Predictive factors of periodontal regeneration outcomes using rhFGF-2: A case-control study.

OBJECTIVE: This study aimed to investigate the factors influencing the clinical outcomes of regenerative therapy using recombinant human fibroblast growth factor-2 (rhFGF-2). BACKGROUND: rhFGF-2 promotes periodontal regeneration, and identifying the factors influencing this regeneration is important for optimizing the effectiveness of rhFGF-2. METHODS AND MATERIALS: This study used a hospital information-integrated database to identify patients who underwent periodontal regenerative therapy with rhFGF-2. Factors included age, smoking status, diabetes mellitus (DM), periodontal inflamed surface area (PISA) at the initial visit, whether the most posterior tooth was involved or not, and preoperative radiological bone defect angle. Periodontal regenerative therapy outcomes were defined as good if radiographic bone fill ≥35% or periodontal pocket closure at 9-15 months after surgery. Bone fill rate (%) and periodontal pocket depth (mm) were also used as outcome measures. Factors were evaluated by simple regression analysis, and then the association between factors and the outcomes was determined by multivariate analysis. RESULTS: PISA and age at the first visit did not significantly influence the success or failure of bone fill rate byrhFGF-2. However, DM, radiographic bone defect angle, and the most posterior tooth significantly influenced the regenerative effect (success/failure in bone fill) of rhFGF-2. The most posterior tooth was significantly associated with bone fill rate by rhFGF-2. Examination of the association between pocket closure and factors shows that the most posterior tooth significantly influenced. The most posterior tooth and preoperative PPD were significantly associated with pocket reduction depth. For the most posterior tooth, a significantly higher bone regeneration rate (p < .05) was observed with a combination of autologous bone graft and rhFGF-2 than with rhFGF-2 alone, and the effect was significant in multivariate analysis. CONCLUSIONS: The radiographic bone defect angle, the involvement of most posterior teeth, and the presence of DM influenced the effectiveness of rhFGF-2 in periodontal regeneration. However, PISA values and age at the initial visit had no significant effect.

Periodontal tissue regeneration with cementogenesis after application of brain-derived neurotrophic factor in 3-wall inflamed intra-bony defect.

OBJECTIVE: The purpose of this study is to investigate regenerative process by immunohistochemical analysis and evaluate periodontal tissue regeneration following a topical application of BDNF to inflamed 3-wall intra-bony defects. BACKGROUND: Brain-derived neurotrophic factor (BDNF) plays a role in the survival and differentiation of central and peripheral neurons. BDNF can regulate the functions of non-neural cells, osteoblasts, periodontal ligament cells, endothelial cells, as well as neural cells. Our previous study showed that a topical application of BDNF enhances periodontal tissue regeneration in experimental periodontal defects of dog and that BDNF stimulates the expression of bone (cementum)-related proteins and proliferation of human periodontal ligament cells. METHODS: Six weeks after extraction of mandibular first and third premolars, 3-wall intra-bony defects were created in mandibular second and fourth premolars of beagle dogs. Impression material was placed in all of the artificial defects to induce inflammation. Two weeks after the first operation, BDNF (25 and 50 µg/mL) immersed into atelocollagen sponge was applied to the defects. As a control, only atelocollagen sponge immersed in saline was applied. Two and four weeks after the BDNF application, morphometric analysis was performed. Localizations of osteopontin (OPN) and proliferating cell nuclear antigen (PCNA)-positive cells were evaluated by immunohistochemical analysis. RESULTS: Two weeks after application of BDNF, periodontal tissue was partially regenerated. Immunohistochemical analyses revealed that cells on the denuded root surface were positive with OPN and PCNA. PCNA-positive cells were also detected in the soft connective tissue of regenerating periodontal tissue. Four weeks after application of BDNF, the periodontal defects were regenerated with cementum, periodontal ligament, and alveolar bone. Along the root surface, abundant OPN-positive cells were observed. Morphometric analyses revealed that percentage of new cementum length and percentage of new bone area of experimental groups were higher than control group and dose-dependently increased. CONCLUSION: These findings suggest that BDNF could induce cementum regeneration in early regenerative phase by stimulating proliferation of periodontal ligament cells and differentiation into periodontal tissue cells, resulting in enhancement of periodontal tissue regeneration in inflamed 3-wall intra-bony defects.

Leucine-rich alpha-2-glycoprotein 1 affects bone destruction via IL-6 in mouse periodontitis model.

