Stimulation of bone formation in cortical bone of mice treated with a receptor activator of nuclear factor-κB ligand (RANKL)-binding peptide that possesses osteoclastogenesis inhibitory activity.

To date, parathyroid hormone is the only clinically available bone anabolic drug. The major difficulty in the development of such drugs is the lack of clarification of the mechanisms regulating osteoblast differentiation and bone formation. Here, we report a peptide (W9) known to abrogate osteoclast differentiation in vivo via blocking receptor activator of nuclear factor-κB ligand (RANKL)-RANK signaling that we surprisingly found exhibits a bone anabolic effect in vivo. Subcutaneous administration of W9 three times/day for 5 days significantly augmented bone mineral density in mouse cortical bone. Histomorphometric analysis showed a decrease in osteoclastogenesis in the distal femoral metaphysis and a significant increase in bone formation in the femoral diaphysis. Our findings suggest that W9 exerts bone anabolic activity. To clarify the mechanisms involved in this activity, we investigated the effects of W9 on osteoblast differentiation/mineralization in MC3T3-E1 (E1) cells. W9 markedly increased alkaline phosphatase (a marker enzyme of osteoblasts) activity and mineralization as shown by alizarin red staining. Gene expression of several osteogenesis-related factors was increased in W9-treated E1 cells. Addition of W9 activated p38 MAPK and Smad1/5/8 in E1 cells, and W9 showed osteogenesis stimulatory activity synergistically with BMP-2 in vitro and ectopic bone formation. Knockdown of RANKL expression in E1 cells reduced the effect of W9. Furthermore, W9 showed a weak effect on RANKL-deficient osteoblasts in alkaline phosphatase assay. Taken together, our findings suggest that this peptide may be useful for the treatment of bone diseases, and W9 achieves its bone anabolic activity through RANKL on osteoblasts accompanied by production of several autocrine factors.

Increased bone mass in mice after single injection of anti-receptor activator of nuclear factor-kappaB ligand-neutralizing antibody: evidence for bone anabolic effect of parathyroid hormone in mice with few osteoclasts.

Receptor activator of nuclear factor-κB ligand (RANKL) is a pivotal osteoclast differentiation factor. To investigate the effect of RANKL inhibition in normal mice, we prepared an anti-mouse RANKL-neutralizing monoclonal antibody (Mab, clone OYC1) and established a new mouse model with high bone mass induced by administration of OYC1. A single subcutaneous injection of 5 mg/kg OYC1 in normal mice significantly augmented the bone mineral density in the distal femoral metaphysis from day 2 to day 28. The OYC1 treatment markedly reduced the serum level of tartrate-resistant acid phosphatase-5b (TRAP-5b, a marker for osteoclasts) on day 1, and this level was undetectable from day 3 to day 28. The serum level of alkaline phosphatase (a marker for osteoblasts) declined significantly following the reduction of TRAP-5b. Histological analysis revealed few osteoclasts in femurs of the treated mice on day 4, and both osteoclasts and osteoblasts were markedly diminished on day 14. Daily injection of parathyroid hormone for 2 weeks increased the bone mineral density in trabecular and cortical bone by stimulating bone formation in the OYC1-treated mice. These results suggest that parathyroid hormone exerted its bone anabolic activity in mice with few osteoclasts. The mouse anti-RANKL neutralizing antibody OYC1 may be a useful tool to investigate unknown functions of RANKL in vivo.

Establishment of a new murine model of hypercalcemia with anorexia by overexpression of soluble receptor activator of NF-κB ligand using an adenovirus vector.

