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Recent advancements in tissue engineering have demonstrated a great potential for the fabrication of three-dimensional tissue structures such as cartilage and bone. However, achieving structural integrity between different tissues and fabricating tissue interfaces are still great challenges. In this study, an in situ crosslinked hybrid, multi-material three-dimensional (3D) bioprinting approach was used for the fabrication of hydrogel structures based on an aspiration-extrusion microcapillary method. Different cell-laden hydrogels were aspirated in the same microcapillary glass and deposited in the desired geometrical and volumetric arrangement directly from a computer model. Alginate and carboxymethyl cellulose (CMC) were modified with tyramine to enhance cell bioactivity and mechanical properties of human bone marrow Mesenchymal Stem Cells (hBMSC)-laden bioinks. Hydrogels were prepared for extrusion by gelling in microcapillary glass utilizing an in situ crosslink approach with ruthenium (Ru) and sodium persulfate (SPS) photo-initiating mechanisms under visible light. The developed bioinks were then bioprinted in precise gradient composition for cartilage-bone tissue interface using microcapillary bioprinting technique. The biofabricated constructs were co-cultured in chondrogenic/osteogenic culture media for three weeks. After cell viability and morphology evaluations of the bioprinted structures, biochemical and histological analyses, and a gene expression analysis for the bioprinted structure were carried out. Analysis of cartilage and bone formation based on cell alignment and histological evaluation indicated that mechanical cues in conjunction with chemical cues successfully induced MSC differentiation into chondrogenic and osteogenic tissues with a controlled interface.

The advent of bioprinting technology into the tissue engineering field has permitted the attainment of complex-shaped tissue constructs with unprecedented degree of precision and reproducibility, promising for the highly demanded tissue substitutes including vascular grafts. However, most of the bioprinted vascular tissue substitutes still lack multicellular composition and hierarchical complexity of native blood vessels. In this study, a multimaterial bioprinting platform incorporating multiple-channel microfluidic printhead was combined with embedded bioprinting technique for the fabrication of vascular-like constructs. Three different bioink formulations targeting intimal, medial, and adventitial zones of the natural vascular tissues were sequentially extruded from the microfluidic channels of printhead into a hydrogel-nanoclay support bath in a controlled manner to reach the biomimicry of vascular tissues. The results demonstrated the successful deposition of three bioink compositions into distinct zones within hollow structures, which would provide an opportunity for the construction of functional vascular substitutes.

Ageing population and new diseases are requiring the development of novel therapeutical strategies. 3D bioprinting an novel application domain of additive manufacturing emerged as a potential transformative strategy for tissue engineering and regenerative medicine. This paper introduces the concept of 3D bioprinting, discussing in detail key requirements of bio-inks and main materials used to encapsulate cells. Recent advances related to the use of smart materials and the concept of 4D printing is also discussed. Main 3D bioprinting techniques are described in detail and key limitations highlighted. Successful cases, demonstrating the relevance of 3D bioprinting are also presented. Finally, the paper addresses the main research challenges and future perspectives in the field of 3D bioprinting. Published version

Tissue interfaces include complex gradient structures formed by transitioning of biochemical and mechanical properties in micro-scale. This characteristic allows the communication and synchronistic functioning of two adjacent but distinct tissues. It is particularly challenging to restore the function of these complex structures by transplantation of scaffolds exclusively produced by conventional tissue engineering methods. Three-dimensional (3D) bioprinting technology has opened an unprecedented approach for precise and graded patterning of chemical, biological and mechanical cues in a single construct mimicking natural tissue interfaces. This paper reviews and highlights biochemical and biomechanical design for 3D bioprinting of various tissue interfaces, including cartilage-bone, muscle-tendon, tendon/ligament-bone, skin, and neuro-vascular/muscular interfaces. Future directions and translational challenges are also provided at the end of the paper.

In this study, highly ionazable protons (pKa 5-6) of urazole were exploited to obtain an anionic hydrogel in two simple and scalable steps. Commercially available multiisocyanate, poly(hexamethylene)diisocyanate, was used to prepare urazole containing gel. Urazole formation was confirmed by FT-IR and 1H-NMR spectroscopy. The hydrogel were characterized by microscopy imaging, spectroscopic and gravimetric analysis. Mechanical analysis and cell viability test were performed for its initial biocompatibility evaluation. The prepared hydrogel is a highly porous hydrogel with a Young’s modulus of 0.91MPa, has swelling ratio of 87% and capable of exchanging ions in a medium. In this report, we demonstrated a strategy to overcome synthetic challenge of incorporating urazole into a material via precursor path rather than attempting to embeding urazole groups directly.

