Dynamic analyses of a soft tissue-implant interface: Biological responses to immediate versus delayed dental implants.

AIM: To qualitatively and quantitatively evaluate the formation and maturation of peri-implant soft tissues around 'immediate' and 'delayed' implants. MATERIALS AND METHODS: Miniaturized titanium implants were placed in either maxillary first molar (mxM1) fresh extraction sockets or healed mxM1 sites in mice. Peri-implant soft tissues were evaluated at multiple timepoints to assess the molecular mechanisms of attachment and the efficacy of the soft tissue as a barrier. A healthy junctional epithelium (JE) served as positive control. RESULTS: No differences were observed in the rate of soft-tissue integration of immediate versus delayed implants; however, overall, mucosal integration took at least twice as long as osseointegration in this model. Qualitative assessment of Vimentin expression over the time course of soft-tissue integration indicated an initially disorganized peri-implant connective tissue envelope that gradually matured with time. Quantitative analyses showed significantly less total collagen in peri-implant connective tissues compared to connective tissue around teeth around implants. Quantitative analyses also showed a gradual increase in expression of hemidesmosomal attachment proteins in the peri-implant epithelium (PIE), which was accompanied by a significant inflammatory marker reduction. CONCLUSIONS: Within the timeframe examined, quantitative analyses showed that connective tissue maturation never reached that observed around teeth. Hemidesmosomal attachment protein expression levels were also significantly reduced compared to those in an intact JE, although quantitative analyses indicated that macrophage density in the peri-implant environment was reduced over time, suggesting an improvement in PIE barrier functions. Perhaps most unexpectedly, maturation of the peri-implant soft tissues was a significantly slower process than osseointegration.

Metformin alleviates junctional epithelium senescence via the AMPK/SIRT1/autophagy pathway in periodontitis induced by hyperglycemia.

The junctional epithelium (JE) serves a crucial protective role in the periodontium. High glucose-related aging results in accelerated barrier dysfunction of the gingival epithelium, which may be associated with diabetic periodontitis. Metformin, an oral hypoglycemic therapeutic, has been proposed as a anti-aging agent. This study aimed to clarify the effect of metformin on diabetic periodontitis and explore its mechanism in ameliorating senescence of JE during hyperglycemia. The db/db mice was used as a diabetic model mice and alterations in the periodontium were observed by hematoxylin-eosin staining and immunohistochemistry. An ameloblast-like cell line (ALC) was cultured with high glucose to induce senescence. Cellular senescence and oxidative stress were evaluated by SA-ß-gal staining and Intracellular reactive oxygen species (ROS) levels. Senescence biomarkers, P21 and P53, and autophagy markers, LC3-II/LC3-I, were measured by western blotting and quantitative real-time PCR. To construct a stable SIRT1 (Sirtuin 1) overexpression cell line, we transfected ALCs with lentiviral vectors overexpressing the mouse SIRT1 gene. Cellular senescence was increased in the JE of db/db mice and the periodontium was destroyed, which could be alleviated by metformin. Moreover, oxidative stress and cellular senescence in a high glucose environment were reduced by metformin in in-vitro assays. The autophagy inhibitor 3-MA and SIRT1 inhibitor EX-527 could dampen the effects of metformin. Overexpression of SIRT1 resulted in increased autophagy and decreased oxidative stress and cellular senescence. Meanwhile, AMPK (AMP-activated protein kinase) inhibition reversed the anti-senescence effects of metformin. Overall, these results suggest that metformin alleviates periodontal damage in db/db mice and cellular senescence in ALCs under high glucose conditions via the AMPK/SIRT1/autophagy pathway.

Influences of standardized clinical probing on peri-implant soft tissue seal in a situation of peri-implant mucositis: A histomorphometric study in dogs.

