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AUTHOR Asulin, Masha and Michael, Idan and Shapira, Assaf and Dvir, Tal
Title One-Step 3D Printing of Heart Patches with Built-In Electronics for Performance Regulation [Abstract]
Year 2021
Journal/Proceedings Advanced Science
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Abstract Three dimensional (3D) printing of heart patches usually provides the ability to precisely control cell location in 3D space. Here, one-step 3D printing of cardiac patches with built-in soft and stretchable electronics is reported. The tissue is simultaneously printed using three distinct bioinks for the cells, for the conducting parts of the electronics and for the dielectric components. It is shown that the hybrid system can withstand continuous physical deformations as those taking place in the contracting myocardium. The electronic patch is flexible, stretchable, and soft, and the electrodes within the printed patch are able to monitor the function of the engineered tissue by providing extracellular potentials. Furthermore, the system allowed controlling tissue function by providing electrical stimulation for pacing. It is envisioned that such transplantable patches may regain heart contractility and allow the physician to monitor the implant function as well as to efficiently intervene from afar when needed.
AUTHOR Gonzalez-Fernandez, T. and Rathan, S. and Hobbs, C. and Pitacco, P. and Freeman, F. E. and Cunniffe, G. M. and Dunne, N. J. and McCarthy, H. O. and Nicolosi, V. and O'Brien, F. J. and Kelly, D. J.
Title Pore-forming bioinks to enable Spatio-temporally defined gene delivery in bioprinted tissues [Abstract]
Year 2019
Journal/Proceedings Journal of Controlled Release
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The regeneration of complex tissues and organs remains a major clinical challenge. With a view towards bioprinting such tissues, we developed a new class of pore-forming bioink to spatially and temporally control the presentation of therapeutic genes within bioprinted tissues. By blending sacrificial and stable hydrogels, we were able to produce bioinks whose porosity increased with time following printing. When combined with amphipathic peptide-based plasmid DNA delivery, these bioinks supported enhanced non-viral gene transfer to stem cells in vitro. By modulating the porosity of these bioinks, it was possible to direct either rapid and transient (pore-forming bioinks), or slower and more sustained (solid bioinks) transfection of host or transplanted cells in vivo. To demonstrate the utility of these bioinks for the bioprinting of spatially complex tissues, they were next used to zonally position stem cells and plasmids encoding for either osteogenic (BMP2) or chondrogenic (combination of TGF-β3, BMP2 and SOX9) genes within networks of 3D printed thermoplastic fibers to produce mechanically reinforced, gene activated constructs. In vivo, these bioprinted tissues supported the development of a vascularised, bony tissue overlaid by a layer of stable cartilage. When combined with multiple-tool biofabrication strategies, these gene activated bioinks can enable the bioprinting of a wide range of spatially complex tissues.
AUTHOR Cunniffe, Gráinne and Gonzalez-Fernandez, Tomas and Daly, Andrew and Nelson Sathy, Binulal and Jeon, Oju and Alsberg, Eben and J. Kelly, Daniel
Title Three-Dimensional Bioprinting of Polycaprolactone Reinforced Gene Activated Bioinks for Bone Tissue Engineering [Abstract]
Year 2017
Journal/Proceedings Tissue Engineering Part A
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Regeneration of complex bone defects remains a significant clinical challenge. Multi-tool biofabrication has permitted the combination of various biomaterials to create multifaceted composites with tailorable mechanical properties and spatially controlled biological function. In this study we sought to use bioprinting to engineer nonviral gene activated constructs reinforced by polymeric micro-filaments. A gene activated bioink was developed using RGD-g-irradiated alginate and nano-hydroxyapatite (nHA) complexed to plasmid DNA (pDNA). This ink was combined with bonemarrow-derived mesenchymal stemcells (MSCs) and then co-printed with a polycaprolactone supporting mesh to provide mechanical stability to the construct. Reporter genes were first used to demonstrate successful cell transfection using this system, with sustained expression of the transgene detected over 14 days postbioprinting. Delivery of a combination of therapeutic genes encoding for bone morphogenic protein and transforming growth factor promoted robust osteogenesis of encapsulated MSCs in vitro, with enhanced levels of matrix deposition and mineralization observed following the incorporation of therapeutic pDNA. Gene activated MSC-laden constructs were then implanted subcutaneously, directly postfabrication, and were found to support superior levels of vascularization andmineralization compared to cell-free controls. These results validate the use of a gene activated bioink to impart biological functionality to three-dimensional bioprinted constructs.
AUTHOR Freeman, Fiona E. and Pitacco, Pierluca and van Dommelen, Lieke H. A. and Nulty, Jessica and Browe, David C. and Shin, Jung-Youn and Alsberg, Eben and Kelly, Daniel J.
