Loss-of-function OGFRL1 variants identified in autosomal recessive cherubism families.

Cherubism (OMIM 118400) is a rare craniofacial disorder in children characterized by destructive jawbone expansion due to the growth of inflammatory fibrous lesions. Our previous studies have shown that gain-of-function mutations in SH3 domain-binding protein 2 (SH3BP2) are responsible for cherubism and that a knock-in mouse model for cherubism recapitulates the features of cherubism, such as increased osteoclast formation and jawbone destruction. To date, SH3BP2 is the only gene identified to be responsible for cherubism. Since not all patients clinically diagnosed with cherubism had mutations in SH3BP2, we hypothesized that there may be novel cherubism genes and that these genes may play a role in jawbone homeostasis. Here, using whole exome sequencing, we identified homozygous loss-of-function variants in the opioid growth factor receptor like 1 (OGFRL1) gene in 2 independent autosomal recessive cherubism families from Syria and India. The newly identified pathogenic homozygous variants were not reported in any variant databases, suggesting that OGFRL1 is a novel gene responsible for cherubism. Single cell analysis of mouse jawbone tissue revealed that Ogfrl1 is highly expressed in myeloid lineage cells. We generated OGFRL1 knockout mice and mice carrying the Syrian frameshift mutation to understand the in vivo role of OGFRL1. However, neither mouse model recapitulated human cherubism or the phenotypes exhibited by SH3BP2 cherubism mice under physiological and periodontitis conditions. Unlike bone marrow-derived M-CSF-dependent macrophages (BMMs) carrying the SH3BP2 cherubism mutation, BMMs lacking OGFRL1 or carrying the Syrian mutation showed no difference in TNF-ɑ mRNA induction by LPS or TNF-ɑ compared to WT BMMs. Osteoclast formation induced by RANKL was also comparable. These results suggest that the loss-of-function effects of OGFRL1 in humans differ from those in mice and highlight the fact that mice are not always an ideal model for studying rare craniofacial bone disorders.

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.

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.

Activation of innate immune receptor TLR9 by mitochondrial DNA plays essential roles in the chemical long-term depression of hippocampal neurons.

Synaptic plasticity is believed to be the cellular basis for experience-dependent learning and memory. Although long-term depression (LTD), a form of synaptic plasticity, is caused by the activity-dependent reduction of cell surface α-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA)-type glutamate receptors (AMPA receptors) at postsynaptic sites, its regulation by neuronal activity is not completely understood. In this study, we showed that the inhibition of toll-like receptor-9 (TLR9), an innate immune receptor, suppresses N-methyl-d-aspartate (NMDA)-induced reduction of cell surface AMPA receptors in cultured hippocampal neurons. We found that inhibition of TLR9 also blocked NMDA-induced activation of caspase-3, which plays an essential role in the induction of LTD. siRNA-based knockdown of TLR9 also suppressed the NMDA-induced reduction of cell surface AMPA receptors, although the scrambled RNA had no effect on the NMDA-induced trafficking of AMPA receptors. Overexpression of the siRNA-resistant form of TLR9 rescued the AMPA receptor trafficking abolished by siRNA. Furthermore, NMDA stimulation induced rapid mitochondrial morphological changes, mitophagy, and the binding of mitochondrial DNA (mtDNA) to TLR9. Treatment with dideoxycytidine and mitochondrial division inhibitor-1, which block mtDNA replication and mitophagy, respectively, inhibited NMDA-dependent AMPA receptor internalization. These results suggest that mitophagy induced by NMDA receptor activation releases mtDNA and activates TLR9, which plays an essential role in the trafficking of AMPA receptors during the induction of LTD.

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.

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.

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.

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.

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.

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.

Masticatory dysfunction in patients with diabetic neuropathy: A cross-sectional study.

INTRODUCTION: Chewing well is essential for successful diet therapy and control of blood glucose level in patients with diabetes. In addition, long-term hyperglycemia is a risk factor for microvascular complications, which are the main cause of morbidity and mortality in these patients. Hence, it is plausible that masticatory disorder may be relevant to diabetic microvascular complications which is caused by long-term hyperglycemia. The aim of this study was to investigate whether masticatory disorders are relevant to diabetic microvascular complications. METHODS: This cross-sectional study included 172 patients with type 2 diabetes who underwent educational hospitalization in the Department of Endocrinology and Diabetic Medicine, Hiroshima University Hospital, from April 2016 to March 2020. Masticatory efficiency was determined quantitatively by using the GLUCO SENSOR GS-Ⅱ. Multivariable linear regression models were constructed to examine which factors were related to masticatory efficiency. Statistical significance was defined as a two-sided p value of < 0.05. RESULTS: According to the bivariable analysis, masticatory efficiency was significantly correlated with duration of diabetes (p = 0. 049), number of remaining teeth (p < 0.0001), the number of moving teeth (p = 0.007) and condition of diabetic neuropathy (p < 0.0001). Moreover, the number of remaining teeth (p < 0.0001) and diabetic neuropathy (p = 0.007) remained significantly correlated with masticatory efficiency in the multivariable analysis. CONCLUSIONS: For the first time, we demonstrated that patients with type 2 diabetes who developed diabetic neuropathy had significantly reduced masticatory efficiency. Effective mastication is an important factor in successful diet therapy for diabetes. To prevent the progression of diabetic complications, especially in patients with diabetic neuropathy, it may be necessary to combine individualized therapies from dentists and nutritionists with consideration for the level of masticatory dysfunction.

