新型复合组织工程瓣膜的构建和体外生物力学研究

目的 构建复合瓣膜进行体外生物力学和脉动流测试.方法 猪主动脉瓣脱细胞处理作为支架,用3-羟基丁酸与3-羟基己酸共聚酯涂层,构建复合瓣膜,用拉伸机进行体外生物力学研究,以新鲜和脱细胞猪瓣作为对照(每组12枚);用脉动流仪进行流体力学研究,以脱细胞猪瓣作为对照(每组6枚).结果 复合瓣膜抗拉强度(12.08±1.72)MPa,与新鲜猪瓣(8.38±0.52)MPa和脱细胞猪瓣(8.16±0.66)MPa比较,差异有统计学意义(P＜0.05);在心输出量2～7L/min时,复合瓣膜与脱细胞猪瓣血流动力学参数差异无统计学意义(P＞0.1).结论 复合瓣膜具有良好的生物力学、流体力学特性.     

组织工程心脏瓣膜内皮细胞的整合素β1表达

目的体外构建组织工程心脏瓣膜（TEHV）,初步探讨内皮细胞黏附生长的分子机制。方法猪主动脉瓣膜经胰酶-EDTA、表面活性剂、核酸酶处理,去除猪主动脉瓣叶的细胞成分,测定瓣叶脱细胞后的生物力学特性;将扩增的人脐静脉血管内皮细胞（HUVECs）种植在瓣叶上,体外静态构建TEHV,观察内皮细胞的生长状态。消化瓣膜内皮细胞,半定量RT-PCR检测内皮细胞整合素＆mRNA的表达,Western-Blot检测内皮细胞膜上整合素＆蛋白的表达。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;种植的HUVECs在瓣叶表面生长状态良好,长成一层连续的细胞层。瓣膜内皮细胞可检测到整合素岛mRNA和蛋白的表达。结论脱细胞猪主动脉瓣膜作为支架,HUVECs做种子细胞可以成功构建TEHV,瓣膜内皮细胞可以表达整合素岛。     

人骨髓基质干细胞构建组织工程心脏瓣膜的研究

目的探讨人骨髓基质干细胞种植在去细胞猪主动脉瓣叶上体外构建组织工程心脏瓣膜的可行性。方法经1%TritonX100、0.01%胰酶0.02%EDTA、DNaseI及RNaseI处理制备去细胞猪主动脉瓣叶支架,测定瓣叶去细胞前、后的生物力学特性;人骨髓基质干细胞体外分离、培养、扩增后种植在去细胞瓣叶表面,观察细胞生长情况。结果猪主动脉瓣叶去细胞后获得完整无细胞的纤维网状支架,瓣叶去细胞前、后的断裂强度和断裂伸长率差异无统计学意义(P>0.05);体外培养的人骨髓基质干细胞(MSCs)在去细胞瓣叶表面形成一层基本连续的细胞层。结论去除细胞成分的猪主动脉瓣叶组织是一种良好的纤维支架,可以用于构建组织工程心脏瓣膜;人骨髓基质干细胞种植在去细胞猪主动脉瓣叶上构建组织工程心脏瓣膜是可行的。     

血流力学对组织工程心脏瓣膜种子细胞形态及功能的影响

目的观察血流力学对组织工程心脏瓣膜种子细胞形态及功能的影响。方法从犬骨髓中分离得到骨髓间充质细胞（MSCs），经DiI标记后接种于去细胞猪主动脉瓣膜支架，培养7d后将所得瓣叶种植于犬腹主动脉内，术后4、8、12周取材，进行HE染色、天狼猩红染色、免疫组织化学染色及超微电镜检查等，观察细胞生长、形态及功能变化。设体外培养组为对照组。结果接种骨髓间充质细胞的去细胞猪主动脉瓣膜支架，移植于犬腹主动脉内后，仍可检测到所种植细胞，细胞生长良好。与对照组比较，细胞形态有所伸长，整体上沿流体方向改变形态，同时，瓣叶组织中Ⅰ、Ⅲ型胶原含量明显增加，并充满中性和酸性黏多糖。结论构建组织工程心脏瓣膜时，血流力学对种子细胞维持形态、保持功能起着重要作用。     