BACKGROUND: Clinical probing is commonly recommended to evaluate peri-implant conditions. In a situation of peri-implant mucositis or peri-implantitis, the peri-implant seal healing from the disruption of soft tissue caused by probing has not yet been studied. This study aimed to investigate soft tissue healing after standardized clinical probing around osseointegrated implants with peri-implant mucositis in a dog model. METHODS: Three transmucosal implants in each hemi-mandible of six dogs randomly assigned to the peri-implant healthy group or peri-implant mucositis group were probed randomly in the mesial or distal site as probing groups (PH or PM), the cross-sectional opposite sites as unprobed control groups. Histomorphometric measurements of implant shoulder (IS)-most coronal level of alveolar bone contact to the implant surface (BCI), apical termination of the junctional epithelium (aJE)-BCI, mucosal margin (MM)-BCI, and MM-aJE were performed at 1 day, 1 week, and 2 weeks after probing. Apoptosis, proliferation, proinflammatory cytokines, and matrix metalloproteinases (MMPs) of peri-implant soft tissue were estimated by immunofluorescent analysis. RESULTS: In the PM group, apical migration of junctional epithelium was revealed by significantly decreased aJE-BCI from 1 day to 2 weeks in comparison to unprobed sites (p < 0.05), while no significant differences were found in the PH group. Immunofluorescent analysis showed higher levels of interleukin-1ß (IL-1ß), IL-6, tumor necrosis factor-α (TNF-α), MMP-1, and MMP-8, together with exaggerated apoptosis and proliferation of peri-implant soft tissue in the PM group. CONCLUSION: Within the limitations, standardized clinical probing might lead to apical migration of the junctional epithelium in a situation of peri-implant mucositis.

Histological Techniques for the Study of the Dentogingival Junction: A Scoping Review Using the Anatomical Quality Assurance Checklist (AQUA) / Técnicas Histológicas para el Estudio de la Unión Dentogingival: Una Revisión de Alcance Utilizando el Anatomical Quality Assurance Checklist (AQUA)

SUMMARY: The dentogingival junction (DGJ) is an adaptation of the oral mucosa composed of epithelial and connective tissues intimately related with the mineralised tissues of the tooth. The histological evidence available is mainly based on studies in animals, separate evaluations of hard and soft tissues, and studies using conventional histological techniques that eliminate the enamel from preparations. The aim of this study was to carry out a review of the existing evidence on histological techniques available for study of the tooth and periodontium in conjunction in humans. A scoping review was carried out of the available literature referring to study of the tooth and the periodontium in conjunction in humans, in the Web of Science (WoS), EMBASE, Scopus and SciELO databases, using the terms "Histological Techniques"[Mesh]) and "Epithelial Attachment"[Mesh]. One hundred and fifty-nine articles were found, of which 54 were selected for full- text reading. Ten were finally included in the qualitative synthesis, and we applied the Anatomical Quality Assurance (AQUA) checklist for analysis the methodological quality of the selected articles. The results showed that the only articles with a low risk of bias in all five domains according to the AQUA criteria corresponded to Silva et al. (2011) and Agustín-Panadero et al. (2020). Finally, we conclude that the quality of the histological sections to observe tissues that simultaneously contain the tooth and the periodontium, is conditioned by the selected technique and by the care required in certain specific tasks during the histological processing of the samples.

La unión dentogingival (DGJ) es una adaptación de la mucosa oral compuesta por tejidos epitelial y conectivo íntimamente relacionados con los tejidos mineralizados del diente. La evidencia histológica disponible se basa principalmente en estudios en animales, evaluaciones separadas de tejidos duros y blandos y estudios utilizando técnicas histológicas convencionales que eliminan el esmalte de las preparaciones. El objetivo de este estudio fue realizar una revisión de la evidencia existente sobre las técnicas histológicas disponibles para el estudio del diente y el periodonto en conjunto en humanos. Se realizó un scoping review de la literatura disponible referente al estudio del diente y el periodonto en conjunto en humanos, en las bases de datos Web of Science (WoS), EMBASE, Scopus y SciELO, utilizando los términos "Histological Techniques"[Mesh]) y "Epithelial Attachment"[Mesh]. Se encontraron 159 artículos, de los cuales 54 fueron seleccionados para lectura de texto completo. Diez fueron finalmente incluidos en la síntesis cualitativa, y se aplicó la lista de verificación Anatómica Quality Assurance (AQUA) para el análisis de la calidad metodológica de los artículos seleccionados. Los resultados mostraron que los únicos artículos con bajo riesgo de sesgo en los cinco dominios según los criterios AQUA correspondían a Silva et al. (2011) y Agustín-Panadero et al. (2020). Finalmente, concluimos que la calidad de los cortes histológicos para observar los tejidos que contienen simultáneamente el diente y el periodonto, está condicionada por la técnica seleccionada y por el cuidado requerido en ciertas tareas específicas durante el procesamiento histológico de las muestras.

