ORCID Profile
0000-0002-3123-9861
Current Organisation
University of Adelaide
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Publisher: Springer Science and Business Media LLC
Date: 18-08-2011
DOI: 10.1007/S00198-011-1740-9
Abstract: It is now well accepted that the molecule receptor activator of NFκB ligand (RANKL) and osteoprotegerin play key roles in regulating physiological and pathological bone turnover. There are a large number of published reports of circulating RANKL levels in both health and pathology. However, interpretation of these data has been elusive, and the relationship between circulating RANKL and RANKL levels in bone is still not clear. This review explores this subject, documenting the possible origins of circulating RANKL and suggesting additional information that is required before serum RANKL levels can provide useful diagnostic or research information.
Publisher: Wiley
Date: 09-2008
DOI: 10.1359/JBMR.080412
Abstract: In the course of attempting to define the bone "secretome" using a signal-trap screening approach, we identified a gene encoding a small membrane protein novel to osteoblasts. Although previously identified in silico as ifitm5, no localization or functional studies had been undertaken on this gene. We characterized the expression patterns and localization of this gene in vitro and in vivo and assessed its role in matrix mineralization in vitro. The bone specificity and shown role in mineralization led us to rename the gene bone restricted ifitm-like protein (Bril). Bril encodes a 14.8-kDa 134 amino acid protein with two transmembrane domains. Northern blot analysis showed bone-specific expression with no expression in other embryonic or adult tissues. In situ hybridization and immunohistochemistry in mouse embryos showed expression localized on the developing bone. Screening of cell lines showed Bril expression to be highest in osteoblasts, associated with the onset of matrix maturation/mineralization, suggesting a role in bone formation. Functional evidence of a role in mineralization was shown by adenovirus-mediated Bril overexpression and lentivirus-mediated Bril shRNA knockdown in vitro. Elevated Bril resulted in dose-dependent increases in mineralization in UMR106 and rat primary osteoblasts. Conversely, knockdown of Bril in MC3T3 osteoblasts resulted in reduced mineralization. Thus, we identified Bril as a novel osteoblast protein and showed a role in mineralization, possibly identifying a new regulatory pathway in bone formation.
Publisher: Elsevier BV
Date: 06-2020
Publisher: Cold Spring Harbor Laboratory
Date: 13-09-2021
DOI: 10.1101/2021.09.13.459432
Abstract: Over-activity of transforming growth factor β1 (TGFβ1) in subchondral bone has a direct causal role in rodent models of knee osteoarthritis (OA), which can be blocked by TGFβ1 neutralisation. In this study, we investigated whether the spatially distributed level of active TGFβ1 in human subchondral bone associates with the characteristic structural, cellular and molecular parameters of human knee OA. Subchondral bone s les (35 OA arthroplasty patients, aged 69±9 years) were obtained from regions below either macroscopically present or denuded cartilage. Bone s les were processed to determine the concentration of active TGFβ1 (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, s les were histologically examined for cartilage OARSI grading, quantification of tartrate resistant acid phosphatase positive cells and bone marrow micro-vasculature. Subchondral bone below severely degenerated/depleted cartilage, characterised by impaired bone matrix quality due to sclerotic microarchitecture, disorganised collagen, high heterogeneity of the mineral distribution, contained increased concentrations of active TGFβ1, compared to adjacent areas with more intact cartilage. In addition, increased levels of active TGFβ1 related directly to increased bone volume while increased OARSI grade associated directly with morphometric characteristics (size, shape and orientation) of osteocyte lacunae. These results indicate that increased active TGFβ1 associates spatially with impaired bone quality and the disease severity of human OA. This study therefore suggests that TGFβ1 could be a therapeutic target to prevent or reduce human disease progression.
Publisher: Elsevier BV
Date: 07-2013
DOI: 10.1016/J.JSBMB.2012.11.016
Abstract: In osteoblast cultures, 1,25-dihydroxyvitamin D (1,25D) has been shown to play either catabolic or anabolic roles on differentiation and mineralisation. We have employed osteoblast-like cells extracted from neonatal mouse calvariae and cells derived from juvenile mouse long bones to compare the biological effects of 1,25D on differentiation and mineralisation in vitro. 1,25D exerts differential effects on osteoblast-like cells depending on their stage of maturation and possibly their skeletal origin. This article is part of a Special Issue entitled 'Vitamin D Workshop'.
Publisher: Springer Science and Business Media LLC
Date: 26-05-2022
DOI: 10.1007/S00223-022-00988-8
Abstract: Osteopetrosis is a heterogeneous group of rare hereditary diseases characterized by increased bone mass of poor quality. Autosomal-dominant osteopetrosis type II (ADOII) is most often caused by mutation of the CLCN7 gene leading to impaired bone resorption. Autosomal recessive osteopetrosis (ARO) is a more severe form and is frequently accompanied by additional morbidities. We report an adult male presenting with classical clinical and radiological features of ADOII. Genetic analyses showed no amino-acid-converting mutation in CLCN7 but an apparent haploinsufficiency and suppression of CLCN7 mRNA levels in peripheral blood mononuclear cells. Next generation sequencing revealed low-frequency intronic homozygous variations in CLCN7 , suggesting recessive inheritance. In silico analysis of an intronic duplication c.595-120_595-86dup revealed additional binding sites for Serine- and Arginine-rich Splicing Factors (SRSF), which is predicted to impair CLCN7 expression. Quantitative backscattered electron imaging and histomorphometric analyses revealed bone tissue and material abnormalities. Giant osteoclasts were present and additionally to lamellar bone, and abundant woven bone and mineralized cartilage were observed, together with increased frequency and thickness of cement lines. Bone mineralization density distribution (BMDD) analysis revealed markedly increased average mineral content of the dense bone (CaMean T -score + 10.1) and frequency of bone with highest mineral content (CaHigh T -score + 19.6), suggesting continued mineral accumulation and lack of bone remodelling. Osteocyte lacunae sections (OLS) characteristics were unremarkable except for an unusually circular shape. Together, our findings suggest that the reduced expression of CLCN7 mRNA in osteoclasts, and possibly also osteocytes, causes poorly remodelled bone with abnormal bone matrix with high mineral content. This together with the lack of adequate bone repair mechanisms makes the material brittle and prone to fracture. While the skeletal phenotype and medical history were suggestive of ADOII, genetic analysis revealed that this is a possible mild case of ARO due to deep intronic mutation.
Publisher: Cold Spring Harbor Laboratory
Date: 29-10-2020
DOI: 10.1101/2020.10.29.360024
Abstract: Osteocyte produced fibroblast growth factor 23 (FGF23) is the key regulator of serum phosphate (Pi) homeostasis. The interplay between parathyroid hormone (PTH), FGF23 and other proteins that regulate FGF23 production and serum Pi levels is complex and incompletely characterised. Evidence suggests that the protein product of the SOST gene, sclerostin (SCL), also a PTH target and also produced by osteocytes, plays a role in FGF23 expression, however the mechanism for this effect is unclear. Part of the problem of understanding the interplay of these mediators is the complex multi-organ system that achieves Pi homeostasis in vivo . In the current study, we sought to address this using a unique cell line model of the osteocyte, IDG-SW3, known to express FGF23 at both the mRNA and protein levels. In cultures of differentiated IDG-SW3 cells, both PTH 1-34 and recombinant human (rh) SCL remarkably induced Fgf23 mRNA expression dose-dependently within 3 hours. Both rhPTH 1-34 and rhSCL also strongly induced C-terminal FGF23 protein secretion. Secreted intact FGF23 levels remained unchanged, consistent with constitutive post-translational cleavage of FGF23 in this cell model. Both rhPTH 1-34 and rhSCL treatments significantly suppressed mRNA levels of Phex, Dmp1 and Enpp1 mRNA, encoding putative negative regulators of FGF23 levels, and induced Galnt3 mRNA expression, encoding N-acetylgalactosaminyl-transferase 3 (GalNAc-T3), which protects FGF23 from furin-like proprotein convertase-mediated cleavage. The effect of both rhPTH 1-34 and rhSCL was antagonised by pre-treatment with the NF-κβ signalling inhibitors, BAY11 and TPCK. RhSCL also stimulated FGF23 mRNA expression in ex vivo cultures of human bone. These findings provide evidence for the direct regulation of FGF23 expression by sclerostin. Locally expressed sclerostin via the induction of FGF23 in osteocytes thus has the potential to contribute to the regulation of Pi homeostasis.
Publisher: Elsevier BV
Date: 11-2016
DOI: 10.1016/J.JSBMB.2015.11.015
Abstract: The association between increased serum 25-hydroxyvitamin D (25D) and reduced osteoclastic bone resorption is well known. Previously, we have demonstrated that mechanism by which this occurs, may include the conversion of 25D to 1,25-dihydroxyvitamin D (1,25D) by osteoclasts, catalysed by the CYP27B1 enzyme. Local 1,25D synthesis in osteoclasts was shown to regulate osteoclastogenesis and moderating resorptive activity. Thus, we hypothesised that osteoclasts differentiated from mice with global deletion of the Cyp27b1 gene (Cyp27b1 KO) would display enhanced resorptive capacity due to the lack of an ameliorating effect of 1,25D. Splenocytes isolated from Cyp27b1 KO mice or their wild-type (WT) littermates between 6 and 8 weeks of age were cultured under osteoclast-forming conditions for up to 14 days. Osteoclast formation was measured by staining for the osteoclast marker tartrate resistant acid phosphatase (TRAP). Bone resorption activity was measured by plating the cells on a bone-like substrate. In Cyp27b1 KO cultures, osteoclastogenesis was reduced, as indicated by fewer TRAP-positive multinucleated cells at all time points measured (p<0.05) when compared to wild-type (WT) levels. However, Cyp27b1 KO osteoclasts demonstrated greater resorption on a per cell basis than their WT counterparts (p<0.03). In addition, the ratio of expression of the pro-apoptotic gene Bax to the pro-survival gene Bcl-2 was decreased in Cyp27b1 KO cultures, implying that these smaller osteoclasts survive longer than WT osteoclasts. Our data indicate abnormal osteoclastogenesis due to the absence of CYP27B1 expression, consistent with the notion that endogenous metabolism of 25D optimises osteoclastogenesis and ameliorates the resulting activity of mature osteoclasts.
Publisher: Elsevier BV
Date: 04-2001
DOI: 10.1016/S8756-3282(01)00404-5
Abstract: Osteolysis is a common complication of tumors that arise in, or metastasize to, bone. The recent discovery of key regulators of osteoclast formation and activity, including receptor activator of nuclear factor of kappaB ligand (RANKL), RANK, and osteoprotegerin (OPG), may facilitate new treatment regimes for certain tumors associated with excessive bone loss. We recently showed that the stromal cells of osteolytic giant cell tumors (GCT) of bone express high levels of mRNA encoding RANKL, relative to mRNA for the RANKL antagonist, OPG, compared with the expression patterns of other lytic and nonlytic bone tumors. In this study, we found that expression of RANKL and OPG mRNA continued by the stromal element of these tumors in a constitutive manner for at least 9 days in the absence of giant cells. Immunostaining of unfractionated GCT cultured in vitro revealed punctate cytoplasmic/membranous staining for RANKL and both cytoplasmic and extracellular matrix staining for OPG in stromal cells. Giant cells (osteoclasts) were negative for RANKL staining, but stained brightly for cytoplasmic OPG protein. We also investigated the functional relevance of these molecules for GCT osteolysis by adding recombinant OPG and RANKL to cultured GCT cells. We found that OPG treatment potently and dose-dependently inhibited resorption of bone slices by GCT, and could also inhibit the formation of multinucleated osteoclasts from precursors within the GCT. These effects of OPG were reversed by stoichiometric concentrations of exogenous RANKL. These data indicate that both the processes of osteoclast formation and activation in GCT are promoted by RANKL. Therefore, GCT represent a paradigm for the direct stimulation of osteoclast formation and activity by tumor stromal cells, in contrast to the mechanisms described for osteolytic breast tumors and multiple myeloma. The demonstration of these relationships is important in developing approaches to limit tumor-induced osteolysis.
Publisher: Cold Spring Harbor Laboratory
Date: 20-01-2023
DOI: 10.1101/2023.01.18.524641
Abstract: It is unclear if different factors influence osteoarthritis (OA) progression and the changes characterising OA disease in hip and knee. We investigated the difference between hip OA and knee OA at the subchondral bone tissue and cellular level, relative to the degree of cartilage degeneration. Bone s les were collected from 11 patients (aged 70±8 years) undergoing knee arthroplasty and 8 patients (aged 64±12 years) undergoing hip arthroplasty surgery. Bone microstructure, osteocyte-lacunar network and bone matrix vascularity were evaluated using synchrotron micro-CT imaging. S les were additionally examined histologically to determine osteocyte density, viability, and connectivity. After adjustment for donor gender and age, associations between the extent of cartilage degeneration, bone volume fraction [8.7, 95% CI (3.4, 14.1)], trabecular number [1.5, 95% CI (0.8, 2.3)], osteocyte lacunar density [4714.9 95% CI (2079.1, 7350.6)] and trabecular separation [-0.06, 95% CI (0.01, 0.1)] were found in both knee and hip OA. When compared to knee OA, hip OA was characterised by higher trabecular thickness [0.006, 95% CI (-4, 0.01)], larger but less spheric osteocyte lacunae [47.3 95% CI (11.2, 83.4), -0.04 95% CI (-0.6, -0.01), respectively], lower vascular canal density [-22.8 95% CI (-35.4, -10.3)] lower osteocyte density [-84.9 95% CI (-102.4, -67.4)], and less senescent but more apoptotic osteocytes [-2.4 95% CI (-3.6, -1.2), 24.9 95% CI (17.7, 32.1)], respectively. Subchondral bone from hip OA and knee OA exhibits different characteristics at the tissue and cellular levels, suggesting different mechanisms of OA progression between the hip and knee joints.
Publisher: Elsevier BV
Date: 11-2016
DOI: 10.1016/J.JSBMB.2015.08.009
Abstract: Overexpression of the human vitamin D receptor (hVDR) transgene under control of the human osteocalcin promoter in FVB/N mice (OSVDR) was previously demonstrated to exhibit increased cortical and trabecular bone volume and strength due to decreased bone resorption and increased bone formation. An important question to address is whether the OSVDR bone phenotype persists on an alternative genetic background such as C57Bl6/J. OSVDR mice (OSV3 line) were backcrossed onto the C57Bl6/J genetic background for at least 6 generations to produce OSVDR mice with 98.4% C57Bl6/J congenicity (ObVDR-B6 mice). Hemizygous male and female ObVDR-B6 and littermate wild-type (WT) mice were fed a standard laboratory chow diet and killed at 3, 9 and 20 weeks of age for analyses of biochemical and structural variables and dynamic indices of bone histomorphometry. At 9 weeks of age, both cortical and trabecular femoral bone volumes were increased in both male and female ObVDR-B6 mice, when compared to WT levels (P<0.05), without systemic changes to calciotropic parameters. The increase in femoral trabecular bone volume was associated with increase in MAR (P<0.01) and reduced osteoclast size (P<0.05). However, in female mice trabecular bone volume was unchanged in femoral metaphysis of 20 weeks mice and in vertebra both at 9 and 20 weeks of age. Increased cortical bone in both male and female ObVDR-B6 mice was due largely to increased periosteal expansion and was associated with increased cortical strength at 20 weeks of age. Overexpression of the human VDR gene in mature osteoblasts of C57Bl6/J mice increases cortical and trabecular bone volumes and confirms the previous reports of increased bone in OSVDR mice on the FVB/N background. However, site-specific and gender-related differences in bone volume suggest that the effects of osteoblast-specific VDR overexpression are more complex than hitherto recognised.
Publisher: Royal Society of Chemistry (RSC)
Date: 2018
DOI: 10.1039/C7TB03251J
Abstract: 3D printing technology combined with electrochemical nano-structuring and HA modification is a promising approach for the fabrication of Ti implants with improved osseointegration.
Publisher: Elsevier BV
Date: 07-2017
DOI: 10.1016/J.INJURY.2017.05.024
Abstract: Tibial plateau fractures are complex and the current evidence for postoperative rehabilitation is weak, especially related to the recommended postoperative weight bearing. The primary aim of this study was to investigate if loading in the first 12 weeks of recovery is associated with patient reported outcome measures at 26 and 52 weeks postoperative. We hypothesized that there would be no association between loading and patient reported outcome measures. Seventeen patients, with a minimum of 52-week follow-up following fragment-specific open reduction and internal fixation for tibial plateau fracture, were selected for this retrospective analysis. Postoperatively, patients were advised to load their limb to a maximum of 20kg during the first 6 weeks. Loading data were collected during walking using force platforms. A ratio of limb loading (affected to unaffected) was calculated at 2, 6 and 12 weeks postoperative. Knee Injury and Osteoarthritis Scores were collected at 6, 12, 26 and 52 weeks postoperative. The association between loading ratios and patient reported outcomes were investigated. Compliance with weight bearing recommendations and changes in the patient reported outcome measures are described. Fracture reduction and migration were assessed on plain radiographs. No fractures demonstrated any measurable postoperative migration at 52 weeks. Significant improvements were seen in all patient reported outcome measures over the first 52 weeks, despite poor adherence to postoperative weight bearing restrictions. There were no associations between weight bearing ratio and patient reported outcomes at 52 weeks postoperative. Significant associations were identified between the loading ratio at 2 weeks and knee-related quality of life at six months (R
Publisher: Wiley
Date: 06-2001
DOI: 10.1359/JBMR.2001.16.6.1015
Abstract: The determinants of cancellous bone turnover and trabecular structure are not understood in normal bone or skeletal disease. Bone remodeling is initiated by osteoclastic resorption followed by osteoblastic formation of new bone. Receptor activator of nuclear factor kappaB ligand (RANKL) is a newly described regulator of osteoclast formation and function, the activity of which appears to be a balance between interaction with its receptor RANK and with an antagonist binding protein osteoprotegerin (OPG). Therefore, we have examined the relationship between the expression of RANKL, RANK, and OPG and indices of bone structure and turnover in human cancellous bone from the proximal femur. Bone s les were obtained from in iduals with osteoarthritis (OA) at joint replacement surgery and from autopsy controls. Histomorphometric analysis of these s les showed that eroded surface (ES/BS) and osteoid surface (OS/BS) were positively associated in both control (p < 0.001) and OA (p < 0.02), indicating that the processes of bone resorption and bone formation remain coupled in OA, as they are in controls. RANKL, OPG, and RANK messenger RNA (mRNA) were abundant in human cancellous bone, with significant differences between control and OA in iduals. In coplotting the molecular and histomorphometric data, strong associations were found between the ratio of RANKL/OPG mRNA and the indices of bone turnover (RANKL/OPG vs. ES/BS: r = 0.93, p < 0.001 RANKL/OPG vs. OS/BS: r = 0.80, p < 0.001). These relationships were not evident in trabecular bone from severe OA, suggesting that bone turnover may be regulated differently in this disease. We propose that the effective concentration of RANKL is related causally to bone turnover.
Publisher: The Endocrine Society
Date: 10-1999
Abstract: We investigated the mechanisms by which calcitonin (CT) suppresses cellular proliferation, using HEK-293 cells stably transfected with either the rat C1a CT receptor (CTR) or the insert-negative form of the human CTR. CT treatment of clonal cell lines expressing either receptor type, but not untransfected HEK-293 cells, strongly suppressed cell growth in a concentration-dependent manner. The reduction in cell growth with CT treatment could not be attributed to cellular necrosis or apoptotic cell death, the latter assessed by both DNA fragmentation analysis and caspase 3 (CPP-32) assay. Growth inhibition was associated with an accumulation of cells in the G2 phase of the cell cycle. CT treatment of the human and rat CTR-expressing cell lines resulted in a rapid and sustained induction of mRNA encoding the cyclin-dependent kinase inhibitor, p21WAF1/CIP1, increased levels of which were maintained at least 48 h after initiation of treatment. Western blot analysis showed a rapid corresponding increase in p21WAF1/CIP1 protein, whereas protein levels of another member of the cyclin-dependent kinase inhibitor family, p27kip1, were unchanged. In parallel with the induction of p21, CT treatment reduced levels of p53 mRNA and protein. CT treatment resulted in a specific cell cycle block in G2, which was associated with inhibition of Cdc2/cyclin B kinase activity as measured by histone H1 phosphorylation. There was no evidence for p21 association with this complex despite the inhibition of Cdc2 activity. Evidence that p21 induction was causative of cell growth suppression was obtained from p21 antisense oligonucleotide experiments. Treatment with a p21 antisense oligonucleotide blocked induction of p21 expression and significantly reduced the CT-mediated growth inhibition. These observations suggest that p21 is required for the G2 arrest in response to CT, but argue against a direct role of p21 in the inhibition of Cdc2 activity. These studies suggest a novel regulation of cell cycle progression by CT and will provide a basis for detailed examination of the molecular mechanisms involved.
