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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 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
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
Abstract Extrusion additive manufacturing is widely used to fabricate polymer-based 3D bone scaffolds. However, the insight views of crystal growths, scaffold features and eventually cell-scaffold interactions are still unknown. In this work, melt and solvent extrusion additive manufacturing techniques are used to produce scaffolds considering highly analogous printing conditions. Results show that the scaffolds produced by these two techniques present distinct physiochemical properties, with melt-printed scaffolds showing stronger mechanical properties and solvent-printed scaffolds showing rougher surface, higher degradation rate, and faster stress relaxation. These differences are attributed to the two different crystal growth kinetics, temperature-induced crystallization (TIC) and strain-induced crystallization (SIC), forming large/integrated spherulite-like and a small/fragmented lamella-like crystal regions respectively. The stiffer substrate of melt-printed scaffolds contributes to higher ratio of nuclear Yes-associated protein (YAP) allocation, favoring cell proliferation and differentiation. Faster relaxation and degradation of solvent-printed scaffolds result in dynamic surface, contributing to an early-stage faster osteogenesis differentiation.

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
Large bone loss injuries require high-performance scaffolds with an architecture and material composition resembling native bone. However, most bone scaffold studies focus on three-dimensional (3D) structures with simple rectangular or circular geometries and uniform pores, not able to recapitulate the geometric characteristics of the native tissue. This paper addresses this limitation by proposing novel anatomically designed scaffolds (bone bricks) with nonuniform pore dimensions (pore size gradients) designed based on new lay-dawn pattern strategies. The gradient design allows one to tailor the properties of the bricks and together with the incorporation of ceramic materials allows one to obtain structures with high mechanical properties (higher than reported in the literature for the same material composition) and improved biological characteristics.

Abstract
Polycaprolactone (PCL) is one of the most recognized polymeric materials used for bone tissue engineering scaffold fabrication. This study aims to evaluate the effects of the molecular weight (Mn) of PCL on the degradation kinematics, surface, microstructural, thermal, mechanical, and biological properties of 3D printed bone scaffolds. Surface properties were investigated considering water-in-air contact angle and nanoindentation tests, while morphological characteristics and degradation kinematics (accelerated degradation tests) were examined using scanning electron microscopy (SEM), pairing with thermal and mechanical properties monitored at each considered time point. A set of mathematical equations describing the variation of fiber diameter, porosity, mechanical properties, and weight, as a function of molecular weight and degradation time, were obtained based on the experimental results. Human adipose-derived stem cells (hADSCs) proliferation and differentiation tests were also conducted using in vitro colorimetric assay. All results indicated that molecular weight had impacts on the surface, mechanical and biological properties of PCL scaffolds, while no significant effects were observed on the degradation rate. Scaffolds with lower molecular weight presented better bio-mechanical properties. These findings provide useful information for the design of polymeric bone tissue engineering scaffolds.

Abstract
The design of scaffolds with optimal biomechanical properties for load-bearing applications is an important topic of research. Most studies have addressed this problem by focusing on the material composition and not on the coupled effect between the material composition and the scaffold architecture. Polymer–bioglass scaffolds have been investigated due to the excellent bioactivity properties of bioglass, which release ions that activate osteogenesis. However, material preparation methods usually require the use of organic solvents that induce surface modifications on the bioglass particles, compromising the adhesion with the polymeric material thus compromising mechanical properties. In this paper, we used a simple melt blending approach to produce polycaprolactone/bioglass pellets to construct scaffolds with pore size gradient. The results show that the addition of bioglass particles improved the mechanical properties of the scaffolds and, due to the selected architecture, all scaffolds presented mechanical properties in the cortical bone region. Moreover, the addition of bioglass indicated a positive long-term effect on the biological performance of the scaffolds. The pore size gradient also induced a cell spreading gradient.

