移植骨髓间充质干细胞对兔退变椎间盘髓核细胞凋亡的影响

目的 观察骨髓间充质干细胞(MSCs)移植对兔退变椎间盘髓核细胞凋亡的影响.方法 以各兔L2/3、L3/4、L4/5、L5/6节段分为正常组、退变组、成纤维细胞(SFs)移植对照组、MSCs移植治疗组.MSCs和SFs分别经绿色荧光蛋白(GFP)转染后,注射植入退变椎间盘的髓核.通过透射电镜观察退变椎间盘凋亡髓核细胞形态;用实时定量聚合酶链反应(PCR)检测退变组织中髓核细胞凋亡相关基因bcl-2和box mRNA的表达;免疫荧光法标记髓核细胞凋亡相关蛋白Caspase-3,并通过TUNEL法标记凋亡髓核细胞,激光共聚焦显微镜检测髓核细胞凋亡蛋白表达率和细胞凋亡比率.结果 透射电镜下,退变椎间盘中凋亡髓核细胞呈现出核染色质边集,空泡形成,核膜断裂,凋亡小体形成等变化.MSCs移植治疗组bcl-2 mRNA的表达量高于退变组和SFs移植对照组(P<0.05),bax mRNA的表达量与退变组差异无统计学意义(P>0.05).MSCs移植治疗组细胞凋亡率和Caspase-3表达率均高于正常组[细胞凋亡率分别为(16.75±2.14)%和(6.86±1.08)%;Caspase-3表达率分别为[(20.34±1.03)%和(6.09±0.77)%](P<0.05),低于退变组和SFs移植对照组[细胞凋亡率分别为(31.87±4.16)%和(29.02±2.16)%;Caspase-3表达率分别为(31.50±3.78)%和(30.20±4.93)%](P<0.05).结论 髓核细胞凋亡在椎间盘退变过程中起重要作用.MSCs移植能有效抑制椎间盘髓核细胞凋亡,延缓椎间盘退变过程.     

Anti-apoptotic effects of caspase inhibitors on rat intervertebral disc cells

BACKGROUND: Apoptosis is thought to be a critical component of disc degeneration. Two main pathways of Fas-mediated apoptosis have been identified: Type I, which is the death-inducing signaling complex pathway, and Type II, which is the mitochondrial pathway. The apoptotic pathway for anulus fibrosus cells, which is phenotypically different from that of nucleus pulposus cells, has not been elucidated to our knowledge. The ultimate initiators or executioners of apoptosis are caspases. There are also inhibitors of caspases, which have the potential of being used as anti-apoptotic therapeutic agents. We therefore undertook this study to determine (1) the apoptotic pathway of anulus fibrosus cells and (2) the anti-apoptotic potential of caspase inhibitors. METHODS: Rat anulus fibrosus cells were isolated, cultured, and placed in either 0% (apoptosis-promoting condition) or 10% (normal control) fetal bovine serum. We identified and quantified the presence of apoptotic cell death, caspase activities, and loss of mitochondrial membrane potential. In addition, we examined the cells for the expression of Fas, procaspases, and cytochrome-c. Finally, we analyzed the degree of anti-apoptotic effects of caspase inhibitors on the cells in 1% fetal bovine serum. RESULTS: The percentage of apoptosis and Fas expression in the cells incubated in 0% fetal bovine serum were increased compared with those in the cells incubated in 10% fetal bovine serum (both p < 0.001). Caspase-8, 9, and 3 activities were increased and expression of procaspases was decreased in the 0% fetal bovine serum compared with those in the 10% fetal bovine serum (all p < 0.001). In contrast, the loss of mitochondrial membrane potential and cytochrome-c release into the cytosol were unchanged in the 0% fetal bovine serum. Pancaspase and caspase-8 inhibitors reduced apoptotic cell death (p < 0.001 and p < 0.05, respectively), but caspase-9 inhibitor did not reduce apoptotic cell death. CONCLUSIONS: Our results suggest that, unlike nucleus pulposus cells, anulus fibrosus cells are Fas Type-I cells, which undergo apoptosis through the death-inducing signaling complex. We also found that apoptosis of intervertebral disc cells can be attenuated by caspase inhibitors.     

