Biofabrication

Bioprinting has evolved into a thriving technology for the fabrication of cell-laden scaffolds. Bioinks are the most critical component for bioprinting. Recently, microgels have been introduced as a very promising bioink, enabling cell protection and the control of the cellular microenvironment. However, the fabrication of the bioinks involves the microfluidic production of the microgels, with a subsequent multistep process to obtain the bioink, which so far has limited its application potential. Here we introduce a direct coupling of microfluidics and 3D-printing for the continuous microfluidic production of microgels with direct in-flow printing into stable scaffolds. The 3D-channel design of the microfluidic chip provides access to different hydrodynamic microdroplet formation regimes to cover a broad range of droplet and microgel diameters. After exiting a microtubing the produced microgels are hydrodynamically jammed into thin microgel filaments for direct 3D-printing into two- and three-dimensional scaffolds. The methodology enables the continuous on-chip encapsulation of cells into monodisperse microdroplets with subsequent in-flow cross-linking to produce cell-laden microgels. The method is demonstrated for different cross-linking methods and cell lines. This advancement will enable a direct coupling of microfluidics and 3D-bioprinting for scaffold fabrication.

Engineered myogenic microtissues derived from human skeletal myoblasts offer unique opportunities for varying skeletal muscle tissue engineering applications, such as in vitro drug-testing and disease modelling. However, more complex models require the incorporation of vascular structures, which remains to be challenging. In this study, myogenic spheroids were generated using a high-throughput, non-adhesive micropatterned surface. Since monoculture spheroids containing human skeletal myoblasts were unable to remain their integrity, co-culture spheroids combining human skeletal myoblasts and human adipose-derived stem cells were created. When using the optimal ratio, uniform and viable spheroids with enhanced myogenic properties were achieved. Applying a pre-vascularization strategy, through addition of endothelial cells, resulted in the formation of spheroids containing capillary-like networks, lumina and collagen in the extracellular matrix, whilst retaining myogenicity. Moreover, sprouting of endothelial cells from the spheroids when encapsulated in fibrin was allowed. The possibility of spheroids, from different maturation stages, to assemble into a more large construct was proven by doublet fusion experiments. The relevance of using three-dimensional microtissues with tissue-specific microarchitecture and increased complexity, together with the high-throughput generation approach, makes the generated spheroids a suitable tool for in vitro drug-testing and human disease modeling.

This bioprinting roadmap features salient advances in selected applications of the technique and highlights the status of current developments and challenges, as well as envisioned advances in science and technology, to address the challenges to the young and evolving technique. The topics covered in this roadmap encompass the broad spectrum of bioprinting; from cell expansion and novel bioink development to cell/stem cell printing, from organoid-based tissue organization to bioprinting of human-scale tissue structures, and from building cell/tissue/organ-on-a-chip to biomanufacturing of multicellular engineered living systems. The emerging application of printing-in-space and an overview of bioprinting technologies are also included in this roadmap. Due to the rapid pace of methodological advancements in bioprinting techniques and wide-ranging applications, the direction in which the field should advance is not immediately clear. This bioprinting roadmap addresses this unmet need by providing a comprehensive summary and recommendations useful to experienced researchers and newcomers to the field.

The advent of 3D bioprinting technologies in tissue engineering has unlocked the potential to fabricate in vitro tissue models, overcoming the constraints associated with the shape limitations of preformed scaffolds. However, achieving an accurate mimicry of complex tissue microenvironments, encompassing cellular and biochemical components, and orchestrating their supramolecular assembly to form hierarchical structures while maintaining control over tissue formation, is crucial for gaining deeper insights into tissue repair and regeneration. Building upon our expertise in developing competent three-dimensional tissue equivalents (e.g. skin, gut, cervix), we established a two-step bottom-up approach involving the dynamic assembly of microtissue precursors (μTPs) to generate macroscopic functional tissue composed of cell-secreted extracellular matrix (ECM). To enhance precision and scalability, we integrated extrusion-based bioprinting technology into our established paradigm to automate, control and guide the coherent assembly of μTPs into predefined shapes. Compared to cell-aggregated bioink, our μTPs represent a functional unit where cells are embedded in their specific ECM. μTPs were derived from human dermal fibroblasts dynamically seeded onto gelatin-based microbeads. After 9 days, μTPs were suspended (50% v/v) in Pluronic-F127 (30% w/v) (µTP:P30), and the obtained formulation was loaded as bioink into the syringe of the Dr.INVIVO-4D6 extrusion based bioprinter. µTP:P30 bioink showed shear-thinning behavior and temperature-dependent viscosity (gel at T > 30 °C), ensuring µTPs homogenous dispersion within the gel and optimal printability. The bioprinting involved extruding several geometries (line, circle, and square) into Pluronic-F127 (40% w/v) (P40) support bath, leveraging its shear-recovery property. P40 effectively held the bioink throughout and after the bioprinting procedure, until µTPs fused into a continuous connective tissue. µTPs fusion dynamics was studied over 8 days of culture, while the resulting endogenous construct underwent 28 days culture. Histological, immunofluorescence analysis, and second harmonic generation reconstruction revealed an increase in endogenous collagen and fibronectin production within the bioprinted construct, closely resembling the composition of the native connective tissues.

