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Type-H capillary endothelial cells control bone formation during embryogenesis and postnatal growth but few signalling mechanisms underpinning this influence have been characterised. Here, we identify a highly expressed type-H endothelial cell protein, Clec14a, and explore its role in coordinating osteoblast activity. Expression of Clec14a and its ligand, Mmrn2 are high in murine type-H endothelial cells but absent from osteoblasts. Clec14a -/- mice have premature condensation of the type-H vasculature and expanded distribution of osteoblasts and bone matrix, increased long-bone length and bone density indicative of accelerated skeletal development, and enhanced osteoblast maturation. Antibody-mediated blockade of the Clec14a-Mmrn2 interaction recapitulates the Clec14a -/- phenotype. Endothelial cell expression of Clec14a regulates osteoblast maturation and mineralisation activity during postnatal bone development in mice. This finding underscores the importance of type-H capillary control of osteoblast activity in bone formation and identifies a novel mechanism that mediates this vital cellular crosstalk., (© 2024. The Author(s).)

Chronic wounds are a drain on global health services and remain a major area of unmet clinical need. Chronic wounds are characterised by a stable and stubborn bacterial biofilm which hinders innate immune response and delays or prevents wound healing. Bioactive glass (BG) fibres offer a promising novel treatment for chronic wounds by targeting the wound-associated biofilm. In this study, the antimicrobial properties of silver-doped BG fibres were tested against Pseudomonas aeruginosa biofilms, which are commonly found in chronic wound infections. Results showed that BG fibres doped with silver resulted in a 5log10 reduction in biofilm formation whereas silver-free fibres only reduced formation by log10, therefore silver-doped fibres possess stronger antimicrobial effects. Moreover, there appeared to be a synergistic effect between the fibres and the silver as the application of the silver-doped fibres placed directly in contact with the forming biofilm resulted in a higher reduction in biofilm formation compared to treatments either: using the dissolution ions, using BG powder, or when the fibres were placed in an insert above the biofilm, inhibiting physical contact, instead. This suggests that the physical properties of the fibres, as well as silver, influence biofilm formation. Finally, results demonstrated that silver chloride, which is not antimicrobial, forms and the concentrations of antimicrobial silver species, namely silver ions and nanoparticles, reduce over time when fibres are soaked in cell culture media, which partially explains why the silver-doped dissolution ions contained lower antimicrobial activity compared to the fibres. As silver chloride is more likely to form with increased temperature and time, the antimicrobial activity of silver-containing dissolution ions is highly dependent on the length of ageing and storage conditions. Many studies investigate the antimicrobial and cytotoxic properties of biomaterials through their dissolution products. However, instability of antimicrobial silver species due to silver chloride formation and its effect on antimicrobial properties of silver-based biomaterials has not been reported before and could influence past and future dissolution-based assays as results showed that the antimicrobial activity of silver-based dissolution ions can vary greatly depending on post processing steps and can therefore produce misleading data., Competing Interests: The authors declare the following financial interests/personal relationships which may be considered as potential competing interests:Sandeep Shirgill reports financial support was provided by 10.13039/501100000268Biotechnology and Biological Sciences Research Council. Gowsihan Poologasundarampillai reports financial support was provided by 10.13039/501100000266Engineering and Physical Sciences Research Council., (© 2023 The Authors.)

We propose a novel image analysis framework to automate analysis of X-ray microtomography images of sintering ceramics and glasses, using open-source toolkits and machine learning. Additive manufacturing (AM) of glasses and ceramics usually requires sintering of green bodies. Sintering causes shrinkage, which presents a challenge for controlling the metrology of the final architecture. Therefore, being able to monitor sintering in 3D over time (termed 4D) is important when developing new porous ceramics or glasses. Synchrotron X-ray tomographic imaging allows in situ, real-time capture of the sintering process at both micro and macro scales using a furnace rig, facilitating 4D quantitative analysis of the process. The proposed image analysis framework is capable of tracking and quantifying the densification of glass or ceramic particles within multiple volumes of interest (VOIs) along with structural changes over time using 4D image data. The framework is demonstrated by 4D quantitative analysis of bioactive glass ICIE16 within a 3D-printed scaffold. Here, densification of glass particles within 3 VOIs were tracked and quantified along with diameter change of struts and interstrut pore size over the 3D image series, delivering new insights on the sintering mechanism of ICIE16 bioactive glass particles in both micro and macro scales., (© 2021 The Authors. Journal of the American Ceramic Society published by Wiley Periodicals LLC on behalf of American Ceramic Society.)

Hydrogels constructed from naturally derived polymers provide an aqueous environment that encourages cell growth, however, mechanical properties are poor and degradation can be difficult to predict. Whilst, synthetic hydrogels exhibit some improved mechanical properties, these materials lack biochemical cues for cells growing and have limited biodegradation. To produce hydrogels that support 3D cell cultures to form tissue mimics, materials must exhibit appropriate biological and mechanical properties. In this study, novel organic-inorganic hybrid hydrogels based on chitosan and silica were prepared using the sol-gel technique. The chemical, physical and biological properties of the hydrogels were assessed. Statistical analysis was performed using One-Way ANOVAs and independent-sample t -tests. Fourier transform infrared spectroscopy showed characteristic absorption bands including amide II, Si-O and Si-O-Si confirming formation of hybrid networks. Oscillatory rheometry was used to characterise the sol to gel transition and viscoelastic behaviour of hydrogels. Furthermore, in vitro degradation revealed both chitosan and silica were released over 21 days. The hydrogels exhibited high loading efficiency as total protein loading was released in a week. There were significant differences between TC 2 G and C 2 G at all-time points ( p < 0.05). The viability of osteoblasts seeded on, and encapsulated within, the hydrogels was >70% over 168 h culture and antimicrobial activity was demonstrated against Pseudomonas aeruginosa and Enterococcus faecalis . The hydrogels developed here offer alternatives for biopolymer hydrogels for biomedical use, including for application in drug/cell delivery and for bone tissue engineering.

