Abstract
We sought to investigate site‐specific expression of bone‐regulatory factors expressed by human dental follicles and to compare the stimulated expression of tumour necrosis factor (ligand) superfamily, member 11/tumour necrosis factor receptor superfamily, member 11b (RANKL/OPG) in human dental follicle cells (HDFCs) from different patients. Analysis of bone‐regulatory markers in follicles from 12 different study participants was performed using RT‐qPCR and immunofluorescence; apical and coronal segments from each dental follicle were processed independently. Four additional dental follicles were used for cell cultures; HDFCs were precultured in osteogenic medium to initiate differentiation and thereafter cultured with 10−6 M forskolin (FSK) to activate the protein kinase cAMP (PKA/cAMP) signalling pathway and induce RANKL/OPG expression. We demonstrate that RANKL expression is significantly higher in the coronal part of follicles than in the apical part. High levels of collagen type 1 (COL1), alkaline phosphatase (ALP) and Gap‐junction protein, alpha 1, 43 kDa (CX43) were expressed, whereas expression of Sp7 transcription factor (OSX), bone morphogenetic protein 2 (BMP2), colony‐stimulating factor 1 (CSF‐1), chemokine (C‐C motif) ligand 2 (MCP1), and OPG was low in all samples. The immunofluorescence localization of CSF‐1, MCP1, osteocalcin (OCN), RANKL, and BMP2 was not specific for either part of the follicles. In conclusion, a consistently high expression of CX43 suggests that gap‐junction communication in HDFCs is essential for the eruption process. Furthermore, the induced expression of RANKL in HDFCs varies significantly between individuals and may relate to clinical variations in tooth eruption.
Highlights
Expression of differentiation factors and signalling molecules essential for osteoclastogenesis and osteogenesis has been demonstrated in dental follicles obtained from rats and mice [1–9]
Examining the expression levels of runt-related transcription factor 2 (RUNX2), Sp7 transcription factor (OSX), CX43, bone morphogenetic protein 2 (BMP2), and alkaline phosphatase (ALP), which are osteoblast-associated transcription factors and differentiation markers, allowed us to assess the regulation of new bone formation and to determine whether the expression of these factors was higher in the apical part of the dental follicles (Fig. 4)
Significant differential spatial gene expression could only be demonstrated for RANKL in the coronal part of the dental follicle, compared with the corresponding apical part
Summary
Expression of differentiation factors and signalling molecules essential for osteoclastogenesis and osteogenesis has been demonstrated in dental follicles obtained from rats and mice [1–9]. Colony-stimulating factor 1 (CSF-1), chemokine (C-C motif) ligand 2 (MCP-1), tumour necrosis factor (ligand) superfamily, member 11 (RANKL), and tumour necrosis factor receptor superfamily, member 11b (OPG), which are key regulators of osteoclastogenesis, are differentially expressed in the coronal part of the dental follicle at specific stages during the eruption process [10–12]. Alveolar bone formation occurs in the basal region of the alveolar crypt. This may, in part, be stimulated by bone morphogenetic protein-2 (BMP-2) [13, 14]. The precise temporal and spatial patterns of gene expression that regulate site-specific osteoclast and osteoblast activities during tooth eruption have not been studied in human follicles. Isolation of the coronal and apical portions of the dental follicle was performed after removal of the tooth from the bone crypt. Specimens were stored at À80°C until used in the gene-expression and histology/immunofluorescence analyses
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