Local drug delivery using drug-releasing implants located inside bone is recognised as a promisin... more Local drug delivery using drug-releasing implants located inside bone is recognised as a promising strategy to address the limitations of systemic drug administration to bone. However, the studies of drug-release kinetics are not possible to perform using existing in-vitro drug releasing systems and 2-D bone cell models. The aim of this work is to demonstrate the use of a 3-D bone bioreactor for studying the drug-release kinetics and distribution of drugs in the ex-vivo cancellous bone environment. Bovine trabecular bone was used as the bone substrate, in which drug-releasing implants were embedded in the form of nano-engineered titanium wires covered with a layer of titania nanotube (TNT) arrays. A hydrophilic fluorescent dye (rhodamine B) was used as a model drug, loaded inside the TNT/Ti implants to monitor drug release and transport in trabecular bone under ex-vivo conditions. In order to better understand how the bioreactor perfusion rate and the local drug release from the titanium wire affect overall drug distribution in the bone sample we utilize 3D finite element modelling. This model is based on porous media theory and takes into account advective-diffusive transport of the drug through the micropores of bone. The results showed a consistent, gradual release of model drug from the TNT/Ti implants, with a characteristic three-dimensional distribution into the surrounding bone over a period of 5 days. These results demonstrate the utility of this system for ex-vivo drug release studies in bone, which can be applied to optimise the administration therapeutics in bone for specific therapies and design of new drug delivery systems
Sclerostin is expressed almost exclusively by mature osteocytes in bone. Recent findings from thi... more Sclerostin is expressed almost exclusively by mature osteocytes in bone. Recent findings from this lab indicate that sclerostin targets pre-osteocytes and osteocytes to regulate bone mineralisation(1), osteoclast activity(2), and, potentially, osteocytic osteolysis(3). Sclerostin expression in vivo is associated with the response of osteocytes to mechanical loading and unloading. The aim of this study was to examine the direct effects of sclerostin on loading-induced bone growth ex vivo. For this, 10x5mm bovine sternum trabecular bone cores were perfused with osteogenic media at 37°C for up to 3 weeks in individual bone culture chambers. The cores were divided into 3 groups; a) mechanically loaded (300 cycles, 4000 µstrain, 1 Hz/day), b) identical loading regime with continuous perfusion of 50 ng/ml recombinant human sclerostin and c) unloaded controls. Loading was accomplished using the Zetos™ long-term bone organ culture and piezo-electric bone loading system. Daily measurements of the bone core stiffness, media pH and ionic calcium concentrations were made. Histomorphometric assessment, including fluorochrome labelling analysis, was made at the end of the experiment. Bone stiffness increased greatly with mechanical loading but this was attenuated significantly with the addition of sclerostin. The pH of the media after 24 hours decreased and ionic calcium concentrations increased in the presence of sclerostin when compared to mechanical loading alone. Sclerostin also completely abrogated loading-induced calcium/calcein uptake by the bone cores. Together, the results suggest that an osteocyte/osteoclast response to sclerostin was responsible for these effects. Our results are the first direct evidence for a negative effect of sclerostin on the anabolic response of bone to mechanical loading.
ABSTRACT Aim: Osteocytes and osteoblasts produce the humoral factor, FGF23, which maintains serum... more ABSTRACT Aim: Osteocytes and osteoblasts produce the humoral factor, FGF23, which maintains serum levels of phosphate and 1, 25 dihydroxy vitamin D within the physiological range. This implies the ability to sense the levels of phosphate in blood and interstitial fluid, however there is no phosphate sensing mechanism has been identified in humans. In this study we investigated the ability of bone tissue to sense phosphate. Methods: We obtained human trabecular bone samples from patients undergoing total hip replacement. Samples were incubated with different concentrations of phosphate with or without dihydroxy vitamin D in the media (phosphate: 1.0mM to 10mM, 1, 25 dihydroxy vitamin D: 0 or 10nM). After 24 to 72 hours, RNA was extracted for measurement by qPCR of mRNA corresponding to phosphate homeostasis or bone formation related genes, such as FGF23, PHEX, DMP1, GALNT3 and SOST. Results: Gene expression in human bone samples was differentially regulated by phosphate in a time and dose dependent. The clearest effects were seen after 72 h treatment and the co-presence of 1, 25 dihydroxy vitamin D (10nM) increased the responses of FGF23, PHEX, DMP1 and SOST mRNA levels to phosphate. After 72 hours treatment with phosphate at 2.5 or 5.0mM, expression of each of FGF23, PHEX, DMP1 and SOST mRNA was increased. GALNT3 was up-regulated by phosphate at 24 hour. Conclusions: These results showed the possibility that extracellular phosphate can be sensed by cells in the bone and that a high phosphate level can stimulate the production of FGF23 or perhaps prevent the cleavage of FGF23 via up-regulation of GALNT3. High levels of 1, 25 dihydroxy vitamin D enhanced these responses. The results also suggested that high phosphate may prevent bone formation by increasing sclerostin expression.
Osteocytes play a major role in normal bone maintenance and have recently been implicated in a nu... more Osteocytes play a major role in normal bone maintenance and have recently been implicated in a number of conditions that affect bone. Sclerostin is an osteocyte secreted protein, deficiency of which was found to be causative of the high bone mass phenotype in patients with sclerosteosis and van Buchem's disease. Mechanical loading of bone within the anabolic range decreases osteocyte sclerostin expression. An anti-sclerostin antibody, currently undergoing clinical trials, has been developed as a bone anabolic agent. Recent evidence from in vitro and in vivo experiments shows that osteocytes, via sclerostin, downregulate osteoblastogenesis (anti-anabolic) and potentiate osteoclastogenesis procatabolic) activity. PURPOSE OF STUDY In this study, the interaction between sclerostin and mechanical loading was investigated in bovine trabecular bone cores cultured ex vivo. METHODS Using a novel bone organ culture system, fresh bovine trabecular bone cores were cultured for up to 3 weeks. The cores were divided into 3 groups; A (loaded, using the Zetos™ piezoelectric loading system), which were subjected to daily mechanical loading (300 cycles of 4,000 μstrain at 1 Hz), B (loaded + sclerostin), which had an identical loading regime and were perfused continuously with 50 ng/ml recombinant human sclerostin, and C (unloaded), which were not loaded and were perfused with medium in the absence of sclerostin. Daily measurements of stiffness, pH and ionic calcium were made. Calcein was introduced both one week and one day before the experiment was ended. RESULTS The stiffness of the cores increased in response to mechanical loading in A. Samples in B had a significantly lower response to mechanical loading. The pH of medium from both B and C was significantly lower than that from A. The ionic calcium concentration present in the culture media after 24 hours was higher in B than in A. Calcein uptake in B was also observed to be less than in A. CONCLUSIONS This is the first direct evidence that sclerostin blunts the anabolic effect of mechanical loading via reduced bone formation, which is in agreement with current in vitro and in vivo published work. The ex vivo culture of trabecular bone appears to be an ideal model for the demonstration of direct effects of bone acting agents in large mammals.
