热塑性淀粉材料的改性及生物医用探索
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摘要
淀粉是一种来源丰富的可降解型天然多糖类高分子化合物。由于其具有原料来源丰富,无毒,可降解且降解产物无毒等诸多优点,在生物材料领域具有良好的应用前景。本论文以热塑性淀粉作为主要原材料,以临床医用为目的,在特定条件下研究了高甘油含量的热塑性淀粉弹性体的老化机理及在相关体液模拟液中的降解机理,针对传统的甘油增塑的热塑型淀粉易老化,吸水性强及在体液模拟液中易崩裂,难于维持力学性能等缺陷,对其进行一系列改性(新型增塑剂共增塑、共混、酯化、交联)。此外,还针对一些特定的应用目标(诱导组织再生膜,骨修复用注射型水凝胶)制备出相关的热塑性淀粉复合材料,研究了复合材料本身的结构与性能,体外降解性能及生物相容性。此外,还对制备的可注射型骨修复用水凝胶进行了系统的体内外生物相容性评价,并通过动物实验考察了它对于颌骨修复的效果。
本论文主要有以下几个方面的贡献:
(1)通过高甘油含量增塑的方法制备了具备一定弹性的热塑性淀粉,首次提出该热塑性淀粉弹性体可潜在应用于软组织工程支架及治疗口腔溃疡用贴膜材料。在保存过程中发现甘油增塑热塑性淀粉(GTPS)容易发生老化,老化是影响GTPS应用的主要因素。本论文针对潜在的应用目标,研究了在人体正常体温(37℃)以及人体感觉比较舒适的相对湿度(RH,50%)条件下,高甘油含量GTPS这个复杂的三元材料体系的老化机理。首次对GTPS弹性体在血浆及唾液模拟液中的降解行为及降解机理进行了研究。研究结果表明当甘油含量高于40%(占总重)时,材料表现出弹性体性征,高甘油含量有助于抑制双螺旋结构的B-型结晶产生但有利于直链淀粉单螺旋V-型结晶结构的生成,以往有关老化机理的研究都未涉及甘油含量对单螺旋结晶生成的影响。当甘油含量高于40%(占总重)时,材料在54天内力学性能变化不大,不同测试结果均证明了高甘油含量有明显的抑制老化作用。首次提出GTPS弹性体在血浆及唾液模拟液中的降解机理。在血浆模拟液中的降解机理是典型的本体降解机理,先溶胀后降解。唾液模拟液中的降解机理是先从酶对于材料表面的侵蚀开始,由表及里逐步发生降解,降解的过程中同时伴随溶胀,前2h主要以表面降解为主,之后表面降解与本体降解同时进行。唾液模拟液中的降解速度远大于血浆模拟液中的降解速度。细胞毒性试验证明GTPS在血液模拟液中的短期浸提物及长期降解产物对L929小鼠成纤细胞的生长都无不利影响,且降解产物有促进细胞分裂的作用。该结论对于GTPS作为体内埋植材料的具有十分重要的价值。通过对GTPS在唾液模拟液中降解行为的研究,发现其表面快速降解性征非常适合作为潜在的短期伤口敷料应用于口腔溃疡的治疗中。
(2)基于第一部分的研究,GTPS存在的易老化,难以在体液中维持力学和尺寸稳定性等缺陷,采用柠檬酸与甘油共同增塑淀粉(CGTPS),深入研究了CGTPS的结构与性能。研究结果表明与传统甘油增塑的TPS相比,CGTPS具有以下特殊的结构和性质:部分酯化,低分子量以及较强的淀粉/增塑剂间相互作用。部分酯化可以防止材料的老化,在淀粉分子链上引入新的基团,从而为潜在改性提供反应位点及提高与可降解聚酯的相容性。分子量的降低可以提高材料的加工性能,调节降解速度,提高CGTPS在聚酯材料中的分散性。此外,该CGTPS材料可以通过熔融共混在短时间内方便制备。CGTPS所具有的这些特殊性质将会使其在环保材料和生物材料领域得到广泛的应用。
(3)制备了厚度在0.05-0.1mm的淀粉/PVA薄膜(SP)膜,首次提出将SP膜用于诱导组织再生(GTR)技术,并针对GTR膜的要求对SP膜的力学性能、溶胀性能、血浆及唾液模拟液中的降解行为及相关生物相容性进行了研究。结果表明SP膜具备很好的亲水性和柔韧性,力学及降解速度可调性和良好的生物相容性,能够在30天内保持其完整性和力学性能稳定性,具备GTR应用要求的潜质,但仍然存在吸水率高溶胀快的缺陷,且吸水后力学性能下降迅速。因此,我们选择了柠檬酸(CA)交联法对SP膜进行了进一步改性,在140℃高温成型条件下制备出一系列不同CA(5-30wt%)含量的SP薄膜,并对其结构进行了细致的研究。发现在高温成型条件下,一部分CA能够与淀粉和PVA之间发生了酯化反应,酯化反应的发生和CA的多羧基结构又导致了体系的化学交联;此外,体系中残留的CA能起到淀粉与PVA的增塑剂作用,且与甘油相比,CA上的羧基能够与淀粉和PVA形成更稳定的氢键作用,从而有效抑制材料老化;酯化和交联还使得SP膜吸水率的下降热稳定性升高。细胞毒性试验证明CA含量在20%以内对细胞毒试验评级结果为0-1级,仍然具有良好的细胞相容性。
(4)对淀粉/海藻酸钠可注射型物理水凝胶进行了6种体外及体内生物相容性评价(溶血实验、细胞毒性实验、经静脉和经口途径全身急性毒性实验、皮下埋植及骨埋植实验)。评价结果为细胞毒性分级为0-1级,不会导致急性溶血反应,无急性毒反应,有轻微皮下组织反应,无骨吸收和骨破坏现象,是一种生物相容性良好的水凝胶材料。通过添加纳米羟基磷灰石制备了淀粉/海藻酸钠/纳米羟基磷灰石复合材料,并通过动物实验观察了其在兔颌骨缺损中的修复效果,实验表明复合纳米羟基磷灰石的淀粉/海藻酸钠注射型水凝胶的修复速度、修复区骨密度、下颌骨体边缘的光滑度均优于空白或单纯使用淀粉/SA材料的修复效果,组织学显示试验侧成骨的方式是周围和中心成骨两种并存,总体来说该水凝胶具有良好的生物安全性和较好的成骨效果。
Starch is a kind of high-yield biodegradable natrual polysaccharid. Because of its nontoxicity, biodegradability and nontoxicity of the degradation products, starch is a good candidate in biomedical field. In this paper, based on the the aim of biomedical application, thermoplastic starch was chosen as the main raw material. The ageing and degradation mechanisms of glycerol plasticed thermolplastic starch (GTPS) with high glycerol