镁基生物材料的表面改性和生物相容性研究
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摘要
生物材料与人类身体健康水平和生活质量息息相关,已成为各国经济发展新的增长点。镁基材料有着优越的生物相容性和力学相容性,并具可腐蚀降解,有望成为可降解的金属硬组织替代材料,成为了生物材料领域新的研究热点。
本研究以镁基材料为对象,对99.99%的纯镁(4N-Mg)以及Mg-Ca系合金在仿生体液中腐蚀降解过程和机制进行了研究。为改善其腐蚀行为和生物活性,对其进行了硬脂酸自组装膜改性处理,并对改性工艺机理和改性后的腐蚀行为进行探讨。为评价镁基材料的生物相容性和成骨行为,对镁基材料进行了溶血率、细胞毒性等体外相容性评价以及全身急性毒性和骨植入试验的体内相容性评价。
方法:
①使用模拟人体生理环境的仿生体液(SBF),对铸态4N-Mg和自行熔炼的铸态Mg-Ca系合金进行体外浸泡研究。利用XRD、SEM、EDS、FTIR、原子吸收光谱、电化学分析系统等设备和手段,分析讨论其腐蚀过程、速度和机制。
②使用硬脂酸自组装膜(SAM)改性方法对4N-Mg以及Mg-0.88Ca进行改性处理研究。首先通过正交试验筛选了包括试样的前处理方法、硬脂酸浓度和在硬脂酸乙醇溶液中的浸泡时间等工艺参数并进行验证;然后对所选用的高浓度短时间浸泡的方法与传统的低浓度长时间浸泡方法进行对比研究,并比较了SAM改性后再进行超声清洗的改性层的抗腐蚀性能。以最优工艺对4N-Mg以及Mg-0.88Ca进行改性处理,对改性后试样的腐蚀过程进行研究。
③对SAM改性前后的4N-Mg和Mg-0.88Ca材料的浸提液以及未处理4N-Mg浸提液的稀释液,依照ISO 10993.4的标准进行溶血率测试,并分析pH值、溶液中的镁离子浓度与溶血率关系。
④以L929成纤维细胞和MC-3T3E1成骨细胞与4N-Mg和Mg-0.88Ca材料的浸提液和未处理4N-Mg浸提液的稀释液,进行MTT细胞毒性测试,并测定稀释浸提液中的镁离子浓度。再以成骨细胞与SAM改性前后的4N-Mg和Mg-0.88Ca材料共培养,讨论材料对细胞增殖和活性状态的影响。
⑤对SAM改性前后4N-Mg浸提液作全身急性毒性试验,以纯种小白鼠为对象施以腹腔注射,给药后7d内观察小鼠的症状和活动状况以及体重变化。
⑥将SAM改性前后的4N-Mg和Mg-0.88Ca材料以及Ti6Al4V对照样植入白兔股骨,植入后各组2周、6周、12周时任取1只处死。进行X线片检查,血镁浓度测试,并对标本进行扫描电镜(SEM)观察、能谱分析(EDS)以及组织切片光镜观察。
结果:
①未处理4N-Mg和Mg-Ca系合金研究
1)Mg-0.88Ca、Mg-1.42Ca、Mg-2.0Ca三种镁钙合金中,钙含量最低的Mg-0.88Ca在SBF中显示较好的耐腐蚀性,4N-Mg显示更强的腐蚀抗力; 4N-Mg及Mg-0.88Ca损伤形式主要是腐蚀坑和网状裂纹,后者在个别处有颗粒腐蚀脱落的局部不均匀腐蚀现象;试样表面被Mg(OH)2、HA、CaMg(CO3)2等白色颗粒和微小的半球状的沉积物覆盖。
2)E-pH图分析表明,当pH值超过11.38后纯镁才进入到钝化区。SBF中HCO3-、HPO42-等阴离子也可以与镁发生反应导致其腐蚀。4N-Mg及Mg-0.88Ca材料细胞毒性满足要求,没有引起全身急性毒性反应,但溶血率不合要求。
②在0.5 mol/L硬脂酸乙醇溶液中浸泡1.5h所得自组装膜经过超声清洗后,对4N-Mg及Mg-0.88Ca的抗腐蚀行为有明显提高。其耐蚀性要优于在稀溶液中长时间浸泡所得膜层,并显示出更好的生物活性。其细胞毒性和溶血率均满足对生物材料的要求。对成骨细胞增殖的促进作用超过未处理试样,没有引起全身急性毒性反应。
③在本实验条件下,当pH值不超过10.61,Mg2+浓度不超过42mg/L时,溶液的溶血率不超过5%。Mg2+浓度小于202.5mg/L满足细胞毒性要求,而Mg2+为不超过156.2mg/L的稀释浸提液对成骨细胞增殖显示出促进作用。
④骨内植入试验中,动物血镁浓度均在正常值范围之内。镁基植入材料显示出较好的骨结合性和骨诱导性,新生骨组织矿化明显,新生骨层上有排列整齐的骨细胞。未改性材料降解快于改性材料,成骨活性上未见明显差别。结果表明镁基材料初步显示了较好的骨诱导性能,证实了SAM对镁基材料腐蚀降解的调控作用。
Biomaterials not only have important social effect in bettering the living quality of human beings, improving their health and saving their lives, but also become a new growth point in economic and scientific-technological areas. The potential worth of magnesium in biomaterials field has caused great interests of research, for its excellent biocompatiblity and similar mechnical properties to bone. Moreover, magnesium-based materials can become novel degradable biomaterials making use of its corrodibility.
In this paper, the corrosion degradation process and mechanism of 4N-Mg and Mg-Ca alloys in SBF were studied. In order to improve the corrosion behavior and biological activity of Mg-based materials, surface modification of self-assembled monolayers (SAM) using of stearic acid was studied, meanwhile the mechanism of SAM and the corrosion behavior of samples were studied. To evaluate the biocompatibility and behavior of bone healing of Mg-based materials, the biocompatibility tests in vitro such as hemolysis ratio, cytotoxicity, and systemic acute toxicity and in vivo test were taken.
