Lorne 2009: Shedding light on tissue engineering

Biomaterials and regenerative medicine is a very hot field at the moment, although it has actually been around for a few decades.

In the early days there was a lot of hype and flurry that was not backed up by too many sound results, with many companies starting up and then finishing just as quickly.

According to biomaterials specialist and cell biologist Dr Jerome Werkmeister, in recent years the field has started to take stock and now it is progressing in a more careful and scientifically rational manner, developing specific ways of looking at the problems.

“And now with the advent of stem cells, the area is becoming intriguing,” he says.

“The whole idea of tissue engineering is really now the persuasion of the body to heal itself. So, we provide the scaffold and the cells and hopefully get proper regenerative tissue that can perform or function normally in the body.”

Werkmeister leads a team of polymer chemists, synthetic chemists and matrix biologists in the biomaterials and regenerative medicine theme at CSIRO in Melbourne.

Together, these scientists are developing new biomaterials scaffolds for regenerative medicine and working out ways these scaffolds can actually be used to make damaged tissues or organs as good as new.

One of their current thrusts centres on naturally occurring biological components being made into scaffolds. “The structure and properties of the scaffolds are critical to ensure controlled cell behaviour and tissue regeneration,” he says.

Synthetic materials are also used as supporting scaffolds for cell growth. However, naturally found proteins have clear advantages in terms of biocompatibility and tissue remodelling abilities as well as generally being biodegradable through natural cell mechanisms when the supporting scaffold is no longer necessary.

Many biological proteins have been tried in the field, he says. “Collagen is probably the prime example, but there are many proteins that can be used and that we have used, alone and in combination.”

At the Lorne Protein Structure and Function conference this week, Werkmeister will talk about a novel photochemical cross-linking mechanism being designed to re-form tissues from these biological materials.

The process was developed by CSIRO scientist Dr Chris Elvin, who is also part of the biomedical materials team. In work published in Nature in 2005, Elvin took apart and then put back together a structural protein called resilin, which forms a rubber-like material that is a key part of the insect cuticle.

“Resilin has the most fantastic elastomeric properties – low stiffness, high strain and highly efficient energy storage properties,” Werkmeister says.

“It plays important roles in many insect functions such as flight, jumping mechanisms and even in generating those annoying sounds that cicadas make.”

In exploring the molecular nature of resilin, Elvin discovered a cross-linking technique based on covalent bonds between tyrosine amino acids that recapitulated resilin’s natural properties, but in a controlled and simple-to-use package.

The cross-linking process itself is remarkably simple. It needs only three components – the protein, a metal ligand complex (typically ruthenium is used) and an electron acceptor. The mixture is then flashed with visible light of 452 nm wavelength to form the polymer – within 20 seconds, the proteins will be cross-linked into a matrix with remarkable tensile strength.

“Chris went on to show that the same cross-linking mechanism he used to make the resilin polymers would work equally effectively on other proteins,” Werkmeister says. This now the subject of two patents.

---PB--- Matrix proteins

Fibrinogen was the first protein used to form CSIRO’s PhotoSeal scaffolds using Elvin’s photochemical technology. This clotting protein is already used fairly successfully as a tissue sealant or glue, usually via a two-pack system that must be transported and kept frozen and which takes 15-20 minutes to cure.

The same result can be achieved with PhotoSeal, however, without adding other clotting components such as thrombin, and the process is about 50 times faster and with five times the adhesive or bond strength in the final product.

The unpolymerised gel is also stable at room temperature as nothing happens until exposed to the right wavelength of light. At least for this application then, the CSIRO product is extremely simple, cost-effective and easy to use compared to all the fibrin-based glues on the market.

The team has now looked at several other matrix proteins as potential scaffold components. “What we have now found is that while fibrinogen works really well as a sealant and a scaffold, so does gelatin, and it is much cheaper and easier to use.

“We are currently testing these different scaffolds in our in vitro models in the lab – doing adhesion and mechanical testing – and getting really good results.”

Werkmeister also reported some very promising preliminary results with the team’s newer scaffolds, mostly as tissue sealants or haemostats, in a number of clinically and commercially relevant animal models.

Incorporating cells and cellular components such as growth factors into the scaffolds is also an obvious interest that the group at CSIRO is pursuing, particularly since the photochemical curing process is non-toxic to cells and the formulations are non-immunogenic and biodegradable.

Initial work has involved adding fibroblasts, chondrocytes or myoblasts into the scaffolds before applying the photochemical process, then doing in vitro testing to assess the matrix properties. These mixtures are also being injected into nude mice to see firstly whether the cells get rejected and then to see if new tissues are forming.

“We have evidence of normal myotube or muscle formation with vascularisation in these mice, and everything looks pretty normal – the results are very preliminary but are looking very exciting.”

---PB--- Matrix engineering

The only problem being encountered so far with the cell/matrix mixtures is that with such a high concentration of proteins cross-linking so quickly, the cells are having trouble moving around.

This sort of defeats the whole idea if the new cells can’t migrate their way out of the matrix and start to regrow tissue or fix the damage.

“To address this, we are finding ways to engineer the scaffold such as by introducing holes or pores into the structure,” Werkmeister says.

“We can do this by various means, but the best way seems to be by simply adding catalase and peroxide, which react to produce oxygen and thus produce bubbles in the matrix. We can then regulate the size and shape of these bubbles to make channels and pores – it is remarkably simple.”

In practical terms, the scaffolding material may be applied in various ways. Generally, the material has been tested by syringe application, but pre-formed matrices are also being looked at.

In this way the scaffold is moulded and set into different shapes in vitro, rather than doing so in situ, making the application possibilities in tissue engineering almost endless.

“Using the photoactivated procedure, soluble proteins could be moulded, cast or extruded into various shapes including sheets, tubes, rings, spheres, rods and fibres. And of course, nothing sets until you shine the light.”

Werkmeister admitted that the competition for such products is fierce – a lot of scaffolds are on the market already and some companies have started producing cell-based therapies as well.

“What we are hoping is that this technology provides that quantum leap you need to get over all these other products out there. It is extremely user-friendly, simple, cheap and instant – shine a bit of light on it and it will actually set in place where you want it to.”