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ephrin-B interactions assist in MSC niche maintenance and contribute to bone remodelling following injury
Bone 44 (2009) S142–S161
Contents lists available at ScienceDirect
Bone j o u r n a l h o m e p a g e : w w w. e l s e v i e r. c o m / l o c a t e / b o n e
Abstracts
Category 6. Bone formation, cartilage and bone matrix
401 Keratin 18 is over-expressed in osteoblasts derived from Pagetic lesions but is not involved in the formation of disrupted matrix D. Naota, B.G. Matthewsa, R.M. Locklinb, Z. Xiab, P.A. Hulleyb, J. Cornisha a Department of Medicine, University of Auckland, Auckland, New Zealand b Nuffield Department of Orthopaedic Surgery, Botnar Research Centre, Oxford, United Kingdom Paget's disease is a common focal bone disorder. Pagetic lesions, which result from over-activity of osteoclasts and osteoblasts, appear lytic at early stages and later turn sclerotic, with areas of irregular, disorganised bone matrix. Pagetic osteoclasts are grossly abnormal, and have been the focus of most of the research on the cellular mechanisms of Paget's. Given the tight coupling between osteoclasts and osteoblasts, we characterised changes in the pagetic osteoblast that could contribute to the development of the disease. We compared gene expression in osteoblasts and bone marrow cells from pagetic and non-pagetic patients. Microarray analysis identified a number of differentially regulated genes, and the intermediate filament protein keratin 18 (KRT18) was one of the most highly upregulated genes in pagetic osteoblasts. Real-time RT-PCR comparing 28 pagetic samples to 49 controls confirmed that KRT18 expression is more than three times higher in pagetic cells. In the present study we investigated the effects of KRT18 over-expression on osteoblasts and mesenchymal cells. Primary human osteoblasts were transduced with a KRT18 adenoviral vector and compared to cells transduced with a control vector. Real-time RT-PCR showed that KRT18 over-expressing cells have increased levels of interleukin 1β, Dkk1, and the chemokine MCP1, genes that had previously been identified as up-regulated in pagetic osteoblasts. In order to study the possible effects of KRT18 over-expression on cell morphology and extracellular matrix formation, we cultured virally transduced human primary mesenchymal cells in 3-dimensional collagen scaffolds. The scaffolds provide an extracellular structure which resembles the bone environment and is therefore better suited for the study of cell morphology and matrix formation and mineralisation. Cells were analysed using confocal imaging. Cells were also stained for alkaline phosphatase activity and calcium deposition was measured by alizarin red staining. Cultures of KRT18 over-expressing cells were not significantly different from the controls. In conclusion, these results suggest that KRT18 plays a role in osteoblast biology, and overexpression of this gene can reproduce 8756–3282/$ – see front matter
some of the features of pagetic osteoblasts, however, this does not appear to include the disrupted matrix formation. doi:10.1016/j.bone.2009.01.454
402 EphB/ephrin-B interactions assist in MSC niche maintenance and contribute to bone remodelling following injury A. Arthura, C. Stylianoub, S.A. Koblarc, K. Matsuod, S. Gronthosa a Bone and Cancer Laboratories, Division of Haematology, Institute of Medical and Veterinary Science/Hanson Institute, Adelaide, SA, Australia b The Institute of Molecular and Cell Biology, Singapore's Agency for Science, Technology and Research, Singapore, Singapore c School of Molecular and Biomedical Science, School of Medicine, University of Adelaide, Adelaide, SA, Australia d Department of Microbiology and Immunology, Graduate School of Medicine, Keio University, Shinjuku-ku, Tokyo, Japan The bone marrow contains mesenchymal stromal/stem cells (MSCs) that reside within a perivascular niche. This precursor population is essential for regulating skeletal tissue homeostasis, including bone formation and repair. Bone remodelling is mediated by bone forming osteoblasts derived from MSC, and by bone resorbing osteoclasts that originate from the haematopoietic