Integrating microfluidic and bioprinting technologies: advanced strategies for tissue vascularization.

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.

Progress in Organ Bioprinting for Regenerative Medicine

Bio-printing is a trending technology in the field of tissue engineering and regenerative medicine, creating complex three-dimensional structures. The advancements in bio-printing, including the steps and materials used in bio-printers, have been highlighted. Various bio-printing techniques such as micro-extrusion, inkjet printing, and laser-based approaches are discussed. Moreover, the roles of biomaterials and their importance in bio-printing cannot be overstated. Characteristics such as cell viability, adhesion, differentiation, biodegradability, and biocompatibility are paramount in ensuring the success of bio-printed tissues. Additionally, the interdisciplinary nature of bio-printing research fosters collaborations between biologists, engineers, material scientists, and medical professionals. This synergy facilitates the development of innovative bio-printing technologies and accelerates their translation into clinical applications. Furthermore, the evolution of bio-printing technology is not only reshaping medical practice but also opening new frontiers in scientific exploration. By mimicking the complex architectures and functionalities of native tissues, bio-printed constructs serve as valuable tools for studying tissue development, disease mechanisms, and drug responses. The ability to recreate physiologically relevant microenvironments in vitro provides researchers with unprecedented insights into cellular behavior and tissue dynamics. The bio-printing represents a transformative approach in tissue engineering and regenerative medicine, offering unparalleled opportunities for tissue fabrication, disease modeling, and drug discovery. As research in this field continues to progress, we can anticipate even more remarkable breakthroughs that will revolutionize healthcare and biomedical research.

Abstract: Bioprinting technology has emerged as a powerful tool for building tissue and organ structures for drug discovery and regenerative medicine applications. In general, bioprinting uses a computer-controlled three-dimensional (3D) printing device to accurately deposit cells and biomaterials into precise geometries with the goal of creating anatomically correct biological structures. While traditional 3D printing uses metals, plastics, and polymers as printing materials or "ink", bioprinting deals with living cells and biological matrix. Hence, there are significant challenges to make a transition from traditional 3D printing to bioprinting, and ultimately achieve functional outcomes in bioprinted tissues. Therefore, it is critical that there is new technology development and in-depth basic research in bioprinted tissues, such as developing novel biomaterials specifically for use in bioprinting and biofabrication techniques, understanding the cell–matrix remodeling for the desired mechanical properties, and functional outcomes, establishing proper vascular perfusion, etc. Currently, there is active research going on bioprinting technology and its potential as a future source for tissue implants. This review paper overviews the current state of the art in bioprinting technology and focuses on the outcomes of the bioprinted tissues and their potential applications in drug discovery and regenerative medicine. Current challenges and limitations are highlighted, and future directions for next-generation bioprinting technology are also presented. Keywords: 3D bioprinting, vascularization, tissue regeneration, 3D tissue model, biofabrication, regenerative medicine

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.

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 technology provides effective alternative treatments for tracheal reconstruction. The formation of a functional microvascular network is essential to support cell metabolism and ensure the long-term survival of grafts. Although several tracheal replacement therapy strategies have been developed in the past, the critical significance of the formation of microvascular networks in 3Dscaffolds has not attracted sufficient attention. Here, we review key technologies and related factors of microvascular network construction in tissue-engineered trachea and explore optimized preparation processes of vascularized functional tissues for clinical applications.

Bioprinting for stem cell research

The reconstruction of dermal blood vessels is essential for skin regeneration process. However, the lack of vascular structure, insufficient angiogenesis induction, and ineffective graft-host anastomosis of the existing skin substitutes are major bottle-necks for permanent skin replacement in tissue engineering. In this study, the uniform strontium silicate (SS) microcylinders are successfully synthesized and integrated into the biomaterial ink to serve as stable cell-induced factors for angiogenesis, and then a functional skin substitute based on a vascularization-induced biomimetic multicellular system is prepared via a "cell-writing" bioprinting technology. With an unprecedented combination of vascularized skin-mimicking structure and vascularization-induced function, the SS-containing multicellular system exhibits outstanding angiogenic activity both in vitro and in vivo. As a result, the bioprinted skin substitutes significantly accelerate the healing of both acute and chronic wounds by promoting the graft-host integration and vascularized skin regeneration in three animal models. Therefore, the study provides a referable strategy to fabricate biomimetic multicellular constructs with angiogenesis-induced function for regeneration of vascularized complex and hierarchical tissues.

