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.

Rare root canal morphology of maxillary second molars: A report of three cases.

Key Clinical Message: Endodontists should be aware that some maxillary second molars can have more than three roots. If any unusual anatomical features are detected during dental radiography or endodontic procedures, it is necessary to conduct cone-beam computed tomography (CBCT) scanning to prevent procedural mishaps. Abstract: CBCT can provide three-dimensional reconstructed images of the root canal system. With the help of CBCT, variations in tooth root number and root canal morphology, such as extra canals, apical ramifications, apical deltas, and lateral canals, can be identified. Knowledge of the variations is very important for the success of endodontic treatment. This report suggests that endodontists must not assume that a MSM has only three tooth roots, which is the most prevalent number.

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.

Successful regenerative response of a severe bone defect in a right lower central incisor affected by a cemental tear.

Cone-beam computed tomography and clinical examinations including pulp vital testing and pocket probing depth showed a cemental tear with a severe labial alveolar bony defect, but no endodontic lesions, in #25, which had a sinus tract at the labial site, in a 75-year-old woman.

Cytokines regulate stemness of mesenchymal stem cells via miR-628-5p during periodontal regeneration.

BACKGROUND: Cytokines play key roles in stimulating periodontal regeneration; however, their exact mechanisms of action remain unclear. Mesenchymal stem cells (MSCs) are multipotent cells that have self-renewal abilities and can differentiate into periodontal tissues such as bone, cementum, and periodontal ligaments following transplantation, like periodontal progenitor cells. Here, we used MSCs to identify the regulatory genes induced by periodontal regenerative cytokines. METHODS: Human MSCs (hMSCs) were cultured under conditions of periodontal regenerative cytokine stimulation or silencing of undifferentiated hMSC transcription factors. To characterize the changes associated with periodontal regenerative cytokine-regulated microRNAs (miRNAs), miRNA, and mRNA expression was evaluated using miRNA arrays and quantitative real-time polymerase chain reaction, respectively. One of the identified miRNAs, miR-628-5p, was then overexpressed or suppressed in hMSCs during osteogenesis; the effect of these changes on osteogenesis was investigated. RESULTS: Cytokine-stimulated MSCs showed characteristic miRNA profiles and mRNA levels of undifferentiated hMSC transcription factors ETV1, SOX11, and GATA6. Next, we silenced these transcription factors in MSCs and examined the miRNA profiles. The levels of miR-628-5p were decreased upon all cytokine treatments and were increased upon silencing of ETV1, SOX11, and GATA6. Overexpression of miR-628-5p suppressed osteogenesis; however, its inhibition enhanced OPN, ALP, OC, BMP2, and RUNX2 mRNA levels, and bone matrix mineralization, but not OSX mRNA or ALP activity. CONCLUSIONS: miR-628-5p negatively regulates MSC stemness during periodontal regeneration. Periodontal regenerative cytokines act as miR-628-5p suppressors to support periodontal regeneration. Thus, selection of effective cytokines for different MSCs, based on miRNA profiling, is important for advancing regenerative therapies.

Endodontic Approach and Periodontal Regenerative Therapy for a Mandibular Right Central Incisor Affected by a Perforation and Cemental Tear.

A cemental tear involves complete or incomplete separation of the cementum on the root surface along the cementodentinal junction. Because a cemental tear can lead to periodontal breakdown and mimic endodontic and periodontal lesions, diagnosing clinical cases can be difficult and requires special examinations. A 72-year-old woman presented with a localized periodontal defect on the labial and interproximal surfaces of the mandibular right central incisor. Performing CBCT scans and a biopsy during periodontal surgery allowed definitive diagnosis of a cemental tear and perforation of the site. First, the perforation was repaired with endodontic therapy. Periodontal regenerative therapy using recombinant human fibroblast growth factor-2 (rhFGF-2) was then performed after removing granulomatous tissue and cementum fragments. Examination of the biopsy specimen showed bacterial colonies. This case showed successful clinical and radiographic outcomes at the 18-month follow-up.

Identification of regulatory mRNA and microRNA for differentiation into cementoblasts and periodontal ligament cells.

