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.

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.

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.

Lithium chloride attenuates suppressed differentiation induced by mechanical strain in cementoblasts.

Aim: The purpose of this study was to investigate the influence of mechanical strain on OCCM-30 cementoblast differentiation and Wnt/ß-catenin pathway activity. Materials and Methods: Mechanical tension in the form of 2500-µ strain was applied to the cells using the Forcel four-point bending system, with or without the Wnt signaling activator, lithium chloride. Changes in cell differentiation and the expression of Wnt/ß-catenin pathway components in response to strain and lithium chloride were assessed by real-time PCR, immunofluorescence, and western blotting. Results: The mRNA expression levels of the cementoblastogenesis-related genes alkaline phosphatase, runt-related transcription factor 2, and collagen 1, were decreased with mechanical strain. Similarly, the Wnt signaling pathway component genes LRP5, AXIN2, and LEF1 were decreased. The immunofluorescence assay demonstrated that scant ß-catenin underwent nuclear translocation after the cells were subjected to mechanical strain. Moreover, western blotting showed that the protein levels of both ß-catenin and phosphorylated ß-catenin were increased after mechanical strain. In the presence of lithium chloride, the differentiation that was suppressed by mechanical strain was attenuated. Conclusions: 2500-µ strain mechanical strain inhibited cementoblast differentiation activity in vitro, which could be alleviated by actviating Wnt/ß-catenin signaling using lithium chloride.

Establishment of a Primary Culture System of Human Periodontal Ligament Cells that Differentiate into Cementum Protein 1-expressing Cementoblast-like Cells.

BACKGROUND/AIM: A better understanding of cementogenesis and cementoblast differentiation would be useful for periodontal therapy. The aim of this study was to establish a cell culture system that reflects cementum formation in periodontal tissue and determine whether or not isolated and cultured primary human periodontal ligament (PDL) cells could be used for the study of the differentiation of cementoblast. MATERIALS AND METHODS: PDL cells were isolated from the outgrowths of tissue fragments of human PDL. PDL cells were incubated for up to 21 days in differentiation medium containing ß-glycerophosphate and ascorbic acid. The changes in the cells were detected by alkaline phosphatase (ALP) and von Kossa staining. Real-time polymerase chain reaction was also performed for cementum protein 1 (CEMP1), which is a specific marker of cementoblasts and their progenitors. RESULTS: On day 5, a small number of PDL cells, which were fibrous, were positive for ALP. On day 7, almost all cells were positive for ALP. On day 14, mineralization nodules appeared, as seen by positive von Kossa staining; the nodules increased in number and size by day 21. The expression of CEMP1 was detected on day 5, and its expression level increased gradually by day 7, reached a peak on day 14, and decreased by day 21. CONCLUSION: Human PDL cells were used to establish a culture system that reflects cementum formation. Our results suggested that this culture method is convenient and useful for the study of cementogenesis and cementoblast differentiation.

Effect of FGF-2, TGF-ß-1, and BMPs on Teno/Ligamentogenesis and Osteo/Cementogenesis of Human Periodontal Ligament Stem Cells.

The periodontal ligament (PDL) is the connective tissue between tooth root and alveolar bone containing mesenchymal stem cells (MSC). It has been suggested that human periodontal ligament stem cells (hPDLSCs) differentiate into osteo/cementoblast and ligament progenitor cells. The periodontitis is a representative oral disease where the PDL tissue is collapsed, and regeneration of this tissue is important in periodontitis therapy. Fibroblast growth factor-2 (FGF-2) stimulates proliferation and differentiation of fibroblastic MSCs into various cell lineages. We evaluated the dose efficacy of FGF-2 for cytodifferentiation of hPDLSCs into ligament progenitor. The fibrous morphology was highly stimulated even at low FGF-2 concentrations, and the expression of teno/ligamentogenic markers, scleraxis and tenomodulin in hPDLSCs increased in a dose dependent manner of FGF-2. In contrast, expression of the osteo/cementogenic markers decreased, suggesting that FGF-2 might induce and maintain the ligamentogenic potential of hPDLSCs. Although the stimulation of tenocytic maturation by TGF-ß1 was diminished by FGF-2, the inhibition of the expression of early ligamentogenic marker by TGF-ß1 was redeemed by FGF-2 treatment. The stimulating effect of BMPs on osteo/cementogenesis was apparently suppressed by FGF-2. These results indicate that FGF-2 predominantly differentiates the hPDLSCs into teno/ligamentogenesis, and has an antagonistic effect on the hard tissue differentiation induced by BMP-2 and BMP-4.

