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BACKGROUND:Intervertebral disc degeneration is the basis of spinal degenerative diseases;however,there is no effective treatment. OBJECTIVE:To investigate whether sinomenine can inhibit interleukin-1β-induced apoptosis in nucleus pulposus cells and its molecular mechanism. METHODS:Rat nucleus pulposus cells were cultured in vitro by trypsin combined with type II collagenase digestion,and the cell growth curve was plotted.An appropriate sinomenine concentration was determined using the cell counting kit-8 kit.Nucleus pulposus cells were divided into control group,sinomenine group,interleukin-1β group,sinomenine+interleukin-1β group,zinc protoporphyrin group,zinc protoporphyrin+sinomenine group,zinc protoporphyrin+interleukin-1β group,and sinomenine+zinc protoporphyrin+interleukin-1β group.Proliferative activity,reactive oxygen species content,apoptosis rate,and heme oxygenase-1 expression in nucleus pulposus cells were detected. RESULTS AND CONCLUSION:The rat nucleus pulposus cells cultured in vitro were polygonal,triangular,and short wedge-shaped,and the cell growth showed an"S"curve.The cells grew slowly in the first 3 days of culture,rapidly in 4-6 days,and slowly again in 7-8 days.The cells then entered the"platform stage"where the number of cells no longer increased.The proliferative activity of myeloid cells showed no significant changes when the concentration of sinomenine was≤80 μmol/L(P>0.05).Interleukin-1β significantly reduced the proliferative activity of nucleus pulposus cells,increased the content of reactive oxygen species and led to apoptosis(P<0.01).Sinomenine intervention not only promoted heme oxygenase-1 expression(P<0.05)but also inhibited interleukin-1β-induced decrease in proliferative activity and increase in reactive oxygen species content and apoptosis rate in nucleus pulposus cells(P<0.05).These effects could be reversed by zinc protoporphyrin(P<0.01).
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BACKGROUND:Salvianolic acid B can inhibit cell damage induced by H2O2,effectively remove excess reactive oxygen species,and exert antioxidant properties.It has been used in the treatment of many diseases.However,there are relatively few studies on the role and mechanism of salvianolic acid B in intervertebral disc degeneration. OBJECTIVE:To observe the effect and mechanism of salvianolic acid B on oxidative stress-induced intervertebral disc degeneration by using gelatin methacryloyl hydrogel as a carrier through the in vitro cell experiment and the in vivo animal experiment. METHODS:The gelatin methacryloyl hydrogel(drug-loaded hydrogel)loaded with salvianolic acid B was prepared.(1)In vitro cell experiment:The lumbar nucleus pulposus cells of adult SD rats were isolated and extracted,and passage 3 nucleus pulposus cells were selected and divided into groups:Group A was added complete medium.In group B,a complete medium containing H2O2 was added.Group C was inoculated on methylacrylylated gelatin hydrogel and added with a complete medium containing H2O2.Group D was inoculated on methyl acrylyl gelatin hydrogel loaded with salvianolic acid B and added into a complete medium containing H2O2.The E group was inoculated on the methylacrylyl gelatin hydrogel loaded with salvianolic acid B,and the complete medium containing H2O2 and the complete medium containing TLR4 signaling pathway inhibitor were added.Cell proliferation,oxidative stress,inflammatory response,gene expression of cell matrix-associated proteins and the protein expression of TLR4/nuclear factor-kB signaling pathway were detected.