Objective Neuron purification is essential to procedure of various nerve cell experimental research, however, at present there is few reports on the effect of various factors on neural axons during purification. To find out a simple method of neuron purification, and to investigate the influence factors of corresponding purification culture in dorsal root gangl ion (DRG) tissue culture on β3-tubul in positive axon. Methods The DRGs were obtained from the 3 days neonatal SD rat microscopically and were made into cell suspension. Then, the amount of attached DRG neurons and non neuronal cells in poly-D-lysine (PDL) group, PDL/Laminin (PDL/LN) group and collagen-I (Col I) group was observed from 10 to 100 minutes. Then, the extension and arborization of β3-tubul in positive axons were observed after 72 hours completely randomised DRG tissue culture for the research of the influences among culture substrates (PDL, PDL/LN, and Col I), FBS (0, 5%, and 10%), 5 fluorouracil (5-Fu, 0, 20, and 40 μmol/L), and cytrarabine (Ara-C, 0, 10, and 20 μmol/L). Results Adherent cells were observed instantly after inoculation by inverted phase contrast microscope and inverted fluoresence microscope; after cell suspension was removed, adherent growth of DRGn cells and non-DRGn cells were still seen. In PDL group, the amount of NSE negative cells was significantly higher than that of NSE positive cells at 10 and 30 minutes (P lt; 0.05); the amount of NSE positive cells was significantly higher than that of NSE negative cells at 80, 90 and 100 minutes (P lt; 0.05). In PDL/LN gruop, there was no significant difference (P gt; 0.05) in the amount of NSE negative cells and NSE positive cells at 10, 20, 30, 40, and 50 minutes; the amount of NSE positive cells was significantly higher (P lt; 0.05) than that of NSE negative cells at 60, 70, 80, 90, 100 minutes. In Col I group, the amount of NSE negative cells was higher than that of NSE positive cells at 10-40 minutes, but showing no significant difference (P gt; 0.05); the amount of NSE positive cells was significantly higher (P lt; 0.05) than that of NSE negative cells at 70-100 minutes. At 72 hours after DRG tissue culture, the best result of β3-tubul in positive axon extension and arborization was obtained when the substrate level was PDL/LN, and the average length of PDL/LN level was significantly larger than that of other two substrates (P lt; 0.05). The highest number of β3-tubul in positive axon distal end was obtained at 5% concentration level of FBS (P lt; 0.05), but showing no significant differences in β3-tubul in positive axon length among three levels (P gt; 0.05). Both the most of β3-tubul in positive axon distal ends and the longest β3-tubul in positive axon average length were obtained at 0 μmol/L concentration level of 5-Fu, showing significant differences between 0 μmol/L level and 20, 40 μmol/L levels (P lt; 0.05). A similar result of β3-tubul in positive axon distal end was got at the 0 μmol/L level and 10 μmol/L level of Ara-C, which was significantly higher than that of 20 μmol/L level (Plt; 0.05). Conclusion? A purified DRG neuron suspension for neuron culture could be obtained via PDL differential attachment for 30 minutes. When DRG neuron culture, neuron special medium, PDL/LN substrate and 10 μmol/L Ara-C are recommended in β3-tubul in positive axon research.
Objective To study the cl inical effects of modified Galveston technology in the treatment of lumbosacral tuberculosis. Methods From January 2001 to May 2008, 19 patients with lumbosacral tuberculosis were treated, including13 males and 6 females aged 21-58 years old (average 38 years old). The course of disease was 8-22 months. The tuberculosis was at the L4-S1 level in 3 cases, the L5, S1 level in 10 cases, the L5-S2 level in 5 cases, and the S1, 2 level in 1 case. Seven cases were compl icated with neural symptom of the lower l imbs, 3 cases of them were grade C and 4 cases were grade D according to the Frankel scale of nerve function. The preoperative JOA score of lower back pain was 5-22 (average 19). Six cases were compl icated with il iac abscess, 3 cases with psoas abscess, 3 cases with sacroil iac joint tuberculosis, and 2 cases with pulmonary tuberculosis. For 12 patients, the operation of modified Galveston internal fixation via the posterior approach, focus debridement via vertebral canal, and interbody fusion with autogeneous il iac bone fragment grafting was performed; for 7 cases, the operation of modified Galveston internal fixation via the posterior approach, vertebral lamina fusion with autogeneous il iac bone fragment grafting, and anterior focus debridement was performed. Results The incision of 18 cases was healed by first intention, and 1 case had sinus 3 weeks after operation and healed 3 months after operation. Nineteen patients were followed up for 12-82 months (average 21 months). There was no recurrence of the local tuberculosis, and the common toxic symptom of tuberculosis disappeared 6-12 months after operation. All the patients achieved bony fusion 4-6 months postoperatively, and 3 patients with sacroil iac joint tuberculosis achieved sacroil iac joint fusion. For those 7 patients with combinations of the neural symptomof the lower l imbs, the symptoms disappeared and their Frankel scales were improved to grade E. The JOA score of low back pain at the final follow-up was 22-29 (average 26). There was a significant difference between preoperation and postoperation (P lt; 0.05). Conclusion The modified Galveston technology is helpful to reconstruct the stabil ity of lumbosacral vertebrae, improve bony fusion rate, reduce the postoperative in-bed time.
