Differences in calcification and osteogenic potential of herniated discs according to the severity of degeneration based on Pfirrmann grade: a cross-sectional study

Our μ-CT results indicate a significant increase in the degree of intervertebral disc calcification with increasing severity of degeneration based on the Pfirrmann grading system, and this finding is consistent with the results of a previous study [18]. The lack of significant differences in calcification between discs of Pfirrmann grades III–V may have been due to the use of calcification grading system that is semi-quantitative, focusing only on the number of calcified spots and not considering the size of such regions. There was one specimen of Pfirrmann grade V that appeared to have a “-” grade of calcification. Although no calcification spot may have formed in this sample or the formed calcification was below the resolution of μ-CT, positive Osterix expression on immunohistochemical staining indicated the osteogenic potential of the disc. Our μ-CT results also showed that the posterior part of the removed intervertebral discs showed much greater calcification than the anterior part.

Dystrophic calcification is a passive pathologic process caused by necrosis or malnutrition, but it has been reported that the calcification of intervertebral discs is an active process that occurs along with hypertrophic differentiation [16]. Our observation of cells expressing BMP-2, Osterix, Osteocalcin, and ALP around and/or within calcified tissue as well as the increased gene expression of BMP-2, Osterix, and Osteocalcin in discs exhibiting greater calcification indicate that intervertebral disc calcification is an active process linked to osteogenesis. Moreover, based on the expression of Runx-2 in degenerated discs [11], the ossification of intervertebral discs is an endochondral process.

BMP-2 is a type of multifunctional growth factor involved in the regulation and control of development, differentiation, and apoptosis of osteoblasts, chondroblasts, neurocytes, and epithelial cells [19]. Takae et al. [4] first reported the expression of BMP-2 and its receptors in degenerated cervical intervertebral discs of mice in 1999. Although their results indicated a relationship between BMP-2 expression and disc degeneration, the exact relationship remained unclear. A series of in vitro and in vivo studies has elucidated the roles of chondrogenesis and the regenerative effect of BMP-2 in the intervertebral disc degeneration process [20, 21]. However, another study [7] demonstrated Osteocalcin expression and calcium deposition after treatment of intervertebral disc cells with BMP-2, indicating that BMP-2 has an osteogenic effect in intervertebral disc tissue. Our findings indicate that the level of BMP-2 expression in intervertebral disc tissue is related to the severity of degeneration, in accordance with the results of a previous study [22]. Our data also reveal that the expression levels of Osterix and Osteocalcin follow trends similar to BMP-2, confirming that the BMP-2 level is consistent with the osteogenic potential of degenerated discs. We hypothesize that BMP-2 plays two roles in degenerated discs: one is compensatory in which BMP-2 accelerates cartilage repair, and because this compensatory expression cannot completely reverse the degeneration, overexpression of BMP-2 finally leads to the ossification of discs as a side effect. The underlying mechanisms by which BMP-2 regulates chondrogenesis and osteogenesis remain unknown, and in our future studies, we will investigate methods to block the osteogenic effect of BMP-2 to ensure proper intervertebral disc regeneration upon treatment with BMP-2.

Our μ-CT and qPCR results both demonstrated that the posterior part of removed intervertebral discs possesses greater osteogenic potential than the anterior part. This phenomenon may be caused by differences in the local environments of these regions of discs. Interestingly, the posterior part of intervertebral discs has also been shown to exhibit more severe inflammation, fibrosis, and angiogenesis than the anterior part [18]. However, the presence of some degree of osteogenic potential in the anterior part indicates that the differences in the local environment are not the only cause for this phenomenon, and some other undefined factors likely participate in the induction of intervertebral disc ossification.

Our immunohistochemical staining experiments revealed the presence of the osteogenic markers BMP-2, Osterix, Osteocalcin, and especially ALP, which hydrolyzes calcium pyrophosphate [23] to initiate an irreversible process of calcification, and even in discs that did not stain positively for ALP, Osterix expression was observed. These findings suggest that osteogenesis in the intervertebral disc is a very slow process first involving the formation of dispersed, amorphous regions of calcification or osteoid tissue with eventual formation of mature bone in the long term. The formation of these calcified tissues may be related to the inflammation and angiogenesis that occur after disc degeneration or herniation. The origin of osteoprogenitor cells responsible for intervertebral disc ossification is still not clear. These cells may be derived from primitive cells in the peripheral blood and/or from osteoprogenitor cells in the degenerated disc [24]. Jin et al. reported that the origin of these cells is the pre-existing primitive cells in the disc. However, other studies have shown that the cells responsible for ossification in aortic calcification and tendinopathy are derived from the two different origins, which suggests this possibility also in intervertebral discs [25, 26].