(2)Animal in vivo experiment:Sixty adult SD rats were randomly divided into normal group,acupuncture group,acupuncture + salvianolic acid group,acupuncture + hydrogel group and acupuncture + loading potion gel group,with 12 rats in each group.The last four groups were treated with acupuncture to establish models of intervertebral disc degeneration and then injected with normal saline,salvianolic acid B solution,non-drug loaded gel and drug-loaded gel in turn.Imaging examination and pathological observation were performed 4 weeks after surgery. RESULTS AND CONCLUSION:(1)In vitro cell experiment:Compared with group A,the cell proliferation was decreased;the oxidative stress reaction and inflammation reaction were enhanced;the expression of extracellular matrix degrading enzymes(matrix metalloproteinase 3,matrix metalloproteinase 13,ADAMTS4,ADAMTS5)was increased in group B(P<0.05),and the synthesis of extracellular matrix(type Ⅱ collagen,proteoglycan)was decreased(P<0.05).The protein expression of the TLR4/nuclear factor-kB signaling pathway was increased(P<0.05).Compared with group B,the cell proliferation of groups D and E was increased,the oxidative stress response and inflammatory response were weakened,and the expression of extracellular matrix degrading enzymes(matrix metalloproteinase 3,matrix metalloproteinase 13,ADAMTS4,ADAMTS5)was decreased(P<0.05),and the synthesis of extracellular matrix was increased(P<0.05).The protein expression of TLR4/nuclear factor-kB signaling pathway was decreased(P<0.05),and the effect was more significant in group E.(2)Animal in vivo experiment:4 weeks after surgery,intervertebral disc height index,index of MRI and pathological and histological grading of the intervertebral disc had improved significantly in the acupuncture+drug-loaded hydrogel group,and simply injecting hydrogel or salvianolic acid B solution can to a certain extent improve the intervertebral disc degeneration,but they are not as good as the injection of the drug-loaded hydrogel.(3)It is concluded that gelatin methacryloyl hydrogel loaded with salvianolic acid B can inhibit oxidative stress and inflammation in the degenerated intervertebral disc tissue,inhibit the degradation of extracellular matrix,and alleviate the process of intervertebral disc degeneration,which may be accomplished by inhibiting the TLR4/nuclear factor-kB signaling pathway.
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BACKGROUND:Intervertebral disk degeneration is a pathological change caused by a series of complex molecular mechanisms that result in the aging and damage of intervertebral discs,ultimately leading to severe clinical symptoms.Traditional Chinese medicine has unique advantages in the treatment of intervertebral disk degeneration due to its low cost,non-addictive nature,multi-target effects,minimally toxic and side effects,and high patient acceptance. OBJECTIVE:To review the latest research results of traditional Chinese medicine monomer intervention-related signaling pathways in the treatment of intervertebral disk degeneration,describe and analyze the action mechanism of traditional Chinese medicine monomer on intervertebral disk degeneration,and provide a new approach and theoretical basis for future basic research and clinical treatment. METHODS:The first author searched for relevant literature from January 2018 to February 2023 in CNKI,PubMed,VIP,and WanFang using the search terms"intervertebral disc,signal pathway".The articles that did not meet the criteria were excluded after preliminary screening of the titles and abstracts.Finally,72 articles were selected for review and analysis. RESULTS AND CONCLUSION:Traditional Chinese medicine monomers can regulate multiple classical signaling pathways such as Wnt/β-catenin,PI3K/Akt,mTOR,NF-κB,and MAPK.They achieve this by regulating oxidative stress,adjusting the expression of pro/anti-apoptotic proteins in cells,stimulating cellular autophagy function,reducing stimulation of cell inflammatory factors,increasing the expression of extracellular matrix markers,reducing the production of matrix-degrading enzymes,maintaining the synthesis and stability of extracellular matrix,inducing differentiation of mesenchymal stem cells in the nucleus pulposus into nucleus pulposus cells,promoting endogenous repair and reconstruction,controlling apoptosis and aging of nucleus pulposus cells,and increasing the activity of nucleus pulposus cells.These actions improve the microenvironment within the intervertebral disc,maintain the normal physiological function of the intervertebral disc,and delay intervertebral disc degeneration.
