An in vitro assessment of teething gels' effects on human gingival mesenchymal stem cells.

BACKGROUND: The aim of this study is to examine the cytotoxic effects of dental gels with different contents, which are frequently used during teething, on gingival mesenchymal stem cells (G-MSCs). METHOD: The teething gels used in this study were Dentinox, Gengigel, Osanite, and Jack and Jill. The human gingival mesenchimal stem cells (hG-MSCs) were incubated with these teething gel solutions (0.1%, 50% and 80% concentrations). Reproductive behavior of G-MSCs was monitored in real time for 72 h using the xCELLigence real-time cell analyzer (RTCA) system. Two-way repeated Anova test and post hoc Bonferroni test were used to evaluate the effect of concentration and dental gel on 0-hour and 72-hour viability. Significance was evaluated at p < 0.05 level. RESULTS: Teething gels prepared at 50% concentration are added to the G-MSC culture, the "cell index" value of G-MSCs to which Dentinox brand gel is added is significantly lower than all other groups (p = 0.05). There is a statistically significant difference between the concentrations in terms of cell index values at the 72nd hour compared to the 0th hour (p = 0.001). CONCLUSIONS: The local anesthetic dental gels used in children have a more negative effect on cell viability as concentration increases.

Viability and Proliferation Assessment of Gingival Fibroblasts Cultured on Silver Nanoparticle-Doped Ti-6Al-4V Surfaces.

PURPOSE: To investigate the biocompatibility of silver nanoparticle (AgNP)-doped Ti-6Al-4V surfaces by evaluating the viability and proliferation rate of human gingival fibroblasts (HGFs)-as the dominant cells of peri-implant soft tissues-seeded on the modified surfaces. MATERIALS AND METHODS: AgNPs (sizes 8 nm and 30 nm) were incorporated onto Ti-6Al-4V specimen surfaces via electrochemical deposition, using colloid silver dispersions with increasing AgNP concentrations of 100 ppm, 200 ppm, and 300 ppm. One control and six experimental groups were included in the study: (1) control (Ti-6Al-4V), (2) 8 nm/100 ppm, (3) 8 nm/200 ppm, (4) 8 nm/300 ppm, (5) 30 nm/100 ppm, (6) 30 nm/200 ppm, and (7) 30 nm/300 ppm. HGF cell primary cultures were isolated from periodontally healthy donor patients and cultured in direct contact with the group specimens for 24 and 72 hours. The cytotoxicity of AgNP-doped Ti-6Al-4V specimens toward HGF was assessed by the MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide) and BrdU (5-bromo-2'-deoxyuridine) assay tests. Calcein AM and ethidium homodimer (EthD-1) fluorescent stains were used to determine the live and dead cells. The morphology and attachment properties of the HGFs were determined via scanning electron microscopy (SEM). RESULTS: Energy dispersive x-ray (EDX) analysis confirmed the presence of AgNPs on the specimens. The MTT test revealed that AgNPs of both sizes and all concentrations presented a decreased cellular metabolic activity compared to the control discs. All concentrations of both sizes of AgNPs affected the cell proliferation rate compared to the control group, as revealed by the BrdU assay. Overall, cytotoxicity of the modified Ti-6Al-4V surfaces depended on cell exposure time. Observation via confocal microscopy confirmed the results of the MTT and BrdU assay tests. Specifically, most cells remained alive throughout the 72-hour culture period. SEM images revealed that adjacent cells form bonds with each other, creating confluent layers of conjugated cells. CONCLUSIONS: The findings of the present study indicate that Ti-6Al-4V surfaces modified with 8 nm and 30 nm AgNPs at concentrations of 100 ppm, 200 ppm, and 300 ppm do not produce any serious cytotoxicity toward HGFs. The initial arrest of the HGF proliferation rate recovered at 72 hours. These results on the antibacterial activity against common periodontal pathogens, in combination with the results found in a previous study by the same research group, suggest that AgNP-doped Ti-6Al-4V surfaces are potential candidates for use in implant abutments for preventing peri-implant diseases.

Cytotoxicity of universal dental adhesive systems: Assessment in vitro assays on human gingival fibroblasts.

