Supplementary Figure 2 from Loss of Osteoclasts Contributes to Development of Osteosarcoma Pulmonary Metastases

Supplementary Methods, Figure Legends 1-2 from Loss of Osteoclasts Contributes to Development of Osteosarcoma Pulmonary Metastases

Supplementary Figure 1 from Loss of Osteoclasts Contributes to Development of Osteosarcoma Pulmonary Metastases

Event Abstract Back to Event Tocopherol and ascorbic acid protect the mechanical properties of bone allograft during gamma irradiation Athena R. Brunt1, 2, Huynh Nguyen1, 3, A I. Cassady1, 2, Nigel A. Morrison1, 2, David A. Morgan3, 4 and Mark R. Forwood1, 2 1 Griffith University, School of Medical Science, Australia 2 Menzies Health Institute Queensland, Australia 3 The Queensland Tissue Bank, Australia 4 The University of Queensland, Australia Introduction: Despite advances in biomaterials, bone tissues remain necessary allograft materials in a wide range of surgical procedures. Total joint arthroplasty is the most commonly performed surgical procedure in orthopaedics. In revision joint arthroplasty, allografts unite biologically with bone and achieve mechanical properties that provide good long-term clinical outcomes[1]. Following total joint replacement, the strength of bone tissue used for structural allografts is a key factor in long-term success. Gamma irradiation is used to terminally sterilise bone allografts, but this damages the mechanical properties of bone[2],[3] and hence adversely impacts clinical outcomes of revision joint arthroplasty. We have established that tocopherol and ascorbic acid provide potent radioprotection of cortical bone allograft[4]. However, morselized cancellous bone (MCB) is the primary allograft material employed in revision joint arthroplasty. This study sought to determine if tocopherol and ascorbic acid can preserve the mechanical properties of morselized cancellous bone during gamma irradiation. Materials and Methods: Cancellous bone was collected from ten paired femora from the Queensland Tissue Bank (QTB), processed and milled according to standard QBSB protocols. Specimens were infused with a mixture of tocopherol and ascorbic acid, and saline (controls), for 4 h at room temperature. Processed and vitamin-infused samples were equally divided into five groups of varying irradiation levels (0, 10, 15, 25 and 50 kGy), and irradiated under frozen conditions. Two methods of confined compaction testing were carried out to characterise the mechanical responses of MCB samples using an Instron 5655A materials testing machine. MCB was packed into a cylindrical test chamber in five layers, of one gram each. Each layer underwent minimal preconditioning by free-dropping the piston on top of the MCB. The first test modelled cyclic loading by subjecting the MCB to 150 cycles of 100 N at 400 N/min[5]. The second test involved the displacement-controlled compaction of MCB in steps of 0.5 mm at 0.5 mm/min (up to 14.5 mm)[6]. Mechanical properties were calculated from load-displacement curves, and normalised according to dimensions. Results and Discussion: The mechanical responses of MCB treated with tocopherol and ascorbic acid were significantly enhanced compared to the controls. Most notably, the untreated MCB was significantly stiffer compared to the MCB treated with tocopherol and ascorbic acid (p < 0.005). Gamma irradiation has been shown to increase stiffness of morselized bone during compaction[6], affirming our findings that treatment of MCB with tocopherol and ascorbic acid protects the mechanical properties of MCB allograft. Conclusion: Our findings provide evidence to support the use of the combination of antioxidants, tocopherol and ascorbic acid, as a promising radioprotectant to improve the mechanical performance of bone allograft, and thereby promote superior clinical outcomes for patients. This research has in part been made possible through the award of an Enid Dowling Foundation Bursary. I thank the Enid Dowling Foundation for their generosityReferences:[1] D.A.F. Morgan and D.W. Robinson, "Allograft bone in total hip arthroplasty" Current Opinions in Orthopaedics. Vol. 6, 1995.