CXXC5 mitigates P. gingivalis-inhibited cementogenesis by influencing mitochondrial biogenesis.

BACKGROUND: Cementoblasts on the tooth-root surface are responsible for cementum formation (cementogenesis) and sensitive to Porphyromonas gingivalis stimulation. We have previously proved transcription factor CXXC-type zinc finger protein 5 (CXXC5) participates in cementogenesis. Here, we aimed to elucidate the mechanism in which CXXC5 regulates P. gingivalis-inhibited cementogenesis from the perspective of mitochondrial biogenesis. METHODS: In vivo, periapical lesions were induced in mouse mandibular first molars by pulp exposure, and P. gingivalis was applied into the root canals. In vitro, a cementoblast cell line (OCCM-30) was induced cementogenesis and submitted for RNA sequencing. These cells were co-cultured with P. gingivalis and examined for osteogenic ability and mitochondrial biogenesis. Cells with stable CXXC5 overexpression were constructed by lentivirus transduction, and PGC-1α (central inducer of mitochondrial biogenesis) was down-regulated by siRNA transfection. RESULTS: Periapical lesions were enlarged, and PGC-1α expression was reduced by P. gingivalis treatment. Upon apical inflammation, Cxxc5 expression decreased with Il-6 upregulation. RNA sequencing showed enhanced expression of osteogenic markers, Cxxc5, and mitochondrial biogenesis markers during cementogenesis. P. gingivalis suppressed osteogenic capacities, mitochondrial biogenesis markers, mitochondrial (mt)DNA copy number, and cellular ATP content of cementoblasts, whereas CXXC5 overexpression rescued these effects. PGC-1α knockdown dramatically impaired cementoblast differentiation, confirming the role of mitochondrial biogenesis on cementogenesis. CONCLUSIONS: CXXC5 is a P. gingivalis-sensitive transcription factor that positively regulates cementogenesis by influencing PGC-1α-dependent mitochondrial biogenesis. Video Abstract.

Activation of periodontal ligament cell cytodifferentiation by juxtacrine signaling from cementoblasts.

BACKGROUND: New cementum forms from existing cementum during periodontal tissue regeneration, indicating that cementoblasts may interact with progenitor cells in the periodontal ligament to enhance cementogenesis. However, the molecular mechanisms of this process are currently unknown. This study aims to clarify the role of cell-cell interactions between cementoblasts and periodontal ligament cells in differentiation into cementoblasts. METHODS: To analyze the role of human cementoblast-like cells (HCEMs) on human periodontal ligament cells (HPDLs), we mixed cell suspensions of enhanced green fluorescent protein-tagged HPDLs and HCEMs, and then seeded and cultured them in single wells (direct co-cultures). We sorted co-cultured HPDLs and analyzed their characteristics, including the expression of cementum-related genes. In addition, we cultured HPDLs and HCEMs in a non-contact environment using a culture system composed of an upper insert and a lower well separated by a semi-permeable membrane (indirect co-cultures), and similar analysis was performed. Gene expression of integrin-binding sialoprotein (IBSP) in cementoblasts was confirmed in mouse periodontal tissues. We also investigated the effect of Wingless-type (Wnt) signaling on the differentiation of HPDLs into cementoblasts. RESULTS: Direct co-culture of HPDLs with HCEMs significantly upregulated the expression of cementoblast-related genes in HPDLs, whereas indirect co-culture exerted no effect. Wnt3A stimulation significantly upregulated IBSP expression in HPDLs, whereas inhibition of canonical Wnt signaling suppressed the effects of co-culture. CONCLUSION: Our results suggest that direct cell interactions with cementoblasts promote periodontal ligament cell differentiation into cementoblasts. Juxtacrine signaling via the canonical Wnt pathway plays a role in this interaction.

Effect of doxycycline doped nanoparticles on osteogenic/cementogenic and anti-inflammatory responses of human cells derived from the periodontal ligament.