Hypercalcemia is a significant complication of certain human malignancies that is primarily caused by the release of calcium from bone due to marked bone resorption by osteoclast activation. Osteoclast differentiation and activation is mediated by receptor activator of NF-κB ligand (RANKL). Transgenic mice overexpressing murine soluble RANKL (sRANKL) that we generated previously exhibited severe osteoporosis accompanied with enhanced osteoclastogenesis, but never exhibited hypercalcemia. To analyze the relationship between serum concentration of sRANKL and hypercalcemia and generate a simple and quick hypercalcemia model, an adenovirus vector harboring murine sRANKL cDNA (Ad-sRANKL) was injected i.p. into male C57BL/6 mice. Sera were collected to measure the levels of sRANKL, calcium and biochemical markers of bone turnover. Food intake and body weight were measured every 3 or 4 days. All the mice were killed 2 weeks after the injection, and femurs were collected to measure bone structure and bone mineral density (BMD). Serum sRANKL and calcium increased, peaking on day 7. Food intake and body weight significantly declined on day 7. These results indicated that the mice had anorexia as a symptom of hypercalcemia. Increases in bone resorption and formation markers with a marked decrease in BMD were observed on day 14. These results reflect accelerated bone formation following activation of osteoclasts, indicating coupling between bone formation and resorption. In conclusion, a new murine model of hypercalcemia with anorexia was established by overexpressing sRANKL. This model would be useful for studies of hypercalcemia and coupling between bone formation and resorption.

Discrete wavelength tuning characteristics of a single-frequency fiber laser with dual-wavelength external frequency-stable light injection.

We investigate discrete wavelength tuning characteristics of a single-frequency fiber laser locked to either of two external lights. Frequency locking is achieved by the cooperatively induced spatial-hole burning (SHB) of a saturable absorber in the laser cavity. We show that lasing frequency is well locked to either of the two external lights when the lasing wavelength of the fiber laser is adjusted to the corresponding wavelength of the external light by tuning the bandpass filter in the laser cavity. The locked frequency stability of the fiber laser is as high as that of the employed external light source.

[Basic experimental study of intraosseous venography using carbon dioxide contrast agent in percutaneous vertebroplasty].

PURPOSE: In percutaneous vertebroplasty (PVP), intraosseous venography is performed using water-soluble ionic iodinated contrast media (iodine contrast media) before injecting bone cement. However, because of contrast medium remaining in the fractured cleft, we experienced the persistence of intravertebral opacification that obscured visualization of the cement under X-ray fluoroscopy. To solve this problem, we examined carbon dioxide (CO(2)) as a contrast medium. METHODS: We measured the contrast of iodine contrast media and CO(2) and performed a subjective assessment of imaging by enforcing intraosseous venography by changing the tube voltage and image intensifier (I.I.) entrance dose in a bone sample of a cadaver using iodine contrast media and CO(2). In vivo study, we performed a subjective assessment of images obtained by intraosseous venography using CO(2) under two kinds of X-ray views. RESULTS: We determined that the contrast of CO(2) is 1/6-1/7.5 that of iodine contrast media, and that CO(2) fluctuates less in its contrast value with changes in tube voltage. In our assessment of the image of the bone sample, CO(2) performed worse that iodine contrast media. However, if the I.I. entrance dose is kept above 2.5 microGy/F, CO(2) is considered to be of clinical use. In the clinical image assessment, the best conditions were an image collection rate of 7.5 F/S and matrix of 1024 x 1024. CONCLUSIONS: CO(2) did not cause any obstacles when we injected bone cement, and its I.I. entrance dose, image collection rate, and matrix size indicate that it can be used for intraosseous venography.

Diagnosis of cell death induced by methylglyoxal, a metabolite derived from glycolysis, in Saccharomyces cerevisiae.

Methylglyoxal (MG) is a ubiquitous metabolite derived from glycolysis; however, this aldehyde kills all types of cell. We analyzed the properties of MG-induced cell death of the budding yeast Saccharomyces cerevisiae. The MCA1 gene encodes a caspase homologue that is involved in H2O2-induced apoptosis in yeast, although the disruption of MCA1 did not repress sensitivity to MG. In addition, the intracellular oxidation level did not increase under conditions in which MG kills the cell. Furthermore, the disruption of genes encoding antioxidant enzymes did not affect the susceptibility to MG. Here, we demonstrate that yeast cells killed by MG do not exhibit the characteristics of apoptosis in a TUNEL assay or an annexin V staining, but show those of necrosis upon propidium iodide staining. We demonstrate that MG at high concentrations provokes necrotic cell death without the generation of reactive oxygen species in S. cerevisiae.

