Combination of losartan with pirfenidone: a protective anti-fibrotic against pulmonary fibrosis induced by bleomycin in rats.

Pirfenidone (PFD), one acceptable medication for treating idiopathic pulmonary fibrosis (IPF), is not well tolerated by patients at full doses. Hence, employing of some approaches such as combination therapy may be applicable for increasing therapeutic efficacy of PFD. Losartan (LOS), an angiotensin II receptor antagonist, could be a suitable candidate for combination therapy because of its stabilizing effect on the pulmonary function of IPF patients. Therefore, this study aimed to investigate the effects of LOS in combination with PFD on bleomycin (BLM)-induced lung fibrosis in rats. BLM-exposed rats were treated with LOS alone or in combination with PFD. The edema, pathological changes, level of transforming growth factor-ß (TGF-ß1), collagen content, and oxidative stress parameters were assessed in the lung tissues. Following BLM exposure, the inflammatory response, collagen levels, and antioxidant markers in rat lung tissues were significantly improved by PFD, and these effects were improved by combination with LOS. The findings of this in vivo study suggest that the combined administration of PFD and LOS may provide more potent protection against IPF than single therapy through boosting its anti-inflammatory, anti-fibrotic, and anti-oxidant effects. These results hold promise in developing a more effective therapeutic strategy for treating of lung fibrosis.

High inflammatory cytokines gene expression can be detected in workers with prolonged exposure to silver and silica nanoparticles in industries.

Occupational health must be strictly considered in industries particularly in nanoparticle factories where workers were exposed to different types of chemicals. We measured the serum levels of inflammatory cytokines in workers who developed skin lesions after exposure to silver and silica nanoparticles. Using a questionnaire in this cross-sectional study, we identified 110 workers in nanoparticle industries who were exposed to silver and silica nanoparticles. We also included 40 healthy subjects as controls from the administrative department of the same factories who were not exposed to nanoparticles. Peripheral blood samples used to measure the mRNA levels of inflammatory cytokines by qRT-PCR. In comparison with the control group, the workers who developed skin lesions had significantly higher levels of interleukin IL4, IL6, IL8, and TNF-α, particularly after two or three decades of exposure to silver and silica nanoparticles. Participants who were exposed to silver had higher levels of IL6 and IL8 compared with those who were exposed to silica. Necessary measures must be considered to protect workers in nanoparticle industries against the potential toxic effects of these compounds. Our network pharmacology study suggests corresponding biochemical pathways for these disorders.

Sterility and bioactivity evaluation of two types of bone graft substitutes after removing the original packaging.

Background: Xenograft and allograft bone substitutes are widely used to replace the missing bone in defects. Since removing the packaging of these grafts can nullify their sterilization, this study aimed to evaluate the sterility and bioactivity changes of an allograft and a xenograft following uncapping/recap. Methods: Two types of commercial allograft and xenograft vials were unpacked and further exposed to operating room air, where implant surgery was performed for one second, ten minutes, and one hour. After three repetitions, samples were analyzed using microbiological tests and scanning electron microscopy (SEM) with energy dispersive x-ray analysis (EDX) for sterility and bioactivity evaluation. Results: None of the bone graft samples showed microbial growth or bioactivity-negative changes after seven days of unpacking the vials. Conclusion: Despite the positive results of this study, future studies and more analysis considering influential factors are required. Also, disinfection and air exchange must still be observed during biomaterial application and bone grafting procedures.

Inhalation of multi-wall carbon nanotubes changes the expression of apoptosis and cancer genes in rat brain and lungs.

