纳米银的细胞生物学及透过血脑屏障的体外研究
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摘要
银作为一种长效性和耐候性的抗菌材料，由于其优良的抗菌效果，已被广泛的运用于各种护理产品和医疗器械中。由于这些产品大多是与人体体液或血液直接接触的，所以必须考虑到它对成纤维细胞、内皮细胞等与之接触的细胞的毒性，而目前对纳米生物材料与细胞间的相互作用及其对细胞结构与功能的影响的研究甚少。另一方面，纳米级的银粒子由于其超微性，会产生很多特殊的理化性质，例如高表面活性，强的吸附能力和高化学活性，使得它与常规生物材料相比更容易透过细胞膜上的孔隙进入细胞内或细胞内的各种细胞器内，还有可能会透过特殊的生理屏障，进一步蓄积于常规材料无法到达的深部组织。动物实验显示，将纳米银按临床给药方式给予大鼠后，给药组与阴性对照组相比有明显脑部神经元固缩，质核素生成更严重，血脑屏障(BBB)有轻微损伤。由于体内实验影响的因素较多，因而关于纳米银是否能透BBB尚缺乏定性的实验来证明，纳米银粒子是否会对构成屏障的脑微血管内皮细胞(BMVEC)和星形胶质细胞(AC)产生毒性，是否会对紧密连接(TJ)造成不可逆的损伤正等待研究去证明。
本课题以纳米银为主要研究对象，选择了与之粒径有不同程度差异的微米级材料作为对照材料，确定粒径分布后，采用医疗器械评价标准中有关固体材料的试验方法进行体外细胞毒性试验。结果表明，按常规的细胞增殖度法(浸提液法)和琼脂覆盖法(间接接触法)都不能测定到几种银材料的毒性，也不能区别纳米生物医药材料与常规医药材料之间生物相容性的差异。将标准中的方法进行改进，采用材料与细胞直接接触培养，分别以MTT生成和LDH释放作为检测指标进行银粒子的体外细胞毒性试验，考查其毒性效应和银粒子浓度、粒径之间的关系，确定可以引起机体毒性反应的银粒子的剂量并揭示纳米材料与常规材料之间体外细胞毒性的差异。结果表明，银材料的粒径分布对的体外细胞毒性试验结果有明显的影响。相同浓度的纳米级银粒子和与之粒径接近的微米级银粒子，比粒径较大的微米级银粒子的细胞毒性大。其中，粒径较大的微米银材料仅在高浓度(500μg/ml)时对细胞生长有显著影响，而纳米银细胞毒性与银粒子的浓度呈量—效关系，当纳米银浓度控制在100μg／ml以内时，其毒性较小。本实验的结果提示了，在对纳米生物医药材料进行安全性评价时不能局限于传统方法，应结合产品的性质和用途建立能反映其特性的评价方法来进行体外细胞毒性试验。
本课题进行了BMVEC和AC原代培养，并采用两种细胞共同培养建立了BBB体外模型，通过细胞间连接的形态学观察和跨膜电阻值(TEER值)测量对模型进行验证。建立的BBB体外模型细胞间的致密性高，在形态、结构上能较好地模拟在体状态的BBB，可为研究纳米粒子透过BBB提供稳定的实验工具。运用transwell系统，对两组银粒子进行透过BBB体外模型的研究，采用透射电镜进行细胞超微结构观察，分别检测了纳米级的银粒子和微米级的银粒子对BBB细胞的损伤、对细胞间TJ的破坏和细胞吞饮囊泡的影响。建立了培养液中银浓度测定的的火焰原子吸收(AAS)法：及痕量银测定的感应耦合等离子体质谱(ICP—MS)法。通过银浓度测定对细胞内外的银分布及在体外BBB中的透过进行研究，揭示两种银粒子材料进入细胞内和透过BBB能力的差异。结果表明纳米银能够通过胞饮进入到BMVEC和AC细胞内并引发细胞水肿，损伤细胞间紧密连接，造成BBB的完整性丢失，进而跨越BBB进入中枢神经系统。而微米银则不能被细胞所吞噬，也不能透过BBB。提示了，虽然纳米粒可消除或减弱BBB的限制，在中枢神经系统疾病的诊断和治疗方面有良好的前景，但是它对BBB的细胞的损伤和细胞间TJ的破坏等负面效应也是不可忽视的。
As an antibacterial materials of long-effective and good weatherability, silver had been applied to a broad range of products from health care to medical instruments. These agents were applied directly to living tissue or plasma, their cytotoxicity should be investigated, whereas the interactions between nano- biomaterial and target cell, and its influences on the cellular structures and functions were poorly studied. On the other hand, silver particles would give rise to some specific physical and chemical interactions for their super micro scales, such as high surface activity and chemical activity, and high adsorption. Because of those properties, nanoparticles may penetrate through the cellular gaps into the cell and organelles more easily than regular materials. Even, it could probably went through those physiological barrier, and accumulated in some deep tissue which regular materials could never reach. During the animal test, silver nanoparticles were given to SD rats as same as the clinical trail. Results showed that the blood-brain barrier (BBB) was injured , the nerve cell of the experiment group was pyknosis and nuclide was formed more signifficant than the control negative group. However there were so many unstable factors that would impact the in vivo test, whether the nanoparticles could penetrate through the BBB was still unknown. Still, more tests were needed to investigate wether nanoparticles would cause cytotoxicity to brain microvessel vescular endothelial cell (BMVEC) and astrocyte cell (AC) , wether it could lead nonreversible damage to the tight junction (TJ).