Circulating 1 α ,25-dihydroxyvitamin D₃ (1,25D) derives from conversion of 25-hydroxyvitamin D₃ (... more Circulating 1 α ,25-dihydroxyvitamin D₃ (1,25D) derives from conversion of 25-hydroxyvitamin D₃ (25D) by the kidney 1 α -hydroxylase (CYP27B1). 1,25D exerts a number of effects on human osteoblasts, including inhibition of proliferation and differentiation, and effects on gene expression, including stimulation of RANKL, osteocalcin (OCN) and bone sialoprotein (BSP). We have examined vitamin D₃ metabolism in human osteoblastic cells, and found that primary normal human osteoblasts (NHBC) and human osteosarcoma cell lines, including MG-63, SaOS-2, HOS, G-292, all up-regulate the negative regulator of 1,25D, the 24-hydroxylase (CYP24), in response to 1,25D exposure. Additionally, all of these cell types expressed CYP27B1 mRNA, implying that human osteoblasts are capable of metabolising 25D into 1,25D. We have investigated this possibility and found that NHBC exposed to physiological concentrations of 25D (10 - 100 nM) in the absence of serum, exhibit up-regulated transcription of the downstream genes RANKL and OCN. Unlike 1,25D, 25D did not elicit a vigorous CYP24 response except at high concentrations (10⁻⁷ - 10⁻⁶ M). Consistent with this, NHBC treated with high concentrations of 25D secreted detectable 1,25D into the culture supernatant. We also found that NHBC express the 25-hydroxylase, and treatment with 1-hydroxyvitamin D₃ (1D), resulted in a gene expression response qualitatively similar to 25D. Inhibition of CYP activity using ketoconazole (10 μ M) resulted in an elevated response to 1,25D, probably due to inhibition of the catabolic activity of CYP24. Results to date indicate that the activity of 1D is CYP-dependent, since ketoconazole abolished its effects. However, 25D effects at low concentrations were unaffected by ketoconazole, indicating that 25D may have direct effects in osteoblasts independent of its conversion to 1,25D. Our results suggest that vitamin D₃ metabolism represents an intrinsic autocrine/paracrine pathway in these cells. Thus, vitamin D metabolites may regulate key functions in human osteoblasts independently of circulating levels of 1,25D
Journal of Bone and Joint Surgery-british Volume, Jul 1, 2014
Introduction Sclerostin has been implicated in mechanotransduction in bone and recent data show a... more Introduction Sclerostin has been implicated in mechanotransduction in bone and recent data show a lack of response to loading in the sclerostin transgenic mouse. Sclerostin, the protein product of the SOST gene, is an attractive therapeutic target for low bone mass conditions, including osteoporosis. It is expressed exclusively by mature osteocytes in bone and we have shown that sclerostin targets pre-osteocytes/osteocytes to regulate bone mineralization and osteoclast activity, as well as inducing catabolic gene expression in osteocytes themselves and promoting osteocyte-mediated bone loss (osteocytic osteolysis). The aim of this study was to examine the direct effects of sclerostin on anabolic responses to loading in bone ex vivo. Methods 10 × 5mm bovine sternum trabecular bone cores were perfused with osteogenic media at 37°C for up to 3 weeks in individual bone culture chambers. The cores were divided into 3 groups; a) mechanically loaded (300 cycles, 4000 μstrain, 1 Hz/day), b) identical loading regime with continuous perfusion of 50 ng/ml recombinant human sclerostin and c) unloaded controls. Loading was accomplished using a second-generation Zetos™ bone loading system. Daily measurements of bone stiffness (Young9s modulus), media pH and ionic calcium concentrations were made. Histomorphometric assessment, including fluorochrome labelling analysis, was made of resin-embedded, non-decalcified samples at the end of the experiment. Gene expression in the bovine bone was examined by real-time RT-PCR. Results Bovine bone cores showed a steady increase in Young9s modulus with daily application of mechanical loading. This increase in stiffness was blocked by the co-addition of sclerostin. Sclerostin also induced bone acidification and a net release of bone calcium, indicated by the decrease in media pH and the relative increase in ionic calcium concentrations in the presence of sclerostin. Sclerostin also completely abrogated loading-induced calcium/calcein uptake. Sclerostin induced an increase in the expression of the bone resorption genes, tartrate resistant acid phosphatase (TRAP), carbonic anhydrase and cathepsin K and induced the release of β-CTX. Histological examination revealed a significant increase in the size of the osteocyte lacunae in sclerostin-treated bone cores, suggesting a role for osteocytic osteolysis in this effect. Discussion/Conclusion The observation that sclerostin abrogated the loading-induced increase in bone stiffness constitutes direct evidence for a negative effect of sclerostin on the anabolic response to mechanical loading. Our findings may be explained in part by the observation that sclerostin negatively controls mineralization by late osteoblasts and pre-osteocytes (1). It is also possible that osteocytes themselves are capable of releasing bone mineral in response to sclerostin. This study demonstrates that sclerostin directly antagonises the anabolic effects of mechanical loading in the absence of external (circulating, neural, hormonal) influences. The mechanisms, by which sclerostin exerts these effects, warrant further study.
The aim of this study was evaluation of osteoinductive properties of demineralized bovine foetal ... more The aim of this study was evaluation of osteoinductive properties of demineralized bovine foetal growth plate in submuscular transplantation (ectopic osteoinduction). Demineralized bovine foetal growth plate was ectopically implanted in 18 male Sprague-Dawley rats. In 18 of the animals under aseptic conditions two submuscular pouches were created between external and internal oblique abdominal muscles in the two flanks: the right was left empty (sham) and the left was filled with 20 mg of demineralized bovine foetal growth plate powder. Radiographs were taken in 2, 4 and 6 weeks after the surgery, then six animals were pharmacologically euthanized after 2, 4 and 6 weeks for histopathological evaluation. Results showed: (1) osteoinductivity of xenogenic demineralized bovine foetal growth plate powder, and (2) earlier mineralization of ectopically implanted demineralized bovine foetal growth plate in the submuscular implanted area. Our results show that submuscular implantation of xenogenic demineralized bovine foetal growth plate has osteoinductive properties in a rat model.
OBJECTIVE The association between the spatially distributed level of active TGFβ1 in human subcho... more OBJECTIVE The association between the spatially distributed level of active TGFβ1 in human subchondral bone, and the characteristic structural and cellular parameters of human knee OA, was assessed. DESIGN Paired subchondral bone samples from 35 OA arthroplasty patients, (15 men and 20 women, aged 69±9 years) were obtained from beneath macroscopically present (CA+) or denuded cartilage (CA-) to determine the concentration of active TGFβ1 (ELISA) and its relationship to bone quality (synchrotron micro-CT), cellularity, and vascularization (histology). RESULTS Bone samples beneath (CA-) regions had significantly increased concentrations of active TGFβ1 protein (mean difference: 26.4; 95% CI: [3.2, 49.7]), when compared to bone in CA+ regions. Trabecular Bone below (CA-) regions had increased bone volume (median difference: 4.3; 96.49% CI: [-1.7, 17.8]), increased trabecular number (1.5 [0.006, 2.6], decreased trabecular separation (-0.05 [-0.1,-0.005]), and increased bone mineral density (394.5 [65.7, 723.3]) comparing to (CA+) regions. Further, (CA-) bone regions showed increased osteocyte density (0.012 [0.006, 0.018]), with larger osteocyte lacunae (39.8 [7.8, 71.7]) that were less spherical (-0.02 [-0.04, -0.003]), and increased bone matrix vascularity (12.4 [0.3, 24.5]) compared to (CA+). In addition, increased levels of active TGFβ1 related to increased bone volume (0.04 [-0.11, 0.9]), while increased OARSI grade associated with lacunar volume (-44.1 [-71.1, -17.2]), and orientation (2.7 [0.8, 4.6]). CONCLUSION Increased concentration of active TGFβ1 in the subchondral bone of human knee OA associates spatially with impaired bone quality and disease severity, suggesting that TGFβ1 is a potentional therapeutic target to prevent or reduce human OA disease progression.
