Effectiveness of novel oral hygiene instruction avoiding inattentional blindness using an application for unique plaque control record calculation: A randomized clinical trial.

OBJECTIVE: This study was performed to estimate the effectiveness of novel oral hygiene instruction (OHI) focusing on areas with deep periodontal pockets for reduction of periodontal inflammation. BACKGROUND DATA DISCUSSING THE PRESENT STATUS OF THE FIELD: Because stained areas on the plaque chart do not always correspond to the areas with deep periodontal pockets, conventional OHI based on O'Leary's plaque control record (PCR) often provides guidance inconsistent with the target area. METHODS: This randomized clinical trial involved two groups: (1) OHI based on the PCR limited in deep pocket sites (novel OHI group) and (2) OHI based on O'Leary's PCR (conventional OHI group). The unique PCR (aggressive target for PCR [agPCR]; only counting the plaque-stained areas with PD at ≥4 mm sites) for the novel OHI was calculate by dedicated expression program. The probing depth (PD), bleeding on probing (BOP), and periodontal inflamed surface area (PISA) were obtained at the baseline and 5 to 6 months later. RESULTS: The approximation curve with PISA before and after instruction indicated that the PISA converged to a lower value after instruction in the novel OHI group. The approximation curve with the improvement rate of the PISA and agPCR showed a positive correlation in the novel OHI group but no correlation in the conventional OHI group. CONCLUSION: Control of inflammation was more effective in the novel OHI group. These results suggest that this novel OHI technique using our developed application could be used as a strategy to improve the effectiveness of brushing instruction.

Suppression of subgingival bacteria by antimicrobial photodynamic therapy using transgingival irradiation: A randomized clinical trial.

BACKGROUND: Antimicrobial photodynamic therapy (aPDT) is an effective method for eradicating bacteria in periodontal therapy. Standard aPDT requires the insertion of a laser tip into a periodontal pocket, in which the direction of irradiation is limited. Therefore, we devised an aPDT method that uses a transgingival near-infrared wavelength and indocyanine green-encapsulated and chitosan-coated nanoparticles as a photosensitizer. METHODS: Forty patients undergoing supportive periodontal therapy, who had a single root tooth with a pocket of 5 mm or deeper, were used as subjects. In the test group, aPDT was performed by laser irradiation from outside the gingiva using photosensitizer nanoparticles. In the control group, pseudo aPDT without photosensitizer was performed by transgingival irradiation. Subgingival plaque was sampled from inside the pocket before, immediately after, and 1 week after treatment, and evaluated by colony counting and real-time polymerase chain reaction. RESULTS: There were no significant differences in age, sex, periodontal pocket depth, and bleeding on probing between the test and control groups. Compared with the colony count before treatment, the count in the test group was significantly reduced immediately after treatment. The number of patients with colony reduction to ≤50% and ≤10% was significantly higher in the test group than in the control group. None of the participants reported pain, although one participant reported discomfort. CONCLUSION: As a bacterial control method for residual pockets in patients undergoing supportive periodontal therapy, transgingival aPDT is a promising treatment strategy that is not generally accompanied by pain or discomfort.

Photodynamic Inactivation of an Endodontic Bacteria Using Diode Laser and Indocyanine Green-Loaded Nanosphere.

Apical periodontitis, an inflammatory lesion causing bone resorption around the apex of teeth, is treated by eradicating infectious bacteria from the root canal. However, it has a high recurrence rate and often requires retreatment. We investigated the bactericidal effect of antimicrobial photodynamic therapy (aPDT)/photodynamic antimicrobial chemotherapy (PACT) using indocyanine green (ICG)-loaded nanospheres coated with chitosan and a diode laser on a biofilm of Enterococcus faecalis, a pathogen of refractory apical periodontitis. Biofilm of E. faecalis was cultured in a porcine infected root canal model. ICG solution was injected into the root canal, which was then irradiated with a laser (810 nm wavelength) from outside the root canal. The bactericidal effect was evaluated by colony counts and scanning electron microscopy. The result of the colony counts showed a maximum 1.89 log reduction after irradiation at 2.1 W for 5 min. The temperature rise during aPDT/PACT was confirmed to be within a safe range. Furthermore, the light energy transmittance through the root was at a peak approximately 1 min after the start of irradiation, indicating that most of the ICG in the root canal was consumed. This study shows that aPDT/PACT can suppress E. faecalis in infected root canals with high efficiency.

