Engineering the stuff of life

A person’s voice is one of their most defining characteristics, something voice disorder surgeon Nathan Welham knows only too well. “It’s an essential part of being human, the way we sound. How we recognise others and how we communicate with our loved ones is very much tied to our voice. So when that’s gone it’s very challenging for patients and when it’s restored it’s a big deal,” he says.

But for a subset of patients whose vocal cords have been ravaged by disease or removed there are few options. This prompted Welham, assistant professor at the University of Wisconsin School of Medicine and Public Health, to turn to tissue engineering. His team made headlines in November 2015 after engineering functional human vocal cord tissue in the laboratory.

The researchers harvested cells from donated human larynxes, before seeding them onto a specially designed 3D scaffold made from collagen – the main structural component of biological tissue. This was placed in a bioreactor – an oven-like device that simulates bodily conditions to promote growth – and once it reached the size of human vocal cords its was implanted in a dog larynx. Welham was surprised to find that not only did it vibrate like natural tissue but it even matched the sound made by human vocal cords. “We never anticipated this system might actually be useful as a therapy in its own right,” he says. While there’s a long way to go, the team believes the breakthrough could lead to practical treatments.

The success is the latest in a long line of advances in the field of bioengineering. Not only is the prospect of widely available lab-grown organs getting closer but engineers are bringing rational design to the inner workings of cells, while progress in materials and nanotechnology is rapidly improving our ability to manipulate biological systems.

Tissue factories

Welham is surprised at how much attention his work received considering recent break­throughs. Japanese researchers announced last September that they had grown tiny functional kidneys from stem cells, and last August, US scientists published a paper on fully-formed miniature human brains. In June 2015, a team from Massachusetts General Hospital in Boston grew an entire rat forelimb using decellularisation – where cells are chemically washed from a donor limb leaving only the collagen frame. This was re-seeded with various cell types before being coated with skin grafts. Electrical pulses made the paw clench and when grafted to a live rat blood circulated through the limb.

“It’s pretty impressive,” says Welham. “But it’s complicated because there are a lot of kinds of tissue, so I’m not sure how far that can be taken with current technology.”

Reproducibility and cost are big problems. Italian surgeon Paolo Macchiarini has been transplanting decellularised tracheas re-seeded with patients’ stem cells since 2008, but each one is hand-engineered and costs hundreds of thousands of pounds so only a handful of procedures have been carried out. Engineered skin has been available for burn victims for two decades, but skin is a flat structure made from a single cell type. Welham says hollow structures like blood vessels, tracheas and bladders are considerably more complex, while solid organs like livers are years from the clinic.

Anthony Atala, director of the Wake Forest Institute for Regenerative Medicine in North Carolina, hopes to transform this cottage industry. The surgeon has been working in tissue engineering for 25 years and transplanted the world’s first lab-grown bladder in 1999, but his main focus is now bioprinting. Using images from computed tomography (CT) scanners and computer-aided design his lab produces 3D models of organs before specialised 3D printers lay down cells a layer at a time using liquid cell suspensions called bio-inks. It’s complex, but Atala says they are making solid progress towards their long-term goal of printing entire human kidneys. “We realised for us to do this with a larger number of patients we needed machines to take what we’ve done by hand and automate the process,” he adds. “But it’s not just scalability we are looking for. It also offers precision and reproducibility.”

Life’s building blocks

While Atala studies nature’s macrostructures, others are focusing on the cells that put these macrostructures together. MIT’s Jim Collins is one of the founders of synthetic biology, which he says puts the engineering into genetic engineering. “Genetic engineering is introducing a gene from species A to species B,” he says. “That’s the equivalent of replacing a red light bulb with a green light bulb. Synthetic biology is focused on designing the underlying circuitry expressing that red or green light bulb.”

The cracking of the genetic code in the 1960s and subsequent advances in DNA sequencing and recombinant DNA technology in the 1970s opened the door to reading and writing life’s blueprint. The advent of high-throughput biology in the 1990s – which uses robotics and data analytics to automate this process – allowed scientists to start tweaking the genetic circuitry governing organisms’ characteristics and behaviour. Genetic engineering is now well-established and has brought us everything from pest-resistant crops to glow-in-the-dark rabbits. But the promise of synthetic biology is the ability to design biological systems from scratch.

Using analogies with electrical engineering, standardised DNA parts – sequences of genes with a specific function – are combined to build ‘cellular circuits’ with more complex functions like influencing cell development, engineering responses to environmental conditions or modifying cellular function. But the field has been “bedevilled by hype and wild promises”, says Collins. When it took off in the 2000s optimists saw it as a shortcut to bulk biofuels, industrial chemicals and drugs, but there are still only a few revenue-generating applications. “We are in the very early stages of the field. Our ability to engineer biological systems is very rudimentary,” Collins says.

As the field matured, focus switched to lower volume high-value compounds like pharmaceutical precursors, fragrances and flavourings. “Nature offers a huge array of valuable chemicals but sometimes only nature knows how to produce them,” says Matthew Chang, a synthetic biologist from the National University of Singapore. “It uses very sophisticated biological pathways to produce chemicals that are sometimes extremely hard to produce through chemical synthesis.”

Microbes have also been engineered to act as low-cost biosensors to detect chemicals in the environment or genetic markers for pathogens in medical samples. Collins’ lab has even freed these capabilities from the cell by freeze-drying synthetic gene circuits onto paper. These systems can be stored at room temperature and are activated by rehydration; removing the complications of introducing components into cells makes them easy to modify.