Methods:
①Simulated body fluid (SBF) which closed to inorganic composition of human plasma was used to immerse casting 4N-Mg alloy which contains 99.99 % magnesium and casting Mg-Ca alloys. Electronic balance, Scanning electron microscope (SEM), energy dispersive x-ray spectroscopy (EDS), X-ray diffraction (XRD) and Fourier Transform Infrared Spectroscopy (FTIR) and digital electronic pH meter and atomic absorption spectrometry were used to analysis the corrosion process, corrosion rate and mechanism.
②Modification treatment was carried out for 4N-Mg and Mg-0.88Ca alloy by stearic acid alcohol solution SAM. Firstly, pre-treatment method, concentration of stearic acid, the time immersing in stearic acid solution were determined by orthogonal test. Secondly, high concentration and short time immersing method was compared with traditional low concentration and long time immersing one, and corrosion resistant property was also compared for the samples being SAM modified and by supersonic cleaned. 4N-Mg and Mg-0.88Ca alloy was modified by SAM method and the corrosion process of samples were studied.
③According to ISO 10993.4 standard to test the rate of hemolysis of 4N-Mg and Mg-0.88Ca alloy with and without modified by SAM, and the relationship between pH value and concentration of Mg2+ ion with hemolysis were analysis..
④L929 fibroblast and Osteoblast cell were employed to co-cultrue with the extracts of 4N-Mg and Mg-0.88Ca alloy, and MTT cytotoxicity tests were done and the concentration of Mg2+ ion is measured. Cytotoxicity of Mg-based materials was further evaluated by direct contact method, 4N-Mg and Mg-0.88Ca alloy SAM modified before and after were cultured with Osteoblast cell, and the effects of material on reproduction and activity of cell were discussed.
⑤Acute toxicity test was done by extracts of 4N-Mg with and without SAM modified. The experiment was carried out on mice, and they were injected into the abdominal cavity. Symptom, moving state and weight change of mouse were observed and recorded within 7 days after injection.
⑥In vivo test was carried on white rabbit’s, and the 4N-Mg and Mg-0.88Ca alloy with and without SAM modified were embedded in thigh bone. Examination by X ray method and the concerntration of Mg2+ ion in blood were done after 2, 6, and 12 weeks, respectively. The specimens were analyzed by SEM and EDS, and tissue slices were observed by optical microscope.
