Mineralising and antibacterial effects of modified calcium phosphate treatment on human root cementum.

BACKGROUND: Aging population will lead to the increase of incidence of root caries globally. The clinical management of root caries is challenging due to the difficulty in moisture isolation. The root caries is caused by the release of organic acids from cariogenic bacteria which results in the dissolution of cementum and dentin of the root. The purpose of this study is to study the efficacy of modified saturated calcium phosphate solution (CaP) supplement with zinc (Zn(2+)) and/or fluoride (F(-)) in providing root cementum surfaces less susceptible to acid dissolution and bacterial colonization. METHODS: Human root cementum sections from extracted premolars were treated with three modified calcium phosphate solutions (M/A-CaPs) respectively: (A) CaP-F/Zn, supplemented with F(-) and Zn(2+); (B) CaP-F, supplemented with F(-) only; (C) CaP-Zn, supplemented with Zn(2+) only. The surface characteristics of treated cementum sections were investigated using scanning electron microscopy (SEM) and fourier transform infrared spectroscopy (FT-IR). Following the acid attack and Streptococcus mutans challenge, M/A-CaPs treated cementum surfaces were analysed using inductive coupled plasma (ICP) and SEM respectively. RESULTS: Compared with the control group, M/A-CaPs treated cementum presented significant improvements in resistance to acid dissolution and bacterial colonization. Among M/A-CaPs, the CaP-F/Zn treated cementum surfaces released the lowest amount of Ca(2+) ions (2.11 ± 0.51 ppm) upon acid challenge (n = 3, p < 0.01) and also presented the most significant inhibiting effect against the colonization of S. mutans (n = 180, p < 0.05). CONCLUSIONS: Saturated calcium phosphate solution CaP supplemented with both F(-) and Zn(2+) could be applied as an effective coating material providing acid resistance and antibacterial property on cementum surfaces. The modified calcium phosphate-based solution could be a new treatment strategy to prevent the development of root caries and arrest the further progression of root caries.

A histological study of peripheral dental caries of equine cheek teeth.

Peripheral caries of equine cheek teeth is a poorly described disorder and, in particular little is known of its histopathology. Histological examinations of decalcified sections of 21 equine peripheral caries-affected cheek teeth showed two different patterns of cemental lesions; including progressive enlargement of focal, flask-like lesions leading to breakdown of the adjacent cementum, and secondly; a more generalized flaking-off of thin layers of under-run, surface cementum. A thick layer of plaque and food material usually lay on the surface of affected cementum and also within cemental defects. Gram-stained sections showed large numbers of bacteria within the lacunae and canaliculi of affected peripheral cementum and within associated plaque. Pioneer bacteria were also seen within dentinal tubules of adjacent, normal-appearing dentin. Subgingival extension of peripheral caries lesions with localized periodontal destruction was rare. Grading of peripheral caries lesions by gross examination was found to underestimate the severity of the disorder as compared to histological grading.

The CEJ: a biofilm and calculus trap.

There is a direct correlation between increasing periodontal probing depth and increasing presence of residual biofilms and calculus. One of the more common areas to find postscaling and root-planing residual biofilm and calculus is the cemento-enamel junction (CEJ)--an area that in most cases is easily accessible. Yet few studies have addressed the question of why the CEJ is a potential biofilm and calculus trap. This article discusses the various anatomical relationships of enamel, dentin, and cementum at the CEJ, the role of CEJ anatomy in the retention of biofilm (with SEM photographs as supporting evidence), and the biological and clinical implications of subgingival residual biofilm and calculus.

CXXC5 orchestrates Stat3/Erk/Akt signaling networks to modulate P. gingivalis-elicited autophagy in cementoblasts.

