Functional assessment of peptide-modified PLGA nanoparticles against oral biofilms in a murine model of periodontitis.

The interaction of the periodontal pathogen Porphyromonas gingivalis (Pg) with commensal streptococci promotes Pg colonization of the oral cavity. Previously, we demonstrated that a peptide (BAR) derived from Streptococcus gordonii (Sg) potently inhibited adherence of Pg to streptococci and reduced Pg virulence in a mouse model of periodontitis. Thus, BAR may represent a novel therapeutic to control periodontitis by preventing Pg colonization of the oral cavity. However, while BAR inhibited the initial formation of Pg/Sg biofilms, much higher concentrations of peptide were required to disrupt an established Pg/Sg biofilm. To improve the activity of the peptide, poly(lactic-co-glycolic acid) (PLGA) nanoparticles were surface-modified with BAR and shown to more potently disrupt Pg/Sg biofilms relative to an equimolar amount of free peptide. The goal of this work was to determine the in vivo efficacy of BAR-modified NPs (BNPs) and to assess the toxicity of BNPs against human gingival epithelial cells. In vivo efficacy of BNPs was assessed using a murine model of periodontitis by measuring alveolar bone resorption and gingival IL-17 expression as outcomes of Pg-induced inflammation. Infection of mice with Pg and Sg resulted in a significant increase in alveolar bone loss and gingival IL-17 expression over sham-infected animals. Treatment of Pg/Sg infected mice with BNPs reduced bone loss and IL-17 expression almost to the levels of sham-infected mice and to a greater extent than treatment with an equimolar amount of free BAR. The cytotoxicity of the maximum concentration of BNPs and free BAR used in in vitro and in vivo studies (1.3 and 3.4 µM), was evaluated in telomerase immortalized gingival keratinocytes (TIGKs) by measuring cell viability, cell lysis and apoptosis. BNPs were also tested for hemolytic activity against sheep erythrocytes. TIGKs treated with BNPs or free BAR demonstrated >90% viability and no significant lysis or apoptosis relative to untreated cells. In addition, neither BNPs nor free BAR exhibited hemolytic activity. In summary, BNPs were non-toxic within the evaluated concentration range of 1.3-3.4 µM and provided more efficacious protection against Pg-induced inflammation in vivo, highlighting the potential of BNPs as a biocompatible platform for translatable oral biofilm applications.

In vitro assessment of stainless steel orthodontic brackets coated with titanium oxide mixed Ag for anti-adherent and antibacterial properties against Streptococcus mutans and Porphyromonas gingivalis.

Orthodontic brackets made from stainless steel were introduced in dentistry, though they have less ability in reducing enamel demineralization and are not successful in preventing microbial as well as biofilm growth. In this study, we evaluated the significant role of different brackets in reducing enamel demineralization indirectly. Results from different tests indicate the significant reduction in adhesion, biofilm formation and slow growth of tested bacterial species on brackets coated with Ag + TiO2 and found to be statistically significant lower than control. There was no loss in cell viability in all brackets indicating that the cells are biocompatible with different bracket materials. Scanning electron microscopy showed less bacteria attached with the surface coated with Ag + TiO2 indicated that bacteria were losing adherent nature on coated surface. In conclusion, TiO2+Ag coating on stainless steel brackets possessed anti-adherent properties and also have demonstrable antibacterial properties therefore helps in preventing dental caries and plaque accumulation indirectly. The cell compatibility of TiO2+Ag coated brackets is superior to the uncoated samples therefore can be used in orthodontics as it not only provide suitable antimicrobial activity and resistance to biofilm formation but also sustained the cell viability of human gingival fibroblast (HGF) cell lines.

Assessment of Photodynamic Inactivation against Periodontal Bacteria Mediated by a Chitosan Hydrogel in a 3D Gingival Model.

