| The purpose of the current experiment in vitro was to investigate the effect of basic fibroblast growth factor ( bFGF) on rabbit articular chondrocytes cultured in three - dimensional chitosan scaffolds combined with collagen and lecithin , to explore a suitable carrier for tissue engineering and to provide preclini-cal base for the repair of articular cartilages.Materials and methodsTransparent cartilage slices were harvested, immediately after euthanasia, under sterile conditions, from the femoral condyles of 4 - week Japanese white rabbits. The cartilage was chopped into chips of 0. 5 - lmm in volume and incubated with collagenase - II for 3.5 hours. After incubation, the undigested cartilage segments were removed, using centrifugation and washing with physiological saline. The isolated chondrocytes were gathered and seeded in 50ml culture flasks, 3. 0 x 105 per flask. The chondrocytes were cultured at 37℃ in a humidified atmosphere with 5% C02. When 90% fusion of chondrocytes was observed, the cells were trysined and transferred the second passage. The second passage was collected after 12 days and suspended at 5. 0 x 107/ml for the next experiment.After the porous chitosan membrane was tailored as round texture, 7. 0mm in diameter, the chitosan scaffolds were embedded by 1% lecithin and 1mg/ml collagen, under sterile conditions. Each scaffold was seeded by injecting 100 ul rabbit chondrocyte suspension, and then was cultured for 2 hours. Following this, the cells were placed to the 96 - well tissue culture plate with 200ul DMEM each example. The culture medium was changed every day. bFGF wasadded to the culture medium in experimental group ( bFGF group) , chondro-cytes not treated with growth factors in the same culture situations served as controls.ResultsThe synthetic chitosan scaffolds expended significantly. The cells adhered to the scaffolds gradually. Collagen matrices generated like spider in the bFGF group and the transparency of chitosan scaffolds decreased.HE staining indicated that the cells were round or polygonal. The plasma was stained slightly, nuclei were blue staining, and the scaffolds were irregular net red staining. The cell count in scaffolds in bFGF group was higher than that in controls.With scanning electron - microscope, the irregular cells with protruded peudopods and secreted matrices were observed on the surface and internal structure of scaffolds at the third week of culture. Two weeks later, the matrix increased significantly, the cells were nearly enveloped in the matrix. The chon-drocytes adhered to each other by the processes, and clung to the scaffold. The density of chondrocytes and secretion in bFGF group was significant higher than that of controls.Immunohistochemical staining showed positive collagen II in both cells and scaffolds. The cytoplasmic staining intensity in experimental group was stranger than that of controls.MTT assay denoted that the OD was significantly different in different time periods of both bFGF group and controls ( p < 0. 01). The similar result was present between both groups (p < 0.01).ConclusionThe chitosan scaffolds combined with lecithin and collagen were demonstrated as the ideal templates for chondrocytes seeding in our study. bFGF was able to facilitate the adhesion, differentiation and proliferation of chondrocytes in ar-ticular cartilage culture of tissue engineering.
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