The enigma that is the nucleus pulposus cell: the search goes on

The development of an effective treatment for degenerative disc disease has been hampered for many years by what seems a fundamental problem; what exactly defines a nucleus pulposus (NP) cell? The paper by Gilson and colleagues elegantly re-opens the debate concerning the lineage and identity of NP cells that are alike yet different from chondrocytes. As we pursue novel investigations and treatment strategies for degenerative disc disease, how do we isolate these unique cells and what is the role of the primordial notochordal cell that may well linger within the NP far longer and perhaps in a different phenotypic appearance than previously thought? The paper by Gilson and colleagues that is the subject of the present editorial presents compelling data concerning the heterogeneity of the cells of the NP, and their origin, development, maturation and function.A recent issue of Arthritis Research and Therapy contains a report by Gilson and colleagues describing their investigation of the differential cell surface marker expression found in samples of bovine intervertebral disc (IVD) and articular chondrocytes [1]. This report raises interesting questions about the identity of the residents within the nucleus pulposus (NP) and has broad implications with respect to regenerative medicine and tissue engineering of the IVD.A recent search of PubMed using the search term ''tissue engineering and intervertebral disc'' returned 263 hits, with the oldest publication dating to 1989. Although investigators have clearly been interested in developing a biological treatment for degenerative disc disease for over 30 years, we must be in the early days since we have yet to characterize the ubiquitous NP cell or to really understand the composition of the NP cellular milieu.The flexible model of cell and tissue classification whereby expression patterns reflect a functional approach rather than strict germ layer derivation suggests that, with respect to the identity of NP cells, there may be more than meets the eye [2].The notochord derives from all three germ layers as it originates in a blended fashion from primitive ectoderm, sharing mesodermal and endodermal attributes as it develops as an outgrowth from Hensen''s node between the ectoderm and endoderm [3]. This co-joined origin is particularly poignant in human and other mammals, as distinct from lower animals, because in higher mammals the developing notochord provides a pathway for migration of ectoderm to endoderm [3]. The presence of vimentin in NP cells suggests that motility may play a role in the development of the NP cellular composition; perhaps including cells that migrate inwards from the vertebral endplates [4]. As Gilson and colleagues have reported, however, the cells occupying the NP may change their appearance over time, masking their original phenotype but perhaps retaining some of their original capacity. Have these cells altered their phenotype as a consequence of maturation or pathological events, or as an adaptive response to life in the disc over time?The IVD is a hypoxic, isolated, immune-privileged compartment, the cells of which must necessarily be highly specialized in order to survive. Classically it has been thought that once the NP has been formed the notochordal cells disappear, leaving behind the fibrocartilagenous NP cell. But then along come Gilson and colleagues - who find that within adult bovine caudal discs (a tissue compartment formerly thought to be fairly homogeneous) there exists a small percentage of notochordal holdouts that continue to express their notochordal lineage markers. Is it that a small, primordial notochordal cell reservoir may linger longer than was previously thought within the mature NP?These observations raise a number of questions - notably, is the protection from degenerative disc disease seen in species that retain their notochordal cell-rich appearance, such as the nonchondrodystrophic canine and rabbit, due to the differential extracellular matrix synthesized by these cells as compared with the NP cell? [5]. Is it a dose-response issue whereby the discs that are relatively deficient in notochordal cells are therefore lacking in the necessary and sufficient molecules synthesized by these cells that may act upon the NP cell [6,7]? It is thought that the notochordal cell-rich disc NP phenotype confers superior biomechanical properties [5,8]. Do notochordal cell-deficient discs therefore fail to resist the loads imposed by daily life over time due to biomechanical or biochemical reasons - or both? Also, and importantly from the perspective of evaluating putative therapies, which cells are the best to use for in vitro assays? Should future NP cell experiments exclude cytokeratin-8⁺ cells or does this matter when evaluating the mechanisms of the IVD NP as an organ?In terms of the progression of degenerative disc disease, the NP could arguably represent the lynchpin in the degenerative cascade since many investigators consider the NP as the area demonstrating the earliest degenerative changes [9-11]. We may therefore need to look ever closer at the question of what really defines the cells within the disc. Are the current models of events leading to failure of the disc as an organ correct? What role(s) do the cells play within the NP that may mitigate or contribute to the progression of organ failure?As we look to the future and contemplate cell-based therapeutics for the treatment of degenerative disc disease, one must wonder what might be the most appropriate source of cells. Bone marrow-derived stem cells originate within an entirely different niche to cells that have adapted to survive within the NP with its tenuous nutrient supply and hypoxic environment. Along which lineage should stem cells or progenitor cells therefore be directed in order to potentially repopulate the disc and how would they best be able to restore homeostasis? For now, given that the mature disc nucleus contains holdouts of the primitive notochordal cell perhaps the best perspective from which to answer these questions is one where we take a fresh look back at the origin, development and maturation of the IVD.     