Bacteria are one of the significant causes of infection in the body after scaffold implantation. Effective use of nanotechnology to overcome this problem is an exciting and practical solution. Nanoparticles can cause bacterial degradation by the electrostatic interaction with receptors and cell walls. Simultaneously, the incorporation of antibacterial materials such as zinc and graphene in nanoparticles can further enhance bacterial degradation. In the present study, zinc-doped hydroxyapatite/graphene was synthesized and characterized as a nanocomposite material possessing both antibacterial and bioactive properties for bone tissue engineering. After synthesizing the zinc-doped hydroxyapatite nanoparticles using a mechanochemical process, they were composited with reduced graphene oxide. The nanoparticles and nanocomposite samples were extensively investigated by transmission electron microscopy, X-ray diffraction, and Raman spectroscopy. Their antibacterial behaviors against Escherichia coli and Staphylococcus aureus were studied. The antibacterial properties of hydroxyapatite nanoparticles were found to be improved more than 2.7 and 3.4 times after zinc doping and further compositing with graphene, respectively. In vitro cell assessment was investigated by a cell viability test and alkaline phosphatase activity using mesenchymal stem cells, and the results showed that hydroxyapatite nanoparticles in the culture medium, in addition to non-toxicity, led to enhanced proliferation of bone marrow stem cells. Furthermore, zinc doping in combination with graphene significantly increased alkaline phosphatase activity and proliferation of mesenchymal stem cells. The antibacterial activity along with cell biocompatibility/bioactivity of zinc-doped hydroxyapatite/graphene nanocomposite are the highly desirable and suitable biological properties for bone tissue engineering successfully achieved in this work.

Bone defect treatment is still a challenge in clinics, and synthetic bone scaffolds with adequate mechanical and biological properties are highly needed. Adequate waste and nutrient exchange of the implanted scaffold with the surrounded tissue is a major concern. Moreover, the risk of mechanical instability in the defect area during regular activity increases as the defect size increases. Thus, scaffolds with better mass transportation and mechanical properties are desired. This study introduces 3D printed polymeric scaffolds with a continuous pattern, ZigZag-Spiral pattern, for bone defects treatments. This pattern has a uniform distribution of pore size, which leads to uniform distribution of wall shear stress which is crucial for uniform differentiation of cells attached to the scaffolds. The mechanical, mass transportation, and biological properties of the 3D printed scaffolds are evaluated. The results show that the presented scaffolds have permeability similar to natural bone and, with the same porosity level, have higher mechanical properties than scaffolds with conventional lay-down patterns 0-90° and 0-45°. Finally, human mesenchymal stem cells are seeded on the scaffolds to determine the effects of geometrical microstructure on cell attachment and morphology. The results show that cells in scaffold with ZigZag-Spiral pattern infilled pores gradually, while the other patterns need more time to fill the pores. Considering mechanical, transportation, and biological properties of the considered patterns, scaffolds with ZigZag-Spiral patterns can mimic the properties of cancellous bones and be a better choice for treatments of bone defects.

The mesostructure of additively manufactured nanocomposite parts can be tailored by the manipulation of process parameters to improve the properties of the final part. The effects of contributing process parameters and their interactions must be identified to be able to tune the properties of additively manufactured (3D-printed) nanocomposites based on their intended applications. Herein, we present the characterization of the effect of three major building parameters, namely layer thickness, infill percentage, and infill pattern, on mechanical and thermal properties of 3D-printed poly(lactic acid) (PLA)/carbon nanotube (CNT) nanocomposites. The characterization of printed parts showed that increasing layer thickness had a deteriorating effect on the mechanical properties regardless of the CNT concentration; however, the Young's modulus and tensile strength of parts were improved by increasing the CNT content in the same design of mesostructure. Moreover, the thermomechanical analysis showed that the residual thermal stresses of 3D-printed nanocomposites increased by increasing the layer thickness. Furthermore, it was shown that decreasing infill percentage resulted in a non-linear reduction of stiffness, strength, and dimensional stability. Moreover, a honeycomb-shaped infill pattern was introduced for the manufacturing of parts, which has shown more isotropic mechanical properties. The contributions of CNTs in enhancing mechanical properties of 3D-printed nanocomposites were investigated, by considering the induced reinforcing effects as well as the alteration of the crystallization behavior of PLA.

