Ko Yasura

PGE2 Signal Through EP2 Promotes the Growth of Articular Chondrocytes

EP2 was identified as the major PGE2 receptor expressed in articular cartilage. An EP2 agonist increased intracellular cAMP in articular chondrocytes, stimulating DNA synthesis in both monolayer and 3D cultures. Hence, the EP2 agonist may be a potent therapeutic agent for degenerative cartilage diseases.

Methylation in the core-promoter region of the chondromodulin-I gene determines the cell-specific expression by regulating the binding of transcriptional activator Sp3.

Transcriptional regulation of cell- and stage-specific genes is a crucial process in the development of mesenchymal tissues. Here we have investigated the regulatory mechanism of the expression of the chondromodulin-I (ChM-I) gene, one of the chondrocyte-specific genes, in osteogenic cells using osteosarcoma (OS) cells as a model. Methylation-specific sequence analyses revealed that the extent of methylation in the core-promoter region of the ChM-I gene was correlated inversely with the expression of the ChM-I gene in OS primary tumors and cell lines. 5-Aza-deoxycytidine treatment induced the expression of the ChM-I gene in ChM-I-negative OS cell lines, and the induction of expression was associated tightly with the demethylation of cytosine at -52 (C(-52)) in the middle of an Sp1/3 binding site to which the Sp3, but not Sp1, bound. The replacement of C(-52) with methyl-cytosine or thymine abrogated Sp3 binding and also the transcription activity of the genomic fragment including C(-52). The inhibition of Sp3 expression by small interfering RNA reduced the expression of the ChM-I gene in ChM-I-positive normal chondrocytes, indicating Sp3 as a physiological transcriptional activator of the ChM-I gene. These results suggest that the methylation status of the core-promoter region is one of the mechanisms to determine the cell-specific expression of the ChM-I gene through the regulation of the binding of Sp3.

Mutation analysis of the RECQL4 gene in sporadic osteosarcomas

Osteosarcoma (OS) is the most prevalent malignant tumor among cases of Rothmund‐Thomson syndrome (RTS) with germline mutations of the RECQL4 gene, a member of the RecQ helicase family. We investigated the involvement of the RECQL4 gene in the development of OS unrelated to RTS. RECQL4 mRNA was detected in 9 of 9 OS cell lines by Northern blotting and 26 of 26 OS tumors by RT‐PCR. Direct sequencing of the entire coding region along with flanking splice junctions and 13 small (<100 bp) introns in 71 OS tumors revealed 2 sites with a single‐base change causing an amino acid change (G1814A for R355Q and C2474T for P441S) and one site with a 6 bp inframe deletion (4837‐42delTGCACC for CT857‐8del). Identical genotypes were found in corresponding normal tissues in all cases, and the frequency of each allele was not significantly different between OS and control populations. Our data indicate that the RECQL4 gene is not a frequent target for somatic mutations in sporadic OS unrelated to RTS.

Ultrasound properties of articular cartilage in the tibio-femoral joint in knee osteoarthritis: relation to clinical assessment (International Cartilage Repair Society grade)

IntroductionThere is a lack of data relating the macroscopic appearance of cartilage to its ultrasound properties. The purpose of the present study was to evaluate degenerated cartilage and healthy-looking cartilage using an ultrasound system.MethodsUltrasound properties – signal intensity (a measure of superficial cartilage integrity), echo duration (a parameter related to the surface irregularity) and the interval between signals (that is, time of flight – which is related to the thickness and ultrasound speed of cartilage) – of 20 knees were measured at seven sites: the lateral femoral condyle (site A, anterior; site B, posterior), the medial condyle (site C), the lateral tibial plateau (site D, center; site E, under the meniscus) and the medial tibial plateau (site F, anterior; site G, posterior). The sites were evaluated macroscopically and classed using the International Cartilage Repair Society (ICRS) grading system.ResultsThe signal intensity of grade 0 cartilage was significantly greater than the intensities of grade 1, grade 2 or grade 3 cartilage. Signal intensity decreased with increasing ICRS grades. The signal intensity was greater at site B than at site C, site D, site F and site G. The signal intensity of grade 0 was greater at site B than at site E. The echo duration did not differ between the grades and between the sites. The interval between signals of grade 3 was less than the intervals of grade 0, grade 1 or grade 2. The interval between signals at site C was less than the intervals at site A, site B, site D, and site E.ConclusionSite-specific differences in signal intensity suggest that a superficial collagen network may be maintained in cartilage of the lateral condyle but may deteriorate in cartilage of the medial condyle and the medial tibial plateau in varus knee osteoarthritis. Signal intensity may be helpful to differentiate ICRS grades, especially grade 0 cartilage from grade 1 cartilage.

