组织工程血管支架材料的研究进展

组织工程血管支架是血管组织工程学中的一个重要组成部分.过去几十年时间里,组织工程血管支架材料由简单的天然材料发展到高分子可降解材料和生物材料的复合物,设计和加工的方式由单纯的手工发展到电镀旋压成型技术,取得了很大进步.目前组织工程血管支架材料的设计加工方法还不够成熟,性能还有待完善,应主要着眼于支架材料的机械性能和生物活性的完善方面的研究.     

Electrospun scaffolds for tissue engineering of vascular grafts

There is a growing demand for off-the-shelf tissue engineered vascular grafts (TEVGs) for the replacement or bypass of damaged arteries in various cardiovascular diseases. Scaffolds from the decellularized tissue skeletons to biopolymers and biodegradable synthetic polymers have been used for fabricating TEVGs. However, several issues have not yet been resolved, which include the inability to mimic the mechanical properties of native tissues, and the ability for long-term patency and growth required for in vivo function. Electrospinning is a popular technique for the production of scaffolds that has the potential to address these issues. However, its application to human TEVGs has not yet been achieved. This review provides an overview of tubular scaffolds that have been prepared by electrospinning with potential for TEVG applications.     

Composition of intraperitoneally implanted electrospun conduits modulates cellular elastic matrix generation

Improving elastic matrix generation is critical to developing functional tissue engineered vascular grafts. Therefore, this study pursued a strategy to grow autologous tissue in vivo by recruiting potentially more elastogenic cells to conduits implanted within the peritoneal cavity. The goal was to determine the impacts of electrospun conduit composition and hyaluronan oligomer (HA-o) modification on the recruitment of peritoneal cells, and their phenotype and ability to synthesize elastic matrix. These responses were assessed as a function of conduit intra-peritoneal implantation time. This study showed that the blending of collagen with poly(ε-caprolactone) (PCL) promotes a faster wound healing response, as assessed by trends in expression of macrophage and smooth muscle cell (SMC) contractile markers and in matrix deposition, compared to the more chronic response for PCL alone. This result, along with the increase in elastic matrix production, demonstrates the benefits of incorporating as little as 25% w/w collagen into the conduit. In addition, PCR analysis demonstrated the challenges in differentiating between a myofibroblast and an SMC using traditional phenotypic markers. Finally, the impact of the tethered HA-o is limited within the inflammatory environment, unlike the significant response found previously in vitro. In conclusion, these results demonstrate the importance of both careful control of implanted scaffold composition and the development of appropriate delivery methods for HA-o.     

Mechanical property characterization of electrospun recombinant human tropoelastin for vascular graft biomaterials

The development of vascular grafts has focused on finding a biomaterial that is non-thrombogenic, minimizes intimal hyperplasia, matches the mechanical properties of native vessels and allows for regeneration of arterial tissue. In this study, the structural and mechanical properties and the vascular cell compatibility of electrospun recombinant human tropoelastin (rTE) were evaluated as a potential vascular graft support matrix. Disuccinimidyl suberate (DSS) was used to cross-link electrospun rTE fibers to produce a polymeric recombinant tropoelastin (prTE) matrix that is stable in aqueous environments. Tubular 1 cm diameter prTE samples were constructed for uniaxial tensile testing and 4 mm small-diameter prTE tubular scaffolds were produced for burst pressure and cell compatibility evaluations from 15 wt.% rTE solutions. Uniaxial tensile tests demonstrated an average ultimate tensile strength (UTS) of 0.36 ± 0.05 MPa and elastic moduli of 0.15 ± 0.04 and 0.91 ± 0.16 MPa, which were comparable to extracted native elastin. Burst pressures of 485 ± 25 mm Hg were obtained from 4 mm internal diameter scaffolds with 453 ± 74 μm average wall thickness. prTE supported endothelial cell growth with typical endothelial cell cobblestone morphology after 48 h in culture. Cross-linked electrospun rTE has promising properties for utilization as a vascular graft biomaterial with customizable dimensions, a compliant matrix and vascular cell compatibility.     

