Expansão ex vivo de células uroteliais em scaffolds 3D

Malformations and possible damages to the urogenital system can be originated in the embryonic period. Moreover, fire guns, knives and accidents, where there is the disruption of the urethra, also cause these lesions. The objective was to analyze the contribution of tissue engineering in the construction of neo-urethra, developed by bioengineering. We performed an urothelial ex vivo expansion of cells in 3D scaffolds (platelet gel matrix and acellular porcine aorta) to assess the contribution of this technique in the construction of a neo-urethra. Mechanical dissociation was made of the inner wall of 10 North Folk rabbit’s bladder, weighing 2.5 to 3.0 kg. After dissociation the cell content was centrifuged and obtained a pellet of urothelial cells. The pellet was ressuspended in culture medium DMEM F12 and cells were maintained in culture for 15 days. Immunohistochemical analysis characterized the urothelial culture. The cells were then implanted in the scaffold - platelet gel. In a second experiment using aortic porcine acellular matrix were implanted urothelial cells alone and urothelial cells on platelet gel, on the inner wall of the scaffold - aorta, with space for setting bordered by a urethral probe. The complex probe - cells - aorta and probe - cells in platelet gel - aorta, were sealed with suture material and culture were maintained in a humidified 37ºC incubator with 5% CO2 in air for 12 days to subsequent histological analysis of urothelium cell adhesion to the scaffolds. By observation under an optical microscope, we could see the growth of cells in the scaffold platelet gel, from a monolayer in to a three-dimensional structure. In the acellular porcine aortic matrix containing the platelet gel, we could observe a few quantity of urothelial cells adhered. However with the acellular porcine aortic matrix in which was implanted only the urothelial cells, we have obtained adhesion to the wall

Avaliação de novas propostas em arcabouços tridimencionais (3D) para cultura de células-tronco mesenquinas e condogênese

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

Gel de plaquetas: arcabouço 3D para cultura celular

INTRODUÇÃO: O reparo tissular é o objetivo final da cirurgia. A cultura celular requer arcabouço mecânico que dê suporte ao crescimento celular e difusão dos nutrientes. O uso do plasma rico em plaquetas (PRP) como um arcabouço 3D possui diversas vantagens: é material biológico, de fácil absorção pós-transplante, rico em fatores de crescimento, em especial PDGF- ββ e TGF-β que estimula síntese de matriz extracelular na cartilagem. OBJETIVO: Desenvolver arcabouço 3D à base de PRP. MATERIAIS E MÉTODOS: Duas formas foram idealizadas: Sphere e Carpet. Condições estéreis foram utilizadas. O gel de plaquetas permaneceu em cultura celular, observado diariamente em microscópio invertido. RESULTADOS: Ambos arcabouços obtiveram sucesso, com aspectos positivos e negativos. DISCUSSÃO: A forma Sphere não aderiu ao plástico. Observou-se retração do gel e investigação ao microscópio dificultada devido às áreas opacas no campo visual. A forma Carpet não aderiu ao plástico e apresentou-se translúcida. O tempo de estudo foi de 20 dias. CONCLUSÕES: A produção de um arcabouço 3D PRP foi um sucesso, e trata-se de uma alternativa que necessita ser mais utilizado e investigado para que se consolide em uma rota eficiente e confiável na tecnologia de engenharia tissular, particularmente em cultura de tecido cartilaginoso.

In vitro evaluation of three different biomaterials as scaffolds for canine mesenchymal stem cells

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

Platelet lysate 3D scaffold supports mesenchymal stem cell chondrogenesis: An improved approach in cartilage tissue engineering

Articular lesions are still a major challenge in orthopedics because of cartilage's poor healing properties. A major improvement in therapeutics was the development of autologous chondrocytes implantation (ACI), a biotechnology-derived technique that delivers healthy autologous chondrocytes after in vitro expansion. To obtain cartilage-like tissue, 3D scaffolds are essential to maintain chondrocyte differentiated status. Currently, bioactive 3D scaffolds are promising as they can deliver growth factors, cytokines, and hormones to the cells, giving them a boost to attach, proliferate, induce protein synthesis, and differentiate. Using mesenchymal stem cells (MSCs) differentiated into chondrocytes, one can avoid cartilage harvesting. Thus, we investigated the potential use of a platelet-lysate-based 3D bioactive scaffold to support chondrogenic differentiation and maintenance of MSCs. The MSCs from adult rabbit bone marrow (n=5) were cultivated and characterized using three antibodies by flow cytometry. MSCs (1×105) were than encapsulated inside 60μl of a rabbit platelet-lysate clot scaffold and maintained in Dulbecco's Modified Eagle Medium Nutrient Mixture F-12 supplemented with chondrogenic inductors. After 21 days, the MSCs-seeded scaffolds were processed for histological analysis and stained with toluidine blue. This scaffold was able to maintain round-shaped cells, typical chondrocyte metachromatic extracellular matrix deposition, and isogenous group formation. Cells accumulated inside lacunae and cytoplasm lipid droplets were other observed typical chondrocyte features. In conclusion, the usage of a platelet-lysate bioactive scaffold, associated with a suitable chondrogenic culture medium, supports MSCs chondrogenesis. As such, it offers an alternative tool for cartilage engineering research and ACI. © 2013 Informa UK Ltd.

Análises físico-químicas e biológicas de diferentes scaffolds à base de polihidroxibutirato e polihidroxibutirato-co-valerato

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)