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Abstract
To progress cardiac tissue engineering strategies closer to the clinic, thicker constructs are required to meet the functional need following a cardiac event. Consequently, pre-vascularization of these constructs needs to be investigated to ensure survival and optimal performance of implantable engineered heart tissue. The aim of this research is to investigate the potential of combining extrusion-based bioprinting (EBB) and melt electrowriting for the fabrication of a myocardial construct with a precisely patterned pre-vascular pathway. Gelatin methacryloyl (GelMA) was investigated as a base hydrogel for the respective myocardial and vascular bioinks with collagen, Matrigel and fibrinogen as interpenetrating polymers to support myocardial functionality. Subsequently, extrusion-based printability and viability were investigated to determine the optimal processing parameters for printing into melt electrowritten meshes. Finally, an anatomically inspired vascular pathway was implemented in a dual EBB set-up into melt electrowritten meshes, creating a patterned pre-vascularized myocardial construct. It was determined that a blend of 5% GelMA and 0.8 mg·ml−1 collagen with a low crosslinked density was optimal for myocardial cellular arrangement and alignment within the constructs. For the vascular fraction, the optimized formulation consisted of 5% GelMA, 0.8 mg·ml−1 collagen and 1 mg·ml−1 fibrinogen with a higher crosslinked density, which led to enhanced vascular cell connectivity. Printability assessment confirmed that the optimized bioinks could effectively fill the microfiber mesh while supporting cell viability (∼70%). Finally, the two bioinks were applied using a dual EBB system for the fabrication of a pre-vascular pathway with the shape of a left anterior descending artery within a myocardial construct, whereby the distinct cell populations could be visualized in their respective patterns up to D14. This research investigated the first step towards developing a thick engineered cardiac tissue construct in which a pre-vascularization pathway is fabricated within a myocardial construct.

Abstract
Abstract In living tissues, cells express their functions following complex signals from their surrounding microenvironment. Capturing both hierarchical architectures at the micro- and macroscale, and anisotropic cell patterning remains a major challenge in bioprinting, and a bottleneck towards creating physiologically-relevant models. Addressing this limitation, we introduced a novel technique, termed Embedded Extrusion-Volumetric Printing (EmVP), converging extrusion-bioprinting and layer-less, ultra-fast volumetric bioprinting, allowing to spatially pattern multiple inks/cell types. Light-responsive microgels were developed for the first time as bioresins (μResins) for light-based volumetric bioprinting, providing a microporous environment permissive for cell homing and self-organization. Tuning the mechanical and optical properties of gelatin-based microparticles enables their use as support bath for suspended extrusion printing, in which features containing high cell densities can be easily introduced. μResins can be sculpted within seconds with tomographic light projections into centimetre-scale, granular hydrogel-based, convoluted constructs. Interstitial microvoids enhanced differentiation of multiple stem/progenitor cells (vascular, mesenchymal, neural), otherwise not possible with conventional bulk hydrogels. As proof-of-concept, EmVP was applied to create complex synthetic biology-inspired intercellular communication models, where adipocyte differentiation is regulated by optogenetic-engineered pancreatic cells. Overall, EmVP offers new avenues for producing regenerative grafts with biological functionality, and for developing engineered living systems and (metabolic) disease models. This article is protected by copyright. All rights reserved

Abstract
Neuroblastoma is an extracranial solid tumor which develops in early childhood and still has a poor prognosis. One strategy to increase cure rates is the identification of patient-specific drug responses in tissue models that mimic the interaction between patient cancer cells and tumor environment. We therefore developed a perfused and micro-vascularized tumor-environment model that is directly bioprinted into custom-manufactured fluidic chips. A gelatin-methacrylate/fibrin-based matrix containing multiple cell types mimics the tumor-microenvironment that promotes spontaneous micro-vessel formation by embedded endothelial cells. We demonstrate that both, adipocyte- and iPSC-derived mesenchymal stem cells can guide this process. Bioprinted channels are coated with endothelial cells post printing to form a dense vessel - tissue barrier. The tissue model thereby mimics structure and function of human soft tissue with endothelial cell-coated larger vessels for perfusion and micro-vessel networks within the hydrogel-matrix. Patient-derived neuroblastoma spheroids are added to the matrix during the printing process and grown for more than two weeks. We demonstrate that micro-vessels are attracted by and grow into tumor spheroids and that neuroblastoma cells invade the tumor-environment as soon as the spheroids disrupt. In summary, we describe the first bioprinted, micro-vascularized neuroblastoma – tumor-environment model directly printed into fluidic chips and a novel medium-throughput biofabrication platform suitable for studying tumor angiogenesis and metastasis in precision medicine approaches in future.

Abstract
Abstract There is a need for long-lived hepatic in vitro models to better predict drug induced liver injury (DILI). Human liver-derived epithelial organoids are a promising cell source for advanced in vitro models. Here, organoid technology is combined with biofabrication techniques, which holds great potential for the design of in vitro models with complex and customizable architectures. Here, porous constructs with human hepatocyte-like cells derived from organoids are generated using extrusion-based printing technology. Cell viability of bioprinted organoids remains stable for up to ten days (88–107% cell viability compared to the day of printing). The expression of hepatic markers, transporters, and phase I enzymes increased compared to undifferentiated controls, and is comparable to non-printed controls. Exposure to acetaminophen, a well-known hepatotoxic compound, decreases cell viability of bioprinted liver organoids to 21–51% (p < 0.05) compared to the start of exposure, and elevated levels of damage marker miR-122 are observed in the culture medium, indicating the potential use of the bioprinted constructs for toxicity testing. In conclusion, human liver-derived epithelial organoids can be combined with a biofabrication approach, thereby paving the way to create perfusable, complex constructs which can be used as toxicology- and disease-models.

Abstract
Three-dimensional (3D) bioprinted culture systems allow to accurately control microenvironment components and analyze their effects at cellular and tissue levels. The main objective of this study was to identify, quantify and localize the effects of physical-chemical communication signals between tumor cells and the surrounding biomaterial stiffness over time, defining how aggressiveness increases in SK-N-BE(2) neuroblastoma (NB) cell line. Biomimetic hydrogels with SK-N-BE(2) cells, methacrylated gelatin and increasing concentrations of methacrylated alginate (AlgMA 0%, 1% and 2%) were used. Young’s modulus was used to define the stiffness of bioprinted hydrogels and NB tumors. Stained sections of paraffin-embedded hydrogels were digitally quantified. Human NB and 1% AlgMA hydrogels presented similar Young´s modulus mean, and orthotopic NB mice tumors were equally similar to 0% and 1% AlgMA hydrogels. Porosity increased over time; cell cluster density decreased over time and with stiffness, and cell cluster occupancy generally increased with time and decreased with stiffness. In addition, cell proliferation, mRNA metabolism and antiapoptotic activity advanced over time and with stiffness. Together, this rheological, optical and digital data show the potential of the 3D in vitro cell model described herein to infer how intercellular space stiffness patterns drive the clinical behavior associated with NB patients.

Abstract
Abstract For patients with soft tissue defects, repair with autologous in vitro engineered adipose tissue could be a promising alternative to current surgical therapies. A volume-persistent engineered adipose tissue construct under in vivo conditions can only be achieved by early vascularization after transplantation. The combination of 3D bioprinting technology with self-assembling microvascularized units as building blocks can potentially answer the need for a microvascular network. In the present study, co-culture spheroids combining adipose-derived stem cells (ASC) and human umbilical vein endothelial cells (HUVEC) were created with an ideal geometry for bioprinting. When applying the favourable seeding technique and condition, compact viable spheroids were obtained, demonstrating high adipogenic differentiation and capillary-like network formation after 7 and 14 days of culture, as shown by live/dead analysis, immunohistochemistry and RT-qPCR. Moreover, we were able to successfully 3D bioprint the encapsulated spheroids, resulting in compact viable spheroids presenting capillary-like structures, lipid droplets and spheroid outgrowth after 14 days of culture. This is the first study that generates viable high-throughput (pre-)vascularized adipose microtissues as building blocks for bioprinting applications using a novel ASC/HUVEC co-culture spheroid model, which enables both adipogenic differentiation while simultaneously supporting the formation of prevascular-like structures within engineered tissues in vitro.

Abstract
One of the most damaging pathologies that affects the health of both soft and hard tissues around the tooth is periodontitis. Clinically, periodontal tissue destruction has been managed by an integrated approach involving elimination of injured tissues followed by regenerative strategies with bone substitutes and/or barrier membranes. Regrettably, a barrier membrane with predictable mechanical integrity and multifunctional therapeutic features has yet to be established. Herein, we report a fiber-reinforced hydrogel with unprecedented tunability in terms of mechanical competence and therapeutic features by integration of highly porous poly(ε-caprolactone) fibrous mesh(es) with well-controlled 3D architecture into bioactive amorphous magnesium phosphate-laden gelatin methacryloyl hydrogels. The presence of amorphous magnesium phosphate and PCL mesh in the hydrogel can control the mechanical properties and improve the osteogenic ability, opening a tremendous opportunity in guided bone regeneration (GBR). Results demonstrate that the presence of PCL meshes fabricated via melt electrowriting can delay hydrogel degradation preventing soft tissue invasion and providing the mechanical barrier to allow time for slower migrating progenitor cells to participate in bone regeneration due to their ability to differentiate into bone-forming cells. Altogether, our approach offers a platform technology for the development of the next-generation of GBR membranes with tunable mechanical and therapeutic properties to amplify bone regeneration in compromised sites.

