RESOURCES

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
Regeneration of complex bone defects remains a significant clinical challenge. Multi-tool biofabrication has permitted the combination of various biomaterials to create multifaceted composites with tailorable mechanical properties and spatially controlled biological function. In this study we sought to use bioprinting to engineer nonviral
gene activated constructs reinforced by polymeric micro-filaments. A gene activated bioink was developed using RGD-g-irradiated alginate and nano-hydroxyapatite (nHA) complexed to plasmid DNA (pDNA). This ink was combined with bonemarrow-derived mesenchymal stemcells (MSCs) and then co-printed with a polycaprolactone supporting mesh to provide mechanical stability to the construct. Reporter genes were first used to demonstrate successful cell transfection using this system, with sustained expression of the transgene detected over 14 days postbioprinting. Delivery of a combination of therapeutic genes encoding for bone morphogenic protein and transforming growth factor promoted robust osteogenesis of encapsulated MSCs in vitro, with enhanced levels of matrix deposition and mineralization observed following the incorporation of therapeutic pDNA. Gene activated MSC-laden constructs were then implanted subcutaneously, directly postfabrication, and were found to support superior levels of vascularization andmineralization compared to cell-free controls. These results validate the use of a gene activated bioink to impart biological functionality to three-dimensional bioprinted constructs.

Abstract
Therapeutic growth factor delivery typically requires supraphysiological dosages, which can cause undesirable off-target effects. The aim of this study was to 3D bioprint implants containing spatiotemporally defined patterns of growth factors optimized for coupled angiogenesis and osteogenesis. Using nanoparticle functionalized bioinks, it was possible to print implants with distinct growth factor patterns and release profiles spanning from days to weeks. The extent of angiogenesis in vivo depended on the spatial presentation of vascular endothelial growth factor (VEGF). Higher levels of vessel invasion were observed in implants containing a spatial gradient of VEGF compared to those homogenously loaded with the same total amount of protein. Printed implants containing a gradient of VEGF, coupled with spatially defined BMP-2 localization and release kinetics, accelerated large bone defect healing with little heterotopic bone formation. This demonstrates the potential of growth factor printing, a putative point of care therapy, for tightly controlled tissue regeneration.

Abstract
Ceramics and metals are important materials that modern technologies are constructed from. The capability to produce such materials in a complex geometry with good mechanical properties can revolutionize the way we engineer our devices. Current curing techniques pose challenges such as high energy requirements{,} limitations of materials with high refractive index{,} tedious post-processing heat treatment processes{,} uneven drying shrinkages{,} and brittleness of green bodies. In this paper{,} a novel modified self-curable epoxide–amine 3D printing system is proposed to print a wide range of ceramics (metal oxides{,} nitrides{,} and carbides) and metals without the need for an external curing source. Through this technique{,} complex multi-material structures (with metal–ceramic and ceramic–ceramic combinations) can also be realized. Tailoring and matching the sintering temperatures of different materials through sintering additives and dopants{,} combined with a structural design providing maximum adhesion between interfaces{,} allow us to successfully obtain superior quality sintered multi-material structures. High-quality ceramic and metallic materials have been achieved (e.g.{,} zirconia with >98% theoretical density). Also{,} highly conductive metals and magnetic ceramics were printed and shaped uniquely without the need for a sacrificial support. With the addition of low molecular weight plasticizers and a multi-stage heat treatment process{,} crack-free and dense high-quality integrated multi-material structures fabricated by 3D printing can thus be a reality in the near future.

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
Abstract Fabrication of dense ceramic articles with intricate fine features and geometrically complex morphology by using a relatively simple and the cost-effective process still remains a challenge. Ceramics, either in its green- or sintered-form, are known for being hard yet brittle which limits further shape reconfiguration. In this work, a combinatorial process of ceramic robocasting and photopolymerization is demonstrated to produce either flexible and/or stretchable ceramic green-body (Flex-Body or Stretch-Body) that can undergo a postprinting reconfiguration process. Secondary shaping may proceed through: i) self-assembly-assisted shaping and ii) mold-assisted shaping process, which allows a well-controlled ceramic structure morphology. With a proposed well-controlled thermal heating process, the ceramic Sintered-Body can achieve >99.0% theoretical density with good mechanical rigidity. Complex and dense ceramic articles with fine features down to 65 μm can be fabricated. When combined with a multi-nozzle deposition process, i) self-shaping ceramic structures can be realized through anisotropic shrinkage induced by suspensions' composition variation and ii) technical and functional multiceramic structures can be fabricated. The simplicity of the proposed technique and its inexpensive processing cost make it an attractive approach for fabricating geometrically complex ceramic articles with unique macrostructures, which complements the existing state of-the-art ceramic additive manufacturing techniques.

Abstract
Despite recent advances to control the spatial composition and dynamic functionalities of bacteria embedded in materials, bacterial localization into complex three-dimensional (3D) geometries remains a major challenge. We demonstrate a 3D printing approach to create bacteria-derived functional materials by combining the natural diverse metabolism of bacteria with the shape design freedom of additive manufacturing. To achieve this, we embedded bacteria in a biocompatible and functionalized 3D printing ink and printed two types of {textquotedblleft}living materials{textquotedblright} capable of degrading pollutants and of producing medically relevant bacterial cellulose. With this versatile bacteria-printing platform, complex materials displaying spatially specific compositions, geometry, and properties not accessed by standard technologies can be assembled from bottom up for new biotechnological and biomedical applications.

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
Repair of large oral bone defects such as vertical alveolar ridge augmentation could benefit from the rapidly developing additive manufacturing technology used to create personalized osteoconductive devices made from porous tricalcium phosphate/hydroxyapatite (TCP/HA)-based bioceramics. These devices can be also used as hydrogel carriers to improve their osteogenic potential. However, the TCP/HA constructs are prone to brittle fracture, therefore their use in clinical situations is difficult. As a solution, we propose the protection of this osteoconductive multi-material (herein called “core”) with a shape-matched “cover” made from biocompatible poly-ɛ-caprolactone (PCL), which is a ductile, and thus more resistant polymeric material. In this report, we present a workflow starting from patient-specific medical scan in Digital Imaging and Communications in Medicine (DICOM) format files, up to the design and 3D printing of a hydrogel-loaded porous TCP/HA core and of its corresponding PCL cover. This cover could also facilitate the anchoring of the device to the patient's defect site via fixing screws. The large, linearly aligned pores in the TCP/HA bioceramic core, their sizes, and their filling with an alginate hydrogel were analyzed by micro-CT. Moreover, we created a finite element analysis (FEA) model of this dual-function device, which permits the simulation of its mechanical behavior in various anticipated clinical situations, as well as optimization before surgery. In conclusion, we designed and 3D-printed a novel, structurally complex multi-material osteoconductive-osteoprotective device with anticipated mechanical properties suitable for large-defect oral bone regeneration.

