Immunohistochemical and biochemical assay of versican in human sound predentine/dentine matrix

Abstract

Aim of this study was to investigate the distribution of versican proteoglycan within the human dentine organic matrix by means of a correlative immunohistochemical analysis with field emission in-lens scanning electron microscope (FEI-SEM), transmission electron microscope (TEM), fluorescence microscope (FM) and biochemical assay. Specimens containing dentine and predentine were obtained from non carious human teeth and divided in three groups: 1) FEI-SEM group: sections were exposed to a pre-embedding immunohistochemical procedure; 2) TEM group: specimens were fixed, demineralised, embedded and submitted to a post-embedding immunohistochemical procedure; 3) FM group: sections mineralised and submitted to a pre-embedding immunohistochemical procedure with fluorescence labelling. Specimens were exposed to two different antibodies to assay distribution of versican fragments and whole versican molecule.Western Blotting analysis of dentine and pulp extracts was also performed. The correlative FEI-SEM,TEM and FM analysis revealed positive immunoreaction for versican fragments both in predentine and dentine, while few gold particles identifying the whole versican molecule were found in predentine only under TEM. No labelling of versican whole molecule was detected by FEI-SEM and FM analysis. The immunoblotting analysis confirmed the morphological findings. This study suggests that in fully developed human teeth versican fragments are significant constituents of the human dentine and predentine organic matrix, while versican whole molecule can be visualised in scarce amount within predentine only. The role of versican fragments within human dentine organic matrix should be further elucidated.

Figure 1

Micrographs of human dentine preparations obtained by means of FEISEM and TEM, revealing the immunoreaction for LF-99 polyclonal antibody, which identifies versican fragments. (a) FEISEM predentine micrograph revealing an intricate network of collagen fibril bundles moderately labeled for LF-99 (arrows). (b) TEM image showing gold nanoparticles (arrows) in the predentine layer: a diffuse and moderately intense labeling can be seen over the collagen fibrils (Bar: 10 µm). (c) FEISEM image of the dentine revealing a generally weak labeling mainly distributed over the peritubular zone (arrows). (d) TEM image of the dentine matrix revealing a moderate to weak labeling over peritubular and intertubular regions; Od: odontoblast process (Bar: 10 µm).

Figure 2

FEISEM and TEM micrographs of human dentine showing immunoreactivity for versican whole molecule by using 12C5 monoclonal antibody. (a) FEISEM micrograph of the predentine zone revealing a very weak reaction for versican whole molecule (arrows) over the collagen fibrils. (b) TEM image revealing several randomly dispersed gold nanoparticles (arrows) over the predentine layer sometimes associated with the collagen fibrils (Bar: 10 µm). (c, d) In both images a few randomly scattered gold particles were detected within the dentine matrix, being almost absent in the FEISEM micrograph, while slightly more numerous in the TEM micrograph (Bar: 10 µm), mainly localised over the intertubular dentine and also present over the odontoblast process (arrows).

Figure 3

FM micrographs of human predentine and dentine showing fluorescence immunoreactivity for versican by using both antibodies. (a) FM micrograph reveals a positive activity in both portions of the predentine (Pd) and dentine (D) layers; bar = 100 µm. (b) FM image revealing positive fluorescence immunostaining in particular in predentine (Pd) layer, typically of 12C5 antibody recognising molecula of versican proteoglycan, while fluorescence decrease towards dentine tissue (D); bar = 100 µm. (c) Control FM micrograph of human predentine and dentine, with no positive reaction; bar = 100 µm.

Figure 4

Western Blot analysis of the human EDTA-decalcified pulverized dentine and human pulp fibroblasts. Positive reaction was found for LF99 antibody within the dentine extracts (lane 1), while only a faint staining was detectable for the 12C5 antibody. Conversely, pulp extracts revealed positive immunoreactions for both tested antibodies, thus demonstrating presence of fragments and whole versican proteoglycan molecules (lane 2).