Age-related fragility fractures are highly correlated with the loss of bone integrity and deteriorated morphology of the osteocytes. Previous studies have reported low-magnitude high-frequency vibration(LMHFV) promotes osteoporotic diaphyseal fracture healing to a greater extent than in age-matched normal fracture healing, yet how osteoporotic fractured bone responds to the mechanical signal has not been explored. As osteocytes are prominent for mechanosensing and initiating bone repair, we hypothesized that LMHFV could enhance fracture healing in ovariectomized metaphyseal fracture through morphological changes and mineralisation in the osteocyte Lacuno-canalicular Network(LCN). As most osteoporotic fractures occur primarily at the metaphysis, an osteoporotic metaphyseal fracture model was established. A total of 72 six-month old female Sprague-Dawley rats (n=72) were obtained(animal ethical approval ref: 16–037-MIS). Half of the rats underwent bilateral ovariectomy(OVX) and kept for 3 months for osteoporosis induction. Metaphyseal fracture on left distal femur was created by osteotomy and fixed by a plate. Rats were then randomized to (1) OVX+LMHFV(20 mins/day and 5 days/week, 35Hz, 0.3g), (2) OVX control, (3) SHAM+LMHFV, (4) SHAM control. Assessments of morphological structural changes, functional markers of the LCN(Scanning Electron Microscopy, FITC-Imaris, immunohistochemistry), mineralization status(EDX, dynamic histomorphometry) and healing outcomes(X-ray, microCT, mechanical testing) were performed at week 1, 2 and 6 post-fracture. One‐way ANOVA with post-hoc test was performed. Statistical significance was set at p < 0.05. Our results showed LMHFV could significantly enhance the morphology of the LCN. There was a 65.3% increase in dendritic branch points(p=0.03) and 93% increase in canalicular length(p=0.019) in the OVX-LMHFV group at week 2 post-fracture. Besides, a similar trend was also observed in the SHAM+LMHFV group, with a 43.4% increase in branch points and 53% increase in canaliculi length at week 2. A significant increase of E11 and DMP1 was observed in the LMHFV groups, indicating the reconstruction of the LCN. The decreasing sclerostin and increasing FGF23 at week 1 represented the active bone formation phase while the gradual increase at week 6 signified the remodelling phase. Furthermore, Ca/P ratio, mineral apposition rate and bone formation rate were all significantly enhanced in the OVX+LMHFV group. The overall bone mineral density in BV was significantly raised in the OVX+LMHFV group at week 2(p=0.043) and SHAM+LMHFV at week 6(p=0.04). Quantitative analysis of microCT showed BV/TV was significantly increased at week 2 in OVX+LMHFV group(p=0.008) and week 6(p=0.001) in both vibration groups. In addition, biomechanical testing revealed that the OVX+LMHFV group had a significantly higher ultimate load(p=0.03) and stiffness(p=0.02) at week 2. To our best knowledge, this is the first report to illustrate LMHFV could enhance osteocytes' morphology, mineralisation status and healing outcome in a new osteoporotic metaphyseal fracture animal model. Our cumulative data supports that the mechanosensitivity of bone would not impair due to osteoporosis. The revitalized osteocyte LCN and upregulated osteocytic protein markers implied a better connectivity and transduction of signals between osteocytes, which may foster the osteoporotic fracture healing process through an enhanced mineralisation process. This could stimulate further mechanistic investigations with potential translation of LMHFV to our fragility fracture patients

