Requirements for growing tissue-engineered vascular grafts.

Small diameter (< 6 mm) vascular grafts are in large demand for coronary and peripheral bypass procedures. Although synthetic grafts have been developed, tissue-based vascular grafts that can better mimic native vessels will likely yield superior results. The success of a tissue-based graft depends on its ability to meet several requirements. First, a graft must possess a confluent, adherent and quiescent endothelium to resist thrombosis in vivo. The mechanical behavior of the graft must mimic the mechanical properties of a native vessel. Hence, a graft must have a highly organized collagen matrix to impart tissue strength. Finally, a graft must contain an elastin network to provide compliance and recoil.     

小口径生物型人工血管移植后的血管壁重构

背景：人工血管相对人体血管最大的优势就是来源丰富,经过生物化改造的人工血管,其特性更接近人体血管,移植后自体化程度也较高。 目的：观察新型小口径生物型人造血管移植后1.5年内不同时期实验犬的生存、生活状况,移植材料的组织相容性、移植血管壁重构的组织病理学变化。 方法：以猪血管为基材,经交联固定,多方位去抗原,共价结合肝素,以及偶联可黏附、富集生长因子的特定多肽等系列生化处理而制成的一种高抗凝的人造血管,管径3.5~4.5 mm。建立犬颈总动脉-人造血管端端连续缝合的动物模型,1.5年内不同时期切取标本,做病理组织学检查。 结果与结论：切取标本发现,移植血管与周围组织粘连少、疏松。病理组织学检查：移植后8周,镜下开始发现宿主组织通过人造血管孔隙长入血管腔内参与移植血管新内膜的形成,移植后12周,镜下于吻合口处,可见新内膜表面有不连续的内皮细胞生长,移植后6个月,通畅的人造血管整段管腔内面均可见内皮细胞生长。移植后12个月,移植血管管壁VG染色尚可见支架层内有大量胶原纤维和毛细血管生长,原先的支架结构已部分被宿主血管壁组织取代。移植后18个月,原先的支架结构已大部分被宿主血管壁组织取代。说明新型小口径生物型人造血管新内膜形成早且完整,自然内皮化相对满意,血管壁重构和血管支架的再生能力强,生物相容和稳定性好。     

Bilayered vascular grafts based on silk proteins

A major block in the development of small diameter vascular grafts is achieving suitable blood vessel regeneration while minimizing the risk of thrombosis, intimal hyperplasia, suture retention, and mechanical failure. Silk-based tubular vessels for tissue engineering have been prepared by molding, dipping, electrospinning, or gel spinning, however, further studies are needed to improve the mechanical and blood compatibility properties. In the present study a bilayered vascular graft based on silk fibroin (SF) was developed. The graft was composed of an inner silk fiber-reinforced SF tube containing heparin and a highly porous SF external layer. Compared with previously fabricated SF tubes the fiber-reinforcement provided a comparable or higher mechanical strength, burst pressure, and suture retention strength, as well as mechanical compliance, to saphenous veins for vascular grafts. Heparin release was sustained for at least 1 month, affording blood compatibility to the grafts. The outer layer of the grafts prepared through lyophilization had a highly porous structure in which the macropore walls were composed of nanofibers similar to extracellular matrix, which offered an excellent environment for cell growth. In vitro studies showed good cytocompatibility and hemocompatibility.     

