Tag Archives: collagen

Researchers weave stem cells into most lifelike bone yet

Detail of bone tissue of tibia (malleolus medialis. Credit: Wikimedia Commons.

Scientists have coaxed stem cells to transform into bone tissue in the lab. The approach mimics real early bone formation and could be used to perform custom-made treatments, as well as pave the way for the development of lab-made fully functional bones.

“Having a fully functional bone is still far away, but we are sure that it will be achieved one day,” Anat Akiva, assistant professor of Cell Biology at Radboud University in the Netherlands, told ZME Science.

Along with colleagues from the Eindhoven University of Technology, the researchers combined their expertise to form an interdisciplinary team. This proved highly useful for tackling such a big challenge as growing bone in the lab.

Bone is a very complex material and how exactly it is formed in the body is not yet fully understood. Bone is essentially a complex matrix of collagen and minerals, with a lot of blood vessels.

To make bone, three types of cells come together in unison: osteoblasts (which build bone tissue), osteoclasts (which take bone away) and osteocytes (which regulate the building and breaking down of bone). 

Other research groups looking to grow bone tended to focus on only one of these types of cells. For their new study, the team from the Netherlands used stem cells to form two types of these cells (osteoblasts and osteocytes), which were ‘woven’ in a collagen matrix to form early-stage bone.

“Our bone from the lab is exactly like newly formed biological bone,” Akiva said, with the research showing that the mineralized matrix is similar in structure and chemistry.

This was the culmination of four years of extensive research, during which the researchers had to overcome a number of challenges.

“When we presented this research in one of the biggest bone conferences when we just started. We, of course, were thrilled with our study, but the audience, composed of bone scientists from every field, started to question our model and whether it is not good enough because we did not show specific things they were considering important. One of them told us: come back when you show that you have osteocytes – so we did. This means that actually already in 2017 we were the first ones to show that human stem cells can differentiate into osteocytes in the lab,” Akiva said.

Both the lab-grown bone and the biological bone that it mimics produce proteins that are required for the healthy functioning of bone. This means that the lab-grown version is ideal for use as a model system of human bone which can be used to test novel therapies.

From a research perspective, being able to replicate bone at the molecular level with such accuracy may be a new milestone. Bone is made of 99% collagen and minerals, but the remaining 1% of proteins is critical for functioning bone formation.

Many bone disorders, such as brittle bone disease, have their origin at the molecular level. Having a reliable model of the human bone could thus lead to great progress in medicine.

“We are now working in several directions: we are using genomic editing to label specific proteins to know when they are produced and to follow in real-time their journey from the cells to the collagen. By that, we hope to unravel the exact function of these different proteins in bone formation,” Akiva said.

“Another topic will be to grow and follow diseased osteoblast cells (such as in brittle bone disease/osteogenesis imperfecta) to understand at the molecular level why the bones of osteogenesis imperfecta patients are so fragile.”

“And last but not least we would like to go further than only the earliest form of bone formation, so integrating other cells will also be one of the next steps we envisage.”

The findings were reported in the journal Advanced Functional Materials.

Elephant skin.

Collagen networks and hyaluronic acid literally keep you in shape, new study reports

Our tissues have to handle stress and deformation every day. New research is looking into how it copes so well.

Elephant skin.

Image via Pixabay.

We tend to take our bodies for granted but even the most mundane of actions — walking around, breathing — exerts mechanical stress and subsequent deformation of it. And yet, day after day, our tissues take the brunt of it with grace and even heal themselves without any conscious effort on our part.

Curious to see how it pulls off such a feat, researchers from the Wageningen University & Research (WUR) and the AMOLF Institute found that two components in soft tissue, collagen and hyaluronic acid, work together to underpin how tissues respond to mechanical stress.

Feeling stressed

Pull on your earlobe and it will be soft and flexible. Keep pulling, increase the force you apply, and it will become progressively stiffer. It’s not just your earlobes — skin, muscles, the cartilage in your joints and ribs behave the same way.

They’re designed to work like that. While soft, such tissues are easily traversed by cells — but they also need to provide mechanical protection to underlying structures, meaning it has to be tough too, and hard to break. Finally, it’s vital that such tissue is able to transition between the two states.

