Ryuji Sakagami

FGF-2 Stimulates Periodontal Regeneration Results of a Multi-center Randomized Clinical Trial

The efficacy of the local application of recombinant human fibroblast growth factor-2 (FGF-2) in periodontal regeneration has been investigated. In this study, a randomized, double-blind, placebo-controlled clinical trial was conducted in 253 adult patients with periodontitis. Modified Widman periodontal surgery was performed, during which 200 µL of the investigational formulation containing 0% (vehicle alone), 0.2%, 0.3%, or 0.4% FGF-2 was administered to 2- or 3-walled vertical bone defects. Each dose of FGF-2 showed significant superiority over vehicle alone (p < 0.01) for the percentage of bone fill at 36 wks after administration, and the percentage peaked in the 0.3% FGF-2 group. No significant differences among groups were observed in clinical attachment regained, scoring approximately 2 mm. No clinical safety problems, including an abnormal increase in alveolar bone or ankylosis, were identified. These results strongly suggest that topical application of FGF-2 can be efficacious in the regeneration of human periodontal tissue that has been destroyed by periodontitis.

Graphene oxide coating facilitates the bioactivity of scaffold material for tissue engineering

Carbon-based nanomaterials are being investigated for biomedical applications. Graphene oxide (GO), a monolayer of carbon, holds promise as a tissue engineering substrate due to its unique physicochemical properties. The aim of this study was to evaluate the effect of a GO coating on cell proliferation and differentiation in vitro. We also assessed the bioactivities of collagen scaffolds coated with different concentrations of GO in rats. The results showed that GO affects both cell proliferation and differentiation, and improves the properties of collagen scaffolds. Subcutaneous implant tests showed that low concentrations of GO scaffold enhances cell in-growth and is highly biodegradable, whereas high concentrations of GO coating resulted in adverse biological effects. Consequently, scaffolds modified with a suitable concentration of GO are useful as a bioactive material for tissue engineering.

Randomized Placebo-Controlled and Controlled Non-Inferiority Phase III Trials Comparing Trafermin, a Recombinant Human Fibroblast Growth Factor 2, and Enamel Matrix Derivative in Periodontal Regeneration in Intrabony Defects

We investigated the efficacy, safety, and clinical significance of trafermin, a recombinant human fibroblast growth factor (rhFGF)‐2, for periodontal regeneration in intrabony defects in Phase III trials. Study A, a multicenter, randomized, double‐blind, placebo‐controlled study, was conducted at 24 centers. Patients with periodontitis with 4‐mm and 3‐mm or deeper probing pocket depth and intrabony defects, respectively, were included. A total of 328 patients were randomly assigned (2:1) to receive 0.3% rhFGF‐2 or placebo, and 323 patients received the assigned investigational drug during flap surgery. One of the co‐primary endpoints, the percentage of bone fill at 36 weeks after drug administration, was significantly greater in the rhFGF‐2 group at 37.131% (95% confidence interval [CI], 32.7502 to 41.5123; n = 208) than it was in the placebo group at 21.579% (95% CI, 16.3571 to 26.8011; n = 100; p < 0.001). The other endpoint, the clinical attachment level regained at 36 weeks, was not significantly different between groups. Study B, a multicenter, randomized, blinded (patients and evaluators of radiographs), and active‐controlled study was conducted at 15 centers to clarify the clinical significance of rhFGF‐2. Patients with 6‐mm and 4‐mm or deeper probing pocket depth and intrabony defects, respectively, were included. A total of 274 patients were randomly assigned (5:5:2) to receive rhFGF‐2, enamel matrix derivative (EMD), or flap surgery alone. A total of 267 patients received the assigned treatment during flap surgery. The primary endpoint, the linear alveolar bone growth at 36 weeks, was 1.927 mm (95% CI, 1.6615 to 2.1920; n = 108) in the rhFGF‐2 group and 1.359 mm (95% CI, 1.0683 to 1.6495; n = 109) in the EMD group, showing non‐inferiority (a prespecified margin of 0.3 mm) and superiority of rhFGF‐2 to EMD. Safety problems were not identified in either study. Therefore, trafermin is an effective and safe treatment for periodontal regeneration in intrabony defect, and its efficacy was superior in rhFGF‐2 compared to EMD treatments.

