3D-printed blood vessels bring artificial organs closer to reality

Growing functional human organs outside the body is a long-sought "holy grail" of organ transplantation medicine that remains elusive. New research from Harvard's Wyss Institute for Biologically Inspired Engineering and John A. Paulson School of Engineering and Applied Science (SEAS) brings that quest one big step closer to completion.

A team of scientists created a new method to 3D print vascular networks that consist of interconnected blood vessels possessing a distinct "shell" of smooth muscle cells and endothelial cells surrounding a hollow "core" through which fluid can flow, embedded inside a human cardiac tissue. This vascular architecture closely mimics that of naturally occurring blood vessels and represents significant progress toward being able to manufacture implantable human organs. The achievement is published in Advanced Materials.

"In prior work, we developed a new 3D bioprinting method, known as "sacrificial writing in functional tissue" (SWIFT), for patterning hollow channels within a living cellular matrix. Here, building on this method, we introduce coaxial SWIFT (co-SWIFT) that recapitulates the multilayer architecture found in native blood vessels, making it easier to form an interconnected endothelium and more robust to withstand the internal pressure of blood flow," said first author Paul Stankey, a graduate student at SEAS in the lab of co-senior author and Wyss Core Faculty member Jennifer Lewis, Sc.D.

The key innovation developed by the team was a unique core-shell nozzle with two independently controllable fluid channels for the "inks" that make up the printed vessels: a collagen-based shell ink and a gelatin-based core ink. The interior core chamber of the nozzle extends slightly beyond the shell chamber so that the nozzle can fully puncture a previously printed vessel to create interconnected branching networks for sufficient oxygenation of human tissues and organs via perfusion. The size of the vessels can be varied during printing by changing either the printing speed or the ink flow rates.

To confirm the new co-SWIFT method worked, the team first printed their multilayer vessels into a transparent granular hydrogel matrix. Next, they printed vessels into a recently created matrix called uPOROS composed of a porous collagen-based material that replicates the dense, fibrous structure of living muscle tissue. They were able to successfully print branching vascular networks in both of these cell-free matrices. After these biomimetic vessels were printed, the matrix was heated, which caused collagen in the matrix and shell ink to crosslink, and the sacrificial gelatin core ink to melt, enabling its easy removal and resulting in an open, perfusable vasculature.

Moving into even more biologically relevant materials, the team repeated the printing process using a shell ink that was infused with smooth muscle cells (SMCs), which comprise the outer layer of human blood vessels. After melting out the gelatin core ink, they then perfused endothelial cells (ECs), which form the inner layer of human blood vessels, into their vasculature. After seven days of perfusion, both the SMCs and the ECs were alive and functioning as vessel walls -- there was a three-fold decrease in the permeability of the vessels compared to those without ECs.

Finally, they were ready to test their method inside living human tissue. They constructed hundreds of thousands of cardiac organ building blocks (OBBs) -- tiny spheres of beating human heart cells, which are compressed into a dense cellular matrix. Next, using co-SWIFT, they printed a biomimetic vessel network into the cardiac tissue. Finally, they removed the sacrificial core ink and seeded the inner surface of their SMC-laden vessels with ECs via perfusion and evaluated their performance.

Not only did these printed biomimetic vessels display the characteristic double-layer structure of human blood vessels, but after five days of perfusion with a blood-mimicking fluid, the cardiac OBBs started to beat synchronously -- indicative of healthy and functional heart tissue. The tissues also responded to common cardiac drugs -- isoproterenol caused them to beat faster, and blebbistatin stopped them from beating. The team even 3D-printed a model of the branching vasculature of a real patient's left coronary artery into OBBs, demonstrating its potential for personalized medicine.

"We were able to successfully 3D-print a model of the vasculature of the left coronary artery based on data from a real patient, which demonstrates the potential utility of co-SWIFT for creating patient-specific, vascularized human organs," said Lewis, who is also the Hansjörg Wyss Professor of Biologically Inspired Engineering at SEAS.

In future work, Lewis' team plans to generate self-assembled networks of capillaries and integrate them with their 3D-printed blood vessel networks to more fully replicate the structure of human blood vessels on the microscale and enhance the function of lab-grown tissues.

