Agarose-based spheroid culture enhanced stemness and promoted odontogenic differentiation potential of human dental follicle cells in vitro.

Human dental follicle cells (HDFCs) are an ideal cell source of stem cells for dental tissue repair and regeneration and they have great potential for regenerative medicine applications. However, the conventional monolayer culture usually reduces cell proliferation and differentiation potential due to the continuous passage during in vitro expansion. In this study, primary HDFC spheroids were generated on 1% agarose, and the HDFCs spontaneously formed cell spheroids in the agarose-coated dishes. Compared with monolayer culture, the spheroid-derived HDFCs exhibited increased proliferative ability for later passage HDFCs as analysed by Cell Counting Kit-8 (CCK-8). The transcription-quantitative polymerase chain reaction (qRT-PCR), western blot and immunofluorescence assay showed that the expression of stemness marker genes Sox2, Oct4 and Nanog was increased significantly in the HDFC spheroids. Furthermore, we found that the odontogenic differentiation capability of HDFCs was significantly improved by spheroid culture in the agarose-coated dishes. On the other hand, the osteogenic differentiation capability was weakened compared with monolayer culture. Our results suggest that spheroid formation of HDFCs in agarose-coated dishes partially restores the proliferative ability of HDFCs at later passages, enhances their stemness and improves odontogenic differentiation capability in vitro. Therefore, spheroid formation of HDFCs has great therapeutic potential for stem cell clinical therapy.

p53 inhibits the osteogenic differentiation but does not induce senescence in human dental follicle cells.

Replicative senescence causes a reduced osteogenic differentiation potential of senescent dental follicle cells (DFCs). The transcription factor p53 is often involved in the induction of cellular senescence, but little is known about its role in DFCs. This study examined for the first time the role of p53 compared to its pro-proliferative antagonist E2F-1 in terms of osteogenic differentiation potential and induction of senescence. Protein expression of E2F-1 decreased during cell aging, while p53 was expressed constitutively. Gene silencing of E2F1 (E2F-1) inhibited the proliferation rate of DFCs and increased the induction of cellular senescence. The induction of cellular senescence is regulated independently of the gene expression of TP53 (p53), since its gene expression depends on the expression of E2F1. Moreover, gene silencing of TP53 induced E2F1 gene expression and increased cell proliferation, but did not affect the rate of induction of cellular senescence. TP53 knockdown further induced the alkaline phosphatase and mineralization in DFCs. However, the simultaneous silencing of TP53 and E2F1 did not inhibit the inductive effect of TP53 knockdown on osteogenic differentiation, indicating that this effect is independent of E2F-1. In summary, our results suggest that p53 inhibits osteogenic differentiation and cell proliferation in senescent DFCs, but is not significantly involved in senescence induction.

Comparison of long non­coding RNA expression profiles in human dental follicle cells and human periodontal ligament cells.

The dental follicle develops into the periodontal ligament, cementum and alveolar bone. Human dental follicle cells (hDFCs) are the precursor cells of periodontal development. Long non­coding RNAs (lncRNAs) have been revealed to be crucial factors that regulate a variety of biological processes; however, whether lncRNAs serve a role in human periodontal development remains unknown. Therefore, the present study used microarrays to detect the differentially expressed lncRNAs and mRNAs between hDFCs and human periodontal ligament cells (hPDLCs). A total of 845 lncRNAs and 1,012 mRNAs were identified to be differentially expressed in hDFCs and hPDLCs (fold change >2.0 or <­2.0; P<0.05). Microarray data were validated by reverse transcription­quantitative polymerase chain reaction. Bioinformatics analyses, including gene ontology, pathway analysis and coding­non­coding gene co­expression network analysis, were performed to determine the functions of the differentially expressed lncRNAs and mRNAs. Bioinformatics analysis identified that a number of pathways may be associated with periodontal development, including the p53 and calcium signaling pathways. This analysis also revealed a number of lncRNAs, including NR_033932, T152410, ENST00000512129, ENST00000540293, uc021sxs.1 and ENST00000609146, which may serve important roles in the biological process of hDFCs. In addition, the lncRNA termed maternally expressed 3 (MEG3) was identified to be differentially expressed in hDFCs by reverse transcription­quantitative polymerase chain reaction. The knockdown of MEG3 was associated with a reduction of pluripotency makers in hDFCs. In conclusion, for the first time, to the best of our knowledge, the current study determined the different expression profiles of lncRNAs and mRNAs between hDFCs and hPDLCs. The observations made may provide a solid foundation for further research into the molecular mechanisms of lncRNAs in human periodontal development.