OBJECTIVE: To investigate the production of leucine-rich α-2-glycoprotein-1 (LRG1) in periodontitis patients and its effectiveness as a new diagnostic marker for periodontitis. SUBJECTS AND METHODS: In vitro experiments were conducted to analyze LRG1 mRNA expression in human gingival epithelial cells and fibroblasts via quantitative real-time PCR. In vivo experiments were conducted to analyze LRG1 localization in periodontitis patients. The correlation between the serum LRG1 levels and alveolar bone resorption in the mouse periodontitis model was also investigated. RESULTS: A positive correlation existed between the periodontal inflamed surface area and serum LRG1 levels (Spearman's rank correlation coefficient: 0.60). LRG1 mRNA expression in human gingival epithelial cells and fibroblasts was upregulated by Porphyromonas gingivalis stimulation or tumor necrosis factor-α stimulation. Interleukin-6 in human gingival epithelial cells and fibroblasts induced the production of LRG1 and transforming growth factor-ß. LRG1 levels in the periodontal tissue and serum in the periodontitis model were higher than those in control mice. LRG1 local administration resulted in alveolar bone resorption, whereas the administration of interleukin-6R antibody inhibited bone resorption. CONCLUSIONS: LRG1 levels in serum and periodontal tissue are upregulated in periodontitis and are implicated in periodontal tissue destruction through interleukin-6 production.

Regulation of osteogenesis in bone marrow-derived mesenchymal stem cells via histone deacetylase 1 and 2 co-cultured with human gingival fibroblasts and periodontal ligament cells.

OBJECTIVE: This study aimed to determine the regulatory mechanism of bone marrow-derived mesenchymal stem cell (BM-MSC) differentiation mediated by humoral factors derived from human periodontal ligament (HPL) cells and human gingival fibroblasts (HGFs). We analyzed histone deacetylase (HDAC) expression and activity involved in BM-MSC differentiation and determined their regulatory effects in co-cultures of BM-MSCs with HPL cells or HGFs. BACKGROUND: BM-MSCs can differentiate into various cell types and can, thus, be used in periodontal regenerative therapy. However, the mechanism underlying their differentiation remains unclear. Transplanted BM-MSCs are affected by periodontal cells via direct contact or secretion of humoral factors. Therefore, their activity is regulated by humoral factors derived from HPL cells or HGFs. METHODS: BM-MSCs were indirectly co-cultured with HPL cells or HGFs under osteogenic or growth conditions and then analyzed for osteogenesis, HDAC1 and HDAC2 expression and activity, and histone H3 acetylation. BM-MSCs were treated with trichostatin A, or their HDAC1 or HDAC2 expression was silenced or overexpressed during osteogenesis. Subsequently, they were evaluated for osteogenesis or the effects of HDAC activity. RESULTS: BM-MSCs co-cultured with HPL cells or HGFs showed suppressed osteogenesis, HDAC1 and HDAC2 expression, and HDAC phosphorylation; however, histone H3 acetylation was enhanced. Trichostatin A treatment remarkably suppressed osteogenesis, decreasing HDAC expression and enhancing histone H3 acetylation. HDAC1 and HDAC2 silencing negatively regulated osteogenesis in BM-MSCs to the same extent as that achieved by indirect co-culture with HPL cells or HGFs. Conversely, their overexpression positively regulated osteogenesis in BM-MSCs. CONCLUSION: The suppressive effects of HPL cells and HGFs on BM-MSC osteogenesis were regulated by HDAC expression and histone H3 acetylation to a greater extent than that mediated by HDAC activity. Therefore, regulation of HDAC expression has prospects in clinical applications for effective periodontal regeneration, mainly, bone regeneration.

Periapical lesion following Cnm-positive Streptococcus mutans pulp infection worsens cerebral hemorrhage onset in an SHRSP rat model.

Cerebral hemorrhage severely affects the daily life of affected individuals. Streptococcus mutans and its adhesion factor Cnm increase the adverse effects of cerebral hemorrhages. However, the mechanism by which Cnm-positive bacteria migrate from apical lesions to cerebral hemorrhage sites is unclear. Therefore, we established an S. mutans-infected apical lesion in a rat model of hypertension and investigated the neurological symptoms associated with cerebral hemorrhage. Eighteen 12-week-old stroke-prone spontaneously hypertensive rats were randomly divided into three groups, i.e. the no infection (control), dental infection with S. mutans KSM153 wild type (Cnm positive), and KSM153 Δcnm groups. Immunofluorescent staining was performed to visualize S. mutans protein. Serum interleukin-1ß levels were measured. The adhesion of S. mutans to the extracellular matrix and human fibroblast cells was also analyzed. Serum antibody titers against S. mutans were comparable between Cnm positive and knockout mutants. However, 3-10 days post-infection, neurological symptom scores and cerebral hemorrhage scores were higher in Cnm-positive rats than in knockout mutants. The localization of S. mutans-derived protein was observed in the vicinity of disrupted blood vessels. Serum interleukin-1ß levels significantly increased post-KSM153 WT infection. Cnm-positive S. mutans clinical isolates showed increased adhesion to the extracellular matrix, human dental pulp cells, and human umbilical vein endothelial cells compared with the Cnm-negative S. mutans isolates. In conclusion, Cnm-positive bacteria colonize the apical lesion site using the extracellular matrix as a foothold and affect cerebral hemorrhage via the bloodstream.

