脊索细胞维持椎间盘髓核软骨样细胞增殖与表型的研究进展

BACKGROUND: The immature disc nucleus pulposus is composed of notochordal cells, but there is no notochordal cell in the mature human intervertebral disc, in which the notochordal cells are replaced by chondrocyte-like cells. It is very important to comprehend the disappearance of the notochordal cells; however, it is still unknown at present. OBJECTIVE: To elaborate the feasibility of notochordal cells to maintain the proliferation and phenotype of chondrocyte-like cells and to induce the cartilage-like differentiation of bone marrow mesenchymal stem cells. METHODS: The first author used the computer to retrieve PubMed and Wanfang databases using the key words of “notochord cells; nucleus pulposus cells; identify” in English and Chinese, respectively. Totally 9 896 relevant articles published from January 1999 to August 2015 were retrieved. Repetitive studies were excluded, and finally 36 articles were in accordance with the inclusion criteria. RESULTS AND CONCLUSION: Now, the main functions of notochordal cells are to promote synthesis of extracellular matrix in the nucleus pulposus, induce directional differentiation of mesenchymal cells into nucleus pulposus cells or act as “seed cells” to form the nucleus pulposus cells. The presence and disappearance of notochordal cells is related to intervertebral disc degeneration. Cell apoptosis is involved in static compression via death receptor signals, and then leads to intervertebral disc degeneration. fas ligand can mediate the reduction of notochordal cells, and hypoxia-inducible factor can induce spinal cord injury thereby triggering cell death and complete disappearance of nucleus pulposus. The measurement and verification of immune makers of notochordal cells, CK-8, CK-18 and galectin-3, can benefit to the identification and isolation of notochordal cells, and thereby help the studies on cell growth and differentiation, function and its mechanism of apoptosis.       

Identification of nucleus pulposus precursor cells and notochordal remnants in the mouse: Implications for disk degeneration and chordoma formation

A classically identified “notochordal” cell population in the nucleus pulposus is thought to regulate disk homeostasis. However, the embryonic origin of these cells has been under dispute for >60 years. Here we provide the first direct evidence that all cell types in the adult mouse nucleus pulposus are derived from the embryonic notochord. Additionally, rare isolated embryonic notochord cells remained in the vertebral column and resembled “notochordal remnants,” which in humans have been proposed to give rise to a rare type of late‐onset cancer called chordoma. Previously, this cell type had not been identified in the mouse model system. The development and characterization of a mouse model that can be used to fate map nucleus pulposus precursor cells in any mutant background will be useful for uncovering the cellular and molecular mechanisms of disk degeneration. In addition, the identification of notochordal remnants in mice is the first step towards generating an in vivo model of chordoma. Developmental Dynamics 237:3953–3958, 2008. © 2008 Wiley‐Liss, Inc.     

The notochordal cell in the nucleus pulposus: a review in the context of tissue engineering

An understanding of developmental biology can provide useful insights into how different tissue-engineered repairs might be designed. During embryogenesis of the intervertebral disk, the cells of the notochord play a critical role in initiating tissue formation, and may be responsible for development of the nucleus pulposus. In some species, including humans, these notochordal cells may eventually be lost, either through apoptosis or terminal differentiation, and are replaced by chondrocyte-like cells. However, there is some evidence that the notochordal cells may persist in at least some humans. This review discusses some of the potential applications of notochordal cells in tissue engineering of the nucleus pulposus.     

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.     

腰椎间盘的超微结构研究

本实验通过透射电镜对大鼠、家兔及人(胎儿及成人)腰椎间盘的观察,发现纤维环由纤维软骨组成。在640 A周期性的胶原原纤维之间,可见位于隐窝内的典型软骨细胞。这些细胞常随所在部位而异。靠近周边部位的细胞更接近成纤维细胞,细胞狭长,细胞膜的扇蛤样外形渐不明显,以至消失。靠内侧的细胞类似脊索细胞,细胞核固缩,细胞质内有浓集的糖原及脂滴。纤维环呈现细胞过渡的特点。纤维环内可见弹力纤维。髓核由包含细的原纤维、颗粒及特殊结构的疏松基质所组成。在各标本中,基质内均有脊索细胞出现。细胞可单独或成堆存在,细胞质内有众多的空泡,浓集的糖原颗粒及微丝。     

The three-dimensional architecture of the notochordal nucleus pulposus: novel observations on cell structures in the canine intervertebral disc