To explore the advantage and indication of combined anterior and posterior surgeries for lumbarsacral junction tuberculosis. Methods Eleven cases of the lumbarsacral junction tuberculosis were treated with combined anterior (radical debridement and autograft) and posterior (instrumentation and fusion) surgeries in one stage between January 2002 and December 2006. There were 9 males and 2 females with the age of 20-56 years old. The courseof disease was 4 to 15 months, 6 months on average. The lessons were located at L5, S1 in 7 patients, at L4,5, S1 in 2 patients and at L5, S2 in 2 patients. The involved vertebral bodies were at 2 segments in 7 patients; and 3 segments in 5 patients. The preoperative kyphosis was 5 to 8° with an average 9°. The sinus was associated in 3 patients, 3 patients had radiculopathy; 4 had paeumonophthisis and 9 had abscess. Results The followed-up period was from 6 months to 3 years, 18 months on average. According to Chen score, among the 11 cases, there were excellent in 9, good in 2. All incisions were healed up primarily. After operation, spinal fusion was achieved in 10 cases within 5 months to 7 months, 6 months on average, and pseudoarthrosis in 1 case was found by the CT examination. The postoperative kyphosis was 0 to 4° with the mean of 2° and the radiculopathy in 3 cases all got nerve function recovery. Conclusion Lumbarsacral junction tuberculosis treated with this surgical technique can achieve a high satisfactory rate with restoring the spinal stabil ity, arresting the disease early, providing early fusion, correcting the kyphosis and preventing progression of kyphosis particularly if lumbosacral spine tuberculosis is associated with sinus or preoperative diagnosis cannot exclude suppurative spondyl itis.
Objective To investigate the therapeutic effects of transplanting allogeneic marrow mesenchymal stem cells (MSCs) via subarachnoid space on spinal cord injury(SCI) and the T cell subpopulation. Methods Density gradient centrifugation was used to isolate and expand MSCs from bone marrow of 10 six-week-old SD rats. The SCI model was produced by weightbeating from 60 eight-week-old female SD rats. Forty survival SCI rats,which BBB scores were zero, were divided randomly into 2 groups:experimental group(group A) and control group(group B). In addition, 20 normal eightweekold SD ratswere used as blank group (group C). In group A, 1 ml cells suspention containing MSCs(the 6th generation, 2×106/ml) was injected via subarachnoid space. Ingroup B, equal volume of L-DMEM was injected in the same way. The BBB score was obtained after 1st,2nd and 3rd weeks of injection. At the same time,T cell subpopulation was detected by flow cytometry. Results The BBB score in group A was better than that in group B, but fewer than that in group C in the 3rd week. CD4+T cells in group A were less than those in groups B and C in the 1st, 2nd, and 3rd weeks. CD8+T cells in group A were less than those in groups B and C in the 2nd and 3rd weeks. The ratio of CD4+/CD8+T cells in group A was less than those in groups B and C in the 1st week. Above differences showed statistically significant difference(P＜0.05). However, there were no statistically significant differences in the ratio of CD4+/CD8+T cells between group A and groups B, C in the 2nd and 3rd weeks (P＞0.05). Conclusion The above results suggest that allogeneic MSCs transplantation via subarachnoid space is beneficial to SCI to some extend, do not result in rejection in vivo. Furthermore, it can lead to immunosuppression in short time. So, it provides clues to apply MSCs to treat SCI and other diseases.
