Scientists grow cartilage to reconstruct nose

Made from a probe of the nasal septum: white, glossy cartilage was made in the laboratory.
Credit: Department of Biomedicine at the University of Basel

Scientists at the University of Basel report first ever successful nose reconstruction surgery using cartilage grown in the laboratory. Cartilage cells were extracted from the patient's nasal septum, multiplied and expanded onto a collagen membrane. The so-called engineered cartilage was then shaped according to the defect and implanted. The results will be published in the current edition of the academic journal The Lancet.

A research team from the University of Basel in Switzerland has reported that nasal reconstruction using engineered cartilage is possible. They used a method called tissue engineering where cartilage is grown from patients' own cells. This new technique was applied on five patients, aged 76 to 88 years, with severe defects on their nose after skin cancer surgery. One year after the reconstruction, all five patients were satisfied with their ability to breathe as well as with the cosmetic appearance of their nose. None of them reported any side effects.

Cells from the nasal septum

The type of non-melanoma skin cancer investigated in this study is most common on the nose, specifically the alar wing of the nose, because of its cumulative exposure to sunlight. To remove the tumor completely, surgeons often have to cut away parts of cartilage as well. Usually, grafts for reconstruction are taken from the nasal septum, the ear or the ribs and used to functionally reconstruct the nose. However, this procedure is very invasive, painful and can, due to the additional surgery, lead to complications at the site of the excision.

Together with colleagues from the University Hospital, the research team from the Department of Biomedicine at the University of Basel has now developed an alternative approach using engineered cartilage tissue grown from cells of the patients' nasal septum. 

They extracted a small biopsy, isolated the cartilage cells (chondrocytes) and multiplied them. The expanded cells were seeded onto a collagen membrane and cultured for two additional weeks, generating cartilage 40 times the size of the original biopsy. The engineered grafts were then shaped according to the defect on the nostril and implanted.

New possibilities for facial reconstruction

According to Ivan Martin, Professor for Tissue Engineering at the Department of Biomedicine at the University and University Hospital of Basel, "The engineered cartilage had clinical results comparable to the current standard surgery. This new technique could help the body to accept the new tissue better and to improve the stability and functionality of the nostril. Our success is based on the long-standing, effective integration in Basel between our experimental group at the Department of Biomedicine and the surgical disciplines at the University Hospital. The method opens the way to using engineered cartilage for more challenging reconstructions in facial surgery such as the complete nose, eyelid or ear."

The same engineered grafts are currently being tested in a parallel study for articular cartilage repair in the knee. Despite the optimistic perspectives, the use of these procedures in the clinical practice is still rather distant. "We need rigorous assessment of efficacy on larger cohorts of patients and the development of business models and manufacturing paradigms that will guarantee cost-effectiveness," says Martin.

Source: University of Basel

Charged graphene gives DNA a stage to perform molecular gymnastics

DNA interacts with charged graphene and contorts into sequence-specific shapes when the charge is changed. Credit: Photo courtesy Alek Aksimentiev

When Illinois researchers set out to investigate a method to control how DNA moves through a tiny sequencing device, they did not know they were about to witness a display of molecular gymnastics.

Fast, accurate and affordable DNA sequencing is the first step toward personalized medicine. Threading a DNA molecule through a tiny hole, called a nanopore, in a sheet of graphene allows researchers to read the DNA sequence; however, they have limited control over how fast the DNA moves through the pore. In a new study published in the journal Nature Communications, University of Illinois physics professor Aleksei Aksimentiev and graduate student Manish Shankla applied an electric charge to the graphene sheet, hoping that the DNA would react to the charge in a way that would let them control its movement down to each individual link, or nucleotide, in the DNA chain.

"Ideally, you would want to step the DNA through the nanopore one nucleotide at a time," said Aksimentiev. "Take a measurement and then have another nucleotide in the sensing hole. That's the goal, and it hasn't been realized yet. We show that, to some degree, we can control the process by charging the graphene."

