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1.
Bratisl Lek Listy ; 120(10): 757-763, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31663351

RESUMO

AIM: The study was aimed at design a good fusion construct that would successfully express the recombinant proteins and produce peptides in Escherichia coli. Two different constructs including human epidermal growth factor (hEGF) gene were designed to obtain an efficient expression level of hEGF. The hEGF sequence was inserted in pET32a vector containing thioredoxin (Trx) sequence and modified pET15b vector containing intein and elastin-like polypeptide (ELP). METHODS: The vectors were transformed into E. coli TOP10F' for multiplication and further into E. coli BL21 (DE3) to express protein. The hEGF expression was induced by isopropyl ß-D-1-thiogalactopyranoside (IPTG) while the expression levels were evaluated by SDS-PAGE and western blotting and compared by ImageJ analysis, BCA and Elisa assays. RESULTS: The expression level after 2 hours of IPTG induction was significantly higher than after other induction times. ImageJ, BCA and Elisa analyses demonstrated that the Trx presence enhanced protein expression significantly when compared to ELP-intein-based construct. CONCLUSION: The pET32a-Trx-hEGF construct had a higher expression than pET15b-ELP-intein-hEGF. Overall, considering Trx, the fusion protein in construct design can make it suitable to significantly express hEGF compared to ELP-intein while its combination with ELP-intein may improve the expression of the ELP-intein construct (Tab. 2, Fig. 7, Ref. 34).


Assuntos
Elastina , Fator de Crescimento Epidérmico/biossíntese , Escherichia coli , Inteínas , Humanos , Peptídeos , Proteínas Recombinantes de Fusão/biossíntese
2.
Bratisl Lek Listy ; 119(6): 391-396, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-29947241

RESUMO

OBJECTIVE: Platelet-derived growth factor (PDGF-BB) is an important factor in the regeneration and wound healing. One of the problems is that there is not enough efficiency in the transmission or delivery of such factors in the wound site. In this study, alginate sulfate hydrogel was synthesized with recombinant PDGF-BB growth factor for achieving a quick method in wound healing. METHODS: In this study, Alginate sulfate hydrogel was made by photo-crosslinking method and methacrylate. Its toxicity was evaluated by MTT assay. Transforming growth factor was studied in releasing from synthesized alginate sulfate hydrogels and also, lack of toxicity was confirmed, and hydrogel was made with a recombinant human growth factor. Wounds were created with a diameter of 10 mm on the back of rats in order to check the wound healing. RESULTS: This study showed that alginate sulfate hydrogels-PDGF-BB were faster in wound healing than non-sulfate alginate hydrogels-PDGF-BB. Therefore, the controlled delivery of growth factor system can be a powerful idea for therapeutic applications for wound healing. CONCLUSION: Alginate sulfate hydrogel with recombinant growth factor 4 µg/cm2 (PDGF-BB) was very suitable for wound healing (Tab. 1, Fig. 3, Ref. 34).


Assuntos
Alginatos/farmacologia , Indutores da Angiogênese/farmacologia , Hidrogéis/farmacologia , Proteínas Proto-Oncogênicas c-sis/farmacologia , Proteínas Recombinantes/farmacologia , Sulfatos/farmacologia , Cicatrização/efeitos dos fármacos , Animais , Becaplermina , Ratos , Regeneração/efeitos dos fármacos
3.
Hum Exp Toxicol ; 37(7): 714-724, 2018 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-28920465

RESUMO

BACKGROUND: Defects in modulating wild-type (wt) p53 and survivin are associated with a resistant disease in acute lymphoblastic leukemia (ALL). Yet, no wt-p53 and survivin modulating drugs have been approved for clinical application in ALL. Here, we investigated if in vitro eicosapentaenoic acid (EPA) concentrations equal to human plasma levels are able to target wt-p53 and survivin. METHODS: Wt-p53 Molt-4 cells (ALL cell line) were treated with 50, 100, 150, and 200 µM of EPA after which cell number, viability, proliferation rate, survivin expression, wt-p53 accumulation, caspase-3 activation, and apoptosis were evaluated. RESULTS: After 48- and 72-h treatments with EPA at concentrations ranging from 50 to 200 µM, cell proliferation rates were measured to be 71.5-32.6% and 68.2-13.7% and metabolic activities were measured to be 77-44% and 71-26%, respectively. Treatment with 50-200 µM of EPA for 48 h resulted in 14.1-74.6% and 69.5-45.5% decreases in survivin mRNA and protein levels, respectively. EPA induced 1.3-6 and 1.9-20-fold increases in caspase-3 activation and wt-p53 accumulation, respectively. Increase in wt-p53/survivin and caspase-3/survivin ratios from 1 in untreated cells to 20.3 and 5.8 was measured for 150 µM of EPA. Low necrotic rates ranging from 0.3% to 2.8% and an increase in the number of total apoptotic cells (early + late) ranging from 9.8% to 81% were also observed with increasing EPA concentrations. CONCLUSION: EPA induces strongly wt-p53 with a remarkable decrease in survivin expression, representing an attractive compound to modulate wt-p53 and survivin in ALL cells.


