Impact of antibiotics on the proliferation and differentiation of human adipose-derived mesenchymal stem cells

Adipose tissue is a promising source of mesenchymal stem cells. Their potential to differentiate and regenerate other types of tissues may be affected by several factors. This may be due to in vitro cell-culture conditions, especially the supplementation with antibiotics. The aim of our study was to evaluate the effects of a penicillin-streptomycin mixture (PS), amphotericin B (AmB), a complex of AmB with copper (II) ions (AmB-Cu2+) and various combinations of these antibiotics on the proliferation and differentiation of adipose-derived stem cells in vitro. Normal human adipose-derived stem cells (ADSC, Lonza) were routinely maintained in a Dulbecco’s Modified Eagle Medium (DMEM) that was either supplemented with selected antibiotics or without antibiotics. The ADSC that were used for the experiment were at the second passage. The effect of antibiotics on proliferation was analyzed using the 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) and sulforhodamine-B (SRB) tests. Differentiation was evaluated based on Alizarin Red staining, Oil Red O staining and determination of the expression of ADSC, osteoblast and adipocyte markers by real-time RT-qPCR. The obtained results indicate that the influence of antibiotics on adipose-derived stem cells depends on the duration of exposure and on the combination of applied compounds. We show that antibiotics alter the proliferation of cells and also promote natural osteogenesis, and adipogenesis, and that this effect is also noticeable in stimulated osteogenesis

Psoriasis Treatment Changes the Expression Profile of Selected Caspases and their Regulatory MicroRNAs

Background/Aims: Psoriasis, an autoimmune diseases of the skin, characterized by patches of abnormal/inflammed skin, although not usually life-threatening, it causes severe discomfort, esthetic impairments, and may lead to impaired social functions and social withdrawal. Besides UV-phototherapy, various anti-inflammatory treatments are applied, depending on the severity of symptoms. In 2008, adalimumab (fully humanized human anti-TNF antibody) was launched for the treatment of psoriasis. In the quest to better understand the pathomechanism of adalimumab’s therapeutic effects, and the acquired resistance to the drug, we have investigated how its administration affect the regulation of the expression of selected caspases, including those activated by inflammosome. Methods: The research was initially carried out on normal human dermal fibroblasts (NHDF) treated with adalimumab for 2, 8 and 24 hours in vitro. Then, expression profile of genes encoding caspases and their regulatory micro-RNAs was determined with the use of oligonucleotide microarray. The validation of the microarray results was carried out by qRT-PCR. The in vitro study was followed by ex-vivo investigation of adalimumab’s effects on the expression of caspase-6 in blood of the psoriatic patients. The samples were collected before, and 2 hours after adalimumab’s administration and the analysis was determined by qRT-PCR. Results: The result of the analysis indicated that introduction of adalimumab to the NHDF culture resulted in the change of the transcription activity of genes encoding caspases and genes encoding miRNAs. The analysis revealed 5 different miRNA molecules regulating the expression of: CASP2, CASP3 and CASP6. There were no statistically significant differences in the expression of gene encoding caspase-6 in the patients’ blood before and 2 hours after the anti-TNF drug administration. Conclusion: We have found that adalimumab administration affects caspases expression, thus they may be used as molecular markers for monitoring the therapy with the use of an anti-TNF drugs, including adalimumab. It is likely that the mechanisms responsible for changed expression profiles of genes encoding caspase-2,-3, and -6, may be caused by the upregulation of the respective microRNA molecules. Increased expression of genes encoding specific caspases may induce inflammatory processes, as well as trigger apoptosis. Furthermore, the proapoptotic activity of caspases may be enhanced by miRNA molecules, which exhibit proapoptotic function. The overexpression of such miRNAs was observed in our study

Comparison of Physicochemical, Mechanical, and (Micro-)Biological Properties of Sintered Scaffolds Based on Natural- and Synthetic Hydroxyapatite Supplemented with Selected Dopants

