Language：English   Publisher：エルゼビア・ジャパン(株)  

Language：English   Publishing type：Research paper (scientific journal)   Publisher：Journal of Dental Sciences  

BACKGROUND/PURPOSE: Actin alpha 2, smooth muscle (ACTA2) is an actin isoform that forms the cytoskeleton. Actin plays a crucial role in numerous cellular functions. ACTA2 is a marker of functional periodontal ligament (PDL) fibroblasts and is upregulated by transforming growth factor-β1 (TGF-β1); however, the underlying function of ACTA2 in PDL tissue is unknown. We aimed to examine the localization and potential function of ACTA2 in PDL tissues and cells. MATERIALS AND METHODS: RNA expression was determined using semi-quantitative reverse transcription-polymerase chain reaction (RT-PCR) and quantitative RT-PCR. Protein expression was determined using immunofluorescence staining and Western blot analysis. Soluble and insoluble collagen production was examined using the Sircol collagen assay and picrosirius red staining, respectively. Small interfering RNA (siRNA) was used for knockdown assay to examine the effect of ACTA2 in human PDL cells. RESULTS: ACTA2 expression was observed in human primary PDL cells and PDL cell line (2-23 cells). TGF-β1 upregulated ACTA2, collagen type Ⅰ alpha1 chain (COL1A1), periostin (POSTN), and fibrillin-Ⅰ(FBN1) expression and soluble and insoluble collagen production in 2-23 cells. However, ACTA2 depletion by siRNA strongly suppressed PDL-related gene expression and collagen production compared with those of TGF-β1-stimulated control cells. Furthermore, ACTA2 knockdown significantly suppressed the phosphorylation of Smad2 and Smad3. CONCLUSION: ACTA2 plays a crucial role in PDL-related marker expression and collagen production via Smad2/3 phosphorylation. Our findings might contribute to the development of novel and effective periodontal therapies.

DOI： 10.1016/j.jds.2022.08.030

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Language：Japanese   Publisher：日本総合歯科学会  

定期的な口腔内清掃を希望する患者が来院するが,歯周疾患の治癒・病状安定を維持している患者が多い一方,病状が進行し予後不良の歯が出現しているものの,様々な理由で治療や抜歯を希望しない患者もいる。今回,患者情報を参考に,様々なコミュニケーション技法を応用して患者と治療方針を検討した。その結果,抜歯を含めた全顎的な治療に取り組むことができた症例を経験したため報告する。症例は76歳,男性。「右上の歯が痛い。」という主訴で来院した。患者は九州大学病院口腔総合診療科で全顎的治療を行った後にSupportive Periodontal Therapy(SPT)へ移行したが,その後約5年にわたり「簡単に掃除をやってほしい。」との訴えが続いた。そのため,定期的な口内法X線写真撮影や歯周基本検査を含む,十分なSPTを行えていなかった。そこで,過去の患者情報から患者の考え方を考察し,(1)目標設定理論を応用し,何か処置をする前には,処置内容,所要時間,その処置が必要な理由をその都度伝えること,(2)"But You Are Free"(BYAF) compliance-gaining techniqueを応用した質問形式を取ること,(3)"Self Persuasion"自主説得理論を応用した治療の提案を行うことのコミュニケーション技法を応用して治療を開始した。その結果,患者は現症や治療方法を十分理解・納得し,予後不良と判断した歯の抜歯と,上顎即時義歯を製作することができた。以上のことから,患者情報や様々なコミュニケーション技法を応用することで,治療へ移行ができる可能性があると考えられた。(著者抄録)

Publisher：Lasers in Dental Science  

Introduction: Dental caries and apical periodontitis are ones of the most prevalent chronic diseases and involve infection by cariogenic and endodontic bacteria. It can be said that the most required to cure is disinfection. We hypothesized that NB-UVB could be used for intraoral disinfection without affecting cells, and that it could be used in combination with TiO2 to disinfect complex root canals. The objectives were to investigate the effects of UV on dental pulp cells and oral bacteria and to evaluate the enhancement effect of a photocatalyst on bactericidal effects of UV irradiation. Methods: UV irradiation devices of UVB (310, 285 nm) and UVC (265 nm) were prepared. Cell proliferation and cytotoxicity assays after UV irradiation were performed using human dental pulp cells. The antibacterial activity of UV irradiation was investigated in Streptococcus mutans, Staphylococcus aureus, Enterococcus faecalis, Actinomyces naeslundii, Porphyromonas gingivalis, and Fusobacterium nucleatum. A curved simulated root canal with plastic training block was used to evaluate the effect of combined UV and TiO2 treatment. Data were analyzed by one-way ANOVA (p < 0.05) followed by Tukey’s post hoc tests (p < 0.05). Results: Human dental pulp cell proliferation was decreased by 265 nm, 285 nm, and 310 nm UV irradiation, although 310 nm UV irradiation did not show cytotoxic effects on these cells. Oral bacterial growth was suppressed following UV irradiation at 285 nm and 265 nm. Viability of all bacteria significantly decreased with UV irradiation. In the curved simulated root canal, viability of E. faecalis in UV irradiation at 285 nm with long taper fibers was significantly decreased in the 300 s irradiation group. E. faecalis proliferation was inhibited by combined UV irradiation and TiO2 application with long taper fibers in the curved simulated root canal. Conclusions: The wavelength of UVB and UVC showed bactericidal effects on oral bacteria including caries-related bacteria and apical periodontitis–related bacteria while NB-UVB did not. UVB with longer fibers was more effective in disinfection on E. faecalis in curved simulated root canal, and the combined use of photocatalyst further improved the disinfection effect.