lineage. However, the molecular signals that maintain multi-potential MSC populations within the stem cell niche and the mechanisms that drive their mobilization towards the bone surfaces to facilitate bone formation are not well defined. The Eph/ephrin family of receptor tyrosine kinases have been implicated in the maintenance of stem cell niches including neural, intestinal and dental tissue, and more recently in the regulation of bone homeostasis. We have characterized the gene and protein expression of EphB/ephrin-B molecules on human culture expanded MSC populations and found that the EphB1, B2 and B4 receptors and ephrin-B1/B2 ligand were the highest expressed B class Eph/ephrin molecules. Functional studies using Fc fusion proteins bound to the extracellular portion of EphB2 or ephrin-B1 showed that reverse ephrin-B signalling inhibited human MSC attachment and spreading, while forward EphB signalling promoted MSC migration. Furthermore, EphB expressing MSC were found to restrict their capacity for selfrenewal while activated ephrin-B expressing MSC promoted osteogenic differentiation. A murine femoral fracture model was used to elucidate the contribution of EphB/ephrin-B molecules to skeletal tissue repair and bone remodelling. Gene expression for
Abstracts / Bone 44 (2009) S142–S161
both EphB and ephrin-B significantly increased during the early stage of bone repair and returned to steady-state levels at the later stages of bone remodelling. EphB2 and ephrin-B1 were found to be most abundantly expressed within the callus site, including blood vessels, hypertrophic and calcified chondrocytes, osteoblasts, osteocytes, and newly forming bone. In this study we have demonstrated the role of EphB/ephrin-B family members in human MSC self-renewal, migration and osteogenic differentiation, and appear to be differentially expressed in skeletal tissues during bone fracture healing using a murine femoral fracture model. These observations suggest that EphB/ephrin-B interaction potentially maintain human MSC within their niche under steady state conditions, while promoting migration and skeletal tissue homeostasis following injury. doi:10.1016/j.bone.2009.01.309
403 Receptor activator NF-kappab ligand (RANKL) expression in synovial tissue (ST) and bone mineral density (BMD) in rheumatoid arthritis (RA) patients A.R. Balanescua, C. Iosifb, C. Ardeleanub, F. Bergheaa, V.C. Bojincaa, V. Predescuc, D. Oprisa, D. Predeteanua, R. Ionescua a Internal Medicine and Rheumatology, Carol Davila University— Sf. Maria Hospital, Bucharest, Romania b Victor Babes— National Research Institute, Bucharest, Romania c Ortopedics, Sf. Pantelimon Hospital, Bucharest, Romania Background: RA is commonly characterized by localized and generalized bone loss. Excessive release of cytokines and growth factors due to inflammation associated with the effect of antirheumatic therapy (mainly corticosteroids) significantly influence total bone mass. Recent evidence indicates that RANKL has a pivotal role in osteoclastogenesis and inflammation-induced bone destruction in RA. Purpose: To compare RANKL expression in the Synovial Tissue (ST) from patients with active and inactive RA and from osteoarthritis patients and to investigate the relationship between RANKL levels and BMD. Methods: 26 patients with RA, were recruited and clinical status was scored using the DAS28. The control group included 22 matched osteoarthritis patients. All RA patients were on immunosuppressive agents, but none of them received corticosteroids or TNF-α antagonists. Immunohistological analysis of ST biopsy specimens was performed according to the Avidin–Biotin-Complex method, using a monoclonal antibody to detect RANKL. Sections were evaluated by semiquantitative analysis to compare RANKL expression between groups. Results: Higher levels of RANKL were expressed in ST from patients with active RA than in ST from patients with inactive RA and osteoarthritis. DXA measurements revealed that all patients with RA showed significant decreases in BMD compared with controls. A comparison of BMD between patients with active and inactive disease did not reveal a significant effect of clinical disease activity