Direct and Indirect Effect of Chlorhexidine on Survival of Stem Cells from the Apical Papilla and Its Neutralization.

INTRODUCTION: Several irrigants have been used for disinfection in regenerative endodontic procedures including chlorhexidine (CHX). In this context, the antibacterial properties of disinfectants are mainly in focus of research even though they may have an undesirable impact on the fate of stem cells. In this study, we hypothesized that CHX has both a direct effect when applied to stem cells of the apical papilla (SCAPs) and an indirect effect when SCAPs are exposed to dentin previously conditioned with CHX. METHODS: Cell toxicity was evaluated in vitro using the CellTox green fluorescence assay (Promega, Madison, WI) and CellTiter-Glo (Promega) after SCAPs were exposed directly to a dynamic concentration range of CHX; apical papilla explant cultures were stained with ApopTag (Merck Millipore, Billerica, MA) after culture with CHX. Furthermore, standardized slabs from human dentin were treated with CHX and consecutively rinsed in EDTA, L-α-lecithin (Sigma-Aldrich, St Louis, MO), or L-α-lecithin followed by EDTA. After that, SCAPs were cultured on the slabs for 5 days, and cellular viability was determined (indirect effect). Data were treated nonparametrically and analyzed using the Krukal-Wallis test (P ≤ .05). RESULTS: Direct exposure of SCAPs to CHX highly affected cell viability at concentrations above 10-3%, whereas lower concentrations had no adverse effect. During the initial 60 minutes, concentrations of 10-2% CHX or higher resulted in early pronounced toxicity with a maximum effect within 15 minutes after exposure. Likewise, CHX-conditioned dentin slabs were detrimental to SCAP survival; however, the deleterious effects were completely reversed by neutralization with L-α-lecithin. CONCLUSIONS: Chlorhexidine is toxic to SCAPs when applied directly or indirectly via conditioned dentin. If applied for a short time and neutralized by L-α-lecithin, it can be a gentle and cell-preserving disinfectant before endodontic regeneration.

Effect of dentin powder on the antimicrobial properties of hyperpure chlorine-dioxide and its comparison to conventional endodontic disinfecting agents.

INTRODUCTION: Previously we found that the high purity chlorine-dioxide(ClO2) has a very potent disinfectant efficacy on oral pathogenic microorganisms and as a root canal irrigant it is able to eliminate the experimental Enterococcus faecalis(E. faecalis) infection from the root canal system. This study examines whether the presence of dentin powder influences the antibacterial efficacy of ClO 2. METHODS: In an in vitro dentin powder model the following irrigants were tested against planktonic E. faecalis: 2% chlorhexidine (CHX), 2.5% sodium hypochlorite (NaOCl), 0.12%ClO2 (Solumium) and one local root canal medicament: saturated Ca(OH)2. Survival of bacteria exposed to agents without and with human dentin powder or preincubated with dentin powder was investigated. The effect of the dentin powder on ClO2 concentration was investigated by titrations. RESULTS: Without dentin powder ClO 2 killed all E. faecalis and delivered the best result already after 1 minute; however, after longer contact time with dentin the difference between the disinfectants disappeared. The presence of dentin powder decreased the concentration of ClO 2 and attenuated the antibacterial efficiency of ClO2 and Ca(OH)2, but did not decrease of CHX and NaOCl.Preincubation with dentin powder caused significant loss of antibacterial activity of all investigated agents, ClO2 and Ca(OH)2 having the highest reduction. CONCLUSION: As the presence of dentin powder had a negative effect on the efficacy of disinfectants, the importance of elimination of dentin scrapings and smear layer from the root canal system during endodontic treatments is highly recommended. ClO 2 can be effective for a final rinse.

Dentin inhibits the antibacterial effect of new and conventional endodontic irrigants.