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The more you walk, the lower your risk of early death, even if you walk fewer than 5,000 steps

The number of steps you should walk every day to start seeing benefits to your health is lower than previously thought, according to the largest analysis to investigate this.

The study, published in the European Journal of Preventive Cardiology [1] today (Wednesday), found that walking at least 3967 steps a day started to reduce the risk of dying from any cause, and 2337 steps a day reduced the risk of dying from diseases of the heart and blood vessels (cardiovascular disease).

However, the new analysis of 226,889 people from 17 different studies around the world has shown that the more you walk, the greater the health benefits. The risk of dying from any cause or from cardiovascular disease decreases significantly with every 500 to 1000 extra steps you walk. An increase of 1000 steps a day was associated with a 15% reduction in the risk of dying from any cause, and an increase of 500 steps a day was associated with a 7% reduction in dying from cardiovascular disease.

The researchers, led by Maciej Banach, Professor of Cardiology at the Medical University of Lodz, Poland, and Adjunct Professor at the Ciccarone Center for the Prevention of Cardiovascular Disease, Johns Hopkins University School of Medicine, found that even if people walked as many as 20,000 steps a day, the health benefits continued to increase. They have not found an upper limit yet.

"Our study confirms that the more you walk, the better," says Prof. Banach. "We found that this applied to both men and women, irrespective of age, and irrespective of whether you live in a temperate, sub-tropical or sub-polar region of the world, or a region with a mixture of climates. In addition, our analysis indicates that as little as 4,000 steps a day are needed to significantly reduce deaths from any cause, and even fewer to reduce deaths from cardiovascular disease."

There is strong evidence that a sedentary lifestyle may contribute to an increase in cardiovascular disease and a shorter life. Studies have shown that insufficient physical activity affects more than a quarter of the world's population. More women than men (32% versus 23%), and people in higher income countries compared to low-income countries (37% versus 16%) do not undertake a sufficient amount of physical activity. According to World Health Organization data, insufficient physical activity is the fourth most frequent cause of death in the world, with 3.2 million deaths a year related to physical inactivity. The COVID-19 pandemic also resulted in a reduction in physical activity, and activity levels have not recovered two years on from it.

Dr Ibadete Bytyçi from the University Clinical Centre of Kosovo, Pristina, Kosovo, senior author of the paper, says: "Until now, it's not been clear what is the optimal number of steps, both in terms of the cut-off points over which we can start to see health benefits, and the upper limit, if any, and the role this plays in people's health. However, I should emphasise that there were limited data available on step counts up to 20,000 a day, and so these results need to be confirmed in larger groups of people."

This meta-analysis is the first not only to assess the effect of walking up to 20,000 steps a day, but also to look at whether there are any differences depending on age, sex or where in the world people live.

The studies analysed by the researchers followed up participants for a median (average) of seven years. The mean (average) age was 64, and 49% of participants were female.

In people aged 60 years or older, the size of the reduction in risk of death was smaller than that seen in people aged younger than 60 years. In the older adults, there was a 42% reduction in risk seen in those who walked between 6,000 and 10,000 steps a day, while there was a 49% reduction in risk in younger adults who walked between 7,000 and 13,000 steps a day.

Prof. Banach says: "In a world where we have more and more advanced drugs to target specific conditions such as cardiovascular disease, I believe we should always emphasise that lifestyle changes, including diet and exercise, which was a main hero of our analysis, might be at least as, or even more effective in reducing cardiovascular risk and prolonging lives. We still need good studies to investigate whether these benefits may exist for intensive types of exertion, such as marathon running and iron man challenges, and in different populations of different ages, and with different associated health problems. However, it seems that, as with pharmacological treatments, we should always think about personalising lifestyle changes."

Strengths of the meta-analysis include its size and that it was not restricted to looking at studies limited to a maximum of 16,000 steps a day. Limitations include that it was an observational study and so cannot prove that increased step counts cause the reduction in the risk of death, only that it is associated with it. The impact of step counts was not tested on people with different diseases; all the participants were generally healthy when they entered the studies analysed. The researchers were not able to account for differences in race and socioeconomic status, and the methods for counting steps were not identical in all the studies included in this meta-analysis.

