Objective To explore the osteogenic potential of cervical intervertebral disc fibroblasts in vitro, to investigate the regulatory factors of recombinant human bone morphogenetic protein 2(rhBMP-2) and tumor necrosis factor α(TNF-α) on osteogenic phenotype of fibroblasts and to discuss the condition that facilitates osteogenesis of fibroblasts. Methods Theannulus fibroblasts cell lines of experiment goats were established in vitro and the biologicspecificity was found. According to different medias, 4 groups were included in this experiment: control group, TNF-α group ( 50 U/ml TNF-α), rhBMP-2 group (0.1 μg/ml rhBMP-2) and TNF-α+rhBMP-2 group (50 U/ml TNF-α+0.1 μg/ml rhBMP-2). Thefibroblasts were incubated in the media for about 3 weeks,and then the markers for osteogenic features were investigated by biochemistry, histochemistry observations. Results rhBMP-2 and TNF-α had no effect on the proliferation of fibroblasts from the experiment goats. rhBMP-2 or TNF-α could stimulate fibroblasts to secrete alkaline phosphatase and collagen type Ⅰ. The combined use of rhBMP-2 and TNF-α or the single use of rhBMP-2 could make fibroblasts to secrete osteocalin and the morphological changes of the fibroblasts were very obvious. Histochemical study of the nodules with specific new bone labeler(Alizarin red S) revealed positive reaction, denoting that the nodules produced by the fibroblasts werebone tissues. There was statistically significant difference(Plt;0.05) inALP activity between 3 experimental groups and control group and in secretion of osteocalcin between rhBMP-2 group, TNF-α+rhBMP-2 group and control group. Conclusion The results point out clearly that rhBMP-2 can induce theosteogenic potential of annulus fibroblasts in vitro.
Objective To detect the cell density, apoptotic rate, and the expressions of BNIP3 in nucleus pulposus of degenerative intervertebral disc of rabbits, so as to further understand the mechanism of intervertebral disc degeneration. Methods Thirty male New Zealand white rabbits, aging 3 months and weighing (2.3 ± 0.2) kg, were divided into sham operation group (control group, n=10) and intervertebral disc degeneration model group (experimental group, n=20). Interbertebral disc degeneration models were establ ished by puncture of L3,4, L4,5, and L5,6 intervertebral discs in the experimental group; intervertebral discs were exposed only and then sutured in the control group. The degree of intervertebral disc degeneration was evaluated according to Pfirrmann classification by MRI at 4 and 8 weeks after establ ishing models. Apototic cells were determined by TUNEL and histological methods, and the immunohistochemical staining was performed to detect the expressions of BNIP3 in nucleus pulposus of intervertebral disc. Results MRI examination showed that the signal intensity decreased gradually at 4 and 8 weeks in the experimental group. There wassignificant difference in the degree of intervertebral disc degeneration between at 4 weeks and at 8 weeks in the experimental group (P lt; 0.05). The histological observation and TUNEL test showed that high density of nucleus pulposus cells and only a few apoptotic cells were observed in the control group; at 4 and 8 weeks, the density of nucleus pulposus cells decreased gradually with more apoptotic cells in the experimental group. There were significant differences in the nucleus pulposus cell density and positive rate of TUNEL staining between 2 groups, and between at 4 weeks and at 8 weeks in the experimental group (P lt; 0.05). The expression of BNIP3 of nucleus pulposus was negative in the control group; however, in the experimental group, the positive expression rates of BNIP3 of nucleus pulposus (the gray values) were 13.45% ± 1.16% and 32.00% ± 1.82% (194.32 ± 4.65 and 117.54 ± 2.11) at 4 and 8 weeks respectively, showing significant differences (P lt; 0.05). Conclusion The decrease of cell density in nucleus pulposus is involved in the development of intervertebral disc degeneration. Cell apoptosis is one of reasons in the decrease of nucleus pulposus cell; BNIP3 is involved in nucleus pulposus cell apoptosis in the degenerative intervertebral disc.