Chitosan hydrogels containing hydroxypropyl methylcellulose (HPMC) and toluidine blue O were prepared and assessed for their mucoadhesive property and antimicrobial efficacy of photodynamic inactivation (PDI). Increased HPMC content in the hydrogels resulted in increased mucoadhesiveness. Furthermore, we developed a simple In Vitro 3D gingival model resembling the oral periodontal pocket to culture the biofilms of Staphylococcus aureus (S. aureus), Aggregatibacter actinomycetemcomitans (A. actinomycetemcomitans), and Porphyromonas gingivalis (P. gingivalis). The PDI efficacy of chitosan hydrogel was examined against periodontal biofilms cultured in this 3D gingival model. We found that the PDI effectiveness was limited due to leaving some of the innermost bacteria alive at the non-illuminated site. Using this 3D gingival model, we further optimized PDI procedures with various adjustments of light energy and irradiation sites. The PDI efficacy of the chitosan hydrogel against periodontal biofilms can significantly improve via four sides of irradiation. In conclusion, this study not only showed the clinical applicability of this chitosan hydrogel but also the importance of the light irradiation pattern in performing PDI for periodontal disease.

Assessment of the antibacterial, cytotoxic and mutagenic potential of the phenolic-rich hydroalcoholic extract from Copaifera trapezifolia Hayne leaves.

Copaifera trapezifolia Hayne occurs in the Atlantic Rainforest, which is considered one of the most important and endangered tropical forests on the planet. Although literature works have described many Copaifera spp., their biological activities remain little known. In the present study, we aimed to evaluate (1) the potential of the hydroalcoholic extract from C. trapezifolia leaves (CTE) to act against the causative agents of tooth decay and apical periodontitis and (2) the cytotoxicity and mutagenicity of CTE to ensure that it is safe for subsequent application. Concerning the tested bacteria, the MIC and the minimum bactericidal concentration of CTE varied between 100 and 400 µg ml-1. The time-kill assay conducted at a CTE concentration of 100 µg ml-1 evidenced bactericidal activity against Porphyromonas gingivalis (ATCC 33277) and Peptostreptococcus micros (clinical isolate) within 72 h. CTE at 200 µg ml-1 inhibited Porphyromonas gingivalis and Peptostreptococcus micros biofilm formation by at least 50 %. A combination of CTE with chlorhexidine dichlorohydrate did not prompt any synergistic effects. The colony-forming assay conducted on V79 cells showed that CTE was cytotoxic at concentrations above 156 µg ml-1. CTE exerted mutagenic effect on V79 cells, but the micronucleus test conducted on Swiss mice and the Ames test did not reveal any mutagenicity. Therefore, the use of standardized and safe extracts could be an important strategy to develop novel oral care products with antibacterial action. These extracts could also serve as a source of compounds for the discovery of new promising biomolecules.

Assessment of the effect of chemical agents used in dentistry on the removal of Porphyromonas gingivalis and Escherichia coli from sandblasted acid-etched titanium dental implants: an in vitro study.

PURPOSE: The aim of this study was to evaluate the capability of chemicals to decontaminate Escherichia coli (E coli) or Porphyromonas gingivalis (P gingivalis) from sandblasted acid-etched (SAE) titanium dental implants. MATERIALS AND METHODS: SAE titanium dental implants were contaminated with E coli or P gingivalis and incubated in a sterile bacterial culture media under aerobic and anaerobic conditions, respectively. The implants were treated with 10 different conditions: calcium hydroxide [Ca(OH)2] paste for 1 minute and saline irrigation for 1 minute; Ca(OH)2 paste for 1 minute and 0.2% chlorhexidine digluconate (CHXD) irrigation for 1 minute; 0.2% CHXD for 1 minute; Dakin's solution for 1 minute; tetracycline hydrochloride (T-HCl) as a 1 g per 20 mL solution for 1, 2, and 3 minutes; and T-HCl paste for 1, 2, and 3 minutes. All implants were irrigated with 1 mL of saline solution and incubated under aerobic and anaerobic conditions for 24 hours or 48 hours for E coli- and P gingivalis-contaminated implants, respectively. The control group was submitted to all procedures except for the chemical treatments. Aliquots were removed, and turbidity was measured by spectrophotometry. The level of bacterial growth in control cultures was considered to have a decontamination percentage (DC%) of 0. RESULTS: Spectrophotometric analysis showed that all chemical treatments resulted in significantly higher DC% compared to controls for SAE implants contaminated with E coli (P < .05) or P gingivalis (P < .05). For the P gingivalis experiments, SAE implants treated with Ca(OH)2 paste and saline solution had a lower DC% (39.3%) than those in the other treatment groups. In the E coli experiments, DC% was significantly lower for SAE implants treated with Ca(OH)2 paste and saline solution (48.7%), Dakin's solution (92.7%), or T-HCl paste for 1 minute (96.6%) than those in the other groups. CONCLUSION: The DC% of SAE implants contaminated with E coli or P gingivalis by means of chemicals commonly used in dentistry is high, with the exception of Ca(OH)2 paste burnished for 1 minute and then irrigated with saline solution for 1 minute.