A novel junctional epithelial cell line, mHAT-JE01, derived from incisor epithelial cells.

OBJECTIVES: Junctional epithelium (JE) connects the tooth surface and gingival epithelium and adheres directly to the tooth enamel. JE plays an important role as a barrier preventing the invasion of exogenous bacteria and substances. However, the cellular characteristics of this epithelium have not been adequately described, because no useful in vitro experimental model exists for JE. METHODS: We generated a novel JE cell line, mHAT-JE01, using naturally immortalized dental epithelium derived from incisor labial cervical cells and by selecting cells that adhered to apatite. mHAT-JE01 was characterized by immunohistochemistry and quantitative reverse transcription-polymerase chain reaction and compared with the gingival epithelial cell line, mOE-PE01. RESULTS: The mHAT-JE01 cells had a higher capacity for producing JE-specific markers than oral mucous epithelial cells. In addition, the presence of lipopolysaccharides from Porphyromonas gingivalis downregulated the expression of JE protein markers in mHAT-JE01 cells. CONCLUSIONS: This cell line is stable and presents the opportunity to characterize JE efficiently, which is essential for the prevention and treatment of periodontal disease.

Downregulation of odontogenic ameloblast-associated protein in the progression of periodontal disease affects cell adhesion, proliferation, and migration.

OBJECTIVE: This work aimed to examine changes in odontogenic ameloblast-associated protein (ODAM) expression during the progression of periodontal disease and to investigate the effect of ODAM deficiency in vitro by RNA sequencing. DESIGN: Gingival tissue samples were collected from three groups, including healthy control, gingivitis and periodontitis patients, and ODAM expression was assessed by immunohistochemistry and quantitative reverse transcription PCR (qRT-PCR). Then, an Odam-knockdown cell line was established by lentiviral infection of small guide RNAs (sgRNAs) into an immortalized ameloblast-lineage cell line. RNA sequencing was carried out in Odam-knockdown and empty lentiviral vector-transfected cells. Differentially expressed genes were compared between these two cell groups to analyze functional enrichment, and a protein-protein interaction network was built. RESULTS: ODAM expression levels in gingival tissue samples were significantly lower in patients with periodontitis than in healthy controls as determined by immunohistochemistry and qRT-PCR. Transcriptomic analysis of Odam-knockdown cells identified 2801 differentially expressed genes, which were enriched in cell-substrate adhesion, proliferation, and migration pathways. The expression of a core of differentially expressed genes was confirmed by qRT-PCR in Odam-knockdown cells and by immunohistochemistry in clinical samples. Knocking down Odam significantly reduced cell adhesion but increased cell proliferation and migration capacity in vitro. CONCLUSIONS: ODAM is important in cell adhesion, proliferation, and migration, and its downregulation may contribute to periodontitis progression in humans.

The versatile roles of odontogenic ameloblast-associated protein in odontogenesis, junctional epithelium regeneration and periodontal disease.

Junctional epithelium (JE) is a vital epithelial component which forms an attachment to the tooth surface at the gingival sulcus by the adhesion of protein complexes from its basal layer. Disruption of the JE is associated with the development of gingivitis, periodontal disease, and alveolar bone loss. Odontogenic ameloblast-associated (ODAM) is comprised of a signal peptide and an ODAM protein with 12 putative glycosylation sites. It is expressed during odontogenesis by maturation stage ameloblasts and is incorporated into the enamel matrix during the formation of outer and surface layer enamel. ODAM, as a secreted protein which is accumulated at the interface between basal lamina and enamel, mediates the adhesion of the JE to the tooth surface; and is involved with extracellular signalling of WNT and ARHGEF5-RhoA, as well as intracellular signalling of BMP-2-BMPR-IB-ODAM. ODAM is also found to be highly expressed in salivary glands and appears to have implications for the regulation of formation, repair, and regeneration of the JE. Bioinformatics and research data have identified the anti-cancer properties of ODAM, indicating its potential both as a prognostic biomarker and therapeutic target. Understanding the biology of ODAM will help to design therapeutic strategies for periodontal and dental disorders.

