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Bovine fetal growth plate powder as a new osteoinduction biomaterial
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Abstracts / Bone 48 (2011) S104–S123
FCT/SFRH/BD/36220/2007 grant and Projects Bonamidi PTDC/CTM/100120/2008 and CRUP E46-09 are acknowledged. This article is part of a Special Issue entitled ECTS 2011. Disclosure of interest: None declared.
doi:10.1016/j.bone.2011.03.166
PP008-M Bovine fetal growth plate powder as a new osteoinduction biomaterial A. Bighamsadegh a,⁎, S. Mohammad b, Z. Shafieisarvestani c a Department of Veterinary Surgery and Radiology, Shahrekord University, Shahrekord, Islamic Republic of Iran b Shahrekord University, Shahrekord, Islamic Republic of Iran c Shiraz University, Shiraz, Islamic Republic of Iran Abstract: The aim of this study was evaluation of osteoinductive properties of demineralized bovine foetal growth plate in submuscular transplantation (ectopic osteoinduction). Demineralized bovine foetal growth plate was ectopically implanted in 18 male Sprague–Dawley rats. In 18 of the animals under aseptic conditions two submuscular pouches were created between external and internal oblique abdominal muscles in the two flanks: the right was left empty (sham) and the left was filled with 20 mg of demineralized bovine foetal growth plate powder. Radiographs were taken in 2, 4 and 6 weeks after the surgery, then six animals were pharmacologically euthanized after 2, 4 and 6 weeks for histopathological evaluation. Results showed: (1) osteoinductivity of xenogenic demineralized bovine foetal growth plate powder, and (2) earlier mineralization of ectopically implanted demineralized bovine foetal growth plate in the submuscular implanted area. Our results show that submuscular implantation of xenogenic demineralized bovine foetal growth plate has osteoinductive properties in a rat model. This article is part of a Special Issue entitled ECTS 2011. Keywords: Ectopic, Osteoinductionin rat, Bovine, Demineralized fetus growth plate. Disclosure of interest: A. Bighamsadegh Grant/Research Support from There is not any conflict of interest, S. Mohammad: none declared, and Z. Shafieisarvestani: none declared.
doi:10.1016/j.bone.2011.03.167
PP009-T The vitamin E analogue Γ-tocotrienol blocks osteoclastogenesis and promotes osteogenesis: Evidence for combined anti-osteolytic and bone forming activity A. Evdokiou a,⁎, V. Liapis a, S. Hay a, A. Keramidas b, E. Maltezou b, Y. Christou c, G.J. Atkins a, D.M. Findlay a, A. Odysseos c a Orthopaedics and Trauma, University of Adelaide, Adelaide, Australia b Chemistry, University of Cyprus, Nicosia, Cyprus c EPOS-LASIS, Nicosia, Cyprus Abstract: Oxidative stress perturbs bone remodelling by modulating the differentiation and survival of osteoblasts and reducing bone formation, while promoting osteoclast formation and activation. Recent compelling evidence from a number of clinical studies has shown that reactive oxygen species (ROS) are involved in the pathogenesis of bone loss and that antioxidant therapy may be useful in the prevention and treatment of bone loss associated with osteoporosis and other bone disorders, including cancer-induced bone loss. In this study we investigated the effects of g-tocotrienol, a potent anti-oxidant vitamin E analogue, on osteoclast differentiation, bone resorption and osteoblast function in-vitro. When human peripheral blood mononuclear cells (PBMCs) or murine monocytic RAW264.7 cells were cultured with the receptor activator of nuclear factor kappa B-ligand (RANKL), γ-tocotrienol dose-dependently inhibited the formation of tartrate-resistant acid phosphatase positive multinucleated cells and bone resorption when these cells were plated on bone matrix. Similarly, g-tocotrienol inhibited bone resorption by mature osteoclasts that were isolated from human Giant Cell Tumours (GCT) of bone. The effect of γ-tocotrienol on osteoblast activity was investigated using mineralizing primary human osteoblast cultures. Under osteo-inductive conditions, γ-tocotrienol reproducibly increased in vitro mineralisation, compared to untreated cells, with a concomitant increase in alizarin red and von Kossa staining. These results have important implications for bone biology and osteoporosis and suggest that targeting of antioxidant therapy to bone can prevent pathological bone loss. This article is part of a Special Issue entitled ECTS 2011. Disclosure of interest: None declared.
