH gradients inside plaque have been demonstrated and plaque pH experimentally controlled. Plaque growth curves can be accurately measured and procedures established for measuring antiplaque and anticaries agents. These studies exemplify the value of the fundamental approach adopted by Dr Cutress--that integrated, basic, applied, and public-health lines of research reinforce each other.

Dental calculus is plaque mineralised by deposition of calcium and phosphate resulting from interactions between the oral microbial plaque flora and components of oral fluids. An artificial-mouth microcosm dental plaque culture system has been developed to study aspects of plaque mineralisation, including pH control. Five plaques were grown from saliva under simulated oral conditions in a mucin-containing medium, and sucrose was applied to mimic meals. The plaques were mineralised with a urea-based, calcium-phosphate-monofluorophosphate-urea (CPMU) mineralising solution. Alkaline pH oscillations were generated by the plaques in response to CPMU applications, and an acidic oscillation followed sucrose applications. Plaque mineralisation by the CPMU procedure was almost totally dependent on the urea present in the mineralising solution, but total mineralisation also increased as the resting pH increased as a result of urea in the medium. Following four CPMU applications with a sucrose application every 12 hours improved plaque viability and mineralisation. The plaque mineral formed resembled a carbonated hydroxyapatite; other potential calcium phosphate minerals were undetectable except for calcium carbonate. A wide range of mineral deposition patterns in plaque were seen by electron microscopy.

Calprotectin is a calcium binding protein produced by leukocytes, macrophages and epithelial cells, and its levels in several tissues increase during infections and in many inflamed areas, suggesting that it may be an indicator of inflammatory activity. Osteopontin is a prominent phosphorylated glycoprotein in bone matrix, having calcium binding capacity. Recently, it has been reported that calprotectin and osteopontin are present in urinary stones (pathological mineralized masses in the body), and that these proteins may be involved in their formation. Dental calculus formed by mineralization of dental plaque is an inflammatory factor which may contribute to periodontal disease. It contains many organic components involved in mineralization. We recently found osteopontin molecules in human dental calculus and suggested that the components of its matrix may be similar to those of urinary stones. In this study, we investigated the presence of calprotectin in human dental calculus by immunohistochemical and immunoblotting analyses using a specific antibody for calprotectin. After fixation and demineralization of dental calculi adhered to tooth roots, sections embedded in paraffin were immunoreacted with the antibody for calprotectin and positive immunostaining for calprotectin was observed. Dental calculus proteins were then extracted with EDTA and separated by electrophoresis on 15% polyacrylamide gels. By immunoblotting analysis, 3 or 4 bands were observed at 11, 14.5, 22-25, 28 or 36.5 kDa and these patterns corresponded to those of calprotectin subunits. When non-immune rabbit serum was used instead of calprotectin-specific antibody as a negative control, no immunoreactivity was observed. These findings indicate that calprotectin is associated not only with antibacterial action but also with calcium binding capacity during dental calculus formation.

The aim was to establish if there is a correlation between the rate of formation of supragingival calculus and the degree of supersaturation of saliva with respect to apatite, brushite and other calcium phosphates. The rate of calculus formation was estimated by measuring the calculus formed on lingual surfaces of the lower incisors in 15 individuals during 30 days. Submandibular saliva stimulated with citric acid and resting whole saliva, alter pH determination and ultrafiltration to remove protein was analysed for calcium, phosphate, sodium, potassium and carbonate. The degree of saturation with respect to calcium phosphates was calculated using a computer program. The rate of calculus formation among the 15 participants ranged widely, from a score of 0.5 to a high of 15. A close correlation (r = 0.91) between salivary pH and degree of supersaturation was found A weak correlation between calcium and calculus formation was also found. But neither for unstimulated nor stimulated saliva was a significant relation between supersaturation with respect to any of the calcium phosphates and the rate of calculus formation observed. Therefore, taking into consideration the background of the wide range of rates of calculus formation and of degrees of supersaturation, it was concluded that relation between them is unlikely, and the use of degree of saturation as a diagnostic tool seems unreliable.

Streptococcus mutans mineralization was studied in vitro, with the use of various metastable calcium phosphate solutions, fluoride-containing or otherwise. Degeneration of bacteria always occurred before their mineralization. After complete mineralization of the cytoplasmic area, growth of crystals was observed both in length and in thickness within the extracellular environment. Composition of the calcifying medium was an essential factor in the S. mutans mineralization process. The present study shows that, contrary to carbonate and magnesium ions that prevent intra- and extracellular deposits, fluoride ions promote crystal growth in the above mentioned mediums. Clinical implications of the role of fluoride, at the plaque level, are foreseen.

The precipitation of Ca-compounds in ageing dental plaque leads to the formation of calculus. Bacterial lipoteichoic acid (LTA) has been discussed as Ca-carrier in this process (Zoon et al., 1989). Therefore in this study, LTA was isolated from oral streptococci and tested for its influence on the precipitation of Ca-phosphate. LTA-containing bacteria, LTA-free bacteria and isolated LTA were added to the reaction mixtures for Ca-phosphate precipitation and the amount of precipitated mineral was measured. LTA-containing cells and, above all, LTA, inhibited Ca-phosphate precipitation in a distinct pH-range just above the acid solubility of the mineral by complexing Ca(2+)-ions. Experiments with Ca-fluoride in place of Ca-phosphate showed that LTA did not affect the precipitation of this mineral.

