Extrusion-based 3D Bioprinting Research Articles

Optimization of Mechanical Stiffness and Cell Density of 3D Bioprinted Cell-Laden Scaffolds Improves Extracellular Matrix Mineralization and Osteocyte-Like Cell Organization for Bone Tissue Engineering

Bioprinting is an emerging technology in which cell-laden biomaterials are precisely dispersed to engineer artificial tissues that mimic aspects of the anatomical and structural complexity of relatively soft tissues such as skin, vessels, and cartilage. However, reproducing the highly mineralized and cellular diversity of bone tissue is still not easily achievable and is yet to be demonstrated. Here, an extrusion-based 3D bioprinting strategy is utilized to fabricate 3D bone-like tissue constructs containing osteoblast- and osteocyte-like cell organization. A simple and low-cost bioink for 3D bioprinting of bone-like tissue is prepared based on two unmodified FDA-compliant polymers (alginate and gelatin) and combined with human mesenchymal stem cells (hMSCs). To form 3D bone-like tissue and bone cell phenotype, the influence of different scaffold stiffness and cell density of 3D bioprinted cell-laden porous scaffolds on osteogenic differentiation and bone-like tissue formation was investigated over time. Our results showed that soft scaffolds (0.8%alg, 0.66 ± 0.08 kPa) had higher DNA content, enhanced ALP activity and stimulated osteogenic differentiation than stiff scaffolds (1.8%alg, 5.4 ± 1.2 kPa). At day 42, significantly more mineralized tissue was formed in soft scaffolds than in stiff scaffolds (43.5 ± 7.1 mm3 vs 22.6 ± 6.0 mm3). Importantly, immunohistochemistry staining demonstrated more osteocalcin protein expression in high mineral compared to low mineral regions. Additionally, cells in soft scaffolds exhibited osteocyte-related gene expression at day 42. Furthermore, the results showed that cell density in 15M cells/ml can promote cell-cell connections at day 7 and mineral formation at day 14, while 5M cells/ml had the significantly higher mineral formation rate than 15M cells/ml from day 14 to day 21. In summary, this work reports the formation of 3D bioprinted bone-like tissue using a simple and low-cost cell-laden bioink, which was optimized for stiffness and cell density, showing great promise for bone tissue engineering applications.

Three-Dimensional Bioprinting of Perfusable Hierarchical Microchannels with Alginate and Silk Fibroin Double Cross-linked Network.

Vascularization is essential for the regeneration of three-dimensional (3D) bioprinting organs. As a general method to produce microfluidic channels in 3D printing constructs, coaxial extrusion has attracted great attention. However, the biocompatible bioinks are very limited for coaxial extrusion to fabricate microchannels with regular structure and enough mechanical properties. Herein, a hybrid bioink composed of alginate (Alg) and silk fibroin (SF) was proposed for 3D bioprinting of microchannel networks based on coaxial extrusion. The rheological properties of the bioink demonstrated that the hybrid Alg/SF bioink exhibited improved viscosity and shear thinning behavior compared with either pure Alg or SF bioink and had similar storage and loss modulus in a wide range of shear frequency, indicating a sound printability. Using a coaxial extrusion system with calcium ions and Pluronic F127 flowing through the core nozzle as cross-linkers, the Alg/SF bioink could be extruded and deposited to form a 3D scaffold with interconnected microchannels. The regular structure and smooth pore wall of microchannels inside the scaffold were demonstrated by optical coherence tomography. Micropores left by the rinse of F127 were observed by scanning electron microscope, constituting a hierarchical structure together with the microchannels and printed macropores. Fourier transform infrared spectroscopy analysis proved the complete rinse of F127 and the formation of β-sheet SF structure. Thus, Alg/SF could form a double cross-linked network, which was much stronger than the pure Alg network. Moreover, cells in the Alg/SF scaffold showed higher viability and proliferation rate than in the Alg scaffold. Therefore, Alg/SF is a promising bioink for coaxial extrusion-based 3D bioprinting, with the printed microchannel network beneficial for complex tissue and organ regeneration.

Bioprinting 101: Design, Fabrication, and Evaluation of Cell-Laden 3D Bioprinted Scaffolds.

