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Chlorhexidine diacetate and povidone-iodine cytotoxicity to canine embryonic fibroblasts and Staphylococcus aureus.

Chlorhexidine diacetate and povidone-iodine were evaluated for fibroblast toxicity on a primary line of canine embryonic fibroblasts, and for bactericidal efficacy against Staphylococcus aureus. The cultured fibroblasts or S. aureus were exposed for 30 minutes to incremental dilutions of 0.5 and 0.0005% chlorhexidine diacetate, 5.0 to 0.05% povidone-iodine, or physiologic buffered saline as a control. To determine survival, fibroblasts were trypsinized and counted; S. aureus colonies were counted on brain-heart infusion agar. Survival for both groups was expressed by calculating the number of living cells in test dilutions as a percentage of the number in control cultures. Fibroblast survival occurred at chlorhexidine concentrations less than 0.013% and at povidone-iodine concentrations less than 0.5% (p less than 0.05). Significant S. aureus survival (p less than 0.05) was noted at chlorhexidine concentrations less than 0.05% and povidone-iodine concentrations less than 1.0%. These data showed that all bactericidal concentrations of chlorhexidine diacetate and povidone-iodine were lethal to canine embryonic fibroblasts in vitro, whereas non-lethal concentrations allowed significant bacterial survival.

Animals↗

Effects of chlorhexidine gluconate and chlorous acid-chlorine dioxide on equine fibroblasts and Staphylococcus aureus.

Equine fibroblasts and Staphylococcus aureus were exposed for 30 minutes to six dilutions of chlorhexidine gluconate, a chlorous acid-chlorine dioxide irrigation solution, a chlorous acid-chlorine dioxide disinfectant, and phosphate buffered saline controls. Cell viability was determined by trypsinizing the cells, staining them with trypan blue, and counting cells that did not take the stain. All fibroblasts were killed when exposed to 1.0% and 0.5% chlorhexidine. The survival rate of fibroblasts increased linearly with decreasing concentrations of chlorhexidine gluconate, with a peak survival of 50% at 0.005% chlorhexidine. The chlorous acid-chlorine dioxide irrigation solution was the least toxic to fibroblasts, with survival rates equivalent to those of controls. The chlorous acid-chlorine dioxide disinfectant was 100% cytotoxic even when diluted 1:1 with phosphate buffered saline. S. aureus growth was inhibited by 1.0% and 0.5% chlorhexidine gluconate; concentrations of 0.05%, 0.01%, and 0.005% did not differ from sterile water controls. The chlorous acid-chlorine dioxide irrigation solution did not inhibit growth of S. aureus in brain-heart infusion broth. The chlorous acid-chlorine dioxide disinfectant inhibited growth of S. aureus.

Animals↗

Comparison of the in vivo and in vitro antibacterial properties of providone iodine and chlorhexidine gluconate mouthrinses.

Clinical and laboratory studies were carried out to compare the antibacterial properties of two antiseptic mouthwashes, namely 1% povidone iodine and 0.2% chlorhexidine gluconate. In a group of 10 subjects after a single rinse with povidone iodine, an immediate mean fall in total salivary aerobes and anaerobes occurred, followed by a return to normal levels by 1-hour postrinsing. With chlorhexidine gluconate a similar but greater reduction in salivary bacterial counts was observed, which was still present up to the 7-h postrinsing period. Saliva samples obtained from the subjects 2 min after rinsing with providone iodine produced little or no inhibition to the growth of a test organism in vitro, whereas following chlorhexidine gluconate, antibacterial activity was present in the saliva specimens up to the 3-h sampling time. Using culture media containing comparable levels of soluble protein to saliva, the minimum inhibitory concentrations of povidone iodine against several standard test organisms were much higher than those of chlorhexidine gluconate. The results suggest that povidone iodine, as a mouthwash, exerts only an immediate antibacterial effect and unlike chlorhexidine, is not retained at antibacterial levels within the oral cavity after expectoration. This lack of prolonged action of povidone iodine in the oral cavity would appear to be relevant to its reported lack of antiplaque activity.

Anti-Infective Agents, Local↗

Effects of subgingival chlorhexidine irrigation of periodontal inflammation.