OBJECTIVES: This work aimed to evaluate if doxycycline-doped polymeric nanoparticles possessed any anti-inflammatory effect and promote osteogenic/cementogenic differentiation of stem cells from human periodontal ligament (PDLSCs). METHODS: The polymeric nanoparticles (NPs) were produced by a polymerization/precipitation process and doped with doxycycline (Dox-NPs). PDLSCs were cultured in the presence or absence of the NPs under osteogenic medium or IL-1ß treatment. Cells' differentiation was assessed by gene expression analysis of osteogenic/cementogenic markers alkaline phosphatase (ALP) and Runt-related transcription factor 2 (RUNX2). An anti-inflammatory effect was also ascertained by analyzing IL-1ß gene expression. Adipogenic and chondrogenic differentiation was used to confirm the multipotency of PDLSCs. RESULTS: Gene expression of ALP and RUNX2 in PDLSCs was significantly upregulated by the osteogenic medium (ALP: p<0.001; RUNX2: p = 0.005) while Dox-NPs further enhanced ALP gene expression of PDLSCs treated with the osteogenic medium. Furthermore, Dox-NPs suppressed the up-regulation of IL-1ß when cells were subjected to an inflammatory challenge. CONCLUSIONS: Dox-NPs enhanced PDLSCs differentiation into osteoblasts/cementoblasts lineages while providing an anti-inflammatory effect. CLINICAL SIGNIFICANCE: Due to their biocompatibility as well as anti-inflammatory and osteogenic/cementogenic effects, Dox-NPs are potential candidates for being used in periodontal regeneration.

LITTIP/Lgr6/HnRNPK complex regulates cementogenesis via Wnt signaling.

Orthodontically induced tooth root resorption (OIRR) is a serious complication during orthodontic treatment. Stimulating cementum repair is the fundamental approach for the treatment of OIRR. Parathyroid hormone (PTH) might be a potential therapeutic agent for OIRR, but its effects still lack direct evidence, and the underlying mechanisms remain unclear. This study aims to explore the potential involvement of long noncoding RNAs (lncRNAs) in mediating the anabolic effects of intermittent PTH and contributing to cementum repair, as identifying lncRNA-disease associations can provide valuable insights for disease diagnosis and treatment. Here, we showed that intermittent PTH regulates cell proliferation and mineralization in immortalized murine cementoblast OCCM-30 via the regulation of the Wnt pathway. In vivo, daily administration of PTH is sufficient to accelerate root regeneration by locally inhibiting Wnt/ß-catenin signaling. Through RNA microarray analysis, lncRNA LITTIP (LGR6 intergenic transcript under intermittent PTH) is identified as a key regulator of cementogenesis under intermittent PTH. Chromatin isolation by RNA purification (ChIRP) and RNA immunoprecipitation (RIP) assays revealed that LITTIP binds to mRNA of leucine-rich repeat-containing G-protein coupled receptor 6 (LGR6) and heterogeneous nuclear ribonucleoprotein K (HnRNPK) protein. Further co-transfection experiments confirmed that LITTIP plays a structural role in the formation of the LITTIP/Lgr6/HnRNPK complex. Moreover, LITTIP is able to promote the expression of LGR6 via the RNA-binding protein HnRNPK. Collectively, our results indicate that the intermittent PTH administration accelerates root regeneration via inhibiting Wnt pathway. The lncRNA LITTIP is identified to negatively regulate cementogenesis, which activates Wnt/ß-catenin signaling via high expression of LGR6 promoted by HnRNPK.

The long non-coding RNA, miRNA and mRNA landscapes of cementoblasts during cementogenesis.

OBJECTIVE: Stimulation of cementogenesis is essential to cementum regeneration and root restoration. Long non-coding RNAs (lncRNAs) participate in the regulatory networks of periodontal regeneration processes. We identified and analysed differentially expressed lncRNAs, miRNAs and mRNAs associated with cementogenic differentiation of cementoblasts. MATERIALS AND METHODS: OCCM-30 immortalized mouse cementoblast cells were induced in cementogenic medium for 7 and 14 days. Total RNA was extracted and subjected to RNA sequencing to screen for differentially expressed lncRNAs, miRNAs and mRNAs. Quantitative reverse-transcription polymerase chain reaction (qRT-PCR) was performed to determine the expression levels of RNAs. Gene Ontology (GO) term and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analyses were used to clarify the potential functions of differentially expressed genes in biological processes and pathways. lncRNA-miRNA-mRNA networks were constructed based on correlation and algorithmic analyses. RESULTS: In all, 461 lncRNAs, 89 miRNAs and 2157 mRNAs showed differential expression in OCCM-30 cells after cementoblast differentiation. At day 7, upregulation of 248 lncRNAs, 30 miRNAs and 905 mRNAs was observed, along with downregulation of 127 lncRNAs, 34 miRNAs and 960 mRNAs. At day 14, 197 lncRNAs, 13 miRNAs and 847 mRNAs were upregulated, while 74 lncRNAs, 12 miRNAs and 760 mRNAs were downregulated. The results of qRT-PCR showed that four candidate lncRNAs, H19, Gdap10, Foxo6os and Ipw, were significantly upregulated after 7 and 14 days of cementogenic induction. The lncRNA-miRNA-mRNA network illustrated a possible competitive endogenous RNA regulatory mechanism. GO analysis showed that consistently differentially expressed mRNAs were involved in blood vessel morphogenesis, cell-substrate adhesion, cell adhesion, ossification and extracellular matrix organization. KEGG analysis indicated that extracellular matrix-receptor interaction, focal adhesion, and the PI3K-Akt, Rap1, mitogen-activated protein kinase, and Ras signalling pathways varied significantly during cementogenesis. CONCLUSION: The expressions of lncRNA, miRNA and mRNA were significantly altered in cementoblasts after cementogenesis. This study highlighted the effect of lncRNAs in the process of cementogenesis and revealed their potential for the discovery of novel biomarkers and therapeutic targets for cementum regeneration.

