Study On Construction And Properties Of A Bionic Scaffold For Neucleus Pulposus Tissue Engineering

Low back pain is one of common and frequently occurring diseases in modern life with increasingly large health-care and socioeconomic costs. It also causes the labor force lose. Disc degeneration diseases, such as lumbar disc herniation, are the major etiological factors. The ideal resolvent for the treatment degenerated disc diseases maybe is to develop the transplants to re-establish the structure and functions of degenerated discs. Following the development of tissue engineering, to construct the ideal disc implants in vivo is coming true. But there are few studies on lumbar disc tissue engineering. At present, agarose, alginate gel, collagen typeⅠ, chitin, fibrin, PLA and PGA are still major scaffold materials for the tissue engineering neucleus pulposus. These scaffolds have been examined their biocompatibility. But the type of collagen in extracellar matrix of neucleus pulposus is collagen typeⅡ, there are differences in structure and water binding capacity between these biological macromolecules and collagen typeⅡ. Although these artificial matrices maintain cell phenotype, but as many of the signals between scaffolds and cells are matrices dependent, these are not true substitutes for neucleus pulposus extracellar matrix itself. To explore the construction of ideal scaffold materials is one of key points in the study of neucleus pulposus tissue engineering. In this paper, collagen typeⅡ, hyaluronic acid (HyA) and 6-chondroitin sulfate (6-CS), which are main components of neucleus pulposus extracellar matrix, were used to construct a ideal bionic scaffold for tissue engineering neucleus pulposus. Sequentially, the properties of the scaffold with potential use in tissue engineering were examined, and the feasibility was discussed. There were four experimental parts in this paper. The first one: study on the preparetion and physico -chemical characterization of the novel bionic scaffold for tissue engineering neucleus pulposus. In this study, porous type Ⅱcollagen-HyA matrices were prepared by lyophilization, then crosslinked using 1-ethyl-3-(3-dimethyl aminopropyl)carbodiimide (EDC) within the presence of 6-CS. EDC covalently attached CS to collagen. The bionic scaffold was named as CHCS-scaffold. The physico-chemical characterization, including GAG content, pore diameter, porosity, water binding capacity, degradation by collagenase, denaturation temperature and elastic modulus, was determinated. The second one: study on the immunogenicity of the CHCS-scaffold. The scaffold was implanted into the rat subcutaneous tissue. The inflammatory responses, which the host responses to the scaffold, were examined. The structural changes of the scaffold in vivo were observed. The expression of Th1/Th2 mRNA and the anti-porcine collagen type Ⅱantibody in serum were determined. The third one: study on the biocompatibility of the CHCS-scaffold. The neucleus pulposus cells were cultured in vitro. The effections on the scaffolds to the cells were examined. The changes of DNA and 3H-proline content, and the expression of aggrecan, biglycan, and decorin mRNA, aggrecan protein were determined. The last one: study on the histological characteristic of the scaffold in vivo. The cultured program 1st cells were the seed cells. The tissue engineering neucleus pulposus was constructed in vitro, and was implanted into the rabbit lumbar disc that the neucleus pulposus had been cut. The changes of the Bodner disc index (BDI), biomechanics of the spine and histological reaction were observed. The main results and conclusion are following 1. The bionic scaffold for neucleus pulposus tissue engineering –CHCS-scaffold has been constructed by using collagen type Ⅱ, HyA and 6-CS by lyophilization combined with chemical cross-linked. 2. The porous CHCS-scaffold has interconnective channels, which pore diameter is from 89 nm to 132 nm, the porosity is 94.8%, the water binding capacity is 79.2%. The scaffold also has high denaturation temperature, anti-degradation by collagenase, and elastic modulus. These results mean the CHCS scaffold has ideal structural properties. 3. The CHCS scaffold that transplanted into rat subcutaneous tissue does not cause obvious inflammatory. The anti porcine collgen type Ⅱantibody is not detected. These mean the CHCS only causes little immunogenicity. 4. The CHCS scaffold does not have toxicity to the neucleus pulposus. The cells canadhere to the surface and into the inner of the scaffold growthed and proliferated. These mean the scaffold has ideal biocompatibility. 5. After 8 weeks, the complex of the CHCS scaffold and the cells implanted into rabbit disc space can make the decrease of disc space slow down, and increase the lumbar biomechanics. At 12 weeks, the neucleus pulposus –like tissue is observed in vivo. These mean the scaffold has ideal histological characteristic in vivo.