Silk bio-ink could help advance tissue engineering with 3-D printers

Scientists have developed a silk-based, 3-D printer ink for use in biomedical implants or tissue engineering. Credit: American Chemical Society

Scientists have developed a silk-based, 3-D printer ink for use in biomedical implants or tissue engineering.
Credit: American Chemical Society

“Polyol-Silk Bioink Formulations as Two-Part Room-Temperature Curable Materials for 3D Printing”

Advances in 3-D printing have led to new ways to make bone and some other relatively simple body parts that can be implanted in patients. But finding an ideal bio-ink has stalled progress toward printing more complex tissues with versatile functions — tissues that can be loaded with pharmaceuticals, for example. Now scientists, reporting in the journal ACS Biomaterials Science & Engineering, have developed a silk-based ink that could open up new possibilities toward that goal.

Most inks currently being developed for 3-D printing are made of thermoplastics, silicones, collagen and gelatin or alginate. But there are limits to how these inks can be used. For example, the temperatures, pH changes and crosslinking methods that may be required to toughen some of these materials can damage cells or other biological components that researchers would want to add to the inks. Additives, such as cytokines and antibiotics, are useful for directing stem cell functions and controlling infections, respectively. To address these bio-ink limitations, David L. Kaplan and colleagues turned to silk protein and developed a way to avoid these harsh processing conditions.

The researchers combined silk proteins, which are biocompatible, and glycerol, a non-toxic sugar alcohol commonly found in food and pharmaceutical products. The resulting ink was clear, flexible, stable in water, and didn’t require any processing methods, such as high temperatures, that would limit its versatility. The researchers say the novel material could potentially be used in biomedical implants and tissue engineering.

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New “Tissue Velcro” could help repair damaged hearts

This GIF (slightly sped up) shows the honeycomb mesh of cells being compressed by contracting heart cells growing along the scaffold (Image: Boyang Zhang).

This GIF (slightly sped up) shows the honeycomb mesh of cells being compressed by contracting heart cells growing along the scaffold (Image: Boyang Zhang).

Engineers at the University of Toronto just made assembling functional heart tissue as easy as fastening your shoes. The team has created a biocompatible scaffold that allows sheets of beating heart cells to snap together just like Velcro™.

“One of the main advantages is the ease of use,” says Professor Milica Radisic (ChemE, IBBME), who led the project. “We can build larger tissue structures immediately before they are needed, and disassemble them just as easily. I don’t know of any other technique that gives this ability.”

Growing heart muscle cells in the lab is nothing new. The problem is that too often, these cells don’t resemble those found in the body. Real heart cells grow in an environment replete with protein scaffolds and support cells that help shape them into long, lean beating machines. In contrast, lab-grown cells often lack these supports, and tend to be amorphous and weak. Radisic and her team focus on engineering artificial environments that more closely imitate what cells see in the body, resulting in tougher, more robust cells.

Two years ago, Radisic and her team invented the Biowire, in which heart cells grew around a silk suture, imitating the way real muscle fibres grow in the heart. “If you think of single fibre as a 1D structure, then the next step is to create a 2D structure and then assemble those into a 3D structure,” says Boyang Zhang a PhD candidate in Radisic’s lab. Zhang and Miles Montgomery, another PhD student in the lab, were co-lead authors on the current work, published today in Science Advances.

Zhang and his colleagues used a special polymer called POMaC to create a 2D mesh for the cells to grow around. It somewhat resembles a honeycomb in shape, except that the holes are not symmetrical, but rather wider in one direction than in another. Critically, this provides a template that causes the cells to line up together. When stimulated with an electrical current, the heart muscle cells contract together, causing the flexible polymer to bend.

Next the team bonded T-shaped posts on top of the honeycomb. When a second sheet is placed above, these posts act like tiny hooks, poking through the holes of honeycomb and clicking into place. The concept the same as the plastic hooks and loops of Velcro™, which itself is based on the burrs that plants use to hitch their seeds to passing animals.

Amazingly, the assembled sheets start to function almost immediately. “As soon as you click them together, they start beating, and when we apply electrical field stimulation, we see that they beat in synchrony,” says Radisic. The team has created layered tissues up to three sheets thick in a variety of configurations, including tiny checkerboards.

The ultimate goal of the project is to create artificial tissue that could be used to repair damaged hearts. The modular nature of the technology should make it easier to customize the graft to each patient. “If you had these little building blocks, you could build the tissue right at the surgery time to be whatever size that you require,” says Radisic. The polymer scaffold itself is biodegradable; within a few months it will gradually break down and be absorbed by the body.

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On the frontiers of cyborg science

Objektfoto'Pantoffeltierchen-Cyborg II' (Photo credit: Wikipedia)

Objektfoto ‘Pantoffeltierchen-Cyborg II’ (Photo credit: Wikipedia)

No longer just fantastical fodder for sci-fi buffs, cyborg technology is bringing us tangible progress toward real-life electronic skin, prosthetics and ultraflexible circuits.

Now taking this human-machine concept to an unprecedented level, pioneering scientists are working on the seamless marriage between electronics and brain signaling with the potential to transform our understanding of how the brain works — and how to treat its most devastating diseases.

