Bone infections (osteomyelitis) are difficult to treat due to the formation of biofilms, antibiotic resistance, and limited penetration of systemic antibiotics to infection site(1). Highly-porous collagen-hydroxyapatite (C-HA) scaffolds have proven capacity to regenerate critical-sized bone defects in vivo and human clinical trials(2). However, they still lack appropriate mechanical properties to support larger defects and weight-bearing applications(2). The objective of this study is to develop a reinforced non-antibiotic antimicrobial-doped hydroxyapatite scaffold to overcome issues surrounding antibiotic resistance, while providing a suitable environment for bone regeneration. The aims are to (i) optimise the delivery of metal-based antimicrobials from the scaffold without impeding pro-osteogenic properties in vitro, (ii) reinforce the metal-based antimicrobial scaffold using 3D printing to enhance the mechanical properties, and (iii) evaluate the reinforced scaffold system in a weight bearing rat femoral defect in vivo.
A range of metal-based antimicrobial doped hydroxyapatite (MBA-HA) doses (formulations not disclosed due to IP restrictions) were incorporated into type-1 collagen matrix to fabricate collagen-MBA-HA (C-MBA-HA) scaffolds. The C-MBA-HA scaffolds were evaluated for cell viability and osteogenesis using rat mesenchymal stromal cells in vitro up to 28 days. The scaffold’s antibacterial properties against S. aureus were assessed in vitro. A 3D-printed polymer framework was combined with the collagen matrix for reinforcement. The compressive modulus, porosity, and microarchitecture were assessed. The reinforced scaffolds were further evaluated in vitro to ensure there were no negative affect of reinforcement on the osteogenic and antimicrobial capacity. Ongoing evaluation of the bone healing potential of C-MBA-HA is being assessed in a 5mm long critical-sized rat femoral defect and will be compared against C-HA and empty defects at 2,4, and 8 weeks using micro-computerisation tomography (μCT) and histology at 8 weeks.
Biomimetic C-HA scaffolds were successfully functionalized with MBA-HA to achieve antimicrobial properties while continuing to support osteogenesis. Specifically, the C-MBA-HA scaffolds resulted in equivalent calcium deposition to the C-HA scaffolds at 28 days, which was further validated with the homogenous distribution of alizarin red staining, i.e., revealing cell-mediated mineralization throughout the scaffolds. The C-MBA-HA scaffolds achieved a 50% reduction of S. aureus, as well as the development of inhibition zones on S. aureus agar plates after 24 hours. The 3D-printed polymer framework was successfully integrated into the C-MBA-HA scaffold to significantly enhance its mechanical properties to better mimic cancellous bone, while maintaining high porosity and an microarchitectural structure favourable for cellular infiltration and bone formation. In vitro cellular and microbial assessment of the reinforced C-MBA-HA scaffolds demonstrated no reduction in beneficial properties when compared to the non-reinforced scaffold.
The successful development of this non-antibiotic antimicrobial and osteoconductive scaffold with enhanced mechanical properties for treatment of weight bearing large defects has the potential to be a one-step local treatment for osteomyelitis.
- Genoud K.J. et al. Mat Tod. 46, 136-154 (2021) 2. Gleeson J.P. et al. Eur Cell Mater. 20, 218-230 (2010)
Funding through Science Foundation Ireland (grant 12/RC/2278 and 17/SP/4721). Co-funded by Johnson & Johnson 3D Printing Innovation & Customer Solutions, Johnson & Johnson Services Inc.