Publisher: Wiley
Date: 20-07-2021
DOI: 10.1002/JBMR.4402
Abstract: Cognitive decline and osteoporosis often coexist and some evidence suggests a causal link. However, there are no data on the longitudinal relationship between cognitive decline, bone loss and fracture risk, independent of aging. This study aimed to determine the association between: (i) cognitive decline and bone loss and (ii) clinically significant cognitive decline (≥3 points) on Mini Mental State Examination (MMSE) over the first 5 years and subsequent fracture risk over the following 10 years. A total of 1741 women and 620 men aged ≥65 years from the population‐based Canadian Multicentre Osteoporosis Study were followed from 1997 to 2013. Association between cognitive decline and (i) bone loss was estimated using mixed‐effects models and (ii) fracture risk was estimated using adjusted Cox models. Over 95% of participants had normal cognition at baseline (MMSE ≥ 24). The annual % change in MMSE was similar for both genders (women −0.33, interquartile range [IQR] −0.70 to +0.00 and men −0.34, IQR: −0.99 to 0.01). After multivariable adjustment, cognitive decline was associated with bone loss in women (6.5% 95% confidence interval [CI], 3.2% to 9.9% for each percent decline in MMSE from baseline) but not men. Approximately 13% of participants experienced significant cognitive decline by year 5. In women, fracture risk was increased significantly (multivariable hazard ratio [HR], 1.61 95% CI, 1.11 to 2.34). There were too few men to analyze. There was a significant association between cognitive decline and both bone loss and fracture risk, independent of aging, in women. Further studies are needed to determine mechanisms that link these common conditions. © 2021 American Society for Bone and Mineral Research (ASBMR).
Publisher: Elsevier BV
Date: 12-2016
DOI: 10.1016/J.MSEC.2016.07.047
Abstract: There is an ongoing demand for new approaches for treating localized bone pathologies. Here we propose a new strategy for treatment of such conditions, via local delivery of hormones/drugs to the trauma site using drug releasing nano-engineered implants. The proposed implants were prepared in the form of small Ti wires/needles with a nano-engineered oxide layer composed of array of titania nanotubes (TNTs). TNTs implants were inserted into a 3D collagen gel matrix containing human osteoblast-like, and the results confirmed cell migration onto the implants and their attachment and spread. To investigate therapeutic efficacy, TNTs/Ti wires loaded with parathyroid hormone (PTH), an approved anabolic therapeutic for the treatment of severe bone fractures, were inserted into 3D gels containing osteoblast-like cells. Gene expression studies revealed a suppression of SOST (sclerostin) and an increase in RANKL (receptor activator of nuclear factor kappa-B ligand) mRNA expression, confirming the release of PTH from TNTs at concentrations sufficient to alter cell function. The performance of the TNTs wire implants using an ex le of a drug needed at relatively higher concentrations, the anti-inflammatory drug indomethacin, is also demonstrated. Finally, the mechanical stability of the prepared implants was tested by their insertion into bovine trabecular bone cores ex vivo followed by retrieval, which confirmed the robustness of the TNT structures. This study provides proof of principle for the suitability of the TNT/Ti wire implants for localized bone therapy, which can be customized to cater for specific therapeutic requirements.
Publisher: Elsevier BV
Date: 07-2000
DOI: 10.1016/S8756-3282(00)00292-1
Abstract: Fibrillin-containing microfibrils are structural components of extracellular matrices of a erse range of tissues, including bone. Their importance in bone biology is illustrated by the skeletal abnormalities manifest in the congenital disorder, Marfan syndrome, which results from mutations in the fibrillin-1 gene. We investigated the expression of fibrillins and other microfibril-associated proteins in human bone and bone-derived osteoblasts. Analysis of RNA extracted from cancellous bone showed expression of mRNAs encoding fibrillin-1 and -2, MAGP-1 and -2, LTBP-2, and MP78/70 (Big-h3). In demineralized normal mature bone, fibrillin-1 was immunolocalized to fibrils within the bone matrix and pericellularly to cells lining the endosteal surfaces of trabecular bone, some osteocytes, and cells associated with blood vessels. LTBP-2 was also identified at the endosteal surface and within the bone matrix in a lamellar fashion. In addition, primary osteoblast-like cells cultured from human trabecular bone (obtained from patients at joint replacement surgery) were found to express abundant mRNA for fibrillins and associated glycoproteins. Moreover, using western blot analysis, fibrillin-1 protein was shown to be secreted into the medium and to be deposited into the cell layer. Immunofluorescence staining of the cell layer visualized fibrillin-1 in the matrix as a three-dimensional network of fine filaments. Expression of fibrillin-1 by osteoblast-like cells was constitutive, and a number of skeletally active agents had little effect on mRNA or protein levels. These results show that human osteoblasts from mature bone express fibrillins and other microfibril-associated proteins, and suggest a role for these molecules in adult human bone.
Publisher: Springer Science and Business Media LLC
Date: 15-10-2013
DOI: 10.1007/S10787-013-0192-6
Abstract: Periprosthetic osteolysis is a serious complication of total hip replacement (THR) in the medium to long term. Although often asymptomatic, osteolysis can lead to prosthesis loosening and periprosthetic fracture. These complications cause significant morbidity and require complex revision surgery. Here, we review advances in our understanding of the cell and tissue response to particles produced by wear of the articular and non-articular surfaces of prostheses. We discuss the molecular and cellular regulators of osteoclast formation and bone resorptive activity, a better understanding of which may lead to pharmacological treatments for periprosthetic osteolysis. We describe the development of imaging techniques for the detection and measurement of osteolysis around THR prostheses, which enable improved clinical management of patients, provide a means of evaluating outcomes of non-surgical treatments for periprosthetic osteolysis, and assist in pre-operative planning for revision surgery. Finally, there have been advances in the materials used for bearing surfaces to minimise wear, and we review the literature regarding the performance of these new materials to date.
Publisher: Springer Science and Business Media LLC
Date: 24-03-2011
DOI: 10.1186/AR3294
Publisher: Elsevier BV
Date: 07-2016
DOI: 10.1016/J.BONE.2016.04.017
Abstract: Osteocytes are essential regulators of bone homeostasis. However, they are difficult to study due to their location within the bone mineralised matrix. Although several techniques have been published for the isolation of osteocytes from mouse bone, no such technique has been described for human osteocytes. We have therefore developed a protocol for the isolation of osteocytes from human trabecular bone s les acquired during surgery. The cells were digested from the bone matrix by sequential collagenase and ethylenediaminetetraacetic acid (EDTA) digestions and the cells from later digests displayed characteristic dendritic osteocyte morphology when cultured ex vivo. Furthermore, the cells expressed characteristic osteocyte marker genes, such as E11, dentin matrix protein 1 (DMP1), SOST, matrix extracellular phosphoglycoprotein (MEPE) and phosphate regulating endopeptidase homologue, X-linked (PHEX). In addition, genes associated with osteocyte perilacunar remodelling, including matrix metallopeptidase-13 (MMP13), cathepsin K (CTSK) and carbonic anhydrase 2 (CAR2) were expressed. The cells also responded to parathyroid hormone (PTH) by downregulating SOST mRNA expression and to 1α,25-dihydroxyvitamin D3 (1,25D) by upregulating fibroblast growth factor 23 (FGF23) mRNA expression. Therefore, the cells behave in a similar manner to osteocytes in vivo. These cells represent an important tool in enhancing current knowledge in human osteocyte biology.
Publisher: Springer Science and Business Media LLC
Date: 31-05-2011
DOI: 10.1007/S00198-011-1672-4
Abstract: Histomorphometric assessment of trabecular bone in osteoporotic sheep showed that bone volume, osteoid surface area, bone formation rate, and osteocyte density were reduced. In contrast, eroded surface area and empty lacunae density were increased. Changes in osteocyte density correlated with changes in osteoblast and osteoclast activity. Osteocytes contribute to the regulation of the activity of osteoclasts and osteoblasts that together control bone mass. Osteocytes therefore likely play a role in the loss of bone mass associated with osteoporosis. The purpose of this study was to investigate the relationships between osteocyte lacunar density and other bone histomorphometric parameters in the iliac crest (IC) and lumbar spine (LS) of osteoporotic sheep. Osteoporosis was induced in ten mature ewes by an established protocol involving a combination of ovariectomy, dexamethasone injection, and low calcium diet for 6 months. Five ewes were used as controls. Post-mortem IC and LS biopsies were collected and processed for further histomorphometric assessment. Bone volume, osteoid surface, and bone formation rate in the IC and LS of osteoporotic sheep were reduced compared to those of the controls. In contrast, eroded surface area was increased in osteoporotic sheep. In the osteoporotic group, osteocyte density was reduced in the LS region and to a greater extent in the IC region. The empty osteocyte lacunae were increased 1.7-fold in LS and 2.1-fold in IC in the osteoporotic group. The osteocyte density correlated positively with markers of osteoblast activity and negatively with those of osteoclast activity. Depletion of osteocytes and an increase in the empty lacunae could be important factors contributing to bone loss in this model since they may adversely affect intercellular communication between osteoblasts and osteoclasts. The regional differences in histology suggest that there may be different pathological mechanisms operating at different anatomical sites.
Publisher: Springer Science and Business Media LLC
Date: 04-03-2009
DOI: 10.1007/S10856-009-3718-0
Abstract: The osteoclast (OC) is the cell type responsible for the resorption of bone. The activity of this cell is important in the aetiology of a large number of skeletal pathologies, and also for the biocompatibility and osseointegration of orthopaedic implant materials. OC mediated acid hydrolysis of calcium phosphate from the bone matrix offers a prime means of studying the biology and activity of this cell type. We have developed a method of coating glass coverslips with a hydroxyapatite (HA)-like mineral, using a biomimetic approach. Hydroxylation followed by formation of a self assembled monolayer (SAM) using the surfactant triethoxysilylpropyl succinic anhydride (TESPSA), allowed biomimetic deposition of HA-like mineral from a simulated body fluid (SBF). The biocompatibility of the TESPSA SAM-HA coated glass coverslips was tested by culturing human mature OC present in s les of giant cell tumour of bone (GCT). Parameters of OC activity were assayed, including F-actin ring formation, release of calcium and formation of osteoclastic resorption pits, confirming that OC were able to attach to and resorb the coated surface. This approach for the preparation of HA coatings on glass coverslips could have wide applicability for the study of osteoclast behaviour in vitro.
Publisher: Elsevier BV
Date: 09-2022
DOI: 10.1016/J.JMBBM.2022.105311
Abstract: Estimating strain distribution in the acetabulum before and after the development of peri-prosthetic osteolytic lesions secondary to total hip arthroplasty may assist with understanding the pathogenesis of this condition. This could be achieved by performing patient-specific finite element analysis of (1) total hip arthroplasty recipients with developed acetabular osteolytic lesions, and (2) models simulating the patient's pelvis and implant immediately after primary surgery. State of the art patient-specific total hip arthroplasty finite element analysis simulations obtain trabecular bone material properties from Hounsfield units within computed tomography (CT) scans of patients. However, this is not feasible when an implant is already in situ due to metal artefact disruption and, in turn, incorrectly reproduced Hounsfield units. Therefore, alternative methods of assigning trabecular bone material properties within such models were tested and strain results compared. It was found that assigning set material properties throughout the trabecular bone geometry was sufficient for the desired application. Simulating the primary implant and pelvis requires geometric and material based assumptions. Therefore, comparisons were made between strain values obtained from simulated primary models, from state of the art methods using material properties obtained from intact bone within a CT scan, and from models with osteolytic lesions. Strain values found using the finite element models simulating the pelvis before osteolytic lesion developed were considerably closer to those found using state of the art methods than those found for the bone loss models. These models could be used to determine relationships between strain distribution and factors such as bone loss.
Publisher: SPIE
Date: 11-09-2013
DOI: 10.1117/12.2027151
Publisher: Elsevier BV
Date: 05-2004
DOI: 10.1016/J.BIOMATERIALS.2003.09.005
Abstract: Tantalum (Ta) is increasingly used in orthopaedics, although there is a paucity of information on the interaction of human osteoblasts with this material. We investigated the ability of Ta to support the growth and function of normal human osteoblast-like cells (NHBC). Cell responses to polished and textured Ta discs were compared with responses to other common orthopaedic metals, titanium and cobalt-chromium alloy, and tissue culture plastic. No consistent differences, that could be attributed to the different metal substrates or to the surface texture, were found in several measured parameters. Attachment of NHBC to each substrate was similar, as was cell morphology, as determined by confocal microscopy. Cell proliferation was slightly faster on plastic than on Ta at 3 days, but by 7 days neither the absolute cell numbers, nor the number of cell isions, was different between Ta and the other substrates. No consistent, substrate-dependent differences were seen in the expression of a number of mRNA species corresponding to the pro-osteoclastic or the osteogenic activity of osteoblasts. No substrate-dependent differences were seen in the extent of in vitro mineralisation by NHBC. These results indicate that Ta is a good substrate for the attachment, growth and differentiated function of human osteoblasts.
Publisher: American Physiological Society
Date: 2018
DOI: 10.1152/AJPCELL.00175.2017
Abstract: Sclerostin has emerged as an important regulator of bone mass. We have shown that sclerostin can act by targeting late osteoblasts/osteocytes to inhibit bone mineralization and to upregulate osteocyte expression of catabolic factors, resulting in osteocytic osteolysis. Here we sought to examine the effect of exogenous sclerostin on osteocytes in trabecular bone mechanically loaded ex vivo. Bovine trabecular bone cores, with bone marrow removed, were inserted into in idual chambers and subjected to daily episodes of dynamic loading. Cores were perfused with either osteogenic media alone or media containing human recombinant sclerostin (rhSCL) (50 ng/ml). Loaded control bone increased in apparent stiffness over time compared with unloaded bone, and this was abrogated in the presence of rhSCL. Loaded bone showed an increase in calcein uptake as a surrogate of mineral accretion, compared with unloaded bone, in which this was substantially inhibited by rhSCL treatment. Sclerostin treatment induced a significant increase in the ionized calcium concentration in the perfusate and the release of β-CTX at several time points, an increased mean osteocyte lacunar size, indicative of osteocytic osteolysis, and the expression of catabolism-related genes. Human primary osteocyte-like cultures treated with rhSCL also released β-CTX from their matrix. These results suggest that osteocytes contribute directly to bone mineral accretion, and to the mechanical properties of bone. Moreover, it appears that sclerostin, acting on osteocytes, can negate this effect by modulating the dimensions of the lacunocanalicular porosity and the composition of the periosteocyte matrix.
Publisher: Springer Science and Business Media LLC
Date: 12-06-2014
DOI: 10.1007/S00223-014-9879-Y
Abstract: The central importance of osteocytes in regulating bone homeostasis is becoming increasingly apparent. However, the study of these cells has been restricted by the relative paucity of cell line models, especially those of human origin. Therefore, we investigated the extent to which SaOS2 human osteosarcoma cells can differentiate into osteocyte-like cells. During culture under the appropriate mineralising conditions, SaOS2 cells reproducibly synthesised a bone-like mineralised matrix and temporally expressed the mature osteocyte marker genes SOST, DMP1, PHEX and MEPE and down-regulated expression of RUNX2 and COL1A1. SaOS2 cells cultured in 3D collagen gels acquired a dendritic morphology, characteristic of osteocytes, with multiple interconnecting cell processes. These findings suggest that SaOS2 cells have the capacity to differentiate into mature osteocyte-like cells under mineralising conditions. PTH treatment of SaOS2 cells resulted in strong down-regulation of SOST mRNA expression at all time points tested. Interestingly, PTH treatment resulted in the up-regulation of RANKL mRNA expression only at earlier stages of differentiation. These findings suggest that the response to PTH is dependent on the differentiation stage of the osteoblast/osteocyte. Together, our results demonstrate that SaOS2 cells can be used as a human model to investigate responses to osteotropic stimuli throughout differentiation to a mature osteocyte-like stage.
Publisher: Elsevier BV
Date: 07-2013
DOI: 10.1016/J.JSBMB.2012.09.029
Abstract: 1α,25-dihydroxy vitamin D3 (1,25D) is reported to up-regulate the expression of the osteocyte-derived phosphatonin, fibroblast growth factor 23 (FGF23), an effect increased by high concentrations of extracellular phosphate (Pi). Osteocytes therefore appear to sense Pi directly and this may be an important means, by which FGF23 production is regulated. The intriguing possibility is that the Pi response and 1,25D pathways interact in additional ways. 1,25D also modulates the expression of other genes related to phosphate handling in cells of the osteoblast lineage. These include receptor activator of nuclear factor kappa-B ligand (RANKL) and dentin matrix acidic phosphoprotein 1 (DMP1). These cells are also capable of synthesising 1,25D due to their expression of the 25-hydroxyvitamin D 1α-hydroxylase (CYP27B1). In this study, the mouse cell line, IDG-SW3, which differentiates into an osteocyte-like phenotype expressing Fgf23 mRNA, was utilised to address this question. Cells were differentiated for 35d and the expression level of several Pi handling or vitamin D-related genes was then evaluated in response to short-term culture with varying concentrations of extracellular Pi, in the presence or absence of 1,25D. Pi and 1,25D both increased Fgf23 mRNA expression, as well as that of N-acetylgalactosaminyltransferase 3 (Galnt3), Dmp1, phosphate-regulating gene with homologies to endopeptidases on the X chromosome (Phex), ectonucleotide pyrophosphatase hosphodiesterase family member 1 (Enpp1) and matrix extracellular phosphoglycoprotein (Mepe). Overall, there was a non-additive, competitive interaction between Pi and 1,25D, which was especially evident with Pi at 10mM. Pi also modulated the 1,25D metabolic pathway, up-regulating Cyp27b1 expression and attenuating 1,25D induction of 25-hydroxyvitamin D 24-hydroxylase (Cyp24a1) mRNA. This study provides evidence that the Pi and 1,25D response in osteocytes is linked in terms of the expression of genes related to phosphate and vitamin D metabolism. This article is part of a Special Issue entitled 'Vitamin D Workshop'.
Publisher: Springer Science and Business Media LLC
Date: 24-01-2014
Publisher: American Society for Microbiology
Date: 02-05-2018
Abstract: Periprosthetic joint infection (PJI) is a potentially devastating complication of orthopedic joint replacement surgery. PJI with associated osteomyelitis is particularly problematic and difficult to cure. Whether viable osteocytes, the predominant cell type in mineralized bone tissue, have a role in these infections is not clear, although their involvement might contribute to the difficulty in detecting and clearing PJI. Here, using Staphylococcus aureus , the most common pathogen in PJI, we demonstrate intracellular infection of human-osteocyte-like cells in vitro and S. aureus adaptation by forming quasi-dormant small-colony variants (SCVs). Consistent patterns of host gene expression were observed between in vitro -infected osteocyte-like cultures, an ex vivo human bone infection model, and bone s les obtained from PJI patients. Finally, we confirm S. aureus infection of osteocytes in clinical cases of PJI. Our findings are consistent with osteocyte infection being a feature of human PJI and suggest that this cell type may provide a reservoir for silent or persistent infection. We suggest that elucidating the molecular/cellular mechanism(s) of osteocyte-bacterium interactions will contribute to better understanding of PJI and osteomyelitis, improved pathogen detection, and treatment. IMPORTANCE Periprosthetic joint infections (PJIs) are increasing and are recognized as one of the most common modes of failure of joint replacements. Osteomyelitis arising from PJI is challenging to treat and difficult to cure and increases patient mortality 5-fold. Staphylococcus aureus is the most common pathogen causing PJI. PJI can have subtle symptoms and lie dormant or go undiagnosed for many years, suggesting persistent bacterial infection. Osteocytes, the major bone cell type, reside in bony caves and tunnels, the lacuno-canalicular system. We report here that S. aureus can infect and reside in human osteocytes without causing cell death both experimentally and in bone s les from patients with PJI. We demonstrate that osteocytes respond to infection by the differential regulation of a large number of genes. S. aureus adapts during intracellular infection of osteocytes by adopting the quasi-dormant small-colony variant (SCV) lifestyle, which might contribute to persistent or silent infection. Our findings shed new light on the etiology of PJI and osteomyelitis in general.