The researchers found that a positive charge in the graphene speeds up DNA movement through the nanopore, while a negative charge stops the DNA in its tracks. However, as they watched, the DNA seemed to dance across the graphene surface, pirouetting into shapes they had never seen, specific to the sequence of the DNA nucleotides.

"It reminds me of Swan Lake," Aksimentiev said. "It's very acrobatic. We were very surprised by the variety of DNA conformations that we can observe at the surface of graphene when we charge it. There is one sequence that starts out laying down on the surface, and when we change the charge, they all tilt on the side like they are doing a one-armed push-up. Then we also have nucleotides that would lay back, or go up like a ballerina en pointe."

Aksimentiev hypothesizes that the conformations are so different and so specific to the sequence because each nucleotide has a slightly different distribution of electrons, the negatively charged parts of the atoms. There is even a visible difference when a nucleotide is methylated, a tiny chemical change that can turn a gene on or off.

By switching the charge in the graphene, the researchers can control not only the DNA's motion through the pore, but also the shape the DNA contorts into.

"Because it's reversible, we can force it to adopt one conformation and then force it to go back. That's why we call it gymnastics," Aksimentiev said.

The researchers extensively used the Blue Waters supercomputer at the National Center for Supercomputing Applications, housed at the University of Illinois. They mapped each individual atom in the complex DNA molecule and ran numerous simulations of many different DNA sequences. Supercomputing power was essential to carrying out the work, Aksimentiev said.

"This is a really computationally intensive project," he said. "Having access to Blue Waters was essential because with the sheer number of simulations, we would not have been able to finish them. It would have taken too long."

The next step is to combine a charged nanopore setup with a sensor to build a DNA sequencing device that would incorporate both motion control and nucleotide recognition. The researchers also hope to explore the unexpected conformational changes for insights into epigenetics, the field that studies how genes are expressed and moderated.

"DNA is much more complicated than just a double helix. It's a complex molecule that has many properties, and we are still uncovering them," Aksimentiev said.

Video animation of DNA dancing as the graphene charge changes:


Source: University of Illinois at Urbana-Champaign

Genetic factors behind surviving or dying from Ebola shown in mouse study

In an emerging disease research lab at the University of Washington, Chris Williams, a research scientist who specializes in microbiology laboratory robotics, programs a piece of equipment that can be programmed to performs many lab tasks. Credit: Brian Donohue

A newly developed mouse model suggests that genetic factors are behind the mild-to-deadly range of reactions to the Ebola virus.

People exposed to Ebola vary in how the virus affects them. Some completely resist the disease, others suffer moderate to severe illness and recover, while those who are most susceptible succumb to bleeding, organ failure and shock.

In earlier studies of populations of people who have contracted Ebola, these differences are not related to any specific changes in the Ebola virus itself that made it more or less dangerous; instead, the body's attempts to fight infection seems to determine disease severity.

In the Oct. 30 edition of Science, scientists describe strains of laboratory mice bred to test the role of an individual's genetic makeup in the course of Ebola disease. Systems biologists and virologists Angela Rasmussen and Michael Katze from the Katze Laboratory at the University of Washington Department of Microbiology led the study in collaboration with the National Institutes of Health's Rocky Mountain Laboratories in Montana and University of North Carolina at Chapel Hill.

Research on Ebola prevention and treatment has been hindered by the lack of a mouse model that replicates the main characteristics of human Ebola hemorrhagic fever. The researchers had originally obtained this genetically diverse group of inbred laboratory mice to study locations on mouse genomes associated with influenza severity.

The research was conducted in a highly secure, state-of-the-art biocontainment safety level 4 laboratory in Hamilton, Mont. The scientists examined mice that they infected with a mouse form of the same species of Ebola virus causing the 2014 West Africa outbreak. The study was done in full compliance with federal, state, and local safety and biosecurity regulations. This type of virus has been used several times before in research studies. Nothing was done to change the virus.

Interestingly, conventional laboratory mice previously infected with this virus died, but did not develop symptoms of Ebola hemorrhagic fever.