颈椎终板软骨细胞凋亡的检测及相关意义

目的检测颈椎终板软骨细胞的细胞凋亡指数,探讨其在椎间盘退变中可能的作用机制。方法颈椎间盘终板及髓核取自我院行颈椎前路手术的35例颈椎椎间盘退变患者（退变组）和19例颈椎外伤患者（外伤组）。光镜观察退变组和外伤组终板和髓核的细胞密度,TUNEL法检测两组终板软骨细胞和髓核细胞的细胞凋亡指数,咔唑分光光度法比较两组髓核蛋白多糖含量。结果退变组终板细胞密度较外伤组减少（P〈0.05）,TUNEL染色显示退变组终板细胞凋亡指数为（34.6±16.1）%,外伤组为（20.1±9.3）%,两组间比较差异有统计学意义（P〈0.05）。Pearson相关分析显示,颈椎终板TUNEL染色阳性细胞率与终板细胞密度、髓核蛋白多糖含量之间呈负相关（r=—0.805,P=0.001;r=—0.677,P=0.023）,与髓核TUNEL阳性细胞率之间呈正相关（r=0.758,P=0.003）。结论颈椎退变终板软骨细胞凋亡率较高,推测在椎间盘退变过程中可能发挥重要作用;软骨细胞凋亡可能与髓核细胞凋亡增加、终板细胞密度与髓核蛋白多糖含量降低密切相关。     

Apoptosis,senescence, and autophagy in rat nucleus pulposus cells: Implications for diabetic intervertebral disc degeneration

This research was aimed to study the mechanisms by which diabetes aggravates intervertebral disc degeneration (IDD) and to discuss the relationship between autophagy and IDD in nucleus pulposus (NP) cells. Sixteen weeks after injecting streptozotocin (STZ), the intervertebral discs (IVDs) were studied by histology, Alcian blue, 1,9‐dimethylmethylene blue (DMMB), immunohistochemistry, and RT‐PCR to explore the IDD. The apoptosis and senescence of NP cells was investigated by terminal deoxyribonucleotidyl transferase (TDT)‐mediated dUTP‐digoxigenin nick end labeling (TUNEL) assay, immunohistochemistry, and Western blot for caspase3, caspase8, caspase9, and p16lnk4A (increased in cellular senescence). The level of autophagy in NP cells was detected by Western blot, immunohistochemistry, and transmission electron microscopy (TEM). The proteoglycan and collagen II in the extracellular matrix and the aggrecan and collagen II mRNA expression in NP cells of diabetic rats were decreased compared with the control group. Diabetes increased apoptosis of NP cells and led to activations of initiators of intrinsic (caspases‐9) and extrinsic (caspase‐8) pathways as well as their common executioner (caspase‐3). Cellular senescence was increased about twofold in NP of diabetic rats. In addition, the Western blot, immunohistochemistry, and TEM demonstrated higher level of autophagy in NP cells of diabetic rats than control rats to a statistically significant extent. These findings support that diabetes induced by STZ can cause IDD by accelerating the apoptosis and senescence of NP cells excluding the overweight influence. And the results suggest that the autophagy may be a response mechanism to the change of NP cells in diabetic rats. © 2012 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 31: 692–702, 2013     

瘦素缺乏小鼠椎间盘退变的组织学观察

目的探讨瘦素在椎间盘退变中的可能作用。方法采用HE染色观察6月龄雄性ob/ob小鼠（瘦素缺乏小鼠）和野生型小鼠（C57BL小鼠）椎间盘的形态学;免疫组织化学检测Ⅱ型胶原、蛋白聚糖的表达;Real-time PCR检测Ⅱ型胶原、Ⅹ型胶原及蛋白聚糖的基因表达。结果与野生型小鼠相比,ob/ob小鼠椎间盘HE染色表现为椎间盘组织的胶原结构紊乱、髓核碎裂、椎间盘高度降低,免疫组化检测显示Ⅱ型胶原、蛋白聚糖表达减少,Real-time PCR检测显示Ⅱ型胶原、蛋白聚糖基因表达下调而Ⅹ型胶原基因表达上调,差异有统计学意义（P〈0.05）。结论活体内瘦素缺乏可能加速小鼠椎间盘退变。     

