S. Kubo

SIRT1 regulation of apoptosis of human chondrocytes.

OBJECTIVE SIRT1 is known to inhibit apoptosis and to promote survival of various types of cells. However, the roles of SIRT1 in apoptosis of human chondrocytes have never been reported. We undertook this study to investigate the relationship of SIRT1 to apoptosis of human chondrocytes, which is a characteristic feature of osteoarthritis (OA). METHODS The expression of SIRT1 in human chondrocytes was examined by reverse transcription-polymerase chain reaction, immunoblotting, and immunohistology of human cartilage samples. The expression of SIRT1 under catabolic, mechanical, and nutritional stresses was investigated by immunoblotting. To examine the effect of SIRT1 on apoptosis, SIRT1 was inhibited by small interfering RNA (siRNA) and activated by resveratrol during nitric oxide (NO)-induced apoptosis. TUNEL staining and immunoblotting of cleaved poly(ADP-ribose) polymerase (PARP) were performed to detect apoptosis. To examine the mechanisms of apoptosis, we used immunoblotting to determine the levels of cleaved caspases and mitochondria-related apoptotic signaling proteins, Bax and Bcl-2, in the mitochondrial fraction. RESULTS SIRT1 expression was confirmed in human chondrocytes and human cartilage samples. All catabolic, mechanical, and nutritional stresses inhibited SIRT1 expression. SIRT1 inhibition by siRNA for SIRT1 increased the percentage of TUNEL-positive cells and increased the amounts of cleaved PARP and cleaved caspases 3 and 9 induced by NO. In contrast, treatment with resveratrol decreased the percentage of TUNEL-positive cells and decreased the amounts of cleaved PARP and cleaved caspases 3 and 9 induced by NO. Furthermore, in the mitochondrial fraction, SIRT1 inhibition by siRNA for SIRT1 increased the amount of Bax but reduced the amount of Bcl-2, while resveratrol reduced the amount of Bax but increased the amount of Bcl-2. CONCLUSION These results indicate that SIRT1 regulates apoptosis in human chondrocytes through the modulation of mitochondria-related apoptotic signals. Further research on SIRT1 might contribute to resolving the pathogenesis of OA.

Initial physics achievements of large helical device experiments

The Large Helical Device (LHD) experiments [O. Motojima, et al., Proceedings, 16th Conference on Fusion Energy, Montreal, 1996 (International Atomic Energy Agency, Vienna, 1997), Vol. 3, p. 437] have started this year after a successful eight-year construction and test period of the fully superconducting facility. LHD investigates a variety of physics issues on large scale heliotron plasmas (R=3.9 m, a=0.6 m), which stimulates efforts to explore currentless and disruption-free steady plasmas under an optimized configuration. A magnetic field mapping has demonstrated the nested and healthy structure of magnetic surfaces, which indicates the successful completion of the physical design and the effectiveness of engineering quality control during the fabrication. Heating by 3 MW of neutral beam injection (NBI) has produced plasmas with a fusion triple product of 8×1018 keV m−3 s at a magnetic field of 1.5 T. An electron temperature of 1.5 keV and an ion temperature of 1.4 keV have been achieved. The maximum s...

Autophagy modulates osteoarthritis-related gene expression in human chondrocytes

OBJECTIVE Autophagy, an evolutionarily conserved process for the bulk degradation of cytoplasmic components, serves as a cell survival mechanism. The purpose of this study was to elucidate the role of autophagy in human chondrocytes and pathophysiology of osteoarthritis (OA). METHODS Autophagy in articular cartilage and primary chondrocytes was assessed using antibodies for the autophagy markers light chain 3 and beclin 1. The states of autophagy under catabolic and nutritional stresses were examined. We also examined the effects of inhibition or induction of autophagy under stimulation with interleukin-1β. Autophagy was inhibited by small interfering RNA targeting ATG5, and autophagy was induced by rapamycin. The effects of inhibition or induction of autophagy were examined by real-time polymerase chain reaction for aggrecan, COL2A1, MMP13, and ADAMTS5 messenger RNA. To further examine the mechanism of autophagy regulation in OA human chondrocytes, we investigated whether autophagy modulates apoptosis and reactive oxygen species (ROS). RESULTS Autophagy was increased in OA chondrocytes and cartilage. Catabolic and nutritional stresses increased autophagy. In addition, the inhibition of autophagy caused OA-like gene expression changes, while the induction of autophagy prevented them. Furthermore, the inhibition of autophagy increased the amount of cleaved poly(ADP-ribose) polymerase and cleaved caspase 9, while the induction of autophagy inhibited these increases. ROS activity was also decreased by induction of autophagy. CONCLUSION These observations suggested that increased autophagy is an adaptive response to protect cells from stresses, and that autophagy regulates OA-like gene expression changes through the modulation of apoptosis and ROS. Further studies about autophagy in chondrocytes will provide novel insights into the pathophysiology of OA.