The keystone pathogen Porphyromonas gingivalis (P. gingivalis) elicits inflammation and autophagy in periodontal tissues. Transcription factor CXXC-type zinc finger protein 5 (CXXC5) and various signals are sensitive to P. gingivalis invasion. Herein, we investigated the P. gingivalis-elicited autophagy activity, the contribution of CXXC5, and the involvement of signals in cementoblasts, tooth root surface cells crucial in periodontal and periapical regions. After coculture with P. gingivalis, cementoblasts exhibited inflammatory cytokine increase, light chain 3(LC3)-I/II conversion, autophagosome activation, and CXXC5 reduction. Cementoblasts with loss and gain of CXXC5 were developed. CXXC5 silencing suppressed autophagy and inflammation, thereby partially compensating for the effects of P. gingivalis, and vice versa. We then screened potential signals and verified the positive participation of Stat3/Akt/Erk networks through specific inhibitor employment. P. gingivalis and CXXC5 induced autophagy through Beclin1 and Atg5 activation. Intriguingly, Annexin V/PI assay and EdU detection revealed that P. gingivalis promoted apoptosis and repressed cell proliferation. In sum, coculture with P. gingivalis enhanced autophagy activity in cementoblasts, which was partially suppressed by CXXC5 downregulation and mediated by Jak/Stat3, PI3K-Akt, and Erk1/2 signaling. This process probably influenced cell apoptosis and proliferation.

Antimicrobial action of intracanal medicaments on the external root surface.

OBJECTIVES: To assess in vitro the antimicrobial action of intracanal medicaments in the external root surface, with or without of cementum. METHODS: After the placement of intracanal medicaments, the coronal and apical openings were sealed. The antimicrobial action of 2% chlorhexidine gel (2%CHX); calcium hydroxide+2% chlorhexidine gel (CH+2%CHX); calcium hydroxide+2% chlorhexidine gel+zinc oxide (CH+2%CHX+ZnO); calcium hydroxide+saline solution (CH+SS) was evaluated against Enterococcus faecalis, Candida albicans, Actinomyces viscosus and Porphyromonas gingivalis. The samples were placed over the agar, and the zones of inhibition of microbial growth were measured after the incubation period. RESULTS: The antimicrobial effects of the medicaments could be ranked from strongest to weakest as follows: 2%CHX, CH+2%CHX, CH+2%CHX+ZnO, CH+SS. No differences among groups were verified in the presence or absence of cementum. CONCLUSIONS: 2%CHX containing medicaments diffused into the dentin and reached the outer surface, exerting antimicrobial action.

Incipient caries lesions on cementum by mono- and co-culture oral bacteria.

OBJECTIVES: There is increasing prevalence of root caries. We hypothesized different biofilms will cause varying demineralization in cementum. This study investigated the extent of demineralization of cementum by oral biofilm formed from three major cariogenic microorganisms: Streptococcus mutans, Lactobacillus acidophilus, and Actinomyces israelii. Sound cementum tooth blocks were incubated with mono-, bi-, and tri-species combinations of the bacteria under investigation. MATERIALS AND METHODS: The matrix (amide I) and phosphate content of the lesions was analyzed by Fourier-transform infrared spectroscopy (FTIR), and calcium and phosphorus levels were analyzed by scanning electron microscopy and energy-dispersive spectroscopy (SEM-EDX). RESULTS: The log[amide I:HPO(4)(2-) absorbance] values showed that A. israelii mono-culture caused significantly more demineralization than the other bacterial cultures. log[Ca:P] showed that all carious lesions were confined to the cementum. CONCLUSIONS: Oral biofilm arising from bacterial species A. israelii alone was the most cariogenic of those tested and produced the most demineralization in incipient carious lesions in cementum.

Er:Yag laser irradiation of the microbiological apical biofilm.

One problem in cases of healing-resistant periapical lesions is to eradicate the contamination at the periapical area. This contamination is due to the microbiological biofilm formed by microorganisms and their subproducts lodged in apical third of the root, on both cementum and dentin surface. Paraendodontic surgery consists of the mechanical removal of harmful agents to promote healing and periapical health. The purpose of this study was to assess the results of Er:YAG laser irradiation on the apical root third of newly extracted teeth to eliminate microbial contamination on root apex surface. Apical irradiation was performed with an Er:YAG laser device using an experimental contact tip, at 100 mJ, 10 Hz, 1 W, 39 J/cm(2), 3 times on the target area. SEM analysis showed the elimination of part of the irradiated cementum and the formation of small roughened without exposing the subjacent dentin. Vaporization of the remaining periodontal tissue and removal of microbiological apical biofilm (MAB) were also observed on the irradiated areas. Under the tested conditions and based on the findings of this study, Er:YAG laser may be considered effective for removal of microbiological apical biofilm.

