Adult Intervertebral Disc Research Articles

High glucose accelerates autophagy in adult rat intervertebral disc cells.

Study DesignIn vitro cell culture.PurposeThe purpose of this study was to investigate the effect of high glucose on autophagy in adult rat intervertebral disc cells.Overview of LiteratureDiabetes mellitus is considered to be an important etiologic factor for intervertebral disc degeneration, resulting in degenerative disc diseases. A glucose-mediated increase of autophagy is a major causative factor for the development of diseases associated with diabetes mellitus. However, no information is available for the effect of high glucose on autophagy in adult intervertebral disc cells.MethodsNucleus pulposus and annulus fibrosus cells were isolated from 24-week-old adult rats, cultured and placed in either 10% fetal bovine serum (normal control) or 10% fetal bovine serum plus two different high glucose concentrations (0.1 M and 0.2 M) (experimental conditions) for one and three days, respectively. The expressions of autophagy markers, such as beclin-1, light chain 3-I (LC3-I) and LC3-II, autophagy-related gene (Atg) 3, 5, 7 and 12, were identified and quantified.ResultsTwo high glucoses significantly increased the expressions of beclin-1, LC3-II, Atg3, 5, 7, and 12 in adult rat nucleus pulposus and annulus fibrosus cells in a dose- and time-dependent manner. The ratio of LC3-II/LC3-I expression was also increased in a dose-respectively time-dependent manner.ConclusionsThe results suggest that autophagy of adult nucleus pulposus and annulus fibrosus cells might be a potential mechanism for the intervertebral disc degeneration in adult patients with diabetes mellitus. Thus, the prevention of autophagy in adult intervertebral disc cells might be considered as a novel therapeutic target to prevent or to delay the intervertebral disc degeneration in adult patients with diabetes mellitus.

AB0674 Spine osteoarthritis - clinical and morphological comparisons

BackgroundSpine osteoarthritisis one of the leading places in the structure of the Rheumatic Diseases. Until recently, the degeneration of the intervertebral disc (IVD), reflected in the reduction of the number...

The Fuzzy Logic of Degenerative Disc Disease: From a Lorenz Attractor to a Percolation Threshold Model

Apart from a small number of capillaries that penetrate only a few millimeters into the outermost annulus, the normal adult human intervertebral disc is virtually avascular (9). Therefore, the nutrients necessary for the survival of the cells inside the disc (especially glucose and oxygen) are derived mainly from diffusion from vascular channels (which have been called “vascular buds”) present in the limit between the intervertebral disc and the vertebral cartilagenous endplate (3). In a very elegant study, a research group from the Biomedical Engineering Department of the University of Miami investigated the effects of mechanical deformation and anisotropy on the transport of oxygen and glucose in the annulus fibrosus of human intervertebral discs obtained from a tissue bank (7). Using custom-designed chambers (Figure 1A and B), the diffusivities of oxygen and the partition coefficient of glucose were measured under 3 levels of confined uniaxial compression (0%, 10%, and 20%) and in 3 directions (axial, circumferential, and radial) (Figure 1C). The results demonstrated that glucose

