Long-term in vivo integrity and safety of 3D-bioprinted cartilaginous constructs

Long-term stability and biological safety are crucial for translation of 3D-bioprinting technology into clinical applications. Here, we addressed the long-term safety and stability issues associated with 3D-bioprinted constructs comprising a cellulose scaffold and human cells (chondrocytes and stem cells) over a period of 10 months in nude mice. Our findings showed that increasing unconfined compression strength over time significantly improved the mechanical stability of the cell-containing constructs relative to cell-free scaffolds. Additionally, the cell-free constructs exhibited a mean compressive stress and stiffness (compressive modulus) of 0.04 +/- 0.05 MPa and 0.14 +/- 0.18 MPa, respectively, whereas these values for the cell-containing constructs were 0.11 +/- 0.08 MPa (p= .019) and 0.53 +/- 0.59 MPa (p= .012), respectively. Moreover, histomorphologic analysis revealed that cartilage formed from the cell-containing constructs harbored an abundance of proliferating chondrocytes in clusters, and after 10 months, resembled native cartilage. Furthermore, extension of the experiment over the complete lifecycle of the animal model revealed no signs of ossification, fibrosis, necrosis, or implant-related tumor development in the 3D-bioprinted constructs. These findings confirm the in vivo biological safety and mechanical stability of 3D-bioprinted cartilaginous tissues and support their potential translation into clinical applications

Simulations of 3D bioprinting: predicting bioprintability of nanofibrillar inks

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

3D Printing Wood Tissue

Biomass from forests provides society with energy, materials and chemicals, thus contributing to the circular bioeconomy. The majority of biomass is found in the wood tissue of trees. Its composition and hierarchical structure originates from the synthesis and bottom-up assembly of biopolymers which involve numerous genes, hormones and exogenous factors. A technology for bottom-up fabrication of materials is 3D printing. In 3D printing, material is assembled layer-by-layer and thereby offers the potential to build up hierarchical complex structures with control of design and material properties. 3D printing wood is not as straight forward as for plastics since wood can’t be processed by melting. Also, printing wood involves the assembly of multiple polymers since wood is a composite material. Inspired by the composition, crosslinking mechanism, anisotropy and structural design of natural wood tissue, this work has established a platform for 3D printing wood biopolymers into hierarchical wood-like structures. The platform consists of extrusion-based 3D printers, designed printing pathways, and wood based solutions and dispersions which are called inks. We found that inks of both cellulose dissolved in ionic liquid and dispersions of cellulose nanofibrils (CNF) were printable due to their shear thinning properties. Good printing fidelity of cellulose solutions required a continuous gel formation. Printing on a coagulating gel allowed non-solvent to diffuse through the print and instantly regenerate cellulose. Diffusion through multiple layers was however challenging making it difficult to 3D print large constructs. CNF (1-4 wt%) exhibits a yield stress, and stops flowing when leaving the nozzle which facilitated the printing of multilayered structures, i.e. an ear. This also contributed to the printing resolution (≈ 300 μm). However, without crosslinking, the printed CNF could not withstand mechanical force. Hence, CNF was mixed with crosslinkable biopolymers. The mixed inks remained printable for CNF concentrations above 2 wt%. The crosslinking time was below 10 minutes and gel strength increased with the concentration of crosslinkable biopolymers. Inks containing alginate were ionically crosslinked and formed reversible hydrogels.\ua0 Enzymatic crosslinking, similar to the polymerization of monolignols in the wood cell wall was obtained by substituting carboxylic groups (COOH) of hemicelluloses with tyramine. Hydrogels with tunable mechanical properties were obtained by varying the degree of substitution by using xylan, or TEMPO oxidized galactoglucomannan with degrees of oxidation from 10 to 60%. A computational fluid dynamics simulation tool was studied as a complement to 3D printing tests of new inks to evaluate printability.\ua0 By simulation, it was easy to isolate parameters such as printing speed and printing height to study their influence on printing fidelity. Finally, natural bottom up assembly of wood tissue was substituted with 3D printing. G-code substituted genome and the cellulose was extruded by a printer head instead of the rosette. Structures that resemble morphological features found in wood were prepared by computer aided design and printed with all wood based inks. Control of printing paths provided anisotropic features resembling the micro fibril angle of the cell wall. The breakthrough of this work is the 3D shaping of wood by a bottom up process. Consequently, products assembled by wood biopolymers can transform from 2D (paper, board, films, textiles) to 3D. The concepts developed in this work can be employed in future applications of 3D printing with wood based materials, such as garments, electronics, wound dressings and packaging

