Showing papers in "Biofabrication in 2018"

A definition of bioinks and their distinction from biomaterial inks.

Abstract: Biofabrication aims to fabricate biologically functional products through bioprinting or bioassembly (Groll et al 2016 Biofabrication 8 013001). In biofabrication processes, cells are positioned at defined coordinates in three-dimensional space using automated and computer controlled techniques (Moroni et al 2018 Trends Biotechnol. 36 384-402), usually with the aid of biomaterials that are either (i) directly processed with the cells as suspensions/dispersions, (ii) deposited simultaneously in a separate printing process, or (iii) used as a transient support material. Materials that are suited for biofabrication are often referred to as bioinks and have become an important area of research within the field. In view of this special issue on bioinks, we aim herein to briefly summarize the historic evolution of this term within the field of biofabrication. Furthermore, we propose a simple but general definition of bioinks, and clarify its distinction from biomaterial inks.

Optimization of gelatin-alginate composite bioink printability using rheological parameters: a systematic approach.

Abstract: Three-dimensional bioprinting has emerged as a promising technique in tissue engineering applications through the precise deposition of cells and biomaterials in a layer-by-layer fashion. However, the limited availability of hydrogel bioinks is frequently cited as a major issue for the advancement of cell-based extrusion bioprinting technologies. It is well known that highly viscous materials maintain their structure better, but also have decreased cell viability due to the higher forces which are required for extrusion. However, little is known about the effect of the two distinct components of dynamic modulus of viscoelastic materials, storage modulus (G') and loss modulus (G″), on the printability of hydrogel-based bioinks. Additionally, 'printability' has been poorly defined in the literature, mostly consisting of gross qualitative measures which do not allow for direct comparison of bioinks. This study developed a framework for evaluating printability and investigated the effect of dynamic modulus, including storage modulus (G'), loss modulus (G″), and loss tangent (G″/G') on the printing outcome. Gelatin and alginate as model hydrogels were mixed at various concentrations to obtain hydrogel formulations with a wide range of storage and loss moduli. These formulations were then evaluated for the quantitatively defined values of extrudability, extrusion uniformity, and structural integrity. For extrudability, increasing either the loss or storage modulus increased the pressure required to extrude the bioink. A mathematical model relating the G' and G″ to the required extrusion pressure was derived based on the data. A lower loss tangent was correlated with increased structural integrity while a higher loss tangent correlated with increased extrusion uniformity. Gelatin-alginate composite hydrogels with a loss tangent in the range of 0.25-0.45 exhibited an excellent compromise between structural integrity and extrusion uniformity. In addition to the characterization of a common bioink, the methodology introduced in this paper could also be used to evaluate the printability of other bioinks in the future.

Bio-resin for high resolution lithography-based biofabrication of complex cell-laden constructs

Abstract: Lithography-based three-dimensional (3D) printing technologies allow high spatial resolution that exceeds that of typical extrusion-based bioprinting approaches, allowing to better mimic the complex architecture of biological tissues. Additionally, lithographic printing via digital light processing (DLP) enables fabrication of free-form lattice and patterned structures which cannot be easily produced with other 3D printing approaches. While significant progress has been dedicated to the development of cell-laden bioinks for extrusion-based bioprinting, less attention has been directed towards the development of cyto-compatible bio-resins and their application in lithography-based biofabrication, limiting the advancement of this promising technology. In this study, we developed a new bio-resin based on methacrylated poly(vinyl alcohol) (PVA-MA), gelatin-methacryloyl (Gel-MA) and a transition metal-based visible light photoinitiator. The utilization of a visible light photo-initiating system displaying high molar absorptivity allowed the bioprinting of constructs with high resolution features, in the range of 25-50 μm. Biofunctionalization of the resin with 1 wt% Gel-MA allowed long term survival (>90%) of encapsulated cells up to 21 d, and enabled attachment and spreading of endothelial cells seeded on the printed hydrogels. Cell-laden hydrogel constructs of high resolution with complex and ordered architecture were successfully bioprinted, where the encapsulated cells remained viable, homogenously distributed and functional. Bone and cartilage tissue synthesis was confirmed by encapsulated stem cells, underlining the potential of these DLP-bioprinted hydrogels for tissue engineering and biofabrication. Overall, the PVA-MA/Gel-MA bio-resin is a promising material for biofabrication and provides important cues for the further development of lithography-based bioprinting of complex, free-form living tissue analogues.

Coaxial extrusion bioprinting of 3D microfibrous constructs with cell-favorable gelatin methacryloyl microenvironments.

Abstract: Bioinks with shear-thinning/rapid solidification properties and strong mechanics are usually needed for the bioprinting of three-dimensional (3D) cell-laden constructs. As such, it remains challenging to generate soft constructs from bioinks at low concentrations that are favorable for cellular activities. Herein, we report a strategy to fabricate cell-laden constructs with tunable 3D microenvironments achieved by bioprinting of gelatin methacryloyl (GelMA)/alginate core/sheath microfibers, where the alginate sheath serves as a template to support and confine the GelMA pre-hydrogel in the core during the extrusion process, allowing for subsequent UV crosslinking. This novel strategy minimizes the bioprinting requirements for the core bioink, and facilitates the fabrication of cell-laden GelMA constructs at low concentrations. We first showed the capability of generating various alginate hollow microfibrous constructs using a coaxial nozzle setup, and verified the diffusibility and perfusability of the bioprinted hollow structures that are important for the tissue engineering applications. More importantly, the hollow alginate microfibers were then used as templates for generating cell-laden GelMA constructs with soft microenvironments, by using GelMA pre-hydrogel as the bioink for the core phase during bioprinting. As such, GelMA constructs at extremely low concentrations (<2.0%) could be extruded to effectively support cellular activities including proliferation and spreading for various cell types. We believe that our strategy is likely to provide broad opportunities in bioprinting of 3D constructs with cell-favorable microenvironments for applications in tissue engineering and pharmaceutical screening.

