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.

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.].

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.

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.

Efeitos antibiofilme e citotóxico de um novo nanosistema carreador de clorexidina e fluconazol / Antibiofilm and cytotoxic effects of a new nanocarrier of chlorhexidine and fluconazole

Os objetivos do presente estudo foram montar e caracterizar um novo nanocarreador dual de clorexidina (CLX) e fluconazol (FLZ), bem como avaliar seu efeito sobre biofilmes microcosmos e sua citotoxicidade sobre queratinócitos orais. Para montar o nanocarreador dual, CLX e FLZ foram adicionados a nanopartículas de óxido de ferro (NPsOF) previamente revestidas por quitosana (QTS), seguido de um processo de solubilização sob agitação magnética. O nanocarreador foi, então, caracterizado por microscopia eletrônica de transmissão, difração de raios X, espectroscopia no infravermelho por transformada de Fourier e análise termogravimétrica. A suscetibilidade de Candida albicans e Candida glabrata no estado planctônico ao nanocarreador dual foi determinada pelos valores de concentração inibitória mínima, utilizando o método da microdiluição em caldo. Um pool de saliva de 2 doadores saudáveis suplementado com espécies de Candida foi usado como inóculo para a formação de biofilmes microcosmos. Os biofilmes foram cultivados (72 h) sobre discos de vidro posicionados no Amsterdam Active Attachment model e tratados (24 h) com NPsOF-QTS carreando 39 µg/mL de CLX + 156 µg/mL de FLZ (NPsOF-QTS-CLX39-FLZ156), 78 µg/mL de CLX + 312 µg/mL de FLZ (NPsOF-QTS-CLX78-FLZ312) e 156 µg/mL de CLX + 624 µg/mL de FLZ (NPsOF-QTS-CLX156-FLZ624). NPsOF (218,5 µg/mL), QTS (218,5 µg/mL) e 156 µg/mL de CLX + 624 µg/mL de FLZ (CLX156-FLZ624) foram testados como controles. Posteriormente, foram realizadas as análises de quantificação das unidades formadoras de colônias (UFCs), produção de ácido lático (LA), composição da matriz extracelular (ME) e viabilidade celular por microscopia confocal de varredura a laser (MCVL). Para o ensaio de citotoxicidade, queratinócitos orais humanos (linhagem NOKsi) foram expostos a diferentes concentrações do nanocarreador dual, por 24 ou 48 h, e a viabilidade celular foi determinada pelo ensaio de redução de MTT. Os dados foram analisados por ANOVA ou teste de Kruskal-Wallis, seguidos dos testes de Fisher LSD ou Tukey (α = 0,05). Os testes de caracterização físico-química mostraram que um nanocarreador dual com dimensões em torno de 6 nm foi obtido, sem comprometer a propriedade cristalina e a estabilidade de NPsOF. Os compostos que formam o nanocarreador estabeleceram uma interação sinérgica em relação ao efeito sobre células planctônicas de Candida. Para os ensaios de biofilme, NPsOF-QTS-CLX156-FLZ624 foi o composto mais eficaz na redução de UFCs de Streptococcus mutans, Lactobacillus spp., C. albicans e C. glabrata, diferindo significativamente dos outros grupos, e esses achados foram confirmados por MCVL. NPsOF-QTS-CLX39-FLZ156, NPsOF-QTS-CLX78-FLZ312 e CLX156- FLZ624 mostraram efeitos antibiofilme similares. O nanocarreador dual também reduziu significativamente a produção de AL e a quantidade de carboidratos e ácidos nucleicos da ME. Um efeito citotóxico dose-dependente sobre queratinócitos orais foi observado para o nanocarreador dual, independentemente do período de exposição testado (24 ou 48 h). NPsOF-QTS-CLX-FLZ e CLX-FLZ reduziram significativamente a viabilidade dos queratinócitos em concentrações de CLX e FLZ iguais ou superiores a 7,8 e 31,25 µg/mL, respectivamente. Por fim, a nanoterapia testada no presente estudo é promissora e constitui um grande avanço dentro dos métodos alternativos aos antimicrobianos tradicionais para o controle da candidíase oral(AU)

