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Expanded web feature: Protein 'spaghetti': a new approach to tissue engineering

A novel biologically-inspired track for the production of synthetic hydrogels

Autumn 2009

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Researchers at the University of Bristol have taken a novel biologically-inspired track for the production of synthetic biocompatible hydrogels, which could be used to grow new tissues tailored to the needs of individual patients.

The term ‘biocompatible hydrogel’ is always likely to struggle to make it as a household name. In many ways this just isn’t fair. We scrub up in shower gels made from these structures, and then add moisturisers, creams and fragrances to our bodies that are also set in hydrogels.

But hydrogels are much more than the carrier donkeys of the cosmetics industry. They deliver drugs inside melting capsules and warming patches; scaffolds can be constructed to culture cells, over which novel drugs can be tested to reduce animal testing. Within years, scientists hope to use to hydrogels grow new tissues such as nerves, blood vessels and cartilage, tailored to the needs of individual patients.

Traditionally, hydrogels have been made from inexpensive synthetic polymers, or recovered from natural sources such as seaweed. However, BBSRC-funded researchers at the University of Bristol led by Professor Dek Woolfson have taken an alternative route.

“To make hydrogels you need something long and thin that will interact with copies of itself and form meshes, but is also water soluble,” says Woolfson. “However, rather than using natural proteins, which are complex, we’ve tried to make something as simple as possible that we fully understand using peptides and self-assembling proteins.”

Woolfson says the ultimate goal is to try and build structures and eventually tissues that mimic the extracellular matrix from the bottom up. But this type of rational design means you have to understand the physical chemistry of every building block, from amino acid to peptide to protein, and engineer them from scratch.

 

 

Spaghetti junction

At this junction, Woolfson’s lab has taken a different road to other researchers. His group make proteins from alpha helices; one of the fundamental ways that strings of amino acids fold, rather than the beta-pleated sheets more commonly investigated. “Ten years ago we were just interested in what kind of assemblies you can make from protein blocks,” says Woolfson. “Because many others were looking into [the beta-structured] amyloid, we decided to be different and use the alpha helix.” After their first publication of note in 2000, his group has led the way in this area.

Over the next decade, Woolfson’s team tinkered with their alpha helices, placing the water-loving (hydrophilic) and water-repelling (hydrophobic) amino acids on different sides to make amphipathic helices. By then making the chain ends unequal, the properties of these ‘sticky ends’ cause the short protein filaments to join together like Lego bricks, creating longer strings known as a supramolecular structures (ref 1, ref 2).

Surprisingly, these self-repeating supramolecular fibres thickened as well as lengthened. Woolfson realised that they could promote or destroy this crystallisation and this way manage the tendency of the filaments to form fatter fibres. The result was, Woolfson says, filaments that weren’t strong enough to crystallise but could interact with each other to form networks of long fibres that looked like over-cooked spaghetti (ref 3).

Woolfson hopes that the resulting structures, hydrogelating self-assembling fibres (hSAFs), are well worth dining out on. But the practical applications won’t be found in shampoos or drug delivery. “The downside of using peptides or proteins is that they are expensive compared with the synthetic polymers,” he says. “We’re almost certainly looking at high-end biomedical applications.”

Taking lessons from nature

And this means constructing novel 3-dimensional scaffolds to grow cells, which is crucial to properly understanding how they behave in living systems. “We’re trying to mimic biology by growing cells in 3 dimensions as you might find in tissue,” says Woolfson. “There’s a lot of evidence that cells behave differently in 3 dimensions to 2 dimensions and all the experiments you can imagine doing in a 2-dimensional tissue culture you can imagine doing again with a good 3-dimensional scaffold.”

Now that living tissues are being added to the system, Woolfson says he is happy to have taken the alpha-helix route because we have far superior abilities to design alpha helices than beta-structured systems.

Woolfson’s co-author Martin Birchall, who was part of the team that carried out the first transplant of an organ (windpipe) grown from stem cells (see ‘Trachea changes’), also favours the alpha helix. “Of concern to me as a clinician is that there are a number of well-known diseases associated with beta-pleated sheets that are serious, including Alzheimer’s, Parkinson’s and Huntington’s disease,” says Birchall. “There are good a priori reasons to get away from them toward something we can manipulate better but doesn’t have the theoretical potential to have unwanted diseases.”

Woolfson adds that his hSAFs are not derived from ex-vivo materials like bovine collagen, or Matrigel, a gel used for cell culture derived from mouse tumours widely-used in industry. “We’re trying to find an alternative to synthetic and ex-vivo amyloid-like materials.”

Although Woolfson’s hSAFs are synthetic, an approach that can bring its own toxicity problems, he hopes they will one day be made by engineered bacteria. “Another beauty of our system is that it’s proteinogenic, which means that you can go into this new area of synthetic biology and make them using recombinant DNA.”

The synthetic biology approach could bring real advantages. Cell scaffolds are often ‘decorated’ with molecules such as growth factors. But if the scaffold is created by bacteria, desirable compounds such as hormones or cytokines could be fused into the substrate as they are manufactured. That could bring a further level of control and specificity to tissue culture; the kind of manipulability required to grow new organs and tissues matched to a person’s genetic or aesthetic needs. 

Trachea changes

University of Bristol surgeon Professor Martin Birchall made history in 2008 when, along with colleagues from Barcelona, he provided 30-year-old Claudia Castillo with a new windpipe, restoring her ability to breathe that had been destroyed by a severe case or tuberculosis – an operation that allowed her to once again read stories to her two children.

Professor Birchall and colleagues performed the first transplant of an organ made from the patient’s own stem cells

The patient’s own bone marrow stem cells were harvested, replicated, and then matured into cartilage cells using an adapted technique originally devised by BBSRC-funded scientist Professor Anthony Hollander, also at the University of Bristol.

The cartilage cells were then placed over a decellularised trachea from a dead human donor which acted as a protein scaffold for the stem cells to grow on. It worked, but Birchall says that donors are in short supply and might not be the right fit. “It’s a very lengthy process. It would be far better if in future we could have highly responsive biomaterials in which we can embed cells of all sorts.”

Birchall also says the alpha-helix system is preferable to the beta-pleated sheets, which is limited in the number of ‘messages’ that can be built in to persuade cells to grow or differentiate. Another advantage is biodegradability which could be useful for nerve regeneration when it would be desirable for the scaffold to disintegrate after a certain time.

It’s technology that could enable people to regain speech or perhaps one day help restore feeling to limbs following paralysing injuries. “You can use this as nerve conduit to join two ends, say of spinal column, and fill with this material and appropriate cues, such as adhesion molecules, and Schwann cells and varieties of stem cells,” Birchall explains. “Whether one could use this for delayed damage [to spinal chords] depends on what we find we are capable of persuading stem cells to do, but it could certainly a good vehicle for administering stem cells for [immediate] spinal chord injury.”

References

1. Engineering nanoscale order into a designed protein fiber (external link)
2. Sticky-end Assembly of a Designed Peptide Fiber Provides Insight Into Protein Fibrillogenesis (external link)
3. Rational design and application of responsive -helical peptide hydrogels (external link)

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