Gene Expression Profiling of Early Intervertebral Disk Degeneration

IntroductionEarly degeneration of the IVD involves a change in the nucleus pulposus (NP) cell population, characterized by the replacement/differentiation of the native notochordal cells (NCs) by/into chondrocyte-like cells (CLCs). In this respect, the NC has received considerable attention as a potential NP progenitor cell and as a target to realize disk regeneration.1,2 The canine species is affected by naturally occurring IVD degeneration, like humans.3 Moreover, with regard to the maintenance of NCs and the occurrence of IVD degeneration, the canine species reveals a unique phenomenon. In chondrodystrophic dogs, NCs are replaced by CLCs before the age of 1 year, with a concurrent onset of IVD degeneration at all spinal levels. By contrast, in nonchondrodystrophic dogs the NC remains the predominant cell type of the NP during the majority of life; replacement of NCs by CLCs and concurrent IVD degeneration only occur at old age. The aim of this study is to investigate gene expression profiles in the process of early NP degeneration, thereby revealing potential biomolecular signaling pathways for IVD regeneration.Materials and MethodsCanine IVD samples from chondrodystrophic and nonchondrodystrophic dogs were classified into (1) NP rich in NCs, (2) NP containing both NCs and CLC-cells (Mixed), and (3) early degenerated NP rich in CLCs (Fig. 1). Gene expression profiling of the NP of these three groups was performed using a two-color cDNA-microarray, with subsequent validation (qPCR and immunohistochemistry) in IVD samples and cultures of primary NCs.ResultsEarly IVD degeneration involved significant regulations of various pathways, including extracellular matrix (ECM) remodeling, plasmin, and plasminogen activator urokinase. Bone Morphogenetic Protein (BMP)-, and Wnt/β-catenin-signaling and cytoskeleton remodeling. Wnt/β-catenin signaling plays a crucial role in maintaining the notochordal fate during embryogenesis and stem-cell fate determination, but is also involved in IVD degeneration and regeneration. Whether the differential expression of Wnt/β-catenin-signaling is associated with degeneration or is an attempt to repair the damaged IVD remains uncertain. Both NCs and CLCs showed nuclear and cytoplasmic expression of the Wnt effector protein β-catenin4; axin2 gene expression, a specific read-out for Wnt signaling, revealed a dual response: in the initial stages of NP degeneration tended to increase (Mixed group), but diminished with ongoing degeneration (CLC-rich group). A concurrent increase in Wnt inhibitory factor 1 gene expression was observed, which may be a significant factor involved in the downregulation of canonical Wnt signaling in the chondrodystrophic dogs.5 Caveolin-1, which is known to regulate canonical Wnt signaling,6 emerged as a significantly regulated gene. NCs in culture showed robust caveolin-1 gene and protein expression and IVD degeneration involved significant decrease in Caveolin-1 gene and protein expression.ConclusionEarly IVD degeneration involves significant alterations in biomolecular pathways involved in ECM synthesis, cell-matrix interactions, and cytoskeletal remodeling. Subclinical, early IVD degeneration is associated with diminished Wnt/β-catenin signaling activity. In this process, caveolin-1 expression appears to be a relevant factor in NC physiology and thus maintenance of the healthy NP.I confirm having declared any potential conflict of interest for all authors listed on this abstractNoDisclosure of InterestNone declaredRisbud MV, Shapiro IM. Notochordal cells in the adult intervertebral disc: new perspective on an old question. Critical Reviews in Eukaryotic Gene Expression 2011;21(1):29–41Erwin WM. The enigma that is the nucleus pulposus cell: the search goes on. Arthritis Research and Therapy 2010;12(3):118Bergknut N, et al. The dog as an animal model for intervertebral disc degeneration? Spine (Phila Pa 1976) 2011; Epub ahead of printSmolders LA, et al. Basal canonical Wnt signaling of the notochordal cells is upregulated during the process of intervertebral disc degeneration. Journal of orthopaedic Research 2011; Epub ahead of printSurmann-Schmitt C, et al. Wif-1 is expressed at cartilage-mesenchyme interfaces and impedes Wnt3a-mediated inhibition of chondrogenesis. Journal of cell Science 2009;122(Pt 20):3627–3637Yamamoto H, et al. Caveolin is necessary for Wnt-3a-dependent internalization of LRP6 and accumulation of beta-catenin. Developmental Cell 2006;11(2):213–223

