Comparison of early wound healing using modified papilla preservation technique between enamel matrix derivative and recombinant human fibroblast growth factor.

PURPOSE: Enamel matrix derivative (EMD) has demonstrated beneficial effects on wound healing following surgery. However, the effects of recombinant human fibroblast growth factor 2 (rhFGF-2) in periodontal regeneration therapy have not been extensively studied. This retrospective study was conducted to compare the wound healing outcomes of the modified papilla preservation technique (mPPT) between EMD and rhFGF-2 therapies. METHODS: A total of 79 sites were evaluated for early wound healing using the modified early wound healing index (mEHI), which included 6 items: incision, fibrin clotting, step, redness, swelling, and dehiscence. A numeric analog scale, along with postoperative images of the 6 mEHI items, was established and used for the evaluations. The inter-rater reliability of the mEHI was assessed via intraclass correlation coefficients (ICCs). After adjusting for factors influencing the mPPT, the differences in mEHI scores between the EMD and rhFGF-2 groups were statistically analyzed. Additionally, radiographic bone fill (RBF) was evaluated 6 months after surgery. RESULTS: The ICC of the mEHI was 0.575. The mEHI, redness score, and dehiscence scores were significantly higher in the rhFGF-2 group (n=33) than in the EMD group (n=46). Similar results were observed in the subgroup of patients aged 50 years or older, but not in those younger than 50 years. In the subgroup with non-contained bone defects, related results were noted, but not in the subgroup with contained bone defects. However, early wound healing did not correlate with RBF at 6 months after surgery. CONCLUSIONS: Within the limitations of this study, the findings suggest that early wound healing following the use of mPPT with rhFGF-2 is somewhat superior to that observed after mPPT with EMD. However, mEHI should be improved for use as a predictive tool for early wound healing and to reflect clinical outcomes after surgery.

Impact of COVID-19 spread on visit intervals and clinical parameters for patients with periodontitis in supportive periodontal therapy: a retrospective study.

PURPOSE: This study investigated the relationship between the number of days that hospital visits were postponed and changes in clinical parameters due to the spread of coronavirus disease 2019 (COVID-19), after the Japanese government declared a state of emergency in April 2020. METHODS: Regarding the status of postponement of appointments, we analyzed the patients who had visited the Nihon University Hospital at Matsudo for more than 1 year for supportive periodontal therapy (SPT) and classified them into low-, moderate- and high-risk subgroups according to the periodontal risk assessment (PRA). Clinical parameters for periodontal disease such as probing depth (PD), full-mouth bleeding score (FMBS), full-mouth plaque score, periodontal inflamed surface area (PISA), and periodontal epithelial surface area (PESA) were analyzed in 2 periods, from October 2019 to March 2020 and after April 2020. Correlation coefficients between days of deferral and the degree of changes in clinical parameters were calculated. RESULTS: The mean age of the 749 patients was 67.56±10.85 years, and 63.82% were female. Out of 749 patients, 33.24% deferred their SPT appointments after April 2020. The average total of postponement days was 109.49±88.84. The number of postponement days was positively correlated with changes in average PD (rs=0.474) and PESA (rs=0.443) in the high-risk subgroup of FMBS, and average PD (rs=0.293) and PESA (rs=0.253) in the high-risk subgroup of tooth number (TN). Patients belonging to the high-risk subgroups for both FMBS and TN had a positive correlation between postponement days and PISA (rs=0.56). CONCLUSIONS: The findings, the spread of COVID-19 appears to have extended the visit interval for some SPT patients. Moreover, longer visit intervals were correlated with the worsening of some clinical parameters for SPT patients with high PRA.

Changes in the components of salivary exosomes due to initial periodontal therapy.

PURPOSE: Exosomes are membrane vesicles that are present in body fluids and contain proteins, lipids, and microRNA (miRNA). Periodontal tissue examinations assess the degree of periodontal tissue destruction according to the probing depth (PD), clinical attachment loss (CAL), bleeding on probing, and X-ray examinations. However, the accurate evaluation of the prognosis of periodontitis is limited. In this study, we collected saliva from patients before and after initial periodontal therapy (IPT) and compared changes in the clinical parameters of periodontitis with changes in the components of salivary exosomes. METHODS: Saliva was collected from patients with stage III and IV periodontitis at the first visit and post-IPT. Exosomes were purified from the saliva, and total protein and RNA were extracted. Changes in expression levels of C6, CD81, TSG101, HSP70, and 6 kinds of miRNA were analyzed by western blots and real-time polymerase chain reaction. RESULTS: Patients with increased C6 expression after IPT had significantly higher levels of periodontal inflamed surface area (PISA), miR-142, and miR-144 before and after IPT than patients with decreased C6 expression after IPT. Patients with decreased and unchanged CD81 expression after IPT showed significantly higher PD, CAL, and PISA before IPT than after IPT. Patients with decreased and unchanged TSG101 expression after IPT had significantly higher PD before IPT than after IPT. Patients with increased HSP70 expression after IPT had significantly higher PD and PISA before and after IPT than patients with unchanged HSP70 after IPT. The expression levels of miR-142, miR-144, miR-200b, and miR-223 changed with changes in the levels of C6, CD81, TSG101, and HSP70 in the salivary exosomes of periodontitis patients before and after IPT. CONCLUSIONS: The expression levels of proteins and miRNAs in salivary exosomes significantly changed after IPT in periodontitis patients, suggesting that the components of exosomes could serve as biomarkers for periodontitis.

