Osteogenic Differentiation Of Dental Follicle Cells Research Articles

Evaluation of Current Studies to Elucidate Processes in Dental Follicle Cells Driving Osteogenic Differentiation.

When research on osteogenic differentiation in dental follicle cells (DFCs) began, projects focused on bone morphogenetic protein (BMP) signaling. The BMP pathway induces the transcription factor DLX3, whichh in turn induces the BMP signaling pathway via a positive feedback mechanism. However, this BMP2/DLX3 signaling pathway only seems to support the early phase of osteogenic differentiation, since simultaneous induction of BMP2 or DLX3 does not further promote differentiation. Recent data showed that inhibition of classical protein kinase C (PKCs) supports the mineralization of DFCs and that osteogenic differentiation is sensitive to changes in signaling pathways, such as protein kinase B (PKB), also known as AKT. Small changes in the lipidome seem to confirm the participation of AKT and PKC in osteogenic differentiation. In addition, metabolic processes, such as fatty acid biosynthesis, oxidative phosphorylation, or glycolysis, are essential for the osteogenic differentiation of DFCs. This review article attempts not only to bring the various factors into a coherent picture of osteogenic differentiation in DFCs, but also to relate them to recent developments in other types of osteogenic progenitor cells.

Curcumin attenuates replicative senescence in human dental follicle cells and restores their osteogenic differentiation

ObjectiveThis study aimed to examine the therapeutic effects of curcumin against replicative senescence in dental follicle cells (DFCs). MethodsHuman DFCs were cultured in Dulbecco's Modified Eagle Medium with growth supplements. Replicative senescence in DFCs at different passages was assessed using β-galactosidase activity assay. Cell proliferation and size of DFCs at different passages were determined by CCK-8 kit and microscopy method, respectively. In addition, curcumin's effect on replicative senescence, cell proliferation, and size of DFCs at different passages was analyzed. Using western-blot analysis and siRNA-mediated gene silencing, we determined the molecular mechanisms involved in curcumin's effect against replicative senescence and osteogenic differentiation in DFCs at different passages. ResultsWe observed decreased proliferation and increased cell size and replicative senescence in cultured human DFCs at higher passages. Intriguingly, despite not showing any effect on cell size, curcumin (50 μM) significantly restored proliferation ability in DFCs and inhibited their replicative senescence. Concerning mechanisms, we found that curcumin inhibits replicative senescence in DFCs via down-regulation of senescence markers (P16 & P21) and restoration of proliferation markers (E2F1 & P53). Additionally, curcumin also rescued the osteogenic differentiation potential in higher-passage DFCs via restoration of osteogenic markers RUNX2 and OPN. ConclusionOur findings reveal for the first time that curcumin could act as a potential anti-senescence therapeutic for DFCs via regulation of proliferation, senescence, and osteogenic differentiation markers.

TRPM7 is Involved in the Regulation of Proliferation, Migration and Osteogenic Differentiation of Human Dental Follicle Cells.

Dental follicle cells (DFCs) are promising candidates for tissue engineering. However, the molecular mechanisms that regulate the biological characteristics of DFCs are still unclear. Transient receptor potential melastatin 7 (TRPM7) is a Ca2+- and Mg2+-permeable cation channel. The aim of this study was to determine the impact of TRPM7 on the proliferation, migration and osteogenic differentiation of DFCs. PCR, Western blotting, Immunocytochemical staining and Patch clamp methods were used to identify the gene and protein expression of TRPM7 in DFCs. DFCs were infected with lentiviruses that expressed either TRPM7 specific shRNA or scrambled non-effective shRNA to investigate its functional role. Cell proliferation and migration were assessed using Cell Counting Kit-8 assays and transwell cell culture chambers separately. Cell osteogenic differentiation were determined by ALP assay kit and Alizarin Red staining. Gene and protein expression of TRPM7 were detected in DFCs, but not of TRPM6, which is a closely related channel with similar function. In the absence of Mg2+, typical whole cell TRPM7-like currents were recorded by patch clamp. These were inhibited by low concentrations of 2-APB, but activated by high concentrations of 2-APB. Functional studies demonstrated that suppression of TRPM7 expression inhibited the proliferation and migration of DFCs, and promoted their osteogenic differentiation. Furthermore, Mg2+ deficiency mimicked the effects of TRPM7 knockdown in terms of osteogenic differentiation of DFCs. These results demonstrate that TRPM7 is involved in regulating the proliferation, migration and osteogenic differentiation of DFCs.

