Allevi Publications List

 

Allevi 2 Publications

The applicability of furfuryl-gelatin as a novel bioink for tissue engineering applications

Abstract: Three‐dimensional bioprinting is an innovative technique in tissue engineering, to create layer‐by‐layer structures, required for mimicking body tissues. However, synthetic bioinks do not generally possess high printability and biocompatibility at the same time. So, there is an urgent need for naturally derived bioinks that can exhibit such optimized properties. We used furfuryl‐gelatin as a novel, visible‐light crosslinkable bioink for fabricating cell‐laden structures with high viability. Hyaluronic acid was added as a viscosity enhancer and either Rose Bengal or Riboflavin was used as a visible‐light crosslinker. Crosslinking was done by exposing the printed structure for 2.5 min to visible light and confirmed using Fourier transform infrared spectroscopy and rheometry. Scanning electron microscopy revealed a highly porous networked structure. Three different cell types were successfully bioprinted within these constructs. Mouse mesenchymal stem cells printed within monolayer and bilayer sheets showed viability, network formation and proliferation (∼5.33 times) within 72 h of culture. C2C12 and STO cells were used to print a double layered structure, which showed evidence of the viability of both cells and heterocellular clusters within the construct. This furfuryl‐gelatin based bioink can be used for tissue engineering of complex tissues and help in understanding how cellular crosstalk happens in vivo during normal or diseased pathology. © 2018 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 2018.

Anilkumar S. et al. Journal of Biomedical Materials Research Part B: Applied Biomaterials(2018).

Fabrication of custom PCL scaffold for nasal septal perforation repair

In this publication,Dr. Chamith S. Rajapakse‘s Lab at the University of Pennsylvania uses an Allevi 2 (previously BioBot 1) to fabricate 3D models of patient-specific bone grafts for nasal septal perforation repair with polcaprolactone (PCL). 

Abstract: Nasal septal perforations (NSPs) are relatively common. They can be problematic for both patients and head and neck reconstructive surgeons who attempt to repair them. Often, this repair is made using an interpositional graft sandwiched between bilateral mucoperichondrial advancement flaps. The ideal graft is nasal septal cartilage. However, many patients with NSP lack sufficient septal cartilage to harvest. Harvesting other sources of autologous cartilage grafts, such as auricular cartilage, adds morbidity to the surgical case and results in a graft that lacks the ideal qualities required to repair the nasal septum. Tissue engineering has allowed for new reconstructive protocols to be developed. Currently, the authors are unaware of any new literature that looks to improve repair of NSP using custom tissue-engineered cartilage grafts. The first step of this process involves developing a protocol to print the graft from a patient's pre-operative CT. In this study, CT scans were converted into STereoLithography (STL) file format. The subsequent STL files were transformed into 3D printable G-Code using the Slic3r software. This allowed us to customize the parameters of our print and we were able to choose a layer thickness of 0.1mm. A desktop 3D bioprinter (BioBot 1) was then used to construct the scaffold. This method resulted in the production of a PCL scaffold that precisely matched the patient’s nasal septal defect, in both size and shape. This serves as the first step in our goal to create patient-specific tissue engineered nasal septal cartilage grafts for NSP repair.

Dominick Gadaleta et al "Fabrication of custom PCL scaffold for nasal septal perforation repair", Proc. SPIE 10579, Medical Imaging 2018: Imaging Informatics for Healthcare, Research, and Applications, 1057908 (6 March 2018); https://doi.org/10.1117/12.2293820

3D printed UV Light Cured Polydimethylsiloxane Devices for Drug Delivery

This work published in International Journal of Pharmaceutics uses an Allevi 2 to characterize a new pectin-based bioink.

 

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.

Holländer, Jenny, et al. "3D printed UV light cured polydimethylsiloxane devices for drug delivery." International journal of pharmaceutics (2017).

