降温速度对冷冻保存异体小静脉通畅率的影响

目的：探讨不同降温速度对异体小静脉内皮细胞代谢活性，超微结构和移植通畅率的影响。材料和方法；采用快速冷冻和二步冷冻两种不同的降温速度对异体小静脉进行冷冻，在液氮中保存４８小时后施行移植。观察方法四唑盐还原试验，扫描电镜、静脉造影及病理检查。结果：经二步冷冻保存的异体小静脉，其代谢活性和内此细胞形态得到良好保持。移植后排斥反应减弱，９０天通畅率８０％。结论：二小冷冻降温是异体小静脉冷冻保存理想的方法     

冷冻保存异体软骨移植免疫排斥反应的研究状况

背景：目前多采用冷冻保存方法来降低异体软骨移植免疫排斥反应,但有关异体软骨的取材、冷冻保存方法、冷冻保存条件仍然需要深入的研究探讨。 目的：对于冷冻保存异体软骨移植后免疫排斥反应机制的研究进行回顾分析,并对不同保存方法的特点进行比较分析。 方法：由第一作者检索1990/2008 PubMed数据及万方数据库有关异体软骨移植后免疫排斥反应及冷冻保存方法对软骨移植影响等方面的相关文献。 结果与结论：异体软骨组织移植治疗关节软骨缺损治疗效果明显优于其他治疗方法。冷冻保存异体关节软骨保持了软骨组织的性状和生物学活性,而且可择期完成关节重建,并且有充裕的时间完成多项指标检测,防止供体携带细菌病毒和传染性疾病的传播,并且降低了软骨组织的抗原性,具有较大的临床应用价值。但冷冻保存的各个环节,如:低温保护剂的应用、降温和复温速度等方面还存在诸多问题,软骨移植后仍然会出现软骨吸收、退变等现象。随着冷冻生物学的不断进步,冷冻损伤机制的不断揭示,这些问题终将会解决,软骨组织冷冻保存技术会得到进一步的完善。     

圆头精子的冷冻保存

目的:探讨圆头精子的冷冻保存效果及其精子功能改变。方法:6例圆头精子症患者各提供1-3份精液标本,应用不含卵黄冷冻保护剂和二步降温方法冷冻保存圆头精子症标本,检测冷冻精子活动率、头-尾膜完整率、存活时间和顶体蛋白酶活性。结果:圆头精子标本冷冻保存后,精子活动率显著减少,膜完整率显著下降,头膜损伤-尾膜完整的精子率显著升高(n=13,P＜0.01)。体外存活时间缩短。精子冷冻前后无顶体蛋白酶活性。不含卵黄冷冻保护剂与含卵黄冷冻保护剂,以及两步冷冻与多步冷冻的效果比较未见显著差异(n=7,P＞0.05)。结论:圆头精子可经历冷冻保存后存活,但冷冻复苏率低。冷冻-解冻过程显著降低精子功能。不含卵黄冷冻保护剂和两步降温可应用于冷冻保存圆头精子症标本。     

人骨软骨组织体外冷冻保存的效果

背景：异体骨软骨移植是治疗关节软骨缺损的有效方法,然而由于移植物体外有效保存时间短,限制了临床应用以及移植物的利用效率。 目的：观察梯度降温冷冻保存同种异体关节软骨的保存效果。 方法：将关节软骨块经体积分数10%二甲基亚砜预处理后,应用程序冷冻仪以-1 ℃/min行梯度降温至-4 ℃,-10 ℃,     -20 ℃,-40 ℃各30 min,最后至-80 ℃冷冻保存。 结果与结论：保存1,3,6个月及1年后检测发现,与新鲜组相比,冷冻后软骨细胞存活率、细胞活性以及浅、中、深层的蛋白多糖水平下降(P < 0.05）,且软骨细胞存活率、细胞活性以及浅、中、深层的蛋白多糖水平随冷冻保存时间延长而降低(P < 0.05）,提示冷冻保存可有效地延长骨软骨移植物存活时间。     