A Reciprocal Interaction between ß-Catenin and Osterix in Cementogenesis.

Although accumulating evidence indicates that both ß-catenin and osterix (Osx) are essential for bone and tooth development, few studies have investigated the interaction of these two key proteins in the context of cementogenesis. In this study, we used transgenic mice with constitutively active ß-catenin and inactive Osx in the dental mesenchyme to address this question. We found that cementoblasts with constitutively active ß-catenin require Osx to produce excessive cellular cementum, and that ablation of Osx prevents this abnormal accumulation. Importantly, cementoblasts transduced with retrovirus expressing constitutively active ß-catenin exhibited upregulation of Osx expression through direct binding to the promoter region of Osx. Osx regulates Lef1 expression and consequently could regulate T-cell factor/lymphoid enhancer factor (Tcf/Lef) binding activity in Wnt/ß-catenin signaling. However, the loss of Tcf/Lef binding activity by Osx ablation was not rescued by transduction of retrovirus expressing constitutively active ß-catenin or ectopic Lef1 overexpression. These results suggest that the Tcf/Lef binding activity of Wnt/ß-catenin signaling is Osx-dependent during cementogenesis. Moreover, Osx differentially regulates the expression of various Tcf family members, suggesting that Osx regulates cementogenesis by utilizing various Tcf/Lef-dependent mechanisms. This is the first report to show that downstream Osx signaling through Tcf/Lefs is critical for cementogenesis.

Intermittent parathyroid hormone (PTH) promotes cementogenesis and alleviates the catabolic effects of mechanical strain in cementoblasts.

BACKGROUND: External root resorption, commonly starting from cementum, is a severe side effect of orthodontic treatment. In this pathological process and repairing course followed, cementoblasts play a significant role. Previous studies implicated that parathyroid hormone (PTH) could act on committed osteoblast precursors to promote differentiation, and inhibit apoptosis. But little was known about the role of PTH in cementoblasts. The purpose of this study was to investigate the effects of intermittent PTH on cementoblasts and its influence after mechanical strain treatment. RESULTS: Higher levels of cementogenesis- and differentiation-related biomarkers (bone sialoprotein (BSP), osteocalcin (OCN), Collagen type I (COL1) and Osterix (Osx)) were shown in 1-3 cycles of intermittent PTH treated groups than the control group. Additionally, intermittent PTH increased alkaline phosphatase (ALP) activity and mineralized nodules formation, as measured by ALP staining, quantitative ALP assay, Alizarin red S staining and quantitative calcium assay. The morphology of OCCM-30 cells changed after mechanical strain exertion. Expression of BSP, ALP, OCN, osteopontin (OPN) and Osx was restrained after 18 h mechanical strain. Furthermore, intermittent PTH significantly increased the expression of cementogenesis- and differentiation-related biomarkers in mechanical strain treated OCCM-30 cells. CONCLUSIONS: Taken together, these data suggested that intermittent PTH promoted cementum formation through activating cementogenesis- and differentiation-related biomarkers, and attenuated the catabolic effects of mechanical strain in immortalized cementoblasts OCCM-30.

Cementogenic genes in human periodontal ligament stem cells are downregulated in response to osteogenic stimulation while upregulated by vitamin C treatment.

