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We investigate whether the expression of the receptor activator of nuclear factor kappa-B ligand (RANKL) and osteoprotegerin (OPG) in human dental follicle cells (HDFCs) regulated by colony stimulating factor 1 (CSF-1), parathyroid hormone-related protein (PTHrP) and bone morphogenetic protein-2 (BMP-2) contributes to osteoclastogenesis. Adolescent human impacted third mandibular molars were used to separate HDFCs. These cells were incubated with PTHrP (10 ng/ml), CSF-1 (25 ng/ml), or BMP-2 (100 ng/ml) for 0.5, 1, 3, 6 and 12 h. The expression of OPG and RANKL was investigated by immunohistochemistry and reverse transcription polymerase chain reaction (RT-PCR). Two co-culture systems and tartrate-resistant acid phosphatase (TRAP) staining were used to examine osteoclast formation. Scanning electron microscopy was utilized for the resorption pit assay. RANKL and OPG were expressed innately in HDFCs. Exogenous PTHrP, CSF-1 and BMP-2 chronologically regulated the expression of RANKL and OPG in HDFCs. PTHrP and CSF-1 had similar regulative patterns leading to the up-regulated expression of RANKL and the down-regulated expression of OPG and opposite for BMP-2. The number of TRAP-positive peripheral blood mononuclear cells (PBMCs) slightly increased in contacted co-culture of HDFCs and PBMCs, whereas secreted OPG from HDFCs inhibited osteoclastogenesis in the transwell co-culture system. Contacted co-culture of HDFCs and PBMCs exhibited small and shallow resorption pits, whereas in the transwell co-culture system, secreted OPG from HDFCs reduced the resorption pits, reflecting the difference in osteoclast production. Collectively, we found a dual action of HDFCs in osteoclastogenesis; moreover, PTHrP, CSF-1 and BMP-2 might influence osteoclastogenesis by regulating the expression of RANKL and OPG in HDFCs. [ABSTRACT FROM AUTHOR]

Dental follicle cells (DFCs) can be artificially differentiated into mineralizing cells. With a dexamethasone-based differentiation protocol, transcription factors ZBTB16 and NR4A3 are highly upregulated but Runx2 and other osteogenic marker genes are not. Previous studies have suggested the involvement of a Runx2-independent differentiation pathway. The objective of this study is to further elucidate this mechanism. Differentiation of DFCs was examined by alkaline phosphatase (ALP) staining and ALP activity measurement, by Alizarin Red S staining and by real-time reverse transcription plus the polymerase chain reaction. ZBTB16 was overexpressed by using a transient transfection method. Resulting genome-wide gene expression changes were assessed by microarray. ZBTB16 and Runx2 were inhibited by short interfering RNA transfection. Promoter binding of ZBTB16 was evaluated by chromatin immunoprecipitation. Downregulation of Runx2 had no effect on dexamethasone-induced differentiation but was effective on BMP2-induced differentiation. Downregulation of ZBTB16, however, impaired dexamethasone-induced differentiation. Genes that were upregulated by dexamethasone induction were also upregulated by ZBTB16 overexpression. Genes that were not upregulated during dexamethasone-induced differentiation were also not regulated by ZBTB16 overexpression. ZBTB16 bound directly to the promoter regions of osterix and NR4A3 but not that of Runx2. Overexpression of ZBTB16 led to changes in the gene expression profile, whereby upregulated genes were overrepresented in osteogenesis-associated biological processes. Our findings suggest that, in DFCs, a Runx2-independent differentiation mechanism exists that is regulated by ZBTB16. [ABSTRACT FROM AUTHOR]

We investigated porcine dental follicle cells at the early crown-formation stage and examined the behavior of cells grown in a collagen type I (Col-I) matrix. Clone-porcine dental follicle cells (DFC-I) and controls, viz., dental follicle itself, nonclone-dental follicle cells, periodontal ligament cells (PDLC), and bone marrow stromal cells, were obtained from 6-month-old pigs. DFC-I showed a different gene expression pattern from controls by reverse-transcription polymerase chain reaction analysis. In addition, Col-I treatment enhanced DFC-I proliferation and increased their alkaline phosphatase activity compared with nontreated DFC-I. The expression of periostin, biglycan, and osteocalcin (OCN) in cells growing on collagen was upregulated, similar to the pattern seen in PDLC. DFC-I with and without Col-I treatment were combined with β-tricalcium phosphate particles and implanted into immunodeficient mice. Significant differences were found in the gene expression patterns of bone sialoprotein, OCN, and periostin in both treated and non-treated implants at 2 and/or 4 weeks. The results showed that Col-I induced the mineralization pathway in these cells. Hard tissue formation was observed in both implant types at 8 weeks. Our results suggest that Col-I facilitates the differentiation of DFC-I along the mineralization process. [ABSTRACT FROM AUTHOR]