Complex Apical Intraradicular Infection and Extraradicular Mineralized Biofilms as the Cause of Wet Canals and Treatment Failure: Report of 2 Cases.

This article describes 2 cases that showed persistent intracanal exudation (wet canal) even after several visits of antimicrobial endodontic treatment. Histologic and histobacteriologic investigation was conducted for determination of the cause. The 2 cases involved teeth with apical periodontitis lesions, which presented persistent exudation refractory to treatment after several visits. In case 1, it was not possible to achieve a dry canal, and surgery had to be performed. In case 2, attempts to dry the canal succeeded and the canal was filled, but follow-up examination showed an enlarged apical periodontitis lesion and extraction was performed. Biopsy specimens consisting of the root apex and apical periodontitis lesion for case 1 and the whole root for case 2 were subjected to histologic and histobacteriologic analyses. Both cases showed complex bacterial infection in the apical root, affecting both the intraradicular space and the outer root surface. Case 1 showed bacterial biofilms in ramifications, on untouched walls, and extending to the external root surface to form a thick and partially mineralized structure with high bacterial density. Different bacterial morphotypes were evidenced. Case 2 had a ledge on the apical canal wall created during instrumentation, which was filled with necrotic debris, filling material, and bacteria. The walls of the apical portion of the canal were covered by a bacterial biofilm, which was continuous with a thick extraradicular biofilm covering the cementum and dentin in resorptive defects. The extraradicular biofilm showed areas of mineralization and was dominated by filamentous bacteria. The 2 cases with wet canals and treatment failure were associated with complex persistent infection in the apical part of the root canal system extending to form thick and partially mineralized biofilm structures (calculus) on the outer apical root surface.

A model for producing caries-like lesions in enamel and dentin using oral bacteria in vitro.

Subsurface enamel lesions and root surface caries-like lesions were consistently produced in vitro using Streptococcus mutans FA1 cultured in thioglycollate broth containing 3.5% w/v dextrose and 2% w/v gelatin. When viewed in polarized light and after imbibition in water, the enamel lesions had a negatively birefringent surface zone and positively birefringent body of the lesion. Those lesions produced after six weeks, after imbibition in quinoline, exhibited a dark zone. The root surface caries-like lesions exhibited a less-radiolucent surface zone above a heavily demineralized body of the lesion. However, no reactionary dentin was seen in the in vitro lesions.

An ultrastructural study of bacterial invasion and tissue breakdown in human experimental root-surface caries.

This study describes the structural features of the interface between microbial deposits and root cementum in actively progressing root-surface caries lesions developed experimentally in six elderly individuals. A total of 18 specimens was examined by microradiography, and a further 18 by light and transmission electron microscopy after intra-oral periods of one, two, and three months. All specimens showed various degrees of subsurface dissolution of mineral and bacterial invasion of the cementum. Although the microradiographic pattern of mineral loss was subsurface in nature, transmission electron microscopy showed dissolution of crystals in the outermost layers of the cementum, with a distinct gradient inward. Bacterial invasion occurred along the borders between bundles of relatively well-mineralized extrinsic collagen fibers in which the characteristic cross-banding remained intact. The pattern of bacterial invasion was influenced by the incremental lines and the cemento-dentinal junction. The invading bacteria were almost exclusively Gram-positive, of various shapes, and possessed thick, moderately electron-dense cell walls and electron-lucent "vacuoles" in the cytoplasm. It is concluded that because of pronounced mineral loss of the outermost cementum, accompanied by bacterial invasion, the surface of an active cementum caries lesion, as observed by transmission electron microscopy, is not identical to that seen in microradiograms.

Localization of Porphyromonas gingivalis-carrying fimbriae in situ in human periodontal pockets.

Fimbriae, which are involved in adherence, constitute an important pathogenic factor of Porphyromonas gingivalis. In vivo, however, the distribution of P. gingivalis-carrying fimbriae is unknown. The localization of P. gingivalis-carrying fimbriae was examined in situ. From 19 patients with severe periodontitis and P. gingivalis, we obtained 20 teeth with periodontal tissue attached, with and without immunolocalized fimbriae. Eleven teeth were subjected to light microscopy, 9 to electron microscopy. In 6 of the 11 samples examined, we detected positive reactions with an anti-P. gingivalis-fimbriae serum, located in the cementum-attached plaque area in the deep pocket zones. In the so-called 'plaque-free zones', P. gingivalis-carrying fimbriae were immunocytochemically observed to reside in contact with the dental cuticle in 6 of the 9 samples examined. These findings suggest that P. gingivalis-carrying fimbriae are strongly related to adherence to the root surface at the bottoms of human periodontal pockets.