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BACKGROUND:Stem cell transplantation is a new way to prevent and cure intervertebral disc degeneration.However,whether the transplanted stem cells can survive,proliferate,differentiate,and restore the function of nucleus pulposus cells after transplantation,is the key and difficult point to overcome. OBJECTIVE:To explore the effects of Bushenhuoxue decoction on survival,proliferation,and nucleus pulposus-like differentiation of adipose-derived stem cells. METHODS:A Transwell chamber was used to construct a co-culture model of human adipose-derived stem cells and human degenerative nucleus pulposus cells.The experiment was divided into control group,model group,drug-containing serum group,and drug-free serum group.Except for the control group,the co-culture system of other groups was treated with 50 μmol/L tert-butyl hydrogen peroxide for 24 hours.The drug-containing serum group and drug-free serum group were treated with DMEM low-glucose complete culture medium containing drug-containing serum of Bushenhuoxue decoction or drug-free serum with 20％volume fraction for 48 hours.The sublayer adipose-derived stem cells were taken.Toluidine blue staining was used to detect proteoglycan synthesis levels.Real-time PCR method was used to detect mRNA expression of type Ⅱ collagen,proteoglycan and SRY-box transcription factor 9.The protein expression of SOX9 was detected by western blot assay.Lactate dehydrogenase assay was used to detect cytotoxicity.Flow cytometry was used to detect reactive oxygen species,and β-galactosidase staining was used to detect cell senescence. RESULTS AND CONCLUSION:(1)Compared with the control group,the proportion of necrotic cells in the model group increased;toluidine blue staining became lighter,and the expression levels of type Ⅱ collagen,proteoglycan,SOX9 mRNA and SOX9 protein decreased(P<0.05).Compared with the model group,the drug-containing serum of Bushenhuoxue decoction could significantly reduce cell injury and promote the expression of type Ⅱ collagen,proteoglycan,SOX9 mRNA,and SOX9 protein(P<0.05),but the improvement in the drug-free serum group was not significant(P>0.05).(2)Compared with the control group,the contents of cytotoxicity,reactive oxygen species,and cell senescence in the model group were significantly increased.Compared with the model group,the microenvironment of the coculture system was significantly improved by drug-containing serum of Bushenhuoxue decoction(P<0.05),while drug-free serum had no significant effect on the microenvironment of the co-culture system(P>0.05).(3)The results show that Bushenhuoxue decoction can promote the survival,proliferation,and nucleus pulposus-like differentiation of adipose-derived stem cells.
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BACKGROUND:Nucleus pulposus cell apoptosis is the main pathological basis for intervertebral disc degeneration,and inflammation and peroxidation are important factors leading to apoptosis in the nucleus pulposus.Studies have shown that matrine has antioxidant,senescent,inflammatory and apoptotic effects,and may be a potential drug for the treatment of disc degeneration. OBJECTIVE:To investigate the effect of matrine on apoptosis of nucleus pulposus cells in rats with intervertebral disc degeneration by regulating the cyclic GMP-AMP synthase(cGAS)-stimulator of interferon genes(STING)signaling pathway. METHODS:(1)Nucleus pulposus cells of rats at a logarithmic phase were randomly separated into a control group,a model group,a low-dose matrine group,a high-dose matrine group,an empty group,and a high-dose matrine+cGAS overexpression group.Except for the control group,cell models of intervertebral disc degeneration were established in the other groups through oxygen-glucose deprivation.At the same time of modeling,the low-dose and high-dose groups were treated with 0.4 and 0.8 mmol/L matrine,respectively,and the empty group was transfected with the empty plasmid,while the high-dose+cGAS overexpression group was treated with 0.8 mmol/L matrine with the transfection of the cGAS overexpression plasmid.After 24 hours of treatment,cell activity and apoptosis,intracellular levels of reactive oxygen species,superoxide dismutase,tumor necrosis factor α and interleukin 1β,and intracellular expression of apoptotic proteins and cGAS-STING pathway proteins were detected.