Abstract
Purpose
In patients suffering from unilateral osteoarthritis in the knee, an osteotomy can provide symptomatic relief and postpone the need for replacement of the joint. Nevertheless, open-wedge osteotomies (OWO) around the knee joint face several challenges like postoperative pain and bone non-union. In this study, the aim was to design, fabricate, and evaluate a gap-filling implant for OWO using an osteoinductive and degradable biomaterial.
Methods
Design of porous wedge-shaped implants was based on computed tomography (CT) scans of cadaveric legs. Implants were 3D printed using a magnesium strontium phosphate-polycaprolactone (MgPSr-PCL) biomaterial ink. Standardized scaffolds with different inter-fibre spacing (IFS) were mechanically characterized and osteoinductive properties of the biomaterial were assessed in vitro. Finally, human-sized implants with different heights (5 mm, 10 mm, 15 mm) were designed and fabricated for ex vivo implantation during three OWO procedures in human cadaveric legs.
Results
Implants printed with an interior of IFS-1.0 resulted in scaffolds that maintained top and bottom porosity, while the interior of the implant exhibited significant mechanical stability. Bone marrow concentrate and culture expanded mesenchymal stromal cells attached to the MgPSr-PCL material and proliferated over 21 days in culture. The production of osteogenic markers alkaline phosphatase activity, calcium, and osteocalcin was promoted in all culture conditions, independent of osteogenic induction medium. Finally, three OWO procedures were planned and fabricated wedges were implanted ex vivo during the procedures. A small fraction of one side of the wedges was resected to assure fit into the proximal biplanar osteotomy gap. Pre-planned wedge heights were maintained after implantation as measured by micro-CT.
Conclusion
To conclude, personalized implants for implantation in open-wedge osteotomies were successfully designed and manufactured. The implant material supported osteogenesis of MSCs and BMC in vitro and full-size implants were successfully implemented into the surgical procedure, without compromising pre-planned wedge height.

Abstract
This research investigates the accelerated hydrolytic degradation process of both anatomically designed bone scaffolds with a pore size gradient and a rectangular shape (biomimetically designed scaffolds or bone bricks). The effect of material composition is investigated considering poly-ε-caprolactone (PCL) as the main scaffold material, reinforced with ceramics such as hydroxyapatite (HA), β-tricalcium phosphate (TCP) and bioglass at a concentration of 20 wt%. In the case of rectangular scaffolds, the effect of pore size (200 μm, 300 μm and 500 μm) is also investigated. The degradation process (accelerated degradation) was investigated during a period of 5 days in a sodium hydroxide (NaOH) medium. Degraded bone bricks and rectangular scaffolds were measured each day to evaluate the weight loss of the samples, which were also morphologically, thermally, chemically and mechanically assessed. The results show that the PCL/bioglass bone brick scaffolds exhibited faster degradation kinetics in comparison with the PCL, PCL/HA and PCL/TCP bone bricks. Furthermore, the degradation kinetics of rectangular scaffolds increased by increasing the pore size from 500 μm to 200 μm. The results also indicate that, for the same material composition, bone bricks degrade slower compared with rectangular scaffolds. The scanning electron microscopy (SEM) images show that the degradation process was faster on the external regions of the bone brick scaffolds (600 μm pore size) compared with the internal regions (200 μm pore size). The thermal gravimetric analysis (TGA) results show that the ceramic concentration remained constant throughout the degradation process, while differential scanning calorimetry (DSC) results show that all scaffolds exhibited a reduction in crystallinity (Xc), enthalpy (Δm) and melting temperature (Tm) throughout the degradation process, while the glass transition temperature (Tg) slightly increased. Finally, the compression results show that the mechanical properties decreased during the degradation process, with PCL/bioglass bone bricks and rectangular scaffolds presenting higher mechanical properties with the same design in comparison with the other materials.

Abstract
Synthetic bone substitutes which can be adapted preoperatively and patient specific may be helpful in various bony defects in the field of oral- and maxillofacial surgery. For this purpose, composite grafts made of self-setting and oil-based calcium phosphate cement (CPC) pastes, which were reinforced with 3D-printed polycaprolactone (PCL) fiber mats were manufactured.

Abstract
The development of advanced biomaterials and manufacturing processes to fabricate biologically and mechanically appropriate scaffolds for bone tissue is a significant challenge. Polycaprolactone (PCL) is a biocompatible and degradable polymer used in bone tissue engineering, but it lacks biofunctionalization. Bioceramics, such as hydroxyapatite (HA) and β tricalcium phosphate (β-TCP), which are similar chemically to native bone, can facilitate both osteointegration and osteoinduction whilst improving the biomechanics of a scaffold. Carbon nanotubes (CNTs) display exceptional electrical conductivity and mechanical properties. A major limitation is the understanding of how PCL-based scaffolds containing HA, TCP, and CNTs behave in vivo in a bone regeneration model. The objective of this study was to evaluate the use of three-dimensional (3D) printed PCL-based composite scaffolds containing CNTs, HA, and β-TCP during the initial osteogenic and inflammatory response phase in a critical bone defect rat model. Gene expression related to early osteogenesis, the inflammatory phase, and tissue formation was evaluated using quantitative real-time PCR (RT-qPCR). Tissue formation and mineralization were assessed by histomorphometry. The CNT+HA/TCP group presented higher expression of osteogenic genes after seven days. The CNT+HA and CNT+TCP groups stimulated higher gene expression for tissue formation and mineralization, and pro- and anti-inflammatory genes after 14 and 30 days. Moreover, the CNT+TCP and CNT+HA/TCP groups showed higher gene expressions related to M1 macrophages. The association of CNTs with ceramics at 10wt% (CNT+HA/TCP) showed lower expressions of inflammatory genes and higher osteogenic, presenting a positive impact and balanced cell signaling for early bone formation. The association of CNTs with both ceramics promoted a minor inflammatory response and faster bone tissue formation.

