Notochordal Nucleus Pulposus Cells Research Articles

Chondrocyte-like cells in nucleus pulposus and articular chondrocytes have similar transcriptomic profiles and are paracrine-regulated by hedgehog from notochordal cells and subchondral bone.

Objective: The nucleus pulposus (NP) comprises notochordal NP cells (NCs) and chondrocyte-like NP cells (CLCs). Although morphological similarities between CLCs and chondrocytes have been reported, interactions between CLCs and NCs remain unclear. In this study, we aimed to clarify regulatory mechanisms of cells in the NP and chondrocytes. Design: We performed single-cell RNA sequencing (scRNA-seq) analysis of the articular cartilage (AC) and NP of three-year-old cynomolgus monkeys in which NCs were present. We then performed immunohistochemical analysis of NP and distal femur. We added sonic hedgehog (SHH) to primary chondrocyte culture. Results: The scRNA-seq analysis revealed that CLCs and some articular chondrocytes had similar gene expression profiles, particularly related to GLI1, the nuclear mediator of the hedgehog pathway. In the NP, cell-cell interaction analysis revealed SHH expression in NCs, resulting in hedgehog signaling to CLCs. In contrast, no hedgehog ligands were expressed by chondrocytes in AC samples. Immunohistochemical analysis of the distal end of femur indicated that SHH and Indian hedgehog (IHH) were expressed around the subchondral bone that was excluded from our scRNA-seq sample. scRNA-seq data analysis and treatment of primary chondrocytes with SHH revealed that hedgehog proteins mediated an increase in hypoxia-inducible factor 1-alpha (HIF-1α) levels. Conclusion: CLCs and some articular chondrocytes have similar transcriptional profiles, regulated by paracrine hedgehog proteins secreted from NCs in the NP and from the subchondral bone in the AC to promote the HIF-1α pathway.

Human iPS cell-derived cartilaginous tissue spatially and functionally replaces nucleus pulposus

The loss of nucleus pulposus (NP) precedes the intervertebral disk (IVD) degeneration that causes back pain. Here, we demonstrate that the implantation of human iPS cell-derived cartilaginous tissue (hiPS-Cart) restores this loss by replacing lost NP spatially and functionally. NP cells consist of notochordal NP cells and chondrocyte-like NP cells. Single cell RNA sequencing (scRNA-seq) analysis revealed that cells in hiPS-Cart corresponded to chondrocyte-like NP cells but not to notochordal NP cells. The implantation of hiPS-Cart into a nuclectomized space of IVD in nude rats prevented the degeneration of the IVD and preserved its mechanical properties. hiPS-Cart survived and occupied the nuclectomized space for at least six months after implantation, indicating spatial and functional replacement of lost NP by hiPS-Cart. Further scRNA-seq analysis revealed that hiPS-Cart cells changed their profile after implantation, differentiating into two lineages that are metabolically distinct from each other. However, post-implanted hiPS-Cart cells corresponded to chondrocyte-like NP cells only and did not develop into notochordal NP cells, suggesting that chondrocyte-like NP cells are nearly sufficient for NP function. The data collectively indicate that hiPS-Cart is a candidate implant for regenerating NP spatially and functionally and preventing IVD degeneration.

Effects of Oxygen Concentration and Culture Time on Porcine Nucleus Pulposus Cell Metabolism: An in vitro Study.

Low back pain is a common ailment that affects millions of individuals each year and is linked to degeneration of the intervertebral discs in the spine. Intervertebral disc degeneration is known to result from an imbalance in anabolic and catabolic activity by disc cells. Due to the avascular nature of the intervertebral disc, oxygen deficiency may occur in the central nucleus pulposus (NP). The resulting hypoxia affects matrix regulation and energy metabolism of disc cells, although the mechanisms are not fully understood. This study investigates in vitro glucose consumption and gene expression by NP cells over time under varying oxygen tensions. Notochordal porcine NP cells were cultured in agarose discs at 21, 5, or 1% oxygen tension for 1, 5, or 10 days. The expression of 10 key matrix genes, as well as Brachyury (T), by NP cells was analyzed using RT-PCR. Glucose consumption was measured using a two-point method. Results show that culture time and oxygen tension significantly affect glucose consumption rates by porcine NP cells. There were also significant changes in T expression based on oxygen level and culture time. The 1% oxygen tension had a significantly higher T expression on day 10 than the other two groups, which may indicate a better maintenance of the notochordal phenotype. MMP 1 and 13 expression increased over time for all groups, while only the 5% group showed an increase over time for MMP 3. TIMP expression followed the direction of MMPs but to a lesser magnitude. Five percent and twenty-one percent oxygen tensions led to decreases in anabolic gene expression while 1% led to increases. Oxygen concentration and culture time significantly impacted glucose consumption rate and the gene expression of matrix regulatory genes with hypoxic conditions most accurately maintaining the proper NP phenotype. This information is valuable not only for understanding disc pathophysiology, but also for harnessing the potential of notochordal NP cells in therapeutic applications.