Loss of ß-catenin causes cementum hypoplasia by hampering cementogenic differentiation of Axin2-expressing cells.

BACKGROUND AND OBJECTIVE: Although cementum plays an essential role in tooth attachment and adaptation to occlusal force, the regulatory mechanisms of cementogenesis remain largely unknown. We have previously reported that Axin2-expressing (Axin2+ ) mesenchymal cells in periodontal ligament (PDL) are the main cell source for cementum growth, and constitutive activation of Wnt/ß-catenin signaling in Axin2+ cells results in hypercementosis. Therefore, the aim of the present study was to further evaluate the effects of ß-catenin deletion in Axin2+ cells on cementogenesis. MATERIALS AND METHODS: We generated triple transgenic mice to conditionally delete ß-catenin in Axin2-lineage cells by crossing Axin2CreERT2/+ ; R26RtdTomato/+ mice with ß-cateninflox/flox mice. Multiple approaches, including X-ray analysis, micro-CT, histological stainings, and immunostaining assays, were used to analyze cementum phenotypes and molecular mechanisms. RESULTS: Our data revealed that loss of ß-catenin in Axin2+ cells led to a cementum hypoplasia phenotype characterized by a sharp reduction in the formation of both acellular and cellular cementum. Mechanistically, we found that conditional removal of ß-catenin in Axin2+ cells severely impaired the secretion of cementum matrix proteins, for example, bone sialoprotein (BSP), dentin matrix protein 1 (DMP1) and osteopontin (OPN), and markedly inhibited the differentiation of Axin2+ mesenchymal cells into osterix+ cementoblasts. CONCLUSIONS: Our findings confirm the vital role of Axin2+ mesenchymal PDL cells in cementum growth and demonstrate that Wnt/ß-catenin signaling shows a positive correlation with cementogenic differentiation of Axin2+ cells.

Connective tissue growth factor promotes cementogenesis and cementum repair via Cx43/ß-catenin axis.

BACKGROUND: Orthodontic tooth movement inevitably induces cementum resorption, which is an urgent problem for orthodontists to confront. Human periodontal ligament stem cells (hPDLSCs) exert an important role in the orthodontic tooth movement and exhibit multidirectional differentiation ability in cementum regeneration. Connective tissue growth factor (CTGF) is an important extracellular matrix protein for bone homeostasis and cell differentiation. The purpose of our study was to explore the role of CTGF in cementum repair and cementogenesis and to elucidate its underlying mechanism. METHODS: A cementum defect model was established by tooth movement with heavy forces, and the cementum repair effect of CTGF was observed via micro-CT, HE staining and immunohistochemical staining. RT‒qPCR, western blotting (WB), alizarin red staining and ALP activity experiments verified the mineralization ability of hPDLSCs stimulated with CTGF. The expression of Cx43 in periodontal ligament cells was detected by WB and immunofluorescence (IF) experiments after CTGF stimulation in vivo and in vitro. Subsequently, the mineralization ability of hPDLSCs was observed after application of CTGF and the small interfering RNA Si-Cx43. Additionally, co-intervention via application of the small interfering RNA Si-CTGF and the Cx43 agonist ATRA in hPDLSCs was performed to deepen the mechanistic study. Next, WB, IF experiments and co-immunoprecipitation were conducted to confirm whether CTGF triggers the Cx43/ß-catenin axis to regulate cementoblast differentiation of hPDLSCs. RESULTS: Local oral administration of CTGF to the cementum defects in vivo facilitated cementum repair. CTGF facilitated the cementogenesis of hPDLSCs in a concentration-dependent manner. Cx43 acted as a downstream effector of CTGF to regulate cementoblast differentiation. Si-Cx43 reduced CTGF-induced cementoblast differentiation. The Cx43 agonist ATRA restored the low differentiation capacity induced by Si-CTGF. Further mechanistic studies showed that CTGF triggered the activation of ß-catenin in a dose-dependent manner. In addition, co-localization IF analysis and co-immunoprecipitation demonstrated that Cx43 interacted with ß-catenin at cell‒cell connections. Si-Cx43 attenuated the substantial expression of ß-catenin induced by CTGF. The Cx43 agonist reversed the inhibition of ß-catenin induced by Si-CTGF. IF demonstrated that the nuclear importation of ß-catenin was related to the immense expression of Cx43 at cell‒cell junctions. CONCLUSIONS: Taken together, these data demonstrate that CTGF promotes cementum repair and cementogenesis through activation of the Cx43/ß-catenin signalling axis.

