Immunohistochemical localization of mesenchymal stem cells in ossified human spinal ligaments

Mesenchymal stem cells (MSCs) have been isolated from various tissues and used for elucidating the pathogenesis of numerous diseases. In our previous in vitro study, we showed the existence of MSCs in human spinal ligaments and hypothesized that these MSCs contributed to the pathogenesis of ossification of spinal ligaments. The purpose of this study was to use immunohistochemical techniques to analyze the localization of MSCs in ossified human spinal ligaments in situ. Ossified (OLF) or non-ossified ligamentum flavum (non-OLF) samples from the thoracic vertebra were obtained from patients who had undergone posterior spinal surgery. Serial sections were prepared from paraffin-embedded samples, and double immunofluorescence staining was performed using antibodies against markers for MSCs (CD73, CD90 and CD105), endothelial cells (CD31), pericytes (α-smooth muscle actin), and chondrocytes (S100). Immunolocalization of MSCs was observed in the perivascular area and collagenous matrix in spinal ligaments. Markers for MSCs and pericytes were co-expressed in the perivascular area. Compared with non-OLF, OLF had a large amount of neovascularization in the fragmented ligament matrix, and a high accumulation of MSCs around blood vessels. The prevalence of MSCs in OLF within collagenous matrix was significantly higher than that in non-OLF. Chondrocytes near the ossification front in OLF also presented expression of MSC markers. MSCs may contribute to the ectopic ossification process of OLF through endochondral ossification.