Interleukin-23 is constitutively expressed in the human annulus in vivo and in vitro,and is up-regulated in vitro by TNF-α

ABSTRACT

Interleukin-23 (IL-23, IL-23p19) is a proinflammatory cytokine in the IL-12-related family. Although inflammatory cells in herniated discs have been shown to contain IL-23, little is known about the presence and role of IL-23 in human disc cells. We analyzed disc specimens for IL-23 localization using immunohistochemistry in control, herniated and non-herniated discs from which annulus fibrosus (annulus) cells were isolated and cultured to identify IL-23 gene expression and production. Microarray analysis was used to assess the expression of IL-23 in disc tissue and in cells exposed to two proinflammatory cytokines, IL-1ß and TNF-α. IL-23 was present in annulus cells at the protein level and its expression was up-regulated significantly in herniated compared to control disc tissue. Direct measurement of medium components confirmed production of IL-23 and its receptor, IL-23R, by annulus cells in vitro. Annulus cells in three-dimensional culture exposed to TNF-α, but not IL-1ß, resulted in significant up-regulation of IL-23 expression compared to control cells. Our findings are evidence for the constitutive presence of IL-23 in the human disc and that its expression in vitro is modified by exposure to TNF-α.     

Degeneration affects the fiber reorientation of human annulus fibrosus under tensile load

The angled, lamellar structure of the annulus fibrosus is integral to its load-bearing function. Reorientation of this fiber structure with applied load may contribute to nonlinear mechanical behavior and to large increases in tensile modulus. Fiber reorientation has not yet been quantified for loaded non-degenerated and degenerated annulus fibrosus tissue. The objective of this study was to measure fiber reorientation and mechanical properties (toe- and linear-region modulus, transition strain, and Poisson's ratio) of loaded outer annulus fibrosus tissue using a new application of FFT image processing techniques. This method was validated for quantification of annulus fiber reorientation during loading in this study. We hypothesized that annulus fibrosus fibers would reorient under circumferential tensile load, and that fiber reorientation would be affine. Additionally, we hypothesized that degeneration would affect fiber reorientation, toe-region modulus and Poisson's ratio. Annulus fibrosus fibers were found to reorient toward the loading direction, and degeneration significantly decreased fiber reorientation (the fiber reorientation parameter, m(FFT)=-1.70 degrees /% strain for non-degenerated and -0.95 degrees /% strain for degenerated tissue). Toe-region modulus was significantly correlated with age (r=0.6). Paired t-tests showed no significant difference in the fiber reorientation parameter calculated experimentally with that calculated using an affine prediction. Thus, an affine prediction is a good approximation of fiber reorientation. The findings of this study add to the understanding of overall disc mechanical behavior and degeneration.     

The chemokine,CXCL16, and its receptor,CXCR6, are constitutively expressed in human annulus fibrosus and expression of CXCL16 is up-regulated by exposure to IL-1ß in vitro

Chemokines are an important group of soluble molecules with specialized functions in inflammation. The roles of many specialized chemokines and their receptors remain poorly understood in the human intervertebral disc. We investigated CXCL16 and its receptor, CXCR6, to determine their immunolocalization in disc tissue and their presence following exposure of cultured human annulus fibrosus cells to proinflammatory cytokines. CXCL16 is a marker for inflammation; it also can induce hypoxia-inducible factor 1α (HIF-1α), which is a phenotypic marker of heathy nucleus pulposus tissue. We found CXCL16 and CXCR6 immunostaining in many cells of the annulus portion of the disc. Molecular studies showed that annulus fibrosus cells exposed to IL-1ß, but not TNF-α, exhibited significant up-regulation of CXCL16 expression vs. control cells. There was no significant difference in the percentage of annulus cells that exhibited immunolocalization of CXCL16 in grade I/II, grade III or grade IV/V specimens. The presence of CXCL16 and its receptor, CXCR6, in the annulus in vivo suggests the need for future research concerning the role of this chemokine in proinflammatory functions, HIF-1α expression and disc vascularization.     