Regeneration of periodontal tissues, particularly cementum, is key to regaining periodontal attachment and health. Human periodontal ligament stem cells (hPDLSCs) have been shown to be a good cell source to regenerate periodontal tissues. However, their subpopulations and the differentiation induction in relation to cementogenic lineages is unclear. Thus, we aim to examine the expression of cementum-associated genes in PDLSC subpopulations and determine the effect of broadly used osteogenic stimulus or vitamin C (VC) on the expression of cementogenic and osteogenic genes in PDLSCs. Our real-time quantitative polymerase chain reaction (qPCR) analysis showed that cementogenic marker cementum attachment protein (CAP) expressed only slightly higher in STRO-1+/CD146+, STRO-1-/CD146+ and STRO-1-/CD146- subpopulations than in the original cell pool, while cementum protein 1 (CEMP1) expression in these subpopulations was not different from the original pool. Notably, under the stimulation with osteogenic differentiation medium, CAP and CEMP1 were downregulated while osteogenic markers bone sialoprotein (BSP) and osteocalcin (OCN) were upregulated. Both CAP and CEMP1 were upregulated by VC treatment. Transplantation of VC-treated PDLSCs into immunocompromised mice resulted in forming significantly more ectopic cementum- and bone-like mineral tissues in vivo. Immunohistochemical analysis of the ectopic growth showed that CAP and CEMP1 were mainly expressed in the mineral tissue and in some cells of the fibrous tissues. We conclude that osteogenic stimulation is not inductive but appears to be inhibitory of cementogenic pathways, whereas VC induces cementogenic lineage commitment by PDLSCs and may be a useful stimulus for cementogenesis in periodontal regeneration.

Response to light compressive force in human cementoblasts in vitro.

The objective of this study is to investigate the responses of human cementoblasts to light compressive force in vitro. A human cementoblast cell line (HCEM) was loaded for 12 h by mounting coverslips (0.25 gf/cm2). The coverslips were removed and the cells were cultured for up to 21 days. Cells without glass loading were used as controls. Cell growth, morphological changes, and the mRNA expression of RUNX2, ALP, WNT5A and SPON1 were investigated. No significant differences were observed in cell numbers between the compressed group and control group. Morphology of the compressed cells was slightly flattened on day 0; however, no indications of cell death were detected. Expression of differentiation markers including RUNX2, ALP and WNT5A was significantly lower in the compressed group (0.7, 0.75 and 0.75-fold respectively, P < 0.05) than in the control group on day 7. The expression levels of SPON1, a differentiation marker of cementoblasts, were higher on days 7 and 14 than on day 0, but were lower in the compressed group than in the control group (P < 0.01). These results suggest that light compressive force does not affect cell growth and morphology, but restrains higher expression of cementogenic differentiation markers in human cementoblasts in vitro.

TGF-ß Signaling Regulates Cementum Formation through Osterix Expression.

TGF-ß/BMPs have widely recognized roles in mammalian development, including in bone and tooth formation. To define the functional relevance of the autonomous requirement for TGF-ß signaling in mouse tooth development, we analyzed osteocalcin-Cre mediated Tgfbr2 (OC(Cre)Tgfbr2(fl/fl)) conditional knockout mice, which lacks functional TGF-ß receptor II (TßRII) in differentiating cementoblasts and cementocytes. Strikingly, OC(Cre)Tgfbr2(fl/fl) mutant mice exhibited a sharp reduction in cellular cementum mass with reduced matrix secretion and mineral apposition rates. To explore the molecular mechanisms underlying the roles of TGF-ß signaling through TßRII in cementogenesis, we established a mouse cementoblast model with decreased TßRII expression using OCCM-30 cells. Interestingly, the expression of osterix (Osx), one of the major regulators of cellular cementum formation, was largely decreased in OCCM-30 cells lacking TßRII. Consequently, in those cells, functional ALP activity and the expression of genes associated with cementogenesis were reduced and the cells were partially rescued by Osx transduction. We also found that TGF-ß signaling directly regulates Osx expression through a Smad-dependent pathway. These findings strongly suggest that TGF-ß signaling plays a major role as one of the upstream regulators of Osx in cementoblast differentiation and cementum formation.

