Inhomogeneity of stiffness and density of the extracellular matrix within the leukoplakia of human oral mucosa as potential physicochemical factors leading to carcinogenesis.

Oral leukoplakia is a clinical term relating to various morphological lesions, including squamous cell hyperplasia, dysplasia and carcinoma. Leukoplakia morphologically manifested as hyperplasia with epithelial dysplasia is clinically treated as precancerous condition. Nevertheless, there is a lack of good markers indicating the transformation of premalignancies towards cancer. A better understanding of the mechanical environment within the tissues where tumors grow might be beneficial for the development of prevention, diagnostic, and treatment methods in cancer management. Atomic force microscopy (AFM) and immunohistology techniques were used to assess changes in the stiffness and morphology of oral mucosa and leukoplakia samples at different stages of their progression towards cancer. The Young's moduli of the tested leukoplakia samples were significantly higher than those of the surrounding mucus. Robust inhomogeneity of stiffness within leukoplakia samples, reflecting an increase in regeneration and collagen accumulation (increasing density) in the extracellular matrix (ECM), was observed. Within the histologically confirmed cancer samples, Young's moduli were significantly lower than those within the precancerous ones. Inhomogeneous stiffness within leukoplakia might act as "a mechanoagonist" that promotes oncogenesis. In contrast, cancer growth might require the reorganization of tissue structure to create a microenvironment with lower and homogenous stiffness. The immunohistology data collected here indicates that changes in tissue stiffness are achieved by increasing cell/ECM density. The recognition of new markers of premalignancy will aid in the development of new therapies and will expand the diagnostic methods.

Bacteria Residing at Root Canals Can Induce Cell Proliferation and Alter the Mechanical Properties of Gingival and Cancer Cells.

Understanding the importance of oral microbiota in human health and disease also leads to an expansion of the knowledge on functional, metabolic, and molecular alterations directly contributing to oral and systemic pathologies. To date, a compelling number of studies have documented the crucial role of some oral cavity-occurring microbes in the initiation and progression of cancers. Although this effect was noted primarily for Fusobacterium spp., the potential impact of other oral microbes is also worthy of investigation. In this study, we aimed to assess the effect of Enterococcus faecalis, Actinomyces odontolyticus, and Propionibacterium acnes on the proliferation capability and mechanical features of gingival cells and cell lines derived from lung, breast, and ovarian cancers. For this purpose, we incubated selected cell lines with heat-inactivated bacteria and supernatants collected from biofilms, cultured in both anaerobic and aerobic conditions, in the presence of surgically removed teeth and human saliva. The effect of oral bacteria on cell population growth is variable, with the highest growth-promoting abilities observed for E. faecalis in relation to human primary gingival fibroblasts (HGF) and lung cancer A549 cells, and P. acnes in relation to breast cancer MCF-7 and ovarian cancer SKOV-3 cells. Notably, this effect seems to depend on a delicate balance between the pro-stimulatory and toxic effects of bacterial-derived products. Regardless of the diverse effect of bacterial products on cellular proliferation capability, we observed significant alterations in stiffness of gingival and lung cancer cells stimulated with E. faecalis bacteria and corresponding biofilm supernatants, suggesting a novel molecular mechanism involved in the pathogenesis of diseases in oral cavities and tooth tissues. Accordingly, it is proposed that analysis of cancerogenic features of oral cavity bacteria should be multivariable and should include investigation of potential alterations in cell mechanical properties. These findings corroborate the important role of oral hygiene and root canal treatment to assure the healthy stage of oral microbiota.

Antimicrobial and Physicochemical Properties of Artificial Saliva Formulations Supplemented with Core-Shell Magnetic Nanoparticles.

Saliva plays a crucial role in oral cavity. In addition to its buffering and moisturizing properties, saliva fulfills many biofunctional requirements, including antibacterial activity that is essential to assure proper oral microbiota growth. Due to numerous extra- and intra-systemic factors, there are many disorders of its secretion, leading to oral dryness. Saliva substitutes used in such situations must meet many demands. This study was design to evaluate the effect of core-shell magnetic nanoparticles (MNPs) adding (gold-coated and aminosilane-coated nanoparticles NPs) on antimicrobial (microorganism adhesion, biofilm formation), rheological (viscosity, viscoelasticity) and physicochemical (pH, surface tension, conductivity) properties of three commercially available saliva formulations. Upon the addition of NPs (20 µg/mL), antibacterial activity of artificial saliva was found to increase against tested microorganisms by 20% to 50%. NPs, especially gold-coated ones, decrease the adhesion of Gram-positive and fungal cells by 65% and Gram-negative bacteria cells by 45%. Moreover, the addition of NPs strengthened the antimicrobial properties of tested artificial saliva, without influencing their rheological and physicochemical properties, which stay within the range characterizing the natural saliva collected from healthy subjects.

