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Sox9, a master regulator of cartilage development, controls the cell fate decision to differentiate from mesenchymal to chondrogenic cells. In addition, Sox9 regulates the proliferation and differentiation of chondrocytes, as well as the production of cartilage-specific proteoglycans. The existence of Sox9-independent mechanisms in cartilage development remains to be determined. Here, we attempted to identify genes involved in such putative mechanisms via microarray analysis using a mouse chondrogenic cell line, N1511. We first focused on transcription factors that exhibited upregulated expression following Bmp2 treatment, which was not altered by subsequent treatment with Sox9 siRNA. Among these, we selected positive regulators for chondrogenesis and identified Iroquois-related homeobox 3 ( Irx3) as one of the candidate genes. Irx3 expression gradually increased with chondrocyte terminal differentiation in a reciprocal manner to Sox9 expression, and promoted the chondrogenic differentiation of mesenchymal cells upon Bmp2 treatment. Furthermore, Irx3 partially rescued impaired chondrogenesis by upregulating the expression of epiphycan and lumican under reduced Sox9 expression. Finally, Irx3 was shown to act in concert with Bmp2 signaling to activate the p38 MAPK pathway, which in turn stimulated Sox9 expression, as well as the expression of epiphycan and lumican in a Sox9-independent manner. These results indicate that Irx3 represents a novel chondrogenic factor of mesenchymal cells, acts synergistically with Bmp2-mediated signaling, and regulates chondrogenesis independent of the transcriptional machinery associated with Sox9-mediated regulation. [ABSTRACT FROM AUTHOR]

Aim Cardiovascular disease is one of the complications of rheumatoid arthritis ( RA). We researched the morbidity and severity of existing carotid atherosclerosis plaque and associated risk factors in patients with RA. Method This study included 413 participants, including 208 patients with RA and 205 age- and sex-matched healthy volunteers. Carotid ultrasound, clinical data collection and assessment of cardiovascular risk factors were performed. Atherosclerotic plaque was defined as an intima-media thickness ≥ 1.1 mm. Severity of plaque was assessed by plaque score, defined as the sum of the maximal thickness of all plaques in bilateral carotid arteries. Results Data were analyzed from 200 patients with RA and 202 controls. Carotid plaque was observed more frequently in patients with RA than controls (47.0 vs. 36.1%, P = 0.027). Moreover, plaque score was significantly higher in RA patients ( P = 0.032). In logistic regression analysis, RA represented an independent risk factor for the presence of plaque (adjusted odds ratio, 1.68; 95% confidence interval, 1.03-2.74). Comparing RA patients with and without plaque, anti-cyclic citrullinated peptide (anti- CCP) antibodies titer was significantly higher in patients with plaque (315.8 ± 454.1 U/ mL) than in patients without (165.7 ± 281.1 U/ mL; P = 0.005). Moreover, multiple linear regression analysis clarified that anti- CCP antibody titer was associated with plaque score in patients with RA. Conclusion High prevalence of any carotid plaques and severe carotid plaques were more frequent in patients with RA. High titer of anti- CCP antibodies represented a risk factor for severe carotid atherosclerotic plaque in patients with RA. [ABSTRACT FROM AUTHOR]

Objective. To determine whether and how the transcription factor Erg participates in the genesis, establishment, and maintenance of articular cartilage. Methods. Floxed Erg mice were mated with Gdf5-Cre mice to generate conditional mutants lacking Erg in their joints. Joints of mutant and control mice were subjected to morphologic and molecular characterization and also to experimental surgically induced osteoarthritis (OA). Gene expression, promoter reporter assays, and gain- and loss-of-function in vitro tests were used to characterize molecular mechanisms of Erg action. Results. Conditional Erg ablation did not elicit obvious changes in limb joint development and overall phenotype in juvenile mice. However, as mice aged, joints of mutant mice degenerated spontaneously and exhibited clear OA-like phenotypic defects. Joints in juvenile mutant mice were more sensitive to surgically induced OA and became defective sooner than operated joints in control mice. Global gene expression data and other studies identified parathyroid hormone–related protein (PTHrP) and lubricin as possible downstream effectors and mediators of Erg action in articular chondrocytes. Reporter assays using control and mutated promoter-enhancer constructs indicated that Erg acted on Ets DNA binding sites to stimulate PTHrP expression. Erg was up-regulated in severely affected areas in human OA articular cartilage but remained barely appreciable in areas of less affected cartilage. Conclusion. The study shows for the first time that Erg is a critical molecular regulator of the endurance of articular cartilage during postnatal life and that Erg can mitigate spontaneous and experimental OA. Erg appears to do this through regulating expression of PTHrP and lubricin, factors known for their protective roles in joints. [ABSTRACT FROM AUTHOR]

