In vitro evaluation of the antimicrobial properties of terpinen-4-ol on apical periodontitis-associated bacteria.

Manuka oil and tea tree oil are essential oils with known antibacterial properties that are believed to be caused by one main component: terpinen-4-ol. Terpinen-4-ol has potent antibacterial activity against caries-related microorganisms. However, few studies have investigated the antimicrobial effects of terpinen-4-ol on bacteria in apical periodontitis. Thus, the objective of the present study was to evaluate the antibacterial and antibiofilm potential of terpinen-4-ol against Enterococcus faecalis, Porphyromonas gingivalis, Prevotella intermedia, and Fusobacterium nucleatum, which have all been detected in apical periodontitis. The minimum inhibitory and minimum bactericidal concentrations of terpinen-4-ol were determined to assess its activity against biofilms. The minimum inhibitory concentration of terpinen-4-ol was 0.25% against E. faecalis and F. nucleatum, 0.05% against P. gingivalis, and 0.1% against P. intermedia. The minimum bactericidal concentration of terpinen-4-ol was 1.0% against E. faecalis, 0.2% against P. gingivalis and P. intermedia, and 0.5% against F. nucleatum. In the biofilm evaluations, all terpinen-4-ol-treated bacteria had significant reductions in biofilm viability compared with controls in experiments assessing attachment inhibitory activity. Furthermore, structural alterations and decreased bacterial cell clumping were observed under scanning electron microscopy, and significantly decreased cell survival was noted using fluorescence microscopy. Together, these results suggest that terpinen-4-ol is a potential antibacterial agent with bactericidal properties, and can also act on established biofilms.

Actin alpha 2, smooth muscle, a transforming growth factor-ß1-induced factor, regulates collagen production in human periodontal ligament cells via Smad2/3 pathway.

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-ß1 (TGF-ß1); 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-ß1 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-ß1-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.

Functions of beta2-adrenergic receptor in human periodontal ligament cells.

Adrenergic receptors (ARs) are receptors of noradrenalin and adrenalin, of which there are nine different subtypes. In particular, ß2 adrenergic receptor (ß2-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 ß2-AR in periodontal ligament (PDL) tissue has not been fully examined. In this report, we investigated that ß2-AR expression in PDL tissues and their features in PDL cells. ß2-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 ß2-AR expression, while its expression was downregulated in that without loading. In HPDLCs, ß2-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 ß2-AR. Their gene expression and intracellular cyclic adenosine monophosphate (cAMP) levels were also investigated in HPDLCs treated with a specific ß2-AR agonist, fenoterol (FEN). Overexpression of ß2-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 ß2-AR expression in PDL tissue and ß2-AR is involved in fibroblastic differentiation and collagen synthesis of PDL cells. The signaling through ß2-AR might be important for restoration and homeostasis of PDL tissue.

A Screening System for Evaluating Cell Extension Formation, Collagen Compaction, and Degradation in Drug Discovery.

The generation of cell extensions is critical for matrix remodeling in tissue invasion by cancer cells, but current methods for identifying molecules that regulate cell extension formation and matrix remodeling are not well adapted for screening purposes. We applied a grid-supported, floating collagen gel system (~100 Pa stiffness) to examine cell extension formation, collagen compaction, and collagen degradation in a single assay. With the use of cultured diploid fibroblasts, a fibroblast cell line, and two cancer cell lines, we found that compared with attached collagen gels (~2800 Pa), the mean number and length of cell extensions were respectively greater in the floating gels. In assessing specific processes in cell extension formation, compared with controls, the number of cell extensions was reduced by latrunculin B, ß1 integrin blockade, and a formin FH2 domain inhibitor. Screening of a kinase inhibitor library (480 compounds) with the floating gel assay showed that compared with vehicle-treated cells, there were large reductions of collagen compaction, pericellular collagen degradation, and number of cell extensions after treatment with SB431542, SIS3, Fasudil, GSK650394, and PKC-412. These data indicate that the grid-supported floating collagen gel model can be used to screen for inhibitors of cell extension formation and critical matrix remodeling events associated with cancer cell invasion.

Calcium-sensing receptor-ERK signaling promotes odontoblastic differentiation of human dental pulp cells.

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.

GDNF From Human Periodontal Ligament Cells Treated With Pro-Inflammatory Cytokines Promotes Neurocytic Differentiation of PC12 Cells.

