Antifungal and genotoxic effects of Thymus serpyllum as a root canal irrigant.

OBJECTIVES: The aim of this study was the assessment of the efficiency of the ethyl acetate (EthOAc) extract of Thymus serpyllum against Candida albicans and to compare it with sodium hypochlorite (NaOCl) and chlorhexidine (CHX), as well as their genotoxic effect. MATERIAL AND METHODS: The antifungal effectiveness of the EthOAc extract of Thymus serpyllum was determined using the agar disk diffusion method. The inhibition zones induced by the EthOAc extract were compared after 5 min, 60 min, and 24 h to those induced by standard solutions (2% CHX and 2% NaOCl). An in vitro genotoxicity assay was performed in cultured lymphocytes from the blood of human volunteers to observe micronuclei formation. Statistical analysis of the results was performed using the Kruskal-Wallis test and one-way analysis of variance. RESULTS: The inhibition zone of combination of CHX with EthOAc extract of Thymus serpyllum against C. albicans was 29.7 mm after 5 min, 28.3 mm after 60 min, and 29 mm after 24 h. The inhibition zone of NaOCl in combination with EthOAc extract of Thymus serpyllum against C. albicans was 0 mm. The EthOAc extract of Thymus serpyllum did not show a genotoxic effect on lymphocyte cells. CONCLUSIONS: The EthOAc extract of Thymus serpyllum in combination with CHX may be a useful root canal disinfection in endodontic therapy.

Detrimental Effects of Chlorhexidine on Articular Cartilage Viability, Matrix, and Mechanics.

BACKGROUND: Chlorhexidine gluconate (CHG) solution is commonly used as an antiseptic irrigation for bacterial decontamination during orthopaedic surgery. Although the chondrotoxicity of CHG on articular cartilage has been reported, the full extent of CHG-related chondrotoxicity and its effects on the extracellular matrix and mechanical properties are unknown. PURPOSE: To investigate the in vitro effects of a single 1-minute CHG exposure on the viability, biochemical content, and mechanics of native articular cartilage explants. STUDY DESIGN: Controlled laboratory study. METHODS: Articular cartilage explants (6 per group) were harvested from femoral condyles of the porcine stifle and sectioned at tidemark. Explants were bathed in CHG solution (0.05% CHG in sterile water) at varying concentrations (0% control, 0.01% CHG, and 0.05% CHG) for 1 minute, followed by complete phosphate-buffered saline wash and culture in chondrogenic medium. At 7 days after CHG exposure, cell viability, matrix content (collagen and glycosaminoglycan [GAG]), and compressive mechanical properties (creep indentation testing) were assessed. RESULTS: One-minute CHG exposure was chondrotoxic to explants, with both 0.05% CHG (2.6% ± 4.1%) and 0.01% CHG (76.3% ± 8.6%) causing a decrease in chondrocyte viability compared with controls (97.5% ± 0.6%; P < .001 for both). CHG exposure at either concentration had no significant effect on collagen content, while 0.05% CHG exposure led to a significant decrease in mean GAG per wet weight compared with the control group (2.6% ± 1.7% vs 5.2% ± 1.9%; P = .029). There was a corresponding weakening of mechanical properties in explants treated with 0.05% CHG compared with controls, with decreases in mean aggregate modulus (177.8 ± 90.1 kPa vs 280.8 ± 19.8 kPa; P < .029) and shear modulus (102.6 ± 56.5 kPa vs 167.9 ± 16.2 kPa; P < .020). CONCLUSION: One-minute exposure to CHG for articular cartilage explants led to dose-dependent decreases in chondrocyte viability, GAG content, and compressive mechanical properties. This raises concern for the risk of mechanical failure of the cartilage tissue after CHG exposure. CLINICAL RELEVANCE: Clinicians should be judicious regarding the use of CHG irrigation at these concentrations in the presence of native articular cartilage.

Harmful effects of chlorhexidine on hepatic metabolism.