Mechanical Behavior of Alginate-Gelatin Hydrogels for 3D Bioprinting

Abstract: Hydrogels comprised of alginate and gelatin have demonstrated potential as biomaterials in three dimensional (3D) bioprinting applications. However, as with all hydrogel-based biomaterials used in extrusion-based bioprinting, many parameters influence their performance and there is limited data characterising the behaviour of alginate-gelatin (Alg-Gel) hydrogels. Here we investigated nine Alg-Gel blends by varying the individual constituent concentrations. We tested samples for printability and print accuracy, compressive behaviour and change over time, and viability of encapsulated mesenchymal stem cells in bioprinted constructs. Printability tests showed a decrease in strand width with increasing concentrations of Alg-Gel. However due to the increased viscosity associated with the higher Alg-Gel concentrations, the minimum width was found to be 0.32mm before blends became too viscous to print. Similarly, printing accuracy was increased in higher concentrations, exceeding 90% in some blends. Mechanical properties were assessed through uniaxial compression testing and it was found that increasing concentrations of both Alg and Gel resulted in higher compressive modulus. We also deemed 15min crosslinking in calcium chloride to be sufficient. From our data, we propose a blend of 7%Alg-8%Gel that yields high printability, mechanical strength and stiffness, and cell viability. However, we found the compressive behaviour of Alg-Gel to reduce rapidly over time and especially when incubated at 37°C. Here we have reported relevant data on Alg-Gel hydrogels for bioprinting. We tested for biomaterial properties and show that these hydrogels have many desirable characteristics that are highly tunable. Though further work is needed before practical use in vivo can be achieved.

Guiseppe MD et al. J Mech Behav Biomed Mater. 2018 Mar;79:150-157. doi: 10.1016/j.jmbbm.2017.12.018. Epub 2017 Dec 21.

Bioprinting 3-Dimensional Skeletal Muscle Tissue Models Using Decellularized Extracellular Matrix

This thesis, published by Zeid Yousef Nawas at University of Washington, utilizes an Allevi 2, Allevi PCL and Allevi software to fabricate physiologically relevant skeletal muscle tissues.

Abstract: Developing biologically relevant models of human tissues and organs is an important enabling step for many applications within biological research and medicine. A specific application that physiologically relevant tissue models can be implemented in is drug discovery. In this study, we represent a platform and methodology of generating 3D humanized physiologically relevant skeletal muscle tissues that recapitulate aspects of the native cellular microenvironment found in the native skeletal muscles for the development of a reproducible and high-throughput drug-screening model. This is achieved by utilizing a 3D bioprinting platform in conjugation with human myoblasts-laden decellularized extracellular matrix (dECM) bioinks to form skeletal muscle tissue constructs. Structures that feature a skeletal muscle tissue that is anchored on both sides by rigid structures are printed. The rigid anchor structures would induce passive tension along the skeletal muscle tissue, which influences cellular alignment and orientation along the anchors’ axis of tension. The results described in this study demonstrate our ability to generate human 3D skeletal muscle tissues in a rapid, high-throughput, and reproducible manner, which can be implemented as a predictive drug screening tool for determining the effects that a novel drug may have in the human body.

Nawas, Zeid Yousef, “Bioprinting 3-Dimensional Skeletal Muscle Tissue Models Using Decellularized Extracellular Matrix” (2017). http://hdl.handle.net/1773/40840

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 (previously BioBot 1)  and Allevi’s 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 1 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 (previously BioBot 1) 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 (previously BioBot 1) 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

 

Allevi Beta Publications

Patient-Specific Bioinks for 3D Bioprinting of Tissue Engineering Scaffolds

Abstract: Bioprinting has emerged as a promising tool in tissue engineering and regenerative medicine. Various 3D printing strategies have been developed to enable bioprinting of various biopolymers and hydrogels. However, the incorporation of biological factors has not been well explored. As the importance of personalized medicine is becoming more clear, the need for the development of bioinks containing autologous/patient-specific biological factors for tissue engineering applications becomes more evident. Platelet-rich plasma (PRP) is used as a patient-specific source of autologous growth factors that can be easily incorporated to hydrogels and printed into 3D constructs. PRP contains a cocktail of growth factors enhancing angiogenesis, stem cell recruitment, and tissue regeneration. Here, the development of an alginate-based bioink that can be printed and crosslinked upon implantation through exposure to native calcium ions is reported. This platform can be used for the controlled release of PRP-associated growth factors which may ultimately enhance vascularization and stem cell migration.

