Vascularization strategies for tissue engineering for tracheal reconstruction.

Tissue engineering is an exciting field that has arisen amidst projections of revolutionizing reconstructive surgery. Fabricating tissue with a patient-specific, three-dimensional geometry from a cocktail of autologous cells, growth factors, and biocompatible/biodegradable polymers would address donor site morbidity concerns, alleviate tissue shortages, and improve patient quality of life. Achieving these rather lofty goals requires the collaborative effort of scientists, engineers, and surgeons in recognizing and addressing the unique concerns of engineered tissue. Tissue engineering is an exciting field that has arisen amidst projections of revolutionizing reconstructive surgery. Fabricating tissue with a patient-specific, three-dimensional geometry from a cocktail of autologous cells, growth factors, and biocompatible/biodegradable polymers would address donor site morbidity concerns, alleviate tissue shortages, and improve patient quality of life. Achieving these rather lofty goals requires the collaborative effort of scientists, engineers, and surgeons in recognizing and addressing the unique concerns of engineered tissue. One of the most important design constraints to providing clinically translatable engineered tissue is the development of a patent microvascular network with properly structured geometry within the tissue engineered constructs. Microvascular networks control the mass transport of nutrients, ensure tissue viability, control tissue differentiation, and allow long-term tissue maintenance. It has been shown that cells cannot survive greater then 200 μm from a blood supply. It is also recognized that angiogenesis occurring in an unregulated fashion produces undesirable results. This is most recognized in the oftstudied tumor angiogenesis; however, little is known about the formation, growth-kinetics, and architecture of microvascular networks in tissue engineered constructs. A complete understanding of angiogenesis in any system is impossible with current assays and imaging modalities. Many techniques have been developed to study angiogenesis and microvascular architecture. These methods include in vitro gels, animal preparations, vascular casts, micro-CT, histology, and immunohistochemistry. Microvascular networks are complex, exhibiting wide variation among environments and can be greatly complicated due to various pathologies. These assays require broad assumptions about or oversimplification of the networks. Angiogenesis can be adequately understood only with a high resolution, threedimensional, quantifiable assay. Result images allow measurement of many parameters, including vascular network density, vessel diameters, void space, and tortuosity. We have developed a novel, sophisticated method with resolution to the endothelial cell level that allows visualization and quantitation of microvascular networks in three dimensions. Briefly, tissue is excised, cryofixed, and six micron serial sections are cut on a cryostat. Each section is immunostained for CD-31, an endothelial cell specific antigen, and stained sections are imaged in 2D using a CCD camera. Immunohistochemistry used in combination with microscopy allows high resolution images of antigenically distinct features of tissue. Although quantitative data can be obtained using other methods, only immunohistochemistry allows quantitation at the cellular level while maintaining the relationship between stained cells and overall tissue morphology. The 2D images are aligned using automated image registration techniques and volume rendered to produce a 3D, quantifiable image of the microvascular networks within the tissue. journals/ijps/vol1n1/3d.rm Quantitative 3D Analysis Of Microvascular Networks In Tissue Engineering 2 of 3 Currently, we are applying this technique to the study of angiogenesis in model tissue engineered constructs. The movie depicts capillary invasion into a fibrin gel following implantation of a model tissue engineered construct within a rat model. The image is 1200 x 500 x 240 μm with 1 μm resolution. Vessels and endothelial cells are pseudo-colored red and the fibrin white. Vessels can be seen growing within the fibrin and endothelial cells along the periphery of the fibrin indicate the leading edge of angiogenesis. The inosculation mechanisms by which surgical free flaps (the tissue engineering gold standard) establish and maintain a patent vascular network are also being studied. This work will lead to a better understanding of angiogenesis in tissue engineering and will contribute to the development of clinically useful tissue engineered products.