Gelatin Methacryloyl-Riboflavin (GelMA-RF) Hydrogels for Bone Regeneration.

Gelatin methacryloyl (GelMA) is a versatile biomaterial that has been used in various biomedical fields. UV light is commonly used to photocrosslink such materials; however, its use has raised several biosafety concerns. We investigated the mechanical and biological properties of a visible-wavelength (VW)-light-crosslinked gelatin-based hydrogel to evaluate its viability as a scaffold for bone regeneration in bone-destructive disease treatment. Irgacure2959 or riboflavin was added as a photoinitiator to create GelMA solutions. GelMA solutions were poured into a mold and exposed to either UV or VW light. KUSA-A1 cell-laden GelMA hydrogels were crosslinked and then cultured. Mechanical characterization revealed that the stiffness range of GelMA-RF hydrogel was suitable for osteoblast differentiation. KUSA-A1 cells encapsulated in GelMA hydrogels photopolymerized with VW light displayed significantly higher cell viability than cells encapsulated in hydrogels photopolymerized with UV light. We also show that the expression of osteogenesis-related genes at a late stage of osteoblast differentiation in osteoblasts encapsulated in GelMA-RF hydrogel was markedly increased under osteoblast differentiation-inducing conditions. The GelMA-RF hydrogel served as an excellent scaffold for the encapsulation of osteoblasts. GelMA-RF hydrogel-encapsulated osteoblasts have the potential not only to help regenerate bone mass but also to treat complex bone defects associated with bone-destructive diseases such as periodontitis.

Angiopoietin-like protein 2 regulates Porphyromonas gingivalis lipopolysaccharide-induced inflammatory response in human gingival epithelial cells.

Angiopoietin-like protein 2 (ANGPTL2) maintains tissue homeostasis by inducing inflammation and angiogenesis. It is produced in infiltrating immune cells or resident cells, such as adipocytes, vascular endothelial cells, and tumor cells. We hypothesized that ANGPTL2 might play an important role as a unique mediator in both systemic and periodontal disease. We demonstrated an increased ANGPTL2 concentration in gingival crevicular fluid from chronic periodontitis patients. Porphyromonas gingivalis lipopolysaccharide (LPS) treatment strongly induced ANGPTL2 mRNA and protein levels in Ca9-22 human gingival epithelial cells. Recombinant human ANGPTL2 increased interleukin 1ß (IL-1ß), IL-8, and tumor necrosis factor-α (TNF-α) mRNA and protein levels in Ca9-22 cells. Small-interfering (si)RNA-mediated ANGPTL2 knockdown in Ca9-22 cells reduced IL-1ß, IL-8 and TNF-α mRNA and protein levels compared with control siRNA (p<0.01) in P. gingivalis LPS-stimulated Ca9-22 cells. Antibodies against integrin α5ß1, an ANGPTL receptor, blocked induction of these inflammatory cytokines in P. gingivalis LPS-treated Ca9-22 cells, suggesting that secreted ANGPTL induces inflammatory cytokines in gingival epithelial cells via an autocrine loop. The classic sequential cascade of P. gingivalis LPS → inflammatory cytokine induction is well established. However, in the current study, we reveal a novel cascade comprising sequential P. gingivalis LPS → ANGPTL2 → integrin α5ß1 → inflammatory cytokine induction, which might be responsible for inducing potent periodontal disorganization activity in gingival epithelial cells. Via this pathway, ANGPTL2 functions in the pathogenesis of periodontitis and contributes to prolonging chronic inflammation in patients with systemic disease.

