Articular Osteocartilage Defect Repaired With The Three-dimentional Acellular Cartilage Matrix Scaffold

Articular cartilage is hard to repair because it lacks a direct blood supply,lymph circulation ,innervation and has a low metabolism.Presnt clinical common repair method has certain limitation.Acullular matrix which has good biocompatibility and hydrophilicity becomes another choice for tissue engineering scaffold.Compared with artificial material,natural materials have an advantage of good biocompatibility,similarity to structure composition of ECM and suitability for cell's growth,proliferation and differentiation.Compared to other natural materials,a acellular matrix scaffold can get more closer to cartilage's complex natural structure and biological characteristics.In this study we prepared a cartilage acellular matrix scaffold and repaired the articular osteochondral defect.PartⅠ. Preparation of a three-dimentional acellular cartilage matrix scaffold and its characteristics.Objective: To prepare a cartilage acellular matrix scaffold and to evaluate its characteristics. Methods: Calf cartilage microparticles were prepared after being physically shattered and gradient centrifugation, and then treated by a modified Courtman's four-step method which is improved to produce the acellular cartilage matrix.3-D cartilage acellular matrix were prepared with the freeze-drying method.The scaffolds were cross-linked by a neotype crosslinking agent Genepin for 48h,and then placede into glycine solution server times for removing redundant Genepin.The freeze-drying method was used to prepare the CACM.The scaffolds were investigated with gross observation,histological staining(hematoxylin-eosin,toluidine blue),Scan electronic microscope(SEM) and porosity measurement,water absorption rate and degradation rate analysis. Results: Gross observation showed the scaffolds were loose porous and dark blue cross-linked by Genepin.The histological staining(haematoxylin- eosin, toluidine blue staining)showed that there were no chondrocyte fragments in the scaffolds.SEM showed that the scaffolds were porous.The CACM scaffold had 90% porosity,(1314±337)% water absorption rate and (13.69±7.3)%, (25.99±8.9)% degradation rate(2w,4w). Conclusion: The Courtman's four-step method which is improved makes acellular effects more thoroughly and a retention of the natural cartilage extracellular matrix components.The neotype crosslinking agent Genepin makes scaffolds'mechanical strength and resistance to degradation be enhanced.PartⅡ.Evaluation of Biocompatibility of acellular cartilage matrix scaffoldsObjective: Evaluate the biocompatibility of acellular cartilage matrix scoffolds by use of bone marrow stromal cells seeded onto scoffolds. Methods: After being cultivated for ten days,BMSCs of rabbit were seeded onto the scaffold with a cell density of 2.0×106/ml. MTT test and SEM were done to assess the growth and proliferation of BMSCs. Results: MTT test showed that BMSCs grew well in the 3-D CACM scaffolds of logarithmic trend, Absorbance compared with the control group,the difference was not statistically significant(P>0.05) ,supporting that the scaffolds had no cytotoxic effect on BMSCs.SEM micrographs indicated that cells covered the scaffolds firmly with cell processes. Conclusion: After being cross-linked by Genepin,the 3-D CACM scaffold has good biocompatibility and can be a good choice for osteochondral tissue engineering.PartⅢ.Repair of the articular osteochondral defects in rabbits with the bBMP-scaffold complexesObjective: To repair the articular osteochondral defects in rabbits with the bBMP-scaffold complexes. Methods: After dissolving 40mg bBMP in 4ml,4M guanidine hydrochloride,put the scaffolds into the solution.Put the scaffolds into a dialysisi-membrane and dialyze to distilled water for 4 days,and then use the freeze-drying method to prepare the bBMP-scaffold complexes.Seed BMSCs onto complexes at a concentration of 2.0×106/ml.9d later,SEM was used to assess the growth of BMSCs. The Articular osteochondral defects of rabbits with 4mm in diameter and reaching medullary cavity were made in the femoral condyles.18 bBMP-scaffold complexes were implanted into articular osteochondral defects of rabbits,6 articular osteochondral defects without treatment were the control group.At 12th and 24th week, Reparative effect of the defects was investigated by gross observation and histological staining (hematoxylin-eosim,toluidine blue). Results: SEM showed that BMSCs grew very well onto bBMP-scaffold complexes and the structure of the scaffold was the same as before. At 12th week,the defects with bBMP-scaffold complexes were filled with hyaline tissue. Histological staining indicated that the defects were mostly repaired with chondrogenesis and subchondral osteogenesis regeneration.The scaffolds were almost degraded.The defects were still existed in control group filled with fibrous tissue.At 24th week, defects with bBMP-scaffold complexes were completely repaired with chondrogenesis and subchondral osteogenesis fully regenerated indicated by histological staining.The control group were filled with fibrous tissue at the surface of the defects and still had deep defects. Conclusion: We found that BMSCs could differentiated into bone and cartilage induced by vivo different environment.The bBMP-scaffold complexes shows a good effect in repairing the articular osteochondral defects in rabbits.This experiment indicate that the Courtman's four-step method which is improved makes acellular effects more thoroughly.The 3-D CACM scaffold reserves most of extracelluar matrix.After being cross-linked by Genepin,the 3-D CACM scaffold has good biocompatibility and degradation rate of the scaffolds is decreased,which make it a suitable carrier for osteochondral tissue engineering.After being combined with bBMP, the bBMP-scaffold complexes repair osteochondral defects better.