目的:探讨后路内固定治疗脊柱结核的必要性及适应证。方法:2002年1月~2008年12月采用后路器械固定、融合结合前路彻底病灶清除、植骨治疗脊柱结核17例。病变位于胸椎3例,胸腰段2例,腰椎4例,腰骶椎8例;累及2个节段7例,3个节段7例,4个节段3例;有3例伴窦道形成;9例伴不同程度的脊髓和(或)神经根受压症状;术前后凸成角10°~72°,平均31°。所有患者均一期手术。结果:术后随访6个月~5年,平均3.1年,术后切口均Ⅰ期愈合,后凸成角7°~58°,平均16°,椎间植骨平均在5个月融合,植骨融合率95.6%,优良率达89.6%,无一例复发。结论:后路器械固定结合前路彻底病灶清除、植骨治疗脊柱结核主要适用于多个节段受累、腰骶段及伴窦道者,利于恢复脊柱的稳定性、提高植骨融合率、纠正和预防后凸畸形。
Objective To explore the possibility of constructing tissue engineered cartilage complex three-dimensional nano-scaffold with collagen type II and hyaluronic acid (HA) by electrospinning. Methods The three-dimensional porous nano-scaffolds were prepared by electrospinning techniques with collagen type II and HA (8 ∶ 1, W ∶ W), which was dissolved in mixed solvent of 3-trifluoroethanol and water (1 ∶ 1, V ∶ V). The morphology were observed by light microscope and scanning electron microscope (SEM). And the porosity, water absorption rate, contact angle, and degradation rate were detected. Chondrocytes were harvested from 1-week-old Japanese white rabbit, which was disgested by 0.25% trypsin 30 minutes and 1% collagenase overlight. The passage 2 chondrocytes were seeded on the nano-scaffold. The cell adhesion and proliferation were evaluated by cell counting kit 8 (CCK-8). The cell-scaffold composites were cultured for 2 weeks in vitro, and the biological morphology and extracelluar matrix (ECM) secretion were observed by histological analysis. Results The optimal electrospinning condition of nano-scaffold was 10% electrospinning solution concentration, 10 cm receiver distance, 5 mL/ h spinning injection speed. The scaffold had uniform diameter and good porosity through the light microscope and SEM. The diameter was 300-600 nm, and the porosity was 89.5% ± 25.0%. The contact angle was (35.6 ± 3.4)°, and the water absorption was 1 120% ± 34% at 24 hours, which indicated excellent hydrophilicity. The degradation rate was 42.24% ± 1.51% at 48 days. CCK-8 results showed that the adhesive rate of cells with scaffold was 169.14% ± 11.26% at 12 hours, and the cell survival rate was 126.03% ± 4.54% at 7 days. The histological and immunohistochemical staining results showed that the chondrocytes could grow well on the scaffold and secreted ECM. And the similar cartilage lacuma structure could be found at 2 weeks after co-culture, which suggested that hyaline cartilage formed. Conclusion The collage type II and HA complex three-dimensional nano-scaffold has good physicochemical properties and excellent biocompatibility, so it can be used as a tissue engineered cartilage scaffold.
Object ive To summa r i z e the advanc ement of cytoske l e ton and axon outgrowth of neuron. Methods The recent l iterature concerning cytoskeleton and axon outgrowth of neuron was reviewed and summarized. Results The actin filaments and microtubules in neuron were highly polarized and dynamic structures confined to the ti ps of axons and the reci procal interactions between these two major cytoskeletal polymers was also dynamic. Attractive or a repulsive cue whose final common path of action was the growth cone cytoskeleton mediated the growth of axons of neuron by intracellular signaling cascades. Regulating the actin filament and microtubule dynamics as well as their interactions in growth cones played a key role in neurite outgrowth and axon guidance. Rho-GTPases and glycogen synthase kinase 3β (GSK-3β), the two major intracellular signal ing pathways had emerged in recent years as candidates for regulating the dynamics of actin filaments and microtubules. Conclusion The axon outgrowth and guidance depend on well-coordinated cytoskeletal and reciprocal interaction dynamics which also mediate axon regeneration after spinal cord injury. Regulating activity of Rho-GTPases and GSK- 3β simultaneously may acts as key role to regulate the dynamics of cytoskeletal and to determine axon outgrowth.