Scaffold-free vascular tissue engineering using bioprinting

Current limitations of exogenous scaffolds or extracellular matrix based materials have underlined the need for alternative tissue-engineering solutions. Scaffolds may elicit adverse host responses and interfere with direct cell–cell interaction, as well as assembly and alignment of cell-produced ECM. Thus, fabrication techniques for production of scaffold-free engineered tissue constructs have recently emerged. Here we report on a fully biological self-assembly approach, which we implement through a rapid prototyping bioprinting method for scaffold-free small diameter vascular reconstruction. Various vascular cell types, including smooth muscle cells and fibroblasts, were aggregated into discrete units, either multicellular spheroids or cylinders of controllable diameter (300–500 μm). These were printed layer-by-layer concomitantly with agarose rods, used here as a molding template. The post-printing fusion of the discrete units resulted in single- and double-layered small diameter vascular tubes (OD ranging from 0.9 to 2.5 mm). A unique aspect of the method is the ability to engineer vessels of distinct shapes and hierarchical trees that combine tubes of distinct diameters. The technique is quick and easily scalable.     

Textile-templated electrospun anisotropic scaffolds for regenerative cardiac tissue engineering

For patients with end-stage heart disease, the access to heart transplantation is limited due to the shortage of donor organs and to the potential for rejection of the donated organ. Therefore, current studies focus on bioengineering approaches for creating biomimetic cardiac patches that will assist in restoring cardiac function, by repairing and/or regenerating the intrinsically anisotropic myocardium. In this paper we present a simplified, straightforward approach for creating bioactive anisotropic cardiac patches, based on a combination of bioengineering and textile-manufacturing techniques in concert with nano-biotechnology based tissue-engineering stratagems. Using knitted conventional textiles, made of cotton or polyester yarns as template targets, we successfully electrospun anisotropic three-dimensional scaffolds from poly(lactic-co-glycolic) acid (PLGA), and thermoplastic polycarbonate-urethane (PCU, Bionate^®). The surface topography and mechanical properties of textile-templated anisotropic scaffolds significantly differed from those of scaffolds electrospun from the same materials onto conventional 2-D flat-target electrospun scaffolds. Anisotropic textile-templated scaffolds electrospun from both PLGA and PCU, supported the adhesion and proliferation of H9C2 cardiac myoblasts cell line, and guided the cardiac tissue-like anisotropic organization of these cells in vitro. All cell-seeded PCU scaffolds exhibited mechanical properties comparable to those of a human heart, but only the cells on the polyester-templated scaffolds exhibited prolonged spontaneous synchronous contractility on the entire engineered construct for 10 days in vitro at a near physiologic frequency of ∼120 bpm. Taken together, the methods described here take advantage of straightforward established textile manufacturing strategies as an efficient and cost-effective approach to engineering 3D anisotropic, elastomeric PCU scaffolds that can serve as a cardiac patch.     

New biodegradable small-diameter artificial vascular prosthesis: a feasibility study

An in-house built instrument was used to fabricate a small internal diameter (2 mm) artificial vascular prosthesis from biodegradable chitosan. This new artificial vascular prosthesis has shown a good biocompatibility based on the studies of its cell compatibility, inflammatory reaction, and platelet adhesion. In an animal test, the prosthesis was used to replace a 4-cm-long section of femoral artery in each of the seven tested dogs. The patency of the replacement was monitored at regular intervals using Doppler ultrasound diagnostics. Nine months after the implantation, hematoxylin and eosin staining, immunohistochemical study, and scanning electron microscope observation were carried out. Complete decomposition of the prosthesis and replacement by a natural blood vessel were observed. The results suggests that the artificial vascular prosthesis displays many characteristics of the "ideal" small-diameter artificial vascular, and have the biocompatibility that can be tailored to match those desired in vascular replacement application.     

Mechanical properties of electrospun bilayer fibrous membranes as potential scaffolds for tissue engineering

Bilayer fibrous membranes of poly(l-lactic acid) (PLLA) were fabricated by electrospinning, using a parallel-disk mandrel configuration that resulted in the sequential deposition of a layer with fibers aligned across the two parallel disks and a layer with randomly oriented fibers, both layers deposited in a single process step. Membrane structure and fiber alignment were characterized by scanning electron microscopy and two-dimensional fast Fourier transform. Because of the intricacies of the generated electric field, bilayer membranes exhibited higher porosity than single-layer membranes consisting of randomly oriented fibers fabricated with a solid-drum collector. However, despite their higher porosity, bilayer membranes demonstrated generally higher elastic modulus, yield strength and toughness than single-layer membranes with random fibers. Bilayer membrane deformation at relatively high strain rates comprised multiple abrupt microfracture events characterized by discontinuous fiber breakage. Bilayer membrane elongation yielded excessive necking of the layer with random fibers and remarkable fiber stretching (on the order of 400%) in the layer with fibers aligned in the stress direction. In addition, fibers in both layers exhibited multiple localized necking, attributed to the nonuniform distribution of crystalline phases in the fibrillar structure. The high membrane porosity, good mechanical properties, and good biocompatibility and biodegradability of PLLA (demonstrated in previous studies) make the present bilayer membranes good scaffold candidates for a wide range of tissue engineering applications.     

