Oral supplementation with p-coumaric acid protects mice against diabetes-associated spontaneous destruction of periodontal tissue.

OBJECTIVE: Dietary bioactive materials having anti-inflammatory and antioxidant potentials are able to inhibit diabetes-associated periodontal complications. Although numerous studies indicate that administration of p-coumaric acid (p-CA) ameliorates diabetes and diabetes-related complications, the roles of p-CA on periodontal tissue destruction in diabetic mice and the possible mechanisms therein are not completely understood. In this study, we evaluated whether supplementation with p-CA protects mice against diabetes-associated spontaneous periodontal destruction and also explored the associated mechanism therein using in vivo and in vitro experimental systems. MATERIALS AND METHODS: C57BL/6 male mice were divided into sham, streptozotocin (STZ), and STZ+CA groups (n = 5/group). Sham group was intraperitoneally injected with sodium buffer, whereas other two groups were injected with the buffer containing 160 mg/kg of STZ. STZ-induced diabetic mice received oral gavage with p-CA (50 mg/kg) (STZ+CA group) or with buffer only (STZ group) daily for 6 weeks. The effect of p-CA on diabetes-associated spontaneous periodontal destruction was evaluated using µCT analysis, hematoxylin and eosin staining, tartrate-resistant acid phosphatase staining, and immunohistochemical staining methods. The efficacies of p-CA on cell proliferation, osteoblast differentiation, reactive oxygen species (ROS) accumulation, and antioxidant-related marker expression were examined using human periodontal ligament fibroblasts (hPLFs) cultured under high glucose condition. RESULTS: Streptozotocin group exhibited periodontal tissue destruction along with increased inflammation, oxidative stress, and osteoclast formation, as well as with decreased osteogenesis. However, oral administration with p-CA protected mice against STZ-induced periodontal destruction by inhibiting inflammation and osteoclastic activation. STZ+CA group also showed higher expression of antioxidant and osteogenic markers in periodontal tissue than did STZ group. Treatment with high glucose concentration (30 mmol/L) impaired proliferation and osteoblast differentiation of hPLFs along with cellular ROS accumulation, whereas these impairments were almost completely disappeared by supplementation with p-CA. CONCLUSION: These findings demonstrate that supplementation with p-CA inhibits diabetes-associated spontaneous destruction of periodontal tissue by enhancing anti-inflammatory, anti-osteoclastogenic, and antioxidant defense systems in STZ-treated mice.

Local delivery of recombinant human FGF7 enhances bone formation in rat mandible defects.

Fibroblast growth factor 7 (FGF7) plays an important role in regulating the proliferation, migration, and differentiation of cells. However, the role of FGF7 in bone formation is not yet fully understood. We examined the effect of FGF7 on bone formation using a rat model of mandible defects. Rats underwent mandible defect surgery and then either scaffold treatment alone (control group) or FGF7-impregnated scaffold treatment (FGF7 group). Micro-CT and histological analyses revealed that the FGF7 group exhibited greater bone formation than did the control group 10 weeks after surgery. With the exception of total porosity (%), all bone parameters had higher values in the FGF7 group than in the control group at each follow-up after surgery. The FGF7 group showed greater expression of osteogenic markers, such as runt-related transcription factor 2, osterix, osteocalcin, bone morphogenetic protein 2, osteopontin, and type I collagen in newly formed bone than did the control group. The delivery of FGF7 also increased the messenger RNA expression of stromal-cell-derived factor 1 (SDF-1) and CXCR4 in newly formed bone in the FGF7 group compared with the control group. Further, addition of exogenous FGF7 induced migration of rat bone marrow stromal cells and increased the expression of SDF-1 and CXCR4 in the cells. Furthermore, the addition of FGF7 augmented mineralization in the cells with increased expression of osteogenic markers, and this augmentation was significantly suppressed by an inhibitor specific for c-Jun N-terminal kinase (SP600125) or extracellular-signal-regulated kinase (PD98059). Collectively, these results suggest that local delivery of FGF7 increases bone formation in a mandible defect with enhanced osteogenesis and chemoattraction.

Evaluation of osseointegration of Ti-6Al-4V alloy orthodontic mini-screws with ibandronate-loaded TiO2 nanotube layer.

