Mature primary human osteocytes in mini organotypic cultures secrete FGF23 and PTH1-34-regulated sclerostin.

Introduction: For decades, functional primary human osteocyte cultures have been crucially needed for understanding their role in bone anabolic processes and in endocrine phosphate regulation via the bone-kidney axis. Mature osteocyte proteins (sclerostin, DMP1, Phex and FGF23) play a key role in various systemic diseases and are targeted by successful bone anabolic drugs (anti-sclerostin antibody and teriparatide (PTH1-34)). However, cell lines available to study osteocytes produce very little sclerostin and low levels of mature osteocyte markers. We have developed a primary human 3D organotypic culture system that replicates the formation of mature osteocytes in bone. Methods: Primary human osteoblasts were seeded in a fibrinogen / thrombin gel around 3D-printed hanging posts. Following contraction of the gel around the posts, cells were cultured in osteogenic media and conditioned media was collected for analysis of secreted markers of osteocyte formation. Results: The organoids were viable for at least 6 months, allowing co-culture with different cell types and testing of bone anabolic drugs. Bulk RNAseq data displayed the developing marker trajectory of ossification and human primary osteocyte formation in vitro over an initial 8- week period. Vitamin D3 supplementation increased mineralization and sclerostin secretion, while hypoxia and PTH1-34 modulated sclerostin. Our culture system also secreted FGF23, enabling the future development of a bone-kidney-parathyroid-vascular multi-organoid or organ-on-a-chip system to study disease processes and drug effects using purely human cells. Discussion: This 3D organotypic culture system provides a stable, long-lived, and regulated population of mature human primary osteocytes for a variety of research applications.

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.

Reducing MRI susceptibility artefacts in implants using additively manufactured porous Ti-6Al-4V structures.

Magnetic Resonance Imaging (MRI) is critical in diagnosing post-operative complications following implant surgery and imaging anatomy adjacent to implants. Increasing field strengths and use of gradient-echo sequences have highlighted difficulties from susceptibility artefacts in scan data. Artefacts manifest around metal implants, including those made from titanium alloys, making detection of complications (e.g. bleeding, infection) difficult and hindering imaging of surrounding structures such as the brain or inner ear. Existing research focusses on post-processing and unorthodox scan sequences to better capture data around these devices. This study proposes a complementary up-stream design approach using lightweight structures produced via additive manufacturing (AM). Strategic implant mass reduction presents a potential tool in managing artefacts. Uniform specimens of Ti-6Al-4V structures, including lattices, were produced using the AM process, selective laser melting, with various unit cell designs and relative densities (3.1%-96.7%). Samples, submerged in water, were imaged in a 3T MRI system using clinically relevant sequences. Artefacts were quantified by image analysis revealing a strong linear relationship (RR2 = 0.99) between severity and relative sample density. Likewise, distortion due to slice selection errors showed a squared relationship (RR2 = 0.92) with sample density. Unique artefact features were identified surrounding honeycomb samples suggesting a complex relationship exists for larger unit cells. To demonstrate clinical utility, a honeycomb design was applied to a representative cranioplasty. Analysis revealed 10% artefact reduction compared to traditional solid material illustrating the feasibility of this approach. This study provides a basis to strategically design implants to reduce MRI artefacts and improve post-operative diagnosis capability. STATEMENT OF SIGNIFICANCE: MRI susceptibility artefacts surrounding metal implants present a clinical challenge for the diagnosis of post-operative complications relating to the implant itself or underlying anatomy. In this study for the first time we demonstrate that additive manufacturing may be exploited to create lattice structures that predictably reduce MRI image artefact severity surrounding titanium alloy implants. Specifically, a direct correlation of artefact severity, both total signal loss and distortion, with the relative material density of these functionalised materials has been demonstrated within clinically relevant MRI sequences. This approach opens the door for strategic implant design, utilising this structurally functionalised material, that may improve post-operative patient outcomes and compliments existing efforts in this area which focus on data acquisition and post-processing methods.

Reactive oxygen: A novel antimicrobial mechanism for targeting biofilm-associated infection.