Artificial Saliva: Challenges and Future Perspectives for the Treatment of Xerostomia.

The chronic sensation of a dry mouth is a disease condition called xerostomia and affects a large part of the population. Xerostomia is associated with decreased secretion, or more often, qualitative changes in saliva proteins and immunoglobulin concentrations that develop as a result of salivary gland dysfunction. Several reasons causing dry mouth were described, and usually, they include taking medications, diseases or radiotherapy. In some situations, when it is difficult to use salivary stimulants or salivary gland damage is irreversible, the only option might seem to be saliva substitutes. The paper presents the most important aspects considering saliva preparations. The rheological and lubricating properties and the reconstruction of the complex saliva structure has been the main purpose of research. The biological properties of saliva preparations were also widely discussed. As part of the work, the antimicrobial effect of three commercial saliva preparations was tested. Finally, inadequate antimicrobial properties against the strains isolated from the oral cavity were demonstrated. The development of salivary substitutes, in particular, the improvement of antimicrobial properties, can be achieved using nanotechnology, including drug delivery systems containing nanocarriers.

Beneficial Effects of Vitamins K and D3 on Redox Balance of Human Osteoblasts Cultured with Hydroxyapatite-Based Biomaterials.

Hydroxyapatite-based biomaterials are commonly used in surgery to repair bone damage. However, the introduction of biomaterials into the body can cause metabolic alterations, including redox imbalance. Because vitamins D3 and K (K1, MK-4, MK-7) have pronounced osteoinductive, anti-inflammatory, and antioxidant properties, it is suggested that they may reduce the adverse effects of biomaterials. The aim of this study was to investigate the effects of vitamins D3 and K, used alone and in combination, on the redox metabolism of human osteoblasts (hFOB 1.19 cell line) cultured in the presence of hydroxyapatite-based biomaterials (Maxgraft, Cerabone, Apatos, and Gen-Os). Culturing of the osteoblasts in the presence of hydroxyapatite-based biomaterials resulted in oxidative stress manifested by increased production of reactive oxygen species and decrease of glutathione level and glutathione peroxidase activity. Such redox imbalance leads to lipid peroxidation manifested by an increase of 4-hydroxynonenal level, which is known to influence the growth of bone cells. Vitamins D3 and K were shown to help maintain redox balance and prevent lipid peroxidation in osteoblasts cultured with hydroxyapatite-based biomaterials. The strongest effect was observed for the combination of vitamin D3 and MK-7. Moreover, vitamins promoted growth of the osteoblasts, manifested by increased DNA biosynthesis. Therefore, it is suggested that the use of vitamins D3 and K may protect redox balance and support the growth of osteoblasts affected by hydroxyapatite-based biomaterials.

Cross talk between redox signalling and metabolic activity of osteoblasts and fibroblasts in the presence of hydroxyapatite-based biomaterials influences bone regeneration.

Regeneration of bone tissue defects that result from metabolic disorders, including periodontal diseases, can be supported by biomaterials based on hydroxyapatite. Despite of good biocompatibility of biomaterials they can cause oxidative stress and inflammatory processes as a result of mechanical interaction with surrounding tissues. Because osteoblasts are responsible for bone regeneration process in which gingival fibroblasts may also participate, the aim of the work was to investigate the influence of hydroxyapatite-based biomaterials (allogeneic and xenogeneic) and biomaterials combined with enamel matrix derivative (Emdogain) on osteoblast and fibroblast redox balance in the context of osteoblast proliferation and differentiation. The results showed that examined substitutes were not cytotoxic in vitro, but affected redox balance of osteoblasts and fibroblasts (ROS level increase and GSH level decrease) which led to oxidative stress (MDA and protein carbonyl groups level increase) resulting in an increase of the Nrf2 and NFκB expression. The consequence of these changes was partial inhibition of proliferation and osteoblast differentiation. Emdogain alone and combined with biomaterials decreased ROS generation and increased GSH level in both osteoblasts and fibroblasts leading to reduction of transcription factors expression especially proinflammatory NFκB, which promoted osteoblast differentiation and mineralization process.