(2)Sixty Sprague-Dawley rats were randomized into six groups(n=10 per group):control group,model group,low-dose matrine group,high-dose matrine group,empty group,and high-dose+cGAS overexpression group.After 12 weeks of modeling,60 and 120 mg/kg matrine were given by gavage in the low-dose and high-dose matrine groups,respectively(once a day),and the empty plasmid was injected into the tail vein in the empty group(2 times/week),while the high-dose+cGAS overexpression group was given 120 mg/kg matrine by gavage and injected with cGAS overexpression plasmid to the tail vein.Treatment in each group was given consecutively for 3 weeks.Samples were taken after drug administration and assayed for apoptosis,levels of reactive oxygen species,superoxide dismutase,tumor necrosis factor α and interleukin 1β,as well as apoptotic protein and cGAS-STING pathway protein expression. RESULTS AND CONCLUSION:Compared with the control group,in the model group,cell activity and superoxide dismutase levels were decreased(P<0.05),and apoptosis rate,levels of reactive oxygen species,tumor necrosis factor α and interleukin 1β,and the expression of cGAS,STING,cleaved caspase-3 and Bax proteins were elevated(P<0.05).Matrine dose-dependently ameliorated the above changes in each index due to cellular modeling(P<0.05),whereas cGAS overexpression partially antagonized the ameliorative effect of high-dose matrine.Similar results to the in vitro cellular experiments were obtained in animal experiments.These results indicate that matrine could inhibit inflammation and oxidative stress by blocking the cGAS-STING signaling,which in turn attenuates apoptosis and elevates the activity of nucleus pulposus cells in rats with intervertebral disc degeneration.
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BACKGROUND:Intervertebral disc degeneration is a condition caused by the combined effects of various factors,such as cellular apoptosis,oxidative stress,and inflammatory responses.Currently,it is believed that the core of this condition lies in the degeneration and apoptosis of nucleus pulposus cells(NPCs).However,the specific pathological mechanisms remain unclear. OBJECTIVE:By investigating the expression of the phosphatidylinositol-3 kinase(PI3K)/protein kinase B(Akt)/hypoxia-inducible factor 1-alpha(HIF-1α)signaling pathway and the apoptosis of NPCs under different oxygen concentrations,to clarify the biological characteristics of NPCs under varying oxygen levels,and to explore the mechanisms of intervertebral disc degeneration. METHODS:Normal and degenerated NPCs were collected.The second-generation cells were used for imaging identification.The third-generation cells were cultured in the following conditions:37℃,air,100%humidity.Leibovitz's medium containing 100 μmol/L CoCl2 and 10%fetal bovine serum medium containing 100 μmol/L H2O2 were used to create distinct oxygen concentration environments for the third-generation cells,allowing for the investigation of cellular responses and behaviors under normal,low oxygen,and oxidative stress conditions.The third-generation cells were divided into six groups:normal NPCs+hypoxia,normal NPCs+normoxia,normal NPCs+oxidative stress,degenerated NPCs+hypoxia,degenerated NPCs+normoxia,and degenerated NPCs+oxidative stress groups.Cell viability and proliferation were assessed using the cell counting kit-8 method.Cell apoptosis was detected using flow cytometry.RT-PCR and western blot assay were utilized to examine the protein and mRNA expression levels of PI3K,AKT,and HIF-1α. RESULTS AND CONCLUSION:At different oxygen concentrations,the cell proliferation rate of both normal and degenerated NPCs decreased as the oxygen concentration increased.Conversely,the apoptosis rate increased as the oxygen concentration rose(P<0.05).With the normal NPCs+hypoxia group as the control group,the effect of degenerated NPCs on the apoptosis rate was highly significant(P<0.001).Oxygen concentration had a highly significant impact on both NPC proliferation rate and apoptosis rate(P<0.001).The interaction between cell degeneration and oxygen concentration significantly affected both NPC proliferation and apoptosis rates(P<0.05).At different oxygen concentrations,the protein and mRNA expression levels of PI3K,AKT,and HIF-1α in normal NPCs were highest under low oxygen concentration,followed by oxidative stress environment,and lowest under normoxia.In degenerated NPCs,the protein and mRNA expression