Research Progress in the Field of Tumor Model Construction Using Bioprinting: A Review.

The development of therapeutic drugs and methods has been greatly facilitated by the emergence of tumor models. However, due to their inherent complexity, establishing a model that can fully replicate the tumor tissue situation remains extremely challenging. With the development of tissue engineering, the advancement of bioprinting technology has facilitated the upgrading of tumor models. This article focuses on the latest advancements in bioprinting, specifically highlighting the construction of 3D tumor models, and underscores the integration of these two technologies. Furthermore, it discusses the challenges and future directions of related techniques, while also emphasizing the effective recreation of the tumor microenvironment through the emergence of 3D tumor models that resemble in vitro organs, thereby accelerating the development of new anticancer therapies.

Bioprinting Cell-Laden Hydrogels for Studies of Epithelial Tissue Morphogenesis.

The extracellular matrix (ECM) provides dynamic structural and molecular signals that affect the form and function of developing tissues. In order to parse how the individual features of the ECM impact cell- and tissue-level behavior during development, engineered culture models should reproduce key structural and molecular features of native ECM. Here, we describe a protocol for bioprinting epithelial cell aggregates embedded within a collagen-Matrigel ink in order to study the dynamic interplay between epithelial tissues and aligned networks of type I collagen fibers. Collagen fiber alignment and geometry can be spatially controlled by modulating the printing speed, nozzle geometry, surface chemistry, and degree of molecular crowding in the printing ink. We provide detailed procedures for generating epithelial cell aggregates, microextrusion printing collagen-Matrigel bioinks, culturing the three-dimensional (3D)-printed tissues, and imaging 3D-printed collagen-Matrigel constructs.

Voxelated bioprinting of modular double-network bio-ink droplets.

Analogous of pixels to two-dimensional pictures, voxels-in the form of either small cubes or spheres-are the basic building blocks of three-dimensional objects. However, precise manipulation of viscoelastic bio-ink voxels in three-dimensional space represents a grand challenge in both soft matter science and biomanufacturing. Here, we present a voxelated bioprinting technology that enables the digital assembly of interpenetrating double-network hydrogel droplets made of polyacrylamide/alginate-based or hyaluronic acid/alginate-based polymers. The hydrogels are crosslinked via additive-free and biofriendly click reaction between a pair of stoichiometrically matched polymers carrying norbornene and tetrazine groups, respectively. We develop theoretical frameworks to describe the crosslinking kinetics and stiffness of the hydrogels, and construct a diagram-of-state to delineate their mechanical properties. Multi-channel print nozzles are developed to allow on-demand mixing of highly viscoelastic bio-inks without significantly impairing cell viability. Further, we showcase the distinctive capability of voxelated bioprinting by creating highly complex three-dimensional structures such as a hollow sphere composed of interconnected yet distinguishable hydrogel particles. Finally, we validate the cytocompatibility and in vivo stability of the printed double-network scaffolds through cell encapsulation and animal transplantation.

Single-Step 3D Bioprinting of Alginate-Collagen Type I Hydrogel Fiber Rings to Promote Angiogenic Network Formation.

In the advent of tissue engineering and regenerative medicine, the demand for innovative approaches to biofabricate complex vascular structures is increasing. We describe a single-step 3D bioprinting method leveraging Aspect Biosystems RX1 technology, which integrates the crosslinking step at a flow-focusing junction, to biofabricate immortalized adult rat brain endothelial cell (SV-ARBEC)-encapsulated alginate-collagen type I hydrogel rings. This single-step biofabrication process involves the strategic layer-by-layer assembly of hydrogel rings, encapsulating SV-ARBECs in a spatially controlled manner while optimizing access to media and nutrients. The spatial arrangement of the SV-ARBECs within the rings promotes spontaneous angiogenic network formation and the constrained deposition of cells within the hydrogel matrix facilitates tissue-like organized vascular-like network development. This approach provides a platform that can be adapted to many different endothelial cell types and leveraged to better understand the mechanisms driving angiogenesis and vascular-network formation in 3D bioprinted constructs supporting the development of more complex tissue and disease models for advancing drug discovery, tissue engineering, and regenerative medicine applications.

Overcoming big bottlenecks in vascular regeneration.

Bioengineering and regenerative medicine strategies are promising for the treatment of vascular diseases. However, current limitations inhibit the ability of these approaches to be translated to clinical practice. Here we summarize some of the big bottlenecks that inhibit vascular regeneration in the disease applications of aortic aneurysms, stroke, and peripheral artery disease. We also describe the bottlenecks preventing three-dimensional bioprinting of vascular networks for tissue engineering applications. Finally, we describe emerging technologies and opportunities to overcome these challenges to advance vascular regeneration.

