Effect of a Ca(OH)2 solution and a chlorhexidine based detergent on the microbial activity of human carious teeth

Occlusal carious lesions from human molars, preserved in continuous humidity after extraction, were removed using conventional clinical techniques. Bacteriological samples were taken after rinsing the cavity with water only, after experimentally infecting the cavity and after treating uninfected and infected cavities either with a saturated Ca(OH)₂ solution or with a chlorhexidine based detergent. The samples were cultivated on blood agar plates aerobically and anaerobically. Cavities rinsed with water only showed very sparse bacterial growth. After experimental infection the growth was significant, but decreased radically after treatment with the test materials. In order to describe the effect of the two test materials on the microbial enzyme activity in infected dentin, cryostat sections of 10 μm were prepared from undemineralized carious dentin fragments excavated from freshly extracted human teeth. Conventional histochemical techniques were applied to demonstrate the aminopeptidase activity in the sections using N-L-leucyl-2-naphthylamide as a substrate. The aminopeptidase activity of carious dentin was inhibited totally with the Ca(OH)₂ solution, whereas the chlorhexidine based detergent had no effect on the enzyme activity.     

Tissue-dissolution capacity and dentin-disinfecting potential of calcium hydroxide mixed with irrigating solutions

OBJECTIVE: The goal of this study was to compare the tissue-dissolution potential and antibacterial effectiveness of a conventional Ca(OH)(2)/saline paste with equivalent Ca(OH)(2)/NaOCl and Ca(OH)(2)/chlorhexidine digluconate medications. STUDY DESIGN: Tissue specimens were obtained from freshly dissected pig palates. Tissue pieces of similar form and weight were incubated in air-tight containers with Ca(OH)(2) pastes or solutions proper for up to 7 days. Antimicrobial testing was performed in dentin blocks infected with Enterococcus faecalis. Medicated, sealed dentin specimens were incubated for 1 and 5 days, and bacterial growth was tested at different dentin depths. RESULTS: Up to day 4, the Ca(OH)(2)/irrigating solution mixtures dissolved tissue more effectively than the conventional Ca(OH)(2)/saline paste. After 7 days, however, no statistically significant differences were found between the saline and hypochlorite mixtures, but the Ca(OH)(2)/chlorhexidine medication was significantly less effective. Dentin block disinfection was quicker and more thorough with the Ca(OH)(2)/chlorhexidine or the Ca(OH)(2)/NaOCl than with the Ca(OH)(2)/saline paste. CONCLUSION: Ca(OH)(2)/irrigant mixtures under investigation appear more advantageous than the conventional Ca(OH)(2)/saline mixture, and merit further investigation in a clinical study.     

In vitro effect of cavity disinfectants on the bond strength of dentin bonding systems.

OBJECTIVE: To evaluate the effect of two cavity disinfectants, a 2% chlorhexidine and a 1% benzalkonium chloride solution, on the shear and tensile bond strengths of dentin bonding systems to dentin. METHOD AND MATERIALS: Superficial dentin was exposed from 84 freshly extracted human third molars. The teeth were randomly assigned to two main groups according to the bonding agent used, either One Step or Optibond Solo. Each dentin bonding system had six test groups (three for shear, three for tensile testing), and each test group had a control and two cavity disinfectant groups. In the control groups, dentin bonding systems were applied after etching the dentin, whereas in the cavity disinfectant groups, dentin was conditioned and treated for 20 seconds with the disinfectants before applying the dentin bonding systems. A hybrid resin composite then was applied to all treated samples. After storage in distilled water at 37 degrees C for 24 hours, shear and tensile tests were performed. Data were analyzed using Kruskall-Wallis and Mann-Whitney U tests. RESULTS: The mean shear and tensile bond strengths of One Step and Optibond Solo were not significantly different from each other, and the cavity disinfectants also had no significant effects on shear and tensile bond strength values versus the controls. CONCLUSION: The results indicate that the use of 2% chlorhexidine and 1% benzalkonium chloride solutions as cavity disinfectants after etching the dentin did not affect the shear and tensile bond strengths of One Step and Optibond Solo.     

