Inhibition of vertebral endplate perfusion results in decreased intervertebral disc intranuclear diffusive transport

Impaired nutrition of the intervertebral disc has been hypothesized to be one of the causes of disc degeneration. However, no causal relationship between decreased endplate perfusion and limited nutrient transport has been demonstrated to support this pathogenic mechanism. To determine the importance of endplate perfusion on solute diffusion into the nucleus pulposus and to show causality of endplate perfusion on intranuclear diffusion in large animal lumbar intervertebral discs, diffusive transport into ovine lumbar intervertebral discs was evaluated after inhibiting adjacent vertebral endplate perfusion. Partial perfusion blocks were created in vertebrae close and parallel to both endplates of lumbar discs of anaesthetized sheep. To assess diffusivity of small molecules through the endplate, N2O was introduced into the inhalation gas mixture and concentrations of intranuclear N2O were measured for 35 min thereafter. Post mortem, procion red was infused through the spinal vasculature and perfusion through the endplate was assessed by quantifying the density of dye-perfused endplate vascular buds in histology sections. Perfusion of the endplates overlying the nucleus pulposus was inhibited by almost 50% in the partially blocked discs relative to the control discs. There was also a nine-fold decreased transport rate of intranuclear N2O in partially blocked discs compared with control discs. The density of perfused endplate vascular buds correlated significantly to the amount of transported intranuclear N2O (r2 = 0.52, P = 0.008). The vertebral endplate was demonstrated to be the main route of intravascular solute transport into the nucleus pulposus of intervertebral discs, and inhibition of endplate perfusion can cause inhibited solute transport into the disc intranuclear tissue.     

Development of a bioreactor for axially loaded intervertebral disc organ culture

Whole disc organ culture is needed for preclinical testing of biological repair of the degenerate intervertebral disc. Such organ culture is hampered by two major limitations: first obtaining adequate nutrition though the calcified cartilage endplates adjacent to the vertebral bone, and second by loss of tissue integrity if the endplates are removed from the discs. In this work we utilize a recently described technique for whole disc isolation that overcomes these problems, by removal of vertebral bone and the adjacent calcified portion of the endplate, and the construction of a bioreactor that permits long-term viability of these discs in loaded organ culture. The bioreactor consists of a culture chamber in which the disc can be dynamically loaded in a uniform manner. The culture chamber is large enough to accommodate discs up to 60 mm in diameter, and so is amendable to study both bovine and human discs. The discs are loaded in the culture chamber by upper and lower platens, which conform to the shape of the remaining cartilaginous endplate and permit fluid flow across its surface. The bioreactor is able to load the disc under a variety of conditions ranging from static to dynamic and from physiological to pathological, and monitor induced changes in disc height. To date, bovine caudal discs have been maintained viable in the bioreactor for up to 4 weeks without any appreciable loss of disc height under physiological cyclic load and, in principle, could be maintained in such a manner for several months. Such long-term organ culture is essential for studying biological repair of the disc.     

骨保护素基因敲除小鼠腰椎间盘退变与腰椎骨质疏松

背景：骨保护素基因敲除小鼠已被证明会表现出明显的骨质疏松及骨关节炎表型。 目的：观察骨保护素基因敲除小鼠随年龄增长腰椎间盘退变和骨质疏松的动态变化关系。 方法：分别取出生后4,8,12周的骨保护素基因敲除纯合子小鼠及正常对照组小鼠的L3椎体和L4/5椎间盘,运用Micro-CT检测L3椎体松质骨微结构指标;用苏木精-伊红染色法观察L4/5椎间盘形态学,测量椎间盘及软骨终板高度。 结果与结论：骨保护素基因敲除纯合子小鼠组L3椎体骨小梁数量、骨小梁厚度、骨体积分数较正常组均明显下降(P < 0.05),而骨小梁分离度、结构模型指数较正常小鼠增高(P < 0.05)。8周及12周的骨保护素基因敲除纯合子小鼠的L4/5椎间盘软骨终板出现退变征象,软骨终板排列不规则,并有骨髓腔组织进入软骨终板及外层纤维环。提示骨保护素基因在维持椎间盘正常的结构和功能方面起到重要作用,骨保护素基因缺失后可导致椎间盘退变和椎体骨质疏松。     