Chemotherapy is an established treatment modality for bone sarcomas such as osteosarcoma (OS). Ho... more Chemotherapy is an established treatment modality for bone sarcomas such as osteosarcoma (OS). However, the use of chemotherapy in high-grade soft tissue sarcomas remains controversial, with the most active chemotherapeutic agent, doxorubicin (DOX), reported to have a response rate of, at best only 34% and most studies reporting lower response rates. Apo2L/TRAIL is a member of the tumour necrosis factor (TNF) family of cytokines and induces death of tumour cells, but not normal cells. Its potent apoptotic activity is mediated through cell surface death domain-containing receptors, DR4/TRAIL-R1 and DR5/TRAIL-R2. We investigated the efficacy of Apo2L/TRAIL as a single agent, and in combination with clinically relevant chemotherapeutic drugs, in fresh isolates of primary malignant cells obtained from biopsy material. The data presented here demonstrate that, in a range of primary bone related tumours, as well as soft tissue sarcomas, chemotherapeutic agents were only moderately effective, in terms of induction of cell death. Apo2L/TRAIL alone had little or no effect on any bone-related tumour or sarcoma in culture. In contrast, the combination of Apo2L/TRAIL and chemotherapeutic drugs produced a significant increase in tumour cell death, with DOX and Apo2L/TRAIL proving to be the most effective combination. These data suggest the potential for Apo2L/TRAIL to increase the effectiveness of chemotherapeutic drugs in bone and soft tissue sarcomas, while perhaps concurrently allowing a reduction in the exposure to drugs such as DOX, and a consequent reduction in toxicity. The synergistic action between these two different classes of agents has yet to be
A nontransformed rat clonal cell line (UMR-201) with phenotypic characteristics of osteoblastic p... more A nontransformed rat clonal cell line (UMR-201) with phenotypic characteristics of osteoblastic precursor cells was found to respond to insulinlike growth factor 1 (IGF-1) by increased osteonectin and pro-a,(I)-collagen mRNA expression. Cells were treated for 24 h with insulin, growth hormone, or IGF-1 to study the regulation of messenger RNA for osteonectin and pro-a,(I)-collagen using Northern blot hybridization. UMR-201 cells possess specific high-affinity receptors for growth hormone, although there were no significant effects of growth hormone (10-9-10-7 M) or insulin (10-9-10-6 M) on mRNA species for osteonectin or pro-a,(I)-collagen. However, IGF-1 increased both mRNA species from a concentration of M. The effect on osteonectin mRNA expression was likely due to increased transcription; when 5' flanking osteonectin (ON) genomic fragments were linked to the bacterial reporter gene chloramphenicol acetyltransferase (CAT) and introduced by transfection into UMR-201 cells, the transcriptional activity of the ON-CAT construct was increased 235 and 270% by lo-* and M IGF-1, respectively. In contrast, growth hormone did not change the transcriptional activity of the ON-CAT construct. In confirmation of other work, transforming growth factor (TGF-0, 0.1-2.5 ng/ml) increased mRNA for osteonectin and pro-cyl(I)-collagen in a dose-dependent manner. Transforming growth factor a (TGF-a) at 0.1-10 ng/ml had no consistent effects in repeated experiments on osteonectin and pro-a,(I)-collagen mRNA. The positive stimulatory effect of both IGF-1 and TGF-0 provides insights on the regulation of bone matrix proteins and suggests a major role of these growth factors in the remodeling process of bone.
The Journal of Steroid Biochemistry and Molecular Biology, Oct 1, 2014
The metabolism of 25-hydroxyvitamin D (25D) to active 1α,25-dihydroxyvitamin D (1,25D) by endogen... more The metabolism of 25-hydroxyvitamin D (25D) to active 1α,25-dihydroxyvitamin D (1,25D) by endogenous expression of 25D 1-α hydroxylase (CYP27B1) in bone cells appears to have functional effects in both osteoclasts and osteoblasts. To examine relationships between CYP27B1 expression in bone and its potential function in vivo, we examined the expression of vitamin D metabolism genes (CYP27B1, CYP24A1, VDR) in human trabecular bone samples and compared them by linear regression analysis with the expression of osteoclast (TRAP, CA2, CATK, NFATC1), osteoblast (TNAP, COL1A1, OCN, MEPE, BRIL), osteocyte (DMP1, SOST, PHEX, MEPE, FGF23)-related gene markers, genes associated with osteoblast/osteocyte control of osteoclastogenesis (RANKL, M-CSF, OPG, IL-8, TWEAK) and transcription factors (NFATC1, RUNX2, OSX, MSX2, HIF1A). This revealed multiple significant gene expression relationships between CYP27B1 and the transcription factors RUNX2, NFATC1, consistent with the coordinated expression of this gene by both osteoblast and osteoclast-lineage cells, and with MSX2 and the hypoxia-inducible transcription factor, HIF1A. CYP27B1 expression associated mainly with gene markers of bone resorption. VDR mRNA expression was also associated with resorption-related genes. Against expectations, CYP27B1 expression did not associate with bone expressed genes known to be 1,25D responsive, such as OCN, RANKL and DMP1. The major implication of these relationships in gene expression is that endogenous 1,25D synthesis and the response to 1,25D in human trabecular bone is linked with coordinated functions in both the osteoclastic and osteoblastic compartments towards the control of bone remodelling. This article is part of a Special Issue entitled '16th Vitamin D Workshop'.
We have reported that short calcitonin (CT) treatment of mature mouse osteoclast-like cells (OCLs... more We have reported that short calcitonin (CT) treatment of mature mouse osteoclast-like cells (OCLs) in culture induced prolonged downregulation of the CT receptor (CTR) and desensitization to CT rechallenge, at the level of adenylate cyclase activity. In this study, we have extended those studies to examine the bone resorbing activity of OCLs pretreated with CT. OCLs, which formed on gelled type I collagen, were pretreated with salmon CT (sCT) (lo-' M, 1 h) and 24 h later were replated onto plastic dishes or dentine slices after removal from the gel by collagenase digestion. The number and population of either mononuclear or multinuclear OCLs that adhere to either surface was not affected by sCT pretreatment. It was found that OCLs pretreated with sCT regained reduced but significant bone resorbing capacity, which was quantitated as the surface area resorbed by OCLs on dentine slices. However, compared with control, the number of resorption pits produced by sCT-pretreated OCLs was slightly reduced, and the total pit area was decreased by approximately 40-50%.
