title: Enzymatic isolation of viable human odontoblasts.
abstract: OBJECTIVE
  To improve an enzymatic method previously used for isolation of rat odontoblasts to isolate viable mature human odontoblasts.
  
  
  METHODS
  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.
  
  
  RESULTS
  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.
  
  
  CONCLUSIONS
  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.
authors.affiliation: Institute of Dentistry, University of Oulu, Oulu, Finland.
authors.forename: H M
authors.initials: HM
authors.lastname: Cuffaro
authors.affiliation: Institute of Dentistry, University of Oulu, Oulu, Finland.
authors.forename: V
authors.initials: V
authors.lastname: Pääkkönen
authors.affiliation: Institute of Dentistry, University of Oulu, Oulu, Finland.|Medical Research Center Oulu, Oulu University Hospital and University of Oulu, Oulu, Finland.
authors.forename: L
authors.initials: L
authors.lastname: Tjäderhane
mesh_terms: D000818:Animals; D002454:Cell Differentiation; D003804:Dentin / Q000201:enzymology; D015870:Gene Expression; D006801:Humans; D009804:Odontoblasts / Q000201:enzymology*; D051381:Rats; D060888:Real-Time Polymerase Chain Reaction
keywords: collagenase; dentine; hyaluronic acid; hyaluronidase; odontoblast isolation