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1.  Intra-articular injection of synthetic microRNA-210 accelerates avascular meniscal healing in rat medial meniscal injured model 
Introduction
The important functions of the meniscus are shock absorption, passive stabilization and load transmission of the knee. Because of the avascularity of two-thirds of the meniscal center region, the treatment of tears in this area is hard. Recently, microRNAs have been proven to play an important role in the pathogenesis of diseases. We focused on microRNA (miR)-210, which plays a wide spectrum of roles comprising mitochondrial metabolism, angiogenesis, DNA repair and cell survival. This study aimed to investigate the effect of intra-articular injection of synthetic miR-210 on the injured meniscus in the avascular zone.
Methods
The middle segments of the medial meniscus of Spraque Dawley rats were incised longitudinally with a scalpel. An intra-articular injection of double-stranded (ds) miR-210 (for control group using control dsRNA) with atelocollagen was administered immediately after injury. Four weeks and 12 weeks after the injection, we conducted a histologic evaluation, immunohistochemical evaluation and Real-time PCR analysis. In vitro, the inner meniscus and synovial cells were isolated from rat knee joint, and were transfected with ds miR-210 or control dsRNA. Real-time PCR and immunohistochemical evaluations were performed.
Results
Twenty-four hours after the injection, FAM (Fluorescein amidite) labeled miR-210 was observed in the cells around the injured site. Four weeks after the injection, the injured site of the miR-210 group was filled with repaired tissue while that of the control was not repaired. In gene expression analysis of the meniscus, the expression of miR-210, Collagen type 2 alpha 1 (Col2a1), Vascular endothelial growth factor (VEGF), and Fibroblast growth factor-2 (FGF2) in the miR-210 group was significantly higher than that in the control. At 12 weeks, the intra-articular injection of miR-210 had healed the injured site of the meniscus and had prevented articular cartilage degeneration. In vitro, miR-210 upregulated Col2a1 expression in the meniscus cells and VEGF and FGF2 expression in the synovial cells.
Conclusions
An intra-articular injection of ds miR-210 was effective in the healing of the damaged white zone meniscus through promotion of the collagen type 2 production from meniscus cells and through upregulated of VEGF and FGF2 from synovial cells.
doi:10.1186/s13075-014-0488-y
PMCID: PMC4265493  PMID: 25430980
2.  Exosomes from IL-1β stimulated synovial fibroblasts induce osteoarthritic changes in articular chondrocytes 
Arthritis Research & Therapy  2014;16(4):R163.
Introduction
Osteoarthritis (OA) is a whole joint disease, and characterized by progressive degradation of articular cartilage, synovial hyperplasia, bone remodeling and angiogenesis in various joint tissues. Exosomes are a type of microvesicles (MVs) that may play a role in tissue-tissue and cell-cell communication in homeostasis and diseases. We hypothesized that exosomes function in a novel regulatory network that contributes to OA pathogenesis and examined the function of exosomes in communication among joint tissue cells.
Methods
Human synovial fibroblasts (SFB) and articular chondrocytes were obtained from normal knee joints. Exosomes isolated from conditioned medium of SFB were analyzed for size, numbers, markers and function. Normal articular chondrocytes were treated with exosomes from SFB, and Interleukin-1β (IL-1β) stimulated SFB. OA-related genes expression was quantified using real-time PCR. To analyze exosome effects on cartilage tissue, we performed glycosaminoglycan release assay. Angiogenic activity of these exosomes was tested in migration and tube formation assays. Cytokines and miRNAs in exosomes were analyzed by Bio-Plex multiplex assay and NanoString analysis.
