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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
DOI/URL DOI
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 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
DOI/URL DOI
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 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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Abstract
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 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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Abstract
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 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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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 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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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 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
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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 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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Abstract
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 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 Kamdem Tamo, Arnaud and Doench, Ingo and Morales Helguera, Aliuska and Hoenders, Daniel and Walther, Andreas and Madrazo, Anayancy Osorio
Title Biodegradation of Crystalline Cellulose Nanofibers by Means of Enzyme Immobilized-Alginate Beads and Microparticles [Abstract]
Year 2020
Journal/Proceedings Polymers
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Recent advances in nanocellulose technology have revealed the potential of crystalline cellulose nanofibers to reinforce materials which are useful for tissue engineering, among other functions. However, the low biodegradability of nanocellulose can possess some problems in biomedical applications. In this work, alginate particles with encapsulated enzyme cellulase extracted from Trichoderma reesei were prepared for the biodegradation of crystalline cellulose nanofibers, which carrier system could be incorporated in tissue engineering biomaterials to degrade the crystalline cellulose nanoreinforcement in situ and on-demand during tissue regeneration. Both alginate beads and microparticles were processed by extrusion-dropping and inkjet-based methods, respectively. Processing parameters like the alginate concentration, concentration of ionic crosslinker Ca2+, hardening time, and ionic strength of the medium were varied. The hydrolytic activity of the free and encapsulated enzyme was evaluated for unmodified (CNFs) and TEMPO-oxidized cellulose nanofibers (TOCNFs) in suspension (heterogeneous conditions); in comparison to solubilized cellulose derivatives (homogeneous conditions). The enzymatic activity was evaluated for temperatures between 25–75 °C, pH range from 3.5 to 8.0 and incubation times until 21 d. Encapsulated cellulase in general displayed higher activity compared to the free enzyme over wider temperature and pH ranges and for longer incubation times. A statistical design allowed optimizing the processing parameters for the preparation of enzyme-encapsulated alginate particles presenting the highest enzymatic activity and sphericity. The statistical analysis yielded the optimum particles characteristics and properties by using a formulation of 2% (w/v) alginate, a coagulation bath of 0.2 M CaCl2 and a hardening time of 1 h. In homogeneous conditions the highest catalytic activity was obtained at 55 °C and pH 4.8. These temperature and pH values were considered to study the biodegradation of the crystalline cellulose nanofibers in suspension. The encapsulated cellulase preserved its activity for several weeks over that of the free enzyme, which latter considerably decreased and practically showed deactivation after just 10 d. The alginate microparticles with their high surface area-to-volume ratio effectively allowed the controlled release of the encapsulated enzyme and thereby the sustained hydrolysis of the cellulose nanofibers. The relative activity of cellulase encapsulated in the microparticles leveled-off at around 60% after one day and practically remained at that value for three weeks.
AUTHOR 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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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 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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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 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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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 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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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 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 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 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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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 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 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
DOI/URL DOI
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 Angelopoulos, Ioannis and Allenby, Mark C. and Lim, Mayasari and Zamorano, Mauricio
Title Engineering inkjet bioprinting processes toward translational therapies [Abstract]
Year 2019
Journal/Proceedings Biotechnology and Bioengineering
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DOI/URL DOI
Abstract
Abstract Bioprinting is the assembly of three-dimensional (3D) tissue constructs by layering cell-laden biomaterials using additive manufacturing techniques, offering great potential for tissue engineering and regenerative medicine. Such a process can be performed with high resolution and control by personalized or commercially available inkjet printers. However, bioprinting's clinical translation is significantly limited due to process engineering challenges. Upstream challenges include synthesis, cellular incorporation, and functionalization of “bioinks,” and extrusion of print geometries. Downstream challenges address sterilization, culture, implantation, and degradation. In the long run, bioinks must provide a microenvironment to support cell growth, development, and maturation and must interact and integrate with the surrounding tissues after implantation. Additionally, a robust, scaleable manufacturing process must pass regulatory scrutiny from regulatory bodies such as U.S. Food and Drug Administration, European Medicines Agency, or Australian Therapeutic Goods Administration for bioprinting to have a real clinical impact. In this review, recent advances in inkjet-based 3D bioprinting will be presented, emphasizing on biomaterials available, their properties, and the process to generate bioprinted constructs with application in medicine. Current challenges and the future path of bioprinting and bioinks will be addressed, with emphasis in mass production aspects and the regulatory framework bioink-based products must comply to translate this technology from the bench to the clinic.
AUTHOR Apelgren, Peter and Karabulut, Erdem and Amoroso, Matteo and Mantas, Athanasios and Martínez Ávila, Héctor and Kölby, Lars and Kondo, Tetsuo and Toriz, Guillermo and Gatenholm, Paul
Title In Vivo Human Cartilage Formation in Three-Dimensional Bioprinted Constructs with a Novel Bacterial Nanocellulose Bioink [Abstract]
Year 2019
Journal/Proceedings ACS Biomaterials Science & Engineering
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DOI/URL DOI
Abstract
Bacterial nanocellulose (BNC) is a 3D network of nanofibrils exhibiting excellent biocompatibility. Here, we present the aqueous counter collision (ACC) method of BNC disassembly to create bioink with suitable properties for cartilage-specific 3D-bioprinting. BNC was disentangled by ACC, and fibril characteristics were analyzed. Bioink printing fidelity and shear-thinning properties were evaluated. Cell-laden bioprinted grid constructs (5 × 5 × 1 mm3) containing human nasal chondrocytes (10 M mL-1) were implanted in nude mice and explanted after 30 and 60 days. Both ACC and hydrolysis resulted in significantly reduced fiber lengths, with ACC resulting in longer fibrils and fewer negative charges relative to hydrolysis. Moreover, ACC-BNC bioink showed outstanding printability, postprinting mechanical stability, and structural integrity. In vivo, cell-laden structures were rapidly integrated, maintained structural integrity, and showed chondrocyte proliferation, with 32.8 ± 13.8 cells per mm2 observed after 30 days and 85.6 ± 30.0 cells per mm2 at day 60 (p = 0.002). Furthermore, a full-thickness skin graft was attached and integrated completely on top of the 3D-bioprinted construct. The novel ACC disentanglement technique makes BNC biomaterial highly suitable for 3D-bioprinting and clinical translation, suggesting cell-laden 3D-bioprinted ACC-BNC as a promising solution for cartilage repair. Bacterial nanocellulose (BNC) is a 3D network of nanofibrils exhibiting excellent biocompatibility. Here, we present the aqueous counter collision (ACC) method of BNC disassembly to create bioink with suitable properties for cartilage-specific 3D-bioprinting. BNC was disentangled by ACC, and fibril characteristics were analyzed. Bioink printing fidelity and shear-thinning properties were evaluated. Cell-laden bioprinted grid constructs (5 × 5 × 1 mm3) containing human nasal chondrocytes (10 M mL-1) were implanted in nude mice and explanted after 30 and 60 days. Both ACC and hydrolysis resulted in significantly reduced fiber lengths, with ACC resulting in longer fibrils and fewer negative charges relative to hydrolysis. Moreover, ACC-BNC bioink showed outstanding printability, postprinting mechanical stability, and structural integrity. In vivo, cell-laden structures were rapidly integrated, maintained structural integrity, and showed chondrocyte proliferation, with 32.8 ± 13.8 cells per mm2 observed after 30 days and 85.6 ± 30.0 cells per mm2 at day 60 (p = 0.002). Furthermore, a full-thickness skin graft was attached and integrated completely on top of the 3D-bioprinted construct. The novel ACC disentanglement technique makes BNC biomaterial highly suitable for 3D-bioprinting and clinical translation, suggesting cell-laden 3D-bioprinted ACC-BNC as a promising solution for cartilage repair.