Hyperelastic anisotropic microplane constitutive model for annulus fibrosus

In a recent paper, Peng et al. (2006, "An Anisotropic Hyperelastic Constitutive Model With Fiber-Matrix Interaction for the Human Annulus Fibrosis," ASME J. Appl. Mech., 73(5), pp. 815-824) developed an anisotropic hyperelastic constitutive model for the human annulus fibrosus in which fiber-matrix interaction plays a crucial role in simulating experimental observations reported in the literature. Later, Guo et al. (2006, "A Composites-Based Hyperelastic Constitutive Model for Soft Tissue With Application to the Human Fibrosis," J. Mech. Phys. Solids, 54(9), pp. 1952-1971) used fiber reinforced continuum mechanics theory to formulate a model in which the fiber-matrix interaction was simulated using only composite effect. It was shown in these studies that the classical anisotropic hyperelastic constitutive models for soft tissue, which do not account for this shear interaction, cannot accurately simulate the test data on human annulus fibrosus. In this study, we show that the microplane model for soft tissue developed by Caner and Carol (2006, "Microplane Constitutive Model and Computational Framework for Blood Vessel Tissue," ASME J. Biomech. Eng., 128(3), pp. 419-427) can be adjusted for human annulus fibrosus and the resulting model can accurately simulate the experimental observations without explicit fiber-matrix interaction because, in microplane model, the shear interaction between the individual fibers distributed in the tissue provides the required additional rigidity to explain these experimental facts. The intensity of the shear interaction between the fibers can be adjusted by adjusting the spread in the distribution while keeping the total amount of the fiber constant. A comparison of results obtained from (i) a fiber-matrix parallel coupling model, which does not account for the fiber-matrix interaction, (ii) the same model but enriched with fiber-matrix interaction, and (iii) microplane model for soft tissue adapted to annulus fibrosus with two families of fiber distributions is presented. The conclusions are (i) that varying degrees of fiber-fiber and fiber-matrix shear interaction must be taking place in the human annulus fibrosus, (ii) that this shear interaction is essential to be able to explain the mechanical behavior of human annulus fibrosus, and (iii) that microplane model can be fortified with fiber-matrix interaction in a straightforward manner provided that there are new experimental data on distribution of fibers, which indicate a spread so small that it requires an explicit fiber-matrix interaction to be able to simulate the experimental data.     

Extracellular matrix in development of the intervertebral disc.

Intervertebral discs allow bending and twisting of the spine whilst resisting compression from gravity and muscle action, and are composite structures of the peripheral annulus fibrosus enclosing the nucleus pulposus. Their development is complex, involving several different connective tissue types, yet little is known of the developing extracellular matrix (ECM). We report the ECM composition of foetal rat discs from their first appearance to birth. The earliest collagen detected was type III, which was subsequently replaced by type II in the cartilaginous inner annulus and joined by type I in the fibrous outer annulus. Type IV collagen appeared in outer annulus, associated with myofibroblast-like cells of the orienting collagenous lamellae. Laminin and fibronectin co-distributed here in later stages, although overall they had a wider distribution. Aggrecan occurred in early nucleus pulposus and then appeared in the inner annulus, in association with cartilage differentiation. Versican appeared later in the inner annulus, and also in the dorsal region of the outer annulus. Comparisons of glycosaminoglycan and proteoglycan label allowed extrapolations to be made as to likely glycosaminoglycan components of the large proteoglycans, and of other proteoglycans that may be present - thus differential distribution of aggrecan and keratan sulfate label suggested the presence of fibromodulin and/or lumican. Functionally aggrecan would confer compression resistance to cartilaginous structures. Versican may also contribute, but along with the small proteoglycans is likely to be associated with various stages of control of cell differentiation, tissue morphogenesis and collagen fibre formation in the assembly of the annulus fibrosus.     

A special theory of biphasic mixtures and experimental results for human annulus fibrosus tested in confined compression

A finite deformation mixture theory is used to quantify the mechanical properties of the annulus fibrosus using experimental data obtained from a confined compression protocol. Certain constitutive assumptions are introduced to derive a special mixture of an elastic solid and an inviscid fluid, and the constraint of intrinsic incompressibility is introduced in a manner that is consistent with results obtained for the special theory. Thirty-two annulus fibrosus specimens oriented in axial (n = 16) and radial (n = 16) directions were obtained from the middle-lateral portion of intact intervertebral discs from human lumbar spines and tested in a stress-relaxation protocol. Material constants are determined by fitting the theory to experimental data representing the equilibrium stress versus stretch and the surface stress time history curves. No significant differences in material constants due to orientation existed, but significant differences existed due to the choice of theory used to fit the data. In comparison with earlier studies with healthy annular tissue, we report a lower aggregate modulus and a higher initial permeability constant. These differences are explained by the choice of reference configuration for the experimental studies.     