Double crosslinked hyaluronic acid and collagen as a potential bioink for cartilage tissue engineering.

The avascular nature of hyaline cartilage results in limited spontaneous self-repair and regenerative capabilities when damaged. Recent advances in three-dimensional bioprinting have enabled the precise dispensing of cell-laden biomaterials, commonly referred to as 'bioinks', which are emerging as promising solutions for tissue regeneration. An effective bioink for cartilage tissue engineering needs to create a micro-environment that promotes cell differentiation and supports neocartilage tissue formation. In this study, we introduced an innovative bioink composed of photocurable acrylated type I collagen (COLMA), thiol-modified hyaluronic acid (THA), and poly(ethylene glycol) diacrylate (PEGDA) for 3D bioprinting cartilage grafts using human nasal chondrocytes. Both collagen and hyaluronic acid, being key components of the extracellular matrix (ECM) in the human body, provide essential biological cues for tissue regeneration. We evaluated three formulations - COLMA, COLMA+THA, and COLMA+THA+PEGDA - for their printability, cell viability, structural integrity, and capabilities in forming cartilage-like ECM. The addition of THA and PEGDA significantly enhanced these properties, showcasing the potential of this bioink in advancing applications in cartilage repair and reconstructive surgery.

[Research progress of three-dimensional bioprinting technology in auricle repair and reconstruction].

Objective: To review the research progress on the application of three-dimensional (3D) bioprinting technology in auricle repair and reconstruction. Methods: The recent domestic and international research literature on 3D printing and auricle repair and reconstruction was extensively reviewed, and the concept of 3D bioprinting technology and research progress in auricle repair and reconstruction were summarized. Results: The auricle possesses intricate anatomical structure and functionality, necessitating precise tissue reconstruction and morphological replication. Hence, 3D printing technology holds immense potential in auricle reconstruction. In contrast to conventional 3D printing technology, 3D bioprinting technology not only enables the simulation of auricular outer shape but also facilitates the precise distribution of cells within the scaffold during fabrication by incorporating cells into bioink. This approach mimics the composition and structure of natural tissues, thereby favoring the construction of biologically active auricular tissues and enhancing tissue repair outcomes. Conclusion: 3D bioprinting technology enables the reconstruction of auricular tissues, avoiding potential complications associated with traditional autologous cartilage grafting. The primary challenge in current research lies in identifying bioinks that meet both the mechanical requirements of complex tissues and biological criteria.

Nanomaterials-Based Hybrid Bioink Platforms in Advancing 3D Bioprinting Technologies for Regenerative Medicine.

3D bioprinting is recognized as the ultimate additive biomanufacturing technology in tissue engineering and regeneration, augmented with intelligent bioinks and bioprinters to construct tissues or organs, thereby eliminating the stipulation for artificial organs. For 3D bioprinting of soft tissues, such as kidneys, hearts, and other human body parts, formulations of bioink with enhanced bioinspired rheological and mechanical properties were essential. Nanomaterials-based hybrid bioinks have the potential to overcome the above-mentioned problem and require much attention among researchers. Natural and synthetic nanomaterials such as carbon nanotubes, graphene oxides, titanium oxides, nanosilicates, nanoclay, nanocellulose, etc. and their blended have been used in various 3D bioprinters as bioinks and benefitted enhanced bioprintability, biocompatibility, and biodegradability. A limited number of articles were published, and the above-mentioned requirement pushed us to write this review. We reviewed, explored, and discussed the nanomaterials and nanocomposite-based hybrid bioinks for the 3D bioprinting technology, 3D bioprinters properties, natural, synthetic, and nanomaterial-based hybrid bioinks, including applications with challenges, limitations, ethical considerations, potential solution for future perspective, and technological advancement of efficient and cost-effective 3D bioprinting methods in tissue regeneration and healthcare.

Scaffolding fundamentals and recent advances in sustainable scaffolding techniques for cultured meat development.