3D bioprinting is an additive manufacturing technique that recapitulates the native architecture of tissues. This is accomplished through the precise deposition of cell-containing bioinks. The spatiotemporal control over bioink deposition permits for improved communication between cells and the extracellular matrix, facilitates fabrication of anatomically and physiologically relevant structures. The physiochemical properties of bioinks, before and after crosslinking, are crucial for bioprinting complex tissue structures. Specifically, the rheological properties of bioinks determines printability, structural fidelity, and cell viability during the printing process, whereas postcrosslinking of bioinks are critical for their mechanical integrity, physiological stability, cell survival, and cell functions. In this review, we critically evaluate bioink design criteria, specifically for extrusion-based 3D bioprinting techniques, to fabricate complex constructs. The effects of various processing parameters on the biophysical and biochemical characteristics of bioinks are discussed. Furthermore, emerging trends and future directions in the area of bioinks and bioprinting are also highlighted. Graphical abstract [Figure: see text] Impact statement Extrusion-based 3D bioprinting is an emerging additive manufacturing approach for fabricating cell-laden tissue engineered constructs. This review critically evaluates bioink design criteria to fabricate complex tissue constructs. Specifically, pre- and post-printing evaluation approaches are described, as well as new research directions in the field of bioink development and functional bioprinting are highlighted.

Extrusion-based 3D bioprinting of alginate-based tissue constructs

Tissues engineering technology led to the development of biomedical scaffolds which are mainly used to biofabricate different artificial human organs. Bioprinting is a direct method to fabricate tissue constructs that can support cell growth. The development and formation of bioinks for 3D bioprinting is always considered as a challenge in the field of biofabrication and tissue engineering. Numerous hydrogels have been discovered to solve the problem of bioinks. An appropriate hydrogel used as bioink should have numerous properties for building scaffolds and tissue constructs. A blend of appropriate rheological and mechanical properties is desired. This paper presents comparisons of different hydrogels, their crosslinking mechanisms, an experimental procedure to investigate best proportions of alginate-based hydrogel and crosslinking solution and mathematical model to predict the process parameters of the extrusion-based 3D bioprinting process. The mathematical model gives the reader values of process parameters like average velocity, pressure ranges to obtain a definite geometry of the construct, and shear stress developed for cell viability prediction. The trials were taken using sodium alginate and cross-linking agent as calcium lactate. The process variables considered to design the experiments were nozzle diameter, pressure range, concentration of sodium alginate and concentration of crosslinking solution. The tissue constructs were evaluated based on their geometry (non-continuous, stable, aggregating and over-flowing) and increase in print area. The mathematical model used the rheological data to find out the shear thinning behavior of different concentrations of bioinks. The experimental data and theoretical data obtained from mathematical model were merged to speculate the printability of sodium alginate when used as a bioink. Taken together, these assessment techniques revealed significant insights into the requirements for printable inks and shear conditions present during the extrusion process and allow the rapid and reproducible characterization of a wide variety of inks for 3D bioprinting.

Characterizing Bioinks for Extrusion Bioprinting: Printability and Rheology.

In recent years, new technologies based on 3D bioprinting have emerged as ideal tools with which to arrange cells and biomaterials in three dimensions and so achieve tissue engineering's original goals. The simplest and most widely used form of bioprinting is based on pneumatic extrusion, where 3D structures are built up by drawing patterns of cell-laden or non-cell-laden material through a robotically manipulated syringe. Developing and characterizing new biomaterials for 3D bioprinting (i.e., bioinks) is critical for the progress of the field. This chapter describes a series of protocols for developing, optimizing, and testing new bioinks for extrusion-based 3D bioprinting.

3D bioprinting of cell-laden electroconductive MXene nanocomposite bioinks.