Fracture of the vertebral endplates, but not equienergetic impact load, promotes disc degeneration in vitro

Vertebral endplate damage is associated with intervertebral disc (IVD) degeneration (DD) in vivo as confirmed by in‐vitro investigations. Our aims were to further characterize the process of DD using an in vitro full‐organ culture model and to elucidate whether significant endplate damage or impact loading alone is pivotal for the initiation of DD. Rabbit spinal segments (n = 80) were harvested, subjected to pure axial impact loading (n = 40) using a custom‐made device, and cultured for 28 days. The applied threshold energy (0.76 J) induced endplate fractures in 21 specimens (group A); 19 remained intact (group B). Markers for DD (cell viability, apoptosis, necrosis, matrix remodeling, and inflammation) were monitored for 28 days post‐trauma in the annulus fibrosus (AF) and nucleus pulposus and compared to non‐impacted control discs. Cell viability in both groups stayed at a control level. Group A compared to group B showed enhanced lactate dehydrogenase (LDH) and caspase‐3/7 activity, reduced glycosaminoglycan content, reduced aggrecan mRNA, but elevated mRNA for collagen‐2, catabolic enzymes (MMP‐1/‐3/‐13), and pro‐inflammatory (TNFα, IL‐6, IL‐8, MCP‐1) and pro‐apoptotic (fas ligand, caspase‐3) proteins. Group B compared to control only showed small changes in mRNA levels. Our findings demonstrate that burst endplates, but not equienergetic loading, promotes DD. © 2011 Orthopaedic Research Society Published by Wiley Periodicals, Inc. J Orthop Res 30:809–816, 2012     

Nucleus pulposus allograft retards intervertebral disc degeneration

Autogenous implantation of nucleus pulposus or nucleus pulposus cells that were activated by coculture retards intervertebral disc degeneration, but harvesting such grafts causes disc degeneration at the donor site. This study examined whether nucleus pulposus allografts similarly retard disc degeneration and whether such allografting induces immunologic rejection. Japanese White rabbits served as donors and recipients for allografts. Lumbar disc degeneration was induced by aspirating the nucleus pulposus. Two weeks later, intact nucleus pulposus or nucleus pulposus cells were injected and compared with a sham procedure and normal control. The recipients' discs were examined histologically and immunologically at intervals for 16 weeks. Discs receiving an intact nucleus pulposus showed the least degeneration, followed by discs receiving nucleus pulposus cells, both of which were better than no treatment. These findings correlated directly with the intensity of immunochemical staining for Type II collagen. Allogeneic grafts did not induce any appreciable host-versus-graft response. Injection of nucleus pulposus and nucleus pulposus cells retards intervertebral disc degeneration. However, injection of intact nucleus pulposus is more effective than injection of nucleus pulposus cells alone. The intercellular matrix plays an important, but poorly understood, role in preserving intervertebral discs.     

骨髓间充质干细胞-壳聚糖凝胶复合体移植治疗椎间盘退变

目的 观察兔骨髓间充质干细胞.壳聚糖凝胶复合体移植修复椎间盘髓核缺损退变的效果.方法 建立兔椎间盘髓核缺损退变模型,将兔骨髓间充质干细胞-壳聚糖凝胶复合体注射移植入缺损退变模型中,兔继续培养4周后处死,取出移植修复的椎间盘进行组织HE染色、Aggre-can番红O染色及Ⅱ-collagen免疫组织化学染色,与正常椎间盘及未行移植的缺损退变椎间盘进行随机对照,检测移植修复的效果.结果 骨髓间充质干细胞-壳聚糖凝胶复合体可以在缺损的椎间盘中正常生长,并呈现向类髓核细胞分化的趋势,合成分泌Ⅱ-collagen和Aggrecan,维持原椎间盘髓核组织的生物学特性,而缺损退变组髓核组织纤维化、完整性丧失,水分丢失,Ⅱ-collagen合成明显减少(P<0.05).结论 兔骨髓间充质干细胞-壳聚糖凝胶复合体能够修复椎间盘缺损退变.     