In cultured meat (CM) production, Scaffolding plays an important role by aiding cell adhesion, growth, differentiation, and alignment. The existence of fibrous microstructure in connective and muscle tissues has attracted considerable interest in the realm of tissue engineering and triggered the interest of researchers to implement scaffolding techniques. A wide array of research efforts is ongoing in scaffolding technologies for achieving the real meat structure on the principality of biomedical research and to replace serum free CM production. Scaffolds made of animal-derived biomaterials are found efficient in replicating the extracellular matrix (ECM), thus focus should be paid to utilize animal byproducts for this purpose. Proper identification and utilization of plant-derived scaffolding biomaterial could be helpful to add diversified options in addition to animal derived sources and reduce in cost of CM production through scaffolds. Furthermore, techniques like electrospinning, modified electrospinning and 3D bioprinting should be focused on to create 3D porous scaffolds to mimic the ECM of the muscle tissue and form real meat-like structures. This review discusses recent advances in cutting edge scaffolding techniques and edible biomaterials related to structured CM production.

Nylons with Applications in Energy Generators, 3D Printing and Biomedicine.

Linear polyamides, known as nylons, are a class of synthetic polymers with a wide range of applications due to their outstanding properties, such as chemical and thermal resistance or mechanical strength. These polymers have been used in various fields: from common and domestic applications, such as socks and fishing nets, to industrial gears or water purification membranes. By their durability, flexibility and wear resistance, nylons are now being used in addictive manufacturing technology as a good material choice to produce sophisticated devices with precise and complex geometric shapes. Furthermore, the emergence of triboelectric nanogenerators and the development of biomaterials have highlighted the versatility and utility of these materials. Due to their ability to enhance triboelectric performance and the range of applications, nylons show a potential use as tribo-positive materials. Because of the easy control of their shape, they can be subsequently integrated into nanogenerators. The use of nylons has also extended into the field of biomaterials, where their biocompatibility, mechanical strength and versatility have paved the way for groundbreaking advances in medical devices as dental implants, catheters and non-absorbable surgical sutures. By means of 3D bioprinting, nylons have been used to develop scaffolds, joint implants and drug carriers with tailored properties for various biomedical applications. The present paper aims to collect evidence of these recently specific applications of nylons by reviewing the literature produced in recent decades, with a special focus on the newer technologies in the field of energy harvesting and biomedicine.

Advanced optical assessment and modeling of extrusion bioprinting.

In the context of tissue engineering, biofabrication techniques are employed to process cells in hydrogel-based matrices, known as bioinks, into complex 3D structures. The aim is the production of functional tissue models or even entire organs. The regenerative production of biological tissues adheres to a multitude of criteria that ultimately determine the maturation of a functional tissue. These criteria are of biological nature, such as the biomimetic spatial positioning of different cell types within a physiologically and mechanically suitable matrix, which enables tissue maturation. Furthermore, the processing, a combination of technical procedures and biological materials, has proven highly challenging since cells are sensitive to stress, for example from shear and tensile forces, which may affect their vitality. On the other hand, high resolutions are pursued to create optimal conditions for subsequent tissue maturation. From an analytical perspective, it is prudent to first investigate the printing behavior of bioinks before undertaking complex biological tests. According to our findings, conventional shear rheological tests are insufficient to fully characterize the printing behavior of a bioink. For this reason, we have developed optical methods that, complementarily to the already developed tests, allow for quantification of printing quality and further viscoelastic modeling of bioinks.

3D-bioprinted GelMA/gelatin/amniotic membrane extract (AME) scaffold loaded with keratinocytes, fibroblasts, and endothelial cells for skin tissue engineering.

Gelatin-methacryloyl (GelMA) is a highly adaptable biomaterial extensively utilized in skin regeneration applications. However, it is frequently imperative to enhance its physical and biological qualities by including supplementary substances in its composition. The purpose of this study was to fabricate and characterize a bi-layered GelMA-gelatin scaffold using 3D bioprinting. The upper section of the scaffold was encompassed with keratinocytes to simulate the epidermis, while the lower section included fibroblasts and HUVEC cells to mimic the dermis. A further step involved the addition of amniotic membrane extract (AME) to the scaffold in order to promote angiogenesis. The incorporation of gelatin into GelMA was found to enhance its stability and mechanical qualities. While the Alamar blue test demonstrated that a high concentration of GelMA (20%) resulted in a decrease in cell viability, the live/dead cell staining revealed that incorporation of AME increased the quantity of viable HUVECs. Further, gelatin upregulated the expression of KRT10 in keratinocytes and VIM in fibroblasts. Additionally, the histological staining results demonstrated the formation of well-defined skin layers and the creation of extracellular matrix (ECM) in GelMA/gelatin hydrogels during a 14-day culture period. Our study showed that a 3D-bioprinted composite scaffold comprising GelMA, gelatin, and AME can be used to regenerate skin tissues.