Effects Of Oxygen Concentration And Culture Time On Porcine Nucleus Pulposus Cell Metabolism: An In Vitro Study

Low back pain is a common ailment that affects millions of individuals each year and is linked to degeneration of the intervertebral discs in the spine. Intervertebral disc degeneration is known to result from an imbalance in anabolic and catabolic activity by disc cells. Due to the avascular nature of the intervertebral disc, oxygen deficiency may occur in the central nucleus pulposus (NP). The resulting hypoxia affects matrix regulation and energy metabolism of disc cells, although the mechanisms are not fully understood. This study investigates in vitro glucose consumption and gene expression by NP cells over time under varying oxygen tensions. Notochordal porcine NP cells were cultured in agarose discs at 21, 5, or 1% oxygen tension for 1, 5, or 10 days. The expression of 10 key matrix genes, as well as Brachyury (T), by NP cells was analyzed using RT-PCR. Glucose consumption was measured using a two-point method. Results show that culture time and oxygen tension significantly affect glucose consumption rates by porcine NP cells. There were also significant changes in T expression based on oxygen level and culture time. The 1% oxygen tension had a significantly higher T expression on day 10 than the other two groups, which may indicate a better maintenance of the notochordal phenotype. MMP 1 and 13 expression increased over time for all groups, while only the 5% group showed an increase over time for MMP 3. TIMP expression followed the direction of MMPs but to a lesser magnitude. Five percent and twenty-one percent oxygen tensions led to decreases in anabolic gene expression while 1% led to increases. Oxygen concentration and culture time significantly impacted glucose consumption rate and the gene expression of matrix regulatory genes with hypoxic conditions most accurately maintaining the proper NP phenotype. This information is valuable not only for understanding disc pathophysiology, but also for harnessing the potential of notochordal NP cells in therapeutic applications.

Dynamic Hydrostatic Pressure Regulates Nucleus Pulposus Phenotypic Expression and Metabolism in a Cell Density-Dependent Manner.

Dynamic hydrostatic pressure (HP) loading can modulate nucleus pulposus (NP) cell metabolism, extracellular matrix (ECM) composition, and induce transformation of notochordal NP cells into mature phenotype. However, the effects of varying cell density and dynamic HP magnitude on NP phenotype and metabolism are unknown. This study examined the effects of physiological magnitudes of HP loading applied to bovine NP cells encapsulated within three-dimensional (3D) alginate beads. Study 1: seeding density (1 M/mL versus 4 M/mL) was evaluated in unloaded and loaded (0.1 MPa, 0.1 Hz) conditions. Study 2: loading magnitude (0, 0.1, and 0.6 MPa) applied at 0.1 Hz to 1 M/mL for 7 days was evaluated. Study 1: 4 M/mL cell density had significantly lower adenosine triphosphate (ATP), glycosaminoglycan (GAG) and collagen content, and increased lactate dehydrogenase (LDH). HP loading significantly increased ATP levels, and expression of aggrecan, collagen I, keratin-19, and N-cadherin in HP loaded versus unloaded groups. Study 2: aggrecan expression increased in a dose dependent manner with HP magnitude, whereas N-cadherin and keratin-19 expression were greatest in low HP loading compared to unloaded. Overall, the findings of the current study indicate that cell seeding density within a 3D construct is a critical variable influencing the mechanobiological response of NP cells to HP loading. NP mechanobiology and phenotypic expression was also found to be dependent on the magnitude of HP loading. These findings suggest that HP loading and culture conditions of NP cells may require complex optimization for engineering an NP replacement tissue.

Chordoma‐derived cell line U‐CH1‐N recapitulates the biological properties of notochordal nucleus pulposus cells

ABSTRACTIntervertebral disc degeneration proceeds with age and is one of the major causes of lumbar pain and degenerative lumbar spine diseases. However, studies in the field of intervertebral disc biology have been hampered by the lack of reliable cell lines that can be used for in vitro assays. In this study, we show that a chordoma‐derived cell line U‐CH1‐N cells highly express the nucleus pulposus (NP) marker genes, including T (encodes T brachyury transcription factor), KRT19, and CD24. These observations were further confirmed by immunocytochemistry and flow cytometry. Reporter analyses showed that transcriptional activity of T was enhanced in U‐CH1‐N cells. Chondrogenic capacity of U‐CH1‐N cells was verified by evaluating the expression of extracellular matrix (ECM) genes and Alcian blue staining. Of note, we found that proliferation and synthesis of chondrogenic ECM proteins were largely dependent on T in U‐CH1‐N cells. In accordance, knockdown of the T transcripts suppressed the expression of PCNA, a gene essential for DNA replication, and SOX5 and SOX6, the master regulators of chondrogenesis. On the other hand, the CD24‐silenced cells showed no reduction in the mRNA expression level of the chondrogenic ECM genes. These results suggest that U‐CH1‐N shares important biological properties with notochordal NP cells and that T plays crucial roles in maintaining the notochordal NP cell‐like phenotype in this cell line. Taken together, our data indicate that U‐CH1‐N may serve as a useful tool in studying the biology of intervertebral disc. © 2016 The Authors. Journal of Orthopaedic Research Published by Wiley Periodicals, Inc. on behalf of Orthopaedic Research Society. J Orthop Res 34:1341–1350, 2016.