Cells from the nucleus pulposus of young (< 2 years) and old (> 5 years) non‐chondrodystrophoid dogs were studied using routine histology, confocal laser scanning microscopy and transmission electron microscopy. The architecture of cell structures – from the tissue scale down to subcellular scale – was reported. Clusters of notochordal cells were observed in young nuclei pulposi, ranging from 10 to 426 cells each. These clusters resisted mechanical disruption and showed evidence of cell–cell signalling via gap junctions. Cells (30–40 µm in diameter) within the clusters had a physaliferous appearance, containing numerous large inclusions which ranged from 1 to 20 µm in diameter. The inclusions were surrounded by a dense actin cortex but were not contained by a lipid bilayer. The contents of the inclusions were determined not to be predominantly carbohydrate or neutral lipid as assessed by histochemical staining, but the exact composition of the contents remained uncertain. There were striking differences in the cell architecture of young vs. old nuclei pulposi, with a loss of both cell clusters and physaliferous cells during ageing. These observations demonstrate unique cell structures, which may influence our understanding of the differences between notochordal and chondrocytic cells in the nucleus pulposus. Such differences could have substantial impact upon how we think about development, degeneration and repair of the intervertebral disc.     

Three-dimensional morphology of the pericellular matrix of intervertebral disc cells in the rat

Intervertebral disc cells are surrounded by a pericellular matrix that is biochemically and morphologically distinct from other extracellular matrix regions. Although the function of the pericellular matrix is not fully understood, prior studies of pericellular matrix-chondrocyte regions in articular cartilage (termed 'chondrons') suggest that the size, shape, and mechanical properties of the pericellular matrix significantly influence the micromechanical environment of the contained cells. A first step in understanding the role of the pericellular matrix in the intervertebral disc is to quantify the three-dimensional morphology and zonal variations of these regions across the disc. In this study, three-dimensional reconstructions and morphometric measurements of pericellular matrix-cell regions were obtained in situ using fluorescence confocal microscopy of en bloc sections of nucleus pulposus and anulus fibrosus of the rat disc immunolabeled for type VI collagen. The morphology of the pericellular matrix and cells varied significantly across regions, with distinct pericellular matrix aspect ratios (largest/smallest diameter) showing shapes that were generally large and rounded in the nucleus pulposus (average of 1.9), and ellipsoidal and discoidal in the inner (2.4) and outer anulus fibrosus (2.8). The average pericellular matrix volume per cell was found to be significantly larger in the nucleus (6424 microm(3)) than that of inner (1903 microm(3)) and outer (1433 microm(3)) anulus. Pericellular matrix regions containing 1 or 2 cells were the dominant subgroup in the rat intervertebral disc at both 1 and 12 months of age. Multicellular pericellular matrix regions were present more often in the younger nucleus pulposus and outer anulus fibrosus. The orientation of the pericellular matrix regions further varied significantly across the disc, reflecting local collagen matrix architecture. These studies provide new information on the organization and shape of intervertebral disc cells and their surrounding pericellular matrix, which may provide new insights into the mechanisms that regulate cell-matrix interactions.     

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.     

Zonal variations in cytoskeletal element organization,mRNA and protein expression in the intervertebral disc

The intervertebral disc is important in maintaining flexibility and dissipating loads applied to the spine. The disc comprises a heterogeneous population of cells, including those of the nucleus pulposus and annulus fibrosus, which are diverse in phenotype, partly due to the different mechanical loads they experience. Several studies have implicated the cytoskeleton in mechanotransduction, but little characterization of the three major cytoskeletal elements – actin, tubulin and vimentin – in the intervertebral disc has been undertaken. In this study we show that there are differences in both the organization and the amounts of these cytoskeletal proteins across the regions of immature bovine intervertebral disc (nucleus pulposus and outer annulus fibrosus), which differs with skeletal maturity. These differences are likely to reflect the diverse mechanical characteristics of the disc regions, and the loads that they experience, i.e. tension in the annulus fibrosus and compression in the nucleus pulposus. Alterations to the organization and amount of cytoskeletal element proteins may change the ability of the cells to respond to mechanical signals, with a loss of tissue homeostasis, suggesting that the cytoskeleton has a potential role in intervertebral disc degeneration.     

