脱除人主动脉瓣膜细胞的研究

目的探讨人主动脉瓣叶去细胞后作为组织工程心脏瓣膜支架的可行性。方法经胰酶-EDTA、表面活性剂和核酸酶处理,去除人主动脉瓣叶的细胞成分,测定瓣叶去细胞前、后的生物力学特性。结果人主动脉瓣去细胞后,经光镜和电镜观察,脱细胞后血管壁无细胞残留,胶原纤维和弹性纤维保留完整,形态学无明显变化;生物学性能也没有明显改变。结论去细胞效果良好,初步制造了同种主动脉血管壁脱细胞基质材料,为构建同种带瓣管道提供了较合适的材料。     

组织工程心脏瓣膜体外构建的初步研究

目的:初步体外构建组织工程人工心脏瓣膜。方法:种子细胞选用胸骨骨髓,抽取后分离出人骨髓间充质干细胞,体外培养,扩增待用。脱细胞瓣膜支架的构建应用破膜剂曲拉通-脱氧胆酸钠对新鲜猪主动脉瓣膜36片(实验组)进行处理48h后,与对照组未经处理的猪主动脉瓣膜(36片)进行对比,在光镜和电镜下观察脱细胞效果。将人骨髓间充质干细胞接种于脱细胞瓣膜支架上静态培养7d,观察细胞-支架复合体生长状况。结果:曲拉通-脱氧胆酸钠法脱细胞彻底,保持原有纤维结构。种植的人骨髓间充质干细胞7d后可生长于瓣膜支架的表面。结论:曲拉通-脱氧胆酸钠法是一种较为理想的猪瓣膜脱细胞方法;人骨髓间充质干细胞能在脱细胞猪瓣膜支架上较好地黏附和生长,从而初步构建组织工程人工心脏瓣膜。     

组织工程心脏瓣膜支架构建的研究

目的：构建脱细胞猪主动脉瓣膜支架，为体外构建组织工程心脏瓣膜提供适宜的支架材料。方法：新鲜猪主动脉瓣叶，采用“低渗液-0．5％SDS-核酸酶”处理，进行大体、光镜、电镜观察和DNA含量测定。结果：“低渗液-0．5％SDS-核酸酶”可以完全脱去瓣膜细胞，同时较好地保持了胶原纤维和弹力纤维的原有排列和形态。结论：本方法可以成功构建脱细胞猪主动脉瓣膜支架，可进一步用于体外构建组织工程心脏瓣膜的研究。     

人脐静脉血管内皮细胞构建组织工程心脏瓣膜及其生理功能

目的 探讨人脐静脉血管内皮细胞(HUVECs)构建组织工程心脏瓣膜(TEHV)及其生理功能.方法 以脱细胞猪主动脉瓣作支架;将扩增的HUVECs种植在瓣膜上,体外静态构建TEHV,观察内皮细胞的形态和生长状况.收集瓣膜培养液,检测瓣膜内皮细胞分泌组织型纤溶酶原激活物(t-PA)、组织型纤溶酶原激活物抑制物(PAI-1)、前列环素(PGI2)、一氧化氮(NO)、内皮素(ET-1)的功能.结果 猪主动脉瓣膜中的细胞成分能完全去除,脱细胞瓣叶的生物力学特性同新鲜瓣叶相比无明显变化以HUVECs做种子细胞,成功构建TEHV;瓣膜表面的内皮细胞生长状态良好,长成连续的细胞层.细胞能够分泌t-PA、PAI-1、PGI2、NO、ET-1等.结论 以脱细胞猪主动脉瓣膜为支架,种植HUVECs成功构建TEHV,其表面HUVECs具有正常内皮细胞的生理功能.     

