Hayatoshi Shibuya

The inhibitory effect of microRNA-146a expression on bone destruction in collagen-induced arthritis.

OBJECTIVE MicroRNA, a class of noncoding RNA, play a role in human diseases. MicroRNA-146a (miR-146a) is a negative regulator of immune and inflammatory responses, and is strongly expressed in rheumatoid arthritis (RA) synovium and peripheral blood mononuclear cells (PBMCs). This study was undertaken to examine whether miR-146a expression inhibits osteoclastogenesis, and whether administration of miR-146a prevents joint destruction in mice with collagen-induced arthritis (CIA). METHODS PBMCs from healthy volunteers were isolated and seeded in culture plates. The following day, double-stranded miR-146a was transfected and cultured in the presence of macrophage colony-stimulating factor and either tumor necrosis factor α or RANKL. After 3 weeks, tartrate-resistant acid phosphatase (TRAP)-positive multinucleated cells were counted. Three days after miR-146a culture, the expression of c-Jun, nuclear factor of activated T cells c1 (NF-ATc1), PU.1, and TRAP was evaluated by quantitative reverse transcriptase-polymerase chain reaction. After the onset of distinct arthritis in mice with CIA, double-stranded miR-146a or nonspecific double-stranded RNA was administered twice by intravenous injection. Radiographic and histologic examinations were performed at 4 weeks. RESULTS The number of TRAP-positive multinucleated cells in human PBMCs was significantly reduced by miR-146a in a dose-dependent manner. The expression of c-Jun, NF-ATc1, PU.1, and TRAP in PBMCs was significantly down-regulated by miR-146a. Administration of miR-146a prevented joint destruction in mice with CIA, although it did not completely ameliorate inflammation. CONCLUSION Our findings indicate that expression of miR-146a inhibits osteoclastogenesis and that administration of double-stranded miR-146a prevents joint destruction in arthritic mice. Administration of miR-146a has potential as a novel therapeutic target for bone destruction in RA.

Acceleration of muscle regeneration by local injection of muscle‐specific microRNAs in rat skeletal muscle injury model

MicroRNA (miRNA)s are a class of non‐coding RNAs that regulate gene expression post‐transcriptionally. Muscle‐specific miRNA, miRNA (miR)‐1, miR‐133 and miR‐206 play a crucial role in the regulation of muscle development and homeostasis. Muscle injuries are a common muscloskeletal disorder, and the most effective treatment has not been established yet. The purpose of this study was to demonstrate that a local injection of double‐stranded (ds) miR‐1, miR‐133 and 206 can accelerate muscle regeneration in a rat skeletal muscle injury model. After the laceration of the rat tibialis anterior muscle, ds miR‐1, 133 and 206 mixture mediated atelocollagen was injected into the injured site. The control group was injected with control siRNA. At 1 week after injury, an injection of miRNAs could enhance muscle regeneration morphologically and physiologically, and prevent fibrosis effectively compared to the control siRNA. Administration of exogenous miR‐1, 133 and 206 can induce expression of myogenic markers, MyoD1, myogenin and Pax7 in mRNA and expression in the protein level at 3 and 7 days after injury. The combination of miR‐1, 133 and 206 can promote myotube differentiation, and the expression of MyoD1, myogenin and Pax7 were up‐regulated in C2C12 cells in vitro. Local injection of miR‐1, 133 and 206 could be a novel therapeutic strategy in the treatment of skeletal muscle injury.

Induction of apoptosis in the synovium of mice with autoantibody-mediated arthritis by the intraarticular injection of double-stranded microRNA-15a

OBJECTIVE MicroRNA is a family of noncoding RNAs that exhibit tissue-specific or developmental stage-specific expression patterns and are associated with human diseases. MicroRNA-15a (miR-15a) is reported to induce cell apoptosis by negatively regulating the expression of Bcl-2, which suppresses the apoptotic processes. The purpose of this study was to investigate whether double-stranded miR-15a administered by intraarticular injection could be taken up by cells and could induce Bcl-2 dysfunction and cell apoptosis in the synovium of arthritic mice in vivo. METHODS Autoantibody-mediated arthritis was induced in male DBA/1J mice. In the experimental group, double-stranded miR-15a labeled with FAM-atelocollagen complex was injected into the knee joint. In the control group, control small interfering RNA-atelocollagen complex was injected into the knee joint. Synovial expression of miR-15a was analyzed by quantitative polymerase chain reaction, FAM by fluorescence microscopy, Bcl-2 by Western blotting, and Bcl-2 and caspase 3 by immunohistochemistry. RESULTS The expression of miR-15a in the synovium of the experimental group was significantly higher than that in the control group. Green fluorescence emission of FAM was observed in the synovium of the experimental group. Bcl-2 protein was down-regulated and the expression of caspase 3 was increased as compared with that in the control group. CONCLUSION These results indicate that the induction of cell apoptosis after intraarticular injection of double-stranded miR-15a occurs through inhibition of the translation of Bcl-2 protein in arthritic synovium.

