yamada harumoto

Objective: We performed comprehensive proteomic analyses of articular cartilage by using the isobaric tags for relative and absolute quantitation (iTRAQ) method, and searched for candidate biomarkers for osteoarthritis (OA). Methods: Articular cartilage was collected from patients with OA or femoral neck fracture for the control group. Molecular variations were detected by the iTRAQ method, and quantitative analyses were performed by western blot. Results: Using the iTRAQ method, we identified 76 proteins with different expression levels in OA patients and the control group. Among these proteins, we selected LECT2 (leukocyte cell-derived chemotaxin-2), BAALC (brain and acute leukemia, cytoplasmic), and PRDX6 (peroxiredoxin-6), which had not been reported as biomarkers for OA. Conclusions: Use of these proteins in combination with conventional OA biomarkers may better reflect the grade and prognosis of OA.

Heterotopic ossification (HO) is defined as the formation of ectopic bone in soft tissue outside the skeletal tissue. HO is thought to result from aberrant differentiation of osteogenic progenitors within skeletal muscle. However, the precise origin of HO is still unclear. Skeletal muscle contains two kinds of progenitor cells, myogenic progenitors and mesenchymal progenitors. Myogenic and mesenchymal progenitors in human skeletal muscle can be identified as CD56(+) and PDGFR alpha(+) cells, respectively. The purpose of this study was to investigate the osteogenic differentiation potential of human skeletal muscle-derived progenitors. Both CD56(+) cells and PDGFR alpha(+) cells showed comparable osteogenic differentiation potential in vitro. However, in an in vivo ectopic bone formation model, PDGFR alpha(+) cells formed bone-like tissue and showed successful engraftment, while CD56(+) cells did not form bone-like tissue and did not adapt to an osteogenic environment. Immunohistological analysis of human HO sample revealed that many PDGFR alpha(+) cells were localized in proximity to ectopic bone formed in skeletal muscle. MicroRNAs (miRNAs) are known to regulate many biological processes including osteogenic differentiation. We investigated the participation of miRNAs in the osteogenic differentiation of PDGFR alpha(+) cells by using microarray. We identified miRNAs that had not been known to be involved in osteogenesis but showed dramatic changes during osteogenic differentiation of PDGFR alpha(+) cells. Upregulation of miR-146b-5p and -424 and downregulation of miR-7 during osteogenic differentiation of PDGFR alpha(+) cells were confirmed by quantitative real-time RT-PCR. Inhibition of upregulated miRNAs, miR-146b-5p and -424, resulted in the suppression of osteocyte maturation, suggesting that these two miRNAs have the positive role in the osteogenesis of PDGFR alpha(+) cells. Our results suggest that PDGFR alpha(+) cells may be the major source of HO and that the newly identified miRNAs may regulate osteogenic differentiation process of PDGFR alpha(+) cells.

Synovial tissue in rheumatoid arthritis (RA) shows dense infiltration of plasmacytes. The purpose of the present study is to identify and localize autoantibodies produced in these immunocytes in RA synovitis. We developed a novel screening system for detecting specific autoantigens. Protein antigens recognized by antibodies in the serum and synovial tissue extract from five RA patients were screened with the AlphaScreen method. For screening, a biotinylated human autoantigen library was constructed by the wheat germ cell-free protein synthesis system. The AlphaScreen analysis of 2183 proteins detected a limited number of antigens reactive with the serum and synovial tissue extract. Eighteen biotinylated proteins, containing top five showing high signals in each synovitis tissue extract, were utilized as probes for the enzyme-labeled antigen method, in order to visualize the site of specific antibody production in synovial lesions. Specific antibodies against two proteins, tripartite motif-containing 21 (TRIM21, also known as SSA/Ro52) and F-box only protein 2 (FBXO2), were visualized in the cytoplasm of plasmacytes in two RA synovitis lesions, respectively. Absorption experiments using unlabeled proteins confirmed the specificity of staining. No positive signals against these two proteins were identified in the additionally evaluated RA and osteoarthritis synovial lesions. The present study indicated 1) the usefulness of screening the human autoantigen library with the AlphaScreen assay for detecting autoantibodies in RA synovitis, and 2) the applicability of biotinylated proteins to the enzyme-labeled antigen method for visualizing the site of autoantibody production within the lesion.

Diabetes mellitus causes neuromusculoskeletal disorders characterized by abnormalities of nervous tissue, joint and bone. Early diagnosis and prevention of disease progression is difficult in cases of neuropathic arthropathy of the knee in diabetes. We report the case of a patient with type 1 diabetes mellitus who developed an insufficiency fracture of the medial part of the proximal tibia, which was viewed as an early finding of neuropathic arthropathy of the knee. In surgical treatment of the fracture, allograft bone transplantation and internal fixation were performed after curettage of the pathologically fragile lesion. Postoperatively, radiological findings have showed complete allograft bone incorporation and no evidence of degenerative changes. Recognition of an insufficiency fracture of the knee as an early indicator of neuropathic arthropathy and prompt treatment of the fracture using allograft bone transplantation could result in joint preservation. (C) 2012 Elsevier B.V. All rights reserved.

