目的:建立可重复使用超声刀手术剪(简称超声刀)的清洗工艺确认方案,并对清洗有效性进行评价,为使用单位进行清洁方法确认和确保安全使用提供依据.方法:选择模拟血液污染物,按照确定的清洗工艺,选择最严苛的清洁方法挑战条件,通过对超声刀的可复用部件刀杆进行污染物负载-清洗数次后,提取残留污染物,并检测残留污染物中总蛋白和血红蛋白残留量.根据美国食品药品监督管理局(FDA)相关指导原则、美国医疗器械促进协会(AAMI)及美国材料和试验协会(ASTM)相关标准,设定可复用超声刀满足清洁要求的残留物限量指标(总蛋白残留量<6.4μg/cm2,血红蛋白残留量<2.2μg/cm2).结果:所建立的确认方案体系中,残留污染物的提取回收率均>79％;彻底提取验证研究中,单次提取回收率达到4次彻底提取回收的90％以上,满足实验要求.负载-清洗1次、4次、6次和8次清洗后,可重复使用超声刀总蛋白残留量均未检出,低于方法检测限(0.43μg/cm2);血红蛋白残留量均<0.34μg/cm2,低于确认方案体系的残留污染物中血红蛋白检测下限(1.10μg/cm2).结论:本研究建立的可复用超声刀手术剪清洁方法确认方案及清洁有效性评价方法;经验证,可重复使用超声刀8次,污染物负载-清洗后仍能满足所规定的清洁要求.

目的:研究乙醇工艺对同种异体肌腱的病毒灭活效果.方法:选择伪狂犬病毒(PRV)、猪细小病毒(PPV)、牛腹病毒性腹泻病毒(BVDV)和人类免疫缺陷病毒(HIV-1)为指示病毒,将同种异体肌腱(3个批号)分别负载指示病毒后,用75％乙醇浸泡灭活处理;分别在不同时间点取样,用96孔板的细胞病变法和Karber法测定,计算乙醇工艺处理后同种异体肌腱的病毒残留量(LgTCID50/0.1mL)和计算病毒降低量(log).结果:负载病毒的3个批号样品经过75％乙醇浸泡2h后,PRV、BVDV和HIV-1滴度降低量分别为≥5.971 logs、≥4.400 logs和≥8.000 logs;病毒灭活动力曲线显示:病毒残留量很快降到最低检出限度值.负载PPV的3个批号样品,经75％乙醇浸泡2h后,病毒滴度降低量平均为0.850 logs,浸泡延长至24 h后,病毒滴度降低量平均为1.350 logs,病毒灭活动力曲线显示病毒滴度随时间呈缓慢降低趋势.结论:75％乙醇工艺浸泡处理同种异体肌腱2h,对PRV、BVDV和HIV-1为有效工艺,对PPV无效.

目的:建立猪细小病毒(PPV)的细胞培养结合荧光定量PCR( ICC-qPCR)方法并进行优化,结合传统的病毒滴定(细胞培养法)及ICC-qPCR方法,考察将其应用于病毒灭活验证研究的可行性.方法:将系列10倍稀释的PPV病毒接种于猪睾丸(ST)细胞,分别扩增培养0、12、18、24 h,考察理想的病毒扩增培养时间,确定ICC-qPCR定量检测区间及获得病毒扩增倍数(K);将病毒平行接种于病毒扩增组及非扩增的对照组,通过扩增组和对照组的系列病毒接种量与PCR反应周期(Ct)值的拟合曲线,分别获得病毒扩增组及对照组的ICC-qPCR检测病毒滴度(Ta、Tc),结合扩增倍数(K),根据公式[Log10( l0Ta-10fc)/(K-l)]直接计算样本中的感染性病毒滴度.最后,将ICC-qPCR、优化ICC-qPCR方法和细胞培养法分别用于模拟样本(由不同比例感染性病毒和非感染性病毒组成)中的感染性病毒滴度测定,考察将其应用于病毒灭活验证研究的可行性.结果:PPV接种后的最佳扩增培养时间为24 h,检测下限为-1 Log10TCID50 · 100μL-1(Logs),定量区间为0～5.00 Logs,扩增倍数为83.11.当模拟样本中的非感染性病毒含量较低或与感染性病毒等量时,ICC-qPCR、优化ICC-qPCR方法和细胞培养法测得的感染性病毒滴度没有显著性差异;然而,当模拟样本中的非感染性病毒含量较高时,由优化的ICC-qPCR方法和细胞培养法测得的感染性病毒滴度分别是1.46、1.50 Logs,二者高度一致,而常规ICC-qPCR方法测得的结果(1.75 Logs )存在极显著性差异.结论:本研究所优化的ICC-qPCR方法作为细胞培养法的替代方法,具有用于病毒灭活验证研究的前景.

