Ⅰ聚丙烯酸（钠盐）调控碳酸钙结晶机制及其复合材料的制备 Ⅱ再生丝素蛋白修饰聚丙烯补片及其应用的初步探索
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摘要
自然界中贝壳等生物材料由于其优异的机械性能日益成为材料学研究的热点。软体动物的贝壳由碳酸钙和有机基质(由几丁质和类丝素蛋白组装形成)构成,其中碳酸钙以方解石或文石等形式存在。在酸性蛋白等生物分子的调控下,贝壳的珍珠质中形成了由厚度为0.5-1.0μm的文石层和厚度为50-100 nm的有机基质层构成的复合材料。珍珠质的层状结构使其具有极其优异的机械性能,成为高性能复合材料制备的蓝本。研究珍珠质中文石的形成过程以及酸性蛋白和有机基质等生物分子在文石形成过程中的调控作用,对于模拟珍珠质的结构人工制备高性能复合材料具有重要意义。
本论文首先以再生丝素蛋白膜为有机基质,利用分子量为2000的聚丙烯酸(以下称PAA 2kDa)作为碳酸钙结晶的调控剂,在碳酸钙饱和溶液中制备了碳酸钙/再生丝素蛋白复合材料。研究发现:(1)当PAA 2kDa的浓度控制在5-200μg/ml时,可以在再生丝素蛋白膜表面形成连续而平整的碳酸钙薄膜。当PAA 2kDa的浓度高于20μg/ml时,碳酸钙薄膜主要由方解石晶体组成。(2)在体系中加入高浓度的镁离子(Ca~(2+)/Mg~(2+)=6/1)并结合20μg/ml PAA2kDa的调控作用,可以在再生丝素蛋白膜表面形成连续、平整而光滑的文石薄膜。重复进行再生丝素蛋白成膜/碳酸钙结晶,可以制备类似于珍珠层结构的文石/再生丝素蛋白复合材料。
为了研究聚丙烯酸或其钠盐对碳酸钙结晶的调控作用,我们将不同分子量的聚丙烯酸或其钠盐加入碳酸钙饱和溶液中,研究其对碳酸钙结晶过程的影响。研高于100μg/ml时改变形成的碳酸钙晶体的形貌。(2)中等分子量的聚丙烯酸钠(如PAAs 5.1kDa、PAAs 8kDa和PAAs 15kDa)可以诱导形成直径为0.1-3.0μm的文石复合微球。(3)高分子量的聚丙烯酸钠(如PAAs 100kDa)可以诱导形成直径为0.4-1.5μm的无定形碳酸钙复合微球。动态光散射和XRD等研究发现:文石和无定形碳酸钙复合微球由直径为400-600nm的无定形碳酸钙粒子聚集形成,碳酸钙粒子聚集过程中不同分子量的聚丙烯酸钠盐对无定形碳酸钙结晶抑制能力不同,中等分子量的聚丙烯酸钠不能完全抑制无定形碳酸钙的结晶,但可以稳定文石的形成,最终形成文石复合微球;而高分子量的聚丙烯酸钠可以完全抑制碳酸钙的结晶,最终形成无定形碳酸钙复合微球。
腹股沟部位的切口疝是腹部手术的常见并发症,采用腹腔镜技术可以将切口疝无张力修复手术的复发率降至5%以下。无张力修复手术一般采用聚丙烯补片(以下称PP mesh)作为修补材料,取得了良好的治疗效果。但PP mesh的使用易引起严重的组织粘连,可能引发肠道损伤、坏死、瘘管的形成等严重的并发症。我们采用低温等离子体对PP mesh进行处理,提高其亲水性。再通过再生丝素蛋白溶液浸涂,对PP mesh进行修饰。实验发现,对PP mesh通过两分钟的等离子体处理,再生丝素蛋白在其表面浸涂效果良好。初步的动物试验表明,通过再生丝素蛋白对PP mesh进行表面修饰,可以有效地降低其在Wistar rat腹壁修复手术中与网膜和腹腔内小肠等脏器间的粘连。通过低温等离子体对PP mesh进行单面处理,再通过再生丝素蛋白浸涂,可以制备单面再生丝素蛋白修饰的PPmesh,可能在腹股沟切口疝的无张力修复手术中有重要的应用。
The biomaterials,such as shell of mullosc are well-known for their outstanding mechanical propertites,which have been popular field of material science.Shell of mullosc is constituted of calcium carbonate and organic matrix,and the organic matrix is assembled from chitin and silk-fibroin-like protein.With the control of some biological macromolecules such as acidic proteins,calcium carbonate in shell of mollusc exists as calcite,aragonite and amorphous calcium carbonate,etc.The layered structure of nacre in shell of mullosc is formed from brick and motar,the brick is flat crystal of aragonite,the thickness of which is less than 1.0μm.The aragonite crystals show a well defined preferred orientation,which are separated by a 50-100 nm thick interlayer of organic biomacromolecules.The fine structure of nacre in shell of mollusk results in the unique properties and become a nice example for designing of the high mechanical hybrid biomaterials.It is very important for us to study on the forming process of flat aragonite layer in the nacre of mullosc shell and the mediated functions in the crystallization of calcium carbonate associated with the organic matrix and acidic proteins,in order to biomimetic synthese of the high mechanical hybrid composite on the basis of mimicking the structure and forming process of nacre.