Needless to say, that’s quite a list of demands. But, our soft tissues rise to the challenge through the use of collagen, hyaluronic acid, and the interactions between the two, a new study reports. The findings not only help us better understand how our bodies function, but may also point the way towards new, synthetic polymers that mimic these impressive properties.

It’s the particular way in which collagen proteins order themselves in soft tissues that give them their resilience. They arrange in a structure known as a sparse network (meaning the proteins don’t form the maximum possible number of bonds between each other). Previous research has gauged the physical resilience of this sparse network in in-vitro conditions: collagen networks were extracted from samples of animal skin and reformed inside a rheometer, an instrument which measures the response of a material during deformation. Such efforts, the team explains, only capture part of the image, however.

“Real tissues are far more complex: they are composed of different molecules that have different sizes and interact with each other in still unknown ways,” says Simone Dussi, postdoc in the WUR Physical Chemistry and one of the study’s corresponding authors. “Because of this complexity, real tissues are way more adaptive than the networks studied so far, made of only collagen.”

“[The presence of hyaluronic acid] significantly changed the mechanical response of the composite networks and we were eager to understand why.”

The team reports that, unlike collagen, hyaluronic acid — a polymer made of much smaller and more flexible molecules — is electromagnetically charged. Because of this, electrostatic interactions generate stress between its individual blocks which accumulates as the tissue is subjected to deformation. According to the team, this buildup of stress basically opposes the deformation.

Networks with a larger amount of hyaluronic acid are “already stiffer at small deformation[s]”, explains co-author Justin Tauber. They also become stiffer in response to larger deformation than networks poor in the acid, the adds.

“We managed to construct a theoretical model and performed computer simulations that matched the experimental results. The key ingredients were identified: In addition to the network structure and the bending rigidity of the collagen fibres, the elasticity and the internal stress generated by the hyaluronic acid are crucial,” Tauber explains.

“The model allows us to make a step further in understanding how real tissues exploit the balance of all these effects. In addition, our findings can be translated into material science to create novel synthetic polymeric materials with more tunable properties.”

The paper “Stress management in composite biopolymer networks” has been published in the journal Nature.

Fox spine.

Bones have a fractal-like structure making them super strong and flexible

Zoom in close enough and bones betray an incredible structural sophistication.

Fox spine.

Fragment of fox spine.
Image via Pixabay.

Researchers from the University of York and the Imperial College London have produced a 3D, nanoscale reconstruction of bone’s mineral structure. Their work reveals a surprising ‘hierarchical organisation’ which underpins the material’s mechanical versatility.

Bred in the bones

Bone is a surprisingly versatile material. Different varieties of bone can be both strong and flexible, maintaining the lithe form of cheetas, the impressive bulk of elephants, or the lightweight frames of birds alike.

These enviable properties are owed to a sophisticated internal structure. However, the exact nature of this structure and of the interactions between the main components of bone — collagen protein strands and the mineral hydroxyapatite — has so far been unknown. According to new research, however, the ‘hierarchical organization’ of bone is based on small elements coming together to form larger and larger structures.

Their results have shown that individual mineral crystals inside bone tissue come together into larger, more complex structures — ones that come together into even more complex levels of organization, the team reports.

For the findings, the team used advanced 3D nanoscale imaging of the mineral component of human bone. They used a combination of electron microscopy-based techniques to reveal its main mineral building blocks. These nanometer-sized crystals of apatite take on a curved, needle-like shape and merge together into larger, twisted platelets that resemble the shape of propeller blades.

These blades, in turn, merge together and split apart throughout the protein phase of the bone. This overarching weaving pattern of mineral and protein is what provides the material’s strength and flexibility.

“Bone is an intriguing composite of essentially two materials, the flexible protein collagen and the hard mineral called apatite,” Lead author, Associate Professor Roland Kröger, says Associate Professor the University of York’s Department of Physics, lead author of the paper.

“The combination of the two materials in a hierarchical manner provides bone with mechanical properties that are superior to those of its individual components alone and we find that there are 12 levels of hierarchy in bone.”

The paper describes the structure as “fractal-like”, containing 12 different levels of complexity. The needle-like crystals merge into the propeller-like platelets in a roughly parallel arrangement with gaps of roughly 2 nanometers between them. These stacks of platelets, along with some single platelets and acicular crystals, come together into larger “polycrystalline aggregates”. These latter ones are larger laterally than the collagen fibers, and can even span several adjacent fibers — providing a continuous, cross-fiber mineral structure that lends resilience to the bone.

