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.

Analysis of the phosphoproteome in human dental follicle cells during osteogenic differentiation.

Dental follicle cells (DFCs) are osteogenic progenitor cells and are well suited for molecular studies of differentiation of alveolar osteoblasts. A recent study examined the metabolism in DFCs during osteogenic differentiation and showed that energy metabolism is increased after 14 days of differentiation (mid phase). However, previous studies have examined proteomes at early (2 h, 24 h) or very late (28 days) stages of differentiation, but not during the phase of increased metabolic activity. In this study, we examined the phosphoproteome at the mid phase (14 days) of osteogenic differentiation. Analysis of DFC phosphoproteomes showed that during this phase of osteogenic differentiation, proteins that are part of signal transduction are significantly regulated. Proteins involved in the regulation of the cytoskeleton and apoptosis were also increased in expression. As osteogenic differentiation induced oxidative stress and apoptosis in DFCs, the oxidative stress defense protein, catalase, was also upregulated during osteogenic differentiation, which supports the biomineralization of DFCs. In summary, this study revealed that during the middle phase (14 days) of osteogenic differentiation, processes in DFCs related to the control of cell organization, apoptosis, and oxidative stress are regulated.

Dental stem cells for tooth regeneration: how far have we come and where next?

INTRODUCTION: Human dental stem cells are promising for tooth repair because of their differentiation potential. In 2018, this journal published a report on dental stem cell treatment options that had been attempted since the early 2000s. Although it is very difficult to follow every trend since then, new achievements have been made in the last 5 years. This review summarizes selected developments in dental stem cell research. AREAS COVERED: This article provides an overview of new developments with human dental stem cells and parts of these cells like extracellular vesicles for regenerative medicine. Preclinical research, clinical trials, and other works in the field of dental stem cells research for whole tooth engineering, dental pulp regeneration, periodontitis, and tooth root regeneration are summarized. In addition, works with dental stem cells for the regeneration of diseases that cannot be cured with the regeneration of dental tissues, such as diabetes, will be presented. EXPERT OPINION: Over the past five years, a number of studies using dental stem cells have improved new strategies for tooth repair. In addition, there are new dental stem cell products such as extracellular vesicles which, in combination with findings from basic research, will lead to new treatment options in the future.

DNA protein kinase promotes cellular senescence in dental follicle cells.

OBJECTIVE: Short telomeres and genomic DNA damage are causes of cellular senescence in dental follicle cells (DFCs). DESIGN: This study examined the role of the DNA damage response (DDR) during cellular senescence of DFCs by ß-galactosidase activity and DNA damage by comet assay. Expression of genes/proteins was determined by Western Blots and reverse transcription-quantitative polymerase chain reaction, while glycolysis was enzymatically estimated. Cell cycle stages and reactive oxygen species (ROS) were investigated by flow cytometry. RESULTS: During the induction of cellular senescence gene expression of DDR genes were down-regulated, while DNA double-strand breaks occurred at the same time. Furthermore, inhibition of DNA protein kinase (DNA-PK) reduced senescence and ROS, both of which are associated with cellular senescence. In contrast, while these data suggest that inhibition of DDR is associated with the induction of cellular senescence, inhibition of DNA-PK did not result in renewal of DFCs, as inhibition resulted in typical features of depleted cells such as increased cell size and reduced cell proliferation rate. DNA-PK repression inhibited both osteogenic differentiation potential and glycolysis, which are typical features of cellular exhaustion. Moreover, DNA-PK affects cellular senescence via activation of AKT1 (protein kinase B). CONCLUSION: Our results suggest that DNA-PK promotes cellular senescence, but DFCs may control the induction of cellular senescence via down-regulation of DDR genes. However, we also showed that inhibition of DNA-PK cannot renew senescent DFCs.

Changes in AMPK activity induces cellular senescence in human dental follicle cells.

Dental Follicle Cells (DFCs) are somatic stem cells with a limited lifespan, but little is known about a possible mechanism of cellular senescence. Previous studies have shown that cellular senescence is associated with increased demand of glycolsis or the "glycolytic metabotype", which can be induced by activation of 5' adenosine monophosphate-activated protein kinase (AMPK), and decreased autophagy. This study examined the role of AMPK in inducing senescence in DFCs. During the induction of cellular senescence, AMPK activity was impaired, suggesting a negative impact on senescence induction. In line with this assumption, cellular senescence was induced upon inhibition of AMPK with a specific siRNA. In addition, after this inhibition, autophagy was also inhibited. Moreover, specific inhibition of autophagy promoted cellular senescence. However, inducers of AMPK such as metformin or AICAR surprisingly increased senescence in DFCs. Interestingly, autophagy was impaired after long-term induction of AMPK with AICAR and metformin. Moreover, activation of AMPK induces the consumption of glucose but decreases NAD/NADH ratio in DFCs that suggest not only "glycolytic metabotype" of DFCs but also Mitochondrial Dysfunction Associated Senescence (MiDAS). Both changes are highly associated with the induction of cellular senescence. Hence, both AMPK activation and inhibition promote the induction of cellular senecence of DFCs.