Silencing microRNA-34a inhibits chondrocyte apoptosis in a rat osteoarthritis model in vitro

OBJECTIVE miRNAs, which are non-coding RNAs, play a role in the pathogenesis of disease including OA. miRNA (miR)-34a is induced by p53, subsequently leading to cell apoptosis, which is one of the major factors in the pathogenesis of OA. The purpose of this study is to investigate the effect of silencing miR-34a on IL-1β-induced chondrocyte apoptosis in a rat OA model in vitro. METHODS Locked nucleotide analogue (LNA)-modified miR-34a-specific anti-sense was transfected into rat chondrocyte monolayer culture. After that, IL-1β was added to the chondrocytes to create an OA model in vitro. The effect of silencing miR-34a on the prevention of chondrocyte apoptosis was analysed by assessment of the expression levels of Col2a1 and iNOS, also through assessment of cell viability and TUNEL staining. RESULTS The expression of miR-34a was significantly up-regulated by IL-1β. Silencing of miR-34a significantly prevented IL-1β-induced down-regulation of Col2a1, as well as IL-1β-induced up-regulation of iNOS. Finally, MiR-34a inhibitor could also reduce TUNEL-positive cells. CONCLUSION Silencing of miR-34a by LNA-modified anti-sense could effectively reduce rat chondrocyte apoptosis induced by IL-1β. This present study revealed that silencing of miR-34a might develop a novel intervention for OA treatment through the prevention of cartilage degradation.

Potential Risks of Femoral Tunnel Drilling Through the Far Anteromedial Portal: A Cadaveric Study

PURPOSE The purpose of this study was to estimate the potential risks when drilling femoral tunnels through the far anteromedial portal in double-bundle anterior cruciate ligament reconstruction in cadaveric knees. METHODS Ten cadaveric knees were used. We drilled the anteromedial bundle (AMB) and posterolateral bundle (PLB) through the far anteromedial portal at 3 different knee flexion angles: 70 degrees, 90 degrees, and 110 degrees. We measured the shortest distance to the common peroneal nerve and the posterior articular cartilage of the lateral femoral condyle and the femoral tunnel length. RESULTS At 70 degrees, the distance to the nerve was less than 10 mm in 7 AMB cases and in 9 PLB cases, and the distance to the cartilage was less than 10 mm in all the AMB and PLB cases. At 90 degrees, the distance to the nerve was less than 10 mm in 1 AMB and 5 PLBs, and the distance to the cartilage was less than 10 mm in 2 AMBs and all the PLBs. On the other hand, at 110 degrees , the distance to the nerve was greater than 10 mm in all the AMBs and PLBs, and the distance to the cartilage did not exceed 10 mm in just 2 of the PLBs. CONCLUSIONS In our cadaveric study we found that the low knee flexion angles when drilling femoral tunnels through the far anteromedial portal might have the potential risks of damage to the common peroneal nerve and the posterior articular cartilage, and the risks would be decreased at higher degrees of knee flexion. However, we found there was a 20% risk of damage to the cartilage while drilling the PLB at 110 degrees. CLINICAL RELEVANCE High knee flexion angles are recommended to avoid damage to the nerve and the cartilage when drilling femoral tunnels through the far anteromedial portal in double-bundle anterior cruciate ligament reconstruction.