The specific combinations of materials and dopants presented in this work have not been previously described. The main goal of the presented work was to prepare and compare the different properties of newly developed composite materials manufactured by sintering. The synthetic-(SHAP) or natural- (NHAP) hydroxyapatite serves as a matrix and was doped with: (i) organic: multiwalled carbon nanotubes (MWCNT), fullerenes C60, (ii) inorganic: Cu nanowires. Research undertaken was aimed at seeking novel candidates for bone replacement biomaterials based on hydroxyapatite—the main inorganic component of bone, because bone reconstructive surgery is currently mostly carried out with the use of autografts; titanium or other non-hydroxyapatite -based materials. The physicomechanical properties of the developed biomaterials were tested by Scanning Electron Microscopy (SEM), Dielectric Spectroscopy (BSD), Nuclear Magnetic Resonance (NMR), and Differential Scanning Calorimetry (DSC), as well as microhardness using Vickers method. The results showed that despite obtaining porous sinters. The highest microhardness was achieved for composite materials based on NHAP. Based on NMR spectroscopy, residue organic substances could be observed in NHAP composites, probably due to the organic structures that make up the tooth. Microbiology investigations showed that the selected samples exhibit bacteriostatic properties against Gram-positive reference bacterial strain S. epidermidis (ATCC 12228); however, the property was much less pronounced against Gram-negative reference strain E. coli (ATCC 25922). Both NHAP and SHAP, as well as their doped derivates, displayed in good general compatibility, with the exception of Cu-nanowire doped derivates

Effects of electro-conductive, biomaterial-based tissue scaffolds on stem cells and transdifferentiation-derived somatic cells

This is the final version of the article, which has been published in final form at / http://www.lestudium-ias.com/content/effects-electro-conductive-biomaterial-based-tissue-scaffolds-stem-cells-and-1The combination of stem cell therapy with a supportive scaffold is a promising approach to improving tissue engineering. We aim producing novel material composites that may serve as artificial Extracellular Matrix (ECM). The natural ECM is composed of an organic (protein, polysaccharide) and inorganic (i.e. hydroxy-apatite) components that when combined with the cells form a tissue. ECM is an integral part of every tissue that besides providing the environment for cells to grow, it also improves tissue’s mechanical properties. It provides elasticity, flexibility and durability for the tissue. Tissue engineering approaches utilize artificial materials (biomaterials) as a substitute of natural ECM. The process of producing tissue scaffolds obtained from biodegradable polymers has become a very intensively researched area for the past several years. Most of the current work focuses on the design and preparation of scaffolds with use of various production technologies and different natural materials like chitosan, collagen, elastin and different synthetic ones, like polymer polycaprolactone (PCL), poly(lactic acid) (PLA), poly(ethylene oxide) (PEO). The objective of this study was to check the impact of the biomaterials on various cell types, and compare their growth pattern. Biodegradable PCL, and five of its hybrids: PCL+SHAP (SHAP, synthetic hydroxyapatite), PCL+NHAP (NHAP, natural hydroxyapatite), PCL+PLGA (PLGA, poly(lactide-co-glycolide), PCL+CaCO3, PCL+SHAP+NHAP+CaCO3 as well as one non degradable biomaterial: polyacrylonitryl (PAN), were tested. For the experiments four different cell types were used: human dermal skin fibroblasts, B16F10 (mouse melanoma cells), HSkMEC (Human Skin Microvascular Endothelial Cells) and HEPC-CB1 (Human Endothelial Progenitor Cells –Cord Blood 1). Impacts of the biomaterials on cells were assessed: 1) by measuring cytotoxic effect of the biomaterials liquid extracts and 2) by direct contact test. The ability of cells to attach to the biomaterials was tested as well as cells’ potential to growth and proliferate on the surface of the biomaterials. None of the tested biomaterials was cytotoxic towards the tested cells, making them a potential valuable raw ingredient for 3D scaffold development that would find its applications in tissue engineering. The differences in efficiency of cells attachment and proliferation between tested biomaterials and cells lines were observed. In addition, a stimulating effect of the biomaterials on cells growth was also detected

