EXPERIMENTAL STUDY OF INFLUENCE OF LIQUID NITROGEN PRESERVATION ON ANTIGENICITY OF HUMAN HOMOGRAFT AORTIC VALVE

To illustrate the effect of liquid nitrogen preservation on antigenicity of human homograft aortic valve (HAV), human aortic valve tissue were cocultured together with peripheral blood mononuclear cells (PBMCs). Following detections were done to show the difference of antigenicity of HAV indirectly at different period after being preserved in liquid nitrogen: ① 3H TdR incorporation (cpm) was observed after tissue cell cocultured and stimulation index (SI) was calculated;② Density of IL 2 in medium was measured by MTT means. The results indicated that antigenicity of fresh HAV was the strongest; with the prolong of being preserved, antigenicity decreased gradually. It decreased significantly within first 48 h preserved at 4℃, then decreased significantly after preserved in liquid nitrogen for 24 h. 2 weeks later, antigenicity decreased to the lowest level.

Comparative investigation of the calcification of the cryopreserved aortic and pulmonary homografts after implantation

Objective:Thisexperimentwasperformedto investigatethecalcificationof thecryopreservedaorticand pulmonaryhomograftsquantitivelyafterimplantation.Methods:Inoperationtheheterotopicimplantationof homo-graftconduitsintotheabdominalaortawasadopted.Thecalcificationof thehomograftconduitswasdeterminedby SX microelectronicprobeanalysis8wk afterimplantation.Results:Beforeimplantationthecalciumcontentof aor-tichomografts[(0.24±0.05)μg/mg]was significantlyhigherthanthatof pulmonaryhomograft[(0.13±0.02)μg/mg,P<0.01].Afterimplantationthecalciumcontentof arotichomografts[(0.53±0.09)μg/mg]was significantlyhigher thanthatbeforeimplantation[(0.24±0.05)μg/mg], withan increaseby(123.4±53.0)%(P<0.01),andso was thatof pulmonaryhomograftsfrom [(0.13±0.02)to(0.21±0.30)μg/mg],withan increaseby(63.8±35.8)%(P<0.01).And thecalciumcontentof aortichomograft[(0.53±0.09)μg/mg]wasstillsignificantlyhigherthanthatof pulmonaryhomo-graft[(0.21±0.03)μg/mg,P<0.01].Conclusion:In homograftconduitimplantationtheaortichomograftsaremore easilycalcifiedthanpulmonaryhomografts.TheMicroelectronicprobetechniquehasa greatsignificanceinelement componentanalysisof biologicalpreparation.

Long-Term Structural Valve Degeneration in Cryopreserved Mitral and Aortic Homograft: Is Pregnancy a Factor?

Objective: This study reports the outcomes of the cryopreserved mitral homograft in 119 patients prospectively followed with clinical, echocardiographic and structural valve deterioration assessments. Methods: 119 patients undergoing mitral and aortic homograft implantation. Patient’s causes of mitral disease were: rheumatic disease (n = 75), endocarditis (n = 37) and others (n = 7). There were 40 partial homografts and 88 total homografts. Mitro-aortic homograft valve replacement was performed in 29 cases. Results: Mean follow-up was 9.8 ± 6.3 years (up to 19.2 yrs). There were 7 early (2

