Three-dimensional bioprinting uses additive manufacturing techniques for the automated fabrication of hierarchically organized living constructs. The building blocks are often hydrogel-based bioinks, which need to be printed into structures with high shape fidelity to the intended computer-aided design. For optimal cell performance, relatively soft and printable inks are preferred, although these undergo significant deformation during the printing process, which may impair shape fidelity. While the concept of good or poor printability seems rather intuitive, its quantitative definition lacks consensus and depends on multiple rheological and chemical parameters of the ink. This review discusses qualitative and quantitative methodologies to evaluate printability of bioinks for extrusion- and lithography-based bioprinting. The physicochemical parameters influencing shape fidelity are discussed, together with their importance in establishing new models, predictive tools and printing methods that are deemed instrumental for the design of next-generation bioinks, and for reproducible comparison of their structural performance.

Printability and Shape Fidelity of Bioinks in 3D Bioprinting.

Polymers for additive manufacturing and 4D-printing: Materials, methodologies, and biomedical applications

Osteochondral defects are prone to induce osteoarthritic degenerative changes. Many tissue-engineering approaches that aim to generate osteochondral implants suffer from poor tissue formation and compromised integration. This illustrates the need for further improvement of heterogeneous tissue constructs. Engineering of these structures is expected to profit from strategies addressing the complexity of tissue organization and the simultaneous use of multiple cell types. Moreover, this enables the investigation of the effects of three-dimensional (3D) organization and architecture on tissue function. In the present study, we characterize the use of a 3D fiber deposition (3DF) technique for the fabrication of cell-laden, heterogeneous hydrogel constructs for potential use as osteochondral grafts. Changing fiber spacing or angle of fiber deposition yielded scaffolds of varying porosity and elastic modulus. We encapsulated and printed fluorescently labeled human chondrocytes and osteogenic progenitors in alginate hydrogel yielding scaffolds of 1×2 cm with different parts for both cell types. Cell viability remained high throughout the printing process, and cells remained in their compartment of the printed scaffold for the whole culture period. Moreover, distinctive tissue formation was observed, both in vitro after 3 weeks and in vivo (6 weeks subcutaneously in immunodeficient mice), at different locations within one construct. These results demonstrate the possibility of manufacturing viable centimeter-scaled structured tissues by the 3DF technique, which could potentially be used for the repair of osteochondral defects.

Biofabrication of osteochondral tissue equivalents by printing topologically defined, cell-laden hydrogel scaffolds

Print me an organ! Why we are not there yet

Extrusion-based bioprinting is one of the leading manufacturing techniques for tissue engineering and regenerative medicine. Its primary limitation is the lack of materials, known as bioinks, which are suitable for the bioprinting process. The degree to which a bioink is suitable for bioprinting has been described as its 'printability.' However, a lack of clarity surrounding the methodologies used to evaluate a bioink's printability, as well as the usage of the term itself, have hindered the field. This article presents a review of measures used to assess the printability of extrusion-based bioinks in an attempt to assist researchers during the bioink development process. Many different aspects of printability exist and many different measurements have been proposed as a consequence. Researchers often do not evaluate a new bioink's printability at all, while others simply do so qualitatively. Several quantitative measures have been presented for the extrudability, shape fidelity, and printing accuracy of bioinks. Different measures have been developed even within these aspects, each testing the bioink in a slightly different way. Additionally, other relevant measures which had little or no examples of quantifiable methods are also to be considered. Looking forward, further work is needed to improve upon current assessment methodologies, to move towards a more comprehensive view of printability, and to standardize these printability measurements between researchers. Better assessment techniques will naturally lead to a better understanding of the underlying mechanisms which affect printability and better comparisons between bioinks. This in turn will help improve upon the bioink development process and the bioinks available for use in bioprinting.

/pdf/assessment-methodologies-for-extrusion-based-bioink-20nple3pt2.pdf

Assessment methodologies for extrusion-based bioink printability.