Recently, the use of orthodontic mini-screws as an anchorage for orthodontic treatment is increasing, and the degree of osseointegration of the mini-screws affects the performance of orthodontic treatment. This study aimed to evaluate the biocompatibility and osseointegration of Titanium 6Aluminum 4Vanadium (Ti-6Al-4V) alloy orthodontic mini-screws with an ibandronate-loaded TiO2 nanotube (TNT) layer. The TNT layer was formed on the surface of the Ti-6Al-4V alloy orthodontic mini-screws and loaded with ibandronate. The TNT formed by anodic oxidation formed a completely self-organized and compact structure and was stably released for 7 days after loading with ibandronate. Mini-screws loaded with ibandronate were implanted into both tibias of rats, confirming rapid initial bone regeneration. We demonstrate that the release of stable ibandronate from the TNT layer of Ti-6Al-4V alloy orthodontic mini-screws can effectively improve biocompatibility and osseointegration.

Streptococcus mutans GS-5 antigen I/II stimulates cell survival in serum deprived-cultures through PI3K/Akt pathways.

The antigen I/II (AgI/II) protein is a major surface protein that mediates the attachment of Streptococcus mutans (S. mutans) to the saliva-coated pellicle. Numerous studies have investigated not only the mechanisms by which AgI/II signaling is transduced within cells, but have also attempted to use AgI/II-specific antibodies to treat dental caries and host immune responses. However, little information is available about the effects of AgI/II on basic cellular events in bone cells. In this study, we examined the effects of the His-tagged recombinant N-terminal half of the AgI/II protein (rAgI/II-N) generated from S. mutans GS-5 on the viability, proliferation, and cell cycle progression of primary calvarial osteoblasts. We also investigated the mechanisms involved in the rAgI/II-N-mediated survival of serum-starved osteoblasts. We found that rAgI/II treatment attenuated the serum deprivation-induced decrease in cell viability and proliferation of osteoblasts. rAgI/II-N also prevented the loss of mitochondrial membrane potential (MMP), alterations in levels of two key mitochondrial Bcl-2 family proteins, and the accumulation of numerous cells into the sub-G(1) phase that were observed in serum-starved osteoblasts. Pharmacological inhibitors of phosphoinositide 3-kinase (PI3K), but not of extracellular signal-regulated kinase or Ras, blocked the rAgI/II-N-mediated protection against serum deprivation-induced cell death. Additional experiments revealed that the integrin α5ß1-mediated PI3K pathway is required for rAgI/II-N-mediated Akt phosphorylation in osteoblasts. Collectively, these results suggest that rAgI/II-N induces survival signals in serum-starved osteoblasts through integrin-induced PI3K/Akt signaling pathways.

Sodium fluoride induces apoptosis in mouse embryonic stem cells through ROS-dependent and caspase- and JNK-mediated pathways.

Sodium fluoride (NaF) is used as a source of fluoride ions in diverse applications. Fluoride salt is an effective prophylactic for dental caries and is an essential element required for bone health. However, fluoride is known to cause cytotoxicity in a concentration-dependent manner. Further, no information is available on the effects of NaF on mouse embryonic stem cells (mESCs). We investigated the mode of cell death induced by NaF and the mechanisms involved. NaF treatment greater than 1mM reduced viability and DNA synthesis in mESCs and induced cell cycle arrest in the G(2)/M phase. The addition of NaF induced cell death mainly by apoptosis rather than necrosis. Catalase (CAT) treatment significantly inhibited the NaF-mediated cell death and also suppressed the NaF-mediated increase in phospho-c-Jun N-terminal kinase (p-JNK) levels. Pre-treatment with SP600125 or z-VAD-fmk significantly attenuated the NaF-mediated reduction in cell viability. In contrast, intracellular free calcium chelator, but not of sodium or calcium ion channel blockers, facilitated NaF-induced toxicity in the cells. A JNK specific inhibitor (SP600125) prevented the NaF-induced increase in growth arrest and the DNA damage-inducible protein 45α. Further, NaF-mediated loss of mitochondrial membrane potential was apparently inhibited by pifithrin-α or CAT inhibitor. These findings suggest that NaF affects viability of mESCs in a concentration-dependent manner, where more than 1mM NaF causes apoptosis through hydroxyl radical-dependent and caspase- and JNK-mediated pathways.

