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.

CKIP-1 mediates P. gingivalis-suppressed osteogenic/cementogenic differentiation of periodontal ligament cells partially via p38 signaling pathway.

Objectives: Casein kinase 2 interacting protein-1 (CKIP-1) is a versatile player involved in various biological processes. However, whether CKIP-1 mediates the osteogenic/cementogenic differentiation of periodontal ligament cells (PDLCs) under Porphyromonas gingivalis (Pg) stimulation remains unknown. Material and Methods: The effect of Pg on PDLC differentiation was first verified. CKIP-1 expression in Pg-infected PDLCs or in PDL of apical periodontitis (AP) mice was detected. The changes of CKIP-1 during PDLC differentiation was also determined. PDLC differentiation capacity in CKIP-1 knockout (KO) mice and CKIP-1-silenced PDLCs with or without Pg stimulation were further studied. Inhibitor was finally applied to verify the involvement of p38 signaling pathway in PDLC differentiation. Results: The suppression effect of Pg on PDLC differentiation was demonstrated. CKIP-1 increased in the PDL of AP mice and Pg-induced PDLCs, and decreased gradually during PDLC differentiation. Increased OSX and RUNX2 expression in PDL were observed in CKIP-1 KO mice. Also, CKIP-1 silencing facilitated and rescued Pg-inhibited PDLC differentiation. Inhibitor for p38 signaling pathway blocked CKIP-1 silencing-facilitated PDLC differentiation. Conclusions: CKIP-1 mediated the osteogenic/cementogenic differentiation of PDLCs partially through p38 signaling pathway, which may provide evidence for the regeneration of periodontal hard tissues damaged by Pg.

Effect of human periodontal ligament stem cell-derived exosomes on cementoblast activity.

OBJECTIVES: Exosomes derived from stem cells are a potential cell-free tool for tissue regeneration with therapeutic potential. However, its application in cementum repair is unclear. This study aimed to investigate the effect of human periodontal ligament stem cell-derived exosomes on the biological activity of cementoblasts, the main effector cells in cementum synthesis. MATERIALS AND METHODS: OCCM-30 cementoblasts were cultured with various human periodontal ligament stem cell-derived exosome concentrations. OCCM-30 cells proliferation, migration, and cementogenic mineralization were examined, along with the gene and protein expression of factors associated with cementoblastic mineralization. RESULTS: Exosomal promoted the migration, proliferation, and mineralization of OCCM-30 cells. The exosome-treated group significantly increased the expression of cementogenic-related genes and proteins. Furthermore, the expression of p-PI3K and p-AKT was enhanced by exosome administration. Treatment with a PI3K/AKT inhibitor markedly attenuated the gene and protein expression of cementoblastic factors, and this effect was partially reversed by exosome administration. CONCLUSIONS: Human periodontal ligament stem cell-derived exosomes can promote the activity of cementoblasts via the PI3K/AKT signaling pathway, providing a scientific basis for promoting the repair process in orthodontically induced inflammatory root resorption.

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.

Human Dental Pulp Stem Cells Differentiate into Cementoid-Like-Secreting Cells on Decellularized Teeth Scaffolds.

Periodontitis is a common inflammatory disease that in some cases can cause tooth loss. Cementum is a mineralized tissue that forms part of the insertion periodontium and serves to fix the teeth to the alveolar bone. In addition, it acts as a reservoir of different growth and differentiation factors, which regulate the biology of the teeth. Cementogenesis is a complex process that is still under investigation and involves different factors, including dentin sialophosphoprotein (DSPP). In this work we studied the role of surface microtopography in the differentiation of human dental pulp stem cells (hDPSCs) into cementoid-like secreting cells. We cultured hDPSCs on decellularized dental scaffolds on either dentin or cementum surfaces. Cell morphology was evaluated by light and electron microscopy. We also evaluated the DSPP expression by immunohistochemistry. The hDPSCs that was cultured on surfaces with accessible dentinal tubules acquired an odontoblastic phenotype and emitted characteristic processes within the dentinal tubules. These cells synthesized the matrix components of a characteristic reticular connective tissue, with fine collagen fibers and DSPP deposits. The hDPSCs that was cultured on cementum surfaces generated a well-organized tissue consisting of layers of secretory cells and dense fibrous connective tissue with thick bundles of collagen fibers perpendicular to the scaffold surface. Intra- and intercellular deposits of DSPP were also observed. The results presented here reinforce the potential for hDPSCs to differentiate in vitro into cells that secrete a cementoid-like matrix in response to the physical stimuli related to the microtopography of contact surfaces. We also highlight the role of DSPP as a component of the newly formed matrix.