The hybrid composite of calcium carbonate/regenerated silk fibroin was prepared successfully in supersaturated solution of calcium carbonated with polyacrylic acid (Mw 2000,as PAA 2kDa below) as crystallization mediator and regenerated silk fibroin membrane as organic matrix.It revealed that with control of the concertration of PAA 2kDa in the range of 5-200μg/ml,the continuous flat and smooth film of calcium carbonate is formed on the surface of regernated silk fibroin membrane, which is composed of calcite within the concentration of PAA 2kDa above 20μg/ml. The continuous flat and smooth film of aragonite on the surface of regenerated silk fibroin membrane could be synthesized with the mediated function of 20μg/ml PAA 2kDa and concentrated solution of Mg~(2+) ions(Mg~(2+)/Ca~(2+)=6/1),which resembles to the layer of aragonite crystal in the nacre of mollusc shell.
Poly(acrylic acid) or its sodium salt with different Mw is added into the supersaturated solution of calcium carbonate as substitute of acidic proteins to study their mediated functions in the crystallization of calcium carbonate.It is concluded that:(1) The crystallization of calcium carbonate is prohibited by lower Mw PAA, such as PAA 2kDa.The distorted crystals of calcium carbonate were obtained with the concentration of PAA 2kDa above 100μg/ml.(2) The formation of calcium carbonate hybrid micro-spheres the diameter of which is about 0.1-3.0μm can be induced by the medium Mw sodium polyacrylate(such as PAAs 5.1kDa,PAAs 8kDa and PAAs 15kDa).The inorganic mineral in the hybrid micro-spheres existed as aragonite under the control of sodium polyacrylate with high concentration.(3) The hybrid micro-spheres of amorphous calcium carbonate were formed under the control of high Mw sodium polyacrylate(such as PAAs 100kDa),the diameter of which is in the range of 0.4-1.5μm.The crystallization of calcium carbonate in the hybrid micro-spheres is inhibited entirely by PAAs 100kDa with high concentration(above 100μg/ml).Dynamic light scatter and XRD investigations of the mechanics of formation of hybrid micro-spheres of calcium carbonate revealed that the hybrid micro-spheres are formed from aggregation of amorphous calcium carbonate particles with diameter of 400-600 nm.The difference of inhibiting the amorphous calcium carbonate from crystallization under sodium sodium polyacrylate with different Mw resulted in the formation of amorphous calcium carbonate or aragonite hybrid micro-spheres.
The inguinal incisional hernias occur in 5 to 20%of patitents after abdominal surgery.The introduction of tension-free incisional hernias repair by laparoscopic technology has reduced the recurrence rate from up to 50%to less than 5%.Due to its good mechanical properties,polypropylene mesh has been proved to be a suitable prosthetic material in hernias repair.However,it can induce the serious adhesion between polypropylene mesh and bowel,omentum or the other viscera,which results in severe complications,such as bowel erosion,intestinal obstruction and enterocutaneous fistulas,etc.In the disseration,the surface of PP mesh was treated by plasma at low temperature to improve its hydrophilicity and then coated in the solution of regenerated silk fibroin.The primary animal test revealed that the adhesion between polypropylene mesh and omentum,bowel and the other viscera in the abdominal wall repair of Wistar rat is significantly decreased with polypropylene mesh modified by regenerated silk fibroin.The dual-side polypropylene mesh of maximizing peritoneal in -growth on one side while mimizing visceral adhesion on the other side modified by regenerated silk fibroin can be fabricated by treating polypropylene mesh with plasma and coating on one side,which can be used in the inguinal incisional hernias repair.
