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AUTHOR Wei, Zhengxi and Liu, Xue and Ooka, Masato and Zhang, Li and Song, Min Jae and Huang, Ruili and Kleinstreuer, Nicole C. and Simeonov, Anton and Xia, Menghang and Ferrer, Marc
Title Two-Dimensional Cellular and Three-Dimensional Bio-Printed Skin Models to Screen Topical-Use Compounds for Irritation Potential [Abstract]
Year 2020
Journal/Proceedings Frontiers in Bioengineering and Biotechnology
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Assessing skin irritation potential is critical for the safety evaluation of topical drugs and other consumer products such as cosmetics. The use of advanced cellular models, as an alternative to replace animal testing in the safety evaluation for both consumer products and ingredients, is already mandated by law in the European Union (EU) and other countries. However, there has not yet been a large-scale comparison of the effects of topical-use compounds in different cellular skin models. This study assesses the irritation potential of topical-use compounds in different cellular models of the skin that are compatible with high throughput screening (HTS) platforms. A set of 451 topical-use compounds were first tested for cytotoxic effects using two-dimensional (2D) monolayer models of primary neonatal keratinocytes and immortalized human keratinocytes. Forty-six toxic compounds identified from the initial screen with the monolayer culture systems were further tested for skin irritation potential on reconstructed human epidermis (RhE) and full thickness skin (FTS) three-dimensional (3D) tissue model constructs. Skin irritation potential of the compounds was assessed by measuring tissue viability, trans-epithelial electrical resistance (TEER), and secretion of cytokines interleukin 1 alpha (IL-1α) and interleukin 18 (IL-18). Among known irritants, high concentrations of methyl violet and methylrosaniline decreased viability, lowered TEER, and increased IL-1α secretion in both RhE and FTS models, consistent with irritant properties. However, at low concentrations, these two compounds increased IL-18 secretion without affecting levels of secreted IL-1α, and did not reduce tissue viability and TEER, in either RhE or FTS models. This result suggests that at low concentrations, methyl violet and methylrosaniline have an allergic potential without causing irritation. Using both HTS-compatible 2D cellular and 3D tissue skin models, together with irritation relevant activity endpoints, we obtained data to help assess the irritation effects of topical-use compounds and identify potential dermal hazards.
AUTHOR Derr, Kristy and Zou, Jinyun and Luo, Keren and Song, Min Jae and Sittampalam, G. Sitta and Zhou, Chao and Michael, Samuel and Ferrer, Marc and Derr, Paige
Title Fully 3D Bioprinted Skin Equivalent Constructs with Validated Morphology and Barrier Function [Abstract]
Year 2019
Journal/Proceedings Tissue Engineering Part C: Methods
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Development of high throughput, reproducible, three-dimensional bioprinted skin equivalents that are morphologically and functionally comparable to native skin tissue is advancing research in skin diseases, and providing a physiologically relevant platform for the development of therapeutics, transplants for regenerative medicine, and testing of skin products like cosmetics. Current protocols for the production of engineered skin rafts are limited in their ability to control three dimensional geometry of the structure and contraction leading to variability of skin function between constructs. Here we describe a method for the biofabrication of skin equivalents that are fully bioprinted using an open market bioprinter, made with commercially available primary cells and natural hydrogels. The unique hydrogel formulation allows for the production of a human-like skin equivalent with minimal lateral tissue contraction in a multiwell plate format, thus making them suitable for high throughput bioprinting in a single print with fast print and relatively short incubation times. The morphology and barrier function of the fully three-dimensional bioprinted skin equivalents are validated by immunohistochemistry staining, optical coherence tomography, and permeation assays.