doi:10.1016/j.bone.2011.03.168
PP010-S Adhesion and proliferation of mesenchymal stem cells on micropatterned thin films modified with nanohydroxyapatite particles A. Carvalho a,b,⁎, A. Pelaez-Vargas a,b, D. Gallego-Perez c, M.H. Fernandes d, D. Hansford c, F.J. Monteiro a,b a INEB/Instituto Nacional de Engenharia Biomédica, Universidade do Porto, Porto, Portugal b DEMM, Faculdade de Engenharia, Universidade do Porto, Porto, Portugal c Department of Biomedical Engineering, The Ohio State University, Columbus, OH, USA d Laboratório de Farmacologia e Biocompatibilidade Celular, Faculdade de Medicina Dentária, Universidade do Porto, Porto, Portugal Abstract: Introduction: Guided tissue regeneration for dentistry is a therapy focused on the re-growth of bone to fill infra-bony defects produced by periodontal diseases. The ability to add microtexture to implant surfaces through a simple process, such as sol-gel, can aid in the guiding of tissue alignment through contact guidance. Objectives: The aim of the work was to evaluate the adhesion, proliferation and metabollic activity of Mesenchymal Stem Cells (MSCs) on microfabricated silica thin films modified with nanoHydroxyapatite particles (nanoHA). Methods: Microtextured silica thin films were produced using a microstamping technique that is a combination of soft lithography and sol-gel processing. Nanoscale hydroxyapatite particles were introduced into the sol for incorporation into the thin films at three difference weight ratios (0, 1, and 5%). Material characterization of the films was conducted using SEM/EDS. Cell cultures of pulp-derived MSCs were conducted under standardized conditions at three time-points (1, 7 and 14 days). TCPS was used as control group for all experiments. Morphological analysis was conducted via fluorescence (staining for F-actin and nuclei) and electron microscopy. Metabollic activity was conducted using Resazurin and ALP assays. Results: Anisotropic silica micropatterned thin films containing different percentages of nanoHA particles were successfully produced. Lineshaped micropatterns faithfully reproduced the mold features with ~5 μm width and ~10 μm interspacing. MSCs showed alignment following the texture of micropatterned surfaces starting at Day 1. This behavior increased in the subsequent time points on all the microtextured surfaces, compared to the TCPS control group. Intragroup analysis using Resazurin assay showed increased cell numbers at all time points. Intergroups analysis showed that MSCs exhibited increased proliferation on the 5% nanoHA group micropatterns compared to the 0% and 1% groups (p<0.05) at Day 7. MSCs on all micropatterned surfaces showed a significantly higher ALP activity (p<0.05) when compared to the flat surface of control group at Day 14. Conclusions: The preliminary results show that all the micropatterned thin films induced the alignment of MSCs and led to improved cellular responses, which were maintained for prolonged periods of time. FCT/SFRH/BD/36220/2007 grant and Projects Bonamidi PTDC/CTM/100120/2008 and CRUP E46-09 are acknowledged. This article is part of a Special Issue entitled ECTS 2011. Disclosure of interest: None declared.
doi:10.1016/j.bone.2011.03.169
PP011-M Involvement of PiT1 and PiT2 in the response to phosphate in osteoblasts in vitro A. Bourgine ⁎, L. Beck, S. Beck-Cormier, G. Couasnay, M. Perré, M. Masson, S. Khoshniat, P. Weiss, J. Guicheux INSERM U791 LIOAD group STEP, University of Nantes, France Abstract: Inorganic phosphate (Pi) is acknowledged as a specific signaling molecule regulating the expression of mineralization-associated genes including matrix gla protein (MGP) and osteopontin (OPN) via the activation of the ERK1/2 pathway in osteoblasts [1]. However, the functional relevance of the Pi-dependent effects in the cellular and molecular mechanisms involved in this regulation has not been completely deciphered. This could involve the intracellular import of Pi through dedicated transporters or the detection of extracellular Pi through some as yet unknown Pi-sensing mechanism. PiT1 and PiT2 are multifunctional membrane proteins capable of transporting Pi. Many in vitro studies have suggested the involvement of PiT1 in bone mineralization, consistent with its preferential in vivo expression in growth plate chondrocytes [2]. PiT2, a closed parent to PiT1, has described roles in mineralized tissues, especially teeth [3]. To gain insights into the role of PiT1 and PiT2 in the ERK1/2-mediated regulation of MGP/OPN in response to Pi in osteoblasts, we used a RNA interference approach. Osteoblastic MC3T3-E1 cells were stably transfected with shRNA directed against PiT1 or PiT2. Protein and mRNA analyses were performed by Western blotting and real-time PCR, respectively. Pi transport activity was measured using radioactive Pi tracer uptake. Our data indicate that the ERK1/2-dependent MGP/OPN activation was blunted in MC3T3-E1 cells knocked-down for PiT1 or PiT2. This inhibitory effect occurs in the presence of a normal Pi transport activity suggesting that the Pi-sensing mechanism is not dependent on Pi entry into cells. These results show that PiT1 and PiT2 play a regulatory role in the osteoblastic responsiveness to extracellular Pi and provide new insights into a possible role of these proteins
Abstracts / Bone 48 (2011) S104–S123
FCT/SFRH/BD/36220/2007 grant and Projects Bonamidi PTDC/CTM/100120/2008 and CRUP E46-09 are acknowledged. This article is part of a Special Issue entitled ECTS 2011. Disclosure of interest: None declared.