Assuntos
Caspase 3/metabolismo , Ácido Eicosapentaenoico/farmacologia , Óleos de Peixe/farmacologia , Proteínas Inibidoras de Apoptose/metabolismo , Leucemia-Linfoma Linfoblástico de Células Precursoras/metabolismo , Proteína Supressora de Tumor p53/metabolismo , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Humanos , Proteínas Inibidoras de Apoptose/genética , Leucemia-Linfoma Linfoblástico de Células Precursoras/genética , RNA Mensageiro/metabolismo , Survivina
4.
Cell Mol Biol (Noisy-le-grand) ; 62(8): 45-51, 2016 Jul 31.
Artigo em Inglês | MEDLINE | ID: mdl-27545214

RESUMO

The PDGF-BB plays a key role in several pathogenesis diseases and it is believed to be an important mediator for wound healing. The recombinant human PDGF-BB is safe and effective to stimulate the healing of chronic, full thickness and lower extremity diabetic neurotrophic ulcers. In the present study, we attempted to produce a PDGF-BB growth factor and also, evaluate its functionality in cell proliferation in yeast host Pichia pink. Pichia pink yeast was used as a host for evaluation of the rhPDGF-BB expression. The coding sequence of PDGF-BB protein was synthesized after optimization and packed into the pGEM. Recombinant proteins were produced and purified. The construct of pPinkα-HC-pdgf was confirmed by sequence, the PDGF-BB protein was expressed and purified with using a nickel affinity chromatography column and then characterized by SDS-PAGE electrophoresis. The biological activity of PDGF-BB was estimated with using human fibroblast cell line. The measurement of protein concentration was determined by Bradford and human PDGF-BB ELISA kit. Purified rhPDGF-BB showed similar biological activity (as the standard PDGF-BB) and suggested that the recombinant protein has a successful protein expression (as well as considerable biological activity in P. pink host). The exact amount of recombinant PDGF-BB concentrations were measured by specific ELISA test which it was about 30 µg/ml. Our study suggested that efficiency of biological activity of PDGF-BB protein may be related to its conformational similarity with standard type and also, it practically may be important in wound healing and tissue regeneration.


Assuntos
Pichia/metabolismo , Proteínas Proto-Oncogênicas c-sis/genética , Proteínas Recombinantes/genética , Células 3T3 , Animais , Becaplermina , Western Blotting , Clonagem Molecular , Eletroforese em Gel de Poliacrilamida , Humanos , Camundongos , Plasmídeos/metabolismo , Reação em Cadeia da Polimerase , Proteínas Proto-Oncogênicas c-sis/isolamento & purificação , Proteínas Proto-Oncogênicas c-sis/metabolismo , Proteínas Recombinantes/isolamento & purificação , Padrões de Referência , Reprodutibilidade dos Testes , Transformação Genética
5.
Res Pharm Sci ; 10(1): 59-67, 2015.
Artigo em Inglês | MEDLINE | ID: mdl-26430458

RESUMO

Anaphase promoting complex (APC) controls cell cycle and chromosome segregation. The APC activation occurs after binding of co-activators, cdh1 and cdc20. Cdh1 plays a role in cancer pathogenesis and is known as a potential drug target. The main aim of this study was prediction of 3D structure of cdh1 and designing the inhibitory compounds based on the structural model. First, 3D structure of cdh1 was predicted by means of homology modelling and molecular dynamics tools, MODELLER and Gromacs package, respectively. Then, inhibitory compounds were designed using virtual screening and molecular docking by means AutoDock package. The overall structure of cdh1 is propeller like and each DW40 repeat contains four anti-parallel beta-sheets. Moreover, binding pocket of the inhibitory compounds was determined. The results might be helpful in finding a suitable cdh1 inhibitor for the treatment of cancer.