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Custom, 3D-printed heart replicas look and pump just like the real thing

No two hearts beat alike. The size and shape of the the heart can vary from one person to the next. These differences can be particularly pronounced for people living with heart disease, as their hearts and major vessels work harder to overcome any compromised function.

MIT engineers are hoping to help doctors tailor treatments to patients' specific heart form and function, with a custom robotic heart. The team has developed a procedure to 3D print a soft and flexible replica of a patient's heart. They can then control the replica's action to mimic that patient's blood-pumping ability.

The procedure involves first converting medical images of a patient's heart into a three-dimensional computer model, which the researchers can then 3D print using a polymer-based ink. The result is a soft, flexible shell in the exact shape of the patient's own heart. The team can also use this approach to print a patient's aorta -- the major artery that carries blood out of the heart to the rest of the body.

To mimic the heart's pumping action, the team has fabricated sleeves similar to blood pressure cuffs that wrap around a printed heart and aorta. The underside of each sleeve resembles precisely patterned bubble wrap. When the sleeve is connected to a pneumatic system, researchers can tune the outflowing air to rhythmically inflate the sleeve's bubbles and contract the heart, mimicking its pumping action.

The researchers can also inflate a separate sleeve surrounding a printed aorta to constrict the vessel. This constriction, they say, can be tuned to mimic aortic stenosis -- a condition in which the aortic valve narrows, causing the heart to work harder to force blood through the body.

Doctors commonly treat aortic stenosis by surgically implanting a synthetic valve designed to widen the aorta's natural valve. In the future, the team says that doctors could potentially use their new procedure to first print a patient's heart and aorta, then implant a variety of valves into the printed model to see which design results in the best function and fit for that particular patient. The heart replicas could also be used by research labs and the medical device industry as realistic platforms for testing therapies for various types of heart disease.

"All hearts are different," says Luca Rosalia, a graduate student in the MIT-Harvard Program in Health Sciences and Technology. "There are massive variations, especially when patients are sick. The advantage of our system is that we can recreate not just the form of a patient's heart, but also its function in both physiology and disease."

Rosalia and his colleagues report their results in a study appearing today in Science Robotics. MIT co-authors include Caglar Ozturk, Debkalpa Goswami, Jean Bonnemain, Sophie Wang, and Ellen Roche, along with Benjamin Bonner of Massachusetts General Hospital, James Weaver of Harvard University, and Christopher Nguyen, Rishi Puri, and Samir Kapadia at the Cleveland Clinic in Ohio.

Print and pump

In January 2020, team members, led by mechanical engineering professor Ellen Roche, developed a "biorobotic hybrid heart" -- a general replica of a heart, made from synthetic muscle containing small, inflatable cylinders, which they could control to mimic the contractions of a real beating heart.

Shortly after those efforts, the Covid-19 pandemic forced Roche's lab, along with most others on campus, to temporarily close. Undeterred, Rosalia continued tweaking the heart-pumping design at home.

"I recreated the whole system in my dorm room that March," Rosalia recalls.

Months later, the lab reopened, and the team continued where it left off, working to improve the control of the heart-pumping sleeve, which they tested in animal and computational models. They then expanded their approach to develop sleeves and heart replicas that are specific to individual patients. For this, they turned to 3D printing.

"There is a lot of interest in the medical field in using 3D printing technology to accurately recreate patient anatomy for use in preprocedural planning and training," notes Wang, who is a vascular surgery resident at Beth Israel Deaconess Medical Center in Boston.

An inclusive design

In the new study, the team took advantage of 3D printing to produce custom replicas of actual patients' hearts. They used a polymer-based ink that, once printed and cured, can squeeze and stretch, similarly to a real beating heart.

As their source material, the researchers used medical scans of 15 patients diagnosed with aortic stenosis. The team converted each patient's images into a three-dimensional computer model of the patient's left ventricle (the main pumping chamber of the heart) and aorta. They fed this model into a 3D printer to generate a soft, anatomically accurate shell of both the ventricle and vessel.

