异体冻干颗粒骨复合骨髓基质干细胞源性成骨细胞体外生物相容性研究  

Study of the biocompatibility on allogeneic freeze-dried bone particles and bone marrow stromal cells-derived osteoblasts in vitro

作　　者：曹基婷[1] 李琪佳[1] 宋会平[2] 王志强[2] 白俊清[2] 

机构地区：[1]华北煤炭医学院实验中心 [2]华北煤炭医学院附属医院骨科,唐山063000

出　　处：《中国矫形外科杂志》2010年第12期1023-1027,共5页Orthopedic Journal of China

基　　金：河北省医学科学研究重点课题计划(编号:20090182)

摘　　要：[目的]评价异体冻干颗粒骨体外生物相容性,为其作为骨组织工程支架材料的临床应用提供实验依据。[方法]4周龄SD大鼠,雌雄不限,采用全骨髓贴壁法体外培养骨髓基质干细胞(BMSCs),取生长状态良好的第3代BMSCs行成骨诱导培养并鉴定。制备异体冻干颗粒骨浸提液,将成骨诱导7d的BMSCs加入浸提液作为实验组,未加浸提液的为对照组,培养1、3、5d,采用MTT法检测异体冻干颗粒骨浸提液对细胞增殖的影响。成骨诱导7d的BMSCs按1×106/ml接种于异体冻干颗粒骨上,倒置相差显微镜、扫描电镜观察与其复合培养的细胞在材料上的生长情况;酶消化法消化复合于冻干颗粒骨上的成骨细胞,流式细胞仪检测细胞周期变化。[结果]BMSCs成骨诱导7d,ALP染色示胞浆内可见阳性蓝染颗粒,胞核呈红色。MTT法检测结果显示细胞在浸提液中生长与增殖状态良好,实验组与对照组差异无统计学意义(P>0.05);倒置相差显微镜可见颗粒骨表面粘附多数成骨细胞;扫描电镜观察可见细胞在材料表面粘附、伸展并能增殖、分泌产生细胞外基质;流式细胞术检测显示,异体颗粒骨对细胞周期无影响,实验组细胞均为正常2倍体细胞。实验组与对照组差异无统计学意义(P>0.05)。[结论]异体冻干颗粒骨无细胞毒性并具有良好的组织细胞相容性,为其临床应用提供了实验依据。[Objective]To access the biocompatibility of allogeneic freeze-dried bone marrow stromal cells in vitro,so as to provide the experimental data for bone tissue engineering scaffold material in the clinical application.[Method]The bone marrow stromal cells were isolated from one-month-old SD rats and cultured with holo-bone marrow direct inoculation methods in vitro.The 3rd generation of BMSCs were induced into osteoblasts.The leaching liquor of allogeneic freeze-dried bone marrow stromal cells was prepared,and the osteoblasts after being induced for 7 days were cultured in leaching liquor（experimental group）.The osteoblasts cultured without leaching liquor were used as controls （control group）.The growth and proliferation ability were analyzed by MTT examination at 1,3 and 5 days.The osteoblasts after being induced for 7 days were implanted in allogeneic freeze-dried bone marrow stromal cells at 1×106/ml.The growth of cells on the materials were observed under inverted microscope and scanning electron microscope.The osteoblasts implanted in allogeneic freeze-dried bone marrow stromal cells were digested by trypsin.And the cell circle was detected by flow cytometry.[Result]ALP staining of osteoblasts showed that there were positive blue grains in cytoplasm and nucleui were red on the 7th day.The MTT test showed that cell proliferation and growth were normally in the leaching liquor.There were no significant differences between experimental group and control group （P0.05）.Many of osteoblasts could adhere to the surface of materials under inverted microscope.The osteoblasts spread well on the surface of allogeneic freeze-dried bone marrow stromal cells and could produce some extracellular matrix under scanning electron microscope.Flow cytometry showed that allogeneic freeze-dried bone marrow stromal cells had no effect on the cell cirle.There were normal diploid cells in experimental group,but no significant difference between experimental group and control group（P0.05）.[Conclusion]Allogeneic freeze-

关 键 词：骨髓基质干细胞 异体骨 生物相容性 成骨细胞 骨组织工程 

分 类 号：R687.3[医药卫生—骨科学]