The role of nuclear receptor 4A1 (NR4A1) in drug-induced gingival overgrowth.

Drug-induced gingival overgrowth (DIGO) is a side effect of cyclosporine A (CsA), nifedipine (NIF), and phenytoin (PHT). Nuclear receptor 4A1 (NR4A1) plays a role in fibrosis in multiple organs. However, the relationship between NR4A1 and DIGO remains unclear. We herein investigated the involvement of NR4A1 in DIGO. In the DIGO mouse model, CsA inhibited the up-regulation of Nr4a1 expression induced by periodontal disease (PD) in gingival tissue, but not that of Col1a1 and Pai1. We detected gingival overgrowth (GO) in Nr4a1 knock out (KO) mice with PD. A NR4A1 agonist inhibited the development of GO in DIGO model mice. TGF-ß increased Col1a1 and Pai1 expression levels in KO mouse gingival fibroblasts (mGF) than in wild-type mice, while the overexpression of NR4A1 in KO mGF suppressed the levels. NR4A1 expression levels in gingival tissue were significantly lower in DIGO patients than in PD patients. We also investigated the relationship between nuclear factor of activated T cells (NFAT) and NR4A1. NFATc3 siRNA suppressed the TGF-ß-induced up-regulation of NR4A1 mRNA expression in human gingival fibroblasts (hGF). CsA suppressed the TGF-ß-induced translocation of NFATc3 into the nuclei of hGF. Furthermore, NIF and PHT also decreased NR4A1 mRNA expression levels and suppressed the translocation of NFATc3 in hGF. We confirmed that CsA, NIF, and PHT reduced cytosolic calcium levels increased by TGF-ß, while CaCl2 enhanced the TGF-ß-up-regulated NR4A1 expression. We propose that the suppression of the calcium-NFATc3-NR4A1 cascade by these three drugs plays a role in the development of DIGO.

Involvement of Rac1 in macrophage activation by brain-derived neurotrophic factor.

Brain-derived neurotrophic factor (BDNF) enhances periodontal tissue regeneration. Tissue regeneration is characterized by inflammation, which directs the quality of tissue repair. This study aimed to investigate the effect of BDNF on the phagocytic activity of RAW264.7 cells. In addition, we studied the effect of BDNF on guanosine triphosphatase (GTP)-RAS-related C3 botulinus toxin substrate (Rac)1 and phospho-Rac1 levels in RAW264.7 cells. Rac1 inhibitor inhibited BDNF-induced phagocytosis of latex-beads. In addition, BDNF enhanced Porphyromonas gingivalis (Pg) phagocytosis by RAW264.7 cells as well as latex-beads. We demonstrated for the first time that BDNF enhances phagocytic activity of RAW264.7 cells through Rac1 activation. The present study proposes that BDNF may reduce inflammatory stimuli during BDNF-induced periodontal tissue regeneration through enhanced phagocytic activity of macrophages.

Aggressive periodontitis and NOD2 variants.

Aggressive periodontitis (AgP) occurs at an early age and causes rapid periodontal tissue destruction. Nucleotide-binding oligomerization domain-containing protein 2 (NOD2) encodes a protein with two caspase recruitment domains and eleven leucine-rich repeats. This protein is expressed mainly in peripheral blood leukocytes and is involved in immune response. NOD2 variants have been associated with increased susceptibility to Crohn's disease, and recently, NOD2 was reported as a causative gene in AgP. The present study aimed to identify potential NOD2 variants in an AgP cohort (a total of 101 patiens: 37 patients with positive family histories and 64 sporadic patients). In the familial group, six patients from two families had a reported heterozygous missense variant (c.C931T, p.R311W). Four patients in the sporadic group had a heterozygous missense variant (c.C1411T, p.R471C), with no reported association to the disease. Overall, two NOD2 variants, were identified in 10% of our AgP cohort. These variants were different from the major variants reported in Crohn's disease. More cases need to be investigated to elucidate the role of NOD2 variants in AgP pathology.

