丝蛋白及其复合材料的制备
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摘要
蚕丝因具有特殊的光泽、透气性好、吸湿性强、手感好及高强度等优点而一直应用于纺织领域。丝蛋白主要来源于家蚕茧丝,具有良好的生物相容性和可调整的生物降解性能,目前已经被广泛地应用于组织工程领域。
本文采用定向冷冻法(冰模板法)结合冷冻干燥法制备了孔洞具有一定方向性的丝蛋白多孔支架,由于在定向冷冻过程中冰的剪切力和体积膨胀效应未能引起丝蛋白的构象变化,因此使用甲醇进行后继处理使其不溶于水。我们采用了扫描电镜、流变、拉曼光谱和压缩测试等方法,考察了丝蛋白原液在不同pH值和初始浓度的情况下对定向支架的成因、内部形貌及力学性能的影响。结果表明,当溶液的pH值为4.4,浓度由低到高时,所制备的丝蛋白支架内部将分别出现纤维状结构、轴向片层状和梭状多孔结构。特别是由初始浓度为15%(及以上)的丝蛋白溶液定向冷冻得到的支架,在压缩试验中表现出较好的力学性能,其轴向压缩模量和屈服应力分别达122.6 MPa和6.9 MPa,满足了进一步应用的基本需求。
自然界中软体动物的壳和脊椎动物的骨骼及牙齿等生物矿物由于其优异的机械性能日益成为材料学研究的热点。脊椎动物的骨骼是由有机的胶原蛋白和无机的羟基磷灰石复合而成。作为动物骨的主要无机成分,羟基磷灰石具有良好的生物相容性和生物活性和较好的力学强度,但是由于缺乏足够的韧性使其的医用价值受到一定的限制。而作为有机物的丝蛋白材料具有可靠的力学性能和生物相容性,在某种程度上可以弥补羟基磷灰石的缺点。因此,结合两者的优点,所制备的羟基磷灰石／丝蛋白复合材料有可能在骨修复领域中具有一定的实际意义。
由于羟基磷灰石微溶于纯水,因此使用传统的机械共混的方法一般不能使羟基磷灰石和丝蛋白混合均匀(羟基磷灰石趋向于形成大块的聚集体)。考虑到丝蛋白链段中含有天冬氨酸等酸性氨基酸能够诱导无机矿物的沉积,在本文中,我们改进了共混的步骤,将羟基磷灰石先分散在再生丝蛋白的水溶液中,采用动态光散射对所制得的白色乳状液体进行分析的结果表明,与羟基磷灰石水溶液相比,丝蛋白溶液中无机物颗粒分散得比较均匀,粒径也较小。在得到了分散性良好的羟基磷灰石和丝蛋白复合支架材料的基础上,我们对其性能进行了初步的研究,发现多孔复合材料中均匀分散于丝蛋白基质相约2.5%左右的羟基磷灰石颗粒对于材料的压缩模量及屈服强度均有一定的贡献,材料的模量由纯丝蛋白的6.8 MPa提高到了35.9 MPa,屈服强度由0.58 MPa提高到了0.93 MPa。
此外,采用高浓度再生丝蛋白溶液浓缩方法,获得了浓度为8-20 wt%的再生丝蛋白浓溶液。通过与无机物离子溶液的共混,同时利用正丁醇调控丝蛋白溶液的构象并结合低温冷冻,制备了无机物含量较高的再生丝蛋白/磷酸钙(主要为透钙磷石晶型)多孔复合材料,并且支架中无机物晶体的形貌随其含量而发生变化；同时,样品的模量和屈服强度随着样品中无机物含量的增多而增大。在丝蛋白浓度为10%,加入的氯化钙和磷酸氢二钠浓度为0.20 mol/L,丝蛋白溶液与正丁醇体积为1：1时,多孔材料的模量可达40.1 MPa,屈服强度为0.58 MPa。
Natural animal silks, mainly from Bombyx mori silkworm, have been in practically used by human beings for centuries because of its unique luster, fineness, and mechanical properties. In recent years, extensive studies have been carried on the biomedical application of regenerated silk fibroin (RSF), which is the main component of silk protein.
In the present work, the directional freezing followed by freeze-drying techniques was used to successfully prepare RSF scaffolds with ordered porous morphology and considerable mechanical properties. In order to investigate pH and concentration effect of the initial RSF solution on the morphology and mechanical properties of the scaffold, various techniques including the scanning electron microscopy (SEM), rheology, Raman spectroscopy and mechanical test were employed. The results show that the morphology of the scaffold changes from fibrils to lamellar structures and then to cellular structures as the initial concentration of RSF solution (at pH 4.4) increases. The RSF scaffolds made by the 15 wt% of RSF aqueous solution or above show good mechanical properties, which beard axial compression modulus of 122.6 MPa and the yield strength of 6.9 MPa, receptively. This would make the directional RSF scaffolds possible for further applications.