Identifying molecular phenotype of nucleus pulposus cells in human intervertebral disc with aging and degeneration.

Previous study claimed that disc degeneration may be preceded by structure and matrix changes in the intervertebral disc (IVD) which coincide with the loss of distinct notochordally derived nucleus pulposus (NP) cells. However, the fate of notochordal cells and their molecular phenotype change during aging and degeneration in human are still unknown. In this study, a set of novel molecular phenotype markers of notochordal NP cells during aging and degeneration in human IVD tissue were revealed with immunostaining and flow cytometry. Furthermore, the potential of phenotype juvenilization and matrix regeneration of IVD cells in a laminin-rich pseudo-3D culture system were evaluated at day 28 by immunostaining, Safranin O, and type II collagen staining. Immunostaining and flow cytometry demonstrated that transcriptional factor Brachyury T, neuronal-related proteins (brain abundant membrane attached signal protein 1, Basp1; Neurochondrin, Ncdn; Neuropilin, Nrp-1), CD24, and CD221 were expressed only in juvenile human NP tissue, which suggested that these proteins may be served as the notochordal NP cell markers. However, the increased expression of CD54 and CD166 with aging indicated that they might be referenced as the potential biomarker for disc degeneration. In addition, 3D culture maintained most of markers in juvenile NP, and rescued the expression of Basp1, Ncdn, and Nrp 1 that disappeared in adult NP native tissue. These findings provided new insight into molecular profile that may be used to characterize the existence of a unique notochordal NP cells during aging and degeneration in human IVD cells, which will facilitate cell-based therapy for IVD regeneration. © 2016 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 34:1316-1326, 2016.

TGFβ regulates Galectin-3 expression through canonical Smad3 signaling pathway in nucleus pulposus cells: implications in intervertebral disc degeneration

Galectin-3 is highly expressed in notochordal nucleus pulposus (NP) and thought to play important physiological roles; however, regulation of its expression remains largely unexplored. The aim of the study was to investigate if TGFβ regulates Galectin-3 expression in NP cells. TGFβ treatment resulted in decreased Galectin-3 expression. Bioinformatic analysis using JASPAR and MatInspector databases cross-referenced with published ChIP-Seq data showed nine locations of highly probable Smad3 binding in the LGALS3 proximal promoter. In NP cells, TGFβ treatment resulted in decreased activity of reporters harboring several 5′ deletions of the proximal Galectin-3 promoter. While transfection of NP cells with constitutively active (CA)-ALK5 resulted in decreased promoter activity, DN-ALK5 blocked the suppressive effect of TGFβ on the promoter. The suppressive effect of Smad3 on the Galectin-3 promoter was confirmed using gain- and loss-of-function studies. Transfection with DN-Smad3 or Smad7 blocked TGFβ mediated suppression of promoter activity. We also measured Galectin-3 promoter activity in Smad3 null and wild type cells. Noteworthy, promoter activity was suppressed by TGFβ only in wild type cells. Likewise, stable silencing of Smad3 in NP cells using sh-Smad3 significantly blocked TGFβ-dependent decrease in Galectin-3 expression. Treatment of human NP cells isolated from tissues with different grades of degeneration showed that Galectin-3 expression was responsive to TGF-β-mediated suppression. Importantly, Galectin-3 synergized effects of TNF-α on inflammatory gene expression by NP cells. Together these studies suggest that TGFβ, through Smad3 controls Galectin-3 expression in NP cells and may have implications in the intervertebral disc degeneration.

Notochordal Nucleus Pulposus Cells: Prospective Strategies for Intervertebral Disc Repair and Regeneration

Background: Notochordal cells (NCs) from the central nucleus pulposus (NP) region of the intervertebral discs (IVD) in the spine have an important function in IVD development, as well as in maintaining disc homeostasis. Indeed, the disappearance of NCs, which occurs early in life in humans, is associated with the earliest stages of disc degeneration, suggesting a link between the two events. Given the vital role NCs play in the IVD, a more complete knowledge of NC behavior and function, as well as how NCs may be employed in the development of new treatment methods for IVD degeneration, is necessary. The purpose of this paper is to provide an overview of NCs, including their phenotypic expression, their loss and consequences thereof in the disc, and possible strategies to use NCs in disc regeneration and repair. Methods: Background information regarding the phenotypic expression and role of NCs in the IVD is reviewed. An overview of the current knowledge on possible factors contributing to NC cell loss is presented, along with resulting consequences of the shift in cell population in the NP. Finally, the current research on methods to employ NCs in regenerative treatment strategies is provided. Results: Notochordal NP cells have a particular phenotype that is unique from aged or degenerated NP cells which is important for IVD development and maintenance. Thus, the disappearance of NCs has detrimental effects on the health and function of the IVD. Numerous factors may contribute to change in NC activity and/or viability, including microenvironmental conditions such as hypoxia, hyperosmolarity, and mechanical loading. Research is ongoing in determining how the positive effects of NCs can be harnessed into new strategies for IVD restoration, including use of NCs in cell transplantation as a cell source, via co-culture of NCs with other cells, or through the use of NC conditioned media. Conclusion: Given the important role NCs play in the health and functionality of the IVD, as well as the positive effects of NCs on other cells, notochordal cells show great potential in the development of new cellular based strategies to prevent and/or treat disc degeneration. Keywords: Cell-based therapy, co-culture, disc degeneration, notochord phenotype, notochordal cell conditioned media, nutrition.