Gene Expression Profiling of Early Intervertebral Disk Degeneration

Introduction Early degeneration of the IVD involves a change in the nucleus pulposus (NP) cell population, characterized by the replacement/differentiation of the native notochordal cells (NCs) by/into chondrocyte-like cells (CLCs). In this respect, the NC has received considerable attention as a potential NP progenitor cell and as a target to realize disk regeneration.1,2 The canine species is affected by naturally occurring IVD degeneration, like humans.3 Moreover, with regard to the maintenance of NCs and the occurrence of IVD degeneration, the canine species reveals a unique phenomenon. In chondrodystrophic dogs, NCs are replaced by CLCs before the age of 1 year, with a concurrent onset of IVD degeneration at all spinal levels. By contrast, in nonchondrodystrophic dogs the NC remains the predominant cell type of the NP during the majority of life; replacement of NCs by CLCs and concurrent IVD degeneration only occur at old age. The aim of this study is to investigate gene expression profiles in the process of early NP degeneration, thereby revealing potential biomolecular signaling pathways for IVD regeneration. Materials and Methods Canine IVD samples from chondrodystrophic and nonchondrodystrophic dogs were classified into (1) NP rich in NCs, (2) NP containing both NCs and CLC-cells (Mixed), and (3) early degenerated NP rich in CLCs (Fig. 1). Gene expression profiling of the NP of these three groups was performed using a two-color cDNA-microarray, with subsequent validation (qPCR and immunohistochemistry) in IVD samples and cultures of primary NCs. Results Early IVD degeneration involved significant regulations of various pathways, including extracellular matrix (ECM) remodeling, plasmin, and plasminogen activator urokinase. Bone Morphogenetic Protein (BMP)-, and Wnt/β-catenin-signaling and cytoskeleton remodeling. Wnt/β-catenin signaling plays a crucial role in maintaining the notochordal fate during embryogenesis and stem-cell fate determination, but is also involved in IVD degeneration and regeneration. Whether the differential expression of Wnt/β-catenin-signaling is associated with degeneration or is an attempt to repair the damaged IVD remains uncertain. Both NCs and CLCs showed nuclear and cytoplasmic expression of the Wnt effector protein β-catenin4; axin2 gene expression, a specific read-out for Wnt signaling, revealed a dual response: in the initial stages of NP degeneration tended to increase (Mixed group), but diminished with ongoing degeneration (CLC-rich group). A concurrent increase in Wnt inhibitory factor 1 gene expression was observed, which may be a significant factor involved in the downregulation of canonical Wnt signaling in the chondrodystrophic dogs.5 Caveolin-1, which is known to regulate canonical Wnt signaling,6 emerged as a significantly regulated gene. NCs in culture showed robust caveolin-1 gene and protein expression and IVD degeneration involved significant decrease in Caveolin-1 gene and protein expression. Conclusion Early IVD degeneration involves significant alterations in biomolecular pathways involved in ECM synthesis, cell-matrix interactions, and cytoskeletal remodeling. Subclinical, early IVD degeneration is associated with diminished Wnt/β-catenin signaling activity. In this process, caveolin-1 expression appears to be a relevant factor in NC physiology and thus maintenance of the healthy NP. I confirm having declared any potential conflict of interest for all authors listed on this abstract No Disclosure of Interest None declared Risbud MV, Shapiro IM. Notochordal cells in the adult intervertebral disc: new perspective on an old question. Critical Reviews in Eukaryotic Gene Expression 2011;21(1):29–41 Erwin WM. The enigma that is the nucleus pulposus cell: the search goes on. Arthritis Research and Therapy 2010;12(3):118 Bergknut N, et al. The dog as an animal model for intervertebral disc degeneration? Spine (Phila Pa 1976) 2011; Epub ahead of print Smolders LA, et al. Basal canonical Wnt signaling of the notochordal cells is upregulated during the process of intervertebral disc degeneration. Journal of orthopaedic Research 2011; Epub ahead of print Surmann-Schmitt C, et al. Wif-1 is expressed at cartilage-mesenchyme interfaces and impedes Wnt3a-mediated inhibition of chondrogenesis. Journal of cell Science 2009;122(Pt 20):3627–3637 Yamamoto H, et al. Caveolin is necessary for Wnt-3a-dependent internalization of LRP6 and accumulation of beta-catenin. Developmental Cell 2006;11(2):213–223

A Cre knock‐in mouse line on the Sickle tail locus induces recombination in the notochord and intervertebral disks

Sickle tail (Skt) was originally identified by gene trap mutagenesis in mice, and the trapped gene is highly expressed in the notochord, intervertebral discs (IVD), and mesonephros. Here, we report the generation of Skt(cre) mice expressing Cre recombinase in the IVD due to target insertion of the cre gene into the Skt locus by recombinase-mediated cassette exchange. Crossing a conditional lacZ Reporter (R26R), Cre expression from the Skt(cre) allele specifically activates β-galactosidase expression in the whole notochord from E9.5 onwards. In E15.5 Skt(cre);R26R embryos, reporter activity was detected in the nucleus pulposus and in a portion of the annulus fibrosus, resulting in expansion of Cre-expressing cells in the adult IVD. Reporter activity was also seen in the Skt(cre);R26R mesonephros at E15.5. These results suggest that Skt(cre) mice are useful for exploring the fate specification of notochordal cells and creating models for IVD-related skeletal diseases.

Transforming growth factor β controls CCN3 expression in nucleus pulposus cells of the intervertebral disc