AUTHOR Markstedt, Kajsa and Håkansson, Karl and Toriz, Guillermo and Gatenholm, Paul
Title Materials from trees assembled by 3D printing – Wood tissue beyond nature limits [Abstract]
Year 2019
Journal/Proceedings Applied Materials Today
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DOI/URL URL DOI
Abstract
Materials from trees have the potential to replace fossil based and other non-sustainable materials in everyday products, thus transforming the society back to a bioeconomy. This paper presents a 3D printing platform which mimics wood biogenesis for the assembly of wood biopolymers into wood-like hierarchical composites. The genome was substituted with G-code, the programming language which controls how the 3D printer assembles material. The rosette was replaced by the printer head for extrusion of cellulose. Instead of microtubules guiding the alignment of cellulose, the printing direction was guided by an x/y stage, thus mimicking the microfibril angle. The printed structures were locked by an enzymatic crosslinking reaction similar to what occurs in the cell wall upon lignification. Hierarchical structures characteristic for wood were designed and printed with control of density, swelling and directional strength. Accelerating the development of the 3D printing technology helps realize the circular bioeconomy where garments, packaging, furniture and entire houses are manufactured by 3D printing wood.
AUTHOR Filardo, G. and Petretta, M. and Cavallo, C. and Roseti, L. and Durante, S. and Albisinni, U. and Grigolo, B.
Title Patient-specific meniscus prototype based on 3D bioprinting of human cell-laden scaffold [Abstract]
Year 2019
Journal/Proceedings Bone and Joint Research
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DOI/URL DOI
Abstract
Objectives Meniscal injuries are often associated with an active lifestyle. The damage of meniscal tissue puts young patients at higher risk of undergoing meniscal surgery and, therefore, at higher risk of osteoarthritis. In this study, we undertook proof-of-concept research to develop a cellularized human meniscus by using 3D bioprinting technology. Methods A 3D model of bioengineered medial meniscus tissue was created, based on MRI scans of a human volunteer. The Digital Imaging and Communications in Medicine (DICOM) data from these MRI scans were processed using dedicated software, in order to obtain an STL model of the structure. The chosen 3D Discovery printing tool was a microvalve-based inkjet printhead. Primary mesenchymal stem cells (MSCs) were isolated from bone marrow and embedded in a collagen-based bio-ink before printing. LIVE/DEAD assay was performed on realized cell-laden constructs carrying MSCs in order to evaluate cell distribution and viability. Results This study involved the realization of a human cell-laden collagen meniscus using 3D bioprinting. The meniscus prototype showed the biological potential of this technology to provide an anatomically shaped, patient-specific construct with viable cells on a biocompatible material. Conclusion This paper reports the preliminary findings of the production of a custom-made, cell-laden, collagen-based human meniscus. The prototype described could act as the starting point for future developments of this collagen-based, tissue-engineered structure, which could aid the optimization of implants designed to replace damaged menisci. Cite this article: G. Filardo, M. Petretta, C. Cavallo, L. Roseti, S. Durante, U. Albisinni, B. Grigolo. Patient-specific meniscus prototype based on 3D bioprinting of human cell-laden scaffold. Bone Joint Res 2019;8:101–106. DOI: 10.1302/2046-3758.82.BJR-2018-0134.R1.
AUTHOR Pedrotty, Dawn M. and Volodymyr, Kuzmenko and Erdem, Karabulut and Sugrue Alan, M. and Christopher, Livia and Vaidya Vaibhav, R. and McLeod Christopher, J. and Asirvatham Samuel, J. and Paul, Gatenholm and Suraj, Kapa
Title Three-Dimensional Printed Biopatches With Conductive Ink Facilitate Cardiac Conduction When Applied to Disrupted Myocardium
Year 2019
Journal/Proceedings Circulation: Arrhythmia and Electrophysiology
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DOI/URL DOI
AUTHOR Gretzinger, Sarah and Beckert, Nicole and Gleadall, Andrew and Lee-Thedieck, Cornelia and Hubbuch, Jürgen
Title 3D bioprinting – Flow cytometry as analytical strategy for 3D cell structures [Abstract]
Year 2018
Journal/Proceedings Bioprinting
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DOI/URL URL DOI
Abstract
The importance of 3D printing technologies increased significantly over the recent years. They are considered to have a huge impact in regenerative medicine and tissue engineering, since 3D bioprinting enables the production of cell-laden 3D scaffolds. Transition from academic research to pharmaceutical industry or clinical applications, however, is highly dependent on developing a robust and well-known process, while maintaining critical cell characteristics. Hence, a directed and systematic approach to 3D bioprinting process development is required, which also allows for the monitoring of these cell characteristics. This work presents the development of a flow cytometry-based analytical strategy as a tool for 3D bioprinting research. The development was based on a model process using a commercially available alginate-based bioink, the β-cell line INS-1E, and direct dispensing as 3D bioprinting method. We demonstrated that this set-up enabled viability and proliferation analysis. Additionally, use of an automated sampler facilitated high-throughput screenings. Finally, we showed that each process step, e.g. suspension of cells in bioink or 3D printing, cross-linking of the alginate scaffold after printing, has a crucial impact on INS-1E viability. This reflects the importance of process optimization in 3D bioprinting and the usefulness of the flow cytometry-based analytical strategy described here. The presented strategy has a great potential as a cell characterisation tool for 3D bioprinting and may contribute to a more directed process development.
AUTHOR Shi, Pujiang and Tan, Yong Sheng Edgar and Yeong, Wai Yee and Li, Hoi Yeung and Laude, Augustinus
Title A bilayer photoreceptor‐retinal tissue model with gradient cell density design: A study of microvalve‐based bioprinting [Abstract]
Year 2018
Journal/Proceedings Journal of Tissue Engineering and Regenerative Medicine
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DOI/URL DOI
Abstract
Abstract ARPE‐19 and Y79 cells were precisely and effectively delivered to form an in vitro retinal tissue model via 3D cell bioprinting technology. The samples were characterized by cell viability assay, haematoxylin and eosin and immunofluorescent staining, scanning electrical microscopy and confocal microscopy, and so forth. The bioprinted ARPE‐19 cells formed a high‐quality cell monolayer in 14 days. Manually seeded ARPE‐19 cells were poorly controlled during and after cell seeding, and they aggregated to form uneven cell layer. The Y79 cells were subsequently bioprinted on the ARPE‐19 cell monolayer to form 2 distinctive patterns. The microvalve‐based bioprinting is efficient and accurate to build the in vitro tissue models with the potential to provide similar pathological responses and mechanism to human diseases, to mimic the phenotypic endpoints that are comparable with clinical studies, and to provide a realistic prediction of clinical efficacy.