In the present study, all the mice lost weight in the first few days after infection. Nineteen percent of the mice were unfazed. They not only survived, but also fully regained their lost weight within two weeks. They had no gross pathological evidence of disease. Their livers looked normal.

Eleven percent were partially resistant and less than half of these died. Seventy percent of the mice had a greater than 50 percent mortality. Nineteen percent of this last group had liver inflammation without classic symptoms of Ebola, and thirty-four percent had blood that took too long to clot, a hallmark of fatal Ebola hemorrhagic fever in humans. Those mice also had internal bleeding, swollen spleens and changes in liver color and texture.

The scientists correlated disease outcomes and variations in mortality rates to specific genetic lines of mice.

"The frequency of different manifestations of the disease across the lines of these mice screened so far are similar in variety and proportion to the spectrum of clinical disease observed in the 2014 West African outbreak," Rasmussen said.

While acknowledging that recent Ebola survivors may have had immunity to this or a related virus that saved them during this epidemic, Katze said, "Our data suggest that genetic factors play a significant role in disease outcome."

In general, when virus infection frenzied the genes involved in promoting blood vessel inflammation and cell death, serious or fatal disease followed. On the other hand, survivors experienced more activity in genes that order blood vessel repair and the production of infection-fighting white blood cells.

The scientists note that certain specialized types of cells in the liver could also have limited virus reproduction and put a damper on systemic inflammation and blood clotting problems in resistant mice. Susceptible mice had widespread liver infection, which may explain why they had more virus in their bodies and poorly regulated blood coagulation. The researchers also noticed that spleens in the resistant and susceptible mice took alternate routes to try to ward off infection.

"We hope that medical researchers will be able to rapidly apply these findings to candidate therapeutics and vaccines," Katze said. They believe this mouse model can be promptly implemented to find genetic markers, conduct meticulous studies on how symptoms originate and take hold, and evaluate drugs and that have broad spectrum anti-viral activities against all Zaire ebolaviruses, including the one responsible for the current West African epidemic.

Source: University of Washington Health Sciences/UW Medicine

Visualizing DNA double-strand break process for the first time

The enzyme I-DmoI (purple) is specifically associated to the double strand of DNA (yellow and green). Credit: CNIO

Scientists from the Spanish National Cancer Research Centre (CNIO), led by Guillermo Montoya, have developed a method for producing biological crystals that has allowed scientists to observe --for the first time-- DNA double chain breaks. They have also developed a computer simulation that makes this process, which lasts in the order of millionths of a second, visible to the human eye. The study is published today by the journal Nature Structural & Molecular Biology.

"We knew that enzymes, or proteins, endonucleases, are responsible for these double strand breaks, but we didn't know exactly how it worked until now," said Montoya. "In our study, we describe in detail the dynamics of this basic biological reaction mediated by the enzyme I-Dmol. Our observations can be extrapolated to many other families of endonucleases that behave identically."

DNA breaks occur in several natural processes that are vital for life: mutagenesis, synthesis, recombination and repair. In the molecular biology field, they can also be generated synthetically. Once the exact mechanism that produces these breaks has been uncovered, this knowledge can be used in multiple biotechnological applications: from the correction of mutations to treat rare and genetic diseases, to the development of genetically modified organisms.

Slow-motion reaction

Enzymes are highly specialised dynamic systems. Their nicking function could be compared, said Montoya, to a specially designed fabric-cutting machine that "it would only make a cut when a piece of clothing with a specific combination of colours passed under the blade."

In this case, researchers concentrated on observing the conformational changes that occurred in the I-Dmol active site; the area that contains the amino acids that act as a blade and produces DNA breaks.

By altering the temperature and pH balance, the CNIO team has managed to delay a chemical reaction that typically occurs in microseconds by up to ten days. Under those conditions, they have created a slow-motion film of the whole process.

"By introducing a magnesium cation we were able to trigger the enzyme reaction and subsequently to produce biological crystals and freeze them at -200ºC," said Montoya. "In that way, we were able to collect up to 185 crystal structures that represent all of the conformational changes taking place at each step of the reaction."