Xenotransplantation of cryopreserved human clumps of mesenchymal stem cells/extracellular matrix complexes pretreated with IFN-γ induces rat calvarial bone regeneration.

Introduction: Three-dimensional (3D) clumps of mesenchymal stem cells (MSCs)/extracellular matrix (ECM) complexes, composed with cells and self-produced intact ECM, can be grafted into defect areas without artificial scaffold to induce successful bone regeneration. Moreover, C-MSCs pretreated with IFN-γ (C-MSCsγ) increased the immunomodulatory enzyme indoleamine 2,3-dioxygenase (IDO) expression and thereby inhibited T cell activity. Xenotransplantation of human C-MSCsγ suppressed host T cell immune rejection and induced bone regeneration in mice. Besides, we have also reported that C-MSCs retain the 3D structure and bone regenerative property even after cryopreservation. To develop the "off-the-shelf" cell preparation for bone regenerative therapy that is promptly provided when needed, we investigated whether C-MSCsγ can retain the immunosuppressive and osteogenic properties after cryopreservation. Methods: Confluent human MSCs that had formed on the cellular sheet were scratched using a micropipette tip and then torn off. The sheet was rolled to make a round clump of cells. The round cell clumps were incubated with a growth medium for 3 days, and then C-MSCs were obtained. To generate C-MSCsγ, after 2 days' culture, C-MSCs were stimulated with 50 ng/ml of IFN-γ. Both C-MSCs and C-MSCsγ were cryopreserved for 2 days and then thawed to obtain Cryo-C-MSCs and Cryo-C-MSCsγ, respectively. The biological properties of those cell clumps were assessed in vitro. In addition, to test whether human Cryo-C-MSCsγ attenuates immune rejection to induce bone regeneration, a xenograft study using a rat calvarial defect was performed. Results: Both IFN-γ pretreatment and cryopreservation process did not affect the 3D structure and cell viability in all human cell clumps. Interestingly, Cryo-C-MSCsγ showed significantly increased IDO mRNA expression equivalent to C-MSCsγ. More importantly, xenotransplantation of human C-MSCsγ and Cryo-C-MSCsγ induced rat calvarial bone regeneration by suppressing rat T cells infiltration and the grafted human cells reduction in the grafted area. Finally, there were no human donor cells in the newly formed bone, implying that the bone reconstruction by C-MSCsγ and Cryo-C-MSCsγ can be due to indirect host osteogenesis. Conclusion: These findings implied that Cryo-C-MSCsγ can be a promising bone regenerative allograft therapy that can be certainly and promptly supplied on demand.

Cancellous bone-like tissue replacement from calcinosis in patients with systemic sclerosis with multiple external root resorption.

Calcinosis is frequently observed in patients with systemic sclerosis (SSc). The fundamental treatment of calcinosis has not yet been established. During follow-up, calcinosis in the subcutaneous surface is often spontaneously extracted or remains confined by fibrous tissues. We previously identified a new symptom in SSc patients, multiple external root resorption (MERR), and these patients had calcifications in the nasal spine. Here, we report for the first time that calcinosis at the nasal spine in patients with MERR can be replaced by cancellous bone-like tissue. Patients 1 and 2 were a 62-year-old Japanese female and a 45-year-old Japanese female (respectively) with MERR who had been previously treated for SSc (Patient 1: limited type, positive for anti-centromere antibody; Patient 2: diffuse type, positive for anti-Scl70 and anti-SS-A antibodies). Patient 3 was a 57-year-old female with MERR who had been previously treated for SSc (diffuse type, positive anti-Scl-70 antibody) and underwent denosumab injection for osteoporosis. Cone-beam computed tomography (CBCT) and CT images in the calcifications at the nasal spine in Patient 1 and 2 were replaced with cancellous bone-like tissue, but not in Patient 3. Serum laboratory examination was performed to assess the systemic bone disease. All three patients had normal clinical data within the references, apart from slightly higher 1,25-dihydroxyvitamin D levels in Patient 1. SSc patients with calcinosis in the maxillofacial area need to be examined carefully for bone replacement using CBCT or CT.

