Chinese Journal of Tissue Engineering Research ›› 2016, Vol. 20 ›› Issue (47): 6887-7005.doi: 10.3969/j.issn.2095-4344.2016.47.001
Received:
2016-09-01
Online:
2016-11-18
Published:
2016-11-18
Contact:
Wei Kun, Professor, School of Material Science and Engineering, South China University of Technology, Guangzhou 510640, Guangdong Province, China
About author:
Hu Lu, Master, School of Material Science and Engineering, South China University of Technology, Guangzhou 510640, Guangdong Province, China; National Engineering Research Center for Tissue Restoration and Reconstruction, Guangzhou 510006, Guangdong Province, China; Guangdong Provincial Key Laboratory of Biomedical Engineering, Guangzhou 510006, Guangdong Province, China
Supported by:
the Guangdong Provincial Science and Technology Program, No. 2015A020214005
CLC Number:
Hu Lu, Wei Kun, Zou Fen. Construction and characteristics of poly(lactic-co-glycolic acid)/calcium silicate scaffolds with three-dimensional pores for bone tissue engineering[J]. Chinese Journal of Tissue Engineering Research, 2016, 20(47): 6887-7005.
2.1 PLGA/硅酸钙复合微球的组成成分分析 图1中圆点标注的位置依次为(400)、(002)、(-202)、(202)、(320),对照X射线衍射标准JCPDS卡片数据可知,它们是硅酸钙的特征峰,在PLGA/硅酸钙谱图中,硅酸钙的特征峰均存在,说明微球中存在硅酸钙;由图2可以看出,在PLGA/硅酸钙复合微球的谱图中,1 762, 1 170 cm-1附近出现的2个峰分别为酯键和C-O键的特征吸收峰,而3 000,2 956 cm-1附近出现的2个较大的吸收峰,分别为-CH3和-CH2-的伸缩振动吸收,且PLGA的其他特征峰均存在,说明复合微球中含有PLGA;而在1 077,477 cm-1附近出现的2个吸收峰,分别为Si-O-Si键的伸缩振动吸收和弯曲振动吸收[24],1 006,945,891,682,643,567,477 cm-1附近出现的特征吸收峰与β-CaSiO3的谱图基本一致[25-26],说明复合微球中也存在硅酸钙。由于PLGA有机组分的存在,硅酸钙在复合微球中的特征峰没有单一组分的尖锐。"
2.3 硅酸钙组分对PLGA降解pH值的影响 图4比较了PLGA微球和PLGA/硅酸钙复合微球的体外降解介质pH值变化情况,降解实验的前15 d,两组微球的pH值基本维持在7.4左右,且相差不大;15 d后,PLGA微球介质的pH值迅速下降,而PLGA/硅酸钙复合微球的下降较为缓慢;在第40天时,PLGA微球介质的pH值下降至2.6左右,而PLGA/硅酸钙复合微球的仍可维持在6左右,结果表明,硅酸钙组分的加入对PLGA的酸性降解产物有一定的中和作用。图5进一步比较了不同含量硅酸钙对PLGA微球体外降解介质pH值的影响,在降解的前15 d内,20%组和30%组的降解介质pH值都有一个明显的升高然后再降低的过程,而10%组基本维持在7.4左右;15 d后,3组pH值都开始缓慢降低,其中10%组下降最为缓慢;到降解的第60天,10%组的pH值降低至3左右,而20%和30%组的pH值比10%略高0.5。说明硅酸钙对PLGA微球体外降解介质pH值的中和作用主要发生在前15 d内。"
2.4 PLGA三维多孔支架和PLGA/硅酸钙复合多孔支架的结构特征 如图6所示,PLGA/硅酸钙支架的尺寸比PLGA支架略小(图6A);PLGA支架的纤维直径和孔径约为600 μm和800 μm(图6B),而PLGA/硅酸钙支架的纤维直径和支架孔径约为500 μm和750 μm(图6C),均小于PLGA支架,且两种支架的纤维直径均大于所用针头直径0.4 mm,说明在打印过程中出现了纤维挤出胀大现象,低温融合技术制备PLGA/硅酸钙复合多孔支架的过程中,支架尺寸又出现了一定程度的回缩;从支架的扫描电镜图片可以看出,PLGA支架保持良好的三维连通多孔结构(图6D),在PLGA/硅酸钙支架中,微球均匀地分散在支架的孔道之间,同时与支架保持较好的孔隙结构(图6E);在PLGA/硅酸钙支架200倍扫描电镜图中,微球已与支架融合,同时单一微球表面仍良好地保留着多孔形貌(图6F)。"
2.5 Micro-CT构建三维多孔支架模型和孔隙特征 利用Micro-CT对支架扫描重建,得到支架的结构模型和孔径分布图(图7),PLGA支架的平均孔隙率、孔隙连通率及平均孔径分别为31.35%、100%、0.32 mm,PLGA/CS支架的平均孔隙率、孔隙连通率及平均孔径分别为24.09%、99.88%、0.17 mm。由图7可知,两支架的孔隙均完全连通,孔隙在支架中分布较均匀;PLGA支架的孔径主要分布在0.4-0.5 mm,同时还出现较多的小于0.1 mm的孔洞(图7C),这部分小孔可能是由于边缘效应造成的;而在PLGA/CS支架中,由于多孔微球的加入,除了0.4-0.5 mm孔洞外,更多的出现了小于0.2 mm的孔洞(图7D);对两支架的孔隙率、孔隙连通率和平均孔径加以比较发现,PLGA/硅酸钙支架的孔隙率和平均孔径均比PLGA支架小,而两支架的孔隙连通率几乎都为100%。以上结果说明,利用低温融合技术将PLGA/硅酸钙复合多孔微球与PLGA三维多孔支架成功组装成具有完全连通的梯度多级孔结构的复合支架。"
2.7 材料的体外细胞相容性 图9A比较了骨髓间充质干细胞在不同硅酸钙含量PLGA/硅酸钙微球浸提液中的增殖情况,第1-5天,所有组别吸光度一直升高,随着微球中硅酸钙含量的增加,浸提液对细胞增殖率的作用总体呈略微增加趋势,但与不含硅酸钙的PLGA微球比较差异均无显著性意义,说明细胞可以在不同硅酸钙含量的PLGA微球浸提液中正常生长,硅酸钙组分的加入对骨髓间充质干细胞的增殖有一定的促进作用,但效果不显著;图9B为骨髓间充质干细胞在PLGA支架和PLGA/硅酸钙支架上的增殖情况,以传统热烧结法制备的PLGA微球支架作为对照组,由图可知,细胞在3组支架上均可稳定生长,说明3组支架均具有良好的生物相容性;从单个时间点来看,3D-Bioplotter制备PLGA组的细胞数明显高于其他两组,PLGA/硅酸钙组的细胞数也显著高于传统热烧结法制备的PLGA微球支架,说明PLGA三维多孔支架和PLGA/硅酸钙复合多孔支架比传统热烧结法制备的PLGA微球支架更有利于骨髓间充质干细胞的增殖,其原因可能是高的孔隙率、孔隙连通率和较大的孔径更有利于细胞的长入。 "
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