Ciliary Neurotrophic Factor (CNTF) Inhibits In Vitro Cementoblast Mineralization and Induces Autophagy, in Part by STAT3/ERK Commitment.

In animal models, the administration of ciliary neurotrophic factor (CNTF) was demonstrated to reduce bone mass and to participate in bone remodeling. Cementoblasts, a cell type embedded in the cementum, are the main cells to produce and mineralize the extracellular matrix. The effect of CNTF on cementoblasts has not yet been addressed. Thus, the goal of this in vitro study was to investigate possible influences of exogenous CNTF on cementogenesis, as well as autophagy regulation and subsequent mechanisms in cementoblasts. Cementoblasts (OCCM-30) were stimulated with exogenous CNTF. Alizarin Red staining was performed to analyze the functional differentiation (mineralization) of OCCM-30 cells. The release of OPG was quantified by ELISA. The expression of cementogenesis markers (RUNX-2, OCN, BMP-7, BSP, and SPON-2) was evaluated by RT-qPCR. Western blotting (WB) was performed for the protein expression of STAT3, COX-2, SHP-2, cPLAα, cPLAß; ERK1/2, P38, and JNK. The autophagic flux was assessed using WB and RT-qPCR analysis of LC3A/B, Beclin-1, and Atg-5, and the autophagosome was investigated by immunofluorescence staining (IF). The ERK1/2 (FR180204) or STAT3 (sc-202818) antagonist was added, and the cellular response was analyzed using flow cytometry. Exogenous CNTF significantly attenuated mineralized nodule formation, impaired OPG release, and downregulated the mRNA levels of RUNX-2, OCN, BMP-7, and BSP. Moreover, CNTF induced the phosphorylation of STAT3 and activated a transient activation of SHP-2, cPLAß, ERK1/2, P38, and JNK protein. CNTF also induced autophagosome formation and promoted autophagy-associated gene and protein expressions. Additionally, the inhibition of ERK1/2 or STAT3 reversed a CNTF-induced mineralization impairment and had regulatory effects on CNTF-induced autophagosome formation. Our data revealed that CNTF acts as a potent inhibitor of cementogenesis, and it can trigger autophagy, in part by ERK1/2 and STAT3 commitment in the cementoblasts. Thus, it may play an important role in inducing or facilitating inflammatory root resorption during orthodontic tooth movement.

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.

Effect of Low-Level Er: YAG (2940 nm) laser irradiation on the photobiomodulation of mitogen-activated protein kinase cellular signaling pathway of rodent cementoblasts.

BACKGROUNDS: Dental avulsion due to trauma, especially in young patients, is a worldwide problem, requiring tooth replacement. Delayed replantation could cause tooth loss when the cementum is severely damaged. A small number of studies has reported that photobiomodulation (PBM) therapy using Er: YAG laser irradiation activates cellular signaling responses in different cell types, resulting in a variety of favorable biological effects. The aim of this in vitro study was to evaluate the potential biostimulatory effect of low-level Er: YAG laser irradiation on the biological responses of cultured mouse cementoblasts (OCCM-30), including the mitogen-activated protein kinases (MAPKs). METHODS: OCCM-30 cells were exposed to 2940 nm Er: YAG laser irradiation for 15 s at 0.34 W (pulse duration of 100 or 1000 µs, 17 mJ/pulse) at energy densities of 1 or 2 J/cm2. Irradiated and non-irradiated OCCM-30 cells were tested for migration (Scratch assay), proliferation (MTS assay) and functional differentiation (Alizarin Red S assay). Lumican (Lum) and Fibromodulin (Fmod) gene expression, and activation of MAPKs, were assessed by RT-PCR and Western blotting, respectively. RESULTS: Low-level Er: YAG laser irradiation at 2 J/cm2 and pulse duration of 1000 µs resulted in the highest migration rate and proliferation. Moreover, the pulse duration irradiation of 100 µs increased Lum expression. Fmod expression was increased after 1000 µs pulse duration laser stimulation. Low-level Er: YAG laser irradiation increased the mineralization of OCCM-30 cells after 7 days and activated ERK1/2, P38 and JNK signaling. CONCLUSIONS: Low-level Er: YAG laser irradiation induces OCCM-30 cell migration, proliferation and differentiation, and activates the MAPK signaling pathway.