小口径组织工程血管支架：如何产生一种具有生理重塑活性的材料

文题释义：小口径血管支架材料：①机械强度：包括生理的顺应性,即可承受长期的血流动力学压力,不会导致动脉瘤形成;②良好的生物相容性;③愈合时不会发生炎症、增生及纤维囊形成;④易于手术的操作和缝合;⑤低免疫原性。 丝素蛋白：存在于产丝节肢动物的腺体中,通过蜕变过程纺成纤维,是一种天然的生物衍生材料。其具有良好的机械性能、生物相容性、稳定化学性能等优点,因此丝素蛋白在血管组织工程领域中应用比较广泛。 背景：心血管疾病目前临床上常用的治疗方法是血管重建术,包括经支架介入治疗、冠状动脉旁路移植术和血管成形术。 目的：总结天然衍生支架材料、人工合成高分子材料和复合材料等组织工程血管支架材料的最新研究进展,为小口径血管置换提供理论依据。 方法：检索PubMed 数据库、万方数据库和中国全文期刊数据库2008年1月至2019年7月相关文献,以检索词为“组织工程;生物材料;支架材料;血管”“Tissue engineering,biological material,scaffold material,blood vessel”进行中英文检索。排除内容较陈旧及结论重复性文献,对最终入选的52篇文献进行探讨。 结果与结论：自体血管移植物,如隐静脉和胸内动脉,是小口径血管的最佳替代物,然而在移植后可能诱发血管腔再次狭窄,以及导致血栓形成、感染和移植失败发生率增加,因而严重阻碍在临床上的应用。考虑到存在的这些局限性,研究者将组织工程血管移植物嵌入细胞,从而产生一种具有生理重塑活材料,这一潜在的解决方案可为血管移植物的未来带来希望。 ORCID: 0000-0002-0841-0464(杨磊) 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     

Controlled fabrication of a biological vascular substitute

Autologous and synthetic vessel grafts have been used as a vascular substitute for cardiovascular bypass procedures. However, these materials are limited by the availability of appropriate caliber autologous vessels, increased susceptibility to thrombosis and intimal hyperplasia following surgery. Electrospinning technology offers the potential for controlling composition, structure and mechanical properties of biomaterials. Vascular graft scaffolds have been fabricated using electrospun polymer blends of Type I collagen, elastin from ligamentum nuchae, and poly (d,l-lactide-co-glycolide). This study demonstrates improved electrospinning characteristics versus previous studies by increasing polymer concentration and adding PLGA to the polymer blend. Additionally, new in vitro biocompatibility and mechanical testing data is presented. The scaffolds possess tissue composition and mechanical properties similar to native vessels. The electrospun vessel matrix is biocompatible and does not elicit local or systemic toxic effects when implanted in vivo. This study demonstrates the promise of electrospinning as a fabrication process for a functional vascular graft for clinical use.     

Anisotropic tensile viscoelastic properties of vascular graft materials tested at low strain rates

Mechanical matching of vascular grafts and host vessels has been suggested to be important in determining graft patency rates. In this context, we have examined the anisotropic viscoelastic properties of natural vessels and some synthetic replacements using low strain rate tensile testing of circumferential and longitudinal strips. The canine iliac artery and iliac vein were compared with 6 mm diameter woven and knitted Dacron® grafts, expanded polytetrafluoroethylene (PTFE) grafts, and helically constructed prototype Polyurethane grafts at wrap angles of 45, 60 and 75°. A thick-walled pressure vessel analysis was used to approximate physiological stress levels, and SEM was used to correlate anisotropic properties with graft wall structure.     

小口径生物型人工血管移植后的血管内皮再生

背景：经过生物化改造的人工血管特性更接近人体血管,移植后自体化程度也较高,但人工血管的内皮再生是解决血管长期通畅的关键。 目的：观察新型小口径生物型人造血管移植后不同时期移植材料的组织相容性及移植血管壁内膜再生的组织病理学变化。 方法：建立犬颈总动脉-人造血管端端连续缝合的动物模型。 结果与结论：①光镜：移植后12周于吻合口处见新内膜表面有不连续的内皮细胞生长;移植后6个月通畅的整段管腔内面均可见内皮细胞生长;移植后1.5年管腔通畅,部分内膜组织呈慢性炎症表现。②电镜：移植后12周新生血管内皮细胞排列规则,从吻合口向移植血管中段爬行;移植后6个月内皮细胞从吻合口向移植血管中段爬行,移植血管中段呈跳跃式片状生长的内皮细胞群落,细胞排列更致密,形态更接近成熟血管内皮细胞;移植后1.5年整段血管内壁均有致密内皮细胞覆盖,部分内膜组织呈慢性炎症表现。说明新型小口径生物型人造血管新生内皮形成早,血管内膜重构能力强,生物相容和稳定性好。     