Numerical investigations of bone remodelling around the mouse mandibular molar primordia.

The formation of the alveolar bone, which houses the dental primordia, and later the roots of tooth, may serve as a model to approach general questions of alveolar bone formation. In this respect, this study aimed to investigate the potential interactions between the alveolar bone formation and tooth eruption by using finite element (FE) methods, and to figure out whether the expanding tooth systems induce shear stresses that lead to alveolar bone formation. 3D geometric surface models were generated from the 3D histological data of the heads of mice (C57 Bl/6J) ranging from stages embryonic (E) to postnatal (P) stages E15 to P20 using the reconstruction software 3-Matic. Bone, dentin, enamel and dental follicle around the primordia were generated and converted into 3D FE models. Models were imported into the FE software package MSC.Marc/Mentat. As material parameters of embryonic dentine, pulp, enamel, dental follicle, and bony structures basically are unknown, these were varied from 1% to 100% of the corresponding known material parameters for humans and a sensitivity analysis was performed. Surface loads were applied to the outside surface of dental follicle ranging from 0.1 to 5.0N/mm2. The validity of the model was analysed by comparing the activity pattern of the alveolar bone as determined in the histological study with the loading pattern from the numerical analysis. The results show that when varying the surface loads, the distribution of shear stresses remained same, and while varying the material properties of the hard tissues, the location of highest shear stresses remained stable. Comparison of the histologically determined growth regions with the distribution of shear stresses computed in the numerical model showed a very close agreement. The results provide a strong proof to support Blechschmidt's hypothesis that the bone in general is created under the influence of shear forces.

Parathyroid hormone-related peptide (1-34) promotes tooth eruption and inhibits osteogenesis of dental follicle cells during tooth development.

Dental follicle cells (DFCs) activate and recruit osteoclasts for tooth development and tooth eruption, whereas DFCs themselves differentiate into osteoblasts to form alveolar bone surrounding tooth roots through the interaction with Hertwig's epithelial root sheath (HERS). Also during tooth development, parathyroid hormone-related peptide (PTHrP) is expressed surrounding the tooth germ. Thus, we aimed to investigate the effect of PTHrP (1-34) on bone resorption and osteogenesis of DFCs in vitro and in vivo. In vitro studies demonstrated that DFCs cocultured with HERS cells expressed higher levels of BSP and OPN than the DFCs control group, whereas cocultured DFCs treated with PTHrP (1-34) had lower expressions of ALP, RUNX2, BSP, and OPN than the cocultured DFCs control group. Moreover, we found PTHrP (1-34) inhibited osteogenesis of cocultured DFCs by inactivating the Wnt/ß-catenin pathway. PTHrP (1-34) also increased the expression of RANKL/OPG ratio in DFCs. Consistently, in vivo study found that PTHrP (1-34) accelerated tooth eruption and inhibited alveolar bone formation. Therefore, these results suggest that PTHrP (1-34) accelerates tooth eruption and inhibits osteogenesis of DFCs by inactivating Wnt/ß-catenin pathway.

SCAPs Regulate Differentiation of DFSCs During Tooth Root Development in Swine.

The tooth root transmits and balances occlusal forces through the periodontium to the alveolar bone. The periodontium, including the gingiva, the periodontal ligament, the cementum and the partial alveolar bone, derives from the dental follicle (DF), except for the gingiva. In the early developmental stages, the DF surrounds the tooth germ as a sphere and functions to promote tooth eruption. However, the morphological dynamics and factors regulating the differentiation of the DF during root elongation remain largely unknown. Miniature pigs are regarded as a useful experimental animal for modeling in craniofacial research because they are similar to humans with respect to dentition and mandible anatomy. In the present study, we used the third deciduous incisor of miniature pig as the model to investigate the factors influencing DF differentiation during root development. We found that the DF was shaped like a crescent and was located between the root apical and the alveolar bone. The expression levels of WNT5a, ß-Catenin, and COL-I gradually increased from the center of the DF (beneath the apical foramen) to the lateral coronal corner, where the DF differentiates into the periodontium. To determine the potential regulatory role of the apical papilla on DF cell differentiation, we co-cultured dental follicle stem cells (DFSCs) with stem cells of the apical papilla (SCAPs). The osteogenesis and fibrogenesis abilities of DFSCs were inhibited when being co-cultured with SCAPs, suggesting that the fate of the DF can be regulated by signals from the apical papilla. The apical papilla may sustain the undifferentiated status of DFSCs before root development finishes. These data yield insight into the interaction between the root apex and surrounding DF tissues in root and periodontium development and shed light on the future study of root regeneration in large mammals.