An SCPPPQ1/LAM332 protein complex enhances the adhesion and migration of oral epithelial cells: Implications for dentogingival regeneration.

Common periodontal disease treatment procedures often fail to restore the structural integrity of the junctional epithelium (JE), the epithelial attachment of the gum to the tooth, leaving the tooth-gum interface prone to bacterial colonization. To address this issue, we introduced a novel bio-inspired protein complex comprised of a proline-rich enamel protein, SCPPPQ1, and laminin 332 (LAM332) to enhance the JE attachment. Using quartz crystal microbalance with dissipation monitoring (QCM-D), we showed that SCPPPQ1 and LAM332 interacted and assembled into a protein complex with high-affinity adsorption of 5.9e-8 [M] for hydroxyapatite (HA), the main component of the mineralized tooth surfaces. We then designed a unique shear device to study the adhesion strength of the oral epithelial cells to HA. The SCPPPQ1/LAM332 complex resulted in a twofold enhancement in adhesion strength of the cells to HA compared to LAM332 (from 31 dyn/cm2 to 63 dyn/cm2). In addition, using a modified wound-healing assay, we showed that gingival epithelial cells demonstrated a significantly high migration rate of 2.7 ± 0.24 µm/min over SCPPPQ1/LAM332-coated surfaces. Our collective data show that this protein complex has the potential to be further developed in designing a bioadhesive to enhance the JE attachment and protect the underlying connective tissue from bacterial invasion. However, its efficacy for wound healing requires further testing in vivo. STATEMENT OF SIGNIFICANCE: This work is the first functional study towards understanding the combined role of the enamel protein SCPPPQ1 and laminin 332 (LAM332) in the epithelial attachment of the gum, the junctional epithelium (JE), to the tooth hydroxyapatite surfaces. Such studies are essential for developing therapeutic approaches to restore the integrity of the JE in the destructive form of gum infection. We have developed a model system that provided the first evidence of the strong interaction between SCPPPQ1 and LAM332 on hydroxyapatite surfaces that favored protein adsorption and subsequently oral epithelial cell attachment and migration. Our collective data strongly suggested using the SCPPPQ1/LAM332 complex to accelerate the reestablishment of the JE after surgical gum removal to facilitate gum regeneration.

Junctional epithelium and hemidesmosomes: Tape and rivets for solving the "percutaneous device dilemma" in dental and other permanent implants.

The percutaneous device dilemma describes etiological factors, centered around the disrupted epithelial tissue surrounding non-remodelable devices, that contribute to rampant percutaneous device infection. Natural percutaneous organs, in particular their extracellular matrix mediating the "device"/epithelium interface, serve as exquisite examples to inspire longer lasting long-term percutaneous device design. For example, the tooth's imperviousness to infection is mediated by the epithelium directly surrounding it, the junctional epithelium (JE). The hallmark feature of JE is formation of hemidesmosomes, cell/matrix adhesive structures that attach surrounding oral gingiva to the tooth's enamel through a basement membrane. Here, the authors survey the multifaceted functions of the JE, emphasizing the role of the matrix, with a particular focus on hemidesmosomes and their five main components. The authors highlight the known (and unknown) effects dental implant - as a model percutaneous device - placement has on JE regeneration and synthesize this information for application to other percutaneous devices. The authors conclude with a summary of bioengineering strategies aimed at solving the percutaneous device dilemma and invigorating greater collaboration between clinicians, bioengineers, and matrix biologists.

Leaky Gum: The Revisited Origin of Systemic Diseases.