Activation of AMP-activated protein kinase by MAPO1 and FLCN induces apoptosis triggered by alkylated base mismatch in DNA

O₆-methylguanine produced in DNA by the action of simple alkylating agents, such as N-methyl-N-nitrosourea (MNU), causes base-mispairing during DNA replication, thus leading to mutations and cancer. To prevent such outcomes, the cells carrying O⁶-methylguanine undergo apoptosis in a mismatch repair protein-dependent manner. We previously identified MAPO1 as one of the components required for the induction of apoptosis triggered by O⁶-methylguanine. MAPO1, also known as FNIP2 and FNIPL, forms a complex with AMP-activated protein kinase (AMPK) and folliculin (FLCN), which is encoded by the BHD tumor suppressor gene. We describe here the involvement of the AMPK-MAPO1-FLCN complex in the signaling pathway of apoptosis induced by O⁶-methylguanine. By the introduction of siRNAs specific for these genes, the transition of cells to a population with sub-G₁ DNA content following MNU treatment was significantly suppressed. After MNU exposure, phosphorylation of AMPKα occurred in an MLH1-dependent manner, and this activation of AMPK was not observed in cells in which the expression of either the Mapo1 or the Flcn gene was downregulated. When cells were treated with AICA-ribose (AICAR), a specific activator of AMPK, activation of AMPK was also observed in a MAPO1- and FLCN-dependent manner, thus leading to cell death which was accompanied by the depolarization of the mitochondrial membrane, a hallmark of the apoptosis induction. It is therefore likely that MAPO1, in its association with FLCN, may regulate the activation of AMPK to control the induction of apoptosis triggered by O⁶-methylguanine.

Prevalence and risk factors for peri-implant diseases in Japanese adult dental patients

We investigated the prevalences and risk factors for peri-implant diseases in Japanese adult dental patients attending a follow-up visit at dental hospitals or clinics as part of their maintenance program. This cross-sectional multicenter study enrolled patients with dental implants who attended regular check-ups as part of a periodontal maintenance program during the period from October 2012 through September 2013. Patients with implants with at least 3 years of loading time were included in the study. The condition of peri-implant tissue was examined and classified into the following categories: healthy, peri-implant mucositis, and peri-implantitis. Patients were also evaluated for implant risk factors. A total of 267 patients (110 men, 157 women; mean age: 62.5 ± 10.7 years) were analyzed. The prevalence of patient-based peri-implant mucositis was 33.3% (n = 89), and the prevalence of peri-implantitis was 9.7% (n = 26). Poor oral hygiene and a history of periodontitis were strong risk factors for peri-implant disease. The present prevalences were lower than those previously reported. The quality of periodontal therapy before and after implant installation and patient compliance and motivation, as indicated by plaque control level, appear to be important in maintaining peri-implant tissue health.

Comparison of Fracture Sites and Post Lengths in Longitudinal Root Fractures

INTRODUCTION Comparing the epidemiology of fractures originating in the cervical and apical regions may help to understand the causes and risk of a vertical root fracture. We aimed to determine the frequency of vertical root fractures in different fracture sites and how the fracture site relates to fracture direction and post length. METHODS Teeth diagnosed with a vertical root fracture were retrospectively surveyed for age and sex of the patient, type of tooth, a fracture region in the longitudinal axial direction, site of the fracture, and presence of a post. The fracture region in the longitudinal axial direction was classified as an incomplete fracture, complete fracture, and uncertain. Incomplete fractures were further classified into a fracture originating in the cervical region, a fracture originating in the midregion, and a fracture originating in the apical region. Posts were evaluated by loss of post and length of post. RESULTS Fractures originating in the cervical and apical region occurred around the same frequency, whereas fractures originating in the midregion were extremely scarce. Of the fractures originating in the cervical region, 36.2% were in a mesial and/or distal site and 57.4% in a buccal and/or lingual site. Of the fractures originating in the apical region, 90.8% were in the buccal and/or lingual site. The number of cases of fractures originating in the apical region decreased with increased post length. CONCLUSIONS Sites of fracture and post length differed greatly between fractures originating in the cervical region and the apical region, suggesting that risk factors for fractures originating in the cervical and apical regions are different.

Cell viabilities and biodegradation rates of DNA/protamine complexes with two different molecular weights of DNA.

Two types of DNA/protamine complexes were prepared from protamine sulfate and 7000 base pair (bp) DNA or original DNA to investigate the effect of the molecular weight of DNA on zeta potential, cell viability, flowability, soft tissue response, and biodegradation rate. The 7000 bp DNA/protamine complex had a negative charge while the original DNA/protamine complex had a positive charge. The cell viabilities (90.4-106.8%) of these complexes were close to each other. The 7000 bp DNA/protamine complex became a softer dough than that of the original DNA/complex when both were kneaded with water. In vivo, the original DNA/protamine complex showed a milder tissue response. The original DNA/protamine complex almost disappeared 30 days after implantation. The 7000 bp DNA/complex disappeared approximately 2 weeks after implantation and areas where samples were implanted became empty. Thereafter, the empty space was gradually replaced by new soft tissues. The original DNA/protamine complex showed low intercalation and groove binding ratios of daunorubicin hydrochloride. Results indicate that high DNA condensation by cationic protamine protected the penetration of degradation enzymes into these complexes. It was found that a high molecular weight of DNA reduced the biodegradation rate and flowability. This study suggests that DNA/protamine complexes could be candidates for biomaterials that control biodegradation rates and flowability.