Calcium hydroxide's association with different vehicles: In vitro action on some dentinal components

OBJECTIVE: Calcium hydroxide, Ca(OH)(2), is a common intracanal medicament. Ca(OH)(2) powder can be mixed with different vehicles and used as a paste for temporary intracanal treatment. The vehicle may influence the dissociation of calcium hydroxide into ions. We sought to evaluate the level of pH and to quantitatively estimate the release of proteins, hydroxyproline, and phosphorus from pieces of radicular dentin kept in different Ca(OH)(2) solutions. STUDY DESIGN: Twenty-eight extracted incisors were maintained for 35 days in Ca(OH)(2) aqueous solutions prepared in chlorhexidine digluconate, propylene glycol (PG), anesthetic solution, camphorated monochlorophenol (CMCP), and CMCP-PG. The control solution contained Ca(OH)(2) without vehicle. RESULTS: The pH values changed little during the experiment. The concentrations of proteins, hydroxyproline, and phosphorus rose for all the solutions under study. Statistical analysis of the data from the control and the experimental groups revealed an increase in the concentration of proteins when chlorhexidine, anesthetic solution, and PG were used; a rise in hydroxyproline levels when CMCP-PG, CMCP, and PG solutions were used; and an increase in phosphorus when PG and chlorhexidine vehicles were used. CONCLUSION: The test solutions with the root dentin remained alkaline. A release of proteins, hydroxyproline, and phosphorus was observed.     

Substantive antimicrobial activity in chlorhexidine-treated human root dentin

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

In vitro antimicrobial activity of various medication preparations on E. faecalis in root canal dentin

The purpose of this study was to evaluate the antimicrobial activity of several medication preparations in root canal dentin infected with Enterococcus faecalis. Roots of extracted bovine incisors were prepared to standardized cylindrical test specimens, 5 mm in height. The smear layer was removed and the samples were autoclaved and then incubated at 37 degrees C/5% CO2 for 24 h in brain-heart infusion (BHI) broth containing 7.0 x 10(4) colony forming units per ml of E. faecalis. The samples were washed in phosphate buffered saline and mounted to individual culture wells with sticky wax. Test medications were applied to fill the canal lumina; medication groups were: (a) sterile H2O (positive control); (b) a 10% mixture of 1.0 g Ca(OH)2 USP in 10 ml sterile H2O; (c) 10% Ca(OH)2 in 0.12% chlorhexidine gluconate (Peridex); (d) Peridex; and (e) uninoculated BHI (negative control). The samples were incubated at 37 degrees C/5% CO2 for 24 h. Dentin samples for quantitative microbiology were then obtained with consecutive sterile burs (ISO 029, 035, 042). All three experimental groups demonstrated significantly greater antimicrobial activity than the positive control (p < 0.001). Group 2 demonstrated significantly greater antimicrobial activity than Group 3 or Group 4 at all dentin depths (p < 0.05). These results suggest that 10% Ca(OH)2 may be more effective than Peridex or 10% Ca(OH)2 in Peridex for the elimination of E. faecalis from dentin tubules.     

Bacteria in experimentally infected cavity preparations.

The presence of bacteria on cavity walls was assessed histologically following experimental infection by inserting soft carious human dentin or guttapercha temporary filings in 40 monkey teeth. Eighty-five infected cavities which had been restored using zinc oxide-eugenol cement (34 teeth), Ca(OH)2 (39 teeth) or amalgam (12 teeth) were also evaluated. Bacteria could regularly be demonstrated in cavities where soft carious human dentin had remained in the cavities for 82 d, but not, or only very rarely, in any of the other series. It was concluded that vital dentin has considerable resistance against infection and that cavity sterilization appears to be of questionable value.     

Application of DIAGNOdent as a guide for removing carious dentin with Er:YAG laser

OBJECTIVES: The purpose of this study was to investigate the DIAGNOdent values applicable as a guide for removal of outer layer of carious dentin with Er:YAG laser. METHODS: Carious dentin of human extracted molars was removed by bur with Caries Detector (Group 1: red stained dentin was left, Group 2: light pink stained dentin was left) and by Er:YAG laser with DIAGNOdent value as a guide (30>Group 3>21, 20>Group 4>11, 10>Group 5). The cavity floor dentin after removing carious dentin was examined using microradiograms, EPMA and SEM. RESULTS: The cavity floor dentin of group 1,3 showed the decreases in the degree of X-ray absorption. In group 2,4 and 5, decrease of Ca, P were not observed. The decrease of Mg was observed on the more superficial part of the cavity floor for group 3 than for group 4. CONCLUSIONS: When carious dentin was removed using Er:YAG laser, the carious dentin outer layer was ideally removed with DIAGNOdent values: 11-20. DIAGNOdent values: 11-20, could indicate the completion of outer layer removal of carious dentin with Er:YAG laser, but there are some problems that must be addressed before the clinical application.     

