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PubMed | Yamanashi University, Pathology, Vanderbilt University and Center for Bone Biology
Type: Journal Article | Journal: Molecular cancer research : MCR | Year: 2014

Osteosarcoma is the most common primary bone malignancy and accounts for more than half of primary skeletal malignancies in children and young adults. Although vascular endothelial growth factor (VEGF) expression in osteosarcoma has been associated with poor outcome, its role in the pathogenesis of osteosarcoma remains controversial. Here, VEGF and VEGFR1 expression in both human and murine osteosarcoma cells associated with increasing malignant potential. Autocrine VEGF/VEGFR1 signaling resulted in constitutive activation of VEGFR1 in highly aggressive osteosarcoma cells. In addition, survival and proliferation of highly aggressive osteosarcoma cells was dependent on autocrine VEGF/R1 signaling in vitro. The effect of VEGFR1 expression on in vivo tumor growth and angiogenesis was evaluated by immunoselecting subpopulations of osteosarcoma cells that express high or low levels of VEGFR1. Cell enriched for high VEGFR1 expression showed increased VEGF production, tumor growth, tumor angiogenesis, and osteolysis in vivo. In addition, it was demonstrated that VEGF and VEGFR1 are coexpressed by a subset of tumor cells in human osteosarcoma, similar to what was observed in the murine osteosarcoma cells. These results suggest that autocrine VEGF/VEGFR1 signaling in a subpopulation of tumor cells plays a pivotal role in osteosarcoma progression.Aggressive osteosarcoma phenotypes are mediated by autocrine VEGF/VEGFR1 signaling and improved stratification measures and novel anti-angiogenic strategies may benefit this specific tumor type.

Harmata A.J.,Vanderbilt University | Harmata A.J.,Center for Bone Biology | Ma Y.,Center for Bone Biology | Ma Y.,Vanderbilt Medical Center | And 7 more authors.
Clinical Orthopaedics and Related Research | Year: 2015

Background: Infectious complications of musculoskeletal trauma are an important factor contributing to patient morbidity. Biofilm-dispersive bone grafts augmented with d-amino acids (d-AAs) prevent biofilm formation in vitro and in vivo, but the effects of d-AAs on osteocompatibility and new bone formation have not been investigated. Questions/purposes: We asked: (1) Do d-AAs hinder osteoblast and osteoclast differentiation in vitro? (2) Does local delivery of d-AAs from low-viscosity bone grafts inhibit new bone formation in a large-animal model? Methods: Methicillin-sensitive Staphylococcus aureus and methicillin-resistant S aureus clinical isolates, mouse bone marrow stromal cells, and osteoclast precursor cells were treated with an equal mass (1:1:1) mixture of d-Pro:d-Met:d-Phe. The effects of the d-AA dose on biofilm inhibition (n = 4), biofilm dispersion (n = 4), and bone marrow stromal cell proliferation (n = 3) were quantitatively measured by crystal violet staining. Osteoblast differentiation was quantitatively assessed by alkaline phosphatase staining, von Kossa staining, and quantitative reverse transcription for the osteogenic factors a1Col1 and Ocn (n = 3). Osteoclast differentiation was quantitatively measured by tartrate-resistant acid phosphatase staining (n = 3). Bone grafts augmented with 0 or 200 mmol/L d-AAs were injected in ovine femoral condyle defects in four sheep. New bone formation was evaluated by μCT and histology 4 months later. An a priori power analysis indicated that a sample size of four would detect a 7.5% difference of bone volume/total volume between groups assuming a mean and SD of 30% and 5%, respectively, with a power of 80% and an alpha level of 0.05 using a two-tailed t-test between the means of two independent samples. Results: Bone marrow stromal cell proliferation, osteoblast differentiation, and osteoclast differentiation were inhibited at d-AAs concentrations of 27 mmol/L or greater in a dose-responsive manner in vitro (p < 0.05). In methicillin-sensitive and methicillin-resistant S aureus clinical isolates, d-AAs inhibited biofilm formation at concentrations of 13.5 mmol/L or greater in vitro (p < 0.05). Local delivery of d-AAs from low-viscosity grafts did not inhibit new bone formation in a large-animal model pilot study (0 mmol/L d-AAs: bone volume/total volume = 26.9% ± 4.1%; 200 mmol/L d-AAs: bone volume/total volume = 28.3% ± 15.4%; mean difference with 95% CI = −1.4; p = 0.13). Conclusions: d-AAs inhibit biofilm formation, bone marrow stromal cell proliferation, osteoblast differentiation, and osteoclast differentiation in vitro in a dose-responsive manner. Local delivery of d-AAs from bone grafts did not inhibit new bone formation in vivo at clinically relevant doses. Clinical Relevance: Local delivery of d-AAs is an effective antibiofilm strategy that does not appear to inhibit bone repair. Longitudinal studies investigating bacterial burden, bone formation, and bone remodeling in contaminated defects as a function of d-AA dose are required to further support the use of d-AAs in the clinical management of infected open fractures. © 2015, The Association of Bone and Joint Surgeons®.