The team also fabricated sleeves to wrap around the printed forms. They tailored each sleeve's pockets such that, when wrapped around their respective forms and connected to a small air pumping system, the sleeves could be tuned separately to realistically contract and constrict the printed models.

The researchers showed that for each model heart, they could accurately recreate the same heart-pumping pressures and flows that were previously measured in each respective patient.

"Being able to match the patients' flows and pressures was very encouraging," Roche says. "We're not only printing the heart's anatomy, but also replicating its mechanics and physiology. That's the part that we get excited about."

Going a step further, the team aimed to replicate some of the interventions that a handful of the patients underwent, to see whether the printed heart and vessel responded in the same way. Some patients had received valve implants designed to widen the aorta. Roche and her colleagues implanted similar valves in the printed aortas modeled after each patient. When they activated the printed heart to pump, they observed that the implanted valve produced similarly improved flows as in actual patients following their surgical implants.

Finally, the team used an actuated printed heart to compare implants of different sizes, to see which would result in the best fit and flow -- something they envision clinicians could potentially do for their patients in the future.

"Patients would get their imaging done, which they do anyway, and we would use that to make this system, ideally within the day," says co-author Nyugen. "Once it's up and running, clinicians could test different valve types and sizes and see which works best, then use that to implant."

Ultimately, Roche says the patient-specific replicas could help develop and identify ideal treatments for individuals with unique and challenging cardiac geometries.

Read more at Science Daily

Why whales don't get brain damage when they swim

Special blood vessels in whale brains may protect them from pulses, caused by swimming, in their blood that would damage the brain, new UBC research has suggested.

There are many theories as to the exact use of these networks of blood vessels cradling a whale's brain and spine, known as 'retia mirabilia', or 'wonderful net', but now UBC zoologists believe they've solved the mystery, with computer modeling backing their predictions.

Land mammals such as horses experience 'pulses' in their blood when galloping, where blood pressures inside the body go up and down on every stride. In a new study, lead author Dr. Margo Lillie and her team have suggested for the first time that the same phenomenon occurs in marine mammals that swim with dorso-ventral movements; in other words, whales. And, they may have found out just why whales avoid long-term damage to the brain for this.

In all mammals, average blood pressure is higher in arteries, or the blood exiting the heart, than in veins. This difference in pressure drives the blood flow in the body, including through the brain, says Dr. Lillie, a research associate emerita in the UBC department of zoology. However, locomotion can forcefully move blood, causing spikes in pressure, or 'pulses' to the brain. The difference in pressure between the blood entering and exiting the brain for these pulses can cause damage.

Long-term damage of this kind can lead to dementia in human beings, says Dr. Lillie. But while horses deal with the pulses by breathing in and out, whales hold their breath when diving and swimming. "So if cetaceans can't use their respiratory system to moderate pressure pulses, they must have found another way to deal with the problem," says Dr. Lillie.

Dr. Lillie and colleagues theorized that the retia use a 'pulse-transfer' mechanism to ensure there is no difference in blood pressure in the cetacean's brain during movement, on top of the average difference. Essentially, rather than dampening the pulses that occur in the blood, the retia transfer the pulse in the arterial blood entering the brain to the venous blood exiting, keeping the same 'amplitude' or strength of pulse, and so, avoiding any difference in pressure in the brain itself.

The researchers collected biomechanic parameters from 11 cetacean species, including, fluking frequency, and input these data into a computer model.

"Our hypothesis that swimming generates internal pressure pulses is new, and our model supports our prediction that locomotion-generated pressure pulses can be synchronized by a pulse transfer mechanism that reduces the pulsatility of resulting flow by up to 97 per cent,"says senior author Dr. Robert Shadwick, professor emeritus in the UBC department of zoology.

The model could potentially be used to ask questions about other animals and what's happening with their blood pressure pulses when they move, including humans, says Dr. Shadwick. And while the researchers say the hypothesis still needs to be tested directly by measuring blood pressures and flow in the brain of swimming cetaceans, this is currently not ethically and technically possible, as it would involve putting a probe in a live whale.

