脱细胞肝素处理犬颈动脉的异种移植研究

目的：研究用去污剂-酶消化联合肝素结合处理法制备小口径异种移植血管.方法：犬颈动脉管壁经脱细胞后部分再行肝素结合处理,样本经组织学及电镜观察脱细胞效果,机械性能研究观察脱细胞对管壁弹性和强度的影响,甲苯胺蓝染色观察肝素结合效果和结合程度.将结合与未结合肝素的脱细胞血管同时植入16只兔双侧颈动脉,术后21 d B超观察移植血管的通畅情况.结果：犬颈动脉血管壁细胞被完全去除,细胞外基质保存完好,机械性能无明显破坏,肝素结合于管壁全层,植入兔颈动脉后两侧均无血管阻塞,而肝素结合组血栓形成少于未结合组.结论：去污剂-酶消化法联合肝素结合处理可以作为制备脱细胞小口径异种移植血管的新方法.     

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.     

兔颈动脉移植后血管内皮型一氧化氮合酶基因表达的变化及意义

目的探讨兔颈动脉移植后移植血管段内皮型一氧化氮合酶信使核糖核酸(eNOSmRNA)表达的变化及其意义。方法建立大白兔颈动脉移植模型。将30只兔按随机数字表法分为4组。A组(n=3)自体血管移植;B组(n=9)新鲜异体血管移植;C组(n=9)同种异体血管经青霉素和链霉素处理常温保存后移植;D组(n=9)同种异体血管经青霉素和链霉素处理冷冻保存后移植。观察比较移植后1～3周移植血管段组织形态学变化和eNOS mRNA的表达。结果光学显微镜下观察A组结构正常,仅有炎性细胞浸润;其余各组术后1周内膜开始增生,2周内膜明显增厚,3周内膜、中膜增厚最明显,同时伴有血栓形成。其中B组改变最严重,B组血管移植后eNOS mRNA表达明显低于其他三组(P<0.05)。结论同种异体动脉血管移植后,血管eNOS基因表达明显下调,可造成移植后血管内膜增生和血栓形成。     

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。     

利用同种异体脱细胞血管基质预构小口径血管的研究

目的通过观察狗腹腔内预植同种异体脱细胞血管基质（ACVM）所形成的结缔组织管作为替代血管的力学性能，以及移植于自体股动脉后的组织结构变化，探讨利用同种异体ACVM预构小口径血管的可行性。方法在狗腹腔内埋植长8～12cm用硅胶棒支撑的已制备好的同种异体ACVM，3周后将硅胶棒周围形成的管状物作为血管替代物桥接移植于自体股动脉后，进行力学、组织学检查及与颈动脉、股静脉自体移植的对比分析。于移植术后6个月观测作为替代血管的通畅情况，组织学观察其组织结构变化。结果血管替代物的力学性能弱于正常动脉而强于正常静脉。组织学观察：形成的管状物内腔有少量的间皮细胞黏附；弹性纤维、胶原纤维结构较完整；纤维网中充满成纤维细胞。6个月后内皮细胞在替代物内壁连续覆盖，平滑肌细胞与对照组相近。移植6个月后异体ACVM组血管全部通畅。结论用同种异体ACVM预构的血管替代物，力学性能符合血管移植、血运重建的需求。所形成的结缔组织管作为血管替代物移植6个月后，管壁厚度及组织结构已接近正常血管。     

兔颈动脉移植同种主动脉带瓣血管模型的建立

目的 建立兔两侧颈动脉移植同种带瓣血管模型。方法 成年新西兰兔１０只作为受体分５组。１个月龄的新西兰白兔２０只作为供体，取其主动脉带瓣血管，移植于受体的两侧颈动脉，术后７、１４、２８、６０、１２０ｄ分别取出每组受体所移植的同种瓣并观察血流通常情况及有无血栓形成。结果 受体全部存活，１０只成年兔的两侧颈动脉血流均通畅，大体标本无血栓形成。结论 此模型对动物损伤小，显露好，且双侧移植有利于分组间对比研     

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.     

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。     

硫酸寡聚糖复合物对实验性腹主动脉瘤的抑制作用

目的 观察乙酰肝素酶抑制剂PI-88对腹主动脉瘤形成的抑制作用。方法 利用豚鼠-SD大鼠异种移植腹主动脉瘤模型，于移植术后4周内连续PI-88治疗；术后4周利用Northem blot杂交和免疫组化技术，检测移植腹主动脉直径、乙酰肝素酶表达、微血管增生及炎性细胞浸润程度。结果 与非治疗组相比，治疗组腹主动脉乙酰肝素酶表达、移植血管直径、微血管增生及炎性细胞浸润程度均显著降低，但仍高于阴性对照组。结论 PI-88可通过抑制乙酰肝素酶作用，控制腹主动脉瘤形成。     

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.     

