Bacterial bonds assist skin grafts
Biological adhesions between bacterial cellulose nanofibrils can improve the performance of biomedical skins and grafts.
Bacterial cellulose (BC) discovered by A J Brown in 1886 is somewhat similar to plant cellulose. It has the same molecular formula, but is chemically more pure as it is free from other plant polysaccharides such as hemicellulose and lignin. It has a higher tensile strength, can be grown into almost any shape, has a superior water retention capacity, and a considerably finer architecture.
Properties such as these have made bacterial cellulose an ideal ingredient in foods such as nata de coco, a sweet jelly with increased fibrousness, chewiness and texture through the addition of bacterial cellulose. It has also been used as a thickener and stabiliser to help retain the viscosity of softer foods.
More recently, the properties of BC have gained the attention of materials scientists in the biomedical sectors where it is used as a wound dressing material, in the pulp and paper industries where it is used as an ultra-stiff paper base, and in coatings industries, where it can be used to stabilise coating suspensions.
BC is sourced naturally in South East Asia from microorganisms such as Acetobacter xyllinum, to cater for the multifaceted Asian food industries. It is a renewable, sustainable and biodegradable nanofibril, and has a nanoscale filamentous structure with very high crystallinity and outstanding properties of stiffness ranging on average between 78-143GPa. Separating the nanofibrils of BC is far simpler and less process-intensive than it is for plant-derived cellulose nanofibrils, making it an attractive ‘green nanomaterial’ with considerable potential in biomaterials applications.
The bulk matter of BC is effectively networked by β conform hydrogen bonds, which help align cellulose molecules into its tight crystalline structure. The β-conformation, as it is known, is a straight-backed molecular chain. The straightness of the molecules allows them to pack densely together, which can improve molecule to molecule interactions through hydrogen bonding. Therefore, BC surfaces are dense with hydrogen bonding sites, however the stiffness of the individual nanofibrils makes it difficult for interfibrilar alignment. BC nanofibrils can therefore create a weak network continuum when packed together with other BC nanofibrils, however, the true mechanical potential of BC is never reached in this way. This is reflected in the stiffness of BC sheets, which is reported to be between 2-15GPa, and is therefore a factor of 10 lower than the nanofibrillar stiffness. How can we scale up BC in a way that reduces losses from its nanofibrillar stiffness? Natural bio-adhesives may have some of the answers.
For materials scientists, the natural world is a constant source of design inspiration. Topics such as structural hierarchy, adhesion and material morphology are of particular interest, since they elucidate novel design guidelines in the advancement of mechanical materials. Biological adhesion is ubiquitous in the natural world, where it exists in a plethora of different function- specific chemical forms.
The design and utility of bio-adhesives in natural materials technologies also constitute an environmentally responsible engineering practice. Synthetic adhesives that could be used to connect BC nanofibrils are often toxic to the environment and do not easily degrade. This is why, when incredible examples of stickiness are found in the natural world - for example, aggregate and flagelliform silks, extracellular polymeric substances (EPS) from biofouling diatoms and mussels - attempts should be made to mimic at least their function and design strategies by using similar, naturally sourced bio-adhesives, preferably with minimal chemical modification.
When how bio-adhesive functions are considered, it is possible to some degree to simplify their mechanisms of adhesion to those dominated by intermolecular secondary forces such as electrostatic and van der Waals, and those dominated by off axis sidechains that obstruct intermolecular shear. A good example of the former can be observed in the nanocrystals of structural biological silks. The stiffness of such nanocrystals is derived primarily from a large concentration of hydrogen bonds that arrange in a quasi-planar manner between β-sheet layers within the nanocrystal. The result is a nanostructure with an exceptionally high stiffness for a polymer.
Sidechains described in the latter case are different, as secondary interactions that arise between side chains and molecules tend to be off-axis, and therefore less likely to have the same cumulative level of quasi-planar resistance described in the first example.