The purposes of this study were to investigate the effect of direct application of chlorhexidine to periodontal pockets and the practicability of patient self-therapy using a technique of subgingival irrigation. Patients received no other oral hygiene instruction. After initial assessment of parameters, patients were given scaling and polishing and then instruction in the irrigation of designated pockets with chlorhexidine or a placebo using a disposable syringe and blunt needle. During the 28-day irrigation period with chlorhexidine there was highly significant reduction in periodontal inflammation which was maintained at levels significantly below the baseline values for a further 28-day period without irrigation. There was a deterioration in the periodontal state of those patients who had used the placebo, The irrigation technique itself caused no discernible injury in this group of routine periodontal patients. Also, staining in the chlorhexidine group was minimal. It is concluded that subgingival irrigation with chlorhexidine is effective in reducing periodontal inflammation and in controlling subgingival plaque. Intermittent treatment of this kind by the patient at home might reduce to more manageable levels the frequency of hygiene visits and the need for rigorous interdental oral hygiene.

Adult↗

The effect of single morning and evening rinses of chlorhexidine on the development of tooth staining and plaque accumulation. A blind cross-over trial.

A dietary aetiology for tooth staining associated with the use of chlorhexidine has been postulated. The time related activity of chlorhexidine would therefore suggest that evening rinsing would result in less staining than morning rinsing. This study measured tooth and tongue staining and plaque accumulation in a group of 18 volunteers, rinsing once at night or once in the morning with chlorhexidine in a blind cross-over design. Beverage intake during two 10-day periods was prescribed. Significantly more tooth staining, but not tongue staining, was seen with morning rinsing. Plaque accumulation showed a trend to more plaque with morning rinsing, which was not significant. The findings would be consistent with a dietary aetiology for tooth staining. However, these results emphasise the considerable duration of activity of chlorhexidine on surfaces in the oral cavity and the theory of progressive desorption of chlorhexidine from the tooth surface is questioned.

Adult↗

Effect of chlorhexidine rinses on the morphology of early dental plaque formed on plastic film.

The effect of chlorhexidine on early plaque formation was studied in vivo in order to assess potential structural and quantitative changes. 3 subjects with healthy gingiva were used. Plastic films were applied to the buccal surfaces of the maxillary right first premolars and plaque was allowed to form for either 4 or 24 h. A single 1-min rinse with 0.2% chlorhexidine digluconate was used for the 4-h experiments, 1 min after film placement, whereas, for the 24-h experiments, an additional 1 min rinse was used at 12 h. In total, 62 films were obtained, half treated and half controls. These were processed for light and transmission electron microscopy, using standardized techniques for area sampling and cell counting. All plastic films were covered by a surface coating of acellular material in or on which bacteria, epithelial cells and leukocytes were observed. In the controls, an important increase in bacterial count was seen between 4 and 24 h, whereas the increase with chlorhexidine was slight. There were fewer bacteria in the chlorhexidine as compared to the water specimens, for both time periods. Many microorganisms in the chlorhexidine samples appeared to be undergoing degeneration with coagulated cytoplasm and disrupted cell walls.

Chlorhexidine↗

Effect on plaque growth and salivary micro-organisms of amine fluoride-stannous fluoride and chlorhexidine-containing mouthrinses.

Amine fluoride-stannous fluoride mouthrinse was tested in a double-blind cross-over clinical trial on 16 healthy subjects. Chlorhexidine mouthrinse prepared in the same colour and taste was used as the control. After a preparatory phase of 2 weeks, during which the subjects' plaque and gingival indices were reduced to zero by professional cleaning, 1-week test periods without mechanical cleaning were started in random order. The fluoride test solution did not prevent plaque accumulation and growth of salivary S. mutans and lactobacilli as did chlorhexidine. Furthermore, the plaque wet weight was statistically highly significantly greater during test periods of fluoride rinsing than during the chlorhexidine phase. The cleansing effect, as subjectively estimated by questionnaire, was in favour of chlorhexidine, but discolorations of teeth were assessed as significantly worse during chlorhexidine periods. Neither of the test solutions showed any effect on salivary flow rate, pH, buffering capacity, or lysosomal enzyme activity.

