| Objective and Background DataTissue engineering techniques that involved the use of articular chondro-cytes , cultured in vitro and implanted after a certain period of time, has been an area of extensive studies. There were, however, critical hurdles that need to be overcome. The purpose of current study was to investigate the effect of basic fi-broblast growth factor (bFGF) and insulin -like growth factor 1 (IGF - 1) on human articular chondrocytes in vitro, and to explore the sequential interaction of bFGF and IGF - 1 on articular condrocytes culture.Materials and methodsChondrocytes isolationArticular chondrocytes was obtained from cartilage chips scraped from the tibial plateaus and femoral condyles from the patients who underwent the surgery of knee joint in the Department of Orthopedic Surgery, the First Hospital affiliated to China Medical University, under sterile conditions. The chips were washed with phosphate buffered saline (PBS) and chopped into chips of 1 mm in DMEM medium. Chips of cartilage were incubated at 37 C for 20 minutes with 0.25% trypsin. This was followed by overnight incubation with 0. 02% col-lagenase II. Then, the undigested cartilage segments were removed using a 200um filter. The isolated chondrocytes were suspended, repeating for 3 times. Cells were counted and cell viability was tested.Chondrocytes Culture ProtocolsThe chondrocytes were seeded, at an initial density of 105cell/ ml, in culture flasks, and cultured at 37 C in a humidified atmosphere with 5% CO2. At confluency (80 -90% fusion) , the cells were were trypsinized with trypsin and transferred the second passage. The second passage was cultured as previously described for the further experiment.3/4 of culture flakes were used to test the growth factors, even for single bFGF (10ng/ml) , single IGF - 1 (100ng/ml) , sequential bFGF and IGF - 1, and 1/4 of culture flakes were used as controls. All above chondrocytes were cultured for 1 week at the same culture conditions.EvaluationThe morphology of chondrocytes and extracellar matrix after a certain period of time was observed with the inverted microscope.The second passage cells were suspended with DMEM and 10% newborn calf serum and transfered to the 96 - well tissue culture plate, at the density of 3 105cell per pore. Cells were cultured for a period of time. Then, MTT assay was conducted. Cells were managed by 5mg/ml MTT and DMSO, the OD was recorded by enzyme linked inmmunosorbent instrument.Chondrocytes were transferred slides for immunohistochemical staining. The sections were incubated with monoclonal antibody against collagen type II. Next, goat against mouse IGg conjugated with biotin was used. This was followed by incubation with SABC for 20 minutes. Sections were counterstained with hematoxylin. In every staining, controls were performed by omitting the primary antibody. For each slide, MetaMorph imaging analysis software was used to determine the gray value of cells staining positive for collagen type II.ResultsbFGF (10ng/ml) was effective to promote the proliferation and dedifferen-tiation of human articular chondrocytes culted in vitro. IGF - 1 ( 100ng/ml) played an obvious role on perliferation of extracellar matrix and expression of chondrocyte phenotypes, but it failed to facilitate multiplication of chondrocytes.Sequential exertion of bFGF ( 10ng/ml) and IGF - 1 (100ng/ml) in theculture medium of human articular chondrocytes in vitro were proved to be effective, and they helped to harvest abundant perliferative chondrocytes with ideal phenotypes in a limited period of culture time.ConclusionsThe present experiment study demonstrates a method to promote perlifera-tion and regain ideal cartilage phenotype for human articular chondrocytes cultured in vitro. Sequential exertion of bFGF and IGF - 1 was effective, bFGF was added to stimulate cell multiplication and de - differentiation, and IGF - 1 was supplemented to increase generation of extracellar matrix and expression of phenotype. Thi...
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