Cardiovascular tissue engineering: state of the art

In patients requiring coronary or peripheral vascular bypass procedures, autogenous arterial or vein grafts remain as the conduit of choice even in the case of redo patients. It is in this class of redo patients that often natural tissue of suitable quality becomes unavailable; so that prosthetic material is then used. Prosthetic grafts are liable to fail due to graft occlusion caused by surface thrombogenicity and lack of elasticity. To prevent this, seeding of the graft lumen with endothelial cells has been undertaken and recent clinical studies have evidenced patency rates approaching reasonable vein grafts. Recent advances have also looked at developing a completely artificial biological graft engineered from the patient's cells with surface and viscoelastic properties similar to autogenous vessels. This review encompasses both endothelialisation of grafts and the construction of biological cardiovascular conduits.     

骨髓间充质干细胞复合蛛丝蛋白血管支架构建小直径组织工程血管的研究

应用动态培养的方式,将骨髓间充质干细胞和蛛丝蛋白血管支架复合培养构建小直径组织工程血管,为心血管疾病修复提供新的血管移植物来源。将间充质干细胞种植到管状的血管支架内腔,应用动态培养的方法构建小直径组织工程血管,并根据扫描电子显微镜观察、HE染色、缝合强度测试、DNA含量检测、羟脯氨酸测定等指标评价组织工程血管的形成程度。复合培养7 d后,细胞在纳米纤维支架表面充分铺展,并且可以迁移到纤维内部生长,细胞与血管支架表现出较好的相容性。组织工程血管的缝合强度经测试为(0.95±0.12) N/针,是天然血管的29.6%。随着时间的持续,组织工程血管中的羟脯氨酸含量和DNA含量不断增加,羟脯氨酸含量在第14和第28 d分别达到0.16和0.2 μg/mg, 且与对照组相比在统计学上有显著性差异。成功构建了一种小直径组织工程血管,各指标较为优良,为其临床应用奠定了基础。     

Experimental study of ectopic-free tissue transfer of rabbit epigastric flap using small-caliber vascular grafts.

Ectopic intraperitoneal free-tissue grafting using small-caliber artificial vascular grafts is reported. The vascular grafts were polyurethane tubes (inner diameter 1.4 mm; outer diameter 1.9 mm; length 15 mm) with a coating of the antithrombotic agents argatroban and 2-hydroxyethylmethacrylate-styrene block copolymer (HS)on the inner surface. The patency of the artificial vessels was investigated in an ectopic-free rabbit epigastric flap in which grafts were used for anastomosis between the femoral and renal vessels. The mean patent duration of uncoated controls (n = 10) was 128 +/- 37 min. Flap viability after one week (n = 17) and the patency of the coated grafts were investigated histologically. The flap take rate was 65%, the arterial graft patency rate 41%, and the venous graft patency rate 24%. The HS-treated surface combined with the argatroban slow-release system exhibited improvement of flap survival in an intraperitoneal ectopic -free grafting model.     

Development of vessels in the foetal cortical transplant depending on the place of grafting in the rat brain.

Formation of new blood vessels within the graft is crucial for the survival of brain grafts. Moreover, it must occur rapidly to prevent ischaemic changes in the grafted neurons. A study was made of the development of the vascular system in the foetal cortical grafts depending on the place of grafting in the rat brain. Pieces of neocortical tissue from an 18-day old rat foetus were transplanted into the lateral ventricle, the striatum or the corpus callosum of 2 months old Wistar rats. The vascular system of the graft was visualized from coronal sections of the brain by means of Pickworth's technique 3, 7, 14 and 28 days after transplantation. After 3 days the vessels in the graft were absent. After 7 days the vessel pattern was poor and very simple and after 14 days the vessels formed large number of branches in the graft. After 28 days the pattern of the vascular network in the graft was similar to that of the vessels of the host brain. The size and branching of the vessels showed considerable variations depending on the localization of the graft.     