Cadherin 2 is Required for Intervertebral Disc Homeostasis and Maintenance of Vacuolated Phenotype in Nucleus Pulposus Cells

Introduction Integrity of the nucleus pulposus (NP) has been implicated in the function and homeostasis of intervertebral disc (IVD). Understanding the regulation of NP cells would contribute to their engineering and development of therapeutics for treating IVD degeneration. Studies in mouse models indicate that early events of IVD degeneration involve segregation of the notochordal NP cell clusters,1 suggesting that disc degeneration may be associated with cell adhesion molecule activities. Cadherins are transmembrane glycoproteins that mediate calcium dependent cell adhesion.2 Based on microarray analysis, we have revealed specific expression of Cdh2 gene, encoding cadherin 2/N-cadherin, in rodent NP cells, suggesting a potential regulatory role of cadherins in IVD homeostasis. To date, the function of cadherin 2 in IVD and its relationship to IVD degeneration remain elusive. We hypothesize that cadherin 2 has a regulatory role in NP cells and that a deregulation of its activities has adverse effects on IVD homeostasis. We aimed to study the expression pattern of cadherin 2 in rodent discs during development, aging, and degeneration, and to investigate its function in the NP via a gene and protein ablation strategies. Materials and Methods Animal experiments were approved by local ethics committee. The vertebral columns of wild-type C57BL/6N mice were collected at different ages ( n = 4): embryonic day (E) 12.5, E14.5, postnatal day 0 (P0), 3 months old, 6 months old, 1 year old, 1.5 and 2 years old. Progressive disc degeneration was induced by annulus puncture of 4-month-old inbred Lewis rats ( n = 6) with 25G needle, and the discs were harvested after 2-, 4-, and 8-weeks of operation. Lumbar IVD of scoliosis patients and lower lumbar spine of the aborted fetus in the second trimester were used as controls. Immunohistochemistry was performed on paraffin sections to study the cadherin 2 expression pattern. For protein ablation study, 4-month-old inbred Lewis rats were anesthetized and the tail IVDs were exposed for injection of rabbit anti-cadherin 2 antibody or control rabbit IgG ( n = 6) into the NP via 34G hypodermic needle. Cadherin 2 gene (Cdh2) was knocked out in the NP using notochord-specific Foxa2-Cre recombination strategy. Disc height was measured and expressed as disc height index (%DHI). The IVD were harvested by 2 and 8 weeks after operation (protein ablation) and from P0 and 1 month-old mutants (gene knockout) for histological analysis. Results By immunofluorescence, cadherin 2 was weakly detected in murine embryonic notochord and newborn NP. At 3- and 6-month old, strong cadherin 2 signals were specifically detected as foci along the cell-cell junctions of the vacuolated NP cells (notochordal cells). Annulus fibrosus showed no signals. In aged IVD, the notochordal cells were replaced by small chondrocyte-like cells with lower expression of cadherin 2. In puncture-induced degenerative rodent discs, the notochordal NP cells were replaced by rounded chondrocyte-like cells, showing reduced level of cadherin 2 expression. Human NP showed heterogeneous cadherin 2 expression. By injecting cadherin 2 antibody into rat NP to perturb its function in vivo, cadherin 2 expression was reduced along with reduction of disc height. Compared with the IgG injection control, cadherin 2 antibody ablation group showed a transformation of the notochordal NP cells into less vacuolated chondrocyte-like phenotype with upregulation of collagen II. Cdh2 conditional knockout mice (CKO) showed absence of cadherin 2 and a loss of vacuolated phenotype in the NP cells (Fig. 1A), displaying significantly smaller body size by 1-month old (Fig. 1B). Moreover, the mutant exhibited irregular annulus organization and reduced disc height in IVD (V). Conclusion Our study suggests cadherin 2 as a marker of notochordal NP cells. The lower expression of cadherin 2 in the aged and puncture-injured rodent IVD substantiates its association with IVD degeneration. The reduction of cadherin 2 positive NP cells in mature human IVD is consistent with gradual loss of notochordal cells after the first decade of life. Our parallel in vivo studies of gene/protein ablation support that cadherin 2 is essential to the maintenance of a vacuolated phenotype of the notochordal NP cells, and that a loss of cadherin 2 function may initiate degenerative changes in the IVD. In summary, our study implicates an important role of cadherin 2 in notochordal cell function and regulating IVD homeostasis. Acknowledgments This work is funded by the Area of Excellence grant (AoE/M-04/04) and the General Research Fund (HKU763712M) from the Research Grant Council of Hong Kong. Disclosure of Interest None declared References Yang F, Leung VY, Luk KD, Chan D, Cheung KM. Injury-induced sequential transformation of notochordal nucleus pulposus to chondrogenic and fibrocartilaginous phenotype in the mouse. J Pathol 2009;218(1):113–121 Leckband D, Prakasam A. Mechanism and dynamics of cadherin adhesion. Annu Rev Biomed Eng 2006;8:259–287