AUTHOR Liu, Xue and Michael, Samuel and Bharti, Kapil and Ferrer, Marc and Song, Min Jae
Title A biofabricated vascularized skin model of atopic dermatitis for preclinical studies [Abstract]
Year 2020
Journal/Proceedings Biofabrication
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Three-dimensional (3D) biofabrication techniques enable the production of multicellular tissue models as assay platforms for drug screening. The increased cellular and physiological complexity in these 3D tissue models should recapitulate the relevant biological environment found in the body. Here we describe the use of 3D bioprinting techniques to fabricate skin equivalent tissues of varying physiological complexity, including human epidermis, non-vascularized and vascularized full-thickness skin tissue equivalents, in a multi-well platform to enable drug screening. Human keratinocytes, fibroblasts, and pericytes, and induced pluripotent stem cell (iPSC)-derived endothelial cells were used in the biofabrication process to produce the varying complexity. The skin equivalents exhibit the correct structural markers of dermis and epidermis stratification, with physiological functions of the skin barrier. The robustness, versatility and reproducibility of the biofabrication techniques are further highlighted by the generation of atopic dermatitis (AD)-disease like tissues. These AD models demonstrate several clinical hallmarks of the disease, including: (i) spongiosis and hyperplasia; (ii) early and terminal expression of differentiation proteins; and (iii) increases in levels of pro-inflammatory cytokines. We show the pre-clinical relevance of the biofabricated AD tissue models to correct disease phenotype by testing the effects of dexamethasone, an anti-inflammatory corticosteroid, and three Janus Kinase inhibitors from clinical trials for AD. This study demonstrates the development of a versatile and reproducible bioprinting approach to create human skin equivalents with a range of cellular complexity for disease modelling. In addition, we establish several assay readouts that are quantifiable, robust, AD relevant, and can be scaled up for compound screening. The results show that the cellular complexity of the tissues develops a more physiologically relevant AD disease model. Thus, the skin models in this study offer an in vitro approach for the rapid understanding of pathological mechanisms, and testing for efficacy of action and toxic effects of drugs.
AUTHOR Ng, Wei Long and Qi, Jovina Tan Zhi and Yeong, Wai Yee and Naing, May Win
Title Proof-of-concept: 3D bioprinting of pigmented human skin constructs [Abstract]
Year 2018
Journal/Proceedings Biofabrication
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Three-dimensional (3D) pigmented human skin constructs have been fabricated using a 3D bioprinting approach. The 3D pigmented human skin constructs are obtained from using three different types of skin cells (keratinocytes, melanocytes and fibroblasts from three different skin donors) and they exhibit similar constitutive pigmentation (pale pigmentation) as the skin donors. A two-step drop-on-demand bioprinting strategy facilitates the deposition of cell droplets to emulate the epidermal melanin units (pre-defined patterning of keratinocytes and melanocytes at the desired positions) and manipulation of the microenvironment to fabricate 3D biomimetic hierarchical porous structures found in native skin tissue. The 3D bioprinted pigmented skin constructs are compared to the pigmented skin constructs fabricated by conventional a manual-casting approach; in-depth characterization of both the 3D pigmented skin constructs has indicated that the 3D bioprinted skin constructs have a higher degree of resemblance to native skin tissue in term of the presence of well-developed stratified epidermal layers and the presence of a continuous layer of basement membrane proteins as compared to the manually-cast samples. The 3D bioprinting approach facilitates the development of 3D in vitro pigmented human skin constructs for potential toxicology testing and fundamental cell biology research.
AUTHOR Tan, Edgar Y. S. and Suntornnond, Ratima and Yeong, Wai Yee
Title High-Resolution Novel Indirect Bioprinting of Low-Viscosity Cell-Laden Hydrogels via Model-Support Bioink Interaction [Abstract]
Year 2021
Journal/Proceedings 3D Printing and Additive Manufacturing
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Abstract Bioprinting of unmodified soft extracellular matrix into complex 3D structures has remained challenging to fabricate. Herein, we established a novel process for the printing of low-viscosity hydrogel by using a unique support technique to retain the structural integrity of the support structure. We demonstrated that this process of printing could be used for different types of hydrogel, ranging from fast crosslinking gelatin methacrylate to slow crosslinking collagen type I. In addition, we evaluated the biocompatibility of the process by observing the effects of the cytotoxicity of L929 and the functionality of the human umbilical vein endothelium primary cells after printing. The results show that the bioprinted construct provided excellent biocompatibility as well as supported cell growth and differentiation. Thus, this is a novel technique that can be potentially used to enhance the resolution of the extrusion-based bioprinter.
