In vitro and in vivo hemocompatibility evaluation of graphite coated polyester vascular grafts

Attempts have been made in this study to prepare a homogeneous and stable coating of graphite on polyester vascular grafts (GPVG) using an electrophoresis method to evaluate thromboresistant and blood compatibility of GPVG in comparison to non-coated PVG and InterGard (collagen sealed PVG) as control. Lactate dehydrogenase (LDH) activity measurement was carried out on all PVG types to evaluate platelet adhesion. To examine tissue reaction GPVG and non-coated sheets of knitted polyester fabric were implanted simultaneously in the dorsal flank of rats subcutaneously. The GPVG, non-coated and control were implanted in descending aorta as end-to-end or end-to-side implantation substitution in 25 sheep for 4-60 weeks. Results showed that the graphite coating on polyester vascular grafts reduced the number of adherent platelets and prevent platelet activation and spreading on the surface in comparison with non-coated and control. Pathological investigation showed inflammatory reactions were totally resolved after 12 weeks and there was no difference in the tissue reaction between graphite coated, non-coated and control patches. All grafts remained patent and there was no significant difference in patency rate between these three types of PVG. We found that GPVG has no need for pre-clotting and it showed lower platelet aggregation, thinner capsule formation and lower calcification after 15 months. However, suturing of GPVG was more difficult in comparison with the other types.     

Construction of tissue-engineered homograft bioprosthetic heart valves in vitro

Human mesenchymal stem cells (MSCs) differentiate into multiple cell-lineages and may serve as an alternative source of seed cells for tissue engineering. We investigated whether MSCs could be induced to differentiate into endothelial cells (ECs) and function as seed cells for the in vitro construction of tissue-engineered heart valves (TEHVs). Aortic or pulmonary valve homografts were decellularized with 0.1% sodium dodecylsulphate and used as scaffolds for TEHVs. The MSCs were isolated from human bone marrow by Percoll gradient centrifugation (1.073 g/ml), differentiated into ECs with vascular endothelial growth factor (10 ng/ml), and seeded onto a decellularized scaffold (high-density seeding, >10(5) cells/cm2) and grown in static culture for 14 days. Over 90% of the differentiated cells from MSCs stained positively for von Willebrand factor and Tie-2-related antigen. Additionally, Weibel-Palade corpuscle was observed in the cytoplasm of these cells. Levels of reendothelialization in static culture on days 7, 14, and 20, were 73%, 85%, and 95%, respectively. These results show that MSCs from human bone marrow can differentiate, in vitro, into ECs that can then be used to construct TEHVs. Reendothelialization in static culture can be used to provide the basic material for pulsatile-flow cultivation.     

脂肪间充质干细胞分化为内皮细胞并体外构建组织工程心脏瓣膜

背景：组织工程心脏瓣膜是利用组织工程技术将种子细胞种植于瓣膜支架上所构建的一种人工瓣膜,目前国内外研究主要集中于种子细胞来源及支架选择上。 目的：探讨人脂肪间充质干细胞体外向内皮细胞诱导分化后的细胞作为种子细胞,脱细胞猪主动脉瓣膜作为支架体外构建组织工程心脏瓣膜的可行性。 方法：利用吸脂术采集脂肪组织,分离、培养脂肪间充质干细胞,流式细胞仪鉴定细胞表型;免疫细胞化学方法及RT-PCR检测细胞分化标志物;应用Triton X-100联合胰蛋白酶的方法制备脱细胞猪主动脉瓣支架,将体外培养扩增的诱导分化后的内皮细胞种植于支架上构建组织工程心脏瓣膜,光镜及电镜下观察组织工程心脏瓣膜的组织学结构。 结果与结论：脂肪组织分离培养的脂肪间充质干细胞向内皮细胞诱导分化后表达CD31、CD34、CD144、Ⅷ因子和内皮型一氧化氮合成酶等内皮细胞特异性抗原;脱细胞猪主动脉瓣膜支架脱细胞完全,弹力纤维及胶原纤维保持完整;构建的组织工程心脏瓣膜可见支架上排列连续的单细胞层。提示脂肪间充质干细胞在体外向内皮细胞诱导分化后已初步具有内皮细胞功能,在脱细胞猪主动脉瓣膜支架上生长良好,可以在体外初步构建组织工程心脏瓣膜。     