Comparison of cellular response in bovine intervertebral disc cells and articular chondrocytes: effects .of lipopolysaccharide on proteoglycan metabolism

Lipopolysaccharide (LPS) induces matrix degradation and markedly stimulates the production of several cytokines, i.e., interleukin-1β, −6, and −10, by disc cells and chondrocytes. We performed a series of experiments to compare cellular responses of cells from the bovine intervertebral disc (nucleus pulposus and annulus fibrosus) and from bovine articular cartilage to LPS. Alginate beads containing cells isolated from bovine intervertebral discs and articular cartilage were cultured with or without LPS in the presence of 10% fetal bovine serum. The DNA content and the rate of proteoglycan synthesis and degradation were determined. In articular chondrocytes, LPS strongly suppressed cell proliferation and proteoglycan synthesis in a dose-dependent manner and stimulated proteoglycan degradation. Compared with articular chondrocytes, nucleus pulposus cells responded in a similar, although less pronounced manner. However, treatment of annulus fibrosus cells with LPS showed no significant effects on proteoglycan synthesis or degradation. A slight, but statistically significant, inhibition of cell proliferation was observed at high concentrations of LPS in annulus fibrosus cells. Thus, LPS suppressed proteoglycan synthesis and stimulated proteoglycan degradation by articular chondrocytes and nucleus pulposus cells. The effects of LPS on annulus fibrosus cells were minor compared with those on the other two cell types. The dissimilar effects of LPS on the various cell types suggest metabolic differences between these cells and may further indicate a divergence in pathways of LPS signaling and a differential sensitivity to exogenous stimuli such as LPS.This work was supported in part by NIH grants 2-P50-AR39239 and 1-P01-AR48152.     

Modeling interlamellar interactions in angle-ply biologic laminates for annulus fibrosus tissue engineering

Mechanical function of the annulus fibrosus of the intervertebral disc is dictated by the composition and microstructure of its highly ordered extracellular matrix. Recent work on engineered angle-ply laminates formed from mesenchymal stem cell (MSC)-seeded nanofibrous scaffolds indicates that the organization of collagen fibers into planes of alternating alignment may play an important role in annulus fibrosus tissue function. Specifically, these engineered tissues can resist tensile deformation through shearing of the interlamellar matrix as layers of collagen differentially reorient under load. In the present work, a hyperelastic constitutive model was developed to describe the role of interlamellar shearing in reinforcing the tensile response of biologic laminates, and was applied to experimental results from engineered annulus constructs formed from MSC-seeded nanofibrous scaffolds. By applying the constitutive model to uniaxial tensile stress–strain data for bilayers with three different fiber orientations, material parameters were generated that characterize the contributions of extrafibrillar matrix, fibers, and interlamellar shearing interactions. By 10 weeks of in vitro culture, interlamellar shearing accounted for nearly 50% of the total stress associated with uniaxial extension in the anatomic range of ply angle. The model successfully captured changes in function with extracellular matrix deposition through variations in the magnitude of model parameters with culture duration. This work illustrates the value of engineered tissues as tools to further our understanding of structure–function relations in native tissues and as a test-bed for the development of constitutive models to describe them.     

Expression of serglycin in human disc is increased in degenerated discs and up-regulated in vitro by exposure to IL-1ß or TNF-α

We investigated whether the multifunctional intercellular proteoglycan, serglycin, is expressed in human intervertebral disc cells and assessed its localization. We also investigated expression levels of serglycin in human annulus fibrosus (annulus) cells exposed to IL-1ß and TNF-α, which are two proinflammatory cytokines that are expressed during disc degeneration. Immunolocalization of serglycin was common in many cells of the human annulus, but less common in the nucleus pulposus (nucleus). Both intracellular and cell membrane localization were observed. Annulus cells from Thompson grades III, IV and V degenerated discs exhibited a 4.69 fold up-regulation in serglycin expression vs. cells from healthier grades I and II discs. In monolayer annulus cell culture, cells from more degenerated discs exhibited a 9.4 fold up-regulation of serglycin expression compared to cells from healthier discs. Exposure of cultured cells to IL-1ß or TNF-α caused significant up-regulation of serglycin expression. We found that serglycin expression increased with increasing disc degeneration both in vivo and in vitro, and also increased with exposure in vitro to IL-1ß and TNF-α.     

