Telocytes: current methods of research, challenges and future perspectives.

Telocytes (TCs) are CD34-positive interstitial cells that have long cytoplasmic projections, called telopodes; they have been identified in several organs and in various species. These cells establish a complex communication network between different stromal and epithelial cell types, and there is growing evidence that they play a key role in physiology and pathology. In many tissues, TC network impairment has been implicated in the onset and progression of pathological conditions, which makes the study of TCs of great interest for the development of novel therapies. In this review, we summarise the main methods involved in the characterisation of these cells as well as their inherent difficulties and then discuss the functional assays that are used to uncover the role of TCs in normal and pathological conditions, from the most traditional to the most recent. Furthermore, we provide future perspectives in the study of TCs, especially regarding the establishment of more precise markers, commercial lineages and means for drug delivery and genetic editing that directly target TCs.

Laser texturing of additively manufactured implants: A tool to programme biological response.

The advent of additive manufacturing (AM) is rapidly shaping healthcare technologies pushing forward personalisation and enhanced implant functionalisation to improve clinical outcomes. AM techniques such as powder bed fusion (PBF) have been adopted despite the need to modify the as-built surface post manufacture. Medical device manufacturers have focused their efforts on refining various physical and chemical surface finishing approaches, however there is little consensus and some methods risk geometry alteration or contamination. This has led to a growing interest in laser texturing technologies to engineer the device surface. Herein, several bioinspired micro and nano textures were applied to laser PBF Ti-6Al-V4 substrates to alter physicochemical properties and in-turn we sought to understand what influences these alterations had on a human osteosarcoma cell line (MG63). Significant variations in roughness and time dependent contact angles were revealed between different patterns provide a tool to elicit desired biological responses. All surface treatments effectively enhanced early cell behaviour and in particular coverage was increased for the micro-textures. Influence of the patterns on cell differentiation was less consistent with alkaline phosphatase content increased only for the channel, grid and dual textures. While long term (21 days) mineralisation was found to be significantly enhanced in grids, dual, triangles and shark skin textures. Further regression analysis of all physicochemical and biological variables indicated that several properties should be used to strongly correlate cell behaviour, resulting in 82 % of the 21 day mineralisation dataset explained through a combination of roughness kurtosis and glycerol contact angle. Overall, this manuscript demonstrates the ability of laser texturing to offer tailored cell-surface interactions, which can be tuned to offer a tool to drive functional customisation of anatomically customised medical devices.

Stakeholder Perspectives on the Current and Future of Additive Manufacturing in Healthcare.

Additive manufacturing (AM) technologies have disrupted many supply chains by making new designs and functionalities possible. The opportunity to realize complex customized structures has led to significant interest within healthcare; however, full utilization critically requires the alignment of the whole supply chain. To offer insights into this process, a survey was conducted to understand the views of different medical AM stakeholders. The results highlighted an agreement between academics, designers, manufacturers, and medical experts, that personalization and design control are the main benefits of AM. Interestingly, surface finish was consistently identified as an obstacle. Nevertheless, there was a degree of acceptance that post-processing was necessary to achieve appropriate quality control. Recommendations were made for extending the use of in situ process monitoring systems to support improved reproducibility. Variations in the future vision of AM were highlighted between stakeholder groups and areas of interest for development noted for each stakeholder. Collectively, this survey indicates that medical stakeholders agree on the capabilities of AM but have different priorities for its implementation and progression. This highlights a degree of disconnection among the supply chain at a ground level; thus, collaboration on AM specific standards and enhancement of communication between stakeholders from project inception is recommended.

Microstructural Evolution, Mechanical Properties, and Preosteoblast Cell Response of a Post-Processing-Treated TNT5Zr ß Ti Alloy Manufactured via Selective Laser Melting.

A Ti-34Nb-13Ta-5Zr (TNT5Zr) ß Ti alloy with a high strength-to-modulus ratio has been developed, showing its potential to become another candidate material in load-bearing implant applications. This work mainly investigates the microstructural evolution, mechanical properties, and biocompatibility of a post-processing-treated TNT5Zr alloy manufactured via selective laser melting (SLM). Transmission electron microscopy observation shows the existence of the single beta grain matrix and alpha precipitates along the grain boundary in the SLM + HIP manufactured TNT5Zr alloy (TNT5Zr-AF + HIP), and ellipsoidal nano-sized intragranular α″ precipitates (approx. 5-10 nm) were introduced after the subsequent low-temperature aging treatment. The precipitation strengthening enables the SLM + HIP + aging manufactured TNT5Zr (TNT5Zr-AF + HIPA) alloy to show a comparable ultimate tensile strength (853 ± 9 MPa) to that of the reference material (Ti64-AF + HIP, 926 ± 23 MPa). Including the inferior notch-like surface of the test pieces, the slip-band cracking that occurs in this ductile TNT5Zr-AF + HIPA alloy is regarded as the main factor in determining its fatigue strength (170 MPa). In vitro short-term biocompatibility evaluation reveals almost no significant difference in the preosteoblast viability, differentiation, and mineralization between TNT5Zr-AF + HIPA and the reference biomaterial (Ti64-AF + HIP).