本论文首先以再生丝素蛋白膜为有机基质,利用分子量为2000的聚丙烯酸(以下称PAA 2kDa)作为碳酸钙结晶的调控剂,在碳酸钙饱和溶液中制备了碳酸钙/再生丝素蛋白复合材料。研究发现:(1)当PAA 2kDa的浓度控制在5-200μg/ml时,可以在再生丝素蛋白膜表面形成连续而平整的碳酸钙薄膜。当PAA 2kDa的浓度高于20μg/ml时,碳酸钙薄膜主要由方解石晶体组成。(2)在体系中加入高浓度的镁离子(Ca~(2+)/Mg~(2+)=6/1)并结合20μg/ml PAA2kDa的调控作用,可以在再生丝素蛋白膜表面形成连续、平整而光滑的文石薄膜。重复进行再生丝素蛋白成膜/碳酸钙结晶,可以制备类似于珍珠层结构的文石/再生丝素蛋白复合材料。
为了研究聚丙烯酸或其钠盐对碳酸钙结晶的调控作用,我们将不同分子量的聚丙烯酸或其钠盐加入碳酸钙饱和溶液中,研究其对碳酸钙结晶过程的影响。研高于100μg/ml时改变形成的碳酸钙晶体的形貌。(2)中等分子量的聚丙烯酸钠(如PAAs 5.1kDa、PAAs 8kDa和PAAs 15kDa)可以诱导形成直径为0.1-3.0μm的文石复合微球。(3)高分子量的聚丙烯酸钠(如PAAs 100kDa)可以诱导形成直径为0.4-1.5μm的无定形碳酸钙复合微球。动态光散射和XRD等研究发现:文石和无定形碳酸钙复合微球由直径为400-600nm的无定形碳酸钙粒子聚集形成,碳酸钙粒子聚集过程中不同分子量的聚丙烯酸钠盐对无定形碳酸钙结晶抑制能力不同,中等分子量的聚丙烯酸钠不能完全抑制无定形碳酸钙的结晶,但可以稳定文石的形成,最终形成文石复合微球;而高分子量的聚丙烯酸钠可以完全抑制碳酸钙的结晶,最终形成无定形碳酸钙复合微球。
腹股沟部位的切口疝是腹部手术的常见并发症,采用腹腔镜技术可以将切口疝无张力修复手术的复发率降至5%以下。无张力修复手术一般采用聚丙烯补片(以下称PP mesh)作为修补材料,取得了良好的治疗效果。但PP mesh的使用易引起严重的组织粘连,可能引发肠道损伤、坏死、瘘管的形成等严重的并发症。我们采用低温等离子体对PP mesh进行处理,提高其亲水性。再通过再生丝素蛋白溶液浸涂,对PP mesh进行修饰。实验发现,对PP mesh通过两分钟的等离子体处理,再生丝素蛋白在其表面浸涂效果良好。初步的动物试验表明,通过再生丝素蛋白对PP mesh进行表面修饰,可以有效地降低其在Wistar rat腹壁修复手术中与网膜和腹腔内小肠等脏器间的粘连。通过低温等离子体对PP mesh进行单面处理,再通过再生丝素蛋白浸涂,可以制备单面再生丝素蛋白修饰的PPmesh,可能在腹股沟切口疝的无张力修复手术中有重要的应用。
The biomaterials,such as shell of mullosc are well-known for their outstanding mechanical propertites,which have been popular field of material science.Shell of mullosc is constituted of calcium carbonate and organic matrix,and the organic matrix is assembled from chitin and silk-fibroin-like protein.With the control of some biological macromolecules such as acidic proteins,calcium carbonate in shell of mollusc exists as calcite,aragonite and amorphous calcium carbonate,etc.The layered structure of nacre in shell of mullosc is formed from brick and motar,the brick is flat crystal of aragonite,the thickness of which is less than 1.0μm.The aragonite crystals show a well defined preferred orientation,which are separated by a 50-100 nm thick interlayer of organic biomacromolecules.The fine structure of nacre in shell of mollusk results in the unique properties and become a nice example for designing of the high mechanical hybrid biomaterials.It is very important for us to study on the forming process of flat aragonite layer in the nacre of mullosc shell and the mediated functions in the crystallization of calcium carbonate associated with the organic matrix and acidic proteins,in order to biomimetic synthese of the high mechanical hybrid composite on the basis of mimicking the structure and forming process of nacre.