content were investigated.It seems like that the GTPS dissolve in water easily. Furthermore, the mechanical properties can not maintain when immerged in body fluid. In order to improve the ageing resistent ability, water resistent ability and mechanical properties, a series of modifications methods (co-plasticization, esterification, crosslinking and and blending with other polymers) were carried out.Moreover,in order to satisfy some special clinical application (guide tissue regeneration membrane , injected hydrogel for bone repair), the starch/PVA(SP) film and starch/sodium alginate/nano-hydroxylapatite(SSH) composites were prapared.The structure and properties,in vitro degradation behaviour and biocompatibility of SP films and SSH were investigated.In addition, the biocompatibility of the SSH was evaluated by six in vitro and in vivo biological experiments. The repairing effects on rabbit mandibular bone defect were also researched.
The main research content and achievements are shown as follows:
(1) The thermoplastic starch with the elastomeric properties was prepared by plasticizing with high glycerol content. The elastomeric property was exhibited when the glycerol content was more than 40% (based on the total weight). The ageing mechanism of the complicated tertiary mixture was investigated under the normal human body temperature (37℃) and a comfortable relative humidity for human being (50 RH %), so as to understand the mechanism of the ageing well and to control it under certain conditions of human body. The degradation mechanism of the GTPS elastomer in simulated body fluid and simulated saliva fluid was firstly investigated. The main results including: high glycerol content is helpful to the formation of the single helix structure, but it is not good for the formation of the double helix structure of B-type. There were no reports focused on the relationship between the single helix structures and ageing before. When the glycerol content was more than 40%, there was only a slight change of the mechanical properties within 54days. The results from all characterizations demonstrated that the high glycerol content can prevent ageing effectively. The degradation process in SBF is typical bulk degradation. The GTPS swelled at first, and then degraded. In SSF, the surface and bulk degradation took place at the same time but the surface degradation played an important role in the first 2 h. The influences of the short-term extractions and the long-term degradation products in SBF were proved to be innocuous, which is very important for GTPS to be applicated in biomedical applications. The quick degradation velocity in SSF made GTPS a promising short-term wound dressing material to deal with the stomatitis in oral.
