Injectable hydrogels with high fixed charge density and swelling pressure for nucleus pulposus repair: Biomimetic glycosaminoglycan analogues

The load-bearing biomechanical role of the intervertebral disc is governed by the composition and organization of its major macromolecular components, collagen and aggrecan. The major function of aggrecan is to maintain tissue hydration, and hence disc height, under the high loads imposed by muscle activity and body weight. Key to this role is the high negative fixed charge of its glycosaminoglycan side chains, which impart a high osmotic pressure to the tissue, thus regulating and maintaining tissue hydration and hence disc height under load. In degenerate discs, aggrecan degrades and is lost from the disc, particularly centrally from the nucleus pulposus. This loss of fixed charge results in reduced hydration and loss of disc height; such changes are closely associated with low back pain. The present authors developed biomimetic glycosaminoglycan analogues based on sulphonate-containing polymers. These biomimetics are deliverable via injection into the disc where they polymerize in situ, forming a non-degradable, nuclear “implant” aimed at restoring disc height to degenerate discs, thereby relieving back pain. In vitro, these glycosaminoglycan analogues possess appropriate fixed charge density, hydration and osmotic responsiveness, thereby displaying the capacity to restore disc height and function. Preliminary biomechanical tests using a degenerate explant model showed that the implant adapts to the space into which it is injected and restores stiffness. These hydrogels mimic the role taken by glycosaminoglycans in vivo and, unlike other hydrogels, provide an intrinsic swelling pressure, which can maintain disc hydration and height under the high and variable compressive loads encountered in vivo.     

The interaction between aggrecan gene VNTR polymorphism and cigarette smoking in predicting incident symptomatic intervertebral disc degeneration

An association between the aggrecan variable number of tandem repeat (VNTR) polymorphism and the disc degeneration has been previously reported in Finnish men, and smoking had previously been suspected of causing disc degeneration. However, the interaction between aggrecan gene VNTR polymorphism and smoking in symptomatic intervertebral disc degeneration (IDD) has not been well studied. To examine the interaction between aggrecan gene VNTR and smoking in the susceptibility of symptomatic IDD of Chinese Han in northern China, intervertebral discs of 132 participants were evaluated on magnetic resonance imaging, using decreased signal intensity. After harvesting the blood samples, the aggrecan gene VNTR region was analyzed using polymerase chain reaction (PCR). The data indicated that between the two groups, participants carrying one or two alleles ≤25 repeats who did not smoke showed a 1.102-fold increased risk for symptomatic IDD (p= 0.855; 95% confidence interval 0.389–3.119), and participants carrying two alleles >25 repeats who smoked more than 1 pack-year showed a 1.013-fold higher risk (p = 0.982; 95% confidence interval 0.333–3.084), whereas participants carrying one or two alleles ≤25 repeats who smoked more than 1 pack-year showed a 4.5-fold increased risk for symptomatic IDD (p = 0.005; 95% confidence interval 1.589–12.743). Overall, we observed an underlying additive and multiplicative interaction between the aggrecan gene VNTR polymorphism and smoking in symptomatic IDD.     

The effects of human intervertebral disc aggrecan on neuronal and endothelial cell growth