A phospholipid-modified polystyrene-polyisobutylene-polystyrene (SIBS) triblock polymer for enhanced hemocompatibility and potential use in artificial heart valves

Poly(styrene-block-isobutylene-block-styrene) ('SIBS') is selected for a novel tri-leaflet heart valve due to its high resistance to oxidation, hydrolysis, and enzyme attack. SIBS is modified using six different phospholipids and its mechanical properties characterized by tensile stress, peel strength, shear strength, contact angle, and surface energy, and then for hemocompatibility by studying the adhesion of fluorescently labeled platelets in a parallel plate chamber under physiological flow conditions. Phospholipid modification decreases SIBS tensile stress (at 45% strain) by 30% and reduces platelet adhesion by a factor of 10, thereby improving its hemocompatibility and its potential use as a synthetic heart valve.     

In vitro evaluation of prosthetic heart valves: towards comparable testing.

The Laboratory of Biomedical Engineering (of the National Health Institute, technical body of the Italian Health Service) performs in vitro testing of prosthetic heart valves for mechanical characteristics of materials, fatigue life, and fluidodynamic performance. Testing of materials is directed towards the physicomechanical characterization of the structural components of the valves, e.g. elasticity and resistance to stress of biological tissues and stents. Long-term fatigue life tests are conducted by means of systems which make valves beat at more than 1200 cycles/min. These tests are preceded and followed by geometrical characterization and by steady flow testing in order to obtain information about stenosis and leakage. Special attention is devoted to pulsatile flow testing which is performed on two pulse duplicators: the Dynatek system and the system developed by the University of Sheffield. The same valve was tested with these systems according to their different possible set-ups within the general requirements established by ISO-DIS 5840. This paper presents significant measurements, taking into account their dependence on the systems adopted. Results show (a) the difficulties in comparing test results because of different operating conditions, and (b) the systems' sensitivity with regards to some parameters which affect measurements under comparable set-up conditions (FDA Interlaboratory Comparison Testing Protocol).     

Presentation of viral antigens by human vascular endothelial cells in vitro

Endothelial cells (EC) separated from the umbilical vein were shown to be free of contaminating monocytes. EC could replace periferal blood-derived macrophages as antigen-presenting cells for in vivo sensitized T cells towards a variety of viral antigens. The T-cell-EC-antigen response was also specificity inhibited by anti-HLA-DR antisera. T cells primed by antigen together with autologous macrophages could be restimulated by antigen pulsed HLA-D/DR identical EC in an antigen specific secondary response, indicating a similar mechanism for antigen presentation by EC or macrophages.     

L-Rap在高糖培养的人视网膜血管内皮细胞的表达变化

目的研究白细胞源性精氨酸氨基肽酶（Leukocyte-derived Arginine Aminopeptidase，L-Rap）在高糖培养的人视网膜血管内皮细胞的表达，分析L-Rap蛋白的功能。方法体外培养人视网膜血管内皮细胞，实时定量PCR检测L-Rap在高糖培养的人视网膜血管内皮细胞的表达变化，建立L-Rap的三维蛋白结构图，分析L-Rap蛋白的功能。结果L-Rap的mRNA在高糖培养的人视网膜血管内皮细胞下降，L-Rap蛋白可能对血管生成因子、免疫炎症因子以及肾素-血管紧张素系统发挥作用。结论L-Rap与糖尿病视网膜病变密切相关，可以从血管生成因子、免疫炎症因子以及肾素-血管紧张素系统这三个途径探索L-Rap对糖尿病视网膜病变的作用。     

Preseeding of human vascular cells in decellularized bovine pericardium scaffold for tissue-engineered heart valve: an in vitro and in vivo feasibility study