The influence of zirconium content on the microstructure, mechanical properties, and biocompatibility of in-situ alloying Ti-Nb-Ta based ß alloys processed by selective laser melting.

This study investigates Ti-Nb-Ta based ß alloys with different zirconium additions (0, 5, 9 wt%) manufactured by SLM. A low level of as-fabricated defects is obtained: the relative density of TNT (Z) alloys is >99.97% with the keyhole size in a range of 3-20 µm. BF TEM images combining SAD patterns of TNT(Z) alloys show single ß phase obtained inside the beta matrix; BF-STEM images reveal potential nano-scale grain boundary alpha phase precipitation. Zirconium functions as a neutral element in these high ß-stabilized Ti-Nb-Ta based alloys. An increase in Vickers hardness and UTS caused by zirconium additions is observed, which is explained by beta grain refinement because higher degree of undercooling occurs. Corrosion ions of TNT(Z) alloys released from immersion testing at each time intervals show extremely small concentrations (<10 µg/L). It indicated that good biocompatibility during culture with the negligible corrosion ions. High strength-to-modulus ratio ß Ti alloys together with excellent biological response show their prospect for biomedical applications.

Methacrylated Silk Fibroin Hydrogels: pH as a Tool to Control Functionality.

The last decade has witnessed significant progress in the development of photosensitive polymers for in situ polymerization and 3D printing applications. Light-mediated sol-gel transitions have immense potential for tissue engineering applications as cell-laden materials can be crosslinked within minutes under mild environmental conditions. Silk fibroin (SF) is extensively explored in regenerative medicine applications due to its ease of modification and exceptional mechanical properties along with cytocompatibility. To efficiently design SF materials, the in vivo assembly of SF proteins must be considered. During SF biosynthesis, changes in pH, water content, and metal ion concentrations throughout the silkworm gland divisions drive the transition from liquid silk to its fiber form. Herein, we study the effect of the glycidyl-methacrylate-modified SF (SilkMA) solution pH on the properties and secondary structure of SilkMA hydrogels by testing formulations prepared at pH 5, 7, and 8. Our results demonstrate an influence of the prepolymer solution pH on the hydrogel rheological properties, compressive modulus, optical transmittance, and network swellability. The hydrogel pH did not affect the in vitro viability and morphology of human dermal fibroblasts. This work demonstrates the utility of the solution pH to tailor the SilkMA conformational structure development toward utility and function and shows the need to strictly control the pH to reduce batch-to-batch variability and ensure reproducibility.

Repeated exposure of nosocomial pathogens to silver does not select for silver resistance but does impact ciprofloxacin susceptibility.

The rise of antimicrobial resistant bacteria coupled with a void in antibiotic development marks Antimicrobial Resistance as one of the biggest current threats to modern medicine. Antimicrobial metals are being developed and used as alternative anti-infectives, however, the existence of known resistance mechanisms and limited data regarding bacterial responses to long-term metal exposure are barriers to widespread implementation. In this study, a panel of reference and clinical strains of major nosocomial pathogens were subjected to serial dosage cycles of silver and ciprofloxacin. Populations exposed to silver initially showed no change in sensitivity, however, increasingly susceptibility was observed after the 25th cycle. A control experiment with ciprofloxacin revealed a selection for resistance over time, with silver treated bacteria showing faster adaptation. Morphological analysis revealed filamentation in Gram negative species suggesting membrane perturbation, while sequencing of isolated strains identified mutations in numerous genes. These included those encoding for efflux systems, chemosensory systems, stress responses, biofilm formation and respiratory chain processes, although no consistent locus was identified that correlated with silver sensitivity. These results suggest that de novo silver resistance is hard to select in a range of nosocomial pathogens, although silver exposure may detrimentally impact sensitivity to antibiotics in the long term. STATEMENT OF SIGNIFICANCE: The adaptability of microbial life continuously calls for the development of novel antibiotic molecules, however, the cost and risk associated with their discovery have led to a drying up in the pipeline, causing antimicrobial resistance (AMR) to be a major threat to healthcare. From all available strategies, antimicrobial metals and, more specifically, silver showcase large bactericidal spectrum and limited toxic effect which coupled with a large range of processes available for their delivery made these materials as a clear candidate to tackle AMR. Previous reports have shown the ability of this metal to enact a synergistic effect with other antimicrobial therapies, nevertheless, the discovery of Ag resistance mechanisms since the early 70s and limited knowledge on the long term influence of silver on AMR poses a threat to their applicability. The present study provides quantitative data on the influence of silver based therapies on AMR development for a panel of reference and clinical strains of major nosocomial pathogens, revealing that prolonged silver exposure may detrimentally impact sensitivity to antibiotics.

Formulation of inherently antimicrobial magnesium oxychloride cement and the effect of supplementation with silver phosphate.