The objectives of the present study were to assemble and characterize a new dual nanocarrier of chlorhexidine (CHX) and fluconazole (FLZ), and evaluate its effect on microcosm biofilms and its cytotoxicity against oral keratinocytes. To assemble the dual nanocarrier, CHX and FLZ were added to iron oxide nanoparticles (IONPs) previously coated by chitosan (CS), followed by a solubilization process under magnetic stirring. The nanocarrier was then characterized by transmission electron microscopy, X-ray diffraction, Fourier transform infrared spectroscopy, and thermogravimetric analysis. The susceptibility of Candida albicans and Candida glabrata in the planktonic state to the dual nanocarrier was determined by the minimum inhibitory concentration values, using the broth microdilution method. A saliva pool from 2 healthy donors supplemented with Candida species was used as an inoculum for microcosm biofilm formation. Biofilms were grown (72 h) on glass discs positioned in the Amsterdam Active Attachment model and treated (24 h) with IONPs-CS carrying 39 µg/mL CHX + 156 µg/mL FLZ (IONPsCS-CHX39-FLZ156), 78 µg/mL CHX + 312 µg/mL FLZ (IONPs-CS-CHX78-FLZ312), and 156 µg/mL CHX + 624 µg/mL FLZ (IONPs-CS-CHX156-FLZ624). IONPs at 218.5 µg/mL, 218.5 µg/mL CS, and 156 µg/mL CHX + 624 µg/mL FLZ (CHX156-FLZ624) were tested as controls. Next, analyses of the quantification of colony-forming units (CFUs), lactic acid production (LA), composition of the extracellular matrix (EM), and viability by confocal laser scanning microscopy (CLSM) were performed. For the cytotoxicity assay, human oral keratinocytes (NOKsi lineage) were exposed to different concentrations of the dual nanocarrier, for 24 or 48 h, and cell viability was determined by the MTT reduction assay. Data were analyzed by ANOVA or Kruskal-Wallis test, followed by Fisher LSD or Tukey tests (α = 0.05). The physico-chemical characterization tests showed that a dual nanocarrier with dimensions around 6 nm was assembled, without compromising the crystalline property and stability of IONPs. The compounds that form the nanocarrier established a synergistic interaction in relation to the effect on Candida planktonic cells. Regarding biofilm assays, IONPs-CS-CHX156-FLZ624 was the most effective compound in reducing CFUs from Streptococcus mutans, Lactobacillus spp., C. albicans, and C. glabrata, differing significantly from the other groups, and these findings were confirmed by CLSM. IONPs-CS-CHX39-FLZ156, IONPs-CS-CHX78-FLZ312, and CHX156-FLZ624 showed similar antibiofilm effects. The dual nanocarrier also significantly reduced LA production and the amount of carbohydrates and nucleic acids from the EM. A dose-dependent cytotoxic effect against oral keratinocytes was observed for the dual nanocarrier, regardless of the exposure period tested (24 or 48 h). IONPs-CSCHX-FLZ and CHX-FLZ significantly reduced keratinocyte viability at CHX and FLZ concentrations equal to or greater than 7.8 and 31.25 µg/mL, respectively. In conclusion, the nanotherapy tested in the current study is promising and constitutes a major advance in alternative methods to traditional antimicrobials for oral candidiasis control(AU)

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.

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.

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.

Itraconazole Attenuates Peritoneal Fibrosis Through Its Effect on the Sonic Hedgehog Signaling Pathway in Mice.

BACKGROUND: Peritoneal fibrosis is a devastating complication of peritoneal dialysis. However, its precise mechanism is unclear, and specific treatments have not yet been established. Recent evidence suggests that the sonic hedgehog (SHH) signaling pathway is involved in tissue fibrogenesis. Drugs that inhibit this pathway are emerging in the field of anti-fibrosis therapy. Itraconazole, an anti-fungal agent, was also recently recognized as an inhibitor of the SHH signaling pathway. In this study, we used a mouse model to investigate whether the SHH signaling pathway is involved in the development of peritoneal fibrosis and the effects of itraconazole on peritoneal fibrosis. METHODS: Peritoneal fibrosis was induced by intraperitoneal (IP) injection of 0.1% chlorhexidine gluconate (CG) solution every other day for 4 weeks, with or without itraconazole treatment (20 mg/kg, IP injection on a daily basis). Male C57BL/6 mice were divided into 4 groups: saline group, saline plus itraconazole group, CG group, and CG plus itraconazole group. Isotonic saline was administered intraperitoneally to the control group. The peritoneal tissues were evaluated for histological changes, expression of fibrosis markers, and the main components of the SHH signaling pathway. RESULTS: Peritoneal thickening was evident in the CG group and was significantly decreased by itraconazole administration (80.4 ± 7.7 vs. 28.2 ± 3.8 µm, p < 0.001). The expression of the following SHH signaling pathway components was upregulated in the CG group and suppressed by itraconazole treatment: SHH, patched, smoothened, and glioma-associated oncogene transcription factor 1. The IP injection of CG solution increased the expression of fibrosis markers such as α-smooth muscle actin and transforming growth factor-ß1 in the peritoneal tissues. Itraconazole treatment significantly decreased the expression of these markers. CONCLUSION: Our study provides the first evidence that the SHH signaling pathway may be implicated in peritoneal fibrosis. It also demonstrates that itraconazole treatment has protective effects on peritoneal fibrosis through the regulation of the SHH signaling pathway. These findings suggest that blockage of the SHH signaling pathway is a potential therapeutic strategy for peritoneal fibrosis.