The concordance of signals based on irregular incremental lines in the human tooth cementum with documented pregnancies: Results from a systematic approach.

BACKGROUND AND OBJECTIVE: There is evidence from previous studies that pregnancies and diseases are recorded in the tooth cementum. This study aims to assess the degree of concordance between signals based on irregular incremental lines (ILs) and reported pregnancies. MATERIAL AND METHODS: 23 recent and 24 archaeological human teeth with known birth history were included in this investigation. 129 histological sections of tooth roots were assessed for irregularities in appearance and width using a standardized protocol. Similarity of observed irregularities at the section level allowed us to define signals at the tooth level. The sensitivity of signals to detect pregnancies was determined and related to the signal prevalence. RESULTS: Pregnancy signals were frequently visually observed. However, applying a standardized process we could only reach signal sensitivities to identify pregnancies up to 20 percentage points above chance level. CONCLUSIONS: Based on a standardized and reproducible method it could be confirmed that some pregnancies leave visible signals in the tooth cementum. The results show the potential of the tooth cementum to support reconstruction of life courses in paleopathology. However, it seems that not all pregnancies affect the cementogenesis in such a way that irregular ILs are identifiable. Further research is needed to better understand which type of pregnancies and other conditions are recorded in the tooth cementum.

Canonical Wnt/ß-catenin signaling has positive effects on osteogenesis, but can have negative effects on cementogenesis.

BACKGROUND: To date, therapeutic approaches for cementum regeneration are limited and outcomes remain unpredictable. A significant barrier to improve therapies for cementum regeneration is that the cementocyte and its intracellular signal transduction mechanisms remain poorly understood. This study aims to elucidate the regulatory mechanism of Wnt pathway in cementogenesis. METHODS: The effects of canonical Wnt signaling were compared in vitro using immortalized murine cementocyte cell line IDG-CM6 and osteocyte cell line IDG-SW3 by quantitative real-time polymerase chain reaction, Western blot, confocal microscopy, alkaline phosphatase (ALP) assay, and Alizarin red S staining. In vivo, histological changes of cementum and bone formation were examined in transgenic mice in which constitutive activation of ß-catenin is driven by Dmp1 promoter. RESULTS: Expression of components of the Wnt/ß-catenin pathway were much greater in the IDG-SW3 cells compared with the IDG-CM6 cells resulting in much lower expression of Sost/sclerostin in the IDG-SW3 cells. In the IDG-CM6 cells, low dose Wnt3a (20 ng/ml) had a modest effect while high dose (200 ng/ml) inhibited runt-related transcription factor 2, osterix, ALP, and osteopontin in contrast to the IDG-SW3 cells where high dose Wnt3a dramatically increased mRNA expression of these same markers. However, high Wnt3a significantly increased mRNA for components of Wnt/ß-catenin signaling pathway in both IDG-CM6 and IDG-SW3 cells. In vivo, constitutive activation of ß-catenin in the Dmp1-lineage cells in mice leads to bone hyperplasia and cementum hypoplasia. CONCLUSION: These findings indicate that Wnt signaling has distinct and different effects on the regulation of long bone as compared with cementum.

