Human disc degeneration is associated with increased MMP 7 expression

During intervertebral disc (IVD) degeneration, normal matrix synthesis decreases and degradation of disc matrix increases. A number of proteases that are increased during disc degeneration are thought to be involved in its pathogenesis. Matrix metalloproteinase 7 (MMP 7) (Matrilysin, PUMP-1) is known to cleave the major matrix molecules found within the IVD, i.e., the proteoglycan aggrecan and collagen type II. To date, however, it is not known how its expression changes with degeneration or its exact location. We investigated the localization of MMP 7 in human, histologically graded, nondegenerate, degenerated and prolapsed discs to ascertain whether MMP 7 is up-regulated during disc degeneration. Samples of human IVD tissue were fixed in neutral buffered formalin, embedded in paraffin, and sections stained with hematoxylin and eosin to score the degree of morphological degeneration. Immunohistochemistry was performed to localize MMP 7 in 41 human IVDs with varying degrees of degeneration. We found that the chondrocyte-like cells of the nucleus pulposus and inner annulus fibrosus were MMP 7 immunopositive; little immunopositivity was observed in the outer annulus. Nondegenerate discs showed few immunopositive cells. A significant increase in the proportion of MMP 7 immunopositive cells was seen in the nucleus pulposus of discs classified as showing intermediate levels of degeneration and a further increase was seen in discs with severe degeneration. Prolapsed discs showed more MMP 7 immunopositive cells compared to nondegenerated discs, but fewer than those seen in cases of severe degeneration.     

Human disc degeneration is associated with increased MMP 7 expression.

During intervertebral disc (IVD) degeneration, normal matrix synthesis decreases and degradation of disc matrix increases. A number of proteases that are increased during disc degeneration are thought to be involved in its pathogenesis. Matrix metalloproteinase 7 (MMP 7) (Matrilysin, PUMP-1) is known to cleave the major matrix molecules found within the IVD, i.e., the proteoglycan aggrecan and collagen type II. To date, however, it is not known how its expression changes with degeneration or its exact location. We investigated the localization of MMP 7 in human, histologically graded, nondegenerate, degenerated and prolapsed discs to ascertain whether MMP 7 is up-regulated during disc degeneration. Samples of human IVD tissue were fixed in neutral buffered formalin, embedded in paraffin, and sections stained with hematoxylin and eosin to score the degree of morphological degeneration. Immunohistochemistry was performed to localize MMP 7 in 41 human IVDs with varying degrees of degeneration. We found that the chondrocyte-like cells of the nucleus pulposus and inner annulus fibrosus were MMP 7 immunopositive; little immunopositivity was observed in the outer annulus. Nondegenerate discs showed few immunopositive cells. A significant increase in the proportion of MMP 7 immunopositive cells was seen in the nucleus pulposus of discs classified as showing intermediate levels of degeneration and a further increase was seen in discs with severe degeneration. Prolapsed discs showed more MMP 7 immunopositive cells compared to nondegenerated discs, but fewer than those seen in cases of severe degeneration.     

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.     

Periostin is expressed by cells of the human and sand rat intervertebral discs

Abstract

Periostin, a matricellular protein in the fasciclin family, is expressed in tissues subjected to constant mechanical stress. Periostin modulates cell-to-extracellular matrix interactions and can bind to collagen, fibronectin, tenascin-C and several integrins. Our objective was to evaluate whether periostin is expressed in the human intervertebral disc. Immunohistochemical localization of periostin was carried out in tissue of human lumbar discs and lumbar discs of the sand rat (Psammomys obesus). Human discs also were examined for periostin gene expression. Immunohistochemical localization demonstrated periostin in the cytoplasm of annulus and nucleus cells, and occasionally in the surrounding pericellular and interterritorial extracellular matrix. Periostin distribution in the human disc was distinctive. Outer annulus contained the highest proportion of periostin-positive cells (88.8%), whereas inner annulus contained only 61.4%. The nucleus pulposus contained the fewest periostin-positive cells (18.5%). There was a significant negative correlation between the percentage of cells positive for periostin in the inner annulus and subject age. Periostin gene expression in the human disc also was confirmed using molecular microarray analysis. Because work by others has shown that periostin plays an important role in the biomechanical properties of other connective tissues (skin, tendon, heart valves), future research is needed to elucidate the role of periostin in disc, loading, aging and degeneration.     