[2] H. Nguyen, D.A.F. Morgan and M.R. Forwood, "Sterilization of allograft bone: effects of gamma irradiation on allograft biology and biomechanics" Cell and Tissue Banking. Vol. 8, 2007.[3] H. Nguyen, A.I. Cassady, M.B. Bennett, E. Gineyts, A. Wu, D.A.F. Morgan and M.R. Forwood, "Reducing the radiation sterilization dose improves mechanical and biological quality while retaining sterility assurance levels of bone allografts" Bone. Vol. 57, 2013.[4] A.R. Brunt, H. Nguyen, N.A. Morrison, D.A.F. Morgan, M.R. Forwood, "Tocopherol and ascorbic acid protect the structural properties of bone allograft during gamma irradiation" 5th International Symposia on Surface and Interface Biomaterials and The 24th Annual Conference of the Australasian Society for Biomaterials and Tissue Engineering, 7-10th April 2015, Sydney, Australia.[5] A.T.M. Phillips, Pankaj, D.T. Brown, T.Z. Oram, C.R. Howie, A.S. Usmani, "The elastic properties of morsellised cortico-cancellous bone graft are dependent on its prior loading" Journal of Biomechanics. Vol. 39, 2006.[6] A.M. Butler, D.A.F. Morgan, R. Verheul, W.R. Walsh, "Mechanical properties of gamma irradiated morselized bone during compaction" Biomaterials. Vol. 26, 2005. Keywords: joint replacement, biomaterial, Clinical relevance, Bone graft Conference: 10th World Biomaterials Congress, Montréal, Canada, 17 May - 22 May, 2016. Presentation Type: Poster Topic: Biomaterials in musculoskeletal orthopeadics and tissues Citation: Brunt AR, Nguyen H, Cassady AI, Morrison NA, Morgan DA and Forwood MR (2016). Tocopherol and ascorbic acid protect the mechanical properties of bone allograft during gamma irradiation. Front. Bioeng. Biotechnol. Conference Abstract: 10th World Biomaterials Congress. doi: 10.3389/conf.FBIOE.2016.01.03020 Copyright: The abstracts in this collection have not been subject to any Frontiers peer review or checks, and are not endorsed by Frontiers. They are made available through the Frontiers publishing platform as a service to conference organizers and presenters. The copyright in the individual abstracts is owned by the author of each abstract or his/her employer unless otherwise stated. Each abstract, as well as the collection of abstracts, are published under a Creative Commons CC-BY 4.0 (attribution) licence (https://creativecommons.org/licenses/by/4.0/) and may thus be reproduced, translated, adapted and be the subject of derivative works provided the authors and Frontiers are attributed. For Frontiers’ terms and conditions please see https://www.frontiersin.org/legal/terms-and-conditions. Received: 27 Mar 2016; Published Online: 30 Mar 2016. Login Required This action requires you to be registered with Frontiers and logged in. To register or login click here. Abstract Info Abstract The Authors in Frontiers Athena R Brunt Huynh Nguyen A I Cassady Nigel A Morrison David A Morgan Mark R Forwood Google Athena R Brunt Huynh Nguyen A I Cassady Nigel A Morrison David A Morgan Mark R Forwood Google Scholar Athena R Brunt Huynh Nguyen A I Cassady Nigel A Morrison David A Morgan Mark R Forwood PubMed Athena R Brunt Huynh Nguyen A I Cassady Nigel A Morrison David A Morgan Mark R Forwood Related Article in Frontiers Google Scholar PubMed Abstract Close Back to top Javascript is disabled. Please enable Javascript in your browser settings in order to see all the content on this page.