MXenes, a new family of burgeoning two-dimensional (2D) transition metal carbides/nitrides, have been extensively explored in recent years owing to their outstanding properties such as a large specific surface area, high electrical conductivity, low toxicity, and biodegradability. Numerous efforts have been devoted to exploring MXenes for various biomedical applications such as cancer therapy, bioimaging, biosensing, and drug delivery. However, the potential application of MXene nanosheets in tissue engineering has been almost overlooked despite their excellent performance in other biomedical applications. The overarching goal of this paper is to demonstrate the potential of MXene cell-laden bioinks for tissue engineering and their ability to assemble functional scaffolds to regenerate damaged tissue via 3D bioprinting. We formulate a new electroconductive cell-laden bioink composed of Ti3C2 MXene nanosheets dispersed homogeneously within hyaluronic acid/alginate (HA/Alg) hydrogels and showed its performance for extrusion-based 3D bioprinting. The prepared hydrogel bioinks with MXenes display excellent rheological properties, which allows the fabrication of multilayered 3D structures with high resolution and shape retention. Moreover, the introduction of Ti3C2 MXene nanosheets within the HA/Alg hydrogel introduces electrical conductivity to the ink, addressing the poor electrical conductivity of the current bioinks that mismatch with the physico-chemical properties of tissue. In addition, the MXene nanocomposite ink with encapsulated Human Embryonic Kidney 293 (HEK-293) cells displayed high cell viability (>95%) in both bulk hydrogel and 3D bioprinted structures. These results suggest that MXene nanocomposite bioinks and their 3D bioprinting with high electrical conductivity, biocompatibility and degradability can synergize some new applications for tissue and neural engineering.

Design and characterisation of multi-functional strontium-gelatin nanocomposite bioinks with improved print fidelity and osteogenic capacity

3D bioprinting of constructs for tissue engineering and regenerative medicine has steadily gained attention due to its potential to fabricate anatomically-precise living constructs, localise specific cell types and enable the regeneration of functional tissues in a clinical setting. However, the limited availability of bioinks that can be successfully 3D bioprinted with high fidelity and simultaneously provide encapsulated cells with a tailored, low-stiffness microenvironment supporting functional tissue formation remains an unmet challenge. To address both the physical and biological limitations of available bioinks, this study aimed to develop a nanocomposite bioink (Sr-GelMA) comprising of strontium-carbonate (Sr) nanoparticles and low concentration (5 w/v%) gelatin-methacryloyl (GelMA) hydrogel for extrusion-based 3D bioprinting of low-stiffness cell-laden scaffolds with high shape fidelity and bone-specific cell signalling factors. We systematically investigated the effect of Sr incorporation on hydrogel physico-chemical properties, print fidelity, scaffold shape retention, as well as cell viability, osteogenic differentiation and in vitro bone formation. Nanocomposite Sr-GelMA hydrogels retained their physical and mechanical properties, while rheological studies revealed a significant increase in viscosity profiles that led to notably enhanced printability compared to GelMA alone. Moreover, bioprinted Sr-GelMA scaffolds exhibited excellent shape fidelity evidenced by a defined pore geometry on the x-y-z axis, resulting in an interconnected bioink filament and pore network that was maintained even after long-term culture and osteogenic differentiation (28 days) of human mesenchymal stromal cells (hMSCs). The presence of clustered Sr nanoparticles in the cell-laden bioink allowed high quality bioprinting combined with high hMSC viability (>95%) post-fabrication. Furthermore, Sr addition resulted in enhanced osteogenic differentiation of hMSCs as revealed by higher alkaline phosphatase (ALP) levels, osteocalcin (OCN) and collagen type-I (Col I) expression, with mineralised nodule formation distributed homogenously throughout the bioprinted construct. This study demonstrated that strontium-based nanocomposite bioinks optimised for extrusion-based 3D bioprinting of osteoconductive scaffolds support long-term shape retention with promising potential for bone tissue regeneration.

Direct process feedback in extrusion-based 3D bioprinting

A major limitation in extrusion-based bioprinting is the lack of direct process control, which limits the accuracy and design complexity of printed constructs. The lack of direct process control results in a number of defects that can influence the functional and mechanical outcomes of the fabricated structures. The machine axes motion cannot be reliably used to predict material placement, and precise fabrication requires additional sensing of the material extrusion. We present an iteration-to-iteration process monitoring system that enables direct process control in the material deposition reference frame. To fabricate parts with low dimensional errors, we integrate a non-contact laser displacement scanner into the printing platform. After fabrication of the initial print using the as-designed reference trajectory, the laser scanner moves across the part to measure the material placement. A custom image processing algorithm compares the laser scanner data to the as-designed reference trajectory to generate an error vector. To compensate for the measured error, the algorithm modifies the axes reference trajectory for the second print iteration. We implement the in situ process monitoring and error compensation technique on an experimental platform to evaluate system performance and demonstrate improvement in spatial material placement.