Bacterial invasion in root cementum and radicular dentin of periodontally diseased teeth in humans. A reservoir of periodontopathic bacteria.

In this study the viability and the distribution of bacteria within the radicular dentin and pulp of periodontally diseased caries-free teeth were studied. Healthy teeth served as controls. Samples were obtained from the pulp tissue and from the radicular dentin. Dentin samples were taken from the interdental surfaces in the subgingival area. Starting from the pulpal side, three to five successive dentin layers of approximately 1 mm thickness were sampled. The samples were processed and cultured using an anaerobic technique. Bacterial growth was detected in 87% of the periodontally diseased teeth. In 83% of the teeth, bacteria were present in at least one of the dentin layers. Fifty-nine percent of the diseased teeth, from which the pulp tissue was cultured, contained bacteria in the pulp samples. The mean bacterial concentrations in the pulp and dentin layers ranged from 1,399 to 16,537 colony-forming units (CFU) per mg of tissue. These concentrations were 259 to 7,190 times greater than concentrations found in healthy teeth. It is suggested that the roots of periodontally diseased teeth could act as bacterial reservoirs from which recolonization of mechanically treated root surfaces can occur, as well as infection of the dental pulp. These findings might change current concepts concerning root surface debridement in periodontal therapy.

Ultrastructural observations on bacterial invasion in cementum and radicular dentin of periodontally diseased human teeth.

In this study the bacterial invasion in root cementum and radicular dentin of periodontally diseased, caries-free human teeth was examined. In addition, structural changes in these tissues, which could be related to the bacterial invasion, were reported. Twenty-one caries-free human teeth with extensive periodontal attachment loss were studied by light and scanning electron microscopy. At the base of the gingival pocket, bacteria were found in the spaces between remnants of Sharpey's fibers and their point of insertion in the cementum. In teeth that had been scaled and root planed, most of the root cementum had been removed. Bacterial invasion was found in the remaining root cementum. The invasion seemed to start as a localized process, often involving only one bacterium. In other areas bacteria were present in lacunar defects in the cementum. These lacunae extended into the radicular dentin. In 11 teeth bacteria had invaded the dentinal tubules. Most bacteria were located in the outer 300 microns of the dentinal tubules, although occasionally they were found in deeper parts. In two of the nontreated teeth, bacteria were detected on the pulpal wall. No correlation was found between the presence of bacterial invasion and the absence of radicular cementum. No bacteria were found in the portion of the root located apically to the epithelial attachment. These data are in agreement with our results from cultural studies of the bacterial flora in these structures. It was also demonstrated that in spite of meticulous scaling and root planning and personal oral hygiene, bacterial plaque remained present on radicular surfaces. Both the invaded dentinal tubules and the lacunae could act as bacterial reservoirs from which recolonization of treated root surfaces occurs. From these reservoirs bacteria could also induce pulpal pathoses. Since these bacterial reservoirs are not eliminated by conventional mechanical periodontal treatment, it seems appropriate to combine mechanical periodontal therapy with the use of chemotherapeutic agents.

Calculus attachment. Review of the literature and new findings.

This study was undertaken in order to provide current information relative to the modes of calculus attachment to tooth surfaces. A total of 63 freshly extracted teeth were fixed, sectioned, and conventionally prepared for light, transmission electron, and scanning electron microscopic examination. Previously reported histologic findings of cuticular attachment, mechanical locking into undercuts, and direct attachment of calculus matrix to the tooth surface were affirmed. The claim of bacterial penetration as a mode of attachment has been rejected. Ultrastructural evidence of cuticular attachment has been presented for the first time via a series of scanning electron micrographs. The most frequently encountered method of attachment was found to be the apparent melding of calculus matrix to the surface of cementum. In many instances, these two substances were virtually indistinguishable.

In vitro effect of laser irradiation on cementum-bound endotoxin isolated from periodontally diseased roots.