INTRODUCTION: This in vitro study aimed to compare the antibacterial effect of a new irrigant, QMiX, with that of conventional irrigation solutions in the presence or absence of dentin powder. METHODS: Dentin powder was prepared from bovine incisors and sterilized. The following irrigants were tested against Enterococcus faecalis (ATCC 29212): 6% sodium hypochlorite (NaOCl), 1% NaOCl, QMiX, 2% chlorhexidine gluconate (CHX), and 17% EDTA. Sterilized saline solution was used as negative control. Survival of bacteria exposed to the irrigants in the presence or absence of dentin was monitored under planktonic conditions. Colony-forming units were counted, and log-transformed numbers were analyzed by using Kruskal-Wallis and Mann-Whitney tests. P values less than .05 were considered significant. RESULTS: In the absence of dentin, after 10 seconds of contact with the bacterial suspension, 6% NaOCl showed the lowest bacterial count; the difference to the negative control was significant. After 30 seconds, 6% NaOCl displayed 0 colony-forming units per milliliter, whereas 1% NaOCl and QMiX showed reduced number of colonies in comparison with the negative control. After 1 minute, both concentrations of NaOCl presented no bacterial growth and QMiX reduced the number of colonies, but EDTA and CHX had bacterial counts similar to the negative control. Dentin had a significant inhibitory effect on 6% NaOCl (10 seconds), 1% NaOCl (10 seconds and 1 minute), and QMiX (10 seconds and 1 minute). After 6 hours, both concentrations of NaOCl, QMiX, and CHX killed all bacteria, regardless of the presence of dentin. CONCLUSIONS: Six percent NaOCl was the most effective irrigant against E. faecalis. Saline and EDTA had no measurable antibacterial effect. Dentin delayed the antibacterial activity of NaOCl and QMiX but did not completely prevent their action.

Pitfalls in efficacy testing--how important is the validation of neutralization of chlorhexidine digluconate?

BACKGROUND: Effective neutralization of active agents is essential to obtain valid efficacy results, especially when non-volatile active agents like chlorhexidine digluconate (CHG) are tested. The aim of this study was to determine an effective and non-toxic neutralizing mixture for a propan-1-ol solution containing 2% CHG. METHODS: Experiments were carried out according to ASTM E 1054-02. The neutralization capacity was tested separately with five challenge microorganisms in suspension, and with a rayon swab carrier. Either 0.5 mL of the antiseptic solution (suspension test) or a saturated swab with the antiseptic solution (carrier test) was added to tryptic soy broth containing neutralizing agents. After the samples were mixed, aliquots were spread immediately and after 3 h of storage at 2 - 8 degrees C onto tryptic soy agar containing a neutralizing mixture. RESULTS: The neutralizer was, however, not consistently effective in the suspension test. Immediate spread yielded a valid neutralization with Staphylococcus aureus, Staphylococcus epidermidis and Corynebacterium jeikeium but not with Micrococcus luteus (p < 0.001) and Candida albicans (p < 0.001). A 3-h storage period of the neutralized active agents in suspension resulted in significant carry-over activity of CHG in addition against Staphylococcus epidermidis (p < 0.001) and Corynebacterium jeikeium (p = 0.044). In the carrier test, the neutralizing mixture was found to be effective and non toxic to all challenge microorganisms when spread immediately. However, after 3 h storage of the neutralized active agents significant carry-over activity of CHG against Micrococcus luteus (p = 0.004; Tukey HSD) was observed. CONCLUSION: Without effective neutralization in the sampling fluid, non-volatile active ingredients will continue to reduce the number of surviving microorganisms after antiseptic treatment even if the sampling fluid is kept cold straight after testing. This can result in false-positive antiseptic efficacy data. Attention should be paid during the neutralization validation process to the amount of antiseptic solution, the storage time and to the choice of appropriate and sensitive microorganisms.

Thymosin beta4 is cytoprotective in human gingival fibroblasts.