In this dissertation, we measured the diameter distributions of several kinds of nano-scale and micro-scale silver particles by SEM , and investigate the in vitro cytotoxity by test of extract and test of indirect contact, which were demonstrated in the International Standard for Biological Evaluation of Medical Devices-part 5: test for invitro cytotoxicity. No significant toxic effect were found in all the experiment group during those tests. Then we made a modification to the test of direct contact method. Cellular morphology, infrastructure changes, mitochondrial function (MTT assay), membrane leakage of lactate dehydrogenase(LDH leakage assay) were assessed under control and exposed conditions. By studying the relationship between the cytotoxicity and the concentration/diameter of the silver particles, we can determine the dose of silver which would cause toxic effects, and make clear the biocompatibility differences between nano-materials and regular biomaterials. The results demonstrated that silver nanoparticles caused more serious damage to L929 cell than silver nanoparticles under the same silver concentration, which meaned the property of the specimen must be considerated fisrtly during the in vitro cytotoxicity test. There was little influence to the cell fonction when the silver concentration was lower than 100μg/ml in nanoparticles group and 500μg/ml in microparticles group. It made a reference to the silver concentration limit of nano-silver products.
In this dissertation a method of coculture of BMVEC and AC of rats was used to establish an BBB model in vitro and evaluate BBB transcytosis and toxicity at the endothelial tight junction. Atomic Absorption Spectroscopy (AAS) methods and Inductively coupled plasma Mass Spaectrometer (ICP-MS) methods were used to determine the concentration of silver in the cell culture media. Silver distribution inside and outside the cytoplasm was determined and the permeation ratio of those two silver particles were calculated to raveal the different biological effects between nanoparticles and regular biomaterials. This in vitro experimental model of rat BBB was close to resemble the in vivo situation for examination of the permeability of nanoparticle and toxicity evaluation. The resullts showed silver nanoparticle was more easier to penetrate through the in vitro BBB model. In the nanoparticles group, the percentage of particles entering into cells were higher and the TJ damage was more signifficant than microparticles. Therefore, the penetration through BBB inferred to the both transcytosis process via transendothelial transport and paracellular permeation. Although the nanopartiles provided a great prospect to diagnosis and treatment of central nervous system (CNS) disease, its nonreversible damage to TJ should be considered as well.
