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1.
Folia Biol (Praha) ; 57(2): 47-56, 2011.
Artigo em Inglês | MEDLINE | ID: mdl-21631961

RESUMO

Soft tissue regeneration with cell and tissue engineering-based approaches has numerous potential applications in plastic and reconstructive surgery. Adipose-derived stem cells (ASC) have been proved as a feasible source for adipose tissue engineering as they possess high proliferative and differentiation capacity. The purpose of our study was to evaluate adipogenic differentiation of human ASC in four different 3D scaffolds of natural origin, namely human platelet-poor plasma, alginate, fibrin gel and collagen sponge, to define their suitability for adipose tissue engineering and potential clinical applications. ASC were isolated from lipoaspirates of three adult female patients, seeded in the scaffolds, and adipogenic differentiation was induced. After two weeks of cultivation, the constructs were assessed for their mechanical and handling properties, cell viability and adipogenic differentiation. Additionally, the expression of vascular endothelial growth factor (VEGF) was analysed in different culture systems. The results indicate that the levels of specific adipogenic markers and VEGF expression were increased in 3D cultures, as compared to 2D culture. Among 3D scaffolds, fibrin gel showed optimal combination of mechanical characteristics and support of adipogenic differentiation; it was easy to handle, allowed high cell viability, and at the same time supported adipogenic differentiation and VEGF expression.


Assuntos
Tecido Adiposo/citologia , Células-Tronco/citologia , Adipócitos/citologia , Tecido Adiposo/crescimento & desenvolvimento , Diferenciação Celular , Sobrevivência Celular , Feminino , Humanos , Pessoa de Meia-Idade , Células-Tronco/metabolismo , Engenharia Tecidual/métodos , Alicerces Teciduais , Fator A de Crescimento do Endotélio Vascular/metabolismo
2.
J Bone Joint Surg Br ; 93(3): 421-6, 2011 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-21357969

RESUMO

The aim of this study was to evaluate the cultivation potential of cartilage taken from the debrided edge of a chronic lesion of the articular surface. A total of 14 patients underwent arthroscopy of the knee for a chronic lesion on the femoral condyles or trochlea. In addition to the routine cartilage biopsy, a second biopsy of cartilage was taken from the edge of the lesion. The cells isolated from both sources underwent parallel cultivation as monolayer and three-dimensional (3D) alginate culture. The cell yield, viability, capacity for proliferation, morphology and the expressions of typical cartilage genes (collagen I, COL1; collagen II, COL2; aggrecan, AGR; and versican, VER) were assessed. The cartilage differentiation indices (COL2/COL1, AGR/VER) were calculated. The control biopsies revealed a higher mean cell yield (1346 cells/mg vs 341 cells/mg), but similar cell proliferation, viability and morphology compared with the cells from the edge of the lesion. The cartilage differentiation indices were superior in control cells: COL2/COL1 (threefold in biopsies (non-significant)); sixfold in monolayer cultures (p = 0.012), and 7.5-fold in hydrogels (non-significant), AGR/VER (sevenfold in biopsies (p = 0.04), threefold (p = 0.003) in primary cultures and 3.5-fold in hydrogels (non-significant)). Our results suggest that the cultivation of chondrocytes solely from the edges of the lesion cannot be recommended for use in autologous chondrocyte implantation.


Assuntos
Cartilagem Articular/patologia , Condrócitos/patologia , Coleta de Tecidos e Órgãos/métodos , Adulto , Artroscopia , Biópsia , Técnicas de Cultura de Células , Forma Celular , Condrócitos/transplante , Desbridamento , Humanos , Articulação do Joelho/patologia , Articulação do Joelho/cirurgia , Adulto Jovem
3.
Biotechnol Bioeng ; 100(4): 773-81, 2008 Jul 01.
Artigo em Inglês | MEDLINE | ID: mdl-18496876

RESUMO

The ability to enhance bone regeneration by implanting autologous osteoblasts in combination with an appropriate scaffold would be of great clinical interest. The aim of our study was to compare the growth and differentiation of alveolar bone cells in tissue-engineered constructs and in monolayer cultures, as the basis for developing procedures for routine preparation of bone-like tissue constructs. Alveolar bone tissue was obtained from four human donors and explant cultures of the cells were established. Expanded cells were seeded on macroporous hydroxyapatite granules, and cultured in medium supplemented with osteogenic differentiation factors for up to 3 weeks. Control monolayer cultures were established in parallel, and cultured in media with or without osteogenic supplements. Cell proliferation, alkaline phosphatase (AP) activity and gene expression of AP, osteopontin and osteocalcin were determined under different culture conditions at weekly intervals. Cells in tissue constructs exhibited growth patterns similar to those in control monolayer cultures: enhanced proliferation was noted during the first 2 weeks of cultivation, followed by a decrease in cell numbers. AP activity at 3 weeks was higher in all cultures in osteogenic medium than in control medium. Gene expression levels were stable in monolayer cultures in both types of media whereas, in tissue constructs, they exhibited patterns of osteogenic differentiation. Light and scanning electron microscopy examination of the cell-seeded constructs showed uniform cell distribution, as well as cell attachment and growth into the interior region of the hydroxyapatite granules. Our results show that bone-like constructs with viable cells exhibiting differentiated phenotype can be prepared by cultivation of alveolar-bone cells on the tested hydroxyapatite granules.


