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This reference work presents the basic principles of angiogenesis induction, including the roles of signaling factors such as hypoxia-inducible factors, biophysical stimulation and angiogenic cells. The book also covers lymphogenesis induction. Both the established fundamentals in the field as well as new trends in the vascularization of engineered tissues are discussed. These include pre-vascularization strategies using preparation of channeled scaffolds and preparation of decellularized blood vessel trees, approaches to inducing formation of microvasculature and approaches to inducing the growth of vascular networks. The authors expand on these concepts with current studies of dual-level approaches to engineer vascularized tissue composites. The book concludes with a discussion of current clinical approaches and the use of vascular grafts in the context of providing clinical practice with new tissue engineering strategies.



Basics of Vascular Biology


Angiogenesis: Basics of Vascular Biology

Angiogenesis occurs by two mechanisms sprouting angiogenesis and intussusceptive angiogenesis. Intussusceptive angiogenesis starts with the formation of an intravascular pillar, which can be extended, so that a vessel tube becomes separated in two parallel branches. Sprouting angiogenesis regards the outward formation of small new vascular branches that starts via invasion of endothelial sprouts into the extracellular matrix. These sprouts are led by a tip cell that strongly responds to exogenous angiogenic factors, of which VEGF-A is the most prominent. This chapter describes molecular steps and metabolic responses that occur within the tip cell and the subsequent signaling that alters the behavior of the adjacent stalk cells. Subsequently, lumen formation, anastomosis, and restoration of perfusion occur, as well as selective removal of excess vascular branches by pruning. The process of angiogenesis is enforced by postnatal vasculogenesis, which represents the recruitment of circulating true endothelial progenitor cells (late outgrowth EPCs or endothelial colony forming cells) to an area in need of blood supply, and is further supported by recruitment of myeloid early-outgrowth EPCs that have an auxiliary function. The detailed studies on the sprouting process itself have to be placed into a (patho)physiological context to be able to generate functional microvascular networks. From combined computational modeling and experimental studies it has become clear that formation of a new microvascular network requires the mutual interplay between sprouting, redistribution (remodeling), and pruning of endothelial tubules into a functional vascular bed.
Victor W. M. van Hinsbergh

Mechanical Regulation of Microvascular Growth and Remodeling

Microvascular growth and remodeling processes are essential to tissue maintenance and repair. While several biochemical regulators have been elucidated, the effects of dynamic mechanical regulatory factors, including local tissue deformation, extracellular matrix mechanical properties, and both luminal and abluminal flow, are incompletely understood. Mechanical regulation is particularly relevant to the field of tissue engineering and regenerative medicine. Recent experimental evidence suggests that microvessels are highly sensitive to changes in local tissue properties like stiffness, ECM density, and external loading. However, an integrated understanding of how microvascular networks are regulated by mechanical factors has not been fully established. In this review, we discuss the microvascular responses to mechanical factors first in terms of cell-based responses and then describe the nuances of these responses when integrated into a multicellular microvessel structure. Finally, we consider the progress in computational modeling approaches to study angiogenesis, wherein the integration of multiple synergistic, antagonistic, or competing stimuli driving the process of microvascular growth and remodeling can be studied, providing better control over experimentally inaccessible variables. By creating a loop wherein experimental data informs computational experiments and vice versa, mechanical effects on microvasculature can be more fully understood and leveraged for engineered tissues.
Laxminarayanan Krishnan, Steven A. LaBelle, Marissa A. Ruehle, Jeffrey A. Weiss, James B. Hoying, Robert E. Guldberg

Angiogenesis Induction


Therapeutic Angiogenesis in Regenerative Medicine

A vascular network along with a sufficient blood supply is not only essential for physiological maintenance of tissue viability but is also a prerequisite for any approaches in regenerative medicine. Previously it was thought that therapeutic angiogenesis can effectively be realized simply by the local release of angiogenic growth factors from matrices, but a more complex correlation between the spatial and temporal distribution of factors and the microenvironmental tissue demands has emerged. After introducing the different kinds and steps of angiogenesis, the review will highlight the key issues of therapeutic angiogenesis and various opportunities for recombinant growth factor delivery strategies. The modifications of binding and release kinetics of angiogenic factors from delivery platforms will be discussed in detail in order to achieve a mature and functional vascular network.
Veronica Sacchi, Rainer Mittermayr, Martin Ehrbar