Energy Metabolism and Lipidome Are Highly Regulated during Osteogenic Differentiation of Dental Follicle Cells.

Dental follicle cells (DFCs) are stem/progenitor cells of the periodontium and give rise to alveolar osteoblasts. However, understanding of the molecular mechanisms of osteogenic differentiation, which is required for cell-based therapies, is delimited. This study is aimed at analyzing the energy metabolism during the osteogenic differentiation of DFCs. Human DFCs were cultured, and osteogenic differentiation was induced by either dexamethasone or bone morphogenetic protein 2 (BMP2). Previous microarray data were reanalyzed to examine pathways that are regulated after osteogenic induction. Expression and activity of metabolic markers were evaluated by western blot analysis and specific assays, relative amount of mitochondrial DNA was measured by real-time quantitative polymerase chain reaction, the oxidative state of cells was determined by a glutathione assay, and the lipidome of cells was analyzed via mass spectrometry (MS). Moreover, osteogenic markers were analyzed after the inhibition of fatty acid synthesis by 5-(tetradecyloxy)-2-furoic acid or C75. Pathway enrichment analysis of microarray data revealed that carbon metabolism was amongst the top regulated pathways after osteogenic induction in DFCs. Further analysis showed that enzymes involved in glycolysis, citric acid cycle, mitochondrial activity, and lipid metabolism are differentially expressed during differentiation, with most markers upregulated and more markedly after induction with dexamethasone compared to BMP2. Moreover, the cellular state was more oxidized, and mitochondrial DNA was distinctly upregulated during the second half of differentiation. Besides, MS of the lipidome revealed higher lipid concentrations after osteogenic induction, with a preference for species with lower numbers of C-atoms and double bonds, which indicates a de novo synthesis of lipids. Concordantly, inhibition of fatty acid synthesis impeded the osteogenic differentiation of DFCs. This study demonstrates that energy metabolism is highly regulated during osteogenic differentiation of DFCs including changes in the lipidome suggesting enhanced de novo synthesis of lipids, which are required for the differentiation process.

Mechanisms during Osteogenic Differentiation in Human Dental Follicle Cells.

Human dental follicle cells (DFCs) as periodontal progenitor cells are used for studies and research in regenerative medicine and not only in dentistry. Even if innovative regenerative therapies in medicine are often considered the main research area for dental stem cells, these cells are also very useful in basic research and here, for example, for the elucidation of molecular processes in the differentiation into mineralizing cells. This article summarizes the molecular mechanisms driving osteogenic differentiation of DFCs. The positive feedback loop of bone morphogenetic protein (BMP) 2 and homeobox protein DLX3 and a signaling pathway associated with protein kinase B (AKT) and protein kinase C (PKC) are presented and further insights related to other signaling pathways such as the WNT signaling pathway are explained. Subsequently, some works are presented that have investigated epigenetic modifications and non-coding ncRNAs and their connection with the osteogenic differentiation of DFCs. In addition, studies are presented that have shown the influence of extracellular matrix molecules or fundamental biological processes such as cellular senescence on osteogenic differentiation. The putative role of factors associated with inflammatory processes, such as interleukin 8, in osteogenic differentiation is also briefly discussed. This article summarizes the most important insights into the mechanisms of osteogenic differentiation in DFCs and is intended to be a small help in the direction of new research projects in this area.

Matrix vesicles from dental follicle cells improve alveolar bone regeneration via activation of the PLC/PKC/MAPK pathway

BackgroundThe regeneration of bone loss that occurs after periodontal diseases is a significant challenge in clinical dentistry. Extracellular vesicles (EVs)-based cell-free regenerative therapies represent a promising alternative for traditional treatments. Developmental biology suggests matrix vesicles (MVs), a subtype of EVs, contain mineralizing-related biomolecules and play an important role in osteogenesis. Thus, we explore the therapeutic benefits and expect to find an optimized strategy for MV application.MethodsHealthy human dental follicle cells (DFCs) were cultured with the osteogenic medium to generate MVs. Media MVs (MMVs) were isolated from culture supernatant, and collagenase-released MVs (CRMVs) were acquired from collagenase-digested cell suspension. We compared the biological features of the two MVs and investigated their induction of cell proliferation, migration, mineralization, and the modulation of osteogenic genes expression. Furthermore, we investigated the long-term regenerative capacity of MMVs and CRMVs in an alveolar bone defect rat model.ResultsWe found that both DFC-derived MMVs and CRMVs effectively improved the proliferation, migration, and osteogenic differentiation of DFCs. Notably, CRMVs showed better bone regeneration capabilities. Compared to MMVs, CRMVs-induced DFCs exhibited increased synthesis of osteogenic marker proteins including ALP, OCN, OPN, and MMP-2. In the treatment of murine alveolar bone defects, CRMV-loaded collagen scaffold brought more significant therapeutic outcomes with less unhealing areas and more mature bone tissues in comparison with MMVs and acquired the effects resembling DFCs-based treatment. Furthermore, the western blotting results demonstrated the activation of the PLC/PKC/MAPK pathway in CRMVs-induced DFCs, while this cascade was inhibited by MMVs.ConclusionsIn summary, our findings revealed a novel cell-free regenerative therapy for repairing alveolar bone defects by specific MV subtypes and suggest that PLC/PKC/MAPK pathways contribute to MVs-mediated alveolar bone regeneration.

