Beyond Islets: A Comprehensive Review of Vascularization Strategies in Tissue Engineering and Regenerative Medicine

Beyond Islets: A Comprehensive Review of Vascularization Strategies in Tissue Engineering and Regenerative Medicine

Abstract

Vascularization, the formation of functional blood vessel networks, is a critical bottleneck in the advancement of tissue engineering and regenerative medicine. The successful integration and long-term survival of engineered tissues and organs necessitate adequate nutrient and oxygen supply, waste removal, and the establishment of dynamic interactions with the host vasculature. While the importance of vascularization is well-established, particularly in the context of islet transplantation, the inherent complexities of replicating the intricate microvasculature found in native tissues present significant challenges. This review provides a comprehensive overview of vascularization strategies employed in tissue engineering, moving beyond the specific context of islet cells to examine the broader landscape of approaches used across diverse tissue types. We delve into various in vitro and in vivo techniques for promoting angiogenesis and vasculogenesis, highlighting the advantages and limitations of each. Furthermore, we discuss the challenges associated with maintaining stable and functional vascular networks, including addressing issues related to vessel maturation, permeability, and immune rejection. Finally, we explore the multifaceted impact of vascularization on cell survival, differentiation, and functionality within engineered tissues, focusing on the interplay between biomechanical cues, growth factors, and cellular interactions. We conclude by identifying key areas for future research and development that hold promise for overcoming current limitations and ultimately realizing the full potential of vascularized tissue engineering for clinical applications.

1. Introduction

Tissue engineering and regenerative medicine strive to repair or replace damaged or diseased tissues and organs using a combination of cells, biomaterials, and growth factors. A major obstacle in this field is the creation of functional, three-dimensional (3D) constructs with sufficient vascularization. The limited diffusion distance of oxygen and nutrients restricts cell survival to within approximately 100-200 μm of a blood vessel. Therefore, the creation of robust and perfused vascular networks within engineered tissues is paramount for their long-term viability and function. While the necessity of vascularization is universally acknowledged, its effective implementation remains a significant hurdle across various tissue engineering applications.

The complexities arise from the multifaceted requirements for functional vasculature. It’s not simply about creating blood vessels; it’s about creating vessels that are stable, permeable enough for nutrient exchange but not excessively leaky, responsive to physiological cues (e.g., metabolic demand), and integrated with the host circulatory system. Moreover, the optimal vascular architecture varies significantly depending on the target tissue. For instance, the sinusoidal capillaries in the liver differ drastically from the glomerular capillaries in the kidney or the microvasculature within bone. Thus, a one-size-fits-all approach to vascularization is unlikely to be successful. The field has witnessed an evolution of vascularization strategies, ranging from simple co-culture models to sophisticated microfabrication techniques and in vivo pre-vascularization approaches. This review aims to provide an in-depth exploration of these methods, highlighting their strengths, weaknesses, and future directions.

2. Biological Principles of Vascularization: Angiogenesis and Vasculogenesis

Vascularization occurs through two primary mechanisms: angiogenesis and vasculogenesis. Understanding the nuances of each is crucial for designing effective vascularization strategies.

Angiogenesis refers to the formation of new blood vessels from pre-existing vasculature. This process is tightly regulated by a balance of pro-angiogenic and anti-angiogenic factors. Vascular endothelial growth factor (VEGF) is a key regulator of angiogenesis, stimulating endothelial cell proliferation, migration, and tube formation. Other important factors include platelet-derived growth factor (PDGF), fibroblast growth factor (FGF), and angiopoietins. Angiogenesis proceeds through several distinct steps: (1) activation of endothelial cells in existing vessels in response to pro-angiogenic stimuli; (2) degradation of the basement membrane surrounding the existing vessel; (3) proliferation and migration of endothelial cells towards the angiogenic stimulus; (4) formation of new capillary sprouts; (5) anastomosis of sprouts to form loops; (6) recruitment of pericytes and smooth muscle cells to stabilize the newly formed vessels; and (7) remodeling of the vascular network. While VEGF is often considered the main driver, the coordinated action of multiple growth factors and signaling pathways is essential for proper angiogenesis. The absence, or over abundance, of any of these factors can lead to disfunctional vasculature.

