Abstract:

[Abstract]   Objective   To investigate the feasibility of construction of tissue engineering nucleus pulposus by com?
bining the novel silk fibroin porous scaffold with PKH26 labeled rabbit nucleus pulposus cells.   Methods   Rabbit nucleus pulposus cells were isolated and cultured, then the passage 1 nucleus pulposus cells were stained with safranin O and typeⅡcollagen immunohistochemical staining. The isolated rabbit nucleus pulposus cells were labeled with PKH26. MTT assay was used for examining the proliferation of the nucleus pulposus cells before and after labeling. Labeled cells were inoculated in the scaffold, cultured for 4 days and then the cell-scaffold complexes were implanted subcutaneously into nude mice. After 12 weeks of in vivo culture, the cell-scaffold complexes were detected by in vivo imaging technology, H & E staining, toluidine blue staining, safranin O staining and collagen type Ⅱ immunohistochemical staining.   Results   Safranin O staining and type Ⅱ collagen immunohistochemical staining of the passage 1 nucleus pulposus cells were positive. The fluorescence intensity of labeled cell was distributed, and the difference of OD value of nucleus pulposus cells was not statistically significant before and after labeling (P > 0.05). The in vivo imaging technique showed a strong fluorescencea in porous scaffold. H &E staining of cell-scaffold complexes showed that the scaffolds were filled with a large number of nucleus pulposus cells and large amount of extracellular matrix. Toluidine blue staining, safranin O staining and type Ⅱ collagen immunohisto?chemical staining were positive, and large amount of extracellular matrix was secreted around the cells.   Conclusion   The new silk fibroin porous scaffold with rabbit nucleus pulposus cells in vivo culture formed nucleus pulposus like tissue, which can be used for construction of tissue engineering nucleus

Key words: silk protein, Tissue engineering, immunohistochemistry, Nucleus pulposus, PKH26