Kenichi Matsuzaka

Background/Objectives: The optimal healing process following root canal treatment involves biological apical sealing through new cementum formation. Bone morphogenetic protein 7 (BMP-7) has recently gained attention as a potential regulator of cementoblast differentiation and periodontal regeneration. However, its effects on periodontal ligament fibroblasts (PDLFs) and the underlying mechanisms remain incompletely understood. This study aimed to investigate whether BMP-7 induces cementoblast-like differentiation of PDLFs both in vivo and in vitro via the BMP-SMAD signaling pathway. Methods: In a rat pulpectomy model, root canals were treated with or without BMP-7 and examined histologically and immunohistochemically for F-spondin (Spon1) expression. In vitro, human PDLFs were stimulated with BMP-7, and analyses of mineralization, cementoblast marker expression, alkaline phosphatase activity, and SMAD-1/5/9 phosphorylation were conducted. Results: Immunohistochemical analysis revealed that Spon1-positive regions increased around the apical area following BMP-7 treatment, suggesting the induction of cementoblast-like differentiation. In vitro, BMP-7 enhanced the expression of cementoblast-associated genes and mineral deposition while activating SMAD-1/5/9 signaling. Phosphorylation was suppressed by the BMP receptor inhibitor LDN-193189, indicating canonical BMP-SMAD pathway involvement. Conclusions: Although the specific concentration range of maximal activity remains to be determined, the findings collectively suggest that BMP-7 can promote cementoblast-like differentiation of PDLFs and may contribute to apical healing through cementum-related mechanisms. These results provide mechanistic and biological insights that support the potential of BMP-7 as a modulator for biologically favorable periapical tissue regeneration following root canal therapy.

The attachment of the oral epithelium to the abutment surface is crucial for the long-term success of dental implants. This study aimed to evaluate the attachment of human epithelial cells to anodized titanium surfaces. Anodized titanium discs were used as the experimental group, while machined titanium discs served as the control. Surface roughness and wettability were first measured for each group. Next, human epithelial cells were seeded onto each disc at a density of 4.0 × 10⁴ cells/cm² and evaluated 3, 6, and 24 h later for cell proliferation, as well as mRNA expression and protein levels of laminin and integrin β₄. Surface roughness was comparable between the two groups; however, wettability was significantly higher in the experimental group. Cell proliferation increased over time in both groups and showed no significant difference. Notably, the expression levels of both laminin and integrin β₄ were significantly higher in the experimental group at 24 h. Furthermore, protein localization of laminin and integrin β₄ was observed along the cell margins on the anodized surface. These findings suggest that anodization enhances epithelial cell attachment by promoting the expression and peripheral organization of key adhesion molecules.

A tricalcium-silicate-nanoparticle-containing cement (Biodentine) was developed to overcome the disadvantages of existing mineral trioxide aggregate (MTA) dental materials. This study aimed at evaluating the effects of Biodentine on the osteogenic differentiation of human periodontal ligament fibroblasts (HPLFs) in vitro and the healing of furcal perforations created experimentally in rat molars in vivo, in comparison to MTA. The in vitro studies performed the following assays: pH measurement using a pH meter, the release of calcium ions using a calcium assay kit, cell attachment and morphology using SEM, cell proliferation using a coulter counter, marker expression using quantitative reverse transcription polymerase chain reaction (qRT-PCR) and cell mineralized deposit formation using Alizarin Red S (ARS) staining. In the in vivo studies, MTA and Biodentine were used to fill the rat molar perforations. Rat molars were processed at 7, 14 and 28 days for analysis of inflammatory processes using hematoxylin and eosin (HE) staining, immunohistochemical staining of Runx2 and tartrate-resistant acid phosphate (TRAP) staining. The results demonstrate that the nanoparticle size distribution of Biodentine is critical for osteogenic potential at an earlier stage compared to MTA. Further studies are required to elucidate the mechanism of action of Biodentine in osteogenic differentiation.

A tricalcium silicate nanoparticle-containing cement (NPC) was developed to overcome the disadvantages of existing mineral trioxide aggregate (MTA) dental materials. This study aimed at evaluating the effects of NPC on the osteogenic differentiation of human periodontal ligament fibroblasts (HPLFs) in vitro, and on the healing of furcal perforations created experimentally in rat molars in vivo, in comparison to MTA. The in vitro studies performed the following assays; pH measurement using a pH meter, the release of calcium ions using a calcium assay kit, cell attachment and morphology using SEM, cell proliferation using a coulter counter, marker expression using quantitative reverse transcription polymerase chain reaction (qRT-PCR) and cell mineralized deposit formation using Alizarin Red S (ARS) staining. In the in vivo studies, MTA and NPC were used to fill the rat molar perforations. Rat molars were processed at 7, 14 and 28 days for analysis of inflammatory processes using hematoxylin and eosin (HE) staining, immunohistochemical staining of Runx2 and tartrate-resistant acid phosphate (TRAP) staining. The results demonstrate that the nanoparticle size distribution of NPC is critical for osteogenic potential at an earlier stage compared to MTA. Further studies are required to elucidate the mechanism of action of NPC in osteogenic differentiation.