椎间盘突出患者椎间盘Fas基因的表达

背景：临床中发现腰椎间盘突出症常在一个家族中多人甚至全部发病,而且发病的部位、原因、症状基本一致,这表明基因在该种病症中扮演着重要的角色。 目的：分析家族性腰椎椎间盘突出症患者椎间盘内Fas凋亡基因表达的特点。 方法：用半定量RT-PCR方法检测15例家族性腰椎椎间盘突出患者、21例普通椎间盘突出患者、5例新鲜尸体椎间盘的软骨终板和髓核组织中Fas基因的表达情况。 结果与结论：家族性椎间盘突出与普通椎间盘突出患者终板内Fas基因的表达均高于新鲜尸体椎间盘终板内Fas基因的表达,差异有显著性意义(P < 0.05),家族性椎间盘突出与普通椎间盘突出患者终板内Fas基因的表达差异无显著性意义(P > 0.05)。家族性椎间盘突出患者髓核内Fas基因表达与普通椎间盘突出患者、新鲜尸体髓核内Fas基因表达相比较,差异无显著性意义(P > 0.05)。结果可见家族性椎间盘终板中Fas基因的表达增加可能是继发性的,可以通过阻止终板退变来达到阻止椎间盘退变的目的。     

脊柱内固定后椎间盘营养途径的实验研究

目的：为了研究脊柱内固定后椎间盘的营养途径。方法：24只日本大耳白兔，行墨汁灌注后，观察同一动物非固定区中椎间盘与固定区域椎间盘的墨汁分布情况，观察相同位置对照组与实验组的墨汁分布情况。结果：软骨终板有血管从中经过，椎间盘营养来源分主要和次要途径，内固定后软骨终板和椎体之间产生分离，椎间盘会新生较多小血管，且血管缓慢而淤积。结论：内固定节段椎间盘处于低应力状态下，一定时间后，营养代谢的改变会导致椎间盘退行样变的发生。     

Matrix metalloproteinase-12 immunolocalization in the degenerating human intervertebral disc and sand rat spine: Biologic implications

Matrix metalloproteinase-12 (MMP-12; macrophage metalloelastase) degrades a number of extracellular matrix components which are present in the intervertebral disc, including type IV collagen, fibronectin, laminin, chondroitin sulfates, elastin and fibrinogen. MMP-12 has recently discovered relationships with cytokines and chemokines which also relate to disc cell biology. To date, no study has assessed immunolocalization of MMP-12 in degenerating human intervertebral disc tissue. Immunocytochemical localization was performed on 18 human disc specimens and on lumbar spines of the sand rat, a small animal model with well-recognized age-related disc degeneration. In the human disc, intracellular localization was present in both the annulus and nucleus portions of the disc. The sand rat degenerating disc also showed MMP-12 disc localization, with additional presence in chondrocytes of the vertebral endplate of older animals. This is the initial characterization of the presence of MMP-12 in the human and sand rat disc, and in chondrocytes of the vertebral endplate in older sand rats with degenerating discs. Findings are important because they document the presence of an additional MMP-12 in disc tissue, thus expanding our understanding of disc extracellular matrix remodeling, and because they provide novel information on the presence of MMP-12 in the cartilage endplate as it undergoes sclerosis during disc degeneration in the aging sand rat.     

Why do some intervertebral discs degenerate,when others (in the same spine) do not?

This review suggests why some discs degenerate rather than age normally. Intervertebral discs are avascular pads of fibrocartilage that allow movement between vertebral bodies. Human discs have a low cell density and a limited ability to adapt to mechanical demands. With increasing age, the matrix becomes yellowed, fibrous, and brittle, but if disc structure remains intact, there is little impairment in function, and minimal ingrowth of blood vessels or nerves. Approximately half of old lumbar discs degenerate in the sense of becoming physically disrupted. The posterior annulus and lower lumbar discs are most affected, presumably because they are most heavily loaded. Age and genetic inheritance can weaken discs to such an extent that they are physically disrupted during everyday activities. Damage to the endplate or annulus typically decompresses the nucleus, concentrates stress within the annulus, and allows ingrowth of nerves and blood vessels. Matrix disruption progresses by mechanical and biological means. The site of initial damage leads to two disc degeneration “phenotypes”: endplate‐driven degeneration is common in the upper lumbar and thoracic spine, and annulus‐driven degeneration is common at L4‐S1. Discogenic back pain can be initiated by tissue disruption, and amplified by inflammation and infection. Healing is possible in the outer annulus only, where cell density is highest. We conclude that some discs degenerate because they are disrupted by excessive mechanical loading. This can occur without trauma if tissues are weakened by age and genetic inheritance. Moderate mechanical loading, in contrast, strengthens all spinal tissues, including discs. Clin. Anat. 28:195–204, 2015. © 2014 Wiley Periodicals, Inc.     