Insulin is an essential regulator of blood glucose homeostasis that is produced exclusively by β cells within the pancreatic islets of healthy individuals. In those affected by diabetes, immune inflammation, damage, and destruction of islet β cells leads to insulin deficiency and hyperglycemia. Current efforts to understand the mechanisms underlying β cell damage in diabetes rely on in vitro-cultured cadaveric islets. However, isolation of these islets involves removal of crucial matrix and vasculature that supports islets in the intact pancreas. Unsurprisingly, these islets demonstrate reduced functionality over time in standard culture conditions, thereby limiting their value for understanding native islet biology. Leveraging a novel, vascularized micro-organ (VMO) approach, we have recapitulated elements of the native pancreas by incorporating isolated human islets within a three-dimensional matrix nourished by living, perfusable blood vessels. Importantly, these islets show long-term viability and maintain robust glucose-stimulated insulin responses. Furthermore, vessel-mediated delivery of immune cells to these tissues provides a model to assess islet-immune cell interactions and subsequent islet killing—key steps in type 1 diabetes pathogenesis. Together, these results establish the islet-VMO as a novel, ex vivo platform for studying human islet biology in both health and disease.

This study endeavors to investigate the progression, research focal points, and budding trends in the realm of skin bioprinting over the past decade from a structural and temporal dynamics standpoint. Scholarly articles on skin bioprinting were obtained from WoSCC. A series of bibliometric tools comprising R software, CiteSpace, HistCite, and an alluvial generator were employed to discern historical characteristics, evolution of active topics, and upcoming tendencies in the area of skin bioprinting. Over the past decade, there has been a consistent rise in research interest in skin bioprinting, accompanied by an extensive array of meaningful scientific collaborations. Concurrently, diverse dynamic topics have emerged during various periods, as substantiated by an aggregate of 22 disciplines, 74 keywords, and 187 references demonstrating citation bursts. Four burgeoning research subfields were discerned through keyword clustering—namely, #3 'in situ bioprinting', #6 'vascular', #7 'xanthan gum', and #8 'collagen hydrogels'. The keyword alluvial map reveals that Module 1, including 'transplantation' etc, has primarily dominated the research module over the previous decade, maintaining enduring relevance despite annual shifts in keyword focus. Additionally, we mapped out the top six key modules from 2023 being 'silk fibroin nanofiber', 'system', 'ionic liquid', 'mechanism', and 'foot ulcer'. Three recent research subdivisions were identified via timeline visualization of references, particularly Clusters #0 'wound healing', #4 'situ mineralization', and #5 '3D bioprinter'. Insights derived from bibliometric analyses illustrate present conditions and trends in skin bioprinting research, potentially aiding researchers in pinpointing central themes and pioneering novel investigative approaches in this field.

Recent advances in the field of mechanobiology have led to the development of methods to characterise single-cell or monolayer mechanical properties and link them to their functional behaviour. However, there remains a strong need to establish this link for three-dimensional (3D) multicellular aggregates, which better mimic tissue function. Here we present a platform to actuate and observe many such aggregates within one deformable micro-device. The platform consists of a single polydimethylsiloxane piece cast on a 3D-printed mould and bonded to a glass slide or coverslip. It consists of a chamber containing cell spheroids, which is adjacent to air cavities that are fluidically independent. Controlling the air pressure in these air cavities leads to a vertical displacement of the chamber's ceiling. The device can be used in static or dynamic modes over time scales of seconds to hours, with displacement amplitudes from a few µm to several tens of microns. Further, we show how the compression protocols can be used to obtain measurements of stiffness heterogeneities within individual co-culture spheroids, by comparing image correlations of spheroids at different levels of compression with finite element simulations. The labelling of the cells and their cytoskeleton is combined with image correlation methods to relate the structure of the co-culture spheroid with its mechanical properties at different locations. The device is compatible with various microscopy techniques, including confocal microscopy, which can be used to observe the displacements and rearrangements of single cells and neighbourhoods within the aggregate. The complete experimental and imaging platform can now be used to provide multi-scale measurements that link single-cell behaviour with the global mechanical response of the aggregates.

