REGENHU-Switzerland-3d-bioprinting-instrument-bio-3d-bioprinter-DevelopmentTeam-0006

SCIENTIFIC PUBLICATIONS

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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 Madiedo-Podvrsan, Sabrina and Belaïdi, Jean-Philippe and Desbouis, Stephanie and Simonetti, Lucie and Ben-Khalifa, Youcef and Soeur, Jérémie and Rielland, Maïté
Title Utilization of patterned bioprinting for heterogeneous and physiologically representative reconstructed epidermal skin models [Abstract]
Year 2021
Journal/Proceedings Scientific Reports
Reftype Madiedo-Podvrsan2021
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Abstract
Organotypic skin tissue models have decades of use for basic research applications, the treatment of burns, and for efficacy/safety evaluation studies. The complex and heterogeneous nature of native human skin however creates difficulties for the construction of physiologically comparable organotypic models. Within the present study, we utilized bioprinting technology for the controlled deposition of separate keratinocyte subpopulations to create a reconstructed epidermis with two distinct halves in a single insert, each comprised of a different keratinocyte sub-population, in order to better model heterogonous skin and reduce inter-sample variability. As an initial proof-of-concept, we created a patterned epidermal skin model using GPF positive and negative keratinocyte subpopulations, both printed into 2 halves of a reconstructed skin insert, demonstrating the feasibility of this approach. We then demonstrated the physiological relevance of this bioprinting technique by generating a heterogeneous model comprised of dual keratinocyte population with either normal or low filaggrin expression. The resultant model exhibited a well-organized epidermal structure with each half possessing the phenotypic characteristics of its constituent cells, indicative of a successful and stable tissue reconstruction. This patterned skin model aims to mimic the edge of lesions as seen in atopic dermatitis or ichthyosis vulgaris, while the use of two populations within a single insert allows for paired statistics in evaluation studies, likely increasing study statistical power and reducing the number of models required per study. This is the first report of human patterned epidermal model using a predefined bioprinted designs, and demonstrates the relevance of bioprinting to faithfully reproduce human skin microanatomy.
AUTHOR Monferrer, Ezequiel and Martín-Vañó, Susana and Carretero, Aitor and García-Lizarribar, Andrea and Burgos-Panadero, Rebeca and Navarro, Samuel and Samitier, Josep and Noguera, Rosa
Title A three-dimensional bioprinted model to evaluate the effect of stiffness on neuroblastoma cell cluster dynamics and behavior [Abstract]
Year 2020
Journal/Proceedings Scientific Reports
Reftype Monferrer2020
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Abstract
Three-dimensional (3D) bioprinted culture systems allow to accurately control microenvironment components and analyze their effects at cellular and tissue levels. The main objective of this study was to identify, quantify and localize the effects of physical-chemical communication signals between tumor cells and the surrounding biomaterial stiffness over time, defining how aggressiveness increases in SK-N-BE(2) neuroblastoma (NB) cell line. Biomimetic hydrogels with SK-N-BE(2) cells, methacrylated gelatin and increasing concentrations of methacrylated alginate (AlgMA 0%, 1% and 2%) were used. Young’s modulus was used to define the stiffness of bioprinted hydrogels and NB tumors. Stained sections of paraffin-embedded hydrogels were digitally quantified. Human NB and 1% AlgMA hydrogels presented similar Young´s modulus mean, and orthotopic NB mice tumors were equally similar to 0% and 1% AlgMA hydrogels. Porosity increased over time; cell cluster density decreased over time and with stiffness, and cell cluster occupancy generally increased with time and decreased with stiffness. In addition, cell proliferation, mRNA metabolism and antiapoptotic activity advanced over time and with stiffness. Together, this rheological, optical and digital data show the potential of the 3D in vitro cell model described herein to infer how intercellular space stiffness patterns drive the clinical behavior associated with NB patients.
AUTHOR Benmeridja, Lara and De Moor, Lise and De Maere, Elisabeth and Vanlauwe, Florian and Ryx, Michelle and Tytgat, Liesbeth and Vercruysse, Chris and Dubruel, Peter and Van Vlierberghe, Sandra and Blondeel, Phillip and Declercq, Heidi
Title High-throughput fabrication of vascularized adipose microtissues for 3D bioprinting [Abstract]
Year 2020
Journal/Proceedings Journal of Tissue Engineering and Regenerative Medicine
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Abstract For patients with soft tissue defects, repair with autologous in vitro engineered adipose tissue could be a promising alternative to current surgical therapies. A volume-persistent engineered adipose tissue construct under in vivo conditions can only be achieved by early vascularization after transplantation. The combination of 3D bioprinting technology with self-assembling microvascularized units as building blocks can potentially answer the need for a microvascular network. In the present study, co-culture spheroids combining adipose-derived stem cells (ASC) and human umbilical vein endothelial cells (HUVEC) were created with an ideal geometry for bioprinting. When applying the favourable seeding technique and condition, compact viable spheroids were obtained, demonstrating high adipogenic differentiation and capillary-like network formation after 7 and 14 days of culture, as shown by live/dead analysis, immunohistochemistry and RT-qPCR. Moreover, we were able to successfully 3D bioprint the encapsulated spheroids, resulting in compact viable spheroids presenting capillary-like structures, lipid droplets and spheroid outgrowth after 14 days of culture. This is the first study that generates viable high-throughput (pre-)vascularized adipose microtissues as building blocks for bioprinting applications using a novel ASC/HUVEC co-culture spheroid model, which enables both adipogenic differentiation while simultaneously supporting the formation of prevascular-like structures within engineered tissues in vitro.
AUTHOR Dubey, Nileshkumar and Ferreira, Jessica A. and Daghrery, Arwa and Aytac, Zeynep and Malda, Jos and Bhaduri, Sarit B. and Bottino, Marco C.
Title Highly Tunable Bioactive Fiber-Reinforced Hydrogel for Guided Bone Regeneration [Abstract]
Year 2020
Journal/Proceedings Acta Biomaterialia
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One of the most damaging pathologies that affects the health of both soft and hard tissues around the tooth is periodontitis. Clinically, periodontal tissue destruction has been managed by an integrated approach involving elimination of injured tissues followed by regenerative strategies with bone substitutes and/or barrier membranes. Regrettably, a barrier membrane with predictable mechanical integrity and multifunctional therapeutic features has yet to be established. Herein, we report a fiber-reinforced hydrogel with unprecedented tunability in terms of mechanical competence and therapeutic features by integration of highly porous poly(ε-caprolactone) fibrous mesh(es) with well-controlled 3D architecture into bioactive amorphous magnesium phosphate-laden gelatin methacryloyl hydrogels. The presence of amorphous magnesium phosphate and PCL mesh in the hydrogel can control the mechanical properties and improve the osteogenic ability, opening a tremendous opportunity in guided bone regeneration (GBR). Results demonstrate that the presence of PCL meshes fabricated via melt electrowriting can delay hydrogel degradation preventing soft tissue invasion and providing the mechanical barrier to allow time for slower migrating progenitor cells to participate in bone regeneration due to their ability to differentiate into bone-forming cells. Altogether, our approach offers a platform technology for the development of the next-generation of GBR membranes with tunable mechanical and therapeutic properties to amplify bone regeneration in compromised sites.
AUTHOR Peiffer, Quentin C. and de Ruijter, Mylène and van Duijn, Joost and Crottet, Denis and Dominic, Ernst and Malda, Jos and Castilho, Miguel
Title Melt electrowriting onto anatomically relevant biodegradable substrates: Resurfacing a diarthrodial joint [Abstract]
Year 2020
Journal/Proceedings Materials & Design
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Three-dimensional printed hydrogel constructs with well-organized melt electrowritten (MEW) fibre-reinforcing scaffolds have been demonstrated as a promising regenerative approach to treat small cartilage defects. Here, we investige how to translate the fabrication of small fibre-reinforced structures on flat surfaces to anatomically relevant structures. In particular, the accurate deposition of MEW-fibres onto curved surfaces of conductive and non-conductive regenerative biomaterials is studied. This study reveals that clinically relevant materials with low conductivities are compatible with resurfacing with organized MEW fibres. Importantly, accurate patterning on non-flat surfaces was successfully shown, provided that a constant electrical field strength and an electrical force normal to the substrate material is maintained. Furthermore, the application of resurfacing the geometry of the medial human femoral condyle is confirmed by the fabrication of a personalised osteochondral implant. The implant composed of an articular cartilage-resident chondroprogenitor cells (ACPCs)-laden hydrogel reinforced with a well-organized MEW scaffold retained its personalised shape, improved its compressive properties and supported neocartilage formation after 28 days in vitro culture. Overall, this study establishes the groundwork for translating MEW from planar and non-resorbable material substrates to anatomically relevant geometries and regenerative materials that the regenerative medicine field aims to create.
AUTHOR Afanasenkau, Dzmitry and Kalinina, Daria and Lyakhovetskii, Vsevolod and Tondera, Christoph and Gorsky, Oleg and Moosavi, Seyyed and Pavlova, Natalia and Merkulyeva, Natalia and Kalueff, Allan V. and Minev, Ivan R. and Musienko, Pavel
Title Rapid prototyping of soft bioelectronic implants for use as neuromuscular interfaces [Abstract]
Year 2020
Journal/Proceedings Nature Biomedical Engineering
Reftype Afanasenkau2020
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Abstract
Neuromuscular interfaces are required to translate bioelectronic technologies for application in clinical medicine. Here, by leveraging the robotically controlled ink-jet deposition of low-viscosity conductive inks, extrusion of insulating silicone pastes and in situ activation of electrode surfaces via cold-air plasma, we show that soft biocompatible materials can be rapidly printed for the on-demand prototyping of customized electrode arrays well adjusted to specific anatomical environments, functions and experimental models. We also show, with the monitoring and activation of neuronal pathways in the brain, spinal cord and neuromuscular system of cats, rats and zebrafish, that the printed bioelectronic interfaces allow for long-term integration and functional stability. This technology might enable personalized bioelectronics for neuroprosthetic applications.
AUTHOR Fanous, Marina and Gold, Sarah and Muller, Silvain and Hirsch, Stefan and Ogorka, Joerg and Imanidis, Georgios
Title Simplification of fused deposition modeling 3D-printing paradigm: Feasibility of 1-step direct powder printing for immediate release dosage form production [Abstract]
Year 2020
Journal/Proceedings International Journal of Pharmaceutics
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Direct powder three-dimensional (3D)-printing (DPP) of tablets to simplify fused deposition modelling (FDM) was explored. The FDM paradigm involving hot-melt extrusion for making 3D-printable drug-loaded filaments as intermediate products for tablet manufacturing has been gaining attention for the decentralized on-site production of personalized dosage forms. For direct 3D-printing, powder blends were loaded into a cartridge-like head and were successfully printed with honeycomb design following heating of the extrusion cartridge. This 1-step DPP with incorporation of in-built porosity providing higher surface area served as proof of concept for manufacture of rapid release dosage forms. Water soluble hydroxypropylcellulose SSL was chosen as matrix former and caffeine as model drug. The effect of PEG4000 as plasticizer/pore former and Kollidon VA64 as rapidly dissolving polymer on DPP processability and dissolution rate was investigated. Directly 3D-printed tablets with low (30%) infill density showed rapid dissolution independently of the formulation, whereas for high (80%) infill density a combination of PEG4000 and Kollidon VA64 was required to achieve rapid release. The obtained tablets demonstrated good uniformity of percent drug content but had variable weight. Caffeine was present in crystalline state and in the stable polymorph in the tablets. Hence, DPP feasibility for immediate release dosage form manufacture was demonstrated. This technique might create an opportunity to avoid hot-melt extrusion allowing 3D-printing independently of mechanical properties of a filament and potentially prolonging product shelf life by reducing thermal stress.
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 Noor, Nadav and Shapira, Assaf and Edri, Reuven and Gal, Idan and Wertheim, Lior and Dvir, Tal
Title 3D Printing of Personalized Thick and Perfusable Cardiac Patches and Hearts [Abstract]
Year 2019
Journal/Proceedings Advanced Science
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Abstract Generation of thick vascularized tissues that fully match the patient still remains an unmet challenge in cardiac tissue engineering. Here, a simple approach to 3D-print thick, vascularized, and perfusable cardiac patches that completely match the immunological, cellular, biochemical, and anatomical properties of the patient is reported. To this end, a biopsy of an omental tissue is taken from patients. While the cells are reprogrammed to become pluripotent stem cells, and differentiated to cardiomyocytes and endothelial cells, the extracellular matrix is processed into a personalized hydrogel. Following, the two cell types are separately combined with hydrogels to form bioinks for the parenchymal cardiac tissue and blood vessels. The ability to print functional vascularized patches according to the patient's anatomy is demonstrated. Blood vessel architecture is further improved by mathematical modeling of oxygen transfer. The structure and function of the patches are studied in vitro, and cardiac cell morphology is assessed after transplantation, revealing elongated cardiomyocytes with massive actinin striation. Finally, as a proof of concept, cellularized human hearts with a natural architecture are printed. These results demonstrate the potential of the approach for engineering personalized tissues and organs, or for drug screening in an appropriate anatomical structure and patient-specific biochemical microenvironment.
AUTHOR Kleger, Nicole and Cihova, Martina and Masania, Kunal and Studart, André R. and Löffler, Jörg F.
Title 3d printing of salt as a template for magnesium with structured porosity [Abstract]
Year 2019
Journal/Proceedings advanced materials
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Abstract Porosity is an essential feature in a wide range of applications that combine light weight with high surface area and tunable density. Porous materials can be easily prepared with a vast variety of chemistries using the salt-leaching technique. However, this templating approach has so far been limited to the fabrication of structures with random porosity and relatively simple macroscopic shapes. Here, a technique is reported that combines the ease of salt leaching with the complex shaping possibilities given by additive manufacturing (AM). By tuning the composition of surfactant and solvent, the salt-based paste is rheologically engineered and printed via direct ink writing into grid-like structures displaying structured pores that span from the sub-millimeter to the macroscopic scale. As a proof of concept, dried and sintered NaCl templates are infiltrated with magnesium (Mg), which is typically highly challenging to process by conventional AM techniques due to its highly oxidative nature and high vapor pressure. Mg scaffolds with well-controlled, ordered porosity are obtained after salt removal. The tunable mechanical properties and the potential to be predictably bioresorbed by the human body make these Mg scaffolds attractive for biomedical implants and demonstrate the great potential of this additive technique.
AUTHOR Daly, Andrew C. and Kelly, Daniel J.
Title Biofabrication of spatially organised tissues by directing the growth of cellular spheroids within 3D printed polymeric microchambers [Abstract]
Year 2019
Journal/Proceedings Biomaterials
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Successful tissue engineering requires the generation of human scale implants that mimic the structure, composition and mechanical properties of native tissues. Here, we report a novel biofabrication strategy that enables the engineering of structurally organised tissues by guiding the growth of cellular spheroids within arrays of 3D printed polymeric microchambers. With the goal of engineering stratified articular cartilage, inkjet bioprinting was used to deposit defined numbers of mesenchymal stromal cells (MSCs) and chondrocytes into pre-printed microchambers. These jetted cell suspensions rapidly underwent condensation within the hydrophobic microchambers, leading to the formation of organised arrays of cellular spheroids. The microchambers were also designed to provide boundary conditions to these spheroids, guiding their growth and eventual fusion, leading to the development of stratified cartilage tissue with a depth-dependant collagen fiber architecture that mimicked the structure of native articular cartilage. Furthermore, the composition and biomechanical properties of the bioprinted cartilage was also comparable to the native tissue. Using multi-tool biofabrication, we were also able to engineer anatomically accurate, human scale, osteochondral templates by printing this microchamber system on top of a hypertrophic cartilage region designed to support endochondral bone formation and then maintaining the entire construct in long-term bioreactor culture to enhance tissue development. This bioprinting strategy provides a versatile and scalable approach to engineer structurally organised cartilage tissues for joint resurfacing applications.
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 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 Laternser, Sandra and Keller, Hansjoerg and Leupin, Olivier and Rausch, Martin and Graf-Hausner, Ursula and Rimann, Markus
Title A Novel Microplate 3D Bioprinting Platform for the Engineering of Muscle and Tendon Tissues [Abstract]
Year 2018
Journal/Proceedings SLAS TECHNOLOGY: Translating Life Sciences Innovation
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Two-dimensional (2D) cell cultures do not reflect the in vivo situation, and thus it is important to develop predictive three-dimensional (3D) in vitro models with enhanced reliability and robustness for drug screening applications. Treatments against muscle-related diseases are becoming more prominent due to the growth of the aging population worldwide. In this study, we describe a novel drug screening platform with automated production of 3D musculoskeletal-tendon-like tissues. With 3D bioprinting, alternating layers of photo-polymerized gelatin-methacryloyl-based bioink and cell suspension tissue models were produced in a dumbbell shape onto novel postholder cell culture inserts in 24-well plates. Monocultures of human primary skeletal muscle cells and rat tenocytes were printed around and between the posts. The cells showed high viability in culture and good tissue differentiation, based on marker gene and protein expressions. Different printing patterns of bioink and cells were explored and calcium signaling with Fluo4-loaded cells while electrically stimulated was shown. Finally, controlled co-printing of tenocytes and myoblasts around and between the posts, respectively, was demonstrated followed by co-culture and co-differentiation. This screening platform combining 3D bioprinting with a novel microplate represents a promising tool to address musculoskeletal diseases.
AUTHOR Khaled, Shaban A. and Alexander, Morgan R. and Irvine, Derek J. and Wildman, Ricky D. and Wallace, Martin J. and Sharpe, Sonja and Yoo, Jae and Roberts, Clive J.
Title Extrusion 3D Printing of Paracetamol Tablets from a Single Formulation with Tunable Release Profiles Through Control of Tablet Geometry [Abstract]
Year 2018
Journal/Proceedings AAPS PharmSciTech
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An extrusion-based 3D printer was used to fabricate paracetamol tablets with different geometries (mesh, ring and solid) from a single paste-based formulation formed from standard pharmaceutical ingredients. The tablets demonstrate that tunable drug release profiles can be achieved from this single formulation even with high drug loading (>{thinspace}80{%} w/w). The tablets were evaluated for drug release using a USP dissolution testing type I apparatus. The tablets showed well-defined release profiles (from immediate to sustained release) controlled by their different geometries. The dissolution results showed dependency of drug release on the surface area/volume (SA/V) ratio and the SA of the different tablets. The tablets with larger SA/V ratios and SA had faster drug release. The 3D printed tablets were also evaluated for physical and mechanical properties including tablet dimension, drug content, weight variation and breaking force and were within acceptable range as defined by the international standards stated in the US Pharmacopoeia. X-ray powder diffraction, differential scanning calorimetry and attenuated total reflectance Fourier transform infrared spectroscopy were used to identify the physical form of the active and to assess possible drug-excipient interactions. These data again showed that the tablets meet USP requirement. These results clearly demonstrate the potential of 3D printing to create unique pharmaceutical manufacturing, and potentially clinical, opportunities. The ability to use a single unmodified formulation to achieve defined release profiles could allow, for example, relatively straightforward personalization of medicines for individuals with different metabolism rates for certain drugs and hence could offer significant development and clinical opportunities.
AUTHOR de Ruijter, Mylène and Ribeiro, Alexandre and Dokter, Inge and Castilho, Miguel and Malda, Jos
Title Simultaneous Micropatterning of Fibrous Meshes and Bioinks for the Fabrication of Living Tissue Constructs [Abstract]
Year 2018
Journal/Proceedings Advanced Healthcare Materials
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Abstract Fabrication of biomimetic tissues holds much promise for the regeneration of cells or organs that are lost or damaged due to injury or disease. To enable the generation of complex, multicellular tissues on demand, the ability to design and incorporate different materials and cell types needs to be improved. Two techniques are combined: extrusion-based bioprinting, which enables printing of cell-encapsulated hydrogels; and melt electrowriting (MEW), which enables fabrication of aligned (sub)-micrometer fibers into a single-step biofabrication process. Composite structures generated by infusion of MEW fiber structures with hydrogels have resulted in mechanically and biologically competent constructs; however, their preparation involves a two-step fabrication procedure that limits freedom of design of microfiber architectures and the use of multiple materials and cell types. How convergence of MEW and extrusion-based bioprinting allows fabrication of mechanically stable constructs with the spatial distributions of different cell types without compromising cell viability and chondrogenic differentiation of mesenchymal stromal cells is demonstrated for the first time. Moreover, this converged printing approach improves freedom of design of the MEW fibers, enabling 3D fiber deposition. This is an important step toward biofabrication of voluminous and complex hierarchical structures that can better resemble the characteristics of functional biological 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 Khaled, Shaban A. and Burley, Jonathan C. and Alexander, Morgan R. and Yang, Jing and Roberts, Clive J.
Title 3D printing of five-in-one dose combination polypill with defined immediate and sustained release profiles [Abstract]
Year 2015
Journal/Proceedings Journal of Controlled Release
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Abstract We have used three dimensional (3D) extrusion printing to manufacture a multi-active solid dosage form or so called polypill. This contains five compartmentalised drugs with two independently controlled and well-defined release profiles. This polypill demonstrates that complex medication regimes can be combined in a single personalised tablet. This could potentially improve adherence for those patients currently taking many separate tablets and also allow ready tailoring of a particular drug combination/drug release for the needs of an individual. The polypill here represents a cardiovascular treatment regime with the incorporation of an immediate release compartment with aspirin and hydrochlorothiazide and three sustained release compartments containing pravastatin, atenolol, and ramipril. X-ray powder diffraction (XRPD) and Attenuated Total Reflectance Fourier Transform Infrared Spectroscopy (ATR-FTIR) were used to assess drug-excipient interaction. The printed polypills were evaluated for drug release using {USP} dissolution testing. We found that the polypill showed the intended immediate and sustained release profiles based upon the active/excipient ratio used.
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 Lee, Ji Seung and Park, Hae Sang and Jung, Harry and Lee, Hanna and Hong, Heesun and Lee, Young Jin and Suh, Ye Ji and Lee, Ok Joo and Kim, Soon Hee and Park, Chan Hum
Title 3D-printable photocurable bioink for cartilage regeneration of tonsil-derived mesenchymal stem cells [Abstract]
Year 2020
Journal/Proceedings Additive Manufacturing
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Abstract
Cartilage regeneration is challenging because of the poor intrinsic self-repair capacity of avascular tissue. Three-dimensional (3D) bioprinting has gained significant attention in the field of tissue engineering and is a promising technology to overcome current difficulties in cartilage regeneration. Although bioink is an essential component of bioprinting technology, several challenges remain in satisfying different requirements for ideal bioink, including biocompatibility and printability based on specific biological requirements. Gelatin and hyaluronic acid (HA) have been shown to be ideal biomimetic hydrogel sources for cartilage regeneration. However, controlling their structure, mechanical properties, biocompatibility, and degradation rate for cartilage repair remains a challenge. Here, we show a photocurable bioink created by hybridization of gelatin methacryloyl (GelMA) and glycidyl-methacrylated HA (GMHA) for material extrusion 3D bioprinting in cartilage regeneration. GelMA and GMHA were mixed in various ratios, and the mixture of 7% GelMA and 5% GMHA bioink (G7H5) demonstrated the most reliable mechanical properties, rheological properties, and printability. This G7H5 bioink allowed us to build a highly complex larynx structure, including the hyoid bone, thyroid cartilage, cricoid cartilage, arytenoid cartilage, and cervical trachea. This bioink also provided an excellent microenvironment for chondrogenesis of tonsil-derived mesenchymal stem cells (TMSCs) in vitro and in vivo. In summary, this study presents the ideal formulation of GelMA/GMHA hybrid bioink to generate a well-suited photocurable bioink for cartilage regeneration of TMSCs using a material extrusion bioprinter, and could be applied to cartilage tissue engineering.
