J.P.H.J. Rutges

Variations in gene and protein expression in human nucleus pulposus in comparison with annulus fibrosus and cartilage cells: potential associations with aging and degeneration

OBJECTIVE Regardless of recent progress in the elucidation of intervertebral disc (IVD) degeneration, the basic molecular characteristics that define a healthy human IVD are largely unknown. Although work in different animal species revealed distinct molecules that might be used as characteristic markers for IVD or specifically nucleus pulposus (NP) cells, the validity of these markers for characterization of human IVD cells remains unknown. DESIGN Eleven potential marker molecules were characterized with respect to their occurrence in human IVD cells. Gene expression levels of NP were compared with annulus fibrosus (AF) and articular cartilage (AC) cells, and potential correlations with aging were assessed. RESULTS Higher mRNA levels of cytokeratin-19 (KRT19) and of neural cell adhesion molecule-1 were noted in NP compared to AF and AC cells. Compared to NP cytokeratin-18 expression was lower in AC, and alpha-2-macroglobulin and desmocollin-2 lower in AF. Cartilage oligomeric matrix protein (COMP) and glypican-3 expression was higher in AF, while COMP, matrix gla protein (MGP) and pleiotrophin expression was higher in AC cells. Furthermore, an age-related decrease in KRT19 and increase in MGP expression were observed in NP cells. The age-dependent expression pattern of KRT19 was confirmed by immunohistochemistry, showing the most prominent KRT19 immunoreaction in the notochordal-like cells in juvenile NP, whereas MGP immunoreactivity was not restricted to NP cells and was found in all age groups. CONCLUSIONS The gene expression of KRT19 has the potential to characterize human NP cells, whereas MGP cannot serve as a characteristic marker. KRT19 protein expression was only detected in NP cells of donors younger than 54 years.

The Dog as an Animal Model for Intervertebral Disc Degeneration

Study Design. Prospective observational and analytic study. Objective. To investigate whether spontaneous intervertebral disc degeneration (IVDD) occurring in both chondrodystrophic (CD) and nonchondrodystrophic dogs (NCD) can be used as a valid translational model for human IVDD research. Summary of Background Data. Different animal models are used in IVDD research, but in most of these models IVDD is induced manually or chemically rather than occurring spontaneously. Methods. A total of 184 intervertebral discs (IVDs) from 19 dogs of different breeds were used. The extent of IVDD was evaluated by macroscopic grading, histopathology, glycosaminoglycan content, and matrix metalloproteinase 2 activity. Canine data were compared with human IVD data acquired in this study or from the literature. Results. Gross pathology of IVDD in both dog types (CD and NCD) and humans showed many similarities, but the cartilaginous endplates were significantly thicker and the subchondral cortices significantly thinner in humans than in dogs. Notochordal cells were still present in the IVDs of adult NCD but were not seen in the CD breeds or in humans. Signs of degeneration were seen in young dogs of CD breeds (<1 year of age), whereas this was only seen in older dogs of NCD breeds (5–7 years of age). The relative glycosaminoglycan content and metalloproteinase 2 activity in canine IVDD were similar to those in humans: metalloproteinase 2 activity increased and glycosaminoglycan content decreased with increasing severity of IVDD. Conclusion. IVDD is similar in humans and dogs. Both CD and NCD breeds may therefore serve as models of spontaneous IVDD for human research. However, as with all animal models, it is important to recognize interspecies differences and, indeed, the intraspecies differences between CD and NCD breeds (early vs. late onset of IVDD, respectively) to develop an optimal canine model of human IVDD.

Increased MMP-2 activity during intervertebral disc degeneration is correlated to MMP-14 levels

