Culture and identification of chondrocytes isolated from the vertebral endplate of patients with type I neurofibromatosis associated with atrophic changesin vitro / 中国组织工程研究

ABSTRACT

BACKGROUND:Previous studies have mainly focused on costal cartilage, articular cartilage, nasal septal cartilage, and auricular cartilage, but in vitro culture of human vertebral endplate cartilage is stil rarely reported. OBJECTIVE: To discuss the feasibility of culture of vertebral endplate chondrocytes from type I neurofibromatosis associated with scoliosis patientsin vitro and to study the biological characters of the chondrocytes. METHODS: Through two-step enzymatic digestion and tissue culture, the chondrocytes from the vertebral endplate of seven type I neurofibromatosis patients isolated and cultured in monolayer and passaged to observe the changes of cel morphology under inverted phase contrast microscope. Colagen type II expression was detected by immunocytochemistry to identify whether the cels had chondrocyte characters. The growth kinetics was detected by using MTT colorimetric assay to draw the growth curve of passage 2 chondrocytes. RESULTS AND CONCLUSION:A few chondrocytes crawled from the cartilage after 2 weeks culture and cels were passaged at 3 weeks. Along with passage going on, the phenotype of chondrocytes was changed from polygonal, round, triangle, and irregular shapes to fusiform. The colagen type II expression in passage 2 cels was positive by immunohistochemical staining. MTT test showed the growth curve of the passage 2 chondrocytes presented a transverse “S”. Cels were found logarithmic growth at days 4-7, reached platform stage at days 8-13, and decreased at day 14. It is an effective and simple procedure by two-step enzymatic digestion and tissue explant method to culture vertebral endplate chondrocytes with high purity and good viability from type I neurofibromatosis patients associated with scoliosisin vitro. Passage 2 chondrocytes from the vertebral endplate exhibit the best viability at days 4-7, which can be used as targets for research of pathogenesis of type I neurofibromatosis with atrophic scoliosis.