AUTHOR Agarwala, Shweta and Lee, Jia Min and Ng, Wei Long and Layani, Michael and Yeong, Wai Yee and Magdassi, Shlomo
Title A novel 3D bioprinted flexible and biocompatible hydrogel bioelectronic platform [Abstract]
Year 2018
Journal/Proceedings Biosensors and Bioelectronics
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DOI/URL URL DOI
Abstract
Abstract Bioelectronics platforms are gaining widespread attention as they provide a template to study the interactions between biological species and electronics. Decoding the effect of the electrical signals on the cells and tissues holds the promise for treating the malignant tissue growth, regenerating organs and engineering new-age medical devices. This work is a step forward in this direction, where bio- and electronic materials co-exist on one platform without any need for post processing. We fabricate a freestanding and flexible hydrogel based platform using 3D bioprinting. The fabrication process is simple, easy and provides a flexible route to print materials with preferred shapes, size and spatial orientation. Through the design of interdigitated electrodes and heating coil, the platform can be tailored to print various circuits for different functionalities. The biocompatibility of the printed platform is tested using C2C12 murine myoblasts cell line. Furthermore, normal human dermal fibroblasts (primary cells) are also seeded on the platform to ascertain the compatibility.
AUTHOR Ng, Wei Long and Goh, Min Hao and Yeong, Wai Yee and Naing, May Win
Title Applying macromolecular crowding to 3D bioprinting: fabrication of 3D hierarchical porous collagen-based hydrogel constructs [Abstract]
Year 2018
Journal/Proceedings Biomaterials Science
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DOI/URL DOI
Abstract
Native tissues and/or organs possess complex hierarchical porous structures that confer highly-specific cellular functions. Despite advances in fabrication processes{,} it is still very challenging to emulate the hierarchical porous collagen architecture found in most native tissues. Hence{,} the ability to recreate such hierarchical porous structures would result in biomimetic tissue-engineered constructs. Here{,} a single-step drop-on-demand (DOD) bioprinting strategy is proposed to fabricate hierarchical porous collagen-based hydrogels. Printable macromolecule-based bio-inks (polyvinylpyrrolidone{,} PVP) have been developed and printed in a DOD manner to manipulate the porosity within the multi-layered collagen-based hydrogels by altering the collagen fibrillogenesis process. The experimental results have indicated that hierarchical porous collagen structures could be achieved by controlling the number of macromolecule-based bio-ink droplets printed on each printed collagen layer. This facile single-step bioprinting process could be useful for the structural design of collagen-based hydrogels for various tissue engineering applications.
AUTHOR Hauser, Daniel and Estermann, Manuela and Milosevic, Ana and Steinmetz, Lukas and Vanhecke, Dimitri and Septiadi, Dedy and Drasler, Barbara and Petri-Fink, Alke and Ball, Vincent and Rothen-Rutishauser, Barbara
Title Polydopamine/Transferrin Hybrid Nanoparticles for Targeted Cell-Killing [Abstract]
Year 2018
Journal/Proceedings Nanomaterials
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Abstract
Polydopamine can form biocompatible particles that convert light into heat. Recently, a protocol has been optimized to synthesize polydopamine/protein hybrid nanoparticles that retain the biological function of proteins, and combine it with the stimuli-induced heat generation of polydopamine. We have utilized this novel system to form polydopamine particles, containing transferrin (PDA/Tf). Mouse melanoma cells, which strongly express the transferrin receptor, were exposed to PDA/Tf nanoparticles (NPs) and, subsequently, were irradiated with a UV laser. The cell death rate was monitored in real-time. When irradiated, the melanoma cells exposed to PDA/Tf NPs underwent apoptosis, faster than the control cells, pointing towards the ability of PDA/Tf to mediate UV-light-induced cell death. The system was also validated in an organotypic, 3D-printed tumor spheroid model, comprising mouse melanoma cells, and the exposure and subsequent irradiation with UV-light, yielded similar results to the 2D cell culture. The process of apoptosis was found to be targeted and mediated by the lysosomal membrane permeabilization. Therefore, the herein presented polydopamine/protein NPs constitute a versatile and stable system for cancer cell-targeting and photothermal apoptosis induction.
AUTHOR Kuzmenko, Volodymyr and Karabulut, Erdem and Pernevik, Elin and Enoksson, Peter and Gatenholm, Paul
Title Tailor-made conductive inks from cellulose nanofibrils for 3D printing of neural guidelines [Abstract]
Year 2018
Journal/Proceedings Carbohydrate Polymers
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DOI/URL URL DOI
Abstract
Neural tissue engineering (TE), an innovative biomedical method of brain study, is very dependent on scaffolds that support cell development into a functional tissue. Recently, 3D patterned scaffolds for neural TE have shown significant positive effects on cells by a more realistic mimicking of actual neural tissue. In this work, we present a conductive nanocellulose-based ink for 3D printing of neural TE scaffolds. It is demonstrated that by using cellulose nanofibrils and carbon nanotubes as ink constituents, it is possible to print guidelines with a diameter below 1 mm and electrical conductivity of 3.8 × 10−1 S cm−1. The cell culture studies reveal that neural cells prefer to attach, proliferate, and differentiate on the 3D printed conductive guidelines. To our knowledge, this is the first research effort devoted to using cost-effective cellulosic 3D printed structures in neural TE, and we suppose that much more will arise in the near future.
AUTHOR Nguyen, Duong and Hägg, Daniel and Forsman, Alma and Ekholm, Josefine and Nimkingratana, Puwapong and Brantsing, Camilla and Kalogeropoulos, Theodoros and Zaunz, Samantha and Concaro, Sebastian and Brittberg, Mats and Lindahl, Anders and Gatenholm, Paul and Enejder, Annika and Simonsson, Stina
Title Cartilage Tissue Engineering by the 3D Bioprinting of iPS Cells in a Nanocellulose/Alginate Bioink [Abstract]
Year 2017
Journal/Proceedings Scientific Reports
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DOI/URL DOI
Abstract
Cartilage lesions can progress into secondary osteoarthritis and cause severe clinical problems in numerous patients. As a prospective treatment of such lesions, human-derived induced pluripotent stem cells (iPSCs) were shown to be 3D bioprinted into cartilage mimics using a nanofibrillated cellulose (NFC) composite bioink when co-printed with irradiated human chondrocytes. Two bioinks were investigated: NFC with alginate (NFC/A) or hyaluronic acid (NFC/HA). Low proliferation and phenotypic changes away from pluripotency were seen in the case of NFC/HA. However, in the case of the 3D-bioprinted NFC/A (60/40, dry weight % ratio) constructs, pluripotency was initially maintained, and after five weeks, hyaline-like cartilaginous tissue with collagen type II expression and lacking tumorigenic Oct4 expression was observed in 3D -bioprinted NFC/A (60/40, dry weight % relation) constructs. Moreover, a marked increase in cell number within the cartilaginous tissue was detected by 2-photon fluorescence microscopy, indicating the importance of high cell densities in the pursuit of achieving good survival after printing. We conclude that NFC/A bioink is suitable for bioprinting iPSCs to support cartilage production in co-cultures with irradiated chondrocytes.