脱细胞天然支架生物力学性能变化及预处理改善组织相容性的实验研究

目的观察脱细胞猪主动脉瓣的生物力学性能变化,探讨不同预处理改善天然支架组织相容性的效果。方法新鲜猪主动脉瓣经酶加去污剂法去除细胞,力学测试仪检测其最大负荷、最大应力、最大应变和弹性模量的变化,苏木精-伊红（HE）染色、Ⅰ型胶原免疫组化染色和扫描电镜观察其病理形态学变化;将脱细胞瓣膜分别予磷酸缓冲液、多聚赖氨酸和未灭活胎牛血清包被处理,然后种植大鼠主动脉肌成纤维细胞,甲基噻唑基四唑试验检测细胞黏附率,HE染色和扫描电镜观察形态学变化。结果酶加去污剂法能完全脱去瓣膜细胞,基本维持胶原纤维的空间结构,但其最大负荷、最大应力及弹性模量下降,最大应变上升（P〈0．05）;胎牛血清预处理去细胞瓣能显著提高肌成纤维细胞的黏附率,促进细胞生长、分化和增殖,并在瓣膜表面形成连续的细胞层（F值=129．26,P=0．000）。结论酶加去污剂法可较完全去除猪主动脉瓣膜细胞并保持细胞外基质的三维结构,但其生物力学性能有所下降;胎牛血清预处理能改善脱细胞瓣天然支架的细胞黏附、生长和繁殖。     

不同试剂脱除猪主动脉瓣细胞的对比研究

目的 用不同方法去除猪主动脉瓣的细胞并比较效果，为组织工程瓣膜提供良好的实验材料。方法 采用十二烷基硫酸钠（SDS）、脱氧胆酯酸钠（SD）、曲拉通（Triton）X-100制备猪主动脉瓣脱细胞基质。标本进行大体、光镜、电镜观察，DNA提取、力学性能的检测并比较。结果 SD无法完全脱除主动脉瓣瓣根内的细胞。TritonX-100和SDS可以完全脱除瓣膜细胞。TritonX-100较好的保持了主动脉瓣胶原纤维和弹性纤维的原有排列和分布，并有较好的力学性能。而SDS对瓣叶和瓣根血管壁中的纤维支架都造成了一定的损伤。SDS和TritonX-100脱细胞时间无明显差别。结论 TritonX-100可以成功脱除猪主动脉瓣细胞，并较好的保持了主动脉瓣的结构和力学性能。     

种植人体活性细胞的生物心脏瓣膜

目前,种植人体活性细胞的生物心脏瓣膜主要有组织工程心脏瓣膜和种植人体活性细胞的猪主动脉瓣两种。组织工程心脏瓣膜是在人体可吸收的聚二醇酸纤维支架上种植人体同种活性细胞,先种植成纤维细胞,再种植单层内皮细胞包裹瓣叶。种植人体活性细胞的猪主动脉瓣是在清除原有细胞的组织内重建人体同种活性细胞。清除新鲜猪主动脉瓣呐原有细胞的方法是将瓣膜先经高、低渗溶液处理,然后用酶溶液处理。细胞经培养分离后,将成纤维细胞植入经处理的瓣膜组织,再植入内皮细胞。种植人体活性细胞的生物心脏瓣膜不会促使受者产生有害的免疫反应,并具有再生能力。     

应用四枝化状聚乙二醇-乙烯砜基交联去细胞带瓣管道的实验研究

目的利用四枝化状聚乙二醇-乙烯砜基（polyethylene glycol-VS,PEG-VS）交联去细胞主动脉带瓣管道,制备新型组织工程复合支架,研究其力学和生物学性能。方法采用胰酶法制备兔主动脉去细胞带瓣管道,利用四枝化状PEG-VS与兔去细胞带瓣管道交联构建复合支架,体外静态条件应用力学测试仪检测去细胞管道和复合支架力学性能。将纯种新西兰大白兔30只随机分为3组,每组10只,对照组：正常兔主动脉带瓣管道;去细胞组：去细胞带瓣管道;复合支架组：PEG复合支架。构建兔颈总动脉瓣膜移植模型,术后28 d分别用超声心动图检测3组移植管道通畅率,用HE染色、扫描电子显微镜观察微观形态和炎症细胞浸润,免疫荧光染色检测复合支架体内内皮化程度。结果体外力学测试结果示：PEG复合支架弹性模量及最大负荷力均较去细胞管道有明显提高（P弹性模量=3.1×10-9,P最大负荷力=1.1×10-6）。术后血管彩色多普勒超声心动图提示：复合支架组管腔通畅率高于对照组（P=0.054）和去细胞组（P=0.019）,腔内血栓形成率、管腔形变率明显降低;HE染色、扫描电子显微镜检测显示：复合支架组体内内皮化程度增高,内皮细胞于支架上均匀附着;免疫荧光检测显示：支架表面内皮细胞标记物CD34阳性率高于对照组和去细胞组。结论利用四枝化状功能化PEG-VS改性去细胞主动脉瓣带瓣管道,可明显改善组织工程支架生物力学性能和组织相容性。     