Functional improvement of collagen-based bioscaffold to enhance periodontal-defect healing via combination with dietary antioxidant and COMP-angiopoietin 1.

Scaffolds combined with bioactive agents can enhance bone regeneration at therapeutic sites. We explore whether combined supplementation with coumaric acid and recombinant human-cartilage oligomeric matrix protein-angiopoietin 1 (rhCOMP-Ang1) is an ideal approach for bone tissue engineering. We developed coumaric acid-conjugated absorbable collagen scaffold (CA-ACS) and investigated whether implanting CA-ACS in combination with rhCOMP-Ang1 facilitates ACS- or CA-ACS-mediated bone formation using a rat model of critically sized mandible defects. We examined the mechanisms by which coumaric acid and rhCOMP-Ang1 regulate behaviors of human periodontal ligament fibroblasts (hPLFs). The CA-ACS exhibits greater anti-degradation and mechanical strength properties than does ACS alone. Implanting CA-ACS loaded with rhCOMP-Ang1 greatly enhances bone regeneration at the defect via the activation of angiogenic, osteogenic, and anti-osteoclastic responses compared with other rat groups implanted with an ACS alone or CA-ACS. Treatment with both rhCOMP-Ang1 and coumaric acid increases proliferation, mineralization, and migration of cultured hPLFs via activation of the Ang1/Tie2 signaling axis at a greater rate than treatment with either of them alone. Collectively, this study demonstrates that CA-ACS impregnated with rhCOMP-Ang1 enhances bone regeneration at therapeutic sites, and this enhancement is associated with a synergistic interaction between rhCOMP-Ang1-mediated angiogenesis and coumaric acid-related antioxidant responses.

Improvement of osseointegration of Ti-6Al-4V ELI alloy orthodontic mini-screws through anodization, cyclic pre-calcification, and heat treatments.

BACKGROUND: Mini-screws are widely used as temporary anchorages in orthodontic treatment, but have the disadvantage of showing a high failure rate of about 10%. Therefore, orthodontic mini-screws should have high biocompatibility and retention. Previous studies have demonstrated that the retention of mini-screws can be improved by imparting bioactivity to the surface. The method for imparting bioactivity proposed in this paper is to sequentially perform anodization, periodic pre-calcification, and heat treatments with a Ti-6Al-4V ELI alloy mini-screw. MATERIALS AND METHODS: A TiO2 nanotube-structured layer was formed on the surface of the Ti-6Al-4V ELI alloy mini-screw through anodization in which a voltage of 20 V was applied to a glycerol solution containing 20 wt% H2O and 1.4 wt% NH4F for 60 min. Fine granular calcium phosphate precipitates of HA and octacalcium phosphate were generated as clusters on the surface through the cyclic pre-calcification and heat treatments. The cyclic pre-calcification treatment is a process of immersion in a 0.05 M NaH2PO4 solution and a saturated Ca(OH)2 solution at 90 °C for 1 min each. RESULTS: It was confirmed that the densely structured protrusions were precipitated, and Ca and P concentrations, which bind and concentrate endogenous bone morphogenetic proteins, increased on the surface after simulated body fluid (SBF) immersion test. In addition, the removal torque of the mini-screw fixed into rabbit tibias for 4 weeks was measured to be 8.70 ± 2.60 N cm. CONCLUSIONS: A noteworthy point in this paper is that the Ca and P concentrations, which provide a scaffold suitable for endogenous bone formation, further increased over time after SBF immersion of the APH group specimens. The other point is that our mini-screws have a significantly higher removal torque compared to untreated mini-screws. These results represent that the mini-screw proposed in this paper can be used as a mini-screw for orthodontics.

Astaxanthin Inhibits Diabetes-Triggered Periodontal Destruction, Ameliorates Oxidative Complications in STZ-Injected Mice, and Recovers Nrf2-Dependent Antioxidant System.