Selected biodegradable and nonbiodegradable materials - their impact on cells growth and proliferation

International audienc

Nuclear expression of Ku70/80 is associated with CHEK2 germline mutations in breast cancer

Ku70/80 protein inhibitors reduce the repair of DNA double-strand breaks via the Ku70/80 pathway, so they can be used to treat cancers with Ku70/80 overexpression. Since the association of Ku70/80 with germline CHEK2 mutations in breast cancer is unknown, in this study we evaluated the expression of Ku70/80 in breast cancers with germline CHEK2 mutations. Immunohistochemistry with a Ku70/80 antibody on tissue microarrays from 225 CHEK2-associated breast cancers was used and automatically assessed with computerized image analysis. We report that the vast majority of breast cancers expressed high level of nuclear Ku70/80 and a small percentage of tumors (3.5%) were negative for Ku70/80 expression. There was a significant difference between the nuclear Ku70/80 expression in CHEK2-associated vs. CHEK2-non-associated breast cancers in all tumors (p = 0.009), and in the estrogen receptor (ER) positive subgroup of breast cancers (p = 0.03). This study is the first reporting an association of Ku70/80 expression with CHEK2 germline mutations in breast cancer. The results suggest that evaluation of Ku70/80 expression in breast cancer may improve the selection of breast cancer patients for Ku70/80 inhibitor therapy, and point to CHEK2-associated breast cancer and a subset of ER-positive breast cancer as potential suitable targets for such therapy

Psoriasis Treatment Changes the Expression Profile of Selected Caspases and their Regulatory MicroRNAs

Background/Aims: Psoriasis, an autoimmune diseases of the skin, characterized by patches of abnormal/inflammed skin, although not usually life-threatening, it causes severe discomfort, esthetic impairments, and may lead to impaired social functions and social withdrawal. Besides UV-phototherapy, various anti-inflammatory treatments are applied, depending on the severity of symptoms. In 2008, adalimumab (fully humanized human anti-TNF antibody) was launched for the treatment of psoriasis. In the quest to better understand the pathomechanism of adalimumab’s therapeutic effects, and the acquired resistance to the drug, we have investigated how its administration affect the regulation of the expression of selected caspases, including those activated by inflammosome. Methods: The research was initially carried out on normal human dermal fibroblasts (NHDF) treated with adalimumab for 2, 8 and 24 hours in vitro. Then, expression profile of genes encoding caspases and their regulatory micro-RNAs was determined with the use of oligonucleotide microarray. The validation of the microarray results was carried out by qRT-PCR. The in vitro study was followed by ex-vivo investigation of adalimumab’s effects on the expression of caspase-6 in blood of the psoriatic patients. The samples were collected before, and 2 hours after adalimumab’s administration and the analysis was determined by qRT-PCR. Results: The result of the analysis indicated that introduction of adalimumab to the NHDF culture resulted in the change of the transcription activity of genes encoding caspases and genes encoding miRNAs. The analysis revealed 5 different miRNA molecules regulating the expression of: CASP2, CASP3 and CASP6. There were no statistically significant differences in the expression of gene encoding caspase-6 in the patients’ blood before and 2 hours after the anti-TNF drug administration. Conclusion: We have found that adalimumab administration affects caspases expression, thus they may be used as molecular markers for monitoring the therapy with the use of an anti-TNF drugs, including adalimumab. It is likely that the mechanisms responsible for changed expression profiles of genes encoding caspase-2,-3, and -6, may be caused by the upregulation of the respective microRNA molecules. Increased expression of genes encoding specific caspases may induce inflammatory processes, as well as trigger apoptosis. Furthermore, the proapoptotic activity of caspases may be enhanced by miRNA molecules, which exhibit proapoptotic function. The overexpression of such miRNAs was observed in our study