小口径异种血管作为冠状动脉旁路移植术桥材料的研究

目的 研制和评价一种新型小口径异种血管，为冠状动脉旁路移植术（CABG）提供新的移植血管来源。方法 将犬颈动脉采用酶-去污剂法进行脱细胞处理，脱细胞后随机分为两组，肝素结合组（n=24）：进一步进行肝素结合处理；肝素未结合组（n=24）：仅脱细胞作为对照。两组的脱细胞异种血管同时分别植入同一只兔左、右侧颈动脉，在3周、3个月和6个月时用B超观察移植血管的通畅情况；3个月和6个月时分别取材行组织学、电子显微镜及免疫组织化学检查。结果 犬颈动脉细胞被完全去除，肝素结合于血管管壁全层。脱细胞异种血管植入兔颈动脉后两组均无血管阻塞，而肝素结合组血栓形成少于肝素未结合组。移植后3个月两组中无血栓形成的血管管壁均出现大量平滑肌细胞，而内腔由内皮细胞覆盖。结论 酶-去污剂联合肝素处理的犬颈动脉，有可能作为CABG的一种新型小口径异种移植血管。     

bFGF预载脱细胞支架构建小口径人工血管的研究

目的目前临床使用的小口径（〈5cm）人工血管因生物相容性差、远期通畅率低，效果不理想。拟通过在脱细胞血管支架表面预载bFGF，制备一种新型的小口径人工血管。方法采用去污剂一酶消化法制备犬颈动脉脱细胞支架，将bFGF预载在经肝素固化（肝素固化组）和未固化的（单脱细胞组）脱细胞支架表面，ELISA法检测结合的bFGF量及体外释放情况。通过与犬BMSCs体外复合培养1～5d，观察bFGF预载肝素固化脱细胞支架（bFGF预载组）和未固化的脱细胞支架（未预载组），以及各自空白对照组细胞生长情况。取8只杂交犬，切断并剪下颈总动脉造成约5cm缺损，随机选取一侧，将预载组支架行端端吻合于缺损中，作为实验侧；将未预载组支架以同样方法植入对侧，作为对照侧。术后8周取材行DSA、HE染色观察移植效果。结果犬颈动脉经脱细胞后大体形态完好、细胞基本去除、纤维结构完整。肝素固化组支架表面结合的bFGF量与bFGF反应浓度成正相关，与相同反应浓度下单脱细胞组比较差异有统计学意义（P〈0．05）；肝素固化组在浓度为100ng／mL下结合的bFGF可在体外持续释放20d。bFGF预载支架促进BMSCs增殖，MTT显示BMSCs在两组支架表面均可黏附生长，复合培养1、2d两组差异无统计学意义（P〉0．05）：3～5d，bFGF预载组支架表面细胞增殖活性明显高于未预载组（P〈0．01）。异体犬颈动脉移植后8周，实验侧支架均通畅，且有细胞覆盖内膜及浸润管壁，而对照侧通畅率仅为12．5％（1／8），闭塞的移植物腔内均为血栓形成，未见细胞覆盖。结论对同种异体血管脱细胞支架表面进行bFGF预载，初步获得一种具有良好生物相容性和通畅性的生物人工血管，其远期通畅率及生物安全性仍待进一步评价。     

肝素处理小口径脱细胞异种血管的移植研究

冠状动脉旁路移植术（CABG）中常因自体动静脉材料有限及曲张、炎症等原因,无法满足需求.我们研究探讨应用脱细胞联合肝素处理的方法在短时间内制备小口径异种移植血管的可行性.     

生物酶连续消化法对犬颈总动脉脱细胞基质材料生物力学的影响研究

目的：探讨利用生物酶连续消化法制备犬颈总动脉脱细胞血管基质时对材料生物力学的影响。方法：利用胰酶和核酸酶连续消化法制备犬颈总动脉脱细胞血管基质材料,通过组织学、扫描电镜观察和DNA含量的检测,判断细胞的脱除情况;检测脱细胞材料的拉伸强度、爆裂强度及顺应性,判断脱细胞过程对材料生物力学性质的影响。结果：组织学染色及扫描电镜的结果表明血管细胞成分被完全去除,血管细胞外基质成分保留完整;与新鲜犬颈总动脉相比,脱细胞材料的拉伸强度和爆裂强度没有明显的改变,而材料的顺应性有所降低。结论：生物酶连续消化法制备的犬颈总动脉脱细胞基质材料保持了与新鲜血管大部分生物力学性质,但材料的顺应性有所降低。     