The growing threat of bacterial resistance to antibiotics is driving an increasing need for new antimicrobial strategies. This work demonstrates the potential of magnesium oxychloride cements (MOC) to be used as inorganic antimicrobial biomaterials for bone augmentation. An injectable formulation was identified at a powder to liquid ratio of 1.4 g mL-1, with an initial setting time below 30 mins and compressive strength of 35 ± 9 MPa. Supplementation with Ag3PO4 to enhance the antimicrobial efficacy of MOC was explored, and shown via real time X-ray diffraction to retard the formation of hydrated oxychloride phases by up to 30%. The antimicrobial efficacy of MOC was demonstrated in vitro against Staphylococcus aureus and Pseudomonas aeruginosa, forming zones of inhibition and significantly reducing viability in broth culture. Enhanced efficacy was seen for silver doped formulations, with complete eradication of detectable viable colonies within 3 h, whilst retaining the cytocompatibility of MOC. Investigating the antimicrobial mode of action revealed that Mg and Ag release and elevated pH contributed to MOC efficacy. Sustained silver release was demonstrated over 14 days, suggesting the Ag3PO4 modified formulation offers two mechanisms of infection treatment, combining the inherent antimicrobial properties of MOC with controlled release of inorganic antimicrobials.

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 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.

Selective Laser Melting of Ti-6Al-4V: The Impact of Post-processing on the Tensile, Fatigue and Biological Properties for Medical Implant Applications.

One of the main challenges in additive manufacturing (AM) of medical implants for the treatment of bone tissue defects is to optimise the mechanical and biological performance. The use of post-processing can be a necessity to improve the physical properties of customised AM processed implants. In this study, Ti-6Al-4V coupons were manufactured using selective laser melting (SLM) in two build orientations (vertical and horizontal) and subsequently post-processed using combinations of hot isostatic pressing (HIP), sandblasting (SB), polishing (PL) and chemical etching (CE). The effect of the different post-manufacturing strategies on the tensile and fatigue performance of the SLMed parts was investigated and rationalised by observing the surface topography. Vertically built samples showed higher yield strength (YS) and ultimate tensile strength (UTS) than the horizontal samples, increasing from 760.9 ± 22.3 MPa and 961.3 ± 50.2 MPa in the horizontal condition to 820.09 ± 16.5 MPa and 1006.7 ± 6.3 MPa in the vertical condition, respectively. After the HIP treatment, the ductility was substantially improved in both orientations; by 2.1 and 2.9 folds in the vertical and horizontal orientations, respectively. The vertically built samples demonstrated a superior ductility of 22% following HIP and polishing. Furthermore, chemical etching was found to be the most effective surface post-processing treatment to improve the fatigue performance after HIP, achieving the highest run-out strength of 450 MPa. Most importantly, chemical etching after HIP enhanced the cellular affinity of the surface, in addition to its good fatigue performance, making it a promising post-processing approach for bone implants where tissue integration is needed.

Tuning the Mechanical and Antimicrobial Performance of a Cu-Based Metallic Glass Composite through Cooling Rate Control and Annealing.

The influence of cooling rate on the wear and antimicrobial performance of a Cu52Z41Al7 (at. %) bulk metallic glass (BMG) composite was studied and the results compared to those of the annealed sample (850 °C for 48 h) and to pure copper. The aim of this basic research is to explore the potential use of the material in preventing the spread of infections. The cooling rate is controlled by changing the mould diameter (2 mm and 3 mm) upon suction casting and controlling the mould temperature (chiller on and off). For the highest cooling rate conditions CuZr is formed but CuZr2 starts to crystallise as the cooling rate decreases, resulting in an increase in the wear resistance and brittleness, as measured by scratch tests. A decrease in the cooling rate also increases the antimicrobial performance, as shown by different methodologies (European, American and Japanese standards). Annealing leads to the formation of new intermetallic phases (Cu10Zr7 and Cu2ZrAl) resulting in maximum scratch hardness and antimicrobial performance. However, the annealed sample corrodes during the antimicrobial tests (within 1 h of contact with broth). The antibacterial activity of copper was proved to be higher than that of any of the other materials tested but it exhibits very poor wear properties. Cu-rich BMG composites with optimised microstructure would be preferable for some applications where the durability requirements are higher than the antimicrobial needs.

Antibacterial Metallic Touch Surfaces.

Our aim is to present a comprehensive review of the development of modern antibacterial metallic materials as touch surfaces in healthcare settings. Initially we compare Japanese, European and US standards for the assessment of antimicrobial activity. The variations in methodologies defined in these standards are highlighted. Our review will also cover the most relevant factors that define the antimicrobial performance of metals, namely, the effect of humidity, material geometry, chemistry, physical properties and oxidation of the material. The state of the art in contact-killing materials will be described. Finally, the effect of cleaning products, including disinfectants, on the antimicrobial performance, either by direct contact or by altering the touch surface chemistry on which the microbes attach, will be discussed. We offer our outlook, identifying research areas that require further development and an overview of potential future directions of this exciting field.