Bioengineered cardiac tissues represent a promising strategy for regenerative medicine. However, methods of vascularization and suitable cell sources for tissue engineering and regenerative medicine have not yet been established. In this study, we developed methods for the induction of vascular endothelial cells from mouse embryonic stem (ES) cells using three-dimensional (3D) suspension culture, and fabricated cardiac cell sheets with a pre-vascularized structure by co-culture of mouse ES cell-derived endothelial cells. After induction, isolated CD31+ cells expressed several endothelial cell marker genes and exhibited the ability to form vascular network structures similar to CD31+ cells from neonatal mouse heart. Co-culture of ES cell-derived CD31+ cells with ES cell-derived cardiomyocytes and dermal fibroblasts resulted in the formation of cardiac cell sheets with microvascular network formation. In contrast, microvascular network formation was reduced in co-cultures without cardiomyocytes, suggesting that cardiomyocytes within the cell sheet might enhance vascular endothelial cell sprouting. Polymerase chain reaction array analysis revealed that the expression levels of several angiogenesis-related genes, including fibroblast growth factor 1 (FGF1), were up-regulated in co-culture with cardiomyocytes compared with cultures without cardiomyocytes. The microvascular network in the cardiac sheets was attenuated by treatment with anti-FGF1 antibody. These results indicate that 3D suspension culture methods may be used to prepare functional vascular endothelial cells from mouse ES cells, and that cardiomyocyte-mediated paracrine effects might be important for fabricating pre-vascularized cardiac cell sheets.

Angiogenesis or neovascularization plays a crucial role in a variety of physiological and pathological conditions including cardiovascular disease and cancer and in tissue engineering. Angiogenesis is a complex process, where emergent behavior of cells results in the formation of microvascular networks. We have begun a systematic development of computation models, starting with models of growth factor interactions with their receptors on the cell surface and the extracellular matrix, proteolysis of the matrix with matrix metalloproteinases and the associated cell signaling. We will proceed toward a physiome model of angiogenesis, i.e., a set of models spanning multiple levels of biological organization.

Tissue engineering offers immense potential for addressing the unmet needs in repairing tissue damage and organ failure. Vascularization, the development of intricate blood vessel networks, is crucial for the survival and functions of engineered tissues. Nevertheless, the persistent challenge of ensuring an ample nutrient supply within implanted tissues remains, primarily due to the inadequate formation of blood vessels. This issue underscores the vital role of the human vascular system in sustaining cellular functions, facilitating nutrient exchange, and removing metabolic waste products. In response to this challenge, new approaches have been explored. Microfluidic devices, emulating natural blood vessels, serve as valuable tools for investigating angiogenesis and allowing the formation of microvascular networks. In parallel, bioprinting technologies enable precise placement of cells and biomaterials, culminating in vascular structures that closely resemble the native vessels. To this end, the synergy of microfluidics and bioprinting has further opened up exciting possibilities in vascularization, encompassing innovations such as microfluidic bioprinting. These advancements hold great promise in regenerative medicine, facilitating the creation of functional tissues for applications ranging from transplantation to disease modeling and drug testing. This review explores the potentially transformative impact of microfluidic and bioprinting technologies on vascularization strategies within the scope of tissue engineering.

Angiogenesis facilitates the formation of microvascular networks and promotes neurological deficit recovery after cerebral ischemia-reperfusion injury (CIRI). This study investigated the angiogenesis effects of 4-methoxy benzyl alcohol (4-MA) on CIRI. The angiogenesis effects of 4-MA and the potential underlying mechanisms were assessed based on a middle cerebral artery occlusion/reperfusion (MCAO/R) rat model and a hind limb ischemic (HLI) mouse model. Immunofluorescence was conducted to detect microvessel density, and Western blotting and polymerase chain reaction were performed to determine the expression of angiogenesis-promoting factors. In addition, we investigated whether the angiogenesis effects of 4-MA caused damage to the blood-brain barrier (BBB). After treatment with 4-MA (20 mg/kg) for 7 days, the neurological deficits recovered and microvessel density in the cerebral cortex increased in the MCAO/R rats. Additionally, 4-MA also regulated the expression of angiogenesis factors, with an increase in vascular endothelial growth factor (VEGF) and vascular endothelial growth factor receptor 2 (VEGFR-2) expression and a decrease in angiopoietin 1 (Ang-1), Ang-2, and Tie-2 expression in both MCAO/R rats and HLI mice. Moreover, 4-MA increased the expression of angiogenesis-promoting factors without exacerbating BBB cascade damage in MCAO/R rats. Our results indicated that 4-MA may contribute to the formation of microvascular networks, thus promoting neurological deficit recovery after CIRI.