6.
Cell Mol Biol (Noisy-le-grand) ; 61(3): 84-91, 2015 Jul 31.
Artigo em Inglês | MEDLINE | ID: mdl-26255136

RESUMO

MicroRNAs are new classes of small non—coding regulatory RNAs which control degradation or suppress translation of its target mRNAs by sequence complementarity. Mature microRNAs are enriched in embryonic stem cells and play important roles in controlling stem cell self—renewal as well as control of differentiation. There is significant evidence that microRNAs are involved in the regulation of stem cell differentiation. The male mouse Embryonic Stem Cell line C57BL6/J with normal karyotype 46, XY was used for profiling microRNA expression in undifferentiated mouse embryonic stem cells (mESCs) and mESCs which were differentiated to germ line cells to determine and compare differences in microRNA expression before and after differentiation. Also, testis tissue samples of a 5—day—old mouse and a mature mouse was used as in vivo control. Profiling was performed by quantitative real—time PCR using locked nucleic acid microRNA—specific LNATM—enhanced primers. After data analysis and comparison of results profiled microRNAs expression, three microRNAs, mmu—miR—21, mmu—miR—21* and mmu—miR—16 showed 50.31, 43.76 and 46.77—fold change increase of expression, respectively, in differentiated mESCs in comparison with undifferentiated state with significant p—value (Average p—value p<0.001 for each members of microRNAs). Expression of Let—7 microRNA family increased in differentiated state when compared with undifferentiated mESCs (Average p—value<0.0001 for each members of family). The levels of expression all other profiled microRNAs were significantly higher in undifferentiated in comparison with differentiated mESCs and their expression was down regulated after differentiation. (Average p—value <0.003 for each members of microRNAs).


Assuntos
Células Germinativas/citologia , MicroRNAs/metabolismo , Células-Tronco Embrionárias Murinas/citologia , Animais , Diferenciação Celular , Células Cultivadas , Análise por Conglomerados , Regulação da Expressão Gênica , Células Germinativas/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Células-Tronco Embrionárias Murinas/metabolismo , Reação em Cadeia da Polimerase em Tempo Real , Testículo/metabolismo
7.
Cell Prolif ; 48(4): 475-85, 2015 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-26132591

RESUMO

OBJECTIVES: Over recent years, constraint-based modelling of metabolic networks has become increasingly popular; the models are suitable for system-level modelling of cell physiology. The goal of the present work was to reconstruct a constraint-based metabolic network model of bone marrow-derived mesenchymal stem cells (BMMSCs). MATERIALS AND METHODS: To reconstruct a BMMSC-specific metabolic model, transcriptomic data of BMMSCs, and additionally, the human generic metabolic network model (Recon1) were used. Then, using the mCADRE algorithm, a draft metabolic network was reconstructed. Literature and proteomic data were subsequently used to refine and improve the draft. From this, iMSC1255 was derived to be the metabolic network model of BMMSCs. RESULTS: iMSC1255 has 1255 genes, 1850 metabolites and 2288 reactions. After including additional constraints based on previously reported experimental results, our model successfully predicted BMMSC growth rate and metabolic phenotypes. CONCLUSIONS: Here, iMSC1255 is introduced to be the metabolic network model of bone marrow-derived mesenchymal stem cells. Based on current knowledge, this is the first report on genome-scale reconstruction and validation of a stem cell metabolic network model.


Assuntos
Células-Tronco Mesenquimais/metabolismo , Redes e Vias Metabólicas , Simulação por Computador , Humanos , Modelos Biológicos , Termodinâmica
8.
Artigo em Inglês | MEDLINE | ID: mdl-26042682

RESUMO

Mesoporous hydroxyapatite with different pore diameters and pore volumes were synthesized by the self-assembly method using Cetyltrimethylammonium bromide (CTAB) as the cationic surfactant and 1-dodecanethiol as the pore expander at different micellization pHs, solvent types and surfactant concentrations. Results of field emission scanning electron microscopy (FESEM) showed a decrease in length/diameter ratio of rod-like particles by an increase in micellization pH and also a sphere to rod transition in morphology by an increase in CTAB concentration. Brunauer-Emmett-Teller (BET) surface area and Low angle X-ray diffraction analysis revealed that the optimized mesoporous hydroxyapatite with controlled pore structure can be obtained under basic micellization pH (about 12, pH of complete ionization of 1-dodecanethiol) by using water as the solvent and a high content of cationic surfactant. The results also show that micellization pH has a strong effect on pore structure and changing the pH can shift the mesostructure to a macroporous structure with morphological changes.