The anisotropic organization of cells and the extracellular matrix (ECM) is essential for the physiological function of numerous biological tissues, including the myocardium. This organization changes gradually in space and time, during disease progression such as myocardial infarction. The role of mechanical stimuli has been demonstrated to be essential in obtaining, maintaining and de-railing this organization, but the underlying mechanisms are scarcely known. To enable the study of the mechanobiological mechanisms involved, in vitro techniques able to spatiotemporally control the multiscale tissue mechanical environment are thus necessary. Here, by using light-sensitive materials combined with light-illumination techniques, we fabricated 2D and 3D in vitro model systems exposing cells to multiscale, spatiotemporally resolved stiffness anisotropies. Specifically, spatial stiffness anisotropies spanning from micron-sized (cellular) to millimeter-sized (tissue) were achieved. Moreover, the light-sensitive materials allowed to introduce the stiffness anisotropies at defined timepoints (hours) after cell seeding, facilitating the study of their temporal effects on cell and tissue orientation. The systems were tested using cardiac fibroblasts (cFBs), which are known to be crucial for the remodeling of anisotropic cardiac tissue. We observed that 2D stiffness micropatterns induced cFBs anisotropic alignment, independent of the stimulus timing, but dependent on the micropattern spacing. cFBs exhibited organized alignment also in response to 3D stiffness macropatterns, dependent on the stimulus timing and temporally followed by (slower) ECM co-alignment. In conclusion, the developed model systems allow improved fundamental understanding of the underlying mechanobiological factors that steer cell and ECM orientation, such as stiffness guidance and boundary constraints.

Biofabrication aims to fabricate biologically functional products through bioprinting or bioassembly (Groll et al 2016 Biofabrication 8 013001). In biofabrication processes, cells are positioned at defined coordinates in three-dimensional space using automated and computer controlled techniques (Moroni et al 2018 Trends Biotechnol. 36 384–402), usually with the aid of biomaterials that are either (i) directly processed with the cells as suspensions/dispersions, (ii) deposited simultaneously in a separate printing process, or (iii) used as a transient support material. Materials that are suited for biofabrication are often referred to as bioinks and have become an important area of research within the field. In view of this special issue on bioinks, we aim herein to briefly summarize the historic evolution of this term within the field of biofabrication. Furthermore, we propose a simple but general definition of bioinks, and clarify its distinction from biomaterial inks.

The existing 3D printing methods exhibit certain fabrication-dependent limitations for printing curved constructs that are relevant for many tissues. Four-dimensional (4D) printing is an emerging technology that is expected to revolutionize the field of tissue engineering and regenerative medicine (TERM). 4D printing is based on 3D printing, featuring the introduction of time as the fourth dimension, in which there is a transition from a 3D printed scaffold to a new, distinct, and stable state, upon the application of one or more stimuli. Here, we present an overview of the current developments of the 4D printing technology for TERM, with a focus on approaches to achieve temporal changes of the shape of the printed constructs that would enable biofabrication of highly complex structures. To this aim, the printing methods, types of stimuli, shape-shifting mechanisms, and cell-incorporation strategies are critically reviewed. Furthermore, the challenges of this very recent biofabrication technology as well as the future research directions are discussed. Our findings show that the most common printing methods so far are stereolithography (SLA) and extrusion bioprinting, followed by fused deposition modelling, while the shape-shifting mechanisms used for TERM applications are shape-memory and differential swelling for 4D printing and 4D bioprinting, respectively. For shape-memory mechanism, there is a high prevalence of synthetic materials, such as polylactic acid (PLA), poly(glycerol dodecanoate) acrylate (PGDA), or polyurethanes. On the other hand, different acrylate combinations of alginate, hyaluronan, or gelatin have been used for differential swelling-based 4D transformations. TERM applications include bone, vascular, and cardiac tissues as the main target of the 4D (bio)printing technology. The field has great potential for further development by considering the combination of multiple stimuli, the use of a wider range of 4D techniques, and the implementation of computational-assisted strategies.

Regenerative healing of spinal cord injury (SCI) poses an ongoing medical challenge by causing persistent neurological impairment and a significant socioeconomic burden. The complexity of spinal cord tissue presents hurdles to successful regeneration following injury, due to the difficulty of forming a biomimetic structure that faithfully replicates native tissue using conventional tissue engineering scaffolds. 3D bioprinting is a rapidly evolving technology with unmatched potential to create 3D biological tissues with complicated and hierarchical structure and composition. With the addition of biological additives such as cells and biomolecules, 3D bioprinting can fabricate preclinical implants, tissue or organ-like constructs, and in vitro models through precise control over the deposition of biomaterials and other building blocks. This review highlights the characteristics and advantages of 3D bioprinting for scaffold fabrication to enable SCI repair, including bottom–up manufacturing, mechanical customization, and spatial heterogeneity. This review also critically discusses the impact of various fabrication parameters on the efficacy of spinal cord repair using 3D bioprinted scaffolds, including the choice of printing method, scaffold shape, biomaterials, and biological supplements such as cells and growth factors. High-quality preclinical studies are required to accelerate the translation of 3D bioprinting into clinical practice for spinal cord repair. Meanwhile, other technological advances will continue to improve the regenerative capability of bioprinted scaffolds, such as the incorporation of nanoscale biological particles and the development of 4D printing.

