MTT Test Assay Research Articles

Long-term proliferation and delayed senescence of bone marrow-derived human mesenchymal stem cells on metformin co-embedded HA/Gel electrospun composite nanofibers

The current work was going to assess the efficacy of hyaluronic acid/Gelatin (HA/GEL) encapsulated with metformin-loaded mesoporous silica nanoparticles (MET@MSNs) on the prolonged in vitro proliferation of bone marrow-derived human mesenchymal stem cells (BM-hMSCs) without prompting aging. For this purpose, composite scaffolds consisting of MET and MET@MSNs in combination with HA were manufactured by convenient and green electrospinning technique. The surface morphology and biochemical characteristics of manufactured scaffolds were evaluated using FE-SEM, TGA, and FTIR. The enhanced proliferation rate and metabolic activity of the BM-hMSCs seeded on MET@MSNs incorporated into HA/Gel scaffolds (MET@MSNs HA/Gel NFs) were confirmed via PicoGreen and MTT test assay, respectively, over 21 days of culture. Moreover, the mRNA expression levels of senescence and stemness marker after a long-term in vitro culturing by qPCR. According to the high proliferation rate and proper metabolic activity of the BM-hMSCs after 21 days of culture on the MET@MSNs HA/Gel NFs, the prepared NFs represented great capacity for both initial and controlled release of MET. The obtained data from MTT and PicoGreen examination also revealed improved metabolic activity and expansion rate for BM-hMSCs grown on the MET@MSNs-NFs compared to the other treated groups after 1, 14 and 21 days of incubation. On the other hand, the hTERT and telomerase expression was significantly increased in BM-hMSCs cultured on MET@MSNs HA/Gel NFs compared to the control group. These results confirmed the capability of MET@MSNs HA/Gel NFs for persistent and controlled discharge of MET in the designing of a novel platform for neutralizing cellular aging and attaining adequate quantities of fresh BM-hMSCs for tissue engineering applications.

Sustainable Rabbit Skin Glue to Produce Bioactive Nanofibers for Nonactive Wound Dressings.

This paper assessed the collagen glue (Col) from rabbit skin for use as a raw material in combination with different water-based dispersants of antimicrobial agents such as ZnO NPs, TiO2 NPs doped with nitrogen and Ag NPs (TiO2-N-Ag NPs), and chitosan (CS) for the production of biocompatible and antimicrobial nanofibers. The electrospun nanofibers were investigated by scanning electron microscopy (SEM), attenuated total reflectance in conjunction with Fourier-transform infrared spectroscopy (ATR-FT-IR) analyses and antioxidant activity. The biocompatibility of electrospun nanofibers was investigated on cell lines of mouse fibroblast NCTC (clone L929) using MTT test assays. Antimicrobial activity was performed against Escherichia coli and Staphylococcus aureus bacteria and Candida albicans pathogenic fungus. Electrospun antimicrobial nanofibers based on collagen glue achieved reduction in the number of viable microorganisms against both fungi and bacteria and exhibited multiple inhibitory actions of fungal and bacterial strains. The electrospun nanofibers showed average dimension sizes in the range of 30–160 nm. The results indicated that both Col/TiO2-N-Ag NPs and Col/CS formulations are suitable for cell proliferation and may be useful for producing of nonactive wound dressings.

Effects of polyethylene glycols on intestinal efflux pump expression and activity in Caco-2 cells

The present study was planned to investigate the influence of polyethylene glycols (PEGs) on the activity and expression of P-glycoprotein (P-gp). Sub-toxic concentrations of PEGs in Caco-2 cells were determined using the MTT test assay. Then the measurement of Rhodamine-123 (Rho-123) uptake, a P-gp fluorescence substrate, in Caco-2 cells confronting PEG 400 (1% and 2% w/v), PEG 4000 (2% and 4% w/v), PEG 6000 (2% and 4% w/v), PEG 10000 (2% and 4% w/v), PEG 15000 (1% and 2% w/v), and PEG 35000 (2% and 4% w/v) overnight was taken to elucidate whether non-toxic concentrations of PEGs are able to impact P-gp activity. Furthermore, western blotting was carried out to investigate P-gp protein expression. The results showed that PEG 400 at concentrations of 1% (w/v) and 2% (w/v) and PEG 6000 at the concentration of 4% (w/v) are notably capable of blocking P-gp. Based on the obtained results it is concluded that the mentioned excipients could be used to obstruct P-gp efflux transporter in order to increase the bioavailability of co-administered substrate drug.

