改性丝素蛋白/纳米羟基磷灰石复合材料
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摘要
天然高分子丝素具有独特的力学性能,优良的生物相容性,极好的吸湿保湿和抗微生物能力,并且来源广泛,其与纳米羟基磷灰石复合,有望克服纯羟基磷灰石力学性能的不足,适用于硬组织尤其是负重骨的修复与替换。但再生丝素蛋白内部大量次级交联点(分子内氢键作用)的存在,导致其变硬变脆,进而造成了羟基磷灰石/丝素复合材料力学性能的不足。目前已有众多研究者通过不同的方法制备了羟基磷灰石/丝素蛋白复合材料,除了颗粒尺寸、复合方式不同之外,也均不可避免的存在力学强度不够的问题。为了弥补该缺陷,本实验致力于减少这种次级交联点,并构建纳米羟基磷灰石针晶在丝素网络中分布的仿生结构的研究。
首先,为了得到最佳的工艺参数,我们在不同反应条件下原位合成了纳米羟基磷灰石(n-HA)/丝素复合材料。通过对合成过程中各种参数的考察,发现pH值高低及热处理过程对复合材料的微观形貌及构象有显著的影响;同时分析了复合材料中,羟基磷灰石晶体与丝素蛋白的结合方式及相互的作用。然后引入定量的交联剂聚乙二醇二缩水甘油醚(polyethylene glycoldiglycidyl ether,简称PGDE),希望其与丝素分子发生交联反应,从而抑制丝素分子内部的氢键作用,减少次级交联点。我们通过透射电镜、X射线衍射、热分析、红外光谱、力学性能测试、扫描电镜等手段对材料进行了检测与表征。
结果表明:本实验中以丝素蛋白、CaCl_2和(NH_4)_2HPO_4等为反应原料用原位共沉淀法成功合成了纳米羟基磷灰石/丝素蛋白复合材料。该复合材料的颗粒尺寸尺寸为100-150nm,HA相在丝素溶液中成功合成,FTIR检测结果证实,该复合材料中无规线团结构的丝素蛋白与羟基磷灰石晶体之间存在着强烈的化学相互作用,而没有与HA相结合的丝素蛋白易溶于水中,在洗涤过程中流失。反应中,pH值对复合材料的颗粒形态、成分及构象均有显著的影响,高pH利于分散性好且纯度高的羟基磷灰石的形成,同时诱导了丝素构象向β折叠转变。
再生丝素蛋白十分脆弱易碎,不易加工成样品作力学性能检测,而加入交联剂后,丝素蛋白力学性能得到明显改善,并且随着交联剂加入量的增加,丝素的柔韧性也越好,但加入量过高,丝素蛋白的拉伸强度会降低。
在纳米羟基磷灰石/丝素蛋白复合材料中,加入交联剂PGDE对丝素蛋白进行改性,可以显著提高该复合材料的力学性能。力学测试数据显示加入交联剂的复合材料拥有更高的抗压强度及弹性模量,这揭示了交联剂对内交联的有效抑制作用,扫描电镜结果反映出加入交联剂的材料内部缺陷明显减少,致密度提高,脆性得到了削弱。PGDE的引入,改变了复合材料颗粒的形貌,促进了针状纳米羟基磷灰石的形成,提高了复合材料中丝素蛋白的结晶性,复合材料的韧性也得到了一定程度的改善。SBF浸泡实验及细胞毒性实验证明,交联剂PGDE的引入并没有降低复合材料的生物相容性。因此PGDE交联改性的纳米羟基磷灰石/丝素蛋白复合材料能较好的满足承载部位骨填充的需求。
Fibroin fibers from well-defined silkworm silk, which have unique mechanical properties and excellent biocompatibility in vitro and in vivo, have been used as biomedical suture material for centuries. The nano-hydroxyapatite/silk fibroin (n-HA/SF) composite is supposed to be a novel biomaterial that is fit for hard tissues even as load-bearing bone implants. However, the regenerated fibroin contains generally lots of molecule inner links, which extremely decreases the tenacity of solid silk fibroin materials. Therefore, the composite n- HA/SF without interface bonding is restricted on mechanical property and hard to meet the requirement of load-bearing application.