Introduction The development of ‘discogenic’ low back pain has been associated with increased nerve growth into degenerated intervertebral discs, particularly in neovascularized areas. A marked loss of proteoglycans, particularly from the disc's inner regions, is also a marked feature of the degenerative process. As proteoglycans from other tissues can have inhibitory effects on nerve growth, we have hypothesized that the alterations in proteoglycan content seen in disc degeneration may affect the growth of nerves and blood vessels into the disc. To test our hypothesis, we have established cell culture assays to determine the effects of human intervertebral disc aggrecan, which forms the major proteoglyan found in the disc, on neuronal and endothelial cell growth. Materials and methods Aggrecan (A1D1 preparations) isolated from the outer (anulus fibrosus, AF) and inner (nucleus pulposus, NP) regions of human lumbar intervertebral discs was incorporated into culture substrata, using methods previously described ( Snow et al. 1990 ). Chick dorsal root ganglia (DRG) and the cell line SH‐SY5Y were used as models of nerve growth. HMEC‐1 and Eahy‐926 cell lines were used as models of endothelial cell growth. Results Human disc aggrecan inhibited SHSY‐5Y cell attachment, SHSY5Y neurite outgrowth and induced sensory DRG neurite growth cone turning in a concentration‐dependent manner. Sensory neurites emanating from DRG across permissive substrates (collagen/laminin) were repelled by high (1 mg/ml), but not low (0.01 mg/ml) concentrations of disc aggrecan and became aligned to the aggrecan border. Disc aggrecan similarly inhibited endothelial cell adhesion, cell spreading and migration. HMEC‐1 and Eahy926 cells migrated over collagen substrates (type I) until they encountered disc aggrecan, where they either stopped migrating or, more commonly, changed their direction of movement and aligned to the aggrecan border. In general, aggrecan isolated from the AF was more inhibitory than that isolated from the NP. The inhibitory effects were partially abrogated following enzymic deglycosylation of the aggrecan, or if aggrecan was added in solution (i.e. was nonsubstrate bound). Conclusions This study provides evidence that disc aggrecan inhibits neuronal and endothelial growth and migration and therefore supports a hypothesis that a loss of aggrecan from degenerated discs predispose the tissue to vascular and neuronal invasion.     

The use of SA-β-Gal to assess cell senescence in intervertebral disc cells

Introduction The intervertebral disc is reported to age faster than other connective tissues with significant degenerative changes already seen in the second decade of life. In other tissues, senescent cells have an altered phenotype, often with a decreased synthetic ability and response to anabolic cytokines. They appear to contribute to age‐related pathologies such as the degeneration of articular cartilage in osteoarthritis. Little is known of cell senescence in the intervertebral disc. In this study, we have investigated the production of senescence‐associated‐ß‐galactosidase (SA‐ß‐Gal), a ‘biomarker’ of cell senescence, in intervertebral disc cells both in cultured populations in vitro, and in vivo, in pathological human discs. Material and methods Intervertebral disc cells were extracted from coccygeal bovine discs and cultured for 3 months. Confluent cell preparations were stained for SA‐β‐Gal ( Fenton et al. 2001 ) at passages 0, 1, 2, 3 and 4. Preparations were fixed in 3% paraformaldehyde in phosphate‐buffered saline (PBS) for 5 min at room temperature and incubated for 24 h at 37 °C in SA‐ß‐Gal solution containing 1 mg/ml 5‐bromo‐4‐chloro‐3‐indolyl ß‐d ‐galactopyranoside (X‐Gal, Sigma, Poole, UK), 5 mmol/l potassium ferrocyanide, 5 mmol/l potassium ferricyanide, 150 mmol/l NaCl, 2 mmol/l MgCl₂ and 40 mmol/l trisodium citrate, titrated with NaH₂PO₄ to pH 6.0. Lysosomal (nonsenescent) ß‐galactosidase activity was detected using the same solution but titrated to pH 4.0. After staining, preparations were rinsed in ice‐cold PBS, dehydrated and mounted. Pathological human disc from patients with disc herniations or discogenic back pain were also stained for SA‐β‐Gal (immersing the tissue in the stain solution overnight) then cryosectioning (10 µm thick) and fixing. Articular cartilage was studied for comparison ( Price et al. 2002 ). Results Cultured intervertebral discs demonstrated some staining for SA‐ß‐Gal at all passages investigated, but there was little change in staining with passage number. Cells in most pathological human discs demonstrated staining for SA‐ß‐Gal. Positive cells were seen more commonly in herniated discs [7/9 (78%); mean age: 46 ± 13] than in those removed from patients with discogenic back pain [2/6 (33%); mean age: 32 ± 5]. Discussion To our knowledge, this is the first study of cell senescence in intervertebral disc cells. The greatest level was seen in tissue from herniated discs where cell cluster formation and cell proliferation are common (but the mean age of the herniation group was slightly higher than the discogenic back pain group). However, for a tissue demonstrating such significant age‐related degenerative changes, there is surprisingly little expression of SA‐ß‐Gal in comparison with that found in other pathological cartilages. These preliminary data suggest that, unlike the situation in osteoarthritis, early cell senescence is not a major contributing factor in the pathogenesis of disc degeneration.     