目的 验证猪源脱细胞结膜基质中病毒灭活工艺的灭活效果.方法 用不同浓度次氯酸钠溶液对猪睾丸(ST)细胞和牛肾(MDBK)细胞行细胞毒性实验;选择猪眼结膜、脱细胞结膜基质各3个批号,用ST细胞和MDBK细胞进行次氯酸钠灭活处理和钴-60(25 kGy)辐照处理后的样品/终止产物的细胞毒性实验;以猪细小病毒(PPV)和牛病毒性腹泻病毒(BVDV)为指示病毒,采用细胞病变法分别测定次氯酸钠工艺和钴-60(25 kGy)辐照工艺对猪脱细胞结膜基质(中间品)病毒灭活效果,并进行细胞盲传实验.结果 次氯酸钠溶液稀释至质量浓度100.00 mg/L(1.0‰)时,ST细胞增殖率大于70％;质量浓度50.00 mg/L(0.5 ‰)次氯酸钠溶液的MDBK细胞增殖率大于70％.1.0‰次氯酸钠溶液处理的猪眼结膜样品在10倍体积稀释后,对ST细胞和MDBK细胞均无抑制作用.脱细胞结膜基质辐照处理,经100倍体积稀释后对MDBK细胞无毒性,经10倍体积稀释后对ST细胞无毒性影响.3个批号猪眼结膜样品经过次氯酸钠溶液处理30 min后,PPV和BVDV的灭活平均降低系数分别为≥4.542 logs和≥4.333 logs;负载PPV病毒样品的盲传3代为阳性,即仍能检测到PPV.3个批号脱细胞结膜基质样品经钴-60辐照后,PPV和BVDV的灭活平均降低系数分别为≥4.792 logs和≥3.667 logs,负载BVDV的3个批号样品盲传结果为阴性,负载PPV病毒的3个批号样品盲传结果为阳性.结论 用次氯酸钠溶液和钴-60辐照两个工艺分别处理脱细胞结膜基质(中间品)后的病毒灭活降低系数累加为:BVDV总降低系数大于8 logs,PPV总降低系数大于9 logs.两个工艺联合可以有效灭活猪源脱细胞结膜基质中污染的PPV和BVDV.

To develop a precise and convenient method to evaluate the virus transmission risk of biologically sourced materials, an integrated cell culture-qPCR (ICC-qPCR) method for Pseudorabies virus (PRV) was established and revised for applications to this new field. The optimized post-infection period was found at 12-hr to achieve a reasonable detection limit ( - 0.25 Log(10)TCID(50)/100 mu L, Logs) and a quantitative range (0.75-3.75 Logs). The results of mimic samples suggested that three 10-fold dilutions at the time of virus inoculation combined with three washes after virus absorption, and the sets of non-amplified samples as controls could efficiently eliminate the false positive signals caused by high levels of noninfectious viruses. The virus inactivation validation studies of acellular porcine corneas suggested that the logs inactivation of PRV at 12 kGy irradiation dose obtained by general ICC-qPCR, revised ICC-qPCR and cell culture were 2.49, 4.85 and 5.08, respectively. At 25 kGy, those were 2.31, 4.85 and 5.08, respectively. The results obtained by the revised ICC-qPCR were consistent with cell culture and more precise than general ICC-qPCR. Therefore, the revised ICC-qPCR proposed in this study has an application prospect in the PRV inactivation validation studies of biologically sourced materials.