Assuntos
Materiais Biocompatíveis/química , Durapatita/química , Nanopartículas/química , Compostos de Sulfidrila/química , Cetrimônio , Compostos de Cetrimônio , Concentração de Íons de Hidrogênio , Micelas , Nanopartículas/ultraestrutura , Tamanho da Partícula , Porosidade
9.
Sci Rep ; 5: 9322, 2015 Mar 30.
Artigo em Inglês | MEDLINE | ID: mdl-25818803

RESUMO

Islet transplantation has been hampered by loss of function due to poor revascularization. We hypothesize that co-transplantation of islets with human embryonic stem cell-derived mesenchymal stromal cells that conditionally overexpress VEGF (hESC-MSC:VEGF) may augment islet revascularization and reduce the minimal islet mass required to reverse diabetes in mice. HESC-MSCs were transduced by recombinant lentiviruses that allowed conditional (Dox-regulated) overexpression of VEGF. HESC-MSC: VEGF were characterized by tube formation assay. After co-transplantation of hESC-MSC:VEGF with murine islets in collagen-fibrin hydrogel in the omental pouch of diabetic nude mice, we measured blood glucose, body weight, glucose tolerance and serum C-peptide. As control, islets were transplanted alone or with non-transduced hESC-MSCs. Next, we compared functional parameters of 400 islets alone versus 200 islets co-transplanted with hESC-MSC:VEGF. As control, 200 islets were transplanted alone. Metabolic function of islets transplanted with hESC-MSC:VEGF significantly improved, accompanied by superior graft revascularization, compared with control groups. Transplantation of 200 islets with hESC-MSC:VEGF showed superior function over 400 islets alone. We conclude that co-transplantation of islets with VEGF-expressing hESC-MSCs allowed for at least a 50% reduction in minimal islet mass required to reverse diabetes in mice. This approach may contribute to alleviate the need for multiple donor organs per patient.


Assuntos
Diabetes Mellitus/terapia , Células-Tronco Embrionárias Humanas/transplante , Transplante das Ilhotas Pancreáticas , Transplante de Células-Tronco Mesenquimais , Fator A de Crescimento do Endotélio Vascular/genética , Animais , Diabetes Mellitus/metabolismo , Diabetes Mellitus/patologia , Expressão Gênica , Humanos , Ilhotas Pancreáticas/patologia , Células-Tronco Mesenquimais/citologia , Células-Tronco Mesenquimais/metabolismo , Camundongos , Fator A de Crescimento do Endotélio Vascular/biossíntese
10.
J Biomed Mater Res A ; 102(2): 506-13, 2014 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-23533050

RESUMO

Since spinal cord injury is a complicated problem, neural tissue repair, and regeneration strategies have received a great deal of attention. In this study, a three-dimensional (3D) nanofibrous core-sheath scaffold with nanorough sheath and aligned core were fabricated by a combined electrospinning method with water vortex and two-nozzle system. In vitro and in vivo biological tests were carried out on the poly(lactic-co-glycolic acid) (PLGA) scaffolds. The cell morphology and proliferation evaluation of nerve cells on 3D PLGA scaffolds were studied. Cells were properly orientated along the aligned fiber direction of the scaffold. In animal studies, adult rats received a complete lateral hemisection at the T9-T10 level. Scaffolds were engrafted to bridge 3 mm defects of 10 adult rat spinal cords; 10 rats were used as controls. For 8 weeks, motor and sensory recovery by open field locomotor scale, narrow beam and tail flick tests were assessed. Locomotor and sensory scores of grafted animals were significantly better than the control group. Histological findings demonstrated that the scaffold supports the axonal regeneration of injured spinal cords and regenerating axons were seen to enter the graft and extend along its length.