应用猪主动脉去细胞瓣膜支架体外构建组织工程心脏瓣膜

目的　探讨应用猪去细胞瓣膜支架体外构建组织工程心脏瓣膜的可行性。　方法　采用去垢剂、渗透压改变和核酸酶消化的方法制备猪主动脉瓣去细胞瓣膜支架 (实验组 ) ,用去内皮细胞主动脉瓣作对照 (对照组 ) ,并对两组含水量、可溶性蛋白含量、热皱缩温度、力学性能和组织相容性进行测定。培养犬主动脉壁间质细胞和内皮细胞 ,将其种植于实验组去细胞支架上 ,观察细胞生长情况 ,并测定内皮细胞合成前列环素的功能。　结果　实验组瓣膜细胞成分完全从瓣膜中去除 ,与对照组新鲜瓣膜相比 ,含水量增高 (P<0 .0 5 ) ,可溶性蛋白含量减少 (P<0 .0 5 ) ,热皱缩温度和抗张强度无明显变化。实验组瓣膜组织相容性试验显示 ,材料组织相容性好 ,体内降解时间为 10周 ;犬主动脉壁间质细胞和内皮细胞在瓣膜表面生长良好 ,内皮细胞具有合成分泌前列环素的功能。　结论　采用去垢剂、渗透压改变和核酸酶消化的方法制备猪主动脉瓣去细胞瓣膜支架 ,在去除细胞和可溶性蛋白质的同时保持了瓣叶的基本结构和力学性能 ;以其为支架体外构建组织工程心脏瓣膜的细胞不仅能在材料表面生长 ,还能合成、分泌血管活性物质 ,是具有生理功能的组织工程心脏瓣膜。     

十二烷基肌氨酸钠法制备带瓣管道支架及其生物学性能分析

目的尝试用十二烷基肌氨酸钠作为脱细胞试剂，制备脱细胞组织工程支架材料，并分析其生物学性能。方法用0．25％十二烷基肌氨酸钠溶液及核酸酶对猪的带瓣膜管道进行脱细胞处理，并对其进行DNA、可溶性蛋白含量测定，HE、Movat染色和电镜观察及生物力学测试；大鼠皮下包埋实验分析生物相容性。结果经脱细胞处理后，带瓣膜管道的瓣叶和管壁中DNA含量仅为正常的1．35％和2．81％，可溶性蛋白含量仅为正常的8．46％和12．65％，细胞成分几乎完全去除。HE和Movat染色及电镜观察显示组织结构完整、细胞结构完全消失，胶原、弹性纤维、蛋白聚糖等主要基质成分充分保留；瓣膜生物力学性能与正常瓣膜差异无统计学意义；支架生物相容性良好，并具有低免疫原性和可降解性。结论十二烷基肌氨酸钠是一种理想的新型脱细胞试剂，以此可以建立一套低毒、高效、对组织结构无损伤的脱细胞方法，并制备出生物学性能优异的脱细胞支架材料。     

Study on decellularized porcine aortic valve/poly (3-hydroxybutyrate-co-3-hydroxyhexanoate) hybrid heart valve in sheep model

To overcome shortcomings of current heart valve prostheses, novel hybrid valves were fabricated from decellularized porcine aortic valves coated with poly (3-hydroxybutyrate-co-3-hydroxyhexanoate [PHBHHx]). In the mechanical test in vitro, the biomechanical performance of hybrid valve was investigated. In an in vivo study, hybrid valve conduits were implanted in pulmonary position in sheep without cardiopulmonary bypass. Uncoated grafts were used as control. The valves were explanted and examined histologically and biochemically 16 weeks after surgery. The hybrid valve conduits maintained original shapes, were covered by a confluent layer of cells, and had less calcification than uncoated control. The mechanical test in vitro revealed that PHBHHx coating improved tensile strength. The results in vivo indicated that PHBHHx coating reduced calcification and promoted the repopulation of hybrid valve with the recipient's cells resembling native valve tissue. The hybrid valve may provide superior valve replacement with current techniques.     

