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Embryonic stem cells (ESCs) have the potential to be used as an unlimited cell source for cell transplantation therapy, as well as for studying mechanisms of disease and early mammalian development. However, applications involving ESCs have been limited by the lack of reliable differentiation methods in many cases. Mesenchymal stem cells (MSCs) have also emerged as a promising cell source, but as suggested in recent studies, these cells display limited potential for proliferation and differentiation, thereby limiting their usefulness in the clinic and in the laboratory. Unfortunately, effective methods for induction of MSCs from pluripotent stem cells have not been established, and the development of such methods remains a major challenge facing stem cell biologists. Oxygen concentration is one of the most important factors regulating tissue development. It has profound effects on cell metabolism and physiology and can strongly influence stem cell fate. Here we demonstrate that severe low O2concentrations (1%) can function as a selective pressure for removing undifferentiated pluripotent cells during the induction of MSCs from rabbit ESCs (rESCs) and that MSCs induced under severe hypoxic conditions function as normal MSCs; that is, they repopulate after cloning, express specific markers (vimentin, CD29, CD90, CD105, and CD140a) and differentiate into adipocytes, osteoblasts, and chondrocytes. Furthermore, we demonstrate that these cells can contribute to cartilage regeneration in an in vivo rabbit model for joint cartilage injury. These results support the notion that exposing ESCs to severe hypoxic conditions during differentiation can be used as a strategy for the preparation of functional MSCs from ESCs.

Purpose The bi-malleolar technique for the extramedullary tibial guide is a representative method for determining the ankle center in total knee arthroplasty (TKA). The purpose of this study is to estimate three-dimensionally the lateral errors (difference between the real ankle center and the bi-malleolar center) and the varus angular errors of this technique under the condition that the malleolar prominences were correctly identified. Methods Magnetic resonance images of 51 lower limbs from 51 healthy volunteers were analyzed. The lateral errors were measured, including or excluding the subcutaneous thickness, along the line perpendicular to the transmalleolar axis (TMA) or along the tibial anteroposterior (AP) axis. Furthermore, we evaluated the effects of the tibial torsion and the difference between the subcutaneous thicknesses on the malleoli on the lateral error. Results When including the skin, the mean lateral errors of the ankle center observed along the line perpendicular to the TMA and along the tibial AP axis were 3.7 ± 1.4 mm and 1.2 ± 1.5 mm, respectively. The mean angular errors were 0.6 ± 0.2° and 0.2 ± 0.3°, respectively. A significant correlation between the tibial torsion and the lateral error was noted when observed along the tibial AP axis. The difference between the subcutaneous thicknesses on the malleoli affected the lateral error. Conclusion The errors were small enough to determine the mechanical axis of the tibia if the tibial guide could catch the bi-malleolar prominences of the ankle accurately and align along the tibial AP axis.

Recently, an additional type of pluripotent stem cell-line derived from mouse embryos has been established and termed epiblast stem cell (EpiSC), and is expected to be an important tool for studying the mechanisms of maintenance of pluripotency since they depend on basic fibroblast growth factor-MAPK and Activin A-Smad2/3 signaling to maintain pluripotency, unlike mouse embryonic stem cells (ESCs). Further, because of the similarities between mouse EpiSCs and human ESCs, EpiSCs are expected to be effective experimental models for human stem cell therapy. Recently, study for conversion from ESC state to EpiSC state or reversion from EpiSC state to ESC state has attracted interest since these techniques may lead to increasing the potential of pluripotent stem cells and our knowledge about their developmental status. In the present study, we find that a low oxygen concentration in culture environment accelerated, improved, and stabilized the EpiSC state of the converted cells from the ESC state using Oct4ΔPE-GFP transgenic ESCs. Induced EpiSCs (iEpiSCs) in hypoxia possess closer gene expression patterns to native EpiSCs, and bisulfite sequences for the promoter regions of Stella and Oct4 genes have elucidated that the iEpiSC gain EpiSC-specific methylation patterns in hypoxia. Our data provide evidence that oxygen concentration is an important factor for establishment of the EpiSC-specific state.

Mechanical stimulation has been shown to regulate the proliferation and differentiation of stem cells. However, the effects of the mechanical stress on the stemness or related molecular mechanisms have not been well determined. Pluripotent stem cells such as embryonic stem (ES) cells and induced pluripotent stem (iPS) cells are used as good materials for cell transplantation therapy and research of mammalian development, since they can self-renew infinitely and differentiate into various cell lineages. Here we demonstrated that the mechanical stimulation to human iPS cells altered alignment of actin fibers and expressions of the pluripotent related genes Nanog, POU5f1 and Sox2. In the mechanically stimulated iPS cells, small GTPase Rho was activated and interestingly, AKT phosphorylation was decreased. Inhibition of Rho-associated kinase ROCK recovered the AKT phosphorylation and the gene expressions. These results clearly suggested that the Rho/ROCK is a potent primary effector of mechanical stress in the pluripotent stem cells and it participates to pluripotency-related signaling cascades as an upper stream regulator.

Fabrication of a new ball-shaped titanium material for vertebral compression fracture treatment has been studied using micro-arc oxidation in an electrolyte containing hydroxyapatite (HAp). To prevent microstructural disturbance on the surface, ethanol was used as a co-solvent. The material thus obtained was found to have high porosity with many micropores, fixed HAp particles on the surface, and elasticity comparable to cancellous bone. Furthermore, it was demonstrated that the materials have osteoconductivity and the elasticity was kept under cycled stress. These indicate that they can be new bone filling materials for percutaneous vertebroplasty (PV).