Classical isoforms of protein kinase C (PKC) and Akt regulate the osteogenic differentiation of human dental follicle cells via both \u03b2-catenin and NF-\u03baB

BackgroundHuman dental follicle cells (DFCs) are the precursor cells of the periodontium with a high potential for regenerative therapies of (alveolar) bone. However, the molecular mechanisms of osteogenic differentiation are inadequately understood. Classical isoforms of protein kinase C (PKC) are reported to inhibit osteogenesis of stem/precursor cells. This study evaluated the role of classical PKCs and potential downstream targets on the osteogenic differentiation of DFCs.MethodsDFCs were osteogenic differentiated with dexamethasone or bone morphogenetic protein 2 (BMP2). Expression of PKC and potential upstream/downstream regulators was manipulated using activators, inhibitors, and small interfering ribonucleic acid (siRNA). Expression of proteins was examined by Western blot analysis, while the activation levels of enzymes and transcription factors were examined by their phosphorylation states or by specific activation assays. Expression levels of osteogenic markers were examined by RT-qPCR (reverse transcription-quantitative polymerase chain reaction) analysis. Activity of alkaline phosphatase (ALP) and accumulation of calcium nodules by Alizarin Red staining were measured as indicators of mineralization.ResultsClassical PKCs like PKCα inhibit the osteogenic differentiation of DFCs, but do not interfere with the induction of differentiation. Inhibition of classical PKCs by Gö6976 enhanced activity of Akt after osteogenic induction. Akt was also regulated during differentiation and especially disturbed BMP2-induced mineralization. The PKC/Akt axis was further shown to regulate the canonical Wnt signaling pathway and eventually nuclear expression of active β-catenin during dexamethasone-induced osteogenesis. Moreover, the nuclear factor “kappa-light-chain-enhancer” of activated B cells (NF-κB) pathway is regulated during osteogenic differentiation of DFCs and via the PKC/Akt axis and disturbs the mineralization. Upstream, parathyroid hormone-related protein (PTHrP) sustained the activity of PKC, while Wnt5a inhibited it.ConclusionsOur results demonstrate that classical PKCs like PKCα and Akt regulate the osteogenic differentiation of DFCs partly via both β-catenin and NF-κB.

MiRNA-101 supports the osteogenic differentiation in human dental follicle cells

ObjectiveHuman dental follicle cells (DFCs) are genuine precursor cells of cementoblasts and alveolar bone osteoblasts. MicroRNAs (miRNAs) represent a class of non-coding endogenous RNAs that silence gene expression post-transcriptionally. miRNA101 actively regulates the osteogenic differentiation of periodontal ligament cells. Therefore the aim of this study was to investigate the role of miRNA101 during the osteogenic differentiation in DFCs. Materials and methodsDFCs were isolated, cultivated and osteogenic differentiated in differentiation medium. Total RNA including miRNAs was isolated and the expression of miRNA101 was examined by real-time RT-PCRs. The expression of miRNA101 was induced by miRNA101-mimic transfection and the gene expression of osteogenic transcription factors was obtained by real-time RT-PCRs. Moreover the induction of the osteogenic differentiation was evaluated by the activity of alkaline phosphatase. ResultsmiRNA101 was regulated in DFCs during the osteogenic differentiation. After miRNA101-mimic transfection the alkaline phosphatase was increased and the gene expression of typical osteogenic transcription factors such as SP7 (osterix) was up-regulated. ConclusionOur results suggest that miRNA101 sustains the osteogenic differentiation of DFCs.

The induction of cellular senescence in dental follicle cells inhibits the osteogenic differentiation.