Large bone defects, usually associated to victims of natural disasters, wars and severe accidents, represent a major clinical problem. The search for an effective and efficient treatment is a key area of research. Our group is exploring a novel and fully automatic approach to produce synthetic grafts anatomically designed to fit on the defect site and able to promote tissue regeneration.

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 bricksarchitecture 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.

The field of bone tissue engineering, which is rapidly evolving, aims at the regeneration of the bone structure and its functions. In regenerative medicine, the use of 3D porous structures aims to mimic the bone structure and to promote cell attachment and tissue regeneration. Between the materials used for bone tissue engineering, bioactive glasses have shown high bioactivity and potential stimulation of osteogenesis and angiogenesis. This book chapter discusses key characteristics of bioglass materials and presents the main technologies being used to weak scaffolds incorporating bioglass materials.

This study investigates the use of low cost, customizable, biodegradable, polymer-ceramic composite porous structures (bone bricks) for large bone tissue regeneration. Different ceramic materials (hydroxyapatite (HA), β-tri-calcium phosphate (TCP) and Bioglass (45S5) were mixed with poly-ε-caprolactone (PCL). Bone bricks with different material compositions were produced using an extrusion-based additive manufacturing system. Produced bone bricks were morphologically and mechanically assessed. Results allowed to establish a correlation between scaffolds architecture and material composition and scaffolds performance. Reinforced scaffolds showed improved mechanical properties. Best mechanical properties were obtained with PCL/TCP bone bricks and topologies based on 38 double zig zag filaments and 14 spirals.

Scaffold-based tissue engineering approaches have been commonly used for skin regeneration or wound healings caused by diseases or trauma. For an ideal complete healing process, scaffold structures need to meet the criteria of biocompatibility, biodegradability, and antimicrobial properties, as well as to provide geometrical necessities for the regeneration of damaged tissue. In this study, design, synthesis and characterization of a three dimensional (3D) printable copolymer based on polycaprolactone-block-poly(1,3-propylene succinate) (PCL-PPSu) including anti-microbial silver particles is presented. 3D printing of PCL-PPSu copolymers provided a lower processing temperature compared to neat PCL, hence, inclusion of temperature-sensitive bioactive reagents into the developed copolymer could be realized. In addition, 3D printed block copolymer showed an enhanced hydrolytic and enzymatic degradation behavior. Cell viability and cytotoxicity of the developed copolymer were evaluated by using human dermal fibroblast (HDF) cells. The addition of silver nitrate within the polymer matrix resulted in a significant decrease in the adhesion of different types of microorganisms on the scaffold without inducing any cytotoxicity on HDF cells in vitro. The results suggested that 3D printed PCL-PPSu scaffolds containing anti-microbial silver particles could be considered as a promising biomaterial for emerging skin regenerative therapies, in the light of its adaptability to 3D printing technology, low-processing temperature, enhanced degradation behavior and antimicrobial properties.

Emerging biomanufacturing technologies have revolutionized the field of tissue engineering by offering unprecedented possibilities. Over the past few years, new opportunities arose by combining traditional and novel fabrication techniques, shaping the hybrid designs in biofabrication. One of the potential application fields is skin tissue engineering, in which a combination of traditional principles of wound dressing with advanced biofabrication methods could yield more efficient therapies. In this study, a hybrid design of fiber-reinforced scaffolds combined with gel casting is developed and the efficiency for in vivo wound healing applications is assessed. For this purpose, 3D fiber meshes produced by melt electrowriting are selectively filled with photocrosslinkable gelatin hydrogel matrices loaded with different growth factor carrier microspheres. Additionally, the influence of the inclusion of inorganic bioactive glass particles within the composite fibrous mesh is evaluated. Qualitative evaluation of secondary wound healing criteria and histological analysis shows that hybrid scaffolds containing growth factors and bioactive glass enhances the healing process significantly, compared to the designs merely providing a fiber-reinforced bioactive hydrogel matrix as the wound dressing. This study aims to explore a new application area for melt electrowriting as a powerful tool in fabricating hybrid therapeutic designs for skin tissue engineering.