Genotoxicity of non-alcoholic mouth rinses: A micronucleus and nuclear abnormalities study with fluorescent microscopy.

AIM: The aim of the present study was to evaluate the genotoxicity of non-alcoholic mouth rinses on buccal epithelial cells using a micronucleus test. METHODS: A total of 105 patients were selected and randomly divided into five groups. Four different mouth rinses and normal saline were given for 2 weeks' duration, and cytological smears were collected before and after exposure. These smears were subjected to micronucleus (MN) and other nuclear abnormalities (ONA) tests using acridine orange stain, and their frequencies were obtained in 500 buccal epithelial cells. The statistical analysis included mean, χ2 -test, analysis of variance, and post-hoc analysis by Bonferroni test. RESULTS: Micronucleated cells (P < .00) and MN (P < .00) were higher in individuals exposed to chlorhexidine (CHX), followed by chlorine dioxide (ClO2 ), potassium nitrate (KNO3 ), and sodium fluoride (NaF), amine fluoride (AmF), and normal saline. ONA were greater (P < .00) in individuals exposed to CHX, followed by ClO2 , AmF, KNO3 , and NaF and normal saline. Overall, the results showed that genotoxic damage was greater in the case of CHX, followed by ClO2 , KNO3 , and NaF, AmF, and normal saline. CONCLUSION: Chronic exposure to mouth rinses can cause genotoxic damage to buccal epithelial cells. Long-term injudicious and inadvertent use of mouth rinses should be discouraged.

Calcium-silicate mesoporous nanoparticles loaded with chlorhexidine for both anti- Enterococcus faecalis and mineralization properties.

BACKGROUND: In infected periapical tissues, Enterococcus faecalis is one of the most common dominant bacteria. Chlorhexidine has been proved to show strong antibacterial ability against E. faecalis but is ineffective in promoting mineralization for tissues around root apex. Mesoporous calcium-silicate nanoparticles are newly synthesized biomaterials with excellent ability to promote mineralization and carry-release bioactive molecules in a controlled manner. In this study, mesoporous calcium-silicate nanoparticles were functionalized with chlorhexidine and their releasing profile, antibacterial ability, effect on cell proliferation and in vitro mineralization property were evaluated. RESULTS: The chlorhexidine was successfully incorporated into mesoporous calcium-silicate nanoparticles by a mixing-coupling method. The new material could release chlorhexidine as well as Ca2+ and SiO32- in a sustained manner with an alkaline pH value under different conditions. The antimicrobial ability against planktonic E. faecalis was dramatically improved after chlorhexidine incorporation. The nanoparticles with chlorhexidine showed no negative effect on cell proliferation with low concentrations. On dentin slices, the new synthesized material demonstrated a similar inhibitory effect on E. faecalis as the chlorhexidine. After being immersed in SBF for 9 days, numerous apatite crystals could be observed on surfaces of the material tablets. CONCLUSIONS: Mesoporous calcium-silicate nanoparticles loaded with chlorhexidine exhibited release of ions and chlorhexidine, low cytotoxicity, excellent antibacterial ability and in vitro mineralization. This material could be developed into a new effective intra-canal medication in dentistry or a new bone defect filling material for infected bone defects.

Corneal Injury from Presurgical Chlorhexidine Skin Preparation.