Assuntos
Axônios/metabolismo , Ácido Láctico/química , Nanofibras/química , Ácido Poliglicólico/química , Regeneração , Traumatismos da Medula Espinal/terapia , Alicerces Teciduais/química , Animais , Masculino , Copolímero de Ácido Poliláctico e Ácido Poliglicólico , Ratos , Ratos Wistar
11.
Res Pharm Sci ; 8(1): 9-15, 2013 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-24459471

RESUMO

Reteplase is a potent thrombolytic agent which is widely used in the management of acute myocardial infarction and stroke. It belongs to the third generation of the thrombolytic drugs and has been derived from native human tissue plasminogen activator by removing three domains of it and keeping the Kringle 2 and Serine protease domains. However, the high cost of this drug, has limited the application of this drug especially in the developing and third world countries. The most laborious steps in the bacterial production of this drug is its purification and refolding steps which keep the process yield low and the cost high. Therefore, in the present study we evaluated the expression of reteplase by a non-lytic insect cell expression system. Following cloning and transfection procedures, recombinant Sf9 insect cell clones expressing the reteplase protein were selected. Primarily, the expression was verified by dot-blot analysis and subsequently it was confirmed by Western Blotting showing a band of about 45 kD on nitrocellulose membrane. The biological activity of the expressed protein was also evaluated and showed to be about 29 IU/ml. This confirmed the possibility of expression and the correct folding of the expressed protein. Hence, optimization of the expression followed by purification of the protein could be the next steps of the study.

12.
Res Pharm Sci ; 7(3): 133-40, 2012 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-23181091

RESUMO

One of the emerging therapeutic strategies for targeted treatment of most cancers is the use of immunotoxins which are fusion proteins consisted of a targeting and a toxic moieties. We previously showed that the recombinant A254-GMCSF fusion protein selectively kills acute myeloblastic leukemia cells which harbor a large number of granulocyte-macrophage colony stimulating factor (GMCSF) receptors. Since further in vitro and preclinical studies require large amounts of this fusion protein free from any troublesome material like lipopolysacharide, we selected the insect cell expression system. Thus, the coding sequences of the A254-GMCSF and its truncated form, A247-GMCSF, were cloned and expressed by Sf9 cells. Subsequently, specific cytotoxicity of the purified proteins was evaluated on GMCSF receptor positive cell lines. SDS-PAGE and Western blot analysis of the expressed A254GMCSF and A247GMCSF fragments revealed bands of about 60 kD which were larger than the theoretically predicted size of about 47 kD. Deglycosylation analysis showed that these proteins are N-glycosylated by the insect cells. However, any other post-translation modification of the proteins by insect cells could be the reason for higher molecular weight of the fragments. Cytotoxicity assays showed specific killing activity of these proteins on HL60 and U937 cell lines with IC(50)s ranging 2-2.5 µg/ml. These IC(50) values are much higher than those obtained from bacterially expressed A254-GMCSF (80 ng/ml) which could be due to any modification performed by insect cells on the fusion proteins.

13.
J Mater Sci Mater Med ; 23(10): 2483-8, 2012 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-22736051

RESUMO

Open-pore titanium scaffolds were fabricated by sintering of compressed mixtures of TiH(1.924) and urea. Spherical and irregular shaped space holders were used to investigate the effect of pore shape on cellular behavior. After removal of the space holder, the shape of the spacers was replicated to the pores. Average diameter of the pores was in the range of 300-600 µm. SEM images showed that titanium hydride resulted in higher surface roughness and larger micro porosities than pure titanium. In vitro evaluations were carried out by using MTT assay, measuring alkaline phosphatase activity and alizarin red staining in flow perfusion bioreactor for cell culture. Observations revealed excellent attachment and proliferation of G-292 cells to the highly porous scaffolds fabricated with titanium hydride and urea of this research.