The oral cavity is the gateway for microorganisms into your body where they disseminate not only to the directly connected respiratory and digestive tracts but also to the many remote organs. Oral microbiota, travelling to the end of the intestine and circulating in our bodies through blood vessels, not only affect a gut microbiome profile but also lead to many systemic diseases. By gathering information accumulated from the era of focal infection theory to the age of revolution in microbiome research, we propose a pivotal role of "leaky gum", as an analogy of "leaky gut", to underscore the importance of the oral cavity in systemic health. The oral cavity has unique structures, the gingival sulcus (GS) and the junctional epithelium (JE) below the GS, which are rarely found anywhere else in our body. The JE is attached to the tooth enamel and cementum by hemidesmosome (HD), which is structurally weaker than desmosome and is, thus, vulnerable to microbial infiltration. In the GS, microbial biofilms can build up for life, unlike the biofilms on the skin and intestinal mucosa that fall off by the natural process. Thus, we emphasize that the GS and the JE are the weakest leaky point for microbes to invade the human body, making the leaky gum just as important as, or even more important than, the leaky gut.

Macrophage migration inhibitory factor regulates specific innate immune sensor responses in gingival epithelial cells.

BACKGROUND: The gingival epithelium protects periodontal tissues and the alveolar bone by maintaining a steady state of regulated inflammatory surveillance, also known as healthy homeostasis. Accordingly, the repertoire of receptors present within the gingival epithelium showcases its ability to recognize microbial colonization and contribute to bacterial sensing. Macrophage migration inhibitory factor (MIF) is one of many cytokines that are expressed in this protective state and is involved in neutrophil regulation. However, its role in the maintenance of healthy gingival tissue has not been described. METHODS: Gingival tissues from wild-type (WT) and Mif knock-out (KO) mice were stained for neutrophils and three key neutrophil chemoattractants: MIF, Gro-α/CXCL1, and Gro-ß/CXCL2 in the junctional epithelium (JE). In addition, gene silencing studies were performed using gingival epithelial cells (GECs) to examine the role of MIF on transcription of key bacterial recognition receptors Toll-like receptors (TLR)-1, -2, -4, -6, -9 and interleukin-1 receptors (IL-1R1 and IL-1R2) in response to oral bacterial stimulation. RESULTS: WT murine gingival tissues demonstrated high expression of MIF in the JE. In Mif KO mice, despite the significant reduction of Gro-α/CXCL1 and Gro-ß/CXCL2, there was a slight increase in neutrophils. Gene silencing experiments showed that MIF down-regulated the mRNA expression of TLR4, IL-1R1, and IL-1R2 in GEC, in addition to decreasing secreted IL-8/CXCL8 in response to bacteria. CONCLUSIONS: MIF regulates the expression of TLR4, IL-1Rs, and IL-8/CXCL8, components that are all involved in maintaining oral health. Our data demonstrate that MIF is a significant contributor to the maintenance of healthy oral homeostasis.

Tapping basement membrane motifs: Oral junctional epithelium for surface-mediated soft tissue attachment to prevent failure of percutaneous devices.

Teeth, long-lasting percutaneous organs, feature soft tissue attachment through adhesive structures, hemidesmosomes, in the junctional epithelium basement membrane adjacent to teeth. This soft tissue attachment prevents bacterial infection of the tooth despite the rich - and harsh - microbial composition of the oral cavity. Conversely, millions of percutaneous devices (catheters, dental, and orthopedic implants) fail from infection yearly. Standard of care antibiotic usage fuels antimicrobial resistance and is frequently ineffective. Infection prevention strategies, like for dental implants, have failed in generating durable soft tissue adhesion - like that seen with the tooth - to prevent bacterial colonization at the tissue-device interface. Here, inspired by the impervious natural attachment of the junctional epithelium to teeth, we synthesized four cell adhesion peptide (CAPs) nanocoatings, derived from basement membranes, to promote percutaneous device soft tissue attachment. The two leading nanocoatings upregulated integrin-mediated hemidesmosomes, selectively increased keratinocyte proliferation compared to fibroblasts, which cannot form hemidesmosomes, and expression of junctional epithelium adhesive markers. CAP nanocoatings displayed marked durability under simulated clinical conditions and the top performer CAP nanocoating was validated in a percutaneous implant murine model. Basement membrane CAP nanocoatings, inspired by the tooth and junctional epithelium, may provide an alternative anti-infective strategy for percutaneous devices to mitigate the worldwide threat of antimicrobial resistance. STATEMENT OF SIGNIFICANCE: Prevention and management of medical device infection is a significant healthcare challenge. Overzealous antibiotic use has motivated alternative material innovations to prevent infection. Here, we report implant cell adhesion peptide nanocoatings that mimic a long-lasting, natural "medical device," the tooth, through formation of cell adhesive structures called hemidesmosomes. Such nanocoatings sidestep the use of antimicrobial or antibiotic elements to form a soft-tissue seal around implants. The top performing nanocoatings prompted expression of hemidesmosomes and defensive factors to mimic the tooth and was validated in an animal model. Application of cell adhesion peptide nanocoatings may provide an alternative to preventing, rather that necessarily treating, medical device infection across a range of device indications, like dental implants.

