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.

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.

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.

Long noncoding RNA expression profiles in intermittent parathyroid hormone induced cementogenesis.

The aim of this study was to explore the involvement of long noncoding RNAs (lncRNAs) during intermittent parathyroid hormone (PTH) induced cementogenesis. Expression profiles of lncRNAs and mRNAs were obtained using high-throughput microarray. Gene Ontology enrichment analysis, Kyoto Encyclopedia of Genes and Genomes pathway analysis, and coding-noncoding gene coexpression networks construction were performed. We identified 190 lncRNAs and 135 mRNAs that were differentially expressed during intermittent PTH-induced cementogenesis. In this process, the Wnt signaling pathway was negatively regulated, and eight lncRNAs were identified as possible core regulators of Wnt signaling. Based on the results of microarrray analysis, we further verified the repressed expression of Wnt signaling crucial components ß-catenin, APC and Axin2. Above all, we speculated that lncRNAs may play important roles in PTH-induced cementogenesis via the negative regulation of Wnt pathway.

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.

Yes-associated protein promotes tumour necrosis factor α-treated cementoblast mineralization partly by inactivating NF-κB pathway.

Cementum regeneration, as one of the most difficult challenges of periodontal regeneration, is influenced by inflammatory factors. Inflammation may hamper or promote periodontal tissue repair under different circumstances, as it is found to do in dentin-pulp complex and bone tissue. Our team demonstrated that YAP promotes mineralization of OCCM, a cementoblast cell line. However, the effect of YAP on its mineralization under inflammatory microenvironment is unclear. In this study, cementogenesis in vitro was up-regulated after transient TNF-α treatment for 30 minutes. YAP expression also was increased by TNF-α treatment. YAP overexpression promoted OCCM mineralization after the cells were transiently treated with TNF-α because YAP overexpression inhibited NF-κB pathway activity, while YAP knockdown elevated it. The inhibited mineralization potential and activated NF-κB pathway activity by YAP knockdown also were partly rescued by the application of the NF-κB inhibitor Bay 11-7082. These results demonstrated that YAP plays a positive role in the mineralization of TNF-α transiently treated cementoblast, partly by inhibiting the NF-κB pathway activity.

Effect of interleukin-33 on cementoblast-mediated cementum repair during orthodontic tooth movement.

OBJECTIVE: This study aims to uncover the role of interleukin-33 on cementoblast-mediated cementum repair. METHODS: 6-8-week-old C57BL/6 mice were used to establish the model of orthodontic tooth movement. Interleukin-33 and suppression of tumorigenicity2 (ST2) expressions were immunohistochemically detected in the periodontal tissue. In vitro, cementoblast-like (OCCM-30) cells were cultured in the presence of recombinant mouse interleukin-33 protein (rmIL-33) at a 1-14 d time frame. ST2 expressions were immunofluorescently labeled and quantitatively examined. The effects of interleukin-33 on cementoblast differentiation, mineralization and proliferation were examined by alkaline phosphatase, alizarin red staining and cell counting kit-8, respectively. To further clarify the effect of interleukin-33 on cementogenesis-related protein expressions, runt-related transcription factor 2 (RUNX2), osterix, osteopontin, bone sialoprotein(BSP), osteocalcin, osteoprotegerin (OPG) and receptor activator of NF-КB ligand (RANKL) expressions were examined by western blot. RESULTS: Orthodontic load of high magnitude induces external apical root resorption, and increases interleukin-33 expression in the periodontal tissue of mice. Cells in the cementum express ST2. Interleukin-33 initially down-regulates but later recovers ST2 mRNA and protein levels in OCCM-30 cells. Interleukin-33 abates cementoblast differentiation and mineralization, and suppresses RUNX2, osterix, BSP and osteopontin expressions in OCCM-30 cells at the later stage of the culture period. Interleukin-33 enhances RANKL expression, and reduces the ratio of OPG/RANKL in OCCM-30 cells. CONCLUSION: Orthodontic load of high magnitude induces interleukin-33 expression in the periodontal tissue. Interleukin-33 has a negative effect on cementogenesis via suppressing cementoblast differentiation, mineralization and cementogenesis-related protein expressions.