Results
Exosomes from IL-1β stimulated SFB significantly up-regulated MMP-13 and ADAMTS-5 expression in articular chondrocytes, and down-regulated COL2A1 and ACAN compared with SFB derived exosomes. Migration and tube formation activity were significantly higher in human umbilical vein endothelial cells (HUVECs) treated with the exosomes from IL-1β stimulated SFB, which also induced significantly more proteoglycan release from cartilage explants. Inflammatory cytokines, IL-6, MMP-3 and VEGF in exosomes were only detectable at low level. IL-1β, TNFα MMP-9 and MMP-13 were not detectable in exosomes. NanoString analysis showed that levels of 50 miRNAs were differentially expressed in exosomes from IL-1β stimulated SFB compared to non-stimulated SFB.
Conclusions
Exosomes from IL-1β stimulated SFB induce OA-like changes both in vitro and in ex vivo models. Exosomes represent a novel mechanism by which pathogenic signals are communicated among different cell types in OA-affected joints.
doi:10.1186/ar4679
PMCID: PMC4261911  PMID: 25092378
3.  Regenerative medicine in orthopedics using cells, scaffold, and microRNA 
Journal of Orthopaedic Science  2014;19(4):521-528.
Cells, scaffold, and growth factors are crucially important in regenerative medicine and tissue engineering. Progress in science and technology has enabled development of these three factors, with basic research being applied clinically. In the past decade, we have investigated tissue regeneration in animal models of musculoskeletal disorders by using cells, scaffold, and delivery systems which has been relatively easy to apply and develop in clinical settings. Moreover, microRNA (miRNA), which are important in biological processes and in the pathogenesis of human diseases, have been used in research on regenerative medicine. For the cell source, we focused on mesenchymal stem cells (MSC) and CD34+ and CD133+ cells as endothelial progenitor cells for regeneration of musculoskeletal organs. These cells are accessible and safe. For less invasive and more effective therapy, we developed a novel cell-delivery system using magnetic force to accumulate cells at a desired site. Furthermore, administration of synthetic miRNA could enhance tissue regeneration. In our studies, use of these cells combined with a cell-delivery system, miRNA, scaffold, and cytokines has led to effective regeneration of musculoskeletal tissues including cartilage, bone, ligaments, muscle, peripheral nerves, and spinal cord. The current and future objective is more effective and less invasive cell-based therapy with spatial control of transplanted cells by use of an external magnetic force. Analysis of efficiency, safety, and the mechanism of tissue regeneration by cells, scaffold, and miRNA will lead to more promising regenerative medicine, involving the development of a new generation of therapy. This review will focus on our regenerative medicine research, which focuses on clinical application of cells, scaffold, and miRNA.
doi:10.1007/s00776-014-0575-6
PMCID: PMC4111855  PMID: 24819307
4.  High flexion knee arthroplasty: the relationship between rotational angles and flexion angle after total knee arthroplasty 
The current patients required high flexion total knee arthroplasty (TKA). We hypothesized the patients who would get the high rotational angle just after TKA could gain high flexion knee 1 year after TKA. Sixty-three patients (average age: 70.6 years) were examined. In order to examine between the intraoperative rotational angles and the gained flexion angles after TKA, the patients were divided into two groups: the H group (more than 120°) and the L groups (less than 120°) by the gained flexion angles. The relationship between the flexion angles at 1 year after surgery and the intraoperative rotational angle had no significant correlation. But the rotational angles in the L group tended to be higher than the ones in the H group, and at only 30°, the L group gained significantly more rotational angle than the H group. These results might show that a tighter rotational stability induces a gain of higher flexion knee after TKA.
doi:10.1007/s12178-014-9215-1
PMCID: PMC4012152  PMID: 24658882
Total knee arthroplasty; Rotational angle; Flexion angle; PS type
5.  High flexion knee arthroplasty: the relationship between rotational angles and flexion angle after total knee arthroplasty 
The current patients required high flexion total knee arthroplasty (TKA). We hypothesized the patients who would get the high rotational angle just after TKA could gain high flexion knee 1 year after TKA. Sixty-three patients (average age: 70.6 years) were examined. In order to examine between the intraoperative rotational angles and the gained flexion angles after TKA, the patients were divided into two groups: the H group (more than 120°) and the L groups (less than 120°) by the gained flexion angles. The relationship between the flexion angles at 1 year after surgery and the intraoperative rotational angle had no significant correlation. But the rotational angles in the L group tended to be higher than the ones in the H group, and at only 30°, the L group gained significantly more rotational angle than the H group. These results might show that a tighter rotational stability induces a gain of higher flexion knee after TKA.
