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Spinal cord injuries is an extremely serious central nervous system injury. The clinical prognosis is very poor. Patients are often associated with lifelong disabilities or paralysis. Regulating NSCs to repair spinal cord injuries is unanimously considered to be a very potential option for the treatment of this type of disease. China has a large population and a large number of patients with spinal cord injuries. Actively regulating spinal cord NSCs is of great significance for the regeneration and repair of spinal cord injuries. Endogenous NSCs avoid many disadvantages of exogenous stem cell transplantation, and have a broader prospect in the treatment of spinal cord injuries. The Wnt signaling pathway plays a very important role in the differentiation of NSCs and the development of the nervous system. However, the molecular mechanism of the proliferation and differentiation of NSCs during regeneration and repair after spinal cord injuries is still not fully understood. This article mainly describes the research progress of Wnt pathway regulating NSCs in the regeneration and repair of spinal cord injuries.
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Objective To construct tissue engineering nucleus pulposus by culture of rabbit bone marrow mesenchymal stem cells (rBMSCs)-nucleus pulposus acellular matrix scaffold (NPAMS)complexes (rBMSCs-NPAMS).Methods Several NPAMS were prepared,and rBMSCs was seeded into NPAMS.The scaffolds and complex were detected by general observation,HE stai-ning,immunohistochemical,qRT-PCR,scanning electron microscopy.Results The scanning electron microscopy showed the seed cell in nucleus pulposus ECM-derived scaffold could adhesion and growth.The cell attachment and proliferation were observed by HE staining.Immunohistochemical examination with typeⅡ collagen showed positive results.qRT-PCR revealed the time-depend-ent of the mRNA expression of collagen Ⅱ,and which was smaller than positive control(P0.05).Con-clusion Natural nucleus pulposus acellular matrix scaffold composite allogeneic bone marrow mesenchymal stem cells can be suc-cessfully built into tissue engineering nucleus pulposus.
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BACKGROUND:At present there is no specific therapy for the treatment of degenerateive disc diseases. The development of tissue engineering technology provides a new therapy idea for it. OBJECTIVE:To review the research progress of acelular matrix scaffold to construct tissue-engineered intervertebral disc. METHODS:A computer-based online search of PubMed, CNKI, Wanfang and Chinese Biomedical Database was performed for relevant articles published between 2005 to 2014 using the keywords of “intervertebral disc degeneration, extracelular matrix, tissue engineering, scaffold, nucleus pulposus, annulus fibrosus” in English and Chinese, respectively. RESULTS AND CONCLUSION:Construction of tissue-engineered intervertebral disc includes three essential elements: cel scaffolds, seed cels and cytokines. Scaffolds are the key. Acelular matrix scaffold is an important scaffold which has been a hot area of research in scaffolds for tissue-engineered intervertebral disc for several years, which can be divided into three types: acelular annulus fibrosus scaffold, acelular nucleus pulposus scaffold and integrated intervertebral disc scaffold. Different scaffolds have a variety of preparation methods. Acelular matrix scaffold has its own significant advantages, such as low antigenicity, good histocompatibility, and a most similarity with the three-dimensional structure of normal nucleus pulposus that can provide a proper microenvironment for cel growth. However, there are stil some disadvantages of acelular matrix scaffold in tissue-engineered intervertebral disc. So, it needs to be further improved.
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Objective To use detergents and nucleic acid enzyme to prepare scaffold of extracellular ma-trix , then assess the morphological and cytotoxic changes in vitro , and explore the feasibility of this type of scaffold as an ideal tissue-engineering scaffold. Methods Fifty pieces of fresh nucleus pulposus were randomly divided into a fresh control group and a decellularized group. The specimens in decellularized group were treated with 0.3%Tri-ton X-100, 0.5%sodium deoxycholate, and nuclease for 24 h. Morphological changes were studied by macroscopy, pathological staining and scanning electron microscopy. Cytotoxicity was determined by CCK-8 and LIVE/DEAD Viability/Cytotoxicity Assay Kit in vitro. Results The shape of scaffold was maintained,and the extracellular ma-trix was presented while the cells disappeared after decellularization. As compared with the fresh tissue , the scaffold and its extracts had no cytotoxicity to rabbit bone marrow stem cells. Conclusions Almost all the cells have been removed while the extracellular matrix is reserved , and the scaffold has no cytotoxicity to the seed cells. The decel-lularized scaffold can be used as an ideal substance to fabricate tissue-engineering nucleus pulposus.