Assuntos
Materiais Biocompatíveis , Alicerces Teciduais , Titânio , Fosfatase Alcalina/metabolismo , Linhagem Celular , Linhagem Celular Tumoral , Proliferação de Células , Humanos , Microscopia Eletrônica de Varredura , Difração de Raios X
14.
Chang Gung Med J ; 35(1): 28-37, 2012.
Artigo em Inglês | MEDLINE | ID: mdl-22483425

RESUMO

BACKGROUND: Recently, tissue engineering has been introduced as a regenerative treatment for bone defects. There is some evidence showing bone regeneration from mesenchymal stem cells (MSC) loaded on hydroxyapatite ß-tricalcium phosphate (HA/TCP) as a scaffold in large defects. This study aimed to compare the quality and quantity of regenerated bone using Bio-Oss, HA/TCP and MSC loaded HA/TCP scaffolds. METHODS: Mesenchymal stem cells were aspirated from iliac crest bone marrow after extracting the first, second and third premolars and the first molar in five mature hybrid dogs. The cells were cultured and their osteogenic differentiation potential was evaluated after the third cell passage using Alizarin red staining in experimental conditions. The HA/TCP scaffold (3 × 3 × 3 mm) was loaded with undifferentiated mesenchymal stem cells. Bilateral bone defects were then prepared in the jaws using trephine burs. The defects were randomly filled with HA/TCP, Bio-Oss, or HA/TCP + MSCs. One defect served as a control and was left as an empty cavity. All defects except the control defect were covered with an absorbable membrane. Histological and histomorphometric evaluations were conducted after 6 weeks and data were subjected to analysis of variance (ANOVA) (p < 0.05). RESULTS: The empty cavity demonstrated more bone formation (60.80%) than the HA/TCP (44.93%) and Bio-Oss (40.60%) (p < 0.05) groups. However, the difference from the HA/TCP + MSCs group was not significant (46.38%) (p > 0.05). CONCLUSION: An MSC-loaded HA/TCP scaffold is a more effective alternative than Bio-OSS or HA/TCP in inducing bone regeneration.


Assuntos
Regeneração Óssea/efeitos dos fármacos , Fosfatos de Cálcio/farmacologia , Durapatita/farmacologia , Células-Tronco Mesenquimais , Minerais/farmacologia , Animais , Técnicas de Cultura de Células , Diferenciação Celular , Células Cultivadas , Cães , Masculino , Mandíbula/cirurgia , Células-Tronco Mesenquimais/citologia , Alicerces Teciduais
15.
J Biomed Mater Res A ; 100(4): 1051-60, 2012 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-22323426

RESUMO

Porous nanocomposites based on poly(ε-caprolactone fumarate) (PCLF) resin matrix; N-vinyl pyrrolidone (NVP) as a reactive diluents and nanohydroxyapatite (nHA) filler were developed for bone tissue engineering applications. Nanocomposite scaffolds with three different contents of nHA [5, 10, and 20 (w/w %)] were prepared by thermal crosslinking of PCLF followed by particulate leaching and characterized in terms of mechanical properties (cyclic loading) and in vitro cell-material interaction by MTT assay and alkaline phosphatase activity measurements. Five osteoblastic cell lines were used to investigate the ability of the nanocomposites to support cell attachment, spreading, and proliferation after 3, 7, and 14 days. By adding the nHA filler phase, elastic modulus of the nanocomposites increased significantly. Scaffolds showed comparable biocompatibility to neat nHA particles, commercial bone graft (Bio-Oss) and tissue culture polystyrene as control groups. According to the results it can be concluded that these scaffolds are potential candidates for bone substitution because of their mechanical strength and bioactivity.


Assuntos
Osso e Ossos , Durapatita/química , Fumaratos/química , Nanopartículas , Poliésteres/química , Engenharia Tecidual , Fosfatase Alcalina/metabolismo , Materiais Biocompatíveis , Osso e Ossos/enzimologia , Linhagem Celular Tumoral , Humanos , Microscopia Eletrônica de Varredura
16.
J Biomed Mater Res A ; 99(2): 240-8, 2011 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-21976449

