Introduction

Due to their biocompatibility and toughness, spider silks have been well documented in medical applications beginning with their use in bandages by the ancient Greeks. Additionally, their versatility and processability creates potential to fine tune their properties to address specific issues via recombinant technologies ^[1]. Here we evaluate and propose applying our composite material to various biomedical applications within tissue regeneration. The conductivity afforded to our material through the binding of reduced graphene oxide (rGO) creates potential for applications within peripheral nerve regeneration or synthetic nerve creation. Additionally, the natural strength of synthetic spidroin-based silk, combined with additional mechanical support from bound rGO could be applied to film creation, which would then act as a reconstructive mesh for ligament or tendon repair.

Peripheral Nerve Regeneration and Synthetic Nerves

The non-immunogenicity of spidroin-based biomaterials has enabled broad usage in tissue engineering and regenerative medicine. Specific applications include bone and cartilage regeneration, lung tissue engineering, as well as peripheral nerve repair ^[1].

Peripheral nerves can be damaged from injuries resulting in the compressing, stretching or cutting of nerves. We are aiming to create an insertable graft to support the regeneration and recovery of these injury sites. Our composite material would enable the functional reconstruction of damaged nerve tissue by promoting cell adhesion and subsequent proliferation, leading to vessel and nerve recovery. Our product will take the form of a non woven mesh of nanofibrils, which will help to allow sufficient space for substance exchange between cells and the repairing tissues. Additionally, creating our graft from non woven mesh will result in the highest potential for fibroblast adhesion compared to film or hydrogel forms, due to the increased availability of cell attachment surfaces ^[3]. The synthetic silk component of our product makes it suitable for the basis of a nerve scaffold due to the neuron affinity and speedy Schwann cell adhesion that this material facilitates ^[1]. Insertion of our graft in an area of peripheral nerve damage will act to encourage axon regeneration and remyelination creating overall regenerated tissue. Additionally, the propensity of silk to be readily broken down in the body by the photoimmune response enables the inserted constructs to be replaced by an autologous matrix.

End Users

With nervous system injuries affecting over 90,000 people each year ^[2], we hope to alleviate a strain on both the economy and the healthcare system with our biocomposite material. Utilising our insertable skeleton we hope to contribute to the rapidly growing field of nerve tissue regeneration. Currently, our product is designed for individuals with nervous system injuries obtained from an accident, a fall, or sports. The implementation of our product within traumatic injury sites will help to speed up recovery times as well as ensure a more thorough recovery. Enabling deep tissue repair will result in wounds with greater tissue regeneration and less scarring, resulting in wounds that are less likely to cause pain and reduce mobility in the future.

Scaffolds

Ligaments

The knee joint is the second most commonly injured body site after the ankle and the leading cause of sport-related surgeries. Knee injuries, especially of the Anterior Cruciate Ligament (ACL), are among the most costly sport injuries, frequently requiring expensive surgery and rehabilitation ^[4]. Rather than damage from repetitive strain, as is the case with tendons, ligaments tend to rupture from a single impact event. With up to a year of relevant strength training, ligaments can regrow and initial mobility can be achieved again, but surgery is often required for optimum rehabilitation. Autografts are currently a common surgery to speed up recovery, but these can have issues with rejection, despite the tissue being taken from the same person. Therefore, using an alternate reconstructive material, such as our biocompatible composite could revolutionise the effectiveness of these invasive surgeries.

Unlike inflexible tendons, ligaments require a degree of elasticity; therefore the elasticity of our material would be more suited to this purpose. Ligaments, such as the ACL in the knee, stabilise joint movements so also need to have a high tensile strength. Our composite aims to have a combination of these properties, so should be relevant for application as a rehabilitative scaffold or taken further, as a synthetic ligament. Integration would be via graft or keyhole surgery depending on the scale and location of the injury, but both methods are commonly practised.

Tendons

Tendon issues can result from trauma or repetitive strain and treating them involves the use of external braces and an extensive recovery time. As a more prolific area of injury area than the upper body, our focus when implementing our product is on the lower body. Around 230,000 people injure their Achilles tendon each year in the US, and with numbers continually on the rise ^[5] finding a faster solution for rehabilitation will only become more prudent.

The strain-stress curve of spider silk illustrates a similar variation tendency like that of the tendon, and the curve has both low-strain modulus and high-strain modulus stages. Interestingly, spider silk also shows the shape memory effect which allows it to recover the initial form after deformation, such as a tendon ^[1]. Thus, there is a clear and relevant target application and consumer for our graft.

Current methods of surgical reconstruction include skin grafts or flap surgery. Benefits of grafts include their availability and ease of harvesting. However, their success is largely dependent on blood vessel availability in the wound bed, whereas flap surgery provides this. Nevertheless, other challenges arise with the technicalities of microsurgery, as required for flap surgery, alongside the risk of more serious complications ^[3]. As autografts currently only exist for ligaments, we propose implementing an internal graft to support the repairing tendon. This graft would work in combination with (or in place of) the hefty and disruptive braces, with the aim of decreasing the six to nine month rehab period. It should provide mechanical relief during rehabilitation by reinforcing the joint and therefore reducing risk of reinjury. The surgery required to implement it would be similar to that of a graft, but would harness more stabilising and reinforcing properties. With an optimised method, our composite skeleton itself should be easy to harvest and pair the benefits of current treatments. Depending on the size and location, integration would be via graft or keyhole surgery.

End Users

Pronounced end users for both ligament and tendon rehabilitative therapies are sports people, who require agility, strength and quick changes in direction. Activities such as football and basketball have the highest injury rates among both male and female athletes ^[6]. Female athletes participating in contact sports sustain ACL injuries at 3 times the rate of male athletes in these same sports ^[7]. This skewed ratio is due to women’s joints generally having more range of motion and less muscle mass around the knee, contributing to more instability and the increased likelihood of the ligament tear ^[8]. However, when considering producing a final product, the difference in male and female knee physiology is similar enough to not have a vast impact on the basis of the graft design.