Enhancing scaffold-free spheroid models: 3D cell bioprinting method for metastatic HSC3-Oral squamous carcinoma cell line.

3D in vitro systems offer advantages over the shortcomings of two-dimensional models by simulating the morphological and functional features of in vivo-like environments, such as cell-cell and cell-extracellular matrix interactions, as well as the co-culture of different cell types. Nevertheless, these systems present technical challenges that limit their potential in cancer research requiring cell line- and culture-dependent standardization. This protocol details the use of a magnetic 3D bioprinting method and other associated techniques (cytotoxicity assay and histological analysis) using oral squamous cell carcinoma cell line, HSC3, which offer advantages compared to existing widely used approaches. This protocol is particularly timely, as it validates magnetic bioprinting as a method for the rapid deployment of 3D cultures as a tool for compound screening and development of heterotypic cultures such as co-culture of oral squamous cell carcinoma cells with cancer-associated fibroblasts (HSC3/CAFs).

Revitalizing liver function in mice with liver failure through transplantation of 3D-bioprinted liver with expanded primary hepatocytes.

The utilization of three-dimensional (3D) bioprinting technology to create a transplantable bioartificial liver emerges as a promising remedy for the scarcity of liver donors. This study outlines our strategy for constructing a 3D-bioprinted liver, using in vitro-expanded primary hepatocytes recognized for their safety and enhanced functional robustness as hepatic cell sources for bioartificial liver construction. In addition, we have developed bioink biomaterials with mechanical and rheological properties, as well as printing capabilities, tailored for 3D bioprinting. Upon heterotopic transplantation into the mesentery of tyrosinemia or 90% hepatectomy mice, our 3D-bioprinted liver effectively restored lost liver functions, consequently extending the life span of mice afflicted with liver injuries. Notably, the inclusion of an artificial blood vessel in our 3D-bioprinted liver allowed for biomolecule exchange with host blood vessels, demonstrating, in principle, the rapid integration of the bioartificial liver into the host vascular system. This model underscores the therapeutic potential of transplantation for the treatment of liver failure diseases.

Alternative Therapies in Transplantology as a Promising Perspective in Medicine.

Despite continuous and rapid progress in the transplantation of cells, tissues, and organs, many patients die before receiving them. This is because of an insufficient number of donors, which leads to a significant disproportion between the need for donors and their availability. This review aims to present the possibilities offered by alternative therapies. We use the term "functional transplantology" to describe such alternative methods of transplantation that could help change the current state of transplantation medicine. Its purpose is not to replace a defective or removed organ with another but to replace its functions using complementary biological, mechanical, or biomechanical structures or devices. Implementation of many innovative solutions shown in the work for clinical applications is already a fact. In the case of others, it should be considered a future vision. We hope that the role of a defective or damaged tissue or a group of tissues will be taken over by different structures that are functionally complementary with the organ being substituted. Undoubtedly, developing the described methods based on functional transplantology will change the face of transplantation medicine. Thus, we show current trends and new directions of thinking and actions in transplantation medicine that combine technology and transplantology. The review considers the latest technologies, including 3D bioprinting, nanotechnology, cell encapsulation, and organoids. We discuss not only the advantages of new approaches but also the limitations and challenges that must be overcome to achieve significant progress in transplantation. That is the only option to provide a safe and efficient way of improving the quality of life of many patients.

Characterization of two different alginate-based bioinks and the influence of melanoma growth within.

Extrusion-based bioprinting is an established method in biofabrication. Suitable bioinks have fundamentally different compositions and characteristics, which should be examined, in order to find a perfect model system. Here, we investigate the effect of two alginate-based, yet unalike 3D-printed bioinks, pre-crosslinked alginate-dialdehyde gelatin (ADA-GEL) and a mixture of alginate, hyaluronic acid, and gelatin (Alg/HA/Gel), on the melanoma cell line Mel Im and vice versa in terms of stiffness, shrinkage, cellular behavior and colony formation over 15 days. Rheological stiffness measurements revealed two soft gels with similar storage moduli. The cells did not have a significant impact on the overall stiffness, whereas ADA-GEL (2.5/2.5%) was significantly stiffer than Alg/HA/Gel (0.5/0.1/3%). Regarding the shrinkage of printed constructs, cells had a significant influence, especially in ADA-GEL, which has covalent bonds between the oxidized alginate and gelatin. Multi-photon microscopy exhibited proliferation, cell spreading and migration in ADA-GEL with cell-cell and cell-matrix interaction, dissimilarly to Alg/HA/Gel, in which cells formed spherical, encapsulated colonies. Scanning electron microscopy and histology showed degradation and multi-layered growth on ADA-GEL and fewer examples of escaped cells on Alg/HA/Gel. Both gels serve as proliferation bioink for melanoma with more necrosis in deeper Alg/HA/Gel colonies and differences in spreading and matrix interaction. These findings show the importance of proper characterization of the bioinks for different applications.

