| Objective: To establish a rolling depression load culture model of chondrocytes within agarose gel and evaluate the feasibility of the cartilage tissue cultured by the rolling depression load.Method:1 Cell culture and identification: Articular cartilage was harvested from knee joints of 4 week old healthy New Zealand rabbit by aseptic operation. Free chondrocytes were isolated from the articular cartilage in the way of enzymatic digestion by stages and cultured in the HG-DMEM supplemented with 20% FBS at 37℃in a 5%CO2 incubator. The culture medium was changed every 3 day. After 3 passage, chondrocytes were tested by Toluidine Blue stain, Safranin-O/Fast green and immunohistochemical collagenⅡ.2 The construction of cell-scaffold composites were mixed with articular chondrocytes as seed cells and low-melt agarose as carrier. Distribution and adhere of chondrocytes in agarose scaffold was tested by SEM 48 hours later,16 cell-scaffold composites were divided into experimental group (A group, n=8) and control group (B group, n=8) respectively, group A was treated by dynamic rolling depression load(20minutes everyday).Group B was incubated in the same medium without rolling depression load. Four weeks later, gross appearance of the composites were investigated, and the composites were assessed by histological staining of HE respectively. Type-Ⅱcollagen of the cell-scaffold composites were detected by immunohistochemistry in group A and B. The amount of type-Ⅱcollagen was analyzed in the method of Image-pro plus.Result:1 Morphology and identification of chondrocytes: The term of adherence of initial chondrocytes was long. The cells began to adhere from 24 hour to 36 hour. Most chondrocytes were polygonal cells, and there were lots of cytolymph. There were 2 or 3 clear nucleoli in the centre round caryon. Cells were easily distinguished from fusiform fibroblasts when they grew like slabstones. After the first passage, some cells died and the rest grew slowly with the shape of fusiform, polygon. With increasing passage, the colony formation of chondrocytes present triangle and long fusiform. After 3 passage, the fixed sample revealed that Toluidine blue, Safranin-O/Fast green andⅡcollagen stain were obviously positive.2 The samples in group A, surrounded with semitransparent white excreta, were more smooth and tramosericeous than the samples in group B. After 48 hours rolling depression load culture, It was observed by SEM that some chondrocytes had adhered to the macropores of scaffolds. After 4 weeks, the cells with much cytolymph, get bigger and excrete a lot of ECM. Some cells ingrowth in the agarose had established cell-cell junction. On the HE staining sections, It was found that in the experimental group, round chondrocytes in the scaffolds, arranging tight and proliferating actively, developed much ECM; However, It was also found that in the group B, there were less ECM surrounding chondrocytes and the parce cells proliferated generally. The expression of type-Ⅱcollagen was positive in both group A and group B by immunohistochemical detection. The positive area of experimental group was larger than that of control group. According to the experimental group (60.67士6.41)% and the control group (19.92士2.34)%, there was significantly difference (P<0.01).Conclusion: Under the rolling depression load, articular chondrocytes in agarose gel scaffolds possess typical character and secrete increasing ECM . The rolling depression load can promotes chondrocytes to develop type-Ⅱcollagen, and it is a preferable in vitro method for chondrocyte tissue engineering. In addition, the detailed theory of the method needs investigating.
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