Relationship between periodontal inflammation and calcium channel blockers induced gingival overgrowth-a cross-sectional study in a Japanese population.

OBJECTIVES: Periodontal inflammation is regarded as a risk factor for drug-induced gingival overgrowth (DIGO). In order to elucidate the involvement of periodontal inflammation in DIGO, the periodontal status of subjects who do not develop DIGO despite receiving causative drugs (non-responders) needs to be examined. Therefore, the aim of the present study which was a pilot study was to assess periodontal inflammatory variables in responders (calcium channel blocker induced-GO patients), non-responders, and patients who did not receive causative drugs (non-consumers). MATERIALS AND METHODS: The following parameters were measured: (1) existence of gingival overgrowth, (2) number of teeth, (3) mean periodontal pocket depth (PPD), and (4) percentage of positive sites for bleeding on probing (BOP). The periodontal inflamed surface area (PISA) and periodontal epithelial surface area (PESA) and the PISA/PESA ratio which indicated the degree of periodontal inflammation in each patient were also used to evaluate periodontal inflammation. RESULTS: Thirteen responders, 32 non-responders, and 83 non-consumers were included in the analyses. The mean PPD, percentage of BOP, PESA, and PISA, and the PISA/PESA ratio were significantly higher in responders than in non-responders and non-consumers (p < 0.01). The BOP, PISA, and PISA/PESA ratio were significantly lower in non-responders than in non-consumers (p < 0.05). A positive correlation was found between PPD and age in non-consumers. On the other hand, a negative correlation was noted between PPD and age in non-responders. CONCLUSIONS: Periodontal inflammation may be associated with the initiation of DIGO. CLINICAL RELEVANCE: It could be speculated that periodontal therapy before the administration of calcium channel blockers may prevent the development of gingival overgrowth.

Aggregatibacter actinomycetemcomitans outer membrane protein 29 (Omp29) induces TGF-ß-regulated apoptosis signal in human gingival epithelial cells via fibronectin/integrinß1/FAK cascade.

Gingival junctional epithelial cell apoptosis caused by periodontopathic bacteria exacerbates periodontitis. This pathological apoptosis is involved in the activation of transforming growth factor ß (TGF-ß). However, the molecular mechanisms by which microbes induce the activation of TGF-ß remain unclear. We previously reported that Aggregatibacter actinomycetemcomitans (Aa) activated TGF-ß receptor (TGF-ßR)/smad2 signalling to induce epithelial cell apoptosis, even though Aa cannot bind to TGF-ßR. Additionally, outer membrane protein 29 kDa (Omp29), a member of the Aa Omps family, can induce actin rearrangements via focal adhesion kinase (FAK) signalling, which also plays a role in the activation of TGF-ß by cooperating with integrin. Accordingly, we hypothesized that Omp29-induced actin rearrangements via FAK activity would enhance the activation of TGF-ß, leading to gingival epithelial cell apoptosis in vitro. By using human gingival epithelial cell line OBA9, we found that Omp29 activated TGF-ßR/smad2 signalling and decreased active TGF-ß protein levels in the extracellular matrix (ECM) of cell culture, suggesting the transactivation of TGF-ßR. Inhibition of actin rearrangements by cytochalasin D or blebbistatin and knockdown of FAK or integrinß1 expression by siRNA transfection attenuated TGF-ßR/smad2 signalling activity and reduction of TGF-ß levels in the ECM caused by Omp29. Furthermore, Omp29 bound to fibronectin (Fn) to induce its aggregation on integrinß1, which is associated with TGF-ß signalling activity. All the chemical inhibitors and siRNAs tested blocked Omp29-induced OBA9 cells apoptosis. These results suggest that Omp29 binds to Fn in order to facilitate Fn/integrinß1/FAK signalling-dependent TGF-ß release from the ECM, thereby inducing gingival epithelial cell apoptosis via TGF-ßR/smad2 pathway.

Distinction Between Cell Proliferation and Apoptosis Signals Regulated by Brain-Derived Neurotrophic Factor in Human Periodontal Ligament Cells and Gingival Epithelial Cells.