Peripheral Nerve Regeneration-Adipose-Tissue-Derived Stem Cells Differentiated by a Three-Step Protocol Promote Neurite Elongation via NGF Secretion.

The lack of supportive Schwann cells in segmental nerve lesions seems to be one cornerstone for the problem of insufficient nerve regeneration. Lately, adipose-tissue-derived stem cells (ASCs) differentiated towards SC (Schwann cell)-like cells seem to fulfill some of the needs for ameliorated nerve recovery. In this study, three differentiation protocols were investigated for their ability to differentiate ASCs from rats into specialized SC phenotypes. The differentiated ASCs (dASCs) were compared for their expressions of neurotrophins (NGF, GDNF, BDNF), myelin markers (MBP, P0), as well as glial-marker proteins (S100, GFAP) by RT-PCR, ELISA, and Western blot. Additionally, the influence of the medium conditioned by dASCs on a neuron-like cell line was evaluated. The dASCs were highly diverse in their expression profiles. One protocol yielded relatively high expression rates of neurotrophins, whereas another protocol induced myelin-marker expression. These results were reproducible when the ASCs were differentiated on surfaces potentially used for nerve guidance conduits. The NGF secretion affected the neurite outgrowth significantly. It remains uncertain what features of these SC-like cells contribute the most to adequate functional recovery during the different phases of nerve recovery. Nevertheless, therapeutic applications should consider these diverse phenotypes as a potential approach for stem-cell-based nerve-injury treatment.

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.

Protein kinase A is activated during bone morphogenetic protein 2-induced osteogenic differentiation of dental follicle stem cells via endogenous parathyroid hormone-related protein.

OBJECTIVE: The aim of this study was to investigate the mechanisms of how protein kinase A (PKA) is activated during bone morphogenetic protein 2 (BMP2)-induced osteogenic differentiation in dental follicle stem cells. DESIGN: Human dental follicle stem cells were cultured and treated with a BMP2-containing osteogenic differentiation medium or differentiation medium without BMP2. Specific siRNAs and substances/proteins were used to modulate pathways. PKA activity and activity of alkaline phosphatase were determined. Expression of targets was measured by Western Blots and reverse transcription-quantitative polymerase chain reaction, while protein interactions were investigated by immunoprecipitation. Immunofluorescence staining was used for subcellular target localization. RESULTS: PKA activity is stimulated after osteogenic induction by BMP2. Differentiation medium without BMP2 strongly induces BMP2 gene expression, which correlates with downstream target expression. Elevation of cAMP levels does not affect alkaline phosphatase activity and PKA does not directly interact with Smad 4. However, PKA activation requires expression of parathyroid hormone-related protein (PTHrP), which is stimulated after BMP2-induced differentiation. Furthermore, neither supplementation with PTHrP nor with the receptor antagonist parathyroid hormone (7-34) affects PKA activity. Thus, endogenous PTHrP expression is required for PKA activation and immunofluorescence staining shows that PTHrP is mainly located in the nucleus of dental follicle stem cells. Beyond, knockdown of PKA stimulates the BMP2 signaling pathway and down-stream expression of PTHrP. CONCLUSIONS: BMP2-induced osteogenic differentiation activates PKA in dental follicle stem cells via endogenous expression of PTHrP. Additionally, PKA inhibits BMP2 signaling and expression of PTHrP in a negative feedback loop.

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

BACKGROUND: Human 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. METHODS: DFCs 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. RESULTS: Classical 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 ß-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. CONCLUSIONS: Our results demonstrate that classical PKCs like PKCα and Akt regulate the osteogenic differentiation of DFCs partly via both ß-catenin and NF-κB.

Effects of Cellular Senescence on Dental Follicle Cells.

The dental follicle is part of the tooth germ, and isolated stem cells from this tissue (dental follicle cells; DFCs) are considered, for example, for regenerative medicine and immunotherapies. However somatic stem cells can also improve pharmaceutical research. Cell proliferation is limited by the induction of senescence, which, while reducing the therapeutic potential of DFCs for cell therapy, can also be used to study aging processes at the cellular level that can be used to test anti-aging pharmaceuticals. Unfortunately, very little is known about cellular senescence in DFCs. This review presents current knowledge about cellular senescence in DFCs.