IL1ß inhibits differentiation of cementoblasts via microRNA-325-3p.

Cementum regeneration is considered the gold standard for the treatment of periodontitis. As one of the most important primary proinflammatory cytokines, interleukin 1ß (IL1ß) plays an essential role during the early stage of periodontitis and its amounts simultaneously increase dramatically during this stage. Though promising, the differentiation of cementoblasts upon IL1ß-induced inflammation of the microenvironment and the relative interaction mechanism are still unknown. Here, we found that IL1ß inhibited cementoblast differentiation and microRNA-325-3p (miR-325-3p) was increased during IL1ß-stimulated cementoblasts. Bioinformatics analysis and luciferase reporter assay demonstrated miR-325-3p targeted runt-related transcription factor 2 directly. Transfection of miR-325-3p suppressed cementoblast differentiation in vitro and the formation of cementum-like tissues in vivo. The inhibitor of miR-325-3p could mitigate the above effects induced by IL1ß. Accordingly, our finding suggests a critical role of miR-325-3p in linking inflammation to impaired cementum regeneration and provides a potential possibility for applying miR-325-3p inhibitors in the treatment of periodontitis-related bone loss.

Axin2+-Mesenchymal PDL Cells, Instead of K14+ Epithelial Cells, Play a Key Role in Rapid Cementum Growth.

To date, attempts to regenerate functional periodontal tissues (including cementum) are largely unsuccessful due to a lack of full understanding about the cellular origin (epithelial or mesenchymal cells) essential for root cementum growth. To address this issue, we first identified a rapid cementum growth window from the ages of postnatal day 28 (P28) to P56. Next, we showed that expression patterns of Axin2 and ß-catenin within cementum-forming periodontal ligament (PDL) cells are negatively associated with rapid cementum growth. Furthermore, cell lineage tracing studies revealed that the Axin2+-mesenchymal PDL cells and their progeny rapidly expand and directly contribute to postnatal acellular and cellular cementum growth. In contrast, the number of K14+ epithelial cells, which were initially active at early stages of development, was reduced during rapid cementum formation from P28 to P56. The in vivo cell ablation of these Axin2+ cells using Axin2CreERT2/+; R26RDTA/+ mice led to severe cementum hypoplasia, whereas constitutive activation of ß-catenin in the Axin2+ cells resulted in an acceleration in cellular cementogenesis plus a transition from acellular cementum to cellular cementum. Thus, we conclude that Axin2+-mesenchymal PDL cells, instead of K14+ epithelial cells, significantly contribute to rapid cementum growth.

Transcription factor 7-like 2-associated signaling mechanism in regulating cementum generation by the NF-κB pathway.

Cementum regeneration is an important and challenging stage in periodontal tissue engineering and regeneration. Pathosis of the periodontium, including cementum, is important in precision diagnosis and obstinate treatment of systemic diseases, such as diabetes, leukemia, and Acquired Immune Deficiency Syndrome. Here, we found that during periodontium development, transcription factor 7-like 2 (Tcf7l2) was widely expressed in the periodontium and dental sac. In mouse cementoblast cell line (OCCM-30), the activation of NF-κB and cementoblast mineralization was significantly reduced when Tcf7l2 gene was silenced. Moreover, Tcf7l2 has a positive effect on NF-κB and cementoblast mineralization. Therefore, Tcf7l2 promotes cementum formation through the NF-κB pathway. In addition, we found a decreased expression of phosphorylated p65 and a thin layer of cementum in Tcf7l2fl/fl mice. These results suggest that Tcf7l2, which accelerates cementum formation by activating NF-κB, has great potential in the treatment of periodontitis and provide guidance for periodontal tissue regeneration.

Developmental pathways of periodontal tissue regeneration: Developmental diversities of tooth morphogenesis do also map capacity of periodontal tissue regeneration?