Previously, we reported that brain-derived neurotrophic factor (BDNF) enhances periodontal tissue regeneration by inducing periodontal ligament cell proliferation in vivo. In addition, the down growth of gingival epithelial cells, which comprises a major obstacle to the regeneration, was not observed. However, the underlying molecular mechanism is still unclear. Therefore, this study aimed to investigate the effect of BDNF on cell proliferation and apoptosis in human periodontal ligament (HPL) cells and human gingival epithelial cells (OBA9 cells) and to explore the molecular mechanism in vitro. HPL cells dominantly expressed a BDNF receptor, TrkB, and BDNF increased cell proliferation and ERK phosphorylation. However, its proliferative effect was diminished by a MEK1/2 inhibitor (U0126) and TrkB siRNA transfection. Otherwise, OBA9 cells showed a higher expression level of p75, which is a pan-neurotrophin receptor, than that of HPL cells. BDNF facilitated not cell proliferation but cell apoptosis and JNK phosphorylation in OBA9 cells. A JNK inhibitor (SP600125) and p75 siRNA transfection attenuated the BDNF-induced cell apoptosis. Moreover, OBA9 cells pretreated with SP600125 or p75 siRNA showed cell proliferation by BDNF stimulation, though it was reduced by U0126 and TrkB siRNA. Interestingly, overexpression of p75 in HPL cells upregulated cell apoptosis and JNK phosphorylation by BDNF treatment. These results indicated that TrkB-ERK signaling regulates BDNF-induced cell proliferation, whereas p75-JNK signaling plays roles in cell apoptotic and cytostatic effect of BDNF. Overall, BDNF activates periodontal ligament cells proliferation and inhibits the gingival epithelial cells growth via the distinct pathway. J. Cell. Biochem. 117: 1543-1555, 2016. © 2015 Wiley Periodicals, Inc.

Mobilization of TLR4 Into Lipid Rafts by Aggregatibacter Actinomycetemcomitans in Gingival Epithelial Cells.

BACKGROUND: An investigation of the mechanisms underlying the production of inflammatory cytokines through the stimulation of microorganisms on gingival epithelial cells may provide insights into the pathogenesis of the initiation of periodontitis. Lipid rafts, microdomains in the cell membrane, include a large number of receptors, and are centrally involved in signal transduction. We herein examined the involvement of lipid rafts in the expression of interleukin (IL-6) and IL-8 in gingival epithelial cells stimulated by periodontal pathogens. METHODS: OBA9, a human gingival cell line, was stimulated by Aggregatibacter actinomycetemcomitans or tumor necrosis factor (TNF)-α in the presence of methyl-ß-cyclodextrin (MßCD). RESULTS: A. actinomycetemcomitans or TNF-α increased IL-8 and IL-6 mRNA levels, and promoted the phosphorylation of ERK and p38 MAP kinase in OBA9. The pretreatment with MßCD abolished increases in IL-6 and IL-8 mRNA levels and the phosphorylation induced by A. actinomycetemcomitans, but did not suppress the response induced by TNF-α. The transfection of TLR4 inhibited A. actinomycetemcomitans-induced increases in IL-8 and IL-6 mRNA levels. Confocal microscopy revealed that MßCD inhibited the mobilization of TLR4 into lipid rafts. CONCLUSION: The mobilization of TLR4 into lipid rafts is involved in the expression of inflammatory cytokines and phosphorylation of MAP kinase in human gingival epithelial cells stimulated by A. actinomycetemcomitans.

Involvement of smad2 and Erk/Akt cascade in TGF-ß1-induced apoptosis in human gingival epithelial cells.