Abstract
Three-dimensional printed hydrogel constructs with well-organized melt electrowritten (MEW) fibre-reinforcing scaffolds have been demonstrated as a promising regenerative approach to treat small cartilage defects. Here, we investige how to translate the fabrication of small fibre-reinforced structures on flat surfaces to anatomically relevant structures. In particular, the accurate deposition of MEW-fibres onto curved surfaces of conductive and non-conductive regenerative biomaterials is studied. This study reveals that clinically relevant materials with low conductivities are compatible with resurfacing with organized MEW fibres. Importantly, accurate patterning on non-flat surfaces was successfully shown, provided that a constant electrical field strength and an electrical force normal to the substrate material is maintained. Furthermore, the application of resurfacing the geometry of the medial human femoral condyle is confirmed by the fabrication of a personalised osteochondral implant. The implant composed of an articular cartilage-resident chondroprogenitor cells (ACPCs)-laden hydrogel reinforced with a well-organized MEW scaffold retained its personalised shape, improved its compressive properties and supported neocartilage formation after 28 days in vitro culture. Overall, this study establishes the groundwork for translating MEW from planar and non-resorbable material substrates to anatomically relevant geometries and regenerative materials that the regenerative medicine field aims to create.

Abstract
Successful tissue engineering requires the generation of human scale implants that mimic the structure, composition and mechanical properties of native tissues. Here, we report a novel biofabrication strategy that enables the engineering of structurally organised tissues by guiding the growth of cellular spheroids within arrays of 3D printed polymeric microchambers. With the goal of engineering stratified articular cartilage, inkjet bioprinting was used to deposit defined numbers of mesenchymal stromal cells (MSCs) and chondrocytes into pre-printed microchambers. These jetted cell suspensions rapidly underwent condensation within the hydrophobic microchambers, leading to the formation of organised arrays of cellular spheroids. The microchambers were also designed to provide boundary conditions to these spheroids, guiding their growth and eventual fusion, leading to the development of stratified cartilage tissue with a depth-dependant collagen fiber architecture that mimicked the structure of native articular cartilage. Furthermore, the composition and biomechanical properties of the bioprinted cartilage was also comparable to the native tissue. Using multi-tool biofabrication, we were also able to engineer anatomically accurate, human scale, osteochondral templates by printing this microchamber system on top of a hypertrophic cartilage region designed to support endochondral bone formation and then maintaining the entire construct in long-term bioreactor culture to enhance tissue development. This bioprinting strategy provides a versatile and scalable approach to engineer structurally organised cartilage tissues for joint resurfacing applications.

Abstract
Two-dimensional (2D) cell cultures do not reflect the in vivo situation, and thus it is important to develop predictive three-dimensional (3D) in vitro models with enhanced reliability and robustness for drug screening applications. Treatments against muscle-related diseases are becoming more prominent due to the growth of the aging population worldwide. In this study, we describe a novel drug screening platform with automated production of 3D musculoskeletal-tendon-like tissues. With 3D bioprinting, alternating layers of photo-polymerized gelatin-methacryloyl-based bioink and cell suspension tissue models were produced in a dumbbell shape onto novel postholder cell culture inserts in 24-well plates. Monocultures of human primary skeletal muscle cells and rat tenocytes were printed around and between the posts. The cells showed high viability in culture and good tissue differentiation, based on marker gene and protein expressions. Different printing patterns of bioink and cells were explored and calcium signaling with Fluo4-loaded cells while electrically stimulated was shown. Finally, controlled co-printing of tenocytes and myoblasts around and between the posts, respectively, was demonstrated followed by co-culture and co-differentiation. This screening platform combining 3D bioprinting with a novel microplate represents a promising tool to address musculoskeletal diseases.

Abstract
Abstract Fabrication of biomimetic tissues holds much promise for the regeneration of cells or organs that are lost or damaged due to injury or disease. To enable the generation of complex, multicellular tissues on demand, the ability to design and incorporate different materials and cell types needs to be improved. Two techniques are combined: extrusion-based bioprinting, which enables printing of cell-encapsulated hydrogels; and melt electrowriting (MEW), which enables fabrication of aligned (sub)-micrometer fibers into a single-step biofabrication process. Composite structures generated by infusion of MEW fiber structures with hydrogels have resulted in mechanically and biologically competent constructs; however, their preparation involves a two-step fabrication procedure that limits freedom of design of microfiber architectures and the use of multiple materials and cell types. How convergence of MEW and extrusion-based bioprinting allows fabrication of mechanically stable constructs with the spatial distributions of different cell types without compromising cell viability and chondrogenic differentiation of mesenchymal stromal cells is demonstrated for the first time. Moreover, this converged printing approach improves freedom of design of the MEW fibers, enabling 3D fiber deposition. This is an important step toward biofabrication of voluminous and complex hierarchical structures that can better resemble the characteristics of functional biological tissues.

Abstract
Abstract Herein, we report the first study to create a three-dimensional (3D) bioprinted artificial larynx for whole-laryngeal replacement. Our 3D bio-printed larynx was generated using extrusion-based 3D bioprinter with rabbit's chondrocyte-laden gelatin methacryloyl (GelMA)/glycidyl-methacrylated hyaluronic acid (GMHA) hybrid bioink. We used a polycaprolactone (PCL) outer framework incorporated with pores to achieve the structural strength of printed constructs, as well as to provide a suitable microenvironment to support printed cells. Notably, we established a novel fluidics supply (FS) system that simultaneously supplies basal medium together with a 3D bioprinting process, thereby improving cell survival during the printing process. Our results showed that the FS system enhanced post-printing cell viability, which enabled the generation of a large-scale cell-laden artificial laryngeal framework. Additionally, the incorporation of the PCL outer framework with pores and inner hydrogel provides structural stability and sufficient nutrient/oxygen transport. An animal study confirmed that the transplanted 3D bio-larynx successfully maintained the airway. With further development, our new strategy holds great potential for fabricating human-scale larynxes with in vivo-like biological functions for laryngectomy patients.

Abstract
Abstract Liver parenchymal microtissues (LPMTs) are three-dimensional (3D) aggregates of hepatocytes that recapitulate in vivo-like cellular assembly. They are considered as a valuable model to study drug metabolism, disease biology, and serve as ideal building blocks for liver tissue engineering. However, their integration into the mainstream drug screening process has been hindered due to the lack of simple, rapid techniques to produce a large number of uniform microtissues and preserve their structural–functional integrity over the long term. Here, we present a high-throughput methodology to produce LPMTs in a novel, economic, and reusable Hanging-drop Culture Chamber (HdCC). A drop-on-demand bioprinting approach was optimized to generate droplets of HepG2 cell suspension on a polyethylene terephthalate substrate. The substrates carrying droplets were placed inside a novel HdCC and incubated to obtain 1600 LPMTs having a size of 200–300 μm. Tissue size, cell viability, cellular arrangement and polarity, and insulin-mediated glucose uptake by LPMTs were analyzed. The microtissues were viable and exhibited an active response to insulin stimulation. Cells within the microtissue reorganized to form hepatic plate-like structures and expressed apical (Multidrug Resistance Protein 2 [MRP2]) and epithelial (Zonula Occludens 1 [ZO1]) markers. Further to maintain the structural integrity and enhance the functional capabilities, LPMTs were sandwiched within gelatin methacrylamide (GelMA) hydrogel and the liver-specific functions were monitored for 2 weeks. The results showed that the 3D structure of LPMTs in GelMA sandwich was maintained while the albumin secretion, urea synthesis, and cytochrome P450 activity were enhanced compared with LPMTs in suspension. In conclusion, this study presents a novel culture chamber for mass production of microtissues and a method for enhancing organ-specific functions of LPMTs in vitro.

Abstract
Bioprinting technologies are powerful new bioengineering tools that can spatially reproduce multiple microenvironmental cues in a highly controlled, tunable, and precise manner. In this study, microvalve bioprinting technology was successfully used to print in close proximity endothelial and tumor cells at higher concentrations than previously thought possible, while preserving their viability. We propose that the resulting multicellular models, bioprinted in a controlled extracellular matrix microenvironment, are well-suited to study endothelial and cancer cell crosstalk within a cancer niche. As proof of concept, microvalve bioprinting was applied to the bioengineering of a simplified glioblastoma model in which biological processes involved in tumor expansion, such as tumor cell invasion patterns, cell proliferation, and senescence could be easily visualized and quantified. In this model, U251 glioblastoma cells and primary human umbilical vein endothelial cells (HUVECs) exhibited good printability and high viability after printing. U251 cells formed physiologically relevant clusters and invasion margins, while HUVECs generated vascular-like networks when primary fibroblasts were added to the model. An oxidative stress mimicking the one encountered within a tumor microenvironment during radiotherapy or genotoxic chemotherapy was shown to both diminish endothelial cells proliferation and to increase their senescence. Results also suggested that stressed glioblastoma cells may alter normal endothelial cell proliferation but not impact their senescence. This data demonstrates the potential of microvalve bioprinting to fabricate in vitro models that can help decipher endothelial and tumor cell crosstalk, within controlled and modulable microenvironments, and can then be used to address critical questions in the context of cancer recurrence.