One of the important issues in urban areas is air pollution which causes respiratory disorders. A significant association between exposure to inhaled particulate matter (PM), mainly ultrafine particles, and increased neurological and pulmonary morbidity and mortality was observed in some research. This study aimed to demonstrate the relation between multi-wall carbon nanotubes (MWCNTs) inhalation and the carcinogenic effect of these materials in the brain and lungs. For this purpose, we investigated gene expression in rat brain and lung tissues induced by exposure to MWCNTs. Rats were exposed to MWCNTs in diameters of 10 and 100 nm (pure and impure) at a concentration of 5 mg/m3. Exposure was done through a whole-body exposure chamber for 5 h/day, 5 days/week for 14 days. After exposure, both brain and lung tissues were isolated to evaluate certain gene expressions including Bax, Bcl2, Rac1, Tp53, Mmp12, and Arc. The results showed that exposure to impure and pure MWCNTs (10 and 100 nm) at a concentration of 5 mg/m3 causes up-regulation or down-regulation of some of these genes. The results suggest that impure and pure MWCNTs (10 and 100 nm) can increase the risk of central nervous system disorders such as Alzheimer's disease and increase the risk of carcinogenesis in the lung tissues of rats exposed to MWCNTs (Tab. 2, Fig. 2, Ref. 64). Text in PDF www.elis.sk Keywords: multi-wall carbon nanotube, inhalation, gene expression, carcinogenicity, brain, lung.

Losartan enhances the suppressive effect of pirfenidone on the bleomycin-induced epithelial-mesenchymal transition and oxidative stress in A549 cell line.

Objectives: Idiopathic pulmonary fibrosis (IPF) is a fatal lung disease. Despite the promising anti-fibrotic effect, the toleration of pirfenidone (PFD) by the patients in full dose is low. Combination therapy is a method for enhancing the therapeutic efficiency of PFD and decreasing its dose. Therefore, the present study evaluated the effect of a combination of losartan (LOS) and PFD on oxidative stress parameters and the epithelial-mesenchymal transition (EMT) process induced by bleomycin (BLM) in human lung adenocarcinoma A549 cells. Materials and Methods: The non-toxic concentrations of BLM, LOS, and PFD were assessed by the MTT assay. Malondialdehyde (MDA) and anti-oxidant enzyme activity including catalase (CAT) and superoxide dismutase (SOD) were assessed after co-treatment. Migration and western blot assays were used to evaluate EMT in BLM-exposed A549 after single or combined treatments. Results: The combination treatment exhibited a remarkable decrease in cellular migration compared with both single and BLM-exposed groups. Furthermore, the combination treatment significantly improved cellular anti-oxidant markers compared with the BLM-treated group. Moreover, combined therapy markedly increased epithelial markers while decreasing mesenchymal markers. Conclusion: This in vitro study revealed that the combination of PFD with LOS might be more protective in pulmonary fibrosis (PF) than single therapy because of its greater efficacy in regulating the EMT process and oxidative stress. The current results might offer a promising therapeutic strategy for the future clinical therapy of lung fibrosis.

Preparation of Citrus reticulata peel nano-encapsulated essential oil and in vitro assessment of its biological properties.

Dental caries is the most common biofilm-dependent oral disease. Streptococcus mutans is among the main microorganisms responsible for the development of dental caries. Nano-suspension of Citrus reticulata (tangerine) peel essential oil in 0.5% (v/v) concentration was prepared and its antibacterial effect on S. mutans in planktonic and biofilm forms as well as its cytotoxic and antioxidant effects were assessed and compared with chlorhexidine (CHX). The minimum inhibitory concentration (MIC) of free essential oil, nano-encapsulated essential oil, and CHX was 5.6% (v/v), 0.0005% (v/v), and 0.0002% (w/v), respectively. The percentage of biofilm inhibition by the free essential oil, nano-encapsulated essential oil, and CHX at half-MIC was 67.3%, 24%, and 90.6%, respectively. The nano-encapsulated essential oil had no cytotoxicity and showed significant antioxidant effects in different concentrations. Nano-encapsulation of tangerine peel essential oil significantly enhanced its biological activities in much lower concentrations than the free essential oil (11,000 times diluted). It also showed lower cytotoxicity and higher antibiofilm effects in sub-MICs compared with CHX, indicating the optimal potential of tangerine nano-encapsulated essential oil for incorporation in the composition of organic antibacterial and antioxidant mouth rinses.