A Structurally and Functionally Biomimetic Biphasic Scaffold for Intervertebral Disc Tissue Engineering

Tissue engineering offers high hopes for the treatment of intervertebral disc (IVD) degeneration. Whereas scaffolds of the disc nucleus and annulus have been extensively studied, a truly biomimetic and mechanically functional biphasic scaffold using naturally occurring extracellular matrix is yet to be developed. Here, a biphasic scaffold was fabricated with collagen and glycosaminoglycans (GAGs), two of the most abundant extracellular matrix components in the IVD. Following fabrication, the scaffold was characterized and benchmarked against native disc. The biphasic scaffold was composed of a collagen-GAG co-precipitate making up the nucleus pulposus-like core, and this was encapsulated in multiple lamellae of photochemically crosslinked collagen membranes comprising the annulus fibrosus-like lamellae. On mechanical testing, the height of our engineered disc recovered by ~82-89% in an annulus-independent manner, when compared with the 99% recovery exhibited by native disc. The annulus-independent nature of disc height recovery suggests that the fluid replacement function of the engineered nucleus pulposus core might mimic this hitherto unique feature of native disc. Biphasic scaffolds comprised of 10 annulus fibrosus-like lamellae had the best overall mechanical performance among the various designs owing to their similarity to native disc in most aspects, including elastic compliance during creep and recovery, and viscous compliance during recovery. However, the dynamic mechanical performance (including dynamic stiffness and damping factor) of all the biphasic scaffolds was similar to that of the native discs. This study contributes to the rationalized design and development of a biomimetic and mechanically viable biphasic scaffold for IVD tissue engineering.     

Expression and regulation of neurotrophins in the nondegenerate and degenerate human intervertebral disc

Introduction

The neurotrophins nerve growth factor (NGF) and brain-derived neurotrophic factor (BDNF) have been identified in the human intervertebral disc (IVD) and have been implicated in the mechanisms associated with nerve ingrowth and nociception in degeneration of the IVD. The aim of the current study was to investigate an association between neurotrophin expression in the IVD and the severity of disc degeneration, including the effect of disc-related proinflammatory cytokines on neurotrophin and neuropeptide expression in cells derived from the human IVD.

Methods

Immunohistochemical analysis was performed to examine the expression of NGF, BDNF and their high-affinity receptors Trk-A and Trk-B in human IVD samples, divided into three categories: non-degenerate, moderate degeneration and severe degeneration. In order to study the effect of disc-related cytokines on neurotrophin/neuropeptide gene expression, nucleus pulposus cells derived from non-degenerate and degenerate IVD samples were seeded in alginate and were stimulated with either IL-1β or TNFα for 48 hours. RNA was extracted, cDNA was synthesised and quantitative real-time PCR was performed to examine the expression of NGF, BDNF and substance P.

Results

Immunohistochemistry showed expression of NGF and BDNF in the native chondrocyte-like cells in all regions of the IVD and in all grades of degeneration. Interestingly only BDNF significantly increased with the severity of degeneration (P < 0.05). Similar expression was observed for Trk-A and Trk-B, although no association with disease severity was demonstrated. In cultured human nucleus pulposus cells, stimulation with IL-1β led to significant increases in NGF and BDNF gene expression (P < 0.05). Treatment with TNFα was associated with an upregulation of substance P expression only.

Conclusion

Our findings show that both the annulus fibrosus and nucleus pulposus cells of the IVD express the neurotrophins NGF and BDNF, factors that may influence and enhance innervation and pain in the degenerate IVD. Expression of Trk-A and Trk-B by cells of the nondegenerate and degenerate IVD suggests an autocrine role for neurotrophins in regulation of disc cell biology. Furthermore, modulation of neurotrophin expression by IL-1β and modulation of substance P expression by TNFα, coupled with their increased expression in the degenerate IVD, highlights novel roles for these cytokines in regulating nerve ingrowth in the degenerate IVD and associated back pain.     