Periodontitis is the most prevalent infectious disease caused by periodontopathic bacteria and is also a chronic inflammatory disease. Gingival crevicular fluid (GCF) is an inflammatory exudate that seeps into the gingival crevices or periodontal pockets around teeth with inflamed gingiva, and contains various materials including leukocytes and cytokines. Since gingival epithelial cells, which form a barrier against bacterial challenges, are affected by GCF, cytokines or other materials contained within GCF are engaged in the maintenance and disruption of the epithelial barrier. Accordingly, its compositional pattern has been employed as a reliable objective index of local inflammation. Transforming growth factor ß1 (TGF-ß1) levels in GCF were previously shown to be markedly higher in patients with periodontitis than in healthy subject. However, it currently remains unclear how TGF-ß1 affects gingival epithelial cell growth or apoptosis; therefore, elucidating the mechanism responsible may lead to a deeper understanding of pathogenic periodontitis. In the present study, the human gingival epithelial cell line, OBA9 cells were stimulated with recombinant TGF-ß1. Apoptosis-related protein levels were determined by Western blotting. Caspase-3/7 activity was measured with a Caspase-Glo assay kit. Surviving and apoptotic cells were detected using an MTS assay and TUNEL staining, respectively. TGF-ßRI siRNA and smad2 siRNA were transfected into cells using the lipofectamine RNAiMAX reagent. TGF-ß1 elevated caspase-3 activity and the number of TUNEL-positive apoptotic cells in OBA9 cells. Furthermore, while the levels of the pro-apoptotic proteins Bax, Bak, Bim, and Bad were increased in OBA9 cells stimulated with TGF-ß1, the TGF-ß1 treatment also decreased the levels of anti-apoptotic proteins such as Bcl-2 and Bcl-xL in a time-dependent manner. Additionally, TGF-ß1 up-regulated the protein levels of cleaved caspase-9. These results indicated that TGF-ß1-induced apoptosis was involved in a mitochondria-related intrinsic pathway. TGF-ß1 phosphorylated smad2 in OBA9 cells and this phosphorylation was clearly reduced by SB431542 (a TGF-ß type I receptor inhibitor). Consistent with this result, SB431542 or smad2 siRNA-induced reductions in smad2 protein expression levels attenuated TGF-ß1-induced apoptosis. On the other hand, the ligation of TGF-ß1 on its receptor also stimulated the phosphorylation of Erk and Akt, which are smad2-independent pathways. However, the inhibition of Erk/Akt signaling pathways by U0126, a MEK-Erk inhibitor and LY294002, a PI3Kinase-Akt inhibitor, augmented TGF-ß1-induced apoptosis in OBA9 cells. Taken together, the results of present study demonstrated that TGF-ß1 activated both the smad2 and Erk/Akt cascades via its receptor on gingival epithelial cells, even though these two pathways have opposite roles in cell death and survival, and the culmination of these signaling events induced mitochondria-dependent apoptosis in gingival epithelial cells. Based on the results of the present study, we herein proposed for the first time, that TGF-ß1 is a novel target cytokine for monitoring the progression of periodontal disease.

Amphotericin B down-regulates Aggregatibacter actinomycetemcomitans-induced production of IL-8 and IL-6 in human gingival epithelial cells.

Gingival epithelium is the primary barrier against microorganism invasion and produces inflammatory cytokines. Amphotericin B, a major antifungal drug, binds to cholesterol in the mammalian cell membrane in addition to fungal ergosterol. Amphotericin B has been shown to regulate inflammatory cytokines in host cells. To investigate the suppressive effect of amphotericin B on the gingival epithelium, we examined the expression of interleukin (IL)-8 and IL-6 and involvement of MAP kinase in human gingival epithelial cells (HGEC) stimulated by Aggregatibacter actinomycetemcomitans. Amphotericin B and the p38 MAP kinase inhibitor down-regulated the A. actinomycetemcomitans-induced increase in the expression of IL-8 and IL-6 at the mRNA. The ERK inhibitor suppressed the A. actinomycetemcomitans-induced IL-8 mRNA expression. Amphotericin B inhibited the A. actinomycetemcomitans-induced phosphorylation of ERK and p38 MAP kinase. Furthermore, amphotericin B inhibited the A. actinomycetemcomitans-induced production of prostaglandin E2. These results suggest that amphotericin B regulate inflammatory responses in HGEC.

Periostin regulates integrin expression in gingival epithelial cells.

OBJECTIVE: Human gingival epithelial cells (HGECs) function as a mechanical barrier against invasion by pathogenic organisms through epithelial cell-cell junction complexes, which are complex components of integrin. Integrins play an important role in the protective functions of HGECs. Human periodontal ligament (HPL) cells regulate periodontal homeostasis. However, periodontitis results in the loss of HPL cells. Therefore, as replenishment, HPL cells or mesenchymal stem cells (MSCs) can be transplanted. Herein, HPL cells and MSCs were used to elucidate the regulatory mechanisms of HGECs, assuming periodontal tissue homeostasis. METHODS: Human gingival fibroblasts (HGFs), HGECs, HPL cells, and MSCs were cultured, and the conditioned medium was collected. With or without silencing periostin mRNA, HGECs were cultured under normal conditions or in a conditioned medium. Integrin and periostin mRNA expression was determined using real-time polymerase chain reaction. Integrin protein expression was analyzed using flow cytometry, and periostin protein expression was determined via western blotting. RESULTS: The conditioned medium affected integrin expression in HGECs. Higher expression of periostin was observed in MSCs and HPL cells than in HGFs. The conditioned medium that contained periostin protein regulated integrin expression in HGECs. After silencing periostin in MSCs and HPL cells, periostin protein was not detected in the conditioned medium, and integrin expression in HGECs remained unaffected. CONCLUSIONS: Integrins in HGECs are regulated by periostin secreted from HPL cells and MSCs. This result suggests that periostin maintains gingival cell adhesion and regulates bacterial invasion/infection. Therefore, the functional regulation of periostin-secreting cells is important in preventing periodontitis.

