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Allevi 2 Publications

 3D printed UV light cured polydimethylsiloxane devices for drug delivery

This paper, published by researchers from  Åbo Akademi University and the University of Helsinki in Finland, use the Allevi 2 platform to fabricate drug-containing PDMS structures.

Abstract: The goal of this work was to study the printability of PDMS with a semi-solid extrusion printer in combination with the UV-assisted crosslinking technology using UV-LED light to manufacture drug containing structures. Structures with different pore sizes and different drug loadings were prepared containing prednisolone as a model drug. The work showed that it was possible to print drug-free and drug-loaded drug delivery devices of PDMS with the 3D printing technique used in this study. The required UV-curing time to get sufficient crosslinking yield and mechanical strength was minimum three minutes. The microgram drug release from the printed structures was highest for the most drug loaded structures regardless of the porosity of the devices. By altering the surface area/volume ratio it was possible to print structures with differences in the release rate. This study shows that room-temperature semi-solid extrusion printing 3D printing technique in combination with UV-LED crosslinking is an applicable method in the production of prednisolone containing PDMS devices. Both the extrusion 3D printing and the UV-crosslinking was done at room temperature, which make this manufacturing method an interesting alternative for manufacturing controlled release devices containing temperature susceptible drugs.

Quantitative criteria to benchmark new and existing bio-inks for cell compatibility

This paper, published by Professor Sarah C. Heilshorn’s lab at Stanford University, utilizes an Allevi 2 and Allevi LAP to develop a novel bioink hydrogel.

Abstract: Recent advancements in 3D bioprinting have led to the fabrication of more complex, more precise, and larger printed tissue constructs. As the field continues to advance, it is critical to develop quantitative benchmarks to compare different bio-inks for key cell-biomaterial interactions, including (1) cell sedimentation within the ink cartridge, (2) cell viability during extrusion, and (3) cell viability after ink curing. Here we develop three simple protocols for quantitative analysis of bio-ink performance. These methods are used to benchmark the performance of two commonly used bio-inks, poly(ethylene glycol) diacrylate (PEGDA) and gelatin methacrylate (GelMA), against three formulations of a novel bio-ink, Recombinant-protein Alginate Platform for Injectable Dual-crosslinked ink (RAPID ink). RAPID inks undergo peptide-self-assembly to form weak, shear-thinning gels in the ink cartridge and undergo electrostatic crosslinking with divalent cations during curing. In the one hour cell sedimentation assay, GelMA, the RAPID inks, and PEGDA with xanthan gum prevented appreciable cell sedimentation, while PEGDA alone or PEGDA with alginate experienced significant cell settling. To quantify cell viability during printing, 3T3 fibroblasts were printed at a constant flow rate of 75 μl min-1 and immediately tested for cell membrane integrity. Less than 10% of cells were damaged using the PEGDA and GelMA bio-inks, while less than 4% of cells were damaged using the RAPID inks. Finally, to evaluate cell viability after curing, cells were exposed to ink-specific curing conditions for five minutes and tested for membrane integrity. After exposure to light with photoinitiator at ambient conditions, over 50% of cells near the edges of printed PEGDA and GelMA droplets were damaged. In contrast, fewer than 20% of cells found near the edges of RAPID inks were damaged after a 5 min exposure to curing in a 10 mM CaCl2solution. As new bio-inks continue to be developed, these protocols offer a convenient means to quantitatively benchmark their performance against existing inks.
Dubbin, Karen et al. Quantitative criteria to benchmark new and existing bio-inks for cell compatibility. Biofabrication. 2017 Sep 1;9(4):044102. doi: 10.1088/1758-5090/aa869f.

Characterization of Pluronic F-127/Pectin Hydrogel for Potential Tissue Engineering Applications

This work from Milwaukee School of Engineering uses an Allevi 2 to characterize a new pectin-based bioink.