Thymosin beta4 (Tbeta(4)) is a naturally occurring, ubiquitous, non-toxic protein with documented wound-healing, anti-inflammatory, anti-apoptotic, and tissue-repair properties in skin, the ocular surface, and the heart. The ability of Tbeta(4) to demonstrate similar protective properties in cells of the oral cavity was analyzed using an in vitro model of cultured human gingival fibroblasts. Thymosin beta 4 significantly suppressed the secretion of interleukin-8 (IL-8) following stimulation with tumor necrosis factoralpha (TNF-alpha), suggesting that it may suppress the inflammatory response initiated by pro-inflammatory cytokines. By contrast, Tbeta(4) was not effective in protecting fibroblasts from challenge with lipopolysaccharide purified from Porphyromonas gingivalis or Escherichia coli. Thymosin beta 4 was able to protect gingival fibroblasts against the known cytotoxic effects of chlorhexidine digluconate, a mouthrinse containing chlorhexidine digluconate, and carbamide peroxide. Additionally, Tbeta(4) was able to protect gingival fibroblasts from the apoptosis that is induced by stimulation with TNF-alpha or by exposure to chlorhexidine. Because of its multifunctional roles in protecting cells against damage, Tbeta(4) may have significant potential for use as an oral heathcare aid with combined antimicrobial, anti-inflammatory, anti-apoptotic, and cytoprotective properties.

Effect of a neutralising agent on the evaluation of the antimicrobial activity of chlorhexidine on the bacterial salivary flora.

OBJECTIVE: To test the efficacy of a previously described neutralising agent to counteract any antimicrobial activity of 0.2% of chlorhexidine (CHX) mouthrinse on the salivary flora, which is only exhibited after sampling of surviving bacteria, resulting in false positive efficacy data. METHODS: Unstimulated salivary samples were collected of 20 volunteers under basal conditions and at 30s and 1h after of a single mouthrinse of 0.2% CHX. Each salivary sample was divided into 2 equal aliquots; one was mixed with neutralising agent (3% Tween 80, 0.3% lecithin and 0.1% cysteine) and the other with a control solution. The colony forming units (cfu/mL) were determined and expressed as logarithms (log(10)cfu/mL). RESULTS: At baseline, the total bacterial concentrations were similar, independently of the addition of neutralising solution or control solution (8.419+/-0.346log(10)cfu/mL and 8.462+/-0.474log(10)cfu/mL, respectively, p=0.440). At 30s performing the CHX mouthrinse, the bacterial load reduction was statistically significant between both sampling methods (1.917+/-1.275log(10)cfu/mL, p<0.001). One hour after performing the CHX mouthrinse, the bacterial load reduction was statistically significant between both sampling methods (0.537+/-0.706log(10)cfu/mL, p=0.003). CONCLUSIONS: Neutralising agent was not toxic to the bacterial salivary flora and effectively deactivated the "residual antimicrobial activity" of the 0.2% CHX (after exposure and during processing of samples). We propose the use of this neutralising agent when evaluating the antibacterial activity of CHX mouthrinses.

Inactivation of the antibacterial activity of iodine potassium iodide and chlorhexidine digluconate against Enterococcus faecalis by dentin, dentin matrix, type-I collagen, and heat-killed microbial whole cells.

The antibacterial activity of chlorhexidine digluconate and iodine potassium iodide on Enterococcus faecalis A197A was tested in the presence of dentin, dentin matrix, dentin pretreated by EDTA and citric acid, collagen, and heat-killed cells of Enterococcus faecalis and Candida albicans. Medications were preincubated for 1 h with each of the potential inhibitors and tested for their antibacterial activity against E. faecalis, strain A197A. Surviving bacteria were sampled after 1 and 24 h of incubation. Dentin matrix and heat-killed microbial cells were the most effective inhibitors of chlorhexidine, whereas dentin pretreated by citric acid or EDTA showed only slight inhibition. Dentin and skin collagen showed some inhibition at 1 h but not after 24 h. Iodine potassium iodide was effectively inhibited by dentin, dentin matrix, and heat-killed microbial cells. Skin collagen and dentin pretreated by EDTA or by citric acid showed little or no inhibitory effect on iodine potassium iodide. Different components of dentin are responsible for the divergent patterns of inhibition of the antibacterial activity of chlorhexidine digluconate and iodine potassium iodide. Chemical treatment of dentin before applying the medication into the root canal may alter the antibacterial effect of the medication.