Abstract
Critical bone defects are the most difficult challenges in the area of tissue repair. Polycaprolactone (PCL) scaffolds, associated with hydroxyapatite (HA) and tricalcium phosphate (TCP), are reported to have an enhanced bioactivity. Moreover, the use of electrical stimulation (ES) has overcome the lack of bioelectricity at the bone defect site and compensated the endogenous electrical signals. Such treatments could modulate cells and tissue signaling pathways. However, there is no study investigating the effects of ES and bioceramic composite scaffolds on bone tissue formation, particularly in the view of cell signaling pathway. This study aims to investigate the application of HA/TCP composite scaffolds and ES and their effects on the Wingless-related integration site (Wnt) pathway in critical bone repair. Critical bone defects (25 mm2) were performed in rats, which were divided into four groups: PCL, PCL + ES, HA/TCP and HA/TCP + ES. The scaffolds were grafted at the defect site and applied with the ES application twice a week using 10 µA of current for 5 min. Bone samples were collected for histomorphometry, immunohistochemistry and molecular analysis. At the Wnt canonical pathway, HA/TCP and HA/TCP + ES groups showed higher Wnt1 and β-catenin gene expression levels, especially HA/TCP. Moreover, HA/TCP + ES presented higher Runx2, Osterix and Bmp-2 levels. At the Wnt non-canonical pathway, HA/TCP group showed higher voltage-gated calcium channel (Vgcc), calmodulin-dependent protein kinase II, and Wnt5a genes expression, while HA/TCP + ES presented higher protein expression of VGCC and calmodulin (CaM) at the same period. The decrease in sclerostin and osteopontin genes expressions and the lower bone sialoprotein II in the HA/TCP + ES group may be related to the early bone remodeling. This study shows that the use of ES modulated the Wnt pathways and accelerated the osteogenesis with improved tissue maturation.

Abstract
Bone tissue engineering scaffolds have made significant progress in treating bone defects in recent decades. However, the lack of a vascular network within the scaffold limits bone formation after implantation in vivo. Recent research suggests that leonurine hydrochloride (LH) can promote healing in full-thickness cutaneous wounds by increasing vessel formation and collagen deposition. Gelatin and Sodium Alginate are both polymers. ATP is a magnesium silicate chain mineral. In this study, a Gelatin/Sodium Alginate/Nano-Attapulgite composite hydrogel was used as the base material first, and the Gelatin/Sodium Alginate/Nano-Attapulgite composite polymer scaffold loaded with LH was then created using 3D printing technology. Finally, LH was grafted onto the base material by an amide reaction to construct a scaffold loaded with LH to achieve long-term LH release. When compared to pure polymer scaffolds, in vitro results showed that LH-loaded scaffolds promoted the differentiation of BMSCs into osteoblasts, as evidenced by increased expression of osteogenic key genes. The results of in vivo tissue staining revealed that the drug-loaded scaffold promoted both angiogenesis and bone formation. Collectively, these findings suggest that LH-loaded Gelatin/Sodium Alginate/Nano-Attapulgite composite hydrogel scaffolds are a potential therapeutic strategy and can assist bone regeneration.

Abstract
The treatment of bone defects caused by diseases, trauma, or tumor has always been a great clinical challenge. Implantation of bone biomaterials into bone defect areas is an effective method for bone injury repair. In this study, we used three-dimensional (3D) printing technology to prepare nano-hydroxyapatite (nHA)/sodium alginate (SA)/gelatin (Gel) hydrogel scaffolds loaded with different ratios (0, 0.13, 0.26, 0.39, 0.53, and 0.79‰) of emodin (EM) (EM/nHA/SA/Gel). Scanning electron microscopy showed that the scaffolds had a smooth surface without fracture and nHA was evenly distributed on the surface. The cell proliferation and migration results showed that the 0.39‰ EM group, in particular, could significantly promote the proliferation and migration of mouse embryonic osteoblast precursor (MC3T3-E1) cells and significantly increase the mRNA expression of osteogenic differentiation-related genes (bone morphogenetic protein/BMP-2, BMP-9, osteocalcin). In addition, the 0.39‰ EM group exhibited the best effect on osteogenic differentiation-related proteins (alkaline phosphatase, Runx 2, OSX). The expression of M2 polarization-related genes (arginase-1, CD206) also significantly increased after the treatment with the 0.39‰ EM group. Micro-CT showed that in the rat skull defect model, the EM/nHA/SA/Gel scaffold group significantly promoted bone regeneration after being implanted into the skull for 30 days. Our results indicate that the EM/nHA/SA/Gel hydrogel scaffolds can not only directly promote the proliferation and differentiation of osteoblasts but also indirectly promote osteogenic differentiation by supporting M2 polarization of macrophages. EM/nHA/SA/Gel hydrogel scaffolds are potential bone tissue engineering materials for bone regeneration.

Abstract
Layered nanoparticles with surface charge are explored as rheological modifiers for extrudable materials, utilizing their ability to induce electrostatic repulsion and create a house-of-cards structure. These nanoparticles provide mechanical support to the polymer matrix, resulting in increased viscosity and storage modulus. Moreover, their advantageous aspect ratio allows for shear-induced orientation and decreased viscosity during flow. In this work, we present a synthesis and liquid-based exfoliation procedure of phenylphosphonate-phosphate particles with enhanced ability to be intercalated by hydrophilic polymers. These layered nanoparticles are then tested as rheological modifiers of sodium alginate. The effective rheological modification is proved as the viscosity increases from 101 up to 103 Pa·s in steady state. Also, shear-thinning behavior is observed. The resulting nanocomposite hydrogels show potential as an extrudable bioink for 3D printing in tissue engineering and other biomedical applications, with good shape fidelity, nontoxicity, and satisfactory cell viability confirmed through encapsulation and printing of mouse fibroblasts.

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
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
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
The middle ear bones (‘ossicles’) may become severely damaged due to accidents or to diseases. In these situations, the most common current treatments include replacing them with cadaver-derived ossicles, using a metal (usually titanium) prosthesis, or introducing bridges made of biocompatible ceramics. Neither of these solutions is ideal, due to the difficulty in finding or producing shape-matching replacements. However, the advent of additive manufacturing applications to biomedical problems has created the possibility of 3D-printing anatomically correct, shape- and size-personalized ossicle prostheses. To demonstrate this concept, we generated and printed several models of ossicles, as solid, porous, or soft material structures. These models were first printed with a plottable calcium phosphate/hydroxyapatite paste by extrusion on a solid support or embedded in a Carbopol hydrogel bath, followed by temperature-induced hardening. We then also printed an ossicle model with this ceramic in a porous format, followed by loading and crosslinking an alginate hydrogel within the pores, which was validated by microCT imaging. Finally, ossicle models were printed using alginate as well as a cell-containing nanocellulose-based bioink, within the supporting hydrogel bath. In selected cases, the devised workflow and the printouts were tested for repeatability. In conclusion, we demonstrate that moving beyond simplistic geometric bridges to anatomically realistic constructs is possible by 3D printing with various biocompatible materials and hydrogels, thus opening the way towards the in vitro generation of personalized middle ear prostheses for implantation.