The parathyroid hormone-related protein is secreted during the osteogenic differentiation of human dental follicle cells and inhibits the alkaline phosphatase activity and the expression of DLX3.

The dental follicle is involved in tooth eruption and it expresses a great amount of the parathyroid hormone-related protein (PTHrP). PTHrP as an extracellular protein is required for a multitude of different regulations of enchondral bone development and differentiation of bone precursor cells and of the development of craniofacial tissues. The dental follicle contains also precursor cells (DFCs) of the periodontium. Isolated DFCs differentiate into periodontal ligament cells, alveolar osteoblast and cementoblasts. However, the role of PTHrP during the human periodontal development remains elusive. Our study evaluated the influence of PTHrP on the osteogenic differentiation of DFCs under in vitro conditions for the first time. The PTHrP protein was highly secreted after 4days of the induction of the osteogenic differentiation of DFCs with dexamethasone (2160.5pg/ml±345.7SD. in osteogenic differentiation medium vs. 315.7pg/ml±156.2SD. in standard cell culture medium; Student's t Test: p<0.05 (n=3)). We showed that the supplementation of the osteogenic differentiation medium with PTHrP inhibited the alkaline phosphatase activity and the expression of the transcription factor DLX3, but the depletion of PTHrP did not support the differentiation of DFCs. Previous studies have shown that Indian Hedgehog (IHH) induces PTHrP and that PTHrP, in turn, inhibits IHH via a negative feedback loop. We showed that SUFU (Suppressor Of Fused Homolog) was not regulated during the osteogenic differentiation in DFCs. So, neither the hedgehog signaling pathway induced PTHrP nor PTHrP suppressed the hedgehog signaling pathway during the osteogenic differentiation in DFCs. In conclusion, our results suggest that PTHrP regulates independently of the hedgehog signaling pathway the osteogenic differentiated in DFCs.

Six1 is required for mouse dental follicle cell and human periodontal ligament-derived cell proliferation.

The periodontal ligament (PDL) is a connective tissue that attaches the tooth cementum to the alveolar bone and is derived from dental follicle cells (DFCs). The DFCs form fibroblasts, osteoblasts, cementoblasts, and PDL stem cells (PDLSCs). We previously reported homeobox transcription factor Six1 expression in mouse DFCs. However, the role of Six1 in periodontal tissue development is largely unknown. In this study, we analyzed SIX1 expression in mouse periodontal tissue cells during postnatal development and adulthood. We also addressed the role of SIX1 in mouse periodontium development and in human cultured PDL-derived cells (PDLCs). In mouse development, SIX1 production was abundant in DFCs and PDL cells by 2 weeks, but it was greatly diminished in the PDL at 4 weeks and in adults. Although the SIX1-positive cell distribution was sparse in the adult PDL, SIX1-positive cells were observed with low expression levels. We used 5-ethynyl-2'-deoxyuridine (EdU) for cell labeling to reveal numerous EdU/SIX1-double positive cells at 2 weeks; however, a few EdU-positive cells remained at 4 weeks. The proportion of DFCs that incorporated EdU was significantly lower in Six1-deficient mice compared with wild-type mice at E18.5. In human PDLCs, SIX1 was intensely expressed, and SIX1-knockdown using siRNA reduced proliferating PDLCs. Our results suggest that SIX1 is a key proliferation regulator in mouse DFCs and human PDLCs, which provides novel insight into Six family gene function in mammals.

Wnt5a regulates dental follicle stem/progenitor cells of the periodontium.