The Role of Oral Cavity Biofilm on Metallic Biomaterial Surface Destruction-Corrosion and Friction Aspects.

Metallic biomaterials in the oral cavity are exposed to many factors such as saliva, bacterial microflora, food, temperature fluctuations, and mechanical forces. Extreme conditions present in the oral cavity affect biomaterial exploitation and significantly reduce its biofunctionality, limiting the time of exploitation stability. We mainly refer to friction, corrosion, and biocorrosion processes. Saliva plays an important role and is responsible for lubrication and biofilm formation as a transporter of nutrients for microorganisms. The presence of metallic elements in the oral cavity may lead to the formation of electro-galvanic cells and, as a result, may induce corrosion. Transitional microorganisms such as sulfate-reducing bacteria may also be present among the metabolic microflora in the oral cavity, which can induce biological corrosion. Microorganisms that form a biofilm locally change the conditions on the surface of biomaterials and contribute to the intensification of the biocorrosion processes. These processes may enhance allergy to metals, inflammation, or cancer development. On the other hand, the presence of saliva and biofilm may significantly reduce friction and wear on enamel as well as on biomaterials. This work summarizes data on the influence of saliva and oral biofilms on the destruction of metallic biomaterials.

Histone deacetylases affect transcriptional regulation of CCL2 and CXCL8 expression by pulmonary fibroblasts in vitro.

INTRODUCTION: Chemokines have been shown to play an important role in tissue remodeling and fibrosis in the respiratory system. In this study we wanted to evaluate the mechanisms, which regulate the expression of selected chemokines by pulmonary fibroblasts in vitro. MATERIAL AND METHODS: Pulmonary fibroblasts were cultured with and without bacterial lipopolysaccharide (LPS) for 6 hours. In addition some of the cultures were pre-treated with histone deacetylase inhibitor Trichostatin A (TSA). Real-time PCR reaction was performed to estimate the expression of chemokines CCL2, CCL3 and CXCL8. RESULTS: In unstimulated cultures detectable expression of CCL2 and CXCL8 was observed, while CCL3 expression could not be detected. After stimulation with LPS, TSA and both agents together CCL2 expression rose by 1.52, 1.62 and 1.8 times in comparison to control cultures respectively. CXCL8 mRNA expression levels after stimulation with LPS, TSA and LPSTSA increased by 1.53, 1.91 and 2.4 times accordingly. CONCLUSION: Epigenetic mechanisms related to histone acetylation affects transcriptional regulation of CCL2 and CXCL8 expression by pulmonary fibroblasts. Those mechanisms may play a role in tissue repair and pathologic remodeling.

Use of magnetic nanoparticles as a drug delivery system to improve chlorhexidine antimicrobial activity.

Nanotechnology offers new tools for developing therapies to prevent and treat oral infections, particularly biofilm-dependent disorders, such as dental plaques and endodontic and periodontal diseases. Chlorhexidine (CHX) is a well-characterized antiseptic agent used in dentistry with broad spectrum activity. However, its application is limited due to inactivation in body fluid and cytotoxicity toward human cells, particularly at high concentrations. To overcome these limitations, we synthesized nanosystems composed of aminosilane-coated magnetic nanoparticles functionalized with chlorhexidine (MNP@CHX). In the presence of human saliva, MNPs@CHX displayed significantly greater bactericidal and fungicidal activity against planktonic and biofilm-forming microorganisms than free CHX. In addition, CHX attached to MNPs has an increased ability to restrict the growth of mixed-species biofilms compared to free CHX. The observed depolarization of mitochondria in fungal cells treated with MNP@CHX suggests that induction of oxidative stress and oxidation of fungal structures may be a part of the mechanism responsible for pathogen killing. Nanoparticles functionalized by CHX did not affect host cell proliferation or their ability to release the proinflammatory cytokine, IL-8. The use of MNPs as a carrier of CHX has great potential for the development of antiseptic nanosystems.

Formulation and candidacidal activity of magnetic nanoparticles coated with cathelicidin LL-37 and ceragenin CSA-13.