Finally, using computational analysis, the researchers illustrated the seven intermediate stages of the DNA chain separation process. "It is very exciting, because the elucidation of this mechanism will give us the information we need to redesign these enzymes and provide precise molecular scissors, which are essential tools for modifying the genome," he concluded.

Source: Centro Nacional de Investigaciones Oncologicas (CNIO)

On a safari through the genome: Genes offer new insights into the distribution of giraffes

Three young, male Angola giraffes. Credit: © Julian Fennessy, GCF

The Giraffe (Giraffa camelopardalis), a symbol of the African savanna and a fixed item on every safari's agenda, is a fascinating animal. However, contrary to many of the continent's other wild animals, these long-necked giants are still rather poorly studied. Based on their markings, distribution and genome, nine subspecies are recognized -- including the two subspecies Angola Giraffe (Giraffa c. angolensis) and South African Giraffe (Giraffa c. giraffa).

South African Giraffes occur farther north than previously assumed

Like most other giraffes, these subspecies are now mainly found in nature reserves. Until recently, scientists assumed a clear demarcation of their ranges: Angola Giraffes occur in Namibia and northern Botswana, while South African Giraffes reside in southern Botswana and South Africa. "However, according to our studies, the distribution areas prove to be much more complex. South African Giraffes also occur in northeastern Namibia and northern Botswana, and Angola Giraffes can be found in northwestern Namibia and southern Botswana, as well," explains the study's author, Friederike Bock from the Biodiversity and Climate Research Center (BiK-F). A look at the new distribution map reveals the presence of a population of Angola Giraffes in the Central Kalahari Game Reserve, the world's second-largest national park, quasi nestled between two populations of the South African Giraffe, with both subspecies living side by side.

Subspecies were the result of early geographic separation

According to the research team, the fact that two genetically distinct subspecies could develop within the same region may be explained by the local geographic conditions that prevailed approximately 500,000 to two million years ago. Back then, the mountain range along the East African Rift Valley was sinking, creating vast wetlands and lakes, such as the paleo lake Makgadikgadi. According to Professor Dr. Axel Janke from the BiK-F, "these large bodies of water may have separated the populations for long periods of time. Moreover, female giraffes likely do not migrate across long distances, thereby contributing to a clear separation of the maternal lines." Today, there no longer exist any barriers that prevent the possible mingling of both subspecies; an investigation of these processes is however subject to further genetic analyses.

Angola and South African Giraffes can be uniquely identified by their maternal gene profile

For the study, the researchers created a profile of the subspecies' mitochondrial DNA, using tissue samples from about 160 giraffes from various populations across the entire African continent. On the basis of this genetic material, inherited from the maternal side, the often similarly marked subspecies can be uniquely identified genetically and the relationships between various populations can be clearly demonstrated. "Our focus was on giraffes in southern Africa, in particular in Botswana and South Africa. There, we sampled populations that had not been genetically analyzed before," says Bock.

New insights enable improved protection measures for the giraffe

According to estimates by the World Conservation Organization IUCN, the world's giraffe population is about 100,000 individuals -- showing a decreasing trend. In Botswana alone, the population has dwindled by more than half in recent years. In order to achieve effective protection measures that will preserve the majority of the giraffe's subspecies, it is indispensable to gain knowledge that allows their reliable identification as well as detailed information regarding their distribution. The surprising results concerning the distribution of the two subspecies in Namibia and Botswana emphasize the importance of additional taxonomic research on all giraffe subspecies.

Source: Senckenberg Research Institute and Natural History Museum

Controlling genes with your thoughts

Thoughts control a near-infrared LED, which starts the production of a molecule in a reaction chamber. Credit: Martin Fussenegger et al., Copyright ETH Zurich

It sounds like something from the scene in Star Wars where Master Yoda instructs the young Luke Skywalker to use the force to release his stricken X-Wing from the swamp: Marc Folcher and other researchers from the group led by Martin Fussenegger, Professor of Biotechnology and Bioengineering at the Department of Biosystems (D-BSSE) in Basel, have developed a novel gene regulation method that enables thought-specific brainwaves to control the conversion of genes into proteins -- called gene expression in technical terms.

