Vascularization in Tissue Engineering E-Book
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- Herausgeber: Bentham Science Publishers
- Kategorie: Wissenschaft und neue Technologien
- Sprache: Englisch
Vascularization in Tissue Engineering presents a comprehensive picture of blood vessel development and the recent developments on the understanding of the role of angiogenesis in regenerating biological tissues. The first-half of this book, consists of three chapters, emphasizing the fundamental knowledge about cell pathways, growth factors, co-culture strategies, cell interactions, and vascularization in pathological scenarios. The second half takes this knowledge a step further and explains the vascular microenvironment, scaffolds, and related applications in regenerative medicines. This section also provides information about biomaterial scaffolds and stem cell cultures for wound-healing and tissue regeneration. Readers will learn about cutting edge technologies in this field. This volume is a handy reference for students and researchers seeking information about the angiogenic processes and applied biotechnology in tissue engineering.
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Seitenzahl: 262
Veröffentlichungsjahr: 2020
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FOREWORD
When Dr. Cai asked me to write this foreword, I was greatly honored to have the opportunity to introduce this eBook.
As one of the research hotspots of life science in the 21st century, tissue engineering is a new subject combining cell biology and material science to construct tissue or organs in vitro or in vivo. The method is to plant seed cells on scaffold materials to form an active complex, which finally establishes tissues and organs with normal physiological functions and is applied to the treatment and repair of human diseases. At present, the construction of skin, bone and cartilage in vitro has made great progress, and has been widely used in clinical. However, how to build a blood supply system that can transport nutrients, oxygen and remove metabolize waste for the body, to ensure the survival of implanted artificial tissue in the body and play its normal physiological function, which is a major difficulty of tissue engineering, as well as a key issue of regenerative medicine.
Vascularization, existing in both physiological and pathological procedures, is a considerable complicated process which is regulated and controlled by a variety of biological factors via diverse pathways and mechanism. It’s a complex and multi-step physiological process, which must be carried out under a strict micro-environment. At present, in addition to conventional treatment methods, plenty of vascular diseases such as arterial ischemic disease, peripheral arterial disease, pulmonary hypertension, limb ischemia, myocardial infarction and cerebral infarction were treated with endothelial progenitor cells possessing the characteristics of stem cells in clinical work, which have a very broad prospect. Therefore, the exploration of the vascularization of tissue engineering will greatly promote the research results of regenerative medicine to clinical application.
It is generally known that seeding cells, growth factors, scaffold materials and microenvironment are the four basic elements of tissue engineering. This eBook is a comprehensive and systematic introduction to the vascularization of tissue engineering from these four aspects. Firstly, it details the whole process of physical vascularization (vascularization and angiogenesis) and the role of various growth factors. Then, the effect of the physiological microenvironment and pathological microenvironment on the vascularization in tissue engineering is elaborated, such as related mechanism, pathophysiological features and so on. Besides that, various common seeding cells, typical co-culture system model and multifarious scaffold materials of vascularization in tissue engineering are also introduced in detail.
In conclusion, I am excited about this eBook. Because it not only systematically and comprehensively introduces the mature theoretical knowledge, methods and technology of vascularization in tissue engineering, but also shows the latest progress achievements and future development in the world. I believe it will be beneficial to all those who have an interest in vascularization in tissue engineering and will lay a crucial and solid theoretical foundation for making future progress of tissue engineering vascularization.
PREFACE
Angiogenesis refers to the process of forming a new vascular system with an existing vascular network through the proliferation and migration of vascular endothelial cells on the basis of existing capillaries and/or venules. Angiogenesis is of great significance in tissue engineering, wound healing and regeneration repair. The core of tissue engineering is to establish a three-dimensional space complex of cells and biological materials, which is used to reconstruct the morphology, structure and function of the damaged tissues and achieve permanent replacement, to achieve the purpose of repairing wounds and rebuilding functions. However, there are few tissue engineering products that can be applied to clinics at present. One of the main reasons is the early vascularization of tissue engineering products. Evidence shows that when the cell mass is greater than 3 mm3, the diffusion of interstitial fluid cannot be used to maintain the cell's survival, and the supply of oxygen and nutrients must be achieved through the regeneration of blood vessels. Because tissue engineering scaffolds do not have a vascular network, vascular regeneration has become an important factor limiting the ability of tissue engineering constructs to form tissues after implantation. Therefore, when using tissue engineering technology to construct large and complex artificial tissues or organs, how to avoid ischemic necrosis or poor healing of the central part of the defect has become the primary task of tissue engineering vascularization. At present, by constructing scaffold material-seed cell-growth factor complex, based on the research foundation of angiogenesis mechanism, the formation of neovascularization in vitro three-dimensional microenvironment or in vivo transplantation culture is the most common method for tissue engineering vascularization. Therefore, it is very important to understand the occurrence, development, physiological and pathological processes of angiogenesis for tissue engineering vascularization. In this book, the authors focus on the biological and pathological conditions of vascularization, microenvironment factors on angiogenesis, co-culture systems and scaffold materials used for angiogenesis, including: (1) biological basis of vascularization; (2) effects of microenvironment factors on angiogenesis; (3) microenvironment of pathological vascularization; (4) vascularization in co-culture systems; (5) vascularization and scaffold material.
