A wound is a wet and messy place. The healing process can be long and painful, and while skin-grafting has developed into a common procedure, it can leave scarring and stenosis behind. An alternative to stapled or glued-on sutures to hold skin grafts in place that could promote healing and even potentially deliver medicine in one fell swoop would be an appealing development.
Mussels, the bivalves that cling heroically to seaside rocks withstanding wave after punishing wave, have something sticky figured out despite all the moisture they put up with. They stick to rocks by secreting mussel adhesive proteins (MAP) from their “feet.” Because of their incredible stickiness under the wettest of conditions, their biocompatibility, and their easy biodegradation, MAPs have become hot targets for medical research, appearing in applications as diverse as hydrogels, nanofibers, and drug delivery systems.
To improve drug delivery in the complex space of a healing wound, researchers based at the Pohang University of Science and Technology reported in the Chemical Engineering Journal that they had created a new kind of skin graft made from a complex mixture of MAPs and other molecules (1).
“This is an interesting use of the technology,” said Bruce P. Lee, a biomedical engineer at Michigan Technological University, who was not involved with the study. “This is research that's highly sought after. There’re a lot of people doing this.”
MAPs contain L-DOPA, a non-coded amino acid derived from tyrosine that often replaces tyrosine in proteins as a stabilizer in cells. MAPs’ inherent stickiness is proportional to the number of L-DOPA residues they contain. The study authors converted every occurrence of tyrosine in the coded sequence of MAPs into L-DOPA to increase their grafts’ stickiness even further. They expressed the resulting MAP sequence in E. coli, purified the protein, and dried it into a powder instead of harvesting minute amounts of protein directly from mussels, which is a laborious and difficult technique tried in the past.
The researchers next mixed the MAP proteins with hyaluronic acid (HA), a polysaccharide used by a wide cross section of all life as a lubricant and shock-absorber. HA is also ubiquitous in cosmetics and medicine since it stabilizes broken skin structures and promotes rebuilding the extracellular matrix after a wound. Lee pointed out that HA is a good choice for a skin graft application, with some pricing drawbacks. “It's a naturally available polysaccharide from your body, but then it’s relatively expensive compared to synthetic polymers,” like heparosan, a synthetic peptide often used to replace HA in drug delivery applications.
To test the MAPs’ ability to promote a scarless, quick-healing skin graft, the researchers created a coacervate, a mixture of droplets composed of proteins and other biomolecules in a liquid-liquid suspension. These suspensions have been used as drug delivery systems in the past because of their ability to stably encapsulate and deliver drugs; one notable example is the localized delivery of antitumor radionuclides, where the heat of the body melts the suspensions and releases the therapeutics (2).
For this study, the researchers made coacervates with MAPs, differing amounts of HA, and two medicines: allantoin and epidermal growth factor (EGF). Allantoin is a topical moisturizer common in skin creams that assists with wound healing, and EGF is a naturally occurring mammalian protein that also helps in wound healing by increasing cell proliferation (2).
The scientists measured the rate at which both drugs diffused into surrounding areas using an in vitro model. The group immersed the coacervate in a buffer and allowed the drugs to diffuse out of suspension. Afterward, they separated the drugs released from the coacervate and quantified them using liquid chromatography.
Two interesting things happened. First, the two drugs diffused at different rates. 100% of the allantoin was released within four days, while EGF diffused slowly, with only 80% of the total load diffusing in 14 days. This suggests that these grafts can deliver both quick- and slow-acting drugs.
Next, the team cultured fibroblasts with coacervates and measured their effects on cell growth. The dual delivery of drugs rapidly increased the proliferation of cells over those cultured with coacervates that didn’t contain drugs or coacervates holding either of the drugs individually. Cells cultured with the dual-drug coacervate proliferated at almost 160% of the rate of cells grown in the presence of an empty coacervate.
They then built an in vitro model of wound healing by placing two populations of keratinocytes side by side in a dish and measuring the rate at which they closed the gap. The dual-drug coacervate closed 98% of the gap in 24 hours, compared to allantoin alone (83% closed) or EGF alone (35% closed, which was indistinguishable from cells cultured with a coacervate that didn’t contain drugs).
There are multiple goals involved in creating new kinds of skin grafts that stick in different ways and can be outfitted to deliver medications. A skin graft that sticks well without a suture can heal a wound or surgical incision without producing a scar.
“Plastic surgeons will use skin grafts because they know they're going to get 100% closure. And usually, they work really, really well,” said Robin Martin, a freelance scientific consultant in wound healing and reconstructive surgery who was not involved in the study. Surgeons use a variety of approaches to get grafts to stay in place. In an area that doesn’t see much motion, a suture will do just fine. In high motion areas, they often use fibrin glue, a surgical formulation that creates fibrin clots. This is useful but has drawbacks. Most importantly, it must be prevented from seeping into the bloodstream where it can cause off-target clotting. Since MAP does not promote clotting on its own, it can simply biodegrade after grafting.
The researchers next compared the ability of a MAP skin graft to heal and then biodegrade in a mouse model. To test this, they cut a circular piece of skin and then reattached it in the same place using either a suture, fibrin glue, an empty MAP-coacervate, or the full coacervate with both drugs included. There was significant scarring when sutures were used, although less occurred with fibrin glue and the empty coacervate. The dual drug MAP coacervate, however, reduced scarring significantly, and unlike the fibrin glue, fully degraded after 10 days of healing.
In addition to aesthetic concerns, scars can be stiff, painful, and can reduce a patient’s range of motion, particularly if it develops on an area like the neck, knee, or elbow. While this can be problematic for adults, these drawbacks are especially worrisome in children where scar tissue does not grow and stretch like undamaged skin. This can cause lifelong difficulties after a wound heals.
Mussels have been a source of inspiration for biomedical engineers for some time, especially those working on wound healing in wet places. For instance, in 2021 a research group based in Hong Kong used another mussel-derived molecule called N-(3,4-dihydroxyphenethyl)methacrylamide to improve the stability and coloration of tooth restorations used by dentists, which often stick well at first but degrade over time in the moist environment of the mouth.
But a laboratory incision in one animal doesn’t translate directly to another. “The mouse model, the dermal model, is anatomically very different from the human. So, I think there's still a way to go to have demonstrated that it's suitable even for a larger animal like a pig,” said Lee.
- Park, W. H., Lee, J., Kim, H. J., Joo, K. I. & Cha, H. J. Sutureless full-thickness skin grafting using a dual drug-in-bioadhesive coacervate. Chemical Engineering Journal 446, 137272 (2022).
- Johnson, N. R. & Wang, Y. Coacervate delivery systems for proteins and small molecule drugs. Expert Opinion on Drug Delivery 11, 1829–1832 (2014).
- Li, K. et al. Enhancing resin-dentin bond durability using a novel mussel-inspired monomer. Materials Today Bio 12, 100174 (2021).