Abstract
Cartilage regeneration is challenging because of the poor intrinsic self-repair capacity of avascular tissue. Three-dimensional (3D) bioprinting has gained significant attention in the field of tissue engineering and is a promising technology to overcome current difficulties in cartilage regeneration. Although bioink is an essential component of bioprinting technology, several challenges remain in satisfying different requirements for ideal bioink, including biocompatibility and printability based on specific biological requirements. Gelatin and hyaluronic acid (HA) have been shown to be ideal biomimetic hydrogel sources for cartilage regeneration. However, controlling their structure, mechanical properties, biocompatibility, and degradation rate for cartilage repair remains a challenge. Here, we show a photocurable bioink created by hybridization of gelatin methacryloyl (GelMA) and glycidyl-methacrylated HA (GMHA) for material extrusion 3D bioprinting in cartilage regeneration. GelMA and GMHA were mixed in various ratios, and the mixture of 7% GelMA and 5% GMHA bioink (G7H5) demonstrated the most reliable mechanical properties, rheological properties, and printability. This G7H5 bioink allowed us to build a highly complex larynx structure, including the hyoid bone, thyroid cartilage, cricoid cartilage, arytenoid cartilage, and cervical trachea. This bioink also provided an excellent microenvironment for chondrogenesis of tonsil-derived mesenchymal stem cells (TMSCs) in vitro and in vivo. In summary, this study presents the ideal formulation of GelMA/GMHA hybrid bioink to generate a well-suited photocurable bioink for cartilage regeneration of TMSCs using a material extrusion bioprinter, and could be applied to cartilage tissue engineering.

Abstract
The increasing number of mastectomies results in a greater demand for breast reconstruction characterized by simplicity and a low complication profile. Reconstructive surgeons are investigating tissue engineering (TE) strategies to overcome the current surgical drawbacks. 3D bioprinting is the rising technique for the fabrication of large tissue constructs which provides a potential solution for unmet clinical needs in breast reconstruction building on decades of experience in autologous fat grafting, adipose-derived mesenchymal stem cell (ASC) biology and TE. A scaffold was bioprinted using encapsulated ASC spheroids in methacrylated gelatin ink (GelMA). Uniform ASC spheroids with an ideal geometry and diameter for bioprinting were formed, using a high-throughput non-adhesive agarose microwell system. ASC spheroids in adipogenic differentiation medium (ADM) were evaluated through live/dead staining, histology (HE, Oil Red O), TEM and RT-qPCR. Viable spheroids were obtained for up to 14 days post-printing and showed multilocular microvacuoles and successful differentiation toward mature adipocytes shown by gene expression analysis. Moreover, spheroids were able to assemble at random in GelMA, creating a macrotissue. Combining the advantage of microtissues to self-assemble and the controlled organization by bioprinting technologies, these ASC spheroids can be useful as building blocks for the engineering of soft tissue implants.

Abstract
Bone tissue engineering strategies that recapitulate the developmental process of endochondral ossification offer a promising route to bone repair. Clinical translation of such endochondral tissue engineering strategies will require overcoming a number of challenges, including the engineering of large and often anatomically complex cartilage grafts, as well as the persistence of core regions of avascular cartilage following their implantation into large bone defects. Here 3D printing technology is utilized to develop a versatile and scalable approach to guide vascularisation during endochondral bone repair. First, a sacrificial pluronic ink was used to 3D print interconnected microchannel networks in a mesenchymal stem cell (MSC) laden gelatin-methacryloyl (GelMA) hydrogel. These constructs (with and without microchannels) were next chondrogenically primed in vitro and then implanted into critically sized femoral bone defects in rats. The solid and microchanneled cartilage templates enhanced bone repair compared to untreated controls, with the solid cartilage templates (without microchannels) supporting the highest levels of total bone formation. However, the inclusion of 3D printed microchannels was found to promote osteoclast/immune cell invasion, hydrogel degradation, and vascularisation following implantation. In addition, the endochondral bone tissue engineering strategy was found to support comparable levels of bone healing to BMP-2 delivery, whilst promoting lower levels of heterotopic bone formation, with the microchanneled templates supporting the lowest levels of heterotopic bone formation. Taken together, these results demonstrate that 3D printed hypertrophic cartilage grafts represent a promising approach for the repair of complex bone fractures, particularly for larger defects where vascularisation will be a key challenge.

Abstract
Abstract Hydrogels are excellent mimetics of mammalian extracellular matrices and have found widespread use in tissue engineering. Nanoporosity of monolithic bulk hydrogels, however, limits mass transport of key biomolecules. Microgels used in 3D bioprinting achieve both custom shape and vastly improved permissivity to an array of cell functions, however spherical-microbead-based bioinks are challenging to upscale, are inherently isotropic, and require secondary crosslinking. Here, bioinks based on high-aspect-ratio hydrogel microstrands are introduced to overcome these limitations. Pre-crosslinked, bulk hydrogels are deconstructed into microstrands by sizing through a grid with apertures of 40–100 µm. The microstrands are moldable and form a porous, entangled structure, stable in aqueous medium without further crosslinking. Entangled microstrands have rheological properties characteristic of excellent bioinks for extrusion bioprinting. Furthermore, individual microstrands align during extrusion and facilitate the alignment of myotubes. Cells can be placed either inside or outside the hydrogel phase with >90% viability. Chondrocytes co-printed with the microstrands deposit abundant extracellular matrix, resulting in a modulus increase from 2.7 to 780.2 kPa after 6 weeks of culture. This powerful approach to deconstruct bulk hydrogels into advanced bioinks is both scalable and versatile, representing an important toolbox for 3D bioprinting of architected hydrogels.

Abstract
Acquired muscle diseases such as cancer cachexia are responsible for the poor prognosis of many patients suffering from cancer. In vitro models are needed to study the underlying mechanisms of those pathologies. Extrusion bioprinting is an emerging tool to emulate the aligned architecture of fibers while implementing additive manufacturing techniques in tissue engineering. However, designing bioinks that reconcile the rheological needs of bioprinting and the biological requirements of muscle tissue is a challenging matter. Here we formulate a biomaterial with dual crosslinking to modulate the physical properties of bioprinted models. We design 3D bioprinted muscle models that resemble the mechanical properties of native tissue and show improved proliferation and high maturation of differentiated myotubes suggesting that the GelMA-AlgMA-Fibrin biomaterial possesses myogenic properties. The electrical stimulation of the 3D model confirmed the contractile capability of the tissue and enhanced the formation of sarcomeres. Regarding the functionality of the models, they served as platforms to recapitulate skeletal muscle diseases such as muscle wasting produced by cancer cachexia. The genetic expression of 3D models demonstrated a better resemblance to the muscular biopsies of cachectic mouse models. Altogether, this biomaterial is aimed to fabricate manipulable skeletal muscle in vitro models in a non-costly, fast and feasible manner.

Abstract
Bone tissue engineering (BTE) has proven to be a promising strategy for bone defect repair. Due to its excellent biological properties, gelatin methacrylate (GelMA) hydrogels have been used as bioinks for 3D bioprinting in some BTE studies to produce scaffolds for bone regeneration. However, applications for load-bearing defects are limited by poor mechanical properties and a lack of bioactivity. In this study, 3D printing technology was used to create nano-attapulgite (nano-ATP)/GelMA composite hydrogels loaded into mouse bone mesenchymal stem cells (BMSCs) and mouse umbilical vein endothelial cells (MUVECs). The bioprintability, physicochemical properties, and mechanical properties were all thoroughly evaluated. Our findings showed that nano-ATP groups outperform the control group in terms of printability, indicating that nano-ATP is beneficial for printability. Additionally, after incorporation with nano-ATP, the mechanical strength of the composite hydrogels was significantly improved, resulting in adequate mechanical properties for bone regeneration. The presence of nano-ATP in the scaffolds has also been studied for cell-material interactions. The findings show that cells within the scaffold not only have high viability but also a clear proclivity to promote osteogenic differentiation of BMSCs. Besides, the MUVECs-loaded composite hydrogels demonstrated increased angiogenic activity. A cranial defect model was also developed to evaluate the bone repair capability of scaffolds loaded with rat BMSCs. According to histological analysis, cell-laden nano-ATP composite hydrogels can effectively improve bone regeneration and promote angiogenesis. This study demonstrated the potential of nano-ATP for bone tissue engineering, which should also increase the clinical practicality of nano-ATP.

Abstract
The advent of improved fabrication technologies{,} particularly 3D printing{,} has enabled the engineering of bone tissue for patient-specific healing and the fabrication of in vitro tissue models for ex vivo testing. However{,} inks made from natural polymers often fall short in terms of mechanical strength{,} stability{,} and the induction of osteogenesis. Our research focused on developing novel printable formulations using a gelatin/pectin polymeric matrix that integrate synergistic reinforcement components i.e. graphene oxide (GO) and oxidized nanocellulose fibers (CNF). Using 3D printing technology and the aforementioned biomaterial composite inks{,} bone-like scaffolds were created. To simulate critical-sized flaws and demonstrate scaffold fidelity{,} 3D scaffolds were successfully printed using formulations with varied GO concentrations (0.25{,} 0.5{,} and 1% wt with respect to polymer content). The addition of GO to hydrogel inks enhanced not only the compressive modulus but also the printability and scaffold fidelity compared to the pure colloid-gelatin/pectin system. Due to its strong potential for 3D bioprinting{,} the sample containing 0.5% GO is shown to have the greatest perspectives for bone tissue models and tissue engineering applications.

