Alginate Bioink Research Articles

Direct writing alginate bioink inside pre-polymers of hydrogels to create patterned vascular networks

We describe a strategy to fabricate a hydrogel-based microvascular construct by direct writing alginate bioink inside the viscous pre-polymer of hydrogels, which acts as a support bath. As the print needle translates through the polymers, the extruded alginate instantaneously forms calcium alginate hydrogel (Ca-Alg) templates deposited within the bath. This phase change allows the formed templates to be anchored within the pre-polymers, while maintaining their structure. After the printing process, the pre-polymers are solidified to form a mechanically robust hydrogel. Finally, a hydrogel construct with embedded microchannels is generated by liquefying and removing the Ca-Alg templates. Using this method, not only the alginate ink alone can be directly printed within the engineered constructs, but also the size and shape of the formed microchannels are controllable. Furthermore, a confluent endothelial layer for the generation of vascular networks can be constructed by adhering and proliferating endothelial cells on the channel linings. This strategy demonstrates a promising technique for rapid construction of in vitro vasculatures, which would provide a versatile platform for a wide array of applications such as tissue engineering, organ-on-a-chip and drug screening.

Fiber Reinforced Cartilage ECM Functionalized Bioinks for Functional Cartilage Tissue Engineering.

Focal articular cartilage (AC) defects, if left untreated, can lead to debilitating diseases such as osteoarthritis. While several tissue engineering strategies have been developed to promote cartilage regeneration, it is still challenging to generate functional AC capable of sustaining high load-bearing environments. Here, a new class of cartilage extracellular matrix (cECM)-functionalized alginate bioink is developed for the bioprinting of cartilaginous tissues. The bioinks are 3D-printable, support mesenchymal stem cell (MSC) viability postprinting and robust chondrogenesis in vitro, with the highest levels of COLLII and ACAN expression observed in bioinks containing the highest concentration of cECM. Enhanced chondrogenesis in cECM-functionalized bioinks is also associated with progression along an endochondral-like pathway, as evident by increases in RUNX2 expression and calcium deposition in vitro. The bioinks loaded with MSCs and TGF-β3 are also found capable of supporting robust chondrogenesis, opening the possibility of using such bioinks for direct "print-and-implant" cartilage repair strategies. Finally, it is demonstrated that networks of 3D-printed polycaprolactone fibers with compressive modulus comparable to native AC can be used to mechanically reinforce these bioinks, with no loss in cell viability. It is envisioned that combinations of such biomaterials can be used in multiple-tool biofabrication strategies for the bioprinting of biomimetic cartilaginous implants.

Design and development of alginate: Poly-l-lysine scaffolds by 3D bio printing and studying their mechanical, structural and cell viability properties.

3D bio-printing is a very rapid and vigorous process which is used for fabricating the in-vitro biological functional tissues. For printing an effective scaffold, it is very imperative; the printed structure must have adequate strength and stiffness for keeping structural veracity. Alginates are such a biomaterial which can be used as a bio-ink for printing the 3D biological scaffolds. Also the bio-ink gel must not be very strong because more viscous results huge shear force for extruding the bio-ink from the nozzle which may results in cell fracture and death. Alginate is such a biocompatible and healthy material for cell. There are many applications of alginate bio-ink in bone, adipose tissue engineering and in vascular field. In this paper we have investigated the biological properties of alginate bio-ink based 3D bio-printed scaffold by using different percentage of honey mixing with same percentage of alginate. Then we characterized the bio-printed scaffolds with the help of UTM, Surfacetensiometer, MTT and SEM. We have studied the mechanical, physic-chemical and the cell viability properties of alginate 5%, alginate 5%:poly-l-lysine 1.5% and alginate 5%:poly-l-lysine 2.5% cross-linked with 1 molar CaCl2 with printing speed of 50mm/s.

