Three Wistar rats (postnatal 15, 18, 21, 25 and 30 days old) were used for histological procedures. Experimental procedure involving the extraction of counterpart teeth has been explained previously [1]. In brief, the right lower first and second molars at P15 were extracted under anaesthesia by an intraperitoneal injection of chloral hydrate (400 mg/kg), with the extracted socket left untreated. The control contained normal molars that had not undergone an extraction. Materials were collected from the two groups at intervals of 10 (postnatal 25 days old: P25) and 15 (postnatal 30 days old: P30) days after extraction. Rats were perfused with physiological saline transcardially followed with 4% paraformaldehyde in a 0.1 M phosphate buffer (pH 7.4) under deep anaesthesia by an intraperitoneal injection of chloral hydratre (400 mg/kg). The maxillae was removed en bloc and immersed in the same fixative for 24 hours.

Following decalcification of the tissues with 10% EDTA at 4˚C, the specimens were embedded in paraffin. Cut at a thickness of 5 μm, sagittal sections were mounted on MAS-coated glass slides (Matsunami Glass Ind, Osaka, Japan). Deparaffined sections were stained with H-E. Using these sections, the numbers of inner and outer cells of HERS were calculated.

For microarray analysis, four upper first molar of experimental and control groups at P30 were used. All samples were extracted under anaesthesia by an intraperitoneal injection of chloral hydratre (400 mg/kg). Immediately after sampling, molars were dipped into 1000 μl of an RNA stabilization regent, RNA later^® (QIAGEN, Valentia, CA). The roots and pulp floors were resected with a razor blade and only root portions except for the coronal portion were homogenised by another razor blade. These samples were then stored at −20˚C until use. An RNeassy mini kit (QIAGEN, Valentia, CA) was used to isolate total RNA from the tissue samples according to the manufacturer’s instructions. The quality and quantity of total RNA samples were determined prior to the microarray experiments by the Agilent 2100 Bioanalyzer (Agilent Technologies, Palo Alto, CA). A quantity of more than 25 ng, and density of 13.5 ng/μl were used for further microarray analysis. Doublestranded cDNA was synthesised from high-quality RNA obtained by using the Low Input Quick Amp labeling kit/lit (Agilent technologies, Palo Alto, CA). 100 ng of total RNA derived from each pool was converted to double-stranded cDNA using the reverse transcriptase and a poly dT primer containing a T7 promoter (Agilent technologies, Palo Alto, CA). Cyamine dye-labelled cRNA was synthesised from cDNA and T7 RNA polymerase (Agilent technologies, Palo Alto, CA). Following fragmentation, 480 ng of cRNA was hybridised for 17 h at 65˚C on Sureprint G3 Rat Gene Expression 8 × 60 k (Agilent technologies). GeneChips were washed and stained in Gene Expression Hybridization kit (Agilent technologies). Fluorescence intensities for each chip were captured on a Scanner G2539A (Agilent technologies), and the data obtained were processed and analysed with the perfect match/mismatch algorithm using Scan Control, version A.8.5.1 (Agilent technologies).

Microarray data were initially processed using Feature Extraction (Agilent technologies). The MAS5.0 algorithm was used to normalise the signals to correct for non-specific hybridization, taking into account perfect match and mismatch probe pairs. Comparison analyses were then used to differentiate the real changes in gene expression levels between normal and experimental groups. Genes that revealed at least two-fold changes in their gene expression were selected, and the obtained data were filtered to ensure both statistical and biological significance. 59 Genes which indicated a statistically significant gap compared with the control group were selected by a paired t-test (P < 0.05).

The numbers of inner and outer cells in HERS were noted as the means ± standard deviation (SD). The significant difference was evaluated using Student’s t-test.

HERS of normal molars at 15 and 21 days old At P15, tooth cusps have not emerged into the oral cavity, but tooth root formation has commenced (Figure 1(a)). In this stage, cells from the inner enamel epithelium (IEE) and outer enamel epithelium (OEE) extended in an apical direction (Figure 1(b)). IEE cells were square, and OEE cells were rectangular. At P21, the tooth has already erupted into the oral cavity. In this stage, root development continues to an apical direction (Figures 1(c) and (d)). During a period of 6 days, cells from IEE and OEE decreased (Table 1).

Table 1. Comparison of number of HERS cells between normal and experimental groups.

Data are given as mean ± SD. n = 3. ^*P < 0.05 versus P21, ^**P < 0.05 versus normal group at P25. ^†P < 0.05 versus experimental group at P30.; Student’s t-test.

At P25, normal tooth roots continued to be formed. The morphological difference of both normal and experimental root dentin were not observed (Figures 2(a) and (c)). However, Sharpey’s fibers displayed an irregular orientation in experimental molar as in our previous report (Figure 2(c)). We compared the number of HERS cells among P21, normal group at P25 (N25) and experimental group at P25 (E25). The number of HERS cells in the normal group was significantly fewer than in the experimental group (Table 1). The cell number from inner and outer enamel epithelium in the normal group at P25 was significantly lower than that at P21, in contrast to an insignificant difference noted in the experimental

group (Table 1).

At P30, normal teeth completed root elongation, but incomplete apical root dentin formation was observed as in a previous report [19]. Sharpey’s fiber findings were identical to those at P25 in our previous report (Figures 3(a) and (c)) [1]. The number of HERS cells in normal groups (N30) was significantly fewer than that in experimental groups (E30) (Table 1), as was observed at P25.

Microarray was used to compare multiple gene expression profiles between normal and non-occlusal groups during root development. Genes that expressed more than two folds were selected. 59 genes displayed either upor down-regulation between normal and experimental molars. A total of 55 genes, including the especially prominent surfactant protein D, were highly expressed in the normal group. Table 2 shows the genes that were down-regulated in the experimental group compared to

the normal group at P30. In the same way, we also found that the expression of four genes was significantly increased in the experimental group (Table 3). Highlyexpressed genes in the experimental group included natriuretic peptide precursor C (Nppc), NAC alpha domain containing (Nacad), somatostatin (Sst), and nuclear receptor subfamily 1, group D, member 1 (Nr1d1), and transcript variant 2. The number of down-regulated genes exceeded that of up-regulated genes in this study.