Hyaluronan distribution in the human and canine intervertebral disc and cartilage endplate

A biotinylated complex of aggrecan G1-domain and link protein was used to characterize the distribution of hyaluronan in paraffin-embedded sections of adult human and canine intervertebral disc and cartilage endplate. Limited chondroitinase ABC and trypsin digestions of the sections before staining was utilized to expose hyaluronan potentially masked by aggrecan. Hyaluronan concentration and hyaluronan to uronic acid ratio in different parts of the discs were measured as a background for the histological analysis.Hyaluronan staining was strong in the nucleus pulposus and inner parts of annulus fibrosus of both species, corroborated by biochemical assays of the same compartments. Particularly in human samples, hyaluronan in the interterritorial matrix of nucleus pulposus and annulus fibrosus was readily accessible to the probe without enzyme treatments. In contrast, the cell-associated hyaluronan signal was enhanced after trypsin or limited chondroitinase ABC-treatment of the sections, suggesting that pericellular hyaluronan was more masked by aggrecan than in the distant matrix. A puzzling feature of canine cartilage endplate cells was their intensive cell-associated hyaluronan signal, part of which appeared intracellular. Hyaluronan was abundant between the collagenous lamellae in annulus fibrosus, perhaps important in the plasticity of this tissue.     

The response of foetal annulus fibrosus cells to growth factors: modulation of matrix synthesis by TGF-β1 and IGF-1

The annulus fibrosus of the intervertebral disc is a complex radial-ply tissue that derives initially from segmental condensations of axial mesenchyme surrounding the notochord. These mesenchymal condensations differentiate into the early annulus fibrosus during foetal development—their outer part becoming fibrous, containing collagen type I; and their inner part cartilaginous, containing type II collagen and aggrecan. With post-natal growth and ageing, there is a switch from type I to type II collagen and an increase in proteoglycan synthesis in the outer annulus. This fibrocartilaginous metaplasia appears to occur in response to compressive loading of the tissue as occurs in tendons that wrap around bony pulleys, and driven by growth factors, such as TGF-β. In this study, using high-density micromass cultures, we have assessed the response of foetal outer annulus cells to growth factor stimulation with TGF-β1 and IGF-1, growth factors known to occur within the developing disc. We qualitatively and quantitatively describe the stimulatory effects of these growth factors, both alone and in combination, on the synthesis of sulphated glycosaminoglycan, and collagen types I and II by annulus cells. We show a potential role for TGF-β1 in pushing cells towards a fibrocartilaginous phenotype, with possible complementary effects of IGF-1.     

Constitutive expression of IL-22 in the human intervertebral disc and its reduction by exposure to pro-inflammatory cytokines in vitro

The importance of cytokines in disc degeneration is well recognized. Little is known about IL-22 expression in the human intervertebral disc. We investigated IL-22 immuno-localization in disc tissue, and molecular expression and production of IL-22 by annulus cells cultured in three-dimensional (3D) culture. We examined human disc tissue using immunohistochemistry and we cultured isolated annulus cells in 3D to analyze IL-22 expression and production, and its receptor, IL-22R, in conditioned media. Ingenuity pathway analysis (IPA) also was used to identify significant gene expression networks within the molecular data. IL-22 and IL-22R were immunolocalized in many cells in the human outer and inner annulus; fewer cells exhibited localization in the nucleus. Three-dimensional culture of annulus cells demonstrated production of IL-22 in conditioned media; exposure to IL-1ß or TNF-α significantly reduced IL-22 levels. Significant decreases also were identified in conditioned media assayed for IL-22R in TNF-α treated cells. IPA analysis showed that IL-22 ranked among the top canonical pathways. We found constitutive expression and production of IL-22 and IL-22R in the disc, which expands our understanding of the effect of pro-inflammatory cytokines on IL-22 expression and production. Three-dimensional cultured annulus cells exposed to IL-1ß or TNF produced significantly lower levels of IL-22 into their conditioned media compared to levels produced by control cells. Our findings have clinical relevance because of the elevated pro-inflammatory milieu within the degenerating human disc.     