Abstract
Three-dimensional (3D) liver bioprinting is a promising technique for creating 3D liver models that can be used for in vitro drug testing, hepatotoxicity studies, and transplantation. The functional performance of 3D bioprinted liver constructs are limited by the lack of cell–cell interactions, which calls for the creation of bioprinted tissue constructs with high cell densities. This study reports the fabrication of 3D bioprinted liver constructs using a novel photocrosslinkable gelatin methacrylamide (GelMA)-based bioink formulation. However, the formation of excess free radicals during photoinitiation poses a challenge, particularly during photocrosslinking of large constructs with high cell densities. Hence, we designed a bioink formulation comprising the base polymer GelMA loaded with an antioxidant cocktail containing vitamin C (L-ascorbic acid (AA)) and vitamin E (α-tocopherol (α-Toc)). We confirmed that the combination of antioxidants loaded in GelMA enhanced the ability to scavenge intracellular reactive oxygen species formed during photocrosslinking. The GelMA formulation was evaluated for biocompatibility in vitro and in vivo. These results demonstrated that the bioink had adequate rheological characteristics and was biocompatible. Furthermore, when compared to bioprinted constructs with lower cell density, high-density primary rat hepatocyte constructs demonstrated improved cell-cell interactions and liver-specific functions like albumin and urea secretion, which increased 5-fold and 2.5-fold, respectively.

Abstract
This work reports the construction of a bicomponent scaffold co-loaded with both a prodrug and a drug (BiFp@Ht) as an efficient platform for wound dressing, by combining the electrospinning and 3D-printing technologies. The outer component consisted of a chitosan/polyethylene oxide-electrospun membrane loaded with the indomethacin–polyethylene glycol–indomethacin prodrug (Fp) and served as a support for printing the inner component, a gelatin methacryloyl/sodium alginate hydrogel loaded with tetracycline hydrochloride (Ht). The different architectural characteristics of the electrospun and 3D-printed layers were very well highlighted in a morphological analysis performed by Scanning Electron Microscopy (SEM). In vitro release profile studies demonstrated that both Fp and Ht layers were capable to release the loaded therapeutics in a controlled and sustained manner. According to a quantitative in vitro biological assessment, the bicomponent BiFp@Ht scaffold showed a good biocompatibility and no cytotoxic effect on HeLa cell cultures, while the highest proliferation level was noted in the case of HeLa cells seeded onto an Fp nanofibrous membrane. Furthermore, the BiFp@Ht scaffold presented an excellent antimicrobial activity against the E. coli and S. aureus bacterial strains, along with promising anti-inflammatory and proangiogenic activities, proving its potential to be used for wound dressing.

Abstract
Fibrous structures with anisotropic fillers as composites have found increasing interest in the field of biofabrication since they can mimic the extracellular matrix of anisotropic tissues such as skeletal muscle or nerve tissue. In the present work, the inclusion of anisotropic fillers in hydrogel-based filaments with an interpenetrating polymeric network (IPN) was evaluated and the dynamics of such fillers in the composite flow were analyzed using computational simulations. In the experimental part, microfabricated rods (200 and 400 μm length, 50 μm width) were used as anisotropic fillers in extrusion of composite filaments using two techniques of wet spinning and 3D printing. Hydrogels such as oxidized alginate (ADA) and methacrylated gelatin (GelMA) were used as matrices. In the computational simulation, a combination of computational fluid dynamics and coarse-grained molecular dynamics was used to study the dynamics of rod-like fillers in the flow field of a syringe. It showed that, during the extrusion process, microrods are far from being well aligned. Instead, many of them tumble on their way through the needle leading to a random orientation in the fiber which was confirmed experimentally.

Abstract
Introduction: Three-dimensional (3D) bioprinting is a promising technique for the development of neuronal in vitro models because it controls the deposition of materials and cells. Finding a biomaterial that supports neural differentiation in vitro while ensuring compatibility with the technique of 3D bioprinting of a self-standing construct is a challenge.Methods: In this study, gelatin methacryloyl (GelMA), methacrylated alginate (AlgMA), and hyaluronic acid (HA) were examined by exploiting their biocompatibility and tunable mechanical properties to resemble the extracellular matrix (ECM) and to create a suitable material for printing neural progenitor cells (NPCs), supporting their long-term differentiation. NPCs were printed and differentiated for up to 15 days, and cell viability and neuronal differentiation markers were assessed throughout the culture.Results and Discussion: This composite biomaterial presented the desired physical properties to mimic the ECM of the brain with high water intake, low stiffness, and slow degradation while allowing the printing of defined structures. The viability rates were maintained at approximately 80% at all time points. However, the levels of β-III tubulin marker increased over time, demonstrating the compatibility of this biomaterial with neuronal cell culture and differentiation. Furthermore, these cells showed increased maturation with corresponding functional properties, which was also demonstrated by the formation of a neuronal network that was observed by recording spontaneous activity via Ca2+ imaging.

Abstract
(1) Background: Synovial tissue plays a fundamental role in inflammatory processes. Therefore, understanding the mechanisms regulating healthy and diseased synovium functions, as in rheumatic diseases, is crucial to discovering more effective therapies to minimize or prevent pathological progress. The present study aimed at developing a bioartificial synovial tissue as an in vitro model for drug screening or personalized medicine applications using 3D bioprinting technology. (2) Methods: The volumetric extrusion technique has been used to fabricate cell-laden scaffolds. Gelatin Methacryloyl (GelMA), widely applied in regenerative medicine and tissue engineering, was selected as a bioink and combined with an immortalized cell line of fibroblast-like synoviocytes (K4IM). (3) Results: Three different GelMA formulations, 7.5–10–12.5% w/v, were tested for the fabrication of the scaffold with the desired morphology and internal architecture. GelMA 10% w/v was chosen and combined with K4IM cells to fabricate scaffolds that showed high cell viability and negligible cytotoxicity for up to 14 days tested by Live & Dead and lactate dehydrogenase assays. (4) Conclusions: We successfully 3D bioprinted synoviocytes-laden scaffolds as a proof-of-concept (PoC) towards the fabrication of a 3D synovial membrane model suitable for in vitro studies. However, further research is needed to reproduce the complexity of the synovial microenvironment to better mimic the physiological condition.

Abstract
The field of bone tissue engineering has seen significant advancements in recent years. Each year, over two million bone transplants are performed globally, and conventional treatments, such as bone grafts and metallic implants, have their limitations. Tissue engineering offers a new level of treatment, allowing for the creation of living tissue within a biomaterial framework. Recent advances in biomaterials have provided innovative approaches to rebuilding bone tissue function after damage. Among them, gelatin methacryloyl (GelMA) hydrogel is emerging as a promising biomaterial for supporting cell proliferation and tissue regeneration, and GelMA has exhibited exceptional physicochemical and biological properties, making it a viable option for clinical translation. Various methods and classes of additives have been used in the application of GelMA for bone regeneration, with the incorporation of nanofillers or other polymers enhancing its resilience and functional performance. Despite promising results, the fabrication of complex structures that mimic the bone architecture and the provision of balanced physical properties for both cell and vasculature growth and proper stiffness for load bearing remain as challenges. In terms of utilizing osteogenic additives, the priority should be on versatile components that promote angiogenesis and osteogenesis while reinforcing the structure for bone tissue engineering applications. This review focuses on recent efforts and advantages of GelMA-based composite biomaterials for bone tissue engineering, covering the literature from the last five years.

Abstract
Fluid pressure develops transiently within mechanically-loaded, cell-embedding hydrogels, but its magnitude depends on the intrinsic material properties of the hydrogel and cannot be easily altered. The recently developed melt-electrowriting (MEW) technique enables three-dimensional printing of structured fibrous mesh with small fibre diameter (20 μm). The MEW mesh with 20 μm fibre diameter can synergistically increase the instantaneous mechanical stiffness of soft hydrogels. However, the reinforcing mechanism of the MEW meshes is not well understood, and may involve load-induced fluid pressurisation. Here, we examined the reinforcing effect of MEW meshes in three hydrogels: gelatin methcryloyl (GelMA), agarose and alginate, and the role of load-induced fluid pressurisation in the MEW reinforcement. We tested the hydrogels with and without MEW mesh (i.e., hydrogel alone, and MEW-hydrogel composite) using micro-indentation and unconfined compression, and analysed the mechanical data using biphasic Hertz and mixture models. We found that the MEW mesh altered the tension-to-compression modulus ratio differently for hydrogels that are cross-linked differently, which led to a variable change to their load-induced fluid pressurisation. MEW meshes only enhanced the fluid pressurisation for GelMA, but not for agarose or alginate. We speculate that only covalently cross-linked hydrogels (GelMA) can effectively tense the MEW meshes, thereby enhancing the fluid pressure developed during compressive loading. In conclusion, load-induced fluid pressurisation in selected hydrogels was enhanced by MEW fibrous mesh, and may be controlled by MEW mesh of different designs in the future, thereby making fluid pressure a tunable cell growth stimulus for tissue engineering involving mechanical stimulation.

Abstract
Gelatin methacryloyl (GelMA) hydrogels have been extensively used for drug delivery and tissue engineering applications due to their good biocompatibility, biodegradability, and controllable photocurable efficiency. Phosphate buffer solution (PBS) is the most widely used reaction system for GelMA synthesis. However, carbonate-bicarbonate buffer solution (CBS) has been tried recently for synthesizing GelMA due to its high reaction efficiency. However, there is a lack of systematic investigation into possible differences in the structure and properties of GelMA synthesized in PBS and CBS, respectively. Therefore, in the current study, GelMA molecules with two degrees of methacryloylation (∼20 and ∼80%) were synthesized under PBS and CBS reaction systems, respectively, in comparable conditions. The results showed that because of the functionalization of methacrylate groups in gelatin chains, which could interfere with the intrachain and interchain interactions, such as hydrogen bonding, the GelMA molecules synthesized in PBS had distinct physical structures and exhibited different properties in comparison with those produced in CBS. GelMA hydrogels synthesized in PBS exhibited higher gel-sol transition temperatures and better photocurable efficiencies, mechanical strength, and biological properties. In contrast, GelMA hydrogels produced in CBS showed advantages in swelling performance and microstructures, such as pore sizes and porosities. In addition, GelMA synthesized in PBS and possessing a high degree of methacryloylation (the “GelMA-PH” polymer) showed great potential for three-dimensional (3D) bioprinting. This focused study has gained helpful new insights into GelMA and can provide guidance on the application of GelMA in 3D printing and tissue engineering.

