Toshifumi Ozaki

Mechanical Stretch Enhances COL2A1 Expression on Chromatin by Inducing SOX9 Nuclear Translocalization in Inner Meniscus Cells

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 α1(II) collagen (COL2A1) were assessed by real‐time PCR analyses after cyclic tensile strain (CTS) treatment (0.5u2009Hz, 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 stretch‐induced COL2A1 expression. In inner meniscus cells, mechanical stretch may have an essential role in the epigenetic regulation of COL2A1 expression.

An anatomical study of the watershed line on the volar, distal aspect of the radius: implications for plate placement and avoidance of tendon ruptures.

PURPOSEnThe watershed line is a useful surgical landmark for positioning a volar locking plate. Implants placed on or distal to it can impinge on flexor tendons and cause injury. However, the details of the anatomy of this line are unclear. We studied macroscopically and histologically the structures of the volar aspect of the distal radius.nnnMETHODSnWe studied 20 distal forearm regions of 10 cadavers (5 males and 5 females; mean age, 79 y [range, 56-88 y]) to clarify the details of the watershed line. In 16 specimens, we investigated the macroscopic appearance of the volar aspect of the radius and the relationships among the bone, the volarradiocarpal ligaments, and the pronator quadratus. Histological analyses were performed in 4 specimens of 2 cadavers to examine the morphology of the margin of the bony structures.nnnRESULTSnIn the medial half of the distal volar radius, 2 lines were identified by direct macroscopic visualization; one was the proximal line that corresponded to the distal ridge of the pronator fossa, and the other was the distal line, which was more prominent. A medial bony prominence was situated on the distal line. In the lateral half, the distal and proximal lines of the medial half merged to form a single line. A lateral prominence was situated on this line.nnnCONCLUSIONSnThe watershed line might not be a distinct line, and it corresponds to the distal margin of the pronator fossa in the lateral half of the volar radius and to a hypothetical line between the distal and proximal lines in the medial half. The medial and lateral bony prominences on the volar radius should be key structures for accurate plate placement to avoid flexor tendon injury.nnnCLINICAL RELEVANCEnThe present study suggests bony landmarks for positioning a volar locking plate.

Transplantation of osteogenically differentiated mouse iPS cells for bone repair

Induced pluripotent stem (iPS) cells are a type of undifferentiated cell that can be obtained from differentiated cells and have the pluripotent potential to differentiate into the musculoskeletal system, the myocardium, vascular endothelial cells, neurons, and hepatocytes. We therefore cultured mouse iPS cells in a DMEM containing 15% FBS, 10−7 M dexamethasone, 10 mM β-glycerophosphate, and 50 μg/ml ascorbic acid for 3 weeks, in order to induce bone differentiation, and studied the expression of the bone differentiation markers Runx2 and osteocalcin using RT-PCR in a time-dependent manner. Osteocalcin, a bone differentiation marker in bone formation, exhibited the highest expression in the third week. In addition, the deposition of calcium nodules was observed using Alizarin red S staining. iPS cells cultured for bone differentiation were transplanted into severe combined immunodeficiency (SCID) mice, and the osteogenic potential exhibited after 4 weeks was studied. When bone differentiation-induced iPS cells were transplanted into SCID mice, bone formation was confirmed in soft X-ray images and tissue specimens. However, teratoma formation was confirmed in 20% of the transplanted models. When mouse iPS cells were treated with irradiation of 2 Gray (Gy) prior to transplantation, teratoma formation was inhibited. When mouse iPS cells treated in a likewise manner were xenotransplanted into rats, bone formation was confirmed but teratoma formation was not observed. It is believed that irradiation before transplantation is an effective way to inhibit teratoma formation.

ROCK inhibitor prevents the dedifferentiation of human articular chondrocytes.

Chondrocytes lose their chondrocytic phenotypes in vitro. The Rho family GTPase ROCK, involved in organizing the actin cytoskeleton, modulates the differentiation status of chondrocytic cells. However, the optimum method to prepare a large number of un-dedifferentiated chondrocytes is still unclear. In this study, we investigated the effect of ROCK inhibitor (ROCKi) on the chondrogenic property of monolayer-cultured articular chondrocytes. Human articular chondrocytes were subcultured in the presence or absence of ROCKi (Y-27632). The expression of chondrocytic marker genes such as SOX9 and COL2A1 was assessed by quantitative real-time PCR analysis. Cellular morphology and viability were evaluated. Chondrogenic redifferentiation potential was examined by a pellet culture procedure. The expression level of SOX9 and COL2A1 was higher in ROCKi-treated chondrocytes than in untreated cells. Chondrocyte morphology varied from a spreading form to a round shape in a ROCKi-dependent manner. In addition, ROCKi treatment stimulated the proliferation of chondrocytes. The deposition of safranin O-stained proteoglycans and type II collagen was highly detected in chondrogenic pellets derived from ROCKi-pretreated chondrocytes. Our results suggest that ROCKi prevents the dedifferentiation of monolayer-cultured chondrocytes, and may be a useful reagent to maintain chondrocytic phenotypes in vitro for chondrocyte-based regeneration therapy.

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

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 α1(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.