levels of PI3K,AKT,and HIF-1α decreasd as the oxygen concentration increased.The protein and mRNA expression levels of PI3K and Akt in normal NPCs were significantly higher than those in degenerated NPCs(P<0.05).With the normal NPCs+hypoxia group as the control group,the effects of normal or degenerated NPCs,as well as oxygen concentration,on the protein and mRNA expression of PI3K,AKT,and HIF-1α were highly significant.The interaction between cell degeneration and oxygen concentration also had an extremely significant effect on the protein and mRNA expression of PI3K,AKT,and HIF-1α(P<0.001).To conclude,there is a close correlation between the proliferation and apoptosis of NPCs and both oxygen concentration and the cellular functional state.The PI3K/Akt/HIF-1α signaling pathway exhibits antagonistic regulatory effects under various cellular functional states and different oxygen concentration environments.The mechanism may be associated with the activation of the PI3K/Akt signaling pathway,which consequently transcribes HIF-1α,thus maintaining the balance of reactive oxygen species metabolism.This pathway plays a significant role in inhibiting oxidative stress,antagonizing NPCs apoptosis,and consequently delaying intervertebral disc degeneration.
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BACKGROUND:Intervertebral disc degeneration is caused by damage and degeneration of the nucleus pulposus and annulus fibrosus tissues inside the intervertebral disc,resulting in structural and functional changes of the intervertebral disc.However,there is yet no effective drug treatment for intervertebral disc degeneration. OBJECTIVE:To investigate the inhibitory effect of syringin on intervertebral disc degeneration. METHODS:A total of 10 male Sprague-Dawley rats were selected,and the coccygeal intervertebral disc(Co4/Co5)of each rat was set as model group,Co5/Co6 intervertebral disc as syringin group,and Co6/Co7 intervertebral disc as control group.The control group did not receive any treatment.In the model group and syringin group,a miniature puncture needle was used to puncture the annulus fibrosus to establish an intervertebral disc degeneration model.Immediately after modeling,2.5 μL of normal saline and syringin solution(5 μmol/L)were given in the model and syringin groups,respectively.Four weeks after injection,the samples were taken.The degree of intervertebral disc degeneration in rats was observed by hematoxylin-eosin and safranine O-fast green staining.The expressions of type Ⅱ collagen,aggrecan and matrix metalloproteinases 3 and 13 in intervertebral disc tissue were analyzed by immunohistochemical staining. RESULTS AND CONCLUSION:Hematoxylin-eosin staining showed that in the model group,the height of intervertebral disc decreased,the cartilage endplate became thinner and cracked,the fibrous ring structure was disordered and cracked,and the nucleus pulposus disappeared;in the syringin group,the height of intervertebral disc was normal or slightly lower than that in the control group,the degree of cartilage endplate degeneration was lighter than that in the model group,the fiber circle permutation was relatively regular with no cracks,and the nucleus pulposus was partially shrunk.Safranine O-fast green staining showed that in the model group,the cartilage endplate of the intervertebral disc was defective and the calcified layer of cartilage became thinner,showing obvious degeneration.The structure and morphology of intervertebral disc cartilage endplate in the syringin group recovered to some extent.Immunohistochemical staining showed that,compared with the control group,the expressions of type Ⅱ collagen and aggrecan in the intervertebral disc cartilage were decreased in the model group(P<0.000 1),while the expressions of matrix metalloproteinases 3 and 13 increased(P<0.000 1).Compared with the model group,the expressions of type Ⅱ collagen and aggrecan in the intervertebral disc cartilage tissue were increased in the syringin group(P<0.001,P<0.000 1),while the expressions of matrix metalloproteinases 3 and 13 decreased(P<0.001,P<0.000 1).These results showed that syringin could improve the structure and function of intervertebral disc by inhibiting the expression of matrix metalloproteinases 3 and 13 and increasing the expression of type Ⅱ collagen and aggrecan,thus preventing and slowing down the procession of intervertebral disc degeneration.
