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The WP9QY peptide (W9) consists of nine amino acids. It binds to RANKL and blocks RANKL-induced increases in bone resorption and osteoclastogenesis. W9 has a unique effect on the coupling mechanism between osteoclasts and osteoblasts, which promotes bone formation while working to suppress bone resorption. In this study, with the aim of clinical application of W9 for fracture treatment, we aimed to clarify the bone repair-promoting effect of W9 when administered locally to a rat femur model of delayed union. Using Sprague-Dawley rats, a model of delayed union was created in the right femur by cauterizing the periosteum. Injection of W9 (1 mg in 100 μl) or phosphate-buffered saline (PBS) (100 μl) at the fracture site was performed at the operation and every week thereafter until death (sacrifice). The bone union rate was 14% in the PBS group and 57% in the W9 group at 8 weeks postoperatively. The X-ray score of the W9 group was significantly higher than that of the PBS group at 8 weeks postoperatively. When bone morphometry was analyzed by micro-computed tomography (CT), total callus volume (TV) and mineralized callus bone volume (BV) were measured. TV showed no significant difference between the two groups, but BV/TV was significantly higher in the W9 group. This finding suggests that local administration of W9 can promote bone maturation from callus and can be considered to contribute to fracture healing. These results reveal that W9 has an effect on fractures of promoting healing and could be applied as a fracture treatment. [ABSTRACT FROM AUTHOR]

Cartilage regeneration treatments using stem cells are associated with problems due to the cell source and the difficulty of delivering the cells to the cartilage defect. We consider labeled induced pluripotent stem (iPS) cells to be an ideal source of cells for tissue regeneration, and if iPS cells could be delivered only into cartilage defects, it would be possible to repair articular cartilage. Consequently, we investigated the effect of magnetically labeled iPS (m-iPS) cells delivered into an osteochondral defect by magnetic field on the repair of articular cartilage. iPS cells were labeled magnetically and assessed for maintenance of pluripotency by their ability to form embryoid bodies in vitro and to form teratomas when injected subcutaneously into nude rats. These cells were delivered specifically into cartilage defects in nude rats using a magnetic field. The samples were graded according to the histologic grading score for cartilage regeneration. m-iPS cells differentiated into three embryonic germ layers and formed teratomas in the subcutaneous tissue. The histologic grading score was significantly better in the treatment group compared to the control group. m-iPS cells maintained pluripotency, and the magnetic delivery system proved useful and safe for cartilage repair using iPS cells. [ABSTRACT FROM AUTHOR]

Aiming for the clinical application of cartilage regeneration, the xeno-free cultivation method to obtain a scaffold-free cartilage-like disc-shaped cell sheet using mesenchymal stem cells (MSCs) derived from human bone marrow without the shrinkage of the sheet was investigated. MSCs were inoculated into Cell Culture Insert (0.3 cm2, pore size; 0.4 μm, pore density; 1.0 × 108/cm2) using serum-free chondrogenic differentiation medium containing TGF-β3, IGF-1 and dexamethasone or other modified media, and cultured at 37°C in 5% CO2 for 3 weeks. Sheet thickness, cartilage specific genes expression, ECM accumulation were determined, and the sections of sheets were stained with alcian blue. A novel mixed medium consisting of a growth medium (10% FCS) with a serum-free chondrogenic differentiation medium could prevent the shrinkage of the sheet and produced a disc-shaped cell sheet. The depth of the sheet was approximately 0.7 mm and the gene expression levels were higher than those in cells in normal human cartilage. The use of human serum instead of FCS did not cause shrinkage and did not decrease the accumulation levels of sGAG and type 2 collagen in the sheet. The cultivation of MSCs grown with completely xeno-free materials using the mixed medium containing human serum in a cell culture insert showed a sheet depth of 1.0 mm and gene expression levels higher than those in normal cartilage. The scaffold-free and xeno-free cartilage-like cell sheet was successfully formed without shrinkage using human bone marrow MSCs and the chondrogenic differentiation medium containing human serum. [ABSTRACT FROM AUTHOR]

Purpose: Although objective evaluation of articular cartilage is important for assessing the outcome of surgical treatment, no reliable method has yet been developed. It has recently been reported that quantitative ultrasound is applicable for assessment of living human cartilage. The purpose of this study was to investigate whether quantitative ultrasound is able to detect subtle changes in articular cartilage, as well as age-related changes in normal cartilage during arthroscopic surgery. Methods: Thirty-six patients with knee injury underwent ultrasonic evaluation of the articular cartilage during arthroscopy. The reflex echogram from the cartilage was converted to a wavelet map using wavelet transformation. As a quantitative index on the wavelet map, the maximum magnitude was selected. Whether or not the cartilage was damaged was judged from the arthroscopic view of the articular surface. Both normal sites (33 sites) and damaged areas (Outerbridge grade I-II, 11 sites) were measured. Results: The average maximum magnitude values for normal and damaged cartilage were 4.2 ± 1.6 and 1.4 ± 0.6, respectively. The maximum magnitude was significantly higher in intact, than in injured, cartilage ( P < 0.01). The maximum magnitude for intact cartilage of the medial femoral condyle showed a significant correlation with patient age ( r = −0.66, P < 0.01). Conclusions: The present ultrasound measurement system offers potential for the detection of subtle change in cartilage. The maximum magnitude is particularly useful for quantitative assessment of medial femoral condyle articular cartilage. This ultrasound measurement system is useful for diagnosis of degenerative cartilage at an early stage. Level of evidence: Diagnostic, Level III. [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]

Effective therapies for the regeneration of large osteochondral defects are still lacking; however, various approaches have been used. We evaluated the efficacy of Escherichia coli-derived dimeric recombinant human BMP-2 (E-rhBMP-2) for the repair of large osteochondral defects in a rabbit model. Osteochondral defects made in the femoral patellar groove of the knee were treated by transplanting gelatin sponges onto which no or various doses of E-rhBMP-2 were loaded. The outcomes were compared with those of an untreated control group four, 12 and 24 weeks after transplantation. At early time points, the cartilage tissue was repaired in a dose-dependent manner, and bone repair was accelerated in the defects treated with high doses of E-rhBMP-2. At 24 weeks, the repair of cartilage tissue was better with E-rhBMP-2 treatment, even at low doses, than without E-rhBMP-2 treatment. Our findings suggest that the use of E-rhBMP-2 improves and accelerates the repair of osteochondral defects in a rabbit model. [ABSTRACT FROM AUTHOR]

