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      Tuning Alginate-Gelatin Bioink Properties by Varying Solvent and Their Impact on Stem Cell Behavior

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          Abstract

          Bioink optimization is considered as one of main challenges in cell-laden 3D bioprinting. Alginate-Gelatin (Alg-Gel) hydrogel have been extensively used as bioink. However, its properties could be influenced by various parameters, and little is known about the evidence featuring the impact of solvent. Here we investigated four Alg-Gel bioink by varying solvent ionic strength (named B-1, B-2, B-3 and B-4). Mechanical properties and printability of bioink samples and their impacts on behaviors of encapsulated epidermal stem cells (ESCs) were tested. Bioink with increased ionic strength of solvent showed decreased stiffness and viscosity, and increased swelling and degradation by printability and mechanical property tests. Due to the increased swelling and degradation was associated with shape-maintenance of post-printing constructs, B-3 and B-4 were hardly observable after 14 days. Cellular behaviors were assessed through viability, proliferation, aggregation and differentiation tests. B-2 with optimal properties resulted in higher viability and proliferation of ESCs, and further facilitated cellular aggregation and lineage differentiation. We demonstrated that the solvent can be tuned by ionic strength to control the properties of Alg-Gel bioink and post-printing constructs, which represented a promising avenue for promotion of therapeutic stem cell behaviors in 3D bioprinting.

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          3D bioprinting for engineering complex tissues.

          Bioprinting is a 3D fabrication technology used to precisely dispense cell-laden biomaterials for the construction of complex 3D functional living tissues or artificial organs. While still in its early stages, bioprinting strategies have demonstrated their potential use in regenerative medicine to generate a variety of transplantable tissues, including skin, cartilage, and bone. However, current bioprinting approaches still have technical challenges in terms of high-resolution cell deposition, controlled cell distributions, vascularization, and innervation within complex 3D tissues. While no one-size-fits-all approach to bioprinting has emerged, it remains an on-demand, versatile fabrication technique that may address the growing organ shortage as well as provide a high-throughput method for cell patterning at the micrometer scale for broad biomedical engineering applications. In this review, we introduce the basic principles, materials, integration strategies and applications of bioprinting. We also discuss the recent developments, current challenges and future prospects of 3D bioprinting for engineering complex tissues. Combined with recent advances in human pluripotent stem cell technologies, 3D-bioprinted tissue models could serve as an enabling platform for high-throughput predictive drug screening and more effective regenerative therapies.
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            Effect of bioink properties on printability and cell viability for 3D bioplotting of embryonic stem cells.

            3D cell printing is an emerging technology for fabricating complex cell-laden constructs with precise and pre-designed geometry, structure and composition to overcome the limitations of 2D cell culture and conventional tissue engineering scaffold technology. This technology enables spatial manipulation of cells and biomaterials, also referred to as 'bioink', and thus allows study of cellular interactions in a 3D microenvironment and/or in the formation of functional tissues and organs. Recently, many efforts have been made to develop new bioinks and to apply more cell sources for better biocompatibility and biofunctionality. However, the influences of printing parameters on the shape fidelity of 3D constructs as well as on cell viability after the cell printing process have been poorly characterized. Furthermore, parameter optimization based on a specific cell type might not be suitable for other types of cells, especially cells with high sensibility. In this study, we systematically studied the influence of bioink properties and printing parameters on bioink printability and embryonic stem cell (ESC) viability in the process of extrusion-based cell printing, also known as bioplotting. A novel method was established to determine suitable conditions for bioplotting ESCs to achieve both good printability and high cell viability. The rheological properties of gelatin/alginate bioinks were evaluated to determine the gelation properties under different bioink compositions, printing temperatures and holding times. The bioink printability was characterized by a newly developed semi-quantitative method. The results demonstrated that bioinks with longer gelation times would result in poorer printability. The live/dead assay showed that ESC viability increased with higher printing temperatures and lower gelatin concentrations. Furthermore, an exponential relationship was obtained between ESC viability and induced shear stress. By defining the proper printability and acceptable viability ranges, a combined parameters region was obtained. This study provides guidance for parameter optimization and the fine-tuning of 3D cell printing processes regarding both bioink printability and cell viability after bioplotting, especially for easily damaged cells, like ESCs.
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              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.
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                Author and article information

                Contributors
                stellarahuang@sina.com
                fuxiaobing@vip.sina.com
                Journal
                Sci Rep
                Sci Rep
                Scientific Reports
                Nature Publishing Group UK (London )
                2045-2322
                22 May 2018
                22 May 2018
                2018
                : 8
                : 8020
                Affiliations
                [1 ]ISNI 0000 0004 1761 8894, GRID grid.414252.4, Institute of Basic Medical Sciences, , General Hospital of PLA, ; Beijing, P. R. China
                [2 ]ISNI 0000 0004 1761 8894, GRID grid.414252.4, Key Laboratory of Tissue Repair and Regeneration of PLA, and Beijing Key Research Laboratory of Skin Injury, Repair and Regeneration, First Hospital Affiliated to General Hospital of PLA, ; Beijing, P. R. China
                [3 ]ISNI 0000 0000 9878 7032, GRID grid.216938.7, Medical College, , Nankai University, ; Tianjin, P. R. China
                [4 ]ISNI 0000 0004 0644 7196, GRID grid.458502.e, National Research Center of Engineering Plastics, , Technical Institute of Physics and Chemistry, Chinese Academy of Sciences, ; Beijing, P. R. China
                Article
                26407
                10.1038/s41598-018-26407-3
                5964146
                29789674
                2786da8d-4f06-42a2-aeef-943f8bf6e5f1
                © The Author(s) 2018

                Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.

                History
                : 30 January 2018
                : 11 May 2018
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