Circumferentially aligned fibers guided functional neoartery regeneration in vivo

An ideal vascular graft should have the ability to guide the regeneration of neovessels with structure and function similar to those of the native blood vessels. Regeneration of vascular smooth muscle cells (VSMCs) with circumferential orientation within the grafts is crucial for functional vascular reconstruction in vivo. To date, designing and fabricating a vascular graft with well-defined geometric cues to facilitate simultaneously VSMCs infiltration and their circumferential alignment remains a great challenge and scarcely reported in vivo. Thus, we have designed a bi-layered vascular graft, of which the internal layer is composed of circumferentially aligned microfibers prepared by wet-spinning and an external layer composed of random nanofibers prepared by electrospinning. While the internal circumferentially aligned microfibers provide topographic guidance for in vivo regeneration of circumferentially aligned VSMCs, the external random nanofibers can offer enhanced mechanical property and prevent bleeding during and after graft implantation. VSMCs infiltration and alignment within the scaffold was then evaluated in vitro and in vivo. Our results demonstrated that the circumferentially oriented VSMCs and longitudinally aligned ECs were successfully regenerated in vivo after the bi-layered vascular grafts were implanted in rat abdominal aorta. No formation of thrombosis or intimal hyperplasia was observed up to 3 month post implantation. Further, the regenerated neoartery exhibited contraction and relaxation property in response to vasoactive agents. This new strategy may bring cell-free small diameter vascular grafts closer to clinical application.     

Rapid endothelialization of PhotoFix natural biomaterial vascular grafts

To date, no off-the-shelf graft has performed better than the autologous vessel in applications requiring small-bore (< 6-mm diameter) vascular grafts. Much research has been devoted to seeding endothelial cells on synthetic grafts to improve their long-term clinical performance. One key challenge is the ability to retain the endothelium on the graft lumen for extended times. The goal of this research was to develop a process to seed endothelial cells inside a vascular graft and to quickly condition the cells so as to minimize their damage or removal under physiological flow. In addition, the use of PhotoFix(R) natural biomaterial grafts as an improved substrate for human umbilical vein endothelial cells has been evaluated. A motorized system that provides uniform cell seeding of a small-diameter graft (4-mm inner diameter, 10-cm length) by automated radial rotation has been developed. The same system is subsequently adapted for gradual increases in flow rates to strengthen the endothelium, which ultimately was exposed to a final flow rate of 300 ml/min. This process is accomplished without graft transfer, decreasing risks of contamination and physical damage. Cell coverage and cell morphology were evaluated with the use of fluorescence microscopy and scanning-electron microscopy to determine the effectiveness of the flow conditioning process. It was found that endothelial cells exhibit roughly 20-50% improved adhesion to PhotoFix vessels compared to fibrin-treated polytetrafluoroethylene (PTFE) synthetic grafts. Flow conditioning for 6 h enhanced in vitro cell retention by 24% and 40% on PhotoFix and PTFE grafts, respectively.     

Improved endothelialization and reduced thrombosis by coating a synthetic vascular graft with fibronectin and stem cell homing factor SDF-1α

Failure of synthetic small-diameter vascular grafts is determined mainly by the lack of endothelial cells, as these cells inhibit thrombosis and intimal hyperplasia. Coating of graft material with homing factors for circulating stem cells has the potential to improve endogenous endothelialization of these grafts and to reduce graft failure. Synthetic knitted polyester grafts (6mm diameter) were coated with FN and SDF-1α before surgical interposition in the carotid artery of sheep. Similar uncoated vascular grafts were implanted in the contralateral side as internal controls. To study the early attraction of stem cells, grafts were implanted in a first series of nine sheep and explanted after 1 or 3 days. In coated grafts, four times higher fractions of CD34(+) and three to four times higher fractions of CD117(+) cells adhering to the vessel walls were found than in control grafts (P<0.05). When such coated and non-coated grafts were implanted in 12 other sheep and explanted after 3 months, all coated grafts were patent, while one control graft was occluded. EcNOS staining revealed that FN-SDF-1α coating significantly increased coverage with endothelial cells from 27 ± 4% of the graft to 48 ± 4% compared with the controls (P=0.001). This was associated with a significant reduction of intimal hyperplasia (average thickness 1.03 ± 0.09 mm in controls vs. 0.69 ± 0.04 mm in coated grafts; P=0.009) and significantly less adhesion of thrombotic material in the middle part of the graft (P=0.029). FN-SDF-1α coating of synthetic small-caliber vascular grafts stimulated the attraction of stem cells and was associated with improved endothelialization and reduced intimal hyperplasia and thrombosis.     