Objective
To investigate the expression and significance of growth-associated protein 43 (GAP-43) in the dorsal root ganglion (DRG) and intervertebral disc in the rat model of intervertebral disc inflammation.
Methods
A total of 103 adult male Sprague Dawley rats (weighing, 200-250 g) were randomly divided into the experimental group (n=48), the control group (n=48), and the blank control group (n=7). Fluoro-gold (F-G) as tracer was injected into the L5, 6 intervertebral disc of 3 groups; after 7 days of F-G injection, complete Freund’s adjuvant (50 μL) and the same volume of saline were injected in the experimental group (to prepare the model of intervertebral disc inflammation) and the control group, respectively, and the blank control group had no further treatment. After 1, 3, 7, and 14 days, T13-L6 DRG and L5, 6 intervertebral disc of experimental group and control group were harvested to detect the GAP-43 by using fluorescent immunohistochemistry, in situ hybridization, and RT-PCR. The DRG and intervertebral disc of blank control group were also harvested after 8 days of F-G injection.
Results
Fluorescent immunohistochemistry results showed that the number of F-G-labeled GAP-43 immunoreaction (GAP-43-IR) cells of the DRGs in the experimental group was significantly higher than that in the control group (P lt; 0.05) at 3 days, and no significant difference was found at the other time points (P gt; 0.05). There was no significant difference in the cross-sectional area of F-G-labeled GAP-43-IR cells between the experimental group and the control group at each time point (P gt; 0.05). The co-expression of GAP-43 with calcitonin gene-related peptide (CGRP) and isolectin B4 (IB4)-binding glycoprotein exhibited that the expression of CGRP was 91.4% ± 7.4% in the control group and was 87.6% ± 7.8% in the experimental group, showing no significant difference between 2 groups (P gt; 0.05). There was no IB4-binding glycoprotein expression in GAP-43-IR cells of the DRGs in 2 groups. The expressions of GAP-43, CGRP, and IB4-positive nerve fibers in the intervertebral disc exhibited that the GAP-43-IR nerve fibers in the experimental group were significantly more than that in the control group (P lt; 0.05), but no significant difference was found in the expression of CGRP between 2 groups (P gt; 0.05); and there was no IB4-binding glycoprotein expression in GAP-43-IR nerve fibers of the intervertebral disc in 2 group. In situ hybridization and RT-PCR detection showed that the positive expression cells ratio of GAP-43 mRNA and the level of GAP- 43 mRNA were significantly higher in the experimental group than in the control group at 1 day (P lt; 0.05), and no significant difference was found at the other time points (P gt; 0.05).
Conclusion
Intradiscal inflammatory environment may induce the expression of GAP-43, and potentially promote the nerve fiber ingrowth of rat.
Objective To develop a high-accuracy, better-safety and low-cost cervical pedicle locator system for guiding cervical pedicle screw placement. Methods Cervical pedicle screw locator system was made of stainless steel. Ten cervical specimens from voluntary donation were divided into two groups according to compatibil ity design: control group inwhich 60 screws were planted into C2-7 by free hand; and experimental group in which 60 screws were planted into C2-7 under the guidance of three-dimensional locator system. The condition of screw insertion was observed and the accuracy was evaluated by the integrity of pedicle walls. Results In the control group, 32 screws (53.33%) were placed inside the pedicles and 28 (46.67%) were outside; 9 screws (15.00%) led to nerve root injury, 5 screws (8.33%) caused vertebral artery injury and no spinal cord injury occurred; and the qual ification ratio of screw insertion was 76.67% (excellent 32, fair 14, poor 14). While in the experimental group, 54 screws (90.00%) were placed inside the pedicles and 6 (10.00%) were outside; 1 screw (1.67%) caused vertebral artery injury and no nerve root injury and spinal cord injury occurred; and the qual ification ratio of screw insertion was 98.33% (excellent 54, fair 5, poor 1). There was significant difference between the two groups (P lt; 0.05). Conclusion Cervical pedicle screw locator system has the advantages of easy manipulation, high accuracy of screw placement and low cost. With further study, it can be appl ied to the cl inical.