Abstract
The main objective was to produce 3D printable hydrogels based on GelMA and hydroxyapatite doped with cerium ions with potential application in bone regeneration. The first part of the study regards the substitution of Ca2+ ions from hydroxyapatite structure with cerium ions (Ca10-xCex(PO4)6(OH)2, xCe = 0.1, 0.3, 0.5). The second part followed the selection of the optimal concentration of HAp doped, which will ensure GelMA-based scaffolds with good biocompatibility, viability and cell proliferation. The third part aimed to select the optimal concentrations of GelMA for the 3D printing process (20%, 30% and 35%). In vitro biological assessment presented the highest level of cell viability and proliferation potency of GelMA-HC5 composites, along with a low cytotoxic potential, highlighting the beneficial effects of cerium on cell growth, also supported by Live/Dead results. According to the 3D printing experiments, the 30% GelMA enriched with HC5 was able to generate 3D scaffolds with high structural integrity and homogeneity, showing the highest suitability for the 3D printing process. The osteogenic differentiation experiments confirmed the ability of 30% GelMA-3% HC5 scaffold to support and efficiently maintain the osteogenesis process. Based on the results, 30% GelMA-3% HC5 3D printed scaffolds could be considered as biomaterials with suitable characteristics for application in bone tissue engineering.

Abstract
As bone diseases and defects are constantly increasing, the improvement of bone regeneration techniques is constantly evolving. The main purpose of this scientific study was to obtain and investigate biomaterials that can be used in tissue engineering. In this respect, nanocomposite inks of GelMA modified with hydroxyapatite (HA) substituted with Mg and Zn were developed. Using a 3D bioprinting technique, scaffolds with varying shapes and dimensions were obtained. The following analyses were used in order to study the nanocomposite materials and scaffolds obtained by the 3D printing technique: Fourier transform infrared spectrometry and X-ray diffraction (XRD), scanning electron microscopy (SEM), and micro-computed tomography (Micro-CT). The swelling and dissolvability of each scaffold were also studied. Biological studies, osteopontin (OPN), and osterix (OSX) gene expression evaluations were confirmed at the protein levels, using immunofluorescence coupled with confocal microscopy. These findings suggest the positive effect of magnesium and zinc on the osteogenic differentiation process. OSX fluorescent staining also confirmed the capacity of GelMA-HM5 and GelMA-HZ5 to support osteogenesis, especially of the magnesium enriched scaffold.

Abstract
Methacryloyl gelatin (GelMA) is a versatile material for bioprinting because of its tunable physical properties and inherent bioactivity. Bioprinting of GelMA is often met with challenges such as lower viscosity of GelMA inks due to higher methacryloyl substitution and longer physical gelation time at room temperature. In this study, a tunable interpenetrating polymer network (IPN) hydrogel was prepared from gelatin-hyaluronan dialdehyde (Gel-HDA) Schiff’s polymer, and 100% methacrylamide substituted GelMA for biofabrication through extrusion based bioprinting. Temperature sweep rheology measurements show a higher sol-gel transition temperature for IPN (30 °C) compared to gold standard GelMA (27 °C). Furthermore, to determine the tunability of the IPN hydrogel, several IPN samples were prepared by combining different ratios of Gel-HDA and GelMA achieving a compressive modulus ranging from 20.6 ± 2.48 KPa to 116.7 ± 14.80 KPa. Our results showed that the mechanical properties and printability at room temperature could be tuned by adjusting the ratios of GelMA and Gel-HDA. To evaluate cell response to the material, MC3T3-E1 mouse pre-osteoblast cells were embedded in hydrogels and 3D-printed, demonstrating excellent cell viability and proliferation after 10 d of 3D in vitro culture, making the IPN an interesting bioink for the fabrication of 3D constructs for tissue engineering applications.

Abstract
The tumor extracellular matrix (ECM) plays a vital role in tumor progression and drug resistance. Previous studies have shown that breast tissue-derived matrices could be an important biomaterial to recreate the complexity of the tumor ECM. We have developed a method for decellularizing and delipidating a porcine breast tissue (TDM) compatible with hydrogel formation. The addition of gelatin methacrylamide and alginate allows this TDM to be bioprinted by itself with good printability, shape fidelity, and cytocompatibility. Furthermore, this bioink has been tuned to more closely recreate the breast tumor by incorporating collagen type I (Col1). Breast cancer cells (BCCs) proliferate in both TDM bioinks forming cell clusters and spheroids. The addition of Col1 improves the printability of the bioink as well as increases BCC proliferation and reduces doxorubicin sensitivity due to a downregulation of HSP90. TDM bioinks also allow a precise three-dimensional printing of scaffolds containing BCCs and stromal cells and could be used to fabricate artificial tumors. Taken together, we have proven that these novel bioinks are good candidates for biofabricating breast cancer models.

Abstract
Extracellular vesicles (EVs) have garnered growing attention as promising acellular tools for bone repair. Although EVs’ potential for bone regeneration has been shown, issues associated with their therapeutic potency and short half-life in vivo hinders their clinical utility. Epigenetic reprogramming with the histone deacetylase inhibitor Trichostatin A (TSA) has been reported to promote the osteoinductive potency of osteoblast-derived EVs. Gelatin methacryloyl (GelMA) hydrogels functionalised with the synthetic nanoclay laponite (LAP) have been shown to effectively bind, stabilise, and improve the retention of bioactive factors. This study investigated the potential of utilising a GelMA-LAP hydrogel to improve local retention and control delivery of epigenetically enhanced osteoblast-derived EVs as a novel bone repair strategy. LAP was found to elicit a dose-dependent increase in GelMA compressive modulus and shear-thinning properties. Incorporation of the nanoclay was also found to enhance shape fidelity when 3D printed compared to LAP-free gels. Interestingly, GelMA hydrogels containing LAP displayed increased mineralisation capacity (1.41-fold) (p ≤ 0.01) over 14 days. EV release kinetics from these nanocomposite systems were also strongly influenced by LAP concentration with significantly more vesicles being released from GelMA constructs as detected by a CD63 ELISA (p ≤ 0.001). EVs derived from TSA-treated osteoblasts (TSA-EVs) enhanced proliferation (1.09-fold), migration (1.83-fold), histone acetylation (1.32-fold) and mineralisation (1.87-fold) of human bone marrow stromal cells (hBMSCs) when released from the GelMA-LAP hydrogel compared to the untreated EV gels (p ≤ 0.01). Importantly, the TSA-EV functionalised GelMA-LAP hydrogel significantly promoted encapsulated hBMSCs extracellular matrix collagen production (≥1.3-fold) and mineralisation (≥1.78-fold) in a dose-dependent manner compared to untreated EV constructs (p ≤ 0.001). Taken together, these findings demonstrate the potential of combining epigenetically enhanced osteoblast-derived EVs with a nanocomposite photocurable hydrogel to promote the therapeutic efficacy of acellular vesicle approaches for bone regeneration.

Abstract
Abstract 3D-printed artificial skeletal muscle, which mimics the structural and functional characteristics of native skeletal muscle, is a promising treatment method for muscle reconstruction. Although various fabrication techniques for skeletal muscle using 3D bio-printers are studied, it is still challenging to build a functional muscle structure. A strategy using microvalve-assisted coaxial 3D bioprinting in consideration of functional skeletal muscle fabrication is reported. The unit (artificial muscle fascicle: AMF) of muscle mimetic tissue is composed of a core filled with medium-based C2C12 myoblast aggregates as a role of muscle fibers and a photo cross-linkable hydrogel-based shell as a role of connective tissue in muscles that enhances printability and cell adhesion and proliferation. Especially, a microvalve system is applied for the core part with even cell distribution and strong cell–cell interaction. This system enhances myotube formation and consequently shows spontaneous contraction. A multi-printed AMF (artificial muscle tissue: AMT) as a piece of muscle is implanted into the anterior tibia (TA) muscle defect site of immunocompromised rats. As a result, the TA-implanted AMT responds to electrical stimulation and represents histologically regenerated muscle tissue. This microvalve-assisted coaxial 3D bioprinting shows a significant step forward to mimicking native skeletal muscle tissue.