Bone structure.

The model of crystal organization in bone proposed by the team.
Patterns specified by the model at the top alongside the mineral organization in different directions (bottom).
Image credits N. Reznikov et al., 2018, Science.

These nanostructures woven into the bone also show a slight curvature, twisting the overall geometry, the team further reports. For example, the individual crystals are curved, the protein (collagen) strands are braided together, mineralized collagen fibrils twist, and the bone themselves have a twist (such as a curvature of a rib).

The team concludes that this fractal-like structure they discovered embedded in our bones is one of the cornerstones of their remarkable physical properties.

The paper “Fractal-like hierarchical organization of bone begins at the nanoscale” has been published in the journal Science.

Meet your new organ: the interstitium

Doctors have identified a previously unknown feature of human anatomy with many implications for the functions of most organs and tissues, and for the mechanisms of most major diseases.

Structural evaluation of the interstitial space. (A) Transmission electron microscopy shows collagen bundles (asterisks) that are composed of well-organized collagen fibrils. Some collagen bundles have a single flat cell along one side (arrowheads). Scale bar, 1 μm. (B) Higher magnification shows that cells (arrowhead) lack features of endothelium or other types of cells and have no basement membrane. Scale bar, 1 μm. (C) Second harmonics generation imaging shows that the bundles are fibrillar collagen (dark blue). Cyan-colored fibers are from autofluorescence and are likely elastin, as shown by similar autofluorescence in the elastic lamina of a nearby artery (inset) (40×). (D) Elastic van Gieson stain shows elastin fibers (black) running along collagen bundles (pink) (40×).

A new paper published on March 27th in Scientific Reports, shows that layers of the body long thought to be dense, connective tissues — below the skin’s surface, lining the digestive tract, lungs, and urinary systems, and surrounding arteries, veins, and the fascia between muscles — are instead interconnected, fluid-filled spaces.

Scientists named this layer the interstitium — a network of strong (collagen) and flexible (elastin) connective tissue fibers filled with fluids, that acts like a shock absorber to keep tissues from rupturing while organs, muscles, and vessels constantly pump and squeeze throughout the day.

This fluid layer that surrounds most organs may explain why cancer spreads so easily. Scientists think this fluid is the source of lymph, the highway of the immune system.

In addition, cells that reside in the interstitium and collagen bundles they line, change with age and may contribute to the wrinkling of skin, the stiffening of limbs, and the progression of fibrotic, sclerotic and inflammatory diseases.

Scientists have long known that more than half the fluid in the body resides within cells, and about a seventh inside the heart, blood vessels, lymph nodes, and lymph vessels. The remaining fluid is “interstitial,” and the current paper is the first to define the interstitium as an organ in its own right and, the authors write, one of the largest of the body, the authors write.

A team of pathologists from NYU School of Medicine thinks that no one saw these spaces before because of the medical field’s dependence on the examination of fixed tissue on microscope slides. Doctors examine the tissue after treating it with chemicals, slicing it thinly, and dyeing it in various colorations. The “fixing” process allows doctors to observe vivid details of cells and structures but drains away all fluid. The team found that the removal of fluid as slides are made makes the connective protein meshwork surrounding once fluid-filled compartments to collapse and appear denser.

“This fixation artifact of collapse has made a fluid-filled tissue type throughout the body appear solid in biopsy slides for decades, and our results correct for this to expand the anatomy of most tissues,” says co-senior author Neil Theise, MD, professor in the Department of Pathology at NYU Langone Health. “This finding has potential to drive dramatic advances in medicine, including the possibility that the direct sampling of interstitial fluid may become a powerful diagnostic tool.”

Researchers discovered the interstitium by using a novel medical technology — Probe-based confocal laser endomicroscopy. This new technology combines the benefits of endoscopy with the ones of lasers. The laser lights up the tissues, sensors analyze the reflected fluorescent patterns, offering a microscopic real-time view of the living tissues.

When probing a patient’s bile duct for cancer spread, endoscopists and study co-authors Dr. David Carr-Locke and Dr. Petros Benias observed something peculiar — a series of interconnected spaces in the submucosa level that was never described in the medical literature.

