RESUMO
AIM OF THE STUDY: Cartilage has a limited capacity for healing after trauma. Autologous chondrocyte implantation is widely used for the treatment of patients with focal damage to articular cartilage. Chondrocytes are isolated from biopsy specimen, cultured in monolayers on plastic then transplanted over the cartilage defect. However, chondrocyte amplification on plastic triggers their dedifferentiation. This phenomenon is characterized by loss of expression of type II collagen, the most abundant cartilage protein. The challenge for autologous chondrocyte implantation is to provide patients with well-differentiated cells. The aim of the present study was to test the capability of bone morphogenetic protein (BMP)-2 to promote redifferentiation of human chondrocytes after their expansion on plastic. MATERIALS AND METHODS: Chondrocytes extracted from nasal cartilage obtained after septoplasty were serially cultured in monolayers. After one, two or three passages, BMP-2 was added to the culture medium. The cellular phenotype was characterized at the gene level by using RT-PCR. The expression of genes coding for type II procollagen with the ratio of IIB/IIA forms, aggrecan, Sox9, osteocalcin and type I procollagen was monitored. RESULTS: Our results show that BMP-2 can stimulate chondrogenic expression of the chondrocytes amplified on plastic, without inducing osteogenic expression. However, this stimulatory effect decreases with the number of passages. CONCLUSION: The efficiency of autologous chondrocyte implantation could be improved by using chondrocytes treated with BMP-2 during their in vitro preparation.
Assuntos
Proteína Morfogenética Óssea 2/farmacologia , Condrócitos/efeitos dos fármacos , Proteínas da Matriz Extracelular/biossíntese , Adolescente , Adulto , Agrecanas/biossíntese , Agrecanas/genética , Desdiferenciação Celular/efeitos dos fármacos , Terapia Baseada em Transplante de Células e Tecidos/métodos , Células Cultivadas/citologia , Células Cultivadas/efeitos dos fármacos , Células Cultivadas/metabolismo , Condrócitos/citologia , Condrócitos/metabolismo , Colágeno Tipo II/biossíntese , Colágeno Tipo II/genética , Proteínas da Matriz Extracelular/genética , Feminino , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Masculino , Pessoa de Meia-Idade , Osteocalcina/biossíntese , Osteocalcina/genética , Pró-Colágeno/biossíntese , Pró-Colágeno/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Fatores de Transcrição SOX9/biossíntese , Fatores de Transcrição SOX9/genética , Adulto JovemRESUMO
Type I and II collagen (native-type) fibrils, positively stained with uranyl acetate, present typical periodic (D = 67 nm) cross-striation patterns. Although the two patterns are similar, the distributions of charged amino acids along the type I and II collagen molecules are different. After optical diffraction analysis or computer image processing of electron micrographs, different Fourier transforms were obtained from type I and II collagen fibrils, either as native fibrils or after in vitro reconstitution from purified molecules. With tissues such as tendon and cartilage, better results were obtained after mild trypsin treatment, which allowed better isolation and staining of the collagen fibrils. The main difference observed in the Fourier transforms was the presence in type II collagen fibrils of a strong tenth-order peak (corresponding to the tenth harmonic of the fundamental frequency). In order to discriminate between the two collagens, we measured the ratio (R) of the areas under the ninth- and tenth-order peaks. In trypsin treated tissues, the distributions of these ratios were clearly separated: below 1.0 for type II collagen fibrils and above 1.5 for type I collagen fibrils. This method appears to be suitable for rapid typing of type I and II collagen fibrils and might be useful for determining the exact composition of fibrils in tissues, such as intervertebral discs, that contain these both types of collagen.