Comparative effect of anthocyanin on proliferation and migration of human gingival fibroblasts in the absence or presence of nicotine.

Background: Oral fibroblast malfunction can result in periodontal diseases. Nicotine can prolong the healing process as an irritant of oral tissues. Anthocyanins have been demonstrated to have potential benefits in preventing or treating smoking-related periodontal diseases. Cyanidin chloride's (CC's) potential in oral wound healing and the viability, proliferation, and migration of human gingival fibroblasts (HGFs) were examined in the presence and absence of nicotine by an in vitro study. Methods: The effects of different nicotine concentrations (1, 2, 3, 4, and 5 mM) on the viability and proliferation of HGF cells were evaluated in the presence and absence of different CC concentrations (5, 10, 25, and 50 µM) using the quantitative MTT assay. The scratch test was performed to evaluate the migration of CC-treated cells in the presence of 2.5-mM nicotine. Results: No cytotoxicity was observed at 1‒100 µM CC concentrations after 24, 48, and 72 hours of exposure to HGF cells. However, a concentration of 200 µM significantly reduced cell viability by about 20% at all the three-time intervals (P<0.05). Also, 3‒5-mM concentrations of nicotine significantly reduced cell viability in a dose- and time-dependent manner. Moreover, the understudied CC concentrations decreased nicotine's adverse effects on cell migration to some extent. Conclusion: Although the understudied CC concentrations could not significantly reduce the adverse effects of understudied nicotine concentrations on the viability and proliferation of HGF cells, they were able to reduce the detrimental effects of nicotine on cell migration significantly.

Applications of Novel and Nanostructured Drug Delivery Systems for the Treatment of Oral Cavity Diseases.

PURPOSE: Novel drug delivery systems (DDSs) hold great promise for the treatment of oral cavity diseases. The main objective of this article was to provide a detailed overview regarding recent advances in the use of novel and nanostructured DDSs in alleviating and treating unpleasant conditions of the oral cavity. Strategies to maximize the benefits of these systems in the treatment of oral conditions and future directions to overcome these issues are also discussed. METHODS: Publications from the last 10 years investigating novel and nanostructured DDSs for pathologic oral conditions were browsed in a systematic search using the PubMed/MEDLINE, Web of Science, and Scopus databases. Research on applications of novel DDSs for periodontitis, oral carcinomas, oral candidiasis, xerostomia, lichen planus, aphthous stomatitis, and oral mucositis is summarized. A narrative exploratory review of the most recent literature was undertaken. FINDINGS: Conventional systemic administration of therapeutic agents could exhibit high clearance of drugs from the bloodstream and low accumulation at the target site. In contrast, conventional topical systems face problems such as short residence time in the affected region and low patient compliance. Novel and nanostructured DDSs are among the most effective and commonly used methods for overcoming the problems of conventional DDSs. The main advantages of these systems are that they possess the ability to protect active agents from systemic and local clearance, enhance bioavailability and cellular uptake, and provide immediate or modified release of therapeutic agents after administration. In the design of local drug delivery devices such as nanofiber mats, films, and patches, components and excipients can significantly affect factors such as drug release rate, residence time in the oral cavity, and taste in the mouth. Choosing appropriate additives is therefore essential. IMPLICATIONS: Local drug delivery devices such as nanofiber mats, nanoparticles, liposomes, hydrogels, films, and patches for oral conditions can significantly affect drug efficacy and safety. However, more precise clinical studies should be designed and conducted to confirm promising in vitro and in vivo results. In recent years, novel and nanostructured DDSs increasingly attracted the attention of researchers as a means of treatment and alleviation of oral diseases and unpleasant conditions. However, more clinical studies should be performed to confirm promising in vitro and in vivo results. To transform a successful laboratory model into a marketable product, the long-term stability of prepared formulations is essential. Also, proper scale-up methods with optimum preparation costs should be addressed.

