摘要目的 探讨一体化灌注法——灌注接种-灌注培养体外构建活化人工骨的效果. 方法 以多孔β-磷酸三钙(β-TCP)支架为载体,采用自行设计的灌注式生物反应器进行人胚成骨细胞的PSPC,并设静态接种-静态培养(sssc)和静态接种-灌注培养(SSPC)为对照组,通过葡萄糖日耗量、细胞活力测定、扫描电镜(SEM)、组织学观察与形态计量分析等检测方法,比较3种构建方法的细胞增殖和分布情况. 结果 培养2、4、6、8d,3组细胞葡萄糖日耗量均随培养时间的延长而增加,SSPC组和PSPC组均明显高于SSSC组,差异有统计学意义(P＜0.05),培养6d内PSPC组明显高于SSPC组,差异有统计学意义(P＜0.05),培养8d时两组葡萄糖日耗量差异无统计学意义(P＞0.05).SSPC组(1.597±0.103)和PSPC组(1.668±0.129)细胞活力差异无统计学意义(P＞0.05),但均明显高于SSSC组(0.347±0.034),差异有统计学意义(P＜0.05).SEM及组织学观察显示SSSC组细胞仅分布在支架周缘,而SSPC组和PSPC组细胞在支架内部及周边均有分布,SSPC组、PSPC组细胞占孑L率(18.66％±4.41％、19.34％±3.55％)均明显高于SSSC组(4.07％±1.78％),差异有统计学意义(p＜0.05). 结论 一体化灌注法体外构建活化人工骨的效果优于传统的静态方法；与静态接种-灌注培养相比,一体化灌注法可显著促进培养初期细胞增殖从而加快组织构建速度.

abstractsObjective To investigate the feasibility of integrated cell seeding and culture in a perfusion bioreactor for reconstruction of bioartificial bone grafts in vitro.Methods A perfusion bioreactor system with integrated seeding and long-term culture functions was utilized for production of bioartificial bone constructs.In the perfusion seeding and perfusion culture (PSPC) group,human fetal osteoblasts were dynamically seeded in porous β-tricalcium phosphate (β-TCP) scaffolds and the cell/scaffold constructs were cultured under flow perfusion conditions for 8 days.Cell proliferation and distribution were assessed in terms of daily D-glucose consumption,cell viability (MTI assay),histological evaluation and scanning electron microcopy (SEM) observation as compared with the conventional method of static seeding and static culture (SSSC) and the method of static seeding and perfusion culture (SSPC).Results The daily glucose consumption increased with time in all the 3 groups after culture for 2,4,6 and 8 days,but it was much higher in the SSPC and PSPC groups than in the SSSC group (P ＜ 0.05).Although the daily glucose consumption was significantly higher in PSPC group than in SSPC group after 2,4 and 6 days of incubation (P ＜0.05),the results of the 8th day were similar in both groups (P ＞ 0.05).The results of cell viability by MIT colorimetry after 8 days of incubation showed no significant difference between the SSPC and PSPC groups (1.597±0.103 versus 1.668±0.129) but significant differences between them and the SSSC group (0.347 ±0.034) (both P ＜ 0.05).SEM and histological analyses showed that cells were distributed throughout the entire scaffolds by 8 days of peffusion culture in both the SSPC and PSPC groups whereas they were located only along the scaffold perimeters in the SSSC group.The histomorphometry showed the rates of cellular porous occupancy in the SSPC (18.66％ ±4.41％) and PSPC groups (19.34％ ± 3.55％) were significantly higher than in the SSSC group (4.07％ ± 1.78％) (P ＜ 0.05).Conclusions The PSPC method may be superior to the SSSC and SSPC methods in reconstruction of bioartificial bone grafts,because the perfusion seeding can result in a high efficiency and a uniform distribution of cells throughout a scaffold,accelerating engineered tissue reconstruction.Moreover,this superiority may become more obvious as the size and porosity volume of a scaffold increase.