RESUMO
Rabbit bone marrow-derived mesenchymal stem cells (MSCs) were stably transfected with the TGF-beta1 gene in monolayer culture using Lipofectamine 2000. After transfection, the expression of cartilage-specific extracellular matrix was upregulated, whereas matrix metalloproteinases 1 and 3 (MMP 1 and 3) protein expressions and enzymatic activities were downregulated. Autologous MSCs modified with the TGF-beta1 gene were seeded into chitosan scaffolds to construct gene-modified cartilage, which was then implanted into the full-thickness articular cartilage defects of rabbits' knees. Twelve weeks after implantation, the defects were filled with regenerated hyaline-like cartilage tissue as confirmed by the positive immunohistochemical staining of collagen type II and intense toluidine blue staining of proteoglycan. Our findings suggest that the repair of cartilage defects can be enhanced by TGF-beta1 gene-modified-tissue engineering of cartilage on the basis of a strategy using MSCs, chitosan, and liposomal transfection.
Assuntos
Cartilagem Articular/citologia , Cartilagem Articular/metabolismo , Células-Tronco Mesenquimais/citologia , Células-Tronco Mesenquimais/metabolismo , Transfecção , Fator de Crescimento Transformador beta1/genética , Agrecanas/genética , Animais , Sequência de Bases , Cartilagem Articular/cirurgia , Quitosana , Colágeno Tipo II/genética , Colágeno Tipo II/metabolismo , Primers do DNA/genética , Portadores de Fármacos , Feminino , Fibronectinas/genética , Fibronectinas/metabolismo , Lipídeos , Lipossomos , Masculino , Metaloproteinases da Matriz/metabolismo , Transplante de Células-Tronco Mesenquimais , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Coelhos , Engenharia Tecidual , Fator de Crescimento Transformador beta1/metabolismoRESUMO
OBJECTIVE: To elongate human nerve axon in culture and search for suitable support matrices for peripheral nervous system transplantation. METHODS: Human embryo cortical neuronal cells, seeded on poly (D,L-lactide-co-glycolide) (PLGA) membrane scaffolds, were elongated with a self-made neuro-axon extending device. The growth and morphological changes of neuron axons were observed to measure axolemmal permeability after elongation. Neurofilament protein was stained by immunohistochemical technique. RESULTS: Human embryo neuron axon could be elongated and cultured on the PLGA membrane and retain their normal form and function. CONCLUSIONS: Three dimensional scaffolds with elongated neuron axon have the basic characteristics of artificial nerves, indicating a fundemental theory of nerve repair with elongated neuron axon.
Assuntos
Axônios/transplante , Ácido Láctico , Ácido Poliglicólico , Polímeros , Engenharia Tecidual/métodos , Axônios/efeitos dos fármacos , Técnicas de Cocultura , Humanos , Imuno-Histoquímica , Copolímero de Ácido Poliláctico e Ácido PoliglicólicoRESUMO
We investigated the influence of the microgravity rotating culture system on the chondrogenic differentiation of bone marrow mesenchymal stem cells (MSCs). During chondrogenic induction, MSCs combined with polyglycolic acid (PGA) were cultured by static culture or microgravity rotating culture and chondrocyte formation was confirmed by toluidine blue staining. Furthermore, the mRNA and protein expressions of a specific cartilage extracellular matrix protein (collagen type II and Aggrecan) were evaluated by real-time RT-PCR and western blot, respectively. Toluidine blue staining indicated the OD values of proteoglycans semi-determination were higher in the microgravity rotating culture group than the static culture group. Following chondrogenic induction, mRNA and proteins of collagen type II and Aggrecan were more significantly expressed in cells of the microgravity rotating culture group compared with the controls. Compared with routine three-dimensional static culture, the microgravity rotating culture system was more effective for the construction of tissue-engineered cartilage in vitro.