本研究の目的は，人工膝関節置換術における術後関節位置覚の経時的推移を明らかにし，膝関節位置覚の変化に影響を与える因子を抽出することである。人工膝関節置換術（以下TKA）を施行した患者30名を対象とした。術前後の位置覚を測定，得点化し，推移を明らかにした。その結果位置覚得点は3週で最も低下し，その後時間の経過に伴い改善することが分かった。術後3週での関節位置覚低下に関与する要因を検討するため，位置覚を高値群，中間群，低値群の3群に分けて関連要因を検討した。その結果，関節位置覚得点が低い群では，年齢が高い，日本整形外科学会膝関節スコアが低い，BMIが高い，術中・術後の出血量が多い，術時間が長い，皮切が大きい傾向が認められた。術前の日常生活活動性の維持，および改善が術後の膝関節位置覚の早期回復に重要と思われた。

SummaryObjectiveUsing human cartilage samples and cultured chondrocytes, to assess the possible involvement of oxidized low-density lipoprotein (ox-LDL) and lectin-like ox-LDL receptor-1 (LOX-1) in pathogenesis and progression of osteoarthritis (OA).MethodsThirty-two cartilage samples were obtained from 16 patients with knee OA, and 12 Control samples from six with femoral neck fracture. LOX-1 mRNA expressions in 12 OA and six Control samples were analyzed by reverse transcription-polymerase chain reaction (RT-PCR). Immunohistochemistry for ox-LDL and LOX-1 was performed in all samples. The histological OA grade was assessed with the modified Mankin score. The relative percentage of the ox-LDL and LOX-1 immunopositive chondrocytes was calculated in all samples. The effects of ox-LDL on cell viability in cultured human chondrocytes were investigated by the 3-(4,5-dimethylthiazolyl-2)-2,5-diphenyltetrazolium bromide (MTT) assay and on proteoglycan synthesis by monitoring [35S] sulfate incorporation.ResultsThere was a statistically significant difference between mean LOX-1/GAPDH (LOX-1/human glyceraldehyde-3-phosphate dehydrogenase) ratio of OA samples and that of Control samples (40.6%±10.3 and 11.9%±2.8, respectively, P

It has been reported that vascular endothelial growth factor (VEGF) and its receptors play an important role in the destruction of articular cartilage in osteoarthritis through increased production of matrix metalloproteinases. We investigated whether the oxidized low-density lipoprotein (ox-LDL) binding to lectin-like ox-LDL receptor-1 (LOX-1) upregulates VEGF expression in cultured bovine articular chondrocytes (BACs). Ox-LDL markedly increased VEGF mRNA expression and protein release in time- and dose-dependent manners, which was significantly suppressed by anti-LOX-1 antibody pretreatment. Activation of peroxisome proliferator-activated receptor (PPAR)-gamma was evident in BACs with ox-LDL addition and was attenuated by anti-LOX-1 antibody. The specific PPAR-gamma inhibitor GW9662 suppressed ox-LDL-induced VEGF expression. These results suggest that the ox-LDL/LOX-1 system upregulates VEGF expression in articular cartilage, at least in part, through activation of PPAR-gamma and supports the hypothesis that ox-LDL is involved in cartilage degradation via LOX-1.

Articular cartilage is an avascular tissue in which chondrocytes are exposed to hypoxic conditions. We previously demonstrated that reactive oxygen species (ROS) induced apoptosis of chondrocytes. We also demonstrated that nitric oxide (NO) was induced when chondrocytes were exposed to hypoxia and that NO inhibited the ROS-induced apoptosis. Hyaluronan (HA) is a high molecular weight glycosaminoglycan whose antioxidative effects have been reported. The purpose of the present study was to determine whether HA synthesis was induced in chondrocytes exposed to hypoxia, and, if so, whether the hypoxia-induced HA synthesis is regulated by NO.Bovine articular chondrocytes were used in this study. Levels of HA were determined by the sandwich enzyme-binding assay. Expression of HA synthase (HAS) was determined with reverse transcription-polymerase chain reaction. The production of NO was examined using the Griess reaction. We also determined inducible nitric oxide synthase (iNOS) enzyme synthesis using the histochemistry and Western blot analysis.Chondrocytes cultured under hypoxic conditions exhibited enhanced HA synthesis. When the NO inhibitors, L-NMMA and L-NAME, were added, the hypoxia-enhanced HA levels in the culture medium were significantly inhibited.Endogenous NO synthesis plays an important role in hypoxia-enhanced HA synthesis.

Mechanical stimulation is known to be an essential factor in the regulation of cartilage metabolism. We tested the hypothesis that expression of lectin-like oxidized low-density lipoprotein receptor-1 (LOX-1) can be modulated by cyclic tensile stretch load in chondrocytes. Cyclic loading of repeated stretch stress at 10 cycles per minute with 10 kPa of stress for 6 h induced expression of LOX-1 to 2.6 times control in cultured bovine articular chondrocytes, equivalent to the addition of 10 microg/mL oxidized low density lipoprotein (ox-LDL) (2.4 times control). Application of the cyclic load to the chondrocytes along with 10 microg/mL ox-LDL resulted in synergistically increased LOX-1 expression to 6.3 times control. Individual application of cyclic loading and 10 microg/mL ox-LDL significantly suppressed chondrocytes viability (84.6% +/- 3.4% and 80.9% +/- 3.2% of control at 24 h, respectively; n = 3; p < 0.05) and proteoglycan synthesis [81.0% +/- 7.1% and 85.7% +/- 5.2% of control at 24 h, respectively; p < 0.05 when compared with 94.6% +/- 4.6% for native-LDL (n = 3)]. Cyclic loading and 10 microg/mL ox-LDL synergistically affected cell viability and proteoglycan synthesis, which were significantly suppressed to 45.6% +/- 4.9% and 48.7% +/- 6.7% of control at 24 h, respectively (n = 3; p < 0.01 when compared with individual application of cyclic loading or 10 microg/mL ox-LDL). In this study, we demonstrated synergistic effects of cyclic tensile stretch load and ox-LDL on cell viability and proteoglycan synthesis in chondrocytes, which may be mediated through enhanced expression of LOX-1 and which has important implications in the progression of cartilage degeneration in osteoarthritis.