In the case of molecules such as chitin, this has been shown to be beneficial to intermolecular adhesion, as the acetyl sidechains laterally stabilise the crystalline form of chitin, resulting in both higher stiffness and fracture toughness than in deacetylated chitin, chitosan, which lacks the side chains. Both of the mechanisms of adhesion described in the examples above can significantly improve the mechanical properties of biopolymers. It would stand to reason, therefore, that it is possible to improve the properties of ‘other’ materials by mimicking similar functional designs.
The materials design team at The University of Edinburgh focuses on biological and biomimetic composites design. They employed both of the bio-adhesive mechanisms previously described to improve the adhesion strength between BC nanofibrils. The specific amino acid monomers alanine and glycine were chosen as they are known to enable hydrogen bonding dominated stickiness in materials such as silk.
In the first instance, the amino acids were used as free-moving secondary bond-forming bio-adhesives between BC nanofibrils. During sheet-forming, the amino acid monomers were able to energetically optimise their locations and orientations between the BC nanofibrils, and in doing so, migrate into the most stable electrostatic attachment. The team also grafted the same amino acids to BC surfaces via esterification reaction on TEMPO-oxidised BC nanofibrils. TEMPO is a free radical reagent used to oxidise primary alcohols aldehydes.
The TEMPO-oxidisation reaction uses 2,2,6,6- tetramethylpiperidine-1-oxyl radicals to oxidise the C6 primary hydroxyl on the cellulose chains to form C6 carboxylate groups. This then allows for the grafting of alanine or glycine as sidechains to the surface of the BC nanofibril. Unlike the free-moving amino acids, when amino acids are grafted to BC surfaces in this way, they are constrained at one end, creating a hairy surface at the molecular-level.
Using experimental tests on manufactured BC sheets using both methods described, coupled with molecular dynamics simulations, the team learned that free-moving amino acids favour adhering BC surfaces together by aligning themselves parallel with the BC. Amino acids covalently attached to the TEMPO-oxidised BC surfaces, on the other hand, are considerably more constrained. The limited mobility in such instances, forces them to electrostatically bond to the nearest available hydrogen-bonding sites.
As such, the mobility of the free amino acid monomers gives them an advantage over pinned sidechains, raising the stiffness of BC sheets to more than 100% that of unglued BC sheets. The sheets made in the laboratory are about the size of a person’s hand – bigger sheets can be made by scaling up the manufacturing technology, which already exists, so sheet size depends solely on the size of the machine.
The researchers also found TEMPO-oxidation and amino acid grafting of BC gives rise to about 50% improvement in stiffness. Clearly, the free movement of bio-adhesives trumps molecular pinned sidechains in this instance, and if BC sheets are to go closer to their theoretical maximum (nanofibrillar) stiffness, bio-adhesives are a good starting point. Considerably more research needs to be done to tighten that hydrogen bonding network between BC nanofibrils to reach their theoretical maximum stiffness. In the future, clever molecular design strategies will need to be applied to bio-adhesive technologies, with the aim of maximising the potential for strong secondary interactions.
Next, this research will be taken to a more applied environment by engaging industrial support from the paper, board, packaging, and biomedical engineering sectors. The future of TEMPO-oxidised BC needs to be revisited using a variety of different bioglues and bonding methods, given that an end goal is to mechanically enhance BC. The design strategies of molecular materials should be guided by atomistic models, and the most promising of them, chemically replicated and tested.
This research approach is believed to elucidate the larger spectrum of possibilities for the control of the mechanical behavior of BC materials.
Bacterial cellulose is a natural, non-toxic, biodegradable material with exceptional mechanical properties and characteristics. It is, however, a nanomaterial and as such must be conjoined with others of its kind to form larger bodies of engineering material. The properties of the nanofibrils are reduced when they are collected together into larger structures and as such a challenge for materials researchers is to deduce novel methods for combining them without reducing their green-materials character. Bioadhesives are a material choice that potentialises the recovery of nanofibrillar mechanical character by improving stress transfer between the nanofibrils. Free-moving bioglues and bioglues pinned through TEMPO-oxidation show promise in the development of stiffer bacterial cellulose materials, however, research efforts in this area are still sparse and applied research will be needed to control and manipulate their mechanical properties for specific applications.
Parvez Alam is Senior Lecturer, University of Edinburgh School of Engineering, Institute for Materials and Processes, UK.