Abstract: The noninvasive discrimination of cancer cells from normal cells in adherent culture by the measuring of the phase shift using phase-shifting laser microscopy (PLM) was investigated with the aim of noninvasive quality control of cell processing for transplantation. A human prostatic carcinoma epithelial cell line (PC-3) and human hepatocellular carcinoma cell lines (Hep3B, PLC, HLF, and Huh7) showed markedly lower phase shifts as measured by PLM than those of human prostate epithelial cells (PREC) and cryopreserved human hepatocytes, respectively, although there was no apparent difference in cell morphology between these sets of cells. Mixed cultures of PC-3 and PREC, in which the percentages of PC-3 cells were determined to be 43.3%, 14.0%, and 10.6% by immunofluorescence staining, were prepared, and the percentages were calculated to be 50.0%, 8.0%, and 2.5% using the distribution of the phase shift data of the cells, respectively. In conclusion, these adherent cancer cells can be noninvasively discriminated from normal adherent cells by phase shift measurement using PLM, and the sensitivity of the method of detecting cancer cell contamination reached 10%. [Copyright &y& Elsevier]

Etanercept (ETN), which is a recombinant human soluble tumor necrosis factor (TNF) receptor that inhibits TNF activity, is effective in the treatment of rheumatoid arthritis. We investigated the effect of ETN on recombinant human bone morphogenetic protein-2 (rhBMP-2)-induced ectopic bone formation in vivo. A block copolymer composed of poly-D,L-lactic acid with random insertion of p-dioxanone and polyethylene glycol (PLA-DX-PEG polymer) was used as the delivery system. Polymer discs (6 mm, 30 mg) containing 5 microg rhBMP-2 were implanted into the left dorsal muscle pouch of mice (n = 50). In the systemic administration groups (n = 5 per group), ETN was subcutaneously injected (25 mg/human = 12.5 microg/mouse) twice per week in a dose-dependent manner (placebo, 12.5 x 10(-3), 12.5 x 10(-1), 12.5, 125 microg), whereas a single dose of ETN (placebo, 12.5 x 10(-3), 12.5 x 10(-1), 12.5, 125 microg) was embedded in each rhBMP-2 polymer disc in the local administration groups (n = 5 per group). Three weeks after implantation, the mice were killed and the implants were analyzed. Implants in the optimally dosed groups had increased radiodensity, which was consistent with a significant increase in bone mineral content of the ossicles. Bone histomorphology revealed a significant increase in bone volume/total volume, number of osteoblasts, osteoblast surface/bone surface, and a significant decrease in the number of osteoclasts, osteoclast surface/bone surface in the optimal dosed systemic and locally administered groups. These data suggest that the optimal dose of ETN, administered either systemically or locally, enhanced the bone-inducing capacity of BMP with no apparent adverse systemic effects. [ABSTRACT FROM AUTHOR]

Objective. There have been a large number of reports on alterations in the serum level of keratan sulphate (KS), a potential marker of articular cartilage degeneration in patients with arthropathy. Such studies have commonly employed ELISA using the anti-KS monoclonal antibody 1/20/5D4 (5D4-ELISA) to determine KS levels. Recently, a highly sensitive KS ELISA (HS-ELISA) kit has been developed, allowing determination of serum KS levels even in small animals, which were formerly undetectable with 5D4-ELISA. However, the effectiveness of this kit in humans has not been demonstrated. The objective of this study was to assess the usefulness of the HS-ELISA for the analysis of human serum samples. Methods. Serum samples were collected from 28 patients with knee OA and 23 healthy volunteers. KS was determined by 5D4-ELISA and HS-ELISA, and measurements were compared with those obtained by HPLC. KS levels in serum samples with protease pretreatment were also determined by HS-ELISA. Results. KS levels determined by HS-ELISA exhibited a better correlation with those determined by HPLC, and a higher diagnostic sensitivity for OA compared with 5D4-ELISA. Protease pretreatment of serum further improved the correlation between the values obtained by HS-ELISA and HPLC, as well as the diagnostic sensitivity of HS-ELISA for OA. Conclusions. HS-ELISA proved useful for determining KS level in serum and the diagnosis of OA. Pretreatment of serum samples with a protease further improved the performance of HS-ELISA. [ABSTRACT FROM AUTHOR]

The promotion of osteoblastic differentiation by bone morphogenetic proteins (BMPs) is accelerated by chemical compounds that increase the intracellular concentration of cyclic 3',5'-adenosine monophosphate (cAMP). cAMP is synthesized from adenosine triphosphate (ATP) by adenyl cyclase and degraded by phosphodiesterase (PDE) family enzymes. Inhibition of PDEs leads to prolonged accumulation of cAMP within cells and Camp-mediated reactions. Rolipram, a specific inhibitor of PDE4, is a compound effective in inducing osteoblastic differentiation. Four PDE4 family members are transcribed from four distinct genes (4A, 4B, 4C, and 4D). Expression of PDE4A and PDE4D has been observed in osteoblastic cells. We identified PDE4D splicing variants that expressed in ST2 or primary calvarial osteoblasts by rapid amplification of the 5'-ends of cDNA when they were cultured with BMP. PDE4D9 mRNA was identified from ST2, and PDE4D1 and -4D2 mRNAs were identified from primary calvarial osteoblasts. Expression of these three variants of PDE4D mRNA was found in ST2, MC3T3-E1, C3H10T1/2, C2C12, and primary calvarial osteoblasts by RT-PCR, but not PDE4D1 or -4D2 in ST2 or PDE4D2 in MC3T3-E1. Expression of these three variants was detectable in brain, heart, lung, liver, kidney, placenta, and femur, and was thus ubiquitous. Purified recombinant PDE4D9 protein exhibited phosphodiesterase activity, which degraded cAMP to AMP, and this activity was inhibited by rolipram. These findings suggest that PDE4D1, -2, and -9 play some roles in bone formation. [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]

To assess the association between HLA-DRB1*0901 and Japanese rheumatoid arthritis (RA) patients, we analyzed the frequency of HLA-DRB1*0901 in 852 Japanese RA patients. We found that the homozygote of DRB1*0901 was associated with Japanese RA patients, while the heterozygote of DRB1*0901 was not. These findings suggest that DRB1*0901 is a weakly susceptible allele of RA, which in our investigation was not associated with RA by a single allele, but can be by a homozygote. DRB1*0901 does not have the shared epitope, and it is suggested that there may be some mechanism ofthe association between HLA-DRB1 and RA other than the shared epitope, which was not strong. [ABSTRACT FROM AUTHOR]