Alginate-honey bioinks with improved cell responses for applications as bioprinted tissue engineered constructs

Abstract

Tuning Alginate Bioink Stiffness and Composition for Controlled Growth Factor Delivery and to Spatially Direct MSC Fate within Bioprinted Tissues

Alginate is a commonly used bioink in 3D bioprinting. Matrix stiffness is a key determinant of mesenchymal stem cell (MSC) differentiation, suggesting that modulation of alginate bioink mechanical properties represents a promising strategy to spatially regulate MSC fate within bioprinted tissues. In this study, we define a printability window for alginate of differing molecular weight (MW) by systematically varying the ratio of alginate to ionic crosslinker within the bioink. We demonstrate that the MW of such alginate bioinks, as well as the choice of ionic crosslinker, can be tuned to control the mechanical properties (Young’s Modulus, Degradation Rate) of 3D printed constructs. These same factors are also shown to influence growth factor release from the bioinks. We next explored if spatially modulating the stiffness of 3D bioprinted hydrogels could be used to direct MSC fate inside printed tissues. Using the same alginate and crosslinker, but varying the crosslinking ratio, it is possible to bioprint constructs with spatially varying mechanical microenvironments. Moreover, these spatially varying microenvironments were found to have a significant effect on the fate of MSCs within the alginate bioinks, with stiffer regions of the bioprinted construct preferentially supporting osteogenesis over adipogenesis.

A cell-printing approach for obtaining hASC-laden scaffolds by using a collagen/polyphenol bioink

In the cell-printing process, bioink has been considered as an extremely important component for successful fabrication of macroscale cell-laden structures. Bioink should be non-toxic, biocompatible, and printable. To date, alginate has been widely used as a whole or partial component of bioink because it is non-toxic to embedded cells and even it can provide good printability with rapid gelation under calcium ions. However, alginate bioinks do not possess cell-activating ability. To overcome the shortcomings of alginate-based bioinks, a new collagen bioink, which was mixed with human adipose stem cells (hASCs) and crosslinked with a polyphenol (tannic acid), was proposed. The feasibility of the bioink was demonstrated using several in vitro assessments for comparison of the macroscale porous cell-laden collagen/polyphenol structure containing the hASCs with the conventional alginate-based cell-laden structure. The levels of the metabolic activity, including the cell viability and cell proliferation, of the cell-laden collagen structure were significantly higher than those of the control (alginate-based cell-laden structure). The results show that the newly designed bioink and cell-laden structure are potentially new outstanding components for regeneration of various tissues.

Dual In Situ Crosslinking of Polymer Bioinks for 3D Tissue Biofabrication

During this research, a 3D bioprinted ear structure has been produced from a hybrid gelatin and alginate bioink hydrogel. Using dual in situ crosslinking, a high-resolution bioprinted structure in the shape of the ear has been formed which is capable of maintaining live cells for 3 weeks of culture. Staining has shown the presence of cells and the formation of extracellular matrix proteins in postbioprinted tissue constructs. This research shows that chondrogenesis and successful matrix formation have occurred. This process has the potential to enhance the field of reconstructive surgery in medicine, by enabling cellular tissue constructs to be fabricated on demand through precise deposition of crosslinkable 3D biomaterials.

Cell-laden 3D bioprinting hydrogel matrix depending on different compositions for soft tissue engineering: Characterization and evaluation

Cell-printing techniques that can construct three-dimensional (3D) structures with biocompatible materials and cells are of great interest for various biomedical applications, such as tissue engineering and drug-screening studies. For successful cell-printing with cells, bioinks are critical for both the processability of printing and the viability of printed cells. However, the influence of composition on 3D bio-printing with cells has not been well explored. In this study, we investigated different compositions of alginate bioinks by varying the concentrations of high molecular weight alginate (High Alg) and low molecular weight alginate (Low Alg). Bioinks of 3wt% alginate containing High Alg alone or a 1:2 (Low Alg:High Alg) composite allowed for the construction of 3D scaffolds with good processability and shapes. Cell-printing with fibroblasts and in vitro culture studies revealed good viability and growth of the printed cells after up to 7days of culture. Bioinks prepared with High and Low Alg at a 2:1 ratio exhibited better cell growth compared with those of other compositions. This study progresses the design and applications of alginate-based bioinks for cell-printing platforms in soft tissue engineering.

A comparison of different bioinks for 3D bioprinting of fibrocartilage and hyaline cartilage