To investigate transforming growth factor β (TGFβ) regulation of CCN3 expression in cells of the nucleus pulposus. Real-time reverse transcription-polymerase chain reaction and Western blot analyses were used to measure CCN3 expression in the nucleus pulposus. Transfections were used to measure the effect of Smad3, MAPKs, and activator protein 1 (AP-1) on TGFβ-mediated CCN3 promoter activity. Lentiviral knockdown of Smad3 was performed to assess the role of Smad3 in CCN3 expression. CCN3 was expressed in embryonic and adult intervertebral discs. TGFβ decreased the expression of CCN3 and suppressed its promoter activity in nucleus pulposus cells. DN-Smad3, Smad3 small interfering RNA, or DN-AP-1 had little effect on TGFβ suppression of CCN3 promoter activity. However, p38 and ERK inhibitors blocked suppression of CCN3 by TGFβ, suggesting involvement of these signaling pathways in the regulation of CCN3. Interestingly, overexpression of Smad3 in the absence of TGFβ increased CCN3 promoter activity. We validated the role of Smad3 in controlling CCN3 expression in Smad3-null mice and in nucleus pulposus cells transduced with lentiviral short hairpin Smad3. In terms of function, treatment with recombinant CCN3 showed a dose-dependent decrease in the proliferation of nucleus pulposus cells. Moreover, CCN3-treated cells showed a decrease in aggrecan, versican, CCN2, and type I collagen expression. The opposing effect of TGFβ on CCN2 and CCN3 expression and the suppression of CCN2 by CCN3 in nucleus pulposus cells further the paradigm that these CCN proteins form an interacting triad, which is possibly important in maintaining extracellular matrix homeostasis and cell numbers.

Notochordal cells in the adult intervertebral disc: new perspective on an old question.

The intervertebral disc is a tissue positioned between each of the vertebrae that accommodates applied biomechanical forces to the spine. The central compartment of the disc contains the nucleus pulposus (NP) which is enclosed by the annulus fibrosus and the endplate cartilage.The NP is derived from the notochord, a rod-like structure of mesodermal origin. Development of the notochord is tightly regulated by interactive transcription factors and target genes. Since a number of these molecules are unique they have be used for cell lineage and fate mapping studies of tissues of the intervertebral disc. These studies have shown that in a number of species including human, NP tissue retains notochordal cells throughout life. In the adult NP, there are present both large and small notochordal cells, as well as a progenitor cell population which can differentiate along the mesengenic pathway. Since tissue renewal in the intervertebral disc is dependent on the ability of these cells to commit to the NP lineage and undergo terminal differentiation, studies have been performed to assess which signaling pathways may regulate these activities. The notch signaling pathway is active in the intervertebral disc and is responsive to hypoxia, probably through HIF-1a. From a disease viewpoint, it is hypothesized that an oxemic shift, possibly mediated by alterations in the vascular supply to the tissues of the disc would be expected to lead to a failure in notochordal progenitor cell activation and a decrease in the number of differentiated cells. In turn, this would lead to decrements in function and enhancement of the effect of agents that are known to promote disc degeneration.

Fibronectin splicing variants in human intervertebral disc and association with disc degeneration.

In this study, normal intervertebral disc (IVD) tissues and degenerative human IVD tissues were compared for presence of fibronectin (FN) mRNA splice variants and for FN fragments (FN-f). To further understand FN RNA splice forms and protein fragments in disc degeneration. FN splice variants play important roles in regulating cell-matrix and matrix-matrix interactions in skeletogenesis and skeletal function in limbs and other sites. However, presence and possible roles of FN splice variants and fragments in human IVD have not been determined. Normal infant and adult IVD tissues were obtained from organ donors, and degenerative human IVD tissues were obtained from patients undergoing spinal surgeries. FN splice patterns were assessed by reverse transcriptase polymerase chain reaction. Relative expression levels were semiquantified by densitometry. FN and its fragments were studied by Western blot analysis. Both the EDB and EDB splice variants were present in normal and degenerative IVD tissues. The EDB to EDB ratio was highest in moderately degenerative tissue. The EDA domain was only expressed in infant but not adult tissue. Variable-region (V) splice forms were present in all tissues studied. A splice form with the entire V-region, the 15th type III domain, and 10th type I domain adjacent to the 3'end of V region omitted (referred to as [V+III-15+I-10], also known as [V+C] splice form) was present at higher levels in adult than in infant samples. FN-f were also detected in degenerating tissue, but not in normal IVD tissue samples. The data indicate that higher levels of EDB isoform and FN-f are associated with IVD degeneration. This shift in alternative splicing may reflect an attempt of tissue repair and remodeling. Novel information gathered in this study will lead to a better understanding of pathologic processes associated with disc malfunction and degeneration.

Toward an understanding of the role of notochordal cells in the adult intervertebral disc: From discord to accord

The goal of this mini-review is to address the long standing argument that the pathogenesis of disc disease is due to the loss and/or the replacement of the notochordal cells by other cell types. We contend that, although cells of different size and morphology exist, there is no strong evidence to support the view that the nucleus pulposus contains cells of distinct lineages. Based on lineage mapping studies and studies of other notochordal markers, we hypothesize that in all animals, including human, nucleus pulposus retains notochordal cells throughout life. Moreover, all cells including chondrocyte-like cells are derived from notochordal precursors, and variations in morphology and size are representative of different stages of maturation, and or, function. Thus, the most critical choice for a suitable animal model should relate more to the anatomical and mechanical characteristics of the motion segment than concerns of cell loss and replacement by non-notochordal cells.