Faramarzi, Negar et al. Adv. Healthcare Mat. (2018) 1701347

Cage-Like Proteins as Bioink Components for 3D Bioprinting

This thesis, published by Adrian Delgado at the University of Waterloo, uses an Allevi Beta to characterize a novel PEGDA-based bioink for use with E. coli. 

Abstract: Hydrogel matrices have been used as structural surrogates in 3D bioprinting as a mechanism to provide the appropriate environment for cell adhesion and proliferation. In this research, the preparation and optimization of a hydrogel bioink containing a cage protein was investigated; specifically a Horse Spleen Ferritin (HSF)-poly (ethyleneglycol) diacrylate (PEGDA)-based bioink was developed. Studies were also undertaken to optimize the formulation of these bioinks for use in 3D bioprinting strategies, to develop techniques to precisely deposit cage proteins in hydrogels while maintaining their quaternary protein structures. In addition, the rheological properties of these various bioinks were evaluated. Finally, an optimized set of hydrogels was studied with respect to their effects on the growth of E. coli expressing a green fluorescent protein variant (His-tag GFP-S65T). Confocal microscopy experiments employed the presence of the bacterially expressed GFP fluorescence to follow bacterial cell migration in bioprinted and casted hydrogel constructs. Evaluation of cell viability within these constructs was also determined. Results indicated that the system had good potential for fabricating hydrogel scaffolds with high accuracy, fidelity and resolution.

Delgado, Adrian. “Cage-Like Proteins as Bioink Components for 3D Bioprinting” (2016). https://uwspace.uwaterloo.ca/handle/10012/12952

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 (previously BioBots 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 BioBot’s 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 BioBot’s 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).

 

Allevi Bioink Publications

Hyperelastic 'bone': A highly versatile, growth factor-free, osteoregenerative, scalable, and surgically friendly biomaterial

Check out this publication from Northwestern University to learn more about a novel osteoregenerative biomaterial, which is now commercially available on the Allevi platform. 

Abstract: Despite substantial attention given to the development of osteoregenerative biomaterials, severe deficiencies remain in current products. These limitations include an inability to adequately, rapidly, and reproducibly regenerate new bone; high costs and limited manufacturing capacity; and lack of surgical ease of handling. To address these shortcomings, we generated a new, synthetic osteoregenerative biomaterial, hyperelastic “bone” (HB). HB, which is composed of 90 weight % (wt %) hydroxyapatite and 10 wt % polycaprolactone or poly(lactic-co-glycolic acid), could be rapidly three-dimensionally (3D) printed (up to 275 cm3/hour) from room temperature extruded liquid inks. The resulting 3D-printed HB exhibited elastic mechanical properties (~32 to 67% strain to failure, ~4 to 11 MPa elastic modulus), was highly absorbent (50% material porosity), supported cell viability and proliferation, and induced osteogenic differentiation of bone marrow–derived human mesenchymal stem cells cultured in vitro over 4 weeks without any osteo-inducing factors in the medium. We evaluated HB in vivo in a mouse subcutaneous implant model for material biocompatibility (7 and 35 days), in a rat posterolateral spinal fusion model for new bone formation (8 weeks), and in a large, non-human primate calvarial defect case study (4 weeks). HB did not elicit a negative immune response, became vascularized, quickly integrated with surrounding tissues, and rapidly ossified and supported new bone growth without the need for added biological factors.

Jakus et al. Hyperelastic 'bone': A highly versatile, growth factor-free, osteoregenerative, scalable, and surgically friendly biomaterial. Science Translational Medicine. 8(358). 2016.

Generating vascular channels within hydrogel constructs using an economical open-source 3D bioprinter and thermoreversible gels

This publication from University of Toronto utilizes Allevi GelMA as an industry standard to test their open-source bioprinter. 