Damage to the human lumbar cartilage endplate and its clinical implications

The cartilaginous endplate (CEP) is a thin layer of hyaline cartilage, and plays an important role in the diffusion of nutrients into the intervertebral discs. Its damage may seriously affect the disc degeneration, and result in low back pain (LBP). However, the structural features of damaged CEPs have not been well characterized, and this hinders our understanding of the etiology of disc degeneration and pain. To present the structural features of micro-damaged CEPs in patients with disc degeneration and LBP that might even be regarded as an initial factor for disc degeneration, we performed a histological study of micro-damaged CEPs harvested from human lumbar intervertebral discs and analyzed its clinical implications. Human lumbar CEPs were excised from 35 patients (mean age 60.91 years) who had disc degeneration and LBP. Control tissue was obtained from 15 patients (mean age 54.67 years) with lumbar vertebral burst fractures. LBP and disability were assessed clinically, and all patients underwent anterior vertebral body fusion surgery. CEPs together with some adjacent nucleus pulposus (NP) were sectioned at 4 µm, and stained using H&E, Safranin O/Fast Green, and Alcian Blue. Immunostaining and PCR were used to identify various markers of degeneration, innervation, and inflammation. Histology demonstrated physical micro-damage in 14/35 CEPs from the disc degeneration group. Six major types of damage could be distinguished: fissure, traumatic nodes, vascular mimicry, incorporation of NP tissue within the CEP, incorporation of bone within the CEP, and incorporation of NP and bone within the CEP. Pain and disability scores (ODI: p = 0.0190; JOA: p = 0.0205; JOABPEQ: p = 0.0034) were significantly higher in those with micro-damaged CEPs (N = 14) than in those with non-damaged CEPs (N = 21). CEP damage was significantly associated with elevated MMP3 (p = 0.043), MMP13 (p = 0.0191), ADAMTS5 (p = 0.0253), TNF-α (p = 0.0011), and Substance P (p = 0.0028), and with reduced Sox9 (p = 0.0212), aggrecan (p = 0.0127), and type II collagen (p = 0.0139). In conclusion, we presented a new classification of human lumbar micro-damaged CEPs. Furthermore, we verify disc degeneration, innervation, and discogenic pain in micro-damaged CEPs.     

腰椎间盘退变MRI：与Modic改变相关的影像学分析

背景：部分椎间盘源性下腰痛患者MRI可出现Modic改变,但Modic改变的相关因素及Modic改变与椎间盘退变之间因果关系目前尚不十分清楚。 目的：分析存在腰椎间盘Modic改变的下腰痛患者性别、年龄分布特点及腰椎间盘发生Modic改变的相关因素。 方法：回顾性分析634例(2 536个椎间盘)存在腰椎间盘Modic改变患者的性别、年龄分布特点,并分析腰椎间盘Modic改变与椎间盘突出或膨出、Schmorl结节、椎体滑脱、椎间盘解剖水平及椎间盘退行性改变程度的相关性。 结果与结论：634例患者中,女性患者ModicⅡ、Ⅲ型出现率均较男性高,而ModicⅠ型出现率小于男性患者(P < 0.001);40岁以上患者较40岁以下患者Modic各型改变的出现率均高(P < 0.001)。2 536个腰椎间盘中,有椎体滑脱、出现Schmorl结节、有椎间盘突出或膨出者Modic各型改变的出现率均比无此类表现者高(P < 0.001);L4/5、L5/S1水平(低位)Modic各型改变的出现率均比L2/3、L3/4水平(高位)高(P < 0.001);椎间盘退行性改变越严重,Modic各型改变的出现率越高(P < 0.001)。椎间盘退行性改变分级、Schmorl结节与Modic改变有显著相关性。结果说明,腰椎间盘Modic改变与患者性别、年龄、椎间盘有无突出或膨出、有无Schmorl结节、椎体有无滑脱、椎间盘解剖水平及椎间盘退行性改变分级均有相关性。其中,椎间盘退行性改变分级、Schmorl结节与腰椎间盘Modic改变间的相关性最高,且椎间盘退行性改变分级较Schmorl结节与之相关性更高。     