DOI： 10.1007/s41547-021-00145-8

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Language：English   Publishing type：Research paper (scientific journal)  

Language：English   Publishing type：Research paper (scientific journal)  

Adrenergic receptors (ARs) are receptors of noradrenalin and adrenalin, of which there are nine different subtypes. In particular, β2 adrenergic receptor (β2-AR) is known to be related to the restoration and maintenance of homeostasis in bone and cardiac tissues; however, the functional role of signaling through β2-AR in periodontal ligament (PDL) tissue has not been fully examined. In this report, we investigated that β2-AR expression in PDL tissues and their features in PDL cells. β2-AR expressed in rat PDL tissues and human PDL cells (HPDLCs) derived from two different patients (HPDLCs-2G and -3S). Rat PDL tissue with occlusal loading showed high β2-AR expression, while its expression was downregulated in that without loading. In HPDLCs, β2-AR expression was increased exposed to stretch loading. The gene expression of PDL-related molecules was investigated in PDL clone cells (2-23 cells) overexpressing β2-AR. Their gene expression and intracellular cyclic adenosine monophosphate (cAMP) levels were also investigated in HPDLCs treated with a specific β2-AR agonist, fenoterol (FEN). Overexpression of β2-AR significantly promoted the gene expression of PDL-related molecules in 2 to 23 cells. FEN led to an upregulation in the expression of PDL-related molecules and increased intracellular cAMP levels in HPDLCs. In both HPDLCs, inhibition of cAMP signaling by using protein kinase A inhibitor suppressed the FEN-induced gene expression of α-smooth muscle actin. Our findings suggest that the occlusal force is important for β2-AR expression in PDL tissue and β2-AR is involved in fibroblastic differentiation and collagen synthesis of PDL cells. The signaling through β2-AR might be important for restoration and homeostasis of PDL tissue.

Language：Japanese   Publishing type：Research paper (scientific journal)  

OBJECTIVE:
In this study, we measured the expression of R-spondin 2 (RSPO2) in periodontal ligament (PDL) tissue and cells. Further, we examined the effects of RSPO2 on osteoblastic differentiation of immature human PDL cells (HPDLCs).
BACKGROUND:
R-spondin (RSPO) family proteins are secreted glycoproteins that play important roles in embryonic development and tissue homeostasis through activation of the Wnt/β-catenin signaling pathway. RSPO2, a member of the RSPO family, has been reported to enhance osteogenesis in mice. However, little is known regarding the roles of RSPO2 in PDL tissues.
METHODS:
Expression of RSPO2 in rat PDL tissue and primary HPDLCs was examined by immunohistochemical and immunofluorescence staining, as well as by semiquantitative RT-PCR. The effects of stretch loading on the expression of RSPO2 and Dickkopf-related protein 1 (DKK1) were assessed by quantitative RT-PCR. Expression of receptors for RSPOs, such as Leucine-rich repeat-containing G-protein-coupled receptors (LGRs) 4, 5, and 6 in immature human PDL cells (cell line 2-14, or 2-14 cells), was investigated by semiquantitative RT-PCR. Mineralized nodule formation in 2-14 cells treated with RSPO2 under osteoblastic inductive condition was examined by Alizarin Red S and von Kossa stainings. Nuclear translocation of β-catenin and expression of active β-catenin in 2-14 cells treated with RSPO2 were assessed by immunofluorescence staining and Western blotting analysis, respectively. In addition, the effect of Dickkopf-related protein 1 (DKK1), an inhibitor of Wnt/β-catenin signaling, was also examined.
RESULTS:
Rat PDL tissue and HPDLCs expressed RSPO2, and HPDLCs also expressed RSPO2, while little was found in 2-14 cells. Expression of RSPO2 as well as DKK1 in HPDLCs was significantly upregulated by exposure to stretch loading. LGR4 was predominantly expressed in 2-14 cells, which expressed low levels of LGR5 and LGR6. RSPO2 enhanced the Alizarin Red S and von Kossa-positive reactions in 2-14 cells. In addition, DKK1 suppressed nuclear translocation of β-catenin, activation of β-catenin, and increases of Alizarin Red S and von Kossa-positive reactions in 2-14 cells, all of which were induced by RSPO2 treatment.
CONCLUSION:
RSPO2, which is expressed in PDL tissue and cells, might play an important role in regulating the osteoblastic differentiation of immature human PDL cells through the Wnt/β-catenin signaling pathway.