新型材料PLLA-脱细胞软骨粒支架的制备及性状研究

目的:以聚左旋乳酸(PLLA)和猪的脱细胞软骨粒混合物为材料制备一种新型的支架,即PLLA-脱细胞软骨粒支架,并对其性状进行研究,从而为软骨组织工程提供一种新的支架材料。方法:利用热相致分法(T IPS法)制备PLLA与脱细胞软骨粒在不同混合比例、不同温度下的聚合物支架,并对各种支架的孔径大小、孔隙率、水结合力、生物力学性状、降解率等进行探讨。结果:PLLA-脱细胞软骨粒支架具有与天然软骨基质相近的成分与结构,其各种性状基本符合软骨支架的要求。结论:PLLA-脱细胞软骨粒支架可作为软骨组织工程良好的细胞外基质材料。     

人主动脉壁脱细胞血管基质的制备

目的 探讨应用同种去细胞主动脉血管壁支架体外构建组织工程管道的可行性.方法 取液氮保存的同种主动脉带瓣管道.首先置于Tris低渗缓冲液室温下溶解血管壁细胞,血管壁用D-Hanks液充分清洗后,再用含十二烷基硫酸钠(SDS)的Tris低渗缓冲液室温下再孵育24h,洗涤脱去细胞;最后用Tris等渗缓冲液充分冲洗残留细胞,完成脱细胞支架制备.并对脱细胞后的管壁进行光镜及电镜形态学观察以及进行理化性能测定.结果 人主动脉血管壁经SDS法去细胞后,经光镜和电镜观察,脱细胞后血管壁无细胞残留,胶原纤维和弹性纤维保留完整,形态学无明显变化;生物学性能也没有明显改变.结论 SDS法去细胞效果良好,初步制造了同种主动脉血管壁脱细胞基质材料,为构建同种带瓣管道提供了较合适的材料.     

组织工程细胞支架与种子细胞复合培养的研究进展

组织工程的核心是建立由生物材料与细胞结合的三维空间复合体,即以合适的生物材料构建与所需要修复的组织或器官结构相同或类似的支架并复合特定细胞,用以对病损组织进行形态、结构和功能的重建而达到永久性替代[1].支架材料是组织工程的基础,它既要为细胞提供生长的空间又要被植入体内,故材料的性质会对细胞的生长、繁殖、分化和宿主内环境产生重要影响.因此,选择具有良好的生物相容性及细胞亲和性好的支架材料尤为重要.另外,要构建细胞支架复合体,又必须考虑种子细胞黏附于支架的问题,支架材料、种子细胞以及复合体的构建在组织工程中被称为三大基本要素.     

海藻酸钠在组织工程中的应用

摘要：组织工程是以细胞生物学和材料科学为基础,在体内或体外构建组织或器官的新兴学科。组织工程主要研究种子细胞和支架材料,支架材料为种子细胞提供三维立体空间结构,并且可以为细胞的气体交换、营养物质获取等提供通路,本文将针对一种具有良好安全性、可生物降解性等的天然材料—海藻酸钠(SA)的性质及在组织工程中的应用进行综述。     

组织工程支架材料聚乙醇酸的体外细胞贴壁性和降解性能研究

目的:选择可降解材料PGA作为研究对象,对其细胞贴壁性和体外及体内降解性能进行研究。方法:通过将L929细胞与支架材料共培养的方法对PGA支架材料的细胞贴壁性进行研究。设计试验计算出细胞在材料上的粘附率,并且通过扫描电子显微镜观察了细胞在材料表面的贴壁形态。在体外降解试验中,分别在去离子水和磷酸盐缓冲液中,在37℃和70℃的条件下对材料的降解性能进行观察。用大鼠皮下包埋试验对PGA在大鼠皮下包埋后1周、4周、8周、12周这4个时间点的降解表现进行了观察。结果:试验结果表明,共培养72 h后,扫描电镜观察到L929细胞在PGA材料上良好贴壁,细胞粘附率大于80%;降解试验表明PGA组织工程支架在动物体内12周内基本降解。结论:体外细胞贴壁性和降解试验的数据表明PGA组织工程支架符合组织工程产品支架材料的需求,是一种较好的组织工程支架材料。     

