| The articular cartilage defects caused by injuries or degenerative events are very common in clinic practice, yet it has a limited capacity of self-regeneration, which bring dysfunction and inferior life quality to patients. It is still a difficult problem to repair and reconstruct articular cartilage defects in orthopaedics, and it is also one of the research foci in recent years. Untill now, there have been a few methods to repair articular cartilage defects including"microfracturing","masaicplasty", transplantations of periosteum, periochondrium and isolated chondrocytes. Although these methods have their advantages and disadvantages respectively, they can not meet the needs of perfect reconstruction in the end. Tissue engineering that aims to"replicate tissue and organ"provide another innovative choice for repair and regeneration of articular cartilage defects.Based on the handling properties of scaffolds, the tissue-engineered cartilage are classified into either preformed cartilage or injectable cartilage. Recently, much progress has been achieved to repair articular cartilage defects with preformed cartilage. Some of prefabricted cartilage have been successfully applicted in clinic and obtained wonderful therapeutic effects. However their clinical applications are much limited. Implantation of the preformed cartilage implies a wide arthrotomic exposure of the joint. Open implantation results in sacrificing some capsular, synovial or ligamentous structure of the joint. It can augment pain, prolong the rehabilitated period for the patients and some complications have been reported with this procedure, including infection and stiffness of the joint. Furthermore, complicated fabrication procedure, hard to model and integrate with chondral defects of preformed cartilage make it not easy for filling irregularly shaped defects. Development of injectable catilage that can be injected arthroscopically has number of advantages in tissue engineering as against prefabricated scaffolds. A major advantage would be the possibility of administering the injectable catilage through minimally invasive surgical procedures in many cases. It also has the advantage of filling cavities with complex geometries, and to provide good bonding to tissue. Injectable cartilage would greatly improve the manipulation characteristics, so it becomes the developed trend for the treatment of chondral defects clinically with tissue-engineered cartilage. Research on injectable cartilage is still in primary stage, mainly focusing on the choice of biomaterials, fabrication of scaffolds and ectopic construction of cartilage. Better seeding cells and injectable scaffolds have not been found, fewer efforts have been made to develop in situ gelation injectable cartilage for repair of articular cartilage defects. If we want to make this technique a successful transition from an animal experiment to a clinical practice, the following essential questions need to be solved: 1. Sources of seeding cells; 2. Choice of suitable biomaterials and fabrication of injectable scaffolds; 3. The feasibility of repairing articular cartilage defects with in situ gelation injectable cartilage. Aimed at this problems, using articular cartilage defects in swines as subjects, we studies the effects of chitosan/β-glycerophosphate disodium (C/GP) gel on the growth, proliferation and chondrogenic differentiation of bone marrow mesenchymal stem cells (BMSCs), the feasibility of the C/GP gel acts as cell vehicle to construct tissue-engineered cartilage in vitro and in situ gelation to repair articular cartilage defects in vivo.Firstly, BMSCs were harvested from bone marrow by puncture and expanded-cultured in vitro. Chitosan and glycerophosphate disodium solutions were mixed in appropriate volume ratios, and the BMSCs were incorporated into the C/GP mixture to construct C/GP-BMSCs gel complex, then 3-D cultured and induced chondrogenesis in vitro. The growth, proliferation and chondrogenic differentiation of BMSCs into C/GP gel and chondrogenesis of C/GP-BMSCs gel complex when chondrogenic cultured in vitro were estimated. Results showed: 1. It took 5-6 days for BMSCs to be confluent with plating concentration of 1×105/cm2; 2. C/GP gel supports the growth, proliferation and chondrogenic differentiation of BMSCs cultured in vitro; 3. C/GP-BMSCs gel complex can keep its original shape after long-term culture; 4. C/GP-BMSCs gel complex can form cartilage tissue when chondrogenic cultured in vitro; 5. BMSCs have the capacity to differentiate into chondrocytes, osteoblasts and fat cells.Secondly, the C/GP-BMSCs mixtures were autologous injected into the articular cartilage defects covered with collagen membrane and gelled in situ to construct tissue-engineered cartilage in order to repair articular cartilage defects in mini-pigs. Gross observation, histology, immunohistochemistry, glycosaminoglycan(GAG) quantification and magnetic resonance imaging(MRI) were applied to analyze the results. Results showed: 1. Defective regions were occupied by hyaline cartilage and Safranin-O histological staining and collagenⅡimmunohistochemistrical staining were positive at 8 and 16 weeks after transplantation; 2. Neither lymphocyte infiltration nor vascular invasion could be seen in or around the transplanted tissue; 3. GAGs contents in tissue-engineered cartilage were significant higher than the control and MRI demonstrated normal shape and signal of cartilage in repair sites at 16 week after surgery.Conclusions can be drawn as follows: 1. Injectable C/GP-BMSCs gel complex exhibit excellent cytocompatibility and support the growth, proliferation and chondrogenic differentiation of BMSCs; 2. Injectable C/GP-BMSCs gel complex can form cartilage tissue when chondrogenic cultured in vitro; 3. Injectable C/GP gel can be used as a good carrier to delivery BMSCs to articular cartilage defects; 4. reconstruction of articular cartilage defects with injectable autologous C/GP-BMSCs gel complex can be obtained.
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