Actinomycin D-mediated sensitization of AIDS-Kaposi's sarcoma cells to Fas-mediated apoptosis: involvement of the mitochondrion-dependent pathway.

Fas engagement rapidly induces formation of the death-inducing signaling complex (DISC) that consists of Fas, FADD and pro-caspase-8. Activated caspase-8 at the DISC directly activates downstream caspases, resulting in induction of apoptosis of the independent mitochondria. In this study, we have obtained evidence demonstrating that Fas-mediated apoptosis in AIDS-KS cells takes place in a mitochondria-dependent manner. FADD and pro-caspase-8 were detected in immunoprecipitates with anti-Fas antibody in anti-Fas mAb (CH-11)-treated Hut 78, a typical Fas-sensitive cell line. On the other hand, DISC formation by CH-11 was markedly reduced in AIDS-KS cells. In addition, CH-11-induced activation of caspase-8-like protease in AIDS-KS cells was much less pronounced compared with that in Hut 78; however, a caspase-8 inhibitor, zIETD-fmk, completely blocked the apoptosis. Further, a caspase-9 inhibitor, zLEHD-fmk, markedly inhibited Fas-mediated apoptosis in AIDS-KS cells. Several apoptotic stimuli induce mitochondria activation allowing cytochrome c release from the mitochondria. In the apoptosome, cytochrome c and Apaf-1 activate caspase-9 which subsequently leads to the activation of caspase-3. In AIDS-KS cells, CH-11 triggered cytochrome c release, an event which was inhibited by zIETD-fmk. Further, a caspase-3 inhibitor, zDEVD-fmk completely inhibited the apoptosis. Altogether, the present data provide evidence that the Fas signal in AIDS-KS cells is preferentially transduced through the mitochondria-dependent pathway, which is initiated by caspase-8 activation.

Carpal tunnel release through a small incision using a special knife guide.

There have been controversial opinions regarding whether the release of the carpal tunnel should be performed arthroscopically or through an open incision. The authors present a method of open release through a small incision using a set of specially designed instruments, retaining advantages of observing the pathology under direct vision and avoiding complications of hazardous injuries to important structures. The instruments consist of a thin metal guide with a groove in the center to accommodate an angled knife holder. The procedure has been performed since 1997 with no complications.

Evaluation of pharmaceuticals with a novel 50-hour animal model of bone loss.

Osteoporosis remains a major public health problem through its associated fragility fractures. Several animal models for the study of osteoporotic bone loss, such as ovariectomy (OVX) and denervation, require surgical skills and several weeks to establish. Osteoclast differentiation and activation is mediated by RANKL. Here we report the establishment of a novel and rapid bone loss model by the administration of soluble RANKL (sRANKL) to mice. Mice were injected intraperitoneally with sRANKL and used to evaluate existing anti-osteoporosis drugs. sRANKL decreased BMD within 50 h in a dose-dependent manner. The marked decrease in femoral trabecular BMD shown by pQCT and the 3D images obtained by microCT were indistinguishable from those observed in the OVX model. Histomorphometry showed that osteoclastic activity was significantly increased in the sRANKL-injected mice. In addition, serum biochemical markers of bone turnover such as Ca, C-telopeptide of type 1 collagen (CTX), and TRACP5b were also significantly increased in the sRANKL-injected mice in a dose-dependent manner. Bisphosphonates (BPs), selective estrogen receptor modulators (SERMs), and PTH are commonly used for the treatment of osteoporosis. We successfully evaluated the effects of anti-bone-resorbing agents such as BPs, a SERM, and anti-RANKL-neutralizing antibody on bone resorption in a couple of weeks. We also evaluated the effects of PTH on bone formation in 2 wk. A combination of sRANKL injections and OVX made it possible to evaluate a SERM. The sRANKL model is the simplest, fastest, and easiest of all osteoporosis models and could be useful in the evaluation of drug candidates for osteoporosis.