Assuntos
Processo Alveolar/citologia , Substitutos Ósseos , Técnicas de Cultura de Células/métodos , Engenharia Tecidual/métodos , Fosfatase Alcalina/análise , Fosfatase Alcalina/genética , Processo Alveolar/crescimento & desenvolvimento , Diferenciação Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Meios de Cultura/farmacologia , Durapatita , Expressão Gênica , Humanos , Osteocalcina/análise , Osteogênese/efeitos dos fármacos , Osteopontina/análise
4.
Folia Biol (Praha) ; 54(6): 177-9, 2008.
Artigo em Inglês | MEDLINE | ID: mdl-19393130

RESUMO

ACI is the most widely used cell-based surgical procedure for the repair of articular cartilage defects. The method is based on in vitro chondrocyte cultivation. Two different culture conditions, rotating-wall-vessel bioreactor and static culture, were assessed by their effect on the re-differentiation potential of human articular chondrocytes seeded into a hydrogel scaffold. Gene expression analysis of the tissue-engineered construct revealed no significant difference between the tested systems.


Assuntos
Cartilagem Articular/citologia , Diferenciação Celular , Condrócitos/citologia , Engenharia Tecidual/métodos , Agrecanas/metabolismo , Reatores Biológicos , Cartilagem Articular/metabolismo , Técnicas de Cultura de Células , Diferenciação Celular/genética , Células Cultivadas , Condrócitos/metabolismo , Condrócitos/transplante , Colágeno Tipo I/metabolismo , Colágeno Tipo II/metabolismo , Expressão Gênica , Humanos , Hidrogel de Polietilenoglicol-Dimetacrilato , Alicerces Teciduais , Versicanas/metabolismo
5.
Cell Biol Int ; 31(6): 620-5, 2007 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-17293128

RESUMO

Auricular cartilage is an attractive potential source of cells for many tissue engineering applications. However, there are several requirements that have to be fulfilled in order to develop a suitable tissue engineered implant. Animal experiments serve as important tools for validating novel concepts of cartilage regeneration; therefore rabbit auricular chondrocytes were studied. Various parameters including isolation procedures, passage number, rate of proliferation and gene expression profile for major extracellular matrix components were evaluated in order to assess the potential use of elastic chondrocytes for tissue engineering. Chondrocytes were isolated from rabbit ear cartilage and grown in monolayer cultures over four passages. Yields of harvested cells and proliferation were analysed from the digestion step to the fourth passage, and changes in phenotype were monitored. The proliferation capacity of cell cultures decreased during cultivation and was accompanied by enlargement of cells, this phenomenon being especially evident in the third and fourth passages. The expression of cartilage specific genes for collagen type II, aggrecan and cartilage non-specific collagen type I was determined. The mRNA levels for all three genes were obviously lower in the primo culture than immediately after isolation. During subsequent cultivation the expression of collagen type II decreased further, while there were only slight changes in expression of aggrecan and collagen type I. This study provides a valuable basis for testing of different tissue engineering applications in rabbit model, where auricular chondrocytes are considered as cell source.


Assuntos
Separação Celular , Condrócitos/citologia , Cartilagem da Orelha/citologia , Agrecanas/genética , Agrecanas/metabolismo , Animais , Contagem de Células , Técnicas de Cultura de Células , Proliferação de Células , Sobrevivência Celular , Colágeno Tipo I/genética , Colágeno Tipo I/metabolismo , Colágeno Tipo II/genética , Colágeno Tipo II/metabolismo , Citometria de Fluxo , Regulação da Expressão Gênica , Coelhos , Fatores de Tempo
6.
Hum Exp Toxicol ; 24(11): 573-80, 2005 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-16323574

RESUMO

Although Hoechst 33342 (H342) is frequently used to label donor cells in cell transplantation research, it has been noted that it might secondarily label the host cells. Furthermore, its potential toxicity leading to cell death has been described. We studied the time course of H342 redistribution from the primary labeled rat bone marrow stromal cells (rBMSC) into the non-labeled rBMSC population over 7 days in culture; we evaluated the nuclear H342 fluorescence intensity as a possible criterion for distinguishing the primary from the secondary labeled cells, and determined the viability of rBMSC after an overnight incubation in 1 microg/mL of H342. H342 labeled >50% of the initially non-labeled cells within the first 6 hours and almost 90% within a week. Nuclear fluorescence intensity was a reliable criterion for distinguishing primary and secondary labeled cells within the first 24 hours, but less so at later time points. The percentage of either apoptotic or necrotic cells did not rise acutely after the overnight incubation in 1 microg/mL of H342. Although a 12-hour incubation of rBMSC in 1 microg/mL of H342 did not cause acute cell death, H342 rapidly and extensively redistributed into non-labeled cells, which makes H342 a relatively unsuitable marker for cell transplantation research.


Assuntos
Benzimidazóis/farmacologia , Células da Medula Óssea/efeitos dos fármacos , Corantes Fluorescentes/farmacologia , Animais , Área Sob a Curva , Benzimidazóis/metabolismo , Células da Medula Óssea/metabolismo , Transplante de Medula Óssea/métodos , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Corantes Fluorescentes/metabolismo , Curva ROC , Ratos , Ratos Wistar , Coloração e Rotulagem , Fatores de Tempo
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