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Intervertebral disc degeneration(IDD)is a pathological change characterized by abnormal intervertebral disc me-tabolism,reduction of nucleus pulposus(NP)and degradation of extracellular matrix(ECM).IDD is closely related to age and ab-normal stress loads on the spine,which often lead to spinal degenerative diseases such as lumbar disc herniation,lumbar spinal stenosis,and ultimately lower back pain and walking impairment.Due to the complex pathogenesis of IDD,it is difficult to slow disease progression,and the treatment is still mainly based on surgery and symptomatic treatment.Therefore,it is urgent to seek a new drug treatment method to alleviate IDD.As an extracellular vesicle with intercellular material transfer and cell function regulation,exosomes have received more and more attention in the repair of degenerative tissues,which provides a new feasible way for the treatment of intervertebral disc degeneration.This article will analyze the mechanism and function of different cell-derived exosomes in the treatment of intervertebral disc degeneration,in order to provide a comprehensive understanding and reference for the subsequent exosome-related clinical treatment for IDD.
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BACKGROUND: The mitochondrial apoptotic pathway is an important pathway in cell apoptosis. Previous studies have found that Bushen Zhuangdu Fang can improve intervertebral disc degeneration by inhibiting the mitochondrial apoptotic pathway in animal experiments. However, its mechanism of action is to be clarified. OBJECTIVE: To observe the effect of serum containing Bushen Zhuangdu Fang on mitochondrial apoptotic pathway key proteins of human nucleus pulposus cells, and to explore the mechanism by which this drug-containing serum improves intervertebral disc degeneration. METHODS: Thirty-seven male Sprague-Dawley rats were randomly divided into blank group, low-dose Chinese medicine group (0.506 g/kg per day), medium-dose Chinese medicine group (1.012 g/kg per day) and high-dose Chinese medicine group (2.024 g/kg per day). After 2 weeks of continuous administration, drug-containing serum was prepared. Human nucleus pulposus cells were randomly divided into normal group, cell model group, low-dose drug-containing serum group, medium-dose drug-containing serum group, and high-dose drug-containing serum group. The cell model group was treated with 200 μmol/L H2O2 for 6 hours, and the normal group received no treatment. The three drug-containing serum groups were treated with corresponding treatments for 48 hours. The pathological changes of nucleus pulposus cells were observed by transmission electron microscopy. Apoptotic rate of nucleus pulposus cells was detected by flow cytometry and mitochondrial membrane potential was detected by flow cytometry. Apaf1, Bcl-2, Bax and Cytc expressions were detected by real-time quantitative PCR and western blot assay. RESULTS AND CONCLUSION: Compared with the normal group, the apoptotic rate of nucleus pulposus cells with obvious apoptotic morphology was significantly increased (P < 0.05), the expression of Apaf1, Cytc, and Bax were significantly increased at mRNA and protein levels (P < 0.05), and the mitochondrial membrane potential and expression of Bcl-2 mRNA and protein were significantly decreased in the cell model group (P < 0.05). After treatment with drug-containing serum, the apoptotic rate of nucleus pulposus cells decreased significantly (P < 0.05), the expression of Apaf1, Cytc, Bax and their proteins decreased significantly (P < 0.05), and the mitochondrial membrane potential, Bcl-2 and their proteins increased significantly (P < 0.05). Therefore, the serum containing Bushen Zhuangdu Fang can effectively inhibit apoptosis of nucleus pulposus cells in a dose-dependent manner. The drug-containing serum may alleviate intervertebral disc degeneration by reducing the expression of Apaf1, Cytc and Bax and increasing the expression of Bcl-2 at protein and gene levels, and inhibiting the mitochondrial apoptotic pathway.