(2) In order to prevent ageing and to promote some other properties, a novel citric acid (CA)-glycerol co-plasticized thermoplastic starch (CGTPS) was prepared by melt blending. The structure and properties was investigated. The novel CGTPS has some new characteristics compared with the traditional glycerol plasticized TPS: namely, partial esterification, low molecular weight, and strong interaction between starch and CA. Partial esterification could have some effect on reducing retrogradation, introducing some new groups onto the starch chains for potential modification, and improving the compatibility with some other polyesters. The decrease in the molecular weight can improve the processing properties, adjust the degradation speed, and improve the diffusion of the CGTPS in the polyesters. Furthermore, the CGTPS can easily be prepared by melt blending in a short time. All of these special properties would make CGTPS a good candidate in environment and biomedical applications.
(3) A series of starch / PVA (SP) films with 0.05-0.1mm thickness were cast by solvent method. The application of SP films in guide tissue regenaration (GTR) technology was firstly proposed. The in vitro degradation and biocompatibility of the SP films were investigated. Generally speaking, the SP films possess high hydrophilic properties, flexible, strong and adjustable mechanical properties, suitable degradation rate, and excellent biocompatibility. Within 30 days, the integrity of the SP films kept well. The mechanical properties didn't change so much from 3 to 30 days, and SP films showed excellent mechanical stability both in SBF and SSF. It is also very important for its potential use as GTR membrane. Furthermore, in order to overcome the high water absorptence and quick swelling velosity, The SP films were modificated by adding CA and molded at 140℃. The esterification happened between CA and starch (or PVA) as the function of CA. The esterification and the multi-carboxyl structure of CA may result in chemical cross-linking in the blending system.The esterification occured more easily between the starch and CA than that between the PVA and CA. The residual free CA acted as the plasticizer of the starch and PVA. Compared to the hydroxyl groups on glycerol, the carboxyi groups on CA can form stronger hydrogen bonds between CA and other components. The crosslinking and strong hydrogen bonding enhanced the thermal stability and decreased the water absorbance. The cell Relative Growth Rates of all samples with different CA concentration exceeded 80% after 7 day's incubation. This result demonstrated that there was no significant toxicity on cell's growth when the CA content was less than 20%.
(4) The starch/sodium alginate/n-HAP (SSH) rejected hydrogel was prepared by melt blending method. According to the Chinese and international standard, six biological evaluation (cytotoxicity, acute toxicity, haemocompatibility, embed in skin and bone )tests pluse a repairing both sides of rabbit mandibular bone defect were investigated. Results demonstrated that the rate of haemocompatibility is 2.06%, no more different between trail and control in acute toxicity, the grade of cytotoxicity is under 1. In the subcutaneous test, there was only a slight subcutaneous tissue reaction. At the same time, complex with n-HA could lead good repairing result in mandibular bone defect. In the bone implant experiment, no absorptance and destruction phenomenons of bone were observed. The SSH could lead good repairing in mandibular bone defect. The SSH is acceptable biological martial and could be a media in repairing bone defect.