Compressive force-induced LincRNA-p21 inhibits mineralization of cementoblasts by impeding autophagy.

The mineralization capability of cementoblasts is the foundation for repairing orthodontic treatment-induced root resorption. It is essential to investigate the regulatory mechanism of mineralization in cementoblasts under mechanical compression to improve orthodontic therapy. Autophagy has a protective role in maintaining cell homeostasis under environmental stress and was reported to be involved in the mineralization process. Long noncoding RNAs are important regulators of biological processes, but their functions in compressed cementoblasts during orthodontic tooth movement remain unclear. In this study, we showed that compressive force downregulated the expression of mineralization-related markers. LincRNA-p21 was strongly enhanced by compressive force. Overexpression of lincRNA-p21 downregulated the expression of mineralization-related markers, while knockdown of lincRNA-p21 reversed the compressive force-induced decrease in mineralization. Furthermore, we found that autophagy was impeded in compressed cementoblasts. Then, overexpression of lincRNA-p21 decreased autophagic activity, while knockdown of lincRNA-p21 reversed the autophagic process decreased by mechanical compression. However, the autophagy inhibitor 3-methyladenine abolished the lincRNA-p21 knockdown-promoted mineralization, and the autophagy activator rapamycin rescued the mineralization inhibited by lincRNA-p21 overexpression. Mechanistically, the direct binding between lincRNA-p21 and FoxO3 blocked the expression of autophagy-related genes. In a mouse orthodontic tooth movement model, knockdown of lincRNA-p21 rescued the impeded autophagic process in cementoblasts, enhanced cementogenesis, and alleviated orthodontic force-induced root resorption. Overall, compressive force-induced lincRNA-p21 inhibits the mineralization capability of cementoblasts by impeding the autophagic process.

Ckip-1 Mediates P. gingivalis-Suppressed Cementoblast Mineralization.

Porphyromonas gingivalis is responsible for the destruction of cementum in patients with periodontitis and periapical periodontitis. However, research about the effects of P. gingivalis on cementoblast mineralization and the underlying mechanism is still lacking. Casein kinase 2 interacting protein 1 (Ckip-1) is a scaffold protein that interacts with various proteins and signals to regulate different cell functions, such as cell morphology, apoptosis, and differentiation. In this study, we verified the suppressive effects of P. gingivalis and lipopolysaccharide (Pg-LPS) on OCCM-30 mineralization. We also showed that Ckip-1 gradually decreased during OCCM-30 mineralization but increased with the aggravation of Pg-induced inflammation. However, it remained unchanged when cells were stimulated with Pg-LPS, regardless of the concentration and incubation time. Then, more cellular cementum and enhanced Osterix expression were observed in Ckip-1 knockout mice when compared with the wild-type mice. Meanwhile, Ckip-1 silencing significantly enhanced cementoblast mineralization with or without P. gingivalis-associated inflammation. The trend was opposite when Ckip-1 was overexpressed. Finally, we found that the p38, Akt, and Wnt pathways were activated, while the Erk1/2 pathway was inhibited when Ckip-1 was silenced. The opposite results were also observed in the Ckip-1 overexpression group. Furthermore, we proved that cell mineralization was weakened when p38, Akt inhibitors were applied and strengthened when the Erk1/2 pathway was inhibited. In summary, Ckip-1 is upregulated underP. gingivalis-induced inflammation and negatively regulates cementoblast mineralization partially through mitogen-activated protein kinases and Akt signaling pathways, which may contribute to the restoration of cementum destroyed by P. gingivalis.