Their presentation is taking place at the 248th National Meeting & Exposition of the American Chemical Society (ACS), the world’s largest scientific society. The meeting features nearly 12,000 presentations on a wide range of science topics and is being held here through Thursday.

“By focusing on the nanoelectronic connections between cells, we can do things no one has done before,” says Charles M. Lieber, Ph.D. “We’re really going into a new size regime for not only the device that records or stimulates cellular activity, but also for the whole circuit. We can make it really look and behave like smart, soft biological material, and integrate it with cells and cellular networks at the whole-tissue level. This could get around a lot of serious health problems in neurodegenerative diseases in the future.”

These disorders, such as Parkinson’s, that involve malfunctioning nerve cells can lead to difficulty with the most mundane and essential movements that most of us take for granted: walking, talking, eating and swallowing.

Scientists are working furiously to get to the bottom of neurological disorders. But they involve the body’s most complex organ — the brain — which is largely inaccessible to detailed, real-time scrutiny. This inability to see what’s happening in the body’s command center hinders the development of effective treatments for diseases that stem from it.

By using nanoelectronics, it could become possible for scientists to peer for the first time inside cells, see what’s going wrong in real time and ideally set them on a functional path again.

For the past several years, Lieber has been working to dramatically shrink cyborg science to a level that’s thousands of times smaller and more flexible than other bioelectronic research efforts. His team has made ultrathin nanowires that can monitor and influence what goes on inside cells. Using these wires, they have built ultraflexible, 3-D mesh scaffolding with hundreds of addressable electronic units, and they have grown living tissue on it. They have also developed the tiniest electronic probe ever that can record even the fastest signaling between cells.

Rapid-fire cell signaling controls all of the body’s movements, including breathing and swallowing, which are affected in some neurodegenerative diseases. And it’s at this level where the promise of Lieber’s most recent work enters the picture.

In one of the lab’s latest directions, Lieber’s team is figuring out how to inject their tiny, ultraflexible electronics into the brain and allow them to become fully integrated with the existing biological web of neurons. They’re currently in the early stages of the project and are working with rat models.

“It’s hard to say where this work will take us,” he says. “But in the end, I believe our unique approach will take us on a path to do something really revolutionary.”

This research will be presented at a meeting of the American Chemical Society

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A significant breakthrough could revolutionize surgical practice and regenerative medicine

Phase 1 Skin injury Phase 2 Application of the solution Phase 3 Using pressure to hold the edges together Phase 4 Skin closure Illustration of the first experiment conducted by the resear chers on rats: a deep wound is repaired by applying the aqueous nanoparticle solution. The wound closes in thirty seconds. Credit: “Matière Molle et Chimie” Laboratory (CNRS/ESPCI Paris Tech)

Innovative strategy to facilitate organ repair

A significant breakthrough could revolutionize surgical practice and regenerative medicine. A team led by Ludwik Leibler from the Laboratoire Matière Molle et Chimie (CNRS/ESPCI Paris Tech) and Didier Letourneur from the Laboratoire Recherche Vasculaire Translationnelle (INSERM/Universités Paris Diderot and Paris 13), has just demonstrated that the principle of adhesion by aqueous solutions of nanoparticles can be used in vivo to repair soft-tissue organs and tissues. This easy-to-use gluing method has been tested on rats. When applied to skin, it closes deep wounds in a few seconds and provides a esthetic, high quality healing. It has also been shown to successfully repair organs that are difficult to suture, such as the liver. Finally, this solution has made it possible to attach a medical device to a beating heart, demonstrating the method’s potential for delivering drugs and strengthening tissues. This work has just been published on the website of the journal Angewandte Chemie.

In an issue of Nature published in December last year, a team led by Ludwik Leibler 1 presented a novel concept for gluing gels and biological tissues using nanoparticles 2. The principle is simple: nanoparticles contained in a solution spread out on surfaces to be glued bind to the gel’s (or tissue’s) molecular network. This phenomenon is called adsorption. At the same time the gel (or tissue) binds the particles together. Accordingly, myriad connections form between the two surfaces. This adhesion process, which involves no chemical reaction, only takes a few seconds. In their latest, newly published study, the researchers used experiments performed on rats to show that this method, applied in vivo , has the potential to revolutionize clinical practice.

In a first experiment, the researchers compared two methods for skin closure in a deep wound: traditional sutures, and the application of the aqueous nanoparticle solution with a brush. The latter is easy to use and closes skin rapidly until it heals completely, without inflammation or necrosis. The resulting scar is almost invisible.

In a second experiment, still on rats, the researchers applied this solution to soft-tissue organs such as the liver, lungs or spleen that are difficult to suture because they tear when the needle passes through them. At present, no glue is sufficiently strong as well as harmless for the organism. Confronted with a deep gash in the liver with severe bleeding, the researchers closed the wound by spreading the aqueous nanoparticle solution and pressing the two edges of the wound toget her. The bleeding stopped. To repair a sectioned liver lobe, the researchers also used nanoparticles: they glued a film coated with nanoparticles onto the wound, and stopped the bleeding. In both situations, organ function was unaffected and the animals survived.