Brain-derived neurotrophic factor and its receptor in the human and the sand rat intervertebral disc

Introduction

Brain-derived neurotrophic factor (BDNF) was first identified in the intervertebral disc (IVD) when its molecular upregulation was observed in sections of nucleus pulposus cultured under conditions of increased osmolarity. BDNF is now known to be involved in a number of biologic functions, including regulation of differentiation/survival of sensory neurons, regulation of nociceptive function and central pain modulation, and modulation of inflammatory pain hypersensitivity. In addition, more recent investigations show that BDNF can induce the recruitment of endothelial cells and the formation of vascular structures. The objectives of the present study were to use immunocytochemistry to determine the distribution of BDNF and its receptor (BDNF-tropomyosine receptor kinase B) in the human IVD, and to test for gene expression of BDNF and its receptor in cultured human annulus fibrosus cells.

Methods

We studied immunohistochemical localization of BDNF and its receptor in the human annulus, quantified the percentage of outer annulus and inner annulus cells and nucleus cells positive for BDNF immunolocalization, and studied the gene expression of BDNF and its receptor using microarray analysis.

Results

The percentage (mean ± standard error of the mean) of cells positive for BDNF localization was significantly greater in the outer annulus (32.3 ± 2.7%, n = 22) compared with either the inner annulus (8.1 ± 1.5%, n = 6) or the nucleus (10.4 ± 2.8%, n = 3) (P < 0.0001). BDNF-receptor immunolocalization showed a pattern similar to that of BDNF, but was not quantitatively assessed. BDNF gene expression levels from cultured annulus cells showed a significant positive correlation with increasing levels of IVD degeneration (P = 0.011).

Conclusion

These findings provide data on the presence of BDNF and its receptor in the human IVD at the translational level, and on the expression of BDNF and its receptor by cultured human annulus cells. Our findings point to the need for further studies to define the role of BDNF in the human IVD and to investigate regulatory events within the disc that control the expression of BDNF and its receptor.     

椎间盘微环境对干细胞疗法修复椎间盘的影响的研究概况

椎间盘(IVD)退变是一种常见的病理状态,保守治疗往往失败,IVD变性的患者最后往往需要手术干预。已经提出的几种治疗方案中,只有椎间盘切除术和关节融合术被证明可以达到预料的效果。生物治疗的目的是预防和控制椎间盘变性,改善椎间盘的功能、髓核和纤维环细胞的合成代谢和修复能力,并抑制基质降解。目前,临床应用仍处于起步阶段。间充质干细胞和基因治疗在预防和治疗IVD变性的作用还需要进一步的研究。最近的研究向我们展示了一种新的保护椎间盘的结构和功能的治疗策略:间充质干细胞(MSCs)移植,尤其是骨髓间充质干细胞(BM-MSCs)。而了解MSCs是否可以以及如何在有排拆性的退化的椎间盘中存活并繁殖是十分重要的。因此,本文着重讨论内源性蛋白酶、细胞因子、低氧、低营养、机械负荷及渗透压的调节对移植的MSCs的影响。     

Nucleus pulposus repair with cultured autologous elastic cartilage derived chondrocytes

Low back pain is one of the most common medical conditions in the Western world. Disc degeneration, an inevitable process of ageing, is one of the major causes of low back pain. Autologous chondrocyte transplantation (ACT) is an increasingly popular method of addressing pathological disorders of cartilage. The purpose of our study was to determine whether autologous chondrocytes from elastic cartilage could survive and synthesise a cartilage specific matrix in the intervertebral disc of rabbits. Sixteen lumbar intervertebral discs (IVD) of New Zealand White rabbits were analysed. In 6 IVD, the nucleus pulposus was evacuated and replaced with tissue engineered autologous chondrocytes from auricular cartilage. In the second group, only the nucleus pulposus was evacuated from 6 IVD, with no chondrocytes implantation. Four non-operated IVD were used as a control. Six months after the operation, the animals were euthanized and the IVD were analysed histologically. Autologous cartilage implants were well tolerated by the host for up to six months in vivo. There was only hyaline-like cartilage in the place of the nucleus pulposus. We could not detect any elastic fibres in the new cartilage matrix. In IVD from which only the nucleus pulposus was evacuated and no chondrocytes were implanted, just fibrous tissue was found instead of nucleus pulposus. The overall histological analysis of new cartilage produced after implantation in our study confirmed the hypothesis that ACT from auricular cartilage can be implanted into the IVD instead of the nucleus pulposus and that a significant percentage of implanted chondrocytes survive and produce hyaline-like cartilage.     

