RESOURCES

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
Abstract Hundreds of new electrochemical sensors are reported in literature every year. However, only a few of them makes it to the market. Manufacturability, or rather the lack of it, is the parameter that dictates if new sensing technologies will remain forever in the laboratory in which they are conceived. Inkjet printing is a low-cost and versatile technique that can facilitate the transfer of nanomaterial-based sensors to the market. Herein, an electroactive and self-assembling inkjet-printable ink based on protein-nanomaterial composites and exfoliated graphene is reported. The consensus tetratricopeptide proteins (CTPRs), used to formulate this ink, are engineered to template and coordinate electroactive metallic nanoclusters (NCs), and to self-assemble upon drying, forming stable films. The authors demonstrate that, by incorporating graphene in the ink formulation, it is possible to dramatically improve the electrocatalytic properties of the ink, obtaining an efficient hybrid material for hydrogen peroxide (H2O2) detection. Using this bio-ink, the authors manufactured disposable and environmentally sustainable electrochemical paper-based analytical devices (ePADs) to detect H2O2, outperforming commercial screen-printed platforms. Furthermore, it is demonstrated that oxidoreductase enzymes can be included in the formulation, to fully inkjet-print enzymatic amperometric biosensors ready to use.

Abstract
Tissue-engineered bone material is one of the effective methods to repair bone defects, but the application is restricted in clinical because of the lack of excellent scaffolds that can induce bone regeneration as well as the difficulty in making scaffolds with personalized structures. 3D printing is an emerging technology that can fabricate bespoke 3D scaffolds with precise structure. However, it is challenging to develop the scaffold materials with excellent printability, osteogenesis ability, and mechanical strength. In this study, graphene oxide (GO), attapulgite (ATP), type I collagen (Col I) and polyvinyl alcohol were used as raw materials to prepare composite scaffolds via 3D bioprinting. The composite materials showed excellent printability. The microcosmic architecture and properties was characterized by scanning electron microscopy, Fourier transform infrared and thermal gravimetric analyzer, respectively. To verify the biocompatibility of the scaffolds, the viability, proliferation and osteogenic differentiation of Bone Marrow Stromal Cells (BMSCs) on the scaffolds were assessed by CCK-8, Live/Dead staining and Real-time PCR in vitro. The composited scaffolds were then implanted into the skull defects on rat for bone regeneration. Hematoxylin-eosin staining, Masson staining and immunohistochemistry staining were carried out in vivo to evaluate the regeneration of bone tissue.The results showed that GO/ATP/COL scaffolds have been demonstrated to possess controlled porosity, water absorption, biodegradability and good apatite-mineralization ability. The scaffold consisting of 0.5% GO/ATP/COL have excellent biocompatibility and was able to promote the growth, proliferation and osteogenic differentiation of mouse BMSCs in vitro. Furthermore, the 0.5% GO/ATP/COL scaffolds were also able to promote bone regeneration of in rat skull defects. Our results illustrated that the 3D printed GO/ATP/COL composite scaffolds have good mechanical properties, excellent cytocompatibility for enhanced mouse BMSCs adhesion, proliferation, and osteogenic differentiation. All these advantages made it potential as a promising biomaterial for osteogenic reconstruction.

Abstract
In this paper, the Ti3C2Tx/GO frame with vertical pore gradient is constructed by using 3D printing technology. The TiO2-Ti3C2Tx heterojunctions is generated in situ by thermal annealing to control the oxidation of 3D frames. TiO2-Ti3C2Tx/rGO/PDMS composites with high EMI SE and excellent thermal management performance are assembled by curing the annealed 3D frame with polydimethylsiloxane (PDMS). Notably, the composites have a unique multilayer-scale structure that rod-shaped TiO2 particles are decorated on Ti3C2Tx substrate and TiO2-Ti3C2Tx/rGO stack to form an amorphous porous gradient pore size structure. The effect of gradient pore size on EMI SE of composites is studied by simulation. Under the synergistic effect of multiple loss mechanism, the designed composites show conductivity of up to 173.1 S/m, the thickness of the composite is 2 mm and the density is 67mg/cm3, which shows excellent EMI SE of 58 dB. The composites also have excellent thermal management performance.

