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3-D printed ‘building blocks’ of life

Images of printed embryonic stem cells, or embryoid bodies (credit: Liliang Ouyang et al./Biofabrication)

Chinese and U.S. scientists have developed a 3-D printing method capable of producing embryoid bodies — highly uniform “blocks” of embryonic stem cells. These cells, which are capable of generating all cell types in the body, could be used to build tissue structures and potentially even micro-organs.

The results were published Wednesday Nov. 4 in an open-access paper in the journal Biofabrication. “The embryoid body is uniform and homogenous, and serves as a much better starting point for further tissue growth,” explains Wei Sun, a lead author on the paper.

The researchers, based at Tsinghua University, Beijing, China, and Drexel University, Philadelphia, used extrusion-based 3-D printing to produce a grid-like 3-D structure to grow an embryoid body that demonstrated cell viability and rapid self-renewal for 7 days while maintaining high pluripotentcy.

“Two other common methods of printing these cells are two-dimensional (in a petri dish) or via the ‘suspension’ method [see 'Better bioprinting with stem cells'], where a ‘stalagmite’ of cells is built up by material being dropped via gravity,” said Sun. ”However, these don’t show the same cell uniformity and homogenous proliferation. I think that we’ve produced a 3-D microenvironment that is much more like that found in vivo for growing embryoid bodies, which explains the higher levels of cell proliferation.”

The researchers hope that this technique can be developed to produce embryoid bodies at high throughput, providing the basic building blocks for other researchers to perform experiments on tissue regeneration and/or for drug screening studies.

The researchers say the next step is to find out more about how to vary the size of the embryoid body by changing the printing and structural parameters, and how varying the embryoid body size leads to “manufacture” of different cell types.

“In the longer term, we’d like to produce controlled heterogeneous embryonic bodies,” said Sun. “This would promote different cell types developing next to each other, which would lead the way for growing micro-organs from scratch within the lab.”

Abstract of Three-dimensional bioprinting of embryonic stem cells directs highly uniform embryoid body formation

With the ability to manipulate cells temporarily and spatially into three-dimensional (3D) tissue-like construct, 3D bioprinting technology was used in many studies to facilitate the recreation of complex cell niche and/or to better understand the regulation of stem cell proliferation and differentiation by cellular microenvironment factors. Embryonic stem cells (ESCs) have the capacity to differentiate into any specialized cell type of the animal body, generally via the formation of embryoid body (EB), which mimics the early stages of embryogenesis. In this study, extrusion-based 3D bioprinting technology was utilized for biofabricating ESCs into 3D cell-laden construct. The influence of 3D printing parameters on ESC viability, proliferation, maintenance of pluripotency and the rule of EB formation was systematically studied in this work. Results demonstrated that ESCs were successfully printed with hydrogel into 3D macroporous construct. Upon process optimization, about 90% ESCs remained alive after the process of bioprinting and cell-laden construct formation. ESCs continued proliferating into spheroid EBs in the hydrogel construct, while retaining the protein expression and gene expression of pluripotent markers, like octamer binding transcription factor 4, stage specific embryonic antigen 1 and Nanog. In this novel technology, EBs were formed through cell proliferation instead of aggregation, and the quantity of EBs was tuned by the initial cell density in the 3D bioprinting process. This study introduces the 3D bioprinting of ESCs into a 3D cell-laden hydrogel construct for the first time and showed the production of uniform, pluripotent, high-throughput and size-controllable EBs, which indicated strong potential in ESC large scale expansion, stem cell regulation and fabrication of tissue-like structure and drug screening studies.

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A new 3-​​D printing method for creating patient-​​specific medical devices

The 3D magnetic printing process systematically aligns and selectively polymerizes groupings of voxels (volume “pixels”) programmed to have specific reinforcement orientation within each layer of printed material based upon a shifting field. The 3-D printer build plate peels after a layer is complete to print additional layers. (credit: Joshua J. Martin et al./Nature Communications)

Northeastern University engineers have devel­oped a 3-D printing process that uses mag­netic fields to shape com­posite materials (mixes of plas­tics and ceramics) into patient-specific biomedical devices, such as catheters.

The devices are intended to be stronger and lighter than cur­rent models and the cus­tomized design could ensure an appro­priate fit, said Ran­dall Erb, assis­tant pro­fessor in the Depart­ment of Mechan­ical and Indus­trial Engi­neering.

The magnetic field enables the engineers to con­trol how the ceramic fibers are arranged, allowing for con­trol of the mechan­ical prop­er­ties of the mate­rial. That con­trol is crit­ical if you’re crafting devices with com­plex archi­tec­tures, such as cus­tomized minia­ture bio­med­ical devices. Within a single patient-specific device, the cor­ners, the curves, and the holes must all be rein­forced by ceramic fibers arranged in just the right con­fig­u­ra­tion to make the device durable.