Coculture of endothelial and smooth muscle cells on a collagen membrane in the development of a small-diameter vascular graft

In this study, we have evaluated the feasibility of developing a biodegradable collagenous small diameter vascular graft of 2mm diameter and 1cm length. In brief, bi-layer type I collagen membrane was fabricated under vacuum suction and lyophilization methods. The smooth muscle cells were inoculated into the lower side of the porous membrane, while endothelial cells were seeded onto upper smooth side of the membrane. After cultured for 7 days, the vascular substitute was either harvested for in vitro examination or in vivo implanted in the subcutaneous layer for biocompatibility test. The tubular vascular prosthesis was then used as a temporary absorbable guide that served as an in vivo vascular graft to promote the complete regeneration of rat inferior vena cava. After implantation for 12 weeks, a thin continuous layer of endothelial cells and smooth muscle cells were lined with the vascular lumen and tunic media, respectively. Histology results showed that there were no signs of significant thrombogeneity and intima hyperplasia. This tissue engineered vascular substitute not only had enough tensile strength and good biocompatibility, but also advanced vascular regeneration. In the future, we suggest that this biodegradable vascular substitute will provide with the possibility in application on small diameter prosthetic grafts in artificial blood vessels.     

Compliant design of artificial graft: compliance determination by new digital X-ray imaging system-based method

The development of an artificial graft requires formulation of biomechanical design criteria. The compliance of artifical grafts, based on the intraluminal pressure-internal diameter (Pi-Di) relationship, was measured by a novel method using a digital X-ray imaging system coupled with an edge detection algorithm and a pressure transducer. The Pi-Di values were obtained from digital angiographic images under continuous inflation of a canine femoral artery anastomosed with an expanded poly(tetrafluoroethylene) (ePTFE) vascular graft as a model vessel with a pressurized contrast medium. The Di at Pi using an NIH Image software specially programmed for the entropy filter method, which enables the detection of the edge of the vessel phantoms of the images, was determined. The Pi-Di relationships showed a "J-shape" curve for the artery, a steeper line with a very low pressure-dependent distensibility for the ePTFE graft, and an intermediate curve for the anastomosis protion. The two indices for the vessel compliance, the stiffness parameter (beta value) and the diameter compliance (Cd), both of which were calculated from the Pi-Di relationships, were 10.6 and 6.8%/mmHg x 10(-2) for the artery, 164 and 0.51%/mmHg x 10(-2) for the ePTFE, and 14.4 and 5.5%/mmHg x 10(-2) for the anastomosis portion, respectively. This method can measure compliance at any portions of the sampling vessel in a single experiment on a real-time basis with very high accuracy, compared with conventional methods, and even in cases of intimal thickening and/or connective tissues-adhered vessels, and may serve to provide information on compliant design criteria of artificial and tissue-engineered graft.     

Readily available tissue-engineered vascular grafts

Autologous or synthetic vascular grafts are used routinely for providing access in hemodialysis or for arterial bypass in patients with cardiovascular disease. However, some patients either lack suitable autologous tissue or cannot receive synthetic grafts. Such patients could benefit from a vascular graft produced by tissue engineering. Here, we engineer vascular grafts using human allogeneic or canine smooth muscle cells grown on a tubular polyglycolic acid scaffold. Cellular material was removed with detergents to render the grafts nonimmunogenic. Mechanical properties of the human vascular grafts were similar to native human blood vessels, and the grafts could withstand long-term storage at 4 °C. Human engineered grafts were tested in a baboon model of arteriovenous access for hemodialysis. Canine grafts were tested in a dog model of peripheral and coronary artery bypass. Grafts demonstrated excellent patency and resisted dilatation, calcification, and intimal hyperplasia. Such tissue-engineered vascular grafts may provide a readily available option for patients without suitable autologous tissue or for those who are not candidates for synthetic grafts.     

