Combined Effects of Exercise and Denosumab Treatment on Local Failure in Post-menopausal Osteoporosis–Insights from Bone Remodelling Simulations Accounting for Mineralisation and Damage

Denosumab has been shown to increase bone mineral density (BMD) and reduce the fracture risk in patients with post-menopausal osteoporosis (PMO). Increase in BMD is linked with an increase in bone matrix mineralisation due to suppression of bone remodelling. However, denosumab anti-resorptive action also leads to an increase in fatigue microdamage, which may ultimately lead to an increased fracture risk. A novel mechanobiological model of bone remodelling was developed to investigate how these counter-acting mechanisms are affected both by exercise and long-term denosumab treatment. This model incorporates Frost's mechanostat feedback, a bone mineralisation algorithm and an evolution law for microdamage accumulation. Mechanical disuse and microdamage were assumed to stimulate RANKL production, which modulates activation frequency of basic multicellular units in bone remodelling. This mechanical feedback mechanism controls removal of excess bone mass and microdamage. Furthermore, a novel measure of bone local failure due to instantaneous overloading was developed. Numerical simulations indicate that trabecular bone volume fraction and bone matrix damage are determined by the respective bone turnover and homeostatic loading conditions. PMO patients treated with the currently WHO-approved dose of denosumab (60 mg administrated every 6 months) exhibit increased BMD, increased bone ash fraction and damage. In untreated patients, BMD will significantly decrease, as will ash fraction; while damage will increase. The model predicted that, depending on the time elapsed between the onset of PMO and the beginning of treatment, BMD slowly converges to the same steady-state value, while damage is low in patients treated soon after the onset of the disease and high in patients having PMO for a longer period. The simulations show that late treatment PMO patients have a significantly higher risk of local failure compared to patients that are treated soon after the onset of the disease. Furthermore, overloading resulted in an increase of BMD, but also in a faster increase of damage, which may consequently promote the risk of fracture, specially in late treatment scenarios. In case of mechanical disuse, the model predicted reduced BMD gains due to denosumab, while no significant change in damage occurred, thus leading to an increased risk of local failure compared to habitual loading.

Proton pump inhibitors inhibit PHOSPHO1 activity and matrix mineralisation in vitro

Proton pump inhibitors (PPIs) have been associated with an increased risk of fragility fractures in pharmaco-epidemiological studies. The mechanism is unclear but it has been speculated that by neutralising gastric acid, they may reduce intestinal calcium absorption, causing secondary hyperparathyroidism and bone loss. Here we investigated that hypothesis that the skeletal effects of PPI might be mediated by inhibitory effects on the bone-specific phosphatase PHOSPHO1. We found that the all PPI tested potential inhibited the activity of PHOSPHO1 with IC50 ranging between 0.73μM for esomeprazole to 19.27μM for pantoprazole. In contrast, these PPIs did not inhibit TNAP activity. We also found that mineralisation of bone matrix in primary osteoblast cultures inhibited by several PPI in a concentration dependent manner. In contrast, the histamine-2 receptor antagonists (H2RA) nizatidine, famotidine, cimetidine and ranitidine had no inhibitory effects on PHOSPHO1 activity. Our experiments shown for the first time that PPI inhibit PHOSPHO1 activity and matrix mineralisation in vitro revealing a potential mechanism by which these widely used drugs are associated with the risk of fractures.

RORα Regulates Odontoblastic Differentiation and Mediates the Pro-Odontogenic Effect of Melatonin on Dental Papilla Cells

Dental papilla cells (DPCs), precursors of odontoblasts, are considered promising seed cells for tissue engineering. Emerging evidence suggests that melatonin promotes odontoblastic differentiation of DPCs and affects tooth development, although the precise mechanisms remain unknown. Retinoid acid receptor-related orphan receptor α (RORα) is a nuclear receptor for melatonin that plays a critical role in cell differentiation and embryonic development. This study aimed to explore the role of RORα in odontoblastic differentiation and determine whether melatonin exerts its pro-odontogenic effect via RORα. Herein, we observed that RORα was expressed in DPCs and was significantly increased during odontoblastic differentiation in vitro and in vivo. The overexpression of RORα upregulated the expression of odontogenic markers, alkaline phosphatase (ALP) activity and mineralized nodules formation (p < 0.05). In contrast, odontoblastic differentiation of DPCs was suppressed by RORα knockdown. Moreover, we found that melatonin elevated the expression of odontogenic markers, which was accompanied by the upregulation of RORα (p < 0.001). Utilising small interfering RNA, we further demonstrated that RORα inhibition attenuated melatonin-induced odontogenic gene expression, ALP activity and matrix mineralisation (p < 0.01). Collectively, these results provide the first evidence that RORα can promote odontoblastic differentiation of DPCs and mediate the pro-odontogenic effect of melatonin.