AUTHOR Baumann, Bernhard and Jungst, Tomasz and Stichler, Simone and Feineis, Susanne and Wiltschka, Oliver and Kuhlmann, Matthias and Lindén, Mika and Groll, Jürgen
Title Control of Nanoparticle Release Kinetics from 3D Printed Hydrogel Scaffolds [Abstract]
Year 2017
Journal/Proceedings Angewandte Chemie International Edition
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DOI/URL DOI
Abstract
The convergence of biofabrication with nanotechnology is largely unexplored but enables geometrical control of cell-biomaterial arrangement combined with controlled drug delivery and release. As a step towards integration of these two fields of research, this study demonstrates that modulation of electrostatic nanoparticle–polymer and nanoparticle–nanoparticle interactions can be used for tuning nanoparticle release kinetics from 3D printed hydrogel scaffolds. This generic strategy can be used for spatiotemporal control of the release kinetics of nanoparticulate drug vectors in biofabricated constructs.
AUTHOR Mouser, V. H. M. and Abbadessa, A. and Levato, R. and Hennink, W. E. and Vermonden, T. and Gawlitta, D. and Malda, J.
Title Development of a thermosensitive HAMA-containing bio-ink for the fabrication of composite cartilage repair constructs [Abstract]
Year 2017
Journal/Proceedings Biofabrication
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Abstract
Fine-tuning of bio-ink composition and material processing parameters is crucial for the development of biomechanically relevant cartilage constructs. This study aims to design and develop cartilage constructs with tunable internal architectures and relevant mechanical properties. More specifically, the potential of methacrylated hyaluronic acid (HAMA) added to thermosensitive hydrogels composed of methacrylated poly[ N -(2-hydroxypropyl)methacrylamide mono/dilactate] (pHPMA-lac)/polyethylene glycol (PEG) triblock copolymers, to optimize cartilage-like tissue formation by embedded chondrocytes, and enhance printability was explored. Additionally, co-printing with polycaprolactone (PCL) was performed for mechanical reinforcement. Chondrocyte-laden hydrogels composed of pHPMA-lac-PEG and different concentrations of HAMA (0%–1% w/w) were cultured for 28 d in vitro and subsequently evaluated for the presence of cartilage-like matrix. Young’s moduli were determined for hydrogels with the different HAMA concentrations. Additionally, hydrogel/PCL constructs with different internal architectures were co-printed and analyzed for their mechanical properties. The results of this study demonstrated a dose-dependent effect of HAMA concentration on cartilage matrix synthesis by chondrocytes. Glycosaminoglycan (GAG) and collagen type II content increased with intermediate HAMA concentrations (0.25%–0.5%) compared to HAMA-free controls, while a relatively high HAMA concentration (1%) resulted in increased fibrocartilage formation. Young’s moduli of generated hydrogel constructs ranged from 14 to 31 kPa and increased with increasing HAMA concentration. The pHPMA-lac-PEG hydrogels with 0.5% HAMA were found to be optimal for cartilage-like tissue formation. Therefore, this hydrogel system was co-printed with PCL to generate porous or solid constructs with different mesh sizes. Young’s moduli of these composite constructs were in the range of native cartilage (3.5–4.6 MPa). Interestingly, the co-printing procedure influenced the mechanical properties of the final constructs. These findings are relevant for future bio-ink development, as they demonstrate the importance of selecting proper HAMA concentrations, as well as appropriate print settings and construct designs for optimal cartilage matrix deposition and final mechanical properties of constructs, respectively.
AUTHOR Henriksson, I. and Gatenholm, P. and Hägg, D. A.
Title Increased lipid accumulation and adipogenic gene expression of adipocytes in 3D bioprinted nanocellulose scaffolds [Abstract]
Year 2017
Journal/Proceedings Biofabrication
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DOI/URL URL
Abstract
Compared to standard 2D culture systems, new methods for 3D cell culture of adipocytes could provide more physiologically accurate data and a deeper understanding of metabolic diseases such as diabetes. By resuspending living cells in a bioink of nanocellulose and hyaluronic acid, we were able to print 3D scaffolds with uniform cell distribution. After one week in culture, cell viability was 95%, and after two weeks the cells displayed a more mature phenotype with larger lipid droplets than standard 2D cultured cells. Unlike cells in 2D culture, the 3D bioprinted cells did not detach upon lipid accumulation. After two weeks, the gene expression of the adipogenic marker genes PPAR γ and FABP4 was increased 2.0- and 2.2-fold, respectively, for cells in 3D bioprinted constructs compared with 2D cultured cells. Our 3D bioprinted culture system produces better adipogenic differentiation of mesenchymal stem cells and a more mature cell phenotype than conventional 2D culture systems.
AUTHOR Paxton, Naomi Claire and Smolan, Willi and Böck, Thomas and Melchels, Ferry P. W. and Groll, Juergen and Juengst, Tomasz
Title Proposal to Assess Printability of Bioinks for Extrusion-Based Bioprinting and Evaluation of Rheological Properties Governing Bioprintability [Abstract]
Year 2017
Journal/Proceedings Biofabrication
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DOI/URL DOI
Abstract
Abstract The development and formulation of printable inks for extrusion-based 3D bioprinting has been a major challenge in the field of biofabrication. Inks, often polymer solutions with the addition of crosslinking to form hydrogels, must not only display adequate mechanical properties for the chosen application, but also show high biocompatibility as well as printability. Here we describe a reproducible two-step method for the assessment of the printability of inks for bioprinting, focussing firstly on screening ink formulations to assess fibre formation and the ability to form 3D constructs before presenting a method for the rheological evaluation of inks to characterise the yield point, shear thinning and recovery behaviour. In conjunction, a mathematical model was formulated to provide a theoretical understanding of the pressure-driven, shear thinning extrusion of inks through needles in a bioprinter. The assessment methods were trialled with a commercially-available crème, poloxamer 407, alginate-based inks and an alginate-gelatin composite material. Yield stress was investigated by applying a stress ramp to a number of inks, which demonstrated the necessity of high yield for printable materials. The shear thinning behaviour of the inks was then characterised by quantifying the degree of shear thinning and using the mathematical model to predict the window of printer operating parameters in which the materials could be printed. Furthermore, the model predicted high shear conditions and high residence times for cells at the walls of the needle and effects on cytocompatibility at different printing conditions. Finally, the ability of the materials to recover to their original viscosity after extrusion was examined using rotational recovery rheological measurements. Taken together, these assessment techniques revealed significant insights into the requirements for printable inks and shear conditions present during the extrusion process and allow the rapid and reproducible characterisation of a wide variety of inks for bioprinting.
AUTHOR DeSimone, Elise and Schacht, Kristin and Pellert, Alexandra and Scheibel, Thomas
Title Recombinant spider silk-based bioinks [Abstract]
Year 2017
Journal/Proceedings Biofabrication
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Abstract
Bioinks, 3D cell culture systems which can be printed, are still in the early development stages. Currently, extensive research is going into designing printers to be more accommodating to bioinks, designing scaffolds with stiff materials as support structures for the often soft bioinks, and modifying the bioinks themselves. Recombinant spider silk proteins, a potential biomaterial component for bioinks, have high biocompatibility, can be processed into several morphologies and can be modified with cell adhesion motifs to enhance their bioactivity. In this work, thermally gelled hydrogels made from recombinant spider silk protein encapsulating mouse fibroblast cell line BALB/3T3 were prepared and characterized. The bioinks were evaluated for performance in vitro both before and after printing, and it was observed that unprinted bioinks provided a good platform for cell spreading and proliferation, while proliferation in printed scaffolds was prohibited. To improve the properties of the printed hydrogels, gelatin was given as an additive and thereby served indirectly as a plasticizer, improving the resolution of printed strands. Taken together, recombinant spider silk proteins and hydrogels made thereof show good potential as a bioink, warranting further development.