Osterix controls cementoblast differentiation through downregulation of Wnt-signaling via enhancing DKK1 expression.

Osterix (Osx), a transcriptional factor essential for osteogenesis, is also critical for in vivo cellular cementum formation. However, the molecular mechanism by which Osx regulates cementoblasts is largely unknown. In this study, we initially demonstrated that overexpression of Osx in a cementoblast cell line upregulated the expression of markers vital to cementogenesis such as osteopontin (OPN), osteocalcin (OCN), and bone sialoprotein (BSP) at both mRNA and protein levels, and enhanced alkaline phosphatase (ALP) activity. Unexpectedly, we demonstrated a sharp increase in the expression of DKK1 (a potent canonical Wnt antagonist), and a great reduction in protein levels of ß-catenin and its nuclear translocation by overexpression of Osx. Further, transient transfection of Osx reduced protein levels of TCF1 (a target transcription factor of ß-catenin), which were partially reversed by an addition of DKK1. We also demonstrated that activation of canonical Wnt signaling by LiCl or Wnt3a significantly enhanced levels of TCF1 and suppressed the expression of OPN, OCN, and BSP, as well as ALP activity and formation of extracellular mineralized nodules. Importantly, we confirmed that there were a sharp reduction in DKK1 and a concurrent increase in ß-catenin in Osx cKO mice (crossing between the Osx loxP and 2.3 Col 1-Cre lines), in agreement with the in vitro data. Thus, we conclude that the key role of Osx in control of cementoblast proliferation and differentiation is to maintain a low level of Wnt-ß-catenin via direct up-regulation of DKK1.

Bone morphogenetic protein-2 gene controls tooth root development in coordination with formation of the periodontium.

Formation of the periodontium begins following onset of tooth-root formation in a coordinated manner after birth. Dental follicle progenitor cells are thought to form the cementum, alveolar bone and Sharpey's fibers of the periodontal ligament (PDL). However, little is known about the regulatory morphogens that control differentiation and function of these progenitor cells, as well as the progenitor cells involved in crown and root formation. We investigated the role of bone morphogenetic protein-2 (Bmp2) in these processes by the conditional removal of the Bmp2 gene using the Sp7-Cre-EGFP mouse model. Sp7-Cre-EGFP first becomes active at E18 in the first molar, with robust Cre activity at postnatal day 0 (P0), followed by Cre activity in the second molar, which occurs after P0. There is robust Cre activity in the periodontium and third molars by 2 weeks of age. When the Bmp2 gene is removed from Sp7(+) (Osterix(+)) cells, major defects are noted in root, cellular cementum and periodontium formation. First, there are major cell autonomous defects in root-odontoblast terminal differentiation. Second, there are major alterations in formation of the PDLs and cellular cementum, correlated with decreased nuclear factor IC (Nfic), periostin and α-SMA(+) cells. Third, there is a failure to produce vascular endothelial growth factor A (VEGF-A) in the periodontium and the pulp leading to decreased formation of the microvascular and associated candidate stem cells in the Bmp2-cKO(Sp7-Cre-EGFP). Fourth, ameloblast function and enamel formation are indirectly altered in the Bmp2-cKO(Sp7-Cre-EGFP). These data demonstrate that the Bmp2 gene has complex roles in postnatal tooth development and periodontium formation.

Strontium-containing mesoporous bioactive glass scaffolds with improved osteogenic/cementogenic differentiation of periodontal ligament cells for periodontal tissue engineering.