SummaryThree-dimensional (3D) printing, or additive manufacturing, is transforming pharmaceutical and biomedical fields by enabling personalized medicine. This review highlights advances in 3D printing for customized drug delivery systems, including patient-specific dosage forms, multidrug polypills, and implantable devices that improve adherence and therapeutic outcomes—especially for pediatric and geriatric populations. The intersection of 3D printing with regenerative medicine is also explored, focusing on bioprinting technologies, stem cell-laden scaffolds, and smart biomaterials such as hydrogels and bioinks for localized therapy and tissue repair. These strategies reflect an expanded vision of personalized medicine, merging individualized pharmacotherapy with tissue engineering. Additionally, the review discusses the integration of artificial intelligence, nano-enabled platforms, and decentralized pharmaceutical manufacturing to accelerate development and access. Key regulatory and technical challenges are outlined, along with future directions to promote the clinical translation and scalability of 3D-printed pharmaceutical and regenerative systems.

Novel therapies resulting from regenerative medicine and tissue engineering technology may offer new hope for patients with injuries, end-stage organ failure, degenerative disorders and many other clinical issues. Currently, patients suffering from diseased and injured organs are treated with transplanted organs. However, there is a shortage of donor organs that is worsening yearly as the population ages and new cases of organ failure increase. Scientists in the field of regenerative medicine and tissue engineering are now applying the principles of cell transplantation, material science, and bioengineering to construct biological substitutes that can restore and maintain normal function in diseased and injured tissues. In addition, the stem cell field is a rapidly advancing part of regenerative medicine, and new discoveries in this field create new options for this type of therapy. For example, new types of stem cells, such as amniotic fluid and placental stem cells, that can circumvent the ethical issues associated with embryonic stem cells have been discovered. The process of somatic cell nuclear transfer and the creation of induced pluripotent cells provide still other potential sources of stem cells for cell-based tissue engineering applications. While stem cells are still in the research phase, some therapies arising from tissue engineering endeavors that make use of autologous, adult cells have already entered the clinical setting, indicating that regenerative medicine holds much promise for the future.

Event Abstract Back to Event Functional epilepsy modelling – a novel platform for studying human stem cell -derived neuronal networks Anssi Pelkonen1*, Lassi Sukki2, Tomi Ryynänen3, Tanja Hyvärinen1, Jukka Lekkala3, Pasi Kallio2 and Susanna Narkilahti1 1 University of Tampere, BioMediTech and Faculty of Medicine and Life Sciences, Finland 2 Tampere University of Technology, Faculty of Biomedical Sciences and Engineering, Finland 3 Tampere University of Technology, Faculty of Biomedical Sciences and Engineering, Finland Motivation Epilepsy is characterized by spread of abnormal electrical activity within and between neuronal networks. New epilepsy treatments are tested in rodent models, but less than 10 % of putative drugs are accepted for use[1] and 30 % of epilepsy patients still do not respond to existing treatments[2]. Therefore, new models are needed for epilepsy modelling and drug testing. Human pluripotent stem cell (hPSC) –derived neural networks are the most promising base material for such models, but they alone cannot model the connections, feedback loops and spread of abnormal activity between neuronal networks. Material and Methods Our solution is an innovative new platform which combines human stem cell –derived neural networks, microfluidic devices and microelectrode array (MEA) technology. hPSC–derived cortical neurons are cultured in the platform where they form functional networks. The networks are cultured in a microfluidic cell culture device which isolates the stem cell derived networks and the axonal connections between them. The device is comprised of polydimethylsiloxane (PDMS), and shaped using a micro- and millimeter-scale mold which consists of SU-8 and stainless steel (3D printed using selective laser melting (SLM)). The device is connected to a custom made MEA chip for measuring the electrical activity within and between the networks. The MEA chip is compatible with a commercially available MEA system (MEA2100, Multi Channel Systems, Reutlingen, Germany). Results The produced neuronal networks are highly active and respond to glutamatergic and GABAergic factors. The PDMS device allows the formation of axonal connections between separated neural networks. The PDMS device and the MEA chip are a suitable environment for the neural networks, which remained viable and active up to 12 weeks. The MEA data shows that the network activity in the device develops into synchronous, network-to-network connected bursts in 3-5 weeks. Individual networks can be subjected to seizure-inducing drugs, and the effects of the drug treatments are clearly visible in the MEA-data. Conclusion The platform is suitable for studying network activity and connectivity of human stem cell –derived neural networks. Therefore, it has the potential to be used in disease modelling and drug testing for epilepsy and other central nervous system disorders. Acknowledgements Funding: Business Finland (formerly known as Finnish Funding Agency for Technology and Innovation (TEKES)); Academy of Finland (Decision No. 311017); Finnish Cultural Foundation (personal grant, TR); Juliana von Wendt fund (personal grant, AP). The work was supported by the Imaging Facility, Facility of Electrophysiological Measurements and iPS Cells Facility (Uni. Tampere). Thanks to Arla Tanner for technical assistance and Juha Heikkilä for assistance with measurement and analysis software.