Vasculogenesis, on the other hand, involves the de novo formation of blood vessels from endothelial progenitor cells (EPCs). EPCs originate in the bone marrow and circulate in the peripheral blood. Under the influence of growth factors and cytokines, EPCs differentiate into endothelial cells and coalesce to form primitive vascular networks. Vasculogenesis is the dominant mechanism during embryonic development but also contributes to vascularization in adult tissues, particularly during wound healing and tumor angiogenesis. There has been some debate in the field as to how important vasculogenesis is in adults, and the extent to which purported EPCs are true endothelial progenitors versus simply circulating hematopoietic cells with some endothelial characteristics. Nonetheless, harnessing the potential of EPCs to seed engineered tissues with endothelial cells remains a promising approach.

Furthermore, intussusceptive angiogenesis (also known as splitting angiogenesis) is a less well-understood mechanism of vessel formation. Unlike sprouting angiogenesis, intussusception involves the splitting of an existing vessel into two without the formation of new endothelial cells. Septa containing interstitial tissue push into the vessel lumen, dividing it into smaller vessels. This process is particularly important for increasing the density of existing vascular networks, often seen in lung development and tumor vascularization. Combining strategies to promote both sprouting and intussusceptive angiogenesis may lead to the creation of more efficient and functional vascular networks.

3. In Vitro Vascularization Strategies

Creating vascularized tissues in vitro presents unique challenges. The controlled environment allows for precise manipulation of cellular and molecular cues, but replicating the complex 3D architecture and dynamic interactions of native vasculature is difficult. Several strategies have been developed to address these challenges, each with its own strengths and limitations.

3.1. Co-Culture Systems:

Co-culturing endothelial cells (ECs) with other cell types, such as fibroblasts, smooth muscle cells, or pericytes, is a simple and widely used approach to promote vascularization in vitro. The non-endothelial cells provide structural support and secrete growth factors that stimulate EC proliferation, migration, and tube formation. For example, co-culturing ECs with fibroblasts in a collagen gel matrix leads to the formation of capillary-like structures. The specific cell types and ratios used in the co-culture system can be tailored to mimic the microenvironment of the target tissue. However, these systems often lack the spatial control and complexity of native vasculature.

3.2. Microfluidic Devices:

Microfluidic devices offer precise control over the microenvironment, allowing for the creation of complex vascular networks in vitro. These devices typically consist of microchannels that are used to seed cells, deliver nutrients, and apply shear stress. ECs can be seeded into these microchannels and stimulated to form perfusable microvessels. Microfluidic devices can also be used to study the effects of different growth factors, shear stress levels, and biomaterial properties on vascularization. Moreover, these devices are increasingly being used to model disease states, such as tumor angiogenesis. One of the main challenges with microfluidic devices is scaling them up to create larger, clinically relevant tissues. While several parallelization strategies have been developed, they often come with increased complexity in the design and fabrication.

3.3. Bioprinting:

Bioprinting is an emerging technology that allows for the precise deposition of cells and biomaterials in a layer-by-layer fashion. This technology can be used to create complex 3D structures with defined vascular architecture. ECs can be bioprinted into specific patterns to form microvessels, which can then be perfused with culture medium. Bioprinting offers a high degree of spatial control over vascularization, allowing for the creation of tissue-specific vascular networks. However, bioprinting is still a relatively new technology, and challenges remain in terms of cell viability, resolution, and scalability. The bioinks themselves also need to be carefully designed to provide adequate support for the cells and promote vascularization. Furthermore, ensuring the long-term stability and functionality of bioprinted vascular networks remains a challenge.