AUTHOR Levato, Riccardo and Webb, William R. and Otto, Iris A. and Mensinga, Anneloes and Zhang, Yadan and van Rijen, Mattie and van Weeren, René and Khan, Ilyas M. and Malda, Jos
Title The bio in the ink: cartilage regeneration with bioprintable hydrogels and articular cartilage-derived progenitor cells
Year 2017
Journal/Proceedings Acta Biomaterialia
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AUTHOR Abbadessa, Anna and Landín, Mariana and Oude Blenke, Erik and Hennink, Wim E. and Vermonden, Tina
Title Two-component thermosensitive hydrogels: Phase separation affecting rheological behavior [Abstract]
Year 2017
Journal/Proceedings European Polymer Journal
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Abstract
Abstract Extracellular matrices are mainly composed of a mixture of different biopolymers and therefore the use of two or more building blocks for the development of tissue-mimicking hydrogels is nowadays an attractive strategy in tissue-engineering. Multi-component hydrogel systems may undergo phase separation, which in turn can lead to new, unexpected material properties. The aim of this study was to understand the role of phase separation on the mechanical properties and 3D printability of hydrogels composed of triblock copolymers of polyethylene glycol (PEG) and methacrylated poly(N-(2-hydroxypropyl) methacrylamide-mono/dilactate) (pHPMAlac) blended with methacrylated hyaluronic acid (HAMA). To this end, hydrogels composed of different concentrations of PEG/pHPMAlac and HAMA, were analyzed for phase behavior and rheological properties. Subsequently, phase separation and rheological behavior as function of the two polymer concentrations were mathematically processed to generate a predictive model. Results showed that PEG/pHPMAlac/HAMA hydrogels were characterized by hydrophilic, HAMA-richer internal domains dispersed in a more hydrophobic continuous phase, composed of PEG/pHPMAlac, and that the volume fraction of the dispersed phase increased by increasing HAMA concentration. Storage modulus, yield stress and viscosity increased with increasing HAMA concentration for low/medium HAMA contents (≤0.75% w/w), while a further increase of HAMA resulted in a decrease of the mentioned properties. On the other hand, by increasing the concentration of PEG/pHPMAlac these rheological properties were enhanced. The generated models showed a good fitting with experimental data, and were used to identify an exemplary 3D printability window for PEG/pHPMAlac/HAMA hydrogels, which was verified by rheological characterization and preparation of 3D printed scaffolds. In conclusion, a clear relationship between phase separation and rheological behavior in these two-component hydrogels can be described by complex functions of the two polymer concentrations. The predictive model generated in this study can be used as a valid tool for the identification of hydrogel compositions with desired, selected characteristics.
AUTHOR {{'{A}}}vila, H{'{e}}ctor Mart{'{i}}nez and Schwarz, Silke and Rotter, Nicole and Gatenholm, Paul
Title 3D bioprinting of human chondrocyte-laden nanocellulose hydrogels for patient-specific auricular cartilage regeneration [Abstract]
Year 2016
Journal/Proceedings Bioprinting
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DOI/URL URL DOI
Abstract
Abstract Auricular cartilage tissue engineering (TE) aims to provide an effective treatment for patients with acquired or congenital auricular defects. Bioprinting has gained attention in several {TE} strategies for its ability to spatially control the placement of cells, biomaterials and biological molecules. Although considerable advances have been made to bioprint complex 3D tissue analogues, the development of hydrogel bioinks with good printability and bioactive properties must improve in order to advance the translation of 3D bioprinting into the clinic. In this study, the biological functionality of a bioink composed of nanofibrillated cellulose and alginate (NFC-A) is extensively evaluated for auricular cartilage TE. 3D bioprinted auricular constructs laden with human nasal chondrocytes (hNC) are cultured for up to 28 days and the redifferentiation capacity of hNCs in NFC-A is studied on gene expression as well as on protein levels. 3D bioprinting with NFC-A bioink facilitates the biofabrication of cell-laden, patient-specific auricular constructs with an open inner structure, high cell density and homogenous cell distribution. The cell-laden NFC-A constructs exhibit an excellent shape and size stability as well as an increase in cell viability and proliferation during in vitro culture. Furthermore, NFC-A bioink supports the redifferentiation of hNCs and neo-synthesis of cartilage-specific extracellular matrix components. This demonstrated that NFC-A bioink supports redifferentiation of hNCs while offering proper printability in a biologically relevant aqueous 3D environment, making it a promising tool for auricular cartilage {TE} and many other biomedical applications.
AUTHOR Raphael, Bella and Khalil, Tony and Workman, Victoria L. and Smith, Andrew and Brown, Cameron P. and Streulli, Charles and Saiani, Alberto and Domingos, Marco
Title 3D cell bioprinting of self-assembling peptide-based hydrogels [Abstract]
Year 2016
Journal/Proceedings Materials Letters
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Abstract
Abstract Bioprinting of 3D cell-laden constructs with well-defined architectures and controlled spatial distribution of cells is gaining importance in the field of Tissue Engineering. New 3D tissue models are being developed to study the complex cellular interactions that take place during both tissue development and in the regeneration of damaged and/or diseased tissues. Despite advances in 3D printing technologies, suitable hydrogels or 'bioinks' with enhanced printability and cell viability are lacking. Here we report a study on the 3D bioprinting of a novel group of self-assembling peptide-based hydrogels. Our results demonstrate the ability of the system to print well-defined 3D cell laden constructs with variable stiffness and improved structural integrity, whilst providing a cell-friendly extracellular matrix “like” microenvironment. Biological assays reveal that mammary epithelial cells remain viable after 7 days of in vitro culture, independent of the hydrogel stiffness.
AUTHOR Gudapati, Hemanth and Dey, Madhuri and Ozbolat, Ibrahim
Title A comprehensive review on droplet-based bioprinting: Past, present and future. [Abstract]
Year 2016
Journal/Proceedings Biomaterials
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DOI/URL DOI
Abstract
Droplet-based bioprinting (DBB) offers greater advantages due to its simplicity and agility with precise control on deposition of biologics including cells, growth factors, genes, drugs and biomaterials, and has been a prominent technology in the bioprinting community. Due to its immense versatility, DBB technology has been adopted by various application areas, including but not limited to, tissue engineering and regenerative medicine, transplantation and clinics, pharmaceutics and high-throughput screening, and cancer research. Despite the great benefits, the technology currently faces several challenges such as a narrow range of available bioink materials, bioprinting-induced cell damage at substantial levels, limited mechanical and structural integrity of bioprinted constructs, and restrictions on the size of constructs due to lack of vascularization and porosity. This paper presents a first-time review of DBB and comprehensively covers the existing DBB modalities including inkjet, electrohydrodynamic, acoustic, and micro-valve bioprinting. The recent notable studies are highlighted, the relevant bioink biomaterials and bioprinters are expounded, the application areas are presented, and the future prospects are provided to the reader.