Abstract
The middle ear bones (‘ossicles’) may become severely damaged due to accidents or to diseases. In these situations, the most common current treatments include replacing them with cadaver-derived ossicles, using a metal (usually titanium) prosthesis, or introducing bridges made of biocompatible ceramics. Neither of these solutions is ideal, due to the difficulty in finding or producing shape-matching replacements. However, the advent of additive manufacturing applications to biomedical problems has created the possibility of 3D-printing anatomically correct, shape- and size-personalized ossicle prostheses. To demonstrate this concept, we generated and printed several models of ossicles, as solid, porous, or soft material structures. These models were first printed with a plottable calcium phosphate/hydroxyapatite paste by extrusion on a solid support or embedded in a Carbopol hydrogel bath, followed by temperature-induced hardening. We then also printed an ossicle model with this ceramic in a porous format, followed by loading and crosslinking an alginate hydrogel within the pores, which was validated by microCT imaging. Finally, ossicle models were printed using alginate as well as a cell-containing nanocellulose-based bioink, within the supporting hydrogel bath. In selected cases, the devised workflow and the printouts were tested for repeatability. In conclusion, we demonstrate that moving beyond simplistic geometric bridges to anatomically realistic constructs is possible by 3D printing with various biocompatible materials and hydrogels, thus opening the way towards the in vitro generation of personalized middle ear prostheses for implantation.

Abstract
The middle ear bones (‘ossicles’) may become severely damaged due to accidents or to diseases. In these situations, the most common current treatments include replacing them with cadaver-derived ossicles, using a metal (usually titanium) prosthesis, or introducing bridges made of biocompatible ceramics. Neither of these solutions is ideal, due to the difficulty in finding or producing shape-matching replacements. However, the advent of additive manufacturing applications to biomedical problems has created the possibility of 3D-printing anatomically correct, shape- and size-personalized ossicle prostheses. To demonstrate this concept, we generated and printed several models of ossicles, as solid, porous, or soft material structures. These models were first printed with a plottable calcium phosphate/hydroxyapatite paste by extrusion on a solid support or embedded in a Carbopol hydrogel bath, followed by temperature-induced hardening. We then also printed an ossicle model with this ceramic in a porous format, followed by loading and crosslinking an alginate hydrogel within the pores, which was validated by microCT imaging. Finally, ossicle models were printed using alginate as well as a cell-containing nanocellulose-based bioink, within the supporting hydrogel bath. In selected cases, the devised workflow and the printouts were tested for repeatability. In conclusion, we demonstrate that moving beyond simplistic geometric bridges to anatomically realistic constructs is possible by 3D printing with various biocompatible materials and hydrogels, thus opening the way towards the in vitro generation of personalized middle ear prostheses for implantation.

Abstract
A new prototype of hybrid silk fibroin and sodium alginate (SF-SA) based osteogenic hydrogel scaffold with a concentration of 2.5% magnesium phosphate (MgP) based gel was prepared with the assistance of an extrusion-based three-dimensional (3D) printing machine in this study. To determine the optimum ratio of MgP-based gel in the hydrogel, a series of physical and biochemical experiments were performed to determine the proper concentration of MgP in two-dimensional hydrogel films, as well as the cell compatibility with these materials in sequence. The SF-SA hydrogel with 2.5wt% magnesium phosphate (SF-SA/MgP) stood out and then was used to fabricate 3D hydrogel scaffolds according to the consequences of the experiments, with SF-SA hydrogel as a control. Then the morphology and osteogenic activity of the scaffolds were further characterized by field emission scanning electron microscope (SEM), calcium mineralization staining, and reverse transcription-polymerase chain reaction (rt-PCR). The SF-SA/MgP hydrogel scaffold promoted the adhesion of rat mesenchymal stem cells with higher degrees of efficiency under dynamic culture conditions. After co-culturing in an osteogenic differentiation medium, cells seeded on SF-SA/MgP hydrogel scaffold were shown to have better performance on osteogenesis in the early stage than the control group. This work illustrates that the 3D structures of hybrid SF-SA/MgP hydrogel are promising headstones for osteogenic tissue engineering.

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
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
3D printed bioactive glass or bioceramic particle reinforced composite scaffolds for bone tissue engineering currently suffer from low particle concentration (100% breaking strain) by adding poly(ethylene glycol) which is biocompatible and FDA approved. The scaffolds require no post-printing washing to remove hazardous components. More exposure of HA microparticles on strut surfaces is enabled by incorporating higher HA concentrations. Compared to scaffolds with 72 wt% HA{,} scaffolds with higher HA content (90 wt%) enhance matrix formation but not new bone volume after 12 weeks implantation in rat calvarial defects. Histological analyses demonstrate that bone regeneration within the 3D printed scaffolds is via intramembranous ossification and starts in the central region of pores. Fibrous tissue that resembles non-union tissue within bone fractures is formed within pores that do not have new bone. The amount of blood vessels is similar between scaffolds with mainly fibrous tissue and those with more bone tissue{,} suggesting vascularization is not a deciding factor for determining the type of tissues regenerated within the pores of 3D printed scaffolds. Multinucleated immune cells are commonly present in all scaffolds surrounding the struts{,} suggesting a role of managing inflammation in bone regeneration within 3D printed scaffolds.

Abstract
Chondrogenic models utilizing human mesenchymal stromal cells (hMSCs) are often simplistic, with a single cell type and the absence of mechanical stimulation. Considering the articulating joint as an organ it would be beneficial to include more complex stimulation. Within this study we applied clinically relevant kinematic load to biphasic constructs. In each case, the upper layer consisted of fibrin embedded hMSCs retained within an elastomeric polyurethane (PU) scaffold. These were randomly assigned to five base scaffolds, a cell-free fibrin PU base, viable bone, decellularized bone, 3D printed calcium phosphate or clinically used cement. This allowed the study of cross talk between viable bone and chondrogenically differentiating MSCs, while controlling for the change in stiffness of the base material. Data obtained showed that the bulk stiffness of the construct was not the defining factor in the response obtained, with viable and decellularized bone producing similar results to the softer PU base. However, the stiff synthetic materials led to reduced chondrogenesis and increased calcification in the upper MSC seeded layer. This demonstrates that the underlying base material must be considered when driving chondrogenesis of human cells using a clinically relevant loading protocol. It also indicates that the material used for bony reconstruction of osteochondral defects may influence subsequent chondrogenic potential.

