Organotypic culture of bone‐like structures using composite ceramic‐fibrin scaffolds

We have developed an organotypic culture system that allows the production of bone tissue features at the cm scale. A composite, calcium phosphate-strained fibrin gel system is able to organise itself in the presence of osteoblastic cells, creating basic hierarchical units as seen in vivo, and can be modified to produce a range of other tissues that require such directional structuring. Constructs evolve over time into multi-compositional structures containing a high mineral content and terminally differentiated, osteocyte-like cells. These tissues can be cultured over extended durations (exceeding 1 year) and are responsive to a variety of chemical and biological agents. The platform can reduce the number of animals used in experimentation by acting as an intermediate stage in which more personalised research conditions can be generated. We provide a thorough description of the protocol used to successfully culture and modify this system as well as guidance on compositional characterisation

Platelet rich plasma injection for acute Achilles tendon rupture: PATH-2 randomised, placebo controlled, superiority trial

Objective To determine whether an injection of platelet rich plasma improves outcomes after acute Achilles tendon rupture. Design Randomised, placebo controlled, two arm, parallel group, participant and assessor masked, superiority trial. Setting Secondary care trauma units across 19 hospitals in the United Kingdom’s health service. Participants Recruitment commenced in July 2015 and follow-up was completed in March 2018. 230 adults aged 18 years and over were included, with acute Achilles tendon rupture presenting within 12 days of injury and managed with non-surgical treatment. Exclusions were injury at the insertion or musculotendinous junction, major leg injury or deformity, diabetes mellitus, platelet or haematological disorder, systemic corticosteroids, anticoagulation treatment, and other contraindicating conditions. Interventions Participants were randomised 1:1 to platelet rich plasma (n=114) or placebo (dry needle; n=116) injection. All participants received standard rehabilitation care (ankle immobilisation followed by physiotherapy). Main outcomes and measures Primary outcome was muscle tendon function at 24 weeks, measured objectively with the limb symmetry index (injured/uninjured×100) in maximal work done during the heel rise endurance test (an instrumented measure of repeated single leg heel rises until fatigue). Secondary outcomes included patient reported function (Achilles tendon rupture score), quality of life (short form 12 version 2®), pain (visual analogue scale), goal attainment (patient specific functional scale), and adverse events. A central laboratory analysed the quality and content of platelet rich plasma. Analyses were by modified intention to treat. Results Participants were 46 years old on average, and 57 (25%) of 230 were female. At 24 weeks, 202 (88%) participants completed the heel rise endurance test and 216 (94%) the patient reported outcomes. The platelet rich plasma was of good quality, with expected growth factor content. No difference was detected in muscle tendon function between participants receiving platelet rich plasma injections and those receiving placebo injections (limb symmetry index, mean 34.7% (standard deviation 17.7%) v 38.5% (22.8%); adjusted mean difference −3.9% (95% confidence interval −10.5% to 2.7%)) or in any secondary outcomes or adverse event rates. Complier average causal effect analyses gave similar findings. Conclusions There is no evidence to indicate that injections of platelet rich plasma can improve objective muscle tendon function, patient reported function, or quality of life after acute Achilles tendon rupture compared with placebo, or that they offer any patient benefit. Trial registration isrctn.com identifier: ISRCTN54992179</p

Inhomogeneous response of articular cartilage:a three-1 dimensional multiphasic heterogeneous study

Articular cartilage exhibits complex mechano-electrochemical behaviour due to its anisotropy, inhomogeneity and material non-linearity. In this work, the thickness and radial dependence of cartilage properties are incorporated into a 3D mechano-electrochemical model to explore the relevance of heterogeneity in the behaviour of the tissue. The model considers four essential phenomena: (i) osmotic pressure, (ii) convective and diffusive processes, (iii) chemical expansion and (iv) three-dimensional through-the-thickness heterogeneity of the tissue. The need to consider heterogeneity in computational simulations of cartilage behaviour and in manufacturing biomaterials mimicking this tissue is discussed. To this end, healthy tibial plateaus from pigs were mechanically and biochemically tested in-vitro. Heterogeneous properties were included in the mechano-electrochemical computational model to simulate tissue swelling. The simulation results demonstrated that swelling of the heterogeneous samples was significantly lower than swelling under homogeneous and isotropic conditions. Furthermore, there was a significant reduction in the flux of water and ions in the former samples. In conclusion, the computational model presented here can be considered as a valuable tool for predicting how the variation of cartilage properties affects its behaviour, opening up possibilities for exploring the requirements of cartilage-mimicking biomaterials for tissue engineering. Besides, the model also allows the establishment of behavioural patterns of swelling and of water and ion fluxes in articular cartilage