TNF-α regulates the composition of the basal lamina and cell-matrix adhesions in gingival epithelial cells.

Laminin 5, type 4 collagen, and α6ß4 integrin contribute to the formation of hemidesmosomes in the epithelia of periodontal tissues, which is critical for the development and maintenance of the dentogingival junction. As it is not known whether TNF-α alters the composition of the epithelial pericellular matrix, human gingival epithelial cells were cultured in the presence or absence of TNF-α. Treatment with TNF-α accelerated epithelial cell migration and closure of in vitro wounds. These data indicate unexpectedly, that TNF-α promotes the formation of the pericellular matrix around epithelial cells and enhances adhesion of epithelial cells to the underlying matrix, properties which are important for cell migration and the integrity of the dentogingival junction.

Interleukin-1ß regulates odontogenic ameloblast-associated protein gene transcription in human gingival epithelial cells.

Junction epithelium (JE) is located apical to the bottom of the gingival sulcus and binds enamel to hemidesmosomes to protect the periodontal tissue from bacterial infection. Function of odontogenic ameloblast-associated protein (ODAM) is suggested by its expression sites (JE and maturation stage ameloblasts) to be involved in the adhesion between the JE and enamel, and odontogenesis. To analyze the changes in ODAM gene and protein expressions in inflamed gingiva, Ca9-22 gingival epithelial cells were stimulated with 1 ng/ml interleukin-1ß (IL-1ß) for 3-24 h, and ODAM mRNA and protein levels were analyzed by real-time PCR and Western blotting. Luciferase (LUC) constructs were made ligating various lengths of human ODAM gene promoters and performed LUC analyses in Ca9-22 cells. Gel shift and chromatin immunoprecipitation (ChIP) assays were performed. IL-1ß induced ODAM mRNA and protein levels at 6-24 h. IL-1ß increased LUC activities of the ODAM gene promoter constructs from - 85 to - 950. These activities were blocked by protein kinase A, tyrosine kinase, mitogen-activated protein (MAP) kinase kinase and phosphoinositide 3-kinase inhibitors. Gel shift and ChIP assays showed that IL-1ß induced CCAAT/enhancer-binding protein (C/EBP) ß and Yin Yang1 (YY1) binding to C/EBP1, 2, 3, and YY1 elements. These data indicate that IL-1ß stimulates ODAM gene transcription mediated through C/EBP1, C/EBP2, C/EBP3, and YY1 elements in the human ODAM gene promoter.

Transcriptional regulation of human odontogenic ameloblast-associated protein gene by tumor necrosis factor-α.

OBJECTIVE: Odontogenic ameloblast-associated protein (ODAM) is produced by maturation stage ameloblasts and junctional epithelium (JE). The function of ODAM is thought to be involved in the attachment of teeth and JE. To elucidate transcriptional regulation of human ODAM gene in inflamed gingiva, we have analyzed the effects of TNF-α on the expression of ODAM gene in Ca9-22 and Sa3 gingival epithelial cells. MATERIALS AND METHODS: Total RNAs were extracted from Ca9-22 and Sa3 cells after stimulation by TNF-α (10 ng/ml). ODAM mRNA and protein levels were analyzed by qPCR and Western blotting. Luciferase (LUC) analyses were performed using LUC constructs inserted in various lengths of ODAM gene promoter. Gel shift and chromatin immunoprecipitation (ChIP) assays were carried out. RESULTS: TNF-α increased ODAM mRNA and protein levels at 3 to 24 h. TNF-α induced LUC activities of the ODAM gene promoter constructs, and the activities were inhibited by protein kinase A, tyrosine kinase, MEK1/2, PI3-kinase and NF-κB inhibitors. Gel shift and ChIP assays revealed that TNF-α increased CCAAT/enhancer-binding protein (C/EBP) ß and Yin Yang1 (YY1) binding to three kinds of C/EBPs and YY1 elements. CONCLUSION: These results demonstrate that TNF-α stimulates ODAM gene transcription via C/EBPs and YY1 elements in the human ODAM gene promoter.

MiR-200b suppresses TNF-α-induced AMTN production in human gingival epithelial cells.

