Scaffolds in tissue engineering of blood vessels

Tissue engineering of small diameter (<5?mm) blood vessels is a promising approach for developing viable alternatives to autologous vascular grafts. It involves in vitro seeding of cells onto a scaffold on which the cells attach, proliferate, and differentiate while secreting the components of extracellular matrix that are required for creating the tissue. The scaffold should provide the initial requisite mechanical strength to withstand in vivo hemodynamic forces until vascular smooth muscle cells and fibroblasts reinforce the extracellular matrix of the vessel wall. Hence, the choice of scaffold is crucial for providing guidance cues to the cells to behave in the required manner to produce tissues and organs of the desired shape and size. Several types of scaffolds have been used for the reconstruction of blood vessels. They can be broadly classified as biological scaffolds, decellularized matrices, and polymeric biodegradable scaffolds. This review focuses on the different types of scaffolds that have been designed, developed, and tested for tissue engineering of blood vessels, including use of stem cells in vascular tissue engineering.     

Drug releasing systems in cardiovascular tissue engineering

Heart disease and atherosclerosis are the leading causes of morbidity and mortality worldwide. The lack of suitable autologous grafts has produced a need for artificial grafts; however, current artificial grafts carry significant limitations, including thrombosis, infection, limited durability and the inability to grow. Tissue engineering of blood vessels, cardiovascular structures and whole organs is a promising approach for creating replacement tissues to repair congenital defects and/or diseased tissues. In an attempt to surmount the shortcomings of artificial grafts, tissue-engineered cardiovascular graft (TECVG), constructs obtained using cultured autologous vascular cells seeded onto a synthetic biodegradable polymer scaffold, have been developed. Autologous TECVGs have the potential advantages of growth, durability, resistance to infection, and freedom from problems of rejection, thrombogenicity and donor scarcity. Moreover polymers engrafted with growth factors, cytokines, drugs have been developed allowing drug-releasing systems capable of focused and localized delivery of molecules depending on the environmental requirements and the milieu in which the scaffold is placed. A broad range of applications for compound-releasing, tissue-engineered grafts have been suggested ranging from drug delivery to gene therapy. This review will describe advances in the development of drug-delivery systems for cardiovascular applications focusing on the manufacturing techniques and on the compounds delivered by these systems to date.     

Effect of cell seeding and mechanical loading on vascularization and tissue formation inside a scaffold: A mechano-biological model using a lattice approach to simulate cell activity

Achieving successful vascularization remains one of the main problems in bone tissue engineering. After scaffold implantation, the growth of capillaries into the porous construct may be too slow to provide adequate nutrients to the cells in the scaffold interior and this inhibits tissue formation in the scaffold core. Often, prior to implantation, a controlled cell culture environment is used to stimulate cell proliferation and, once in place, the mechanical environment acting on the tissue construct is determined by the loading conditions at the implantation site. To what extent do cell seeding conditions and the construct loading environment have an effect on scaffold vascularization and tissue growth? In this study, a mechano-biological model for tissue differentiation and blood vessel growth was used to determine the influence of cell seeding on vascular network development and tissue growth inside a regular-structured bone scaffold under different loading conditions. It is predicted that increasing the number of cells seeded homogeneously reduces the rate of vascularization and the maximum penetration of the vascular network, which in turn reduces bone tissue formation. The seeding of cells in the periphery of the scaffold was predicted to be beneficial for vascularization and therefore for bone growth; however, tissue formation occurred more slowly during the first weeks after implantation compared to homogeneous seeding. Low levels of mechanical loading stimulated bone formation while high levels of loading inhibited bone formation and capillary growth. This study demonstrates the feasibility of computational design approaches for bone tissue engineering.     

SPERMIOGENESIS IN THE PULMONATE SNAIL, EUHADRA HICKONIS 1. ACROSOME FORMATION

Electron microscopical observations of the course of acrosomal differentiation in Euhadra hickonis show that the vesicular component of the mature acrosome is produced by early Golgi activity, whereas an equivalent amount of material that forms a basal component is added later to the outside of the vesicle. It is also suggested that similar material which concurrently accumulates against part of the outer surface of the nuclear envelope is finally incorporated into the basal part of the acrosome.
In the early spermatid, which has a highly polymorphic nucleus, material derived from the well-developed Golgi complex accumulates within a network of tubules in its central maturing zone to form a single acrosomal vesicle ca. 150 nm in diameter. The next stage is characterized by the strikingly spherical shape of the nucleus, as well as by the addition of electron-dense material to the outside of the nuclear envelope over the future anterior surface, and to its inside in the posterior region where the centriolar fossa will form.
At mid-spermiogenesis the Golgi complex moves posteriorly away from the acrosomal vesicle, which remains in the anterior cytoplasm. A growing mass of densely filamentous material forms a hollowed hemisphere around one side of the vesicle. This complex approaches the coated anterior part of the nuclear envelope, turning if necessary so that the filamentous material is in the lead, and the latter merges with the electron-dense material at the center of the coated area. As the late spermatid nucleus elongates, this material passes through a series of changes in arrangement and electron density, finally forming a homogeneously particulate element of medium density that surrounds the proximal half of the acrosomal vesicle and caps the slender tip of the nucleus in the mature spermatozoon.     