Axial micromotion of bone fragments enhances callus formation during fracture repair or limb lengthening. To examine this, we used an axial-shortening model of the tibial callus in rabbits and performed histological analyses. After 10-mm lengthening of the left tibia with an external fixator, we shortened the callus by 2 mm. Radiographs and quantitative evaluation of corrected bone mineral density showed a significant increase in mineralization in the shortened callus (57.3 vs. 36.2%, p = 0.001). Histologically, greater osteoblast proliferation and more vigorous trabecular bone formation were noted in the shortened calluses than in the controls. Around the front of membranous bone formation in the shortened callus, there was a significant decrease in mean percentage area of vascular lumens (1.8 vs. 4.5%, p = 0.009), which seemed attributable to compressive force, and a significantly increased production of vascular endothelial growth factor (VEGF; 422.5 vs. 142.7 pg/mg protein, p = 0.007) and its receptors. There were also increased numbers of tartrate-resistant acid phosphatase (TRAP)-positive osteoclasts and proliferating cell nuclear antigen (PCNA)-positive cells. A marked increase of hypoxia inducible factor-1alpha (HIF-1alpha) expression in osteoblasts was also observed in this area. Thus, enhancement of membranous bone formation by static compression or axial dynamization may be at least partly attributable to HIF-1alpha-mediated VEGF induction following the local hypoxia caused by collapse of vascular lumens.

Severe loading and complex kinematics in total knee replacement make wear performance and toughness of the polyethylene in tibial inserts important. We investigated wear of crosslinked polyethylene using a knee simulator and measured toughness using small-punch testing. GUR1050 rods were gamma-irradiated in air at doses from 0 to 200 kGy, annealed in nitrogen, and machined into tibial inserts. The simulator was run to 4 million cycles, and wear rates determined from weight loss. Wear rate decreased by 54, 78, and 95% as radiation dose increased from 50 to 75 to 100 kGy, respectively. At every dose, toughness was significantly less after simulator testing, but the difference between control and wear-tested polyethylene, considered to be due to fatigue damage accumulation, was smallest at 50 kGy. The simulator-tested polyethylene that received 35 to 75 kGy had slightly higher toughness than equivalent material that received no irradiation. However, the toughness of simulator-tested polyethylene that received 150 and 200 kGy was lower than that of the simulator-tested polyethylene that received no irradiation. Our results suggest that an optimal irradiation dose may exist for crosslinked polyethylene for use in TKR and that the optimum dose would be less than the 100 kGy or more that are used in some current crosslinked polyethylene for hip replacement.

In degradative articular diseases such as rheumatoid arthritis and osteoarthritis, loss of the extracellular matrix occurs, resulting in the destruction of joint cartilage. Proteolysis of aggrecan is one of the early events that leads to breakdown of the extracellular matrix. Aggrecanase-1 (ADAMTS--4) is considered to play a pivotal role in the abrasion of cartilage aggrecan in rheumatoid arthritis and osteoarthritis. To identify an endogenous inhibitor of aggrecanase-1, we performed a yeast two-hybrid screen using the catalytic domain of human aggrecanase-1 as a bait and transformed an EGY 48 yeast strain carrying the bait plasmid with a human liver cDNA library plasmid. This screen identified alpha1-antitrypsin, a member of the family of plasma serine proteinase inhibitors, as a prey. Recombinant aggrecanase-1 and alpha1-antitrypsin were expressed in mammalian cells and used in co-immunoprecipitation experiments, which showed that full-length aggrecanase-1 and alpha1-antitrypsin are also associated in vivo. However, aggrecanase-1 did not interfere with the inhibitory activity of alpha1-antitrypsin against elastase, and alpha1-antitrypsin had no effect on the proteolytic activity of aggrecanase-1. Taken together, these data suggest that aggrecanase-1 and alpha1-antitrypsin bind in vivo, although the physiological significance of the interaction between aggrecanase-1 and alpha1-antitrypsin remains unclear.

Anatomic reference axes that determine rotational alignment of the tibial component have not been established. To assess variability of three anatomic reference axes (a new tibial anteroposterior axis that we proposed, the transmalleolar axis of the ankle, and the second metatarsus bone axis of the foot), we measured the angles between a defined anteroposterior axis of the tibia (a line perpendicular to the transepicondylar axis) and each of the three axes in 57 knees of healthy subjects using computed tomography scans. The angle between the defined anteroposterior axis and our proposed anteroposterior axis (a line connecting the middle of the posterior cruciate ligament and the medial edge of the patellar tendon attachment) averaged -0.2 degrees +/- 2.8 degrees (range, -5.5 degrees -6.3 degrees). The angle between the defined anteroposterior axis and the transmalleolar axis averaged 25.9 degrees +/- 9 degrees (range, 8 degrees -49.4 degrees), and the angle between the defined anteroposterior axis and the second metatarsus bone axis averaged 5.2 degrees +/- 10 degrees (range, -21.9 degrees -24 degrees). The variability of the anteroposterior axis was less than than the other reference axes. These data indicate that our proposed tibial anteroposterior axis is more reliable for determining rotational alignment of the tibial component in total knee arthroplasty.