Use of oxygen-loaded nanobubbles to improve tissue oxygenation: bone-relevant mechanisms of action and effects on osteoclast differentiation

Gas-loaded nanobubbles have potential as a method of oxygen delivery to increase tumour oxygenation and therapeutically alleviate tumour hypoxia. However, the mechanism(s) whereby oxygen-loaded nanobubbles increase tumour oxygenation are unknown; with their calculated oxygen-carrying capacity being insufficient to explain this effect. Intra-tumoural hypoxia is a prime therapeutic target, at least partly due to hypoxia-dependent stimulation of the formation and function of bone-resorbing osteoclasts which establish metastatic cells in bone. This study aims to investigate potential mechanism(s) of oxygen delivery and in particular the possible use of oxygen-loaded nanobubbles in preventing bone metastasis via effects on osteoclasts. Lecithin-based nanobubbles preferentially interacted with phagocytic cells (monocytes, osteoclasts) via a combination of lipid transfer, clathrin-dependent endocytosis and phagocytosis. This interaction caused general suppression of osteoclast differentiation via inhibition of cell fusion. Additionally, repeat exposure to oxygen-loaded nanobubbles inhibited osteoclast formation to a greater extent than nitrogen-loaded nanobubbles. This gas-dependent effect was driven by differential effects on the fusion of mononuclear precursor cells to form pre-osteoclasts, partly due to elevated potentiation of RANKL-induced ROS by nitrogen-loaded nanobubbles. Our findings suggest that oxygen-loaded nanobubbles could represent a promising therapeutic strategy for cancer therapy; reducing osteoclast formation and therefore bone metastasis via preferential interaction with monocytes/macrophages within the tumour and bone microenvironment, in addition to known effects of directly improving tumour oxygenation

Single nucleus and spatial transcriptomic profiling of healthy human hamstring tendon

The molecular and cellular basis of health in human tendons remains poorly understood. Among human tendons, hamstring tendon has markedly low pathology and can provide a prototypic healthy tendon reference. The aim of this study was to determine the transcriptomes and location of all cell types in healthy hamstring tendon. Using single nucleus RNA sequencing, we profiled the transcriptomes of 10 533 nuclei from four healthy donors and identified 12 distinct cell types. We confirmed the presence of two fibroblast cell types, endothelial cells, mural cells, and immune cells, and identified cell types previously unreported in tendons, including different skeletal muscle cell types, satellite cells, adipocytes, and undefined nervous system cells. The location of these cell types within tendon was defined using spatial transcriptomics and imaging, and potential transcriptional networks and cell–cell interactions were analyzed. We demonstrate that fibroblasts have the highest number of potential cell–cell interactions in our dataset, are present throughout the tendon, and play an important role in the production and organization of extracellular matrix, thus confirming their role as key regulators of hamstring tendon homeostasis. Overall, our findings underscore the complexity of the cellular networks that underpin healthy human tendon function and the central role of fibroblasts as key regulators of hamstring tendon tissue homeostasis

An in vitro model for the development of mature bone containing an osteocyte network