The dental follicle (DF) surrounding the developing tooth germ is an ectomesenchymal tissue composed of various cell populations derived from the cranial neural crest. Human dental follicle cells (HDFC) are believed to contain precursor cells for cementoblasts, periodontal ligament cells, and osteoblasts. Bone morphogenetic proteins (BMPs) produced by Hertwig’s epithelial root sheath or present in enamel matrix derivatives (EMD) seem to be involved in the control of DF cell differentiation, but their precise function remains largely unknown. We report the immunolocalization of STRO-1 (a marker of multipotential mesenchymal progenitor cells) and BMP receptors (BMPR) in DF in vivo. In culture, HDFC co-express STRO-1/BMPR and exhibit multilineage properties. Incubation with rhBMP-2 and rhBMP-7 or EMD for 24 h increases the expression of BMP-2 and BMP-7 by HDFC. Long-term stimulation of these cells by rhBMP-2 and/or rhBMP-7 or EMD significantly increases alkaline phosphatase activity (AP) and mineralization. Expression of cementum attachment protein (CAP) and cementum protein-23 (CP-23), two putative cementoblast markers, has been detected in EMD-stimulated whole DF and in cultured HDFC stimulated with EMD or BMP-2 and BMP-7. RhNoggin, a BMP antagonist, abolishes AP activity, mineralization, and CAP/CP-23 expression in HDFC cultures and the expression of BMP-2 and BMP-7 induced by EMD. Phosphorylation of Smad-1 and MAPK is stimulated by EMD or rhBMP-2. However, rhNoggin blocks only Smad-1 phosphorylation under these conditions. Thus, EMD may activate HDFC toward the cementoblastic phenotype, an effect mainly (but not exclusively) involving both exogenous and endogenous BMP-dependent pathways. [ABSTRACT FROM AUTHOR]

The dental follicle is a mesenchymal tissue that surrounds the developing tooth germ. During tooth root formation, periodontal components, viz., cementum, periodontal ligament (PDL), and alveolar bone, are created by dental follicle progenitors. Here, we report the presence of PDL progenitors in mouse dental follicle (MDF) cells. MDF cells were obtained from mouse incisor tooth germs and immortalized by the expression of a mutant human papilloma virus type 16 E6 gene lacking the PDZ-domain-binding motif. MDF cells expressing the mutant E6 gene (MDF E6-EGFP cells) had an extended life span, beyond 150 population doublings (PD). In contrast, normal MDF cells failed to proliferate beyond 10 PD. MDF E6-EGFP cells expressed tendon/ligament phenotype-related genes such as Scleraxis ( Scx), growth and differentiation factor-5, EphA4, Six-1, and type I collagen. In addition, the expression of periostin was observed. To elucidate the differentiation capacity of MDF E6-EGFP cells in vivo, the cells were transplanted into severe combined immunodeficiency mice. At 4 weeks, MDF E6-EGFP cell transplants had the capacity to generate a PDL-like tissue that expressed periostin, Scx, and type XII collagen and the fibrillar assembly of type I collagen. Our findings suggest that MDF E6-EGFP cells can act as PDL progenitors, and that these cells may be a useful research tool for studying PDL formation and for developing regeneration therapies. [ABSTRACT FROM AUTHOR]

The c-fos gene is expressed in the dental follicle of the first mandibular molar of rats. Molecules that accelerate tooth eruption, epidermal growth factor (EGF) and colony-stimulating factor-1 (CSF-1), enhance the expression of the c-fos gene in vitro in both a time- and concentration-dependent manner. In vivo, EGF enhances c-fos expression in the follicle from day 0-7 postnatally, but by day 9 the follicle is refractory to this stimulus. Immunostaining reveals that the c-fos gene is translated in the cultured dental follicle cells, with staining seen in the nucleus and the perinuclear region of the cytoplasm. In vivo, immunostaining for c-fos is prominent in the dental follicle early postnatally, with little or no staining seen in the stellate reticulum and dental pulp. By day 10 postnatally, staining for c-fos is greatly reduced in the dental follicle. Thus, the presence of c-fos early postnatally in the tissue required for eruption, the dental follicle, as well as the enhancement of c-fos gene expression in the follicle by EGF or CSF-1, suggests that c-fos plays a role in tooth eruption. That role may be either to promote differentiation of mononuclear cells into osteoclasts needed for eruption or to recruit the mononuclear cells into the follicle. [ABSTRACT FROM AUTHOR]

Because the dental follicle is necessary for the eruption of teeth of limited eruption, it was the objective of this study to determine if the cells of the follicle could be cultured in vitro. To achieve this, dental follicles and associated enamel organs were dissected from the first and second mandibular molars of 6-7-day-old rats (secretory stage of amelogenesis), and then cultured in a medium that promotes fibroblast growth - the predominant cell type of the dental follicle. The cultured cells grew to confluency and were kept through 3 passages before experimentation. The cultured cells were fibroblastic in shape, elongate with processes, and transmission electron microscopy revealed that they contained an abundant rough endoplasmic reticulum, but did not form desmosomes. Immunofluorescent staining for anti-vimentin showed that all the cells stained and electron-microscopic immunogold labeling indicated that the antibody was associated with intermediate filaments. As revealed by SDS-polyacrylamide gel electrophoresis and Western blotting, the cultured cells synthesized and secreted the extracellular matrix molecules fibronectin and procollagens. Subsequent immunofluorescence staining of permeabilized and non-permeabilized cells confirmed the presence of fibronectin and type I collagen both intra- and extracellularly. Thus, based on all the above characteristics, the cultured cells appeared to be fibroblasts derived from the dental follicle, although a few of the fibroblasts may be derived from undifferentiated mesenchymal cells interposed between the alveolar bone and follicle. Experiments now can be conducted to determine how these cultured cells respond directly to growth factors that alter the rates of tooth eruption. [ABSTRACT FROM AUTHOR]