Evidence of Nonnotochordal Origin in Chondrocyte-like Cells of the Nucleus Pulposus Appearing in Early Stage Disk Degeneration in the Mouse Model

IntroductionAlthough degeneration of the intervertebral disk (IVD) accounts for progression of various spinal diseases, not much has been clarified on the changes in cellular environment in the pathology of disk degeneration. Various animal models have been reported for use in different purposes; however, not much variation has been shown in mice. Development of a reproducible mouse model is important since recent molecular regulatory investigations are often performed using transgenic (Tg) mice. To define fate of cells in the IVD that originate from the notochord, we investigated the IVDs of double-transgenic (TG) mouse encoding Protein-0 (P0)-Cre/Floxed-EGFP. Cells originating from the neural crest or notochord express positivity for GFP in this TG mouse. We then developed a new disk degeneration model in this Tg mouse and investigated the changes in the cellular environment of the IVD.Materials and MethodsA total of 40 P0-Cre/Floxed-EGFP mice (8 weeks old, female) were divided in four groups. A loop was created with wires in their tails which induced degeneration in the nucleus pulposus and annulus fibrosus (AF) by lateral compression in all mice except for the normal control group (n = 10). Loop was loosened after 2 weeks and left to regenerate for 2 weeks in group R (n = 10). Loop was kept tightened for four straight weeks in group D (n = 10). Classical annulus puncture by a 27G needle was performed in group P(n = 10). All animals were sacrificed after 4 weeks. Histological evaluations were performed on HE and Safranin-O stained sections using disk degeneration grading system by Nishimura et al and Nomura et al. Immunohistochemistry was performed to detect GFP positive cells and CD24, a defined cell marker for nucleus pulposus cells in the IVD. Furthermore, immunohistochemistry was performed to detect brachyury, a specific marker for the notochord.ResultsWhen histological grades were evaluated as a sum score of AF and nucleus pulposus, Group D showed the most degenerative changes compared to Group R or P. The severity of disk degeneration showed a linear trend in the order of severity. Next, morphology of cells that compose the IVD was investigated, and found an appearance of chondrocyte-like cells in the very lateral portion of nucleus pulposus near inner AF. Unlike the AF cells, chondrocyte-like cells expressed CD24, however, did not express GFP and brachyury, suggesting a nonnotochordal origin of chondrocyte-like cells.ConclusionTransition of notochordal nucleus pulposus cells to chondrocyte-like cells have been documented by many researchers. Kim et al reported that chondrocyte-like NP cells in the nucleus pulposus originated and migrated from the cartilage endplate in vitro and in vivo. Choi and McCann suggest that in the newborn mouse, all cells in the NP region, including smaller chondrocyte-like NP cells, are of notochordal origin. However, combination of disk degeneration model and lineage tracking shows that some of the NP cells may not originate from the notochord in at least three mouse disk degeneration models. Further analysis of these models may prove to be important in understanding the pathology of disk degeneration and possibly regenerative techniques.I confirm having declared any potential conflict of interest for all authors listed on this abstractYesDisclosure of InterestNone declared

Evidence of Nonnotochordal Origin in Chondrocyte-like Cells of the Nucleus Pulposus Appearing in Early Stage Disk Degeneration in the Mouse Model