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
Recently, Deferoxamine (DFO) and magnesium (Mg) have been identified as critical factors for angiogenesis and bone formation. However, in current research studies, there is a lack of focus on whether DFO plus Mg can affect the regeneration of β-tricalcium phosphate (β-TCP) in osteoporosis and through what biological mechanisms. Therefore, the present work was aimed to preparation and evaluate the effect of Deferoxamine/magnesium modified β-tricalcium phosphate promotes (DFO/Mg-TCP) in ovariectomized rats model and preliminary exploration of possible mechanisms. The MC3T3-E1 cells were co-cultured with the exudate of DFO/Mg-TCP and induced to osteogenesis, and the cell viability, osteogenic activity were observed by Cell Counting Kit-8(CCK-8), Alkaline Phosphatase (ALP) staining, Alizarin Red Staining (RES) and Western Blot. In vitro experiments, CCK-8, ALP and ARS staining results show that the mineralization and osteogenic activity of MC3T3-E1increased significantly after intervention by DFO/Mg-TCP, as well as a higher levels of protein expressions including VEGF, OC, Runx-2 and HIF-1α. In vivo experiment, Micro-CT and Histological analysis evaluation show that DFO/Mg-TCP treatment presented the stronger effect on bone regeneration, bone mineralization and biomaterial degradation, when compared with OVX+Mg-TCP group and OVX+TCP group, as well as a higher VEGF, OC, Runx-2 and HIF-1α gene expression. The present study indicates that treatment with DFO/Mg-TCP was associated with increased regeneration by enhancing the function of osteoblasts in an OVX rat.

Abstract
Fabrication of three-dimensional (3D) scaffolds using natural biomaterials introduces valuable opportunities in bone tissue reconstruction and regeneration. The current study aimed at the development of paste-like 3D printing inks with an extracellular matrix-inspired formulation based on marine materials: sodium alginate (SA), cuttlebone (CB), and fish gelatin (FG). Macroporous scaffolds with microporous biocomposite filaments were obtained by 3D printing combined with post-printing crosslinking. CB fragments were used for their potential to stimulate biomineralization. Alginate enhanced CB embedding within the polymer matrix as confirmed by scanning electron microscopy (ESEM) and micro-computer tomography (micro-CT) and improved the deformation under controlled compression as revealed by micro-CT. SA addition resulted in a modulation of the bulk and surface mechanical behavior, and lead to more elongated cell morphology as imaged by confocal microscopy and ESEM after the adhesion of MC3T3-E1 preosteoblasts at 48 h. Formation of a new mineral phase was detected on the scaffold’s surface after cell cultures. All the results were correlated with the scaffolds’ compositions. Overall, the study reveals the potential of the marine materials-containing inks to deliver 3D scaffolds with potential for bone regeneration applications.

Abstract
Hierarchical structures are abundant in almost all tissues of the human body. Therefore, it is highly important for tissue engineering approaches to mimic such structures if a gain of function of the new tissue is intended. Here, the hierarchical structures of the so-called enthesis, a gradient tissue located between tendon and bone, were in focus. Bridging the mechanical properties from soft to hard secures a perfect force transmission from the muscle to the skeleton upon locomotion. This study aimed at a novel method of bioprinting to generate gradient biomaterial constructs with a focus on the evaluation of the gradient printing process. First, a numerical approach was used to simulate gradient formation by computational flow as a prerequisite for experimental bioprinting of gradients. Then, hydrogels were printed in a single cartridge printing set-up to transfer the findings to biomedically relevant materials. First, composites of recombinant spider silk hydrogels with fluorapatite rods were used to generate mineralized gradients. Then, fibroblasts were encapsulated in the recombinant spider silk-fluorapatite hydrogels and gradually printed using unloaded spider silk hydrogels as the second component. Thereby, adjustable gradient features were achieved, and multimaterial constructs were generated. The process is suitable for the generation of gradient materials, e.g., for tissue engineering applications such as at the tendon/bone interface.

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
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
Critical bone defects are a major clinical challenge in reconstructive bone surgery. Polycaprolactone (PCL) mixed with bioceramics, such as hydroxyapatite (HA) and tricalcium phosphate (TCP), create composite scaffolds with improved biological recognition and bioactivity. Electrical stimulation (ES) aims to compensate the compromised endogenous electrical signals and to stimulate cell proliferation and differentiation. We investigated the effects of composite scaffolds (PCL with HA; and PCL with β-TCP) and the use of ES on critical bone defects in Wistar rats using eight experimental groups: untreated, ES, PCL, PCL/ES, HA, HA/ES, TCP, and TCP/ES. The investigation was based on histomorphometry, immunohistochemistry, and gene expression analysis. The vascular area was greater in the HA/ES group on days 30 and 60. Tissue mineralization was greater in the HA, HA/ES, and TCP groups at day 30, and TCP/ES at day 60. Bmp-2 gene expression was higher in the HA, TCP, and TCP/ES groups at day 30, and in the TCP/ES and PCL/ES groups at day 60. Runx-2, Osterix, and Osteopontin gene expression were also higher in the TCP/ES group at day 60. These results suggest that scaffolds printed with PCL and TCP, when paired with electrical therapy application, improve bone regeneration.

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 Hydrogel ink formulations based on rheology additives are becoming increasingly popular as they enable 3-dimensional (3D) printing of non-printable but biologically relevant materials. Despite the widespread use, a generalized understanding of how these hydrogel formulations become printable is still missing, mainly due to their variety and diversity. Employing an interpretable machine learning approach allows the authors to explain the process of rendering printability through bulk rheological indices, with no bias toward the composition of formulations and the type of rheology additives. Based on an extensive library of rheological data and printability scores for 180 different formulations, 13 critical rheological measures that describe the printability of hydrogel formulations, are identified. Using advanced statistical methods, it is demonstrated that even though unique criteria to predict printability on a global scale are highly unlikely, the accretive and collaborative nature of rheological measures provides a qualitative and physically interpretable guideline for designing new printable materials.

