多孔微球包埋rhBMP-2及复合骨髓基质干细胞应用于鼠脊柱融合的实验研究
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摘要
目的:探讨粘附骨髓间充质干细胞和/或包埋rhBMP-2的微球支架对鼠后外侧横突间脊柱融合模型的作用。方法:梯度离心法和差速贴壁法体外分离培大鼠BMSCs;采用复乳发泡法制备聚乳酸-羟基乙酸(PLGA)多孔微球支架,用其包埋rhBMP-2作为缓释载体;同时,用该微球与BMSCs体外共培养,通过荧光染色观察细胞在支架中的生长和分裂增殖状态;以单纯加入rhBMP-2组为对照,对比研究rhBMP-2多孔微球对BMSCs增殖和分化的影响;将rhBMP-2多孔微球单用或联合复合BMSCs的多孔微球用于鼠后外侧横突间脊柱融合模型。结果:梯度离心法和差速贴壁法体外分离培养BMSCs纯度高、细胞损伤小;以PLGA多孔微球为支架材料的体外培养体系适合BMSCs的生长、增殖和分化;体外培养条件下,与单纯加入rhBMP-2组相比,rhBMP-2多孔微球具有更强、更持久的细胞增殖、分化以及成骨效应;单独使用rhBMP-2多孔微球或联合使用复合BMSCs的多孔微球在第4周时就获得了100%的融合率。结论:梯度离心法和差速贴壁法、BMSCs-PLGA多孔微球支架体系以及rhBMP-2微球缓释载体作为骨组织工程领域的研究方法和手段具有良好的推广和应用前景。
[Abstract]Objective:To evaluate the efficacy of bone morphogenetic protein(BMP)-2-microspheres in inducing a posterolateral spine fusion in rat model.Methods:BMSCs were isolated from the bone marrow by density gradient centrifugation and fulling combined with adhering to cuture plastic in different time,Highly open porous biodegradable polymeric microspheres were fabricated for use as scaffold microcarriers for cell and rhBMP-2 delivery.A modified water-in-oil-in-water(W1/O/W2) double emulsion solvent evaporation and gas foaming method was employed for producing the microspheres.Attached cells on the microspheres scaffold were stained with Molecular Probes for cytological observation;BMSCs influenced by rhBMP-2-porous microspheres in cocultured with porous microspheres in vitro studied by the control group of rhBMP-2 adminstered only;Combination or used alone of rhBMP-2-porous microspheres and BMSCs-porous microspheres for posterolateral spine fusion in rats.Results:The method of density gradient centrifugation and fulling combined with adhering to cuture plastic in different time isolating BMSCs from bone marrow is simple,furthermore,the homozygosis of BMSCs is good and the injury of BMSCs is very little;BMSCs are compatible to grow,proliferate and differentiate in these porous microspheres; rhBMP-2-porous microspheres could induce BMSCs resulting in the more persisting and powerful effect of the proliferation,differentiation and bone formation in porous microspheres in vitro;All animals treated with rhBMP-2-porous microspheres alone or combination of BMSCs-porous microspheres demonstrated successful spinal fusion,four weeks postoperatively.Conclusions:The experimental study shows that the method of density gradient centrifugation and fulling combined with adhering to cuture plastic in different time,the porous microspheres for rhBMP-2 and/or BMSCs are the optimal methods and techniques for the construction of the bone tissue engineering.
[Abstract]Objective:To evaluate the efficacy of bone morphogenetic protein(BMP)-2-microspheres in inducing a posterolateral spine fusion in rat model.Methods:BMSCs were isolated from the bone marrow by density gradient centrifugation and fulling combined with adhering to cuture plastic in different time,Highly open porous biodegradable polymeric microspheres were fabricated for use as scaffold microcarriers for cell and rhBMP-2 delivery.A modified water-in-oil-in-water(W1/O/W2) double emulsion solvent evaporation and gas foaming method was employed for producing the microspheres.Attached cells on the microspheres scaffold were stained with Molecular Probes for cytological observation;BMSCs influenced by rhBMP-2-porous microspheres in cocultured with porous microspheres in vitro studied by the control group of rhBMP-2 adminstered only;Combination or used alone of rhBMP-2-porous microspheres and BMSCs-porous microspheres for posterolateral spine fusion in rats.Results:The method of density gradient centrifugation and fulling combined with adhering to cuture plastic in different time isolating BMSCs from bone marrow is simple,furthermore,the homozygosis of BMSCs is good and the injury of BMSCs is very little;BMSCs are compatible to grow,proliferate and differentiate in these porous microspheres; rhBMP-2-porous microspheres could induce BMSCs resulting in the more persisting and powerful effect of the proliferation,differentiation and bone formation in porous microspheres in vitro;All animals treated with rhBMP-2-porous microspheres alone or combination of BMSCs-porous microspheres demonstrated successful spinal fusion,four weeks postoperatively.Conclusions:The experimental study shows that the method of density gradient centrifugation and fulling combined with adhering to cuture plastic in different time,the porous microspheres for rhBMP-2 and/or BMSCs are the optimal methods and techniques for the construction of the bone tissue engineering.
引文
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