Prediction of geometric characteristics in polycaprolactone (PCL) scaffolds produced by extrusion-based additive manufacturing technique for tissue engineering

Purpose Extrusion-based additive manufacturing (AM) has been considered as a promising technique to fabricate scaffolds for tissue engineering due to affordability, versatility and ability to print porous structures. The reliability and controllability of the printing process are necessary to produce 3D-printed scaffolds with desired properties and depend on the geometric characteristics such as porosity and pore diameter. The purpose of this study is to develop an analytical model and explore its effectiveness in the prediction of geometric characteristics of 3D-printed scaffolds. Design/methodology/approach An analytical model was developed to simulate the geometric characteristics of scaffolds produced by extrusion-based AM using fluid mechanics. Polycaprolactone (PCL) was chosen as a scaffold material and was assumed to be a non-Newtonian fluid for the model. The effectiveness of the model was verified through comparison with the experimental results. Findings A comparison study between simulation and experimental results shows that strut diameter, pore size and porosity of scaffolds can be predicted by using extrusion pressure, temperature, nozzle diameter, nozzle length and printing speed. Simulation results demonstrate that geometric characteristics have a strong relationship with processing parameters, and the model developed in this study can be used for predicting the scaffold properties for the extrusion-based 3D bioprinting process. Originality/value The present study provides a prediction model that can simulate the printing process by a simple input of processing parameters. The geometric characteristics can be predicted prior to the experimental verification, and such prediction will reduce the process time and effort when a new material or method is applied.

Liquid-phase 3D bioprinting of gelatin alginate hydrogels: influence of printing parameters on hydrogel line width and layer height

Extrusion-based 3D bioprinting is a direct deposition approach used to create three-dimensional (3D) tissue scaffolds typically comprising hydrogels. Hydrogels are hydrated polymer networks that are chemically or physically cross-linked. Often, 3D bioprinting is performed in air, despite the hydrated nature of hydrogels and the potential advantage of using a liquid phase to provide cross-linking and otherwise functionalize the hydrogel. In this work, we print gelatin alginate hydrogels directly into a cross-linking solution of calcium chloride and investigate the influence of nozzle diameter, distance between nozzle and surface, calcium chloride concentration, and extrusion rate on the dimensions of the printed hydrogel. The hydrogel layer height was generally found to increase with increasing extrusion rate and nozzle distance, according to the increased volume extruded and the available space, respectively. In addition, the hydrogel width was generally found to increase with decreasing nozzle distance and cross-linking concentration corresponding to confinement-induced spreading and low cross-linking regimes, respectively. Width/height ratios of ~ 1 were generally achieved when the nozzle diameter and distance were comparable above a certain cross-linking concentration. Using these relationships, biocompatible 3D multilayer structures were successfully printed directly into calcium chloride cross-linking solution.

3D bioprinting and in vitro study of bilayered membranous construct with human cells-laden alginate/gelatin composite hydrogels

Extrusion-based 3D bioprinting of cell-laden hydrogels is a potential technology for regenerative medicine, which enables the fabrication of constructs with spatially defined cell distribution. However, the limited assessment of rheological behaviors of hydrogel before printing is still a major issue for the advancement of 3D bioprinting. In this work, we systematically investigated the rheological behaviors (i.e. viscosity, storage modulus (G’), and loss modulus (G”)) of alginate/gelatin composite hydrogels first for 3D printing complex constructs. The rheological studies revealed that viscosity of alginate/gelatin hydrogels is temperature-dependent and shear thinning. Sol-gel transition (intersection of G’ and G”) study provided indication for printing temperature, which are in the range of 18.8 °C (H2/7.5) to 24.5 °C (H2/24.5). The alginate (2 wt%) /gelatin (15 wt%) composite hydrogel sample was chosen to print the constructs and subsequent bioprinting. Complex constructs (i.e. nose and ear) were obtained with high printing resolution (151 ± 13.04 μm) in a low temperature (4 °C) chamber and crosslinking with 2 wt% CaCl2 subsequently without extra supports. Human amniotic epithelial cells (AECs) showed superior potential to differentiate into epithelial cells, while Wharton’s jelly derived mesenchymal stem cells (WJMSCs) showed a superior angiogenic potential and fibroblastic phenotype. For the in vitro study, AECs and WJMSCs as seed cells, encapsulated in alginate/gelatin composite hydrogels, were bioprinted to form biomimetic bilayered membranous construct. High cell viability (> 95%) were observed up to 6 days after printing. The presented 3D bioprinting of human AECs and WJMSCs-laden alginate/gelatin composite hydrogels provides promising potentials for future skin tissue engineering.