AMP-activated protein kinase and the down-stream activated process of autophagy regulate the osteogenic differentiation of human dental follicle cells.

OBJECTIVE: Dental follicle cells (DFCs) are progenitors of alveolar osteoblasts. AMP-activated protein kinase (AMPK) and the down-stream activated autophagy process play a key role in cellular energy and metabolic homeostasis and are involved in many biological processes including differentiation. Previous studies showed ambiguous results about the role of AMPK and autophagy in osteogenic differentiation of various osteogenic progenitors, but the role of AMPK and autophagy in DFCs is unknown. This study examined the role of AMPK and autophagy in the osteogenic differentiation of DFCs. MATERIALS AND METHODS: We evaluated the expression of AMPK isoforms and autophagy markers during osteogenic differentiation via Western Blot analyses and the impact of AMPK / autophagy activators and inhibitors and siRNAs on osteogenic differentiation via ALP activity assay, Alizarin Red staining and Real-Time Reverse-Transcription PCR. RESULTS: We have shown that expression of AMPK and autophagy markers are regulated during osteogenic differentiation and that activation of AMPK inhibits the ALP activity and other osteogenic markers after induction of osteogenic differentiation, while inhibition of AMPK and autophagy increased the expression of some osteogenic markers. In long-term cultures with osteogenic differentiation medium, however, both the activation and the inhibition of AMPK significantly inhibited biomineralization of DFCs. In contrast, activation or inhibition of autophagy barely affected early differentiation markers, while autophagy inhibition enhanced biomineralization and autophagy activation diminished mineralization capability of DFCs. CONCLUSIONS: AMPK regulates the osteogenic differentiation in earlier stages while indirectly affecting biomineralization at least partly via autophagy. The osteogenic differentiation of DFCs is sensitive to changes in AMPK and autophagic activity.

High endogenous expression of parathyroid hormone-related protein (PTHrP) supports osteogenic differentiation in human dental follicle cells.

Dental follicle cells (DFCs) are progenitor cells for mineralizing cells such as alveolar osteoblasts, but little is known about the mechanisms of the differentiation. Interestingly, different cell lines sometimes have different potentials to differentiate into mineralizing cells. In this study, we compared two different DFC lines, with one cell line (DFC_B) showing a high alkaline phosphatase (ALP) activity in long-term cultures with standard medium and a reliable mineralizing potential. However, the other cell line DFC_A shows low ALP activity in standard medium and almost no mineralization. Known osteogenic markers such as RUNX2 were similarly expressed in both cell lines. However, the proosteogenic signaling pathway of the bone morphogenetic protein (BMP) is induced in DFC_B, and the parathyroid hormone-related protein (PTHrP), which is involved in tooth root development, was also expressed more strongly. Previous studies have shown that the secreted PTHrP negatively regulate the transition from pre-osteoblastic progenitors to osteoblasts, but we showed that an inhibition of PTHrP gene expression reduced the ALP activity and the BMP-signaling pathway. In addition, endogenously expressed PTHrP is located in the cell nucleus. In contrast, supplementation of PTHrP or an inhibitor for the PTHrP receptor did not affect the ALP activity of DFC_B. In conclusion, our data suggest that a high endogenous expression of PTHrP in DFCs supports the induction of osteogenic differentiation via an intracrine mode.

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.

Short telomeres correlate with a strong induction of cellular senescence in human dental follicle cells.

BACKGROUND: Dental follicle cells (DFCs) are dental stem cells and interesting options for regenerative therapies in dentistry. However, DFCs acquire replicative senescence in long-term cultures, but little is known about molecular processes. In previous studies, we observed that DFC cell lines become senescent at different rates. We hypothesized that short telomere length and increased DNA damage with genomic instability correlate with the accelerated induction of cellular senescence. RESULTS: For this study we compared DFC cell lines that became senescent at different rates (DFC_F: strong senescent phenotype; DFC_S: weak senescent phenotype). The telomeres of DFC_F were shorter than those of the telomeres of DFC_S prior senescence. Interestingly, telomere lengths of both cell lines were nearly unchanged after induction of senescence. Gene expression analyses with genes associated with DNA damage before and after the induction of cellular senescence revealed that almost all genes in DFCs_F were down-regulated while the gene expression in DFC_S was almost constitutive. Moreover, number of aneuploid DFC_F were significantly higher after induction of cellular senescence. CONCLUSION: Our results supported our initial hypothesis that telomere length and genomic instability correlate with the accelerated induction of cellular senescence in DFC_F.

