Characterization of Porphyromonas gingivalis sialidase and disruption of its role in host-pathogen interactions.

Key to onset and progression of periodontitis is a complex relationship between oral bacteria and the host. The organisms most associated with severe periodontitis are the periodontal pathogens of the red complex: Tannerella forsythia, Treponema denticola and Porphyromonas gingivalis. These organisms express sialidases, which cleave sialic acid from host glycoproteins, and contribute to disease through various mechanisms. Here, we expressed and purified recombinant P. gingivalis sialidase SiaPG (PG_0352) and characterized its activity on a number of substrates, including host sialoglycoproteins and highlighting the inability to cleave diacetylated sialic acids - a phenomenon overcome by the NanS sialate-esterase from T. forsythia. Indeed SiaPG required NanS to maximize sialic acid harvesting from heavily O-acetylated substrates such as bovine salivary mucin, hinting at the possibility of interspecies cooperation in sialic acid release from host sources by these members of the oral microbiota. Activity of SiaPG and P. gingivalis was inhibited using the commercially available chemotherapeutic zanamivir, indicating its potential as a virulence inhibitor, which also inhibited sialic acid release from mucin, and was capable of inhibiting biofilm formation of P. gingivalis on oral glycoprotein sources. Zanamivir also inhibited attachment and invasion of oral epithelial cells by P. gingivalis and other periodontal pathogens, both in monospecies but also in multispecies infection experiments, indicating potential to suppress host-pathogen interactions of a mixed microbial community. This study broadens our understanding of the multifarious roles of bacterial sialidases in virulence, and indicates that their inhibition with chemotherapeutics could be a promising strategy for periodontitis therapy.

Six-Month Evaluation of a Sodium Bicarbonate-Containing Toothpaste for Reduction of Established Gingivitis: A Randomized USA-Based Clinical Trial.

OBJECTIVES: Short-term use of sodium bicarbonate (NaHCO3)-containing toothpaste reduces plaque and improves clinical measures of gingivitis. To examine this over a longer period, we compared efficacy and tolerability of twice-daily brushing for 24 weeks with 67% or 0% NaHCO3-containing toothpastes in USA-based participants with moderate gingivitis (Clinicaltrials.gov:NCT02207400). METHODS: This was a six-month, randomized, examiner-blind, parallel-group, clinical trial. Investigators randomized adults with blood in expectorate after brushing and ≥ 20 gingival bleeding sites to 67% NaHCO3 (n = 123; n = 107 completed study) or 0% NaHCO3 (n = 123; n = 109 completed study) toothpastes. Primary efficacy variables included between-treatment differences in number of bleeding sites and Modified Gingival Index (MGI) score at 24 weeks. Secondary efficacy variables included Bleeding Index and Turesky modification of the Quigley-Hein Plaque Index (overall and interproximal sites) at six, 12, and 24 weeks. A subset of 50 participants underwent sampling to assess plaque microbiology over the course of treatment. RESULTS: Compared with the 0% NaHCO3 toothpaste, the 67% NaHCO3 toothpaste produced statistically significant improvements at Week 24 in number of bleeding sites (46.7% difference) and MGI (33.9% difference), and for all other endpoints (all p < 0.0001). There was no significant between-treatment difference in the proportion of participants harboring opportunistic pathogens. Products were generally well tolerated, with two and five treatment-related adverse events reported in the 67% and 0% NaHCO3 toothpaste groups, respectively. CONCLUSIONS: Gingival bleeding, gingivitis, and plaque indices were significantly improved at six, 12, and 24 weeks with twice-daily brushing with 67% NaHCO3-containing toothpaste in participants with moderate gingivitis.

The anti-adhesive mode of action of a purified mushroom (Lentinus edodes) extract with anticaries and antigingivitis properties in two oral bacterial phatogens.