Publisher: Wiley
Date: 29-03-2002
DOI: 10.1002/IJC.10376
Abstract: Apo2L/TRAIL is a member of the tumor necrosis factor (TNF) family of cytokines that induces death of cancer cells but not normal cells. Its potent apoptotic activity is mediated through its cell surface death domain-containing receptors, DR4 and DR5. Apo2L/TRAIL interacts also with 3 "decoy" receptors that do not induce apoptosis, DcR1, DcR2, which lack functional death domains, and osteoprotegerin (OPG). The aim of our study was to investigate the cytotoxic activity of Apo2L/TRAIL on established osteogenic sarcoma cell lines (BTK-143, HOS, MG-63, SJSA-1, G-292 and SAOS2) and in primary cultures of normal human bone (NHB) cells. When used alone, Apo2L/TRAIL at 100 ng/ml for 24 hr induced greater than 80% cell death in only 1 (BTK-143) of the 6 osteogenic sarcoma cell lines. In contrast, Apo2L/TRAIL-resistant cells were susceptible to Apo2L/TRAIL-mediated apoptosis in the presence of the anticancer drugs, Doxorubicin (DOX), Cisplatin (CDDP) and Etoposide (ETP) but not Methotrexate (MTX) or Cyclophosphamide (CPM). Importantly, neither Apo2L/TRAIL alone nor in combination with any of these drugs affected primary normal human bone cells under equivalent conditions. Apo2L/TRAIL-induced apoptosis, and its augmentation by chemotherapy in the resistant cell lines was mediated through caspase-8 and caspase-3 activation. Furthermore, Apo2L/TRAIL-induced apoptosis and its augmentation by chemotherapy was effectively inhibited by caspase-8 zIETD-fmk and caspase-3 zDEVD-fmk protease inhibitors and by the pan-caspase inhibitor zVAD-fmk. The pattern of basal Apo2L/TRAIL receptor mRNA expression, or expression of the intracellular caspase inhibitor FLICE-inhibitory protein, FLIP, could not be readily correlated with resistance or sensitivity to Apo2L/TRAIL-induced apoptosis. However, the augmentation of Apo2L/TRAIL effects by chemotherapy was associated with drug-induced up-regulation of death receptors DR4 and DR5 mRNA and protein. No obvious correlation was seen between the expression of OPG mRNA or protein and susceptibility of cells to Apo2L/TRAIL-induced apoptosis. Stable over-expression of a dominant negative form of the Fas-associated death domain protein (FADD) in the Apo2L/TRAIL-sensitive BTK-143 cells completely inhibited Apo2L/TRAIL-induced cell death. Our results indicate that chemotherapy and Apo2L/TRAIL act synergistically to kill cancer cells but not normal bone-derived osteoblast-like cells, which has implications for future therapy of osteosarcoma.
Publisher: Elsevier BV
Date: 06-2000
DOI: 10.1016/S8756-3282(00)00280-5
Abstract: An in vitro culture system to generate human osteoclasts (OC) was recently described in which OC precursors in the human peripheral blood mononuclear cell (PBMC) population differentiate in the presence of murine ST-2 stromal cells. We used this culture system to define the cytokine environment in which human OC form and to determine the separate contributions of the stromal and hematopoietic elements. We designed a panel of reverse transcriptase-polymerase chain reaction (RT-PCR) primers that specifically lify the respective murine or human mRNA species that correspond to cytokines and their cognate receptors previously shown to promote or inhibit OC differentiation. ST-2 cells were cocultured with human PBMC for up to 21 days in the presence of 1alpha,25(OH)(2) vitamin D(3), dexamethasone, and recombinant human macrophage-colony stimulating factor (M-CSF). OC formation was monitored by the appearance of cells that were positive for tartrate-resistant acid phosphatase (TRAP) and able to form resorption lacunae on slices of dentine. We found that the ST-2 cells in these cultures express messenger RNA (mRNA) encoding a repertoire of many of the reported osteoclastogenic factors (interleukins [IL]-1/IL-1R1, IL-11, IL-6/IL-6R, and IL-17 transforming growth factor [TGF]-beta), as well as the recently described OC differentiation factor (ODF/TRANCE/RANKL). The stromal cells also expressed mRNA encoding two molecules shown to be inhibitory to osteoclastogenesis, osteoprotegerin (OPG) and IL-18. OPG, IL-1, IL-1R1, IL-6, IL-6R, IL-11R, IL-17, IL-18, IL-18R, TGF-beta, and M-CSF were expressed by both the stromal cells and the PBMC. Expression of mRNA encoding RANK, IL-1R2, and c-fms, was specific for the PBMC. In addition, PBMC were found to express sIL-6R, granulocyte macrophage (GM)-CSF, GM-CSFRalpha, and tumor necrosis factor (TNF)-alpha. Whereas this indicated that human OC formation occurs in a complex environment of many positive and negative influences, we identified three apparent features of the cytokine environment that may be a characteristic of normal osteoclast formation. First, the ratio of mouse ODF:OPG mRNA was found to increase during the cocultures, consistent with a key role for ODF in the promotion by stromal cells of OC formation. Second, we found that mRNA encoding IL-1 and IL-17, as well as IL-6 and sIL-6R, were coordinately expressed by the PBMC. Third, analysis of the culture medium showed that the PBMC secreted IL-1, IL-6, and TNF-alpha protein only in coculture with ST-2 cells during the first few days of osteoclast development. We conclude that human OC formation occurs in a complex environment of many positive and negative influences however, these are likely to be strictly regulated by a coordinated cytokine response of both stromal and hematopoietic cells.
Publisher: Elsevier BV
Date: 11-2016
DOI: 10.1016/J.YMGME.2016.09.003
Abstract: Severe, progressive skeletal dysplasia is a major symptom of multiple mucopolysaccharidoses (MPS) types. While a gene therapy approach initiated at birth has been shown to prevent the development of bone pathology in different animal models of MPS, the capacity to correct developed bone disease is unknown. In this study, ex vivo micro-computed tomography was used to demonstrate that bone mass and architecture of murine MPS VII L5 vertebrae were within the normal range at 1month of age but by 2months of age were significantly different to normal. The difference between normal and MPS VII BV/TV increased with age reaching a maximal difference at approximately 4months of age. In mature MPS VII bone BV/TV is increased (51.5% versus 21.5% in normal mice) due to an increase in trabecular number (6.2permm versus 3.8permm in normal mice). The total number of osteoclasts in the metaphysis of MPS VII mice was decreased, as was the percentage of osteoclasts attached to bone. MPS VII osteoblasts produced significantly more osteoprotegerin (OPG) than normal osteoblasts and supported the production of fewer osteoclasts from spleen precursor cells than normal osteoblasts in a co-culture system. In contrast, the formation of osteoclasts from MPS VII spleen monocytes was similar to normal in vitro, when exogenous RANKL and m-CSF was added to the culture medium. Administration of murine β-glucuronidase to MPS VII mice at 4months of age, when bone disease was fully manifested, using lentiviral gene delivery resulted in a doubling of osteoclast numbers and a significant increase in attachment capacity (68% versus 29.4% in untreated MPS VII animals). Bone mineral volume rapidly decreased by 39% after gene therapy and fell within the normal range by 6months of age. Collectively, these results indicate that lentiviral-mediated gene therapy is effective in reversing established skeletal pathology in murine MPS VII.
Publisher: Public Library of Science (PLoS)
Date: 26-09-2019
Publisher: Elsevier BV
Date: 2012
DOI: 10.1016/J.ACTBIO.2011.09.004
Abstract: Bacterial infection, extensive inflammation and poor osseointegration have been identified as the major reasons for [early] orthopaedic implant failures based on titanium. Creating implants with drug-eluting properties to locally deliver drugs is an appealing way to address some of these problems. To improve properties of titanium for orthopaedic applications, this study explored the modification of titanium surfaces with titaniananotube (TNT) arrays, and approach that combines drug delivery into bone and potentially improved bone integration. A titania layer with an array of nanotube structures (∼120 nm in diameter and 50 μm in length) was synthesized on titanium surfaces by electrochemical anodization and loaded with the water-insoluble anti-inflammatory drug indomethacin. A simple dip-coating process of polymer modification formed thin biocompatible polymer films over the drug-loaded TNTs to create TNTs with predictable drug release characteristics. Two biodegradable and antibacterial polymers, chitosan and poly(lactic-co-glycolic acid), were tested for their ability to extend the drug release time of TNTs and produce favourable bone cell adhesion properties. Dependent on polymer thickness, a significant improvement in the drug release characteristics was demonstrated, with reduced burst release (from 77% to >20%) and extended overall release from 4 days to more than 30 days. Excellent osteoblast adhesion and cell proliferation on polymer-coated TNTs compared with uncoated TNTs were also observed. These results suggest that polymer-modified implants with a TNT layer are capable of delivering a drug to a bone site over an extended period and with predictable kinetics. In addition, favourable bone cell adhesion suggests that such an implant would have good biocompatibility. The described approach is broadly applicable to a wide range of drugs and implants currently used in orthopaedic practice.
Publisher: Cold Spring Harbor Laboratory
Date: 04-11-2020
DOI: 10.1101/2020.11.02.365866
Abstract: Neck of femur (NOF) fracture is a prevalent fracture type amongst the ageing and osteoporotic populations, commonly requiring total hip replacement (THR) surgery. Increased fracture risk has also been associated with Alzheimer disease (AD) in the aged. Here, we sought to identify possible relationships between the pathologies of osteoporosis and dementia by analysing bone expression of neurotropic or dementia-related genes in patients undergoing THR surgery for NOF fracture. Femoral bone s les from 66 NOF patients were examined for expression of the neurotropic genes amyloid precursor protein ( APP ), APP-like protein-2 ( APLP2 ), Beta Secretase Cleaving Enzyme-1 ( BACE1 ) and nerve growth factor (NGF). Relationships were examined between the expression of these and of bone regulatory genes, systemic factors and bone structural parameters ascertained from plain radiographs. We found strong relative levels of expression and positive correlations between APP, APLP2, BACE1 and NGF levels in NOF bone. Significant correlations were found between APP, APLP2, BACE1 mRNA levels and bone remodelling genes TRAP, RANKL , and the RANKL:OPG mRNA ratio, indicative of potential functional relationships at the time of fracture. Analysis of the whole cohort, as well as non-dementia and dementia sub-groups, revealed structural relationships between APP and APLP2 mRNA expression and lateral femoral cortical thickness. These findings suggest that osteoporosis and AD may share common molecular pathways of disease progression, perhaps explaining the common risk factors associated with these diseases. The observation of a potential pathologic role for AD-related genes in bone may also provide alternative treatment strategies for osteoporosis and fracture prevention.
Publisher: Wiley
Date: 21-06-2011
DOI: 10.1002/JBMR.345
Publisher: Wiley
Date: 08-2008
DOI: 10.1359/JBMR.080310
Publisher: Springer New York
Date: 22-10-2010
Publisher: Royal Society of Chemistry (RSC)
Date: 2015
DOI: 10.1039/C5TB00150A
Abstract: A nanoengineered drug releasing aluminium wire implant has been developed and inserted into viable bone by a needle puncturing approach to directly deliver therapeutics inside the bone.
Publisher: Institute of Electrical and Electronics Engineers (IEEE)
Date: 07-2022
Publisher: Cold Spring Harbor Laboratory
Date: 19-11-2020
DOI: 10.1101/2020.11.19.385138
Abstract: Differentiation of multi-potent mesenchymal progenitor cells give rise to several tissue types including bone, cartilage, and adipose. In addition to the complication arising from the numerous spatial, temporal, and hormonal factors that regulate lineage allocation, targeting of these cells in vivo is challenging, making mesenchymal progenitor cell lines valuable tools to study both tissue development and the differentiated cell types. Mesenchymal stem cells (MSCs) can be isolated from humans and animals however, obtaining homogenous, responsive cells in a reproducible fashion can be problematic. As such, we have developed two novel mesenchymal progenitor cell (MPC) lines, MPC1 and MPC2, which were generated from the bone marrow of male C57BL/6 mice. These cells were immortalized using the temperature sensitive large T-antigen, allowing for thermal control of proliferation and differentiation. Both MPC1 and MPC2 cell lines are capable of osteogenic, adipogenic, and chondrogenic differentiation. Under osteogenic conditions both cell lines formed discrete mineralized nodules, staining for alizarin red and alkaline phosphatase, while expressing high levels of osteogenic genes including Sost , Fgf23 , and Dmp1 . Sost and Dmp1 mRNA levels were drastically reduced with parathyroid hormone, thus recapitulating in vivo responses. MPC cells secreted both the intact (iFGF23) and C -terminal (cFGF23) forms of endocrine hormone FGF23, which was upregulated in the presence of 1,25 dihydroxy vitamin D (1,25D). In addition to osteogenic differentiation, both cell lines also rapidly entered the adipogenic lineage, expressing several adipose markers after only 4 days in adipogenic media. MPC cells were also capable of chondrogenic differentiation, displaying increased expression of common cartilage genes including aggrecan, sox9, and cartilage oligomeric matrix protein. With the ability to differentiate into multiple mesenchymal lineages and mimic in vivo responses of key regulatory genes roteins, MPC cells are a valuable model to study factors that regulate mesenchymal lineage allocation as well as the mechanisms that dictate transcription, protein modification, and secretion of these factors.
Publisher: Portico
Date: 25-07-2012
Publisher: Cold Spring Harbor Laboratory
Date: 29-09-2021
DOI: 10.1101/2021.09.27.21264218
Abstract: The primary aim of this study was to assess the effect of time to surgery on fracture reduction, assessed as residual articular step, in cases of tibial plateau fracture (TPF). The secondary aim was to assess the effect of pre-operative demographics and residual articular step on patient reported outcomes (PROMs) following TPF. Between 2006 and 2017 all surgically treated TPF patients managed by a single surgeon at our institution were prospectively consented for the study of fracture outcomes. Timing to surgical intervention, reduction of articular step, age, gender, medical background, fracture classification, mechanism of injury and PROMs (Lysholm Scores and Knee Injury and Osteoarthritis Outcome Scores (KOOS)) were recorded and analysed. Reduction of articular step, defined as mm, was assessed by a single blinded examiner using measurements on plain radiographs on PACS. One hundred seventeen patients were enrolled, 52 with Schatzker II, four with Schatzker IV and 61 with Schatzker VI fractures. Patients were followed-up to a mean time of 3.9 years. The ability to achieve fracture reduction was negatively influenced by time to theatre with the odds of achieving reduction decreasing 17% each day post-injury (p = 0.002). An increased time to theatre was associated with reduced Lysholm scores at the one-year mark (p = 0.01). The ability to achieve fracture reduction did not influence PROMs within the study period. Delay in surgical fixation negatively affects fracture reduction in TPF and may delay recovery. However, residual articular step did not influence the investigated PROMs in the cohort investigated over the mid-term (mean of 3.9 years).
Publisher: Springer Science and Business Media LLC
Date: 22-02-2021
Publisher: Springer Science and Business Media LLC
Date: 03-01-2013
Publisher: Elsevier BV
Date: 2016
DOI: 10.1016/J.ACTBIO.2015.11.025
Abstract: Peri-prosthetic osteolysis (PPO) occurs in response to prosthetic wear particles causing an inflammatory reaction in the surrounding tissue that leads to subsequent bone loss. Semaphorin-3a (SEM3A), neuropilin-1 (NRP1) and plexin-A1 (PLEXA1) are axonal guidance molecules that have been recently implicated in regulating bone metabolism. This study investigated SEM3A, NRP1 and PLEXA1 protein and mRNA expression in human PPO tissue and polyethylene (PE) particle-stimulated human peripheral blood mononuclear cell (PBMC)-derived osteoclasts in vitro. In addition, the effects of tumour necrosis factor alpha (TNFα) on cultured osteoclasts was assessed. In PPO tissues, a granular staining pattern of SEM3A and NRP1 was observed within large multi-nucleated cells that contained prosthetic wear particles. Immunofluorescent staining confirmed the expression of SEM3A, NRP1 and PLEXA1 in large multi-nucleated human osteoclasts in vitro. Furthermore, SEM3A, NRP1 and PLEXA1 mRNA levels progressively increased throughout osteoclast differentiation induced by receptor activator of nuclear factor κB ligand (RANKL), and the presence of PE particles further increased mRNA expression of all three molecules. Soluble SEM3A was detected in human osteoclast culture supernatant at days 7 and 17 of culture, as assessed by ELISA. TNFα treatment for 72h markedly decreased the mRNA expression of SEM3A, NRP1 and PLEXA1 by human osteoclasts in vitro. Our findings suggest that SEM3A, NRP1 and PLEXA1 may have important roles in PPO, and their interactions, alone or as a complex, may have a role in pathological bone loss progression. Peri-prosthetic osteolysis occurs in response to prosthetic wear particles causing an inflammatory reaction in the surrounding tissue that leads to subsequent bone loss. The rate of hip and knee arthroplasty is increasing by at least 5% per year. However, these joint replacements have a finite lifespan, with data from the National Joint Replacement Registry (Australia) showing that the major cause of failure of total hip replacements is aseptic loosening. In aseptic loosening, wear particles liberated from prostheses are phagocytosed by macrophages, leading to release of inflammatory cytokines and up-regulation of osteoclast formation and activity. Semaphorin-3a, neuropilin-1 and plexin-A1 are axonal guidance molecules that have been recently implicated in regulating bone metabolism. This is the first report to show that these molecules may be involved in the implant failure.
Publisher: Springer Science and Business Media LLC
Date: 03-02-2012
DOI: 10.1007/S00198-012-1915-Z
Abstract: Osteocytes actively participate in almost every phase of mineral handling by bone. They regulate the mineralisation of osteoid during bone formation, and they are also a major RANKL-producing cell. Osteocytes are thus able to liberate bone mineral by regulating osteoclast differentiation and activity in response to a range of stimuli, including bone matrix damage, bone disuse and mechanical unloading, oestrogen deficiency, high-dose glucocorticoid and chemotherapeutic agents. At least some of these activities may be regulated by the osteocyte-secreted product, sclerostin. There is also mounting evidence that in addition to regulating phosphate homeostasis systemically, osteocytes contribute directly to calcium homeostasis in the mature skeleton. Osteocyte cell death and the local loss of control of bone mineralisation may be the cause of focal hypermineralisation of bone and osteopetrosis, as seen in aging and pathology. The sheer number of osteocytes in bone means that "a little give and take" in terms of regulation of bone mineral content translates into a powerful whole organism effect.
Publisher: Wiley
Date: 10-06-2010
DOI: 10.1111/J.1600-0765.2010.01275.X
Abstract: Host-derived enzymes, cytokines and other proinflammatory mediators play an integral role in periodontal destruction. The levels of tumor necrosis factor-like weak inducer of apoptosis (TWEAK) and its receptor, fibroblast growth factor-inducible 14 protein (Fn14), are elevated in tissues from a number of chronic inflammatory diseases. The aim of the present study was to investigate the expression of TWEAK and Fn14 at the protein and mRNA levels in gingival biopsies from periodontitis patients and from clinically healthy patients. Gingival biopsies were obtained from healthy sites (n = 7) and from sites affected by periodontitis (n = 27). The expression of TWEAK and Fn14 was investigated by immunohistochemistry in formalin-fixed, paraffin-embedded tissues. The levels of mRNA for TWEAK and Fn14 were also investigated by RT-PCR. The expression of TWEAK and Fn14 proteins was significantly higher in periodontitis tissue than in healthy tissue. In periodontitis tissues, TWEAK and Fn14 proteins were mainly expressed by mononuclear leukocytes (morphologically resembling lymphocytes and plasma cells), by cells lining blood vessels, by spindle-shaped cells resembling fibroblasts and by multinucleated cells. The Fn14 mRNA level in periodontitis tissue was significantly higher than that in healthy tissue. A moderate correlation between TWEAK/Fn14 expression and inflammation and bone loss, but not pocket depth, was noted. This study demonstrates higher expression of TWEAK protein and of Fn14 mRNA and protein in periodontitis tissues than in clinically healthy controls. Our data support the concept that TWEAK/Fn14 signaling is an additional player in the pathogenesis of periodontitis and adds to the increasing number of cytokine networks involved in periodontal inflammation.