Conclusions:
①Research on untreated 4N-Mg and Mg-Ca alloy
1) 4N-Mg alloy had better corrosion resistance property than Mg-Ca alloy. And Mg-0.88Ca has the lowest corrosion speed among Mg-0.88Ca, Mg-1.42Ca and Mg-2.0Ca. The form of crrosion mainly exhibited the corrosion pit and the crack.
2) Though the analyse of the E-pH chart, only when the pH is higher than 11.38 the magnesium come into the passive region.The anion such as HCO3-、HPO42- in the SBF also could induce the corrosion of magnesium.
3) The rate of hemolysis of 4N-Mg and Mg-0.88Ca alloy was not qualified, and cytotoxicity of 4N-Mg and Mg-0.88Ca alloy meet the demand of biomaterial.
②The SAM which formed on 4N-Mg and Mg-0.88Ca in the 0.5 mol/L ethanol slution of stearic acid after soaked 1.5h had the best effect to the behavior of anti-corrosion to the samples. The SAM which formed in the higher concentration of stearic acid slution after little soaking time excelled in the one which formed in the lower concentration of stearic acid slution after long soaking time, and showed better bioactivity. The hemolysis and cytotoxicity of those samples treated by SAM were qulified,and had not iduced acute toxicity.
③In my test, the extract which pH under 10.61 and concentration of Mg~(2+) ion not exceed 42 mg/L, the hemolysis rate was qualified, and the exacts while the concentration of Mg~(2+) ion equal to 202.5mg/L met the cytotoxicity requirement, while no more than 156.2mg/L promoted the cell proliferation.
④In vivo study, the magnesium concentration in blood of two groups of animal subjects were in the normal scale, Mg-based materials have good bone inducement and showed better capabilty of contact with bone. The new bone mineralizes obviously on the interface. And on the new bone, the osteoclast cells array orderly. Untreated magnesium corroded faster than SAM treated magnesium-based materials. All results indicated that Mg-based materials showed better biocompobility and the capacity of inducing new bone, proved that SAM had an effect on controlling corrsion rate of Mg-based materials.
本研究以镁基材料为对象,对99.99%的纯镁(4N-Mg)以及Mg-Ca系合金在仿生体液中腐蚀降解过程和机制进行了研究。为改善其腐蚀行为和生物活性,对其进行了硬脂酸自组装膜改性处理,并对改性工艺机理和改性后的腐蚀行为进行探讨。为评价镁基材料的生物相容性和成骨行为,对镁基材料进行了溶血率、细胞毒性等体外相容性评价以及全身急性毒性和骨植入试验的体内相容性评价。
方法:
①使用模拟人体生理环境的仿生体液(SBF),对铸态4N-Mg和自行熔炼的铸态Mg-Ca系合金进行体外浸泡研究。利用XRD、SEM、EDS、FTIR、原子吸收光谱、电化学分析系统等设备和手段,分析讨论其腐蚀过程、速度和机制。
②使用硬脂酸自组装膜(SAM)改性方法对4N-Mg以及Mg-0.88Ca进行改性处理研究。首先通过正交试验筛选了包括试样的前处理方法、硬脂酸浓度和在硬脂酸乙醇溶液中的浸泡时间等工艺参数并进行验证;然后对所选用的高浓度短时间浸泡的方法与传统的低浓度长时间浸泡方法进行对比研究,并比较了SAM改性后再进行超声清洗的改性层的抗腐蚀性能。以最优工艺对4N-Mg以及Mg-0.88Ca进行改性处理,对改性后试样的腐蚀过程进行研究。
③对SAM改性前后的4N-Mg和Mg-0.88Ca材料的浸提液以及未处理4N-Mg浸提液的稀释液,依照ISO 10993.4的标准进行溶血率测试,并分析pH值、溶液中的镁离子浓度与溶血率关系。