Chlorhexidine (CHX) is an over-the-counter antiseptic amply used by the population. There are reports that CHX acts in mitochondria as an uncoupler and inhibitor. The purpose of this study was to investigate the short-term effects of CHX on hepatic metabolic pathways linked to energy metabolism in the perfused rat liver. The compound inhibited both glucose synthesis and the urea cycle. Oxygen consumption was raised at low concentrations (up to 10 µM) and diminished at higher ones. A pronounced diminution in the cellular ATP content was observed. Conversely, CHX stimulated glycolysis and enhanced leakage of cellular enzymes (lactate dehydrogenase and fumarase). In isolated mitochondria, this antiseptic inhibited pyruvate carboxylation, oxidases, and oxygen uptake at very low concentrations (2 µM) and promoted uncoupling. The results described herein raise great concerns about the safety of CHX, as the observed effects can induce hypoglycemia, lactic acidosis, ammonemia as well as cell membrane disruption.

Myelin toxicity of chlorhexidine in zebrafish larvae.

BACKGROUND: Chlorhexidine gluconate (CHG) is a topical antiseptic solution recommended for skin preparation before central venous catheter placement and maintenance in adults and children. Although CHG is not recommended for use in children aged <2 months owing to limited safety data, it is commonly used in neonatal intensive care units worldwide. We used zebrafish model to verify the effects of early-life exposure to CHG on the developing nervous system, highlighting its impact on oligodendrocyte development and myelination. METHODS: Zebrafish embryos were exposed to different concentrations of CHG from 4 h post fertilization to examine developmental toxicity. The hatching rate, mortality, and malformation of the embryos/larvae were monitored. Oligodendrocyte lineage in transgenic zebrafish embryos was used to investigate defects in oligodendrocytes and myelin. Myelin structure, locomotor behavior, and expression levels of genes involved in myelination were investigated. RESULTS: Exposure to CHG significantly induced oligodendrocyte defects in the central nervous system, delayed myelination, and locomotor alterations. Ultra-microstructural changes with splitting and fluid-accumulated vacuoles between the myelin sheaths were found. Embryonic exposure to CHG decreased myelination, in association with downregulated mbpa, plp1b, and scrt2 gene expression. CONCLUSION: Our results suggest that CHG has a potential for myelin toxicity in the developing brain. IMPACT: To date, the neurodevelopmental toxicity of chlorhexidine gluconate (CHG) exposure on the developing brains of infants remains unknown. We demonstrated that CHG exposure to zebrafish larvae resulted in significant defects in oligodendrocytes and myelin sheaths. These CHG-exposed zebrafish larvae exhibited structural changes and locomotor alterations. Given the increased CHG use in neonates, this study is the first to identify the risk of early-life CHG exposure on the developing nervous system.

Chlorhexidine residues in sludge from municipal wastewater treatment plants: analytical determination and toxicity evaluation.

In this work, a procedure for the sensitive and selective determination of chlorhexidine in sludge from municipal sewage treatment plants (STPs) based on matrix solid-phase dispersion (MSPD) and liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) was optimized and validated. Analysis of sewage sludge samples, obtained from different STPs in Northwest Spain from 2018 to 2021, showed that chlorhexidine was ubiquitous in this environmental compartment with concentrations between 0.3 and 16 µg g-1. The toxicity of this pollutant was assessed in in vitro assays considering three different model organisms: Candida albicans, Escherichia coli, and Staphylococcus aureus. C. albicans was the most sensitive of the tested microorganisms to chlorhexidine with a lethal threshold concentration of 0.1 mg L-1. Thus, the lowest observed sludge residue was 3 times higher than the acute toxicity threshold measured for C. albicans. Moreover, E. coli and S. aureus were also affected at chlorhexidine concentrations around 1.8 mg L-1 and 0.5 mg L-1, respectively. So, chlorhexidine residues might affect the population of microorganisms existing in STPs. In addition, the potential phytotoxicity of the compound was evaluated with germination experiments using different model seeds. At the evaluated dose (10 µg g-1 dried soil), chlorhexidine did not affect the germination of Sorghum saccharatum, Lepidium sativum, or Sinapis alba seeds. Thus, amending agriculture soils with chlorhexidine containing sludge is unlikely to affect the germination of plants.