Medial Patellofemoral Ligament Reconstruction Fixed With a Cylindrical Bone Plug and a Grafted Semitendinosus Tendon at the Original Femoral Site for Recurrent Patellar Dislocation

Background: The medial patellofemoral ligament (MPFL) is the most important factor for stabilizing the patella and preventing lateral patellar dislocation. Medial patellofemoral ligament reconstruction is an accepted surgical technique to restore patellofemoral stability after lateral patellar dislocation. The authors recently developed a new anatomical MPFL reconstruction method using a cylindrical bone plug and grafted semitendinosus tendon at the anatomical femoral attachment site to mimic the native MPFL. This study evaluated the new technique for stabilizing recurrent patellar dislocation. Hypothesis: This new MPFL reconstruction technique will improve knee symptoms and function with excellent clinical results. Study Design: Case series; Level of evidence, 4. Method: Thirty-one knees were evaluated from 29 cases of recurrent patellar dislocation that were surgically treated using the anatomical MPFL reconstruction technique. The average patient age was 22.2 years (range, 12-34 years); postsurgery follow-up was 2 to 5 years (average, 3.2 years). The patients were clinically evaluated based on the Kujala score, range of motion, and signs of apprehension. The Merchant view was used to measure congruence and tilting angles. Results: Of the 31 knees, 30 showed good clinical results after surgery, while 1 patient showed remaining signs of apprehension. The Kujala score improved from an average of 64 points (range, 35-70) initially to an average of 94.5 points (range, 79-100) at the final follow-up. Range of motion improved for all patients, with an average knee extension of 0° ± 2° and knee flexion of 145° ± 3° at final follow-up. No patellar redislocation was reported. Radiological assessment indicated significant improvement to the congruence angle from 13° ± 4° before surgery to −5° ± 5° at the final follow-up, while the tilting angle went from 8° ± 7° before surgery to 7° ± 4° at the final follow-up. Conclusion: This study demonstrated excellent results using the new procedure for recurrent dislocation of the patella, with instability in only 1 of 31 knees (3.2%).

Changes in microRNA expression in peripheral mononuclear cells according to the progression of osteoarthritis

MicroRNAs (miRNAs) are a family of non-coding RNAs that play an important role in human diseases, including osteoarthritis (OA). The objective of this study was to investigate the expression patterns of miRNAs in the peripheral blood mononuclear cells (PBMCs) of OA patients. PBMCs were isolated from 36 patients with OA, 6 RA patients, and 36 healthy controls. The expression patterns of miR-146a, 155, 181a, and 223 in PBMCs were analyzed using quantitative reverse transcription-polymerase chain reaction (qPCR). We investigated the expression patterns of the miRNAs in OA progression, and their relationships with the parameters of age, body mass index (BMI), the femorotibial angle (FTA), and serum keratan sulfate (KS). The relative expression levels of miR-146a, 155, 181a, and 223 in the OA patients were significantly higher than those found in healthy controls. In the early stages of OA, miR-146a and 223 expressions were significantly higher than they were at later stages. There was a significant correlation between the expression of miR-223 and KS. This study demonstrated that high expression levels of miR-146a, 155, 181a, and 223 in the PBMCs of OA patients might be related to the pathogenesis of OA. This evidence could lead to the elucidation of the mechanism underlying OA pathogenesis and hence to a novel therapeutic strategy for OA.

Clinical outcomes of second-look arthroscopic evaluation after anterior cruciate ligament augmentation comparison with single- and double-bundle reconstruction

We report the clinical outcome and findings at second-look arthroscopy of 216 patients (mean age 25 years (11 to 58)) who underwent anterior cruciate ligament (ACL) reconstruction or augmentation. There were 73 single-bundle ACL augmentations (44 female, 29 male), 82 double-bundle ACL reconstructions (35 female, 47 male), and 61 single-bundle ACL reconstructions (34 female, 27 male). In 94 of the 216 patients, proprioceptive function of the knee was evaluated before and 12 months after surgery using the threshold to detect passive motion test. Second-look arthroscopy showed significantly better synovial coverage of the graft in the augmentation group (good: 60 (82%), fair: 10 (14%), poor: 3 (4%)) than in the other groups (p = 0.039). The mean side-to-side difference measured with a KT-2000 arthrometer was 0.4 mm (-3.3 to 2.9) in the augmentation group, 0.9 mm (-3.2 to 3.5) in the double-bundle group, and 1.3 mm (-2.7 to 3.9) in the single-bundle group: the result differed significantly between the augmentation and single-bundle groups (p = 0 .013). No significant difference in the Lysholm score or pivot-shift test was seen between the three groups (p = 0.09 and 0.65, respectively). In patients with good synovial coverage, three of the four measurements used revealed significant improvement in proprioceptive function (p = 0.177, 0.020, 0.034, and 0.026). We conclude that ACL augmentation is a reasonable treatment option for patients with favourable ACL remnants.