Fungal infections caused by Candida spp. represent an emerging problem during treatment of immunocompromised patients and those hospitalized with serious principal diseases. The ever-growing number of fungal strains exhibiting drug resistance necessitates the development of novel antimicrobial therapies including those based on membrane-permeabilizing agents and nanomaterials as drug carriers. In this study, the fungicidal activities of LL-37 peptide, ceragenin CSA-13 and its magnetic derivatives (MNP@LL-37, MNP@CSA-13) against laboratory and clinical strains of C. albicans, C. glabrata and C. tropicalis were evaluated. These experiments confirm the high anti-fungal activity of these well-characterized agents mediated by their interaction with the fungal membrane and demonstrate elevated activity following immobilization of LL-37 and CSA-13 on the surface of magnetic nanoparticles (MNPs). Furthermore, MNP-based nanosystems are resistant to inhibitory factors present in body fluids and effectively inhibit formation of fungal biofilm. Simultaneously, synthesized nanostructures maintain immunomodulatory properties, described previously for free LL-37 peptide and CSA-13 substrate and they do not interfere with the proliferation and viability of osteoblasts, confirming their high biocompatibility.

Sporicidal activity of ceragenin CSA-13 against Bacillus subtilis.

Spore-forming bacteria are a class of microorganisms that possess the ability to survive in extreme environmental conditions. Morphological features of spores assure their resistance to stress factors such as high temperature, radiation, disinfectants, and drying. Consequently, spore elimination in industrial and medical environments is very challenging. Ceragenins are a new class of cationic lipids characterized by a broad spectrum of bactericidal activity resulting from amphipathic nature and membrane-permeabilizing properties. To assess the impact of ceragenin CSA-13 on spores formed by Bacillus subtilis (ATCC 6051), we performed the series of experiments confirming that amphipathic and membrane-permeabilizing properties of CSA-13 are sufficient to disrupt the structure of B. subtilis spores resulting in decreased viability. Raman spectroscopy analysis provided evidence that upon CSA-13 treatment the number of CaDPA-positive spores was clearly diminished. As a consequence, a loss of impermeability of the inner membranes of spores, accompanied by a decrease in spore resistance and killing take place. In addition to their broad antimicrobial spectrum, ceragenins possess great potential for development as new sporicidal agents.

Magnetic nanoparticles as a drug delivery system that enhance fungicidal activity of polyene antibiotics.

This study was designed to assess the antifungal/anti-biofilm and hemolytic properties of two polyene antibiotics, amphotericin B (AMF) and nystatin (NYS), attached to the surface of magnetic nanoparticles (MNP) against clinical isolates of Candida species and human red blood cells, respectively. The developed nanosystems, MNP@AMF and MNP@NYS, displayed stronger fungicidal activity than unbound AMF or NYS. Synergistic activity was observed with a combination of polyenes and MNPs against all tested Candida strains. Nanosystems were more potent than unbound agents when tested against Candida strains in the presence of pus, and as agents able to prevent Candida biofilm formation. The observed inactivation of catalase Cat1 in Candida cells upon treatment with the nanosystems suggests that disruption of the oxidation-reduction balance is a mechanism leading to inhibition of Candida growth. The significant decrease of polyenes lytic activity against host cells after their attachment to MNPs surface indicates improvement in their biocompatibility.

The assessment of sIgA, histatin-5, and lactoperoxidase levels in saliva of adolescents with dental caries.

BACKGROUND: Saliva contains a number of protective factors such as mucins, immunoglobulins (e.g., IgA, IgG, and IgM), and enzymes (e.g., lysozyme and lactoperoxidases) that play an important role in the maintenance of oral health. The aim of this study was to compare levels of sIgA, histatin-5, and lactoperoxidase in saliva of adolescents with dental caries. MATERIAL AND METHODS: Thirty-five adolescents (age 18 years) from high school were examined. Eight subjects with DMF=3 (Group I) and 27 adolescents with DMF>11 (Group II) were enrolled for this study. Clinical evaluation procedures comprised oral examination (including tooth, periodontal, and oral mucosal status) and collection of saliva samples. Saliva was collected for enzyme-linked immunosorbent assay (ELISA) and was used for determination of sIgA, histatin-5, and lactoperoxidase levels. RESULTS: Our results showed that adolescents with very high intensity of dental caries (DMF>11) had increased levels of sIgA, histatin-5, and lactoperoxidase compared to adolescents with lower intensity of caries. The increase was statistically significant (p<0.05). CONCLUSIONS: We suggest that high intensity of caries is associated with increased levels of some salivary components - sIgA, histatin-5 and lactoperoxidase - that possess strong bactericidal or bacteriostatic effects, resulting in aggregation of oral bacteria and their clearance from the oral cavity.