Human vascular cells from saphenous veins have been used for cell seeding on the synthetic scaffolds for constructing tissue-engineered heart valve (TEHV). However, little is known about the seeding of human vascular cells on bovine pericardium, a potential natural scaffold for TEHV. This study was aimed to assess the basic in vitro and in vivo characteristics of the human vascular cells seeded on decellularized bovine pericardium. In vitro, bovine pericardium samples with cell seeding were inspected on day 7, 14, and 21 by histology, scanning electron microscopy, and immunohistochemistry. In vivo, experiments were performed in nude mice by bilateral dorsal incision for the implantation of decellularized bovine pericardium with and without cell seeding. Results demonstrated that a total of 8-10 × 10(6) cells were obtained within 4-5 wk by the primary co-culture, which were detected positive for von Willebrand factor, α-smooth muscle actin antibodies, and fibronectin, indicating the presence of endothelial cells, smooth muscle cells, and fibroblasts, respectively. In vitro, the seeded cells showed a steady increase of endothelial activity from day 1 to day 7 and remained stable until day 21. After 30 days of implantation in vivo, the cells on the decellularized bovine pericardium could differentiate directionally and show all the identities of human endothelial cells, smooth muscle cells, and fibroblasts. These results indicate that the human vascular cells from the saphenous vein are an optional cell source for seeding on decellularized bovine pericardium scaffold for constructing TEHV.     

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.     

雌二醇对原代人牙周膜细胞生物学特性的影响

目的 探讨不同浓度17β-雌二醇对原代人牙周膜细胞(human periodontal ligament cells,hPDLCs)增殖、细胞周期以及碱性磷酸酶活性的影响.方法 无菌条件下刮取人牙周膜组织,采用组织块结合酶消化法进行原代培养并用免疫组化法鉴定.处于对数生长期的第4代hPDLCs分别加入不同浓度的17β-...     

体外诱导羊膜来源间充质干细胞分化为血管内皮细胞

背景：羊膜来源间充质干细胞植入机体不同类型组织后是否可以分化为相应组织靶细胞呢？ 目的：检测血管内皮细胞生长因子在体外诱导人羊膜间充质干细胞分化为血管内皮细胞的可行性。 方法：分离培养羊膜间充质干细胞,鉴定其表面抗原表达,用含体积分数2%胎牛血清以及50 μg/L血管内皮生长因子的条件培养基诱导,诱导后细胞通过内皮细胞标志物血管内皮生长因子受体2以及v-WF染色鉴定。 结果与结论：羊膜间充质干细胞表面抗原CD29、CD44、CD105阳性,CD34、CD45、CD106以及HLA-DR阴性。诱导后细胞形态明显改变,内皮细胞标志物血管内皮细胞生长因子受体2以及v-WF染色结果阳性。提示羊膜间充质干细胞在体外具有分化为血管内皮细胞的能力。     

In vitro susceptibility of human vascular wall cells to infection with Chlamydia pneumoniae.

Chlamydia pneumoniae is a common respiratory pathogen. Recent studies have demonstrated the presence of C. pneumoniae in coronary and aortic atherosclerotic lesions. To study the role of C. pneumoniae in atherosclerosis, we investigated the susceptibilities of three different cells of the human vascular wall to infection with C. pneumoniae AR-39. These cell types were endothelial cells, smooth muscle cells, and macrophages derived from peripheral blood monocytes. Infection was assessed by using a direct fluorescent antibody to assess inclusion counts. Duplicate cell samples were harvested 3 days postinfection and were passed in HL cells, a susceptible human epithelial cell line, to determine if infectious organisms were produced. Endothelial cells, smooth muscle cells, and macrophages were capable of supporting C. pneumoniae growth in vitro. These results showed that three different cell types known to be important in atherogenesis are susceptible to infection with C. pneumoniae.     

New pulsatile bioreactor for in vitro formation of tissue engineered heart valves

Two potential obstacles to the creation of implantable tissue engineered heart valves are inadequate mechanical properties (ability to withstand hemodynamic stresses) and adverse host-tissue reactions due to the presence of residual nondegraded polymer scaffold. In an attempt to address these problems, we developed an in vitro cell culture system that provides physiological pressure and flow of nutrient medium to the developing valve constructs. It is anticipated that in vitro physical stress will stimulate the tissue engineered heart valve construct to develop adequate strength prior to a possible implantation. Long-term in vitro development will be realized by an isolated and thereby contamination-resistant system. Longer in vitro development will potentially enable more complete biodegradation of the polymeric scaffold during in vitro cultivation. This new dynamic bioreactor allows for adjustable pulsatile flow and varying levels of pressure. The system is compact and easily fits into a standard cell incubator, representing a highly isolated dynamic cell culture setting with maximum sterility, optimal gas supply and stable temperature conditions especially suited for long-term experiments.     