本课题以纳米银为主要研究对象，选择了与之粒径有不同程度差异的微米级材料作为对照材料，确定粒径分布后，采用医疗器械评价标准中有关固体材料的试验方法进行体外细胞毒性试验。结果表明，按常规的细胞增殖度法(浸提液法)和琼脂覆盖法(间接接触法)都不能测定到几种银材料的毒性，也不能区别纳米生物医药材料与常规医药材料之间生物相容性的差异。将标准中的方法进行改进，采用材料与细胞直接接触培养，分别以MTT生成和LDH释放作为检测指标进行银粒子的体外细胞毒性试验，考查其毒性效应和银粒子浓度、粒径之间的关系，确定可以引起机体毒性反应的银粒子的剂量并揭示纳米材料与常规材料之间体外细胞毒性的差异。结果表明，银材料的粒径分布对的体外细胞毒性试验结果有明显的影响。相同浓度的纳米级银粒子和与之粒径接近的微米级银粒子，比粒径较大的微米级银粒子的细胞毒性大。其中，粒径较大的微米银材料仅在高浓度(500μg/ml)时对细胞生长有显著影响，而纳米银细胞毒性与银粒子的浓度呈量—效关系，当纳米银浓度控制在100μg／ml以内时，其毒性较小。本实验的结果提示了，在对纳米生物医药材料进行安全性评价时不能局限于传统方法，应结合产品的性质和用途建立能反映其特性的评价方法来进行体外细胞毒性试验。
本课题进行了BMVEC和AC原代培养，并采用两种细胞共同培养建立了BBB体外模型，通过细胞间连接的形态学观察和跨膜电阻值(TEER值)测量对模型进行验证。建立的BBB体外模型细胞间的致密性高，在形态、结构上能较好地模拟在体状态的BBB，可为研究纳米粒子透过BBB提供稳定的实验工具。运用transwell系统，对两组银粒子进行透过BBB体外模型的研究，采用透射电镜进行细胞超微结构观察，分别检测了纳米级的银粒子和微米级的银粒子对BBB细胞的损伤、对细胞间TJ的破坏和细胞吞饮囊泡的影响。建立了培养液中银浓度测定的的火焰原子吸收(AAS)法：及痕量银测定的感应耦合等离子体质谱(ICP—MS)法。通过银浓度测定对细胞内外的银分布及在体外BBB中的透过进行研究，揭示两种银粒子材料进入细胞内和透过BBB能力的差异。结果表明纳米银能够通过胞饮进入到BMVEC和AC细胞内并引发细胞水肿，损伤细胞间紧密连接，造成BBB的完整性丢失，进而跨越BBB进入中枢神经系统。而微米银则不能被细胞所吞噬，也不能透过BBB。提示了，虽然纳米粒可消除或减弱BBB的限制，在中枢神经系统疾病的诊断和治疗方面有良好的前景，但是它对BBB的细胞的损伤和细胞间TJ的破坏等负面效应也是不可忽视的。
As an antibacterial materials of long-effective and good weatherability, silver had been applied to a broad range of products from health care to medical instruments. These agents were applied directly to living tissue or plasma, their cytotoxicity should be investigated, whereas the interactions between nano- biomaterial and target cell, and its influences on the cellular structures and functions were poorly studied. On the other hand, silver particles would give rise to some specific physical and chemical interactions for their super micro scales, such as high surface activity and chemical activity, and high adsorption. Because of those properties, nanoparticles may penetrate through the cellular gaps into the cell and organelles more easily than regular materials. Even, it could probably went through those physiological barrier, and accumulated in some deep tissue which regular materials could never reach. During the animal test, silver nanoparticles were given to SD rats as same as the clinical trail. Results showed that the blood-brain barrier (BBB) was injured , the nerve cell of the experiment group was pyknosis and nuclide was formed more signifficant than the control negative group. However there were so many unstable factors that would impact the in vivo test, whether the nanoparticles could penetrate through the BBB was still unknown. Still, more tests were needed to investigate wether nanoparticles would cause cytotoxicity to brain microvessel vescular endothelial cell (BMVEC) and astrocyte cell (AC) , wether it could lead nonreversible damage to the tight junction (TJ).