An effective method of inactivating chlorhexidine.

OBJECTIVE: The purpose of this study was to find an effective inactivating agent for chlorhexidine that would facilitate removal of all residual antimicrobial effect, which may cause false-negative results during microbiologic culturing. STUDY DESIGN: L-alpha-lecithin, Tween 80, and sodium thiosulfate were used in different proportions to prepare 6 potential inactivating solutions. Nine mL of each inactivating solution was mixed with 1 mL of 2% chlorhexidine solution. After 5 minutes of equilibration, 0.1 mL of bacterial cell suspension containing 2 x 10(4) viable cell of Enterococcus faecalis was added to the mixture. At 10 and 60 minutes, 0.1-mL aliquots were withdrawn and spread over blood agar plates and incubated at 37 degrees C for 72 hours. The number of colony-forming units on the blood agar plates was determined and recorded. RESULTS: The combination of 3% Tween 80 and 0.3% L-alpha-lecithin was found to be the most effective inactivating agent, allowing full recovery of the test organisms in the presence of chlorhexidine. CONCLUSION: The present study demonstrated a method to predictably inactivate chlorhexidine.

The effect of toothpaste on the propensity of chlorhexidine and cetyl pyridinium chloride to produce staining in vitro: a possible predictor of inactivation.

BACKGROUND/AIMS: Cationic antiseptics such as chlorhexidine (CHX) and cetyl pyridinium chloride (CPC) interact with dietary chromogens to produce extrinsic stain, and this can be used as a measure of activity of products. The aim of these studies in vitro was to determine if toothpaste influenced the tea staining effects of CHX and CPC as a predictor of action in vivo. METHOD: Clear acrylic specimens were soaked in pooled human saliva followed by sequential 2-min soaks in pairs of agents, namely 0.05% CHX, 0.05% CPC, 0.2% CHX, water (W) and toothpaste slurry (TP). The combinations were; TP/CHX, CHX/TP, TP/CPC, CPC/TP, W/CHX, CHX/W, W/CPC, CPC/W, TP/W, W/TP, W/W. These treatments were followed by a 60 min soak in tea. Optical density readings were taken at baseline and after each of 8 cycles. RESULTS: In the separate CHX and CPC studies by comparison with W/CHX, TP/0.05% CHX had little effect on staining, but TP/0.2% CHX showed a reduction in staining of 18%. 0.05% CHX/TP reduced staining by >40%, and 0.2% CHX by >78%. TP/CPC reduced staining by >26% and CPC/TP by 80%. Water after 0.2% CHX, 0.05% CHX and CPC reduced staining by 18%, 13% and 17% respectively. Little staining was seen with TP and W combinations. The data for CHX are in agreement with a study in vivo except TP followed by CHX reduced the activity of CHX. CONCLUSION: Toothpaste appears to adversely affect the activity of CHX and CPC particularly if used immediately after the antiseptics. The data further supports the concept of separating the use of antiseptics until sometime after the use of toothpaste, and the idea of developing mouthwash friendly toothpastes.

Inactivation of root canal medicaments by dentine, hydroxylapatite and bovine serum albumin.