Optimization of collagen type I-hyaluronan hybrid bioink for 3D bioprinted liver microenvironments.

Abstract: Current 3D printing of tissue is restricted by the use of biomaterials that do not recapitulate the native properties of the extracellular matrix (ECM). These restrictions have thus far prevented optimization of composition and structure of the in vivo tissue microenvironment. The artificial nature of currently used biomaterials affects cellular phenotype and function of the bioprinted tissues, and results in inaccurate modeling of disease and drug metabolism significantly. Collagen type I is the major structural component in the ECM, and is widely used as a 3D hydrogel, but is less applicable for 3D bioprinting due to low viscosity and slow polymerization. We have hypothesized that a combination of hyaluronic acid with collagen I yields a bioink with the properties required for extrusion bioprinting, while supporting native cell-matrix interactions and preservation of the native microenvironment properties. To test this hypothesis, we tested the viscoelastic properties of three bioink formulations -2:1, 3:1, and 4:1 collagen type I to hyaluronic acid, and examined cellular behavior in order to determine an optimal formulation that allows for bioprinting while supporting biological activity. We then employed this formulation to bioprint 3D liver tissue constructs containing primary human hepatocytes and liver stellate cells and tested the effects of acetaminophen, a common liver toxicant. Our results have shown that the combination of methacrylated collagen type I and thiolated hyaluronic acid yield a simple, printable bioink that allows for modulation that was directly related to stromal cell elongation. Further, the bioink adequately allowed for implementation as a support hydrogel for hepatocytes which were able to remain viable over two weeks and responded to drug treatment appropriately.

Proof-of-concept: 3D bioprinting of pigmented human skin constructs

Abstract: Three-dimensional (3D) pigmented human skin constructs have been fabricated using a 3D bioprinting approach. The 3D pigmented human skin constructs are obtained from using three different types of skin cells (keratinocytes, melanocytes and fibroblasts from three different skin donors) and they exhibit similar constitutive pigmentation (pale pigmentation) as the skin donors. A two-step drop-on-demand bioprinting strategy facilitates the deposition of cell droplets to emulate the epidermal melanin units (pre-defined patterning of keratinocytes and melanocytes at the desired positions) and manipulation of the microenvironment to fabricate 3D biomimetic hierarchical porous structures found in native skin tissue. The 3D bioprinted pigmented skin constructs are compared to the pigmented skin constructs fabricated by conventional a manual-casting approach; in-depth characterization of both the 3D pigmented skin constructs has indicated that the 3D bioprinted skin constructs have a higher degree of resemblance to native skin tissue in term of the presence of well-developed stratified epidermal layers and the presence of a continuous layer of basement membrane proteins as compared to the manually-cast samples. The 3D bioprinting approach facilitates the development of 3D in vitro pigmented human skin constructs for potential toxicology testing and fundamental cell biology research.

Automated 3D bioassembly of micro-tissues for biofabrication of hybrid tissue engineered constructs

Abstract: Bottom-up biofabrication approaches combining micro-tissue fabrication techniques with extrusion-based 3D printing of thermoplastic polymer scaffolds are emerging strategies in tissue engineering. These biofabrication strategies support native self-assembly mechanisms observed in developmental stages of tissue or organoid growth as well as promoting cell-cell interactions and cell differentiation capacity. Few technologies have been developed to automate the precise assembly of micro-tissues or tissue modules into structural scaffolds. We describe an automated 3D bioassembly platform capable of fabricating simple hybrid constructs via a two-step bottom-up bioassembly strategy, as well as complex hybrid hierarchical constructs via a multistep bottom-up bioassembly strategy. The bioassembly system consisted of a fluidic-based singularisation and injection module incorporated into a commercial 3D bioprinter. The singularisation module delivers individual micro-tissues to an injection module, for insertion into precise locations within a 3D plotted scaffold. To demonstrate applicability for cartilage tissue engineering, human chondrocytes were isolated and micro-tissues of 1 mm diameter were generated utilising a high throughput 96-well plate format. Micro-tissues were singularised with an efficiency of 96.0 ± 5.1%. There was no significant difference in size, shape or viability of micro-tissues before and after automated singularisation and injection. A layer-by-layer approach or aforementioned bottom-up bioassembly strategy was employed to fabricate a bilayered construct by alternatively 3D plotting a thermoplastic (PEGT/PBT) polymer scaffold and inserting pre-differentiated chondrogenic micro-tissues or cell-laden gelatin-based (GelMA) hydrogel micro-spheres, both formed via high-throughput fabrication techniques. No significant difference in viability between the construct assembled utilising the automated bioassembly system and manually assembled construct was observed. Bioassembly of pre-differentiated micro-tissues as well as chondrocyte-laden hydrogel micro-spheres demonstrated the flexibility of the platform while supporting tissue fusion, long-term cell viability, and deposition of cartilage-specific extracellular matrix proteins. This technology provides an automated and scalable pathway for bioassembly of both simple and complex 3D tissue constructs of clinically relevant shape and size, with demonstrated capability to facilitate direct spatial organisation and hierarchical 3D assembly of micro-tissue modules, ranging from biomaterial free cell pellets to cell-laden hydrogel formulations.