④以L929成纤维细胞和MC-3T3E1成骨细胞与4N-Mg和Mg-0.88Ca材料的浸提液和未处理4N-Mg浸提液的稀释液,进行MTT细胞毒性测试,并测定稀释浸提液中的镁离子浓度。再以成骨细胞与SAM改性前后的4N-Mg和Mg-0.88Ca材料共培养,讨论材料对细胞增殖和活性状态的影响。
⑤对SAM改性前后4N-Mg浸提液作全身急性毒性试验,以纯种小白鼠为对象施以腹腔注射,给药后7d内观察小鼠的症状和活动状况以及体重变化。
⑥将SAM改性前后的4N-Mg和Mg-0.88Ca材料以及Ti6Al4V对照样植入白兔股骨,植入后各组2周、6周、12周时任取1只处死。进行X线片检查,血镁浓度测试,并对标本进行扫描电镜(SEM)观察、能谱分析(EDS)以及组织切片光镜观察。
结果:
①未处理4N-Mg和Mg-Ca系合金研究
1)Mg-0.88Ca、Mg-1.42Ca、Mg-2.0Ca三种镁钙合金中,钙含量最低的Mg-0.88Ca在SBF中显示较好的耐腐蚀性,4N-Mg显示更强的腐蚀抗力; 4N-Mg及Mg-0.88Ca损伤形式主要是腐蚀坑和网状裂纹,后者在个别处有颗粒腐蚀脱落的局部不均匀腐蚀现象;试样表面被Mg(OH)2、HA、CaMg(CO3)2等白色颗粒和微小的半球状的沉积物覆盖。
2)E-pH图分析表明,当pH值超过11.38后纯镁才进入到钝化区。SBF中HCO3-、HPO42-等阴离子也可以与镁发生反应导致其腐蚀。4N-Mg及Mg-0.88Ca材料细胞毒性满足要求,没有引起全身急性毒性反应,但溶血率不合要求。
②在0.5 mol/L硬脂酸乙醇溶液中浸泡1.5h所得自组装膜经过超声清洗后,对4N-Mg及Mg-0.88Ca的抗腐蚀行为有明显提高。其耐蚀性要优于在稀溶液中长时间浸泡所得膜层,并显示出更好的生物活性。其细胞毒性和溶血率均满足对生物材料的要求。对成骨细胞增殖的促进作用超过未处理试样,没有引起全身急性毒性反应。
③在本实验条件下,当pH值不超过10.61,Mg2+浓度不超过42mg/L时,溶液的溶血率不超过5%。Mg2+浓度小于202.5mg/L满足细胞毒性要求,而Mg2+为不超过156.2mg/L的稀释浸提液对成骨细胞增殖显示出促进作用。
④骨内植入试验中,动物血镁浓度均在正常值范围之内。镁基植入材料显示出较好的骨结合性和骨诱导性,新生骨组织矿化明显,新生骨层上有排列整齐的骨细胞。未改性材料降解快于改性材料,成骨活性上未见明显差别。结果表明镁基材料初步显示了较好的骨诱导性能,证实了SAM对镁基材料腐蚀降解的调控作用。
Biomaterials not only have important social effect in bettering the living quality of human beings, improving their health and saving their lives, but also become a new growth point in economic and scientific-technological areas. The potential worth of magnesium in biomaterials field has caused great interests of research, for its excellent biocompatiblity and similar mechnical properties to bone. Moreover, magnesium-based materials can become novel degradable biomaterials making use of its corrodibility.
In this paper, the corrosion degradation process and mechanism of 4N-Mg and Mg-Ca alloys in SBF were studied. In order to improve the corrosion behavior and biological activity of Mg-based materials, surface modification of self-assembled monolayers (SAM) using of stearic acid was studied, meanwhile the mechanism of SAM and the corrosion behavior of samples were studied. To evaluate the biocompatibility and behavior of bone healing of Mg-based materials, the biocompatibility tests in vitro such as hemolysis ratio, cytotoxicity, and systemic acute toxicity and in vivo test were taken.
Methods:
①Simulated body fluid (SBF) which closed to inorganic composition of human plasma was used to immerse casting 4N-Mg alloy which contains 99.99 % magnesium and casting Mg-Ca alloys. Electronic balance, Scanning electron microscope (SEM), energy dispersive x-ray spectroscopy (EDS), X-ray diffraction (XRD) and Fourier Transform Infrared Spectroscopy (FTIR) and digital electronic pH meter and atomic absorption spectrometry were used to analysis the corrosion process, corrosion rate and mechanism.