The Thermochemotherapy Effect of Mn<sub>0.5</sub>Zn<sub>0.5</sub>Fe<sub>2</sub>O<sub>4</sub> Magnetic Fluid Hyperthermia Combined with As<sub>2</sub>O<sub>3</sub> on GLC-82 Cells

AIM: To study the preparation and characterization of the Mn0.5Zn0.5Fe2O4 (MZF) magnetic nanoparticles and the antitumor function of MZF nanomagnetic fluid hyperthermia (NMFH) combined with As2O3 on GLC-82 cells.METHODS: The MZF magnetic nanoparticles were prepared by the method of chemical coprecipitation.,which were detected by transmission electron microscopy (TEM) and energy dispersive spectrometry (EDS). In vitro heating test of MZF magnetic fluid in an alternating magnetic field (AMF) was carried out. After that, we studied the therapeutic effects on cultured GLC-82 cells treated by thermochemotherapy of NMFH combined with As2O3 at various concentrations (2,5,8μM) were evaluated by MTT assay and flow cytometry assay (FCM).RESULTS: The MZF magnetic nanoparticles were prepared successfully. Their shapes are approximately global and their diameters are about 30 nm. EDS showed MZF magnetic nanoparticle contained Mn, Zn, Fe elements. The corresponding fluid of MZF magnetic nanoparticle could rise to 40~50°C due to different concentration in a AMF and keep stable from then on. MTT assay and FCM revealed that the thermochemotherapy effect of the MZF magnetic nanoparticles combined with As2O3 can cause significantly inhibit cellular growth observed by MTT test assay and obviously induce cellular apoptosis by FCM detecting (p<0.05). The apoptotic indexes of GLC-82 cells of the pure As2O3 group,pure MZF magnetic nanoparticles hyperthermia group and thermochemotherapy of NMFH combined with As2O3 group were 4.98%,16.30%,22.53%,respectively,while the apoptotic index of the negative control group was 0.56%.CONCLUSION: The MZF magnetic nanoparticles successfully were prepared through improved co-precipitation process and have good power absorption capabilities in the high frequency AMF, then they can rise to a steady temperature. MZF magnetic nanoparticles have hyperthermia effects on tumor and their antitumor effect significantly increase when combined with As2O3.

Preparation of PEI-Fe<sub>3</sub>O<sub>4</sub>/Survivin-siRNA Magnetic Complex and its Anti-Tumor Effect Combined with MFH on Lung Carcinoma Cells

Objective: To prepare and characterize the PEI-Fe3O4 nanosized magnetic complex and investigate the therapeutic effects of the PEI-Fe3O4 /survivin-siRNA magnetic complex combined with magnetic fluid hyperthermia (MFH) on human lung carcinoma cells . Methods: Fe3O4 magnetic nanoparticles (MNPs) were prepared by the technique of chemical co-precipitation and the surface of Fe3O4 MNPs was modified by PEI. Transmission electron microscope(TEM)，X-ray diffraction(XRD)and Fourier transform infrared spectroscopy(FTIR) were applied to characterize the PEI-Fe3O4 magnetic complex. Thermodynamic test was used to observe temperature change of various concentration of Fe3O4 suspension under alternating magnetic field(AMF). The DNA binding efficiency of PEI-Fe3O4 was observrd by electrophoresis experiment. Transfection efficiency was determined by delivering reporter gene(pEGFP-encoding luciferase)to GLC-82 cell lines using PEI-Fe3O4 as a vector. Inhibitory effect of the expression of survivin mRNA in GLC-82 cells was detected by RT-PCR after transfection with PEI-Fe3O4/survivin-siRNA. The therapeutic effects of PEI-Fe3O4/survivin-siRNA combined with MFH on GLC-82 cells was evaluated by MTT test. Results: The prepared Fe3O4 MNPs and PEI-Fe3O4 magnetic complex were approximately spherical，high electron dense，good dispersibility uniform in size observed by TEM, with an average diameter of about 20 nm.The XRD pattern of the sample showed the characteristic peaks of their cubic spinel structure,The observed diffraction lines were found to correspond to those of a standard ferrite(Fe3O4) pattern. The results of FTIR demonstrated the adsorption effect of PEI. The corresponding suspension with different concentrations of Fe3O4 MNPs could rise to a steady temperature ranging from 42°C～59°C under AMF within 40 minutes and keep stable from then on. The PEI-Fe3O4 magnetic complex had good binding ability with DNA which could efficiently deliver the foreign gene to GLC-82 cell.Inhibitory ratio of survivin mRNA expressions by survivin-siRNA was 35.6%and MTT test assay showed that the cell growth inhibition rate(IR) of the combination therapy(58.71%) is higher than that(31.87%,28.45%) of the single therapy. Conclusion: The experiment results suggested that PEI-Fe3O4 magnetic complex was an novel non-virus gene vector, which has multiple benefits in combination of thermotherapy and gene therapy on lung carcinoma.

Adenovirus-mediated bone morphogenetic protein-2 gene transfection of bone marrow mesenchymal stem cells combined with nano-hydroxyapatite to construct bone graft material in vitro

To study the adhesion, proliferation and expression of bone marrow mesenchymal stem cells (BMSCs) on nano-hydroxyapatite (Nano-HA) bone graft material after transfection of adenovirus-mediated human bone morphogenetic protein-2 expression vector (Ad-BMP-2). BMSCs were transfected using Ad-BMP-2. Immunohistochemistry and Western blot were used to detect BMP-2 expression in transfected cells. After transfection, BMP-2 protein was highly expressed in BMSCs; MTT test assay showed that the Nano-HA bone graft material could not inhibit in vitro proliferation of BMSCs. Ad-BMP-2-transfected BMSCs are well biocompatible with Nano-HA bone graft material, the transfected cells in material can secrete BMP-2 stably for a long time.