Contact guidance for cardiac tissue engineering using 3D bioprinted gelatin patterned hydrogel

Abstract: Here, we have developed a 3D bioprinted microchanneled gelatin hydrogel that promotes human mesenchymal stem cell (hMSC) myocardial commitment and supports native cardiomyocytes (CMs) contractile functionality. Firstly, we studied the effect of bioprinted microchanneled hydrogel on the alignment, elongation, and differentiation of hMSC. Notably, the cells displayed well defined F-actin anisotropy and elongated morphology on the microchanneled hydrogel, hence showing the effects of topographical control over cell behavior. Furthermore, the aligned stem cells showed myocardial lineage commitment, as detected using mature cardiac markers. The fluorescence-activated cell sorting analysis also confirmed a significant increase in the commitment towards myocardial tissue lineage. Moreover, seeded CMs were found to be more aligned and demonstrated synchronized beating on microchanneled hydrogel as compared to the unpatterned hydrogel. Overall, our study proved that microchanneled hydrogel scaffold produced by 3D bioprinting induces myocardial differentiation of stem cells as well as supports CMs growth and contractility. Applications of this approach may be beneficial for generating in vitro cardiac model systems to physiological and cardiotoxicity studies as well as in vivo generating custom designed cell impregnated constructs for tissue engineering and regenerative medicine applications.

A dentin-derived hydrogel bioink for 3D bioprinting of cell laden scaffolds for regenerative dentistry

Abstract: Recent studies in tissue engineering have adopted extracellular matrix (ECM) derived scaffolds as natural and cytocompatible microenvironments for tissue regeneration. The dentin matrix, specifically, has been shown to be associated with a host of soluble and insoluble signaling molecules that can promote odontogenesis. Here, we have developed a novel bioink, blending printable alginate (3% w/v) hydrogels with the soluble and insoluble fractions of the dentin matrix. We have optimized the printing parameters and the concentrations of the individual components of the bioink for print accuracy, cell viability and odontogenic potential. We find that, while viscosity, and hence printability of the bioinks, was greater in the formulations containing higher concentrations of alginate, a higher proportion of insoluble dentin matrix proteins significantly improved cell viability; where a 1:1 ratio of alginate and dentin (1:1 Alg-Dent) was most suitable. We further demonstrate high retention of the soluble dentin molecules within the 1:1 Alg-Dent hydrogel blends, evidencing renewed interactions between these molecules and the dentin matrix post crosslinking. Moreover, at concentrations of 100 μg ml-1, these soluble dentin molecules significantly enhanced odontogenic differentiation of stem cells from the apical papilla encapsulated in bioprinted hydrogels. In summary, the proposed novel bioinks have demonstrable cytocompatibility and natural odontogenic capacity, which can be a used to reproducibly fabricate scaffolds with complex three-dimensional microarchitectures for regenerative dentistry in the future.

3D bioprinting of scaffolds with living Schwann cells for potential nerve tissue engineering applications.

Abstract: Three-dimensional bioprinting of biomaterials shows great potential for producing cell-encapsulated scaffolds to repair nerves after injury or disease. For this, preparation of biomaterials and bioprinting itself are critical to create scaffolds with both biological and mechanical properties appropriate for nerve regeneration, yet remain unachievable. This paper presents our study on bioprinting Schwann cell-encapsulated scaffolds using composite hydrogels of alginate, fibrin, hyaluronic acid, and/or RGD peptide, for nerve tissue engineering applications. For the preparation of composite hydrogels, suitable hydrogel combinations were identified and prepared by adjusting the concentration of fibrin based on the morphological spreading of Schwann cells. In bioprinting, the effects of various printing process parameters (including the air pressure for dispensing, dispensing head movement speed, and crosslinking conditions) on printed structures were investigated and, by regulating these parameters, mechanically-stable scaffolds with fully interconnected pores were printed. The performance of Schwann cells within the printed scaffolds were examined in terms of viability, proliferation, orientation, and ability to produce laminin. Our results show that the printed scaffolds can promote the alignment of Schwann cells inside scaffolds and thus provide haptotactic cues to direct the extension of dorsal root ganglion neurites along the printed strands, demonstrating their great potential for applications in the field of nerve tissue engineering.

3D bioprinting of a hyaluronan bioink through enzymatic-and visible light-crosslinking.