Bone fracture healing is regulated by a series of complex physicochemical and biochemical processes. One of these processes is bone mineralisation, which is vital for normal bone development, its biomechanical competence and fracture healing. Phosphatase, orphan 1 (PHOSPHO1), a bone-specific phosphatase, has been shown to be involved in the mineralisation of the extracellular matrix in bone. It can hydrolyse phosphoethanolamine and phosphocholine to generate inorganic phosphate, which is crucial for bone mineralisation. Phospho1−/− mice show hypomineralised bone and spontaneous fractures. All these data led to the hypothesis that PHOSPHO1 is essential for bone mineralisation and its structural integrity. However, no study to our knowledge has shown the effects of PHOSPHO1 on bone fracture healing. In this study, we examined how PHOSPHO1-deficiency might affect the healing and quality of the fractured bones in Phospho1−/− mice. We performed rodded immobilised fracture surgery on the right tibia of control wild type (WT) and Phospho1−/− mice (n=16 for each group) at eight weeks of age. Bone was left to heal for four weeks and then the mice were euthanised and their tibias were analysed using Faxitron X-ray analyses, microCT, histology and histomorphometry and three-point bending test. Our microCT and X-ray analyses revealed that the appearance of the callus and several static parameters of bone remodeling at the fracture sites were markedly different in WT and Phospho1−/− mice. We observed a significant increase of BS/BV, BS/TV and trabecular number and decrease in trabecular thickness and separation in Phospho1−/− callus in comparison to the WT callus. These observations were further confirmed by histomorphometry. The increased bone mass at the fracture sites of Phospho1−/− mice appears to be caused by increased bone formation as there is a significant increase of osteoblast number, while osteoclast numbers remained unchanged. There was a marked increase of osteoid volume over bone volume (OV/BV) in the Phospho−/− callus. Interestingly, the amount of osteoid was markedly higher at the fracture sites than that of normal trabecular bones. The three-point bending test showed that Phospho 1 −/− fractured bone had more of an elastic characteristics than the WT bone as they underwent more of a plastic deformity before the breakage point compare to the WT. Our work suggests that PHOSPHO1 plays an integral role during bone fracture repair. PHOSPHO1 can be an interesting target to improve the fracture healing process

Calcification of the intervertebral disc (IVD) has been correlated with degenerative disc disease (DDD), a common cause of low back pain. The appearance of calcium deposits has been shown to increase with age, and its occurrence has been associated with several other disorders such as hyperparathyroidism, chondrocalcinosis, and arthritis. Trauma, vertebral fusion and infection have also been shown to increase the incidence of IVD calcification. The role of IVD calcification in the development DDD is unknown. Our preliminary data suggest that ionic calcium content and expression of the extracellular calcium-sensing receptor (CaSR), a G protein-coupled receptor (GPCR) and regulator of calcium homeostasis, are increased in the degenerated discs. However, its role in DDD remains unclear. IVD Cells: Bovine and normal human IVD cells were incubated in PrimeGrowth culture medium (Wisent Bioproducts, Canada; Cat# 319–510-CL, −S1, and S2) and supplemented with various concentrations of calcium (1.0, 1.5, 2.5, 5.0 mM), a CaSR agonist [5 µM], or IL-1β [10 ng/ml] for 7 days. Accumulated matrix protein was quantitated for aggrecan and type II collagen (Col II) by Western blotting. Conditioned medium was also collected from cells treated for 24h and measured for the synthesis and release of total proteoglycan using the DMMB assay and Western blotting for Col II content. IVD Cultures: Caudal IVDs from tails of 20–24 month old steers were isolated with the PrimeGrowth Isolation kit (Wisent Bioproducts, Canada). IVDs were cultured for 4 weeks in PrimeGrowth culture medium supplemented with calcium (1.0, 2.5, or 5.0 mM), or a CaSR agonist [5 µM]. Cell viability was measured in NP and AF tissue using Live/Dead Imaging kit (ThermoFisher, Waltham, MA), to determine if Ca2+ effects cell viability end the expression of aggrecan and Col II was evaluated in the IVD tissue by Western blotting. Histological sections were prepared to determine total proteoglycan content, alkaline phosphatase expression and degree of mineralisation by von Kossa staining. The accumulation of aggrecan and Col II decreased dose-dependently in IVD cells following supplementation with calcium or the CaSR agonist. Conditioned medium also demonstrated decreases in the synthesis and release of proteoglycan and collagen with increasing Ca2+ dose or direct activation of the CaSR with agonist. A similar phenomenon was observed for total proteoglycan and aggrecan and Col II in IVDs following calcium supplementation or the CaSR agonist. In addition to decreases in Col II and aggrecan, increases in alkaline phosphatase expression and mineralisation was observed in IVDs cultured in elevated Ca2+ concentrations without affecting cell viability. Our results suggest that changes in the local concentrations of calcium are not benign, and that activation of the CaSR may be a contributing factor in IVD degeneration. Determining ways to minimise Ca2+ infiltration into the disc may mitigate disc degeneration