Therapeutic Angiogenesis: Translational and Clinical Experience

Therapeutic angiogenesis aims to target major public health issues, such as coronary artery disease and peripheral artery disease. Angiogenesis is often induced by gene therapy-derived transgene overexpression. Myriad of preclinical studies have been published during the last three decades, and over two thousand clinical trials have been concluded. Despite of the great enthusiasm on the research field, evolution to clinical applications has been slow. This chapter will discuss the accomplishments and challenges encountered on the path from bench to bedside.
Henna Korpela, Jaakko Lampela, Jussi Nurro, Juho Pajula, Seppo Ylä-Herttuala

Targeting the Cellular “Oxygen Sensors”: Hypoxia Pre-conditioning and Stabilization of Hypoxia-Inducible Factors

The development of novel strategies in tissue engineering and regenerative medicine is inspired by the knowledge on the cell biological processes underlying regeneration. A clear key element in the early phase of healing is the cellular response to hypoxia. Novel therapeutic approaches target the cellular “oxygen sensors” by applying hypoxic pre-conditioning and pharmacologically simulated hypoxia. The cellular response to hypoxia is highly conserved, is well-orchestrated, and relies on hypoxia-inducible factors, which require labile transcription factor subunits and initiate among other pathways the cellular adaption to hypoxia and increase the production of pro-angiogenic factors. Hence, targeting cellular oxygen sensors is considered to be a promising strategy for tissue engineering and regenerative medicine. In this chapter, an overview of the current knowledge on the biology of hypoxia is given. Furthermore, we will review current research in the application of hypoxia-based strategies such as hypoxia pre-conditioning and prolyl hydroxylase (PHD) inhibitors for tissue engineering and regenerative medicine.
Hermann Agis

Probing Vasculature by In Vivo Phage Display for Target Organ-Specific Delivery in Regenerative Medicine

Different mammalian tissues in their normal and pathological state have distinct molecular features in the luminal side of the vessels. These features can be targeted with peptides that concentrate, i.e., home to their target tissue. The homing peptides can be used to deliver cargo, such as therapeutic molecules, to the target tissue, or they can help in detecting pathological changes in the tissue. Some of the homing peptides are capable of penetrating to the target tissue and bringing their cargo with them. One of the most studied homing peptides, iRGD, penetrates to its target tissue with the help of the CendR sequence. The homing and penetrating peptides are discovered by screening a peptide library encoded on the surface of bacteriophages. In in vivo biopanning, the library is injected to the circulation of a living animal, and later, unbound phages are washed off during circulation and bound phages are eluted from the target tissue. The eluted phages are amplified and form the library for the second biopanning round. After a few rounds, the phages containing sequences that home to the target tissue are concentrated in the library.
Toini Pemmari, Tiia Koho, Tero A. H. Järvinen

From Secondary Intent to Accelerated Regenerative Healing: Emergence of the Bio-intelligent Scaffold Vasculogenic Strategy for Skin Reconstruction

This chapter introduces the concept of bio-intelligent scaffolds and discusses the development of a pro-angiogenic porous fibrin-alginate synthetic skin replacement scaffold termed Smart Matrix. This was developed through a biologically-led series of assays to optimize endothelial cell ingress, which identified fibrin as Fibrin was identified as a pro-migratory material. To fabricate scaffold structures, a formulation incorporating alginate as a bulking agent and a compatible surfactant mix was developed for foam formation. Stabilization was achieved with glutaraldehyde-borohydride cross-linking. Smart Matrix prototypes supported conduction of endothelial cells, fibroblasts, and MSC over 48 h in vitro. Extended fibroblast culture within the scaffold did not induce a myofibroblast phenotype. In a porcine full-thickness wound model, Smart Matrix prototypes integrated over 7 days, with rapid vascularization, partly due to vasculogenesis deep within the scaffold structure. Single stage skin reconstruction with a split-thickness overgraft was achieved, with a non-scarring outcome. In a novel delayed porcine wound model, both Matriderm and Smart Matrix activated healing, but only Smart Matrix produced a normal histological neodermis. This bio-intelligent biomaterial scaffold can promote integrative vasculogenesis, leading to a rapid regenerative-type healing. This could offer important clinical benefit and enhance the quality of patient’s lives.
J. F. Dye

