Kenichi Matsuzaka

Prevention of biofilm formation on titanium surfaces modified with conjugated molecules comprised of antimicrobial and titanium-binding peptides

Specific binding of antimicrobial peptides to titanium (Ti) surfaces may serve to prevent biofilm formation, leading to a reduction in peri-implantitis. This study evaluated the binding behavior of conjugated molecules consisting of antimicrobial and hexapeptidic Ti-binding peptides (minTBP-1) using the quartz crystal microbalance (QCM-D) technique, and investigated the effect of modification of Ti surfaces with these peptides on the bioactivity of Porphyromonas gingivalis. Four kinds of peptide were prepared: histatin 5 (DSHAKRHHGYKRKFHEKHHSHRGY), minTBP-1 + histatin 5 (RKLPDAPDSHAKRHHGYKRKFHEKHHSHRGY), lactoferricin (FQWQRNMRKVR), and minTBP-1 + lactoferricin (RKLPDAPGGFQWQRNMRKVR). The QCM-D analysis demonstrated that significantly larger increases in peptide adsorption were observed in the conjugated peptides than in antimicrobial peptides alone. In addition, ATP activity in P. gingivalis in peptide-modified specimens significantly decreased compared to that in the Ti control. These results indicate that surface modification with conjugated molecules consisting of antimicrobial and Ti-binding peptides is a promising method for reduction of biofilm formation on Ti surfaces.

Analysis of side population cells derived from dental pulp tissue

Kenmotsu M, Matsuzaka K, Kokubu E, Azuma T, Inoue T. Analysis of side population cells derived from dental pulp tissue. International Endodontic Journal. Aim To investigate the characteristics of side population (SP) cells derived from the dental pulp of young and aged rats. Methodology Maxillary and mandibular incisors were extracted from 5-week-old (young) rats and 60- to 80-week-old (aged) rats. Coronal pulp tissue was removed mechanically, and single-cell suspensions were prepared using collagenase and dispase. Cells were stained with Hoechst 33342 and sorted with an fluorescence-activated cell sorter (FACS). Isolated SP and main population (MP) cells were analysed by real-time reverse transcription polymerase chain reaction, immunohistochemical localization and cell cycle determination. Two-way analysis of variance and the multiple comparison Scheffè test were used for statistical analysis (P < 0.05). Results Approximately 0.40% of pulp cells in young rats and 0.11% in aged rats comprised SP cells. SP cells expressed a higher mRNA level of ATP-binding cassette transporter G2 (ABCG2), but lower mRNA levels of nestin, alkaline phosphatase, p16 and p57 than MP cells in both age groups. Immunohistochemical observation revealed ABCG2-positive cells localized in the cell-rich zone and nestin in the odontoblastic layer in both groups. Furthermore, the majority of both young and aged SP and MP cells were in growth arrest of the G0/G1 phase. Conclusion The FACS analysis revealed a decrease in the proportion of SP cells with age, whilst p16 mRNA expression indicated an increase in cell senescence. The cell cycles of SP and MP cells from both young and aged dental pulp were generally in the G0/G1 phase.

Characteristics of Newly Formed Bone During Guided Bone Regeneration: Analysis of cbfa-1, Osteocalcin, and VEGF Expression

This study investigated the expression of core-binding factor alpha-1 (cbfa-1), osteocalcin, and vascular endothelial growth factor (VEGF) relative to new bone formation during guided bone regeneration; cbfa-1 is a prerequisite transcription factor for osteoblastic differentiation. Osteocalcin, a bone-specific extracellular matrix protein, is a marker of mature osteoblasts, whereas VEGF, a mitogen for endothelial cells, is a polypeptide thought to stimulate new blood vessel formation. Membranes (expanded polytetrafluoroethylene) were applied to defects created in the left tibiae of rats, while right tibial defects remained uncovered as a control group. Animals were killed 6, 8, or 10 days later. The cbfa-1 was detected by immunohistochemistry, and reverse transcriptase-polymerase chain reaction was used to detect osteocalcin and VEGF. The ratio of cbfa-1 positive cells in experimental bone defects was higher than in the control group. Osteocalcin mRNA expression increased gradually in the control group but significantly in the experimental group over time. The VEGF mRNA expression in the experimental group at 10 days was significantly lower than in the control group. These findings suggested that osteogenic cells differentiated into osteoblasts in the membrane-covered defects and that the bone healing process would be completed at an early stage.