Bacteria in experimentally infected cavity preparations

ABSTRACT— The presence of bacteria on cavity walls was assessed histologically following experimental infection by inserting soft carious human dentin or guttapercha temporary fillings in 40 monkey teeth. Eighty-five infected cavities which had been restored using zinc oxide-eugenol cement (34 teeth), Ca(OH)₂ (39 teeth) or amalgam (12 teeth) were also evaluated. Bacteria could regularly be demonstrated in cavities where soft carious human dentin had remained in the cavities for 82 d, but not, or only very rarely, in any of the other series. It was concluded that vital dentin has considerable resistance against infection and that cavity sterilization appears to be of questionable value.     

Residual antibacterial activity of chlorhexidine digluconate and camphorated p-monochlorophenol in calcium hydroxide-based root canal dressings

The purpose of this study was to evaluate the residual antibacterial activity of several calcium hydroxide [Ca(OH)2]-based pastes, placed in root canals of dogs' teeth with induced chronic periapical lesions. Root canals were instrumented with the ProFile rotary system and filled with 4 pastes: G1 (n=16): Ca(OH)2 paste + anesthetic solution; G2 (n=20): Calen paste + camphorated p-monochlorophenol (CMCP); G3 (n=18): Calen; and G4 (n=18): Ca(OH)2 paste + 2% chlorhexidine digluconate. After 21 days, the pastes were removed with size 60 K-files and placed on Petri plates with agar inoculated with Micrococcus luteus ATCC 9341. Pastes that were not placed into root canals served as control. After pre-diffusion, incubation and optimization, the inhibition zones of bacterial growth were measured and analyzed by Mann-Whitney U test at 5% significance level. All pastes showed residual antibacterial activity. The control samples had larger halos (p<0.05). The mean residual antibacterial activity halos in G1, G2, G3 and G4 were 7.6; 10.4; 17.7 and 21.4 mm, respectively. The zones of bacterial growth of G4 were significantly larger than those of G1 and G2 (p<0.05). In conclusion, regardless of the vehicle and antiseptic, all Ca(OH)2-based pastes showed different degrees of measurable residual antibacterial activity. Furthermore, unlike CMCP, chlorhexidine increased significantly the antibacterial activity of Ca(OH)2.     

Pronase digestion of carious dentin.

The aim of this study was to investigate the individual capabilities of the proteolytic enzyme preparation Pronase, the enzyme collagenase and sodium hypochlorite to disintegrate and solubilize carious dentin. Samples of carious dentin, and samples of sound dentin for comparison, were extracted 4 times in succession for 24 h with buffered solutions of Pronase. Separate carious dentin samples were extracted in the same manner with buffered solutions of collagenase or with aqueous sodium hypochlorite. The extracts, the solid residues left over after the extractions and untreated samples of carious and sound dentin were digested with sulfuric acid-H(2)O(2) and then analyzed for nitrogen content by a special adaptation of the Berthelot color reaction. Although Pronase did not attack sound dentin, it solubilized more than 90% of the nitrogen present in carious dentin. Collagenase solubilized approximately 66% of the nitrogen, whereas sodium hypochlorite released only 12-20% of the nitrogen of carious dentin. In clinical dentistry, chemical disintegration of carious dentin may reduce the need for mechanical removal of sound tooth structure.     

Physiological recalcification of carious dentin.

In order to confirm the ability of physiological recalcification of the human carious dentin, the first layer of carious dentin was removed from the symmetric cavities of bilateral pairs of human teeth, disclosing it by 0.5% basic fuchsin-propylene glycol solution staining. One of the pair teeth was immediately extracted and the other was left in the mouth after filling the cavity with polycarboxylate cement. The Ca content and hardness of the remaining second layer immediately and three months after the operation were compared by an electron probe microanalyzer and microhardness tester. They increased markedly after three months returning to the normal level from inside, proving physiological recalcification. A similar experiment was performed by using bilateral pairs of dog teeth with cavities having artificially decalcified dentin floor. After removing the fuchsin-stainable first layer, one of the pair was immediately extracted and the other was left in the mouth for three months after exposing or filling the cavity with various cements. As the Ca content was compared, a marked recalcification of the second layer of softened dentin was observed after three months returning to the normal level from the inside. The effect of different cavity treatment was slight.     