Ex Vivo Toxicity of Commonly Used Topical Antiseptics and Antibiotics on Human Chondrocytes.

Topical povidone-iodine, chlorhexidine, bacitracin, and vancomycin are commonly used antiseptic and antimicrobial agents to reduce risk and treat surgical site infections in numerous orthopedic procedures. Chondrocytes potentially may be exposed to these agents during operative procedures. The impact of these topical agents on chondrocyte viability is unclear. The goal of this study is to determine human chondrocyte viability ex vivo after exposure to commonly used concentrations of these topical antiseptic and antimicrobial agents. Human osteochondral plugs were harvested from the knee joint of a human decedent within 36 hours of death. Individual human osteochondral plugs were exposed to normal saline as a control; a range of concentrations of povidone-iodine (0.25%, 0.5%, and 1%), chlorhexidine (0.01% and 0.5%), and bacitracin (10,000 units/L, 50,000 units/L, and 100,000 units/L) for 1-minute lavage; or a 48-hour soak in vancomycin (0.16 mg/mL, 0.4 mg/mL, and 1.0 mg/mL) with nutrient media. Chondrocyte viability was evaluated with a live/dead viability assay at 0, 2, 4, and 6 days after exposure to bacitracin at 0, 3, and 6 days). Control subjects showed greater than 70% viability at all time points. Povidone-iodine, 0.5% chlorhexidine, and vancomycin showed significant cytotoxicity, with viability dropping to less than 40% by day 6. Chondrocytes exposed to 0.01% chlorhexidine maintained viability. Chondrocytes exposed to bacitracin showed viability until day 3, when there was a large drop in viability. Commonly used topical concentrations of povidone-iodine, vancomycin, and bacitracin are toxic to human chondrocytes ex vivo. A low concentration of chlorhexidine appears safe. Caution should be used when articular cartilage may be exposed to these agents during surgery. [Orthopedics. 2022;45(5):e263-e268.].

The cytotoxic effects of various endodontic irrigants on the viability of dental mesenchymal stem cells.

This study aimed to evaluate cytotoxic effects of various irrigation solutions used in regenerative endodontic treatments (RETs) on mesenchymal stem cells, and further examine the long-term effect of hypochlorous acid (HOCl) on the cell viability and alkaline phosphatase (ALP) activity. Stem cells were exposed to various concentrations of NaOCl, EDTA, chlorhexidine (CHX), etidronic acid (HEDP)/NaOCl combination and HOCl. HOCl was tested for its effects on ALP activity up to 21 days. Additionally, cell viability was measured fluorescently using calcein AM. The most cytotoxic irrigant was CHX even with the lowest concentration. NaOCl and HEDP/NaOCl with 1:100 dilution decreased viability to around 40%. HOCl showed the lowest cytotoxicity among all tested irrigants. HOCl also showed no significant reduction in ALP activity compared with the controls. The cytotoxicity of endodontic irrigants was time and concentration dependent. HOCl demonstrated promising results regarding viability and ALP activity, since RETs require host stem cell survival.

Evaluation of in vitro activity of five antimicrobial agents on Acanthamoeba isolates and their toxicity on human corneal epithelium.

