Toshiaki Nakamura

Bone morphogenetic protein 9 (BMP9) has strong osteogenic potential and has emerged as a promising therapeutic target for periodontal wound healing and regeneration. However, although BMP9 induces osteoblastic differentiation, its effects on cementoblasts and potential synergistic interactions with FK506, an immunosuppressive drug, are unknown. This study investigated the combined effects of BMP9 and FK506 treatment on cementoblast differentiation using a human cementoblast-like cell line, HCEM. BMP9 significantly upregulated alkaline phosphatase activity, mineralization, and the expression of mineralized tissue-related genes, including IBSP and SPP1, in HCEM cells, with FK506 co-treatment further enhancing these effects. FK506 also augmented BMP9-induced phosphorylation of SMAD1/5/8, ERK1/2, and p38, as shown by western blotting. Moreover, silencing of FKBP1A, which encodes FK506-binding protein 12 (FKBP12)-a key regulator of BMP signaling-enhanced BMP9 responsiveness, confirming its regulatory role. Neither BMP9 nor FK506 affected cell proliferation. In conclusion, BMP9 induces cementoblast differentiation via SMAD1/5/8, ERK1/2, and p38 pathways in HCEM cells, which are further enhanced through the regulation of FKBP12 by FK506. The combination of BMP9 and FK506 may offer a promising strategy for periodontal wound healing and regeneration, warranting further preclinical and clinical investigations.

Aim: To histologically evaluate the effects of cross‐linked hyaluronic acid (xHyA) with or without a collagen matrix (CM) on periodontal wound healing/regeneration in class III furcation defects in dogs.Materials and Methods: Class III furcation defects were surgically created in the mandibular premolars in six beagle dogs. The defects were randomly treated as follows: open flap debridement (OFD) + CM (CM), OFD + xHyA (xHyA), OFD + xHyA + CM (xHyA/CM) and OFD alone (OFD). At 10 weeks, the animals were euthanized for histological evaluation.Results: The newly formed bone areas in the xHyA (4.04 ± 1.51 mm²) and xHyA/CM (4.32 ± 1.14 mm²) groups were larger than those in the OFD (3.25 ± 0.81 mm²) and CM (3.31 ± 2.26 mm²) groups. The xHyA (6.25 ± 1.45 mm) and xHyA/CM (6.40 ± 1.35 mm) groups yielded statistically significantly (p < .05) greater formation of new connective tissue attachment (i.e., new cementum, with inserting connective tissue fibres) compared with the OFD (1.47 ± 0.85 mm) group. No significant differences were observed in any of the histomorphometric parameters between the xHyA and xHyA/CM groups. Complete furcation closure was not observed in any of the four treatment modalities.Conclusions: Within their limits, the present results suggest that the use of xHyA with or without CM positively influences periodontal wound healing in surgically created, acute‐type class III furcation defects.

Aim: To evaluate periodontal wound healing/regeneration of one‐wall intra‐bony defects treated with recombinant human fibroblast growth factor‐2 (rhFGF‐2) and beta‐tricalcium phosphate (β‐TCP), carbonate apatite (CO₃Ap), or deproteinized bovine bone mineral (DBBM) in dogs.Materials and Methods: The stability of rhFGF‐2 adsorbed onto the bone substitutes was evaluated by Enzyme‐Linked Immunosorbent Assay (ELISA). One‐wall intra‐bony defects (5 × 5 × 5 mm) created in five adult male beagle dogs were treated with rhFGF‐2 alone (rhFGF‐2), rhFGF‐2 with β‐TCP (rhFGF‐2/β‐TCP), rhFGF‐2 with CO₃Ap (rhFGF‐2/CO₃Ap), or rhFGF‐2 with DBBM (rhFGF‐2/DBBM). Histological outcomes (e.g., linear length of new cementum adjacent to the newly formed bone with inserting collagen fibres [NA] as the primary outcome) were evaluated at 10 weeks post surgery.Results: Significantly higher amount of rhFGF‐2 was adsorbed onto CO₃Ap compared with β‐TCP. Among the treatment groups, the rhFGF‐2/DBBM group showed the highest amount of periodontal tissue regeneration. The rhFGF‐2/DBBM group showed significantly greater formation of NA (3.22 ± 0.40 mm) compared with rhFGF‐2 (1.17 ± 1.00 mm, p < .01) group. Additionally, new bone area in the rhFGF‐2/DBBM group (9.78 ± 2.30 mm²) was significantly higher than that in the rhFGF‐2 (5.08 ± 1.26 mm², p < .01), rhFGF‐2/β‐TCP (5.91 ± 1.27 mm², p < .05), and rhFGF‐2/CO₃Ap (6.51 ± 1.49 mm², p < .05) groups. Slight ankylosis was found in the rhFGF‐2/β‐TCP (1/9 sites), rhFGF‐2/CO₃Ap (3/10 sites), and rhFGF‐2/DBBM (1/9 sites) groups.Conclusions: Within their limitations, the present data indicate that DBBM seems to be a suitable carrier for rhFGF‐2 and that rhFGF‐2/DBBM treatment promotes favourable periodontal regeneration compared with rhFGF‐2, rhFGF‐2/β‐TCP, and rhFGF‐2/CO₃Ap treatments in one‐wall intra‐bony defects.