Nothing is known on the impact of developmental divergence on periodontal tissue regeneration in vertebrate animals. Molecularly, the induction of tooth morphogenesis is highly conserved deploying across animal phyla a constant and reproducible set of gene pathways, which result in morphogenesis of multiple odontode forms and shapes. Genetic mutations positively affect animal speciation via evolving biting and masticatory forces as well as dietary habits selectively imprinted in animal phyla during evolutionary speciation. The geometry of the attachment apparatus of a tooth is important for the interpretation of the induction of cementogenesis with de novo Sharpey's fibres as in thecodonty, ie, a tripartite attachment of alveolar bone, periodontal ligament and cementum. This review addresses the tooth implantation in different animal clades from the fibrous attachment of the Elasmobranch Carcharinus obscurus dusky shark, reviewing the evolution and functional significance of cementum with functionally inserted Sharpey's fibres. In sharks there is a continuous tooth replacement mechanistically supported by the continuously erupting dental lamina. We show that the arching of the continuously erupting dental lamina, a critical step for the selachians' tooth differentiation, is prominently characterized by transforming growth factor-ß3 (TGF-ß3 ) expression not only within the dental lamina but also in cellular condensations in the mesenchymal tissues of the erupting tooth. Such findings indicate the pleiotropic multifaceted activity of a highly conserved mammalian gene across genera, masterminding tooth morphogenesis in both selachians and mammals as well as periodontal tissue induction in the non-human primate Papio ursinus. In P. ursinus, the induction of cementogenesis entails the expression of TGF-ß3 and osteocalcin with fine-tuning and regulation of bone morphogenetic proteins BMP-2 and BMP-7, and upregulation of TGF-ß3 . TGF-ß3 autoinduction and upregulation during the induction of cementogenesis and osteogenesis in P. ursinus provide novel insights into the induction of cementogenesis. It is hypothesized that the evolutionary expression and upregulation of the TGF-ß3 gene may provide the mechanistic insights into the induction of extensive cementogenesis as seen in stem mammals and the induction of trabecular-like cementum formation in mosasaurs' tooth attachment. Aspidin, the precursor of cementum, was reported to appear 310-330 million years ago (Ma) in Odontostraci armoured fish. Studies showed that the differentiation of cementum with inserted Sharpey's fibres is also present in lower amniotes such as Diatectomorpha or Diadectidae, the first herbivorous tetrapods, 323 Ma. In mosasaurs, 168-165 Ma, there is the induction of extensive trabeculation of cementum though nothing is known on the phylogenetic temporo-spatial evolution of cementum before Diadectidae and stem mammals. The large trabeculations of cementum as seen in the attachment of extinct mosasaurs invocates a pleiotropic capacity of cemental growth previously unknown. The appearance of cementum facing a vascularized and innervated periodontal ligament space with Sharpey's fibres inserting on to mineralized cementum provides a multiform pleiotropic masticatory apparatus adapted to multiple biting and lacerating forces as well as finely tuned and controlled forces beyond mastication and deglutition. The remarkable cementogenesis as seen in stem mammals but particularly in mosasaurs with cemental trabeculations across the ligament space invocates the developmental capacity of cementum. The large cemental trabeculations as seen in mosasaurs and the cemental growth in stem mammals, together with regenerating scenarios in P. ursinus with large seams of cellular cementum and cementoid populated by contiguous cementoblasts indicate the continuous molecular cross-talk between cementum, newly formed cementoid matrix, cementoblasts and extracellular matrix soluble molecular signals. This molecular cross-talk may control the biomolecular homeostasis of both cementum and periodontal ligament, including angiogenesis. A further molecular scenario is invocated by the tight and exquisite anatomical relationships between the cementoid surfaces and the newly formed capillaries. The primitiveness of the craniate masticatory mineralized craniofacial apparatus has been controlled by several yet ancestral common genes not lastly the TGF-ß3 gene. The TGF-ß3 might have been responsible for the induction of cementogenesis not only in extant P. ursinus but also in Diatectomorpha and mosasaurs, thus providing continuous evolutionary mechanisms for the induction of tissue morphogenesis across animal phyla for almost a billion years of evolution, epitomizing Nature's parsimony in controlling tissue induction and morphogenesis. TGF-ß receptor II regulates osterix expression via Smad-dependent pathways indicating that TGF-ß signalling acts as an upstream regulator of osterix during cementoblast differentiation. The presence of morphogenetic signals within the cemental matrix capable of inducing bone formation needs now to be assigned: bone induction initiated by extracted and partially purified cemental matrices may be the result of a slow release of embryonic remnants of osteogenic signals required and deployed during cementogenesis. The cementum may thus rule the periodontal ligament space homeostasis, remodelling and repair by releasing sequestered morphogenetic signals that were deployed during embryogenesis.

