不同钙化模式对经导管主动脉瓣膜植入效果影响的数值模拟研究

目的通过有限元方法评估不同钙化模式对经导管主动脉瓣膜植入效果的影响。方法根据钙化斑块在主动脉瓣叶上的位置不同,建立对合线钙化模型、附着线钙化模型和圆圈钙化模型3种不同钙化形式的主动脉根部模型。使用ABAQUS软件仿真自膨胀经导管主动脉瓣膜植入3个钙化模型中的过程,分析不同钙化模型对主动脉根部应力、瓣架变形以及瓣周间隙的影响。结果圆圈钙化模型中钙化斑块的最大主应力最大,为18.42 MPa,可能导致假体植入后发生脑卒中的风险更高;圆圈钙化模型的瓣架变形程度也最大,可能导致更差的假体耐久性;附着线钙化模型的瓣周间隙面积为37.2 mm~2,超过其他模型的2倍,植入后出现严重瓣周返流的风险可能性更高。结论不同的主动脉瓣叶钙化模式与经导管主动脉瓣膜植入后的主动脉根部应力、瓣架变形以及瓣周间隙有关,对术后并发症和假体耐久性产生影响。研究结果为临床上经导管主动脉瓣膜植入术术后效果的预测提供参考。     

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。     

抗菌处理对猪肺动脉瓣膜细胞活性及组织结构的影响

目的 探讨抗菌处理对肺动脉瓣膜细胞活性和组织结构的影响因素及优化同种瓣膜的制备方法。方法 3组猪肺动脉瓣叶(n=6)浸泡在含抗生素的DMEM液中，在不同温度下孵育6h或24h。然后通过XTT比色法测定瓣膜细胞活性，用免疫组织化学荧光染色检测瓣膜细胞和细胞外基质，并行光镜观察。结果 抗菌时间为24h，37℃抗菌组与4℃抗菌组的瓣膜细胞活性与瓣膜组织结构无明显区别。抗菌6h组的瓣膜细胞活性与瓣膜组织结构明显优于抗菌24h组。结论 抗菌时间为24h时，抗菌温度对瓣膜细胞活性及结构完整性无明显影响。抗菌时间缩短至6h，有利于瓣膜细胞活性与结构完整性的保持。     

人工生物瓣膜研究进展

人工心脏瓣膜置换术是目前治疗末期心脏瓣膜疾病的有效手段。临床上广泛使用的人工生物瓣虽有血流近似生理流型、不易形成血栓等优点,但却有容易钙化、撕裂、有残留免疫原性及缓慢释放交联剂所带来的细胞毒性等缺点。该文综述了目前为克服这些缺点而进行的相关研究进展。     

液氮冷冻保存对主动脉瓣膜细胞活性及细胞外基质的影响

液氮冷冻保存是目前最好的同种心瓣膜保存方法 ,但液氮冷冻保存也可导致瓣膜的结构损害。为了探讨液氮冷冻保存对主动脉瓣膜组织结构的影响情况 ,本实验通过采用XTT比色法、形态学观察、免疫组织化学荧光染色法研究了不同冷冻保存时间对猪主动脉瓣膜细胞活性及细胞外基质的影响。研究结果显示 ,液氮保存后瓣膜细胞活性明显下降 (P <0 .0 5 ) ,细胞超微结构损害增加 ,可溶性细胞外基质纤维连接蛋白与硫酸软骨素不同程度流失 ,间质胶原纤维排列紊乱。但在冻存时间小于 3月时 ,损害的程度与冻存时间无明显关系。     

脱细胞猪主动脉瓣叶的生物力学特性和免疫反应性

目的：探讨脱细胞猪主动脉瓣叶构建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、核酸酶处理，去除瓣叶的细胞成分，测定脱细胞瓣叶的生物力学特性，同时行大鼠皮下包埋实验，观察其免疫反应性。结果：瓣叶中的细胞成分能完全去除，获得无细胞的纤维网状支架。脱细胞瓣叶与新鲜瓣叶有基本相同的应力-应变曲线及应力-松弛曲线，而弹性模量、面积比、松弛强度、断裂强度和断裂伸长率两者无显著差异。脱细胞瓣叶的免疫反应性明显降低。结论：猪主动脉瓣叶经脱细胞处理后可以作为组织工程心脏瓣膜的支架材料。     