Abstract: Human compatible organs are needed because there is a greater requirement for organ transplants than there is availability of organ donors. Even though the number of people in need of a transplant has been increasing the number of donors recovered remains the same. Biofabrication, including bioprinting, provides novel methods to print tissue containing the cells and a biomaterial scaffold. Advances in biofabrication is closing the gap between the number of people waiting for a transplant and those receiving one. This research focused on the use of a novel pectin-based bioink. Pluronic® F-127, the other component of the bioink, is used to obtain the desired shape during the initial bio-printing process at 37 ᵒC. In order for the pectin/Pluronic solution to maintain its structure at a lower temperature, e.g., 4 ᵒC, a cross-linker solution needs to be introduced to gel the pectin. The crosslinkers tested were Ca2+ , oligochitosan, and Zn2+ . The scaffold structural integrity after bioprinting was tested, determining the cross-linker Ca2+ to be the most effective. Moreover, the post-printing treatment of the scaffold using chitosan was also investigated to increase its surface charge property. Stability testing was conducted, during testing period all samples remained stable. The results of this research provide further insight into the process of bioprinting with a pectin-based bioink.
Bryant, Elizabeth A.  Characterization of Pluronic F-127/Pectin Hydrogel for Potential Tissue Engineering Applications. Proceedings of the National Conference on Undergraduate Research. 2018. Oklahoma City, Oklahoma.

Feasibility of Fabricating Personalized 3D-printed Bone Grafts Guided by High-Resolution Imaging

This publication from Dr. Chamith S. Rajapakse‘s Lab at the University of Pennsylvania uses an Allevi 2 to fabricate 3D models of patient-specific bone grafts guided by medical imaging.

Abstract: Current methods of bone graft treatment for critical size bone defects can give way to several clinical complications such as limited available bone for autografts, non-matching bone structure, lack of strength which can compromise a patient’s skeletal system, and sterilization processes that can prevent osteogenesis in the case of allografts. We intend to overcome these disadvantages by generating a patient-specific 3D printed bone graft guided by high-resolution medical imaging. Our synthetic model allows us to customize the graft for the patients’ macro- and microstructure and correct any structural deficiencies in the re-meshing process. These 3D-printed models can presumptively serve as the scaffolding for human mesenchymal stem cell (hMSC) engraftment in order to facilitate bone growth. We performed high resolution CT imaging of a cadaveric human proximal femur at 0.030-mm isotropic voxels. We used these images to generate a 3D computer model that mimics bone geometry from micro to macro scale represented by STereoLithography (STL) format. These models were then reformatted to a format that can be interpreted by the 3D printer. To assess how much of the microstructure was replicated, 3D-printed models were re-imaged using micro-CT at 0.025-mm isotropic voxels and compared to original high-resolution CT images used to generate the 3D model in 32 sub-regions. We found a strong correlation between 3D-printed bone volume and volume of bone in the original images used for 3D printing (R2 = 0.97). We expect to further refine our approach with additional testing to create a viable synthetic bone graft with clinical functionality.
Hong, Abigail L. et al. Proceedings Volume 10138, Medical Imaging 2017: Imaging Informatics for Healthcare, Research, and Applications; 101380O (2017); doi: 10.1117/12.2254475

 

3D Printing Vegemite and Marmite: Redefining “Breadboards”

This publication from the University of Wollongong uses an Allevi 2 to 3D print “breadboards” out of Vegemite and Marmite for educational purposes.

Abstract: The ability to use Food Layered Manufacturing (FLM) to fabricate attractive food presentations and incorporate additives that can alter texture, nutrition, color, and flavor have made it widely investigated for combatting various issues in the food industry. For a food item to be FLM compatible, it must possess suitable rheological properties to allow for its extrusion and to keep its 3D printed structure. Here, we present a rheological analysis of two commercially available breakfast spreads, Vegemite and Marmite, and show their compatibility with FLM in producing 3D structures onto bread substrates. Furthermore, we demonstrated that these materials can be used to fabricate attractive food designs that can be used for educational activities. The inherent conductivity of the breakfast spreads was used to print edible circuits onto a “breadboard.”
Hamilton, Charles C. et al. Journal of Food Engineering xxx (2017) 1-6

 

BioBot Beta Publications

Dual-Stage Crosslinking of a Gel-Phase Bioink Improves Cell Viability and Homogeneity for 3D Bioprinting

This paper, published in Advanced Healthcare Materials by Professor Sarah C. Heilshorn’s lab at Stanford University, utilizes an Allevi Beta to develop a novel bioink hydrogel.