INTRODUCTION: Dental follicle gives rise to one or several tissues of the periodontium including the periodontal ligament, cementum and/or alveolar bone. Whether Wnt5a is expressed in the postnatal periodontium or regulates dental follicle stem/progenitor cells is unknown. METHODS: Dental follicle stem/progenitor cells were isolated from postnatal day 1 (p1) to p11 from rat mandibular first molars. Immunolocalization mapped Wnt5a expression in the alveolar bone, periodontal ligament, and the developing ameloblast and odontoblast layers. Mononucleated and adherent cells were isolated from p7 dental follicle. Wnt5a was overexpressed in dental follicle stem/progenitor cells to study their proliferation, osteogenic differentiation and migration behavior, with subpopulations of native dental follicle stem/progenitor cells as controls, using real-time PCR (Taqman), Lenti-viral transfection, Western blotting and immunofluorescence. RESULTS: Wnt5a was expressed consistently in p1 to p11 rat peridontium. Native, p7 dental follicle stem/progenitor cells had modest ability to mineralize in the tested 14 days. Even in chemically defined osteogenesis medium, dental follicle stem/progenitor cells only showed modest mineralization. Upon addition of 300 ng/mL Wnt5a protein in osteogenesis medium, dental follicle stem/progenitor cells displayed mineralization that was still unremarkable. Chemically induced or Wnt5a-induced mineralization of dental follicle cells only occurred sparsely. Combination of Wnt5a with 100 ng/mL BMP2 finally prompted dental follicle stem/progenitor cells to produce robust mineralization with elevated expression of Runx2, alkaline phosphatase, collagen 1α1 and osteocalcin. Thus, native dental follicle stem/progenitor cells or some of their fractions may be somewhat modest in mineralization. Strikingly, Wnt5a protein significantly augmented RANKL ligand, suggesting putative regulatory roles of dental follicle stem/progenitor cells for the monocyte/osteoclast lineage and potential involvement in alveolar bone remodeling and/or resorption. P-Jnk1/2 was activated in Wnt5a overexpressed dental follicle cells; conversely, exposure to SP600125, a c-Jun N-terminal kinase (JNK) inhibitor attenuated Runx2, collagen 1α1 and osteocalcin expression either in the presence or absence of Wnt5a. Wnt5a overexpression in dental follicle stem/progenitor cells significantly reduced their proliferation rates, but robustly augmented their migration capacity. CONCLUSIONS: These findings provide a glimpse of Wnt5a's putative roles in dental follicle stem/progenitor cells and the periodontium with implications in periodontal disease, tooth eruption, dental implant bone healing and orthodontic tooth movement.

Contribution of mesenchymal proliferation in tooth root morphogenesis.

In mouse tooth development, the roots of the first lower molar develop after crown formation to form 2 cylindrical roots by post-natal day 5. This study compared the morphogenesis and cellular events of the mesial-root-forming (MRF) and bifurcation-forming (BF) regions, located in the mesial and center of the first lower molar, to better define the developmental mechanisms involved in multi-rooted tooth formation. We found that the mesenchyme in the MRF showed relatively higher proliferation than the bifurcation region. This suggested that spatially regulated mesenchymal proliferation is required for creating cylindrical root structure. The mechanism may involve the mesenchyme forming a physical barrier to epithelial invagination of Hertwig's epithelial root sheath. To test these ideas, we cultured roots in the presence of pharmacological inhibitors of microtubule and actin polymerization, nocodazole and cytochalasin-D. Cytochalasin D also inhibits proliferation in epithelium and mesenchyme. Both drugs resulted in altered morphological changes in the tooth root structures. In particular, the nocodazole- and cytochalasin-D-treated specimens showed a loss of root diameter and formation of a single-root, respectively. Immunolocalization and three-dimensional reconstruction results confirmed these mesenchymal cellular events, with higher proliferation in MRF in multi-rooted tooth formation.

c-kit positive cells and networks in tooth germs of human midterm fetuses.

Numerous studies have attempted to characterize the dental pulp stem cells. However, studies performed on prenatal human tissues have not been performed to evaluate the in situ characterization and topography of progenitor cells. We aimed to perform such a study using of antibodies for CD117/c-kit and multiplex antibody for Ki67+ caspase 3. Antibodies were applied on samples dissected from five human midterm fetuses. Positive CD117/c-kit labeling was found in mesenchymal derived tissues, such as the dental follicle and the dental papilla. The epithelial tissues, that is, dental lamina, enamel organ and oral epithelia, also displayed isolated progenitor cells which were CD117/c-kit positive. Interestingly, CD117/c-kit positive cells of mesenchymal derived tissues extended multiple prolongations building networks; the most consistent of such networks were those of the dental follicle and the perivascular networks of the dental papilla. However, the mantle of the dental papilla was also positive for CD117/c-kit positive stromal networks. The CD117/c-kit cell populations building networks appeared mostly with a Ki67 negative phenotype. The results suggest that CD117/c-kit progenitor cells of the prenatal tooth germ tissues might be involved in intercellular signaling.

