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A significant stumbling block in the attempt to create artificial organs has been developing working capillaries – the miniature blood vessels needed to deliver oxygen, water and nutrients to organs and carry away waste.
Leon Bellan, assistant professor of mechanical engineering at Vanderbilt University in Tennessee, says he saw this as an interesting engineering problem and believes the solution lies in a rather unlikely piece of kit – a candy-floss machine.
His graduate work at the university had previously focused on nanoscale fibres formed using electrospinning, whereby a very high voltage produces a fibre-forming jet that deposits nanofibres in a chaotic mat on a surface.
Bellan believed that the nanofibre-formed nanochannels he had been able to create looked like capillaries, but were far too small. “I started thinking about the analogies people used for electrospinning, such as Silly String [aerosol string] or cotton candy [candy floss],” says Bellan. “Cotton candy seemed a promising ‘sacrificial template’, and the machine was inexpensive and easily obtained.”
Modelling capillaries
After picking up a candy-floss machine for around $40, Bellan made channels in the lab that looked a lot like capillaries, using the spun sugar as a sacrificial template.
Years later, his team have worked out a combination of materials that allows the technique to be applied to biomaterials, to keep living cells alive.
To make the sacrificial template, the engineers use PNIPAM, a thermo-responsive polymer that is insoluble in water above 32ºC, but soluble below that temperature.
A fibrous mesh is made by spinning the material, using a machine similar to a candy-floss machine. Larger sticks of the material are attached to the fibrous mesh to form inlets to which tubing can be attached. Gelatin, cell-culture media, cells, and an enzyme called transglutaminase are all mixed in and then poured over the PNIPAM structure. Everything is then placed in an incubator at 37ºC.
Stable gel
The enzyme causes bonds to form between the gelatin molecules, slowly forming an irreversible gel that won’t dissolve again if heated. Once the gel has set, everything is removed from the incubator and allowed to cool to room temperature, at which point the PNIPAM structures dissolve and leave a complex fluidic network behind. This network is within a gelatin gel containing cells, which are nourished by the vessel system made by the fibres.
So far, the team has demonstrated that the combination of materials can form complex 3D networks of channels that mimic capillaries. By flowing appropriate cell-culture media through these channels, the researchers can keep cells embedded within the gel alive for at least a week, with around 90% viability.
Without these channels – or with channels that have no flow – cell viability drops substantially to around 60%, highlighting the importance of keeping the cells nourished.
Endothelial experiment
The team is now exploring techniques to get endothelial cells – which form the lining of blood vessels – in the channels.
The team hope this approach will allow the fabrication of channels on the capillary-size scale. The approach already improves on 3D printing techniques that use biomaterials, which typically can make features only 10 times larger than a capillary. The next steps will be to use the technique with other types of cells to form more complex, functional artificial tissues.
“The ultimate goal is to develop a scaleable technique to produce capillaries in a cell-laden biomaterial, so tissues of unlimited thickness can be produced and the cells embedded within thrive and proliferate,” says Bellan.
While the process is still a long way from market, the team hopes it will become part of a toolbox that engineers can use to build artificial tissue.