Abstract
A bottleneck limiting the practical application of lithium metal anodes is the uncontrolled growth of lithium dendrites caused by gradient distributed Li+ from separators to collectors. Herein{,} 3D-printed frameworks with a gradient distributed heterojunction and fast Li+ conductivity interfaces are developed to regulate the Li+ distribution and the direction of dendrite growth. More importantly{,} the effect of different Li+ concentration gradient frameworks on Li+ deposition behavior was analyzed in detail. Synchrotron X-ray tomography demonstrates that macropores dominate the framework{,} which effectively suppresses the volume change caused by lithium deposition. DFT calculations confirm the high lithiophilicity of γ-Al2O3 and the graphene heterojunction. Synchrotron radiation-based soft X-ray absorption spectroscopy illustrates the fast Li+ conductivity Li–Al–O interface resulting from the shortened Al–O bond distance. Benefiting from the higher Li+ concentration differences during the dissolution process and Li–Al–O interfaces{,} the gradient framework can achieve a high rate performance of ∼40 mV overpotential at 10 mA cm−2 and long cycle stability of ∼1500 h at 1 mA cm−2.

Abstract
The treatment of tumour-related bone defects should ideally combine bone regeneration with tumour treatment. Additive manufacturing (AM) could feasibly place functional bone-repair materials within composite materials with functional-grade structures, giving them bone repair and anti-tumour effects. Magnetothermal therapy is a promising non-invasive method of tumour treatment that has attracted increasing attention. In this study, we prepared novel hydrogel composite scaffolds of polyvinyl alcohol/sodium alginate/hydroxyapatite (PVA/SA/HA) at low temperature via AM. The scaffolds were loaded with various concentrations of magnetic graphene oxide (MGO) @Fe3O4 nanoparticles. The scaffolds were characterised by fourier transform infrared spectroscopy (FTIR), scanning electron microscope (SEM) and thermal gravimetric analysis (TGA), which showed that the scaffolds have good moulding qualities and strong hydrogen bonding between the MGO/PVA/SA/HA components. TGA analysis demonstrated the expected thermal stability of the MGO and scaffolds. Thermal effects can be adjusted by varying the contents of MGO and the strength of an external alternating magnetic field. The prepared MGO hydrogel composite scaffolds enhance biological functions and support bone mesenchymal stem cell differentiation in vitro. The scaffolds also show favourable anti-tumour characteristics with effective magnetothermal conversion in vivo.

Abstract
Self-assembling bioinks offer the possibility to biofabricate with molecular precision, hierarchical control, and biofunctionality. For this to become a reality with widespread impact, it is essential to engineer these ink systems ensuring reproducibility and providing suitable standardization. We have reported a self-assembling bioink based on disorder-to-order transitions of an elastin-like recombinamer (ELR) to co-assemble with graphene oxide (GO). Here, we establish reproducible processes, optimize printing parameters for its use as a bioink, describe new advantages that the self-assembling bioink can provide, and demonstrate how to fabricate novel structures with physiological relevance. We fabricate capillary-like structures with resolutions down to ∼10 µm in diameter and ∼2 µm thick tube walls and use both experimental and finite element analysis to characterize the printing conditions, underlying interfacial diffusion-reaction mechanism of assembly, printing fidelity, and material porosity and permeability. We demonstrate the capacity to modulate the pore size and tune the permeability of the resulting structures with and without human umbilical vascular endothelial cells. Finally, the potential of the ELR-GO bioink to enable supramolecular fabrication of biomimetic structures was demonstrated by printing tubes exhibiting walls with progressively different structure and permeability.

Abstract
Abstract With the advent of flexible electronics, the old fashioned and conventional solid-state technology will be replaced by conductive inks combined with low-cost printing techniques. Graphene is an ideal candidate to produce conductive inks, due to its excellent conductivity and zero bandgap. The possibility to chemically modify graphene with active molecules opens up the field of responsive conductive inks. Herein, a bioresponsive, electroactive, and inkjet-printable graphene ink is presented. The ink is based on graphene chemically modified with selected enzymes and an electrochemical mediator, to transduce the products of the enzymatic reaction into an electron flow, proportional to the analyte concentration. A water-based formulation is engineered to be respectful with the enzymatic activity while matching the stringent requirements of inkjet printing. The efficient electrochemical performance of the ink, as well as a proof-of-concept application in biosensing, is demonstrated. The versatility of the system is demonstrated by modifying graphene with various oxidoreductases, obtaining inks with selectivity toward glucose, lactate, methanol, and ethanol.