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
Polycaprolactone (PCL) scaffolds have been widely investigated for tissue engineering applications, however, they exhibit poor cell adhesion and mechanical properties. Subsequently, PCL composites have been produced to improve the material properties. This study utilises a natural material, Bombyx mori silk microparticles (SMP) prepared by milling silk fibre, to produce a composite to enhance the scaffolds properties. Silk is biocompatible and biodegradable with excellent mechanical properties. However, there are no studies using SMPs as a reinforcing agent in a 3D printed thermoplastic polymer scaffold. PCL/SMP (10, 20, 30 wt%) composites were prepared by melt blending. Rheological analysis showed that SMP loading increased the shear thinning and storage modulus of the material. Scaffolds were fabricated using a screw-assisted extrusion-based additive manufacturing system. Scanning electron microscopy and X-ray microtomography was used to determine scaffold morphology. The scaffolds had high interconnectivity with regular printed fibres and pore morphologies within the designed parameters. Compressive mechanical testing showed that the addition of SMP significantly improved the compressive Young's modulus of the scaffolds. The scaffolds were more hydrophobic with the inclusion of SMP which was linked to a decrease in total protein adsorption. Cell behaviour was assessed using human adipose derived mesenchymal stem cells. A cytotoxic effect was observed at higher particle loading (30 wt%) after 7 days of culture. By day 21, 10 wt% loading showed significantly higher cell metabolic activity and proliferation, high cell viability, and cell migration throughout the scaffold. Calcium mineral deposition was observed on the scaffolds during cell culture. Large calcium mineral deposits were observed at 30 wt% and smaller calcium deposits were observed at 10 wt%. This study demonstrates that SMPs incorporated into a PCL scaffold provided effective mechanical reinforcement, improved the rate of degradation, and increased cell proliferation, demonstrating potential suitability for bone tissue engineering applications.

Abstract
There is a significant unmet clinical need to prevent amputations due to large bone loss injuries. We are addressing this problem by developing a novel, cost-effective osseointegrated prosthetic solution based on the use of modular pieces, bone bricks, made with biocompatible and biodegradable materials that fit together in a Lego-like way to form the prosthesis. This paper investigates the anatomical designed bone bricks with different architectures, pore size gradients, and material compositions. Polymer and polymer-composite 3D printed bone bricks are extensively morphological, mechanical, and biological characterized. Composite bone bricks were produced by mixing polycaprolactone (PCL) with different levels of hydroxyapatite (HA) and β-tri-calcium phosphate (TCP). Results allowed to establish a correlation between bone bricks architecture and material composition and bone bricks performance. Reinforced bone bricks showed improved mechanical and biological results. Best mechanical properties were obtained with PCL/TCP bone bricks with 38 double zig-zag filaments and 14 spiral-like pattern filaments, while the best biological results were obtained with PCL/HA bone bricks based on 25 double zig-zag filaments and 14 spiral-like pattern filaments.

Abstract
Scaffolds play a key role in tissue engineering applications. In the case of bone tissue engineering, scaffolds are expected to provide both sufficient mechanical properties to withstand the physiological loads, and appropriate bioactivity to stimulate cell growth. In order to further enhance cell–cell signaling and cell–material interaction, electro-active scaffolds have been developed based on the use of electrically conductive biomaterials or blending electrically conductive fillers to non-conductive biomaterials. Graphene has been widely used as functioning filler for the fabrication of electro-active bone tissue engineering scaffolds, due to its high electrical conductivity and potential to enhance both mechanical and biological properties. Nitrogen-doped graphene, a unique form of graphene-derived nanomaterials, presents significantly higher electrical conductivity than pristine graphene, and better surface hydrophilicity while maintaining a similar mechanical property. This paper investigates the synthesis and use of high-performance nitrogen-doped graphene as a functional filler of poly(ɛ-caprolactone) (PCL) scaffolds enabling to develop the next generation of electro-active scaffolds. Compared to PCL scaffolds and PCL/graphene scaffolds, these novel scaffolds present improved in vitro biological performance.