Journal of Bone and Mineral Research, Jun 21, 2011
The identity of the cell type responsive to sclerostin, a negative regulator of bone mass, is unk... more The identity of the cell type responsive to sclerostin, a negative regulator of bone mass, is unknown. Since sclerostin is expressed in vivo by mineral-embedded osteocytes, we tested the hypothesis that sclerostin would regulate the behavior of cells actively involved in mineralization in adult bone, the preosteocyte. Differentiating cultures of human primary osteoblasts exposed to recombinant human sclerostin (rhSCL) for 35 days displayed dose-and time-dependent inhibition of in vitro mineralization, with late cultures being most responsive in terms of mineralization and gene expression. Treatment of advanced (day 35) cultures with rhSCL markedly increased the expression of the preosteocyte marker E11 and decreased the expression of mature markers DMP1 and SOST. Concomitantly, matrix extracellular phosphoglycoprotein (MEPE) expression was increased by rhSCL at both the mRNA and protein levels, whereas PHEX was decreased, implying regulation through the MEPE-ASARM axis. We confirmed that mineralization by human osteoblasts is exquisitely sensitive to the triphosphorylated ASARM-PO4 peptide. Immunostaining revealed that rhSCL increased the endogenous levels of MEPE-ASARM. Importantly, antibody-mediated neutralization of endogenous MEPE-ASARM antagonized the effect of rhSCL on mineralization, as did the PHEX synthetic peptide SPR4. Finally, we found elevated Sost mRNA expression in the long bones of HYP mice, suggesting that sclerostin may drive the increased MEPE-ASARM levels and mineralization defect in this genotype. Our results suggest that sclerostin acts through regulation of the PHEX/MEPE axis at the preosteocyte stage and serves as a master regulator of physiologic bone mineralization, consistent with its localization in vivo and its established role in the inhibition of bone formation.
Sclerostin is a product of mature osteocytes embedded in mineralised bone and is a negative regul... more Sclerostin is a product of mature osteocytes embedded in mineralised bone and is a negative regulator of bone mass and osteoblast differentiation. While evidence suggests that sclerostin has an anti-anabolic role, the possibility also exists that sclerostin has catabolic activity. To test this we treated human primary pre-osteocyte cultures, cells we have found are exquisitely sensitive to sclerostin, or mouse osteocyte-like MLO-Y4 cells, with recombinant human sclerostin (rhSCL) and measured effects on pro-catabolic gene expression. Sclerostin dose-dependently up-regulated the expression of receptor activator of nuclear factor kappa B (RANKL) mRNA and down-regulated that of osteoprotegerin (OPG) mRNA, causing an increase in the RANKL:OPG mRNA ratio. To examine the effects of rhSCL on resulting osteoclastic activity, MLO-Y4 cells plated onto a bone-like substrate were primed with rhSCL for 3 days and then either mouse splenocytes or human peripheral blood mononuclear cells (PBMC) were added. This resulted in cultures with elevated osteoclastic resorption (approximately 7-fold) compared to untreated co-cultures. The increased resorption was abolished by co-addition of recombinant OPG. In co-cultures of MLO-Y4 cells with PBMC, SCL also increased the number and size of the TRAP-positive multinucleated cells formed. Importantly, rhSCL had no effect on TRAP-positive cell formation from monocultures of either splenocytes or PBMC. Further, rhSCL did not induce apoptosis of MLO-Y4 cells, as determined by caspase activity assays, demonstrating that the osteoclastic response was not driven by dying osteocytes. Together, these results suggest that sclerostin may have a catabolic action through promotion of osteoclast formation and activity by osteocytes, in a RANKLdependent manner.
Objective: Over-activity of transforming growth factor β1 in subchondral bone has a direct causal... more Objective: Over-activity of transforming growth factor β1 in subchondral bone has a direct causal role in rodent models of knee osteoarthritis (OA), which can be blocked by β1 neutralisation. In this study, we investigated whether the spatially distributed level of active β 1 in human subchondral bone associates with the characteristic structural, cellular and molecular parameters of human knee OA. Design: Subchondral bone samples (35 OA arthroplasty patients, aged 69±9 years) were obtained from regions below either macroscopically present or denuded cartilage. Bone samples were processed to determine the concentration of active β 1 (ELISA) and gene-specific mRNA expression (RT-PCR). Synchrotron micro-CT imaging was utilised to assess the bone microstructure, bone mineralization, the osteocyte lacunar network and bone matrix vascularity. Finally, samples were histologically examined for cartilage OARSI grading, quantification of tartrate resistant acid phosphatase positive cells and...
respond to particles of clinically-relevant conventional and cross-linked polyethylene and metal ... more respond to particles of clinically-relevant conventional and cross-linked polyethylene and metal alloys by up-regulation of resorptive and inflammatory pathways, Acta Biomaterialia (2019), doi:
In this paper, a comprehensive framework is proposed to estimate the anisotropic permeability mat... more In this paper, a comprehensive framework is proposed to estimate the anisotropic permeability matrix in trabecular bone specimens based on micro-computed tomography (microCT) imaging combined with porescale fluid dynamics simulations. Two essential steps in the proposed methodology are the selection of (i) a representative volume element (RVE) for calculation of trabecular bone permeability and (ii) a converged mesh for accurate calculation of pore fluid flow properties. Accurate estimates of trabecular bone porosities are obtained using a microCT image resolution of approximately 10 lm. We show that a trabecular bone RVE in the order of 2 × 2 × 2 mm 3 is most suitable. Mesh convergence studies show that accurate fluid flow properties are obtained for a mesh size above 125,000 elements. Volume averaging of the pore-scale fluid flow properties allows calculation of the apparent permeability matrix of trabecular bone specimens. For the four specimens chosen, our numerical results show that the so obtained permeability coefficients are in excellent agreement with previously reported experimental data for both human and bovine trabecular bone samples. We also identified that bone samples taken from long bones generally exhibit a larger permeability in the longitudinal direction. The fact that all coefficients of the permeability matrix were different from zero indicates that bone samples are generally not harvested in the principal flow directions. The full permeability matrix was diagonalized by calculating the eigenvalues, while the eigenvectors showed how strongly the bone sample's orientations deviated from the principal flow directions. Porosity values of the four bone specimens range from 0.83 to 0.86, with a low standard deviation of ±0.016, principal permeability values range from 0.22 to 1.45 • 10 −8 m 2 , with a high standard deviation of ±0.33. Also, the anisotropic ratio ranged from 0.27 to 0.83, with high standard deviation. These results indicate that while the four specimens are quite similar in terms of average porosity, large variability exists with respect to permeability and specimen anisotropy. The utilized computational approach compares well with semi-analytical models based on homogenization theory. This methodology can be applied in bone tissue engineering applications for generating accurate pore morphologies of bone replacement materials and to consistently select similar bone specimens in bone bioreactor studies.