BACKGROUND: Acanthamoeba keratitis (AK) is an important cause of ocular morbidity in both contact lens wearers and non wearers. Medical management comprises prolonged empiric treatment with multiple drugs, leading to adverse effects and suboptimal cure. The present study evaluated the efficiency and safety of common antimicrobial agents used in treatment of AK. METHODS: Six Acanthamoeba isolates (four AK, two water samples) were axenized and subjected to in vitro susceptibility testing against chlorhexidine, pentamidine isethionate, polymyxin B, miltefosine, and fluconazole to check for trophocidal and cysticidal activity. The safety profile was analysed by observing the cytotoxicity of the highest cidal concentration toward human corneal epithelial cell (HCEC) line. RESULTS: Chlorhexidine had the lowest cidal concentration against both cysts and trophozoites (range 4.16-25 µg/ml) followed by pentamidine isethionate (range 25-166.7 µg/ml). Both agents were nontoxic to HCEC. Polymyxin B (range 25-200 µg/ml) and fluconazole (range 64-512 µg/ml) had relatively higher minimum inhibitory concentrations (MIC); fluconazole was nontoxic even at 1024 µg/ml, but cytotoxicity was observed at 400 µg/ml with polymyxin B. Miltefosine was not effective against cysts at tested concentrations. A. castellanii were more susceptible to all agents (except pentamidine isethionate) than A. lenticulata. Clinical isolates were less susceptible to polymyxin B and fluconazole than environmental isolates, reverse was true for miltefosine. CONCLUSION: Chlorhexidine and pentamidine isethionate were the most effective and safe agents against both trophozoites and cysts forms of our Acanthamoeba isolates. Fluconazole had higher MIC but was nontoxic. Polymyxin B was effective at high MIC but therapeutic dose was found toxic. Miltefosine, at tested concentrations, could not inhibit cysts of Acanthamoeba. Clinical isolates had higher MICs for polymyxin B and fluconazole.

Interaction of epigallocatechin-gallate and chlorhexidine with Streptococcus mutans stimulated odontoblast-like cells: Cytotoxicity, Interleukin-1ß and co-species proteomic analyses.

OBJECTIVES: The dentin therapeutic agent chlorhexidine has inflammatory and cytotoxic characteristics urging investigation of alternatives like the natural compound epigallocatechin-gallate. The aim is to verify the effect of epigallocatechin-gallate and chlorhexidine on viability, interleukin-1ß (IL-1ß) and differential protein expression of MDPC-23 odontoblast-like cells stimulated by Streptococcus mutans. DESIGN: Cells were stimulated with heat-killed S. mutans at multiplicity of infection (MOI) of 100-1000 and subsequently treated with 100-1 µM of epigallocatechin-gallate. Cells with no treatment or chlorhexidine were controls. Combined stimulated/treated cells were tested for cytotoxicity (Alamar-Blue, N = 3, n = 3), total protein (N = 3, n = 3), IL-1ß (ELISA, N = 3, n = 3), and differential protein expression by liquid chromatography-tandem mass spectrometry (LC-MS/MS, n = 2). RESULTS: Cells stimulated at MOI 100/1000 and treated with 10 µM epigallocatechin-gallate and chlorhexidine did not present cytotoxicity. IL-1ß significantly increased in both un-stimulated and stimulated chlorhexidine 10 µM groups when compared to un-treated control (p < 0.05). MOI 100 chlorhexidine 10 µM group significantly increased IL-1ß compared to un-stimulated chlorhexidine 10 µM and epigallocatechin-gallate 10 µM groups, as well as to MOI 100 epigallocatechin-gallate 10 µM group (p < 0.05). LC-MS/MS revealed S. mutans and mammalian proteins, with tooth-specific proteins exhibiting different abundance levels, depending on the tested condition. CONCLUSIONS: Odontoblast-like cells stimulated with S. mutans at different MOI combined with epigallocatechin-gallate treatment did not cause cytotoxicity. S. mutans stimulation combined with chlorhexidine 100 µM treatment decreased cell viability, while treatment with chlorhexidine 10 µM concentration significantly increased IL-1ß. S. mutans stimulation and treatment of cells resulted in varied protein expression.

Dynamic and quantitative characterization of antagonism within disinfectant mixtures by a modified area-concentration ratio method.

