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

Yang Yu-ming; Yuan Feng; Lu Hai-tao; Zhang Jun-wei; Sheng Xiao-lei; Li Zhi-duo

doi:10.3969/j.issn.2095-4344.2015.37.003

Abstract：BACKGROUND:Periosteal cel s have been used in bone repair, but whether nucleus puplousus cel s co-cultured with autologous periosteal cel s can differentiate into osteoblasts in spinal fusion is rarely reported. OBJECTIVE:To isolate nucleus puplousus cel s and periosteal cel s so as to observe the osteogenic ability of nucleus puplousus cel s co-cultured with periosteal cel s or not. METHODS:Type II col agenase digestion method was used to isolate and purify nucleus pulposus cel s, which were confirmed by toluidine blue and immunohistochemical staining. Periosteal cel s were isolated histological y and cultured in complete medium, and cel surface antigens CD90, CD105 were identified by immunofluorescence staining. According to the experimental needs, the cel s were assigned into two groups. Nucleus pulposus cel s and periosteal cel s were co-cultured by osteogenic induction medium in the experimental group. Nucleus pulposus cel s in the control group were cultured alone in osteogenic induction medium. Cel morphology was observed by inverted microscopy, and cel proliferation was detected by cel counting kit-8. The osteogenic differentiation indexes of cel s in each group were measured using alkaline phosphatase staining, alizarin red staining, and type I col agen immunohistoehemical staining. The expression of osteopontin was tested by western blot assay. RESULTS AND CONCLUSION:CD105 and CD90 expressions of the periosteal cel s were positive. Nucleus puplousus cel s were positive for toluidine blue and col agen type II immunohistochemical staining. The proliferative ability of nucleus puplousus cel s was significantly higher in the experimental group than the control group at days 1, 3, 5, 7, 9. After 2 weeks of induction, the cel s were positive for alkaline phosphatase staining, alizarin red staining, and type I col agen 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 cel s possess osteogenic ability, but have lower proliferative ability in vitro. After co-culture with periosteal cel s, the proliferative ability of nucleus puplousus cel s can be increased. Under osteogenic induction, nucleus puplousus cel s co-cultured with periosteal cel s have good compatibility and adhere with each other, which have stronger osteogenic ability than cel s cultured alone.