Numerous studies highlight that astaxanthin (ASTX) ameliorates hyperglycemic condition and hyperglycemia-associated chronic complications. While periodontitis and periodontic tissue degradation are also triggered under chronic hyperglycemia, the roles of ASTX on diabetes-associated periodontal destruction and the related mechanisms therein are not yet fully understood. Here, we explored the impacts of supplemental ASTX on periodontal destruction and systemic complications in type I diabetic mice. To induce diabetes, C57BL/6 mice received a single intraperitoneal injection of streptozotocin (STZ; 150 mg/kg), and the hyperglycemic mice were orally administered with ASTX (12.5 mg/kg) (STZ+ASTX group) or vehicle only (STZ group) daily for 60 days. Supplemental ASTX did not improve hyperglycemic condition, but ameliorated excessive water and feed consumptions and lethality in STZ-induced diabetic mice. Compared with the non-diabetic and STZ+ASTX groups, the STZ group exhibited severe periodontal destruction. Oral gavage with ASTX inhibited osteoclastic formation and the expression of receptor activator of nuclear factor (NF)-κB ligand, 8-OHdG, γ-H2AX, cyclooxygenase 2, and interleukin-1ß in the periodontium of STZ-injected mice. Supplemental ASTX not only increased the levels of nuclear factor erythroid 2-related factor 2 (Nrf2) and osteogenic transcription factors in the periodontium, but also recovered circulating lymphocytes and endogenous antioxidant enzyme activity in the blood of STZ-injected mice. Furthermore, the addition of ASTX blocked advanced glycation end products-induced oxidative stress and growth inhibition in human-derived periodontal ligament cells by upregulating the Nrf2 pathway. Together, our results suggest that ASTX does not directly improve hyperglycemia, but ameliorates hyperglycemia-triggered periodontal destruction and oxidative systemic complications in type I diabetes.

Surgical approach and orthodontic treatment of mandibular condylar osteochondroma.

Osteochondroma is a common benign tumor of bones, but it is rare in the mandibular condyle. With its outgrowth it manifests clinically as deviation of the mandible limitation of mouth opening, and facial asymmetry. After the tumor is diagnosed on the basis of clinical symptoms and radiographic examination including cone-beam computed tomography (CBCT) analysis, an appropriate surgery and treatment plan should be formulated. Herein, we present the case of a 44-year-old female patient who visited our dental hospital because her chin point had been deviating to the left side slowly but progressively over the last 3 years and she had difficulty masticating. Based on CBCT, she was diagnosed with skeletal Class III malocclusion accompanied by osteochondroma of the right mandibular condyle. Maxillary occlusal cant with the right side down was observed, but it was confirmed to be an extrusion of the molars associated with dental compensation. Therefore, after intrusion of the right molars with the use of temporary anchorage devices, sagittal split ramus osteotomy was used to remove the tumor and perform orthognathic surgery simultaneously. During 6 months after the surgery, continuous bone resorption and remodeling were observed in the condyle of the affected side, which led to a change in occlusion. During the postoperative orthodontic treatment, intrusive force and buccal torque were applied to the molars on the affected side, and a proper buccal overjet was created. After 18 months, CBCT revealed that the rate of bone absorption was continuously reduced, bone corticalization appeared, and good occlusion and a satisfying facial profile were achieved.

Growth observation and orthodontic treatment of a hemifacial microsomia patient treated with distraction osteogenesis.

Hemifacial microsomia (HFM) patients may experience emotional withdrawal during their growth period due to their abnormal facial appearance. Distraction osteogenesis at an early age to improve their appearance can encourage these patients. Some abnormalities of the affected side can be overcome by distraction osteogenesis at an early age. However, differences in the growth rate between the affected and unaffected sides during the rest of the growth period are inevitable due to the characteristics of HFM. Therefore, re-evaluation should be performed after completion of growth in order to achieve stable occlusion through either orthognathic surgery or camouflage orthodontic treatment. An eight-year-old patient visited the clinic exhibiting features of HFM with slight mandibular involvement. He received phase I treatment with distraction osteogenesis and a functional appliance. Distraction osteogenesis was performed at the right ramus, which resulted in an open bite at the right posterior dentition. After distraction osteogenesis, a functional appliance and partial fixed appliance were used to achieve extrusion of the affected posterior dentition and settlement of the occlusion adjustment on the unaffected posterior dentition. The patient visited the clinic regularly for follow-up assessments, and at the age of 20 years, he showed facial asymmetry of the mandible, which had deviated to the right side. He received orthodontic treatment to improve the occlusion of his posterior dentition after the growth period. Without orthognathic surgery, stable occlusion and a satisfactory facial appearance were obtained through camouflage orthodontic treatment.