Title 3D bioprinting spatiotemporally defined patterns of growth factors to tightly control tissue regeneration [Abstract]
Year 2020
Journal/Proceedings Science Advances
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Therapeutic growth factor delivery typically requires supraphysiological dosages, which can cause undesirable off-target effects. The aim of this study was to 3D bioprint implants containing spatiotemporally defined patterns of growth factors optimized for coupled angiogenesis and osteogenesis. Using nanoparticle functionalized bioinks, it was possible to print implants with distinct growth factor patterns and release profiles spanning from days to weeks. The extent of angiogenesis in vivo depended on the spatial presentation of vascular endothelial growth factor (VEGF). Higher levels of vessel invasion were observed in implants containing a spatial gradient of VEGF compared to those homogenously loaded with the same total amount of protein. Printed implants containing a gradient of VEGF, coupled with spatially defined BMP-2 localization and release kinetics, accelerated large bone defect healing with little heterotopic bone formation. This demonstrates the potential of growth factor printing, a putative point of care therapy, for tightly controlled tissue regeneration.
AUTHOR Fisch, Philipp and Broguiere, Nicolas and Finkielsztein, Sergio and Linder, Thomas and Zenobi-Wong, Marcy
Title Bioprinting of Cartilaginous Auricular Constructs Utilizing an Enzymatically Crosslinkable Bioink [Abstract]
Year 2021
Journal/Proceedings Advanced Functional Materials
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Abstract Bioprinting of functional tissues could overcome tissue shortages and allow a more rapid response for treatments. However, despite recent progress in bioprinting, and its outstanding ability to position cells and biomaterials in a precise 3D manner, its success has been limited, due to insufficient maturation of constructs into functional tissue. Here, a novel calcium-triggered enzymatic crosslinking (CTEC) mechanism for bioinks based on the activation cascade of Factor XIII is presented and utilized for the biofabrication of cartilaginous constructs. Hyaluronan transglutaminase (HA-TG), an enzymatically crosslinkable material, has shown excellent characteristics for chondrogenesis and builds the basis of the CTEC bioink. The bioink supports tissue maturation with neocartilage formation and stiffening of constructs up to 400 kPa. Bioprinted constructs remain stable in vivo for 24 weeks and bioprinted auricular constructs transform into cartilaginous grafts. A major limitation of the current study is the deposition of collagen I, indicating the maturation toward fibrocartilage rather than elastic cartilage. Shifting the maturation process toward elastic cartilage will therefore be essential in order for the developed bioinks to offer a novel tissue engineered treatment for microtia patients. CTEC bioprinting furthermore opens up use of enzymatically crosslinkable biopolymers and their modularity to support a multitude of tissues.
AUTHOR Li, Zuxi and Zhang, Xiao and Yuan, Tao and Zhang, Yi and Luo, Chunyang and Zhang, Jiyong and Liu, Yang and Fan, Weimin
Title Addition of Platelet-Rich Plasma to Silk Fibroin Hydrogel Bioprinting for Cartilage Regeneration [Abstract]
Year 2020
Journal/Proceedings Tissue Engineering Part A
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The recent advent of 3D bioprinting of biopolymers provides a novel method for fabrication of tissue-engineered scaffolds and also offers a potentially promising avenue in cartilage regeneration. Silk fibroin (SF) is one of the most popular biopolymers used for 3D bioprinting, but further application of SF is hindered by its limited biological activities. Incorporation of growth factors (GFs) has been identified as a solution to improve biological function. Platelet-rich plasma (PRP) is an autologous resource of GFs, which has been widely used in clinic. In this study, we have developed SF-based bioinks incorporated with different concentrations of PRP (12.5%, 25%, and 50%; vol/vol). Release kinetic studies show that SF-PRP bioinks could achieve controlled release of GFs. Subsequently, SF-PRP bioinks were successfully fabricated into scaffolds by bioprinting. Our results revealed that SF-PRP scaffolds possessed proper internal pore structure, good biomechanical properties, and a suitable degradation rate for cartilage regeneration. Live/dead staining showed that 3D, printed SF-PRP scaffolds were biocompatible. Moreover, in vitro studies revealed that tissue-engineered cartilage from the SF-PRP group exhibited improved qualities compared with the pure SF controls, according to histological and immunohistochemical findings. Biochemical evaluations confirmed that SF-PRP (50% PRP, v/v) scaffolds allowed the largest increases in collagen and glycosaminoglycan concentrations, when compared with the pure SF group. These findings suggest that 3D, printed SF-PRP scaffolds could be potential candidates for cartilage tissue engineering.
