Asiatic Acid Attenuates Osteoporotic Bone Loss in Ovariectomized Mice Through Inhibiting NF-kappaB/MAPK/ Protein Kinase B Signaling Pathway.

Osteoporosis is a condition associated with osteolytic bone disease that is primarily characterized by inordinate osteoclast activation. Protein kinase B (Akt) pathways activated by receptor activator of nuclear factor kappa-B ligand (RANKL) are essential for osteoclastogenesis. Asiatic acid (AA) is a natural pentacyclic triterpenoid compound extracted from a traditional Chinese herb that exhibits a wide range of biological activities. AA has been found to alleviate the hypertrophic and fibrotic phenotype of chondrocytes via the Akt signaling pathway. In this study, we investigated whether AA alleviated bone loss by inhibiting the Akt signaling pathway during osteoclastogenesis and its effect on osteoblasts. The effect of AA cytotoxicity on mouse bone marrow-derived macrophages/monocytes (BMMs) was evaluated in vitro using a Cell Counting Kit-8 assay. The effects of AA on osteoclast differentiation and function were detected using tartrate-resistant acid phosphatase (TRAP) staining and a pit formation assay. A Western blot and qRT-PCR were conducted to evaluate the expression of osteoclast-specific genes and protein signaling molecules. In addition, alkaline phosphatase and alizarin red staining were performed to assess osteoblast differentiation and mineralization. The bone protective effect of AA was investigated in vivo using ovariectomized mice. we found that AA could dose-dependently inhibit RANKL-induced osteoclastogenesis. Moreover, the pit formation assay revealed that osteoclast function was suppressed by treatment with AA. Moreover, the expression of osteoclast-specific genes was found to be substantially decreased during osteoclastogenesis. Analysis of the molecular mechanisms showed that AA could inhibit NF-kappaB/MAPK/Akt signaling pathway, as well as the downstream factors of NFATc1 in the osteoclast signaling pathway activated by RANKL. However, AA did not significantly promote osteoblast differentiation and mineralization. The in vivo experiments suggested that AA could alleviate ovariectomy-induced bone loss in ovariectomized mice. Our results demonstrate that AA can inhibit osteoclastogenesis and prevent ovariectomy-induced bone loss by inhibiting the NF-kappaB/MAPK/Akt signaling pathway. The discovery of the new molecular mechanism that AA inhibits osteoclastogenesis provides essential evidence to support the use of AA as a potential drug for the treatment of osteoclast-related diseases.

Spleen tyrosine kinase (SYK) inhibitor PRT062607 protects against ovariectomy-induced bone loss and breast cancer-induced bone destruction.

Osteolytic diseases, including breast cancer-induced osteolysis and postmenopausal osteoporosis, are attributed to excessive bone resorption by osteoclasts. Spleen tyrosine kinase (SYK) is involved in osteoclastogenesis and bone resorption, whose role in breast cancer though remains controversial. Effects of PRT062607 (PRT), a highly specific inhibitor of SYK, on the osteoclast and breast cancer functionalities are yet to be clarified. This study demonstrated the in vitro inhibitory actions of PRT on the osteoclast-specific gene expression, bone resorption, and osteoclastogenesis caused by receptor activator of nuclear factor kappa B ligand (RANKL), as well as its in vitro suppressive effects on the growth, migration and invasion of breast carcinoma cell line MDA-MB-231, which were achieved through PLCγ2 and PI3K-AKT-mTOR pathways. Further, we proved that PRT could prevent post-ovariectomy (OVX) loss of bone and breast cancer-induced bone destruction in vivo, which agreed with the in vitro outcomes. In conclusion, our findings suggest the potential value of PRT in managing osteolytic diseases mediated by osteoclasts.

An Experimental Study on the Safety and Mechanism of Reduction of Subaxial Cervical Facet Dislocation Using Z-Shape Elevating-Pulling Reduction Technique.