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@#Objective To explore the changes of rabbit adipose stem cells(ASCs)and bone mesenchymal stem cells(BMSCs)cultured in vitro and the anabolism under basic fibroblast growth factor(bFGF).Methods BMSCs and ASCs were cultured with DMEM,DMEM/F12(2∶1)or α-MEM respectively.The 3rd generation ASCs and BMSCs were divided into 2 groups respectively:group A:ASCs cultured in chondrogenic medium(CM),group B:ASCs cultured in CM supplemented with bFGF 5 ng/ml,group C:BMSCs cultured in CM,group D:BMSCs cultured in CM supplemented with bFGF 5 ng/ml.Morphological changes were observed under inverted microscope.The 35SO42-incorporation and total hydroxyproline were measured.Results BMSCs and ASCs showed much higher growth rate when cultured in α-MEM medium comparison with that in DMEM or in DMEM/F12(2:1).Both stem cells attachment cultured in monolayer greatly increased and cell clones were abundant,while the cells attachment became rather difficult and cell clones were less after cutured in CM.All stem cells possessed a round-like morphology,and the cells in group B and D were more than that in the other 2 groups.The 35SO42-incorporation and total hydroxyproline synthesis of group B or D increased compared with that of group A or C,but there was no diference between group D and B.Conclusion The rabbit ASCs and BMSCs cultured in CM suppling with bFGF grow well and their metabolism increased.
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Objective To investigate the effect of direct intercell contact on the bone mesenchymal stem cells(MSCs)differentiate into nucleus pulposus cells(NPs)when cocultured with NPs in constant magnetic field.Methods The primary NPs labeled by DAP1 were cocultured with the 3 rd generation of MSCs through direct and indirect intercell contact in the presence or absence of constant magnetic field(0.05,0.10,0.50 and 1.00 mT,respectively). Observation of morphological changes was performed every 24 hours.The method of MTT was employed to evaluate the level of proliferation.The gene expression of collagen Ⅱ,Sox-9 and Aggrecan was measured by using RT-PCR. Results MSCs cocultured with direct intercell contact with the NPs rounded up and presented a round ring-like structure appearance.The expression of marker genes including Collagen type Ⅱ,Aggrecan and Sox-9 were significantly increased when cells cocultured in constant magnetic field of 0.05 mT compared with those without constant magnetic field(P＜0.05).There were no significantly changes with regard to the expression of the above genes in 0.10 mT field(P＞0.05).The growth of NP-like cells was suppressed when the intensity of magnetic field was higher than 0.10 mT(P＜0.05).Conclusion It is suggested that 0.05 mT constant magnetic field and direct intercell contact facilitate differentiation of MSCs into NPs.
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[Objective]To evaluate whether transplanted marrow mesenchymal stem cells interfered in TGF-?1 can differentiate to nucleus pulposus cells and increase the amount of proteoglycan and collagenase Ⅱ content in intervertebral discs.[Method]We used an in vivo model to investigate the feasibility of marrow mesenchymal stem cells that cultured in vitro and interfered in TGF-?1 delivery,retention,and survival in the degeneratived disc space.In 2,4,6,8 weeks we used immunohistochemical staining to determine the change of collagenase Ⅱ content;spectrophotometry to determine the change of amount of proteoglycan with Phlorglucinol;the experiment date were analyzed by SPSS 11.5 soft ware.[Result]We found MSCs could maintain viability and proliferate within the rabbit inter vertebral disc.The amount of proteoglycan and collagen Type Ⅱ content of the intervertebral in matrix synthesis in the experiment group was increased in 8 weeks.We found no changes in the modle group.[Conclusion]Our data suggest that transplanted marrow mesenchymal stem cells in vivo can survive and increase proteoglycan and collagen Type Ⅱ amount interfered in TGF-?1 in some periods,which support its potential use as a treatment of intervertebral disc degeneration.