Cancer is one of the leading causes of death in the 21st century, with metastasis of cancer attributing to 90% of cancer-related deaths. Therefore, to improve patient outcomes there is a need for better preclinical models to increase the success of translating oncological therapies into the clinic. Current traditional static in vitro models lack a perfusable network which is critical to overcome the diffusional mass transfer limit to provide a mechanism for the exchange of essential nutrients and waste removal, and increase their physiological relevance. Furthermore, these models typically lack cellular heterogeneity and key components of the immune system and tumour microenvironment. This review explores rapidly developing strategies utilising perfusable microphysiological systems (MPS) for investigating cancer cell metastasis. In this review we initially outline the mechanisms of cancer metastasis, highlighting key steps and identifying the current gaps in our understanding of the metastatic cascade, exploring MPS focused on investigating the individual steps of the metastatic cascade before detailing the latest MPS which can investigate multiple components of the cascade. This review then focuses on the factors which can affect the performance of an MPS designed for cancer applications with a final discussion summarising the challenges and future directions for the use of MPS for cancer models.

With the advent of personalized medicine, the drug delivery system will be changed significantly. The development of personalized medicine needs the support of many technologies, among which three-dimensional printing (3DP) technology is a novel formulation-preparing process that creates 3D objects by depositing printing materials layer-by-layer based on the computer-aided design method. Compared with traditional pharmaceutical processes, 3DP produces complex drug combinations, personalized dosage, and flexible shape and structure of dosage forms (DFs) on demand. In the future, personalized 3DP drugs may supplement and even replace their traditional counterpart. We systematically introduce the applications of 3DP technologies in the pharmaceutical industry and summarize the virtues and shortcomings of each technique. The release behaviors and control mechanisms of the pharmaceutical DFs with desired structures are also analyzed. Finally, the benefits, challenges, and prospects of 3DP technology to the pharmaceutical industry are discussed.

The anisotropic organization of cells and the extracellular matrix (ECM) is essential for the physiological function of numerous biological tissues, including the myocardium. This organization changes gradually in space and time, during disease progression such as myocardial infarction. The role of mechanical stimuli has been demonstrated to be essential in obtaining, maintaining and de-railing this organization, but the underlying mechanisms are scarcely known. To enable the study of the mechanobiological mechanisms involved, in vitro techniques able to spatiotemporally control the multiscale tissue mechanical environment are thus necessary. Here, by using light-sensitive materials combined with light-illumination techniques, we fabricated 2D and 3D in vitro model systems exposing cells to multiscale, spatiotemporally resolved stiffness anisotropies. Specifically, spatial stiffness anisotropies spanning from micron-sized (cellular) to millimeter-sized (tissue) were achieved. Moreover, the light-sensitive materials allowed to introduce the stiffness anisotropies at defined timepoints (hours) after cell seeding, facilitating the study of their temporal effects on cell and tissue orientation. The systems were tested using cardiac fibroblasts (cFBs), which are known to be crucial for the remodeling of anisotropic cardiac tissue. We observed that 2D stiffness micropatterns induced cFBs anisotropic alignment, independent of the stimulus timing, but dependent on the micropattern spacing. cFBs exhibited organized alignment also in response to 3D stiffness macropatterns, dependent on the stimulus timing and temporally followed by (slower) ECM co-alignment. In conclusion, the developed model systems allow improved fundamental understanding of the underlying mechanobiological factors that steer cell and ECM orientation, such as stiffness guidance and boundary constraints.

Recent advances in the field of mechanobiology have led to the development of methods to characterise single-cell or monolayer mechanical properties and link them to their functional behaviour. However, there remains a strong need to establish this link for three-dimensional (3D) multicellular aggregates, which better mimic tissue function. Here we present a platform to actuate and observe many such aggregates within one deformable micro-device. The platform consists of a single polydimethylsiloxane piece cast on a 3D-printed mould and bonded to a glass slide or coverslip. It consists of a chamber containing cell spheroids, which is adjacent to air cavities that are fluidically independent. Controlling the air pressure in these air cavities leads to a vertical displacement of the chamber's ceiling. The device can be used in static or dynamic modes over time scales of seconds to hours, with displacement amplitudes from a few µm to several tens of microns. Further, we show how the compression protocols can be used to obtain measurements of stiffness heterogeneities within individual co-culture spheroids, by comparing image correlations of spheroids at different levels of compression with finite element simulations. The labelling of the cells and their cytoskeleton is combined with image correlation methods to relate the structure of the co-culture spheroid with its mechanical properties at different locations. The device is compatible with various microscopy techniques, including confocal microscopy, which can be used to observe the displacements and rearrangements of single cells and neighbourhoods within the aggregate. The complete experimental and imaging platform can now be used to provide multi-scale measurements that link single-cell behaviour with the global mechanical response of the aggregates.