脱细胞血管基质制备和异体移植的实验研究

目的研究建立无细胞血管基质制备的新方法,并探讨脱细胞血管异体移植的可行性。方法采用不同去垢剂[1％Triton X-100、1％十二烷基磺酸钠(SDS)]多步骤对血管进行脱细胞处理,并通过组织学和力学观测,建立兔动脉血管脱细胞的方法;并进行脱细胞血管的异体移植。结果经低渗溶液和去垢剂1％TritonX-100、1％SDS等多步骤处理,兔胸主动脉血管的细胞基本脱除,细胞外基质保持完好,血管的弹性、韧性和力学强度仅轻度降低;用该法制备的兔颈动脉(直径约2．0mm)进行异体移植,经2个月观察,全部通畅。结论经去垢剂TritonX-100、SDS加低渗溶液和蛋白酶抑制剂处理的多步法,可以脱除血管的细胞成分,细胞外基质和力学特性保持完好,是一种较好的方法;用该法制备的兔颈动脉(直径约2．0mm)进行异体移植,初步取得成功。     

氯化镁缓释抗钙化组织工程小口径血管体外性能测试

目的:构建氯化镁(MgCl 2)微米缓释抗钙化的组织工程小口径血管。 方法:联合应用Triton X-100+脱氧胆酸钠盐、DNA/RNA核酶对绵羊颈动脉行脱细胞处理,制成组织工程小口径血管支架,HE染色,电镜下观察脱细胞情况和血管支架性能。采用复乳法超声破碎高速搅拌挥发法,制备载有MgCl ...     

胎猪脱细胞主动脉作为小口径组织工程血管替代物的体内试验研究

目的:研究胎猪脱细胞主动脉(decellularized aorta of fetal pigs,DAFP)的生物相容性,确定其是否有作为支架材料用于小口径组织工程血管移植的潜力。方法:利用胰酶和核酸酶联合的脱细胞方法来制备胎猪主动脉脱细胞基质(DAFP),将其作为小口径组织工程血管的生物支架材料移植在成年犬单侧颈总动脉处,并监测其移植处的血流通畅情况,后期又通过组织学染色观察组织工程血管的组织学结构;扫描电镜观察血管的内表面结构;透射电镜观察其内表面的内皮细胞再生情况。结果:组织学染色结果表明构建的小口径组织工程血管具有完整的内膜层及中膜层结构;扫描电镜结果显示组织工程血管内表面覆盖着完整的内皮细胞层。结论:DAFP有作为小直径组织工程血管支架用于体内移植的潜力。     

脱细胞血管基质和间充质干细胞构建组织工程血管

目的 探讨利用异种脱细胞血管基质和间充质干细胞体外构建小口径血管移植物的方法.方法 采用去垢剂和胰蛋白酶去除猪髂动脉血管壁的细胞成分,对脱细胞基质进行组织学、力学检测及孔隙率评估.分离培养犬骨髓问充质干细胞,种植到脱细胞基质上,并进一步在搏动性生物反应器内培养,采用HE染色和扫描电镜对构建的组织工程血管进行检测.结果 脱细胞处理后,猪髂动脉的细胞成分完全去除,细胞外基质保存完好,力学强度轻度下降;脱细胞基质的孔隙率为94.9%.间充质干细胞能够种植到脱细胞基质上,在剪切力的作用下细胞基本融合,高度伸长并且其排列与流体的方向一致.结论 小口径血管移植物可以通过将间充质干细胞种植到异种脱细胞血管基质并在搏动性生物反应器内培养的方法进行构建.     

牛心包组织工程心脏瓣膜支架脱细胞方法的比较

目的对比去污剂-酶消化法、胰蛋白酶消化法和去氧胆酸钠法去除新鲜牛心包组织上细胞的效果和保护基质的能力，为组织工程心脏瓣膜的构建提供较满意的平台。方法应用3种方法处理新鲜牛心包组织，用光学显微镜、扫描电镜观察脱细胞效果和胶原纤维、弹力纤维改变；用热皱缩实验、拉力测试观察基质的物理性能变化；用DNA抽提比较脱细胞前后细胞数量差异。结果3种方法均能完全去除细胞，与去污剂-酶消化法比较，另2种方法对基质破坏明显。结论去污剂-酶消化法脱细胞效果好，且有良好的保护基质的能力。     

