PLLA/PLGA/HA复合材料的制备与评价
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摘要
目的
本研究为一种改进的以PLLA为基体的复合材料,通过对其物相组成、力学性能、降解性能、显微结构及其生物学行为进行较系统深入的研究,以探求是否符合生物工程材料的要求,而可运用于临床。
方法
以新制备的L-丙交酯和乙交酯(5:1的摩尔比)作单体,在140℃和真空条件下采用开环聚合法制备共聚物PLGA。以HA颗粒与PLLA和PLGA为原料制备复合材料,并采用模压成型的方法制备条形样品。分别测试含不同质量比的HA试样三点抗弯强度及弹性模量、剪切强度、抗扭转强度。
为了解PLLA、PLGA和复合材料对降解的影响,三者的试样在PBS液中进行降解产物自催化24周,分期检测降解液中的酸度pH值、失重率、吸水率、力学性能变化、分子量分布及变化,并对复合材料不同时间点表面和断面的形貌做扫描电镜检查,以探求降解机制。
成骨样细胞株MG-63以10~5个/ml与复合材料共同培养,倒置显微镜观察细胞形态变化,电镜观察细胞在复合材料表面黏附情况。连续培养后行Von Kossa染色、碱性磷酸酶染色及碱性磷酸酶含量测定。将复合材料置于兔的皮下肌肉组织中,分别在1周、2周、4周、8周、16周和32周内取材行材料-组织界面的HE染色,按国家医疗器械生物学评价标准分别对复合材料进行急性毒性试验、亚急性毒性试验、热原试验、溶血试验、抑菌试验等检测。
27只兔,分3组,每组实验动物各9只,在术后12、24、36周分别处死实验动物,取出含植入标本的腰椎行病理切片观察复合材料成骨情况和降解情况;经X线透射检测,自体髂骨植骨、复合材料植入、骨水泥植入三组术后4周、16周、24周,36周时的正侧位片。对植入复合材料中的3只行术后4、12、24、36周时MRI观察。扫描电镜结合能谱观察体内降解12、24、36周后断面和骨-材料界面的形貌变化。所有动物观察椎间植入后的融合情况。
结果
复合材料的抗弯强度随着HA添加量的增加而增加,而抗弯模量随着HA添加量的增加而降低。当HA含量为15%时,复合材料的抗弯强度和抗弯模量分别达到220MPa和5.35GPa以上;复合材料中HA颗粒和聚合物基体之间形成良好的界面,后随着HA添加量的减少,复合材料发生了从脆性断裂向韧性断裂转化的过程。PLLA、PLGA和复合材料在PBS溶液中降解时,其吸水率和失重率都逐渐增加,而pH值则逐渐的降低,但复合材料的变化慢于前二者。PLLA、PLGA和复合材料的力学性能随降解时间的增加都呈现出下降的趋势,但复合材料的力学性能衰减速率明显低于前二者;在降解初期PLLA、PLGA和复合材料的分子量下降速率较快,随着降解地进行,下降地速率逐渐的变慢。PLLA在PBS溶液中的降解属于本体水解,由于基体内部水解所产生的低聚物难以通过扩散排出,引起内部链端羧基浓度增加,产生“自催化水解”,从而导致材料内部的降解速率比表面快,材料中心产生空洞,表层形成外壳,即“双态降解”。复合材料中由于HA的碱性中和了PLLA降解中的酸性产物,加快了内部降解产物的扩散,减缓了酸对PLLA的催化降解作用。
成骨细胞样细胞贴壁生长,成多角形、星形,形态正常贴附、铺展,与复合材料表面紧密连接,电镜下见细胞有伪足伸出,成为细胞在材料表面适宜生长提供了一个有力的证据。细胞生长良好、增殖性能佳,细胞毒性小。骨矿化培养7天胞体出现云雾状钙化灶,培养4周Von kossa染色形成大的黑色结节;碱性磷酸酶钙-钴法染色示胞浆内出现呈深蓝色细颗粒样物质,细胞呈梭形、多角形、椭圆形等,形状各异,大小不等,细胞之间连接成网状,定量检测示第12天达到最高峰14.7U/L,两者结合均说明复合材料可引导部分细胞表达成骨活性。
将三者的复合材料、自体髂骨、骨水泥三者分别植入3组兔体内,从组织学改变可以看出骨组织随着时间的延长,由分散的块状逐步变为团聚的现象,于术后36周时新生的骨岛间彼此连接,长出新骨。复合材料由最初的大片状逐渐形成小块状,24周时形成“瓦片状”和“鱼鳞状”排列,36周时材料降解彻底的“空泡状”痕迹遍布整个视野,难以与骨质绝然分开,表示降解良好。X线检查显示术后36周时植入材料组动物的侧位片可基本达到与自体髂骨等同良好的融合效果,未见植入的材料沉陷、松动、脱出,移位等,椎间隙高度与术前相比,基本保持不变。磁共振显示L5、L6椎间隙处的T_2等信号影,由4周时的大小约为30mm~2逐步缩小到约为6mm~2,体现了显影的无机材料逐步变少的这一个动态过程。扫描电镜观察骨与材料间的“阶梯状”改变随着时间的延长逐渐变得不明显,甚至消失;高倍镜下,骨与复合材料的界面逐渐结合后难以区分,更进一步说明了降解性能良好。从复合材料、自体髂骨、骨水泥三组实验动物术后36周时,与椎骨结合的扫描电镜观察结果显示可以看出:自体髂骨的结合情况最好,复合材料次之,而骨水泥组基本不结合。
细胞毒性试验显示成骨细胞在复合材料浸提液中生长良好,标准在0～1级之间,增殖良好。全身急性、亚急性、慢性毒性试验、溶血试验、热原试验、抑菌试验均显示复合材料无明显毒性反应。皮内试验炎症反应轻,肌肉植入试验随着时间的变化,炎症程度先增强后减弱,然后在Ⅰ级趋于稳定;而首先无囊壁形成,然后囊壁逐渐增厚至Ⅳ级,最后维持在Ⅰ级趋于稳定,显示了良好的生物相容性。
结论
PLLA/PLGA/HA复合材料由于良好的力学性能和生物降解性能,细胞毒性小,在随时间降解的同时能促进成骨,是一种可供安全、有效使用的内置材料。
objective