TGF-β3修饰退变髓核细胞移植修复兔退变椎间盘的效果观察

目的探讨TGF-β3基因修饰后退变髓核细胞生物学效应以及植入兔退变椎间盘后对退变椎间盘的影响。方法将重组腺病毒载体Ad-TGF-β3与第2代退变髓核细胞按10∶1比例混合培养转染(Ad-TGF-β3组),待细胞融合后传代,MTT检测转染细胞增殖活性,Western blot检测TGF-β3蛋白含量,免疫细胞化学染色观察对数生长期转染细胞Ⅱ型胶原染色阳性率;采用病毒空载体转染髓核细胞(Adv组)和未经转染髓核细胞(空白组)作为对照。取30只新西兰兔,体重3.2～3.5 kg,雌雄不限,通过针刺L3、4、L4、5和L5、6椎间盘制备椎间盘退变模型。将实验动物按照随机数字法分为3组,转染细胞组(A组,n=12)、退变细胞组(B组,n=12)和空白对照组(C组,n=6)。A、B组将100μL浓度为1×105个/mL对应细胞悬液注射入退变椎间盘,C组同法注入等量PBS。注射后6、10、14周取A、B组各4只、C组2只实验动物处死,取L3、4、L4、5和L5、6椎间盘行组织学观察,RT-PCR检测Ⅱ型胶原和蛋白多糖mRNA表达。结果 Ad-TGF-β3转染后髓核细胞活性明显改善;转染后3、7、14 d,TGF-β3在髓核细胞内表达逐渐升高;Ad-TGF-β3组髓核细胞细胞质内见棕黄色Ⅱ型胶原阳性染色,阳性率显著高于Adv组及空白组(P<0.05)。组织学观察示,A组椎间盘退变程度较B、C组明显减轻。6、10、14周A组Ⅱ型胶原和蛋白多糖mRNA表达显著高于B、C组,差异均有统计学意义(P<0.05)。结论 TGF-β3基因修饰退变髓核细胞后可明显改善细胞生物活性,转染后髓核细胞植入兔体内可明显增加退变椎间盘的基质分泌。     

Activation of rat nucleus pulposus cells by coculture with whole bone marrow cells collected by the perfusion method

Cell proliferation and matrix synthesis were compared for rat nucleus pulposus cells cocultured with mesenchymal stem cells (MSCs) or fresh whole bone marrow cells (BMCs), harvested by the perfusion or aspiration methods. Nucleus pulposus cells were isolated from tail intervertebral discs of F344/slc rats, and BMCs were obtained from femora. Proteoglycan synthesis, DNA synthesis, and aggrecan mRNA expression were measured. The level of transforming growth factor‐β in supernatants from the culture system was also measured. Cell number, aggrecan mRNA expression, and uptake of [³⁵S]‐sulfate and [³H]‐thymidine by nucleus pulposus cells cocultured with fresh whole BMCs all increased significantly compared with nucleus pulposus cells cocultured with MSCs. TGF‐β secreted by nucleus pulposus cells cocultured with fresh whole BMCs also significantly increased when compared with cocultures with MSCs. The perfusion method was superior to the aspiration method for preventing contamination of BMCs with peripheral red blood cells and lymphocytes, which may cause an autoimmune response in the disc. In conclusion, we suggest that fresh whole BMCs harvested by the perfusion method are more effective for increasing the proliferative and matrix synthesis capacity of nucleus pulposus cells. © 2008 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 27:222–228, 2009     

Therapeutic effect of co-culture of rat bone marrow mesenchymal stem cells and degenerated nucleus pulposus cells on intervertebral disc degeneration