Protease-activated receptor type 1 (PAR1) increases CEMP1 gene expression through MAPK/ERK pathway

Abstract: PAR1 is a G-coupled protein receptor that regulates several cellular metabolism processes, including differentiation and proliferation of osteogenic and cementogenic related cells and our group previously demonstrated the regenerative potential of PAR1 in human periodontal ligament stem cells (hPDLSCs). In this study, we hypothesized that PAR1 regulates the cementogenic differentiation of hPDLSCs. Our goal was to identify the intracellular signaling pathway underlying PAR1 activation in hPDSLC differentiation. hPDLSCs were isolated using the explant technique. Cells were cultured in an osteogenic medium (OST) (α-MEM, 15% fetal bovine serum, L-glutamine, penicillin, streptomycin, amphotericin B, dexamethasone, and beta-glycerophosphate). The hPDLSCs were treated with a specific activator of PAR1 (PAR1 agonist) and blockers of the MAPK/ERK and PI3K pathways for 2 and 7 days. The gene expression of CEMP1 was assessed by RT-qPCR. The activation of PAR1 by its agonist peptide led to an increase in CEMP1 gene expression when compared with OST control. MAPK/ERK blockage abrogated the upregulation of CEMP1 gene expression induced by PAR1 agonist (p < 0.05). PI3K blockage did not affect the gene expression of CEMP1 at any experimental time (p > 0.05). We concluded that CEMP1 gene expression increased by PAR1 activation is MAPK/ERK-dependent and PI3K independent, suggesting that PAR1 may regulate cementogenetic differentiation of hPDLSCs.

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.

Delivery of Alkaline Phosphatase Promotes Periodontal Regeneration in Mice.

Factors regulating the ratio of pyrophosphate (PPi) to phosphate (Pi) modulate biomineralization. Tissue-nonspecific alkaline phosphatase (TNAP) is a key promineralization enzyme that hydrolyzes the potent mineralization inhibitor PPi. The goal of this study was to determine whether TNAP could promote periodontal regeneration in bone sialoprotein knockout mice (Ibsp-/- mice), which are known to have a periodontal disease phenotype. Delivery of TNAP was accomplished either systemically (through a lentiviral construct expressing a mineral-targeted TNAP-D10 protein) or locally (through addition of recombinant human TNAP to a fenestration defect model). Systemic TNAP-D10 delivered by intramuscular injection at 5 d postnatal (dpn) increased circulating alkaline phosphatase (ALP) levels in Ibsp-/- mice by 5-fold at 30 dpn, with levels returning to normal by 60 dpn when tissues were evaluated by micro-computed tomography and histology. Local delivery of recombinant human TNAP to fenestration defects in 5-wk-old wild type (WT) and Ibsp-/- mice did not alter long-term circulating ALP levels, and tissues were evaluated by micro-computed tomography and histology at postoperative day 45. Systemic and local delivery of TNAP significantly increased alveolar bone volume (20% and 37%, respectively) and cementum thickness (3- and 42-fold) in Ibsp-/- mice, with evidence for periodontal ligament attachment and bone/cementum marker localization. Local delivery significantly increased regenerated cementum and bone in WT mice. Addition of 100-µg/mL bovine intestinal ALP to culture media to increase ALP in vitro increased media Pi concentration, mineralization, and Spp1 and Dmp1 marker gene expression in WT and Ibsp-/- OCCM.30 cementoblasts. Use of phosphonoformic acid, a nonspecific inhibitor of sodium Pi cotransport, indicated that effects of bovine intestinal ALP on mineralization and marker gene expression were in part through Pi transport. These findings show for the first time through multiple in vivo and in vitro approaches that pharmacologic modulation of Pi/PPi metabolism can overcome periodontal breakdown and accomplish regeneration.