To explore the advanced composite material whose matrix ingredient is polylactic acid,which is systematically and deeply investigated by its phase component,mechanical property,degradation property,microstructure, biological behavior,in order to find out whether it can be applicated to Clinical work or not,if in accordance with the need of biological engineering materials.
method
The new monomer is made up of the L-lactide and L-glycolide with the mol ratio(5:1),which is adopted ring opening polymerization process to preparate multipolymer PLGA in vacuum and in 140℃.The composite materialis consisted raw material of PLLA,PLGA and HA kernel, adopted compression moulding forming method with column sample. Test different mass ratio HA type with flexure strength,flexure modulus, shear(ing) strength and anti-torsional strength separately.
To understand the influence of degradation with PLLA,PLGA and composite material,and autocatalysis 24 weeks in simulated-body fluid. The degradated-liquids tests are carried out in different periods with PH, rate of weight loss,water absorption,the changeable of mechanical property, the changeable or distribution of molecular weight,and concentration of calciumion.Further the surface and section appearance in SEM were detected in different time in order to explore the machine processed of degradation.
The Osteoplast MG-63 with contents of 10~5/ml and composite material are co-cultivation,the forms of cells are detected by inverted microscope and the adhesive are detected by electron microscope.The cells are stained by Von Kossa,alkaline phosphatase,and the determination of alkaline also measured.The composite materials are put into the musculature of the rabbits,which are taken out and stained in the stuff-tissue inter face at 1w、2w、4w、8w、16w and 32w.Biological safty tests of the composite materials,such as acute toxicity,subacute toxicity, pyrogen,hemolysis,and antibacterial,which were performed according to the biomedical safty assessment standards.
Twenty-seven rabbits are divided into three groups which are nine ones,it turns out the osteogenesis fusion and degradation of composite materials procedures by histological observation at 4w、12w、24w and 36w.
The iliac bone group,the composite material group,bone cement group were taken anterioposterior and lateral film at 4w、12w、24w and 36w.The three rabbits of composite material group were implement by MRI at 4w、12w、24w and 36w.The section and material-bone surface appearance in SEM and energy spectrum were detected at 12w、24w and 36w.The intervertebral bone fusion were observed by all the rabbits.