Abstract: The advent of 3D bioprinting offers new opportunities to create complex vascular structures within engineered tissues. However, the most suitable sacrificial material for producing branching vascular conduits within hydrogel-based constructs has not yet been resolved. Here, we assess two leading contenders, gelatin and Pluronic F-127, for a number of characteristics relevant to their use as sacrificial materials (printed filament diameter and its variability, toxicity, rheological properties, and compressive moduli). To aid in our assessment and help accelerate the adoption of 3D bioprinting by the biomedical field, we custom-built an inexpensive (< $3000 CAD) 3D bioprinter. This open-source 3D printer was designed to be fabricated in a modular manner with 3D printed/laser-cut components and off-the-shelf electronics to allow for easy assembly, iterative improvements, and customization by future adopters of the design. We found Pluronic F-127 to produce filaments with higher spatial resolution, greater uniformity, and greater elastic modulus than gelatin filaments, and with low toxicity despite being a surfactant, making it particularly suitable for engineering smaller vascular conduits. Notably, the addition of hyaluronan to gelatin increased its viscosity to achieve filament resolutions and print uniformity approaching that with Pluronic F-127. Gelatin-hyaluronan was also more resistant to plastic deformation than Pluronic F-127, and therefore may be advantageous in situations in which the sacrificial material provides structural support. We expect that this work to establish an economical 3D bioprinter and assess sacrificial materials will assist the ongoing development of vascularized tissues and will help accelerate the widespread adoption 3D bioprinting to create engineered tissues.

Fitzsimmons, Ross EB, et al. "Generating vascular channels within hydrogel constructs using an economical open-source 3D bioprinter and thermoreversible gels." Bioprinting 9 (2018): 7-18.

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

This publication from Drexel University uses Allevis 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’s 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. Dinh, R. Luo, M. T. A. Christine, W. N. Lin, W.-C. Shih, J. C.-H. Goh, C.-H. Chen, Small 2017, 13, 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’s 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

 

Allevi Review Articles

Microphysiological Systems: Automated Fabrication via Extrusion Bioprinting

This publication by Allevi in Microphysiological Systems reviews cutting-edge advancements in microphysiological systems and advantages of fabricating these systems with automated biofabrication.

Abstract: Microphysiological systems (MPS) offer great potential for improving pre-clinical testing for pharmaceutical treatments and novel therapies. These advanced in vitro models are designed to recapitulate the basic functions of living tissues or organs through dynamic culture and biomimetic microarchitecture. Increasing advancements in MPS design require advanced fabrication methods, such as extrusion bioprinting. Extrusion bioprinting presents the ability to automate fabrication of these systems in a simplified, one-step process, while providing the ability to fabricate complex, reproducible designs that incorporate dynamic culture, 3D microtissues and integrated sensors for analysis into a single system. This work reviews the main components that constitute these systems, current state-of-the-art MPS fabricated via extrusion bioprinting, and future considerations for the development of MPS.

Prendergast ME, Montoya G, Pereira T, Lewicki J, Solorzano R, Atala A. Microphysiological Systems: automated fabrication via extrusion bioprinting. Microphysiol Syst 2018;2:3.

Bioinks for Biofabrication: Current State and Future Perspectives

This publication by Allevi in the Journal of 3D Printing in Medicine reviews current state-of-the-art in bioinks for biofabrication as well as future perspectives on development of bioinks.

Abstract: Recent progress in 3D printing technologies is leading a revolution in cell culture methods. These systems rely heavily on bioinks, the raw cells and biomaterials used to create these 3D cultures. These formulations, ranging from cell suspensions to hard acellular thermoplastics, must function with 3D systems and offer biocompatible environments that mimic in vivo tissue characteristics. Although much progress has been made for the production of increasingly complex reproducible 3D tissues, a full biofabrication platform with both improved technology and superior bioinks must be developed. Here, important properties for these materials are examined; recent advances in bioinks are summarized; and finally, future considerations for 3D biofabrication are discussed. 

ME Prendergast et al. Bioinks for biofabrication: current state and future perspectives Journal of 3D printing in medicine, 2017