Local drug delivery using drug-releasing implants located inside bone is recognised as a promisin... more Local drug delivery using drug-releasing implants located inside bone is recognised as a promising strategy to address the limitations of systemic drug administration to bone. However, the studies of drug-release kinetics are not possible to perform using existing in-vitro drug releasing systems and 2-D bone cell models. The aim of this work is to demonstrate the use of a 3-D bone bioreactor for studying the drug-release kinetics and distribution of drugs in the ex-vivo cancellous bone environment. Bovine trabecular bone was used as the bone substrate, in which drug-releasing implants were embedded in the form of nano-engineered titanium wires covered with a layer of titania nanotube (TNT) arrays. A hydrophilic fluorescent dye (rhodamine B) was used as a model drug, loaded inside the TNT/Ti implants to monitor drug release and transport in trabecular bone under ex-vivo conditions. In order to better understand how the bioreactor perfusion rate and the local drug release from the titanium wire affect overall drug distribution in the bone sample we utilize 3D finite element modelling. This model is based on porous media theory and takes into account advective-diffusive transport of the drug through the micropores of bone. The results showed a consistent, gradual release of model drug from the TNT/Ti implants, with a characteristic three-dimensional distribution into the surrounding bone over a period of 5 days. These results demonstrate the utility of this system for ex-vivo drug release studies in bone, which can be applied to optimise the administration therapeutics in bone for specific therapies and design of new drug delivery systems
Sclerostin is expressed almost exclusively by mature osteocytes in bone. Recent findings from thi... more Sclerostin is expressed almost exclusively by mature osteocytes in bone. Recent findings from this lab indicate that sclerostin targets pre-osteocytes and osteocytes to regulate bone mineralisation(1), osteoclast activity(2), and, potentially, osteocytic osteolysis(3). Sclerostin expression in vivo is associated with the response of osteocytes to mechanical loading and unloading. The aim of this study was to examine the direct effects of sclerostin on loading-induced bone growth ex vivo. For this, 10x5mm bovine sternum trabecular bone cores were perfused with osteogenic media at 37°C for up to 3 weeks in individual bone culture chambers. The cores were divided into 3 groups; a) mechanically loaded (300 cycles, 4000 µstrain, 1 Hz/day), b) identical loading regime with continuous perfusion of 50 ng/ml recombinant human sclerostin and c) unloaded controls. Loading was accomplished using the Zetos™ long-term bone organ culture and piezo-electric bone loading system. Daily measurements of the bone core stiffness, media pH and ionic calcium concentrations were made. Histomorphometric assessment, including fluorochrome labelling analysis, was made at the end of the experiment. Bone stiffness increased greatly with mechanical loading but this was attenuated significantly with the addition of sclerostin. The pH of the media after 24 hours decreased and ionic calcium concentrations increased in the presence of sclerostin when compared to mechanical loading alone. Sclerostin also completely abrogated loading-induced calcium/calcein uptake by the bone cores. Together, the results suggest that an osteocyte/osteoclast response to sclerostin was responsible for these effects. Our results are the first direct evidence for a negative effect of sclerostin on the anabolic response of bone to mechanical loading.
ABSTRACT Aim: Osteocytes and osteoblasts produce the humoral factor, FGF23, which maintains serum... more ABSTRACT Aim: Osteocytes and osteoblasts produce the humoral factor, FGF23, which maintains serum levels of phosphate and 1, 25 dihydroxy vitamin D within the physiological range. This implies the ability to sense the levels of phosphate in blood and interstitial fluid, however there is no phosphate sensing mechanism has been identified in humans. In this study we investigated the ability of bone tissue to sense phosphate. Methods: We obtained human trabecular bone samples from patients undergoing total hip replacement. Samples were incubated with different concentrations of phosphate with or without dihydroxy vitamin D in the media (phosphate: 1.0mM to 10mM, 1, 25 dihydroxy vitamin D: 0 or 10nM). After 24 to 72 hours, RNA was extracted for measurement by qPCR of mRNA corresponding to phosphate homeostasis or bone formation related genes, such as FGF23, PHEX, DMP1, GALNT3 and SOST. Results: Gene expression in human bone samples was differentially regulated by phosphate in a time and dose dependent. The clearest effects were seen after 72 h treatment and the co-presence of 1, 25 dihydroxy vitamin D (10nM) increased the responses of FGF23, PHEX, DMP1 and SOST mRNA levels to phosphate. After 72 hours treatment with phosphate at 2.5 or 5.0mM, expression of each of FGF23, PHEX, DMP1 and SOST mRNA was increased. GALNT3 was up-regulated by phosphate at 24 hour. Conclusions: These results showed the possibility that extracellular phosphate can be sensed by cells in the bone and that a high phosphate level can stimulate the production of FGF23 or perhaps prevent the cleavage of FGF23 via up-regulation of GALNT3. High levels of 1, 25 dihydroxy vitamin D enhanced these responses. The results also suggested that high phosphate may prevent bone formation by increasing sclerostin expression.
Osteocytes play a major role in normal bone maintenance and have recently been implicated in a nu... more Osteocytes play a major role in normal bone maintenance and have recently been implicated in a number of conditions that affect bone. Sclerostin is an osteocyte secreted protein, deficiency of which was found to be causative of the high bone mass phenotype in patients with sclerosteosis and van Buchem's disease. Mechanical loading of bone within the anabolic range decreases osteocyte sclerostin expression. An anti-sclerostin antibody, currently undergoing clinical trials, has been developed as a bone anabolic agent. Recent evidence from in vitro and in vivo experiments shows that osteocytes, via sclerostin, downregulate osteoblastogenesis (anti-anabolic) and potentiate osteoclastogenesis procatabolic) activity. PURPOSE OF STUDY In this study, the interaction between sclerostin and mechanical loading was investigated in bovine trabecular bone cores cultured ex vivo. METHODS Using a novel bone organ culture system, fresh bovine trabecular bone cores were cultured for up to 3 weeks. The cores were divided into 3 groups; A (loaded, using the Zetos™ piezoelectric loading system), which were subjected to daily mechanical loading (300 cycles of 4,000 μstrain at 1 Hz), B (loaded + sclerostin), which had an identical loading regime and were perfused continuously with 50 ng/ml recombinant human sclerostin, and C (unloaded), which were not loaded and were perfused with medium in the absence of sclerostin. Daily measurements of stiffness, pH and ionic calcium were made. Calcein was introduced both one week and one day before the experiment was ended. RESULTS The stiffness of the cores increased in response to mechanical loading in A. Samples in B had a significantly lower response to mechanical loading. The pH of medium from both B and C was significantly lower than that from A. The ionic calcium concentration present in the culture media after 24 hours was higher in B than in A. Calcein uptake in B was also observed to be less than in A. CONCLUSIONS This is the first direct evidence that sclerostin blunts the anabolic effect of mechanical loading via reduced bone formation, which is in agreement with current in vitro and in vivo published work. The ex vivo culture of trabecular bone appears to be an ideal model for the demonstration of direct effects of bone acting agents in large mammals.
