一体化灌注法体外构建活化人工骨的优化研究  

Optimization of bioartificial bone graft construction in vitro

作　　者：王林[1] 马真胜[1] 雷伟[1] 胡蕴玉[1] 王臻[1] 冯亚非[1] 张扬[1] 裴国献[1] 

机构地区：[1]第四军医大学西京骨科医院,西安710032

出　　处：《中华生物医学工程杂志》2014年第2期87-93,共7页Chinese Journal of Biomedical Engineering

基　　金：国家自然科学基金（31170913）

摘　　要：目的探讨人胚成骨细胞在多孔β-磷酸三钙（β-TCP）支架内灌注性接种及培养的影响因素及其作用机制，对一体化灌注法体外构建活化人工骨进行优化研究。方法应用自行设计的灌注式生物反应器进行多孔β-TCP支架内人胚成骨细胞的一体化灌注性接种和培养，以静态接种为对照。通过细胞活力（Mrrr法）测定、活细胞接种率、组织形态学观察和计量学分析等分别检测细胞接种时间、接种密度、灌注速率等因素对支架内细胞黏附和生长的作用。结果细胞灌注接种效果优于静态接种；灌注接种-灌注培养法细胞生长及分布优于静态接种-灌注培养法。接种时间、接种密度、灌注速率等因素均显著影响细胞接种及培养效果。灌注法构建活化人工骨的最优条件：接种时间12h～24h，接种密度2×10^5／ml一5×10^5／ml，灌注接种速率1ml／min，灌注培养速率0．5ml／min～2ml／min（灌注初期24h）及2ml／min（灌注24h后）。结论一体化灌注法利于多孔β-TCP支架内人胚成骨细胞的黏附和生长。接种时间、接种密度、灌注速率等因素均影响活化人工骨体外构建效果，对其进行优化有助于人工骨移植物的临床转化和推广应用。Objective To investigate the influencing factors and mechanisms of human fetal osteoblasts seeding and culture in porous β-tricalcium phosphate （β-TCP） particles, and to optimize the bioartifical bone graft construction in vitro. Methods A newly designed perfusion bioreactor was employed for human fetal osteoblasts seeding and culture in porous β-TCP particles. Static seeding was served as the negative control. The effects of different conditions, including the time of seeding, density of cell suspension, flow rate of cell seeding and culture, on cell adhesion and growth were evaluated by the cell viability assay （MTT） and measurement of the cell seeding efficiency, histological and histomorphometric study. Results Perfusion seeding technique was superior to static seeding. Perfusion seeding and culture of fetal cells in β-TCP scaffolds resulted in significantly higher cell viability and growth rates than those by static seeding followed by perfusion culture. The time of seeding, density of cell suspension and flow rate of seeding and culture were determinants of the cell seeding and growth. The optimal conditions for constructing bioartificial bone included the time of seeding between 12 and 24 hours, the density of cell suspension between 2× 10^5/ml and 5× 10^5/ml, the flow rate of seeding I ml/min and the perfusion flow rate for incubation between 0.5 ml/min and 2 ml/min within the first 24 hours and 2 ml/min thereafter. Conclusions The in vitro perfusion technique is effective for seeding and culture of human fetal osteoblasts in porous β- TCP particles. The time of seeding, cell density and flow rate markedly impact on the outcomes of bioartificialbone construction in vitro. The optimization of conditions for bioartificial bone construction may facilitate clinical translation and promotion.

关 键 词：骨代用品 生物反应器 细胞骨架 

分 类 号：R318.17[医药卫生—生物医学工程]