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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

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.

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.

In this paper a neural network based modeling approach is proposed for localization and estimation of loads acting on aircraft wings from full field depth measurements. These measurements can be provided by a multitude of sensors such as depth cameras. Depth cameras have many advantages over other intensity sensors in that they can work in low light conditions and they are invariant to texture and color changes. First, an autoencoder is proposed to extract maximum informative data from the depth images and encode them at a much smaller dimension. Next, to develop the models for localization and estimation of loads, supervised multinomial classification and logistic regression networks are proposed, where the encoded depth features are utilized as input in both networks. The performance of the proposed method is validated on a composite wing subject to concentrated and distributed loads, during which the proposed methods for localization and estimation of loads achieved high accuracies of 90.6% and 90.5%, respectively.

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.

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.

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.

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.

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.

Multimaterial additive manufacturing or three-dimensional (3D) printing of hydrogel structures provides the opportunity to engineer geometrically dependent functionalities. However, current fabrication methods are mostly limited to one type of material or only provide one type of functionality. In this paper, we report a novel method of multimaterial deposition of hydrogel structures based on an aspiration-on-demand protocol, in which the constitutive multimaterial segments of extruded filaments were first assembled in liquid state by sequential aspiration of inks into a glass capillary, followed by in situ gel formation. We printed different patterned objects with varying chemical, electrical, mechanical, and biological properties by tuning process and material related parameters, to demonstrate the abilities of this method in producing heterogeneous and multi-functional hydrogel structures. Our results show the potential of proposed method in producing heterogeneous objects with spatially controlled functionalities while preserving structural integrity at the switching interface between different segments. We anticipate that this method would introduce new opportunities in multimaterial additive manufacturing of hydrogels for diverse applications such as biosensors, flexible electronics, tissue engineering and organ printing.

Three dimensional (3D) bioprinting is a rapidly advancing tissue engineering technology that holds great promise for the regeneration of several tissues, including bone. However, to generate a successful 3D bone tissue engineering construct, additional complexities should be taken into account such as nutrient and oxygen delivery, which is often insufficient after implantation in large bone defects. We propose that a well-designed tissue engineering construct, that is, an implant with a specific spatial pattern of cells in a matrix, will improve the healing outcome. By using a computational model of bone regeneration we show that particular cell patterns in tissue engineering constructs are able to enhance bone regeneration compared to uniform ones. We successfully bioprinted one of the most promising cell-gradient patterns by using cell-laden hydrogels with varying cell densities and observed a high cell viability for three days following the bioprinting process. In summary, we present a novel strategy for the biofabrication of bone tissue engineering constructs by designing cell-gradient patterns based on a computational model of bone regeneration, and successfully bioprinting the chosen design. This integrated approach may increase the success rate of implanted tissue engineering constructs for critical size bone defects and also can find a wider application in the biofabrication of other types of tissue engineering constructs.

This paper presents a new optimization methodology of material blending for heterogeneous object modeling by matching the material governing features for designing a heterogeneous object. The proposed method establishes point-to-point correspondence represented by a set of connecting lines between two material directrices. To blend the material features between the directrices, a heuristic optimization method developed with the objective is to maximize the sum of the inner products of the unit normals at the end points of the connecting lines and minimize the sum of the lengths of connecting lines. The geometric features with material information are matched to generate non-self-intersecting and non-twisted connecting surfaces. By subdividing the connecting lines into equal number of segments, a series of intermediate piecewise curves are generated to represent the material metamorphosis between the governing material features. Alternatively, a dynamic programming approach developed in our earlier work is presented for comparison purposes. Result and computational efficiency of the proposed heuristic method is also compared with earlier techniques in the literature. Computer interface implementation and illustrative examples are also presented in this paper.