USING CRYOPRESERVED HOMOGRAFT PERICARDIAL PATCH IN STAGED COMPLETE REPAIR FOR TETRALOGY OF FALLOT

Objective To compare the difference of effect while using homograt pericardium patch and Gore-rex patch in staged complete repair for Tetralogy of Fallot （TOF） to enlarge the right ventricular outflow tract （RVOT）. Methods Twenty-eight patients with TOF underwent the staged complete repair. Gore-rex patches were used to enlarge the RVOT of 13 patients, and cryopreserved homograft pericardium patches were used to enlarge the RVOT of 15 patients. The patients were followed up with 2-dimensional echocardiography and chest x-ray. Results One operative death in Gore-rex patch group, the mortality was 7. 7% ; 1 early postoperative death in cryopreserved homograft pericardium patch group, the mortality was 6. 7%. Between 2 groups, hemostasia time in operation room with significant difference （ P 〈 0. 01 ）, pericardial cavity drainage volume with difference （ P 〈 0. 05 ）. Gore-rex patch group was followed up 2 to 4.5 years, homograft pericardium patch group was followed up 0. 8 to 2.1 years. Echocardiography showed that there was significant difference of the residual obstruction at RVOT level （ P 〈 0. 01 ）. No calcification shadow was discovered on the chest x-ray of both groups. Conclusion Homograft pericardium is tissue with high density and intensity, its elasticity and compliance are good. Using homograft pericardium patch maybe helpful to decrease the residual obstruction of RVOT after operation. It can be adapted as a repairing material in cardiac surgery.

同种异体带瓣管道的研究进展

根治复杂性心瓣膜病需采用心外管道,目前心外管道有人工材料、同种异体带瓣管道(VHC)及异种带瓣管道。VHC具有符合人体的抗钙化、抗感染和无需终生抗凝等生理特点,然而VHC仍存在供体短缺和无法长期保存等问题,导致其无法被大力推广,但深低温保存技术有望解决长期保存这一问题。笔者综述了VHC供体的筛选、保存方法及其临床应用的研究进展,为VHC在临床上对左、右心室流出道的重建提供参考。

Quantitative analysis of host cells growing into canine homograft valved aortic and pulmonary artery

Background Cryopreserved conduit valved homografts （CVH） have been widely used in surgical treatment of cardiac disease. This study aimed to determine the extent of host cell ingrowth and the durability and immunogenicity of CVH,and to compare the performance of CVH stored at 4℃ and CVH cryopreserved in liquid nitrogen at -196℃.Methods Heterotopic transplants of canine CVH stored at 4℃ （n=14） and cryopreserved in liquid nitrogen （n=14） were made onto the abdominal aorta of recipient dogs. Animals were sacrificed at 7 and 15 days and at 1, 3, 6, 9, and 12months after transplantation to excise the implanted CVHs. Tissue DNA extraction and quantitative polymerase chain reaction （PCR） were performed to calculate the ratio of donor cells and host cells in the CVH. The tissue viability of CVH after implantation was analyzed by detecting alkaline fibroblast growth factor 2 （FGF-2） using immunohistochemical staining and by observation under transmission electron microscope and scanning electron microscope.Results All the animals survived and recovered well. There were few repopulating host cells （0.04-0.83%） in the implanted CVH at 7 or 15 days. The ratio of ingrowing host cells into the CVH continued rising after implantation and reached 40%-47% in the 12th month postoperation. Histology, transmission electron microscopy and FGF-2immunohistochemical staining indicated that fibroblasts and the host＇s endothelial cells were the main cellular elements invading the CVH. There were no significant differences in results between CVH stored at 4℃ and CVH cryopreserved in liquid nitrogen.Conclusions Host cells growing into CVH are very important for maintaining the long-term structure and function of the implanted CVH. There is no significant difference between CVH storing at 4℃ or in liquid nitrogen in regard to the ingrowth of host cells or of morphologic features after CVH allografting.

CLINICAL USE OF HOMOGRAFT STORED BY VITRIFICATION

Using 20% DMSO and 6% propylene glycol in Kreb' Ringer phosphate solution as cryopotective agent for homograft vitrification storage, the viability of stored bomograft (79.2%) was higher than that of slow cooling storage (59.7%). About 540000 cm2 of vitrified homograft were used to cover the wounds after excision of burn eschar in 135 pateints with major burns. The take-rate was over 94%. The cryopreservation of skin by vitrification can improve the viability and quality of skin and save time and cost.