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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DOI/URL DOI
Abstract
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 Colle, Julien and Blondeel, Phillip and De Bruyne, Axelle and Bochar, Silke and Tytgat, Liesbeth and Vercruysse, Chris and Van Vlierberghe, Sandra and Dubruel, Peter and Declercq, Heidi
Title Bioprinting predifferentiated adipose-derived mesenchymal stem cell spheroids with methacrylated gelatin ink for adipose tissue engineering [Abstract]
Year 2020
Journal/Proceedings Journal of Materials Science: Materials in Medicine
Reftype Colle2020
DOI/URL DOI
Abstract
The increasing number of mastectomies results in a greater demand for breast reconstruction characterized by simplicity and a low complication profile. Reconstructive surgeons are investigating tissue engineering (TE) strategies to overcome the current surgical drawbacks. 3D bioprinting is the rising technique for the fabrication of large tissue constructs which provides a potential solution for unmet clinical needs in breast reconstruction building on decades of experience in autologous fat grafting, adipose-derived mesenchymal stem cell (ASC) biology and TE. A scaffold was bioprinted using encapsulated ASC spheroids in methacrylated gelatin ink (GelMA). Uniform ASC spheroids with an ideal geometry and diameter for bioprinting were formed, using a high-throughput non-adhesive agarose microwell system. ASC spheroids in adipogenic differentiation medium (ADM) were evaluated through live/dead staining, histology (HE, Oil Red O), TEM and RT-qPCR. Viable spheroids were obtained for up to 14 days post-printing and showed multilocular microvacuoles and successful differentiation toward mature adipocytes shown by gene expression analysis. Moreover, spheroids were able to assemble at random in GelMA, creating a macrotissue. Combining the advantage of microtissues to self-assemble and the controlled organization by bioprinting technologies, these ASC spheroids can be useful as building blocks for the engineering of soft tissue implants.
AUTHOR Zhang, Danwei and Jonhson, Win and Herng, Tun Seng and Ang, Yong Quan and Yang, Lin and Tan, Swee Ching and Peng, Erwin and He, Hui and Ding, Jun
Title A 3D-printing method of fabrication for metals{,} ceramics{,} and multi-materials using a universal self-curable technique for robocasting [Abstract]
Year 2019
Journal/Proceedings Materials Horizons
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DOI/URL DOI
Abstract
Ceramics and metals are important materials that modern technologies are constructed from. The capability to produce such materials in a complex geometry with good mechanical properties can revolutionize the way we engineer our devices. Current curing techniques pose challenges such as high energy requirements{,} limitations of materials with high refractive index{,} tedious post-processing heat treatment processes{,} uneven drying shrinkages{,} and brittleness of green bodies. In this paper{,} a novel modified self-curable epoxide–amine 3D printing system is proposed to print a wide range of ceramics (metal oxides{,} nitrides{,} and carbides) and metals without the need for an external curing source. Through this technique{,} complex multi-material structures (with metal–ceramic and ceramic–ceramic combinations) can also be realized. Tailoring and matching the sintering temperatures of different materials through sintering additives and dopants{,} combined with a structural design providing maximum adhesion between interfaces{,} allow us to successfully obtain superior quality sintered multi-material structures. High-quality ceramic and metallic materials have been achieved (e.g.{,} zirconia with >98% theoretical density). Also{,} highly conductive metals and magnetic ceramics were printed and shaped uniquely without the need for a sacrificial support. With the addition of low molecular weight plasticizers and a multi-stage heat treatment process{,} crack-free and dense high-quality integrated multi-material structures fabricated by 3D printing can thus be a reality in the near future.
AUTHOR Khaled, Shaban A. and Alexander, Morgan R. and Wildman, Ricky D. and Wallace, Martin J. and Sharpe, Sonja and Yoo, Jae and Roberts, Clive J.
Title 3D extrusion printing of high drug loading immediate release paracetamol tablets [Abstract]
Year 2018
Journal/Proceedings International Journal of Pharmaceutics
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DOI/URL URL DOI
Abstract
The manufacture of immediate release high drug loading paracetamol oral tablets was achieved using an extrusion based 3D printer from a premixed water based paste formulation. The 3D printed tablets demonstrate that a very high drug (paracetamol) loading formulation (80% w/w) can be printed as an acceptable tablet using a method suitable for personalisation and distributed manufacture. Paracetamol is an example of a drug whose physical form can present challenges to traditional powder compression tableting. Printing avoids these issues and facilitates the relatively high drug loading. The 3D printed tablets were evaluated for physical and mechanical properties including weight variation, friability, breaking force, disintegration time, and dimensions and were within acceptable range as defined by the international standards stated in the United States Pharmacopoeia (USP). X-ray Powder Diffraction (XRPD) was used to identify the physical form of the active. Additionally, XRPD, Attenuated Total Reflectance Fourier Transform Infrared spectroscopy (ATR-FTIR) and differential scanning calorimetry (DSC) were used to assess possible drug-excipient interactions. The 3D printed tablets were evaluated for drug release using a USP dissolution testing type I apparatus. The tablets showed a profile characteristic of the immediate release profile as intended based upon the active/excipient ratio used with disintegration in less than 60 s and release of most of the drug within 5 min. The results demonstrate the capability of 3D extrusion based printing to produce acceptable high-drug loading tablets from approved materials that comply with current USP standards.
AUTHOR Daly, Andrew C. and Pitacco, Pierluca and Nulty, Jessica and Cunniffe, Gráinne M. and Kelly, Daniel J.
Title 3D printed microchannel networks to direct vascularisation during endochondral bone repair [Abstract]
Year 2018
Journal/Proceedings Biomaterials
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DOI/URL URL DOI
Abstract
Bone tissue engineering strategies that recapitulate the developmental process of endochondral ossification offer a promising route to bone repair. Clinical translation of such endochondral tissue engineering strategies will require overcoming a number of challenges, including the engineering of large and often anatomically complex cartilage grafts, as well as the persistence of core regions of avascular cartilage following their implantation into large bone defects. Here 3D printing technology is utilized to develop a versatile and scalable approach to guide vascularisation during endochondral bone repair. First, a sacrificial pluronic ink was used to 3D print interconnected microchannel networks in a mesenchymal stem cell (MSC) laden gelatin-methacryloyl (GelMA) hydrogel. These constructs (with and without microchannels) were next chondrogenically primed in vitro and then implanted into critically sized femoral bone defects in rats. The solid and microchanneled cartilage templates enhanced bone repair compared to untreated controls, with the solid cartilage templates (without microchannels) supporting the highest levels of total bone formation. However, the inclusion of 3D printed microchannels was found to promote osteoclast/immune cell invasion, hydrogel degradation, and vascularisation following implantation. In addition, the endochondral bone tissue engineering strategy was found to support comparable levels of bone healing to BMP-2 delivery, whilst promoting lower levels of heterotopic bone formation, with the microchanneled templates supporting the lowest levels of heterotopic bone formation. Taken together, these results demonstrate that 3D printed hypertrophic cartilage grafts represent a promising approach for the repair of complex bone fractures, particularly for larger defects where vascularisation will be a key challenge.
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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DOI/URL DOI
Abstract
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 Khaled, Shaban A. and Burley, Jonathan C. and Alexander, Morgan R. and Yang, Jing and Roberts, Clive J.
Title 3D printing of tablets containing multiple drugs with defined release profiles [Abstract]
Year 2015
Journal/Proceedings International Journal of Pharmaceutics
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DOI/URL URL DOI
Abstract
Abstract We have employed three-dimensional (3D) extrusion-based printing as a medicine manufacturing technique for the production of multi-active tablets with well-defined and separate controlled release profiles for three different drugs. This ‘polypill’ made by a 3D additive manufacture technique demonstrates that complex medication regimes can be combined in a single tablet and that it is viable to formulate and ‘dial up’ this single tablet for the particular needs of an individual. The tablets used to illustrate this concept incorporate an osmotic pump with the drug captopril and sustained release compartments with the drugs nifedipine and glipizide. This combination of medicines could potentially be used to treat diabetics suffering from hypertension. The room temperature extrusion process used to print the formulations used excipients commonly employed in the pharmaceutical industry. Attenuated Total Reflectance Fourier Transform Infrared Spectroscopy (ATR-FTIR) and X-ray powder diffraction (XRPD) were used to assess drug–excipient interaction. The printed formulations were evaluated for drug release using {USP} dissolution testing. We found that the captopril portion showed the intended zero order drug release of an osmotic pump and noted that the nifedipine and glipizide portions showed either first order release or Korsmeyer–Peppas release kinetics dependent upon the active/excipient ratio used.
AUTHOR Kessel, Benjamin and Lee, Mihyun and Bonato, Angela and Tinguely, Yann and Tosoratti, Enrico and Zenobi-Wong, Marcy
Title 3D Bioprinting of Macroporous Materials Based on Entangled Hydrogel Microstrands [Abstract]
Year 2020
Journal/Proceedings Advanced Science
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DOI/URL DOI
Abstract
Abstract Hydrogels are excellent mimetics of mammalian extracellular matrices and have found widespread use in tissue engineering. Nanoporosity of monolithic bulk hydrogels, however, limits mass transport of key biomolecules. Microgels used in 3D bioprinting achieve both custom shape and vastly improved permissivity to an array of cell functions, however spherical-microbead-based bioinks are challenging to upscale, are inherently isotropic, and require secondary crosslinking. Here, bioinks based on high-aspect-ratio hydrogel microstrands are introduced to overcome these limitations. Pre-crosslinked, bulk hydrogels are deconstructed into microstrands by sizing through a grid with apertures of 40–100 µm. The microstrands are moldable and form a porous, entangled structure, stable in aqueous medium without further crosslinking. Entangled microstrands have rheological properties characteristic of excellent bioinks for extrusion bioprinting. Furthermore, individual microstrands align during extrusion and facilitate the alignment of myotubes. Cells can be placed either inside or outside the hydrogel phase with >90% viability. Chondrocytes co-printed with the microstrands deposit abundant extracellular matrix, resulting in a modulus increase from 2.7 to 780.2 kPa after 6 weeks of culture. This powerful approach to deconstruct bulk hydrogels into advanced bioinks is both scalable and versatile, representing an important toolbox for 3D bioprinting of architected hydrogels.
AUTHOR Zhang, Danwei and Peng, Erwin and Borayek, Ramadan and Ding, Jun
Title Controllable Ceramic Green-Body Configuration for Complex Ceramic Architectures with Fine Features [Abstract]
Year 2019
Journal/Proceedings Advanced Functional Materials
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DOI/URL DOI
Abstract
Abstract Fabrication of dense ceramic articles with intricate fine features and geometrically complex morphology by using a relatively simple and the cost-effective process still remains a challenge. Ceramics, either in its green- or sintered-form, are known for being hard yet brittle which limits further shape reconfiguration. In this work, a combinatorial process of ceramic robocasting and photopolymerization is demonstrated to produce either flexible and/or stretchable ceramic green-body (Flex-Body or Stretch-Body) that can undergo a postprinting reconfiguration process. Secondary shaping may proceed through: i) self-assembly-assisted shaping and ii) mold-assisted shaping process, which allows a well-controlled ceramic structure morphology. With a proposed well-controlled thermal heating process, the ceramic Sintered-Body can achieve >99.0% theoretical density with good mechanical rigidity. Complex and dense ceramic articles with fine features down to 65 μm can be fabricated. When combined with a multi-nozzle deposition process, i) self-shaping ceramic structures can be realized through anisotropic shrinkage induced by suspensions' composition variation and ii) technical and functional multiceramic structures can be fabricated. The simplicity of the proposed technique and its inexpensive processing cost make it an attractive approach for fabricating geometrically complex ceramic articles with unique macrostructures, which complements the existing state of-the-art ceramic additive manufacturing techniques.
AUTHOR Azim, N. and Hart, C. and Sommerhage, F. and Aubin, M. and Hickman, J. J. and Rajaraman, S.
Title Precision Plating of Human Electrogenic Cells on Microelectrodes Enhanced With Precision Electrodeposited Nano-Porous Platinum for Cell-Based Biosensing Applications [Abstract]
Year 2019
Journal/Proceedings Journal of Microelectromechanical Systems
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DOI/URL URL DOI
Abstract
Microelectrode Arrays are established platforms for biosensing applications; however, limitations in electrode impedance and cell-electrode coupling still exist. In this paper, the SNR of 25 μm diameter gold (Au) microelectrodes was improved by decreasing the impedance with precision electrodeposition. SEM determined that N-P Pt. microelectrodes had nanoporous structures that filled the insulation cylinders. EIS, CV, and RMS noise measurements concluded that the optimized electrodeposition of N-P Pt. led to a lowered impedance of 18.36 kΩ ± 2.6 kΩ at 1 kHz, a larger double layer capacitance of 73 nF, and lowered RMS noise of 2.08±0.16 μV as compared to the values for Au of 159 kΩ ± 28 kΩ at 1 kHz, 17nF, and 3.14 ± 0.42 μV, respectively. Human motoneurons and human cardiomyocytes were cultured on N-P Pt. devices to assess their biocompatibility and signal quality. In order to improve the cell-electrode coupling, a precision plating technique was used. Both cell types were electrically active on devices for up to 10 weeks, demonstrated improved SNR, and expected responses to precision chemical and electrical stimulation. The modification of Au microelectrodes with nanomaterials in combination with precision culturing of human cell types provides cost effective, highly sensitive, well coupled and relevant biosensing platforms for medical and pharmaceutical research.
AUTHOR Kokkinis, Dimitri and Bouville, Florian and Studart, André R.
Title 3D Printing of Materials with Tunable Failure via Bioinspired Mechanical Gradients [Abstract]
Year 2018
Journal/Proceedings Advanced Materials
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DOI/URL DOI
Abstract
Abstract Mechanical gradients are useful to reduce strain mismatches in heterogeneous materials and thus prevent premature failure of devices in a wide range of applications. While complex graded designs are a hallmark of biological materials, gradients in manmade materials are often limited to 1D profiles due to the lack of adequate fabrication tools. Here, a multimaterial 3D‐printing platform is developed to fabricate elastomer gradients spanning three orders of magnitude in elastic modulus and used to investigate the role of various bioinspired gradient designs on the local and global mechanical behavior of synthetic materials. The digital image correlation data and finite element modeling indicate that gradients can be effectively used to manipulate the stress state and thus circumvent the weakening effect of defect‐rich interfaces or program the failure behavior of heterogeneous materials. Implementing this concept in materials with bioinspired designs can potentially lead to defect‐tolerant structures and to materials whose tunable failure facilitates repair of biomedical implants, stretchable electronics, or soft robotics.
AUTHOR Schaffner, Manuel and Faber, Jakob A. and Pianegonda, Lucas and Rühs, Patrick A. and Coulter, Fergal and Studart, André R.
Title 3D printing of robotic soft actuators with programmable bioinspired architectures [Abstract]
Year 2018
Journal/Proceedings Nature Communications
Reftype Schaffner2018
DOI/URL DOI
Abstract
Soft actuation allows robots to interact safely with humans, other machines, and their surroundings. Full exploitation of the potential of soft actuators has, however, been hindered by the lack of simple manufacturing routes to generate multimaterial parts with intricate shapes and architectures. Here, we report a 3D printing platform for the seamless digital fabrication of pneumatic silicone actuators exhibiting programmable bioinspired architectures and motions. The actuators comprise an elastomeric body whose surface is decorated with reinforcing stripes at a well-defined lead angle. Similar to the fibrous architectures found in muscular hydrostats, the lead angle can be altered to achieve elongation, contraction, or twisting motions. Using a quantitative model based on lamination theory, we establish design principles for the digital fabrication of silicone-based soft actuators whose functional response is programmed within the material's properties and architecture. Exploring such programmability enables 3D printing of a broad range of soft morphing structures.
AUTHOR Schaffner, Manuel and R{"u}hs, Patrick A. and Coulter, Fergal and Kilcher, Samuel and Studart, Andr{'e} R.
Title 3D printing of bacteria into functional complex materials [Abstract]
Year 2017
Journal/Proceedings Science Advances
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DOI/URL URL DOI
Abstract
Despite recent advances to control the spatial composition and dynamic functionalities of bacteria embedded in materials, bacterial localization into complex three-dimensional (3D) geometries remains a major challenge. We demonstrate a 3D printing approach to create bacteria-derived functional materials by combining the natural diverse metabolism of bacteria with the shape design freedom of additive manufacturing. To achieve this, we embedded bacteria in a biocompatible and functionalized 3D printing ink and printed two types of {textquotedblleft}living materials{textquotedblright} capable of degrading pollutants and of producing medically relevant bacterial cellulose. With this versatile bacteria-printing platform, complex materials displaying spatially specific compositions, geometry, and properties not accessed by standard technologies can be assembled from bottom up for new biotechnological and biomedical applications.
AUTHOR Schroeder, Thomas B. H. and Guha, Anirvan and Lamoureux, Aaron and VanRenterghem, Gloria and Sept, David and Shtein, Max and Yang, Jerry and Mayer, Michael
Title An electric-eel-inspired soft power source from stacked hydrogels [Abstract]
Year 2017
Journal/Proceedings Nature
Reftype
DOI/URL DOI
Abstract
Progress towards the integration of technology into livingo ganisms requires electrical power sources that are biocompatible, mechanically flexible, and able to harness the chemical energy available inside biological systems. Conventional batteries were not designed with these criteria in mind. The electric organ of the knifefish Electrophorus electricus (commonly known as the electric eel) is, however, an example of an electrical power source that operates within biological constraints while featuring power characteristics that include peak potential differences of 600 volts and currents of 1 ampere1,2. Here we introduce an electric eel-inspired power concept that uses gradients of ions between miniature polyacrylamide hydrogel compartments bounded by a repeating sequence of cation- and anion-selective hydrogel membranes. The system uses a scalable stacking or folding geometry that generates 110 volts at open circuit or 27 milliwatts per square metre per gel cell upon simultaneous, self-registered mechanical contact activation of thousands of gel compartments in series while circumventing power dissipation before contact. Unlike typical batteries, these systems are soft, flexible, transparent, and potentially biocompatible. These characteristics suggest that artificial electric organs could be used to power next-generation implant materials such as pacemakers, implantable sensors, or prosthetic devices in hybrids of living and non-living systems3–6.�
AUTHOR Nulty, Jessica and Freeman, Fiona E. and Browe, David C. and Burdis, Ross and Ahern, Daniel P. and Pitacco, Pierluca and Lee, Yu Bin and Alsberg, Eben and Kelly, Daniel J.
Title 3D Bioprinting of prevascularised implants for the repair of critically-sized bone defects [Abstract]
Year 2021
Journal/Proceedings Acta Biomaterialia
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DOI/URL URL DOI
Abstract
For 3D bioprinted tissues to be scaled-up to clinically relevant sizes, effective prevascularisation strategies are required to provide the necessary nutrients for normal metabolism and to remove associated waste by-products. The aim of this study was to develop a bioprinting strategy to engineer prevascularised tissues in vitro and to investigate the capacity of such constructs to enhance the vascularisation and regeneration of large bone defects in vivo. From a screen of different bioinks, a fibrin-based hydrogel was found to best support human umbilical vein endothelial cell (HUVEC) sprouting and the establishment of a microvessel network. When this bioink was combined with HUVECs and supporting human bone marrow stem/stromal cells (hBMSCs), these microvessel networks persisted in vitro. Furthermore, only bioprinted tissues containing both HUVECs and hBMSCs, that were first allowed to mature in vitro, supported robust blood vessel development in vivo. To assess the therapeutic utility of this bioprinting strategy, these bioinks were used to prevascularise 3D printed polycaprolactone (PCL) scaffolds, which were subsequently implanted into critically-sized femoral bone defects in rats. Microcomputed tomography (µCT) angiography revealed increased levels of vascularisation in vivo, which correlated with higher levels of new bone formation. Such prevascularised constructs could be used to enhance the vascularisation of a range of large tissue defects, forming the basis of multiple new bioprinted therapeutics. Statement of Significance This paper demonstrates a versatile 3D bioprinting technique to improve the vascularisation of tissue engineered constructs and further demonstrates how this method can be incorporated into a bone tissue engineering strategy to improve vascularisation in a rat femoral defect model.
AUTHOR Zhang, Yi and Wang, Bin and Hu, Junchao and Yin, Tianyuan and Yue, Tao and Liu, Na and Liu, Yuanyuan
Title 3D Composite Bioprinting for Fabrication of Artificial Biological Tissues. [Abstract]
Year 2021
Journal/Proceedings International journal of bioprinting
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DOI/URL
Abstract
Three-dimensional (3D) bioprinting is an important technology for fabricating artificial tissue. To effectively reconstruct the multiscale structure and multi-material gradient of natural tissues and organs, 3D bioprinting has been increasingly developed into multi-process composite mode. The current 3D composite bioprinting is a combination of two or more printing processes, and oftentimes, physical field regulation that can regulate filaments or cells during or after printing may be involved. Correspondingly, both path planning strategy and process control all become more complex. Hence, the computer-aided design and computer-aided manufacturing (CAD/CAM) system that is traditionally used in 3D printing system is now facing challenges. Thus, the scale information that cannot be modeled in the CAD process should be considered in the design of CAM by adding a process management module in the traditional CAD/CAM system and add more information reflecting component gradient in the path planning strategy.
AUTHOR Rößler, Sina and Brückner, Andreas and Kruppke, Iris and Wiesmann, Hans-Peter and Hanke, Thomas and Kruppke, Benjamin
Title 3D Plotting of Silica/Collagen Xerogel Granules in an Alginate Matrix for Tissue-Engineered Bone Implants [Abstract]
Year 2021
Journal/Proceedings Materials
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DOI/URL URL DOI
Abstract
Today, materials designed for bone regeneration are requested to be degradable and resorbable, bioactive, porous, and osteoconductive, as well as to be an active player in the bone-remodeling process. Multiphasic silica/collagen Xerogels were shown, earlier, to meet these requirements. The aim of the present study was to use these excellent material properties of silica/collagen Xerogels and to process them by additive manufacturing, in this case 3D plotting, to generate implants matching patient specific shapes of fractures or lesions. The concept is to have Xerogel granules as active major components embedded, to a large proportion, in a matrix that binds the granules in the scaffold. By using viscoelastic alginate as matrix, pastes of Xerogel granules were processed via 3D plotting. Moreover, alginate concentration was shown to be the key to a high content of irregularly shaped Xerogel granules embedded in a minimum of matrix phase. Both the alginate matrix and Xerogel granules were also shown to influence viscoelastic behavior of the paste, as well as the dimensionally stability of the scaffolds. In conclusion, 3D plotting of Xerogel granules was successfully established by using viscoelastic properties of alginate as matrix phase.
AUTHOR Shende, Pravin and Trivedi, Riddhi
Title 3D Printed Bioconstructs: Regenerative Modulation for Genetic Expression [Abstract]
Groups
Year 2021
Journal/Proceedings Stem Cell Reviews and Reports
Reftype Shende2021
DOI/URL DOI
Abstract
Layer-by-layer deposition of cells, tissues and similar molecules provided by additive manufacturing techniques such as 3D bioprinting offers safe, biocompatible, effective and inert methods for the production of biological structures and biomimetic scaffolds. 3D bioprinting assisted through computer programmes and software develops mutli-modal nano- or micro-particulate systems such as biosensors, dosage forms or delivery systems and other biological scaffolds like pharmaceutical implants, prosthetics, etc. This review article focuses on the implementation of 3D bioprinting techniques in the gene expression, in gene editing or therapy and in delivery of genes. The applications of 3D printing are extensive and include gene therapy, modulation and expression in cancers, tissue engineering, osteogenesis, skin and vascular regeneration. Inclusion of nanotechnology with genomic bioprinting parameters such as gene conjugated or gene encapsulated 3D printed nanostructures may offer new avenues in the future for efficient and controlled treatment and help in overcoming the limitations faced in conventional methods. Moreover, expansion of the benefits from such techniques is advantageous in real-time delivery or in-situ production of nucleic acids into the host cells.