Abstract: Current bioinks for cell-based 3D bioprinting are not suitable for technology scale-up due to the challenges of cell sedimentation, cell membrane damage, and cell dehydration. A novel bioink hydrogel is presented with dual-stage crosslinking specifically designed to overcome these three major hurdles. This bioink enables the direct patterning of highly viable, multicell type constructs with long-term spatial fidelity.
Dubbin, K., Hori, Y., Lewis, K. K. and Heilshorn, S. C. (2016), Dual-Stage Crosslinking of a Gel-Phase Bioink Improves Cell Viability and Homogeneity for 3D Bioprinting. Advanced Healthcare Materials. doi: 10.1002/adhm.201600636

 

Characterisation of Hyaluronic Acid Methylcellulose Hydrogels for 3D Bioprinting

This publication from the Harry Perkins Institute of Medical Research uses an Allevi Beta printer to characterize hyaluronic acid methylcellulose bioionks.

Abstract: Hydrogels containing hyaluronic acid (HA) and methylcellulose (MC) have shown promising results for three dimensional (3D) bioprinting applications. However, several parameters influence the applicability bioprinting and there is scarce data in the literature characterising HAMC. We assessed eight concentrations of HAMC for printability, swelling and stability over time, rheological and structural behaviour, and viability of mesenchymal stem cells. We show that HAMC blends behave as viscous solutions at 4 °C and have faster gelation times at higher temperatures, typically gelling upon reaching 37 °C. We found the storage, loss and compressive moduli to be dependent on HAMC concentration and incubation time at 37 °C, and show the compressive modulus to be strain-rate dependent. Swelling and stability was influenced by time, more so than pH level. We demonstrated that mesenchymal stem cell viability was above 75% in bioprinted structures and cells remain viable for at least one week after 3D bioprinting. The mechanical properties of HAMC are highly tuneable and we show that higher concentrations of HAMC are particularly suited to cell-encapsulated 3D bioprinting applications that require scaffold structure and delivery of cells.
Nicholas Law et al. Characterisation of Hyaluronic Acid Methylcellulose Hydrogels for 3D Bioprinting, Journal of the Mechanical Behavior of Biomedical Materials, http://dx.doi.org/10.1016/j.jmbbm.2017.09.031

 

3D Bioprinting Hydrogel for Tissue Engineering an Aortic Scaffold

This thesis work by Benjamin Stewart from the University of Denver uses an Allevi Beta printer to fabricate analyze scaffolds for thoracic aortic grafts.

Abstract: The gold standard in 2016 for thoracic aortic grafts is Dacron®, polyethylene terephthalate, due to the durability over time, the low immune response elicited and the propensity for endothelialization of the graft lumen over time. These synthetic grafts provide reliable materials that show remarkable long term patency. Despite the acceptable performance of Dacron® grafts, it is noted that autographs still outperform other types of vascular grafts when available due to recognition of the host’s cells and adaptive mechanical properties of a living graft. 3-D bioprinting patient-specific scaffolds for tissue engineering (TE) brings the benefits of non-degrading synthetic grafts and autologous grafts together by constructing a synthetic scaffold that supports cell infiltration, adhesion, and development in order to promote the cells to build the native extracellular matrix in response to biochemical and physical cues. Using the BioBots 3-D bioprinter, scaffold materials we tested non-Newtonian photosensitive hydrogel that formed a crosslinked matrix under 365 nm UV light with appropriate water content and mechanical properties for cell infiltration and adhesion to the bioprinted scaffold. Viscometry data on the PEGDA-HPMC 15%-2% w/v hydrogel (non-Newtonian behavior) informed CFD simulation of the extrusion system in order to exact the pressure-flow rate relationship for every hydrogel and geometry combination. Surface tension data and mechanical properties were obtained from material testing and provide content to further characterize each hydrogel and resulting crosslinked scaffold. The goal of this work was to create a basis to build a database of hydrogels with corresponding print settings and resulting mechanical properties.
Stewart, Benjamin, “3D Bioprinting Hydrogel for Tissue Engineering an Ascending Aortic Scaffold” (2017). Electronic Theses and Dissertations. 1269. http://digitalcommons.du.edu/etd/1269