A novel rat tail disc degeneration model induced by static bending and compression

BackgroundA new rat tail intervertebral disc degeneration model was established to observe the morphologic and biologic changes of static bending and compression applied to the discs.MethodsIn total, 20 Sprague‐Dawley rats with similar weight were randomly divided into 4 groups. Group 1 served as a control group for a baseline assessment of normal discs. Group 2 underwent a sham surgery, using an external device to bend the vertebrae of coccygeal 8‐10. Groups 3 and 4 were the loaded groups, and external devices were instrumented to bend the spine with a compression level of 1.8 N and 4.5 N, respectively. Magnetic resonance imaging (MRI), histological, and quantitative real‐time PCR (qRT‐PCR) analysis were performed on all animals on day 14 of the experiment.ResultsMagnetic resonance imaging and histological results showed that the changes of intervertebral disc degeneration increased with the size of compression load. Some architecture disorganizations in nucleus pulposus and annulus fibrosus were found on both of the convex and concave side in the groups of 1.8 N and 4.5 N. An upregulation of MM‐3, MM‐13, and collagen 1‐α1 mRNA expression and a downregulation of collagen 2‐α1 and aggrecan mRNA expression were observed in the sham and loading groups. Significant changes were found between the loading groups, whereas the sham group showed similar results to the control group.ConclusionsStatic bending and compression could induce progressive disc degeneration, which could be used for biologic study on disc degeneration promoted by static complex loading.     

Resveratrol Has Anabolic Effects on Disc Degeneration in a Rabbit Model

This study was done to evaluate whether injections of resveratrol, a natural compound found in the skin of grapes, had anabolic effects on degenerated intervertebral discs in a rabbit model. Two non-continuous lumbar discs were punctured in rabbits to induce disc degeneration. Four weeks and 6 weeks after puncture, the rabbits were treated by injections with dimethylsulfoxide (DMSO) or resveratrol. At 4, 8, and 16 weeks after initial injection, rabbits were sacrificed and the spine was extracted for magnetic resonance image (MRI), mRNA expression, and histological staining. Resveratrol treatment resulted in stronger signal intensity in T2-weighted images. MRI grade showed significantly lower in the resveratrol group than the DMSO group (P = 0.039). In the resveratrol group, aggrecan gene expression was significantly increased than that in the DMSO group at 16 weeks after injection (P = 0.027). MMP-13 mRNA levels in the resveratrol group were significantly decreased than those in the DMSO group at 8 and 16 weeks (P = 0.006 and P = 0.048, respectively). In hematoxylin and eosin stain, resveratrol-treated discs showed the features of regeneration. Histologic grade revealed improvement in resveratrol-treated discs, compared with DMSO-treated discs (P = 0.024). These anabolic effects on degenerated discs indicate that resveratrol is a promising candidate for treatment of degenerative disc disease.     

Expression of chondrocyte markers by cells of normal and degenerate intervertebral discs.