AIM: This study examined and compared the inhibition of the antibacterial effect of saturated calcium hydroxide solution, chlorhexidine acetate and iodine potassium iodide by dentine, hydroxylapatite and bovine serum albumin. METHODOLOGY: Enterococcus faecalis strain A197A prepared to a suspension of 3 x 10(8) cells per ml in 0.5% peptone water was used. Fifty microL of saturated calcium hydroxide solution, 0.05% chlorhexidine acetate or 0.2/0.4% iodine potassium iodide were incubated at 37 degrees C with 28 mg dentine powder (DP), hydroxylapatite (HA) or bovine serum albumin (BSA) in 50 microL water for 1 h before adding 50 microL of the bacterial suspension. Samples for bacterial culturing were taken from the suspension 1 and 24 h after adding the bacteria. In further experiments, the amount of dentine was stepwise reduced from 28 mg 150 microL-1 to 2.8 mg 150 microL-1. RESULTS: Calcium hydroxide was totally inactivated by the presence of 28 mg of DP, HA or BSA. Chlorhexidine (0.05%) was strongly inhibited by BSA and slowed down by dentine. However, HA had little or no inhibitory effect on chlorhexidine. The antibacterial effect of 0.2/0.4% iodine potassium iodide on E. faecalis was totally inhibited by dentine (28 mg), but was practically unaffected by HA or BSA. A stepwise reduction of dentine from 28 mg 150 microL-1 to 2.8 mg 150 microL-1 was followed by a similar reduction of the inhibition of the antibacterial activity of chlorhexidine. Iodine potassium iodide was not inhibited at all with dentine amounts less than 28 mg. However, the effect of saturated calcium hydroxide solution was totally eliminated by dentine, in all four concentrations. CONCLUSION: Inhibition by dentine of the antibacterial activity of calcium hydroxide, chlorhexidine and iodine potassium iodide occurs by different mechanisms. Different components of dentine may be responsible for the inhibition of these three medicaments. Calcium hydroxide was particularly sensitive to inhibition by both inorganic and organic compounds.

Inactivation of local root canal medicaments by dentine: an in vitro study.

AIMS: The aim of the study was to investigate the inactivation by dentine of the antibacterial activity of various commonly used local root canal medicaments. METHODOLOGY: The medicaments tested were saturated calcium hydroxide solution, 1% sodium hypochlorite, 0.5% and 0.05% chlorhexidine acetate, and 2/4% and 0.2/0.4% iodine potassium iodide. Dentine was sterilized by autoclaving and crushed into powder with a particle size of 0.2-20 microns. Aliquots of dentine suspension were incubated with the medicaments in sealed test tubes at 37 degrees C for 24 h or 1 h before adding the bacteria. In some experiments bacteria were added simultaneously with dentine powder and the medicament. Enterococcus faecalis A197A was used as a test organism. Samples for bacterial culturing were taken from the suspensions at 5 min, 1 h and 24 h after adding the bacteria. RESULTS: Dentine powder had an inhibitory effect on all medicaments tested. The effect was dependent on the concentration of the medicament as well as on the length of the time the medicament was preincubated with dentine powder before adding the bacteria. The effect of calcium hydroxide on E. faecalis was totally abolished by the presence of dentine powder. Similarly, 0.2/0.4% iodine potassium iodide lost its effect after preincubation for 1 h with dentine before adding the bacteria. The effect of 0.05% chlorhexidine and 1% sodium hypochlorite on E. faecalis was reduced but not totally eliminated by the presence of dentine. No inhibition could be measured when full strength solutions of chlorhexidine and iodine potassium iodide were used in killing E. faecalis. CONCLUSIONS: The dentine powder model appears to be an efficient tool for the study of interactions between local endodontic medicaments, dentine, and microbes.

Development of resistance to chlorhexidine diacetate and cetylpyridinium chloride in Pseudomonas stutzeri and changes in antibiotic susceptibility.