Optimisation des matériaux hybrides à base de cellules souches pour la réparation de grands défauts osseux

The limitations imposed to both autogenous and allogenous bone grafts led to the development of new strategies for the treatment of large bone defects. The approach of bone tissue engineering aims to restore damaged bone tissue by combining osteocompetent cells such as mesenchymal stromal cells (MSC), and material scaffolds like ceramics. However, the therapeutic effectiveness of cell constructs has not yet met that of autologous bone grafts, in part due to the high death rate of cells (loaded onto the material scaffold) upon their implantation into the injured site. In order to improve the therapeutic functionality of these cell constructs, different strategies can be implemented. In this context, the Glassbone project aimed to optimize the conditions for preparation of tissue engineered products by approaching three aspects: identification of optimal ceramic scaffold relevant to bone formation; survival of implanted cells post-implantation, and finally cell preconditioning to promote cell viability in vivo. Such project will pave the way for the development of new “pro-survival” tissue engineered materials for optimal tissue regeneration.     

国产真丝涤纶人造血管的动物应用研究

目的：探讨国产真丝涤纶人造血管动物应用的可行性。方法：以真丝涤纶人造血管行犬的腹主动脉移植。结果：移植后１个月血管腔内表面形成不完善的内膜,外膜纤维组织明显增生;移植后３个月血管腔内表面完全内皮化,外膜部分肉芽组织胶原化;半年至１年内皮细胞排列更加整齐有序,吻合口轻度内膜增生,但无狭窄,外周为致密的纤维组织。结论：真丝涤纶人造血管作为新一代血管代用品是可行的,但其远期通畅率是有待进一步观察。     

Tissue engineering of vascular grafts.

The challenge of tissue engineering blood vessels with the mechanical properties of native vessels, and with the anti-thrombotic properties required is immense. Recent advances, however, indicate that the goal of providing a tissue-engineered vascular graft that will remain patent in vivo for substantial periods of time, is achievable. For instance, collagen gels have been used to fabricate a tissue in vitro that is representative of a native vessel: an acellular collagen tubular structure, when implanted as a vascular graft, was able to function, and to become populated with host cells. A completely cellular approach culturing cells into tissue sheets and wrapping these around a mandel was able to form a layered tubular structure with impressive strength. Culture of cells onto a biodegradable scaffold within a dynamic bioreactor, generated a tissue-engineered vascular graft with substantial stiffness and, when lined with endothelial cells, was able to remain patent for up to 4 weeks in vivo. In our experiments, use of a non-degradable polyurethane scaffold and culture with smooth muscle cells generated a construct with mechanical properties similar to native vessels. This composite tissue engineered vascular graft with an endothelial layer formed using fluid shear stress to align the endothelial cells, was able to remain patent with an neointima for up to 4 weeks. These results show that tissue engineering of vascular grafts has true potential for application in the clinical situation.     

组织器官三维构建及原位组织工程概念——组织工程连载之四

组织器官三维构建就是把种子细胞和支架材料结合而获得设计的组织或器官,属于组织工程的核心内容,也最能体现组织工程的技术水平,如血管、气管的构建。由于传统组织工程存在缺陷,Shimizu于1998年首先提出了原位组织工程的概念,它是运用组织工程学基本原理,通过各种方法诱导移植的外源性的种子细胞或内源性的缺损组织局部细胞发生迁移、增殖、分化形成新生组织修复缺损。原位组织工程最大的特点是不依赖体外的细胞培养装置--生物反应器。原位组织工程是传统离体组织工程的有益补充。离体组织工程仍具有广阔的发展前景。     

Evaluation of biological protein-based collagen scaffolds in cartilage and musculoskeletal tissue engineering--a systematic review of the literature