The biomaterials, such as shell of mollusk, bone and teeth of vertebrate, are well-known for their outstanding mechanical properties, which have been the popular field of material science. The bone of vertebrate is a composite consociated of collagen and hydroxyapatite (HAP). As he main component of bones and teeth, HAP has potential applications in the replacement of hard tissues in medical field due to its bioactivity and biocompatibility, however, it also has drawbacks such as fragile and brittle. Therefore, after combination of HAP with silk protein, the mechanical properties of the composite may be greatly improved. The porous RSF/HAP hybrid scaffold can be used to heal bone defects.
Since the suspension of HAP is limited in the water, the traditional method to mix HAP and RSF would result in the congeries of HAP in the solution and than in the obtained material. Considering the Asp and Glu residue which contains side group of -COO- in silk fibroin, it is expected that the silk fibroin would combine with Ca2+ to induce the deposition of mineral. Therefore, a new method for preparation of RSF/HAP hybrid material was developed in our experiments recently. We modified the mixed procedure of HAP and RSF solution and found that the suspension of HAP particles in the solution was much better than that in water, by the DLS. The morphology of the material was observed by SEM. It showed that the HAP was well dispersed and 2.5% of HAP is more than enough to provide a good contribution to the compression performances of the hybrid materials. The hybrid materials beard compression modulus of 35.9 MPa and yield stress of 0.93 MPa, while the pure RSF scaffold showed 6.8 MPa and 0.58 MPa, respectively.
Moreover, RSF/calcium phosphate composite scaffold was fabricated by integrating conformational transition of regenerated silk fibroin with inorganic ions and n-butanol. After characterization on the morphology of the composite, the crystallization of calcium phosphate (mainly dicalcium phosphate dehydrate) as well as its different shapes were further discussed, and some of the mechanical properties of the products obtained were measured. The study on the hybrid materials showed that the compression modulus and yield stress of the composite were proportion to the amounts of the mineral. The hybrid scaffold represented compression modulus of 40.1 MPa and yield stress of 0.58 MPa, when the concentration of RSF solution was 10%, the concentration of CaCl2 and Na2HPO4 solutions were 0.20 mol/L, and the volume ratio of RSF and n-butanol was 1:1.
本文采用定向冷冻法(冰模板法)结合冷冻干燥法制备了孔洞具有一定方向性的丝蛋白多孔支架,由于在定向冷冻过程中冰的剪切力和体积膨胀效应未能引起丝蛋白的构象变化,因此使用甲醇进行后继处理使其不溶于水。我们采用了扫描电镜、流变、拉曼光谱和压缩测试等方法,考察了丝蛋白原液在不同pH值和初始浓度的情况下对定向支架的成因、内部形貌及力学性能的影响。结果表明,当溶液的pH值为4.4,浓度由低到高时,所制备的丝蛋白支架内部将分别出现纤维状结构、轴向片层状和梭状多孔结构。特别是由初始浓度为15%(及以上)的丝蛋白溶液定向冷冻得到的支架,在压缩试验中表现出较好的力学性能,其轴向压缩模量和屈服应力分别达122.6 MPa和6.9 MPa,满足了进一步应用的基本需求。
自然界中软体动物的壳和脊椎动物的骨骼及牙齿等生物矿物由于其优异的机械性能日益成为材料学研究的热点。脊椎动物的骨骼是由有机的胶原蛋白和无机的羟基磷灰石复合而成。作为动物骨的主要无机成分,羟基磷灰石具有良好的生物相容性和生物活性和较好的力学强度,但是由于缺乏足够的韧性使其的医用价值受到一定的限制。而作为有机物的丝蛋白材料具有可靠的力学性能和生物相容性,在某种程度上可以弥补羟基磷灰石的缺点。因此,结合两者的优点,所制备的羟基磷灰石／丝蛋白复合材料有可能在骨修复领域中具有一定的实际意义。
由于羟基磷灰石微溶于纯水,因此使用传统的机械共混的方法一般不能使羟基磷灰石和丝蛋白混合均匀(羟基磷灰石趋向于形成大块的聚集体)。考虑到丝蛋白链段中含有天冬氨酸等酸性氨基酸能够诱导无机矿物的沉积,在本文中,我们改进了共混的步骤,将羟基磷灰石先分散在再生丝蛋白的水溶液中,采用动态光散射对所制得的白色乳状液体进行分析的结果表明,与羟基磷灰石水溶液相比,丝蛋白溶液中无机物颗粒分散得比较均匀,粒径也较小。在得到了分散性良好的羟基磷灰石和丝蛋白复合支架材料的基础上,我们对其性能进行了初步的研究,发现多孔复合材料中均匀分散于丝蛋白基质相约2.5%左右的羟基磷灰石颗粒对于材料的压缩模量及屈服强度均有一定的贡献,材料的模量由纯丝蛋白的6.8 MPa提高到了35.9 MPa,屈服强度由0.58 MPa提高到了0.93 MPa。
此外,采用高浓度再生丝蛋白溶液浓缩方法,获得了浓度为8-20 wt%的再生丝蛋白浓溶液。通过与无机物离子溶液的共混,同时利用正丁醇调控丝蛋白溶液的构象并结合低温冷冻,制备了无机物含量较高的再生丝蛋白/磷酸钙(主要为透钙磷石晶型)多孔复合材料,并且支架中无机物晶体的形貌随其含量而发生变化；同时,样品的模量和屈服强度随着样品中无机物含量的增多而增大。在丝蛋白浓度为10%,加入的氯化钙和磷酸氢二钠浓度为0.20 mol/L,丝蛋白溶液与正丁醇体积为1：1时,多孔材料的模量可达40.1 MPa,屈服强度为0.58 MPa。