Characterization and antibacterial performance of ZrNO–Ag coatings

Composite metal nitride–metal (MeN–Me) physical vapor deposited (PVD) films (e.g. TaN–Cu, TiN–Cu, ZrN–Ag, etc.) are often considered for applications such as tribological and antibacterial coatings. In this study, ZrN, ZrO2–Ag and ZrNO–Ag coatings with different Ag contents were deposited by reactive magnetron sputtering. Monoclinic ZrO2 and fcc Ag composite structures were obtained when Zr and Ag were cosputtered to react with oxygen. ZrNO–Ag showed a porous structure containing monoclinic ZrO2, fcc Ag and Zr2ON2. Actinobacillus actinomycetemcomitans (A. actinomycetemcomitans) and Staphylococcus aureus (S. aureus), major pathogens frequently found in the implant-associated infections, were cultured on the coated samples. The antibacterial effects were determined using in vitro anti-bacterial analyses by a fluorescence staining method employing Syto9 and bacterial viability agar tests. In addition, the biocompatibility of human gingival fibroblast (HGF) cells on coatings was also evaluated by a MTT test assay. It showed that the nanostructure and Ag content of the ZrO2–Ag and ZrNO–Ag coatings were correlated with the biocidal property. The ZrN coating deposited by magnetron sputtering did not improve the bacterial reduction of A. actinomycetemcomitans and S. aureus. The ZrO2–Ag and ZrNO–Ag coated samples had lower bacterial retention, and the ZrNO–Ag coatings with silver content of 11.8at.% possessed the best antibacterial performance with excellent HGF cell compatibility as well.

Leishmanicidal activity and cytotoxicity of compounds from two Annonacea species cultivated in Northeastern Brazil

Visceral leishmaniasis is endemic in 88 countries, with a total of 12 million people infected and 350 million at risk. In the search for new leishmanicidal agents, alkaloids and acetogenins isolated from leaves of Annona squamosa and seeds of Annona muricata were tested against promastigote and amastigote forms of Leishmania chagasi. Methanol-water (80:20) extracts of A. squamosa leaves and A. muricata seeds were extracted with 10% phosphoric acid and organic solvents to obtain the alkaloid and acetogenin-rich extracts. These extracts were chromatographed on a silica gel column and eluted with a mixture of several solvents in crescent order of polarity. The compounds were identified by spectroscopic analysis. The isolated compounds were tested against Leishmania chagasi, which is responsible for American visceral leishmaniasis, using the MTT test assay. The cytotoxicity assay was evaluated for all isolated compounds, and for this assay, RAW 264.7 cells were used. O-methylarmepavine, a benzylisoquinolinic alkaloid, and a C37 trihydroxy adjacent bistetrahydrofuran acetogenin were isolated from A. squamosa, while two acetogenins, annonacinone and corossolone, were isolated from A. muricata. Against promastigotes, the alkaloid showed an IC50 of 23.3 µg/mL, and the acetogenins showed an IC50 ranging from 25.9 to 37.6 µg/mL; in the amastigote assay, the IC50 values ranged from 13.5 to 28.7 µg/mL. The cytotoxicity assay showed results ranging from 43.5 to 79.9 µg/mL. These results characterize A. squamosa and A. muricata as potential sources of leishmanicidal agents. Plants from Annonaceae are rich sources of natural compounds and an important tool in the search for new leishmanicidal therapies.

DNA microarray analysis of gene expression of MC3T3-E1 osteoblast cell cultured on anodized- or machined titanium surface

Purpose: The aim of this study was to evaluate adhesion and gene expression of the MC3T3-E1 cells cultured on machined titanium surface (MS) and anodized titanium surface (AS) using MTT test, Scanning electron micrograph and cDNA microarray. Materials and Methods: The MTT test assay was used for examining the proliferation of MC3T3-E1 cells, osteoblast like cells from Rat calvaria, on MS and AS for 24 hours and 48 hours. Cell cultures were incubated for 24 hours to evaluate the influence of the substrate geometry on both surfaces using a Scanning Electron Micrograph (SEM). The cDNA microarray Agilent Rat 22K chip was used to monitor expressions of genes. Results: After 24 hours of adhesion, the cell density on AS was higher than MS (p 0.05). AS had the irregular, rough and porous surface texture. After 48 hours incubation of the MC3T3-E1 cells, connective tissue growth factor (CTGF) was up-regulated on AS than MS (more than 2 fold) and the insulin-like growth factor 1 receptor was down-regulated (more than 2 fold) on AS than MS. Conclusion: Microarray assay at 48 hours after culturing the cells on both surfaces revealed that osteoinductive molecules appeared more prominent on AS, whereas the adhesion molecules on the biomaterial were higher on MS than AS, which will affect the phenotype of the plated cells depending on the surface morphology. (J Korean Acad Periodontol 2008;38:299-308)