Mechanical and Biochemical Effect of Monopolar Radiofrequency Energy on Human Articular Cartilage An In Vitro Study

Background There are growing concerns about thermal chondroplasty using radiofrequency energy to treat partial-thickness cartilage defects. However, most studies emphasize effects on chondrocyte viability, and other factors such as mechanical properties are less studied. Hypothesis Radiofrequency energy may cause significant effects on articular cartilage other than chondrocyte viability. Study Design Controlled laboratory study. Methods Human osteoarthritic cartilage samples were obtained from total knee arthroplasty, and monopolar radiofrequency energy was applied using commercially available equipment. Material properties (compressive stiffness, surface roughness, and thickness) just before and after thermal treatment were determined using ultrasound. A series of biochemical analyses were also performed after explant culture of the samples. Results The cartilage surface became smoother by radiofrequency energy, whereas cartilage stiffness or thickness was not altered significantly. Collagen fibrils, especially in the superficial layers, were converted to denatured form, whereas proteoglycan contents released in the media as well as retained in the tissue remained unchanged. The concentrations of matrix metalloproteinases (MMP-1 and MMP-2) were reduced remarkably. Conclusion Radiofrequency energy is able to create a smooth cartilage surface and reduce catabolic enzymes at the cost of collagen denaturation and chondrocyte death in the superficial layers. The stiffness of the cartilage is not changed at time zero. Clinical Relevance Further animal as well as clinical studies will be necessary to fully evaluate the long-term effects of radiofrequency energy.

Ultrasound has the potential to detect degeneration of articular cartilage clinically, even if the information is obtained from an indirect measurement of intrinsic physical characteristics

This letter is the response to the concern shown by Zheng [1] regarding our article [2]. Because our ultrasound system obtains indirect information on intrinsic physical characteristics of living human articular cartilage in vivo settings, we consider that the signal intensity obtains information including the intrinsic physical characteristics. We do not disregard to measure the intrinsic physical characteristics. Indeed, the signal intensity correlated with the aggregate modulus of articular cartilage significantly [3]. We mentioned the equations of Young modulus, indicating speed of sound, density of a material, and acoustic impedance of a material [4] and presented Gabor function as the mother wavelet and equations [2]. The signal intensity did not depend on the surface curvature, in radiuses of more than 40-mm, and mainly reflects the condition from surface of cartilage to one wavelength depth [5]. The tip of the probe with an ultrasonic transducer is designed to achieve uniform distance between the transducer and cartilage surface. Our ultimate goal is not to measure the intrinsic physical characteristics but to improve the diagnostic use of an arthroscopic ultrasound system and the method to detect early stage of degeneration of human articular cartilage. It is not easy to calculate the intrinsic physical characteristics from ultrasonic echo obtained under arthroscopy. True it is ideal that the intrinsic physical characteristics of cartilage are measured accurately, but it is still difficult in clinical settings using existing devices. We consider that weakness to measure the intrinsic physical characteristics accurately does not interfere with our final purpose, i.e. quantitative evaluation of human cartilage in clinical settings such as arthroscopy. Information on the signal intensity is valuable for clinicians who want to know mechanism of degeneration of cartilage without performing a tissue biopsy. We believe that the ultrasound information that our technique provides will help clinicians to understand degeneration of articular cartilage, even if the information is obtained from an indirect measurement of the intrinsic physical characteristics.