BACKGROUND: In a previous study, we evaluated the in vivo effects of an Nd:YAG laser on periodontal disease by measuring crevicular interleukin (IL)-1beta levels before and after laser application. It was found that laser therapy was less effective than traditional scaling and root planing. These results might be due to incomplete removal of microbial residues and cementum-bound endotoxin on root surfaces by the laser. In this study, we explored the in vitro effectiveness of an Nd:YAG laser for the elimination of cementum-bound endotoxin by measuring IL-1beta changes in stimulated monocytes. METHODS: Fresh human monocytes were harvested from adults without periodontitis and grown in RPMI 1640 medium. Diseased cementum particles were collected and prepared from teeth with untreated periodontitis and were irradiated with 5 levels of laser energy. Cementum particles were subjected to endotoxin testing by a limulus amebocyte lysate (LAL) assay and then were incubated with cultured monocytes. Production of IL-1beta in stimulated monocytes was measured by enzyme-linked immunosorbent assay and quantified by spectrophotometry. RESULTS: The endotoxin unit (EU) of diseased cementum was 18.4 EU/mg, which seemed to be remarkably lower than that of common periodontal pathogens including Porphyromonas gingivalis (381) at 15,300 EU/mg/ml, Prevotella intermedia (ATCC 25611) at 227 EU/mg/ml, and Fusobacterium nucleatum (ATCC 25586) at 1,987 EU/mg/ml. Monocytes subjected to stimulation by diseased cementum particles without laser irradiation produced 124 to 145 pg/ml IL-1beta, 9- to 18-fold higher than that of unstimulated monocytes (7.07 to 15.95 pg/ml). Diseased cementum particles after irradiation with various energy levels of the Nd:YAG laser could still stimulate monocytes to secrete 89 to 129 pg/ml IL-1beta. No statistically significant difference was found in the production of IL-1beta induced by diseased-bound cementum with or without laser irradiation. CONCLUSIONS: The Nd:YAG laser varying from 50 mJ, 10 pps to 150 mJ, 20 pps, for 2 minutes, did not seem to be effective in destroying diseased cementum endotoxin.

Histological differences between teeth with adult periodontitis and prepubertal periodontitis.

Thirty-nine permanent teeth from three siblings aged 11 to 14 years were extracted because of generalized prepubertal periodontitis (PP) and studied histologically with light microscopy. Twenty healthy teeth (HT) removed for orthodontic procedures from 10 children of similar ages and 22 teeth affected by adult periodontitis (AP) extracted from patients over 50 years old were used as controls. The cementum of the PP teeth was much thicker than that of the healthy teeth. A high percentage of resorption, unrelated to inflammation, was found in all the teeth studied. In 24 PP teeth the cementum was infiltrated by microorganisms and bacterial substances. All PP teeth showed a cuticle ranging from 10 to 80 microns in thickness on the diseased cementum. No similar structure was noted on the diseased cementum of the AP teeth. The PP teeth did not show any structural changes which could account for the severity of the periodontal lesions.

Ultrastructure and histochemistry of the dental cuticle in adult periodontitis.

This study examined the dental cuticle (DC) at the interface with cementum surface, as well as its relationship to the overlying subgingival plaque (SP), the so-called plaque-free zone (PFZ), the junctional epithelium (JE), and the coronal fibers of the residual periodontal ligament (PL) by transmission electron microscopy (TEM) and histochemistry. Material comprised of 41 extracted, adult periodontitis-affected teeth (AP). Following extraction, 20 teeth were fixed in 3% glutaraldehyde in 0.1M sodium cacodylate, post-fixed in 1% osmium tetroxide, embedded in araldite, decalcified in EDTA, re-embedded in araldite, and sectioned. En bloc histochemistry was undertaken on the remaining 21 teeth, using ruthenium red, alcian blue-lanthanum nitrate, or safranin-O, and processed as above. Results show that the DC covered the cementum surface from the SP to the JE, and formed an interface with these structures. No DC was observed at the interface with PL. Morphological variations in DC surface were observed at the interface with the SP and at the so-called PFZ where bacteria were always in close contact with or surrounded by the DC. At the interface with JE, the DC appeared homogeneous, although layers varying in electron density were distinguishable. Teeth treated histochemically revealed a positive reaction of DC and bacteria to the three methods, suggesting the presence of anionic polymers including glycoproteins in the DC. It was concluded that on adult periodontitis affected teeth, the DC always covers exposed cementum and may mediate bacterial adhesion, and adsorb components from the periodontal pocket.

