Rapid generation of three‐dimensional microchannels for vascularization using a subtractive printing technique

The development of tissue‐engineered products has been limited by lack of a perfused microvasculature that delivers nutrients and maintains cell viability. Current strategies to promote vascularization such as additive three‐dimensional printing techniques have limitations. This study validates the use of an ultra‐fast laser subtractive printing technique to generate capillary‐sized channels in hydrogels prepopulated with cells by demonstrating cell viability relative to the photodisrupted channels in the gel. The system can move the focal spot laterally in the gel at a rate of 2500 mm/s by using a galvanometric scanner to raster the in plane focal spot. A Galilean telescope allows z‐axis movement. Blended hydrogels of polyethylene glycol and collagen with a range of optical clarities, mechanical properties and swelling behavior were tested to demonstrate that the subtractive printing process for writing vascular channels is compatible with all of the blended hydrogels tested. Channel width and patterns were controlled by adjusting the laser energy and focal spot positioning, respectively. After treatment, high cell viability was observed at distances greater than or equal to 18 μm from the fabricated channels. Overall, this study demonstrates a flexible technique that has the potential to rapidly generate channels in tissue‐engineered constructs.      

Deconvolution of images from 3D printed cells in layers on a chip

Layer‐by‐layer cell printing is useful in mimicking layered tissue structures inside the human body and has great potential for being a promising tool in the field of tissue engineering, regenerative medicine, and drug discovery. However, imaging human cells cultured in multiple hydrogel layers in 3D‐printed tissue constructs is challenging as the cells are not in a single focal plane. Although confocal microscopy could be a potential solution for this issue, it compromises the throughput which is a key factor in rapidly screening drug efficacy and toxicity in pharmaceutical industries. With epifluorescence microscopy, the throughput can be maintained at a cost of blurred cell images from printed tissue constructs. To rapidly acquire in‐focus cell images from bioprinted tissues using an epifluorescence microscope, we created two layers of Hep3B human hepatoma cells by printing green and red fluorescently labeled Hep3B cells encapsulated in two alginate layers in a microwell chip. In‐focus fluorescent cell images were obtained in high throughput using an automated epifluorescence microscopy coupled with image analysis algorithms, including three deconvolution methods in combination with three kernel estimation methods, generating a total of nine deconvolution paths. As a result, a combination of Inter‐Level Intra‐Level Deconvolution (ILILD) algorithm and Richardson‐Lucy (RL) kernel estimation proved to be highly useful in bringing out‐of‐focus cell images into focus, thus rapidly yielding more sensitive and accurate fluorescence reading from the cells in different layers. © 2017 American Institute of Chemical Engineers Biotechnol. Prog., 34:445–454, 2018     

Simple and rapid technique to detect PVY presence in some Solanaceae plants

The study of plant-virus interactions requires a method which enables not only the detection of virus presence, but also its distribution in the infected plant tissue. We adopted a tissue print immunoblot technique for localization of potato virus Y (PVY) coat protein (CP) in infected potato and tobacco tissue. In potato stem cross-sections PVY CP was detected on the whole surface of the prints, however significantly higher concentration was observed in epidermis and phloem tissue. In the petiole of infected tobacco leaves the presence of viral protein was confined mainly to peripheral layers of parenchyma and epidermis. In phloem tissue the signal was also visible but it was significantly weaker. In our hands this approach is highly specific and gives resolution on the level of individual cells, thus it can be applied in several fields. As potyviral capsid proteins are involved in several events of pathogenesis the technique for immunolocalization of PVY CP could provide information which will shed new light on the virology of PVY.     