Abstract
The investigation of novel hydrogel systems allows for the study of relationships between biomaterials, cells, and other factors within osteochondral tissue engineering. Three-dimensional (3D) printing is a popular research method that can allow for further interrogation of these questions via the fabrication of 3D hydrogel environments that mimic tissue-specific, complex architectures. However, the adaptation of promising hydrogel biomaterial systems into 3D-printable bioinks remains a challenge. Here, we delineated an approach to that process. First, we characterized a novel methacryloylated gelatin composite hydrogel system and assessed how calcium phosphate and glycosaminoglycan additives upregulated bone- and cartilage-like matrix deposition and certain genetic markers of differentiation within human mesenchymal stem cells (hMSCs), such as RUNX2 and SOX9. Then, new assays were developed and utilized to study the effects of xanthan gum and nanofibrillated cellulose, which allowed for cohesive fiber deposition, reliable droplet formation, and non-fracturing digital light processing (DLP)-printed constructs within extrusion, inkjet, and DLP techniques, respectively. Finally, these bioinks were used to 3D print constructs containing viable encapsulated hMSCs over a 7 d period, where DLP printed constructs facilitated the highest observed increase in cell number over 7 d (∼2.4×). The results presented here describe the promotion of osteochondral phenotypes via these novel composite hydrogel formulations, establish their ability to bioprint viable, cell-encapsulating constructs using three different 3D printing methods on multiple bioprinters, and document how a library of modular bioink additives affected those physicochemical properties important to printability.

Abstract
Bioprinting is a biofabrication technology which allows efficient and large-scale manufacture of 3D cell culture systems. However, the available biomaterials for bioinks used in bioprinting are limited by their printability and biological functionality. Fabricated constructs are often homogeneous and have limited complexity in terms of current 3D cell culture systems comprising multiple cell types. Inspired by the phenomenon that hydrogels can exchange liquids under the infiltration action, infiltration-induced suspension bioprinting (IISBP), a novel printing technique based on a hyaluronic acid (HA) suspension system to modulate the properties of the printed scaffolds by infiltration action, was described in this study. HA served as a suspension system due to its shear-thinning and self-healing rheological properties, simplicity of preparation, reusability, and ease of adjustment to osmotic pressure. Changes in osmotic pressure were able to direct the swelling or shrinkage of 3D printed gelatin methacryloyl (GelMA)-based bioinks, enabling the regulation of physical properties such as fiber diameter, micromorphology, mechanical strength, and water absorption of 3D printed scaffolds. Human umbilical vein endothelial cells (HUVEC) were applied as a cell culture model and printed within cell-laden scaffolds at high resolution and cell viability with the IISBP technique. Herein, the IISBP technique had been realized as a reliable hydrogel-based bioprinting technique, which enabled facile modulation of 3D printed hydrogel scaffolds properties, being expected to meet the scaffolds requirements of a wide range of cell culture conditions to be utilized in bioprinting applications.

Abstract
Compared to other conventional scaffold fabrication techniques, three-dimensional (3D) printing is advantageous in producing bone tissue engineering scaffolds with customized shape, tailored pore size/porosity, required mechanical properties and even desirable biomolecule delivery capability. However, for scaffolds with a large volume, it is highly difficult to get seeded cells to migrate to the central region of the scaffolds, resulting in an inhomogeneous cell distribution and therefore lowering the bone forming ability. To overcome this major obstacle, in this study, cell-laden bone tissue engineering scaffolds consisting of osteogenic peptide (OP) loaded β-tricalcium phosphate (TCP)/poly(lactic-co-glycolic acid) (PLGA) (OP/TCP/PLGA, designated as OTP) nanocomposite struts and rat bone marrow derived mesenchymal stem cell (rBMSC)-laden gelatin/GelMA hydrogel rods were produced through ‘dual-nozzle’ low temperature hybrid 3D printing. The cell-laden scaffolds exhibited a bi-phasic structure and had a mechanical modulus of about 19.6 MPa, which was similar to that of human cancellous bone. OP can be released from the hybrid scaffolds in a sustained manner and achieved a cumulative release level of about 78% after 24 d. rBMSCs encapsulated in the hydrogel rods exhibited a cell viability of about 87.4% right after low temperature hybrid 3D printing and could be released from the hydrogel rods to achieve cell anchorage on the surface of adjacent OTP struts. The OP released from OTP struts enhanced rBMSCs proliferation. Compared to rBMSC-laden hybrid scaffolds without OP incorporation, the rBMSC-laden hybrid scaffolds incorporated with OP significantly up-regulated osteogenic differentiation of rBMSCs by showing a higher level of alkaline phosphatase expression and calcium deposition. This ‘proof-of-concept’ study has provided a facile method to form cell-laden bone tissue engineering scaffolds with not only required mechanical strength, biomimetic structure and sustained biomolecule release profile but also excellent cell delivery capability with uniform cell distribution, which can improve the bone forming ability in the body.

Abstract
Abstract Bioprinting is gaining importance for the manufacturing of tailor-made hydrogel scaffolds in tissue engineering, pharmaceutical research and cell therapy. However, structure fidelity and geometric deviations of printed objects heavily influence mass transport and process reproducibility. Fast, three-dimensional and nondestructive quality control methods will be decisive for the approval in larger studies or industry. Magnetic resonance imaging (MRI) meets these requirements for characterizing heterogeneous soft materials with different properties. Complementary to the idea of decentralized 3D printing, magnetic resonance tomography is common in medicine, and image data processing tools can be transferred system-independently. In this study, a MRI measurement and image analysis protocol was evaluated to jointly assess the reproducibility of three different hydrogels and a reference material. Critical parameters for object quality, namely porosity, hole areas and deviations along the height of the scaffolds are discussed. Geometric deviations could be correlated to specific process parameters, anomalies of the ink or changes of ambient conditions. This strategy allows the systematic investigation of complex 3D objects as well as an implementation as a process control tool. Combined with the monitoring of metadata this approach might pave the way for future industrial applications of 3D printing in the field of biopharmaceutics.

Abstract
Methacrylated gelatin (GelMA) in the form of methacryloyl, methacrylate, and methacrylamide is an established and widely accepted photocrosslinkable bioink, for three dimensional bioprinting of various tissues. One of the limitations of photocrosslinkable bioinks is the inability to control the free radicals generated by photoinitiators and ultraviolet (UV) rays. The presence of excess free radicals compromises the viability and functionality of cells during crosslinking. In this study, ascorbic acid, a known free radical scavenger (FRS) molecule, was introduced into the GelMA bioink formulation to protect the cell viability, proliferation, and tissue functions of 3D bioprinted parenchymal liver constructs. The concentration of FRS in the bioink was optimized and used for 3D bioprinting of HepG2 cells. The results confirmed that the inclusion of 3.4 mM FRS in the GelMA bioink formulation nullified the excess ROS formed inside the cells. Furthermore, the optimized GelMA formulation containing FRS preserved and improved the cell activity, albumin, and urea synthesis in the 3D construct over 7 days in culture. In the future, this concept could be implemented in the biofabrication of large liver constructs that require multiple or longer durations of UV irradiation.

Abstract
Engineering scaffolds with a structure mimicking that of native cornea allows for addressing the severe donor shortage for the corneal blindness treatment, which, however, remains challenging. In the light that corneal stromal (CS) cells can play a key role in corneal stroma formation, in this study we incorporated CS cells into three-dimensional (3D) scaffolds printed from hyaluronic acid-modified gelatin-methacrylate (GelMA-HA) scaffolds and characterized the scaffolds in terms of remodeled extracellular matrix (ECM) in vitro. Our results illustrated that the modification of GelMA by HA allowed for 3D printing of corneal scaffolds and further improved the characteristics of primary rabbit-derived corneal stromal cells for remodelling scaffolds. After 60 days, we decellularized the remodeled corneal scaffolds and examined their optical properties; and our results demonstrated that the 3D printed corneal scaffolds provided CS cells with cues that guided them toward the directional and spatial organization and facilitated the ECM remodelling.

Abstract
Understanding the optimal conditions required for bone healing can have a substantial impact to target the problem of non–unions and large bone defects. The combination of bioactive factors, regenerative progenitor cells and biomaterials to form a tissue engineered (TE) complex is a promising solution but translation to the clinic has been slow. We hypothesized the typical material testing algorithm used is insufficient and leads to materials being mischaracterized as promising. In the first part of this study, human bone marrow – derived mesenchymal stromal cells (hBM-MSCs) were embedded in three commonly used biomaterials (hyaluronic acid methacrylate, gelatin methacrylate and fibrin) and combined with relevant bioactive osteogenesis factors (dexamethasone microparticles and polyphosphate nanoparticles) to form a TE construct that underwent in vitro osteogenic differentiation for 28 days. Gene expression of relevant transcription factors and osteogenic markers, and von Kossa staining were performed. In the second and third part of this study, the same combination of TE constructs were implanted subcutaneously (cell containing) in T cell-deficient athymic Crl:NIH-Foxn1rnu rats for 8 weeks or cell free in an immunocompetent New Zealand white rabbit calvarial model for 6 weeks, respectively. Osteogenic performance was investigated via MicroCT imaging and histology staining. The in vitro study showed enhanced upregulation of relevant genes and significant mineral deposition within the three biomaterials, generally considered as a positive result. Subcutaneous implantation indicates none to minor ectopic bone formation. No enhanced calvarial bone healing was detected in implanted biomaterials compared to the empty defect. The reasons for the poor correlation of in vitro and in vivo outcomes are unclear and needs further investigation. This study highlights the discrepancy between in vitro and in vivo outcomes, demonstrating that in vitro data should be interpreted with extreme caution. In vitro models with higher complexity are necessary to increase value for translational studies.
Statement of significance
Preclinical testing of newly developed biomaterials is a crucial element of the development cycle. Despite this, there is still significant discrepancy between in vitro and in vivo test results. Within this study we investigate multiple combinations of materials and osteogenic stimulants and demonstrate a poor correlation between the in vitro and in vivo data. We propose rationale for why this may be the case and suggest a modified testing algorithm.