Handmade tri-leaflet ePTFE conduits versus homografts for right ventricular outflow tract reconstruction

Background This study aimed to investigate the performance of handmade tri-leaflet expanded polytetrafluoroethylene(ePTFE)conduits in the absence of a suitable homograft.Methods Patients who underwent right ventricular outflow tract reconstruction with tri-leaflet ePTFE conduits or homografts between December 2016 and August 2020 were included.The primary endpoint was the incidence of moderate or severe conduit stenosis(>36 mmHg)and/or moderate or severe insufficiency.The secondary endpoint was the incidence of severe conduit stenosis(>64 mmHg)and/or severe insufficiency.Results There were 102 patients in the ePTFE group and 52 patients in the homograft group.The median age was younger[34.5(interquartile range:20.8-62.8)vs.60.0(interquartile range:39.3-81.0)months,P=0.001]and the median weight was lower[13.5(10.0-19.0)vs.17.8(13.6-25.8)kg,P=0.003]in the ePTFE group.The conduit size was smaller(17.9±2.2 vs.20.5±3.0 mm,P<0.001)and the conduit Z score was lower(1.48±1.04 vs.1.83±1.05,P—0.048)in the ePTFE group.There was no significant difference in the primary endpoints(log rank,T3=0.33)and secondary endpoints(log rank,P=0.35).Multivariate analysis identified lower weight at surgery[P=0.01;hazard ratio:0.75;95%confidence interval(CI)0.59-0.94]and homograft conduit use(P=0.04;hazard ratio:8.43;95%CI 1.14—62.29)to be risk factors for moderate or severe conduit insufficiency.No risk factors were found for moderate or severe conduit stenosis or conduit dysfunction on multivariate analysis.Conclusion Handmade tri-leaflet ePTFE conduits showed acceptable early and midterm outcomes in the absence of a suitable homograft,but a longer follow-up is needed.

两种细胞洗涤剂制备脱细胞同种生物带瓣管道支架的研究

目的 比较去氧胆酸钠(deoxycholic acid,DOA)和十二烷基硫酸钠(sodium dodecylsulphate,SDS)两种细胞洗涤剂制备脱细胞的同种生物带瓣管道(homograft bioprosthetic tube valved,HBTV)支架的效果,为体外构建组织工程瓣(tissue- engineering heart valve,TEHV)提供同种生物瓣支架材料。 方法 对同种带瓣主动脉管道和带瓣肺动脉管道,分别用含0 .5 % DOA或0 .0 3% SDS的Tris低渗缓冲液共同孵育4 8h,脱去经液氮保存、具有活性的HBTV表面的内皮细胞,固定剂固定后分别进行HE、胶原纤维和弹力纤维染色的光镜观察,以及扫描电镜观察,摄片。 结果 两种洗涤剂均完全地脱去了HBTV表面的内皮细胞,中心部位的成纤维细胞有部分存留;细胞外基质,如胶原纤维和弹力纤维等均较完整地保留下来,其形态结构与脱细胞前无明显改变。 结论 0 .0 3%～0 .1% SDS或0 .5 % DOA的低渗缓冲液对制备HBTV脱内皮细胞支架效果均较佳,有利于HBTV再内皮化时种植细胞的黏附和扩增,可进一步应用于体外构建TEHV的研究。对于脱主动脉壁的细胞,SDS浓度应增至0 .1%为佳。

经液氮保存具有活性的同种生物瓣脱细胞支架的制备

目的为体外构建组织工程瓣提供脱细胞的同种生物瓣支架材料,并建立相应的技术方法。方法选取经液氮保存、具有活性的同种生物带瓣管道作为实验材料,用含十二烷基硫酸钠(SDS)的Tris低渗缓冲液脱去同种带瓣管道存在的细胞;固定剂固定后,分别行HE染色、胶原纤维和弹力纤维染色的光镜和扫描电镜观察、摄片。结果用质量浓度为0.03%SDS的Tris低渗缓冲液与同种瓣膜共同孵育48h,完全脱去了表面的内皮细胞(ECs),又较完整地保留了胶原纤维和弹力纤维等细胞外基质主要成分,其形态学结构在脱细胞前后无明显改变;瓣叶中心部位有部分成纤维细胞存留。而对于脱主动脉壁细胞,质量浓度为0.1%SDS更合适。结论质量浓度为0.03%～0.1%SDS的低渗缓冲液对制备脱细胞的同种生物瓣支架效果较佳,有利于同种瓣再内皮化时种植细胞的粘附和扩增,可进一步应用于组织工程瓣体外构建的研究。