BACKGROUND: In previous works we have shown that a low-molecular-mass (LMM) fraction from mushroom (Lentinus edodes) homogenate interferes with binding of Streptococcus mutans to hydroxyapatite and Prevotella intermedia to gingival cells. Additionally, inhibition of biofilm formation of both odonto- and periodonto-pathogenic bacteria and detachment from preformed biofilms have been described for this compound. Further purification of mushroom extract has been recently achieved and a sub-fraction (i.e. # 5) has been identified as containing the majority of the mentioned biological activities. The aim of this study was to characterise the bacterial receptors for the purified mushroom sub-fraction #5 in order to better elucidate the mode of action of this compound when interfering with bacterial adhesion to host surfaces or with bacteria-bacteria interactions in the biofilm state. METHODS: Candidate bacterial molecules to act as target of this compound were bacterial surface molecules involved in cell adhesion and biofilm formation, and, thus, we have considered cell wall associated proteins (CWPs), teichoic acid (TA) and lipoteichoic acid (LTA) of S. mutans, and outer membrane proteins (OMPs) and lipopolysaccharide (LPS) of P. intermedia. RESULTS: Fifteen S. mutans CWPs and TA were capable of binding sub-fraction #5, while LTA did not. As far as P. intermedia is concerned, we show that five OMPs interact with sub-fraction # 5. Capacity of binding to P. intermedia LPS was also studied but in this case negative results were obtained. CONCLUSIONS: Binding sub-fraction # 5 to surface molecules of S. mutans or P. intermedia may result in inactivation of their physiological functions. As a whole, these results indicate, at molecular level, the bacterial surface alterations affecting adhesion and biofim formation. For these antimicrobial properties, the compound may find use in daily oral hygiene.

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.

Biological Evaluation of Oral Care Products Using 3D Tissue-Engineered In Vitro Models of Plaque-Induced Gingivitis.

BACKGROUND: The aim of this study was to investigate and visualize the anti-inflammatory and anti-bacterial effects of different oral care products using an infected and inflamed 3D tissue-engineered gingival mucosal model. METHODS: A 3D full-thickness oral mucosal model was engineered inside tissue culture inserts using collagen hydrogels populated with human gingival fibroblasts and THP-1 monocytes and layered with oral epithelial cell lines. Oral saliva bacteria were cultured and added to the surface of the models and inflammation was further simulated with lipopolysaccharide (LPS) of Escherichia coli. The 3D models were exposed to three different types of toothpastes, a chlorhexidine antiseptic mouthwash, different antibiotics, and a mechanical rinse with phosphate-buffered saline (PBS) prior to biological evaluation using the PrestoBlue tissue viability assay, histology, optical coherence tomography (OCT), confocal microscopy, and measurement of the release of the inflammatory markers IL-1ß, IL-6, and IL-8 with ELISA. RESULTS: Multiple-endpoint analyses of the infected oral mucosal models treated with different anti-bacterial agents showed consistent outcomes in terms of tissue viability, histology, OCT, and confocal microscopy findings. In terms of anti-inflammatory testings, the positive control group showed the highest level of inflammation compared with all other groups. Depending on the anti-bacterial and anti-inflammatory potential of the test groups, different levels of inflammation were observed in the test groups. CONCLUSIONS: The inflamed 3D oral mucosal model developed in this study has the potential to be used as a suitable in vitro model for testing the biocompatibility, anti-inflammatory, and anti-bacterial properties of oral care products including mouthwashes and toothpastes. The results of this study indicate that the chlorhexidine mouthwash has both anti-bacterial and cytotoxic effects on the 3D oral mucosal model. Hyaluronic-acid-containing toothpaste has significant anti-bacterial and anti-inflammatory effects on the 3D oral mucosal model.

Effects of mushroom and chicory extracts on the shape, physiology and proteome of the cariogenic bacterium Streptococcus mutans.