目的:建立细胞培养-核酸扩增荧光定量PCR(integrated cell culture quantitative PCR,ICC-qPCR)病毒灭活验证方法;结合传统细胞病变效应(cytopathic effect,CPE)法,考察ICC-qPCR方法的适用性;利用这2种方法验证复用电生理导管环氧乙烷病毒灭活工艺的有效性.方法:通过制备指示病毒(伪狂犬病毒,PRV)质粒DNA作为定量标准品,用PRV特异性引物建立定量PCR方法;再用PRV病毒侵染宿主细胞,同时用热灭活PRV作为对照,确定PRV活病毒全部进入宿主细胞但不扩增的最佳样本收获时间,通过检测细胞中活病毒,建立ICC-qPCR活病毒检测方法;确立CPE法测定滴度与ICC-qPCR法检测DNA拷贝数之间的线性关系.最后,模拟临床使用的最坏情况对一次性电生理导管进行含指示病毒(PRV)生物污染物负载,模拟环氧乙烷病毒灭活工艺,通过ICC-qPCR和CPE法验证病毒灭活工艺的有效性.结果:本研究中建立的ICC-qPCR方法的标准曲线线性关系r2＞0.99;定量下限为104 copies·μL-1,约相当于2 logs·mL-1,检测下限为103copies·μL-1,约相当于-1 logs·mL-1;特异性结果表明只能检测到PRV的扩增曲线,无非特异性扩增;重复性试验的RSD＜5％.确定ICC-qPCR方法中PRV最佳收获时间为宿主细胞染毒后1～2h.ICC-qPCR检测拷贝数与病毒滴度间具有良好的线性关系(r2＞0.99).ICC-qPCR法病毒灭活工艺验证结果表明:经环氧乙烷灭活后病毒负载导管其病毒滴度降低＞9.0 logs;阴性对照组(负载不合PRV生物污染物)和空白导管样品均未检出病毒DNA.CPE法的病毒滴定结果表明,经环氧乙烷灭活后病毒负载导管其病毒滴度降低≥8.5 logs·mL-1;阴性对照组和空白导管样品均未检出PRV.结论:本研究建立的ICC-qPCR方法应用于PRV病毒灭活验证,可以有效检测活病毒,比传统CPE法(-0.5 logs· mL-1)灵敏度高.将此方法应用于复用电生理导管的环氧乙烷病毒灭活工艺验证,同时与CPE法进行比较,验证了ICC-qPCR方法的适用性以及环氧乙烷灭活电生理导管负载的PRV病毒的工艺有效性.

由于胶原蛋白具有良好的生物相容性、可降解性、生物活性及可加工性等,作为再生医疗产品中重要的生物材料,被广泛应用于神经、骨、软骨、肌腱、韧带、血管植入物和皮肤修复中.为了保证胶原蛋白基再生医疗产品的安全和有效,有必要建立适当的质量控制体系.本文重点从原材料、产品性能、病毒去除/灭活、包装和灭菌等方面来探讨胶原蛋白基再生医疗产品质量控制的考虑要点,为该类产品的研发、生产及监管提供技术参考.

目的:动物源性医疗器械的主要安全风险是外源因子污染和不可预测的免疫学及毒理学反应.本文概述最新动物源性医疗器械的精准风险评价关键技术,为相关产品研发和注册审评提供参考,为科学监管提供技术支持.方法:总结自主研发的针对动物源性医疗器械安全风险和质量控制的检测与评价关键技术、方法、标准,以及免疫毒理学评价的几点专家共识.结果 和结论:动物源性生物材料DNA残留量测定、残留α-Gal抗原检测方法及标准已发布实施;研究建立了淋巴细胞增殖试验、采用Gal抗原缺失小鼠的免疫学评价方法等免疫毒理学评价技术手段.部分检测项目被《动物源性医疗器械注册技术审查指导原则》(2017修订版)收录.这些技术为动物源性医疗器械的精准风险评价和风险控制提供了技术支持.同时,作者展望了正在研究的风险评价新技术,提出了系统的精准风险评价体系仍需不断完善.