Universal adhesives are the most important innovation in restorative dentistry. They are composed of different monomers, solvents and fillers. The potential cytotoxic effect of these materials is an important scientific aspect in recent literature. The aim of this study was to determine, using different in vitro techniques, the cytotoxicity evaluation of seven universal enamel-dental adhesives on human gingival fibroblasts. For this purpose, seven universal dental enamel adhesives have been evaluated by in vitro cytotoxicity tests using direct contact tests (an unpolymerized and a polymerized method) and an indirect contact test: preparation of extracts. The polymerized method showed a cytotoxicity range from 36% (G-PremioBond, GPB) to 79% (FuturaBond M+, FB). With the unpolymerized direct methods the range was from 4% (Prime&Bond Active, PBA) to 40% (Ibond Universal, IB) for undiluted adhesives; generally passing to the major dilutions the test showed a strong inhibitory activity by all the adhesives. Whereas with the indirect method by diluting the extracts of all dental adhesives the cell viability increased. The data obtained from the work has shown a lower cytotoxic effect of Optibond Solo Plus (OB) and Adhesive Universal (AU) with more reliable results with the extracts technique. The choice of reliable in vitro cytotoxic technique could represent, in dental practice, an important aid for clinical procedures in the use of adhesive systems.

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.

[Use of integral indices in the assessment of bucсal cytology in health and in the oral cavity pathology.]

Cytological analysis of buccal epithelium (BE) - the buccal cytogram allows to select about 20 parameters reflecting cytogenetic, inflammatory, proliferative and other disorders in epithelial cells used for biomonitoring and laboratory diagnostics. In order to simplify and increase the information content of this study, we conducted a comparative analysis of various integral indices when interpreting the buccal cytogram. 40 practically healthy volunteers, 35 patients with chronic periodontitis, 22 patients with chronic gingivitis and 20 patients with partial loss of teeth were examined. The obtained data revealed an increase in the degree of disorders of proliferation and apoptosis, their ratio as the inflammation increased from gingival tissue lesions to inflammation periodontal tissue. In assessing the condition of the mucous membranes in chronic gingivitis, the apoptosis index turned out to be more informative.In chronic periodontitis the index of cytogenetic disorders is indicative. In this category of patients, the accumulation index of cytogenetic disorders did not have any advantages over the others, and the reparative index was less informative.

Assessment of toxicity and oxidative DNA damage of sodium hypochlorite, chitosan and propolis on fibroblast cells.

The objective of this study was to evaluate and compare the cytotoxicity and genotoxicity on human fibroblast cell lines of sodium hypochlorite (NaOCl), chitosan and propolis as root canal irrigating solutions. Human fibroblast cells were exposed to chitosan, propolis and NaOCl for 4 and 24 h. Cell viability was assessed by 2,3-bis-(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide, and oxidative DNA damage was assessed by determination of 8-hydroxydeoxyguanosine (8-OHdG) level with an ELISA kit. The data of cell cytotoxicity were analysed statistically using a test of one-way analysis of variance at a significance level of p < 0.05. In the NaOCI group, the 8-OHdG level was higher than in the chitosan group, but there was no statistical difference when compared with the other groups (p < 0.05). It was determined that the irrigation solutions were cytotoxic, depending on the dose and time. NaOCl was the most toxic solution after both 4 and 24 h of exposure (p < 0.05). Chitosan and propolis may be alternatives to NaOCl for irrigation solutions, because they are both less toxic and produce less oxidative DNA damage.

Assessment of toxicity and oxidative DNA damage of sodium hypochlorite, chitosan and propolis on fibroblast cells

Abstract The objective of this study was to evaluate and compare the cytotoxicity and genotoxicity on human fibroblast cell lines of sodium hypochlorite (NaOCl), chitosan and propolis as root canal irrigating solutions. Human fibroblast cells were exposed to chitosan, propolis and NaOCl for 4 and 24 h. Cell viability was assessed by 2,3-bis-(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide, and oxidative DNA damage was assessed by determination of 8-hydroxydeoxyguanosine (8-OHdG) level with an ELISA kit. The data of cell cytotoxicity were analysed statistically using a test of one-way analysis of variance at a significance level of p < 0.05. In the NaOCI group, the 8-OHdG level was higher than in the chitosan group, but there was no statistical difference when compared with the other groups (p < 0.05). It was determined that the irrigation solutions were cytotoxic, depending on the dose and time. NaOCl was the most toxic solution after both 4 and 24 h of exposure (p < 0.05). Chitosan and propolis may be alternatives to NaOCl for irrigation solutions, because they are both less toxic and produce less oxidative DNA damage.