BACKGROUND: Dental caries is an infectious disease which results from the acidic demineralisation of the tooth enamel and dentine as a consequence of the dental plaque (a microbial biofilm) accumulation. Research showed that several foods contain some components with antibacterial and antiplaque activity. Previous studies indicated antimicrobial and antiplaque activities in a low-molecular-mass (LMM) fraction of extracts from either an edible mushroom (Lentinus edodes) or from Italian red chicory (Cichorium intybus). METHODS: We have evaluated the antimicrobial mode of action of these fractions on Streptococcus mutans, the etiological agent of human dental caries. The effects on shape, macromolecular syntheses and cell proteome were analysed. RESULTS: The best antimicrobial activity has been displayed by the LMM mushroom extract with a bacteriostatic effect. At the MIC of both extracts DNA synthesis was the main macromolecular synthesis inhibited, RNA synthesis was less inhibited than that of DNA and protein synthesis was inhibited only by roughly 50%. The partial inhibition of protein synthesis is compatible with the observed significant increase in cell mass. The increase in these parameters is linked to the morphological alteration with transition from cocci of the untreated control to elongated cells. Interestingly, these modifications were also observed at sub-MIC concentrations. Finally, membrane and cytosol proteome analysis was conducted under LMM mushroom extract treatment in comparison with untreated S. mutans cells. Significant changes were observed for 31 membrane proteins and 20 of the cytosol fractions. The possible role of the changed proteins is discussed. CONCLUSIONS: This report has shown an antibiotic-like mode of action of mushroom and chicory extracts as demonstrated by induced morphogenetic effects and inhibition of specific macromolecular synthesis. This feature as well as the safe use of this extract as result of its natural origin render the LMM both mushroom and chicory extracts suitable for the formulation into products for daily oral hygiene such as mouthwashes or toothpastes.

The anticaries effect of a food extract (shiitake) in a short-term clinical study.

The main objective was to investigate whether low-molecular-weight fraction of edible mushroom shiitake extract (Lentinus edodes) possesses caries-preventive properties. The study was designed as a double-blind, three-leg, cross-over, randomized, controlled clinical trial carried out on two series of volunteers at the University of Gothenburg, and the Academic Centre for Dentistry Amsterdam. Volunteers rinsed twice daily with a solution containing low-molecular-weight fraction of edible mushroom, placebo (negative control without active ingredients), or Meridol (positive control, AmF-SnF(2)) for two weeks, with a two-week washout period between each rinsing period. Changes in the acidogenicity of dental plaque before and after a sucrose challenge, shifts in microbial composition, and plaque scores were determined. Frequent rinses with shiitake reduced the metabolic activity of dental plaque. No reduction of plaque scores and no inhibition of the production of organic acids in plaque was found. Minor differences in microbial composition between test sessions were found. To conclude, the results indicate that shiitake extract has anticariogenic potential, but not to the same extent as the positive control.

The Impact on Dental Staining Caused by Beverages in Combination with Chlorhexidine Digluconate.

OBJECTIVES: There are several hypotheses regarding how chlorhexidine (CHX) digluconate causes staining with the role of beverages, specifically the precipitation of anionic dietary chromogens onto adsorbed cations, the most probable cause. The aim of this study was to investigate and compare the staining potential of common beverages using an in vitro staining and brushing model to better understand the interactions between chromogens from different beverage categories and the teeth. MATERIALS AND METHODS: Human enamel samples were exposed to a cyclic treatment of artificial saliva and 0.2% CHX mouthwash combined with a range of beverages, with and without brushing, simulating a period equivalent to 2 weeks. Eleven beverages were tested: diet coke, diet lemonade, white wine, red wine, lager beer, black tea, coffee, black tea with milk, coffee with milk, ginger and lemon infusion, and water. Toothbrushing was performed in a brushing simulator with toothpaste and also with water. Colorimetric differences were determined by ΔE using a VITA Easyshade dental spectrophotometer. Statistical analyses were performed by one-way analysis of variance with post hoc Tukey's honestly significant difference test and Levene's test. RESULTS: Black tea and red wine produced highest staining, which agrees with the literature. Significant staining was also observed for a ginger and lemon infusion, coffee, coffee with milk, tea with milk, and lager beer compared with water (p < 0.05). The staining potential of diet coke in combination with brushing appeared to be connected to its low pH. Both white wine and diet lemonade produced stain comparable to the water control. After treatment with high staining beverages, scanning electron microscope evaluation confirmed the formation of a surface layer. The mechanical resistance of the stain differed depending on the beverage, black tea stain was the most resistant. The addition of milk to tea and coffee considerably modified the stain layer and the adhesion to the tooth surface. CONCLUSION: The data may help demonstrate that appropriate user guidance can avoid stain and in turn help improve user compliance during short-term use of this gold standard antimicrobial treatment.