Regenerative healing of spinal cord injury (SCI) poses an ongoing medical challenge by causing persistent neurological impairment and a significant socioeconomic burden. The complexity of spinal cord tissue presents hurdles to successful regeneration following injury, due to the difficulty of forming a biomimetic structure that faithfully replicates native tissue using conventional tissue engineering scaffolds. 3D bioprinting is a rapidly evolving technology with unmatched potential to create 3D biological tissues with complicated and hierarchical structure and composition. With the addition of biological additives such as cells and biomolecules, 3D bioprinting can fabricate preclinical implants, tissue or organ-like constructs, and in vitro models through precise control over the deposition of biomaterials and other building blocks. This review highlights the characteristics and advantages of 3D bioprinting for scaffold fabrication to enable SCI repair, including bottom–up manufacturing, mechanical customization, and spatial heterogeneity. This review also critically discusses the impact of various fabrication parameters on the efficacy of spinal cord repair using 3D bioprinted scaffolds, including the choice of printing method, scaffold shape, biomaterials, and biological supplements such as cells and growth factors. High-quality preclinical studies are required to accelerate the translation of 3D bioprinting into clinical practice for spinal cord repair. Meanwhile, other technological advances will continue to improve the regenerative capability of bioprinted scaffolds, such as the incorporation of nanoscale biological particles and the development of 4D printing.

Cancer is one of the leading causes of death in the 21st century, with metastasis of cancer attributing to 90% of cancer-related deaths. Therefore, to improve patient outcomes there is a need for better preclinical models to increase the success of translating oncological therapies into the clinic. Current traditional static in vitro models lack a perfusable network which is critical to overcome the diffusional mass transfer limit to provide a mechanism for the exchange of essential nutrients and waste removal, and increase their physiological relevance. Furthermore, these models typically lack cellular heterogeneity and key components of the immune system and tumour microenvironment. This review explores rapidly developing strategies utilising perfusable microphysiological systems (MPS) for investigating cancer cell metastasis. In this review we initially outline the mechanisms of cancer metastasis, highlighting key steps and identifying the current gaps in our understanding of the metastatic cascade, exploring MPS focused on investigating the individual steps of the metastatic cascade before detailing the latest MPS which can investigate multiple components of the cascade. This review then focuses on the factors which can affect the performance of an MPS designed for cancer applications with a final discussion summarising the challenges and future directions for the use of MPS for cancer models.

With the advent of personalized medicine, the drug delivery system will be changed significantly. The development of personalized medicine needs the support of many technologies, among which three-dimensional printing (3DP) technology is a novel formulation-preparing process that creates 3D objects by depositing printing materials layer-by-layer based on the computer-aided design method. Compared with traditional pharmaceutical processes, 3DP produces complex drug combinations, personalized dosage, and flexible shape and structure of dosage forms (DFs) on demand. In the future, personalized 3DP drugs may supplement and even replace their traditional counterpart. We systematically introduce the applications of 3DP technologies in the pharmaceutical industry and summarize the virtues and shortcomings of each technique. The release behaviors and control mechanisms of the pharmaceutical DFs with desired structures are also analyzed. Finally, the benefits, challenges, and prospects of 3DP technology to the pharmaceutical industry are discussed.

The application of additive manufacturing (AM) technology plays a significant role in various fields, incorporating a wide range of cutting-edge technologies such as aerospace, medical treatment, electronic information, and materials. It is currently widely adopted for medical services, national defense, and industrial manufacturing. In recent years, AM has also been extensively employed to produce bone scaffolds and implant materials. Through AM, products can be manufactured without being constrained by complex internal structures. AM is particularly advantageous in the production of macroscopically irregular and microscopically porous biomimetic bone scaffolds, with short production cycles required. In this paper, AM commonly used to produce bone scaffolds and orthopedic implants is overviewed to analyze the different materials and structures adopted for AM. The applications of antibacterial bone scaffolds and bone scaffolds in biologically relevant animal models are discussed. Also, the influence on the comprehensive performance of product mechanics, mass transfer, and biology is explored. By identifying the reasons for the limited application of existing AM in the biomedical field, the solutions are proposed. This study provides an important reference for the future development of AM in the field of orthopedic healthcare. In conclusion, various AM technologies, the requirements of bone scaffolds and the important role of AM in building bridges between biomaterials, additives, and bone tissue engineering scaffolds are described and highlighted. Nevertheless, more caution should be exercised when designing bone scaffolds and conducting in vivo trials, due to the lack of standardized processes, which prevents the accuracy of results and reduces the reliability of information.

