Preliminary Construction of Tissue Engineering Nucleus Pulposus Combining Silk Fibroin Porous Scaffold with Rabbit Nucleus Pulposus Cells

ZHAO Jianing; XU Baoshan; ZENG Chao; YANG Qiang; MA Xinlong; ZHANG Chunqiu; LI Xiulan; ZHANG Yang

doi:10.3969/j.issn.0253-9896.2014.11.006

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 inoculat?ed 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 typeⅡimmunohistochemical staining. Results Safranin O stain?ing and typeⅡcollagen immunohistochemical staining of the passage 1 nucleus pulposus cells were positive. The fluores?cence 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 scaf?fold. 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 typeⅡcollagen 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.