Release and diffusion of hydroxyl ion from calcium hydroxide-based medicaments

The release and diffusion of hydroxyl ions (OH(-)) of calcium hydroxide (Ca(OH)(2))-based intracanal medications may be affected by the association with other substances. The aim of this study was to evaluate the diffusion of OH- ions through root dentin by the medications: G1, Ca(OH)(2)/saline; G2, Calen; G3, Calen/camphorated p-monochlorophenol (CMCP); and G4, Calen/0.4% chlorhexidine (CHX). Root canals from bovine teeth were prepared in a standardized manner. A cavity until dentin was prepared in the middle third of the root surface of each specimen. The external surface of the root was made impermeable using a layer of adhesive, except the prepared cavity. The root canals were filled with different medications, and teeth were individually stored in flasks containing 10 ml distilled water at 37°C. The water pH was measured at 1, 3, 7, 14, 21, 30, and 60 days. Data obtained were subjected to anova and Tukey's tests. Increase in pH was observed at 3 days for Calen/CHX and from 7 to 14 days for the other mixtures. Calen paste promoted pH increase up to 21 days. Calen/CMCP had the highest pH up to 21 days, and all groups had similar results at 30 days. At 60 days, the greatest pH values were observed for Calen/CMCP and Calen alone. All different formulations of Ca(OH)(2)-based medications tested release hydroxyl ion that can diffuse through the dentin.     

An in vitro study of affected dentin as a risk factor for the development of secondary caries

The objective of the study was to investigate the association between the presence of residual caries (inner affected dentin) on the cavity walls of cavity preparations and the further development of secondary caries lesions. Two 2 x 5 x 2 mm cavities, one in the apical portion (A) and one in the cervical portion (C) of the root, were prepared on both the lingual (L) and buccal (B) surfaces of 18 extracted human roots. The apical cavities (AB and AL) were artificially demineralized for 30 min using a decalcifying solution, followed by staining with a caries detector dye and then excavation of irreversibly demineralized dentin, leaving behind a layer of inner carious dentin. The cervical cavities (CB and CL) remained intact with sound dentin on the cavity walls. All cavities were then restored with composite resin. Following restoration, lingual specimens (AL and CL) were completely covered by an acid-resistant varnish to prevent further demineralization. All specimens were then incubated in an in vitro microbial artificial mouth model for 3 days in order to develop secondary carious lesions. At the end of the study all specimens were processed for energy-dispersive X-ray analysis of Ca concentration adjacent to the border between dentin and restoration. Statistical analysis of Ca concentrations revealed that the presence of affected inner dentin does not increase the susceptibility to secondary caries. Therefore, it was concluded that conservative cavity preparations leaving behind affected dentin do not increase the risk of secondary caries development.     

Partial caries removal in primary teeth: association of clinical parameters with microbiological status

The relationship between clinical characteristics of carious dentin and bacterial colonization after partial caries removal is not completely understood. The aim of this study was to compare microbial counts between categories of carious dentin color, consistency and humidity, and to evaluate the correlation between these characteristics and the presence of cariogenic microorganisms in deep cavities (2/3 or more of the dentin thickness) submitted to partial caries removal. Sixteen primary teeth were submitted to the removal of all carious tissue from the lateral walls of the cavity, whereas carious tissue of the pulp wall was removed superficially. Dentin in the pulp wall was classified according to color, consistency and humidity immediately after cavity preparation and 3-6 months after cavity sealing and a tissue sample was collected on the same occasion for microbiological evaluation. Before sealing, Streptococcus mutans (p = 0.033) and Lactobacillus spp. (p = 0.048) counts were higher in cavities with humid dentin compared to cavities with dry dentin. A negative correlation was observed between carious dentin consistency and S. mutans count during this phase (r(s) = -0.571; p = 0.020). Arrest of dentinal caries lesions was observed after sealing, which was characterized by a reduction of bacterial counts and changes in dentin color, consistency and humidity, irrespectively of baseline dentin characteristics. The clinical characteristics of carious dentin change after the period of cavity sealing and cannot be applied as absolute indicators to limit the excavation of carious dentin when minimally invasive techniques are used.     

In vitro evaluation of the antimicrobial activity of calcium hydroxide combined with chlorhexidine gel used as intracanal medicament

The aim of this study was to investigate the antimicrobial activity of calcium hydroxide (Ca(OH)2) combined with 2% chlorhexidine gluconate (CHX) gel against endodontic pathogens and to compare the results with the ones achieved by Ca(OH)2 mixed with sterile water and by CHX gel alone. Two methods were used: the agar diffusion test and the direct contact test. Ca(OH)2 + 2% CHX gel produced inhibitory zones ranging from 2.84 to 6.5 mm, and required from 30 seconds to 6 hours to eliminate all tested microorganisms. However, 2% CHX gel showed the largest microbial growth zones from 4.33 to 21.67 mm, and required 1 minute or less to inhibit all tested microorganisms. A paste of Ca(OH)2 plus sterile water inhibited only the microorganisms with which it was in direct contact and required from 30 seconds to 24 hours to kill all tested microorganisms. In conclusion, 2% CHX gel + Ca(OH)2 showed better antimicrobial activity than Ca(OH)2 manipulated with sterile water.     