OBJECTIVE: We sought to clarify the safety and unlocking mechanism of the Z-shape elevating-pulling closed reduction (ZR) technique and to analyze the differences in facet contact force and intraspinal pressure during subaxial facet dislocation reduction using the ZR technique and traditional skull traction closed reduction (SR). METHODS: In 15 human cadaveric skull-neck-thorax specimens, reproducible unilateral and bilateral facet dislocations (UFDs/BFDs) were created at the C5-C6 level and then reduced by applying the ZR and SR techniques, respectively. Tekscan FlexiForce A-201 pressure sensors were used to measure the anterior and posterior intraspinal pressure and injured facet contact force under physiological conditions and before and after reduction. The maximum pressures during the reduction process were recorded. RESULTS: After creation of the facet dislocation, the anterior and posterior intraspinal pressure and facet contact force were significantly increased relative to normal (P < 0.001). The UFDs and BFDs of all specimens were successfully reduced by both ZR and SR, and the intraspinal pressure and facet contact force were significantly reduced compared with before reduction (P < 0.001). Compared with SR, the maximum posterior intraspinal pressure during BFD reduction (P = 0.027) and the maximum facet contact force during UFD reduction (P < 0.001) were lower when ZR was used for closed reduction. CONCLUSIONS: Our findings suggest that ZR and SR can both be used to reduce subaxial facet dislocation and decompress the spinal cord. However, the ZR technique appears to safer and more effective than the SR technique for closed reduction of subaxial facet dislocations.

Compound phenotype of osteogenesis imperfecta and Ehlers-Danlos syndrome caused by combined mutations in COL1A1 and COL5A1.

Osteogenesis imperfecta (OI) is an inherited connective tissue disorder with a broad clinical spectrum that can overlap with Ehlers-Danlos syndrome (EDS). To date, patients with both OI and EDS have rarely been reported. In the present study, we investigated a family with four members, one healthy individual, one displaying OI only, and two displaying the compound phenotype of OI and EDS, and identified the pathogenic mutations. Whole exome sequencing was applied to the proband and her brother. To verify that the mutations were responsible for the pathogenesis, conventional Sanger sequencing was performed for all members of the family. We identified a known COL1A1 (encoding collagen type I α 1 chain) mutation (c.2010delT, p.Gly671Alafs*95) in all three patients (the proband, her brother, and her mother) in this family, but also a novel heterozygous COL5A1 (encoding collagen type V α 1 chain) mutation (c.5335A>G, p.N1779D) in the region encoding the C-terminal propeptide domain in the proband and her mother, who both had the compound phenotype of OI and EDS. The results of the present study suggested that the proband and her mother presented with the compound OI-EDS phenotype caused by pathogenic mutations in COL5A1 and COL1A1.

In Vitro Biomechanical Study of Epidural Pressure during the Z-shape Elevating-Pulling Reduction Technique for Cervical Unilateral Locked Facets.

Objective: To analyze the mechanism of the halo vest-assisted Z-shape elevating-pulling reduction technique for cervical unilateral locked facets, and confirm the safety of the spinal cord under the epidural pressure that occurs during the reduction process. Methods: Eleven osteoligamentous whole coronal and cervical spine specimens were established as skull-neck-thorax models of cervical unilateral locked facets at the C5/6 level. The halo vest-assisted Z-shape elevating-pulling reduction technique was then applied to reduce the locked facets. The changes in the epidural pressure in five cervical positions (cervical physiological curvature, cervical lateral bending, cervical unilateral locked facets, cervical unilateral perched facets, and reduction) were measured by a pressure sensor during the reduction procedure. The models simultaneously underwent multi-angle radiographic examination and CT scanning. Results: Successful closed reduction was achieved via the halo vest-assisted Z-shape elevating-pulling reduction technique in all 11 models. The epidural pressure in the cervical unilateral locked facets position was significantly higher than that in the other four cervical positions (P < 0.005). There was no significant difference in the epidural pressures measured during cervical lateral bending, cervical unilateral perched facets, and reduction. Conclusions: Maximum epidural pressures were measured in the position of cervical unilateral locked facets. The halo vest-assisted Z-shape elevating-pulling reduction technique achieved spinal decompression without causing secondary spinal cord injury. The halo vest-assisted Z-shape elevating-pulling reduction technique is safe and effective, and has a high success rate of reduction.