Investigating wound healing characteristics of gingival and skin keratinocytes in organotypic cultures.

OBJECTIVES: The gingiva heals at an accelerated rate with reduced scarring when compared to skin. Potential well-studied factors include immune cell number, angiogenesis disparities and fibroblast gene expression. Differential keratinocyte gene expression, however, remains relatively understudied. This study explored the contrasting healing efficiencies of gingival and skin keratinocytes, alongside their differential gene expression patterns. METHODS: 3D organotypic culture models of human gingiva and skin were developed using temporarily immortalised primary keratinocytes. Models were wounded for visualisation of re-epithelialisation and analysis of keratinocyte migration to close the wound gap. Concurrently, differentially expressed genes between primary gingival and skin keratinocytes were identified, validated, and functionally assessed. RESULTS: Characterisation of the 3D cultures of gingiva and skin showed differentiation markers that recapitulated organisation of the corresponding in vivo tissue. Upon wounding, gingival models displayed a significantly higher efficiency in re-epithelialisation and stratification versus skin, repopulating the wound gap within 24 hours. This difference was likely due to distinct patterns of migration, with gingival cells demonstrating a form of sheet migration, in contrast to skin, where the leading edge was typically 1-2 cells thick. A candidate approach was used to identify several genes that were differentially expressed between gingival and skin keratinocytes. Knockdown of PITX1 resulted in reduced migration capacity of gingival cells. CONCLUSION: Gingival keratinocytes retain in vivo superior wound healing capabilities in in vitro 2D and 3D environments. Intrinsic gene expression differences could result in gingival cells being 'primed' for healing and play a role in faster wound resolution. CLINICAL SIGNIFICANCE STATEMENT: The successful development of organotypic models, that recapitulate re-epithelialisation, will underpin further studies to analyse the oral response to wound stimuli, and potential therapeutic interventions, in an in vitro environment.

An In Vitro Evaluation of Denture Cleansing Regimens against a Polymicrobial Denture Biofilm Model.

Denture stomatitis (DS) is an inflammatory disease resulting from a polymicrobial biofilm perturbation at the denture surface-palatal mucosa interface. Recommendations made by dental health care professionals often lack clarity for appropriate denture cleaning. This study investigated the efficacy of brushing with off-the-shelf denture cleanser (DC) tablets (Poligrip®) vs. two toothpastes (Colgate® and Crest®) in alleviating the viable microorganisms (bacteria and fungi) in an in vitro denture biofilm model. Biofilms were grown on poly(methyl)methacrylate (PMMA) discs, then treated daily for 7 days with mechanical disruption (brushing), plus Poligrip® DC, Colgate® or Crest® toothpastes. Weekly treatment with Poligrip® DC on day 7 only was compared to daily modalities. All treatment parameters were processed to determine viable colony forming units for bacteria and fungi using the Miles and Misra technique, and imaged by confocal laser scanning microscopy (CLSM). Brushing with daily DC therapy was the most effective treatment in reducing the viable biofilm over 7 days of treatment. Brushing only was ineffective in controlling the viable bioburden, which was confirmed by CLSM imaging. This data indicates that regular cleansing of PMMA with DC was best for polymicrobial biofilms.

