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We aimed to evaluate the materials based on 4‐methacryloxyethyl trimellitate anhydride/methyl methacrylate tri‐n‐butylborane (Super‐bond [SB]) and nano hydroxyapatite (naHAp) for the repair of perforation at pulp chamber floor (PPF) in vitro and in vivo models. SB and naHAp were mixed in the mass ratio of 10% or 30% to produce naHAp/SB. Human periodontal ligament stem cells (HPDLSCs) were cultured on resin discs of SB or naHAp/SB to analyze the effects of naHAp/SB on cell adhesion, proliferation, and cementoblastic differentiation. A rat PPF model was treated with SB or naHAp/SB to examine the effects of naHAp/SB on the healing of defected cementum and periodontal ligament (PDL) at the site of PPF. HPDLSCs were spindle‐shaped and adhered to all resin discs. Changing the resin from SB to naHAp/SB did not significantly alter cell proliferation. Both 10% and 30% naHAp/SB were more effective than SB in promoting cementoblastic differentiation of HPDLSCs. In the rat PPF model, 30% naHAp/SB was more effective than SB in promoting the formation Sharpey's fiber‐like structures with expression of the PDL‐related marker and cementum‐like structures with expression of cementum‐related markers. In conclusion, 30% naHAp/SB can be the new restorative material for PPF because it exhibited the abilities of adhering to dentin and healing of defected periodontal tissue. Significance Statement: Perforation at pulp chamber floor (PPF) induces inflammatory reactions in surrounding periodontal tissues and leads to tooth loss. An ideal restorative material should exhibit biocompatibility, provide an adequate bond strength to dentin, and induce the healing of defected periodontal tissues. However, the materials meet all requirements were not reported. We aimed to develop and evaluate new materials based on 4‐methacryloxyethyl trimellitate anhydride/methyl methacrylate tri‐n‐butylborane (super‐bond [SB]) and nano hydroxyapatite (naHAp) and mixed them (naHAp/SB). The naHAp/SB exhibited biocompatible properties with adhering ability to dentin and induced the healing of natural periodontal tissues. Our results suggested that naHAp/SB may be the new material meeting all requirements for the repair of PPF and it seems to lead to long‐term preservation of tooth. [ABSTRACT FROM AUTHOR]

Objective: The purpose of this study was to evaluate the function of Schwann cells in wound healing of periodontal tissue. Background: In our previous study, glial cell line‐derived neurotrophic factor (GDNF) promoted the migration of human periodontal ligament (PDL) cells and that GDNF expression increased in wounded periodontal tissue. GDNF reportedly induces the migration of Schwann cell precursors. Schwann cells play a crucial role in the regeneration of peripheral tissues, including bone tissue. However, the role of Schwann cells on periodontal tissue regeneration remains unclear. Methods: A transwell assay and a WST‐1 (water‐soluble tetrazolium compound‐1) proliferation assay were used to determine whether GDNF promotes the migration and proliferation of Schwann cells, respectively. Quantitative RT‐PCR and Alizarin Red S staining were performed to examine the effect of these cells on the differentiation of human preosteoblast (Saos2 cells) using conditioned medium from YST‐1 (YST‐1‐CM). Western blotting analysis was performed to determine whether YST‐1‐CM activates ERK signaling pathway in Saos2 cells. The expression of Schwann cell markers, S100 calcium‐binding protein B (S100‐B) and growth associated protein 43 (GAP‐43), was determined in normal and wounded periodontal tissue by immunofluorescent staining. Results: Glial cell line‐derived neurotrophic factor promoted the migration of YST‐1 cells but did not affect the proliferation of YST‐1 cells. Saos2 cells cultured with YST‐1‐CM increased the expression of osteoblastic markers and mineralization. YST‐1‐CM also induced phosphorylation of ERK1/2 in Saos2 cells. The number of S100‐B‐immunoreactive cells which also expressed GAP‐43 was increased in rat wounded periodontal tissue during healing process. Conclusion: The accumulation of Schwann cells in wounded periodontal tissue suggests that they play a significant role in wound healing of this tissue, especially alveolar bone tissue. [ABSTRACT FROM AUTHOR]

Dopamine (DA) is produced from tyrosine by tyrosine hydroxylase (TH). A recent study has reported that DA promotes the mineralization of murine preosteoblasts. However, the role of DA in odontoblasts has not been examined. Therefore, in this investigation, we researched the expression of TH and DA in odontoblasts and the effects of DA on the differentiation of preodontoblasts (KN‐3 cells). Immunostaining showed that TH and DA were intensely expressed in odontoblasts and preodontoblasts of rat incisors and molars. KN‐3 cells expressed D1‐like and D2‐like receptors for DA. Furthermore, DA promoted odontoblastic differentiation of KN‐3 cells, whereas an antagonist of D1‐like receptors and a PKA signaling blocker, inhibited such differentiation. However, antagonists of D2‐like receptors promoted differentiation. These results suggested that DA in preodontoblasts and odontoblasts might promote odontoblastic differentiation through D1‐like receptors, but not D2‐like receptors, and PKA signaling in an autocrine or paracrine manner and plays roles in dentinogenesis. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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. These data could argue the possible involvement of different signaling pathways in tumor necrosis factor‐α‐inducing events depending on the cell aging stage, which might cause different cell responses. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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. J. Cell. Physiol. 9999: 2647-2660, 2015. © 2015 Wiley Periodicals, Inc. [ABSTRACT FROM AUTHOR]

Appropriate mechanical loading during occlusion and mastication play an important role in maintaining the homeostasis of periodontal ligament (PDL) tissue. Connective tissue growth factor (CTGF/CCN2), a matricellular protein, is known to upregulate extracellular matrix production, including collagen in PDL tissue. However, the underlying mechanisms of CTGF/CCN2 in regulation of PDL tissue integrity remain unclear. In this study, we investigated the effect of CTGF/CCN2 on osteo/cementoblastic and fibroblastic differentiation of human PDL stem cells using the cell line 1-11. CTGF/CCN2 expression in rat PDL tissue and human PDL cells (HPDLCs) was confirmed immunohisto/cytochemically. Mechanical loading was found to increase gene expression and secretion of CTGF/CCN2 in HPDLCs. CTGF/CCN2 upregulated the proliferation and migration of 1-11 cells. Furthermore, increased bone/cementum-related gene expression in this cell line led to mineralization. In addition, combined treatment of 1-11 cells with CTGF/CCN2 and transforming growth factor-β1 (TGF-β1) significantly promoted type I collagen and fibronectin expression compared with that of TGF-β1 treatment alone. Thus, these data suggest the underlying biphasic effects of CTGF/CCN2 in 1-11 cells, inducible osteo/cementoblastic, and fibroblastic differentiation dependent on the environmental condition. CTGF/CCN2 may contribute to preservation of the structural integrity of PDL tissue, implying its potential use as a therapeutic agent for PDL regeneration. J. Cell. Physiol. 229: 150-159, 2014. © 2014 Wiley Periodicals, Inc. [ABSTRACT FROM AUTHOR]