组织工程骨的研究与应用

骨组织工程研究的目的是为了将细胞和材料构建成工程骨用作骨移植替代材料。骨髓基质干细胞作为种子细胞的培养技术较为成熟,基因转移技术的应用使得其分化增殖能力得到增强,更利于骨组织的再生。现有多种人工合成或天然的支架材料均未能达到理想的要求,需应用新技术工艺对其进行修饰和改性以降低免疫原性,增强细胞在支架材料上的吸附、分化、增殖能力,并在工程骨构建过程中引入生长因子,利于新骨形成。组织工程骨初步进入临床应用取得了良好的效果,并将逐步实现产品产业化生产。     

Glutaraldehyde residues in heart valve prostheses

Bioprosthetic heart valves prepared from glutaraldehyde-pretreated bovine pericardium are used to replace diseased human cardiac valves. Mineralisation in the course of time and toxic effects are possibly caused by glutaraldehyde residues. Different washing methods carried out before transplantation are compared with regard to the different ability of glutaraldehyde extraction.     

Controlled-release drug delivery of diphosphonates to inhibit bioprosthetic heart valve calcification: release rate modulation with silicone matrices via drug solubility and membrane coating

Calcification (CALC) is the most frequent cause of the clinical failure of bioprosthetic heart valves (BHV) fabricated from glutaraldehyde pretreated porcine aortic valves or bovine pericardium. The present investigation describes the formulation, characterization, and the in vivo efficacy of prolonged controlled-release silicone matrices containing the anticalcification agent disodium 1,1-hydroxyethylidene diphosphonate (Na2EHDP). Controlled release of EHDP was regulated by codispersions of Na2EHDP and the less soluble salt Ca2EHDP. Prolonged and constant release rates (zero-order) were obtained by coating silicone matrices with permeable silicone membranes, which were prepared by leaching with acetone pre-embedded polyethyleneglycol. All EHDP-containing matrices (co-implanted subdermally with BHV cusps in rats) significantly inhibited BHV CALC without detectable adverse effects on bone mineral and calcium metabolism. Matrices containing Na2EHDP:Ca2EHDP ratios of 10:90 or greater with respect to Na2EHDP completely inhibited CALC. Significant inhibition of BHV CALC was also observed with prereleased matrices (5 months in vitro), thus demonstrating prolonged efficacy. It is concluded that sustained release of effective anticalcification therapy without side effects was achieved by using codispersions of calcium and sodium EHDP salts, and that a delayed and/or constant release rate of EHDP was obtained by coating reservoir-type matrices with silicone membranes that were pre-embedded with polyethyleneglycol.     

Prevention of calcification of bioprosthetic heart valve leaflets by Ca2+ diphosphonate pretreatment

Calcification frequently causes failure of bioprosthetic heart valves (BHV) fabricated from glutaraldehyde-pretreated porcine aortic valve or bovine pericardium. Systemic diphosphonate therapy inhibits this disease process, but with adverse effects on overall growth, bone development, and calcium metabolism. The present study was designed to examine the hypothesis that the immobilization of ethanehydroxydiphosphonate (EHDP) within BHV as the poorly soluble Ca2+ salt would inhibit calcification at drug levels insufficient to produce side effects. Glutaraldehyde-pretreated pericardial BHV tissue was exposed to a physiologic concentration of Na2EHDP (0.14 M in 0.05 M HEPES, pH 7.4) and subsequently washed in a NaCl or CaCl2 (0.14 M in 0.05 M HEPES, pH 7.4) solution to precipitate the Na or Ca2+ salts of EHDP on/within the tissue, respectively. Incorporation of CaEHDP into BHV ranged from 74.8 nM/mg (after 1 h, 37 degrees C) to 353 nM/mg (2 weeks, 22 degrees C). None of the Na2EHDP was incorporated into BHV without exposure to CaCl2. In vitro release of CaEHDP from BHV into a physiologic buffer not containing Ca2+ was rapid, with greater than 95% removed after 4 d, while release of CaEHDP into buffer containing a physiologic concentration of Ca2+ ion (1.5 mM) was markedly reduced, with 30% of the precipitated CaEHDP remaining immobilized on or within the tissue matrix following 21 d.(ABSTRACT TRUNCATED AT 250 WORDS)     