 

Differences in Time-Dependent Mechanical Properties Between Extruded and Molded Hydrogels

This publication in Biofabrication by Dr. Kara Spiller’s lab at Drexel University utilizes an Allevi Beta to analyze and compare mechanical and swelling properties of gelatin methacrylate hydrogels prepared with conventional molding techniques and 3D printing.

Abstract: The mechanical properties of hydrogels used in biomaterials and tissue engineering applications are critical determinants of their functionality. Despite the recent rise of additive manufacturing, and specifically extrusion-based bioprinting, as a prominent biofabrication method, comprehensive studies investigating the mechanical behavior of extruded constructs remain lacking. To address this gap in knowledge, we compared the mechanical properties and swelling properties of crosslinked gelatin-based hydrogels prepared by conventional molding techniques or by 3D bioprinting using a BioBots Beta pneumatic extruder. A preliminary characterization of the impact of bioprinting parameters on construct properties revealed that both Young’s modulus and optimal extruding pressure increased with polymer content, and that printing resolution increased with both printing speed and nozzle gauge. High viability (>95%) of encapsulated NIH 3T3 fibroblasts confirmed the cytocompatibility of the construct preparation process. Interestingly, the Young’s moduli of extruded and molded constructs were not different, but extruded constructs did show increases in both the rate and extent of time-dependent mechanical behavior observed in creep. Despite similar polymer densities, extruded hydrogels showed greater swelling over time compared to molded hydrogels, suggesting that differences in creep behavior derived from differences in microstructure and fluid flow. Because of the crucial roles of time-dependent mechanical properties, fluid flow, and swelling properties on tissue and cell behavior, these findings highlight the need for greater consideration of the effects of the extrusion process on hydrogel properties.
N Ersumo, C E Witherel and K L Spiller. “Differences in Time-Dependent Mechanical Properties Between Extruded and Molded Hydrogels,” Biofabrication. 8(3).

 

Bioprinted Thrombosis on a Chip

This publication from Dr. Ali Khademhosseini’s Lab in Lab on a Chip constructs a biomimetic thrombosis-on-a-chip model with pluronic, GelMA and an Allevi Beta.

Abstract: Thrombosis and its complications are among the most prevalent medical problems. Despite advancements in medical therapies, there is often incomplete resolution of these issues. The residual thrombus can undergo fibrotic changes over time through invaded fibroblasts from the surrounding tissues and eventually lead to the formation of a permanent clot. In order to understand the importance of cellular interactions and the impact of potential therapeutics to treat thrombosis, an in vitro platform using human cells and blood components would be beneficial. Towards achieving this aim, there have been studies utilizing the capabilities of microdevices to study the hemodynamics associated with thrombosis. In this work, we have further exploited the utilization of 3D bioprinting technology, for the construction of a highly biomimetic thrombosis-on-a-chip model. The model consisted of microchannels coated with a layer of confluent human endothelium embedded in a gelatin methacryloyl (GelMA) hydrogel, where human whole blood was infused and induced to form thrombi. Continuous perfusion with tissue plasmin activator led to dissolution of non-fibrotic clots, revealing clinical relevance of the model. Further encapsulating fibroblasts in the GelMA matrix demonstrated the potential migration of these cells into the clot and subsequent deposition of collagen type I over time, facilitating fibrosis remodeling that resembles the in vivo scenario. Our study suggests that in vitro 3D bioprinted blood coagulation models can be used to study the pathology of fibrosis, and particularly, in thrombosis. This versatile platform may be conveniently extended to other vascularized fibrosis models.
Zhang, Y. Shrike, et al. “Bioprinted Thrombosis-on-a-Chip.” Lab on a Chip(2016).