A prospective randomised study of anatomical single-bundle versus double-bundle anterior cruciate ligament reconstruction: quantitative evaluation using an electromagnetic measurement system

We conducted a prospective randomised study of anatomical single-bundle (A-SB group) versus double-bundle (A-DB group) anterior cruciate ligament (ACL) reconstruction using the hamstrings tendons. Twenty patients with unilateral ACL deficiency were randomised into two groups. We created the bone tunnels at the position of the original insertion of the anteromedial bundle footprint and posterolateral bundle footprint in the A-DB group and at the central position between these two bundles in the A-SB group. All of the patients were tested before ACL reconstruction and one year after surgery. The KT-1000 measurements, isokinetic muscle peak torque and heel-height difference were evaluated and the general knee condition was assessed by Lysholm score. For pre- and postoperative stability assessment, we used the six-degrees-of-freedom of knee kinematic measurement system using an electromagnetic device (the EMS) for quantitative assessment during the Lachman test and the pivot shift test. There were no significant differences in the KT-1000 measurements, isokinetic muscle peak torque, heel-height difference, and Lysholm score at one-year follow-up between these two groups. The EMS data showed there were significant differences in the acceleration of the pivot shift test between the operated knee and the contralateral normal knees in the A-SB group. In conclusion, clinical outcomes were equally good in both groups. However, the EMS data showed the anatomical double-bundle ACL reconstruction tended to be biomechanically superior to the single-bundle reconstruction.

Enhancement of Tendon-Bone Osteointegration of Anterior Cruciate Ligament Graft Using Granulocyte Colony-Stimulating Factor

Background Whereas anterior cruciate ligament rupture usually requires reconstruction, the attachment between the tendon and the bone is the weakest region in the early posttransplantation period. In this process, the acquisition of appropriate vascularity is a key for early bone-tendon healing. Hypothesis Granulocyte colony-stimulating factor has an effect on the maturation of bone-tendon integration of anterior cruciate ligament reconstruction. Study Design Controlled laboratory study. Methods Twenty-eight healthy adult beagle dogs underwent bilateral anterior cruciate ligament reconstruction using the ipsilateral flexor digitorum superficialis tendon and were divided into 2 groups. A granulocyte colony-stimulating factor-incorporated gelatin surrounded the graft in the granulocyte colony-stimulating factor group, and the same gelatin without granulocyte colony-stimulating factor was used as the control group. Assessment was done at 2 and 4 weeks. Results Histological analysis at week 2 demonstrated that, in addition to more Sharpey fibers, microvessels were significantly enhanced in the granulocyte colony-stimulating factor groups grafts. Computed tomography at week 4 showed a significantly smaller tibial bone tunnel in the granulocyte colony-stimulating factor group. Real-time polymerase chain reaction revealed significantly elevated messenger ribonucleic acid expression levels of vascular endothelial growth factor and osteocalcin in the tibial bone tunnel and graft compared with controls. Furthermore, biomechanical testing of force during loading to ultimate failure at week 4 demonstrated a significant increase in strength in the granulocyte colony-stimulating factor group. Conclusion This study demonstrated that a local application of granulocyte colony-stimulating factor-incorporated gelatin significantly accelerates bone-tendon interface strength via enhanced angiogenesis and osteogenesis. Clinical Relevance Granulocyte colony-stimulating factor has therapeutic potential in promoting an environment conductive to angiogenesis and osteogenesis in bone tunnels.

Reliability and usefulness of a new in vivo measurement system of the pivot shift.

Residual pivot shift after ACL reconstruction is a crucial factor related to poor clinical outcome. However, no method exists that is able to evaluate pivot shift quantitatively and noninvasively. We propose a new measurement system for the pivot shift test using an electromagnetic device and have evaluated its reliability and clinical usefulness. Posterior translation, lateral translation and maximum velocity during the reduction phase of pivot shift were calculated and used as parameters for evaluation. In measurement system analysis, discrepancies of motion between the bones and the sensors were minimal, while reproducibility in repeated measurement was acceptable. Next, clinical usefulness was evaluated by correlating the values obtained by kinematic measurement with the clinical grade. We found differences in each of the measured parameters among clinical grades. These data suggest the system is a valuable measurement tool for clinical evaluation of the pivot shift test.Level of Evidence: Level II, diagnostic study. See Guidelines for Authors for a complete description of levels of evidence.

Potential involvement of SIRT1 in the pathogenesis of osteoarthritis through the modulation of chondrocyte gene expressions.