Tenascin in the human intervertebral disc: alterations with aging and disc degeneration

Our objective for this study was to determine the presence and distribution of tenascin in the human intervertebral disc. The tenascins are a family of extracellular matrix proteins with repeated structural domains homologous to epidermal growth factor, fibronectin type III and the fibrinogens. Little is known about the presence of this protein in the disc. Ten normal human discs donated from subjects newborn to 15 years old, 10 control discs from adult donors aged 24-41 years, and 11 surgical disc specimens from patients aged 26-76 years were examined for immunolocalization of tenascin. In young discs, tenascin was localized throughout the annulus; in the nucleus, localization was confined to pericellular matrix. In adult control and degenerating disc specimens, tenascin in the annulus was localized primarily in pericellular matrix regions encircling either single cells or clusters of disc cells; in rare instances localization was more diffuse in the intraterritorial matrix. In young, healthy disc, tenascin was abundant throughout the annulus. In contrast, degenerating discs in adults showed a localization restricted to the pericellular, and rarely, more restricted intraterritorial matrix. These observations indicate that changes in the amount and distribution of tenascin may have a role in disc aging and degeneration, possibly by modulating fibronectin-disc-cell interactions, and causing alterations in the shape of disc cells.     

An in vitro study investigating the survival and phenotype of mesenchymal stem cells following injection into nucleus pulposus tissue

Introduction  

The decreased disc height characteristic of intervertebral disc (IVD) degeneration has often been linked to low back pain, and thus regeneration strategies aimed at restoring the disc extracellular matrix and ultimately disc height have been proposed as potential treatments for IVD degeneration. One such therapy under investigation by a number of groups worldwide is the use of autologous mesenchymal stem cells (MSCs) to aid in the regeneration of the IVD extracellular matrix. To date, however, the optimum method of application of these cells for regeneration strategies for the IVD is unclear, and few studies have investigated the direct injection of MSCs alone into IVD tissues. In the present article, we investigated the survival and phenotype of human MSCs, sourced from aged individuals, following injection into nucleus pulposus (NP) tissue explant cultures.     

Moderately degenerated lumbar motion segments: Are they truly unstable?

The two main load bearing tissues of the intervertebral disc are the nucleus pulposus and the annulus fibrosus. Both tissues are composed of the same basic components, but differ in their organization and relative amounts. With degeneration, the clear distinction between the two tissues disappears. The changes in biochemical content lead to changes in mechanical behaviour of the intervertebral disc. The aim of the current study was to investigate if well-documented moderate degeneration at the biochemical and fibre structure level leads to instability of the lumbar spine. By taking into account biochemical and ultrastructural changes to the extracellular matrix of degenerating discs, a set of constitutive material parameters were determined that described the individual tissue behaviour. These tissue biomechanical models were then used to simulate dynamic behaviour of the degenerated spinal motion segment, which showed instability in axial rotation, while a stabilizing effect in the other two principle bending directions. When a shear load was applied to the degenerated spinal motion segment, no sign of instability was found. This study found that reported changes to the nucleus pulposus and annulus fibrosus matrix during moderate degeneration lead to a more stable spinal motion segment and that such biomechanical considerations should be incorporated into the general pathophysiological understanding of disc degeneration and how its progress could affect low back pain and its treatments thereof.     

Notochordal cell disappearance and modes of apoptotic cell death in a rat tail static compression-induced disc degeneration model

Introduction

The intervertebral disc has a complex structure originating developmentally from both the mesenchyme and notochord. Notochordal cells disappear during adolescence, which is also when human discs begin to show degenerative signs. During degeneration later in life, disc cells decline because of apoptosis. Although many animal models have been developed to simulate human disc degeneration, few studies have explored the long-term changes in cell population and phenotype. Our objective was to elucidate the time-dependent notochordal cell disappearance and apoptotic cell death in a rat tail static compression-induced disc degeneration model.

Methods

Twenty-four 12-week-old male Sprague–Dawley rat tails were instrumented with an Ilizarov-type device and loaded statically at 1.3 MPa for up to 56 days. Loaded and distal-unloaded discs were harvested. Changes in cell number and phenotype were assessed with histomorphology and immunofluorescence. Apoptosis involvement was determined with terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL) staining and immunohistochemistry.