Dental stem cells such as human dental follicle cells (DFCs) have opened new promising treatment alternatives for today's dental health issues such as periodontal tissue regeneration. However, cellular senescence represents a restricting factor to cultured stem cells, resulting in limited lifespan and reduced cell differentiation potential. Therefore, this study evaluated if and how DFCs exhibit features of cellular senescence after being expanded in cell culture. The cell proliferation of DFCs decreased, while the cell size increased during prolonged cell culture. Moreover, DFCs expressed the senescence-associated β-galactosidase after a prolonged cell culture. The onset of senescence inhibited both the induction of osteoblast markers RUNX2 and osteopontin and the biomineralization of DFCs after stimulation of the osteogenic differentiation. In conclusion, we showed that a prolonged cell culture induces cellular senescence and inhibits the osteogenic differentiation in DFCs.

Osteogenic differentiation and gene expression profile of human dental follicle cells induced by human dental pulp cells.

Dental follicle cells (DFCs) differentiate into cementoblasts or osteoblasts under appropriate triggering. However, the mechanism(s) for osteogenic differentiation of DFCs are still unclear. The purpose of this study was to examine the effects of dental papilla-derived human dental pulp cells (hDPCs) on osteogenic differentiation of human DFCs (hDFCs) in vitro and in vivo and to compare gene expression in hDFCs in the presence or absence of hDPCs. To evaluate the osteogenic differentiation of hDFCs induced by hDPCs, hDFCs were cultured in osteogenic medium with or without hDPCs-conditioned medium (CM) in vitro and the cells transplanted into the subcutaneous tissue of immunodeficient mice in vivo. The hDPCs-CM enhanced alkaline phosphatase promoter activity of hDFCs in osteogenic culture. The expression of several osteoblast marker genes was increased in hDFCs treated with hDPCs-CM compared to hDFCs in normal medium. The hDFCs induced by hDPCs-CM also produced more calcified nodules than hDFCs in normal medium. In transplantation experiments, hDPCs-CM promoted the osteogenic induction and bone formation of hDFCs. Microarray analysis and quantitative real-time PCR showed that osteogenesis-related genes including WNT2, VCAN, OSR2, FOSB, and POSTN in hDFCs were significantly upregulated after induction by hDPCs-CM compared to hDFCs in normal medium. These findings indicate that hDPCs could increase the expression of osteogenic genes in hDFCs and stimulate their osteogenesis and could be a cellular resource for bone regeneration therapy when induced by hDPCs-derived factors.

Laminin regulates the osteogenic differentiation of dental follicle cells via integrin-α2/-β1 and the activation of the FAK/ERK signaling pathway.

Dental follicle cells (DFCs) are ideal for studies concerning the differentiation of dental precursor cells into alveolar osteoblasts and cementoblasts. Previous investigations have suggested that the extracellular matrix (ECM) protein laminin and the ECM receptor integrin-α2/-β1 play regulatory roles during the osteogenic differentiation of DFCs. Our present data indicate that laminin impairs alkaline phosphatase (ALP) activity following osteogenic induction while inducing integrin-α2/-β1 expression, osteogenic differentiation marker elevation, and DFC biomineralization. Integrin-α2/-β1 facilitates the laminin-dependent expression of osteogenic differentiation markers and the laminin-dependent inhibition of ALP activity. Moreover, these laminin-dependent effects on the osteogenic differentiation of DFCs can be reversed by the inhibition of the FAK/ERK signaling pathway. Thus, laminin regulates the inhibition of early osteogenic differentiation markers and the induction of late osteogenic differentiation markers via integrin-α2/-β1 and the activation of the FAK/ERK signaling pathway.

NOTCH1 signaling regulates the BMP2/DLX-3 directed osteogenic differentiation of dental follicle cells

Dental follicle cells (DFCs) are dental stem/progenitor cells and the genuine precursors of alveolar osteoblasts and dental cementoblasts. A previous study showed that the transcription factor DLX3 (distal less homeobox 3) supports the osteogenic differentiation in DFCs via a positive feedback loop with the bone morghogenetic protein (BMP) 2. Until today, however, the control of this BMP2/DLX3 pathway by additional signaling pathways remains elusive. Previous studies also suggested that the NOTCH signaling pathway plays a role in the osteogenic differentiation of DFCs. In this study we showed that DLX3 overexpression and the initiation of the osteogenic differentiation by BMP2 or dexamethasone induced the NOTCH signaling pathway in DFCs. However, the induction of NOTCH-signaling impaired not only the osteogenic differentiation (ALP activity and mineralized nodules) but also the expression of the transcription factor DLX3 and the activation of the BMP-signaling pathway. So, NOTCH signaling plays a regulatory role for the osteogenic differentiation of DFCs. In conclusion, results of our study suggest that the NOTCH-signaling pathway, which is activated during the osteogenic differentiation of DFCs, regulates the BMP2/DLX3 directed differentiation of DFCs via a negative feed-back loop.

