Ossification process involving the human thoracic ligamentum flavum: role of transcription factors

Abstract

Introduction: Ossification of the ligamentum flavum (OLF) of the spine is associated with serious neurologic compromise, but the pathomechanism of this process remains unclear. The objective of this study was to investigate the pathomechanism of the ossification process, including the roles of various transcriptional factors in the ossification of human thoracic ligamentum flavum.

Methods: Sections of the thoracic ligamentum flavum were obtained from 31 patients with OLF who underwent posterior thoracic decompression, and from six control patients free of OLF. Cultured ligamentum flavum cells (n = 6, each) were examined with real-time reverse transcription-polymerase chain reaction (RT-PCR) analysis for Sry-type high-mobility group box 9 (Sox9), runt-related transcription factor 2 (Runx2), muscle segment homeobox 2 (Msx2), Osterix, distal-less homeobox 5 (Dlx5), and AP-1. The harvested sections were examined with hematoxylin-eosin, the terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end-labeling (TUNEL) method, and immunohistochemistry for the transcriptional factors.

Results: Compared with the control, the OLF showed disorganization of the elastic fiber bundles and abundant hypertrophic chondrocytes in the ossification front. TUNEL-positive chondrocytes were found near the ossified plaques. The mRNA expression levels of Sox9, Runx2, Msx2, and AP-1 in cultured cells from the ligamentum flavum of OLF patients were significantly different from those of the control. OLF samples were strongly immunoreactive to Sox9, Runx2, and Msx2 at proliferating chondrocytes in the fibrocartilage area. Hypertrophic chondrocytes were positive for Runx2, Osterix, Dlx5, and AP-1.

Conclusions: The ossification process in OLF seems to involve chondrocyte differentiation under the unique expression of transcriptional factors. Accumulation of hypertrophic chondrocytes was evident around the calcified area at the ossification front, and we suggest that the differentiation of these cells seems to be concerned with the ossification process.

Figure 1

Representative case of tuberous type OLF. Sagittal (a) and axial (b) CT images obtained in a 72-year-old woman with T11-12 OLF. Note the fusing of the ossified plaque at midline and the progression of this process into the spinal canal. The resected sample (c) contains a hard mass from dura mater aspect (arrow, ossification). CT, computed tomography; OLF, ossification of the ligamentum flavum.

Figure 2

Histologic examination of the ossification front. Section of the control ligamentum flavum (a) showing a thin ossification front and regular arrangement of fiber bundles. In OLF samples (representative case of fused type), note the expanding ossification front, gathering many chondrocytes, and featuring an irregular of calcification front (b). In addition to the ossification front, note the presence of osteoblasts as well as small blood cells with mesenchymal cells (c, d). In the EVG image, elastic fibers exhibit a regular arrangement (e), whereas the OLF samples contained irregular, fragmented fibers or no elastic fibers (f). (a-d) H&E; (e, f) EVG staining. CCA, calcified cartilage area; EVG, Elastica van Gieson; FA, fiber area; FCA, fibrocartilage area; OA, ossified area; OLF, ossification of the ligamentum flavum. Scale bar = 100 μm.

Figure 3

TEM examination and TUNEL staining of the ossification front. The typical structure of the elastic fibers had disappeared with an increase in the number of collagen fibers (a: control, b: OLF). Note the presence of matrix vesicles containing electron-dense material (c) and also the presence of pigmented chondrocytes (d) near the ossified layer (a, b: magnification ×1,000, c; magnification ×4,000, d; magnification ×2000). Note the presence of TUNEL-positive chondrocytes near the ossified layer (arrow) (e, scale bar = 100 μm). OLF: ossification of the ligamentum flavum; TEM: transmission electron microscopy; TUNEL: terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end-labeling.

Figure 4

Morphologic findings of cultured cells and relative expression levels of mRNA of transcriptional factors. Cultured cells in OLF (a) and control (b) showed spindle shape (scale bar = 100 μm). RT-PCR analysis of the ossified and nonossified cultured ligament cells showed that the mRNA expression levels of Sox9, Runx2, Msx2, and AP-1 in OLF were significantly different from those of the control (c). Data are expressed as mean ± SEM values of six samples each. Differences between the two groups were examined with the Student t test. *P < 0.05, compared with the control. mRNA, messenger RNA; OLF, ossification of the ligamentum flavum; RT-PCR, reverse transcription-polymerase chain reaction.

Figure 5

Immunostaining for Sox9, Runx2, and Msx2 of the OLF. Sox9 (a) was expressed in proliferating chondrocytes. Runx2 (b) was expressed in hypertrophic chondrocytes. Msx2 (c) was expressed in proliferating chondrocytes (scale bar = 100 μm; n.c., negative control; black arrow, hypertrophic chondrocytes; black arrowheads, proliferating chondrocytes).

Figure 6

Immunostaining for Osterix, Dlx5, and AP-1 in the OLF. Osterix (a), Dlx5 (b), and AP-1 (c) were strongly positive in hypertrophic chondrocytes (scale bar = 100 μm; n.c., negative control; black arrow, hypertrophic chondrocytes; black arrowheads, proliferating chondrocytes). OLF, ossification of the ligamentum flavum.