AUTHOR Chen, Shengyang and Jang, Tae-Sik and Pan, Houwen Matthew and Jung, Hyun-Do and Sia, Ming Wei and Xie, Shuying and Hang, Yao and Chong, Seow and Wong, Dongan
Title 3D Freeform Printing of Nanocomposite Hydrogels through in situ Precipitation in Reactive Viscous Fluid
Year 2020
Journal/Proceedings International Journal of Bioprinting
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AUTHOR Lin, Che-Wei and Su, Yu-Feng and Lee, Chih-Yun and Kang, Lin and Wang, Yan-Hsiung and Lin, Sung-Yen and Wang, Chih-Kuang
Title 3D printed bioceramics fabricated using negative thermoresponsive hydrogels and silicone oil sealing to promote bone formation in calvarial defects [Abstract]
Year 2020
Journal/Proceedings Ceramics International
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The purpose of the present work was to investigate the potential for application and the effectiveness of osteoconductive scaffolds with bicontinuous phases of 3D printed bioceramics (3DP-BCs) based on reverse negative thermoresponsive hydrogels (poly[(N-isopropylacrylamide)-co-(methacrylic acid)]; p(NiPAAm-MAA)). 3DP-BCs have bioceramic objects and microchannel pores when created using robotic deposition additive manufacturing. We evaluated the benefits of silicone oil sealing on the 3DP-BC green body during the sintering process in terms of densification and structural stability. The shrinkage, density, porosity, element composition, phase structure and microstructural analyses and compression strength measurements of sintered 3DP-BC objects are presented and discussed in this study. In addition, the results of cell viability assays and bone healing analyses of the calvarial bone defects in a rabbit model were used to evaluate 3DP-BC performance. The main results indicated that these 3DP-BC scaffolds have optimal continuous pores and adequate compressive strength, which can enable the protection of calvarial defects and provide an environment for cell growth. Therefore, 3DP-BC scaffolds have better new bone regeneration efficiency in rabbit calvarial bone defect models than empty scaffolds and mold-forming bioceramic scaffolds (MF-BCs).
AUTHOR Browning, James R. and Derr, Paige and Derr, Kristy and Doudican, Nicole and Michael, Sam and Lish, Samantha R. and Taylor, Nicholas A. and Krueger, James G. and Ferrer, Marc and Carucci, John A. and Gareau, Daniel S.
Title A 3D biofabricated cutaneous squamous cell carcinoma tissue model with multi-channel confocal microscopy imaging biomarkers to quantify antitumor effects of chemotherapeutics in tissue [Abstract]
Year 2020
Journal/Proceedings Oncotarget; Vol 11, No 27
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// James R. Browning 1 , Paige Derr 2 , Kristy Derr 2 , Nicole Doudican 3 , Sam Michael 2 , Samantha R. Lish 1 , Nicholas A. Taylor 3 , James G. Krueger 1 , Marc Ferrer 2 , John A. Carucci 3 and Daniel S. Gareau 1 1 Laboratory for Investigative Dermatology, The Rockefeller University, New York, New York, USA 2 National Center for Advancing Translational Sciences, National Institutes of Health, Rockville, Maryland, USA 3 The Ronald O. Perelman Department of Dermatology, New York University School of Medicine, New York, New York, USA Correspondence to: Daniel S. Gareau, email: dgareau@rockefeller.edu Keywords: squamous cell carcinoma; screening; 3D printing; in vitro model; confocal microscopy Received: January 05, 2020     Accepted: April 03, 2020     Published: July 07, 2020 ABSTRACT Cutaneous squamous cell carcinoma (cSCC) causes approximately 10,000 deaths annually in the U. S. Current therapies are largely ineffective against metastatic and locally advanced cSCC. There is a need to identify novel, effective, and less toxic small molecule cSCC therapeutics. We developed a 3-dimensional bioprinted skin (3DBPS) model of cSCC tumors together with a microscopy assay to test chemotherapeutic effects in tissue. The full thickness SCC tissue model was validated using hematoxylin and eosin (H&E) and immunohistochemical histological staining, confocal microscopy, and cDNA microarray analysis. A nondestructive, 3D fluorescence confocal imaging assay with tdTomato-labeled A431 SCC and ZsGreen-labeled keratinocytes was developed to test efficacy and general toxicity of chemotherapeutics. Fluorescence-derived imaging biomarkers indicated that 50% of cancer cells were killed in the tissue after 1?M 5-Fluorouracil 48-hour treatment, compared to a baseline of 12% for untreated controls. The imaging biomarkers also showed that normal keratinocytes were less affected by treatment (11% killed) than the untreated tissue, which had no significant killing effect. Data showed that 5-Fluorouracil selectively killed cSCC cells more than keratinocytes. Our 3DBPS assay platform provides cellular-level measurement of cell viability and can be adapted to achieve nondestructive high-throughput screening (HTS) in bio-fabricated tissues.