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.

Soft matrix supports osteogenic differentiation of human dental follicle cells

The differentiation of stem cells can be directed by the grade of stiffness of the developed tissue cells. For example a rigid extracellular matrix supports the osteogenic differentiation in bone marrow derived mesenchymal stem cells (MSCs). However, less is known about the relation of extracellular matrix stiffness and cell differentiation of ectomesenchymal dental precursor cells. Our study examined for the first time the influence of the surface stiffness on the proliferation and osteogenic differentiation of human dental follicle cells (DFCs). Cell proliferation of DFCs was only slightly decreased on cell culture surfaces with a bone-like stiffness. The osteogenic differentiation in DFCs could only be initiated with a dexamethasone based differentiation medium after using varying stiffness. Here, the softest surface improved the induction of osteogenic differentiation in comparison to that with the highest stiffness. In conclusion, different to bone marrow derived MSCs, soft ECMs have a superior capacity to support the osteogenic differentiation of DFCs.

The Differentiation and Gene Expression Profile of Human Dental Follicle Cells

Human dental follicle cells (DFCs) are progenitor cells. Recent studies supposed that osteogenic differentiation of DFCs is controlled by growth factors such as BMP2 and IGF2, but their influence on the differentiation of DFCs has not been investigated in detail. We examined DFCs after the induction of osteogenic differentiation with BMP2, IGF2 and a standard osteogenic differentiation medium (ODM) with dexamethasone. The alkaline phosphatase (ALP) activity and the calcium accumulation demonstrated osteogenic differentiation after all treatments, but with the most effective differentiation by ODM. Interestingly, markers of the process of osteoblast differentiation were much higher up-regulated in BMP2- or IGF2-treated cells than in ODM-treated cells. To evaluate the reason of these differences, we compared genome-wide expression profiles at an early stage of differentiation. Chondroblast markers in BMP2-differentiated cells and general markers for cell differentiation/proliferation in IGF2-treated cells were significantly regulated. However, ODM-treated DFCs expressed late markers of osteogenic-differentiated DFCs such as the transcription factor ZBTB16 that is not expressed in BMP2- or IGF2-differentiated cells. Importantly, although the BMP-antagonist noggin (NOG) diminishes the phosphorylation of SMAD1 in DFCs, it did not inhibit osteogenic differentiation by ODM and the expression of ZBTB16. In conclusion, this study demonstrates that osteogenic differentiation of DFCs can be stimulated with all tested inducers but also independently of BMP signaling. To evaluate this mechanism, the transcription factor ZBTB16 is a target for further investigations.

Gene expression profiles of dental follicle cells before and after osteogenic differentiation in vitro

Recently, osteogenic precursor cells were isolated from human dental follicles, which differentiate into cementoblast- or osteoblast-like cells under in vitro conditions after the induction with dexamethasone or insulin. However, mechanisms for osteogenic differentiation are not understood in detail. In a previous study, real-time RT-PCR results demonstrated molecular mechanisms in dental follicle cells (DFCs) during osteogenic differentiation that are different from those in bone-marrow-derived mesenchymal stem cells. We analysed gene expression profiles in DFCs before and after osteogenic differentiation with the Affymetrix GeneChip(R) Human Gene 1.0 ST Array. Transcripts of 98 genes were up-regulated after differentiation. These genes could be clustered into subcategories such as cell differentiation, cell morphogenesis, and skeletal development. Osteoblast-specific transcription factors like osterix and runx2 were constitutively expressed in differentiated DFCs. In contrast, the transcription factor ZBTB16, which promotes the osteoblastic differentiation of mesenchymal stem cells as an up-stream regulator of runx2, was differentially expressed after differentiation. Transcription factors NR4A3, KLF9 and TSC22D3, involved in the regulation of cellular development, were up-regulated as well. In conclusion, we present the first transcriptome of human DFCs before and after osteogenic differentiation. This study sheds new light on the complex mechanism of osteogenic differentiation in DFCs.