AIMS: To investigate the phenotype of cells in normal and degenerate intervertebral discs by studying the expression of molecules characteristic of chondrocytes in situ. METHODS: Human intervertebral discs taken at surgery were graded histologically, and classified on this basis as normal or degenerate. Eighteen of each type were selected, and in situ hybridisation was performed for the chondrocytic markers Sox9 and collagen II using (35)S labelled cDNA probes. Aggrecan was located by immunohistochemistry, using the monoclonal antibody HAG7E1, and visualised with an avidin-biotin peroxidase system. RESULTS: In the normal discs, strong signals for Sox9 and collagen II mRNA, and strong staining for the aggrecan protein were seen for the cells of the nucleus pulposus (NP), but reactions were weak or absent over the cells of the annulus fibrosus (AF). In degenerate discs, the Sox9 and collagen II mRNA signals remained visible over the cells of the NP and were again absent in the AF. Aggrecan staining was not visible in the NP cells, and was again absent in the AF. CONCLUSIONS: Cells of the normal NP showed expression of all three markers, clearly indicating a chondrocytic phenotype. In degeneration, there was evidence of a loss of aggrecan synthesis, which may contribute to the pathogenesis of disc degeneration. AF cells showed no evidence of a chondrocytic phenotype in either normal or degenerate discs.     

An injectable cross-linked scaffold for nucleus pulposus regeneration

Incorporation of scaffolds has long been recognized as a critical element in most tissue engineering strategies. However with regard to intervertebral disc tissue engineering, the use of a scaffold containing the principal extracellular matrix components of native disc tissue (i.e. collagen type II, aggrecan and hyaluronan) has not been investigated. In this study the behavior of bovine nucleus pulposus cells that were seeded within non-cross-linked and enzymatically cross-linked, atelocollagen type II based scaffolds containing varying concentrations of aggrecan and hyaluronan was investigated. Cross-linking atelocollagen type II based scaffolds did not cause any negative effects on cell viability or cell proliferation over the 7-day culture period. The cross-linked scaffolds retained the highest proteoglycan synthesis rate and the lowest elution of sulfated glycosaminoglycan into the surrounding medium. From confined compression testing and volume reduction measurements, it was seen that the cross-linked scaffolds provided a more stable structure for the cells compared to the non-cross-linked scaffolds. The results of this study indicate that the enzymatically cross-linked, composite collagen-hyaluronan scaffold shows the most potential for developing an injectable cell-seeded scaffold for nucleus pulposus treatment in degenerated intervertebral discs.     

糖尿病与椎间盘退变：对其结构、细胞凋亡、终板供血及细胞外基质的影响

背景：糖尿病作为一种全身代谢性疾病,不但会引起碳水化合物以及脂肪、蛋白质代谢的紊乱,同时极易发生周围微血管的病变,影响包括椎间盘在内的全身多器官组织的营养代谢过程。目的：综述近年来国内外关于糖尿病对椎间盘退变的影响的研究进展。方法：应用计算机由第一作者在PubMed数据库和中国知网CNKI数据库,以“intervertebral disc,degeneration,diabetes mellitus”为英文检索词,以“椎间盘,退变,糖尿病”为中文检索词,在1981年1月至2014年1月间共检索到8 414篇相关文献,排除重复研究,共34篇文献符合纳入标准。结果与结论：有关糖尿病慢性并发症的发病机制极为复杂,细胞凋亡是近年来关注的又一热点,研究证明高血糖为椎间盘细胞凋亡的诱导因素之一。糖尿病易引起全身的微小血管病变,因此也极有可能引起椎体终板内血管芽收缩,造成局部血流降低或中断,继而沿终板途径运输的营养物质减少,最终导致椎间盘营养障碍,从而引起椎间盘的退变。椎间盘细胞外基质的减少是椎间盘退变的主要原因。关于糖尿病对椎间盘退变影响的机制尚未清楚,尚需更进一步的研究加以阐述和证实。中国组织工程研究杂志出版内容重点：组织构建;骨细胞;软骨细胞;细胞培养;成纤维细胞;血管内皮细胞;骨质疏松;组织工程     