With the aim to increase type II collagen content in the scaffold-free cartilage-like cell sheet using human bone marrow mesenchymal stem cells, we examined the effect of epigallocatechin-3-gallate (EGCG) addition to the chondrogenic medium for the cell sheet culture. The addition of EGCG (10 μM) increased the content of type II collagen 2-fold, while the addition did not markedly change the expression level of the genes encoding type II collagen and Sox 9. The reactive oxygen species level in the cells in cell sheets was thought to be too low to suppress the accumulation of type II collagen. On the other hand, the addition of EGCG markedly decreased both the matrix metalloproteinase-13 concentration in the supernatant of cell sheet culture and the type II collagen degradation activity in that supernatant. Taken together, EGCG may enhance the accumulation of type II collagen by suppressing type II collagen degradation. Addition of EGCG to the sheet culture of MSC markedly increased not only wet weight (A) and sGAG amount (B) but also type II collagen amount (C) in the cartilage-like cell sheet. [ABSTRACT FROM PUBLISHER]

WP9QY (W9) is a receptor activator of nuclear factor-κB ligand (RANKL)-binding peptide that inhibits osteoclastogenesis by blunting the RANKL-RANK interaction, and also increases osteoblastogenesis via RANKL reverse signaling. W9 has dual effects on osteoclasts and osteoblasts; however, it is unknown whether the peptide has an effect on chondrocytes. Here, we report that W9 induces proliferation and differentiation of chondrocytes in vitro and repairs full-thickness articular cartilage defects in vivo. W9 stimulated chondrocyte differentiation in a two-dimensional (2D) culture of human mesenchymal stem cells (hMSCs), and transforming growth factor β3 (TGF-β3) showed synergistic effects with W9 on chondrogenesis. W9 enlarged the size of 3D pellet cultures of hMSCs and produced chondrocyte-specific matrices, especially in combined treatment with TGF-β3. The peptide also stimulated proliferation of hMSCs with induction of expression of chondrogenesis-related genes. Several RANKL inhibitors had no effect on chondrocytic differentiation. RANKL-knockdown experiments showed that W9 did not induce chondrogenesis through RANKL, but did induce osteoblastogenesis through RANKL. Intraarticular injection of W9 resulted in significant repair of full-thickness articular cartilage defects in rabbits. Taken together, these results suggest that W9 ameliorates the articular cartilage defects by increasing the volume of cartilaginous matrices with accompanying induction of proliferation and differentiation of chondrocytes via mechanisms independent of RANKL inhibition and RANKL reverse signaling. Since no pharmaceuticals are clinically available for treatment of cartilage damage such as osteoarthritis, our findings demonstrate the potential of W9 to address the unmet medical needs. • Accumulation of cartilaginous matrices in chondrocytes with WP9QY peptide. • Synergy between WP9QY and TGF-β3 in chondrogenesis. • Unknown mechanisms independent of RANKL inhibition and RANKL reverse signaling. • Potential to address unmet medical needs for cartilage damage such as osteoarthritis. • Important clues for understanding regulation of cartilage regeneration. [ABSTRACT FROM AUTHOR]

Background: Aiming to construct an osteochondral-like structure, the combination of a β-tricalcium phosphate (βTCP) block with a scaffold-free sheet formed using mesenchymal stem cells (MSCs) was investigated. Methods: Human bone marrow MSCs in a cell culture insert that was set in a 24-well plate were cultivated using a chondrogenic medium containing dexamethasone, IGF-1, and TGFβ3 for 3 weeks during which a cylindrical βTCP block was put on the sheet at day 1, and the cell sheet construct was harvested. In other experiments, at day 14, the construct was put on a cell sheet that was prepared the day before and cultivated for 3 weeks. Results: The addition of a βTCP block resulted in a combined osteochondral-like construct and comparable staining intensity by Alcian blue, while the expression levels of the aggrecan and type II collagen genes decreased a little. During the culture with the βTCP block, the expression levels of the aggrecan gene increased monotonically. The increase in the inoculum cell number from 1.86 to 3.72 × 10 cells resulted in marked increases in the thickness of cell sheet parts in the βTCP block and expression levels of the aggrecan and type II collagen genes, while the thickness of cell sheet parts on the βTCP block scarcely changed. On the other hand, the addition of a cell sheet that was prepared a day before to the construct at day 14 resulted in the marked increase in thickness of the cell sheet part on the βTCP block, while the thickness of that in the βTCP block did not increase. Conclusion: A combined osteochondral-like structure was produced by putting a βTCP block on the sheet of MSC. The thickness of the cell sheet parts in and on the βTCP block could be increased by the increase in inoculum cell number and by providing an additional cell sheet, respectively. [ABSTRACT FROM AUTHOR]

Because bone morphogenetic protein 2 gene transfected Escherichia coli (E-BMP-2) produce recombinant human BMP-2 (rhBMP-2) more efficiently than mammalian cells (Chinese hamster ovary [CHO]-BMP-2), they may be a more cost-effective source of rhBMP-2 for clinical use. However, use of E-BMP-2 for regenerating long bones in large animals has not been reported. In the current study, we evaluated the healing efficacy of E-BMP-2 in a canine model. We created 2.5-cm critical-size segmental ulnar defects in test animals, then implanted E-BMP-2 and 700 mg of artificial bone (beta-tricalcium phosphate; β-TCP) into the wounds. We examined the differential effects of 5 E-BMP-2 treatments (0, 35, 140, 560, and 2240 μg) across 5 experimental groups (control, BMP35, BMP140, BMP560, and BMP2240). Radiography and computed tomography were used to observe the regeneration process. The groups in which higher doses of E-BMP-2 were administered (BMP560 and BMP2240) displayed more pronounced bone regeneration; the regenerated tissues connected to the host bone, and the cross-sectional areas of the regenerated bone were larger than those of the originals. The groups in which lower doses of E-BMP-2 were administered (BMP35 and BMP140) experienced relatively less bone regeneration; furthermore, the regenerated tissues failed to connect to the host bone. In these groups, the cross-sectional areas of the regenerated bone were equal to or smaller than those of the originals. No regeneration was observed in the control group. These findings suggest that, like CHO-BMP-2, E-BMP-2 can be used for the regeneration of large defects in long bones and that its clinical use might decrease the cost of bone regeneration treatments. [ABSTRACT FROM AUTHOR]