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Objective To compare the cellular characteristics of the nucleus pulposus(NP) cells of young and adult rabbits.Methods The structure and arrangement of the NP cells from young and adult rabbits were investigated on both tissue and ultrastructure level by confocal laser scanning microscope and transmission electron microscope. Results The NP cells from the young rabbit discs,which were round and contained numerous large inclusion bodies,often formed clusters with a diameter of(266?167)??m and a density of(8.0?2.4) per high power(?40);The NP cells from the adult rabbit discs were round or oval shape and had no large size inclusion bodies,formed less clusters or were singly diffusedly distributed,with the diameter of clusters to be(94?42)??m(single cell excluded) and density to be(14.9?4.3) per high power(?40).Conclusion There were remarkable morphological differences in the cell architecture of nuclei pulposi between the young and adult rebbits.These features of the aging and degenerating disc contribute to decreased biological function.
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STUDY DESIGN: In-vitro experimental study. OBJECTIVES: To determine the proteoglycan synthesis of the rabbit nucleus pulposus cells in various concentration of extracellular collagen type I and II under the stimulation of TGF-beta1. SUMMARY OF LITERATURE REVIEW: Therapeutic effect of growth factor and gene therapy can be altered by composition of extracellular matrix. However, the effect of extracellular collagen types I and II on synthetic activity of intervertebral disc cells is not thoroughly studied before. MATERIALS AND METHODS: The nucleus pulposus cells were isolated and cultured from 10 skeletally mature rabbits. Cultures were trypsinized and incorporated into alginate beads with different concentration of extracellular collagen type I and II (0.5%, 1.0% and 1.5%). Those cultures with TGF-beta1 (10 ng/ml) served stimulated condition of matrix synthesis. Newly synthesized proteoglycans were assessed by 35 S-sulfate incorporation using chromatography on Sephadex G-25 in PD-10 columns. Scintillation count was normalized with DNA content by Hoechst dye method. RESULTS: In basal condition, difference in proteoglycan synthesis in given concentration of extracellular collagen type I and II were statistically insignificant. In stimulated condition with TGF-beta1, difference in proteoglycan synthesis in given concentration of extracellular collagen type I and II was also statistically insignificant. However, cultures in stimulated condition with TGF-beta1 showed increased amount of newly synthesized proteoglycans compared to those of basal condition regardless of the concentration of extracellular collagen type I and II (p < 0.05). CONCLUSION: Anabolic response of rabbit nucleus pulposus cells is relatively insensitive to extracellular matrix composition, which facilitates application of gene therapy in various conditions of disc degeneration.
Sujets)
Lapins , Chromatographie , Collagène de type I , Collagène , ADN , Matrice extracellulaire , Thérapie génétique , Dégénérescence de disque intervertébral , Disque intervertébral , Protéoglycanes , Facteur de croissance transformant bêta-1 , TrypsineRésumé
Objective To explore morphologic characterizatics of cellular processes from adult human nucleus pulposus cells. Methods The nucleus pulposus of adult human intervertebral disc were obtained from 8 patients(Thompson's grade Ⅰ-Ⅱ) and then the tissues specimens were carried out by frozen section and electron microscopic section as well as cell isolation and cultured,processes of nucleus pulposus cells were examined using light microscopy,laser scanning confocal microscopy and transmission electron microscopy. Results When examined at both the confocal and electron microscope level,all the cells possessed the processes and adjacent nucleus pulposus cells processes possessed a gap junction.The elongated and round cells were examined when NP cells became monolayer in vitro.The rate of elongated cells to round cells was 2.3 to 1.The elongated cells protruded along with the long axis of cell body without second processes.Dendritic processes of round cells protruded to all directions from the cell body with multiple-level processes.Conclusion Processes are one of the morphologic characteristics of intervertebral disc cells.The research on processes functions could be helpful to understand pathomechanism of intervertebral disc degradation.