Abstract
In the present study, 3D histochemistry and imaging methodology is described for human gingiva to analyze its vascular network. Fifteen human gingiva samples without signs of inflammation were cleared using a mixture of 2-parts benzyl benzoate and 1-part benzyl alcohol (BABB), after being immunofluorescently stained for CD31, marker of endothelial cells to visualize blood vessels in combination with fluorescent DNA dyes. Samples were imaged in 3D with the use of confocal microscopy and light-sheet microscopy and image processing. BABB clearing caused limited tissue shrinkage 13 ± 7% as surface area and 24 ± 1% as volume. Fluorescence remained intact in BABB-cleared gingiva samples and light-sheet microscopy was an excellent tool to image gingivae whereas confocal microscopy was not. Histochemistry on cryostat sections of gingiva samples after 3D imaging validated structures visualized in 3D. Three-dimensional images showed the vascular network in the stroma of gingiva with one capillary loop in each stromal papilla invading into the epithelium. The capillary loops were tortuous with structural irregularities that were not apparent in 2D images. It is concluded that 3D histochemistry and imaging methodology described here is a promising novel approach to study structural aspects of human gingiva in health and disease.
Highlights
In the present study, 3D histochemistry and imaging methodology is described for human gingiva to analyze its vascular network
The present study describes our investigations to optimize the methodology to stain, clear and image human gingiva using fluorescent markers, the benzyl alcohol (BA)- and benzyl benzoate (BB)-containing clearing solution BABB and light-sheet microscopy
3D images of capillaries in stromal papillae invading stratified epithelium of human gingiva are shown for the first time
Summary
3D histochemistry and imaging methodology is described for human gingiva to analyze its vascular network. It is concluded that 3D histochemistry and imaging methodology described here is a promising novel approach to study structural aspects of human gingiva in health and disease. Novel methods to clear tissues have been reported (for review, see Azaripour et al.[9]; Lee et al.4) and have been developed either for the removal of lipid bilayers of cell membranes[3,5,10,11,12,13,14,15] or by impregnation of the tissue by a solvent with high RI15–18 to eliminate differences in RI. The present study describes our investigations to optimize the methodology to stain, clear and image human gingiva using fluorescent markers, the benzyl alcohol (BA)- and BB-containing clearing solution BABB and light-sheet microscopy
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