Immunohistochemical identification of type I and type III collagen and chondroitin sulphate in human pre‐dentine: a correlative FEI‐SEM/TEM study

Abstract

To identify type I- (I-CF) and type III-collagen fibrils (III-CF) and chondroitin 4/6 sulphate (CS) within human pre-dentine by means of a correlative analysis under field emission in-lens-scanning electron microscopy (FEI-SEM) and transmission electron microscopy (TEM). Human-extracted teeth were obtained and submitted to either a pre-embedding or a post-embedding immunolabelling procedure using monoclonal primary antibodies anti-I-CF, anti-III-CF and anti-CS. Gold-conjugated secondary antibodies were coupled to primary antibodies to visualize labelling under the electron beam. Correlative labelling patterns were obtained for I-CF and CS under both FEI-SEM and TEM. Field emission in lens-SEM analysis revealed an intricate three-dimensional network of I-CF and CS clarifying the intimate relationship between the two main components of the pre-dentine organic matrix. TEM analysis revealed odontoblasts exhibiting intracellular labelling for CS, which became more intense and diffuse over the pre-dentine organic matrix. The same diffuse immunoreaction was revealed for I-CF, whereas a weak immunolocalization of III-CF was found scattered throughout the pre-dentine layer and over the collagen fibrils. Both the pre- and post-embedding immunohistochemical approaches have led to the visualization of CF- and CS-labelling distribution within the pre-dentine layer, adding further knowledge on the elucidation of collagen-proteoglycans interaction in the organic matrix of human dental roots.