Noise Level and Perception of the Closing Click After Heart Valve Replacement with St. Jude Medical and Björk Shiley Monostrut Prostheses

Abstract: The metallic click generated by the closure of mechanical heart valve prostheses may severely bother patients, but generated sound energy and the extent of complaints after implantation are not known. In 62 patients, after valve Replacement with St. Jude Medical (SJM) (n = 35) and Björk Shiley Monostrut (BSM) (n = 27) prostheses, sound energy was recorded with a calibrated noise level analyzer at 5, 10, and 100 cm distance from patients and correlated with their complaints. At a distance of 100 cm, the BSM valves produced a significantly higher sound pressure level, 30.5 ± 5 db(A), compared to the SJM valves, 24.1 ±4 db(A) (p = 0.0001). There was no significant difference at shorter distances. After splitting into frequency bands the highest sound pressure levels were observed in the high frequency ranges (8 to 16 kHz) representing the metallic click. BSM valves produced higher sound levels in all frequency ranges at 1 m distance. Seventy-three percent of all patients were aware of the noise generated by the valve; 20% had disturbed sleep; and 26% preferred a less noisy valve type. Twelve of 27 patients with BSM valves wanted less noisy valves, whereas only 4 of 35 patients with SJM valves wished to have a less noisy valve type (Chi-square p = 0.003). In patients who could hear their valve measured, sound level was higher than in patients who could not. In 9 of 27 patients with BSM (33%), versus 3 of 35 with SJM prostheses (9%), the clicking caused sleep disturbances. In 85% of BSM, versus 63% SJM, patients could hear their valve (p = 0.05). Since higher sound pressure levels generated by the closing click resulted in an increased rate of complaints, noise level should be regarded as a criterion when a mechanical heart valve prosthesis is selected.     

Experimental evaluation and early clinical results of a new low-profile bileaflet aortic valve

We performed an experimental and clinical evaluation of a new low-profile bileaflet aortic valve (Regent, St. Jude Medical Inc., St. Paul, MN, U.S.A.). Common valve sizes were experimentally tested for leakage volume, pressure drop, and transvalvular hemodynamics using a pulse duplicator. Thirty patients (mean age 60 +/- 7 years, predominant valve stenosis n = 25) received the Regent prosthesis for initial clinical evaluation. In vitro evaluation revealed equivalent leakage volumes, larger performance indices (0.552 versus 0.513), and lower pressure drops in comparison to SJM hemodynamic plus valve controls. Clinically, 21 mm (n = 9), 23 mm (n = 12), and 25 mm (n = 9) valves were implanted with no significant perioperative complications. Echocardiography revealed low transvalvular flow velocities (2.2 +/- 0.4 m/s) and low pressure gradients (20 +/- 6 mm Hg) postoperatively and at 6 months follow-up. In vitro testing and early clinical results are promising; however, long-term performance has to be proven.     

Flow Characteristics of the St. Jude Prosthetic Valve: An In Vitro and In Vivo Study

The St. Jude cardiac prosthetic aortic valve was evaluated in vitro and in vivo in an attempt to establish flow characteristics and to correlate them with clinical findings. In vitro, a fluid vehicle (6% Polyol V-10, 32°C) with viscosity similar to blood (0.035 dyne-sec/cm²) was used under conditions of steady flow through a flow chamber simulating the aortic root. Gradient, velocity, and shear stress were measured 5.79 mm, 26.79 mm, 44.79 mm, and 77.79 mm downstream from 25-mm and 27-mm valves using a laser-Doppler anemometer. At 417 ml/sec, the valve gradient was 6.2 mmHg with the 25-mm valve, and 5.2 mmHg with the 27-mm prosthesis. Velocity was maximum at the orifice center, and wall shear stress was low (maximum 600 dyne/cm²). In vivo, six patients with 25-mm St. Jude aortic valves were studied within 48 hours after surgery to determine cardiac output, valve flow, and gradient. The gradient was 3.3 ± 1.9 mmHg (M ± SD) at 249 ± 96 ml/sec and the effective valve area was as large as the geometric area (2.58 vs. 3.09 cm²). Thus, flow through the St. Jude valve is unobstructed and central, has low turbulence, and achieves optimal effective valve area for a given available orifice area.     

Hexsyn Trileaflet Valve: Application to Temporary Blood Pumps

A trileaflet valve, compression molded of Hexsyn rubber, was evaluated as a component in temporary blood pumps. A pair of valves was integrated into an LVAD which was tested in vitro and in vivo. The valve's hemodynamic performance was satisfactory and the blood compatibility was excellent in a three-week, in vivo experiment conducted to demonstrate performance in a short-term application. Hexsyn, which has excellent properties and produces high quality, complex components with ease of fabrication, offers a potential for low-cost blood pump valves.     