The effect of naproxen patches on relieving orthodontic pain by evaluation of VAS and IL-1β inflammatory factor: a split-mouth study

ABSTRACT Introduction: Pain related to orthodontic tooth movement is common and cause dissatisfaction and discomfort. Objective: The present study aimed to compare the efficacy of naproxen patches in pain control during orthodontic tooth separation, by means of visual analogue scale (VAS) and interleukin 1β (IL-1β) levels in gingival crevicular fluid (GCF). Methods: In this split-mouth triple-blind clinical trial, with 40 patients following separation, 5% naproxen or placebo patches were randomly placed on the upper right or left first molars every 8 hours. Pain intensity scores were determined after 2 and 6 hours, sleep time, 24 hours, days 2, 3 and 7 by the patients using a 100-mm VAS ruler. IL-1β levels in GCF were evaluated by ELISA at baseline, 1 and 24 hours and 7 days. Paired samples t-tests and two-way repeated measures ANOVA analysis of variance with a significance level of 0.05 were applied. Results: A total number of 30 patients (13 males and 17 females) finished the trial. Significant differences were found in pain scores (p< 0.0001) and IL-1β levels (p= 0.047) between naproxen and placebo groups. Lower pain scores were reported for the patients using naproxen patches at all time points, except 1 hour after separation. IL-1β levels were lower for the patients using naproxen patches only 1 hour after separation (p= 0.047). The peak of pain scores and IL-1β levels were calculated at 24 hours. Conclusion: In the light of VAS scores and IL-1β levels, naproxen patches reduced the pain caused by separator placement.

RESUMO Introdução: a dor relacionada à movimentação dentária ortodôntica é comum e causa insatisfação e desconforto. Objetivo: o presente estudo teve como objetivo avaliar a eficácia de curativos de naproxeno no controle da dor durante a separação ortodôntica dos dentes, por meio de escalas visuais analógicas (EVA) e dos níveis de interleucina 1β (IL-1β) no fluido crevicular gengival (FCG). Métodos: neste ensaio clínico, triplo-cego, boca dividida, com 40 pacientes após a separação dos dentes, foram aplicados, de forma aleatória, curativos com naproxeno a 5% ou placebo, nos primeiros molares superiores, direito ou esquerdo, a cada 8 horas. Os escores de intensidade da dor foram registrados pelos pacientes após 2 e 6 horas, durante o sono, após 24 horas, 2, 3 e 7 dias, usando uma EVA de 100 mm. Os níveis de IL-1β no FCG foram avaliados pelo ELISA no momento inicial, e após 1 e 24 horas e 7 dias. Foram aplicados testes t para amostras pareadas e ANOVA de duas vias para medidas repetidas, com nível de significância de 0,05. Resultados: no total, 30 pacientes (13 homens e 17 mulheres) terminaram o ensaio clínico. Diferenças significativas foram encontradas nos escores de dor (p< 0,0001) e níveis de IL-1β (p= 0,047) entre os grupos naproxeno e placebo. Índices mais baixos de dor foram relatados pelos pacientes que usaram curativos de naproxeno em todos os tempos avaliados, com exceção de 1 hora após a separação. Os níveis de IL-1β foram menores nos pacientes que usaram os curativos de naproxeno apenas 1 'hora após a separação (p= 0,047). Os picos dos escores de dor e dos níveis de IL-1β foram registrados 24 horas após a separação. Conclusão: considerando-se os escores das EVAs e os níveis de IL-1β, pode-se concluir que os curativos de naproxeno reduziram a dor causada pela instalação dos separadores ortodônticos.

Truncated forms of RUNX3 Unlike Full Length Protein Alter Cell Proliferation in a TGF-ß Context Dependent Manner.