The capabilities of additive manufacturing for fabrication of complex and thin-walled ceramic-based objects are restricted by the availability of ceramics inks. Formulations of current ink systems strictly depend on using a high content of organic additives (5-30 wt%). The high amounts of additives affect uniformity and dimensional accuracy of the final object. Here, we designed a single additive that enables printing of high aspect ratio and thin-walled structures (height/width = 58) from an ink of alumina nanoparticles that comprises very low organic content (i.e., 1.25 wt % of nanoparticles mass). In addition to the generally exploited electrostatic effect, this additive has purpose-driven tailoring to harness steric hindrance to control the viscoelastic response of ceramic suspensions and realize an optimum ink for extrusion-based 3D printing. We pursued a stepwise approach in developing such an additive through synthesis of series of copolymers with backbone monomers of 2-acrylamido-2-methylpropanesulfonic acid and acrylic acid and side chains of poly(ethylene glycol). When the optimized additive is used, the suspension attains ∼80 wt % solid loading−99% of the theoretical limit calculated by the Krieger−Dougherty equation. The shrinkage and deflection of the printed patterns as well as compactness and sinter-ability of dried structures are monitored. The printed structures did not experience any deformation or deflection during printing and reached 68% of theoretical density (TD: 3.98 g/cm3) after drying. This compactness allowed sintering at lower temperatures and improved dimensional control of the final product. Our approach to formulate ceramic inks enables the embodiment of fully aqueous systems with the utmost material content and has the potential to expand the limited portfolio of ceramic inks.

Three-dimensional (3D) melt electrospinning writing (MEW) is a promising technique for 3D printing of porous scaffolds with well-defined geometrical features. The diameter of electrospun fibers strongly affect the achievable resolution and consequently several other physical, mechanical, and structural properties of the fabricated scaffold. However, there are a few process parameters which significantly affect the size of electrospun fibers. In this study, response surface methodology (RSM) was used to investigate the critical and optimized process parameters and their interaction effects on the desired fiber diameter. Four process parameters, including collector speed, tip-to-collector distance, applied pressure, and voltage were studied considering their practical ranges. The results showed that all the parameters except the applied voltage had a significant effect on the printed fiber diameters. A generalized model for the interaction effects of the parameters was introduced which can be used as a framework for selecting the process parameters to achieve the desired fiber diameter. The developed model was validated by choosing random process parameters and printing three-dimensional scaffolds. The results confirm that the predicted fiber diameters match closely with the actual fiber diameters measured directly from the printed scaffold. © 2018 Elsevier Ltd 213M687 Türkiye Bilimsel ve Teknolojik AraÅ?tirma Kurumu This work was supported by the Scientific and Technological Research Council of Turkey (TUBITAK) [grant number 213M687 ]. We thank Adnan Tasdemir for assistance with the SEM work, Navid Khani for suggestions and comments that greatly improved the concept, and Ali Nadernezhad for his comments on the manuscript.

Processing and composition can significantly affect the mechanobiology, biodegradability, and cellular behavior of polymer-based bone scaffolds to replace damaged bone tissue. In this research, hydroxyapatite (HA), zinc-doped HA (ZnHA), and ZnHA-graphene (ZnHA-rGO) nanoparticles are composed in a polycaprolactone (PCL) matrix. After compositing PCL with nanoparticles, 3D bone scaffolds were built by a custom-built 3D printing system. The characterization of nanoparticles was extensively investigated by TEM, EDX-MAP, XRD, and ATR-FTIR. Simultaneously, 3D-printed scaffolds with different compositions were studied in terms of structure, morphology, thermogravimetry, biodegradability, and mechanical behaviors. The FE-SEM images of the scaffolds showed a highly regular structure and good printability of the developed material system. Moreover, the stiffness modulus of the samples increased due to the presence of the nanoparticles, especially in the ZnHA-rGO nanocomposite. In vitro cell assessment of 3D bone scaffolds was investigated by cell viability tests, cell attachment, and alizarin red staining via mesenchymal stem cells (MSCs). For differentiation capacity of the developed scaffolds, stem cell osteogenesis differentiation was studied by RT-PCR to analyze the ALP, RUNX2, BMP2, TGFβ, and OCN genes. The cellular assessments revealed an increase in PCL scaffold's cell osteogenesis due to the HA nanoparticles in the scaffold matrix. Zinc doping in the HA nanoparticles and rGO addition significantly increased the osteogenesis of MSCs. In particular, the nanocomposite of ZnHA-rGO in PCL scaffold matrix significantly improved the osteogenic differentiation and, thus, it is a viable option for effective regeneration of damaged bone tissue.

