以生物活性基質進行軟骨及骨軟骨之組織工程

Abstract

Abstract Articular cartilage injury is a great challenge for orthopedic surgeons. Tissue engineering had merged as a new method for cartilage repair. The tissue engineering triads consisted of cells, biomaterial scaffold and bioactive factors and environment (provided by bioreactor). In our study, all these three aspects had been explored. Part I. Cartilage tissue engineering: Hypothesis: Current synthetic scaffolds for cartilage tissue engineering have many shortcomings. We hypothesized that a tri-copolymer formed from gelatin, chondroitin, and hyaluronan might mimic cartilage matrix and provide the necessary information for cartilage tissue engineering. Methods: Porcine chondrocytes seeded tricopolymer were cultured in spinner flasks and Petri dish for 2, 3, 4 and 5 weeks for in vitro cartilage tissue engineering. Large scale animal study with 15 miniature pigs is well designed, with randomized control study to compare the therapeutic effect of allogenous chondrocytes seeded tricopolymer scaffold based tissue engineering, autogenous osteochondral transplantation and spontaneous healing process for full thickness articular defects and osteochondral defects. Another 6 pigs were treated with defects creation for spontaneous healing or filled with scaffold without cell seeding. Results: In culturing results of the tissue-engineered constructs in spinner flask, we found chondrocytes are distributed more evenly than in the scaffold in Petri dish. The constructs were found with new extracellular matrix synthesis with type II collagen contents. And the chondrocytes still retain their phenotype as demonstrated by immunohistochemistry. In animal study, after exclusion of the cases with infection and secondary arthritis, the best results comes from the autogenous osteochondral transplantation except the integration to host cartilage was poor. The results of tissue engineering treated group were satisfactory with repair tissue varied from hyaline cartilage to fibrocartilage. The spontaneous healing response is not enough for good repair. Filling of scaffolds without cell seeding can not offer good repair. For osteochondral defects, the subchondral bone plate was not restored through cartilage tissue engineering, thus osteochondral tissue engineering is necessary. Part II. Osteochondral tissue engineering: Hypothesis: Osteochondral tissue engineering is necessary for the treatment of osteochondral defect. We hypothesized through a biphasic scaffold and double-chamber bioreactor, tissue engineered osteochondral constructs can be developed. Methods: Gelatin sponge was formed on the top of the calcined bovine bone to fabricate a biphasic scaffold. Porcine chondrocytes were seeded on the biphasic scaffold and cultured in specially designed double chamber bioreactor for 2 and 4 weeks. Then the human mesenchymal stem cells (hMSCs) were induced separately for chondrogenesis and osteogenesis, and co-cultured in double chamber bioreactor for fabrication of tissue engineered osteochondral constructs. Results: Cartilage formation with porcine chondrocytes seeded biphasic scaffold on the surface of the calcined bovine bone was successful demonstrated. The induction of hMSC is successful, except that there is a lower efficiency for chondrogenic induction. Cell pellets were too aggregated, which can not cover the whole surface of calcined bovine bone. The calcined bovine bone is easily cracked in injection seeding of cells, further modification of the procedure in fabrication of osteochondral constructs is necessary.