The presence and absence of lymphatic vessels in the adult human intervertebral disc: relation to disc pathology

Although the normal adult human intervertebral disc is considered to be avascular, vascularised cellular fibrous tissue can be found in pathological conditions involving the disc such as disc herniation. Whether lymphatics vessels form a component of this reparative tissue is not known as the presence or absence of lymphatics in herniated and normal disc tissue is not known. We examined spinal tissues and discectomy specimens for the presence of lymphatics. The examination used immunohistochemistry to identify the specific lymphatic endothelial cell markers,podoplanin and LYVE1. Lymphatic vessels were not found in the nucleus pulposus or annulus fibrosus of intact, non-herniated lumbar and thoracic discs but were present in the surrounding ligaments. Ingrowth of fibrous tissue was seen in 73% of herniated disc specimens of which 36% contained LYVE1+/podoplanin + lymphatic vessels. Lymphatic vessels were not seen in the sacrum and coccyx or biopsies of four sacrococcygeal chordomas, but they were noted in surrounding extra-osseous fat and fibrous tissue at the edge of the infiltrating tumour. Our findings indicate that lymphatic vessels are not present in the normal adult intervertebral disc but that, when there is extrusion of disc material into surrounding soft tissue, there is ingrowth of reparative fibrous tissue containing lymphatic vessels. Our findings also indicate that chordoma, a tumour of notochordal origin, spreads to regional lymph nodes via lymphatics in para-spinal soft tissues.

Isolation and in vitro chondrogenic potential of human foetal spine cells

Cell therapy for nucleus pulposus (NP) regeneration is an attractive treatment for early disc degeneration as shown by studies using autologous NP cells or stem cells. Another potential source of cells is foetal cells. We investigated the feasibility of isolating foetal cells from human foetal spine tissues and assessed their chondrogenic potential in alginate bead cultures. Histology and immunohistochemistry of foetal tissues showed that the structure and the matrix composition (aggrecan, type I and II collagen) of foetal intervertebral disc (IVD) were similar to adult IVD. Isolated foetal cells were cultured in monolayer in basic media supplemented with 10% Fetal Bovine Serum (FBS) and from each foetal tissue donation, a cell bank of foetal spine cells at passage 2 was established and was composed of around 2000 vials of 5 million cells. Gene expression and immunohistochemistry of foetal spine cells cultured in alginate beads during 28 days showed that cells were able to produce aggrecan and type II collagen and very low level of type I and type X collagen, indicating chondrogenic differentiation. However variability in matrix synthesis was observed between donors. In conclusion, foetal cells could be isolated from human foetal spine tissues and since these cells showed chondrogenic potential, they could be a potential cell source for IVD regeneration.

Aggrecan, versican and type VI collagen are components of annular translamellar crossbridges in the intervertebral disc

The aim of this study was to undertake a detailed analysis of the structure of the inter and intra-lamellar regions of the annulus fibrosus. A total of 30 newborn to 6 year-old lumbar ovine intervertebral discs (IVDs) were fixed and decalcified en-bloc to avoid differential swelling artifacts during processing and vertical mid-sagittal, and horizontal 4 mum sections were cut. These were stained with toluidine blue to visualise anionic proteoglycan (PG) species, H & E for cellular morphology and picro-sirius red (viewed under polarized light) to examine collagenous organization. Immunolocalisations were also undertaken using anti-PG core-protein and glycosaminoglycan (GAG) side chain antibodies to native chondroitin sulphate (CS), Delta C-4-S and C-6-S unsaturated stubs generated by chondroitinase ABC digestion of CS, keratan sulphate (KS), and with antibodies to type I, II, VI, IX and X collagens. Trans-lamellar cross bridges (TLCBs), discontinuities in annular lamellae's which provide transverse interconnections, stained prominently with toluidine blue in the adult IVDs but less so in the newborn IVDs. In adult discs TLCBs were evident in both the posterior and anterior AF where they extended from the outermost annular lamellae almost to the transitional zone extending across as many as eight lamellar layers displaying a characteristic circuitous, meandering, serpentine type course. There were significantly fewer TLCBs in 2 week-old compared with skeletally mature sheep and there was a further increase from 2 to 6 years. Immunolocalisation of perlecan delineated blood vessels in the TLBs of the newborn but not adult IVDs extending into the mid AF. In contrast adult but not 2 week-old TLCBs were immunopositive for C-4-S, C-6-S, KS, aggrecan, versican and type VI collagen. The change in number and matrix components of the trans-lamellar cross bridges with skeletal maturity and ageing suggest that they represent an adaptation to the complex biomechanical forces occurring in the annulus fibrosus.

