Biomedical subjects

Michiaki Takagi

Publications and source records attributed to Michiaki Takagi.

10 recordsLinked to original sources

PubMed2007

Upregulation of matrix metalloproteinase (MMP)-1 and its activator MMP-3 of human osteoblast by uniaxial cyclic stimulation.

Proper mechanical loading is essential for bone remodeling and maintenance of human skeletal system. Matrix metalloproteinases (MMPs) are secreted by mesenchymal stromal lining cells and osteoblasts to prepare the initiation sites for osteoclastic bone resorption at the beginning of the remodeling cycle. However, only a few studies have addressed the effect of mechanical stress on MMPs and their endogenous tissue inhibitors of matrix metalloproteinases (TIMPs) in osteoblasts. In this study, the response of human osteoblasts to uniaxial cyclic stretching was investigated to clarify this more in detail. Stretching affected the orientation of the osteoblasts, and quantitative reverse transcription-polymerase chain reaction revealed coordinated upregulation of MMP-1 and its activator MMP-3 mRNA by cyclic 5% stretching at 3 h (p < 0.01). Upregulation of cyclooxygenase-2 mRNA was also found in response to cyclic 1 and 5% stretchings at 1, 3, and 6 h (p < 0.01). No changes were found in MMP-2, TIMP-1, and -2. The mRNA expression of MMP-9 was low and MMP-13 was not detected. This study suggests that MMP-1 and -3, enhanced by uniaxial cyclic mechanical stimulation of osteoblasts, are candidate key enzymes in the processing of collagen on bone surface, which might be necessary to allow osteoclastic recruitment leading to bone resorption. The strain might also play a role in cleaning of demineralized bone surface during the reversal phase, before bone formation starts.

Base Sequence↗

PubMed2006

Gene expression and localization of diacylglycerol kinase isozymes in the rat spinal cord and dorsal root ganglia.

The dorsal root ganglion (DRG) and dorsal horn of the spinal cord are areas through which primary afferent information passes enroute to the brain. Previous studies have reported that, during normal neuronal activity, the regional distribution of a second messenger, diacylglycerol (DG), which is derived from phosphoinositide turnover, is diverse in these areas. However, the way that DG is regulated in these organs remains unknown. The present study was performed to investigate mRNA expression and protein localization of DG kinase (DGK) isozymes, which play a central role in DG metabolism. Gene expression for DGK isozymes was detected with variable regional distributions and intensities in the spinal cord. Among the isozymes, most intense signals were found for DGKzeta and DGKiota in the DRG. By immunohistochemical analysis, DGKzeta immunoreactivity was detected heterogeneously in the nucleus and cytoplasm of small DRG neurons with variable levels of distribution, whereas it was detected exclusively in the cytoplasm of large neurons. On the other hand, DGKiota immunoreactivity was distributed solely in the cytoplasm of most of the DRG neurons. Double-immunofluorescent imaging of these isozymes showed that they coexisted in a large population of DRG neurons at distinct subcellular sites, i.e., DGKzeta in the nucleus and DGKiota in the cytoplasm. Thus, DGK isozymes may have different functional roles at distinct subcellular sites. Furthermore, the heterogeneous subcellular localization of DGKzeta between the nucleus and cytoplasm implies the possible translocation of this isozyme in small DRG neurons under various conditions.

Animals↗

PubMed2005

Developmental abnormalities in rat embryos leading to tibial ray deficiencies induced by busulfan.