Expression and localization of amelotin, laminin γ2 and odontogenesis-associated phosphoprotein (ODAPH) on the basal lamina and junctional epithelium.

At maturation stage of enamel development, a specialized basal lamina (sBL) was built between ameloblasts and enamel. After the teeth eruption, the ameloblasts transform into the inner cell layer of junctional epithelium. The inner cell layer forms the internal basal lamina of junctional epithelium. However, the composition of the sBL and internal basal lamina was not clarified. The objective of our study was to make a description of the localization of amelotin (AMTN), laminin γ2 (LAMC2) and Odontogenesis-associated phosphoprotein (ODAPH) on the sBL and internal basal lamina. In immunohistochemical study, AMTN, LAMC2 and ODAPH were detected on the sBL at maturation stage. AMTN was also detected in ameloblasts at maturation stage. The expression of AMTN decreased from early-to-late maturation stage. In contrast, the expression of LAMC2 and ODAPH was stable. Immunofluorescence double-staining showed the localization of AMTN was close to enamel surface. However, the localization of ODAPH was close to ameloblasts. LAMC2 and ODAPH were observed on internal basal lamina of junctional epithelium. In contrast, no expression of AMTN was detected on internal basal lamina of junctional epithelium. Our results suggested that ODAPH might participate in enamel maturation and periodontal health, which might provide a better understanding of enamel defects and periodontal disease in clinic.

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.

The Role of Lactic Acid on Wound Healing, Cell Growth, Cell Cycle Kinetics, and Gene Expression of Cultured Junctional Epithelium Cells in the Pathophysiology of Periodontal Disease.

Lactic acid (LA) is short-chain fatty acid, such as butyric acid and propionic acid, that is produced as a metabolite of lactic acid bacteria, including periodontopathic bacteria. These short-chain fatty acids have positive effects on human health but can also have negative effects, such as the promotion of periodontal disease (PD), which is caused by periodontal pathogens present in the gingival sulcus. PD is characterized by apical migration of junctional epithelium, deepening of pockets, and alveolar bone loss. Thus, the junctional epithelial cells that form the bottom of the gingival sulcus are extremely important in investigating the pathophysiology of PD. The aim of this study was to investigate the effect of LA on wound healing, cell growth, cell cycle kinetics, and gene expression of cultured junctional epithelium cells. The results showed that stimulation with 10 mM LA slowed wound healing of the junctional epithelial cell layer and arrested the cell cycle in the G0/G1 (early cell cycle) phase, thereby inhibiting cell growth. However, cell destruction was not observed. LA also enhanced mRNA expression of integrin α5, interleukin (IL)-6, IL-8, intercellular adhesion molecule-1, and receptor activator of nuclear factor kappa-B ligand. The results of this study suggest that stimulation of junctional epithelial cells with high concentrations of LA could exacerbate PD, similarly to butyric acid and propionic acid.

[Expression and distribution of calprotectin in healthy and inflamed periodontal tissues].

