| Articular cartilage plays an important role in reducing friction and cushioning vibration of the articulation. The major cause of various types of joint disease is the destroyed articular cartilage. How to repair cartilage defects and restore cartilage function, is the main focus of treatment of joint disease. Autologous cartilage allograft transplantation, condylar joint replacement, and cartilage tissue engineering techniques have been used to repair the cartilage defect. The most promising techniques is the cartilage tissue engineering.Tissue engineering technology is a kind of technique that promoting appropriate seed cells into the targeting tissue, using corresponding scaffold combined with certain growth factors and given the appropriate biological microenvironment. The mainly source of seed cells cartilage tissue engineering are chondrocytes and stem cells. The clinical application of chondrocytes is limited due to its deficiency; in the contrary, the stem cells, especially bone marrow mesenchymal stem cells(BMSCs), is widely used in cartilage tissue engineering due to their extensive source and strong proliferation ability. Platelet-rich fibrin(PRF), a kind of plasma extract that are newly studied in recent years, is rich in various growth factors and overcome the poor biocompatibility of other kinds of scaffolds. Therefore, the PRF has an unparalleled prospect in the research and clinical applications.Previous studies of our research group have done a number of researches on cartilage tissue engineering. We have successfully synthesized cartilage in vitro by constructing BMSCs/PRF composite using the BMSCs as seed cells and the PRF as scaffold. In order to simulate the under-pressure environment of cartilage in vivo, we have developed a new type of cellular stress loading system. By giving BMSCs/PRF composite certain pressure in vitro, we found that the pressure can promote the synthesis of cartilage of BMSCs/PRF composite. Meanwhile, we have found that NF-κB signaling pathway plays an important role in the process of cartilage synthesis using pressure-regulated BMSCs/PRF composite in vitro studies. Whether the NF-κB signaling pathway also play a role in the in vivo study, is still not known.Previously, there have been differences biomechanical properties between the tissue engineered cartilage and the normal cartilage, often leading to stress concentration on the contact surface between grafts and the defect regions, resulting in negative influences of tissue engineered cartilage function. So, good biomechanical properties of tissue engineered cartilage is necessary. Whether the new synthetized cartilage by pressure-regulated BMSCs/PRF has the same biomechanical properties as the normal one, directly determines the function of the new cartilage.In the present study, by establishing the animal models of condylar process in rabbits, we use the pressure-regulated BMSCs/PRF composite to restore the defect region. And, after we used PDTC to block the NF-κB signaling pathway, by observing the repair condition of cartilage defects, detecting the expression of NF-κB signaling molecules and relative gene expression, we explored the involvement of NF-κB signaling pathway in the repairation of condylar cartilage defects using pressure-regulated BMSCs/PRF. Also, using the elastic modulus measurement, we evaluated the biomechanical properties of the new cartilage.The present study was divided into three parts:Experiment 1: first, we extracted bone marrow from rabbits, isolated and cultured BMSCs. By the osteogenic and adipogenic differentiation examinations and surface marker analysis, we proved that the isolated and cultured cells are BMSCs. By producing PRF membrane using extracting ear artery blood of rabbit, we successfully constructed BMSCs/PRF composite. Secondly, we did experiments about screening the proper concentration of PDTC to inhibiting the NF-κB signaling pathway. The results showed that 20μmol/L of PDTC can effectively block the NF-κB signaling pathway while maintaining cell sheet activity. Thirdly, we used the pressure-regulated BMSCs/PRF composite(with or without PDTC treatment) to filling the experimental condylar cartilage defect in rabbits, and evaluating the repair results by histopathological staining. HE staining showed that the pressure-regulated BMSCs/PRF group had a better repairation of condylar cartilage defect than that in BMSCs/PRF group, the former group almost achieved the best in 8 weeks. The PDTC treatment significantly made worse the condylar cartilage repairation of both group.We also used the toluidine blue staining and safranin O fast green staining to evaluate the cartilage repairation in the chondrocytes number and cartilage synthesis perspectives. The results are consistent with the HE staining. The results show that the pre-pressure regulation to BMSCs/PRF could promote the repair of cartilage defects while blocking the NF-κB signaling pathway coul significantly reduce the promotion of pre-pressure regulation.Experiment 2: in this part, we used the Western Blotting and PCR to detect the NF-κB signaling molecules I-κB and P-65, and cartilage formation-related gene Aggrecan and Sox-9. The results showed that the I-κB phosphorylation and P-65 gene expression in pressure-regulated BMSCs/PRF group were significantly higher than that in the BMSCs/PRF group. After PDTC treatment, the I-κB phosphorylation and P-65 gene expression had no statistical difference between the two groups. The gene expression of Aggrecan and Sox-9 had the similar tendency with that of NF-κB signaling molecules. Both the pressure-regulated BMSCs/PRF group and BMSCs/PRF group had higher gene expression of Aggrecan and Sox-9 than that in control group, while the pressure-regulated BMSCs/PRF group was even higher than BMSCs/PRF group. The PDTC treatment significantly decreased the gene expression. Thus, in the perspective of molecular biology, we proved that pre-pressure regulation to BMSCs/PRF could promote the repair of cartilage defects while the NF-κB signaling pathway play an important role in it.Experiment 3: In this part, we did comparative analysis of the biomechanical properties of newborn cartilage in defect region using mechanics Load Test System. By giving pressure on the newborn cartilage zone with at a constant speed, we detected the strain in the recording pressure head and drew the stress-strain curves of every group. Then we could obtain the elastic modulus of samples and evaluate the biomechanical characteristics of each group. The results showed that the elastic modulus of the pressure-regulated BMSCs/PRF group at 8 weeks was the most similar as the normal one. The elastic modulus of all groups grew up gradually as time went on. The elastic modulus of the pressure-regulated BMSCs/PRF group was higher than that of BMSCs/PRF group. PDTC treatment could significantly reduce the elastic modulus of newborn cartilage in every group, especially in the pressure-regulated BMSCs/PRF group.In summary, our study showed that the cartilage repair in the pressure-regulated BMSCs/PRF group was better than that in the BMSCs/PRF group, indicating that the pressure regulating promotes the BMSCs/PRF composite to differentiate into cartilage. Furthermore, the NF-κB pathway inhibition made the cartilage repair worse, suggesting the NF-κB signaling pathway participate in the process of cartilage repair by BMSCs/PRF composite. Finally, the cartilage repair in the pressure-regulated BMSCs/PRF group was even more slowed down by NF-κB pathway inhibition, indicating that the NF-κB signaling pathway play an important role in the cartilage repair by the pressure-regulated BMSCs/PRF composite. Conclusion: 1. The pressure-regulated BMSCs/PRF composite is an ideal restoration to repair the condylar cartilage defect. The NF-κB signaling pathway participate in the process of cartilage repair by BMSCs/PRF composite. 2. Pressure regulating promotes the BMSCs/PRF composite to differentiate into cartilage from the perspectives of chondrocytes number, cartilage matrix amount, integration ability with the remaining cartilage and the biomechanical properties of new cartilage. NF-κB signaling pathway play an important role in the cartilage repair by the pressure-regulated BMSCs/PRF composite. |
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