腰椎间盘的扩散张量成像观测及临床应用

目的　为腰椎间盘退变导致的腰腿痛等病症的临床诊断提供影像学依据。 方法　选取脊柱腰段扩散张量成像（DTI）扫描检查正常者200例和腰椎间盘退变者100例,在工作站划分腰椎间盘和腰椎间盘退变的感兴趣区,测量感兴趣区的表观扩散系数（ADC）和各向异性分数（FA）,比较不同解剖部位、年龄组腰椎间盘和不同Pfirrmann分级腰椎间盘退变的FA、ADC值。 结果　不同解剖部位腰椎间盘的FA值和ADC值均有统计学差异（P<0.05）,L1~2、L2~3、L3~4椎间盘的FA值逐渐降低,L4~5、L5~S1椎间盘的FA值则逐渐增高;L1~2、L2~3、L3~4椎间盘的ADC值逐渐增高,L3~4、L4~5、L5~S1椎间盘的ADC值则无明显变化。不同年龄组腰椎间盘的FA值无统计学差异（P>0.05）,ADC值有统计学差异（P<0.05）,随着年龄增长ADC值逐渐降低。不同Pfirrmann分级腰椎间盘退变的FA值和ADC值均有统计学差异（P<0.05）,随着Pfirrmann分级增高,FA值逐渐增高,ADC值逐渐降低。 结论　解剖部位、年龄均影响腰椎间盘的ADC值,DTI的FA值和ADC值可以定量评估腰椎间盘及其退变程度,为早期腰椎间盘退变的临床诊断提供影像学依据。     

腰椎间盘的扩散张量成像观测及临床应用

目的　为腰椎间盘退变导致的腰腿痛等病症的临床诊断提供影像学依据。 方法　选取脊柱腰段扩散张量成像（DTI）扫描检查正常者200例和腰椎间盘退变者100例,在工作站划分腰椎间盘和腰椎间盘退变的感兴趣区,测量感兴趣区的表观扩散系数（ADC）和各向异性分数（FA）,比较不同解剖部位、年龄组腰椎间盘和不同Pfirrmann分级腰椎间盘退变的FA、ADC值。 结果　不同解剖部位腰椎间盘的FA值和ADC值均有统计学差异（P<0.05）,L1~2、L2~3、L3~4椎间盘的FA值逐渐降低,L4~5、L5~S1椎间盘的FA值则逐渐增高;L1~2、L2~3、L3~4椎间盘的ADC值逐渐增高,L3~4、L4~5、L5~S1椎间盘的ADC值则无明显变化。不同年龄组腰椎间盘的FA值无统计学差异（P>0.05）,ADC值有统计学差异（P<0.05）,随着年龄增长ADC值逐渐降低。不同Pfirrmann分级腰椎间盘退变的FA值和ADC值均有统计学差异（P<0.05）,随着Pfirrmann分级增高,FA值逐渐增高,ADC值逐渐降低。 结论　解剖部位、年龄均影响腰椎间盘的ADC值,DTI的FA值和ADC值可以定量评估腰椎间盘及其退变程度,为早期腰椎间盘退变的临床诊断提供影像学依据。     

多次胶原酶消化法培养小鼠椎间盘髓核细胞

背景：椎间盘为无血运组织,椎间盘髓核细胞为分化终末细胞,细胞增殖能力较差,体外培养难度较大。 目的：探索小鼠椎间盘髓核细胞体外分离培养的方法。 方法：取小鼠椎间盘髓核组织,使用多次胶原酶消化的方法,分离培养髓核组织细胞,接种,传代,取第2代细胞,分别采用免疫细胞化学和RT-PCR方法检测椎间盘髓核细胞特征性分泌物Ⅱ型胶原和聚合蛋白的分泌量及mRNA的表达,并与软骨细胞,成骨细胞及成纤维细胞进行比较。 结果与结论：椎间盘髓核细胞贴壁后呈现软骨细胞的形态;Ⅱ型胶原和聚合蛋白染色均为阳性;Ⅱ型胶原和聚合蛋白mRNA表达与软骨细胞相同,与成纤维细胞和成骨细胞存在明显差别。说明多次胶原酶消化的方法可以获得大量纯净的椎间盘髓核细胞,性状稳定。     

Effect of Severity of Intervertebral Disc Injury on Mesenchymal Stem Cell-Based Regeneration