Mobility-limiting injuries are more likely to be operated on in young or active people. This is because the lifestyles of these demographics tend to involve stretching or pressuring their joints to a greater extent than the older population. While widely prolific within professional athletes, ligament and tendon injuries commonly occur within the general population, creating a broad scope for a meaningful therapeutic and economic benefit.Therefore, whilst our implementation will be targeted towards some specific demographics, we conclude that applications of our product span multiple sexes, ages and lifestyles.

Safety and Challenges

Laparoscopic (“keyhole”) surgery is regularly performed on sports injury patients, but waiting lists are often months long due to ACL ruptures being among the most commonly treated injuries. This issue was echoed by a consultation with physiotherapist Adam Davies (OceanPhysio). Due to the nature of our composite, surgery similar to laparoscopy would be required for implementation of ligament therapies. Issues arise as if surgeries are already oversubscribed, then we would only be increasing the pressure and demand on them. This is mirrored for all applications of our product as surgery is essential for integration in the body.

Additionally, despite the biocompatibility of our composite, rejection by the body remains a real risk, especially considering the challenges that remain with autograft immune responses. This was brought to our attention by stakeholders Silvia Perrone (MovMedix) and Adam Davies (OceanPhysio). Silk and graphene have both been seen to be independently biocompatible ^{[9, 10]}, but due to conformational changes when combined, the variety of effects of this are unknown.

Envisioning the potential implementation of our product from scaffolds to biomaterials, we aim for it to be meaningful, beneficent and safe. Strong accountability will be upheld by the fact that the patient would not undergo any procedure with our synthetic scaffold without it passing rigorous preliminary testing first and not consenting to do so. This mirrors the approach we took during our inclusivity hardware testing, ensuring patient autonomy via detailed discussions with the patients and thorough safety considerations.

For the industrial synthesis, we are using Rosetta E. coli as a chassis. Whilst this is a non-virulent strain of bacteria, the organisms will undergo a lysing process as well as thorough extraction of the synthetic silk. Therefore, there is no risk of contamination of live GM organisms into our medical implants. Finally, any modifications being made to bacteria are specific to protein production and have no associated health risks to humans, in the unlikely event that contamination were to occur.

When assessing environmental safety and sustainability, the carbon dioxide produced by E. coli fermentation and the land required for the factory must be considered. Both silk farms and our synthetic sites will likely be equal in size, but sites will produce a higher silk yield with no animal element, so will be more efficient and ethical. These considerations were brought to our attention during Ian Archer’s (IBiolC) bioprocess costing workshop. This helped to introduce how to evaluate impacts from an industrial lens. Likewise, production would likely be on a scale to not produce significant carbon emissions. Silk and graphene are also both easily recycled materials.

References

1. Zhang, Q., Li, M., Hu, W., Wang, X. and Hu, J., 2021. Spidroin-Based Biomaterials in Tissue Engineering: General Approaches and Potential Stem Cell Therapies. Stem Cells International, 2021.
2. Stabenfeldt SE, García AJ, LaPlaca MC (June 2006). "Thermoreversible laminin-functionalized hydrogel for neural tissue engineering". Journal of Biomedical Materials Research Part A. 77 (4): 718
3. Leal-Egaña A., Lang G., Mauerer C., et al. Interactions of Fibroblasts with Different Morphologies Made of an Engineered Spider Silk Protein. Advanced Engineering Materials . 2012;14(3):B67–B75. https://doi.org/10.1002/adem.201180072.
4. Joseph AM, Collins CL, Henke NM, Yard EE, Fields SK, Comstock RD. A multisport epidemiologic comparison of anterior cruciate ligament injuries in high school athletics. J Athl Train. 2013 Nov-Dec;48(6):810-7. https://doi.org/10.4085/1062-6050-48.6.03
5. McFadden M. Achilles injuries on the rise in the US. WNDU. 2017 April 20.
6. Joseph AM, Collins CL, Henke NM, Yard EE, Fields SK, Comstock RD. A multisport epidemiologic comparison of anterior cruciate ligament injuries in high school athletics. J Athl Train. 2013;48(6):810-817. https://doi.org/10.4085/1062-6050-48.6.03
7. Alicia MM, Daniel KS, Kate EW, Laura Y, Marc TG, Robert SH et al. Anterior Cruciate Ligament Injury Risk in Sport: A Systematic Review and Meta-Analysis of Injury Incidence by Sex and Sport Classification. J Athl Train 1 May 2019; 54 (5): 472–482. https://doi.org/doi:10.4085/1062-6050-407-16
8. Cosgaria A. F Tears in Female Athletes: Q&A with a Sports Medicine Expert. The Johns Hopkins University. 2022 https://www.hopkinsmedicine.org/health/conditions-and-diseases/acl-injury-or-tear/acl-tears-in-female-athletes-qa-with-a-sports-medicine-expert#:~:text=Why%20are%20ACL%20tears %20more,range%20of%20motion%20than%20mens. [Accessed 31/08/22]
9. Long Y, Cheng X, Tang Q, Chen L. The antigenicity of silk-based biomaterials: sources, influential factors and applications. Journal of Materials Chemistry B. 2021;9(40):8365-77. [10] Ren J, Braileanu G, Gorgojo P, Valles C, Dickinson A, Vijayaraghavan A, Wang T. On the biocompatibility of graphene oxide towards vascular smooth muscle cells. Nanotechnology. 2020 Nov 11;32(5):055101.