人椎间盘髓核细胞增殖活性与益肾活血通络方的干预调控

背景:NF-κB信号通路与椎间盘退变的关系是骨科学术界研究热点,深入研究椎间盘中各种信号通路的作用,有助于了解椎间盘退变的发生机制。目的:通过研究不同静水压压力下益肾活血通络方含药血清对人椎间盘髓核细胞NF-κB信号通路的调控作用,以期能从分子生物学角度探讨益肾活血通络方治疗椎间盘退变性疾病的可能机制及作用靶点。方法:将第3代人椎间盘髓核细胞分为8组,分别加入益肾活血通络方含药血清,在体外不同静水压加载条件(0.3,1,3 MPa)下作用2,4,6 h后,使用倒置相差显微镜观察椎间盘髓核细胞的形态及生长状况;使用透射电镜观察椎间盘髓核细胞的超微结构;采用CCK-8法检测各组髓核细胞的增殖活性;采用Annexin V-FITC/Propidium Iodide凋亡试剂盒检测髓核细胞的凋亡率;采用Western blot法检测髓核细胞内NF-κB p65、CollagenⅡ、ADAMTS-4、MMP-13、Caspase-3的表达。结果与结论:①在同一压力及作用时间下压力+中药血清组髓核细胞形态较常压组、单纯压力组更完整,生长状况更好,其中0.3,1 MPa压力+中药血清组优于3 MPa压力+中药血清组;②压力+中药血清组髓核细胞增殖活性更高,其中0.3 MPa压力+中药血清组与0.3 MPa单纯压力组比较差异有显著性意义(P<0.05);③压力+中药血清组椎间盘髓核细胞的凋亡率较常压组、单纯压力干预组低(P<0.05);④与单纯压力组比较,压力+中药血清组CollagenⅡ、Caspase-3表达增加,NF-κB p65、ADAMTS-4、MMP-13表达减少(P<0.05);⑤结果表明,益肾活血通络方能增加细胞活性,减少细胞凋亡,有效延缓髓核细胞的退变,其机制可能与椎间盘髓核细胞NF-κB信号通路促进CollagenⅡ、Caspase-3表达,抑制NF-κB p65、ADAMTS-4、MMP-13表达有关。     

An ultrastructural study of chondroptosis: programmed cell death in degenerative intervertebral discs in vivo

Apoptosis has been regarded to mediate intervertebral disc degeneration (IDD); however, the basic question of how the apoptotic bodies are cleared in the avascular intervertebral disc without phagocytes, which are essential to apoptosis, remains to be elucidated. Our goals were to investigate the ultrastructure of nucleus pulposus (NP) cells undergoing chondroptosis, a variant of apoptotic cell death, in a rabbit annular needle‐puncture model of IDD. Experimental IDD was induced by puncturing discs with a 16‐G needle in New Zealand rabbits. At 4 and 12 weeks after puncture, progressive degeneration was demonstrated by X‐ray, magnetic resonance imaging and histological staining. TUNEL staining suggested a significant increase in the apoptosis index in the degenerated NP. However, the percentage of apoptotic cells with the classic ultrastructure morphology was much less than that with chondroptotic ultrastructure morphology under transmission electron microscopy (TEM). The chondroptotic cells from the early to late stage were visualized under TEM. In addition, the percentage of chondroptotic cells was significantly enhanced in the degenerated NP. Furthermore, ‘paralyzed’ cells were found in the herniated tissue. Western blotting revealed an increase in caspase3 expression in the degenerated NP. The expression of the Golgi protein (58K) was increased by the fourth week after puncture but decreased later. These findings indicate that chondroptosis is a major type of programmed cell death in the degenerated rabbit NP that may be related to the progressive development of IDD.     

Reflections on notochordal differentiation arising from a study of chordomas

This study of thirteen cases of chordoma serves to emphasize the occurrence of three different histological patterns; classical, seven; chondroid, three; and intermediate or mesenchymal, three. The study also suggests that more adequate sampling of these tumours detects the chondroid variant more readily. These varying patterns of differentiation in tumours of notochordal origin suggest that the parent tissue may have the potential to develop along similar lines in the embryo. Thus mesenchymal and cartilaginous tissue formed from notochordal cells could contribute to the formation of the nucleus pulposus and inner portion of the intervertebral disc cartilages. This concept contrasts with the previously held view that the notochord atrophies at an early stage in embryonic development.     