Abstract
Additive manufactured scaffolds are widely used as 3D support structures for tissue engineering. This paper investigates the mechanisms behind bone regeneration due to the combined use of 3D printed poly (ϵ-caprolactone)/graphene (PCL/G) electro-active scaffolds and electrical stimulation. A comprehensive in vivo study was conducted to assess the proposed approach, using a rat model. Results show that the combined use of electro-active scaffolds and electrical stimulation therapy accelerates the bone regeneration process and the formation of more organized new bone, through fast angiogenesis, and a rapid transition to the mineralization and bone remodelling phase. The mechanism is investigated and explained.

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
Abstract 3D printing of multicomponent materials as an advantageous method over traditional mold casting methods is demonstrated, developing small core–shell capsule composites fabricated by a two-step 3D printing process. Using a two-print-head system (fused deposition modeling extruder and a liquid inkjet print head), micro-sized capsules are manufactured in sizes ranging from 100 to 800 µm. The thermoplastic polymer poly(ε-caprolactone) (PCL) is chosen as matrix/shell material due to its optimal interaction with the embedded hydrophobic liquids. First, the core–shell capsules are printed with model liquids and pure PCL to optimize the printing parameters and to ensure fully enclosed capsules inside the polymer. As a proof of concept, novel “click” reaction systems, used in self-healing and stress-detection applications, are manufactured in which PCL composites with nano- and micro-fillers are combined with reactive, encapsulated liquids. The so generated 3D printed core–shell capsule composite can be used for post-printing reactions and damage sensing when combined with a fluorogenic dye.

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
Graphene and its derivatives have been extensively explored in various fields and have shown great promise toward energy harvesting, environmental protection, and health care. 3D graphene-containing structures (3DGCSs) are especially endowed with useable features relating to physicochemical properties within the hierarchical architectures. Thus, 3DGCSs are increasingly being applied for tissue engineering because of their supportability of human cells and functionalization potential. This review focuses on recent progress in tissue engineering utilizing 3DGCSs, providing insights into fabrication, application, and constraints in bionic research.

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
Scaffolds are important physical substrates for cell attachment, proliferation and differentiation. Multiple factors could influence the optimal design of scaffolds for a specific tissue, such as the geometry, the materials used to modulate cell proliferation and differentiation, its biodegradability and biocompatibility. The optimal design of a scaffold for a specific tissue strongly depends on both materials and manufacturing processes. Previous studies of human adipose-derived stem cells (hADSCs) seeded on poly(ε-caprolactone) (PCL)/graphene scaffolds have proved that the addition of small concentrations of graphene to PCL scaffolds improves cell proliferation. Based on such results, this paper further investigates, for the first time, both in vitro and in vivo characteristics of 3D printed PCL/graphene scaffolds. Scaffolds were evaluated from morphological, biological and short term immune response points of view. Results show that the produced scaffolds induce an acceptable level of immune response, suggesting high potential for in vivo applications. Finally, the scaffolds were used to treat a rat calvaria critical size defect with and without applying micro electrical stimulation (10 μA). Quantification of connective and new bone tissue formation and the levels of ALP, RANK, RANKL, OPG were considered. Results show that the use of scaffolds containing graphene and electrical stimulation seems to increase cell migration and cell influx, leading to new tissue formation, well-organized tissue deposition and bone remodelling.

Abstract
Tissue engineering embraces the potential of recreating and replacing defective body parts by advancements in the medical field. Being a biocompatible nanomaterial with outstanding physical, chemical, optical, and biological properties, graphene-based materials were successfully employed in creating the perfect scaffold for a range of organs, starting from the skin through to the brain. Investigations on 2D and 3D tissue culture scaffolds incorporated with graphene or its derivatives have revealed the capability of this carbon material in mimicking in vivo environment. The porous morphology, great surface area, selective permeability of gases, excellent mechanical strength, good thermal and electrical conductivity, good optical properties, and biodegradability enable graphene materials to be the best component for scaffold engineering. Along with the apt microenvironment, this material was found to be efficient in differentiating stem cells into specific cell types. Furthermore, the scope of graphene nanomaterials in liver tissue engineering as a promising biomaterial is also discussed. This review critically looks into the unlimited potential of graphene-based nanomaterials in future tissue engineering and regenerative therapy.