Ablation of Pyrophosphate Regulators Promotes Periodontal Regeneration.

Biomineralization is regulated by inorganic pyrophosphate (PPi), a potent physiological inhibitor of hydroxyapatite crystal growth. Progressive ankylosis protein (ANK) and ectonucleotide pyrophosphatase/phosphodiesterase 1 (ENPP1) act to increase local extracellular levels of PPi, inhibiting mineralization. The periodontal complex includes 2 mineralized tissues, cementum and alveolar bone (AB), both essential for tooth attachment. Previous studies demonstrated that loss of function of ANK or ENPP1 (reducing PPi) resulted in increased cementum formation, suggesting PPi metabolism may be a target for periodontal regenerative therapies. To compare the effects of genetic ablation of Ank, Enpp1, and both factors concurrently on cementum and AB regeneration, mandibular fenestration defects were created in Ank knockout (Ank KO), Enpp1 mutant (Enpp1asj/asj), and double KO (dKO) mice. Genetic ablation of Ank, Enpp1, or both factors increased cementum regeneration compared to controls at postoperative days (PODs) 15 and 30 (Ank KO: 8-fold, 3-fold; Enpp1asj/asj: 7-fold, 3-fold; dKO: 11-fold, 4-fold, respectively) associated with increased fluorochrome labeling and expression of mineralized tissue markers, dentin matrix protein 1 (Dmp1/DMP1), osteopontin (Spp1/OPN), and bone sialoprotein (Ibsp/BSP). Furthermore, dKO mice featured increased cementum thickness compared to single KOs at POD15 and Ank KO at POD30. No differences were noted in AB volume between genotypes, but osteoblast/osteocyte markers were increased in all KOs, partially mineralized osteoid volume was increased in dKO versus controls at POD15 (3-fold), and mineral density was decreased in Enpp1asj/asj and dKOs at POD30 (6% and 9%, respectively). Increased numbers of osteoclasts were present in regenerated AB of all KOs versus controls. These preclinical studies suggest PPi modulation as a potential and novel approach for cementum regeneration, particularly targeting ENPP1 and/or ANK. Differences in cementum and AB regeneration in response to reduced PPi conditions highlight the need to consider tissue-specific responses in strategies targeting regeneration of the entire periodontal complex.

Identification of regulatory mRNA and microRNA for differentiation into cementoblasts and periodontal ligament cells.

OBJECTIVE: Periodontitis causes periodontal tissue destruction and results in physiological tooth dysfunction. Therefore, periodontal regeneration is ideal therapy for periodontitis. Mesenchymal stem cells (MSCs) are useful for periodontal regenerative therapy as they can differentiate into periodontal cells; however, the underlying regulatory mechanism is unclear. In this study, we attempted to identify regulatory genes involved in periodontal cell differentiation and clarify the differentiation mechanism for effective periodontal regenerative therapy. BACKGROUND: The cementum and periodontal ligament play important roles in physiological tooth function. Therefore, cementum and periodontal ligament regeneration are critical for periodontal regenerative therapy. Mesenchymal stem cell transplantation can be a common periodontal regenerative therapy because these cells have multipotency and self-renewal ability, which induces new cementum or periodontal ligament formation. Moreover, MSCs can differentiate into cementoblasts. Cementoblast- or periodontal ligament cell-specific proteins have been reported; however, it is unclear how these proteins are regulated. MicroRNA (miRNA) can also act as a key regulator of MSC function. Therefore, in this study, we identified regulatory genes involved in cementoblast or periodontal cell differentiation and commitment. METHODS: Human MSCs (hMSCs), cementoblasts (HCEM), and periodontal ligament cells (HPL cells) were cultured, and mRNA or miRNA expression was evaluated. Additionally, cementoblast-specific genes were overexpressed or suppressed in hMSCs and their expression levels were investigated. RESULTS: HCEM and HPL cells expressed characteristic genes, of which we focused on ets variant 1 (ETV1), miR-628-5p, and miR-383 because ETV1 is a differentiation-related transcription factor, miR-628-5p was the second-highest expressed gene in HCEM and lowest expressed gene in HPL cells, and miR-383 was the highest expressed gene in HCEM. miR-628-5p and miR-383 overexpression in hMSCs regulated ETV1 mRNA expression, and miR-383 overexpression downregulated miR-628-5p expression. Moreover, miR-383 suppression decreased miR-383 expression and enhanced ETV1 mRNA expression, but miR-383 suppression also decreased miR-628-5p. Furthermore, silencing of ETV1 expression in hMSCs regulated miR-628-5p and miR-383 expression. Concerning periodontal cell commitment, miR-628-5p, miR-383, and ETV1 regulated the expression of HCEM- or HPL cell-related genes by adjusting the expression of these miRNAs. CONCLUSION: HCEM and HPL cells show characteristic mRNA and miRNA profiles. In particular, these cells have specific miR-383, miR-628-5p, and ETV1 expression patterns, and these genes interact with each other. Therefore, miR-383, miR-628-5p, and ETV1 are key genes involved in cementogenesis or HPL cell differentiation.

