Biomimetic generation of the strongest known biomaterial found in limpet tooth

Limpet tooth has the highest known tensile strength of any known biological composite material. In our latest manuscript on limpet tooth, we chart new territory in marine invertebrate cell culture, developmental biology, transcriptomics and biomimetics with implications for sustainable technology.
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The Common Limpet (Patella vulgata) is a frequent sight along the seashore, where it clings to rocks at low tide. When the tide comes in these small creatures graze on algae using a specialised feeding organ known as the radula which is coated with teeth strong enough to leave marks on the rocky surface beneath. Based on this observation it has long been suspected that limpets must possess teeth of incredible robustness, and in 2015 this was confirmed in a study which demonstrated a high elastic modulus combined with a tensile strength equivalent to synthetic carbon fibre (Barber, Lu, and Pugno 2015). This combination of mechanical properties is attributable to a unique composite structure of tightly packed chitin fibres interspersed with filamentous crystals of iron oxide in the form of goethite (α-FeO(OH)).

The overarching aim of our latest study was to develop a biomimetic composite based on limpet tooth with comparable mechanical properties (Rumney et al. 2022). To achieve this, we set about developing novel cell culture techniques suitable for isolating, maintaining and differentiating cells from the limpet radula. After extensive troubleshooting for each basic step and fundamental process, we were able to achieve our milestone for maintaining limpet radula cells in vitro. However, the cells had surprises in store for us that were far beyond our expectations. Not only were the cells capable of depositing iron oxide crystals and making chitin and secreting chitinases, as we would expect for generating limpet tooth material, but they also demonstrated a fantastic regenerative capacity, which provided additional insight into the developmental biology of the radula. As the radula grows continuously to replace rows of teeth throughout the lifespan of the limpet, we reasoned that the bulbous sack (Formation Zone, FZ) at the start of the radula must contain stem cells, and found that isolated FZ maintained in culture conditions generate ribbons of radula scaffold containing limpet tooth structures. Furthermore, we found that cells isolated from the FZ and maintained for 6 weeks in culture spontaneously developed into individual teeth. To our astonishment, we found that cells isolated from whole radula could regroup in culture conditions, with distinct ‘head’ and tail’ structures reminiscent of the FZ and radula.

In parallel with developing our limpet cell culture assays, we also carried out a detailed transcriptomic analysis on each stage of the limpet radula. Differential gene expression revealed differences in key patterning genes, providing yet more insight into the developmental biology of the limpet radula. We were able to dig deep into the transcriptomic data to identify sequential changes in expression for genes associated with chitin synthesis, but also chitinases suggesting a remodelling of the chitin scaffold at specific developmental stages. We also found complementary gradients of genes associated with iron reduction and iron oxidation along the length of the radula.

When it came to developing our own biomimetic material, we developed an improved method to solubilise chitin for electrospinning. We found that limpet cells would readily attach to the chitin scaffold, but without much mineralisation. Then it occurred to us that in the native limpet tooth material, the proximity of the chitin fibres to each other was far too dense for a cell to be able to make its way in, so any mineralisation processes would have to be secreted by the limpet cells from outside a scaffold. Electrospun chitin scaffolds were incubated with limpet radula cell conditioned media, and this was sufficient to allow deposition of biogenic iron onto the chitin structures, significantly increasing the tensile strength of the material.

This means that after five years we have our proof-of-concept material, which could present many advantages. Chitin currently exists as a by-product of the fishing industry, with an estimated 1x106 tonnes generated per annum (Hamed, Özogul, and Regenstein 2016). It would be beneficial to have this waste repurposed into a usable, high tensile strength but ultimately biodegradable material, where it could take the place of many synthetics which will never degrade within our lifetimes and are harming the health of the world’s ecosystems. Chitin can also be sourced from fungi, meaning that an even more sustainable ‘fish-free’ model is possible.

As for the limpet cells, our research has shown so much but raised many more questions, such as what are all of the individual cell types that require characterisation and how do they function? Answering these questions will help lead us to the target genes we need for full scalability, by insertion into a suitable host vector that will allow mass production of the mineralising limpet cell secretions.

The mineral component of limpet tooth is named in honour of the philosopher and polymath Johann Wolfgang von Goethe whose famous quotes include “Knowing is not enough; we must apply. Willing is not enough; we must do”. Little did he know that nearly two centuries after his death we would be applying the same tenacious philosophy to overcome technical hurdles and generate a material with the potential to help solve the key 21st Century challenge of sustainability.

References

Barber, Asa H., Dun Lu, and Nicola M. Pugno. 2015. “Extreme Strength Observed in Limpet Teeth.” Journal of the Royal Society Interface 12 (105). https://doi.org/10.1098/rsif.2014.1326.

Hamed, Imen, Fatih Özogul, and Joe M. Regenstein. 2016. “Industrial Applications of Crustacean By-Products (Chitin, Chitosan, and Chitooligosaccharides): A Review.” Trends in Food Science and Technology. https://doi.org/10.1016/j.tifs.2015.11.007.

Rumney, Robin M. H., Samuel C. Robson, Alexander P. Kao, Eugen Barbu, Lukasz Bozycki, James R. Smith, Simon M. Cragg, et al. 2022. “Biomimetic Generation of the Strongest Known Biomaterial Found in Limpet Tooth.” Nature Communications 2022 13:1 13 (1): 1–13. https://doi.org/10.1038/s41467-022-31139-0.