The Runt related transcription factors (RUNX) are recognized as key players in suppressing or promoting tumor growth. RUNX3, a member of this family, is known as a tumor suppressor in many types of cancers, although such a paradigm was challenged by some researchers. The TGF-ß pathway governs major upstream signals to activate RUNX3. RUNX3 protein consists of several regions and domains. The Runt domain is a conserved DNA binding domain and is considered as the main part of RUNX proteins. Herein, we compared the effects of Runt domains and full-Runx3 in cell viability by designing two constructs of Runx3, including N-terminal region and Runt domain. We investigated the effect of full-Runx3, N-t, and RD on growth inhibition in AGS, MCF-7, A549, and HEK293 cell lines which are different in TGF-ß sensitivity, in the absence and presence of TGF-ß. The full length RUNX3 did not notably inhibit growth of these cell lines while, the N-t and RD truncates showed different trends in these cell lines. Cell proliferation in the TGF-ß impaired context cell lines (AGS and MCF-7) significantly decrease while in the A549 significantly increase. On the other hand, transfection of N-t and RD did not considerably affect the cell proliferation in the HEK293.Our results show that full-lenght RUNX3 did not affect the cell viability. Conversely, the N-t and RD constructs significantly changed cell proliferation. Therefore, therapeutic potentials for these truncated proteins are suggested in tumors with RUNX proteins dysfunction, even in the TGF-ß impair context.

Efficacy of vitamins E and C for reversing the cytotoxic effects of nicotine and cotinine.

Nicotine has adverse cellular and molecular effects on oral mucosa, bone, and teeth. Vitamin E (α-tocopherol) and vitamin C (ascorbic acid) are biological antioxidants with positive effects on wound healing and bone formation. This in vitro study sought to assess the cytotoxic effects of different concentrations of nicotine and cotinine (a metabolite of nicotine) on MG-63 osteoblast-like cells and human gingival fibroblasts (HGFs) in the presence and absence of antioxidant vitamins E and C (separately and combined). Cell viability and proliferation were assessed using the methyl thiazol tetrazolium (MTT) assay. Cell migration was assessed using the scratch test, and expression of apoptosis-related genes was quantitatively analyzed using real-time PCR. Dose-dependent negative effects of nicotine on the morphology, viability, proliferation, and migration of MG-63 and HGF cells were statistically significantly greater than those of cotinine. Vitamin E (separately and combined with vitamin C) was statistically significantly more effective than vitamin C (at the concentration used in this study) at improving cell viability, proliferation, and migration, and at reducing apoptosis of cells exposed to nicotine or cotinine. Based on the positive results of this study, vitamin C and especially vitamin E (systemically and/or locally) may be useful in the repair and regeneration of oral hard and soft tissues in smokers.

Identification and in vitro characterization of novel nanobodies against human granulocyte colony-stimulating factor receptor to provide inhibition of G-CSF function.

It has been shown that Granulocyte colony-stimulating factor (G-CSF) has a higher expression in malignant tumors, and anti-G-CSF therapy considerably decreases tumor growth, tumor vascularization and metastasis. Thus, blocking the signaling pathway of G-CSF could be beneficial in cancer therapy. This study is aimed at designing and producing a monoclonal nanobody that could act as an antagonist of G-CSF receptor. Nanobodies are the antigen binding fragments of camelid single-chain antibodies, also known as VHH. These fragments have exceptional properties which makes them ideal for tumor imaging and therapeutic applications. We have used our previously built nanobody phage libraries to isolate specific nanobodies to the G-CSF receptor. After a series of cross-reactivity and affinity experiments, two unique nanobodies were selected for functional analysis. Proliferation assay, real-time PCR and immunofluorescence assays were used to characterize these nanobodies. Finally, VHH26 nanobody that was able to specifically bind G-CSF receptor (G-CSF-R) on the surface of NFS60 cells and efficiently block G-CSF-R downstream signaling pathway in a dose-dependent manner was selected. This nanobody could be further developed into a valuable tool in tumor therapy and it forms a basis for additional studies in preclinical animal models.

In vitro proliferation and osteogenic differentiation of endometrial stem cells and dental pulp stem cells.