3.4. Decellularized Matrices:

Decellularized matrices (DCMs) are derived from native tissues by removing all cellular components while preserving the extracellular matrix (ECM) architecture and composition. DCMs provide a natural scaffold for cell attachment, proliferation, and differentiation. Seeding ECs into decellularized scaffolds can promote vascularization by providing a biomimetic environment that supports EC adhesion, migration, and tube formation. DCMs can be derived from a variety of tissues, including blood vessels, heart, and liver, providing a tissue-specific microenvironment for vascularization. However, the decellularization process can alter the ECM structure and composition, which can affect cell behavior. Furthermore, removing all cellular components while maintaining the structural integrity of the ECM can be technically challenging. Careful optimization of the decellularization protocol is crucial to ensure that the DCM retains its bioactivity and supports vascularization. The use of human-derived decellularized matrices is often preferred to remove the issue of xenograft rejection, but it is challenging to create these matrices due to scarcity of tissue.

4. In Vivo Vascularization Strategies

While in vitro models offer valuable insights into vascularization, ultimately, engineered tissues must be integrated with the host vasculature in vivo. Several strategies have been developed to promote in vivo vascularization, ranging from direct implantation of cell-seeded scaffolds to pre-vascularization techniques.

4.1. Scaffold Design and Material Properties:

The design and material properties of the scaffold play a crucial role in promoting in vivo vascularization. Scaffolds with interconnected pores allow for cell infiltration and vascular ingrowth. The pore size and architecture of the scaffold should be optimized to promote both cell attachment and angiogenesis. The scaffold material should also be biocompatible and biodegradable, allowing for gradual replacement by newly formed tissue. Several materials have been used for tissue engineering scaffolds, including collagen, gelatin, alginate, and synthetic polymers such as poly(lactic-co-glycolic acid) (PLGA). The choice of material depends on the specific application and the desired mechanical and degradation properties. In recent years, the incorporation of bioactive molecules, such as growth factors or cell-adhesive peptides, into the scaffold has been shown to further enhance vascularization. For instance, incorporating VEGF into a PLGA scaffold can promote angiogenesis and improve the survival of implanted cells.

4.2. Growth Factor Delivery:

Controlled delivery of growth factors, particularly VEGF, is a common strategy to promote angiogenesis in vivo. Growth factors can be delivered locally via direct injection, encapsulation within microspheres, or incorporation into the scaffold material. The release kinetics of the growth factor can be tailored to optimize its angiogenic effect. For example, a sustained release of VEGF may be more effective than a bolus injection in promoting long-term vascularization. Gene therapy approaches can also be used to deliver growth factors. In this approach, cells are transfected with a gene encoding a pro-angiogenic factor, which is then expressed by the cells in vivo. While growth factor delivery can be effective in promoting angiogenesis, it is important to carefully control the dosage and timing of delivery to avoid unwanted side effects, such as excessive vascular permeability or tumor growth.

4.3. Angiogenic Cell Transplantation:

Transplantation of angiogenic cells, such as EPCs or mesenchymal stem cells (MSCs), can promote vascularization in vivo. These cells secrete pro-angiogenic factors and can differentiate into endothelial cells, contributing to the formation of new blood vessels. Angiogenic cells can be delivered directly to the implantation site or seeded onto the scaffold prior to implantation. MSCs are particularly attractive for cell-based therapies due to their ease of isolation, high proliferation rate, and immunomodulatory properties. However, the survival rate of transplanted cells is often low, which limits their therapeutic efficacy. Strategies to improve cell survival, such as preconditioning cells with growth factors or genetically modifying them to express anti-apoptotic proteins, are being actively investigated.