BACKGROUND: Chlorhexidine skin preparation has been shown to provide highly effective antimicrobial presurgical skin cleansing. However, there is a significant risk of ocular toxicity when it is used in periocular areas. CASE DESCRIPTION: We describe 2 cases of significant corneal damage resulting from 4% chlorhexidine gluconate preoperative skin cleanser, despite the use of protective occlusive dressing over the eyes. Because of the potential for severe corneal toxicity resulting from use of chlorhexidine, alternative agents such as 10% povidone-iodine should be considered for skin preparation near periocular areas whenever possible. CONCLUSIONS: If chlorhexidine gluconate must be employed near periocular areas, great care must be exercised to avoid contact with the eyes, and additional protective measures (e.g., absorbent eye pads along with tightly occlusive dressings) must be used whenever possible.

Determination of mutagenicity of the precipitate formed by sodium hypochlorite and chlorhexidine using the Ames test.

The aim of this study was to determine the direct mutagenic potential of any precipitate formed by combining sodium hypochlorite (NaOCl) and chlorhexidine (CHX). The precipitates formed by NaOCl and CHX were dissolved in 100% dimethyl sulfoxide and cultured with mutant Salmonella Typhimurium strains. The cells were observed for reverse mutation. The numbers of positive/mutated wells were statistically compared with those in the background plates using the two-sample proportion independent t-test. The precipitates were not found to be significantly more mutagenic than the background plates. Within the limitations of this study, the results suggest that the precipitates formed when sodium hypochlorite and chlorhexidine contact did not show mutagenic (and are therefore carcinogenic) potential.

Cytokine expression in human osteoblasts after antiseptic treatment: a comparative study between polyhexanide and chlorhexidine.

PURPOSE/AIM OF THE STUDY: Chlorhexidine and polyhexanide are frequently used antiseptics in clinical practice and have a broad antimicrobial range. Both antiseptics are helpful medical agents for septic wound treatment with a high potential for defeating joint infections. Their effect on human osteoblasts has, so far, not been sufficiently evaluated. The aim of this study was to investigate the activating potential of polyhexanide and chlorhexidine on inflammatory cytokines/chemokines in human osteoblasts in vitro. MATERIALS AND METHODS: Human osteoblasts were isolated and cultivated in vitro and then treated separately with 0.1% and 2% chlorhexidine and 0.04% polyhexanide as commonly applied concentrations in clinical practice. Detection of cell structure and cell morphology was performed by light microscopic inspection. Cytokine and chemokine secretion was determined by using a multiplex suspension array. RESULTS: Cell shrinking, defective cell membrane, and the loss of cell adhesion indicated cell damage of human osteoblasts after treatment with both antiseptics was evaluated by using light microscopy. Polyhexanide, but not chlorhexidine, caused human osteoblasts to secrete various interleukins (1ß, 6, and 7), interferon γ, tumor necrosis factor α, vascular endothelial growth factor, eotaxin, fibroblast growth factor basic, and granulocyte macrophage colony-stimulating factor as quantified by multiplex suspension array. CONCLUSIONS: Both antiseptics induced morphological cell damage at an optimum exposure between 1 and 10 min. But only polyhexanide mediated a pronounced secretion of inflammatory cytokines and chemokines in human osteoblasts. Therefore, we recommend a preferred usage of chlorhexidine in septic surgery to avoid the induction of an inflammatory reaction.

Is cetrimide-chlorhexidine risky for secondary sclerosing cholangitis?

BACKGROUND: The liver is the most frequent organ for placement of hydatid cyst disease. All known protoscolicidals that are used for echinococcus degeneration have a risk of caustic secondary sclerosing cholangitis. The cetrimide-chlorhexidine combination is an effective protoscolicidal agent for treatment of hydatid liver cysts. OBJECTIVES: The aim of this experimental study was to examine this combination for potential risks of caustic secondary sclerosing cholangitis. MATERIAL AND METHODS: Thirty rats were enrolled and divided into two groups. In the study group, 0.15 mL of a cetrimide (0.5%) and chlorhexidine (0.05%) combination was injected into the bile ducts for five minutes. The control group included the same amount of normal saline and waiting period. The rats were followed for 120 days and the living rats were examined for biliary injury by biochemical analysis and histopathology. RESULTS: No specific histopathological findings for caustic sclerosing cholangitis such as bile duct stricture or periductal fibrosis were present in any groups. Other pathological criteria demonstrating inflammation including portal inflammation, bile duct proliferation and necrosis were similar in both groups. Biochemical analysis including a liver function test (aspartate aminotransferase, alanine aminotransferase, alkaline phosphatase, gamma-glutamyltranspeptidase) appeared similar to the control group. CONCLUSIONS: A cetrimide 0.5% and chlorhexidine 0.05% combination has similar liver function results and histopathological effects to normal saline on bile ducts and it appears to be safe for bile ducts.