Strain transfer in the annulus fibrosus under applied flexion

A detailed understanding of the anatomical and mechanical environment in the intervertebral disc at the scale of the cell is necessary for the design of tissue engineering repair strategies and to elucidate the role of mechanical factors in pathology. The objective of this study was to measure and compare the macroscale to microscale strains in the outer annulus fibrosus in various cellular regions of intact discs over a range of applied flexion. Macroscale strains were measured on the annulus fibrosus surface, and contrasted to in situ microscale strains using novel confocal microscopy techniques for dual labeling of the cell and the extracellular matrix. Fiber oriented surface strains were significantly higher than in situ fiber strains, which implies a mechanism of load redistribution that minimizes strain along the fibers. Non-uniformity of the strains and matrix distortion occurred immediately and most interestingly varied little with increase in flexion (3–16°), suggesting that inter-fiber shear is important in the initial stages of strain redistribution. Fiber oriented intercellular strains were significantly larger and compressive compared to in situ strains in other regions of the extracellular matrix indicating that the mechanical environment in this region may be unique. Further examination of the structural morphology in this pericellular region is needed to fully understand the pathway of strain transfer from the tissue to the cell. This study provides new knowledge on the complex in situ micro-mechanical environment of the annulus fibrosus that is essential to understanding the mechanobiological behavior of this tissue.     

Sustained cell proliferation in denervated skeletal muscle of mice

Summary Cellular proliferation in skeletal muscle was measured throughout the first 4 weeks after denervation. Twenty four mice had one leg denervated, and 4 groups of 6 of these mice were injected with tritiated thymidine once daily for 7 days, either during the first, second, third or fourth week after denervation. Autoradiographic labelling of muscle and connective tissue nuclei in denervated muscles was compared with innervated muscles from the opposite innervated legs of the same mice. Labelling of connective tissue and muscle (myonuclear and satellite cell) nuclei was significantly higher in denervated muscles, compared with innervated muscles on the unoperated side. There were no significant differences among labelling of nuclei in muscles denervated for 1, 2, 3 or 4 weeks. However, connective tissue labelling after 1 week of denervation was significantly higher than at later times. This study shows that nuclei of muscle and connective tissue cells proliferate and turnover at high levels for at least one month after denervation.     

Contribution of collagen fibers to the compressive stiffness of cartilaginous tissues

Cartilaginous tissues such as the intervertebral disk are predominantly loaded under compression. Yet, they contain abundant collagen fibers, which are generally assumed to contribute to tensile loading only. Fiber tension is thought to originate from swelling of the proteoglycan-rich nucleus. However, in aged or degenerate disk, proteoglycans are depleted, whereas collagen content changes little. The question then rises to which extend the collagen may contribute to the compressive stiffness of the tissue. We hypothesized that this contribution is significant at high strain magnitudes and that the effect depends on fiber orientation. In addition, we aimed to determine the compression of the matrix. Bovine inner and outer annulus fibrosus specimens were subjected to incremental confined compression tests up to 60 % strain in radial and circumferential direction. The compressive aggregate modulus was determined per 10 % strain increment. The biochemical composition of the compressed specimens and uncompressed adjacent tissue was determined to compute solid matrix compression. The stiffness of all specimens increased nonlinearly with strain. The collagen-rich outer annulus was significantly stiffer than the inner annulus above 20 % compressive strain. Orientation influenced the modulus in the collagen-rich outer annulus. Finally, it was shown that the solid matrix was significantly compressed above 30 % strain. Therefore, we concluded that collagen fibers significantly contribute to the compressive stiffness of the intervertebral disk at high strains. This is valuable for understanding the compressive behavior of collagen-reinforced tissues in general, and may be particularly relevant for aging or degenerate disks, which become more fibrous and less hydrated.     

Glycation increases human annulus fibrosus stiffness in both experimental measurements and theoretical predictions

One of the primary age-related changes to collagenous tissues is the increased concentration of advanced glycation endproducts (AGEs). Although AGEs have been shown to increase the mechanical stiffness of many tissues, their influence on the mechanical properties of the annulus fibrosus has not been measured experimentally. In previous theoretical work, we hypothesized that the mechanical influence of AGEs on the annulus could be represented in an additive strain energy function with a separate crosslinking term, but the material coefficients associated with this term were not correlated with AGE concentration. In the current study, we measured the tensile stress-strain response of the human annulus in the axial direction both before and after glycation with methylglyoxal. Using nonlinear regression, the strain energy function was simultaneously applied to these new data and to data from a wide range of experimental protocols reported in the literature to determine values for the material coefficients appearing in the constitutive equation. Nonenzymatic collagen crosslinking induced a statistically significant change in annular material properties. Furthermore, the concentration of AGEs correlated positively with the material coefficients found in the terms of the strain energy function that we associate with collagen crosslinking. These data suggest that AGEs contribute to age-related disc stiffening as well as validate the hypothesis that biochemical constituents can be related mathematically to tissue behavior. In the future, this structurally guided constitutive relationship may provide further insight into the structure-function relationships of the annulus fibrosus.     