The coexistence of various typical disinfectant pollutants has the potential to produce toxicity interaction towards organisms in the environment. A suitable model is necessary to evaluate the interaction quantitatively. Hence, the area-concentration ratio (ACR) method was modified (MACR) by combing confidence intervals to dynamically and quantitatively evaluate the toxicity interactions within disinfectant mixture pollutants. Disinfectant mixtures were designed by the direct equipartition design ray method using three guanidine disinfectants, chlorhexidine diacetate (CD), chlorhexidine (CHL), and polyhexamethylene biguanidine (POL) and one chlorine-containing disinfectant calcium hypochlorite (CAL). The toxicities of the four disinfectants and their mixtures towards Vibrio qinghaiensis sp.-Q67 (Q67) were determined by the time-dependent toxicity microplate analysis method. And the toxicity mechanism was analyzed by determining the effects of four disinfectants and their binary mixtures on the structure of cell, DNA and proteins (Pro) for Q67. The results show that the toxicities of CD and CHL to Q67vary little with time, but POL and CAL show the obvious time-dependent toxicity. The toxicities of CD, CHL and POL to Q67 are significantly stronger than that of CAL at the same exposure time. The toxicities of three binary mixture systems don't have significant difference in different exposure time. MACR can dynamically, quantitatively and accurately characterize toxicity interactions compared with ACR. According to MACR, the antagonism intensity dynamically changes with the prolongation of exposure time for binary mixture rays of three guanidine disinfectants and CAL, and linearly correlates with the components' concentration ratios. Four disinfectants all can destroy cell membrane and cause desaturation DNA of test organism, and CAL even can destroy the structure of DNA and protein. The probably reason for the antagonism within binary mixtures is the reaction between guanidine group and ClO-, which is called chemical antaogism.

Assessment of the cytotoxic effects and chemical composition of the insoluble precipitate formed from sodium hypochlorite and chlorhexidine gluconate.

AIM: To investigate (1) the cytotoxic potential of the brown precipitate (BP) formed with sodium hypochlorite (NaOCl) and chlorhexidine gluconate (CHX), using both a small animal model of Caenorhabditis elegans (C. elegans) and cultured human gingival fibroblasts; and (2) the chemical composition of BP using Time-of-Flight Secondary Ion Mass Spectrometry (ToF-SIMS). METHODOLOGY: Brown precipitate was obtained by mixing equal volumes of 6% NaOCl and 2% CHX and separating the BP from clear supernatant by centrifugation. The brown precipitate was weighed and solubilized in dimethyl sulfoxide for cytotoxicity experiments. The cytotoxic effect of BP was assessed using C. elegans larvae and primary immortalized human gingival fibroblasts-hTERT (hTERT-hNOF) cells. Various dilutions of BP (25 ng/µL-150 ng/µL), supernatant (0.15% v/v), NaOCl (1:100-1:1000 dilutions of 6% NaOCl) or CHX (1:500-1:1000 dilutions of 2% CHX) along with vehicle control (0.5% v/v ethanol and 0.15% v/v DMSO) or untreated control (growth medium) were tested on C. elegans larvae and hTERT-hNOF cells. Viability was assessed in C. elegans larvae using stereomicroscopy and in hTERT-hNOF cells using dehydrogenase-based colorimetric assay. ToF-SIMS was used to assess the chemical composition of BP in comparison with CHX and para-chloroaniline (PCA). The C. elegans and cell line data were analysed using Log-Rank test and Student's t-test, respectively (p < .05). RESULTS: BP-75 ng/µL and BP-150 ng/µL were significantly more toxic to C. elegans larvae than the untreated, vehicle, supernatant or CHX treatment groups (p < .0001). Similarly, in hTERT-hNOF cells, BP-50 ng/µL, BP-75 ng/µL and BP-150 ng/µL induced significant cytotoxicity within 2 h compared with untreated, vehicle, supernatant and CHX treatments (p < .05). ToF-SIMS analysis of BP revealed ion composition characteristic of both CHX and the carcinogen PCA. CONCLUSIONS: Brown precipitate was toxic in both C. elegans larvae and hTERT-hNOF cells. The ToF-SIMS analysis of BP revealed ions characteristic of CHX and PCA that could account for the toxicities observed in C. elegans larvae and human gingival fibroblasts. Because of the insoluble and toxic nature of BP, consecutive use of CHX and NaOCl irrigants should be avoided in root canal treatment.

Effects of TGF-ß1 Receptor Inhibitor GW788388 on the Epithelial to Mesenchymal Transition of Peritoneal Mesothelial Cells.