Abstract: Extrusion-based three-dimensional bioprinting relies on bioinks engineered to combine viscoelastic properties for extrusion and shape retention, and biological properties for cytocompatibility and tissue regeneration. To satisfy these conflicting requirements, bioinks often utilize either complex mixtures or complex modifications of biopolymers. In this paper we introduce and characterize a bioink exploiting a dual crosslinking mechanism, where an enzymatic reaction forms a soft gel suitable for cell encapsulation and extrusion, while a visible light photo-crosslinking allows shape retention of the printed construct. The influence of cell density and cell type on the rheological and printability properties was assessed correlating the printing outcomes with the damping factor, a rheological characteristic independent of the printing system. Stem cells, chondrocytes and fibroblasts were encapsulated, and their viability was assessed up to 14 days with live/dead, alamar blue and trypan blue assays. Additionally, the impact of the printing parameters on cell viability was investigated. Owing to its straightforward preparation, low modification, presence of two independent crosslinking mechanisms for tuning shear-thinning independently of the final shape fixation, the use of visible green instead of UV light, the possibility of encapsulating and sustaining the viability of different cell types, the hyaluronan bioink here presented is a valid biofabrication tool for producing 3D printed tissue-engineered constructs.

Development of a functional airway-on-a-chip by 3D cell printing.

Abstract: We used 3D cell printing to emulate an airway coupled with a naturally-derived blood vessel network in vitro. Decellularized extracellular matrix bioink derived from porcine tracheal mucosa (tmdECM) was used to encapsulate and print endothelial cells and fibroblasts within a designated polycarprolactone (PCL) frame. Providing a niche that emulates conditions in vivo, tmdECM gradually drives endothelial re-orientation, which leads to the formation of a lumen and blood vessel network. A fully-differentiated in vitro airway model was assembled with the printed vascular platform, and collectively reproduced a functional interface between the airway epithelium and the vascular network. The model presented respiratory symptoms including asthmatic airway inflammation and allergen-induced asthma exacerbation in physiological context. Because of the adaptable and automated nature of direct 3D cell printing, we expect that this will have relevance in vivo and high reproducibility for production of high-content platforms for preclinical trials in biomedical research.

Simulations of 3D bioprinting: predicting bioprintability of nanofibrillar inks.

Abstract: 3D bioprinting with cell containing bioinks show great promise in the biofabrication of patient specific tissue constructs. To fulfil the multiple requirements of a bioink, a wide range of materials and bioink composition are being developed and evaluated with regard to cell viability, mechanical performance and printability. It is essential that the printability and printing fidelity is not neglected since failure in printing the targeted architecture may be catastrophic for the survival of the cells and consequently the function of the printed tissue. However, experimental evaluation of bioinks printability is time-consuming and must be kept at a minimum, especially when 3D bioprinting with cells that are valuable and costly. This paper demonstrates how experimental evaluation could be complemented with computer based simulations to evaluate newly developed bioinks. Here, a computational fluid dynamics simulation tool was used to study the influence of different printing parameters and evaluate the predictability of the printing process. Based on data from oscillation frequency measurements of the evaluated bioinks, a full stress rheology model was used, where the viscoelastic behaviour of the material was captured. Simulation of the 3D bioprinting process is a powerful tool and will help in reducing the time and cost in the development and evaluation of bioinks. Moreover, it gives the opportunity to isolate parameters such as printing speed, nozzle height, flow rate and printing path to study their influence on the printing fidelity and the viscoelastic stresses within the bioink. The ability to study these features more extensively by simulating the printing process will result in a better understanding of what influences the viability of cells in 3D bioprinted tissue constructs.

Laser bioprinting of human induced pluripotent stem cells-the effect of printing and biomaterials on cell survival, pluripotency, and differentiation.

Abstract: Research on human induced pluripotent stem cells (hiPSCs) is one of the fastest growing fields in biomedicine. Generated from patient's own somatic cells, hiPSCs can be differentiated towards all functional cell types and returned to the patient without immunological concerns. 3D printing of hiPSCs could enable the generation of functional organs for replacement therapies or realization of organ-on-chip systems for individualized medicine. Printing of living cells was demonstrated with immortalized cell lines, primary cells, and adult stem cells with different printing technologies and biomaterials. However, hiPSCs are more sensitive to handling procedures, in particular, when dissociated into single cells. Both pluripotency and directed differentiation are influenced by numerous environmental factors including culture media, biomaterials, and cell density. Notably, existing literature on the effect of applied biomaterials on pluripotency is rather ambiguous. In this study, laser bioprinting of undifferentiated hiPSCs in combination with different biomaterials was performed and the impact on cells' behavior, pluripotency, and differentiation was investigated. Our findings suggest that hiPSCs are indeed more sensitive to the applied biomaterials, but not to laser printing itself. With appropriate biomaterials, such as the hyaluronic acid based solutions applied in this study, hiPSCs can be successfully laser printed without losing their pluripotency.

Bioprinting of mineralized constructs utilizing multichannel plotting of a self-setting calcium phosphate cement and a cell-laden bioink.