OBJECTIVE: Periodontitis causes periodontal tissue destruction and results in physiological tooth dysfunction. Therefore, periodontal regeneration is ideal therapy for periodontitis. Mesenchymal stem cells (MSCs) are useful for periodontal regenerative therapy as they can differentiate into periodontal cells; however, the underlying regulatory mechanism is unclear. In this study, we attempted to identify regulatory genes involved in periodontal cell differentiation and clarify the differentiation mechanism for effective periodontal regenerative therapy. BACKGROUND: The cementum and periodontal ligament play important roles in physiological tooth function. Therefore, cementum and periodontal ligament regeneration are critical for periodontal regenerative therapy. Mesenchymal stem cell transplantation can be a common periodontal regenerative therapy because these cells have multipotency and self-renewal ability, which induces new cementum or periodontal ligament formation. Moreover, MSCs can differentiate into cementoblasts. Cementoblast- or periodontal ligament cell-specific proteins have been reported; however, it is unclear how these proteins are regulated. MicroRNA (miRNA) can also act as a key regulator of MSC function. Therefore, in this study, we identified regulatory genes involved in cementoblast or periodontal cell differentiation and commitment. METHODS: Human MSCs (hMSCs), cementoblasts (HCEM), and periodontal ligament cells (HPL cells) were cultured, and mRNA or miRNA expression was evaluated. Additionally, cementoblast-specific genes were overexpressed or suppressed in hMSCs and their expression levels were investigated. RESULTS: HCEM and HPL cells expressed characteristic genes, of which we focused on ets variant 1 (ETV1), miR-628-5p, and miR-383 because ETV1 is a differentiation-related transcription factor, miR-628-5p was the second-highest expressed gene in HCEM and lowest expressed gene in HPL cells, and miR-383 was the highest expressed gene in HCEM. miR-628-5p and miR-383 overexpression in hMSCs regulated ETV1 mRNA expression, and miR-383 overexpression downregulated miR-628-5p expression. Moreover, miR-383 suppression decreased miR-383 expression and enhanced ETV1 mRNA expression, but miR-383 suppression also decreased miR-628-5p. Furthermore, silencing of ETV1 expression in hMSCs regulated miR-628-5p and miR-383 expression. Concerning periodontal cell commitment, miR-628-5p, miR-383, and ETV1 regulated the expression of HCEM- or HPL cell-related genes by adjusting the expression of these miRNAs. CONCLUSION: HCEM and HPL cells show characteristic mRNA and miRNA profiles. In particular, these cells have specific miR-383, miR-628-5p, and ETV1 expression patterns, and these genes interact with each other. Therefore, miR-383, miR-628-5p, and ETV1 are key genes involved in cementogenesis or HPL cell differentiation.

A mandibular second molar with a middle mesial root canal.

The present case report describes the clinical detection and root canal management of a rare middle mesial canal of a Japanese mandibular second molar by troughing preparation using an operating microscope and cone-beam computed tomography.

Periodontal ligament cells regulate osteogenesis via miR-299-5p in mesenchymal stem cells.

BACKGROUND: The periodontal ligament contains periodontal ligament cells, which is a heterogeneous cell population, and includes progenitor cells that can differentiate into osteoblasts/cementoblasts. Mesenchymal stem cells (MSCs) can differentiate into various cells and can be used for periodontal regenerative therapy. Therefore, transplanted MSCs can be affected by humoral factors from periodontal ligament cells via the transcription factors or microRNAs (miRNAs) of MSCs. In addition, periostin (POSTN) is secreted from HPL cells and can regulate periodontal regeneration and homeostasis. To clarify the regulatory mechanism of humoral factors from periodontal ligament cells, we attempted to identify key genes, specifically microRNAs, involved in this process. METHODS: Human MSCs (hMSCs) were indirectly co-cultured with human periodontal ligament cells (HPL cells) and then evaluated for osteogenesis, undifferentiated MSCs markers, and miRNA profiles. Furthermore, hMSCs were indirectly co-cultured with HPL cells in the presence of anti-POSTN monoclonal antibody (anti-POSTN Ab) to block the effect of POSTN from HPL cells, and then evaluated for osteogenesis or undifferentiated MSC markers. Moreover, hMSCs showed alterations in miRNA expression or cultured with HPL were challenged with POSTN during osteogenesis, and cells were evaluated for osteogenesis or undifferentiated MSC markers. RESULTS: hMSCs co-cultured with HPL cells showed suppressed osteogenesis and characteristic expression of SOX11, an undifferentiated MSC marker, as well as miR-299-5p. Overexpression of miR-299-5p regulated osteogenesis and SOX11 expression as observed with indirect co-culture with HPL cells. Furthermore, MSCs co-cultured with HPL cells were recovered from the suppression of osteogenesis and SOX11 mRNA expression by anti-POSTN Ab. However, POSTN induced miR-299-5p and SOX11 expression, and enhanced osteogenesis. CONCLUSION: Humoral factors from HPL cells suppressed osteogenesis in hMSCs. The suppressive effect was mediated by miR-299-5p and SOX11 in hMSCs.

Regulation of osteogenesis via miR-101-3p in mesenchymal stem cells by human gingival fibroblasts.