Background: Massive frozen stocked allogeneic bone grafts are often used to reconstruct large bone defects caused by trauma or tumor resections. However, the long-term failure rate of such massive allografts was reported to be 25% because of infection, fracture, and nonunion. In this study, we evaluated the ability of a recombinant human bone morphogenetic protein (rhBMP)-2-retaining paste to promote the osteogenic potential of frozen stocked allogeneic bone grafts to repair intercalated femoral shaft defects in a rat model. Methods: After confirming the transplantation intolerance between two rat strains (Wistar and Lewis) by skin transplantation from Lewis rats to Wistar rats, an 8-mm-long bone segment was removed from the Wistar rats, and a frozen stocked allograft coated with the rhBMP-2-retaining paste from the Lewis rats was placed into the defect and subjected to intramedullary fixation with an 18-gauge injection needle pin. The allografted femurs were evaluated by radiographic, histologic, and biomechanical examinations at specified time points. Results: The results revealed successful repair of critical-size cortical bone defects by implanting frozen stocked allografts coated with the rhBMP-2-retaining synthetic biodegradable carrier paste from an immunologically intolerant host. Conclusions: This experimental study suggest that allogeneic bone grafting in combination with rhBMP-2 and its local delivery system may represent an innovative approach to the reconstruction of bone defects. [ABSTRACT FROM AUTHOR]

Abstract: An ex vivo cartilage defect model for the evaluation of cartilage regeneration using mesenchymal stem cells (MSCs) was developed. Porcine chondrocytes and human MSCs were transplanted into cartilage defects created on the porcine osteochondral and chondral discs and cultivated for 3weeks. Although the regeneration of cartilage-like tissues was observed after the transplantation of chondrocytes to defects on both of the osteochondral and chondral discs, the transplanted MSCs formed cartilage-like tissues only in the defect on the chondral disc, in which an in vivo cartilage-like structure was partly observed, and a degraded tissue was observed in the defect on the osteochondral disc. The effects of medium additives such as serum, transforming growth factor-β3 (TGF-β3), and fibroblast growth factor-2, and the transfection of TGF-β3 gene to MSCs could be clearly estimated using the cartilage defect model. In conclusion, a chondral disc with defects is useful for evaluating cartilage regeneration using MSCs. [Copyright &y& Elsevier]

Abstract: The effect of the addition of monosaccharides composing glycosaminoglycans (GAGs) on the accumulation of type 2 collagen (COL(II)) in a three-dimensional (3D) culture of porcine chondrocyte cells was investigated for possible application to cartilage regenerative medicine. Primary chondrocytes from porcine cartilage were cultivated in three-dimension employing atelo collagen gel for 3 weeks with the addition of several saccharides. The addition of d-glucuronic acid (d-GlcA), N-acetyl-d-galactosamine (d-GalNAc), chondroitin sulfate C (CSC), d-galactose, N-acetyl-d-glucosamine, and l-iduronic acid increased markedly not aggrecan but COL(II) accumulation although the addition of d-fructose and d-mannose not composing GAGs did not show such an effect. The addition of d-GlcA and d-GalNAc had no synergistic effect. The addition of CSC, d-GlcA, and d-GalNAc also increased COL(II) mRNA expression while aggrecan mRNA expression was not increased by these compounds. In conclusion, the addition of monosaccharides composing GAGs might be valuable for increasing COL(II) accumulation in the 3D culture of chondrocytes. [Copyright &y& Elsevier]

The effect of glycosaminoglycan addition on a three-dimensional (3D) culture of porcine chondrocyte cells was investigated with a view to use in cartilage regenerative medicine. Chondroitin sulfate C increased the mRNA expression of type 2 collagen, while chondroitin sulfate A did not. Hyaluronic acid of high molecular weight markedly decreased the mRNA expression of both aggrecan and type 2 collagen, although hyaluronic acid of low molecular weight showed no apparent effect. [Copyright &y& Elsevier]

Collagen gel and a copolymer mesh of polylactate and polyglucuronic acid (PLGA) were combined for a three-dimensional (3D) culture of chondrocyte cells having both uniform cell distribution and mechanical strength. Although the 3D culture in 96-multi-wells caused decreases in the glucose consumption rate and cell density in the latter stages of cultivation, transfer of the culture gel from a 96-multi-well plate to a 24-multi-well plate and an increase in medium volume effectively increased the glucose consumption rate and the accumulation of glycosaminoglycan (GAG) in the gel. The reason for the decrease in glucose consumption rate in a 96-multi-well plate was not the depletion of glucose or the accumulation of lactate in the gel, but the accumulation of degradation products of PLGA. [Copyright &y& Elsevier]

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]

Quality evaluation of mesenchymal stem cells (MSCs) based on efficacy would be helpful for their clinical application. In this study, we aimed to find the factors of human bone marrow MSCs relating to cartilage repair. The expression profiles of humoral factors, messenger RNAs (mRNAs), and microRNAs (miRNAs) were analyzed in human bone marrow MSCs from five different donors. We investigated the correlations of these expression profiles with the capacity of the MSCs for proliferation, chondrogenic differentiation, and cartilage repair in vivo. The mRNA expression of MYBL1 was positively correlated with proliferation and cartilage differentiation. By contrast, the mRNA expression of RCAN2 and the protein expression of TIMP-1 and VEGF were negatively correlated with proliferation and cartilage differentiation. However, MSCs from all five donors had the capacity to promote cartilage repair in vivo regardless of their capacity for proliferation and cartilage differentiation. The mRNA expression of HLA-DRB1 was positively correlated with cartilage repair in vivo. Meanwhile, the mRNA expression of TMEM155 and expression of miR-486-3p, miR-148b, miR-93, and miR-320B were negatively correlated with cartilage repair. The expression analysis of these factors might help to predict the ability of bone marrow MSCs to promote cartilage repair. [ABSTRACT FROM AUTHOR]

Abstract: Porcine articular cartilage tissue was crushed using Multi-Beads-Shocker™ and digested with trypsin and collagenase type II. Chondrocyte cell yield was maximized by automatic crushing for 10 s, which was 4 times that by manual dicing with a surgical blade and might be useful for the automation of the cell processing. [Copyright &y& Elsevier]

Predicting the responses of patients with rheumatoid arthritis (RA) to tocilizumab is difficult, because inflammatory markers such as C-reactive protein rapidly normalize regardless of clinical efficacy. We aimed to identify factors that could predict response to tocilizumab. Sixty-five patients completed 52 weeks of tocilizumab therapy. Serum fibrinogen, D-dimer and interleukin (IL)-1β levels were measured at baseline and after 4 weeks of therapy. Clinical responses to tocilizumab were assessed using disease activity score 28-erythrocyte sedimentation rate and the clinical disease activity index at baseline and after 52 weeks of therapy (UMIN Clinical Trials Registry No. UMIN000002246). Mean age was 60.5 years (range 22-85 years). Mean disease duration was 11.2 years (range 0-45 years). All patients had moderate-to-severe disease activity and were resistant to disease-modifying anti-rheumatic drugs and/or other biologics. Baseline IL-1β levels were significantly lower in responders than in non-responders ( p = 0.045), but multiple logistic regression analysis found no significant difference (adjusted odds ratio 2.74; 95 % confidence interval 0.84-8.95; p = 0.096). Low D-dimer and IL-1β levels at 4 weeks predicted greater decrease in disease activity after 52 weeks of treatment ( p = 0.005 and p < 0.001, respectively). Effects of tocilizumab at 52 weeks could be predicted from D-dimer and IL-1β levels after 4 weeks of tocilizumab treatment. These markers might be more useful than current inflammatory markers for early-stage prediction of response to tocilizumab in 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]