Menopause causes vertebral endplate degeneration and decrease in nutrient diffusion to the intervertebral discs

The vasculature in the outer annulus supplies only the periphery of the disc so that nutrition to the bulk of the disc, including all the inner annulus and nucleus pulposus, is derived from the vertebral epiphyseal end arteries where nutrients diffuse across the cartilaginous endplate to reach the disc. In this regard the vertebral endplate plays an important role in disc nutrition. Compromise of diffusion of nutrients to the disc cells may play a large part in the progression or even initiation of disc degeneration. Increasing evidence suggests that estrogen deficiency also influence the severity of disc degeneration in post-menopausal females. Structural disorganization of the vertebral endplate occurs with disc degeneration, with the most common endplate changes observed clinically being Schmorl’s node. Schmorl’s node is more commonly seen in post-menopausal women than younger women. Osteosclerosis, osteonecrosis and fibrosis associated with Schmorl’s nodes can impede nutrient diffusion into the disc as well as removal of metabolites from the disc. We hypothesize that menopause negatively affects vertebral endplate quality and induces endplate degeneration. This endplate degeneration decreases nutrients diffusion from vertebral body into discs, and also impedes removal of metabolites, leads to further disc degeneration. To confirm our hypothesis, a cross-sectional post-contrast MRI study can be performed in pre-menopausal and post-menopausal women. If the hypothesis is confirmed, then low dose hormone replacement treatment may retard disc degeneration in post menopausal women and thereby limit the consequences associated with disc degeneration such as low back pain.     

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     

Pathological changes in the cartilaginous plates in relation to intervertebral disc lesions

Three hundred sixty-eight intervertebral discs (T11/12-L5/S1) were obtained at autopsy from 61 individuals (36 male, 25 female) ranging from 25 to 85 years of age, and subsequently examined histopathologically as sagittal-sectioned specimens with special reference to the cartilaginous plates. The numbers of cartilaginous foci found in fissured and ruptured regions of the plates were found to increase with age, and were considered to represent a restoration mechanism. Measurement of the cartilaginous plate/intervertebral disc antero-posterior length ratio showed a decrease with age in intervertebral discs from the same spinal level. Therefore, cartilage cell proliferation in the vertebral body rim was found following rupture of the outer layer of the annulus fibrosus and was thought to be one of the causes of spur formation in spondylosis deformans. When the changes in a cartilaginous plate with aging were accompanied by destructive processes of the vertebrae such as osteoporosis or metastatic cancer, an increase in the height of the disc, or ballooning, developed. On the other hand, when degeneration of the intervertebral disc increased and the nucleus pulposus collapsed, the height of the disc decreased. Thus, although the cartilaginous plate exhibits a restoration mechanism, degeneration with age progresses, resulting in various disc lesions.     

Intervertebral disc height in treated and untreated overweight post-menopausal women

BACKGROUND: The effect of the menopause and HRT on the intervertebral discs has not been investigated. METHODS: One hundred women were recruited, comprising of 44 post-menopausal women on HRT, 33 untreated post-menopausal women and 23 pre-menopausal women. The height of the intervertebral discs between the 12th thoracic vertebra and the 3rd lumbar vertebra was measured by utilizing the bone densitometer height cursors. RESULTS: The untreated menopausal group of women had the lowest total disc height (D1-D3: 1.95 0.31 cm). This was significantly lower than the pre-menopausal group D1-D3: 2.16 0.24 cm) and the hormone-treated group (2.2 0.26 cm) (P > 0.02). The 2nd intervertebral disc consistently maintained a significant difference between the untreated menopausal group (D2: 0.63 0.13) and the other two groups (pre-menopausal group (D2: 0.72 0.09 cm) and treated menopausal group (D2: 0.73 0.12 cm) (P > 0.02). CONCLUSIONS: Estrogen-replete women appear to maintain higher intervertebral discs compared to untreated post-menopausal women. The estrogenic milieu may be relevant because of the significant impact it has on the hydrophilic glycosaminoglycans, the water content, collagen and elastin of the intervertebral discs. The maintenance of adequate disc height may allow the intervertebral discs to retain their discoid shape and viscoelastic function, containing vertical forces which may threaten spinal architecture leading to vertebral body compression fractures.     