This investigation assessed the effectiveness of an in vitro ultrasonic debridement regime on periodontally involved teeth with visually-detectable calculus deposits, using the LAL assay for lipopolysaccharides (LPS) as a marker of bacterial products. 34 single rooted teeth with extensive amounts of calcified accretions were selected, of which 10 served as uninstrumented controls for the estimation of existing LPS levels. The periodontally involved roots of the 24 experimental teeth were debrided with a Cavitron TF-10 tip for a limited time using light pressure and overlapping strokes which ensured complete surface instrumentation without reference to the presence of calculus. Marked reductions in root surface area coverage by calculus were nevertheless achieved as assessed planimetrically from photographic records of the teeth before and after debridement. The root surfaces of each tooth were then stripped away and the harvested material subjected to 'Westphal' extraction and LAL assay for LPS. The resulting LPS yields from the experimental teeth ranged from less than 0.08 to 22.387 ng compared to 1,900 to 29,200 ng from the uninstrumented controls. The extremely small residual LPS yields from the experimental teeth not only confirm the efficacy of ultrasonic debridement but, when coupled with the lack of a relationship with the widely varying amounts of the remaining calculus, support the contention that bacterial plaque rather than calculus has the greater pathogenic potential. These results reinforce the growing impression of the superficial location of bacterial toxic products associated with periodontally involved root surfaces.

A pilot study was carried out to obtain an idea of the strength of the relations between early mandibular calculus production in humans and amounts and concentrations of bound calcium and phosphate in three salivary fractions. The salivary fractions were: (1) the spontaneous precipitate from incubated whole saliva, (2) the spontaneous precipitate from incubated centrifugal (20,000 X g) saliva supernatant and (3) the fresh centrifugal saliva sediment. Strong and highly significant correlations were found for the amount and concentration of calcium in the fresh sediment and whole saliva precipitate fractions. Although the correlations for the calcium parameters of the saliva supernatant precipitate were not significant, there were significant differences in these parameters between low and high calculus formers as determined by Student's t test. Relations between calculus production and amount and concentration of phosphate were weaker and not significant for all fractions. The data suggest that the amount or concentration of calcium in fresh saliva sediment is an important determinant of early mandibular calculus production.

Acidic proline-rich proteins (APRP) were quantitated immunochemically in salivary secretions from groups of: caries-resistant (CR) and caries-susceptible (CS) subjects; heavy- and light-calculus-formers; and patients with Sjögren's Syndrome, drug-induced xerostomia, and recurrent parotitis. In all groups except the parotitis patients, there were comparable levels of APRP, about 40-50 mg%, with similar values in parotid and submandibular saliva. In chronic recurrent parotitis, the values were somewhat higher (about 60 mg%). There were no differences in the proportion of APRP-A to C in a subset of CR and CS. Taken as a whole, the data support the view that the secretion of APRP is stable and that caries status and propensity to calculus formation are not associated with abnormal levels of these phosphoproteins.

The initiator of calculus matrix calcification, in vitro, was isolated. Crude phospholipid, known to contain the factor, was separated into five fractions by column chromatography. A single protein-containing fraction induced apatite formation during incubation. The nucleating fraction was indentified as a proteolipid.

Calculus plays an important role in chronic inflammatory periodontal disease and tooth loss. Patients can suffer from calculus formation despite good oral hygiene. The strength of adhesion between calculus and the enamel surface, though not determinant for the formation of calculus itself, determines whether calculus remains on the teeth during eating and toothbrushing. In this study, the amount and the strength of adhesion of calcium-rich deposits formed in vivo on different materials are related to substrate surface free energies (sfe). In 4 beagle dogs, fenestrated crowns were made on the upper fourth premolars. Smooth facings of glass (sfe 120 mJ.m-2), polished bovine enamel (sfe 85 mJ.m-2), polymethylmethacrylate (PMMA sfe 56 mJ.m-2) and polytetafluorethylene (PTFE sfe 20 mJ.m-2) were inserted in the crowns for 1, 3, 7, 14 or 28 days. The amount of deposit was evaluated both gravimetrically and planimetrically using Alizarin Red S for staining. Adhesion of calcium-rich deposits was evaluated planimetrically by studying their removal in a brushing machine. Dry weight increased linearly with time (approximately 0.18 mg.cm-2 per day) and was slightly less on PTFE than on the other materials. After 1 to 3 days, staining already revealed a 100% coverage by calcium-rich deposits. The number of strokes required to reduce the planimetric scores by 63% was extremely small on PTFE and PMMA and related with substrate surface free energies. This study shows that a possible way to reduce calculus formation in vivo is to decrease the surface free energy of the enamel using appropriate surfactants in, e.g., toothpastes.