 

BioBots Bioink Publications

Nanosecond Pulsed Dielectric Barrier Discharge induced Anti-Tumor Effects Propagate Through the depth of Tissue via Intracellular Signaling

This publication from Drexel University uses Allevi LAP to fabricate 3D cell-laden extracellular matrix tissue models to examine the propagation of plasma effects.

Abstract: Studies utilizing xenograft mouse models have shown that plasma applied to the skin overlying tumors results in their shrinkage. Plasma is considered a non-penetrating treatment; however, these studies demonstrate plasma effects beyond the postulated depth of physical penetration of plasma components. The present study examines the propagation of plasma effects through a tissue model using 3-D, cell-laden extracellular matrices (ECM). These matrices are used as barriers against direct plasma penetration. By placing them onto a monolayer of target cancer cells to create an in-vitro analogue to the in-vivo studies, we distinguished between cellular effects from direct plasma exposure and cellular effects due to cell-to-cell signaling stimulated by plasma. We show that nanosecond pulsed dielectric barrier discharge (nspDBD) plasma treatment applied atop an acellular barrier impedes the externalization of Calreticulin (CRT) in the target cells. In contrast, when a barrier is populated with cells, CRT externalization is restored. Thus, we demonstrate that plasma components stimulate signaling between cells embedded in the barrier to transfer plasma effects to the target cells.
Pietro Ranieri et al. Nanosecond Pulsed Dielectric Barrier Discharge induced Anti-Tumor Effects Propagate Through the depth of Tissue via Intracellular Signaling. Plasma Medicine. 2017 DOI: 10.1615/PlasmaMed.2017019883

 

Effective Light Directed Assembly of Building Blocks with Microscale Control

This paper, published by Dr. C.-H. Chen’s Lab at University of Singapore uses Allevi GelMA to pattern cell-laden tissue blocks.

Abstract: Light-directed forces have been widely used to pattern micro/nanoscale objects with precise control, forming functional assemblies. However, a substantial laser intensity is required to generate sufficient optical gradient forces to move a small object in a certain direction, causing limited throughput for applications. A high-throughput light-directed assembly is demonstrated as a printing technology by introducing gold nanorods to induce thermal convection flows that move microparticles (diameter = 40 µm to several hundreds of micrometers) to specific light-guided locations, forming desired patterns. With the advantage of effective light-directed assembly, the microfluidic-fabricated monodispersed biocompatible microparticles are used as building blocks to construct a structured assembly (≈10 cm scale) in ≈2 min. The control with microscale precision is approached by changing the size of the laser light spot. After crosslinking assembly of building blocks, a novel soft material with wanted pattern is approached. To demonstrate its application, the mesenchymal stem-cell-seeded hydrogel microparticles are prepared as functional building blocks to construct scaffold-free tissues with desired structures. This light-directed fabrication method can be applied to integrate different building units, enabling the bottom-up formation of materials with precise control over their internal structure for bioprinting, tissue engineering, and advanced manufacturing.
N.-D. DinhR. LuoM. T. A. ChristineW. N. LinW.-C. ShihJ. C.-H. GohC.-H. ChenSmall 201713, 1700684. https://doi.org/10.1002/smll.201700684

 

Guidelines for Standardization of Bioprinting: A Systematic Study

This publication from Dr. Marcy Zenobi-Wong’s Lab in BioNanoMaterials  uses Allevi Gelatin Methacrylate (GelMA) to create a set of standardized guidelines suggested for the development of bioinks.

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 fulfill 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.
Kesti, M. et al. Guidelines for Standardization of Bioprinting: A Systematic Study BioNanoMaterials 17(3) · January 2016
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