Result
With the increasing of the HA,the flexure strength is raising,while the flexure modulus is reducing.When the HA is 15%,the flexure strength of the composite materials are reaching 220MPa and 5.35GPa respectively.With degradation of the PLLA,PLGA and composite material in PBS,the rate of weight loss and water absorption are increasing,and the pH is decreasing.The changeable of the composite material and mechanical property are slower than the former two.After twelve week's degradation The mechanical property of the PLLA can offer Define spongy bone fixation,and the composite material still can supplied cortex bone fixation.The changeable of molecular weight are reducing fast at the beginning,and slowly in the end.The degradation kinetics of the composite material were indicative of an autocatalysis process simple bulk hydrolysis,and presented the modal of "biomodal degradation".
The Osteoplast MG-63 growth adherence in the composite materials with,polygon Shape,star pattern,outstretch pseudopod in electron microscope. Seven days after cultivation,there was nebulous calcific focus in the corpus neuroni.Four weeks later,big black nodus was occurred by Von Kossa staining.The corpus neuroni were blue with kernels by alkaline phosphatase staining,there were polygon Shape,fusiformis,oval pattern.The alkaline phosphatase was 14.7U/L in the twelve days after cultivation.
The composite material,iliac bone and bone cement were put into three group rabbits.Thirty-six weeks later,the scattered-lump bone were turned to union with the new bone formation.At the same time,the big mass of composite material were degradation thoroughly,like "vacuolated-shape" There was no signs of subsidence,prolapsus,loosen, dislocation and no loss of the intervertebral height.The fusion is satisfying in the 36 weeks X-ray after operation.The area of middle-signal image in T2 are from 30mm~2 in the four weeks postoperatively to 6mm~2 in the Thirty-six weeks postoperatively.The fusion of which is good in the composite material group,is fair in the iliac bone group,and is poor in the bone cement group by SEM in thirty-six weeks postoperatively.
No toxicity of the composite material,such as acute toxicity、subacute toxicity、pyrogen、hemolysis、antibacterial were detected by the toxicology studies.
Conclusion
The composite material of which is made up of PLLA,PLGA and HA is a safety,effective internal fixation,because it has a good mechanical property,degradation property and no toxicity.Furthermore,it can promote osteogenesis in vivo.
本研究为一种改进的以PLLA为基体的复合材料,通过对其物相组成、力学性能、降解性能、显微结构及其生物学行为进行较系统深入的研究,以探求是否符合生物工程材料的要求,而可运用于临床。
方法
以新制备的L-丙交酯和乙交酯(5:1的摩尔比)作单体,在140℃和真空条件下采用开环聚合法制备共聚物PLGA。以HA颗粒与PLLA和PLGA为原料制备复合材料,并采用模压成型的方法制备条形样品。分别测试含不同质量比的HA试样三点抗弯强度及弹性模量、剪切强度、抗扭转强度。