To investigate new methods that can decrease the duration of bone transport (BT) distraction osteogenesis, we injected composite materials containing recombinant human bone morphogenetic protein-2 (BMP-2) and induced the generation of a callus bridge by rapid segmental transport (4 mm/day) in a rabbit bone defect model. The composite materials consisted of BMP-2 (0, 30, or 100 µg), β-tricalcium phosphate powder (βTCP, 100 mg/animal; particle size, <100 µm), and polyethylene glycol (PEG; 40 mg/animal). A paste of equivalent composition was percutaneously injected at the lengthening and the docking sites after surgery and after BT, respectively. The radiographic, mechanical, and histological examinations 12 weeks post-operative revealed that the generation of bridging callus in the presence and in the absence of BMP-2 was significantly different. The callus mass in the bone defect region was adequately and consistently developed in the presence of 100 µg of BMP (administered for 6 weeks), and the bones were consolidated in 12 weeks. Such an adequate callus formation was not observed in the control animals without BMP-2 treatment. The result of this experimental study suggests the potential application of BMP-2 in accelerating callus formation and in enabling rapid bone transporting, thereby shortening the treatment period for the repair of diaphyseal bone defects by distraction osteogenesis. © 2010 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 29:452-456, 2011 [ABSTRACT FROM AUTHOR]

Articular cartilage defects that do not repair spontaneously induce osteoarthritic changes in joints over a long period of observation. In this study, we examined the usefulness of transplanting culture-expanded bone marrow mesenchymal cells into osteochondral defects of joints with cartilage defects. First, we performed experiments on rabbits and up on obtaining good results proceeded to perform the experiments on humans. Macroscopic and histological repair with this method was good, and good clinical results were obtained although there was no significant difference with the control group. Recent reports have indicated that this procedure is comparable to autologous chondrocyte implantation, and concluded that it was a good procedure because it required one step less than that required by surgery, reduced costs for patients, and minimized donor site morbidity. Although some reports have previously shown that progenitor cells formed a tumor when implanted into immune-deficient mice after long term in vitro culture, the safety of the cell transplantation was confirmed by our clinical experience. Thus, this procedure is useful, effective, and safe, but the repaired tissues were not always hyaline cartilage. To obtain better repair with this procedure, treatment approaches using some growth factors during in vitro culture or gene transfection are being explored. J. Cell. Physiol. 225: 291–295, 2010. © 2010 Wiley-Liss, Inc. [ABSTRACT FROM AUTHOR]

The objective of this study was to evaluate the effects of the mechanical environment on the formation of cartilage tissue in transplanted embryonic stem (ES) cells. Full-thickness osteochondral defects were created on the patella groove of SD rats, and ES cells (CCE ES cells obtained from 129/Sv/Ev mice and Green ES FM260 ES cells obtained from 129SV [D3] - Tg [NCAG-EGFP] CZ-001-FM260Osb mice) were transplanted into the defects embedded in collagen gel. The animals were randomly divided into either the joint-free group (JF group) or the joint-immobilized group (JI group) for 3 weeks after a week postoperatively. The results showed that cartilage-like tissue formed in the defects of the JF group whereas large teratomatous masses developed in the defects of the JI group. Some parts of the cartilage-like tissue and the teratomatous masses were positively stained with immunostain for GFP when the Green ES FM260 ES cells were transplanted. It is suggested that the environment plays an important role for ES cells in the process of repairing cartilage tissue in vivo. © 2007 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 26:10-17, 2008 [ABSTRACT FROM AUTHOR]