Biomaterial-based 3D bioprinting strategy for orthopedic tissue engineering.

Tissue engineering (TE) is a modern approach to improve or restore tissues that has been diseased or damaged by combining the factors in growth signaling and appropriate cells to form compatible biomaterial scaffold. Bone repair has been a major global health challenge in recent decades. Bone transplantation is widely used as an effective clinical treatment for this purpose. However, there are several serious issues with transplantation, including a shortage of autologous bone, immune rejection, the risk of virus transmission with allogeneic bone, and postoperative complications. In recent years, scaffolds for bone tissue engineering have emerged as a promising alternative for bone repair. These scaffolds have porous structures that mimic the extracellular matrix, which can enhance the migration, proliferation, and differentiation of osteoblasts. This process accelerates the repair of bone defects.The usage of bioactive materials has become important tool in tissue engineering and regenerative medicine application as they are to mimic tissues mechanically, chemically, and physically. Polymeric scaffold provides numerous advantageous for tissue engineering since the physicochemical properties such as porosity, pore size, solubility and biocompatibility can be controlled. Therefore, research works were carried out to explore the potential of various materials in producing bone scaffolds in tissue engineering technology. As for both newcomers and experts, this review paper will help them to find information on various types of biomaterials in imparting or enhancing their biological properties such as biocompatibility, bacterial inhibition, tissue regeneration and cell growth, bio inertness, bioactive and resorbability for bone scaffold tissue engineering application.

The knowledge related to angiogenesis has grown exponentially over the past few decades with the recognition that angiogenesis is essential for numerous normal and pathological processes. Very importantly, angiogenesis is required for the growth and metastasis of solid tumors in human beyond the size of 1–2 mm³ (Arap et al. 1998; Folkman 1990, 1995). Angiogenesis is the process of developing buds and outgrowth of capillaries from existing blood vessels that are derived as extensions due to hypoxia or other forms of signaling that occurs in the microenvironment that surrounds a tissue or tumors. On the other hand neovascularization is defined as the formation of functional microvascular network with red blood cell perfusion. Angiogenesis is required for invasive tumor growth and metastasis and therefore constitutes an important step in the control of cancer progression. In general vascular tumors are severely restricted in their growth potential because of the lack of a blood supply. To achieve the new blood vessel formation, endothelial cells must first escape from their stable location by breaking through the basement membrane, and this degradation is associated with migration of endothelial cells out of the vascular channel toward the angiogenic stimulus. During this process, the subendothelial basement membrane, a dense meshwork of collagen, glycoproteins, and proteoglycans are proteolytically disrupted to allow formation of new capillaries. Though it is an integral component of normal processes such as reproduction and wound healing, angiogenesis is known to play an important role in other pathological processes ranging from tumor growth and metastasis to inflammation and ocular diseases. During angiogenesis tumor cells exploit their microenvironment by releasing cytokines and growth factors to activate normal, quiescent cells around them and initiate a cascade of events that quickly becomes dysregulated. For example, tumor cell-released vascular endothelial growth factor (VEGF) stimulates the sprouting and proliferation of endothelial cells and thereby play a crucial role in neovascularization of solid tumors (Leung et al. 1989). The expression of VEGF has been shown to correlate with the density of microvessels in various tumors and exhibit higher metastatic ability (Folkman 1995; Leung et al. 1989; Toi et al. 1994). Therefore, inhibition of angiogenesis triggered by VEGF or other factors is accepted as a valuable approach to cancer therapy.

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Tracheal replacement

The formation of microvascular networks plays essential roles in regenerative medicine and tissue engineering. Nevertheless, the self-organization mechanisms underlying the dynamic morphogenic process are poorly understood due to a paucity of effective tools for mapping the spatiotemporal dynamics of single cell behaviors. By establishing a single cell nanobiosensor along with live cell imaging, we perform dynamic single cell analysis of the morphology, displacement, and gene expression during microvascular self-organization. Dynamic single cell analysis reveals that endothelial cells self-organize into subpopulations with specialized phenotypes to form microvascular networks and identifies the involvement of Notch1-Dll4 signaling in regulating the cell subpopulations. The cell phenotype correlates with the initial Dll4 mRNA expression level and each subpopulation displays a unique dynamic Dll4 mRNA expression profile. Pharmacological perturbations and RNA interference of Notch1-Dll4 signaling modulate the cell subpopulations and modify the morphology of the microvascular network. Taken together, a nanobiosensor enables a dynamic single cell analysis approach underscoring the importance of Notch1-Dll4 signaling in microvascular self-organization.