Though this book, readers will have a better understanding of the occurrence, development, physiological and pathological processes of angiogenesis, and know more about ways and means of angiogenesis. The authors sincerely hope that this book will add further insight into basic and applied researchers as well as clinicians involved in tissue engineering vascularization, thus contributing to further advances in regenerative medicine.
List of Contributors
Biological Basis of Vascularization
Yichen Ge,Yuting Wen,Xiaoxiao Cai*Abstract
Vascularization, existing in both physiological and pathological procedures, is a considerable complicated process which is regulated and controlled by a variety of biological factors via diverse pathways and mechanism. It is crucial to understand these processes and factors well if we intend to unveil the mystery of vascularization. More importantly, understanding basic processes offer tools and thinking directions with which researchers are able to design various methods to achieve vascularization, which is called vascular tissue engineering. In this part, major procedures of physical vascularization (vasculogenesis and angiogenesis) and growth factors are introduced, as well as their roles and new research outcomes in vascularization. These factors include vascular endothelial growth factor(VEGF), basic fibroblast growth factor(bFGF), platelet-derived growth factor(PDGF), angiopoietin1, angiopoietin2 and others(junctional molecules, integrals etc.). Lastly, some frequently used markers and testing methods about vascularization research are briefly introduced.
1. Introduction of Biological Vascularization
It is generally acknowledged that the mechanism of neovascularization comprises two aspects---vasculogenesis and angiogenesis [1, 2]. Supposed as the main mechanism of the construction of vascular networks in the embryonic stage, vasculogenesis occurs in the bone marrow as endothelial progenitor cells (EPCs) gradually migrating, differentiating and finally reforming new vessels, besides it also exists in adult tissue especially in the ischemic area [3, 4]. In the embryo, the first vascular network is built when somites beginning to form by the process of vasculogenesis. Locating between two germ layers, the first blood islands which form the inner layer of the yolk sac occur by in situ differentiation from the extra-embryonic mesoderm. In the embryonic stage, vascular network remodeling is characterized as the change of number and/or location of vascular segments to improve functional adaptability without evident network expansion. Vascular fusion can reduce the number of vascular segments and therefore give rise to larger vessels. Larger vessels in other regions are remodeled into a network of smaller vessels which subsequently increase the number of vessels segments. These biological processes result in a big transformation of primary plexus to enter into a more complexly structured secondary stage. Further expansion of primary and secondary vascular plexus during postnatal life occurs with the process of angiogenesis. Angiogenesis generally refers to the process of new blood vessels formation based on pre-existing ones (Shown as Fig. 1a-c). The process of angiogenesis comprises two distinct mechanisms, sprouting of endothelial cells (ECs) and splitting of vessel lumens by intussusceptive microvascular growth (IMG).
1.1. Vasculogenesis
1.1.1. Embryonic Vasculogenesis
Vasculogenesis is a process in which blood vessels differentiate from in situ into ECs. Initially, the term was used for embryonic development and the presence of angioblasts, which were entirely associated with prenatal growth. “Vasculogenesis” was first brought out by Werner Risau [5] to term the development of mesodermal vascular plexus by differentiation of vascular fibroblasts. Angioblasts are thought to accumulate in the angiogenesis area. Vasculogenesis that occuring embryogenesis and extraembryonic are often accompanied by hematopoiesis, thus the term “angioblasts” has also been proposed. Elementary observations in Sabin [6] strongly suggest that both hematopoietic cells and ECs origins from the angioblasts. The existence of hemangioblasts in vivo has developed. At present, instead of defined as an actual ordinary cell precursor, it’s rather more like a competitive cell that can generate hematopoietic cells and ECs to local environmental signals [7].