AUTHOR Vyas, Cian and Zhang, Jun and Øvrebø, Øystein and Huang, Boyang and Roberts, Iwan and Setty, Mohan and Allardyce, Benjamin and Haugen, Håvard and Rajkhowa, Rangam and Bartolo, Paulo
Title 3D printing of silk microparticle reinforced polycaprolactone scaffolds for tissue engineering applications [Abstract]
Year 2021
Journal/Proceedings Materials Science and Engineering: C
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DOI/URL URL DOI
Abstract
Polycaprolactone (PCL) scaffolds have been widely investigated for tissue engineering applications, however, they exhibit poor cell adhesion and mechanical properties. Subsequently, PCL composites have been produced to improve the material properties. This study utilises a natural material, Bombyx mori silk microparticles (SMP) prepared by milling silk fibre, to produce a composite to enhance the scaffolds properties. Silk is biocompatible and biodegradable with excellent mechanical properties. However, there are no studies using SMPs as a reinforcing agent in a 3D printed thermoplastic polymer scaffold. PCL/SMP (10, 20, 30 wt%) composites were prepared by melt blending. Rheological analysis showed that SMP loading increased the shear thinning and storage modulus of the material. Scaffolds were fabricated using a screw-assisted extrusion-based additive manufacturing system. Scanning electron microscopy and X-ray microtomography was used to determine scaffold morphology. The scaffolds had high interconnectivity with regular printed fibres and pore morphologies within the designed parameters. Compressive mechanical testing showed that the addition of SMP significantly improved the compressive Young's modulus of the scaffolds. The scaffolds were more hydrophobic with the inclusion of SMP which was linked to a decrease in total protein adsorption. Cell behaviour was assessed using human adipose derived mesenchymal stem cells. A cytotoxic effect was observed at higher particle loading (30 wt%) after 7 days of culture. By day 21, 10 wt% loading showed significantly higher cell metabolic activity and proliferation, high cell viability, and cell migration throughout the scaffold. Calcium mineral deposition was observed on the scaffolds during cell culture. Large calcium mineral deposits were observed at 30 wt% and smaller calcium deposits were observed at 10 wt%. This study demonstrates that SMPs incorporated into a PCL scaffold provided effective mechanical reinforcement, improved the rate of degradation, and increased cell proliferation, demonstrating potential suitability for bone tissue engineering applications.
AUTHOR Seydel, Caroline
Title 3D-Bioprinted Cell Therapy and Disease Modeling Applications
Year 2021
Journal/Proceedings Genetic Engineering & Biotechnology News
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DOI/URL DOI
AUTHOR Shin, Crystal S. and Cabrera, Fernando J. and Lee, Richard and Kim, John and Ammassam Veettil, Remya and Zaheer, Mahira and Adumbumkulath, Aparna and Mhatre, Kirti and Ajayan, Pulickel M. and Curley, Steven A. and Scott, Bradford G. and Acharya, Ghanashyam
Title 3D-Bioprinted Inflammation Modulating Polymer Scaffolds for Soft Tissue Repair [Abstract]
Year 2021
Journal/Proceedings Advanced Materials
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DOI/URL DOI
Abstract
Abstract Development of inflammation modulating polymer scaffolds for soft tissue repair with minimal postsurgical complications is a compelling clinical need. However, the current standard of care soft tissue repair meshes for hernia repair is highly inflammatory and initiates a dysregulated inflammatory process causing visceral adhesions and postsurgical complications. Herein, the development of an inflammation modulating biomaterial scaffold (bioscaffold) for soft tissue repair is presented. The bioscaffold design is based on the idea that, if the excess proinflammatory cytokines are sequestered from the site of injury by the surgical implantation of a bioscaffold, the inflammatory response can be modulated, and the visceral adhesion formations and postsurgical complications can be minimized. The bioscaffold is fabricated by 3D-bioprinting of an in situ phosphate crosslinked poly(vinyl alcohol) polymer. In vivo efficacy of the bioscaffold is evaluated in a rat ventral hernia model. In vivo proinflammatory cytokine expression analysis and histopathological analysis of the tissues have confirmed that the bioscaffold acts as an inflammation trap and captures the proinflammatory cytokines secreted at the implant site and effectively modulates the local inflammation without the need for exogenous anti-inflammatory agents. The bioscaffold is very effective in inhibiting visceral adhesions formation and minimizing postsurgical complications.
AUTHOR Leu Alexa, Rebeca and Iovu, Horia and Ghitman, Jana and Serafim, Andrada and Stavarache, Cristina and Marin, Maria-Minodora and Ianchis, Raluca
Title 3D-Printed Gelatin Methacryloyl-Based Scaffolds with Potential Application in Tissue Engineering [Abstract]
Year 2021
Journal/Proceedings Polymers
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The development of materials for 3D printing adapted for tissue engineering represents one of the main concerns nowadays. Our aim was to obtain suitable 3D-printed scaffolds based on methacrylated gelatin (GelMA). In this respect, three degrees of GelMA methacrylation, three different concentrations of GelMA (10%, 20%, and 30%), and also two concentrations of photoinitiator (I-2959) (0.5% and 1%) were explored to develop proper GelMA hydrogel ink formulations to be used in the 3D printing process. Afterward, all these GelMA hydrogel-based inks/3D-printed scaffolds were characterized structurally, mechanically, and morphologically. The presence of methacryloyl groups bounded to the surface of GelMA was confirmed by FTIR and 1H-NMR analyses. The methacrylation degree influenced the value of the isoelectric point that decreased with the GelMA methacrylation degree. A greater concentration of photoinitiator influenced the hydrophilicity of the polymer as proved using contact angle and swelling studies because of the new bonds resulting after the photocrosslinking stage. According to the mechanical tests, better mechanical properties were obtained in the presence of the 1% initiator. Circular dichroism analyses demonstrated that the secondary structure of gelatin remained unaffected during the methacrylation process, thus being suitable for biological applications.
AUTHOR Jiahui Lai and Xinliang Ye and Jia Liu and Chong Wang and Junzhi Li and Xiang Wang and Mingze Ma and Min Wang
Title 4D printing of highly printable and shape morphing hydrogels composed of alginate and methylcellulose [Abstract]
Year 2021
Journal/Proceedings Materials & Design
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Abstract
4D printing of swellable/shrinkable hydrogels has been viewed as an appealing approach for fabricating dynamic structures for various biomedical applications. However, 4D printing of precise hydrogel structures is still highly challenging due to the relatively poor printability of hydrogels and high surface roughness of printed patterns, when micro extrusion-based 3D printers are used. In this study, a highly printable and shape morphing hydrogel was investigated for 4D printing by blending alginate (Alg) and methylcellulose (MC). The optimized Alg/MC hydrogel exhibited excellent rheological properties, extrudability and shape fidelity of printed structures. The printable Alg/MC hydrogel was 4D printed into a series of patterned 2D architectures which were encoded with anisotropic stiffness and swelling behaviors by strategically controlling the network density gradients vertical to the orientation of the patterned strips. By controlling the strip interspacing and angle, these 2D architectures could transform into various prescribed simple 3D morphologies (e.g., tube-curling and helix) and complex 3D morphologies (e.g., double helix and flowers) after immersion in a calcium chloride solution. This shape morphing Alg/MC hydrogel with excellent printability has high potential for 4D printing of delicate hydrogel patterns, which are increasingly needed in the tissue engineering, biomedical device and soft robotics fields.
AUTHOR Chelsea Twohig and Mari Helsinga and Amin Mansoorifar and Avathamsa Athirasala and Anthony Tahayeri and Cristiane Miranda França and Silvia Amaya Pajares and Reyan Abdelmoniem and Susanne Scherrer and Stéphane Durual and Jack Ferracane and Luiz E. Bertassoni
Title A dual-ink 3D printing strategy to engineer pre-vascularized bone scaffolds in-vitro [Abstract]
Year 2021
Journal/Proceedings Materials Science and Engineering: C
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Abstract
A functional vascular supply is a key component of any large-scale tissue, providing support for the metabolic needs of tissue-remodeling cells. Although well-studied strategies exist to fabricate biomimetic scaffolds for bone regeneration, success rates for regeneration in larger defects can be improved by engineering microvascular capillaries within the scaffolds to enhance oxygen and nutrient supply to the core of the engineered tissue as it grows. Even though the role of calcium and phosphate has been well understood to enhance osteogenesis, it remains unclear whether calcium and phosphate may have a detrimental effect on the vasculogenic and angiogenic potential of endothelial cells cultured on 3D printed bone scaffolds. In this study, we presented a novel dual-ink bioprinting method to create vasculature interwoven inside CaP bone constructs. In this method, strands of a CaP ink and a sacrificial template material was used to form scaffolds containing CaP fibers and microchannels seeded with vascular endothelial and mesenchymal stem cells (MSCs) within a photo-crosslinkable gelatin methacryloyl (GelMA) hydrogel material. Our results show similar morphology of growing vessels in the presence of CaP bioink, and no significant difference in endothelial cell sprouting was found. Furthermore, our initial results showed the differentiation of hMSCs into pericytes in the presence of CaP ink. These results indicate the feasibility of creating vascularized bone scaffolds, which can be used for enhancing vascular formation in the core of bone scaffolds.
AUTHOR Kwak, Chaesu and Young Ryu, Seoung and Park, Hyunsu and Lim, Sehyeong and Yang, Jeewon and Kim, Jieun and Hyung Kim, Jin and Lee, Joohyung
Title A pickering emulsion stabilized by chlorella microalgae as an eco-friendly extrusion-based 3D printing ink processable under ambient conditions [Abstract]
Year 2021
Journal/Proceedings Journal of Colloid and Interface Science
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Abstract
Three-dimensional (3D) printing technology is actively utilized in various industrial fields because it facilitates effective and customizable fabrication of complex structures. An important processing route for 3D printing is the extrusion of inks in the form of colloidal suspensions or emulsions, which has recently attracted considerable attention because it allows for selection of a wide range of printing materials and is operable under ambient processing conditions. Herein, we investigate the 3D printability of complex fluids containing chlorella microalgae as an eco-friendly material for 3D printing. Two possible ink types are considered: aqueous chlorella suspensions and emulsions of oil and water mixtures. While the aqueous chlorella suspensions at high particle loading display the 3D-printable rheological properties such as high yield stress and good shape retention, the final structures after extruding and drying the suspensions under ambient conditions show a significant number of macroscopic defects, limiting their practical application. In contrast, the 3D structures produced from the oil-in-water Pickering emulsions stabilized by chlorella microalgae, which are amphiphilic and active at the oil–water interface, show significantly reduced defect formation. Addition of a fast-evaporable oil phase, hexane, is crucial in the mechanisms of enhanced cementation between the individual microalgae via increased inter-particle packing, capillary attraction, and hydrophobic interaction. Furthermore, addition of solid paraffin wax, which is crystalline but well-soluble in the hydrocarbon oil phase under ambient conditions, completely eliminates the undesirable defect formation via enhanced inter-particle binding, while maintaining the overall rheological properties of the emulsion. The optimal formulation of the Pickering emulsion is finally employed to produce a 3D scaffold of satisfactory structural integrity, suggesting that the chlorella-based ink, in the form of an emulsion, has potential as an eco-friendly 3D printing ink processable under ambient conditions.
AUTHOR Bin Wang and Pedro J. Díaz-Payno and David C. Browe and Fiona E. Freeman and Jessica Nulty and Ross Burdis and Daniel J. Kelly
Title Affinity-bound growth factor within sulfated interpenetrate network bioinks for bioprinting cartilaginous tissues [Abstract]
Year 2021
Journal/Proceedings Acta Biomaterialia
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3D bioprinting has emerged as a promising technology in the field of tissue engineering and regenerative medicine due to its ability to create anatomically complex tissue substitutes. However, it still remains challenging to develop bioactive bioinks that provide appropriate and permissive environments to instruct and guide the regenerative process in vitro and in vivo. In this study alginate sulfate, a sulfated glycosaminoglycan (sGAG) mimic, was used to functionalize an alginate-gelatin methacryloyl (GelMA) interpenetrating network (IPN) bioink to enable the bioprinting of cartilaginous tissues. The inclusion of alginate sulfate had a limited influence on the viscosity, shear-thinning and thixotropic properties of the IPN bioink, enabling high-fidelity bioprinting and supporting mesenchymal stem cell (MSC) viability post-printing. The stiffness of printed IPN constructs greatly exceeded that achieved by printing alginate or GelMA alone, while maintaining resilience and toughness. Furthermore, given the high affinity of alginate sulfate to heparin-binding growth factors, the sulfated IPN bioink supported the sustained release of transforming growth factor-β3 (TGF-β3), providing an environment that supported robust chondrogenesis in vitro, with little evidence of hypertrophy or mineralization over extended culture periods. Such bioprinted constructs also supported chondrogenesis in vivo, with the controlled release of TGF-β3 promoting significantly higher levels of cartilage-specific extracellular matrix deposition. Altogether, these results demonstrate the potential of bioprinting sulfated bioinks as part of a ‘single-stage’ or ‘point-of-care’ strategy for regenerating cartilaginous tissues. Statement of Significance: This study highlights the potential of using sulfated interpenetrating network (IPN) bioink to support the regeneration of phenotypically stable articular cartilage. Construction of interpenetrate networks in the bioink enables unique high-fidelity bioprinting and unique synergistic mechanical properties. The presence of alginate sulfate provided the capacity of high affinity-binding of TGF-β3, which promoted robust chondrogenesis.
AUTHOR Leu Alexa, Rebeca and Iovu, Horia and Trica, Bogdan and Zaharia, Catalin and Serafim, Andrada and Alexandrescu, Elvira and Radu, Ionut-Cristian and Vlasceanu, George and Preda, Silviu and Ninciuleanu, Claudia Mihaela and Ianchis, Raluca
Title Assessment of Naturally Sourced Mineral Clays for the 3D Printing of Biopolymer-Based Nanocomposite Inks [Abstract]
Year 2021
Journal/Proceedings Nanomaterials
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The present study investigated the possibility of obtaining 3D printed composite constructs using biomaterial-based nanocomposite inks. The biopolymeric matrix consisted of methacrylated gelatin (GelMA). Several types of nanoclay were added as the inorganic component. Our aim was to investigate the influence of clay type on the rheological behavior of ink formulations and to determine the morphological and structural properties of the resulting crosslinked hydrogel-based nanomaterials. Moreover, through the inclusion of nanoclays, our goal was to improve the printability and shape fidelity of nanocomposite scaffolds. The viscosity of all ink formulations was greater in the presence of inorganic nanoparticles as shear thinning occurred with increased shear rate. Hydrogel nanocomposites presented predominantly elastic rather than viscous behavior as the materials were crosslinked which led to improved mechanical properties. The inclusion of nanoclays in the biopolymeric matrix limited hydrogel swelling due the physical barrier effect but also because of the supplementary crosslinks induced by the clay layers. The distribution of inorganic filler within the GelMA-based hydrogels led to higher porosities as a consequence of their interaction with the biopolymeric ink. The present study could be useful for the development of soft nanomaterials foreseen for the additive manufacturing of customized implants for tissue engineering.
AUTHOR Otto, I. A. and Capendale, P. E. and Garcia, J. P. and de Ruijter, M. and van Doremalen, R. F. M. and Castilho, M. and Lawson, T. and Grinstaff, M. W. and Breugem, C. C. and Kon, M. and Levato, R. and Malda, J.
Title Biofabrication of a shape-stable auricular structure for the reconstruction of ear deformities [Abstract]
Year 2021
Journal/Proceedings Materials Today Bio
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Bioengineering of the human auricle remains a significant challenge, where the complex and unique shape, the generation of high-quality neocartilage, and shape preservation are key factors. Future regenerative medicine–based approaches for auricular cartilage reconstruction will benefit from a smart combination of various strategies. Our approach to fabrication of an ear-shaped construct uses hybrid bioprinting techniques, a recently identified progenitor cell population, previously validated biomaterials, and a smart scaffold design. Specifically, we generated a 3D-printed polycaprolactone (PCL) scaffold via fused deposition modeling, photocrosslinked a human auricular cartilage progenitor cell–laden gelatin methacryloyl (gelMA) hydrogel within the scaffold, and cultured the bioengineered structure in vitro in chondrogenic media for 30 days. Our results show that the fabrication process maintains the viability and chondrogenic phenotype of the cells, that the compressive properties of the combined PCL and gelMA hybrid auricular constructs are similar to native auricular cartilage, and that biofabricated hybrid auricular structures exhibit excellent shape fidelity compared with the 3D digital model along with deposition of cartilage-like matrix in both peripheral and central areas of the auricular structure. Our strategy affords an anatomically enhanced auricular structure with appropriate mechanical properties, ensures adequate preservation of the auricular shape during a dynamic in vitro culture period, and enables chondrogenically potent progenitor cells to produce abundant cartilage-like matrix throughout the auricular construct. The combination of smart scaffold design with 3D bioprinting and cartilage progenitor cells holds promise for the development of clinically translatable regenerative medicine strategies for auricular reconstruction.
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 Bagnol, Romain and Sprecher, Christoph and Peroglio, Marianna and Chevalier, Jerome and Mahou, Redouan and Büchler, Philippe and Richards, Geoff and Eglin, David
Title Coaxial micro-extrusion of a calcium phosphate ink with aqueous solvents improves printing stability, structure fidelity and mechanical properties [Abstract]
Year 2021
Journal/Proceedings Acta Biomaterialia
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Micro-extrusion-based 3D printing of complex geometrical and porous calcium phosphate (CaP) can improve treatment of bone defects through the production of personalized bone substitutes. However, achieving printing and post-printing shape stabilities for the efficient fabrication and application of rapid hardening protocol are still challenging. In this work, the coaxial printing of a self-setting CaP cement with water and ethanol mixtures aiming to increase the ink yield stress upon extrusion and the stability of fabricated structures was explored. Printing height of overhang structure was doubled when aqueous solvents were used and a 2 log increase of the stiffness was achieved post-printing. A standard and fast steam sterilization protocol applied as hardening step on the coaxial printed CaP cement (CPC) ink resulted in constructs with 4 to 5 times higher compressive moduli in comparison to extrusion process in the absence of solvent. This improved mechanical performance is likely due to rapid CPC setting, preventing cracks formation during hardening process. Thus, coaxial micro-extrusion-based 3D printing of a CPC ink with aqueous solvent enhances printability and allows the use of the widespread steam sterilization cycle as a standalone post-processing technique for production of 3D printed personalized CaP bone substitutes. Statement of Significance Coaxial micro-extrusion-based 3D printing of a self-setting CaP cement with water:ethanol mixtures increased the ink yield stress upon extrusion and the stability of fabricated structures. Printing height of overhang structure was doubled when aqueous solvents were used, and a 2 orders of magnitude log increase of the stiffness was achieved post-printing. A fast hardening step consisting of a standard steam sterilization was applied. Four to 5 times higher compressive moduli was obtained for hardened coaxially printed constructs. This improved mechanical performance is likely due to rapid CPC setting in the coaxial printing, preventing cracks formation during hardening process.
AUTHOR Fenelon, Mathilde and Etchebarne, Marion and Siadous, Robin and Grémare, Agathe and Durand, Marlène and Sentilhes, Loic and Catros, Sylvain and Gindraux, Florelle and L'Heureux, Nicolas and Fricain, Jean-Christophe
Title Comparison of amniotic membrane versus the induced membrane for bone regeneration in long bone segmental defects using calcium phosphate cement loaded with BMP-2 [Abstract]
Year 2021
Journal/Proceedings Materials Science and Engineering: C
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Thanks to its biological properties, the human amniotic membrane (HAM) combined with a bone substitute could be a single-step surgical alternative to the two-step Masquelet induced membrane (IM) technique for regeneration of critical bone defects. However, no study has directly compared these two membranes. We first designed a 3D-printed scaffold using calcium phosphate cement (CPC). We assessed its suitability in vitro to support human bone marrow mesenchymal stromal cells (hBMSCs) attachment and osteodifferentiation. We then performed a rat femoral critical size defect to compare the two-step IM technique with a single-step approach using the HAM. Five conditions were compared. Group 1 was left empty. Group 2 received the CPC scaffold loaded with rh-BMP2 (CPC/BMP2). Group 3 and 4 received the CPC/BMP2 scaffold covered with lyophilized or decellularized/lyophilized HAM. Group 5 underwent a two- step induced membrane procedure with insertion of a polymethylmethacrylate (PMMA) spacer followed by, after 4 weeks, its replacement with the CPC/BMP2 scaffold wrapped in the IM. Micro-CT and histomorphometric analysis were performed after six weeks. Results showed that the CPC scaffold supported the proliferation and osteodifferentiation of hBMSCs in vitro. In vivo, the CPC/BMP2 scaffold very efficiently induced bone formation and led to satisfactory healing of the femoral defect, in a single-step, without autograft or the need for any membrane covering. In this study, there was no difference between the two-step induced membrane procedure and a single step approach. However, the results indicated that none of the tested membranes further enhanced bone healing compared to the CPC/BMP2 group.
AUTHOR Zhang, Xiao and Liu, Yang and Luo, Chunyang and Zhai, Chenjun and Li, Zuxi and Zhang, Yi and Yuan, Tao and Dong, Shilei and Zhang, Jiyong and Fan, Weimin
Title Crosslinker-free silk/decellularized extracellular matrix porous bioink for 3D bioprinting-based cartilage tissue engineering [Abstract]
Year 2021
Journal/Proceedings Materials Science and Engineering: C
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As cartilage tissue lacks the innate ability to mount an adequate regeneration response, damage to it is detrimental to the quality of life of the subject. The emergence of three-dimensional bioprinting (3DBP) technology presents an opportunity to repair articular cartilage defects. However, widespread adoption of this technique has been impeded by difficulty in preparing a suitable bioink and the toxicity inherent in the chemical crosslinking process of most bioinks. Our objective was to develop a crosslinker-free bioink with the same biological activity as the original cartilage extracellular matrix (ECM) and good mechanical strength. We prepared bioinks containing different concentrations of silk fibroin and decellularized extracellular matrix (SF-dECM bioinks) mixed with bone marrow mesenchymal stem cells (BMSCs) for 3D bioprinting. SF and dECM interconnect with each other through physical crosslinking and entanglement. A porous structure was formed by removing the polyethylene glycol from the SF-dECM bioink. The results showed the SF-dECM construct had a suitable mechanical strength and degradation rate, and the expression of chondrogenesis-specific genes was found to be higher than that of the SF control construct group. Finally, we confirmed that a SF-dECM construct that was designed to release TGF-β3 had the ability to promote chondrogenic differentiation of BMSCs and provided a good cartilage repair environment, suggesting it is an ideal scaffold for cartilage tissue engineering.
AUTHOR Curti, Filis and Drăgușin, Diana-Maria and Serafim, Andrada and Iovu, Horia and Stancu, Izabela-Cristina
Title Development of thick paste-like inks based on superconcentrated gelatin/alginate for 3D printing of scaffolds with shape fidelity and stability [Abstract]
Year 2021
Journal/Proceedings Materials Science and Engineering: C
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Shape fidelity and integrity are serious challenges in the 3D printing of hydrogel precursors, as they can influence the overall performance of 3D scaffolds. This work reports the development of superconcentrated inks based on sodium alginate and fish gelatin as an appealing strategy to satisfy such challenges and dictate the quality of the printed scaffolds, without using crosslinking strategies during 3D printing. SEM micrographs and micro-CT images indicate the homogeneous distribution of the polysaccharide in the gelatin-based matrix, suggesting its potential to act as a reinforcing additive. The high concentration of gelatin aqueous solution (50 wt%) and substantial incorporation of alginate have facilitated the highly accurate printability and influence the in vitro stability and mechanical properties of the printed scaffolds. An improvement of the stiffness is dictated by the increase of alginate concentration from 20 wt% to 25 wt%, and an increase of Young modulus with about 46% is reached, confirming the reinforcing effect of polysaccharide. This study highlights the potential of paste-type inks to provide high resolution 3D printed structures with appealing structural and dimensional stability, in vitro degradability and mechanical properties for biomedical applications.
