The expressions of tooth eruption relevant genes are different in incisors and molars dental follicle cells in rat: an in vitro study

Background The incisors and molars showed different patterns of tooth eruption in rodents and the dental follicle cells play key roles in tooth eruption. Little is known about the differences in incisors and molars dental follicle cells during tooth eruption in rodents. The purpose of this study was to investigate the differences between incisor dental follicle cells and molar dental follicle cells during tooth eruption in rat. Methods Incisor dental follicle cells and molar dental follicle cells were obtained as previously described. Immunofluorescence was used to identify the cells. Gene expression was measured by real-time qPCR and western blot. Results Compared with molar dental follicle cells, the incisor dental follicle cells showed higher expression of OPG, BMP-2 and BMP-3. The molar dental follicle cells showed higher expression of MCP-1 and RANKL. Conclusions The expression patterns of genes related to tooth eruption were different in incisors and molars dental follicle cells in rat.

between incisor dental follicle cells and molar dental follicle cells during tooth eruption in rat.
Methods Incisor dental follicle cells and molar dental follicle cells were obtained as previously described. Immunofluorescence was used to identify the cells. Gene expression was measured by real-time qPCR and western blot.
Results Compared with molar dental follicle cells, the incisor dental follicle cells showed higher expression of OPG, BMP-2 and BMP-3. The molar dental follicle cells showed higher expression of MCP-1 and RANKL.
Conclusions The expression patterns of genes related to tooth eruption were different in incisors and molars dental follicle cells in rat.

Background
Many diseases can cause abnormal tooth eruption such as delayed tooth eruption (DTE) [1] and cleidocranial dysplasia (CCD) [2] . Understanding the mechanism of tooth eruption is very important for solving the problem of abnormal tooth eruption. Tooth eruption is a complex process, in which there are not only the histological changes of alveolar bone and tooth germ, but also cytologic changes of dental follicle cells, osteoblasts, osteoclasts and other cells, and a variety of cytokines are involved [3] .
The dental follicle cells play an important role in the process of tooth eruption [4] .
In the process of tooth eruption, the tooth dental follicle cells not only regulate bone resorption, but also regulate bone formation. The basal part of dental follicle showed bone formation [14] . Bone morphogenetic protein-2 (BMP-2) and bone morphogenetic protein-3 (BMP-3) can promote osteogenic differentiation of dental follicle cells [15] . BMP-2 can induce osteogenic differentiation of tooth dental follicle through NOTCH hedgehog signaling pathways [16,17] . In order to coordinate the opposite biological processes of tooth eruption, namely osteoclastogenesis and 5 osteogenesis, the expression of CSF-1, MCP-1, RANKL, OPG, BMP-2 and BMP-3 in dental follicle cells may be different in space-time.
The incisors and molars of rodent exhibit two different development patterns. The incisors can erupt throughout their life, but the molars will not erupt initiatively after the root have developed [18] . Therefore, the incisor dental follicle and molar dental follicle may play different roles in tooth eruption in rats. Exploring the difference of IF and MF cells during eruption can provide a theoretical basis for the occurrence of abnormal tooth eruption.

Cell culture and identification
Purified IF and MF cells of rats were obtained after twice differential trypsin digestion. IF and MF cells were characterized by a typical fibroblasts-like morphology of stellate or spindle shape ( Fig. 1 A, B). Both IF and MF cells were positive for the mesenchymal cell maker vimentin, but negative for the epithelial cell marker CK14 ( Fig. 1 C-F). These results indicated that the methods to isolate IF and MF cells are effective with a high purification.

IF and MF cells exhibited approximate proliferation ability
CCK-8 analysis showed that there was no significant difference in proliferation rates between IF and MF cells. Both IF and MF cells proliferation rates reached the peak at the 6 th day and the numbers of IF and MF cells started decreasing. (Fig. 2).

IF and MF cells produced different amount of the minerals.
Alkaline phosphatase staining showed different amount of the minerals between the IF and MF cells (Fig. 3). Numerous minerals were formed in IF group and connected together while minerals were relatively few in the MF group. IF cells presented 6 higher mineral formation than MF cells.
IF and MF cells showed differential expression patterns of eruption related genes.
There was a significantly different genes expression profile between IF and MF cells.
Compared with MF cells, IF cells showed higher expression of CSF-1, but lower expression of MCP-1, which were monocytes recruitment relevant genes. IF showed higher expression of OPG, BMP-2, BMP-3 which were osteogenesis relevant genes, while MF showed higher expression of RANKL which was osteoclastogenesis (Fig. 4).