In early mouse embryos, angioblasts originated from decentralized progenitors in the lateral plate mesoderm and expressed Flk-1 (VEGFR-2) and Brachyury (Bry)genes in turn [8]. Bry gene was inhibited and Flk-1 was activated as development progressed. The dynamic changes of these progenitor cell differentiation markers were as follows: Bry +/Flk-1 -, Bry-/ Flk-1 -, Bry +/Flk-1-, Bry +/Flk-1 -, and Bry +/Flk-1 -. These groups represent different phases of differentiation of vascular mother cells. In addition to Flk-1 and Bry genes, they also display diverse sequences of other genic groups [9]. The existence of vascular mother cells has been verified in vitro though collecting endothelial and hematopoietic descendants of colony-forming cells (CFCs) derived from embryoid [10, 11]. Embryoid bodies obtained from suspension cultured embryonic stem cells established a model that simulates many cell differentiation in multi-aspect during early somatic embryogenesis [12]. In vitro, fast breeding CFCs that produce hematopoietic colonies functioned equally to vascular mother cells. The vascular mother cells have multi-directional differentiation potentials to form ECs, smooth muscle cells and hematopoietic cells under specific conditions [8, 9, 11]. The commitment of angiogenic cells is controlled by transcription factor of etv2/er71gene, the upstream of core genes in the development of EC lineage [13], and activation of endothelial cell line specific genes (endothelin, endothelin and VE cadherin) and hematopoietic and/or hematopoietic (hematopoietic, hematopoietic and SCL) lineages [14]. ETv2 guide differentiation of endothelial and hematopoietic lineage via regulating ETS associate genes necessary for downstream stimulation of hematopoietic and endothelial differentiation [14].
Members of the TGF-beta superfamily participated in mesoembryonic expression of Bry, for instance BMP (bone morphogenetic protein) and lymph node and activin signals, as a guide between self-renewal of pluripotent stem cells [15, 16]. Fibroblast Growth Factor-2 (FGF-2) and BMP4 are key signaling ingredients that stimulate the development of embryonic mesoderm, thus accelerate ECs and blood cells production [17, 18]. TFIIS, subtype of TCEA3 which express in mesoderm was proved to drive the production mesoderm EC. As shRNA transfection reduced the expression of TCEA3 in mouse embryonic stem cells, the expression of Bry marker in mesoderm increased, the expression of multipotent genes in mesoderm decreased, the differentiation of EC boosted and the production of vascular endothelial growth factor (VEGF) A increased [19]. Therefore, ECs can bypass a hemangioblast intermediate directly from mesodermal angioblasts. EC differentiation in the period of embryonic process has been demonstrated to be produced by vascular mother cells directly from the mesoderm [20, 21]. These ECs can further form tubules during mesoderm culture in vitro. Although current evidence supports the existence of angioblasts, it has been a challenge to isolate these cells and determine their exact location in developing embryos.
Embryonic EC are considered be descendants of angioblasts [22]. Angioblasts were found to transform phenotype in mice: initially expressed tal-1/flk-1, then CD31 was obtained, while the expression of tal-1 was reduced [21]. This phenotypic change of angioblasts was observed during the formation of cardiac ducts, dorsal aortas, interlaminar vessels and main veins in different embryos. During these events, cells processes migration, isolation and assemble into tube/vessel as they differentiate into mature ECs. Instead of classical growth factors such as platelet-derived growth factor (PDGF), VEGF and FGF, notch and ephb2/ephb4 signaling pathways was considered the key components to regulate dorsal aortic angiogenesis [23, 24]. It has been proved that basement-membrane (BM) and extracellular matrix (ECM) components for instance collagen XVIII, laminin, including beads and sulfate side chains, can dynamically regulate the development of brand blood vessels. The above ECM components are important accumulator for growth factors, which can be fluid into surrounding tissues by selective stimulation thus regulate activity of cell migration, assembly and angiogenesis [25, 26].
1.1.2. Postpartum Vasculogenesis
The discovery of circulating endothelial progenitor cells (CEPC) are a real breakthrough in the field of adult vasculogenesis [27]. The majority of CEPs were bone marrow (BM) derived bone marrow cells or circulating cells of lymphoid lineage, stimulating angiogenesis, and expressing EC markers while stimulating vascular growth factor in vitro, but not differentiating into ECs to develop blood vessels [28]. In fact, the term of EPCs has been debated and a new method for selecting these cells has been proposed [29].