AUTHOR Shiwarski,Daniel J. and Hudson,Andrew R. and Tashman,Joshua W. and Feinberg,Adam W.
Title Emergence of FRESH 3D printing as a platform for advanced tissue biofabrication
Year 2021
Journal/Proceedings APL Bioengineering
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AUTHOR Kjar, Andrew and McFarland, Bailey and Mecham, Keetch and Harward, Nathan and Huang, Yu
Title Engineering of tissue constructs using coaxial bioprinting [Abstract]
Year 2021
Journal/Proceedings Bioactive Materials
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Bioprinting is a rapidly developing technology for the precise design and manufacture of tissues in various biological systems or organs. Coaxial extrusion bioprinting, an emergent branch, has demonstrated a strong potential to enhance bioprinting's engineering versatility. Coaxial bioprinting assists in the fabrication of complex tissue constructs, by enabling concentric deposition of biomaterials. The fabricated tissue constructs started with simple, tubular vasculature but have been substantially developed to integrate complex cell composition and self-assembly, ECM patterning, controlled release, and multi-material gradient profiles. This review article begins with a brief overview of coaxial printing history, followed by an introduction of crucial engineering components. Afterward, we review the recent progress and untapped potential in each specific organ or biological system, and demonstrate how coaxial bioprinting facilitates the creation of tissue constructs. Ultimately, we conclude that this growing technology will contribute significantly to capabilities in the fields of in vitro modeling, pharmaceutical development, and clinical regenerative medicine.
AUTHOR Oliveira, Hugo and Médina, Chantal and Stachowicz, Marie-Laure and Paiva dos Santos, Bruno and Chagot, Lise and Dusserre, Nathalie and Fricain, Jean-Christophe
Title Extracellular matrix (ECM)-derived bioinks designed to foster vasculogenesis and neurite outgrowth: Characterization and bioprinting [Abstract]
Year 2021
Journal/Proceedings Bioprinting
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The field of bioprinting has shown a tremendous development in recent years, focusing on the development of advanced in vitro models and on regeneration approaches. In this scope, the lack of suitable biomaterials that can be efficiently formulated as printable bioinks, while supporting specific cellular events, is currently considered as one of the main limitations in the field. Indeed, extracellular matrix (ECM)-derived biomaterials formulated to enable printability and support cellular response, for instance via integrin binding, are eagerly awaited in the field of bioprinting. Several bioactive laminin sequences, including peptides such as YIGSR and IKVAV, have been identified to promote endothelial cell attachment and/or neurite outgrowth and guidance, respectively. Here, we show the development of two distinct bioinks, designed to foster vasculogenesis or neurogenesis, based on methacrylated collagen and hyaluronic acid (CollMA and HAMA, respectively), both relevant ECM-derived polymers, and on their combination with cysteine-flanked laminin-derived peptides. Using this strategy, it was possible to optimize the bioink printability, by tuning CollMA and HAMA concentration and ratio, and modulate their bioactivity, through adjustments in the cell-active peptide sequence spatial density, without compromising cell viability. We demonstrated that cell-specific bioinks could be customized for the bioprinting of both human umbilical vein cord endothelial cells (HUVECs) or adult rat sensory neurons from the dorsal root ganglia, and could stimulate both vasculogenesis and neurite outgrowth, respectively. This approach holds great potential as it can be tailored to other cellular models, due to its inherent capacity to accommodate different peptide compositions and to generate complex peptide mixtures and/or gradients.
AUTHOR Wibowo, Arie and Tajalla, Gusti U. N. and Marsudi, Maradhana A. and Cooper, Glen and Asri, Lia A.T.W. and Liu, Fengyuan and Ardy, Husaini and Bartolo, Paulo J.D.S.
Title Green Synthesis of Silver Nanoparticles Using Extract of Cilembu Sweet Potatoes (Ipomoea batatas L var. Rancing) as Potential Filler for 3D Printed Electroactive and Anti-Infection Scaffolds [Abstract]
Year 2021
Journal/Proceedings Molecules
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Electroactive biomaterials are fascinating for tissue engineering applications because of their ability to deliver electrical stimulation directly to cells, tissue, and organs. One particularly attractive conductive filler for electroactive biomaterials is silver nanoparticles (AgNPs) because of their high conductivity, antibacterial activity, and ability to promote bone healing. However, production of AgNPs involves a toxic reducing agent which would inhibit biological scaffold performance. This work explores facile and green synthesis of AgNPs using extract of Cilembu sweet potato and studies the effect of baking and precursor concentrations (1, 10 and 100 mM) on AgNPs’ properties. Transmission electron microscope (TEM) results revealed that the smallest particle size of AgNPs (9.95 ± 3.69 nm) with nodular morphology was obtained by utilization of baked extract and ten mM AgNO3. Polycaprolactone (PCL)/AgNPs scaffolds exhibited several enhancements compared to PCL scaffolds. Compressive strength was six times greater (3.88 ± 0.42 MPa), more hydrophilic (contact angle of 76.8 ± 1.7°), conductive (2.3 ± 0.5 × 10−3 S/cm) and exhibited anti-bacterial properties against Staphylococcus aureus ATCC3658 (99.5% reduction of surviving bacteria). Despite the promising results, further investigation on biological assessment is required to obtain comprehensive study of this scaffold. This green synthesis approach together with the use of 3D printing opens a new route to manufacture AgNPs-based electroactive with improved anti-bacterial properties without utilization of any toxic organic solvents.
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 e Silva, Edney P. and Huang, Boyang and Helaehil, Júlia V. and Nalesso, Paulo R. L. and Bagne, Leonardo and de Oliveira, Maraiara A. and Albiazetti, Gabriela C. C. and Aldalbahi, Ali and El-Newehy, Mohamed and Santamaria-Jr, Milton and Mendonça, Fernanda A. S. and Bártolo, Paulo and Caetano, Guilherme F.
Title In vivo study of conductive 3D printed PCL/MWCNTs scaffolds with electrical stimulation for bone tissue engineering [Abstract]
Year 2021
Journal/Proceedings Bio-Design and Manufacturing
Reftype e Silva2021
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Critical bone defects are considered one of the major clinical challenges in reconstructive bone surgery. The combination of 3D printed conductive scaffolds and exogenous electrical stimulation (ES) is a potential favorable approach for bone tissue repair. In this study, 3D conductive scaffolds made with biocompatible and biodegradable polycaprolactone (PCL) and multi-walled carbon nanotubes (MWCNTs) were produced using the extrusion-based additive manufacturing to treat large calvary bone defects in rats. Histology results show that the use of PCL/MWCNTs scaffolds and ES contributes to thicker and increased bone tissue formation within the bone defect. Angiogenesis and mineralization are also significantly promoted using high concentration of MWCNTs (3 wt%) and ES. Moreover, scaffolds favor the tartrate-resistant acid phosphatase (TRAP) positive cell formation, while the addition of MWCNTs seems to inhibit the osteoclastogenesis but present limited effects on the osteoclast functionalities (receptor activator of nuclear factor κβ ligand (RANKL) and osteoprotegerin (OPG) expressions). The use of ES promotes the osteoclastogenesis and RANKL expressions, showing a dominant effect in the bone remodeling process. These results indicate that the combination of 3D printed conductive PCL/MWCNTs scaffold and ES is a promising strategy to treat critical bone defects and provide a cue to establish an optimal protocol to use conductive scaffolds and ES for bone tissue engineering.
AUTHOR Zamani, Yasaman and Amoabediny, Ghassem and Mohammadi, Javad and Zandieh-Doulabi, Behrouz and Klein-Nulend, Jenneke and Helder, Marco N.
Title Increased Osteogenic Potential of Pre-Osteoblasts on Three-Dimensional Printed Scaffolds Compared to Porous Scaffolds for Bone Regeneration [Abstract]
Year 2021
Journal/Proceedings Iranian Biomedical Journal
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Abstract
Background: One of the main challenges with conventional scaffold fabrication methods is the inability to control scaffold architecture. Recently, scaffolds with controlled shape and architecture have been fabricated using three-dimensional printing (3DP). Herein, we aimed to determine whether the much tighter control of microstructure of 3DP poly(lactic-co-glycolic) acid/β-tricalcium phosphate (PLGA/β-TCP) scaffolds is more effective in promoting osteogenesis than porous scaffolds produced by solvent casting/porogen leaching. Methods: Physical and mechanical properties of porous and 3DP scaffolds were studied. The response of pre-osteoblasts to the scaffolds was analyzed after 14 days. Results: The 3DP scaffolds had a smoother surface (Ra: 22 ± 3 µm) relative to the highly rough surface of porous scaffolds (Ra: 110 ± 15 µm). Water contact angle was 112 ± 4° on porous and 76 ± 6° on 3DP scaffolds. Porous and 3DP scaffolds had the pore size of 408 ± 90 and 315 ± 17 µm and porosity of 85 ± 5% and 39 ± 7%, respectively. Compressive strength of 3DP scaffolds (4.0 ± 0.3 MPa) was higher than porous scaffolds (1.7 ± 0.2 MPa). Collagenous matrix deposition was similar on both scaffolds. Cells proliferated from day 1 to day 14 by fourfold in porous and by 3.8-fold in 3DP scaffolds. Alkaline phosphatase (ALP) activity was 21-fold higher in 3DP scaffolds than porous scaffolds. Conclusion: The 3DP scaffolds show enhanced mechanical properties and ALP activity compared to porous scaffolds in vitro, suggesting that 3DP PLGA/β-TCP scaffolds are possibly more favorable for bone formation.
AUTHOR Daskalakis, Evangelos and Liu, Fengyuan and Huang, Boyang and Acar, Anil A. and Cooper, Glen and Weightman, Andrew and Blunn, Gordon and Koç, Bahattin and Bartolo, Paulo
Title Investigating the Influence of Architecture and Material Composition of 3D Printed Anatomical Design Scaffolds for Large Bone Defects [Abstract]
Year 2021
Journal/Proceedings International Journal of Bioprinting; Vol 7, No 2 (2021)
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Abstract
There is a significant unmet clinical need to prevent amputations due to large bone loss injuries. We are addressing this problem by developing a novel, cost-effective osseointegrated prosthetic solution based on the use of modular pieces, bone bricks, made with biocompatible and biodegradable materials that fit together in a Lego-like way to form the prosthesis. This paper investigates the anatomical designed bone bricks with different architectures, pore size gradients, and material compositions. Polymer and polymer-composite 3D printed bone bricks are extensively morphological, mechanical, and biological characterized. Composite bone bricks were produced by mixing polycaprolactone (PCL) with different levels of hydroxyapatite (HA) and β-tri-calcium phosphate (TCP). Results allowed to establish a correlation between bone bricks architecture and material composition and bone bricks performance. Reinforced bone bricks showed improved mechanical and biological results. Best mechanical properties were obtained with PCL/TCP bone bricks with 38 double zig-zag filaments and 14 spiral-like pattern filaments, while the best biological results were obtained with PCL/HA bone bricks based on 25 double zig-zag filaments and 14 spiral-like pattern filaments.
AUTHOR Wang, Weiguang and Chen, Jun-Xiang and Hou, Yanhao and Bartolo, Paulo and Chiang, Wei-Hung
Title Investigations of Graphene and Nitrogen-Doped Graphene Enhanced Polycaprolactone 3D Scaffolds for Bone Tissue Engineering [Abstract]
Year 2021
Journal/Proceedings Nanomaterials
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Scaffolds play a key role in tissue engineering applications. In the case of bone tissue engineering, scaffolds are expected to provide both sufficient mechanical properties to withstand the physiological loads, and appropriate bioactivity to stimulate cell growth. In order to further enhance cell–cell signaling and cell–material interaction, electro-active scaffolds have been developed based on the use of electrically conductive biomaterials or blending electrically conductive fillers to non-conductive biomaterials. Graphene has been widely used as functioning filler for the fabrication of electro-active bone tissue engineering scaffolds, due to its high electrical conductivity and potential to enhance both mechanical and biological properties. Nitrogen-doped graphene, a unique form of graphene-derived nanomaterials, presents significantly higher electrical conductivity than pristine graphene, and better surface hydrophilicity while maintaining a similar mechanical property. This paper investigates the synthesis and use of high-performance nitrogen-doped graphene as a functional filler of poly(ɛ-caprolactone) (PCL) scaffolds enabling to develop the next generation of electro-active scaffolds. Compared to PCL scaffolds and PCL/graphene scaffolds, these novel scaffolds present improved in vitro biological performance.
AUTHOR King, William E. and Bowlin, Gary L.
Title Near-Field Electrospinning and Melt Electrowriting of Biomedical Polymers—Progress and Limitations [Abstract]
Year 2021
Journal/Proceedings Polymers
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Abstract
Near-field electrospinning (NFES) and melt electrowriting (MEW) are the process of extruding a fiber due to the force exerted by an electric field and collecting the fiber before bending instabilities occur. When paired with precise relative motion between the polymer source and the collector, a fiber can be directly written as dictated by preprogrammed geometry. As a result, this precise fiber control results in another dimension of scaffold tailorability for biomedical applications. In this review, biomedically relevant polymers that to date have manufactured fibers by NFES/MEW are explored and the present limitations in direct fiber writing of standardization in published setup details, fiber write throughput, and increased ease in the creation of complex scaffold geometries are discussed.
AUTHOR Iria Seoane-Viaño and Patricija Januskaite and Carmen Alvarez-Lorenzo and Abdul W. Basit and Alvaro Goyanes
Title Semi-solid extrusion 3D printing in drug delivery and biomedicine: Personalised solutions for healthcare challenges [Abstract]
Year 2021
Journal/Proceedings Journal of Controlled Release
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Abstract
Three-dimensional (3D) printing is an innovative additive manufacturing technology, capable of fabricating unique structures in a layer-by-layer manner. Semi-solid extrusion (SSE) is a subset of material extrusion 3D printing, and through the sequential deposition of layers of gel or paste creates objects of any desired size and shape. In comparison to other extrusion-based technologies, SSE 3D printing employs low printing temperatures which makes it suitable for drug delivery and biomedical applications, and the use of disposable syringes provides benefits in meeting critical quality requirements for pharmaceutical use. Besides pharmaceutical manufacturing, SSE 3D printing has attracted increasing attention in the field of bioelectronics, particularly in the manufacture of biosensors capable of measuring physiological parameters or as a means to trigger drug release from medical devices. This review begins by highlighting the major printing process parameters and material properties that influence the feasibility of transforming a 3D design into a 3D object, and follows with a discussion on the current SSE 3D printing developments and their applications in the fields of pharmaceutics, bioprinting and bioelectronics. Finally, the advantages and limitations of this technology are explored, before focusing on its potential clinical applications and suitability for preparing personalised medicines.
AUTHOR De Moor, Lise and Minne, Mendy and Tytgat, Liesbeth and Vercruysse, Chris and Dubruel, Peter and Van Vlierberghe, Sandra and Declercq, Heidi
Title Tuning the Phenotype of Cartilage Tissue Mimics by Varying Spheroid Maturation and Methacrylamide-Modified Gelatin Hydrogel Characteristics [Abstract]
Year 2021
Journal/Proceedings Macromolecular Bioscience
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Abstract
Abstract In hybrid bioprinting of cartilage tissue constructs, spheroids are used as cellular building blocks and combined with biomaterials for dispensing. However, biomaterial intrinsic cues can deeply affect cell fate and to date, the influence of hydrogel encapsulation on spheroid viability and phenotype has received limited attention. This study assesses this need and unravels 1) how the phenotype of spheroid-laden constructs can be tuned through adjusting the hydrogel physico–chemical properties and 2) if the spheroid maturation stage prior to encapsulation is a determining factor for the construct phenotype. Articular chondrocyte spheroids with a cartilage specific extracellular matrix (ECM) are generated and different maturation stages, early-, mid-, and late-stage (3, 7, and 14 days, respectively), are harvested and encapsulated in 10, 15, or 20 w/v% methacrylamide-modified gelatin (gelMA) for 14 days. The encapsulation of immature spheroids do not lead to a cartilage-like ECM production but when more mature mid- or late-stage spheroids are combined with a certain concentration of gelMA, a fibrocartilage-like as well as a hyaline cartilage-like phenotype can be induced. As a proof of concept, late-stage spheroids are bioprinted using a 10 w/v% gelMA–Irgacure 2959 solution with the aim to test the processing potential of the spheroid-laden bioink.
AUTHOR Lagatuz, M. and Vyas, R. J. and Predovic, M. and Lim, S. and Jacobs, N. and Martinho, M. and Valizadegan, H. and Kao, D. and Oza, N. and Theriot, C. A. and Zanello, S. B. and Taibbi, G. and Vizzeri, G. and Dupont, M. and Grant, M. B. and Lindner, D. J. and Reinecker, H.-C. and Pinhas, A. and Chui, T. Y. and Rosen, R. B. and Moldovan, N. and Vickerman, M. B. and Radhakrishnan, K. and Parsons-Wingerter, P.
Title Vascular Patterning as Integrative Readout of Complex Molecular and Physiological Signaling by VESsel GENeration Analysis [Abstract]
Year 2021
Journal/Proceedings J Vasc Res
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The molecular signaling cascades that regulate angiogenesis and microvascular remodeling are fundamental to normal development, healthy physiology, and pathologies such as inflammation and cancer. Yet quantifying such complex, fractally branching vascular patterns remains difficult. We review application of NASA’s globally available, freely downloadable VESsel GENeration (VESGEN) Analysis software to numerous examples of 2D vascular trees, networks, and tree-network composites. Upon input of a binary vascular image, automated output includes informative vascular maps and quantification of parameters such as tortuosity, fractal dimension, vessel diameter, area, length, number, and branch point. Previous research has demonstrated that cytokines and therapeutics such as vascular endothelial growth factor, basic fibroblast growth factor (fibroblast growth factor-2), transforming growth factor-beta-1, and steroid triamcinolone acetonide specify unique “fingerprint” or “biomarker” vascular patterns that integrate dominant signaling with physiological response. In vivo experimental examples described here include vascular response to keratinocyte growth factor, a novel vessel tortuosity factor; angiogenic inhibition in humanized tumor xenografts by the anti-angiogenesis drug leronlimab; intestinal vascular inflammation with probiotic protection by Saccharomyces boulardii, and a workflow programming of vascular architecture for 3D bioprinting of regenerative tissues from 2D images. Microvascular remodeling in the human retina is described for astronaut risks in microgravity, vessel tortuosity in diabetic retinopathy, and venous occlusive disease.
AUTHOR Jhinuk Rahman and Julian Quodbach
Title Versatility on demand – The case for semi-solid micro-extrusion in pharmaceutics [Abstract]
Year 2021
Journal/Proceedings Advanced Drug Delivery Reviews
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Since additive manufacturing of pharmaceuticals has been introduced as viable method to produce individualized drug delivery systems with complex geometries and release profiles, semi-solid micro-extrusion has shown to be uniquely beneficial. Easy incorporation of actives, room-temperature processability and avoidance of cross-contamination by using disposables are some of the advantages that led many researchers to focus their work on this technology in the last few years. First acceptability and in-vivo studies have brought it closer towards implementation in decentralized settings. This review covers recently established process models in light of viscosity and printability discussions to help develop high quality printed medicines. Quality defining formulation and process parameters to characterize the various developed dosage forms are presented before critically discussing the role of semi-solid micro-extrusion in the future of personalized drug delivery systems. Remaining challenges regarding regulatory guidance and quality assurance that pose the last hurdle for large scale and commercial manufacturing are addressed.
AUTHOR Hamid, Omar A. and Eltaher, Hoda M. and Sottile, Virginie and Yang, Jing
Title 3D bioprinting of a stem cell-laden, multi-material tubular composite: An approach for spinal cord repair [Abstract]
Year 2020
Journal/Proceedings Materials Science and Engineering: C
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Abstract
Development of a biomimetic tubular scaffold capable of recreating developmental neurogenesis using pluripotent stem cells offers a novel strategy for the repair of spinal cord tissues. Recent advances in 3D printing technology have facilitated biofabrication of complex biomimetic environments by precisely controlling the 3D arrangement of various acellular and cellular components (biomaterials, cells and growth factors). Here, we present a 3D printing method to fabricate a complex, patterned and embryoid body (EB)-laden tubular scaffold composed of polycaprolactone (PCL) and hydrogel (alginate or gelatine methacrylate (GelMA)). Our results revealed 3D printing of a strong, macro-porous PCL/hydrogel tubular scaffold with a high capacity to control the porosity of the PCL scaffold, wherein the maximum porosity in the PCL wall was 15%. The method was equally employed to create spatiotemporal protein concentration within the scaffold, demonstrating its ability to generate linear and opposite gradients of model molecules (fluorescein isothiocyanate-conjugated bovine serum albumin (FITC-BSA) and rhodamine). 3D bioprinting of EBs-laden GelMA was introduced as a novel 3D printing strategy to incorporate EBs in a hydrogel matrix. Cell viability and proliferation were measured post-printing. Following the bioprinting of EBs-laden 5% GelMA hydrogel, neural differentiation of EBs was induced using 1 μM retinoic acid (RA). The differentiated EBs contained βIII-tubulin positive neurons displaying axonal extensions and cells migration. Finally, 3D bioprinting of EBs-laden PCL/GelMA tubular scaffold successfully supported EBs neural differentiation and patterning in response to co-printing with 1 μM RA. 3D printing of a complex heterogeneous tubular scaffold that can encapsulate EBs, spatially controlled protein concentration and promote neuronal patterning will help in developing more biomimetic scaffolds capable of replicating the neural patterning which occurs during neural tube development.
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 Rupp, Harald and Binder, Wolfgang H.
Title 3D Printing of Core–Shell Capsule Composites for Post-Reactive and Damage Sensing Applications [Abstract]
Year 2020
Journal/Proceedings Advanced Materials Technologies
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Abstract 3D printing of multicomponent materials as an advantageous method over traditional mold casting methods is demonstrated, developing small core–shell capsule composites fabricated by a two-step 3D printing process. Using a two-print-head system (fused deposition modeling extruder and a liquid inkjet print head), micro-sized capsules are manufactured in sizes ranging from 100 to 800 µm. The thermoplastic polymer poly(ε-caprolactone) (PCL) is chosen as matrix/shell material due to its optimal interaction with the embedded hydrophobic liquids. First, the core–shell capsules are printed with model liquids and pure PCL to optimize the printing parameters and to ensure fully enclosed capsules inside the polymer. As a proof of concept, novel “click” reaction systems, used in self-healing and stress-detection applications, are manufactured in which PCL composites with nano- and micro-fillers are combined with reactive, encapsulated liquids. The so generated 3D printed core–shell capsule composite can be used for post-printing reactions and damage sensing when combined with a fluorogenic dye.
AUTHOR Critchley, Susan and Sheehy, Eamon J. and Cunniffe, Gráinne and Diaz-Payno, Pedro and Carroll, Simon F. and Jeon, Oju and Alsberg, Eben and Brama, Pieter A. J. and Kelly, Daniel J.