Multimaterial bioprinting is a promising technology integrating multimaterial setups into bioprinting platforms for the fabrication of multicellular, heterogeneous and functional tissue constructs. By simultaneously or sequentially dispensing different categories of materials including cell-laden hydrogels or extracellular matrix components, sacrificial materials and scaffolding polymers with hierarchical microarchitecture, multimaterial bioprinting has achieved several milestones for the native tissue biomimicry. Particularly, reconstruction of multiscale, hierarchically branched vascular networks within engineered tissues has been residing as a grand challenge for the fabrication of tissues with long term viability and functionality, which hampers the transition of engineered constructs from research to clinic. To date, various multimaterial bioprinting approaches have been proposed to address the need for fabrication of vascular or vascularized tissues. In this review, a comprehensive overview is given to demonstrate the potential of multimaterial bioprinting technology for the biofabrication of vascular and vascularized tissues. In this regard, different multimaterial bioprinting approaches developed so far are described thoroughly, following the introduction of bioprinting modalities employed in tissue engineering and organization of vascular tissues. Subsequently, multimaterial bioprinting of vascular and vascularized tissues are presented with the main emphasis on the developed fabrication strategies and applied techniques. Comparative evaluation of the multimaterial bioprinting approaches and future perspectives are also delivered to reader.

In this paper, a numerical model based on a systematic study of electrified jet printing is presented. The Volume of Fluid (VOF) method which suits for modeling multiphase flows with a continuous interface is used. The surface tension force is calculated with the Continuum Surface Force (CSF) method and the electric forces are added to the momentum equation by taking the divergence of the Maxwell stress tensor. A systematic study is carried out by introducing three dimensionless numbers, namely Reynolds, Electro-Weber and Weber numbers. Employing these dimensionless numbers, the number of effective parameters is reduced, and a relative comparison of the importance of competing forces on the process becomes possible. It is observed that the electric forces contribute to the formation of the jet by acting on its tip, and by pulling the jet towards the deposition surface. The results show that an increase in Reynolds and Electro-Weber numbers both lead to form a thinner jet. It is also observed that further increase in Electro-Weber and Reynolds numbers leads to the formation of an unstable jet. (C) 2017 Elsevier B.V. All rights reserved.

Three-dimensional (3D) bioprinting is an additive manufacturing process in which the combination of biomaterials and living cells, referred to as a bioink, is deposited layer-by-layer to form biologically active 3D tissue constructs. Recent advancements in the field show that the success of this technology requires the development of novel biomaterials or the improvement of existing bioinks. Polyethylene glycol (PEG) is one of the well-known synthetic biomaterials and has been commonly used as a photocrosslinkable bioink for bioprinting; however, other types of cell-friendly crosslinking mechanisms to form PEG hydrogels need to be explored for bioprinting and tissue engineering. In this work, we proposed micro-capillary based bioprinting of a novel molecularly engineered PEG-based bioink that transiently incorporates low molecular weight gelatin (LMWG) fragments. The rheological properties and release profile of the LMWG fragments were characterized, and their presence during hydrogel formation had no effect on the swelling ratio or sol fraction when compared to PEG hydrogels formed without the LMWG fragments. For bioprinting, PEG was first functionalized with cell-adhesive RGD ligands and was then crosslinked using protease-sensitive peptides via a Michael-type addition reaction inside the micro-capillary. The printability was assessed by the analysis of extrudability, shape fidelity, and printing accuracy of the hydrogel filaments after the optimization of the gelation conditions of the PEG-based bioink. The LMWG fragments supplemented into the bioink allowed the extrusion of smooth and uniform cylindrical strands of the hydrogel and improved shape fidelity and printing accuracy. Encapsulated cells in both bioprinted and non-bioprinted PEG-based hydrogels showed high viability and continued to proliferate over time in culture with a well-defined cell morphology depending on the presence of the cell adhesive peptide RGD. The presented micro-capillary based bioprinting process for a novel PEG-based bioink can be promising to construct complex 3D structures with micro-scale range and spatiotemporal variations without using any cytotoxic photoinitiator, UV light, or polymer support.

In this paper, a feature-based bio-CAD modeling of three-dimensional tissue scaffolds by considering spatial distribution of biologically active materials is presented for biomanufacturing and tissue engineering applications. Proposed model is based on uniform distribution of bio-active particles in different regions of scaffold, which is constrained by geometrical and biological features. The proposed method was integrated with a recently developed method of multi-material additive manufacturing of hydrogel structures, for bio-additive manufacturing of the heterogeneous scaffolds. 3D bioprinted heterogeneous scaffolds were provided as an example for physical implementation of developed algorithm to validate the model.