DOI： 10.1111/jre.12611

Language：Japanese   Publishing type：Research paper (scientific journal)  

Language：English   Publishing type：Research paper (scientific journal)  

Wnt5a, a non-canonical Wnt protein, is known to play important roles in several cell functions. However, little is known about the effects of Wnt5a on osteoblastic differentiation of periodontal ligament (PDL) cells. Here, we examined the effects of Wnt5a on osteoblastic differentiation and associated intracellular signaling in human PDL stem/progenitor cells (HPDLSCs). We found that Wnt5a suppressed expression of bone-related genes (ALP, BSP, and Osterix) and alizarin red-positive mineralized nodule formation in HPDLSCs under osteogenic conditions. Immunohistochemical analysis revealed that a Wnt5a-related receptor, receptor tyrosine kinase-like orphan receptor 2 (Ror2), was expressed in rat PDL tissue. Interestingly, knockdown of Ror2 by siRNA inhibited the Wnt5a-induced downregulation of bone-related gene expression in HPDLSCs. Moreover, Western blotting analysis showed that phosphorylation of the intracellular signaling molecule, c-Jun N-terminal kinase (JNK) was upregulated in HPDLSCs cultured in osteoblast induction medium with Wnt5a, but knockdown of Ror2 by siRNA downregulated the phosphorylation of JNK. We also examined the effects of JNK inhibition on Wnt5a-induced suppression of osteoblastic differentiation of HPDLSCs. The JNK inhibitor, SP600125 inhibited the Wnt5a-induced downregulation of bone-related gene expression in HPDLSCs. Additionally, SP600125 inhibited the Wnt5a-induced suppression of the alizarin red-positive reaction in HPDLSCs. These results suggest that Wnt5a suppressed osteoblastic differentiation of HPDLSCs through Ror2/JNK signaling. Non-canonical Wnt signaling, including Wnt5a/Ror2/JNK signaling, may function as a negative regulator of mineralization, preventing the development of non-physiological mineralization in PDL tissue.

Language：English   Publishing type：Research paper (scientific journal)  

Wnt5a, a non-canonical Wnt protein, is known to play important roles in several cell functions. However, little is known about the effects of Wnt5a on osteoblastic differentiation of periodontal ligament (PDL) cells. Here, we examined the effects of Wnt5a on osteoblastic differentiation and associated intracellular signaling in human PDL stem/progenitor cells (HPDLSCs). We found that Wnt5a suppressed expression of bone-related genes (ALP, BSP, and Osterix) and alizarin red-positive mineralized nodule formation in HPDLSCs under osteogenic conditions. Immunohistochemical analysis revealed that a Wnt5a-related receptor, receptor tyrosine kinase-like orphan receptor 2 (Ror2), was expressed in rat PDL tissue. Interestingly, knockdown of Ror2 by siRNA inhibited the Wnt5a-induced downregulation of bone-related gene expression in HPDLSCs. Moreover, Western blotting analysis showed that phosphorylation of the intracellular signaling molecule, c-Jun N-terminal kinase (JNK) was upregulated in HPDLSCs cultured in osteoblast induction medium with Wnt5a, but knockdown of Ror2 by siRNA downregulated the phosphorylation of JNK. We also examined the effects of JNK inhibition on Wnt5a-induced suppression of osteoblastic differentiation of HPDLSCs. The JNK inhibitor, SP600125 inhibited the Wnt5a-induced downregulation of bone-related gene expression in HPDLSCs. Additionally, SP600125 inhibited the Wnt5a-induced suppression of the alizarin red-positive reaction in HPDLSCs. These results suggest that Wnt5a suppressed osteoblastic differentiation of HPDLSCs through Ror2/JNK signaling. Non-canonical Wnt signaling, including Wnt5a/Ror2/JNK signaling, may function as a negative regulator of mineralization, preventing the development of non-physiological mineralization in PDL tissue.

DOI： 10.1002/jcp.26086

Language：English   Publishing type：Research paper (scientific journal)  

The periodontal ligament (PDL) plays an important role in anchoring teeth in the bone socket. Damage to the PDL, such as after severe inflammation, can be treated with a therapeutic strategy that uses stem cells derived from PDL tissue (PDLSCs), a strategy that has received intense scrutiny over the past decade. However, there is an insufficient number of PDLSCs within the PDL for treating such damage. Therefore, we sought to induce the differentiation of induced pluripotent stem (iPS) cells into PDLSCs as an initial step toward PDL therapy. To this end, we first induced iPS cells into neural crest (NC)-like cells. We then captured the p75 neurotrophic receptor-positive cells (iPS-NC cells) and cultured them on an extracellular matrix (ECM) produced by human PDL cells (iPS-NC-PDL cells). These iPS-NC-PDL cells showed reduced expression of embryonic stem cell and NC cell markers as compared with iPS and iPS-NC cells, and enrichment of mesenchymal stem cell markers. The cells also had a higher proliferative capacity, multipotency, and elevated expression of PDL-related markers than iPS-NC cells cultured on fibronectin and laminin (iPS-NC-FL cells) or ECM produced by human skin fibroblast cells (iPS-NC-SF cells). Overall, we present a culture method to produce high number of PDLSC-like cells from iPS cells as a first step toward a strategy for PDL regeneration.

