| ObjectivesThe purpose of the persent study in vitro was to investigate the effect of three - dimensional scaffolds made of chitosan ploylysine as well as lecithin, and transforming growth factor (TGF - 1) on rabbit articular chondrocytes culture, to explore the action for tissue engineering and to provide preclinical base for the repair of articular cartilages injuries.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 - 1mm3in volume and incubated with collagenase for 3.5 -4 hours. After incubation, the undigested cartilage segments were removed, using centrifugation and washing with physiological saline. The isolated chondrocytes were gathered and seeded in culture flasks at 4. 8 106/ml per flask. The chondrocytes were cultured with 4ml DMEM at 37 C in a humidified atmosphere with 5% CO2. When 90% fusion of chondrocytes was observed, the cells were trypsined and transferred the second passage using. The second passage was collected and suspended at 4.5 107/ml for the further perliferation.Each porous chitosan scaffold was seeded by injecting 100 l rabbit chon-drocyte suspension, and then was cultured for 4 hours. Following this, the cells were transfered to the 96 -well tissue culture plate with 100 l DMEM each example. The culture medium was changed every day. TGF - 1 (10ng/ml) wasadded to the culture medium in experimental group (TGF - 1 group) , chondro-cytes not treated with growth factors in the same culture situations served as controls.Gross and invert microscope, histological light microscope, immunohisto-chemical stainning of collagen II, scanning electron microscope, MTT assay of cell content were used to evaluate the perliferation of chondrocytes.ResultsTrie cells suspension adhered to the surface of the synthetic chitosan scaffolds , and then diffused into the inner of the scaffolds rapidly, indicating the hy-drophilic property and adsorbability of sacffolds. Hie cells adhered to the scaffolds gradually. Collagen matrices generated like spider and the transparency of chitosan scaffolds decreased. With the prolong of cells culture, the contour of the cell became unclear, matrix lacuna was able to be observed.HE staining indicated that the cells were round or polygonal. Hie plasma was stained slightly, nuclei were blue staining, and the scaffolds were irregular net red staining. The cell count in scaffolds in TGF - 1 group was higher than that in controls.Immunohistochemical staining showed positive collagen II in both plasma and scaffolds. The cytoplasmic staining intensity in experimental group was stranger than that of controls.With scanning electron - microscope, uneven grooves were found on the chitosan scaffold. The irregular cells with protruded peudopods and secreted matrices were observed on the surface and internal structure of scaffolds at the forth week of culture. The matrix increased significantly, the cells were nearly enveloped in the matrix. The chondrocytes adhered to each other by the processes, and clung to the scaffold. The density of chondrocytes and secretion in TGF - 1 group was significant higher than that of controls.MTT assay denoted that the OD was significantly different in different time periods of both TGF - 1 group and controls ( p <0.01). Trie similar result was present between both groups ( p < 0.05 ).ConclusionThe chitosan scaffolds combined with lecithin and collagen were demonstrated as the ideal templates for chondrocytes seeding in the current study. Because of good hydrophile and adhesive properties, the synthetic chitosan scaffold played no inhibition role. TGF - β1was able to facilitate the adhesion, differentiation and proliferation of chondrocytes in articular cartilage culture of tissue engineering. |
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