Avaliação dos eixos ECA / ANG II / AT1 e ECA-2 / ANG (1-7) / MAS em fibroblastos de gengiva e ligamento periodontal humanos estimulados por IL-1ß e sua contribuição na produção e regulação de citocinas e quimiocinas / Evaluation ACE /ANG II /AT1 and ACE-2 /ANG (1-7) /MAS axis in gingival and periodontal ligament human fibroblasts stimulated with IL-1ß and its contribution in the production of cytokines and chemokines

O Sistema renina-angiotensina (SRA) tem sido relatado como um importante modulador de processos inflamatórios e imunológicos, incluindo a doença periodontal (DP). Estudos sugerem neste sistema um eixo alternativo (ECA-2 /ANG(1-7) /MAS) que atuaria como um contra-regulador de efeitos mediados pelo clássico eixo (ECA /ANGII /AT1). Sabe-se que bactérias periodontopatogênicas, como a Porphyromonas gingivalis (Pg), possuem componentes bioativos de membrana (ex. lipopolissacarídeos-LPS) capazes de induzir uma forte resposta imune no hospedeiro devido à liberação de citocinas nas células, entre elas Interleucina (IL)- 1ß. Neste contexto, fibroblastos são as células mais abundantes nos tecidos periodontais e possuem em sua superfície celular receptores necessários para o reconhecimento da invasão bacteriana, ativando cascatas intracelulares, que levam à produção de citocinas. O objetivo deste estudo foi verificar se os eixos ECA/ ANGII/ AT1 e ECA-2/ ANG(1-7)/ MAS contribuem para a produção e/ ou regulação de citocinas inflamatórias (CI) por fibroblastos de gengiva humana (HGF) e ligamento periodontal humano (HPLF) estimulados por IL-1ß. Após o pré-tratamento com Losartan e Ang (1-7) ou silenciamento mediado por RNA de interferência (RNAi) de AT1, HGF e HPLF foram estimulados por IL-1ß por 3 horas (RNAm) ou 24 horas (proteína). Expressão de RNAm para AT1, MAS, ECA, ECA-2, IL-1ß, TNF-α, IL-6, IL-8, IL-10, TGF-ß, CXCL12, RANK-L e OPG foram avaliados por RT-qPCR e das proteínas IL-6, IL-8, ECA e ECA-2 por ELISA. Foi realizado também Western Blot para detecção de AT1 e ECA nos extratos celulares e dosagem de nitrito no sobrenadante das culturas. Ambos os subtipos de fibroblastos mostraram aumento da expressão de RNAm para AT1, IL-1ß, IL-6, IL-8, TNF-α e OPG, quando estimulados por IL-1ß. No entanto, apenas em HPLF foi observado aumento para MAS, ECA e TGF-ß. Losartan e Ang (1-7) não modularam o transcrito, a secreção de CI e nem a produção de nitrito no sobrenadante das culturas, tanto em HGF como em HPLF. O silenciamento do receptor AT1 reduziu a secreção de IL-6 e IL-8 induzida por IL-1ß em cultura de HGF e HPLF e aumentou a expressão gênica de OPG somente em HGF. Estes resultados sugerem que o silenciamento de AT1, mas não o bloqueio farmacológico deste receptor pelo antagonista Losartan, em HGF e HPLF, pode controlar a produção de IL-6 e IL-8, que por sua vez contribuem para a patogênese periodontal.(AU)

