A ‘Horse-on-a-Chip’? The Future Of Equine Drug Research Could Look Very Different

The research process for drug toxicology in horses has always been long, slow, and expensive. Too often, when veterinarians want to more about the way a drug behaves in horses, they find themselves relying on limited data collected from a small number of horses. That's because there is a lot of expense and regulation associated with using live animals for research of any kind, even a simple drug administration study aimed at determining how quickly horses' bodies metabolize a therapeutic substance. It's also expensive for universities to maintain horse research herds of significant size year after year, awaiting their use in a short study.

A research group at the Gluck Equine Research Center is hopeful they have a solution that will make it quicker and easier for scientists to understand how drugs behave in horses, and it sounds like something out of a sci-fi drama: microscopic equine organ systems.

It's no longer science fiction. Dr. Carrie Shaffer said researchers aren't reconstructing full-size organs, but rather are using defined layers of cells that mirror what you'd find in an equine kidney, liver, lung, or intestine. The cells come from tissue-specific stem cells collected from a Thoroughbred foal that had to be euthanized due to an unrelated structural deformity. Stem cells have the ability to become any kind of differentiated cell upon command, so the researchers are able to direct the cells to form a particular organ tissue.

“We can prove, using a variety of different methods, that our equine microscopic organ systems are stem-cell derived and have the same characteristics and architecture as the corresponding tissue in the horse.”

These microscopic organ systems are grown in clear, plastic microfluidic chips that are about the size of a AA battery. In human medicine, similar microfluidic chips have been developed to mimic the human liver, lung, intestine, kidney, and blood/brain barrier and are used to study various aspects of cell biology and tissue responses to therapeutics.

The metabolism of a drug isn't dependent on the full-size physical structure of an equine liver or kidney, according to Shaffer – it's how the cells of those organs interact with drugs they encounter as the substance passes through an animal's bloodstream and into the organ tissue. Shaffer is able to grow specific liver cells in one channel of the microfluidic chip while creating artificial blood vessels and blood-like fluid flow on the opposite channel of the chip. This simulates a continuous blood supply interfaced with the mucous membranes that are normally found in the body. The blood flow can go in only one direction, which also mimics the horse's body, where veins and arteries carry blood through an organ in only one direction at a time.

“In the case of the lung chip and the intestine chip, we can also introduce relevant biomechanical forces that simulate complex biological processes,” she said. “We can introduce physical stresses into the chip that mimic breathing and lung inflation, or recreate defined patterns of stretch across the intestine chip that simulate the wave-like pattern of nutrients and waste products moving along the equine intestinal system.”

These forces have been shown to direct gene expression in the cells, which create small, but critical, changes that make the microfluidic chips behave more like the cells found in a live animal.

Previous iterations of this technology didn't include biomechanical forces like stretch, so the tissue wasn't as true to that in a horse's body. Additionally, previous tissue culture systems did not allow for directional fluid flow, but rather exposed a single type of liver or kidney cell to static fluid containing a drug at a fixed concentration. That's not how real kidneys and livers actually work, said Shaffer – the organs contain multiple cell types that are exposed to blood flowing at a relatively high rate. Therapeutics within the bloodstream pass through various organ systems within seconds, and carry metabolized drug away from one organ system for delivery to another.

“Under normal drug testing conditions, we are able to analyze a blood sample from a horse after a drug is administered, but we cannot tell in that blood sample where the drug metabolism occurred,” she said. “We don't know whether the drug was liver-metabolized, intestinal-metabolized, or metabolized in the lung. Our horse-on-a-chip microfluidic technology allows us to isolate exactly where drug metabolism occurs within the horse.”

Some drugs metabolize at different rates in different organs, and organs probably take turns at metabolizing a drug but there's currently no way to know in what order metabolism occurs for a given therapeutic. That information could be useful because some drugs linger longer in the body than expected, and scientists often don't know where the hold-up is.

Shaffer said her lab has performed only a handful of studies with the technology because it's so new. So far, the team has pulsed a drug through an equine lung-chip and a liver-chip for sample collection from the apparatus at defined times post-administration to see how much of the drug had been metabolized by specific tissues in a set timeframe.