This is the strategy taken by many nat­ural com­pos­ites from bones to trees. Fibers of cal­cium phos­phate, the min­eral com­po­nent of bone, are nat­u­rally ori­ented precisely around the holes for blood ves­sels to ensure the bone’s strength and sta­bility to enable, say, your femur to with­stand a daily jog.

Aligning fibers with magnets

The 3D magnetic-printer setup. A digital light processor (DLP) photo-polymerizes resin with UV while a magnetic field is simultaneously applied via electromagnetic solenoids. (credit: Joshua J. Martin et al./Nature Communications)

Erb ini­tially described the role of magnets in the composite-making process in a 2012 paper in the journal Sci­ence. First the researchers “mag­ne­tize” the ceramic fibers by dusting them very lightly with iron oxide, which has been FDA-approved for drug-delivery appli­ca­tions.

They then apply ultra-low mag­netic fields to indi­vidual sec­tions of the com­posite material — the ceramic fibers immersed in liquid plastic — to align the fibers according to the exacting spec­i­fi­ca­tions dic­tated by the product they are printing.

In a video accom­pa­nying the Sci­ence article, you can see the fibers spring to atten­tion when the mag­netic field is turned on. “Mag­netic fields are very easy to apply,” says Erb. “They’re safe, and they pen­e­trate not only our bodies but many other materials.”

Finally, in a process called “stere­olith­o­g­raphy,” they build the product, layer by layer, using a computer-controlled laser beam that hardens the plastic. Each six-by-six inch layer takes a minute to complete.

Using mag­nets, the new printing method aligns each minus­cule fiber in the direc­tion that con­forms pre­cisely to the geom­etry of the item being printed.

“If you can print a catheter whose geom­etry is spe­cific to the indi­vidual patient, you can insert it up to a cer­tain crit­ical spot, you can avoid punc­turing veins, and you can expe­dite delivery of the contents.”

The engineers’ open-access paper on the new tech­nology appears in the Oct. 23 issue of Nature Com­mu­ni­ca­tions.

Custom-designing neonatal catheters

Erb has received a $225,000 Small Busi­ness Tech­nology Transfer grant from the National Institutes of Health to develop neonatal catheters with a local com­pany. “Another of our goals is to use cal­cium phos­phate fibers and bio­com­pat­ible plas­tics to design sur­gical implants.”

Neonatal preemie with catheters (credit: March of Dimes Foundation)

The new technology is especially valuable for prema­ture babies (“preemies”) in neonatal care units, some weighing just a bit over a pound, with plastic tubes snaking through their nose or mouth, or dis­ap­pearing into veins or other parts of the body. Those tubes, or “catheters,” are how the babies get the nec­es­sary oxygen, nutri­ents, fluid, and med­ica­tions to stay alive.

The problem is, today’s catheters only come in stan­dard sizes and shapes, which means they cannot accom­mo­date the needs of all pre­ma­ture babies. “With neonatal care, each baby is a dif­ferent size, each baby has a dif­ferent set of prob­lems,” says Erb.

Worldwide, “15 million babies are born too soon every year” and of those, “1 million children die each year due to complications of preterm birth,” according to a report by the World Health Organization. This data was cited in the “March of Dimes Premature Birth Report Card,” issued today (Nov. 5) by March of Dimes. “Babies who survive an early birth often face serious and lifelong health problems, including breathing problems, jaundice, vision loss, cerebral palsy, and intellectual delays,” the March of Dimes report noted.

The report provides rates and grades for major cities or counties in each U.S. state and Puerto Rico. It also provides preterm birth rates by race and ethnicity. The U.S. preterm birth rate ranks among the worst of high-resource countries, the March of Dimes says.

Abstract of Designing bioinspired composite reinforcement architectures via 3D magnetic printing

Discontinuous fibre composites represent a class of materials that are strong, lightweight and have remarkable fracture toughness. These advantages partially explain the abundance and variety of discontinuous fibre composites that have evolved in the natural world. Many natural structures out-perform the conventional synthetic counterparts due, in part, to the more elaborate reinforcement architectures that occur in natural composites. Here we present an additive manufacturing approach that combines real-time colloidal assembly with existing additive manufacturing technologies to create highly programmable discontinuous fibre composites. This technology, termed as ‘3D magnetic printing’, has enabled us to recreate complex bioinspired reinforcement architectures that deliver enhanced material performance compared with monolithic structures. Further, we demonstrate that we can now design and evolve elaborate reinforcement architectures that are not found in nature, demonstrating a high level of possible customization in discontinuous fibre composites with arbitrary geometries.