Cartilage is a dense connective tissue with limited self-repair capabilities. Mesenchymal stem cell (MSC) laden hydrogels are commonly used for fibrocartilage and articular cartilage tissue engineering, however they typically lack the mechanical integrity for implantation into high load bearing environments. This has led to increased interested in 3D bioprinting of cell laden hydrogel bioinks reinforced with stiffer polymer fibres. The objective of this study was to compare a range of commonly used hydrogel bioinks (agarose, alginate, GelMA and BioINK™) for their printing properties and capacity to support the development of either hyaline cartilage or fibrocartilage in vitro. Each hydrogel was seeded with MSCs, cultured for 28 days in the presence of TGF-β3 and then analysed for markers indicative of differentiation towards either a fibrocartilaginous or hyaline cartilage-like phenotype. Alginate and agarose hydrogels best supported the development of hyaline-like cartilage, as evident by the development of a tissue staining predominantly for type II collagen. In contrast, GelMA and BioINK™ (a PEGMA based hydrogel) supported the development of a more fibrocartilage-like tissue, as evident by the development of a tissue containing both type I and type II collagen. GelMA demonstrated superior printability, generating structures with greater fidelity, followed by the alginate and agarose bioinks. High levels of MSC viability were observed in all bioinks post-printing (∼80%). Finally we demonstrate that it is possible to engineer mechanically reinforced hydrogels with high cell viability by co-depositing a hydrogel bioink with polycaprolactone filaments, generating composites with bulk compressive moduli comparable to articular cartilage. This study demonstrates the importance of the choice of bioink when bioprinting different cartilaginous tissues for musculoskeletal applications.

3D Bioprinting of Developmentally Inspired Templates for Whole Bone Organ Engineering.

The ability to print defined patterns of cells and extracellular-matrix components in three dimensions has enabled the engineering of simple biological tissues; however, bioprinting functional solid organs is beyond the capabilities of current biofabrication technologies. An alternative approach would be to bioprint the developmental precursor to an adult organ, using this engineered rudiment as a template for subsequent organogenesis in vivo. This study demonstrates that developmentally inspired hypertrophic cartilage templates can be engineered in vitro using stem cells within a supporting gamma-irradiated alginate bioink incorporating Arg-Gly-Asp adhesion peptides. Furthermore, these soft tissue templates can be reinforced with a network of printed polycaprolactone fibers, resulting in a ≈350 fold increase in construct compressive modulus providing the necessary stiffness to implant such immature cartilaginous rudiments into load bearing locations. As a proof-of-principal, multiple-tool biofabrication is used to engineer a mechanically reinforced cartilaginous template mimicking the geometry of a vertebral body, which in vivo supported the development of a vascularized bone organ containing trabecular-like endochondral bone with a supporting marrow structure. Such developmental engineering approaches could be applied to the biofabrication of other solid organs by bioprinting precursors that have the capacity to mature into their adult counterparts over time in vivo.

Granular gel support-enabled extrusion of three-dimensional alginate and cellular structures

Freeform fabrication of soft structures has been of great interest in recent years. In particular, it is viewed as a critical step toward the grand vision of organ printing—the on-demand design and fabrication of three-dimensional (3D) human organ constructs for implantation and regenerative medicine. The objective of this study is to develop a novel granular gel support material-enabled, two-step gelation-based ‘printing-then-gelation’ approach to fabricate 3D alginate structures using filament extrusion. Specifically, a granular Carbopol microgel bath holds the ungelled alginate structure being extruded, avoiding the instantaneous gelation of each printed layer as well as resultant surface tension-induced nozzle clogging. Since Carbopol microgels react with multivalent cations, which are needed for alginate crosslinking, gelatin is introduced as a sacrificial material to make an alginate and gelatin bioink for extrusion, which gels thermally (step-one gelation) to initially stabilize the printed structure for removal from Carbopol. Then gelatin is melted and diffused away while alginate is ionically crosslinked in a 37 °C calcium chloride bath (step-two gelation), resulting in an alginate structure. The proposed ‘printing-then-gelation’ approach works for alginate structure fabrication, and it is also applicable for the printing of cellular constructs and other similar homogeneous soft structures using a two-step or even multi-step approach. The main conclusions are: (1) 0.8% (w/v) Carbopol bath with a neutral pH value may be most suitable for soft structure printing; (2) it is most effective to use a 0.9% (w/v) NaCl solution to facilitate the removal of residual Carbopol; and (3) alginate structures fabricated using the proposed approach demonstrate better mechanical properties than those fabricated using the conventional ‘gelation-while-printing’ approach.

Bioprinting three-dimensional cell-laden tissue constructs with controllable degradation.