The primary bottleneck in bioprinting cell-laden structures with carefully controlled spatial relation is a lack of biocompatible inks and printing conditions. In this regard, we explored using thermogelling chitosan-gelatin (CG) hydrogel as a novel bioprinting ink; CG hydrogels are unique in that it undergoes a spontaneous phase change at physiological temperature, and does not need post-processing. In addition, we used a low cost (<$800) compact 3D printer, and modified with a new extruder to print using disposable syringes and hypodermic needles. We investigated (i) the effect of concentration of CG on gelation characteristics, (ii) solution preparation steps (centrifugation, mixing, and degassing) on printability and fiber formation, (iii) the print bed temperature profiles via IR imaging and grid-based assessment using thermocouples, (iv) the effect of feed rate (10-480 cm min-1), flow rate (15-60 μl min-1) and needle height (70-280 μm) on fiber size and characteristics, and (v) the distribution of neuroblastoma cells in printed fibers, and the viability after five days in culture. We used agarose gel to create uniform print surfaces to maintain a constant gap with the needle tip. These results showed that degassing the solution, and precooling the solution was necessary for obtaining continuous fibers. Fiber size decreased from 760, to 243 μm as the feed rate increased from 10 to 100 cm min-1. Bed temperature played the greatest role in fiber size, followed by feed rate. Increased needle height initially decreased fiber size but then increased showing an optimum. Cells were well distributed within the fibers and exhibited excellent viability and no contamination after 5 d. Overall we printed 3D, sterile, cell-laden structures with an inexpensive bioprinter and a novel ink, without post-processing. The bioprinter described here and the novel CG hydrogels have significant potential as an ink for bioprinitng various cell-laden structures.

https://iopscience.iop.org/article/10.1088/1758-5090/aa96dd/pdf

Bioprinted chitosan-gelatin thermosensitive hydrogels using an inexpensive 3D printer.

Developing a scaffold from porcine adipose tissue

As an emerging sterilization technology, cold atmospheric plasma offers a dry, non-thermal, rapid process that is minimally damaging to a majority of substrates. However, the mechanisms by which plasma interacts with living cells are poorly understood and the plasma generation apparatuses are complex and resource-intensive. In this study, the roles of reactive oxygen species (ROS), nitric oxide (NO), and charged particles (ions) produced by surface dielectric barrier discharge (SDBD) plasma on prokaryotic (Listeria monocytogenes (Gram-positive)) and eukaryotic (human umbilical vein endothelial cells (HUVEC)) cellular function were evaluated. HUVEC and bacterial oxidative stress responses, the accumulation of nitrite in aqueous media, air ion density, and bacterial inactivation at various distances from SDBD actuators were measured. SDBD actuator designs were also varied in terms of electrode number and length to evaluate the cellular effects of plasma volume and power distribution. NO and ions were found to contribute minimally to the observed cellular effects, whereas ROS were found to cause rapid bacterial inactivation, induce eukaryotic and prokaryotic oxidative stress, and result in rapid oxidation of bovine muscle tissue. The results of this study underscore the dominance of ROS as the major plasma generated species responsible for cellular effects, with ions and RNS having a secondary, complimentary role.

/pdf/investigation-of-the-roles-of-plasma-species-generated-by-54id2k9tyg.pdf

Investigation of the Roles of Plasma Species Generated by Surface Dielectric Barrier Discharge.

This study evaluated a novel approach to decellularizing porcine adipose tissue while preserving its 3-D architecture. An ethanol-water mixture was used as a solvent to remove lipids and the number of freeze-thaw cycles (1-7), ethanol concentration, and tissue thickness were tested. Trypsin incubation time (1-3 h) and xylene immersion time were investigated separately. Processed sample microarchitecture was analyzed via scanning electron microscope, cellular content was analyzed via hematoxylin and eosin (HE H&E micrographs confirmed unaltered morphology and absence of cells. Incubation for 1.5 h in trypsin removed 99.5% DNA from delipidized samples. An average of 40% rehydration swelling, an elastic modulus of 324(±141) kPa, and an ultimate tensile strength of 87.4(±23.1) kPa were observed. The matrix exhibited strain hardening behavior similar to small intestinal submucosa. Cells successfully infiltrated and spread in the decellularized scaffold. Removal of lipids significantly reduced incubation in trypsin EDTA. In summary, the acellular matrix shows significant potential as a new template for tissue regeneration. © 2016 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 104A: 3127-3136, 2016.

In vitro characterization of acelluar porcine adipose tissue matrix for use as a tissue regenerative scaffold

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Bioprinted chitosan-gelatin thermosensitive hydrogels using an inexpensive 3D printer.

Developing a scaffold from porcine adipose tissue

Investigation of the Roles of Plasma Species Generated by Surface Dielectric Barrier Discharge.

In vitro characterization of acelluar porcine adipose tissue matrix for use as a tissue regenerative scaffold