BackgroundAfter non-contact co-culture of bone marrow mesenchymal stem cells (BMSCs) with nucleus pulposus cells (NPCs), exosomes secreted by BMSCs were able to ameliorate the degree of disc degeneration. The reason for this is, at least in part, that exosomes from BMSCs achieve by affecting the level of autophagy in NPCs, while the components in exosomes are diverse and their specific mechanism of action is still unclear.PurposeHere, we aimed to explore the therapeutic effect of co-culture of BMSCs and NPCs on NPCs and explore its specific mechanism of action.Study design/SettingIn vitro study.MethodsRat NPCs and BMSCs were isolated and cultured in vitro. The serum deprivation experiment (using oxygen, glucose, and serum deprivation [OGD]) simulates the pathological state of low blood supply of the intervertebral disc in vivo. We used apoptotic cell staining and flow cytometry to study the effect of BMSCs on the apoptosis rate of rat NPCs, and the apoptotic proteins active-caspase-3, active-caspase-9, autophagy marker proteins LC3 and Beclin 1 were further detected using Western blot analysis. The expression levels of the pro-apoptotic protein Bax and the apoptosis-inhibiting protein Bcl2 were measured. The differentially expressed miRNAs were screened in a gene expression profiling chip. Then qRT-PCR was used to detect the effect of different treatment methods on miR-155 expression. The effect of anti-miR-155 antibodies on autophagy was studied by flow cytometry and transmission electron microscopy. A luciferase reporter assay was used to study the direct interaction between miR-155 and BACH1 mRNA, which was analyzed by TargetScan software, and the results were verified by Western blotting.ResultsCompared with the OGD group, the expression level of miR-155 and the NPC autophagy level significantly increased; the HO-1 protein expression increased; and the Bach1 protein expression, degeneration index, and apoptosis index all significantly decreased in the co-culture group. After BMSCs transfected with anti-miR-155 were co-cultured with NPCs, the miR-155 expression in the cells was significantly reduced, the HO-1 protein expression and the level of cell autophagy was reduced. However, Bach1 protein expression, NPC degeneration index, and apoptosis index increased. After being inhibited by the autophagy inhibitor wortmannin, the cell degeneration index and apoptosis rate significantly improved.ConclusionIn the OGD model, BMSCs can significantly increase the viability, the level of autophagy, and reduce the level of apoptosis in rat NPCs. BMSC exosomes increase miR-155 expression in NPCs, which targets Bach1 and in turn upregulates HO-1 expression, activates autophagy in NPCs, inhibits the apoptosis level, and improves intervertebral disc degeneration.Clinical SignificanceOur experiment shows that it is maybe feasible to treat disc degeneration with drugs. At the same time, compared with BMSC injection method of treatment, side effects of drug therapy are smaller, and can be controlled, it also provides a new way for intervertebral disc degeneration drug treatment.     

Bone morphogenetic protein-7 protects human intervertebral disc cells in vitro from apoptosis.

BACKGROUND CONTEXT: Disc degeneration includes dysfunction and loss of disc cells leading to a decrease in extracellular matrix (ECM) components. Apoptosis has been identified in degenerated discs. Bone morphogenetic protein-7 (BMP-7) has been reported to stimulate ECM synthesis in the intervertebral disc (IVD), but its effect on disc cell viability is unknown. PURPOSE: To investigate whether BMP-7 can protect disc cells from programmed cell death while enhancing ECM production. STUDY DESIGN: An in vitro study to examine the effect of BMP-7 on apoptosis of IVD cells. METHODS: Human nucleus pulposus (NP) cells were cultured in monolayer, and human recombinant pure BMP-7 (rhBMP-7) was added to the medium when the cells were in the second passage. Thereafter, apoptosis was induced by either tumor necrosis factor-alpha (TNF-alpha) or hydrogen peroxide (H(2)O(2)). Cellular apoptosis was evaluated by terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling assay and caspase-3 activity. ECM synthesis was assessed by immunofluorescence for collagen-2 and aggrecan. To study the possibility of bone induction by rhBMP-7 in disc cells, alkaline phosphatase activity and Alizarin red-S staining were evaluated. RESULTS: Apoptosis was induced by both TNF-alpha and H(2)O(2). Addition of rhBMP-7 resulted in inhibition of the apoptotic effects caused by both inducers. Further, BMP-7 decreased caspase-3 activity. In the presence of BMP-7, ECM production was maintained by the cells despite being in an apoptotic environment. No osteoblastic induction of the disc cells was seen. CONCLUSIONS: BMP-7 was demonstrated to prevent apoptosis of human disc cells in vitro. One of the antiapoptotic effects of BMP-7 on NP cells might be a result of its inactivation of caspase-3. Collagen production was maintained by addition of rhBMP-7 in an apoptotic environment.     