Enhanced rheological behaviors of alginate hydrogels with carrageenan for extrusion-based bioprinting

Three-dimensional (3D) bioprinting using biocompatible materials is widely used in the field of tissue engineering and regenerative medicine. However, precise printing of 3D structures is challenging due to weak and uncontrollable mechanical properties of various hydrogels, thus limiting their potential in preclinical and clinical applications. In this study, our goal is to demonstrate the feasibility of precise fabrication of alginate/carrageenan composite scaffolds using extrusion-based 3D bioprinting. At first, the proper concentration of crosslinking agents was determined by the assessment of shear modulus of alginate-based hydrogels. Moreover, alginate/carrageenan composite hydrogels were prepared with different concentrations of carrageenan and used to measure their rheological properties. Based on the assessed viscosities and shear moduli of alginate and alginate/carrageenan hydrogels, printing resolutions in different printing parameters were simulated and presented in the printability maps. In addition, alginate and alginate/carrageenan scaffolds were bioprinted with various printing parameters and used to compare their printability with the simulated results. Also, 3D deposition of both alginate and alginate/carrageenan hydrogels were assessed and compared with each other by continuous monitoring of shape fidelity in 3D structures in ten layers and similar printing resolution. Finally, the cell viability of the 3D alginate/carrageenan composite scaffolds, printed using optimized printing parameters, was evaluated using live/dead staining and confocal fluorescence imaging. Thus, the results in the study show the potential uses of carrageenan for a prospective bioink with remarkable mechanical properties suitable for precise fabrication of 3D hydrogel scaffolds using bioprinting techniques.

Evaluation of sterilisation methods for bio-ink components: gelatin, gelatin methacryloyl, hyaluronic acid and hyaluronic acid methacryloyl

Reliable and scalable sterilisation of hydrogels is critical to the clinical translation of many biofabrication approaches, such as extrusion-based 3D bioprinting of cell-laden bio-inks. However sterilisation methods can be destructive, and may have detrimental effects on the naturally-derived hydrogels that constitute much of the bio-ink palette. Determining effective sterilisation methods requires detailed analysis of the effects of sterilisation on relevant properties such as viscosity, printability and cytocompatibility. Yet there have been no studies specifically exploring the effects of sterilisation on bio-inks to date. In this work, we explored the effects of various sterilisation techniques on four of the most widely used bio-ink components: gelatin, gelatin methacryloyl, hyaluronic acid, and hyaluronic acid methacrylate. Autoclaving was the most destructive sterilisation method, producing large reductions in viscosity and in mechanical properties following crosslinking. Filter sterilisation caused some reduction in rheological properties of GelMA due to removal of higher molecular weight components, but did not affect photocrosslinking. Ethylene oxide (EtO) was the least destructive sterilisation method in terms of rheological properties for all materials, had no detrimental effect on the photocrosslinkable methacrylate/methacrylamide groups, and so was chosen for more detailed examination. In biological analyses, we found that EtO treatment successfully eradicated a bacterial challenge of E. coli, caused no decrease in viability of human mesenchyman stem cells (hMSCs), and had no effect on their rate of proliferation. Finally, we found that EtO-treated hydrogels supported encapsulated hMSCs to differentiate towards the chondrogenic lineage, and to produce new cartilage matrix. Our results bring to light various effects that sterilisation can have on bio-inks, as well as highlighting EtO sterilisation as a method which minimises degradation of properties, while still promoting biological function.

