Arthritic degenerative disorders of the spinal intervertebral disc are the most common of the musculoskeletal conditions and have a major impact on society because of the frequency of occurrence and the economic consequences (1). Environmental factors such as physical activity and mechanical loading may explain only a subset of intervertebral disc disorders when compared with inherited genetic factors (2–5). Intervertebral disc disorders and degeneration have been associated with mutations or polymorphisms in genes encoding matrix proteins, including type IX collagen (6) and aggrecan (7), as well as with genes encoding interleukin-1 (IL-1) (8), IL-6 (9), cartilage intermediate-layer protein (10), and vitamin D receptor (11).
Mice with genetic mutations in select extracellular matrix proteins, including type II collagen (12) and type I collagen (13), have been shown to acquire structural and functional matrix alterations in the intervertebral disc. In one study, mice carrying an inactivated allele of the Col2a1 gene showed early vertebral end plate ossification and decreased glycosaminoglycan concentration in the vertebrae, end plate, and annulus fibrosus beginning at 1 month, with differences between mutant mice and wild-type (WT) controls no longer evident by the age of 9 months (12). In another study, mice heterozygous for a type I collagen mutation (Mov13 strain) were reported to show decreased compressive and tensile stiffness of the intervertebral discs, thus providing evidence of functional changes associated with collagen deficiency (13). Taken together, the results of these studies demonstrate that collagens are an important contributor to the organization and function of the intervertebral disc extracellular matrix and may represent a precipitating factor, or key partner, in the cascade of degeneration events.
Genetic analyses of human populations have identified substitution mutations in the chains of the type IX collagen molecules as being linked to a predisposition to intervertebral disc disorders (6, 14–16). Type IX collagen is a member of the fibril-associated collagen with interrupted triple helix group of collagens. These collagens act as molecular bridges between fibrillar collagens and other extracellular matrix components (17, 18). The type IX collagen molecule, in combination with type XI collagen, is a key player in the type II/IX/XI heterofibril that is an important stabilizing element in cartilaginous tissue composed of type II collagen (19). Mice expressing a transgene for Col9a1 associated with a shortened collagen α1(IX) chain exhibit degenerative disc changes that include shortening of vertebrae, reduced matrix staining for mucous material, matrix disorganization, and end plate irregularities (20). Mice homozygous for an inactivated Col9a1 gene have also been generated and present with degenerative changes in articular cartilage, beginning at an early time point (6 months of age) (21, 22). These mice also exhibit changes in the expression of proteases and mechanical function that are consistent with human osteoarthritis (OA) (23, 24).
Although type IX collagen is a major component of intervertebral disc extracellular matrix collagen (18, 25, 26), no corresponding information is available regarding the impact of a type IX collagen deletion on intervertebral disc structure and function. In this study, the lumbar spines of mice homozygous for the inactivated Col9a1 gene (Col9a1−/−) were evaluated to identify age-related and genotype-related changes in key spinal structures. The intervertebral disc and end plate regions of spines from WT and type IX collagen–deficient mice were evaluated using histologic and immunohistochemical methods. A semiquantitative grading scheme was used to quantify degeneration of the intervertebral disc and vertebral end plates. Differences noted between WT and knockout mouse spines suggest that the type IX collagen deletion leads to premature intervertebral disc degeneration.
- Top of page
- MATERIALS AND METHODS
- AUTHOR CONTRIBUTIONS
Early signs of degeneration were observed in both the intervertebral disc and end plate regions of the type IX collagen–deficient mice beginning at 3 months of age and 6 months of age, respectively, using the grading scheme described by Boos and coworkers (29). According to this system, features of morphologic change are evaluated in the intervertebral disc and end plate regions that are associated with aging and atypical changes in humans. A degenerative or pathologic tissue is thus classified as one that is graded as older or younger than would be expected for its chronologic age. Many of the observed changes in the intervertebral disc region were common to human intervertebral disc degenerative disease, including mucous degeneration, granular changes, and cell death. Evidence of chondrocyte proliferation involving multiple chondrocytes in small groups, and regions of staining for Safranin O, were also observed but are more consistent with OA cartilage changes and disc herniation changes than the senescent changes often reported for intervertebral disc aging and degeneration (33, 34).
Cell death appeared to be a factor of interest only at the 6-month time point and was noted as areas in which we could not identify any cell nuclei. There was minimal evidence of tear or cleft formation in the intervertebral disc in the annulus fibrosus or nucleus pulposus regions and no differences in these categories between mutant and WT mice. The accelerated degeneration of the intervertebral disc regions in Col9a1−/− mouse spines was apparent at the earliest time point (3 months) and certainly preceded that in the end plate, supporting the hypothesis that disc degeneration initiates in the central intervertebral disc (35). The extent of intervertebral disc degeneration in the mice, even at 12 months, was relatively mild when compared with the full range of the grading scheme (22 points). This observation is consistent with the ages of the mice (3–12 months), as compared with a maximum age for C57BL mice of 30 months (36, 37). In comparison, severe degeneration of the intervertebral disc in humans is seen primarily in those beyond the fifth decade of life, as demonstrated via macroscopic (35, 38) and microscopic (29) analysis of the spine motion segments.