The renin-angiotensin system (RAS) has been reported as an important modulator of inflammatory and immune responses, including periodontal disease (PD). Studies suggest an alternative axis as part of this system (ACE-2 / ANG (1-7) / MAS) that would act as counter-regulatory to the classical axis (ECA / ANGII / AT1). It is known that periodontal bacteria such as Porphyromonas gingivalis (Pg) have bioactive components in their membrane (such as lipopolysaccharide-LPS) capable of inducing a strong immune response in the host due to the release of cytokines in cells, including interleukin (IL) - 1ß. In this regard, fibroblasts are the most abundant cells in periodontal tissues and receptors needed for the recognition of bacterial invasion by activating intracellular cascades that lead to cytokine production. The aim of this study was to determine whether the axes ACE / ANGII / AT1 and ACE-2 / ANG (1-7) / MAS contribute to the production and / or regulation of inflammatory cytokines (IC) by fibroblasts of human gingiva (HGF) and human periodontal ligament (HPLF) stimulated IL-1ß. After pre-treatment with Losartan, Ang (1-7) or silencing mediated by RNA interference (RNAi) of AT1, HGF and HPLF were stimulated by IL-1ß for 3 hours (RNAm) or 24 hours (protein). Expression mRNA for AT1, MAS, ACE, ACE-2, IL-1ß, TNF-α, IL-6, IL-8, IL-10, TGF-ß, CXCL12, RANK-L and OPG was assessed by RT- qPCR and proteins IL-6, IL-8, ACE and ACE-2 by ELISA. Western Blot for the detection of AT1 and ECA and dosage of nitrite was also performed. Experiments stimulated by IL-1ß showed a positive control for gene expression AT1, IL-1ß, IL-6, IL-8, TNF-α and OPG in HGF and HPLF and MAS, ACE and TGF-ß only HPLF. Losartan and Ang (1-7) did not modulate the transcription and secretion of IC and no nitrite production in the culture supernatant of HGF and HPLF. The silencing AT1 reduced IL-6 secretion and IL-8 induced by IL- ß in cultured HGF and HPLF and increased OPG gene expression only HGF. These results suggest that silencing AT1, but not pharmacological blockade of this receptor by Losartan in HPLF and HGF, can control the production of IL-6 and IL-8, which in turn contribute to the pathogenesis of periodontal disease.(AU)

Assessment of human gingival fibroblast interaction with dental implant abutment materials.

The biocompatibility of dental implant abutment materials depends on numerous factors including the nature of the material, its chemical composition, roughness, texture, hydrophilicity and surface charge. The aim of the present study was to compare the viability and adhesion strength of human gingival fibroblasts (HGFs) grown on several dental materials used in implant prosthodontics. Surfaces of the tested materials were assessed using an optical imaging profiler. For material toxicity and cellular adhesion evaluation, primary human gingival fibroblast cells were used. To evaluate the strength of cellular adhesion, gingival fibroblasts were cultured on the tested materials and subjected to lateral shear forces by applying 300 and 500 rpm shaking intensities. Focal adhesion kinase (FAK) expression and phosphorylation in cells grown on the specimens were registered by cell-based ELISA. There was a tendency of fibroblast adhesion strength to decrease in the following order: sandblasted titanium, polished titanium, sandblasted zirconium oxide, polished zirconium oxide, gold-alloy, chrome-cobalt alloy. Higher levels of total as well as phospho-FAK protein were registered in HGFs grown on roughened titanium. Material type and surface processing technique have an impact on gingival fibroblast interaction with dental implant abutment materials.

Isolation and multiple differentiation potential assessment of human gingival mesenchymal stem cells.

The aim of this study was to isolate human mesenchymal stem cells (MSCs) from the gingiva (GMSCs) and confirm their multiple differentiation potentials, including the odontogenic lineage. GMSCs, periodontal ligament stem cells (PDLSCs) and dermal stem cells (DSCs) cultures were analyzed for cell shape, cell cycle, colony-forming unit-fibroblast (CFU-F) and stem cell markers. Cells were then induced for osteogenic and adipogenic differentiation and analyzed for differentiation markers (alkaline phosphatase (ALP) activity, mineralization nodule formation and Runx2, ALP, osteocalcin (OCN) and collagen I expressions for the osteogenic differentiation, and lipid vacuole formation and PPARγ-2 expression for the adipogenic differentiation). Besides, the odontogenic differentiation potential of GMSCs induced with embryonic tooth germ cell-conditioned medium (ETGC-CM) was observed. GMSCs, PDLSCs and DSCs were all stromal origin. PDLSCs showed much higher osteogenic differentiation ability but lower adipogenic differentiation potential than DSCs. GMSCs showed the medial osteogenic and adipogenic differentiation potentials between those of PDLSCs and DSCs. GMSCs were capable of expressing the odontogenic genes after ETGC-CM induction. This study provides evidence that GMSCs can be used in tissue engineering/regeneration protocols as an approachable stem cell source.

Nonwoven-based gelatin/polycaprolactone membrane proves suitability in a preclinical assessment for treatment of soft tissue defects.