Alumina-on-alumina bearings for total hip arthroplasty have been introduced as a promising alternative to reduce wear debris and to increase the life expectancy of the prosthesis. We report a case of a late dissociation of an alumina-on-alumina bearing modular acetabular component, which occurred 2 years, 8 months after surgery. Detailed analysis of the retrieved prosthesis suggested that the cause of the failure may be strong rotational torque developed by a roughening of the bearing's alumina surface caused by edge loading (microseparation). The strong friction torque in articulation may explain the higher reported rate of mechanical loosening of the acetabular component in alumina-on-alumina bearing total hip prostheses.

Ebselen (2-phenyl-1,2-benzisoselenazol-3(2H)-one), a synthetic heterocyclic seleno-organic compound, has been shown to act as a scavenger of reactive oxygen species (ROS). We have previously reported that interleukin-1 (IL-1) inhibited proteoglycan (PG) synthesis and induced the production of ROS in cartilage explants and isolated chondrocyte cultures. In this study, we report the protective effect of ebselen against IL-1-mediated inhibition of PG synthesis and ROS induction in cultured cartilage explants and chondrocytes. Ebselen also reversed the inhibition of PG synthesis in mechanically stressed cultured chondrocytes. These data suggest that the use of the antioxidant ebselen may be a useful tool for studying the mechanisms of cartilage degradation.

The objective of the current study was to identify a new extraarticular anatomic landmark indicating the anteroposterior orientation of the tibia using computed tomography. In 39 volunteers (20 males, 19 females), computed tomography scans for healthy right knees in extension were done perpendicular to the tibial shaft axis. The anteroposterior axis of the tibia was defined as a line perpendicular to the transepicondylar axis and passing through the middle of the posterior cruciate ligament. At the level of the tibial plateau and the patellar tendon attachment, the mean medial percentage width of intersecting point of the patellar tendon and the anteroposterior axis was 10.8% +/- 9.8% (range, -9.3% - +30.0%) and -0.2% +/- 10.4% (range, -23.6% -+23.0%), respectively. The mean angle between the anteroposterior axis and a line connecting the middle of the posterior cruciate ligament to the medial border of the patellar tendon attachment was 0.0 degrees +/- 2.8 degrees (range, -6.3 degrees -+5.2 degrees). The medial border of this attachment therefore can serve as a reliable anterior anatomic landmark to determine the anteroposterior axis of the tibia, and the line connecting the middle of the posterior cruciate ligament and the medial border of the attachment may be useful as a reference axis indicating the anteroposterior orientation of the tibia.

We have previously demonstrated that reactive oxygen species (ROS) are involved in cartilage degradation. A decrease in the size of hyaluronan (HA), which is the major macromolecule in synovial fluid and is responsible for imparting viscosity to it, is reported in arthritis patients. The purpose of this study is to determine the ROS that depolymerize HA. The luminol derivative, L-012, was used to determine the generation of ROS. To generate hydroxyl radicals, a mixture of hydrogen peroxide (H 2 O 2 ) and ferrous ions (Fe 2+ ) was added to HA. The antioxidants and the depolymerization of HA were studied in this system. The hydroxyl radical is one of the ROS, causing the depolymerization of HA, which reacts with L-01. These data suggest that hydroxyl radicals play an important role at the site of inflammation.

A posterior-stabilizing knee prosthesis, called the Bisurface knee (Kyocera Corp, Kyoto, Japan), with a ball-and-socket joint installed in the midposterior portion of the tibiofemoral joint, has been developed to satisfy 2 conflicting demands in knee joint design: kinematics and wear resistance. To confirm if the prosthesis has achieved its design objectives, a contact area and stress study was done throughout the range of motion and compared with results obtained for the Insall-Burstein 2 knee. The posterior-stabilizing ability of the ball-and-socket joint also was assessed. This study showed that the ball-and-socket joint could provide sufficient posterior stability, earlier start of flexion, and lower contact stress in the tibial polyethylene insert in flexion. The design of the Bisurface knee could provide a good balance between kinematics, stability, and wear resistance.

Hyaluronic acid (HA) has been shown to be clinically effective, and is currently used for the treatment of arthropathy. We previously reported that HA of molecular weight 90 kDa (90-HA) inhibits the fibrinolytic factors in human synovial fibroblasts. In the present study, we investigated the effect of high molecular weight (190 kDa) HA (190-HA) compared with 90-HA on the pericellular fibrinolytic system of human synovial fibroblasts in osteoarthritis (OA) and rheumatoid arthritis (RA). Human synovial fibroblasts were obtained from synovial tissues of OA and RA, and were cultured in the presence and absence of 90-HA and 190-HA. Antigens of urokinase-type plasminogen activator (u-PA) and PA inhibitor-1 (PAI-1) were measured by ELISA, and the u-PA activity of the cell surface fraction was evaluated by electrophoretic enzymography. The binding assay of u-PA and the immunocytochemical analysis of u-PA were performed to detect u-PA receptor (u-PAR). HA inhibited the secretion of both u-PA and PAI-1 antigens from the synovial fibroblasts of OA to their conditioned medium, and the degree of inhibition was more effective in OA than in RA. The u-RA binding assay to these cells showed that both 90-HA and 190-HA slightly decreased the maximal number of binding sites (Bmax) in OA. However, in RA, stimulation with 90-HA and 190-HA decreased Bmax by a half and a quarter, respectively. Immunohistochemical analysis showed that u-PAR was constitutively expressed in both synovial fibroblasts, but if these cells were treated with HA, the decrease in the staining of u-PAR was more pronounced in RA than in OA. Furthermore, the degree was more effective with 190-HA than with 90-HA. HA inhibited the pericellular fibrinolytic activity mediated by the u-PA/u-PAR system in synovial fibroblasts of OA and RA, and 190-HA inhibited it more effectively than 90-HA.

