A chance meeting between a spider expert and a chemist has led to the development of antibiotic synthetic spider silk.
After five years’ work an interdisciplinary team of scientists at The University of Nottingham has developed a technique to produce chemically functionalised spider silk that can be tailored to applications used in drug delivery, regenerative medicine and wound healing.
The Nottingham research team has shown for the first time how ‘click-chemistry’ can be used to attach molecules, such as antibiotics or fluorescent dyes, to artificially produced spider silk synthesised by E.coli bacteria. The research, funded by the Biotechnology and Biological Sciences Research Council (BBSRC) has been published in the online journal Advanced Materials.
The chosen molecules can be ‘clicked’ into place in soluble silk protein before it has been turned into fibres, or after the fibres have been formed. This means that the process can be easily controlled and more than one type of molecule can be used to ‘decorate’ individual silk strands.
In a laboratory in the Centre of Biomolecular Sciences, Professor Neil Thomas from the School of Chemistry in collaboration with Dr Sara Goodacre from the School of Life Sciences, has led a team of BBSRC DTP-funded PhD students starting with David Harvey who was then joined by Victor Tudorica, Leah Ashley and Tom Coekin. They have developed and diversified this new approach to functionalising ‘recombinant’ — artificial — spider silk with a wide range of small molecules.
They have shown that when these ‘silk’ fibres are ‘decorated’ with the antibiotic levofloxacin it is slowly released from the silk, retaining its anti-bacterial activity for at least five days.
Neil Thomas, a Professor of Medicinal and Biological Chemistry, said: “Our technique allows the rapid generation of biocompatible, mono or multi-functionalised silk structures for use in a wide range of applications. These will be particularly useful in the fields of tissue engineering and biomedicine.”
Remarkable qualities of spider silk
Spider silk is strong, biocompatible and biodegradable. It is a protein-based material that does not appear to cause a strong immune, allergic or inflammatory reaction. With the recent development of recombinant spider silk, the race has been on to find ways of harnessing its remarkable qualities.
The Nottingham research team has shown that their technique can be used to create a biodegradable mesh which can do two jobs at once. It can replace the extra cellular matrix that our own cells generate, to accelerate growth of the new tissue. It can also be used for the slow release of antibiotics.
Professor Thomas said: “There is the possibility of using the silk in advanced dressings for the treatment of slow-healing wounds such as diabetic ulcers. Using our technique infection could be prevented over weeks or months by the controlled release of antibiotics. At the same time tissue regeneration is accelerated by silk fibres functioning as a temporary scaffold before being biodegraded.”
The medicinal properties of spider silk recognised for centuries.
The medicinal properties of spider silk have been recognised for centuries but not clearly understood. The Greeks and Romans treated wounded soldiers with spider webs to stop bleeding. It is said that soldiers would use a combination of honey and vinegar to clean deep wounds and then cover the whole thing with balled-up spider webs.
There is even a mention in Shakespeare’s Midsummer Night’s Dream: “I shall desire you of more acquaintance, good master cobweb,” the character ‘Bottom’ said. “If I cut my finger, I shall make bold of you.”
‘I think we could make that!’
The idea came together at a discipline bridging university ‘sandpit’ meeting five years ago. Dr Goodacre says her chance meeting at that event with Professor Thomas proved to be one of the most productive afternoons of her career.
Dr Goodacre, who heads up the SpiderLab in the School of Life Sciences, said: “I got up at that meeting and showed the audience a picture of some spider silk. I said ‘I want to understand how this silk works, and then make some.’
“At the end of the session Neil came up to me and said ‘I think my group could make that.’ He also suggested that there might be more interesting ‘tweaks’ one could make so that the silk could be ‘decorated’ with different, useful, compounds either permanently or which could be released over time due to a change in the acidity of the environment.”
The approach required the production of the silk proteins in a bacterium where an amino acid not normally found in proteins was included. This amino acid contained an azide group which is widely used in ‘click’ reactions that only occur at that position in the protein. It was an approach that no-one had used before with spider silk — but the big question was — would it work?