Three-dimensional bioprinting of cell-laden hydrogels in a sacrificial support-bath has recently emerged as a potential solution for fabricating complex biological structures. Physical properties of the support-bath strongly influence the bioprinting process and the outcome of the fabricated constructs. In this study, we reported the application of a composite Pluronic-nanoclay support-bath including calcium ions as the crosslinking agent for bioprinting of cell-laden alginate-based hydrogels. By tuning the rheological properties, a shear-thinning composite support-bath with fast self-recovery behavior was yielded, which allowed continuous printing of complex and large-scale structures. The printed structures were easily and efficiently harvested from the support-bath without disturbing their shape fidelity. Moreover, the results showed that support-bath assisted bioprinting process did not influence the viability of cells encapsulated within hydrogel. This study demonstrates that Pluronic-nanoclay support-bath can be utilized for bioprinting of complex, cell-laden constructs for vascular and other tissue engineering applications.

Melt electrospinning writing has been emerged as a promising technique in the field of tissue engineering, with the capability of fabricating controllable and highly ordered complex three-dimensional geometries from a wide range of polymers. This three-dimensional (3D) printing method can be used to fabricate scaffolds biomimicking extracellular matrix of replaced tissue with the required mechanical properties. However, controlled and homogeneous cell attachment on melt electrospun fibers is a challenge. The combination of melt electrospinning writing with other tissue engineering approaches, called hybrid biomanufacturing, has introduced new perspectives and increased its potential applications in tissue engineering. In this review, principles and key parameters, challenges, and opportunities of melt electrospinning writing, and particularly, recent approaches and materials in this field are introduced. Subsequently, hybrid biomanufacturing strategies are presented for improved biological and mechanical properties of the manufactured porous structures. An overview of the possible hybrid setups and applications, future perspective of hybrid processes, guidelines, and opportunities in different areas of tissue/organ engineering are also highlighted.

There is a significant unmet clinical need for modular and customized porous biodegradable constructs (scaffolds) for non-union large bone loss injuries. This paper proposes modelling and biomanufacturing of modular and customizable porous constructs for patient-specific critical bone defects. A computational geometry-based algorithm was developed to model modular porous constructs using a parametric femur model based on the frequency of common injuries. The generated modular constructs are used to generate biomimetic path planning for three-dimensional (3D) printing of modular scaffold pieces. The developed method can be used for regenerating bone tissue for treating non-union large bone defects.

Three-dimensional (3D) bioprinting enables the controlled fabrication of complex constructs for tissue engineering applications and has been actively explored in recent years. However, its progress has been limited by the existing difficulties in the development of bioinks with suitable biocompatibility and mechanical properties and at the same time adaptability to the process. Herein, we describe the engineering of a nanocomposite agarose bioink with tailored properties using 2D nanosilicate additives. The suitability of agarose for 3D bioprinting has been debated due to its bioinert nature and compatibility with extrusion-based bioprinting. Nanosilicates were used to tailor the flow behavior of agarose solutions, and detailed rheological characterization of different bioink formulations was performed to elucidate the effect of nanosilicates on the flow behavior and gelation of agarose bioinks. The proper selection of nanosilicate concentration resulted in extrusion 3D printed structures with high shape fidelity and structural integrity. Moreover, the influence of addition of nanosilicates on the bioactivity of agarose was studied, and nanocomposite bioinks showed significant improvement in metabolic activity of encapsulated cells. The bioactivity of the nanocomposite bioinks was also evaluated through a cell spreading assay; the charged surfaces of nanosilicates resulted in full spreading and elongation of fibroblasts, and the extent of change in morphology of cells was found to be directly correlated with the nanosilicate concentration. Our findings suggested that engineered agarose-nanosilicate bioinks can be exploited as a new generation of hydrogel bioinks for extrusion 3D bioprinting with tunable flow properties and bioactivity.

Bioprinting is a relatively new technology where living cells with or without biomaterials are printed layer-by-layer in order to create three-dimensional (3D) living structures. In this article, novel bioprinting methodologies are developed to fabricate 3D biological structures directly from computer models using live multicellular aggregates. Multicellular aggregates made out of at least two cell types from fibroblast, endothelial and smooth muscle cells are prepared and optimized. A novel bioprinting approach is proposed in order to continuously extrude cylindrical multicellular aggregates through the bioprinter's glass microcapillaries. The multicellular aggregates are first aspirated into a capillary and then compressed to form a continuous cylindrical multicellular bioink. To overcome surface tension-driven droplet formation, the required compression ratio is calculated. Based on the developed bioprinting strategies, multicellular aggregates and their support structures are bioprinted to form 3D tissue constructs with predefined shapes. The effect of the bioprinting process was examined for fusion, cell viability at different compression ratios, and f-actin cytoskeletal organization. The results show that the bioprinted 3D constructs fuse rapidly and have high cell viability after printing. © 2016 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 105B: 2530-2544, 2017.

