Effect of rat brain tissue extracts on osteoblast proliferation and differentiation.

PURPOSE: The reason for enhanced fracture healing in traumatic brain injury patients is not clearly understood. It is possible that factors inherent in the brain passing through the blood-brain barrier to the peripheral circulation, or a disruption of central nervous system (CNS) control of the sympathetic nervous system (SNS), stimulates the process of fracture healing. METHODS: In this study, we assessed proliferation [using the 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) assay] and differentiation [using alkaline phosphatase (ALP)] in rat osteoblasts incubated with gray matter or other tissue extracts with and without the addition of an α- or ß-adrenergic receptor blocker (phentolamine or propranolol). RESULTS: Gray matter extract from normal brain caused a dose-dependent increase in osteoblast proliferation and differentiation. Serum from normal rats enhanced differentiation but not proliferation. Alpha-receptor blockade had no effect on proliferation or differentiation. Beta-receptor blockade caused a partial, but statistically significant, decrease in gray matter stimulation of osteoblast differentiation. CONCLUSION: The results of this study indicate that gray matter extract from normal brain increases osteoblast proliferation and differentiation and that ß receptors may be involved in differentiation under these conditions.

Somatosensory evoked potential from S1 nerve root stimulation.

The objective of this study was to detect cerebral potentials elicited by proximal stimulation of the first sacral (S1) nerve root at the S1 dorsal foramen and to investigate latency and amplitude of the first cerebral potential. Tibial nerve SEP and S1 nerve root SEP were obtained from 20 healthy subjects and 5 patients with unilateral sciatic nerve or tibial nerve injury. Stimulation of the S1 nerve root was performed by a needle electrode via the S1 dorsal foramen. Cerebral potentials were recorded twice to document reproducibility. Latencies and amplitudes of the first cerebral potentials were recorded. Reproducible cerebral evoked potentials were recorded and P20s were identified in 36 of 40 limbs in the healthy subjects. The mean latency of P20 was 19.8 ± 1.6 ms. The mean amplitude of P20-N30 was 1.2 ± 0.9 µV. In the five patients, P40 of tibial nerve SEP was absent, while well-defined cerebral potentials of S1 nerve root SEP were recorded and P20 was identified from the involved side. This method may be useful in detecting S1 nerve root lesion and other disorders affecting the proximal portions of somatosensory pathway. Combined with tibial nerve SEP, it may provide useful information for diagnosis of lesions affecting the peripheral nerve versus the central portion of somatosensory pathway.

[Comparison of 3D C-arm fluoroscopy-based and CT-based navigation systems in the lumbar pedicle puncture: in vitro experiment on a cadaveric lumbar trunk specimen].

OBJECTIVE: To compare the accuracy and operating features of 3D C-arm fluoroscopy-based and CT-based navigation systems in the lumbar pedicle punctures. METHODS: A specimen of cadaveric lumbar trunk underwent lumbar pedicle punctures at the levels of L3, L4, and L5 under the guidance of the 3D C-arm fluoroscopy-based and CT-based navigation systems. During the procedure C-arm fluoroscopy was used to monitor the accuracy of the puncture. Generally, in comparison with the 3D C-arm fluoroscopy-based navigation system, the best operation route and protocol could be drawn up pre-operatively, matched registration needed to be renewed for each vertebra, and the images thus obtained were of high quality in CT-based navigation. RESULTS: Both navigation systems had excellent accuracy in the guidance of lumbar pedicle punctures, and had different operating features. CONCLUSION: Both navigation systems had its special advantages. The operating process of the 3D C-arm fluoroscopy-based navigation system was more convenient and rapid, and suitable for percutaneous vertebral puncture. CT based navigation system had clearer pictures, especially for the osteoporotic vertebral bodies, and it had less requirements for the equipments.

[Effect of EPHA2-siRNA plasmid on biological behavior of human osteosarcoma cells in vitro].

OBJECTIVE: To investigate the role of EPHA2 in regulating apoptosis, proliferation and vasculogenic mimicry of osteosarcoma cells, by gene silencing through RNA interference. METHODS: EPHA2-siRNA plasmids were achieved by gene cloning. The plasmids were transfected into human osteosarcoma cells (MG63). The expression level of EPHA2 protein was measured by Western blotting. The proliferation, apoptosis and vasculogenic mimicry features of osteosarcoma MG63cells were assessed by light microscopy, MTIP assay, flow cytometry, annexin V-FITC/PI and HE staining, respectively. RESULTS: The EPHA2-siRNA plasmid was confirmed by DNA sequencing. After treatment with Sequence-specific siRNA targeted EPHA2, the protein level of the transfected group decreased significantly. As compared to non-siRNA transfected cells, the transfected group showed lower proliferation, higher and earlier apoptosis and less osteosarcoma-generated vasculogenic mimicry. CONCLUSION: EPHA2 gene may be involoved in apoptosis and proliferation of osteosarcoma cells, and may be necessary for vasculogenic mimicry. Down-regulation of EPHA2 expression by sequence-specific siRNA may be considered as a new option in the treatment of EPHA2 over-expressing cancer including osteosarcoma in future.

