Diferenciação condrogênica de células-tronco em matriz 3D de quitosana e gelatina: avaliações in vitro e in vivo

• Ano de defesa: 2010
• Autor(a) principal: Natalia Martins Breyner
• Tipo de documento: Tese
• Tipo de acesso: Acesso aberto
• Idioma: por
• Instituição de defesa: Universidade Federal de Minas Gerais; UFMG
• Palavras-chave em Português: quitosana; células-tronco; matriz 3D; Quitosana; Células tronco; Tecido adiposo; Células-tronco mesenquimais; Gelatina
• Link de acesso: http://hdl.handle.net/1843/UCSD-8H8QFH

Cartilage is composed of specialized cells called chondrocytes which produce a large amount of extracellular matrix composed mainly of collagen fibers. Unlike other connective tissues, cartilage does not contain blood vessels, and therefore, presents limited repair capability. If damaged, cartilage doesnt heal. Over the last years, surgeons and scientists have elaborated a series of cartilage repair procedures that help to postpone the need of joint replacement. Nowadays, bioengineering and tissue engineering techniques are being developed aiming the generation of cartilage tissue using a cellular scaffolding material to support cell growth and promoting new cartilage tissue formation. Following the same principle, this work aims to create two types of chitosan/gelatin composed 3D matrices in order to support cartilage tissue formation. Matrices differ only in the crosslinker used (glutaraldehyde or genipin). Chitosan and gelatin were chosen for their resemblance with the cartilages extracellular matrix, their capacity to promote cell adhesion, migration, communication and differentiation. Both biomaterials also present the property of forming a porous scaffold when united, which facilitates the diffusion of nutrients and the removal of metabolites. The promotion of guided tissue regeneration requires the presence of 3D support and biologically active factors, or cells. In the present study, rat adipose tissue derived stem cells were used. These cells have the ability to differentiate into several cell lines, when stimulated properly, thus present an attractive potential for the treatment desired. Cells were cultured in chondrogenic induction medium containing TGF-â and dexamethasone. Those molecules were employed as they activate intracellular signaling cascades that promote the differentiation of stem cells towards the chondrogenic lineage. Tests were performed in vitro, in which the cells were cultured in induction medium and evaluated for the ability of differentiation. It was noted that from the 3rd week of culture on, there was a reduction in cell proliferation and alkaline phosphatase activity, as well as increased synthesis of collagen and expression of type II collagen. In vivo, reabsorption rate of the scaffolds was assessed and both were completely absorbed by the 9th week of implantation, presenting total tissue remodeling. In addition, we evaluated the ability of these scaffolds to maintain the differentiated phenotype of the cells for prolonged periods after implantation, since the scaffolds were colonized by cells cultured in induction medium for 3 weeks in advance. After 6 weeks of implantation, type II collagen expression was still present, indicating maintenance of the chondrogenic phenotype. Taken together, the data obtained in this study revealed that the rat adipose tissue derived cells were able to differentiate into chondrocytes in vitro when cultured in monolayer and in 3D chitosan-gelatin based scaffolds. Data indicates as well that even after 6 weeks of implantation cells maintained the differentiated phenotype, demonstrating that the matrices studied adequately mimic the native ECM. Further studies are required to enhance 3D matrices capability of shape manipulation.