Intervertebral disc (IVD) degeneration is associated with the increased expression of several matrix metalloproteinases (MMPs), in particular MMP‐2. However, little is known about the actual activity of MMP‐2 in healthy and degenerated discs, or what mechanisms are involved in its activation. A major activation pathway involves complex formation with MMP‐14 and a tissue inhibitor of metalloproteinases‐2 (TIMP‐2). In a series of 56 human IVDs, obtained at autopsy and graded according to the Thompson score (I–V), we analysed whether MMP‐2 activity was increased in different stages of IVD degeneration and to what extent activation was related to the production of MMP‐14 and TIMP‐2. MMP‐2 activation and production were quantified by gelatin zymography. Immunohistochemical staining of MMP‐14 and TIMP‐2 was quantified with a video overlay‐based system. A positive correlation was observed between the amount of active MMP‐2 and pro‐MMP‐2 and degeneration grade (p < 0.001, correlation coefficient (CC) 0.557; and p < 0.001, CC 0.556, respectively). MMP‐2 activity correlated positively with MMP‐14 and less strongly with TIMP‐2 (p = 0.001, CC 0.436; and p = 0.03, CC 0.288, respectively). Moreover, immunopositivity for MMP‐14 correlated to degeneration grade (p = 0.002, CC 0.398). IVD degeneration was associated with the activity of MMP‐2 and the correlation of its activation with MMP‐14 production suggests MMP‐14 activates MMP‐2 during degeneration. As MMP‐14 is capable of activating several other enzymes that are also thought to be involved in IVD degeneration, it may be a key mediator of the degenerative process. Copyright

Identification of Cell Surface-Specific Markers to Target Human Nucleus Pulposus Cells Expression of Carbonic Anhydrase XII Varies With Age and Degeneration

OBJECTIVE Back pain is a major cause of disability, affecting millions of people worldwide. One cause of axial back pain is degeneration of the nucleus pulposus (NP) of the intervertebral disc. This study was undertaken to investigate associations of NP cells with cell surface-specific proteins that differ from proteins in closely related cell types, i.e., intervertebral disc anulus fibrosus (AF) cells and articular cartilage (AC) chondrocytes, in order to identify potential surface molecules for directed delivery of therapeutic agents. METHODS We conducted a complementary DNA microarray analysis of 16 human samples from 6 donors, followed by gene list reduction using a systematic approach. Genes that were more highly expressed in NP than AC cells, contained transmembrane domains, and appeared attractive for targeting were assessed by quantitative reverse transcription-polymerase chain reaction (RT-PCR). As a viable candidate, carbonic anhydrase XII (CAXII) was analyzed at the protein level by immunohistochemistry and functional study. RESULTS Microarray results demonstrated a clear divide between the AC and AF and between the AC and NP samples. However, the transcriptomic profile of AF and NP samples displayed a greater intersubject similarity. Of the 552 genes with up-regulated expression in NP cells, 90 contained transmembrane domains, and 28 were quantified by RT-PCR. Most intense CAXII labeling was observed in the NP of discs from young subjects and in degenerative tissue. CONCLUSION CAXII may be considered for detection or targeting of degenerating disc cells. Furthermore, CAXII may be involved in pH regulation of NP cells. Its potential for directed delivery of regenerative factors and its functional role in NP cell homeostasis warrant further investigation.

Hypertrophic differentiation and calcification during intervertebral disc degeneration.

BACKGROUND In degenerative intervertebral discs (IVDs) collagen type X expression and calcifications have been demonstrated, resembling advanced osteoarthritis (OA), which is associated with hypertrophic differentiation, characterized by the production of collagen type X, Runt-related transcription factor 2 (Runx2), osteoprotegerin (OPG), alkaline phosphatase (ALP) and calcifications. OBJECTIVE The aim of this study was to determine if hypertrophic differentiation occurs during IVD degeneration. METHODS IVDs from all Thompson degeneration grades were prepared for histology, extraction of nucleus pulposus (NP) and annulus fibrosis (AF) tissue (N=50) and micro-CT (N=27). The presence of collagen type X, OPG and Runx2 was determined by immunohistochemistry, with OPG levels also determined by Enzyme-linked immunosorbent assay (ELISA). The presence of calcification was determined by micro-CT, von Kossa and Alizarin Red staining. RESULTS Immunohistochemical staining for collagen type X, OPG, Runx2 appeared more intense in the NP of degenerative compared to healthy IVD samples. OPG levels correlated significantly with degeneration grade (NP: P<0.000; AF: P=0.002) and the number of microscopic calcifications (NP: P=0.002; AF: P=0.008). The extent of calcifications on micro-CT also correlated with degeneration grade (NP: P<0.001, AF: P=0.001) as did von Kossa staining (NP: P=0.015, AF: P=0.016). ALP staining was only incidentally seen in the transition zone of grades IV and V degenerated IVDs. CONCLUSION This study for the first time demonstrates that hypertrophic differentiation occurs during IVD degeneration, as shown by an increase in OPG levels, the presence of ALP activity, increased immunopositivity of Runx2 and collagen type X.