"As interesting as they are, they're essentially inaccessible," he says. "They are the biggest animals on the planet, possibly ever, and understanding how they manage to survive and live and do what they do is a fascinating piece of basic biology."

Read more at Science Daily

First time genome editing made possible on cells lining blood vessel walls

The lab of Youyang Zhao, PhD, from Stanley Manne Children's Research Institute at Ann & Robert H. Lurie Children's Hospital of Chicago developed a unique nanoparticle to deliver genome editing technology, including CRISPR/Cas9, to endothelial cells, which are cells that line blood vessel walls. This is the first time that vascular endothelial cells could be reached for genome editing, since the usual way to deliver CRISPR/Cas9 -- through a virus -- does not work for this cell type. Findings were published in the journal Cell Reports.

"The nanoparticle we developed is a powerful new delivery system for genome editing in vascular endothelial cells, and could be used to treat many diseases, including acute respiratory distress syndrome from severe COVID-19," said senior author Dr. Zhao from Lurie Children's. "With this nanoparticle we can introduce genes to inhibit vascular injury and/or promote vascular repair, correct gene mutations and turn genes on or off to restore normal function. It also allows us to edit multiple genes at the same time. This is an important advance for treating any disease caused by endothelial dysfunction."

Endothelial dysfunction is at the root of many diseases, such as coronary artery disease, stroke, bronchopulmonary dysplasia and pulmonary artery hypertension. Dr. Zhao explained that genome editing in endothelial cells could even treat cancers by cutting off the blood supply to the tumor or blocking cancer metastasis.

At this stage, Dr. Zhao and colleagues achieved excellent results in a mouse model. The nanoparticle carrying CRISPR/Cas9 plasmid DNA was introduced via a one-time IV injection and required a few days to be effective. Preclinical testing will be necessary before clinical trials can begin.

"Our nanoparticle delivery system for genome editing and transgene expression also is a huge advance for cardiovascular research," added Dr. Zhao.

From Science Daily

New discovery explains antihypertensive properties of green and black tea

 A new study from the University of California, Irvine shows that compounds in both green and black tea relax blood vessels by activating ion channel proteins in the blood vessel wall. The discovery helps explain the antihypertensive properties of tea and could lead to the design of new blood pressure-lowering medications.

Published in Cellular Physiology and Biochemistry, the discovery was made by the laboratory of Geoffrey Abbott, PhD, a professor in the Department of Physiology and Biophysics at the UCI School of Medicine. Kaitlyn Redford, a graduate student in the Abbott Lab, was first author of the study titled, "KCNQ5 potassium channel activation underlies vasodilation by tea."

Results from the research revealed that two catechin-type flavonoid compounds (epicatechin gallate and epigallocatechin-3-gallate) found in tea, each activate a specific type of ion channel protein named KCNQ5, which allows potassium ions to diffuse out of cells to reduce cellular excitability. As KCNQ5 is found in the smooth muscle that lines blood vessels, its activation by tea catechins was also predicted to relax blood vessels -- a prediction confirmed by collaborators at the University of Copenhagen.

"We found by using computer modeling and mutagenesis studies that specific catechins bind to the foot of the voltage sensor, which is the part of KCNQ5 that allows the channel to open in response to cellular excitation. This binding allows the channel to open much more easily and earlier in the cellular excitation process," explained Abbott.

Because as many as one third of the world's adult population have hypertension, and this condition is considered to be the number one modifiable risk factor for global cardiovascular disease and premature mortality, new approaches to treating hypertension have enormous potential to improve global public health. Prior studies demonstrated that consumption of green or black tea can reduce blood pressure by a small but consistent amount, and catechins were previously found to contribute to this property. Identification of KCNQ5 as a novel target for the hypertensive properties of tea catechins may facilitate medicinal chemistry optimization for improved potency or efficacy.

In addition to its role in controlling vascular tone, KCNQ5 is expressed in various parts of the brain, where it regulates electrical activity and signaling between neurons. Pathogenic KCNQ5 gene variants exist that impair its channel function and in doing so cause epileptic encephalopathy, a developmental disorder that is severely debilitating and causes frequent seizures. Because catechins can cross the blood-brain barrier, discovery of their ability to activate KCNQ5 may suggest a future mechanism to fix broken KCNQ5 channels to ameliorate brain excitability disorders stemming from their dysfunction.