ObjectiveTo evaluate the feasibility and short-term effectiveness of bilateral percutaneous balloon kyphoplasty through unilateral transverse process-extrapedicular approach for osteoporotic vertebral compression fracture (OVCF) of lumbar.MethodsA retrospective analysis was made on the clinical data of 93 patients with OVCF of lumbar who met the selection criteria between January 2018 and June 2019. According to the different surgical methods, they were divided into group A (44 cases, treated with bilateral percutaneous balloon kyphoplasty through unilateral transverse process-extrapedicular approach) and group B [49 cases, treated with percutaneous kyphoplasty (PKP) via bilateral transpedicle approach]. There was no significant difference in gender, age, body mass index, T value of bone mineral density, injury cause, fractured level, time from injury to operation, comorbidities, and preoperative Cobb angle of injured vertebra, visual analogue scale (VAS) score, and Oswestry disability index (ODI) between the two groups (P>0.05). The operation time, intraoperative fluoroscopy times, bone cement injection amount, and incidence of bone cement leakage were recorded and compared between the two groups; Cobb angle of the injured vertebrae, VAS score, and ODI were measured before operation, at 2 days and 1 year after operation. The contralateral distribution ratio of bone cement was calculated according to the anteroposterior X-ray film at 2 days after operation.ResultsThe operation time and the intraoperative fluoroscopy times in group A were significantly less than those in group B (P<0.05). There was no bone cement adverse reactions, cardiac and cerebrovascular adverse events, and no complications such as puncture needles erroneously inserted into the spinal canal and nerve injuries occurred in the two groups. Bone cement leakage occurred in 6 cases and 8 cases in groups A and B, respectively, all of which were asymptomatic paravertebral or intervertebral leakage, and no intraspinal leakage occurred; the bone cement injection amount and incidence of bone cement leakage between the two groups showed no significant differences (P>0.05). The contralateral distribution ratio of bone cement in group A was significantly lower than that in group B (t=2.685, P=0.009). Patients in both groups were followed up 12-20 months, with an average of 15.3 months. The Cobb angle of the injured vertebrae, VAS score, and ODI in the two groups were significantly improved at 2 days after operation, however, the Cobb angle of the injured vertebra at 1 year after operation was significantly lost when compared with the 2 days after operation, the VAS score and ODI at 1 year after operation were significantly further improved when compared with the 2 days after operation, the differences were all significant (P<0.05). There was no significant difference in the Cobb angle of the injured vertebrae, VAS score, and ODI between the two groups at each time point after operation (P>0.05).ConclusionBilateral percutaneous balloon kyphoplasty through unilateral transverse process-extrapedicular approach is comparable to bilateral PKP in short-term effectiveness with regard to fracture reduction, reduction maintenance, pain relief, and functional improvement. It has great advantages in reducing operation time and radiation exposure, although it is inferior in bone cement distribution.