In order to improve the weakness, the research is concentrated to reduce the molecule inner links in regenerated fibroin and construct a web structure to guide n-HA formation in the composite. Firstly, a series of composites were prepared under different reaction conditions in order to search optimal technical parameters for synthesizing of HA/SF composite. Secondly, a crosslinking agent named polyethylene glycol diglycidyl ether (PGDE) was adopted to improve the interface between n-HA and SF in the composite. By the connecting reaction between PGDE and SF molecules, the molecule inner links were expected to be reduced. Finally, the microstructure, phase composition and conformation of in-site synthesized composite were determined by Transmission Electron Microscope (TEM), X-Ray Diffraction (XRD), Fourier Transform Infra Red (FTIR) and so on.
Nano-HA/SF composites with the particle size of 100~150nm were prepared by an in situ co-precipitation method, with CaCl_2 and (NH_4)_2HPO_4 as reaction agents for HA. The XRD test showed HA phase was successfully synthesized with the existence of SF. FTIR results validated that the random coil SF has strong chemical interactions with HA in the composites, while the part without bonding with HA is easy to dissolve in water and gets lost. The pH value in the reaction had significant effect on morphologies, components and conformations of HA/SF. At high pH value, nano-HA/SF crystallites formed in high length/diameter ratio and the random coil SF easily convert to 6 sheet structure, while at low pH value additional phase than HA may appear.
The regenerated fibroin is too frangible to test mechanical property. With the adopting of PGDE, its flexility is obviously improved, but superfluous coupling agent will decrease the tensile strength.
PGDE was introduced to the HA/SF composite by in situ co-precipitation method, in order to obtain cross-linked structure by reacting with SF and enhance the interface bonding between n- HA particles and SF matrix. The coupling agent can strengthen the directional growth of HA particles in the composite and decrease the brittleness of solid SF. Various tests have proved that PGDE can improve the crystallization of SF in the composite, simultaneously the mechanical properties of HA/SF was significantly enhanced by 100%. The fracture surface of PGDE coupled composite took on traces of plastic deformation. This reveals the bonds between SF molecules by PGDE can reduce the inner molecule links, which is the main cause of SF's brittleness. And the results of SBF soaking and Cell culture experiments showed PGDE haven't decreased the biocompatibility of composite. So the cross-linked HA/SF by PGDE can meet the requirement of load-bearing application.
首先,为了得到最佳的工艺参数,我们在不同反应条件下原位合成了纳米羟基磷灰石(n-HA)/丝素复合材料。通过对合成过程中各种参数的考察,发现pH值高低及热处理过程对复合材料的微观形貌及构象有显著的影响;同时分析了复合材料中,羟基磷灰石晶体与丝素蛋白的结合方式及相互的作用。然后引入定量的交联剂聚乙二醇二缩水甘油醚(polyethylene glycoldiglycidyl ether,简称PGDE),希望其与丝素分子发生交联反应,从而抑制丝素分子内部的氢键作用,减少次级交联点。我们通过透射电镜、X射线衍射、热分析、红外光谱、力学性能测试、扫描电镜等手段对材料进行了检测与表征。
结果表明:本实验中以丝素蛋白、CaCl_2和(NH_4)_2HPO_4等为反应原料用原位共沉淀法成功合成了纳米羟基磷灰石/丝素蛋白复合材料。该复合材料的颗粒尺寸尺寸为100-150nm,HA相在丝素溶液中成功合成,FTIR检测结果证实,该复合材料中无规线团结构的丝素蛋白与羟基磷灰石晶体之间存在着强烈的化学相互作用,而没有与HA相结合的丝素蛋白易溶于水中,在洗涤过程中流失。反应中,pH值对复合材料的颗粒形态、成分及构象均有显著的影响,高pH利于分散性好且纯度高的羟基磷灰石的形成,同时诱导了丝素构象向β折叠转变。
再生丝素蛋白十分脆弱易碎,不易加工成样品作力学性能检测,而加入交联剂后,丝素蛋白力学性能得到明显改善,并且随着交联剂加入量的增加,丝素的柔韧性也越好,但加入量过高,丝素蛋白的拉伸强度会降低。
在纳米羟基磷灰石/丝素蛋白复合材料中,加入交联剂PGDE对丝素蛋白进行改性,可以显著提高该复合材料的力学性能。力学测试数据显示加入交联剂的复合材料拥有更高的抗压强度及弹性模量,这揭示了交联剂对内交联的有效抑制作用,扫描电镜结果反映出加入交联剂的材料内部缺陷明显减少,致密度提高,脆性得到了削弱。PGDE的引入,改变了复合材料颗粒的形貌,促进了针状纳米羟基磷灰石的形成,提高了复合材料中丝素蛋白的结晶性,复合材料的韧性也得到了一定程度的改善。SBF浸泡实验及细胞毒性实验证明,交联剂PGDE的引入并没有降低复合材料的生物相容性。因此PGDE交联改性的纳米羟基磷灰石/丝素蛋白复合材料能较好的满足承载部位骨填充的需求。
