Role of Implant Nanotopography and Bioactive Coating on Osseointegration and Bacterial Growth

Abstract

Titanium (Ti) is widely used for orthopaedic and dental implants. Osseointegration of implants is critical to success. Surface topography, and chemistry can be important in maximising material influence on bone forming mesenchymal stem cells (MSCs). Implant infection is also a serious issue with many implants failing due to septic loosening. High aspect ratio topographies have shown a potential antibacterial effect that could be utilised to help prevent implant failure. Engineering these surface characteristics has been proved hard to balance without detriment to each other – i.e. antibacterial surfaces are not good for osseointegration. The focus of this thesis is the development of novel antimicrobial/osteogenic surfaces to increase implant lifetime and also tackle the modulus mismatch problem between Ti and bone, which leads to implant micromotion and failure, using a 3D printing approach. Alkaline hydrothermal treatment was applied to produce antimicrobial high- aspect ratio nanotopographies on the Ti scaffolds. A polyethylacrylate (PEA) coating was applied via plasma polymerisation that interacts specifically with fibronectin (FN) to adsorb ultra-low doses of bone morphogenic protein 2 (BMP2) coating to help deliver osteogenesis on the antimicrobial features. MSC bone mineralisation in response to the test substrates was examined using qRT-PCR, Raman spectroscopy, calcein blue, Alizarin red and Giemsa staining. Pseudomonas aeruginosa were cultured on the substrates, and the number of viable microbial cells was determined by quantitation of the ATP present and live/dead staining. In addition, changes in bacterial metabolomics during culture on different implants were investigated. Hence, another aim of this project was to elucidate the effect of P . aeruginosa and their quorum-sensing signal molecules (QSSMs) on MSCs adhesion and growth when cultured on the Ti implants. Two different QSSMs were used in this study, N-(3-oxododecanoyl)homoserine lactone (C12-HSL) and N- butanoylhomoserine lactone (C4-HSL) and MSC viability was analysed by flow cytometry using annexin V, JC-1 and cell cycle analysis. Ti surfaces with the polymer coating (pPEA/FN/BMP2) showed an improvement in MSCs growth, adhesion and bone mineralisation compared with uncoated substrates. Moreover, Ti nanowire coated surfaces displayed a decrease of P. aeruginosa adhesion based on the ATP present and a downregulation for some of 2 the amino acids and nucleotides that are responsible for the biofilm formation. A significant reduction in MSCs viability was observed when cultured on the flat surfaces in the presence of C12-HSL but not in the cells cultured on the coated nanowires substrate. 3D Ti scaffolds with different diameters (300, 600, and 900 μm) were produced using the selective laser melting technique (SLM). Compression and cell testing showed that the 3D Ti lattices with 900 μm diameter struts showed a better cell response and also the closest modulus match to cortical bone, while the 3D nanowire scaffolds showed a potential bactericidal effect on P. aeruginosa. In conclusion, this work represents a new strategy that has a potential to fabricate Ti implant materials with topographies that reduce microbial viability and polymer coatings that capture bone driving growth factors to enhance osteogenesis of MSCs in vitro.