Multiscale molecular profiling of pathological bone resolves sexually dimorphic control of extracellular matrix composition

ABSTRACTCollagen assembly during development is essential for successful matrix mineralisation, which determines bone quality and mechanocompetence. However, the biochemical and structural perturbations that drive pathological skeletal collagen configuration remain unclear. Deletion of vascular endothelial growth factor (VEGF; also known as VEGFA) in bone-forming osteoblasts (OBs) induces sex-specific alterations in extracellular matrix (ECM) conformation and mineralisation coupled to vascular changes, which are augmented in males. Whether this phenotypic dimorphism arises as a result of the divergent control of ECM composition and its subsequent arrangement is unknown and is the focus of this study. Herein, we used murine osteocalcin-specific Vegf knockout (OcnVEGFKO) and performed ex vivo multiscale analysis at the tibiofibular junction of both sexes. Label-free and non-destructive polarisation-resolved second-harmonic generation (p-SHG) microscopy revealed a reduction in collagen fibre number in males following the loss of VEGF, complemented by observable defects in matrix organisation by backscattered electron scanning electron microscopy. This was accompanied by localised divergence in collagen orientation, determined by p-SHG anisotropy measurements, as a result of OcnVEGFKO. Raman spectroscopy confirmed that the effect on collagen was linked to molecular dimorphic VEGF effects on collagen-specific proline and hydroxyproline, and collagen intra-strand stability, in addition to matrix carbonation and mineralisation. Vegf deletion in male and female murine OB cultures in vitro further highlighted divergence in genes regulating local ECM structure, including Adamts2, Spp1, Mmp9 and Lama1. Our results demonstrate the utility of macromolecular imaging and spectroscopic modalities for the detection of collagen arrangement and ECM composition in pathological bone. Linking the sex-specific genetic regulators to matrix signatures could be important for treatment of dimorphic bone disorders that clinically manifest in pathological nano- and macro-level disorganisation.This article has an associated First Person interview with the first author of the paper.

Sintered S53P4 bioactive glass scaffolds have anti-inflammatory properties and stimulate osteogenesis in vitro

Bioactive glasses (BAG) are used as bone-graft substitutes in orthopaedic surgery. A specific BAG scaffold was developed by sintering BAG-S53P4 granules. It is hypothesised that this scaffold can be used as a bone substitute to fill bone defects and induce a bioactive membrane (IM) around the defect site. Beyond providing the scaffold increased mechanical strength, that the initial inflammatory reaction and subsequent IM formation can be enhanced by coating the scaffolds with poly(DL-lactide-co-glycolide) (PLGA) is also hypothesised. To study the immunomodulatory effects, BAG-S53P4 (± PLGA) scaffolds were placed on monolayers of primary human macrophage cultures and the production of various pro- and anti-inflammatory cytokines was assessed using reverse transcriptase quantitative polymerase chain reaction (RT-qPCR) and ELISA. To study the osteogenic effects, BAG-S53P4 (± PLGA) scaffolds were cultured with rabbit mesenchymal stem cells and osteogenic differentiation was evaluated by RT-qPCR and matrix mineralisation assays. The scaffold ion release was quantified and the BAG surface reactivity visualised. Furthermore, the pH of culture media was measured. BAG-S53P4 scaffolds had both anti-inflammatory and osteogenic properties that were likely attributable to alkalinisation of the media and ion release from the scaffold. pH change, ion release, and immunomodulatory properties of the scaffold could be modulated by the PLGA coating. Contrary to the hypothesis, the coating functioned by attenuating the BAG surface reactions and subsequent anti-inflammatory properties, rather than inducing an elevated inflammatory response compared to BAG-S53P4 alone. These results further validated the use of BAG-S53P4 (± PLGA) scaffolds as bone substitutes and indicate that scaffold properties can be tailored to a specific clinical need.

Citrate Supplementation Restores the Impaired Mineralisation Resulting from the Acidic Microenvironment: An In Vitro Study

Chronic metabolic acidosis leads to bone-remodelling disorders based on excessive mineral matrix resorption and inhibition of bone formation, but also affects the homeostasis of citrate, which is an essential player in maintaining the acid–base balance and in driving the mineralisation process. This study aimed to investigate the impact of acidosis on the osteogenic properties of bone-forming cells and the effects of citrate supplementation in restoring the osteogenic features impaired by the acidic milieu. For this purpose, human mesenchymal stromal cells were cultured in an osteogenic medium and the extracellular matrix mineralisation was analysed at the micro- and nano-level, both in neutral and acidic conditions and after treatment with calcium citrate and potassium citrate. The acidic milieu significantly decreased the citrate release and hindered the organisation of the extracellular matrix, but the citrate supplementation increased collagen production and, particularly calcium citrate, promoted the mineralisation process. Moreover, the positive effect of citrate supplementation was observed also in the physiological microenvironment. This in vitro study proves that the mineral matrix organisation is influenced by citrate availability in the microenvironment surrounding bone-forming cells, thus providing a biological basis for using citrate-based supplements in the management of bone-remodelling disorders related to chronic low-grade acidosis.