静水压对人椎间盘髓核细胞形态及基质表达的影响*☆

背景：加载在椎间盘上的重力引起的静态压力刺激是椎间盘细胞代谢的重要调节因素。 目的：观察静水压对体外单层培养人椎间盘髓核细胞形态学及基质代谢的影响。 方法：在静水压加载系统中对体外单层培养的传4代人髓核细胞施以0.3,0.7,3 MPa的静压,分别加压30,60,90,120 min,以常压0.1 MPa为对照。 结果与结论：①髓核细胞形态：在静水压干预下细胞体积均变小。0.3,0.7 MPa静水压下轻微缩小,细胞形态相对完整;3 MPa静水压下缩小最明显,且细胞形态不完整。②髓核细胞存活率：在持续静水压刺激下开始的30 min,不论压力大小存活率都偏低,在0.3,0.7 MPa时随作用时间延长而增加或维持稳定,细胞增殖逐渐增强,在3 MPa时随时间趋于下降,最终细胞总体数量减少。③髓核细胞蛋白多糖：各组表达量随时间增加逐渐增加,当持续加压120 min时,在0.3,0.7 MPa静水压下合成量呈高表达状态,在0.1,3 MPa静压表达相对较少。表明静水压会对髓核细胞形态学、存活率及基质表达产生影响。 关键词：静水压;髓核细胞;形态学;蛋白多糖;组织构建细胞学实验  doi:10.3969/j.issn.1673-8225.2012.13.001     

Diversity of intervertebral disc cells: phenotype and function

The intervertebral disc (IVD) is a moderately moving joint that is located between the bony vertebrae and provides flexibility and load transmission throughout the spinal column. The disc is composed of different but interrelated tissues, including the central highly hydrated nucleus pulposus (NP), the surrounding elastic and fibrous annulus fibrosus (AF), and the cartilaginous endplate (CEP), which provides the connection to the vertebral bodies. Each of these tissues has a different function and consists of a specific matrix structure that is maintained by a cell population with distinct phenotype. Although the healthy IVD is able to balance the slow matrix turnover of synthesis and degradation, this balance is often disturbed, leading to degenerative disorders. Successful therapeutic management of IVD degeneration requires a profound understanding of the cellular and molecular characteristics of the functional IVD. Hence, the phenotype of IVD cells has been of significant interest from multiple perspectives, including development, growth, remodelling, degeneration and repair. One major challenge that complicates our understanding of the disc cells is that both the cellular phenotype and the extracellular matrix strongly depend on disc maturity and health and as a consequence are continuously evolving. This review delineates the diversity of the cell types found in the intervertebral disc, with emphasis on human, but with reference to other species. The cells of the NP appear rounded and express a proteoglycan-rich matrix, whereas the more elongated AF cells are embedded in a collagen fibre matrix and the CEPs represent a layer of cartilage. Even though all disc cells have often been referred to as ‘intervertebral disc chondrocytes’, distinct phenotypical differences in comparison with articular chondrocytes exist and have been reported recently. The availability of more specific markers has also improved our understanding of progenitor cell differentiation towards an IVD cell phenotype. Ultimately, new cell- and tissue-engineering approaches to regenerative therapies will only be successful if the specific characteristics of the individual tissues and their context in the function of the whole organ, are taken into consideration.     

Reversible DNA damage checkpoint activation at the presenescent stage in telomerase‐deficient cells of Saccharomyces cerevisiae

The telomere protects the ends of eukaryotic linear chromosomes, and its shortening or erosion is recognized as DNA damage, leading to loss of proliferation activity and, thus, cellular senescence at the population level. Here, using a GFP‐based DNA damage checkpoint marker suited for single‐cell observation of Saccharomyces cerevisiae cells, we correlated the checkpoint status of telomere‐shortened cells with their behavior. We show that some cells possessing short telomeres retain proliferation capacity even after the DNA damage checkpoint is activated. At the presenescent stage, the activation of the checkpoint causes cell cycle delay, but does not induce permanent cell cycle arrest, eventually leading to the expansion of cell size that is characteristic of cellular senescence. Moreover, the proliferation capacity of checkpoint‐activated cells is not dependent on homologous recombination or the checkpoint adaptation pathway. The retention of proliferation capacity is specific to the telomere‐derived DNA damage response, suggesting that damaged telomeres differ functionally from other types of DNA damage. Our data establish the role of the presenescent stage in telomere shortening‐induced senescence, which proceeds gradually and is associated with a variety of changes, including altered cell morphology and metabolism.     

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。