Introduction Although degeneration of the intervertebral disk (IVD) accounts for progression of various spinal diseases, not much has been clarified on the changes in cellular environment in the pathology of disk degeneration. Various animal models have been reported for use in different purposes; however, not much variation has been shown in mice. Development of a reproducible mouse model is important since recent molecular regulatory investigations are often performed using transgenic (Tg) mice. To define fate of cells in the IVD that originate from the notochord, we investigated the IVDs of double-transgenic (TG) mouse encoding Protein-0 (P0)-Cre/Floxed-EGFP. Cells originating from the neural crest or notochord express positivity for GFP in this TG mouse. We then developed a new disk degeneration model in this Tg mouse and investigated the changes in the cellular environment of the IVD. Materials and Methods A total of 40 P0-Cre/Floxed-EGFP mice (8 weeks old, female) were divided in four groups. A loop was created with wires in their tails which induced degeneration in the nucleus pulposus and annulus fibrosus (AF) by lateral compression in all mice except for the normal control group ( n = 10). Loop was loosened after 2 weeks and left to regenerate for 2 weeks in group R ( n = 10). Loop was kept tightened for four straight weeks in group D ( n = 10). Classical annulus puncture by a 27G needle was performed in group P( n = 10). All animals were sacrificed after 4 weeks. Histological evaluations were performed on HE and Safranin-O stained sections using disk degeneration grading system by Nishimura et al and Nomura et al. Immunohistochemistry was performed to detect GFP positive cells and CD24, a defined cell marker for nucleus pulposus cells in the IVD. Furthermore, immunohistochemistry was performed to detect brachyury, a specific marker for the notochord. Results When histological grades were evaluated as a sum score of AF and nucleus pulposus, Group D showed the most degenerative changes compared to Group R or P. The severity of disk degeneration showed a linear trend in the order of severity. Next, morphology of cells that compose the IVD was investigated, and found an appearance of chondrocyte-like cells in the very lateral portion of nucleus pulposus near inner AF. Unlike the AF cells, chondrocyte-like cells expressed CD24, however, did not express GFP and brachyury, suggesting a nonnotochordal origin of chondrocyte-like cells. Conclusion Transition of notochordal nucleus pulposus cells to chondrocyte-like cells have been documented by many researchers. Kim et al reported that chondrocyte-like NP cells in the nucleus pulposus originated and migrated from the cartilage endplate in vitro and in vivo. Choi and McCann suggest that in the newborn mouse, all cells in the NP region, including smaller chondrocyte-like NP cells, are of notochordal origin. However, combination of disk degeneration model and lineage tracking shows that some of the NP cells may not originate from the notochord in at least three mouse disk degeneration models. Further analysis of these models may prove to be important in understanding the pathology of disk degeneration and possibly regenerative techniques. I confirm having declared any potential conflict of interest for all authors listed on this abstract Yes Disclosure of Interest None declared

Cadherin 2 as Human Notochordal Cell Marker and its Expression Pattern in Disk Degeneration

Introduction Cadherins are transmembrane glycoproteins that mediate calcium-dependent cell adhesion. Recent studies suggest that cadherins also have nonadhesive functions. Maturation of human intervertebral disk (IVD) as well as early events of IVD degeneration in a mouse model1 is associated with segregation of nucleus pulposus (NP) cells suggesting that disk degeneration may be associated with cell adhesion molecule activities. From our microarray data analysis and subsequent immunohistochemical assays, we have revealed specific expression of Cdh2 gene and its product, cadherin 2/N-cadherin, in rodent notochordal NP cells, suggesting a potential regulatory role of cadherins in IVD homeostasis. Cadherin 2 belongs to the classic cadherin family. To date, the expression and function of cadherin 2 in IVD and its relationship to disk degeneration remain elusive. We aim to study the expression pattern of cadherin 2 in human IVD using aborted fetus and clinical samples, and to investigate its function in the rodent NP via a protein ablation strategy. Materials and Methods Degenerated lumbar disks (grade 3, Schneiderman's classification) ( n = 5) were obtained from 45 to 58-year-old patients. Scoliotic lumbar disks ( n = 5) from patients were obtained as nondegenerated samples. Lower lumbar spine of the aborted fetus in the second trimester were used as controls. Compartmental origin and degeneration status of the samples was validated by our established multicolor FAST staining method,2 in which the disk sections were sequentially stained with alcian blue, safranin-O, tartrazine, and fast green. Immunohistochemistry for cadherin 2 was performed on paraffin sections with antigen retrieval by hyaluronidase and citrate buffer method and counterstained by hematoxylin to visualize cell nuclei. The percentage of positive cells was calculated and analyzed by nonparametric u test. For protein ablation study, 4-month-old inbred Lewis rats were anesthetized and the tail IVDs were exposed for injection of rabbit anti-cadherin 2 antibody or control IgG ( n = 6 for each) into the NP via 34G hypodermic needle. By 2 weeks and 8 weeks after operation, the disks were harvested for histological examination. Results Human fetal NP cells showed homogeneous cadherin 2 expression. Annulus fibrosus (AF), cartilaginous endplates, and vertebral bodies showed no signal of cadherin 2. However, adult NP showed heterogeneous cadherin 2 expression, where only a subpopulation of the NP cells expressed the protein. Nondegenerated and degenerated NP exhibited different cell distribution with the later tended to contain cell clusters. The frequency of cadherin 2 expressing cells was significantly higher in the degenerated NP (51.9%) than in nondegenerated NP (20.0%). FAST staining assay showed that degenerated NP samples were intensely stained by safranin-O instead of alcian blue, validating a loss of sulfated acidic glycosaminoglycan due to degeneration. Cadherin 2 protein ablation study showed that the vacuolated notochordal cells in the NP partially transformed into elongated or rounded nonvacuolated cells, which are commonly presented in degenerative disks. Conclusion Human fetal NP mainly contains notochordal cells. In this study, we showed that cadherin 2 is expressed homogeneously in fetal NP suggesting cadherin 2 is a marker of notochordal cells. The reduction of cadherin 2 positive NP cells in mature human IVD is consistent with gradual loss of notochordal cells after the first decade of life. Under this notion, our findings imply that a subpopulation (20%) of NP cells retain a phenotype resembling notochordal cells in mature human IVD, although morphologically they are not vacuolated. The higher frequency of cadherin 2 expressing cells in degenerated NP implicates that proliferation of cells of notochordal origin occurs during disk degeneration. Protein ablation study suggests that cadherin 2 has a regulatory function in maintenance of notochordal cells and a loss of its function may initiate disk degeneration. Overall, our study implies a function of cadherin 2 in NP homeostasis. I confirm having declared any potential conflict of interest for all authors listed on this abstract No Disclosure of Interest None declared Yang F, et al. Injury-induced sequential transformation of notochordal nucleus pulposus to chondrogenic and fibrocartilaginous phenotype in the mouse. Journal of Pathology 2009;218(1):113–121 Leung VY, et al. Matrix remodeling during intervertebral disk growth and degeneration detected by multichromatic FAST staining. Journal of Histochemistry and Cytochemistry 2009;57(3):249–256