Bone morphogenetic protein‐9 (BMP‐9) has been shown to potently induce osteoblastic differentiation of periodontal ligament fibroblasts (PDLFs) and may be a candidate therapeutic agent for periodontal tissue healing/regeneration, but the effect of the inflammatory environment of periodontitis on such approaches is unclear. We investigated whether interleukin‐1β (IL‐1β) affected BMP‐9‐mediated osteoblastic differentiation of human (h) PDLFs. IL‐1β suppressed BMP‐9‐induced osteogenic differentiation of hPDLFs, as evidenced by reduced alkaline phosphatase (ALP) activity and mineralization, and the downregulated expression of BMP‐9‐mediated bone‐related genes, RUNX2, SP7, IBSP, and SPP1. In hPDLFs, with or without BMP‐9, IL‐1β increased the protein expression of activin A, a BMP‐9 antagonist, and decreased follistatin protein, an antagonist of activin A. Similarly, IL‐1β upregulated the expression of the activin A gene and downregulated that of the follistatin gene. Notably, follistatin re‐established BMP‐9‐induced ALP activity suppressed by IL‐1β. Activin A inhibited the expression of BMP‐9‐responsive genes and BMP‐9‐induced ALP activity, while follistatin re‐established them. Finally, extracellular signal‐regulated kinase 1/2 (ERK1/2), p38, and nuclear factor‐kappa B (NF‐κB) inhibition significantly blocked IL‐1β‐induced activin A gene expression. Our data indicate that IL‐1β inhibits BMP‐9‐induced osteoblastic differentiation of hPDLFs, possibly by promoting activin A production via the ERK1/2, p38, and NF‐κB pathways.

Periodontal disease is a chronic inflammatory disease of the periodontal tissue. The periodontal inflamed surface area (PISA) is a proposed index for quantifying the inflammatory burden resulting from periodontitis lesions. This study aimed to investigate longitudinal changes in the periodontal status as evaluated by the PISA following the active periodontal treatment. To elucidate the prognostic factors of PISA, mixed-effect modeling was performed for clinical parameters, tooth-type, and levels of periodontal pathogens as independent variables. One-hundred-twenty-five patients with chronic periodontitis who completed the active periodontal treatment were followed-up for 24 months, with evaluations conducted at 6-month intervals. Five-times repeated measures of mean PISA values were 130+/−173, 161+/−276, 184+/−320, 175+/−417, and 209+/−469 mm². Changes in clinical parameters and salivary and subgingival periodontal pathogens were analyzed by mixed-effect modeling. Plaque index, clinical attachment level, and salivary levels of Porphyromonas gingivalis were associated with changes in PISA at the patient- and tooth-level. Subgingival levels of P. gingivalis and Prevotella intermedia were associated with changes in PISA at the sample site. For most patients, changes in PISA were within 10% of baseline during the 24-month follow-up. However, an increase in the number of bleeding sites in a tooth with a deep periodontal pocket increased the PISA value exponentially.

The periodontal inflamed surface area (PISA) is a useful index for clinical and epidemiological assessments, since it can represent the inflammation status of patients in one contentious variable. However, calculation of the PISA is difficult, requiring six point probing depth measurements with or without bleeding on probing on 28 teeth, followed by data input in a calculation program. More simple methods are essential for screening periodontal disease or in epidemiological studies. In this study, we tried to establish a convenient partial examination method to estimate PISA. Cross-sectional data of 254 subjects who completed active periodontal therapy were analyzed. Teeth that represent the PISA value were selected by an item response theory approach. The maxillary second molar, first premolar, and lateral incisor and the mandibular second molar and lateral incisor were selected. The sum of the PISAs of these teeth was significantly correlated with the patient’s PISA (R² = 0.938). More simply, the sum of the maximum values of probing pocket depth with bleeding for these teeth were also significantly correlated with the patient’s PISA (R² = 0.6457). The simple model presented in this study may be useful to estimate PISA.

