P38 Mitogen-Activated Protein Kinase Inhibitor, FR167653, Inhibits Parathyroid Hormone Related Protein-Induced Osteoclastogenesis and Bone Resorption

p38 mitogen-activated protein kinase (MAPK) acts downstream in the signaling pathway that includes receptor activator of NF-κB (RANK), a powerful inducer of osteoclast formation and activation. We investigated the role of p38 MAPK in parathyroid hormone related protein (PTHrP)-induced osteoclastogenesis in vitro and PTHrP-induced bone resorption in vivo. The ability of FR167653 to inhibit osteoclast formation was evaluated by counting the number of tartrate-resistant acid phosphatase positive multinucleated cells (TRAP-positive MNCs) in in vitro osteoclastgenesis assays. Its mechanisms were evaluated by detecting the expression level of c-Fos and nuclear factor of activated T cells c1 (NFATc1) in bone marrow macrophages(BMMs) stimulated with sRANKL and M-CSF, and by detecting the expression level of osteoprotegerin (OPG) and RANKL in bone marrow stromal cells stimulated with PTHrP in the presence of FR167653. The function of FR167653 on bone resorption was assessed by measuring the bone resorption area radiographically and by counting osteoclast number per unit bone tissue area in calvaria in a mouse model of bone resorption by injecting PTHrP subcutaneously onto calvaria. Whole blood ionized calcium levels were also recorded. FR167653 inhibited PTHrP-induced osteoclast formation and PTHrP-induced c-Fos and NFATc1 expression in bone marrow macrophages, but not the expression levels of RANKL and OPG in primary bone marrow stromal cells treated by PTHrP. Furthermore, bone resorption area and osteoclast number in vivo were significantly decreased by the treatment of FR167653. Systemic hypercalcemia was also partially inhibited. Inhibition of p38 MAPK by FR167653 blocks PTHrP-induced osteoclastogenesis in vitro and PTHrP-induced bone resorption in vivo, suggesting that the p38 MAPK signaling pathway plays a fundamental role in PTHrP-induced osteoclastic bone resorption

Spinal biomechanics modeling and finite element analysis of surgical instrument interaction

When the spinal surgery robot assists the surgeon perform the surgery, the patient is prone on the operating table. However, due to the force of the surgical instruments on the spine, there is a corresponding deformation in the surgical field, which affects the accuracy of the operation. In order to improve the accuracy and safety of the operation, this paper reconstructs the three-dimensional model of the lumbar spine which includes the vertebral body and the intervertebral disc based on the CT scan data, and then the lumbar spine is analyzed by the finite element method. The mathematical model of the relationship between force and displacement is established by using response surface methodology based on the simulation results. After that, the position control system is constructed based on the mathematical model. Through the simulation of the control system, the trajectory curve of the end of the manipulator is compared and the validity of the mathematical model is verified

Postoperative pulmonary complications following posterior spinal instrumentation and fusion for congenital scoliosis.

BACKGROUND:Congenital scoliosis (CS) may lead to more serious pulmonary complications compared with idiopathic scoliosis after spinal fusion surgery. However, little has been reported about postoperative pulmonary complication events in patients with CS after spinal fusion surgery. OBJECTIVE:To investigate the incidence of and predictive factors of postoperative pulmonary complications following posterior spinal instrumentation and fusion surgery for the treatment of CS. METHODS:We retrospectively reviewed the records of 174 patients with CS (128 females and 46 males, mean age 16.4 years) treated with posterior spinal instrumentation and fusion surgery between January 2012 and April 2017. We extracted demographic, medical history, and clinical data, and investigated the major predictive factors for postoperative pulmonary complications by logistic regression and receiver-operating characteristic curves analyses. RESULTS:A total of 26 (14.9%) patients developed postoperative pulmonary complications, consisting of pleural effusion (10.9%), pneumonia (6.9%), pneumothorax (1.1%), atelectasis (2.3%), hypoxemia (6.3%), and respiratory failure (1.1%). Logistic regression analysis revealed that the predictive factors for postoperative pulmonary complications were age > 18.1 years (P = 0.039), a Cobb angle of > 77° (P = 0.011), operation time of > 430 min (P = 0.032), and blood transfusion volume > 1500 ml (P = 0.015). CONCLUSIONS:Postoperative pulmonary complications are among the main complications following posterior spinal instrumentation and fusion surgery in patients with CS. Such patients aged >18.1 years, with Cobb angles > 77°, operation times > 430 min, and/or blood transfusion volume of > 1500 ml may be more likely to develop postoperative pulmonary complications