The team is still validating these emerging  methods and drafting papers for peer-reviewed journals describing the process they've used to create this technology. Shaffer said they're still a few months away from using the organ chips en masse for huge studies – and they need to expand to include tissues from other breeds – but she thinks the microfluidic chips could be useful for pre-clinical analysis of new therapeutic drugs.

“The big sell with our horse-on-a-chip technology is that it's going to significantly reduce animal use for studies – reduce euthanasia, reduce the need for research herds,” she said. “We can now perform the majority of upstream pre-clinical analyses  in the lab using our technology that recreates the dynamic environment within the horse. Before, we'd study the effects of a new drug using expensive and limited research herds. Now, we can perform critical toxicity and safety studies before the candidate drug is ever injected into a horse.

“The key to our technology is that we don't need to euthanize additional horses.  We can go back to our cryobank of Thoroughbred tissue and enrich for tissue-specific stem cells to essentially grow equine microfluidic organ-chips indefinitely. My research team has developed several innovative methods that allow us to keep using and expanding these diverse equine tissues indefinitely.”

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Grayson-Jockey Club Research Foundation To Hold Second Photo Contest To Celebrate Healthy Horses

Grayson-Jockey Club Research Foundation announced today that it will again be hosting an online photo contest for horse lovers to celebrate their equine companions.

The contest opens February 1, and entries will be accepted through February 28. Horse enthusiasts are encouraged to submit original photos of horses representing all breeds, backgrounds, and disciplines on Grayson's website at grayson-jockeyclub.org/default.asp?section=2&area=PHOTOINFO.

Finalists will be selected by the Grayson team, and the winning photo will be chosen by votes from the public on Grayson's Facebook page. The winner will receive a Grayson “swag bag,” and each finalist will also receive a prize. Selected photos submitted to the contest will be shared on Grayson's social media accounts using the hashtag #ilovehealthyhorses.

“We received an enthusiastic response to last year's photo contest and are looking forward to seeing submissions this year as horse lovers continue to increase awareness of the importance of equine veterinary research and how it leads to healthy horses,” said Jamie Haydon, president of Grayson-Jockey Club Research Foundation.

For the contest's official rules, please visit grayson-jockeyclub.org/default.asp?section=2&area=PHOTORULES&menu=1.

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TERF Awards $20k to the University Of Pennsylvania School Of Veterinary Medicine

The Thoroughbred Education and Research Foundation (TERF) has awarded $20,000 to the University Of Pennsylvania School Of Veterinary Medicine to be used for equine research.

In alignment with their mission to support and promote equine research, TERF annually provides grant funding to organizations that are engaged in the research of issues which impact equine health.

Funding will be used to support research by Mary Robinson, VMD, PhD in a study of the use of bisphosphonates to medically treat equines. Bisphosphonates inhibit bone breakdown and are useful for treating bone disorders in horses.

The study will provide evidence for a better understanding of the impact of the use of bisphosphonates in the treatment of Thoroughbreds for these issues which can significantly impact an equine’s quality of life.

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Sesamoid Bones: They Take A Lot Of Pressure And Raise A Lot Of Questions For Researchers

As racing continues its quest to reduce injury rates, one key area of interest for many experts is the proximal sesamoid bones.

Most racing fans who have heard of sesamoid bones know about the two small, triangular bones held inside the suspensory ligament that form the back of the equine ankle, but horses (and humans) actually have other sets of sesamoids in the body. The two that form each ankle are called the proximal sesamoid bones. The human kneecap or patella is present in the horse as a component of the stifle and is also considered a type of sesamoid bone. The navicular bone in the internal structures of the hoof is also a type of sesamoid. Sesamoids exist because they reduce friction on joints by gliding over the joint's surface, helping to pull the limb back and forth.

The proximal sesamoid bones are part of the ankle or fetlock, which drops down toward the racing surface to absorb the horse's weight during a footfall. The joint flexes farther down the harder the foot falls. The elastic tendons and ligaments (particularly the suspensory ligament) are crucial during this shock absorption procedure, and the proximal sesamoids are hard at work in this moment too – which may mean it's not surprising that they're a common source of injury.