Meta-analysis of caries microbiome studies can improve upon disease prediction outcomes.

As one of the most prevalent infective diseases worldwide, it is crucial that we not only know the constituents of the oral microbiome in dental caries but also understand its functionality. Herein, we present a reproducible meta-analysis to effectively report the key components and the associated functional signature of the oral microbiome in dental caries. Publicly available sequencing data were downloaded from online repositories and subjected to a standardized analysis pipeline before analysis. Meta-analyses identified significant differences in alpha and beta diversities of carious microbiomes when compared to healthy ones. Additionally, machine learning and receiver operator characteristic analysis showed an ability to discriminate between healthy and disease microbiomes. We identified from importance values, as derived from random forest analyses, a group of genera, notably containing Selenomonas, Aggregatibacter, Actinomyces and Treponema, which can be predictive of dental caries. Finally, we propose the most appropriate study design for investigating the microbiome of dental caries by synthesizing the studies, which had the most accurate differentiation based on random forest modelling. In conclusion, we have developed a non-biased, reproducible pipeline, which can be applied to microbiome meta-analyses of multiple diseases, but importantly we have derived from our meta-analysis a key group of organisms that can be used to identify individuals at risk of developing dental caries based on oral microbiome inhabitants.

In vitro assessment of shiitake mushroom (Lentinula edodes) extract for its antigingivitis activity.

Gingivitis is a preventable disease characterised by inflammation of the gums due to the buildup of a microbial biofilm at the gingival margin. It is implicated as a precursor to periodontitis, a much more serious problem which includes associated bone loss. Unfortunately, due to poor oral hygiene among the general population, gingivitis is prevalent and results in high treatment costs. Consequently, the option of treating gingivitis using functional foods, which promote oral health, is an attractive one. Medicinal mushrooms, including shiitake, have long been known for their immune system boosting as well as antimicrobial effects; however, they have not been employed in the treatment of oral disease. In the current study, the effectiveness of shiitake mushroom extract was compared to that of the active component in the leading gingivitis mouthwash, containing chlorhexidine, in an artificial mouth model (constant depth film fermenter). The total bacterial numbers as well as numbers of eight key taxa in the oral community were investigated over time using multiplex qPCR. The results indicated that shiitake mushroom extract lowered the numbers of some pathogenic taxa without affecting the taxa associated with health, unlike chlorhexidine which has a limited effect on all taxa.

The effects of fractions from shiitake mushroom on composition and cariogenicity of dental plaque microcosms in an in vitro caries model.

The aim of the current study was to investigate the anticariogenic potential of the (sub)fractions obtained from the edible mushroom shiitake (Lentinula edodes) in in vitro caries model. We used a modified constant depth film fermentor (CDFF) with pooled saliva as the inoculum and bovine dentin as a substratum. The test compounds were low molecular weight fraction (MLMW) of the shiitake extract and subfractions 4 and 5 (SF4 and SF5) of this fraction. Chlorhexidine (CHX) and water served as a positive and a negative control, respectively. Dentin mineral loss was quantified (TMR), microbial shifts within the microcosms were determined (qPCR), and the acidogenicity of the microcosms was assessed (CIA). From the compounds tested, the SF4 of shiitake showed strong inhibiting effect on dentin demineralization and induced microbial shifts that could be associated with oral health. The acid producing potential was increased, suggesting uncoupling of the glycolysis of the microbiota by the exposure to SF4. In conclusion, the results suggest that SF4 of shiitake has an anticariogenic potential.

Effects of fruit and vegetable low molecular mass fractions on gene expression in gingival cells challenged with Prevotella intermedia and Actinomyces naeslundii.