4.4. Arteriovenous (AV) Loop Anastomosis:

AV loop anastomosis is a surgical technique that involves creating a direct connection between an artery and a vein. This creates a high-flow vascular pedicle that can be used to vascularize an engineered tissue. The engineered tissue is wrapped around the AV loop, which provides a source of nutrients and oxygen. Over time, the engineered tissue becomes vascularized by the ingrowth of blood vessels from the AV loop. AV loop anastomosis is a well-established technique that has been used to vascularize a variety of tissues, including skin, muscle, and bone. However, it is a complex surgical procedure that requires specialized expertise. Furthermore, the size and shape of the engineered tissue are limited by the size and location of the AV loop. The AV loop can also be prone to thrombosis or stenosis, which can compromise the vascularization of the engineered tissue.

4.5. Pre-Vascularization Strategies:

Pre-vascularization involves creating a vascular network within the engineered tissue prior to implantation. This can be achieved by implanting the tissue into a highly vascularized site, such as the omentum or the subcutaneous space, for a period of time before transplanting it to the final target location. The pre-vascularized tissue is then harvested and transplanted to the desired site. Pre-vascularization can improve the survival and integration of the engineered tissue by providing a pre-existing vascular network that can quickly connect with the host vasculature. However, pre-vascularization requires a second surgical procedure to harvest the pre-vascularized tissue, which can increase the morbidity and cost of the procedure. The degree of vascularization achieved during the pre-vascularization period can also be variable, depending on the implantation site and the duration of pre-vascularization.

5. Challenges in Maintaining Stable Vascular Networks

Creating functional vascular networks is not simply about inducing angiogenesis; it’s also about maintaining the stability and integrity of those networks over the long term. Several factors can contribute to vascular instability, including vessel regression, excessive permeability, and immune rejection.

5.1. Vessel Regression:

Vessel regression is the process by which newly formed blood vessels are pruned or eliminated. This can occur due to a lack of sufficient pericyte coverage, inadequate growth factor stimulation, or unfavorable hemodynamic conditions. Vessel regression can compromise the perfusion of the engineered tissue, leading to cell death and tissue failure. Strategies to prevent vessel regression include promoting pericyte recruitment, providing sustained growth factor stimulation, and optimizing the mechanical properties of the scaffold.

5.2. Vascular Permeability:

Excessive vascular permeability can lead to edema, inflammation, and impaired tissue function. Vascular permeability is regulated by a variety of factors, including endothelial cell junctions, glycocalyx, and inflammatory mediators. Strategies to reduce vascular permeability include strengthening endothelial cell junctions, restoring the glycocalyx, and inhibiting inflammatory signaling pathways. Some studies suggest that carefully tuning the pore size of the engineered tissues can also reduce permeability.

5.3. Immune Rejection:

Immune rejection is a major challenge in tissue engineering, particularly when using allogeneic or xenogeneic cells or tissues. The immune system can recognize the engineered tissue as foreign and mount an immune response, leading to graft rejection and vascular destruction. Strategies to prevent immune rejection include using autologous cells, immunosuppression, and encapsulation of the engineered tissue in a biocompatible membrane. Modifying the surface of endothelial cells to express immune checkpoint ligands such as PD-L1 can also prevent the recruitment of T cells and therefore reduce rejection.

5.4. Mechanical Cues and Hemodynamics:

The mechanical environment plays a critical role in vascular stability. Shear stress, generated by blood flow, influences endothelial cell alignment, gene expression, and secretion of vasoactive factors. Insufficient or irregular shear stress can lead to vascular remodeling and instability. The mechanical properties of the scaffold also influence vascularization. Stiff scaffolds can promote angiogenesis but can also lead to vessel compression and impaired perfusion. Conversely, soft scaffolds may not provide sufficient support for vascular network formation. Balancing the mechanical properties of the scaffold to provide adequate support without compromising vessel function is a key consideration.

6. Impact of Vascularization on Cell Survival, Differentiation, and Function

The presence of a functional vascular network has a profound impact on the survival, differentiation, and function of cells within engineered tissues. Adequate perfusion provides cells with the nutrients and oxygen they need to survive and thrive. Vascularization also facilitates the removal of waste products, preventing the accumulation of toxic metabolites. The interplay between vascularization and cellular processes is complex and multifaceted.