Background. Current treatments for the prevention of thromboembolism include heparin and low-molecular weight heparins (LMWHs). A number of studies have suggested that long term administration of these drugs may adversely affect osteoblasts and therefore, bone metabolism. Xarelto(tm) (Rivaroxaban) is a new anti-thrombotic drug for the prevention of venous thromboembolism in adult patients undergoing elective hip and knee replacement surgery. The aim of this in vitro study was to investigate the possible effects of rivaroxaban on osteoblast proliferation, function, matrix mineralisation and gene expression compared to enoxaparin, a commonly used LMWH. Methods. Primary human osteoblast cultures were treated with varying concentrations of rivaroxaban (0.013, 0.13, 1.3 and 13 μg/ml) or enoxaparin (0.1, 1.0 and 10 international units/ml). The effect of each drug on osteoblast function and matrix mineralisation was evaluated by measuring alkaline phosphatase activity and calcium deposition, respectively. The MTS assay was used to assess the effect of drug treatments on cell proliferation. Changes in osteocalcin, Runx2 and BMP-2 messenger RNA (mRNA) expression following drug treatments were measured by real-time polymerase chain reaction (PCR). Results. Rivaroxaban and enoxaparin treatment did not adversely affect osteoblast proliferation. However, both drugs caused a significant reduction in osteoblast function, as measured by alkaline phosphatase activity, with a moderate reduction in calcium deposition also observed. This reduction in osteoblast function was associated with a reduction in the mRNA expression of the bone marker, osteocalcin, the transcription factor, Runx2, and the osteogenic factor, BMP-2. Conclusion. These data show that rivaroxaban treatment may negatively affect bone through a reduction in osteoblast function. The increased duration of recommended Rivaroxaban therapy (2 and 5 weeks) post-arthroplasty compared to Enoxaparin therapy (average one week) may have a more pronounced effect on bone homeostasis

The quality of bone in the skeleton depends on the amount of bone, geometry, microarchitecture and material properties, and the molecular and cellular regulation of bone turnover and repair. This study aimed to identify material and structural factors that alter in fragility hip fracture patients treated with antiresorption therapies (FxAr) compared to fragility hip fracture patients not on treatment (Fx). Bone from the intertrochanteric site, femoral head (FH: FxAr = 5, Fx = 8), compression screw cores and box chisel were obtained from patients undergoing hemi-arthroplasty surgery, FxAr (6f, 2m, mean 79 and range [64–89] years), and Fx (7f, 1m, age 85 [75–93] years). Control bone was obtained at autopsy (9f, 4m, 77 [65–88] years). Treated patients were on various bisphosphonates. Samples were resin-embedded, for quantitative backscattered electron imaging of the degree of mineralisation and assessment of bone architecture. Trabecular bone volume fraction (BV/TV) and architectural parameters were not significantly different between FxAr and Fx groups. Both groups showed normal distributions of weight (wt) % Ca; however, the FxAr was less mineralised than the Fx and the control group (mean wt % Ca: FxAr = 24.3%, Fx = 24.8%, Control = 24.9%). When comparing the FH specimens only, we found that BV/TV in the FxAr was greater than the Fx group (18% vs 15%). All other parameters were not significantly different. In addition, the mineralisation was greater in the FxAr group compared to the Fx group (25.5 % vs 25.0%) but was not significantly different. Collectively, these data suggest the effect on bone of antiresorptives may be different for patients on antiresorptive treatment that do not subsequently fracture. Assessment of bone material property data together with other bone quality measures may hold the key to better understanding of antiresorptive treatment efficacy