More than 30 different grading schemes have been used to assess the degenerative state of the lumbar intervertebral disc and adjacent cartilaginous end plate regions (39). For the assessment of macroscopic anatomy or histologic sections, one of the most widely used systems is the grading scale proposed by Thompson and coworkers (38), which assigns a degeneration score from grade I to grade V to the intervertebral disc, end plate, and vertebral body regions and is associated with good to excellent interobserver agreement. However, it is not well-suited to the grading of histologic sections and does not assess cellular changes apparent by histology.
Multiple grading schemes developed for evaluation of histologic sections are available (40, 41), although information on observer reliability measures via either kappa statistics or intraclass correlation coefficient measures is not always available with these schemes. The comprehensive and well-validated grading system developed by Boos and coworkers (29) was chosen for the current evaluation of mouse spines. Use of that approach has demonstrated good correlation with the scale of macroscopic degeneration grading described by Thompson et al and found utility in predicting age-related degeneration in human samples. Although that system was not specifically developed for evaluation of animal histology, the histologic features appear to be equally relevant to the murine spine with age and degeneration, as noted in this study. With our use of the grading scale proposed by Boos et al, interobserver variability values in the grader evaluations were in the “almost perfect” range and were consistent with those reported for human tissue evaluation (29). For these reasons, it was concluded that the intervertebral disc and end plate grading schemes described by Boos and coworkers were appropriate for use in the current study.
An observation of note was that summed grades across individual criteria could not be expected to correlate with progression of degeneration in all cases, and that the full range of the scheme (e.g., 0–22 for the intervertebral disc region) would not be expected for even the most severely degenerative changes in humans. Thus, the finding of relatively small grade differences, as observed for the type IX collagen mutation model in this study, should not be interpreted as “mild” degenerative changes but rather identification of specific changes in the intervertebral disc or end plate. Use of a statistical approach designed to test for differences in specific features of this grading scheme, rather than the cumulative score, may be useful in future studies.
Intervertebral disc and end plate degeneration related to a type IX collagen mutation may have important consequences in the clinical setting with respect to understanding and treating intervertebral disc degeneration and low back pain. The type IX collagen–deficient mice may be a useful model of premature and spontaneous intervertebral disc and end plate degeneration, with the benefit of age-matched and littermate controls. Furthermore, models of spontaneous degenerative disease require no surgical, mechanical, or chemical injury for initiation of intervertebral disc changes (42). This mouse model may also provide additional insight into mechanisms of intervertebral disc and end plate degeneration in humans. There are morphologic features reported in humans having single-point mutations in genes encoding α2(IX) or α3(IX) chains that coincide with those observed in the Col9a1−/− mice studied. These common features (assessed using magnetic resonance imaging in humans) include annular tears (16, 43) and end plate fracture or irregularity (16, 44).
Current findings suggest that end plate changes may be only a minor contributor to the pathology, with metabolic changes in the cell population being a major factor. In a related study of knee OA changes in this same animal model, elevated staining and expression of collagenases relevant to collagen and proteoglycan degradation were expressed in cartilage of the Col9a1−/− mice at a young age, suggesting that biochemical changes and subsequent structural changes could be precipitating factors for the progression of OA (24). Related to these changes was a later loss of compressive stiffness and an elevated level of permeability, which are consistent with functional changes that occur in OA cartilage. Whether these same mechanisms contribute to changes in the intervertebral disc matrix or end plate regions in the Col9a1−/− mouse spine are not known, although the implications for proteolytic involvement could be meaningful in designing treatment strategies for prevention of progressive intervertebral disc degeneration. Ongoing studies will explore the expression of proteolytic enzymes in the spines of mice carrying this type IX collagen gene mutation, as well as studies directed at understanding a role for the collagen deficiency in modulating nutrient and metabolism of the intervertebral disc cells.
- Top of page
- MATERIALS AND METHODS
- AUTHOR CONTRIBUTIONS
Dr. Setton had full access to all of the data in the study and takes responsibility for the integrity of the data and the accuracy of the data analysis.
Study design. Boyd, Jing, Li, Chen, Setton.
Acquisition of data. Boyd, Richardson, Allen, Flahiff, Jing, Chen.
Analysis and interpretation of data. Boyd, Allen, Flahiff, Li, Chen, Setton.
Manuscript preparation. Boyd, Richardson, Chen, Setton.
Statistical analysis. Boyd, Setton.