The knee meniscus is the cushioning fibro-cartilage tissue present in between the femoral condyles and tibial plateau of the knee joint. It is largely avascular in nature and suffers from a wide range of tears and injuries caused by accidents, trauma, active lifestyle of the populace and old age of individuals. Healing of the meniscus is especially difficult due to its avascularity and hence requires invasive arthroscopic approaches such as surgical resection, suturing or implantation. Though various tissue engineering approaches are proposed for the treatment of meniscus tears, three-dimensional (3D) printing/bioprinting, injectable hydrogels and physical stimulation involving modalities are gaining forefront in the past decade. A plethora of new printing approaches such as direct light photopolymerization and volumetric printing, injectable biomaterials loaded with growth factors and physical stimulation such as low-intensity ultrasound approaches are being added to the treatment portfolio along with the contemporary tear mitigation measures. This review discusses on the necessary design considerations, approaches for 3D modeling and design practices for meniscal tear treatments within the scope of tissue engineering and regeneration. Also, the suitable materials, cell sources, growth factors, fixation and lubrication strategies, mechanical stimulation approaches, 3D printing strategies and injectable hydrogels for meniscal tear management have been elaborated. We have also summarized potential technologies and the potential framework that could be the herald of the future of meniscus tissue engineering and repair approaches.

Cancer is one of the leading causes of death in the 21st century, with metastasis of cancer attributing to 90% of cancer-related deaths. Therefore, to improve patient outcomes there is a need for better preclinical models to increase the success of translating oncological therapies into the clinic. Current traditional static in vitro models lack a perfusable network which is critical to overcome the diffusional mass transfer limit to provide a mechanism for the exchange of essential nutrients and waste removal, and increase their physiological relevance. Furthermore, these models typically lack cellular heterogeneity and key components of the immune system and tumour microenvironment. This review explores rapidly developing strategies utilising perfusable microphysiological systems (MPS) for investigating cancer cell metastasis. In this review we initially outline the mechanisms of cancer metastasis, highlighting key steps and identifying the current gaps in our understanding of the metastatic cascade, exploring MPS focused on investigating the individual steps of the metastatic cascade before detailing the latest MPS which can investigate multiple components of the cascade. This review then focuses on the factors which can affect the performance of an MPS designed for cancer applications with a final discussion summarising the challenges and future directions for the use of MPS for cancer models.

With the advent of personalized medicine, the drug delivery system will be changed significantly. The development of personalized medicine needs the support of many technologies, among which three-dimensional printing (3DP) technology is a novel formulation-preparing process that creates 3D objects by depositing printing materials layer-by-layer based on the computer-aided design method. Compared with traditional pharmaceutical processes, 3DP produces complex drug combinations, personalized dosage, and flexible shape and structure of dosage forms (DFs) on demand. In the future, personalized 3DP drugs may supplement and even replace their traditional counterpart. We systematically introduce the applications of 3DP technologies in the pharmaceutical industry and summarize the virtues and shortcomings of each technique. The release behaviors and control mechanisms of the pharmaceutical DFs with desired structures are also analyzed. Finally, the benefits, challenges, and prospects of 3DP technology to the pharmaceutical industry are discussed.

The anisotropic organization of cells and the extracellular matrix (ECM) is essential for the physiological function of numerous biological tissues, including the myocardium. This organization changes gradually in space and time, during disease progression such as myocardial infarction. The role of mechanical stimuli has been demonstrated to be essential in obtaining, maintaining and de-railing this organization, but the underlying mechanisms are scarcely known. To enable the study of the mechanobiological mechanisms involved, in vitro techniques able to spatiotemporally control the multiscale tissue mechanical environment are thus necessary. Here, by using light-sensitive materials combined with light-illumination techniques, we fabricated 2D and 3D in vitro model systems exposing cells to multiscale, spatiotemporally resolved stiffness anisotropies. Specifically, spatial stiffness anisotropies spanning from micron-sized (cellular) to millimeter-sized (tissue) were achieved. Moreover, the light-sensitive materials allowed to introduce the stiffness anisotropies at defined timepoints (hours) after cell seeding, facilitating the study of their temporal effects on cell and tissue orientation. The systems were tested using cardiac fibroblasts (cFBs), which are known to be crucial for the remodeling of anisotropic cardiac tissue. We observed that 2D stiffness micropatterns induced cFBs anisotropic alignment, independent of the stimulus timing, but dependent on the micropattern spacing. cFBs exhibited organized alignment also in response to 3D stiffness macropatterns, dependent on the stimulus timing and temporally followed by (slower) ECM co-alignment. In conclusion, the developed model systems allow improved fundamental understanding of the underlying mechanobiological factors that steer cell and ECM orientation, such as stiffness guidance and boundary constraints.