Nerve regeneration and elastin formation within poly(glycerol sebacate)-based synthetic arterial grafts one-year post-implantation in a rat model

The objective of this study was to evaluate the long-term performance of cell-free vascular grafts made from a fast-degrading elastic polymer. We fabricated small arterial grafts from microporous tubes of poly(glycerol sebacate) (PGS) reinforced with polycaprolactone (PCL) nanofibers on the outer surface. Grafts were interpositioned in rat abdominal aortas and characterized at 1 year post-implant. Grafts remodeled into “neoarteries” (regenerated arteries) with similar gross appearance to native rat aortas. Neoarteries mimic arterial tissue architecture with a confluent endothelium and media and adventita-like layers. Patent vessels (80%) showed no significant stenosis, dilation, or calcification. Neoarteries contain nerves and have the same amount of mature elastin as native arteries. Despite some differences in matrix organization, regenerated arteries had similar dynamic mechanical compliance to native arteries in vivo. Neoarteries responded to vasomotor agents, albeit with different magnitude than native aortas. These data suggest that an elastic vascular graft that resorbs quickly has potential to improve the performance of vascular grafts used in small arteries. This design may also promote constructive remodeling in other soft tissues.     

新型组织工程血管材料：静电纺复合纳米纤维小口径管状支架

背景：小口径人工血管对生物相容性和抗凝血的要求远远高于普通大口径人工血管,因此血管移植体内原位诱导组织再生成为了新的研究方向。 目的：总结近几年静电纺复合纳米纤维小口径管状支架的主要研究进展,并讨论其在体内原位诱导血管再生方面的重要应用。 方法：由第一作者检索中国期刊网CNKI全文数据库、万方数据库及ISI Web of Knowledge外文数据库,有关复合纳米纤维小口径管状支架的制备方法、血管支架仿生天然细胞外基质微环境的表面修饰以及种植体植入后生物相容性和安全性评价等方面的文献。 结果与结论：静电纺复合纳米纤维制备小口径管状支架,即将天然材料和合成材料共纺在一起,这样既能克服天然生物高分子材料力学性能的不足,又能避免合成材料在生物相容性和安全性的缺陷,成为制备小口径血管组织工程支架的必然趋势。同时制备多层血管,进行功能化修饰,模拟天然细胞外基质的结构和功能,将成为用于心血管组织修复及再生小口径血管组织工程研究的新方向。在获得上述新进展的同时,经动物实验检验的静电纺血管支架以聚合物为主。尽管这类支架采用了各种手段避免血栓、炎症等不良反应,其生物相容性仍旧无法与天然材料相比。由此可见,在天然材料与合成材料之间找到一个最佳比例,使复合材料的力学性能和血管相容性达到一个平衡,将会显著提高静电纺复合纳米纤维支架在小口径血管组织再生中的应用。     

血管组织工程的发展现状和趋势

心脑血管疾病是全球发病率和死亡率最高的疾病,其主要病因是动脉粥样硬化。临床上主要采用血管移植物重建病损血管,人造合成血管在大口径血管修复中取得了满意的效果,但在小口径血管修复中效果并不理想。近30年来,血管组织工程发展极其迅速,从再生的角度为血管修复提供了新的途径。本文综述血管组织工程的最新进展(体外、体内、原位血管组织工程),并对未来发展趋势进行了前瞻性展望。     

Lectin and antibody-based histochemical techniques for cardiovascular tissue engineering

Abstract

Tissue engineering holds immense potential for treatment of cardiovascular diseases by creating living structures to replace diseased blood vessels, heart valves, and cardiac muscle. In a traditional approach, scaffolds are seeded with stem cells and subjected to stimuli in bioreactors that mimic physiologic conditions or are directly implanted into target sites in animal models. The expected results are significant cell changes, extensive remodeling of the scaffolds and creation of surrogate structures that would be deemed acceptable for tissue regeneration. Histochemical techniques are increasingly becoming essential tools in tissue engineering research. In our studies, we used lectin and antibody-based techniques to characterize novel collagen and elastin scaffolds and to ensure efficient removal of xenoantigens. Scaffolds were implanted in animals and infiltrated host cells were identified using antibodies to activated fibroblasts, macrophages, and lymphocytes. Stem cell-seeded scaffolds were subjected to mechanical strains and tested for differentiation into cardiovascular cells using antibody-based double immunofluorescence methods. Finally, living heart valves were constructed from scaffolds and stem cells, subjected to conditioning in a bioreactor and stem cell differentiation evaluated by immunofluorescence. Overall, these techniques have proven to be outstanding companions to biochemical, molecular biology and cell analysis methods used in tissue engineering research and development.     

