Search results for: Cetyltrimethylammonium Bromide (CTAB)

Authors: Hilal Turkoglu Sasmazel, Ozan Ozkan, Seda Surucu

Abstract:

Tissue engineering is the field of treating defects caused by injuries, trauma or acute/chronic diseases by using artificial scaffolds that mimic the extracellular matrix (ECM), the natural biological support for the tissues and cells within the body. The main aspects of a successful artificial scaffold are (i) large surface area in order to provide multiple anchorage points for cells to attach, (ii) suitable porosity in order to achieve 3 dimensional growth of the cells within the scaffold as well as proper transport of nutrition, biosignals and waste and (iii) physical, chemical and biological compatibility of the material in order to obtain viability throughout the healing process. By hybrid scaffolds where two or more different materials were combined with advanced fabrication techniques into complex structures, it is possible to combine the advantages of individual materials into one single structure while eliminating the disadvantages of each. Adding this to the complex structure provided by advanced fabrication techniques enables obtaining the desired aspects of a successful artificial tissue scaffold. In this study, fibroblast compatibility of poly(ε-caprolactone) (PCL)/chitosan core-shell electrospun hybrid scaffolds with proper mechanical, chemical and physical properties successfully developed in our previous study was investigated. Standard 7-day cell culture was carried out with L929 fibroblast cell line. The viability of the cells cultured with the scaffolds was monitored with 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) viability assay for every 48 h starting with 24 h after the initial seeding. In this assay, blank commercial tissue culture polystyrene (TCPS) Petri dishes, single electrospun PCL and single electrospun chitosan mats were used as control in order to compare and contrast the performance of the hybrid scaffolds. The adhesion, proliferation, spread and growth of the cells on/within the scaffolds were observed visually on the 3rd and the 7th days of the culture period with confocal laser scanning microscopy (CSLM) and scanning electron microscopy (SEM). The viability assay showed that the hybrid scaffolds caused no toxicity for fibroblast cells and provided a steady increase in cell viability, effectively doubling the cell density for every 48 h for the course of 7 days, as compared to TCPS, single electrospun PCL or chitosan mats. The cell viability on the hybrid scaffold was ~2 fold better compared to TCPS because of its 3D ECM-like structure compared to 2D flat surface of commercially cell compatible TCPS, and the performance was ~2 fold and ~10 fold better compared to single PCL and single chitosan mats, respectively, even though both fabricated similarly with electrospinning as non-woven fibrous structures, because single PCL and chitosan mats were either too hydrophobic or too hydrophilic to maintain cell attachment points. The viability results were verified with visual images obtained with CSLM and SEM, in which cells found to achieve characteristic spindle-like fibroblast shape and spread on the surface as well within the pores successfully at high densities.

Keywords: chitosan, core-shell, fibroblast, electrospinning, PCL

Procedia PDF Downloads 152

Authors: Jolene M. Helena, Annie M. Joubert, Peace Mabeta, Magdalena Coetzee, Roy Lakier, Anne E. Mercier

Abstract:

Targeting the distant tumour and its microenvironment whilst preserving bone density is important in improving the outcomes of patients with bone metastases. 2-Ethyl-3-O-sulphamoyl-estra1,3,5(10)16-tetraene (ESE-16) is an in-silico-designed 2- methoxyestradiol analogue which aimed at enhancing the parent compound’s cytotoxicity and providing a more favourable pharmacokinetic profile. In this study, the potential radiosensitization effects of ESE-16 were investigated in an in vitro bone metastasis model consisting of murine pre-osteoblastic (MC3T3-E1) and pre-osteoclastic (RAW 264.7) bone cells, metastatic prostate (DU 145) and breast (MDA-MB-231) cancer cells, as well as human umbilical vein endothelial cells (HUVECs). Cytotoxicity studies were conducted on all cell lines via spectrophotometric quantification of 3-(4,5-dimethylthiazol-2-yl)-2,5- diphenyltetrazolium bromide. The experimental set-up consisted of flow cytometric analysis of cell cycle progression and apoptosis detection (Annexin V-fluorescein isothiocyanate) to determine the lowest ESE-16 and radiation doses to induce apoptosis and significantly reduce cell viability. Subsequent experiments entailed a 24-hour low-dose ESE-16-exposure followed by a single dose of radiation. Termination proceeded 2, 24 or 48 hours thereafter. The effect of the combination treatment was investigated on osteoclasts via tartrate-resistant acid phosphatase (TRAP) activity- and actin ring formation assays. Tumour cell experiments included investigation of mitotic indices via haematoxylin and eosin staining; pro-apoptotic signalling via spectrophotometric quantification of caspase 3; deoxyribonucleic acid (DNA) damage via micronuclei analysis and histone H2A.X phosphorylation (γ-H2A.X); and Western blot analyses of bone morphogenetic protein-7 and matrix metalloproteinase-9. HUVEC experiments included flow cytometric quantification of cell cycle progression and free radical production; fluorescent examination of cytoskeletal morphology; invasion and migration studies on an xCELLigence platform; and Western blot analyses of hypoxia-inducible factor 1-alpha and vascular endothelial growth factor receptor 1 and 2. Tumour cells yielded half-maximal growth inhibitory concentration (GI50) values in the nanomolar range. ESE-16 concentrations of 235 nM (DU 145) and 176 nM (MDA-MB-231) and a radiation dose of 4 Gy were found to be significant in cell cycle and apoptosis experiments. Bone and endothelial cells were exposed to the same doses as DU 145 cells. Cytotoxicity studies on bone cells reported that RAW 264.7 cells were more sensitive to the combination treatment than MC3T3-E1 cells. Mature osteoclasts were more sensitive than pre-osteoclasts with respect to TRAP activity. However, actin ring morphology was retained. The mitotic arrest was evident in tumour and endothelial cells in the mitotic index and cell cycle experiments. Increased caspase 3 activity and superoxide production indicated pro-apoptotic signalling in tumour and endothelial cells. Increased micronuclei numbers and γ-H2A.X foci indicated increased DNA damage in tumour cells. Compromised actin and tubulin morphologies and decreased invasion and migration were observed in endothelial cells. Western blot analyses revealed reduced metastatic and angiogenic signalling. ESE-16-induced radiosensitization inhibits metastatic signalling and tumour cell survival whilst preferentially preserving bone cells. This low-dose combination treatment strategy may promote the quality of life of patients with metastatic bone disease. Future studies will include 3-dimensional in-vitro and murine in-vivo models.

Keywords: angiogenesis, apoptosis, bone metastasis, cancer, cell migration, cytoskeleton, DNA damage, ESE-16, radiosensitization.

Procedia PDF Downloads 137