| Part.1Improvement of chondrocyte adhering ability investigation on the chitosan membrane by CD44monoclonal antibody-biotin-avidin binding techniqueObjective:To indicate whether the chondrocyte adhering ability can be improved by CD44monoclonal antibody-biotin-avidin binding technique while being cultivated on the2D chitosan membrane.Methods:The chondrocyte were separated, cultivated and then indentified. The2D chondrocyte-chitosan cultivating system was prepared after the chitosan membrane was available. There are three groups as follow:A, chitosan membrane+chondrocyte (general group); B, chitosan membrane bound with biotin-advidin+chondrocyte (BA tech group); C, chitosan membrane bound with CD44monoclonal antibody-biotin-avidin+chondrocyte (CBA tech group). The adhering and developing of the chondrocyte were tracked by phase contrast microscope. The images were analyzed after the chondrocyte were planted on the membrane for1h,2h,4h,12h and24h. The flatten area of the chondrocyte was calculated by the special image soft, and the adhering ability of the chondtocyte on the chitosan membrane were analyzed.Results:Most of the chondrocyte planted on the chtosan membrane in group C are flatten after cultivated for1h. But it took chondrocyte in group B2h, while12h for group A to achieve the roughly same shape. The flatten area of chondrocyte in group C is distinct larger than that of group B and A. The size of25%chontrocyte planted in group C are around1500-2000μm2after cultivated for4h. While chondrocyte with the same size in group B at the identical stage is near15%, and it took group A24h to reach15%. The flatten area increased with the cultivated time prolong. Statistically, the average area of the chondrocyte in each group exhibited the following pattern: group C> group B> group A, with a statistical significance (P<0.05)Conclusion:The adhering ability of chondrocyte on the chitosan membrane can be improved by the Biotin-Avidin technique compared with the general method, the improvement, however, is remarkable by CD44monoclonal antibody-biotin-avidin binding technique. Part.2Improvement of chondrocyte adhering ability investigation on the chitosan3D scaffold and the preparation of in vitro tissue engineering cartilage by CD44monoclonal antibody-biotin-avidin binding techniqueObjective:To indicate whether the chondrocyte adhering ability in the3D chitosan scaffold can be improved by CD44monoclonal antibody-biotin-avidin binding technique, and investigate whether the proliferation and phenotypic expression of chondrocyte in the prepared tissue engineering cartilage (TEC) can be enhanced.Methods:The chondrocyte-scaffold complex cultivated system was prepared in3groups:A, chitosan scaffold+chondrocyte (general group); B, chitosan scaffold bound with biotin-advidin+chondrocyte (BA tech group); C, chitosan scaffold bound with CD44monoclonal antibody-biotin-avidin+chondrocyte (CBA tech group). The detached chondrocyte after planted were counted by flow cytometer, and the detaching ratio was caculated. The growth curve was draw by MTT test method. Scanning electron microscope (SEM) images were employed to analyze the development of the chondrocyte on the chondrocyte-scaffold complex. The DNA and GXG contents of TEC in the scaffold were tasted by the biochemical method, and the histology section was prepared to investigate the proliferation of the chondrocyte and expression of the extracellular matrix. The expression of type-Ⅱ collagen (Col Ⅱ), aggrecan, sox9and mRNA were tested by RT-PCR.Results:Detaching ratio of the planted chondrocyte:the detaching rate of the group A is3.71times of that of group C, while2.17times of group B. group A> group B> group C, with a statistical significance (P<0.05). The most chondrocyte exhibite in the images obtained from group C, and their phenotype are dispersed and even. Rare chondrocyte, if any, they were clustered and uneven, has been observed in the images of group A. The results obtained from images of group B is fall in between group A and C. Results of histology sections revealed that chondrocyte in group C show a vigorous proliferation and the most extracellular matrix expression. Both the chondrocyte proliferation and extracellular matrix expression are very weak, and results obtained from group B are fall in between group A and C. The contents of DNA, GXG, mRNA of type-Ⅱ collagen (Col Ⅱ), aggrecan, and sox9all show the following pattern:group C> group B> group A, with a statistical significance (P <0.05).Conclusion:Compared with Biotin-Avidin technique, the adhering ability and proliferation of the planted chondrocyte in the chitosan scaffold are more remarkable, when CD44monoclonal antibody-biotin-avidin binding technique is employed, and the cyto-phenotype expression has been improved more. Part.3Repairing effect of the tissue engineering cartilage constructed by CD44monoclonal antibody-biotin-avidin binding technique for the damaged load bearing section of pig knee-jointObjective:To indicate Repairing effect of the tissue engineering cartilage, which is constructed either by CD44monoclonal antibody-biotin-avidin binding technique, for the damaged load bearing section of pig knee-joint.Methods:The tissue engineering cartilage was prepared as described in section3, part2. The detached rate was measured, and the expression of type-Ⅱ collagen (Col II), aggrecan, sox9and mRNA were tested by Real-time PCR. The level-through cartilage damage of bearing section was divided in four groups:group A, chitosan scaffold+chondrocyte (general group); group B, chitosan scaffold bound with biotin-advidin+chondrocyte (BA tech group); group C, chitosan scaffold bound with CD44monoclonal antibody-biotin-avidin+chondrocyte (CBA tech group); group D, planted with the autologous cartilage (autologous group). Samples were taken after cultivated for12or24weeks, and estimated by Moran grade system. The modulus of elasticity and rigidity were been estimated by biomechanics method. The protein content of col II and aggrecan were tested by Western blot. The histology test, such as HE, TB and immunohistochemistry dye were taken for the samples and estimated by Wakitani histology grade method.Results:Detaching ratio of the planted chondrocyte:group A> group B> group C, and the results have a statistical significance (P<0.05). The mRNA of type-Ⅱ collagen (Col Ⅱ), aggrecan, and sox9obtained from Real-time PCR results all show the following pattern:group C> group B> group A, with a statistical significance (P<0.05). In the Moran estimate system that taken after12or24weeks, a greater grade means a better repair. Group C and group D show no statistical significance (P>0.05), and with the following pattern:group C> group B> group A, with a statistical significance (P<0.05). After12weeks or24weeks, the modulus of elasticity and rigidity results are as follow:group D> group C> group B> group A, with a statistical significance (P<0.05). The Western blot results of Col II and aggrecan revealed:group D> group C> group B> group A, with a statistical significance (P<0.05). In the Wakitani histology estimate system, which is taken after12or24weeks, a lower grade means a better repair. Group C and group D show no statistical significance (P>0.05). While group C<group B<group A, with a statistical significance (P<0.05).Conclusion:The repair effect of tissue engineering cartilage constructed by the Biotin-Avidin technique for the damaged load bearing section of pig knee-joint is better than the general method. The effect, however, is much better by CD44monoclonal antibody-biotin-avidin binding technique. Though it can not be equal to the autologous repair totally, the CBA technique can achieve nearly the same repair effect. |
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