INTRODUCTION: Mesenchymal stem cells (MSCs) can differentiate into various types of cells and can thus be used for periodontal regenerative therapy. However, the mechanism of differentiation is still unclear. Transplanted MSCs are, via their transcription factors or microRNAs (miRNAs), affected by periodontal cells with direct contact or secretion of humoral factors. Therefore, transplanted MSCs are regulated by humoral factors from human gingival fibroblasts (HGF). Moreover, insulin-like growth factor (IGF)-1 is secreted from HGF and regulates periodontal regeneration. To clarify the regulatory mechanism for MSC differentiation by humoral factors from HGF, we identified key genes, specifically miRNAs, involved in this process, and determined their function in MSC differentiation. MATERIALS AND METHODS: Mesenchymal stem cells were indirectly co-cultured with HGF in osteogenic or growth conditions and then evaluated for osteogenesis, undifferentiated MSC markers, and characteristic miRNAs. MSCs had their miRNA expression levels adjusted or were challenged with IGF-1 during osteogenesis, or both of which were performed, and then, MSCs were evaluated for osteogenesis or undifferentiated MSC markers. RESULTS: Mesenchymal stem cells co-cultured with HGF showed suppression of osteogenesis and characteristic expression of ETV1, an undifferentiated MSC marker, as well as miR-101-3p. Over-expression of miR-101-3p regulated osteogenesis and ETV1 expression as well as indirect co-culture with HGF. IGF-1 induced miR-101-3p and ETV1 expression. However, IGF-1 did not suppress osteogenesis. CONCLUSIONS: Humoral factors from HGF suppressed osteogenesis in MSCs. The effect was regulated by miRNAs and undifferentiated MSC markers. miR-101-3p and ETV1 were the key factors and were regulated by IGF-1.

Combined endodontic and periodontal management of a class 3 invasive cervical resorption in a mandibular first molar.

Dental radiography and cone-beam computed tomography revealed the left mandibular first molar in a 68-year-old female patient with Heithersay Class 3 invasive cervical resorption (ICR). The inhibition of ICR progression and environmental improvement in and around the affected tooth through combined endodontic and periodontal treatments led to a favorable clinical outcome.

Characteristics of high-molecular-weight hyaluronic acid as a brain-derived neurotrophic factor scaffold in periodontal tissue regeneration.

Brain-derived neurotrophic factor (BDNF), for which bovine collagen-derived atelocollagen is used as a scaffold, enhances periodontal tissue regeneration. However, a scaffold that does not contain unknown ingredients is preferable. Since the synthesized high-molecular-weight (HMW)-hyaluronic acid (HA) is safe and inexpensive, we evaluated the efficacy of HMW-HA as a BDNF scaffold. CD44, a major receptor of HA, was expressed in cultures of human periodontal ligament cells, and HMW-HA promoted the adhesion and proliferation of human periodontal ligament cells, although it did not influence the mRNA expression of bone (cementum)-related proteins. The in vitro release kinetics of BDNF from HMW-HA showed that BDNF release was sustained for 14 days. Subsequently, we examined the effect of BDNF/HMW-HA complex on periodontal tissue regeneration in dogs. A greater volume of newly formed alveolar bone and a longer newly formed cementum were observed in the BDNF/HMW-HA group than in the HMW-HA group, suggesting that HMW-HA assists the regenerative capacity of BDNF, although HMW-HA itself does not enhance periodontal tissue regeneration. Neither the poly (lactic-co-glycolic acid) group nor the BDNF/poly (lactic-co-glycolic acid) group enhanced periodontal tissue regeneration. In conclusion, HMW-HA is an adequate scaffold for the clinical application of BDNF.

Humoral factors released from human periodontal ligament cells influence calcification and proliferation in human bone marrow mesenchymal stem cells.

BACKGROUND: Transplantation of bone marrow mesenchymal stem cells (MSCs) is believed to be a new modality for periodontal tissue regeneration. However, little is known about the factors that influence the functions of the transplanted MSCs. Periodontal ligament cells may be implicated in regulating MSC functions. METHODS: To examine the effect of humoral factors (HFs) produced by human periodontal ligament (HPL) cells on human MSC (hMSC) gene expression, hMSCs were cocultured separately with HPL cells. The gene expression of the hMSCs was analyzed by microarray. Moreover, the effect of conditioned medium (CM) from cultures of HPL cells (CM-HPL cells) on the proliferation and mineralizing capacity of hMSCs was examined. RESULTS: Thirty-five genes whose expressions were upregulated more than two-fold and 32 genes whose expressions were downregulated more than two-fold were identified in hMSCs cocultured separately with HPL cells. CM-HPL cells prevented calcification in hMSCs cultured with bone morphogenetic protein-2 but not dexamethasone. CM-HPL cells stimulated cell proliferation in hMSCs, whereas CM from culture of human osteoblasts did not influence the proliferation. CONCLUSIONS: To the best of our knowledge, this is the first study on the global gene expression of MSCs cocultured with periodontal ligament cells. A particular humoral factor released from periodontal ligament cells is suggested to affect differentiation and proliferation in MSCs.