3D Bioprinting of Cellulose Structures from an Ionic Liquid

This article reports on 3D bioprinting of dissolved cellulose to produce small feature structures with a tailored design ofregenerated cellulose. The process consists of dissolving cellulose with different origins and molecular weight in an ionic liquid(1 - ethyl - 3 - methylimidazolium acetate), controlled multilayered dispensing, and coagulation. The printability was examined bystudying the viscosity of cellulose solutions and by varying the settings of the printer setup regarding flow rate and needledimensions. Water was added as a nonsolvent, enabling a coagulation process to form a gel structure of the printed solutions.By printing on a coagulating gel, the printed solutions were regenerated within a few seconds. Rheology analysis showed thathigher concentrations of cellulose and cellulose of a high molecular weight were shear thinning, providing favorable printingproperties. Printing 3D structures of cellulose dissolved in an ionic liquid followed by coagulation by a nonsolvent was possible.Both complex patterns of 2D structures as well as multilayered prints were created to obtain 3D structures. This novel methodallows for the production of spatially tailored 3D gels or membrane structures made from cellulose

Biomimetic Inks Based on Cellulose Nanofibrils and Cross-Linkable Xylans for 3D Printing

This paper presents a sustainable all-wood-based ink which can be used for 3D printing of constructs for a large variety of applications such as clothes, furniture, electronics, and health care products with a customized design and versatile gel properties. The 3D printing technologies where the material is dispensed in the form of liquids, so called inks, have proven suitable for 3D printing dispersions of cellulose nanofibrils (CNFs) because of their unique shear thinning properties. In this study, novel inks were developed with a biomimetic approach where the structural properties of cellulose and the cross-linking function of hemicelluloses that are found in the plant cell wall were utilized. The CNF was mixed with xylan, a hemicellulose extracted from spruce, to introduce cross-linking properties which are essential for the final stability of the printed ink. For xylan to be cross-linkable, it was functionalized with tyramine at different degrees. Evaluation of different ink compositions by rheology measurements and 3D printing tests showed that the degree of tyramine substitution and the ratio of CNFs to xylan-tyramine in the prepared inks influenced the printability and cross-linking density. Both two-layered gridded structures and more complex 3D constructs were printed. Similarly to conventional composites, the interactions between the components and their miscibility are important for the stability of the printed and cross-linked ink. Thus, the influence of tyramine on the adsorption of xylan to cellulose was studied with a quartz crystal microbalance to verify that the functionalization had little influence on xylan\u27s adsorption to cellulose. Utilizing xylan-tyramine in the CNF dispersions resulted in all-wood-based inks which after 3D printing can be cross-linked to form freestanding gels while at the same time, the excellent printing properties of CNFs remain intact

Synthesis of tunable hydrogels based on O-acetyl-galactoglucomannans from spruce

© 2016 Elsevier LtdHydrogels with tunable mechanical properties based on O-acetyl-galactoglucomannans (GGMs) from spruce functionalized with tyramine, a molecule containing crosslinkable phenolic groups, were prepared. Gel formation was induced by enzymatic crosslinking at the addition of horse radish peroxidase and hydrogen peroxide to the modified GGMs. The degree of substitution determined the hydrogels final properties, and was varied by TEMPO oxidation of GGM to a degree of oxidation from 10 to 60%. GGM and its derivatives were characterized by gas chromatography and high pressure size exclusion chromatography to analyze sugar composition and molar mass, respectively. Tyramine-conjugated GGM was evaluated by nuclear magnetic resonance, fourier transform infrared spectroscopy and elemental analysis. Measurements of moduli over time showed crosslinking within 20 s and maximum stress of the prepared gels were compared by compression testing. Overall this system presents a cell friendly hydrogel from a renewable, low cost resource which could be applied in cell delivery, wound dressings, and biofabrication

Materials from trees assembled by 3D printing – Wood tissue beyond nature limits

Materials from trees have the potential to replace fossil based and other non-sustainable materials in everyday products, thus transforming the society back to a bioeconomy. This paper presents a 3D printing platform which mimics wood biogenesis for the assembly of wood biopolymers into wood-like hierarchical composites. The genome was substituted with G-code, the programming language which controls how the 3D printer assembles material. The rosette was replaced by the printer head for extrusion of cellulose. Instead of microtubules guiding the alignment of cellulose, the printing direction was guided by an x/y stage, thus mimicking the microfibril angle. The printed structures were locked by an enzymatic crosslinking reaction similar to what occurs in the cell wall upon lignification. Hierarchical structures characteristic for wood were designed and printed with control of density, swelling and directional strength. Accelerating the development of the 3D printing technology helps realize the circular bioeconomy where garments, packaging, furniture and entire houses are manufactured by 3D printing wood