on the lumbar spine and total proximal femur BMD. No relation was found between the lumbar and femoral BMD and the expression of RANKL in ST in patients with inactive RA. However, a good correlation was found between the decrease in BMD and the increase in RANKL expression in ST from patients with active disease. Conclusion: The highest expression of RANKL was detected in RA patients with active synovitis. Our data showed a significant correlation between the increased RANKL expression in ST from RA patients with active disease and the decrease in BMD, suggesting that RANKL may have a role in generalized bone loss as well. Normal level of RANKL in the ST of patients with inactive disease at the time of synovial biopsy raise de possibility that effective treatment of the disease m doi:10.1016/j.bone.2009.01.310
S143
404 Can serum markers of bone metabolism predict the progression of knee OA? P.A. Berrya, R.A. Maciewiczb, F.M. Cicuttinia, M.D. Downey-Jonesb, E.A. Millsc, C.J. Oakleyb, A.E. Wlukaa,d a Epidemiology and Preventive Medicine, Monash University, Melbourne, VIC, Australia b Respiratory and Inflammation Research Area, Astra Zeneca, Macclesfield, Cheshire, UK c Statistical Sciences, Astra Zeneca, Macclesfield, Cheshire, UK d Baker Heart Research Institute, Melbourne, VIC, Australia Introduction: Cartilage and bone are affected simultaneously as osteoarthritis progresses, but this relationship is incompletely understood. This study aimed to examine the relationship between serum markers of bone formation and resorption, and change in cartilage quantity over 2 years, and to determine whether ratios of markers of bone formation to resorption provide additional information. Methods: Change in cartilage volume over 2 years was measured in 117 subjects with symptomatic knee osteoarthritis using MRI. The relationships between change in cartilage volume and baseline levels of serum markers of bone formation (intact N-terminal propeptide of type I procollagen (PINP) and osteocalcin), resorption (N-telopeptide of type I collagen (NTX-I), the C-telopeptide of type I collagen (CTX-I) and the C-telopeptide of type I collagen (ICTP)) and ratios of markers of bone formation to resorption were examined. Results: Individually, lower levels of PINP (p = 0.02), osteocalcin (p = 0.01), NTX-I (p = 0.02), and CTX-I (p = 0.02) were associated with elevated medial cartilage volume loss. No significant associations were obtained between marker ratios of bone metabolism and change in cartilage volume. Conclusions: The results of this study suggest that overall, lower serum levels of bone metabolism in osteoarthritis may be predictive of cartilage loss. Further insight into understanding how individual markers of bone metabolism affect cartilage loss may facilitate understanding of the pathogenesis of this disease and aid in predicting disease progression. doi:10.1016/j.bone.2009.01.311
405 Association between pre-existing microdamage, collagen cross-links, bone volume fraction and compressive mechanical properties of human vertebral trabecular bone H.C. Folleta, B. Burt-Pichata, S. Viguet-Carrina, B. Depallea, E. Gineytsa, M. Arlota, R.D. Chapurlata, P.D. Delmasa, M.L. Bouxseinb a Inserm U831, University of Lyon, Lyon, France b Department of Orthopedic Surgery, Harvard Medical School, Boston, USA Studies have suggested that age-related increases in fatigue microdamage and changes in the profile of collagen crosslink concentration may contribute to skeletal fragility. To further explore this notion, we determined the influence of pre-existing microdamage, collagen cross-link concentration and bone volume fraction (BV/TV) on the mechanical behavior of human trabecular bone. To do this we removed two cylindrical trabecular specimens (8.2 mm diameter, 10 mm height) from L2 vertebrae of 52 recently deceased donors (54–93 yrs of age; 22 men and 30 women). One core was prepared for histologic analysis of pre-existing microdamage by bulk staining in xylenol, whereas the other core was used for assessment of 3D morphology by microCT and compressive mechanical properties. The central part of the vertebral body, containing exclusively trabecular bone, was retained for analysis of pyridinoline (PYD), deoxypyridinoline (DPD) and pentosidine (PEN) content by high