玻璃化法处理同种异体动脉的移植

背景:体外血管预处理及保存方法有多种,经过学者们长期大量的研究观察,各种方法均得到了改进,但是仍然存在弊端。因此找到一种更有效的或者几种体外血管预处理保存方法的结合,还需要大量的研究实践。目的:拟通过比较玻璃化法和传统冷冻法保存处理的同种异体血管移植后效果,找出一种更为实用且制作简便的血管处理方法。方法:选用健康青紫兰兔96只,手术切除双侧股动脉,根据不同体外血管预处理方法将实验分为3组,即新鲜血管自体移植组、冷冻+辐照预处理血管同种异体移植组及玻璃化+辐照预处理血管同种异体移植组。血管移植后1,2,8,12周,每组取6只动物进行腹主动脉数字减影血管造影、扫描电子显微镜、组织病理学观察,观察移植血管通畅率、动脉瘤形成情况及组织形态学变化。结果与结论:移植后12周,新鲜血管自体移植组血管的累计通畅率显著高于冷冻+辐照预处理组(P0.05),玻璃化+辐照预处理组与其他两组比较,差异无显著性意义(P0.05)。组织病理学检查显示,玻璃化+辐照处理的同种异体血管内膜及中层平滑肌增生较冷冻+辐照预处理组轻,管腔狭窄不明显,炎症反应较轻。经玻璃化法+辐照保存的同种异体血管制作程序简单,移植血管通畅率高,组织反应轻,是一种比较理想的同种异体血管体外处理方法。     

不同保存方法对后交叉韧带保存后组织形态学影响*

目的观察不同的低温保存方法对异体骨-后交义韧带-骨复合组织形态学的影响。方法新西兰白兔膝关节96个随机分成4组:新鲜对照组、二步冷冻保存组、-80℃冷冻保存组、-196℃冷冻保存组,后三组经保存二周后于37℃水浴快速复温,随即与新鲜对照组一起进行光镜、电镜观察组织形态学方面的变化。结果HE染色光镜观察:新鲜对照组在胶原纤维形态、排列整齐性及分布致密度均优于其它各组,二步冷冻保存组较新鲜对照组稍差,但明显优于其他二组,-196℃冷冻保存组织学表现最差。电镜观察:新鲜对照组在胶原微纤维排列整齐性及分布致密度方面优于其它各组,二步冷冻组较新鲜对照组差,但优于其它二组,-196℃冷保存组最差。结论二步冷冻保存能够较好地维持后交叉韧带的组织形态学特性。     

狗带血管坐骨神经的动物模型建立及冷冻保存的实验研究

目的：建立狗带血管坐骨神经的动物模型，探讨神经、动脉和静脉三种组织同时冷冻保存的可行性及不同维持温度对冷冻保存神经和血管的影响。方法：采用二步冷冻步骤，选择4种不同的维持温度(-40℃、-50℃、-60℃、-70℃)对狗带血管坐骨神经的动物模型进行冷冻，在液氮中保存21d，取出标本用37℃水浴复温。观察方法包括光镜下HE常规染色和髓鞘染色，四氮唑兰还原试验。结果：经-60℃维持温度冷冻后再保存于液氮中的动脉、静脉、神经，其血管代谢活性，内皮细胞和神经雪旺细胞的保持较好：结论：狗带血管坐骨神经的动物模型，解剖结构清晰，可操作性较好。神经、动脉和静脉三种组织同时冷冻保存是可行的，-60℃是带血管神经冷冻保存较理想的维持温度.     