Dr Goodacre said: “It was the start of a fascinating adventure that saw a postdoc undertake a very preliminary study to construct the synthetic silks. He was a former SpiderLab PhD student who had previously worked with our tarantulas. Thanks to his ground work we showed we could produce the silk proteins in bacteria. We were then joined by David Harvey, a new PhD student, who not only made the silk fibres, incorporating the unusual amino acid, but also decorated it and demonstrated its antibiotic activity. He has since extended those first ideas far beyond what we had thought might be possible.”
David Harvey’s work is described in this paper but Professor Thomas and Dr Goodacre say this is just the start. There are other joint SpiderLab/Thomas lab students working on uses for this technology in the hope of developing it further.
David Harvey, the lead author on this their first paper, has just been awarded his PhD and is now a postdoctoral researcher on a BBSRC follow-on grant so is still at the heart of the research. His current work is focused on driving the functionalised spider silk technology towards commercial application in wound healing and tissue regeneration.
Where will we be in 5 years’ time?
Dr Goodacre said: “It is likely that this paper is just the start of a very exciting range of studies using the new spider silk material. Some of the future work will also be supported by other, neat ideas from the world of spiders and their silk, which the SpiderLab is currently trying to unravel.”
A new study of one of our closest invertebrate relatives, the acorn worm, reveals that this feat might one day be possible.
Acorn worms burrow in the sand around coral reefs, but their ancestral relationship to chordates means they have a genetic makeup and body plan surprisingly similar to ours.
A study led by the University of Washington and published in the December issue of the journal Developmental Dynamics has shown that acorn worms can regrow every major body part – including the head, nervous system and internal organs – from nothing after being sliced in half.
“This could have implications for central nervous system regeneration in humans if we can figure out the mechanism the worms use to regenerate.”
The new study finds that when an acorn worm – one of the few living species of hemichordates – is cut in half, it regrows head or tail parts on each opposite end in perfect proportion to the existing half.
Each half of the worm continues to thrive, and subsequent severings also produce vital, healthy worms once all of the body parts regrow.
The researchers also analyzed the gene expression patterns of acorn worms as they regrew body parts, which is an important first step in understanding the mechanisms driving regeneration.
It’s as if the cells are independently reading road signs that tell them how far the mouth should be from the gill slits, and in what proportion to other body parts and the original worm’s size.
Humans can regrow parts of organs and skin cells to some degree, but we have lost the ability to regenerate complete body parts.
Scientists suspect several reasons for this: Our immune systems – in a frenzy to staunch bleeding or prevent infection – might inhibit regeneration by creating impenetrable scar tissue over wounds, or perhaps our relatively large size compared with other animals might make regeneration too energy intensive.
Learn and see more: Our closest worm kin regrow body parts, raising hopes of regeneration in humans
Vitamins A and C aren’t just good for your health, they affect your DNA too. Researchers at the Babraham Institute and their international collaborators have discovered how vitamins A and C act to modify the epigenetic ‘memory’ held by cells; insight which is significant for regenerative medicine and our ability to reprogramme cells from one identity to another. The research is published today in Proceedings of the National Academy of Science (PNAS).
For regenerative medicine, the holy grail is to be able to generate a cell that can be directed to become any other cell, such as brain cells, heart cells and lung cells. Cells with this ability are present in the early embryo (embryonic stem cells, ESC) and give rise to the many different cell types in the body. For the purposes of regenerative medicine, we need to be able to force adult cells from a patient to regress back to possessing embryonic-like capabilities and to ‘forget’ their previous identity.
A cell’s identity is established at the DNA level by epigenetic changes to the DNA. These changes don’t alter the order of the DNA letters but control which parts of the genome can be read and accessed. Consequently, every different cell type has a unique epigenetic fingerprint, enforcing and maintaining specific patterns of gene expression appropriate to the cell type. To reverse cells back to the naïve pluripotent state this epigenetic layer of information has to be lost to open up the full genome again.
Researchers from the Babraham Institute, UK, University of Stuttgart, Germany and University of Otago, New Zealand worked together to uncover how vitamins A and C affect the erasure of epigenetic marks from the genome. They looked in particular at the epigenetic modification where a methyl chemical tag is added to the C letters in the DNA sequence. Embryonic stem cells show low levels of this C tagging, called cytosine methylation, but in established cell types much more of the genome is marked by this modification. Removing the methyl tags from the DNA, called demethylation, is a central part of achieving pluripotency and wiping epigenetic memory.