This paper deals with the development of a realtime structural health monitoring system for airframe structures to localize and estimate the magnitude of the loads causing deflections to the critical components, such as wings. To this end, a framework that is based on artificial neural networks is developed where features that are extracted from a depth camera are utilized. The localization of the load is treated as a multinomial logistic classification problem and the load magnitude estimation as a logistic regression problem. The neural networks trained for classification and regression are preceded with an autoencoder, through which maximum informative data at a much smaller scale are extracted from the depth features. The effectiveness of the proposed method is validated by an experimental study performed on a composite unmanned aerial vehicle (UAV) wing subject to concentrated and distributed loads, and the results obtained by the proposed method are superior when compared with a method based on Castigliano&rsquo, s theorem.

This paper reports the synthesis of nanocomposite agarose hydrogels with improved bioactivity with the incorporation of anisotropic 2D nanosilicates (Laponite) to promote cell binding, growth and proliferation. Rheological measurements showed that the incorporation of nanosilicates slightly increased the gelation temperature (T-gel). The use of higher nanosilicate content at the constant agarose concentration improved the mechanical properties of the gels. Due to the non-swelling nature of agarose, the addition of nanosilicates did not result in any remarkable change in the swelling properties of the agarose gels, while collapsed agarose nanofibers were observed with the incorporation of nanosilicates. EDX analysis confirmed the presence of the embedded nanosilicates in the gel matrix. The existence of physical interactions between nanosilicate and agarose was demonstrated by FTIR over the shifting of Si-O stretching band to a lower frequency. The encapsulated NIH/3T3 fibroblast cells showed enhanced proliferation and spreading in the presence of nanosilicates.

A single additive, a grafted copolymer, is designed to ensure the stability of suspensions of highly loaded iron oxide nanoparticles (IOPs) and to facilitate three-dimensional (3D) printing of these suspensions in the filament form. This poly (ethylene glycol)-grafted copolymer of N-[3(dimethylamino)propyl]methacrylamide and acrylic acid harnesses both electrostatic and steric repulsion to realize an optimum formulation for 3D printing. When used at 1.15 wt % (by the weight of IOPs), the suspension attains ∼81 wt % solid loading-96% of the theoretical limit as calculated by the Krieger-Dougherty equation. Rectangular, thick-walled toroidal, and thin-walled toroidal magnetic cores and a porous lattice structure are fabricated to demonstrate the utilization of this suspension as an ink for 3D printing. The electrical and magnetic properties of the magnetic cores are characterized through impedance spectroscopy (IS) and vibrating sample magnetometry (VSM), respectively. The IS indicates the possibility of utilizing wire-wound 3D printed cores as the inductive coils. The VSM verifies that the magnetic properties of IOPs before and after the ink formulation are kept almost unchanged because of the low dosage of the additive. This particle-targeted approach for the formulation of 3D printing inks allows embodiment of a fully aqueous system with utmost target material content.

Using decellularized extracellular matrix (dECM) hydrogels as bioinks has been an important step forward for bioprinting of functional tissue constructs, considering their rich microenvironment and their high degree of biomimicry. However, directly using dECM hydrogels as bioinks may not be suitable for bioprinting processes because of the loss of shape fidelity and geometrical precision of bioprinted structure due to their slow gelation kinetics. In this article, the development and direct bioprinting of dECM hydrogel bioink from bovine Achilles tendon were presented. The developed bioink is used for a microcapillary-based bioprinting process without any support structure and/or any additional cross-linker components. The reported decellularization and solubilization methods yield dECM pre-gels which turn into stable hydrogels in a short time at physiological conditions. The gelation kinetics and mechanical strength of bioinks with different concentrations and digestion times are characterized. A support structure-free 3D bioprinting of the developed bioink is shown by aspirating dECM bioinks and then in situ gelation and extrusion through a fine microcapillary nozzle. The viability assays indicate that the developed dECM bioink has no cytotoxic effect on encapsulated NIH 3T3 cells and the cells show lineage-specific morphology in the early days of culture as well.

