低压细胞灌注法提高多孔人工骨成骨能力的体外研究  

Enhancement of in vitro tissue engineered bone formation by a low-pressure system of cell perfusion into porous scaffold

作　　者：王居勇[1] 张怀华[2] 沈慧良[1] 麻生义则[3] 陈佳妮[3] 张庆明[1] 李旗[1] 王居强[4] 雍宜民[1] 四宮谦一 

机构地区：[1]首都医科大学宣武医院骨科,北京市100053 [2]中国华北电力集团公司北京电力医院骨科,北京市100073 [3]东京医科齿科大学医学部骨科 [4]中国医科大学第一临床学院骨科,辽宁省沈阳市110001

出　　处：《中国组织工程研究与临床康复》2007年第31期6130-6134,共5页Journal of Clinical Rehabilitative Tissue Engineering Research

摘　　要：目的:多孔生物材料中心部所存在的气泡有可能限制了细胞和培养液进入中心部,从而限制了材料内细胞的进一步增殖和分化,继而影响移植后的体内骨形成。实验设计使用低压方法排除多孔材料生物材料β-TCP内的气泡,检测细胞/β-TCP复合体的成骨能力,以验证其可行性。方法:实验于2003-04/2005-06在东京医科齿科大学医学部骨科实验室完成。在体外培养扩增骨髓间充质细胞后,将细胞置于成骨分化的培养液中,通过常压(大气压,101.1kPa)和低压(13.3kPa)两种方法将细胞灌注种植入72个多孔β-TCP人工骨中。细胞种植后检测β-TCP人工骨内含有的细胞悬浊液量,细胞生存率和细胞种植效率;应用扫描电镜观察细胞种植后人工骨内细胞附着情况。并在继续细胞分化培养1,2,3,4周时检测人工骨的DNA含量和碱性磷酸酶活性。结果:①低压组人工骨的细胞悬浊液量、全细胞种植效率和活细胞种植效率均高于常压组(P<0.01);两种方法细胞种植后的细胞生存率没有明显不同(P>0.05)。②扫描电镜观察显示低压组人工骨内附着更多的骨髓间充质细胞。③低压组细胞分化培养过程中不同时点的人工骨碱性磷酸酶活性和DNA含量均高于常压组(P<0.01)。结论:13.3kPa低压细胞灌注法可以明显提高多孔人工骨的细胞种植和成骨能力。低压细胞种植法是一个既简单方便又实用有效的方法。AIM： The air bubble in the central part of porous biomaterials may prevent the cells and culturing liquid from entering the central part and restrict the proliferation and differentiation of cells in biomaterials, which will further influence the in vivo bone formation after implantation. This study is designed to eliminate the air bubble in porous biomaterials β-tricalcium phosphate （β-TCP） by a low pressure system, detect the bone formation of cell/β-TCP composites, and verify the feasibility of this method. METHODS： The experiment was carried out in the laboratory of Orthopaedics Department of Medicine School in Tokyo Medical and Dental University from April 2003 to June 2005. Bone marrow stromal cells （BMSCs） were cultured in vitro and placed in osteogenic medium, then 72 porous blocks of β-TCP were soaked in the cell suspension by low pressure （13.3 kPa） and normal pressure （atmospheric pressure 101.1 kPa）, respectively. The cell suspension volume, cell viability and cell seeding efficiency of BMSCs in β-TCP blocks were examined, and scanning electron microscopy was used to observe the cell adhesion in β-TCP blocks after cell seeding; Alkaline phosphate activity and DNA content of composites were measured at 1, 2, 3 and 4 weeks of subculture. RESULTS： ①Cell suspension volume, total cell seeding efficiency and viable cell seeding efficiency were higher in low pressure group than in the controls （P〈 0.01）; there was no difference in cell viability between the two groups after cell seeding （P 〉 0.05）.②Scanning electron microscopy demonstrated that a greater number of BMSCs covered the central parts of the composites in low pressure group. ③The alkaline phosphate activity and DNA content of composites were higher in the low pressure group than in the controls at different phases of culture （P 〈 0.01）. CONCLUSION： The application of low pressure （13.3 kPa） during the seeding of BMSCs in perfusing medium into a porous scaffold is useful for tissue enginee

关 键 词：低压 骨髓基质细胞 骨形成 磷酸三钙 

分 类 号：R318[医药卫生—生物医学工程]