Abstract
Bioink-formulations based on gelatin methacrylate combined with oxidized cellulose nanofibrils are employed in the present study. The parallel investigation of the printing performance, morphological, swelling, and biological properties of the newly developed hydrogels was performed, with inks prepared using methacrylamide-modified gelatins of fish or bovine origin. Scaffolds with versatile and well-defined internal structure and high shape fidelity were successfully printed due to the high viscosity and shear-thinning behavior of formulated inks and then photo-crosslinked. The biocompatibility of 3D-scaffolds was surveyed using human adipose stem cells (hASCs) and high viability and proliferation rates were obtained when in contact with the biomaterial. Furthermore, bioprinting tests were performed with hASCs embedded in the developed formulations. The results demonstrated that the designed inks are a versatile toolkit for 3D bioprinting and further show the benefits of using fish-derived gelatin for biofabrication.

Abstract
For 3D bioprinted tissues to be scaled-up to clinically relevant sizes, effective prevascularisation strategies are required to provide the necessary nutrients for normal metabolism and to remove associated waste by-products. The aim of this study was to develop a bioprinting strategy to engineer prevascularised tissues in vitro and to investigate the capacity of such constructs to enhance the vascularisation and regeneration of large bone defects in vivo. From a screen of different bioinks, a fibrin-based hydrogel was found to best support human umbilical vein endothelial cell (HUVEC) sprouting and the establishment of a microvessel network. When this bioink was combined with HUVECs and supporting human bone marrow stem/stromal cells (hBMSCs), these microvessel networks persisted in vitro. Furthermore, only bioprinted tissues containing both HUVECs and hBMSCs, that were first allowed to mature in vitro, supported robust blood vessel development in vivo. To assess the therapeutic utility of this bioprinting strategy, these bioinks were used to prevascularise 3D printed polycaprolactone (PCL) scaffolds, which were subsequently implanted into critically-sized femoral bone defects in rats. Microcomputed tomography (µCT) angiography revealed increased levels of vascularisation in vivo, which correlated with higher levels of new bone formation. Such prevascularised constructs could be used to enhance the vascularisation of a range of large tissue defects, forming the basis of multiple new bioprinted therapeutics.
Statement of Significance
This paper demonstrates a versatile 3D bioprinting technique to improve the vascularisation of tissue engineered constructs and further demonstrates how this method can be incorporated into a bone tissue engineering strategy to improve vascularisation in a rat femoral defect model.

Abstract
The development of materials for 3D printing adapted for tissue engineering represents one of the main concerns nowadays. Our aim was to obtain suitable 3D-printed scaffolds based on methacrylated gelatin (GelMA). In this respect, three degrees of GelMA methacrylation, three different concentrations of GelMA (10%, 20%, and 30%), and also two concentrations of photoinitiator (I-2959) (0.5% and 1%) were explored to develop proper GelMA hydrogel ink formulations to be used in the 3D printing process. Afterward, all these GelMA hydrogel-based inks/3D-printed scaffolds were characterized structurally, mechanically, and morphologically. The presence of methacryloyl groups bounded to the surface of GelMA was confirmed by FTIR and 1H-NMR analyses. The methacrylation degree influenced the value of the isoelectric point that decreased with the GelMA methacrylation degree. A greater concentration of photoinitiator influenced the hydrophilicity of the polymer as proved using contact angle and swelling studies because of the new bonds resulting after the photocrosslinking stage. According to the mechanical tests, better mechanical properties were obtained in the presence of the 1% initiator. Circular dichroism analyses demonstrated that the secondary structure of gelatin remained unaffected during the methacrylation process, thus being suitable for biological applications.

Abstract
A functional vascular supply is a key component of any large-scale tissue, providing support for the metabolic needs of tissue-remodeling cells. Although well-studied strategies exist to fabricate biomimetic scaffolds for bone regeneration, success rates for regeneration in larger defects can be improved by engineering microvascular capillaries within the scaffolds to enhance oxygen and nutrient supply to the core of the engineered tissue as it grows. Even though the role of calcium and phosphate has been well understood to enhance osteogenesis, it remains unclear whether calcium and phosphate may have a detrimental effect on the vasculogenic and angiogenic potential of endothelial cells cultured on 3D printed bone scaffolds. In this study, we presented a novel dual-ink bioprinting method to create vasculature interwoven inside CaP bone constructs. In this method, strands of a CaP ink and a sacrificial template material was used to form scaffolds containing CaP fibers and microchannels seeded with vascular endothelial and mesenchymal stem cells (MSCs) within a photo-crosslinkable gelatin methacryloyl (GelMA) hydrogel material. Our results show similar morphology of growing vessels in the presence of CaP bioink, and no significant difference in endothelial cell sprouting was found. Furthermore, our initial results showed the differentiation of hMSCs into pericytes in the presence of CaP ink. These results indicate the feasibility of creating vascularized bone scaffolds, which can be used for enhancing vascular formation in the core of bone scaffolds.

Abstract
3D bioprinting has emerged as a promising technology in the field of tissue engineering and regenerative medicine due to its ability to create anatomically complex tissue substitutes. However, it still remains challenging to develop bioactive bioinks that provide appropriate and permissive environments to instruct and guide the regenerative process in vitro and in vivo. In this study alginate sulfate, a sulfated glycosaminoglycan (sGAG) mimic, was used to functionalize an alginate-gelatin methacryloyl (GelMA) interpenetrating network (IPN) bioink to enable the bioprinting of cartilaginous tissues. The inclusion of alginate sulfate had a limited influence on the viscosity, shear-thinning and thixotropic properties of the IPN bioink, enabling high-fidelity bioprinting and supporting mesenchymal stem cell (MSC) viability post-printing. The stiffness of printed IPN constructs greatly exceeded that achieved by printing alginate or GelMA alone, while maintaining resilience and toughness. Furthermore, given the high affinity of alginate sulfate to heparin-binding growth factors, the sulfated IPN bioink supported the sustained release of transforming growth factor-β3 (TGF-β3), providing an environment that supported robust chondrogenesis in vitro, with little evidence of hypertrophy or mineralization over extended culture periods. Such bioprinted constructs also supported chondrogenesis in vivo, with the controlled release of TGF-β3 promoting significantly higher levels of cartilage-specific extracellular matrix deposition. Altogether, these results demonstrate the potential of bioprinting sulfated bioinks as part of a ‘single-stage’ or ‘point-of-care’ strategy for regenerating cartilaginous tissues. Statement of Significance: This study highlights the potential of using sulfated interpenetrating network (IPN) bioink to support the regeneration of phenotypically stable articular cartilage. Construction of interpenetrate networks in the bioink enables unique high-fidelity bioprinting and unique synergistic mechanical properties. The presence of alginate sulfate provided the capacity of high affinity-binding of TGF-β3, which promoted robust chondrogenesis.

Abstract
There is an enduring need for vascularization of bioprinted constructs with vascular networks optimized for distribution of nutrient-containing fluids, both for in vitro applications and in vivo implantation. However, most of the efforts in this field were directed so far towards generation of simple linear channels, often lined with endothelial cells only, and thus lacking the anatomical details of real vascular networks. To start addressing this need, here we explored the possibility of using actual vascular patterns derived from human ocular fundus for instructing the 3D printing activity. In order to assign to these patterns the organ-specific topology, and eventually vessel branch-defined cellular composition, we describe the use of the branching analysis program VESGEN 2D for planning a workflow that links the primary vascular images with their 3D printing with bioinks. To this end, we show how to process flat vascular images and, for an even more realistic representation, how to retro-engineer concave retinal patterns from flat images and to print them in a supporting hydrogel. This work opens the possibility of bioprinting more anatomically realistic vascular networks, and thus to eventually improve the vascularization of living tissue-engineered constructs.

Abstract
There is an enduring need for vascularization of bioprinted constructs with vascular networks optimized for distribution of nutrient-containing fluids, both for in vitro applications and in vivo implantation. However, most of the efforts in this field were directed so far towards generation of simple linear channels, often lined with endothelial cells only, and thus lacking the anatomical details of real vascular networks. To start addressing this need, here we explored the possibility of using actual vascular patterns derived from human ocular fundus for instructing the 3D printing activity. In order to assign to these patterns the organ-specific topology, and eventually vessel branch-defined cellular composition, we describe the use of the branching analysis program VESGEN 2D for planning a workflow that links the primary vascular images with their 3D printing with bioinks. To this end, we show how to process flat vascular images and, for an even more realistic representation, how to retro-engineer concave retinal patterns from flat images and to print them in a supporting hydrogel. This work opens the possibility of bioprinting more anatomically realistic vascular networks, and thus to eventually improve the vascularization of living tissue-engineered constructs.

Abstract
There is an enduring need for vascularization of bioprinted constructs with vascular networks optimized for distribution of nutrient-containing fluids, both for in vitro applications and in vivo implantation. However, most of the efforts in this field were directed so far towards generation of simple linear channels, often lined with endothelial cells only, and thus lacking the anatomical details of real vascular networks. To start addressing this need, here we explored the possibility of using actual vascular patterns derived from human ocular fundus for instructing the 3D printing activity. In order to assign to these patterns the organ-specific topology, and eventually vessel branch-defined cellular composition, we describe the use of the branching analysis program VESGEN 2D for planning a workflow that links the primary vascular images with their 3D printing with bioinks. To this end, we show how to process flat vascular images and, for an even more realistic representation, how to retro-engineer concave retinal patterns from flat images and to print them in a supporting hydrogel. This work opens the possibility of bioprinting more anatomically realistic vascular networks, and thus to eventually improve the vascularization of living tissue-engineered constructs.