A novel tissue scaffold design technique has been proposed with controllable heterogeneous architecture design suitable for additive manufacturing processes. The proposed layer-based design uses a bi-layer pattern of radial and spiral layers consecutively to generate functionally gradient porosity, which follows the geometry of the scaffold. The proposed approach constructs the medial region from the medial axis of each corresponding layer, which represents the geometric internal feature or the spine. The radial layers of the scaffold are then generated by connecting the boundaries of the medial region and the layer's outer contour. To avoid the twisting of the internal channels, reorientation and relaxation techniques are introduced to establish the point matching of ruling lines. An optimization algorithm is developed to construct sub-regions from these ruling lines. Gradient porosity is changed between the medial region and the layer's outer contour. Iso-porosity regions are determined by dividing the sub-regions peripherally into pore cells and consecutive iso-porosity curves are generated using the iso-points from those pore cells. The combination of consecutive layers generates the pore cells with desired pore sizes. To ensure the fabrication of the designed scaffolds, the generated contours are optimized for a continuous, interconnected, and smooth deposition path-planning. A continuous zig-zag pattern deposition path crossing through the medial region is used for the initial layer and a biarc fitted iso-porosity curve is generated for the consecutive layer with C^1 continuity. The proposed methodologies can generate the structure with gradient (linear or non-linear), variational or constant porosity that can provide localized control of variational porosity along the scaffold architecture. The designed porous structures can be fabricated using additive manufacturing processes.

Porous scaffolds with interconnected and continuous pores have recently been considered as one of the most successful tissue engineering strategies. In the literature, it has been concluded that properly interconnected and continuous pores with their spatial distribution could contribute to perform diverse mechanical, biological and chemical functions of a scaffold. Thus, there has been a need for reproducible and fabricatable scaffold design with controllable and functional gradient porosity. Improvements in Additive Manufacturing (AM) processes for tissue engineering and their design methodologies have enabled the development of controlled and interconnected scaffold structures. However homogeneous scaffolds with uniform porosity do not capture the intricate spatial internal micro architecture of the replaced tissue and thus are not capable of capturing the design. In this work, a novel heterogeneous scaffold modeling is proposed for layered-based additive manufacturing processes. First, layers are generated along the optimum build direction considering the heterogeneous micro structure of tissue. Each layer is divided into functional regions based on the spatial homogeneity factor. An area weight based method is developed to generate the spatial porosity function that determines the deposition pattern for the desired gradient porosity. To design a multi-functional scaffold, an optimum deposition angle is determined at each layer by minimizing the heterogeneity along the deposition path. The proposed methodology is implemented and illustrative examples are also provided. The effective porosity is compared between the proposed design and the conventional uniform porous scaffold design. Sample designed structures have also been fabricated with a novel micro-nozzle biomaterial deposition system. The result has shown that the proposed methodology generates scaffolds with functionally gradient porosity.

Porous scaffolds with interconnected and continuous pores have recently been developed to stimulate tissue regeneration. Even though few researches have focused on the internal architecture of porous scaffolds but concluded that properly interconnected and continuous pores with spatial distribution might perform diverse mechanical, biological and chemical functions of a scaffold. Thus the need for reproducible and fabricatable scaffold design with controllable gradient porosity is obvious but is hardly achieved because of design and fabrication limitations. In this paper, a novel functionally gradient variational porosity architecture has been proposed with continuous material deposition planning scheme. The medial axis transformation for the scaffold has been calculated to generate an internal feature of the geometric domain. The medial axis is then used as a base to develop the medial boundary to define the medial regions. Then the complex internal architecture of scaffolds is divided into sub-regions using the ruling lines that are generated between the slice’s contour and the medial boundary. The desired controlled variational porosity along the scaffold architecture has been achieved with the combination of two geometrically oriented consecutive layers while meeting the tissue scaffold design constraints. This ensures truly porous structures in every direction as well as controllable porosity with interconnected pores along the scaffold architecture. The proposed methodology has been implemented and illustrative examples are also provided. A sample designed structure has been fabricated with a NC motion controlled micro-nozzle deposition system.