Recent advances in the field of mechanobiology have led to the development of methods to characterise single-cell or monolayer mechanical properties and link them to their functional behaviour. However, there remains a strong need to establish this link for three-dimensional (3D) multicellular aggregates, which better mimic tissue function. Here we present a platform to actuate and observe many such aggregates within one deformable micro-device. The platform consists of a single polydimethylsiloxane piece cast on a 3D-printed mould and bonded to a glass slide or coverslip. It consists of a chamber containing cell spheroids, which is adjacent to air cavities that are fluidically independent. Controlling the air pressure in these air cavities leads to a vertical displacement of the chamber's ceiling. The device can be used in static or dynamic modes over time scales of seconds to hours, with displacement amplitudes from a few µm to several tens of microns. Further, we show how the compression protocols can be used to obtain measurements of stiffness heterogeneities within individual co-culture spheroids, by comparing image correlations of spheroids at different levels of compression with finite element simulations. The labelling of the cells and their cytoskeleton is combined with image correlation methods to relate the structure of the co-culture spheroid with its mechanical properties at different locations. The device is compatible with various microscopy techniques, including confocal microscopy, which can be used to observe the displacements and rearrangements of single cells and neighbourhoods within the aggregate. The complete experimental and imaging platform can now be used to provide multi-scale measurements that link single-cell behaviour with the global mechanical response of the aggregates.

The advent of 3D bioprinting technologies in tissue engineering has unlocked the potential to fabricate in vitro tissue models, overcoming the constraints associated with the shape limitations of preformed scaffolds. However, achieving an accurate mimicry of complex tissue microenvironments, encompassing cellular and biochemical components, and orchestrating their supramolecular assembly to form hierarchical structures while maintaining control over tissue formation, is crucial for gaining deeper insights into tissue repair and regeneration. Building upon our expertise in developing competent three-dimensional tissue equivalents (e.g. skin, gut, cervix), we established a two-step bottom-up approach involving the dynamic assembly of microtissue precursors (μTPs) to generate macroscopic functional tissue composed of cell-secreted extracellular matrix (ECM). To enhance precision and scalability, we integrated extrusion-based bioprinting technology into our established paradigm to automate, control and guide the coherent assembly of μTPs into predefined shapes. Compared to cell-aggregated bioink, our μTPs represent a functional unit where cells are embedded in their specific ECM. μTPs were derived from human dermal fibroblasts dynamically seeded onto gelatin-based microbeads. After 9 days, μTPs were suspended (50% v/v) in Pluronic-F127 (30% w/v) (µTP:P30), and the obtained formulation was loaded as bioink into the syringe of the Dr.INVIVO-4D6 extrusion based bioprinter. µTP:P30 bioink showed shear-thinning behavior and temperature-dependent viscosity (gel at T > 30 °C), ensuring µTPs homogenous dispersion within the gel and optimal printability. The bioprinting involved extruding several geometries (line, circle, and square) into Pluronic-F127 (40% w/v) (P40) support bath, leveraging its shear-recovery property. P40 effectively held the bioink throughout and after the bioprinting procedure, until µTPs fused into a continuous connective tissue. µTPs fusion dynamics was studied over 8 days of culture, while the resulting endogenous construct underwent 28 days culture. Histological, immunofluorescence analysis, and second harmonic generation reconstruction revealed an increase in endogenous collagen and fibronectin production within the bioprinted construct, closely resembling the composition of the native connective tissues.

Conventional gut-on-chip (GOC) models typically represent the epithelial layer of the gut tissue, neglecting other important components such as the stromal compartment and the extracellular matrix (ECM) that play crucial roles in maintaining intestinal barrier integrity and function. These models often employ hard, flat porous membranes for cell culture, thus failing to recapitulate the soft environment and complex 3D architecture of the intestinal mucosa. Alternatively, hydrogels have been recently introduced in GOCs as ECM analogs to support the co-culture of intestinal cells in in vivo-like configurations, and thus opening new opportunities in the organ-on-chip field. In this work, we present an innovative GOC device that includes a 3D bioprinted hydrogel channel replicating the intestinal villi architecture containing both the epithelial and stromal compartments of the gut mucosa. The bioprinted hydrogels successfully support both the encapsulation of fibroblasts and their co-culture with intestinal epithelial cells under physiological flow conditions. Moreover, we successfully integrated electrodes into the microfluidic system to monitor the barrier formation in real time via transepithelial electrical resistance measurements.

Extrusion-based bioprinting is a promising technology for the fabrication of complex three-dimensional (3D) tissue-engineered constructs. To further improve the printing accuracy and provide mechanical support during the printing process, hydrogel-based support bath materials have been developed. However, the gel structure of some support bath materials can be compromised when exposed to certain bioink crosslinking cues, hence their compatibility with bioinks can be limited. In this study, a xanthan gum-based composite support material compatible with multiple crosslinking mechanisms is developed. Different support bath materials can have different underlying polymeric structures, for example, particulate suspensions and polymer solution with varying supramolecular structure) and these properties are governed by a variety of different intermolecular interactions. However, common rheological behavior can be expected because they have similar demonstrated performance and functionality. To provide a detailed exploration/identification of the common rheological properties expressed by different support bath materials from a unified perspective, benchmark support bath materials from previous studies were prepared. A comparative rheological study revealed both the structural and shear behavior characteristics shared by support bath materials, including yield stress, gel complex moduli, shear-thinning behavior, and self-healing properties. Gel structural stability and functionality of support materials were tested in the presence of various crosslinking stimuli, confirming the versatility of the xanthan-based support material. We further investigated the effect of support materials and the diameter of extrusion needles on the printability of bioinks to demonstrate the improvement in bioink printability and structural integrity. Cytotoxicity and cell encapsulation viability tests were carried out to confirm the cell compatibility of the xanthan gum-based support bath material. We propose and demonstrate the versatility and compatibility of the novel support bath material and provide detailed new insight into the essential properties and behavior of these materials that serve as a guide for further development of support bath-based 3D bioprinting.