To achieve the ultimate goal of periodontal tissue engineering, it is of great importance to develop bioactive scaffolds which can stimulate the osteogenic/cementogenic differentiation of periodontal ligament cells (PDLCs) for the favorable regeneration of alveolar bone, root cementum and periodontal ligament. Strontium (Sr) and Sr-containing biomaterials have been found to induce osteoblast activity. However, there has been no systematic report about the interaction between Sr or Sr-containing biomaterials and PDLCs for periodontal tissue engineering. The aims of this study were to prepare Sr-containing mesoporous bioactive glass (Sr-MBG) scaffolds and investigate whether the addition of Sr could stimulate osteogenic/cementogenic differentiation of PDLCs in a tissue-engineering scaffold system. The composition, microstructure and mesopore properties (specific surface area, nanopore volume and nanopore distribution) of Sr-MBG scaffolds were characterized. The proliferation, alkaline phosphatase (ALP) activity and osteogenesis/cementogenesis-related gene expression (ALP, Runx2, Col I, OPN and CEMP1) of PDLCs on different kinds of Sr-MBG scaffolds were systematically investigated. The results show that Sr plays an important role in influencing the mesoporous structure of MBG scaffolds in which high contents of Sr decreased the well-ordered mesopores as well as their surface area/pore volume. Sr(2+) ions could be released from Sr-MBG scaffolds in a controlled way. The incorporation of Sr into MBG scaffolds has significantly stimulated ALP activity and osteogenesis/cementogenesis-related gene expression of PDLCs. Furthermore, Sr-MBG scaffolds in a simulated body fluid environment still maintained excellent apatite-mineralization ability. The study suggests that the incorporation of Sr into MBG scaffolds is a viable way to stimulate the biological response of PDLCs. Sr-MBG scaffolds are a promising bioactive material for periodontal tissue-engineering applications.

The stimulation of proliferation and differentiation of periodontal ligament cells by the ionic products from Ca7Si2P2O16 bioceramics.

The ultimate goal of periodontal tissue engineering is to produce predictable regeneration of alveolar bone, root cementum, and periodontal ligament, which are lost as a result of periodontal diseases. To achieve this goal, it is of great importance to develop novel bioactive materials which could stimulate the proliferation, differentiation and osteogenic/cementogenic gene expression of periodontal ligament cells (PDLCs) for periodontal regeneration. In this study, we synthesized novel Ca(7)Si(2)P(2)O(16) ceramic powders for the first time by the sol-gel method and investigated the biological performance of PDLCs after exposure to different concentrations of Ca(7)Si(2)P(2)O(16) extracts. The original extracts were prepared at 200 mg ml(-1) and further diluted with serum-free cell culture medium to obtain a series of diluted extracts (100, 50, 25, 12.5 and 6.25 mg ml(-1)). Proliferation, alkaline phosphatase (ALP) activity, Ca deposition, and osteogenesis/cementogenesis-related gene expression (ALP, Col I, Runx2 and CEMP1) were assayed for PDLCs on days 7 and 14. The results showed that the ionic products from Ca(7)Si(2)P(2)O(16) powders significantly stimulated the proliferation, ALP activity, Ca deposition and osteogenesis/cementogenesis-related gene expression of PDLCs. In addition, it was found that Ca(7)Si(2)P(2)O(16) powders had excellent apatite-mineralization ability in simulated body fluids. This study demonstrated that Ca(7)Si(2)P(2)O(16) powders with such a specific composition possess the ability to stimulate the PDLC proliferation and osteoblast/cemenoblast-like cell differentiation, indicating that they are a promising bioactive material for periodontal tissue regeneration application.

Genetic evidence for the vital function of Osterix in cementogenesis.

