Objective The biological effects of fibroblast growth factor (FGF) may be different under different intensities and durations. To investigate the impact of sustained increasing FGF signal upon the development of epiphyseal plate. Methods Epiphyseal plates cultured in vitro were obtained from embryonic C57BL/6J mice, and were divided into control group (0.1% DMSO), basic FGF (bFGF) group (100 μg/L bFGF and 0.1% DMSO), and PD98059 group (100 μg/L bFGF and 50 μmol/L PD98059 with 0.1% DMSO). The total length (TL) and ossified tissue length (OSL) of the cultured bones weremeasured with Calcein staining 6 days after culture. The expressions of Indian hedgehog (Ihh), collagen type II (Col II), and Col X genes were detected by real-time fluorescent quantative PCR 7 days after culture. Results The embryonic bones cultured in vitro continued growth. At 6 days after culture, there was no significant difference in increased percentage of TL between bFGF group and control group (P gt; 0.05), the increased percentage of OSL in bFGF group was significantly less than that in control group (P lt; 0.05). There was no significant difference in the increased percentage of TL and OSL between PD98059 group and control group (P gt; 0.05), but they were significantly higher than those of bFGF group (P lt; 0.05). At 7 days after culture, the gene expressions of Ihh, Col II, and Col X in bFGF group significantly decreased when compared with those in control group (P lt; 0.05). There was no significant difference in the gene expressions of Col II and Col X between PD98059 group and control group (P gt; 0.05), but the gene expressions were significantly higher than those of bFGF group (P lt; 0.05); the expression of Ihh in PD98059 group was significantly higher than that in control group and bFGF group (P lt; 0.05). Conclusion Sustained increasing FGF signal may affect the Col II and Col X expressions by down-regulating Ihh, which may lead to the development retardation of epiphyseal plate cultured in vitro. The external signal regulated kinase pathway may play an important role in the process.
Objective To observe the efficiency and biological characteristics in regenerating in vitro tissue-engineered cartilage from epiphyseal chondrocyte-scaffold complex. MethodsThe first passage epiphyseal chondrocytes were collected and mixed with the biological gel-matrix, the chondrocyte-gel fluid wasdropped into the scaffold to form a complex. The complexes were in vitro cultivated. The changes of complexes in morphology and synthesis of collagens type ⅡandtypeⅠ and aggrecan were observed under the gross and the inverted and light microscopes. The sulfate GAG content in complexes was measured by the the modified dimethylmethylene blue method. Results During cultivation, thecomplexes could keep its original shape with the stable homogeneous three-dimensional distribution of chondrocytes，gradually became milk white and translucence with their rigidity increasing. In the 1st week, the chondrocytic lacunae formed in the complexes. After 2 weeks, the complex was gradually reorganized into the mature engineered cartilage with rich collagen typeⅡand aggrecan and typical cartilage histological structure, but with negative immunological staining of collagen typeⅠ. In the 4th week, the engineered cartilage resembled the nature epiphyseal plate in the characteristic of histological structure, and had over 34% of the sulfate GAG content of the natural epiphyseal plate. Conclusion Theepiphyseal chondrocyte-scaffold complex can be reorganized into typical cartilage with the epiphyseallike histological structure, and be fit for repairing the epiphyseal defect. The tissue engineered cartilage cultivated for 1-2 weeks may be a good choice for repairing epiphyseal defect.