We investigated the effectiveness of the transforming growth factor beta-1 (TGF-ß) receptor inhibitor GW788388 on the epithelial to mesenchymal transition (EMT) using human peritoneal mesothelial cells (HPMCs) and examined the effectiveness of GW788388 on the peritoneal membrane using a peritoneal fibrosis mouse model. HPMCs were treated with TGF-ß with or without GW788388. Animal experiments were conducted on male C57/BL6 mice. Peritoneal fibrosis was induced by intraperitoneal injection of chlorhexidine gluconate. GW788388 was administered by once-daily oral gavage. The morphological change, cell migration, and invasion resulted from TGF-ß treatment, but these changes were attenuated by cotreatment with GW788388. TGF-ß-treated HPMCs decreased the level of the epithelial cell marker and increased the levels of the mesenchymal cell markers. Cotreatment with GW788388 reversed these changes. Phosphorylated Smad2 and Smad3 protein levels were stimulated with TGF-ß and the change was attenuated by cotreatment with GW788388. For the peritoneal fibrosis mice, thickness and collagen deposition of parietal peritoneum was increased, but this change was attenuated by cotreatment with GW788388. GW788388, an orally available potent TGF-ß receptor type 1 inhibitor, effectively attenuated TGF-ß-induced EMT in HPMCs. Cotreatment with GW788388 improved peritoneal thickness and fibrosis, and recovered peritoneal membrane function in a peritoneal fibrosis mouse model.

Comparative evaluation of the cytotoxic effects of different oral antiseptics: A primary culture study.

BACKGROUND: Dental plaque is a major oral health problem with severe consequences. Oral antiseptics provide important means for controlling dental plaque formation and are widely used by the public. However, some of these antiseptics have been shown to have side effects on oral tissues. AIM: In this study, we aimed to investigate the time and dose-dependent cytotoxic effects of various antiseptics on primary human gingival fibroblasts (HGF). METHODS: HGF cells were obtained using primary culture techniques. The effects of various doses of 5 antiseptics containing Chlorhexidine-Gluconate (CHX), CHX with Benzydamine-Hydrochloride (Benzydamine-HCl), Povidone-Iodine (PVP-I), Benzydamine-HCl and Essential-Oil on HGFs were analyzed by using 2,3-bis (2-metoxy-4-nitro-5-sulfophenyl)-5-[(phenylamino) carbonyl]-2H-tetrazolium hydroxide cell viability assay after 30, 60, and 180 s of exposure. Results: Cell viability analyses showed that cell death increased in an application time and dose-dependent manner. There was a statistically significant difference in the effects of each antiseptic on live-cell densities compared to the control group and each other (P < 0.001). Antiseptic containing 0.2% CHX showed the highest cytotoxicity on cells. The remaining viable cell density after administration of 0.2% CHX at a dose of 12.5% for 30 s is 35.19%. The high cytotoxic effect of 0.2% CHX was followed by 0.12% CHX with 0.15% Benzydamine-HCl, PVP-I and 0.15% Benzydamine-HCl groups. The lowest cytotoxic effect was observed for the Essential-Oil containing antiseptic solution. CONCLUSIONS: The results of this study show that these five antiseptic agents have variable effects on in vitro HGF proliferation. The doses and administration times of antiseptics should be controlled carefully during dental applications.

Biocompatibility and stability during storage of Foley urinary catheters coated chlorhexidine loaded nanoparticles by nanocoating: in vitro and in vivo evaluation.

Foley urinary catheters were coated by chlorhexidine-loaded micelles and chlorhexidine-loaded nanospheres. In our prior study, the nanocoating of Foley urinary catheter was investigated for chlorhexidine-release study, degradation, antibacterial evaluation, and cytotoxicity assessment. These studies presented the 1 month antibacterial property of nanocoating deposited via the layers of micelles and nanospheres. In this study, we evaluated the biocompatibility of these catheters, including hemocompatibility, skin irritation, skin sensitization, and stability during the age of coated urinary catheter. Results demonstrated that coated urinary catheters presented slight hemolysis, whereas skin irritation on rabbit and skin sensitization on Dunkin Hartley guinea pig showed no signs of dermal toxicity, which indicated that inflammation, redness, and swelling did not occur. Moreover, the stability of coated urinary catheters during storage indicated no change in chlorhexidine peaks by high performance liquid chromatography. Information from these studies supports the biocompatibility of coated urinary catheters via nanocoating and their use as indwelling devices to prevent urinary tract infections.