doi:10.1016/j.bone.2011.03.166
PP008-M Bovine fetal growth plate powder as a new osteoinduction biomaterial A. Bighamsadegh a,⁎, S. Mohammad b, Z. Shafieisarvestani c a Department of Veterinary Surgery and Radiology, Shahrekord University, Shahrekord, Islamic Republic of Iran b Shahrekord University, Shahrekord, Islamic Republic of Iran c Shiraz University, Shiraz, Islamic Republic of Iran Abstract: The aim of this study was evaluation of osteoinductive properties of demineralized bovine foetal growth plate in submuscular transplantation (ectopic osteoinduction). Demineralized bovine foetal growth plate was ectopically implanted in 18 male Sprague–Dawley rats. In 18 of the animals under aseptic conditions two submuscular pouches were created between external and internal oblique abdominal muscles in the two flanks: the right was left empty (sham) and the left was filled with 20 mg of demineralized bovine foetal growth plate powder. Radiographs were taken in 2, 4 and 6 weeks after the surgery, then six animals were pharmacologically euthanized after 2, 4 and 6 weeks for histopathological evaluation. Results showed: (1) osteoinductivity of xenogenic demineralized bovine foetal growth plate powder, and (2) earlier mineralization of ectopically implanted demineralized bovine foetal growth plate in the submuscular implanted area. Our results show that submuscular implantation of xenogenic demineralized bovine foetal growth plate has osteoinductive properties in a rat model. This article is part of a Special Issue entitled ECTS 2011. Keywords: Ectopic, Osteoinductionin rat, Bovine, Demineralized fetus growth plate. Disclosure of interest: A. Bighamsadegh Grant/Research Support from There is not any conflict of interest, S. Mohammad: none declared, and Z. Shafieisarvestani: none declared.
doi:10.1016/j.bone.2011.03.167
PP009-T The vitamin E analogue Γ-tocotrienol blocks osteoclastogenesis and promotes osteogenesis: Evidence for combined anti-osteolytic and bone forming activity A. Evdokiou a,⁎, V. Liapis a, S. Hay a, A. Keramidas b, E. Maltezou b, Y. Christou c, G.J. Atkins a, D.M. Findlay a, A. Odysseos c a Orthopaedics and Trauma, University of Adelaide, Adelaide, Australia b Chemistry, University of Cyprus, Nicosia, Cyprus c EPOS-LASIS, Nicosia, Cyprus Abstract: Oxidative stress perturbs bone remodelling by modulating the differentiation and survival of osteoblasts and reducing bone formation, while promoting osteoclast formation and activation. Recent compelling evidence from a number of clinical studies has shown that reactive oxygen species (ROS) are involved in the pathogenesis of bone loss and that antioxidant therapy may be useful in the prevention and treatment of bone loss associated with osteoporosis and other bone disorders, including cancer-induced bone loss. In this study we investigated the effects of g-tocotrienol, a potent anti-oxidant vitamin E analogue, on osteoclast differentiation, bone resorption and osteoblast function in-vitro. When human peripheral blood mononuclear cells (PBMCs) or murine monocytic RAW264.7 cells were cultured with the receptor activator of nuclear factor kappa B-ligand (RANKL), γ-tocotrienol dose-dependently inhibited the formation of tartrate-resistant acid phosphatase positive multinucleated cells and bone resorption when these cells were plated on bone matrix. Similarly, g-tocotrienol inhibited bone resorption by mature osteoclasts that were isolated from human Giant Cell Tumours (GCT) of bone. The effect of γ-tocotrienol on osteoblast activity was investigated using mineralizing primary human osteoblast cultures. Under osteo-inductive conditions, γ-tocotrienol reproducibly increased in vitro mineralisation, compared to untreated cells, with a concomitant increase in alizarin red and von Kossa staining. These results have important implications for bone biology and osteoporosis and suggest that targeting of antioxidant therapy to bone can prevent pathological bone loss. This article is part of a Special Issue entitled ECTS 2011. Disclosure of interest: None declared.