Stem-cell-based therapies were introduced aiming to overcome the limitations of the existing procedures for regeneration of mineralized tissues. Stem cells isolated from the endometrial tissue and dental pulp have the capacity to differentiate into various functional cells including osteoblasts. However, studies comparing their ability to regenerate mineralized tissue are lacking. The purpose of this study was to compare the proliferation and osteogenic differentiation potential of endometrial stem cells (EnSCs) and dental pulp stem cells (DPSCs) using in vitro cell culture technique. The DPSCs and EnSCs were isolated from human dental pulp and endometrium, respectively. Their proliferation and osteogenic potential were compared in the same osteogenic medium (OM) after 3, 5, 7 and 10 days using the methyl thiazol tetrazolium assay, alizarin red staining, and real-time quantitative reverse transcription polymerase chain reaction (Real-Time qRT-PCR). The EnSCs showed higher proliferation rate compared to DPSCs. Regarding osteogenesis, alizarin red-positive colonies appeared earlier and in greater amounts in DPSCs group. The real-time qRT-PCR demonstrated significantly greater osteogenic potential of DPSCs compared to EnSCs. Our findings revealed significant differences in stem cell properties based on the tissue source. The EnSCs had greater proliferation rate than DPSCs, while DPSCs showed greater osteogenic potential compared to EnSCs in the same OM.

In vitro comparison of the efficacy of TGF-ß1 and PDGF-BB in combination with freeze-dried bone allografts for induction of osteogenic differentiation in MG-63 osteoblast-like cells.

Predictable regeneration of alveolar bone defects has always been a challenge in implant dentistry. Bone allografts are widely used bone substitutes with controversial osteoinductive activity. This in vitro study aimed to assess the osteogenic potential of some commercially available freeze-dried bone allografts supplemented with human recombinant platelet-derived growth factor-BB and transforming growth factor beta-1. Cell viability, mineralization, and osteogenic gene expression of MG-63 osteoblast-like cells were compared among the allograft alone, allograft/platelet-derived growth factor-BB, allograft/transforming growth factor beta-1, and allograft/platelet-derived growth factor-BB/transforming growth factor beta-1 groups. The methyl thiazol tetrazolium assay, real-time quantitative reverse transcription polymerase chain reaction and alizarin red staining were performed, respectively, for assessment of cell viability, differentiation, and mineralization at 24-72 h post treatment. The allograft with greater cytotoxic effect on MG-63 cells caused the lowest differentiation among the groups. In comparison with allograft alone, allograft/transforming growth factor beta-1, and allograft/transforming growth factor beta-1/platelet-derived growth factor-BB caused significant upregulation of bone sialoprotein and osteocalcin osteogenic mid-late marker genes, and resulted in significantly higher amounts of calcified nodules especially in mineralized non-cytotoxic allograft group. Supplementation of platelet-derived growth factor-BB alone in 5 ng/mL concentration had no significant effect on differentiation or mineralization markers. According to the results, transforming growth factor beta-1 acts synergistically with bone allografts to enhance the osteogenic differentiation potential. Therefore, this combination may be useful for rapid transformation of undifferentiated cells into bone-forming cells for bone regeneration. However, platelet-derived growth factor-BB supplementation did not support this synergistic ability to enhance osteogenic differentiation and thus, further investigations are required.

Surface characterization and biological properties of regular dentin, demineralized dentin, and deproteinized dentin.