AUTHOR Somasekharan, Lakshmi and Kasoju, Naresh and Raju, Riya and Bhatt, Anugya
Title Formulation and Characterization of Alginate Dialdehyde, Gelatin, and Platelet-Rich Plasma-Based Bioink for Bioprinting Applications [Abstract]
Year 2020
Journal/Proceedings Bioengineering
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Layer-by-layer additive manufacturing process has evolved into three-dimensional (3D) “bio-printing” as a means of constructing cell-laden functional tissue equivalents. The process typically involves the mixing of cells of interest with an appropriate hydrogel, termed as “bioink”, followed by printing and tissue maturation. An ideal bioink should have adequate mechanical, rheological, and biological features of the target tissues. However, native extracellular matrix (ECM) is made of an intricate milieu of soluble and non-soluble extracellular factors, and mimicking such a composition is challenging. To this end, here we report the formulation of a multi-component bioink composed of gelatin and alginate -based scaffolding material, as well as a platelet-rich plasma (PRP) suspension, which mimics the insoluble and soluble factors of native ECM respectively. Briefly, sodium alginate was subjected to controlled oxidation to yield alginate dialdehyde (ADA), and was mixed with gelatin and PRP in various volume ratios in the presence of borax. The formulation was systematically characterized for its gelation time, swelling, and water uptake, as well as its morphological, chemical, and rheological properties; furthermore, blood- and cytocompatibility were assessed as per ISO 10993 (International Organization for Standardization). Printability, shape fidelity, and cell-laden printing was evaluated using the RegenHU 3D Discovery bioprinter. The results indicated the successful development of ADA–gelatin–PRP based bioink for 3D bioprinting and biofabrication applications.
AUTHOR Eltaher, Hoda M. and Abukunna, Fatima E. and Ruiz-Cantu, Laura and Stone, Zack and Yang, Jing and Dixon, James E.
Title Human-scale tissues with patterned vascular networks by additive manufacturing of sacrificial sugar-protein composites [Abstract]
Year 2020
Journal/Proceedings Acta Biomaterialia
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Combating necrosis, by supplying nutrients and removing waste, presents the major challenge for engineering large three-dimensional (3D) tissues. Previous elegant work used 3D printing with carbohydrate glass as a cytocompatible sacrificial template to create complex engineered tissues with vascular networks (Miller et al. 2012, Nature Materials). The fragile nature of this material compounded with the technical complexity needed to create high-resolution structures led us to create a flexible sugar-protein composite, termed Gelatin-sucrose matrix (GSM), to achieve a more robust and applicable material. Here we developed a low-range (25–37˚C) temperature sensitive formulation that can be moulded with micron-resolution features or cast during 3D printing to produce complex flexible filament networks forming sacrificial vessels. Using the temperature-sensitivity, we could control filament degeneration meaning GSM can be used with a variety of matrices and crosslinking strategies. Furthermore by incorporation of biocompatible crosslinkers into GSM directly, we could create thin endothelialized vessel walls and generate patterned tissues containing multiple matrices and cell-types. We also demonstrated that perfused vascular channels sustain metabolic function of a variety of cell-types including primary human cells. Importantly, we were able to construct vascularized human noses which otherwise would have been necrotic. Our material can now be exploited to create human-scale tissues for regenerative medicine applications. Statement of Significance Authentic and engineered tissues have demands for mass transport, exchanging nutrients and oxygen, and therefore require vascularization to retain viability and inhibit necrosis. Basic vascular networks must be included within engineered tissues intrinsically. Yet, this has been unachievable in physiologically-sized constructs with tissue-like cell densities until recently. Sacrificial moulding is an alternative in which networks of rigid lattices of filaments are created to prevent subsequent matrix ingress. Our study describes a biocompatible sacrificial sugar-protein formulation; GSM, made from mixtures of inexpensive and readily available bio-grade materials. GSM can be cast/moulded or bioprinted as sacrificial filaments that can rapidly dissolve in an aqueous environment temperature-sensitively. GSM material can be used to engineer viable and vascularized human-scale tissues for regenerative medicine applications.
