The presence of extracellular matrix degrading metalloproteinases during fetal development of the intervertebral disc

Abstract

Matrix metalloproteinases (MMPs) regulate connective tissue architecture and cell migration through extracellular matrix (ECM) degradation and are associated with both physiological and pathological processes. Although they are known to play a role in skeletal development, little is known about the role of MMPs in intervertebral disc (IVD) development. Sixteen fetal human lumbar spine segments, obtained at autopsy, were compared with five normal, non-fetal L4-L5 IVDs. Intensity and/or localization of immunohistochemical staining for MMP-1, -2, -3 and -14 were evaluated by three independent observers. MMP-2 production and activation was quantified by gelatin zymography. MMP-1 and -14 were abundantly present in the nucleus pulposus (NP) and notochordal (NC) cells of the fetal IVDs. In non-fetal IVDs, MMP-1 and -14 staining was significantly less intense (p = 0.001 and p < 0.001, respectively). MMP-3 was found in almost the entire IVD with no significant difference from non-fetal IVDs. MMP-2 staining in the NC and NP cells of the fetal IVD was moderate, but weak in the non-fetal IVD. Gelatin zymography showed a negative correlation of age with MMP-2 activity (p < 0.001). MMP-14 immunostaining correlated positively with MMP-2 activity (p = 0.001). For the first time, the presence of MMP-1, -2, -3 and -14 in the fetal human IVD is shown and the high levels of MMP-1, -2 and -14 suggest a role in the development of the IVD. In particular, the gradual decrease in MMP-2 activation during gestation pinpoints this enzyme as key player in fetal development, possibly through activation by MMP-1 and -14.

Fig. 1

Hematoxylin–eosin staining of NC cells in the fetal intervertebral disc (a). Identification of NC cells by positive immunohistochemical staining for EMA (b) and CK AE1/AE3 (c). Scale bars represent 25 μm (a, b) and 100 μm (c)

Fig. 2

Immunohistochemical staining for MMP-1 (a, b), MMP-2 (c, d), MMP-3 (e, f) and MMP-14 (g, h) in the nucleus pulposus of fetal (a, c, e, g) and non-fetal IVDs (b, d, f, h). Arrows accompanied by “NC” indicate clusters of notochordal cells and within the nucleus pulposus and “NP” indicates the nucleus pulposus containing chondrocyte-like NP cells. Scale bars represent 100 μm (a, b, e, f), 50 μm (g, h) and 25 μm (c, d)

Fig. 3

Intensity of immunohistochemical staining for MMP-1, -2, -3 and -14 in fetal and non-fetal IVDs. An intense staining for the antigen in >50% of the cells was scored as ++, an intense staining in 10–50% of the cells was scored +, ± was scored if the staining was seen in less than 10% of the cells and − was scored when no staining was observed, **p < 0.005

Fig. 4

Amount of active and pro-MMP-2 in fetal and non-fetal IVDs. Error bars represent standard error of the mean, *p < 0.05