机械应力对牛心包瓣原发性损坏的影响

引起牛心包瓣原发性损坏的生理学和病理学因素与机械学因素同等重要。从我院再次手术中收集到的22只失效瓣膜来看,在瓣架尖端区和侧边区的损坏发生率分别占30.3％和36％,撕裂、磨损和钙化的发生率分别占25.8％、18％和31.6％。牛心包片的弯曲试验和牛心包瓣的显微镜观察表明,由于经戊二醛处理的牛心包片的拉伸弹性模量是压缩模量的25倍,因此当牛心包片弯曲变形时,弯曲内侧必然出现挤压屈服,并逐渐发展到楔状挤压。牛心包瓣临床失效的形式及其分布提示,瓣叶损坏主要是由于瓣叶在启闭活动过程中的这种弯曲挤压引起的。因此在改进牛心包瓣时,应考虑机械应力,特别是弯曲变形的应力对瓣膜损坏的影响。     

纳米羟基磷灰石薄膜与人脐静脉血管内皮细胞的增殖

背景：合肥大学材料系和中国科学院安徽光学密精机械研究所联合研究应用脉冲激光沉积合成技术制备出一种新型的纳米羟基磷灰石人工心脏机械瓣膜。 目的：观察纳米羟基磷灰石人工心脏机械瓣膜与人脐静脉血管内皮细胞的相容性。 方法：将体外分离培养的传2-4代人脐静脉血管内皮细胞悬液接种于纳米羟基磷灰石人工心脏机械瓣膜上,培养3,7,21 d后,扫描电子显微镜下观察细胞在纳米羟基磷灰石人工心脏机械瓣膜上的生长情况。分别采用纳米羟基磷灰石人工心脏机械瓣膜常温浸提液、纳米羟基磷灰石人工心脏机械瓣膜高温浸提液、高密度聚乙烯浸提液及苯酚溶液培养人脐静脉血管内皮细胞,72 h后采用MTT法检测细胞增殖情况。 结果与结论：扫描电镜观察培养3 d时,人脐静脉血管内皮细胞呈梭形或多边形,伸出突起黏附于纳米羟基磷灰石人工心脏机械瓣膜上;7 d时,可见瓣膜表面细胞伸展充分,连接融合;21 d时,细胞成片融合,牢固覆盖于瓣膜表面,部分区域形成细胞外基质。MTT检测结果显示,纳米羟基磷灰石人工心脏机械瓣膜对人脐静脉血管内皮细胞无细胞毒性,具有良好的细胞相容性。中国组织工程研究杂志出版内容重点：生物材料;骨生物材料; 口腔生物材料; 纳米材料; 缓释材料; 材料相容性;组织工程全文链接：     

主动脉瓣二瓣化畸形的血流动力学研究进展

主动脉瓣二瓣化畸形(bicuspid aortic valuve,BAV)是最常见的先天性心脏病,会导致升主动脉扩张、主动脉瘤、主动脉夹层等并发症,给致命性临床事件发生埋下隐患。与正常三叶式主动脉瓣相比,主动脉瓣二瓣化畸形会极大地改变血液从左心室射入主动脉的状态,破坏主动脉中生理状态的旋动流,改变主动脉的正常几何形态及生理功能。血流动力学环境的异常改变会引起瓣膜钙化、血管壁细胞衰退,致使瓣膜的力学性能显著降低,从而加速BAV疾病恶化。本研究旨在综述血流动力学因素在主动脉瓣二瓣化畸形及其并发症发展中的作用。     

个体化治疗预防小主动脉瓣环瓣膜置换后瓣膜与患者的不匹配

背景：小主动脉瓣环主动脉瓣置换是心外科手术的难点,治疗不当可能出现瓣膜与患者不匹配现象,使左室流出道狭窄、跨瓣压差增大,引起左室后负荷增加致心肌肥厚甚至充血性心力衰竭。 目的：总结预防小主动脉瓣环瓣膜置换后发生人工心脏瓣膜与患者不匹配的治疗策略。 方法：小主动脉瓣环均主动脉瓣置换患者85例。瓣口直径>17 mm,≤19 mm的患者,选19 mm SJM Regent 瓣;对瓣口直径≤17 mm的患者,用牛心包补片加宽瓣环,再选19 mm SJM Regent 瓣行瓣膜置换;对于瓣口直径>19 mm,≤21 mm,选21 mm Hancock II ultra生物瓣置换。治疗后应用超声心动图测量有效瓣口面积指数、左心室重量指数、室间隔厚度、左心室后壁厚度、跨瓣峰速、跨瓣压差和跨瓣平均压。出院后通过门诊对患者进行随访,定期复查超声心动图。 结果与结论：治疗后早期无死亡病例,均治愈出院。随访时间为6个月-3年。主要并发症为低心排综合征2例、二次开胸止血1例、呼吸机依赖2例。所以患者均未出现脑栓塞或脑出血等脑部并发症。无瓣膜功能失调或卡瓣。未发现牛心包补片撕裂、瘤样膨出、钙化、血栓形成、免疫反应和感染等情况。81例获随访,随访率为 95%(81/85)。NYHA心功能分级Ⅰ级65例,Ⅱ级16例。各不同瓣环直径患者治疗后跨主动脉瓣峰速和平均压差均明显降低,有效瓣口面积指数明显增加,左心室重量指数、室间隔厚度和左心室后壁厚度均明显降低,均未出现人工心脏瓣膜与患者不匹配。置换21 mm Hancock II ultra 生物瓣和21 mm SJM Regent 瓣组间的比较,前者获得了更好的跨瓣峰速和平均压差,以及更好的左心室重塑指标。19 mm Regent 瓣患者治疗后体质量和体表面积较治疗前明显增加。结果提示对于小主动脉瓣环的患者应采取个体化的治疗策略预防主动脉瓣置换后瓣膜与患者不匹配的发生。 中国组织工程研究杂志出版内容重点：肾移植;肝移植;移植;心脏移植;组织移植;皮肤移植;皮瓣移植;血管移植;器官移植;组织工程全文链接：     