Abstract: Due to their characteristic resemblance of the mineral component of bone, calcium phosphates are widely accepted as optimal bone substitute materials. Recent research focused on the development of pasty calcium phosphate cement (CPC) formulations, which can be fabricated into various shapes by low-temperature extrusion-based additive manufacturing, namely 3D plotting. While it could be demonstrated that sensitive substances like growth factors can be integrated in such printed CPC scaffolds without impairment of their biological activity live cells cannot be suspended in CPC as they may not be functional when enclosed in a solid and stiff matrix. In contrast, 3D bioprinting of soft cell-laden hydrogels (bioinks) enables the fabrication of constructs with spatially defined cell distribution, which has the potential to overcome problems of conventional cell seeding techniques-but such objects lack mechanical stability. Herein, we combine 3D plotting of CPC and bioprinting of a cell-laden bioink for the first time. As model bioink, an alginate-methylcellulose blend (alg/mc) was used, previously developed by us. Firstly, a fabrication regime was established, enabling optimal setting of CPC and cell survival inside the bioink. As the cells are exposed to the chemical changes of CPC precursors during setting, we studied the compatibility of the complex system of CPC and cell-laden alg/mc for a combined extrusion process and characterized the cellular behavior of encapsulated human mesenchymal stroma cells within the bioink at the interface and in direct vicinity to the CPC. Furthermore, biphasic scaffolds were mechanically characterized and a model for osteochondral tissue grafts is proposed. The manuscript discusses possible impacts of the CPC setting reaction on cells within the bioink and illustrates the advantages of CPC in bioprinting as alternative to the commonly used thermoplasts for bone tissue engineering.

Photolithographic-stereolithographic-tandem fabrication of 4D smart scaffolds for improved stem cell cardiomyogenic differentiation.

Abstract: 4D printing is a highly innovative additive manufacturing process for fabricating smart structures with the ability to transform over time. Significantly different from regular 4D printing techniques, this study focuses on creating novel 4D hierarchical micropatterns using a unique photolithographic-stereolithographic-tandem strategy (PSTS) with smart soybean oil epoxidized acrylate (SOEA) inks for effectively regulating human bone marrow mesenchymal stem cell (hMSC) cardiomyogenic behaviors. The 4D effect refers to autonomous conversion of the surficial-patterned scaffold into a predesigned construct through an external stimulus delivered immediately after printing. Our results show that hMSCs actively grew and were highly aligned along the micropatterns, forming an uninterrupted cellular sheet. The generation of complex patterns was evident by triangular and circular outlines appearing in the scaffolds. This simple, yet efficient, technique was validated by rapid printing of scaffolds with well-defined and consistent micro-surface features. A 4D dynamic shape change transforming a 2-D design into flower-like structures was observed. The printed scaffolds possessed a shape memory effect beyond the 4D features. The advanced 4D dynamic feature may provide seamless integration with damaged tissues or organs, and a proof of concept 4D patch for cardiac regeneration was demonstrated for the first time. The 4D-fabricated cardiac patch showed significant cardiomyogenesis confirmed by immunofluorescence staining and qRT-PCR analysis, indicating its promising potential in future tissue and organ regeneration applications.

High-throughput fabrication of vascularized spheroids for bioprinting.

Abstract: Overcoming the problem of vascularization remains the main challenge in the field of tissue engineering. As three-dimensional (3D) bioprinting is the rising technique for the fabrication of large tissue constructs, small prevascularized building blocks were generated that can be incorporated throughout a printed construct, answering the need for a microvasculature within the small micron range (<10 μm). Uniform spheroids with an ideal geometry and diameter for bioprinting were formed, using a high-throughput non-adhesive agarose microwell system. Since monoculture spheroids of endothelial cells were unable to remain stable, coculture spheroids combining endothelial cells with fibroblasts and/or adipose tissue derived mesenchymal stem cells (ADSC) as supporting cells, were created. When applying the favorable coculture ratio, viable spheroids were obtained and endothelial cells spontaneously formed a capillary-like network and lumina, as shown by immunohistochemistry and transmission electron microscopy. Especially the presence of ADSC led to a higher vascularization and extracellular matrix production of the microtissue. Moreover, spheroids were able to assemble at random in suspension and in a hydrogel, creating a macrotissue. During at random assembly, cells reorganized, creating a branched capillary-network throughout the entire fused construct by inoculating with capillaries of adjacent spheroids. Combining the advantage of this natural capacity of microtissues to self-assemble and the controlled organization by bioprinting technologies, these prevascularized spheroids can be useful as building blocks for the engineering of large vascularized 3D tissues.

Co-axial wet-spinning in 3D bioprinting: state of the art and future perspective of microfluidic integration.

Abstract: Nowadays, 3D bioprinting technologies are rapidly emerging in the field of tissue engineering and regenerative medicine as effective tools enabling the fabrication of advanced tissue constructs that can recapitulate in vitro organ/tissue functions. Selecting the best strategy for bioink deposition is often challenging and time consuming process, as bioink properties-in the first instance, rheological and gelation-strongly influence the suitable paradigms for its deposition. In this short review, we critically discuss one of the available approaches used for bioprinting-namely co-axial wet-spinning extrusion. Such a deposition system, in fact, demonstrated to be promising in terms of printing resolution, shape fidelity and versatility when compared to other methods. An overview of the performances of co-axial technology in the deposition of cellularized hydrogel fibres is discussed, highlighting its main features. Furthermore, we show how this approach allows (i) to decouple the printing accuracy from bioink rheological behaviour-thus notably simplifying the development of new bioinks-and (ii) to build heterogeneous multi-materials and/or multicellular constructs that can better mimic the native tissues when combined with microfluidic systems. Finally, the ongoing challenges and the future perspectives for the ultimate fabrication of functional constructs for advanced research studies are highlighted.