Mechanical force inhibits osteoclastogenic potential of human periodontal ligament fibroblasts through OPG production and ERK-mediated signaling.

Periodontal ligament and gingival fibroblasts play important roles in bone remodeling. Periodontal ligament fibroblasts stimulate bone remodeling while gingival fibroblasts protect abnormal bone resorption. However, few studies had examined the differences in stimulation of osteoclast formation between the two fibroblast populations. The precise effect of mechanical forces on osteoclastogenesis of these populations is also unknown. This study revealed that more osteoclast-like cells were induced in the co-cultures of bone marrow cells with periodontal ligament than gingival fibroblasts, and this was considerably increased when anti-osteoprotegerin (OPG) antibody was added to the co-cultures. mRNA levels of receptor activator of nuclear factor-kappaB ligand (RANKL) were increased in both populations when they were cultured with dexamethasone and vitamin D(3). Centrifugal forces inhibited osteoclastogenesis of both populations, and this was likely related to the force-induced OPG up-regulation. Inhibition of extracellular signal-regulated kinase (ERK) signaling by a pharmacological inhibitor (10 microM PD98059) or by siERK transfection suppressed the force-induced OPG up-regulation along with the augmentation of osteoclast-like cells that were decreased by the force. These results suggest that periodontal ligament fibroblasts are naturally better at osteoclast induction than gingival fibroblasts, and that centrifugal force inhibited osteoclastogenesis of the periodontal fibroblasts through OPG production and ERK activation.

Role of MAPK in mechanical force-induced up-regulation of type I collagen and osteopontin in human gingival fibroblasts.

In addition to periodontal ligament, the gingival plays an important role in alveolar bone remodeling induced by physiological and mechanical stimuli. However, there are few reports showing the cellular responses of human gingival fibroblasts (HGF) to a mechanical force. This study examined the effects of centrifugal force on the proliferation of the bone tissue components, such as type I collagen (COL I), osteopontin (OPN), and osteonectin (ONN) in the HGF. The roles of extracellular signal-regulated kinase (ERK), c-Jun-N-terminal kinase (JNK), and p-38 kinase were also investigated. Centrifugal force induced cell cycle arrest in the G(1) phase without any cytotoxic effects and increased the levels of COL I and OPN expression in the cells but had no effect on ONN. The force-induced up-regulation of COL I was found to be mediated by both the ERK-c-Fos-COL I and JNK-c-Jun-COL I pathways, while that of OPN was mediated only by the ERK-mediated pathway. Our present findings suggest that centrifugal force up-regulates COL I and OPN expression in HGF, where both ERK and JNK play indispensable roles.

Plasma-arc generated light inhibits proliferation and induces apoptosis of human gingival fibroblasts in a dose-dependent manner.

OBJECTIVE: This study examined the effects of blue light exposure on the proliferation and cytotoxicity of human gingival fibroblasts (HGF). Cellular mechanism by which blue light causes cytotoxic effects was also investigated. METHODS: HGF were exposed to the plasma-arc generated blue light with various energy densities ranging from 2 to 48J/cm(2). After light exposure of the cells, they were processed for analyzing tritium incorporation, succinate dehydrogenase (SDH) activity, trypan blue exclusion, and DNA fragmentation. In addition, possible mechanism of the light-mediated cytotoxicity was investigated through flow cytometric and Western blot analyses. RESULTS: Blue light exposure significantly inhibited proliferation and SDH activity of HGF in a dose-dependent manner; exposure more than 12J/cm(2) had a toxic effect on the cells. The blue light-induced cytotoxicity of the cells resulted from apoptosis, as proven by the migration of many cells to the sub-G(1) phase of cell cycle and the appearance of DNA ladders. Additional experiments revealed that blue light induces apoptosis of HGF through mitochondrial stress and poly (ADP ribose) polymerase cleavage. SIGNIFICANCE: This study suggests that plasma-arc generated blue light exerts some harm to cells, particularly damaging effect to DNA, and thus a long curing time more than recommended can cause biological damage on the oral tissue.