Baffled by their findings, they asked Dr. Neil Theise, professor in the Department of Pathology at NYU Langone Health and co-author of the paper for help in resolving the mystery. When Theise made biopsy slides out of the same tissue, the reticular pattern found by endomicroscopy vanished. The pathology team would later discover that the spaces seen in biopsy slides, traditionally dismissed as tears in the tissue, were instead the remnants of collapsed, previously fluid-filled, compartments.

Researchers collected tissues samples of bile ducts from 12 cancer patients during surgery. Before the pancreas and the bile duct were removed, patients underwent confocal microscopy for live tissue imaging. After recognizing this new space in images of bile ducts, the team was able to quickly spot it throughout the body.

Theise believes that the protein bundles seen in the space are likely to generate electrical current as they bend with the movements of organs and muscles, and may play a role in techniques like acupuncture.

Another scientist involved in the study was first author Rebecca Wells of the Perelman School of Medicine at the University of Pennsylvania, who determined that the skeleton in the newfound structure was comprised of collagen and elastin bundles.

Scientists find 80-million-year dinosaur collagen

Utilizing rigorous, state-of-the-art methods, researchers have confirmed the presence of collagen in the fossil of an 80-million-year-old Brachylophosaurus.

An artistic reconstruction of Brachylophosaurus. Image via Wiki Commons.

Some 80 million years ago, during the Cretaceous, most Brachylophosaurus were likely having a pretty good time. They featured a tough bony crest and reached sizes of up to 11 meters (36 feet), making them inaccessible for most predators.

We’ve found plenty of Brachylophosaurus fossils and we know quite a bit about their anatomy and lifestyle. In 2003, researchers even found evidence of tumors, likely caused by environmental factors or genetic propensity. But now, Elena Schroeter, NC State postdoctoral researcher, and Mary Schweitzer, professor of biological sciences with a joint appointment at the North Carolina Museum of Natural Sciences, have found something even more exciting: collagen.

The discovery was made in 2009, but they wanted to confirm it, using the latest available technology. With today’s equipment, they can strongly claim it is indeed collagen.

“Mass spectrometry technology and protein databases have improved since the first findings were published, and we wanted to not only address questions concerning the original findings, but also demonstrate that it is possible to repeatedly obtain informative peptide sequences from ancient fossils,” Schroeter says.

“We collected B. canadensis with molecular investigation in mind,” Schweitzer says. “We left a full meter of sediment around the fossil, used no glues or preservatives, and only exposed the bone in a clean, or aseptic, environment. The mass spectrometer that we used was cleared of contaminants prior to running the sample as well.”

Collagen is the main structural protein found in skin and other connective tissues — it basically holds everything together. It’s the most abundant protein in the human body and also has numerous medical uses in treating complications of the bones and skin. The fact that the collagen was preserved for millios of years is impressive in itself, but it could be even more significant. Recovering the protein in such fossils could shed some valuable light on the evolutionary relationships between dinosaurs and modern animals, and teach us a lot about collagen itself.

Brachylophosaur canadensis fossil femur (MOR 2598) in field jacket, showing area of sampling for molecular analyses.
Credit: Mary Schweitzer

The sample was taken from the dinosaur’s femur and so far, the tests seem to confirm predictions — namely, it resembles the collagen from crocodiles and birds.

“We are confident that the results we obtained are not contamination and that this collagen is original to the specimen,” Schroeter says. “Not only did we replicate part of the 2009 results, thanks to improved methods and technology we did it with a smaller sample and over a shorter period of time.”

Now, researchers are wondering whether this is an isolated, exceptional case, or if other fossils might contain collagen as well. If this is true, then the existing fossil record is teeming with valuable evolutionary and anatomical information we’ve yet to find.

“Our purpose here is to build a solid scientific foundation for other scientists to use to ask larger questions of the fossil record,” Schweitzer adds. “We’ve shown that it is possible for these molecules to preserve. Now, we can ask questions that go beyond dinosaur characteristics. For example, other researchers in other disciplines may find that asking why they preserve is important.”