Growth Factors in Periodontal Complex Regeneration.

The ultimate goal of periodontal treatments is the regeneration of all lost periodontal tissues including bone, cementum and the periodontal ligament (PDL). Until now, the clinical methods for periodontal regeneration have been associated with significant failure or incomplete success. Various studies have reported the promising effects of growth factors/cytokines on periodontal regeneration. Growth factors/cytokines include proteins or steroid hormones that bind to cellular receptors, known as signalling molecules, and that trigger cellular responses that eventually stimulate cell proliferation and differentiation. The present review aims to provide an overview of the main growth factors that play an important role in and have been used in the regeneration of periodontal components.

Axin2-expressing cells in the periodontal ligament are regulated by bone morphogenetic protein signalling and play a pivotal role in periodontium development.

AIM: To date, controversies still exist regarding the exact cellular origin and regulatory mechanisms of periodontium development, which hinders efforts to achieve ideal periodontal tissue regeneration. Axin2-expressing cells in the periodontal ligament (PDL) have been shown to be a novel progenitor cell population that is essential for periodontal homeostasis. In the current study, we aimed to elucidate the regulatory role of bone morphogenetic protein receptor type 1A (BMPR1A)-mediated BMP signalling in Axin2-expressing cells during periodontium development. MATERIALS AND METHODS: Two strains of Axin2 gene reporter mice, Axin2lacZ/+ and Axin2CreERT2/+ ; R26RtdTomato/+ mice, were used. We next generated Axin2CreERT2/+ ; R26RDTA/+ ; R26RtdTomato/+ mice to genetically ablate of Axin2-lineage cells. Axin2CreERT2/+ ; Bmpr1afl/fl ; R26RtdTomato/+ mice were established to conditionally knock out Bmpr1a in Axin2-lineage cells. Multiple approaches, including micro-computed tomography, calcein green, and alizarin red double-labelling, scanning electron microscopy, and histological and immunostaining assays, were used to analyse periodontal phenotypes and molecular mechanisms. RESULTS: X-gal staining revealed that Axin2-expressing cells in the PDL were mainly distributed along the alveolar bone and cementum surface. Cell lineage tracing and cell ablation assays further demonstrated the indispensable role of Axin2-expressing cells in periodontium development. Next, we found that conditional knockout of Bmpr1a in Axin2-lineage cells led to periodontal defects, which were characterized by alveolar bone loss, impaired cementogenesis, and abnormal Sharpey's fibres. CONCLUSIONS: Our findings suggest that Axin2-expressing cells in the PDL are essential for periodontium development, which is regulated by BMP signalling.

Identification of Dental Stem Cells Similar to Skeletal Stem Cells.

Stem and progenitor cells play important roles in the development and maintenance of teeth and bone. Surface markers expressed in bone marrow-derived mesenchymal stem cells are also expressed in dental tissue-derived stem cells. Mouse skeletal stem cells (mSSCs, CD45-Ter119-Tie2-CD51+Thy-6C3-CD105-CD200+) and human skeletal stem cells (hSSCs, CD45-CD235a-TIE2-CD31-CD146-PDPN+CD73+CD164+) have been identified in bone and shown to play important roles in skeletal development and regeneration. However, it is unclear whether dental tissues also harbor mSSC or hSSC populations. Here, we employed rainbow tracers and found that clonal expansion occurred in mouse dental tissues similar to that in bone. We sorted the mSSC population from mouse periodontal ligament (mPDL) tissue and mouse dental pulp (mDP) tissue in the lower incisors by fluorescence-activated cell sorting (FACS). In addition, we demonstrated that mPDL-derived skeletal stem cells (mPDL-SSCs) and mDP-derived skeletal stem cells (mDP-SSCs) have similar clonogenic capacity, as well as cementogenic and odontogenic potential, but not adipogenic potential, similar to the characteristics of mSSCs. Moreover, we found that the dental tissue-derived mSSC population plays an important role in repairing clipped incisors. Importantly, we sorted the hSSC population from human periodontal ligament (hPDL) and human dental pulp (hDP) tissue in molars and identified its stem cell characteristics. Finally, hPDL-like and hDP-like structures were generated after transplanting hPDL-SSCs and hDP-SSCs beneath the renal capsules. In conclusion, we demonstrated that mouse and human PDL and DP tissues harbor dental stem cells similar to mSSCs and hSSCs, respectively, providing a precise stem cell population for the exploration of dental diseases.