The Use of Chlorhexidine as a Skin Preparation on the Head and Neck: A Systematic Review of Ocular and Ototoxicity.

BACKGROUND: Chlorhexidine gluconate is one of the most effective surgical preparations, but it has known potential ocular and ototoxicity. OBJECTIVE: To review reported cases of ocular and ototoxicity caused by chlorhexidine and summarize the clinical situations in which chlorhexidine toxicity occurred. METHODS: We performed a systematic review of PubMed and the Web of Science. RESULTS: Fourteen cases reported sensorineural hearing loss from chlorhexidine instilled into the ear. Of the 38 cases of ocular toxicity, 8 cases were caused by direct instillation in the eye and 17 involved periocular surgical preparation. In the remaining cases, the area prepped was less defined. Seven cases involved preparation of the face, 1 for the scalp, 2 cases were drips from distant sites, and 3 cases did not specify the means of exposure. CONCLUSION: The vast majority of toxicity occurred in patients undergoing general anesthesia and was rarely seen in situations where surgery was performed by dermatologists. Ultimately, it should be up to the individual physician to decide whether chlorhexidine is the best choice for a particular outpatient procedure.

Antimicrobial, antibiofilm and cytotoxic effects of a colloidal nanocarrier composed by chitosan-coated iron oxide nanoparticles loaded with chlorhexidine.

OBJECTIVES: This study evaluated the antimicrobial and antibiofilm effects of a colloidal nanocarrier for chlorhexidine (CHX) on Candida glabrata and Enterococcus faecalis, as well as tested its cytotoxic effect on murine fibroblasts. METHODS: Iron oxide nanoparticles (IONPs) were coated with chitosan (CS) and loaded with CHX at 31.2, 78 and 156 µg/mL. Antimicrobial effects were assessed by determining the minimum inhibitory concentration (MIC), using the broth microdilution method, and fractional inhibitory concentration index (FICI). Preformed biofilms (48 h) were treated with different concentrations of the nanocarrier (24 h) and quantified by colony-forming units (CFUs), total biomass and metabolic activity. For cytotoxicity, the viability of L929 cells was evaluated by MTT assay after 24 and 48 h of exposure to the nanocarrier. Data were submitted to ANOVA and Fisher LSD or Tukey post-hoc tests (α = 0.05). RESULTS: MIC and FICI results showed an indifferent interaction among the components of the nanocarrier for all strains evaluated. CHX alone and nanocarrier containing 156 µg/mL CHX did not differ from each other in reducing the number of CFUs. However, the nanocarrier containing 156 µg/mL CHX promoted the highest reductions in total biofilm biomass and metabolism, surpassing the effect of CHX alone. After 24 and 48 h of exposure, the nanocarrier reduced CHX toxicity to the L929 cell at low concentrations. CONCLUSION: These findings suggest that the CHX nanocarrier has potential to be used in the control of oral diseases associated with C. glabrata and E. faecalis. CLINICAL RELEVANCE: CHX has improved the antibiofilm effect and reduced the cytotoxicity (at low concentrations) when conjugated to CS-coated IONPs. This new colloidal formulation has potential as an alternative antimicrobial agent to pure CHX for the control of biofilm-related oral diseases, such as oral candidiasis and endodontic infections.

Connective tissue growth factor is correlated with peritoneal lymphangiogenesis.