For Collins the most exciting near-term applications lie in engineering microbes to serve as living diagnostics and therapeutics. Bacteriophages – viruses that target bacteria – are popular tools. They can be engineered to provide diagnostic outputs like bioluminescence, but Collins’ lab has designed them to attack bacterial colonies with enzymes that degrade their protective biofilms or neutralise their ability to develop resistance. The technology is being developed for clinical applications by EnBiotix, which Collins helped found.

In a similar vein, Chang’s lab has reengineered E coli bacteria to detect and destroy the multi-?drug-resistant pathogen Pseudomonas aeruginosa. They have completed pre-clinical trials and are seeking funding for full clinical trials.

But neat analogies to electrical circuits can only go so far. “There aren’t any engineered systems that come close to even the smallest living cell,” says Collins. Advances in molecular biology have given scientists fine-?grained understanding of many cellular processes, but our comprehension of how they fit together is still basic. Interfering in biological systems often has unpredictable outcomes and bioengineering is as much trial and error as rational design, he says. And then there’s the elephant in the room – evolution. “Engineers like to build in designed features to a system. They don’t like the idea that you’ve got this external set of factors that harness randomness to modify the system,” he adds.

Problem-solving

These issues present problems, but that’s exactly what engineers can contribute to biology, says Kostas Kostarelos, who leads the Nanomedicine Lab at the University of Manchester. “I think it’s an innate capacity of engineers to solve problems. If medicine and biology have problems they back themselves to solve them,” he says. While synthetic biology’s approach is to manipulate the genetic basis of biological systems, Kostarelos belongs to another camp exploiting biological and synthetic materials, including nanomaterials, to directly influence biological processes.

In 1995 the anti-cancer drug-delivery system Doxil became the first nanomedicine to be approved by the US Food and Drug Administration. Cancer’s complexity and localised nature, and the toxicity of drugs used to treat it, have made it a major focus of nanomedicine.

Nanoparticles can be designed to target tumours, sense their local environment and deliver drugs in a specific sequence, says Ben Almquist, who leads a bioengineering lab at Imperial College London, while another promising approach is designing materials that recruit and reprogram immune cells to target tumours.

Kostarelos’s lab, based at Manchester’s Graphene Institute, is also investigating the medical potential of graphene and other 2D materials. Among their unique properties, these one-atom-thick materials have extremely high electron mobility, lending them to high-precision bio-sensing. “My lab is trying to clarify whether these unique properties translate into the context of a physiological environment,” says Kostarelos. Often they don’t because the environment affects the material’s morphology and chemistry, but many believe the technology holds great promise.

Traditionally bioengineers have made medical implants biologically inert due to the body’s immune response, but Almquist says materials are now being designed to interact with biological systems. Bioactive bone implant coatings have been shown to increase mechanical stability of the bone-implant interface by driving its growth and maturation, he says.

Almquist is particularly excited about ‘inorganic transmembrane proteins’. His lab is using semiconductor fabrication techniques to create silicon nanostructures that fuse into cell membranes just like natural membrane proteins, opening up new possibilities for interfacing silicon-based devices with cells, he says.

Scaling up

Good results in the lab mean little if they don’t translate to the clinic. Nanomedicine has had some success – a review in Nanomedicine: Nanotechnology, Biology and Medicine found 247 nanomedicine products had been approved by regulators or were undergoing clinical trials by 2013. Bioprinting, on the other hand, has yet to achieve the acceptance of other additive manufacturing technologies.

Dan Thomas, founder of 3Dynamic Systems, which sells a high-precision bioprinter called Omega, says the field faces big challenges: designing low-shear deposition mechanisms that maintain the viability of living cells; rapid biofabrication speeds to prevent nutrient or oxygen deprivation, and integration with specialised bioreactors. “Many of these technologies have yet to be invented,” he says.

Early applications are likely to be in engineering tissues for medical compound screening. Using bio-printed tissue rather than animals or humans could dramatically speed up development. Leading the way is US firm Organovo, which already provides pharmaceutical companies with bioprinted liver tissue for drug toxicity testing.

Synthetic biology on the other hand is carving out a niche as a design house for the biotech industry. John Cumbers, formerly a synthetic biology researcher at Nasa’s Ames Research Centre, is the founder of SynBioBeta – an ‘activity hub’ for synthetic biology providing networking, courses, consulting and conferences. He believes the field is about to take off thanks to automation of design processes. “The Wright brothers didn’t solve flying by mimicking birds flapping their wings,” he says. “They solved it by working out how to rapidly iterate through plane designs so they could fail early and fail often.”

This is the approach taken by Chain Biotech, based at the Imperial College Incubator in London. Founder Edward Green started eco-friendly chemicals company Green Biologics in 2003, but his new venture is focused on re-engineering the Clostridium microbe at the heart of his first company’s processes to broaden the chemicals it can produce. They have already automated every step of the design stages and are now working to integrate them into one seamless process. “We want to accelerate the strain development process so that rather than taking months or years it takes days,” says Green.

Boston-based Ginkgo Bioworks, which has built what it calls “the world’s first organism engineering foundry”, also uses automation to rapidly iterate through designs, but forward engineering is still an essential part of its work. “Brute force without insight doesn’t scale,” says Gingko’s creative director Christina Agapakis. The firm has raised $54m and has worked with the US Department of Energy to design microbes that convert greenhouse gases into valuable chemicals.

Ginkgo’s tagline is “replacing technology with biology” and Agapakis thinks this makes perfect sense. “Biology can do incredible things,” she says “It can self-assemble incredibly complex nanometre-scale systems; it can do incredible chemical transformations at ambient pressures and temperatures; and biology does this using renewable resources with no externalities. We can learn a lot from biology.”