Standard preclinical assessments in vitro often have limitations regarding their transferability to human beings, mainly evoked by their nonhuman and tissue-different/nontissue-specific source. Here, we aimed at employing tissue-authentic simple and complex interactive fibroblast-epithelial cell systems and their in vivo-relevant biomarkers for preclinical in vitro assessment of nonwoven-based gelatin/polycaprolactone membranes (NBMs) for treatment of soft tissue defects. NBMs were composed of electrospun gelatin and polycaprolactone nanofiber nonwovens. Scanning electron microscopy in conjunction with actin/focal contact integrin fluorescence revealed successful adhesion and proper morphogenesis of keratinocytes and fibroblasts, along with cells' derived extracellular matrix deposits. The "feel-good factor" of cells under study on the NBM was substantiated by forming a confluent connective tissue entity, which was concomitant with a stratified epithelial equivalent. Immunohistochemistry proved tissue authenticity over time by abundance of the biomarker vimentin in the connective tissue entity, and chronological increase of keratins KRT1/10 and involucrin expression in epithelial equivalents. Suitability of the novel NBM as wound dressing was evidenced by an almost completion of epithelial wound closure in a pilot mini-pig study, after a surgical intervention-caused gingival dehiscence. In summary, preclinical assessment by tissue-authentic cell systems and the animal pilot study revealed the NBM as an encouraging therapeutic medical device for prospective clinical applications.

Micronucleus assay assessment of possible genotoxic effects in patients treated with titanium alloy endosseous implants or miniplates.

The use of titanium and its alloys (Ti-6Al-4V) for oral surgery has increased dramatically in recent years. Ti is a stable biocompatible metal suitable for oral applications and it has been used for endosseous subperiosteal implants and miniplate fixation for more than 25 years. Dental implants are typically made of Ti or Ti alloys. The alloys are potentially toxic due to release of vanadium and aluminum. We tested the possible genotoxicity of Ti alloy endosseous implants and miniplates on the oral mucosal tissues of two groups of patients: 17 patients receiving Ti miniplate and screw fixation, and 37 endosseous dental implant placement patients. Preoperative and postoperative mucogingival cell samples were collected. Genotoxicity was assessed by the micronucleus assay (MN). There were slight but not statistically significant increases in the frequencies of MN (p=0.087 and p=0.047) post-operation in both groups. In summary, neither of the applications showed genotoxicity in the oral epithelial cells of patients.

Nature-inspired antimicrobial polymers--assessment of their potential for biomedical applications.

We explored the potential of poly(oxonorbornene)-based synthetic mimics of antimicrobial peptides (SMAMPs), a promising new class of antimicrobial polymers with cell-selectivity and low resistance development potential, for clinical applications. We evaluated their antimicrobial activity against a panel of seven clinical and regulatory relevant bacteria strains, and tested their toxicity with two different kinds of primary human cells. For the antimicrobial activity, we performed the minimum inhibitory concentration (MIC) assay and determined the minimum bactericidal concentration (MBC) according to the NCCLS guidelines. The results revealed specific problems that may occur when testing the antimicrobial activity of amphiphilic cationic polymers, and confirmed the working hypothesis that the more hydrophilic SMAMP polymers in our portfolio were 'doubly selective', i.e. they are not only selective for bacteria over mammalian cells, but also for Gram-positive over Gram-negative bacteria. The data also showed that we could improve the broad-band activity of one SMAMP, and in combination with the results from the cell toxicity experiments, identified this polymer as a promising candidate for further in-vitro and in-vivo testing. Transmission electron studies revealed that the cellular envelopes of both E. coli and S. aureus were severely damaged due to SMAMP action on the bacterial membrane, which strengthened the argument that SMAMPs closely resemble antimicrobial peptides. To test cell toxicity, we used the traditional hemolysis assay with human red blood cells, and the novel xCelligence assay with primary human fibroblasts. The data reported here is the first example in which a hemolysis assay is benchmarked against the xCelligence assay. It revealed that the same trends were obtained using these complementary methods. This establishes the xCelligence assay with primary human cells as a useful tool for SMAMP characterization.

A molecular survey of S. mutans and P. gingivalis oral microbial burden in human saliva using relative endpoint polymerase chain reaction (RE-PCR) within the population of a Nevada dental school revealed disparities among minorities.