OBJECTIVE: Calprotectin, the heterdimer of S100A8 and S100A9, is the major cytoplasmic protein of neutrophils, which is also expressed or induced in gingival epithelial cells, activated mononuclear macrophages and vascular endothelial cells. Calprotectin is intimately associated with the initiation and progression of periodontitis, but the in vivo expression patterns of calprotectin in healthy and inflamed periodontal tissue are not fully understood. To observe the expression, distribution and cellular localization of calprotectin in the samples of healthy periodontal tissues and experimental periodontitis tissues of Beagles and to explore their relationship with periodontal inflammation and possible effect. METHODS: Experimental periodontitis model was established by ligation around the mandibular second molar of the Beagle dogs, while the contralateral teeth were healthy controls. Induction duration was 12 weeks, before the dogs were executed. Tissue specimens were demineralized and serial sections were made conventionally. The in vivo expression of calprotectin in the healthy and inflamed periodontal tissues were examined by immunohistochemistry. The in vitro expression of calprotectin in human primary gingival fibroblasts (GFs) and periodontal ligament (PDL) cells were detected by immunocytochemistry. RESULTS: Immunohistochemistry analysis indicated that calprotectin was expressed in gingival epithelial cells and infiltrated neutrophils in the healthy periodontium within the gingival epithelium, S100A8/A9 was most strongly expressed in the junctional epithelium, followed by surface epithelium, and least expressed in the sulcular epithelium. The S100A8/A9 expression levels were sharply defined at the junction between the junctional epithelium and the sulcular epithelium. In periodontal inflammatory lesions, the expression level of calprotectin in sulcular epithelium and junctional epithelium was up-regulated than that in the healthy gingival epithelium. Calprotectin was inducibly expressed in fibroblast-like cells in gingival connective tissue and periodontal ligament tissue, microvascular endothelial cells (ECs) and bone marrow fibroblasts under inflammatory conditions. Additionally, the expression of calprotectin in primary human GFs and PDL cells was confirmed by immunnocytochemistry staining. CONCLUSION: Constitutively expressed in neutrophils and gingival epithelial cells, and calprotectin might maintain the homeostasis and integrity of periodontium. Inflammation-induced expression of calprotectin in GFs, PDL cells, microvascular ECs and bone marrow fibroblasts might process anti-microbial function and promote leukocytes transmigration to defend the host against the microorganisms.

Effect of Butyric Acid in the Proliferation and Migration of Junctional Epithelium in the Progression of Periodontitis: An In Vitro Study.

PURPOSE: To elucidate the effects of butyric acid (BA), a metabolite of bacteria involved in periodontitis, and a possible enhancer of the junctional epithelial cells. METHODS: A murine junctional epithelial cell line, JE-1, was used to assess the effects of sodium butyrate (NaB) as BA. Cell proliferation, migration and attachment were analyzed. Additionally, gene and promoter expression analysis was performed, i.e., cap analysis of gene expression (CAGE) and gene ontology (GO) term enrichment analysis. RESULTS: NaB affected junctional epithelial cell proliferation, migration and attachment. A high concentration of NaB caused cell death and a low concentration tended to promote migration and adhesion. CAGE analysis revealed 75 upregulated and 96 downregulated genes in the cells after 0.2 mM NaB stimulation for 3 h. Regarding GO term enrichment, the genes upregulated >4-fold participated predominantly in cell migration and proliferation. The results of this study suggest that BA produced from periodontopathic bacteria is involved in periodontal tissue destruction at high concentrations. Furthermore, at low concentrations, BA potentially participates in periodontal disease progression by increasing proliferation, migration and attachment of the junctional epithelium and thereby increasing epithelial down-growth.

Runx2 deficiency in junctional epithelium of mouse molars decreases the expressions of E-cadherin and junctional adhesion molecule 1.