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
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
Emerging 3D printing technologies can provide exquisite control over the external shape and internal architecture of scaffolds and tissue engineered constructs, enabling systematic studies to explore how geometric design features influence the regenerative process. Here we used fused deposition modelling (FDM) and melt electrowriting (MEW) to investigate how scaffold microarchitecture influences the healing of large bone defects. FDM was used to fabricate scaffolds with relatively large fibre diameters and low porosities, while MEW was used to fabricate scaffolds with smaller fibre diameters and higher porosities, with both scaffolds being designed to have comparable surface areas. Scaffold microarchitecture significantly influenced the healing response following implantation into critically sized femoral defects in rats, with the FDM scaffolds supporting the formation of larger bone spicules through its pores, while the MEW scaffolds supported the formation of a more round bone front during healing. After 12 weeks in vivo, both MEW and FDM scaffolds supported significantly higher levels of defect vascularisation compared to empty controls, while the MEW scaffolds supported higher levels of new bone formation. Somewhat surprisingly, this superior healing in the MEW group did not correlate with higher levels of angiogenesis, with the FDM scaffold supporting greater total vessel formation and the formation of larger vessels, while the MEW scaffold promoted the formation of a dense microvasculature with minimal evidence of larger vessels infiltrating the defect region. To conclude, the small fibre diameter, high porosity and high specific surface area of the MEW scaffold proved beneficial for osteogenesis and bone regeneration, demonstrating that changes in scaffold architecture enabled by this additive manufacturing technique can dramatically modulate angiogenesis and tissue regeneration without the need for complex exogenous growth factors. These results provide a valuable insight into the importance of 3D printed scaffold architecture when developing new bone tissue engineering strategies.

Abstract
Hydroxyapatite (HA) is a promising scaffold material for the treatment of bone defects. However{,} the lack of angiogenic properties and undesirable mechanical properties (such as fragility) limits the application of HA. Nanoattapulgite (ATP) is a nature-derived clay mineral and has been proven to be a promising bioactive material for bone regeneration due to its ability to induce osteogenesis. In this study{,} polyvinyl alcohol/collagen/ATP/HA (PVA/COL/ATP/HA) scaffolds were printed. Mouse bone marrow mesenchymal stem/stromal cells (BMSCs) and human umbilical vein endothelial cells (HUVECs) were used in vitro to assess the biocompatibility and the osteogenesis and vascularization induction potentials of the scaffolds. Subsequently{,} in vivo micro-CT and histological staining were carried out to evaluate new bone formation in a rabbit tibial defect model. The in vitro results showed that the incorporation of ATP increased the printing fidelity and mechanical properties{,} with values of compressive strengths up to 200% over raw PC-H scaffolds. Simultaneously{,} the expression levels of osteogenic-related genes and vascularization-related genes were significantly increased after the incorporation of ATP. The in vivo results showed that the PVA/COL/ATP/HA scaffolds exhibited synergistic effects on promoting vascularization and bone formation. The combination of ATP and HA provides a promising strategy for vascularized bone tissue engineering.

Abstract
Objective
Alveolar bone defects can be highly variable in their morphology and, as the defect size increases, they become more challenging to treat with currently available therapeutics and biomaterials. This investigation sought to devise a protocol for fabricating customized clinical scale and patient-specific, bioceramic scaffolds for reconstruction of large alveolar bone defects.
Methods
Two types of calcium phosphate (CaP)-based bioceramic scaffolds (alginate/β-TCP and hydroxyapatite/α-TCP, hereafter referred to as hybrid CaP and Osteoink™, respectively) were designed, 3D printed, and their biocompatibility with alveolar bone marrow stem cells and mechanical properties were determined. Following scaffold optimization, a workflow was developed to use cone beam computed tomographic (CBCT) imaging to design and 3D print, defect-specific bioceramic scaffolds for clinical-scale bone defects.
Results
Osteoink™ scaffolds had the highest compressive strength when compared to hybrid CaP with different infill orientation. In cell culture medium, hybrid CaP degradation resulted in decreased pH (6.3) and toxicity to stem cells; however, OsteoInk™ scaffolds maintained a stable pH (7.2) in culture and passed the ISO standard for cytotoxicity. Finally, a clinically feasible laboratory workflow was developed and evaluated using CBCT imaging to engineer customized and defect-specific CaP scaffolds using OsteoInk™. It was determined that printed scaffolds had a high degree of accuracy to fit the respective clinical defects for which they were designed (0.27 mm morphological deviation of printed scaffolds from digital design).
Significance
From patient to patient, large alveolar bone defects are difficult to treat due to high variability in their complex morphologies and architecture. Our findings shows that Osteoink™ is a biocompatible material for 3D printing of clinically acceptable, patient-specific scaffolds with precision-fit for use in alveolar bone reconstructive procedures. Collectively, emerging digital technologies including CBCT imaging, 3D surgical planning, and (bio)printing can be integrated to address this unmet clinical challenge.

Abstract
Biocompatibility, biodegradability, shear tinning behavior, quick gelation and an easy crosslinking process makes alginate one of the most studied polysaccharides in the field of regenerative medicine. The main purpose of this study was to obtain tissue-like materials suitable for use in bone regeneration. In this respect, alginate and several types of clay were investigated as components of 3D-printing, nanocomposite inks. Using the extrusion-based nozzle, the nanocomposites inks were printed to obtain 3D multilayered scaffolds. To observe the behavior induced by each type of clay on alginate-based inks, rheology studies were performed on composite inks. The structure of the nanocomposites samples was examined using Fourier Transform Infrared Spectrometry and X-ray Diffraction (XRD), while the morphology of the 3D-printed scaffolds was evaluated using Electron Microscopy (SEM, TEM) and Micro-Computed Tomography (Micro-CT). The swelling and dissolvability of each composite scaffold in phosfate buffer solution were followed as function of time. Biological studies indicated that the cells grew in the presence of the alginate sample containing unmodified clay, and were able to proliferate and generate calcium deposits in MG-63 cells in the absence of specific signaling molecules. This study provides novel information on potential manufacturing methods for obtaining nanocomposite hydrogels suitable for 3D printing processes, as well as valuable information on the clay type selection for enabling accurate 3D-printed constructs. Moreover, this study constitutes the first comprehensive report related to the screening of several natural clays for the additive manufacturing of 3D constructs designed for bone reconstruction therapy.

Abstract
Bioactive composites made of ∽85 wt% poly(ε-caprolactone) (PCL) and ∽15 wt% nanometric hydroxyapatite (HA) produced from biogenic sources were 3D printed by an extrusion-based process to obtain porous scaffolds suitable for bone regeneration. Three different composite formulations were considered by using HA synthesized from three distinct natural sources, which were collected as food wastes: cuttlefish bones, mussel shells and chicken eggshells. Composition and thermal properties of the materials were analysed by thermogravimetric analysis (TGA), differential scanning calorimetry (DSC) and x-ray spectroscopy (XRD), while the morphological and mechanical properties of the 3D scaffolds were studied by means of electron microscopy (SEM) and compression tests. Bioactivity was tested by seeding human osteoblast cell line (MG63) onto the scaffolds which were analysed by confocal microscopy and Alamar Blue and PicoGreen® tests after 1 to 7 culture days. The elastic modulus (177–316 MPa) is found to be within the range reported for typical trabecular bones being increased by the presence of the bio-HA particles. Moreover, cells adhesion, viability and proliferation are largely promoted in the scaffolds containing nanometric HA with respect to pure PCL, the best results being revealed when mussel shell-derived HA is used. Indeed, different biological sources result in different cell proliferation rates, pointing that the biological origin has an impact on the cells-scaffold interaction. In general, the results show that PCL/bio-HA scaffolds possess improved mechanical properties and enhanced bioactivity when compared with pure PCL ones.

