Construction Of Tissue Engineering Interverbral Disc And Animal Experiment In Vivo

Objective: To study the feasibility of construction of intervertebral disc using tissue engineering methods and to find out the effect of varing the concentration of intervertebral disc cells seeded on the PLGA scaffolds using following investigations:MTT assay, scanning electron microscopy observation, gross morphology, histology, immunohistochem- ical analysis and biochemical analysis of glycosaminoglycans.Methods: (1) A 3-dimensional porous PLGA scaffold was fabricated by temperature induced phase separation method.(2) Cells were isolated from a 20 week old abortus. The 2nd generation of cells were used to construct cell-scaffold composites and the composites was observer in vitro by MTT assay and scanning electron microscopy. (3) 40 cell-scaffold composites were constructed varing the cell concentration of 0, 104/150μl,105/150μl and 106/150μl. Composites were incubated in vitro for 48 hours then implanted subcutaneously onto the dorsom of nude mice. The mice were killed and the implants were harvested 4 weeks after surgery, followed by a series of investigations: Gross morphology,histology, immunohisto- chemical analysis and biochemical analysis of glycosaminoglycans.Results: (1) A well-regulated array of microtubules orientation- structured scaffold with 50-300μm of diameter was fabricated by an temperature -induced phase separation technique. (2)Cells attached to the PLGA scaffolds well and synthesis extra cellular matrices. MTT dying observation showed the color of the composite increased with the increasing cell number.(3) Histology, immunohistochemical analysis and biochemical analysis of glycosaminoglycans indicated that composites loading cells of 105 and 106 showed a better synthesis function than the other two groups, espesically in types II collagen and GAG. Immunohistochemical analysis of BrdU marked cells proved that the cells in the composites were the human disc cells seeded in 4 weeks ago.Conclution: (1) PLGA scaffold was suitable for tissue engineering interverbral disc and abortus intervertebral disc cells attached to the PLGA scaffolds well. (2) Composites loading cells of 10~5 and 10~6 might be helpful in the treatment of disc degeneration disease in future. (3)The potential feasible cell quantity for tissue engineering interverbral disc might be 10~5 or higher than 10~5.