Publisher: Elsevier BV
Date: 2004
Publisher: Bioscientifica
Date: 12-2002
Abstract: We recently reported that calcitonin (CT) can profoundly inhibit the growth of HEK-293 cells transfected with the human calcitonin receptor (hCTR). We also obtained preliminary evidence that suggested a role for CT in cell survival, and in the present study we have investigated the pro-apoptotic action of CT, which we observe in conditions of low serum concentration. Under these conditions, we have found that CT treatment of HEK-293 cells stably transfected with the insert-negative form of the human CTR (HR12 cells) caused a time-dependent decrease in cell number associated with loss of cellular attachment. Loss of cellular adherence in CT-treated cultures caused programmed cell death, as shown by Annexin V staining of cells, failure of cells to exclude Trypan Blue dye, condensation and cleavage of nuclear DNA, and appearance of hypodiploid cells in fluorescence-activated cell sorting (FACS) analysis. The accumulation of non-adherent cells and cell death was concomitant with increased intracellular activity of caspase-3. However, inhibition of caspase activation in HR12 cells did not prevent CT-mediated loss of attachment and did not maintain the viability of non-adherent cells, indicating that caspase activation accompanied, but was probably not the cause of, the loss of cell viability. Neither the effects of CT on cell survival nor the activation of caspase-3 were observed in serum-replete conditions, suggesting that serum-derived factors provide protection of cells from CT-induced apoptosis. The inhibitory effects of CT on cell growth were found previously to be related to activation of Erk1/2 MAP kinase. In the present experiments, it was found that the Erk1/2 inhibitor, PD 98059, inhibited the CT-induced loss of cellular adherence and the consequent reduction in cell numbers. These results demonstrate that CT can negatively affect cell survival and they identify roles for cell adherence and MAP kinase activation in this process.
Publisher: Springer Science and Business Media LLC
Date: 28-01-2014
Publisher: American Chemical Society (ACS)
Date: 25-01-2021
Publisher: Royal Society of Chemistry (RSC)
Date: 2015
DOI: 10.1039/C5RA10418A
Abstract: In this study, drug-releasing aluminium (Al) wire implants featuring nanoporous alumina (NPA) layers produced by different anodization approaches are systematically investigated as potential platforms for localized drug delivery and bone therapy.
Publisher: Elsevier BV
Date: 03-2019
DOI: 10.1016/J.ACTBIO.2019.01.047
Abstract: Periprosthetic osteolysis is a major cause of implant failure in total hip replacements. Aseptic loosening caused by osteolytic lesions is associated with the production of bioactive wear particles from the articulations of implants. Wear particles infiltrate the surrounding tissue of implants, promoting inflammation as well as bone resorption. Osteocytes have been shown to both regulate physiological osteoclastogenesis and directly remodel their perilacunar bone matrix by the process of osteocytic osteolysis. We hypothesise that osteocytes respond to wear debris of orthopaedic implant materials by adopting a pro-catabolic phenotype and thus contribute to periprosthetic osteolysis through the known pathways of bone loss. Osteocyte responses to particles derived from clinically relevant materials, ultra-high molecular weight polyethylene (UHMWPE), highly cross-linked polyethylene (XLPE) and metal alloys, Ti6Al4V and CoCrMo, were examined in vitro in human primary osteocyte-like cultures. Osteocyte-like cells exposed to both polyethylene and metal wear particle types showed upregulated expression of catabolic markers associated with osteocytic osteolysis, MMP13, carbonic anhydrase 2 (CA2) and cathepsin K (CTSK). In addition, pro-osteoclastogenesis markers RANKL and M-CSF were induced, as well as the expression of pro-inflammatory cytokines, IL-6 and TNFα, albeit with different kinetics. These findings suggest a previously unrecognised action of wear particles of multiple orthopaedic materials on osteocytes, and suggest a multifaceted role for osteocytes in periprosthetic osteolysis. STATEMENT OF SIGNIFICANCE: This study addresses periprosthetic osteolysis, a major clinical problem leading to aseptic loosening of orthopaedic implants. It is well accepted that wear particles of polyethylene and of other implant materials stimulate the activity of bone resorbing osteoclasts. Our recent work provided evidence that commercial particles of ultra-high molecular weight polyethylene (UHMWPE) stimulated osteocytes to adopt a bone catabolic state. In this study we demonstrate for the first time that particles derived from materials in clinical use, conventional UHMWPE, highly cross-linked polyethylene (XLPE), and Ti6Al4V and CoCrMo metal alloys, all stimulate human osteocyte activities of osteocyte-regulated osteoclastogenesis, osteocytic osteolysis, proinflammatory responses, osteocyte apoptosis, albeit to varying extents. This study provides further mechanistic insight into orthopaedic wear particle mediated bone disease in terms of the osteocyte, the most abundant and key controlling cell type in bone.
Publisher: AMPCo
Date: 10-2014
DOI: 10.5694/MJA13.00220
Publisher: MDPI AG
Date: 15-11-2019
DOI: 10.3390/JCM8111988
Abstract: Vitamin D, along with calcium, is generally considered necessary for bone health and reduction of fractures. However, he effects of improving vitamin D status have not always been observed to improve bone mineral density (BMD). We have investigated whether varying vitamin D status in humans, as measured by serum 25(OH)D levels, relate to micro-structural and histomorphetric measures of bone quality and quantity, rather than density. Intertrochanteric trabecular bone biopsies and serum s les were collected from patients undergoing hip arthroplasty (65 females, 38 males, mean age 84.8 ± 8.3 years) at Royal Adelaide Hospital. Estimated GFR, serum ionized calcium, alkaline phosphatase, albumin, supplement and medication intake prior to surgery were taken from patient case records. Serum 25(OH)D, 1,25(OH)2D, and parathyroid hormone (PTH) levels were measured by immunoassays. Trabecular bone structural indices were determined by high-resolution micro-CT. Mean wall thickness (MWT) was measured on toluidine blue-stained histological sections. Bone mRNA levels for vitamin D metabolising enzymes CYP27B1 and CYP24A1 were measured by qRT-PCR. While serum 25(OH)D levels did not associate with bone volume/tissue volume (BV/TV%), serum 25(OH)D levels were strongly and independently associated with MWT (r = 0.81 p 0.0001) with values significantly greater in patients with higher serum 25(OH)D levels. Furthermore, serum 25(OH)D levels were negatively associated with Bone Surface/Bone Volume (BS/BV) (r = −0.206, p 0.05) and together with bone CYP27B1 and CYP24A1 mRNA accounted for 10% of the variability of BS/BV (p = 0.001). These data demonstrate that serum 25(OH)D is an independent positive predictor of micro-structural and bone formation measures and may be dependent, in part, on its metabolism within the bone.
Publisher: Springer Science and Business Media LLC
Date: 07-2003
Publisher: Elsevier BV
Date: 02-2014
Publisher: Wiley
Date: 13-07-2018
DOI: 10.1002/JOR.24057
Abstract: We investigated if time between injury and surgery affects cancellous bone properties in patients suffering tibial plateau fractures (TPF), in terms of structural integrity and gene expression controlling bone loss. A cohort of 29 TPF, operated 1–17 days post‐injury, had biopsies from the fracture and an equivalent contralateral limb site, at surgery. S les were assessed using micro‐computed tomography and real‐time RT‐PCR analysis for the expression of genes known to be involved in bone remodeling and fracture healing. Significant decreases in the injured vs control side were observed for bone volume fraction (BV/TV, −13.5 ± 6.0%, p = 0.011), trabecular number (Tb.N, −10.5 ± 5.9%, p = 0.041) and trabecular thickness (Tb.Th, −4.6 ± 2.5%, p = 0.033). Changes in these parameters were more evident in patients operated 5–17 days post‐injury, compared to those operated in the first 4 days post‐injury. A significant negative association was found between Tb.Th ( r = −0.54, p 0.01) and BV/TV ( r = −0.39, p 0.05) in relation to time post‐injury in the injured limb. Both BV/TV and Tb.Th were negatively associated with expression of key molecular markers of bone resorption, CTSK , ACP5 , and the ratio of RANKL : OPG mRNA. These structure/gene expression relationships did not exist in the contralateral tibial plateau of these patients. This study demonstrated that there is a significant early time‐dependent bone loss in the proximal tibia after TPF. This bone loss was significantly associated with altered expression of genes typically involved in the process of osteoclastic bone resorption but possibly also bone resorption by osteocytes. The mechanism of early bone loss in such fractures should be a subject of further investigation. © 2018 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 36:2865–2875, 2018.
Publisher: Elsevier BV
Date: 03-2018
DOI: 10.1016/J.JSBMB.2017.07.033
Abstract: The indirect action of 1α,25(OH)
Publisher: Wiley
Date: 16-07-2007
DOI: 10.1002/JBM.A.31336
Abstract: Porous tantalum (Ta) has found application in orthopedics, although the interaction of human osteoblasts (HOB) with this material has not been reported. The aim of this study was to investigate the interaction of primary HOB with porous tantalum, using 5-mm thick discs of porous tantalum. Comparison was made with discs of solid tantalum and tissue culture plastic. Confocal microscopy was used to investigate the attachment and growth of cells on porous Ta, and showed that HOB attached successfully to the metal "trabeculae," underwent extensive cell ision, and penetrated into the Ta pores. The maturation of HOB on porous Ta was determined in terms of cell expression of the osteoblast phenotypic markers, STRO-1, and alkaline phosphatase. Despite some donor-dependent variation in STRO-1/AlkPhos expression, growth of cells grown on porous Ta either promoted, or did not impede, the maturation of HOB. In addition, the expression of key osteoblastic genes was investigated after 14 days of culture. The relative levels of mRNA encoding osteocalcin, osteopontin and receptor activator of NFkappaB ligand (RANKL) was not different between porous or solid Ta or plastic, although these genes were expressed differently by cells of different donors. However, bone sialoprotein and type I collagen mRNA species showed a decreased expression on porous Ta compared with expression on plastic. No substrate-dependent differences were seen in the extent of in vitro mineralization by HOB. These results indicate that porous Ta is a good substrate for the attachment, growth, and differentiated function of HOB.
Publisher: Elsevier BV
Date: 07-2013
DOI: 10.1016/J.JSBMB.2012.09.008
Abstract: We have reported the metabolism of 25(OH) vitamin D3 (25D) into active 1α,25(OH)2 vitamin D3 (1,25D) by osteoclasts derived from human peripheral blood mononuclear cells (PBMC), RAW 264.7cells or giant cell tumor of bone (GCT), which appears to optimize osteoclast differentiation but inhibit their activity. In this study, to elucidate the mechanism by which 25D reduces osteoclast resorption, we further examined the effect of 25D on osteoclast function by using GCT-derived osteoclasts. 25D treated cells on dentine slices resulted in decreased resorption volume and depth in 3D image analysis. Tartrate-resistant acid phosphatase (TRAP) has been reported to enhance the dephosphorylation of substrate binding proteins, resulting in reduced osteoclast attachment. Therefore, we next investigated the effect of 25D on cell migration. Treatment of GCT cells with 25D augmented cell migration, as determined by live cell imaging. These observations suggest that 25D metabolism by osteoclasts reduces their resorptive capacity, in part by modifying their surface adhesion and migration properties. This article is part of a Special Issue entitled "Vitamin D Workshop".
Publisher: Elsevier BV
Date: 10-2014
DOI: 10.1016/J.JSBMB.2014.01.002
Abstract: There are several lines of evidence that demonstrate the ability of 1,25-dihydroxyvitamin D (1,25(OH)2D3), acting via the vitamin D receptor (VDR) to mediate negative or positive effects in bone. Transgenic over-expression of VDR in osteoblasts and osteocytes in a mouse model (OSVDR) has been previously shown to inhibit processes of bone resorption and enhance bone formation, under conditions of adequate calcium intake. While these findings suggest that vitamin D signalling in osteoblasts and osteocytes promotes bone mineral accrual, the vitamin D requirement for this action is not well understood. In this study, 4 week old female OSVDR and wild-type (WT) mice were fed either a vitamin D-replete (1000IU/kg diet, D+) or vitamin D-deficient (D-) diet for 4 months to observe changes to bone mineral homeostasis. Tibial bone mineral volume was analysed by micro-CT and changes to bone cell activities were measured using standard dynamic histomorphometric techniques. While vitamin D-deplete WT mice demonstrated a reduction in periosteal bone accrual and overall bone mineral volume, OSVDR mice, however, displayed increased cortical and cancellous bone volume in mice which remained higher during vitamin D-depletion due to a reduced osteoclast number and increased bone formation rate. These data suggest that increased VDR-mediated activity in osteoblast and osteocytes prevents bone loss due to vitamin D-deficiency. This article is part of a Special Issue entitled '16th Vitamin D Workshop'.
Publisher: Elsevier BV
Date: 10-2014
DOI: 10.1016/J.JSBMB.2013.10.003
Abstract: Maintenance of an adequate vitamin D status, as indicated by the level of circulating 25-hydroxyvitamin D (25(OH)D), is associated with higher bone mass and decreased risk of fracture. However, the molecular actions of vitamin D hormone (1,25(OH)2D3) in bone are complex, and include stimulation of osteoclastogenesis via RANK-ligand up-regulation, as well as the inhibition of mineralisation. We hypothesise that these ergent data may be reconciled by autocrine actions of 1,25(OH)2D3 which effect skeletal maintenance, as opposed to endocrine 1,25(OH)2D3 which acts to maintain serum calcium homeostasis. We have previously described local metabolism of 1,25(OH)2D3 within osteoblasts, with effects on gene expression and cell function. The aim of the current study was to investigate potential autocrine actions of 1,25(OH)2D3 within cells that exhibit osteocyte-like properties. Late osteoblastic MLO-A5 cells were cultured in the presence of 25(OH)D for 9 days with gene expression analysed pre- and post-mineralisation. Gene expression analysis revealed maturation within this time frame to an osteocyte-like stage, evidenced by increased Dmp1 and Phex mRNA expression. Expression of Cyp27b1 in 25(OH)D treated MLO-A5 cells was associated with elevated media levels of 1,25(OH)2D3 (p<0.05), induction of Cyp24a1 (p<0.001) and elevated ratios of Opg:Rankl mRNA (p<0.01). Chronic 25(OH)D exposure also increased osteocalcin mRNA in MLO-A5 cells, which contrasted with the dose-dependent inhibition of osteocalcin mRNA observed with acute treatment in MLO-Y4 cells (p<0.01). Treatment of MLO-Y4 cells with 25(OH)D also inhibited Phex mRNA expression (p<0.05), whilst Enpp1 gene expression was induced (p<0.01). Overall, the current study demonstrates that osteocyte-like cells convert physiological levels of 25(OH)D to 1,25(OH)2D3, with changes in gene expression that are consistent with increased osteocyte maturation. Although the physiological role of local metabolism of 1,25(OH)2D3 within osteocytes requires further investigation, the abundance and erse functions of this cell type within bone underscore its potential importance. This article is part of a Special Issue entitled '16th Vitamin D Workshop'.
Publisher: MDPI AG
Date: 02-09-2022
DOI: 10.3390/PROSTHESIS4030039
Abstract: Polyethylene (PE) liners are a common bearing surface of orthopaedic prostheses. Wear particles of ultra-high molecular weight PE (UHMWPE) contribute to periprosthetic osteolysis, a major cause of aseptic loosening. Vitamin E is added to some PE liners to prevent oxidative degradation. Osteocytes, an important cell type for controlling both bone mineralisation and bone resorption, have been shown to respond UHMWPE particles by upregulating pro-osteoclastogenic and osteocytic osteolysis. Here, we examined the effects of the vitamin E analogues α-tocopherol and γ-tocotrienol alone or in the context of UHMWPE particles on human osteocyte gene expression and mineralisation behaviour. Human osteoblasts differentiated to an osteocyte-like stage were exposed to UHMWPE wear particles in the presence or absence of either α-Tocopherol or γ-Tocotrienol. Both α-Tocopherol and γ-Tocotrienol induced antioxidant-related gene expression. UHMWPE particles independently upregulated antioxidant gene expression, suggesting an effect of wear particles on oxidative stress. Both vitamin E analogues strongly induced OPG mRNA expression and γ-Tocotrienol also inhibited RANKL mRNA expression, resulting in a significantly reduced RANKL:OPG mRNA ratio (p 0.01) overall. UHMWPE particles reversed the suppressive effect of α-Tocopherol but not of γ-Tocotrienol on this pro-osteoclastogenic index. UHMWPE particles also upregulated osteocytic-osteolysis related gene expression. Vitamin E analogues alone or in combination with UHMWPE particles also resulted in upregulation of these genes. Consistent with this, both vitamin E analogues promoted calcium release from mineralised cultures of osteocyte-like cells. Our findings suggest that while vitamin E may suppress osteocyte support of osteoclastogenesis in the presence of UHMWPE particles, the antioxidant effect may induce osteocytic osteolysis, which could promote periprosthetic osteolysis. It will be important to conduct further studies of vitamin E to determine the long-term effects of its inclusion in prosthetic materials.
Publisher: MDPI AG
Date: 24-12-2019
DOI: 10.3390/JCM9010053
Abstract: Osteolysis adjacent to total hip replacement (THR) prostheses is a major cause of their eventual failure. Periprosthetic osteolysis is associated with the production of bioactive particles, produced by the wear of articulating prosthesis surfaces. Wear particles invade the periprosthetic tissue, inducing inflammation and bone resorption. Previous studies have shown that osteocytes, the most numerous cell type in mineralised bone, can respond to wear particles of multiple orthopaedic material types. Osteocytes play important roles in bone resorption, regulating bone resorption by osteoclasts and directly through osteocytic osteolysis, also known as perilacunar remodelling. In this study, we perform a histological analysis of bone biopsies obtained from cohorts of male and female patients undergoing either primary THR surgery or revision THR surgery for aseptic loosening. The osteocyte lacunae area (Ot.Lac.Ar) and percentage lacunar area/bone area (%Ot.Lac.Ar/B.Ar) were significantly larger overall in revision THR bone than bone from similar sites in primary THR. Analysis by patient gender showed that increased Ot.Lac.Ar, indicative of increased perilacunar remodelling, was restricted to female revision s les. No significant differences in osteoclast parameters were detectable between the cohorts. These findings suggest previously unrecognised gender-specific mechanisms of bone loss in orthopaedic wear particle-induced osteolysis in humans.
Publisher: Elsevier BV
Date: 03-2007
DOI: 10.1016/J.JSBMB.2006.12.084
Abstract: Although local synthesis of 1,25D has been postulated to regulate parameters of cell growth and differentiation in non-renal cells, the physiological role of 1,25D production in bone cells remains unclear. We used the technique of RNA interference to inhibit the mRNA encoding the enzyme responsible for 1,25D synthesis, 25-hydroxyvitamin D 1alpha-hydroxylase (CYP27B1). Human osteosarcoma (HOS) cells were transfected with siRNA for CYP27B1 or non-silencing RNA before being treated with 25D for 48h under normal growth conditions. De novo synthesis of 1,25D was measured in the media as well as mRNA levels for CYP27B1, osteocalcin (OCN) and 25-hydroxyvitamin D 24-hydroxylase (CYP24). We demonstrated that HOS cells express CYP27B1 mRNA, metabolize 25D and secrete detectable levels of de novo synthesized 1,25D. CYP27B1 mRNA silencing by RNAi, resulted in the suppression of 1,25D production and subsequent reduction of OCN and CYP24 mRNA expression. Our findings suggest that local 1,25D synthesis has paracrine effects in the bone microenvironment implying that vitamin D metabolism in human osteoblasts represents a physiologically important pathway, possibly regulating the maturation of osteoblasts.
Publisher: Elsevier BV
Date: 03-2018
DOI: 10.1016/J.JSBMB.2017.09.005
Abstract: Previous studies have shown that 1α,25-dihydroxyvitamin D
Publisher: The Endocrine Society
Date: 25-08-2010
DOI: 10.1210/EN.2010-0334
Abstract: The extrarenal synthesis of 1α,25 dihydroxyvitamin D3 (1,25D) has been demonstrated in a number of cell types including osteoblasts and cells of the monocyte/macrophage lineage. The skeleton appears responsive to serum levels of the 1,25D precursor, 25 hydroxyvitamin D3 (25D), in terms of bone mineralization parameters. The effect of metabolism of 25D into active 1,25D by osteoclast lineage cells is unknown. We found that CYP27B1 mRNA expression increased with exposure of human peripheral blood mononuclear cells (PBMCs) to macrophage colony-stimulating factor in the presence or absence of receptor activator of nuclear factor-κB ligand. Consistent with this, human osteoclast cultures incubated with 25D produced measurable quantities of 1,25D. Osteoclast formation from either mouse RAW264.7 cells or human PBMCs in the presence of physiological concentrations of 25D resulted in significant up-regulation of the key osteoclast transcription factor, nuclear factor of activated T cells-c1 in PBMCs and a number of key osteoclast marker genes in both models. The expression of the osteoblast coupling factor, ephrin-b2, was also increased in the presence of 25D. Levels of CYP27B1 and nuclear factor of activated T cells-1 mRNA correlated during osteoclastogenesis and also in a cohort of human bone s les. CYP27B1 short-hairpin RNA knockdown in RAW264.7 cells decreased their osteoclastogenic potential. 25D dose dependently reduced the resorptive capacity of PBMC-derived osteoclasts without compromising cell viability. 25D also reduced resorption by RAW264.7- and giant cell tumor-derived osteoclasts. Conversely, osteoclasts formed from vitamin D receptor-null mouse splenocytes had increased resorptive activity compared with wild-type cells. We conclude that 25D metabolism is an important intrinsic mechanism for optimizing osteoclast differentiation, ameliorating osteoclast activity, and potentially promoting the coupling of bone resorption to formation.