To date, attempts to regenerate a complete tooth, including the critical periodontal tissues associated with the tooth root, have not been successful. Controversy still exists regarding the origin of the cell source for cellular cementum (epithelial or mesenchymal). This disagreement may be partially due to a lack of understanding of the events leading to the initiation and development of the tooth roots and supportive tissues, such as the cementum. Osterix (OSX) is a transcriptional factor essential for osteogenesis, but its role in cementogenesis has not been addressed. In the present study, we first documented a close relationship between the temporal- and spatial-expression pattern of Osx and the formation of cellular cementum. We then generated 3.6-kilobase (kb) collagen type I (3.6-kb Col 1)-Osx transgenic mice, which displayed accelerated cementum formation versus wild-type (WT) controls. Importantly, the conditional deletion of Osx in the mesenchymal cells with two different Cre systems (the 2.3-kb Col 1 and an inducible CAG-Cre estrogen receptor [CreER]) led to a sharp reduction in cellular cementum formation (including the cementum mass and mineral deposition rate) and gene expression of dentin matrix protein 1 (DMP1) by cementocytes. However, the deletion of the Osx gene after cellular cementum formed did not alter the properties of the mature cementum as evaluated by backscattered scanning electron microscopy (SEM) and resin-casted SEM. Transient transfection of Osx in the cementoblasts in vitro significantly inhibited cell proliferation and increased cell differentiation and mineralization. Taken together, these data support: (1) the mesenchymal origin of cellular cementum (from periodontal ligament [PDL] progenitor cells); (2) the vital role of OSX in controlling the formation of cellular cementum; and (3) the limited remodeling of cellular cementum in adult mice.

Effect of PGE2 induced by compressive and tensile stresses on cementoblast differentiation in vitro.

OBJECTIVE: The aim of the study was to clarify the mechanisms underlying orthodontically induced root resorption by characterizing the role of PGE(2) induced by compressive stress (CS) and tensile stress (TS) on cementoblast metabolism in vitro. DESIGN: Mouse cementoblast cell line OCCM-30 was continuously stimulated with 0.2 KPa CS or 5.0 KPa TS. COX-2 mRNA expression and PGE(2) production were thus quantified. In addition, cells were treated with COX-2 inhibitor and the role of PGE(2) induced by CS or TS on the expression of genes related to cementoblast differentiation was examined. PGE(2) receptors mRNA expression induced by CS or TS was also evaluated. Moreover, cells were treated with exogenous PGE(2) and the role of PGE(2) concentration on matrix mineralization was verified. RESULTS: CS and TS enhanced COX-2 mRNA expression and PGE(2) production. PGE(2) synthesis, however, was markedly induced by CS. Gene expression of bone morphogenetic protein 2 (BMP-2), osteocalcin (OCN) and receptor activator for nuclear factor kappaB ligand (RANKL) was enhanced by CS on an endogenous PGE(2)-mediated manner. Osteoprotegerin (OPG) expression was not affected by CS. Meanwhile, TS up-regulated the expression of BMP-2 and alkaline phosphatase (ALP) on an endogenous PGE(2)-mediated manner. TS down-regulated RANKL mRNA expression, whilst OPG expression was not affected. Moreover, EP4 mRNA expression was considerably enhanced by TS. Regarding PGE(2) concentration, only cells treated with low concentration presented anabolic response. CONCLUSIONS: Gene expression was differentially regulated according to the type of mechanical stimulation applied to cementoblasts. In addition, it is shown that PGE(2) plays an important role on mediating cementoblast mechanosensitivity.

Aberrant cementum phenotype associated with the hypophosphatemic hyp mouse.

BACKGROUND: Cementogenesis is sensitive to altered local phosphate levels; thus, we hypothesized a cementum phenotype, likely of decreased formation, would be present in the teeth of X-linked hypophosphatemic (Hyp) mice. Mutations in the phosphate-regulating gene with homologies to endopeptidases on the X chromosome (Phex) cause X-linked hypophosphatemia, characterized by rickets, osteomalacia, and hypomineralized dentin formation, a phenotype recapitulated in the Hyp mouse homolog. Here, we report a developmental study of tooth root formation in Hyp mouse molars, focusing on dentin and cementum. METHODS: Light and transmission electron microscopy were used to study molar tissues from wild-type (WT) and Hyp mice. Demineralized and hematoxylin and eosin-stained tissues at developmental stages 23 to 96 days postcoital (dpc) were examined by light microscopy. Immunohistochemistry methods were used to detect bone sialoprotein (BSP) distribution in Hyp and WT mouse molar tissues, and transmission electron microscopy was used to study similar molar tissues in the non-demineralized state. RESULTS: Dentin in Hyp mice exhibited mineralization defects by 33 dpc, as expected, but this defect was partially corrected by 96 dpc. In support of our hypothesis, a cementum phenotype was detected using a combination of immunohistochemistry and transmission electron microscopy, which included thinner BSP-positive staining within the cementum, discontinuous mineralization, and a globular appearance compared to WT controls. CONCLUSION: Mutations in the phosphate-regulating Phex gene of the Hyp mouse resulted in defective cementum development.