Responses and adaptations of intervertebral disc cells to microenvironmental stress: a possible central role of autophagy in the adaptive mechanism

Intervertebral discs comprise the largest avascular cartilaginous organ in the body, and its nutrient condition can be impaired by degeneration, aging and even metabolic disease. The unique microenvironment brings special stresses to various disc cell types, including nucleus pulposus cells, notochordal cells, annulus fibrosus cells and endplate chondrocytes. These cells experience nutrient starvation, acidic stress, hypoxic stress, hyperglycemic stress, osmotic stress and mechanical stress. Understanding the detailed responses and complex adaptive mechanisms of disc cells to various stresses might provide some clues to guide therapy for disc degeneration. By reviewing the published literatures describing disc cells under different hostile microenvironments, we conclude that these cells exhibit different responses to microenvironmental stresses with different mechanisms. Moreover, the interaction and combination of these stresses create a complex environment that synergistically increase or decrease influences on disc cells, compared with the effects of a single stress. Interestingly, most of these stresses activate autophagy, a self-protective mechanism by which dysfunctional protein and organelles are degraded. It is becoming clear that autophagy facilitates the cellular adaptation to stresses and might play a central role in regulating the adaptation of disc cells under stress. Therefore, autophagy modulation might be a potential therapeutic method to treat disc degeneration.     

Geometry of the Intervertebral Volume and Vertebral Endplates of the Human Spine

Replacement of a degenerated vertebral disc with an artificial intervertebral disc (AID) is currently possible, but poses problems, mainly in the force distribution through the vertebral column. Data on the intervertebral disc space geometry will provide a better fit of the prosthesis to the vertebrae, but current literature on vertebral disc geometry is very scarce or not suitable. In this study, existing CT-scans of 77 patients were analyzed to measure the intervertebral disc and vertebral endplate geometry of the lumbar spine. Ten adjacent points on both sides of the vertebrae (S1-superior to T12-inferior) and sagittal and transverse diameters were measured to describe the shape of the caudal and cranial vertebral planes of the vertebrae. It was found that the largest endplate depth is located in the middle or posterior regions of the vertebra, that there is a linear relationship between all inferior endplate depths and the endplate location (p < 0.0001) within the spinal column, and that the superior endplate depth increases with age by about 0.01 mm per year (p < 0.02). The wedge angle increases from T12-L1 to L5-S1. The results allow for improvement of the fit of intervertebral disc-prostheses to the vertebrae and optimized force transmission through the vertebral column.     

Improving the fixation of an artificial intervertebral disc

The fixation of an artificial intervertebral disc has been studied especially with respect to the dimensions, the convexity of the endplates and the size of the fixation elements. From literature and cadaveric vertebrae, the dimensions and shape of the lumbar vertebral endplates were determined and the dimensions of fixation ribs for the artificial intervertebral disc were calculated. To withstand shear forces and prevent dislocation, two sagittal ribs, each 3.5 mm in height and at least 20 mm in length and four transversal ribs, each 1.5 mm in height and with a total length of 60 mm are sufficient. A range of five different sagittal diameters was selected as sufficient for all patients. At least 72.6 % of the endplate of the vertebrae is covered. A convexity with a radius of 140 mm limits the gap to 0.62 mm.     

Influence of age and gender on thoracic vertebral body shape and disc degeneration: an MR investigation of 169 cases

There are limited data detailing the pattern of age and gender-related changes to the thoracic vertebral bodies and intervertebral discs. A retrospective MR investigation, involving T1-weighted midsagittal images from 169 cases, was undertaken to examine age influences on the anterior wedge (anteroposterior height ratio or Ha/Hp), biconcavity (midposterior height ratio or Hm/Hp), and compression indices (posterior height/anteroposterior diameter or Hp/D) of the thoracic vertebral bodies. Disc degenerative changes in the annulus, nucleus, end-plate and disc margin were noted on T2-weighted sagittal images for the 169 cases, based on a 3-level grading system. A linear age-related decline in the Ha/Hp and Hm/Hp indices was noted. The Hp/D index increased during the first few decades of life, then decreased gradually thereafter. The prevalence of abnormal findings in the annuli, nuclei and disc margins increased with increasing age, particularly in the mid and lower thoracic discs. Greater disc degenerative changes were observed in males. These findings provide further insight into the nature of thoracic vertebral shape changes across the lifespan, and the typical patterns of degeneration of the thoracic intervertebral discs.     

腰痹舒干预腰椎间盘退变模型兔髓核组织水通道蛋白1、水通道蛋白3的表达

BACKGROUND: The aging and lesions of the intervertebral disc are closely related to the lack of nutritional blood supply to the disc. Aquaporin plays an important role in the nutritional supply to the intervertebral discs, but the specific mechanism has not been fully defined.     