Reactive oxygen species (ROS) is a novel therapeutic strategy for topical or local application to wounds, mucosa or internal structures where there may be heavy bacterial bioburden with biofilm and chronic inflammation. Bacterial biofilms are a significant problem in clinical settings owing to their increased tolerance towards conventionally prescribed antibiotics and their propensity for selection of further antibacterial resistance. There is therefore a pressing need for the development of alternative therapeutic strategies that can improve antibiotic efficacy towards biofilms. ROS has been successful in treating chronic wounds and in clearing multidrug-resistant organisms, including methicillin-resistant Staphylococcus aureus (MRSA), and carbapenemase-producing isolates from wounds and vascular line sites. There is significant antifungal activity of ROS against planktonic and biofilm forms. Nebulised ROS has been evaluated in limited subjects to assess reductions in bioburden in chronically colonised respiratory tracts. The antibiofilm activity of ROS could have great implications for the treatment of a variety of persistent respiratory conditions. Use of ROS on internal prosthetic devices shows promise. A variety of novel delivery mechanisms are being developed to apply ROS activity to different anatomical sites.

Preparation and characterisation of nanophase Sr, Mg, and Zn substituted hydroxyapatite by aqueous precipitation.

Hydroxyapatite (HA) substituted with 2 mol% Sr, 10 mol% Mg, and 2 mol% Zn were precipitated under identical alkaline conditions (pH 11) at 20°C from an aqueous solution. As-synthesised materials were confirmed to be phase pure by XRD and samples prepared in air contained surface adsorbed CO2 as observed by FTIR. SEM studies revealed a globular morphology and agglomeration behaviour, typical of precipitated nHA. EDS spectra confirmed nominal compositions and substitution of Sr, Mg and Zn. At the levels investigated cationic doping was not found to radically influence particle morphology. An indication of the potential in-vivo bioactivity of samples was achieved by analysing samples immersed in SBF for up to 28 days by interferometry and complementary SEM micrographs. Furthermore, a live/dead assay was used and confirmed the viability of seeded MC3T3 osteoblast precursor cells on HA and substituted HA substrates up to 7 days of culture.

Structural changes to resorbable calcium phosphate bioceramic aged in vitro.

This work investigates the effect of mammalian cell culture conditions on 3D printed calcium phosphate scaffolds. The purpose of the studies presented was to characterise the changes in scaffold properties in physiologically relevant conditions. Differences in crystal morphologies were observed between foetal bovine serum-supplemented media and their unsupplemented analogues, but not for supplemented media containing tenocytes. Scaffold porosity was found to increase for all conditions studied, except for tenocyte-seeded scaffolds. The presence of tenocytes on the scaffold surface inhibited any increase in scaffold porosity in the presence of extracellular matrix secreted by the tenocytes. For acellular conditions the presence or absence of sera proteins strongly affected the rate of dissolution and the distribution of pore diameters within the scaffold. Exposure to high sera protein concentrations led to the development of significant numbers of sub-micron pores, which was otherwise not observed. The implication of these results for cell culture research employing calcium phosphate scaffolds is discussed.

Monitoring sinew contraction during formation of tissue-engineered fibrin-based ligament constructs.

The ability to study the gross morphological changes occurring during tissue formation is vital to producing tissue-engineered structures of clinically relevant dimensions in vitro. Here, we have used nondestructive methods of digital imaging and optical coherence tomography to monitor the early-stage formation and subsequent maturation of fibrin-based tissue-engineered ligament constructs. In addition, the effect of supplementation with essential promoters of collagen synthesis, ascorbic acid (AA) and proline (P), has been assessed. Contraction of the cell-seeded fibrin gel occurs unevenly within the first 5 days of culture around two fixed anchor points before forming a longitudinal ligament-like construct. AA+P supplementation accelerates gel contraction in the maturation phase of development, producing ligament-like constructs with a higher collagen content and distinct morphology to that of unsupplemented constructs. These studies highlight the importance of being able to control the methods of tissue formation and maturation in vitro to enable the production of tissue-engineered constructs with suitable replacement tissue characteristics for repair of clinical soft-tissue injuries.