Mesenchymal stem cell (MSC) implantation has been shown previously to arrest disc degeneration. This study aims to assess the effect of severity of disc degeneration on the ability of MSCs to arrest the degeneration. Disc degeneration was induced in New Zealand white rabbits at lumbar levels by annular puncture. The degeneration was allowed to progress for 1 month (early group) or 7 months (late group), followed by intradiscal injection of autologous MSCs. For disc levels that received MSCs treatment, 1 × 10⁵ BrdU-labeled MSCs were injected per disc level. For the early group, MSC-injection had no significant effects on disc height or the progression of disc degeneration. For the late group, although the MSC-injected discs displayed lower disc heights than the control discs, they were significantly less degenerated together with near normal level of proteoglycan in localized areas. This is the first pilot study to demonstrate that severity of degeneration can influence the therapeutic effect of MSCs. Future studies of cell-based intervertebral disc regeneration should be carefully controlled in the context of stage of disc degeneration.     

人颈椎间盘退变与细胞凋亡及基质金属蛋白酶11的表达

背景：前期研究发现基质金属蛋白酶11基因在人退变颈、腰椎间盘组织中明显上调。 目的：观察人退变颈椎间盘髓核组织中基质金属蛋白酶11的表达与细胞凋亡的关系。 方法：纳入30个经MRI确认的退变颈椎间盘髓核组织和20个因颈椎创伤治疗获得的正常颈椎间盘髓核组织。 结果与结论：苏木精-伊红染色显示退变的颈椎间盘髓核组织中髓核细胞较正常髓核组织明显减少(P < 0.01),而凋亡细胞较正常髓核组织明显增多(P < 0.01)。免疫组化染色显示退变的颈椎间盘髓核组织中基质金属蛋白酶11的表达明显高于正常髓核组织(P < 0.01),且基质金属蛋白酶11表达与TUNEL染色检测到的细胞凋亡正相关(r=0.44,P < 0.05)。说明高表达的基质金属蛋白酶11不仅可直接破坏细胞外基质尚可诱导髓核细胞凋亡,在椎间盘退变的过程中发挥重要作用。     

Human annulus cells regulate PAPP-A and IGFBP-4 expression,and thereby insulin-like growth factor bioavailability,in response to proinflammatory cytokine exposure in vitro

Abstract

Pregnancy-associated plasma protein-A (PAPP-A) is a metalloproteinase which cleaves IGF binding protein (BP)-4 in the extracellular matrix, making IGF available to nearby cells. We have shown that PAPP-A is present in the human intervertebral disc, and is significantly upregulated in more degenerated discs where increased proinflammatory cytokine levels are present. We hypothesized that increased proinflammatory cytokines present in the degenerating disc might be related to PAPP-A expression. Experiments exposed human annulus cells to IL-1-β or TNF-α to test this hypothesis. Treated cells showed significantly increased PAPP-A in conditioned media versus controls (p?<?0.001). PAPP-A production following exposure to IL-1β was significantly greater in cells derived from more degenerated versus healthier discs (p?=?0.05). PAPP-A gene expression (microarray analysis) was significantly upregulated in IL-1β- or TNF-α-exposed cells (p?=?0.01–0.004). Quantitative RT-PCR confirmed significant upregulation of IGFBP-4 in IL-1β- or TNF-α-exposed cells. Data have potential relevance to future cell-based biologic therapies for disc degeneration.     