Title 3D printing of fibre-reinforced cartilaginous templates for the regeneration of osteochondral defects [Abstract]
Year 2020
Journal/Proceedings Acta Biomaterialia
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Abstract
Successful osteochondral defect repair requires regenerating the subchondral bone whilst simultaneously promoting the development of an overlying layer of articular cartilage that is resistant to vascularization and endochondral ossification. During skeletal development articular cartilage also functions as a surface growth plate, which postnatally is replaced by a more spatially complex bone-cartilage interface. Motivated by this developmental process, the hypothesis of this study is that bi-phasic, fibre-reinforced cartilaginous templates can regenerate both the articular cartilage and subchondral bone within osteochondral defects created in caprine joints. To engineer mechanically competent implants, we first compared a range of 3D printed fibre networks (PCL, PLA and PLGA) for their capacity to mechanically reinforce alginate hydrogels whilst simultaneously supporting mesenchymal stem cell (MSC) chondrogenesis in vitro. These mechanically reinforced, MSC-laden alginate hydrogels were then used to engineer the endochondral bone forming phase of bi-phasic osteochondral constructs, with the overlying chondral phase consisting of cartilage tissue engineered using a co-culture of infrapatellar fat pad derived stem/stromal cells (FPSCs) and chondrocytes. Following chondrogenic priming and subcutaneous implantation in nude mice, these bi-phasic cartilaginous constructs were found to support the development of vascularised endochondral bone overlaid by phenotypically stable cartilage. These fibre-reinforced, bi-phasic cartilaginous templates were then evaluated in clinically relevant, large animal (caprine) model of osteochondral defect repair. Although the quality of repair was variable from animal-to-animal, in general more hyaline-like cartilage repair was observed after 6 months in animals treated with bi-phasic constructs compared to animals treated with commercial control scaffolds. This variability in the quality of repair points to the need for further improvements in the design of 3D bioprinted implants for joint regeneration. Statement of Significance Successful osteochondral defect repair requires regenerating the subchondral bone whilst simultaneously promoting the development of an overlying layer of articular cartilage. In this study, we hypothesised that bi-phasic, fibre-reinforced cartilaginous templates could be leveraged to regenerate both the articular cartilage and subchondral bone within osteochondral defects. To this end we used 3D printed fibre networks to mechanically reinforce engineered transient cartilage, which also contained an overlying layer of phenotypically stable cartilage engineered using a co-culture of chondrocytes and stem cells. When chondrogenically primed and implanted into caprine osteochondral defects, these fibre-reinforced bi-phasic cartilaginous grafts were shown to spatially direct tissue development during joint repair. Such developmentally inspired tissue engineering strategies, enabled by advances in biofabrication and 3D printing, could form the basis of new classes of regenerative implants in orthopaedic medicine.
AUTHOR Wibowo, Arie and Vyas, Cian and Cooper, Glen and Qulub, Fitriyatul and Suratman, Rochim and Mahyuddin, Andi Isra and Dirgantara, Tatacipta and Bartolo, Paulo
Title 3D Printing of Polycaprolactone-Polyaniline Electroactive Scaffolds for Bone Tissue Engineering. [Abstract]
Year 2020
Journal/Proceedings Materials
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Electrostimulation and electroactive scaffolds can positively influence and guide cellular behaviour and thus has been garnering interest as a key tissue engineering strategy. The development of conducting polymers such as polyaniline enables the fabrication of conductive polymeric composite scaffolds. In this study, we report on the initial development of a polycaprolactone scaffold incorporating different weight loadings of a polyaniline microparticle filler. The scaffolds are fabricated using screw-assisted extrusion-based 3D printing and are characterised for their morphological, mechanical, conductivity, and preliminary biological properties. The conductivity of the polycaprolactone scaffolds increases with the inclusion of polyaniline. The in vitro cytocompatibility of the scaffolds was assessed using human adipose-derived stem cells to determine cell viability and proliferation up to 21 days. A cytotoxicity threshold was reached at 1% wt. polyaniline loading. Scaffolds with 0.1% wt. polyaniline showed suitable compressive strength (6.45 ± 0.16 MPa) and conductivity (2.46 ± 0.65 × 10(-4) S/cm) for bone tissue engineering applications and demonstrated the highest cell viability at day 1 (88%) with cytocompatibility for up to 21 days in cell culture.
AUTHOR García-Astrain, Clara and Lenzi, Elisa and Jimenez de Aberasturi, Dorleta and Henriksen-Lacey, Malou and Binelli, Marco R. and Liz-Marzán, Luis M.
Title 3D-Printed Biocompatible Scaffolds with Built-In Nanoplasmonic Sensors [Abstract]
Year 2020
Journal/Proceedings Advanced Functional Materials
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Abstract 3D printing strategies have acquired great relevance toward the design of 3D scaffolds with precise macroporous structures, for supported mammalian cell growth. Despite advances in 3D model designs, there is still a shortage of detection tools to precisely monitor in situ cell behavior in 3D, thereby allowing a better understanding of the progression of diseases or to test the efficacy of drugs in a more realistic microenvironment. Even if the number of available inks has exponentially increased, they do not necessarily offer the required functionalities to be used as internal sensors. Herein the potential of surface-enhanced Raman scattering (SERS) spectroscopy for the detection of biorelevant analytes within a plasmonic hydrogel-based, 3D-printed scaffold is demonstrated. Such SERS-active scaffolds allow for the 3D detection of model molecules, such as 4-mercaptobenzoic acid. Flexibility in the choice of plasmonic nanoparticles is demonstrated through the use of gold nanoparticles with different morphologies, gold nanorods showing the best balance between SERS enhancement and scaffold transparency. Detection of the biomarker adenosine is also demonstrated as a proof-of-concept toward the use of these plasmonic scaffolds for SERS sensing of cell-secreted molecules over extended periods of time.
AUTHOR Zamani, Yasaman and Amoabediny, Ghassem and Mohammadi, Javad and Seddiqi, Hadi and Helder, Marco N. and Zandieh-Doulabi, Behrouz and Klein-Nulend, Jenneke and Koolstra, Jan Harm
Title 3D-printed poly(Ɛ-caprolactone) scaffold with gradient mechanical properties according to force distribution in the mandible for mandibular bone tissue engineering [Abstract]
Year 2020
Journal/Proceedings Journal of the Mechanical Behavior of Biomedical Materials
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Abstract
In bone tissue engineering, prediction of forces induced to the native bone during normal functioning is important in the design, fabrication, and integration of a scaffold with the host. The aim of this study was to customize the mechanical properties of a layer-by-layer 3D-printed poly(ϵ-caprolactone) (PCL) scaffold estimated by finite element (FE) modeling in order to match the requirements of the defect, to prevent mechanical failure, and ensure optimal integration with the surrounding tissue. Forces and torques induced on the mandibular symphysis during jaw opening and closing were predicted by FE modeling. Based on the predicted forces, homogeneous-structured PCL scaffolds with 3 different void sizes (0.3, 0.6, and 0.9 mm) were designed and 3D-printed using an extrusion based 3D-bioprinter. In addition, 2 gradient-structured scaffolds were designed and 3D-printed. The first gradient scaffold contained 2 regions (0.3 mm and 0.6 mm void size in the upper and lower half, respectively), whereas the second gradient scaffold contained 3 regions (void sizes of 0.3, 0.6, and 0.9 mm in the upper, middle and lower third, respectively). Scaffolds were tested for their compressive and tensile strength in the upper and lower halves. The actual void size of the homogeneous scaffolds with designed void size of 0.3, 0.6, and 0.9 mm was 0.20, 0.59, and 0.95 mm, respectively. FE modeling showed that during opening and closing of the jaw, the highest force induced on the symphysis was a compressive force in the transverse direction. The compressive force was induced throughout the symphyseal line and reduced from top (362.5 N, compressive force) to bottom (107.5 N, tensile force) of the symphysis. Compressive and tensile strength of homogeneous scaffolds decreased by 1.4-fold to 3-fold with increasing scaffold void size. Both gradient scaffolds had higher compressive strength in the upper half (2 region-gradient scaffold: 4.9 MPa; 3 region-gradient scaffold: 4.1 MPa) compared with the lower half (2 region-gradient scaffold: 2.5 MPa; 3 region-gradient scaffold: 2.7 MPa) of the scaffold. 3D-printed PCL scaffolds had higher compressive strength in the scaffold layer-by-layer building direction compared with the side direction, and a very low tensile strength in the scaffold layer-by-layer building direction. Fluid shear stress and fluid pressure distribution in the gradient scaffolds were more homogeneous than in the 0.3 mm void size scaffold and similar to the 0.6 mm and 0.9 mm void size scaffolds. In conclusion, these data show that the mechanical properties of 3D-printed PCL scaffolds can be tailored based on the predicted forces on the mandibular symphysis. These 3D-printed PCL scaffolds had different mechanical properties in scaffold building direction compared with the side direction, which should be taken into account when placing the scaffold in the defect site. Our findings might have implications for improved performance and integration of scaffolds with native tissue.
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 Mancini, I. A. D. and Schmidt, S. and Brommer, H. and Pouran, B. and Schäfer, S. and Tessmar, J. and Mensinga, A. and van Rijen, M. H. P. and Groll, J. and Blunk, T. and Levato, R. and Malda, J. and van Weeren, P. R.
Title A composite hydrogel-3D printed thermoplast osteochondral anchor as example for a zonal approach to cartilage repair: in vivo performance in a long-term equine model [Abstract]
Year 2020
Journal/Proceedings Biofabrication
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Recent research has been focusing on the generation of living personalized osteochondral constructs for joint repair. Native articular cartilage has a zonal structure, which is not reflected in current constructs and which may be a cause of the frequent failure of these repair attempts. Therefore, we investigated the performance of a composite implant that further reflects the zonal distribution of cellular component both in vitro and in vivo in a long-term equine model. Constructs constituted of a 3D-printed poly(ϵ-caprolactone) (PCL) bone anchor from which reinforcing fibers protruded into the chondral part of the construct over which two layers of a thiol-ene cross-linkable hyaluronic acid/poly(glycidol) hybrid hydrogel (HA-SH/P(AGE-co-G)) were fabricated. The top layer contained Articular Cartilage Progenitor Cells (ACPCs) derived from the superficial layer of native cartilage tissue, the bottom layer contained mesenchymal stromal cells (MSCs). The chondral part of control constructs were homogeneously filled with MSCs. After six months in vivo, microtomography revealed significant bone growth into the anchor. Histologically, there was only limited production of cartilage-like tissue (despite persistency of hydrogel) both in zonal and non-zonal constructs. There were no differences in histological scoring; however, the repair tissue was significantly stiffer in defects repaired with zonal constructs. The sub-optimal quality of the repair tissue may be related to several factors, including early loss of implanted cells, or inappropriate degradation rate of the hydrogel. Nonetheless, this approach may be promising and research into further tailoring of biomaterials and of construct characteristics seems warranted.
AUTHOR Jung, Harry and Lee, Ji Seung and Lee, Jun Ho and Park, Ki Joon and Lee, Jae Jun and Park, Hae Sang
Title A Feasibility Study for 3D-printed Poly(methyl methacrylate)-resin Tracheostomy Tube Using a Hamster Cheek Pouch Model
Year 2020
Journal/Proceedings In Vivo
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AUTHOR Wang, Zehao and Hui, Aiping and Zhao, Hongbin and Ye, Xiaohan and Zhang, Chao and Wang, Aiqin and Zhang, Changqing
Title A Novel 3D-bioprinted Porous Nano Attapulgite Scaffolds with Good Performance for Bone Regeneration [Abstract]
Year 2020
Journal/Proceedings International Journal of Nanomedicine
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BACKGROUND: Natural clay nanomaterials are an emerging class of biomaterial with great potential for tissue engineering and regenerative medicine applications, most notably for osteogenesis. MATERIALS AND METHODS: Herein, for the first time, novel tissue engineering scaffolds were prepared by 3D bioprinter using nontoxic and bioactive natural attapulgite (ATP) nanorods as starting materials, with polyvinyl alcohol as binder, and then sintered to obtain final scaffolds. The microscopic morphology and structure of ATP particles and scaffolds were observed by transmission electron microscope and scanning electron microscope. In vitro biocompatibility and osteogenesis with osteogenic precursor cell (hBMSCs) were assayed using MTT method, Live/Dead cell staining, alizarin red staining and RT-PCR. In vivo bone regeneration was evaluated with micro-CT and histology analysis in rat cranium defect model. RESULTS: We successfully printed a novel porous nano-ATP scaffold designed with inner channels with a dimension of 500 µm and wall structures with a thickness of 330 µm. The porosity of current 3D-printed scaffolds ranges from 75% to 82% and the longitudinal compressive strength was up to 4.32±0.52 MPa. We found firstly that nano-ATP scaffolds with excellent biocompatibility for hBMSCscould upregulate the expression of osteogenesis-related genes bmp2 and runx2 and calcium deposits in vitro. Interestingly, micro-CT and histology analysis revealed abundant newly formed bone was observed along the defect margin, even above and within the 3D bioprinted porous ATP scaffolds in a rat cranial defect model. Furthermore, histology analysis demonstrated that bone was formed directly following a process similar to membranous ossification without any intermediate cartilage formation and that many newly formed blood vessels are within the pores of 3D-printed scaffolds at four and eight weeks. CONCLUSION: These results suggest that the 3D-printed porous nano-ATP scaffolds are promising candidates for bone tissue engineering by osteogenesis and angiogenesis.
AUTHOR Moxon, Sam and Ferreira, Miguel and Santos, Patricia and Popa, Bogdan and Gloria, Antonio and Katsarava, Ramaz and Tugushi, David and Serra, Armenio and Hooper, Nigel and Kimber, Susan and Fonseca, Ana and Domingos, Marco
Title A Preliminary Evaluation of the Pro-Chondrogenic Potential of 3D-Bioprinted Poly(ester Urea) Scaffolds
Year 2020
Journal/Proceedings Polymers
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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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Abstract
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 Cui, Xiaolin and Li, Jun and Hartanto, Yusak and Durham, Mitchell and Tang, Junnan and Zhang, Hu and Hooper, Gary and Lim, Khoon and Woodfield, Tim
Title Advances in Extrusion 3D Bioprinting: A Focus on Multicomponent Hydrogel-Based Bioinks [Abstract]
Groups Bioinks
Year 2020
Journal/Proceedings Advanced Healthcare Materials
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DOI/URL DOI
Abstract
Abstract 3D bioprinting involves the combination of 3D printing technologies with cells, growth factors and biomaterials, and has been considered as one of the most advanced tools for tissue engineering and regenerative medicine (TERM). However, despite multiple breakthroughs, it is evident that numerous challenges need to be overcome before 3D bioprinting will eventually become a clinical solution for a variety of TERM applications. To produce a 3D structure that is biologically functional, cell-laden bioinks must be optimized to meet certain key characteristics including rheological properties, physico-mechanical properties, and biofunctionality; a difficult task for a single component bioink especially for extrusion based bioprinting. As such, more recent research has been centred on multicomponent bioinks consisting of a combination of two or more biomaterials to improve printability, shape fidelity and biofunctionality. In this article, multicomponent hydrogel-based bioink systems are systemically reviewed based on the inherent nature of the bioink (natural or synthetic hydrogels), including the most current examples demonstrating properties and advances in application of multicomponent bioinks, specifically for extrusion based 3D bioprinting. This review article will assist researchers in the field in identifying the most suitable bioink based on their requirements, as well as pinpointing current unmet challenges in the field.
AUTHOR Huang, Boyang and Vyas, Cian and Byun, Jae Jong and El-Newehy, Mohamed and Huang, Zhucheng and Bártolo, Paulo
Title Aligned multi-walled carbon nanotubes with nanohydroxyapatite in a 3D printed polycaprolactone scaffold stimulates osteogenic differentiation [Abstract]
Year 2020
Journal/Proceedings Materials Science and Engineering: C
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DOI/URL URL DOI
Abstract
The development of highly biomimetic scaffolds in terms of composition and structures, to repair or replace damaged bone tissues, is particularly relevant for tissue engineering. This paper investigates a 3D printed porous scaffold containing aligned multi-walled carbon nanotubes (MWCNTs) and nano-hydroxyapatite (nHA), mimicking the natural bone tissue from the nanoscale to macroscale level. MWCNTs with similar dimensions as collagen fibres are coupled with nHA and mixed within a polycaprolactone (PCL) matrix to produce scaffolds using a screw-assisted extrusion-based additive manufacturing system. Scaffolds with different material compositions were extensively characterised from morphological, mechanical and biological points of views. Transmission electron microscopy and polarised Raman spectroscopy confirm the presence of aligned MWCNTs within the printed filaments. The PCL/HA/MWCNTs scaffold are similar to the nanostructure of native bone and shows overall increased mechanical properties, cell proliferation, osteogenic differentiation and scaffold mineralisation, indicating a promising approach for bone tissue regeneration.
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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DOI/URL URL DOI
Abstract
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 Estermann, Manuela and Bisig, Christoph and Septiadi, Dedy and Petri-Fink, Alke and Rothen-Rutishauser, Barbara
Title Bioprinting for Human Respiratory and Gastrointestinal In Vitro Models [Abstract]
Year 2020
Journal/Proceedings
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DOI/URL DOI
Abstract
Increasing ethical and biological concerns require a paradigm shift toward animal-free testing strategies for drug testing and hazard assessments. To this end, the application of bioprinting technology in the field of biomedicine is driving a rapid progress in tissue engineering. In particular, standardized and reproducible in vitro models produced by three-dimensional (3D) bioprinting technique represent a possible alternative to animal models, enabling in vitro studies relevant to in vivo conditions. The innovative approach of 3D bioprinting allows a spatially controlled deposition of cells and biomaterial in a layer-by-layer fashion providing a platform for engineering reproducible models. However, despite the promising and revolutionizing character of 3D bioprinting technology, standardized protocols providing detailed instructions are lacking. Here, we provide a protocol for the automatized printing of simple alveolar, bronchial, and intestine epithelial cell layers as the basis for more complex respiratory and gastrointestinal tissue models. Such systems will be useful for high-throughput toxicity screening and drug efficacy evaluation.
AUTHOR Zamani, Yasaman and Mohammadi, Javad and Amoabediny, Ghassem and Helder, Marco N. and Zandieh-Doulabi, Behrouz and Klein-Nulend, Jenneke
Title Bioprinting of Alginate-Encapsulated Pre-osteoblasts in PLGA/β-TCP Scaffolds Enhances Cell Retention but Impairs Osteogenic Differentiation Compared to Cell Seeding after 3D-Printing [Abstract]
Year 2020
Journal/Proceedings Regenerative Engineering and Translational Medicine
Reftype Zamani2020
DOI/URL DOI
Abstract
In tissue engineering, cellularization of scaffolds has typically been performed by seeding the cells after scaffold fabrication. 3D-printing technology now allows bioprinting of cells encapsulated in a hydrogel simultaneously with the scaffold material. Here, we aimed to investigate whether bioprinting or cell seeding post-printing is more effective in enhancing responses of pre-osteoblastic MC3T3-E1 cell line derived from mouse calvaria.
AUTHOR Lee, J. M. and Sing, S. L. and Yeong, W. Y.
Title Bioprinting of Multimaterials with Computer-aided Design/Computer -aided Manufacturing [Abstract]
Year 2020
Journal/Proceedings International Journal of Bioprinting; Vol 6, No 1 (2020)
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DOI/URL URL
Abstract
Multimaterials deposition, a distinct advantage in bioprinting, overcomes material’s limitation in hydrogel-based bioprinting. Multimaterials are deposited in a build/support configuration to improve the structural integrity of three-dimensional bioprinted construct. A combination of rapid cross-linking hydrogel has been chosen for the build/support setup. The bioprinted construct was further chemically cross-linked to ensure a stable construct after print. This paper also proposes a file segmentation and preparation technique to be used in bioprinting for printing freeform structures.
AUTHOR Huang, Yen-Lin and Liang, Ching-Yeu and Ritz, Danilo and Coelho, Ricardo and Septiadi, Dedy and Estermann, Manuela and Cumin, Cécile and Rimmer, Natalie and Schötzau, Andreas and Núñez López, Mónica and Fedier, André and Konantz, Martina and Vlajnic, Tatjana and Calabrese, Diego and Lengerke, Claudia and David, Leonor and Rothen-Rutishauser, Barbara and Jacob, Francis and Heinzelmann-Schwarz, Viola
Title Collagen-rich omentum is a premetastatic niche for integrin α2-mediated peritoneal metastasis [Abstract]
Year 2020
Journal/Proceedings eLife
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DOI/URL DOI
Abstract
The extracellular matrix (ECM) plays critical roles in tumor progression and metastasis. However, the contribution of ECM proteins to early metastatic onset in the peritoneal cavity remains unexplored. Here, we suggest a new route of metastasis through the interaction of integrin alpha 2 (ITGA2) with collagens enriched in the tumor coinciding with poor outcome in patients with ovarian cancer. Using multiple gene-edited cell lines and patient-derived samples, we demonstrate that ITGA2 triggers cancer cell adhesion to collagen, promotes cell migration, anoikis resistance, mesothelial clearance, and peritoneal metastasis in vitro and in vivo. Mechanistically, phosphoproteomics identify an ITGA2-dependent phosphorylation of focal adhesion kinase and mitogen-activated protein kinase pathway leading to enhanced oncogenic properties. Consequently, specific inhibition of ITGA2-mediated cancer cell-collagen interaction or targeting focal adhesion signaling may present an opportunity for therapeutic intervention of metastatic spread in ovarian cancer.
AUTHOR Diloksumpan, Paweena and de Ruijter, Myl{`{e}}ne and Castilho, Miguel and Gbureck, Uwe and Vermonden, Tina and van Weeren, P. Ren{'{e}} and Malda, Jos and Levato, Riccardo
Title Combining multi-scale 3D printing technologies to engineer reinforced hydrogel-ceramic interfaces [Abstract]
Year 2020
Journal/Proceedings Biofabrication
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DOI/URL DOI
Abstract
Multi-material 3D printing technologies that resolve features at different lengths down to the microscale open new avenues for regenerative medicine, particularly in the engineering of tissue interfaces. Herein, extrusion printing of a bone-biomimetic ceramic ink and melt electrowriting (MEW) of spatially organized polymeric microfibres are integrated for the biofabrication of an osteochondral plug, with a mechanically reinforced bone-to-cartilage interface. A printable physiological temperature-setting bioceramic, based on α-tricalcium phosphate, nanohydroxyapatite and a custom-synthesized biodegradable and crosslinkable poloxamer, was developed as bone support. The mild setting reaction of the bone ink enabled us to print directly within melt electrowritten polycaprolactone meshes, preserving their micro-architecture. Ceramic-integrated MEW meshes protruded into the cartilage region of the composite plug, and were embedded with mechanically soft gelatin-based hydrogels, laden with articular cartilage chondroprogenitor cells. Such interlocking design enhanced the hydrogel-to-ceramic adhesion strength >6.5-fold, compared with non-interlocking fibre architectures, enabling structural stability during handling and surgical implantation in osteochondral defects ex vivo. Furthermore, the MEW meshes endowed the chondral compartment with compressive properties approaching those of native cartilage (20-fold reinforcement versus pristine hydrogel). The osteal and chondral compartment supported osteogenesis and cartilage matrix deposition in vitro, and the neo-synthesized cartilage matrix further contributed to the mechanical reinforcement at the ceramic-hydrogel interface. This multi-material, multi-scale 3D printing approach provides a promising strategy for engineering advanced composite constructs for the regeneration of musculoskeletal and connective tissue interfaces.
AUTHOR Müller, Michael and Fisch, Philipp and Molnar, Marc and Eggert, Sebastian and Binelli, Marco and Maniura-Weber, Katharina and Zenobi-Wong, Marcy
Title Development and thorough characterization of the processing steps of an ink for 3D printing for bone tissue engineering [Abstract]
Year 2020
Journal/Proceedings Materials Science and Engineering: C
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DOI/URL URL DOI
Abstract
Achieving reproducibility in the 3D printing of biomaterials requires a robust polymer synthesis method to reduce batch-to-batch variation as well as methods to assure a thorough characterization throughout the manufacturing process. Particularly biomaterial inks containing large solid fractions such as ceramic particles, often required for bone tissue engineering applications, are prone to inhomogeneity originating from inadequate mixing or particle aggregation which can lead to inconsistent printing results. The production of such an ink for bone tissue engineering consisting of gellan gum methacrylate (GG-MA), hyaluronic acid methacrylate and hydroxyapatite (HAp) particles was therefore optimized in terms of GG-MA synthesis and ink preparation process, and the ink's printability was thoroughly characterized to assure homogeneous and reproducible printing results. A new buffer mediated synthesis method for GG-MA resulted in consistent degrees of substitution which allowed the creation of large 5 g batches. We found that both the new synthesis as well as cryomilling of the polymer components of the ink resulted in a decrease in viscosity from 113 kPa·s to 11.3 kPa·s at a shear rate of 0.1 s−1 but increased ink homogeneity. The ink homogeneity was assessed through thermogravimetric analysis and a newly developed extrusion force measurement setup. The ink displayed strong inter-layer adhesion between two printed ink layers as well as between a layer of ink with and a layer without HAp. The large polymer batch production along with the characterization of the ink during the manufacturing process allows ink production in the gram scale and could be used in applications such as the printing of osteochondral grafts.