"For the first time, we have been able to tap into human brainwaves, transfer them wirelessly to a gene network and regulate the expression of a gene depending on the type of thought. Being able to control gene expression via the power of thought is a dream that we've been chasing for over a decade," says Fussenegger.

A source of inspiration for the new thought-controlled gene regulation system was the game Mindflex, where the player wears a special headset with a sensor on the forehead that records brainwaves. The registered electroencephalogram (EEG) is then transferred into the playing environment. The EEG controls a fan that enables a small ball to be thought-guided through an obstacle course.

Wireless Transmission to Implant

The system, which the Basel-based bioengineers recently presented in the journal Nature Communications, also makes use of an EEG headset. The recorded brainwaves are analysed and wirelessly transmitted via Bluetooth to a controller, which in turn controls a field generator that generates an electromagnetic field; this supplies an implant with an induction current.

A light then literally goes on in the implant: an integrated LED lamp that emits light in the near-infrared range turns on and illuminates a culture chamber containing genetically modified cells. When the near-infrared light illuminates the cells, they start to produce the desired protein.

Thoughts Control Protein Quantity

The implant was initially tested in cell cultures and mice, and controlled by the thoughts of various test subjects. The researchers used SEAP for the tests, an easy-to-detect human model protein which diffuses from the culture chamber of the implant into the mouse's bloodstream.

To regulate the quantity of released protein, the test subjects were categorised according to three states of mind: bio-feedback, meditation and concentration. Test subjects who played Minecraft on the computer, i.e. who were concentrating, induced average SEAP values in the bloodstream of the mice. When completely relaxed (meditation), the researchers recorded very high SEAP values in the test animals. For bio-feedback, the test subjects observed the LED light of the implant in the body of the mouse and were able to consciously switch the LED light on or off via the visual feedback. This in turn was reflected by the varying amounts of SEAP in the bloodstream of the mice.

New Light-sensitive Gene Construct

"Controlling genes in this way is completely new and is unique in its simplicity," explains Fussenegger. The light-sensitive optogenetic module that reacts to near-infrared light is a particular advancement. The light shines on a modified light-sensitive protein within the gene-modified cells and triggers an artificial signal cascade, resulting in the production of SEAP. Near-infrared light was used because it is generally not harmful to human cells, can penetrate deep into the tissue and enables the function of the implant to be visually tracked.

The system functions efficiently and effectively in the human-cell culture and human-mouse system. Fussenegger hopes that a thought-controlled implant could one day help to combat neurological diseases, such as chronic headaches, back pain and epilepsy, by detecting specific brainwaves at an early stage and triggering and controlling the creation of certain agents in the implant at exactly the right time.

Source: ETH Zurich

Making lab-grown tissues stronger

Connective tissues like cartilage are made of cross-linked bundles of collagen fibers. UC Davis biomedical engineers have discovered that reducing oxygen or adding an enzyme called LOX can make these bundles stronger. The technique can be used to strengthen both natural cartilage kept in the lab for transplant, and artificial cartilage grown in culture. Credit: Eleftherios Makris and Kyriacos Athanasiou, UC Davis

Lab-grown tissues could one day provide new treatments for injuries and damage to the joints, including articular cartilage, tendons and ligaments.

Cartilage, for example, is a hard material that caps the ends of bones and allows joints to work smoothly. UC Davis biomedical engineers, exploring ways to toughen up engineered cartilage and keep natural tissues strong outside the body, report new developments this week in the journal Proceedings of the National Academy of Sciences.

"The problem with engineered tissue is that the mechanical properties are far from those of native tissue," said Eleftherios Makris, a postdoctoral researcher at the UC Davis Department of Biomedical Engineering and first author on the paper. Makris is working under the supervision of Professor Kyriacos A. Athanasiou, a distinguished professor of biomedical engineering and orthopedic surgery, and chair of the Department of Biomedical Engineering.