同种带瓣管道用于右心室流出道重建的中期效果

目的:评价同种带瓣管道用于右心室流出道重建的中期效果。方法:回顾性收集我院2007-10至2014-07应用同种带瓣管道重建右心室流出道的122例患者资料。随诊患者术后死亡、再次手术以及同种带瓣管道衰败情况。根据手术方式分为Ross手术组(n=38)和非Ross手术组(n=84);根据中位年龄分为≤6岁组(n=61)和>6岁组(n=61);按带瓣管道类型分为主动脉带瓣管道组(n=21)和肺动脉管道组(n=101);按管道直径大小分为≤19mm组(n=31)和>19mm组(n=91)。分析患者术前一般信息、不同管道类型、直径、应用病种与预后的关系。结果:平均随访(35.4±22.2)个月,有2例(1.6%)患者随访期间死亡。全部患者术后1年、5年、7年管道免于衰败率分别为94.2%、81.2%、75.4%。肺动脉同种瓣组1年、5年、7年管道免于衰败率分别为96.2%、86.1%、79.9%,主动脉同种瓣组分别为80.0%、59.7%、59.7%,两组差异有统计学意义(P=0.011)。Ross手术组1年、5年、7年管道免于衰败率分别为96.4%、89.0%、89.0%,非Ross组分别为91.3%、78.3%、67.1%,两组差异有统计学意义(P=0.045)。而患者年龄、不同管道直径、术前是否紫绀、是否二次及以上手术对于管道免于衰败率差异无统计学意义(P均>0.05)。结论:同种带瓣管道用于先天性心脏病右心室流出道重建中期效果良好,远期防止同种带瓣管道衰败是临床进一步研究的重点。

人同种心包补片在法洛四联症分期手术中的应用

目的比较人同种心包补片和Gore-tex补片在法洛四联症(tetralogy of Fallot,TOF)分期手术中扩大右心室流出道(right ventricular outflow tract,RVOT)的效果.方法 28例已行一期手术再行分期手术的TOF患者,按不同的二期或三期手术时间分为两组,Gore-tex组:13例,以Gore-tex补片扩大RVOT;人同种心包补片组:15例,以深低温冷冻保存的人同种心包补片扩大RVOT.比较两组术后临床结果和随访结果.结果 Gore-tex补片组术中死亡1例(7.7%),人同种心包补片组术后早期死亡1例(6.7%).人同种心包补片组住ICU时间、呼吸机使用时间和心包腔引流量均少于Gore-tex补片组(P＜0.01).所有患者均获得随访,随访时间0.8～4.5年,超声心动图检查提示Gore-tex补片组患者术后RVOT水平残余梗阻的比率高于人同种心包补片组(P＜0.01),胸部X线片未发现钙化影.结论人同种心包组织致密,缝合后不易渗血,止血方便;且具有良好的弹性和顺应性,有助于降低术后RVOT水平的残余梗阻发生,可作为补片材料在心脏外科手术中应用.

保存方法对同种异体肋软骨移植的影响

[目的 ]探讨不同保存方法对同种异体肋软骨移植的影响。 [方法 ]对 4 0只家兔肋软骨进行Co6 0 照射、酒精浸泡、冷冻及中药试剂浸泡四种方法处理后 ,植入不同家兔鼻背部 ;1 2周后取出 ,进行吸收率和组织学观察。 [结果 ]放射组肋软骨的平均吸收率最低 (1 1 .7% ) ,酒精组最高 (2 3.6 8% ) ;移植后的组织反应 ,中药组最弱 ,酒精组最强。 [结论 ]同种异体肋软骨移植后组织学的改变与吸收程度无正相相关联系。传统的冷冻。