AUTHOR Kamdem Tamo, Arnaud and Doench, Ingo and Morales Helguera, Aliuska and Hoenders, Daniel and Walther, Andreas and Madrazo, Anayancy Osorio
Title Biodegradation of Crystalline Cellulose Nanofibers by Means of Enzyme Immobilized-Alginate Beads and Microparticles [Abstract]
Year 2020
Journal/Proceedings Polymers
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Recent advances in nanocellulose technology have revealed the potential of crystalline cellulose nanofibers to reinforce materials which are useful for tissue engineering, among other functions. However, the low biodegradability of nanocellulose can possess some problems in biomedical applications. In this work, alginate particles with encapsulated enzyme cellulase extracted from Trichoderma reesei were prepared for the biodegradation of crystalline cellulose nanofibers, which carrier system could be incorporated in tissue engineering biomaterials to degrade the crystalline cellulose nanoreinforcement in situ and on-demand during tissue regeneration. Both alginate beads and microparticles were processed by extrusion-dropping and inkjet-based methods, respectively. Processing parameters like the alginate concentration, concentration of ionic crosslinker Ca2+, hardening time, and ionic strength of the medium were varied. The hydrolytic activity of the free and encapsulated enzyme was evaluated for unmodified (CNFs) and TEMPO-oxidized cellulose nanofibers (TOCNFs) in suspension (heterogeneous conditions); in comparison to solubilized cellulose derivatives (homogeneous conditions). The enzymatic activity was evaluated for temperatures between 25–75 °C, pH range from 3.5 to 8.0 and incubation times until 21 d. Encapsulated cellulase in general displayed higher activity compared to the free enzyme over wider temperature and pH ranges and for longer incubation times. A statistical design allowed optimizing the processing parameters for the preparation of enzyme-encapsulated alginate particles presenting the highest enzymatic activity and sphericity. The statistical analysis yielded the optimum particles characteristics and properties by using a formulation of 2% (w/v) alginate, a coagulation bath of 0.2 M CaCl2 and a hardening time of 1 h. In homogeneous conditions the highest catalytic activity was obtained at 55 °C and pH 4.8. These temperature and pH values were considered to study the biodegradation of the crystalline cellulose nanofibers in suspension. The encapsulated cellulase preserved its activity for several weeks over that of the free enzyme, which latter considerably decreased and practically showed deactivation after just 10 d. The alginate microparticles with their high surface area-to-volume ratio effectively allowed the controlled release of the encapsulated enzyme and thereby the sustained hydrolysis of the cellulose nanofibers. The relative activity of cellulase encapsulated in the microparticles leveled-off at around 60% after one day and practically remained at that value for three weeks.
AUTHOR Steier, Anke and Schmieg, Barbara and Irtel von Brenndorff, Yannic and Meier, Manuel and Nirschl, Hermann and Franzreb, Matthias and Lahann, Joerg
Title Enzyme Scaffolds with Hierarchically Defined Properties via 3D Jet Writing [Abstract]
Year 2020
Journal/Proceedings Macromolecular Bioscience
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Abstract The immobilization of enzymes into polymer hydrogels is a versatile approach to improve their stability and utility in biotechnological and biomedical applications. However, these systems typically show limited enzyme activity, due to unfavorable pore dimensions and low enzyme accessibility. Here, 3D jet writing of water-based bioinks, which contain preloaded enzymes, is used to prepare hydrogel scaffolds with well-defined, tessellated micropores. After 3D jet writing, the scaffolds are chemically modified via photopolymerization to ensure mechanical stability. Enzyme loading and activity in the hydrogel scaffolds is fully retained over 3 d. Important structural parameters of the scaffolds such as pore size, pore geometry, and wall diameter are controlled with micrometer resolution to avoid mass-transport limitations. It is demonstrated that scaffold pore sizes between 120 µm and 1 mm can be created by 3D jet writing approaching the length scales of free diffusion in the hydrogels substrates and resulting in high levels of enzyme activity (21.2% activity relative to free enzyme). With further work, a broad range of applications for enzyme-laden hydrogel scaffolds including diagnostics and enzymatic cascade reactions is anticipated.
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 Abu Awwad, Hosam Al-Deen M. and Thiagarajan, Lalitha and Kanczler, Janos M. and Amer, Mahetab H. and Bruce, Gordon and Lanham, Stuart and Rumney, Robin M. H. and Oreffo, Richard O. C. and Dixon, James E.