Publisher: Elsevier BV
Date: 10-2002
DOI: 10.1016/S8756-3282(02)00858-X
Abstract: Apo2 ligand (Apo2L/TRAIL) is a member of the tumor necrosis factor (TNF) cytokine family. Apo2L/TRAIL can selectively induce programmed cell death in transformed cells, although its wide tissue distribution suggests potential physiological roles. We have investigated the expression, in human osteoblast-like cells (NHBC), of Apo2L/TRAIL and the known Apo2L/TRAIL death receptors, DR4 and DR5, and the Apo2L/TRAIL decoy receptors, DcR-1, DcR-2, and osteoprotegerin (OPG). NHBC expressed abundant mRNA corresponding to each of these molecular species. Immunofluorescence staining demonstrated that Apo2L/TRAIL protein was abundant within the cytoplasm of NHBC and OPG was strongly expressed at the cell surface. DR5 and DcR-2 were present in the cell membrane and cytoplasm and DcR-1 was confined to the nucleus. DR4 staining was weak. Neither Apo2L/TRAIL alone, nor in combination with chemotherapeutic agents of clinical relevance to treatment of osteogenic sarcoma, induced cell death in NHBC, as assessed morphologically and by activation of caspase-3. In contrast, the human osteogenic sarcoma cell lines, BTK-143 and G-292, were sensitive to exogenous Apo2L/TRAIL alone, and to the combined effect of Apo2L/TRAIL/cisplatin and Apo2L/TRAIL/doxorubicin treatments, respectively. In NHBC, we observed strong associations between the levels of mRNA corresponding to the pro-apoptotic molecules, Apo2L/TRAIL, DR4, and DR5, and those corresponding to pro-survival molecules, DcR-1, DcR-2, OPG, and FLIP, suggesting that the balance between pro-survival and pro-apoptotic molecules is a mechanism by which NHBC can resist Apo2L/TRAIL-mediated apoptosis. In contrast, osteogenic sarcoma cells had low or absent levels of DcR-1 and DcR-2. These results provide a foundation to explore the role of Apo2L/TRAIL in osteoblast physiology. In addition, they predict that therapeutic use of recombinant Apo2L/TRAIL, in combination with chemotherapeutic agents to treat skeletal malignancies, would have limited toxic effects on normal osteoblastic cells.
Publisher: Oxford University Press (OUP)
Date: 08-10-2011
DOI: 10.1093/RHEUMATOLOGY/KER291
Abstract: The study of primary hip OA is continuing to redefine what was once considered a stagnant pathology as one of dynamic change, occurring over a long period of time involving the many composite tissue types of the joint including the bone. Examination of the inverse relationships evident between OA and fracture cohorts, including in iduals with osteoporosis (OP), indicates an imbalance in formation and resorption in the bony component of both pathologies. This review contains an overview of primary OA followed by an assessment of differential gene expression and altered cellular characteristics identified in the bony compartments of primary hip OA, with a focus on the wingless mouse mammary tumor virus integration (Wnt) and TGF-β signalling pathways. The studies reviewed here suggest that OA is a systemic disease involving the bone and validate the assessment of molecular changes to further investigate this complex disease.
Publisher: Wiley
Date: 06-2003
DOI: 10.1359/JBMR.2003.18.6.1088
Abstract: Human osteoblast phenotypes that support osteoclast differentiation and bone formation are not well characterized. Osteoblast differentiation markers were examined in relation to RANKL expression. RANKL expression was induced preferentially in immature cells. These results support an important link between erse osteoblast functions. Cells of the osteoblast lineage support two apparently distinct functions: bone formation and promotion of osteoclast formation. The aim of this study was to examine the relationship between these phenotypes in human osteoblasts (NHBC), in terms of the pre-osteoblast marker, STRO-1, and the mature osteoblast marker, alkaline phosphatase (AP), and the expression of genes involved in osteoclast formation, RANKL and OPG. The osteotropic stimuli, 1alpha,25(OH)2vitamin D3 (vitD3) and dexamethasone, were found to have profound proliferative and phenotypic effects on NHBCs. VitD3 inhibited NHBC proliferation and increased the percentage of cells expressing STRO-1 over an extended culture period, implying that vitD3 promotes and maintains an immature osteogenic phenotype. Concomitantly, RANKL mRNA expression was upregulated and maintained in NHBC in response to vitD3. Dexamethasone progressively promoted the proliferation of AP-expressing cells, resulting in the overall maturation of the cultures. Dexamethasone had little effect on RANKL mRNA expression and downregulated OPG mRNA expression in a donor-dependent manner. Regression analysis showed that RANKL mRNA expression was associated negatively with the percentage of cells expressing AP (p < 0.01) in vitD3- and dexamethasone-treated NHBCs. In contrast, RANKL mRNA expression was associated positively with the percentage of STRO-1+ cells (p < 0.01). In NHBCs sorted by FACS based on STRO-1 expression (STRO-1bright and STRO-1dim populations), it was found that vitD3 upregulated the expression of RANKL mRNA preferentially in STRO-1bright cells. The results suggest that immature osteoblasts respond to osteotropic factors in a potentially pro-osteoclastogenic manner. Additionally, the dual roles of osteoblasts, in supporting osteoclastogenesis or forming bone, may be performed by the same lineage of cells at different stages of their maturation.
Publisher: Wiley
Date: 30-04-2021
DOI: 10.1002/JOR.25051
Abstract: The objectives of this study were to (1) develop a semiautomated method to obtain lesion volume and bone mineral density (BMD) in terms of Hounsfield units from pelvic computed tomography (CT) scans in three regions of interest, and (2) assess accuracy and reliability of the method based on cadaveric CT scans. Image artefacts due to metal implants reduce CT clarity and are more severe with more than one implant in situ. Therefore, accuracy and reliability tests were performed with varying numbers of total hip arthroplasties implanted. To test the accuracy of lesion size measurements, microcomputed tomography was used as a reference. Mean absolute error ranged from 36 to 284 mm 3 after five measurements. Intra‐ and inter‐operator reliability of the entire method was measured for a selection of parameters. All coefficient of variation values were good to excellent for CT scans of the native pelvic anatomy and a CT scans of the same pelvis with one and two implants in situ. Accuracy of quantifying lesion volume decreased with decreasing CT image clarity by 0.6%–3.6% mean absolute relative error. Reliability of lesion volume measurement decreased with decreasing CT clarity. This was also the case for reliability of BMD measurements in the region most disrupted by metal artefact. The presented method proposes an approach for quantifying bone loss which has been proven to be accurate, reliable, and clinically applicable.
Publisher: Oxford University Press (OUP)
Date: 06-2001
DOI: 10.1093/RHEUMATOLOGY/40.6.623
Abstract: This study investigated the involvement of the recently identified regulators of osteoclast formation RANKL [receptor activator of nuclear factor kappaB (RANK) ligand, osteoclast differentiation factor, TRANCE, osteoprotegerin ligand] and its natural inhibitor, osteoprotegerin (OPG), in the bone erosion of rheumatoid arthritis (RA). mRNA was extracted from cells isolated from the pannus and synovial membrane regions of joints of 11 RA patients. Semiquantitative reverse transcription-polymerase chain reaction was carried out, and the isolated cells were also cultured to determine their ability to form osteoclasts. mRNAs encoding RANKL, RANK, OPG and macrophage-colony stimulating factor were expressed by cells isolated from RA joints. In addition, mRNA encoding for tumour necrosis factor apoptosis-inducing ligand and the osteoclast markers tartrate-resistant acid phosphatase and calcitonin receptor were also often expressed. Osteoclasts capable of forming resorption lacunae were generated from cells in the RA joints. At 50 ng/ml, recombinant OPG completely inhibited the resorptive activity of these cells. There was a significant correlation between the ratio of RANKL mRNA to OPG mRNA and the number of resorption pits produced (P = 0.028). These data suggest that RANKL is an essential factor for osteoclast formation by cells in the rheumatic joint and that OPG may prevent the bone erosion seen in RA joints.
Publisher: Wiley
Date: 09-2006
DOI: 10.1359/JBMR.060604
Abstract: RANK expression in vivo on hematopoietic subsets including pre-osteoclasts, identified by monoclonal antibodies, has not been described. We describe the lineages that express RANK in bone marrow, peripheral blood, and GCTs. We show that CD14(+)RANK(high) cells constitute a circulating pre-osteoclast pool. The expression of RANK by subsets of hematopoietic cells has not been adequately studied in humans. While attributed to the monocytoid lineage, the phenotype of the pre-osteoclast (pre-OC) with respect to RANK expression in vivo remains unclear. We tested monoclonal antibodies (MAbs) raised against the extracellular domain of recombinant human RANK for reactivity with normal peripheral blood (PB) and bone marrow (BM) mononuclear cells (PBMNCs and BMMNCs, respectively). We also tested reactivity with giant cell tumor cells (GCT), a confirmed source of pre-OC and mature OCs. Human PBMNCs, BMMNCs, and GCT cells were analyzed for reactivity with anti-RANK MAbs by flow cytometry in combination with hematopoietic lineage restricted markers. GCTs were also analyzed by immunofluorescence. CD14+ monocytoid cells were sorted by fluorescence-activated cell sorting (FACS) based on their relative RANK expression and cultured under OC-forming conditions. RANK+ cells were detected similarly by three independent anti-RANK MAbs. One MAb (80736) immunoprecipitated RANK-RANKL complexes from surface-biotinylated GCT lysates. Using dual-color flow cytometry, RANK was detected on CD14+ (monocytoid), CD19+ (B-lymphoid), CD56+ (NK cell), and glycophorin A+ erythroid progenitors. Minor populations of both CD3+ T lymphocytes and BM CD34+ hematopoietic progenitors also expressed cell surface RANK. In GCTs, RANK expression was identified on mononuclear CD45(+)CD14(+)alphaVbeta3(+)c-Fms+ cells, likely to be committed pre-OC, and on multinucleated CD45(+)alphaVbeta3(+)TRACP(+) OCs. Importantly, sorted CD14(+)RANK(high) PBMNCs treated with recombinant RANKL and macrophage-colony stimulating factor (M-CSF) gave rise to approximately twice the number of osteoclasts than RANK(mid) or RANK(low) cells. These results suggest that committed monocytoid RANK+ pre-OCs are represented in the marrow and circulate in the periphery, forming a pool of cells capable of responding rapidly to RANKL. The ability to reliably detect committed pre-OC in peripheral blood could have important clinical applications in the management of diseases characterized by abnormal osteoclastic activity.
Publisher: Elsevier BV
Date: 10-2013
Publisher: Cold Spring Harbor Laboratory
Date: 10-07-2021
DOI: 10.1101/2021.07.08.21257202
Abstract: Autosomal Dominant Osteopetrosis type II (ADOII), also known as Albers-Schönberg disease, is caused by mutation of the CLCN7 chloride channel gene and is characterized by reduced bone resorption. Here we report on an in idual with the classic features of ADOII, who had a history of fractures from childhood, displayed high bone mass and characteristic “sandwich vertebrae” on x-ray. Our genetic analyses showed no amino acid converting mutation in the patient’s DNA but we did find evidence of haploinsufficiency of CLCN7 mRNA. An iliac crest bone s le from the patient revealed bone tissue and material abnormalities relative to normal controls based on quantitative backscattered electron imaging and histomorphometric analyses. Additionally to lamellar bone, we observed significant amounts of woven bone and mineralised cartilage, as well as an increased frequency and thickness (up to 15 microns) of cement lines. Giant osteoclasts with numerous nuclei were present. The bone mineralisation density distribution (BMDD) of the entire bone area revealed markedly increased average mineral content of the dense bone (CaMean T-score +10.1) and frequency of bone with highest mineral content (CaHigh T-score +19.6), suggesting continued mineral accumulation and lack of bone remodelling. Osteocyte lacunae sections (OLS) characteristics were unremarkable except the OLS shape which was unusually circular. Together, our findings suggest that the reduced expression of CLCN7 mRNA in osteoclasts, and possibly also osteocytes, causes poorly remodelled bone with abnormal bone matrix with high mineral content. This together with the lack of adequate bone repair mechanisms makes the material brittle and prone to fracture.
Publisher: American Physiological Society
Date: 12-2009
DOI: 10.1152/AJPCELL.00216.2009
Abstract: The vitamin K family members phylloquinone (vitamin K1) and the menaquinones (vitamin K2) are under study for their roles in bone metabolism and as potential therapeutic agents for skeletal diseases. We have investigated the effects of two naturally occurring homologs, phytonadione (vitamin K1) and menatetrenone (vitamin K2), and those of the synthetic vitamin K, menadione (vitamin K3), on human primary osteoblasts. All homologs promoted in vitro mineralization by these cells. Vitamin K1-induced mineralization was highly sensitive to warfarin, whereas that induced by vitamins K2 and K3 was less sensitive, implying that γ-carboxylation and other mechanisms, possibly genomic actions through activation of the steroid xenobiotic receptor, are involved in the effect. The positive effect on mineralization was associated with decreased matrix synthesis, evidenced by a decrease from control in expression of type I collagen mRNA, implying a maturational effect. Incubation in the presence of vitamin K2 or K3 in a three-dimensional type I collagen gel culture system resulted in increased numbers of cells with elongated cytoplasmic processes resembling osteocytes. This effect was not warfarin sensitive. Addition of calcein to vitamin K-treated cells revealed vitamin K-dependent deposition of mineral associated with cell processes. These effects are consistent with vitamin K promoting the osteoblast-to-osteocyte transition in humans. To test whether vitamin K may also act on mature osteocytes, we tested the effects of vitamin K on MLO-Y4 cells. Vitamin K reduced receptor activator of NF-κB ligand expression relative to osteoprotegerin by MLO-Y4 cells, an effect also seen in human cultures. Together, our findings suggest that vitamin K promotes the osteoblast-to-osteocyte transition, at the same time decreasing the osteoclastogenic potential of these cells. These may be mechanisms by which vitamin K optimizes bone formation and integrity in vivo and may help explain the net positive effect of vitamin K on bone formation.
Publisher: Elsevier BV
Date: 10-2010
DOI: 10.1016/J.JOCA.2010.07.005
Abstract: This study examined differential gene expression, histomorphometric indices and relationships between these, in femoral trabecular bone from osteoarthritis (OA) patients and control (CTL) subjects, with the aim of identifying potential molecular drivers consistent with changes in structural and remodelling indices in the OA pathology. Bone s les from the intertrochanteric (IT) region were obtained from age and sex-matched cohorts of 23 primary hip OA patients and 21 CTL subjects. Real-time polymerase chain reaction (PCR) and histomorphometric analysis were performed on each s le and correlations between gene expression and histomorphometric variables determined. Alterations in gene expression, structural indices and correlations between these were found in OA bone compared to CTL. In OA bone, expression of critical regulators of osteoblast differentiation (TWIST1) and function (PTEN, TIMP4) were decreased, while genes associated with inflammation (SMAD3, CD14) were increased. Bone structural and formation indices (BV/TV, Tb.N, OS/BS) were increased, whereas resorption indices (ES/BS, ES/BV) were decreased. Importantly, significant correlations in CTL bone between CTNNB1 expression and formation indices (OS/BS, OS/BV, OV/BV) were absent in OA bone, indicating altered WNT/β-catenin signalling. TWIST1 expression and BV/TV were correlated in CTL bone, but not in OA bone, consistent with altered osteoblastogenesis in OA. Matrix metalloproteinase 25 (MMP25) expression and remodelling indices (ES/BS, ES/BV, ES/TV) were correlated only in OA pointing to aberrant bone remodelling in this pathology. These findings indicate an altered state of osteoblast differentiation and function in OA driven by several key molecular regulators. In association with this differential gene expression, an altered state of both trabecular bone remodelling and resulting microarchitecture were also observed, further characterising the pathogenesis of primary hip OA.
Publisher: Cold Spring Harbor Laboratory
Date: 18-04-2022
DOI: 10.1101/2022.04.17.488608
Abstract: Polyethylene (PE) liners are a common bearing surface of orthopaedic prostheses. Wear particles of ultra-high molecular weight PE (UHMWPE) contribute to periprosthetic osteolysis, a major cause of aseptic loosening. Vitamin E is added to some PE liners to prevent oxidative degradation. Osteocytes, an important cell type for controlling both bone mineralisation and bone resorption, have been shown to respond UHMWPE particles by upregulating pro-osteoclastogenic and osteocytic osteolysis. Here, we examined the effects of the vitamin E analogues α-tocopherol and γ-tocotrienol alone or in the context of UHMWPE particles on human osteocyte gene expression and mineralisation behaviour. Human osteoblasts differentiated to an osteocyte-like stage were exposed to UHMWPE wear particles in the presence or absence of either α-Tocopherol or γ-Tocotrienol. Both α-Tocopherol and γ-Tocotrienol induced antioxidant-related gene expression. UHMWPE particles independently upregulated antioxidant gene expression, suggesting an effect of wear particles on oxidative stress. Both vitamin E analogues strongly induced OPG mRNA expression and γ-Tocotrienol also inhibited RANKL mRNA expression, resulting in a significantly reduced RANKL : OPG mRNA ratio (p 0.01) overall. UHMWPE particles reversed the suppressive effect of α-Tocopherol but not of γ-Tocotrienol on this pro-osteoclastogenic index. UHMWPE particles also upregulated osteocytic-osteolysis related gene expression. Vitamin E analogues alone or in combination with UHMWPE particles also resulted in upregulation of these genes. Consistent with this, both vitamin E analogues promoted calcium release from mineralised cultures of osteocyte-like cells. Our findings suggest that while vitamin E may suppress osteocyte support of osteoclastogenesis in the presence of UHMWPE particles, the antioxidant effect may induce osteocytic osteolysis, which could promote periprosthetic osteolysis. It will be important to conduct further studies of vitamin E to determine the long-term effects of its inclusion in prosthetic materials.
Publisher: MDPI AG
Date: 26-02-2020
DOI: 10.3390/JCM9030626
Abstract: Tibial plateau fractures (TPFs) are challenging, requiring complex open reduction and internal fixation (ORIF) and are often associated with complications including surgical site infections (SSIs). In 2007, we introduced a novel management protocol to treat TPFs which consisted of an angiosome- or perforator-sparing (APS) anterolateral approach followed by unrestricted weight bearing and range of motion. The primary aim of this retrospective study was to investigate complication rates and patient outcomes associated with our new management protocol. In total, 79 TPFs treated between 2004 and 2007 through a classic anterolateral surgical approach formed the “Classic Group” while 66 TPFS treated between 2007 and 2013 formed the “APS Group”. Fracture reduction, maintenance of reduction and patient-reported outcomes were assessed. There was a clinically important improvement in the infection incidence with the APS (1.5%) versus the Classic technique (7.6%) (1/66 versus 2/79 for superficial infections 0/66 versus 4/79 for deep infections). Despite a more aggressive rehabilitation, there was no difference in the fracture reduction over time or the functional outcomes between both groups (p 0.05). The APS anterolateral approach improved the rate of SSIs after TPFs without compromising fracture reduction and stabilisation. We continue to use this new management approach and early unrestricted weight bearing when treating amenable TPFs.