冷冻保存胎兔周围神经异体移植

目的 :探讨胚胎兔周围神经经冷冻保存后 ,同种异体移植修复周围神经缺损的作用。方法 :以采用两步冷冻方法制备冷冻保存的胎兔坐骨神经 (维持温度值 - 4 0℃ )为供体 ,移植桥接成年兔腓总神经缺损区 ,并与自体神经移植、新鲜异体神经移植、空白缺损比较 ,16周后作再生神经电生理检测、神经断面图像分析及透射电镜观察。结果 :胎神经移植组神经再生效果不如自体神经好 ,但优于新鲜异体神经组。结论 :冷冻保存胎神经移植桥接周围神经缺损 ,可以促进和引到轴突再生 ,有可能代替自体神经移植     

冷冻保存同种异体软骨移植修复膝关节软骨缺损(英文)

背景:采用传统方法冻存后,关节软骨细胞存活率低,且软骨表层与深层的软骨细胞存活率差别较大,移植物易发生退行性变,导致手术失败。目的:经打孔梯度降温冻存膝关节软骨后并行异体移植,观察打孔梯度降温冻存对兔关节软骨的影响。方法:自2月龄新西兰白兔膝关节股骨膑面取骨软骨移植物,随机分为3组:实验组在软骨面以3mm×3mm矩阵打孔,梯度降温冷冻保存。以非打孔经梯度降温冷冻保存组、非打孔超低温冷冻保存组为对照。复温后移植到成年新西兰白兔相应膝关节缺损部位,观察各组移植物大体形态学、组织化学、免疫组织化学染色效果的差别。结果与结论:实验组、非打孔经梯度降温冷冻保存组大体形态学、组织化学、免疫组织化学染色效果均明显优于非打孔超低温冷冻保存组。实验组与非打孔经梯度降温冷冻保存组效果差别不明显,但实验组明显加强了对中间层软骨组织的保护程度。提示关节软骨的梯度降温冷冻保存效果优于快速降温冷冻保存;关节软骨打孔冷冻保存对深层细胞有一定的保护作用,提高了软骨细胞存活率,延缓了移植软骨组织的退变过程。     

胎兔周围神经低温保存温度值的实验研究

目的 :探讨胚胎兔周围神经低温冷藏后的改变。方法 :以 10 %DMSO(二甲基亚砜 )为低温保护剂 ,采用两步冷冻保存步骤 ,选择 4种不同的冷冻维持温度 (- 2 0℃、- 4 0℃、- 6 0℃、- 80℃ ) ,对孕龄 2 6d～ 2 8d的胎兔坐骨神经进行冷冻 ,维持 30min后 ,放入液氮中保存一周。复温后行四唑盐还原试验、组织学和超微结构观察 ,探讨冷冻维持温度对胎兔周围神经的影响。结果 :- 4 0℃维持温度冷冻后液氮保存的胎兔周围神经代谢活性和超微结构得到良好维持。结论 :两步冷冻法可以较好地保存胎兔周围神经的生物活性 ,为胎周围神经的冷冻保存和移植奠定了基础     

实验性冷冻保存带血管的同种异体骨移值

为探讨应用吻合血管的冷冻保存同种异体肋骨移植修复骨缺损的可能性，以15％二甲基甲砜(DMSO)作为低温保护剂，采用两步玲冻步骤，对狗的含后肋问血管的肋骨段进行冷冻处理，在液氟中保存96h后，施行同种异体移植于髂嵴骨缺损区。术后3周内使用免疫抑制剂。对移植体进行免疫学(白细胞个素Ⅱ、T细胞亚群)监测，SPECT扫描、血管造影和病理学分析。实验结果表明，同种异体移植肋骨段血循环丰富，骨细胞代谢活跃，未发生急性排斥反应，3个月达骨性愈台，取得了类似于吻合血管的自体骨移植的效果。     