Amelotin (AMTN) is an enamel protein that is localized in junctional epithelium (JE) of gingiva and suggested to be involved in the attachment between JE and tooth enamel. MicroRNA is a small non-coding RNA that regulates gene expression at post-transcriptional level by binding to the 3'-untranslated region (3'-UTR) of target mRNAs. In this study, we have analyzed the effects of miR-200b on the expression of AMTN in human gingival epithelial (Ca9-22) cells. Total RNAs and proteins were extracted from Ca9-22 cells transfected with miR-200b expression plasmid or miR-200b inhibitor and stimulated by TNF-α (10 ng/ml, 12 h). AMTN and inhibitor of kappa-B kinase beta (IKKß) mRNA and protein levels were measured by qPCR and Western blot. Human AMTN 3'-UTR that contains putative miR-200b target sites were cloned downstream of -353AMTN luciferase (LUC) plasmid. Ca9-22 cells were transfected with -353AMTN 3'-UTR LUC constructs and miR-200b expression plasmid, and LUC activities were measured with or without stimulation by TNF-α. TNF-α-induced AMTN mRNA levels were partially inhibited by miR-200b overexpression and enhanced by miR-200b inhibitor. TNF-α-induced IKKß mRNA and protein levels were almost completely inhibited by miR-200b. Transcriptional activities of -353AMTN 3'-UTR LUC constructs were induced by TNF-α and partially inhibited by miR-200b. IKKß inhibitor IMD0354 and NF-κB inhibitor triptolide decreased TNF-α-induced LUC activities. Furthermore, both inhibitors reduced AMTN mRNA levels in the presence or absence of TNF-α. These results suggest that miR-200b suppresses AMTN expression by targeting to AMTN and IKKß mRNAs in the human gingival epithelial cells.

IL-1ß enhances cell adhesion through laminin 5 and ß4 integrin in gingival epithelial cells.

The junctional epithelium and dental enamel adhere because of hemidesmosomes containing laminin 5 and α6ß4 integrin, which are important adhesion molecules in the internal basal lamina. Interleukin (IL)-1 is important in the pathogenesis of periodontal disease. IL-1ß induces bone resorption by activating osteoclasts; however, its effects on adhesion of epithelial cells remain to be clarified. Laminin ß3, ß4 integrin, and focal adhesion kinase mRNA levels were higher after 1 h and 3 h of stimulation with IL-1ß (1 ng/mL), and IL-1ß, type I α1, and type IV α1 collagen mRNA levels were higher after 1 h and lower after 3 h of stimulation with IL-1ß. After IL-1ß stimulation, colocalization of laminin 5 and ß4 integrin was increased after 1 h, colocalization of ß4 integrin and plectin was increased after 1 h and decreased after 3 h, and colocalization of ß4 integrin and type IV collagen was decreased after 3 h. Wound healing assays showed that IL-1ß treatment (3 h) delayed wound healing. These results suggest that IL-1ß enhances cell adhesion by altering localization of epithelial adhesion molecules.

Negative feedback by SNAI2 regulates TGFß1-induced amelotin gene transcription in epithelial-mesenchymal transition.

Junctional epithelium (JE) demonstrates biological responses with the rapid turnover of gingival epithelial cells. The state occurs in inflammation of gingiva and wound healing after periodontal therapy. To understand the underlying mechanisms and to maintain homeostasis of JE, it is important to investigate roles of JE-specific genes. Amelotin (AMTN) is localized at JE and regulated by inflammatory cytokines and apoptotic factors that represent a critical role of AMTN in stabilizing the dentogingival attachment, which is an entrance of oral bacteria. In this study, we demonstrated that the AMTN gene expression was regulated by SNAI2 and transforming growth factor ß1 (TGFß1)-induced epithelial-mesenchymal transition (EMT) that occurs in wound healing and fibrosis during chronic inflammation. SNAI2 downregulated AMTN gene expression via SNAI2 bindings to E-boxes (E2 and E4) in the mouse AMTN gene promoter in EMT of gingival epithelial cells. Meanwhile, TGFß1-induced AMTN gene expression was attenuated by SNAI2 and TGFß1-induced SNAI2, without inhibition of the TGFß1-Smad3 signaling pathway. Moreover, SNAI2 small interfering RNA (siRNA) rescued SNAI2-induced downregulation of AMTN gene expression, and TGFß1-induced AMTN gene expression was potentiated by SNAI2 siRNA. Taken together, these data demonstrated that AMTN gene expression in the promotion of EMT was downregulated by SNAI2. The inhibitory effect of AMTN gene expression was an independent feedback on the TGFß1-Smad3 signaling pathway, suggesting that the mechanism can be engaged in maintaining homeostasis of gingival epithelial cells at JE and the wound healing phase.