Organotypic model of the gingiva for studying bacterial and viral pathogens implicated in periodontitis.

Background: Three-dimensional (3D) tissue models bridge the gap between conventional two-dimensional cell cultures and animal models. The aim of this study was to develop an organotypic 3D gingival (OTG) model to provide a tool to investigate bacterial and viral pathogens in periodontitis. Methods: The OTG model composed of gingival fibroblasts (GFs) and telomerase-immortalized gingival keratinocytes (TIGKs) was constructed and applied to study infections by Porphyromonas gingivalis and herpes simplex virus 1 (HSV-1). Immunohistochemical staining, confocal microscopy, qPCR, titration techniques, and colony-forming unit counts were applied to interrogate epithelial markers expression, monitor P. gingivalis and HSV-1 presence, and evaluate the immune response along with the efficiency of antimicrobial drugs. Results: The OTG model resembled the morphology of the human gingiva. During infection, both pathogens penetrated deep into the tissue and persisted for a few days with P. gingivalis also forming a biofilm on the cell surface. The infection triggered the expression of inflammatory mediators in cells and both pathogens were efficiently eliminated by specific antimicrobials. Conclusions: Presented OTG model constitutes a simple and convenient tool to study the interaction between bacterial and viral pathogens within the gingival tissue, including penetration, persistence and biofilm formation. It is also suitable to examine the efficiency of antimicrobial drugs.

Subversion of Lipopolysaccharide Signaling in Gingival Keratinocytes via MCPIP-1 Degradation as a Novel Pathogenic Strategy of Inflammophilic Pathobionts.

Periodontal disease (PD) is an inflammatory disease of the supporting tissues of the teeth that develops in response to formation of a dysbiotic biofilm on the subgingival tooth surface. Although exacerbated inflammation leads to alveolar bone destruction and may cause tooth loss, the molecular basis of PD initiation and progression remains elusive. Control over the inflammatory reaction and return to homeostasis can be efficiently restored by negative regulators of Toll-like receptor (TLR) signaling pathways such as monocyte chemoattractant protein-induced protein 1 (MCPIP-1), which is constitutively expressed in gingival keratinocytes and prevents hyperresponsiveness in the gingiva. Here, we found that inflammophilic periodontal species influence the stability of MCPIP-1, leading to an aggravated response of the epithelium to proinflammatory stimulation. Among enzymes secreted by periodontal species, gingipains-cysteine proteases from Porphyromonas gingivalis-are considered major contributors to the pathogenic potential of bacteria, strongly influencing the components of the innate and adaptive immune system. Gingipain proteolytic activity leads to a rapid degradation of MCPIP-1, exacerbating the inflammatory response induced by endotoxin. Collectively, these results establish a novel mechanism of corruption of inflammatory signaling by periodontal pathogens, indicating new possibilities for treatment of this chronic disease. IMPORTANCE Periodontitis is a highly prevalent disease caused by accumulation of a bacterial biofilm. Periodontal pathogens use a number of virulence strategies that are under intensive study to find optimal therapeutic approaches against bone loss. In our work, we present a novel mechanism utilized by the key periodontal pathogen Porphyromonas gingivalis, based on the selective degradation of the negative regulator of inflammation, MCPIP-1. We found that the diminished levels of MCPIP-1 in gingival keratinocytes-cells at the forefront of the fight against bacteria-cause sensitization to endotoxins produced by other oral species. This results in an enhanced inflammatory response, which promotes the growth of inflammophilic pathobionts and damage of tooth-supporting tissues. Our observation is relevant to understanding the molecular basis of periodontitis and the development of new methods for treatment.

Antimicrobial photodynamic therapy effectively reduces Porphyromonas gingivalis infection in gingival fibroblasts and keratinocytes: An in vitro study.