The adhesion of zirconia and soft tissue is very important for the success of zirconia implants. The aim of this study was to characterize the effects of excimer laser treatment of zirconia on the adhesion of L929 fibroblasts. In this study, polished zirconia disks treated with an excimer laser were the experimental group and untreated zirconia disks were the control group. Surface roughness and contact angles of zirconia disks were measured. mRNA expression levels of integrin β1 and collagen type I α1 in L929 fibroblasts cultured on zirconia disks were measured using qRT-PCR. Cell morphology was evaluated using 3D laser microscopy and the expression of vinculin was characterized using confocal microscopy. There was no significant difference in the surface roughness of zirconia disks, but contact angles were significantly lower. mRNA expression of integrin β1 was significantly higher at 3, 6 and 24 h and of collagen type I α1 was significantly higher at 6 and 24 h. L929 fibroblasts tended to form elongated microspikes and vinculin colocalization in those microspikes. Furthermore, vinculin was strongly expressed in filopodia of L929 fibroblasts at 24 h. These results suggest that excimer laser treatment improves adhesion between zirconia disks and L929 fibroblasts.

Odontoblasts differentiate from dental papilla stem cells, but the genetic changes that occur during this process remain unclear. The aim of this study was to investigate gene expression patterns during differentiation of mouse iPS cells into odontoblast-like cells. Mouse iPS cells were cultured on a collagen type-1 scaffold with bone morphogenetic protein 4 (BMP4) and retinoic acid (RA). The results of immunofluorescence studies for dentin sialoprotein, dentin matrix protein 1 (DMP1), and nestin were positive. A qRT-PCR analysis revealed that mRNA expression levels of neural crest marker sex determining region Y box (Sox)-10, dentin sialophosphoprotein (Dspp), and Dmp1 were up-regulated, but that mRNA expression levels of the mineralization markers bone sialoprotein and osteocalcin were down-regulated. Microarray analysis showed that 2,597 entities were up-regulated and 1,327 down-regulated among a total of 15,330 investigated. Sox11 was among the up-regulated genes identified. The Sox11 mRNA expression level with odontoblast induction after day 11 was higher than that after day 2 (pSox11. The Dspp mRNA expression level in Sox11 siRNA-treated cells was significantly lower than that in the control (pSox11 is an important factor in odontoblastic differentiation.

Objectives: Epigenetic phenomena are changes in gene expression not involving the DNA sequence. DNA methylation is a major occurrence underlying epigenetic changes in human cells. Although aberrant DNA methylation is well documented in malignant lesions, limited information has been shown on the involvement of DNA methylation in oral lichen planus and oral lichenoid lesions (OLP). The present study aimed to investigate DNA methylation of E-cadherin and p16 in OLP, and compare the findings with those in non-inflamed gingiva (Non), radicular cyst (RC), and oral squamous cell carcinoma (SCC). Materials and methods: Paraffin-embedded surgical biopsy specimens were sliced, DNA was extracted, bisulfite treatment was applied, and methylation-specific polymerase chain reaction was performed. Immunohistochemistry was performed to observe the relative expression patterns of these genes. Results: E-cadherin was hypermethylated in OLP (p < 0.01), SCC (p < 0.01), and RC (p < 0.05), when compared with Non; DNA hypermethylation was confirmed in OLP and SCC when compared to Non and RC. Hypermethylation of p16(ink4a) was observed only in SCC (p < 0.01). Conclusion: DNA methylation levels of E-cadherin and p16(ink4a) were significantly higher in OLP than in normal tissues, and may be associated with the pathogenesis and progression of the disease.

Objectives: Transplantation of stem cells into wounds has become popular in regeneration therapies. As stem cells for transplantation, human dental pulp stem cells (hDPSCs) are known to be pluripotent cells that are relatively easy to collect from the pulp of deciduous or wisdom teeth. The purpose of this study was to investigate whether hDPSCs treated with fibroblast growth factor 7 (FGF7) would contribute to the regeneration of wounded rat submandibular glands (SMGs).Materials and methods: In in vitro studies, hDPSCs were treated with or without FGF7 and mRNA expression levels were examined at days 3, 7 and 14 using qRT‐PCR. The target genes analyzed were BMI1 as an undifferentiated marker, AQP5 as an acinar cell marker, CK19 as a ductal epithelial cell marker, αSMA as a myoepithelial cell marker and VIMENTIN as a fibroblast marker. In in vivo studies, hDPSCs treated with or without FGF7 for 14 days were mixed with type I collagen gels and were transplanted into wounded rat SMGs. Hematoxylin–Eosin and immunohistochemical staining were performed at days 3 and 7, and the numbers of positive cells were counted. The primary antibodies used were against BMI1, AQP5, αSMA, PanCK and VIMENTIN.Results: In the in vitro studies, mRNA levels of BMI1 were decreased and αSMA were increased at days 3, 7 and 14, while AQP5 was increased at day 14 in the FGF7 group. In the in vivo studies, the proliferation of hDPSCs and cell islands was observed at day 7 in the FGF7 group. Few BMI1‐positive cells were observed, while numbers of AQP5‐positive and αSMA‐positive cells were increased at days 3 and 7 in the FGF7 group. Moreover, cell islands were AQP5‐positive.Conclusion: These results suggest that FGF7‐treated hDPSCs differentiate into AQP5‐positive and αSMA‐positive cells. Moreover, AQP5‐positive cell aggregations were formed.