doi:10.1007/s12178-014-9215-1
PMCID: PMC4012152  PMID: 24658882
Total knee arthroplasty; Rotational angle; Flexion angle; PS type
6.  Treatment of cartilage defects by subchondral drilling combined with covering with atelocollagen membrane induces osteogenesis in a rat model 
Journal of Orthopaedic Science  2013;18(4):627-635.
Background
The coverage of the atelocollagen membrane at the chondral defect after subchondral drilling might improve the beneficial effects for cartilage repair because of the prevention of scattering and accumulation of cells and growth factors from bone marrow within the chondral defect. On the other hand, it might block cells and factors derived from the synovium or cause high pressure in the chondral defect, resulting in prevention of cells and growth factors gushing out from the bone marrow, which leads to disadvantages for cartilage repair.
Method
We tested this hypothesis in a 2-mm-diameter chondral defect created in the articular cartilage of the patellar groove in a rat models. Defects were left untreated, or were drilled or drilled and covered with an atelocollagen membrane; healing was evaluated by histology and gene expression analysis using real-time polymerase chain reaction and immunohistochemistry.
Results
Membrane coverage induced bone tissue ingrowth into the punched chondral defect. At 1 week, expression of TGFβ, Sox9, Runx2, osteocalcin, Col1a1, and Col2a1 in the drilling group was significantly higher than in the covering group. At 4 weeks, expressions of TGFβ, Runx2, and Col1a1 were all significantly higher in the drilling group, while Sox9, osteocalcin, and Col2a1 were significantly higher in the covering group. Immunohistochemistry demonstrated Sox9, osteocalcin, and type II collagen on the bony reparative tissue in the covering group.
Conclusions
These results suggest that the atelocollagen membrane coverage resulted in inhibition of cartilage repair.
doi:10.1007/s00776-013-0379-0
PMCID: PMC3726928  PMID: 23564076
7.  MicroRNA-146a expresses in interleukin-17 producing T cells in rheumatoid arthritis patients 
Background
Interleukin (IL)-17 is an important factor in rheumatoid arthritis (RA) pathogenesis. MicroRNA (miRNA)s are a family of non coding RNAs and associated with human diseases including RA. The purpose of this study is to identify the miRNAs in the differentiation of IL-17 producing cells, and analyze their expression pattern in the peripheral blood mononuclear cells (PBMC) and synovium from RA patients.
Methods
IL-17 producing cells were expanded from CD4+T cell. MiRNA microarray was performed to identify the miRNAs in the differentiation of IL-17 producing cells. Quantitative polymerase chain reaction was performed to examine the expression patterns of the identified miRNAs in the PBMC and synovium from RA and osteoarthritis (OA) patients. Double staining combining in situ hybridization and immunohistochemistry of IL-17 was performed to analyze the expression pattern of identified miRNA in the synovium.
Results
Six miRNAs, let-7a, miR-26, miR-146a/b, miR-150, and miR-155 were significantly up regulated in the IL-17 producing T cells. The expression of miR-146a and IL-17 was higher than in PBMC in the patients with low score of Larsen grade and short disease duration. MiR-146a intensely expressed in RA synovium in comparison to OA. MiR-146a expressed intensely in the synovium with hyperplasia and high expression of IL-17 from the patients with high disease activity. Double staining revealed that miR-146a expressed in IL-17 expressing cells.