The term tissue engineering is the technology that combines cells, engineering and biological/synthetic material in order to repair, replace or regenerate biological tissues such as bone, muscle, tendons and cartilage. The major human applications of tissue engineering are: skin, bone, cartilage, corneas, blood vessels, left mainstem bronchus and urinary structures. In this systematic review several criteria were identified as the most desirable characteristics of an ideal scaffold. These state that an ideal scaffolds needs to be biodegradable, possess mechanical strength, be highly porous, biocompatible, non-cytotoxic, non antigentic, stuitable for cell attachment, proliferation and differentiation, flexible and elastic, three dimensional, osteoconductive and support the transport of nutrients and metabolic waste. Subsequently, studies reporting on the various advantages and disadvantages of using collagen based scaffolds in musculoskeletal and cartilage tissue engineering were identified. The purpose of this review is to 1) provide a list of ideal characteristics of a scaffold as identified in the literature 2) identify different types of biological protein-based collagen scaffolds used in musculoskeletal and cartilage tissue engineering 3) assess how many of the criteria each scaffold type meets 4) weigh different scaffolds against each other according to their relative properties and shortcomings. The rationale behind this approach is that the ideal scaffold material has not yet been identified. Hence, this review will define how many of the identified ideal characteristics are fulfilled by natural collagen-based scaffolds and address the shortcomings of its use as found in the literature.     

维管植物木质部输导特性以及仿生应用的研究

本文从细胞壁、导管(管胞)、木质部三个不同的层次分别论述了维管植物木质部的耐压性和韧性机理,并对木质部强有力的输导性机理进行了阐述。从仿生学角度出发,分别提出了仿生耐压管道和一次性超强榆导毛细管束的仿生结构模型。其中仿生耐压管道自内向外分别由内管、纤维层、增厚层、均压层和保护层组成,具有很好的耐压性和一定的保温性;一次性超强输导毛细管束采用许多根微细的毛细管加以穿孔板组成,能最大限度地维持水的内聚张力。     

Role of growth factors and endothelial cells in therapeutic angiogenesis and tissue engineering

To achieve the goals of engineering large complex tissues, and possibly internal organs, vascularization of the regenerating tissue is essential. To maintain the initial volume after implantation of regenerated tissue, improved vascularization is considered to be important. Recent advances in understanding the process of blood vessel growth has offered significant tools for the neovascularization of bioengineered tissues and therapeutic angiogenesis. Several angiogenic growth factors including vascular endothelial growth factor (VEGF), basic fibroblast growth factor (bFGF) and hepatocyte growth factor (HGF) were used for vascularization of ischemic tissues. Other approaches such as prevascularization of the scaffold, prior to cell seeding, and incorporation of endothelial cells in the bioengineered tissue showed encouraging results. In this article, we will review recent advances in angiogenic growth factors, and discuss the role of these growth factors and endothelial cells in therapeutic angiogenesis and tissue engineering.     

Morphological characterization of a novel scaffold for anterior cruciate ligament tissue engineering

Tissue engineering offers an interesting alternative to current anterior cruciate ligament (ACL) surgeries. Indeed, a tissue-engineered solution could ideally overcome the long-term complications due to actual ACL reconstruction by being gradually replaced by biological tissue. Key requirements concerning the ideal scaffold for ligament tissue engineering are numerous and concern its mechanical properties, biochemical nature, and morphology. This study is aimed at predicting the morphology of a novel scaffold for ligament tissue engineering, based on multilayer braided biodegradable copoly(lactic acid-co-(e-caprolactone)) (PLCL) fibers The process used to create the scaffold is briefly presented, and the degradations of the material before and after the scaffold processing are compared. The process offers varying parameters, such as the number of layers in the scaffold, the pitch length of the braid, and the fibers' diameter. The prediction of the morphology in terms of pore size distribution and pores interconnectivity as a function of these parameters is performed numerically using an original method based on a virtual scaffold. The virtual scaffold geometry and the prediction of pore size distribution are evaluated by comparison with experimental results. The presented process permits creation of a tailorable scaffold for ligament tissue engineering using basic equipment and from minimum amounts of raw material. The virtual scaffold geometry closely mimics the geometry of real scaffolds, and the prediction of the pore size distribution is found to be in good accordance with measurements on real scaffolds. The scaffold offers an interconnected network of pores the sizes of which are adjustable by playing on the process parameters and are able to match the ideal pore size reported for tissue ingrowth. The adjustability of the presented scaffold could permit its application in both classical ACL reconstructions and anatomical double-bundle reconstructions. The precise knowledge of the scaffold morphology using the virtual scaffold will be useful to interpret the activity of cells once it will be seeded into the scaffold. An interesting perspective of the present work is to perform a similar study aiming at predicting the mechanical response of the scaffold according to the same process parameters, by implanting the virtual scaffold into a finite element algorithm.     