Development of an in vitro periodontal biofilm model for assessing antimicrobial and host modulatory effects of bioactive molecules.

BACKGROUND: Inflammation within the oral cavity occurs due to dysregulation between microbial biofilms and the host response. Understanding how different oral hygiene products influence inflammatory properties is important for the development of new products. Therefore, creation of a robust host-pathogen biofilm platform capable of evaluating novel oral healthcare compounds is an attractive option. We therefore devised a multi-species biofilm co-culture model to evaluate the naturally derived polyphenol resveratrol (RSV) and gold standard chlorhexidine (CHX) with respect to anti-biofilm and anti-inflammatory properties. METHODS: An in vitro multi-species biofilm containing S. mitis, F. nucleatum, P. gingivalis and A. actinomycetemcomitans was created to represent a disease-associated biofilm and the oral epithelial cell in OKF6-TERT2. Cytotoxicity studies were performed using RSV and CHX. Multi-species biofilms were either treated with either molecule, or alternatively epithelial cells were treated with these prior to biofilm co-culture. Biofilm composition was evaluated and inflammatory responses quantified at a transcriptional and protein level. RESULTS: CHX was toxic to epithelial cells and multi-species biofilms at concentrations ranging from 0.01-0.2%. RSV did not effect multi-species biofilm composition, but was toxic to epithelial cells at concentrations greater than 0.01%. In co-culture, CHX-treated biofilms resulted in down regulation of the inflammatory chemokine IL-8 at both mRNA and protein level. RSV-treated epithelial cells in co-culture were down-regulated in the release of IL-8 protein, but not mRNA. CONCLUSIONS: CHX possesses potent bactericidal properties, which may impact downstream inflammatory mediators. RSV does not appear to have bactericidal properties against multi-species biofilms, however it did appear to supress epithelial cells from releasing inflammatory mediators. This study demonstrates the potential to understand the mechanisms by which different oral hygiene products may influence gingival inflammation, thereby validating the use of a biofilm co-culture model.

Inhibitory effects of sword bean extract on alveolar bone resorption induced in rats by Porphyromonas gingivalis infection.

BACKGROUND: The domesticated legume, Canavalia gladiata (commonly called the sword bean), is known to contain canavanine. The fruit is used in Chinese and Japanese herbal medicine for treating the discharge of pus, but its pharmacological mechanisms are still unclear. OBJECTIVES: This study examined the effect of sword bean extract (SBE) on (i) oral bacteria and human oral epithelial cells in vitro, and (ii) the initiation and progression of experimental Porphyromonas gingivalis-induced alveolar bone resorption in rats. MATERIAL AND METHODS: A high-performance liquid chromatography/ultraviolet method was applied to quantitate canavanine in SBE. By assessing oral bacterial growth, we estimated the minimum inhibitory concentration and minimum bactericidal concentration of SBE, canavanine, chlorhexidine gluconate (CHX) solution. The cytotoxicity of SBE, canavanine, CHX, leupeptin and cystatin for KB cells was determined using a trypan blue assay. The effects of SBE, canavanine, leupeptin and cystatin on Arg-gingipain (Rgp) and Lys-gingipain (Kgp) were evaluated by colorimetric assay using synthetic substrates. To examine its effects on P. gingivalis-associated periodontal tissue breakdown, SBE was orally administered to P. gingivalis-infected rats. RESULT: Sword bean extract contained 6.4% canavanine. SBE and canavanine inhibited the growth of P. gingivalis and Fusobacterium nucleatum. The cytotoxicity of SBE, canavanine and cystatin on KB cells was significantly lower than that of CHX. Inhibition of Rgp with SBE was comparable to that with leupeptin, a known Rgp inhibitor, and inhibition of Kgp with SBE was significantly higher than that with leupeptin at 500 µg/mL ( p < 0.05). P. gingivalis-induced alveolar bone resorption was significantly suppressed by administration of SBE, with bone levels remaining comparable to non-infected animals ( p < 0.05). CONCLUSION: The present study suggests that SBE might be effective against P. gingivalis-associated alveolar bone resorption.