Adult↗

The in vitro effects of chlorhexidine on subgingival plaque bacteria.

The purpose of this study was to determine the susceptibility to chlorhexidine of a range of bacteria which may be isolated from subgingival plaque. In addition, the effect of chlorhexidine on the survival of bacteria in subgingival plaque samples from patients with chronic inflammatory periodontal disease was investigated. The minimum inhibitory concentration (MIC) of chlorhexidine for 52 strains of bacteria ranged from 8 to 500 micrograms/ml. The modal value of the MIC was found to be 62 micrograms/ml, 64% of the strains tested being inhibited at this concentration. A concentration of 250 micrograms/ml of chlorhexidine inhibited the growth of all bacteria in the 25 subgingival plaque samples investigated. The MIC of chlorhexidine for the samples ranged from 31 to 250 micrograms/ml, the modal value being 125 micrograms/ml.

Adult↗

The use of an oxidising mouthwash to reduce staining associated with chlorhexidine. Studies in vitro and in vivo.

Discolouration of the teeth, tongue and dental restorative materials associated with the use of cationic antiseptics is still the limiting factor in long-term usage of antiplaque agents such as chlorhexidine. Oxidising agents have been used successfully to remove dental stain in animals, but products and regimens have not been evaluated in humans. These pilot studies in vitro and in vivo assessed a long-established oxidising mouthwash, based on peroxyborate, for stain removal from teeth and acrylic. The laboratory studies measured chlorhexidine tea stain removal from tooth and acrylic specimens. The oxidising agent markedly reduced staining on specimens. In a short-term clinical experiment, dental staining was forced over a 2 1/2-day period by reciprocal chlorhexidine and tea rinsing. 5 rinses with the oxidising mouthwash during the next 1 1/2 days dramatically reduced staining compared with water after rinses. Additionally, a single rinse with peroxyborate following the 5 water after rinses, again considerably reduced the residual staining. Effects on tongue staining were less impressive. Both experimental models produced findings consistent with those from the animal model. The results suggest proprietary oxidising mouthwashes could have a place in the control of dental stain associated with the use of chlorhexidine. Further studies to evaluate such products in a more conventional chlorhexidine regimen are indicated.

Adult↗

Effects of chlorhexidine on proteolytic and glycosidic enzyme activities of dental plaque bacteria.

Chlorhexidine was tested for its ability to inhibit a wide range of glycosidic and proteolytic enzyme activities produced by Treponema denticola, Porphyromonas gingivalis, Bacteroides intermedius, Actinobacillus actinomycemcomitans, Capnocytophaga sputigena, Capnocytophaga gingivalis, Capnocytophaga orchracea, Capnocytophaga sp., Actinomyces viscosus, Streptococcus mitior, Streptococcus mutans, Streptococcus sobrinus, Streptococcus mitis, Streptococcus anginosus, Streptococcus oralis and Streptococcus sanguis. The enzymes produced by Capnocytophaga spp. were the most resistant to inhibition by chlorhexidine while the hydrolysis of proteolytic substrates by all the other species was markedly susceptible to inhibition with less than 0.125 mM chlorhexidine inhibiting enzyme activities by greater than or equal to 50%. Glycosidase activities, of all species, were generally more resistant to inhibition, especially neuraminidase activity. Chlorhexidine at less than 0.032 mM inhibited the degradation of bovine serum albumin by suspensions of dental plaque bacteria. These observations support an hypothesis that chlorhexidine exerts a bacteristatic effect in vivo, in part, by reducing the ability of dental plaque bacteria to degrade host-derived proteins and glycoproteins which normally provide essential nutrients for growth.

Acetylglucosaminidase↗

A comparison of cetylpyridinium chloride, triclosan and chlorhexidine mouthrinse formulations for effects on plaque regrowth.