Objectives. The deformed rheumatoid forefoot may be treated with resection of lesser metatarsal heads combined with arthrodesis or resection of the first metatarsophalangeal joint. Recurrent hallux valgus deformity has been reported by resection. We performed a prospective, randomized, internal-controlled study to compare results between arthrodesis and resection. Methods. We resected the lesser metatarsal heads bilaterally and performed arthrodesis of the first metatarsophalangeal joint on one side and resection on the opposite side. We investigated 26 patients (52 feet) who were followed at least one year. Patients were assessed for clinical score, hallux valgus angle (HVA), angle between first and second metatarsals, and angle between first and fifth metatarsals preoperatively, postoperatively and at final follow-up. We evaluated callosities, claw toes, recurrences, and procedure preferences. Results. The mean follow-up period was 4.1 years. No significant differences between arthrodesis and resection were seen, with the exception of HVA. That was significantly less on arthrodesis side (11.5°) than on resection side (17.0°, p < 0.05). Seven callosities on resection side and four on arthrodesis side were observed. On resection side, hallux valgus deformity often recurred (15.3%). Patients expressed a significant preference for arthrodesis over resection ( p = 0.008). Conclusions. Arthrodesis provides better results for maintaining HVA. [ABSTRACT FROM AUTHOR]

Background: The efficacy of autologous bone grafting in repairing nonunion fractures, large bone defects and spinal instability is widely accepted. However, the cellular and molecular mechanisms underlying new bone formation in bone grafting have yet to be fully elucidated. The purpose of this study was to clarify the fate, origin and the contribution of the cells within the grafted bone. Methods: This study was designed to investigate the role and fate of cells contained in the grafted bone and their contribution to new bone formation in the graft in an animal model. Middiaphyseal cylindrical bone samples obtained from green fluorescent protein (GFP) transgenic and wild-type rats were transplanted into the back muscle of wild-type and GFP rats, respectively. The transplanted bones were evaluated by immunohistochemistry, in situ hybridization and quantitative reverse transcription polymerase chain reaction. Results: Immunohistochemical analyses showed that all the cells in the newly formed bone originated from the grafted bone, and osteoblasts were gradually replaced by host cells. Conversely, osteoclasts were immediately replaced by host cells 2 weeks after the bone graft. In addition, expression of bone morphogenetic protein (Bmp)- 4, Bmp receptors and Noggin in the grafted bone was significantly upregulated before new bone formation occurred, indicating that the grafted cells might contribute to the recruitment of mesenchymal cells into the graft bed. Conclusion: This study revealed the possible molecular mechanisms of the contribution of cells contained in grafted bone to facilitate new bone formation. [ABSTRACT FROM AUTHOR]

We investigated the effects of biologics for rheumatoid arthritis (RA) patients on bone mineral density (BMD) and bone metabolic markers (BMM), retrospectively, and also clarified the effects of bisphosphonates (alendronate or risedronate 35 mg/week) and glucocorticoids. Participants in this study comprised 219 patients with RA, including 117 patients treated with biologics (infliximab, n = 90; etanercept, n = 27) and 102 patients with conventional disease-modifying anti-rheumatic drugs (DMARDs) for 1 year. Changes in BMD at the lumbar spine and total hip and BMMs [urinary type I collagen cross-linked N-telopeptide (NTX) and bone-specific alkaline phosphatase] were measured. BMD of the lumbar spine in both groups and total hip BMD in the biologics group were unchanged during treatment with biologics. However, BMD of the total hip was significantly decreased in the DMARDs group (from 0.731 ± 0.135 to 0.706 ± 0.135 g/cm). Patients receiving glucocorticoids without bisphosphonates showed significant decrease in BMD of the total hip compared with patients not receiving glucocorticoids or receiving glucocorticoids with bisphosphonates in both biologics and DMARDs groups. Furthermore, BMD of the lumbar spine increased ( p < 0.05) for patients in the biologics group who received bisphosphonates. NTX was significantly decreased only in the biologics group. Multiple regression analysis showed that BMD and bone metabolic marker levels correlated positively with bisphosphonate and biologics use and negatively with glucocorticoid use. BMD of the total hip was maintained in the patients using biologics without glucocorticoids or with bisphosphonates, but it was not maintained in the DMARDs patients, even without glucocorticoids or with bisphosphonates. Even if biologics have protective effect against bone loss of RA patients, we should consider reducing the dose of glucocorticoids and adding bisphosphonates for the treatment of osteoporosis. [ABSTRACT FROM AUTHOR]

The effects of epigallocatechin-3-o-gallate (EGCG) and quercetin on the contents of extracellular matrix (ECM) in porcine cartilage at 4 °C were investigated. The addition of quercetin at 0.01 mM for the incubation of porcine cartilage disks at 4 °C for 2 week could suppress the decrease in ECM and the compliance of the disks, markedly greater than those of EGCG (1.0 mM). [ABSTRACT FROM PUBLISHER]

Objective. To evaluate the prevention of joint destruction and clinical efficacy of low-dose etanercept (ETN) (25 mg/week) compared with standard-dose ETN (50 mg/week) in RA.Methods. In this prospective, randomized, open-label study, 70 patients were assigned to receive ETN at either 50 or 25 mg/week for 52 weeks. The primary endpoint was the variation in modified total Sharp score (mTSS), and secondary endpoints were variations in disease activity score in 28 joints (DAS-28), modified HAQ and adverse event rate. Values of mTSS were calculated at baseline and after 52 weeks. Non-progression was estimated as ΔmTSS &leq;0.5, and the non-progression rate was compared between groups.Results. Mean values at baseline were as follows: disease duration 9.2 years; DAS-28 5.45; and annual progression of mTSS 26.1. No significant differences in background were seen between groups. At 52 weeks, the non-progression rate was significantly less in the 25 mg/week group (36.7%) than in the 50 mg/week group (67.7%) (P = 0.041). Mean ΔmTSS was higher at 25 mg/week (1.03) than at 50 mg/week (−0.13). DAS-28 was significantly improved at 4 weeks, and the effect of treatment lasted for 52 weeks in both groups. No differences in adverse event rates were seen between groups.Conclusion. Low-dose ETN is not inferior to standard-dose ETN in terms of effects on clinical manifestations. However, in terms of the radiographic non-progression rate, the effects of low-dose ETN may be inferior to the effects of standard-dose ETN.Trial Registration: UMIN Clinical Trials Registry, http://www.umin.ac.jp/ctr/, UMIN000001798. [ABSTRACT FROM PUBLISHER]