A 54-year-old woman complained of a painful, swollen, and clicking left wrist for 1 year. She had an 8-year history of systemic lupus erythematosus (SLE), treated with oral prednisolone. Flexion and extension of all fingers were difficult to initiate on wrist extension, and movement was accompanied by a palpable click at the wrist. Magnetic resonance imaging (MRI) and tenography revealed the expanded sheath of the flexor tendons and well-defined round free bodies known as rice bodies. Synovectomy and excision of rice bodies resulted in complete disappearance of swelling and triggering of the wrist.

In earlier work, we obtained evidence for the presence of histamine H1 and H2 receptors on chondrocytes. Activation of the H1 receptor enhanced keratan sulfate synthesis, and protein kinase C (PKC) inhibitors antagonized histamine-stimulated keratan sulfate (KS) synthesis. These data do indicate the involvement of PKC in activation of H1 receptor, but precise mechanisms remained to be clarified. Human articular chondrocytes were treated with different concentrations of histamine and its antagonist. Intracellular Ca 2+ and proteoglycan synthesis was measured, using the fluorescent indicator dye Fura-2 AM and [ 35 S]-sulfate incorporation, respectively. Activation of the H1 receptor led to stimulation of proteoglycan synthesis and evoked increases in levels of intracellular Ca 2+ . Activity of PKC was also enhanced with activation of the H1 receptor. Intracellular Ca 2+ and activation of PKC are involved in the signal transduction pathway of H1 receptor-mediated stimulation of proteoglycan synthesis.

Interleukin-1 (IL-1) causes cartilage degradation through nitric oxide (NO) synthesis. Although Interleukin-4 (IL-4) antagonizes the IL-1-mediated cartilage degradation, the precise mechanisms are not clear. We examined the effect of IL-4 on NO synthesis in parallel with intracellular Ca levels ([Ca 2+ ]i) and proteoglycan (PG) synthesis. IL-4-inhibited IL-1-enhanced NO release in a dose–dependent manner. IL-1-enhanced [Ca 2+ ]i in the chondrocytes, and IL-4 attenuated this increase. IL-4 reversed IL-1-inhibited PG synthesis. Accordingly, IL-4 reversed the IL-1-inhibited PG synthesis through the inhibition of NO release. An increase in [Ca 2+ ]i with IL-1 is possibly involved in this action.

Twelve patients with osteoarthritis of the knee were administrated hyaluronic acid (HA), and measured for fibrinolytic factors on synovial fluids. It was observed that urokinase-type plasminogen activator (u-PA) activity is enhanced 3 h after administration of HA, and that the activity gradually decreased from the 2nd to the 4th week on the patients whose clinical parameters showed improvement. Antigen of u-PA and PA inhibitor-1 (PAI-1) were increased 3 h after the administration of HA in the cases that improved. However, u-PA antigen gradually decreased from the 2nd to the 4th week similarly to u-PA activity. On the other hand PAI-1 antigen was increased from the 2nd to the 4th week in the improved cases. These results demonstrate that the decrease of fibrinolytic activity in synovial fluid is associated with the improvement of osteoarthritis by treatment with HA.

A β-tricalcium phosphate-monocalcium phosphate monohydrate (β-TCP-MCPM) cement was evaluated as an effective carrier of recombinant human bone morphogenetic protein-2 (rhBMP-2) in rat femoral critical-size defects. Hard cement cylinders (4 × 5 mm) impregnated with two different doses of rhBMP-2 (1.26 or 6.28 μg) were implanted into each defect, and the results were compared with those in rats that had implantations of cylinders only. Implantation of the 6.28 μg dose of rhBMP-2 caused a large bone shell to form around the defect, resulting in osseous union in all cases within 3 weeks. Except for β-TCP granules, the cement was resorbed and replaced by bone tissue at 6 weeks. A torsion test at 9 weeks showed that the failure torque and bone stiffness had recovered 99% and 141%, respectively, compared with the intact contralateral femur. The defects that received 1.26 μg of rhBMP-2 resulted in 40% union and 41% of the failure torque at 9 weeks. However, no instances of union were observed in the defects implanted with cylinders only. In conclusion, the β-TCP-MCPM cement was shown to be effective as a rhBMP-2 carrier. Combined with rhBMP-2, this cement was rapidly resorbed and completely healed the defects. © 1999 John Wiley & Sons, Inc. J Biomed Mater Res, 44, 168–175, 1999.

Objectives: Osteoarthritis (OA) is a common joint deterioration initiated by multiple factors. To better understand related factors in the development of this disease, we focused on the mechanical stress loaded on articular cartilage. Materials and Methods: The anterior cruciate ligaments of rabbit knee joints were transected, and expression of protein kinase C (PKC) examined immunohistochemically. The PKC activator 12-o-tetradecanoyl-phorbol-13-acetate (TPA) was then administered intraarticularly. To determine the involvement of gas mediators, a cartilage defect was made on the medial femoral condyle of rabbit knee joints. Hydrostatic pressure was loaded on the cartilage taken from the surrounding defects, and levels of superoxide anion and nitric oxide (NO) were measured. Bovine chondrocytes were subjected to cyclic mechanical stretch using a Flexercell Strain Instrument. Proteoglycan synthesis and PKC activity were measured. Expression of matrix metal loproteinase (MMP)-3 and tissue inhibitors of metalloproteinase (TIMP)-1 in articular cartilages obtained from OA patients were examined using Northern blots. Results: Chondrocytes from experimentally induced OA were stained positively with anti-α-PKC antibody. Intraarticular administration of TPA prevented the development of OA changes. Cyclic tensile stretch loaded on chondrocytes decreased proteoglycan synthesis and PKC activity. Thus, PKC is involved in the stress-mediated degradation of articular cartilage. Cartilage defects led to degradation of surrounding cartilage and to enhanced superoxide anion and NO synthesis. We also noted increased and decreased expressions of MMP-3 and TIMP-1 mRNA in human OA cartilage, respectively. Conclusion: PKC, gas mediators (superoxide anion, NO), and proteinases are all involved in OA.

