TNF-alpha and IL-1beta suppress N-cadherin expression in MC3T3-E1 cells.

Excessive production of tumor necrosis factor (TNF) and interleukin-1 (IL-1) secondary to estrogen deficiency have been implicated as the cause of osteoporosis in postmenopausal woman. These cytokines appear to stimulate osteoclast precursor proliferation and activate mature osteoclast formation directly and possibly indirectly via osteoblasts. To investigate the other possible roles that these cytokines may play in stimulating the bone resorption process, we examined the effect of TNF-alpha and IL-1beta on cell-cell adhesion molecules, cadherins, in osteoblastic MC3T3-E1 cells. In this study, we investigated cadherin expression and the effect of TNF-alpha, IL-1beta, and parathyroid hormone (PTH) on the expression of cadherins in MC3T3-E1 cells. Confluent cultures of MC3T3-E1 cells were challenged with recombinant human TNF-alpha (1-100 U/ml), recombinant human IL-1beta (1-100 ng/ml) and human PTH(1-34) (1-100 ng/ml), respectively. The results show that MC3T3-E1 cells express functional cadherin molecules, N-cadherin and OB-cadherin. TNF-alpha (10-100 U/ml) and IL-1beta (10-100 ng/ml) suppressed N-cadherin without changing OB-cadherin expression, while PTH (1-100 ng/ml) had no effect on cadherin expression. These results raise the possibility that TNF-alpha and IL-1beta may compromise the cell-cell adhesion of osteoblasts which cover the bone surface. The ensuing compromised cell-cell adhesion of osteoblasts may in turn facilitate the direct adhesion of osteoclasts on the calcified bone matrix surface. These results implicate an indirect role for osteoblasts in the promotion of bone resorption by TNF-alpha and IL-1beta.

Colocalization of noggin and bone morphogenetic protein-4 during fracture healing.

The regulation of callus formation during fracture repair involves the coordinate expression of growth factors and their receptors. This article describes the temporal and spatial expression of noggin gene, an antagonist to bone morphogenetic protein (BMP), during the fracture repair process. Noggin expression was examined by means of Northern blotting and in situ hybridization and compared with the expression pattern of BMP-4 in a model of fracture repair in adult mice. Expression levels of noggin messenger RNA (mRNA) were enhanced in the early phase of fracture callus formation. The localization of the noggin mRNA was similar to that of BMP-4 mRNA. Distinct noggin mRNA signals were located predominantly in cells lining the periosteum and the cortical endosteum near the fracture site at 2 days after fracture. At 5, 10, and 21 days after fracture, noggin mRNA was detected in the chondrocytes and osteoblasts in the newly formed callus. The pattern of localization was indistinguishable from that of BMP-4. These results suggest that the noggin/BMP-4 balance could be an important factor in the regulation of callus formation during fracture healing.

A phosphodiesterase inhibitor, pentoxifylline, enhances the bone morphogenetic protein-4 (BMP-4)-dependent differentiation of osteoprogenitor cells.

Bone morphogenetic protein-4 (BMP-4), a member of the transforming growth factor-beta superfamily, is capable of initiating differentiation of uncommitted mesenchymal cells into a chondro/osteogenic pathway. This study reports the effects of pentoxifylline (PTX), a nonspecific inhibitor of phosphodiesterases (PDEs), that causes elevation of the intracellular cyclic adenosine monophosphate (cAMP) level on the BMP-4-induced chondro/osteogenic differentiation of a mesenchymal cell line, C3H10T1/2; a bone marrow stromal cell line, ST2; and an osteoblastic cell line, MC3T3-E1. It was found that PTX enhanced BMP-4-induced chondro/osteogenic differentiation in C3H10T1/2 and ST2 cells. Similar effects were observed when adding dibutyryl-cAMP and forskolin. These results indicate that cAMP may potentiate the action of BMP-4 on osteoprogenitor cells, highlighting the possibility that PDE inhibitors could be used as therapeutic agents to enhance bone formation through this effect.

Phosphodiesterase inhibitors, pentoxifylline and rolipram, increase bone mass mainly by promoting bone formation in normal mice.