Genomic structure and promoter analysis of the mouse neutral ceramidase gene.

We report here the molecular cloning of the mouse neutral ceramidase gene and its promoter analysis. The gene, composed of 27 exons ranging in size from 40 to 292 bp, spans more than 70 kb. Analysis of the 5(')-flanking region of the ceramidase genes revealed that the first exon of the gene of mouse liver was exactly the same as that of mouse kidney and Swiss 3T3 fibroblasts but completely different from that of mouse brain. The putative promoter regions of liver and brain ceramidase genes contained several well-characterized promoter elements such as GATA-2, C/EBP, and HNF3beta but lacked TATA and CAAT boxes, a typical feature of a housekeeping gene, although the expression is regulated in a tissue-specific manner. Interestingly, a GC box was exclusively found in the putative promoter of mouse liver whereas potential AP1 and AP4 binding sites were present in that of mouse brain. By a luciferase reporter gene assay, it was shown that the GC-rich region, which exists just upstream of the first exon, conferred the promoter activity in Swiss 3T3 cells.

Radiographic and magnetic resonance imaging findings of polylevolactic acid screws after rotational acetabular osteotomy.

INTRODUCTION: Biodegradable polylevolactic acid implants have become more commonly used for the treatment of fractures and osteotomies over the past few years. In the present study, the biocompatibility and degradation of polylevolactide screws used for rotational acetabular osteotomy were assessed on the basis of radiographic and MRI findings. MATERIALS AND METHODS: Forty-nine hips of 47 patients were analyzed for this study. The average age of the patients at the time of surgery was 38.0 years (range 18-62 years). The original diagnosis was osteoarthritis in 43 hips and osteonecrosis in 6 hips. The mean duration of follow-up was 2.8 years (range 1.5-5.5 years). Anteroposterior radiographs obtained at 1 week, 6 months, 1 year after the operation, as well as the most recent radiographs, were used to assess bony union, the radiolucent tract of each screw, and the sclerotic rim around each screw. Fifteen patients were chosen randomly to undergo MRI. RESULTS: Union of the osteotomized surfaces occurred in all patients within 6 months of surgery. Radiographs showed no osteolysis, no formation of bone cysts, and no displacement of the osteotomized acetabulum in any of the 49 hips. None of the patients was found to have any complications caused by problems with the biocompatibility of the implants. At the final postoperative examination, a central radiolucent tract and a sclerotic rim around the screws were seen in 34/49 hips (69%) and 23/49 hips (47%), respectively. On MRI findings, we were able to detect significant resorption of the screws in two patients after 1.2 and 1.7 years of follow-up. Though localized low-intensity areas on T1-weighted images and high-intensity areas on T2-weighted images were observed at the top of the screws in 5 patients, there was no development of pain, tenderness, or a sinus during the follow-up period. CONCLUSION: Although absorption of polylevolactic acid screws did not occur in a large majority of cases, rotational acetabular osteotomy appears to be a good indication for the use of polylevolactic acid screws.

Chronological changes of serum chromium levels after modern metal-on-metal total hip arthroplasty.

We determined changes in serum chromium concentration every 6 months up to 3 years after implantation in 44 patients (mean age 63 (49-79) years, 35 women) who underwent modern metal-on-metal total hip arthroplasty (Metasul). The serum chromium levels increased between the first and second year after implantation (1 year: 1.05 (SD 0.76) microg/L, 2 years: 1.46 (SD 0.91) microg/L). A small increase was observed during the third year (1.61 (SD 1.31) microg/L). The clinical relevance of this finding is uncertain and should be determined in long-term studies on a large scale.