Cardiovasculardiseasesaretheleadingcauseofdeaths throughout the world. Vascular diseases are mostly treated with autografts and blood vessel transplantations. However, traditional grafting methods have several problems including lack of suitable harvest sites, additional surgical costs for harvesting procedure, pain, infection, lack of donors, and even no substitutes at all. Recently, tissue engineering and regenerative medicine approaches are used to regenerate damaged or diseased tissues. Most of the tissueengineeringinvestigationshavebeen basedonthecell seeding into scaffolds by providing a suitable environment for cell attachment, proliferation, and differentiation. Because of the challenges such as difficulties in seeding cells spatially, rejection, andinflammationofbiomaterialsused,therecenttissueengineering studies focus on scaffold-free techniques. In this paper, the development of novel computer aided algorithms and methods are developed for 3D bioprinting of scaffold-free biomimetic macro- vascular structures. Computer model mimicking a real human aorta is generated using imaging techniques and the proposed computa- tional algorithms. An optimized three-dimensional bioprinting path planning are developed with the proposed self-supported model. Mouse embryonic fibroblast (MEF) cell aggregates and support structures (hydrogels) are 3D bioprinted layer-by-layer according to the proposed self-supported method to form an aortic tissue construct. Biotechnol. Bioeng. 2015;112: 811-821.

In this paper, we present the biomanufacturing of patterned electrically conductive and cell-embedded hydrogel structures, in which selectively patterned regions of hydrogels including carbon nanotubes (CNTs) and NIH 3T3 cells were demonstrated. We employed a recently developed method of multimaterial additive manufacturing of hydrogels, by using an aspiration-on-demand protocol. The printed structures were characterized by electrical resistivity measurements and microscopy techniques. The results showed that by selectively patterning the electrical conductivity, each region can be considered as a resistor in series circuit. Moreover, due to the novel approach in assembly of cell-patterned segments in low viscosity, the cell viability in printed structures was high after printing. The prospects of combining the functionalities of two fabricated structures were further discussed and highlighted. (C) 2016 The Authors. Published by Elsevier B.V.

In this paper, a novel method of integrated modelling and bio-additive manufacturing of hybrid bioinspired structures is presented. An algorithm is developed to generate optimized and continuous path plan while changing material and internal composition spatially and hierarchically based on the assigned functionality. Biodegradable polymers and hydrogels are used as reinforcing and biological functional bioinks respectively. A new hybrid multi-head 3D bioprinter is developed to manufacture designed three-dimensional constructs depositing bioinks layer-by-layer. Simultaneous incorporation of multiple deposition heads and integrated path planning provide the benefits of using the deposition-on-demand of multi-material bio-inks. The modelled constructs are analyzed and bioprinted. (C) 2017 Published by Elsevier Ltd on behalf of CIRP.

In this research, a novel development of bioink from cell sheets is presented for scaffold free bioprinting applications. Poly(N-isopropylacrylamide) (PNIPAAm) coated surfaces were first prepared by using initiated chemical vapor deposition method. Cell-sheets were then grown on these thermoresponsive pNIPAAm coated surfaces and easily detached without disturbing delicate cell–extracellular matrix (ECM) and cell–cell interactions. The detached cells sheets were used to prepare cell sheet based bioink and bioprinted to form various shapes. The results showed that the prepared cell-sheet based bioink shows an increase in the structural integrity compare to cell-aggregates suggesting that the cell sheet aggregates preserved interconnected ECM proteins. The viability of cell sheet based aggregates was also compared to single cell aggregates at three different time points in a seven-day period after printing. The developed cell-sheet based bioink has several advantages in terms of shape fidelity, reproducibility and automated deposition for bioprinting applications. The results also demonstrated that the bioprinted constructs secreted collagen type I which is a strong indication of starting ECM deposition. Moreover, the immunostaining results showed that the fibronectin in cell sheets was preserved during and after the preparation process of bioink.

This research focuses on the development of a layered-based novel fabrication methodology for three-dimensional tissue scaffolds with heterogeneous structure of varying concentrations of drug molecules and/or multiple biomaterials. A pressure assisted multi-syringe single nozzle deposition (MSSND) system has been developed for the scaffold fabrication. A custom-designed nozzle has been developed to combine the flows from two or more syringes into a mixing chamber. The presented work also details the fluid model for pressure requirements and material volume flow for a biomaterial. Sodium alginic acid is selected as scaffold biomaterial and cross-linked with calcium chloride solution for gelation. The system designed is able to fabricate three-dimensional (3D) structures from hydrogels with varying concentration of drug microspheres depending on the design requirements. Sample scaffolds are successfully fabricated with different nozzles of diameter 100, 150, and 200 μm with varying porosities. In this research, 3D heterogeneous scaffolds are developed that are capable of releasing proteins and growth factors spatially and temporally in addition to their function as supporting structures.