A novel tissue scaffold design technique has been proposed with controllable heterogeneous architecture design suitable for additive manufacturing processes. The proposed layer-based design uses a bi-layer pattern of radial and spiral layer consecutively to generate functionally gradient porosity, which follows the geometric shape of the scaffold. The proposed approach constructs the medial region from the medial axis of each corresponding layer. The radial layers of the scaffold are then generated by connecting the boundaries of the medial region and the layer's outer contour. Gradient porosity is changed between the medial region and the layer's outer contour. Iso-porosity regions are determined by dividing the sub-regions peripherally into pore cells and consecutive iso-porosity curves are generated using the iso-points from those pore cells. The combination of consecutive layers generates the pore cells with desired pore sizes. To ensure the fabrication of the designed scaffolds, the generated contours are optimized for a continuous, interconnected, and smooth deposition path-planning. The proposed methodologies can generate the structure with gradient (linear or non-linear), variational or constant porosity that can provide localized control of variational porosity along the scaffold architecture. The designed porous structures can be fabricated using additive Manufacturing processes.

This paper presents localized and temporal control of release kinetics over 3-dimensional (3D) hybrid wound devices to improve wound-healing process. Imaging study is performed to extract wound bed geometry in 3D. Non-Uniform Rational B-Splines (NURBS) based surface lofting is applied to generate functionally graded regions. Diffusion-based release kinetics model is developed to predict time-based release of loaded modifiers for functionally graded regions. Multi-chamber single nozzle solid freeform dispensing system is used to fabricate wound devices with controlled dispensing concentration. Spatiotemporal control of biological modifiers thus enables a way to achieve target delivery to improve wound healing.

Reconstructing or repairing the damaged or diseased tissues with porous scaffolds to restore the mechanical, biological and chemical functions is one of the major tissue engineering strategies. Development of Solid Free Form (SFF) techniques and improvement in biomaterial properties by synergy have provided the leverage to fabricate controlled and interconnected porous scaffold structures. But homogeneous scaffolds with regular porosity do not provide all the biological and mechanical requirements of an ideal tissue scaffold. Thus achieving controllable, continuous, interconnected gradient porosity with reproducible and fabricatable design is critical for successful regeneration of the replaced tissue. In this research, a novel scaffold modeling approach has been proposed to achieve bio-mimetic tissue scaffolds. Firstly, the optimum filament deposition angle has been determined based on the internal heterogeneous regions and their locations. Then an area-weight based approach has been applied to generate the spatial porosity function to determine the filament deposition location for the desired bio-mimetic porosity. The proposed methodology has been implemented using computer simulation. A micro-nozzle biomaterial deposition system driven by NC motion control has been used to fabricate a sample designed structure.

Porous membranes/scaffolds such as guided tissue regeneration (GTR) membranes, cell sheets, tissue matrices or polymeric meshes are being widely used in soft tissue engineering to regenerate damaged, diseased tissue or wound. These membranes are mostly regular porous structures with repeating internal architecture. When they are applied onto wound area, various forces caused by bandage, contraction and self weight might cause deformation. As a result, the geometry and the designed porosity changes which eventually alters the desired choreographed functionality. To avoid the negative effect cause by such deformation and its associated consequences, a novel design methodology has been proposed to determine and include the resultant deformation. The proposed design will minimize the variation in effective porosity while ensuring its surface conformity. Thus the proposed design will provide a better functionality by providing both structural integrity and proper biological properties. The proposed methodology has been implemented and results will be shown with illustrative examples. Also a comparison study showing effective porosity for both the proposed method and conventional regular porosity will be presented for a free-form surface mimicking a wound.

This paper presents a new multi-material based toolpath planning methodology for porous tissue scaffolds with multiple hollowed features. Ruled surface with hollowed features generated in our earlier work is used to develop toolpath planning. Ruling lines are reoriented to enable continuous and uniform size multi-material printing through them in two steps. Firstly, all ruling lines are matched and connected to eliminate start and stops during printing. Then, regions with high number of ruling lines are relaxed using a relaxation technique to eliminate over deposition. A novel layer-by-layer deposition process is progressed in two consecutive layers: The first layer with hollow shape based zigzag pattern and the next layer with spiral pattern deposition. Heterogeneous material properties are mapped based on the parametric distances from the hollow features.