Heart valve replacement surgery: past,present and future

1. The present review covers the historical and present role of valvular surgery in treating heart disease and speculates what the future may be in this very important field of cardiac surgery. 2. One of the important elements of tissue valve replacement is durability and how to analyse the valves mathematically, so that the projected durability could be described from the observed period to an extended period. We found that the Weibull distribution is a very good mathematical method of describing the deterioration of tissue valves. 3. We also can see a very marked difference among different pericardial valves. The Carpentier-Edwards pericardial valve has tremendous durability compared with the previous generation of pericardial valves, namely the Ionescu-Shiley. 4. The incidence of structural valve deterioration varies according to the age of the patient at the time of implant. A very important feature to analyse in making a clinical decision with regard to the type of valve to implant is the age of the patient at the time of implantation, because this determines their long-term survival. 5. Another very important feature of valve replacement is the problem of valve thrombosis. This was the problem that we encountered in experimental animals and why it is so difficult to obtain a long-term survivor with mechanical valve replacement in dogs. Now, in humans, the incidence of thrombosis of these artificial valves is extremely low. 6. Another important feature of valve replacement is the incidence of late thromboembolism (TE). Differences in reports using the same valve are greater than differences between different prostheses and the average is approximately 1.5-2%/year incidence of TE. 7. We can conclude from this review that there has been significant progress in diminishing operative mortality and enhanced long-term survival following valve replacement over the past four decades. Increased durability of tissue valves has now become apparent, from the earliest application to the current time with glutaraldehyde-preserved pericardial valves. The late complication rates are approximately the same with all valve types, except that there is a dimunition in the possibility for valve thrombosis for tissue valves versus mechanical valves. Finally, anticoagulation continues to be required for mechanical valve replacement and we still require more durable tissue valves and freedom from anticoagulation for mechanical valves.     

风湿性心脏病合并二尖瓣乳头状弹力纤维瘤 1例

目的探究发生于心脏瓣膜的乳头状弹力纤维瘤的临床及病理特征,为临床治疗提供参考。方法对 1例风湿性心脏病合并二尖瓣腱索乳头状弹力纤维瘤病人,进行手术治疗,对二尖瓣肿物的标本行病理检查进行形态学研究,并复习文献。结果心脏乳头状弹力纤维瘤病人多无特殊症状。心脏超声表现为二尖瓣腱索见一条索状稍强回声。组织学表现为:肿瘤呈乳头状突起,表面被覆单层立方上皮细胞,中心为无血管的轴心区,主要部分是特征性的淡红色的弹力纤维组织。结论风湿性心脏病是一种常见的心脏疾病,而乳头状弹力纤维瘤是一种少见且发病隐匿的心脏肿瘤,病人常无明显症状,可引起栓塞风险。该例肿瘤位于二尖瓣乳头肌腱索处很罕见。术前行超声心动图及手术完整切除肿瘤是提高心脏乳头状弹力纤维瘤病人诊断率及治疗的关键。     

Current progress on scaffolds of tissue engineering heart valves

Tissue engineering heart valves (TEHV) may be the most promising valve substitute, but the study has been relatively stagnant in the recent five years due to the special position, function and mechanical property of heart valves. It is one of the key factors to select an ideal scaffold material in the construction of TEHV. And this article will briefly review the current research and progress on the scaffolds of TEHV, especially based on Chinese works. __________ Translated from Chinese Journal of Experimental Surgery, 2007, 24(3): 261–263 [译自: 中华实验外科杂志]