| Background: Injury of articular cartilage continues to present a major problem despite many advances in orthopedic basic and clinical science. It is well known that partial thickness damage to articular cartilage does not spontaneously heal. However, full thickness osteochondral injury undergoes a fibrocartilaginous repair that does not possess the same biological, biochemical, or biomechanical characteristics of native hyaline cartilage. This fibrocartilagenous repair ultimately failed under normal stresses, leading to degenerative arthritis. Therefore, marrow stimulation techniques (drilling, arthroplasty, microfracture), which were used in clinical treatment, result in fibro- cartilage repair tissue that rapidly deteriorates if subjected to sheer or compressive loads. In this experimental study, we selected china goat as animal model, because the anatomy of knee in the goat is similar to human body. The purpose of this study was to establish the method of isolating and expansion chondrocytes in vitro quickly;observe the characteristic of chondrocytes cultured in alginate gel;investigate the results of the goat articular cartilage repaired by implantation autologous chondrocytes suspended in calcium alginate, and examine the results of regeneration when use CPM (continuous passive motion) after operation.Methods: (1) Chondrocytes isolated from cartilage, which was harvested under sterile condition from China goat shoulder joint, cultured in vitro, and determined the PHenotype of chondrocytes by staining with Safranin"O" Toluidine blue and immunohistochemistry of collagen type II. (2) Normal chondrocytes cultured in RCCS(rotary cell culture system), which contained the Cytodex-3 microcarriers in the culture medium (DMEM). Growth of chondrocytes on Cytodex-3 microcarriers was observed dynamically under PHase contrast microscope.compare the total DNA cultured by monolayer with cultured by RCCS, to determine the PHenotype of chondrocytes cultured in RCCS. (3) Normal chondrocytes suspended in calcicum alginate were observed dynamically under PHase contrast microscope, the proliferates of cells evaluated by quantitation of DNA> the quantity of proteoglycan was evaluated by biochemical method with ultraviolet spectroPHotometer and with Toluidine blues Safranin "CTstaining and immunohistochemistry, PHenotype of cells was examined. (4) Tissue engineered cartilage was constructed by cultured chondrocytes in alginate glu, also characterized by histological and immunohistochemistry. (5) A full-thickness articular-cartilage defect (6mm in diameter) was created in the femoral condyle of goats. The goats were divided into five groups. (D the defects were left empty, (2) covered with periosteum, (3) filled with calcium alginate and covered with periosteum, ? filled with autologous chondrocytes suspended in alginate and covered with periosteum, (5) filled with autologous chondrocytes suspended in alginate covered with periosteum and treated by CPM for two weeks postoperatively. Results of cartilage repair were assessed after 3,6 months ( and 12 months those treated by CPM group).Results: Chondrocytes isolated by shoulder joint, cultured in monolayer and RCCS, as well as in calcium alginate, all cells preserved the differentiated PHenotype of chondrocytes, which stained intensively with Safranin"O" s Toluidine blue and immunohistochemistry of collagen type II . When the articular chondrocytes cultured in RCCCS attached rapidly to the surface of Cytodex-3 microcarriers. Quickly growth of these cells was observed after they fully spread onto the microcarriers. The chondrocytes increased 15-17 times in twelfth days of culture as compared with the initial density. In alginate, many chondrocytes clusters appeared, maintained their characteristically differentiated, rounded, chondrocyte morPHology throughout the culture period. Glycosaminoglycan (GAG) production and total DNA increased with time. Histological examination of this cartilaginous tissue in vitro revealed it containeda cartilage cell and matrix strongly stained with Safranin "0" - Toluidine blue and Type II collage. In animal experimental, results indicated that every group have been repaired by fibrocartilage or hyaline cartilage in different degree. By histological and histochemical grading scale evaluation of area of defect, the method filled with autologous chondrocytes suspended in alginate covered with periosteum and treated by CPM was the best way to repair full-thicks cartilage defect in weight-bearing areas of goat knee. The mean difference is significant analyzed by one -way ANOVA between the CPM group with others. Conclusion :1) The methods to isolate and culture goat chondrocytes have been established successfully, and the cell cultured in RCCS were proliferated quickly than in monolaye culture.2) Chondrocytes cultured in alginate gel over a short-time, not only could maintained their characteristically differentiated, rounded, chondrocyte morPHology but also secreted glycosaminoglycan (GAG) and Type II collagen. Another characteristic is chondrocytes could proliferate throughout the culture period.3) Using chondrocytes, which were cultured in alginate for a short time and harvested by remove alginate calcium, articular cartilage was formed successfully. The tissue has the histological of native cartilage and has the biochemical composition of native cartilage.4) The defect created in the femoral condyle of goats could be repaired by hyaline cartilage filled with autologous chondrocytes suspended in alginate> covered with periosteum and treated by CPM postoperatively. The tissue repaired by this way, haven not degenerated after one year.
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