A validated new histological classification for intervertebral disc degeneration

UNLABELLED Histology is an important outcome variable in basic science and pre-clinical studies regarding intervertebral disc degeneration (IVD). Nevertheless, an adequately validated histological classification for IVD degeneration is still lacking and the existing classifications are difficult to use for inexperienced observers. OBJECTIVE Therefore the aim of this study was to develop and to validate a new histological classification for IVD degeneration. Moreover, the new classification was compared to the frequently used non-validated classification. METHODS The new classification was applied to human IVD sections. The sections were scored twice by two independent inexperienced observers, twice by two experienced IVD researchers and once by a pathologist. For comparison, the sections were also scored according to the classification described by Boos et al. by two experienced IVD researchers. Macroscopic grading according Thompson et al., glycosaminoglycan (GAG) content and age were used for validation. RESULTS The new classification had an excellent intra- and a good inter-observer reliability. Intraclass Correlation Coefficients (ICC) were 0.83 and 0.74, respectively. Intra- and inter-observer reliability were comparable for experienced and inexperienced observers. Statistically significant correlations were found between the new classification, macroscopic score, GAG content in the nucleus pulposus (NP) and age; Correlation coefficient (CC) 0.79, -0.62 and 0.68, respectively. The CCs of the Boos classification were all lower compared to the new classification. CONCLUSION the new histological classification for IVD degeneration is a valid instrument for evaluating IVD degeneration in human IVD sections and is suitable for inexperienced and experienced researchers.

Micro-CT quantification of subchondral endplate changes in intervertebral disc degeneration

BACKGROUND The intervertebral disc (IVD) is dependent on nutrient provision through a cartilage layer with underlying subchondral bone, analogous to joint cartilage. In the joint, subchondral bone remodeling has been associated with osteoarthritis (OA) progression due to compromised nutrient and gas diffusion and reduced structural support of the overlaying cartilage. However, subchondral bone changes in IVD degeneration have never been quantified before. OBJECTIVE The aim of this study is to determine the subchondral bone changes at different stages of IVD degeneration by micro-CT. METHODS Twenty-seven IVDs including the adjacent vertebral endplates were obtained at autopsy. Midsagittal slices, graded according the Thompson score, were scanned. Per scan 12 standardized cylindrical volumes of interest (VOI) were selected. Six VOIs contained the bony endplate and trabeculae (endplate VOIs) and six accompanying VOIs only contained trabecular bone (vertebral VOIs). Bone volume as percentage of the total volume (BV/TV) of the VOI, trabecular thickness (TrTh) and connectivity density (CD) were determined. RESULTS An increase in BV/TV and TrTh was found in endplate VOIs of IVDs with higher Thompson score whereas these values remained stable or decreased in the vertebral VOIs. CONCLUSION The increase in bone volume combined with the increase in TrTh in endplate VOIs strongly suggest that the subchondral endplate condenses to a more dense structure in degenerated IVDs. This may negatively influence the diffusion and nutrition of the IVD. The endplate differences between intact and mild degenerative IVDs (grade II) indicate an early association of subchondral endplate changes with IVD degeneration.

Histological characteristics of diffuse idiopathic skeletal hyperostosis.

Diffuse idiopathic skeletal hyperostosis (DISH) is a predominantly radiographic diagnosis and histological knowledge of DISH is limited. The aim of this study was to describe the histological characteristics of DISH in the spinal column and to study the relation between DISH and intervertebral disc (IVD) degeneration. Therefore, 10 human cadaveric spines with fluoroscopic evidence of DISH were compared with 10 controls. Plain radiographs and computed tomography (CT) scans were obtained and tissue blocks were resected from three predefined levels of all specimens. The microscopic sections were scored by two blinded observers using a newly developed scoring system specific for characteristics of DISH and a validated scoring system for IVD degeneration. Maximum IVD height was measured on the CT scans. Analyses were performed using Fishers exact test and Students t‐test. When compared to controls, the right sided sections from DISH specimens showed partial or complete bone bridges, consisting of cortical woven bone, accompanied by morphological changes in the adjoining part of the IVD. Using the histological scoring system for DISH, all parameters were significantly different between the DISH and control group (p < 0.01). The contralateral location did not show differences between the groups. The overall degree of IVD degeneration and height of IVD was comparable for the two groups. The histopathological changes observed in spines with DISH corresponded to the fluoroscopic images and CT scans. The degree of IVD degeneration and IVD height was comparable for both groups, suggesting a limited role for IVD degeneration in the pathogenesis of DISH.