Contents lists available at ScienceDirect
Bone j o u r n a l h o m e p a g e : w w w. e l s e v i e r. c o m / l o c a t e / b o n e
Abstracts
Category 6. Bone formation, cartilage and bone matrix
401 Keratin 18 is over-expressed in osteoblasts derived from Pagetic lesions but is not involved in the formation of disrupted matrix D. Naota, B.G. Matthewsa, R.M. Locklinb, Z. Xiab, P.A. Hulleyb, J. Cornisha a Department of Medicine, University of Auckland, Auckland, New Zealand b Nuffield Department of Orthopaedic Surgery, Botnar Research Centre, Oxford, United Kingdom Paget's disease is a common focal bone disorder. Pagetic lesions, which result from over-activity of osteoclasts and osteoblasts, appear lytic at early stages and later turn sclerotic, with areas of irregular, disorganised bone matrix. Pagetic osteoclasts are grossly abnormal, and have been the focus of most of the research on the cellular mechanisms of Paget's. Given the tight coupling between osteoclasts and osteoblasts, we characterised changes in the pagetic osteoblast that could contribute to the development of the disease. We compared gene expression in osteoblasts and bone marrow cells from pagetic and non-pagetic patients. Microarray analysis identified a number of differentially regulated genes, and the intermediate filament protein keratin 18 (KRT18) was one of the most highly upregulated genes in pagetic osteoblasts. Real-time RT-PCR comparing 28 pagetic samples to 49 controls confirmed that KRT18 expression is more than three times higher in pagetic cells. In the present study we investigated the effects of KRT18 over-expression on osteoblasts and mesenchymal cells. Primary human osteoblasts were transduced with a KRT18 adenoviral vector and compared to cells transduced with a control vector. Real-time RT-PCR showed that KRT18 over-expressing cells have increased levels of interleukin 1β, Dkk1, and the chemokine MCP1, genes that had previously been identified as up-regulated in pagetic osteoblasts. In order to study the possible effects of KRT18 over-expression on cell morphology and extracellular matrix formation, we cultured virally transduced human primary mesenchymal cells in 3-dimensional collagen scaffolds. The scaffolds provide an extracellular structure which resembles the bone environment and is therefore better suited for the study of cell morphology and matrix formation and mineralisation. Cells were analysed using confocal imaging. Cells were also stained for alkaline phosphatase activity and calcium deposition was measured by alizarin red staining. Cultures of KRT18 over-expressing cells were not significantly different from the controls. In conclusion, these results suggest that KRT18 plays a role in osteoblast biology, and overexpression of this gene can reproduce 8756–3282/$ – see front matter
some of the features of pagetic osteoblasts, however, this does not appear to include the disrupted matrix formation. doi:10.1016/j.bone.2009.01.454
402 EphB/ephrin-B interactions assist in MSC niche maintenance and contribute to bone remodelling following injury A. Arthura, C. Stylianoub, S.A. Koblarc, K. Matsuod, S. Gronthosa a Bone and Cancer Laboratories, Division of Haematology, Institute of Medical and Veterinary Science/Hanson Institute, Adelaide, SA, Australia b The Institute of Molecular and Cell Biology, Singapore's Agency for Science, Technology and Research, Singapore, Singapore c School of Molecular and Biomedical Science, School of Medicine, University of Adelaide, Adelaide, SA, Australia d Department of Microbiology and Immunology, Graduate School of Medicine, Keio University, Shinjuku-ku, Tokyo, Japan The bone marrow contains mesenchymal stromal/stem cells (MSCs) that reside within a perivascular niche. This precursor population is essential for regulating skeletal tissue homeostasis, including bone formation and repair. Bone remodelling is mediated by bone forming osteoblasts derived from MSC, and by bone resorbing osteoclasts that originate from the haematopoietic lineage. However, the molecular signals that maintain multi-potential MSC populations within