The notochord in the mammalian adult: A paradox

This issue of Arthritis & Rheumatism contains a report by Erwin et al describing the authors’ investigation of a population of early embryonic cells from the notochord—by analyzing not embryos, but intervertebral discs from adult dogs (1). How is this possible? The fine chord defining the body axis in the early embryo of endoskeletal animals (vertebrates) is the basis of an evolutionary explosion, giving rise to everything from ascidians and amphioxus (2) to fish and humans (3). Its power lies in the specific mix of mesenchymal stem cells that ultimately are the origin of the central skeleton, including the spine and intervertebral bodies. Should there be a source of chordal stem cells, those cells might approach the ideal of embryonic stem cells residual in an adult organism. But is there a notochord cell within an adult spine? And how might one discriminate such a cell within the spine tissue cells? Current opinion holds that a certain subpopulation of intervertebral disc cells are remainders of the embryonic notochord and are therefore termed notochord cells (4). In the embryo, those cells are well defined by location in the notochord, by morphology (1,5,6), and by gene expression pattern (3,4). In the adult, morphology and location are the only criteria used to discriminate notochord cells from chondrocytes of the nucleus pulposus in the intervertebral disc. Since the notochord cells are thought to possess a buoyant density different from that of chondrocytes, density gradient centrifugation is applied to separate the 2 populations (7). As described in the report by Erwin et al (1), the cytoplasm of notochord cells is “foamy” (leading to the term “physaliphorous cells”), and their nucleus is amorphous and extended in size. These features may contribute to reduced buoyant density. An extensive endoplasmic reticulum (ER) is the origin of the physaliphorous morphology of that subpopulation in the embryonic notochord (5), and pioneering studies of the ER have involved investigations of its function in the embryonic notochord (8). Most adult mammals do not possess disc cells with similar morphology, but instead possess only fibroblastic or chondrocytic cells. However, a physaliphorous cytoplasm is expressed by a population of disc cells in some domestic animals, including dogs, sheep, and goats (4,9). Interestingly, Jun seems to prevent notochord cell apoptosis, and induced Jun deletion (in the Cre/lox system) causes scoliosis in the axial skeleton (10). In mongrel dogs, the presence of notochord cells is associated with resistance to agerelated disc degeneration, and the cells are able to promote in vitro proteoglycan production by chondrocytes (11). At least part of this capability is attributable to the tissue hormone connective tissue growth factor, as identified also in the study by Erwin and colleagues (1). But are these cells residual notochord cells, or successors, or cells that opportunistically and simply express the endoplasmic morphology of heavily active cells involved in the maintenance of disc tissue? The implications for preventive measures or curative approaches against disc degeneration are obvious. The critical issue is the definition of the term “notochord cells.” The biochemical findings presented by Erwin et al describe those cells as supportive for chondrocytes. Beyond the key hormonal function, the elaborate ER reflects intense metabolic activities, but which ones? Embryonic notochord cells could be identified via the expression of embryogenetic gene families such as Fox and Sox (3), but the current literature includes no information on Fox or Sox expression in those adult notochordal cells. Quite obviously, gene expression profiling would be beneficial. There is a major difference in perspectives: on the one side, there could be a supportive cell type enabling adult intervertebral discs to sustain the Juergen A. Mollenhauer, PhD, DSc: Naturwissenschaftliches und Medizinisches Institut an der Universitaet Tuebingen, Reutlingen, Germany, and Rush University Medical Center, Chicago, Illinois. Address correspondence and reprint requests to Juergen A. Mollenhauer, PhD, DSc, NMI, Markwiesenstr. 55, D-72770, Reutlingen, Germany. E-mail: juergen.mollenhauer@nmi.de. Submitted for publication June 19, 2006; accepted in revised form August 24, 2006.