Low molecular mass (LMM) fractions obtained from extracts of raspberry, red chicory, and Shiitake mushrooms have been shown to be an useful source of specific antibacterial, antiadhesion/coaggregation, and antibiofilm agent(s) that might be used for protection towards caries and gingivitis. In this paper, the effects of such LMM fractions on human gingival KB cells exposed to the periodontal pathogens Prevotella intermedia and Actinomyces naeslundii were evaluated. Expression of cytokeratin 18 (CK18) and ß4 integrin (ß4INT) genes, that are involved in cell proliferation/differentiation and adhesion, and of the antimicrobial peptide ß2 defensin (HßD2) in KB cells was increased upon exposure to either live or heat-killed bacteria. All LMM fractions tested prevented or reduced the induction of gene expression by P. intermedia and A. naeslundii depending on the experimental conditions. Overall, the results suggested that LMM fractions could modulate the effects of bacteria associated with periodontal disease in gingival cells.

Effects of mushroom and chicory extracts on the physiology and shape of Prevotella intermedia, a periodontopathogenic bacterium.

Contrary to the common assumption that food has a negative impact on oral health, research has shown that several foods contain a number of components with antibacterial and antiplaque activity. These natural compounds may be useful for improving daily oral hygiene. In this study we evaluate the mode of antimicrobial action of fractions of mushroom and red chicory extracts on Prevotella intermedia, a periodontopathogenic bacterium. The minimal inhibitory concentration corresponded to 0.5x compared to the natural food concentration for both extracts. This concentration resulted in a bacteriostatic effect in mushroom extract and in a slightly bactericidal effect in chicory extract. Cell mass continued to increase even after division stopped. As regards macromolecular synthesis, DNA was almost totally inhibited upon addition of either mushroom or chicory extract, and RNA to a lesser extent, while protein synthesis continued. Cell elongation occurred after septum inhibition as documented by scanning electron microscopy and cell measurement. The morphogenetic effects are reminiscent of the mode of action of antibiotics such as quinolones or ß-lactams. The discovery of an antibiotic-like mode of action suggests that these extracts can be advantageously employed for daily oral hygiene in formulations of cosmetic products such as mouthwashes and toothpastes.

Testing a low molecular mass fraction of a mushroom (Lentinus edodes) extract formulated as an oral rinse in a cohort of volunteers.

Although foods are considered enhancing factors for dental caries and periodontitis, laboratory researches indicate that several foods and beverages contain components endowed with antimicrobial and antiplaque activities. A low molecular mass (LMM) fraction of an aqueous mushroom extract has been found to exert these activities in in vitro experiments against potential oral pathogens. We therefore conducted a clinical trial in which we tested an LMM fraction of shiitake mushroom extract formulated in a mouthrinse in 30 young volunteers, comparing the results with those obtained in two identical cohorts, one of which received water (placebo) and the other Listerine. Plaque index, gingival index and bacterial counts in plaque samples were determined in all volunteers over the 11 days of the clinical trial. Statistically significant differences (P < 0.05) were obtained for the plaque index on day 12 in subjects treated with mushroom versus placebo, while for the gingival index significant differences were found for both mushroom versus placebo and mushroom versus Listerine. Decreases in total bacterial counts and in counts of specific oral pathogens were observed for both mushroom extract and Listerine in comparison with placebo. The data suggest that a mushroom extract may prove beneficial in controlling dental caries and/or gingivitis/periodontitis.

Plant and fungal food components with potential activity on the development of microbial oral diseases.

This paper reports the content in macronutrients, free sugars, polyphenols, and inorganic ions, known to exert any positive or negative action on microbial oral disease such as caries and gingivitis, of seven food/beverages (red chicory, mushroom, raspberry, green and black tea, cranberry juice, dark beer). Tea leaves resulted the richest material in all the detected ions, anyway tea beverages resulted the richest just in fluoride. The highest content in zinc was in chicory, raspberry and mushroom. Raspberry is the richest food in strontium and boron, beer in selenium, raspberry and mushroom in copper. Beer, cranberry juice and, especially green and black tea are very rich in polyphenols, confirming these beverages as important sources of such healthy substances. The fractionation, carried out on the basis of the molecular mass (MM), of the water soluble components occurring in raspberry, chicory, and mushroom extracts (which in microbiological assays revealed the highest potential action against oral pathogens), showed that both the high and low MM fractions are active, with the low MM fractions displaying the highest potential action for all the fractionated extracts. Our findings show that more compounds that can play a different active role occur in these foods.