3D printing strategies for peripheral nerve regeneration.

Abstract: After many decades of biomaterials research for peripheral nerve regeneration, a clinical product (the nerve guide), is emerging as a proven alternative for relatively short injury gaps. This review identifies aspects where 3D printing can assist in improving long-distance nerve guide regeneration strategies. These include (1) 3D printing of the customizable nerve guides, (2) fabrication of scaffolds that fill nerve guides, (3) 3D bioprinting of cells within a matrix/bioink into the nerve guide lumen and the (4) establishment of growth factor gradients along the length a nerve guide. The improving resolution of 3D printing technologies will be an important factor for peripheral nerve regeneration, as fascicular-like guiding structures provide one path to improved nerve guidance. The capability of 3D printing to manufacture complex structures from patient data based on existing medical imaging technologies is an exciting aspect that could eventually be applied to treating peripheral nerve injury. Ultimately, the goal of 3D printing in peripheral nerve regeneration is the automated fabrication, potentially customized for the patient, of structures within the nerve guide that significantly outperform the nerve autograft over large gap injuries.

Scaffold-free, label-free and nozzle-free biofabrication technology using magnetic levitational assembly

Abstract: Tissue spheroids have been proposed as building blocks in 3D biofabrication. Conventional magnetic force-driven 2D patterning of tissue spheroids requires prior cell labeling by magnetic nanoparticles, meanwhile a label-free approach for 3D magnetic levitational assembly has been introduced. Here we present first time report on rapid assembly of 3D tissue construct using scaffold-free, nozzle-free and label-free magnetic levitation of tissue spheroids. Chondrospheres of standard size, shape and capable to fusion have been biofabricated from primary sheep chondrocytes using non-adhesive technology. Label-free magnetic levitation was performed using a prototype device equipped with permanent magnets in presence of gadolinium (Gd3+) in culture media, which enables magnetic levitation. Mathematical modeling and computer simulations were used for prediction of magnetic field and kinetics of tissue spheroids assembly into 3D tissue constructs. First, we used polystyrene beads to simulate the assembly of tissue spheroids and to determine the optimal settings for magnetic levitation in presence of Gd3+. Second, we proved the ability of chondrospheres to assemble rapidly into 3D tissue construct in the permanent magnetic field in the presence of Gd3+. Thus, scaffold- and label-free magnetic levitation of tissue spheroids is a promising approach for rapid 3D biofabrication and attractive alternative to label-based magnetic force-driven tissue engineering.

Homogeneous hydroxyapatite/alginate composite hydrogel promotes calcified cartilage matrix deposition with potential for three-dimensional bioprinting.

Abstract: Calcified cartilage regeneration plays an important role in successful osteochondral repair, since it provides a biological and mechanical transition from the unmineralized cartilage at the articulating surface to the underlying mineralized bone. To biomimic native calcified cartilage in engineered constructs, here we test the hypothesis that hydroxyapatite (HAP) stimulates chondrocytes to secrete the characteristic matrix of calcified cartilage. Sodium citrate (SC) was added as a dispersant of HAP within alginate (ALG), and homogeneous dispersal of HAP within ALG hydrogel was confirmed using sedimentation tests, electron microscopy, and energy dispersive spectroscopy. To examine the biological performance of ALG/HAP composites, chondrocyte survival and proliferation, extracellular matrix production, and mineralization potential were evaluated in the presence or absence of the HAP phase. Chondrocytes in ALG/HAP constructs survived well and proliferated, but also expressed higher levels of calcified cartilage markers compared to controls, including Collagen type X secretion, alkaline phosphatase (ALP) activity, and mineral deposition. Compared to controls, ALG/HAP constructs also showed an elevated level of mineralized matrix in vivo when implanted subcutaneously in mice. The printability of ALG/HAP composite hydrogel precursors was verified by 3D printing of ALG/HAP hydrogel scaffolds with a porous structure. In summary, these results confirm the hypothesis that HAP in ALG hydrogel stimulates chondrocytes to secrete calcified matrix in vitro and in vivo and reveal that ALG/HAP composites have the potential for 3D bioprinting and osteochondral regeneration.

Biofabrication of human articular cartilage: a path towards the development of a clinical treatment.

Abstract: Cartilage injuries cause pain and loss of function, and if severe may result in osteoarthritis (OA). 3D bioprinting is now a tangible option for the delivery of bioscaffolds capable of regenerating the deficient cartilage tissue. Our team has developed a handheld device, the Biopen, to allow in situ additive manufacturing during surgery. Given its ability to extrude in a core/shell manner, the Biopen can preserve cell viability during the biofabrication process, and it is currently the only biofabrication tool tested as a surgical instrument in a sheep model using homologous stem cells. As a necessary step toward the development of a clinically relevant protocol, we aimed to demonstrate that our handheld extrusion device can successfully be used for the biofabrication of human cartilage. Therefore, this study is a required step for the development of a surgical treatment in human patients. In this work we specifically used human adipose derived mesenchymal stem cells (hADSCs), harvested from the infra-patellar fat pad of donor patients affected by OA, to also prove that they can be utilized as the source of cells for the future clinical application. With the Biopen, we generated bioscaffolds made of hADSCs laden in gelatin methacrylate, hyaluronic acid methacrylate and cultured in the presence of chondrogenic stimuli for eight weeks in vitro. A comprehensive characterisation including gene and protein expression analyses, immunohistology, confocal microscopy, second harmonic generation, light sheet imaging, atomic force mycroscopy and mechanical unconfined compression demonstrated that our strategy resulted in human hyaline-like cartilage formation. Our in situ biofabrication approach represents an innovation with important implications for customizing cartilage repair in patients with cartilage injuries and OA.