To study the role of protein kinase C (PKC) in cartilage tissue in osteoarthritis, experimental osteoarthritis was induced in the knee joints of rabbits by resection of the anterior cruciate ligament (ACL). At 4 weeks after the operation, osteoarthritic changes varying from surface irregularities and cleft formation to loss of the tangential layer were observed, and cloning or hypocellularity of the chondrocytes was observed mainly in the transitional and radial layers. The PKC activator 12-o-tetradecanoyl-phorbol-13-acetate (TPA) or non-PKC-activating phorbol ester 4 α-phorbol-12,13-didecanate (PDD) was administered intraarticularly once a week from the day of the operation for 3 weeks. Histologic evaluation with a rating scale was carried out. In the TPA-administered group, cartilage structures were preserved almost completely, and score of the cartilage lesion was significantly less than that in animals administered PDD or in nonadministered controls. A chondroprotective role of PKC under mechanical stress was suggested.

Vancomycin (VCM), a methiciline-cefem resistant Staphylococcus aureus (MRSA)-specific antibiotic, was incorporated in a self-setting tetracalcium phosphate (TTCP)-dicalcium phosphate dihydrate (DCPD) apatite cement that hardened isothermally into a hydroxyapatite (HAP) phase with crystallinity similar to that of host bone. Effective release of VCM into PBS lasted for 2 weeks from cements containing 1% VCM and for longer than 9 weeks from cements containing 5% VCM. The rate of release of VCM differed between cements with different crystallinities as well as between the two dissolution media, PBS and simulated body fluid. Mean concentration of VCM in the bone marrow tissue released from cements containing 5% VCM was 20 times the minimum inhibitory concentration 3 weeks after implantation in bone. Direct contact with new bone was observed with the cements containing 1% VCM. Slow delivery of VCM from a self-setting TTCP-DCPD apatite cement with low crystallinity could be used to treat MRSA osteomyelitis.

Aims An immunohistochemical screen for mouse embryos showed that cell adhesion molecule 1 (CADM1), which is an immunoglobulin superfamily member, was expressed in developing bones. Here, we determined the cell types expressing CADM1 and examined its usefulness in the differential diagnosis of osteosarcoma. Main methods Serial sections of murine developing mandibles were stained with anti-CADM1 antibody, by a coloring substrate reactive to alkaline phosphatase (ALP), a broad osteoblastic marker for preosteoblasts to osteoblasts, and by in situ hybridization for osteopontin (OPN), a marker for mature osteoblasts. CADM1 immunohistochemistry was also performed on human remodeling bones, osteosarcomas and other soft tissue tumors. Key findings CADM1 immunohistochemistry for the mandible revealed that morphologically identifiable osteoblasts expressed CADM1 on their plasma membranes, but neither osteocytes nor bone lining cells did. At the mandibular margin, not only OPN-positive cells but also OPN-negative, ALP-positive cells were CADM1-positive, whereas inside the mandible, OPN-positive cells were often CADM1-negative. Clear membranous staining was detected in the majority of osteosarcomas (46/57), whereas only 13% (6/46) of the other soft tissue tumors were CADM1-positive ( P Significance These results indicated that CADM1 was a novel osteoblastic adhesion molecule that is expressed transiently during osteoblastic maturation, and a useful diagnostic marker for osteosarcoma cells.

One hundred and eight neonates and infants who showed the clinical triad of a head turned to one side, adduction contracture of the hip joint on the occipital side of the turned head, and truncal curvature, which we named TAC syndrome, were studied. These cases included seven with congenital and five with late infantile dislocations of the hip joint and 14 who developed muscular torticollis. Forty one were among 7103 neonates examined by one of the authors. An epidemiological analysis confirmed the aetiology of the syndrome to be environmental. The side to which the head was turned and that of the adducted hip contracture showed a high correlation with the side of the maternal spine on which the fetus had been lying. TAC syndrome is an important asymmetrical deformity that should be kept in mind during neonatal examination, and may be aetiologically related to the unilateral dislocation of the hip joint, torticollis, and infantile scoliosis which develop after a vertex presentation.

Excessive mechanical stress on the cartilage causes the degradation of the matrix, leading to the osteoarthritis (OA). Matrix metalloproteinases 13 (MMP13) is a major catalytic enzyme in OA and p38 plays an important role in its induction. However, precise pathway inducing p38 activation has not been elucidated. We hypothesized here that the small GTPase Rho and its effector ROCK might function in upper part of the mechanical stress-induced matrix degeneration pathway. Bovine metacarpal phalangeal articular cartilage explants were loaded with 1 MPa dynamic compression for 6 h with or without a ROCK specific inhibitor Y27632 or/and a p38 specific inhibitor SB202190. Then p38 phosphorylation and MMP13 expression were assessed by western blot or/and quantitative RT-PCR. Rho-activity was measured by pull-down assay using glutathione S-transferase fusion protein of Rho binding domain. Cyclic compression caused Rho activation, p38 phosphorylation and MMP13 expression. Both Y27632 and SB202190 were found to block the mechanical stress-enhanced p38 phosphorylation and subsequent MMP13 expression. The present results show that p38 phosphorylation and MMP13 expression are regulated by Rho/ROCK activation, and support the potential novel pathway that Rho/ROCK is in the upper part of the mechanical stress-induced matrix degeneration cascade in cartilage comprised of p38 and MMP13.