AUTHOR Shapira, Assaf and Noor, Nadav and Oved, Hadas and Dvir, Tal
Title Transparent support media for high resolution 3D printing of volumetric cell-containing ECM structures [Abstract]
Year 2020
Journal/Proceedings Biomedical Materials
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3D bioprinting may revolutionize the field of tissue engineering by allowing fabrication of bio-structures with high degree of complexity, fine architecture and heterogeneous composition. The printing substances in these processes are mostly based on biomaterials and living cells. As such, they generally possess weak mechanical properties and thus must be supported during fabrication in order to prevent the collapse of large, volumetric multi-layered printouts. In this work, we characterize a uniquely formulated media used to support printing of extracellular matrix-based biomaterials. We show that a hybrid material, comprised of calcium-alginate nanoparticles and xanthan gum, presents superb qualities that enable printing at high resolution of down to 10 microns, allowing fabrication of complex constructs and cellular structures. This hybrid also presents an exclusive combination of desirable properties such as biocompatibility, high transparency, stability at a wide range of temperatures and amenability to delicate extraction procedures. Moreover, as fabrication of large, volumetric biological structures may require hours and even days to accomplish, we have demonstrated that the hybrid medium can support prolonged, precise printing for at least 18 hours. All these qualities make it a promising support medium for 3D printing of tissues and organs.
AUTHOR Pan, Houwen Matthew and Chen, Shengyang and Jang, Tae-Sik and Han, Win Tun and Jung, Hyun-do and Li, Yaning and Song, Juha
Title Plant seed-inspired cell protection, dormancy, and growth for large-scale biofabrication [Abstract]
Year 2019
Journal/Proceedings Biofabrication
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Biofabrication technologies have endowed us with the capability to fabricate complex biological constructs. However, cytotoxic biofabrication conditions have been a major challenge for their clinical application, leading to a trade-off between cell viability and scalability of biofabricated constructs. Taking inspiration from nature, we proposed a cell protection strategy which mimicks the protected and dormant state of plant seeds in adverse external conditions and their germination in response to appropriate environmental cues. Applying this bioinspired strategy to biofabrication, we successfully preserved cell viability and enhanced the seeding of cell-laden biofabricated constructs via a cytoprotective pyrogallol (PG)-alginate encapsulation system. Our cytoprotective encapsulation technology utilizes PG-triggered sporulation and germination processes to preserve cells, is mechanically robust, chemically resistant, and highly customizable to adequately match cell protectability with cytotoxicity of biofabrication conditions. More importantly, the facile and tunable decapsulation of our PG-alginate system allows for effective germination of dormant cells, under typical culture conditions. With this approach, we have successfully achieved a biofabrication process which is reproducible, scalable, and provided a practical solution for off-the-shelf availability, shipping and temporary storage of fabricated bio-constructs.
AUTHOR Petta, D. and Armiento, A. R. and Grijpma, D. and Alini, M. and Eglin, D. and D'Este, M.