F-spondin negatively regulates dental follicle differentiation through the inhibition of TGF-ß activity.

OBJECTIVE: F-spondin is an extracellular matrix (ECM) protein that belongs to the thrombospondin type I repeat superfamily and is a negative regulator of bone mass. We have previously shown that f-spondin is specifically expressed in the dental follicle (DF), which gives rise to the periodontal ligament (PDL) during the tooth root formation stage. To investigate the molecular mechanism of PDL formation, we investigated the function of f-spondin in DF differentiation. DESIGN: The expression patterning of f-spondin in the developing tooth germ was compared with that of periodontal ligament-related genes, including runx2, type I collagen and periostin, by in situ hybridization analysis. To investigate the function of f-spondin during periodontal ligament formation, an f-spondin adenovirus was infected into the bell stage of the developing tooth germ, and the effect on dental differentiation was analyzed. RESULTS: F-spondin was specifically expressed in the DF of the developing tooth germ; by contrast, type I collagen, runx2 and periostin were expressed in the DF and in the alveolar bone. F-spondin-overexpresssing tooth germ exhibited a reduction in gene expression of periostin and type I collagen in the DF. By contrast, the knockdown of f-spondin in primary DF cells increased the expression of these genes. Treatment with recombinant f-spondin protein functionally inhibited periostin expression induced by transforming growth factor-ß (TGF-ß). CONCLUSION: Our data indicated that f-spondin inhibits the differentiation of DF cells into periodontal ligament cells by inhibiting TGF-ß. These data suggested that f-spondin negatively regulates PDL differentiation which may play an important role in the immature phenotype of DF.

New Irradiation Method with Indocyanine Green-Loaded Nanospheres for Inactivating Periodontal Pathogens.

Antimicrobial photodynamic therapy (aPDT) has been proposed as an adjunctive strategy for periodontitis treatments. However, use of aPDT for periodontal treatment is complicated by the difficulty in accessing morphologically complex lesions such as furcation involvement, which the irradiation beam (which is targeted parallel to the tooth axis into the periodontal pocket) cannot access directly. The aim of this study was to validate a modified aPDT method that photosensitizes indocyanine green-loaded nanospheres through the gingivae from outside the pocket using a diode laser. To establish this trans-gingival irradiation method, we built an in vitro aPDT model using a substitution for gingivae. Irradiation conditions and the cooling method were optimized before the bactericidal effects on Porphyromonas gingivalis were investigated. The permeable energy through the gingival model at irradiation conditions of 2 W output power in a 50% duty cycle was comparable with the transmitted energy of conventional irradiation. Intermittent irradiation with air cooling limited the temperature increase in the gingival model to 2.75 °C. The aPDT group showed significant bactericidal effects, with reductions in colony-forming units of 99.99% after 5 min of irradiation. This effect of aPDT against a periodontal pathogen demonstrates the validity of trans-gingival irradiation for periodontal treatment.

Serum Amyloid A Promotes E-Selectin Expression via Toll-Like Receptor 2 in Human Aortic Endothelial Cells.

Periodontitis is a chronic inflammatory disease that affects the periodontium. Recent studies suggest an association between periodontal and cardiovascular diseases. However, the detailed molecular mechanism is unknown. A previous study has demonstrated that experimental periodontitis induces serum amyloid A (SAA) in the liver and peripheral blood of ApoE-deficient mice as an atherosclerosis model. SAA is an acute-phase protein that affects systemic inflammation. The aim of this study is to investigate the atherosclerosis-onset mechanism using human aortic endothelial cells (HAECs) stimulated by SAA in vitro. Atherosclerosis PCR array and qPCR analyses showed upregulation of adhesion molecules such as intercellular adhesion molecule-1, vascular cell adhesion molecule-1, and E-selectin in HAECs upon SAA stimulation. In addition, the results demonstrated that Toll-like receptor, TLR2, could serve as an important receptor of SAA in HAECs. Furthermore, small interfering RNA (siRNA) against TLR2 inhibited the upregulation of adhesion molecules in HAECs stimulated by SAA. Our results suggest that SAA stimulates the expression of adhesion molecules via TLR2. SAA could be an important molecule for atherosclerosis induced by periodontal disease.