Temporo-spatial distribution of blood vessels in human lumbar intervertebral discs

While there is consensus in the literature that blood vessels are confined to the outer anulus fibrosus of normal adult intervertebral disc, debate continues whether there is a vascular in-growths into inner parts of the intervertebral disc during degeneration. We therefore tested the hypothesis that vascular in-growth is not a distinct feature of disc degeneration. The specific endothelial cell marker CD 31 (PECAM) was used to immunohistochemically investigate 42 paraffin-embedded complete mid-sagittal human intervertebral disc sections of various ages (0-86 years) and varying extent of histomorphological degeneration. Additionally, 20 surgical disc samples from individuals (26-69 years) were included in this study. In discs of fetal to infantile age, blood vessels perforated the cartilaginous end plate and extended into the inner and outer anulus fibrosus, but not into the nucleus pulposus. In adolescents and adults, no blood vessels were seen except for the outer zone of the anulus fibrosus adjacent to the insertion to ligaments. The cartilaginous end plate remained free of vessels, except for areas with circumscribed destruction of the end plate. In advanced disc degeneration, no vessels were observed except for those few cases with complete, scar-like disc destruction. However, some rim lesions and occasionally major clefts were surrounded by a small network of capillary blood vessels extending into deeper zones of the anulus fibrosus. A subsequent morphometric analysis, revealed slightly "deeper" blood vessel extension in juvenile/adolescent discs when compared to young, mature and senile adult individuals with significantly "deeper" extension in the posterior than anterior anulus. The analysis of the surgical specimens showed that only sparse capillary blood vessels which did not extend into the nucleus pulposus even in major disc disruption. Our results show that vascular invasion deeper than the periphery was not observed during disc degeneration, which supports the hypothesis that vascular in-growth is not a distinct feature of disc degeneration.

Cells From Different Regions of the Intervertebral Disc

This study examined how the culture system and region of cellular origin affect disc cell morphology and extracellular matrix production. To determine the role of the cell populations in the different regions of the adult intervertebral disc in maintaining gradients in composition across the disc. It is not known whether the steep profiles in composition across the intervertebral disc are maintained by distinct cell populations or whether differences in cell metabolism are determined by changes in the physical environment across the disc. Very little information exists on the matrix produced by cells from the mature, non-notochordal nucleus pulposus. Cells were extracted from articular cartilage, nucleus pulposus, and the inner and outer anulus fibrosus of caudal discs from 18- to 24-month-old steers cultured in alginate or collagen gels or in monolayer. The effect of culture system and cell origin on cell morphology and matrix synthesis was measured using 35S-sulphate labeling and indirect immunolocalization. Distinct morphologic differences between cells from different regions cultured in monolayer were retained through two passages. The rate of sulfate incorporation varied with cell type. Immediately after isolation, it was two- to threefold greater for nucleus cells than for cells from the disc inner anulus or articular cartilage. The rate was lowest for outer anulus cells. It also varied with culture system. For all cell types, the incorporation rate was highest in alginate and lowest in monolayer. Immunolocalization showed that nucleus cells stained strongly for all proteoglycan epitopes, whereas outer anulus cells stained least and in monolayer produced little proteoglycan. The disc has at least three distinct cell populations, which differ in morphology and in amount and type of matrix they produce. Cells from mature nucleus pulposus produced sulfated glycosaminoglycans at a high rate in contrast to reported results for notochordal nucleus cells. Alginate, although an appropriate culture system for inner anulus and nucleus cells, may not be a suitable medium for outer anulus cells.

The characterization of versican and its message in human articular cartilage and intervertebral disc

Splicing variation of the versican message and size heterogeneity of the versican core protein were analyzed in human articular cartilage and intervertebral disc. Splicing variation of the message was studied by PCR analysis to detect the presence or absence of exons 7 and 8, which encode large chondroitin sulfate attachment regions. At all ages in normal cartilage from the third trimester fetus to the mature adult, the presence of the versican isoform possessing exon 8 but not exon 7 (V 1) could be readily detected. The message isoforms possessing neither exon 7 nor 8 (V 3) or both exons 7 and 8 (V 0) were only detectable in the fetus, and the isoform possessing only exon 7 (V 2) was never detected. In osteoarthritic cartilage and in adult intervertebral disc the versican message pattern was the same as that observed in the normal adult with only the isoform possessing exon 8 being detected. Core protein heterogeneity was studied by immunoblotting following enzymic removal of the glycosaminoglycan chains from the proteoglycan, using an antibody recognizing the globular G1 region of versican. All articular cartilage extracts from the fetus to the mature adult contained multiple core protein sizes of greater than 200 kDa. The adult cartilage extracts tended to have an increased proportion of the smaller sized core proteins and osteoarthritic cartilage possessed similar core protein sizes to the normal adult. In contrast, intervertebral disc at all post-natal ages showed a greater range of size heterogeneity with a prominent component of about 50 kDa. The abundance of this component increased if the samples were treated with keratanase prior to analysis, suggesting that the G1 region of versican in disc can be substituted with keratan sulfate. The increased presence of versican in the disc relative to articular cartilage may suggest a more pronounced functional role for this proteoglycan, particularly in the nucleus pulposus.