Recombinant amelogenin regulates the bioactivity of mouse cementoblasts in vitro.

Amelogenin (AMG) is a cell adhesion molecule that has an important role in the mineralization of enamel and regulates events during dental development and root formation. The purpose of the present study was to investigate the effects of recombinant human AMG (rhAMG) on mineralized tissue-associated genes in cementoblasts. Immortalized mouse cementoblasts (OCCM-30) were treated with different concentrations (0.1, 1, 10, 100, 1000, 10,000, 100,000 ng · mL-1) of recombinant human AMG (rhAMG) and analyzed for proliferation, mineralization and mRNA expression of bone sialoprotein (BSP), osteocalcin (OCN), collagen type I (COL I), osteopontin (OPN), runt-related transcription factor 2 (Runx2), cementum attachment protein (CAP), and alkaline phosphatase (ALP) genes using quantitative RT-PCR. The dose response of rhAMG was evaluated using a real-time cell analyzer. Total RNA was isolated on day 3, and cell mineralization was assessed using von Kossa staining on day 8. COL I, OPN and lysosomal-associated membrane protein-1 (LAMP-1), which is a cell surface binding site for amelogenin, were evaluated using immunocytochemistry. F-actin bundles were imaged using confocal microscopy. rhAMG at a concentration of 100,000 ng · mL-1 increased cell proliferation after 72 h compared to the other concentrations and the untreated control group. rhAMG (100,000 ng · mL-1) upregulated BSP and OCN mRNA expression levels eightfold and fivefold, respectively. rhAMG at a concentration of 100,000 ng · mL-1 remarkably enhanced LAMP-1 staining in cementoblasts. Increased numbers of mineralized nodules were observed at concentrations of 10,000 and 100,000 ng · mL-1 rhAMG. The present data suggest that rhAMG is a potent regulator of gene expression in cementoblasts and support the potential application of rhAMG in therapies aimed at fast regeneration of damaged periodontal tissue.

Yes-associated protein 1 promotes the differentiation and mineralization of cementoblast.

Yes-associated protein 1 (YAP1) transcriptional coactivator is a mediator of mechanosensitive signaling. Cementum, which covers the tooth root surface, continuously senses external mechanical stimulation. Cementoblasts are responsible for the mineralization and maturation of the cementum. However, the effect of YAP1 on cementoblast differentiation remains largely unknown. In this study, we initially demonstrated that YAP1 overexpression enhanced the mineralization ability of cementoblasts. YAP1 upregulated the mRNA and protein expression of several cementogenesis markers, such as alkaline phosphatase (ALP), runt-related transcription factor 2 (Runx2), osteocalcin (OCN), and dentin matrix acidic phosphoprotein 1 (DMP1). The YAP1 overexpression group showed higher intensities of ALP and Alizarin red stain than the YAP1-knockdown group. Unexpectedly, a sharp increase in the expression of dentin sialophosphoprotein (DSPP) was induced by the overexpression of YAP1. Knockdown of YAP1 suppressed DSPP transcriptional activity. YAP1 overexpression activated Smad-dependent BMP signaling and slightly inhibited Erk1/2 signaling pathway activity. Treatment with specific BMP antagonist (LDN193189) prevented the upregulation of the mRNA levels of ALP, RUNX2, and OCN, as well as intensity of ALP-stained and mineralized nodules in cementoblasts. The Erk1/2 signaling pathway inhibitor (PD 98,059) upregulated these cementogenesis markers. Thus, our study suggested that YAP1 enhanced cementoblast mineralization in vitro. YAP1 exerted its effect on the cementoblast partly by regulating the Smad-dependent BMP and Erk1/2 signaling pathways.

MicroRNA-155-3p Mediates TNF-α-Inhibited Cementoblast Differentiation.

