利用干细胞过滤富集器快速构建生物材料的实验研究

Rapid construction of bioactive materials by self-designed enriched filtrator of stem cells

目的通过研究干细胞过滤富集器对人骨髓间充质干细胞（BMSCs）的富集作用，探讨干细胞过滤富集器制备构建生物材料的可行性。方法32例患者骨髓血在干细胞过滤富集器中循环滤过β-磷酸三钙（β-TCP），对比滤过前、后BMSCs的数量，测定富集作用；对比滤过前、后骨髓中有核细胞、红细胞和血小板的数量，检测干细胞过滤富集器对血细胞的影响；通过骨髓样本的细菌学检查，检测干细胞过滤富集器的安全性；应用扫描电镜观察BMSCs在B．TCP上的黏附情况。结果滤过前、后骨髓培养生成的集落形成单位分别为（232．8±128．6）／mL、（37．8±34．9）／mL，差异有统计学意义（t=10．743，P〈0．001）。富集器对BMSCs的富集率为85．6％±8．4％（63．8％，97．1％）。滤过前、后骨髓中有核细胞数量[（16．8±5．4）×109／mL、（16．3±5．0）×109／mL]、红细胞数量[（3．9±0．6）×1012／mL、（3．8±0．6）×1012／mLl和血小板数量[（120．4±54．6）×109／mL、（123．0±51．8）×109／mL]无显著变化，差异无统计学意义（P〉0.05）。滤过前、后的骨髓样本细菌学检测均为阴性；体外成骨诱导培养后，扫描电镜下可见β-TCP上有多量BMSCs集落的生成。结论干细胞过滤富集器能有效富集BMSCs，并一期与β—TCP复合，快速、简便、安全地制备活性生物材料。

Objective To evaluate the efficiency of our self-designed enriched filtrator of stem cells in screening and collecting enriched human bone marrow stem cells （BMSCs） for rapid construction of bioactive materials. Methods Bone marrow blood from 32 patients was circulated in our self-designed enriched filtrator and filtered through beta-tricalcium phosphate（β-TCP） . The numbers of BMSCs were counted pre- and post-filtration to assess the enrichment efficiency. The numbers of nucleated cells, red blood cells and platelets were compared pre- and post-filtration to assess the effects of the enriched filtrator of stem cells on blood cells. Bacteriological examinations were performed to test the safety of the device. Scanning electron microscopy was carried out to observe the adhesion of BMSCs on β-TCP. Results There was a significant difference between the pre-filtration colony number of BMSCs cultured （232. 8 ± 128.6/mL） and the post-fihration one （37.8 ± 34. 9/mL） （ t = 10. 743, P 〈 0. 001）. The efficiency of BMSCs enrichment of the device was 85.6% ± 8.4% （from 63.8% to 97.1% ）. There were no significant differences between pre-fihration and post-filtration in the numbers of nucleated cells [ （16. 8±5.4）×109/mL versus （16.3± 5.0） ×109/roLl, red blood cells [ （3.9 ±0.6）×1012/mL versus （3.8 ＋ 0. 6）×1012/mL] and platelets [ （ 120. 4± 54. 6）×109 /mL versus （ 123.0 ± 51.8 ）×109/mL] （ P 〉 0. 05）. All the bacteriological examinations of the bone marrow samples were negative. After in vitro osteogenic induction culture, formation of numerous BMSCs colonies was observed on the β-TCP by scanning electron microscopy. Conclusion Since our self-designed enriched fihrator can efficiently collect enriched BMSCs and combine them with β-TCP simul- taneously, the device is capable of rapid and safe construction of bioactive materials.