RESUMO

The study investigates the photo-polymerization shrinkage behavior, dynamic mechanical properties, and biocompatibility of cyanoacrylate bioadhesives containing POSS nanostructures and TMPTMA as crosslinking agents. Adhesives containing 2-octyl cyanoacrylate (2-OCA) and different percentages of POSS nanostructures and TMPTMA as crosslinking agents were prepared. The 1-phenyl-1, 2-propanedione (PPD) was incorporated as photo-initiator into the adhesive in 1.5, 3, and 4 wt %. The shrinkage strain of the specimens was measured using bonded-disk technique. Shrinkage strain, shrinkage strain rate, maximum and time at maximum shrinkage strain rate were measured and compared. Mechanical properties of the adhesives were also studied using dynamic mechanical thermal analysis (DMTA). Biocompatibility of the adhesives was examined by MTT method. The results showed that shrinkage strain increased with increasing the initiator concentration up to 3 wt % in POSS-containing and 1.5 wt % in TMPTMA-containing specimens and plateaued out at higher concentrations. By increasing the crosslinking agent, shrinkage strain, and shrinkage strain rate increased and the time at maximum shrinkage strain rate decreased. The study indicates that the incorporation of crosslinking agents into the cyanoacrylate adhesives resulted in improved mechanical properties. Preliminary MTT studies also revealed better biocompatibility profile for the adhesives containing crosslinking agents comparing to the neat specimens.


Assuntos
Adesivos/química , Materiais Biocompatíveis/química , Reagentes de Ligações Cruzadas/química , Cianoacrilatos/química , Metacrilatos/química , Nanoestruturas/química , Compostos de Organossilício/química , Linhagem Celular , Proliferação de Células , Humanos , Luz , Teste de Materiais , Osteoblastos/citologia , Osteoblastos/fisiologia , Polímeros/química , Estresse Mecânico , Propriedades de Superfície
17.
Pak J Biol Sci ; 14(7): 441-8, 2011 Apr 01.
Artigo em Inglês | MEDLINE | ID: mdl-21902056

RESUMO

To achieve higher titer of rabies virus higher density of host cells will need. In this study, capability of FibraCel disks packed in 500 mL spinner basket versus Cytodex-1 in 500 mL spinner flask was investigated for propagation of Vero cells and PV rabies virus proliferation. Minimal Essential Medium (MEM) + 10% Foetal Calf Serum (FCS) and Virus Production- Serum Free Medium (VP-SFM) +4 mM L-glutamine were used in growth phase and MEM+ 0.2% Bovine Serum Albumin (BSA) and VP-SFM were used in virus production phase. Adapted Vero cells grown in VP-SFM were used in all SFM experiments while batch and stepwise perfusion modes were applied and compared in growth stage. The highest Vero cell density were achieved in the trials with 10 g FibraCel disk in stepwise perfusion mode equal to 6.12 x 10(6) and 5.87 x 10(6) cells mL(-1) in MEM and VP-SFM, respectively while with 2.73 g Cytodex-1 lower density equal to 4.2 x 10(6) and 4.0 x 10(6) cells mL(-1) were achieved. The highest titer of rabies virus and overall virus production rate were resulted in VP-SFM and on 10 g disks equal to 2.9 x 10(7) Fluorescent Focus Unit (FFU) mL(-1) and 0.14 FFU/Cell/h, respectively versus 1.7 x 10(7) FFU mL(-1) and 0.08 FFU/cell/h on cytodex-1 in similar conditions. The second harvest of virus was also satisfactory in experiment with 10 g disks (1.7 x 10(7) FFU mL(-1)) in compare to Cytodex-1 (0.51 x 10(7) FFU mL(-1)). An equal surface area at 6600 and 12000 cm(-2) were provided in all comparable trials with seeding density of 12.5 x 10(3) cells cm(-2). Adapted Vero cells grown in VP-SFM were used in all SFM experiments while batch and stepwise perfusion modes were applied and compared in growth stage.


Assuntos
Técnicas de Cultura de Células/métodos , Dextranos , Vírus da Raiva/crescimento & desenvolvimento , Células Vero/citologia , Cultura de Vírus/métodos , Animais , Proliferação de Células , Chlorocebus aethiops , Meios de Cultura Livres de Soro , Compostos Orgânicos , Replicação Viral
18.
J Mater Sci Mater Med ; 22(6): 1555-67, 2011 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-21526410

RESUMO

Nerve tissue engineering is one of the most promising methods in nerve tissue regeneration. The development of blended collagen and glycosaminoglycan scaffolds can potentially be used in many soft tissue engineering applications. In this study an attempt was made to develop two types of random and aligned electrospun, nanofibrous scaffold using collagen and a common type of glycosaminoglycan. Ion chromatography test, MTT and attachment assays were conducted respectively to trace the release of glycosaminoglycan, and to investigate the biocompatibility of the scaffold. Cell cultural tests showed that the scaffold acted as a positive factor to support connective tissue cell outgrowth. The positive effect of fiber orientation on cell outgrowth organization was traced through SEM images. Porosity percentage calculation and tensile strength measurement of the webs specified analogous properties to the native neural matrix tissue. These results suggested that nanostructured porous collagen-glycosaminoglycan scaffold is a potential cell carrier in nerve tissue engineering.