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.

Isolation and characterization of dental follicle-derived Hertwig's epithelial root sheath cells.

OBJECTIVES: The aim of this study was the isolation and characterization of dental follicle-derived Hertwig's epithelial root sheath cells (DF-HERSCs). MATERIALS AND METHODS: DF-HERSCs were isolated from dental follicle (DF)-derived single-cell suspensions. Their epithelial phenotypes were analyzed by Western blotting, polymerase chain reaction (PCR), and quantitative polymerase chain reaction (qPCR). Epithelial-mesenchymal transition (EMT) was induced in DF-HERSCs by treatment with transforming growth factor-ß (TGF-ß) or fetal bovine serum (FBS)-added medium. Characteristics of DF-HERSCs were compared with normal human oral keratinocytes (NHOKs) and normal human epidermal keratinocytes (NHEKs). Osteogenic differentiation and mineralization of DF-HERSCs were analyzed by alkaline phosphatase (ALP) and Alizarin red staining. All experiments were conducted in triplicate. RESULTS: Primary DF-HERSCs were isolated from DF. Epithelial phenotypes of DF-HERSCs were confirmed by morphological and Western blot analysis. PCR results demonstrated that the origin of DF-HERSCs was neither endothelial nor hematopoietic. Enamel matrix derivative (EMD)-associated genes were not expressed in DF-HERSCs. Treatment with TGF-ß and FBS-added medium triggered the progression of EMT in DF-HERSCs. The acquired potency of differentiation and mineralization was shown in EMT-progressed DF-HERSCs. CONCLUSIONS: DF contains putative populations of HERSC, named DF-HERSC. DF-HERSCs shared common characteristics with NHOKs and NHEKs.

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.

Tooth Formation: Are the Hardest Tissues of Human Body Hard to Regenerate?

With increasing life expectancy, demands for dental tissue and whole-tooth regeneration are becoming more significant. Despite great progress in medicine, including regenerative therapies, the complex structure of dental tissues introduces several challenges to the field of regenerative dentistry. Interdisciplinary efforts from cellular biologists, material scientists, and clinical odontologists are being made to establish strategies and find the solutions for dental tissue regeneration and/or whole-tooth regeneration. In recent years, many significant discoveries were done regarding signaling pathways and factors shaping calcified tissue genesis, including those of tooth. Novel biocompatible scaffolds and polymer-based drug release systems are under development and may soon result in clinically applicable biomaterials with the potential to modulate signaling cascades involved in dental tissue genesis and regeneration. Approaches for whole-tooth regeneration utilizing adult stem cells, induced pluripotent stem cells, or tooth germ cells transplantation are emerging as promising alternatives to overcome existing in vitro tissue generation hurdles. In this interdisciplinary review, most recent advances in cellular signaling guiding dental tissue genesis, novel functionalized scaffolds and drug release material, various odontogenic cell sources, and methods for tooth regeneration are discussed thus providing a multi-faceted, up-to-date, and illustrative overview on the tooth regeneration matter, alongside hints for future directions in the challenging field of regenerative dentistry.