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-1ß) 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-1ß antibody. Compared with untreated cells, HPDLCs treated with IL-1ß or tumor necrosis factor-alpha showed an increase in the secretion of GDNF protein. Conditioned medium of IL-1ß-treated HPDLCs (IL-1ß-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-1ß-CM-treated PC12 cells. These stimulatory effects of IL-1ß-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. © 2016 Wiley Periodicals, Inc.

Wnt5a Induces Collagen Production by Human Periodontal Ligament Cells Through TGFß1-Mediated Upregulation of Periostin Expression.

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 TGFß1 function by neutralizing antibody suppressed the Wnt5a-induced PDL-related gene expression and collagen production in HPDLCs. Interestingly, in HPDLCs cultured with Wnt5a, TGFß1 neutralizing antibody significantly suppressed periostin expression, while periostin siRNA had no effect on TGFß1 expression. These results suggest that Wnt5a expressed in PDL tissue plays specific roles in inducing collagen production by PDL cells through TGFß1-mediated upregulation of periostin expression.

Effect of CTGF/CCN2 on osteo/cementoblastic and fibroblastic differentiation of a human periodontal ligament stem/progenitor cell line.

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-ß1 (TGF-ß1) significantly promoted type I collagen and fibronectin expression compared with that of TGF-ß1 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.

Effects of Activin A on the phenotypic properties of human periodontal ligament cells.

Periodontal ligament (PDL) tissue plays an important role in tooth preservation by structurally maintaining the connection between the tooth root and the bone. The mechanisms involved in the healing and regeneration of damaged PDL tissue, caused by bacterial infection, caries and trauma, have been explored. Accumulating evidence suggests that Activin A, a member of the transforming growth factor-ß (TGF-ß) superfamily and a dimer of inhibinßa, contributes to tissue healing through cell proliferation, migration, and differentiation of various target cells. In bone, Activin A has been shown to exert an inhibitory effect on osteoblast maturation and mineralization. However, there have been no reports examining the expression and function of Activin A in human PDL cells (HPDLCs). Thus, we aimed to investigate the biological effects of Activin A on HPDLCs. Activin A was observed to be localized in HPDLCs and rat PDL tissue. When PDL tissue was surgically damaged, Activin A and IL-1ß expression increased and the two proteins were shown to be co-localized around the lesion. HPDLCs treated with IL-1ß or TNF-α also up-regulated the expression of the gene encoding inhibinßa. Activin A promoted chemotaxis, migration and proliferation of HPDLCs, and caused an increase in fibroblastic differentiation of these cells while down-regulating their osteoblastic differentiation. These osteoblastic inhibitory effects of Activin A, however, were only noted during the early phase of HPDLC osteoblastic differentiation, with later exposures having no effect on differentiation. Collectively, our results suggest that Activin A could be used as a therapeutic agent for healing and regenerating PDL tissue in response to disease, trauma or surgical reconstruction.

Expression and effects of epidermal growth factor on human periodontal ligament cells.

Repair of damaged periodontal ligament (PDL) tissue is an essential challenge in tooth preservation. Various researchers have attempted to develop efficient therapies for healing and regenerating PDL tissue based on tissue engineering methods focused on targeting signaling molecules in PDL stem cells and other mesenchymal stem cells. In this context, we investigated the expression of epidermal growth factor (EGF) in normal and surgically wounded PDL tissues and its effect on chemotaxis and expression of osteoinductive and angiogenic factors in human PDL cells (HPDLCs). EGF as well as EGF receptor (EGFR) expression was observed in HPDLCs and entire PDL tissue. In a PDL tissue-injured model of rat, EGF and IL-1ß were found to be upregulated in a perilesional pattern. Interleukin-1ß induced EGF expression in HPDLCs but not EGFR. It also increased transforming growth factor-α (TGF-α) and heparin-binding EGF-like growth factor (HB-EGF) expression. Transwell assays demonstrated the chemotactic activity of EGF on HPDLCs. In addition, EGF treatment significantly induced secretion of bone morphogenetic protein 2 and vascular endothelial growth factor, and gene expression of interleukin-8 (IL-8), and early growth response-1 and -2 (EGR-1/2). Human umbilical vein endothelial cells developed well-formed tube networks when cultured with the supernatant of EGF-treated HPDLCs. These results indicated that EGF upregulated under inflammatory conditions plays roles in the repair of wounded PDL tissue, suggesting its function as a prospective agent to allow the healing and regeneration of this tissue.