Stimulation by Light

Tissue engineering techniques, to replace wounded or missing tissue, are advancing rapidly to ensure the speedy recovery of patients. However, this field faces limitations of cells and biomaterials which prevents the acceleration of regeneration. Low level light therapy, a physical therapy, shows potential in enhancing and supporting the existing medicinal treatments. Visible light in the red and near-infrared range has shown to have positive stimulatory effects on various types of cells involved in wound healing and tissue regeneration. As angiogenesis is an essential part of this process, light therapy was investigated in multiple studies to see its beneficial effect on vessel formation. In vitro, in vivo, and in a clinical setup, LLLT therapy proved that it is capable of stimulating not only endothelial cells but other cells such as fibroblasts, smooth muscle cells, and lymphocytes which are involved in the vessel formation process. It triggers the activation of cytochrome c oxidase, which leads to the production of NO, ROS, and ATP in the mitochondria. These molecules appear to act as secondary messengers initiating ERK/Sp1 and PI3K signaling pathway, which in turn leads to proliferation, migration, and the synthesis of proangiogenic factors. This data indicates that LLLT could be a promising adjuvant treatment in the future.
S. Chaudary, S. Rieger, Heinz Redl, P. Dungel

Approaches for Generation of Lymphatic Vessels

The lymphatic system plays an important role in fluid homeostasis, immune cell trafficking, and fat absorption. Due to injury, diseases, or surgery, the lymphatic system can be disrupted which often leads to lymphedema in the adjacent extremities. Tissue engineering is an emerging research field dealing with the substitution of nonfunctional parts of the human body with in vitro engineered tissues. Regenerative approaches try to stimulate the formation of functional tissues in situ. During the last few decades, the construction of blood vessels in vitro to supply engineered tissues with nutrients gained more and more interest. However, research in the field of lymphatic development stayed behind, but several approaches for lymphatics engineering were developed so far. Lymphatic endothelial cells can be seeded to scaffold materials and afterwards implanted into sites of disrupted lymphatic vasculature. Several regenerative approaches describe the stimulation of lymph vessel growth in vivo. Although the methods developed so far hold promise for the clinical use of engineered lymphatics, the optimal parameters for lymphatic engineering remain a challenge for future studies.
Sabrina Rohringer, Mira Schaupper, Wolfgang Holnthoner

Biomimetic Models of the Microcirculation for Scientific Discovery and Therapeutic Testing

Tissue engineering and regenerative medicine therapies require understanding how molecules and cells coordinate together to influence system level behavior. A key obstacle to advancing our understanding of physiological systems is the inability to probe the specific component-level effects when biological experiments fall short of providing the necessary spatial and temporal resolution over the time course of a response. Hence, a critical question emerges: How can we gain new views not possible with in vivo experiments? The objective of this chapter will be to highlight the impact of biomimetic models, including in vitro, ex vivo, and computational approaches for advancing our understanding of the cellular dynamics involved in microvascular remodeling, which is needed for engineering thick tissues and a common denominator for many pathologies. This overview emphasizes the multiscale cellular complexity of microvascular growth and provides examples of integrative models that offer novel perspectives.
Robert M. Dolan, Jessica Motherwell, Nicholas Hodges, Ariana D. Suarez-Martinez, Peter McFetridge, Cheryl Gomillion, Malisa Sarntinoranont, Shayn M. Peirce, Chris Anderson, Walter L. Murfee



Microvascular Networks and Models: In Vitro Formation

The microvasculature involves the part of the vascular system made of vessels with diameters inferior to 100 μm. There are many culture models allowing for the formation of microvascular networks in vitro, developed either to study cellular and/or molecular aspects of angiogenesis and vasculogenesis or to prevascularize engineered tissues. In this chapter, we describe the cellular (Sect. 2) and material (Sect. 3) components used to generate such in vitro models. Innovative, advanced bioengineering processes, based on bioprinting or microfluidics, to create microvascular networks are also described (Sect. 4).
Ulrich Blache, Julien Guerrero, Sinan Güven, Agnes Silvia Klar, Arnaud Scherberich