Lymphatic absorption in the peritoneal cavity may contribute to ultrafiltration failure in peritoneal dialysis (PD). Lymphatic vessels develop during PD-related peritoneal fibrosis. Connective tissue growth factor (CTGF, also called CCN2) is an important determinant of fibrotic tissue remodeling, but little is known about its possible involvement in lymphangiogenesis. In this study, we investigated the relationship between CTGF and peritoneal lymphangiogenesis. A positive correlation was observed between vascular endothelial growth factor-C (VEGF-C), a major lymphangiogenic growth factor, and the CTGF concentration in human PD effluents. CTGF expression was positively correlated with expression of lymphatic markers and VEGF-C in human peritoneal biopsies. We found a positive correlation between the increase in CTGF and the increase in VEGF-C in cultured human peritoneal mesothelial cells (HPMCs) treated with transforming growth factor-ß1 (TGF-ß1). The diaphragm is a central player in peritoneal lymphatic absorption. CTGF expression was also correlated with expression of VEGF-C and lymphatics in a rat diaphragmatic fibrosis model induced by chlorhexidine gluconate (CG). Furthermore, CTGF gene deletion reduced VEGF-C expression and peritoneal lymphangiogenesis in the mouse CG model. Inhibition of CTGF also reduced VEGF-C upregulation in HPMCs treated with TGF-ß1. Our results suggest a close relationship between CTGF and PD-associated lymphangiogenesis.

Enzyme responsive copolymer micelles enhance the anti-biofilm efficacy of the antiseptic chlorhexidine.

Staphylococcal biofilms cause many infectious diseases and are highly tolerant to the effects of antimicrobials; this is partly due to the biofilm matrix, which acts as a physical barrier retarding the penetration and reducing susceptibility to antimicrobials, thereby decreasing successful treatment outcomes. In this study, both single and mixed micellar systems based on poly vinyl caprolactam (PCL)-polyethylene glycol (PEG) copolymers were optimised for delivery of chlorhexidine (CHX) to S. aureus, MRSA and S. epidermidis biofilms and evaluated for their toxicity using Caenorhabditis elegans. The respective polyethylene glycol (PEG) and poly vinyl caprolactam (PCL) structural components promoted stealth properties and enzymatic responsive release of CHX inside biofilms, leading to significantly enhanced penetration (56%) compared with free CHX and improving the efficacy against Staphylococcus aureus biofilms grown on an artificial dermis (2.4 log reduction of CFU). Mixing Soluplus-based micelles with Solutol further enhanced the CHX penetration (71%) and promoted maximum reduction in biofilm biomass (>60%). Nematodes-based toxicity assay showed micelles with no lethal effects as indicated by their high survival rate (100%) after 72 h exposure. This study thus demonstrated that bio-responsive carriers can be designed to deliver a poorly water-soluble antimicrobial agent and advance the control of biofilm associated infections.

Comparison of anti-peritoneal fibrotic effects between an mTORC1-specific blocker and a PI3K/mTOR dual-blocker.

OBJECTIVE: To compare the anti-peritoneal fibrotic effects between a mammalian target of rapamycin complex 1-specific blocker and a phosphatidyl-inositol 3-kinase/mammalian target of rapamycin dual-blocker. METHODS: A total of 40 male Sprague-Dawley rats were randomly divided into five groups with eight animals per group. The normal group (N group) did not receive any intervention. The normal saline group (NS group) received an intraperitoneal injection of normal saline at 1 ml/100 g daily. The model group (3 W group), rapamycin (RAPA) group and BEZ235 (PI3K/mTOR dual-blocker) group all received an intraperitoneal injection of 0.1% chlorhexidine gluconate at 1 ml/100g daily. And the RAPA and BEZ235 groups also received a 0.5 mg/d RAPA or 2.5 mg/d BEZ235 gavage every day, respectively. Rats in each group were sacrificed after 3 weeks. RESULTS: Immunohistochemistry, real-time PCR and western blotting analysis of fibrosis-related indicators (FN, Col 1, and α-SMA) confirmed that RAPA and BEZ235 significantly inhibited peritoneal fibrosis and that these two drugs had similar effects. The p-Akt, p-mTOR, p-p70S6K expression levels were significantly up-regulated in the 3 W group compared to the NS group, confirming that the mTOR pathway was significantly activated during peritoneal fibrosis. RAPA significantly inhibited the phosphorylation of mTOR and p70S6K but did not have significant effects on p-Akt upstream of mTOR. BEZ235 had significant inhibitory effects on all signaling molecules (p-Akt, p-mTOR, and p-p70S6K) in the mTOR pathway. CONCLUSION: RAPA did not up-regulate p-Akt in a negative feedback fashion. Both drugs effectively inhibited peritoneal fibrosis.

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.