Circulating 1 α ,25-dihydroxyvitamin D₃ (1,25D) derives from conversion of 25-hydroxyvitamin D₃ (... more Circulating 1 α ,25-dihydroxyvitamin D₃ (1,25D) derives from conversion of 25-hydroxyvitamin D₃ (25D) by the kidney 1 α -hydroxylase (CYP27B1). 1,25D exerts a number of effects on human osteoblasts, including inhibition of proliferation and differentiation, and effects on gene expression, including stimulation of RANKL, osteocalcin (OCN) and bone sialoprotein (BSP). We have examined vitamin D₃ metabolism in human osteoblastic cells, and found that primary normal human osteoblasts (NHBC) and human osteosarcoma cell lines, including MG-63, SaOS-2, HOS, G-292, all up-regulate the negative regulator of 1,25D, the 24-hydroxylase (CYP24), in response to 1,25D exposure. Additionally, all of these cell types expressed CYP27B1 mRNA, implying that human osteoblasts are capable of metabolising 25D into 1,25D. We have investigated this possibility and found that NHBC exposed to physiological concentrations of 25D (10 - 100 nM) in the absence of serum, exhibit up-regulated transcription of the downstream genes RANKL and OCN. Unlike 1,25D, 25D did not elicit a vigorous CYP24 response except at high concentrations (10⁻⁷ - 10⁻⁶ M). Consistent with this, NHBC treated with high concentrations of 25D secreted detectable 1,25D into the culture supernatant. We also found that NHBC express the 25-hydroxylase, and treatment with 1-hydroxyvitamin D₃ (1D), resulted in a gene expression response qualitatively similar to 25D. Inhibition of CYP activity using ketoconazole (10 μ M) resulted in an elevated response to 1,25D, probably due to inhibition of the catabolic activity of CYP24. Results to date indicate that the activity of 1D is CYP-dependent, since ketoconazole abolished its effects. However, 25D effects at low concentrations were unaffected by ketoconazole, indicating that 25D may have direct effects in osteoblasts independent of its conversion to 1,25D. Our results suggest that vitamin D₃ metabolism represents an intrinsic autocrine/paracrine pathway in these cells. Thus, vitamin D metabolites may regulate key functions in human osteoblasts independently of circulating levels of 1,25D
Journal of Bone and Joint Surgery-british Volume, Jul 1, 2014
Introduction Sclerostin has been implicated in mechanotransduction in bone and recent data show a... more Introduction Sclerostin has been implicated in mechanotransduction in bone and recent data show a lack of response to loading in the sclerostin transgenic mouse. Sclerostin, the protein product of the SOST gene, is an attractive therapeutic target for low bone mass conditions, including osteoporosis. It is expressed exclusively by mature osteocytes in bone and we have shown that sclerostin targets pre-osteocytes/osteocytes to regulate bone mineralization and osteoclast activity, as well as inducing catabolic gene expression in osteocytes themselves and promoting osteocyte-mediated bone loss (osteocytic osteolysis). The aim of this study was to examine the direct effects of sclerostin on anabolic responses to loading in bone ex vivo. Methods 10 × 5mm bovine sternum trabecular bone cores were perfused with osteogenic media at 37°C for up to 3 weeks in individual bone culture chambers. The cores were divided into 3 groups; a) mechanically loaded (300 cycles, 4000 μstrain, 1 Hz/day), b) identical loading regime with continuous perfusion of 50 ng/ml recombinant human sclerostin and c) unloaded controls. Loading was accomplished using a second-generation Zetos™ bone loading system. Daily measurements of bone stiffness (Young9s modulus), media pH and ionic calcium concentrations were made. Histomorphometric assessment, including fluorochrome labelling analysis, was made of resin-embedded, non-decalcified samples at the end of the experiment. Gene expression in the bovine bone was examined by real-time RT-PCR. Results Bovine bone cores showed a steady increase in Young9s modulus with daily application of mechanical loading. This increase in stiffness was blocked by the co-addition of sclerostin. Sclerostin also induced bone acidification and a net release of bone calcium, indicated by the decrease in media pH and the relative increase in ionic calcium concentrations in the presence of sclerostin. Sclerostin also completely abrogated loading-induced calcium/calcein uptake. Sclerostin induced an increase in the expression of the bone resorption genes, tartrate resistant acid phosphatase (TRAP), carbonic anhydrase and cathepsin K and induced the release of β-CTX. Histological examination revealed a significant increase in the size of the osteocyte lacunae in sclerostin-treated bone cores, suggesting a role for osteocytic osteolysis in this effect. Discussion/Conclusion The observation that sclerostin abrogated the loading-induced increase in bone stiffness constitutes direct evidence for a negative effect of sclerostin on the anabolic response to mechanical loading. Our findings may be explained in part by the observation that sclerostin negatively controls mineralization by late osteoblasts and pre-osteocytes (1). It is also possible that osteocytes themselves are capable of releasing bone mineral in response to sclerostin. This study demonstrates that sclerostin directly antagonises the anabolic effects of mechanical loading in the absence of external (circulating, neural, hormonal) influences. The mechanisms, by which sclerostin exerts these effects, warrant further study.
The aim of this study was evaluation of osteoinductive properties of demineralized bovine foetal ... more The aim of this study was evaluation of osteoinductive properties of demineralized bovine foetal growth plate in submuscular transplantation (ectopic osteoinduction). Demineralized bovine foetal growth plate was ectopically implanted in 18 male Sprague-Dawley rats. In 18 of the animals under aseptic conditions two submuscular pouches were created between external and internal oblique abdominal muscles in the two flanks: the right was left empty (sham) and the left was filled with 20 mg of demineralized bovine foetal growth plate powder. Radiographs were taken in 2, 4 and 6 weeks after the surgery, then six animals were pharmacologically euthanized after 2, 4 and 6 weeks for histopathological evaluation. Results showed: (1) osteoinductivity of xenogenic demineralized bovine foetal growth plate powder, and (2) earlier mineralization of ectopically implanted demineralized bovine foetal growth plate in the submuscular implanted area. Our results show that submuscular implantation of xenogenic demineralized bovine foetal growth plate has osteoinductive properties in a rat model.
OBJECTIVE The association between the spatially distributed level of active TGFβ1 in human subcho... more OBJECTIVE The association between the spatially distributed level of active TGFβ1 in human subchondral bone, and the characteristic structural and cellular parameters of human knee OA, was assessed. DESIGN Paired subchondral bone samples from 35 OA arthroplasty patients, (15 men and 20 women, aged 69±9 years) were obtained from beneath macroscopically present (CA+) or denuded cartilage (CA-) to determine the concentration of active TGFβ1 (ELISA) and its relationship to bone quality (synchrotron micro-CT), cellularity, and vascularization (histology). RESULTS Bone samples beneath (CA-) regions had significantly increased concentrations of active TGFβ1 protein (mean difference: 26.4; 95% CI: [3.2, 49.7]), when compared to bone in CA+ regions. Trabecular Bone below (CA-) regions had increased bone volume (median difference: 4.3; 96.49% CI: [-1.7, 17.8]), increased trabecular number (1.5 [0.006, 2.6], decreased trabecular separation (-0.05 [-0.1,-0.005]), and increased bone mineral density (394.5 [65.7, 723.3]) comparing to (CA+) regions. Further, (CA-) bone regions showed increased osteocyte density (0.012 [0.006, 0.018]), with larger osteocyte lacunae (39.8 [7.8, 71.7]) that were less spherical (-0.02 [-0.04, -0.003]), and increased bone matrix vascularity (12.4 [0.3, 24.5]) compared to (CA+). In addition, increased levels of active TGFβ1 related to increased bone volume (0.04 [-0.11, 0.9]), while increased OARSI grade associated with lacunar volume (-44.1 [-71.1, -17.2]), and orientation (2.7 [0.8, 4.6]). CONCLUSION Increased concentration of active TGFβ1 in the subchondral bone of human knee OA associates spatially with impaired bone quality and disease severity, suggesting that TGFβ1 is a potentional therapeutic target to prevent or reduce human OA disease progression.