We have selected heat-treated bone allografts as the graft material since the Tokai Bone Bank, the first regional bone bank in Japan, was established in 1992. In this study, we examined changes in bone mineral density (BMD), and morphology observed by magnetic resonance imaging (MRI), and histological findings of bone grafts in cases followed up for 7-10 years after bone grafting to grasp the remodeling of heat-treated cortical bone allografts for posterior lumber interbody fusion (PLIF). BMD of bone grafts was reduced by half at 10 years after grafting. MRI revealed that bone grafts were indistinguishable initially in only 22.2% of cases, whereas after a lengthy period of 10 years distinguishable in many cases. Histologically, new bone formation at the graft-host interface was observed earlier, at 1 year after grafting, than that at the periphery of canals in the specimens. The laminated structure of the cortical bone eroded over time, and fragmented bone trabeculae were observed in the specimens at 8 years or longer after grafting, though necrotic bone still remained in some sites.

Aim: This study compares the effect of bisphosphonate and intermittent PTH administration on haversian remodeling in cortical bone allografts in rabbits. Materials and methods: An intercalary heat-treated cortical bone allograft was applied to a segment skeletal defect in the left femur of Japanese white rabbits. The rabbits were randomly assigned to one of three groups: the vehicle control group (CNT); the bisphosphonate group (B group); and the intermittent PTH treatment group (P group). Periodic radiographic evaluation was performed and peripheral quantitative computerized tomography (pQCT) was used to evaluate the total bone area (Area), bone mineral density (BMD), and bone mineral content (BMC). The allografts also underwent histological examination. Results: The P group was radiographically superior in the latter stage, compared with the other groups. pQCT analysis of the allografts showed that the B group had a significantly higher Area and BMC. These parameters in the latter stage were significantly lower in the P group than in the other groups. The allograft of the B group was histologically mostly necrotic bone, whereas allograft of the P group showed abundant newly formed bone. Conclusion: In rabbits, bisphosphonate prevents resorption, but suppresses remodeling and incorporation; by contrast, PTH increases resorption and accelerates allograft remodeling and incorporation. Based on our preliminary data, we suggest that further research on the manner of administration of bisphosphonate and PTH - which have contrasting effects can be beneficial in maintaining bone strength and in regulating remodeling and allograft incorporation. (J. Endocrinol. Invest. 35: 139-145, 2012) (C) 2012, Editrice Kurtis

Purpose Human multicentric osteosarcoma (HMOS) is a rare, aggressive variant of osteosarcoma, and its etiology is not clear. We used newly established HMOS cells, which were derived from primary (HMOS-A) and secondary (HMOS-P) lesions, respectively, to perform a basic study analyzing the cellular biology and gene expression of HMOS. Methods We performed a cell growth assay, an invasion assay, DNA microarray analysis, quantitative real-time RT-PCR (Qrt-PCR), and a telomerase assay and compared the results between HMOS-A, HMOS-P, and human osteosarcoma (HOS) cell lines (MNNG-HOS and Saos-2). Results The cell biological analysis revealed that HMOSA and HMOS-P had similar characteristics to Saos-2, and the invasion assay showed that they had similar characteristics to MNNG-HOS. The DNA microarray study showed that the gene expression profiles of HMOS-A and HMOS-P were similar to that of MNNG-HOS, but the overexpression of MMP-2, MMP-9, and MT1-MMP was observed in HMOS-A and HMOS-P, which was correlated with the invasiveness of the extracellular matrix, and collagen type-4 (COL-4) and VEGF were also detected. HMOS-A and HMOS-P showed low telomerase activity similar to Saos-2, which are known to be telomerase negative, but a similar telomere length and telomerase protein to MNNG-HOS. Conclusions HMOS-A and HMOS-P demonstrated strong invasive ability, and their gene expression profiles correlated with the invasiveness of the extracellular matrix. Their telomerase activity was low, but they did not shown the typical features of alternative lengthening of telomeres (ALT). HMOS-A and HMOS-P are useful models for further study of various biological aspects and therapeutic manipulation of HMOS.

Background We conducted a nationwide epidemiologic study regarding hip osteoarthritis (OA) in Japan, and a previous report found these patients to be unique in comparison to Caucasians. This report focused on the data regarding each hip joint, and the involvement of acetabular dysplasia with hip OA was analyzed. Methods Seven hundred twenty OA hips were examined. Sixty-five joints with osteonecrosis of the femoral head and 215 non-OA contralateral joints of the unilateral patients were examined as controls. The revised system of stage classification for hip OA of the Japanese Orthopedic Association (JOA) was used according to the reproducibility in order to ensure reliable data from the multiple institutions. The acetabular dysplasia indexes were also chosen according to the reproducibility and measured in the radiograph of bilateral hip joints. The clinical score was assessed using the JOA scoring system. The relative risk of the grade of acetabular dysplasia indexes for hip OA was calculated as the odds ratio and the 95% confidence interval. Results The stage of the OA joints deteriorated with increasing age. The clinical scores also decreased. The grade of the acetabular dysplasia indexes of the OA joints was significantly higher than that of the control joints. Each index of acetabular dysplasia demonstrated significantly increased odds ratios for hip OA. Among the OA joints, the deterioration of the OA stage was found to be significantly associated with an increasing grade of acetabular dysplasia. The odds ratio for OA deterioration in the acetabular dysplasia index was also obtained. The joints of females tended to have a higher grade and prevalence of acetabular dysplasia than those of males. Conclusions These findings confirmed a high prevalence of acetabular dysplasia in hip OA joints in Japan. Acetabular dysplasia was one of the most important factors associated with hip OA.