the stem cell niche and the mechanisms that drive their mobilization towards the bone surfaces to facilitate bone formation are not well defined. The Eph/ephrin family of receptor tyrosine kinases have been implicated in the maintenance of stem cell niches including neural, intestinal and dental tissue, and more recently in the regulation of bone homeostasis. We have characterized the gene and protein expression of EphB/ephrin-B molecules on human culture expanded MSC populations and found that the EphB1, B2 and B4 receptors and ephrin-B1/B2 ligand were the highest expressed B class Eph/ephrin molecules. Functional studies using Fc fusion proteins bound to the extracellular portion of EphB2 or ephrin-B1 showed that reverse ephrin-B signalling inhibited human MSC attachment and spreading, while forward EphB signalling promoted MSC migration. Furthermore, EphB expressing MSC were found to restrict their capacity for selfrenewal while activated ephrin-B expressing MSC promoted osteogenic differentiation. A murine femoral fracture model was used to elucidate the contribution of EphB/ephrin-B molecules to skeletal tissue repair and bone remodelling. Gene expression for
Abstracts / Bone 44 (2009) S142–S161
both EphB and ephrin-B significantly increased during the early stage of bone repair and returned to steady-state levels at the later stages of bone remodelling. EphB2 and ephrin-B1 were found to be most abundantly expressed within the callus site, including blood vessels, hypertrophic and calcified chondrocytes, osteoblasts, osteocytes, and newly forming bone. In this study we have demonstrated the role of EphB/ephrin-B family members in human MSC self-renewal, migration and osteogenic differentiation, and appear to be differentially expressed in skeletal tissues during bone fracture healing using a murine femoral fracture model. These observations suggest that EphB/ephrin-B interaction potentially maintain human MSC within their niche under steady state conditions, while promoting migration and skeletal tissue homeostasis following injury. doi:10.1016/j.bone.2009.01.309
403 Receptor activator NF-kappab ligand (RANKL) expression in synovial tissue (ST) and bone mineral density (BMD) in rheumatoid arthritis (RA) patients A.R. Balanescua, C. Iosifb, C. Ardeleanub, F. Bergheaa, V.C. Bojincaa, V. Predescuc, D. Oprisa, D. Predeteanua, R. Ionescua a Internal Medicine and Rheumatology, Carol Davila University— Sf. Maria Hospital, Bucharest, Romania b Victor Babes— National Research Institute, Bucharest, Romania c Ortopedics, Sf. Pantelimon Hospital, Bucharest, Romania Background: RA is commonly characterized by localized and generalized bone loss. Excessive release of cytokines and growth factors due to inflammation associated with the effect of antirheumatic therapy (mainly corticosteroids) significantly influence total bone mass. Recent evidence indicates that RANKL has a pivotal role in osteoclastogenesis and inflammation-induced bone destruction in RA. Purpose: To compare RANKL expression in the Synovial Tissue (ST) from patients with active and inactive RA and from osteoarthritis patients and to investigate the relationship between RANKL levels and BMD. Methods: 26 patients with RA, were recruited and clinical status was scored using the DAS28. The control group included 22 matched osteoarthritis patients. All RA patients were on immunosuppressive agents, but none of them received corticosteroids or TNF-α antagonists. Immunohistological analysis of ST biopsy specimens was performed according to the Avidin–Biotin-Complex method, using a monoclonal antibody to detect RANKL. Sections were evaluated by semiquantitative analysis to compare RANKL expression between groups. Results: Higher levels of RANKL were expressed in ST from patients with active RA than in ST from patients with inactive RA and osteoarthritis. DXA measurements revealed that all patients with RA showed significant decreases in BMD compared with controls. A comparison of BMD between patients with active and inactive disease did not reveal a significant effect of clinical disease activity on the lumbar spine and total proximal femur BMD. No relation was found between the lumbar