[Transduction of recombinant marrow stem cells using lentiviral vector and adenoviral vector: a comparative study].

OBJECTIVE: To compare the efficiency of transfection of marrow stem cells (MSCs) by using lentiviral vector and adenoviral vector. METHODS: MSCs were obtained from the femur bone marrow of rat and cultured. Recombinant plasmid pHIV-CS-CG-PRE containing green fluorescent protein (GFP) gene, and package plasmids pRSV-Rev, pMDLg/pPRE, and pMD.G were infected into the human embryonic kidney cells of the line 293T. The rat MSCs were transfected with the DFP recombinant lentiviral particles, and flow cytometry was used to detect the expression of GFP 1, 3, and 5 weeks later. Full-length human bone morphogenetic protein = 2 (hBMP2) gene was searched out from the GenBank and isolated from human liver cDNA from a sample of liver tissue resected from a patient with liver rupture and then cloned. Thus the lentiviral vector with BMP-2 gene, pHIV-CS-CDF-CG-PRE-BMP-2 was constructed and transfected into 293 cells. Indirect immunofluorescence assay, ELISA, and Western blotting were used to detect the expression of hBMP2 gene. RESULTS: After infected by the viral vectors the GFP expression was significantly better in the lentiviral vector-infected rMSCs than in the adenoviral vector-infected ones. FCM showed that the GFP expression rates of the lentiviral vector-infected rMSCs 1, 3, and 5 weeks after the infection were 90%, 83.2%, and 79% respectively, and he GFP expression rates of the adenoviral vector-infected rMSCs 1, 3, and 5 weeks after the infection were 71%, 0.13%, and 0.05% respectively, all significantly lower than on the former group. Indirect immunofluorescence assay, ELISA, and Western blotting showed that the recombinant lentivirus successfully expressed the target protein in the transfected 293T cells. CONCLUSION: Lentiviral vector with hBMP2 gene can be constructed successfully. The transfection efficiency of BMP2 gene by lentivirus is significantly higher than that of adenovirus.

[The relation and evaluation of the vertebral artery and the nerve root to posterior lateral mass screw fixation].

OBJECTIVE: To determine the location of the vertebral artery foramens from C(3) to C(6) and their relationship to the point 1 mm medial to the center of the lateral mass and to identify the value of oblique radiograph for cervical lateral mass screw trajectory by a cadaveric study. METHODS: (1) Twenty-eight cervical specimens (C(3)-C(7)) of human cadavers aged from 28 to 79 years were analysed. The transverse radiographs of C(3)-C(6) vertebrae were taken and the angle between the parasagittal plane and the line connecting the point of the lateral mass with the lateral limit of the transverse process foramen of C(3)-C(6) were measured. (2) The K-wires were drilled into lateral mass of C(3)-C(6) starting 1 mm medial to the center of the lateral mass and exiting by the juncture between the transverse process and the facet in ten specimens. Four wire placements under direct visualization, including placement of the wire tip staying the ventral cortex and 2, 4, 6 mm over-penetration of the ventral cortex of lateral mass, were performed separately on each specimen. After each placement, radiographs were taken on 45 degrees oblique left and 45 degrees oblique right views. Each intervertebral foramen on the oblique radiographs was divided into two parts: superior and inferior parts. The former is the true intervertebral foramen, while the latter is the intertransverse foramen on the gross specimen. The number of wire tips in each part was quantified for each placement. All results on the radiographs were compared with those on the gross anatomy. RESULTS: (1) The angles between the parasagittal plane and the line connecting the posterior starting point of the lateral mass with the lateral limit of the transverse foramen (C(3)-C(6)) were lateral to the sagittal plane, ranging from 5 degrees to 12 degrees. Among the vertebrae, there were no statistically significant difference (P > 0.05). (2) 15% of the wires without over-penetration and 41.3% with 2 mm over-penetration were found in the inferior parts of the intervertebral foramen in oblique views, while the wires were not noted in the intervertebral foramen by gross anatomy. with 4 mm over-penetration of the ventral cortex, 35% and 65% of wires were noted in the superior and inferior parts of the intervertebral foramen respectively, while only 28.8% of wires were found in the inferior part approximating the nerve roots in gross specimens. With 6 mm over-penetration, the number in the intervertebral foramen were 63.8% superiorly and 36.2% inferiorly on the oblique radiographs while all the tips were at the inferior part (intertransverse foramens) in gross specimens. The tip of wire crossed the line connecting the posterior borders of the intervertebral foramens in oblique radiographs when it penetrated the ventral cortex of lateral mass 4 mm or more. CONCLUSIONS: (1) There is no risk of damaging the vertebral artery if a screw is directed more than 15 degrees lateral to the sagittal plane at C(3 approximately 6) starting 1 mm medial to the center of the lateral mass. (2) Ideal screw tip position on oblique radiograph may not cross the line connecting the posterior borders of the intervertebral foramen on radiograph. If the screw tip is noted in the superior part of intervertebral foramen on the oblique radiograph, the screw may be identified as dangerous.