Poly(tetrafluoroethylene) (PTFE), a fully fluorinated linear thermoplastic polymer, and in particular the porous form expanded PTFE (ePTFE) has found widespread use in biomaterials application due to its properties of high toughness, non-adhesiveness and hydrophobicity. While it performs ideally for many applications, some challenges have been identified for its use in small diameter vascular grafts and as a tissue space-filler for cosmetic reconstructions where the implant interfaces with bone. For these applications modification of the surface of ePTFE has been investigated as a means to enhance its performance. This review will focus on the applications listed above and will detail methods of evaluating the biological response, methods used to enhance the surface properties of ePTFE, and how the modified materials have performed in their intended applications. This review will focus on work published from 2004 onwards. (c) 2014 Wiley Periodicals, Inc. J. Appl. Polym. Sci. 2014, 131, 40533.

Background: Bone allografts carry a risk of infection, so terminal sterilization by gamma irradiation at 25 kGy is recommended; but is deleterious to bone quality. Contemporary bone banking significantly reduces initial allograft bioburden, questioning the need to sterilize at 25 kGy.Methods: We inoculated allograft bone with Staphylococcus epidermidis and Bacillus pumilus, then exposed them to gamma irradiation at 0, 5, 10, 15,20 and 25 kGy. Mechanical and biological properties of allografts were also assessed. Our aim was to determine an optimal dose that achieves sterility assurance while minimizing deleterious effects on allograft tissue.Results: 20-25 kGy eliminated both organisms at concentrations from 10(1) to 10(3) CFU, while 10-15 kGy sterilized bone samples to a bioburden concentration of 10(2) CFU. Irradiation did not generate pro-inflammatory bone surfaces, as evidenced by macrophage activation, nor did it affect attachment or proliferation of osteoblasts. At doses >= 10 kGy, the toughness of cortical bone was reduced (P < 0.05), and attachment and fusion of osteoclasts onto irradiated bone declined at 20 and 25 kGy (P < 0.05). There was no change in collagen cross-links, but a significant dose-response increase in denatured collagen (P < 0.05).Conclusions: Our mechanical and cell biological data converge on 15 kGy as a threshold for radiation sterilization of bone allografts. Between 5 and 15 kGy, bone banks can undertake validation that provides allografts with an acceptable sterility assurance level, improving their strength and biocompatibility significantly.Clinical relevance: The application of radiation sterilization doses between 5 and 15 kGy will improve bone allograft mechanical performance and promote integration, while retaining sterility assurance levels. Improved quality of allograft bone will promote superior clinical outcomes. (C) 2013 Elsevier Inc. All rights reserved.

A series of surface-modified expanded poly(tetrafluoroethylene) membranes showed varied levels of in vitro macrophage proinflammatory response. Membranes containing a mixture of phosphate and hydroxyl groups (as determined by X-ray photoelectron spectroscopy analysis) stimulate greater macrophage activation than samples containing a mixture of phosphate and carboxylic acid segments. The types of proteins that adsorbed irreversibly from serum onto the two samples with the highest and lowest cellular response were investigated using surface-matrix-assisted laser desorption ionisation time-of-flight mass spectrometry. Distinct differences in the number and type of proteins that adsorbed were observed between these samples. A correlation was found between the main protein components adsorbed onto the surfaces and the resulting in vitro proinflammatory response. This study strongly supports the hypothesis that the cellular response is not controlled directly by surface properties but is mediated by specific protein adsorption events. This in turn highlights the importance of better understanding and controlling the properties of intelligent surface-modified biomaterials. (c) 2012 Wiley Periodicals, Inc. J Biomed Mater Res Part A, 2013.

Bone remodeling is a central event in the maintenance of skeletal tissue, and involves cycles of resorption followed by the formation of bone tissue. The activity of osteoclasts and osteoblasts during these cycles is tightly regulated by systemic and local factors coupling the action of these cells. Tartrate-resistant acid phosphatase (TRAP) is predominantly expressed in bone by osteoclasts but has also been detected in osteoblasts and osteocytes. Moreover, TRAP can stimulate the differentiation of mesenchymal lineage cells, i.e. progenitors of osteoblasts and adipocytes. In order to further explore the effects of TRAP on bone turnover, the structural and molecular phenotypes of osteoclasts and osteoblasts were assessed in TRAP-overexpressing transgenic mice. Transgenic mice of both sexes display increased cortical bone mineral content and density, which cannot be accounted for by decreased bone resorption since osteoclast numbers and resorptive activity do not differ from wild-type mice. Examination of the osteoblast phenotype revealed that markers of bone formation, i.e. procollagen type I N-terminal propeptides, and osteoblast lineage markers as well as the TRAP 1B mRNA transcript are increased in TRAP-overexpressing mice. Expression of the osteoclast-selective TRAP 1C mRNA is not increased in TRAP transgenic mice. Elevated expression of TRAP mRNA and protein were detected in osteoblasts, osteocytes and in the bone matrix of TRAP transgenic mice, suggesting that TRAP overexpression in osteoblast lineage cells is associated with increased cortical bone mineral content and density. The data presented here support the hypothesis that TRAP overexpression in the osteoblastic cell lineage stimulates the differentiation and/or activation of these cells.

It is not known if the radiation sterilisation dose (RSD) of 25 kGy affects mechanical properties and biocompability of allograft bone by alteration of collagen triple helix or cross-links. Our aim was to investigate the mechanical and biological performance, cross-links and degraded collagen content of irradiated bone allografts. Human femoral shafts were sectioned into cortical bone beams (40 × 4 × 2 mm) and irradiated at 0, 5, 10, 15, 20, and 25 kGy for three-point bending tests. Corresponding cortical bone slices were used for in vitro determination of macrophage activation, osteoblast proliferation and attachment, and osteoclast formation and fusion. Subsequently, irradiated cortical bone samples were hydrolised for determination of pyridinoline (PYD), deoxypyridinoline (DPD), and pentosidine (PEN) by high performance liquid chromatography (HPLC) and collagen degradation by the alpha chymotrypsin (i j CT) method. Irradiation up to 25 kGy did not affect the elastic properties of cortical bone, but the modulus of toughness was decreased from 87% to 74% of controls when the gamma dose increased from 15 to 25 kGy. Macrophages activation, the proliferation and attachment of osteoblasts on irradiated bone was not affected. Osteoclast formation and fusion were less than 40% of controls when cultured on bone irradiated at 25 kGy, and 80% at 15 kGy. Increasing radiation dose did not significantly alter the content of PYR, DPD or PEN but increased the content of denatured collagen. Cortical allografts fragility increases at doses above 15 kGy. Decreased osteoclast viability at these doses suggests a reduction in the capacity for bone remodelling. These changes were not correlated with alterations in collagen cross-links but in degradation to the collagen secondary structure as evidenced by increased content of denatured collagen.