为了解PLLA、PLGA和复合材料对降解的影响,三者的试样在PBS液中进行降解产物自催化24周,分期检测降解液中的酸度pH值、失重率、吸水率、力学性能变化、分子量分布及变化,并对复合材料不同时间点表面和断面的形貌做扫描电镜检查,以探求降解机制。
成骨样细胞株MG-63以10~5个/ml与复合材料共同培养,倒置显微镜观察细胞形态变化,电镜观察细胞在复合材料表面黏附情况。连续培养后行Von Kossa染色、碱性磷酸酶染色及碱性磷酸酶含量测定。将复合材料置于兔的皮下肌肉组织中,分别在1周、2周、4周、8周、16周和32周内取材行材料-组织界面的HE染色,按国家医疗器械生物学评价标准分别对复合材料进行急性毒性试验、亚急性毒性试验、热原试验、溶血试验、抑菌试验等检测。
27只兔,分3组,每组实验动物各9只,在术后12、24、36周分别处死实验动物,取出含植入标本的腰椎行病理切片观察复合材料成骨情况和降解情况;经X线透射检测,自体髂骨植骨、复合材料植入、骨水泥植入三组术后4周、16周、24周,36周时的正侧位片。对植入复合材料中的3只行术后4、12、24、36周时MRI观察。扫描电镜结合能谱观察体内降解12、24、36周后断面和骨-材料界面的形貌变化。所有动物观察椎间植入后的融合情况。
结果
复合材料的抗弯强度随着HA添加量的增加而增加,而抗弯模量随着HA添加量的增加而降低。当HA含量为15%时,复合材料的抗弯强度和抗弯模量分别达到220MPa和5.35GPa以上;复合材料中HA颗粒和聚合物基体之间形成良好的界面,后随着HA添加量的减少,复合材料发生了从脆性断裂向韧性断裂转化的过程。PLLA、PLGA和复合材料在PBS溶液中降解时,其吸水率和失重率都逐渐增加,而pH值则逐渐的降低,但复合材料的变化慢于前二者。PLLA、PLGA和复合材料的力学性能随降解时间的增加都呈现出下降的趋势,但复合材料的力学性能衰减速率明显低于前二者;在降解初期PLLA、PLGA和复合材料的分子量下降速率较快,随着降解地进行,下降地速率逐渐的变慢。PLLA在PBS溶液中的降解属于本体水解,由于基体内部水解所产生的低聚物难以通过扩散排出,引起内部链端羧基浓度增加,产生“自催化水解”,从而导致材料内部的降解速率比表面快,材料中心产生空洞,表层形成外壳,即“双态降解”。复合材料中由于HA的碱性中和了PLLA降解中的酸性产物,加快了内部降解产物的扩散,减缓了酸对PLLA的催化降解作用。
成骨细胞样细胞贴壁生长,成多角形、星形,形态正常贴附、铺展,与复合材料表面紧密连接,电镜下见细胞有伪足伸出,成为细胞在材料表面适宜生长提供了一个有力的证据。细胞生长良好、增殖性能佳,细胞毒性小。骨矿化培养7天胞体出现云雾状钙化灶,培养4周Von kossa染色形成大的黑色结节;碱性磷酸酶钙-钴法染色示胞浆内出现呈深蓝色细颗粒样物质,细胞呈梭形、多角形、椭圆形等,形状各异,大小不等,细胞之间连接成网状,定量检测示第12天达到最高峰14.7U/L,两者结合均说明复合材料可引导部分细胞表达成骨活性。
将三者的复合材料、自体髂骨、骨水泥三者分别植入3组兔体内,从组织学改变可以看出骨组织随着时间的延长,由分散的块状逐步变为团聚的现象,于术后36周时新生的骨岛间彼此连接,长出新骨。复合材料由最初的大片状逐渐形成小块状,24周时形成“瓦片状”和“鱼鳞状”排列,36周时材料降解彻底的“空泡状”痕迹遍布整个视野,难以与骨质绝然分开,表示降解良好。X线检查显示术后36周时植入材料组动物的侧位片可基本达到与自体髂骨等同良好的融合效果,未见植入的材料沉陷、松动、脱出,移位等,椎间隙高度与术前相比,基本保持不变。磁共振显示L5、L6椎间隙处的T_2等信号影,由4周时的大小约为30mm~2逐步缩小到约为6mm~2,体现了显影的无机材料逐步变少的这一个动态过程。扫描电镜观察骨与材料间的“阶梯状”改变随着时间的延长逐渐变得不明显,甚至消失;高倍镜下,骨与复合材料的界面逐渐结合后难以区分,更进一步说明了降解性能良好。从复合材料、自体髂骨、骨水泥三组实验动物术后36周时,与椎骨结合的扫描电镜观察结果显示可以看出:自体髂骨的结合情况最好,复合材料次之,而骨水泥组基本不结合。
细胞毒性试验显示成骨细胞在复合材料浸提液中生长良好,标准在0～1级之间,增殖良好。全身急性、亚急性、慢性毒性试验、溶血试验、热原试验、抑菌试验均显示复合材料无明显毒性反应。皮内试验炎症反应轻,肌肉植入试验随着时间的变化,炎症程度先增强后减弱,然后在Ⅰ级趋于稳定;而首先无囊壁形成,然后囊壁逐渐增厚至Ⅳ级,最后维持在Ⅰ级趋于稳定,显示了良好的生物相容性。
结论
PLLA/PLGA/HA复合材料由于良好的力学性能和生物降解性能,细胞毒性小,在随时间降解的同时能促进成骨,是一种可供安全、有效使用的内置材料。
objective
To explore the advanced composite material whose matrix ingredient is polylactic acid,which is systematically and deeply investigated by its phase component,mechanical property,degradation property,microstructure, biological behavior,in order to find out whether it can be applicated to Clinical work or not,if in accordance with the need of biological engineering materials.
method
The new monomer is made up of the L-lactide and L-glycolide with the mol ratio(5:1),which is adopted ring opening polymerization process to preparate multipolymer PLGA in vacuum and in 140℃.The composite materialis consisted raw material of PLLA,PLGA and HA kernel, adopted compression moulding forming method with column sample. Test different mass ratio HA type with flexure strength,flexure modulus, shear(ing) strength and anti-torsional strength separately.
