| Articulus cartilage injury is one of the most common diseases in clinical orthopaedics, its ability of self renovation is very limited. Osteochondral defects, typically derived by traumatic injuries are often associated with mechanical instability of the joint, and therefore with the risk of inducing osteoarthritic degenerative changes. Therefore, to explore a suitable osteochondral substitute transplanting graft is of great importance. This paper centered on a promising osteochondral substitute transplant graft.1. Research on the pore structure and biocompatibility of a novel collagen I / chitosan /Nanoβ-tricalcium phosphate (β-TCP) bilayered osteochondral scaffold.Objective: To examine and analyze the pore structure and biocompatibility of a novel collagen I / chitosan /β-TCP bilayered osteochondral scaffold. Methods: The interval porosity was evaluated by a volumn method. The pore size, porosity and interpores of the scaffold were observed by scanning electron microscopy(SEM). Rabbit bone marrow stem cells (MSC)were isolated and amplified, then seeded onto the scaffold with a cell density of 3.0×10~5 /ml. The proliferation of the cells on the scaffolds was examined using MTT method, and the growth curve was drawn. 2 weeks later, the scaffolds with cells were dehydrated and observed by SEM. Results: The scaffolds have high porosity and proper pore size, the porosity is above 95%. MSC can adhere onto the scaffold well, and the proliferation rate of the cells on the scaffolds was very good. Conclusion: The novel collagen I / chitosan /β-TCP bilayered osteochondral scaffold has good pore structure and biocompatibility, and possibly will become a new biomaterial of TE suitable for osteochondral repair.2. Cartilage induction of cell-scaffold composite by in vitro 3-D culturing and ectopic cartilage differentiation.Objective: To observe the possibility of 3-D cartilage induction of collagen I / chitosan /β-TCP bilayered osteochondral scaffold when loaded with MSC. Methods: Bone marrow were drawn from six 8-week-old mature Japanese white rabbits. Bone marrow from the tibia was obtained and mononuclear cells were separated by centrifugation in a Percoll solution. The cells were cultured to the second passage, then loaded onto the scaffold and cultured in a 24-well plate. The culture medium was then replaced by a cartilage inductive medium containing rhTGFβ1 10μg/L,dexamethasone 1×10~7 mol/L,VitC 50μg/L,newborn bovine serum 100 mL /L. 2 weeks later, the cell-scaffold composites were sectioned 5μm in thickness and stained with HE and toluidine, others were implanted into the muscle pouch of their respected host rabbits. 6 weeks later, the materials were tacken from the muscle pouch, sectioned and stained with HE, toluidine and type II collagen immunohistochemistry. Results: MSC grow fast during the 3-D culture process. Cells cover the surface and internal wall of the material in the form of multi-layers. Toluidine staining indicates positive. The materials degraded partially, but the structure was not obviously changed. After the consequent 6 weeks muscle pouch implant, the materials degraded mostly, cartilage-like tissue were formed. Both toluidine staining and type II collagen immunohistochemistry showed positive. Conclusion: MSC can be induced toward cartilage during 3-D culture when loaded onto collagen I / chitosan /β-TCP bilayered osteochondral scaffold, after the consequent 6 weeks in vivo differentiation, the cell-scaffold composites can further differentiate toward cartilage-like tissue.3. Manufacturing of osteochondral biomaterial incorporated with bovine BMP(bBMP).Objective: Incorporate bBMP into the collagen I / chitosan /β-TCP bilayered osteochondral scaffold to enhance its osteochondral inductive activity. Methods: Dissolve 40mg bovine BMP in 4ml 4M guanidine hydrochloride. Put the collagen I / chitosan /β-TCP bilayered osteochondral scaffolds into the bBMP solution and exhanstion the air entrapment of the scaffold. Put the scaffold absorbed with bBMP into a dialysis-membrane and dialyze to distilled water for 4 days. Then freeze-drying for 12 hours, sterilize with ethylene oxide. Take 3 biomaterials for SEM examination. Load MSC onto six biomaterials at a concentration of 1×10~6/ml. 2 weeks later, SEM examine to observe the growth condition of MSC onto or into the scaffold. Results: When incorporated with bBMP, the scaffold maintained the same structure as before. SEM can clearly see bBMP thin film into or onto the biomaterial. MSC grow onto the biomaterial very well, and large amount of cells can be abserved. Conclusion: The osteochondral biomaterial produced with dialysis method maintained good structure and biocompatibility, while might greatly increased its bioactivity.4. Rabbits osteochondral defects repairing with biomaterials incorporated with bBMP. Objective: To repair rabbits osteochondral defects with the biomaterials incorporated with bBMP. Methods: 8 Japanese white rabbits weighed from 2.3 to 2.7kg were used as experiment animals. Defects with 4mm in diameter and reaching medullary cavity were made in femoral condyles. The right knees were treated with biamaterials combined with bBMP as experimental group, the left knees were either treated with collagen I / chitosan /β-TCP bilayered osteochondral scaffolds or with nothing as control group. 12 weeks later, the animals were killed and the newly formed tissues were observed macroscopically and microscopically. Results: After 12 weeks in vivo repairing, osteochondral defects of the experimental group were filled with hyaline tissue, which is closely unified with the surrouding cartilage. The control 1 group were filled with fibrous tissue, the outline of defects were still obvious. The control 2 group still had deep defects, without much repairing tissue. Histology showed that the experimental group were repaired with hyaline cartilage, toluidine staining also showed positive results. The control 1 group were repaired with fibrous tissue on the surface, while the subchondral bone have had good repair. The control 2 group still have deep defects, the surface of the defects were covered with fibrous tissue. Conclusion: collagen I / chitosan /β-TCP bilayered osteochondral scaffolds have good bioactivity in osteochondral defects repairing, especially when incorporated with bBMP.The above experiments indicate that collagen I / chitosan /β-TCP bilayered osteochondral scaffold has good porosity and biocompatibility as a new scaffold for osteochondral tissue engineering. When combined with bBMP, it obtained much better bioactivity, and can repair osteochondral defects much better. |