Cryopreservation of rat islets of Langerhans by vitrification

Cryopreservation could be a possible means of addressing the shortage of islets of Langerhans. We investigated the effects of EDT324 solution on the vitrification of isolated rat islets of Langerhans. Rat pancreatic islets were cryopreserved in 10% DMSO by a slow-rate freezing method or were cryopreserved in EDT324 solution by vitrification. The cryopreserved islets were compared in terms of viability, stimulation index and metabolic function after transplantation. After cryopreservation, the viability and stimulation of islets stored in EDT324 were 92.4% and 6.4, respectively, and were higher than islets stored by slow freezing (72.5% and 1.5, respectively). Streptozotocin-induced diabetic rats were transplanted with islets cryopreserved in EDT324, which corrected diabetes and achieved euglycemia within 2?days after transplantation. These results indicate that EDT324 allows successful cryopreservation of rat islets for long-term storage as an alternative solution to traditionally used solutions, such as 10% DMSO. Transplantation of cryopreserved islets into diabetic rats can achieve euglycemia.     

Beneficial effects of freezing rate determined by indirect thermophysical calculation on cell viability in cryopreserved tissues

Many types of mammalian cells, such as sperm, blood, embryos, etc., have been successfully cryopreserved for the last few decades, while no optimal method for the cryopreservation of mammalian tissues or organs has been established, showing a poor survival after thawing with a low recovery of function. In this study, the freezing rate was determined by indirect thermodynamic calculation, and its potential effect on the cryoprotection of human saphenous veins and tissue-engineered bones was investigated. The vein segments were frozen according to the calculated freezing rate, using rate-controlled freezing devices, with a freezing solution composed of 10% dimethylsulphoxide and 20% fetal bovine serum in RPMI 1640 media. The efficacy of indirect calculation was assessed by the cell viability measured using fluorescence double-staining methods. The results indicated that the freezing rate determined by indirect calculation significantly (P < 0.05) maintained the post-thaw cellular viability of the blood vessel, particularly in terms of the endothelial cells. However, it exerted relatively less protective effect on the osteoblastic cell-cultured scaffolds. These results suggest that freezing-induced injuries may occur in tissues, and the freezing rate determined by indirect thermophysical calculation can be used for the optimization of tissue cryopreservation by minimizing the injuries.     

Spermatogonial survival after cryopreservation and short-term orthotopic immature human cryptorchid testicular tissue grafting to immunodeficient mice

BACKGROUND: Fertility preservation has become an urgent clinical requisite for prepubertal male cancer patients undergoing gonadotoxic treatment. As these patients do not yet produce spermatozoa for freezing, only immature tissue is available for storage. We studied the survival and proliferative activity of spermatogonia and Sertoli cells after cryopreservation of cryptorchid testicular tissue pieces followed by xenografting for 21 days. METHODS AND RESULTS: Single pieces of tissue from cryptorchid testes (2-9 mm(3)) of young boys (2-12 years) were cryopreserved, thawed and transplanted into the scrotum of mice. Quantitative morphometric and immunohistochemical techniques were used to evaluate the integrity of the tissue, as well as the survival and proliferative capacity of spermatogonia and Sertoli cells before and after freezing/thawing/grafting. Three weeks after grafting, cryopreserved tissue was removed and analysed. Most of the tubules (88.3%) were intact and there was no fibrosis or sclerosis, 14.5% of the initial spermatogonial population remained, as identified by the MAGE A4 antibody, and 32% of these cells showed proliferative activity evidenced by Ki67, compared to 17.8% before cryopreservation and grafting. The number of Sertoli cells was unchanged and 5.1% were Ki67-positive, compared to none at all before freezing and grafting. CONCLUSIONS: Through our orthotopic xenografting model, we have demonstrated the survival and proliferative activity of spermatogonia and Sertoli cells in cryopreserved immature human cryptorchid tissue. Testicular tissue banking may thus prove to be a promising technique for the preservation of fertility in prepubertal boys undergoing oncological treatments. As the stem cell niche is maintained, the cryopreserved tissue can potentially be used for future autotransplantation. In addition, whole tissue freezing does not exclude alternative clinical uses, including isolated cell transplantation after dissociation, selection and enrichment. However, as this work was done on cryptorchid tissue, studies on normal immature testicular tissue, involving longer grafting periods, are needed to demonstrate a differentiation capacity before clinical implementation. Ethical and safety issues should also be addressed.     