doi:10.1016/j.bone.2011.03.168
PP010-S Adhesion and proliferation of mesenchymal stem cells on micropatterned thin films modified with nanohydroxyapatite particles A. Carvalho a,b,⁎, A. Pelaez-Vargas a,b, D. Gallego-Perez c, M.H. Fernandes d, D. Hansford c, F.J. Monteiro a,b a INEB/Instituto Nacional de Engenharia Biomédica, Universidade do Porto, Porto, Portugal b DEMM, Faculdade de Engenharia, Universidade do Porto, Porto, Portugal c Department of Biomedical Engineering, The Ohio State University, Columbus, OH, USA d Laboratório de Farmacologia e Biocompatibilidade Celular, Faculdade de Medicina Dentária, Universidade do Porto, Porto, Portugal Abstract: Introduction: Guided tissue regeneration for dentistry is a therapy focused on the re-growth of bone to fill infra-bony defects produced by periodontal diseases. The ability to add microtexture to implant surfaces through a simple process, such as sol-gel, can aid in the guiding of tissue alignment through contact guidance. Objectives: The aim of the work was to evaluate the adhesion, proliferation and metabollic activity of Mesenchymal Stem Cells (MSCs) on microfabricated silica thin films modified with nanoHydroxyapatite particles (nanoHA). Methods: Microtextured silica thin films were produced using a microstamping technique that is a combination of soft lithography and sol-gel processing. Nanoscale hydroxyapatite particles were introduced into the sol for incorporation into the thin films at three difference weight ratios (0, 1, and 5%). Material characterization of the films was conducted using SEM/EDS. Cell cultures of pulp-derived MSCs were conducted under standardized conditions at three time-points (1, 7 and 14 days). TCPS was used as control group for all experiments. Morphological analysis was conducted via fluorescence (staining for F-actin and nuclei) and electron microscopy. Metabollic activity was conducted using Resazurin and ALP assays. Results: Anisotropic silica micropatterned thin films containing different percentages of nanoHA particles were successfully produced. Lineshaped micropatterns faithfully reproduced the mold features with ~5 μm width and ~10 μm interspacing. MSCs showed alignment following the texture of micropatterned surfaces starting at Day 1. This behavior increased in the subsequent time points on all the microtextured surfaces, compared to the TCPS control group. Intragroup analysis using Resazurin assay showed increased cell numbers at all time points. Intergroups analysis showed that MSCs exhibited increased proliferation on the 5% nanoHA group micropatterns compared to the 0% and 1% groups (p<0.05) at Day 7. MSCs on all micropatterned surfaces showed a significantly higher ALP activity (p<0.05) when compared to the flat surface of control group at Day 14. Conclusions: The preliminary results show that all the micropatterned thin films induced the alignment of MSCs and led to improved cellular responses, which were maintained for prolonged periods of time. FCT/SFRH/BD/36220/2007 grant and Projects Bonamidi PTDC/CTM/100120/2008 and CRUP E46-09 are acknowledged. This article is part of a Special Issue entitled ECTS 2011. Disclosure of interest: None declared.
doi:10.1016/j.bone.2011.03.169
PP011-M Involvement of PiT1 and PiT2 in the response to phosphate in osteoblasts in vitro A. Bourgine ⁎, L. Beck, S. Beck-Cormier, G. Couasnay, M. Perré, M. Masson, S. Khoshniat, P. Weiss, J. Guicheux INSERM U791 LIOAD group STEP, University of Nantes, France Abstract: Inorganic phosphate (Pi) is acknowledged as a specific signaling molecule regulating the expression of mineralization-associated genes including matrix gla protein (MGP) and osteopontin (OPN) via the activation of the ERK1/2 pathway in osteoblasts [1]. However, the functional relevance of the Pi-dependent effects in the cellular and molecular mechanisms involved in this regulation has not been completely deciphered. This could involve the intracellular import of Pi through dedicated transporters or the detection of extracellular Pi through some as yet unknown Pi-sensing mechanism. PiT1 and PiT2 are multifunctional membrane proteins capable of transporting Pi. Many in vitro studies have suggested the involvement of PiT1 in bone mineralization, consistent with its preferential in vivo expression in growth plate chondrocytes [2]. PiT2, a closed parent to PiT1, has described roles in mineralized tissues, especially teeth [3]. To gain insights into the role of PiT1 and PiT2 in the ERK1/2-mediated regulation of MGP/OPN in response to Pi in osteoblasts, we used a RNA interference approach. Osteoblastic MC3T3-E1 cells were stably transfected with shRNA directed against PiT1 or PiT2. Protein and mRNA analyses were performed by Western blotting and real-time PCR, respectively. Pi transport activity was measured using radioactive Pi tracer uptake. Our data indicate that the ERK1/2-dependent MGP/OPN activation was blunted in MC3T3-E1 cells knocked-down for PiT1 or PiT2. This inhibitory effect occurs in the presence of a normal Pi transport activity suggesting that the Pi-sensing mechanism is not dependent on Pi entry into cells. These results show that PiT1 and PiT2 play a regulatory role in the osteoblastic responsiveness to extracellular Pi and provide new insights into a possible role of these proteins