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Objective To construct human acidic fibroblast growth factor (aFGF) recombinant eu karyotic expression vector and transfect it into muscle satellite cells(MSCs) of rat,in purpose of further study the method to set up cell bank.Methods The aFGF gene was cloned from human total RNA which was obtained from human skeletal muscle tissue by RT-PCR method.Human interleukin 2 (IL-2) signal peptide sequence (SPS) was obtained by direct chemosynthesis method.Then aFGF and SPS were fused to obtain SPS-aFGF.Finally,directional cloning SPS-aFGF into pEGFP-N1,the recombinant (pEGFP-N1-SPS-aFGF) was obtained.The recombinant was confirmed by endonuclease digestion and DNA sequencing.MSCs were purified by difference-speed adherence method and were ideontified by immunofluorescence assay.The correct cells were divided into 3 groups:Experimental group (aFGF +N 1),control group (N 1),blank group (blank).All the groups were transfected by Lipofectamine 2000TM Reagent,and pEGFP-N1-SPS-aFGF,pEGFP-N1 were respectively added in experimental group and control group while blank group was added none plasmid.Fluorescence microscope was employed to detect transfection efficiency tendency along with time changes.The expression of target gene was detected by fluorescent quantitation PCR and Western blot.Results (1) The sequencing of pEGFP-N1-SPS-aFGF was completely correct and the outcome of endonuclease was equal to actual ban s-ize.(2)The expression of GFP in transfected cells were observed by fluorescencemicroscope and transfection efficiency reached the peak at 72 h.(3)Real-time fluorescent quantitation PCR proved strong aFGF mRNA expression in transfected cells (the average relative expression of experimental group was 1464.95)with aFGF gene,while it was detected a little in the other groups (the average relative expression of control group was 1.016 and blank group was 1.000) (P ＜ 0.05).Western blot also proved strong expression in Experimental group then the other two groups.Conclusion aFGF eukaryotic expression vector was successfully constructed and transfected into MSCs.This study may be expected to obtain some specific functions cells.
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Objective To observe the clinical effects of the adjustive tractor for cervical dislocation.Methods Forty-seven patients were included between September 2007 and November 2011.There were 36 males and 11 females with age ranged from 7 to 62 (mean,35 years).The mean interval from injury to admission was 8.6 h (range,0.5-72 h).There were atlanto-occipital dislocation in 2 cases,C1.2 in 2 cases,C2.3 dislocation in 5 cases,C3.4 dislocation in 2,C4.5 in 9,C5.6 in 13,C6.7 in 14.Thirty cases were complicated by fracture.No facet locking occurred in sixteen cases.Facet locking was found in ten cases and bilateral facet locking was in 21 cases.After reduction,brace or internal fixation followed.According to American Spinal Injury Association (ASIA) spinal function impairment scale standard,there were 4 cases in level A,10 cases in level B,18 in C,10 in D,and 5 in E.According to Japanese Orthopaedic Association (JOA) spinal function rating standard,the mean JOA score was 9 (range,2-14).Results All 47 cases were reduced successfully without neuronal function aggravation.Traction power ranged from 7 to 60 kg (mean,25.6 kg),the mean time of traction was 8 min (range,3-10 min).The mean follow-up was 38 (range,6-48) months.All the patients achieved normal alignment and intervertebral height.The intervertebral body fusion was observed in all of cases,the mean fusion time was 3.3 months (range,3-6 months).One patient who experienced nonunion of vertebral arch fracture didn't receive further treatment because of absence of symptoms.At last follow-up,there were 3 cases in level A,1 in level B,4 in C,8 in D,and 31 in E according to ASIA scale.The mean JOA score was 12 (range,2-17).Conclusion The adjustive tractor is simple and safe for prompt reduction.