搏动辅助血泵专用的蝴蝶瓣离体性能测试

目的：对已设计制成的低成本搏动血泵专用瓣膜蝴蝶瓣（butterfly valve,B-Y)按ISO5840进行国际规范化的离体性能测试。方法：先对B－Y瓣进行物理性能测试,然后根据ISO5840的要求用特殊仪器对3枚B－Y瓣作静态及动脉性能测试,其均值与进行同样测试的2种美国进口植入型瓣膜Bjork-Shiley(B-S)瓣和Medtronic-Hall(M-H)瓣作比较。结果：B－Y瓣的物理性能符合临床要求,在静态测试中2枚B－Y瓣膜片过薄,在12mmHg（1kPa=7.5mmHg)水压下有下凹内翻产生较多静止泄漏量。动态测试方面无搏动流中B－Y瓣的跨瓣压力落差及搏动流时跨瓣正压压力阶差均较B－S瓣和M－H瓣膜略高,但其关闭回流量远较B－S瓣与M－H瓣为低,其舒张泄漏量与B－S瓣相仿,而明显低于M－H瓣。结论：B－Y瓣的水流阻力较进口瓣膜为高,但在临床允许范围以内。其总反流量（关闭回流量、舒张泄漏量）明显低于进口瓣膜,保证了瓣膜的效率和耐用性,潜在溶血程度亦因而轻微。B－Y瓣由于结构简单,成本低廉,却有与进口植入型瓣膜相仿的功能,是其主要特点,适合短期辅助循环的临床应用。     

染料介导光氧化处理去细胞猪主动脉瓣的生物学特性

目的探讨染料介导光氧化处理猪主动脉瓣膜的生物学特性，以期获得性能良好的新型生物瓣膜材料。方法经去细胞处理的猪主动脉瓣30个，随机分为3组。光氧化处理组（n=10）：去细胞瓣膜经染料介导光氧化处理；戊二醛处理组（n=10）：去细胞瓣膜采用戊二醛处理；去细胞对照组（n=10）：去细胞瓣不做固定处理，置于生理盐水中备用。分别测定并比较3组瓣膜的形态学改变、组织学特点、瓣膜厚度、含水量、热皱缩温度、断裂强度和可溶性蛋白含量。结果光氧化处理组主动脉瓣呈淡蓝色，瓣膜质地柔软，弹性大，瓣叶无皱缩。光氧化处理组主动脉瓣瓣膜厚度较戊二醛处理组薄（0．26±0．09mm vs．0．38±0．08mm；P〈0．05），组织含水量较戊二醛处理组大（86．30％±4．03％VS．71．10％±3．23％；P〈0．05），热皱缩温度低于戊二醛处理组（76．30±0．70℃ vs．87．70±0．30℃；P〈0．05）；瓣膜厚度、组织含水量和热皱缩温度与去细胞对照组比较差异无统计学意义。光氧化处理组断裂强度较去细胞对照组明显增大（17．33±2．65mPa vs．9．11±0．95mPa，P〈0．05），可溶性蛋白含量较去细胞对照组明显减少（0．039％±0．013％VS．0．107％±0．024％；P〈0．05），而与戊二醛处理组比较差异无统计学意义。结论染料介导光氧化处理去细胞猪主动脉瓣有较好的生物学特性。     

Introduction of a flexible polymeric heart valve prosthesis with special design for aortic position

Objective: Current prosthetic heart valves necessitate permanent anticoagulation or have limited durability and impaired hemodynamic performance compared to natural valves. Recently a polymeric valve prostheses with special design for mitral position demonstrated excellent in vitro and in vivo results with improved durability and no need for permanent anticoagulation. In this study, a respective flexible polymeric aortic valve is presented and in vitro and in vivo results are reported. Methods: The aortic prosthesis (ADIAM^® lifescience AG, Erkelenz, Germany) is entirely made of polycarbonaturethane. The tri-leaflet flexible prosthesis mimicks the natural aortic valve and has a diminished pressure loss and reduced stress and strain peaks at the commissures. The valve underwent long-term in vitro testing and in vivo-testing in a growing calve animal model (20 weeks, 7 aortic valves) and was compared to two different commercial bioprostheses. Results: The polymeric aortic heart valve substitute demonstrated excellent in vitro and in vivo hemodynamics. Five/seven animals with aortic PCU-prostheses had an excellent clinical long-term course. The explanted valves showed a variable degree of calcification. Two of the seven animals died at 27 and 77 days due to pannus overgrowth causing severe LVOTO without degeneration of the valve itself. Both animals with commercial bioprostheses had to be sacrificed because of congestive heart failure related to structural degeneration of the bioprosthesis after 10 and 30 days of implantation. There was no increased thrombogenity of the PCU valves compared to bioprostheses. Conclusion: The new flexible polymeric aortic valve prosthesis is superior to current bioprostheses in animal testing.