A comparative analysis of the differential spatial and temporal distributions of the large (aggrecan, versican) and small (decorin, biglycan, fibromodulin) proteoglycans of the intervertebral disc.

This study provides a comparative analysis of the temporal and spatial distribution of 5 intervertebral disc (IVD) proteoglycans (PGs) in sheep. The main PGs in the 2 and 10 y old sheep groups were polydisperse chondroitin sulphate and keratan sulphate substituted species. Their proportions did not differ markedly either with spinal level or disc zone. In contrast, the fetal discs contained 2 slow migrating (by composite agarose polyacrylamide gel electrophoresis, CAPAGE), relatively monodisperse chondroitin sulphate-rich aggrecan species which were also identified by monoclonal antibody 7-D-4 to an atypical chondroitin sulphate isomer presentation previously found in chick limb bud, and shark cartilage. The main small PG detectable in the fetal discs was biglycan, whereas decorin predominated in the 2 and 10 y old IVD samples; its levels were highest in the outer annulus fibrosus (AF). Versican was most abundant in the AF of the fetal sheep group; it was significantly less abundant in the 2 and 10 y old groups. Furthermore, versican was immunolocalised between adjacent layers of annular lamellae suggesting that it may have some role in the provision of the viscoelastic properties to this tissue. Versican was also diffusely distributed throughout the nucleus pulposus of fetal IVDs, and its levels were significantly lower in adult IVD specimens. This is the first study to identify versican in ovine IVD tissue sections and confirmed an earlier study which demonstrated that ovine IVD cells synthesised versican in culture (Melrose et al. 2000). The variable distribution of the PGs identified in this study provides further evidence of differences in phenotypic expression of IVD cell populations during growth and development and further demonstrates the complexity of the PGs in this heterogeneous but intricately organised connective tissue.

Detection and distribution of the carbohydrate binding protein galectin-3 in human notochord, intervertebral disc and chordoma

Galectin-3 is an endogenous carbohydrate-binding protein which plays a role in cell differentiation, morphogenesis and cancer biology. We investigated the occurrence and distribution of galectin-3 in the embryonic and fetal human notochord, the developing human vertebral column, adult intervertebral discs and in six chordomas, which are tumors thought to originate from notochordal remnants. By means of Western blots, the expression of galectin-3 was confirmed in tissue probes from the vertebral column region beginning with the 8th gestational week. These results were supported by immunohistochemical data which revealed the presence of galectin-3 in the cytoplasm of cells of the notochord also from the 8th gestational week onwards. Notochordal immunostaining became stronger with increasing gestational age. A persisting notochordal remnant in an adult intervertebral disc and various cells of the nucleus pulposus also contained galectin-3. All chordomas showed moderate or strong immunoreactivity irrespective of their cellular composition. Subcellularly, galectin-3 was localized mostly in the cytoplasm, while a subset of tumor cells also showed nuclear distribution. Differences in staining patterns of chordoma cells could not, in general, be correlated to any histological features of these tumors.

Histogenesis of clear cell chondrosarcoma

The histogenesis of clear cell chondrosarcoma is still unclear: Apart from typical clear cell tumor areas, extensive production of woven bone formation suggests within the clear cell cartilagenous stroma is an intriguing phenomenon. Three cases of clear cell chondrosarcoma documented in the Bone Tumor Registry of Westphalia were examined for their patterns of osteonectin expression, and compared with other bone tumors of either osseous or cartilaginous origin, and with normal cartilage tissue. Found predominantly in osseous structures, the protein osteonectin takes part in the formation of new bone. The three clear cell chondrosarcomas showed a strong immunoexpression of osteonectin in clear cell, chondroid and in osseous tumor areas. Similarly, evidence of osteonectin was also found in osteoblastic and in chondroblastic osteosarcomas as well as in osteoblastomas. In contrast, osteonectin could not be demonstrated in the chondrosarcomas and mesenchymal chondrosarcomas from our registry that were analysed for comparison, and was found only minimally in the fibroblastic areas of dedifferentiated chondrosarcomas. The chondroblastic tumor components were always negative. There was no immunoexpression of osteonectin either in fetal or adult intervertebral disc tissue. The present immunohistochemical study of osteonectin has distinctly separated clear cell chondrosarcoma from the other variants of chondrosarcoma, and aptly verified the specificity of this entity. Moreover, the study would call for further histogenetic evaluation of clear cell chondrosarcoma, since the pattern of osteonectin expression in that tumor seems to indicate an osteogenic rather than a chondrogenic origin.