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BACKGROUND:intervertebral disc degeneration can reduce nucleus pulposus cells,and peroxiredoxin II involved in the regulation of resist oxidation damage,cell division,differentiation,signal transduction and apoptosis.Peroxiredoxin Ⅱ has promotive effect on intervertebral disc degeneration,whereas the mechanism remains poorly understood.OBJECTIVE:To observe the effects of perexiredoxin Ⅱ on human intevertebral disc cells activity and type Ⅱ collagen synthesis in vitro.METHODS:Human degenerated human lumbar disc cells were cultured in vitro,and assigned into the control and peroxidase Ⅱ groups.Peroxidase Ⅱ with doses of 10,100 and 1 000 ng/L were added into the peroxidase Ⅱ groups.The cells were identified by immunohistochemical staining,and the cell proliferation was detected using cck-8 kit.Cell supematant was collected at days 3 and 7 after operation,and the expression of type Ⅱ collagen was measured by double-antibody sandwich enzyme-linked immunosorbent assay.RESULTS AND CONCLUSION:In vitro cultured human degenerative lumbar intervertebral disc nucleus pulposus cells by adding peroxidase increased with the dose-Ⅱ,the disc nucleus pulposus cells of the volume and type Ⅱ collagen synthesis gradually reduced(P ＜ 0.01).Tips peroxidase Ⅱ on the intervertebral disc nucleus pulposus cells,the number and type Ⅱ collagen synthesis significantly inhibited in a dose-dependent manner.Thus speculated that peroxidase Ⅱ on the nucleus pulposus cells in vitro may lead to disc degeneration as a precipitating factor.
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BACKGROUND:Constructing animaI model of intervertebraI disc degeneration which more faithfully mimics the pathologic hallmarks of human intervertebral disc degeneration can be a beneficial assistance for further intervertebraI disc degeneration therapy.However,there is not an accepted optimal model for intervertebral disc degeneration study OBJECTIVE:To compare the rabbit model of degenerative intervertebral disc constructed by anulus puncture and anulus incision.METHODS:Totally 32 New Zealand white rabbits were randomly divided into the anulus puncture group and annulus incision group.Intervertebral disc of L_(3-6) 6was exposed by extro-peritoneal approach,and the discs were injured by puncturing the anulus or cutting the anulus The deep and direction were controlled.Pathological change of intervertebral disc was checked with MRI and histopathological examination at weeks 2,4,12,and 20 after operation.RESULTS AND CONCLUSION:At week 4 after operation.the area of nucleus gelatinosus was deflated with enlarged anulus fibrosus,T_2-weighted image(T_2WI)declined,blurred,and the height of intervertebral space was also decreased,the grade of T2 value in the anulus puncture group was lower than that of the annulus incision group(P＜0 05):with time prolonged,T2 scores increased,and the intervertebraI space narrowed.which reached a peak at week 20 after operation.The differences had no significance.The histological sections demonstrated that the cell content in nucleus pulposus was increased gradually.The rabbit model of intervertebraI disc degeneration can be successfully constructed by the methods of anulus puncture and annulus incision.The degeneration of incision modelis more severe than that of puncture model.Anulus puncture method can faithfully mimic intervertebral disc degeneration after damage in human being.
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BACKGROUND:For treatment of spinal cord injury, exogenous neural stem cell transplantation still faces many problems.Thus, the strategy of supplementary treatment of activating exogenous neural stem cells has been a hot focus.It has been found that lithium chloride can significantly inhibit differentiation and promote proliferation of neural stem cells, whose effects are correlated to Wnt signal pathway.OBJECTIVE:To investigate the effect of lithium on endogenous neural stem cells after spinal cord injury in rats.DESIGN, TIME AND SETTING:The randomized controlled animal study was performed at the Central Laboratory, Zhujiang Hospital from March to August 2008.MATERIALS:A total of 55 adult female Wistar rats were assigned into normal control group(n=5), simple injury group(n=25), and lithium chloride group(n=25).Lithium chloride was purchased from Guanghua, Guangzhou, China.METHODS:In the simple injury group and lithium chloride group, rat models of acute spinal cord injury at T10 segment were made by Allens method.From 1 hour following model induction, rats in the lithium chloride group received 3 mmol/kg per day lithium chloride through intraperithoneal injection.Samples were directly obtained.In the simple injury group, rats received an equal volume of saline.Rats in the normal control group were left intact.24 hours before samples were obtained, rats in each group were intraperitoneally injected with Brdu solution for labeling, once every 8 hours, totally 3 times.Spinal cord at 5 mm from the center of damage region received Brdu, catenin immunohistochemistry.MAIN OUTCOME MEASURES:BrdU-positive cell number and area of catenin-positive expression were measured.RESULTS:There were a few Brdu-positive cells and less expression of catenin in the center canal and adventitia of spinal cord in the normal control group.Many Brdu-positive cells and a little expression of catenin were found in gray matter and ependyma of injury area in the simple injury group 24 hours after model induction, which reached the peak at 1 week, and declined gradually at 2 weeks.Just a few Brdu-positive cells and little expression of catenin existed at 4 weeks.Compared with the simple injury group, there was no difference in Brdu-positive cells and the expression of catenin at 24 hours following model induction in the lithium chloride group.There were more Brdu-positive cells and the expression of catenin in the center canal and adventitia of spinal cord at 1 week(P
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[Objective]To analyze the mechanism of shape memory alloy imrasegmental fixator for lumbar spondylolysis(WMAIF)with finite element method.[Method]The three-dimensional model of WMAIF was established by using ANSYS 8.0 software for large finite element analysis.The model was extended with restriction in different parts,and to undertake corresponding mechanics calculation.[Result]The finite element model of WMAIF was set up,which could predict the strength and displacement of every node and element of its own structure in the course of deforming.[Conclusion]WMAIF is strong enough against tensile stress,which is a new and reliable intrasegmental instrument for direct repairing of lumbar spondylolysis.