Abstract
There is an enduring need for vascularization of bioprinted constructs with vascular networks optimized for distribution of nutrient-containing fluids, both for in vitro applications and in vivo implantation. However, most of the efforts in this field were directed so far towards generation of simple linear channels, often lined with endothelial cells only, and thus lacking the anatomical details of real vascular networks. To start addressing this need, here we explored the possibility of using actual vascular patterns derived from human ocular fundus for instructing the 3D printing activity. In order to assign to these patterns the organ-specific topology, and eventually vessel branch-defined cellular composition, we describe the use of the branching analysis program VESGEN 2D for planning a workflow that links the primary vascular images with their 3D printing with bioinks. To this end, we show how to process flat vascular images and, for an even more realistic representation, how to retro-engineer concave retinal patterns from flat images and to print them in a supporting hydrogel. This work opens the possibility of bioprinting more anatomically realistic vascular networks, and thus to eventually improve the vascularization of living tissue-engineered constructs.

Abstract
The present study investigated the possibility of obtaining 3D printed composite constructs using biomaterial-based nanocomposite inks. The biopolymeric matrix consisted of methacrylated gelatin (GelMA). Several types of nanoclay were added as the inorganic component. Our aim was to investigate the influence of clay type on the rheological behavior of ink formulations and to determine the morphological and structural properties of the resulting crosslinked hydrogel-based nanomaterials. Moreover, through the inclusion of nanoclays, our goal was to improve the printability and shape fidelity of nanocomposite scaffolds. The viscosity of all ink formulations was greater in the presence of inorganic nanoparticles as shear thinning occurred with increased shear rate. Hydrogel nanocomposites presented predominantly elastic rather than viscous behavior as the materials were crosslinked which led to improved mechanical properties. The inclusion of nanoclays in the biopolymeric matrix limited hydrogel swelling due the physical barrier effect but also because of the supplementary crosslinks induced by the clay layers. The distribution of inorganic filler within the GelMA-based hydrogels led to higher porosities as a consequence of their interaction with the biopolymeric ink. The present study could be useful for the development of soft nanomaterials foreseen for the additive manufacturing of customized implants for tissue engineering.

Abstract
Bioengineering of the human auricle remains a significant challenge, where the complex and unique shape, the generation of high-quality neocartilage, and shape preservation are key factors. Future regenerative medicine–based approaches for auricular cartilage reconstruction will benefit from a smart combination of various strategies. Our approach to fabrication of an ear-shaped construct uses hybrid bioprinting techniques, a recently identified progenitor cell population, previously validated biomaterials, and a smart scaffold design. Specifically, we generated a 3D-printed polycaprolactone (PCL) scaffold via fused deposition modeling, photocrosslinked a human auricular cartilage progenitor cell–laden gelatin methacryloyl (gelMA) hydrogel within the scaffold, and cultured the bioengineered structure in vitro in chondrogenic media for 30 days. Our results show that the fabrication process maintains the viability and chondrogenic phenotype of the cells, that the compressive properties of the combined PCL and gelMA hybrid auricular constructs are similar to native auricular cartilage, and that biofabricated hybrid auricular structures exhibit excellent shape fidelity compared with the 3D digital model along with deposition of cartilage-like matrix in both peripheral and central areas of the auricular structure. Our strategy affords an anatomically enhanced auricular structure with appropriate mechanical properties, ensures adequate preservation of the auricular shape during a dynamic in vitro culture period, and enables chondrogenically potent progenitor cells to produce abundant cartilage-like matrix throughout the auricular construct. The combination of smart scaffold design with 3D bioprinting and cartilage progenitor cells holds promise for the development of clinically translatable regenerative medicine strategies for auricular reconstruction.

Abstract
Abstract Bioprinting of unmodified soft extracellular matrix into complex 3D structures has remained challenging to fabricate. Herein, we established a novel process for the printing of low-viscosity hydrogel by using a unique support technique to retain the structural integrity of the support structure. We demonstrated that this process of printing could be used for different types of hydrogel, ranging from fast crosslinking gelatin methacrylate to slow crosslinking collagen type I. In addition, we evaluated the biocompatibility of the process by observing the effects of the cytotoxicity of L929 and the functionality of the human umbilical vein endothelium primary cells after printing. The results show that the bioprinted construct provided excellent biocompatibility as well as supported cell growth and differentiation. Thus, this is a novel technique that can be potentially used to enhance the resolution of the extrusion-based bioprinter.

Abstract
Abstract In hybrid bioprinting of cartilage tissue constructs, spheroids are used as cellular building blocks and combined with biomaterials for dispensing. However, biomaterial intrinsic cues can deeply affect cell fate and to date, the influence of hydrogel encapsulation on spheroid viability and phenotype has received limited attention. This study assesses this need and unravels 1) how the phenotype of spheroid-laden constructs can be tuned through adjusting the hydrogel physico–chemical properties and 2) if the spheroid maturation stage prior to encapsulation is a determining factor for the construct phenotype. Articular chondrocyte spheroids with a cartilage specific extracellular matrix (ECM) are generated and different maturation stages, early-, mid-, and late-stage (3, 7, and 14 days, respectively), are harvested and encapsulated in 10, 15, or 20 w/v% methacrylamide-modified gelatin (gelMA) for 14 days. The encapsulation of immature spheroids do not lead to a cartilage-like ECM production but when more mature mid- or late-stage spheroids are combined with a certain concentration of gelMA, a fibrocartilage-like as well as a hyaline cartilage-like phenotype can be induced. As a proof of concept, late-stage spheroids are bioprinted using a 10 w/v% gelMA–Irgacure 2959 solution with the aim to test the processing potential of the spheroid-laden bioink.

Abstract
Development of a biomimetic tubular scaffold capable of recreating developmental neurogenesis using pluripotent stem cells offers a novel strategy for the repair of spinal cord tissues. Recent advances in 3D printing technology have facilitated biofabrication of complex biomimetic environments by precisely controlling the 3D arrangement of various acellular and cellular components (biomaterials, cells and growth factors). Here, we present a 3D printing method to fabricate a complex, patterned and embryoid body (EB)-laden tubular scaffold composed of polycaprolactone (PCL) and hydrogel (alginate or gelatine methacrylate (GelMA)). Our results revealed 3D printing of a strong, macro-porous PCL/hydrogel tubular scaffold with a high capacity to control the porosity of the PCL scaffold, wherein the maximum porosity in the PCL wall was 15%. The method was equally employed to create spatiotemporal protein concentration within the scaffold, demonstrating its ability to generate linear and opposite gradients of model molecules (fluorescein isothiocyanate-conjugated bovine serum albumin (FITC-BSA) and rhodamine). 3D bioprinting of EBs-laden GelMA was introduced as a novel 3D printing strategy to incorporate EBs in a hydrogel matrix. Cell viability and proliferation were measured post-printing. Following the bioprinting of EBs-laden 5% GelMA hydrogel, neural differentiation of EBs was induced using 1 μM retinoic acid (RA). The differentiated EBs contained βIII-tubulin positive neurons displaying axonal extensions and cells migration. Finally, 3D bioprinting of EBs-laden PCL/GelMA tubular scaffold successfully supported EBs neural differentiation and patterning in response to co-printing with 1 μM RA. 3D printing of a complex heterogeneous tubular scaffold that can encapsulate EBs, spatially controlled protein concentration and promote neuronal patterning will help in developing more biomimetic scaffolds capable of replicating the neural patterning which occurs during neural tube development.

Abstract
Multimaterials deposition, a distinct advantage in bioprinting, overcomes material’s limitation in hydrogel-based bioprinting. Multimaterials are deposited in a build/support configuration to improve the structural integrity of three-dimensional bioprinted construct. A combination of rapid cross-linking hydrogel has been chosen for the build/support setup. The bioprinted construct was further chemically cross-linked to ensure a stable construct after print. This paper also proposes a file segmentation and preparation technique to be used in bioprinting for printing freeform structures.

Abstract
Multi-material 3D printing technologies that resolve features at different lengths down to the microscale open new avenues for regenerative medicine, particularly in the engineering of tissue interfaces. Herein, extrusion printing of a bone-biomimetic ceramic ink and melt electrowriting (MEW) of spatially organized polymeric microfibres are integrated for the biofabrication of an osteochondral plug, with a mechanically reinforced bone-to-cartilage interface. A printable physiological temperature-setting bioceramic, based on α-tricalcium phosphate, nanohydroxyapatite and a custom-synthesized biodegradable and crosslinkable poloxamer, was developed as bone support. The mild setting reaction of the bone ink enabled us to print directly within melt electrowritten polycaprolactone meshes, preserving their micro-architecture. Ceramic-integrated MEW meshes protruded into the cartilage region of the composite plug, and were embedded with mechanically soft gelatin-based hydrogels, laden with articular cartilage chondroprogenitor cells. Such interlocking design enhanced the hydrogel-to-ceramic adhesion strength >6.5-fold, compared with non-interlocking fibre architectures, enabling structural stability during handling and surgical implantation in osteochondral defects ex vivo. Furthermore, the MEW meshes endowed the chondral compartment with compressive properties approaching those of native cartilage (20-fold reinforcement versus pristine hydrogel). The osteal and chondral compartment supported osteogenesis and cartilage matrix deposition in vitro, and the neo-synthesized cartilage matrix further contributed to the mechanical reinforcement at the ceramic-hydrogel interface. This multi-material, multi-scale 3D printing approach provides a promising strategy for engineering advanced composite constructs for the regeneration of musculoskeletal and connective tissue interfaces.