本论文主要有以下几个方面的贡献:
(1)通过高甘油含量增塑的方法制备了具备一定弹性的热塑性淀粉,首次提出该热塑性淀粉弹性体可潜在应用于软组织工程支架及治疗口腔溃疡用贴膜材料。在保存过程中发现甘油增塑热塑性淀粉(GTPS)容易发生老化,老化是影响GTPS应用的主要因素。本论文针对潜在的应用目标,研究了在人体正常体温(37℃)以及人体感觉比较舒适的相对湿度(RH,50%)条件下,高甘油含量GTPS这个复杂的三元材料体系的老化机理。首次对GTPS弹性体在血浆及唾液模拟液中的降解行为及降解机理进行了研究。研究结果表明当甘油含量高于40%(占总重)时,材料表现出弹性体性征,高甘油含量有助于抑制双螺旋结构的B-型结晶产生但有利于直链淀粉单螺旋V-型结晶结构的生成,以往有关老化机理的研究都未涉及甘油含量对单螺旋结晶生成的影响。当甘油含量高于40%(占总重)时,材料在54天内力学性能变化不大,不同测试结果均证明了高甘油含量有明显的抑制老化作用。首次提出GTPS弹性体在血浆及唾液模拟液中的降解机理。在血浆模拟液中的降解机理是典型的本体降解机理,先溶胀后降解。唾液模拟液中的降解机理是先从酶对于材料表面的侵蚀开始,由表及里逐步发生降解,降解的过程中同时伴随溶胀,前2h主要以表面降解为主,之后表面降解与本体降解同时进行。唾液模拟液中的降解速度远大于血浆模拟液中的降解速度。细胞毒性试验证明GTPS在血液模拟液中的短期浸提物及长期降解产物对L929小鼠成纤细胞的生长都无不利影响,且降解产物有促进细胞分裂的作用。该结论对于GTPS作为体内埋植材料的具有十分重要的价值。通过对GTPS在唾液模拟液中降解行为的研究,发现其表面快速降解性征非常适合作为潜在的短期伤口敷料应用于口腔溃疡的治疗中。
(2)基于第一部分的研究,GTPS存在的易老化,难以在体液中维持力学和尺寸稳定性等缺陷,采用柠檬酸与甘油共同增塑淀粉(CGTPS),深入研究了CGTPS的结构与性能。研究结果表明与传统甘油增塑的TPS相比,CGTPS具有以下特殊的结构和性质:部分酯化,低分子量以及较强的淀粉/增塑剂间相互作用。部分酯化可以防止材料的老化,在淀粉分子链上引入新的基团,从而为潜在改性提供反应位点及提高与可降解聚酯的相容性。分子量的降低可以提高材料的加工性能,调节降解速度,提高CGTPS在聚酯材料中的分散性。此外,该CGTPS材料可以通过熔融共混在短时间内方便制备。CGTPS所具有的这些特殊性质将会使其在环保材料和生物材料领域得到广泛的应用。
(3)制备了厚度在0.05-0.1mm的淀粉/PVA薄膜(SP)膜,首次提出将SP膜用于诱导组织再生(GTR)技术,并针对GTR膜的要求对SP膜的力学性能、溶胀性能、血浆及唾液模拟液中的降解行为及相关生物相容性进行了研究。结果表明SP膜具备很好的亲水性和柔韧性,力学及降解速度可调性和良好的生物相容性,能够在30天内保持其完整性和力学性能稳定性,具备GTR应用要求的潜质,但仍然存在吸水率高溶胀快的缺陷,且吸水后力学性能下降迅速。因此,我们选择了柠檬酸(CA)交联法对SP膜进行了进一步改性,在140℃高温成型条件下制备出一系列不同CA(5-30wt%)含量的SP薄膜,并对其结构进行了细致的研究。发现在高温成型条件下,一部分CA能够与淀粉和PVA之间发生了酯化反应,酯化反应的发生和CA的多羧基结构又导致了体系的化学交联;此外,体系中残留的CA能起到淀粉与PVA的增塑剂作用,且与甘油相比,CA上的羧基能够与淀粉和PVA形成更稳定的氢键作用,从而有效抑制材料老化;酯化和交联还使得SP膜吸水率的下降热稳定性升高。细胞毒性试验证明CA含量在20%以内对细胞毒试验评级结果为0-1级,仍然具有良好的细胞相容性。
(4)对淀粉/海藻酸钠可注射型物理水凝胶进行了6种体外及体内生物相容性评价(溶血实验、细胞毒性实验、经静脉和经口途径全身急性毒性实验、皮下埋植及骨埋植实验)。评价结果为细胞毒性分级为0-1级,不会导致急性溶血反应,无急性毒反应,有轻微皮下组织反应,无骨吸收和骨破坏现象,是一种生物相容性良好的水凝胶材料。通过添加纳米羟基磷灰石制备了淀粉/海藻酸钠/纳米羟基磷灰石复合材料,并通过动物实验观察了其在兔颌骨缺损中的修复效果,实验表明复合纳米羟基磷灰石的淀粉/海藻酸钠注射型水凝胶的修复速度、修复区骨密度、下颌骨体边缘的光滑度均优于空白或单纯使用淀粉/SA材料的修复效果,组织学显示试验侧成骨的方式是周围和中心成骨两种并存,总体来说该水凝胶具有良好的生物安全性和较好的成骨效果。
Starch is a kind of high-yield biodegradable natrual polysaccharid. Because of its nontoxicity, biodegradability and nontoxicity of the degradation products, starch is a good candidate in biomedical field. In this paper, based on the the aim of biomedical application, thermoplastic starch was chosen as the main raw material. The ageing and degradation mechanisms of glycerol plasticed thermolplastic starch (GTPS) with high glycerol content were investigated.It seems like that the GTPS dissolve in water easily. Furthermore, the mechanical properties can not maintain when immerged in body fluid. In order to improve the ageing resistent ability, water resistent ability and mechanical properties, a series of modifications methods (co-plasticization, esterification, crosslinking and and blending with other polymers) were carried out.Moreover,in order to satisfy some special clinical application (guide tissue regeneration membrane , injected hydrogel for bone repair), the starch/PVA(SP) film and starch/sodium alginate/nano-hydroxylapatite(SSH) composites were prapared.The structure and properties,in vitro degradation behaviour and biocompatibility of SP films and SSH were investigated.In addition, the biocompatibility of the SSH was evaluated by six in vitro and in vivo biological experiments. The repairing effects on rabbit mandibular bone defect were also researched.