OBJECTIVES:To establish an experimental method for evaluating material permeability of type I collagen hydrogels.METHODS:Using BSA-FITC as an indicator, by combining BSA-FITC with PBS they were used as permeability media, and using transwell load hydrogen sample to detect BSA-FITC transparent rate.RESULTS:In the concentration range of 100 μg·mL-1~0.781 μg·mL-1, the standard curve R2 ≥ 0.99, Lower Limit of Quantity (LLOQ) is 3.125 μg·mL-1, RSD <5%, detection recovery rate is in the range of 80%~120%.CONCLUSIONS:In this study, we established an experimental method for evaluating material permeability of hydrogel. The BSA-FITC transparent rate of type I collagen hydrogel was 100% at 28 h.

Some single-use medical devices are reprocessed and reused in some countries in the world, but the regulatory approach is different, and in some countries it isn't regulated yet. In this article, the regulatory status quo of single-use medical devices is reviewed. The regulatory development, important regulatory documents and regulatory approaches of single-use medical device reprocessing in the United States, Germany and the UK are introduced. And how to perform scientific risk assessment and effective risk control is discussed. The information is useful to establish China-specific regulations, and to develop relevant standards, guidelines or specifications and the risk control strategies.

The reuse of high-cost single-use medical devices (SUD) is permitted in many countries, such as the United States, Germany and the United Kingdom, but strict regulatory requirements must be met. In addition to regulatory policies and regulations, such as market access mode and special requirements on Good Manufacture Practice (GMP), there are strict technical requirements on the potential risk control and quality assurance system. Therefore, effective risk assessment and risk control technology are the keys to ensure effective quality control and safe use of SUDs. In this article, based on analyzing the technological requirements of the national regulatory on SUDs in the United States, Germany and Britain, and combined with the review from latest relevant literature, to discuss the strategies of how to carry out scientific risk assessment. Some risk control technologies on the reuse of SUDs are introduced, which will provide support for the further study on risk control strategies and regulatory decisions for the reuse of SUDs in China.

目前,有关与人体神经系统直接或者间接接触的植入类医疗器械越来越多.这些植入类医疗器械可能会引起神经系统结构和/或功能的不利反应,导致广泛的副作用,这些副作用则被称为医疗器械产品的神经毒性.由于神经系统有限的修复能力,因此增加了临床前评价神经毒性的重要性.目前,还没有特定的标准或指南来规范医疗器械产品的神经毒性评价要求本文结合国内外最新的相关标准和参考文献,给出植入类医疗器械神经毒性评价的要点,为相关产品的临床前安全性评价、质量控制及注册前技术审评提供技术参考.

由于银具有很好的抗菌作用,自古以来人类就在医疗（如外用药品的磺胺嘧啶银软膏等）、餐具及食品保藏等领域充分利用银的抗菌作用。随着现代科学技术的发展,特别是纳米科学和纳米技术的发展,各种含纳米银或银化合物产品,如:含纳米银或银化合物敷料或者贴剂、银涂层导管等在我国已作为医疗器械在临床上应用。在评价这类产品中银的特殊生物效应和潜在风险时,首先需要充分表征含银产品中银的存在形式（纳米银还是化合态银,即:银离子）以及银的释放动态。银释放的量、释放的速率、纳米银还是化合态银等是预测和分析其中的银在体内暴露情况,及其进入体内后各种潜在风险的基础信息。特别是如果有纳米银颗粒物的释放和脱落时,需要进一步评价纳米银颗粒物进入体内的吸收、分布、代谢和排泄,在体内蓄积的靶器官以及可能存在的长期毒性风险。因此,含银敷料中银的表征和银释放动力学研究是该类产品风险评价的基础。针对产业发展和国家监管的需求,中国食品药品检定研究院（中检院）联合国家纳米科学中心等,起草了《含银敷料中银的表征和体外释放试验技术共识》（以下简称《技术共识》）。《技术共识》中所涉及的敷料中释放试验方法参照了《美国药典》及《中华人民共和国药典》（2015年版）关于药物贴剂的药物释放试验方法。其他表征和银的测定方法都给出了相应的可参考标准。本《技术共识》中给出的方法经过了起草小组不同实验室的验证,充分证实了其可实施性。2018年6月21日,中检院召开了"纳米技术产品质量检测及临床前评价技术论坛",有关国内纳米材料和毒理学领域专家、学者,包括医疗器械监管机构及检测机构、相关研究机构、企业、医疗机构代表,及中检院药品、医疗器械及药包材领域相关技术人员对该《技术共识》进行了充分的研讨,形成了专家共识。本《技术共识》供含银敷料类产品研发、检测及技术审评相关人员参考。

