Absence of biglycan accelerates the degenerative process in mouse intervertebral disc

Abstract

Study design: A study of the histologic changes of the intervertebral discs (IVDs) in biglycan (Bgn)-deficient mice.

Objective: In this study, we investigate whether the absence of Bgn accelerates the degenerative process in mouse intervertebral disc (IVD).

Summary of background data: Proteoglycans and collagen fibrils are major components in the extracellular matrix (ECM) composition of IVD. The ECM of IVD contains several members of the small leucine repeat proteoglycans (SLRPs) family. Bgn is one member of SLRPs family, and showed a unique expression with age and degeneration in the human IVD. To date, there have been no in vivo studies to see whether SLRPs have a role in maintaining the structural integrity of IVD. To explore the functions of Bgn in the IVD, we examined discs in Bgn-deficient mice.

Methods: A total of 30 spine specimens were harvested from wild-type (WT) and Bgn-deficient mice. Five specimens for each genotype at 4-, 6-, and 9-month old were examined in the experiments. Histologic analysis of the IVD was performed. Histologic gradings were performed separately on nucleus pulposus, anulus fibrosus, and endplate according to the classification system proposed by Boos et al.

Results: We found that Bgn-deficient mice developed an early onset of disc degeneration compared with WT mice. The degenerative scores of Bgn-deficient mice were significantly higher than those of WT mice at 4- and 9-month-old. High scores for nucleus pulposus and anulus fibrosus in Bgn-deficient mice significantly affected the difference in total degenerative scores at 9 months of age.

Conclusion: Bgn deficiency significantly accelerated disc degeneration.

Figure 1

Morphometrical analysis of the nucleus pulposus (NP). A, representative picture with the outline of the radiolucent area between the radiopaque margins of the end plate (EP). VB (vertebral body) SC (spinal cord); B, the mean and standard deviation of NP area are shown. Biglycan-deficient mice at 9-month-old had a significantly reduced size compared to age-matched wild-type mice (P<0.05).

Figure 2

Photomicrograph of the intervertebral disc (IVD) from 4-month-old mouse, hematoxylin and eosin staining. NP: nucleus pulposus, AF: annulus fibrosus, EP: end plate, WT: wild-type mouse, Bgn^−/0: Bgn-deficient mouse. Note small rounded cells resembling chondrocytes (white arrows) proliferate in the inner AF from Bgn^−/0 mouse (B), while IVD from WT mouse shows fibroblastic elongated cells (black arrows) in the inner AF (A). New bone formation (arrowheads) is seen in the cartilaginous EP of Bgn^−/0 mice (B).

Figure 3

Photomicrographs of intervertebral disc (IVD) from Bgn-deficient mouse at 6 months of age, Safranin O/Fast green FCF staining. NP: nucleus pulposus, AF: annulus fibrosus, EP: end plate Losses of notochordal cells associated with proliferation of chondrocyte-like cells are seen in the NP. A circle of 60×60 pixels were measured at fixed portions, anterior 1/3, middle 1/3, and posterior 1/3 for the upper and lower calcified EP (total 6 zones). For the NP, 2 fixed portions, upper-middle and lower middle zone, were measured.

Figure 4

Photomicrographs of the intervertebral disc from 9-month-old mouse, hematoxylin and eosin staining. NP: nucleus pulposus, AF: annulus fibrosus, EP: end plate, WT: wild-type mouse, Bgn^−/0: Bgn-deficient mouse. In the AF of WT mouse, the shapes of cells become rounded, resembling chondrocytes. New bone formation is identified in the cartilaginous EP, which is frequently seen in the EP of Bgn^−/0 mouse at 4-month-old (A). In the NP of Bgn^−/0 mouse, there is a regression in the number of chondrocytes. Arrow indicates cleft in the NP (B).

Figure 5

Histological gradings for intervertebral disc in wild-type (WT) and Bgn-deficient (Bgn^−/0) mice. Total degenerative scores of Bgn^−/0 mice are significantly higher than those of WT mice (p=0.0037 at 4M, p=0.098 at 6M, p=0.018 at 9M). The degenerative scores of WT and Bgn^−/0 mice significantly increase with age. High scores of the NP and AF in Bgn^−/0 mice significantly affect the difference in total degenerative scores at 9 months of age.