聚羟基丁酸及其共聚物在组织工程心脏瓣膜中应用的初步研究

应用组织工程支架 ,将活细胞种植于可生物降解的瓣膜支架上 ,制造出一种组织相容性好 ,不需抗凝 ,具有修复能力 ,且支持患者终生的理想心脏瓣膜。对照进行聚羟基丁酸 (polyhydroxybutyrate ,PHB)、聚羟基丁酸 /聚羟基戊酸共聚物 (Poly-hydroxybutyrate/hydroxyvalerate ,PHBV)皮下包埋和种植细胞生长研究实验。结果表明PHBV皮下包埋 8周仍基本保持膜状结构 ,10周完全降解 ,且生物相容性优于PHB。PHBV更适于组织工程心脏瓣膜支架材料的应用。     

Advances towards understanding heart valve response to injury.

BACKGROUND: Composed of endocardial endothelial, valvular interstitial, cardiac muscle, and smooth muscle cells (SMC), heart valves are prone to various pathologic conditions the morphology of which has been well described. The morphology of diseased valves suggest that the "response to injury" process occurs in these valves, and is associated with an accumulation of interstitial cells and matrix, valvular inflammation and calcification, conditions that lead to dysfunction. The purpose of this study is to describe the current knowledge of the regulation of the valvular "response to injury" process, since we feel that this paradigm is essential to understanding valve disease. METHODS: The pertinent literature relating to the cell and molecular biology of valvular repair, and specifically interstitial cell function in valve repair, is reviewed. RESULTS: The cell and molecular biology of valve interstitial cells are poorly understood. Molecules regulating some of the aspects of the "response to injury" process have been studied, however, the signal transduction pathways, gene activation, and interactions of bioactive molecules with each other, with cells, and with the matrix have not been characterized. Initial studies identify the cell and molecular biology of interstitial cells to be an important area of research. Agents that have been studied include nitric oxide (NO) and FGF-2 and several matrix-related proteins including osteopontin. The present review suggests several directions for future study and a working model of valvular repair is presented. DISCUSSION: The regulation of the "response to injury" process in the human heart valve is still largely unknown. The cell and molecular events and processes that occur in heart valve function and repair remain poorly understood. These events and processes are vital to our understanding of the pathobiology of heart valve disease, and to the successful design of tissue engineered replacement valves.     

Viability and enzymatic activity of cryopreserved porcine heart valve.

Fibroblast viability of a natural tissue valve for replacing a defective heart valve through allograft or xenograft has been suggested to affect its clinical durability. In this study, the cell viability and enzymatic activity of porcine heart valve leaflets were examined in regard to concerning to the preservation process [variable warm ischemic time (WIT), cold ischemic time (CIT), and cryopreservation]. Porcine heart enblocs were obtained and valve dissection was performed after 2, 12, 24, or 36 hours, in respective groups A, B, C, and D, as WIT. Each group was stored for 24 hours as CIT and cryopreserved. Leaflets were dissected from a valved conduit after each process, and cell viability and enzymatic activity in the leaflet were investigated using trypan blue staining and API ZYM kits. WIT extension significantly decreased fibroblast viability (p < 0.05, 92.25 +/- 2.7% at 2 hours, 84.9 +/- 6.7% at 12 hours, 57.0 +/- 10.2% at 24 hours, 55.9 +/- 7.9% at 36 hours), while CIT for 24 hours was also influenced significantly (p < 0.05), whereas cryopreservation demonstrated no effect on cellular viability. In enzyme activity observation, several enzymes related to lipid or nucleotide degradation (esterase, esterase lipase, particularly phosphatase, phosphohydrolase) were remarkably changed following the valve-fabrication process. After 24 hours CIT, these enzymatic activities in groups B, C and D significantly increased, but the activities decreased after cryopreservation. Particularly, both the viability and enzymatic activity showed remarkable changes after CIT in group B (WIT = 12 hours). These results suggest that WIT is more important than CIT in maintaining viability of the valve, and that completing all the cryopreservation process within 12 hours after acquisition is recommended.     