Multicentre comparative study of Z-shape elevating-pulling reduction and skull traction reduction for treatment of lower cervical locked facets.

PURPOSE: The aim of this study was to assess the clinical efficacy and safety of Z-shape elevating-pulling reduction as compared to that of conventional skull traction in the treatment of lower cervical locked facet. METHODS: Patients with cervical locked facet (n = 63) were retrospectively enrolled from four medical centers and divided into two groups according to the pre-operative reduction method used: Z-shape elevating-pulling reduction (Z-shape elevating group; n = 20) or traditional skull traction reduction (skull traction group; n = 43). RESULTS: The success rates, efficacy of reduction, and safety were compared between the two groups. The success rates were significantly better in the Z-shape elevating group than in the skull traction group: 87.5% (7/8) vs. 35.3% (6/17) for unilateral locked facet reduction (P = 0.03) and 100% (12/12) vs. 69.2% (18/26) for bilateral locked facet reduction (P = 0.04). There was no obvious change in American Spinal Injury Association (ASIA) grade after the reduction in either group. Combined surgery was necessary in 5% in the Z-shape elevating group vs. 27.9% in the skull traction group. Imaging showed that the segment angle and horizontal displacement were significantly improved after surgery in both groups, with no significant difference between the groups. Follow-up with radiography showed good recovery of the cervical spine sequence; all internal fixation sites were stable, with no loosening, prolapse, or breakage of internal fixators. CONCLUSIONS: Halo vest-assisted Z-shape elevating-pulling reduction appears to be a simple, safe, and effective technique for pre-operative reduction of lower cervical locked facets.

New reduction technique for the treatment of unilateral locked facet joints of the lower cervical spine : A retrospective analysis of 12 cases.

BACKGROUND: Closed reduction of bilateral locked facet joints of the lower cervical spine is possible, but reduction of unilateral locked facet joints of the lower cervical spine (ULFJLCS) is challenging. We explored a new, simple, safe, and effective closed reduction method for the treatment of ULFJLCS. METHODS: A retrospective analysis was done on 12 consecutive cases with traumatic ULFJLCS that underwent closed reduction by Z­shape elevating-pulling reduction through a halo-vest. After reduction, only anterior cervical decompression and internal fixation were performed. The success of reduction and nerve function was assessed, and follow-up data analyzed. RESULTS: All patients using our new reduction technique underwent successful closed reduction; the shortest time of reduction was 40 min and the longest 110 (mean, 65) min. No aggravation of neurological damage was observed, nor were other complications. All patients were followed-up from 28 to 72 (mean, 44) months after surgery. The improvement in Frankel's score (on average) was two levels in most patients. CONCLUSION: These data demonstrate that our new reduction technique is a simple, safe, and effective treatment for ULFJLCS.

Effect of modified compound calcium phosphate cement on the differentiation and osteogenesis of bone mesenchymal stem cells.