Insufficiency fracture occurs when normal or physiological activity stresses a bone that is deficient in mineral or elastic resistance [1]. Insufficiency fractures are most often seen in elderly women with postmenopausal osteoporosis. However, it can occur in a variety of conditions in which the mineral content or the elasticity of bone is impeded, including osteoporosis of any cause, osteomalacia, hyperparathyroidism, rheumatoid arthritis, fluoride treatment, diabetes mellitus, fibrous dysplasia, Paget’s disease of the bone, and also renal osteodystrophy [1–3]. Insufficiency fractures most commonly occur in the pelvic girdle and in the sacrum [2]. In addition, the tibia is also a common site of insufficiency fractures [2]. The tibial insufficiency fractures are often treated by conservative measures, such as adequate rest, immobilization with casts or braces, analgesic agents, and reduced activities in daily living [3–5]; surgical treatments are rarely indicated unless the fractures do not heal or heal incorrectly [6–9]. Therefore, there is very little information available when adopting the surgical treatment to the proximal tibial insufficiency fractures [2]. Here, we report a case of nonunited insufficiency fracture of the proximal tibia with a marked varus deformity in a hemodialysis patient, which was complicated with a supracondylar insufficiency fracture of the ipsilateral femur. To our knowledge, correction osteotomy for such a case has not been reported previously. Our patient was informed and gave the consent that data concerning the case would be submitted for publication.

The purpose of this study was to clarify the causes of the increase in the posterior tibial slope during open-wedge high tibial osteotomy (HTO) and to investigate whether its changes influenced the correction angle in frontal plane. We retrospectively reviewed 20 patients (26 knees) treated with open-wedge HTO. They were divided into the following two groups. Group A consisted of the knees whose opening gaps were fixed using a spacer plate having the trapezoidal block with a 2° posterior slope. In Group B, anterior and posterior opening gaps were fixed separately. The posterior tibial slope and the hip–knee–ankle angle were measured based on CT data. The relationship between the correction rate in frontal plane and the changes of posterior tibial slope was investigated. Increase in the posterior tibial slope was 2.1 ± 2.5° in Group A and 0.2 ± 1.2° in Group B, which showed a statistical difference (P = 0.02). The difference between the hip–knee–ankle angles before and after operation was 5.2 ± 2.3° in Group A and 5.5 ± 2.5° in Group B. The correction rate was statistically correlated with the changes of posterior tibial slope (R = −0.55, P = 0.003). To avoid increase in the posterior tibial slope, the trapezoidal block with a only 2° posterior slope in a spacer plate was not sufficient, and it was necessary to fix anterior and posterior gaps separately. The correction angle in frontal plane had a trade-off relationship with the changes in posterior tibial slope. Thus, we thought that increase in the posterior tibial slope might result in correction loss. Therapeutic study, Retrospective comparative study, Level III.

Preoperative planning of total knee arthroplasty (TKA) based on computerized tomography (CT) data can produce a femoral rotational error due to lack of information on the femoral cartilage thickness. The research question of this study is how much femoral rotational error is expected due to the cartilage remnants when using the posterior condylar angles (PCA, angle between the posterior condylar line and the surgical epicondylar axis (SEA)) on CT data. CT arthrography was performed for 35 consecutive varus osteoarthritic knees in 31 patients who underwent TKA, on which the cartilage thicknesses of the posterior femoral condyles were measured. The PCAs when including or excluding the cartilage remnants were also measured. The cartilage thicknesses of the medial and lateral posterior condyles averaged 0.39mm (SD=0.53) and 1.55mm (SD=0.26), respectively (p0.0001). When the cartilage was included or excluded, the PCA averaged 2.2° (SD=1.5) and 3.3° (SD=1.5), respectively (p=0.002). The cartilage remnants in the posterior femoral condyles produced an average of 1.1° and a maximum of 2.1° of additional femoral external rotation when using CT data for the preoperative planning. CT scan measurements of femoral rotation are subject to error. Although this is said to be small and within the safety margin for setting the femoral component parallel to the trans-epicondylar axis, this difference should be considered by surgeons who use the posterior condylar axis, in order to avoid excessive external rotation of the femoral component.

Despite recent cell-engineering advances, treatment and repair of cartilage remains challenging. Although stem cell transplantation therapy using mesenchymal stem cells (MSCs) is considered a prominent strategy, the major problem of limited proliferative capacity of autologous cells has been unsolved. Recently, an induced pluripotent stem (iPS) cell line was suggested as an alternative way to cure various human diseases due to their potential proliferating infinitely while possessing the capacity to form all types of cells. However, the method to induce lineage-restricted differentiation has not been well examined or established. Here, we suggest a simple method to induce mesenchymal progenitors possessing chondrogenic property from mouse iPS cells. The MSC-like cells produced in our study expressed some MSC markers, and could also differentiate to osteoblast and adipocyte. The present study demonstrates the property of iPS cells as an alternative candidate for treatment of articular disorders, and suggests an effective approach for preparing chondrocyte from iPS cells.

Since 1961 we have used the Spitzy shelf operation combined with a vertical flap osteotomy of the outer cortex of the ilium for early arthrosis secondary to acetabular dysplasia, subluxation, and dislocation. In 124 hips, followed for 10 (5-25) years, clinical improvement was maintained in 67/85 of patients who were operated on under the age of 30 and in 22/39 of patients treated after 30. However, less than half of the hips in the younger group and only one out of ten in the older group were symptom-free.

35 calluses formed during limb lengthening were classified radiographically into 6 types: external, straight, attenuated, opposite, pillar, and agenetic. The healing indexes correlated well to the intrinsic periosteal and endosteal conditions of each type. This classification enabled us to estimate the intrinsic conditions, predict the healing index, control the daily lengthening speed, and decide to apply early augmentation of the callus.