Background: Currently, regenerative repair of large bone defects that result from bone tumor resection or severe trauma is a challenging issue because of the limited regenerative potential of bone and treatment modalities. The aim of this study was to achieve repair of large bone defects to the original three-dimensional (3D) anatomical state by combining computer-aided technologies and local delivery of bone morphogenetic protein (BMP) in a canine model. Methods: Computed tomography (CT) images of the pelvic bone of each dog were obtained, and an imaginary spherical malignant bone tumor of 15-mm diameter was placed in the left ilium of a canine on the 3D CT image. Resection of the whole tumor with a 10-mm margin of healthy bone was planned preoperatively by using computer-aided design (CAD) software. In addition, an image of the implant to be used to fill the resulting bone defect was constructed on the computer image. A porous hydroxyapatite (HA) implant identical to the imaged bone defect was made by shaving a tetragonal porous apatite block (40 × 20 × 10 mm) with a computer-aided manufacturing system operated by using the CT-image data of the bone defect obtained from the CAD system. To resect the iliac bone as planned preoperatively on the 3D CT image, computer-aided surgery was performed using the CT data. The defect was filled with the HA implant fabricated as described and coated with a putty carrier either with BMP-2 (BMP group, n = 6) or without BMP-2 (control group, n = 6). Results: In the BMP group, new bone formation was noted around each implant on CT images at 3 weeks after surgery and was remodeled to restore the original anatomy of the ilium on serial CT images. At 12 weeks, the implant was enclosed within new bone, and histological analysis revealed bone formation on and within the implant. Little bone formation was noted in the control group. Conclusions: This new method may enable efficacious and precise regenerative repair of large bone defects without bone grafting. [ABSTRACT FROM AUTHOR]

Background: Currently, regenerative repair of large bone defects that result from bone tumor resection or severe trauma is a challenging issue because of the limited regenerative potential of bone and treatment modalities. The aim of this study was to achieve repair of large bone defects to the original three-dimensional (3D) anatomical state by combining computer-aided technologies and local delivery of bone morphogenetic protein (BMP) in a canine model. Methods: Computed tomography (CT) images of the pelvic bone of each dog were obtained, and an imaginary spherical malignant bone tumor of 15-mm diameter was placed in the left ilium of a canine on the 3D CT image. Resection of the whole tumor with a 10-mm margin of healthy bone was planned preoperatively by using computer-aided design (CAD) software. In addition, an image of the implant to be used to fill the resulting bone defect was constructed on the computer image. A porous hydroxyapatite (HA) implant identical to the imaged bone defect was made by shaving a tetragonal porous apatite block (40 × 20 × 10 mm) with a computer-aided manufacturing system operated by using the CT-image data of the bone defect obtained from the CAD system. To resect the iliac bone as planned preoperatively on the 3D CT image, computer-aided surgery was performed using the CT data. The defect was filled with the HA implant fabricated as described and coated with a putty carrier either with BMP-2 (BMP group, n = 6) or without BMP-2 (control group, n = 6). Results: In the BMP group, new bone formation was noted around each implant on CT images at 3 weeks after surgery and was remodeled to restore the original anatomy of the ilium on serial CT images. At 12 weeks, the implant was enclosed within new bone, and histological analysis revealed bone formation on and within the implant. Little bone formation was noted in the control group. Conclusions: This new method may enable efficacious and precise regenerative repair of large bone defects without bone grafting. [ABSTRACT FROM AUTHOR]

Introduction: As a powerful bone-inducing cytokine, rhBMP-2 has been used as a bone graft substitute in combination with animal-derived collagen to achieve interbody or posterolateral spinal fusion. Successful interspinous process fusion using rhBMP-2 in combination with synthetic carrier materials would offer a safe, minimally invasive spinal fusion option for the treatment of spinal disorders. The aims of the present study were to achieve interspinous process fusion by implanting rhBMP-2-retaining degradable material instead of bone grafting and to evaluate efficacy for vertebral stabilization.Materials and Methods: A polymer gel (200 mg), β-tricalcium phosphate powder (400 mg), and rhBMP-2 (0, 30, 60 or 120 μg) were mixed to generate a plastic implant, which was then placed during surgery to bridge the L5-6 interspinous processes of 58 rabbits. Control animals received implants either without rhBMP-2 or with autogenous bone chips from the iliac crest. L5-6 vertebrae were recovered 8 weeks postoperatively. Interspinous process fusion was evaluated by radiography, biomechanical bending test, intradiscal pressure (IDP) measurement, and histology.Results: In bending tests, strength of fusion was significantly greater in BMP60 and BMP120 groups than in sham, BMP0, BMP30 or autogenous bone groups. IDP at L5-6 was significantly reduced in BMP60 and BMP120 groups compared to sham, BMP0, BMP30, and autograft groups. Histologically, coronal sections of the fusion mass showed a bone mass bridging both spinous processes.Conclusion: Solid interspinous process fusion was achieved in rabbit models by 8 weeks after implanting the biodegradable bone-inducing material. These results suggest a potential new less-invasive option without bone grafting for the treatment of lumbar disorders. [ABSTRACT FROM AUTHOR]

Introduction: As a powerful bone-inducing cytokine, rhBMP-2 has been used as a bone graft substitute in combination with animal-derived collagen to achieve interbody or posterolateral spinal fusion. Successful interspinous process fusion using rhBMP-2 in combination with synthetic carrier materials would offer a safe, minimally invasive spinal fusion option for the treatment of spinal disorders. The aims of the present study were to achieve interspinous process fusion by implanting rhBMP-2-retaining degradable material instead of bone grafting and to evaluate efficacy for vertebral stabilization. Materials and methods: A polymer gel (200 mg), β-tricalcium phosphate powder (400 mg), and rhBMP-2 (0, 30, 60 or 120 μg) were mixed to generate a plastic implant, which was then placed during surgery to bridge the L5-6 interspinous processes of 58 rabbits. Control animals received implants either without rhBMP-2 or with autogenous bone chips from the iliac crest. L5-6 vertebrae were recovered 8 weeks postoperatively. Interspinous process fusion was evaluated by radiography, biomechanical bending test, intradiscal pressure (IDP) measurement, and histology. Results: In bending tests, strength of fusion was significantly greater in BMP60 and BMP120 groups than in sham, BMP0, BMP30 or autogenous bone groups. IDP at L5-6 was significantly reduced in BMP60 and BMP120 groups compared to sham, BMP0, BMP30, and autograft groups. Histologically, coronal sections of the fusion mass showed a bone mass bridging both spinous processes. Conclusion: Solid interspinous process fusion was achieved in rabbit models by 8 weeks after implanting the biodegradable bone-inducing material. These results suggest a potential new less-invasive option without bone grafting for the treatment of lumbar disorders. [ABSTRACT FROM AUTHOR]