Co-culture Systems for Vasculogenesis

Vascularization is a fundamental aspect of tissue engineering and is one of the main challenges in the field when trying to construct thick tissues. Co-culture systems have demonstrated promising potential in construction of vascularized tissues, and in enhancing graft viability and persistence in vivo. In this chapter, we discuss pivotal studies integrating co-cultures of endothelial with various types of supporting cells, aimed to generate vascularized and functional tissue. The influence of different biomaterial components, construct geometry and external mechanical stimulations on the forming vasculature, is reviewed. A comprehensive understanding of the processes leading to the formation of mature and stable vessel networks within engineered tissues will provide guidelines to enhance current protocols, which will ultimately improve integration prospects and enable the fabrication of clinically relevant, large engineered tissues.
Luba Perry, Shahar Ben-Shaul, Shira Landau, Shulamit Levenberg

Cell Sorting, Culture, Preconditioning, and Modulation/Cell Aggregates: Sheets

A novel cell culture surface grafted with the temperature-responsive polymer poly(N-isopropylacrylamide) has been developed for controlled attachment and detachment of living cells through temperature changes. This temperature-responsive surface allows us to harvest confluent cells in the form of an intact monolayer cell sheet with deposited extra cellular matrices. Different from scaffold-based tissue engineering, cell sheet-based tissue engineering without the use of scaffolds provides another way for the development of tissue engineering. Taking advantage of the unique ability of cell sheets to generate three-dimensional (3D) tissues, we have also developed several methods to stack cell sheets and create functional tissues for therapy of a vast variety of diseases ranging from periodontitis and corneal disease to esophageal ulceration and heart failure. Moreover, we have created functional anisotropic tissues, thick vascularized tissues, and even organ-like systems by using cell sheet-based tissue engineering. In this chapter, we summarize development of temperature-responsive cell culture surfaces, cell sheet manipulation technologies, fabrication of thick tissues, and organ-like structures with cell sheets as well as applications of cell sheet-based tissue engineering.
Botao Gao, Tatsuya Shimizu, Teruo Okano

In Vitro and In Vivo Approaches for Prevascularization of Three-Dimensional Engineered Tissues

A major hurdle in tissue engineering of organs is the incorporation of a functioning blood vessel network integrated throughout the engineered tissue that readily links to the surrounding host blood vessels to provide the oxygen and nutrients required by the engineered construct. In the early years of tissue engineering development, vascularization was not a priority and generally angiogenic ingrowth from neighboring host capillary networks, a process termed extrinsic vascularization was used to vascularize implanted tissue engineering constructs. Extrinsic vascularization takes weeks, and much of the implanted tissue becomes ischemic and dies before capillary ingrowth is complete. In 2000, intrinsic vascularization was devised by Tanaka et al. who isolated a macrovascular pedicle in a plastic chamber which subsequently underwent considerable angiogenic sprouting. A new arteriovenous capillary network was therefore formed within the chamber space which was capable of growing with and supporting the survival of tissue/organ specific cells implanted in the chamber. There was a time lag to development of this pedicle-based angiogenic network, and in recent years a new technique termed pre-vascularization has been developed that involves co-culture of endothelial cells with parenchymal cells or stem cells as they assemble in vitro. Capillary networks are formed throughout the construct, and upon implantation inosculate (functionally join) with host capillaries. Inosculation takes at least 2 days and provides blood flow within this time period within the construct. The most efficient vascularization technique for thick three-dimensional tissue engineering would be the combination of pre-vascularization in vitro with vascularization via angiogenic sprouting of a vascular pedicle, this combination has rarely been successfully utilized.
Geraldine M. Mitchell, Wayne A. Morrison

Perfusion Bioreactors for Prevascularization Strategies in Cardiac Tissue Engineering

Cardiac tissue engineering is currently being pursued with three different applications in mind: drug safety screening, disease modeling, and cardiac repair. Mini- and microengineered heart tissues are well suitable for drug safety screening and disease modeling. But generation of large cardiac patches of clinically relevant thickness, to functionally support the injured heart after myocardial infarction, still needs improvement. The high oxygen and nutrient demand request prevascularization of the engineered tissues in vitro prior to implantation. Vascularization and cardiac tissue development are influenced by several factors such as perfusion velocity, shear stress, coculture, extracellular matrix, mechanical strain, electrical stimulation, and many more. As engineering approaches get ever more sophisticated and bioreactors increasingly complex, cardiac tissue engineering evolves and quality control becomes more prominent. This chapter will focus on different perfusion bioreactors that aim at cultivating highly vascularized and functional engineered heart tissues by, e.g., direct perfusion through the tissue or cultivation on top of an engineered vascular bed.
Ingra Mannhardt, Anna Marsano, Andreas Teuschl

