Investigating the Involvement of C-X-C Motif Chemokine 5 and P2X7 Purinoceptor in Ectopic Calcification in Mouse Models of Duchenne Muscular Dystrophy.

Ectopic calcification of myofibers is an early pathogenic feature in patients and animal models of Duchenne muscular dystrophy (DMD). In previous studies using the Dmd mouse model, we found that the dystrophin-null phenotype exacerbates this abnormality and that mineralised myofibers are surrounded by macrophages. Furthermore, the P2X7 purinoceptor, functioning in immune cells offers protection against dystrophic calcification.

Ablation of the dystrophin Dp71f alternative C-terminal variant increases sarcoma tumour cell aggressiveness.

Alterations in Dp71 expression, the most ubiquitous dystrophin isoform, have been associated with patient survival across tumours. Intriguingly, in certain malignancies, Dp71 acts as a tumour suppressor, while manifesting oncogenic properties in others. This diversity could be explained by the expression of two Dp71 splice variants encoding proteins with distinct C-termini, each with specific properties.

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.

The P2X7 purinoceptor in pathogenesis and treatment of dystrophino- and sarcoglycanopathies.

Dystrophinopathy and sarcoglycanopathies are incurable diseases caused by mutations in the genes encoding dystrophin or members of the dystrophin associated protein complex (DAPC). Restoration of the missing dystrophin or sarcoglycans via genetic approaches is complicated by the downsides of personalised medicines and immune responses against re-expressed proteins. Thus, the targeting of disease mechanisms downstream from the mutant protein has a strong translational potential.

P2X7 Purinoceptor Affects Ectopic Calcification of Dystrophic Muscles.

Ectopic calcification (EC) of myofibers is a pathological feature of muscle damage in Duchenne muscular dystrophy (DMD). Mineralisation of muscle tissue occurs concomitantly with macrophage infiltration, suggesting a link between ectopic mineral deposition and inflammation. One potential link is the P2X7 purinoceptor, a key trigger of inflammation, which is expressed on macrophages but also up-regulated in dystrophic muscle cells.

Biomimetic generation of the strongest known biomaterial found in limpet tooth.

The biomaterial with the highest known tensile strength is a unique composite of chitin and goethite (α-FeO(OH)) present in teeth from the Common Limpet (Patella vulgata). A biomimetic based on limpet tooth, with corresponding high-performance mechanical properties is highly desirable. Here we report on the replication of limpet tooth developmental processes ex vivo, where isolated limpet tissue and cells in culture generate new biomimetic structures.

Genetically-programmed, mesenchymal stromal cell-laden & mechanically strong 3D bioprinted scaffolds for bone repair.

Additive manufacturing processes used to create regenerative bone tissue engineered implants are not biocompatible, thereby restricting direct use with stem cells and usually require cell seeding post-fabrication. Combined delivery of stem cells with the controlled release of osteogenic factors, within a mechanically-strong biomaterial combined during manufacturing would replace injectable defect fillers (cements) and allow personalized implants to be rapidly prototyped by 3D bioprinting. Through the use of direct genetic programming via the sustained release of an exogenously delivered transcription factor RUNX2 (delivered as recombinant GET-RUNX2 protein) encapsulated in PLGA microparticles (MPs), we demonstrate that human mesenchymal stromal (stem) cells (hMSCs) can be directly fabricated into a thermo-sintered 3D bioprintable material and achieve effective osteogenic differentiation.

In vivo delivery of VEGF RNA and protein to increase osteogenesis and intraosseous angiogenesis.

Deficient bone vasculature is a key component in pathological conditions ranging from developmental skeletal abnormalities to impaired bone repair. Vascularisation is dependent upon vascular endothelial growth factor (VEGF), which drives both angiogenesis and osteogenesis. The aim of this study was to examine the efficacy of blood vessel and bone formation following transfection with VEGF RNA or delivery of recombinant human VEGF protein (rhVEGF) across in vitro and in vivo model systems.

Total Absence of Dystrophin Expression Exacerbates Ectopic Myofiber Calcification and Fibrosis and Alters Macrophage Infiltration Patterns.

Duchenne muscular dystrophy (DMD) causes severe disability and death of young men because of progressive muscle degeneration aggravated by sterile inflammation. DMD is also associated with cognitive and bone-function impairments. This complex phenotype results from the cumulative loss of a spectrum of dystrophin isoforms expressed from the largest human gene.

Knockout and Knock-in Mouse Models to Study Purinergic Signaling.