人退变腰椎间盘中细胞凋亡与Bax及半胱胺酸蛋白酶蛋白3基因的表达

目的 检测人腰椎间盘组织中的细胞密度、细胞凋亡率以及Bax和半胱氨酸蛋白酶蛋白3(Caspase-3)的表达,为深入了解人腰椎间盘退变的机制提供实验依据,并为将来通过生物学方法 延缓腰椎间盘退变提供新的思路.方法 实验组30个椎间盘标本(L2～S1)来自27例行后路腰椎间盘切除椎间融合手术自愿捐赠的患者,男18例,女9例;年龄30～72岁,平均51.09岁.所有病变节段均经MRI证实,患者术前均未接受椎间盘造影、胶原酶髓核溶解或椎间盘激光汽化术.对照组20个标本(L2～S1)来自5例青年男性意外死亡者新鲜尸检腰椎间盘;年龄24～37岁,平均30.83岁.采用HE染色观察凋亡软骨细胞、凋亡小体和细胞密度,TUNEL染色法检测人腰椎间盘中的凋亡细胞率,用SP免疫组织化学法检测Bax及Caspase-3表达,图像分析Bax及Caspase-3的平均灰度值.结果 HE染色示对照组、实验组软骨终板和髓核内的平均细胞密度分别为(17.16±1.22)、(12.41±0.95)个/HP,二者差异有统计学意义(P＜0.01).TUNEL染色观察示对照组的软骨终板与髓核内的平均凋亡细胞率为6.97%±0.92%,低于实验组的12.59±0.95%(P＜0.01).SP免疫组织化学染色示对照组髓核内Bax、Caspase-3染色阳性细胞率分别为11.02%4±1.18%和9.01%±1.00%,均低于实验组的19.29%±1.18%和15.07%±0.97%,差异均有统计学意义(P＜0.01).对照组腰椎间盘髓核内Bax、Caspase-3的平均灰度值分别为187.33±7.88和185.68±3.26,实验组分别为124.98±6.69和160.13±4.37,两组间比较差异有统计学意义(P＜0.01).将全部腰椎间盘标本进行Pearson相关分析,对照组和实验组腰椎间盘凋亡细胞率与细胞密度间成负相关,相关系数r分别为-0.88和-0.93(P＜0.01)两组髓核内Bax、Caspase-3阳性细胞率与凋亡细胞率之间成正相关,相关系数r分别为0.83和0.91(P＜0.01).结论 细胞密度下降参与了人腰椎间盘的退变过程,Bax和Caspase-3的表达上调在人腰椎间盘髓核细胞的凋亡过程中有一定作用.     