Aim: To clinically and histologically evaluate in dogs the healing of gingival recessions treated with coronally advanced flap (CAF) with or without cross‐linked hyaluronic acid (HA).Materials and methods: Gingival recession defects were surgically created on the vestibular side of both maxillary canines in 8 dogs. After 8 weeks of plaque accumulation, the 16 chronic defects were randomly treated with either CAF alone or CAF and HA‐gel (CAF/HA). Clinical and histological outcomes were evaluated at 10 weeks post‐surgically.Results: Compared to baseline, the clinical measurements at 10 weeks revealed a statistically significant decrease in gingival recession for both CAF (p < 0.01) and CAF/HA (p < 0.001) groups. Statistically significant differences were found in clinical attachment level (p < 0.05) and width of gingival recession (p < 0.01) favouring the CAF/HA group. Bone formation was statistically significantly greater in the CAF/HA group than in the CAF group (1.84 ± 1.16 mm vs., 0.72 ± 0.62 mm, respectively, p < 0.05). Formation of cementum and connective tissue attachment were statistically significantly higher in the CAF/HA group compared with the CAF group (i.e. 4.31 ± 1.78 mm versus 2.40 ± 1.35 mm and 1.69 ± 0.98 mm versus 0.74 ± 0.68 mm, respectively (p < 0.05)).Conclusions: The present data have for the first time provided histologic evidence for periodontal regeneration of gingival recession defects following treatment with CAF and HA.Clinical relevance: The use of HA in conjunction with CAF may represent a novel modality for treating gingival recession defects.

Periodontal examination data have a complex structure. For epidemiological studies, mass screenings, and public health use, a simple index that represents the periodontal condition is necessary. Periodontal indices for partial examination of selected teeth have been developed. However, the selected teeth vary between indices, and a justification for the selection of examination teeth has not been presented. We applied a graded response model based on the item response theory to select optimal examination teeth and sites that represent periodontal conditions. Data were obtained from 254 patients who participated in a multicenter follow-up study. Baseline data were obtained from initial follow-up. Optimal examination sites were selected using item information calculated by graded response modeling. Twelve sites—maxillary 2nd premolar (palatal-medial), 1st premolar (palatal-distal), canine (palatal-medial), lateral incisor (palatal-central), central incisor (palatal-distal) and mandibular 1st premolar (lingual, medial)—were selected. Mean values for clinical attachment level, probing pocket depth, and bleeding on probing by full mouth examinations were used for objective variables. Measuring the clinical parameters of these sites can predict the results of full mouth examination. For calculating the periodontal index by partial oral examination, a justification for the selection of examination sites is essential. This study presents an evidence-based partial examination methodology and its modeling.

Aim: To evaluate the effects of low‐intensity pulsed ultrasound (LIPUS) with/without intra‐marrow perforation (IMP) on periodontal healing in two‐wall intra‐bony defects in dogs.Materials and Methods: Two‐wall intra‐bony defects (5 mm wide, 5 mm deep) were created at the distal and mesial aspects of mandibular premolars in four beagle dogs (four defects per dog). The 16 defects were divided into four treatment groups: IMP, LIPUS, IMP + LIPUS (IMP/LIPUS) and control (open flap debridement). The LIPUS and IMP/LIPUS sites received daily LIPUS exposure for 3 weeks starting 1 week after surgery. The animals were euthanized 4 weeks after surgery for histologic evaluation.Results: There was significantly greater new bone formation at LIPUS (2.93 ± 0.74 mm) and IMP/LIPUS (3.18 ± 0.52 mm) sites than at control sites (1.65 ± 0.46 mm). New bone area at LIPUS (6.36 ± 2.28 mm²) and IMP/LIPUS (6.13 ± 1.25 mm²) sites was significantly greater than that at control sites (2.15 ± 1.75 mm²). New cementum length at LIPUS sites (4.09 ± 0.75 mm) was significantly greater than that at control (2.29 ± 1.02 mm) and IMP (2.41 ± 0.41 mm) sites. No significant difference was observed between LIPUS and IMP/LIPUS sites in any histomorphometric parameter.Conclusions: These findings suggest that LIPUS effectively promotes periodontal regeneration in two‐wall intra‐bony defects in dogs.