Articular Cartilage Repair With Magnetic Mesenchymal Stem Cells

Background: Cell therapies are hampered by the difficulty of delivering cells to and retaining them in target tissues long enough to repair or regenerate local tissues. Hypothesis: Magnetic-assisted delivery of magnetically labeled mesenchymal stem cells (m-MSCs) would be rapid, allowing for chondrogenic differentiation and functional joint repair without replacement. Study Design: Controlled laboratory study. Methods: Sixteen mini-pigs aged 6 to 7 months were used. A full-thickness cartilage defect was created in the center of the patella with a cylindrical punch (diameter, 6 mm). At 4 weeks after creation of the cartilage defects, the animals were divided into 3 treatment groups: In the M group, m-MSCs (5 × 106 cells) were injected and accumulated to the cartilage defect using an external magnetic force (1.5 T) for 10 minutes; in the G group, the patella was faced upward, filled with MSCs (5 × 106 cells), and held for 10 minutes; and in the C group, only phosphate-buffered saline was injected. The regenerated cartilage was evaluated in 5 knees in each of the 3 groups by arthroscopic surgery at 6 and 12 weeks and histological and ultrasound evaluation at 12 and 24 weeks. Results: The mean arthroscopic scores at 6 weeks were 10.4 ± 1.10 in the M group, 8.8 ± 0.84 in the G group, and 7.4 ± 0.89 in the C group. There was a statistically significant difference between the M group and the other 2 groups. The mean arthroscopic scores at 12 weeks were 12.8 ± 1.30 (M group), 10.5 ± 1.30 (G group), and 9.5 ± 0.58 (C group), with a statistically significant difference between the M and C groups. The mean histological scores using the Wakitani scoring system at 12 weeks were 2.8 ± 0.96 (M group), 5.4 ± 0.55 (G group), and 6.0 ± 2.20 (C group), and the mean histological scores at 24 weeks were 2.4 ± 1.50 (M group), 3.5 ± 0.56 (G group), and 5.3 ± 1.50 (C group). The mean histological scores at 12 weeks were significantly better in the M group than in the other groups, and the M group maintained a significantly better histological score than did the C group at 24 weeks. Conclusion: The m-MSCs had no adverse effect on chondrogenic differentiation, and m-MSCs delivered by magnetic field application repaired cartilage defects. Clinical Relevance: The clinical application of this novel stem cell delivery system is a potential therapeutic option for treating cartilage defects and may be more applicable throughout the body than traditional methods.

Effects of unloading bracing on knee and hip joints for patients with medial compartment knee osteoarthritis

BACKGROUND Osteoarthritis affects the whole body, thus biomechanical effects on other joints should be considered. Unloading knee braces could be effective for knee osteoarthritis, but their effects on the contralateral knee and bilateral hip joints remain unknown. This study investigated the effects of bracing on the kinematics and kinetics of involved and contralateral joints during gait. METHODS Nineteen patients with medial compartment knee osteoarthritis were analysed. Kinematics and kinetics of the knee and hip joints in frontal and sagittal planes were measured during walking without and with bracing on the more symptomatic knee. FINDINGS The ipsilateral hip in the braced condition showed a lower adduction angle by an average of 2.58° (range, 1.05°-4.16°) during 1%-49% of the stance phase, and a lower abduction moment at the second peak during the stance phase than the hip in the unbraced condition (P<0.05 and P<0.005, respectively). With bracing, the contralateral hip showed a more marked peak extension moment and lower abduction moment at the first peak (P<0.05), and the contralateral knee adduction angle increased by an average of 0.32° (range, 0.21°-0.45°) during 46%-55% of the stance phase (P<0.05), compared to no bracing. INTERPRETATION Unloading bracing modified the contralateral knee adduction angle pattern at a specific time point during gait. It also affected the frontal plane on the ipsilateral hip and the frontal and sagittal planes on the contralateral hip joint. Consideration should be provided to other joints when treating knee osteoarthritis.