6.1. Cell Survival:

Vascularization is essential for cell survival in engineered tissues. The limited diffusion distance of oxygen and nutrients restricts cell viability to within approximately 100-200 μm of a blood vessel. Without adequate vascularization, cells in the center of the tissue construct will undergo necrosis or apoptosis. Vascularization can improve cell survival by providing a direct supply of oxygen and nutrients to the cells. Furthermore, vascularization can promote the formation of new tissue by providing a scaffold for cell attachment and migration.

6.2. Cell Differentiation:

Vascularization can influence cell differentiation by providing a microenvironment that supports cell-specific differentiation pathways. For example, the presence of endothelial cells can promote the differentiation of stem cells into endothelial cells. Vascularization can also provide a source of growth factors and cytokines that can stimulate cell differentiation. The interplay between endothelial cells and other cell types is crucial for proper tissue development and function. The lack of vascularization can lead to aberrant differentiation or dedifferentiation of cells, compromising the functionality of the engineered tissue. For instance, in bone tissue engineering, adequate vascularization is crucial for osteoblast differentiation and bone formation.

6.3. Cell Function:

Vascularization is critical for maintaining the function of engineered tissues. Adequate perfusion provides cells with the substrates they need to perform their specific functions. Vascularization also facilitates the removal of waste products, preventing the accumulation of toxic metabolites that can impair cell function. The absence of vascularization can lead to impaired tissue function or even tissue failure. In the context of engineered pancreatic islets, vascularization is essential for glucose-stimulated insulin secretion. Similarly, in engineered liver tissue, vascularization is crucial for drug metabolism and detoxification.

7. Future Directions and Conclusion

Vascularization remains a major challenge in tissue engineering and regenerative medicine, but significant progress has been made in recent years. Future research should focus on developing more sophisticated and biomimetic vascularization strategies that can replicate the complex architecture and dynamic interactions of native vasculature. Several key areas warrant further investigation:

• Advanced biomaterials: Developing biomaterials that actively promote angiogenesis and vasculogenesis by incorporating growth factors, cell-adhesive peptides, or ECM-derived components.
• Microfabrication and bioprinting: Improving the resolution and scalability of microfabrication and bioprinting techniques to create complex vascular networks with defined architecture and functionality.
• Immunomodulation: Developing strategies to modulate the immune response and prevent rejection of engineered tissues, particularly when using allogeneic or xenogeneic cells.
• Longitudinal in vivo imaging: Implementing non-invasive imaging techniques to monitor vascularization in vivo and assess the long-term stability and functionality of engineered tissues.
• Personalized vascularization: Tailoring vascularization strategies to the specific needs of the patient and the target tissue, taking into account factors such as age, disease state, and genetic background.

The successful vascularization of engineered tissues will ultimately depend on a multidisciplinary approach that combines advances in cell biology, biomaterials science, engineering, and surgery. By addressing the challenges outlined above, we can move closer to realizing the full potential of tissue engineering and regenerative medicine for treating a wide range of diseases and injuries. The integration of advanced technologies and a deeper understanding of the biological principles of vascularization will pave the way for the creation of functional, long-lasting engineered tissues and organs that can improve the lives of patients worldwide. While the focus has often been on a few angiogenic factors such as VEGF, more research is needed into the relative importance of other molecules, and how to effectively modulate these at the appropriate location and time. We also need more research into the role of hemodynamic forces and the local mechanochemical environment in guiding vascular network formation and stabilization. The integration of artificial intelligence and machine learning in the analysis of vascular networks is also expected to lead to breakthroughs in understanding how the vasculature adapts to different physiological needs. This approach can help us predict the long-term outcome of different vascularization strategies and design more robust and effective treatments.

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