AUTHOR Abbadessa, Anna and Mouser, Vivian H. M. and Blokzijl, Maarten M. and Gawlitta, Debby and Dhert, Wouter J. A. and Hennink, Wim E. and Malda, Jos and Vermonden, Tina
Title A Synthetic Thermosensitive Hydrogel for Cartilage Bioprinting and Its Biofunctionalization with Polysaccharides [Abstract]
Year 2016
Journal/Proceedings Biomacromolecules
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DOI/URL DOI
Abstract
Hydrogels based on triblock copolymers of polyethylene glycol and partially methacrylated poly[N-(2-hydroxypropyl) methacrylamide mono/dilactate] make up an attractive class of biomaterials because of their biodegradability, cytocompatibility, and tunable thermoresponsive and mechanical properties. If these properties are fine-tuned, the hydrogels can be three-dimensionally bioprinted, to generate, for instance, constructs for cartilage repair. This study investigated whether hydrogels based on the polymer mentioned above with a 10% degree of methacrylation (M10P10) support cartilage formation by chondrocytes and whether the incorporation of methacrylated chondroitin sulfate (CSMA) or methacrylated hyaluronic acid (HAMA) can improve the mechanical properties, long-term stability, and printability. Chondrocyte-laden M10P10 hydrogels were cultured for 42 days to evaluate chondrogenesis. M10P10 hydrogels with or without polysaccharides were evaluated for their mechanical properties (before and after UV photo-cross-linking), degradation kinetics, and printability. Extensive cartilage matrix production occurred in M10P10 hydrogels, highlighting their potential for cartilage repair strategies. The incorporation of polysaccharides increased the storage modulus of polymer mixtures and decreased the degradation kinetics in cross-linked hydrogels. Addition of HAMA to M10P10 hydrogels improved printability and resulted in three-dimensional constructs with excellent cell viability. Hence, this novel combination of M10P10 with HAMA forms an interesting class of hydrogels for cartilage bioprinting.
AUTHOR Abbadessa, A. and Blokzijl, M. M. and Mouser, V. H. M. and Marica, P. and Malda, J. and Hennink, W. E. and Vermonden, T.
Title A thermo-responsive and photo-polymerizable chondroitin sulfate-based hydrogel for 3D printing applications [Abstract]
Year 2016
Journal/Proceedings Carbohydrate Polymers
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DOI/URL URL DOI
Abstract
Abstract The aim of this study was to design a hydrogel system based on methacrylated chondroitin sulfate (CSMA) and a thermo-sensitive poly(N-(2-hydroxypropyl) methacrylamide-mono/dilactate)-polyethylene glycol triblock copolymer (M15P10) as a suitable material for additive manufacturing of scaffolds. {CSMA} was synthesized by reaction of chondroitin sulfate with glycidyl methacrylate (GMA) in dimethylsulfoxide at 50 °C and its degree of methacrylation was tunable up to 48.5%, by changing reaction time and {GMA} feed. Unlike polymer solutions composed of {CSMA} alone (20% w/w), mixtures based on 2% w/w of {CSMA} and 18% of {M15P10} showed strain-softening, thermo-sensitive and shear-thinning properties more pronounced than those found for polymer solutions based on {M15P10} alone. Additionally, they displayed a yield stress of 19.2 ± 7.0 Pa. The 3D printing of this hydrogel resulted in the generation of constructs with tailorable porosity and good handling properties. Finally, embedded chondrogenic cells remained viable and proliferating over a culture period of 6 days. The hydrogel described herein represents a promising biomaterial for cartilage 3D printing applications.
AUTHOR M{"u}ller, Michael and {"O}zt{"u}rk, Ece and Arlov, {O}ystein and Gatenholm, Paul and Zenobi-Wong, Marcy
Title Alginate Sulfate--Nanocellulose Bioinks for Cartilage Bioprinting Applications [Abstract]
Year 2016
Journal/Proceedings Annals of Biomedical Engineering
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DOI/URL DOI
Abstract
One of the challenges of bioprinting is to identify bioinks which support cell growth, tissue maturation, and ultimately the formation of functional grafts for use in regenerative medicine. The influence of this new biofabrication technology on biology of living cells, however, is still being evaluated. Recently we have identified a mitogenic hydrogel system based on alginate sulfate which potently supports chondrocyte phenotype, but is not printable due to its rheological properties (no yield point). To convert alginate sulfate to a printable bioink, it was combined with nanocellulose, which has been shown to possess very good printability. The alginate sulfate/nanocellulose ink showed good printing properties and the non-printed bioink material promoted cell spreading, proliferation, and collagen II synthesis by the encapsulated cells. When the bioink was printed, the biological performance of the cells was highly dependent on the nozzle geometry. Cell spreading properties were maintained with the lowest extrusion pressure and shear stress. However, extruding the alginate sulfate/nanocellulose bioink and chondrocytes significantly compromised cell proliferation, particularly when using small diameter nozzles and valves.
AUTHOR Kesti, Matti and Fisch, Philipp and Pensalfini, Marco and Mazza, Edoardo and Zenobi-Wong, Marcy
Title Guidelines for standardization of bioprinting: a systematic study of process parameters and their effect on bioprinted structures [Abstract]
Year 2016
Journal/Proceedings BioNanoMaterials
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DOI/URL DOI
Abstract
Biofabrication techniques including three-dimensional bioprinting could be used one day to fabricate living, patient-specific tissues and organs for use in regenerative medicine. Compared to traditional casting and molding methods, bioprinted structures can be much more complex, containing for example multiple materials and cell types in controlled spatial arrangement, engineered porosity, reinforcement structures and gradients in mechanical properties. With this complexity and increased function, however, comes the necessity to develop guidelines to standardize the bioprinting process, so printed grafts can safely enter the clinics. The bioink material must firstly fulfil requirements for biocompatibility and flow. Secondly, it is important to understand how process parameters affect the final mechanical properties of the printed graft. Using a gellan-alginate physically crosslinked bioink as an example, we show shear thinning and shear recovery properties which allow good printing resolution. Printed tensile specimens were used to systematically assess effect of line spacing, printing direction and crosslinking conditions. This standardized testing allowed direct comparison between this bioink and three commercially-available products. Bioprinting is a promising, yet complex fabrication method whose outcome is sensitive to a range of process parameters. This study provides the foundation for highly needed best practice guidelines for reproducible and safe bioprinted grafts.
AUTHOR Hou, Xiaochun and Liu, Shiying and Wang, Min and Wiraja, Christian and Huang, Wei and Chan, Peggy and Tan, Timothy and Xu, Chenjie
Title Layer-by-Layer 3D Constructs of Fibroblasts in Hydrogel for Examining Transdermal Penetration Capability of Nanoparticles [Abstract]
Year 2016
Journal/Proceedings Journal of Laboratory Automation
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DOI/URL URL DOI
Abstract
Nanoparticles are emerging transdermal delivery systems. Their size and surface properties determine their efficacy and efficiency to penetrate through the skin layers. This work utilizes three-dimensional (3D) bioprinting technology to generate a simplified artificial skin model to rapidly screen nanoparticles for their transdermal penetration ability. Specifically, this model is built through layer-by-layer alternate printing of blank collagen hydrogel and fibroblasts. Through controlling valve on-time, the spacing between printing lines could be accurately tuned, which could enable modulation of cell infiltration in the future. To confirm the effectiveness of this platform, a 3D construct with one layer of fibroblasts sandwiched between two layers of collagen hydrogel is used to screen silica nanoparticles with different surface charges for their penetration ability, with positively charged nanoparticles demonstrating deeper penetration, consistent with the observation from an existing study involving living skin tissue.