Animal model and treatment protocol.

<p>The PTHrP-induced bone resorption animal model was established by injecting PTHrP (18 µg/day) onto the dorsal surface of the mice calvaria daily for five days (day 1 to 5). Animals in the treatment (n = 7) or the control group (n = 5) were injected with FR167653 (30 mg/kg twice daily) or distilled water, respectively, daily for seven days, initiating from two days before PTHrP injection. 50 µl of whole blood was collected retro-orbitally before treatment, on day three, and on day five to determine whole-blood ionized calcium levels. Mice were sacrificed on day six, 12 hours after the last injection of PTHrP.</p

The effect of FR167653 on PTHrP-induced bone resorption.

<p>The calvarial bone resorption was measured radiographically on the left side (area marked by yellow line) where treated by direct subcutaneous injection of PTHrP. A. Treatment by FR167653 reduced the bone resorption area (*: P<0.005). DW:distilled water. B. Representative X-rays of mouse calvaria with PTHrP-induced bone resorption. a: control group (treated with DW). b: FR167653 treatment group.</p

The effects of FR167653 on sRANKL-induced c-Fos and NFATc1 expression level in M-CSF-dependent bone marrow macrophages (MDBMMs).

<p>Bone marrow macrophages were treated with 100 ng/ml M-CSF and 100 ng/ml sRANKL without or with FR67653 at the indicated concentration. After 24 h incubation, cells were harvested and subjected to RT-PCR (A). For western blot analysis (B), the cells were incubated for 48 h. Expression of c-Fos and NFATc1 in both mRNA and protein levels were increased following the sRANKL treatment. This enhancement was inhibited by FR167653 in a dose dependent manner. The results are shown as mean ±S.D. of three independent experiments. *: p<0.05 versus the group treated only with sRANKL.</p

Effects of FR167653 on osteoclast formation in M-CSF-dependent bone marrow macrophages treated with sRANKL.

<p>A. Osteoclast formation assay using M-CSF-dependent bone marrow macrophages (MDBMMs) prepared as described in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0023199#s2" target="_blank">Materials and Methods</a> section. TRAP-positive MNCs with three or more nuclei were counted as osteoclasts. n = 6. *: p<0.001 versus the group treated only with sRANKL. B. TRAP staining for MDBMMs stimulated by sRANKL (100 ng/ml). a: No treatment. b: FR167653 (0.1 µM). c: FR167653 (1 µM). d: FR167653 (10 µM).</p

The effects of FR167653 on osteoclast number in calvaria from PTHrP-treated animals.

<p>A. Osteoclast number per unit bone area (OcN-BTA) was measured histologically as described in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0023199#s2" target="_blank">Materials and Methods</a> section. Treatment of FR167653 reduces the number of osteoclasts (*: P<0.005). B. TRAP staining and counterstain with hematoxylin on calvaria sections. a: Histological section of animals treated with DW. b: Histological section of animals treated with FR167653. Osteoclasts are indicated by arrowheads.</p

The effect of FR167653 on whole-blood ionized calcium levels in PTHrP-treated mice.

<p>PTHrP induces a significant elevation of whole-blood ionized calcium levels in mice within three hours of injection with PTHrP (at day three and five). The whole-blood ionized calcium levels are lower in the FR167653-treated group than in the DW-treated group (control group) at day three (*: P<0.05). By day five, however, there was no significant difference between two groups.</p