Strains of Pseudomonas stutzeri developed stable resistance to chlorhexidine diacetate (CHA) or cetylpyridinium chloride (CPC) when exposed to gradually increasing concentrations of either antibacterial agent. Such strains showed reduced sensitivity to other non-antibiotics, including triclosan, and to some antibiotics, although this varied from strain to strain. Resistant strains were inactivated less readily by CHA or CPC and were less sensitive to sodium dodecyl sulphate. Some CHA-resistant and some CPC-resistant strains were more hydrophobic than the parent strains. Alterations in the cell envelope are likely to be responsible for non-specific changes in sensitivity to several antibacterial agents. Attempts to transfer CHA or CPC resistance by conjugation were unsuccessful. DNA from some CHA- or CPC-resistant strains could transform Ps. stutzeri strain JM 302, a histidine auxotroph, to prototrophy.

Bactericidal activity of skin disinfectants on methicillin-resistant Staphylococcus aureus.

We studied bactericidal activity of 10% povidone-iodine, 0.5% chlorhexidine gluconate, and 0.5% chlorhexidine in 80% ethanol on four strains of methicillin-resistant and two strains of methicillin-susceptible Staphylococcus aureus. The pathogen was exposed to each of the disinfectants for 15, 30, 60, 120, and 240 s at room temperature. The inocula from these suspensions were cultured 72 h at 37 degrees C after the antimicrobial activity of the disinfectants in the suspensions was inactivated by 1:1000 dilution with neutralizer. No organism grew in any of the strains after exposure to 0.5% chlorhexidine in 80% ethanol. The 15-, 30-, and 60-s exposure to 10% povidone-iodine reduced the mean colony count by 55.2%, 91.2%, and 96.7%, respectively, and the exposures to 0.5% chlorhexidine gluconate reduced the mean colony count by 37.2%, 77.1%, and 93.3%, respectively. The difference in colony count between disinfectants was significant at 15- and 30-s exposures (P < 0.01 and 0.05, respectively). The results suggest that bactericidal activity of 0.5% chlorhexidine in 80% ethanol is more potent and more rapid against methicillin-susceptible and methicillin-resistant strains of S. aureus.

The antibacterial and anti-staining properties of the novel anti-adherent agent M239, 144 alone and in combination with chlorhexidine.

The anti-adherent agent M239,144 has been shown to reduce the adhesion of S. sanguis strains to saliva-coated hydroxyapatite by 27-94%. The purpose of this study was to determine any antibacterial properties of M239,144 which might inhibit replication of the organisms which do adhere and to determine whether M239,144 interferes with the antibacterial properties of the antiseptic agent chlorhexidine. Maximum inhibitory dilutions (MIDs) were calculated using both broth and agar methods. 0.1% M239,144 displayed no antibacterial activity against S. oralis NCTC 7864 or S. sanguis NCTC 7863 using the broth dilution method and had no antibacterial activity against a range of plaque-forming organisms using the agar dilution method. The anti-bacterial activity of chlorhexidine against four of five oral plaque-forming species was reduced by 0.1% M239,144. The effect of a novel anti-adherent agent, MK239,144, on chlorhexidine staining was assessed in an in vitro model. 1% M239,144 completely prevented chlorhexidine staining at concentrations from 0.0002% to 0.2%. M239,144/chlorhexidine formulations show potential for a non-staining anti-plaque mouthwash.

Chlorhexidine inactivation by saliva.

Chlorhexidine mouth rinsing is commonly used for oral flora reduction. Indigenous microorganisms (viridans streptococci) are significantly suppressed, while "hospital-acquired" gram-negative bacilli are not affected in vivo. To explain the discrepancies between good in vitro and poor in vivo activity of chlorhexidine, minimum bactericidal concentration values for 120 isolates were studied by means of a standard dilution method in fresh whole saliva, broth, and glucose 5%. Both saliva and broth significantly reduced the bactericidal activity of chlorhexidine against all microorganisms tested as compared to glucose 5% (p less than 0.01). Minimum bactericidal concentrations for indigenous flora were significantly lower than the values obtained for the "hospital-acquired" microorganisms (p less than 0.05). These observations of chlorhexidine inactivation by saliva may explain why chlorhexidine mouth rinsing is of limited value in decontaminating the oral cavity.