1. We have confirmed theDiabetes Mellitus OLETF type I(Dmo1) effect on hyperphagia, dyslipidaemia and obesity in the Otsuka Long-Evans Tokushima Fatty (OLETF) strain. The critical interval was narrowed down to 570 kb betweenD1Got258top162CA1by segregation analyses using congenic lines.2. Within the critical 570 kb region of theDmo1locus, we identified the G-protein-coupled receptor geneGPR10as the causative gene mutated in the OLETF strain. The ATG translation initiation codon ofGPR10is changed into ATA in this strain and, so, is unavailable for the initiation of translation.3. The GPR10 protein has a cognate ligand, namely prolactin-releasing peptide (PrRP). Centrally administered PrRP suppressed the food intake of congenic rats that have a Brown Norway derivedDmo1region (i.e. with wild-typeGPR10), but did not suppress that of the OLETF strain, indicating that GPR10 is without function and could explain hyperphagia in the OLETF strain.4. Moreover, when restricted in food volume to the same level consumed by the congenic strain, OLETF rats showed few differences in the parameters of dyslipidaemia and obesity compared with congenic strains.5. Taken together, these results demonstrate that the mutated GPR10 receptor is responsible for the hyperphagia leading to obesity and dyslipidaemia in the obese diabetic strain rat. [ABSTRACT FROM AUTHOR]

Examines the single-allele correction of a genetic defect of the Dmo1 locus. Effect of the Dmo1 locus on lipid metabolism; Involvement of Dmo1 in glucose homeostasis after loading; Establishment of congenic rats by the BC4 generation.

Focuses on the disorders of lipid metabolism in diabetic patients. Study on the development diabetes; Examination on the elevation of the plasma levels; Types of diabetes.

1. Dmo1 ( Diabetes Mellitus OLETF type I) is a major quantitative trait locus for dyslipidaemia, obesity and diabetes phenotypes of male Otsuka Long Evans Tokushima Fatty (OLETF) rats. 2. Our congenic lines, produced by transferring Dmo1 chromosomal segments from the non-diabetic Brown Norway (BN) rat into the OLETF strain, have confirmed the strong, wide-range therapeutic effects of Dmo1 on dyslipidaemia, obesity and diabetes in the fourth (BC4) and fifth (BC5) generations of congenic animals. Analysis of a relatively small number of BC5 rats ( n = 71) suggested that the critical Dmo1 interval lies within a < 4.9 cM region between D1Rat461 and D1Rat459. 3. To confirm the assignment of the Dmo1 critical interval , we intercrossed BC5 animals to produce a larger study population (BC5:F1 males; n = 406). For the present study, we used bodyweight at 18 weeks of age as an index of obesity; this phenotype is representative of the closely associated dyslipidaemia and hyperglycaemia phenotypes. 4. Interval mapping assigned logarithm of odds (LOD) peaks at the D1Rat90 marker (LOD = 9.11). One LOD support interval lies within the < 1.7 cM region between D1Rat461 and D1Rat459. 5. This large intercross study confirms that Dmo1 is likely localized within the interval . [ABSTRACT FROM AUTHOR]