Embedded Human Periodontal Ligament Stem Cells Spheroids Enhance Cementogenic Differentiation via Plasminogen Activator Inhibitor 1.

Spheroids reproduce the tissue structure that is found in vivo more accurately than classic two-dimensional (2D) monolayer cultures. We cultured human periodontal ligament stem cells (HPLSCs) as spheroids that were embedded in collagen gel to examine whether their cementogenic differentiation could be enhanced by treatment with recombinant human plasminogen activator inhibitor-1 (rhPAI-1). The upregulated expression of cementum protein 1 (CEMP1) and cementum attachment protein (CAP), established cementoblast markers, was observed in the 2D monolayer HPLSCs that were treated with rhPAI-1 for 3 weeks compared with that in the control and osteogenic-induction medium groups. In the embedded HPLSC spheroids, rhPAI-1 treatment induced interplay between the spheroids and collagenous extracellular matrix (ECM), indicating that disaggregated HPLSCs migrated and spread into the surrounding ECM 72 h after three-dimensional (3D) culture. Western blot and immunocytochemistry analyses showed that the CEMP1 expression levels were significantly upregulated in the rhPAI-1-treated embedded HPLSC spheroids compared with all the 2D monolayer HPLSCs groups and the 3D spheroid groups. Therefore, 3D collagen-embedded spheroid culture in combination with rhPAI-1 treatment may be useful for facilitating cementogenic differentiation of HPLSCs.

REV-ERBs negatively regulate mineralization of the cementoblasts.

OBJECTIVE: The role of circadian clock in cementogenesis is unclear. This study examines the role of REV-ERBs, one of circadian clock proteins, in proliferation, migration and mineralization of cementoblasts to fill the gap in knowledge. METHODS: Expression pattern of REV-ERBα in cementoblasts was investigated in vivo and in vitro. CCK-8 assay, scratch wound healing assay, alkaline phosphatase (ALP) and alizarin red S (ARS) staining were performed to evaluate the effects of REV-ERBs activation by SR9009 on proliferation, migration and mineralization of OCCM-30, an immortalized cementoblast cell line. Furthermore, mineralization related markers including osterix (OSX), ALP, bone sialoprotein (BSP) and osteocalcin (OCN) were evaluated. RESULTS: Strong expression of REV-ERBα was found in cellular cementum around tooth apex. Rev-erbα mRNA oscillated periodically in OCCM-30 and declined after mineralization induction. REV-ERBs activation by SR9009 inhibited proliferation but promoted migration of OCCM-30 in vitro. Results of ALP and ARS staining suggested that REV-ERBs activation negatively regulated mineralization of OCCM-30. Mechanically, REV-ERBs activation attenuated the expression of OSX and its downstream targets including ALP, BSP and OCN. CONCLUSIONS: REV-ERBs are involved in cementogenesis and negatively regulate mineralization of cementoblasts via inhibiting OSX expression. Our study provides a potential target regarding periodontal and cementum regeneration.

Calcitriol and enamel matrix derivative differentially regulated cemento-induction and mineralization in human periodontal ligament-derived cells.

BACKGROUND: Alveolar bone and cementum share many biological and developmental similarities. The mineralizing effect of calcitriol has been previously reported. Yet, its cemento-inductivity has not been confirmed. This study evaluated the potential cemento-inductivity effect of calcitriol and enamel matrix derivative (EMD) on human periodontal ligament-derived cells (hPDLCs). METHODS: The hPDLCs obtained from extracted third molars or premolars were cultured with calcitriol, or EMD. Cementogenic gene expression was examined using real-time quantitative reverse transcription polymerase chain reaction. Expression analysis also included cementoblast-specific markers, cementum protein 1 (CEMP1), cementum attachment protein (CAP), and recently reported cementoblast-enriched genes, secreted frizzled related protein 1 (SFRP1), and Dickkopf-related protein 1 (DKK1). Mineralization capacities were evaluated by alkaline phosphatase (ALP) activity, Alizarin Red, and Von Kossa staining followed by scanning electron microscope imaging and element mapping. RESULTS: Among tested conditions, 10 nM calcitriol enhanced most cementogenic gene expression, transforming growth factor-ß1, bone morphogenetic proteins (BMP-2 and BMP-4), core-binding factor subunit alpha-1/Runt-related transcription factor 2, Type I collagen, ALP, bone sialoprotein, osteopontin), osteocalcin, CEMP1, and CAP, and Wnt signaling negative modulators, SFRP1 and DKK1, along with highest ALP activity and mineralization formation in hPDLCs. However, only moderate CEMP1 protein was observed. In contrast, EMD stimulated stronger CEMP1 and CAP protein, but presented weaker mineralization capacity, hinting at the possibility that strong stimulation of mineralization might dominate cemetogenic specific factors and vice versa. CONCLUSIONS: Calcitriol demonstrated not only great osteoinductivity, but also the potential to induce cementogenic gene expression by initiating hPDLC differentiation and promoting mineralization. Compared with calcitriol, EMD promoted cemento-inductivity in hPDLCs at a later time point via highly expressed CEMP1 and CAP protein, but with less mineralization. Thus, calcitriol and EMD could provide differential enhancement of cemento-induction and mineralization, likely acting at various differentiation stages.