Review: application of stem cells for vascular tissue engineering

As the prevalence of vascular disease has continued to expand, the need for a suitable arterial replacement has prompted researchers to look beyond synthetic and autologous grafts toward the field of tissue engineering. Advances in vascular tissue engineering have utilized both mesenchymal and hematopoietic stem cells as a cell source in an attempt to create a fully engineered small-diameter graft. Stem cells offer enormous potential as a cell source because of their proliferative and growth potential, and the application of stem cell technology has far-reaching implications for future applications. The innovative use of stem cells for vascular tissue engineering has opened new possibilities for a fully engineered blood vessel. The purpose of this review is to summarize the current perspective on the use of stem cells for vascular tissue engineering. It focuses principally on the classes of stem cells used, techniques for differentiation scaffolding technology, and the successes and failures of models.     

The performance of cross-linked acellular arterial scaffolds as vascular grafts; pre-clinical testing in direct and isolation loop circulatory models

There is a significant need for small diameter vascular grafts to be used in peripheral vascular surgery; however autologous grafts are not always available, synthetic grafts perform poorly and allografts and xenografts degenerate, dilate and calcify after implantation. We hypothesized that chemical stabilization of acellular xenogenic arteries would generate off-the-shelf grafts resistant to thrombosis, dilatation and calcification. To test this hypothesis, we decellularized porcine renal arteries, stabilized elastin with penta-galloyl glucose and collagen with carbodiimide/activated heparin and implanted them as transposition grafts in the abdominal aorta of rats as direct implants and separately as indirect, isolation-loop implants. All implants resulted in high patency and animal survival rates, ubiquitous encapsulation within a vascularized collagenous capsule, and exhibited lack of lumen thrombogenicity and no graft wall calcification. Peri-anastomotic neo-intimal tissue overgrowth was a normal occurrence in direct implants; however this reaction was circumvented in indirect implants. Notably, implantation of non-treated control scaffolds exhibited marked graft dilatation and elastin degeneration; however PGG significantly reduced elastin degradation and prevented aneurismal dilatation of vascular grafts. Overall these results point to the outstanding potential of crosslinked arterial scaffolds as small diameter vascular grafts.     

Particle-hemodynamics modeling of the distal end-to-side femoral bypass: effects of graft caliber and graft-end cut

Late-stage occlusions of peripheral synthetic bypass grafts are frequently due to intimal hyperplasia and/or thrombosis at the distal anastomosis, resulting in unacceptably high failure rates. It has been widely established that hemodynamic and blood particle interactions with the vascular surface as well as surgical injury and compliance mismatch are inciting mechanisms capable of eliciting various cellular level responses associated with distal anastomotic intimal hyperplasia (IH) formation. Primary geometric factors influencing anastomotic hemodynamics include the graft-to-artery diameter ratio and graft-hood shape, which are determined by the graft caliber and initial graft-end cut selected by the vascular surgeon. In this study, the particle-hemodynamic effects of graft-end cuts (straight, curved, and S-shaped) and graft-to-artery diameter ratios (2:1 vs. 1.5:1) have been numerically assessed in four common unexpanded anastomotic configurations with respect to vortical flow patterns, wall shear stress based parameters, and platelet interactions with the vascular surface. Sites of significant platelet–wall interactions have been identified by a novel near-wall residence time (NWRT) model, which includes shear stress based factors for platelet activation and endothelial cell expression of anti-thrombogenic compounds. Of the configurations evaluated, straight and curved graft-end cuts with a graft-to-artery diameter ratio of 1.5:1 were found to reduce the particle-hemodynamic potential for IH development at locations critical to flow delivery. Nevertheless, the potential for significant IH occurrence via platelet and/or endothelial response pathways was highly evident in all conventional anastomoses considered, such that a decisively superior configuration was not determined. These results illustrate the need for alternative anastomotic designs with the intent of reducing critical hemodynamic wall parameters and mitigating regions of significant particle–wall interactions.