In this dissertation, we measured the diameter distributions of several kinds of nano-scale and micro-scale silver particles by SEM , and investigate the in vitro cytotoxity by test of extract and test of indirect contact, which were demonstrated in the International Standard for Biological Evaluation of Medical Devices-part 5: test for invitro cytotoxicity. No significant toxic effect were found in all the experiment group during those tests. Then we made a modification to the test of direct contact method. Cellular morphology, infrastructure changes, mitochondrial function (MTT assay), membrane leakage of lactate dehydrogenase(LDH leakage assay) were assessed under control and exposed conditions. By studying the relationship between the cytotoxicity and the concentration/diameter of the silver particles, we can determine the dose of silver which would cause toxic effects, and make clear the biocompatibility differences between nano-materials and regular biomaterials. The results demonstrated that silver nanoparticles caused more serious damage to L929 cell than silver nanoparticles under the same silver concentration, which meaned the property of the specimen must be considerated fisrtly during the in vitro cytotoxicity test. There was little influence to the cell fonction when the silver concentration was lower than 100μg/ml in nanoparticles group and 500μg/ml in microparticles group. It made a reference to the silver concentration limit of nano-silver products.
In this dissertation a method of coculture of BMVEC and AC of rats was used to establish an BBB model in vitro and evaluate BBB transcytosis and toxicity at the endothelial tight junction. Atomic Absorption Spectroscopy (AAS) methods and Inductively coupled plasma Mass Spaectrometer (ICP-MS) methods were used to determine the concentration of silver in the cell culture media. Silver distribution inside and outside the cytoplasm was determined and the permeation ratio of those two silver particles were calculated to raveal the different biological effects between nanoparticles and regular biomaterials. This in vitro experimental model of rat BBB was close to resemble the in vivo situation for examination of the permeability of nanoparticle and toxicity evaluation. The resullts showed silver nanoparticle was more easier to penetrate through the in vitro BBB model. In the nanoparticles group, the percentage of particles entering into cells were higher and the TJ damage was more signifficant than microparticles. Therefore, the penetration through BBB inferred to the both transcytosis process via transendothelial transport and paracellular permeation. Although the nanopartiles provided a great prospect to diagnosis and treatment of central nervous system (CNS) disease, its nonreversible damage to TJ should be considered as well.
引文
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[46] Yasuto K, Ikumi T, Takeo N. Evaluation of Blood-Brain Barrier Transporters by Coculture of Brain Capillary Endothelial Cells with Astrocytes[J]. Drug Metabol Pharmacokin, 2002, 17 (1): 34-41.
[47] Abbott NJ. Inflammatory mediators and modulation of blood-brain barrier permeability[J]. Cell Mol Neurobiol, 2000, 20: 131-147.
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[53] 马丽娜，赵文静，洛涛，等大脑皮质星形胶质细胞体外条件化培养体系的建立[J]．吉林大学学报(医学版)，20004，30(5)，727—729．
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[61] IsobeI, Watanabe T, Yotsuyanagi T, et al. Atrocity contributions to blood-brain barrier formation by endothelial cells: a possible use of aortic endothelial cell for in vitro BBB model. Neurochem Int, 1996, 28(5-6): 523-533.