Abstract
In this study, a tissue-engineered trachea, consisting of multilevel structural electrospun polylactide (PLA) membranes enveloping 3D-printed thermoplastic polyurethane (TPU) skeletons, was developed to create a mechanically robust, antibacterial and bioresorbable graft for the tracheal reconstruction. The study design incorporated two distinct uses of stereocomplex PLA: patterned electrospun fibers to enhance tissue integration compared to the random layered fibers, meanwhile possessing good antibacterial property; and 3D-printed TPU scaffold with elasticity to provide external support and protection. Herein, ionic liquid (IL)-functioned graphene oxide (GO) was synthesized and presented enhanced mechanical and hydrophilicity properties. More interesting, antibacterial activity of the GO-g-IL modified PLA membranes were proved by Escherichia coli and Staphylococcus aureus, showing superior antibacterial effect compared to single GO or IL. The synergistic antibacterial effect could be related to that GO break cytomembrane of bacteria by its extremely sharp edges, while IL works by electrostatic interaction between its cationic structures and electronegative phosphate groups of bacteria membranes, leading to the loss of cell electrolyte and cell death. Hence, after L929 fibroblast cells were seeded on patterned fibrous membranes with phenotypic shape, further effective cell infiltration, cell proliferation and attachment were observed. In addition, the tissue-engineered trachea scaffolds were implanted into rabbit models. The in vivo result confirmed that the scaffolds with patterned membranes manifested favorable biocompatibility and promoted tissue regeneration.
In this study, a tissue-engineered trachea, consisting of multilevel structural electrospun polylactide (PLA) membranes enveloping 3D-printed thermoplastic polyurethane (TPU) skeletons, was developed to create a mechanically robust, antibacterial and bioresorbable graft for the tracheal reconstruction. The study design incorporated two distinct uses of stereocomplex PLA: patterned electrospun fibers to enhance tissue integration compared to the random layered fibers, meanwhile possessing good antibacterial property; and 3D-printed TPU scaffold with elasticity to provide external support and protection. Herein, ionic liquid (IL)-functioned graphene oxide (GO) was synthesized and presented enhanced mechanical and hydrophilicity properties. More interesting, antibacterial activity of the GO-g-IL modified PLA membranes were proved by Escherichia coli and Staphylococcus aureus, showing superior antibacterial effect compared to single GO or IL. The synergistic antibacterial effect could be related to that GO break cytomembrane of bacteria by its extremely sharp edges, while IL works by electrostatic interaction between its cationic structures and electronegative phosphate groups of bacteria membranes, leading to the loss of cell electrolyte and cell death. Hence, after L929 fibroblast cells were seeded on patterned fibrous membranes with phenotypic shape, further effective cell infiltration, cell proliferation and attachment were observed. In addition, the tissue-engineered trachea scaffolds were implanted into rabbit models. The in vivo result confirmed that the scaffolds with patterned membranes manifested favorable biocompatibility and promoted tissue regeneration.

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
Purpose The main purpose of the present work is to study the effect of nano hydroxyapatite (HA) and graphene oxide (GO) particles on thermal and mechanical performances of 3D printed poly(ε-caprolactone) (PCL) filaments used in Bone Tissue Engineering (BTE). Design/methodology/approach Raw materials were prepared by melt blending, followed by 3D printing via 3D Discovery (regenHU Ltd., CH) with all fabricating parameters kept constant. Filaments, including pure PCL, PCL/HA, and PCL/GO, were tested under the same conditions. Several techniques were used to mechanically, thermally, and microstructurally evaluate properties of these filaments, including Differential Scanning Calorimetry (DSC), tensile test, nano indentation, and Scanning Electron Microscope (SEM). Findings Results show that both HA and GO nano particles are capable of improving mechanical performance of PCL. Enhanced mechanical properties of PCL/HA result from reinforcing effect of HA, while a different mechanism is observed in PCL/GO, where degree of crystallinity plays an important role. In addition, GO is more efficient at enhancing mechanical performance of PCL compared with HA. Originality/value For the first time, a systematic study about effects of nano HA and GO particles on bioactive scaffolds produced by Additive Manufacturing (AM) for bone tissue engineering applications is conducted in this work. Mechanical and thermal behaviors of each sample, pure PCL, PCL/HA and PCL/GO, are reported, correlated, and compared with literature.

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.