Clinical evaluation of photodynamic therapy efficacy in the treatment of oral leukoplakia.

BACKGROUND: The aim of the study was clinical evaluation of photodynamic therapy efficacy in the treatment of oral leukoplakia lesions. METHODS: Twenty-three consecutive patients aged 21-79 were included to the study. In all patients 44 homogeneous, flat leukoplakia lesions were clinically diagnosed and confirmed histopathologically. Photodynamic therapy was performed with the use of Photolon(®) photosensitizer, containing 20% Chlorine-e6 and 10% dimethyl sulfoxide and a semiconductor laser, with power up to 300mW and a wavelength of 660nm. Ten illumination sessions were conducted with the use of superficial light energy density of 90J/cm(2). RESULTS: At baseline the mean size of leukoplakia lesion was 6.5±5.10cm(2) while after photodynamic therapy 3±2.99cm(2). Significant reduction (on average by 53.8%) of leukoplakia lesions sizes was observed after therapy. Twelve (27.27%) lesions had been completely cured, 22 (50%) partially cured, although 10 (22.73%) lasted unchanged. The efficacy of PTD was comparable in women and men irrespective of age. There have been no adverse site effects during therapy noted. CONCLUSIONS: Within the limits of the study it can be concluded that photodynamic therapy with the use of Chlorine-e6 can lead to considerable reduction of oral leukoplakia lesions size thus may be useful in clinical practice. However there is a need of further studies on larger number of cases and longer follow-up time.

Clinical assessment of the efficacy of photodynamic therapy in the treatment of oral lichen planus.

The study objective was clinical assessment of the efficacy of photodynamic therapy (PDT) in the treatment of oral lichen planus (OLP). There were 23 patients aged 31-82 included in the study with oral lichen planus diagnosed clinically and histopathologically. In all patients photodynamic therapy was performed with the use of chlorin e6 (Photolon(®)), containing 20 % chlorin e6 and 10 % dimethyl sulfoxide as a photosensitizer. PDT was performed using a semiconductor laser, with power up to 300 mW and a wavelength of 660 nm. A series of illumination sessions was conducted with the use of superficial light energy density of 90 J/cm(2). Changes of lesion size were monitored at one, two, five, and ten PDT appointments from the series of ten according to the authors' own method. The sizes of clinical OLP lesions exposed to PDT were reduced significantly (on average by 55 %). The best effects were observed for the lesions on the lining mucosa (57.6 %). The therapy was statistically significantly less effective when masticatory mucosa was affected (reduction, 30.0 %). Due to substantial efficacy and noninvasiveness, PDT can be useful in the treatment of OLP lesions.

Cytological picture of the oral mucosa in patients with gastric and colon cancer.

The incidence of malignant gastrointestinal cancers in Poland has been constantly growing, which has led to an intensification of the search for new markers of the early clinical stage of this disease. The oral cavity,as the first part of the gastrointestinal tract, has a very important role. The oral cavity presents symptoms of both typically stomatological and systemic diseases. Oral cancers, benign or malignant, may originate and grow in any of the tissues of the mouth, and within this small area they may be of varied clinical, histological and biological features. These can be lesions typically observed in the oral cavity, but also characteristic of cases where the symptoms occur both in the mouth and in other body parts. The aim of this study was to present a cytological picture of the oral mucosa in patients with gastric and colon cancer and to compare the cytological picture with that obtained from a group of patients with no cancer, using the Papanicolaou classification and the Bethesda system. The study was conducted in 126 patients treated surgically in the II General and Gastroenterological Surgery Clinic between 2006 and 2008. All patients were divided into two groups based on the type of lesions. In both of the studied groups, more than half of the patients did not present any abnormalities in the mucosa of the mouth, lips and cheeks in the physical examination. None of the patients had erosion, ulceration or lesions typical of leukoplakia or lichen planus. No malignant cells were detected in either of the studied groups, and there were no well-defined lesions found in the oral cavity that would distinguish the patients with gastrointestinal cancer.