Bioresorbable vascular scaffolds(BVS) are new treatment strategies of percutaneous coronary intervention. They have been introduced to overcome limitations of bare metal stents (BMS) and drug-eluting stents(DES), since they provide temporary scaffolding and then disappear, liberate the treated vessel from cage. In this article, we review the current status and problems of BVS, various tests required before gaining regulatory approval for clinical use.

Objective To study the antibacterial activity of nanosilver on Staphylococcus aureus and the underlying mechanism. Methods The Staphylococcus aureus (ATCC6538), double strength nutrient broth and nutrient broth were provided by National Institute for Food and Drug Control. The diameter of silver powder was less than 100 nm (576832-5G, SIGMA-ALORICH). The minimal inhibitory concentration (MIC) of silver nanoparticles was determined against Staphylococcus aureus in culture at rotation speed of 300 r/min, and shaking-flask method was used to measure the optical density (OD) value of Staphylococcus aureus suspension at 50.00, 100.00, 200.00, and 800.00μg/mL. The structure of Staphylcoccus aureus treated with silver nanoparticles was observed by scanning electric microscopy. Results The results showed that MIC of silver nanoparticles against Staphylococcus aureus was 800.00μg/mL. The silver nanoparticles at 800.00μg/mL inhibited the growth of bacteria in nutrient broth completely. The growth curves showed that silver nanoparticles prolonged the lap phase of Staphylcoccus aureus. After exposure to 50.00μg/mL silver nanoparticles for 4 hours, it was observed that the cell wall was broken and the inner material flowed out, indicatied the death of cells. Conclusion It is demonstrated that silver nanoparticles have antibacterial activity on Staphylcoccus aureus, which could get into the cell and lead to cell death.

BACKGROUND:Hydrophobic acrylic foldable intraocular lens should be evaluated biologicaly with New Zealand rabbits as implant objects prior to clinical trial. OBJECTIVE:To observe the biological safety of hydrophobic acrylic foldable intraocular lens. METHODS: Twelve New Zealand rabbits were enroled. The right eyes were implanted with self-developed hydrophobic acrylic foldable intraocular lens (Shenyang Baiao Medical Device Co., Ltd., China) as experimental group, and the left eyes were implanted with Acrysof IQ SN60WF (Alcon, USA) as control group. RESULTS AND CONCLUSION: After implantation, there were no significant differences in the cornea, anterior chamber, implant position, posterior segment of eyebal between two groups. Tissue proliferation had no obviously difference between the two groups, and there were cornea and lens pouch inflammations. No macrophages and other inflammatory cels were visualized on the surface of intraocular lens, and fibrous tissues were found on the intraocular lens surface and in the haptics root. These findings suggest that the self-developed hydrophobic acrylic foldable intraocular lens has no difference from Acrysof IQ SN60WF widely used in clinic, and it has the biological safety.

