Inner Meniscus Cells Maintain Higher Chondrogenic Phenotype Compared with Outer Meniscus Cells

Journal Orthopaedic Research

Hachioji

et al. 2011

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ABSTRACT:The meniscus plays an important role in controlling the biomechanics of the knee. However, the mechanical stress-related response in meniscus cells remains unclear. We investigated mechanical stretch-regulated gene expression in human meniscus cells. Human inner and outer meniscus cells were prepared from the inner and outer halves of the lateral meniscus. The gene expressions of Sry-type HMG box (SOX) 9 and a1(II) collagen (COL2A1) were assessed by real-time PCR analyses after cyclic tensile strain (CTS) treatment (0.5 Hz, 5% stretch). The localization and phosphorylation of SOX9 were evaluated by immunohistochemical and Western blot (WB) analyses. Chromatin immunoprecipitation (IP) analysis was performed to assess the stretch-related protein-DNA complex formation between SOX9 and the COL2A1 enhancer on chromatin. Type II collagen deposition and SOX9 production were detected only in inner menisci. CTS treatments increased expression of the COL2A1 and SOX9 genes in inner meniscus cells, but not in outer meniscus cells. In addition, CTS treatments stimulated nuclear translocalization and phosphorylation of SOX9 in inner meniscus cells. Chromatin IP analyses revealed that CTS increased the association between SOX9 and its DNA-binding site, included in the COL2A1 enhancer, on chromatin. Our results indicate that inner and outer meniscus cells have different properties in mechanical stretchinduced COL2A1 expression. In inner meniscus cells, mechanical stretch may have an essential role in the epigenetic regulation of COL2A1 expression. ß

Section: Discussionmentioning

confidence: 99%

Section: Cells and Cell Culturementioning

confidence: 99%

Section: Immunohistochemistry Western Blot (Wb) and Immunoprecipitamentioning

confidence: 99%

mentioning

confidence: 99%

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Mechanical stretch enhances COL2A1 expression on chromatin by inducing SOX9 nuclear translocalization in inner meniscus cells

Kanazawa

Journal Orthopaedic Research

Hachioji

et al. 2011

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“…LFC, lateral femoral condyle media for three weeks according to the manufacturer's protocol (Millipore, Billerica, MA). Lipid vacuoles were stained with oil red O solution [20]. Osteogenic induction was performed using MSC osteogenesis kit (Millipore) and 1 ng/ml of BMP-2 for three weeks.…”

Section: In Vitro Differentiation and Histological Analysesmentioning

confidence: 99%

Comparison between normal and loose fragment chondrocytes in proliferation and redifferentiation potential

Sakata

International Orthopaedics (SICOT)

Miyazawa

et al. 2012

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Purpose Loose fragments in osteochondritis dissecans (OCD) of the knee require internal fixation. On the other hand, loose fragments derived from spontaneous osteonecrosis of the knee (SONK) are usually removed. However, the difference in healing potential between OCD-and SONK-related loose fragments has not been elucidated. In this study, we investigated proliferative activity and redifferentiation potential of normal cartilage-derived and loose fragment-derived chondrocytes. Methods Cells were prepared from normal articular cartilages and loose fragment cartilages derived from knee OCD and SONK. Cellular proliferation was compared. Redifferentiation ability of pellet-cultured chondrocytes was assessed by real-time PCR analyses. Mesenchymal differentiation potential was investigated by histological analyses. Positive ratio of a stem cell marker CD166 was evaluated in each cartilaginous tissue. Results Normal and OCD chondrocytes showed a higher proliferative activity than SONK chondrocytes. Chondrogenic pellets derived from normal and OCD chondrocytes produced a larger amount of safranin O-stained proteoglycans compared with SONK-derived pellets. Expression of chondrogenic marker genes was inferior in SONK pellets. The CD166-positive ratio was higher in normal cartilages and OCD loose fragments than in SONK loose fragments. Conclusions The OCD chondrocytes maintained higher proliferative activity and redifferentiation potential compared with SONK chondrocytes. Our results suggest that chondrogenic properties of loose fragment-derived cells and the amount of CD166-positive cells may affect the repair process of osteochondral defects.

Mechanical stretch increases Smad3‐dependent CCN2 expression in inner meniscus cells

Journal Orthopaedic Research

Kanazawa

Miyake

et al. 2012

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The intrinsic zone-specific properties of the menisci are determined by biomechanical environments. In this study, we examined mechanical stretch-dependent expression of multifunctional growth factor CYR61/CTGF/NOV (CCN) 2, and investigated the role of CCN2 in meniscus cells. Uni-axial cyclic tensile strain (CTS) was applied using a STB-140 system. CTS-induced expression of CCN2 and a1(I) collagen (COL1A1) was assessed by quantitative real-time PCR analysis. The distribution of CCN2 and Smad2/3 in stretched cells was investigated by immunohistochemical analysis. Smad2/3-dependent CCN2 transactivation was measured by luciferase reporter assay. The relationship between Smad2/3 and CTS-induced CCN2 transcription was investigated by chromatin immunoprecipitation. CTS stimulated gene expression of CCN2 and COL1A1 in inner meniscus cells, but not in outer meniscus cells. Recombinant CCN2 increased COL1A1 expression only in inner meniscus cells. CCN2 synthesis and nuclear translocalization of phosphorylated Smad2/3 in inner meniscus cells were stimulated by CTS. The CCN2 promoter activity was synergistically enhanced by overexpressed Smad3 in stretched inner meniscus cells, but was not by Smad2. Chromatin immunoprecipitation revealed that CTS increased the association between Smad3 and the Smad-binding element on the CCN2 proximal promoter in inner meniscus cells. Our results suggest that stretch-induced CCN2 may have a crucial role in regulating COL1A1 expression in the inner meniscus.