“Gluing a film to stop leakage” is only one example of the possibilities opened up by adhesion brought by nanoparticles. In an entirely different field, the researchers have succeeded in using anoparticles to attach a biodegradable membrane used for cardiac cell therapy, and to achieve this despite the substantial mechanical constraints due to its beating. They thus showed that it would be possible to attach various medical devices to organs and tissues for therapeutic, repair or mechanical strengthening purposes.

This adhesion method is exceptional because of its potential spectrum of clinical applications. It is simple, easy to use and the nanoparticles employed (silica, iron oxides) can be metabolized by the organism. It can easily be integrated into ongoing research on healing and tissue regeneration and contribute to the development of regenerative medicine.

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Light-guiding Gels Provide New Avenues for Drug Detection and Delivery

A freely moving mouse with a hydrogel implant and fiber optic cable. The blue light delivered through the cable is evenly distributed by the hydrogel. Source: Seok-Hyun Yun, Harvard Medical School.

Potentially transformative ways to use specific types of light for more even complex and direct manipulation of individual cells

In medicine, light therapy is currently used to treat seasonal affective disorder, psoriasis, and other medical conditions, while highly targeted lasers may be used for specific skin disorders, eye diseases, or cancers. Advances in imaging methods and equipment now allow scientists to see the effects of light at the cellular level, leading to research on potentially transformative ways to use specific types of light for more even complex and direct manipulation of individual cells.

Optogenetics is a relatively new technique that harnesses light to activate or inhibit light-responsive proteins that control specific cell functions. Most optogenetics research to date has targeted brain cells, allowing scientists to manipulate individual neurons and observe the effects. While a groundbreaking tool for studying the inner workings of the brain without the need for electrodes or any direct contact with brain tissue, the challenge of shining a light deep within the body has limited broader uses of optogenetics.

For most proposed clinical uses, light needs to be delivered evenly across a number of cells to have a reproducible therapeutic effect, but human tissue is not transparent and scatters, absorbs, or otherwise reduces light penetration, reducing the ability to deliver light below the skin. To address this delivery challenge, NIBIB grantee Seok-Hyun Andy Yun, Ph.D., and researchers at Harvard Medical School and various institutions in Korea, experimented with using transparent hydrogels in combination with optogenetics.

Hydrogels are similar in concept to commonplace, edible gelatin, but are being researched for use in medical implants. Currently, most medical implants are made with rigid materials like plastic and metal. Though such implants are carefully designed to work within the body, by nature, placing a hard object among relatively soft tissues can cause inflammation and other unwanted side effects. In contrast, hydrogels can be easily constructed using more biologically-friendly (“biocompatible”) materials, and their high water content and flexible nature may conform more closely to  muscles, organs, and other internal body parts so that light is guided efficiently. Some fluids can also flow through hydrogels, which may allow for different types of uses than can be achieved with implants made of conventional, impermeable materials.

Experimenting with the hydrogel recipe, Yun and colleagues devised a strong yet flexible, clear hydrogel slab able to guide a laser beam, bouncing the light back and forth within its boundaries. The researchers were also able to seed the hydrogel with cells—like fruit cocktail in a gelatin dessert—which refract and scatter light, creating a uniform glow throughout the slab.

When lit by a fiber optic and implanted just under the skin in mice, the glowing hydrogel could be clearly seen. In a follow-up experiment, the researchers grew cells that glow green in the presence of cadmium, a toxic heavy metal often used to make quantum dot sensors. When cadmium-core quantum dots were injected into mice with hydrogel implants, the cells within the hydrogel glowed green. However, when dots with a more biocompatible zinc-based coating were injected, the hydrogel did not glow, suggesting that the zinc coating effectively shielded the cells in the hydrogel from cadmium toxicity.

To test the hydrogel’s ability to deliver a treatment, the researchers created slabs embedded with cells that glow in the presence of calcium. The slabs were then fitted with a blue light fiber optic and implanted in mice with chemically induced diabetes. When exposed to blue light, a protein called melanopsin sets off a cascade of activity within the cells, including the release of calcium, that help to control the effects of diabetes.

In mice exposed to the blue light, the cells in the implanted hydrogel glowed much more compared to hydrogels in unexposed mice, suggesting the former group had higher levels of intracellular calcium and anti-diabetic activity. To further validate this finding, the mice were given a glucose tolerance test to see how long it took for their blood sugar levels to return to normal—since diabetes impairs the body’s ability to process sugar, blood sugar levels remain abnormally high for longer periods of time than in a person or animal without diabetes. The light-exposed diabetic mice achieved regular blood sugar levels within an hour and a half, while the untreated diabetic mice continued to have elevated blood sugar even after two hours, indicating that light delivered via the hydrogel produced a measurable biological effect and may someday be a useful means of delivering optogenetic treatments.

By delivering light inside the body in a controlled and predictable manner and being able to host genetically engineered cells that respond to light or emit light in response to specific chemical signals, the hydrogels created by Yun and colleagues may help address some of the challenges with using optogenetic approaches in clinical care.

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