ObjectiveTo compare the effect of percutaneous kyphoplasty (PKP) with different phases bone cement for treatment of osteoporotic vertebral compression fracture (OVCF).MethodsThe clinical data of 219 OVCF patients who treated with PKP and met the selection criteria between June 2016 and May 2018 were retrospectively analyzed. According to the different time of intraoperative injection of bone cement, they were divided into observation group [116 cases, intraoperative injection of polymethyl methacrylate (PMMA) bone cement in low-viscosity wet-sand phase)] and control group (103 cases, intraoperative injection of PMMA bone cement in low-viscosity wire-drawing phase). There was no significance in general date of gender, age, disease duration, body mass index, bone mineral density T value, fracture vertebral body, preoperative fracture severity of the responsible vertebral body, anterior height ratio of the responsible vertebral body, preoperative pain visual analogue scale (VAS) score, and Oswestry disability index (ODI) between the two groups (P>0.05). The VAS score and ODI score were used to evaluate the improvement of patients’ symptoms at immediate, 2 days, 3 months after operation and at last follow-up. At 1 day, 3 months after operation, and at last follow-up, X-ray film and CT of spine were reexamined to observe the distribution of bone cement in the vertebral body, bone cement leakage, and other complications. During the follow-up, the refracture rate of the responsible vertebral body and the fracture rate of the adjacent vertebral body were recorded.ResultsThe injection amount of bone cement in the observation group and control group were (4.53±0.45) mL and (4.49±0.57) mL, respectively, showing no significant difference between the two groups (t=1.018, P=0.310). Patients in both groups were followed up 6-18 months (mean, 13.3 months). There were 95 cases (81.9%) and 72 cases (69.9%) of the bone cement distribution range more than 49% of the cross-sectional area of the vertebral body in the observation group and the control group, respectively, showing significant difference in the incidence between the two groups (χ2=4.334, P=0.037). The VAS score and ODI score of the postoperative time points were significantly improved compared with those before operation (P<0.05), and there were significant differences among the postoperative time points (P<0.05). The VAS score and ODI score of the observation group were significantly better than those of the control group (P<0.05) at immediate, 2 days, and 3 months after operation, and there was no significant difference between the two groups at last follow-up (P>0.05). At 1 day after operation, the cement leakage occurred in 18 cases of the observation group (8 cases of venous leakage, 6 cases of paravertebral leakage, 4 cases of intradiscal leakage) and in 22 cases of the control group (9 cases of venous leakage, 8 cases of paravertebral leakage, 5 cases of intradiscal leakage). There was no significant difference between the two groups (P>0.05). During the follow-up, 5 cases (4.3%) in the observation group, 12 cases (11.7%) in the control group had responsible vertebral refracture, and 6 cases (5.2%) in the observation group and 14 cases (13.6%) in the control group had adjacent vertebral fracture, the differences were significant (χ2=4.105, P=0.043; χ2=4.661, P=0.031).ConclusionBone cement injection with wet-sand phase in PKP is beneficial for the bone cement evenly distributed, strengthening the responsible vertebral, relieving the short-term pain after operation, decreasing the rate of responsible vertebral refracture and adjacent vertebral fracture without increasing the incidence of relevant complications and can enhance the effectiveness.
目的 探讨躯体感觉诱发电位（SEP）在颈脊髓损伤术前、术中监测的意义。 方法 纳入2010年1月－2012年4月治疗的241例颈脊髓损伤患者，术前按美国脊柱脊髓损伤协会（ASIA）评分并分级，确定损伤平面。术前与术中SEP监测，分析不同损伤分级以及不同损伤平面术前的波幅及潜伏期的差异，术中SEP监测以波幅下降＞50%和或潜伏期延长＞10%为预警标准。 结果 各损伤分级组术前SEP监测：A级组SEP波消失，呈一直线，而B、C、D、E级组均测出SEP波形，根据是否可测出SEP波形，可将A级与B、C、D、E及组区别。B、C、D级组之间波幅和潜伏期均无统计学意义（P＞0.05）。E级组较B、C、D级组波幅增高、潜伏期缩短，差异有统计学意义（P＜0.05）；不完全性颈脊髓损伤组内不同损伤平面组之间波幅和潜伏期差异均无统计学意义（P＞0.05）。术中SEP对脊髓功能损伤监测的灵敏度83.3%、特异度98.7%。其中术中：SEP阳性8例，真阳性5例，4例术者处理后波幅及潜伏期回复至正常范围，术后无新的神经功能损伤，另1例术者采取各种处理后波幅及潜伏期无恢复，术后神经功能损伤较术前加重；假阳性3例，1例麻醉师给予升高血压后波形恢复至正常，另2例经麻醉师调整麻醉深度后波形恢复正常，此3例术后无新的神经功能损伤。SEP阴性233例，真阴性232例，术后无新的神经功能损伤；假阴性1例，患者术中、术后波形未见异常，术后运动功能损伤程度较术前加重。 结论 ① SEP能准确评估完全性和不完性颈脊髓损伤，但对不完全性颈脊髓损伤的损伤程度不能作出准确评估、也不能区分颈脊髓损伤的损伤平面；② 术中SEP监测能较好地反映颈脊髓功能完整性，对减少颈脊髓损伤术中发生医源性颈脊髓损伤风险具有重要意义。