BACKGROUND: Little is known about the developmental changes associated with tibial ray deficiencies. The aim of this study was to detect cell death, proliferation, and gene expression that result in tibial ray deficiencies. METHODS: We induced tibial ray deficiencies in rat embryos using a teratogenic agent (busulfan) and observed the developmental changes in 1126 hindlimbs. We performed Nile blue staining, whole mount in situ hybridization for fibroblast growth factor 8 (Fgf8), bone morphogenetic protein 4 (Bmp4) and Sonic hedgehog (Shh), terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end-labeling (TUNEL) and assessment of cell proliferation by 5-bromo-2'-deoxy-uridine (BrdU)/anti-BrdU immunohistochemistry. RESULTS: In situ hybridization showed reductions in Fgf8 and Bmp4 expression. Histological examination showed a delay of mesenchymal condensation, increased mesenchymal cell death, decreased mesenchymal cell proliferation, and a reduction in the number of mesenchymal cells. These abnormalities may cause hypoplasia of the limb. Bmp4 expression was markedly reduced in the anterior mesenchyme. Shh was expressed in the posterior mesenchyme. We suggest that the posterior skeletal elements may be fully formed owing to Shh expression, but the anterior skeletal elements may be underdeveloped owing to an intense reduction of Bmp4 expression in the anterior mesenchyme, causing hypoplasia of the tibial ray. CONCLUSIONS: The combined effects of increased cell death, decreased cell proliferation, reduction of Fgf8 expression, and intense reduction of Bmp4 expression in the anterior mesenchyme may play an important role in the development of tibial ray deficiency induced by busulfan.

Animals↗

PubMed2005

The microenvironment around total hip replacement prostheses.

The metal stem of the totally replaced hip carries load and resists fatigue, but it is electrochemically corroded. Metallic atoms act as haptens, induce type 1 T-helper cells/Th1-type immune responses and enhance periprosthetic osteolysis. Stiff metal implants, which do not have the same elasticity as the surrounding bone, cause stress shielding. Cyclic loading and lack of ligamentous support lead to mechanical and ischemia reperfusion injury and particle formation from bone, polymethylmethacrylate, and porous implant surfaces, which accelerate third-body polyethylene wear. Surgical injury and micromotion induce the formation of a fibrous capsule interface. Type-B lining cells produce lubricin and surface-active phospholipids to promote solid-to-solid lubrication but may loosen the implant from bone. The pumping action of the cyclically loaded joint and synovial fluid pressure waves dissect the implant-host interface and transports polyethylene particles and pro-inflammatory mediators to the interface. Hyaluronan induces formation of a synovial lining like layer. Because of its localization close to bone, foreign body inflammation at the interface stimulates osteoclastogenesis and peri-implant bone loss. Metal-on-metal and ceramic-on-ceramic pairs might minimize third body wear, but can lead to high-impact load of the acetabulum. Diamond coating of a metal-on-polyethylene couple might solve both of these problems. The basic biomaterial solutions allow good mechanical performance and relatively long life in-service, but surface modifications (porous coating, hydroxyapatite, diamond, bioglass, and others) may facilitate performance of the implant and improve the biomaterial and body interfaces.

Arthroplasty, Replacement, Hip↗

PubMed2004

Developmental failure of phalanges in the absence of growth/differentiation factor 5.

Growth/differentiation factor 5 (GDF5) is a member of the bone morphogenetic protein (BMP) family, which has been implicated in several skeletogenic events including cartilage and bone formation. To study the role of GDF5, we analyzed digit development in brachypodism (bp) mice, which carry functional null mutations of the Gdf5 gene and exhibit a reduction in the length of digit bones and loss of the middle phalanges. In situ detection of apoptosis and whole-mount detection of cell death showed abnormal apoptosis in the developing phalanges of bp mice. In situ hybridization in bp mice showed overexpression of Gdf5 mRNA in the developing phalanges where apoptotic cells were increased. In addition, bp mice exhibited excessive apoptosis in the interdigital regions. The condensed mesenchymal cells were progressively decreased in the developing phalanges and failed to form cartilage models of the middle phalanges. These findings show that excessive apoptosis in the absence of GDF5 results in developmental failure of the phalanges. We conclude that GDF5 is essential for maintenance and growth of the developing phalanges.

