Development of Multifunctional Bactericidal Micro-nanostructured Titanium Surfaces with Appropriate Osteogenic and Inflammatory Responses
Creators: name: Martins de Sousa, Karolinne
Publisher: name: RMIT University
Year: 2024
Subjects: classificationCode: 510501
subject: Biological physics
classificationCode: 401806
subject: Nanomanufacturing
classificationCode: 401605
subject: Functional materials

description: Ti and its alloys have found widespread applications in the field of orthopaedic materials owing to their distinguished mechanical characteristics. However, despite the advantageous use of Ti, these medical devices still continue to face significant challenges pertaining to the limited interfacial bonding between the biomaterial and host-tissue, as well as their vulnerability to bacterial infections. Inadequate osseointegration and bacterial infections are prevalent causes of revision surgeries, with reported occurrences as high as 20% of all cases. Surface micropatterning is currently being utilized as a commercial standard surface treatment to enhance the attachment of host cells and orthopaedic implants. Nonetheless, microstructured surfaces do not provide effective bactericidal actions. In order to fill this gap, the first scope of this study was to design Ti surfaces composed of micro-nanolevel architectures developed using a cost-effective and commercially scalable methodology, representing a potential alternative method for fabricating orthopaedic implants. Thus far, current existing fabrication approaches have failed to provide surface patterning with the objective to support positive behaviours of osteoblasts cells using microscale patterns while, concomitantly, possess sharp nanostructures to mechanically inactivate bacterial cells. The micro-nanostructured surface developed in this work serve as an excellent support for osteoblast cells, while also demonstrating exceptional antibacterial properties with only 0.6% of the P. aeruginosa and 5% S. aureus bacterial cells remaining viable when compared to control non-structured surfaces. The findings of this study indicate that the synergistic effect of a moderate micro-roughness combined with a more densely packed nanostructure configuration is more propitious for the enhancement of MG-63 osteoblast-like cell responses and the mechanical inactivation of pathogenic bacterial cells.
Several published works on bactericidal surfaces have supported the efficiency of certain nanofeatures to eliminate pathogenic bacterial cells using mechanical means, without the need of chemical functionalisation that can eventually induce antimicrobial resistance. Nevertheless, once the device has been implanted into the host, serum proteins are immediately adsorbed into its surface, potentially compromising the bactericidal action of the surface nanostructures. Presently, the interaction of human serum protein with complex nanostructured surfaces and its effects on the surface’s bactericidal performance remains unclear. This thesis addresses this concern by investigating the adsorption of human serum protein on nanosheet-like Ti surfaces at increasing concentrations of 0.1, 1, 10 and 100%. The adsorption of human serum protein formed aggregates, resulting in an increase in the nanosheet edge thickness from 10 nm (surfaces without protein adsorption) to up to 21 nm (surfaces incubated with 100% human serum protein). Despite protein adsorption and their consequential geometric alterations of the surface topography, the nanostructures were found to remain active, exhibiting high levels of bactericidal properties. Ti nanosheet surfaces incubated with 100% human serum protein were able to efficiently inactivate 81.7 ± 11.0% and 70.1 ± 9.3% of P. aeruginosa and S. aureus cells, respectively. Remarkably, protein-conditioned nanosheet surfaces exhibited an increased antibiofouling behaviour, with a significant reduction of attached bacterial cells for both strains when compared to nanosheet-like Ti surfaces without an adsorbed protein layer. Specifically, when nanostructured surfaces were exposed to 100% human serum protein, the extent of bacterial adhesion was significantly decreased, with reductions of approximately 9-fold and 20-fold observed for S. aureus and P. aeruginosa, respectively, in comparison to surfaces without an adsorbed protein layer.
Following protein adsorption, inflammation is the first hurdle to be overcome after a device has been placed in the body, directly dictating the fate of implantation, as macrophage cells remain present in the implantation sites for long periods of time, influencing the biological actions that will lead to tissue healing or implant failure. When considering mechano-bactericidal surfaces as a potential substitute for conventional antibiotic treatments, significant research efforts have focused predominantly on bacterial inactivation, whereas the potential host immunological responses are often neglected. In order to fulfill the abovementioned gap, this thesis addresses the assessment of the inflammatory response of RAW 264.7 macrophage cells towards Ti nanosheets-like surfaces using an in vitro S. aureus infection model. The Ti nanosheets were able to effectively inactivate attaching bacterial cells, while concomitantly promoting the polarisation of macrophage cells towards M2 phenotype, associated with wound healing and tissue repair. In contrast, macrophage cells grown on non-nanostructured Ti surfaces activated the M1-like polarised phenotype, indicating the persistent inflammation resulting from exposure to S. aureus cells.
The research described in this thesis constitutes an advancement in our comprehension of the interplay between bactericidal micro-nanostructured Ti surfaces and the adherent eukaryotic cells pivotal in the context of wound healing subsequent to orthopaedic implantation. Furthermore, it provides significant insights into the protein adsorption and the immunomodulatory attributes exhibited by the Ti nanosheets here studied and their potential application as a future generation of implantable biomaterials. This work will hopefully inspire the development of advanced multifunctional orthopaedic implant surfaces.