Past and Future Prevascularization Strategies with Clinical Relevance: Leading to a Dual Approach

During the last decades, numerous approaches for the engineering of soft and hard tissues were developed. However, tissues with a thickness larger than 2 mm show limited diffusion of essential nutrients which underlines the need for vascularization (Griffith et al., Tissue Eng 11(1–2):257–266, 2005). It is therefore of utmost importance to generate functional prevascularized thick tissues, which can be surgically connected to host tissues. First prevascularization strategies started in the early 1980s when Judah Folkman published his work about the role of endothelial cells in angiogenesis by trying to identify endothelial cell function during blood vessel formation (Folkman, Lab Investig 51(6):601–604, 1984). This was the decade when first prevascularization strategies began in polymeric scaffolds. The discovery of growth factors and cytokines led to the development of growth factor loaded constructs for delivery during the last decade of the twentieth century. Over the years, protocols for the decellularization or extraction of biological materials were developed which led to a boom of vascularization strategies in these matrices since the early 2000s. Technical progress during the last years makes now the 3D bioprinting of tissues and vasculature possible, although the use of this method in microvascular tissue engineering is still in its infancy. Independent of which matrix is used for the prevascularization strategy, the growing knowledge about cell interactions and pathways in angiogenesis as well as vasculogenesis led to the conclusion that the use of 3D cultures instead of 2D formed networks and the combination of endothelial cells with cells displaying angiogenesis stimulating properties led to the most promising results.
Karl H. Schneider, Sabrina Rohringer, Wolfgang Holnthoner, Severin Mühleder, Heinz Redl

Microsurgical Approaches for In Vivo Prevascularization

The rapid and sufficient vascularization of large tissues is the main obstacle to the broad implementation of tissue engineering (TE) into clinical practices. Typically, the vascularization of engineered tissues is achieved after implantation, by stimulating the ingrowth of surrounding blood vessels via the delivery of angiogenic factors, the addition of angiogenic cells, and the optimization of scaffold properties. Although these approaches showed promising results, the ingrowth of the host’s vasculature into the implant remains slow. In a parallel effort, various prevascularization approaches were developed, which aim at inducing the formation of a vasculature within engineered tissues, before implantation. Such a prevasculature can connect to the host’s vasculature and rapidly perfuse the implant. However, building a patterned, hierarchical, functional vascular tree that can be hooked to the host, possibly via microsurgery, is a long-lasting challenge. Current approaches of prevascularization include the in vitro induction of endothelial cells organization into a microvascular network and the in vivo incubation of an engineered tissue within a surgically prepared angiogenic site (e.g., arteriovenous loop). This last approach, rooted in surgical practices, allows for the ingrowth of a hierarchical, functional vasculature within the construct, which can connect to the host upon transfer to the secondary site of defect. Here, we outline this family of promising surgical strategies aiming at the in vivo formation of vascular networks within engineered tissues.
Christoph Koepple, Ulrich Kneser, Volker J. Schmidt

From Autologous Flaps to Engineered Vascularized Grafts for Bone Regeneration

Replacement of damaged or lost tissue typically relies on the availability of living, functional substitutes and the rapid development of a stable and efficient vascularization upon transplantation, in order to guarantee their survival. These requirements challenge current surgical reconstruction techniques in the clinical practice.
In the past decades, the field of tissue engineering has introduced the possibility to combine materials and living cells to generate functional substitutes, which can be tailored to specific requirements of the implantation site. At the same time, plastic and reconstructive surgery has developed a large armamentarium of grafting possibilities and flaps supporting vascularization of native tissues, especially through progress made in microsurgical techniques.
In this chapter, we describe advances in the two fields and discuss how the principles and techniques independently developed could be combined towards the prefabrication of vascularized tissues. The resulting paradigm of “regenerative surgery,” here exemplified in the specific context of bone regeneration, could represent the future standard for the reconstruction of complex body parts.
Alexander Haumer, Tarek Ismail, Alexander Lunger, Rik Osinga, Arnaud Scherberich, Dirk Johannes Schaefer, Ivan Martin


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