Bioprinted osteon-like scaffolds enhance in vivo neovascularization

Bone tissue engineers are facing a daunting challenge when attempting to fabricate bigger constructs intended for use in the treatment of large bone defects, which is the vascularization of the graft. Cell-based approaches and, in particular, the use of in vitro coculture of human umbilical vein endothelial cells (HUVECs) and human mesenchymal stem cells (hMSCs) has been one of the most explored options. We present in this paper an alternative method to mimic the spatial pattern of HUVECs and hMSCs found in native osteons based on the use of extrusion-based 3D bioprinting (3DP). We developed a 3DP biphasic osteon-like scaffold, containing two separate osteogenic and vasculogenic cell populations encapsulated in a fibrin bioink in order to improve neovascularization. To this end, we optimized the fibrin bioink to improve the resolution of printed strands and ensure a reproducible printing process; the influence of printing parameters on extruded strand diameter and cell survival was also investigated. The mechanical strength of the construct was improved by co-printing the fibrin bioink along a supporting PCL carrier scaffold. Compressive mechanical testing showed improved mechanical properties with an average compressive modulus of 131 ± 23 MPa, which falls in the range of cortical bone. HUVEC and hMSC laden fibrin hydrogels were printed in osteon-like patterns and cultured in vitro. A significant increase in gene expression of angiogenic markers was observed for the biomimetic scaffolds. Finally, biphasic scaffolds were implanted subcutaneously in rats. Histological analysis of explanted scaffolds showed a significant increase in the number of blood vessels per area in the 3D printed osteon-like scaffolds. The utilization of these scaffolds in constructing biomimetic osteons for bone regeneration demonstrated a promising capacity to improve neovascularization of the construct. These results indicates that proper cell orientation and scaffold design could play a critical role in neovascularization.

Formulation and Characterization of a SIS-Based Photocrosslinkable Bioink.

Decellularized extracellular matrices (dECMs) represent a promising alternative as a source of materials to develop scaffolds that closely mimic the native environment of cells. As a result, dECMs have attracted significant attention for their applications in regenerative medicine and tissue engineering. One such application is 3D bioprinting, in which dECMs can be used to prepare bioinks with the biomimicry attributes required for regeneration purposes. Formulating bioinks is, however, challenging, due to difficulties in assuring that the printed materials match the mechanical properties of the tissue which is to be regenerated. To tackle this issue, a number of strategies have been devised, including crosslinking methods, the addition of synthetic materials as excipients, and the use of synthetic matrices for casting. We are particularly interested in extrusion-based 3D bioprinting, mainly due to the ease of rapidly conducting tests for adjusting operating conditions such that the required rheological and mechanical properties are met when using it. Here, we propose a novel bioink that consists of an acid-based precipitation of a small intestinal submucosa (SIS) dECM. The formulated bioink also relies on photocrosslinking reactions to attempt to control gelation and ultimately the mechanical properties of the extruded material. Photoinitiation was explored with the aid of varying concentrations of riboflavin (RF). Manual extrusion and rheological flow tests confirmed the printability and shear-thinning behavior of all formulations. Photocrosslinking reactions, however, failed to promote a substantial increase in gelation, which was attributed to considerable entanglement of undigested collagen molecules. As a result, pendant amine groups thought to be involved in the photo-mediated reactions remain largely inaccessible. In silico computational fluid dynamics (CFD) simulations were implemented to determine shear stress values on the bioink along the exit of the printing nozzle. Moreover, we calculated a stability parameter as a means to estimate changes in the bioink stability during the extrusion process. Future studies should be directed toward assessing the role of temperature-induced gelation in the rheological properties of the bioink and the development of strategies to improve the efficiency of photocrosslinking processes.

Rapid assembling organ prototypes with controllable cell-laden multi-scale sheets

A native organ has heterogeneous structures, strength, and cell components. It is a big challenge to fabricate organ prototypes with controllable shapes, strength, and cells. Herein, a hybrid method is developed to fabricate organ prototypes with controlled cell deposition by integrating extrusion-based 3D printing, electrospinning, and 3D bioprinting. Multi-scale sheets were first fabricated by 3D printing and electrospinning; then, all the sheets were assembled into organ prototypes by sol–gel reaction during bioprinting. With this method, macroscale structures fabricated by 3D printing ensure the customized structures and provide mechanical support, nanoscale structures fabricated by electrospinning offer a favorable environment for cell growth, and different types of cells with controllable densities are deposited in accurate locations by bioprinting. The results show that L929 mouse fibroblasts encapsulated in the structures exhibited over 90% survival within 10 days and maintained a high proliferation rate. Furthermore, the cells grew in spherical shapes first and then migrated to the nanoscale fibers showing stretched morphology. Additionally, a branched vascular structure was successfully fabricated using the presented method. Compared with other methods, this strategy offers an easy way to simultaneously realize the shape control, nanofibrous structures, and cell accurate deposition, which will have potential applications in tissue engineering.

