Experimental Study On LIPUS Optimize The Construction Of Tissue-engineered Cartilage And Its Role In Repair Of Articular Cartilage Defects

Background and Objective:With the expanding traffic, the popularization of sports and the aging population, the morbidity of articular cartilage injury and arthritis has been significantly increasing. This makes the repair of articular cartilage defect become a worldwide problem. Mostly, surgical techniques for hyaline cartilage reparation are impossible to obtain the expected results. In this situation, researchers are forced to find a way to induce complete cartilage repair. Recently, the advent and development of tissue engineering has provided alternative possibilities. This research chooses chondrocytes as the seed cell and alginate as the scaffold. Meanwhile, we also use LIPUS to optimize the construction of tissue engineered cartilage. Finally, we investigate the effectiveness of this artificial cartilage in hyaline cartilage reparation in order to explore a new way for treating articular cartilage defect.Method:1. We get some pieces of articular cartilage from both keens of two week-old New Zealand White rabbits under the aseptic condition. And then we separate the chondrocytes by mechanical method combined with enzyme digestion. To observe biological characteristics including growth and phenotype of rabbit articular chondrocytes cultured in vitro (TB staining and expression of collagen II through immunohistochemistry)2. Put the chondrocytes and sodium alginate together into a special model, adjust the chondrocytes density to5×106/ml. Then add adequate solution of calcium chloride in order to confect tissue-engineered cartilage.3. Divide these tissue-engineered cartilages into four groups:Control group (without LIPUS); LIPUS was applied to next three groups for three days,20minutes per day: Low-intensive group (F0:1MHZ, PRF:1kHZ, Amp:50mv, Cycle:50)、Mid-intensive group (F0:1MHZ, PRF:1kHZ, Amp:75mv, Cycle:50)、High-intensive group (F0:1MHZ, PRF:1kHZ, Amp:100mv, Cycle:50). After that, we choose the best parameter of ultrasound by test cell proliferation through CCK-8and expression of collagen Ⅱ through immunohistochemistry.4. The research chooses27New Zealand rabbits about28～35weeks old. We use appropriate method to produce an articular cartilage defect about3mm in diameter,3mm in depth in both two femoral intercondylar fossas in all rabbits. Then devide the rabbit into3groups: Control group, LIPUS-group, LIPUS+group. We implant different tissue-engineered cartilages into the corresponding group (without Control group). The femoral condyles was harvested at the end of4,8,12weeks for gross observation and histological observation (HE, Safranin O2, collagen Ⅱ immunehisto-chemical staining). We also use wakitani score to evaluate the effect of repair.Result:1. Chondrocytes can be isolate by mechanical method combined with enzyme digestion. In certain culture conditions, the2nd and3rd generation of the chondrocytes may good enough to be seed cell for tissue engineering.2. Chondrocytes growth well in alginate (three-dimensional environment). Low-intensity group and Mid-intensity group can stimulate chondrocytes proliferation and collagen II secretion. Low intensity group has more obviously effect, but the High-intensity group inhibits the cell proliferation and collagen II secretion.3. The4th,8th,12th weeks gross observation and histological observation shows that the cartilage defect area in LIPUS-group and LIPUS+group was filled with hyaline cartilage, whereas the Control group is filled with fibrous cartilage. With the time goes by, the wakitani score in each group showed a decreasing trend. In4weeks, compare with Control group in wakitani score, The LIPUS+group has significant difference (P<0.01), The LIPUS-group has statistical difference (P<0.05). but two treatment group has no statistical difference (P>0.05). In8,12weeks, compare with Control group, the two treatment group has significant difference (P<0.01). compare with each other, the two treatment group has statistical difference (P<0.05).Conclusion:1. We can get a plenty of chondrocytes with high bioactivity by mechanical-enzyme digestion.2. According to this method, we can produce tissue-engineered cartilage in specific shape which meet the experimental requirements, and LIPUS can optimized the construction of the tissue-engineered cartilages.3. Both ordinary and LIPUS optimized tissue-engineered cartilages can repair the articular cartilage defect by promote the chondrocytes proliferation, secretion of extracellular matrix (such as collagen II), decreasing the wakitani score. But in contrast. LIPUS optimized tissue-engineered cartilage is better than the ordinary one.