Barriers to mesenchymal stromal cells for low back pain

Intervertebral disc degeneration is the main cause of low back pain. In the past 20 years, the injection of mesenchymal stromal cells (MSCs) into the nucleus pulposus of the degenerative disc has become the main approach for the treatment of low back pain. Despite the progress made in this field, there are still many barriers to overcome. First, intervertebral disc is a highly complex load-bearing composite tissue composed of annulus fibrosus, nucleus pulposus and cartilaginous endplates. Any structural damage will change its overall biomechanical function, thereby causing progressive degeneration of the entire intervertebral disc. Therefore, MSC-based treatment strategies should not only target the degenerated nucleus pulposus but also include degenerated annulus fibrosus or cartilaginous endplates. Second, to date, there has been relatively little research on the basic biology of annulus fibrosus and cartilaginous endplates, although their pathological changes such as annular tears or fissures, Modic changes, or Schmorl's nodes are more commonly associated with low back pain. Given the high complexity of the structure and composition of the annulus fibrosus and cartilaginous endplates, it remains an open question whether any regeneration techniques are available to achieve their restorative regeneration. Finally, due to the harsh microenvironment of the degenerated intervertebral disc, the delivered MSCs die quickly. Taken together, current MSC-based regenerative medicine therapies to regenerate the entire disc complex by targeting the degenerated nucleus pulposus alone are unlikely to be successful.     

Annulus Fibrosus Cell Characteristics Are a Potential Source of Intervertebral Disc Pathogenesis

In the end stage of intervertebral disc degeneration, cartilage, bone, endothelial cells, and neurons appear in association with the worsening condition. The origin of the abnormal cells is not clear. This study investigated the properties of progenitor cells in the annulus fibrosus (AF) using one in vitro and two in vivo models. Cultivation of rabbit AF cells with chondrogenic media significantly increased expressions of collagen and aggrecan. Upon exposure to osteogenic conditions, the cultures showed increased mineralization and expression of osteopontin, runx2, and bmp2 genes. Two models were used in the in vivo subcutaneous implantation experiments: 1) rabbit AF tissue in a demineralized bone matrix (DBM) cylinder (DBM/AF), and, 2) rat intact and needle punctured lumbar discs. Bone formation in the AF tissue was detected and hypertrophic chondrocytes and osteoblasts were present 1 month after implantation of the DBM/AF to nude mice. In addition to collagen I and II, immunostaining shows collagen X and osteocalcin expression in DBM/AF specimens 4 months after implantation. Similar changes were detected in the injured discs. Almost the entire needle punctured disc had ossified at 6 months. The results suggest that AF cells have characteristics of progenitor cells and, under appropriate stimuli, are capable of differentiating into chondrocytes and osteoblasts in vitro as well as in vivo. Importantly, these cells may be a target for biological treatment of disc degeneration.     

人退变椎间盘软骨终板干细胞多向分化的研究

目的:为了分离和鉴定人退变椎间盘软骨终板干细胞。方法:收集因腰椎间盘退变性疾病行腰椎间盘摘除术并植骨融合的标本。在解剖显微镜下清理软骨终板组织,并消化软骨终板,提取软骨终板细胞。获得的软骨终板细胞经过琼脂糖三维筛选系统培养后,选取细胞克隆团并进行体外扩增,扩增后的细胞行流式细胞术检测干细胞标志物证实退变软骨终板中存在干细胞。结果:共聚焦免疫荧光提示退变椎间盘软骨终板组织中存在干细胞标志物STRO1、CD105、CD73、CD90阳性的细胞。经琼脂糖三维培养基筛选的CESCs在免疫表型上符合干细胞标准。结论:在人退变椎间盘的软骨终板中存在具有多向分化潜能的干细胞。