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Analysis of proteomic patterns of human osteoarthritis synovial fluid using MALDI-TOF mass spectrometry
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Abstracts / Bone 47 (2010) S385–S458
significant traumatic injury. The hip fractures in the two senior groups occurred without significant traumatic injury or car accident. The Singh scoring revealed statistically significance among these three groups: The senior hip fracture female group had an average score of 2.51, 3.31 for the senior male group, and 5.71 for the female control group. Interestingly, the Singh score values were negatively associated with the serum ferritin concentrations. The senior hip fracture group had the highest ferritin value (288.57 ± 20.5), significantly higher than both the male control group (210.12 ± 16.4, p < 0.05), and the female control group (65.23 ± 13.7, p < 0.001). Conclusions: This study has two observations. Firstly, the serum ferritin concentration in the hip-fracture group is associated with the Singh scores and significantly different among study groups. Secondly, the increase of ferritin showed a positive association with age, suggesting a potential more widespread iron overload in the aged women. Iron overload in senior women with hip fracture is of high clinical relevance. The close relationship between iron overload and the bone mineral density is under investigation in a larger sample of patients with hip fracture. It is reasonable that iron overload measurement in old women with hip-fracture and the identification of underlying genetic mutations may be relevant to an appropriate therapeutic approach. doi:10.1016/j.bone.2010.09.240
251 Three-dimensional culture of osteoblast seeded on titanium alloy scaffold with controlled internal structure fabricated using electron beam melting techniques in vitro Hongxun Sang1, Guochen Li1, Lin Wang1, Xiang Li2, Yong Fan1, Lisong Wang1, Wei Lei1, Chengtao Wang2, Xin Huang1, Guoxian Pei1 1 Institute of Orthopaedics, Department of Orthopaedic Surgery, Xijing Hospital, The Fourth Military Medical University, Xian, China 2 School of Mechanical Engineering, Shanghai Jiaotong University, Shanghai, China Objectives: To explore the feasibility of culturing osteoblasts on titanium alloy scaffold with controlled internal structure fabricated using electron beam melting techniques in vitro and to investigate the effects of like-honeycomb scaffold with controlled porous structure. Methods: A direct metal rapid prototyping (RP) fabrication technique, electron beam melting (EBM) process, was utilized to fabricate porous titanium alloy scaffold with fully interconnected and controlled internal pore structure. Osteoblasts were isolated from rabbit skulls, seeded on scaffolds and cultured for up to 1, 7 and 14 days, respectively. The experiment was divided into experiment group (cells were culture with scaffold) and control group (cells alone). The growth of rabbit osteoblasts on the scaffolds was observed by inverted phase contrast microscope, scanning electron microscope, and histological section staining methods. Proliferation and differentiation of osteoblasts were determined at different time points by MTT assay and the activity of alkaline phosphatase. Results: The results showed the osteoblasts on scaffolds grow well in vitro with biological and morphological characteristics similar to those of normal osteoblasts. Good adhesion, proliferation and differentiation of the osteoblasts on the scaffold can be observed with the culture time prolonged. The number of cells on scaffolds was higher than the control group (p < 0.05). Conclusion: The porous scaffold not only has good biocompatibility, but also can improve the adhesion, growth and proliferation of osteoblasts, showing no adverse effects on the cell functions. The controlled like-honeycomb pore structure well adjusted the distribution of osteoblasts on the scaffolds. doi:10.1016/j.bone.2010.09.241
252 Analysis of proteomic patterns of human osteoarthritis synovial fluid using MALDI-TOF mass spectrometry Xiaohua Pan1, Yong Dai2, Liling Huang2, Jiakai Chen1, Bowen Lin1, Xiaofen Chen2, Feng Yi1 1 Department of Orthopaedics, The Second Clinical Medical College of Jinan University (Shenzhen People's Hospital), Shenzhen, China 2 Clinical Medical Research Center, The Second Clinical Medical College of Jinan University (Shenzhen people's hospital), Shenzhen, China Objectives: To utilize matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) in osteoarthritis research. Our aim was to find differentially expressed disease-related and condition-specific peptides in synovial fluid in knee joints of patients suffering from osteoarthritis (OA), and to develop and validate the peptide classification model for OA diagnosis. Methods: Based on the American College of Rheumatology Criteria, 30 OA cases and 10 healthy donors were enrolled and underwent analysis. Magnetic beads-based