Transcriptome analysis of odontoblasts in primary and secondary dentinogenesis.
Authors: Simon S. , Smith A. J. , Lumley P. J. , Millard J. , Berdal A. , Cooper P. R. 
Published: August 2008

Aim  To determine whether odontoblasts demonstrate differential gene expression during primary and secondary dentinogenesis.Methodology  Coronal pulps were removed from immature developing and mature erupted bovine teeth extracted from 30‐month‐old animals. RNA from odontoblasts was selectively extracted [McLachlan et al (2003) Archives of Oral Biology48, 373–83]. Microarray analysis (Affymetrix) was used to compare the transcriptomes between the two odontoblast populations. One hundred nanograms of total RNA were initially double amplified to generate cDNA. Twenty‐five micrograms of the cRNA generated by in vitro transcription reaction was fragmented using 5× Fragmentation buffer and RNase‐free water at 94 °C for 35 min to generate 35–200 base fragments. Fifteen micrograms of fragmented cRNA was reconstitued in a hybridization cocktail, which was applied to each array and hybridized for 16 h at 45 °C. The two samples were analysed in duplicate using four GeneChip Bovine Genome arrays (900561). Bioinformatic analysis was performed using three software programmes (D‐chip, Onto‐Express and Pathway express). In parallel, the expression of 20 genes were compared between two populations of odontoblasts by RT‐PCR [Early stage (ES) and late stage (LS)] (ADM, AMEL, BMP4, Clock Genes, CollagenIII, CollagenI, DMPI, DSPP, MEPE, Msx1, Msx2, OSTEOCALCIN, PGAP I, TGFβ1, TGFβ1R, NESTIN, Alkaline Phosphatase, SHH, NaNog, LPR15).Results  Of the 24 000 genes analysed by microarray, 1338 were differently expressed in both populations by at least twofold (Paired analysis, P < 0.05). A total of 764 genes were up‐regulated and 574 down‐regulated in ES compared with LS odontoblasts. Amongst these genes, several known markers of odontoblasts (including Coll I, DMP1, Amelogenin, Osteoclacin) were found to be differentially expressed, as weel as several other genes not previously studied in odontoblasts. Several of these are components of intracellular signalling, including pathways involved in MAPKinase, apoptosis, TGF‐Beta, or FGF transduction. Ontological analysis revealed that 28.33% of the bovine genes identified by microarray analysis were assignable to biological processes, 26.79% as cellular components and 32.26% in molecular processes. RT‐PCR analysis confirmed the differential expression of some of the cited genes. For example, Amelogenin was only detected in ES, whereas osteocalcin was detected only in LS odontoblasts. Collagen‐I expression was not, however, differentially expressed, although Adrenomedullin and DMP1 were, up‐ and down‐regulated in LS, respectively. These results agreed with the microarray findings.Conclusion  The differential changes in gene expression in primary and secondary dentinogenesis indicate modifications in transcriptional control of the cells and highlight the need to identify the nature of these control mechanisms both to characterize cell phenotype and to better understand how cell secretory behaviour can be controlled during tertiary dentinogenesis.