Angiogenesis, the formation of new capillaries from existing ones, is a fundamental process in regenerative medicine and tissue engineering. While it is known to be affected by circadian rhythms in vivo, its peripheral regulation within the vasculature and the role it performs in regulating the interplay between vascular cells have not yet been investigated. Peripheral clocks within the vasculature have been described in the endothelium and in smooth muscle cells. However, to date, scarce evidence has been presented regarding pericytes, a perivascular cell population deeply involved in the regulation of angiogenesis and vessel maturation, as well as endothelial function and homeostasis. More crucially, pericytes are also a promising source of cells for cell therapy and tissue engineering. Here, we established that human primary pericytes express key circadian genes and proteins in a rhythmic fashion upon synchronization. Conversely, we did not detect the same patterns in cultured endothelial cells. In line with these results, pericytes’ viability was disproportionately affected by circadian cycle disruption, as compared to endothelial cells. Interestingly, endothelial cells’ rhythm could be induced following exposure to synchronized pericytes in a contact co-culture. We propose that this mechanism could be linked to the altered release/uptake pattern of lactate, a known mediator of cell-cell interaction which was specifically altered in pericytes by the knockout of the key circadian regulator Bmal1. In an angiogenesis assay, the maturation of vessel-like structures was affected only when both endothelial cells and pericytes did not express Bmal1, indicating a compensation system. In a 3D tissue engineering scaffold, a synchronized clock supported a more structured organization of cells around the scaffold pores, and a maturation of vascular structures. Our results demonstrate that pericytes play a critical role in regulating the circadian rhythms in endothelial cells, and that silencing this system disproportionately affects their pro-angiogenic function. Particularly, in the context of tissue engineering and regenerative medicine, considering the effect of circadian rhythms may be critical for the development of mature vascular structures and to obtain the maximal reparative effect.

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.

Microfibers, a type of long, thin, and flexible material, can be assembled into functional 3D structures by folding, binding, and weaving. As a novel spinning method, combining microfluidic technology and wet spinning, microfluidic spinning technology can precisely control the size, morphology, structure, and composition of the microfibers. Particularly, the process is mild and rapid, which is suitable for preparing microfibers using biocompatible materials and without affecting the viability of cells encapsulated. Furthermore, owing to the controllability of microfluidic spinning, microfibers with well-defined structures (such as hollow structures) will contribute to the exchange of nutrients or guide cell orientation. Thus, this method is often used to fabricate microfibers as cell scaffolds for cell encapsulation or adhesion and can be further applied to biomimetic fibrous tissues. In this review, the focus is on different fiber structures prepared by microfluidic spinning technology, including solid, hollow, and heterogeneous structures, generated from three essential elements: spinning platform, fiber composition, and solidification methods. Furthermore, the application of microfibers is described with different structures in tissue engineering, such as blood vessels, skeletal muscle, bone, nerves, and lung bronchi. Finally, the challenges and future development prospects of microfluidic spinning technology in tissue engineering applications are discussed.