Periodontitis is a prevalent and chronic inflammatory disease that is interrelated with systemic health. Periodontitis can be promoted by tumor necrosis factor α (TNF-α). Cementum, a vital part of the periodontium, is a bone-like mineralized tissue that is produced by cementoblasts. Our laboratory previously revealed that TNF-α inhibits cementoblast differentiation and mineralization. However, how TNF-α modulates cementoblast differentiation and mineralization remains largely unknown. MicroRNA-155 (miR-155) is induced and regulates TNF-α-inhibited osteogenic differentiation. In this study, we found that miR-155-3p was increased during TNF-α-stimulated OCCM-30 cells and involved in cementoblast differentiation and mineralization. Overexpression of miR-155-3p suppressed cementoblast mineralization. Bioinformatics analysis revealed that potassium channel tetramerization domain containing 1 ( Kctd1) is a candidate target gene of miR-155-3p. Moreover, miR-155-3p overexpression suppressed KCTD1 levels. Meanwhile, its knockdown increased KCTD1 expression. Transfection with miR-155-3p also inhibited the luciferase activity of 3'-untranslated regions in the Kctd1 wild type but not the mutant. These data indicated that Kctd1 is a direct and novel target of miR-155-3p. The Wnt signaling pathway inhibits cementoblast differentiation, and we further demonstrated that miR-155-3p partially modulates cementoblast differentiation through the canonical Wnt signaling pathway. In addition to the gain/loss function assay of miR-155-3p, the luciferase activity assay of canonical Wnt signaling was performed. The assays revealed that miR-155-3p increased ß-catenin-mediated transcriptional activation. Overall, our data clarified that miR-155-3p mediated TNF-α-inhibited cementoblast differentiation by targeting Kctd1, at least partially through canonical Wnt signaling pathway. These findings reveal the expanded function of miRNAs in cementoblast differentiation and mineralization.

Localization of RELM-ß/FIZZ2 Is Associated with Cementum Formation.

Resistin-like molecule-ß/found in inflammatory zone 2 (RELM-ß/FIZZ2) is a cysteine-rich secretory protein that is localized in the epithelium of the gastrointestinal tract and lung alveoli. Previous reports have suggested that this protein regulates glucose metabolism and inflammation. In the present study, to analyze the involvement of RELM-ß/FIZZ2 in tooth development, we immunohistochemically examined the localization of RELM-ß/FIZZ2 in tooth germs of embryonic days (E) 15-20 and postnatal days (P) 7-42 rats. RELM-ß/FIZZ2 was hardly detected in the tooth germ at the bud (E15) stage. However, at the cap (E17) and bell (E20) stages, this protein was detectable in the inner enamel epithelium; whereas cells in the other parts of the enamel organ including the outer enamel epithelium and stellate reticulum did not show the immunoreactivity. During the root formation stage (P14-28), cells in Hertwig's epithelial root sheath (HERS) localized RELM-ß/FIZZ2. Intense immunoreactivity was also seen in the matrix of the root dentin facing the HERS and the dental follicle. This reactivity was not present on the more upwardly located dentin surface. In contrast, cementum matrix positive for osteopontin and bone sialoprotein was observed on the dentin instead of immunoreactivity for RELM-ß/FIZZ2. Osterix-positive cells, indicating cementoblast progenitors, were also detected in the dental follicle near the matrix positive for RELM-ß/FIZZ2. These results suggest that RELM-ß/FIZZ2 secreted by the inner enamel epithelium was mainly localized in the matrix at the surface of the apical root dentin and might be involved in cementogenesis. Anat Rec, 2017. © 2017 Wiley Periodicals, Inc. Anat Rec, 300:1865-1874, 2017. © 2017 Wiley Periodicals, Inc.

Efficacy of stem cells on periodontal regeneration: Systematic review of pre-clinical studies.