Objective: To evaluate whether microbubble-enhanced ultrasound (US) treatment promotes the delivery of methotrexate (MTX) into synovial cells and the enhanced antiinflammatory effects of intraarticular MTX therapy in a rabbit arthritis model., Methods: Arthritis was induced in both knees of 53 rabbits by immunization with ovalbumin. MTX including a microbubble agent was then injected into the left and right knee joints, and the right knees were exposed to US (MTX+/US+ group), while the left knees were not (MTX+/US- group). The knee joints were evaluated histologically in 7 rabbits at 5 time points up to day 56. Quantitative gene expression of interleukin-1beta (IL-1beta) in synovial tissue was measured on days 7 and 28. Eight rabbits were used for the measurement of MTX concentration in synovial tissue 12 hours after treatment. To evaluate the effect of microbubble-enhanced US treatment in the absence of MTX, only the microbubble agent was injected into the left and right knee joints of 10 rabbits with or without US exposure, and these animals were evaluated histologically on days 7 and 28., Results: The MTX concentration in synovial tissue was significantly higher in the MTX+/US+ group than in the MTX+/US- group. Synovial inflammation was less prominent in the MTX+/US+ group compared with the MTX+/US- group, judging from the results of the histologic evaluation and the gene expression levels of IL-1beta in synovial tissue. It also appeared that microbubble-enhanced US exposure itself did not affect inflammation., Conclusion: Microbubble-enhanced US exposure promoted the uptake of MTX into synovial cells, which resulted in enhancement of the antiinflammatory effects of the intraarticular MTX injection. These results suggest that application of this technique may have clinical benefit.

1. We have confirmed the Diabetes Mellitus OLETF type I (Dmo1) effect on hyperphagia, dyslipidaemia and obesity in the Otsuka Long-Evans Tokushima Fatty (OLETF) strain. The critical interval was narrowed down to 570 kb between D1Got258 to p162CA1 by segregation analyses using congenic lines. 2. Within the critical 570 kb region of the Dmo1 locus, we identified the G-protein-coupled receptor gene GPR10 as the causative gene mutated in the OLETF strain. The ATG translation initiation codon of GPR10 is changed into ATA in this strain and, so, is unavailable for the initiation of translation. 3. The GPR10 protein has a cognate ligand, namely prolactin-releasing peptide (PrRP). Centrally administered PrRP suppressed the food intake of congenic rats that have a Brown Norway derived Dmo1 region (i.e. with wild-type GPR10), but did not suppress that of the OLETF strain, indicating that GPR10 is without function and could explain hyperphagia in the OLETF strain. 4. Moreover, when restricted in food volume to the same level consumed by the congenic strain, OLETF rats showed few differences in the parameters of dyslipidaemia and obesity compared with congenic strains. 5. Taken together, these results demonstrate that the mutated GPR10 receptor is responsible for the hyperphagia leading to obesity and dyslipidaemia in the obese diabetic strain rat.

Objective: To examine the ability of cartilage-like tissue, generated ectopically in a diffusion chamber using recombinant human bone morphogenetic protein 2 (rHuBMP-2), to repair cartilage defects in rats., Methods: Muscle-derived mesenchymal cells were prepared by dissecting thigh muscles of 19-day postcoital rat embryos. Cells were propagated in vitro in monolayer culture for 10 days and packed within diffusion chambers (10(6)/chamber) together with type I collagen (CI) and 0, 1, or 10 microg rHuBMP-2, and implanted into abdominal subfascial pockets of adult rats. Tissue pellets were harvested from the diffusion chambers at 2 days to 6 weeks after implantation, and examined by histology, by reverse transcription-polymerase chain reaction (PCR) for aggrecan, CII, CIX, CX, and CXI, MyoD1, and core binding factor a1/runt-related gene 2, and by real-time PCR for CII. Tissue pellets generated in the chamber 5 weeks after implantation were transplanted into a full-thickness cartilage defect made in the patellar groove of the same strain of adult rat., Results: In the presence of 10 microg rHuBMP-2, muscle-derived mesenchymal cells expressed CII messenger RNA at 4 days after transplantation, and a mature cartilage mass was formed 5 weeks after transplantation in the diffusion chamber. Cartilage was not formed in the presence of 1 microg rHuBMP-2 or in the absence of rHuBMP-2. Defects receiving cartilage engineered with 10 microg rHuBMP-2 were repaired and restored to normal morphologic condition within 6 months after transplantation., Conclusion: This method of tissue engineering for repair of articular defects may preclude the need to harvest cartilage tissue prior to mosaic arthroplasty or autologous chondrocyte implantation. Further studies in large animals will be necessary to validate this technique for application in clinical practice.