PPARγ-Induced Global H3K27 Acetylation Maintains Osteo/Cementogenic Abilities of Periodontal Ligament Fibroblasts.

The periodontal ligament is a soft connective tissue embedded between the alveolar bone and cementum, the surface hard tissue of teeth. Periodontal ligament fibroblasts (PDLF) actively express osteo/cementogenic genes, which contribute to periodontal tissue homeostasis. However, the key factors maintaining the osteo/cementogenic abilities of PDLF remain unclear. We herein demonstrated that PPARγ was expressed by in vivo periodontal ligament tissue and its distribution pattern correlated with alkaline phosphate enzyme activity. The knockdown of PPARγ markedly reduced the osteo/cementogenic abilities of PDLF in vitro, whereas PPARγ agonists exerted the opposite effects. PPARγ was required to maintain the acetylation status of H3K9 and H3K27, active chromatin markers, and the supplementation of acetyl-CoA, a donor of histone acetylation, restored PPARγ knockdown-induced decreases in the osteo/cementogenic abilities of PDLF. An RNA-seq/ChIP-seq combined analysis identified four osteogenic transcripts, RUNX2, SULF2, RCAN2, and RGMA, in the PPARγ-dependent active chromatin region marked by H3K27ac. Furthermore, RUNX2-binding sites were selectively enriched in the PPARγ-dependent active chromatin region. Collectively, these results identified PPARγ as the key transcriptional factor maintaining the osteo/cementogenic abilities of PDLF and revealed that global H3K27ac modifications play a role in the comprehensive osteo/cementogenic transcriptional alterations mediated by PPARγ.

Cold Atmospheric Plasma Promotes Regeneration-Associated Cell Functions of Murine Cementoblasts In Vitro.

The aim of the study was to examine the efficacy of cold atmospheric plasma (CAP) on the mineralization and cell proliferation of murine dental cementoblasts. Cells were treated with CAP and enamel matrix derivates (EMD). Gene expression of alkaline phosphatase (ALP), bone gamma-carboxyglutamate protein (BGLAP), periostin (POSTN), osteopontin (OPN), osterix (OSX), collagen type I alpha 1 chain (COL1A1), dentin matrix acidic phosphoprotein (DMP)1, RUNX family transcription factor (RUNX)2, and marker of proliferation Ki-67 (KI67) was quantified by real-time PCR. Protein expression was analyzed by immunocytochemistry and ELISA. ALP activity was determined by ALP assay. Von Kossa and alizarin red staining were used to display mineralization. Cell viability was analyzed by XTT assay, and morphological characterization was performed by DAPI/phalloidin staining. Cell migration was quantified with an established scratch assay. CAP and EMD upregulated both mRNA and protein synthesis of ALP, POSTN, and OPN. Additionally, DMP1 and COL1A1 were upregulated at both gene and protein levels. In addition to upregulated RUNX2 mRNA levels, treated cells mineralized more intensively. Moreover, CAP treatment resulted in an upregulation of KI67, higher cell viability, and improved cell migration. Our study shows that CAP appears to have stimulatory effects on regeneration-associated cell functions in cementoblasts.

Wnt1 Promotes Cementum and Alveolar Bone Growth in a Time-Dependent Manner.