Title 3D bioprinting of a hyaluronan bioink through enzymatic-and visible light-crosslinking [Abstract]
Year 2018
Journal/Proceedings Biofabrication
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Extrusion-based three-dimensional bioprinting relies on bioinks engineered to combine viscoelastic properties for extrusion and shape retention, and biological properties for cytocompatibility and tissue regeneration. To satisfy these conflicting requirements, bioinks often utilize either complex mixtures or complex modifications of biopolymers. In this paper we introduce and characterize a bioink exploiting a dual crosslinking mechanism, where an enzymatic reaction forms a soft gel suitable for cell encapsulation and extrusion, while a visible light photo-crosslinking allows shape retention of the printed construct. The influence of cell density and cell type on the rheological and printability properties was assessed correlating the printing outcomes with the damping factor, a rheological characteristic independent of the printing system. Stem cells, chondrocytes and fibroblasts were encapsulated, and their viability was assessed up to 14 days with live/dead, alamar blue and trypan blue assays. Additionally, the impact of the printing parameters on cell viability was investigated. Owing to its straightforward preparation, low modification, presence of two independent crosslinking mechanisms for tuning shear-thinning independently of the final shape fixation, the use of visible green instead of UV light, the possibility of encapsulating and sustaining the viability of different cell types, the hyaluronan bioink here presented is a valid biofabrication tool for producing 3D printed tissue-engineered constructs.
AUTHOR Aied, Ahmed and Song, Wenhui and Wang, Wenxin and Baki, Abdulrahman and Sigen, A.
Title 3D Bioprinting of stimuli-responsive polymers synthesised from DE-ATRP into soft tissue replicas [Abstract]
Year 2018
Journal/Proceedings Bioprinting
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Synthetic polymers possess more reproducible physical and chemical properties than their naturally occurring counterparts. They have also emerged as an important alternative for fabricating tissue substitutes because they can be molecularly tailored to have vast array of molecular weights, block structures, active functional groups, and mechanical properties. To this date however, there has been very few successful and fully functional synthetic tissue and organ substitutes and with the rapidly spreading 3D printing technology beginning to reshape the tissue engineering and regenerative field, the need for an effective, safe, and bio printable biomaterial is becoming more and more urgent. Here, we have developed a synthetic polymer from controlled living radical polymerisation that can be printed into well-defined structures. The polymer showed low cytotoxicity before and after printing. Additionally, the incorporation of gelatine-methacrylate coated PLGA microparticles within the hydrogel provided cell adhesion surfaces for cell proliferation. The results point to possible application of the microparticle seeded, synthetic hydrogel as a direct printable tissue or organ substitute.
AUTHOR Ng, Wei Long and Goh, Min Hao and Yeong, Wai Yee and Naing, May Win
Title Applying macromolecular crowding to 3D bioprinting: fabrication of 3D hierarchical porous collagen-based hydrogel constructs [Abstract]
Year 2018
Journal/Proceedings Biomaterials Science
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Native tissues and/or organs possess complex hierarchical porous structures that confer highly-specific cellular functions. Despite advances in fabrication processes{,} it is still very challenging to emulate the hierarchical porous collagen architecture found in most native tissues. Hence{,} the ability to recreate such hierarchical porous structures would result in biomimetic tissue-engineered constructs. Here{,} a single-step drop-on-demand (DOD) bioprinting strategy is proposed to fabricate hierarchical porous collagen-based hydrogels. Printable macromolecule-based bio-inks (polyvinylpyrrolidone{,} PVP) have been developed and printed in a DOD manner to manipulate the porosity within the multi-layered collagen-based hydrogels by altering the collagen fibrillogenesis process. The experimental results have indicated that hierarchical porous collagen structures could be achieved by controlling the number of macromolecule-based bio-ink droplets printed on each printed collagen layer. This facile single-step bioprinting process could be useful for the structural design of collagen-based hydrogels for various tissue engineering applications.
AUTHOR Suntornnond, R. and Tan, E. Y. S. and An, J. and Chua, C. K.
Title A highly printable and biocompatible hydrogel composite for direct printing of soft and perfusable vasculature-like structures [Abstract]
Year 2017
Journal/Proceedings Scientific Reports
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Vascularization is one major obstacle in bioprinting and tissue engineering. In order to create thick tissues or organs that can function like original body parts, the presence of a perfusable vascular system is essential. However, it is challenging to bioprint a hydrogel-based three-dimensional vasculature-like structure in a single step. In this paper, we report a new hydrogel-based composite that offers impressive printability, shape integrity, and biocompatibility for 3D bioprinting of a perfusable complex vasculature-like structure. The hydrogel composite can be used on a non-liquid platform and is printable at human body temperature. Moreover, the hydrogel composite supports both cell proliferation and cell differentiation. Our results represent a potentially new vascularization strategy for 3D bioprinting and tissue engineering.