The apical border plaque in severe periodontitis. An ultrastructural study.

This study concerns the apical border (AB) plaque in relation to severe forms of periodontitis (SP), including juvenile, post-juvenile, and rapidly progressing periodontitis. Twenty-four (24) teeth from 16 patients with SP were examined by transmission electron microscopy (TEM). The AB was not discrete, with islands of bacteria in the so-called plaque-free zone (PFZ). Coronal to the AB the established plaque consisted of a layer of Gram-positive cocci and ghost cells and a superficial layer mainly of Gram-negative morphotypes, including cocci, rods, filaments, fusiforms, and spirochetes. The most apical apparently intact organisms in the PFZ were in bacterial islands or in isolation and were predominantly Gram-negative cocci and rods, with ghost cells in abundance. Ruthenium red, alcian blue-lanthanum nitrate, and safranin O were used to label matrix polyanionic macromolecules, and periodic acid (thiosemicarbazide) silver proteinate for intracellular polysaccharide (IPS). The matrix components were mainly fibrillar. Many intact bacteria exhibited extracellular polysaccharides or glycocalyces associated with their cell wall, and cytoplasmic IPS granules. The latter varied in distribution and were evident even in the most apically advanced intact microorganisms. The results indicate that IPS and some matrix features of the apical border plaque in severe periodontitis in certain aspects resemble those of sub-contact area plaque on children's teeth, in health or associated with early chronic gingivitis, and with those in chronic adult periodontitis. They also suggest the establishment of acidic regions in the microniche at the bottom of the periodontal pocket in the various forms of periodontitis differing in rate of progression. It was concluded that there was a limited range of intact bacterial morphotypes in the apical border plaque in severe periodontitis, similar to those in chronic adult periodontitis.

Root surface characteristics of primary teeth from children with prepubertal periodontitis.

This study describes the histologic characteristics of root surfaces of primary teeth from children with prepubertal periodontitis (PP). Fifteen primary teeth from 4 children with PP, and 2 control primary teeth from 2 healthy children were examined. Light microscopy revealed normal root surfaces in the control teeth. In contrast, the PP specimens revealed bacteria inside dentin tubules or covering cementum, a cuticle, or resorbed dentin; normal, wider than normal, or hypoplastic cementum; resorption lacunae with various depths; aplastic root resorption; alternate resorption and repair; and active repair. No cementoclasts were found in the resorption lacunae. Scanning electron microscopy revealed intrabony and suprabony root areas, and a "plaque free zone" (PFZ). Colonies of filaments were evident at the cemento-enamel junction (CEJ). The suprabony root surfaces had resorption lacunae, isolated short rods, calculus, colonies of filaments, or colonies composed by an heterogeneous bacterial population. The coronal boundary of the PFZ was the border of a sheet-like structure, which included isolated rods or filaments. At the PFZ, isolated filaments and rods, and a fibril matrix were evident. The apical boundary of the PFZ consisted of bundles of soft tissue remnants or the insertion of the periodontal fibers. The intrabony surfaces were mostly covered by soft tissue, which included isolated filaments and short rods. Resorption lacunae with or without soft tissue were also evident in this area. Crystals of calcium oxalate dihydrate and erythrocytes in distinct forms were found at various root areas. The present findings are different from those previously reported for hypophosphatasia specimens.

In vitro study of the penetration of Streptococcus sanguis and Prevotella intermedia strains into human dentinal tubules.

The persistent presence of bacteria in the root canal system often leads to the failure of treatment. The purpose of this study was to determine in vitro penetration of Streptococcus sanguis and Prevotella intermedia into dentinal tubules. Samples obtained from human teeth were inoculated with a strain of S. sanguis (NCTC 7853) and P. intermedia (NCTC 93336) for 20 days. Bacterial penetration into tubules was investigated at scanning electron microscopy and light microscopic level. The results showed that S. sanguis could penetrate into dentinal tubules 382.3 microns, whereas P. intermedia could penetrate 25.9 microns. It was observed that P. intermedia had not penetrated into all dentinal tubules. If penetration occurred the depth was quite limited.