Language：English   Publishing type：Research paper (scientific journal)  

The periodontal ligament (PDL) plays an important role in anchoring teeth in the bone socket. Damage to the PDL, such as after severe inflammation, can be treated with a therapeutic strategy that uses stem cells derived from PDL tissue (PDLSCs), a strategy that has received intense scrutiny over the past decade. However, there is an insufficient number of PDLSCs within the PDL for treating such damage. Therefore, we sought to induce the differentiation of induced pluripotent stem (iPS) cells into PDLSCs as an initial step toward PDL therapy. To this end, we first induced iPS cells into neural crest (NC)-like cells. We then captured the p75 neurotrophic receptor-positive cells (iPS-NC cells) and cultured them on an extracellular matrix (ECM) produced by human PDL cells (iPS-NC-PDL cells). These iPS-NC-PDL cells showed reduced expression of embryonic stem cell and NC cell markers as compared with iPS and iPS-NC cells, and enrichment of mesenchymal stem cell markers. The cells also had a higher proliferative capacity, multipotency, and elevated expression of PDL-related markers than iPS-NC cells cultured on fibronectin and laminin (iPS-NC-FL cells) or ECM produced by human skin fibroblast cells (iPS-NC-SF cells). Overall, we present a culture method to produce high number of PDLSC-like cells from iPS cells as a first step toward a strategy for PDL regeneration.

DOI： 10.1089/scd.2017.0077

Language：English   Publishing type：Research paper (scientific journal)  

Cellular senescence has been suggested to be involved in physiological changes of cytokine production. Previous studies showed that the concentration of tumor necrosis factor-α (TNF-α) is higher in the blood of aged people compared with that of young people. So far, the precise effects of TNF-α on the odontoblastic differentiation of pulp cells have been controversial. Therefore, we aimed to clarify how this cytokine affected pulp cells during aging. Human dental pulp cells (HDPCs) were cultured until reaching the plateau of their growth, and the cells were isolated at actively (young HDPCs; yHDPCs) or inactively (senescent HDPCs; sHDPCs) proliferating stages. sHDPCs expressed senescence-related molecules while yHDPCs did not. When these HDPCs were cultured in an odontoblast-inductive medium, both young and senescent cells showed mineralization, but mineralization in sHDPCs was lower compared with yHDPCs. However, the administration of TNF-α to this culture medium altered these responses: yHDPCs showed downregulated mineralization, while sHDPCs exhibited significantly increased mineralization. Furthermore, the expression of tumor necrosis factor receptor 1 (TNFR1), a receptor of TNF-α, was significantly upregulated in sHDPCs compared with yHDPCs. Downregulation of TNFR1 expression led to decreased mineralization of TNF-α-treated sHDPCs, whereas restored the reduction in TNF-α-treated yHDPCs. These results suggested that sHDPCs preserved the odontoblastic differentiation capacity and TNF-α promoted odontoblastic differentiation of HDPCs with the progress of their population doublings through increased expression of TNFR1. Thus, TNF-α might exert a different effect on the odontoblastic differentiation of HDPCs depending on their proliferating activity. In addition, the calcification of pulp chamber with age may be related with increased reactivity of pulp cells to TNF-α.

DOI： 10.1002/jcp.26905

Language：English   Publishing type：Research paper (scientific journal)  

Activation of the G protein-coupled calcium-sensing receptor (CaSR) has crucial roles in skeletal development and bone turnover. Our recent study has identified a role for activated CaSR in the osteogenic differentiation of human periodontal ligament stem cells. Furthermore, odontoblasts residing inside the tooth pulp chamber play a central role in dentin formation. However, it remains unclear how CaSR activation affects the odontoblastic differentiation of human dental pulp cells (HDPCs). We have investigated the odontoblastic differentiation of HDPCs exposed to elevated levels of extracellular calcium (Ca) and strontium (Sr), and the contribution of CaSR and the L-type voltage-dependent calcium channel (L-VDCC) to this process. Immunochemical staining of rat dental pulp tissue demonstrated that CaSR was expressed at high levels in the odontoblastic layer, moderate levels in the sublayer, and low levels in the central pulp tissue. Although normal HDPCs expressed low levels of CaSR, stimulation with Ca or Sr promoted both CaSR expression and odontoblastic differentiation of HDPCs along with increased expression of odontoblastic makers. These effects were inhibited by treatment with a CaSR antagonist, whereas treatment with an L-VDCC inhibitor had no effect. Additionally, knockdown of CaSR with siRNA suppressed odontoblastic differentiation of Ca- and Sr-treated HDPCs. ERK1/2 phosphorylation was observed in Ca- and Sr-treated HDPCs, whereas CaSR antagonist treatment or CaSR knockdown blocked ERK1/2 phosphorylation. Furthermore, inhibition of ERK1/2 suppressed mineralization of Ca- and Sr-treated HDPCs. These results suggest that elevated concentrations of extracellular Ca and Sr induce odontoblastic differentiation of HDPCs through CaSR activation and the ERK1/2 phosphorylation.