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
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
Micro-extrusion-based 3D printing of complex geometrical and porous calcium phosphate (CaP) can improve treatment of bone defects through the production of personalized bone substitutes. However, achieving printing and post-printing shape stabilities for the efficient fabrication and application of rapid hardening protocol are still challenging. In this work, the coaxial printing of a self-setting CaP cement with water and ethanol mixtures aiming to increase the ink yield stress upon extrusion and the stability of fabricated structures was explored. Printing height of overhang structure was doubled when aqueous solvents were used and a 2 log increase of the stiffness was achieved post-printing. A standard and fast steam sterilization protocol applied as hardening step on the coaxial printed CaP cement (CPC) ink resulted in constructs with 4 to 5 times higher compressive moduli in comparison to extrusion process in the absence of solvent. This improved mechanical performance is likely due to rapid CPC setting, preventing cracks formation during hardening process. Thus, coaxial micro-extrusion-based 3D printing of a CPC ink with aqueous solvent enhances printability and allows the use of the widespread steam sterilization cycle as a standalone post-processing technique for production of 3D printed personalized CaP bone substitutes.
Statement of Significance
Coaxial micro-extrusion-based 3D printing of a self-setting CaP cement with water:ethanol mixtures increased the ink yield stress upon extrusion and the stability of fabricated structures. Printing height of overhang structure was doubled when aqueous solvents were used, and a 2 orders of magnitude log increase of the stiffness was achieved post-printing. A fast hardening step consisting of a standard steam sterilization was applied. Four to 5 times higher compressive moduli was obtained for hardened coaxially printed constructs. This improved mechanical performance is likely due to rapid CPC setting in the coaxial printing, preventing cracks formation during hardening process.

Abstract
Thanks to its biological properties, the human amniotic membrane (HAM) combined with a bone substitute could be a single-step surgical alternative to the two-step Masquelet induced membrane (IM) technique for regeneration of critical bone defects. However, no study has directly compared these two membranes. We first designed a 3D-printed scaffold using calcium phosphate cement (CPC). We assessed its suitability in vitro to support human bone marrow mesenchymal stromal cells (hBMSCs) attachment and osteodifferentiation. We then performed a rat femoral critical size defect to compare the two-step IM technique with a single-step approach using the HAM. Five conditions were compared. Group 1 was left empty. Group 2 received the CPC scaffold loaded with rh-BMP2 (CPC/BMP2). Group 3 and 4 received the CPC/BMP2 scaffold covered with lyophilized or decellularized/lyophilized HAM. Group 5 underwent a two- step induced membrane procedure with insertion of a polymethylmethacrylate (PMMA) spacer followed by, after 4 weeks, its replacement with the CPC/BMP2 scaffold wrapped in the IM. Micro-CT and histomorphometric analysis were performed after six weeks. Results showed that the CPC scaffold supported the proliferation and osteodifferentiation of hBMSCs in vitro. In vivo, the CPC/BMP2 scaffold very efficiently induced bone formation and led to satisfactory healing of the femoral defect, in a single-step, without autograft or the need for any membrane covering. In this study, there was no difference between the two-step induced membrane procedure and a single step approach. However, the results indicated that none of the tested membranes further enhanced bone healing compared to the CPC/BMP2 group.

Abstract
Polycaprolactone (PCL) is widely used in additive manufacturing for the construction of scaffolds for tissue engineering because of its good bioresorbability, biocompatibility, and processability. Nevertheless, its use is limited by its inadequate mechanical support, slow degradation rate and the lack of bioactivity and ability to induce cell adhesion and, thus, bone tissue regeneration. In this study, we fabricated 3D PCL scaffolds reinforced with a novel Mg-doped bioactive glass (Mg-BG) characterized by good mechanical properties and biological reactivity. An optimization of the printing parameters and scaffold fabrication was performed; furthermore, an extensive microtopography characterization by scanning electron microscopy and atomic force microscopy was carried out. Nano-indentation tests accounted for the mechanical properties of the scaffolds, whereas SBF tests and cytotoxicity tests using human bone-marrow-derived mesenchymal stem cells (BM-MSCs) were performed to evaluate the bioactivity and in vitro viability. Our results showed that a 50/50 wt% of the polymer-to-glass ratio provides scaffolds with a dense and homogeneous distribution of Mg-BG particles at the surface and roughness twice that of pure PCL scaffolds. Compared to pure PCL (hardness H = 35 ± 2 MPa and Young’s elastic modulus E = 0.80 ± 0.05 GPa), the 50/50 wt% formulation showed H = 52 ± 11 MPa and E = 2.0 ± 0.2 GPa, hence, it was close to those of trabecular bone. The high level of biocompatibility, bioactivity, and cell adhesion encourages the use of the composite PCL/Mg-BG scaffolds in promoting cell viability and supporting mechanical loading in the host trabecular bone.

Abstract
This study aimed to develop printable calcium magnesium phosphate pastes that harden by immersion in ammonium phosphate solution post-printing. Besides the main mineral compound, biocompatible ceramic, magnesium oxide and hydroxypropylmethylcellulose (HPMC) were the crucial components. Two pastes with different powder to liquid ratios of 1.35 g/mL and 1.93 g/mL were characterized regarding their rheological properties. Here, ageing over the course of 24 h showed an increase in viscosity and extrusion force, which was attributed to structural changes in HPMC as well as the formation of magnesium hydroxide by hydration of MgO. The pastes enabled printing of porous scaffolds with good dimensional stability and enabled a setting reaction to struvite when immersed in ammonium phosphate solution. Mechanical performance under compression was approx. 8–20 MPa as a monolithic structure and 1.6–3.0 MPa for printed macroporous scaffolds, depending on parameters such as powder to liquid ratio, ageing time, strand thickness and distance.