Intraoperative malposition of pedicle probe or screws: a potential cause of the acceleration of degeneration in superior adjacent intervertebral disc

Adjacent segment degeneration (ASD) is considered as a long-term complication of spinal fusion procedure. Numerous clinical studies have reported some factors related with ASD, but few could address the reason why the incidence of caudal ASD is significantly lower than that of cranial ASD. Because the pedicle of vertebral arch is closer to the superior endplate of vertebrea and its cranial intervertebral disc, there might be some possibilities of malpositions of pedicle probe or screws into the superior vertebral endplate or disc during the procedure of posterior intervertebral fusion. A number of evidences have showed that puncture of intervertebral disc will result in disc degeneration. Thus the authors put forward the hypothesis that intraoperative malposition of pedicle probe or screws might be a cause of ASD at cranial segments.     

In situ oxygen utilization in the rat intervertebral disc

Nucleus pulposus cells of the intervertebral disc have no endogenous vasculature and have thus been hypothesized to be hypoxic. This hypothesis was tested using 2-nitroimidazole, EF5, a drug that at low oxygen concentrations forms covalent adducts with cellular proteins. After administrating EF5 to rats, sections of the intervertebral disc were analysed for EF5 adducts. Drug adducts were quantified in tissue sections using a fluorescent monoclonal antibody. Although the level of EF5 fluorescence in all intervertebral disc tissues was low, the transition zone at the periphery of the nucleus pulposus exhibited the highest level of EF5 binding. To substantiate this result, tissue nitroreductase levels and drug pharmacology were evaluated. Nitroreductase levels were measured in whole discs under severe hypoxia. We noted that there was robust EF5 binding to cells in the annulus fibrosus and transition zone with modest binding to cells of the nucleus pulposus and endplate. High-performance liquid chromatography analysis indicated limitations in EF5 access to the nucleus pulposus, most probably related to the lack of vasculature and slow drug distribution through the gel-like interior of the disc. However, despite diffusion problems, the drug dose was determined to be sufficient to report the oxygen status of the nucleus pulposus cells. Based on these findings, we conclude that despite poor vascularization, the disc cells accommodate to the local environment by displaying a limited need for oxygen. Accordingly, the cells of the intervertebral disc are not severely hypoxic.     

针刺损伤兔椎间盘退变模型组织病理变化及软骨样细胞的迁移规律**☆

背景：兔椎间盘退变模型间盘退变表现为随时间进展脊索细胞将被软骨样细胞逐渐替代,但兔针刺纤维环间盘退变模型中软骨样细胞的来源和移行规律尚不明确。 目的：观察针刺兔纤维环间盘退变模型椎间盘病理变化过程,并初步探讨软骨样细胞来源及移行规律。 方法：将24只新西兰大白兔随机分为手术组与假手术组。手术组使用16 G穿刺针针刺L2/L3、L3/L4、L4/L5及L5/L6椎间盘纤维环,假手术组暴露至相同椎间盘前方后冲洗闭合伤口。 结果与结论：针刺损伤椎间盘退变过程中的软骨样细胞来源于终板。在髓核与上下终板交界处,软骨细胞脱离终板成串向髓核中心迁移;在髓核与内层纤维环交界处,软骨细胞沿纤维走行迁移并随之向皱缩的髓核边缘迁移。椎间盘退变过程中非钙化层逐渐变薄,非钙化层/钙化层比值逐渐降低。 关键词：椎间盘退变;软骨细胞;软骨终板;纤维环穿刺;新西兰大白兔  doi:10.3969/j.issn.1673-8225.2012.09.018     

Human intervertebral disc cell morphology and cytoskeletal composition: a preliminary study of regional variations in health and disease

Classically, intervertebral disc cells have been described as fibrocytic in the anulus fibrosus and chondrocytic in the nucleus pulposus. Recent animal studies, however, have suggested that disc cell morphology may be more complex than previously considered. Here, by utilizing labelling of components of the cytoskeleton in combination with confocal microscopy, we have examined the detailed morphology of human intervertebral disc cells in pathological and non-pathological tissue. Filamentous-actin- and vimentin-positive cells that appeared either fibrocytic or chondrocytic were observed in all intervertebral discs. However, in localized areas of the disc, stellate cells that extended multiple, branching cytoplasmic processes into their surrounding matrix were also seen. This stellate appearance formed a marked feature of disc cells regionally in certain pathologies, i.e. in cells of the outer anulus fibrosus in scoliotic discs and in inner anulus/nucleus pulposus cells in one spondylolisthetic disc. We conclude that the phenotypic variation of human intervertebral disc cells should be extended to include cells with a stellate appearance, which may be more prevalent in tissue that has been subjected to abnormal load or tension.