Purinergic signaling involves extracellular purines and pyrimidines acting upon specific cell surface purinoceptors classified into the P1, P2X, and P2Y families for nucleosides and nucleotides. This widespread signaling mechanism is active in all major tissues and influences a range of functions in health and disease. Orthologs to all but one of the human purinoceptors have been found in mouse, making this laboratory animal a useful model to study their function.

PyMT-Maclow: A novel, inducible, murine model for determining the role of CD68 positive cells in breast tumor development.

CD68+ tumor-associated macrophages (TAMs) are pro-tumorigenic, pro-angiogenic and are associated with decreased survival rates in patients with cancer, including breast cancer. Non-specific models of macrophage ablation reduce the number of TAMs and limit the development of mammary tumors. However, the lack of specificity and side effects associated with these models compromise their reliability.

Established Models and New Paradigms for Hypoxia-Driven Cancer-Associated Bone Disease.

The five-year survival rate for primary bone cancers is ~ 70% while almost all cases of secondary metastatic bone cancer are terminal. Hypoxia, the deficiency of oxygen which occurs as the rate of tumour growth exceeds the supply of vascularisation, is a key promoter of tumour progression. Hypoxia-driven effects in the primary tumour are wide ranging including changes in gene expression, dysregulation of signalling pathways, resistance to chemotherapy, neovascularisation, increased tumour cell proliferation and migration.

Preclinical models for in vitro mechanical loading of bone-derived cells.

It is well established that bone responds to mechanical stimuli whereby physical forces are translated into chemical signals between cells, via mechanotransduction. It is difficult however to study the precise cellular and molecular responses using in vivo systems. In vitro loading models, which aim to replicate forces found within the bone microenvironment, make the underlying processes of mechanotransduction accessible to the researcher.

The hypoxic cancer secretome induces pre-metastatic bone lesions through lysyl oxidase.

Tumour metastasis is a complex process involving reciprocal interplay between cancer cells and host stroma at both primary and secondary sites, and is strongly influenced by microenvironmental factors such as hypoxia. Tumour-secreted proteins play a crucial role in these interactions and present strategic therapeutic potential. Metastasis of breast cancer to the bone affects approximately 85% of patients with advanced disease and renders them largely untreatable.

The P2Y13 receptor regulates extracellular ATP metabolism and the osteogenic response to mechanical loading.

ATP release and subsequent activation of purinergic receptors has been suggested to be one of the key transduction pathways activated by mechanical stimulation of bone. The P2Y(13) receptor, recently found to be expressed by osteoblasts, has been suggested to provide a negative feedback pathway for ATP release in different cell types. Therefore, we hypothesized that the P2Y(13) receptor may contribute to the mediation of osteogenic responses to mechanical stimulation by regulating ATP metabolism by osteoblasts.

Purinergic signalling in bone.

Purinergic signaling in bone was first proposed in the early 1990s with the observation that extracellular ATP could modulate events crucial to the normal functioning of bone cells. Since then the expression of nearly all the P2Y and P2X receptors by osteoblasts and osteoclasts has been reported, mediating multiple processes including cell proliferation, differentiation, function, and death. This review will highlight the most recent developments in the field of purinergic signaling in bone, with a special emphasis on recent work resulting from the European Framework 7 funded collaboration ATPBone, as well as Arthritis Research UK and Bone Research Society supported projects.

Generation of a novel mouse model for the inducible depletion of macrophages in vivo.

Macrophages play an essential role in tissue homeostasis, innate immunity, inflammation, and wound repair. Macrophages are also essential during development, severely limiting the use of mouse models in which these cells have been constitutively deleted. Consequently, we have developed a transgenic model of inducible macrophage depletion in which macrophage-specific induction of the cytotoxic diphtheria toxin A chain (DTA) is achieved by administration of doxycycline.

Purinergic signalling in osteoblasts.

The skeleton is maintained throughout life via the finely tuned actions of osteoblasts and osteoclasts, with disruption in this balance eventually leading to bone disease. The exact mechanisms balancing these actions are not fully known, although several regulatory systems are known to be involved. The involvement of purinergic signalling in bone has come to light over the past 20 years or so.

Isolation and culture of human osteoblasts.

The skeleton is a dynamic organ that is constantly active throughout life. The highly coordinated actions of bone cells early in life determine the body's shape and form, whilst the constant remodelling (bone resorption followed by an equal amount of bone formation) during adulthood helps to maintain skeletal mass and repair microdamage. When the balance of bone resorption and bone formation becomes unequal, bone diseases, such as osteoporosis, occur.