The administration of either Pentoxifylline (PTX), a methylxanthine derivative and an inhibitor of cyclic AMP (c-AMP) phosphodiesterases (PDEs), or Rolipram, an inhibitor specific to type-4 PDE (PDE4) in normal mice, significantly increased both cortical and cancellous bone mass. Vertebrae and tibiae from mice treated with PTX or Rolipram were analyzed by means of bone densitometry and histomorphometry. The results revealed that both PTX and Rolipram increased bone mass in normal mice mainly through the acceleration of bone formation. These findings suggest that both PTX and Rolipram can enhance physiological bone formation and thereby increase bone mass in normal mice. The possibility that these agents may be of value for the treatment of osteoporosis is discussed.

Advantages of concurrent use of anabolic and antiresorptive agents over single use of these agents in increasing trabecular bone volume, connectivity, and biomechanical competence of rat vertebrae.

The purpose of this study was to evaluate the usefulness of a combination regimen of anabolic and antiresorptive agents in increasing skeletal quantity and quality in comparison to a single-drug regimen with these agents. We examined histomorphometrically and biomechanically the effects of separate and combined administration of intermittent parathyroid hormone (PTH) and estrogen or bisphosphonate on both axial and appendicular skeletons of male Wistar rats, which were 4 months old and weighed approximately 300 g at the beginning of the treatment. The animals were untreated or injected with vehicle, recombinant human PTH(1-84) (PTH; 100 microg/kg daily), 17beta-estradiol (E2, 500 microg/kg every other day), incadronate disodium (YM175, 80 microg/kg every other day), combined PTH and E2 (PTH + E2), or a combination of PTH and YM175 (PTH + YM175). After 1 month of treatment, the three groups in which PTH was administered (PTH, PTH + E2, and PTH + YM175) had significantly higher trabecular bone volume and connectivity in the proximal tibial metaphyses (PTMs) compared with the untreated and vehicle-treated groups, whereas only combination groups (PTH + E2 and PTH + YM175) showed significant increases in these indices in the lumbar vertebrae. This site-related discrepancy was attributed to the fact that PTH significantly elevated bone resorption not in the PTMs but in the vertebrae. This increased bone resorption in the vertebrae was suppressed by the addition of an antiresorptive agent. As a result, trabecular bone mass, connectivity, and mechanical strength of the vertebrae were significantly increased from control levels only in the concurrent treatment groups (PTH + E2 and PTH + YM175). The superior skeletal effects of PTH cotherapy over PTH monotherapy were not seen with regard to bone mass, but with increased connectivity and mechanical strength. The concurrent use of PTH and an antiresorptive agent has been shown to be superior to the single use of PTH for enhancing these properties of rat vertebrae, which encourages future research, especially in larger animals.

Enhancement of bone morphogenetic protein-2-induced new bone formation in mice by the phosphodiesterase inhibitor pentoxifylline.

Porous collagen disks (6 mm diameter, 1 mm thickness) were impregnated with recombinant human bone morphogenetic protein-2 (rhBMP-2) (5 microg/disk) and implanted onto the back muscles of mice. Pentoxifylline (PTX), which is a methylxanthine-derived inhibitor of phosphodiesterases (PDEs), or vehicle, was injected (5, 25, 50, 100, 200, and 300 mg/kg body weight/day) into the mice subcutaneously once a day for 3 weeks from the day of implantation of the bone morphogenetic protein (BMP)-laden disks. The rhBMP-2-induced ectopic ossicles were harvested and examined using radiographic, histological, and biochemical methods to determine size, bone quality, and calcium content. When compared with controls, ossicles from mice treated with >50 mg/kg per day of PTX were significantly larger in size and had a greater calcium content. However, no differences were noted in mice treated with lower doses (5 and 25 mg/kg per day) of PTX. The temporal sequence of the bone-forming process was unchanged by PTX based on histological examination. The histology of the ossicles from high- and low-dose PTX-treated mice was essentially identical to that observed in the control mice. These experimental results indicate that PTX enhanced the bone-inducing capacity of BMP-2. The underlying mechanism of action most likely involves the inhibition of intracellular phosphodiesterases and a resulting elevation of the intracellular content of cyclic nucleotides. Further studies are warranted to understand how BMP-induced bone formation is pharmacologically modified by PTX.