FRESH-based 3D bioprinting of complex biological geometries using chitosan bioink.

Traditional three-dimensional (3D) bioprinting has always been associated with the challenge of print fidelity of complex geometries due to the gel-like nature of the bioinks. Embedded 3D bioprinting has emerged as a potential solution to print complex geometries using proteins and polysaccharides-based bioinks. This study demonstrated the Freeform Reversible Embedding of Suspended Hydrogels (FRESH) 3D bioprinting method of chitosan bioink to 3D bioprint complex geometries. 4.5% chitosan was dissolved in an alkali solvent to prepare the bioink. Rheological evaluation of the bioink described its shear-thinning nature. The power law equation was fitted to the shear rate-viscosity plot. The flow index value was found to be less than 1, categorizing the material as pseudo-plastic. The chitosan bioink was extruded into another medium, a thermo-responsive 4.5% gelatin hydrogel. This hydrogel supports the growing print structures while printing. After this, the 3D bioprinted structure was crosslinked with hot water to stabilize the structure. Using this method, we have 3D bioprinted complex biological structures like the human tri-leaflet heart valve, a section of a human right coronary arterial tree, a scale-down outer structure of the human kidney, and a human ear. Additionally, we have shown the mechanical tunability and suturability of the 3D bioprinted structures. This study demonstrates the capability of the chitosan bioink and FRESH method for 3D bioprinting of complex biological models for biomedical applications.

Innovative technologies for the fabrication of 3D/4D smart hydrogels and its biomedical applications - A comprehensive review.

Repairing and regenerating damaged tissues or organs, and restoring their functioning has been the ultimate aim of medical innovations. 'Reviving healthcare' blends tissue engineering with alternative techniques such as hydrogels, which have emerged as vital tools in modern medicine. Additive manufacturing (AM) is a practical manufacturing revolution that uses building strategies like molding as a viable solution for precise hydrogel manufacturing. Recent advances in this technology have led to the successful manufacturing of hydrogels with enhanced reproducibility, accuracy, precision, and ease of fabrication. Hydrogels continue to metamorphose as the vital compatible bio-ink matrix for AM. AM hydrogels have paved the way for complex 3D/4D hydrogels that can be loaded with drugs or cells. Bio-mimicking 3D cell cultures designed via hydrogel-based AM is a groundbreaking in-vivo assessment tool in biomedical trials. This brief review focuses on preparations and applications of additively manufactured hydrogels in the biomedical spectrum, such as targeted drug delivery, 3D-cell culture, numerous regenerative strategies, biosensing, bioprinting, and cancer therapies. Prevalent AM techniques like extrusion, inkjet, digital light processing, and stereo-lithography have been explored with their setup and methodology to yield functional hydrogels. The perspectives, limitations, and the possible prospects of AM hydrogels have been critically examined in this study.

3D-Bioprinted Hepar-on-a-Chip Implanted in Graphene-Based Plasmonic Sensors.

Precise three-dimensional (3D) bioprinting designs enable the fabrication of unique structures for 3D-cell culture models. There is still an absence of real-time detection tools to effectively track in situ 3D-cell performance, hindering a comprehensive understanding of disease progression and drug efficacy assessment. While numerous bioinks have been developed, few are equipped with internal sensors capable of accurate detection. This study addresses these challenges by constructing a 3D-bioprinted hepar-on-a-chip embedded with graphene quantum dot-capped gold nanoparticle-based plasmonic sensors, featuring strong surface-enhanced Raman scattering (SERS) enhancement, biostability, and signal consistency. Such an integrated hepar-on-a-chip demonstrates excellent capability in the secretion of liver function-related proteins and the expression of drug metabolism and transport-related genes. Furthermore, the on-site detection of cell-secreted biomarker glutathione transferase α (GST-α) was successfully achieved using the plasmonic probe, with a dynamic linear detection range of 20-500 ng/mL, showcasing high anti-interference and specificity for GST-α. Ultimately, this integrated hepar-on-a-chip system offers a high-quality platform for monitoring liver injury.