Bone morphogenetic protein-9 induces osteogenic differentiation of rat dental follicle stem cells in P38 and ERK1/2 MAPK dependent manner.

Dental follicle stem cells are a group of cells possessing osteogenic, adipogenetic and neurogenic differentiations, but the specific mechanism underlying the multilineage differentiation remains still unclear. Great attention has been paid to bone morphogenetic protein-9 (BMP-9) due to its potent osteogenic activity. In the present study, rat dental follicle stem cells were isolated and purified, and cells of passage 3 underwent adenovirus mediated BMP-9 gene transfection to prepare dental follicle stem cells with stable BMP-9 expression. Detection of alkaline phosphatase (ALP) and calcium deposition showed dental follicle stem cells transfected with BMP-9 gene could significantly promote the osteogenesis. In addition, SB203580 and PD98059 were employed to block the p38 mitogen-activated protein kinase (p38MAPK) and extracellular signal-regulated kinase (ERK1/2), respectively. Detection of ALP and calcium deposition revealed the BMP-9 induced osteogenic differentiation of dental follicle stem cells depended on MAPK signaling pathway.

In vitro and in vivo osteogenesis of human mesenchymal stem cells derived from skin, bone marrow and dental follicle tissues.

The present study evaluated the human mesenchymal stem cells (hMSCs) isolated from skin (hSMSC), bone marrow (hBMSC) and dental follicle (hDFMSC) tissues on their in vitro and in vivo osteogenic potential using demineralized bone matrix (DBM) and fibrin glue scaffold. Cells originated from three distinct tissues showed positive expressions of CD44, CD73, CD90, CD105 and vimentin, and differentiation ability into osteocytes, adipocytes and chondrocytes. hMSCs from all tissues co-cultured with a mixed DBM and fibrin glue scaffold in non-osteogenic induction media were positively stained by von Kossa and expressed osteoblast-related genes, such as osteocalcin (OC), osteonectin (ON), runt-related transcription factor 2 (Runx2) and osterix. For in vivo osteogenic evaluation, PKH26 labeled hMSCs were implanted into the subcutaneous spaces of athymic mice with a mixed scaffold. At 4 weeks of implantation, PKH26 labeled cells were detected in all hMSC-implanted groups. Bone formation with OC expression and radio-opacity intensity were observed around DBM scaffold in all hMSC-implanted groups. Interestingly, hDFMSCs-implanted group showed the highest OC expression and calcium content. These findings demonstrated that hDFMSCs could be a potential alternative autologous cell source for bone tissue engineering.

Immunohistochemical analysis of human homologue of drosophila patched (PTCH) in dental follicles of impacted third molars / Análisis imunohistoquímico del homólogo humano del gen parcheado de drosofila (PTCH) en folículos dentales de terceros molares retenidos

This study investigated the immunodetection of PTCH in epithelial components of dental follicles associated with impacted third molars without radiographic signs of pathosis. One hundred and five specimens of dental follicles associated with impacted third molars with incomplete rhizogenesis (between Nolla's stage 6 and 9) were surgically removed from 56 patients. Epithelial cell proliferation was determined by using immunohistochemical labeling. Statistical analysis was performed using Fisher exact test and a level of significance of 5 percent. Of the 105 dental follicles collected, 3 were PTCH-positive. The specimens with squamous metaplasia and epithelial hyperplasia had higher rates of positivity for PTCH, as well as those with active remnants of odontogenic epithelium. This study suggests that the odontogenic cells of the dental follicle might be proliferating during the rhizogenesis, while the squamous metaplasia and hyperplasia of the epithelial lining and proliferative odontogenic epithelial rests show the differentiation potential of dental follicles.

Se investigó la inmunodetección de PTCH en los componentes epiteliales de los folículos dentales asociados a terceros molares retenidos sin signos radiográficos y morfológicos de patología. Fueron quirúrgicamente extraídos de 56 pacientes 105 muestras de folículos dentales asociadas a terceros molares retenidos con rizogénesis incompleta (entre el estadio de Nolla 6 y 9). La proliferación de células epiteliales se deteminó mediante inmunohistoquímica. El análisis estadístico se realizó mediante la prueba exacta de Fisher. De los 105 folículos dentales recogidos, 3 fueron PTCH-positivos. Las muestras con metaplasia escamosa e hiperplasia epitelial tuvieron mayores tasas de positividad para PTCH, así como aquellos con los restos de proliferación del epitélio odontogénico. En conclusión, este estudio sugiere que las células odontogénicas del folículo dental podrían estar proliferando durante la rizogénesis, mientras que la metaplasia escamosa e hiperplasia del epitelio y de restos epiteliales odontogénicos en proliferación muestran el potencial de diferenciación de los folículos dentales.