The family of enzymes responsible for active removal of the methyl tags are called TET. The researchers looked at the molecular signals that control TET activity to understand more about how the activity of the TET enzymes can be manipulated during cellular programming to achieve pluripotency.
They found that vitamin A enhances epigenetic memory erasure in naïve ESC by increasing the amount of TET enzymes in the cell, meaning greater removal of methyl tags from the C letters of the DNA sequence. In contrast, they found that vitamin C boosted the activity of the TET enzymes by regenerating a co-factor required for effective action.
Dr Ferdinand von Meyenn, postdoctoral researcher at the Babraham Institute and co-first author on the paper, explained: “Both vitamins A and C act individually to promote demethylation, enhancing the erasure of epigenetic memory required for cell reprogramming.” Dr Tim Hore, previously a Human Frontier Long Term Research Fellow at the Babraham Institute, now Lecturer at the University of Otago, New Zealand and co-first author on the paper, continued: “We found out that the mechanisms of how vitamins A and C enhance demethylation are different, yet synergistic.”
The improved understanding of the effect of vitamin A on the TET2 enzyme also potentially explains why a proportion of patients with acute promyelocytic leukaemia (once considered the deadliest form of acute leukaemia) are resistant to effective combination treatment with vitamin A. By providing a possible explanation for this insensitivity for further investigation, this work could point the way to better management of the vitamin A resistant cases.
Professor Wolf Reik, Head of the Epigenetics Programme at the Babraham Institute, said: “This research provides an important understanding in order to progress the development of cell treatments for regenerative medicine. It also enhances our understanding of how intrinsic and extrinsic signals shape the epigenome; knowledge that could provide valuable insight into human disease, such as acute promyelocytic leukaemia and other cancers. Putting the full picture together will allow us to understand the full complexity of the epigenetic control of the genome.”
A study from the University of Pittsburgh School of Medicine and the McGowan Institute for Regenerative Medicine identifies a mechanism by which bioscaffolds used in regenerative medicine influence cellular behavior, a question that has remained unanswered since the technology was first developed several decades ago.
The findings were recently published online in Science Advances.
Bioscaffolds composed of extracellular matrix (ECM) derived from pig tissue promote tissue repair and reconstruction. Currently, these bioscaffolds are used to treat a wide variety of illnesses such hernias and esophageal cancer, as well as to regrow muscle tissue lost in battlefield wounds and other serious injuries.
“Bioscaffolds fulfill an unmet medical need, and have already changed the lives of millions of people,” said lead study investigator Stephen Badylak, D.V.M., M.D., Ph.D., professor of surgery at Pitt and deputy director of the McGowan Institute, a joint effort of Pitt and UPMC.
Researchers know that ECM is able to instruct the human body to replace injured or missing tissue, but exactly how the ECM material influences cells to cause functional tissue regrowth has remained a fundamental unanswered question in the field of regenerative medicine.
In the new study, Dr. Badylak and his team showed that cellular communication occurs using nanovesicles, extremely tiny fluid-filled sacs that bud off from a cell’s outer surface and allow cells to communicate by transferring proteins, DNA and other “cargo” from one cell to another.
Exosomes are present in biological fluids such as blood, saliva and urine, where they influence a variety of cellular behaviors, but researchers had yet to identify them in solid body tissues.
“We always thought exosomes are free floating, but recently wondered if they are also present in the solid ECM and might facilitate the cellular communication that is critical to regenerative processes,” Dr. Badylak said.
To explore this possibility, researchers used specialized proteins to break up the ECM, similar to the process that occurs when a bioscaffold becomes incorporated into the recipient’s tissue.
The research team then exposed two different cell types – immune cells and neuronal stem cells – to isolated matrix bound vesicles, finding that they caused both cell types to mimic their normal regrowth behaviors.
“Sure enough, we found that vesicles are embedded within the ECM. In fact, these bioscaffolds are loaded with these vesicles,” Dr. Badylak said. “This study showed us that the matrix bound vesicles are clearly active, can influence cellular behavior and are possibly the primary mechanism by which bioscaffolds cause tissue regrowth in the body.”
Researchers also found that vesicles isolated from different source tissues have distinct molecular signatures, and they are now focused on harnessing this new information for both therapeutic and diagnostic purposes.