A rat tail temporary static compression model reproduces different stages of intervertebral disc degeneration with decreased notochordal cell phenotype

The intervertebral disc nucleus pulposus (NP) has two phenotypically distinct cell types—notochordal cells (NCs) and non‐notochordal chondrocyte‐like cells. In human discs, NCs are lost during adolescence, which is also when discs begin to show degenerative signs. However, little evidence exists regarding the link between NC disappearance and the pathogenesis of disc degeneration. To clarify this, a rat tail disc degeneration model induced by static compression at 1.3 MPa for 0, 1, or 7 days was designed and assessed for up to 56 postoperative days. Radiography, MRI, and histomorphology showed degenerative disc findings in response to the compression period. Immunofluorescence displayed that the number of DAPI‐positive NP cells decreased with compression; particularly, the decrease was notable in larger, vacuolated, cytokeratin‐8‐ and galectin‐3‐co‐positive cells, identified as NCs. The proportion of TUNEL‐positive cells, which predominantly comprised non‐NCs, increased with compression. Quantitative PCR demonstrated isolated mRNA up‐regulation of ADAMTS‐5 in the 1‐day loaded group and MMP‐3 in the 7‐day loaded group. Aggrecan‐1 and collagen type 2α‐1 mRNA levels were down‐regulated in both groups. This rat tail temporary static compression model, which exhibits decreased NC phenotype, increased apoptotic cell death, and imbalanced catabolic and anabolic gene expression, reproduces different stages of intervertebral disc degeneration. © 2013 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 32:455–463, 2014.     

Cyclic mechanical stretch stress increases the growth rate and collagen synthesis of nucleus pulposus cells in vitro

STUDY DESIGN: A rabbit model designed to investigate the effects of applied cyclic tensile stress on the cell division rate and the collagen synthesis in the rabbit nucleus pulposus cells in vitro. OBJECTIVE: To evaluate the effects of mechanical stress on nucleus pulposus cells, thus adding to the understanding of the adaptation of the intervertebral disc to mechanical stress. SUMMARY OF BACKGROUND DATA: Intervertebral disc cells in vivo are exposed to a multitude of physical forces during physical motion. Although it is known that in intervertebral disc disease, a common pathway of disc degeneration is mechanical stress on the nucleus pulposus or the anulus fibrosus or both, the underlying mechanism has been less well defined. METHODS: Nucleus pulposus cells were isolated from 4-week-old Japanese white rabbits. These cells were subjected to the mechanical cyclic stretch stress using a computerized, pressure-operated instrument that physically deformed the cells. The DNA synthesis rate, collagen synthesis rate, and cell cycle progression were measured. RESULTS: Cyclic tensile stretch increased the DNA synthesis rate in nucleus pulposus cells and in the population of cells in the S phase of the cell cycle during 1 to 2 days of subjugation to stress. Cyclic tensile stretch also increased collagenous protein synthesis in nucleus pulposus cells during 1 to 4 days of stress. CONCLUSIONS: Mechanical stress on nucleus pulposus cells promotes the proliferation of cells and alters the properties of intervertebral disc cells. This study may reflect the adaptation of the intervertebral disc to increased motion and stress.     

Reinsertion of stimulated nucleus pulposus cells retards intervertebral disc degeneration: an in vitro and in vivo experimental study.

Reinsertion of autogenous nucleus pulposus, an innovative method to delay further disc degeneration, has been proved with an experimental animal model. This study examined whether coculture of nucleus pulposus cells with annulus fibrosus cells (a) activates annulus fibrosus cells and (b) retards disc degeneration when reinserted into the disc in a rabbit model of disc degeneration. Coculture of the two cell types stimulated proliferation of each, as indicated by increased DNA synthesis measured by increases in DNA polymerase alpha expression and uptake of 5-bromo-2'deoxy-uridine assessed by an enzyme-linked immunosorbent assay. In a model of disc degeneration in rabbits, reinsertion of activated nucleus pulposus cells delayed the formation of clusters of chondrocyte-like cells, the destruction of disc architecture, and the elaboration of type-II collagen as measured immunohistochemically compared with no treatment. The direct reinsertion of activated nucleus pulposus cells into the disc offers a promising line of investigation for delaying intervertebral disc degeneration, although these results obtained with notochordal cells may not necessarily apply when mature central nucleus pulposus cells are used.     