and femoral BMD and the expression of RANKL in ST in patients with inactive RA. However, a good correlation was found between the decrease in BMD and the increase in RANKL expression in ST from patients with active disease. Conclusion: The highest expression of RANKL was detected in RA patients with active synovitis. Our data showed a significant correlation between the increased RANKL expression in ST from RA patients with active disease and the decrease in BMD, suggesting that RANKL may have a role in generalized bone loss as well. Normal level of RANKL in the ST of patients with inactive disease at the time of synovial biopsy raise de possibility that effective treatment of the disease m doi:10.1016/j.bone.2009.01.310
S143
404 Can serum markers of bone metabolism predict the progression of knee OA? P.A. Berrya, R.A. Maciewiczb, F.M. Cicuttinia, M.D. Downey-Jonesb, E.A. Millsc, C.J. Oakleyb, A.E. Wlukaa,d a Epidemiology and Preventive Medicine, Monash University, Melbourne, VIC, Australia b Respiratory and Inflammation Research Area, Astra Zeneca, Macclesfield, Cheshire, UK c Statistical Sciences, Astra Zeneca, Macclesfield, Cheshire, UK d Baker Heart Research Institute, Melbourne, VIC, Australia Introduction: Cartilage and bone are affected simultaneously as osteoarthritis progresses, but this relationship is incompletely understood. This study aimed to examine the relationship between serum markers of bone formation and resorption, and change in cartilage quantity over 2 years, and to determine whether ratios of markers of bone formation to resorption provide additional information. Methods: Change in cartilage volume over 2 years was measured in 117 subjects with symptomatic knee osteoarthritis using MRI. The relationships between change in cartilage volume and baseline levels of serum markers of bone formation (intact N-terminal propeptide of type I procollagen (PINP) and osteocalcin), resorption (N-telopeptide of type I collagen (NTX-I), the C-telopeptide of type I collagen (CTX-I) and the C-telopeptide of type I collagen (ICTP)) and ratios of markers of bone formation to resorption were examined. Results: Individually, lower levels of PINP (p = 0.02), osteocalcin (p = 0.01), NTX-I (p = 0.02), and CTX-I (p = 0.02) were associated with elevated medial cartilage volume loss. No significant associations were obtained between marker ratios of bone metabolism and change in cartilage volume. Conclusions: The results of this study suggest that overall, lower serum levels of bone metabolism in osteoarthritis may be predictive of cartilage loss. Further insight into understanding how individual markers of bone metabolism affect cartilage loss may facilitate understanding of the pathogenesis of this disease and aid in predicting disease progression. doi:10.1016/j.bone.2009.01.311
405 Association between pre-existing microdamage, collagen cross-links, bone volume fraction and compressive mechanical properties of human vertebral trabecular bone H.C. Folleta, B. Burt-Pichata, S. Viguet-Carrina, B. Depallea, E. Gineytsa, M. Arlota, R.D. Chapurlata, P.D. Delmasa, M.L. Bouxseinb a Inserm U831, University of Lyon, Lyon, France b Department of Orthopedic Surgery, Harvard Medical School, Boston, USA Studies have suggested that age-related increases in fatigue microdamage and changes in the profile of collagen crosslink concentration may contribute to skeletal fragility. To further explore this notion, we determined the influence of pre-existing microdamage, collagen cross-link concentration and bone volume fraction (BV/TV) on the mechanical behavior of human trabecular bone. To do this we removed two cylindrical trabecular specimens (8.2 mm diameter, 10 mm height) from L2 vertebrae of 52 recently deceased donors (54–93 yrs of age; 22 men and 30 women). One core was prepared for histologic analysis of pre-existing microdamage by bulk staining in xylenol, whereas the other core was used for assessment of 3D morphology by microCT and compressive mechanical properties. The central part of the vertebral body, containing exclusively trabecular bone, was retained for analysis of pyridinoline (PYD), deoxypyridinoline (DPD) and pentosidine (PEN) content by high