Antibodies against ClC7 inhibit extracellular acidification-induced Cl− currents and bone resorption activity in mouse osteoclasts

The Cl− channel/transporter ClC7 is crucial for osteoclastic bone resorption and might become a therapeutic target for osteoporosis. In this study, we raised anti-ClC7 polyclonal antibodies against three different peptide sequences, including G215, P249, and R286, which are the mutation regions found in autosomal dominant osteopetrosis type II patients and examined the effects of these antibodies on the ClC7 Cl− current induced by extracellular acidification (acid-activated Cl− current) using the whole-cell patch clamp technique and bone resorption activity in mouse osteoclasts. Intracellular dialysis of osteoclasts with antibodies to intracellular G215 (Ab-G215) and extracellular application of antibodies to extracellular P249 (Ab-P249) or R286 (Ab-R286) inhibited the acid-activated Cl− current. These antibodies also suppressed the acid-activated Cl− current in ClC7 overexpressing Raw264.7 cells; however, Cl− currents evoked by hypotonic stimulation and the inherent inwardly rectifying K+ currents in mouse osteoclasts were unaffected by these antibodies. Furthermore, extracellularly applied Ab-P249 and Ab-R286 also reduced bone resorption activity. Our results demonstrate that these antibodies specifically block ClC7 in mouse osteoclasts. Thus, anti-ClC7 antibodies have potential promise for treatment of osteoporosis.

Combination of Root Surface Modification with BMP-2 and Collagen Hydrogel Scaffold Implantation for Periodontal Healing in Beagle Dogs.

Objective : Biomodification of the root surface plays a major role in periodontal wound healing. Root surface modification with bone morphogenetic protein (BMP) stimulates bone and cementum-like tissue formation; however, severe ankylosis is simultaneously observed. Bio-safe collagen hydrogel scaffolds may therefore be useful for supplying periodontal ligament cells and preventing ankylosis. We examined the effects of BMP modification in conjunction with collagen hydrogel scaffold implantation on periodontal wound healing in dogs. Material and Methods: The collagen hydrogel scaffold was composed of type I collagen sponge and collagen hydrogel. One-wall infrabony defects (5 mm in depth, 3 mm in width) were surgically created in six beagle dogs. In the BMP/Col group, BMP-2 was applied to the root surface (loading dose; 1 µg/µl), and the defects were filled with collagen hydrogel scaffold. In the BMP or Col group, BMP-2 coating or scaffold implantation was performed. Histometric parameters were evaluated at 4 weeks after surgery. Results: Single use of BMP stimulated formation of alveolar bone and ankylosis. In contrast, the BMP/Col group frequently enhanced reconstruction of periodontal attachment including cementum-like tissue, periodontal ligament and alveolar bone. The amount of new periodontal ligament in the BMP/Col group was significantly greater when compared to all other groups. In addition, ankylosis was rarely observed in the BMP/Col group. Conclusion: The combination method using root surface modification with BMP and collagen hydrogel scaffold implantation facilitated the reestablishment of periodontal attachment. BMP-related ankylosis was suppressed by implantation of collagen hydrogel.

A novel inhibitory mechanism of nitrogen-containing bisphosphonate on the activity of Cl− extrusion in osteoclasts

Nitrogen-containing bisphosphonates have been well known to be inhibited farnesyl diphosphate synthase (FDPS), an enzyme in mevalonic acid metabolism, resulting in disturbance in polymerization of cytoskeleton structure in bone resorption and promotion of apoptosis in mature osteoclasts. Although bisphosphonates have been reported to activate ion transporters in native epithelium and Xenopus oocytes, little is known whether bisphosphonates affect acid hydrochronic acid extrusion in osteoclasts during bone resorption. The aim of this study was to determine the role of bisphosphonates on inhibition of hydrochronic acid extrusion in osteoclasts. Effects of zoledronic acid, a nitrogen-containing bisphosphonate, on the Cl− current activated by extracellular acidification were examined in two types of osteoclasts derived from RAW264.7 cells and mouse bone marrow macrophages (BMMs). Extracellular acidification induced outwardly rectifying Cl− currents in mouse osteoclasts. Zoledronic acid dose-dependently inhibited the acid-activated Cl− current. The non-nitrogen bisphosphonate etidronic acid had no effect on the acid-activated Cl− current. Tetracycline-induced FDPS silencing caused a significant decrease in the Cl− current. The inhibitor of geranylgeranyl transferase suppressed the Cl− current. By contrast, the inhibitory action of zoledronic acid was rescued by addition of geranylgeranyl acid, a derivative of mevalonic acid. The activity of acid-activated Cl− currents was dependent on expression of ClC-7 during osteoclastogenesis. These results suggest that nitrogen-containing bisphosphonates suppress the activity of osteoclastic acid-activated Cl− currents through FDPS inhibition, suggesting the inhibition of Cl− extrusion activity.