Expression Of Interleukin-10 In Human Dental Follicle Cells And Its Effects On Tooth Eruption

In dental practice, clinicians often face with the treatment of both simple and complex dental complications that arise from abnormal tooth eruption which manifests either as delayed eruption or eruption failure. Studies have reported that the incidence of tooth impaction due to eruption failure ranges from 5.6 to 18.8 percent of the population. Up to now, 25 syndromes have been discovered to be related with abnormal tooth eruption. Tooth eruption is a complex process that involves the timely action and interaction of cells of the dental organ, follicle, and alveolus. Alveolar bone resorption is required to form an eruption pathway for the teeth to exit their bony crypts. Dental follicle, a loose connective tissue sac surrounding the unerupted teeth, is also necessary for tooth eruption to occur. Studies have shown that teeth can not erupt without the dental follicles. The dental follicle regulates the cellular events of tooth eruption, including recruitment of mononuclear cells to the follicle and directing the subsequent osteoclastogenesis needed for alveolar bone resorption .There are many molecules regulating the tooth eruption process, including colony stimulating factor-1 (CSF-1), monocyte chemotactic protein-1 (MCP-1), parathyroid hormone-related protein (PTHrP), interleukin-1α(IL-1α), epidermal growth factor (EGF), transforming growth factor-β1 (TGF-β1), osteoprotegerin (OPG), receptor activator of nuclear factor-kappa B ligand (RANKL), etc. These molecules play important roles in the tooth eruption process. CSF-1 and MCP-1 act to attract the monocytes into the dental follicle, while OPG and RANKL are key factors for osteoclast formation. It has been reported that osteoclastogenesis during tooth eruption is mediated by RANKL-RANK/OPG pathway.Interleukin-10 (IL-10), initially described as cytokine synthesis inhibitory factor, is synthesized by T cells, macrophages, monocytes and B cells. IL-10 is expressed in human periodontal ligament cells and rat dental follicle cells as well. Previous studies have shown that IL-10 not only suppresses inflammatory response and immune reaction, but also inhibits osteoclastogenesis. IL-10 knockout mice have accelerated alveolar bone resorption. So we hoped to find out whether IL-10 was involved in the osteoclastogenesis during tooth eruption and if it was a fact, which molecules IL-10 did regulate in the process.This study was undertaken to determine the expression of IL-10 in human dental follicle cells (HDFCs) and the effects of IL-10 on tooth eruption through cellular and molecular biological methods. We found the expression of IL-10 in HDFCs for the first time, examined the effect of IL-10 on proliferation and activity of alkaline phosphatase (ALP) of HDFCs, and the influence of IL-10 on the expression of CSF-1, MCP-1, OPG and RANKL. The transduction pathways of IL-10-induced OPG and RANKL expression were also discussed. The present study was intended to elucidate the role of IL-10 in the tooth eruption process, providing the theoretical basis for the treatment and prevention of abnormities and diseases related with abnormal tooth eruption. Our study was as follows:Part 1. HDFC culture and identification Objective: To culture HDFCs in vitro and identify the cells. Methods: The third mandibular molars were surgically removed from adolescents for the need of orthodontic treatment with informed consent. Then, HDFCs were cultured from the isolated dental follicles. Immunocytochemistry was used to detect the expression of vimentin (VIM) and cytokeratin (CK) in HDFCs of the 5th passage. Results: HDFCs were immunostained positively for VIM and negatively for CK. Conclusions: The cultured HDFCs were originated from ectomesenchyme and had the morphology of fibroblasts.Part 2. The expression of IL-10 in HDFCsObjective: To study the expression of IL-10 in HDFCs. Methods: RT-PCR and immunocytochemistry were used to detect the mRNA and protein expression of IL-10 in HDFCs of the 5th passage, respectively. Results: IL-10 mRNA and protein were detected in HDFCs. Conclusions: IL-10 was expressed in HDFCs.Part 3. The effect of IL-10 on the biological characteristics of HDFCsObjective: To study the effect of IL-10 on proliferation and activity of ALP of HDFCs. Methods: HDFCs of the 5th passage were co-cultured with 0, 1, 10, 25, 50, 100ng/ml IL-10 or co-cultured with 10ng/mlIL-10 for 0d, 1d, 3d, 5d, 7d. The proliferation and the activity of ALP were assayed, respectively. Results: Different doses of IL-10 acting for the same time or 10ng/mlIL-10 acting for different time had no significant difference on the proliferation of HDFCs. 10,25,50,100ng/mlIL-10 acting for the same time or 10ng/mlIL-10 acting for 3-7d decreased the activity of ALP. Conclusions: IL-10 had no effect on the proliferation of HDFCs. However, IL-10 suppressed the activity of ALP of HDFCs, inhibiting their differentiation to osteoblasts.Part 4. The effect of IL-10 on the expression of osteoclastogenic factors in HDFCs Objective: To study the effect of IL-10 on expression of osteoclastogenic factors: CSF-1, MCP-1, OPG and RANKL in HDFCs. Methods: HDFCs of the 5th passage were co-cultured with 25ng/ml IL-10 for 0h, 1h, 3h, 6h, 9h, 12h. The mRNA of CSF-1, MCP-1, OPG and RANKL was examined in HDFCs by RT-PCR, respectively. Then, the cells were co-cultured with 25ng/ml IL-10 for 0h, 2h, 4h, 6h, 8h. The protein expression of CSF-1 and MCP-1 was examined by ELISA, respectively. The protein expression of OPG and RANKL was examined by Western Blot, respectively. Results: 25ng/ml IL-10 down-regulated the mRNA expression of CSF-1 in 3-12h and the protein expression of CSF-1 in 6-8h. 25ng/ml IL-10 down-regulated the mRNA expression of MCP-1 in 1-6h and the protein expression of MCP-1 in 4-8h. 25ng/ml IL-10 up-regulated the mRNA expression of OPG in 1-6h and the protein expression of OPG in 2-8h. 25ng/ml IL-10 down-regulated the mRNA expression of RANKL in 3-12h and the protein expression of RANKL in 4-8h. Conclusions: IL-10 inhibited the chemotactic function of CSF-1 and MCP-1 by down-regulating the expression of CSF-1 and MCP-1. IL-10 inhibited osteoclastogenesis by enhancement of OPG and suppression of RANKL. This inhibition of osteoclastic activity may be essential to keep the normal morphology of the alveolar bone in the physiological tooth eruption process.Part 5. The transduction pathways of IL-10-induced OPG and RANKL expressionObjective: To study the transduction pathways of IL-10-induced OPG and RANKL expression in HDFCs. Methods: HDFCs were treated with 25ng/ml IL-10 and/or 5μg/ml dbcAMP (PKA activator); 112nM KT5720 (PKA inhibitor); 50ng/ml PMA (PKC activator); 1μM Go6983 (PKC inhibitor). After 6h of stimulation, the cells were harvested for RNA extraction. OPG and RANKL mRNA expression was examined using RT-PCR. Results: dbcAMP and KT5720, did not change IL-10-induced OPG mRNA expression. However, IL-10-induced RANKL mRNA expression was increased by dbcAMP and decreased by KT5720. IL-10-induced OPG mRNA expression was enhanced by PMA and suppressed by Go6983. However, PMA and Go6983, had no effect on IL-10-induced RANKL mRNA expression. Conclusions: IL-10-induced OPG mRNA expression was dependent on PKC pathway, whereas IL-10-induced RANKL mRNA expression was dependent on PKA pathway.