Language：English   Publishing type：Research paper (scientific journal)  

Activation of the G protein-coupled calcium-sensing receptor (CaSR) has crucial roles in skeletal development and bone turnover. Our recent study has identified a role for activated CaSR in the osteogenic differentiation of human periodontal ligament stem cells. Furthermore, odontoblasts residing inside the tooth pulp chamber play a central role in dentin formation. However, it remains unclear how CaSR activation affects the odontoblastic differentiation of human dental pulp cells (HDPCs). We have investigated the odontoblastic differentiation of HDPCs exposed to elevated levels of extracellular calcium (Ca) and strontium (Sr), and the contribution of CaSR and the L-type voltage-dependent calcium channel (L-VDCC) to this process. Immunochemical staining of rat dental pulp tissue demonstrated that CaSR was expressed at high levels in the odontoblastic layer, moderate levels in the sublayer, and low levels in the central pulp tissue. Although normal HDPCs expressed low levels of CaSR, stimulation with Ca or Sr promoted both CaSR expression and odontoblastic differentiation of HDPCs along with increased expression of odontoblastic makers. These effects were inhibited by treatment with a CaSR antagonist, whereas treatment with an L-VDCC inhibitor had no effect. Additionally, knockdown of CaSR with siRNA suppressed odontoblastic differentiation of Ca- and Sr-treated HDPCs. ERK1/2 phosphorylation was observed in Ca- and Sr-treated HDPCs, whereas CaSR antagonist treatment or CaSR knockdown blocked ERK1/2 phosphorylation. Furthermore, inhibition of ERK1/2 suppressed mineralization of Ca- and Sr-treated HDPCs. These results suggest that elevated concentrations of extracellular Ca and Sr induce odontoblastic differentiation of HDPCs through CaSR activation and the ERK1/2 phosphorylation.

DOI： 10.1016/j.bone.2017.05.012

Language：English   Publishing type：Research paper (scientific journal)  

Objective The aim of this study was to investigate transforming growth factor-β-induced gene product-h3 (βig-h3) expression in dental pulp tissue and its effects on odontoblastic differentiation of dental pulp cells (DPCs). Design A rat direct pulp capping model was prepared using perforated rat upper first molars capped with mineral trioxide aggregate cement. Human DPCs (HDPCs) were isolated from extracted teeth. βig-h3 expression in rat dental pulp tissue and HDPCs was assessed by immunostaining. Mineralization of HDPCs was assessed by Alizarin red-S staining. Odontoblast-related gene expression in HDPCs was analyzed by quantitative RT-PCR. Results Expression of βig-h3 was detected in rat dental pulp tissue, and attenuated by direct pulp capping, while expression of interleukin-1β and tumor necrosis factor-α was increased in exposed pulp tissue. βig-h3 expression was also detected in HDPCs, with reduced expression during odontoblastic differentiation. The above cytokines reduced βig-h3 expression in HDPCs, and promoted their mineralization. Recombinant βig-h3 inhibited the expression of odontoblast-related genes and mineralization of HDPCs, while knockdown of βig-h3 gene expression promoted the expression of odontoblast-related genes in HDPCs. Conclusions The present findings suggest that βig-h3 in DPCs may be involved in reparative dentin formation and that its expression is likely to negatively regulate this process.

DOI： 10.1016/j.archoralbio.2017.02.018

Language：English   Publishing type：Research paper (scientific journal)  

Glial cell line-derived neurotrophic factor (GDNF) is known to mediate multiple biological activities such as promotion of cell motility and proliferation, and morphogenesis. However, little is known about its effects on periodontal ligament (PDL) cells. Recently, we reported that GDNF expression is increased in wounded rat PDL tissue and human PDL cells (HPDLCs) treated with pro-inflammatory cytokines. Here, we investigated the associated expression of GDNF and the pro-inflammatory cytokine interleukin-1 beta (IL-1β) in wounded PDL tissue, and whether HPDLCs secrete GDNF which affects neurocytic differentiation. Rat PDL cells near the wounded area showed intense immunoreactions against an anti-GDNF antibody, where immunoreactivity was also increased against an anti-IL-1β antibody. Compared with untreated cells, HPDLCs treated with IL-1β or tumor necrosis factor-alpha showed an increase in the secretion of GDNF protein. Conditioned medium of IL-1β-treated HPDLCs (IL-1β-CM) increased neurite outgrowth of PC12 rat adrenal pheochromocytoma cells. The expression levels of two neural regeneration-associated genes, growth-associated protein-43 (Gap-43), and small proline-rich repeat protein 1A (Sprr1A), were also upregulated in IL-1β-CM-treated PC12 cells. These stimulatory effects of IL-1β-CM were significantly inhibited by a neutralizing antibody against GDNF. In addition, U0126, a MEK inhibitor, inhibited GDNF-induced neurite outgrowth of PC12 cells. These findings suggest that an increase of GDNF in wounded PDL tissue might play an important role in neural regeneration probably via the MEK/ERK signaling pathway. J. Cell. Biochem. 118: 699–708, 2017.