This systematic review aims to evaluate mesenchymal stem cells (MSC) periodontal regenerative potential in animal models. MEDLINE, EMBASE and LILACS databases were searched for quantitative pre-clinical controlled animal model studies that evaluated the effect of local administration of MSC on periodontal regeneration. The systematic review was conducted according to the Preferred Reporting Items for Systematic Reviews and Meta-Analyses statement guidelines. Twenty-two studies met the inclusion criteria. Periodontal defects were surgically created in all studies. In seven studies, periodontal inflammation was experimentally induced following surgical defect creation. Differences in defect morphology were identified among the studies. Autogenous, alogenous and xenogenous MSC were used to promote periodontal regeneration. These included bone marrow-derived MSC, periodontal ligament (PDL)-derived MSC, dental pulp-derived MSC, gingival margin-derived MSC, foreskin-derived induced pluripotent stem cells, adipose tissue-derived MSC, cementum-derived MSC, periapical follicular MSC and alveolar periosteal cells. Meta-analysis was not possible due to heterogeneities in study designs. In most of the studies, local MSC implantation was not associated with adverse effects. The use of bone marrow-derived MSC for periodontal regeneration yielded conflicting results. In contrast, PDL-MSC consistently promoted increased PDL and cementum regeneration. Finally, the adjunct use of MSC improved the regenerative outcomes of periodontal defects treated with membranes or bone substitutes. Despite the quality level of the existing evidence, the current data indicate that the use of MSC may provide beneficial effects on periodontal regeneration. The various degrees of success of MSC in periodontal regeneration are likely to be related to the use of heterogeneous cells. Thus, future studies need to identify phenotypic profiles of highly regenerative MSC populations.

Cementogenesis is inhibited under a mechanical static compressive force via Piezo1.

OBJECTIVE: To investigate whether Piezo1, a mechanotransduction gene mediates the cementogenic activity of cementoblasts under a static mechanical compressive force. MATERIALS AND METHODS: Murine cementoblasts (OCCM-30) were exposed to a 2.0 g/cm2 static compressive force for 3, 6, 12, and 24 hours. Then the expression profile of Piezo1 and the cementogenic activity markers osteoprotegerin (Opg), osteopontin (Opn), osteocalcin (Oc), and protein tyrosine phosphataselike member A (Ptpla) were analyzed. Opg, Opn, Oc, and Ptpla expression was further measured after using siRNA to knock down Piezo1. Real-time PCR, Western blot, and cell proliferation assays were performed according to standard procedures. RESULTS: After mechanical stimulation, cell morphology and proliferation did not change significantly. The expression of Piezo1, Opg, Opn, Oc, and Ptpla was significantly decreased, with a high positive correlation between Opg and Piezo1 expression. After Piezo1 knockdown, the expression of Opg, Opn, Oc, and Ptpla was further decreased under mechanical stimulation. CONCLUSIONS: Cementogenic activity was inhibited in OCCM-30 cells under static mechanical force, a process that was partially mediated by the decrease of Piezo1. This study provides a new viewpoint of the pathogenesis mechanism of orthodontically induced root resorption and repair.

Cemento dental: aspectos atuais de interesse para o clínico / Dental cementum: current aspects to the clinicians

Embora tenha havido avanço no entendimento da homeostase do cemento dental, o papel deste tecido e sua biologia permanecem não completamente elucidados. Este estudo buscou fornecer informações sobre os conhecimentos mais recente relacionados à biologia do cemento dental, com o objetivo de discutir o papel exercido por este tecido em condições não fisiológicas nos tecidos periodontais. Devido aos avanços na exploração do tecido ósseo, que compartilha diversas características similares, a pesquisa abrangente sobre o cemento dental tem sido encorajada, a fim de esclarecer a função completa deste tecido na homeostase periodontal e regeneração. Desta forma, no presente trabalho, sempre que possível será feito um paralelo entre osso alveolar e cemento dental. O desenvolvimento de metodologias e técnicas celulares e moleculares avançadas possibilitou um melhor entendimento do comportamento do cemento em situações diversas, como quando em situações patológicas, como a doença periodontal, e até mesmo frente à regeneração tecidual. Ademais, estudos clínicos e em modelo animal demonstraram resultados em relação à formação de cemento em abordagens regenerativas. No entanto, sugere-se que estudos posteriores possam contribuir para um melhor conhecimento sobre o cemento e o perfil celular dos cementoblastos e cementócitos, bem como suas interações para fornecer novos insights para o desenvolvimento de terapias eficientes e mais previsíveis para regeneração dos tecidos periodontais. Apesar dos avanços dos estudos clínicos e laboratoriais, pôde-se concluir que inúmeras questões referentes à biologia do cemento permanecem não esclarecidas.