Junctional epithelium (JE) attaching to the enamel surface seals gaps around the teeth, functioning as the first line of gingival defense. Runt-related transcription factor 2 (Runx2) plays a role in epithelial cell fate, and the deficiency of Runx2 in JE causes periodontal destruction, while its effect on the barrier function of JE remains largely unexplored. In the present study, hematoxylin-eosin (H&E) staining revealed the morphological differences of JE between wild-type (WT) and Runx2 conditional knockout (cKO) mice. We speculated that these changes were related to the down-regulation of E-cadherin (E-cad), junctional adhesion molecule 1 (JAM1), and integrin ß6 (ITGB6) in JE. Moreover, immunohistochemistry (IHC) was conducted to assess the expressions of these proteins. To verify the relationship between Runx2 and the three above-mentioned proteins, human gingival epithelial cells (HGEs) were cultured for in vitro experiment. The expression of Runx2 in HEGs was depleted by lentivirus. Quantitative real-time PCR (qRT-PCR) and Western blotting analysis were adopted to analyze the differences in mRNA and protein expressions. Taken together, Runx2 played a crucial role in maintaining the structure and function integrality of JE via regulating the expressions of E-cad and JAM1.

ODAM promotes junctional epithelium-related gene expression via activation of WNT1 signaling pathway in an ameloblast-like cell line ALC.

OBJECTIVE: In this study, we investigated the potential and mechanism of odontogenic ameloblast-associated protein (ODAM) in the promoting junctional epithelium-related gene expression in an ameloblast-like cell line ALC. BACKGROUND: ODAM is expressed in ameloblasts and JE and acts as a component of the inner basal lamina (IBL) and intercellular matrix of JE. ODAM KO mice showed destruction of the integrity of the JE, which detaches from teeth. ODAM was confirmed to regulate the cytoskeleton through the ODAM-ARHGEF5-RhoA signaling pathway of the JE. Whether ODAM contributes to the regulation of ameloblast differentiation in JE remains unclear. After the formation of enamel, the ameloblast undergoes a series of morphological changes. Whether ODAM will affect the biological behavior of ameloblasts making them have the characteristics of JE is unclear. METHODS: A murine ameloblast-like cell line, ALC, was used to investigate the effects of ODAM on the JE-like changes of ALC cells in an epithelium-induced environment by generating ODAM overexpression and ODAM knockdown cells through a lentivirus transduction approach. The biomarkers of junctional epithelium CK19, SLPI, and ODAM and the potential regulatory gene WNT1 were investigated by real-time PCR, western blot, immunocytochemistry, immunostaining, luciferase reporter, and rescue assays. RESULTS: ODAM, CK19, and SLPI were significantly upregulated after epithelial induction. Overexpression of ODAM in ALC cells markedly increased CK19 and SLPI expression, while knockdown of ODAM in ALC cells clearly decreased CK19 and SLPI expression. A reporter luciferase assay showed that ODAM activated the WNT signaling pathway, especially through WNT1. Exogenous overexpression of ODAM upregulated WNT1 expression, while knockdown of ODAM reversed this effect. The WNT1 inhibition assay further confirmed the above results and showed that the WNT1 pathway was positively correlated with biomarkers of junctional epithelium CK19 and SLPI expression. Rescue studies showed that knocking down WNT1 in the ODAM-overexpressing ALC cells decreased the expression of CK19 and SLPI. Immunocytochemistry showed that ODAM colocalized with CK19, SLPI, and WNT1 in the cells. CONCLUSION: In conclusion, the research work showed that ODAM promotes junctional epithelium-related gene expression in ALC via the ODAM-WNT1 axis, which may provide new insight into the function of ODAM and JE formation.

Epithelium-Specific Runx2 knockout mice display junctional epithelium and alveolar bone defects.

OBJECTIVE: The aim of this investigation was to study the effects of Runt-related transcription factor 2 (Runx2) on the junctional epithelium and alveolar bone. METHODS: The attachment level of the junctional epithelium and the resorption of alveolar bone were analyzed by histology and scanning electron microscopy. The expression of amelotin was determined by immunohistochemistry, Western blot, and real-time PCR. The ultrastructure of the dentogingival interface was observed by transmission electron microscopy. RESULTS: The cKO mice demonstrated remarkable attachment loss, epithelial hyperplasia, and alveolar bone loss. The relative protein and mRNA expression of amelotin was increased in the junctional epithelium of the cKO mice. The attachment apparatus of the cKO mice showed ultrastructural deficiency. CONCLUSIONS: Loss of Runx2 led to the junctional epithelium and alveolar bone defects in mice. Runx2 may play a crucial role in maintaining the integrity of the dentogingival junction and the normal structure of alveolar bone.