Objective To explore the occurrence condition of the neck axial symptom (AS) after cervical Bryan artificial disc replacement combined with anterior cervical discectomy and fusion (Hybrid surgery) and traditional anterior cervical discectomy and fusion (ACDF surgery) to treat the two-level cervical disease, and to do contrastive analysis. Methods Between August 2006 and March 2010, 18 patients underwent Hybrid surgery (group A) and 30 patients underwent two-level ACDF surgery (group B). There was no significant difference in age, gender, disease duration, type, and operated segment between 2 groups (P gt; 0.05). The Japanese Orthopaedic Association (JOA) score, neck disability index (NDI) score, cervical curvature of the operated segment, total range of motion (ROM) of C2-7, ROM of the adjacent segment, and incidence of neck AS were recorded and compared between before operation and at last follow-up. Results All the patients were followed up 18-34 months (24.1 months on average). In both groups, the JOA and NDI scores at last follow-up had significantly improvement when compared with preoperative scores (P lt; 0.01), but there was no significant difference between 2 groups at preoperation and last follow-up (P gt; 0.05). The kyphosis incidence of the operated segment in group B was significantly higher than that in group A (χ2=5.333, P=0.021). There was no significant difference in the total ROM of C2-7 between at preoperation and last follow-up in group A (t=0.410, P=0.685); the total ROM of C2-7 at last follow-up was significantly lower than that at preoperation in group B (t=3.007, P=0.006); and significant difference was found between 2 groups at last follow-up (t=2.664, P=0.013). At last follow-up, ROM of the superior and inferior adjacent segments in group B increased obviously (P lt; 0.05) and was significantly higher than that in group A (t=2.252, P=0.033; t=2.203, P=0.037). The incidence of neck AS were 16.7% in group A and 46.7% in group B, showing significant difference at last follow-up (χ2=4.427, P=0.035). Conclusion Compared with two-level ACDF surgery, Hybrid surgery has good outcomes. At the same time, it can maintain the curvature of operated segments and total ROM, avoid excessive increased ROM of the adjacent segments, and reduce the incidence of neck AS.
Objective To introduce the latest advances of research on repair of the degenerative intervertebral disc with gene transduction.Methods The recentlypublished articles about the treatment of degenerative disc with gene transduction were reviewed, especially the articles published during the recent 5 years about the application of this therapy to regulating the synthesisand degradation of the extracellular matrix of the degenerative intervertebral disc.Results The shape and function of the normal intervertebral disc were reported to be closely related to the synthesis and degradation of the extracellular matrix of the intervertebral disc. The extracellular matrix of the intervertebral disc was a target for the gene transduction to repair the degenerative intervertebral disc. There was a great development of the treatment with gene transduction, especially in vector choice, target gene transduction, and transgene regulation and safety. Conclusion The advances of the research have indicated that repair of the degenerative intervertebral disc with gene transduction is a keyto curing the disease of the degenerative intervertebral disc.
Objective
To evaluate the influence of PKH26 labeling on the biological function of the goat nucleus pulposus cells and the biological function of seeded cells in nude mice by in vivo imaging techonology.
Methods
Primary nucleus pulposus cells were isolated by enzymatic digestion from the nucleus pulposus tissue of the 1-year-old goat disc. The nucleus pulposus cells at passage 1 were labeled with PKH26 and the fluorescent intensity was observed under the fluorescence microscopy. The labeled cells were stained with toluidine blue and collagen type II immunocytochemistry. The cells viability and proliferation characteristics were assessed by trypan blue staining and MTT assay, respectively. Real-time fluorescent quantitative PCR was used to detect the gene expressions of collagen types I and II, and aggrecan. The fluorescent intensity and scope of the nucleus pulposus cells-scaffold composite in vivo for 6 weeks after implanting into 5 6-week-old male nude mice were measured by in vivo imaging technology.
Results
Primary nucleus pulposus cells were ovoid in cell shape, showing cluster growth, and the cells at passage 1 showed chondrocyte-like morphology under the inverted phase contrast microscope. The results of toluidine blue and collagen type II immunocytochemistry staining for nucleus pulposus cells at passage 1 were positive. The fluorescent intensity was even after labeling, and the cell viability was more than 95% before and after PKH26 labeling. There was no significant difference in cell growth curve between before and after labeling (P gt; 0.05). The real-time fluorescent quantitative PCR showed that there was no significant difference in gene expressions of collagen types I and II, and aggrecan between before and after labeling (P gt; 0.05). Strong fluorescence in nucleus pulposus cells-scaffold composite was detected and by in vivo imaging technology.
Conclusion
The PKH26 labeling has no effect on the activity, proliferation, and cell phenotype gene expression of the nucleus pulposus cells. A combination of PKH26 labeling and in vivo imaging technology can track the biological behavior of the cells in vivo.