Natural animal silks, mainly from Bombyx mori silkworm, have been in practically used by human beings for centuries because of its unique luster, fineness, and mechanical properties. In recent years, extensive studies have been carried on the biomedical application of regenerated silk fibroin (RSF), which is the main component of silk protein.
In the present work, the directional freezing followed by freeze-drying techniques was used to successfully prepare RSF scaffolds with ordered porous morphology and considerable mechanical properties. In order to investigate pH and concentration effect of the initial RSF solution on the morphology and mechanical properties of the scaffold, various techniques including the scanning electron microscopy (SEM), rheology, Raman spectroscopy and mechanical test were employed. The results show that the morphology of the scaffold changes from fibrils to lamellar structures and then to cellular structures as the initial concentration of RSF solution (at pH 4.4) increases. The RSF scaffolds made by the 15 wt% of RSF aqueous solution or above show good mechanical properties, which beard axial compression modulus of 122.6 MPa and the yield strength of 6.9 MPa, receptively. This would make the directional RSF scaffolds possible for further applications.
The biomaterials, such as shell of mollusk, bone and teeth of vertebrate, are well-known for their outstanding mechanical properties, which have been the popular field of material science. The bone of vertebrate is a composite consociated of collagen and hydroxyapatite (HAP). As he main component of bones and teeth, HAP has potential applications in the replacement of hard tissues in medical field due to its bioactivity and biocompatibility, however, it also has drawbacks such as fragile and brittle. Therefore, after combination of HAP with silk protein, the mechanical properties of the composite may be greatly improved. The porous RSF/HAP hybrid scaffold can be used to heal bone defects.
Since the suspension of HAP is limited in the water, the traditional method to mix HAP and RSF would result in the congeries of HAP in the solution and than in the obtained material. Considering the Asp and Glu residue which contains side group of -COO- in silk fibroin, it is expected that the silk fibroin would combine with Ca2+ to induce the deposition of mineral. Therefore, a new method for preparation of RSF/HAP hybrid material was developed in our experiments recently. We modified the mixed procedure of HAP and RSF solution and found that the suspension of HAP particles in the solution was much better than that in water, by the DLS. The morphology of the material was observed by SEM. It showed that the HAP was well dispersed and 2.5% of HAP is more than enough to provide a good contribution to the compression performances of the hybrid materials. The hybrid materials beard compression modulus of 35.9 MPa and yield stress of 0.93 MPa, while the pure RSF scaffold showed 6.8 MPa and 0.58 MPa, respectively.
Moreover, RSF/calcium phosphate composite scaffold was fabricated by integrating conformational transition of regenerated silk fibroin with inorganic ions and n-butanol. After characterization on the morphology of the composite, the crystallization of calcium phosphate (mainly dicalcium phosphate dehydrate) as well as its different shapes were further discussed, and some of the mechanical properties of the products obtained were measured. The study on the hybrid materials showed that the compression modulus and yield stress of the composite were proportion to the amounts of the mineral. The hybrid scaffold represented compression modulus of 40.1 MPa and yield stress of 0.58 MPa, when the concentration of RSF solution was 10%, the concentration of CaCl2 and Na2HPO4 solutions were 0.20 mol/L, and the volume ratio of RSF and n-butanol was 1:1.
引文
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