Results

The number of disc nucleus pulposus and annulus fibrosus cells decreased with the loading period; particularly, the decrease was notable at day 7 in larger, vacuolated, cytokeratin-8- and galectin-3-co-positive cells, indicating notochordal origin. Subsequently, the proportion of cells positive for TUNEL and cleaved caspase-3, markers of apoptosis induction, increased from day 7 through day 56. Although the percentage of cells immunopositive for cleaved caspase-8, a marker of apoptosis initiation through the death-receptor pathway, increased only at day 7, the percentage of cells immunopositive for cleaved caspase-9 and p53-regulated apoptosis-inducing protein 1 (p53AIP1), markers of apoptosis initiation through the p53-mediated mitochondrial pathway, increased from day 7 through day 56. The percentage of cells immunopositive for B-cell lymphoma 2 (Bcl-2) and silent mating type information regulation 2 homolog 1 (SIRT1), antiapoptotic proteins, decreased consistently with compression.

Conclusions

This rat tail model mimics notochordal cell disappearance and apoptotic cell death in human disc aging and degeneration. Sustained static compression induces transient activation of apoptosis through the death-receptor pathway and persistent activation of apoptosis through the p53-mediated mitochondrial pathway in disc cells. The increased proapoptotic and decreased antiapoptotic proteins observed at all time points signify static compression-induced disc cell death and degeneration.     

Immunolocalization of type X collagen in human lumbar intervertebral discs during ageing and degeneration

 Type X collagen has so far not been reported to occur in human intervertebral discs. The objective of this study was therefore to investigate the occurrence of type X collagen in human lumbar intervertebral discs during ageing and degeneration. Ninety intervertebral discs with adjacent endplates were excised in toto from individuals (0–86 years) without known spinal disease and were processed for routine decalcified histology. Appropriate slices of each disc were processed for immunohistochemistry using a type-spec ific, monoclonal antibody raised against human type X collagen. Each intervertebral disc was examined for macroscopic and histomorphological features of disc degeneration. Immunohistochemically, a positive specific type X staining was observed in the hypertrophic zone of the growth plate and only in the interstitial matrix of juvenile (<2 years) nucleus pulposus. In adult discs, type X collagen could be localized in conjunction with advanced disc degeneration and first occurred in the disc matrix (i.e., pericellular region) of a 47-year-old specimen. Positive type X staining of the disc matrix was more frequently found in senile (>70 years) discs with end stages of disc degeneration. This study provides the first evidence for the occurrence of type X collagen in human lumbar intervertebral discs and it appears that type X collagen is re-expressed in late stages of disc degeneration. Accepted: 24 April 1997     

Aquaporin expression in the human intervertebral disc

The nucleus pulposus (NP) of the human intervertebral disc (IVD) is a hyperosmotic tissue that is subjected to daily dynamic compressive loads. In order to survive within this environment the resident chondrocyte-like cells must be able to control their cell volume, whilst also controlling the anabolism and catabolism of their extra-cellular matrix. Recent studies have demonstrated expression of a range of bi-directional, transmembrane water and solute transporters, named aquaporins (AQPs), within chondrocytes of articular cartilage. The aim of this study was to use immunohistochemsitry to investigate the expression of aquaporins 1, 2 and 3 within the human IVD. Results demonstrated expression of both AQP-1 and -3 by cells within the NP and inner annulus fibrosus (AF), while outer AF cells lacked expression of AQP-1 and showed very low numbers of AQP-3 immunopositive cells. Cells from all regions were negative for AQP-2. Therefore this study demonstrates similarities in the phenotype of NP cells and articular chondrocytes, which may be due to similarities in tissue osmolarity and mechanobiology. The decrease in expression of AQPs from the NP to the outer AF may signify changes in cellular phenotype in response to differences in mechanbiology, osmolarity and hydration between the gelatinous NP and the fibrous AF.     

CD24 is expressed specifically in the nucleus pulposus of intervertebral discs

Intervertebral disc (IVD) consists of a soft gelatinous material in its center, the nucleus pulposus (NP), bounded peripherally by fibrocartilage, annulus fibrosus (AF). Despite the number of patients with IVD degeneration, gene expression analysis has not been undertaken in NP and therefore little is known about the molecular markers expressed in NP. Here, we undertook a microarray screen in NP with the other nine tissues to identify the specific cell surface markers for NP. Five membrane associating molecules out of 10,490 genes were identified as highly expressing genes in NP compared with the other tissues. Among them, we identified CD24, a glycosylphosphatidylinositol (GPI) anchor protein as a cell surface marker for NP. CD24 expression was also detected in the herniated NP and chordoma, a malignant primary tumor derived from notochordal cells, while it was absent in chondrosarcoma. Therefore, CD24 is a molecular marker for NP as well as the diseases of IVD.     