Publisher: Elsevier BV
Date: 11-2012
DOI: 10.1016/J.JOCA.2012.07.005
Abstract: This study compared human primary osteoblasts derived from hip osteoarthritis (OA) cases against controls (CTLs) to investigate candidate OA disease genes, twist homologue 1 (TWIST1), wingless MMTV integration site family member 5B (WNT5B), transforming growth factor-β (TGFβ1) and SMAD family member 3 (SMAD3), during osteoblast differentiation, relative to calcium apposition and elemental mineral composition. Primary osteoblast cultures were generated from intertrochanteric trabecular bone s les from five female primary hip OA cases and five age-matched female CTLs. During a 42-day differentiation time-course, alizarin red stains, energy-dispersive X-ray spectroscopy and real-time RT-polymerase chain reaction (PCR) were used to quantify calcium, elemental composition and gene expression, respectively. Data were analysed using linear mixed effects models and Pearson correlation matrices. Significant differences, correlations and associations were found in OA and CTL osteoblasts between gene and mineral measures. The calcium: phosphorous (Ca:P) ratio was significantly more varied in OA compared to CTL. Calcium apposition, mineral composition as well as TWIST1 and TGFβ1 mRNA expression changed significantly over time. TWIST1 mRNA expression was elevated and correlated with SMAD3 mRNA levels in the OA cohort during the time-course. Associations were observed between tissue non-specific alkaline phosphatase (TNAP), osteocalcin (OCN), TWIST1, TGFβ1, SMAD3 mRNA levels and mineral measures in OA against CTL. Temporal differences between SMAD3 mRNA expression and mineral composition were also found in OA. Dysregulated expression of TWIST1, TGFβ1 and SMAD3 mRNA observed in OA bone is reflected in the functionality of the osteoblast when these cells are cultured ex vivo. The results presented here are consistent with at least part of the aetiology of primary hip OA deriving from altered intrinsic properties of the osteoblast.
Publisher: Bentham Science Publishers Ltd.
Date: 31-12-2013
DOI: 10.2174/138945011131400212
Abstract: The active form of vitamin D, 1,25-dihydroxyvitamin D3, carries out its erse range of biological activities by binding to the nuclear vitamin D receptor, present in almost every cell of the body. It is well established that adequate serum 25-hydroxyvitamin D levels correlate with a reduction in the incidence of osteoporosis however, the physiological basis for this relationship remains elusive. Although, the endocrine actions of vitamin D are thoroughly appreciated, the effect of vitamin D on bone tissue and bone cells is yet to be completely understood. There exists a wealth of literature that suggests the VDR within the three major bone cell types, osteoblasts, osteocytes and osteoclasts, is responsible for the regulation of bone homeostasis. The circumstances, under which the action of 1,25-dihydroxyvitamin D3 elicits an anabolic or catabolic role have not been elucidated. However, it would seem that vitamin D can evoke both of these effects and that this is partly mediated by calcium homeostasis. This raises the possibility that dietary calcium intake and vitamin D metabolism act concomitantly at the kidney, intestine and the bone in a coordinated response. Thus, to maintain adequate bone homeostasis and reduce the risk of metabolic bone disease via the diet, it is important to consider this duality of vitamin D action in relation to the overall calcium economy.
Publisher: Wiley
Date: 2005
DOI: 10.1002/JCP.20255
Abstract: While it has been assumed that osteoblasts in the human support osteoclast formation, in vitro evidence of this is currently lacking. We tested the ability of normal human trabecular bone-derived osteoblasts (NHBCs) to support osteoclast formation from human peripheral blood mononuclear cells (PBMC) in response to treatment with either 1alpha,25-dihydroxyvitamin D3 (1,25D) or parathyroid hormone (PTH), using a serum-replete medium previously used to support human osteoclast formation on a stroma of murine ST-2 cells. Under these conditions, NHBC did not support osteoclast formation, as assessed by morphological, histochemical, and functional criteria, despite our previous results demonstrating a link between induction of RANKL mRNA expression and NHBC phenotype in these media. We next tested a defined, serum-free medium (SDM) on NHBC phenotype, their expression of RANKL and OPG, and their ability to support osteoclast formation. SDM, containing dexamethasone (DEX) and 1,25D, induced phenotypic maturation of NHBC, based on the expression of STRO-1 and the bone/liver/kidney isoform of alkaline phosphatase (AP). PTH as a single factor did not induce phenotypic change. 1,25D and DEX induced the greatest ratio of RANKL:OPG mRNA, predictive of supporting osteoclast formation. Consistent with this, co-culture of NHBC with CD14+ PBMC, or bone marrow mononuclear cell (BMMC), or CD34+ BMMC precursors in SDM + 1,25D + DEX, resulted in functional osteoclast formation. Osteoclast formation also occurred in PTH + DEX stimulated co-cultures. Interestingly, SDM supplemented with recombinant RANKL (25-100 ng/ml) and M-CSF (25 ng/ml), did not induce osteoclast formation from any of the osteoclast precursor populations in stromal-free cultures, unlike serum-replete medium. This study demonstrates that under the appropriate conditions, adult human primary osteoblasts can support de novo osteoclast formation, and this model will enable the detailed study of the role of both cell types in this process.
Publisher: Research Square Platform LLC
Date: 27-01-2021
DOI: 10.21203/RS.3.RS-146887/V1
Abstract: Mesenchymal progenitors differentiate into several tissues including bone, cartilage, and adipose. Targeting these cells in vivo is challenging, making mesenchymal progenitor cell lines valuable tools to study tissue development. Mesenchymal stem cells (MSCs) can be isolated from humans and animals however, obtaining homogenous, responsive cells in a reproducible fashion is challenging. As such, we developed two mesenchymal progenitor cell (MPC) lines, MPC1 and MPC2, generated from bone marrow of male C57BL/6 mice. These cells were immortalized using the temperature sensitive large T-antigen, allowing for thermal control of proliferation and differentiation. Both MPC1 and MPC2 cells are capable of osteogenic, adipogenic, and chondrogenic differentiation. Under osteogenic conditions, both lines formed mineralized nodules, and stained for alizarin red and alkaline phosphatase, while expressing osteogenic genes including Sost , Fgf23 , and Dmp1 . Sost and Dmp1 mRNA levels were drastically reduced with parathyroid hormone, thus recapitulating in vivo responses. MPC cells secreted intact (iFGF23) and C -terminal (cFGF23) forms of the endocrine hormone FGF23, which was upregulated by 1,25 dihydroxy vitamin D (1,25D). Both lines also rapidly entered the adipogenic lineage, expressing adipose markers after 4 days in adipogenic media. MPC cells were also capable of chondrogenic differentiation, displaying increased expression of cartilage genes including aggrecan, Sox9 , and Comp . With the ability to differentiate into multiple mesenchymal lineages and mimic in vivo responses of key regulatory genes roteins, MPC cells are a valuable model to study factors that regulate mesenchymal lineage allocation as well as the mechanisms that dictate transcription, protein modification, and secretion of these factors.
Publisher: Elsevier BV
Date: 09-2015
DOI: 10.1016/J.MCE.2015.06.005
Abstract: While vitamin D supplementation is common, the anabolic mechanisms that improve bone status are poorly understood. Under standard mineralising conditions including media ionised calcium of 1.1 mM, 1,25-dihydroxyvitamin D3 (1,25D) enhanced differentiation and mineral deposition by the mature osteoblast re-osteocyte cell line, MLO-A5. This effect was markedly increased with a higher ionised calcium level (1.5 mM). Gene expression analyses revealed that 1,25D-induced mineral deposition was associated with induction of Enpp1 mRNA, coding for nucleotide pyrophosphatase phosphodiesterase 1 (NPP1) and NPP1 protein levels. Since MLO-A5 cells express abundant alkaline phosphatase that was not further modified by 1,25D treatment or exposure to increased calcium, this finding suggested that the NPP1 production of pyrophosphate (PPi) may provide alkaline phosphatase with substrate for the generation of inorganic phosphate (Pi). Consistent with this, co-treatment with Enpp1 siRNA or a NPP1 inhibitor, PPADS, abrogated 1,25D-induced mineral deposition. These data demonstrate that 1,25D stimulates osteoblast differentiation and mineral deposition, and interacts with the extracellular calcium concentration. 1,25D regulates Enpp1 expression, which presumably, in the context of adequate tissue non-specific alkaline phosphatase activity, provides Pi to stimulate mineralisation. Our findings suggest a mechanism by which vitamin D with adequate dietary calcium can improve bone mineral status.
Publisher: Elsevier BV
Date: 06-2022
DOI: 10.1016/J.JOCA.2022.03.004
Abstract: 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. Paired subchondral bone s les 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). Bone s les 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]). 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 potential therapeutic target to prevent or reduce human OA disease progression.
Publisher: Elsevier BV
Date: 08-2012
DOI: 10.1016/J.ACTBIO.2012.04.037
Abstract: Wear particle-induced orthopaedic prosthesis loosening is associated with elevated osteoclast activity. The immunoreceptor tyrosine-based activation motif (ITAM)-related molecules OSCAR, FcRγ, TREM2 and DAP12 are important for osteoclast formation. The aim of this study was to determine if these molecules are involved in peri-implant loosening by investigating their expression in peri-implant tissues obtained at revision of joint replacement components containing polyethylene (PE) wear particles, and in osteoclasts formed in vitro in the presence of PE particles. The results showed that there was a marked and statistically significant increase in protein levels of the ITAM-related molecules in the revision tissues. The levels of OSCAR, FcRγ, TREM2 and DAP12 mRNA in the revision tissues were also increased. In vitro PE particles stimulated osteoclast resorption in the presence of 50 ng ml(-1) receptor activator NFκB (RANKL) and significantly elevated the expression of OSCAR, FcRγ, TREM2 and DAP12 during osteoclast formation. These findings suggest that the ITAM signalling molecules and their co-receptors have a role in pathogenic bone loss associated with implant PE wear.
Publisher: Springer International Publishing
Date: 2015
Publisher: Elsevier BV
Date: 03-2015
Publisher: Elsevier BV
Date: 06-2007
DOI: 10.1016/J.BONE.2007.02.024
Abstract: Circulating 1 alpha,25-dihydroxyvitamin D(3) (1,25D) derives from renal conversion of 25-hydroxyvitamin D(3) (25D), by the 25D 1 alpha-hydroxylase (CYP27B1). Blood 25D levels, but not 1,25D levels, are the best indicator of vitamin D status and predict fracture risk in the elderly. We examined the extent to which osteoblasts can metabolize 25D. Well-characterized human primary osteoblasts and osteosarcoma (OS) cell lines were examined for the expression and regulation of genes associated with vitamin D metabolism, using real-time PCR. Primary osteoblasts and OS cell lines were found to express CYP27B1 mRNA and secreted detectable 1,25D in response to 25D. Of the OS cell lines tested, HOS expressed the most CYP27B1 mRNA and secreted the highest levels of 1,25D. All osteoblastic cells examined up-regulated expression of the catabolic regulator of 1,25D, the 25-hydroxyvitamin D-24-hydroxylase (CYP24), when incubated with either 1,25D or 25D. Exposure to physiological levels of 25D resulted in up-regulated transcription of the 1,25D responsive genes, osteocalcin (OCN), osteopontin (OPN) and RANKL. Specific knockdown of CYP27B1 in HOS cells using siRNA resulted in up to 80% reduction in both 1,25D secretion and the transcription of OCN and CYP24, strongly implying that the 25D effect in osteoblasts is preceded by conversion to 1,25D. Incubation with 25D, like 1,25D, inhibited primary osteoblast proliferation and promoted in vitro mineralization. Finally, we detected expression by osteoblasts of receptors for vitamin D binding protein (DBP), cubilin and megalin, suggesting that osteoblasts are able to internalize DBP-25D complexes in vivo. Together, our results suggest that autocrine, and perhaps paracrine, pathways of vitamin D(3) metabolism may regulate key osteoblast functions independently of circulating, kidney derived 1,25D. Our results are therefore consistent with the reported benefits of maintaining a healthy vitamin D status in the elderly to reduce the risk of fractures.
Publisher: Wiley
Date: 20-11-2016
DOI: 10.1002/JBM.A.35595
Abstract: To treat skeletal conditions such as bone infections, osteoporotic fractures, and osteosarcoma, it would be ideal to introduce drugs directly to the affected site. Localized drug delivery from the bone implants is a promising alternative to systemic drug administration. In this study we investigated electrochemically nanoengineered Ti wire implants with titania nanotubes (TNTs), as minimally invasive drug-releasing implants for the delivery of drugs directly into the bone tissue. Since trabecular bone in vivo contains a highly interconnected bone marrow, we sought to determine the influence of marrow on drug release and diffusion. Electrochemical anodization of Ti wires (length 10 mm) was performed to create an oxide layer with TNTs on the surface, followed by loading with a fluorescent model drug, Rhodamine B (RhB). Cores of bovine trabecular bone were generated from the sternum of a young steer, and were processed to have an intact bone marrow, or the marrow was removed. RhB-loaded TNTs/Ti wires were inserted into the bone cores, which were then cultured ex vivo using the ZetOS™ bioreactor system to maintain bone viability. Release and diffusion of RhB inside the bone was monitored using fluorescence imaging and different patterns of drug transport in the presence or absence of marrow were observed. Scanning electron microscopy of the implants after retrieval from bone cores confirmed survival of the TNTs structures. Histological investigation showed the presence of bone cells adherent on the implants. This study shows a potential of Ti drug-releasing implants based on TNTs technology towards localized bone therapy. © 2015 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 104A: 714-725, 2016.
Publisher: Elsevier
Date: 2017
Publisher: Elsevier BV
Date: 10-2014
DOI: 10.1016/J.JSBMB.2013.09.016
Abstract: 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 s les 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'.
Publisher: Elsevier BV
Date: 07-2004
Publisher: Springer Science and Business Media LLC
Date: 02-09-2008
DOI: 10.1007/S00198-008-0728-6
Abstract: The effect of strontium ranelate (SR) on human osteoblast differentiation was tested. SR induced osteoblastic proliferation, in vitro mineralization, and increased the expression of osteocyte markers. SR also elicited an osteoprotegerin (OPG) secretory response. We conclude that SR promotes the osteoblast maturation and osteocyte differentiation while promoting an additional antiresorptive effect. SR is a new treatment for osteoporosis that reduces the risk of hip and vertebral fractures in postmenopausal women. This study sought to investigate the extent, to which SR modulates human osteoblast differentiation. Adult human primary osteoblasts (NHBC) were exposed to SR under mineralizing conditions in long-term cultures. Osteoblast differentiation status was investigated by cell-surface phenotypic analysis. Expression of genes associated with osteoblast/osteocyte differentiation was examined using real-time RT-PCR. Secreted OPG was assayed by enzyme-linked immunosorbent assay. SR significantly increased osteoblast replication. SR time- and dose-dependently induced an osteocyte-like phenotype, as determined by cell surface alkaline phosphatase and STRO-1 expression. SR at 5 mM or greater dramatically increased in vitro mineralization. In parallel, mRNA levels of dentin matrix protein (DMP)-1 and sclerostin were higher under SR treatment, strongly suggestive of the presence of osteocytes. SR also increased the OPG/RANKL ratio throughout the culture period, consistent with an effect to inhibit osteoblast-induced osteoclastogenesis. This study suggests that SR can promote osteoblast maturation and an osteocyte-like phenotype. Coupled with its effect on the OPG/RANKL system, these findings are consistent with in vivo effects in patients receiving SR for the treatment of osteoporosis.
Publisher: Elsevier BV
Date: 12-2011
DOI: 10.1016/J.MCE.2011.05.024
Abstract: The endocrine activity of 1,25-dihydroxyvitamin D (1,25(OH)(2)D(3)) contributes to maintaining plasma calcium and phosphate homeostasis through actions on the intestine, kidney and bone. A significant body of evidence has been published over the last 10 years indicating that all major bone cells have the capacity to metabolise 25-hydroxyvitamin D (25(OH)D(3)) to 1,25(OH)(2)D(3), which in turn exerts autocrine aracrine actions to regulate bone cell proliferation and maturation as well as bone mineralisation and resorption. In vivo and in vitro studies indicate that these autocrine aracrine activities of 1,25(OH)(2)D(3) in bone tissue contribute to maintaining bone mineral homeostasis and enhancing skeletal health.
Publisher: MDPI AG
Date: 27-12-2021
DOI: 10.3390/JCM11010122
Abstract: Periprosthetic joint infection (PJI) is a serious complication of total hip arthroplasty. Staged revision surgery is considered effective in eradicating PJI. We aimed to determine the rate of infection resolution after each stage of staged revision surgery (first stage, repeat first stage, second stage, excision arthroplasty, and reimplantation) and to assess functional outcomes and the mortality rate at ten years in a consecutive series of 30 chronic PJI of total hip arthroplasties. Infection resolution was defined as no clinical nor laboratory evidence of infection at 24 months after the last surgery and after a minimum of 12 months following cessation of antimicrobial treatment. Four patients died within 24 months of their final surgery. Nineteen patients, 73% (worst-case analysis (wca) 63%), were infection free after 1 surgery 22 patients, 85% (wca 73%), were infection free after 2 surgeries and 26 patients, 100% (wca 87%), were infection free after three and four surgeries. The median Harris Hip Score was 41 prior to first revision surgery and improved to 74 at twelve months and 76 at ten years after the final surgery. Thirteen patients died at a mean of 64 months from first revision, giving a mortality rate of 43% at ten years, which is approximately 25% higher than that of an age-matched general population. The results show that with repeated aggressive surgical treatment, most PJIs of the hip are curable. Ten years after successful treatment of PJI, functional outcomes and pain are improved and maintained compared to before initial surgery, but this must be balanced against the high 10-year mortality. Level of evidence: cohort studies.
Publisher: Elsevier BV
Date: 08-2009
DOI: 10.1016/J.BIOMATERIALS.2009.03.035
Abstract: Polyethylene (PE) wear particles are associated with the osteolysis seen in aseptic loosening that leads to orthopaedic implant failure. While cells of the monocyte/macrophage lineage are implicated, evidence is now emerging that osteoblastic cells may also be affected by PE. In this study we investigated the effect of PE particles on osteoblasts, using a novel in vitro cell culture system that was developed to juxtapose cells and PE particles, replicating the 3-dimensional (3D) environment near implants. This system allowed normal human bone-derived cells (NHBC) to undergo differentiation into a mature osteocyte-like phenotype over a 21-28-day culture period. PE particles induced an increase in mRNA expression of the osteocyte markers E11, DMP-1 and SOST/sclerostin. NHBC responded to PE particles by increasing the mRNA expression of several genes associated with osteoclast formation and activity (RANKL, IL-8 and M-CSF) and decreased the expression of the osteoclast antagonist, OPG. PE also appeared to induce a switch in the RUNX2 control of gene expression from that of promoting matrix production (type I collagen) to inducing the expression of pro-osteoclastogenic genes. These results suggest that PE particles switch mature osteoblastic cells from an anabolic to a more catabolic phenotype. This concept was further supported by the finding that PE-induced expression of RANKL mRNA in the mouse osteocyte cell line, MLO-Y4. Overall, our results suggest that PE particles directly induce a change in the phenotype of mature osteoblasts and osteocytes, consistent with the net loss of bone near orthopaedic implants.
Publisher: Oxford University Press (OUP)
Date: 2003
DOI: 10.1093/RHEUMATOLOGY/KEG047
Abstract: To demonstrate the expression of osteoprotegerin (OPG) and receptor activator of nuclear factor kappaB ligand (RANKL) in synovial tissue from rheumatoid arthritis (RA) patients, establish the cell lineage expressing OPG and compare the expression of OPG in RA, spondyloarthropathies, osteoarthritis and normal synovial tissue. Synovial biopsy specimens were obtained at arthroscopy from 16 RA and 12 spondyloarthropathy patients with active synovitis of a knee joint, six RA patients with no evidence of active synovitis, 10 patients with osteoarthritis and 18 normal subjects. Immunohistological analysis was performed using monoclonal antibodies (mAb) to detect OPG and RANKL expression. In addition, dual immunohistochemical evaluation was performed with lineage-specific monoclonal antibodies (macrophages, fibroblasts and endothelial cells) and OPG to determine the cell lineages expressing OPG. The sections were evaluated by computer-assisted image analysis and semiquantitative analysis. Two patterns of OPG expression were seen, one exclusively in endothelial cells and one expressed predominantly in macrophages in the synovial lining layer. Both patterns of OPG staining could be blocked with excess recombinant OPG. Endothelial and synovial lining expression of OPG was seen in all synovial tissues except those from patients with active RA. In contrast, RANKL expression was seen predominantly in synovial tissue from patients with active disease, mainly in sublining regions, particularly within areas of lymphocyte infiltration. OPG expression on macrophage type synovial lining cells as well as endothelial cells is deficient in RA patients with active synovitis, in contrast to that seen in spondyloarthropathy patients with active synovitis. This deficiency in OPG expression in the inflamed joint of RA patients may be important in the development of radiologically defined joint erosions.
