Novel Antibiotic-Free Scaffold for the Treatment of Infection and Regeneration of Bone

The bone infection osteomyelitis (typically caused by Staphylococcus aureus) usually requires a multistep procedure – long term administration of high-dose systemic antibiotics combined with surgical debridement and bone grafting. However, the disease remains notoriously difficult-to-treat due to the poor penetration of systemic antibiotics into the necrotic bone, antibiotic resistance, and the steep decline in antibiotic discovery. Therefore, osteomyelitis treatment has a dual challenge: ensuring an effective and non-toxic dose of antimicrobial, while ensuring bone regeneration is stimulated. Thus, the overall aim of this thesis was to develop a one-step tissue engineering-based treatment strategy for osteomyelitis that combines local, controlled release of non-antibiotic antibacterials with a regenerative collagen-based scaffold to facilitate bone repair.

In the study presented in Chapter 2 of this thesis, a number of non-antibiotic antibacterial agents (specifically chitosan, copper nanoparticles, silver nanoparticles, zinc nanoparticles, copper chloride salt, silver nitrate salt, and zinc chloride salt) were screened as potential agents for osteomyelitis infection treatment based on their antibacterial effect on three clinically relevant bacterial species (Staphylococcus aureus, Staphylococcus epidermidis, and Escherichia coli) while retaining viable numbers of mammalian cells. The results demonstrate that there is a fine balance between the two, and that in particular chitosan and the metal salts, copper chloride and zinc chloride, may have potential for osteomyelitis infection treatment due to their superior antibacterial activity potential while maintaining mammalian cell viability.

In the study presented in Chapter 3 of this thesis, the optimal antibacterial metal salts identified from the screening process in Chapter 2 were incorporated into 3D collagen/chitosan-based scaffolds via both (i) direct incorporation and (ii) through a chitosan microparticle controlled delivery system, with the aim of minimising toxicity and prolonging bioactivity. The results demonstrated that the two scaffold types were effective in providing different metal salt release quantities and/or profiles which in turn influenced the antibacterial activity of the scaffolds against S. aureus. It was also found

that the scaffolds did not elicit a significant toxic effect towards mammalian cells, some scaffolds supported osteogenesis, and all copper-incorporated scaffolds enhanced angiogenesis.

In the study presented in Chapter 4 of this thesis, a range of silver-doped hydroxyapatites were successfully produced and incorporated into collagen scaffolds. The results demonstrate that the composite silver-doped hydroxyapatite /collagen scaffolds demonstrate enhanced microarchitectural and mechanical properties vs. the collagen control, whilst also demonstrating potent antibacterial activity. Although the scaffolds showed toxicity towards mammalian cells in vitro, an assessment in an in vivo environment may reveal the true cytocompatibility of the scaffolds, as shown previously in the literature for similar scaffolds. Further fine-tuning of the dose of silver-doped hydroxyapatite or its bioactivity through e.g. size or shape changes may also be required to maximise the potential of this nascent system.

In Chapter 5 a second multifunctional material – copper-doped bioactive glass – was tested as an alternative to silver-doped hydroxyapatite due to its potential to enhance both osteogenesis and angiogenesis while retaining antibacterial potency. In addition to promoting osteogenesis and angiogenesis in vitro, the scaffolds developed were capable of significant antibacterial activity. Most promisingly, when tested in an in vivo chick embryo ex ovo model, the copper-doped bioactive glass scaffolds were not only biocompatible, showing no signs of toxicity, but also demonstrated the same pattern of enhanced osteogenesis and angiogenesis as the in vitro studies.

Collectively, the research in this thesis presents a number of potential single-stage solutions for osteomyelitis treatment. Such strategies potentially reduce the need for antibiotics and bone grafting, reducing hospital stays and costs. In addition, the platform systems developed in Chapters 3 – 5 might be further modified and used for the controlled delivery of an array of antimicrobial and therapeutic metal ions depending on the intended application, making them attractive candidates for other indication beyond bone including soft tissue applications such as wound healing.