Bone is a dynamic tissue that remodels continuously in response to local mechanical and chemical stimuli. This process can also result in maladaptive ectopic bone in response to injury, yet pathological differences at the molecular and structural levels are poorly understood. A number of in vivo models exist but can often be too complex to allow isolation of factors which may stimulate disease progression. A self-structuring model of bone formation is presented using a fibrin gel cast between two calcium phosphate ceramic anchors. Femoral periosteal cells, seeded into these structures, deposit an ordered matrix that closely resembles mature bone in terms of chemistry (collagen:mineral ratio) and structure, which is adapted over a period of one year in culture. Raman spectroscopy and X-ray diffraction confirm that the mineral is hydroxyapatite associated with collagen. Second-harmonic imaging demonstrates that collagen is organized similarly to mature mouse femora. Remarkably, cells differentiated to the osteocyte phase are linked by canaliculi (as demonstrated with nano-computed tomography) and remained viable over the full year of culture. It is demonstrated that novel drugs can prevent ossification in constructs. This model can be employed to study bone formation in an effort to encourage or prevent ossification in a range of pathologies

A new method to sort differentiating osteoclasts into defined homogeneous subgroups

Osteoclasts regulate skeletal development but also drive pathological osteolysis, making them prime therapeutic targets. Osteoclast research is limited by the heterogeneity of osteoclast populations generated in vitro, where the mixture of undifferentiated monocytes, binuclear pre-osteoclasts and multinucleated osteoclasts has by necessity been considered a single osteoclast population. This study describes the differentiation of primary human CD14+ monocyte-derived osteoclasts in 3D collagen gels. These osteoclasts remained small (>95% with ≤5 nuclei) but were viable and active; when released from the gel with collagenase, they fused rapidly when reseeded onto solid substrates and resorbed dentine for 2–3 weeks. 3D-generated osteoclasts expressed cell surface markers of osteoclast differentiation (e.g., CD9, RANK, OSCAR, CD63, CD51/61) which, with their small size, enabled live cell sorting of highly enriched viable subpopulations of human osteoclasts that retained full functional resorption capacity. Low-yield osteoclast preparations were strongly enriched to remove undifferentiated cells (e.g., 13.3% CD51/61+ to 84.2% CD51/61+), and subpopulations of CD9+CD51/61− early osteoclasts and CD9+CD51/61+ mature cells were distinguished. This novel approach allows the study of selected populations of differentiating osteoclasts in vitro and opens the door to in-depth transcriptomic and proteomic analysis of these cells, increasing our ability to study human osteoclast molecular mechanisms relevant to development, aging and disease

An in vitro scratch tendon tissue injury model: effects of high frequency low magnitude loading

The healing process of ruptured tendons is suboptimal, taking months to achieve tissue with inferior properties to healthy tendon. Mechanical loading has been shown to positively influence tendon healing. However, high frequency low magnitude (HFLM) loads, which have shown promise in maintaining healthy tendon properties, have not been studied with in vitro injury models. Here, we present and validate an in vitro scratch tendon tissue injury model to investigate effects of HFLM loading on the properties of injured rat tail tendon fascicles (RTTFs). A longitudinal tendon tear was simulated using a needle aseptically to scratch a defined length along individual RTTFs. Tissue viability, biomechanical, and biochemical parameters were investigated before and 7 days after culture . The effects of static, HFLM (20 Hz), and low frequency (1 Hz) cyclic loading or no load were also investigated. Tendon viability was confirmed in damaged RTTFs after 7 days of culture, and the effects of a 0.77 ± 0.06 cm scratch on the mechanical property (tangent modulus) and tissue metabolism in damaged tendons were consistent, showing significant damage severity compared with intact tendons. Damaged tendon fascicles receiving HFLM (20 Hz) loads displayed significantly higher mean tangent modulus than unloaded damaged tendons (212.7 ± 14.94 v 92.7 ± 15.59 MPa), and damaged tendons receiving static loading (117.9 ± 10.65 MPa). HFLM stimulation maintained metabolic activity in 7-day cultured damaged tendons at similar levels to fresh tendons immediately following damage. Only damaged tendons receiving HFLM loads showed significantly higher metabolism than unloaded damaged tendons (relative fluorescence units —7021 ± 635.9 v 3745.1 ± 641.7). These validation data support the use of the custom-made in vitro injury model for investigating the potential of HFLM loading interventions in treating damaged tendons