AUTHOR Tan, Wen See and Juhari, Muhammad Aidil Bin and Shi, Qian and Chen, Shengyang and Campolo, Domenico and Song, Juha
Title Development of a new additive manufacturing platform for direct freeform 3D printing of intrinsically curved flexible membranes [Abstract]
Year 2020
Journal/Proceedings Additive Manufacturing
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DOI/URL URL DOI
Abstract
The wearable technology market has been expanding from wearable medical devices for non-invasive continuous monitoring of patient vital signs to wearable devices for tracking fitness activities that any person can access. Regardless of their form or function, desirable characteristics of wearable devices are the ability to be flexible, conformal, and easily attachable to the human body. However, as the human body is intrinsically curved and irregular, flat devices often have poor interfacial adhesion with the human body. This often leads to interfacial delamination and eventual detachment of the device. Therefore, a new additive manufacturing (AM) platform, a direct freeform 3D printing process (DF3DP), is proposed to allow direct construction of intrinsically curved 3D surfaces during the material deposition phase without the need for any pre-shaped supporting molds or templates. This 3D freeform printing process involves a supporting matrix made up of calcium alginate microgels, printing material made from silicone ink, and freeform printing paths derived from customized G-codes that conform exactly to the scanned human surface profile. Curved meshes mimicking the human elbow were used as a demonstration. A static contact stability test showed that the printed 3D silicone mesh was highly conformal to the model elbow surface as compared to a 2D flat mesh. A dynamic contact stability test was also conducted by subjecting both meshes to 100 cycles of mechanical flexion and extension, proving that intrinsically curved surfaces can provide better contact stability for complex human body surfaces undergoing motion than can flat surfaces. These results have proven that intrinsically curved membranes or structures fabricated by DF3DP can reduce the interfacial shear stress and occurrence of cracks and delamination while maintaining structural integrity and stability during use without compromising the comfort of the users. Our approach can resolve interfacial issues in flexible substrates and has great potential for epidermal devices or soft robotics via its long-term sustainable performance.
AUTHOR Zhang, Hua and Cong, Yang and Osi, Amarachi Rosemary and Zhou, Yang and Huang, Fangcheng and Zaccaria, Remo P. and Chen, Jing and Wang, Rong and Fu, Jun
Title Direct 3D Printed Biomimetic Scaffolds Based on Hydrogel Microparticles for Cell Spheroid Growth [Abstract]
Year 2020
Journal/Proceedings Advanced Functional Materials
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DOI/URL DOI
Abstract
Abstract Biocompatible hydrogel inks with shear-thinning, appropriate yield strength, and fast self-healing are desired for 3D bioprinting. However, the lack of ideal 3D bioprinting inks with outstanding printability and high structural fidelity, as well as cell-compatibility, has hindered the progress of extrusion-based 3D bioprinting for tissue engineering. In this study, novel self-healable pre-cross-linked hydrogel microparticles (pcHμPs) of chitosan methacrylate (CHMA) and polyvinyl alcohol (PVA) hybrid hydrogels are developed and used as bioinks for extrusion-based 3D printing of scaffolds with high fidelity and biocompatibility. The pcHμPs display excellent shear thinning when injected through a syringe and subsequently self-heal into gels as shear forces are removed. Numerical simulations indicate that the pcHμPs experience a plug flow in the nozzle with minimal disturbance, which favors a steady and continuous printing. Moreover, the pcHμPs show a self-supportive yield strength (540 Pa), which is critical for the fidelity of printed constructs. A series of biomimetic constructs with very high aspect ratio and delicate fine structures are directly printed by using the pcHμP ink. The 3D printed scaffolds support the growth of bone-marrow-derived mesenchymal stem cells and formation of cell spheroids, which are most important for tissue engineering.
AUTHOR Sohrabi, Somayeh and kassir, Nour and Keshavarz Moraveji, Mostafa
Title Droplet microfluidics: fundamentals and its advanced applications [Abstract]
Year 2020
Journal/Proceedings RSC Advances
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DOI/URL DOI
Abstract
Droplet-based microfluidic systems have been shown to be compatible with many chemical and biological reagents and capable of performing a variety of operations that can be rendered programmable and reconfigurable. This platform has dimensional scaling benefits that have enabled controlled and rapid mixing of fluids in the droplet reactors{,} resulting in decreased reaction times. This{,} coupled with the precise generation and repeatability of droplet operations{,} has made the droplet-based microfluidic system a potent high throughput platform for biomedical research and applications. In addition to being used as micro-reactors ranging from the nano- to femtoliter (10−15 liters) range; droplet-based systems have also been used to directly synthesize particles and encapsulate many biological entities for biomedicine and biotechnology applications. For this{,} in the following article we will focus on the various droplet operations{,} as well as the numerous applications of the system and its future in many advanced scientific fields. Due to advantages of droplet-based systems{,} this technology has the potential to offer solutions to today{'}s biomedical engineering challenges for advanced diagnostics and therapeutics.
AUTHOR Huang, Boyang and Aslan, Enes and Jiang, Zhengyi and Daskalakis, Evangelos and Jiao, Mohan and Aldalbahi, Ali and Vyas, Cian and Bártolo, Paulo
Title Engineered dual-scale poly (ε-caprolactone) scaffolds using 3D printing and rotational electrospinning for bone tissue regeneration [Abstract]
Year 2020
Journal/Proceedings Additive Manufacturing
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DOI/URL URL DOI
Abstract
Large bone defects due to trauma or disease present a significant clinical challenge with limited efficacy of current therapies. A key aim is to develop biomimetic scaffolds that reflect the native tissue structure with 3D printing being an important enabling technology. However, the incorporation of multiple length scales and anisotropic features, mimicking the native architecture, is difficult with current processes. In this study, we propose a simple and versatile hybrid printing process using a screw-assisted additive manufacturing technique combined with rotational electrospinning to fabricate dual-scale anisotropic scaffolds. 3D microscale porous polycaprolactone (PCL) structures with highly aligned nanoscale fibres were successfully produced layer-by-layer. The scaffolds were morphological, mechanical and biological characterised. Human adipose-derived stem cells (hADSCs) were seeded on the hybrid scaffold to evaluate the effects of structural and anisotropic topographic cues on cell attachment, proliferation and osteogenesis differentiation. Results show that the 3D printed microscale structures have uniform and well-defined geometries and the alignment of nanoscale electrospun fibres increases by increasing the electrospinning rotational velocity. Mechanical results show that there is no significant difference between 3D printed scaffolds with or without electrospun meshes. In vitro results show higher cell seeding efficiency and proliferation in dual-scale scaffolds with high density electrospun meshes. A more stretched and elongated cell morphology was observed in aligned nanofibre scaffolds showing higher anisotropic cytoskeletal organization than 3D printed PCL scaffolds without electrospun meshes. The dual-scale scaffolds present improved overall osteogenic markers expressions (COL-1, ALP and OCN). However, no statistical difference between normalised osteogenic marker expressions were observed between dual-scale scaffolds and 3D printed scaffolds. This might be attributed to the poor bioactivity of the substrate material, PCL, suggesting topographical cues might not be sufficient to stimulate cell fate towards to an osteogenic linage. The results suggest that the proposed fabrication strategy is a promising approach for the design of novel bone scaffolds to modulate cell fates by integrating the topographic cue reported in this paper with biochemical cues associated to the use of more bioactive materials.
AUTHOR Lee, Jia Min and Yeong, Wai Yee
Title Engineering macroscale cell alignment through coordinated toolpath design using support-assisted 3D bioprinting [Abstract]
Year 2020
Journal/Proceedings Journal of The Royal Society Interface
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DOI/URL DOI
Abstract
Aligned cells provide direction-dependent mechanical properties that influence biological and mechanical function in native tissues. Alignment techniques such as casting and uniaxial stretching cannot fully replicate the complex fibre orientation of native tissue such as the heart. In this study, bioprinting is used to direct the orientation of cell alignment. A 0°–90° grid structure was printed to assess the robustness of the support-assisted bioprinting technique. The variation in the angles of the grid pattern is designed to mimic the differences in fibril orientation of native tissues, where angles of cell alignment vary across the different layers. Through bioprinting of a cell–hydrogel mixture, C2C12 cells displayed directed alignment along the longitudinal axis of printed struts. Cell alignment is induced through firstly establishing structurally stable constructs (i.e. distinct 0°–90° structures) and secondly, allowing cells to dynamically remodel the bioprinted construct. Herein reports a method of inducing a macroscale level of controlled cell alignment with angle variation. This was not achievable both in terms of methods (i.e. conventional alignment techniques such as stretching and electrical stimulation) and magnitude (i.e. hydrogel features with less than 100 µm features).
AUTHOR Song, Jie-Liang and Fu, Xin-Ye and Raza, Ali and Shen, Nai-An and Xue, Ya-Qi and Wang, Hua-Jie and Wang, Jin-Ye
Title Enhancement of mechanical strength of TCP-alginate based bioprinted constructs [Abstract]
Year 2020
Journal/Proceedings Journal of the Mechanical Behavior of Biomedical Materials
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DOI/URL URL DOI
Abstract
To overcome the mechanical drawback of bioink, we proposed a supporter model to enhance the mechanical strength of bioprinted 3D constructs, in which a unit-assembly idea was involved. Based on Computed Tomography images of critical-sized rabbit bone defect, the 3D re-construction was accomplished by a sequenced process using Mimics 17.0, BioCAM and BioCAD software. 3D constructs were bioprinted using polycaprolactone (PCL) ink for the outer supporter under extrusion mode, and cell-laden tricalcium phosphate (TCP)/alginate bioink for the inner filler under air pressure dispensing mode. The relationship of viscosity of bioinks, 3D bioprinting pressure, TCP/alginate ratio and cell survival were investigated by the shear viscosities analysis, live/dead cell test and cell-counting kit 8 measurement. The viscosity of bioinks at 1.0 s−1-shear rate could be adjusted within the range of 1.75 ± 0.29 Pa·s to 155.65 ± 10.86 Pa·s by changing alginate concentration, corresponding to 10 kPa–130 kPa of printing pressure. This design with PCL supporter could significantly enhance the compressive strength and compressive modulus of standardized 3D mechanical testing specimens up to 2.15 ± 0.14 MPa to 2.58 ± 0.09 MPa, and 42.83 ± 4.75 MPa to 53.12 ± 1.19 MPa, respectively. Cells could maintain the high viability (over 80%) under the given printing pressure but cell viability declined with the increase of TCP content. Cell survival after experiencing 7 days of cell culture could be achieved when the ratio of TCP/alginate was 1 : 4. All data supported the feasibility of the supporter and unit-assembly model to enhance mechanical properties of bioprinted 3D constructs.
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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DOI/URL DOI
Abstract
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 Dubey, Nileshkumar and Ferreira, Jessica A. and Malda, Jos and Bhaduri, Sarit B. and Bottino, Marco C.
Title Extracellular Matrix/Amorphous Magnesium Phosphate Bioink for 3D Bioprinting of Craniomaxillofacial Bone Tissue [Abstract]
Year 2020
Journal/Proceedings ACS Applied Materials & Interfaces
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DOI/URL DOI
Abstract
Bioprinting, a promising field in regenerative medicine, holds great potential to create three-dimensional, defect-specific vascularized bones with tremendous opportunities to address unmet craniomaxillofacial reconstructive challenges. A cytocompatible bioink is a critical prerequisite to successfully regenerate functional bone tissue. Synthetic self-assembling peptides have a nanofibrous structure resembling the native extracellular matrix (ECM), making them an excellent bioink component. Amorphous magnesium phosphates (AMPs) have shown greater levels of resorption while maintaining high biocompatibility, osteoinductivity, and low inflammatory response, as compared to their calcium phosphate counterparts. Here, we have established a novel bioink formulation (ECM/AMP) that combines an ECM-based hydrogel containing 2% octapeptide FEFEFKFK and 98% water with AMP particles to realize high cell function with desirable bioprintability. We analyzed the osteogenic differentiation of dental pulp stem cells (DPSCs) encapsulated in the bioink, as well as in vivo bone regeneration, to define the potential of the formulated bioink as a growth factor-free bone-forming strategy. Cell-laden AMP-modified bioprinted constructs showed an improved cell morphology but similar cell viability (∼90%) compared to their AMP-free counterpart. In functional assays, the cell-laden bioprinted constructs modified with AMP exhibited a high level of mineralization and osteogenic gene expression without the use of growth factors, thus suggesting that the presence of AMP-triggered DPSCs’ osteogenic differentiation. Cell-free ECM-based bioprinted constructs were implanted in vivo. In comparison with the ECM group, bone volume per total volume for ECM/1.0AMP was approximately 1.7- and 1.4-fold higher at 4 and 8 weeks, respectively. Further, a significant increase in the bone density was observed in ECM/1.0AMP from 4 to 8 weeks. These results demonstrate that the presence of AMP in the bioink significantly increased bone formation, thus showing promise for in situ bioprinting strategies. We foresee significant potential in translating this innovative bioink toward the regeneration of patient-specific bone tissue for regenerative dentistry. Bioprinting, a promising field in regenerative medicine, holds great potential to create three-dimensional, defect-specific vascularized bones with tremendous opportunities to address unmet craniomaxillofacial reconstructive challenges. A cytocompatible bioink is a critical prerequisite to successfully regenerate functional bone tissue. Synthetic self-assembling peptides have a nanofibrous structure resembling the native extracellular matrix (ECM), making them an excellent bioink component. Amorphous magnesium phosphates (AMPs) have shown greater levels of resorption while maintaining high biocompatibility, osteoinductivity, and low inflammatory response, as compared to their calcium phosphate counterparts. Here, we have established a novel bioink formulation (ECM/AMP) that combines an ECM-based hydrogel containing 2% octapeptide FEFEFKFK and 98% water with AMP particles to realize high cell function with desirable bioprintability. We analyzed the osteogenic differentiation of dental pulp stem cells (DPSCs) encapsulated in the bioink, as well as in vivo bone regeneration, to define the potential of the formulated bioink as a growth factor-free bone-forming strategy. Cell-laden AMP-modified bioprinted constructs showed an improved cell morphology but similar cell viability (∼90%) compared to their AMP-free counterpart. In functional assays, the cell-laden bioprinted constructs modified with AMP exhibited a high level of mineralization and osteogenic gene expression without the use of growth factors, thus suggesting that the presence of AMP-triggered DPSCs’ osteogenic differentiation. Cell-free ECM-based bioprinted constructs were implanted in vivo. In comparison with the ECM group, bone volume per total volume for ECM/1.0AMP was approximately 1.7- and 1.4-fold higher at 4 and 8 weeks, respectively. Further, a significant increase in the bone density was observed in ECM/1.0AMP from 4 to 8 weeks. These results demonstrate that the presence of AMP in the bioink significantly increased bone formation, thus showing promise for in situ bioprinting strategies. We foresee significant potential in translating this innovative bioink toward the regeneration of patient-specific bone tissue for regenerative dentistry.
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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DOI/URL URL DOI
Abstract
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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Abstract
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 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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Abstract
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 Hauptstein, Julia and Böck, Thomas and Bartolf-Kopp, Michael and Forster, Leonard and Stahlhut, Philipp and Nadernezhad, Ali and Blahetek, Gina and Zernecke-Madsen, Alma and Detsch, Rainer and Jüngst, Tomasz and Groll, Jürgen and Teßmar, Jörg and Blunk, Torsten
Title Hyaluronic Acid-Based Bioink Composition Enabling 3D Bioprinting and Improving Quality of Deposited Cartilaginous Extracellular Matrix [Abstract]
Year 2020
Journal/Proceedings Advanced Healthcare Materials
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Abstract
Abstract In 3D bioprinting, bioinks with high concentrations of polymeric materials are frequently used to enable fabrication of 3D cell-hydrogel constructs with sufficient stability. However, this is often associated with restricted cell bioactivity and an inhomogeneous distribution of newly produced extracellular matrix (ECM). Therefore, this study investigates bioink compositions based on hyaluronic acid (HA), an attractive material for cartilage regeneration, which allow for reduction of polymer content. Thiolated HA and allyl-modified poly(glycidol) in varying concentrations are UV-crosslinked. To adapt bioinks to poly(ε-caprolactone) (PCL)-supported 3D bioprinting, the gels are further supplemented with 1 wt% unmodified high molecular weight HA (hmHA) and chondrogenic differentiation of incorporated human mesenchymal stromal cells is assessed. Strikingly, addition of hmHA to gels with a low polymer content (3 wt%) results in distinct increase of construct quality with a homogeneous ECM distribution throughout the constructs, independent of the printing process. Improved ECM distribution in those constructs is associated with increased construct stiffness after chondrogenic differentiation, as compared to higher concentrated constructs (10 wt%), which only show pericellular matrix deposition. The study contributes to effective bioink development, demonstrating dual function of a supplement enabling PCL-supported bioprinting and at the same time improving biological properties of the resulting constructs.
AUTHOR De Moor, Lise and Fernandez, Sélina and Vercruysse, Chris and Tytgat, Liesbeth and Asadian, Mahtab and De Geyter, Nathalie and Van Vlierberghe, Sandra and Dubruel, Peter and Declercq, Heidi
Title Hybrid Bioprinting of Chondrogenically Induced Human Mesenchymal Stem Cell Spheroids [Abstract]
Year 2020
Journal/Proceedings Frontiers in Bioengineering and Biotechnology
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Abstract
To date, the treatment of articular cartilage lesions remains challenging. A promising strategy for the development of new regenerative therapies is hybrid bioprinting, combining the principles of developmental biology, biomaterial science, and 3D bioprinting. In this approach, scaffold-free cartilage microtissues with small diameters are used as building blocks, combined with a photo-crosslinkable hydrogel and subsequently bioprinted. Spheroids of human bone marrow-derived mesenchymal stem cells (hBM-MSC) are created using a high-throughput microwell system and chondrogenic differentiation is induced during 42 days by applying chondrogenic culture medium and low oxygen tension (5%). Stable and homogeneous cartilage spheroids with a mean diameter of 116 ± 2.80 μm, which is compatible with bioprinting, were created after 14 days of culture and a glycosaminoglycans (GAG)- and collagen II-positive extracellular matrix (ECM) was observed. Spheroids were able to assemble at random into a macrotissue, driven by developmental biology tissue fusion processes, and after 72 h of culture, a compact macrotissue was formed. In a directed assembly approach, spheroids were assembled with high spatial control using the bio-ink based extrusion bioprinting approach. Therefore, 14-day spheroids were combined with a photo-crosslinkable methacrylamide-modified gelatin (gelMA) as viscous printing medium to ensure shape fidelity of the printed construct. The photo-initiators Irgacure 2959 and Li-TPO-L were evaluated by assessing their effect on bio-ink properties and the chondrogenic phenotype. The encapsulation in gelMA resulted in further chondrogenic maturation observed by an increased production of GAG and a reduction of collagen I. Moreover, the use of Li-TPO-L lead to constructs with lower stiffness which induced a decrease of collagen I and an increase in GAG and collagen II production. After 3D bioprinting, spheroids remained viable and the cartilage phenotype was maintained. Our findings demonstrate that hBM-MSC spheroids are able to differentiate into cartilage microtissues and display a geometry compatible with 3D bioprinting. Furthermore, for hybrid bioprinting of these spheroids, gelMA is a promising material as it exhibits favorable properties in terms of printability and it supports the viability and chondrogenic phenotype of hBM-MSC microtissues. Moreover, it was shown that a lower hydrogel stiffness enhances further chondrogenic maturation after bioprinting.
AUTHOR Strauß, Svenja and Meutelet, Rafaela and Radosevic, Luka and Gretzinger, Sarah and Hubbuch, Jürgen
Title Image analysis as PAT-Tool for use in extrusion-based bioprinting [Abstract]
Year 2020
Journal/Proceedings Bioprinting
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Abstract
The technology of bioprinting is arousing a growing interest in biopharmaceutical research and industry. In order to accelerate process development in the field of bioprinting, image-based analysis methods are non-invasive, time- and cost-saving tools which are usable for printer characterization, bioink printability evaluation, and process optimization. Image processing can also be used for the study of reproducibility, since reliable production is important in the transition from research to industrial application, and more precisely to clinical studies. This study revolves around the establishment of an automated and image-based line analysis method for bioprinting applications which enables an easy comparison of 3D-printed lines. Diverse rheological properties of bioinks and the printing process affect the geometry of the resulting object. The line represents a simple geometry, where the influence of the rheological properties and printing parameters is directly apparent. Therefore, a method for line analysis was developed on the basis of image recognition. At first, the method is tested for several substances such as Nivea®, pure and colored Kolliphor solutions, and two commercially available hydrogel formulations which can be used as bioinks. These are Biogelx™-ink-RGD by Biogelx and Cellink® Bioink by Cellink. The examination of limitations showed that transparent materials such as Kolliphor-based solutions cannot be analyzed with the developed method whereas opaque materials such as Nivea® and both bioinks can be analyzed. In the course of process characterization, the method was used to investigate the shrinkage behavior for both bionks. With the help of the line analysis tool, a shrinkage behavior of both bioinks was demonstrated and thus, process time could be identified as a critical process parameter.
AUTHOR López-Carrasco, Amparo and Martín-Vañó, Susana and Burgos-Panadero, Rebeca and Monferrer, Ezequiel and Berbegall, Ana P. and Fernández-Blanco, Beatriz and Navarro, Samuel and Noguera, Rosa
Title Impact of extracellular matrix stiffness on genomic heterogeneity in MYCN-amplified neuroblastoma cell line [Abstract]
Year 2020
Journal/Proceedings Journal of Experimental & Clinical Cancer Research
Reftype López-Carrasco2020
DOI/URL DOI
Abstract
Increased tissue stiffness is a common feature of malignant solid tumors, often associated with metastasis and poor patient outcomes. Vitronectin, as an extracellular matrix anchorage glycoprotein related to a stiff matrix, is present in a particularly increased quantity and specific distribution in high-risk neuroblastoma. Furthermore, as cells can sense and transform the proprieties of the extracellular matrix into chemical signals through mechanotransduction, genotypic changes related to stiffness are possible.
AUTHOR Fisch, Philipp and Holub, Martin and Zenobi-Wong, Marcy
Title Improved accuracy and precision of bioprinting through progressive cavity pump-controlled extrusion [Abstract]
Year 2020
Journal/Proceedings bioRxiv
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Abstract
3D bioprinting has seen a tremendous growth in recent years in a variety of fields such as tissue and organ models, drug testing and regenerative medicine. This growth has led researchers and manufacturers to continuously advance and develop novel bioprinting techniques and materials. Although new bioprinting methods are emerging (e.g. contactless and volumetric bioprinting), micro-extrusion bioprinting remains the most widely used method. Micro-extrusion bioprinting, however, is still largely dependent on the conventional pneumatic extrusion process, which relies heavily on homogenous biomaterial inks and bioinks to maintain a constant material flowrate. Augmenting the functionality of the bioink with the addition of nanoparticles, cells or biopolymers can induce inhomogeneities resulting in uneven material flow during printing and/or clogging of the nozzle, leading to defects in the printed construct. In this work, we evaluated a novel extrusion technique based on a miniaturized progressive cavity pump. We compared the accuracy and precision of this system to the pneumatic extrusion system and tested both for their effect on cell viability after extrusion. The progressive cavity pump achieved a significantly higher accuracy and precision compared to the pneumatic system while maintaining good viability and was able to maintain its reliability independently of the bioink composition, printing speed or nozzle size. Progressive cavity pumps are a promising tool for bioprinting and could help provide standardized and validated bioprinted constructs while leaving the researcher more freedom in the design of the bioinks with increased functionality.