A relatively small number of agents are used in mouthrinse products, although the possible variability in the final formulations is enormous. The aim of this study was to compare equal concentrations of 3 antimicrobial agents, in simple formulations, for plaque inhibition. This 4-day plaque regrowth study was a 5-cell, randomised, double blind cross-over design, involving 20 healthy human volunteers. The mouthrinse formulations were aqueous 0.05% solutions of cetylpyridinium chloride (CPC), chlorhexidine and triclosan, together with a 0.1% CPC and a minus active control rinse. On Day 1, from a zero plaque baseline, volunteers ceased normal oral hygiene and rinsed 2x daily for 1 min. with 10-ml volumes of the allocated rinses. On Day 5, plaque was scored by index and area. All rinses produced lower mean plaque values compared to control, but unlike the CPC and chlorhexidine rinses, the differences with triclosan did not always reach significance. The CPC and chlorhexidine rinses were always significantly more effective than the triclosan rinse. The greatest plaque inhibition was with 0.1% CPC although rarely significantly greater than the 0.05% CPC and chlorhexidine rinses which were similar in efficacy. The results indicate that further studies on lower concentration chlorhexidine solutions are warranted.

Adult↗

Bacterial penetration in vitro through GTAM membrane with and without topical chlorhexidine application. A light and scanning electron microscopic study.

Premature exposure of membrane in the oral cavity is considered the most common complication as well as a reason for failure or incomplete success of guided tissue regeneration, as the exposed membrane undergoes plaque accumulation. A method to control, or at least to reduce the bacterial invasion of the membrane allowing a delay in the membrane removal, could be of clinical interest. The purpose of the present study was to evaluate the possibility of treating, with topical chlorhexidine application, the bacterial colonization of exposed membranes. A special device was developed in order to evaluate, under an experimental environment, the bacterial colonization. This device was made from a removable acrylic denture base to which 5 gold cups were attached. The cups consisted of an internal compartment, isolated from the oral cavity by a GTAM membrane, and an external compartment exposed to the oral environment. 3 healthy subjects had this device fitted, bilaterally, in the molar-premolar region of the upper jaw. The cups of one side of each subject had 0.2% chlorhexidine gluconate gel applied 2x a day for 1 min, whereas those of the other side served as controls. 12 cups were removed after 2 weeks and the remainder removed after 1 month. The non-treated control specimens were characterized by greater amounts of plaque accumulation. In all the test sites, plaque deposits increased in thickness and quantity during the 4 weeks of the experiment. Complete invasion of the membrane and initial colonization of its internal surface were observed only in 4-week specimens. A relatively simple flora consisting mainly of cocci and short rods, was found in bacterial deposits forming under the influence of chlorhexidine, whereas in non-treated specimens, it was possible to observe a more mature and complex plaque, composed mostly of filamentous bacteria. In conclusion, topical application of chlorhexidine gel is an effective method of reducing plaque and calculus formation on the surface of GTAM membranes exposed to the oral cavity for up to 4 weeks. This study has, however, failed to demonstrate the capacity of chlorhexidine to prevent or to retard bacterial penetration through the thickness of the inner portion of the membranes.

Acrylic Resins↗

A short-term clinical study design to investigate the chemical plaque inhibitory properties of mouthrinses when used as adjuncts to toothpastes: applied to chlorhexidine.

The removal of plaque by toothbrushing with toothpaste is the most common form of plaque control in the developed world. However, the use of chemical adjuncts such as mouthrinses is increasing. In practice mouthrinses and toothpaste are used together, however, in many clinical trials, employed to assess mouthrinse activity, toothpaste use is suspended. This fails to measure the effect of chemical interactions which are known to occur between toothpaste ingredients and mouthrinses. The objective of this trial was to develop a methodology which would assess the adjunctive chemical plaque inhibitory action of mouthrinses, when used with toothpaste but without the indeterminate variable of toothbrushing. The study was a single blind, randomised, 7-way crossover design, based on a variation of a 4 day plaque regrowth model. The 2 x daily rinsing regimens produced increasing plaque scores in the following order: (1) water/chlorhexidine, (2) chlorhexidine/water, (3) chlorhexidine/toothpaste slurry, (4) toothpaste slurry/chlorhexidine, (5) water/toothpaste slurry, (6) toothpaste slurry/water, (7) water/water. Chlorhexidine and water or chlorhexidine and toothpaste slurry combinations produced significantly lower plaque scores than water alone. Slurry and water combinations resulted in less plaque than water alone, but differences were not significant. Toothpaste slurry and chlorhexidine produced significantly increased plaque scores compared to chlorhexidine and water. The study suggests that, outside the Hawthorne effect, chlorhexidine rinses would be less effective in reducing plaque when used with toothpaste than when used alone. The methodology could be employed as a screening tool for the evaluation of mouthrinses expected to be used as adjuncts to normal oral hygiene methods. The same could be used to optimise oral hygiene regimens which include the use of mouthrinses.