While engineered cartilage has yet to be tested or approved for use in humans, a current method for treating serious joint problems is with transplants of native cartilage. But it is well known that this method is not sufficient as a long-term clinical solution, Makris said.
The major component of cartilage is a protein called collagen, which also provides strength and flexibility to the majority of our tissues, including ligaments, tendons, skin and bones. Collagen is produced by the cells and made up of long fibers that can be cross-linked together.

Engineering new cartilage

Researchers in the Athanasiou group have been maintaining native cartilage in the lab and culturing cartilage cells, or chondrocytes, to produce engineered cartilage.

"In engineered tissues the cells produce initially an immature matrix, and the maturation process makes it tougher," Makris said.

Knee joints are normally low in oxygen, so the researchers looked at the effect of depriving native or engineered cartilage of oxygen. In both cases, low oxygen led to more cross-linking and stronger material. They also found that an enzyme called lysyl oxidase, which is triggered by low oxygen levels, promoted cross-linking and made the material stronger.

"The ramifications of the work presented in the PNAS paper are tremendous with respect to tissue grafts used in surgery, as well as new tissues fabricated using the principles of tissue engineering," Athanasiou said. Grafts such as cadaveric cartilage, tendons or ligaments -- notorious for losing their mechanical characteristics in storage -- can now be treated with the processes developed at UC Davis to make them stronger and fully functional, he said.
Athanasiou also envisions that many tissue engineering methods will now be altered to take advantage of this strengthening technique.

Source: University of California - Davis

First step: From human cells to tissue-engineered esophagus

This images shows a tissue-engineered esophagus. Credit: The Saban Research Institute

In a first step toward future human therapies, researchers at The Saban Research Institute of Children's Hospital Los Angeles have shown that esophageal tissue can be grown in vivo from both human and mouse cells. The study has been published online in the journal Tissue Engineering, 
Part A.

The tissue-engineered esophagus formed on a relatively simple biodegradable scaffold after the researchers transplanted mouse and human organ-specific stem/progenitor cells into a murine model, according to principal investigator Tracy C. Grikscheit, MD, of the Developmental Biology and Regenerative Medicine program of The Saban Research Institute and pediatric surgeon at Children's Hospital Los Angeles.

Progenitor cells have the ability to differentiate into specific types of cell, and can migrate to the tissue where they are needed. Their potential to differentiate depends on their type of "parent" stem cell and also on their niche. The tissue-engineering technique discovered by the CHLA researchers required only a simple polymer to deliver the cells, and multiple cellular groupings show the ability to generate a replacement organ with all cell layers and functions.

"We found that multiple combinations of cell populations allowed subsequent formation of engineered tissue. Different progenitor cells can find the right 'partner' cell in order to grow into specific esophageal cell types -- such as epithelium, muscle or nerve cells -- and without the need for exogenous growth factors. This means that successful tissue engineering of the esophagus is simpler than we previously thought," said Grikscheit.

She added that the study shows promise for one day applying the process in children who have been born with missing portions of the organ, which carries food, liquids and saliva from the mouth to the stomach. The process might also be used in patients who have had esophageal cancer -- the fastest growing type of cancer in the U.S. -- or otherwise damaged tissue, for example from accidentally swallowing caustic substances.

"We have demonstrated that a simple and versatile, biodegradable polymer is sufficient for the growth of tissue-engineered esophagus from human cells," added Grikscheit. "This not only serves as a potential source of tissue, but also a source of knowledge, as there are no other robust models available for studying esophageal stem cell dynamics. Understanding how these cells might behave in response to injury and how various donor cell types relate could expand the pool of potential donor cells for engineered tissue."

Additional contributors include first author Ryan G. Spurrier, MD, Allison L. Speer, MD, Xiaogang Hou, PhD and Wael N. El-Nachef, MD, of The Saban Research Institute of Children's Hospital Los Angeles. The study was supported by grants from the California Institute for Regenerative Medicine.

Source: Children's Hospital Los Angeles Saban Research Institute