Gelatin/Chondroitin-6-Sulfate Copolymer Scaffold for Culturing Human Nucleus Pulposus Cells In Vitro with Production of Extracellular Matrix

摘要

Tissue-engineering approaches for treating degenerative intervertebral discs aim to regenerate intervertebral disc tissues in order to retard or even reverse the degenerative process. This study was designed to investigate the feasibility of the glutaraldehyde crosslinked geIatin/chondroitin-6-sulfate copolymer scaffold to serve as a bioactive scaffold for culturing human nucleus pulposus (NP) cells in vitro with preservation of the cell viability, cell proliferation, and production of important extracellular matrix, including glycoaminoglycans (GAG) and Type II collagen. Each experimental sample was seeded with 1 x 10~6 human NP cells, and then the cell-scaffold hybrids were cultured in vitro for 6 or 12 weeks. SEM showed a highly porous structure with an average pore size of 100 mu m in the copolymer scaffold. Immediately after cell seeding, SEM showed that the seeded cells penetrated deeply and distributed evenly in the copolymer scaffold. Water-soluble tetrazolium salt-1 (WST-1) assay showed good viability and active proliferation of cultured human NP cells in the copolymer scaffolds up to 12 weeks. The cell-scaffold hybrids contained significantly higher levels of sulfated GAG than the control samples (41.29 mu g vs 6.04 pig per scaffold). Immunohistochemical study showed Type II collagen fibrils on the surface of scaffold substrate after 6 weeks of cultivation. More abundant deposition of Type II collagen could be detected after 12 weeks. The results achieved in this study indicate that the gelatin/chondroitin-6-suIfate copolymer scaffold is a promising bioactive scaffold for regeneration of nucleus pulposus tissue.