Cadherin 2 as Human Notochordal Cell Marker and its Expression Pattern in Disk Degeneration

IntroductionCadherins are transmembrane glycoproteins that mediate calcium-dependent cell adhesion. Recent studies suggest that cadherins also have nonadhesive functions. Maturation of human intervertebral disk (IVD) as well as early events of IVD degeneration in a mouse model1 is associated with segregation of nucleus pulposus (NP) cells suggesting that disk degeneration may be associated with cell adhesion molecule activities. From our microarray data analysis and subsequent immunohistochemical assays, we have revealed specific expression of Cdh2 gene and its product, cadherin 2/N-cadherin, in rodent notochordal NP cells, suggesting a potential regulatory role of cadherins in IVD homeostasis. Cadherin 2 belongs to the classic cadherin family. To date, the expression and function of cadherin 2 in IVD and its relationship to disk degeneration remain elusive. We aim to study the expression pattern of cadherin 2 in human IVD using aborted fetus and clinical samples, and to investigate its function in the rodent NP via a protein ablation strategy.Materials and MethodsDegenerated lumbar disks (grade 3, Schneiderman's classification) (n = 5) were obtained from 45 to 58-year-old patients. Scoliotic lumbar disks (n = 5) from patients were obtained as nondegenerated samples. Lower lumbar spine of the aborted fetus in the second trimester were used as controls. Compartmental origin and degeneration status of the samples was validated by our established multicolor FAST staining method,2 in which the disk sections were sequentially stained with alcian blue, safranin-O, tartrazine, and fast green. Immunohistochemistry for cadherin 2 was performed on paraffin sections with antigen retrieval by hyaluronidase and citrate buffer method and counterstained by hematoxylin to visualize cell nuclei. The percentage of positive cells was calculated and analyzed by nonparametric u test. For protein ablation study, 4-month-old inbred Lewis rats were anesthetized and the tail IVDs were exposed for injection of rabbit anti-cadherin 2 antibody or control IgG (n = 6 for each) into the NP via 34G hypodermic needle. By 2 weeks and 8 weeks after operation, the disks were harvested for histological examination.ResultsHuman fetal NP cells showed homogeneous cadherin 2 expression. Annulus fibrosus (AF), cartilaginous endplates, and vertebral bodies showed no signal of cadherin 2. However, adult NP showed heterogeneous cadherin 2 expression, where only a subpopulation of the NP cells expressed the protein. Nondegenerated and degenerated NP exhibited different cell distribution with the later tended to contain cell clusters. The frequency of cadherin 2 expressing cells was significantly higher in the degenerated NP (51.9%) than in nondegenerated NP (20.0%). FAST staining assay showed that degenerated NP samples were intensely stained by safranin-O instead of alcian blue, validating a loss of sulfated acidic glycosaminoglycan due to degeneration. Cadherin 2 protein ablation study showed that the vacuolated notochordal cells in the NP partially transformed into elongated or rounded nonvacuolated cells, which are commonly presented in degenerative disks.ConclusionHuman fetal NP mainly contains notochordal cells. In this study, we showed that cadherin 2 is expressed homogeneously in fetal NP suggesting cadherin 2 is a marker of notochordal cells. The reduction of cadherin 2 positive NP cells in mature human IVD is consistent with gradual loss of notochordal cells after the first decade of life. Under this notion, our findings imply that a subpopulation (20%) of NP cells retain a phenotype resembling notochordal cells in mature human IVD, although morphologically they are not vacuolated. The higher frequency of cadherin 2 expressing cells in degenerated NP implicates that proliferation of cells of notochordal origin occurs during disk degeneration. Protein ablation study suggests that cadherin 2 has a regulatory function in maintenance of notochordal cells and a loss of its function may initiate disk degeneration. Overall, our study implies a function of cadherin 2 in NP homeostasis.I confirm having declared any potential conflict of interest for all authors listed on this abstractNoDisclosure of InterestNone declaredYang F, et al. Injury-induced sequential transformation of notochordal nucleus pulposus to chondrogenic and fibrocartilaginous phenotype in the mouse. Journal of Pathology 2009;218(1):113–121Leung VY, et al. Matrix remodeling during intervertebral disk growth and degeneration detected by multichromatic FAST staining. Journal of Histochemistry and Cytochemistry 2009;57(3):249–256

Differential expression level of cytokeratin 8 in cells of the bovine nucleus pulposus complicates the search for specific intervertebral disc cell markers.

