一体化灌注法体外构建活化人工骨的实验研究

Integrated perfusion seeding and culture of human fetal osteoblasts in β-tricalcium phosphate scaffolds for reconstruction of bioartificial bone graft

目的探讨一体化灌注法——灌注接种一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组均明屁高于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尤统计学意义（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±0．054），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剧边均有分布，SSPC组、PSPC组细胞占孔率（18．66％±4．41％、19．34％±3．55％）均明显高于SSSC组（4．07％±1．78％），差异有统汁学意义（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。

Objective Tu investigate the feasibility of integrated cell seeding and cuhure in a perfusion bioreaetor for reconstruction of bioartificial hone grafts in vitro. Methods A perfusion bioreactor system with integrated seeding and long-term cuhure functions was utilized for production of bioartifieial bone constructs. In the perfusion seeding and perfusion euhure （PSPC） group, human fetal osteoblasts were dynamically seeded in polvus β-triealcium phosphate （β-TCP） scaffolds and tbe cell/scaffold eonstrucls were euhured under flow perfusion conditions for 8 days. Cell proliteration and distribution were assessed in terms of daily D-glucose consumption, cell viability （MTT assay）, histologieal evaluation and scanning electron microcopy （SEM） observation as conlpared with the conventional method of static seeding and static： euhure （SSSC） and the method of static seeding and perfusion cuhure （SSPC） . Results The daily glucose consumption increased with time in all tbe 3 groups after cuhure 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 signifieantly higher in PSPC group than in SSPC group after 2, 4 and 6 days of incubation （ P 〈 0.05）, the results of the 8lh clay were similar in both groups （ P 〉 0.05） . The resuhs of cell viability by MTT eolorimetry 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 tbe entire scaffohts by 8 clays of perfusion 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 oceupancy 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.