Rheumatoid arthritis is a common autoimmune disease characterized by chronic inflammation. We report a meta-analysis of genome-wide association studies (GWAS) in a Japanese population including 4,074 individuals with rheumatoid arthritis (cases) and 16,891 controls, followed by a replication in 5,277 rheumatoid arthritis cases and 21,684 controls. Our study identified nine loci newly associated with rheumatoid arthritis at a threshold of P < 5.0 × 10?8, including B3GNT2, ANXA3, CSF2, CD83, NFKBIE, ARID5B, PDE2A-ARAP1, PLD4 and PTPN2. ANXA3 was also associated with susceptibility to systemic lupus erythematosus (P = 0.0040), and B3GNT2 and ARID5B were associated with Graves' disease (P = 3.5 × 10?4 and 2.9 × 10?4, respectively). We conducted a multi-ancestry comparative analysis with a previous meta-analysis in individuals of European descent (5,539 rheumatoid arthritis cases and 20,169 controls). This provided evidence of shared genetic risks of rheumatoid arthritis between the populations. [ABSTRACT FROM AUTHOR]

Background: The combination of a β-tricalcium phosphate (βTCP) block with a scaffold-free chondrocyte sheet formed by the centrifugation of chondrocytes in a well was investigated with the aim of constructing an osteochondral-like structure. Methods: Human and porcine articular cartilage chondrocytes were respectively centrifuged in a 96-well plate or cell culture insert (0.32 cm) that was set in a 24-well plate, cultivated in the respective vessel for 3 weeks, and the cell sheets were harvested. In some cases, a cylindrical βTCP block (diameter 5 mm, height 3 mm) was placed on the sheet on days 1-7. The sheet size, cell number, and sulfated glycosaminoglycan accumulation were determined. Results: The use of a 96-well plate for not suspension but adhesion culture and the initial centrifugation of a well containing cells were crucial to obtaining a uniformly thick cell sheet. The glycosaminoglycan density of the harvested cell sheet was comparable to that of the pellet culture. An inoculum cell number of more than 31 × 10 cells tended to result in a curved cell sheet. Culture involving 18.6 × 10 cells and the 96-well plate for adhesion culture showed no curving of the cell sheet (thickness of 0.85 mm), and these were found to be the best of the culture conditions tested. The timing of the addition of a βTCP block to the cell sheet (1-7 days) markedly affected the balance between the thickness of cell sheet parts on and in the βTCP block. Conclusion: Centrifugation and subsequent cultivation of chondrocytes (18.6 × 10 cells) in a 96-well plate for adhesion culture led to the production of a scaffold-free cartilage-like cell sheet with a thickness of 0.85 mm. A combined osteochondral-like structure was produced by putting a βTCP block on the cell sheet. The thickness of the cell sheet on the βTCP block and the binding strength between the cell sheet and the βTCP block could be optimized by adjusting the inoculum cell number and timing of βTCP block addition. [ABSTRACT FROM AUTHOR]

Abstract: The effects of gravity on bone metabolism are unclear, and little has been reported about the effects of hypergravity on the mature skeleton. Since low gravity has been shown to decrease bone volume, we hypothesized that hypergravity increases bone volume. To clarify this hypothesis, adult female rats were ovariectomized and exposed to hypergravity (2.9G) using a centrifugation system. The rats were killed 28days after the start of loading, and the distal femoral metaphysis of the rats was studied. Bone architecture was assessed by micro-computed tomography (micro-CT) and bone mineral density was measured using peripheral quantitative CT (pQCT). Hypergravity increased the trabecular bone volume of ovariectomized rats. Histomorphometric analyses revealed that hypergravity suppressed both bone formation and resorption and increased bone volume in ovariectomized rats. Further, the cell morphology, activity, proliferation, and differentiation of osteoclasts and osteoblasts exposed to hypergravity were evaluated in vitro. Hypergravity inhibited actin ring formation in mature osteoclasts, which suggested that the osteoclast activity was suppressed. However, hypergravity had no effect on osteoblasts. These results suggest that hypergravity can stimulate an increase in bone volume by suppressing bone resorption in ovariectomized rats. [Copyright &y& Elsevier]

Background: Screening and prophylactic treatment for hepatitis B virus (HBV) reactivation is recommended for patients who receive immunosuppressive or cytotoxic therapy. The aim of this study was to clarify the prevalence of HBV reactivation in rheumatoid arthritis (RA) patients who had received more than 1 year of immunosuppressive therapy. This study also evaluated guidelines for determining HBV reactivation in patients with RA. Methods: This was a prospective non-randomized, non-controlled study. We enrolled 50 patients with RA who had antibodies against hepatitis B core antigen (anti-HBc) and who had started treatment with disease-modifying anti-rheumatic drugs, including those who had additionally received anti-tumor necrosis factor-α (anti-TNF-α). HBV DNA levels were measured every 2-3 months by a real-time, polymerase chain reaction-based method. Entecavir was administered to patients with HBV DNA levels >2.1 log/ml. Results: The mean observation period was 23 months (range 12-32 months). HBV reactivation occurred in 2 of 5 patients with HBV surface antigen (HBsAg) and in 1 of 45 patients without HBsAg. In patients who received anti-TNF-α therapy, antibodies against HBsAg decreased significantly. Entecavir therapy inhibited HBV amplification and prevented HBV-associated flares of hepatitis. Conclusions: The incidence of HBV reactivation was low in RA patients in whom HBV infection had been resolved. Screening for HBV reactivation and prophylactic therapy with entecavir were effective means of preventing HBV-associated hepatic failure in patients with HBsAg, as well as in those with only anti-HBc who received immunosuppressive therapy for RA. [ABSTRACT FROM AUTHOR]

