Osteogenic differentiation of nucleus puplousus cells co-cultured with autologous periosteal cells

doi:10.3969/j.issn.2095-4344.2015.37.003

Abstract

Abstract:

BACKGROUND: Periosteal cells have been used in bone repair, but whether nucleus puplousus cells co-cultured with autologous periosteal cells can differentiate into osteoblasts in spinal fusion is rarely reported.

OBJECTIVE: To isolate nucleus puplousus cells and periosteal cells so as to observe the osteogenic ability of nucleus puplousus cells co-cultured with periosteal cells or not.

METHODS: Type II collagenase digestion method was used to isolate and purify nucleus pulposus cells, which were confirmed by toluidine blue and immunohistochemical staining. Periosteal cells were isolated histologically
and cultured in complete medium, and cell surface antigens CD90, CD105 were identified by immunofluorescence staining. According to the experimental needs, the cells were assigned into two groups. Nucleus pulposus cells and periosteal cells were co-cultured by osteogenic induction medium in the experimental group. Nucleus pulposus cells in the control group were cultured alone in osteogenic induction medium. Cell morphology was observed by inverted microscopy, and cell proliferation was detected by cell counting kit-8. The osteogenic differentiation indexes of cells in each group were measured using alkaline phosphatase staining, alizarin red staining, and type I collagen immunohistoehemical staining. The expression of osteopontin was tested by western blot assay.

RESULTS AND CONCLUSION: CD105 and CD90 expressions of the periosteal cells were positive. Nucleus puplousus cells were positive for toluidine blue and collagen type II immunohistochemical staining. The proliferative ability of nucleus puplousus cells was significantly higher in the experimental group than the control group at days 1, 3, 5, 7, 9. After 2 weeks of induction, the cells were positive for alkaline phosphatase staining, alizarin red staining, and type I collagen immunohistoehemical staining, but the experimental group showed higher positive expressions than the control group (P < 0.05). The expression of osteopontin was also higher in the experimental group than the control group. These findings indicate that nucleus puplousus cells possess osteogenic ability, but have lower proliferative ability in vitro. After co-culture with periosteal cells, the proliferative ability of nucleus puplousus cells can be increased. Under osteogenic induction, nucleus puplousus cells co-cultured with periosteal cells have good compatibility and adhere with each other, which have stronger osteogenic ability than cells cultured alone.

中国组织工程研究杂志出版内容重点：组织构建；骨细胞；软骨细胞；细胞培养；成纤维细胞；血管内皮细胞；骨质疏松；组织工程

Key words: Periosteum, Osteoblasts, Bone Morphogenetic Protein 7

CLC Number:

R318

Yang Yu-ming, Yuan Feng, Lu Hai-tao, Zhang Jun-wei, Sheng Xiao-lei, Li Zhi-duo. Osteogenic differentiation of nucleus puplousus cells co-cultured with autologous periosteal cells[J]. Chinese Journal of Tissue Engineering Research, 2015, 19(37): 5916-5922.

Figures/Tables 6

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