Due to the common markers between endothelial progenitor cells (EPC) and hematopoietic cells, there is no suitable method to identify EPC in vivo environment, for instance bone marrow (BM), peripheral blood (PB), umbilical cord blood (CB) and solid tissue. The absence of a novel marker, or a collection of markers, and the high uncertainty for isolating and culturing EPCs make it obscure to define these cells. Tissue sources can alter the characteristics of EPCs; when separated from in vivo biological cultures, these cells can be influenced by artificial irritation in vitro. One of the current methods for identifying EPCs depends on the ability of adhesion and colony in vitro with auxiliary usage of flow cytometry techniques to select cells according to their surface phenotypes. Generally speaking, CEPCs screened from mononuclear cells (MNC) origin from peripheral blood are defined as “early” and “late” in vitro growth cells. In addition to VEGFR2 (flk-1), the “early” growth cells obtained via short-period culture were defined as hematopoietic adhered cells expressing cell surface marker CD14, CD45 and CD11b.They can support angiogenesis and angiogenesis, but cannot spontaneously form tubules in vitro [30-33]. Early endothelial progenitor cells promote angiogenesis by secreting angiogenesis-promoting molecules in a paracrine manner and can merge into capillaries at perivascular locations [31]. Since proliferation can also be used as a standard for defining these cells, a few groups have constructed a colony formation analysis. Following the initial culture of non-adherent cells for a short-term, EC conditioned colonies were detected; CFCs were also verified to be a origin of medullary lymphoid cells, which stimulated angiogenesis, but could not form tubules [34]. Another type termed “late” endothelial CFCs (ECFCs) are screened following long-term culture in vitro up to 2-4 weeks, which do not express any myeloid/lymphoid surface markers such as CD14, CD45 [35, 36]. These cells can directly integrate into the permanent vascular system, and form spontaneous lumen tubules in matrix gel when transplanted internal, and ultimately conduct blood vessels. ECFC is further characterized by colony formation analysis, which means that a different population can be selected, showing high proliferation potential and high telomerase activity [36-38].
After pre-screening of CEPCs with magnetic beads enriched CD34/CD133 double positive cells, two different culture groups could be separated. One is termed “primitive EPCs”, which forms micro colonies with more proliferation dynamic and stimulated vasculogenesis in postpartum. Preclinical study of human vascular diseases with usage of animal models revealed these primitive EPCs are potential tools for vascular regeneration.
Besides CEPC, there are also a number of resident EPCs. Some permanent EPCs are described as discrete lesions in the endothelium of large vessels, mainly the aorta [39]. Although the conversion rate of EC is relatively low or off the record, there remain some parts in the lining of ECs expressing more proliferation dynamic and high expression of telomerase. Cells situated in these areas are prone to proliferate due to tissue damage. Permanent endothelial progenitor cells were also found in the wall of the artery at the medial edge of the adventitia [40]. These CD34-expressing cells can develop into mature ECs, via vascular tubules in transplantable tumors in vivo, and promote the formation of tubules in the culture of artery explants. The medial edge of adventitia is the same spot where Sca-1 positive precursor cells was detected. It maintains self-renewal characteristics and can differentiate into other cell lines under selective stimulation, for instance mesenchymal stem/stromal cells (MSCs), osteoblasts and ECs etc.
1.1.3. Vasculogenesis in Reparative Process
Current studies have shown that vasculogenesis also happens in adult tissues mostly during wound healing events. The ischemia model revealed that the main source of CEPCs in granulation tissue vasculogenesis is bone marrow(BM). BM-derived progenitor cells can be regulated by SDF-1 (interstitial derived factor), VEGF, erythropoietin, G-CSF (granulocyte colony stimulating factor), statins, bFGF, PLGF (placental growth factor), estrogen, insulin, angiopoietin-1, CXCR4 antagonists and other mediators, such as IL-6 or IL-10 [32, 41, 42]. For example, IL-10 stimulates CEPC present from BM towards wound healing site after myocardial infarction, which seems to be modulated via SDF-1/CXCR4 and STAT-3/VEGF signaling pathways.
It has been widely proved that vascular system is distorted and disordered in inflammatory reactions and permanent wound healing events, such as diabetes, obesity, atherosclerosis, hypercholesterolemia, and dyslipidemia (lipid metabolic changes). This is associated with a lower activity and dysfunction of CEPCs, as shown by a lower response to growth factor/chemokine [43].