Page J.M.,Vanderbilt University | Page J.M.,Center for Bone Biology | Prieto E.M.,Vanderbilt University | Prieto E.M.,Center for Bone Biology | And 7 more authors.
Acta Biomaterialia | Year: 2012

Injectable and settable bone grafts offer significant advantages over pre-formed implants due to their ability to be administered using minimally invasive techniques and to conform to the shape of the defect. However, injectable biomaterials present biocompatibility challenges due to the potential toxicity and ultimate fate of reactive components that are not incorporated in the final cured product. In this study the effects of stoichiometry and triethylenediamine (TEDA) catalyst concentration on the reactivity, injectability, and biocompatibility of two component lysine-derived polyurethane (PUR) biocomposites were investigated. Rate constants were measured for the reactions of water (a blowing agent resulting in the generation of pores), polyester triol, dipropylene glycol (DPG), and allograft bone particles with the isocyanate-terminated prepolymer using an in situ attenuated total reflection Fourier transform infrared spectroscopy technique. Based on the measured rate constants, a kinetic model predicting the conversion of each component with time was developed. Despite the fact that TEDA is a well-known urethane gelling catalyst, it was found to preferentially catalyze the blowing reaction with water relative to the gelling reactions by a ratio >17:1. Thus the kinetic model predicted that the prepolymer and water proceeded to full conversion, while the conversions of polyester triol and DPG were <70% after 24 h, which was consistent with leaching experiments showing that only non-cytotoxic polyester triol and DPG were released from the reactive PUR at early time points. The PUR biocomposite supported cellular infiltration and remodeling in femoral condyle defects in rabbits at 8 weeks, and there was no evidence of an adverse inflammatory response induced by unreacted components from the biocomposite or degradation products from the cured polymer. Taken together, these data underscore the utility of the kinetic model in predicting the biocompatibility of reactive biomaterials. © 2012 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.

Harmata A.J.,Vanderbilt University | Harmata A.J.,Center for Bone Biology | Ward C.L.,U.S. Army | Zienkiewicz K.J.,Vanderbilt University | And 3 more authors.
Journal of Materials Research | Year: 2014

Injectable bone grafts with strength exceeding that of trabecular bone could improve the clinical management of a number of orthopedic conditions. Ceramic/polymer composites have been investigated as weight-bearing bone grafts, but they are typically weaker than trabecular bone due to poor interfacial bonding. We hypothesized that entrapment of surface-initiated poly(ε-caprolactone) (PCL) chains on 45S5 bioactive glass (BG) particles within an in situ-formed polymer network would enhance the mechanical properties of reactive BG/polymer composites. When the surface-initiated PCL molecular weight exceeded the molecular weight between crosslinks of the network, the compressive strength of the composites increased 6- to 10-fold. The torsional strength of the composites exceeded that of human trabecular bone by a factor of two. When injected into femoral condyle defects in rats, the composites supported new bone formation at 8 weeks. The initial bone-like strength of BG/polymer composites and their ability to remodel in vivo highlight their potential for development as injectable grafts for repair of weight-bearing bone defects. Copyright © Materials Research Society 2014.