组织器官脱细胞支架的制备及研究进展北大核心CSCD

脱细胞基质(decellularized extracellular matrix, dECM)旨在去除引起免疫排斥的细胞,保留原组织结构和成分。由于其具有与原组织器官相似的结构和成分,在组织工程和生物医学的应用上受到广泛关注,已成为一种很有前景的生物医学材料。通过适当的脱细胞方法,dECM很容易能够从组织器官中获得。文中总结了脱细胞的方法及最新研究进展,同时对脱细胞后支架灭菌、交联和保存的方式进行综述,概括了不同组织器官获得的脱细胞支架的最新应用及进展。最后对脱细胞支架目前面临的问题和挑战进行分析,并展望了未来的发展趋势。     

Advances in 3D printing of composite scaffolds for the repairment of bone tissue associated defects

The conventional methods of using autografts and allografts for repairing defects in bone, the osteochondral bone, and the cartilage tissue have many disadvantages, like donor site morbidity and shortage of donors. Moreover, only 30% of the implanted grafts are shown to be successful in treating the defects. Hence, exploring alternative techniques such as tissue engineering to treat bone tissue associated defects is promising as it eliminates the above-mentioned limitations. To enhance the mechanical and biological properties of the tissue engineered product, it is essential to fabricate the scaffold used in tissue engineering by the combination of various biomaterials. Three-dimensional (3D) printing, with its ability to print composite materials and with complex geometry seems to have a huge potential in scaffold fabrication technique for engineering bone associated tissues. This review summarizes the recent applications and future perspectives of 3D printing technologies in the fabrication of composite scaffolds used in bone, osteochondral, and cartilage tissue engineering. Key developments in the field of 3D printing technologies involves the incorporation of various biomaterials and cells in printing composite scaffolds mimicking physiologically relevant complex geometry and gradient porosity. Much recently, the emerging trend of printing smart scaffolds which can respond to external stimulus such as temperature, pH and magnetic field, known as 4D printing is gaining immense popularity and can be considered as the future of 3D printing applications in the field of tissue engineering.     

生物支架材料——组织工程连载之二

具有三维结构的支架材料是组织工程的核心内容之一。现有组织工程支架可分为天然生物材料、合成有机材料和无机材料三类。支架材料近年来研究十分活跃,不仅在组织工程的最早产品人工皮肤领域进行了更为完善的研究和开发,同时在诸如人工骨、软骨、神经、血管、皮肤、肝、脾、肾、膀胱等方面进行了大量研究和探索。与普通组织工程支架需要预先制备并在体外成型不同,近年来在骨和软骨组织工程实践中兴起的可注射支架具有许多优势,是未来组织工程支架发展的重要方向之一。     

Tissue reaction to intraperitoneally implanted catheter materials

The limiting factor that introduces long-term complications of intraperitoneal (i.p.) catheters used, for example, with the Programable Implantable Medication System (PIMS) is the encapsulation of the catheter tip with tissues due to tissue reaction. The objective is the development of new catheters for PIMS or other systems for i.p. insulin delivery that allow continued insulin flow. The study is based on two hypotheses: (1) vascularized tissue will grow into a porous end plug mounted at the catheter tip (100–300 μm pore diameter), with sufficient blood supply to carry the insulin to the circulation; (2) use of a narrow pore diameter (25 μm or less) end plug will prevent tissue ingrowth yet allow insulin flow. The biological response to the following materials, all designed for use in catheters, were studied: polyurethane, segmented polyether-urethane, alumina coated on Teflon (proplast regular and micro-pore), pyrolytic carbon, high density polyethylene, ultra high molecular weight polyethylene, hydroxyapatite, bioglass, and expanded Teflon. Some of these materials also are used for several other applications: vascular grafts, in the cardiovascular system, and for dental, orthopaedic, and other purposes. The shape and size of the end plugs made from each of the materials were as similar as possible to minimize size effects. The test materials were implanted i.p. in 12 dogs for a period of 12 weeks. The cylindrical plugs were typically 1.5–2.5 cm long, with an inside diameter of 0.3 cm, and an outside diameter of 0.6 cm. When the explants were retrieved, thin capsules were observed, of varying thickness and blood supply, surrounding the end of the catheters. Pathological evaluation revealed the best large pore material to be proplast regular and the best small pore materials to be carbon and polyurethane.     