A correlative imaging methodology was developed to accurately quantify bone formation in the complex lattice structure of additive manufactured implants. Micro computed tomography (μCT) and histomorphometry were combined, integrating the best features from both, while demonstrating the limitations of each imaging modality. This semi-automatic methodology registered each modality using a coarse graining technique to speed the registration of 2D histology sections to high resolution 3D μCT datasets. Once registered, histomorphometric qualitative and quantitative bone descriptors were directly correlated to 3D quantitative bone descriptors, such as bone ingrowth and bone contact. The correlative imaging allowed the significant volumetric shrinkage of histology sections to be quantified for the first time (~15 %). This technique demonstrated the importance of location of the histological section, demonstrating that up to a 30 % offset can be introduced. The results were used to quantitatively demonstrate the effectiveness of 3D printed titanium lattice implants.

Bioactive glass has high potential for bone regeneration due to its ability to bond to bone and stimulate osteogenesis whilst dissolving in the body. Although three-dimensional (3-D) bioactive glass scaffolds with favorable pore networks can be made from the sol-gel process, compositional and structural evolutions in their porous structures during degradation in vivo, or in vitro, have not been quantified. In this study, bioactive glass scaffolds were put in a simulated body fluid flow environment through a perfusion bioreactor. X-ray microtomography (μCT) was used to non-destructively image the scaffolds at different degradation stages. A new 3-D image processing methodology was developed to quantify the scaffold's pore size, interconnect size and connectivity from μCT images. The accurate measurement of individual interconnect size was made possible by a principal component analysis-based algorithm. During 28 days of dissolution, the modal interconnect size in the scaffold was reduced from 254 to 206 μm due to the deposition of mineral phases. However, the pore size remained unchanged, with a mode of 682 μm. The data presented are important for making bioactive glass scaffolds into clinical products. The technique described for imaging and quantifying scaffold pore structures as a function of degradation time is applicable to most scaffold systems., (Copyright © 2011 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.)

X-ray microtomography (microCT) is a popular tool for imaging scaffolds designed for tissue engineering applications. The ability of synchrotron microCT to monitor tissue response and changes in a bioactive glass scaffold ex vivo were assessed. It was possible to observe the morphology of the bone; soft tissue ingrowth and the calcium distribution within the scaffold. A second aim was to use two newly developed compression rigs, one designed for use inside a laboratory based microCT machine for continual monitoring of the pore structure and crack formation and another designed for use in the synchrotron facility. Both rigs allowed imaging of the failure mechanism while obtaining stress-strain data. Failure mechanisms of the bioactive glass scaffolds were found not to follow classical predictions for the failure of brittle foams. Compression strengths were found to be 4.5-6 MPa while maintaining an interconnected pore network suitable for tissue engineering applications.

The need to shift from tissue replacement to tissue regeneration has led to the development of tissue engineering and in situ tissue regeneration. Both of these strategies often employ the use of scaffolds--templates that allow cells to attach and then guide the new tissue growth. There are many design criteria for an ideal scaffold. These criteria vary depending on the tissue type and location in the body. In any application of a scaffold it is vital to be able to characterise the scaffold before it goes into in vitro testing. In vitro testing allows the cell response to be investigated before its in vivo performance is assessed. A full characterisation of events in vitro and in vivo, in three dimensions (3D), is necessary if a scaffold's performance and effectiveness is to be fully quantified. This paper focuses on porous scaffolds for bone regeneration, suggests appropriate design criteria for a bone regenerating scaffold and then reviews techniques for obtaining the vitally important quantification of its pore structure. The techniques discussed will include newly developed methods of quantifying X-ray microtomography (microCT) images in 3D and for predicting the scaffolds mechanical properties and the likely paths of fluid flow (and hence potential cell migration). The complications in investigating scaffold performance in vitro are then discussed. Finally, the use of microCT for imaging scaffolds for in vivo tests is reviewed.

In tissue engineering, porous scaffolds are often used as three-dimensional (3D) supports for tissue growth. In scaffold design, it is imperative to be able to quantify the pore sizes and more importantly the interconnects between the pores. X-ray micro-computed tomography (microCT) has become a popular tool for obtaining 3D images of scaffold biomaterials, however images are only qualitative. In this work, methods were developed for obtaining pore size distributions for both the macropores and their interconnects. Scaffolds have been developed, by foaming sol-gel derived bioactive glasses, which have the potential to fulfil the criteria for an ideal scaffold for bone tissue engineering. MicroCT images were obtained from scaffolds with different pore structures. The images were thresholded and three algorithms were applied in 3D to identify pores and interconnects and to obtain pore size distributions. The results were validated against mercury intrusion porosimetry and manual 3D image analysis. The microCT data were then meshed such that predictions of permeability as a function of changes in the pore network could be made. Such predictions will be useful for optimising bioreactor conditions for tissue engineering applications. These techniques would be suitable for many other types of scaffolds.