IntroductionDevelopment of cell therapies for repairing the intervertebral disc is limited by the lack of a source of healthy human disc cells. Stem cells, particularly mesenchymal stem cells, are seen as a potential source but differentiation strategies are limited by the lack of specific markers that can distinguish disc cells from articular chondrocytes.MethodsWe searched for markers using the differential in-gel electrophoresis proteomic technology to compare proteins of bovine nucleus pulposus cells, phenotypically similar to mature human nucleus cells, with those of bovine articular chondrocytes. In the cohort of the differentially expressed proteins identified by mass spectrometry, cytokeratin 8 (CK8) was further validated by immunostaining of freshly isolated cells and frozen tissue sections using monoclonal antibodies.ResultsWe identified a set of 14 differentially expressed proteins. Immunohistochemistry showed that only a subset of cells (approximately 10%) was positive for one of these proteins, CK8, an intermediate filament protein present in epithelial but not mesenchymal cells. In tissue sections, CK8-positive cells were seen in all discs examined and appeared as small isolated clusters surrounded by gelatinous matrix. Notochordal nucleus pulposus cells from pig, phenotypically similar to human infant nucleus pulposus cells, were all CK8-positive. The mesenchymal intermediate filament protein vimentin was present in all bovine and porcine nucleus pulposus cells.ConclusionsThe notochordal cell population is reported to disappear from the nucleus pulposus of bovine discs before birth and from human discs in childhood. However our finding of the co-expression of vimentin and CK8 in small isolated clusters of the bovine nucleus pulposus cells indicates that a subpopulation of notochordal-like cells remains in the mature bovine disc. This finding agrees with reports in the literature on co-expression of cytokeratins and vimentin in adult human discs. As notochordal cells produce factors that promote matrix production, the CK8-positive subpopulation could have important implications for activity and survival of the nucleus pulposus, and should be considered in development of cell therapies for disc repair. In addition, the finding of differential expression of proteins in the cell population of nucleus pulposus has implications with regard to the search for specific markers.

A Phenotypic Comparison of Proteoglycan Production of Intervertebral Disc Cells Isolated from Rats, Rabbits, and Bovine Tails; Which Animal Model is Most Suitable to Study Tissue Engineering and Biological Repair of Human Disc Disorders?

The nucleus pulposus (NP) of the intervertebral disc in cattle and humans shows the most dramatic changes with aging of any cartilaginous tissue. In humans, notochordal cells disappear from the NP and are replaced with chondrocytic cells by adolescence. However, notochordal cells of the NP persist into adult life in some species, such as rats and rabbits. Therefore, comparison of the metabolic activity of notochordal and nonnotochordal cells is considered to be important for determining the type of cell to use for transplantation to regenerate intervertebral discs. In this study, we investigated the notochordal NP cells of rats and rabbits, as well as nonnotochordal (chondrocyte-like) bovine NP cells, in a three-dimensional culture system to examine whether proteoglycan metabolism varied among these three cell types. As a result, bovine NP cells produced around 0.18 mg/mL of glycosaminoglycan after culture for 5 days, while rat and rabbit NP cells produced about four and two times more glycosaminoglycan than bovine cells, respectively. In conclusion, this study demonstrated marked differences of energy metabolism and production of matrix components between notochordal and nonnotochordal NP cells. Animals with notochordal cells in the NP, such as rats and rabbits, may not provide good models for investigation of biological repair and tissue engineering for human disc disorders.

Mesenchymal Stem Cells Arrest Intervertebral Disc Degeneration Through Chondrocytic Differentiation and Stimulation of Endogenous Cells

Degenerative disc disease (DDD) is a common disease which affects millions of people. Autograft of the bone marrow derived mesenchymal stem cells (BMSCs) have been shown to have the ability to arrest degeneration in rabbit and canine intervertebral discs. In this study, we have used the mouse model to investigate the mechanism of degeneration arrest. BMSC from Egfp transgenic mice were injected into the degenerated murine intervertebral discs induced by annular puncture. We found that BMSC could arrest the progressive degeneration of the discs with significant regeneration of the nucleus pulposus (NP). In the regeneration, expression of proteoglycan genes were upregulated and extracellular matrix (ECM) progressively accumulated in the NP after BMSC injection. Combined in situ hybridization and immunohistochemistry revealed that BMSC underwent chondrocytic differentiation in the regeneration process. Interestingly, BMSC-induced an increase of endogenous notochordal cells in NP and expression of chondrocytic markers. In this study, we have firstly shown that the BMSC could arrest the degeneration of the murine notochordal NP and contribute to the augmentation of the ECM in the NP by both autonomous differentiation and stimulatory action on endogenous cells.