AUTHOR Lorson, Thomas and Jaksch, Sebastian and Lübtow, Michael M. and Jüngst, Tomasz and Groll, Jürgen and Lühmann, Tessa and Luxenhofer, Robert
Title A Thermogelling Supramolecular Hydrogel with Sponge-Like Morphology as a Cytocompatible Bioink [Abstract]
Year 2017
Journal/Proceedings Biomacromolecules
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Biocompatible polymers that form thermoreversible supramolecular hydrogels have gained great interest in biomaterials research and tissue engineering. When favorable rheological properties are achieved at the same time, they are particularly promising candidates as material that allow for the printing of cells, so-called bioinks. We synthesized a novel thermogelling block copolymer and investigated the rheological properties of its aqueous solution by viscosimetry and rheology. The polymers undergo thermogelation between room temperature and body temperature, form transparent hydrogels of surprisingly high strength (G′ > 1000 Pa) and show rapid and complete shear recovery after stress. Small angle neutron scattering suggests an unusual bicontinuous sponge-like gel network. Excellent cytocompatibility was demonstrated with NIH 3T3 fibroblasts, which were incorporated and bioplotted into predefined 3D hydrogel structures without significant loss of viability. The developed materials fulfill all criteria for future use as bioink for biofabrication.
AUTHOR DeSimone, Elise and Schacht, Kristin and Pellert, Alexandra and Scheibel, Thomas
Title Recombinant spider silk-based bioinks [Abstract]
Year 2017
Journal/Proceedings Biofabrication
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Bioinks, 3D cell culture systems which can be printed, are still in the early development stages. Currently, extensive research is going into designing printers to be more accommodating to bioinks, designing scaffolds with stiff materials as support structures for the often soft bioinks, and modifying the bioinks themselves. Recombinant spider silk proteins, a potential biomaterial component for bioinks, have high biocompatibility, can be processed into several morphologies and can be modified with cell adhesion motifs to enhance their bioactivity. In this work, thermally gelled hydrogels made from recombinant spider silk protein encapsulating mouse fibroblast cell line BALB/3T3 were prepared and characterized. The bioinks were evaluated for performance in vitro both before and after printing, and it was observed that unprinted bioinks provided a good platform for cell spreading and proliferation, while proliferation in printed scaffolds was prohibited. To improve the properties of the printed hydrogels, gelatin was given as an additive and thereby served indirectly as a plasticizer, improving the resolution of printed strands. Taken together, recombinant spider silk proteins and hydrogels made thereof show good potential as a bioink, warranting further development.
AUTHOR Hou, Xiaochun and Liu, Shiying and Wang, Min and Wiraja, Christian and Huang, Wei and Chan, Peggy and Tan, Timothy and Xu, Chenjie
Title Layer-by-Layer 3D Constructs of Fibroblasts in Hydrogel for Examining Transdermal Penetration Capability of Nanoparticles [Abstract]
Year 2016
Journal/Proceedings Journal of Laboratory Automation
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Nanoparticles are emerging transdermal delivery systems. Their size and surface properties determine their efficacy and efficiency to penetrate through the skin layers. This work utilizes three-dimensional (3D) bioprinting technology to generate a simplified artificial skin model to rapidly screen nanoparticles for their transdermal penetration ability. Specifically, this model is built through layer-by-layer alternate printing of blank collagen hydrogel and fibroblasts. Through controlling valve on-time, the spacing between printing lines could be accurately tuned, which could enable modulation of cell infiltration in the future. To confirm the effectiveness of this platform, a 3D construct with one layer of fibroblasts sandwiched between two layers of collagen hydrogel is used to screen silica nanoparticles with different surface charges for their penetration ability, with positively charged nanoparticles demonstrating deeper penetration, consistent with the observation from an existing study involving living skin tissue.