Nuclear receptor 4A1 (NR4A1) upregulated by n-butylidenephthalide via the mitogen-activated protein kinase (MAPK) pathway ameliorates drug-induced gingival enlargement.

Drug-induced gingival enlargement (DIGE) is a side effect of ciclosporin, calcium channel blockers, and phenytoin. DIGE is a serious disease that leads to masticatory and esthetic disorders, severe caries, and periodontitis but currently has no standard treatment. We recently reported that nuclear receptor 4A1 (NR4A1) is a potential therapeutic target for DIGE. This study aimed to evaluate the therapeutic effects of n-butylidenephthalide (BP), which increases the expression of NR4A1, on DIGE. In this study, NR4A1 mRNA expression was analyzed in the patients with periodontal disease (PD) and DIGE. We evaluated the effect of BP on NR4A1 expression in gingival fibroblasts and in a DIGE mouse model. RNA sequencing (RNA-seq) was conducted to identify the mechanisms by which BP increases NR4A1 expression. The results showed that NR4A1 mRNA expression in the patients with DIGE was significantly lower than the patients with PD. BP suppressed the upregulation of COL1A1 expression, which was upregulated by TGF-ß. BP also ameliorated gingival overgrowth in DIGE mice and reduced Col1a1 and Pai1 expression. BP also decreased Il1ß mRNA expression in gingival tissue in DIGE. RNA-seq results showed an increase in the expression of several genes related to mitogen-activated protein kinase including DUSP genes in gingival fibroblasts stimulated by BP. Treatment with ERK and JNK inhibitors suppressed the BP-induced increase in NR4A1 expression. In addition, BP promoted the phosphorylation of ERK in gingival fibroblasts. In conclusion, BP increases NR4A1 expression in gingival fibroblasts through ERK and JNK signaling, demonstrating its potential as a preventive and therapeutic agent against DIGE.

Role of sclerostin deletion in bisphosphonate-induced osteonecrosis of the jaw.

PURPOSE: Bone resorption inhibitors, such as bisphosphonates (BP) and denosumab, are frequently used for the management of osteoporosis. Although both drugs reduce the risk of osteoporotic fractures, they are associated with a serious side effect known as medication-related osteonecrosis of the jaw (MRONJ). Sclerostin antibodies (romosozumab) increase bone formation and decrease the risk of osteoporotic fractures: however, their anti-resorptive effect increases ONJ. Thus, this study aimed to elucidate the role of sclerostin deletion in the development of MRONJ. METHODS: Sclerostin knockout (SostΔ26/Δ26) mice were used to confirm the development of ONJ by performing tooth extractions. To confirm the role of sclerostin deficiency in a more ONJ-prone situation, we used the BP-induced ONJ model in combination with severe periodontitis to evaluate the development of ONJ and bone formation in wild-type (WT) and SostΔ26/Δ26 mice. Wound healing assay using gingival fibroblasts with or without sclerostin stimulation and tooth extraction socket healing were evaluated in the WT and SostΔ26/Δ26 mice. RESULTS: ONJ was not detected in the extraction socket of SostΔ26/Δ26 mice. Moreover, the incidence of ONJ was significantly lower in the SostΔ26/Δ26 mice treated with BP compared to that of the WT mice. Osteogenic proteins, osteocalcin, and runt-related transcription factor 2, were expressed in the bone surface in SostΔ26/Δ26 mice. Recombinant sclerostin inhibited gingival fibroblast migration. The wound healing rate of the extraction socket was faster in SostΔ26/Δ26 mice than in WT mice. CONCLUSION: Sclerostin deficiency did not cause ONJ and reduced the risk of developing BP-induced ONJ. Enhanced bone formation and wound healing were observed in the tooth extraction socket. The use of romosozumab (anti-sclerostin antibody) has proven to be safe for surgical procedures of the jaw.

Effect of Periodontal Treatment on Reducing Chronic Inflammation in Systemically Healthy Patients With Periodontal Disease.