Low-level (gallium-aluminum-arsenide) laser irradiation of Par-C10 cells and acinar cells of rat parotid gland

We investigated cell response, including cell proliferation and expression of heat stress protein and bcl-2, to clarify the influence of low-level [gallium-aluminum-arsenide (Ga-Al-As) diode] laser irradiation on Par-C10 cells derived from the acinar cells of rat parotid glands. Furthermore, we also investigated amylase release and cell death from irradiation in acinar cells from rat parotid glands. The number of Par-C10 cells in the laser-irradiated groups was higher than that in the non-irradiated group at days 5 and 7, and the difference was statistically significant (P < 0.01). Greater expression of heat shock protein (HSP)25 and bcl-2 was seen on days 1 and 3 in the irradiated group. Assay of the released amylase showed no significant difference statistically between the irradiated group and the non-irradiated group. Trypan blue exclusion assay revealed that there was no difference in the ratio of dead to live cells between the irradiated and the non-irradiated groups. These results suggest that low-level laser irradiation promotes cell proliferation and expression of anti-apoptosis proteins in Par-C10 cells, but it does not significantly affect amylase secretion and does not induce rapid cell death in isolated acinar cells from rat parotid glands.

Morphological and molecular changes in denture-supporting tissues under persistent mechanical stress in rats

The purpose of this study was to determine the effects of mechanical compression on the palatal mucosa using an experimental palatal base. The palatal base was either pressed onto (stress group) or not pressed onto (fit group) rat palatal mucosa. Blood flow was measured and the animals were sacrificed 6-72 h later for analysis. The expression of heat shock protein 70 (HSP70), vascular endothelial growth factor (VEGF) and proliferation cell nuclear antigen (PCNA) was characterized by immunohistochemical staining. For morphometric analysis, connective tissues were divided into bone side and epithelial side tissues. The ratio of PCNA-positive cells (PCNA score) was calculated, and the expressions of mRNA encoding HSP70 and VEGF was evaluated. Whereas blood flow in the stress group showed ischaemia, none was found in the fit group. Proliferation cell nuclear antigen scores on the bone side were higher than on the epithelial side in the stress group (P < 0.05). Heat shock protein 70- and VEGF-positive cells were observed under compression conditions, particularly in the periosteum. In the stress group, the expressions of mRNA encoding HSP70 and VEGF were highest at 12 h (P < 0.05). These results suggest that mechanical compression of the palatal plate induces ischaemia, and that cells in the underlying denture-supporting tissue, which includes the periosteum, synthesize HSP70 and VEGF to maintain homeostasis under these conditions.

Effect of Stretching Force on the Cells of Epithelial Rests of Malassez In Vitro

Background and Objective. The aim of this study was to investigate the behavior of cells from epithelial rest of Malassez (ERM) against stretching force. Material and Methods. ERM-cultured cells were stretched for 1 hour, at the cycle of 18% elongation for 1 second followed by 1-second relaxation. The cells without addition of stretching force were used as controls. The cells were observed by immunohistochmical staining using actin 0, 12, 24, 36, 48, and 72 hours. Furthermore, expressions of HSP70-, VEGF-, and OPN-mRNAs of cells were also evaluated using quantitative RT-PCR. Results. Actin filaments were randomly orientated in the cytoplasm in the control group, whereas in the stretching group, actin filaments were orientated comparatively parallel to the stretching direction. Expression of HSP70-mRNA in the stretching group was significantly higher than that of control group at 12, 24, 36 hours (P < .05). Expression of VEGF-mRNA in the stretching group was significantly higher than that of control group at 24, 36, 48, and 72 hours (P < .05). Expression of OPN-mRNA in the stretching group was significantly higher than that of control group at 12 and 24 hours (P < .05). Conclusion. ERM cells response against the stretching force by expressing HSP70, VEGF, and OPN.

