Biofilm viability checker: An open-source tool for automated biofilm viability analysis from confocal microscopy images.

Quantifying biofilm formation on surfaces is challenging because traditional microbiological methods, such as total colony-forming units (CFUs), often rely on manual counting. These are laborious, resource intensive techniques, more susceptible to human error. Confocal laser scanning microscopy (CLSM) is a high-resolution technique that allows 3D visualisation of biofilm architecture. In combination with a live/dead stain, it can be used to quantify biofilm viability on both transparent and opaque surfaces. However, there is little consensus on the appropriate methodology to apply in confocal micrograph processing. In this study, we report the development of an image analysis approach to repeatably quantify biofilm viability and surface coverage. We also demonstrate its use for a range of bacterial species and translational applications. This protocol has been created with ease of use and accessibility in mind, to enable researchers who do not specialise in computational techniques to be confident in applying these methods to analyse biofilm micrographs. Furthermore, the simplicity of the method enables the user to adapt it for their bespoke needs. Validation experiments demonstrate the automated analysis is robust and accurate across a range of bacterial species and an improvement on traditional microbiological analysis. Furthermore, application to translational case studies show the automated method is a reliable measurement of biomass and cell viability. This approach will ensure image analysis is an accessible option for those in the microbiology and biomaterials field, improve current detection approaches and ultimately support the development of novel strategies for preventing biofilm formation by ensuring comparability across studies.

A review of co-culture models to study the oral microenvironment and disease.

Co-cultures allow for the study of cell-cell interactions between different eukaryotic species or with bacteria. Such an approach has enabled researchers to more closely mimic complex tissue structures. This review is focused on co-culture systems modelling the oral cavity, which have been used to evaluate this unique cellular environment and understand disease progression. Over time, these systems have developed significantly from simple 2D eukaryotic cultures and planktonic bacteria to more complex 3D tissue engineered structures and biofilms. Careful selection and design of the co-culture along with critical parameters, such as seeding density and choice of analysis method, have resulted in several advances. This review provides a comparison of existing co-culture systems for the oral environment, with emphasis on progression of 3D models and the opportunity to harness techniques from other fields to improve current methods. While filling a gap in navigating this literature, this review ultimately supports the development of this vital technique in the field of oral biology.

A call for action to the biomaterial community to tackle antimicrobial resistance.

The global surge of antimicrobial resistance (AMR) is a major concern for public health and proving to be a key challenge in modern disease treatment, requiring action plans at all levels. Microorganisms regularly and rapidly acquire resistance to antibiotic treatments and new drugs are continuously required. However, the inherent cost and risk to develop such molecules has resulted in a drying of the pipeline with very few compounds currently in development. Over the last two decades, efforts have been made to tackle the main sources of AMR. Nevertheless, these require the involvement of large governmental bodies, further increasing the complexity of the problem. As a group with a long innovation history, the biomaterials community is perfectly situated to push forward novel antimicrobial technologies to combat AMR. Although this involvement has been felt, it is necessary to ensure that the field offers a united front with special focus in areas that will facilitate the development and implementation of such systems. This paper reviews state of the art biomaterials strategies striving to limit AMR. Promising broad-spectrum antimicrobials and device modifications are showcased through two case studies for different applications, namely topical and implantables, demonstrating the potential for a highly efficacious physical and chemical approach. Finally, a critical review on barriers and limitations of these methods has been developed to provide a list of short and long-term focus areas in order to ensure the full potential of the biomaterials community is directed to helping tackle the AMR pandemic.

Dynamic viscoelastic characterisation of human osteochondral tissue: understanding the effect of the cartilage-bone interface.

BACKGROUND: Despite it being known that subchondral bone affects the viscoelasticity of cartilage, there has been little research into the mechanical properties of osteochondral tissue as a whole system. This study aims to unearth new knowledge concerning the dynamic behaviour of human subchondral bone and how energy is transferred through the cartilage-bone interface. METHODS: Dynamic mechanical analysis was used to determine the frequency-dependent (1-90 Hz) viscoelastic properties of the osteochondral unit (cartilage-bone system) as well as isolated cartilage and bone specimens extracted from human femoral heads obtained from patients undergoing total hip replacement surgery, with a mean age of 78 years (N = 5, n = 22). Bone mineral density (BMD) was also determined for samples using micro-computed tomography as a marker of tissue health. RESULTS: Cartilage storage and loss moduli along with bone storage modulus were found to increase logarithmically (p < 0.05) with frequency. The mean cartilage storage modulus was 34.4 ± 3.35 MPa and loss modulus was 6.17 ± 0.48 MPa (mean ± standard deviation). In contrast, bone loss modulus decreased logarithmically between 1 and 90 Hz (p < 0.05). The storage stiffness of the cartilage-bone-core was found to be frequency-dependent with a mean value of 1016 ± 54.0 N.mm- 1, while the loss stiffness was determined to be frequency-independent at 78.84 ± 2.48 N.mm- 1. Notably, a statistically significant (p < 0.05) linear correlation was found between the total energy dissipated from the isolated cartilage specimens, and the BMD of the isolated bone specimens at all frequencies except at 90 Hz (p = 0.09). CONCLUSIONS: The viscoelastic properties of the cartilage-bone core were significantly different to the tissues in isolation (p < 0.05). Results from this study demonstrate that the functionality of these tissues arises because they operate as a unit. This is evidenced through the link between cartilage energy dissipated and bone BMD. The results may provide insights into the functionality of the osteochondral unit, which may offer further understanding of disease progression, such as osteoarthritis (OA). Furthermore, the results emphasise the importance of studying human tissue, as bovine models do not always display the same trends.