大段负重骨移植中的生物力学实验研究

用大段自体骨和同种异体骨在羊负重的胫骨上移植后,植骨要产生替代和再生过程。由于组织抗原与移植骨固有性的差异,对上述过程要有不同的力学反应。结果表明：植入骨的愈合,生长过程与其力上关。不同类型移植骨在愈合、生长过程生物力学性质和明显差异。同时也也证明深冷冻、辐照处理的同种异体骨是代替自体移植骨最可受的材料。     

Preclinical evaluation of ice-free cryopreserved arteries: structural integrity and hemocompatibility

Objective: Arterial allografts are routinely employed for reconstruction of infected prosthetic grafts. Usually, banked cryopreserved arteries are used; however, existing conventional freezing cryopreservation techniques applied to arteries are expensive. In contrast, a new ice-free cryopreservation technique results in processing, storage and shipping methods that are technically simpler and potentially less costly. The objective of this study was to determine whether or not ice-free cryopreservation causes tissue changes that might preclude clinical use. Methods: Conventionally frozen cryopreserved porcine arteries were compared with ice-free cryopreserved arteries and untreated fresh controls using morphological (light, scanning electron and laser scanning microscopy), viability (alamarBlue assay) and hemocompatibility methods (blood cell adhesion, thrombin/antithrombin-III-complex, polymorphonuclear neutrophil-elastase, β-thromboglobulin and terminal complement complex SC5b-9). Results: No statistically significant structural or hemocompatibility differences between ice-free cryopreserved and frozen tissues were detectable. There were no quantitative differences observed for either autofluorescence (elastin) or second harmonic generation (collagen) measured by laser scanning microscopy. Cell viability in ice-free cryopreserved arteries was significantly reduced compared to fresh and frozen tissues (p < 0.05). Conclusions: The formation of ice in aortic artery preservation did not make a difference in histology, structure or thrombogenicity, but significantly increased viability compared with a preservation method that precludes ice formation. Reduced cell viability should not reduce in vivo performance. Therefore, ice-free cryopreservation is a potentially safe and cost-effective technique for the cryopreservation of blood vessel allografts.     

Cryopreservation of artificial cartilage: viability and functional examination after thawing

In biomedical research and in reconstructive surgery, preservation of intact tissue has been an unsolved problem. In this study, we investigated the viability of cryopreserved artificial cartilage and its synthetic activity of cartilage-specific matrix proteins after thawing for in vitro use. A polymer fleece cylinder (diameter = 3 mm; height = 3 mm) was loaded with a suspension of bovine chondrocytes (25 x 10(6)/ml) and encapsulated with fibrin glue. After a culture period of 1 week, the artificial cartilage units were frozen in a cryoprotection solution containing 10% basal medium (RPMI 1640), 10% DMSO and 80% FCS. The freezing procedure consisted of three steps: a 30-min period at +4 degrees C followed by a 24-hour storage at -80 degrees C. After that, the tissue units were transferred into liquid nitrogen (-196 degrees C) for final storage. Using histochemical staining techniques of cryogenic slices, we investigated the ability of cryopreserved artificial cartilage to produce its specific matrix after thawing. A modified MTT assay was used to determine the viability of frozen tissue units in comparison with unpreserved samples at different moments after thawing. Depending on the chondrocytes used for the formation of artificial cartilage, the viability of cryopreserved tissue varied between 65 and 85%. Both the intensity of alcian blue staining for proteoglycans and the azan staining for collagens increased proportionally with incubation time after thawing. These findings indicate that cryopreservation of small artificial cartilage units is possible with a minor loss of cell viability. Secondly, its synthetic activity of cartilage-specific matrix did not decline after the freezing process.     