AUTHOR Hou, Yanhao and Wang, Weiguang and Bartolo, Paulo Jorge Da Silva
Title Investigating the Effect of Carbon Nanomaterials Reinforcing Poly(Ε-Caprolactone) Scaffolds for Bone Repair Applications [Abstract]
Year 2020
Journal/Proceedings International Journal of Bioprinting
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Abstract
Scaffolds, three-dimensional (3D) substrates providing appropriate mechanical support and biological environments for new tissue formation, are the most common approaches in tissue engineering. To improve scaffold properties such as mechanical properties, surface characteristics, biocompatibility and biodegradability, different types of fillers have been used reinforcing biocompatible and biodegradable polymers. This paper investigates and compares the mechanical and biological behaviors of 3D printed poly(ε-caprolactone) scaffolds reinforced with graphene (G) and graphene oxide (GO) at different concentrations. Results show that contrary to G which improves mechanical properties and enhances cell attachment and proliferation, GO seems to show some cytotoxicity, particular at high contents.
AUTHOR Šimková, Kateřina and Thormann, Ursula and Imanidis, Georgios
Title Investigation of drug dissolution and uptake from low-density DPI formulations in an impactor–integrated cell culture model [Abstract]
Year 2020
Journal/Proceedings European Journal of Pharmaceutics and Biopharmaceutics
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Abstract
Besides deposition, pulmonary bioavailability is determined by dissolution of particles in the scarce epithelial fluid and by cellular API uptake. In the present work, we have developed an experimental in vitro model, which is combining the state-of-the-art next generation impactor (NGI), used for aerodynamic performance assessment of inhalation products, with a culture of human alveolar A549 epithelial cells to study the fate of inhaled drugs following lung deposition. The goal was to investigate five previously developed nano-milled and spray-dried budesonide formulations and to examine the suitability of the in vitro test model. The NGI dissolution cups of stages 3, 4, and 5 were transformed to accommodate cell culture inserts while assuring minimal interference with the air flow. A549 cells were cultivated at the air–liquid interface on Corning® Matrigel® -coated inserts. After deposition of aerodynamically classified powders on the cell cultures, budesonide amount was determined on the cell surface, in the interior of the cell monolayer, and in the basal solution for four to eight hours. Significant differences in the total deposited drug amount and the amount remaining on the cell surface at the end of the experiment were found between different formulations and NGI stages. Roughly 50% of budesonide was taken up by the cells and converted to a large extent to its metabolic conjugate with oleic acid for all formulations and stages. Prolonged time required for complete drug dissolution and cell uptake in case of large deposited powder amounts suggested initial drug saturation of the surfactant layer of the cell surface. Discrimination between formulations with respect to time scale of dissolution and cell uptake was possible with the present test model providing useful insights into the biopharmaceutical performance of developed formulations that may be relevant for predicting local bioavailability. The absolute quantitative result of cell uptake and permeation into the systemic compartment is unreliable, though, because of partly compromised cell membrane integrity due to particle impaction and professed leakiness of A549 monolayer tight junctions, respectively.
AUTHOR Rafiee, Mohammad and Farahani, Rouhollah D. and Therriault, Daniel
Title Multi-Material 3D and 4D Printing: A Survey [Abstract]
Groups
Year 2020
Journal/Proceedings Advanced Science
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Abstract
Abstract Recent advances in multi-material 3D and 4D printing (time as the fourth dimension) show that the technology has the potential to extend the design space beyond complex geometries. The potential of these additive manufacturing (AM) technologies allows for functional inclusion in a low-cost single-step manufacturing process. Different composite materials and various AM technologies can be used and combined to create customized multi-functional objects to suit many needs. In this work, several types of 3D and 4D printing technologies are compared and the advantages and disadvantages of each technology are discussed. The various features and applications of 3D and 4D printing technologies used in the fabrication of multi-material objects are reviewed. Finally, new avenues for the development of multi-material 3D and 4D printed objects are proposed, which reflect the current deficiencies and future opportunities for inclusion by AM.
AUTHOR Ruiz-Cantu, Laura and Gleadall, Andrew and Faris, Callum and Segal, Joel and Shakesheff, Kevin and Yang, Jing
Title Multi-material 3D bioprinting of porous constructs for cartilage regeneration [Abstract]
Year 2020
Journal/Proceedings Materials Science and Engineering: C
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Abstract
The current gold standard for nasal reconstruction after rhinectomy or severe trauma includes transposition of autologous cartilage grafts in conjunction with coverage using an autologous skin flap. Harvesting autologous cartilage requires a major additional procedure that may create donor site morbidity. Major nasal reconstruction also requires sculpting autologous cartilages to form a cartilage framework, which is complex, highly skill-demanding and very time consuming. These limitations have prompted facial reconstructive surgeons to explore different techniques such as tissue engineered cartilage. This work explores the use of multi-material 3D bioprinting with chondrocyte-laden gelatin methacrylate (GelMA) and polycaprolactone (PCL) to fabricate constructs that can potentially be used for nasal reconstruction. In this study, we have investigated the effect of 3D manufacturing parameters including temperature, needle gauge, UV exposure time, and cell carrier formulation (GelMA) on the viability and functionality of chondrocytes in bioprinted constructs. Furthermore, we printed chondrocyte-laden GelMA and PCL into composite constructs to combine biological and mechanical properties. It was found that 20% w/v GelMA was the best concentration for the 3D bioprinting of the chondrocytes without comprising the scaffold's porous structure and cell functionality. In addition, the 3D bioprinted constructs showed neocartilage formation and similar mechanical properties to nasal alar cartilage after a 50-day culture period. Neocartilage formation was also observed in the composite constructs evidenced by the presence of glycosaminoglycans and collagen type II. This study shows the feasibility of manufacturing neocartilage using chondrocytes/GelMA/PCL 3D bioprinted porous constructs which could be applied as a method for fabricating implants for nose reconstruction.
AUTHOR Rupp, Harald and Binder, Wolfgang H.
Title Multicomponent Stress-Sensing Composites Fabricated by 3D-Printing Methodologies [Abstract]
Year 2020
Journal/Proceedings Macromolecular Rapid Communications
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Abstract The preparation and characterization of mechanoresponsive, 3D-printed composites are reported using a dual-printing setup for both, liquid dispensing and fused-deposition-modeling. The here reported stress-sensing materials are based on high- and low molecular weight mechanophores, including poly(ε-caprolactone)-, polyurethane-, and alkyl(C11)-based latent copper(I)bis(N-heterocyclic carbenes), which can be activated by compression to trigger a fluorogenic, copper(I)-catalyzed azide/alkyne “click”-reaction of an azide-functionalized fluorescent dye inside a bulk polymeric material. Focus is placed on the printability and postprinting activity of the latent mechanophores and the fluorogenic “click”-components. The multicomponent specimen containing both, azide and alkyne, are manufactured via a 3D-printer to place the components separately inside the specimen into void spaces generated during the FDM-process, which subsequently are filled with liquids using a separate liquid dispenser, located within the same 3D-printing system. The low-molecular weight mechanophores bearing the alkyl-C11 chains display the best printability, yielding a mechanochemical response after the 3D-printing process.
AUTHOR Plou, Javier and García, Isabel and Charconnet, Mathias and Astobiza, Ianire and García-Astrain, Clara and Matricardi, Cristiano and Mihi, Agustín and Carracedo, Arkaitz and Liz-Marzán, Luis M.
Title Multiplex SERS Detection of Metabolic Alterations in Tumor Extracellular Media [Abstract]
Year 2020
Journal/Proceedings Advanced Functional Materials
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Abstract The composition and intercellular interactions of tumor cells in the tissues dictate the biochemical and metabolic properties of the tumor microenvironment. The metabolic rewiring has a profound impact on the properties of the microenvironment, to an extent that monitoring such perturbations could harbor diagnostic and therapeutic relevance. A growing interest in these phenomena has inspired the development of novel technologies with sufficient sensitivity and resolution to monitor metabolic alterations in the tumor microenvironment. In this context, surface-enhanced Raman scattering (SERS) can be used for the label-free detection and imaging of diverse molecules of interest among extracellular components. Herein, the application of nanostructured plasmonic substrates comprising Au nanoparticles, self-assembled as ordered superlattices, to the precise SERS detection of selected tumor metabolites, is presented. The potential of this technology is first demonstrated through the analysis of kynurenine, a secreted immunomodulatory derivative of the tumor metabolism and the related molecules tryptophan and purine derivatives. SERS facilitates the unambiguous identification of trace metabolites and allows the multiplex detection of their characteristic fingerprints under different conditions. Finally, the effective plasmonic SERS substrate is combined with a hydrogel-based three-dimensional cancer model, which recreates the tumor microenvironment, for the real-time imaging of metabolite alterations and cytotoxic effects on tumor cells.
AUTHOR Lee, Mihyun and Bae, Kraun and Levinson, Clara and Zenobi-Wong, Marcy
Title Nanocomposite bioink exploits dynamic covalent bonds between nanoparticles and polysaccharides for precision bioprinting [Abstract]
Year 2020
Journal/Proceedings Biofabrication
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Abstract
The field of bioprinting has made significant recent progress towards engineering tissues with increasing complexity and functionality. It remains challenging, however, to develop bioinks with optimal biocompatibility and good printing fidelity. Here, we demonstrate enhanced printability of a polymer-based bioink based on dynamic covalent linkages between nanoparticles (NPs) and polymers, which retains good biocompatibility. Amine-presenting silica NPs (ca. 45 nm) were added to a polymeric ink containing oxidized alginate (OxA). The formation of reversible imine bonds between amines on the NPs and aldehydes of OxA lead to significantly improved rheological properties and high printing fidelity. In particular, the yield stress increased with increasing amounts of NPs (14.5 Pa without NPs, 79 Pa with 2 wt% NPs). In addition, the presence of dynamic covalent linkages in the gel provided improved mechanical stability over 7 d compared to ionically crosslinked gels. The nanocomposite ink retained high printability and mechanical strength, resulting in generation of centimeter-scale porous constructs and an ear structure with overhangs and high structural fidelity. Furthermore, the nanocomposite ink supported both in vitro and in vivo maturation of bioprinted gels containing chondrocytes. This approach based on simple oxidation can be applied to any polysaccharide, thus the widely applicability of the method is expected to advance the field towards the goal of precision bioprinting.
AUTHOR Hou, Yanhao and Wang, Weiguang and Bártolo, Paulo
Title Novel Poly(ɛ-caprolactone)/Graphene Scaffolds for Bone Cancer Treatment and Bone Regeneration [Abstract]
Year 2020
Journal/Proceedings 3D Printing and Additive Manufacturing
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Abstract
Scaffold-based bone tissue engineering is the most relevant approach for critical-sized bone defects. It is based on the use of three-dimensional substrates to provide the appropriate biomechanical environment for bone regeneration. Despite some successful results previously reported, scaffolds were never designed for disease treatment applications. This article proposes a novel dual-functional scaffold for cancer applications, comprising both treatment and regeneration functions. These functions are achieved by combining a biocompatible and biodegradable polymer and graphene. Results indicate that high concentrations of graphene enhance the mechanical properties of the scaffolds, also increasing the inhibition on cancer cell viability and proliferation.
AUTHOR Lim, Khoon S. and Abinzano, Florencia and Bernal, Paulina Nuñez and Albillos Sanchez, Ane and Atienza-Roca, Pau and Otto, Iris A. and Peiffer, Quentin C. and Matsusaki, Michiya and Woodfield, Tim B. F. and Malda, Jos and Levato, Riccardo
Title One-Step Photoactivation of a Dual-Functionalized Bioink as Cell Carrier and Cartilage-Binding Glue for Chondral Regeneration [Abstract]
Year 2020
Journal/Proceedings Advanced Healthcare Materials
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Abstract
Abstract Cartilage defects can result in pain, disability, and osteoarthritis. Hydrogels providing a chondroregeneration-permissive environment are often mechanically weak and display poor lateral integration into the surrounding cartilage. This study develops a visible-light responsive gelatin ink with enhanced interactions with the native tissue, and potential for intraoperative bioprinting. A dual-functionalized tyramine and methacryloyl gelatin (GelMA-Tyr) is synthesized. Photo-crosslinking of both groups is triggered in a single photoexposure by cell-compatible visible light in presence of tris(2,2′-bipyridyl)dichlororuthenium(II) and sodium persulfate as initiators. Neo-cartilage formation from embedded chondroprogenitor cells is demonstrated in vitro, and the hydrogel is successfully applied as bioink for extrusion-printing. Visible light in situ crosslinking in cartilage defects results in no damage to the surrounding tissue, in contrast to the native chondrocyte death caused by UV light (365–400 nm range), commonly used in biofabrication. Tyramine-binding to proteins in native cartilage leads to a 15-fold increment in the adhesive strength of the bioglue compared to pristine GelMA. Enhanced adhesion is observed also when the ink is extruded as printable filaments into the defect. Visible-light reactive GelMA-Tyr bioinks can act as orthobiologic carriers for in situ cartilage repair, providing a permissive environment for chondrogenesis, and establishing safe lateral integration into chondral defects.
AUTHOR Diloksumpan, Paweena and Bolaños, Rafael Vindas and Cokelaere, Stefan and Pouran, Behdad and de Grauw, Janny and van Rijen, Mattie and van Weeren, René and Levato, Riccardo and Malda, Jos
Title Orthotopic Bone Regeneration within 3D Printed Bioceramic Scaffolds with Region-Dependent Porosity Gradients in an Equine Model [Abstract]
Year 2020
Journal/Proceedings Advanced Healthcare Materials
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Abstract
Abstract The clinical translation of three-dimensionally printed bioceramic scaffolds with tailored architectures holds great promise toward the regeneration of bone to heal critical-size defects. Herein, the long-term in vivo performance of printed hydrogel-ceramic composites made of methacrylated-oligocaprolactone-poloxamer and low-temperature self-setting calcium-phosphates is assessed in a large animal model. Scaffolds printed with different internal architectures, displaying either a designed porosity gradient or a constant pore distribution, are implanted in equine tuber coxae critical size defects. Bone ingrowth is challenged and facilitated only from one direction via encasing the bioceramic in a polycaprolactone shell. After 7 months, total new bone volume and scaffold degradation are significantly greater in structures with constant porosity. Interestingly, gradient scaffolds show lower extent of remodeling and regeneration even in areas having the same porosity as the constant scaffolds. Low regeneration in distal regions from the interface with native bone impairs ossification in proximal regions of the construct, suggesting that anisotropic architectures modulate the cross-talk between distant cells within critical-size defects. The study provides key information on how engineered architectural patterns impact osteoregeneration in vivo, and also indicates the equine tuber coxae as promising orthotopic model for studying materials stimulating bone formation.
AUTHOR Chen, Grona and Xu, Yihua and Chi Lip Kwok, Philip and Kang, Lifeng
Title Pharmaceutical Applications of 3D Printing [Abstract]
Year 2020
Journal/Proceedings Additive Manufacturing
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Abstract
Although 3D printing (3DP) has long been an integral part of industries such as aviation and automotive, its use in healthcare, especially the pharmaceutical industry, is relatively new and currently receiving close attention. At the beginning of 2018, we reviewed the applications of 3DP for drug delivery and drug testing [1]. Due to the rapid development of this field, it is necessary to summarize the latest development in this field after 2 years. In this article, we reviewed the three major areas in pharmaceutical applications. First, drug delivery system is the most studied subject, including controlled release, polypills, gastrofloating, orodispersibles and microneedles. Second, 3DP also helped the development of pharmaceutical devices, including pharmacy dispensing aids and drug eluting devices. Lastly, we reviewed the pharmaceutical models for drug testing, covering acellular and cellular models. We also summarized the materials used in the mentioned articles and their regulatory status for pharmaceutical applications to provide references for future research.
AUTHOR Bedell, Matthew L. and Navara, Adam M. and Du, Yingying and Zhang, Shengmin and Mikos, Antonios G.
Title Polymeric Systems for Bioprinting [Abstract]
Groups Bioinks
Year 2020
Journal/Proceedings Chemical Reviews
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Abstract
Bioprinting is rapidly being adopted as a major method for fabricating tissue engineering constructs. Through the precise deposition of cell- and bioactive molecule-laden materials, bioprinting offers researchers a means to create biological constructs with enhanced spatial complexity that more closely mimics native tissue. The vast majority of materials used in bioprinting have been polymers due to their suitability toward resembling the cellular environment and the variety of methods available to process polymeric systems in ambient or relatively mild chemical and environmental conditions. In this review, we will discuss in detail the wide variety of natural and synthetic polymers that have been employed as inks in bioprinting. We will review recent bioprinting innovations, such as increasing architectural complexity and cell viability in heterogeneous tissue constructs, which allow for the investigation of biological questions that could not be addressed before. We will also survey nascent fields of study that promise to further advance the development of novel biofabrication technologies in the field, such as 4D bioprinting and the inclusion of nanomaterials. To conclude, we will examine some of the necessary steps that must take place to bring this technology to commercial markets and facilitate its use in clinical therapies. Bioprinting is rapidly being adopted as a major method for fabricating tissue engineering constructs. Through the precise deposition of cell- and bioactive molecule-laden materials, bioprinting offers researchers a means to create biological constructs with enhanced spatial complexity that more closely mimics native tissue. The vast majority of materials used in bioprinting have been polymers due to their suitability toward resembling the cellular environment and the variety of methods available to process polymeric systems in ambient or relatively mild chemical and environmental conditions. In this review, we will discuss in detail the wide variety of natural and synthetic polymers that have been employed as inks in bioprinting. We will review recent bioprinting innovations, such as increasing architectural complexity and cell viability in heterogeneous tissue constructs, which allow for the investigation of biological questions that could not be addressed before. We will also survey nascent fields of study that promise to further advance the development of novel biofabrication technologies in the field, such as 4D bioprinting and the inclusion of nanomaterials. To conclude, we will examine some of the necessary steps that must take place to bring this technology to commercial markets and facilitate its use in clinical therapies.
AUTHOR Cernecu, Alexandra and Lungu, Adriana and Stancu, Izabela Cristina and Vasile, Eugeniu and Iovu, Horia
Title Polysaccharide-Based 3D Printing Inks Supplemented with Additives
Year 2020
Journal/Proceedings University Politechnica of Bucharest Scientific Bulletin
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AUTHOR Schwab, Andrea and Levato, Riccardo and D’Este, Matteo and Piluso, Susanna and Eglin, David and Malda, Jos
Title Printability and Shape Fidelity of Bioinks in 3D Bioprinting [Abstract]
Year 2020
Journal/Proceedings Chemical Reviews
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Abstract
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. 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.
AUTHOR Athanasiadis, Markos and Afanasenkau, Dzmitry and Derks, Wouter and Tondera, Christoph and Murganti, Francesca and Busskamp, Volker and Bergmann, Olaf and Minev, Ivan R.
Title Printed elastic membranes for multimodal pacing and recording of human stem-cell-derived cardiomyocytes [Abstract]
Year 2020
Journal/Proceedings npj Flexible Electronics
Reftype Athanasiadis2020
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Abstract
Bioelectronic interfaces employing arrays of sensors and bioactuators are promising tools for the study, repair and engineering of cardiac tissues. They are typically constructed from rigid and brittle materials processed in a cleanroom environment. An outstanding technological challenge is the integration of soft materials enabling a closer match to the mechanical properties of biological cells and tissues. Here we present an algorithm for direct writing of elastic membranes with embedded electrodes, optical waveguides and microfluidics using a commercial 3D printing system and a palette of silicone elastomers. As proof of principle, we demonstrate interfacing of cardiomyocytes derived from human induced pluripotent stem cells (hiPSCs), which are engineered to express Channelrhodopsin-2. We demonstrate electrical recording of cardiomyocyte field potentials and their concomitant modulation by optical and pharmacological stimulation delivered via the membrane. Our work contributes a simple prototyping strategy with potential applications in organ-on-chip or implantable systems that are multi-modal and mechanically soft.
AUTHOR Figueiredo, Lara and Le Visage, Catherine and Weiss, Pierre and Yang, Jing
Title Quantifying Oxygen Levels in 3D Bioprinted Cell-Laden Thick Constructs with Perfusable Microchannel Networks [Abstract]
Year 2020
Journal/Proceedings Polymers
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The survival and function of thick tissue engineered implanted constructs depends on pre-existing, embedded, functional, vascular-like structures that are able to integrate with the host vasculature. Bioprinting was employed to build perfusable vascular-like networks within thick constructs. However, the improvement of oxygen transportation facilitated by these vascular-like networks was directly quantified. Using an optical fiber oxygen sensor, we measured the oxygen content at different positions within 3D bioprinted constructs with and without perfusable microchannel networks. Perfusion was found to play an essential role in maintaining relatively high oxygen content in cell-laden constructs and, consequently, high cell viability. The concentration of oxygen changes following switching on and off the perfusion. Oxygen concentration depletes quickly after pausing perfusion but recovers rapidly after resuming the perfusion. The quantification of oxygen levels within cell-laden hydrogel constructs could provide insight into channel network design and cellular responses.
AUTHOR Tan, Wen See and Shi, Qian and Chen, Shengyang and Bin Juhari, Muhammad Aidil and Song, Juha
Title Recyclable and biocompatible microgel-based supporting system for positive 3D freeform printing of silicone rubber [Abstract]
Year 2020
Journal/Proceedings Biomedical Engineering Letters
Reftype Tan2020
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Abstract
Additive manufacturing (AM) of biomaterials has evolved from a rapid prototyping tool into a viable approach for the manufacturing of patient-specific implants over the past decade. It can tailor to the unique physiological and anatomical criteria of the patient’s organs or bones through precise controlling of the structure during the 3D printing. Silicone elastomers, which is a major group of materials in many biomedical implants, have low viscosities and can be printed with a special AM platform, known as freeform 3D printing systems. The freeform 3D printing systems are composed of a supporting bath and a printing material. Current supporting matrices that are either commercially purchased or synthesized were usually disposed of after retrieval of the printed part. In this work, we proposed a new and improved supporting matrix comprises of synthesized calcium alginate microgels produced via encapsulation which can be recycled, reused, and recovered for multiple prints, hence minimizing wastage and cost of materials. The dehydration tolerance of the calcium alginate microgels was improved through physical means by the addition of glycerol and chemical means by developing new calcium alginate microgels encapsulated with glycerol. The recyclability of the heated calcium alginate microgels was also enhanced by a rehydration step with sodium chloride solution and a recovery step with calcium chloride solution via the ion exchange process. We envisaged that our reusable and recyclable biocompatible calcium alginate microgels can save material costs, time, and can be applied in various freeform 3D printing systems.
AUTHOR Schipani, Rossana and Scheurer, Stefan and Florentin, Romain and Critchley, Susan E. and Kelly, Daniel John
Title Reinforcing interpenetrating network hydrogels with 3D printed polymer networks to engineer cartilage mimetic composites [Abstract]
Year 2020
Journal/Proceedings Biofabrication
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Engineering constructs that mimic the complex structure, composition and biomechanics of the articular cartilage represents a promising route to joint regeneration. Such tissue engineering strategies require the development of biomaterials that mimic the mechanical properties of articular cartilage whilst simultaneously providing an environment supportive of chondrogenesis. Here three-dimensional (3D) bioprinting is used to develop polycaprolactone (PCL) fibre networks to mechanically reinforce interpenetrating network (IPN) hydrogels consisting of alginate and gelatin methacryloyl (GelMA). Inspired by the significant tension-compression nonlinearity of the collagen network in articular cartilage, we printed reinforcing PCL networks with different ratios of tensile to compressive modulus. Synergistic increases in compressive modulus were observed when IPN hydrogels were reinforced with PCL networks that were relatively soft in compression and stiff in tension. The resulting composites possessed equilibrium and dynamic mechanical properties that matched or approached that of native articular cartilage. Finite Element (FE) modelling revealed that the reinforcement of IPN hydrogels with specific PCL networks limited radial expansion and increased the hydrostatic pressure generated within the IPN upon the application of compressive loading. Next, multiple-tool biofabrication techniques were used to 3D bioprint PCL reinforced IPN hydrogels laden with a co-culture of bone marrow-derived stromal cells (BMSCs) and chondrocytes (CCs). The bioprinted biomimetic composites were found to support robust chondrogenesis, with encapsulated cells producing hyaline-like cartilage that stained strongly for sGAG and type II collagen deposition, and negatively for type X collagen and calcium deposition. Taken together, these results demonstrate how 3D bioprinting can be used to engineer constructs that are both pro-chondrogenic and biomimetic of the mechanical properties of articular cartilage.