To understand the influence of degradation with PLLA,PLGA and composite material,and autocatalysis 24 weeks in simulated-body fluid. The degradated-liquids tests are carried out in different periods with PH, rate of weight loss,water absorption,the changeable of mechanical property, the changeable or distribution of molecular weight,and concentration of calciumion.Further the surface and section appearance in SEM were detected in different time in order to explore the machine processed of degradation.
The Osteoplast MG-63 with contents of 10~5/ml and composite material are co-cultivation,the forms of cells are detected by inverted microscope and the adhesive are detected by electron microscope.The cells are stained by Von Kossa,alkaline phosphatase,and the determination of alkaline also measured.The composite materials are put into the musculature of the rabbits,which are taken out and stained in the stuff-tissue inter face at 1w、2w、4w、8w、16w and 32w.Biological safty tests of the composite materials,such as acute toxicity,subacute toxicity, pyrogen,hemolysis,and antibacterial,which were performed according to the biomedical safty assessment standards.
Twenty-seven rabbits are divided into three groups which are nine ones,it turns out the osteogenesis fusion and degradation of composite materials procedures by histological observation at 4w、12w、24w and 36w.
The iliac bone group,the composite material group,bone cement group were taken anterioposterior and lateral film at 4w、12w、24w and 36w.The three rabbits of composite material group were implement by MRI at 4w、12w、24w and 36w.The section and material-bone surface appearance in SEM and energy spectrum were detected at 12w、24w and 36w.The intervertebral bone fusion were observed by all the rabbits.
Result
With the increasing of the HA,the flexure strength is raising,while the flexure modulus is reducing.When the HA is 15%,the flexure strength of the composite materials are reaching 220MPa and 5.35GPa respectively.With degradation of the PLLA,PLGA and composite material in PBS,the rate of weight loss and water absorption are increasing,and the pH is decreasing.The changeable of the composite material and mechanical property are slower than the former two.After twelve week's degradation The mechanical property of the PLLA can offer Define spongy bone fixation,and the composite material still can supplied cortex bone fixation.The changeable of molecular weight are reducing fast at the beginning,and slowly in the end.The degradation kinetics of the composite material were indicative of an autocatalysis process simple bulk hydrolysis,and presented the modal of "biomodal degradation".
The Osteoplast MG-63 growth adherence in the composite materials with,polygon Shape,star pattern,outstretch pseudopod in electron microscope. Seven days after cultivation,there was nebulous calcific focus in the corpus neuroni.Four weeks later,big black nodus was occurred by Von Kossa staining.The corpus neuroni were blue with kernels by alkaline phosphatase staining,there were polygon Shape,fusiformis,oval pattern.The alkaline phosphatase was 14.7U/L in the twelve days after cultivation.
The composite material,iliac bone and bone cement were put into three group rabbits.Thirty-six weeks later,the scattered-lump bone were turned to union with the new bone formation.At the same time,the big mass of composite material were degradation thoroughly,like "vacuolated-shape" There was no signs of subsidence,prolapsus,loosen, dislocation and no loss of the intervertebral height.The fusion is satisfying in the 36 weeks X-ray after operation.The area of middle-signal image in T2 are from 30mm~2 in the four weeks postoperatively to 6mm~2 in the Thirty-six weeks postoperatively.The fusion of which is good in the composite material group,is fair in the iliac bone group,and is poor in the bone cement group by SEM in thirty-six weeks postoperatively.
No toxicity of the composite material,such as acute toxicity、subacute toxicity、pyrogen、hemolysis、antibacterial were detected by the toxicology studies.
Conclusion
The composite material of which is made up of PLLA,PLGA and HA is a safety,effective internal fixation,because it has a good mechanical property,degradation property and no toxicity.Furthermore,it can promote osteogenesis in vivo.
引文
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