Analysis of type-I collagen fibrils and chondroitin sulfate distribution in human dentin by confocal laser scanning microscopy combined with dual immunofluorescent labeling technique / 中华口腔医学杂志

ABSTRACT

<p><b>OBJECTIVE</b>To introduce the method of dual immunofluorescence labeling of human dentin matrix without demineralization of the whole dentin fragments, and to analyze the distribution of type-I collagen fibrils and chondroitin sulfate in human dentin.</p><p><b>METHODS</b>Forty 30 µm- thick middle coronal dentin sections were obtained from 8 freshly extracted human third molars and etched with 37% phosphoric acid(PA) gel for 15 s. After preconditioning with or without tosyl- phenylalanyl chloromethyl ketone(TPCK) treated trypsin digestion, sections were subjected to dual immunofluorescent labeling and scanned by confocal laser scanning microscopy to identify the type-I collagen fibrils and chondroitin sulfate.</p><p><b>RESULTS</b>Chondroitin sulfate was localized in the lumens of the dentin tubules and peritubular dentin, while the type-I collagen fibrils were localized in intertubular dentin and peritubular dentin. After preconditioning with TPCK treated trypsin digestion, the red fluorescence was decreased or disappeared.</p><p><b>CONCLUSIONS</b>The dual immunofluorescence labeling methodology can be used to study the human dentin matrix without demineralization of the whole dentin fragments. Chondroitin sulfate was localized in the lumens of the dentin tubules and peritubular dentin, while the type-I collagen fibrils were localized in intertubular dentin and peritubular dentin.</p>