Isolation and characterization of the human immature osteoblast culture system from the alveolar bones of aged donors for bone regeneration therapy.

BACKGROUND: Establishment of human osteoblast cultures that retain bone-forming capacity is one of the prerequisites for successful bone regeneration therapy. Because osteoblasts harvested from adults exhibit limited growth, the use of immature osteoblasts that can expand ex vivo should greatly facilitate bone regeneration therapy. In this study, we developed immature human osteoblasts isolated from aged alveolar bone (HAOBs). METHODS: HAOBs obtained after the collagenase digestion of alveolar bones from elderly donors. Then, we assessed osteogenic ability of HAOB after treatment with recombinant human bone morphogenic protein-2 or transplantation into immunodeficient mice. In addition, we performed global gene expression analysis to identify functional marker for HAOB. RESULTS: HAOBs, which can differentiate into osteoblasts and have a robust bone-forming ability, were successfully extracted from donors who were > 60 years of age. We found that the HAOBs exhibited a higher osteogenic ability compared with those of human mesenchymal stem cells and highly expressed NEBULETTE (NEBL) with osteogenic abilities. CONCLUSIONS: HAOBs have properties similar to those of human immature osteoblasts and appear to be a novel material for cell-based bone regeneration therapy. Additionally, the expression level of NEBL may serve as a marker for the osteogenic ability of these cells.

The KK-Periome database for transcripts of periodontal ligament development.

The periodontal ligament (PDL) is a strong connective tissue that surrounds the tooth root, absorbs occlusal forces, and functions as a sense organ. PDL originated from dental follicle (DF), which possessed mesenchymal progenitors in the developing tooth germ. However, as specific marker genes for PDL and DF are currently unavailable, the molecular mechanisms of PDL development are yet to be clarified. To facilitate the identification of such genes, we have previously established a transcriptome database of the human PDL (the KK-Periome database) and screened for specific genes expressed during PDL development. Initial screening of the database revealed two marker genes for distinguishing DF and PDL. The KK-Periome database thus appears to offer a useful resource for investigating genes involved in PDL development.

Transcriptome database KK-Periome for periodontal ligament development: expression profiles of the extracellular matrix genes.

Specialized connective tissues such as tendon/ligament develop through a series of events that require temporal and spatial expression of numerous genes in mesenchymal progenitors. However, the genes required for tendon/ligament development have not been identified yet. To solve this problem, we made a cDNA library from periodontal ligament and sequenced 11,520 cDNA clones, as a model for investigating tendon/ligament development. The resulting sequence data was assembled to 617 expressed sequence tag (EST) clusters, and an EST database for human periodontal ligament (PDL) was constructed (designated as the KK-Periome database). In the KK-Periome database, the top 13 EST clusters were related to extracellular matrix (ECM) genes. The temporal and spatial expression patterns of these genes during mouse PDL development were examined by in situ hybridization. Among these genes, F-spondin was expressed specifically in dental follicle (DF) cells during tooth germ development, whereas tenascin-N was strongly expressed in the terminally differentiated PDL. This characteristic expression profile was confirmed by in vivo differentiation assay of human PDL (hPDL) cells in the mouse transplant. Thus, the KK-Periome database was proven to be a useful resource for PDL-derived ESTs (transcriptome), and in fact, initial evidence indicated that F-spondin and tenascin-N might serve as markers for DF and PDL, respectively.