Osteocytes (OCY) are the end stage differentiation cells of the osteoblast lineage, and are incorporated in the bone matrix during bone formation. In doing so, OCY control the mineralisation of osteoid. OCY form a dense inter-connected network of cell bodies and cell processes throughout the mineralised matrix of bone. OCY viability depends on interstitial fluid flow along the OCY canaliculi, driven by pulsatile blood flow and loading of the skeleton. Maintenance of the density and viability of OCY are essential for bone health because OCY perform many important functions in bone. Firstly, OCY appear to initiate bone repair of bone microdamage. Secondly, OCY are almost certainly the cells, which initiate new bone formation in response to increased loading of bone. Thirdly, OCY are able to regulate the amount of new bone formation in bone remodelling cycles, at least in part by the production of a molecule called sclerostin (SCL). Mutations in the SCL gene, or deletion of the SCL gene in transgenic mice, are associated with particularly dense, fracture resistant bones. This information has led to development of anti-SCL antibodies as a potential anabolic therapy for bones. Bone loss in ovariectomised aged rats was shown recently to be reversed by treatment with neutralising SCL antibodies. There is also some data to suggest that these antibodies may promote fracture healing. Reduced OCY viability and/or density have been reported in association with osteoporotic fracture. OCY viability seems to be dependent on skeletal loading, adequate skeletal blood flow and estrogen in females. OCY viability is adversely affected by hypoxia, unloading of the skeleton and pharmacobiology, such as chronic exposure to glucocorticoids. Both micro and macro-fractures result in disruption of the OCY network, as do procedures such as drilling and cutting of bone. Because of the important roles of OCY in bone, new approaches to bone health may require the identification of agents to protect these cells from harmful influences in disease and ageing

Currently, there is no animal model in which to evaluate the underlying physiological processes leading to the heterotopic ossification (HO) which forms in most combat-related and blast wounds. We sought to reproduce the ossification that forms under these circumstances in a rat by emulating patterns of injury seen in patients with severe injuries resulting from blasts. We investigated whether exposure to blast overpressure increased the prevalence of HO after transfemoral amputation performed within the zone of injury. We exposed rats to a blast overpressure alone (BOP-CTL), crush injury and femoral fracture followed by amputation through the zone of injury (AMP-CTL) or a combination of these (BOP-AMP). The presence of HO was evaluated using radiographs, micro-CT and histology. HO developed in none of nine BOP-CTL, six of nine AMP-CTL, and in all 20 BOP-AMP rats. Exposure to blast overpressure increased the prevalence of HO.

This model may thus be used to elucidate cellular and molecular pathways of HO, the effect of varying intensities of blast overpressure, and to evaluate new means of prophylaxis and treatment of heterotopic ossification.

Cite this article: Bone Joint J 2015;97-B:572–6

Nanotechnology is the study, production and controlled manipulation of materials with a grain size < 100 nm. At this level, the laws of classical mechanics fall away and those of quantum mechanics take over, resulting in unique behaviour of matter in terms of melting point, conductivity and reactivity. Additionally, and likely more significant, as grain size decreases, the ratio of surface area to volume drastically increases, allowing for greater interaction between implants and the surrounding cellular environment. This favourable increase in surface area plays an important role in mesenchymal cell differentiation and ultimately bone–implant interactions.

Basic science and translational research have revealed important potential applications for nanotechnology in orthopaedic surgery, particularly with regard to improving the interaction between implants and host bone. Nanophase materials more closely match the architecture of native trabecular bone, thereby greatly improving the osseo-integration of orthopaedic implants. Nanophase-coated prostheses can also reduce bacterial adhesion more than conventionally surfaced prostheses. Nanophase selenium has shown great promise when used for tumour reconstructions, as has nanophase silver in the management of traumatic wounds. Nanophase silver may significantly improve healing of peripheral nerve injuries, and nanophase gold has powerful anti-inflammatory effects on tendon inflammation.

Considerable advances must be made in our understanding of the potential health risks of production, implantation and wear patterns of nanophase devices before they are approved for clinical use. Their potential, however, is considerable, and is likely to benefit us all in the future.

