Enzymatic isolation of viable human odontoblasts.

Abstract

To improve an enzymatic method previously used for isolation of rat odontoblasts to isolate viable mature human odontoblasts. Collagenase I, collagenase I/hyaluronidase mixture and hyaluronidase were used to extract mature human odontoblasts from the pulp chamber. Detachment of odontoblasts from dentine was determined with field emission scanning electron microscopy (FESEM) and to analyse the significance of differences in tubular diameter, and the t-test was used. MTT-reaction was used to analyse cell viability, and nonparametric Kruskal-Wallis and Mann-Whitney post hoc tests were used to analyse the data. Immunofluorescent staining of dentine sialoprotein (DSP), aquaporin-4 (AQP4) and matrix metalloproteinase-20 (MMP-20) and quantitative PCR (qPCR) of dentine sialophosphoprotein (DSPP) were used to confirm the odontoblastic nature of the cells. MTT-reaction and FESEM demonstrated collagenase I/hyaluronidase resulted in more effective detachment and higher viability than collagenase I alone. Hyaluronidase alone was not able to detach odontoblasts. Immunofluorescence revealed the typical odontoblastic-morphology with one process, and DSP, AQP4 and MMP-20 were detected. Quantitative PCR of DSPP confirmed that the isolated cells expressed this odontoblast-specific gene. The isolation of viable human odontoblasts was successful. The cells demonstrated morphology typical for odontoblasts and expressed characteristic odontoblast-type genes and proteins. This method will enable new approaches, such as apoptosis analysis, for studies using fully differentiated odontoblasts.