Animals↗

PubMed2004

Matrix metalloproteinase-9 expression, tartrate-resistant acid phosphatase activity, and DNA fragmentation in vascular and cellular invasion into cartilage preceding primary endochondral ossification in long bones.

Vascular and cellular invasion into cartilage are essential for endochondral ossification. Recently it has been shown that matrix metalloproteinase-9 (MMP-9)/gelatinase B is a key regulator of growth plate angiogenesis and apoptosis of hypertrophic chondrocytes. To study vascular and cellular invasion into cartilage preceding primary endochondral ossification in long bones, precursor femurs from 13- to 16-day-old murine embryos were sectioned. Tartrate-resistant acid phosphatase (TRAP) activity, in situ hybridization for matrix metalloproteinase-9 (MMP-9), immunostaining for CD31, and in situ detection of apoptosis (TUNEL) were studied. TRAP activity, MMP-9 mRNA, and CD31 expression were initially detected in the intertrabecular spaces of the perichondral collar, and then in cells migrating into the cartilage. The first cells involved in the primary invasion into cartilage were CD31-positive vascular endothelial cells and MMP-9-positive cells, followed by TRAP-positive cells. At the cartilage-marrow interface, CD31-positive vascular endothelial cells and MMP-9-positive cells were predominant. These results suggest that MMP-9-positive cells cooperate with vascular endothelial cells in cartilage angiogenesis. TUNEL-positive staining was detected on chondrocytes attached to the inner surface of the perichondral collar, and also detected in the area where cartilage was removed. These results suggest that chondrocytes separated from the cartilage matrix may undergo apoptosis.

Acid Phosphatase↗

PubMed2004

Hyaluronate inhibits the interleukin-1beta-induced expression of matrix metalloproteinase (MMP)-1 and MMP-3 in human synovial cells.

Intra-articular administration of hyaluronate (HA) is an effective treatment for arthritis. HA injections can decrease not only joint pain but also synovial effusion, although little is known concerning the mechanism of HA action. The aim of this study was to investigate the role of HA on the expression and production of matrix metalloproteinase (MMP) in synovial cells activated by interleukin (IL)-1beta in order to achieve a better understanding of exogenous HA function in the extracellular matrix degradation in arthritic joints. Human synovial cells were incubated with HA (0.1-1000 microg/ml) and/or IL-1beta (1 ng/ml). The expression of MMP-1 and MMP-3 mRNAs was analyzed by quantitative real-time polymerase chain reaction. The protein levels of MMP-1 and MMP-3 in cultured media were measured by immunoblotting. Expression of MMP-1 and MMP-3 mRNAs was induced by IL-1beta. The IL-1beta-mediated induction of MMP-1 mRNA expression was attenuated by 10 microg/ml HA (p=0.026) and that of MMP-3 mRNA was strongly down-regulated in the presence of 10 or 1000 microg/ml HA (p<0.001). The increased protein levels of MMP-1 and MMP-3 were also reduced by 1000 microg/ml HA. These data suggest that HA inhibits the expression and production of MMP-1 and MMP-3 in IL-1beta-stimulated human synovial cells. We therefore prepose that intra-articular HA may rescue inflamed joints from bone and cartilage destruction by reducing the production of MMP-1 and MMP-3.

Cells, Cultured↗

PubMed2002

Acidic cysteine endoproteinase cathepsin K in the degeneration of the superficial articular hyaline cartilage in osteoarthritis.