Antigen clearing from porcine heart valves with preservation of structural integrity

Bioprostheses currently used for replacement of diseased cardiovascular tissue are preserved and partially protected from immune rejection through chemical fixation. However, after implantation, chemically preserved (fixed) material has limited durability and lacks the ability to revitalize through cellular ingrowth and remodeling. As an alternative to fixation, we aimed at thoroughly removing antigens from tissue, leaving an intact scaffold, suitable for integration and revitalization in the host. Extensive washing of porcine heart valves with a mixture of two detergents (SDS and Triton X-100) yielded an intact matrix devoid of cells and depleted of soluble proteins that was minimally immunogenic in rabbits. A detailed characterization of the biomechanics and durability of the tissue is under way. If the lack of immunogenicity is confirmed in primates, our results would suggest that a detergent-washed, unfixed porcine heart valve can be an attractive non-inflammatory scaffold for heart valve regeneration in humans.     

Increased numbers of circulating donor-specific T helper lymphocytes after human heart valve transplantation

Implantation of cryopreserved human donor heart valves for either congenital or acquired cardiac disease has been performed since the last three decades. Although the clinical outcome is good, long-term valve degeneration resulting in dysfunction has been observed. A specific immunological response of the recipient against the allograft has been proposed as one of the factors involved in this process. Helper T lymphocytes play an important intermediate role in cellular and humoral immune response. Increasing numbers of circulating donor-specific helper T lymphocytes precursors (HTLp) correlate with graft rejection after organ transplantation. To investigate whether cryopreserved human donor heart valves are able to induce a donor-specific T helper response, we monitored the HTLp frequencies (HTLpf) in peripheral blood samples of 13 patients after valve allograft transplantation by use of a limiting dilution assay followed by an interleukin-2 bioassay. Prior to transplantation, HTLpf specific for donor and third-party antigens showed individual baseline levels. After allografting, the antidonor frequencies significantly increased in 11 of the 13 patients (P = 0.02). This was not found for stimulation with third-party spleen cells (P = 0.68), which indicates a donor-specific response. Maximal donor-specific HTLpf were already found at 1--2 months after operation. Valve allograft transplantation induces an increase in the numbers of donor-specific HTLp in peripheral blood of the patients. Analogous to organ transplantation, these HTLp may play a crucial role in events that lead to valve damage. Therefore, monitoring of HTLp in peripheral blood samples might be informative for donor valve degeneration (rejection) and subsequently valve allograft failure.     

A broad and strong humoral immune response to donor HLA after implantation of cryopreserved human heart valve allografts

Cryopreserved human heart valves are used for valve replacement in patients with congenital or acquired heart disease. Although no blood group or human leukocyte antigens (HLA) matching is performed and no immunosuppression is administered, the clinical results are relatively good. After valve replacement, the majority of the patients develop HLA antibodies, whereas a smaller group of patients shows valve-related events at the long term after right ventricular outflow tract reconstruction. Therefore, we hypothesized that not the mere presence, but rather the titers of antidonor HLA antibodies may be related to valve allograft failure. The presence and specificity of HLA class I antibodies were determined by complement-dependent microlymphocytotoxicity (CDC) test in longitudinally taken peripheral blood samples of 35 valve allograft recipients. In eight patients with an antibody response specific against donor-HLA class I, the titers were measured by this CDC method after stepwise dilution of the plasma. Panel reactive antibodies of more than 10% were found in 31 of 35 (89%) valve allograft recipients. From these 31 patients, 24 (77%) developed donor-specific HLA class I antibodies. All eight selected patients had detectable donor-specific antibody titers, ranging from 1:2 to 1:8,000. Two donor valve recipients before retransplantation had (donor-specific) HLA antibodies and showed high titers of 1:256 and 1:8,000 shortly after the second allograft valve replacement, which was associated with an early graft failure in the latter patient. We conclude that transplantation of cryopreserved human heart valve allografts leads to a broad and strong humoral response, which is probably the result of a lack of immunosuppressive therapy after valve transplantation. Patients receiving a second or following valve allograft appeared to be sensitized and developed early and high allo-antibody titers after second valve allograft implantation. Valve failure was diagnosed in a patient with extremely high titers. These findings suggest that preoperative cross-matching may identify patients with high donor-specific HLA antibody titers and may reduce the risk for early recurrent graft failure.     