Macrauchenia ("long llama"). Image: Wikimedia Commons

Darwin’s ‘strangest animals’ finally classified thanks to protein sequencing

While in South American during his 1830 expedition with the HMS Beagle, Charles Darwin came across the fossils of two peculiar hoofed species which he was unable to classify properly. One was Macrauchenia, which looked like a camel with the head of an ant eater, and the other was  Toxodon which had the body of rhino, the head of a hippo and the teeth of a rodent. So, was the Macrauchenia related to the camel or the ant eater? Who was Toxodon’s closet cousin, the hippo or the rhino? Darwin was puzzled and to no avail concluded these were  “perhaps one of the strangest animals ever discovered”. But Darwin didn’t have the tools we have today. Now, using a ground breaking technique researchers have sequenced the collagen of a myriad of South American mammals, including Darwin’s ‘strangest animals’ and finally found their real taxonomy.

Macrauchenia ("long llama"). Image: Wikimedia Commons

Macrauchenia (“long llama”). Image: Wikimedia Commons

The two beasts are among 250 mammals that make up a family known as the South American ungulates, which lived for 60 million years on the continent and vanished only 12,000 year ago. Studying Macrauchenia and Toxodon has been difficult because: 1) few and disperse fossil fragments and 2) because researchers have never been able to isolate proper DNA samples. These simply get too damaged because of the wet climate and interfere with conventional genetic screening that is typically used to relate ancient, extinct species with another and uncover their ancestry. Here’s where collagen comes in, though – the fibrous protein that binds cells together into organs and tissues. It can last for at least 10 times as long as DNA and be used to build a collagen family tree.

Toxodon. Digital illustration: martinoraptor

Toxodon. Digital illustration: martinoraptor

A team made up of researchers at the Natural History Museum in London and  University of York, UK extracted, then sequenced collagen from 48 fossils from the remains of Darwin’s animals collected from Argentina and Uruguay. Previously, the South American ungulates were suggested to have belonged to the group Afrotheria, along with elephants and manatees. The protein sequencing, however, clearly shows that both animals belong to  Perissodactyla, a group that includes horses, tapirs and rhinos. The paper appeared in the journal Nature.

So, finally Darwin’s puzzle has been solved, but in doing so the researchers have unlocked a tool that could prove to be a lot more useful. The oldest DNA comes from an 800,000 years old ice core. Collagen can survive for at least four millions years, and in cold conditions maybe even 20 million years. With this in mind, the technique employed in this study could be used study other extinct groups where DNA is not an option, like is the case of dwarf elephants and enormous rodents of the Indonesian island of Flores, or Australia’s giant lizards and kangaroos.

Tilapia Fish May Help Cure Our Wounds in the Future

Scientists believe that collagen extracted from fish (namely tilapia) can be applied as a “wound dressing”, to help clean the wound and accelerate healing.

Tilapia. Image via Wiki Commons.

Collagen is the main structural protein of the various connective tissues in animals. As the main component of connective tissue, it is the most abundant protein in mammals, but can also be found in fish. Collagen from cows and pigs has been used previously, but there are a couple of drawbacks to cow collagen, such as the potential for infectious disease transmission and religious issues in some areas of the world.

Researchers started to focus on other potential sources of collagen, and they concluded that the tilapia would be a great choice. Tilapia is the common name for nearly a hundred species of cichlid fish from the tilapiine cichlid tribe. Tilapia are mainly freshwater fish inhabiting shallow streams, ponds, rivers and lakes and less commonly found living in brackish water. In recent years, tilapia have been grown intensively throughout the world, and the aquacultured tilapia makes a great substitute.

The team studied the issue and found that tilapia collagen doesn’t provoke a negative immune response. Then, they studied its healing properties and noted that tilapia collagen encouraged the growth of fibroblasts and increased the expression of genes involved in wound healing. All in all, the results were encouraging enough to move on to animal testing.

Image credits: Zhou et al.

For this, they inflicted 1.8-cm-wide wounds on the backs of rats. They then treated the wounds with nothing (as a control), an algae based wound dressing (Kaltostat), and tilapia collagen. As seen below, the tilapia collagen was the most effective at treating the wounds – after two weeks, they were basically gone.

Researchers hope to refine their research and ultimately release it as a product, but they have a tough competition ahead of them. For example, the company Eqalix uses soybean protein to promote healing, and they have a couple of years of headstart research; they are currently trying to obtain FDA approval.

