• Anthony J Hayes, Susan M Smith, Mark A Gibson, and James Melrose.
• Confocal Microscopy Unit, Cardiff School of Biosciences, University of Cardiff, United Kingdom.
• Spine. 2011 Oct 1; 36 (21): E1365-72.

Study DesignA comparative immunolocalization study of elastin-associated proteins and established intervertebral disc (IVD) extracellular matrix (ECM) components.ObjectiveTo localize for the first time, elastic fiber–associated proteins with structural fibrillar components in the annulus fibrosus (AF) of the fetal IVD.Summary Of Background DataElastin has been identified histochemically in adult bovine, human, and immature rat IVDs, and in fetal human IVDs using electron microscopy; however, no immunolocalization studies have been undertaken for associated components in human fetal IVDs.MethodsEn-bloc fixation of thoracolumbar spinal segments in formalin and Histochoice followed by standard histochemical processing, paraffin embedding, microtome sectioning, and identification of IVD ECM components using a range of specific mono- and polyclonal antibodies and bright-field and laser scanning confocal microscopy.ResultsThe elastic fiber-associated proteins fibrillin-1, LTBP-2, and MAGP-1 were prominently immunolocalized in the outer lamellar layers of the AF of the human fetal IVD. Dual localization of selected components by confocal microscopy demonstrated that versican and LTBP-2 were colocalized with fibrillin-1 microfibrils in the AF lamellae with a similar distribution to the elastin fibers. LTBP-2 was also associated with pericellular perlecan in the outer AF. These interconnections between elastin-associated proteins resulted in an elastic network, which connected the AF cells with the adjacent cartilaginous vertebral bodies.ConclusionSpecific immunolocalization of fibrillin-1, MAGP-1, and versican with elastin in the outer AF of the fetal human IVD has been demonstrated. We deduce from the established distributions of the elastin-associated proteins and their known interactivities with matrix components that these stabilize and aid in the integration of the elastic fibers in the annular lamellae and may be responsible for the generation of tensional forces in the outer AF, which direct the assembly of this tissue.

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