AUTHOR Sanz-Fraile, Hector and Amorós, Susana and Mendizabal, Irene Isabel and Gálvez-Montón, Carolina and Prat-Vidal, Cristina and Bayés-Genís, Antoni and Navajas, Daniel and Farre, Ramon and Otero, Jorge
Title Silk-reinforced Collagen Hydrogels with Raised Multiscale Stiffness for Mesenchymal Cells 3D Culture [Abstract]
Year 2020
Journal/Proceedings Tissue Engineering Part A
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Type I collagen hydrogels are of high interest in tissue engineering. With the evolution of 3D bioprinting technologies, a high number of collagen-based scaffolds have been reported for the development of 3D cell cultures. A recent proposal was to mix collagen with silk fibroin derived from Bombyx Mori silkworm. Nevertheless, due to the difficulties in the preparation and the characteristics of the protein, several problems like phase separation and collagen denaturation appears during the procedure. Therefore, the common solution is to diminish the concentration of collagen although in that way the most biologically relevant component is reduced. In the present work, we present a new, simple and effective method to develop a collagen-silk hybrid hydrogel with high collagen concentration and with increased stiffness approaching that of natural tissues, which could be of high interest for the development of cardiac patches for myocardial regeneration and for preconditioning of mesenchymal stem cells to improve their therapeutic potential. Sericin in the silk was preserved by using a physical solubilizing procedure which results in a preserved fibrous structure of type I collagen, as shown by ultrastructural imaging. The macro- and micromechanical properties of the hybrid hydrogels measured by tensile stretch and Atomic Force Microscopy respectively, showed a more than two-fold stiffening as compared with collagen-only hydrogels. Rheological measurements showed improved printability properties for the developed biomaterial. The suitability of the hydrogels for 3D cell culture was assessed by 3D bioprinting bone marrow-derived mesenchymal stem cells cultured within the scaffolds. The result was a biomaterial with improved printability characteristics that better resembled the mechanical properties of natural soft tissues while preserving biocompatibility owing to the high concentration of collagen.
AUTHOR Li, Huijun and Tan, Yu Jun and Kiran, Raj and Tor, Shu Beng and Zhou, Kun
Title Submerged and non-submerged 3D bioprinting approaches for the fabrication of complex structures with the hydrogel pair GelMA and alginate/methylcellulose [Abstract]
Year 2020
Journal/Proceedings Additive Manufacturing
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The extrusion-based bioprinting of hydrogels such as gelatin methacrylate (GelMA) into structures with complex shape suffers from poor printability due to their low viscosity. The present study deals with hydrogel materials by using the mixture of cell-laden photopolymerizable GelMA as a main printing material and the mixture of alginate and methylcellulose (Alg/MC) as a support material because of its high viscosity and good thixotropic property. One extrusion-based approach is developed by printing the two mixtures into structures in an alternating layer-by-layer manner, with the electrostatic interactions between polycationic GelMA and polyanionic Alg/MC contributing to the integrity of the structures. The final printed structures are exposed to ultraviolet (UV) light to form crosslinks in GelMA through photopolymerization for further structural strengthening. The one-time UV exposure minimizes cell damage in cell-GelMA, demonstrating an advantage over those in previously reported studies that required repeated UV exposures upon the printing of each layer of a structure. The other approach is developed by submerging the extrusion nozzle into a bath of Alg/MC to print cell-laden GelMA structures, which, upon printing completion, are also subject to one-time UV exposure before the removal of the support material Alg/MC. A flower with living cells is printed to demonstrate the capability of the second approach of fabricating structures with geometric complexity. The structures printed using both approaches demonstrate a well-maintained shape fidelity, structural integrity and cell viability of over 93% up to five culturing days. The proposed two printing approaches based on the cell-GelMA and Alg/MC pair will be beneficial for exploring new opportunities in bioprinting.
AUTHOR Prasopthum, Aruna and Deng, Zexing and Khan, Ilyas M. and Yin, Zhanhai and Guo, Baolin and Yang, Jing
Title Three dimensional printed degradable and conductive polymer scaffolds promote chondrogenic differentiation of chondroprogenitor cells [Abstract]
Year 2020
Journal/Proceedings Biomaterials Science
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Conductive polymers have been used for various biomedical applications including biosensors{,} tissue engineering and regenerative medicine. However{,} the poor processability and brittleness of these polymers hinder the fabrication of three-dimensional structures with desirable geometries. Moreover{,} their application in tissue engineering and regenerative medicine has been so far limited to excitable cells such as neurons and muscle cells. To enable their wider adoption in tissue engineering and regenerative medicine{,} new materials and formulations that overcome current limitations are required. Herein{,} a biodegradable conductive block copolymer{,} tetraaniline-b-polycaprolactone-b-tetraaniline (TPT){,} is synthesised and 3D printed for the first time into porous scaffolds with defined geometries. Inks are formulated by combining TPT with PCL in solutions which are then directly 3D printed to generate porous scaffolds. TPT and PCL are both biodegradable. The combination of TPT with PCL increases the flexibility of the hybrid material compared to pure TPT{,} which is critical for applications that need mechanical robustness of the scaffolds. The highest TPT content shows the lowest tensile failure strain. Moreover{,} the absorption of a cell adhesion-promoting protein (fibronectin) and chondrogenic differentiation of chondroprogenitor cells are found to be dependent on the amount of TPT in the blends. Higher content of TPT in the blends increases both fibronectin adsorption and chondrogenic differentiation{,} though the highest concentration of TPT in the blends is limited by its solubility in the ink. Despite the contradicting effects of TPT concentration on flexibility and chondrogenic differentiation{,} a concentration that strikes a balance between the two factors is still available. It is worth noting that the effect on chondrogenic differentiation is found in scaffolds without external electric stimulation. Our work demonstrates the possibility of 3D printing flexible conductive and biodegradable scaffolds and their potential use in cartilage tissue regeneration{,} and opens up future opportunities in using electric stimulation to control chondrogenesis in these scaffolds.
AUTHOR Zhang, Xihui and Jiang, Tianyan and Chen, Dandan and Wang, Qi and Zhang, Leshuai W.
Title Three-dimensional liver models: state of the art and their application for hepatotoxicity evaluation [Abstract]
Year 2020
Journal/Proceedings Critical Reviews in Toxicology
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AbstractWhile alternative methods for toxicity testing using re-constructed human skin and cornea have been written into guidelines and adopted by regulatory authorities, three-dimensional (3D) liver models are currently applied in the industrial settings for hepatotoxicity screening and prediction. These 3D liver models can recapitulate the architecture, functionality and toxicity response of the native liver, demonstrated by a set of related hallmarks. In this comprehensive review, non-scaffold and scaffold-based methods available for 3D liver model formation are introduced, with an emphasis on their advantages and drawbacks. We then focus on the characteristics of primary human hepatocytes, stem cell derived hepatocyte like cells, and immortalized hepatic cell lines as cell resources for model reconstruction. Primary hepatocytes are generally regarded to be superior to other cell types due to their comparable metabolic profiles to the native liver. Additionally, the application of 3D liver models (mostly liver spheroids) on the evaluation of drug induced liver injury and chronic liver diseases (steatosis, cirrhosis, cholestasis), as well as the potential of nanomaterials to introduce hepatotoxicity are summarized. Finally, the global 3D cell market from 3D liver model manufacturing to the contract service of in vitro hepatotoxicity testing using the models is extensively explored. However, 3D liver models face cultural and regulatory barriers in different countries, and therefore the business development of 3D liver models is not easy. Toxicologists, material scientists, engineers should work together to develop, validate and apply 3D liver models for hepatotoxicity testing under the support from industrial organizations and governmental agencies.
AUTHOR Vyas, Cian and Ates, Gokhan and Aslan, Enes and Hart, Jack and Huang, Boyang and Bartolo, Paulo
Title Three-Dimensional Printing and Electrospinning Dual-Scale Polycaprolactone Scaffolds with Low-Density and Oriented Fibers to Promote Cell Alignment [Abstract]
Year 2020
Journal/Proceedings 3D Printing and Additive Manufacturing
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Complex and hierarchically functionalized scaffolds composed of micro- and nanoscale structures are a key goal in tissue engineering. The combination of three-dimensional (3D) printing and electrospinning enables the fabrication of these multiscale structures. This study presents a polycaprolactone 3D-printed and electrospun scaffold with multiple mesh layers and fiber densities. The results show successful fabrication of a dual-scale scaffold with the 3D-printed scaffold acting as a gap collector with the printed microfibers as the electrodes and the pores a series of insulating gaps resulting in aligned nanofibers. The electrospun fibers are highly aligned perpendicular to the direction of the printed fiber and form aligned meshes within the pores of the scaffold. Mechanical testing showed no significant difference between the number of mesh layers whereas the hydrophobicity of the scaffold increased with increasing fiber density. Biological results indicate that increasing the number of mesh layers improves cell proliferation, migration, and adhesion. The aligned nanofibers within the microscale pores allowed enhanced cell bridging and cell alignment that was not observed in the 3D-printed only scaffold. These results demonstrate a facile method of incorporating low-density and aligned fibers within a 3D-printed scaffold that is a promising development in multiscale hierarchical scaffolds where alignment of cells can be desirable.
AUTHOR Schwab, Andrea and Helary, Christophe and Richards, Geoff and Alini, Mauro and Eglin, David and D{textquoteright}Este, Matteo
Title Tissue mimetic hyaluronan bio-ink containing oriented collagen fibers to modulate hMSC spreading and differentiation [Abstract]
Year 2020
Journal/Proceedings bioRxiv
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Biofabrication is providing scientists and clinicians the ability to produce engineered tissues with desired shapes, chemical and biological gradients. Typical resolutions achieved with extrusion-based bioprinting are at the macroscopic level. However, for capturing the fibrillar nature of the extracellular matrix (ECM), it is necessary to arrange ECM components at smaller scales, down to the sub-micron and the molecular level.In this study, we introduce a (bio)ink containing hyaluronan (HA) as tyramine derivative (THA) and collagen (Col). Similarly to other connective tissues, in this (bio)ink Col is present in fibrillar form and HA as viscoelastic space filler. THA was enzymatically crosslinked under mild conditions allowing simultaneous Col fibrillogenesis, thus achieving a homogeneous distribution of Col fibrils within the viscoelastic HA-based matrix. THA-Col composite displayed synergistic properties in terms of storage modulus and shear-thinning, translating into good printability.Shear-induced alignment of the Col fibrils along the printing direction was achieved and quantified via immunofluorescence and second harmonic generation.Cell-free and cell-laden constructs were printed and characterized, analyzing the influence of the controlled microscopic anisotropy on cell behavior and chondrogenic differentiation.THA-Col showed cell instructive properties modulating hMSC adhesion, morphology and sprouting from spheroids stimulated by the presence and the orientation of Col fibers. Actin filament staining showed that hMSCs embedded into aligned constructs displayed increased cytoskeleton alignment along the fibril direction. Based on gene expression of cartilage/bone markers and matrix production, hMSCs embedded into the bioink displayed chondrogenic differentiation comparable or superior to standard pellet culture by means of proteoglycan production (Safranin O staining and proteoglycan quantification) as well as increase in cartilage related genes.The possibility of printing matrix components with control over microscopic alignment brings biofabrication one step closer to capturing the complexity of native tissues.
AUTHOR Nasim Golafshan and Elke Vorndran and Stefan Zaharievski and Harold Brommer and Firoz Babu Kadumudi and Alireza Dolatshahi-Pirouz and Uwe Gbureck and René {van Weeren} and Miguel Castilho and Jos Malda
Title Tough magnesium phosphate-based 3D-printed implants induce bone regeneration in an equine defect model [Abstract]
Year 2020
Journal/Proceedings Biomaterials
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One of the important challenges in bone tissue engineering is the development of biodegradable bone substitutes with appropriate mechanical and biological properties for the treatment of larger defects and those with complex shapes. Recently, magnesium phosphate (MgP) doped with biologically active ions like strontium (Sr2+) have shown to significantly enhance bone formation when compared with the standard calcium phosphate-based ceramics. However, such materials can hardly be shaped into large and complex geometries and more importantly lack the adequate mechanical properties for the treatment of load-bearing bone defects. In this study, we have fabricated bone implants through extrusion assisted three-dimensional (3D) printing of MgP ceramics modified with Sr2+ ions (MgPSr) and a medical grade polycaprolactone (PCL) polymer phase. MgPSr with 30 wt% PCL (MgPSr-PCL30) allowed the printability of relevant size structures (>780 mm3) at room temperature with an interconnected macroporosity of approximately 40%. The printing resulted in implants with a compressive strength of 4.3 MPa, which were able to support up to 50 cycles of loading without plastic deformation. Notably, MgPSr-PCL30 scaffolds were able to promote in vitro bone formation in medium without the supplementation with osteo-inducing components. In addition, long-term in vivo performance of the 3D printed scaffolds was investigated in an equine tuber coxae model over 6 months. The micro-CT and histological analysis showed that implantation of MgPSr-PCL30 induced bone regeneration, while no bone formation was observed in the empty defects. Overall, the novel polymer modified MgP ceramic material and extrusion-based 3D printing process presented here greatly improved the shape ability and load bearing properties of MgP-based ceramics with simultaneously induction of new bone formation.
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 Döhler, Diana and Kang, Jiheong and Cooper, Chris Brittain and Tok, Jeffrey B.-H. and Rupp, Harald and Binder, Wolfgang H. and Bao, Zhenan
Title Tuning the Self-Healing Response of Poly(dimethylsiloxane)-Based Elastomers [Abstract]
Year 2020
Journal/Proceedings ACS Appl. Polym. Mater.
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We present a comprehensive investigation of mechanical properties of supramolecular polymer networks with rationally developed multistrength hydrogen-bonding interactions. Self-healing poly(dimethylsiloxane) (PDMS)-based elastomers with varying elasticity, fracture toughness, and the ability to dissipate strain energy through the reversible breakage and re-formation of the supramolecular interactions were obtained. By changing the ratio between isophorone diisocyanate (IU), 4,4′-methylenebis(cyclohexyl isocyanate) (MCU), and 4,4′-methylenebis(phenyl isocyanate) (MPU) and by varying the molecular weight of the PDMS precursor, we obtained a library of poly(urea)s to study the interplay of mechanical performance and self-healability. The Young’s moduli of the presented materials ranged between 0.4 and 13 MPa and increased with decreasing molecular weight of the PDMS precursor and increasing content of MCU or MPU units related to the formation of stronger hydrogen-bonding interactions. By exchanging MPU against MCU units, we achieved an optimum balance between mechanical properties and self-healing performance, and by the additional reduction of the molecular weight of the precursor polymer, a minimum recovery of 80% in stress within 12 h at room temperature was observed. Selected poly(urea)s could be processed via 3D printing by the conventional extrusion method, obtaining dimensionally stable and freestanding objects. We present a comprehensive investigation of mechanical properties of supramolecular polymer networks with rationally developed multistrength hydrogen-bonding interactions. Self-healing poly(dimethylsiloxane) (PDMS)-based elastomers with varying elasticity, fracture toughness, and the ability to dissipate strain energy through the reversible breakage and re-formation of the supramolecular interactions were obtained. By changing the ratio between isophorone diisocyanate (IU), 4,4′-methylenebis(cyclohexyl isocyanate) (MCU), and 4,4′-methylenebis(phenyl isocyanate) (MPU) and by varying the molecular weight of the PDMS precursor, we obtained a library of poly(urea)s to study the interplay of mechanical performance and self-healability. The Young’s moduli of the presented materials ranged between 0.4 and 13 MPa and increased with decreasing molecular weight of the PDMS precursor and increasing content of MCU or MPU units related to the formation of stronger hydrogen-bonding interactions. By exchanging MPU against MCU units, we achieved an optimum balance between mechanical properties and self-healing performance, and by the additional reduction of the molecular weight of the precursor polymer, a minimum recovery of 80% in stress within 12 h at room temperature was observed. Selected poly(urea)s could be processed via 3D printing by the conventional extrusion method, obtaining dimensionally stable and freestanding objects.
AUTHOR Iordache, F. and Alexandru, D. and Pisoschi, A. M. and PoP, A.
Title 3D Bioprinting of Blood Vessel Model Using Collagen-Hyaluronic Acid Hydrogel [Abstract]
Year 2019
Journal/Proceedings AgroLife Scientific Journal
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3D bioprinting is a technology that supports fabrication of biomimetic tissues with complex architecture. It has application in drug discovery, tissue development, and regenerative medicine. The aim of this study was to create a blood vessel model correlating properties of collagen-hyaluronic acid hydrogel with bioprinter parameters such as speed rate, pressure, number of layers, nozzle diameter, and temperature. The blood vessel model was created using BioCAD software and bioprinted by extrusion technology using collagen-hyaluronic acid hydrogel. We analyzed the water uptake, enzymatic degradation and morphology by scanning electron microscopy and after staining with Hematoxylin and Eosin (H&E) and Trichromic Masson dyes. The results showed that the blood vessel constructs have 2.46 mm (±0.41) mean diameter, 1.4 mm (±0.10) mean thick wall, and 2.8 mm (±0.05) mean height which is appropriate with the model created in the BioCAD software. The optimal parameters for these constructs were: 1.1 bar pressure, 1mm/sec speed rate, 18°C temperature, 0.2 mm nozzle diameter, and 10 numbers of layers. Increasing hydrogel weight by 22% at 2 hours after immersion in PBS suggesting that is hydrophilic. Furthermore, decreasing by up to 47.2% in the presence of collagenase (50 μg/ml) shows that is biodegradable. H&E and Trichromic Masson staining showed that collagen-hyaluronic acid hydrogel organized in a network with pores dimension that could support cells growth and differentiation. In conclusion, our scaffold mimics the blood vessel structure, further experiment will be addressed for study the biocompatibility of these scaffold with mesenchymal stem cells.
AUTHOR Li, J. and Liu, X. and Crook, J. M. and Wallace, G. G.
Title 3D graphene-containing structures for tissue engineering [Abstract]
Year 2019
Journal/Proceedings Materials Today Chemistry
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Graphene and its derivatives have been extensively explored in various fields and have shown great promise toward energy harvesting, environmental protection, and health care. 3D graphene-containing structures (3DGCSs) are especially endowed with useable features relating to physicochemical properties within the hierarchical architectures. Thus, 3DGCSs are increasingly being applied for tissue engineering because of their supportability of human cells and functionalization potential. This review focuses on recent progress in tissue engineering utilizing 3DGCSs, providing insights into fabrication, application, and constraints in bionic research.
AUTHOR Alison, Lauriane and Menasce, Stefano and Bouville, Florian and Tervoort, Elena and Mattich, Iacopo and Ofner, Alessandro and Studart, André R.
Title 3D printing of sacrificial templates into hierarchical porous materials [Abstract]
Year 2019
Journal/Proceedings Scientific Reports
Reftype Alison2019
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Abstract
Hierarchical porous materials are widespread in nature and find an increasing number of applications as catalytic supports, biological scaffolds and lightweight structures. Recent advances in additive manufacturing and 3D printing technologies have enabled the digital fabrication of porous materials in the form of lattices, cellular structures and foams across multiple length scales. However, current approaches do not allow for the fast manufacturing of bulk porous materials featuring pore sizes that span broadly from macroscopic dimensions down to the nanoscale. Here, ink formulations are designed and investigated to enable 3D printing of hierarchical materials displaying porosity at the nano-, micro- and macroscales. Pores are generated upon removal of nanodroplets and microscale templates present in the initial ink. Using particles to stabilize the droplet templates is key to obtain Pickering nanoemulsions that can be 3D printed through direct ink writing. The combination of such self-assembled templates with the spatial control offered by the printing process allows for the digital manufacturing of hierarchical materials exhibiting thus far inaccessible multiscale porosity and complex geometries.
AUTHOR Rupp, Harald and Döhler, Diana and Hilgeroth, Philipp and Mahmood, Nasir and Beiner, Mario and Binder, Wolfgang H.
Title 3D Printing of Supramolecular Polymers: Impact of Nanoparticles and Phase Separation on Printability [Abstract]
Year 2019
Journal/Proceedings Macromolecular Rapid Communications
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Abstract 3D printing of linear and three-arm star supramolecular polymers with attached hydrogen bonds and their nanocomposites is reported. The concept is based on hydrogen-bonded supramolecular polymers, known to form nano-sized micellar clusters. Printability is based on reversible thermal- and shear-induced dissociation of a supramolecular polymer network, which generates stable and self-supported structures after printing, as checked via melt-rheology and X-ray scattering. The linear and three-arm star poly(isobutylene)s PIB-B2 (Mn = 8500 g mol −1), PIB-B3 (Mn = 16 000 g mol −1), and linear poly(ethylene glycol)s PEG-B2 (Mn = 900 g mol−1, 8500 g mol −1) are prepared and then probed by melt-rheology to adjust the viscosity to address the proper printing window. The supramolecular PIB polymers show a rubber-like behavior and are able to form self-supported 3D printed objects at room temperature and below, reaching polymer strand diameters down to 200–300 µm. Nanocomposites of PIB-B2 with silica nanoparticles (12 nm, 5–15 wt%) are generated, in turn leading to an improvement of their shape persistence. A blend of the linear polymer PIB-B2 and the three-arm star polymer PIB-B3 (ratio ≈ 3/1 mol) reaches an even higher structural stability, able to build free-standing structures.
AUTHOR Mehrotra, Shreya and Moses, Joseph Christakiran and Bandyopadhyay, Ashutosh and Mandal, Biman B.
Title 3D Printing/Bioprinting Based Tailoring of in Vitro Tissue Models: Recent Advances and Challenges [Abstract]
Year 2019
Journal/Proceedings ACS Applied Bio Materials
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Prodigious progress in the past decade has pronounced 3D printing as one of the most promising technique for assembling biological materials in a complex layout that mimics native human tissues. With the advent of technology, several improvements in printing techniques have facilitated the development of intricate strategies and designs that were imaginably distant due to the conventional top-down approaches. Most of these advanced strategies generally follow a thorough coordination and an elaborate biomimetic blueprint due to which it is now possible to fabricate in vitro tissue models with ease. However, much remains to be accomplished at several forefronts for utilizing this technology to its full potential. With several printing strategies at the lead, it has now become essential to systematically analyze and learn from several endeavors such that shortcomings can be understood and future efforts can be made toward negating them. Taking account of all the recent tissue specific developments in this field, this review serves as a framework for bringing together in discussion several strategies and constraints in developing small scaled in vitro tissues. Highlighting the growing popularity of the organ and body on chip platforms and their easy scale up using 3D printing, latest advancements, and the challenges in this field are also discussed. Prodigious progress in the past decade has pronounced 3D printing as one of the most promising technique for assembling biological materials in a complex layout that mimics native human tissues. With the advent of technology, several improvements in printing techniques have facilitated the development of intricate strategies and designs that were imaginably distant due to the conventional top-down approaches. Most of these advanced strategies generally follow a thorough coordination and an elaborate biomimetic blueprint due to which it is now possible to fabricate in vitro tissue models with ease. However, much remains to be accomplished at several forefronts for utilizing this technology to