The WNT/ß-catenin signaling pathway plays a central role in the biology of the periodontium, yet the function of specific extracellular WNT ligands remains poorly understood. By using a Wnt1-inducible transgenic mouse model targeting Col1a1-expressing alveolar osteoblasts, odontoblasts, and cementoblasts, we demonstrate that the WNT ligand WNT1 is a strong promoter of cementum and alveolar bone formation in vivo. We induced Wnt1 expression for 1, 3, or 9 wk in Wnt1Tg mice and analyzed them at the age of 6 wk and 12 wk. Micro-computed tomography (CT) analyses of the mandibles revealed a 1.8-fold increased bone volume after 1 and 3 wk of Wnt1 expression and a 3-fold increased bone volume after 9 wk of Wnt1 expression compared to controls. In addition, the alveolar ridges were higher in Wnt1Tg mice as compared to controls. Nondecalcified histology demonstrated increased acellular cementum thickness and cellular cementum volume after 3 and 9 wk of Wnt1 expression. However, 9 wk of Wnt1 expression was also associated with periodontal breakdown and ectopic mineralization of the pulp. The composition of this ectopic matrix was comparable to those of cellular cementum as demonstrated by quantitative backscattered electron imaging and immunohistochemistry for noncollagenous proteins. Our analyses of 52-wk-old mice after 9 wk of Wnt1 expression revealed that Wnt1 expression affects mandibular bone and growing incisors but not molar teeth, indicating that Wnt1 influences only growing tissues. To further investigate the effect of Wnt1 on cementoblasts, we stably transfected the cementoblast cell line (OCCM-30) with a vector expressing Wnt1-HA and performed proliferation as well as differentiation experiments. These experiments demonstrated that Wnt1 promotes proliferation but not differentiation of cementoblasts. Taken together, our findings identify, for the first time, Wnt1 as a critical regulator of alveolar bone and cementum formation, as well as provide important insights for harnessing the WNT signal pathway in regenerative dentistry.

A Biphasic Feature of Gli1+-Mesenchymal Progenitors during Cementogenesis That Is Positively Controlled by Wnt/ß-Catenin Signaling.

Cementum, a specialized bony layer covering an entire molar root surface, anchors teeth into alveolar bone. Gli1, a key transcriptional activator in Hedgehog signaling, has been identified as a mesenchymal progenitor cell marker in various tissues, including the periodontal ligament (PDL). To address the mechanisms by which Gli1+ progenitor cells contribute to cementogenesis, we used the Gli1lacZ/+ knock-in line to mark Gli1+ progenitors and the Gli1CreERT2/+; R26RtdTomato/+ line (named Gli1Lin) to trace Gli1 progeny cells during cementogenesis. Our data unexpectedly displayed a biphasic feature of Gli1+ PDL progenitor cells and cementum growth: a negative relationship between Gli1+ progenitor cell number and cementogenesis but a positive correlation between Gli1-derived acellular and cellular cementoblast cell number and cementum growth. DTA-ablation of Gli1Lin cells led to a cementum hypoplasia, including a significant reduction of both acellular and cellular cementoblast cells. Gain-of-function studies (by constitutive stabilization of ß-catenin in Gli1Lin cells) revealed a cementum hyperplasia. A loss of function (by conditional deletion of ß-catenin in Gli1+ cells) resulted in a reduction of postnatal cementum growth. Together, our studies support a vital role of Gli1+ progenitor cells in contribution to both types of cementum, in which canonical Wnt/ß-catenin signaling positively regulates the differentiation of Gli1+ progenitors to cementoblasts during cementogenesis.

Intermittent parathyroid hormone promotes cementogenesis via ephrinB2-EPHB4 forward signaling.

Intermittent parathyroid hormone (PTH) promotes periodontal repair, but the underlying mechanisms remained unclear. Recent studies found that ephrinB2-EPHB4 forward signaling mediated the anabolic effect of PTH in bone homeostasis. Considering the similarities between cementum and bone, we aimed to examine the therapeutic effect of PTH on resorbed roots and explore the role of forward signaling in this process. In vivo experiments showed that intermittent PTH significantly accelerated the regeneration of root resorption and promoted expression of EPHB4 and ephrinB2. When the signaling was blocked, the resorption repair was also delayed. In vitro studies showed that intermittent PTH promoted the expression of EPHB4 and ephrinB2 in OCCM-30 cells. The effects of PTH on the mineralization capacity of OCCM-30 cells was mediated through the ephrinB2-EPHB4 forward signaling. These results support the premise that the anabolic effects of intermittent PTH on the regeneration of root resorption is via the ephrinB2-EPHB4 forward signaling pathway.