BACKGROUND: Porphyromonas gingivalis possess the ability to invade host cells which prevents this pathogen from eradication by conventional periodontal therapy. Recently, antimicrobial photodynamic therapy (aPDT) was introduced to periodontal treatment as a complementary antibacterial method. The aim of this study was to evaluate the effect of toluidine blue-O (TBO) mediated aPDT on the viability of P. gingivalis invading gingival fibroblasts and keratinocytes in an in vitro model of infection. METHODS: Primary human gingival fibroblasts (PHGF) and telomerase immortalized gingival keratinocytes (TIGK) were infected with Pg ATCC 33277. Two concentrations of TBO (0.01 mg/mL, TBO-c1 and 0.001 mg/mL, TBO-c2) and a non-laser red light source (λ = 630 nm) were applied to treat both cell-adherent/intracellular Pg (the adhesion/invasion model) or exclusively the intracellular bacteria (the intracellular infection model). RESULTS: The median viability of cell-adherent/intracellular Pg in infected keratinocytes declined from 1.88 × 105 cfu/mL in infected cells treated with TBO without irradiation to 40 cfu/mL upon irradiation for 10 s with TBO-c1. At higher light doses a complete photokilling of P. gingivalis was observed. Pg from exclusively intracellular infection model was also efficiently eradicated as the residual viability dropped from 1.44 × 105 cfu/mL in control samples to 160, 20 and 10 cfu/mL upon irradiation for 10, 20 and 30 s, respectively. In the infected fibroblasts irradiation significantly reduced bacterial viability but did not completely eradicate the intracellular pathogen. CONCLUSIONS: Antimicrobial PDT is effective in reducing the viability of intracellular periopathogens, however those residing within gingival fibroblasts seems to attenuate the photokilling effectiveness of this method.

Evaluation of interleukin-1 beta and the ratio of interleukin-1 beta to interleukin-1 receptor antagonist in gingival crevicular fluid during orthodontic canine retraction.

BACKGROUND: Orthodontic tooth movement (OTM) is a complex phenomenon mediated by cytokines, of which interleukin-1 beta (IL­1ß) is potently involved in the remodeling of the periodontal ligament (PDL) and bone. Whether the pattern of IL­1ß release differs at the sides of tension and compression is not yet clarified. OBJECTIVES: The aim of the present study was to evaluate the level of IL­1ß and the ratio of IL­1ß to interleukin-1 receptor antagonist (IL­1RA) in gingival crevicular fluid (GCF) at the tension and compression sides during orthodontic canine retraction. MATERIAL AND METHODS: Seventeen patients scheduled for orthodontic treatment with bilateral extraction of maxillary first premolars and canine retraction were enrolled. Tooth 2.3 was retracted, teeth 1.3 and 3.3 served as controls. Gingival crevicular fluid samples were collected from the tension and compression sides of each tooth at baseline (before the 1st activation - day 0) and at days 2 and 7, and then again before the 2nd activation (day 28) and at days 30 and 35. The levels of IL­1ß and IL­RA were evaluated with the enzyme-linked immunosorbent assay (ELISA). RESULTS: After the 1st activation, a statistically significant increase in the level of IL­1ß was observed at teeth 2.3 (p < 0.03 mesially and p < 0.05 distally) and 1.3 (p < 0.05 mesially and distally), both at the tension and compression sides. The 2nd activation resulted in a gradual increase in the IL­1ß level at both canines; however, statistical significance was reached only for tooth 2.3 (p < 0.05 mesially and p < 0.02 distally). In terms of the IL­1ß/IL­1RA ratio, a significant increase was observed only at the compression side of the experimental tooth (p < 0.01). CONCLUSIONS: An increase in the IL­1ß level in GCF was observed both at the tension and compression sides of the actively retracted canine 2.3 as well as the contralateral canine 1.3; a significant rise in the IL­1ß/IL­1RA ratio was noted only at the compression side of the experimental tooth 2.3, indicating the zone of active bone resorption.

Evaluation of depression, anxiety and stress levels in patients with oral lichen planus.

The pathogenesis of oral lichen planus (OLP) remains to be fully elucidated; however, certain psychoneurological factors may influence the onset and exacerbation of OLP. The aim of the present study is to evaluate the intensity of negative emotions in patients with OLP. A cross-sectional, questionnaire-based study was performed. The sample consisted of 52 subjects, comprising 26 patients with OLP (OLP group) and 26 controls (CTRL group). The Depression Anxiety Stress Scale 21 (DASS-21) was used for psychometric evaluation. The patients were also asked about their attitude toward the disease, treatment, and interference of the disease on daily life. The mean level of depression was 12.54 ± 6.6 in the OLP group and 7.69 ± 5.22 in the CTRL group (P = 0.006). The mean level of anxiety was 11.15 ± 7.95 in the OLP group and 6.62 ± 4.86 in the CTRL group (P = 0.018). The mean stress levels were 8.69 ± 7.06 and 3.85 ± 3.18 in the OLP and CTRL groups, respectively (P = 0.003). Severe and very severe scores of depression and very severe scores of anxiety and stress were present in the OLP group, whereas these emotions were normal in the majority of controls. Depression, stress, and anxiety may be involved in the pathogenesis and course of OLP.