Chondrocyte‐like nested cells in the aged intervertebral disc are late‐stage nucleus pulposus cells

Aging is a major risk factor of intervertebral disc degeneration and a leading cause of back pain. Pathological changes associated with disc degeneration include the absence of large, vacuolated and reticular‐shaped nucleus pulposus cells, and appearance of smaller cells nested in lacunae. These small nested cells are conventionally described as chondrocyte‐like cells; however, their origin in the intervertebral disc is unknown. Here, using a genetic mouse model and a fate mapping strategy, we have found that the chondrocyte‐like cells in degenerating intervertebral discs are, in fact, nucleus pulposus cells. With aging, the nucleus pulposus cells fuse their cell membranes to form the nested lacunae. Next, we characterized the expression of sonic hedgehog (SHH), crucial for the maintenance of nucleus pulposus cells, and found that as intervertebral discs age and degenerate, expression of SHH and its target Brachyury is gradually lost. The results indicate that the chondrocyte‐like phenotype represents a terminal stage of differentiation preceding loss of nucleus pulposus cells and disc collapse.     

Nonlinear finite element analysis of anular lesions in the L4/5 intervertebral disc

Degenerate intervertebral discs exhibit both material and structural changes. Structural defects (lesions) develop in the anulus fibrosus with age. While degeneration has been simulated in numerous previous studies, the effects of structural lesions on disc mechanics are not well known. In this study, a finite element model (FEM) of the L4/5 intervertebral disc was developed in order to study the effects of anular lesions and loss of hydrostatic pressure in the nucleus pulposus on the disc mechanics. Models were developed to simulate both healthy and degenerate discs. Degeneration was simulated with either rim, radial or circumferential anular lesions and by equating nucleus pressure to zero. The anulus fibrosus ground substance was represented as a nonlinear incompressible material using a second-order polynomial, hyperelastic strain energy equation. Hyperelastic material parameters were derived from experimentation on sheep discs. Endplates were assumed to be rigid, and annulus lamellae were assumed to be vertical in the unloaded state. Loading conditions corresponding to physiological ranges of rotational motion were applied to the models and peak rotation moments compared between models. Loss of nucleus pulposus pressure had a much greater effect on the disc mechanics than the presence of anular lesions. This indicated that the development of anular lesions alone (prior to degeneration of the nucleus) has minimal effect on disc mechanics, but that disc stiffness is significantly reduced by the loss of hydrostatic pressure in the nucleus. With the degeneration of the nucleus, the outer innervated anulus or surrounding osteo-ligamentous anatomy may therefore experience increased strains.     

Isolation and Characterisation of a Recombinant Antibody Fragment That Binds NCAM1-Expressing Intervertebral Disc Cells

Degeneration of the intervertebral discs (IVD) is a leading cause of neck and low back pain. Degeneration begins in the central nucleus pulposus region, leading to loss of IVD osmotic properties. Regeneration approaches include administration of matrix-mimicking scaffolds, cells and/or therapeutic factors. Cell-targeting strategies are likely to improve delivery due to the low cell numbers in the IVD. Single-chain antibody fragments (scFvs) that bind IVD cells were isolated for potential delivery of therapeutics to degenerated IVD. The most cell-distal domain of neural cell adhesion molecule 1 (NCAM1) was cloned and expressed in Escherichia coli. Phage display technology was used to isolate a human scFv against the recombinant domain by panning a scFv library on the immobilised protein. The isolated scFv bound cultured rat astrocytes, as well as bovine nucleus pulposus and annulus fibrosus cells in immunocytochemical studies. The scFv also labelled cells in bovine spinal cord and six-month and two-year old bovine IVD sections by immunohistochemistry. Antibody fragments can provide cell-binding moieties at improved cost, time, yield and functionalisation potential over whole antibodies. The described scFv has potential application in delivery of therapeutics to NCAM1-expressing cells in degenerated IVD.