Extent of Inflammation and Foreign Body Reaction to Porous Polyethylene In Vitro and In Vivo.

BACKGROUND/AIM: High-density porous polyethylene (PP) offers possibilities for reconstruction in craniofacial surgery. The purpose of this study was to evaluate the extent of inflammation and foreign body reactions to PP in vitro and in vivo. MATERIALS AND METHODS: Cell attachment, proliferation and expression of inflammatory cytokines were assessed using murine macrophages (RAW 264.7) on two different PP materials in vitro. In vivo, Balb/c mice received PP implants at their dorsum. After sacrifice, samples were analyzed histologically and real-time PCR was used to assess expression of inflammatory cytokines. RESULTS: Cells showed a significantly decreased proliferation (p<0.001) after 48 h and a significantly increased expression of TNF-α (p<0.05) at 24, 48 and 72 h. All animals showed foreign body cell reactions and signs of chronic inflammation. Expression of all but one of the investigated cytokines dropped to non-significant levels after an initial increase. CONCLUSION: Application of porous polyethylene can cause local chronic inflammatory reactions. Although clinical application seems to be immunologically safe, indication and risks should be evaluated carefully when using PP implants.

Cellular senescence in dental pulp stem cells.

OBJECTIVE: This short review summarizes our current knowledge about dental stem cell aging and about possible targets for the regulation of cellular senescence. DESIGN: A literature search was performed using a combination of keywords, e.g., stem cells, replicative senescence, differentiation potential, dental pulp, dental follicle and periodontal ligament. RESULTS: Previous studies have shown that cellular senescence occurs while the proliferation of dental stem cells. Moreover, the differentiation potential was significantly decreased in senescent stem cells and senescent cells secrete also factors that are harmful to the adjacent tissue cells. Moreover, many targets for the regulation of cellular senescence are considered; for example pathways related to the nutrient sensing such as the 5' adenosine monophosphate-activated protein kinase (AMPK) pathway. CONCLUSIONS: The regulation of cellular senescence will play a crucial role in the clinical use of stem cells. However, there is no cell culture protocol available that prevents dental stem cell senescence. Therefore, more knowledge about molecular processes in stem cells is needed before and after the induction of senescence.

WNT5A supports viability of senescent human dental follicle cells.

The osteogenic differentiation of dental follicle cells (DFCs) is inhibited by the onset of cellular senescence, but the cause for this is largely unknown. Recently it was shown that WNT5a, which is an inductor of the non-canonical WNT pathway, stimulates both cellular senescence and osteogenic differentiation of different cell types. In this study, we investigated the role of WNT5a for viability and osteogenic differentiation in human DFCs after the induction of cellular senescence. DFCs were cultivated until the induction of cellular senescence. The induction of cellular senescence was confirmed by ß-galactosidase staining, estimation of population doubling time, and slightly telomere length shortening. After induction of cellular senescence, the expression of WNT5A and the potential to induce the osteogenic differentiation decreased. Inhibition of WNT5A by specific siRNAs had significant effect on the viability of DFCs. Cell proliferation was reduced, whereas both cellular senescence and cell death were increased in DFCs. However, an inhibition of WNT5A did only slightly effect the osteogenic differentiation of DFCs. Our results suggest that WNT5A supports viability during both cell proliferation and osteogenic differentiation of DFCs.

The cell cycle regulator protein P16 and the cellular senescence of dental follicle cells.

Cellular senescence is a restricting factor for regenerative therapies with somatic stem cells. We showed previously that the onset of cellular senescence inhibits the osteogenic differentiation in stem cells of the dental follicle (DFCs), although the mechanism remains elusive. Two different pathways are involved in the induction of the cellular senescence, which are driven either by the cell cycle protein P21 or by the cell cycle protein P16. In this study, we investigated the expression of cell cycle proteins in DFCs after the induction of cellular senescence. The induction of cellular senescence was proved by an increased expression of ß-galactosidase and an increased population doubling time after a prolonged cell culture. Cellular senescence regulated the expression of cell cycle proteins. The expression of cell cycle protein P16 was up-regulated, which correlates with the induction of cellular senescence markers in DFCs. However, the expression of cyclin-dependent kinases (CDK)2 and 4 and the expression of the cell cycle protein P21 were successively decreased in DFCs. In conclusion, our data suggest that a P16-dependent pathway drives the induction of cellular senescence in DFCs.