Immunohistochemical analysis of versican in human dentin matrix

Objective: Aim of this study was to investigate the immunohistochemical labeling pattern of versican within the dentin matrix of human sound molars by means of a correlative fluorescence microscope (FM), field emission in-lens scanning electron microscope (FEI-SEM) and transmission electron microscope (TEM) analysis.

Methods: Dentin specimens were produced by non carious human teeth. Specimens were randomly assigned to three groups: 1) FM group and 2) FEI-SEM group: unfixed tooth sections were exposed to a pre-embedding immunohistochemical procedure, post fixed, dehydrated and HMDS dried prior to morphological analysis; 2) TEM Group: specimens were fixed, demineralized, embedded in resin and exposed to a post-embedding immunohistochemical procedure. Specimens were exposed to two different antibodies to respectively assay presence and distribution of versican fragments and versican whole molecule (LF-99 and 12C5 DSHB, USA). The immunohistochemical protocol was performed in accordance with Breschi et al., J Biomed Mater Res 2003.

Results: The correlative FM, FEI-SEM and TEM analysis revealed positive immunoreaction for versican fragments which appear as constituents of the dentin organic matrix of mature human teeth mainly localized at the peritubular level. In addition, a close relation between versican fragments and the intricate network of demineralized collagen fibrils was found, since versican fragments were identified approximately at the junction between each collagen periods of the peritubular dentin matrix. Conversely, very weak labeling was found for the whole versican molecule in the dentin matrix of mature human teeth, while no labeling was found for the versican intact molecule.Positive labeling was found in predentin.

Conclusions: This study supports the hypothesis that versican fragments are significant constituents of the dentin matrix of human mature teeth maintaining its antigen integrity either in predentin and in dentin. The role of versican fragments in mineralization or homeostasis within the dentin organic matrix should be elucidated by further biochemical assays.