AUTHOR Ng, Wei Long and Yeong, Wai Yee and Naing, May Win
Title Polyelectrolyte gelatin-chitosan hydrogel optimized for 3D bioprinting in skin tissue engineering [Abstract]
Year 2016
Journal/Proceedings International Journal of Bioprinting
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Bioprinting is a promising automated platform that enables the simultaneous deposition of multiple types of cells and biomaterials to fabricate complex three-dimensional (3D) tissue constructs. Most of the previous bioprinting works focused on collagen-based biomaterial, which has poor printability and long crosslinking time. This posed a immerse challenge to create a 3D construct with pre-determined shape and configuration. There is a need for a functional material with good printability in order to fabricate a 3D skin construct. Recently, the use of chitosan for wound healing applications has attracted huge attention due to its attractive traits such as its antimicrobial properties and ability to trigger hemostasis. In this paper, we report the modification of chitosan-based biomaterials for functional 3D bioprinting. Modification to the chitosan was carried out via the oppositely charged functional groups from chitosan and gelatin at a specific pH of ~pH 6.5 to form polyelectrolyte complexes. The polyelectrolyte hydrogels were evaluated in terms of chemical interactions within polymer blend, rheological properties (viscosities, storage and loss modulus), printing resolution at varying pressures and feed rates and biocompatibility. The chitosan-based hydrogels formulated in this work exhibited good printability at room temperature, high shape fidelity of the printed 3D constructs and good biocompatibility with fibroblast skin cells.
AUTHOR Schacht, Kristin and J{"{u}}ngst, Tomasz and Schweinlin, Matthias and Ewald, Andrea and Groll, J{"{u}}rgen and Scheibel, Thomas
Title Biofabrication of Cell-Loaded 3D Spider Silk Constructs [Abstract]
Year 2015
Journal/Proceedings Angewandte Chemie International Edition
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Biofabrication is an emerging and rapidly expanding field of research in which additive manufacturing techniques in combination with cell printing are exploited to generate hierarchical tissue-like structures. Materials that combine printability with cytocompatibility, so called bioinks, are currently the biggest bottleneck. Since recombinant spider silk proteins are non-immunogenic, cytocompatible, and exhibit physical crosslinking, their potential as a new bioink system was evaluated. Cell-loaded spider silk constructs can be printed by robotic dispensing without the need for crosslinking additives or thickeners for mechanical stabilization. Cells are able to adhere and proliferate with good viability over at least one week in such spider silk scaffolds. Introduction of a cell-binding motif to the spider silk protein further enables fine-tuned control over cell–material interactions. Spider silk hydrogels are thus a highly attractive novel bioink for biofabrication.
AUTHOR Rimann, Markus and Bono, Epifania and Annaheim, Helene and Bleisch, Matthias and Graf-Hausner, Ursula
Title Standardized 3D Bioprinting of Soft Tissue Models with Human Primary Cells. [Abstract]
Year 2015
Journal/Proceedings Journal of laboratory automation
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Cells grown in 3D are more physiologically relevant than cells cultured in 2D. To use 3D models in substance testing and regenerative medicine, reproducibility and standardization are important. Bioprinting offers not only automated standardizable processes but also the production of complex tissue-like structures in an additive manner. We developed an all-in-one bioprinting solution to produce soft tissue models. The holistic approach included (1) a bioprinter in a sterile environment, (2) a light-induced bioink polymerization unit, (3) a user-friendly software, (4) the capability to print in standard labware for high-throughput screening, (5) cell-compatible inkjet-based printheads, (6) a cell-compatible ready-to-use BioInk, and (7) standard operating procedures. In a proof-of-concept study, skin as a reference soft tissue model was printed. To produce dermal equivalents, primary human dermal fibroblasts were printed in alternating layers with BioInk and cultured for up to 7 weeks. During long-term cultures, the models were remodeled and fully populated with viable and spreaded fibroblasts. Primary human dermal keratinocytes were seeded on top of dermal equivalents, and epidermis-like structures were formed as verified with hematoxylin and eosin staining and immunostaining. However, a fully stratified epidermis was not achieved. Nevertheless, this is one of the first reports of an integrative bioprinting strategy for industrial routine application.