Tea has been produced and consumed for more than 4,000 years and upwards of 2 billion cups of tea are currently drunk each day worldwide, second only to water in terms of the volume consumed by people globally. The three commonly consumed caffeinated teas (green, oolong, and black) are all produced from the leaves of the evergreen species Camellia sinensis, the differences arising from different degrees of fermentation during tea production.

Black tea is commonly mixed with milk before it is consumed in countries including the United Kingdom and the United States. The researchers in the present study found that when black tea was directly applied to cells containing the KCNQ5 channel, the addition of milk prevented the beneficial KCNQ5-activating effects of tea. However, according to Abbott, "We don't believe this means one needs to avoid milk when drinking tea to take advantage of the beneficial properties of tea. We are confident that the environment in the human stomach will separate the catechins from the proteins and other molecules in milk that would otherwise block catechins' beneficial effects."

This hypothesis is borne out by other studies showing antihypertensive benefits of tea regardless of milk co-consumption. The team also found, using mass spectrometry, that warming green tea to 35 degrees Celsius alters its chemical composition in a way that renders it more effective at activating KCNQ5.

"Regardless of whether tea is consumed iced or hot, this temperature is achieved after tea is drunk, as human body temperature is about 37 degrees Celsius," explained Abbott. "Thus, simply by drinking tea we activate its beneficial, antihypertensive properties."

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Living skin can now be 3D-printed with blood vessels included

Researchers at Rensselaer Polytechnic Institute have developed a way to 3D print living skin, complete with blood vessels. The advancement, published online today in Tissue Engineering Part A, is a significant step toward creating grafts that are more like the skin our bodies produce naturally.

"Right now, whatever is available as a clinical product is more like a fancy Band-Aid," said Pankaj Karande, an associate professor of chemical and biological engineering and member of the Center for Biotechnology and Interdisciplinary Studies (CBIS), who led this research at Rensselaer. "It provides some accelerated wound healing, but eventually it just falls off; it never really integrates with the host cells."

A significant barrier to that integration has been the absence of a functioning vascular system in the skin grafts.

Karande has been working on this challenge for several years, previously publishing one of the first papers showing that researchers could take two types of living human cells, make them into "bio-inks," and print them into a skin-like structure. Since then, he and his team have been working with researchers from Yale School of Medicine to incorporate vasculature.

In this paper, the researchers show that if they add key elements -- including human endothelial cells, which line the inside of blood vessels, and human pericyte cells, which wrap around the endothelial cells -- with animal collagen and other structural cells typically found in a skin graft, the cells start communicating and forming a biologically relevant vascular structure within the span of a few weeks. You can watch Karande explain this development here.

"As engineers working to recreate biology, we've always appreciated and been aware of the fact that biology is far more complex than the simple systems we make in the lab," Karande said. "We were pleasantly surprised to find that, once we start approaching that complexity, biology takes over and starts getting closer and closer to what exists in nature."

Once the Yale team grafted it onto a special type of mouse, the vessels from the skin printed by the Rensselaer team began to communicate and connect with the mouse's own vessels.

"That's extremely important, because we know there is actually a transfer of blood and nutrients to the graft which is keeping the graft alive," Karande said.

In order to make this usable at a clinical level, researchers need to be able to edit the donor cells using something like the CRISPR technology, so that the vessels can integrate and be accepted by the patient's body.

"We are still not at that step, but we are one step closer," Karande said.

"This significant development highlights the vast potential of 3D bioprinting in precision medicine, where solutions can be tailored to specific situations and eventually to individuals," said Deepak Vashishth, the director CBIS. "This is a perfect example of how engineers at Rensselaer are solving challenges related to human health."

Karande said more work will need to be done to address the challenges associated with burn patients, which include the loss of nerve and vascular endings. But the grafts his team has created bring researchers closer to helping people with more discrete issues, like diabetic or pressure ulcers.

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