②Modification treatment was carried out for 4N-Mg and Mg-0.88Ca alloy by stearic acid alcohol solution SAM. Firstly, pre-treatment method, concentration of stearic acid, the time immersing in stearic acid solution were determined by orthogonal test. Secondly, high concentration and short time immersing method was compared with traditional low concentration and long time immersing one, and corrosion resistant property was also compared for the samples being SAM modified and by supersonic cleaned. 4N-Mg and Mg-0.88Ca alloy was modified by SAM method and the corrosion process of samples were studied.
③According to ISO 10993.4 standard to test the rate of hemolysis of 4N-Mg and Mg-0.88Ca alloy with and without modified by SAM, and the relationship between pH value and concentration of Mg2+ ion with hemolysis were analysis..
④L929 fibroblast and Osteoblast cell were employed to co-cultrue with the extracts of 4N-Mg and Mg-0.88Ca alloy, and MTT cytotoxicity tests were done and the concentration of Mg2+ ion is measured. Cytotoxicity of Mg-based materials was further evaluated by direct contact method, 4N-Mg and Mg-0.88Ca alloy SAM modified before and after were cultured with Osteoblast cell, and the effects of material on reproduction and activity of cell were discussed.
⑤Acute toxicity test was done by extracts of 4N-Mg with and without SAM modified. The experiment was carried out on mice, and they were injected into the abdominal cavity. Symptom, moving state and weight change of mouse were observed and recorded within 7 days after injection.
⑥In vivo test was carried on white rabbit’s, and the 4N-Mg and Mg-0.88Ca alloy with and without SAM modified were embedded in thigh bone. Examination by X ray method and the concerntration of Mg2+ ion in blood were done after 2, 6, and 12 weeks, respectively. The specimens were analyzed by SEM and EDS, and tissue slices were observed by optical microscope.
Conclusions:
①Research on untreated 4N-Mg and Mg-Ca alloy
1) 4N-Mg alloy had better corrosion resistance property than Mg-Ca alloy. And Mg-0.88Ca has the lowest corrosion speed among Mg-0.88Ca, Mg-1.42Ca and Mg-2.0Ca. The form of crrosion mainly exhibited the corrosion pit and the crack.
2) Though the analyse of the E-pH chart, only when the pH is higher than 11.38 the magnesium come into the passive region.The anion such as HCO3-、HPO42- in the SBF also could induce the corrosion of magnesium.
3) The rate of hemolysis of 4N-Mg and Mg-0.88Ca alloy was not qualified, and cytotoxicity of 4N-Mg and Mg-0.88Ca alloy meet the demand of biomaterial.
②The SAM which formed on 4N-Mg and Mg-0.88Ca in the 0.5 mol/L ethanol slution of stearic acid after soaked 1.5h had the best effect to the behavior of anti-corrosion to the samples. The SAM which formed in the higher concentration of stearic acid slution after little soaking time excelled in the one which formed in the lower concentration of stearic acid slution after long soaking time, and showed better bioactivity. The hemolysis and cytotoxicity of those samples treated by SAM were qulified,and had not iduced acute toxicity.
③In my test, the extract which pH under 10.61 and concentration of Mg~(2+) ion not exceed 42 mg/L, the hemolysis rate was qualified, and the exacts while the concentration of Mg~(2+) ion equal to 202.5mg/L met the cytotoxicity requirement, while no more than 156.2mg/L promoted the cell proliferation.
④In vivo study, the magnesium concentration in blood of two groups of animal subjects were in the normal scale, Mg-based materials have good bone inducement and showed better capabilty of contact with bone. The new bone mineralizes obviously on the interface. And on the new bone, the osteoclast cells array orderly. Untreated magnesium corroded faster than SAM treated magnesium-based materials. All results indicated that Mg-based materials showed better biocompobility and the capacity of inducing new bone, proved that SAM had an effect on controlling corrsion rate of Mg-based materials.
引文
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