Structure and mechanical properties of Ank/Ank mutant mouse dental tissues--an animal model for studying periodontal regeneration.

Enamel, dentine and cementum are dental tissues with distinct functional properties associated with their unique hierarchical structures. Some potential ways to repair or regenerate lost tooth structures have been revealed in our studies focused on examining teeth obtained from mice with mutations at the mouse progressive ankylosis (ank) locus. Previous studies have shown that mice with such mutations have decreased levels of extracellular inorganic pyrophosphate (PP(i)) at local sites resulting in ectopic calcification in joint areas and in formation of a significantly thicker cementum layer when compared with age-matched wild-type (WT) tissue [Ho AM, Johnson MD, Kingsley DM. Role of the mouse ank gene in control of tissue calcification and arthritis. Science 2000;289:265-70; Nociti Jr FH, Berry JE, Foster BL, Gurley KA, Kingsley DM, Takata T, et al. Cementum: a phosphate-sensitive tissue. J Dent Res 2002;81:817-21]. As a next step, to determine the quality of the cementum tissue formed in mice with a mutation in the ank gene (ank/ank), we compared the microstructure and mechanical properties of cementum and other dental tissues in mature ank/ank vs. age-matched WT mice. Backscattered scanning electron microscopy (SEM) imaging and transmission electron microscopy (TEM) analyses on mineralized tissues revealed no decrease in the extent of mineralization between ank/ank cementum vs. WT controls. Atomic-force-microscopy-based nanoindentation performed on enamel, dentine or cementum of ank/ank vs. age-matched WT molars revealed no significant difference in any of the tested tissues in terms of hardness and elastic modulus. These results indicate that the tissue quality was not compromised in ank/ank mice despite faster rate of formation and more abundant cementum when compared with age-matched WT mice. In conclusion, these data suggest that this animal model can be utilized for studies focused on defining mechanisms to promote cementum formation without loss of mechanical integrity.

Nfic gene disruption inhibits differentiation of odontoblasts responsible for root formation and results in formation of short and abnormal roots in mice.

BACKGROUND: Nuclear factor I genes play an important role in the development of the brain, lung, and roots of teeth. We had reported that Nfic-deficient mice form normal crowns, but abnormal roots of molar teeth. However, the mechanism by which the disruption of Nfic gene causes abnormal root formation remains unknown. METHODS: To understand this mechanism, the root formation in Nfic-deficient mice was examined and compared to that of wild-type mice by morphological, immunohistochemical, and in situ hybridization analyses. RESULTS: Nfic-deficient mice formed normal Hertwig's epithelial root sheath (HERS) but severely disrupted odontoblast differentiation, leading to the formation of aberrant odontoblasts in the early stage of root formation. They became dissociated and polygonal in shape, lost their orientation and polarity, and did not express dentin sialophosphoprotein. The abnormal roots contained trapped aberrant odontoblasts, thereby resembling osteodentin in overall morphology. No osteoclasts were associated with abnormal roots. Further, the abnormal roots exhibited a decreased number of cementoblasts and cementum formation on the root surface. CONCLUSIONS: The loss of Nfic did not interfere with the formation of HERS, but it caused disrupted odontoblast differentiation, which resulted in the formation of short and abnormal roots, and decreased cementum. This finding suggests that root dentin is required for normal cementum formation. Therefore, Nfic may be a key regulator of root odontoblast differentiation and root formation.