The Construction Of Tissue Engineering Cartilage Graft In Vitro With Cell Sheet Technique

Repair of cartilage defects remains one of the most challenging tasks in plastic and reconstructive surgery. Nowadays, tissue engineering technique provides a promising alternative to solve this difficult problem. Due to the special structure of cartilage which is composed of cell and matrix only and without nerve and vessel in it, cartilage was initially studied in 1990's. Just as other tissue engineering researches, cartilage tissue engineering consists of seed cell, scaffold, growth factor and tissue reconstruction method. By now, cartilage has been successfully regenerated in vivo and in vitro in many researches with tissue engineering technique. Clinical applications of the engineered cartilage have been reported to repair joint cartilage defects and satisfied results were obtained by some groups.However, some obstacles limit the general application of cartilage tissue engineering in clinical practice such as the limited source of seed cells, the unquenchable interference among cells with scaffolds and bodies, inflammatory reaction induced by biomaterials degradation product and long term biological instability of the tissue engineered cartilages ( Maintaining the biological characters of the regenerated cartilage in long time could bring about the truly application in clinical works).Objective: To regenerate tissue engineered cartilage with favorable contour, adequate volume and enough strength for repair of cartilage defects. In details: 1. To explore how to get more seed cells in cell culture and how to improve efficacy in cell usage in tissue fabrication. 2. To regenerate three-dimensional cartilage with cell sheet method which could minimize the disturbance of scaffold materials to tissue regeneration due to its scaffold-free characteristics. 3. To employ a novel cartilage regeneration method with bionic cell aggregate shaped by external and internal supporting system. 4. To study the regeneration of tissue engineered cartilage with characteristics of cartilage hyalina by bioreactor. 5. To observe the differentiative ability and stability of the tissue engineered cartilage which was implanted into the animal body. 6 To explore the application potential of engineered cartilage regenerated with this way in repairing cartilage defects.Methods and results: In present study, rabbit chondrocytes and bone marrow mesenchymal stem cells (BMSCS) were successfully separated and cultured with high-density culture and continuous culture of primary and passage cells. Imitating the formation process of embryo cartilage tissue, the bionic cell sheet composed of cell and matrix produced by the cells themselves was fabricated and its characteristics were studied. Then cartilage was regenerated by chondrocyte sheet which cultured in vitro in bioreactor. The results showed that as differentiated cells, chondrocyte could secrete collagenâ…¡and glycosaminoglycans.The uniformity and mechanical strength of the in vitro reconstructed cartilage kept weak but it could transformed into mature cartilage tissue when implanted into animal body. It was very difficult to get enough volume of cartilage in vitro due to the limited donor site and instability of chondrocyte phenotype in cell culture procedure. We further chose BMSCS as seed cells in the following studies. Firstly, pellet and patch-like cartilage was reconstructed in vitro by centrifugalization and continuous culture and induction. It was convinced that cell seeding protocol and dynamic cultural environment were essential to the formation and maturation of the cartilage tissue. Then, with the chondrocytes transformed from BMSCS as seed cells, PLGA scaffold as the internal supporting structure, cartilage with the shape of human cartilagines tracheales and cartilage alaris major were reconstructed in vitro. It was effective to reconstruct cartilago hyalina like cartilage graft with high homogeneous and good mechanical strength by in vitro dynamic culture of cell sheet shaped by internal scaffold. Furthermore, the efficacy of this method was proved to be successful when the graft was transplanted in vivo. Finally, we used the technique to repair the cartilage defect of 1cm in diameter of rabbit ear new cartilage was found and the defect got repaired in 12 weeks.Conclusions: The current research showed that special contour-bionic tissue engineered cartilage can be reconstructed with the strategy of cell aggregation culture plus external and internal supporting scaffold in bioreactor. High density aggregated culture pattern was helpful to keep the chondrocyte phenotype and can recover the phenotype of the dedifferentiated chondrocytes. Cell sheet was composed of cells and matrix secreted by the cells themselves: it biomimic the way natural cartilage formation both in tissue origination and histological structure. The method could minimize the possible disturbances of the scaffold materials to the formation of newly formed cartilage. To shape the tissue engineered cartilage into special contour for clinical application, outer and inner supporting was proved to be efficient. The strategy was simple and effective, and helpful in shaping cartilage contour. In addition it made the manipulation of cell sheet easy to form cartilage of predetermined shape.