1.2. Angiogenesis
1.2.1. Introduction of Angiogenesis
Angiogenesis is defined as the development of blood vessels from existing vessels which is important for organ regeneration. A false in this process leads to numerous disorders such as ischemic, infectious, inflammatory and malignant. Blood vessels are evolved to permit hematopoietic cells to examine the body for immune supervision, provide nutrition, and dispose circulating waste. Vessels also provide organ-related indicators in a perfusion-independent manner. Although this process is conducive to tissue differentiation and repair, it can stimulate malignant diseases and inflammation to occur, and is taken advantage by cancer metastasize to kill patients. Blood vessels circulate through every organ, thus abnormal vascular growth can cause many diseases. For example, insufficient growth or maintenance of blood vessels can generate myocardial infarction, apoplectic coma, ulcerative diseases and neural degeneration. Deviating from normal growth of blood vessels can also generate pulmonary hypertension and blinding eye diseases. Several angiogenesis patterns have been verified. In growing mammalian embryos, ECs derived from angioblasts assemble into vascular labyrinths. Different signals participated in vessel differentiation [44]. The following germination generates the production of the vascular network, defined as angiogenesis. Then arteriogenesis starts, and ECs arranged channels are covered by pericytes materials including vascular smooth muscle cells (VSMCs), which provides stability and support perfusion. Blood vessels can also form through other mechanisms, the processes require further studies. For instance, predecessor of blood vessels can be referred to as the process of intussusception splitting, resulting in the production of sub-vessels. In other cases, blood vessels co-selectively occur, in which tumor cells hijack existing blood vessels, or tumor cells can arrange blood vessels development, a phenomenon defined as vascular mimicry. It is hypothesis that stem cells derived from cancer can even generate tumor ECs [45]. Despite the controversy, the assemble of bone marrow-derived cells (BMDC) and endothelial progenitor cells into the vascular border contributes to vascular repair or pathological vasodilation in healthy adults. Progenitor cells then integrate into ECs during postnatal angiogenesis. Collateral vessels bring a large amount of blood flow to ischemic tissues during vascular reconstruction, which is generated by various mechanisms, including attraction and stimulation of bone marrow cells [46].
1.2.2. Branching, Maturation and Resting of Blood Vessels in Angiogenesis
In healthy adults, resting ECs processes a long half-life and are guarded by autocrine controlling signals such as NOTCH, fibroblast growth factor (FGFs), angiopoietin-1 (Ang1) and VEGF. Due to vascular oxygen support, ECs are assembled with oxygen sensors and hypoxia sensible factors, including hypoxia inducible factor 2α (HIF-2α) and prolylhydroxylase domain 2 (PHD2), allowing blood vessels to readjust their morphology to optimize blood circulation. Static ECs form a single layer of phalangeal osteocytes with flow-lined surfaces, which are connected by connecting molecules like claudins and VE-cadherin. ECs are enveloped by pericytes, which release survival signals such as VEGF and Ang1 and inhibit the proliferation of ECs.
When resting vessels sense angiogenesis signals released by hypoxia, inflammation or tumor cells, like Ang2, VEGF-C, FGFs, VEGF and chemokines, pericytes separate first from the vascular wall (the process is in response to Ang2) and isolated away from basement membrane via degrading proteolysis regulated by matrix metalloproteinases. VEGF up-regulated the permselectivity of the endothelial cell layer, which led to exosmosis of plasma protein, thus creating an extracellular matrix (ECM) scaffold. ECs managed to transform to the surface of the ECM, via integrin signaling. Angiogenic molecules deposited in extracellular matrix was exposed by protease, for instance fibroblast growth factor (FGF) and VEGF, and remodeling the extracellular matrix into a vasoactive environment. In order to prevent ECs from collectively migrating to angiogenesis signals, an endothelial cell, called a type cell, exists such as VEGF receptors, gap ligands DLL4. The adjacent organs of apical cells take stalk cells as auxiliary sites. Regulated by NOTCH, their division lengthens the stalk and stimulated by NOTCH ankyrin repeat protein (NRRAP), WNTs, placental growth factor (PLGF) and FGFs. VE-cadherin, CD34, VEGF and hedgehog mediated the development of lumen [2]. Tip cells are composed of filamentous cells which can sense environmental signals, for instance adrenaline and signal quantities. Stalk cells transmit spatial information about the location of their extracellular environments via