同种带瓣大动脉重建右心室流出道

目的 应用自行采集和冷冻保存的同种带瓣大动脉 (VHC)完成先天性心脏病右心室流出道的重建 ,并观察其疗效及存在的问题。 方法 用 VHC材料治疗先天性心脏病 95例 ,90例手术根治 ,5例行 VHC右心室流出道与肺动脉连接姑息手术。 结果 院内死亡 13例 ;术后随访 6 8例 ,死亡 2例 ,均为感染。 6 6例长期生存者中 2 5例胸部 X线片示 VHC有钙化 ,多为主动脉材料 ,仅 5例有轻中度压力阶差 (35～ 6 0 mm Hg)。 结论 VHC可广泛用于治疗复杂先天性心脏病。程序降温、超低温保存和两步化冻是 VHC使用质量的保证。 VHC的长期通畅比合成管道好 ,随植入时间延长钙化率增加 ,肺动脉 VHC优于主动脉。有肺动脉高压者宜尽早手术。为了预防 VHC植入后感染 ,应重视、改进其收集和保存的方法。

新生动物同种主动脉带瓣管道移植的免疫反应与钙含量的相关性

目的 :探讨新生动物同种主动脉带瓣管道 (VAHC)移植后免疫反应与钙含量之间关系。方法 :体重 15 0～ 2 0 0g的新生新西兰兔 2 4只作为 VAHC的供体 ,0 .9～ 1.2及 2 .1～ 2 .9kg的幼兔和成年兔各 12只为受体 ,建立新生兔的 VAHC移植模型。术后 1个月和 6个月分别取材作免疫荧光染色及钙含量测定。结果 :免疫荧光检查显示各移植组 VAHC血管壁 Ig G荧光染色结果均为阳性。半定量分析显示植入幼兔体内 1个月及成年兔 1个月和 6个月体内的阳性积分显著高于植入幼兔体内 6个月的 VAHC(P<0 .0 5 )。各移植组 VAHC血管壁的钙含量及植入幼兔体内 6个月和成年兔体内瓣膜的钙含量显著高于对照组 (P<0 .0 5 )。血管壁免疫荧光强度积分结果与钙含量呈明显的正相关 (r=0 .6 2 ,P<0 .0 5 )。结论 :新生兔的

具有活性的同种生物静脉脱细胞支架的制备

目的制备同种异体、无免疫排斥反应的脱细胞生物支架材料,并建立相应的技术方法,为体外构建组织工程胆管提供实验准备。方法选取新鲜具有活性的同种生物静脉作为实验材料,用含0.03%十二烷基硫酸钠(SDS)的Tris低渗缓冲液,以改进的BOOTH法脱去静脉壁的内皮细胞,保留细胞外基质。固定剂固定制成的脱细胞支架,分别行HE染色、胶原纤维染色的光镜和扫描电镜观察、摄片,了解脱细胞支架的形态学和组织学特征并做处理前后的比较。将支架材料进行同种异体间动物皮下接种,结合不同生长因子局部一次性滴入(空白组,bFGF组,VEGF组,bFGF+VEGF组),2周后取出支架,进行免疫组化血管染色(8因子),观察支架新生血管的长入情况。结果用质量浓度为0.03%SDS的Tris低渗缓冲液与静脉壁共同孵育48h后,既完全脱去了静脉壁内膜表面的内皮细胞,又较完整地保留了胶原纤维等细胞外基质主要成分,基质主要成分的形态学结构在脱细胞前后无明显改变。皮下植入的异体支架局部未见明显排斥反应,2周后支架内新生血管有不同程度的长入,以滴入bFGF+VEGF组新生血管长入最显著。结论质量浓度为0.03%的SDS的低渗缓冲液对制备脱细胞的同种生物支架效果较佳,既能完全脱去静脉壁内膜表面可引起免疫排斥反应的内皮细胞,又能较完整地保留能维持支架形态的胶原纤维等细胞外基质主要成分;所制备的脱细胞支架异体植入无排斥反应,bFGF和VEGF的局部使用可促进新生血管在支架内的生长,有助于支架的远期存活;该实验所制备的静脉脱细胞支架可望在组织工程胆管体外构建的研究中应用。