Researchers at the Stanford University School of Medicine have mapped out the sets of biological and chemical signals necessary to quickly and efficiently direct human embryonic stem cells to become pure populations of any of 12 cell types, including bone, heart muscle and cartilage. The ability to make pure populations of these cells within days rather than the weeks or months previously required is a key step toward clinically useful regenerative medicine — potentially allowing researchers to generate new beating heart cells to repair damage after a heart attack or to create cartilage or bone to reinvigorate creaky joints or heal from trauma.
The study also highlights key, but short-lived, patterns of gene expression that occur during human embryo segmentation and confirms that human development appears to rely on processes that are evolutionarily conserved among many animals.
Nicotinamide riboside rejuvenates stem cells, allowing better regeneration processes in aged mice
Nicotinamide riboside (NR) is pretty amazing. It has already been shown in several studies to be effective in boosting metabolism. And now a team of researchers at EPFL’s Laboratory of Integrated Systems Physiology (LISP), headed by Johan Auwerx, has unveiled even more of its secrets. An article written by Hongbo Zhang, a PhD student on the team, was published today in Science and describes the positive effects of NR on the functioning of stem cells. These effects can only be described as restorative.
As mice, like all mammals, age, the regenerative capacity of certain organs (such as the liver and kidneys) and muscles (including the heart) diminishes. Their ability to repair them following an injury is also affected. This leads to many of the disorders typical of aging.
Mitochondria: also useful in stem cells
Hongbo Zhang wanted to understand how the regeneration process deteriorated with age. To do so, he teamed up with colleagues from ETH Zurich, the University of Zurich and universities in Canada and Brazil. Through the use of several markers, he was able to identify the molecular chain that regulates how mitochondria – the “powerhouse” of the cell – function and how they change with age. The role that mitochondria play in metabolism has already been amply demonstrated, “but we were able to show for the first time that their ability to function properly was important for stem cells,” said Auwerx.
Under normal conditions, these stem cells, reacting to signals sent by the body, regenerate damaged organs by producing new specific cells. At least in young bodies. “We demonstrated that fatigue in stem cells was one of the main causes of poor regeneration or even degeneration in certain tissues or organs,” said Hongbo Zhang.
A multidisciplinary research team discovers how cells know to rush to a wound and heal it — opening the door to new treatments for diabetes, heart disease and cancer
Researchers at the University of Arizona have discovered what causes and regulates collective cell migration, one of the most universal but least understood biological processes in all living organisms.
The findings, published in the March 13, 2015, edition of Nature Communications, shed light on the mechanisms of cell migration, particularly in the wound-healing process. The results represent a major advancement for regenerative medicine, in which biomedical engineers and other researchers manipulate cells’ form and function to create new tissues, and even organs, to repair, restore or replace those damaged by injury or disease.
“The results significantly increase our understanding of how tissue regeneration is regulated and advance our ability to guide these processes,” said Pak Kin Wong, UA associate professor of mechanical and aerospace engineering and lead investigator of the research.
“In recent years, researchers have gained a better understanding of the molecular machinery of cell migration, but not what directs it to happen in the first place,” he said. “What, exactly, is orchestrating this system common to all living organisms?”
Leaders of the Pack
The answer, it turns out, involves delicate interactions between biomechanical stress, or force, which living cells exert on one another, and biochemical signaling.
The UA researchers discovered that when mechanical force disappears — for example at a wound site where cells have been destroyed, leaving empty, cell-free space — a protein molecule, known as DII4, coordinates nearby cells to migrate to a wound site and collectively cover it with new tissue. What’s more, they found, this process causes identical cells to specialize into leader and follower cells. Researchers had previously assumed leader cells formed randomly.
Wong’s team observed that when cells collectively migrate toward a wound, leader cells expressing a form of messenger RNA, or mRNA, genetic code specific to the DII4 protein emerge at the front of the pack, or migrating tip. The leader cells, in turn, send signals to follower cells, which do not express the genetic messenger. This elaborate autoregulatory system remains activated until new tissue has covered a wound.
The same migration processes for wound healing and tissue development also apply to cancer spreading, the researchers noted. The combination of mechanical force and genetic signaling stimulates cancer cells to collectively migrate and invade healthy tissue.