The hybrid composite of calcium carbonate/regenerated silk fibroin was prepared successfully in supersaturated solution of calcium carbonated with polyacrylic acid (Mw 2000,as PAA 2kDa below) as crystallization mediator and regenerated silk fibroin membrane as organic matrix.It revealed that with control of the concertration of PAA 2kDa in the range of 5-200μg/ml,the continuous flat and smooth film of calcium carbonate is formed on the surface of regernated silk fibroin membrane, which is composed of calcite within the concentration of PAA 2kDa above 20μg/ml. The continuous flat and smooth film of aragonite on the surface of regenerated silk fibroin membrane could be synthesized with the mediated function of 20μg/ml PAA 2kDa and concentrated solution of Mg~(2+) ions(Mg~(2+)/Ca~(2+)=6/1),which resembles to the layer of aragonite crystal in the nacre of mollusc shell.
Poly(acrylic acid) or its sodium salt with different Mw is added into the supersaturated solution of calcium carbonate as substitute of acidic proteins to study their mediated functions in the crystallization of calcium carbonate.It is concluded that:(1) The crystallization of calcium carbonate is prohibited by lower Mw PAA, such as PAA 2kDa.The distorted crystals of calcium carbonate were obtained with the concentration of PAA 2kDa above 100μg/ml.(2) The formation of calcium carbonate hybrid micro-spheres the diameter of which is about 0.1-3.0μm can be induced by the medium Mw sodium polyacrylate(such as PAAs 5.1kDa,PAAs 8kDa and PAAs 15kDa).The inorganic mineral in the hybrid micro-spheres existed as aragonite under the control of sodium polyacrylate with high concentration.(3) The hybrid micro-spheres of amorphous calcium carbonate were formed under the control of high Mw sodium polyacrylate(such as PAAs 100kDa),the diameter of which is in the range of 0.4-1.5μm.The crystallization of calcium carbonate in the hybrid micro-spheres is inhibited entirely by PAAs 100kDa with high concentration(above 100μg/ml).Dynamic light scatter and XRD investigations of the mechanics of formation of hybrid micro-spheres of calcium carbonate revealed that the hybrid micro-spheres are formed from aggregation of amorphous calcium carbonate particles with diameter of 400-600 nm.The difference of inhibiting the amorphous calcium carbonate from crystallization under sodium sodium polyacrylate with different Mw resulted in the formation of amorphous calcium carbonate or aragonite hybrid micro-spheres.
The inguinal incisional hernias occur in 5 to 20%of patitents after abdominal surgery.The introduction of tension-free incisional hernias repair by laparoscopic technology has reduced the recurrence rate from up to 50%to less than 5%.Due to its good mechanical properties,polypropylene mesh has been proved to be a suitable prosthetic material in hernias repair.However,it can induce the serious adhesion between polypropylene mesh and bowel,omentum or the other viscera,which results in severe complications,such as bowel erosion,intestinal obstruction and enterocutaneous fistulas,etc.In the disseration,the surface of PP mesh was treated by plasma at low temperature to improve its hydrophilicity and then coated in the solution of regenerated silk fibroin.The primary animal test revealed that the adhesion between polypropylene mesh and omentum,bowel and the other viscera in the abdominal wall repair of Wistar rat is significantly decreased with polypropylene mesh modified by regenerated silk fibroin.The dual-side polypropylene mesh of maximizing peritoneal in -growth on one side while mimizing visceral adhesion on the other side modified by regenerated silk fibroin can be fabricated by treating polypropylene mesh with plasma and coating on one side,which can be used in the inguinal incisional hernias repair.
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