超深低温冷冻对大鼠骨力学性能影响的生物力学研究

目的:探讨不同时段超深低温保存对骨力学性能的影响。方法:以15%DMSO为低温保护剂,采用两步冷冻法,对SD大鼠股骨用-55℃的冷冻预处理,分为18d、180d和360d组,在-196℃液氮中保存,测试前用40℃水浴复温,按不同保存天数,每组股骨各10段,与新鲜对照组分别进行抗压和抗折实验。结果:骨压缩强度极限(MPa):对照组、18d、180d、360d组分别为(77.26±11.80)、(76.02±15.00)、(61.90±12.20)和(56.44±4.47)MPa。股骨弯曲最大挠度:对照组、18d、180d、360d组依次为(44.60±10.54)、(44.20±9.50)、(20.01±7.58)和(16.2±3.42)mm。结论:骨的抗折挠度,360d组与对照组有显著差异(P<0.01),表明骨的脆性增加。

不同处理方法对同种带瓣主动脉组织结构及机械抗张强度的影响

对同种带瓣主动脉的取材、处理、保存进行了研究，建立了梯度降温的计算机程序，通过组织学及超微结构观察，组织机械抗张强度测试，证实抗生素灭菌，深低温保存方法可以较好地保持同种移植物的生理特性，保持纤维组织及成纤维细胞的结构完整，保持组织机械抗张强度。

低温保存大鼠同种异体主动脉移植后的排斥反应与治疗

目的 研究经低温保存的同种异体主动脉移植后的排斥反应 ,观察环孢素 A与脾脏切除联合免疫抑制治疗对排斥反应的影响 .方法 经低温保存 6 0 d的 SD大鼠同种异体主动脉以端对端的方式异位植入 SD大鼠的腹主动脉 .免疫抑制治疗组在移植术后摘除脾脏 ,从术后 1d开始给予环孢素A15 mg.kg- 1 .d- 1 ,ip.分别于术后 3,7,14,2 1和 2 8d分批处死受体大鼠 ,取出移植物 ,常规石蜡包埋 ,制成切片 ,进行免疫组织学染色以观察免疫球蛋白 Ig M和 Ig G的沉积情况 .结果 单纯手术组的移植物手术 7d后有明显的 Ig M沉积 ( ) ,手术 2 1d后有明显的 Ig G沉积 ( ) .假移植手术对照组染色均为阴性 ( - ) .免疫抑制治疗组术后第 7日 Ig M沉积为可疑阳性 (± ) ,术后 14,2 1和 2 8d Ig M沉积均为弱阳性 ( +) .免疫抑制治疗组术后 2 1和 2 8d Ig G沉积为弱阳性 ( +) .结论 低温保存的同种异体主动脉移植后有明显的免疫球蛋白 Ig M和 Ig G沉积 ,由此证明排斥反应的存在 .环孢素

液氮保存同种异体血管（动脉）移植的实验研究

目的：通过对经液氮保存的犬同种异体血管体内移植后活性、内皮细胞覆盖及通畅情况的研究，探讨该种血管作为血管材料替代物的优越性。方法：取犬新鲜动脉，经抗生素灭菌后，保存于液氮中８～２９周后，用于同种异体移植２２例，同时分别对冷冻前的血管、复苏血管、移植后的同种异体血管进行组织培养和病理分析，观察低温对血管壁结构、功能和血管活性的影响及经低温保存后供体血管在受体内１～５３周的情况。结果：低温保存的血管，其管壁基本完整。体内移植后累积通畅率为７７．３％，其中６８．２％的移植血管有内皮细胞覆盖。血管栓塞率为１３．６％。体外培养发现移植后有８６．４％的血管有活性。结论：经液氮保存的同种异体血管，保存了血管的活性，移植后内皮细胞重新覆盖，减少了血栓形成，该方法保存的血管为一种较为理想的血管材料替代物。