SIRT1 has been implicated as a key factor in aging‐related diseases. Nevertheless, the role of SIRT1 in the pathogenesis of osteoarthritis (OA) is still unknown. We examined the expression of SIRT1 in cartilage samples and the effect of SIRT1 inhibition on chondrocyte gene expression changes to elucidate the role of SIRT1 in chondrocytes. SIRT1 expression was examined using cartilage samples from patients undergoing total knee arthroplasty and femoral head replacement by immunohistochemistry. The effect of SIRT1 inhibition by siRNA on chondrocyte gene expression was examined by real‐time PCR and Western blotting. SIRT1 expression was barely detectable in the severely degenerated cartilage while SIRT1 was clearly expressed in the less damaged cartilage. The inhibition of SIRT1 by siRNA induced OA‐like gene expression changes, namely the significant down‐regulation of aggrecan and up‐regulation of COL10A1 and ADAMTS‐5. Our observations suggest that SIRT1 expression decreases with development of OA and the reduction of SIRT1 in chondrocytes may cause chondrocyte hypertrophy and cartilage matrix loss. SIRT1 might play important roles in the pathogenesis of OA.

The intra-operative joint gap in cruciate-retaining compared with posterior-stabilised total knee replacement

We have developed a new tensor for total knee replacements which is designed to assist with soft-tissue balancing throughout the full range of movement with a reduced patellofemoral joint. Using this tensor in 40 patients with osteoarthritis we compared the intra-operative joint gap in cruciate-retaining and posterior-stabilised total knee replacements at 0 degrees , 10 degrees , 45 degrees , 90 degrees and 135 degrees of flexion, with the patella both everted and reduced. While the measurement of the joint gap with a reduced patella in posterior-stabilised knees increased from extension to flexion, it remained constant for cruciate-retaining joints throughout a full range of movement. The joint gaps at deep knee flexion were significantly smaller for both types of prosthetic knee when the patellofemoral joint was reduced (p < 0.05).

Femoral component placement changes soft tissue balance in posterior-stabilized total knee arthroplasty

BACKGROUND We developed a new tensor for total knee arthroplasty enabling the soft tissue balance measurement after femoral trial placement with the patello-femoral joint reduced. The purpose of the present study is to compare the measurements of joint gap and ligament balance between osteotomized femoral and tibial surfaces in posterior-stabilized total knee arthroplasty with that between surfaces of femoral trial component and tibial osteotomy. METHODS Using this tensor, the effect of femoral trial placement on the soft tissue balance was analyzed in 80 posterior-stabilized total knee arthroplasties for varus osteoarthritic knees. Both joint gap and varus ligament imbalance were measured with 40 lb of joint distraction force at extension and flexion, and compared between before and after femoral trial placement. FINDINGS In assessing the joint gap, there was significant decrease as much as 5.3mm at extension, not flexion, after femoral trial prosthesis placement. Varus ligament imbalances were significantly reduced with 3.1° at extension and increased with 1.2° in average at flexion after femoral trial placement. INTERPRETATION These changes at extension were caused by tensed posterior structures of the knee with the posterior condyle of the externally rotated aligned femoral trial. At the knee flexion, medial tension in the extensor mechanisms might be increased after femoral trial placement with patello-femoral joint repaired, and increased varus imbalance. Accordingly, we conclude that intensive medial release before femoral component placement to obtain rectangular joint gap depending on the conventional osteotomy gap measurement has a possible risk of medial looseness after total knee arthroplasty.

The overexpression of SIRT1 inhibited osteoarthritic gene expression changes induced by interleukin-1β in human chondrocytes.

In this study, we examined the effects of overexpression of SIRT1 on IL‐1β‐induced gene expression changes in human chondrocytes to explore a protective role of SIRT1 in human chondrocytes. SIRT1 was overexpressed in human chondrocytes by expression plasmid under stimulation with IL‐1β. SIRT1 was also inhibited by siRNA under stimulation with IL‐1β. Gene expression changes were examined by real‐time PCR. The interaction of SIRT1 and p65 (NF‐κB) were examined by Western blotting. SIRT1, MMP‐13, and ADAMTS‐5 expressions in human cartilage were examined by immunohistochemistry. IL‐1β stimulation significantly up‐regulated MMP‐1, 2, 9, and 13 and ADAMTS‐5. Overexpression of SIRT1 significantly inhibited the up‐regulation of those genes caused by IL‐1β while the inhibition of SIRT1 further increased them. In addition, the overexpression of SIRT1 markedly reduced the IL‐1β‐induced acetylation of p65. SIRT1 expression was clearly detected in the non‐OA cartilage while MMP‐13 and ADAMTS‐5 were undetectable. In contrast, in the OA cartilage, SIRT1 expression was decreased while MMP‐13 and ADAMTS‐5 were increased. Our observations suggested that SIRT1 can play a protective role by suppressing IL‐1β‐induced expressions of cartilage‐degrading enzymes partially through the modulation of the NF‐κB pathway. SIRT1 overexpression might be a new therapeutic approach for OA.