Beyond mineralisation: metabolic functions for matrix mineralisation regulators

The physiological mineralisation of skeletal tissues, as well as the pathological mineralisation of soft tissues involves a fine balance between regulators that either promote or inhibit the process. In recent years, several studies have advocated a non-skeletal role for some of these mineralisation regulators in a range of human diseases, including diabetes, cardiovascular disease, obesity and neurodegenerative disease. This is an emerging area of interest and the functional roles and mechanisms of action of these various endocrine factors, phosphatases and phosphodiesterase’s in important pathologies are the focus of this review. Mechanistic insight of the pathways through which these acknowledged regulators of skeletal mineralisation act beyond the skeleton has the potential to identify druggable targets for commonly experienced morbidities, notably those related to metabolism and metabolic syndrome.

Role of offset and gradient architectures of 3-D melt electrowritten scaffold on differentiation and mineralization of osteoblasts

Background Cell-scaffold based therapies have the potential to offer an efficient osseous regenerative treatment and PCL has been commonly used as a scaffold, however its effectiveness is limited by poor cellular retention properties. This may be improved through a porous scaffold structure with efficient pore arrangement to increase cell entrapment. To facilitate this, melt electrowriting (MEW) has been developed as a technique able to fabricate cell-supporting scaffolds with precise micro pore sizes via predictable fibre deposition. The effect of the scaffold’s architecture on cellular gene expression however has not been fully elucidated. Methods The design and fabrication of three different uniform pore structures (250, 500 and 750 μm), as well as two offset scaffolds with different layout of fibres (30 and 50%) and one complex scaffold with three gradient pore sizes of 250–500 - 750 μm, was performed by using MEW. Calcium phosphate modification was applied to enhance the PCL scaffold hydrophilicity and bone inductivity prior to seeding with osteoblasts which were then maintained in culture for up to 30 days. Over this time, osteoblast cell morphology, matrix mineralisation, osteogenic gene expression and collagen production were assessed. Results The in vitro findings revealed that the gradient scaffold significantly increased alkaline phosphatase activity in the attached osteoblasts while matrix mineralization was higher in the 50% offset scaffolds. The expression of osteocalcin and osteopontin genes were also upregulated compared to other osteogenic genes following 30 days culture, particularly in offset and gradient scaffold structures. Immunostaining showed significant expression of osteocalcin in offset and gradient scaffold structures. Conclusions This study demonstrated that the heterogenous pore sizes in gradient and fibre offset PCL scaffolds prepared using MEW significantly improved the osteogenic potential of osteoblasts and hence may provide superior outcomes in bone regeneration applications.

Calcium Polyphosphate Nanoparticles Act as an Effective Inorganic Phosphate Source during Osteogenic Differentiation of Human Mesenchymal Stem Cells

The ability of bone-marrow-derived mesenchymal stem/stromal cells (BM-MSCs) to differentiate into osteoblasts makes them the ideal candidate for cell-based therapies targeting bone-diseases. Polyphosphate (polyP) is increasingly being studied as a potential inorganic source of phosphate for extracellular matrix mineralisation. The aim of this study is to investigate whether polyP can effectively be used as a phosphate source during the in vitro osteogenic differentiation of human BM-MSCs. Human BM-MSCs are cultivated under osteogenic conditions for 28 days with phosphate provided in the form of organic β-glycerolphosphate (BGP) or calcium-polyP nanoparticles (polyP-NP). Mineralisation is demonstrated using Alizarin red staining, cellular ATP content, and free phosphate levels are measured in both the cells and the medium. The effects of BGP or polyP-NP on alkaline phosphatase (ALP) activity and gene expression of a range of osteogenic-related markers are also assessed. PolyP-NP supplementation displays comparable effects to the classical BGP-containing osteogenic media in terms of mineralisation, ALP activity and expression of osteogenesis-associated genes. This study shows that polyP-NP act as an effective source of phosphate during mineralisation of BM-MSC. These results open new possibilities with BM-MSC-based approaches for bone repair to be achieved through doping of conventional biomaterials with polyP-NP.