Title Genetically-programmed, mesenchymal stromal cell-laden & mechanically strong 3D bioprinted scaffolds for bone repair [Abstract]
Year 2020
Journal/Proceedings Journal of Controlled Release
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Additive manufacturing processes used to create regenerative bone tissue engineered implants are not biocompatible, thereby restricting direct use with stem cells and usually require cell seeding post-fabrication. Combined delivery of stem cells with the controlled release of osteogenic factors, within a mechanically-strong biomaterial combined during manufacturing would replace injectable defect fillers (cements) and allow personalized implants to be rapidly prototyped by 3D bioprinting. Through the use of direct genetic programming via the sustained release of an exogenously delivered transcription factor RUNX2 (delivered as recombinant GET-RUNX2 protein) encapsulated in PLGA microparticles (MPs), we demonstrate that human mesenchymal stromal (stem) cells (hMSCs) can be directly fabricated into a thermo-sintered 3D bioprintable material and achieve effective osteogenic differentiation. Importantly we observed osteogenic programming of gene expression by released GET-RUNX2 (8.2-, 3.3- and 3.9-fold increases in OSX, RUNX2 and OPN expression, respectively) and calcification (von Kossa staining) in our scaffolds. The developed biodegradable PLGA/PEG paste formulation augments high-density bone development in a defect model (~2.4-fold increase in high density bone volume) and can be used to rapidly prototype clinically-sized hMSC-laden implants within minutes using mild, cytocompatible extrusion bioprinting. The ability to create mechanically strong 'cancellous bone-like’ printable implants for tissue repair that contain stem cells and controlled-release of programming factors is innovative, and will facilitate the development of novel localized delivery approaches to direct cellular behaviour for many regenerative medicine applications including those for personalized bone repair.
AUTHOR Schmieg, Barbara and Schimek, Adrian and Franzreb, Matthias
Title Development and performance of a 3D‐printable Polyethylenglycol‐Diacrylate hydrogel suitable for enzyme entrapment and long‐term biocatalytic applications [Abstract]
Year 2018
Journal/Proceedings Engineering in Life Sciences
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Physical entrapment of enzymes within a porous matrix is a fast and gentle process to immobilize biocatalysts to enable their recycling and long‐term use. This study introduces the development of a biocompatible 3D‐printing material suitable for enzyme entrapment, while having good rheological and UV‐hardening properties. Three different viscosity‐enhancing additives have been tested in combination with a polyethylenglycol‐diacrylate‐based hydrogel system. The addition of polyxanthan or hectorite clay particles results in hydrogels that degrade over hours or days, releasing entrapped compounds. In contrast, the addition of nanometer‐sized silicate particles ensures processability while preventing disintegration of the hydrogel. Lattice structures with a total height of 6 mm consisting of 40 layers were 3D‐printed with all materials and characterized by image analysis. Rheological measurements identified a shear stress window of 200 < τ < 500 Pa at shear rates of 25 s−1 and 25°C for well‐defined geometries with an extrusion‐based printhead. Enzymes immobilized in these long‐term stable hydrogel structures retained an effective activity of approximately 10% compared to the free enzyme in solution. It could be shown that the reduction of effective activity isn't caused by a significant reduction of the intrinsic enzyme activity but by mass transfer limitations within the printed hydrogel structures. This article is protected by copyright. All rights reserved
AUTHOR Kumari, Sushma and Bargel, Hendrik and Anby, Mette U. and Lafargue, David and Scheibel, Thomas
Title Recombinant Spider Silk Hydrogels for Sustained Release of Biologicals [Abstract]
Year 2018
Journal/Proceedings ACS Biomaterials Science and Engineering
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Therapeutic biologics (i.e., proteins) have been widely recognized for the treatment, prevention, and cure of a variety of human diseases and syndromes. However, design of novel protein-delivery systems to achieve a nontoxic, constant, and efficient delivery with minimal doses of therapeutic biologics is still challenging. Here, recombinant spider silk-based materials are employed as a delivery system for the administration of therapeutic biologicals. Hydrogels made of the recombinant spider silk protein eADF4(C16) were used to encapsulate the model biologicals BSA, HRP, and LYS by direct loading or through diffusion, and their release was studied. Release of model biologicals from eADF4(C16) hydrogels is in part dependent on the electrostatic interaction between the biological and the recombinant spider silk protein variant used. In addition, tailoring the pore sizes of eADF4(C16) hydrogels strongly influenced the release kinetics. In a second approach, a particles-in-hydrogel system was used, showing a prolonged release in comparison with that of plain hydrogels (from days to week). The particle-enforced spider silk hydrogels are injectable and can be 3D printed. These initial studies indicate the potential of recombinant spider silk proteins to design novel injectable hydrogels that are suitable for delivering therapeutic biologics.