Conclusion
These results indicated that miR-146a was associated with IL-17 expression in the PBMC and synovium in RA patients. There is the possibility that miR-146a participates in the IL-17 expression.
doi:10.1186/1471-2474-11-209
PMCID: PMC2950393  PMID: 20840794
8.  MicroRNA-140 is expressed in differentiated human articular chondrocytes and modulates IL-1 responses 
Arthritis and rheumatism  2009;60(9):2723-2730.
OBJECTIVE
MicroRNAs (miRNAs) are a class of noncoding small RNAs that act as negative regulators of gene expression. The miRNAs exhibit tissue-specific expression patterns and changes in their expression may contribute to pathogenesis. The objectives of this study were to identify miRNAs expressed in articular chondrocytes, determine changes in osteoarthritic cartilage and address the function of miR-140.
METHODS
To identify miRNAs specifically expressed in chondrocytes, we performed gene expression profiling using miRNA microarrays and quantitative PCR with human articular chondrocytes compared to human mesenchymal stem cells (MSC). The expression pattern of miR-140 was monitored during chondrogenic differentiation of hMSC in pellet cultures and in human articular cartilage from normal and osteoarthritic knee joints. We tested effects of IL-1β on miR-140 expression. Double-strand (ds) miR-140 was transfected into chondrocytes to analyze changes in the expression of genes associated with osteoarthritis.
RESULTS
Microarray analysis showed that miR-140 has the largest difference in expression between chondrocytes and MSC. During chondrogenesis cultures of MSC miR-140 expression increased in parallel with Sox9 and Col2a1. Normal human articular cartilage expressed miR-140 and this was significantly reduced in OA tissue. In vitro treatment of chondrocytes with IL-1β suppressed miR-140 expression. Transfection of chondrocytes with ds-miR-140 downregulated IL-1β-induced ADAMTS-5 expression and rescued the IL-1β –dependent repression of Aggrecan gene expression.
CONCLUSION
This study shows that miR-140 has a chondrocyte differentiation-related expression pattern. The reduction in miR-140 expression in OA cartilage and in response to IL-1β may contribute to the abnormal gene expression pattern characteristic of OA.
doi:10.1002/art.24745
PMCID: PMC2806094  PMID: 19714579
microRNA; chondrocytes; mesenchymal stem cells; cartilage; osteoarthritis
9.  Expression of microRNA-146 in osteoarthritis cartilage 
Arthritis and rheumatism  2009;60(4):1035-1041.
Objective
A role of microRNAs, which are ∼22- nucleotide non coding RNAs, has recently been recognized in human diseases. The objective of this study was to identify the expression pattern of microRNA-146 (miR-146) in cartilage from patients with osteoarthritis (OA).
Methods
The expression of miR-146 in cartilage from 15 patients with OA was analyzed by quantitative reverse transcription-polymerase chain reaction (RT-PCR) and by in situ hybridization. Induction of the expression of miR-146 by cultures of normal human articular chondrocytes following stimulation with interleukin-1β (IL-1β) was examined by quantitative RT-PCR.
Results
All cartilage samples were divided into three groups according to a modified Mankin scale; grade I: 0 - 5, grade II: 6 - 10, grade III: 11 - 14. In OA cartilage samples of grade I, the expression of miR-146a and Col2a1 was significantly higher than that of other groups (p<0.05). In OA cartilage of grades II and III, the expression of miR-146a and Col2a1 decreased while the expression of MMP13 was elevated in grade II. These data show that miR-146a is expressed intensely in cartilage with a low Mankin grade, and that miR-146a expression decreases in accordance with level of MMP13 expression. Section in situ hybridization of pri-miR-146a revealed that pri-miR-146a is expressed in chondrocytes in all layers, especially in the superficial layer where it is intensely expressed. The expression of miR-146 was markedly elevated by IL-1β stimulation in human chondrocytes in vitro.