Chemotherapy is an established treatment modality for bone sarcomas such as osteosarcoma (OS). Ho... more Chemotherapy is an established treatment modality for bone sarcomas such as osteosarcoma (OS). However, the use of chemotherapy in high-grade soft tissue sarcomas remains controversial, with the most active chemotherapeutic agent, doxorubicin (DOX), reported to have a response rate of, at best only 34% and most studies reporting lower response rates. Apo2L/TRAIL is a member of the tumour necrosis factor (TNF) family of cytokines and induces death of tumour cells, but not normal cells. Its potent apoptotic activity is mediated through cell surface death domain-containing receptors, DR4/TRAIL-R1 and DR5/TRAIL-R2. We investigated the efficacy of Apo2L/TRAIL as a single agent, and in combination with clinically relevant chemotherapeutic drugs, in fresh isolates of primary malignant cells obtained from biopsy material. The data presented here demonstrate that, in a range of primary bone related tumours, as well as soft tissue sarcomas, chemotherapeutic agents were only moderately effective, in terms of induction of cell death. Apo2L/TRAIL alone had little or no effect on any bone-related tumour or sarcoma in culture. In contrast, the combination of Apo2L/TRAIL and chemotherapeutic drugs produced a significant increase in tumour cell death, with DOX and Apo2L/TRAIL proving to be the most effective combination. These data suggest the potential for Apo2L/TRAIL to increase the effectiveness of chemotherapeutic drugs in bone and soft tissue sarcomas, while perhaps concurrently allowing a reduction in the exposure to drugs such as DOX, and a consequent reduction in toxicity. The synergistic action between these two different classes of agents has yet to be
A nontransformed rat clonal cell line (UMR-201) with phenotypic characteristics of osteoblastic p... more A nontransformed rat clonal cell line (UMR-201) with phenotypic characteristics of osteoblastic precursor cells was found to respond to insulinlike growth factor 1 (IGF-1) by increased osteonectin and pro-a,(I)-collagen mRNA expression. Cells were treated for 24 h with insulin, growth hormone, or IGF-1 to study the regulation of messenger RNA for osteonectin and pro-a,(I)-collagen using Northern blot hybridization. UMR-201 cells possess specific high-affinity receptors for growth hormone, although there were no significant effects of growth hormone (10-9-10-7 M) or insulin (10-9-10-6 M) on mRNA species for osteonectin or pro-a,(I)-collagen. However, IGF-1 increased both mRNA species from a concentration of M. The effect on osteonectin mRNA expression was likely due to increased transcription; when 5' flanking osteonectin (ON) genomic fragments were linked to the bacterial reporter gene chloramphenicol acetyltransferase (CAT) and introduced by transfection into UMR-201 cells, the transcriptional activity of the ON-CAT construct was increased 235 and 270% by lo-* and M IGF-1, respectively. In contrast, growth hormone did not change the transcriptional activity of the ON-CAT construct. In confirmation of other work, transforming growth factor (TGF-0, 0.1-2.5 ng/ml) increased mRNA for osteonectin and pro-cyl(I)-collagen in a dose-dependent manner. Transforming growth factor a (TGF-a) at 0.1-10 ng/ml had no consistent effects in repeated experiments on osteonectin and pro-a,(I)-collagen mRNA. The positive stimulatory effect of both IGF-1 and TGF-0 provides insights on the regulation of bone matrix proteins and suggests a major role of these growth factors in the remodeling process of bone.
The Journal of Steroid Biochemistry and Molecular Biology, Oct 1, 2014
The metabolism of 25-hydroxyvitamin D (25D) to active 1α,25-dihydroxyvitamin D (1,25D) by endogen... more The metabolism of 25-hydroxyvitamin D (25D) to active 1α,25-dihydroxyvitamin D (1,25D) by endogenous expression of 25D 1-α hydroxylase (CYP27B1) in bone cells appears to have functional effects in both osteoclasts and osteoblasts. To examine relationships between CYP27B1 expression in bone and its potential function in vivo, we examined the expression of vitamin D metabolism genes (CYP27B1, CYP24A1, VDR) in human trabecular bone samples and compared them by linear regression analysis with the expression of osteoclast (TRAP, CA2, CATK, NFATC1), osteoblast (TNAP, COL1A1, OCN, MEPE, BRIL), osteocyte (DMP1, SOST, PHEX, MEPE, FGF23)-related gene markers, genes associated with osteoblast/osteocyte control of osteoclastogenesis (RANKL, M-CSF, OPG, IL-8, TWEAK) and transcription factors (NFATC1, RUNX2, OSX, MSX2, HIF1A). This revealed multiple significant gene expression relationships between CYP27B1 and the transcription factors RUNX2, NFATC1, consistent with the coordinated expression of this gene by both osteoblast and osteoclast-lineage cells, and with MSX2 and the hypoxia-inducible transcription factor, HIF1A. CYP27B1 expression associated mainly with gene markers of bone resorption. VDR mRNA expression was also associated with resorption-related genes. Against expectations, CYP27B1 expression did not associate with bone expressed genes known to be 1,25D responsive, such as OCN, RANKL and DMP1. The major implication of these relationships in gene expression is that endogenous 1,25D synthesis and the response to 1,25D in human trabecular bone is linked with coordinated functions in both the osteoclastic and osteoblastic compartments towards the control of bone remodelling. This article is part of a Special Issue entitled '16th Vitamin D Workshop'.
We have reported that short calcitonin (CT) treatment of mature mouse osteoclast-like cells (OCLs... more We have reported that short calcitonin (CT) treatment of mature mouse osteoclast-like cells (OCLs) in culture induced prolonged downregulation of the CT receptor (CTR) and desensitization to CT rechallenge, at the level of adenylate cyclase activity. In this study, we have extended those studies to examine the bone resorbing activity of OCLs pretreated with CT. OCLs, which formed on gelled type I collagen, were pretreated with salmon CT (sCT) (lo-' M, 1 h) and 24 h later were replated onto plastic dishes or dentine slices after removal from the gel by collagenase digestion. The number and population of either mononuclear or multinuclear OCLs that adhere to either surface was not affected by sCT pretreatment. It was found that OCLs pretreated with sCT regained reduced but significant bone resorbing capacity, which was quantitated as the surface area resorbed by OCLs on dentine slices. However, compared with control, the number of resorption pits produced by sCT-pretreated OCLs was slightly reduced, and the total pit area was decreased by approximately 40-50%.