Bone autografts are often used for reconstruction of bone defects; however, due to the limitations of autografts, researchers have been in search of bone substitutes. Dentin is of particular interest for this purpose due to high similarity to bone. This in vitro study sought to assess the surface characteristics and biological properties of dentin samples prepared with different treatments. This study was conducted on regular (RD), demineralized (DemD), and deproteinized (DepD) dentin samples. X-ray diffraction and Fourier transform infrared spectroscopy were used for surface characterization. Samples were immersed in simulated body fluid, and their bioactivity was evaluated under a scanning electron microscope. The methyl thiazol tetrazolium assay, scanning electron microscope analysis and quantitative real-time polymerase chain reaction were performed, respectively to assess viability/proliferation, adhesion/morphology and osteoblast differentiation of cultured human dental pulp stem cells on dentin powders. Of the three dentin samples, DepD showed the highest and RD showed the lowest rate of formation and deposition of hydroxyapatite crystals. Although, the difference in superficial apatite was not significant among samples, functional groups on the surface, however, were more distinct on DepD. At four weeks, hydroxyapatite deposits were noted as needle-shaped accumulations on DemD sample and numerous hexagonal HA deposit masses were seen, covering the surface of DepD. The methyl thiazol tetrazolium, scanning electron microscope, and quantitative real-time polymerase chain reaction analyses during the 10-day cell culture on dentin powders showed the highest cell adhesion and viability and rapid differentiation in DepD. Based on the parameters evaluated in this in vitro study, DepD showed high rate of formation/deposition of hydroxyapatite crystals and adhesion/viability/osteogenic differentiation of human dental pulp stem cells, which may support its osteoinductive/osteoconductive potential for bone regeneration.

Effects of Er,Cr:YSGG Laser Treatment on Human Gingival Fibroblast Attachment, Viability and Morphology of Root Surface: An In Vitro Study.

Rehabilitation of periodontal support is the main goal of therapies for periodontitis. Hand instrumentation with curettes, piezoelectric ultrasonic scalers and lasers, such as Er,Cr:YSGG, are used for this purpose. This study was designed to evaluate human gingival fibroblast viability attachment to root surfac after modification with the mentioned therapeutic alternatives. Lasers showed significantly lower cell viability after 72 hours compared to hand instrumentation and ultrasound, probably due to more irregular root surfaces after treatment.

Effects of nicotine in the presence and absence of vitamin E on morphology, viability and osteogenic gene expression in MG-63 osteoblast-like cells.

BACKGROUND: Evidence shows that oxidative stress induced by nicotine plays an important role in bone loss. Vitamin E with its antioxidative properties may be able to reverse the effects of nicotine on bone. This study aimed to assess the effects of nicotine in the presence and absence of vitamin E on morphology, viability and osteogenic gene expression in MG-63 (osteosarcoma) human osteoblast-like cells. METHODS: We treated the cells with 5 mM nicotine. The viability and morphology of cells were evaluated respectively using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium (MTT) and crystal violet assays. The effect of nicotine on osteogenic gene expression in MG-63 cells was assessed by real-time reverse-transcription polymerase chain reaction of osteoblast markers, namely, alkaline phosphatase, osteocalcin and bone sialoprotein. RESULTS: The results revealed that survival and proliferation of MG-63 cells were suppressed following exposure to nicotine, and cytoplasm vacuolization occurred in the cells. Nicotine significantly down-regulated the expression of osteogenic marker genes. Such adverse effects on morphology, viability and osteogenic gene expression of MG-63 cells were reversed by vitamin E therapy. CONCLUSIONS: In conclusion, vitamin E supplementation may play a role in proliferation and differentiation of osteoblasts, and vitamin E can be considered as an anabolic agent to treat nicotine-induced bone loss.

Osteoinductive Activity of DFDBA Materials versus Growth Factors on Gene Expression of MG-63 Cells: An In Vitro Study.