nloaded conducted a transcriptomic screen of osteosarcoma (OS) biopsies and found that expression of last-specific tartrate-resistant acid phosphatase 5 (ACP5/TRAP) is significantly downregulated in OS red with nonmalignant bone (P < 0.0001). Moreover, lesions from OS patients with pulmonary metasad 2-fold less ACP5/TRAP expression (P < 0.018) than lesions from patients without metastases. In ad, we found a direct correlation (P = 0.0166) between ACP5/TRAP expression and time to metastasis. ore, we examined whether metastasis-competent (MC) OS cells could induce loss of ACP5 osteoclasts ntribute to metastasis. We found that MC OS cell lines can inhibit osteoclastogenesis in vitro and . In addition, osteoclasts can inhibit the migration of MC OS cells in vitro. Finally, ablation of osteoclasts oledronic acid increases the number of metastatic lung lesions in an orthotopic OS model, whereas rant treatment increases osteoclast numbers and reduces metastatic lesions. These data indicate that fulvest the metastatic potential of OS is determined early in tumor development and that loss of osteoclasts in the primary lesion enhances OS metastasis. Cancer Res; 70(18); 7063–72. ©2010 AACR.

AbstractWe conducted a transcriptomic screen of osteosarcoma (OS) biopsies and found that expression of osteoclast-specific tartrate-resistant acid phosphatase 5 (ACP5/TRAP) is significantly downregulated in OS compared with nonmalignant bone (P < 0.0001). Moreover, lesions from OS patients with pulmonary metastases had 2-fold less ACP5/TRAP expression (P < 0.018) than lesions from patients without metastases. In addition, we found a direct correlation (P = 0.0166) between ACP5/TRAP expression and time to metastasis. Therefore, we examined whether metastasis-competent (MC) OS cells could induce loss of ACP5+ osteoclasts and contribute to metastasis. We found that MC OS cell lines can inhibit osteoclastogenesis in vitro and in vivo. In addition, osteoclasts can inhibit the migration of MC OS cells in vitro. Finally, ablation of osteoclasts with zoledronic acid increases the number of metastatic lung lesions in an orthotopic OS model, whereas fulvestrant treatment increases osteoclast numbers and reduces metastatic lesions. These data indicate that the metastatic potential of OS is determined early in tumor development and that loss of osteoclasts in the primary lesion enhances OS metastasis. Cancer Res; 70(18); 7063–72. ©2010 AACR.

Information about the development and function of human macrophage lineage populations, such as osteoclasts, is limited because of the lack of defined in vitro systems for their large-scale generation. Two M-CSF-containing cytokine cocktails were found under serum-free conditions to expand dramatically and to differentiate over time human CD34(+) hemopoietic stem cells into nonadherent and adherent macrophage populations. These populations exhibited increasing degrees of maturity over a 3-week period characterized by morphology, surface marker expression (CD11b, CD86, CD64, CD14, and c-Fms), phagocytic function, and gene-expression profiling using quantitative PCR and microarray analysis (principal component analysis, k-means clustering, and gene ontology classification). As assessed by the last criterion, the adherent population obtained at 3 weeks from the one protocol tested had high similarity to the well-studied peripheral blood monocyte-derived macrophages. The one population tested could be induced to differentiate into osteoclasts in the presence of M-CSF and receptor activator of NF-kappa B ligand, as judged by morphology, gene expression, and bone-resorbing ability. In addition to the large numbers of macrophage lineage cells able to be produced, this replicating system may be suitable for the molecular analysis of macrophage lineage commitment and progression and for gene targeting and delivery. J. Leukoc. Biol. 85: 766-778; 2009.