Influence of tooth-polishing pastes and sealants on DIAGNOdent values

OBJECTIVE The purpose of this study was to investigate the influence of tooth-polishing pastes and sealants on values obtained with a caries diagnostic system based on detection of caries-associated fluorescence (DIAGNOdent, KaVo). METHOD AND MATERIALS Ten tooth-polishing pastes and four dentifrices were measured alone, and it was shown that products containing pumice gave high DIAGNOdent values. The deepest occlusal pits of 20 extracted sound premolars (12 unsealed teeth, 8 sealed teeth) were measured before polishing, after rotating-brush polishing with pumice-containing tooth-polishing paste, and after final rotating-brush polishing with water in this in vitro study. In the clinical phase, the deepest occlusal pits of 21 molars and 2 premolars that clinically required application of sealant were measured before polishing, after rotating-brush polishing with one of two pumice-containing tooth-polishing pastes, and after sealant application. All measurements were done five times, and the average values were obtained. Data were statistically analyzed using analysis of variance subsequent to Fishers protected, least significant difference. RESULTS Clinical study showed that the value after polishing with either of the two polishing pastes was significantly higher than the value before polishing. Both in vitro and clinical studies showed that the value was increased by pumice-containing paste polishing and rotating-brush polishing with water after paste polishing could not recover the value to the level before paste polishing. Sealant treatment in the clinical study significantly decreased DIAGNOdent values, and the values after sealant application were lower than the values before polishing. CONCLUSION Polishing pastes and sealants used in this study could interfere with DIAGNOdent values.

Behavior of rat periodontal ligament cells on fibroblast growth factor-2-immobilized titanium surfaces treated by plasma modification

The aim of this study was to investigate the behavior of rat periodontal ligament (PDL) cells cultured on fibroblast growth factor-2 (FGF-2)-immobilized titanium surfaces treated by oxygen (O2) plasma. We used cell disks (15 mm in diameter), and 35-mm culture dishes sputter-coated with titanium. These were treated with oxygen plasma and dipped in FGF-2 solution. Immobilized FGF-2 was visualized with a confocal laser-scanning microscope, and its weight was calculated to be approximately 22.6 ng/cm(2) using a quartz crystal microbalance-dissipation apparatus. The PDL cells were obtained from rat incisors. Cells from fourth subculture were seeded onto the FGF-2-immobilized titanium surface. Proliferation ratio, alkaline phosphatase (ALP) activity, and expressions of type I collagen and vascular endothelial growth factor (VEGF) mRNAs were evaluated. Proliferation ratio and expressions of type I collagen and VEGF mRNAs were significantly higher, whereas ALP activity was significantly lower in FGF-2-immobilized cells than in control group (p < 0.05). These findings suggest that oxygen plasma modification can immobilize FGF-2 onto a titanium surface. Immobilized FGF-2, although inferior to culture medium with FGF-2, influenced the proliferation of PDL cells and might have promoted collagen and vascular synthesis.

Effect on the amount of bone-implant contact when splinting immediate-loaded dental implants.

Purpose:Much attention has been focused on the immediate or early loading of implants with or without splinting. The purpose of this study was to evaluate the contact rate between bones and implants, with or without splinting. Materials and Methods:Under general anesthesia, an 8-mm-deep cavity for a dental implant was drilled in the mandibular ridge of dogs where teeth had been extracted 4 months earlier. Rough-surfaced, cylindrical screw implants (International Team for Implantology [ITI] monotype implants 4 mm diameter and 8 mm long, Straumann, Basel, Switzerland) were placed with splinting on the right side and without splinting on the left side using gold abutment. Resin plates for the maxilla were adjusted to attach to the gold abutment in each mandible. At 4, 8, or 12 weeks after the implantation, specimens were stained using toluidine blue and fuchsin. The sections were observed and morphometric analysis was performed to measure the rates of bone-implant contact and new bone-implant contact. Results:The ratio of bone-implant contact on the lingual side was higher than on the buccal side in both the splinted and the unsplinted groups, and the rates in the splinted group were also higher than in the unsplinted group. The ratio of new bone-implant contact was not significantly different between the splinted and unsplinted groups, except for spongy bone at 4 weeks. Conclusion:Splinting of immediate-loading dental implants can be adequate for osseointegration, particularly in spongy bone.