1H NMR spectroscopy as a tool to evaluate key metabolic functions of primary porcine hepatocytes after cryopreservation

Proton NMR spectroscopy of biological fluids has produced interesting results lately. We used the technique to investigate the effects of cryopreservation on primary porcine hepatocytes as successful cryopreservation of primary porcine hepatocytes is of importance to the development of bioartificial liver support systems. After isolation 10(8) hepatocytes were cryopreserved for 1 week in Williams E/10% DMSO, either by quick freezing (-5 to -30 degrees C/min), slow freezing (-0.3 to -3 degrees C/min) or stepwise freezing protocols on cell suspensions and confluent cell plates. Plating efficiency was assessed by percentage LDH release. Metabolic functions of cryopreserved hepatocytes at 24 h post-thawing were compared with those of fresh hepatocyte cultures at 48 h. 1H nuclear magnetic resonance spectroscopy of the culture medium post-incubation, using the presaturation technique, assessed the following: glucose metabolism, transamination and glutamine synthesis and succinate synthesis. Freshly isolated cells had a viability of 82 +/- 4.3% and a plating efficiency of 87 +/- 3.8%. All cryopreservation protocols resulted in significantly reduced viability and plating efficiency. No significant differences were observed between different cryopreservation media or protocols. When comparing cryopreserved with freshly isolated cells, we observed that metabolism of acetyl-CoA precursors was significantly impaired in cryopreserved cells. Lactate and pyruvate production was also significantly less, although glucose consumption was similar. No differences were observed in gluconeogenic amino acid metabolism, transamination and urea synthesis. 1H NMR spectroscopy can be used to provide information about metabolic activity and functions of cultured primary cells.     

不同冻存方法对羊膜超微结构和上皮细胞活性的影响

背景：羊膜冻存方法众多,对羊膜超微结构和生物活性的影响不一,目前尚无有效的冻存方法。 目的：比较不同冻存方法对羊膜超微结构和活性影响的研究,探寻更为理想的冻存方法。 方法：将新鲜羊膜采用深低温和玻璃化冻存法保存,分别于冻存后3,6个月复苏羊膜,以新鲜羊膜组织为对照组,比较羊膜的超微结构差异、羊膜上皮细胞离体氧分压和乳酸脱氢酶活性。 结果与结论：不同冻存方法保存的羊膜超微结构有明显改变,但玻璃化冻存对其超微结构的影响相对较小;与新鲜羊膜相比较,深低温冻存组3,6个月羊膜的乳酸脱氢酶灰度值和氧分压明显降低(P < 0.05);玻璃化冻存组6个月后的羊膜乳酸脱氢酶灰度值和氧分压明显降低(P < 0.05),而玻璃化冻存组3个月后的上述检测结果与新鲜羊膜相比差异无显著性意义(P > 0.05)。结果证实,羊膜的玻璃化冻存技术优于深低温冻存技术,不仅维持了羊膜的超微结构,而且保持了羊膜上皮细胞的功能和活性。 中国组织工程研究杂志出版内容重点：组织构建;骨细胞;软骨细胞;细胞培养;成纤维细胞;血管内皮细胞;骨质疏松;组织工程全文链接：     

足背浅静脉及其瓣膜的应用解剖

在专供研究用的21具(42侧)成人足标本上,解剖观测了足背浅静脉及其瓣膜,发现大、小隐静脉足背段瓣膜数分别为2.8个和1.4个;在第一～四跖背静脉汇入口处瓣膜出现率分别为70.0%,53.3%,85.3%,77.8%,在共干型的跖背静脉内瓣膜出现率达100%;在直径小于2mm的跖背静脉也观测到瓣膜。本文认为足背静脉(弓)作为移植体(静脉动脉化)来修复掌浅弓缺损可以减少临床修复掌浅弓手术的复杂性。但应充分重视足背静脉(弓)内及跖背静脉汇入口处的静脉瓣。