See related article, pages 466–475 Blood vessel walls are built and maintained by 2 interdependent cell types, endothelial cells and mural cells. Signals exchanged between these 2 cell types control the formation, maturation, remodeling, and function of microvascular networks. Mural cells are either pericytes in the microvasculature or smooth muscle cells (SMCs) in larger vessels. Interactions between endothelial cells and mural cells serve to stabilize nascent capillary vessels, provide endothelial cell survival factors, inhibit endothelial cell proliferation, promote mural cell differentiation, and guide vascular network remodeling.1,2 Yet precisely how endothelial cells recruit mural cells to form a functional vessel wall is still incompletely understood. The initial steps in this process of vessel wall formation, called investment, involve the establishment of cell-cell contacts between endothelium and incoming mural cells, which triggers a self-reinforcing mechanism to maintain the contacts and initiate the next phase of vessel wall maturation. N-cadherin appears to be important for heterotypic cell-cell adhesion between endothelial cells and nascent SMCs in the embryonic day 12.5 dorsal aorta.3 As vascular development proceeds, an iterative investment sequence is set into motion, as is evident by the multilayered structure of the tunica media that is produced in large vessels. In the microvasculature, communication between endothelial cells and mural cells prevents vascular regression and promotes maturation processes in both cell types.2 Investment of microvessels with pericytes is a reversible process that is highly responsive to changes in the rate and direction of blood flow.4 Although these basic steps of vessel wall investment have been known for many years, the molecular mechanisms that are responsible for morphogenesis of this structure are still not well understood. In this issue of Circulation Research , a report by Liu et al begins to identify some of the key early players in this …

3-D bioprinting technologies in tissue engineering and regenerative medicine: Current and future trends

There is critical unmet need for new vascularized tissues to support or replace injured tissues and organs. Various synthetic and natural materials were already established for use of two-dimensional (2D) and three-dimensional (3D) in vitro neovascularization assays, however, they still cannot mimic the complex functions of the sum of the extracellular matrix (ECM) in native intact tissue. Currently, this issue is only addressed by artificial products such as Matrigel™, which comprises a complex mixture of ECM proteins, extracted from animal tumor tissue. Despite its outstanding bioactivity, the isolation from tumor tissue hinders its translation into clinical applications. Since nonhuman ECM proteins may cause immune reactions, as are frequently observed in clinical trials, human ECM proteins represent the best option when aiming for clinical applications. Here, we describe an effective method of isolating a human placenta substrate (hpS) that induces the spontaneous formation of an interconnected network of green fluorescence-labeled human umbilical vein endothelial cells (gfpHUVECs) in vitro. The substrate was biochemically characterized by using a combination of bicinchoninic acid (BCA) assay, DNA, and glycosaminoglycan (GAG) content assays, sodium dodecyl sulfate/polyacrylamide gel electrophoresis (SDS-PAGE) analysis and Western blot, angiogenesis arrays, chromatographic thrombin detection, high performance liquid chromatography (HPLC)-based amino acid quantification analysis, and assessment of antimicrobial properties. 2D in vitro cell culture experiments have been performed to determine the vasculogenic potential of hpS, which demonstrated that cell networks developed on hpS show a significantly higher degree of complexity (number of tubules/junctions; total/mean tube length) when compared with Matrigel. As 3D cell culture techniques represent a more accurate representation of the in vivo condition, the substrate was 3D solidified using various natural polymers. 3D in vitro vasculogenesis assays have been performed by seeding gfpHUVECs in an hpS-fibrinogen clot. In conclusion, hpS provides a potent human/material-based alternative to xenogenic-material-based biomaterials for vascularization strategies in tissue engineering.

Three-dimensional spheroids of adipose-derived mesenchymal stem cells are potent initiators of blood vessel formation in porous polyurethane scaffolds