骨膜细胞与髓核细胞共培养向成骨方向的分化

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

背景:骨膜细胞已被应用于骨缺损及修复,但其可否在成骨诱导环境中与髓核细胞共培养向成骨分化,并应用于椎间隙骨性融合的相关研究报道较少。目的:分离培养髓核细胞与骨膜细胞,观察特定环境下髓核细胞的成骨潜能,以及加入骨膜细胞与其共培养后的成骨能力。方法:Ⅱ型胶原酶消化离心贴壁法分离兔髓核细胞;组织贴壁培养法分离兔骨膜细胞。采用细胞表面抗原CD90、CD105免疫荧光染色法鉴定骨膜细胞,甲苯胺蓝染色及Ⅱ型胶原免疫组织化学鉴定髓核细胞。实验分为两组:成骨诱导环境单独培养的髓核细胞为对照组,加入骨膜细胞与髓核细胞共培养为实验组。CCK8法检测细胞增殖,分别行碱性磷酸酶、茜素红染色、Ⅰ型胶原免疫组织化学染色进行成骨鉴定,Western blot检测两组细胞成骨诱导后骨桥蛋白的表达。结果与结论:骨膜细胞表面抗原CD90、CD105呈阳性,髓核细胞甲苯胺蓝染色及Ⅱ型胶原染色阳性;1,3,5,7,9 d各时间点实验组髓核细胞增殖活性显著高于对照组;诱导2周后2组细胞碱性磷酸酶染色、茜素红染色及Ⅰ型胶原免疫组织化学染色均为阳性,但实验组阳性表达高于对照组(P<0.05);实验组骨桥蛋白表达强于对照组。髓核细胞具有成骨分化的潜能,但体外的增殖能力较低,加入骨膜细胞后,共培养细胞增殖分化能力提高。成骨诱导下骨膜细胞与髓核细胞共培养具有良好相容性,细胞相互黏附,并具有强于单独诱导髓核细胞的成骨能力。

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 histologicallyand 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.