3D printed Lego®-like modular microfluidic devices based on capillary driving

Abstract: The field of how to rapidly assemble microfluidics with modular components continuously attracts researchers' attention, however, extra efforts must be devoted to solving the problems of leaking and aligning between individual modules. This paper presents a novel type of modular microfluidic device, driven by capillary force. There is no necessity for a strict seal or special alignment, and its open structures make it easy to integrate various stents and reactants. The key rationale for this method is to print different functional modules with a low-cost three-dimensional (3D) printer, then fill the channels with capillary materials and assemble them with plugs like Lego® bricks. This rapidly reconstructed modular microfluidic device consists of a variety of common functional modules and other personalized modules, each module having a unified standard interface for easy assembly. As it can be printed by a desktop 3D printer, the manufacturing process is simple and efficient, with controllable regulation of the flow channel scale. Through diverse combinations of different modules, a variety of different functions can be achieved, without duplicating the manufacturing process. A single module can also be taken out for testing and analysis. What's more, combined with basic circuit components, it can serve as a low-cost Lego®-like modular microfluidic circuits. As a proof of concept, the modular microfluidic device has been successfully demonstrated and used for stent degradation and cell cultures, revealing the potential use of this method in both chemical and biological research.

Matrix stiffness and tumor-associated macrophages modulate epithelial to mesenchymal transition of human adenocarcinoma cells.

Abstract: The tumor microenvironment (TME) is gaining increasing attention in oncology, as it is recognized to be functionally important during tumor development and progression. Tumors are heterogeneous tissues that, in addition to tumor cells, contain tumor-associated cell types such as immune cells, fibroblasts, and endothelial cells. These other cells, together with the specific extracellular matrix (ECM), create a permissive environment for tumor growth. While the influence of tumor-infiltrating cells and mechanical properties of the ECM in tumor invasion and progression have been studied separately, their interaction within the complex TME and the epithelial -to-mesenchymal transition (EMT) is still unclear. In this work, we develop a 3D co-culture model of lung adenocarcinoma cells and macrophages in an interpenetrating network hydrogel, to investigate the influence of the macrophage phenotype and ECM stiffness in the induction of EMT. Rising ECM stiffness increases both tumor cell proliferation and invasiveness. The presence of tumor-associated macrophages and the ECM stiffness jointly contribute to an invasive phenotype, and modulate the expression of key EMT-related markers. Overall, these findings support the utility of in vitro 3D cancer models that allow one to study interactions among key components of the TME.

Self-assembled human osseous cell sheets as living biopapers for the laser-assisted bioprinting of human endothelial cells.

Abstract: A major challenge during the engineering of voluminous bone tissues is to maintain cell viability in the central regions of the construct. In vitro prevascularization of bone substitutes relying on endothelial cell bioprinting has the potential to resolve this issue and to replicate the native bone microvasculature. Laser-assisted bioprinting (LAB) commonly uses biological layers of hydrogel, called 'biopapers', to support patterns of printed cells and constitute the basic units of the construct. The self-assembly approach of tissue engineering allows the production of biomimetic cell-derived bone extracellular matrix including living cells. We hypothesized that self-assembled osseous sheets can serve as living biopapers to support the LAB of human endothelial cells and thus guide tubule-like structure formation. Human umbilical vein endothelial cells were bioprinted on the surface of the biopapers following a predefined pattern of lines. The osseous biopapers showed relevant matrix mineralization and pro-angiogenic hallmarks. Our results revealed that formation of tubule-like structures was favored when the cellular orientation within the biopaper was parallel to the printed lines. Altogether, we validated that human osseous cell sheets can be used as biopapers for LAB, allowing the production of human prevascularized cell-based osseous constructs that can be relevant for autologous bone repair applications.

Fabrication of modular hyaluronan-PEG hydrogels to support 3D cultures of hepatocytes in a perfused liver-on-a-chip device.

Abstract: Liver cell culture models are attractive in both tissue engineering and for development of assays for drug toxicology research. To retain liver specific cell functions, the use of adequate cell typ ...

Pre-set extrusion bioprinting for multiscale heterogeneous tissue structure fabrication

Abstract: Recent advances in three-dimensional bioprinting technology have led to various attempts in fabricating human tissue-like structures. However, current bioprinting technologies have limitations for creating native tissue-like structures. To resolve these issues, we developed a new pre-set extrusion bioprinting technique that can create heterogeneous, multicellular, and multimaterial structures simultaneously. The key to this ability lies in the use of a precursor cartridge that can stably preserve a multimaterial with a pre-defined configuration that can be simply embedded in a syringe-based printer head. The multimaterial can be printed and miniaturized through a micro-nozzle without conspicuous deformation according to the pre-defined configuration of the precursor cartridge. Using this system, we fabricated heterogeneous tissue-like structures such as spinal cords, hepatic lobule, blood vessels, and capillaries. We further obtained a heterogeneous patterned model that embeds HepG2 cells with endothelial cells in a hepatic lobule-like structure. In comparison with homogeneous and heterogeneous cell printing, the heterogeneous patterned model showed a well-organized hepatic lobule structure and higher enzyme activity of CYP3A4. Therefore, this pre-set extrusion bioprinting method could be widely used in the fabrication of a variety of artificial and functional tissues or organs.