Reactive calcium-phosphate-containing poly(ester-co-ether) methacrylate bone adhesives: setting, degradation and drug release considerations.

This study has investigated novel bone adhesives consisting of fluid photo-polymerizable poly(lactide-co-propylene glycol-co-lactide)dimethacrylate (PGLA-DMA) mixed with systematically varying fillers of ß-tricalcium phosphate (ß-TCP) and monocalcium phosphate monohydrate (MCPM), for the delivery of an antibacterial drug chlorhexidine (CHX). All formulations were found to polymerize fully within 200 s after exposure to blue light. In addition, water sorption by the polymerized materials catalyzed varying filler conversion to dicalcium phosphate (DCP) (i.e. brushite and monetite). With greater DCP levels, faster degradation was observed. Moreover, increase in total filler content enhanced CHX release, associated with higher antibacterial activity. These findings thus suggest that such rapid-setting and degradable adhesives with controllable drug delivery property could have potential clinical value as bone adhesives with antibacterial activity.

Frictional properties of light-activated antimicrobial polymers in blood vessels.

The adhesion of microbes to catheter surfaces is a serious problem and the resulting infections frequently lead to longer hospitalisation and higher risk for the patient. Several approaches have been developed to produce materials that are less susceptible to microbial colonisation. One such approach is the incorporation of photoactivated compounds, such as Toluidine Blue O (TBO), in the polymeric matrix resulting in 'light-activated antimicrobial materials'. The insertion and removal of catheters can cause tissue damage and patient discomfort through frictional forces; hence the lubricity of a catheter material is also very important. In this work the tribological performance of silicone and polyurethane containing TBO and gold nanoparticles were evaluated using two different surfaces, the inner part of the aorta and the superior vena cava of sheep. Static and kinetic friction coefficients of these materials were measured using a tribometric device developed for in vitro applications using dry materials and those lubricated with blood. It was found that neither the preparation process nor the presence of TBO or gold nanoparticles, had an effect on the friction factors in comparison to those of untreated materials. In all cases, static and kinetic friction coefficients on aorta tissue were higher than those on vena cava due to higher surface roughness of the aorta. The presence of blood as a lubricant resulted in lower friction coefficients.

Cloning, heterologous gene expression and biochemical characterization of the alpha-1,3-glucanase from the filamentous fungus Penicillium purpurogenum.

There has been much recent interest in alpha-1,3-glucanases (mutanases) as they have the potential to be used in the treatment of dental caries. Mutanases have been reported in a number of bacteria, yeast and fungi but remain a relatively uncharacterised family of enzymes. In this study we heterologously expressed the mutanase gene from the filamentous fungus Penicillium purpurogenum to enable further characterization of its enzymatic activity. The mutanase cDNA was cloned and expressed in the methylotrophic yeast Pichia pastoris. The molecular mass of the secreted protein was about 102 kDa. The recombinant enzyme hydrolyzed mutan with a specific activity of 3.9 U/mg of protein. The recombinant enzyme was specific for mutan and could not cleave a variety of other polysaccharides demonstrating a specificity for alpha-1,3-glucosidic linkages. The pH and temperature optima were pH 4.6 and 45 degrees C, respectively. Synthetic compounds were also tested as substrates to assess whether the P. purpurogenum mutanase has an exo- or endo-type mechanism of hydrolysis. The results suggest an endo-hydrolytic mode of action. The type of mechanism was confirmed since mutanase activity was not suppressed in the presence of inhibitors of exo-type enzymes.