Design and evaluation of novel blood incubation systems for in vitro hemocompatibility assessment of planar solid surfaces

Success in the development of hemocompatible biomaterials depends on adequate equipment and procedures for standardized analysis of blood-materials interactions in vitro. In view of the limited standard of knowledge on that important aspect, two novel incubation systems were designed, built, and evaluated for the in vitro assessment of the hemocompatibility of planar solid surfaces: A screening setup was introduced for the comparison of up to 12 different samples. A perfusion setup was developed to model the directed blood flow in the vascular system during incubation by a recirculation circuit, allowing the variation of the wall shear rate at the sample surface. The incubation procedures utilized freshly drawn, heparinized whole human blood. Hemocompatibility in terms of selected aspects of coagulation, thrombogenicity, and immune responses was quantified through plasma levels of characteristic molecules (immunoassays), cell counting, and analysis of adherent cells and fibrin formation (scanning electron microscopy), respectively. Prevention of blood-air contact and mechanical stress, constant temperature and blood pH during incubation, and the suitable choice of reference materials were found to be crucial for reliable testing. Considering those requirements, screening and perfusion system both provided sensitive discrimination between a given set of planar solid surfaces. In conclusion, the suggested methods for an in vitro hemocompatibility assessment permit versatile, sensitive, and efficient analysis of important blood-material interactions despite the unavoidable variability of blood characteristics in different experiments.     

Origin and appearance of HITS induced by prosthetic heart valves: an in vitro study

Patients with mechanical heart valve prostheses show significantly enhanced numbers of HITS detected by transcranial Doppler ultrasound. In order to assess the origin of HITS formation, an in vitro study was set out to quantify valve induced microemboli for mechanical and bioprosthetic valves under various circulatory conditions by means of Ultrasound-Doppler-Sonography. At the same time the influence of CO2 partial pressure on HITS rate vas investigated. It can be summarised that for mechanical heart valve prostheses a strong correlation exists between left ventricular dp/dtmax and the detected HITS rates. It was also demonstrated that a bioprosthesis generates significantly less HITS than a mechanical valve. The origin of HITS is gaseous since the tests were carried out using a cell-free filtered water-glycerol test fluid. The HITS rate could be increased by increasing the amount of dissolved gas within the test fluid. The results support the hypothesis that cavitation is the key factor in the appearance of gaseous microemboli at heart valve prostheses.     

Tissue engineering of heart valves: formation of a three-dimensional tissue using porcine heart valve cells

Tissue engineering is a promising approach to obtaining lifetime durability of heart valves. The goal of this study was to develop a heart valve-like tissue and to compare the ultrastructure with normal valves. Myofibroblasts and endothelial cells were seeded on a type I collagen scaffold. The histologic organization and extracellular matrix were compared in light and electron micrographs. Radiolabeled proteoglycans were characterized by enzymatic degradation experiments. In tissue engineered specimens, cross sectional evaluation revealed that the scaffold (300 microm) was consistently infiltrated with myofibroblasts. Both sides were covered with a multicellular layer of myofibroblasts and overlaid by endothelial cells (50 microm). A newly formed extracellular matrix containing collagen fibrils and proteoglycans was found in the interstitial space. Collagen fibrils with a 60 nm banding pattern were found in both specimens. Small sized proteoglycans (65 nm) were associated and aligned at intervals of 60 nm with collagen fibrils. Large sized proteoglycans (180 nm) were located outside the collagen bundles in amorphous compartments of the extracellular matrix. The majority of glycosaminoglycans were chondroitin/dermatan sulfate, and a minority were heparan sulfate. The morphology and topography of cells and the organization of extracellular matrix in artificial tissues strongly resembles those of native valve tissues.     