Discussion
The dental follicle cells play key roles in the process of tooth eruption [4] . The incisors and molars showed two different patterns of eruption in rodents that have attracted many researches in which the dental follicle may play a crucial role.
Therefore, exploring the difference in the expression of genes related to tooth eruption in incisors and molars dental follicle in rats can further clarify the mechanism of tooth eruption and provide a possible theoretical basis for the occurrence of abnormal tooth eruption.
A variety of signaling molecules and transcription factors have been showed to be expressed in the dental follicle and participate in tooth eruption such as CSF-1, MCP-1, RANKL, OPG, BMP-1 and BMP3 [15,19] . However, the differences in their expression of incisors and molars dental follicle cells in rodents have not been studied. Since incisors and molars in rats showed different patterns of eruption, the expression of tooth eruption related genes in IF and MF cells will not be same. 7 Therefore, a hypothesis was proposed that the expression patterns of genes related to tooth eruption were different in incisors and molars dental follicle cells in rat.
MCP-1 and CSF-1 are effective chemokine of monocytes. Study has shown that MCP-1 gene is expressed in dental follicle. On the third day after the birth of rat, the expression of MCP-1 achieved the peak, which was consistent with the gathering of monocytes. The dental follicle recruits a large number of monocytes, which then differentiation into osteoclasts, is the key to tooth eruption [12] . Injection of CSF-1 can increase the numbers of monocytes and osteoclasts in the dental follicle of normal mice, thus accelerating tooth eruption [20] . Our results showed that the MF cells expressed higher MCP-1 and CSF-1 protein which indicated that MF cells had stronger ability to recruit monocytes. MF cells also expressed higher RANKL which enhanced bone resorption than IF cells. Together, these results revealed that MF cells had stronger osteoclastogenic ability. During tooth eruption, the dental follicle also express genes related to osteogenesis such as OPG, BMP-2, BMP-3. Our results showed that IF cells expressed higher OPG, BMP-2 and BMP-3. The results indicated that the IF cells had stronger osteogenic ability.
In this study, we used the third passages of IF and MF cells of rats. The results showed that the IF cells expressed higher OPG, BMP-2, and BMP-3 than MF cells,

Cell isolation, purification and identification
Incisor dental follicle cells IF molar dental follicle cells MF were isolated from incisor and molar germs of post-natal PN 7-day Sprauge-Dawley SD rats referring to a modified method as previously described [21] . Briefly, 5 post-natal 7-day old SD rats were euthanized by cervical dislocation under over-dose anesthesia (chloral hydrate, 10%, 0.5ml per rat). The mandibles were dissected and then the incisor and first molar germs were isolated with the aid of a stereomicroscope. The Incisor dental follicle was isolated from the incisor germ while the molar dental follicle was isolated from the molar germ. Then minced the tissues into pieces, and digested with a mixture of 625 U/mL type I collagenase (Sigma-Aldrich, USA) and 2.4U/mL Dispase II (Sigma-Aldrich, USA) at 37 °C for 0.5h. Using α-MEM supplemented 10% fetal bovine serum and 1% penicillin/streptomycin solution to terminate digestion.
After centrifugation at 1500 rpm for 5minutes, IF and MF cells were re-suspended 9 and cultured with α-MEM supplemented with 10 FBS (Gibco, USA), 1% penicillin/streptomycin solution (P/S; Solarbio, China). Cells were incubated at 37 °C in a humidified atmosphere with 5% CO2. The medium was changed every 2 days.
After the cells grow and fuse by 70%, a differential digestion method using trypsin/EDTA (Hyclone, USA) was performed to purify the targeting cells [22] . Purified

Consent for publication
Not applicable Availability of data materials All datasets used and analysed during the current study are available from the corresponding author on reasonable request.

Competing interests
There are no conflicts of interest to declare.   Figure 1 Purified IF and MF cells observed under phasecontrast microscope (PH) and identified by imm Differential expression patterns of tooth eruption related genes between IF and MF cells. Com NC3Rs ARRIVE Guidelines Checklist.docx