Building on a previous MRC CiC-funded project, we have the opportunity to accelerate the development of antimicrobial implants for surgical reconstruction after trauma. Unfortunately, bacterial colonisation is very common after implantation of prosthetics or devices following injury as the fragility of traumatised tissue, coupled with the immunocompromised state of trauma survivors, creates a very favourable environment for infection. Implant infection can undo months of painstaking reconstruction, resulting not only in significant costs for the NHS but also a massive psychological downturn for the patient.

Our team previously created antimicrobial peptides capable of disrupting the bacterial cell membrane on contact. We showed that the peptides could be attached to the surface of titanium following chemical and thermal treatments of the surface and attachment of the peptides using titanium binders. This method although successful, it requires conjugation of the peptides with the binders that can interfere with the antimicrobial activity.

Method

Design and production of a biomimetic elastin-like polypeptide scaffold with antibacterial bioactive domains. The polypeptide can be designed to be easily modified by the insertion of a bioactive domain in the C-terminal region. A new macromolecule that will be the result of the fusion between the elastin-like biopolymer and an antibacterial peptide sequence will be produced and fully characterised.

Incorporation of synthetic antibacterial peptides in the biomimetic elastin-like scaffold by an enzymatic route using transglutaminase. The antibacterial peptides will be embedded within the matrix that will be cross-linked by the use of a transglutaminase.

Release of the peptides realized by two ways:

• Release triggered by a thermo-responsive behaviour.
• Release triggered by aproteolytic or elastolytic activity.

The antimicrobial ink will be placed by 3D printing on modified (either by plasma treatment or chemical treatment) titanium surfaces and polymers.

Bone defects can arise through trauma or disease. Conventionally, defects are treated by filling of the cavity space with a bone substitute to restore mechanical integrity and stimulate the regeneration of bone tissue. Autologous bone is the “gold standard” for this application. However, for large defects, it is not a viable approach, as only a limited volume of autologous bone can be harvested. Additional surgery is also required, causing further trauma and increasing the risk of infection transmission. Calcium phosphate-based materials can be produced synthetically in large volumes and are designed to be resorbed over time, eventually becoming replaced completely by new bone. Of particular interest is the development of materials that can additionally influence biological processes resulting in enhanced bone formation. Calcium pyrophosphate has been shown to provide an environment that stimulates new bone formation during resorption, making these materials of great interest for future formulations.

Method

In this study, calcium pyrophosphates are synthesised using precipitation and thermal methods. Precipitation of calcium pyrophosphate is achieved by combining aqueous solutions of calcium and pyrophosphate salts. By closely controlling the pH of the precipitation reaction, the resulting phase and microstructure of the calcium pyrophosphate can be manipulated. High temperatures can also be used to transform calcium phosphate precursors to highly crystalline phases of calcium pyrophosphate that possess different physiochemical properties compared to those synthesised by precipitation. These differences may influence biological interactions, such as the rate of dissolution and resorption in the physiological environment. It is possible to deliver these materials in the form of pre-fabricated granules or as an injectable paste. In vitro cell-material interactions are being investigated to confirm viability and osteogenic capacity. Candidate materials will then be assessed in vivo using suitable defect models.

The extracellular matrix is crucial for both cellular biochemical and biomechanical cues, overall determining tissue type. It is comprised of multiple proteins, of which, collagen is the main component. Collagen is a hierarchical protein secreted by cells into the extracellular space. It is secreted as a triple helix which assembles into staggered parallel arrays to form fibrils. These fibrils then self-assemble into fibres which make up the main components of the extracellular matrix. The orientation of these fibrils/fibres determines how the tissue functions. Collagen is also important during wound healing and is deposited rapidly to allow cells to traverse the wound bed and begin the remodelling phase. However, as this process is rapid the orientation and assembly of the collagen will not match that of the native tissue. This, overall, results in a scar.

Method

Predominate methods for characterisation of collagen matrix assembly have been atomic force microscopy (AFM), scanning electron microscopy (SEM), fibrillogenesis studies and circular dichroism (CD). Both AFM and SEM have been used to study structural changes to overall matrix formation caused either by topographical cues or chemical influence. Fibrillogenesis studies and CD have been used to understand the way in which inorganic ions influence different stages of collagen fibril assembly. This includes changes to the molecular structure of collagen at the nanoscale to overall matrix assembly at the macroscale. Cellular work has also been conducted to see how any changes to the collagen matrix affect cellular growth and proliferation. Metabolism assays and fluorescent imaging have been undertaken to understand these changes. Other methods such as Raman spectroscopy, rheology, differential scanning calorimetry and x-ray fluorescence have also been used to look at changes in collagen assembly due to external influence.

Scars are common, costly and can seriously impair quality of life. In addition to trauma, scarring and fibrosis are associated with broader disease and degenerative conditions. There is no treatment currently proven to control problem fibrosis and lessen the functional impact of scarring.  In the first phase of SRMRC Professors Logan and Grover exploited a potent anti-scarring molecule called Decorin(Esmaeili et al., 2014; Hill et al., 2015). They sourced GMP manufactured recombinant Decorin and demonstrated its translatable potential by securing two fully funded (Wellcome Trust and MRC) clinical trials of our novel formulated anti-scarring dressings to treat skin burns and damaged corneas. We believe this technology has the potential for expansion to other indications to prevent scarring in many more tissues injured by trauma or disease.

Method

The methods we are using in this project including:

• Production of a prototype of the cranial membrane with a defined formulation approved by clinicians routinely undertaking craniotomy procedures.
• Assessing the release amount of the drug and its efficacy using ELISA, collagen fibrillogenesis and appropriate models of human meningeal scarring.

The team have shown in the laboratory that high-intensity blue light (part of the visible light spectrum) is antimicrobial against bacteria and biofilms, and a promising agent for the treatment of contaminated or infected wounds. We wish to do further experiments to see if we can reduce the duration required for bacterial killing, and to investigate whether other wavelengths or doses of light offer improved activity.

Method

In collaboration with the University of Birmingham, the dental school, and Defence Science and Technology Laboratory, the team is planning to perform a range of laboratory tests on key wound bacteria in order to ascertain an optimal wavelength (or combination) of visible light, dose and duration, that can be used as a novel antibacterial technology for the decontamination of wounds. They will also perform host cell toxicity studies in the laboratory to ensure there are no adverse effects on human tissue cells following light delivery.