Cite this article: Bone Joint J 2014; 96-B: 569–73.

Objectives

The purpose of this study was to evaluate in vivo biocompatibility of novel single-walled carbon nanotubes (SWCNT)/poly(lactic-co-glycolic acid) (PLAGA) composites for applications in bone and tissue regeneration.

Peri-prosthetic osteolysis and subsequent aseptic loosening is the most common reason for revising total hip replacements. Wear particles originating from the prosthetic components interact with multiple cell types in the peri-prosthetic region resulting in an inflammatory process that ultimately leads to peri-prosthetic bone loss. These cells include macrophages, osteoclasts, osteoblasts and fibroblasts. The majority of research in peri-prosthetic osteolysis has concentrated on the role played by osteoclasts and macrophages. The purpose of this review is to assess the role of the osteoblast in peri-prosthetic osteolysis.

In peri-prosthetic osteolysis, wear particles may affect osteoblasts and contribute to the osteolytic process by two mechanisms. First, particles and metallic ions have been shown to inhibit the osteoblast in terms of its ability to secrete mineralised bone matrix, by reducing calcium deposition, alkaline phosphatase activity and its ability to proliferate. Secondly, particles and metallic ions have been shown to stimulate osteoblasts to produce pro inflammatory mediators in vitro. In vivo, these mediators have the potential to attract pro-inflammatory cells to the peri-prosthetic area and stimulate osteoclasts to absorb bone. Further research is needed to fully define the role of the osteoblast in peri-prosthetic osteolysis and to explore its potential role as a therapeutic target in this condition.

Cite this article: Bone Joint J 2013;95-B:1021–5.

Periosteum is important for bone homoeostasis through the release of bone morphogenetic proteins (BMPs) and their effect on osteoprogenitor cells. Smoking has an adverse effect on fracture healing and bone regeneration. The aim of this study was to evaluate the effect of smoking on the expression of the BMPs of human periosteum. Real-time polymerase chain reaction was performed for BMP-2,-4,-6,-7 gene expression in periosteal samples obtained from 45 fractured bones (19 smokers, 26 non-smokers) and 60 non-fractured bones (21 smokers, 39 non-smokers). A hierarchical model of BMP gene expression (BMP-2 > BMP-6 > BMP-4 > BMP-7) was demonstrated in all samples. When smokers and non-smokers were compared, a remarkable reduction in the gene expression of BMP-2, -4 and -6 was noticed in smokers. The comparison of fracture and non-fracture groups demonstrated a higher gene expression of BMP-2, -4 and -7 in the non-fracture samples. Within the subgroups (fracture and non-fracture), BMP gene expression in smokers was either lower but without statistical significance in the majority of BMPs, or similar to that in non-smokers with regard to BMP-4 in fracture and BMP-7 in non-fracture samples. In smokers, BMP gene expression of human periosteum was reduced, demonstrating the effect of smoking at the molecular level by reduction of mRNA transcription of periosteal BMPs. Among the BMPs studied, BMP-2 gene expression was significantly higher, highlighting its role in bone homoeostasis.

Lengthening of the humerus is now an established technique. We compared the complications of humeral lengthening with those of femoral lengthening and investigated whether or not the callus formation in the humerus proceeds at a higher rate than that in the femur. A total of 24 humeral and 24 femoral lengthenings were performed on 12 patients with achondroplasia. We measured the pixel value ratio (PVR) of the lengthened area on radiographs and each radiograph was analysed for the shape, type and density of the callus. The quality of life (QOL) of the patients after humeral lengthening was compared with that prior to surgery. The complication rate per segment of humerus and femur was 0.87% and 1.37%, respectively. In the humerus the PVR was significantly higher than that of the femur. Lower limbs were associated with an increased incidence of concave, lateral and central callus shapes. Humeral lengthening had a lower complication rate than lower-limb lengthening, and QOL increased significantly after humeral lengthening. Callus formation in the humerus during the distraction period proceeded at a significantly higher rate than that in the femur.

These findings indicate that humeral lengthening has an important role in the management of patients with achondroplasia.