基于丝素蛋白/壳聚糖的肝组织工程支架材料研究
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摘要
丝素蛋白和壳聚糖均为天然生物材料,原料易得,生物相容性好。但丝素蛋白支架成型困难,抗凝血性较差;壳聚糖支架则降解较快,在水溶液里难以稳定存在。本研究将丝素蛋白与壳聚糖复合,希望通过强烈的氢键作用,改善其理化性能,开发出适合于肝组织工程领域的丝素蛋白/壳聚糖复合支架材料。
丝素蛋白冷冻干燥后易自发形成层片状结构。通过控制温度、浓度、搅拌时间等参数,用冷冻干燥的方法首次成功获得了均质三维多孔的丝素蛋白/壳聚糖支架材料。壳聚糖可以促进丝素蛋白由无规卷曲/α-螺旋向β-折叠的转变,壳聚糖的含量越高,这种转变越明显。对丝素蛋白支架的层片状结构向丝素蛋白/壳聚糖支架的三维多孔结构的转变,提出了丝素蛋白与壳聚糖的自组装模型。
为了考察丝素蛋白/壳聚糖支架的降解性和在水溶液中的稳定性,进行了为期8周的体外降解实验。该支架可以在长达6周的降解时间里保持多孔网络结构,降解速率相对平稳,降解液pH值接近中性,且支架的溶胀性保持稳定。
丝素蛋白/壳聚糖支架的细胞相容性和组织相容性良好。该支架能促进HepG2细胞和原代肝细胞的存活和黏附,可以为细胞提供一个开放的生长环境,促进细胞向支架内部生长,并保证足够的营养和气体交换。支架植入大鼠皮下和大网膜4周的结果表明,其组织相容性较好,仅引起轻微的炎症反应,且体内降解速率适当,在植入4周后仍能保持多孔结构。
在温和的环境下,将肝素引入到丝素蛋白/壳聚糖溶液里,成功制备了含有肝素的丝素蛋白/壳聚糖支架。该支架保持了多孔结构,具有很好的亲水性和力学性能,而且没有细胞毒性。肝素的加入使得丝素蛋白/壳聚糖支架的抗凝血性显著增强,成为可以长期保持肝素活性的有效的肝素缓释系统。
Both silk fibroin (SF) and chitosan (CS) are natural biomaterials with good biocompatibility and abundant resource. It is difficult to prepare pure SF scaffold with porous structure, and the anticoagulant property of SF is poor. Meanwhile, pure CS scaffold degradates relatively quickly, and is not stable in aqueous solution. In this study, aiming to improve the physicochemical properties, we prepared the blend of SF and CS as a water-stable scaffold through intense hydrogen bonding, which may be suitable for liver tissue engineering.
After freeze-drying, pure SF scaffold demonstrated sheet-like structure. Through controlling the temperature, concentration and mixing time, three-dimensional homogeneous porous silk fibroin/chitosan (SFCS) scaffolds were successfully prepared by freeze-drying method. Adding of CS promoted the conformation transition of SF from a random coil/α-helix to aβ-sheet structure. Furthermore, higher content of CS promoted the formation of moreβ-sheet structure. A model about the self-assembly of SF and CS into three-dimensional porous structure has been proposed.
To test the degradation and water-stable properties, in vitro degradation behaviors of SFCS scaffolds have been systematically investigated up to 8 weeks. SFCS scaffolds could maintain its porous structure till 6 weeks of degradation, the degradation rate was relatively stable, the pH value of degradation solution fluctuated in a narrow range near neutrality, and the swelling properties kept steady. SFCS scaffolds demonstrated good cytocompatibility and histocompatibility.
SFCS scaffolds could promote the survival and attachment of HepG2 (human hepatoma cell line) cells and primary hepatocytes. They could provide an open-environment to cells with sufficient nutrition and gas exchange and promote their ingrowth. The in vivo implantation of porous SFCS scaffolds into subcutaneous tissue and omentum of SD rats resulted in slight inflammation and moderate degradation rate. SFCS scaffolds could maintain porous structure after 4 weeks of implantation.