Cellular engineering of vascular bypass grafts: Role of chemical coatings for enhancing endothelial cell attachment

Surgical treatment of vascular disease has become common. The use of synthetic materials is limited to grafts larger than 5–6 mm, because of the frequency of occlusion observed with small-diameter prosthetics. An alternative would be a hybrid or tissue-engineered graft with the surface coated with a monolayer of the patient's own cells. Currently, to be effective, high-density seeding regimens have to be undertaken. This is because endothelial cells (ECs) are washed off the graft lumen once exposed to physiological blood flow. EC attachment has been shown to be significantly improved by pre-coating with substances known to attach ECs selectively. The review examines the various types of coating and bonding technology used to date to enhance endothelial cell attachment onto the surface of prosthetic vascular bypass grafts.     

A novel hyaluronan-based biomaterial (Hyaff-11) as a scaffold for endothelial cells in tissue engineered vascular grafts

Current prosthetic small diameter vascular grafts show poor long-term patency rates, leading to the pursuit of a biological alternative. Hyaff-11 is a hyaluronan-based biodegradable polymer developed for tissue-engineering applications. This study aimed to determine whether human vascular endothelial cells attach to Hyaff-11 scaffolds and produce a subendothelial matrix. Two forms of fibrous, non-woven Hyaff-11 scaffolds: unpressed and pressed felts, were analysed. Attachment of human venous endothelial cells was investigated after 1, 5, 10 and 20 days in culture using SEM and confocal microscopy. The deposition of subendothelial matrix components was investigated by immunofluorescent staining.

We demonstrate that endothelial cells adhere to the individual fibres of both unpressed and pressed scaffolds: with a seeding density of 1×10⁶ cells/cm², 94% of the cells attached to Hyaff-11 fibres after 24 h. The pressed material provided the best environment for cell growth, allowing the formation of a complete endothelial monolayer after 20 days. Furthermore, endothelial cells on Hyaff-11 pressed felts deposited an organised subendothelial matrix containing laminin, fibronectin, type IV and type VIII collagen. This work indicates Hyaff-11 based biopolymers as suitable scaffolds to promote endothelialisation within the next generation of vascular grafts.     

Long-term outcomes using vascular grafts sealed with fragmented autologous adipose tissue for aortoiliac occlusive disease

The aim of this study was to test the safety and efficacy of fragmented autologous adipose tissue (FAT) grafts for revascularization in aortoiliac occlusive disease. Twenty-seven patients with atherosclerotic aortoiliac occlusive disease underwent surgical treatment using FAT grafts. A piece of adipose connective tissue was obtained from the operative wound, cut into small pieces, and pressed into the wall of a fabric vascular prosthesis. Cumulative primary patency rates were 92% at 1 year, 92% at 3 years, and 86% at 6 years. Cumulative secondary patency rates were 96%, 96%, and 90% for the same intervals. In this clinical study, the FAT grafts demonstrated good long-term patency rates and no particular problems. This is the first clinical report of long-term outcomes using FAT grafts for aortofemoral or aortoiliac bypasses. FAT grafts are thus safe for revascularization in aortoiliac occlusive disease.     

Achieving the ideal properties for vascular bypass grafts using a tissue engineered approach: a review

The multiple demands placed on small calibre cardiovascular bypass grafts have meant that a synthetic prosthesis with good long-term patency has not been developed. A tissue-engineered graft could fulfil the ideal characteristics present in an artery. However, the great disadvantage of such a conduit is the time necessary for maturation leading to unacceptable delays once the decision to intervene surgically has been made. This maturation process is essential to produce a graft which can withstand haemodynamic stress. Once implanted, the tissue-engineered graft can contract in response to immediate haemodynamic conditions and remodel in the long term. We review the latest tissue engineering approaches used to give the favourable properties of mechanical strength, arterial compliance, low thrombogenicity, long-term resistance towards biodegradation as well as technological advances which shorten the time required for production of an implantable graft.     