Objective: To investigate the effects of silver nanoparticles( silver NPs) on the gene expression profile of rat embryo cells,and to further analyze the molecular mechanisms of silver- NP- induced toxicity. Methods: Embryo cells were treated with 20 μg·mL- 1silver NPs suspension( suspended in cell culture medium) for 48 hours, and then the RNAs were extracted. The gene expression profile of treated cells was analyzed compared to the control cells using DNA microarray technology. Results: Totally 355 differentially expressed genes which were induced by silver NPs were observed. Among them,132 of the genes were up- regulated,and 223 of the genes were down- regulated. Some biological responses related to the immune system were significantly activated,such as natural killer cell mediated cytotoxicity and B cell receptor signaling pathway. The biological process of phosphorylation of protein amino acids was inhibited. The secretory leukocyte peptidase inhibition factor and MMP3 etc. were increased significantly. Conclusion: Silver NPs induced global biological responses of embryo cells through activating some signal pathways,such as inflammatory and immune system related signal pathways. Embryonic cells protected themselves against injury and damage caused by silver NPs with upregulating secretory leukocyte peptidase inhibitor,etc. Silver NPs inhibited the biological process of protein amino acid phosphorylation and induced the high expression ofMMP3,which might further decrease various biological functions of the cells and cause serious damage to the cell matrix. In contrast to cancer cell lines or other immortal cells,embryo cells have much similar functions and properties to normal human cells. These new toxic mechanisms found in silver NPs exposed normal cells are very important for understanding the potential toxicity of silver NPs to humans. The present finding also contributes to a better understanding of the reproductive toxicity risks of silver NPs for medical applications.

Objective: To investigate the key factors and parameters of the in vitro cytotoxicity test(MTT assay and LDH assay) for assessing nanomaterials referring to ASTM,so that to provide evidences for establishing standards of nanomaterial’s cytotoxicity test.Methods: The detection wavelength of MTT assay and LDH assay were obtained by full-wavelength scanning;the concentration and storage condition of the positive control APAP were tested;the experimental factors of MTT assay were tested by changing the MTT media with or without glycine buffer after adding DMSO;the experimental conditions of LDH assay were measured by changing the concentration of serum(10%,5%,2.5%) in cell culture media.The cytotoxicity of the silver nanoparticle containing hydrogel was assessed by refined MTT assay and LDH assay.Results: The double-wavelength was found at 540-570 /680 nm and 490 /680 nm,which is suitable for MTT assay and LDH assay respectively,in this experimental system.The proper concentration of positive control APAP was 20-30 mmol.L-1,which should be stored at-20 ℃ and will be stable for 14 days.The serum contained in the cell culture media could affect the LDH test with a serum concentration-depended manner,but this kind of interference could be deducted by the blank control.The IC50 of silver nanoparticle-containing hydrogel tested by refined MTT assay and LDH assay were 26.8 mg.mL-1 and 61.8 mg.mL-1,respectively.Conclusions: Two assessment points can be obtained in one experiment by combining re-fined MTT assay and LDH assay,so that the cytotoxicity of nanomaterials can be evaluated more comprehensively.In addition,this study defines the key factors and parameters of the cytotoxicity test(MTT assay and LDH assay) for assessing nanomaterials,and improves the feasibility of the method.

Objective: To investigate the method for evaluating antibacterial activity of silver nanoparticles-containing dressing.Methods: Shaking-flask method was used,and the rotational speed and the culture temperature,which were important factors for bacteria growth,were investigated.The refined protocol was used to evaluate the antibacterial activity for 4 batchs of silver nanoparticles-containing dressing;while,the Inhibition Zone Test was used as comparative study.Results: The results of the culture condition(different rotational speed and different culture temperature) selection tests suggested that the condition of 200 r·min-1 rotational speed and 25 ℃,cultured for 1 hour were better for shaking-flask method.This refined method was further used for evaluating antibacterial activity of silver nanoparticles-containing dressing.The results showed that the bacterial inhibition rate were 97%,80%,80% and 90% for Staphylcoccus aureus;91%,78%,93% and 76% for Candida albicans in 4 batchs of silver nanoparticles-containing dressing,respectively.In contrast,Inhibition Zone Test could not reflect the antibacterial activity accurately to silver nanoparticles-containing dressing.Conclusion: This refined protocol of shaking-flask method is useful for examining antibacterial activity of silver nanoparticles-containing products accurately,and provided scientific evidences for the establishment of the examination standard of silver nanoparticles-containing products antibacterial performance.

To establish sterility testing method for nano-silver dressing,the experiment was conducted according to the method of the Chinese Pharmacopoeia,2005 edition volume Ⅱ.The result was tested by the direct inoculation method to 14 batches of nano-silver dressing.The sample had no obvious antibacterial activity.The sterility testing method for the nano-silver dressing is accurate and reliable.