Assessment of viable periodontal pathogens by reverse transcription quantitative polymerase chain reaction.

BACKGROUND AND OBJECTIVE: Molecular biological methods for the detection of periodontitis-associated bacteria based on DNA amplification have many advantages over classical culture techniques. However, when it comes to assessing immediate therapeutic success, e.g. reduction of viable bacteria, DNA-based polymerase chain reaction is unsuitable because it does not distinguish between live and dead bacteria. Our objective was to establish a simple RNA-based method that is easily set up and allows reliable assessment of the live bacterial load. MATERIAL AND METHODS: We compared conventional quantitative real-time PCR (qPCR), propidium monoazide-qPCR and reverse transcription qPCR (RT-qPCR) for the detection of periodontal pathogens after antibiotic treatment in vitro. Applicability was tested using clinical samples of subgingival plaque obtained from patients at different treatment stages. RESULTS: The bacterial load was remarkably stable over prolonged periods when assessed by conventional qPCR, while both propidium monoazide intercalation as well as cDNA quantitation showed a decline according to decreasing numbers of viable bacteria after antibiotic treatment. Clinical samples of subgingival plaque were directly subjected to DNase I treatment and RT without previous extraction or purification steps. While the results of the DNA- and RNA-based methods are comparable in untreated patients, the classical qPCR frequently detected substantial bacterial load in treated patients where RT-qPCR no longer indicates the presence of those pathogens. The disagreement rates ranged between 4 and 20% in first visit patients and 8-50% in the group of currently treated patients. CONCLUSION: We propose to use RNA-based detection methods to verify the successful eradication of periodontal pathogens.

[Efficient, minimally invasive periodontal therapy. An evidence based treatment concept]. / Effiziente, minimal-invasive Parodontitistherapie. Ein evidenzbasiertes Behandlungskonzept.

A large number of reports indicate beneficial effects of systemic antibiotics for patients with periodontal diseases. Although these advantages are clear in general, the specific relationship of benefit and risk in various clinical situations remains a subject of debate. Uncertainties persist regarding the individual prescription and combination with other procedures. Since the early 1990s it has been pointed out that systemic antibiotics given in the context of non-surgical subgingival debridement may reduce the need for periodontal surgery. Recent studies confirm these findings especially with regard to the combination of amoxicillin and metronidazole. However, these antibiotics should not be viewed as a substitute for thorough debridement, or as a means to compensate for improper oral hygiene. In addition, current evidence does not provide evidence for the indiscriminate use of just any antibiotic in any periodontal patient. A treatment protocol implementing the recent evidence is shown. It is noteworthy to realize that chronic and aggressive periodontitis today can be treated successfully with simple and cost-effective means in most instances.

The clinical relevance of microbiologic testing: a comparative analysis of microbiologic samples secured from the same sites and cultured in two independent laboratories.

A field study using five different private periodontal practices was conducted; it compared two microbiologic culture samples simultaneously secured from the same sites within 23 individual patients and submitted for bacterial identification and antibiotic sensitivity testing to two separate laboratories. The results from the two laboratories were often different. In no instance did both laboratories agree on the presence of identical bacterial species. When only bacteria above threshold levels were compared, agreement was found in only nine of 23 cases. When examining antibiotic sensitivity, using 100% kill of all tested pathogens as the ideal, agreement between the two laboratories was poor. The laboratories agreed on the use of amoxicillin 17% of the time, tetracycline 26% of the time, and metronidazole 48% of the time. The use of amoxicillin and metronidazole in combination yielded a 78% agreement when the results of both laboratories were combined. It would appear from the data that the empirical use of amoxicillin-metronidazole combination therapy may be more clinically sound and cost effective than culturing and antibiotic selection based on the results of culture from any single microbiologic testing laboratory.