Highly flexible and rapidly degradable papaverine-loaded electrospun fibrous membranes for preventing vasospasm and repairing vascular tissue

Vasospasm is a common post-operative complication after vascular anastomosis. Currently, the main treatment is a local injection of antispasmodic drugs. However, this method has a high rate of relapse and is subject to a large degree of individual variation, and repeated injections cause additional pain for patients. In this study, we developed highly flexible and rapidly degradable papaverine-loaded electrospun fibrous membranes to be wrapped around vascular suturing to prevent vasospasm. Poly-l-lactic acid/polyethylene glycol (PLLA/PEG) electrospun fibers containing papaverine maintained a high degree of flexibility and could withstand any folding, and are therefore suitable for wrapping vascular suturing. A rapid release of papaverine, between 2 and 7 days, was achieved by adjusting the proportions of PEG and PLLA. PLLA electrospun fibers containing 40% PEG (PLLA-40%) could control drug release and polymer degradation most effectively during the first 2 weeks post-operation. Testing using an in vivo rabbit model showed that PLLA-40% fibrous membranes produced significant antispasmodic effect without observable inflammation or hyperplasia, and the fibrous membranes were ideally biodegradable, with no impact on regional blood flow, pressure, vessel diameter or surrounding tissue hyperplasia. Therefore, papaverine-loaded electrospun fibrous membranes show the potential to greatly reduce post-operative vasospasm and maintain regular vascular morphology during antispasmodic therapy.     

Preparation and characterization of a novel tobramycin-containing antibacterial collagen film for corneal tissue engineering

Corneal disease is a major cause of blindness and keratoplasty is an effective treatment method. However, clinical treatment is limited due to a severe shortage of high-quality allogeneic corneal tissues and the bacterial infection after corneal transplantation. In this study, we develop a novel artificial and antibacterial collagen film (called Col-Tob) for corneal repair. In the Col-Tob film, the tobramycin, which is an aminoglycoside antibiotic to treat various types of bacterial infections, was cross-linked by 1-ethyl-3-(3-dimethyl aminopropyl) carbodiimide and N-hydroxysuccinimide onto the collagen. Physical properties, antibacterial property and biocompatibility of the films were characterized. The results indicate that the film is basically transparent and has appropriate mechanical properties. Cell experiments show that human corneal epithelial cells could adhere to and proliferate well on the film. Most importantly, the film exhibits excellent antibacterial effect in vitro. Lamellar keratoplasty shows that the Col-Tob film can be sutured in rabbit eyes and are epithelialized completely in15 ± 5 days, and their transparency is restored quickly in the first month. Corneal rejection reaction, neovascularization and keratoconus are not observed within 3 months. This film, which can be prepared in large quantities and at low cost, should have potential application in corneal repair.     

The female-to-male transsexual patient: a source of human ovarian cortical tissue for experimental use

We demonstrated that ovaries removed from female-to-male transsexuals can be used as research material for ovarian cryopreservation, grafting and culture studies. A 21 year old female-to-male transsexual individual, who had been treated with androgens for 12 months, donated her ovaries removed in the sex reversal operation. She had normal numbers of, and proportions of, primordial (98.6%) and primary (1.4%) follicles in her ovarian cortex. After freezing and thawing the ovarian tissue was grafted to immuno-incompetent mice, which were stimulated by human recombinant FSH for 14 days, 10 weeks after transfer. Initiation of growth had occurred in the grafts in which only 79.4% of follicles were primordial, 17.1% primary, and in which there were also secondary and pre-antral follicles. Because long-term androgen treatment does not appear to cause depletion of the primordial follicle pool or affect the developmental capacity of the follicles, ovaries from individuals who undergo sex-reversal operations are an excellent source of tissue for research and maybe even for oocyte donation in the future.     

Fibrin: a natural biodegradable scaffold in vascular tissue engineering

Arterial occlusive disease remains a major health issue in the developed world and a rapidly growing problem in the developing world. Although a growing number of patients are now being effectively treated with minimally invasive techniques, there remains a tremendous pressure on the vascular community to develop a synthetic small-diameter vascular graft with improved long-term patency rates. The field of tissue engineering offers an exciting alternative in the search for living organ replacement structures. Several methodologies have emerged for constructing blood vessel replacements with biological functionality. Common strategies include cell-seeded biodegradable synthetic scaffolds, cell self-assembly, cell-seeded gels and xenogeneic acellular materials. A wide range of materials are being investigated as potential scaffolds for vascular tissue engineering applications. Some are commercialised and others are still in development. Recently, researchers have studied the role of fibrin gel as a three-dimensional scaffold in vascular tissue engineering. This overview describes the properties of fibrin gel in vascular tissue engineering and highlights some recent progress and difficulties encountered in the development of cell fibrin scaffold technology.