Heparin was added into SFCS scaffolds under mild conditions. SFCS scaffolds containing heparin maintained porous structure, good hydrophilicity and mechanical properties, moreover, they were not cytotoxic. Adding of heparin leads the SFCS scaffold to be blood compatible and an effective heparin delivering system for liver tissue engineering.
丝素蛋白冷冻干燥后易自发形成层片状结构。通过控制温度、浓度、搅拌时间等参数,用冷冻干燥的方法首次成功获得了均质三维多孔的丝素蛋白/壳聚糖支架材料。壳聚糖可以促进丝素蛋白由无规卷曲/α-螺旋向β-折叠的转变,壳聚糖的含量越高,这种转变越明显。对丝素蛋白支架的层片状结构向丝素蛋白/壳聚糖支架的三维多孔结构的转变,提出了丝素蛋白与壳聚糖的自组装模型。
为了考察丝素蛋白/壳聚糖支架的降解性和在水溶液中的稳定性,进行了为期8周的体外降解实验。该支架可以在长达6周的降解时间里保持多孔网络结构,降解速率相对平稳,降解液pH值接近中性,且支架的溶胀性保持稳定。
丝素蛋白/壳聚糖支架的细胞相容性和组织相容性良好。该支架能促进HepG2细胞和原代肝细胞的存活和黏附,可以为细胞提供一个开放的生长环境,促进细胞向支架内部生长,并保证足够的营养和气体交换。支架植入大鼠皮下和大网膜4周的结果表明,其组织相容性较好,仅引起轻微的炎症反应,且体内降解速率适当,在植入4周后仍能保持多孔结构。
在温和的环境下,将肝素引入到丝素蛋白/壳聚糖溶液里,成功制备了含有肝素的丝素蛋白/壳聚糖支架。该支架保持了多孔结构,具有很好的亲水性和力学性能,而且没有细胞毒性。肝素的加入使得丝素蛋白/壳聚糖支架的抗凝血性显著增强,成为可以长期保持肝素活性的有效的肝素缓释系统。
Both silk fibroin (SF) and chitosan (CS) are natural biomaterials with good biocompatibility and abundant resource. It is difficult to prepare pure SF scaffold with porous structure, and the anticoagulant property of SF is poor. Meanwhile, pure CS scaffold degradates relatively quickly, and is not stable in aqueous solution. In this study, aiming to improve the physicochemical properties, we prepared the blend of SF and CS as a water-stable scaffold through intense hydrogen bonding, which may be suitable for liver tissue engineering.
After freeze-drying, pure SF scaffold demonstrated sheet-like structure. Through controlling the temperature, concentration and mixing time, three-dimensional homogeneous porous silk fibroin/chitosan (SFCS) scaffolds were successfully prepared by freeze-drying method. Adding of CS promoted the conformation transition of SF from a random coil/α-helix to aβ-sheet structure. Furthermore, higher content of CS promoted the formation of moreβ-sheet structure. A model about the self-assembly of SF and CS into three-dimensional porous structure has been proposed.
To test the degradation and water-stable properties, in vitro degradation behaviors of SFCS scaffolds have been systematically investigated up to 8 weeks. SFCS scaffolds could maintain its porous structure till 6 weeks of degradation, the degradation rate was relatively stable, the pH value of degradation solution fluctuated in a narrow range near neutrality, and the swelling properties kept steady. SFCS scaffolds demonstrated good cytocompatibility and histocompatibility.
SFCS scaffolds could promote the survival and attachment of HepG2 (human hepatoma cell line) cells and primary hepatocytes. They could provide an open-environment to cells with sufficient nutrition and gas exchange and promote their ingrowth. The in vivo implantation of porous SFCS scaffolds into subcutaneous tissue and omentum of SD rats resulted in slight inflammation and moderate degradation rate. SFCS scaffolds could maintain porous structure after 4 weeks of implantation.
Heparin was added into SFCS scaffolds under mild conditions. SFCS scaffolds containing heparin maintained porous structure, good hydrophilicity and mechanical properties, moreover, they were not cytotoxic. Adding of heparin leads the SFCS scaffold to be blood compatible and an effective heparin delivering system for liver tissue engineering.
引文
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