Journal Reference: Tian Zhou, Nanping Wang, Yang Xue, Tingting Ding, Xin Liu, Xiumei Mo, and Jiao Sun. “Development of Biomimetic Tilapia Collagen Nanofibers for Skin Regeneration through Inducing Keratinocytes Differentiation and Collagen Synthesis of Dermal Fibroblasts.” ACS Appl. Mater. Interfaces 7 (5), pp 3253–3262. 19-Jan-2015. DOI: 10.1021/am507990m


Molecules of collagen (top) next to molecules of a crystal of hydroxyapatite (bottom). (c) MIT

Atomic structure of bone deciphered for the first time

Bone is a really awesome material, being hard and flexible at the same time. For something so ubiquitous and studied for so long, it might come as a surprise to some of you to hear that the molecular bone structure of bone has alluded scientists for such a long while. This is because, even though the constituent elements that make up bone have been known for a long time, how they line together to form such an intricate structure has been very difficult to identify, until recently after MIT researchers used supercomputers to reveal with almost atomic precision the precise structure of bone. Their work is a significant step forward in material science, marking a milestone. The researchers hope they can synthesize bone fibers soon enough, while furthering their understanding of how some diseases attack bones.

Molecules of collagen (top) next to molecules of a crystal of hydroxyapatite (bottom).  (c) MIT

Molecules of collagen (top) next to molecules of a crystal of hydroxyapatite (bottom). (c) MIT

Rather curiously, bone is made out of two materials:  a soft, flexible biomolecule called collagen and a hard, brittle form of the mineral apatite (hydroxyapatite). Each substance, taken individually, has nothing to do with bone, but when combined in a very complex manner, they form something that is simultaneously hard, tough and slightly flexible.

For years scientists have been studying bone at a molecular level, using tools such as atomic force microscopes to probe the material at an atomic level, however using such methods imaging is only possible in 2-D. Bone, taken as a whole, is an intricate molecular 3-D structure that can not be decoded by looking from a 2-D perspective.

“It’s easy to get images of bone, but it’s hard to see exactly where the minerals are located inside the collagen,” says Shu-Wei Chang, a CEE graduate student who was a co-author of the paper.

Using a supercomputer, engineers from MIT, led by civil engineer and materials scientist Markus Buehler, calculated what fibers of collagen, strengthened with hydroxyapatite crystals, look like. To verify their findings, they made a comparative study with laboratory findings. This whole process required several iterations before they could come to a sound conclusion and took several months to complete, something very important to note since the same process would have taken years and years to complete not too long ago. Thankfully supercomputing has evolved exponentially and MIT were quick to update their stock.

What makes up bones

According to their results, the MIT researchers found that the key to the bone’s fantastic molecular structure lies in how the hydroxyapatite crystals align with collagen. Hydroxyapatite grains are tiny, thin platelets just a few nanometers (billionths of a meter) across, and are deeply embedded in the collagen matrix. The two constituents are bound together by electrostatic interactions, which allows them to slip somewhat against each other without breaking. Large pieces of hydroxyapatite are brittle, like chalk, but at such small sizes, the mineral is actually ductile. The researchers also found that under stress tests, the mineral parts of the fibers took on four times as much stress as the collagen portions, whereas bending happened almost exclusively in the collagen.

“In this arrangement of tiny hydroxyapatite grains embedded in the collagen matrix, the two materials can each contribute the best of their properties,” Buehler says. “Hydroxyapatite takes most of the forces in the material, whereas collagen takes most of the stretching.”

Buehler and his team hope they can next create bone fibers in the lab. Also, now with a better understanding of what makes up bone and how its constituent elements are arranged, doctors may find what goes wrong in certain diseases, including osteoporosis and brittle bone disease.

“We can use this model to understand how a bone becomes more brittle,” says Arun Nair, a CEE postdoc who was the first author of the paper. For example, collagen is made up of thousands of amino acids, but “if only one of those amino acids is altered, it changes the way the minerals form” inside the bone, Nair says.

“That’s why this model is so critical,” he adds. Without it, you could observe how bone changes as a result of disease, but “you don’t know why. Now, we can see how a very tiny change … changes the way the mineral grows, or how the forces and deformation are distributed.”

The findings were reported in the journal Nature Communications.