Li X.,Vanderbilt Ingram Cancer Center | Sterling J.A.,Vanderbilt Ingram Cancer Center | Sterling J.A.,Center for Bone Biology | Sterling J.A.,Tennessee Valley Healthcare System VISN | And 8 more authors.
Molecular Cancer Research | Year: 2012

Loss of TGF-β type II receptor (TβRII, encoded by Tgfbr2) expression in the prostate stroma contributes to prostate cancer initiation, progression, and invasion. We evaluated whether TβRII loss also affected prostate cancer bone metastatic growth. Immunohistologic analysis revealed that TβRII expression was lost in cancer-associated fibroblasts in human prostate cancer bone metastatic tissues. We recapitulated the human situation with a conditional stromal Tgfbr2 knockout (Tgfbr2-KO) mouse model. Conditioned media from primary cultured Tgfbr2-KO or control Tgfbr2-flox prostatic fibroblasts (koPFCM or wtPFCM, respectively) were applied to C4-2B prostate cancer cells before grafting the cells tibially. We found that koPFCM promoted prostate cancer cell growth in the bone and development of early mixed osteoblastic/osteolytic bone lesions. Furthermore, the koPFCM promoted greater C4-2B adhesion to type-I collagen, the major component of bone matrix, compared to wtPFCM-treated C4-2B. Cytokine antibody array analysis revealed that koPFCM had more than two-fold elevation in granulocyte colony-stimulating factor and CXCL1, CXCL16, and CXCL5 expression relative to wtPFCM. Interestingly, neutralizing antibodies of CXCL16 or CXCL1 were able to reduce koPFCM-associated C4-2B type-I collagen adhesion to that comparable with wtPFCM-mediated adhesion. Collectively, our data indicate that loss of TGF-β responsiveness in prostatic fibroblasts results in upregulation of CXCL16 and CXCL1 and that these paracrine signals increase prostate cancer cell adhesion in the bone matrix. These microenvironment changes at the primary tumor site can mediate early establishment of prostate cancer cells in the bone and support subsequent tumor development at the metastatic site. ©2012 AACR.

Li S.,University of Pittsburgh | Pal R.,University of Pittsburgh | Monaghan S.A.,University of Pittsburgh | Schafer P.,Celgene | And 6 more authors.
Blood | Year: 2011

Immunomodulatory derivatives of thalidomide (IMiD compounds), such as pomalidomide and lenalidomide, are highly active in multiple myeloma (MM) treatment. However, the precise mechanisms of action and resistance in MM are unresolved. Here we show that IMiD compounds down-regulate CCAAT/enhancer- binding protein-β (C/EBPβ) resulting in abrogation of cell proliferation. Overexpression of C/EBPβ rescued MM cells from IMiD-induced inhibition of proliferation, indicating that C/EBPβ is critical in mediating antiproliferative effects. IMiD-induced decrease of C/EBPβ protein led to impaired transcription of interferon regulatory factor 4 (IRF4). Down-regulation of IRF4 by lenalidomide was confirmed by longitudinal studies of bone marrow samples from 23 patients obtained before and during lenalidomide treatment using CD138+/IRF4+ double labeling. In contrast to down-regulation of C/EBPβ protein, IMiD compounds did not alter C/EBPβ mRNA levels or protein stability, suggesting translational regulation of C/EBPβ. We could demonstrate that C/EBPβ protein expression is under eIF4E-translational control in MM. Furthermore, inhibition of the eIF4E-C/EBPβ axis by IMiD compounds was not observed in IMiD-resistant MM cells. However, targeting translation at a different level by inhibiting eukaryotic translation initiation factor 4E-binding protein 1 phosphorylation overcame resistance, suggesting that this pathway is critical and might be a target to overcome drug resistance. © 2011 by The American Society of Hematology.

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