Alginate hydrogel is a popular biologically inert material that is widely used in 3D bioprinting, especially in extrusion-based printing. However, the printed cells in this hydrogel could not degrade the surrounding alginate gel matrix, causing them to remain in a poorly proliferating and non-differentiating state. Here, we report a novel study of the 3D printing of human corneal epithelial cells (HCECs)/collagen/gelatin/alginate hydrogel incubated with a medium containing sodium citrate to obtain degradation-controllable cell-laden tissue constructs. The 3D-printed hydrogel network with interconnected channels and a macroporous structure was stable and achieved high cell viability (over 90%). By altering the mole ratio of sodium citrate/sodium alginate, the degradation time of the bioprinting constructs can be controlled. Cell proliferation and specific marker protein expression results also revealed that with the help of sodium citrate degradation, the printed HCECs showed a higher proliferation rate and greater cytokeratin 3(CK3) expression, indicating that this newly developed method may help to improve the alginate bioink system for the application of 3D bioprinting in tissue engineering.

Time-Resolved Imaging Study of Jetting Dynamics during Laser Printing of Viscoelastic Alginate Solutions.

Matrix-assisted pulsed-laser evaporation direct-write (MAPLE DW) has been successfully implemented as a promising laser printing technology for various fabrication applications, in particular, three-dimensional bioprinting. Since most bioinks used in bioprinting are viscoelastic, it is of importance to understand the jetting dynamics during the laser printing of viscoelastic fluids in order to control and optimize the laser printing performance. In this study, MAPLE DW was implemented to study the jetting dynamics during the laser printing of representative viscoelastic alginate bioinks and evaluate the effects of operating conditions (e.g., laser fluence) and material properties (e.g., alginate concentration) on the jet formation performance. Through a time-resolved imaging approach, it is found that when the laser fluence increases or the alginate concentration decreases, the jetting behavior changes from no material transferring to well-defined jetting to well-defined jetting with an initial bulgy shape to jetting with a bulgy shape to pluming/splashing. For the desirable well-defined jetting regimes, as the laser fluence increases, the jet velocity and breakup length increase while the breakup time and primary droplet size decrease. As the alginate concentration increases, the jet velocity and breakup length decrease while the breakup time and primary droplet size increase. In addition, Ohnesorge, elasto-capillary, and Weber number based phase diagrams are presented to better appreciate the dependence of jetting regimes on the laser fluence and alginate concentration.

3D Bioprinting Human Chondrocytes with Nanocellulose-Alginate Bioink for Cartilage Tissue Engineering Applications.

The introduction of 3D bioprinting is expected to revolutionize the field of tissue engineering and regenerative medicine. The 3D bioprinter is able to dispense materials while moving in X, Y, and Z directions, which enables the engineering of complex structures from the bottom up. In this study, a bioink that combines the outstanding shear thinning properties of nanofibrillated cellulose (NFC) with the fast cross-linking ability of alginate was formulated for the 3D bioprinting of living soft tissue with cells. Printability was evaluated with concern to printer parameters and shape fidelity. The shear thinning behavior of the tested bioinks enabled printing of both 2D gridlike structures as well as 3D constructs. Furthermore, anatomically shaped cartilage structures, such as a human ear and sheep meniscus, were 3D printed using MRI and CT images as blueprints. Human chondrocytes bioprinted in the noncytotoxic, nanocellulose-based bioink exhibited a cell viability of 73% and 86% after 1 and 7 days of 3D culture, respectively. On the basis of these results, we can conclude that the nanocellulose-based bioink is a suitable hydrogel for 3D bioprinting with living cells. This study demonstrates the potential use of nanocellulose for 3D bioprinting of living tissues and organs.

Freeform inkjet printing of cellular structures with bifurcations.

Organ printing offers a great potential for the freeform layer-by-layer fabrication of three-dimensional (3D) living organs using cellular spheroids or bioinks as building blocks. Vascularization is often identified as a main technological barrier for building 3D organs. As such, the fabrication of 3D biological vascular trees is of great importance for the overall feasibility of the envisioned organ printing approach. In this study, vascular-like cellular structures are fabricated using a liquid support-based inkjet printing approach, which utilizes a calcium chloride solution as both a cross-linking agent and support material. This solution enables the freeform printing of spanning and overhang features by providing a buoyant force. A heuristic approach is implemented to compensate for the axially-varying deformation of horizontal tubular structures to achieve a uniform diameter along their axial directions. Vascular-like structures with both horizontal and vertical bifurcations have been successfully printed from sodium alginate only as well as mouse fibroblast-based alginate bioinks. The post-printing fibroblast cell viability of printed cellular tubes was found to be above 90% even after a 24 h incubation, considering the control effect.