DOI： 10.1002/jcb.25662

Language：English   Publishing type：Research paper (scientific journal)  

OBJECTIVE:

The aim of this study was to investigate transforming growth factor-β-induced gene product-h3 (βig-h3) expression in dental pulp tissue and its effects on odontoblastic differentiation of dental pulp cells (DPCs).
DESIGN:

A rat direct pulp capping model was prepared using perforated rat upper first molars capped with mineral trioxide aggregate cement. Human DPCs (HDPCs) were isolated from extracted teeth. βig-h3 expression in rat dental pulp tissue and HDPCs was assessed by immunostaining. Mineralization of HDPCs was assessed by Alizarin red-S staining. Odontoblast-related gene expression in HDPCs was analyzed by quantitative RT-PCR.
RESULTS:

Expression of βig-h3 was detected in rat dental pulp tissue, and attenuated by direct pulp capping, while expression of interleukin-1β and tumor necrosis factor-α was increased in exposed pulp tissue. βig-h3 expression was also detected in HDPCs, with reduced expression during odontoblastic differentiation. The above cytokines reduced βig-h3 expression in HDPCs, and promoted their mineralization. Recombinant βig-h3 inhibited the expression of odontoblast-related genes and mineralization of HDPCs, while knockdown of βig-h3 gene expression promoted the expression of odontoblast-related genes in HDPCs.
CONCLUSIONS:

The present findings suggest that βig-h3 in DPCs may be involved in reparative dentin formation and that its expression is likely to negatively regulate this process.

Language：English   Publishing type：Research paper (scientific journal)  

In cases of pulp exposure due to deep dental caries or severe traumatic injuries, existing pulp-capping materials have a limited ability to reconstruct dentin-pulp complexes and can result in pulpectomy because of their low potentials to accelerate dental pulp cell activities, such as migration, proliferation, and differentiation. Therefore, the development of more effective therapeutic agents has been anticipated for direct pulp capping. Dental pulp tissues are enriched with dental pulp stem cells (DPSCs). Here, the authors investigated the effects of semaphorin 3A (Sema3A) on various functions of human DPSCs in vitro and reparative dentin formation in vivo in a rat dental pulp exposure model. Immunofluorescence staining revealed expression of Sema3A and its receptor Nrp1 (neuropilin 1) in rat dental pulp tissue and human DPSC clones. Sema3A induced cell migration, chemotaxis, proliferation, and odontoblastic differentiation of DPSC clones. In addition, Sema3A treatment of DPSC clones increased β-catenin nuclear accumulation, upregulated expression of the FARP2 gene (FERM, RhoGEF, and pleckstrin domain protein 2), and activated Rac1 in DPSC clones. Furthermore, in the rat dental pulp exposure model, Sema3A promoted reparative dentin formation with dentin tubules and a well-aligned odontoblast-like cell layer at the dental pulp exposure site and with novel reparative dentin almost completely covering pulp tissue at 4 wk after direct pulp capping. These findings suggest that Sema3A could play an important role in dentin regeneration via canonical Wnt/β-catenin signaling. Sema3A might be an alternative agent for direct pulp capping, which requires further study.

Language：English   Publishing type：Research paper (scientific journal)  

In cases of pulp exposure due to deep dental caries or severe traumatic injuries, existing pulp-capping materials have a limited ability to reconstruct dentin-pulp complexes and can result in pulpectomy because of their low potentials to accelerate dental pulp cell activities, such as migration, proliferation, and differentiation. Therefore, the development of more effective therapeutic agents has been anticipated for direct pulp capping. Dental pulp tissues are enriched with dental pulp stem cells (DPSCs). Here, the authors investigated the effects of semaphorin 3A (Sema3A) on various functions of human DPSCs in vitro and reparative dentin formation in vivo in a rat dental pulp exposure model. Immunofluorescence staining revealed expression of Sema3A and its receptor Nrp1 (neuropilin 1) in rat dental pulp tissue and human DPSC clones. Sema3A induced cell migration, chemotaxis, proliferation, and odontoblastic differentiation of DPSC clones. In addition, Sema3A treatment of DPSC clones increased β-catenin nuclear accumulation, upregulated expression of the FARP2 gene (FERM, RhoGEF, and pleckstrin domain protein 2), and activated Rac1 in DPSC clones. Furthermore, in the rat dental pulp exposure model, Sema3A promoted reparative dentin formation with dentin tubules and a well-aligned odontoblast-like cell layer at the dental pulp exposure site and with novel reparative dentin almost completely covering pulp tissue at 4 wk after direct pulp capping. These findings suggest that Sema3A could play an important role in dentin regeneration via canonical Wnt/β-catenin signaling. Sema3A might be an alternative agent for direct pulp capping, which requires further study.