Conclusion
This study shows that miR-146 is intensely expressed in low grade OA cartilage, and that its expression is induced by stimulation of IL-1β. MiR-146 might play a role in OA cartilage pathogenesis.
doi:10.1002/art.24404
PMCID: PMC2670476  PMID: 19333945
10.  The effect of an external magnetic force on cell adhesion and proliferation of magnetically labeled mesenchymal stem cells 
Background
As the strategy for tissue regeneration using mesenchymal stem cells (MSCs) for transplantation, it is necessary that MSCs be accumulated and kept in the target area. To accumulate MSCs effectively, we developed a novel technique for a magnetic targeting system with magnetically labeled MSCs and an external magnetic force. In this study, we examined the effect of an external magnetic force on magnetically labeled MSCs in terms of cell adhesion and proliferation.
Methods
Magnetically labeled MSCs were plated at the bottom of an insert under the influence of an external magnetic force for 1 hour. Then the inserts were turned upside down for between 1 and 24 hours, and the number of MSCs which had fallen from the membrane was counted. The gene expression of MSCs affected magnetic force was analyzed with microarray. In the control group, the same procedure was done without the external magnetic force.
Results
At 1 hour after the inserts were turned upside down, the average number of fallen MSCs in the magnetic group was significantly smaller than that in the control group, indicating enhanced cell adhesion. At 24 hours, the average number of fallen MSCs in the magnetic group was also significantly smaller than that in control group. In the magnetic group, integrin alpha2, alpha6, beta3 BP, intercellular adhesion molecule-2 (ICAM-2), platelet/endothelial cell adhesion molecule-1 (PECAM-1) were upregulated. At 1, 2 and 3 weeks after incubation, there was no statistical significant difference in the numbers of MSCs in the magnetic group and control group.
Conclusions
The results indicate that an external magnetic force for 1 hour enhances cell adhesion of MSCs. Moreover, there is no difference in cell proliferation after using an external magnetic force on magnetically labeled MSCs.
doi:10.1186/1758-2555-2-5
PMCID: PMC2828444  PMID: 20152029
11.  Expression of MicroRNA-146 in Rheumatoid Arthritis Synovial Tissue 
Arthritis and rheumatism  2008;58(5):1284-1292.
Objective
Several microRNA, which are ~22-nucleotide noncoding RNAs, exhibit tissue-specific or developmental stage–specific expression patterns and are associated with human diseases. The objective of this study was to identify the expression pattern of microRNA-146 (miR-146) in synovial tissue from patients with rheumatoid arthritis (RA).
Methods
The expression of miR-146 in synovial tissue from 5 patients with RA, 5 patients with osteoarthritis (OA), and 1 normal subject was analyzed by quantitative reverse transcription–polymerase chain reaction (RT-PCR) and by in situ hybridization and immunohistochemistry of tissue sections. Induction of miR-146 following stimulation with tumor necrosis factor α (TNFα) and interleukin-1β (IL-1β) of cultures of human rheumatoid arthritis synovial fibroblasts (RASFs) was examined by quantitative PCR and RT-PCR.
Results
Mature miR-146a and primary miR-146a/b were highly expressed in RA synovial tissue, which also expressed TNFα, but the 2 microRNA were less highly expressed in OA and normal synovial tissue. In situ hybridization showed primary miR-146a expression in cells of the superficial and sublining layers in synovial tissue from RA patients. Cells positive for miR-146a were primarily CD68+ macrophages, but included several CD3+ T cell subsets and CD79a+ B cells. Expression of miR-146a/b was markedly up-regulated in RASFs after stimulation with TNFα and IL-1β.
Conclusion
This study shows that miR-146 is expressed in RA synovial tissue and that its expression is induced by stimulation with TNFα and IL-1β. Further studies are required to elucidate the function of miR-146 in these tissues.
doi:10.1002/art.23429
PMCID: PMC2749927  PMID: 18438844

Results 1-11 (11)