A prospective study of the incidence and outcomes of incidental dural tears in microendoscopic lumbar decompressive surgery.

Little information is available about the incidence and outcome of incidental dural tears associated with microendoscopic lumbar decompressive surgery. We prospectively examined the incidence of dural tears and their influence on the outcome six months post-operatively in 555 consecutive patients (mean age 47.4 years (13 to 89)) who underwent this form of surgery. The incidence of dural tears was 5.05% (28/555). The risk factors were the age of the patient and the procedure of bilateral decompression via a unilateral approach. The rate of recovery of the Japanese Orthopaedic Association score in patients with dural tears was significantly lower than that in those without a tear (77.7% vs. 87.6%; p < 0.02), although there were no significant differences in the improvement of the Oswestry Disability Index between the two groups. Most dural tears were small, managed by taking adequate care of symptoms of low cerebrospinal fluid pressure, and did not require direct dural repair. Routine MRI scans were undertaken six months post-operatively; four patients with a dural tear had recurrent or residual disc herniation and two had further stenosis, possibly because the dural tear prevented adequate decompression and removal of the fragments of disc during surgery; as yet, none of these patients have undergone further surgery.

The natural history of asymptomatic lumbar canal stenosis in patients undergoing surgery for cervical myelopathy.

We retrospectively examined the prevalence and natural history of asymptomatic lumbar canal stenosis in patients treated surgically for cervical compressive myelopathy in order to assess the influence of latent lumbar canal stenosis on the recovery after surgery. Of 214 patients who had undergone cervical laminoplasty for cervical myelopathy, we identified 69 (32%) with myelographically documented lumbar canal stenosis. Of these, 28 (13%) patients with symptomatic lumbar canal stenosis underwent simultaneous cervical and lumbar decompression. Of the remaining 41 (19%) patients with asymptomatic lumbar canal stenosis who underwent only cervical surgery, 39 were followed up for ≥ 1 year (mean 4.9 years (1 to 12)) and were included in the analysis (study group). Patients without myelographic evidence of lumbar canal stenosis, who had been followed up for ≥ 1 year after the cervical surgery, served as controls (135 patients; mean follow-up period 6.5 years (1 to 17)). Among the 39 patients with asymptomatic lumbar canal stenosis, seven had lumbar-related leg symptoms after the cervical surgery. Kaplan-Meier analysis showed that 89.6% (95% confidence interval (CI) 75.3 to 96.0) and 76.7% (95% CI 53.7 to 90.3) of the patients with asymptomatic lumbar canal stenosis were free from leg symptoms for three and five years, respectively. There were no significant differences between the study and control groups in the recovery rate measured by the Japanese Orthopaedic Association score or improvement in the Nurick score at one year after surgery or at the final follow-up. These results suggest that latent lumbar canal stenosis does not influence recovery following surgery for cervical myelopathy; moreover, prophylactic lumbar decompression does not appear to be warranted as a routine procedure for coexistent asymptomatic lumbar canal stenosis in patients with cervical myelopathy, when planning cervical surgery.

Transcriptional regulation of the mBMP-4 gene through an E-box in the 5'-flanking promoter region involving USF.

We analyzed the promoter activity of murine bone morphogenetic protein-4 (mBMP-4) and determined that the 5'-flanking region of exon I plays a critical role for transcription and position -265 to -246 of 5'-flanking region of mBMP-4 gene acts as a cis-element important for the regulation of BMP4 transcription in MC3T3E1 cells. By use of site-directed mutagenesis, we have established that the E-box, CACGTG, located within this short region of promoter, is essential for transcriptional activation of the mBMP-4 gene. Upstream stimulatory factor (USF), a member of the helix-loop-helix (HLH) group of regulatory proteins, was found to bind to this E-box using supershift gel mobility analysis. It is proposed that the HLH transcription regulatory proteins play an important role in mBMP-4 gene transcription in this osteoblastic cell line.