Although some progress has been made to understand dental cementum homeostasis, its role and biology remains not completely elucidated. This study aimed to provide information on the recent knowledge related to the dental cementum biology, in order to discuss the role of this tissue in physiological and non-physiological conditions in the periodontal tissues. Due to advances in the exploration of bone tissue, which shares several similar features, comprehensive research on dental cementum has been encouraged in order to clarify the complete function of this tissue in periodontal homeostasis and regenerative approach. Novel methodologies and advanced cellular and molecular techniques provided better understanding of cementum in different circumstances, as pathological situations such as periodontal disease and even tissue regeneration. In addition, clinical and animal model designs show positive outcomes to cementum formation in regenerative approaches, however, it is suggested that further studies may contribute to better understand cementum tissue and cementoblasts and cementocytes profile, as well as their interactions, providing new insights to develop efficient and more predictable therapies for periodontal tissue regeneration. Despite advances in clinical and laboratory studies, it can be concluded that many questions regarding the cementum biology remain unclear.

Heterogeneous Human Periodontal Ligament-Committed Progenitor and Stem Cell Populations Exhibit a Unique Cementogenic Property Under In Vitro and In Vivo Conditions.

An undesirable complication that arises during dental treatments is external apical-root resorption, which causes root-cementum and root-dentin loss. To induce de novo cementogenesis, stem cell therapy is required. Cementum-forming cells (cementoblasts) are known to be differentiated from periodontal-lineage mesenchymal stem cells (MSCs), which are derived from the dental follicle (DF) in developing tissues and the periodontal ligament (PDL) in adult tissues, but the periodontal-lineage MSC type that is optimal for inducing de novo cementogenesis remains unidentified, as does the method to isolate these cells from harvested tissues. Thus, we investigated the cementogenic potential of DF- and PDL-derived MSCs that were isolated by using two widely used cell-isolation methods: enzymatic digestion and outgrowth (OG) methods. DF- and PDL-derived cells isolated by using both methods proliferated actively, and all four isolated cell types showed MSC gene/protein expression phenotype and ability to differentiate into adipogenic and chondrogenic lineages. Furthermore, cementogenic-potential analysis revealed that all cell types produced alizarin red S-positive mineralized materials in in vitro cultures. However, PDL-OG cells presented unique cementogenic features, such as nodular formation of mineralized deposits displaying a cellular intrinsic fiber cementum-like structure, as well as a higher expression of cementoblast-specific genes than in the other cell types. Moreover, in in vivo transplantation experiments, PDL-OG cells formed cellular cementum-like hard tissue containing embedded osteocalcin-positive cells, whereas the other cells formed acellular cementum-like materials. Given that the root-cementum defect is likely regenerated through cellular cementum deposition, PDL-OG cell-based therapies might potentially facilitate the de novo cellular cementogenesis required for regenerating the root defect.

Synergistic acceleration in the osteogenic and angiogenic differentiation of human mesenchymal stem cells by calcium silicate-graphene composites.

Recent exciting findings of the biological interactions of graphene materials have shed light on potential biomedical applications of graphene-containing composites. Owing to the superior mechanical properties and low coefficient of thermal expansion, graphene has been widely used in the reinforcement of biocomposites. In the present study, various ratios of graphene (0.25wt%, 0.5wt% and 1.0wt%) were reinforced into calcium silicate (CS) for bone graft application. Results show that the graphene was embedded in the composites homogeneously. Adding 1wt% graphene into CS increased the young's modulus by ~47.1%. The formation of bone-like apatite on a range of composites with graphene weight percentages ranging from 0 to 1 has been investigated in simulated body fluid. The presence of a bone-like apatite layer on the composites surface after immersion in simulated body fluid was considered by scanning electron microscopy. In vitro cytocompatibility of the graphene-contained CS composites was evaluated using human marrow stem cells (hMSCs). The proliferation and alkaline phosphatase, osteopontin and osteocalcin osteogenesis-related protein expression of the hMSCs on the 1wt% graphene-contained specimens showed better results than on the pure CS. In addition, the angiogenesis-related protein (vWF and ang-1) secretion of cells was significantly stimulated when the graphene concentration in the composites was increased. These results suggest that graphene-contained CS bone graft are promising materials for bone tissue engineering applications.