Segmental bone replacement via patient‐specific,three‐dimensional printed bioresorbable graft substitutes and their use as templates for the culture of mesenchymal stem cells under mechanical stimulation at various frequencies

The treatment of large segmental bone defects remains a challenge as infection, delayed union, and nonunion are common postoperative complications. A three‐dimensional printed bioresorbable and physiologically load‐sustaining graft substitute was developed to mimic native bone tissue for segmental bone repair. Fabricated from polylactic acid, this graft substitute is novel as it is readily customizable to accommodate the particular size and location of the segmental bone of the patient to be replaced. Inspired by the structure of the native bone tissue, the graft substitute exhibits a gradient in porosity and pore size in the radial direction and exhibit mechanical properties similar to those of the native bone tissue. The graft substitute can serve as a template for tissue constructs via seeding with stem cells. The biocompatibility of such templates was tested under in vitro conditions using a dynamic culture of human mesenchymal stem cells. The effects of the mechanical loading of cell‐seeded templates under in vitro conditions were assessed via subjecting the tissue constructs to 28 days of daily mechanical stimulation. The frequency of loading was found to have a significant effect on the rate of mineralization, as the alkaline phosphatase activity and calcium deposition were determined to be particularly high at the typical walking frequency of 2 Hz, suggesting that mechanical stimulation plays a significant role in facilitating the healing process of bone defects. Utilization of such patient‐specific and biocompatible graft substitutes, coupled with patient’s bone marrow cells seeded and exposed to mechanical stimulation of 2 Hz have the potential of reducing significant volumes of cadaveric tissue required, improving long‐term graft stability and incorporation, and alleviating financial burdens associated with delayed or failed fusions of long bone defects.     

3D-printed applicators for high dose rate brachytherapy: Dosimetric assessment at different infill percentage

PurposeDosimetric assessment of high dose rate (HDR) brachytherapy applicators, printed in 3D with acrylonitrile butadiene styrene (ABS) at different infill percentage.Materials and methodsA low-cost, desktop, 3D printer (Hamlet 3DX100, Hamlet, Dublin, IE) was used for manufacturing simple HDR applicators, reproducing typical geometries in brachytherapy: cylindrical (common in vaginal treatment) and flat configurations (generally used to treat superficial lesions). Printer accuracy was investigated through physical measurements. The dosimetric consequences of varying the applicator’s density by tuning the printing infill percentage were analysed experimentally by measuring depth dose profiles and superficial dose distribution with Gafchromic EBT3 films (International Specialty Products, Wayne, NJ). Dose distributions were compared to those obtained with a commercial superficial applicator.ResultsMeasured printing accuracy was within 0.5 mm. Dose attenuation was not sensitive to the density of the material. Surface dose distribution comparison of the 3D printed flat applicators with respect to the commercial superficial applicator showed an overall passing rate greater than 94% for gamma analysis with 3% dose difference criteria, 3 mm distance-to-agreement criteria and 10% dose threshold.ConclusionLow-cost 3D printers are a promising solution for the customization of the HDR brachytherapy applicators. However, further assessment of 3D printing techniques and regulatory materials approval are required for clinical application.     

Diffuse Reflectance Infrared Spectroscopic Identification of Dispersant/Particle Bonding Mechanisms in Functional Inks

In additive manufacturing, or 3D printing, material is deposited drop by drop, to create micron to macroscale layers. A typical inkjet ink is a colloidal dispersion containing approximately ten components including solvent, the nano to micron scale particles which will comprise the printed layer, polymeric dispersants to stabilize the particles, and polymers to tune layer strength, surface tension and viscosity. To rationally and efficiently formulate such an ink, it is crucial to know how the components interact. Specifically, which polymers bond to the particle surfaces and how are they attached? Answering this question requires an experimental procedure that discriminates between polymer adsorbed on the particles and free polymer. Further, the method must provide details about how the functional groups of the polymer interact with the particle. In this protocol, we show how to employ centrifugation to separate particles with adsorbed polymer from the rest of the ink, prepare the separated samples for spectroscopic measurement, and use Diffuse Reflectance Fourier Transform Infrared Spectroscopy (DRIFTS) for accurate determination of dispersant/particle bonding mechanisms. A significant advantage of this methodology is that it provides high level mechanistic detail using only simple, commonly available laboratory equipment. This makes crucial data available to almost any formulation laboratory. The method is most useful for inks composed of metal, ceramic, and metal oxide particles in the range of 100 nm or greater. Because of the density and particle size of these inks, they are readily separable with centrifugation. Further, the spectroscopic signatures of such particles are easy to distinguish from absorbed polymer. The primary limitation of this technique is that the spectroscopy is performed ex-situ on the separated and dried particles as opposed to the particles in dispersion. However, results from attenuated total reflectance spectra of the wet separated particles provide evidence for the validity of the DRIFTS measurement.     