weak cation exchange chromatography (MB-WCX) was performed for sample processing and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) was conducted for peptide profile. ClinProt software 2.1 was used for data analysis and a genetic algorithm was modeled for class prediction. Results: Two protein peaks were found which may be characterized as the potential diagnostic markers for OA. Two other significant different protein peaks were fund in OA patients at medium stage comparing to the early and late stage. A Genetic Algorithm (GA) was used to establish differential diagnosis models of OA. As a result, the algorithm models marked 100% of OA, and of 97.92% medium stage of OA. Conclusion: This study demonstrated that the use of proteomics methods to identify potential biomarkers of OA is possible, and the identified potential biomarkers may be potential markers for diagnosis and monitoring the progression of OA. doi:10.1016/j.bone.2010.09.242
253 Trabecular bone phenotype in cartilage-specific inactivation of the Rac1 gene in mice Guoyan Wang, Frank Beier Skeletal Biology Group, Department of Physiology & Pharmacology, University of Western Ontario, London, Ontario, Canada Objectives: To understand the trabecular bone responses to the chondrocyte-specific deletion of Rac1 gene. We investigated the regulation of communication between skeletal cell types, such as chondrocytes with osteoblasts and osteoclasts. Methods: In this study, we used cartilage-specific inactivation Rac1 gene in mice and adenoviral infection of chondrocytes co-cultured with osteoblasts/ osteoclasts in vitro. We characterized the trabecular bone phenotype of Rac1 deficient chondrocytes using immunohistochemistry and, realtime PCR. Results: Cartilage-specific deletion of Rac1 gene results in a loss of trabecular structure and a delay in secondary ossification. Our results showed that six osteoclast marker genes are decreased in Rac1KO mice but not changed when osteoclasts are co-cultured with chondrocytes in vitro. In contrast, four osteoblast markers showed decreased expression in Rac1 deficient mice and ALP activity and mineralization decreased in co-culture with Rac1-deficient chondrocytes in vitro. In contrast, MMP13 and Runx2 expression increased at the interface between hypertrophic cartilage and the trabecular zone. Conclusion: The down-regulation of most osteoclast and osteoblast marker genes in chondrocyte-specific Rac1KO mice suggested that trabecular defects are due to altered communication from chondrocytes to other cells. Rac1KO mice develop a significant bone phenotype
Abstracts / Bone 47 (2010) S385–S458
significant traumatic injury. The hip fractures in the two senior groups occurred without significant traumatic injury or car accident. The Singh scoring revealed statistically significance among these three groups: The senior hip fracture female group had an average score of 2.51, 3.31 for the senior male group, and 5.71 for the female control group. Interestingly, the Singh score values were negatively associated with the serum ferritin concentrations. The senior hip fracture group had the highest ferritin value (288.57 ± 20.5), significantly higher than both the male control group (210.12 ± 16.4, p < 0.05), and the female control group (65.23 ± 13.7, p < 0.001). Conclusions: This study has two observations. Firstly, the serum ferritin concentration in the hip-fracture group is associated with the Singh scores and significantly different among study groups. Secondly, the increase of ferritin showed a positive association with age, suggesting a potential more widespread iron overload in the aged women. Iron overload in senior women with hip fracture is of high clinical relevance. The close relationship between iron overload and the bone mineral density is under investigation in a larger sample of patients with hip fracture. It is reasonable that iron overload measurement in old women with hip-fracture and the identification of underlying genetic mutations may be relevant to an appropriate therapeutic approach. doi:10.1016/j.bone.2010.09.240
251 Three-dimensional culture of osteoblast seeded on titanium alloy scaffold with controlled internal structure fabricated using electron beam melting techniques in vitro Hongxun Sang1, Guochen Li1, Lin Wang1, Xiang Li2, Yong Fan1, Lisong Wang1, Wei Lei1, Chengtao Wang2, Xin Huang1, Guoxian Pei1 1 Institute of Orthopaedics, Department of Orthopaedic Surgery, Xijing Hospital, The Fourth Military Medical University, Xian, China 2 School of Mechanical Engineering, Shanghai Jiaotong University, Shanghai, China Objectives: To explore the feasibility of culturing osteoblasts on titanium alloy scaffold with controlled internal structure fabricated using electron beam melting techniques in vitro and to investigate the effects of like-honeycomb scaffold with controlled porous structure. Methods: A direct metal rapid prototyping (RP) fabrication technique, electron beam melting (EBM) process, was utilized to fabricate porous titanium alloy scaffold with fully interconnected and controlled internal pore structure. Osteoblasts were isolated from rabbit skulls, seeded on scaffolds and cultured for up to 1, 7 and 14 days, respectively. The experiment was divided into experiment group (cells were culture with scaffold) and control group (cells alone). The growth of rabbit osteoblasts on the scaffolds was observed by inverted phase contrast microscope, scanning electron microscope, and histological section staining methods. Proliferation and differentiation of osteoblasts were determined at different time points by MTT assay and the activity of alkaline phosphatase. Results: The results showed the osteoblasts on scaffolds grow well in vitro with biological and morphological characteristics similar to those of normal osteoblasts. Good adhesion, proliferation and differentiation of the osteoblasts on the scaffold can be observed with the culture time prolonged. The number of cells on scaffolds was higher than the control group (p < 0.05). Conclusion: The porous scaffold not only has good biocompatibility, but also can improve the adhesion, growth and proliferation of osteoblasts, showing no adverse effects on the cell functions. The controlled like-honeycomb pore structure well adjusted the distribution of osteoblasts on the scaffolds. doi:10.1016/j.bone.2010.09.241
252 Analysis of proteomic patterns of human osteoarthritis synovial fluid using MALDI-TOF mass spectrometry Xiaohua Pan1, Yong Dai2, Liling Huang2, Jiakai Chen1, Bowen Lin1, Xiaofen Chen2, Feng Yi1 1 Department of Orthopaedics, The Second Clinical Medical College of Jinan University (Shenzhen People's Hospital), Shenzhen, China 2 Clinical Medical Research Center, The Second Clinical Medical College of Jinan University (Shenzhen people's hospital), Shenzhen, China Objectives: To utilize matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) in osteoarthritis research. Our aim was to find differentially expressed disease-related and condition-specific peptides in synovial fluid in knee joints of patients suffering from osteoarthritis (OA), and to develop and validate the peptide classification model for OA diagnosis. Methods: Based on the American College of Rheumatology Criteria, 30 OA cases and 10 healthy donors were enrolled and underwent analysis. Magnetic beads-based weak cation exchange chromatography (MB-WCX) was performed for sample processing and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) was conducted for peptide profile. ClinProt software 2.1 was used for data analysis and a genetic algorithm was modeled for class prediction. Results: Two protein peaks were found which may be characterized as the potential diagnostic markers for OA. Two other significant different protein peaks were fund in OA patients at medium stage comparing to the early and late stage. A Genetic Algorithm (GA) was used to establish differential diagnosis models of OA. As a result, the algorithm models marked 100% of OA, and of 97.92% medium stage of OA. Conclusion: This study demonstrated that the use of proteomics methods to identify potential biomarkers of OA is possible, and the identified potential biomarkers may be potential markers for diagnosis and monitoring the progression of OA. doi:10.1016/j.bone.2010.09.242
253 Trabecular bone phenotype in cartilage-specific inactivation of the Rac1 gene in mice Guoyan Wang, Frank Beier Skeletal Biology Group, Department of Physiology & Pharmacology, University of Western Ontario, London, Ontario, Canada Objectives: To understand the trabecular bone responses to the chondrocyte-specific deletion of Rac1 gene. We investigated the regulation of communication between skeletal cell types, such as chondrocytes with osteoblasts and osteoclasts. Methods: In this study, we used cartilage-specific inactivation Rac1 gene in mice and adenoviral infection of chondrocytes co-cultured with osteoblasts/ osteoclasts in vitro. We characterized the trabecular bone phenotype of Rac1 deficient chondrocytes using immunohistochemistry and, realtime PCR. Results: Cartilage-specific deletion of Rac1 gene results in a loss of trabecular structure and a delay in secondary ossification. Our results showed that six osteoclast marker genes are decreased in Rac1KO mice but not changed when osteoclasts are co-cultured with chondrocytes in vitro. In contrast, four osteoblast markers showed decreased expression in Rac1 deficient mice and ALP activity and mineralization decreased in co-culture with Rac1-deficient chondrocytes in vitro. In contrast, MMP13 and Runx2 expression increased at the interface between hypertrophic cartilage and the trabecular zone. Conclusion: The down-regulation of most osteoclast and osteoblast marker genes in chondrocyte-specific Rac1KO mice suggested that trabecular defects are due to altered communication from chondrocytes to other cells. Rac1KO mice develop a significant bone phenotype