BACKGROUND: The aim of this study is to evaluate the effect of self-invented compound calcium phosphate cement upon the proliferation and osteogenesis of bone mesenchymal stem cells (BMSCs). METHODS: Four groups including traditional calcium phosphate cement, modified calcium phosphate cement, modified calcium phosphate cement plus bone morphogenetic protein (BMP), and control groups were established. The cell proliferation curve was delineated by MTT. The activity of BMSCs to synthesize alkaline phosphatase (AKP) was evaluated. The growth and invasion of BMSCs were observed. The expression levels of aggrecan, collagen I, collagen II, AKP, and OSX messenger RNA (mRNA) were measured by using RT-PCR. RESULTS: Compared with other groups, the BMSCs in the modified calcium phosphate cement group presented with loose microstructure and the BMSCs closely attached to the vector margin. At 7 days after co-culture, the expression of AKP in the modified calcium phosphate cement plus BMP group was significantly upregulated compared with those in other groups. In the modified calcium phosphate cement group, the BMSCs properly proliferated on the surface of bone cement and invaded into the cement space. At 10 days, the expression levels of aggrecan, collagen I, collagen II, AKP, and OSX mRNA in the modified calcium phosphate cement and modified calcium phosphate cement plus BMP groups were significantly upregulated than those in other groups. CONCLUSIONS: Modified compound calcium phosphate cement possesses excellent biocompatibility and osteogenic induction ability. Loose microstructure and large pore size create a favorable environment for BMSCs proliferation and vascular invasion, as an ideal vector for releasing BMP cytokines to mediate the differentiation and osteogenesis of BMSCs.

Hepatoma-derived growth factor-2 is highly expressed during development and in spinal cord injury.

Hepatoma­derived growth factor­2 (HDGF­2) is expressed in neurons, astrocytes and oligodendrocytes of the adult mouse brain. However, it has remained elusive whether HDGF­2 is expressed in the spinal cord and is involved in the its development and repair. In the present study, the expression of HDGF­2 was investigated in rat spinal cords at different developmental stages and following spinal cord injury (SCI). Protein levels of HDGF­2 were examined using western blot analysis, while the distribution pattern and cell populations of HDGF­2 protein expression were characterized using immunohistochemistry. Western blot analysis demonstrated that the levels of HDGF­2 protein expression were the greatest in the spinal cord on embryonic day 19, and were also highly expressed in rat spinal cords on post­natal day 7 (P7); however, they were low at P14 and not detectable at two months. HDGF­2 expression was significantly upregulated in the embryonic spinal cord and injured spinal cord. By contrast, the expression of HDGF­2 was low in uninjured adult spinal cords. HDGF­2 expression in the fetal rat spinal cord and injured spinal cord was significantly higher than that in uninjured adult spinal cord tissues (P<0.05). The number of cells positive for HDGF­2 was 141±62, 107±33 and 92±18 at days 1, 21 and 45 following SCI, respectively, as opposed to 50±9 in uninjured rats, and a significant difference was identified between the different time­points following SCI (P<0.01). In conclusion, the overexpression of HDGF­2 in the embryonic spinal cord and injured spinal cord may be involved in fetal spinal cord development and repair of SCI, respectively.

Reformatted computed tomographic evaluation of the thoracic pedicle in a Chinese population for the surgical application of transpedicular screw placement.

PURPOSE: To study the transverse thoracic pedicle diameter of a Chinese population and to determine the feasibility and safety of transpedicular screw fixation. METHODS: The authors studied the transverse pedicle diameter of the T1-T12 of the thoracic spine in a Chinese population using reformatted computed tomography. The data were compared with Caucasians and other Asians. RESULTS: The mean outer pedicle widths of the thoracic spine from T1 to T12 were 8.43, 6.65, 5.20, 4.44, 4.50, 4.87, 5.04, 5.32, 5.66, 6.65, 8.08 and 8.27 mm in males and 7.91, 6.03, 4.55, 3.91, 4.05, 4.31, 4.39, 4.60, 5.13, 5.67, 7.21 and 7.50 mm in females, respectively. Female patients have smaller dimensions compared with male patients. A significant percentage of patients have an outer pedicle width of less than 4.5 mm from T3 to T8, which is not suitable for transpedicular screw fixation with a 3.5 mm screw. CONCLUSIONS: The results of this study suggest that transpedicular screw fixation may not be suitable for the mid-thoracic regions in most Chinese females and that modified pedicle screw techniques or modified type of fixation is required in these patients. CT evaluation is a must before this procedure is performed.