3D bioprinting of heterogeneous bi- and tri-layered hollow channels within gel scaffolds using scalable multi-axial microfluidic extrusion nozzle.

Abstract: One of the primary focuses in recent years in tissue engineering has been the fabrication and integration of vascular structures into artificial tissue constructs. However, most available methodologies lack the ability to create multi-layered concentric conduits inside natural extracellular matrices (ECMs) and gels that replicate more accurately the hierarchical architecture of biological blood vessels. In this work, we present a new microfluidic nozzle design capable of multi-axial extrusion in order to 3D print and pattern bi- and tri-layered hollow channel structures. This nozzle allows, for the first time, for these structures to be embedded within layers of gels and ECMs in a fast, simple and low-cost manner. By varying flow rates (1-6 ml min-1), printspeeds (1-16 m min-1), and material concentration (25-175 mM and 1.5%-2.5% for calcium chloride and alginate, respectively) we are able to accurately determine the operational printing range as well as achieve a wide range of conduit dimensions (0.69-2.31 mm) that can be printed within a few seconds. Our scalable design allows for multi-axial extrusion and versatility in material incorporation in order to create heterogeneous structures. We demonstrate the ability to print distinct concentric layers of different cell types, namely endothelial cells and fibroblasts. By incorporating various layers of different cell-friendly materials (such as collagen and fibrin) alongside materials with high mechanical strength (i.e. alginate), we were able to increase long-term cell viability and growth without compromising the structural integrity. In this way, we can improve cellular adhesion in our biocompatible constructs as well as allow them to remain structurally sound. We are able to realize complex heterogeneous, hierarchical architectures that have strong potential for use not only in vascular tissue applications, but also in other artificially fabricated tubular or fiber-like structures such as skeletal muscle or nerve conduits.

3D printing human induced pluripotent stem cells with novel hydroxypropyl chitin bioink: scalable expansion and uniform aggregation.

Abstract: Human induced pluripotent stem cells (hiPSCs) are more likely to successfully avoid the immunological rejection and ethical problems that are often encountered by human embryonic stem cells in various stem cell studies and applications. To transfer hiPSCs from the laboratory to clinical applications, researchers must obtain sufficient cell numbers. In this study, 3D cell printing was used as a novel method for iPSC scalable expansion. Hydroxypropyl chitin (HPCH), utilized as a new type of bioink, and a set of optimized printing parameters were shown to achieve high cell survival (>90%) after the printing process and high proliferation efficiency (∼32.3 folds) during subsequent 10 d culture. After the culture, high levels of pluripotency maintenance were recognized by both qualitative and quantitative detections. Compared with static suspension culture, hiPSC aggregates formed in 3D-printed constructs showed a higher uniformity in size. Using a novel dual-fluorescent labeling method, hiPSC aggregates in the constructs were found more inclined to form by in situ proliferation rather than multicellular aggregation. This study revealed unique advantages of non-ionic crosslinking bioink material HPCH, including high gel strength and rapid temperature response in hiPSC printing, and achieved primed state hiPSC printing for the first time. Features achieved in this study, such as high cell yield, high pluripotency maintenance and uniform aggregation provide good foundations for further hiPSC studies on 3D micro-tissue differentiation and drug screening.

Automated fabrication of photopatterned gelatin hydrogels for organ-on-chips applications

Abstract: Organ-on-chip platforms aim to improve preclinical models for organ-level responses to novel drug compounds. Heart-on-a-chip assays in particular require tissue engineering techniques that rely on labor-intensive photolithographic fabrication or resolution-limited 3D printing of micropatterned substrates, which limits turnover and flexibility of prototyping. We present a rapid and automated method for large scale on-demand micropatterning of gelatin hydrogels for organ-on-chip applications using a novel biocompatible laser-etching approach. Fast and automated micropatterning is achieved via photosensitization of gelatin using riboflavin-5'phosphate followed by UV laser-mediated photoablation of the gel surface in user-defined patterns only limited by the resolution of the 15 μm wide laser focal point. Using this photopatterning approach, we generated microscale surface groove and pillar structures with feature dimensions on the order of 10-30 μm. The standard deviation of feature height was 0.3 μm, demonstrating robustness and reproducibility. Importantly, the UV-patterning process is non-destructive and does not alter gelatin micromechanical properties. Furthermore, as a quality control step, UV-patterned heart chip substrates were seeded with rat or human cardiac myocytes, and we verified that the resulting cardiac tissues achieved structural organization, contractile function, and long-term viability comparable to manually patterned gelatin substrates. Start-to-finish, UV-patterning shortened the time required to design and manufacture micropatterned gelatin substrates for heart-on-chip applications by up to 60% compared to traditional lithography-based approaches, providing an important technological advance enroute to automated and continuous manufacturing of organ-on-chips.