Abstract
Electrostimulation and electroactive scaffolds can positively influence and guide cellular behaviour and thus has been garnering interest as a key tissue engineering strategy. The development of conducting polymers such as polyaniline enables the fabrication of conductive polymeric composite scaffolds. In this study, we report on the initial development of a polycaprolactone scaffold incorporating different weight loadings of a polyaniline microparticle filler. The scaffolds are fabricated using screw-assisted extrusion-based 3D printing and are characterised for their morphological, mechanical, conductivity, and preliminary biological properties. The conductivity of the polycaprolactone scaffolds increases with the inclusion of polyaniline. The in vitro cytocompatibility of the scaffolds was assessed using human adipose-derived stem cells to determine cell viability and proliferation up to 21 days. A cytotoxicity threshold was reached at 1% wt. polyaniline loading. Scaffolds with 0.1% wt. polyaniline showed suitable compressive strength (6.45 ± 0.16 MPa) and conductivity (2.46 ± 0.65 × 10(-4) S/cm) for bone tissue engineering applications and demonstrated the highest cell viability at day 1 (88%) with cytocompatibility for up to 21 days in cell culture.

Abstract
The development of highly biomimetic scaffolds in terms of composition and structures, to repair or replace damaged bone tissues, is particularly relevant for tissue engineering. This paper investigates a 3D printed porous scaffold containing aligned multi-walled carbon nanotubes (MWCNTs) and nano-hydroxyapatite (nHA), mimicking the natural bone tissue from the nanoscale to macroscale level. MWCNTs with similar dimensions as collagen fibres are coupled with nHA and mixed within a polycaprolactone (PCL) matrix to produce scaffolds using a screw-assisted extrusion-based additive manufacturing system. Scaffolds with different material compositions were extensively characterised from morphological, mechanical and biological points of views. Transmission electron microscopy and polarised Raman spectroscopy confirm the presence of aligned MWCNTs within the printed filaments. The PCL/HA/MWCNTs scaffold are similar to the nanostructure of native bone and shows overall increased mechanical properties, cell proliferation, osteogenic differentiation and scaffold mineralisation, indicating a promising approach for bone tissue regeneration.

Abstract
Large bone defects due to trauma or disease present a significant clinical challenge with limited efficacy of current therapies. A key aim is to develop biomimetic scaffolds that reflect the native tissue structure with 3D printing being an important enabling technology. However, the incorporation of multiple length scales and anisotropic features, mimicking the native architecture, is difficult with current processes. In this study, we propose a simple and versatile hybrid printing process using a screw-assisted additive manufacturing technique combined with rotational electrospinning to fabricate dual-scale anisotropic scaffolds. 3D microscale porous polycaprolactone (PCL) structures with highly aligned nanoscale fibres were successfully produced layer-by-layer. The scaffolds were morphological, mechanical and biological characterised. Human adipose-derived stem cells (hADSCs) were seeded on the hybrid scaffold to evaluate the effects of structural and anisotropic topographic cues on cell attachment, proliferation and osteogenesis differentiation. Results show that the 3D printed microscale structures have uniform and well-defined geometries and the alignment of nanoscale electrospun fibres increases by increasing the electrospinning rotational velocity. Mechanical results show that there is no significant difference between 3D printed scaffolds with or without electrospun meshes. In vitro results show higher cell seeding efficiency and proliferation in dual-scale scaffolds with high density electrospun meshes. A more stretched and elongated cell morphology was observed in aligned nanofibre scaffolds showing higher anisotropic cytoskeletal organization than 3D printed PCL scaffolds without electrospun meshes. The dual-scale scaffolds present improved overall osteogenic markers expressions (COL-1, ALP and OCN). However, no statistical difference between normalised osteogenic marker expressions were observed between dual-scale scaffolds and 3D printed scaffolds. This might be attributed to the poor bioactivity of the substrate material, PCL, suggesting topographical cues might not be sufficient to stimulate cell fate towards to an osteogenic linage. The results suggest that the proposed fabrication strategy is a promising approach for the design of novel bone scaffolds to modulate cell fates by integrating the topographic cue reported in this paper with biochemical cues associated to the use of more bioactive materials.