Clumps of Mesenchymal Stem Cells/Extracellular Matrix Complexes Generated with Xeno-Free Chondro-Inductive Medium Induce Bone Regeneration via Endochondral Ossification.

Three-dimensional clumps of mesenchymal stem cells (MSCs)/extracellular matrix (ECM) complexes (C-MSCs) can be transplanted into tissue defect site with no artificial scaffold. Importantly, most bone formation in the developing process or fracture healing proceeds via endochondral ossification. Accordingly, this present study investigated whether C-MSCs generated with chondro-inductive medium (CIM) can induce successful bone regeneration and assessed its healing process. Human bone marrow-derived MSCs were cultured with xeno-free/serum-free (XF) growth medium. To obtain C-MSCs, confluent cells that had formed on the cellular sheet were scratched using a micropipette tip and then torn off. The sheet was rolled to make a round clump of cells. The cell clumps, i.e., C-MSCs, were maintained in XF-CIM. C-MSCs generated with XF-CIM showed enlarged round cells, cartilage matrix, and hypertrophic chondrocytes genes elevation in vitro. Transplantation of C-MSCs generated with XF-CIM induced successful bone regeneration in the SCID mouse calvaria defect model. Immunofluorescence staining for human-specific vimentin demonstrated that donor human and host mouse cells cooperatively contributed the bone formation. Besides, the replacement of the cartilage matrix into bone was observed in the early period. These findings suggested that cartilaginous C-MSCs generated with XF-CIM can induce bone regeneration via endochondral ossification.

Generalized periodontitis treated with periodontal, orthodontic, and prosthodontic therapy: A case report.

BACKGROUND: Generalized periodontitis is a severe periodontal disease characterized by rapid periodontal destruction in healthy persons. This case report describes the treatment of a severe crowding, large overjet, and occlusal collapse due to the loss of anterior guidance with generalized periodontitis. CASE SUMMARY: A 35-year-old female patient with a chief complaint of crowding and maxillary protrusion was diagnosed with generalized periodontitis by clinical and radiographic examinations. To improve crowding and overjet, orthodontic treatment was performed after basic periodontal therapy. Severely damaged upper right lateral incisor and left canine were extracted, and lower right first premolar and left second premolar were also removed to treat severe crowding. After orthodontic treatment, periodontal flap surgery for upper left molars and guided tissue regeneration for the lower left second molar was performed. Then, a dental implant was inserted in the upper left canine legion. The esthetics of the maxillary anterior tooth was improved by prosthetic restorations. The treatment result showed a well-improved occlusion with proper anterior guidance and healthy periodontal tissue after a retention period of 10 years. CONCLUSION: Periodontal, orthodontic, and prosthodontic treatments are extremely useful to improve function and stable periodontal tissue for generalized periodontitis.

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.

Subunit-dependent and subunit-independent rules of AMPA receptor trafficking during chemical long-term depression in hippocampal neurons.

Long-term potentiation (LTP) and long-term depression (LTD) of excitatory neurotransmission are believed to be the neuronal basis of learning and memory. Both processes are primarily mediated by neuronal activity-induced transport of postsynaptic AMPA-type glutamate receptors (AMPARs). While AMPAR subunits and their specific phosphorylation sites mediate differential AMPAR trafficking, LTP and LTD could also occur in a subunit-independent manner. Thus, it remains unclear whether and how certain AMPAR subunits with phosphorylation sites are preferentially recruited to or removed from synapses during LTP and LTD. Using immunoblot and immunocytochemical analysis, we show that phosphomimetic mutations of the membrane-proximal region (MPR) in GluA1 AMPAR subunits affect the subunit-dependent endosomal transport of AMPARs during chemical LTD. AP-2 and AP-3, adaptor protein complexes necessary for clathrin-mediated endocytosis and late endosomal/lysosomal trafficking, respectively, are reported to be recruited to AMPARs by binding to the AMPAR auxiliary subunit, stargazin (STG), in an AMPAR subunit-independent manner. However, the association of AP-3, but not AP-2, with STG was indirectly inhibited by the phosphomimetic mutation in the MPR of GluA1. Thus, although AMPARs containing the phosphomimetic mutation at the MPR of GluA1 were endocytosed by a chemical LTD-inducing stimulus, they were quickly recycled back to the cell surface in hippocampal neurons. These results could explain how the phosphorylation status of GluA1-MPR plays a dominant role in subunit-independent STG-mediated AMPAR trafficking during LTD.

External cervical resorption detected via cone-beam computed tomography in a patient with myelin oligodendrocyte glycoprotein antibody-associated disease: A case report.

External cervical resorption may occur in patients with MOG antibody-associated disease, which is clearly detected on cone-beam computed tomography. Therefore, dental screening is essential for these patients before initiating bisphosphonate therapy. Larger sample sizes are crucial to determine any possible association between external cervical resorption and MOG antibody-associated disease.

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.