[1] Hoet PH, Bruske-Hohlfeld I, Salata OV. Nanoparticles-known and unknown health risks [J]. J Nanobiotechnology. 2004; 2: 12
[2] Andre Nel, Tian Xia, Lutz Madler,, et al. Toxic potential of materials at the nanolevel [J]. Science 2006; 311: 622-727
[3] Shvedova A A, Schwegler-berry D, Murray A R, et al. Exposure to carbon nanotube material: assessment of nanotube cytotoxicity using human keratinocyte cell [J]. J Toxicol Environ Health A 2003; 66: 1909-1926
[4] Hiu-Wing Lam, John T, Richard M et al. Pulmonary Toxicity of Single-Wall Carbon Nanotubes in Mice 7 and 90 Days After Intratracheal Instillation [J]. Toxicoi Sci 2004; 77: 126-134
[5] Michael P H, William H.F, Timothy D L, et al. Research Strategies for Safety Evaluation of Nanomaterials, Part Ⅱ: Toxicological and Safely Evaluation of Nanomaterials, Current Challenges and Data Needs [J]. Toxicol Sci 2005; 88(1): 12-17
[6] Missirlis. D, Tirelli. N, Hubbell, JA. Amphiphilic PEG-Based nanoparticles for the delivery of hydrophobic drug [J]. 7th World Biomaterials Congress 2004, 353
[7] Oberdorster G, Sharp Z, Atudorei V, et al. Translocation of inhaled ultrafine particles to brain [J]. Inhal Toxicol 2004; 16: 437-445
[8] Gunter Oberdorster, Eva Oberdorster, Jan Oberdorster, et al. Nanotoxicology: An emerging discipline evolving from studies of ultrafine particles. Environ Health Perspect 2005; 113(7): 823-839
[9] Liao D, Creason J, Shy C, et al. Daily variation of particulate air pollution and poor cardiac autonomic control in the elderly [J]. Environ Health Perspect 1999; 107: 521-525
[10] Hardman R.A Toxicologic review of quantum dots: toxicity depends on physicochemical and environmental factors [J]. Environ Health Perspect. 2006; 114(2): 165-72
[11] Bermudez E, Mangum J B, Wong B A, et al. Pulmonary responses of rats, mice and hamsters to subchronic inhalation of ultrafine titanium dioxide particles [J]. Toxicol Sci. 2004; 77(2): 347-57
[12] Renwick L C, Donaldson K, Clouter A. Impairment of alveolar macrophage phagocytosis by ultrafine particles [J]. Toxicol Appl Pharmacol 2001; 172: 119-127
[13] Warheit DB, Webb TR, Reed KL. Pulmonary toxicity screening studies in male rats with TiO2 particulates substantially encapsulated with pyrogenically deposited, amorphous silica [J]. Part Fibre Toxicol 2006; 26, 3:3
[14] 金一和，孙鹏，张颖花．纳米材料对人体潜在性影响问题[J]．自然 2002；23(5)：306-307
[15] Warheit DB, Webb TR, Reed KL. Pulmonary toxicity screening studies in male rats with TiO2 particulates substantially encapsulated with pyrogenically deposited, amorphous silica [J]. Part Fibre Toxicol. 2006; 26; 3(1): 3
[16] Wei lu, Yu-zhen Tan, Kai-li Hu, et al. Cationic albumin conjugated pegylated nanoparticle with its transcytosis ability and little toxicity against blood-brain barrier [J]. Int J Pharm. 2005; 295(1-2): 247-60
[17] 陈国明，刘岚．柠檬酸包埋的四氧化三铁纳米颗粒安全性研究[J]．江苏药学与临床研究2003；11：4-6
[18] Hansen T, Boutrand JP, Eloy R, et al. Biological tolerance of different nanoparticles in a rat model-clinical and histological findings. 7th World Biomaterials Congress, 2004: 752
