An Advanced Method of DNA Nanostructure Formation Developed

Figure 1: Uni-molecular magnetic tweezers orchestrating the DNA nanostructure formation

Professor Tae-Young Yoon’s research team from the Department of Physics at KAIST has developed a new method to form DNA nanostructures by using magnetic tweezers to observe and to induce the formation of the structure in real time.

Unlike traditional designs of "DNA origami" which relies on thermal or chemical annealing methods, the new technology utilizes a completely different dynamic in DNA folding. This allows the folding to be done within only ten minutes.

Developed in 2006, the "DNA origami" allows a long skeleton of DNA to be folded into an arbitrary structure by using small stapler DNA pieces. This has been a prominent method in DNA nanotechnology.
 

Figure 2: The evolution of DNA nanostructure formation using magnetic tweezers. The DNA nanostructure with a 21-nanometer size was formed in about eight minutes.

However, the traditional technology which adopts thermal processes could not control the DNA formation during the folding because every interaction among DNAs occurs simultaneously. Thus, the thermal processes, which take dozens of hours to complete, had to be repeated multiple times in order to find the optimal condition.

The research team designed a DNA folding using uni-molecular magnetic tweezers that applied force to a single DNA molecule while measuring the state of the DNA. Through this technology, they were able to induce the formation of DNA nanostructure and observe it at the same time.

During high temperature heat treatment, the first stage of conventional thermal processes, the internal structure of the long skeleton DNA untangles. To induce such state, after attaching one side of the skeleton DNA to the surface of glass and the other side to a magnetic material, the team unfolded the internal structure of the DNA by pulling the two sides apart with magnetic force.

Unlike the conventional thermal processes, this method lets the stapler DNA swiftly adhere to the skeleton DNA within a minute because the sites are revealed at room temperature.

After the stapler pieces connected to the skeleton, the team removed the magnetic force. Next, the structure folded through self-assembly as the stapler DNAs stuck to different sites on the skeleton DNA.

Professor Yoon said, “With the existing thermal methods, we could not differentiate the reactions of the DNA because the response of each DNA pieces mutually interacted with each other.” He added that “Using the magnetic tweezers, we were able to sort the process of DNA nanostructure formation into a series of reactions of DNA molecules that are well known, and shorten the time taken for formation in only ten minutes.”

He commented, “This nanostructure formation method will enable us to create more intricate and desirable DNA nanostructures by programming the folding of DNA origami structures.”

Conducted by Dr. Woori Bae under the guidance of Professor Yoon, the research findings were published online in the December 4th issue of Nature Communications.


Source: KAIST

New technology may identify tiny strains in body tissues before injuries occur

The top image shows how the new algorithm is able to identify an area (in red) where stress has created a weak spot in a small piece of plastic wrap. The older method (shown in the bottom half of the picture) is unable to pinpoint the place where the plastic wrap is weakening.
Credit: John Boyle, © The Royal Society (used with permission)

Researchers at Washington University in St. Louis have developed algorithms to identify weak spots in tendons, muscles and bones prone to tearing or breaking. The technology, which needs to be refined before it is used in patients, one day may help pinpoint minor strains and tiny injuries in the body's tissues long before bigger problems occur.

The research is available online Aug. 27 in the Journal of the Royal Society Interface, which publishes research at the nexus of the physical and life sciences.

"Tendons are constantly stretching as muscles pull on them, and bones also bend or compress as we carry out everyday activities," said senior investigator Stavros Thomopoulos, PhD, professor of orthopaedic surgery. "Small cracks or tears can result from these loads and lead to major injuries. Understanding how these tears and cracks develop over time therefore is important for diagnosing and tracking injuries."

To that end, Thomopoulos and his colleagues developed a way to visualize and even predict spots where tissues are weakened. To accomplish this, they stretched tissues and tracked what happened as their shapes changed or became distorted.

The paper's first author, John J. Boyle, a graduate student in biomedical engineering, combined mechanical engineering fundamentals with image-analysis techniques to create the algorithms, which were tested in different materials and in animal models.

"If you imagine stretching Silly Putty or a swimming cap with a picture on it, as you pull, the picture becomes distorted," Boyle said. "This allows us to track how the material responds to an external force."

In one of the experiments described in the paper, Boyle sprayed a pattern of dots on plastic wrap, stretched it and tracked the dots.

"As you pull and stretch the plastic wrap, eventually tears begin to emerge," he explained. 

"The new algorithm allowed us to find the places where the tears were beginning to form and to track them as they extended. Older algorithms are not as good at finding and tracking localized strains as the material stretches."

In fact, one of the two new algorithms is 1,000 times more accurate than older methods at quantifying very large stretches near tiny cracks and tears, the research showed. And a second algorithm has the ability to predict where cracks and failures are likely to form.

"This extra accuracy is critical for quantifying large strains," said Guy Genin, PhD, professor of mechanical engineering and co-senior investigator on the study. "Commercial algorithms that estimate strain often are much less sensitive, and they are prone to detecting noise that can arise from the algorithm itself rather than from the material being examined. The new algorithms can distinguish the noise from true regions of large strains."

Thomopoulos, who also is a professor of biomedical engineering and of mechanical engineering, works with Genin to study the shoulder's rotator cuff, a group of tendons and muscles that connect the upper arm to the shoulder blade. They want to learn why some surgeries to repair rotator cuff injuries ultimately fail. Their goal is to increase the odds that the tissue in the shoulder will heal following surgery, and they believe the new algorithms could help them get closer to that goal.

How soon the new algorithms could be used in patients depends on getting better images of the body's tissues. Current imaging techniques, such as MRI and ultrasound, lack the required clarity and resolution.

Genin also explained that although the goal of the current study is to better understand how forces at work on human tissue cause injury and stress, the algorithms also could help engineers identify vulnerable parts of buildings and other structures. Our muscles and bones, he said, are influenced by the same strains that affect those structures.

"Whether it's a bridge or a tendon, it's vital to understand the ways that physical forces cause structures and tissues to deform so that we can identify the onset of failures and eventually predict them," he said.

In the long run, they want to use the algorithms to prevent additional injuries following surgery to repair knees, shoulders and other tissues. They also said it may be possible some day to predict problems before they occur.

The group, which applied for a provisional patent earlier this year, hopes the algorithms will be useful to researchers in the medical and engineering fields.

As a piece of plastic wrap is stretched, the new algorithms identify the location (in red) where it is weakening, which is where the material eventually breaks.

Source: Washington University in St. Louis

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

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