Alterations of bovine nucleus pulposus cells with aging

Aging is one of the major etiological factors driving intervertebral disc (IVD) degeneration, the main cause of low back pain. The nucleus pulposus (NP) includes a heterogeneous cell population, which is still poorly characterized. Here, we aimed to uncover main alterations in NP cells with aging. For that, bovine coccygeal discs from young (12 months) and old (10–16 years old) animals were dissected and primary NP cells were isolated. Gene expression and proteomics of fresh NP cells were performed. NP cells were labelled with propidium iodide and analysed by flow cytometry for the expression of CD29, CD44, CD45, CD146, GD2, Tie2, CD34 and Stro-1. Morphological cell features were also dissected by imaging flow cytometry. Elder NP cells (up-regulated bIL-6 and bMMP1 gene expression) presented lower percentages of CD29+, CD44+, CD45+ and Tie2+ cells compared with young NP cells (upregulated bIL-8, bCOL2A1 and bACAN gene expression), while GD2, CD146, Stro-1 and CD34 expression were maintained with age. NP cellulome showed an upregulation of proteins related to endoplasmic reticulum (ER) and melanosome independently of age, whereas proteins upregulated in elder NP cells were also associated with glycosylation and disulfide bonds. Flow cytometry analysis of NP cells disclosed the existence of 4 subpopulations with distinct auto-fluorescence and size with different dynamics along aging. Regarding cell morphology, aging increases NP cell area, diameter and vesicles. These results contribute to a better understanding of NP cells aging and highlighting potential anti-aging targets that can help to mitigate age-related disc disease.     

人颈椎间盘髓核细胞的体外培养和鉴定

目的:建立人颈椎间盘髓核细胞体外培养体系,并对其细胞表型进行鉴定。方法:采用酶消化法分离人颈椎间盘髓核细胞,进行单层培养,倒置相差显微镜观察细胞生长和形态,流式细胞仪测定细胞周期和凋亡率,并行甲苯胺蓝、Ⅱ型胶原及CK8免疫组化染色对其细胞表型进行鉴定。结果:原代髓核细胞凋亡率6.1±1.4%,S期细胞比例7.3±0.5%。贴壁后形态为多角形或短楔形,传代后生长加速。细胞呈甲苯胺蓝异染性;Ⅱ型胶原免疫组化染色阳性;只有少量椭圆形大细胞CK8免疫组化染色阳性。结论:成功建立人颈椎间盘髓核细胞体外培养模型,并证实成年后髓核内仍有少量细胞保持脊索细胞表型。     

人颈椎间盘髓核细胞的体外培养和鉴定

目的：建立人颈椎间盘髓核细胞体外培养体系,并对其细胞表型进行鉴定。方法：采用酶消化法分离人颈椎间盘髓核细胞,进行单层培养,倒置相差显微镜观察细胞生长和形态,流式细胞仪测定细胞周期和凋亡率,并行甲苯胺蓝、Ⅱ型胶原及CK8免疫组化染色对其细胞表型进行鉴定。结果：原代髓核细胞凋亡率6.1±1.4%,S期细胞比例7.3±0.5%。贴壁后形态为多角形或短楔形,传代后生长加速。细胞呈甲苯胺蓝异染性;Ⅱ型胶原免疫组化染色阳性;只有少量椭圆形大细胞CK8免疫组化染色阳性。结论：成功建立人颈椎间盘髓核细胞体外培养模型,并证实成年后髓核内仍有少量细胞保持脊索细胞表型。     

Transcriptional profiling of mouse liver in response to chronic heat stress

Mice were used to study the effects of chronic heat stress on hepatic gene expression. Twenty-five mice were allocated to either chronic heat stress (34 °C) or control (24 °C) conditions for a period of 2 weeks from 47 to 60 d of age. Nineteen genes differentially expressed in liver were identified using DNA microarrays. Genes involved in the anti-oxidant pathway and metabolism were up-regulated. Genes involved in generation of reactive oxygen radicals and mitochondrial expressed genes were down-regulated. Enzyme activity measurements confirmed the array results. Mice exposed to chronic heat stress showed signs of increased oxidative stress in liver cells.     

Expression of cytokeratin and vimentin in nucleus pulposus cells

With immunohistochemistry using monoclonal antibodies against intermediate filament proteins, nucleus pulposus cells were found to express cytokeratin(s) simultaneously with vimentin in fetal life and childhood. This finding adds to the series of human tissues showing coexpression of cytokeratins and vimentin. Surprisingly remnants of such cells were also found in the nucleus pulposus of adults, and a possible relationship of such cells to chordoma formation is discussed.     

Determination of the strain-dependent hydraulic permeability of the compressed bovine nucleus pulposus

The hydraulic permeability, k, of the nucleus pulposus (NP) is crucial, both in withstanding compressive stress and for convective transport of nutrients within the disc. Permeability has previously been determined using biphasic mathematical models, but has not been found by direct permeation experiments, which is the objective of this study. Bovine coccygeal nucleus samples (n=64), phi10mm and thickness 683+/-49microm (mean+/-S.D.) were compressed axially to one of lambda=1.0, 0.9, 0.8, 0.7, 0.6, 0.5, 0.4 or 0.3, where lambda is the stretch ratio. Ringer's solution was permeated through the sample, with an o-ring ensuring axial flow. During stress equilibrium, k was determined and fitted to four permeability-strain equations. Permeability decreased exponentially with compression, and was best described by Values of k were comparable to those arising from mathematical models, lending confidence to permeability being determined from such models.