In vitro formation of capillary networks using optical lithographic techniques

Tissue engineering approaches have been developed for vascular grafts, but success has been limited to arterial replacements of large-caliber vessels. We have developed an innovative technique to transplant engineered capillary networks by printing techniques. Endothelial cells were cultured on a patterned substrate, in which network patterns were generated by prior optical lithography. Subsequently, the patterned cells were transferred to extracellular matrix and tissue at which point they changed their morphologies and formed tubular structures. Microinjection of dye showed that the micrometer-scale tubular structure had in vitro flow potential. When capillary-like networks engineered on amnion membranes were transplanted into mice, we found blood cells inside of the lumen of the transplanted capillary-like structure. This is the first report of the in vitro formation of capillary networks using cell transfer technique, and this novel technique may open the way for development of rapid and effective blood perfusion systems in regenerative medicine.     

Osteochondral tissue engineering

Osteochondral defects (i.e., defects which affect both the articular cartilage and underlying subchondral bone) are often associated with mechanical instability of the joint, and therefore with the risk of inducing osteoarthritic degenerative changes. Current surgical limits in the treatment of complex joint lesions could be overcome by grafting osteochondral composite tissues, engineered by combining the patient's own cells with three-dimensional (3D) porous biomaterials of pre-defined size and shape. Various strategies have been reported for the engineering of osteochondral composites, which result from the use of one or more cell types cultured into single-component or composite scaffolds in a broad spectrum of compositions and biomechanical properties. The variety of concepts and models proposed by different groups for the generation of osteochondral grafts reflects that understanding of the requirements to restore a normal joint function is still poor. In order to introduce the use of engineered osteochondral composites in the routine clinical practice, it will be necessary to comprehensively address a number of critical issues, including those related to the size and shape of the graft to be generated, the cell type(s) and properties of the scaffold(s) to be used, the potential physical conditioning to be applied, the degree of functionality required, and the strategy for a cost-effective manufacturing. The progress made in material science, cell biology, mechanobiology and bioreactor technology will be key to support advances in this challenging field.     

A multiscale mechanobiological modelling framework using agent-based models and finite element analysis: application to vascular tissue engineering

Computational models of mechanobiological systems have been widely used to provide insight into these systems and also to predict their behaviour. In this context, vascular tissue engineering benefits from further attention given the challenges involved in developing functional low calibre vascular grafts with long-term patency. In this study, a novel multiscale mechanobiological modelling framework is presented, which takes advantage of lattice-free agent-based models coupled with the finite element method to investigate the dynamics of VSMC growth in vascular tissue engineering scaffolds. The results illustrate the ability of the mechanobiological modelling approach to capture complex multiscale mechanobiological phenomena. Specifically, the framework enabled the study of the influence of scaffold compliance and loading regime in regulating the growth of VSMCs in vascular scaffolds and their role in development of intimal hyperplasia (IH). The model demonstrates that low scaffold compliance compared to host arteries leads to increased luminal ingrowth and IH development. In addition, culture of a tissue-engineered blood vessel under a pulsatile luminal pressure reduced luminal ingrowth and enhanced collagen synthesis within the scaffold compared to non-pulsatile culture. The mechanobiological framework presented provides a robust platform for testing hypotheses in vascular tissue engineering and lends itself to use as an optimisation design tool.     

Numerical Modeling of Interstitial Fluid Flow Coupled with Blood Flow through a Remodeled Solid Tumor Microvascular Network

Modeling of interstitial fluid flow involves processes such as fluid diffusion, convective transport in extracellular matrix, and extravasation from blood vessels. To date, majority of microvascular flow modeling has been done at different levels and scales mostly on simple tumor shapes with their capillaries. However, with our proposed numerical model, more complex and realistic tumor shapes and capillary networks can be studied. Both blood flow through a capillary network, which is induced by a solid tumor, and fluid flow in tumor’s surrounding tissue are formulated. First, governing equations of angiogenesis are implemented to specify the different domains for the network and interstitium. Then, governing equations for flow modeling are introduced for different domains. The conservation laws for mass and momentum (including continuity equation, Darcy’s law for tissue, and simplified Navier–Stokes equation for blood flow through capillaries) are used for simulating interstitial and intravascular flows and Starling’s law is used for closing this system of equations and coupling the intravascular and extravascular flows. This is the first study of flow modeling in solid tumors to naturalistically couple intravascular and extravascular flow through a network. This network is generated by sprouting angiogenesis and consisting of one parent vessel connected to the network while taking into account the non-continuous behavior of blood, adaptability of capillary diameter to hemodynamics and metabolic stimuli, non-Newtonian blood flow, and phase separation of blood flow in capillary bifurcation. The incorporation of the outlined components beyond the previous models provides a more realistic prediction of interstitial fluid flow pattern in solid tumors and surrounding tissues. Results predict higher interstitial pressure, almost two times, for realistic model compared to the simplified model.