DOI： 10.1177/0022034516653085

Language：English   Publishing type：Research paper (scientific journal)  

Wnt5a, a member of the noncanonical Wnt proteins, is known to play important roles in the development of various organs and in postnatal cell functions. However, little is known about the effects of Wnt5a on human periodontal ligament (PDL) cells. In this study, we examined the localization and potential function of Wnt5a in PDL tissue. Immunohistochemical analysis revealed that Wnt5a was expressed predominantly in rat PDL tissue. Semi-quantitative reverse-transcription polymerase chain reaction and Western blotting analysis demonstrated that human PDL cells (HPDLCs) expressed Wnt5a and its receptors (Ror2, Fzd2, Fzd4, and Fzd5). Removal of occlusal pressure by extraction of opposing teeth decreased Wnt5a expression in rat PDL tissue, and the expression of Wnt5a and its receptors in HPDLCs was upregulated by exposure to mechanical stress. Stimulation with Wnt5a significantly enhanced the proliferation and migration of HPDLCs. Furthermore, Wnt5a suppressed osteoblastic differentiation of HPDLCs cultivated in osteogenic induction medium, while it significantly enhanced the expression of PDL-related genes, such as periostin, type-I collagen, and fibrillin-1 genes, and the production of collagen in HPDLCs cultivated in normal medium. Both knockdown of periostin gene expression by siRNA and inhibition of TGFβ1 function by neutralizing antibody suppressed the Wnt5a-induced PDL-related gene expression and collagen production in HPDLCs. Interestingly, in HPDLCs cultured with Wnt5a, TGFβ1 neutralizing antibody significantly suppressed periostin expression, while periostin siRNA had no effect on TGFβ1 expression. These results suggest that Wnt5a expressed in PDL tissue plays specific roles in inducing collagen production by PDL cells through TGFβ1-mediated upregulation of periostin expression.

Language：English   Publishing type：Research paper (scientific journal)  

Wnt5a, a member of the noncanonical Wnt proteins, is known to play important roles in the development of various organs and in postnatal cell functions. However, little is known about the effects of Wnt5a on human periodontal ligament (PDL) cells. In this study, we examined the localization and potential function of Wnt5a in PDL tissue. Immunohistochemical analysis revealed that Wnt5a was expressed predominantly in rat PDL tissue. Semi-quantitative reverse-transcription polymerase chain reaction and Western blotting analysis demonstrated that human PDL cells (HPDLCs) expressed Wnt5a and its receptors (Ror2, Fzd2, Fzd4, and Fzd5). Removal of occlusal pressure by extraction of opposing teeth decreased Wnt5a expression in rat PDL tissue, and the expression of Wnt5a and its receptors in HPDLCs was upregulated by exposure to mechanical stress. Stimulation with Wnt5a significantly enhanced the proliferation and migration of HPDLCs. Furthermore, Wnt5a suppressed osteoblastic differentiation of HPDLCs cultivated in osteogenic induction medium, while it significantly enhanced the expression of PDL-related genes, such as periostin, type-I collagen, and fibrillin-1 genes, and the production of collagen in HPDLCs cultivated in normal medium. Both knockdown of periostin gene expression by siRNA and inhibition of TGFβ1 function by neutralizing antibody suppressed the Wnt5a-induced PDL-related gene expression and collagen production in HPDLCs. Interestingly, in HPDLCs cultured with Wnt5a, TGFβ1 neutralizing antibody significantly suppressed periostin expression, while periostin siRNA had no effect on TGFβ1 expression. These results suggest that Wnt5a expressed in PDL tissue plays specific roles in inducing collagen production by PDL cells through TGFβ1-mediated upregulation of periostin expression.

DOI： 10.1002/jcp.24950

Event date： 2023.11

Language：English   Presentation type：Symposium, workshop panel (public)  

Venue：アクトシティ浜松   Country：Japan  

Event date： 2023.10

Language：Japanese   Presentation type：Symposium, workshop panel (public)  

Venue：日本大学歯学部創設百周年記念講堂   Country：Japan  

Event date： 2022.6

Language：English   Presentation type：Oral presentation (general)  