Intraoral manifestation of systemic AL amyloidosis with unique microscopic presentation of intracellular amyloid deposition in striated muscles.

We report the history of a 59-year old patient with systemic AL amyloidosis of intraoral manifestation. The patient first presented with complaints about dysphagia and remarkable enlargement of the tongue with highly reduced mobility, as well as bilateral submucosal thickenings on the cheeks. Histopathological examination of the incisional biopsy of the buccal mucosa and underlying tissues revealed AL amyloidosis. The microscopic presentation was, however, unique, as the amyloid deposits were present intracellularly in the striated muscles. The subsequent bone marrow biopsy confirmed the diagnosis of primary amyloidosis/multiple myeloma - associated amyloidosis.

Periopathogens differ in terms of the susceptibility to toluidine blue O-mediated photodynamic inactivation.

BACKGROUND: The main goal of periodontal therapy is to eliminate the infection spreading in periodontium. Antimicrobial photodynamic therapy may be applied in order to eradicate pathogens remaining in periodontal tissues after conventional mechanical debridement, to improve the treatment results. The aim of this in vitro study was to evaluate the susceptibility of selected key periopathogens to toluidine blue O-mediated photodynamic inactivation and the influence of photosensitizer's concentration and light dose on the effectiveness of this process. METHODS: Following bacterial strains were used in the experiments: Porphyromonas gingivalis ATCC 33277, Aggregatibacter actinomyctemecomitans ATCC 33384, Fusobacterium nucleatum ATCC 10953. Toluidine blue O (TBO) was used in concentration ranging from 0.004 to 0.5mg/mL. Irradiation was performed by a non-laser red light source. RESULTS: Complete eradication of P. gingivalis was obtained upon the application of TBO in the concentration of 0.1mg/mL and the highest light dose. A, actinomycetemcomitans was, in turn, not susceptible to photodynamic inactivation regardless of the dosimetric parameters applied. High viability reductions were also obtained for F. nucleatum, however no complete eradication. The effectiveness of photodynamic inactivation of susceptible periopathogens was dependent on the light dose and photosensitizer's concentration. CONCLUSIONS: Periopathogens differ in terms of their susceptibility to photodynamic inactivation. Antimicrobial PDT may be valuable in the treatment of those cases of periodontal disease, in which P. gingivalis is a dominating pathogen. Microbiological examination prior to clinical application of aPDT may be recommended.

[Application of photodynamic therapy in dentistry ­ literature review].

Photodynamic therapy (PDT) is based on the principle that the target cells are destroyed by means of toxic reactive oxygen species generated upon the interaction of a photosensitizer, light and oxygen. This method is nowadays widely applied in various branches of medicine, mainly in oncology and dermatology. It is also applied in dentistry in the treatment of oral potentially malignant disorders (like lichen planus or leukoplakia) and infectious conditions (periodontitis, herpetic cheilitis, root canal disinfection). The application of the photodynamic therapy in the abovementioned indications is worth attention, as the method is noninvasive, painless, and the results of the published studies seem promising. The present article aims at presenting the principle of the photodynamic therapy and, based on the literature, the possibilities and results of its application in dentistry.

Antimicrobial photodynamic therapy-A discovery originating from the pre-antibiotic era in a novel periodontal therapy.

Antimicrobial photodynamic therapy (aPDT) involves pathogens' destruction caused by means of toxic Reactive Oxygen Species (ROS) that are generated upon the interaction of a photoactivatable substance (photosensitizer), light of the appropriate wavelength and oxygen. Among many clinical applications, it is also used as a supplementary method of treatment of periodontal disease. Many in vitro studies confirmed, that a major periopathogenic bacterium, Porphyromonas gingivalis is susceptible to this method. Several animal model studies pointed, that even a single application of aPDT adjunctive to conventional scaling and root planning (SRP) promotes better tissue healing, reduces the inflammatory infiltrate and bone loss. The outcomes of clinical trials are, however, inconsistent. Although in several the superiority of combined treatment protocol (SRP+aPDT) over the conventional (SRP alone) was reported, it was not confirmed in other trials. Nonetheless, the reduction of bleeding indices favoring the combined therapy was observed in the majority of the studies. It indicates, that aPDT has an influence on the extent of inflammation and further studies are needed to establish an optimal protocol of treatment combining mechanical debridement with photochemotherapy in order to obtain good treatment outcomes in our patients.