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[Objective]To investigate the influence of wing-like memory alloy intrasegmental fixator(WMAIF)on healing of lumbar spondylolysis.[Method]Eighteen healthy hybrid adult dogs were used in the experiment,regardless of gender,the L_6 isthmic portion were injured by operation randomly and equally divided into three groups.Group Ⅰ were fixed with WMAIF,groupⅡ were fixed with steel wire,group Ⅲ were treated with nothing.Every group was examined through CT at 2,4,6,8 and 10 weeks.[Result]In group Ⅰ,the fracture achieved healing at 6 weeks after operation.In group Ⅱ,the fracture achieved healing at 10 weeks after operation,while no repair of fracture was found in group Ⅲ.[Conclusion]Lumber spondylolysis fixation with WMAIF has stable and continued axial stress,WMAIF is beneficial to fracture healing,and is a new and reliable intrasegmental instrument for direct repair of lumbar spondylolysis.
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<p><b>OBJECTIVE</b>To investigate gene expression of three nitric oxide synthase isozymes in injured spinal cord tissue.</p><p><b>METHODS</b>Thirty-six adult SD rats were randomly divided into six groups: a normal group and five injury groups, with six per each group. Animals in the injury groups were sacrificed at 2, 6, 12, 24, 48 h after injury. A compression injury model on the spinal cord was made according to Nystrom B et al and gene expression of the three NOS isozymes were examined by reverse transcription polymerase chain reaction (RT-PCR).</p><p><b>RESULTS</b>Gene expression of nNOS and eNOS were detectable in the normal group and were up-regulated quickly after injury, reaching a maximum at 6 h: (0.633 +/- 0.012) and (1.236 +/- 0.207). Gene expression of iNOS was detectable only in the injury groups and it was gradually up-regulated after injury, reaching a maximum at 24 h: (1.043 +/- 0.049).</p><p><b>CONCLUSION</b>Injury to the spinal cord leads to early up-regulation of cNOS and late up-regulation of iNOS. Different NOS isozymes may play different roles in secondary spinal cord injury.</p>
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Animais , Feminino , Masculino , Ratos , Regulação Enzimológica da Expressão Gênica , Óxido Nítrico Sintase , Genética , Óxido Nítrico Sintase Tipo I , Óxido Nítrico Sintase Tipo II , Óxido Nítrico Sintase Tipo III , RNA , Genética , Metabolismo , Ratos Sprague-Dawley , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Medula Espinal , Patologia , Traumatismos da Medula Espinal , GenéticaRESUMO
<p><b>OBJECTIVE</b>To investigate the gene expression of two kinds of constitutive nitric oxide synthase (cNOS): neuronal NOS (nNOS) and endothelial NOS (eNOS) in injured spinal cord tissue.</p><p><b>METHODS</b>Thirty-six adult Sprague-Dawley rats were divided randomly into six groups: the normal group and the injury groups (2, 6, 12, 24, 48 h after injury, respectively). A compression injury model of the spinal cord wa s ma de and gene expression of nNOS and eNOS were examined by reverse transcription polymerase chain reaction (RT-PCR).</p><p><b>RESULTS</b>The gene expression of nNOS and eNOS was detected in the normal group and they were up-regulated quickly after injury, reaching the maximum at 6 h. There was no difference between gene expression of nNOS and eNO S in the normal group, but in each injury group the gene expression of eNOS was much higher than that of nNOS.</p><p><b>CONCLUSIONS</b>Expression of constitutive NOS (cNOS) in spinal co rd tissue was up-regulated after injury mainly in the early stage. cNOS as a wh ole offers protection in spinal cord injury, but different cNOS may play different roles.</p>