组织工程角膜生物材料载体制备的比较性研究

目的 比较用不同的方法脱细胞处理异种（猪）角膜基质制备组织工程角膜支架材料，并对其生物相容性进行研究。方法 成年York猪角膜基质材料，以Triton联合0．25％胰酶，处理30min 3h为材料1；Triton联合DNA—RNA酶，处理8～14h为材料2；Triton联合0．25％胰酶、DNA—RNA酶，处理3～5h为材料3。3种材料用无水氯化钙脱水干燥保存。在材料上接种兔角膜基质成纤维细胞，观察细胞生长情况。新西兰大白兔24只，随机分为3组，每组8只。右眼为实验眼，左眼为空白对照眼。将材料植入兔眼角膜基质层中，术后每日裂隙灯检查术眼；1、4、8和12周分别随机取2只兔眼角膜作HE染色，观察材料的生物相容性。结果 材料1、2细胞脱出不完全，处理最佳时间分别是2hN10～12h，角膜基质网状间隙无明显增大；接种兔角膜基质成纤维细胞后，3～4d细胞死亡；植入兔角膜基质中有明显的免疫排斥反应。材料3脱出细胞完全，其脱细胞处理的最佳时间为4h，角膜基质纤维结构无破坏，呈三维网状结构，网状间隙明显增大；接种兔角膜基质成纤维细胞后，细胞在材料上贴附生长良好；将其植入兔角膜基质中无炎性及排斥反应，可逐渐降解吸收，有良好的生物相容性。结论 Triton联合0．25％胰酶、DNA—RNA酶法处理的异种（猪）角膜基质具有良好的生物相容性，可作为一种组织工程角膜的支架材料。     

Implantation of decellularized small-caliber vascular xenografts with and without surface heparin treatment

Heparin treatment of decellularized xenografts has been reported to reduce graft thrombogenicity. However, little is known about the in vivo comparison of heparin-treated with non-heparin-treated xenografts, especially for small-caliber vascular implants. We implanted either a heparin-treated or a non-heparin-treated canine carotid artery as bilateral carotid xenograft in rabbits (n = 24). Small-caliber xenografts (3 approximately 4 mm) were decellularized by enzymatic and detergent extraction and were further covalently linked with heparin. During implantation, thrombosis rate was 4% in the heparin-treated xenografts and 25% in the non-heparin-treated xenografts after 3 weeks (P < 0.05). After 6 months, it was 8 versus 58%, respectively (P < 0.01). Both heparin-treated and non-heparin-treated xenografts harvested at the end of 3 and 6 months showed a satisfactory cellular reconstruction of either smooth muscle cells or endothelial cells. These results indicate that heparin treatment of the small-caliber decellularized xenograft reduces the in vivo thrombogenicity. Both heparin-treated and non-heparin-treated xenografts seem to undergo a similar cellular remodeling process up to 6 months.     

Heparin Coating of Small-Caliber Decellularized Xenografts Reduces Macrophage Infiltration and Intimal Hyperplasia

Small‐caliber decellularized xenografts with surface heparin coating are known to reduce in vivo thrombogenicity. This study was performed to examine whether heparin coating on the small‐caliber decellularized xenografts would reduce macrophage infiltration and intimal hyperplasia. In a rabbit model of bilateral carotid implantation, each of the animals (n = 18) received a heparin‐coated decellularized xenograft from a canine carotid artery on one side and a nonheparin‐coated one on the other side. These experiments were terminated respectively at 1 week (n = 6), 3 weeks (n = 6), and 12 weeks (n = 6). Results showed that, compared with the nonheparin‐coated grafts, the heparin‐coated grafts had significantly less macrophage infiltration 1 week after implantation, identified by the mouse antirabbit macrophage antibody (RAM11)‐positive cells on the vascular wall, covering all the proximal, middle, and distal parts of the grafts (P < 0.01). Moreover, the heparin‐coated grafts also showed less deposition of proliferation cell nuclear antigen (PCNA)‐positive cells on the vascular wall, indicating less cell proliferation, which was significant not only at 1 week (P < 0.01) but also at 12 weeks (P < 0.01). Intimal hyperplasia, measured by the intimal : media (I : M) ratio, was found similar in both groups at 1 and 3 weeks. However, the I : M ratio was significantly lower in the heparin‐coated group than in the nonheparin‐coated group at 12 weeks, especially in the proximal anastomosis area (0.76 ± 0.12 vs. 0.345 ± 0.06, P < 0.01). Heparin coating of small‐caliber decellularized xenografts is associated with an early reduction of macrophage infiltration and intimal hyperplasia in a rabbit model of bilateral carotid artery implantation for 12 weeks. Thus, heparin coating appears to deliver not only the antithrombogeneity but also the antiproliferative property for small‐caliber decellularized xenografts.     