BACKGROUND: We determined the effects and an accurate marker of periodontal treatment on serum interleukin (IL)-6 and high-sensitivity C-reactive protein (HsCRP) levels in systemically healthy individuals with periodontal disease. METHODS: This multicenter study included systemically healthy individuals with periodontal disease who received initial periodontal treatment and had no periodontal treatment history. Periodontal parameters, including periodontal inflamed surface area, masticatory efficiency, and periodontal disease classification; serum IL-6 and HsCRP levels; and serum immunoglobulin (Ig)G titers against periodontal pathogens were evaluated at baseline and after treatment. Subjects were classified as low or high responders (group) based on periodontal inflamed surface area changes. RESULTS: There were 153 participants. Only periodontal inflamed surface area changes were markedly different between low and high responders. Periodontal treatment (time point) decreased both serum IL-6 and HsCRP levels. The interaction between group and time point was remarkable only for serum IL-6 levels. Changes in serum immunoglobulin (Ig)G titers against periodontal pathogens were not associated with IL-6 changes in high responders. We analyzed the indirect effect of serum anti-Porphyromonas gingivalis type 2 IgG titer changes using mediation analysis and found no significance. However, the direct effect of group (low or high responder) on IL-6 changes was considerable. CONCLUSIONS: Periodontal treatment effectively decreased serum IL-6 levels, independent of periodontal pathogen infection, in systemically healthy individuals with periodontal disease.

Anti-Inflammatory Effects of Geniposidic Acid on Porphyromonas gingivalis-Induced Periodontitis in Mice.

Periodontal disease is predominantly caused by the pathogenic bacterium Porphyromonas gingivalis that produces inflammation-inducing factors in the host. Eucommia ulmoides is a plant native to China that has been reported to reduce blood pressure, promote weight loss, and exhibit anti-inflammatory effects. Geniposidic acid (GPA) is the major component of E. ulmoides. Herein, we investigated the effects of GPA on P. gingivalis-induced periodontitis by measuring the inflammatory responses in human gingival epithelial cells (HGECs) after P. gingivalis stimulation and GPA addition in a P. gingivalis-induced periodontitis mouse model. We found that GPA addition suppressed interleukin (IL)-6 mRNA induction (33.8% suppression), IL-6 production (69.2% suppression), toll-like receptor (TLR) 2 induction, and mitogen-activated protein kinase (MAPK) phosphorylation in HGECs stimulated by P. gingivalis. Inoculation of mice with GPA inhibited P. gingivalis-induced alveolar bone resorption (25.6% suppression) by suppressing IL-6 and TLR2 production in the serum and gingiva. GPA suppressed osteoclast differentiation of bone marrow cells induced by M-CSF and sRANKL in mice (56.7% suppression). GPA also suppressed the mRNA expression of OSCAR, NFATc1, c-Fos, cathepsin K, and DC-STAMP. In summary, GPA exerts an anti-inflammatory effect on periodontal tissue and may be effective in preventing periodontal disease.

Clumps of mesenchymal stem cells/extracellular matrix complexes directly reconstruct the functional periodontal tissue in a rat periodontal defect model.

Periodontitis is an inflammatory disease characterized by tooth-supporting periodontal tissue destruction, including the cementum, periodontal ligament, and alveolar bone. To regenerate the damaged periodontal tissue, mesenchymal stem cells (MSCs) have attracted much scientific and medical attention. Recently, we generated clumps of MSCs/extracellular matrix (ECM) complexes (C-MSCs), which consist of cells and self-produced ECM. C-MSCs can be transplanted into lesion areas without artificial scaffold to induce tissue regeneration. To develop reliable scaffold-free periodontal tissue regenerative cell therapy by C-MSCs, this study investigated the periodontal tissue regenerative capacity of C-MSCs and the behavior of the transplanted cells. Rat bone marrow-derived MSCs were isolated from rat femur. Confluent cells were scratched using a micropipette tip and then torn off. The sheet was rolled to make a three-dimensional round clump of cells, C-MSCs. Then, ten C-MSCs were grafted into a rat periodontal fenestration defect model. To trace the grafted cells in the defect, PKH26-labeled cells were also employed. Micro-CT and histological analyses demonstrated that transplantation of C-MSCs induced successful periodontal tissue regeneration in the rat periodontal defect model. Interestingly, the majority of the cells in the reconstructed tissue, including cementum, periodontal ligaments, and alveolar bone, were PKH26 positive donor cells, suggesting the direct tissue formation by MSCs. This study demonstrates a promising scaffold-free MSCs transplantation strategy for periodontal disease using C-MSCs and offers the significance of multipotency of MSCs to induce successful periodontal tissue regeneration.