OBJECTIVE: To measure cartilage pH in patients with osteoarthritis (OA) and to analyze the presence of cathepsin K, the recently discovered acidic endoproteinase, in phenotypically altered chondrocytes. METHODS: Intraoperative measurements of the pH of clinically normal, fibrillated, superficially fissured, and deeply fissured cartilage surfaces (grades 0-3, respectively) in OA patients undergoing primary hip replacement surgery were performed with the use of a sting electrode sterilized with microbicidic plasma. Fluorescent pH probes were used for in situ assessment of cartilage matrix pH. Cathepsin K was assessed using quantitative reverse transcriptase-polymerase chain reaction and immunohistochemistry methods. RESULTS: The pH of grade 0 cartilage surfaces was 7.1 +/- 0.4 (mean +/- SD), compared with 6.2 +/- 0.9 (P < 0.05), 5.7 +/- 1.0 (P < 0.001), and 5.5 +/- 1.0 (P < 0.001) for grades 1-3 cartilage surfaces, respectively. Fluorescent pH probes and acid-dependent autocatalytic conversion of cathepsin K into its active, low molecular weight form in cartilage confirmed these findings. Cathepsin K messenger RNA levels increased in relation to the severity of OA, and the number of cathepsin K-containing chondrocytes increased from a mean +/- SD of 12 +/- 3 in grade 0 cartilage surfaces to 47 +/- 7, 50 +/- 6, and 100 +/- 12 in grades 1-3 cartilage surfaces, respectively (P < 0.001 for all comparisons). CONCLUSION: Acid-activated, but pharmacologically inhibitable, cathepsin K is induced in phenotypically altered chondrocytes in OA. The findings suggest that cathepsin K, rather than neutral matrix metalloproteinases, degrades the superficial gliding surfaces of the articular hyaline cartilage in OA.

Acids↗

PubMed2002

Fundamental imaging properties of transillumination laser computed tomography based on coherent detection imaging method.

The coherent detection imaging (CDI) method uses the optical heterodyne detection technique. CW and single frequency lasers having long coherence lengths are used to exploit the maximum advantages of heterodyne detection, such as high directionality, selectivity and sensitivity. The CDI method based on optical heterodyne detection enables selective filtering of the directional coherence-retaining emergent photons, which leads to image reconstruction from projections, similar to X-ray computed tomography (CT). So far we have demonstrated the advantages and capabilities of the measurement technique for transillumination optical computed tomography in biomedicine. Here, we investigate the fundamental imaging properties of CDI method, such as its high directionality and quantitativeness, with preliminary physical phantom experiments. The results show that the CDI method satisfies the requirements for CT reconstruction under the first order approximation, and enables quantitative measurements in the sense that the relationship between estimated and actual concentration retains a satisfactory linearity.

Equipment Design↗

PubMed2002

Dense innervation in pseudocapsular tissue compared to aneural interface tissue in loose totally replaced hips.

BACKGROUND: The function of many inflammatory cells is in part regulated by neuronal cells, which may lead to so-called neurogenic inflammation. Sensory nerves also mediate the pain sensation. METHODS: This immunohistochemical study focused on visualization of C-sensory and sympathetic innervation in the synovial membrane-like interface and pseudocapsular tissue around loosened total hip replacement. RESULTS: The synovial membrane-like interface did not contain C-sensory peptidergic or sympathetic neural structures. Only limited attempts to neural regeneration were detected. In contrast, pseudocapsule expressed dense innervation with strong CPON-ir sympathetic innervation and osteoarthritis also had C-sensory fibers. Intense neural regeneration was seen in these synovial membranes. Surprisingly, stellate and/or highly dendritic fibroblast-like cells in the fibrotic areas in the interface tissue expressed strong immunoreactivity to the neural marker PGP 9.5, ubiquitin carboxyterminal hydrolase. CONCLUSION: Pain related to aseptic loosening cannot arise in the aneural interface membrane. Inflammation in interface/aseptic loosening seems to be driven by non-neurogenic factors, such as foreign bodies and micromovement. Insufficient lysosomal degradation of denatured proteins causes accumulation of ubiquitinated conjugates and enzymes involved in the process. This leads to insufficient degradation of platelet derived growth factor (PDGF)-receptor complex and can contribute to the accumulation of connective tissue in the interface. Failure in ubiquitin mediated proteolysis might support overgrowth of interface tissue and aseptic loosening.

Adrenergic Fibers↗