人VEGF165基因真核表达质粒的构建及其对血管内皮细胞增殖的影响

目的 :构建人VEGF16 5基因的真核表达质粒pBudCE4 .1/VEGF16 5 ,观察其在血管内皮细胞 (VEC)中的表达和表达产物对VEC增殖的影响。方法 :用RT PCR法从引产胎儿心脏组织中克隆VEGF16 5基因 ,并将其克隆至真核表达质粒pBud CE4 .1中 ,对重组真核表达质粒pBudCE4 .1/VEGF16 5进行酶切鉴定和测序。以脂质体转染法将pBudCE4 .1/VEGF16 5导入VEC中 ,用Northernblot和免疫细胞化学染色法 ,分别从mR NA水平和蛋白质水平检测它们在转染的VEC中的表达 ,并检测表达产物对VEC增殖的影响。结果 :人VEGF16 5基因的RT PCR产物为 5 76bp。测序结果显示 ,扩增的VEGF16 5基因的序列与基因文库中登录的序列完全一致。经HindⅢ和BamHI酶切鉴定证实 ,VEGF16 5基因已成功地克隆至真核表达质粒pBudCE4 .1中。以其转染血管内皮细胞 (VEC)后 ,经Northernblot杂交和免疫细胞化学染色法检测均证实VEGF16 5基因表达。表达产物对VEC增殖有明显的促进作用。结论 :所构建的pBudCE4 .1/VEGF16 5真核表达质粒可在VEC中表达 ,表达产物可明显促进VEC增殖 ,为通过VEGF16 5基因转染防治移植器官内血管的狭窄奠定了基础     

海洋单细胞海藻体外对人脑胶质瘤干细胞生物学活性的影响

背景：海藻具有广阔的药理活性前景,加强其研究对有目的进行应用开发有重要的指导意义。 目的：观察海洋单细胞海藻在体外对人脑胶质瘤干细胞生物学活性的影响。 方法：以酶消化法培养人脑胶质瘤干细胞,流式细胞分选出CD133阳性细胞,细胞传代获得第3代细胞。流式细胞仪检测海洋单细胞海藻作用前后细胞CD133表达变化,免疫组织化学检测贴壁细胞巢蛋白及胶质纤维酸性蛋白的表达。实验组分别加入不同质量浓度的海洋单细胞海藻,阴性对照加不含药的PBS,将稀释成4,6,8,10 g/L的海洋单细胞海藻加入细胞培养液中并作用24,48,72 h,流式细胞仪检测胶质瘤干细胞生长周期,应用酶标仪检测细胞生长抑制情况。 结果与结论：随着浓度和时间的增加,与对照组相比倒置显微镜下可见实验组胶质瘤干细胞不易聚团成球,出现贴壁分化,并逐渐明显;加药后胶质瘤干细胞CD133表达量明显减少;出现的贴壁细胞免疫组织化学染色巢蛋白及胶质纤维酸性蛋白表达阳性;流式细胞仪检测显示,停滞在S、G2/M期细胞数增加,而G0/G1期细胞数减少;随着浓度和时间的增加,胶质瘤干细胞增殖明显抑制,与对照组相比差异有显著性意义(P < 0.05~0.01)。提示海洋单细胞海藻能够抑制人脑胶质瘤干细胞的增殖,并促进其分化,且作用具有浓度和时间依赖性。     

人脂肪干细胞的分离培养与生物学特性

背景：高效、稳定地获得大量较高纯度的人脂肪干细胞,是其在组织工程学及再生医学中广泛应用的基础和前提。 目的：探索体外培养脂肪干细胞的适宜条件,从而提高其增殖能力。 方法：采用胶原酶消化的方法,从人腹部皮下块状脂肪中分离出脂肪干细胞,经贴壁筛选法纯化细胞、低糖培养基体外培养扩增。Giesam染色后观察细胞形态;绘制细胞生长曲线并进行细胞周期分析,观察分析传代后染色体核型改变;选取第3代脂肪干细胞做流式细胞鉴定、EdU细胞增殖能力检测以及克隆形成实验。 结果与结论：分离培养的原代脂肪干细胞形态不一,经传代后的细胞形态趋于长梭形,排列紧密呈漩涡状生长。细胞生长曲线呈S形,细胞周期分析及EdU掺入法结果显示体外培养的脂肪干细胞具有较强的增殖能力。经染色体核型分析结果提示,体外培养不会引起脂肪干细胞的核型改变。第3代脂肪干细胞经流式细胞仪检测CD29,CD44,CD90,CD105均呈阳性表达,而CD34和CD45呈阴性;克隆形成率为8.8%;在一定的诱导条件下,脂肪干细胞能够向脂肪细胞及成骨细胞分化。结果说明采用胶原酶消化法可成功分离培养人脂肪干细胞,且具有较强的增殖能力。