| 【Background】 Temporomandibular joint is more important and complex human joints, Mandibular condylar cartilage damage often be caused due to trauma or Inflammation or O steoarthrit is and so on. Because articular cartilage has no blood vessel tissue, For defect repair itself so weak, and often unable to repair itself and cause further damage. The patient will lost the joint function in severe cases and cause great obstacles. At present, there are mang methods for cartilage defect repair, but there are many limitations to these methods. To explore an effective method for cartilage repair has become a pressing demand. With the emergence of tissue engineering technolog y,more and more people use tissue engineering to repair cartilage defect. The research group has achieved good results by undifferentiated adipose derived mesenchymal stem cells to repair rabbit ear cartilage composite P RF. The purpose of the experiment to investigate the feasibilit y that undifferentiated ADSCs composite PRF repair the condylar cartilage loss in vivo.【Objective】 To observe the repair effect of undifferentiated ADSCs membrane composite PRF implantation in rabbit mandibular condylar cartilage defects and to find a new method for the treatment of mandibular condylar cartilage defect and to solve the problem of clinical patients.【Methods】 1、The ADSCs were obtained by trypsin digestion and centrifugation of adipose tissue from the back of rabbit. We examine the cell growth phenotype and multilineage differentiation capacity and surface antigen to prove that the cells obtained is adipose derived mesenchymal stem cells. If the isolated cells were shown to be ad ipose derived mesenchymal stem cells, We will use the medium containing ascorbic acid to induce cell to become cell membrane and lay the foundation for the next experiment. 2、The method for obtaining PRF colloid is by high speed centrifugal artery blood from rabbit ear margin. We will make the PRF become powder and prepare for the later experiment. 3、Animal experimental model is that a critical-sized osteochondral defect, which was 3mm in width and 3mm in depth, was created in the mandibular condyle. 4、36 male New Zealand rabbits were randomly divided into 3 groups, i.e. ADSCs membrane/PRF group, PRF group and control group. Each packet are filled into the corresponding implants according to the experimental design after the animal model established. The rabbits were killed after 1 month, 2 month and 3 month respectively to obtain the specimens of condyle and then the specimens were analyzed by comparing for gross observation and histological examination. 5、We collect the blood of each rabbit before the operation and 7 days、14 days、30 days after operation and measure the level of CD4、CD8、IL-2、IL4.We calculate the CD4/CD8 by the level of CD4、C D8. We use the data to determine whether the body produces immune rejection after al ogeneic ADSCs transplantation.6、All the data using SPSS17.0 software for statistical. and the dose data record as the mean plus or minus the standard deviation form, multiple samples were compared using analysis of variance, two two compared using the SNK- q inspection, inspection level alpha is 0.05 on both sides.【Results】 1、The colony of ADSCs in the culture dish look like spiral,the nucleus shows blue and the cytoplasm shows red in HE staining. ADSCs were induced into adipocytes and osteoblasts,the level of CD29、CD44 is high and the level of C D31、CD34、C D45 is low in the detection of surface antigens in the experiment. These characteristics consistent with characteristics of ADSCs. 2、The specimens were analyzed by comparing for gross observation and histological examination in the 1 month、2 month、3 month after condylar defect model was established the for the blank group,the result showed that cartilage defect area without new cartilage formation. This shows that experimental animal model is successful. 3 、 The repair condition after transplantion. The result of gross observation and histological examination showed that: in the blank group, cartilage defect area without new cartilage formation in the each observation time point. With the extension of time,there are small amount of new bone tissue at the bottom of the cartilage defect area. At the same time small amount of cartilage cells crawl to the surface of the new bone tissue. In the PRF group, there are no obvious cartilage formation After operation. At both ends of the cartilage defect,there are small amount of cartilage cells crawl formed cartilaginous tissue, but the defect is still obviously.In the ADSCs Membrane and PRF Compound group, there is A thin layer of white new cartilage in the surface of the cartilage defect area in the 1 month after operation. The Cell is irregular and light color in comparison with the normal articular cartilage in histological staining results.The cartilage defect area be filledwith new cartilage tissue in the 2 months after operation and t here are small clearance at the junction of the new tissue and normal tissue. The defect area covered by new cartilage tissue, and it’s surface is smooth, the junction of the new tissue and normal tissue become fuzzy and it can be discovered by identify ca refully. The histological results showed that new cartilage tissue is compact and high density, the new cartilage contact with the bone tissue become fibrosis and ossification. The IPP6.0 statistical results showed that the cartilage defect repair rate was: the blank group(1.32±0.23)%, the PRF group(10.37±0.76)%, the ADSCs Membrane and PRF compound group(87.25±4.12)%, comparison of variance analysis showed that there is no statistical difference among three groups(P<0.01).It showed that : PRF have the role of promoti ng the cartilage formation, but this function will become limited in this limit defect. The experimental group better repair effect proved that feasibility uninduced adipose derived stem cells(ADSCs) and platelet rich fibrin(PRF) composite on repair of cartilage defect in the mandibular condyle 4、the result that the level of CD4、C D8、IL-2、IL4 before the operation and 7 days、14 days、30 days after operation and calculated CD4/CD8 show that there were no statistical difference(p>0.05). In the HE staining,we have not found the immune cell infiltration by high power microscope. those proved that the body didn,t appear rejection to the al ograft ADSCs Transplantation.【Conclusion】 1、The cells from adipose tissue are ADSCs, it has strong ability of the added value. The cell culture process is simple.The cells will become cell membrane by use medium containing ascorbic acid,so the member of cells will be doubled. Extracellular matrix and cell to form a three-dimensional network structure, this structure make the cell stable and is not easy to loss. It is suitable for the repair of tissue defect. 2、The PRF volume of reserves and easy extraction. It contains cell factor that to promote the formation of cartilage tissue. Allogeneic ADSCs membrane composite PRF hasgood effect on the restoration of the function of condylar cartilage defect. At the same time, low immunogenicity of allogenic ADSCs transplantatio n did not cause rejection. |
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