The main research content and achievements are shown as follows:
(1) The thermoplastic starch with the elastomeric properties was prepared by plasticizing with high glycerol content. The elastomeric property was exhibited when the glycerol content was more than 40% (based on the total weight). The ageing mechanism of the complicated tertiary mixture was investigated under the normal human body temperature (37℃) and a comfortable relative humidity for human being (50 RH %), so as to understand the mechanism of the ageing well and to control it under certain conditions of human body. The degradation mechanism of the GTPS elastomer in simulated body fluid and simulated saliva fluid was firstly investigated. The main results including: high glycerol content is helpful to the formation of the single helix structure, but it is not good for the formation of the double helix structure of B-type. There were no reports focused on the relationship between the single helix structures and ageing before. When the glycerol content was more than 40%, there was only a slight change of the mechanical properties within 54days. The results from all characterizations demonstrated that the high glycerol content can prevent ageing effectively. The degradation process in SBF is typical bulk degradation. The GTPS swelled at first, and then degraded. In SSF, the surface and bulk degradation took place at the same time but the surface degradation played an important role in the first 2 h. The influences of the short-term extractions and the long-term degradation products in SBF were proved to be innocuous, which is very important for GTPS to be applicated in biomedical applications. The quick degradation velocity in SSF made GTPS a promising short-term wound dressing material to deal with the stomatitis in oral.
(2) In order to prevent ageing and to promote some other properties, a novel citric acid (CA)-glycerol co-plasticized thermoplastic starch (CGTPS) was prepared by melt blending. The structure and properties was investigated. The novel CGTPS has some new characteristics compared with the traditional glycerol plasticized TPS: namely, partial esterification, low molecular weight, and strong interaction between starch and CA. Partial esterification could have some effect on reducing retrogradation, introducing some new groups onto the starch chains for potential modification, and improving the compatibility with some other polyesters. The decrease in the molecular weight can improve the processing properties, adjust the degradation speed, and improve the diffusion of the CGTPS in the polyesters. Furthermore, the CGTPS can easily be prepared by melt blending in a short time. All of these special properties would make CGTPS a good candidate in environment and biomedical applications.
(3) A series of starch / PVA (SP) films with 0.05-0.1mm thickness were cast by solvent method. The application of SP films in guide tissue regenaration (GTR) technology was firstly proposed. The in vitro degradation and biocompatibility of the SP films were investigated. Generally speaking, the SP films possess high hydrophilic properties, flexible, strong and adjustable mechanical properties, suitable degradation rate, and excellent biocompatibility. Within 30 days, the integrity of the SP films kept well. The mechanical properties didn't change so much from 3 to 30 days, and SP films showed excellent mechanical stability both in SBF and SSF. It is also very important for its potential use as GTR membrane. Furthermore, in order to overcome the high water absorptence and quick swelling velosity, The SP films were modificated by adding CA and molded at 140℃. The esterification happened between CA and starch (or PVA) as the function of CA. The esterification and the multi-carboxyl structure of CA may result in chemical cross-linking in the blending system.The esterification occured more easily between the starch and CA than that between the PVA and CA. The residual free CA acted as the plasticizer of the starch and PVA. Compared to the hydroxyl groups on glycerol, the carboxyi groups on CA can form stronger hydrogen bonds between CA and other components. The crosslinking and strong hydrogen bonding enhanced the thermal stability and decreased the water absorbance. The cell Relative Growth Rates of all samples with different CA concentration exceeded 80% after 7 day's incubation. This result demonstrated that there was no significant toxicity on cell's growth when the CA content was less than 20%.
(4) The starch/sodium alginate/n-HAP (SSH) rejected hydrogel was prepared by melt blending method. According to the Chinese and international standard, six biological evaluation (cytotoxicity, acute toxicity, haemocompatibility, embed in skin and bone )tests pluse a repairing both sides of rabbit mandibular bone defect were investigated. Results demonstrated that the rate of haemocompatibility is 2.06%, no more different between trail and control in acute toxicity, the grade of cytotoxicity is under 1. In the subcutaneous test, there was only a slight subcutaneous tissue reaction. At the same time, complex with n-HA could lead good repairing result in mandibular bone defect. In the bone implant experiment, no absorptance and destruction phenomenons of bone were observed. The SSH could lead good repairing in mandibular bone defect. The SSH is acceptable biological martial and could be a media in repairing bone defect.
引文
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