Abstract
Sensors are crucial in the understanding of machines working under high temperatures and high-pressure conditions. Current devices utilize polymeric materials as electrical insulators which pose a challenge in the device’s lifespan. Ceramics, on the other hand, is robust and able to withstand high temperature and pressure. For such applications, a co-fired ceramic device which can provide both electrical conductivity and insulation is beneficial and acts as a superior candidate for sensor devices. In this paper, we propose a novel fabrication technique of complex multi-ceramics structures via 3D printing. This fabrication methodology increases both the geometrical complexity and the device’s shape precision. Structural ceramics (alumina) was employed as the electrical insulator whilst providing mechanical rigidity while a functional ceramic (alumina-doped zinc oxide) was employed as the electrically conductive material. The addition of sintering additives, tailoring the printing pastes’ solid loadings and heat treatment profile resolves multi-materials printing challenges such as shrinkage disparity and densification matching. Through high-temperature co-firing of ceramics (HTCC) technology, dense high quality functional multi-ceramics structures are achieved. The proposed fabrication methodology paves the way for multi-ceramics sensors to be utilized in high temperature and pressure systems in the near future.

Abstract
Background: One of the main challenges with conventional scaffold fabrication methods is the inability to control scaffold architecture. Recently, scaffolds with controlled shape and architecture have been fabricated using three-dimensional printing (3DP). Herein, we aimed to determine whether the much tighter control of microstructure of 3DP poly(lactic-co-glycolic) acid/β-tricalcium phosphate (PLGA/β-TCP) scaffolds is more effective in promoting osteogenesis than porous scaffolds produced by solvent casting/porogen leaching. Methods: Physical and mechanical properties of porous and 3DP scaffolds were studied. The response of pre-osteoblasts to the scaffolds was analyzed after 14 days. Results: The 3DP scaffolds had a smoother surface (Ra: 22 ± 3 µm) relative to the highly rough surface of porous scaffolds (Ra: 110 ± 15 µm). Water contact angle was 112 ± 4° on porous and 76 ± 6° on 3DP scaffolds. Porous and 3DP scaffolds had the pore size of 408 ± 90 and 315 ± 17 µm and porosity of 85 ± 5% and 39 ± 7%, respectively. Compressive strength of 3DP scaffolds (4.0 ± 0.3 MPa) was higher than porous scaffolds (1.7 ± 0.2 MPa). Collagenous matrix deposition was similar on both scaffolds. Cells proliferated from day 1 to day 14 by fourfold in porous and by 3.8-fold in 3DP scaffolds. Alkaline phosphatase (ALP) activity was 21-fold higher in 3DP scaffolds than porous scaffolds. Conclusion: The 3DP scaffolds show enhanced mechanical properties and ALP activity compared to porous scaffolds in vitro, suggesting that 3DP PLGA/β-TCP scaffolds are possibly more favorable for bone formation.

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
Multifunctional and resistant 3D structures represent a great promise and a great challenge in bone tissue engineering. This study addresses this problem by employing polycaprolactone (PCL)-based scaffolds added with hydroxyapatite (HAp) and superparamagnetic iron oxide nanoparticles (SPION), able to drive on demand the necessary cells and other bioagents for a high healing efficiency. PCL-HAp-SPION scaffolds with different concentrations of the superparamagnetic component were developed through the 3D-printing technology and the specific topographical features were detected by Atomic Force and Magnetic Force Microscopy (AFM-MFM). AFM-MFM measurements confirmed a homogenous distribution of HAp and SPION throughout the surface. The magnetically assisted seeding of cells in the scaffold resulted most efficient for the 1% SPION concentration, providing good cell entrapment and adhesion rates. Mesenchymal Stromal Cells (MSCs) seeded onto PCL-HAp-1% SPION showed a good cell proliferation and intrinsic osteogenic potential, indicating no toxic effects of the employed scaffold materials. The performed characterizations and the collected set of data point on the inherent osteogenic potential of the newly developed PCL-HAp-1% SPION scaffolds, endorsing them towards next steps of in vitro and in vivo studies and validations.

Abstract
The purpose of the present work was to investigate the potential for application and the effectiveness of osteoconductive scaffolds with bicontinuous phases of 3D printed bioceramics (3DP-BCs) based on reverse negative thermoresponsive hydrogels (poly[(N-isopropylacrylamide)-co-(methacrylic acid)]; p(NiPAAm-MAA)). 3DP-BCs have bioceramic objects and microchannel pores when created using robotic deposition additive manufacturing. We evaluated the benefits of silicone oil sealing on the 3DP-BC green body during the sintering process in terms of densification and structural stability. The shrinkage, density, porosity, element composition, phase structure and microstructural analyses and compression strength measurements of sintered 3DP-BC objects are presented and discussed in this study. In addition, the results of cell viability assays and bone healing analyses of the calvarial bone defects in a rabbit model were used to evaluate 3DP-BC performance. The main results indicated that these 3DP-BC scaffolds have optimal continuous pores and adequate compressive strength, which can enable the protection of calvarial defects and provide an environment for cell growth. Therefore, 3DP-BC scaffolds have better new bone regeneration efficiency in rabbit calvarial bone defect models than empty scaffolds and mold-forming bioceramic scaffolds (MF-BCs).

Abstract
BACKGROUND: Natural clay nanomaterials are an emerging class of biomaterial with great potential for tissue engineering and regenerative medicine applications, most notably for osteogenesis. MATERIALS AND METHODS: Herein, for the first time, novel tissue engineering scaffolds were prepared by 3D bioprinter using nontoxic and bioactive natural attapulgite (ATP) nanorods as starting materials, with polyvinyl alcohol as binder, and then sintered to obtain final scaffolds. The microscopic morphology and structure of ATP particles and scaffolds were observed by transmission electron microscope and scanning electron microscope. In vitro biocompatibility and osteogenesis with osteogenic precursor cell (hBMSCs) were assayed using MTT method, Live/Dead cell staining, alizarin red staining and RT-PCR. In vivo bone regeneration was evaluated with micro-CT and histology analysis in rat cranium defect model. RESULTS: We successfully printed a novel porous nano-ATP scaffold designed with inner channels with a dimension of 500 µm and wall structures with a thickness of 330 µm. The porosity of current 3D-printed scaffolds ranges from 75% to 82% and the longitudinal compressive strength was up to 4.32±0.52 MPa. We found firstly that nano-ATP scaffolds with excellent biocompatibility for hBMSCscould upregulate the expression of osteogenesis-related genes bmp2 and runx2 and calcium deposits in vitro. Interestingly, micro-CT and histology analysis revealed abundant newly formed bone was observed along the defect margin, even above and within the 3D bioprinted porous ATP scaffolds in a rat cranial defect model. Furthermore, histology analysis demonstrated that bone was formed directly following a process similar to membranous ossification without any intermediate cartilage formation and that many newly formed blood vessels are within the pores of 3D-printed scaffolds at four and eight weeks. CONCLUSION: These results suggest that the 3D-printed porous nano-ATP scaffolds are promising candidates for bone tissue engineering by osteogenesis and angiogenesis.