Periodontal fibroblasts modulate proliferation and osteogenic differentiation of embryonic stem cells through production of fibroblast growth factors.

BACKGROUND: Periodontal ligament fibroblasts (PLFs) maintain homeostasis of periodontal ligaments by producing paracrine factors that affect various functions of stem-like cells. It is hypothesized that PLFs induce proliferation and differentiation of stem cells more effectively than gingival fibroblasts (GFs) and skin fibroblasts (SFs). METHODS: PLFs and GFs were isolated from extracted teeth and cultured in the presence and absence of osteogenesis-inducing factors. Mouse embryonic stem (mES) cells and SFs were purchased commercially. mES cells were incubated with culture supernatants of these fibroblasts or cocultured directly with the cells. Proliferation and mineralization in mES cells were determined at various times of incubation. Immunostaining and polymerase chain reaction were performed. The activity of mitogen-activated protein kinase and alkaline phosphatase (ALP) was also measured. RESULTS: In cocultures, PLFs stimulated proliferation of mES cells more effectively than GFs or SFs. Similarly, the addition of culture supernatant of PLFs induced the most prominent proliferation of mES cells, and this was significantly inhibited by treatment with antibody against fibroblast growth factor (FGF)4 or the c-Jun N-terminal kinase inhibitor SP600125 (anthra[1,9-cd]pyrazol-6(2H)-one). Supplementation with culture supernatant from the fibroblasts induced osteogenic differentiation of mES cells in the order PLFs > GFs > SFs. These activities of PLFs were related to their potential to produce osteogenic markers, such as ALP and runt-related transcription factor-2 (Runx2), and to secrete FGF7. Pretreatment of mES cells with the extracellular signal-regulated kinase inhibitor PD98059 [2-(2-amino-3-methyoxyphenyl)-4H-1-benzopyran-4-one] or SP600125 clearly attenuated mineralization induced by culture supernatant of PLF with attendant decreases in mRNA levels of Runx2, bone sialoprotein, osteocalcin, and osteopontin. CONCLUSION: PLFs regulate the proliferation and osteogenic differentiation of mES cells more strongly than GFs and SFs via the secretion of FGF through a mechanism that involves mitogen-activated protein kinase-mediated signaling.

Enhanced compatibility and initial stability of Ti6Al4V alloy orthodontic miniscrews subjected to anodization, cyclic precalcification, and heat treatment.

OBJECTIVE: To evaluate the bioactivity, and the biomechanical and bone-regenerative properties of Ti6Al4V miniscrews subjected to anodization, cyclic precalcification, and heat treatment (APH treatment) and their potential clinical use. METHODS: The surfaces of Ti6Al4V alloys were modified by APH treatment. Bioactivity was assessed after immersion in simulated body fluid for 3 days. The hydrophilicity and the roughness of APH-treated surfaces were compared with those of untreated (UT) and anodized and heat-treated (AH) samples. For in vivo tests, 32 miniscrews (16 UT and 16 APH) were inserted into 16 Wistar rats, one UT and one APH-treated miniscrew in either tibia. The miniscrews were extracted after 3 and 6 weeks and their osseointegration (n = 8 for each time point and group) was investigated by surface and histological analyses and removal torque measurements. RESULTS: APH treatment formed a dense surface array of nanotubular TiO2 layer covered with a compact apatite-like film. APH-treated samples showed better bioactivity and biocompatibility compared with UT and AH samples. In vivo, APH-treated miniscrews showed higher removal torque and bone-to-implant contact than did UT miniscrews, after both 3 and 6 weeks (p < 0.05). Also, early deposition of densely mineralized bone around APH-treated miniscrews was observed, implying good bonding to the treated surface. CONCLUSIONS: APH treatment enhanced the bioactivity, and the biomechanical and bone regenerative properties of the Ti6Al4V alloy miniscrews. The enhanced initial stability afforded should be valuable in orthodontic applications.