AUTHOR Håkansson, Karl M. O. and Henriksson, Ida C. and de la Peña Vázquez, Cristina and Kuzmenko, Volodymyr and Markstedt, Kajsa and Enoksson, Peter and Gatenholm, Paul
Title Solidification of 3D Printed Nanofibril Hydrogels into Functional 3D Cellulose Structures [Abstract]
Year 2016
Journal/Proceedings Advanced Materials Technologies
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DOI/URL DOI
Abstract
Cellulose nanofibrils isolated from trees have the potential to be used as raw material for future sustainable products within the areas of packaging, textiles, biomedical devices, and furniture. However, one unsolved problem has been to convert the nanofibril-hydrogel into a dry 3D structure. In this study, 3D printing is used to convert a cellulose nanofibril hydrogel into 3D structures with controlled architectures. Such structures collapse upon drying, but by using different drying processes the collapse can be controlled and the 3D structure can be preserved upon solidification. In addition, a conductive cellulose nanofibril ink is fabricated by adding carbon nanotubes. These findings enable the use of wood derived materials in 3D printing for fabrication of sustainable commodities such as packaging, textiles, biomedical devices, and furniture with conductive parts. Furthermore, with the introduction of biopolymers into 3D printing, the 3D printing technology itself can finally be regarded as sustainable.
AUTHOR Stichler, Simone and Jungst, Tomasz and Schamel, Martha and Zilkowski, Ilona and Kuhlmann, Matthias and Bock, Thomas and Blunk, Torsten and Tessmar, Jorg and Groll, Jurgen
Title Thiol-ene Clickable Poly(glycidol) Hydrogels for Biofabrication. [Abstract]
Year 2016
Journal/Proceedings Annals of biomedical engineering
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DOI/URL DOI
Abstract
In this study we introduce linear poly(glycidol) (PG), a structural analog of poly(ethylene glycol) bearing side chains at each repeating unit, as polymer basis for bioink development. We prepare allyl- and thiol-functional linear PG that can rapidly be polymerized to a three-dimensionally cross-linked hydrogel network via UV mediated thiol-ene click reaction. Influence of polymer concentration and UV irradiation on mechanical properties and swelling behavior was examined. Thiol-functional PG was synthesized in two structural variations, one containing ester groups that are susceptible to hydrolytic cleavage, and the other one ester-free and stable against hydrolysis. This allowed the preparation of degradable and non-degradable hydrogels. Cytocompatibility of the hydrogel was demonstrated by encapsulation of human bone marrow-derived mesenchymal stem cells (hBMSCs). Rheological properties of the hydrogels were adjusted for dispense plotting by addition of high molecular weight hyaluronic acid. The optimized formulation enabled highly reproducible plotting of constructs composed of 20 layers with an overall height of 3.90 mm.
AUTHOR Markstedt, Kajsa and Mantas, Athanasios and Tournier, Ivan and Mart{'{i}}nez {{'{A}}}vila, H{'{e}}ctor and H{"{a}}gg, Daniel and Gatenholm, Paul
Title 3D Bioprinting Human Chondrocytes with Nanocellulose-Alginate Bioink for Cartilage Tissue Engineering Applications [Abstract]
Year 2015
Journal/Proceedings Biomacromolecules
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DOI/URL DOI
Abstract
The introduction of 3D bioprinting is expected to revolutionize the field of tissue engineering and regenerative medicine. The 3D bioprinter is able to dispense materials while moving in X, Y, and Z directions, which enables the engineering of complex structures from the bottom up. In this study, a bioink that combines the outstanding shear thinning properties of nanofibrillated cellulose (NFC) with the fast cross-linking ability of alginate was formulated for the 3D bioprinting of living soft tissue with cells. Printability was evaluated with concern to printer parameters and shape fidelity. The shear thinning behavior of the tested bioinks enabled printing of both 2D gridlike structures as well as 3D constructs. Furthermore, anatomically shaped cartilage structures, such as a human ear and sheep meniscus, were 3D printed using MRI and CT images as blueprints. Human chondrocytes bioprinted in the noncytotoxic, nanocellulose-based bioink exhibited a cell viability of 73% and 86% after 1 and 7 days of 3D culture, respectively. On the basis of these results, we can conclude that the nanocellulose-based bioink is a suitable hydrogel for 3D bioprinting with living cells. This study demonstrates the potential use of nanocellulose for 3D bioprinting of living tissues and organs. The introduction of 3D bioprinting is expected to revolutionize the field of tissue engineering and regenerative medicine. The 3D bioprinter is able to dispense materials while moving in X, Y, and Z directions, which enables the engineering of complex structures from the bottom up. In this study, a bioink that combines the outstanding shear thinning properties of nanofibrillated cellulose (NFC) with the fast cross-linking ability of alginate was formulated for the 3D bioprinting of living soft tissue with cells. Printability was evaluated with concern to printer parameters and shape fidelity. The shear thinning behavior of the tested bioinks enabled printing of both 2D gridlike structures as well as 3D constructs. Furthermore, anatomically shaped cartilage structures, such as a human ear and sheep meniscus, were 3D printed using MRI and CT images as blueprints. Human chondrocytes bioprinted in the noncytotoxic, nanocellulose-based bioink exhibited a cell viability of 73% and 86% after 1 and 7 days of 3D culture, respectively. On the basis of these results, we can conclude that the nanocellulose-based bioink is a suitable hydrogel for 3D bioprinting with living cells. This study demonstrates the potential use of nanocellulose for 3D bioprinting of living tissues and organs.
AUTHOR Schacht, Kristin and J{"{u}}ngst, Tomasz and Schweinlin, Matthias and Ewald, Andrea and Groll, J{"{u}}rgen and Scheibel, Thomas
Title Biofabrication of Cell-Loaded 3D Spider Silk Constructs [Abstract]
Year 2015
Journal/Proceedings Angewandte Chemie International Edition
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DOI/URL DOI
Abstract
Biofabrication is an emerging and rapidly expanding field of research in which additive manufacturing techniques in combination with cell printing are exploited to generate hierarchical tissue-like structures. Materials that combine printability with cytocompatibility, so called bioinks, are currently the biggest bottleneck. Since recombinant spider silk proteins are non-immunogenic, cytocompatible, and exhibit physical crosslinking, their potential as a new bioink system was evaluated. Cell-loaded spider silk constructs can be printed by robotic dispensing without the need for crosslinking additives or thickeners for mechanical stabilization. Cells are able to adhere and proliferate with good viability over at least one week in such spider silk scaffolds. Introduction of a cell-binding motif to the spider silk protein further enables fine-tuned control over cell–material interactions. Spider silk hydrogels are thus a highly attractive novel bioink for biofabrication.