BACKGROUND: The University of Nevada, Las Vegas School of Dental Medicine recently opened an orthodontic treatment clinic to address the needs of the racially and ethnically diverse population of Southern Nevada, primarily focusing on the treatment and care of low-income and minority patients. Although orthodontic treatment and therapy has been shown to induce changes in the oral cavity, much of this evidence was collected from traditional White, teenage orthodontic clinic populations. The primary goal of this study was to describe the microbial burden of the cariogenic and periodontal pathogens, Streptococcus mutans and Porphyromonas gingivalis within the UNLV-SDM patient population. METHODS: Representative saliva samples were collected from healthy adult patients for DNA isolation. Relative endpoint polymerase chain reaction (RE-PCR) was performed to ascertain the presence and relative microbial burden of these oral pathogens. RESULTS: Nearly one quarter (13/56) or 23.3% of these patients had elevated levels of S. mutans, while (10/56) and 17.8% of these samples were found to have elevated levels of P. gingivalis, - with (90%) of P. gingivalis-positive samples from minority patients (X2=17.921, d.f. = 1; p<0.0001). CONCLUSIONS: These findings of elevated P. gingivalis levels, primarily among minority patients, may suggest underlying oral health practices contributing to adverse oral health conditions within this population. Oral health knowledge and practices among minority patients may be strongly influenced by other factors, including education and socioeconomic status, suggesting additional research may be needed to accurately determine the most appropriate standards for care and oral health education within this patient population.

Co-purified gelatinases alter the stability and biological activities of human plasma fibronectin preparations.

BACKGROUND AND OBJECTIVE: Fibronectin (FN) is an important cell adhesion molecule that is used widely to characterize cell behavior. Preparations of FN purified from human plasma by gelatin-Sepharose affinity chromatography typically also contain gelatin-binding gelatinases that may cleave FN, reduce its stability and alter its biological activities. Available methods for separating gelatinases from FN are resource demanding. Therefore, our objective was to devise a time- and cost-efficient protocol for purification of gelatinase-free FN. MATERIAL AND METHODS: Experiments tested the elution profiles for FN and gelatinases from gelatin-Sepharose using a concentration range (1-7%) of dimethyl sulfoxide (DMSO) and 4 m urea as eluants. Subsequently, we explored the sequential application of those eluants for differential elution of gelatinases and FN using a single affinity column. Finally, experiments characterized the stability of purified FN with or without contaminating gelatinases, as well as the effects of FN degradation on cell attachment and migration. RESULTS: Assay optimization demonstrated that pre-elution with 3% DMSO efficiently eliminated gelatinases but not FN from gelatin-Sepharose, whereas subsequent elution with 4 m urea released FN. Sequential elutions with DMSO and urea produced gelatinase-free FN, which was more stable than FN eluted by urea only. Fibronectin degradation did not affect human gingival fibroblast attachment, but increased cell migration significantly. CONCLUSION: The present experiments devised a time- and cost-efficient protocol for eliminating gelatinases during purification of human plasma FN. Gelatinase-free FN preparations had greater stability, which may be essential for experiments because FN fragments have altered biological activities compared with intact FN.

Endodontic antimicrobial photodynamic therapy: safety assessment in mammalian cell cultures.

OBJECTIVES: The purpose of this study was to assess the in vitro synergistic effect of methylene blue (MB) and red light on human gingival fibroblasts and osteoblasts with parameters similar to those that may be applied in a clinical setting for endodontic disinfection. MATERIALS AND METHODS: Both cell types were sensitized with 50 microg/mL MB followed by exposure to red light at 665 nm for 5 minutes with an irradiance of 10, 20, and 40 mW/cm(2). After photodynamic therapy (PDT), cell viability and mitochondrial activity were evaluated by the neutral red and MTT assay, respectively. The assessment of PDT-induced apoptosis was investigated by western blot analysis using cleaved poly(ADP-ribose) polymerase-specific antibodies. RESULTS: Light at 20 and 40 mW/cm(2) with MB had modest effects at 24 hours on osteoblasts in both assays, whereas sodium hypochlorite completely eliminated cells. Western blot analysis revealed no signs of apoptosis in either cell type. CONCLUSION: The data suggest that there is a safe therapeutic window whereby PDT can inactivate endodontic pathogens without affecting host cell viability.