Publisher: Wiley
Date: 09-01-2016
DOI: 10.1002/CAM4.599
Publisher: Springer Science and Business Media LLC
Date: 04-01-2021
Publisher: MDPI AG
Date: 08-07-2022
DOI: 10.3390/BIOM12070960
Abstract: The regulation of vitamin D3 actions in humans occurs mainly through the Cytochrome P450 24-hydroxylase (CYP24A1) enzyme activity. CYP24A1 hydroxylates both 25-hydroxycholecalciferol (25(OH)D3) and 1,25-dihydroxycholecalciferol (1,25(OH)2D3), which is the first step of vitamin D catabolism. An abnormal status of the upregulation of CYP24A1 occurs in many diseases, including chronic kidney disease (CKD). CYP24A1 upregulation in CKD and diminished activation of vitamin D3 contribute to secondary hyperparathyroidism (SHPT), progressive bone deterioration, and soft tissue and cardiovascular calcification. Previous studies have indicated that CYP24A1 inhibition may be an effective strategy to increase endogenous vitamin D activity and decrease SHPT. This study has designed and synthesized a novel C-24 O-methyloxime analogue of vitamin D3 (VD1-6) to have specific CYP24A1 inhibitory properties. VD1-6 did not bind to the vitamin D receptor (VDR) in concentrations up to 10−7 M, assessed by a VDR binding assay. The absence of VDR binding by VD1-6 was confirmed in human embryonic kidney HEK293T cultures through the lack of CYP24A1 induction. However, in silico docking experiments demonstrated that VD1-6 was predicted to have superior binding to CYP24A1, when compared to that of 1,25(OH)2D3. The inhibition of CYP24A1 by VD1-6 was also evident by the synergistic potentiation of 1,25(OH)2D3-mediated transcription and reduced 1,25(OH)2D3 catabolism over 24 h. A further indication of CYP24A1 inhibition by VD1-6 was the reduced accumulation of the 24,25(OH)D3, the first metabolite of 25(OH)D catabolism by CYP24A1. Our findings suggest the potent CYP24A1 inhibitory properties of VD1-6 and its potential for testing as an alternative therapeutic candidate for treating SHPT.
Publisher: Ovid Technologies (Wolters Kluwer Health)
Date: 2019
Abstract: We describe 2 cases of nonagenarians with periprosthetic knee fractures that were not amenable to either standard internal fixation nor prosthesis revision because of infected leg ulcers in the same limb. The fractures were internally fixed by percutaneous insertion of medial and lateral plates that spanned the knee. Both patients returned to their baseline level of activity without developing surgical site infections. Percutaneous bridging plates that span the knee are a useful option for treating these difficult cases.
Publisher: Wiley
Date: 08-2009
DOI: 10.1359/JBMR.090305
Abstract: We have recently shown that TNF-related weak inducer of apoptosis (TWEAK) is a mediator of inflammatory bone remodeling. The aim of this study was to investigate the role of TWEAK in modulating human osteoblast activity, and how TWEAK and TNFalpha might interact in this context. Recombinant TWEAK and TNF were both mitogenic for human primary osteoblasts (NHBC). TWEAK dose- and time-dependently regulated the expression of the osteoblast transcription factors RUNX2 and osterix. TWEAK inhibited in vitro mineralization and downregulated the expression of osteogenesis-associated genes. Significantly, TWEAK and TWEAK/TNF induced the expression of the osteoblast differentiation inhibitor and SOST gene product, sclerostin. Sclerostin induction was mitogen-activated protein kinase (MAPK) dependent. The SOST mRNA levels induced by TWEAK were equivalent to or exceeded those seen in steady-state human bone, and the TWEAK/TNF induction of SOST mRNA was recapitulated in fresh cancellous bone explants. TWEAK-induced sclerostin expression was observed in immature osteoblastic cells, both in cycling (Ki67(+)) primary NHBC and in the cell lines MC3T3-E1 and MG-63, as well as in human osteocyte-like cells and in the osteocyte cell line, MLO-Y4. Treatment of NHBC with recombinant human sclerostin mimicked the effects of TWEAK to suppress RUNX2 and osteocalcin (OCN). TWEAK, TNF, and sclerostin treatment of NHBC similarly altered levels of phosphorylated and total GSK3beta and active and total levels of beta-catenin, implying that the Wnt signaling pathway was affected by all three stimuli. Sclerostin also rapidly activated ERK-1/2 MAPK signaling, indicating the involvement of additional signaling pathways. Together, our findings suggest that TWEAK, alone and with TNF, can regulate osteoblast function, at least in part by inducing sclerostin expression. Our results also suggest new roles and modes of action for sclerostin.
Publisher: Elsevier BV
Date: 03-2018
DOI: 10.1016/J.JSBMB.2017.10.022
Abstract: Mature osteoclasts express the vitamin D receptor (VDR) and are able to synthesise and respond to 1,25(OH)
Publisher: Cold Spring Harbor Laboratory
Date: 25-04-2023
DOI: 10.1101/2023.04.23.537998
Abstract: 1. Few human osteocyte in vitro models exist and the differentiation of immature osteoblasts to an osteocyte stage typically takes at least 4-weeks of culture, making the study of this process challenging and time consuming. The osteosarcoma cell line Saos-2 has proved to be a useful model of human osteoblast differentiation through to a mature osteocyte-like stage. Culture under osteogenic conditions in a standard 5% CO 2 and normoxic (21% O 2 ) atmosphere results in reproducible mineralisation and acquisition of mature osteocyte markers over the expected 28-35 day culture period. In order to expedite experimental assays, we tested whether reducing available oxygen to mimic concentrations experienced by osteocytes in vivo would increase the rate of differentiation of Saos-2 cells. Cells cultured in a 5% CO 2 , 1% O 2 atmosphere exhibited accelerated deposition of mineral, reaching near saturation by 14 days as demonstrated with the Alizarin Red and Von Kossa staining. The gene expression of the major hypoxia-induced transcription factor HIF1α and the key osteogenic transcription factor RUNX2 were both elevated under 1% O 2 . Early ( COLA1, MEPE ) and mature ( PHEX, DMP1 and SOST ) osteocyte markers were also upregulated earlier under hypoxic compared to normoxic growth conditions. Thus, culture under low oxygen accelerates key markers of osteocyte differentiation, resulting in a useful human osteocyte-like in vitro cell model within 14 days.
Publisher: Elsevier BV
Date: 03-2016
DOI: 10.1016/J.ACTBIO.2016.01.016
Abstract: Periprosthetic osteolysis (PO) leading to aseptic loosening, is the most common cause of failure of total hip replacement (THR) in the mid- to long-term. Polyethylene (PE) particulates from the wear of prosthesis liners are bioactive and are implicated in the initiation and or progression of osteolysis. Evidence exists that cells of the osteoblast/osteocyte lineage are affected by PE particles and contribute to the catabolic response by promoting osteoclastic bone resorption. In this study, we hypothesised that osteocytes contribute directly to PO by removing bone from their perilacunar matrix. Osteocyte responses to ultra-high molecular weight PE (UHMWPE) particles were examined in vitro in human primary osteocyte-like cultures, in vivo in the mouse calvarial osteolysis model, and in the acetabulum of patients undergoing revision total hip replacement (THR) surgery for PO. Osteocytes exposed to UHMWPE particles showed upregulated expression of catabolic markers, MMP-13, carbonic anhydrase 2 (CA2), cathepsin K (CTSK) and tartrate resistant acid phosphatase (TRAP), with no effect on cell viability, as assessed by Caspase 3 activity. Consistent with this catabolic activity causing perilacunar bone loss, histological analysis of calvarial sections from mice exposed to UHMWPE revealed a significant (p<0.001) increase in osteocyte lacunar area (Lac.Ar) compared to sham-operated animals. Furthermore, acetabular biopsies from patients with PO also showed significantly (p<0.001) increased osteocyte lacunar size in trabecular bone adjacent to PE particles, compared with osteocyte lacunar size in bone from primary THR patients. Together, these findings suggest a previously unrecognised action of UHMWPE wear particles on osteocytes, which directly results in a loss of osteocyte perilacunar bone. This action may exacerbate the indirect pro-osteoclastic action of UHMWPE-affected osteocytes, previously shown to contribute to aseptic loosening of orthopaedic implants. This study addresses the clinical problem of periprosthetic osteolysis, bone loss in response to polyethylene wear particles derived from materials used in orthopaedic implants. Periprosthetic osteolysis has been thought to be due largely to wear particles stimulating the activity of bone resorbing osteoclasts. However, in this study we demonstrate for the first time that polyethylene particles stimulate another type of bone loss, mediated by the direct activity of bone mineral embedded osteocytes, termed osteocytic osteolysis or osteocyte perilacunar remodelling. This study provides new mechanistic insight into wear-particle mediated bone loss and represents a new paradigm for the way in which bone cells, namely osteocytes, the key controlling cell type in bone, react to biomaterials.
Publisher: Elsevier BV
Date: 2017
DOI: 10.1016/J.MCE.2016.11.007
Abstract: Myeloperoxidase (MPO) and eosinophil peroxidase (EPO) are heme-containing enzymes, well known for their antimicrobial activity, are released in abundance by innate immune infiltrates at sites of inflammation and injury. We have discovered new and previously unrecognised roles for heme peroxidases in extracellular matrix biosynthesis, angiogenesis, and bone mineralisation, all of which play an essential role in skeletal integrity. In this study we used in vitro models of osteoclastogenesis to investigate the effects of heme peroxidase enzymes on osteoclast differentiation and bone resorbing activity, pertinent to skeletal development and remodelling. Receptor activator of nuclear factor kappa B-ligand (RANKL) stimulates the formation of tartate-resistant acid phosphatase (TRAP) positive multinucleated cells and increases bone resorption when cultured with human peripheral blood mononuclear cells (PBMCs) or the RAW264.7 murine monocytic cell line. When RANKL was added in combination with either MPO or EPO, a dose-dependent inhibition of osteoclast differentiation and bone resorption was observed. Notably, peroxidases had no effect on the bone resorbing activity of mature osteoclasts, suggesting that the inhibitory effect of the peroxidases was limited to osteoclast precursor cells. Mechanistically, we observed that osteoclast precursor cells readily internalize peroxidases, and inhibited the phosphorylation of JNK, p38 MAPK and ERK1/2, important signalling molecules central to osteoclastogenesis. Our findings suggest that peroxidase enzymes, like MPO and EPO, may play a fundamental role in inhibiting RANKL-induced osteoclast differentiation at inflammatory sites of bone fracture and injury. Therefore, peroxidase enzymes could be considered as potential therapeutic agents to treat osteolytic bone disease and aberrant bone resorption.
Publisher: Elsevier BV
Date: 08-2013
DOI: 10.1016/J.MCE.2013.06.013
Abstract: Calcium, in combination with vitamin D, is an effective treatment for osteoporosis. Since bone mineralisation occurs concurrently with osteoblast to osteocyte transition, we hypothesised that calcium would stimulate this process. The effect of calcium (1.8-11.8mM) was tested on human primary osteoblast (NHBC) differentiation in vitro. Cultures were assayed for cell-associated mineral and gene expression associated with osteoblast differentiation and mineralisation. Treatment with calcium resulted in a striking dose- and time-dependent increase in cell-associated mineralisation. Calcium appeared to promote osteoblast to osteocyte differentiation, as indicated by increased expression of osteocalcin (OCN), E11, dentin matrix protein 1 (DMP1) and SOST mRNA. The expression of the osteoclast inhibitor, osteoprotegerin, was dramatically enhanced by calcium. Calcium also increased the ratio of PHEX mRNA expression relative to that of MEPE, suggesting a mechanism for the pro-anabolic effect. Consistent with this, calcium-dependent mineralisation was reversed in the presence of MEPE-ASARM peptides. This study suggests that calcium promotes osteoblast to osteocyte transition and concurrent matrix mineralisation, at least in part through the PHEX-MEPE axis.
Publisher: Hindawi Limited
Date: 07-12-2016
DOI: 10.1002/TERM.2239
Abstract: The success of implantation of materials into bone is governed by effective osseointegration, requiring biocompatibility of the material and the attachment and differentiation of osteoblastic cells. To enhance cellular function in response to the implant surface, micro- and nano-scale topography have been suggested as essential. In this study, we present bone implants based on 3D-printed titanium alloy (Ti6Al4V), with a unique dual topography composed of micron-sized spherical particles and vertically aligned titania nanotubes. The implants were prepared by combination of 3D-printing and anodization processes, which are scalable, simple and cost-effective. The osseointegration properties of fabricated implants, examined using human osteoblasts, showed enhanced adhesion of osteoblasts compared with titanium materials commonly used as orthopaedic implants. Gene expression studies at early (day 7) and late (day 21) stages of culture were consistent with the Ti substrates inducing an osteoblast phenotype conducive to effective osseointegration. These implants with the unique combination of micro- and nano-scale topography are proposed as the new generation of multi-functional bone implants, suitable for addressing many orthopaedic challenges, including implant rejection, poor osseointegration, inflammation, drug delivery and bone healing. Copyright © 2016 John Wiley & Sons, Ltd.
Publisher: Public Library of Science (PLoS)
Date: 04-10-2011
Publisher: Elsevier BV
Date: 07-2005
DOI: 10.1016/S0002-9440(10)62959-8
Abstract: Giant cell tumor of bone (GCT) is a generally benign, osteolytic neoplasm comprising stromal cells and osteoclast-like giant cells. The osteoclastic cells, which cause bony destruction, are thought to be recruited from normal monocytic pre-osteoclasts by stromal cell expression of the ligand for receptor activator of nuclear factor kappaB (RANKL). This model forms the foundation for clinical trials in GCTs of novel cancer therapeutics targeting RANKL. Using expression profiling, we identified both osteoblast and osteoclast signatures within GCTs, including key regulators of osteoclast differentiation and function such as RANKL, a C-type lectin, osteoprotegerin, and the wnt inhibitor SFRP4. After ex vivo generation of stromal- and osteoclast-enriched cultures, we unexpectedly found that RANKL mRNA and protein were more highly expressed in osteoclasts than in stromal cells, as determined by expression profiling, flow cytometry, immunohistochemistry, and reverse transcriptase-polymerase chain reaction. The expression patterns of molecules implicated in signaling between stromal cells and monocytic osteoclast precursors were analyzed in both primary and fractionated GCTs. Finally, using array-based comparative genomic hybridization, neither GCTs nor the derived stromal cells demonstrated significant genomic gains or losses. These data raise questions regarding the role of RANKL in GCTs that may be relevant to the development of molecularly targeted therapeutics for this disease.
Publisher: MDPI AG
Date: 27-12-2021
DOI: 10.3390/JCM11010138
Abstract: Surgical management of displaced tibial plateau fracture (TPF) is often delayed due to accompanying soft tissue injuries sustained at the time of injury. The primary aim of this study was to assess the effect of time to surgery on fracture reduction in cases of TPF. The secondary aim was to assess the effect of preoperative demographics and residual articular step on Lysholm Scores and Knee Injury and Osteoarthritis Outcome Scores (KOOS) following fixation. Patients between 2006 and 2017, managed by a single surgeon, were prospectively enrolled in the study. Reduction of articular step, defined as mm, was assessed by a single blinded examiner. A total of 117 patients were enrolled, 52 with Schatzker II, 4 with Schatzker IV, and 61 with Schatzker VI fractures. Patients were followed up to a mean of 3.9 years. Analysis showed that the ability to achieve fracture reduction was negatively influenced by time to theatre, with the odds of achieving reduction decreasing 17% with each subsequent day post injury (p = 0.002). Furthermore, an increased time to theatre was associated with a reduced Lysholm score at one year (p = 0.01). The ability to achieve fracture reduction did not influence PROMs within the study period. We conclude that delay in surgical fixation negatively affects fracture reduction in TPF and may delay recovery. However, residual articular step does not necessarily influence PROMs over the mid-term.
Publisher: Hindawi Limited
Date: 17-01-2019
DOI: 10.1155/2019/9838167
Abstract: The process of osteoblast switching to alternative mesenchymal phenotypes is incompletely understood. In this study, we tested the ability of the osteoblast reosteocyte osteogenic cell line, MLO-A5, to also differentiate into either adipocytes or chondrocytes. MLO-A5 cells expressed a subset of skeletal stem cell markers, including Sca-1, CD44, CD73, CD146, and CD166. Confluent cultures of cells underwent differentiation within 3 days upon the addition of osteogenic medium. The same cultures were capable of undergoing adipogenic and chondrogenic differentiation under lineage-appropriate culture conditions, evidenced by lineage-specific gene expression analysis by real-time reverse-transcription-PCR, and by Oil Red O and alcian blue (pH 2.5) staining, respectively. Subcutaneous implantation of MLO-A5 cells in a gel foam into NOD SCID mice resulted in a woven bone-like structure containing embedded osteocytes and regions of cartilage-like tissue, which stained positive with both alcian blue (pH 2.5) and safranin O. Together, our findings show that MLO-A5 cells, despite being a strongly osteogenic cell line, exhibit characteristics of skeletal stem cells and display mesenchymal lineage plasticity in vitro and in vivo . These unique characteristics suggest that this cell line is a useful model with which to study aging and disease-related changes to the mesenchymal lineage composition of bone.
Publisher: Elsevier BV
Date: 10-2006
DOI: 10.1016/J.BIOMATERIALS.2006.05.054
Abstract: This study investigates receptor activator NF-kappaB (RANK), RANK ligand (RANKL) and tumour necrosis factor (TNFalpha), key factors regulating bone turnover, present in the tissues near peri-prosthetic osteolysis. Tissue was obtained from zones of peri-prosthetic osteolysis from 11 patients undergoing revision of total hip prostheses, analysed preoperatively by high-resolution spiral multislice CT using a metal artefact suppression protocol. Synovial tissue from 10 patients with osteoarthritis undergoing primary hip replacement was used as control tissue. Immunohistochemical analysis of formalin fixed tissue sections demonstrated that RANK, RANKL and TNFalpha were strongly expressed by large multinucleated cells containing polyethylene wear debris in revision tissues. Control tissue stained weakly for RANK, RANKL and TNFalpha. A strong statistical correlation (p<0.02) was found between the five parameters, volume of bone loss, polyethylene wear debris, RANK, RANKL and TNFalpha expression. Importantly, in vitro studies revealed that RANKL and TNFalpha synergise to increase the volume of bone resorbed, by more than seven fold, when compared to the effect of either cytokine treatment alone. This suggests that the interaction of TNFalpha and RANKL promotes osteoclast activity associated with polyethylene wear and therapies targeting TNF activity may be useful to treat peri-implant osteolysis.
Publisher: Elsevier BV
Date: 02-2017
Publisher: Elsevier BV
Date: 12-2008
DOI: 10.1016/J.MAM.2008.05.003
Abstract: The endocrine hormone, 1alpha,25-dihydroxyvitamin D(3) (1,25D) is an important regulator of calcium and phosphorus homeostasis. In this context, 1,25D is generally recognized as necessary for the maintenance of a healthy skeleton through its actions on the small intestine. In this review, we highlight the direct effects of 1,25D on the constituent cells of the bone, actions that are independent of effects on the intestine and kidney. We also consider the evidence that 25D levels, not 1,25D levels, correlate best with parameters of bone health, and that the bone itself is a site of metabolic conversion of 25D into 1,25D, by virtue of its expression of the 25-hydroxyvitamin D 1alpha-hydroxylase, CYP27B1. We review the evidence that at least osteoblasts and chondrocytes, and possibly also bone resorbing osteoclasts, are capable of such metabolic conversion, and therefore that these cells likely participate in autocrine and paracrine loops of vitamin D metabolism. We conclude that the skeleton is an intracrine organ for vitamin D metabolism, challenging the long-held notion that 1,25D is solely an endocrine hormone.
Publisher: Wiley
Date: 19-11-2013
DOI: 10.1002/JBMR.2003
Abstract: The osteocyte product sclerostin is emerging as an important paracrine regulator of bone mass. It has recently been shown that osteocyte production of receptor activator of NF-κB ligand (RANKL) is important in osteoclastic bone resorption, and we reported that exogenous treatment of osteocytes with sclerostin can increase RANKL-mediated osteoclast activity. There is good evidence that osteocytes can themselves liberate mineral from bone in a process known as osteocytic osteolysis. In the current study, we investigated sclerostin-stimulated mineral dissolution by human primary osteocyte-like cells (hOCy) and mouse MLO-Y4 cells. We found that sclerostin upregulated osteocyte expression of carbonic anhydrase 2 (CA2/Car2), cathepsin K (CTSK/Ctsk), and tartrate-resistant acid phosphatase (ACP5/Acp5). Because acidification of the extracellular matrix is a critical step in the release of mineral from bone, we further examined the regulation by sclerostin of CA2. Sclerostin stimulated CA2 mRNA and protein expression in hOCy and in MLO-Y4 cells. Sclerostin induced a decrease in intracellular pH (pHi) in both cell types as well as a decrease in extracellular pH (pHo) and the release of calcium ions from mineralized substrate. These effects were reversed in the co-presence of the carbonic anhydrase inhibitor, acetozolamide. Car2-siRNA knockdown in MLO-Y4 cells significantly inhibited the ability of sclerostin to both reduce the pHo and release calcium from a mineralized substrate. Knockdown in MLO-Y4 cells of each of the putative sclerostin receptors, Lrp4, Lrp5 and Lrp6, using siRNA, inhibited the sclerostin induction of Car2, Catk and Acp5 mRNA, as well as pHo and calcium release. Consistent with this activity of sclerostin resulting in osteocytic osteolysis, human trabecular bone s les treated ex vivo with recombinant human sclerostin for 7 days exhibited an increased osteocyte lacunar area, an effect that was reversed by the co-addition of acetozolamide. These findings suggest a new role for sclerostin in the regulation of perilacunar mineral by osteocytes.