The Upper Devonian tetrapodomorph Gogonasus andrewsae from Western Australia: Reconstruction of the shoulder girdle and opercular series using X-ray Micro-Computed Tomography

The tetrapodomorph fish, Gogonasus andrewsae is a three dimensionally well-preserved sarcopterygian from the Gogo Formation (Frasnian, early Upper Devonian, ∼380 million years ago) in Western Australia. High-resolution X-ray Micro-Computed Tomography and 3D printouts were used to obtain a digital reconstruction of its shoulder girdle and opercular series. Our new findings show the opercular series in a close fit against the upper bones of the shoulder girdle only if the anocleithrum, supracleithrum and post-temporal are aligned more horizontally than in previous reconstructions. The lowermost subopercular bone also differs, in partly covering the clavicle of the shoulder girdle. The ascending process of the clavicle, and the ventral process of the anocleithrum, do not fit closely inside the cleithrum, and perhaps functioned for ligamentous attachment. A rugose area on the anocleithral process is in a similar relative position to the attachment of a muscle ligament on the shoulder girdle of various living actinopterygians. Our manipulation of 3D printouts permits testing of the morphological fit of extremely fragile acid-etched bones, and indicates a new way to investigate the constructional morphology of one or more mechanical units of the vertebrate skeleton. It is suggested that Micro-CT imaging, reconstruction, visualisation and 3D printing techniques will provide a rigorous new test leading to modification of previous reconstructions of extinct vertebrates that were based on graphical methods and 2D imaging.     

Carbon: The Ultimate Electrode Choice for Widely Distributed Polymer Solar Cells

As mass‐produced, low‐cost organic electronics enter our everyday lives, so does the waste from them. The challenges associated with end‐of‐life management must be addressed by careful design and carbon‐based electrodes are central to these developments. Here, the reproducible production of vacuum‐, indium tin oxide (ITO)‐, and silver‐free solar cells in a fully packaged form using only roll‐to‐roll processing is reported. Replacing silver with carbon as electrode material significantly lowers the manufacturing cost and makes the organic photovoltaic (OPV) modules environmentally safe while retaining their flexibility, active area efficiency, and stability. The substitution of silver with carbon does not affect the roll‐to‐roll manufacturing of the modules and allows for the same fast printing and coating. The use of carbon as electrode material is one step closer to the wide release of low‐cost plastic solar cells and opens the door to new possible applications where silver recycling is not manageable.     

NBT-II cell locomotion is modulated by restricting the size of focal contacts and is improved through EGF and ROCK signaling

Focal contacts, large macromolecular complexes that link the extracellular matrix and the internal cell cytoskeleton, are thought to govern cell locomotion. However, the maturation process through which focal contacts control the cellular migratory machinery by changes in size and molecular composition remain unclear. Here, we fabricated cell growth substrates that contained linear ECM strips of micron- or submicron-width in order to limit the enlargement of focal contacts. We found that NBT-II cells plated on the submicron substrate possessed smaller focal complexes that exhibited a highly dynamic turnover. These cells possessed various leading edges at multiple sites of the cell periphery, which prevented the cell from advancing. In contrast, cells grown on the micron-width substrate possessed large and stable focal adhesions. Most of these cells were elongated bipolar cells that were tethered at both ends and were immobile. Further, EGF and ROCK signaling pathways can modulate the cellular migratory responses according to the substrate guidance. On the submicron-width substrate, EGF treatment increased the focal contact size and the contractile force, causing these cells to develop one leading edge and migrate along the submicron-sized ECM paths. In contrast, inhibition of ROCK signaling decreased the focal contact size for cells plated on the micron substrate. These cells became less tethered and were able to migrate along or even across the micron-sized ECM paths. Our results indicate that formation and maturation of focal contacts is controlled by both ECM cues and intracellular signaling and they play a central role in directed cell motion.