Antiplatelet therapy and vascular grafts. Studies in a baboon ex vivo shunt

Antiplatelet therapy is currently recommended in an effort to improve patency rates of small-caliber vascular grafts. The effect of aspirin and heparin on acute platelet deposition was studied in a baboon ex vivo shunt. Two grafts, expanded polytetrafluoroethylene and knitted Dacron, were exposed to a flow rate of 25 mL/min after administration of aspirin or heparin. Indium 111-labeled platelet uptake by the grafts was determined over 2 1/2 hours. The amount of platelet deposition in the treated groups was significantly less than that of controls after 2 1/2 hours. There was no difference between the aspirin and heparin groups. The finding that heparin inhibited platelet deposition to a degree comparable with aspirin suggests that it may not be necessary to start antiplatelet therapy preoperatively. Intraoperative systemic heparinization will provide sufficient inhibition of platelet deposition. A protocol for perioperative antiplatelet therapy is outlined.     

Aorta-coronary bypass grafting with heparinized vascular grafts in dogs. A preliminary study

At present, only the autogenous saphenous vein is acceptable in aorta-coronary bypass grafting. We developed a small-caliber vascular graft and evaluated the potential application for aorta-coronary bypass grafting. Canine carotid arteries were cross-linked with polyepoxy compounds, such as polyglycerol polyglycidyl ether, which is a new cross-linking reagent, and then heparinized by our own method. The polyepoxy compound-cross-linked graft can keep the natural vessel compliance and is stronger than the glutaraldehyde-cross-linked graft; thus, it provides excellent suturability and compliance match. Heparin was gradually released from the graft wall, and thrombus formation was completely prevented during the period before development of the endothelial lining. As a pilot study, the grafts, 2 to 3 mm in internal diameter and 5 to 7 cm in length, were evaluated as bilateral carotid replacements in five dogs. All grafts were patent at intervals of 14 to 177 days. Histologic examinations showed excellent antithrombogenic and healing characteristics, although the endothelialization was delayed by heparin, which inhibits cell adhesion and fibrin deposition. The 3 mm internal diameter graft was evaluated as an aorta-coronary bypass grafting model in eight dogs. Flow within grafts to the right coronary artery ranged from 25 to 35 ml/min, and flow in the circumflex or left anterior descending grafts ranged from 75 to 100 ml/min. Cineangiography was performed to confirm graft patency. Three dogs died of viral infection and one was killed. At necropsy, the grafts remained patent without thrombi along the graft length. Four dogs were allowed to survive for long-term evaluation. All grafts were patent at time intervals to 21 to 113 days with 100% patency. These results led us to conclude that our newly developed small-caliber vascular graft shows great promise in application for aorta-coronary bypass grafting.     

Decellularized vein as a potential scaffold for vascular tissue engineering

PURPOSE: Current strategies to create small-diameter vascular grafts involve seeding biocompatible, compliant scaffolds with autologous vascular cells. Our purpose was to study the composition and strength of decellularized vein to determine its potential as a vascular tissue-engineering scaffold. METHODS: Intact human greater saphenous vein specimens were decellularized by using sodium dodecyl sulfate (SDS). Residual cellular and extracellular matrix composition was studied with light and electron microscopy as well as immunohistochemistry. Burst and suture-holding strength was measured in vitro by insufflation and pull-through techniques. To assess initial handling and durability of decellularized vein in vivo, a canine model was developed wherein decellularized canine jugular veins were implanted as carotid interposition grafts in recipient animals. After two weeks of arterial perfusion, these grafts were studied with duplex imaging and histologic methods. RESULTS: Human saphenous vein decellularized by using SDS was devoid of endothelial cells and >94% of the cells resident within the vein wall. Collagen morphology appeared unchanged, and elastin staining decreased only slightly. Basement membrane collagen type IV remained intact. Compared with fresh vein, decellularized vein had similar in vitro burst (2480 +/- 460 mm Hg vs 2380 +/- 620 mm Hg; P >.05) and suture-holding (185 +/- 30 gm vs 178 +/- 66 gm; P >.05) strength. Decellularized canine vein functioned well in vivo without dilation, anastomotic complication, or rupture over 2 weeks of arterial perfusion. CONCLUSIONS: Vein rendered acellular with SDS has well-preserved extracellular matrix, basement membrane structure, and strength sufficient for vascular grafting. These properties suggest proof of concept for its use as a scaffold for further vascular tissue engineering. CLINICAL RELEVANCE: The following research examines the creation of a new small-diameter bypass graft. It is clinically relevant to patients who need distal arterial bypass, coronary artery bypass, or hemodialysis access, but who do not have adequate autologous vein for their surgeries. Future investigations will involve further tissue engineering of this vascular scaffold (eg, autologous endothelial seeding of its lumen) and testing the clinical usefulness of the completed graft.     