The lumbar intervertebral disc exhibits a complex physiological structure with interactions between various segments, and its components are extremely complex. The material properties of different components in the lumbar intervertebral disc, especially the water content (undergoing dynamic change as influenced by age, degeneration, mechanical loading, and proteoglycan content) - critically determine its mechanical properties. When the lumbar intervertebral disc is under continuous pressure, water seeps out, and after the pressure is removed, water re-infiltrates. This dynamic fluid exchange process directly affects the mechanical properties of the lumbar intervertebral disc, while previous isotropic modeling methods have been unable to accurately reflect such solid-liquid phase behaviors. To explore the load-bearing mechanism of the lumbar intervertebral disc and establish a more realistic mechanical model of the lumbar intervertebral disc, this study developed a solid-liquid biphasic, fiber-reinforced finite element model. This model was used to simulate the four movements of the human lumbar spine in daily life, namely flexion, extension, axial rotation, and lateral bending. The fluid pressure, effective solid stress, and liquid pressure-bearing ratio of the annulus fibrosus and nucleus pulposus of different lumbar intervertebral discs were compared and analyzed under the movements. Under all the movements, the fluid pressure distribution was closer to the nucleus pulposus, while the effective solid stress distribution was more concentrated in the outer annulus fibrosus. In terms of fluid pressure, the maximum fluid pressure of the lumbar intervertebral disc during lateral bending was 1.95 MPa, significantly higher than the maximum fluid pressure under other movements. Meanwhile, the maximum effective solid stress of the lumbar intervertebral disc during flexion was 2.43 MPa, markedly higher than the maximum effective solid stress under other movements. Overall, the liquid pressure-bearing ratio under axial rotation was smaller than that under other movements. Based on the solid-liquid biphasic modeling method, this study more accurately revealed the dominant role of the liquid phase in the daily load-bearing process of the lumbar intervertebral disc and the solid-phase mechanical mechanism of the annulus fibrosus load-bearing, and more effectively predicted the solid-liquid phase co-load-bearing mechanism of the lumbar intervertebral disc in daily life.
ObjectiveTo evaluate the effectiveness of percutaneous monoplanar screw internal fixation via injured vertebrae for treatment of thoracolumbar fracture.MethodsBetween May 2015 and August 2017, 38 cases of thoracolumbar fractures without neurological symptom were treated with percutaneous monoplanar screw internal fixation via injured vertebrae. There were 22 males and 16 females, aged 25-52 years (mean, 32.5 years). There were 23 cases of AO type A3 and 15 cases of AO type A4. The injured vertebrae located at T11 in 4 cases, T12 in 9 cases, L1 in 11 cases, L2 in 10 cases, L3 in 3 cases, and L4 in 1 case. The mean interval between injury and operation was 4.5 days (range, 3-7 days). The pre- and post-operative degrees of lumbodorsal pain were estimated by the visual analogue scale (VAS) score. The X-ray film, CT three-dimensional reconstruction, and MRI were performed, and the ratio of anterior vertebral body height and sagittal Cobb angle were measured to assess the kyphosis of the fractured area.ResultsAll operations in 38 patients successfully completed without complications such as dural sac, nerve root, or vascular injury. The operation time was (56.2±3.7) minutes and the intraoperative blood loss was (42.3±3.5) mL. All incisions healed by first intention without redness, swelling, or exudation. All patients were followed up 17-33 months, with an average of 21.5 months. The VAS score at each time point after operation significantly improved when compared with that before operation (P<0.05), and significantly improved at 3 months and last follow-up when compared with that at 1 week (P<0.05); there was no significant difference between 3 months and last follow-up (P>0.05). There was no internal fixator loosening, breakage, or delayed kyphosis in all patients. The ratio of anterior vertebral body height and sagittal Cobb angle significantly improved postoperatively (P<0.05), and no significant difference was found between the different time points after operation (P>0.05).ConclusionPercutaneous monoplanar screw internal fixation via injured vertebrae is an easy approach to treat thoracolumbar fracture without neurological symptom, which can effectively restore vertebral body height and correct kyphosis, and avoid long-term segmental kyphosis.