Stabilization strategies in extrusion-based 3D bioprinting for tissue engineering

Three dimensional (3D) printing is a revolutionizing technology, which endows engineers, designers, and manufacturers with the ability to rapidly translate digital sketches into physical objects. The advantages that lie in the high resolution and accuracy of this technique were not concealed from the eyes of tissue engineers that soon harnessed this power for fabrication of complex biological structures. Nevertheless, while the conventional 3D printing scheme is oriented to yield durable and sturdy structures, the delicate nature of the substances used in 3D bioprinting results in fragile and mechanically unstable constructs. This poses a significant restriction that needs to be overcome in order to successfully complete the printing of intact, accurate, and biologically relevant constructs with desirable properties. To address these complications, advanced means of stabilization which are applied during and/or following the printing procedure are constantly being developed. In this review, the rational and principles behind widely used stabilization strategies in extrusion-based bioprinting will be covered. Examples of implementation of these strategies in recently published research in the field of tissue engineering will also be presented and discussed.

3D Printed Stem-Cell Derived Neural Progenitors Generate Spinal Cord Scaffolds.

A bioengineered spinal cord is fabricated via extrusion-based multi-material 3D bioprinting, in which clusters of induced pluripotent stem cell (iPSC)-derived spinal neuronal progenitor cells (sNPCs) and oligodendrocyte progenitor cells (OPCs) are placed in precise positions within 3D printed biocompatible scaffolds during assembly. The location of a cluster of cells, of a single type or multiple types, is controlled using a point-dispensing printing method with a 200 μm center-to-center spacing within 150 μm wide channels. The bioprinted sNPCs differentiate and extend axons throughout microscale scaffold channels, and the activity of these neuronal networks is confirmed by physiological spontaneous calcium flux studies. Successful bioprinting of OPCs in combination with sNPCs demonstrates a multicellular neural tissue engineering approach, where the ability to direct the patterning and combination of transplanted neuronal and glial cells can be beneficial in rebuilding functional axonal connections across areas of central nervous system (CNS) tissue damage. This platform can be used to prepare novel biomimetic, hydrogel-based scaffolds modeling complex CNS tissue architecture in vitro and harnessed to develop new clinical approaches to treat neurological diseases, including spinal cord injury.

Ex Vivo Functionality of 3D Bioprinted Corneal Endothelium Engineered with Ribonuclease 5-Overexpressing Human Corneal Endothelial Cells.

Human corneal endothelial cells (HCECs) are scarcely proliferative in vivo. The cultured HCECs engineered to overexpress ribonuclease (RNase) 5 (R5-HCECs) are prepared after transient transfection with RNase 5 plasmid vector. As candidate targets of R5-HCECs for enhancement of cellular proliferation and survival of R5-HCECs, programmed cell death protein 4 is inhibited, and cyclin D1 and cyclin E1 are activated. The cultured R5-HCECs and control HCECs on lyophilized amniotic membrane (AM) are deposited as a carrier by extrusion-based 3D bioprinting to prepare transplantable RNase 5 vector-transfected HCECs-laden AM graft (R5-Graft) and the control HCECs-laden AM graft (Ct-Graft), respectively. The ready-to-use R5-Graft shows clearer basolateral expression of Na+ -K+ ATPase pump and higher cell confluency than Ct-Graft. From 2 weeks after graft transplantation, both R5-Graft and Ct-Graft start restoring clarity of the rabbit corneas, and their central corneal edema are much less than those in the control group at 3 and 4 weeks. The ex vivo expression of corneal endothelial phenotypical markers is clear in R5-Grafs rather than in Ct-Grafts at 4 weeks. In conclusion, the fabricated corneal endothelium with cultured HCECs easily survives and functions as corneal endothelium in vivo. Furthermore, the use of the cultured HCECs engineered to overexpress RNase 5 (R5-HCECs) may be an option to obtain higher graft cellularity and to enhance the function of transplanted grafts.

Searching for the optimal bioink in extrusion-based 3D bioprinting for reconstructive surgery

Searching for the optimal bioink in extrusion-based 3D bioprinting for reconstructive surgery