Abstract
Scaffolds, three-dimensional (3D) substrates providing appropriate mechanical support and biological environments for new tissue formation, are the most common approaches in tissue engineering. To improve scaffold properties such as mechanical properties, surface characteristics, biocompatibility and biodegradability, different types of fillers have been used reinforcing biocompatible and biodegradable polymers. This paper investigates and compares the mechanical and biological behaviors of 3D printed poly(ε-caprolactone) scaffolds reinforced with graphene (G) and graphene oxide (GO) at different concentrations. Results show that contrary to G which improves mechanical properties and enhances cell attachment and proliferation, GO seems to show some cytotoxicity, particular at high contents.

Abstract
Scaffold-based bone tissue engineering is the most relevant approach for critical-sized bone defects. It is based on the use of three-dimensional substrates to provide the appropriate biomechanical environment for bone regeneration. Despite some successful results previously reported, scaffolds were never designed for disease treatment applications. This article proposes a novel dual-functional scaffold for cancer applications, comprising both treatment and regeneration functions. These functions are achieved by combining a biocompatible and biodegradable polymer and graphene. Results indicate that high concentrations of graphene enhance the mechanical properties of the scaffolds, also increasing the inhibition on cancer cell viability and proliferation.

Abstract
Complex and hierarchically functionalized scaffolds composed of micro- and nanoscale structures are a key goal in tissue engineering. The combination of three-dimensional (3D) printing and electrospinning enables the fabrication of these multiscale structures. This study presents a polycaprolactone 3D-printed and electrospun scaffold with multiple mesh layers and fiber densities. The results show successful fabrication of a dual-scale scaffold with the 3D-printed scaffold acting as a gap collector with the printed microfibers as the electrodes and the pores a series of insulating gaps resulting in aligned nanofibers. The electrospun fibers are highly aligned perpendicular to the direction of the printed fiber and form aligned meshes within the pores of the scaffold. Mechanical testing showed no significant difference between the number of mesh layers whereas the hydrophobicity of the scaffold increased with increasing fiber density. Biological results indicate that increasing the number of mesh layers improves cell proliferation, migration, and adhesion. The aligned nanofibers within the microscale pores allowed enhanced cell bridging and cell alignment that was not observed in the 3D-printed only scaffold. These results demonstrate a facile method of incorporating low-density and aligned fibers within a 3D-printed scaffold that is a promising development in multiscale hierarchical scaffolds where alignment of cells can be desirable.

Abstract
Biomanufacturing is a relatively new research domain focusing on the use of additive manufacturing technologies, biomaterials, cells and biomolecular signals to produce tissue constructs for tissue engineering. For bone regeneration, researchers are focusing on the use of polymeric and polymer/ceramic scaffolds seeded with osteoblasts or mesenchymal stem cells. However, the design of high-performance scaffolds in terms of mechanical, cell-stimulation and biological performance is still required. This is the first paper investigating the use of an extrusion additive manufacturing system to produce poly(ε-caprolactone) (PCL), PCL/graphene nanosheet (GNS) and PCL/carbon nanotube (CNT) scaffolds for bone applications. Scaffolds with regular and reproducible architecture were produced and evaluated from chemical, physical and biological points of view. Results suggest that the addition of both graphene and CNT allow the fabrication of scaffolds with improved properties. It also shows that scaffolds containing graphene present better mechanical properties and high cell-affinity improving cell attachment, proliferation and differentiation.