Rock信号通路抑制剂对大鼠髓核细胞生物学特性的影响

【摘要】 目的 探讨Rock信号通路抑制剂对大鼠髓核细胞的基质代谢及凋亡等生物学特性的影响。方法〓qPCR和western blot检测TNF-α与Rock通路抑制剂Y-27632对大鼠原代髓核细胞Cox2、MMP3、MMP13的表达调控;MTS细胞增殖试验检测不同浓度Y-27632刺激下对髓核细胞增殖的影响;Annexin/PI法检测Y-27632对髓核细胞凋亡的影响。结果〓予Rock抑制剂Y-27632刺激后,髓核细胞MMP13的表达水平上调(P<0.05),髓核细胞的凋亡率增加(P<0.05),但予Y-27632刺激后髓核细胞的增殖速度无明显差异(P>0.05)。结论〓Rock信号通路可能通过调控髓核细胞MMP13表达水平及其凋亡等生物学特性从而参与了椎间盘的退变。     

Low‐intensity pulsed ultrasound stimulation enhances TIMP‐1 in nucleus pulposus cells and MCP‐1 in macrophages in the rat

Recent studies have reported that low‐intensity pulsed ultrasound (LIPUS) stimulates cell proliferation and proteoglycan production in rabbit intervertebral disc cells, and moreover promotes the secretion of MCP‐1 (monocyte chemotaxis protein‐1) from macrophages in a disc organ culture model. These findings suggest the possible application of LIPUS for biological repair of disc degeneration and herniation. Although the mechanisms involved are not well understood, several cytokine pathways may play a role. Therefore, in order to evaluate the effect of LIPUS stimulation on cytokine production by nucleus pulposus cells and macrophages, in vitro culture studies were designed. Nucleus pulposus cells and macrophages were collected from Sprague‐Dawley rats, cultured separately in a monolayer, and stimulated with LIPUS for 7 days. After culture, the culture medium and the cells were analyzed by cytokine array, RT‐PCR, and ELISA. Cytokine array showed that LIPUS stimulation significantly upregulated TIMP‐1 (tissue inhibitor of metalloproteinase‐1) in the nucleus pulposus and MCP‐1 in macrophages in comparison with the control. This was confirmed at the gene level by RT‐PCR in nucleus pulposus cells and macrophages after stimulation with LIPUS. Quantitative evaluation of these proteins by ELISA showed higher levels in nucleus pulposus cells and macrophages stimulated by LIPUS than in controls. These results showed that LIPUS stimulation significantly activated TIMP‐1 and MCP‐1 in nucleus pulposus cells and macrophages at both the protein and gene levels, suggesting that LIPUS may be a promising supplemental treatment for intervertebral disc herniation. © 2008 Orthopaedic Society. Published by Wiley Periodicals, Inc. J Orthop Res 26:865–871, 2008     

New strategies for disc repair: novel preclinical trials

Degeneration of lumbar intervertebral discs is a major cause of low back complaints, an irreversible occurrence with no currently available treatment. Furthermore, various surgical procedures can accelerate disc degeneration. On the other hand, recent experimental studies on disc cells have demonstrated an important role for the nucleus pulposus in preserving overall disc structure. The authors group has already found that nucleus pulposus cells activated annulus fibrous cells, and reinsertion of nucleus pulposus cells slowed further disc degeneration. We have designed three subsequent studies that were designed to examine further possibilities for clinical transplantation: (1) activation of nucleus pulposus cells by mesenchymal stem cells; (2) focus on the multilineage differentiation potential of mesenchymal stem cells as an alternative cell source for cell transplantation therapy of disc degeneration; (3) the possibility of a human nucleus pulposus cell line as a cell source for cell transplantation therapy. Activation of nucleus pulposus could be achieved by co-culture with autogenous mesenchymal stem cells allowed to have direct cellular interaction. This would be a useful clinical cell source. Induction of nucleus pulposus cells by autogenous mesenchymal stem cells also would be an important subject for a clinical trial. Clinical application of the cells derived from a human nucleus pulposus cell line is an important project to be undertaken in the near future.This Instructional lecture was presented at the 76th Annual Meeting of the Japanese Orthopaedic Association, May 2003