In vivo study of the effects of cryopreservation on heart valve xenotransplantation.

BACKGROUND: Recent reports have suggested that cryopreservation reduces the immunogenicity of donor tissue. The immunomodulation by cryopreservation might influence on the tissue durability after xenotransplantation. We investigated the in vivo morphologic changes in cryopreserved xenograft (CXG) heart valves. MATERIAL AND METHOD: We transplanted a fresh (fresh xenograft; FXG) and a cryopreserved (CXG) porcine aortic root and a cryopreserved canine (cryopreserved allograft; CAG) aortic root into the abdominal aorta of a dog without any immunosuppressive agents. Explanted grafts on the 21st to 49th days after implantation were analyzed morphologically with light microscopy using some special stains, immunohistochemical analysis, and scanning electron microscopy (SEM). RESULT: Light microscopy showed the absence of smooth muscle cells in the media of the aorta in any group after transplantation. FXG valves did not maintain any cellularity after transplantation. CXG valves contained cellular infiltration in themselves. CAG valves contained numerous fibroblasts, which showed the maintenance of tissue integrity without allowing cellular infiltration. The structure of elastic fibers was well maintained, even in the part of CXG valve with cellular infiltration. Immunohistochemical studies documented the infiltration of T lymphocytes in CXG valves that were labeled by anti-CD3 antibodies. SEM demonstrated that no endothelia were seen on the surface of the valves in any group after transplantation. CONCLUSION: We concluded that the cryopreservation method might provide an immunomodulation of xenogeneic heart valves for transplantation.     

Measurements of the effects of decellularization on viscoelastic properties of tissues in ovine, baboon, and human heart valves

In the development of tissue-engineered heart valves based on allograft decellularized extracellular matrix scaffolds, the material properties of the implant should be ideally comparable to the native semilunar valves. This investigation of the viscoelastic properties of the three functional aortic/pulmonary valve tissues (leaflets, sinus wall, and great vessel wall) was undertaken to establish normative values for fresh samples of human valves and to compare these properties after various steps in creating scaffolds for subsequent bioreactor-based seeding protocols. Torsional wave methods were used to measure the viscoelastic properties. Since preclinical surgical implant validation studies require relevant animal models, the tests reported here also include results for three pairs of both ovine and baboon aortic and pulmonary valves. For human aortic valves, four cryopreserved valves were compared with four decellularized scaffolds. Because of organ and heart valve transplant scarcity for pulmonary valves, only three cryopreserved and two decellularized pulmonary valves were tested. Leaflets are relatively soft. Loss angles are similar for all tissue samples. Regardless of species, the decellularization process used in this study has little effect on viscoelastic properties.     

利用人骨髓基质干细胞构建组织工程心脏瓣膜的体外实验

BACKGROUND:Nowadays, mechanical or biological valve recipients used in the clinic are still at the risk of infection, hemorrhage, thrombosis and reoperation owing to valve stenosis. Tissue-engineered heart valve with biological activity can overcome the disadvantages above. While, the optimal choice of scaffolds and seeding cells remains disputable. OBJECTIVE:To explore the feasibility to construct tissue-engineered heart valve with acellularized porcine aortic valve scaffold and human bone marrow stromal stem cells in vitro. METHODS:The porcine aortic valves were decellularized with the detergent and enzymatic extraction process to remove the cellular components. Human bone marrow stromal stem cells were aspirated from sternum of the patients with simple congenital heart malformation, and then the cells were seeded on the acellularized porcine aortic valve scaffold and cultured for 5 days. RESULTS AND CONCLUSION:Flow cytometry identified that the characteristics of surface antigen of the inoculated seed cells were in line with those of human bone marrow stromal stem cells. Light microscopy and electron microscopy confirmed that the cellular components in the porcine aortic valves could be removed to obtain the complete acellular fiber mesh stent. There was no significant difference in biomechanical property between before and after acellularization. The human bone marrow stromal stem cells implanted on the acellularized porcine aortic valve scaffold could form a continuous cell layer on the surfaces of the scaffold. The inoculated bone marrow stromal stem cells could be differentiated into fibroblasts. The implantation of human bone marrow stromal stem cells on the acellularized porcine aortic valve scaffold can construct the tissue-engineered heart valve.