Aging is associated with increased activities of matrix metalloproteinase-2 and -9 in tenocytes.

BACKGROUND: Most tendon pathology is associated with degeneration, which is thought to involve cyclic loading and cumulative age-related changes in tissue architecture. However, the association between aging and degeneration of the extracellular matrix (ECM) in tendons has not been investigated extensively. METHODS: We examined tenocytes from Achilles tendons taken from rats of three different ages (2, 12, and 24 months). Tenocyte viability was assessed using the 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) assay. Quantitative real-time polymerase chain reaction (PCR) was used to determine the levels of mRNAs that encode type-I collagen, matrix metalloproteinase (MMP)-2 and -9, tissue inhibitor of metalloproteinase (TIMP)-1 and -2 and transforming growth factor (TGF)-ß1. Gelatin zymography was used to evaluate the enzymatic activities of MMP-2 and -9. Furthermore, the concentration of TGF-ß1 in conditioned medium was evaluated using enzyme-linked immunosorbent assay (ELISA). RESULTS: The results of the MTT assay showed that the number of viable tenocytes decreased with age. No differences were observed in the levels of mRNAs that encode type-I collagen and TGF-ß1 among the three age groups, and the TGF-ß1 concentration did not change with age. However, mRNAs that encode MMP-2 and -9 were significantly more abundant in tenocytes from the aging group, and gelatin zymography revealed that the enzymatic activities of MMP-2 and -9 also increased significantly with age. Furthermore, as compared with young group, mRNAs that encode TIMP-1 and -2 were significantly decreased in tenocytes from the aging group. CONCLUSIONS: Activities of MMP-2 and MMP-9 in tenocytes increase with age. This might provide a mechanistic explanation of how aging contributes to tendinopathy or tendon rupture with age.

Optimizing human synovial fluid preparation for two-dimensional gel electrophoresis.

BACKGROUND: Proteome analysis is frequently applied in identifying the proteins or biomarkers in knee synovial fluids (SF) that are associated with osteoarthritis and other arthritic disorders. The 2-dimensional gel electrophoresis (2-DE) is the technique of choice in these studies. Disease biomarkers usually appear in low concentrations and may be masked by high abundant proteins. Therefore, the main aim of this study was to find the most suitable sample preparation method that can optimize the expression of proteins on 2-DE gels that can be used to develop a reference proteome picture for non-osteoarthritic knee synovial fluid samples. Proteome pictures obtained from osteoarthritic knee synovial fluids can then be compared with the reference proteome pictures obtained in this study to assist us in identifying the disease biomarkers more correctly. RESULTS: The proteomic tool of 2-DE with immobilized pH gradients was applied in this study. A total of 12 2-DE gel images were constructed from SF samples that were free of osteoarthritis. In these samples, 3 were not treated with any sample preparation methods, 3 were treated with acetone, 3 were treated with 2-DE Clean-Up Kit, and 3 were treated with the combination of acetone and 2-D Clean-Up Kit prior to 2-DE analysis. Gel images were analyzed using the PDQuest Basic 8.0.1 Analytical software. Protein spots that were of interest were excised from the gels and sent for identification by mass spectrometry. Total SF total protein concentration was calculated to be 21.98 ± 0.86 mg/mL. The untreated SF samples were detected to have 456 ± 33 protein spots on 2-DE gel images. Acetone treated SF samples were detected to have 320 ± 28 protein spots, 2-D Clean-Up Kit treated SF samples were detected to have 413 ± 31 protein spots, and the combined treatment method of acetone and 2-D Clean-Up Kit was detected to have 278 ± 26 protein spots 2-DE gel images. SF samples treated with 2-D Clean-Up Kit revealed clearer presentation of the isoforms and increased intensities of the less abundant proteins of haptoglobin, apolipoprotein A-IV, prostaglandin-D synthase, alpha-1B-glycoprotein, and alpha-2-HS-glycoprotein on 2-DE gel images as compared with untreated SF samples and SF samples treated with acetone. CONCLUSIONS: The acetone precipitation method and the combined treatment effect of acetone and 2-DE Clean-Up Kit are not preferred in preparing SF samples for 2-DE analysis as both protein intensities and numbers decrease significantly. On the other hand, 2-D Clean-Up Kit treated SF samples revealed clearer isoforms and higher intensities for the less abundant proteins of haptoglobin, apolipoprotein A-IV, prostaglandin-D synthase, alpha-1B-glycoprotein, and alpha-2-HS-glycoprotein on 2-DE gels. As a result, it is recommended that SF samples should be treated with protein clean up products such as 2-D Clean-Up Kit first before conducting proteomic research in searching for the relevant biomarkers associated with knee osteoarthritis.

A novel approach to sonographic examination in a patient with a calf muscle tear: a case report.

INTRODUCTION: Rupture of the distal musculotendinous junction of the medial head of the gastrocnemius, also known as "tennis leg", can be readily examined using a soft tissue ultrasound. Loss of muscle fiber continuity and the occurrence of bloody fluid accumulation can be observed using ultrasound with the patient in the prone position; however, some cases may have normal ultrasound findings in this conventional position. We report a case of a middle-aged man with tennis leg. Ultrasound examination had normal findings during the first two attempts. During the third attempt, with the patient's calf muscles examined in an unconventional knee flexed position, sonographic findings resembling tennis leg were detected. CASE PRESENTATION: A 60-year-old man in good health visited our rehabilitation clinic complaining of left calf muscle pain. On suspicion of a ruptured left medial head gastrocnemius muscle, a soft tissue ultrasound examination was performed. An ultrasound examination revealed symmetrical findings of bilateral calf muscles without evidence of muscle rupture. A roentgenogram of the left lower limb did not reveal any bony lesions. An ultrasound examination one week later also revealed negative sonographic findings. However, he still complained of persistent pain in his left calf area. A different ultrasound examination approach was then performed with the patient lying in the supine position with his knee flexed at 90 degrees. The transducer was then placed pointing upwards to examine the muscles and well-defined anechoic fluid collections with areas of hypoechoic surroundings were observed. CONCLUSION: For patients suffering from calf muscle area pain and suspicion of tennis leg, a soft tissue ultrasound is a simple tool to confirm the diagnosis. However, in the case of negative sonographic findings, we recommend trying a different positional approach to examine the calf muscles by ultrasound before the diagnosis of tennis leg can be ruled out.