Assuntos
Materiais Revestidos Biocompatíveis/síntese química , Colágeno/química , Microtecnologia/métodos , Tecido Nervoso , Engenharia Tecidual/métodos , Alicerces Teciduais , Fenômenos Biomecânicos , Células Cultivadas , Materiais Revestidos Biocompatíveis/química , Colágeno/metabolismo , Galvanoplastia/métodos , Humanos , Teste de Materiais , Modelos Biológicos , Nanoestruturas/química , Regeneração Nervosa/fisiologia , Tecido Nervoso/citologia , Tecido Nervoso/fisiologia , Alicerces Teciduais/química
19.
J Biomed Mater Res A ; 95(4): 1074-83, 2010 Dec 15.
Artigo em Inglês | MEDLINE | ID: mdl-20878932

RESUMO

Biocompatibility of ß-TCP/HDPE-UHMWPE nanocomposite as a new bone substitute material was evaluated by using highly purified human osteoblast cells. Human osteoblast cells were isolated from bone tissue and characterized by immunofluorescence Staining before and after purification using magnetic bead system. Moreover, proliferation, alkaline phosphatase production, cell attachment, calcium deposition, gene expression, and morphology of osteoblast cells on ß-TCP/HDPE-UHMWPE nanocomposites were evaluated. The results have shown that the human osteoblast cells were successfully purified and were suitable for subsequent cell culturing process. The high proliferation rate of osteoblast cells on ß-TCP/HDPE-UHMWPE nanocomposite confirmed the great biocompatibility of the scaffold. Expression of bone-specific genes was taken place after the cells were incubated in composite extract solutions. Furthermore, osteoblast cells were able to mineralize the matrix next to composite samples. Scanning electron microscopy demonstrated that cells had normal morphology on the scaffold. Thus, these results indicated that the nanosized ß-TCP/HDPE-UHMWPE blend composites could be potential scaffold, which is used in bone tissue engineering.


Assuntos
Materiais Biocompatíveis/farmacologia , Fosfatos de Cálcio/farmacologia , Teste de Materiais/métodos , Nanocompostos/química , Osteoblastos/citologia , Polietileno/farmacologia , Polietilenos/farmacologia , Fosfatase Alcalina/metabolismo , Adesão Celular/efeitos dos fármacos , Separação Celular , Forma Celular/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Imunofluorescência , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Nanocompostos/ultraestrutura , Osteoblastos/efeitos dos fármacos , Osteoblastos/enzimologia , Osteoblastos/ultraestrutura , Tamanho da Partícula , Pós , Coloração e Rotulagem , Difração de Raios X
20.
Acta Trop ; 109(1): 61-3, 2009 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-18983807

RESUMO

CD26 and CD30 are surface molecules expressed on activated Th1 and Th2 cells, respectively. It is, therefore assumed that plasma levels of CD26 and CD30 (sCD26 and sCD30) correlate with Th1 and Th2 response, respectively. In this study, plasma levels of sCD26 and sCD30 in patients with non-healing form of cutaneous leishmaniasis (CL) were measured and compared with the levels of sCD26 and sCD30 in patients with healing form of CL and healthy control volunteers. The results indicate that the plasma levels of sCD26 and sCD30 are significantly (p<0.05) higher in patients with non-healing form of CL than patients with healing form of CL or healthy control. No significant difference is seen in the levels of sCD26 and sCD30 in patients with healing form of CL in comparison with healthy control group. It is concluded that sCD30 might be used as an indicator of a Th2 response in patients with non-healing form of CL.


Assuntos
Dipeptidil Peptidase 4/sangue , Antígeno Ki-1/sangue , Leishmaniose Cutânea/sangue , Adolescente , Adulto , Feminino , Humanos , Leishmaniose Cutânea/imunologia , Leishmaniose Cutânea/patologia , Masculino , Pessoa de Meia-Idade , Células Th1/imunologia , Células Th2/imunologia , Adulto Jovem
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