[19] Karluss Thomas, Philip Sayre. Research Strategies for Safety Evaluation of Nanomaterials, Part Ⅰ: Evaluating the Human Health Implications of Exposure to Nanoscale Materials [J]. Toxicol Sci 2005; 87(2): 316-321
[20] Hoet P.H.M. Biological tolerance of different nanoparticles in a rat model-clinical and histological finding. 7th World Biomaterials Congress, 2004: 752
[21] 尹其华，刘璐等．59Fe示踪谷氨酸包被三氧化二铁纳米颗粒的制备及其在小鼠体内的生物学分布[J]．医学研究生学报 2005；18(4)：312-316
[22] Revell PA, Gatti AM, Gambarelli A, et al. Detection of cocr particles in the spleen of guinea pigs six weeks after their intra-osseous implantation. 7th World Biomaterials Congress, 2004: 753
[23] Hussain SM, Hess KL, Gearhart JM, et al. In vitro toxicity of nanoparticles in BRL 3A rat liver cells. Toxicol In Vitro. 2005 24
[24] Li Qian, Tang Meng. Study on cytotoxicity and oxidative effects of different size of hematite(Fe_2O_3)nanoparticles on CHL cells in vitro [J]. chin J Public May 2005; 21 (5): 589～591
[25] 孟纯阳，安洪．纳米羟基磷灰石／聚酰胺66与人胚胎成纤维细胞的生物相容性研究[J]．重庆医科大学学报 2005；30(2)，169-172
[26] 戴伯军，李家顺，等．纳米氧化锆强韧化高孔隙率磷酸钙人工古细胞支架生物学评价[J]．中国矫形外科杂志 2004；12(13)：998-1001
[27] 许艳慧，李四群，李志安．纳米羟基磷灰石复合材料的研究-纳米羟基磷灰石的细胞毒性[J]．实用口腔医学杂志 2004；20(2)：147-150
[28] Chen CN, Kuo CC, Chen LT. Evaluation of cellular compatibility and blood compatibility of electrospum bioabsorbable Nanofiber. 7th World Biomaterials Congress, 2004: 1741
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[53] 马丽娜，赵文静，洛涛，等大脑皮质星形胶质细胞体外条件化培养体系的建立[J]．吉林大学学报(医学版)，20004，30(5)，727—729．
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[1] Hoet PH, Bruske-Hohlfeld I, Salata OV. Nanoparticles-known and unknown health risks [J]. J Nanobiotechnology. 2004; 2: 12
[2] Andre Nel, Tian Xia, Lutz Madler,, et al. Toxic potential of materials at the nanolevel [J]. Science 2006; 311: 622-727
[3] Shvedova A A, Schwegler-berry D, Murray A R, et al. Exposure to carbon nanotube material: assessment of nanotube cytotoxicity using human keratinocyte cell [J]. J Toxicol Environ Health A 2003; 66: 1909-1926
[4] Hiu-Wing Lam, John T, Richard M et al. Pulmonary Toxicity of Single-Wall Carbon Nanotubes in Mice 7 and 90 Days After Intratracheal Instillation [J]. Toxicoi Sci 2004; 77: 126-134
[5] Michael P H, William H.F, Timothy D L, et al. Research Strategies for Safety Evaluation of Nanomaterials, Part Ⅱ: Toxicological and Safely Evaluation of Nanomaterials, Current Challenges and Data Needs [J]. Toxicol Sci 2005; 88(1): 12-17
[6] Missirlis. D, Tirelli. N, Hubbell, JA. Amphiphilic PEG-Based nanoparticles for the delivery of hydrophobic drug [J]. 7th World Biomaterials Congress 2004, 353
[7] Oberdorster G, Sharp Z, Atudorei V, et al. Translocation of inhaled ultrafine particles to brain [J]. Inhal Toxicol 2004; 16: 437-445
[8] Gunter Oberdorster, Eva Oberdorster, Jan Oberdorster, et al. Nanotoxicology: An emerging discipline evolving from studies of ultrafine particles. Environ Health Perspect 2005; 113(7): 823-839