Fibroin fibers from well-defined silkworm silk, which have unique mechanical properties and excellent biocompatibility in vitro and in vivo, have been used as biomedical suture material for centuries. The nano-hydroxyapatite/silk fibroin (n-HA/SF) composite is supposed to be a novel biomaterial that is fit for hard tissues even as load-bearing bone implants. However, the regenerated fibroin contains generally lots of molecule inner links, which extremely decreases the tenacity of solid silk fibroin materials. Therefore, the composite n- HA/SF without interface bonding is restricted on mechanical property and hard to meet the requirement of load-bearing application.
In order to improve the weakness, the research is concentrated to reduce the molecule inner links in regenerated fibroin and construct a web structure to guide n-HA formation in the composite. Firstly, a series of composites were prepared under different reaction conditions in order to search optimal technical parameters for synthesizing of HA/SF composite. Secondly, a crosslinking agent named polyethylene glycol diglycidyl ether (PGDE) was adopted to improve the interface between n-HA and SF in the composite. By the connecting reaction between PGDE and SF molecules, the molecule inner links were expected to be reduced. Finally, the microstructure, phase composition and conformation of in-site synthesized composite were determined by Transmission Electron Microscope (TEM), X-Ray Diffraction (XRD), Fourier Transform Infra Red (FTIR) and so on.
Nano-HA/SF composites with the particle size of 100~150nm were prepared by an in situ co-precipitation method, with CaCl_2 and (NH_4)_2HPO_4 as reaction agents for HA. The XRD test showed HA phase was successfully synthesized with the existence of SF. FTIR results validated that the random coil SF has strong chemical interactions with HA in the composites, while the part without bonding with HA is easy to dissolve in water and gets lost. The pH value in the reaction had significant effect on morphologies, components and conformations of HA/SF. At high pH value, nano-HA/SF crystallites formed in high length/diameter ratio and the random coil SF easily convert to 6 sheet structure, while at low pH value additional phase than HA may appear.
The regenerated fibroin is too frangible to test mechanical property. With the adopting of PGDE, its flexility is obviously improved, but superfluous coupling agent will decrease the tensile strength.
PGDE was introduced to the HA/SF composite by in situ co-precipitation method, in order to obtain cross-linked structure by reacting with SF and enhance the interface bonding between n- HA particles and SF matrix. The coupling agent can strengthen the directional growth of HA particles in the composite and decrease the brittleness of solid SF. Various tests have proved that PGDE can improve the crystallization of SF in the composite, simultaneously the mechanical properties of HA/SF was significantly enhanced by 100%. The fracture surface of PGDE coupled composite took on traces of plastic deformation. This reveals the bonds between SF molecules by PGDE can reduce the inner molecule links, which is the main cause of SF's brittleness. And the results of SBF soaking and Cell culture experiments showed PGDE haven't decreased the biocompatibility of composite. So the cross-linked HA/SF by PGDE can meet the requirement of load-bearing application.
引文
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