Objective Findings of recent in vivo and in vitro studies suggest that oxidized low-density lipoprotein (ox-LDL) plays a role in the degeneration of cartilage. The purpose of this study was to determine whether ox-LDL induces chondrocyte senescence through binding to lectin-like ox-LDL receptor 1 (LOX-1). Methods The effects of ox-LDL on senescence of cultured bovine articular chondrocytes (BACs) were investigated by observing senescence-associated (SA) β-galactosidase (β-gal) activity, cell proliferation activity, and telomerase activity. Telomerase activity was measured after adding LY294002 (a specific inhibitor of phosphatidylinositol 3-kinase [PI3K]) or after adding insulin-like growth factor 1 (IGF-1; an activator of PI3K) plus ox-LDL to the culture medium to elucidate the involvement of the PI3K/Akt pathway. Immunoblot analysis was used to investigate whether ox-LDL affects the phosphorylation of Akt. To ascertain whether these effects were attributable to ox-LDL binding to LOX-1, BACs were preincubated with TS-20, an anti-bovine LOX-1 blocking antibody. Results The activity of SA β-gal was increased and the incorporation of bromodeoxyuridine into BACs was decreased by ox-LDL in a dose-dependent manner. The telomerase activity of BACs was suppressed by the addition of ox-LDL in a time- and dose-dependent manner. LY294002 suppressed the telomerase activity of BACs, and IGF-1 reversed the ox-LDL–induced suppression of telomerase activity. In addition, ox-LDL rapidly decreased the amount of phosphorylated Akt in BACs. Pretreatment of cultured BACs with TS-20 recovered these effects. Conclusion These data show that ox-LDL binding to LOX-1 induces stress-induced premature senescence of chondrocytes and results in suppression of telomerase activity by inactivating the PI3K/Akt pathway. Oxidized LDL may play an important role in the pathogenesis of osteoarthritis by inducing chondrocyte senescence.

Recent studies have illustrated multiple differentiation potentials of embryonic stem cells (ESCs), derived from parthenogenetic embryos, to various kinds of cells (all three embryonic germ layers). However, differentiation diversity of the parthenogenetic ESCs (PgESCs) in vivo remains to be elucidated. In the present study, we established mouse PgESC-lines and observed their contribution diversity in vivo by producing chimeric mice using embryos possessing single nucleotide polymorphisms of mitochondrial DNA (mtDNA) as hosts. Based on southern blot analysis using specific probes to detect the SNPs on mtDNA, PgESC-derived mtDNA were contained in many organs such as brain, lung, and heart of the chimeric mouse. We concluded that PgESCs contributed to various internal organs in vivo, and that they were also stably maintained in adult animals.

Abnormal mechanical stress loaded on the cartilage leads to the osteoarthritis (OA). Although intraarticular hyaluronan (HA) injection is an effective treatment for OA, the underlying mechanism has not been made clear. Mechanical compression was loaded on the bovine cartilage using the Biopress system. Proteoglycan (PG) and reactive oxygen species (ROS) synthesis were measured with [35S] incorporation and fluorescent dye, respectively. Accumulation of peroxynitrite was determined with western blotting using nitrotyrosine antibody. Mechanical compression inhibited PG synthesis and enhanced ROS. Externally added HA reversed stress-inhibited PG synthesis and attenuated ROS synthesis. HA also significantly decreased the generation of nitrotyrosine. HA neutralized stress-enhanced ROS synthesis and resulted in the reversing of PG synthesis. These data suggest that HA plays an anabolic effect as an antioxidant.

Background Autologous chondrocyte implantation is an established technique for the repair of degenerated articular cartilage. Recently, the detection of side population (SP) cells, which have the ability to strongly efflux Hoechst 33342 (Ho) fluorescence dye, has attracted attention as a method of stem cell isolation. Although SP cells from synovial tissue were expected to be an excellent source for this tissue engineering, their precise character in the synovial tissue has not been determined. Methods Synovial tissues from bovine metacarpophalangeal joints were used as a stem cell source. For efficient collection of stem cells, we first prepared a preculture before sorting in medium containing FBS at variable concentrations for 4 days. Using a cell sorter and the Ho-dye, a poorly stained population enriched with stem cells was then isolated. To determine the characteristics of the stem cells, specific marker genes such as CD34, Flk-1, c-Kit, Abcg-2 were identified by real-time PCR. Sorted SP cells were cultured in a stem cell medium supplemented with bFGF, SCF and fibronectin, and evaluated for their differentiation potentials into chondrocytes, osteocytes and myocytes. Results SP cells of synovium tissue were increased from 2% of the total cell population to approximately 10% of the total cells by preculture in the 1%FBS contained medium. Sorted SP cells expressed CD34, Flk-1, c-Kit, Abcg-2 and Mdr-1 -all are important marker genes for stem cell characteristics. The SP cells could be further expanded ex vivo while maintaining stem cell potentials such as marker gene expression, Ho-dye efflux potential and multiple differentiation potentials into chondrocyte, osteocyte and myocyte. Conclusion In the present study, we demonstrated that the cells with outstanding stem cell properties were efficiently collected as a SP fraction from bovine synovial membrane. Furthermore, we have described an efficient isolation method and the culture conditions for ex vivo expansion that maintains their important characteristics. Our results suggest that the SP cells of synovium tissue might be important candidates as sources for cell transplantation.

Submicron-sized hydroxyapatite (HAp) particles were loaded on the titanium plate by anodic oxidation over spark discharge voltage in HAp-containing bath. The biocompatibility and bioactivity were drastically improved by HAp deposition on the anodic oxide films. The affinity with living bone was the following order: anodized titanium plate with HAp > anodized titanium plate ≫ titanium. Bone-bonding strength between HAp-loaded titanium plate and living bone after 4 weeks' implant was much higher than that of anodized titanium plate.