[9] Liao D, Creason J, Shy C, et al. Daily variation of particulate air pollution and poor cardiac autonomic control in the elderly [J]. Environ Health Perspect 1999; 107: 521-525
[10] Hardman R.A Toxicologic review of quantum dots: toxicity depends on physicochemical and environmental factors [J]. Environ Health Perspect. 2006; 114(2): 165-72
[11] Bermudez E, Mangum J B, Wong B A, et al. Pulmonary responses of rats, mice and hamsters to subchronic inhalation of ultrafine titanium dioxide particles [J]. Toxicol Sci. 2004; 77(2): 347-57
[12] Renwick L C, Donaldson K, Clouter A. Impairment of alveolar macrophage phagocytosis by ultrafine particles [J]. Toxicol Appl Pharmacol 2001; 172: 119-127
[13] Warheit DB, Webb TR, Reed KL. Pulmonary toxicity screening studies in male rats with TiO2 particulates substantially encapsulated with pyrogenically deposited, amorphous silica [J]. Part Fibre Toxicol 2006; 26, 3:3
[14] 金一和，孙鹏，张颖花．纳米材料对人体潜在性影响问题[J]．自然 2002；23(5)：306-307
[15] Warheit DB, Webb TR, Reed KL. Pulmonary toxicity screening studies in male rats with TiO2 particulates substantially encapsulated with pyrogenically deposited, amorphous silica [J]. Part Fibre Toxicol. 2006; 26; 3(1): 3
[16] Wei lu, Yu-zhen Tan, Kai-li Hu, et al. Cationic albumin conjugated pegylated nanoparticle with its transcytosis ability and little toxicity against blood-brain barrier [J]. Int J Pharm. 2005; 295(1-2): 247-60
[17] 陈国明，刘岚．柠檬酸包埋的四氧化三铁纳米颗粒安全性研究[J]．江苏药学与临床研究2003；11：4-6
[18] Hansen T, Boutrand JP, Eloy R, et al. Biological tolerance of different nanoparticles in a rat model-clinical and histological findings. 7th World Biomaterials Congress, 2004: 752
[19] Karluss Thomas, Philip Sayre. Research Strategies for Safety Evaluation of Nanomaterials, Part Ⅰ: Evaluating the Human Health Implications of Exposure to Nanoscale Materials [J]. Toxicol Sci 2005; 87(2): 316-321
[20] Hoet P.H.M. Biological tolerance of different nanoparticles in a rat model-clinical and histological finding. 7th World Biomaterials Congress, 2004: 752
[21] 尹其华，刘璐等．59Fe示踪谷氨酸包被三氧化二铁纳米颗粒的制备及其在小鼠体内的生物学分布[J]．医学研究生学报 2005；18(4)：312-316
[22] Revell PA, Gatti AM, Gambarelli A, et al. Detection of cocr particles in the spleen of guinea pigs six weeks after their intra-osseous implantation. 7th World Biomaterials Congress, 2004: 753
[23] Hussain SM, Hess KL, Gearhart JM, et al. In vitro toxicity of nanoparticles in BRL 3A rat liver cells. Toxicol In Vitro. 2005 24
[24] Li Qian, Tang Meng. Study on cytotoxicity and oxidative effects of different size of hematite(Fe_2O_3)nanoparticles on CHL cells in vitro [J]. chin J Public May 2005; 21 (5): 589～591
[25] 孟纯阳，安洪．纳米羟基磷灰石／聚酰胺66与人胚胎成纤维细胞的生物相容性研究[J]．重庆医科大学学报 2005；30(2)，169-172
[26] 戴伯军，李家顺，等．纳米氧化锆强韧化高孔隙率磷酸钙人工古细胞支架生物学评价[J]．中国矫形外科杂志 2004；12(13)：998-1001
[27] 许艳慧，李四群，李志安．纳米羟基磷灰石复合材料的研究-纳米羟基磷灰石的细胞毒性[J]．实用口腔医学杂志 2004；20(2)：147-150
[28] Chen CN, Kuo CC, Chen LT. Evaluation of cellular compatibility and blood compatibility of electrospum bioabsorbable Nanofiber. 7th World Biomaterials Congress, 2004: 1741