Biologists have known of the existence of leader cells and the DII4 protein for some years and have suspected they might be important in collective cell migration. But precisely how leader cells formed, what controlled their behavior, and their genetic makeup were all mysteries — until now.
Broad Medical Applications
“Knowing the genetic makeup of leader cells and understanding their formation and behavior gives us the ability to alter cell migration,” Wong said.
With this new knowledge, researchers can re-create, at the cellular and molecular levels, the chain of events that brings about the formation of human tissue. Bioengineers now have the information they need to direct normal cells to heal damaged tissue, or prevent cancer cells from invading healthy tissue.
Scientists hope to give people back the use of disabled arms and legs after a world-first breakthrough allowing them to regrow the missing nerves in rats.
Trials of the technology are still in the early stages, but better-than-expected results raise prospects of saving limbs lost in accidents or eventually overcoming paraplegia and quadriplegia.
St Vincent’s Hospital researchers have built and implanted a bridge between severed nerves in areas too large and complex to be healed by conventional nerve grafts.
After successfully restoring the feeling and partial use of legs in rats missing the main nerve to their limbs, director of neurosciences Prof Mark Cook said new trials had begun to see how far the technology can allow nerves to regrow in the hope it can be expanded to humans studies in the next two to five years.
“We will have the potential to deal with a broader range and bigger injured nerves down the track,” Prof Cook said.
“Some of these gaps in humans are substantial. You can have long sections of nerves in humans because obviously their limbs are a lot longer than in animals such as rats.
“We started by focusing on the large peripheral nerves as it seems the best target and, once we have mastered that, we would be able to move on to the spinal chord. This worked better than we anticipated so we are really pleased it is something we can take to humans in the not-too-distant future.”
In conjunction with ARC Centre of Excellence for Electromaterials Science in Wollongong, the St Vincent’s team built a chamber made of polymer and containing a gel of growth factors, which is placed in a 1cm section of missing nerve to act as a bridge and restore contact between the brain and damaged sections of the body.
In recent trials the scientists were able to see rats regain partial and, when the chambers were removed after four weeks, discovered new nerves had grown to reconnect the disabled limbs to the brain.
Assoc Prof Robert Kapsa said the technology could mitigate the impact of injuries by restoring more feeling.
Researchers have generated functional hepatocytes from human stem cells, transplanted them into mice with acute liver injury, and shown the ability of these stem-cell derived human liver cells to function normally and increase survival of the treated animals.
This promising advance in the development of cell-based therapies to treat liver failure resulting from injury or disease relied on the development of scalable, reproducible methods to produce stem cell-derived hepatocytes in bioreactors, as described in an article in Stem Cells and Development, a peer-reviewed journal from Mary Ann Liebert, Inc., publishers. The article is available free on the Stem Cells and Development website.
Massoud Vosough and coauthors demonstrate a large-scale, integrated manufacturing strategy for generating functional hepatocytes in a single suspension culture grown in a scalable stirred bioreactor. In the article “Generation of Functional Hepatocyte-Like Cells from Human Pluripotent Stem Cells in a Scalable Suspension Culture” the authors describe the method used for scale-up, differentiation of the pluripotent stem cells into liver cells, and characterization and purification of the hepatocytes based on their physiological properties and the expression of liver cell biomarkers.
David C. Hay, MRC Centre for Regenerative Medicine, University of Edinburgh, U.K., comments on the importance of Vosough et al.’s contribution to the scientific literature in his editorial in Stem Cells and Development entitled “Rapid and Scalable Human Stem Cell Differentiation: Now in 3D.” The researchers “developed a system for mass manufacture of stem cell derived hepatocytes in numbers that would be useful for clinical application,” creating possibilities for future “immune matched cell based therapies,” says Hay. Such approaches could be used to correct mutated genes in stem cell populations prior to differentiation and transplantation, he adds.
“The elephant in the room for stem cell therapy rarely even acknowledged let alone addressed in the literature is that of scalable production of cells for translational application,” says Editor-in-Chief Graham C. Parker, PhD, research professor, Carman and Ann Adams Department of Pediatrics, Wayne State University School of Medicine. “Baharvand’s groups’ landmark publication not only demonstrates but exquisitely describes the methodology required to scale up stem cell populations for clinical application with a rigor to satisfy necessary manufacturing standards.”