Publisher: Elsevier BV
Date: 09-2015
DOI: 10.1016/J.MCE.2015.06.021
Abstract: Sclerostin, the SOST gene product, is a negative regulator of bone formation and a positive regulator of bone resorption. In this study, treatment of human primary osteoblasts, including cells differentiated to an osteocyte-like stage, with 1α,25-dihydroxyvitaminD3 (1,25D) resulted in the dose-dependent increased expression of SOST mRNA. A similar effect was observed in human trabecular bone s les cultured ex vivo, and in osteocyte-like cultures of differentiated SAOS2 cells. Treatment of SAOS2 cells with 1,25D resulted in the production and secretion of sclerostin protein. In silico analysis of the human SOST gene revealed a single putative DR3-type vitamin D response element (VDRE) at position -6216 bp upstream of the transcription start site (TSS). This sequence was confirmed to have strong VDRE activity by luciferase reporter assays and electrophoretic mobility shift analysis (EMSA). Sequence substitution in the VDR/RXR half-sites abolished VDRE reporter activity and binding of nuclear proteins. A 6.3 kb fragment of the human proximal SOST promoter demonstrated responsiveness to 1,25D. The addition of the evolutionary conserved region 5 (ECR5), a known bone specific enhancer region, ahead of the 6.3 kb fragment increased basal promoter activity but did not increase 1,25D responsiveness. Site-specific mutagenesis abolished the responsiveness of the 6.3 kb promoter to 1,25D. We conclude that 1,25D is a direct regulator of human SOST gene and sclerostin protein expression, extending the pathways of control of sclerostin expression. At least some of this responsiveness is mediated by the identified classical VDRE however the nature of the transcriptional regulation by 1,25D warrants further investigation.
Publisher: Elsevier BV
Date: 2015
DOI: 10.1016/J.MCE.2014.10.007
Abstract: Fibroblast growth factor-23 (FGF23), produced by osteocytes, is the key physiological regulator of phosphate homeostasis. Sepsis patients often experience transient hypophosphataemia, suggesting the regulation of FGF23 levels by pro-inflammatory factors. Here, we used the osteocyte-like cell line IDG-SW3 to investigate the effect of pro-inflammatory stimuli on FGF23 production. In differentiated IDG-SW3 cultures, basal Fgf23 mRNA was dose-dependently up-regulated by pro-inflammatory cytokines TNF, IL-1β and TWEAK, and bacterial LPS. Similar effects were observed in human bone s les. TNF- and IL-1β-induced Fgf23 expression was NF-κB-dependent. Conversely, mRNA encoding negative regulators of FGF23, Phex, Dmp1 and Enpp1, were suppressed by TNF, IL-1β, TWEAK and LPS, independent of NF-κβ signalling. Galnt3, the protein product of which protects intact FGF23 protein from furin/furin-like proprotein convertase cleavage, increased in response to these treatments. C-terminal FGF23 and intact FGF23 protein levels also increased, the latter only in the presence of Furin inhibitors, suggesting that enzymatic cleavage exerts critical control of active FGF23 secretion by osteocytes. Our results demonstrate in principle that pro-inflammatory stimuli are capable of increasing osteocyte secretion of FGF23, which may contribute to hypophosphataemia during sepsis and possibly other inflammatory conditions.
Publisher: Elsevier
Date: 2005
Publisher: Elsevier BV
Date: 11-2016
DOI: 10.1016/J.JSBMB.2015.12.006
Abstract: The osteocyte expressed gene SOST encodes sclerostin, a potent negative regulator of bone formation and inducer of bone resorption. We have recently demonstrated that the human SOST gene is positively regulated in response to 1α,25-dihydroxyvitamin D
Publisher: Elsevier BV
Date: 11-2016
DOI: 10.1016/J.JSBMB.2015.12.005
Abstract: The role of the vitamin D receptor (VDR) in maintaining skeletal health appears to be complex and dependent on the physiological context. Global Vdr deletion in a mouse model (Vdr
Publisher: Wiley
Date: 29-03-2005
DOI: 10.1002/JCP.20354
Abstract: Recent studies demonstrate roles for osteoprotegerin (OPG) in both skeletal and extra-skeletal tissues. Although its role in preventing osteoclast (OC) formation and activity is well documented, emerging evidence suggests a role of OPG in endothelial cell survival and the prevention of arterial calcification. In this communication, we show that vascular endothelial cells in situ, and human umbilical vein endothelial cells (HUVEC) in vitro, express abundant OPG. In HUVEC, OPG co-localizes with P-selectin and von Willebrand factor (vWF), within the Weibel-Palade bodies (WPB). Treatment of HUVEC with the pro-inflammatory cytokines, tumor necrosis factor (TNF)-alpha and IL-1beta, resulted in mobilization from the WPBs and subsequent secretion of OPG protein into the culture supernatant. Furthermore, TNF-alpha treatment of HUVEC resulted in a sustained increase in OPG mRNA levels and protein secretion over the 24-h treatment period. Reciprocal immunoprecipitation experiments revealed that while not associated with P-Selectin, OPG is physically complexed with vWF both within the WPB and following secretion from endothelial cells. Interestingly, this association was also identified in human peripheral blood plasma. In addition to its interaction with vWF, we show that OPG also binds with high avidity to the vWF reductase, thrombospondin (TSP-1), raising the intriguing possibility that OPG may provide a link between TSP-1 and vWF. In summary, the intracellular localization of OPG in HUVEC, in association with vWF, together with its rapid and sustained secretory response to inflammatory stimuli, strongly support a modulatory role in vascular injury, inflammation and hemostasis.
Publisher: Elsevier BV
Date: 07-2010
DOI: 10.1016/J.JSBMB.2010.03.048
Abstract: Current evidence suggests that levels of 25-(OH)vitamin D3 (25D), rather than 1alpha,25-(OH)2vitamin D3 (1,25D), directly affect bone mineralization and that the skeleton is a site of extra-renal synthesis of 1,25D. Since cells of the monocyte lineage can also metabolise 25D, it is possible that osteoclasts participate in local production of, and the response to, 1,25D. In this study, we investigated the effects of vitamin D metabolism on osteoclastogenesis using both the murine RAW 264.7 cell line and the human peripheral blood mononuclear cell (PBMC) models. PBMC-derived osteoclasts expressed cytoplasmic cyp27b1 and nuclear vdr proteins. PBMC expressed CYP27B1 mRNA, levels of which increased during RANKL induced differentiation into osteoclasts in both cell types. While 1,25D elicited a robust CYP24 transcriptional response in PBMC, the response to 25D was approximately 100-fold less at the concentrations used. Using media devoid of pre-existing vitamin D metabolites, we found that 25D was metabolised by RAW 264.7 cells to 1,25D and resulted in significant elevation in the numbers of TRAP-positive, multinucleated osteoclasts when present in the cultures for the first 3-5 days. These results suggest that vitamin D metabolism by osteoclast lineage cells is an important regulator of osteoclast formation.
Publisher: Wiley
Date: 10-08-2017
DOI: 10.1002/CAM4.1115
Publisher: Elsevier BV
Date: 11-2016
DOI: 10.1016/J.JSBMB.2016.03.004
Abstract: Clinical and animal data indicate that serum 25-hydroxyvitamin D
Publisher: Baishideng Publishing Group Inc.
Date: 2011
Publisher: Springer Science and Business Media LLC
Date: 05-12-2008
DOI: 10.1007/S00774-008-0018-6
Abstract: Osteoclasts are the unique cell type capable of resorbing bone. The discovery of the TNF-ligand family member, RANKL, has allowed more reliable study of these important cells. The mouse monocytic cell line, RAW 264.7, has been shown to readily differentiate into osteoclasts upon exposure to recombinant RANKL. Unlike primary osteoclast precursors, there is no requirement for the addition of macrophage colony stimulating factor (M-CSF). However, to date, their differentiation has always been studied in the context of added foetal calf serum (FCS). FCS is a complex and largely undefined mixture of growth factors and matrix proteins, and varies between batches. For this reason, osteoclastogenesis would ideally be studied in the context of a defined, serum-free medium. RAW 264.7 cells were cultured in serum-replete alpha-MEM or serum-deprived medium (SDM) shown previously to support the growth of human osteoclasts in a co-culture with normal osteoblasts. In SDM, in the presence of recombinant RANKL, RAW 264.7 cells readily differentiated into tartrate resistant acid phosphatase (TRAP) positive multinucleated osteoclast-like cells, a process that was enhanced with the addition of 1alpha,25-dihydroxyvitamin D(3) (1,25D). While the osteoclasts grown in SDM were smaller in size compared with those derived in serum-replete media, their resorptive capacity was significantly increased as indicated by a twofold increase in average resorption pit size. In conclusion, we describe a defined model for studying osteoclast differentiation and activity in the absence of serum, which will be ideal for studying the role of agonistic and antagonistic molecules in this process.
Publisher: Elsevier BV
Date: 06-2015
DOI: 10.1016/J.INJURY.2015.02.019
Abstract: Osteoporotic tibial plateau fractures (TPFs) are difficult to treat with either open reduction internal fixation (ORIF) or acute total knee arthroplasty (TKA). They have high complication rates, poor outcomes and often fail in the short- to mid-term. We investigated the use of impaction bone grafting (IBG) as an adjunct to stabilise the fracture in a cohort of osteoporotic TPFs. Nine consecutive osteoporotic TPFs were surgically stabilised with ORIF augmented with IBG or with IBG alone (one pure depression fracture) using on average allograft from 2 femoral heads/case (range 1-4 heads or 25-100 cm(3)). The median bone mineral density T-score of the patients was -2.9 (-2.5 to -4.5). All patients were mobilised weight-bearing as tolerated immediately after surgery and had regular follow-up to a minimum of 2 years where functional scores were taken and gait was assessed. Fracture reduction was assessed on plain radiographs and computed tomography (CT) scans maintenance of fracture reduction was monitored using plain radiographs, CT and radiostereometric analysis (RSA). Bone graft remodelling was assessed by comparison of immediate post-operative CT scans with scans at a minimum of 1 year. All surgeries were uneventful. All patients progressed to full weight bearing within 6 weeks of surgery and regained a normal gait by 3 months. Seven fractures healed with a cranio-caudal migration of less than 3mm (range 0-2.6mm using RSA and 0-2mm using CT). Two fractures had an isolated posterolateral fragment depression of 13.5mm and 9 mm, respectively, which did not affect the overall joint alignment or clinical outcomes at short-term follow-up. At latest CT follow-up, on average 51% of the graft area (range 36-70%) had remodelled into new host bone. Impaction bone grafting shows promising results as an adjunct to the surgical stabilisation of osteoporotic TPFs. In this case series the technique provided enough fracture stability for patients to mobilise weight-bearing as tolerated immediately after surgery and achieve full weight-bearing by the sixth postoperative week. There was no failure of fixation and 7 of the 9 cases healed with minimal fracture displacement.
Publisher: Elsevier BV
Date: 06-2016
DOI: 10.1016/J.COPH.2016.02.003
Abstract: Bone remodelling is an essential process for shaping and maintaining bone mass in the mature skeleton. During our lifetime bone is constantly being removed by osteoclasts and new bone is formed by osteoblasts. The activities of osteoclasts and osteoblasts must be regulated under a strict balance to ensure that bone homeostasis is maintained. Osteocytes, which form an extensive, multi-functional syncytium throughout the bone, are increasingly considered to be the cells that maintain this balance. Current research is elucidating key signalling pathways by which the osteocyte exerts control over the other cell types in bone and over its own activities, and potential ways in which these pathways may be exploited therapeutically.
Publisher: Royal Society of Chemistry (RSC)
Date: 2015
DOI: 10.1039/C5TB00125K
Abstract: Release behavior and cancer toxicity of different forms of Se loaded into nanoporous AAO were studied.
Publisher: Informa UK Limited
Date: 09-2012
DOI: 10.2147/IJN.S33655
Publisher: MDPI AG
Date: 12-04-2018
DOI: 10.3390/MPS1020014
Publisher: The American Association of Immunologists
Date: 15-08-2006
DOI: 10.4049/JIMMUNOL.177.4.2610
Abstract: TNF-like weak inducer of apoptosis (TWEAK) is a TNF family member with pleiotropic effects on a variety of cell types, one of which is the induction of proinflammatory cytokines by synovial fibroblasts derived from rheumatoid arthritis (RA) patients. In this study, we report that the serum TWEAK level was dramatically elevated during mouse collagen-induced arthritis (CIA) and blocking TWEAK by a neutralizing mAb significantly reduced the clinical severity of CIA. Histological analyses also revealed that TWEAK inhibition diminished joint inflammation, synovial angiogenesis, as well as cartilage and bone erosion. Anti-TWEAK treatment proved efficacious when administered just before the disease onset but not during the priming phase of CIA. Consistent with this, TWEAK inhibition did not affect either cellular or humoral responses to collagen. In contrast, TWEAK inhibition significantly reduced serum levels of a panel of arthritogenic mediators, including chemokines such as MIP-1β (CCL-4), lymphotactin (XCL-1), IFN-γ-inducible protein 10 (IP-10) (CXCL-10), MCP-1 (CCL-2), and RANTES (CCL-5), as well as the matrix metalloprotease-9. Exploring the possible role of the TWEAK/Fn14 pathway in human RA pathogenesis, we showed that TWEAK can target human primary chondrocytes and osteoblast-like cells, in addition to synovial fibroblasts. We further demonstrated that TWEAK induced the production of matrix metalloproteases in human chondrocytes and potently inhibited chondrogenesis and osteogenesis using in vitro models. These results provide evidence for a novel cytokine pathway that contributes to joint tissue inflammation, angiogenesis, and damage, as well as may inhibit endogenous repair, suggesting that TWEAK may be a new therapeutic target for human RA.
Publisher: Elsevier BV
Date: 07-2013
DOI: 10.1016/J.JSBMB.2012.08.008
Abstract: A current controversial question related to vitamin D supplementation is what level of serum 25-hydroxyvitamin D3 (25(OH)D3) is required to reduce the incidence of osteoporotic fractures. The reasoning behind vitamin D supplementation has been mostly derived from the role of vitamin D to promote intestinal calcium absorption and reduce bone resorption. While minimum 25(OH)D3 levels of 20nmol/L are required for sufficient intestinal calcium absorption to prevent osteomalacia, the mechanistic details of how higher 25(OH)D3 levels, well beyond that required for optimal calcium absorption, are able to prevent fractures and increase bone mineral density is unclear. Substantial evidence has arisen over the past decade that conversion of 25(OH)D3 to 1,25(OH)2D3via the 1-alpha hydroxylase (CYP27B1) enzyme in osteoblasts, osteocytes, chondrocytes and osteoclasts regulates processes such as cell proliferation, maturation and mineralization as well as bone resorption, which are all dependent on the presence the of the vitamin D receptor (VDR). We and others have also shown that increased vitamin D activity in mature osteoblasts by increasing levels of VDR or CYP27B1 leads to improved bone mineral volume using two separate transgenic mouse models. While questions remain regarding activities of vitamin D in bone to influence the anabolic and catabolic processes, the biological importance of vitamin D activity within the bone is unquestioned. However, a clearer understanding of the varied mechanisms by which vitamin D directly and indirectly influences mineral bone status are required to support evidence-based recommendations for vitamin D supplementation to reduce the risk of fractures. This article is part of a Special Issue entitled 'Vitamin D workshop'.
Publisher: Spandidos Publications
Date: 27-05-2014
Publisher: Elsevier BV
Date: 09-1999
DOI: 10.1016/S8756-3282(99)00176-3
Abstract: Interleukin-1 (IL-1) has been shown to promote osteoclast (OC) differentiation, in addition to acting as a survival factor for mature osteoclasts. In this study, we investigate the expression of IL-1 during human osteoclast formation, taking advantage of a recently reported in vitro culture system that generates human OC from precursors in the peripheral blood mononuclear cell (PBMC) fraction, in the presence of murine stromal cells. This system enabled us to use species-specific probes and immunoassays to determine the respective cytokine contributions of the stromal cell and hemopoietic cell populations. Formation of functional osteoclasts occurred in cocultures of human PBMC and ST-2 cells for up to 21 days in the presence of 1alpha,25(OH)2-vitamin D3, dexamethasone, and recombinant human macrophage colony-stimulating factor (rhM-CSF). Total RNA was prepared at intervals during the cocultures and reverse transcriptase-polymerase chain reaction (RT-PCR) was performed using primers designed to lify specifically the mRNA species corresponding to the respective murine or human IL-1alpha and IL-1beta isoforms. Using human-specific primers, it was found that the hemopoietic cell component expressed both IL-1alpha and IL-1beta mRNA. Specific measurement of secreted human IL-1beta protein showed greatly augmented levels in coculture compared with hemopoietic cells grown in the absence of ST-2 cells, consistent with the known signaling from stromal cells to hemopoietic cells during osteoclastogenesis. Specific detection of mouse mRNA products showed that the ST-2 stromal cells in the coculture also expressed mRNA corresponding to IL-1alpha and IL-1beta. The expression of both mouse and human IL-1 mRNA was found to decline over the course of the coculture, although the level of IL-1alpha mRNA relative to IL-1beta mRNA remained constant, indicating that the two isoforms were coregulated in both cell populations under these conditions. Importantly, the hemopoietic cells were found to influence strongly the IL-1 mRNA levels in ST-2 cells, such that mouse IL-1alpha and IL-1beta mRNA levels were greatly enhanced in coculture, compared with ST-2 cells alone. Secreted mouse IL-1beta protein was upregulated in coculture in parallel with mRNA levels. However, the absolute levels of mouse IL-1beta achieved were more than 20-fold lower than the human IL-1beta levels. Prostaglandin estradiol (PGE2) levels were measured and found to be greatly increased in the coculture compared with ST-2 cells or hemopoietic cells alone, consistent with evidence that IL-1 action in osteoclastogenesis is mediated by PGE2. These results provide novel evidence that bidirectional signaling between stromal and hemopoietic cells may be important in the generation of human osteoclasts.
Publisher: The Endocrine Society
Date: 02-07-2015
DOI: 10.1210/EN.2015-1345
Abstract: During lactation, the large transfer of calcium from the mother to the milk is primarily sourced from the maternal skeleton. To determine whether the calcitonin receptor (CTR) plays a physiological role to protect the skeleton from excessive resorption during lactation, we assessed the maternal skeleton of global CTR knockout (CTRKO) and littermate control mice at the end of lactation (postnatal day 21). Micro-computed tomography analyses showed no effect on trabecular or cortical bone in the distal femur and L1 vertebra of maternal global CTR deletion at the end of lactation in global CTRKO mice compared with that in control mice. Bone resorption, as assessed by osteoclast number and activity at the end of lactation, was unaffected by maternal CTR deletion. Cathepsin K, carbonic anhydrase 2, matrix metalloproteinase 13, and receptor activator of nuclear factor-κB ligand mRNA levels, however, were markedly elevated by 3- to 6.5-fold in whole bone of lactating global CTRKO females. Because these genes have been shown to be up-regulated in osteocytes during lactation when osteocytes resorb their surrounding bone matrix, together with their reported expression of the CTR, we determined the osteocyte lacunar area in cortical bone. After lactation, the top 20% of osteocyte lacunar area in global CTRKO mice was 10% larger than the top 20% in control mice. These data are consistent with an increased osteocytic osteolysis in global CTRKO mice during lactation, which is further supported by the increased serum calcium observed in global CTRKO mice after lactation. These results provide evidence for a physiological role for the CTR to protect the maternal skeleton during lactation by a direct action on osteocytes to inhibit osteolysis.
Publisher: American Society of Hematology
Date: 06-2008
No related grants have been discovered for Gerald Atkins.