Early postnatal development of the mandibular permanent first molar in infants with isolated cleft palate.

BACKGROUND: Based on measurements on dental casts, smaller permanent teeth in children with cleft palate have previously been reported in the literature; however, the early maturation of teeth and the size of the follicles and crowns have not been investigated. HYPOTHESIS: The maturation of the mandibular permanent first molar (M1(inf)) is delayed, and the mesiodistal diameters of the follicle and crown of M1(inf), respectively, are reduced in children with isolated cleft palate (ICP). DESIGN: Retrospective, longitudinal. Cephalometric X-rays were available for 2 and 22 months old children with clefts (64 children with ICP, and a control group of 38 children with unilateral incomplete cleft lip). The width of the follicle and the crown of M1(inf), and the maturation of M1(inf) were assessed. Intra-observer error was acceptable. RESULTS: M1(inf) maturation was delayed in children with ICP at both 2 and 22 months of age. The mesiodistal diameter of the crown of M1(inf) in the ICP group was reduced. Thus, the two hypotheses could not be refuted. CONCLUSIONS: Children with ICP showed smaller dimensions of the M1(inf) , and in addition, the maturation of M1(inf) was delayed.

Transcription factors TP53 and SP1 and the osteogenic differentiation of dental stem cells.

Dental follicle is a loose connective tissue that surrounds the developing tooth. Dental follicle cells (DFCs) have a promising potential for tissue engineering applications including periodontal and bone regeneration. However, little is known about the molecular mechanisms underlying osteogenic differentiation. In a previous study we detected that more than 35% of genes that are regulated during osteogenic differentiation of DFCs have promoter binding sites for the transcription factors TP53 and SP1. However, the role of these transcription factors in dental stem cells is still unknown. We hypothesize that both factors influence the processes of cell proliferation and differentiation in dental stem cells. Therefore, we transiently transfected DFCs and dental pulp stem cells (SHED; Stem cells from human exfoliated decidiuous teeth) with expression vectors for these transcription factors. After overexpression of SP1 and TP53, SP1 influenced cell proliferation and TP53 osteogenic differentiation in both dental cell types. The effects on cell proliferation and differentiation were less pronounced after siRNA mediated silencing of TP53 and SP1. This indicates that the effects we observed after TP53 and SP1 overexpression are indirect and subject of complex regulation. Interestingly, upregulated biological processes in DFCs after TP53-overexpression resemble the downregulated biological processes in SHED after SP1-overexpression. Here, regulated processes are involved in cell motility, wound healing and programmed cell death. In conclusion, our study demonstrates that SP1 and TP53 influence cell proliferation and differentiation and similar biological processes in both SHED and DFCs.

MT1-MMP expression in the odontogenic region of rat incisors undergoing interrupted eruption.

MT1-MMP (membrane type matrix metalloproteinase-1) has been considered an important membrane-type matrix metalloproteinase involved in the remodeling process in tissue and organ development, including the processes of the tooth and root growth and dental eruption. Therefore, the aims of this study were to evaluate MT1-MMP expression in the odontogenic region, as well as the eruption rate and morphology of the lower-left rat incisor, where the eruption process was interrupted for 14 days by a steel wire attached from the center of the incisor labial face and braced to the first molar. In the interrupted eruption group, the eruption rate was significantly reduced, producing drastic morphological alterations in the tooth germ and socket area. The MT1-MMP expression was widespread in the dental follicle, in both groups studied (normal and interrupted eruption groups); however a significant decrease in immunostaining was observed in the interrupted eruption group. Results indicate that MT1-MMP may have an important role in the process of dental eruption.

Multilineage differentiation of dental follicle cells and the roles of Runx2 over-expression in enhancing osteoblast/cementoblast-related gene expression in dental follicle cells.