Adult↗

The propensity of different brands of tea and coffee to cause staining associated with chlorhexidine.

Dental staining is a well known and probably the most problematic side effect of using chlorhexidine oral products. Whatever mechanisms are involved, there is no doubt that cationic antiseptics, such as chlorhexidine, can precipitate or bind to surfaces anionic chromogens contained in foods and beverages. The aim of this study in vitro was to determine whether, under controlled conditions, different brands of tea or coffee varied in their propensity to cause staining associated with chlorhexidine. Optically clear acrylic specimens were cycled through saliva, chlorhexidine and different tea and coffee solutions. Staining was measured using a spectrophotometer. After 15 cycles, it was apparent that staining varied at the extreme, both within and between the tea and coffee groups. All coffee brands produced less staining than the tea brands. The least staining coffee and least staining tea brands were approximately 3x less chromogenic than the most staining equivalent beverage. Previous randomised controlled clinical trials have indicated that tea and coffee contribute to dental and tongue staining associated with chlorhexidine mouthrinses. Additionally, abstinence from tea and coffee significantly reduces staining. The results of this study in vitro suggest that when abstinence is difficult, tea and coffee brands of low chromogenicity may be recommended. Clearly these data in vitro require validation in vivo.

Analysis of Variance↗

6-month use of 0.2% delmopinol hydrochloride in comparison with 0.2% chlorhexidine digluconate and placebo (II). Effect on plaque and salivary microflora.

This double-blind, randomised, 6-month clinical trial with parallel group design in 68 subjects with gingivitis was conducted to study the effects on the oral flora of delmopinol hydrochloride 2 mg/ml (0.2% w/v, Decapinol Mouthwash), when used for partly supervised mouthrinsing in comparison with placebo and chlorhexidine digluconate 2 mg/ml (0.2% w/v, Hibitane Dental, ICI Pharmaceuticals, UK). Apart from estimating the total cultivable microbial dental plaque flora and salivary flora, analyses were focused on bacterial groups associated with gingivitis/periodontitis and dental caries. Furthermore, the presence of staphylococci, gram-negative enteric bacteria and yeasts in saliva were evaluated. The minimal inhibitory concentration (MIC) was determined for isolates belonging to the predominating micro-organisms in samples of both dental plaque and saliva. In relation to the findings in the placebo group, the use of delmopinol during the rinsing period did not produce an undesirable shift in the bacterial populations considered to be related to dental caries or periodontal diseases. These groups remained virtually unchanged during the study. In relation to the observations in the placebo group, slight reductions in the total cultivable plaque and salivary flora were observed during the study and no change was found in the ratio total anaerobically/aerobically cultivable microbial flora. Furthermore, no increased growth in staphylococci, enteric bacteria or yeasts was observed in the saliva samples. The pattern of changes taking place in the composition of the plaque and salivary microbial flora in samples from the participants rinsing with chlorhexidine were in most aspects similar to that observed in the delmopinol group. In the delmopinol group, no microbiologically significant changes were observed over time in the MIC-values for the isolates, neither in the plaque nor in the saliva samples, which indicates that no adaptation to delmopinol had taken place during the rinsing period. Similar observations were made for the plaque isolates in samples from the participants in the chlorhexidine group. On the other hand, when gram-positive and catalase-negative cocci from the saliva samples of the latter group were tested against chlorhexidine, 4-6 times higher MIC-values were obtained at 3 and 6 months both when compared to baseline and in comparison with the other two rinsing groups (p<0.01 or p<0.05). Neither delmopinol nor chlorhexidine showed any residual effect on the studied microbial groups in the plaque and the saliva samples 3 months after the end of treatment. In conclusion, delmopinol was accompanied by a composition of the plaque and salivary flora associated with healthy conditions in the oral cavity.