Venue：Web開催   Country：Japan  

[OBJECTIVE] ACTA2 (alpha-smooth muscle actin; α-SMA), one of the cytoskeleton protein, is known to be expressed in periodontal ligament (PDL) tissue. It might be involved in PDL function such as proliferation and migration. Also ACTA2 is upregulated with transforming growth factor-β1 (TGF-β1) which exists in PDL tissue predominantly (Fujii et al, Cell Tissue Res. 2010). So it is assumed that ACTA2 is involved in TGF-β1-dependent function in human PDL cells, but little is known about them. Therefore, we focused on ACTA2 and examined its role within the PDL function via interaction with or without TGF-β1 to find its role in the remodeling of the PDL.
[MATERIALS AND METHODS] Human PDL cell line 2-23, which was isolated from heterogeneous immortalized Human PDL cells (Hasegawa et al. J Cell Physiol. 2018) was used. (1) Western blotting analysis was performed to examine the protein expression of ACTA2. (2) ACTA2 or scramble siRNA were transfected to 2-23 for 48 h, and performed the WST-1 assay and scratch wound healing assay to analyze the proliferation and migration. (3) 2-23 cells were cultured with or without human recombinant TGF-β1 (rhTGF-β1: 10 ng/ml) for 24 h in the presence of ACTA2 or scramble siRNA. qRT-PCR was performed to examined the mRNA expression of PDL related genes; collagen1A1 (COL1A1), periostin (POSTN), and fibrillin1 (FBN-1). Picro-sirius red staining and sircol collagen assay were performed to analyze the collagen production. Western blotting analysis was performed to examine the phosphorylation of TGF-β1-related molecules; Smad2, Smad3, and YAP. All procedures were performed in compliance with requirements of the Institutional Review Board for Human Genome / Gene Research (approval number: 30-167) and Research Ethics Committee (approval number: 27-76) at Kyushu University.
[RESULTS] ACTA2 protein expression was observed through Western blotting analysis in 2-23. After transfection with ACTA2 siRNA, cell proliferation and migration levels were significantly downregulated compared with scramble siRNA. The mRNA expression of ACTA2, COL1A1, POSTN, and FBN-1 was upregulated in 2-23 stimulated with TGF-β1. Those mRNA expression was significantly downregulated in the presence of ACTA2 siRNA stimulated with rhTGF-β1 compared with scramble siRNA. The amounts of collagen production were upregulated in 2-23 stimulated with rhTGF-β1 which were analyzed by picro-sirius red staining and sircol collagen assay. But after ACTA2 knockdown, the collagen production stimulated with rhTGF-β1 were significantly downregulated compared with scramble siRNA. We revealed that phosphorylation of Smad2 and Smad3 in 2-23 were observed at 15-min time point with TGF-β1, and phosphorylation of YAP was observed at 30-min time point with TGF-β1 by Western blotting analysis. However, after ACTA2 knockdown, at each time point, the phosphorylation of Smad2, Smad3, and YAP was downregulated after TGF-β1 stimulation.
[DISCUSSION] In this research, ACTA2 was involved in proliferation and migration of human PDL cells. These results suggest that as cytoskeleton protein, ACAT2 itself is crucial for PDL function. In the presence of ACTA2 siRNA, upregulation of PDL related genes, collagen production, and phosphorylation of TGF-β1-related molecules were significantly downregulated, suggesting that ACTA2 might be a key factor for TGF-β1 function.
[CONCLUSION] ACTA2 regulates human PDL function via interaction with or without TGF-β1.

Event date： 2021.10 - 2021.11

Language：Japanese   Presentation type：Symposium, workshop panel (public)  

Venue：Web開催   Country：Japan  

Event date： 2021.10 - 2021.11

Language：Japanese   Presentation type：Symposium, workshop panel (public)  

Venue：Web開催   Country：Japan  

Event date： 2019.3

Language：English   Presentation type：Symposium, workshop panel (public)  

Venue：九州大学   Country：Japan  

Event date： 2018.11

Language：Japanese   Presentation type：Oral presentation (general)  

Country：Japan  

Event date： 2018.10 - 2019.10

Language：English   Presentation type：Oral presentation (general)  

Venue：COEX, Seoul   Country：Korea, Republic of  

Event date： 2018.7

Language：English   Presentation type：Oral presentation (general)  

Venue：London   Country：United Kingdom  

Event date： 2018.7

Language：English   Presentation type：Oral presentation (general)  

Venue：London   Country：United Kingdom  

Event date： 2018.7

Language：English   Presentation type：Oral presentation (general)  

Venue：London   Country：United Kingdom  

Event date： 2018.7

Language：Japanese   Presentation type：Oral presentation (general)  

Country：Japan  

Event date： 2018.7

Language：Japanese   Presentation type：Oral presentation (general)  

Country：Japan  

Event date： 2018.6

Language：Japanese   Presentation type：Oral presentation (general)  

Venue：横浜みなとみらいホール   Country：Japan  

Event date： 2018.6

Language：Japanese   Presentation type：Oral presentation (general)  

Venue：横浜みなとみらいホール   Country：Japan  

Event date： 2018.6

Language：Japanese   Presentation type：Oral presentation (general)  

Venue：横浜みなとみらいホール   Country：Japan  

Event date： 2018.6

Language：Japanese   Presentation type：Oral presentation (general)  

Venue：横浜みなとみらいホール   Country：Japan  

Event date： 2015.6

Language：Japanese   Presentation type：Symposium, workshop panel (public)  

Venue：小倉   Country：Japan  

Language：Japanese  

Language：Japanese  

Language：English