Reduced blood loss and transfusion requirements with low systemic heparinization: preliminary clinical results in coronary artery revascularization

In coronary artery revascularization, low systemic heparinization was compared to full systemic heparinization during perfusion with heparin surface-coated cardiopulmonary bypass equipment. Twelve patients were randomly assigned to two groups and perfused with low [activated clotting time (ACT) > 180 s] or full (ACT > 480 s) systemic heparinization. A standard battery of blood samples was taken before the procedure, after heparinization, and at regular intervals during and after cardiopulmonary bypass. No differences were seen between the two groups in regard to age, body surface area, preoperative hematocrit, duration of bypass, bypass hypothermia, cross-clamp time, and number of bypasses per patient. However, there were more internal thoracic artery (ITA) grafts in the group with low systemic heparinization (1.5 ± 0.8 ITA grafts per patient versus 0.8 ± 0.4 ITA grafts per patient with full heparinization; p < 0.05). The oxygenator gradient at the end of perfusion (before weaning) was 107 ± 40 mmHg for low versus 110 ± 10 mmHg for full heparinization (difference not significant). The total amount of heparin used was 7200 ± 1030 IU for low versus 51 400 ± 9700 IU for full (p < 0.05). Postoperative hematocrit was 35.0 ± 2.0% for low versus 24.7 ± 2.7% for full (p < 0.05). Total chest tube drainage was 428 ± 153 ml/m² for low versus 935 ± 414 ml/m² for full (p < 0.05). Homologous transfusions of blood products were necessary in 3/6 patients for low versus 6/6 patients for full (p < 0.10). The total volume of packed red cells transfused was 221 ± 228 ml/m² for low versus 842 ± 366 ml/m² for full (p < 0.05). Final hematocrit at day 7 was 31.0 ± 2.0% for low versus 33.0 ± 3.0% for full (difference not significant). Full systemic heparinization can be avoided during clinical cardiopulmonary bypass by the use of heparin-coated perfusion equipment. A low dose of heparin, similar to the amounts given during vascular surgery, results in reduced blood loss and transfusion requirements.     

Basic fibroblast growth factor coating and endothelial cell seeding of a decellularized heparin-coated vascular graft

The objective of this study was to determine the effect of basic fibroblast growth factor (bFGF) coating on endothelial cell seeding and proliferation on a decellularized heparin coated vascular graft and to determine the retention of seeded cells on the graft under flow conditions. Disks of heparin coated decellularized grafts were incubated for 24 h as controls or with bFGF. Human microvascular endothelial cells (HMECs) or canine peripheral blood endothelial progenitor cells (CEPC) were seeded onto the disks and incubated for 96 h or 48 h, respectively. HMECs were also seeded onto the luminal surfaces of two heparin-coated decellularized grafts for 3 h. One graft was placed in a perfusion culture system and cultured for an additional 6 h with flow and pressure. After culturing, there were 4.7 +/- 1.4 cells/mm(2) HMECs on control grafts and 11.4 +/- 1.4 cells/mm(2) in bFGF treated grafts (P < 0.05). Likewise, with CEPCs, there were 14.8 +/- 4.8 cells/mm(2) in control grafts and 33.3 +/- 7.3 cells/mm(2) in bFGF treated grafts. After only 3 h of cell attachment, 60% of HMECs were retained in the intact graft exposed flow relative to the static control graft, which is an acceptable level. These data demonstrate that bFGF coating on the heparin bound decellularized grafts significantly increases both HMEC and dog EPC proliferation and that seeded cells are stable under perfusion conditions.