Existing research suggests that sesamoid fractures or suspensory apparatus failures are associated with 30 to 50 percent of fatal injuries in Thoroughbred racehorses. At a recent virtual session of the University of Kentucky's annual Equine Showcase, researchers said that makes them a crucial area of study – but we have to start from the beginning.

Scientists would like to know how the structure of the proximal sesamoids changes in response to intense exercise like racing. We know bones change their shape and structure in proportion to the amount and types of forces placed on them through exercise in a process called bone remodeling. (You can learn more about bone development and remodeling here.) It would be helpful to know if somewhere in that process, sesamoid bones undergo abnormal changes that could signal or predispose an upcoming fracture.

According to Dr. James MacLeod, researcher and faculty member at the Gluck Equine Research Center, scientists first need the answers to more basic questions about proximal sesamoids. In order for researchers to know what is considered an abnormal structural change, they have to know what's normal for these particular bones – what size, shape, and internal structure is typical? How do they develop? When do they develop? How much variation is there in size, shape and structure between individuals, between breeds and between sports?

Unfortunately, MacLeod said, existing science is somewhat light on the answers to these questions.

“It turns out that in the horse, very little information was published about proximal sesamoid bone development and maturation in a normal sense,” he said.

When trying to answer the basic question of when these bones develop, MacLeod and his colleagues dug up two publications in textbooks suggesting that these particular bones don't begin to form in a developing equine fetus until very late in gestation, between Day 290 and 330 in what's typically a 340-day gestation. The end of ossification (hardening) for the bones was, according to these textbooks, complete at around month three or four of the foal's life.

“We had evidence right away that there was much more to know about the development of proximal sesamoid bones,” he said.

Soon after the research team began their inquiries, Dr. Emma Adam, assistant professor at Gluck, used advanced imaging to discover that the very beginnings of cartilage (which would eventually transition to bone) were beginning to form in what would become the fetlock at Day 46 of gestation. At that point the fetus was only three centimeters long, with a tiny forming limb only three millimeters long.

Currently, MacLeod and his colleagues are in the process of learning more about the variability of the bones in adults, assembling lots of samples from horses who have died for reasons independent of development or injury to the sesamoid bones. Researchers want to study them grossly (recording observations detectable without a microscope) as well as at a microscopic level. They're looking at elements like bone volume, which refers to the amount of a bone that is minerals. Researchers already know that sesamoid bone volume increases with age as an animal matures and the bone itself grows. Next, MacLeod said, we need to learn how bone volume may change when the horse grows old enough to begin exercise.

Another element that could be important in microscopic bone changes is the trabeculae, which are the bands or thin rods of tissue that together make up the hard structural elements of the bone. MacLeod hopes researchers will learn more about the orientation of these little beam-like supports – are they isotropic, meaning their orientation creates a look of sameness throughout a sample, or are they anisotropic, meaning many of them lie in a single, similar orientation? This matters because it impacts how easy a substance is to break. If you think about chopping an anisotropic piece of wood, he points out, it's easy to do with the grain because all the strands of the block's interior structure are pointing more or less the same direction. If you chop against that grain, it suddenly becomes tougher. With an isotropic substance like metal, its components are oriented in all different directions at a cellular level, making it equally difficult to cut or split no matter how you approach it — there's no area or angle of weakness on a microscopic level.

The initial step to understanding these elements of the bone's structure is to get as many samples as possible from a wide cross section of ages and breeds. Those breed differences could be really important, too — it won't help racehorses if the team develop their sense of normal sesamoid bones from Shetland ponies.

“You'd certainly expect [to see differences],” he said. “The skeletal system in general matures differently between different breeds. Small horses and ponies actually mature faster than larger horses.”

There could also be important differences in what's “normal” between male and female animals, as well as large, heavy-bodied and fine-boned horses within the same breed.

For now, MacLeod said his team has more questions than answers, but he is hopeful that soon – maybe even by next year's annual equine research showcase – he can provide some.

“I think as we ask the questions, as we generate quality data sets, as we advance imaging technologies, I think we will be able to answer those questions,” he said.

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