Abstract
Carbon nanotubes (CNTs) with exceptional physical and chemical properties are attracting significant interest in the field of tissue engineering. Several reports investigated CNTs biocompatibility and their impact in terms of cell attachment, proliferation and differentiation mainly using polymer/CNTs membranes. However, these 2D membranes are not able to emulate the complex in vivo environment. In this paper, additive manufacturing (3D printing) is used to create composite 3D porous scaffolds containing different loadings of multi-walled carbon nanotubes (MWCNT) (0.25, 0.75 and 3 wt%) for bone tissue regeneration. Pre-processed and processed materials were extensively characterised in terms of printability, morphological and topographic characteristics and thermal, mechanical and biological properties. Scaffolds with pore sizes ranging between 366 μm and 397 μm were successfully produced and able to sustain early-stage human adipose-derived mesenchymal stem cells attachment and proliferation. Results show that MWCNTs enhances protein adsorption, mechanical and biological properties. Composite scaffolds, particularly the 3 wt% loading of MWCNTs, seem to be good candidates for bone tissue regeneration.

Abstract
The use of porous scaffolds created by additive manufacturing is considered a viable approach for the regeneration of critical-size bone defects. This paper investigates the xenotransplantation of polycaprolactone (PCL) tissue constructs seeded with differentiated and undifferentiated human adipose-derived mesenchymal stem cells (hADSCs) to treat calvarial critical-sized defect in Wistar rats. PCL scaffolds without cells were also considered. In vitro and in vivo biological evaluations were performed to assess the feasibility of these different approaches. In the case of cell seeded scaffolds, it was possible to observe the presence of hADSCs in the rat tissue contributing directly (osteoblasts) and indirectly (stimulation by paracrine factors) to tissue formation, organization and mineralization. The presence of bone morphogenetic protein-2 (BMP-2) in the rat tissue treated with cell-seeded PCL scaffolds suggests that the paracrine factors of undifferentiated hADSC cells could stimulate BMP-2 production by surrounding cells, leading to osteogenesis. Moreover, BMP-2 acts synergistically with growth factors to induce angiogenesis, leading to higher numbers of blood vessels in the groups containing undifferentiated and differentiated hADSCs.

Abstract
Abstract Biomanufacturing is a relatively new research domain focusing on the use of additive manufacturing technologies, biomaterials, cells, and biomolecular signals to produce tissue constructs for tissue engineering. For bone regeneration, researchers are focusing on the use of polymeric and polymer/ceramic scaffolds seeded with osteoblasts or mesenchymal stem cells. However, high-performance scaffolds in terms of mechanical, cell stimulation, and biological performance are still required. This article investigates the use of an extrusion additive manufacturing system to produce poly(ɛ-caprolactone) (PCL) and PCL/graphene nanosheet scaffolds for bone applications. Scaffolds with regular and reproducible architecture and uniform dispersion of graphene were produced and coated with P1-latex protein extracted from the Hevea brasiliensis rubber tree. Results show that the obtained scaffolds cultivated with human adipose-derived stem cells present no toxicity effects. The presence of graphene nanosheet and P1-latex protein in the scaffolds increased cell proliferation compared with PCL scaffolds. Moreover, the presence of P1-latex protein promotes earlier osteogenic differentiation, suggesting that PCL/graphene/P1-latex protein scaffolds are suitable for bone regeneration applications.

Abstract
Scaffolds are physical substrates for cell attachment, proliferation, and differentiation, ultimately leading to the regeneration of tissues. They must be designed according to specific biomechanical requirements such as mechanical properties, surface characteristics, biodegradability, biocompatibility, and porosity. The optimal design of a scaffold for a specific tissue strongly depends on both materials and manufacturing processes. Polymeric scaffolds reinforced with electro-active particles could play a key role in tissue engineering by modulating cell proliferation and differentiation. This paper investigates the use of an extrusion additive manufacturing system to produce PCL/pristine graphene scaffolds for bone tissue applications. PCL/pristine graphene blends were prepared using a melt blending process. Scaffolds with regular and reproducible architecture were produced with different concentrations of pristine graphene. Scaffolds were evaluated from morphological, mechanical, and biological view. The results suggest that the addition of pristine graphene improves the mechanical performance of the scaffolds, reduces the hydrophobicity, and improves cell viability and proliferation.