Abstract: Aiming for the clinical application of cartilage regeneration, a culture method for mesenchymal stem cells (MSCs) derived from human bone marrow to obtain scaffold-free cartilage-like disk-shaped sheet of uniform sizes without the shrinkage was investigated. A disk-shaped cell sheet having the same diameter as that of the membrane without the shrinkage was formed after the cultivation of MSCs (18.6×105 cells/well) for 3weeks in a cell culture insert (CCI) containing a flat membrane whose porosity was 12%, while 6.2 and 31.0×105 MSCs/well, respectively, resulted in the shrinkage of the aggregate and the hole formation in the center part of the sheet. Cell aggregates shrunk also in a 96-well plate and CCIs having lower porosity. The disk-shaped cell sheet showed the comparable thickness (1.2mm) and sulfated glycosaminoglycan (sGAG) density to those of the pellet formed in a pellet culture. The gene expression levels of aggrecan and type II collagen in the disk-shaped cell sheet were not lower than those in the pellet. In conclusion, the usage of CCI having 12% porosity and 18.6×105 MSCs/well could avoid the shrinkage from the formation of the scaffold-free cartilage-like disk-shaped cell sheet. [Copyright &y& Elsevier]

Pyoderma gangrenosum (PG) is characterized by ulcerative skin lesions. Infliximab (IFX) may promote PG healing in patients with inflammatory bowel disease, but whether IFX is effective for treating PG in patients with rheumatoid arthritis (RA) has not reported. We report the case of a 53-year-old woman with PG complicated by RA who was treated using IFX therapy. This case suggests that IFX therapy might offer effective treatment for such patients. [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]

Abstract: High-expression plasmid vectors for human mesenchymal stem cells (MSCs) were constructed by combination of cytomegalovirus immediate-early enhancer with cellular promoters. MSCs transfected with the vector showed higher transgene production of a cytokine, which increased the differentiation level to chondrocytes. [ABSTRACT FROM AUTHOR]

Ultra-high molecular weight polyethylene (UHMWPE) has been widely used as a bearing surface of joint implants. Since wear of UHMWPE component has been the primal cause of implant failure, crosslinking of UHMWPE is now widely used to improve its wear property. On the other hand, crosslinking degrades its fatigue property and increase of fatigue related failure, such as delamination, is possible. However, the relationship between degraded fatigue property and implant failure is not clear, primarily because the fatigue properties of failed and retrieved UHMWPE components are not known. In this study, we propose a new test method to evaluate the fatigue property of retrieved UHMWPE components or final products using small specimens. First, the effects of test conditions were investigated using virgin UHMWPE. Then, tests were repeated using specimens from UHMWPE components of retrieved knee implants. The fatigue properties of virgin and retrieved UHMWPE were successfully evaluated by the new test method and a relationship was found among the extent of oxidation, the fatigue property, and occurrence of delamination. This result suggested that evaluation of fatigue property of UHMWPE component can predict the occurrence of fatigue related implant failure during operation in vivo. [ABSTRACT FROM AUTHOR]

Bone morphogenetic proteins (BMPs) were originally identified as osteoinductive proteins. With cloning of BMP genes, studies of BMPs and their clinical application have advanced. However, with increasing clinical applications, drug delivery systems and production costs have become more important issues. To address these issues, we asked whether E. coli-derived rhBMP-2 (E-BMP-2)-adsorbed porous beta-TCP granules could achieve posterolateral lumbar fusion in a rabbit model similar to autogenous bone grafts. Lumbar spinal fusion masses were evaluated by 3-D computed tomography, mechanical testing, and histological analyses 8 weeks after surgery. By these measures E-BMP-2-adsorbed beta-TCP granules achieved lumbar spinal fusion in dose-dependent fashion in a rabbit model as well as autogenous bone graft. Our preliminary findings suggest E-BMP-2-adsorbed porous beta-TCP could be a novel, effective alternative to autogenous bone grafting for generating new bone and promoting regenerative repair of bone, and potentially utilizable in the clinical setting for treating spinal disorders. [ABSTRACT FROM AUTHOR]

Background and purpose Osteochondral defects have a limited capacity for repair. We therefore investigated the effects of tumor necrosis factor (TNF) signal blockade by etanercept (human recombinant soluble TNF receptor) on the repair of osteochondral defects in rabbit knees. Material and methods Osteochondral defects (5 mm in diameter) were created in the femoral patellar groove in rabbits. Soon after the procedure, a first subcutaneous injection of etanercept was performed. This single injection or, alternatively, 4 injections in total (twice a week for 2 weeks) were given. Each of these 2 groups was divided further into 3 subgroups: a low-dose group (0.05 μg/kg), an intermediate-dose group (0.4 μ g/kg), and a high-dose group (1.6 μ g /kg) with 19 rabbits in each. As a control, 19 rabbits were injected with water alone. The rabbits in each subgroup were killed 4 weeks (6 rabbits), 8 weeks (6 rabbits), or 24 weeks (7 rabbits) after surgery and repair was assessed histologically. Results Histological examination revealed that the natural process of repair of the osteochondral defects was promoted by 4 subcutaneous injections of intermediate-dose etanercept and by 1 or 4 injections of high-dose etanercept at the various time points examined postoperatively (4, 8, and 24 weeks). Western blot showed that rabbit TNFα had a high affinity for etanercept. Interpretation Blocking of TNF by etanercept enabled repair of osteochondral defects in rabbit knee. Anti-TNF therapy could be a strategy for the use of tissue engineering for bone and cartilage repair. [ABSTRACT FROM AUTHOR]

Noggin is a major extracellular antagonist to bone morphogenetic proteins (BMPs) which binds to BMPs and blocks binding of them to BMP-specific receptors and negatively regulates BMP-induced osteoblastic differentiation. In this study, we investigated the effect of noggin silencing by transfection of small interfering RNA (siRNA) on BMP-induced osteoblastic differentiation in vitro and ectopic bone formation in vivo induced by recombinant human BMP-2 (rhBMP-2). Noggin mRNA expression was up-regulated in response to rhBMP-2 in C2C12 cells, a myoblastic cell line, in dose- and time-dependent fashion as determined by real-time RT-PCR assay. Silencing of noggin expression by transfection of noggin siRNA suppressed BMP-stimulated noggin expression, resulting in acceleration of BMP-induced osteoblastic differentiation. For in vivo noggin silencing, siRNA was injected locally into back muscles and transfected into local cells by electroporation, where rhBMP-2-retaining (5 microg) collagen disks had been surgically placed. The implants were harvested at 2 weeks after surgery from experimental and control group mice and analyzed by radiological and histological methods. As a result, bone mineral content of ossicles ectopically induced by rhBMP-2 was significantly increased by silencing of noggin. Our findings suggest that silencing of noggin enhances the osteoblastic differentiation of BMP-responding cells in vitro and new bone formation induced by rhBMP-2 in vivo by eliminating negative regulation of the effects of BMP. RNA interference might be useful for intensifying the effects of BMP in promoting new bone (callus) formation in repair of damaged bone. [ABSTRACT FROM AUTHOR]