Control of microorganisms in vitro by endodontic irrigants

The aim of this study was to determine the minimum inhibitory concentration (MIC) and antimicrobial effectiveness by the direct exposure test of 4 endodontic irrigants [1% sodium hypochlorite (NaOCl), 2% chlorhexidine (CHX), 1% calcium hydroxide (Ca(OH)2; prepared with 1 g of Ca(OH)2 and 100 mL of sterile distilled water), a solution of Ca(OH)2 + detergent (HCT20)] for S. aureus, E. faecalis, P. aeruginosa, B. subtilis, C. albicans and a mixed culture. Microbial growth was analyzed by two methods: turbidity of the culture medium that was confirmed by Gram stain and subculture in a specific nutrient broth. In the dilution test, NaOCl solution showed MIC equal to 0.1% for S. aureus, E. faecalis, P. aeruginosa and C. albicans and equal to 1% for B. subtilis and the mixed culture. CHX (2%) presented MIC equal to 0.000002% for S. aureus, 0.02% for E. faecalis, B. subtilis, C. albicans and the mixed culture and 0.002% for P. aeruginosa. Ca(OH)2 solution (1%) showed MIC greater than 1% for all the microorganisms except P. aeruginosa for which it was equal to 1%. Calcium hydroxide solution + detergent showed MIC equal to 4.5 mL for S. aureus, P. aeruginosa, B. subtilis, C. albicans and the mixed culture and greater than 4.5 mL for E. faecalis. In the direct exposure test, NaOCl had better antimicrobial effectiveness for all microorganisms at all times. CHX (2%) was effective for S. aureus, E. faecalis and C. albicans at all times, and ineffective for P. aeruginosa, B. subtilis and the mixed culture. The other solutions showed the worst results.     

Antibacterial activity of resin adhesives, glass ionomer and resin-modified glass ionomer cements and a compomer in contact with dentin caries samples

A total of 103 clinical samples of carious dentin were used to study the antibacterial action of different dental resin adhesive materials (Gluma 2000, Syntac, Prisma Universal Bond 3, Scotchbond Multi-Purpose and Prime&Bond 2.0) glass ionomer cements (Ketac-Cem, Ketac-Bond, Ketac-Silver, Ketac-Fil) resin-modified glass ionomer cements (Fuji II LC, Vitremer and Vitrebond) and a compomer (Dyract). The agar plate diffusion method was used for the microbial cultures and a chlorhexidine control. The growth of the caries-producing microorganisms was effectively inhibited by the Vitremer and Vitrebond cements, and to a lesser extent by the Scotchbond Multi-Purpose adhesive system. Overall, there were statistically significant differences in the antibacterial activity of the products tested.     

Remineralization of carious dentin. I: in vitro microradiographic study in human teeth capped with calcium hydroxide

The objective of this in vitro study was to evaluate a possible remineralization of human carious dentin by calcium hydroxide. Thirty-nine freshly extracted human permanent and deciduous carious teeth were split into two halves. One half was used as control and the other as experimental. In the latter, a cavity was prepared and the remaining bottom layer of demineralized dentin capped with chemically pure calcium hydroxide. The experimental samples were stored at 37 masculine C. Time intervals were 2, 4, 6, 8, 10 and 12 weeks. All tooth halves were sawed and ground to plano-parallel sections with a thickness varying from 75 to 117 micro m. Qualitative microradiographs showed a qualitative increase in radiopacity of the calcium hydroxide treated samples. Quantitative microradiography showed a statistically significant increase in total mineral content in the experimental samples compared to the control samples. These results indicate an in vitro remineralization of carious dentin by calcium hydroxide.     

Histologic demonstration of bacteria subjacent to dental restorations.

Sections of demineralized teeth which had received experimentally inserted restorations using a variety of materials have been stained according to the Brown and Brenn technique. Conventional cavity preparations had been made in intact areas and the cavities were dried gently without additional treatment prior to restoration. Different restorative materials were used and the teeth were extracted after 3-117 days' observation. No bacteria could be demonstrated at the dentin/filling interface or in the dentin if zinc oxide/eugenol, Ca(OH)2 mixed with water or corticonsteroid/antibiotic preparations were employed as base or lining materials. A few bacteria were found subjacent to two of seven unlined amalgam fillings and subjacent to three silicate restorations which had been lined with a polymeric material.