Peripheral nerve injuries can lead to sensory or motor deficits that have a serious impact on a patient's mental health and quality of life. Nevertheless, it remains a major clinical challenge to develop functional nerve conduits as an alternative to autologous grafts. We applied reduced graphene oxide (rGO) as a bioactive conductive material to impart electrophysiological properties to the catheter and the application of a pulsed magnetic field to excite the formation of microcurrents and induce nerve regeneration. In vitro studies showed that the nerve conduit and the pulsed magnetic field made no effect on cell survival, increased S-100β protein expression, enhanced cell adhesion, and increased the expression level of nerve regeneration-related mRNAs. In vivo experiments suggested that the protocol was effective in promoting nerve regeneration, resulting in functional recovery of sciatic nerves in rats, when they were damaged close to that of the autologous nerve graft, and increased expression of S-100β, NF200, and GAP43. These results indicate that rGO composite nerve conduits combined with pulsed magnetic field stimulation have great potential for peripheral nerve rehabilitation.

Owing to its thermoresponsive and photocrosslinking characteristics, gelatin methacryloyl (GelMA)-based biomaterials have gained widespread usage as a novel and promising bioink for three-dimensional bioprinting and diverse biomedical applications. However, the flow behaviors of GelMA during the sol-gel transition, which are dependent on time and temperature, present significant challenges in printing thick scaffolds while maintaining high printability and cell viability. Moreover, the tunable properties and photocrosslinking capabilities of GelMA underscore its potential for localized drug delivery applications. Previous research has demonstrated the successful incorporation of minocycline (MH) into GelMA scaffolds for therapeutic applications. However, achieving a prolonged and sustained release of concentrated MH remains a challenge, primarily due to its small molecular size. The primary aim of this study is to investigate an optimal extrusion printing method for GelMA bioink in extrusion bioprinting, emphasizing its flow behaviors that are influenced by time and temperature. Additionally, this research seeks to explore the potential of GelMA bioink as a carrier for the sustained release of MH, specifically targeting cellular protection against oxidative stress. The material properties of GelMA were assessed and further optimization of the printing process was conducted considering both printability and cell survival. To achieve sustained drug release within GelMA, the study employed a mechanism using metal ion mediation to facilitate the interaction between MH, dextran sulfate (DS), and magnesium, leading to the formation of nanoparticle complexes (MH-DS). Furthermore, a GelMA-based in vitro model was developed in order to investigate the cellular protective properties of MH against oxidative stress. The experimental results revealed that the printability and cell viability of GelMA are significantly influenced by the printing duration, nozzle temperature, and GelMA concentrations. Optimal printing conditions were identified based on a thorough assessment of both printability and cell viability. Scaffolds printed under these optimal conditions exhibited exceptional printability and sustained high cell viability. Notably, it was found that lower GelMA concentrations reduced the initial burst release of MH from the MH-dextran sulfate (MH-DS) complexes, thus favoring more controlled, sustained release profiles. Additionally, MH released under these conditions significantly enhanced fibroblast viability in an in vitro model simulating oxidative stress.

Bioprinting has evolved into a thriving technology for the fabrication of cell-laden scaffolds. Bioinks are the most critical component for bioprinting. Recently, microgels have been introduced as a very promising bioink, enabling cell protection and the control of the cellular microenvironment. However, the fabrication of the bioinks involves the microfluidic production of the microgels, with a subsequent multistep process to obtain the bioink, which so far has limited its application potential. Here we introduce a direct coupling of microfluidics and 3D-printing for the continuous microfluidic production of microgels with direct in-flow printing into stable scaffolds. The 3D-channel design of the microfluidic chip provides access to different hydrodynamic microdroplet formation regimes to cover a broad range of droplet and microgel diameters. After exiting a microtubing the produced microgels are hydrodynamically jammed into thin microgel filaments for direct 3D-printing into two- and three-dimensional scaffolds. The methodology enables the continuous on-chip encapsulation of cells into monodisperse microdroplets with subsequent in-flow cross-linking to produce cell-laden microgels. The method is demonstrated for different cross-linking methods and cell lines. This advancement will enable a direct coupling of microfluidics and 3D-bioprinting for scaffold fabrication.