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.     

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.     

In vivo behavior of decellularized vein allograft

BACKGROUND: We are investigating decellularized vein allograft as a scaffold to engineer a non-synthetic, small-diameter vascular graft. This study examines the in vivo behavior of this scaffolding after implantation into the arterial circulation. MATERIALS AND METHODS: Canine animals underwent bilateral carotid interposition grafting using jugular vein implanted as either: 1) fresh autograft, 2) fresh allograft, or 3) decellularized allograft. Decellularization was achieved using sodium dodecyl sulfate. Grafts were examined with duplex ultrasound biweekly to determine luminal diameter, thrombosis, stenosis, or anastomotic breakdown. After perfusion fixation at 2 or 8 weeks, grafts underwent histological, morphometric, and immunohistochemical examination. RESULTS: All animals survived without neurological or hemorrhagic complication. No deterioration of graft integrity (rupture, aneurysm) was observed in any group. Luminal narrowing was observed in both allograft groups, but secondary to different pathology. Fresh allografts had significant mononuclear cell infiltrate, intimal hyperplasia, and intramural hemorrhage consistent with rejection. Conversely, decellularized allografts had minimal evidence of rejection but instead had a compact fibrin layer formed along their lumen. This fibrin layer was absent in the peri-anastomotic regions where endothelium had migrated from the native artery. By 8 weeks, decellularized grafts had repopulated with cells staining positive for smooth muscle alpha-actin. CONCLUSIONS: After 8 weeks of arterial flow, decellularized vein allograft exhibits satisfactory strength, reduced antigenicity compared to fresh allograft, and supports cellular repopulation. These characteristics make it satisfactory for further tissue engineering; combined with luminal vascular cell seeding, it may prove useful as a small-diameter arterial bypass graft.     

Small-diameter blood vessels engineered with bone marrow-derived cells

OBJECTIVE: The objective of this study is to investigate if bone marrow-derived cells (BMCs) regenerate vascular tissues and improve patency in tissue-engineered small-diameter (internal diameter = 3 mm) vascular grafts. SUMMARY BACKGROUND DATA: BMCs have demonstrated the ability to differentiate into endothelial-like cells and vascular smooth muscle-like cells and may offer an alternative cell source for vascular tissue engineering. Thus, we tissue-engineered small-diameter vascular grafts with BMCs and decellularized arteries. METHODS: Canine BMCs were differentiated in vitro into smooth muscle alpha-actin/smooth muscle myosin heavy-chain-positive cells and von Willebrand factor/CD31-positive cells and seeded onto decellularized canine carotid arteries (internal diameter = 3 mm). The seeded grafts were implanted in cell donor dogs. The vascular-tissue regeneration and graft patency were investigated with immunohistochemistry and angiography, respectively. RESULTS: The vascular grafts seeded with BMCs remained patent for up to 8 weeks in the canine carotid artery interposition model, whereas nonseeded grafts occluded within 2 weeks. Within 8 weeks after implantation, the vascular grafts showed regeneration of the 3 elements of artery (endothelium, media, and adventitia). BMCs labeled with a fluorescent dye prior to implantation were detected in the retrieved vascular grafts, indicating that the BMCs participated in the vascular tissue regeneration. CONCLUSIONS: Here we show that BMCs have the potential to regenerate vascular tissues and improve patency in tissue-engineered small-diameter vascular grafts. This is the first report of a small-diameter neovessel engineered with BMCs as a cell source.     

Decellularized native and engineered arterial scaffolds for transplantation

More than 570,000 coronary artery bypass grafts are implanted each year, creating an important demand for small-diameter vascular grafts. For patients who lack adequate internal mammary artery or saphenous vein, tissue-engineered arteries may prove useful. However, the time needed to tissue engineer arteries (7 weeks or more) is too long for many patients. Decellularized cadaveric human arteries are another possible source of vascular conduit, but limited availability and the potential for disease transmission limit their widespread use. In contrast, decellularized tissue-engineered arteries could serve as grafts for immediate implantation, as scaffolds onto which patients' cells could be seeded, or as carriers for genetically engineered cells to aid cell transplantation. The goal of this study was to quantify the effects of decellularization on vascular matrix and mechanical properties. Specifically, we compared cellular elimination, extracellular matrix retention, and mechanical characteristics of porcine carotid arteries before and after treatment with three decellularization methods. In addition, for the first time, tissue-engineered arteries were decellularized. Decellularized native arteries were also used as a scaffold onto which vascular cells were seeded. These studies identified a decellularization method for native and engineered arteries that maximized cellular elimination, without greatly compromising mechanical integrity. We showed that engineered tissues could be decellularized, and demonstrated the feasibility of reseeding decellularized vessels with vascular cells.     

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.