Journal of Bone and Mineral Research, Jun 21, 2011
The identity of the cell type responsive to sclerostin, a negative regulator of bone mass, is unk... more The identity of the cell type responsive to sclerostin, a negative regulator of bone mass, is unknown. Since sclerostin is expressed in vivo by mineral-embedded osteocytes, we tested the hypothesis that sclerostin would regulate the behavior of cells actively involved in mineralization in adult bone, the preosteocyte. Differentiating cultures of human primary osteoblasts exposed to recombinant human sclerostin (rhSCL) for 35 days displayed dose-and time-dependent inhibition of in vitro mineralization, with late cultures being most responsive in terms of mineralization and gene expression. Treatment of advanced (day 35) cultures with rhSCL markedly increased the expression of the preosteocyte marker E11 and decreased the expression of mature markers DMP1 and SOST. Concomitantly, matrix extracellular phosphoglycoprotein (MEPE) expression was increased by rhSCL at both the mRNA and protein levels, whereas PHEX was decreased, implying regulation through the MEPE-ASARM axis. We confirmed that mineralization by human osteoblasts is exquisitely sensitive to the triphosphorylated ASARM-PO4 peptide. Immunostaining revealed that rhSCL increased the endogenous levels of MEPE-ASARM. Importantly, antibody-mediated neutralization of endogenous MEPE-ASARM antagonized the effect of rhSCL on mineralization, as did the PHEX synthetic peptide SPR4. Finally, we found elevated Sost mRNA expression in the long bones of HYP mice, suggesting that sclerostin may drive the increased MEPE-ASARM levels and mineralization defect in this genotype. Our results suggest that sclerostin acts through regulation of the PHEX/MEPE axis at the preosteocyte stage and serves as a master regulator of physiologic bone mineralization, consistent with its localization in vivo and its established role in the inhibition of bone formation.
Sclerostin is a product of mature osteocytes embedded in mineralised bone and is a negative regul... more Sclerostin is a product of mature osteocytes embedded in mineralised bone and is a negative regulator of bone mass and osteoblast differentiation. While evidence suggests that sclerostin has an anti-anabolic role, the possibility also exists that sclerostin has catabolic activity. To test this we treated human primary pre-osteocyte cultures, cells we have found are exquisitely sensitive to sclerostin, or mouse osteocyte-like MLO-Y4 cells, with recombinant human sclerostin (rhSCL) and measured effects on pro-catabolic gene expression. Sclerostin dose-dependently up-regulated the expression of receptor activator of nuclear factor kappa B (RANKL) mRNA and down-regulated that of osteoprotegerin (OPG) mRNA, causing an increase in the RANKL:OPG mRNA ratio. To examine the effects of rhSCL on resulting osteoclastic activity, MLO-Y4 cells plated onto a bone-like substrate were primed with rhSCL for 3 days and then either mouse splenocytes or human peripheral blood mononuclear cells (PBMC) were added. This resulted in cultures with elevated osteoclastic resorption (approximately 7-fold) compared to untreated co-cultures. The increased resorption was abolished by co-addition of recombinant OPG. In co-cultures of MLO-Y4 cells with PBMC, SCL also increased the number and size of the TRAP-positive multinucleated cells formed. Importantly, rhSCL had no effect on TRAP-positive cell formation from monocultures of either splenocytes or PBMC. Further, rhSCL did not induce apoptosis of MLO-Y4 cells, as determined by caspase activity assays, demonstrating that the osteoclastic response was not driven by dying osteocytes. Together, these results suggest that sclerostin may have a catabolic action through promotion of osteoclast formation and activity by osteocytes, in a RANKLdependent manner.
Objective: Over-activity of transforming growth factor β1 in subchondral bone has a direct causal... more Objective: Over-activity of transforming growth factor β1 in subchondral bone has a direct causal role in rodent models of knee osteoarthritis (OA), which can be blocked by β1 neutralisation. In this study, we investigated whether the spatially distributed level of active β 1 in human subchondral bone associates with the characteristic structural, cellular and molecular parameters of human knee OA. Design: Subchondral bone samples (35 OA arthroplasty patients, aged 69±9 years) were obtained from regions below either macroscopically present or denuded cartilage. Bone samples were processed to determine the concentration of active β 1 (ELISA) and gene-specific mRNA expression (RT-PCR). Synchrotron micro-CT imaging was utilised to assess the bone microstructure, bone mineralization, the osteocyte lacunar network and bone matrix vascularity. Finally, samples were histologically examined for cartilage OARSI grading, quantification of tartrate resistant acid phosphatase positive cells and...
respond to particles of clinically-relevant conventional and cross-linked polyethylene and metal ... more respond to particles of clinically-relevant conventional and cross-linked polyethylene and metal alloys by up-regulation of resorptive and inflammatory pathways, Acta Biomaterialia (2019), doi:
In this paper, a comprehensive framework is proposed to estimate the anisotropic permeability mat... more In this paper, a comprehensive framework is proposed to estimate the anisotropic permeability matrix in trabecular bone specimens based on micro-computed tomography (microCT) imaging combined with porescale fluid dynamics simulations. Two essential steps in the proposed methodology are the selection of (i) a representative volume element (RVE) for calculation of trabecular bone permeability and (ii) a converged mesh for accurate calculation of pore fluid flow properties. Accurate estimates of trabecular bone porosities are obtained using a microCT image resolution of approximately 10 lm. We show that a trabecular bone RVE in the order of 2 × 2 × 2 mm 3 is most suitable. Mesh convergence studies show that accurate fluid flow properties are obtained for a mesh size above 125,000 elements. Volume averaging of the pore-scale fluid flow properties allows calculation of the apparent permeability matrix of trabecular bone specimens. For the four specimens chosen, our numerical results show that the so obtained permeability coefficients are in excellent agreement with previously reported experimental data for both human and bovine trabecular bone samples. We also identified that bone samples taken from long bones generally exhibit a larger permeability in the longitudinal direction. The fact that all coefficients of the permeability matrix were different from zero indicates that bone samples are generally not harvested in the principal flow directions. The full permeability matrix was diagonalized by calculating the eigenvalues, while the eigenvectors showed how strongly the bone sample's orientations deviated from the principal flow directions. Porosity values of the four bone specimens range from 0.83 to 0.86, with a low standard deviation of ±0.016, principal permeability values range from 0.22 to 1.45 • 10 −8 m 2 , with a high standard deviation of ±0.33. Also, the anisotropic ratio ranged from 0.27 to 0.83, with high standard deviation. These results indicate that while the four specimens are quite similar in terms of average porosity, large variability exists with respect to permeability and specimen anisotropy. The utilized computational approach compares well with semi-analytical models based on homogenization theory. This methodology can be applied in bone tissue engineering applications for generating accurate pore morphologies of bone replacement materials and to consistently select similar bone specimens in bone bioreactor studies.
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Papers by David Findlay