Assessment of acute cyto- and genotoxicity of corrosion eluates obtained from orthodontic materials using monolayer cultures of immortalized human gingival keratinocytes.

Whilst a patient is undergoing orthodontic treatment, dental appliances based on non-precious metals or titanium remain in the oral cavity for up to several years. Throughout this period the appliance is in either direct or indirect contact with the oral mucosa. To investigate the possibility of cell damage occurring as a result of appliance corrosion, monolayer cultures of immortalized human gingival keratinocytes were assessed for acute cyto- and genotoxicity using the hexosaminidase assay and the Comet assay respectively. The materials tested included 1. a nickel-free wire, 2. a UK-1 bond, 3. nickel-free as well as nickel-containing brackets with and without color signature and 4. a titanium expansion screw. Each of the test materials was corroded in a solution consisting of equal amounts of lactic acid and sodium chloride (0.1 M) for 1, 3, 7 and 14 days. The cell cultures were then exposed to eluates exhibiting the highest ion concentrations. None of the eluates was found to exhibit acute cytotoxicity, regardless of the type of test system used. Qualitative assessment using neutral red dye for live cells and either trypan blue or propidium iodide to disclose dead cells failed to reveal any significant increase in cell damage when exposed cells were compared to control cultures. Unrestricted cell vitality was confirmed by quantifying viable cells through measurement of hexosaminidase enzyme activity. Furthermore, assessment of genotoxicity revealed no apparent DNA damage to immortalized gingival keratinocytes following exposure to the test eluates. Because the materials tested in this study were corroded using the exacting methods normally applied to precious metals or gold-containing alloys, the lack of either acute cyto- or genotoxic effects following exposure to the test eluates indicates that the materials tested exert no adverse effects on cells similar to those of the target tissue exposed to the materials in situ.

[The comprehensive assessment of studies of the microbiological and cytological indices of the implant-epithelial area]. / Kompleksnaia otsenka issledovanii mikrobiologicheskikh i tsitologicheskikh pokazatelei implantoépitelial'noi zony.

Microbiological and cytological study of the subgingival deposit at the site of introduction of intraosseous cylindrical nickelide-titanium dental implants and comparison of the findings with similar values for the adjacent teeth showed that such studies help assess the status of the implant-epithelial zone.

Modification of the agar overlay assay: assessment of the influence of acrylics used in orthodontics on proliferation and differentiation of primary and transformed fibroblasts.

In orthodontics, removable acrylic appliances are preferably produced from methylmetacrylates. However, in the adjacent oral mucosal tissue, cell damage may be caused by the evaporation of residual monomer. The aim of this study was to modify the classic agar overlay assay in order to apply histochemical methods by comparing conventionally used transformed mouse fibroblasts (L-929) with primary human gingival fibroblasts to further elucidate the term toxicity. While proliferation was assessed via the incorporation of the base analogon bromdesoxyuridine, differentiation was investigated by detection of the fibroblast-specific intermediate filament vimentin. After the monomers had taken effect, reduced proliferation extending over the inhibition area was observed by indirect immunofluorescence, independent of cell type. In contrast to this observation, a few cells within the inhibition area which could not be clearly detected by neutral red staining still exhibited mitotic activity. Detection of the differentiation-specific intermediate filament vimentin was comparable with the degree of neutral red fading visible in the classic agar overlay assay. The study showed that the inhibition areas with primary gingival fibroblasts were smaller (approximately 1/3) after monomer action compared with conventionally applied transformed fibroblasts. The results indicate that the modified assay is comparable with the classic method. Evaluation of neutral red staining with respect to toxic material influence can moreover be supplemented by histochemical studies of typical cell properties.

Application of immunoprinting for assessment of fibroblast secretory heterogeneity.

Fibroblasts are capable of secreting a number of inflammatory mediators and cytokines and may exhibit a marked heterogeneity in this capacity. The relative frequency of cytokine-secreting fibroblasts in chronic inflammatory connective tissue disorders may affect the amount of these molecules secreted locally and dictate the intensity or chronicity of the disease process. We have devised a simple, in situ immunodetection method for assessment of the frequency of actively secreting cells in adherent cell cultures. Our technique is based on the principle of immunoprinting and is coupled with an enzyme-linked immunodetection system. The methodology, sensitivity, specificity and accuracy of this technique are described.