Bacteria↗

Differences in efficacy of two commercial 0.2% chlorhexidine mouthrinse solutions: a 4-day plaque re-growth study.

BACKGROUND: The purpose of this clinical cross-over study was to examine the antibacterial and plaque-inhibiting properties of two chlorhexidine solutions compared with a negative control. MATERIAL AND METHODS: Twenty-one volunteers refrained from all oral hygiene measures, but rinsed instead twice daily with 10 ml of a conventional chlorhexidine solution (0.2%; CHX), a chlorhexidine solution with anti-discolouration system (ADS) (0.2%, alcohol-free chlorhexidine solution (CSP)) or a placebo solution (Pla). Plaque index (PI), plaque area (PA) and bacterial vitality were assessed after 24 h (PI1, vital flora (VF)1) and 96 h (PI2; VF2, PA). After a 10-day wash-out period, a new test cycle was started. RESULTS: Results for Pla were 0.94, 1.59, 27.4 (PI1, PI2, PA) and 79% and 72% (VF1 and VF2). CSP significantly reduced the parameter PI1, PI2 and PA to 0.67 (p=0.012), 1.0 and 15.7 (p<0.001). VF1 and VF2 (63% and 53%) were not significantly affected. The corresponding figures of CHX were 0.42, 0.43, 6.77, 33 and 16%, which were all significantly lower (all p<0.001). On comparing the two chlorhexidine solutions, CHX showed significantly higher reductions of all parameters. CONCLUSION: The results suggest that the 0.2% alcohol-containing solution showed superiority in inhibiting plaque re-growth and reducing bacterial vitality compared with the solution with ADS.

Adult↗

Symptoms of immediate chlorhexidine hypersensitivity in patients with a positive prick test.

There are numerous reports of anaphylaxis from chlorhexidine in surgical operations and other medical procedures, usually due to its application to wounds or mucous membranes. We wanted to analyse the clinical data of patients with a positive chlorhexidine prick test and perform some additional testing. We studied the case records of the patients with a positive chlorhexidine prick test and performed an open application test and tests for specific IgE. We found 33 patients with a positive prick test. 10 of them had had severe symptoms from chlorhexidine, and 11 had had only mild local symptoms. The size of the prick test reaction was mainly in line with the strength of the severest symptoms. Small 3- to 4-mm reactions were usually without obvious clinical relevance. Specific IgE could be demonstrated in 6 patients out of 14 tested by the ImmunoCAP method. Besides severe attacks, patients with a positive prick test often have milder local symptoms, such as exacerbation of dermatitis. Local symptoms from chlorhexidine-containing products may precede severe attacks. We recommend a prick test to be performed routinely when symptoms during medical interventional procedures, e.g. local and general anaesthesia, are investigated.

Adolescent↗

Effect of toothbrushing with chlorhexidine gel on salivary microflora, oral hygiene, and caries.

The effect of daily toothbrushing with 0.5% chlorhexidine-containing gel for 12 months was evaluated in a double-blind study in 37 dental students. The active gel did not markedly influence plaque formation, gingival conditions, or caries as compared with placebo gel treatment. Salivary bacterial counts were performed on subgroups of six subjects using chlorhexidine gel and on six using placebo gel. No differences in the effect of treatment on the microorganisms studied in the two subgroups could be detected except for S. sanguis. The percentage of this species decrease in the placebo group and increased in the chlorhexidine group. The difference became significant after 2 weeks. A tendency to a greater reduction of S. mutans noted in the chlorhexidine group was most marked in individuals who had high initial counts of this species. The proportion of S. sanguis, which could grow on chlorhexidinei-containing mitis salivarius medium, increased and after 12 months of chlorhexidine treatment averaged 34% of cultivable S. sanguis compared with 0.002% prior to treatment. The number of less sensitive S. sanguis decreased in the 12 months following termination of treatment. There was no observed tendency for the selection or proliferation of other streptococci, gram-negative rods, yeasts, or staphylococci.

Adult↗