Guided bone regeneration using demineralized freeze-dried bone allograft (DFDBA) and growth factors (GFs) is a current goal in implant dentistry because of their potential osteoinductive abilities. Regarding controversial results, the purpose of this study was to compare the osteoinductivity of three different DFDBAs from two different banks with two different GFs: transforming growth factor-beta (TGF-ß) and platelet-derived growth factor (PDGF). MG-63 osteoblast-like cells were exposed to two different concentration of commercial DFDBAs (10 and 20 mg/mL) and growth factors (5 and 10 ng/mL). Cell viability and proliferation were evaluated using a quantitative MTT assay (24 and 72 hours after treatment). For the assessment of cell differentiation, the expression of osteogenic marker genes was evaluated using quantitative real-time polymerase chain reaction 72 hours after treatment. Cell viability and proliferation in different concentrations of GFs was similar but significantly different in the DFDBA groups. Although water-soluble materials released from DFDBAs reduced viability and even caused cytotoxicity (viability <70%) in first 24 hours after treatment, increased viability and proliferation were seen after 72 hours. Dose-dependent up-regulation of osteocalcin (OC) was seen in the two DFDBA groups and in TGF-ß-treated cells. In contrast, dose-dependent down-regulation of OC was seen in PDGF-treated cells. The results show that induction of osteogenic differentiation (osteoinduction) at higher concentrations of DFDBAs (with the exception of one group) is more rapid than in the GF groups. In addition, TGF-ß at higher concentrations but PDGF at lower concentrations were associated with better results.

The effect of nicotine and cotinine on human gingival fibroblasts attachment to root surfaces.

BACKGROUND: Different compounds of smoking (e.g., nicotine and cotinine) are risk factors for various diseases such as oral cancer and periodontal diseases. Some studies reported the negative effects of nicotine on cell proliferation and differentiation. The present in vitro study assessed the effects of nicotine and cotinine (long-acting metabolite of nicotine) on the attachment and viability of human gingival fibroblast (HGF) cells to tooth root surfaces. METHODS: A total of 70 teeth specimens were placed into 48-well culture plates and covered with HGF cell suspension, in complete Dulbecco's modified Eagle's medium culture medium containing 1 nM, 1 µm, 1 mM, and 5 mM of nicotine and cotinine concentrations. Cellular attachment and viability measured using an MTT assay and a scanning electron microscope were used for cell morphological evaluation. RESULTS: After 24 h, low (nanomolar and micromolar) and high concentrations (millimolar) of nicotine and cotinine caused a significant reduction in the initial cell adhesion in comparison with the control group, but no significant difference was observed between the nicotine and the cotinine groups (p<0.05). Dentally attached cells with low concentrations of nicotine and cotinine proliferated 48 h after exposure, the same as the control group. However, dentally attached cells with high concentrations of nicotine and cotinine (especially 5 mM) did not proliferate 24 h after exposure (p<0.05). CONCLUSIONS: Low concentrations of nicotine and cotinine caused a reduction in the initial cell adhesion. However, no significant adverse effects on the proliferation of attached cells were seen in the longer period. High concentrations of nicotine and cotinine have adverse effects on the cell adhesion and proliferation of HGF cells.

Indomethacin-enhanced anticancer effect of arsenic trioxide in A549 cell line: involvement of apoptosis and phospho-ERK and p38 MAPK pathways.

BACKGROUND: Focusing on novel drug combinations that target different pathways especially apoptosis and MAPK could be a rationale for combination therapy in successful treatment of lung cancer. Concurrent use of cyclooxygenase (COX) inhibitors with arsenic trioxide (ATO) might be a possible treatment option. METHODS: Cytotoxicity of ATO, dexamethasone (Dex), celecoxib (Cel), and Indomethacin (Indo) individually or in combination was determined at 24, 48, and 72 hrs in A549 lung cancer cells. The COX-2 gene and protein expression, MAPK pathway proteins, and caspase-3 activity were studied for the most cytotoxic combinations. RESULTS: The IC50s of ATO and Indo were 68.7 µmol/L and 396.5 µmol/L, respectively. Treatment of cells with combinations of clinically relevant concentrations of ATO and Indo resulted in greater growth inhibition and apoptosis induction than did either agent alone. Caspase-3 activity was considerably high in the presence of ATO and Indo but showed no difference in single or combination use. Phosphorylation of p38 and ERK1/2 was remarkable in the concurrent presence of both drugs. CONCLUSIONS: Combination therapy with ATO and Indo exerted a very potent in vitro cytotoxic effect against A549 lung cancer cells. Activation of ERK and p38 pathways might be the mechanism of higher cytotoxic effect of ATO-Indo combination.