AUTHOR Horvath, Lenke and Umehara, Yuki and Jud, Corinne and Blank, Fabian and Petri-Fink, Alke and Rothen-Rutishauser, Barbara
Title Engineering an in vitro air-blood barrier by 3D bioprinting. [Abstract]
Year 2015
Journal/Proceedings Scientific reports
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DOI/URL URL
Abstract
Intensive efforts in recent years to develop and commercialize in vitro alternatives in the field of risk assessment have yielded new promising two- and three dimensional (3D) cell culture models. Nevertheless, a realistic 3D in vitro alveolar model is not available yet. Here we report on the biofabrication of the human air-blood tissue barrier analogue composed of an endothelial cell, basement membrane and epithelial cell layer by using a bioprinting technology. In contrary to the manual method, we demonstrate that this technique enables automatized and reproducible creation of thinner and more homogeneous cell layers, which is required for an optimal air-blood tissue barrier. This bioprinting platform will offer an excellent tool to engineer an advanced 3D lung model for high-throughput screening for safety assessment and drug efficacy testing.
AUTHOR M{"{u}}ller, Michael and Becher, Jana and Schnabelrauch, Matthias and Zenobi-Wong, Marcy
Title Nanostructured Pluronic hydrogels as bioinks for 3D bioprinting [Abstract]
Year 2015
Journal/Proceedings Biofabrication
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DOI/URL URL
Abstract
Bioprinting is an emerging technology in the field of tissue engineering as it allows the precise positioning of biologically relevant materials in 3D, which more resembles the native tissue in our body than current homogenous, bulk approaches. There is however a lack of materials to be used with this technology and materials such as the block copolymer Pluronic have good printing properties but do not allow long-term cell culture. Here we present an approach called nanostructuring to increase the biocompatibility of Pluronic gels at printable concentrations. By mixing acrylated with unmodified Pluronic F127 it was possible to maintain the excellent printing properties of Pluronic and to create stable gels via UV crosslinking. By subsequent elution of the unmodified Pluronic from the crosslinked network we were able to increase the cell viability of encapsulated chondrocytes at day 14 from 62% for a pure acrylated Pluronic hydrogel to 86% for a nanostructured hydrogel. The mixed Pluronic gels also showed good printability when cells where included in the bioink. The nanostructured gels were, with a compressive modulus of 1.42 kPa, mechanically weak, but we were able to increase the mechanical properties by the addition of methacrylated hyaluronic acid. Our nanostructuring approach enables Pluronic hydrogels to have the desired set of properties in all stages of the bioprinting process.
AUTHOR Rimann, Markus and Bono, Epifania and Annaheim, Helene and Bleisch, Matthias and Graf-Hausner, Ursula
Title Standardized 3D Bioprinting of Soft Tissue Models with Human Primary Cells. [Abstract]
Year 2015
Journal/Proceedings Journal of laboratory automation
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DOI/URL DOI
Abstract
Cells grown in 3D are more physiologically relevant than cells cultured in 2D. To use 3D models in substance testing and regenerative medicine, reproducibility and standardization are important. Bioprinting offers not only automated standardizable processes but also the production of complex tissue-like structures in an additive manner. We developed an all-in-one bioprinting solution to produce soft tissue models. The holistic approach included (1) a bioprinter in a sterile environment, (2) a light-induced bioink polymerization unit, (3) a user-friendly software, (4) the capability to print in standard labware for high-throughput screening, (5) cell-compatible inkjet-based printheads, (6) a cell-compatible ready-to-use BioInk, and (7) standard operating procedures. In a proof-of-concept study, skin as a reference soft tissue model was printed. To produce dermal equivalents, primary human dermal fibroblasts were printed in alternating layers with BioInk and cultured for up to 7 weeks. During long-term cultures, the models were remodeled and fully populated with viable and spreaded fibroblasts. Primary human dermal keratinocytes were seeded on top of dermal equivalents, and epidermis-like structures were formed as verified with hematoxylin and eosin staining and immunostaining. However, a fully stratified epidermis was not achieved. Nevertheless, this is one of the first reports of an integrative bioprinting strategy for industrial routine application.
AUTHOR Kesti, Matti and M{"{u}}ller, Michael and Becher, Jana and Schnabelrauch, Matthias and D{textquoteright}Este, Matteo and Eglin, David and Zenobi-Wong, Marcy
Title A versatile bioink for three-dimensional printing of cellular scaffolds based on thermally and photo-triggered tandem gelation [Abstract]
Year 2014
Journal/Proceedings Acta Biomaterialia
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DOI/URL URL DOI
Abstract
Abstract Layer-by-layer bioprinting is a logical choice for the fabrication of stratified tissues like articular cartilage. Printing of viable organ replacements, however, is dependent on bioinks with appropriate rheological and cytocompatible properties. In cartilage engineering, photocrosslinkable glycosaminoglycan-based hydrogels are chondrogenic, but alone have generally poor printing properties. By blending the thermoresponsive polymer poly(N-isopropylacrylamide) grafted hyaluronan (HA-pNIPAAM) with methacrylated hyaluronan (HAMA), high-resolution scaffolds with good viability were printed. HA-pNIPAAM provided fast gelation and immediate post-printing structural fidelity, while {HAMA} ensured long-term mechanical stability upon photocrosslinking. The bioink was evaluated for rheological properties, swelling behavior, printability and biocompatibility of encapsulated bovine chondrocytes. Elution of HA-pNIPAAM from the scaffold was necessary to obtain good viability. HA-pNIPAAM can therefore be used to support extrusion of a range of biopolymers which undergo tandem gelation, thereby facilitating the printing of cell-laden, stratified cartilage constructs with zonally varying composition and stiffness.
AUTHOR M{"{u}}ller, Michael and Becher, Jana and Schnabelrauch, Matthias and Zenobi-Wong, Marcy
Title Printing thermoresponsive reverse molds for the creation of patterned two-component hydrogels for 3D cell culture. [Abstract]
Year 2013
Journal/Proceedings Journal of visualized experiments : JoVE
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DOI/URL URL
Abstract
Bioprinting is an emerging technology that has its origins in the rapid prototyping industry. The different printing processes can be divided into contact bioprinting(1-4) (extrusion, dip pen and soft lithography), contactless bioprinting(5-7) (laser forward transfer, ink-jet deposition) and laser based techniques such as two photon photopolymerization(8). It can be used for many applications such as tissue engineering(9-13), biosensor microfabrication(14-16) and as a tool to answer basic biological questions such as influences of co-culturing of different cell types(17). Unlike common photolithographic or soft-lithographic methods, extrusion bioprinting has the advantage that it does not require a separate mask or stamp. Using CAD software, the design of the structure can quickly be changed and adjusted according to the requirements of the operator. This makes bioprinting more flexible than lithography-based approaches. Here we demonstrate the printing of a sacrificial mold to create a multi-material 3D structure using an array of pillars within a hydrogel as an example. These pillars could represent hollow structures for a vascular network or the tubes within a nerve guide conduit. The material chosen for the sacrificial mold was poloxamer 407, a thermoresponsive polymer with excellent printing properties which is liquid at 4 degrees C and a solid above its gelation temperature ~20 degrees C for 24.5% w/v solutions(18). This property allows the poloxamer-based sacrificial mold to be eluted on demand and has advantages over the slow dissolution of a solid material especially for narrow geometries. Poloxamer was printed on microscope glass slides to create the sacrificial mold. Agarose was pipetted into the mold and cooled until gelation. After elution of the poloxamer in ice cold water, the voids in the agarose mold were filled with alginate methacrylate spiked with FITC labeled fibrinogen. The filled voids were then cross-linked with UV and the construct was imaged with an epi-fluorescence microscope.