Assessment of glycosaminoglycan distribution in human lumbar intervertebral discs using chemical exchange saturation transfer at 3 T: feasibility and initial experience

Recent studies have proposed that glycosaminoglycan chemical exchange saturation transfer (gagCEST) is associated with a loss of glycosaminoglycans (GAGs), which may be an initiating factor in intervertebral disc (IVD) degeneration. Despite its promising potential, this application has not been reported in human in vivo IVD studies because of the challenges of B₀ magnetic field inhomogeneity in gagCEST. This study aimed to evaluate the feasibility of quantifying CEST values in IVDs of healthy volunteers using a clinical 3 T scanner. A single‐slice turbo spin echo sequence was used to quantify the CEST effect in various GAG phantoms and in IVDs of 12 volunteers. The phantom results indicated high correlation between gagCEST and GAG concentrations (R² = 0.95). With optimal B₀ inhomogeneity correction, in vivo CEST maps of IVDs showed robust contrast between the nucleus pulposus (NP) and the annulus fibrosus (AF) (p < 0.01), as well as higher signal in the central relative to the peripheral NP. In addition, a trend of decreasing CEST values from upper to lower disc levels was evident in NP. Our results demonstrate that in vivo gagCEST quantification in human lumbar IVDs is feasible at 3 T in combination with successful B₀ inhomogeneity correction, but without significant hardware modifications. Our initial findings suggest that it would be worthwhile to perform direct correlation studies between CEST and GAGs using cadaver samples, and to extend this novel technique to studies on patients with degenerative discs to better understand its distinct imaging features relative to conventional techniques. Copyright © 2011 John Wiley & Sons, Ltd.     

不同代次兔髓核细胞的形态学特征和生物学性状

背景：椎间盘退变是个慢性、复杂的过程,然而椎间盘退变其发生机制尚未完全阐明,很难自行修复。近年来研究细胞移植治疗椎间盘退行性变尚处在实验室阶段。研究髓核细胞的生物学性状可为研究椎间盘退变机制、组织工程构建椎间盘、基因治疗等提供理论依据。 目的：研究兔不同代次髓核细胞的生物学特性,旨在找出合适的种子细胞去治疗椎间盘退变性疾病。 方法：从新西兰大耳白兔椎间盘髓核组织中,分离并培养髓核细胞同时进行培养传代,对原代及第3,4代髓核细胞进行苏木精-伊红染色观察细胞形态学变化;甲苯胺蓝染色和免疫细胞化学法检测髓核细胞内聚集蛋白聚糖和Ⅱ型胶原的表达;反转录PCR法测定Ⅱ型胶原和聚合蛋白聚糖mRNA的表达水平,观察各代髓核细胞生物学特性的变化。 结果与结论：兔椎间盘髓核细胞可以在体外培养并进行传代,原代髓核细胞一般需7 d左右贴壁,形状呈类圆形或多角形,原代和第3代髓核细胞都呈圆形或多角形,活力较强,苏木精-伊红染色后细胞核被染成均一蓝黑色,胞浆呈现淡粉色;髓核细胞经过甲苯胺蓝染色后,胞浆内呈现天蓝色,通过Ⅱ型胶原免疫组织化学染色后,胞浆内表现为黄褐色沉淀。到第4代细胞出现退变,Ⅱ型胶原和聚合蛋白聚糖mRNA的表达水平较前几代细胞显著下降。前3代的髓核细胞代谢旺盛,表型一致,聚集蛋白聚糖和Ⅱ型胶原表达正常,传第4代后髓核细胞开始出现衰老、退变。     

Cell Cluster Formation in Degenerate Lumbar Intervertebral Discs is Associated with Increased Disc Cell Proliferation

Healthy human intervertebral discs contain relatively few cells and these are sparsely distributed. A characteristic feature of disc degeneration, however, is the appearance of cell clusters, particularly in damaged areas. How these clusters form is currently unknown. We have examined excised pathological human discs for evidence of cell proliferation. Disc sections were immunostained for the proliferating cell nuclear antigen (PCNA) and the proliferation-associated Ki-67 antigen. PCNA immunopositive cells were observed within degenerate discs, commonly though not exclusively, in cell clusters. Cells immunopositive for the Ki-67 antigen were less prevalent than those for PCNA, but similarly were observed frequently within clusters in degenerate discs. In contrast, immunopositivity for these markers was not common in less degenerate discs or in areas of the disc where cell clusters were not observed. These observations suggest that disc cell proliferation is associated with disc degeneration and is the likely cause of cell cluster formation.