Effective bone biomaterials provide structural support for bone regeneration and elicit minimal inflammatory or toxic effects in vivo. Poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV) is a bacterially derived biodegradable polymer that possesses suitable mechanical strength for use as a bone biomaterial and has a slow rate of degradation in biological environments. Our previous in vitro study showed that many PHBV preparations are contaminated with bacterial lipopolysaccharide, and we developed a purification procedure to substantially remove it. Here, we have evaluated the in vivo biocompatibility of PHBV purified by H(2)O(2) treatment and solvent extraction. We utilized a murine tibial defect model consisting of a hole drilled through the diameter of the tibial diaphysis into which nonporous cylindrical plugs of purified PHBV were implanted. The animals were sacrificed at 1 week and 4 weeks postsurgery, and tibiae were examined using histological staining. The PHBV implant induced a mild inflammatory response 1 week after injury, which persisted for 4 weeks. Granuloma type tissues formed only when the implant protruded into the overlaying tissue. Woven bone formation occurred adjacent to the implant, which gave rise to lamellar bone and stabilized the implant indicating that the PHBV did not affect this process. Our data validated the murine defect model and indicate that solid PHBV induces a mild tissue reaction with bone deposition adjacent to the implant with no fibrous tissue present at 4 weeks post surgery.

Tartrate-resistant acid phosphatases (TRAcPs), also known as purple acid phosphatases (PAPs), are a family of binuclear metallohydrolases that have been identified in plants, animals and fungi. The human enzyme is a major histochemical marker for the diagnosis of bone-related diseases. TRAcPs can occur as a small form possessing only the ~35 kDa catalytic domain, or a larger ~55 kDa form possessing both a catalytic domain and an additional N-terminal domain of unknown function. Due to its role in bone resorption the 35 kDa TRAcP has become a promising target for the development of anti-osteoporotic chemotherapeutics.

Microphthalmia transcription factor (MITF) regulates bone homeostasis by inducing expression of critical genes associated with osteoclast function. Gpnmb is a macrophage-enriched gene that has also been shown to be expressed in osteoblasts. Here, we have shown gpnmb to be highly induced in maturing murine osteoclasts. Microarray expression profile analysis identified gpnmb as a potential target of MITF in RAW264.7 cells, subclone C4 (RAW/C4), that overexpress this transcription factor. Electrophoretic mobility shift assays identified a MITF-binding site (M-box) in the gpnmb promoter that is conserved in different mammalian species. Anti-MITF antibody supershifted the DNA–MITF complex for the promoter site while MITF binding was abolished by mutation of this site. The gpnmb promoter was transactivated by co-expression of MITF in reporter gene assays while mutation of the gpnmb M-box prevented MITF transactivation. The induction of gpnmb expression during osteoclastogenesis was shown to exhibit similar kinetics to the known MITF targets, acp5 and clcn7. GPNMB expressed in RAW/C4 cells exhibited distinct subcellular distribution at different stages of osteoclast differentiation. At days 5 and 7, GPNMB protein co-localised with the osteoclast/macrophage lysosomal/endocytic marker MAC-3/LAMP-2, suggesting that GPNMB resides in the endocytic pathway of mature macrophages and is possibly targeted to the plasma membrane of bone-resorbing osteoclasts. The inclusion of gpnmb in the MITF regulon suggests a role for GPNMB in mature osteoclast function.

Background Obesity is associated with macrophage infiltration of adipose tissue, which may link adipose inflammation to insulin resistance. However, the impact of inflammatory cells in the pathophysiology of obesity remains unclear. Tartrate resistant acid phosphatase (TRAP) is an enzyme expressed by subsets of macrophages and osteoclasts that exists either as an enzymatically inactive monomer or as an active, proteolytically processed dimer. Principal Findings Using mice over expressing TRAP, we show that over-expression of monomeric, but not the dimeric form in adipose tissue leads to early onset spontaneous hyperplastic obesity i.e. many small fat cells. In vitro, recombinant monomeric, but not proteolytically processed TRAP induced proliferation and differentiation of mouse and human adipocyte precursor cells. In humans, monomeric TRAP was highly expressed in the adipose tissue of obese individuals. In both the mouse model and in the obese humans the source of TRAP in adipose tissue was macrophages. In addition, the obese TRAP over expressing mice exhibited signs of a low-grade inflammatory reaction in adipose tissue without evidence of abnormal adipocyte lipolysis, lipogenesis or insulin sensitivity. Conclusion Monomeric TRAP, most likely secreted from adipose tissue macrophages, induces hyperplastic obesity with normal adipocyte lipid metabolism and insulin sensitivity.