OBJECTIVES: Dental follicle cells (DFCs) provide the origin of periodontal tissues, and Runx2 is essential for bone formation and tooth development. In this study, pluripotency of DFCs was evaluated and effects of Runx2 on them were investigated. MATERIALS AND METHODS: The DFCs were induced to differentiate towards osteoblasts, adipocytes or chondrocytes, and alizarin red staining, oil red O staining or alcian blue staining was performed to reveal the differentiated states. Bone marrow stromal cells (BMSCs) and primary mouse fibroblasts served as controls. DFCs were also infected with recombinant retroviruses encoding either full-length Runx2 or mutant Runx2 without the VWRPY motif. Western blot analysis, real-time real time RT-PCR and in vitro mineralization assay were performed to evaluate the effects of full-length Runx2 or mutant Runx2 on osteogenic/cementogenic differentiation of the cells. RESULTS: The above-mentioned staining methods demonstrated that DFCs were successfully induced to differentiate towards osteoblasts, adipocytes or chondrocytes respectively, confirming the existence of pluripotent mesenchymal stem cells in dental follicle tissues. However, staining intensity in DFC cultures was weaker than in BMSC cultures. Real-time PCR analysis indicated that mutant Runx2 induced a more pronounced increase in expression levels of OC, OPN, Col I and CP23 than full-length Runx2. Mineralization assay also showed that mutant Runx2 increased mineralization nodule formation more prominently than full-length Runx2. CONCLUSIONS: Multipotent DFCs can be induced to differentiate towards osteoblasts, adipocytes or chondrocytes in vitro. Runx2 over-expression up-regulated expression levels of osteoblast/cementoblast-related genes and in vitro enhanced osteogenic differentiation of DFCs. In addition, mutant Runx2-induced changes in DFCs were more prominent than those induced by full-length Runx2.

TNF-alpha upregulates expression of BMP-2 and BMP-3 genes in the rat dental follicle--implications for tooth eruption.

The dental follicle appears to regulate both the alveolar bone resorption and bone formation needed for tooth eruption. Tumor necrosis factor-alpha (TNF-alpha) gene expression is maximally upregulated at postnatal day 9 in the rat dental follicle of the first mandibular molar, a time that correlates with rapid bone growth at the base of the tooth crypt, as well as a minor burst of osteoclastogenesis. TNF-alpha expression is correlated with the expression of bone morphogenetic protein-2 (BMP-2), a molecule expressed in the dental follicle that can promote bone formation. Because BMP-2 signaling may be augmented by bone morphogenetic protein-3 (BMP-3), our objective in this study was to determine if the dental follicle expresses BMP-3 and if TNF-alpha stimulates the dental follicle cells to express BMP-2 and BMP-3. Dental follicles were collected from different postnatal ages of rat pups. Dental follicle cells were incubated with TNF-alpha to study its dosage and time-course effects on gene expression of BMP-2 and BMP-3, as determined by real-time RT-PCR. Next, immunostaining was conducted to confirm if the protein was synthesized and ELISA of the conditioned medium was conducted to determine if BMP-2 was secreted. We found that BMP-3 expression is correlated with the expression of TNF-alpha in the dental follicle and TNF-alpha significantly increased BMP-2 and BMP-3 expression in vitro. Immunostaining and ELISA showed that BMP-2 and BMP-3 were synthesized and secreted. This study suggests that TNF-alpha can upregulate the expression of bone formation genes that may be needed for tooth eruption.

Novel Golgi protein, GoPro49, is a specific dental follicle marker.

UNLABELLED: GoPro49 is a recently identified, novel Golgi protein that is expressed in embryonic mesenchymal tissues, including dental follicle. In the present study, we have tested the hypothesis that the gene is a specific marker for the dental follicle, and examined its expression during the development of mouse incisors and molars. In situ hybridization showed that GoPro49 is expressed in dental follicles from bud to post-eruption stages. The expression is intense throughout the dental follicle during crown development, and persists in the root follicle during root development. In the forming periodontal ligament, GoPro49 expression is down-regulated upon differentiation of the follicle cells to cementoblasts and osteoblasts marked by Bsp1. In cultured dental follicle cells, the GoPro49 protein co-localizes with beta-COP, suggesting that GoPro49 may function in the secretory pathway. We conclude that GoPro49 is a novel, specific marker for the dental follicle and can be used to identify this tissue. ABBREVIATIONS: Bsp1, bone sialoprotein 1; GoPro49, Golgi protein 49 kDa; E16, embryonic day 16; HERS, Hertwig's epithelial root sheath; PDL, periodontal ligament; dpn, day post-natal.