Katsumasa Maeda

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.

Periodontal tissue regeneration using fibroblast growth factor -2:Randomized controlled phase II clinical trial

Background The options for medical use of signaling molecules as stimulators of tissue regeneration are currently limited. Preclinical evidence suggests that fibroblast growth factor (FGF)-2 can promote periodontal regeneration. This study aimed to clarify the activity of FGF-2 in stimulating regeneration of periodontal tissue lost by periodontitis and to evaluate the safety of such stimulation. Methodology/Principal Findings We used recombinant human FGF-2 with 3% hydroxypropylcellulose (HPC) as vehicle and conducted a randomized double-blinded controlled trial involving 13 facilities. Subjects comprised 74 patients displaying a 2- or 3-walled vertical bone defect as measured ≥3 mm apical to the bone crest. Patients were randomly assigned to 4 groups: Group P, given HPC with no FGF-2; Group L, given HPC containing 0.03% FGF-2; Group M, given HPC containing 0.1% FGF-2; and Group H, given HPC containing 0.3% FGF-2. Each patient underwent flap operation during which we administered 200 µL of the appropriate investigational drug to the bone defect. Before and for 36 weeks following administration, patients underwent periodontal tissue inspections and standardized radiography of the region under investigation. As a result, a significant difference (p = 0.021) in rate of increase in alveolar bone height was identified between Group P (23.92%) and Group H (58.62%) at 36 weeks. The linear increase in alveolar bone height at 36 weeks in Group P and H was 0.95 mm and 1.85 mm, respectively (p = 0.132). No serious adverse events attributable to the investigational drug were identified. Conclusions Although no statistically significant differences were noted for gains in clinical attachment level and alveolar bone gain for FGF-2 groups versus Group P, the significant difference in rate of increase in alveolar bone height (p = 0.021) between Groups P and H at 36 weeks suggests that some efficacy could be expected from FGF-2 in stimulating regeneration of periodontal tissue in patients with periodontitis. Trial Registration ClinicalTrials.gov NCT00514657

Molecules interacting with PRIP-2, a novel Ins(1,4,5)P3 binding protein type 2: Comparison with PRIP-1

A family of phospholipase C-related, catalytically inactive proteins (designated PRIP) have been identified as a group of novel inositol 1,4,5-trisphosphate binding proteins with a domain organization similar to phospholipase C-delta but lacking the enzymatic activity. The PRIP family consists of at least two types of proteins (PRIP-1 and PRIP-2 subfamilies). In the present study, we examined the tissue distribution of PRIP-2, its expression in rat brain at the mRNA level, and the characteristics of its binding to inositol compounds, protein phosphatase 1, and gamma-amino butyric acid receptor associated protein. We also compared these characteristics with those of PRIP-1. Northern blot analysis and reverse-transcription polymerase chain reaction showed that PRIP-1 was present mainly in the brain, whereas PRIP-2 was expressed ubiquitously. In situ hybridization studies using rat brain revealed that the mRNA for both PRIP-1 and PRIP-2 was similarly expressed; it was detected in the granular cell and Purkinje cell layers in the cerebellum, and in the hippocampal pyramidal cells, dentate granule cells, and pyramidal and/or granule cells of the cerebral cortex in the cerebrum. PRIP-2 bound inositol 1,4,5-trisphosphate and its parent lipid, phosphatidylinositol 4,5-bisphosphate, with a similar affinity, while PRIP-1 preferentially bound the former ligand by about 10-fold. PRIP-1 and PRIP-2 interacted with protein phosphatase 1 and gamma-amino butyric acid receptor associated protein in a similar manner. These results indicate that, similar to PRIP-1, PRIP-2 may be involved in both inositol 1,4,5-trisphosphate-mediated and gamma-amino butyric acid-related signaling.

Extracellular matrix regulates induction of alkaline phosphatase expression by ascorbic acid in human fibroblasts.

During wound healing and inflammation, fibroblasts express elevated alkaline phosphatase (ALP), but are not in contact with collagen fibrils in the fibronectin (FN)‐rich granulation tissue. We hypothesized that the extracellular matrix (ECM) environment might influence the induction of ALP in fibroblasts. Here we tested this hypothesis by studying the ALP‐inductive response of normal human gingival fibroblasts to ascorbic acid (AsA). AsA induced ALP activity and protein in cells in conventional monolayer culture. This induction was inhibited by blocking‐antibodies to the FN receptor α5β1 integrin and by the proline analog 3,4‐dehydroproline (DHP). DHP prevented cells from arranging FN fibrils into a pericellular network and reduced the activity of cell spreading on FN. Plating of cells on FN facilitated the up‐regulation by AsA of ALP expression, but did not substitute for AsA. In contrast, AsA did not cause ALP induction in cells cultured on and in polymerized type I collagen gels. Collagen fibrils inhibited the up‐regulation by AsA of ALP expression in cells plated on FN. These results indicate that the ECM regulates the induction of ALP expression by AsA in fibroblasts: FN enables them to express ALP in response to AsA through interaction with integrin α5β1, whereas type I collagen fibrils cause the suppression of ALP expression and overcome FN.

Taste responses in mice lacking taste receptor subunit T1R1

•  The taste receptor heterodimer T1R1 + T1R3, metabotropic glutamate receptors (mGluRs) and/or their variants may function as umami taste receptors. •  Here, we used newly developed T1R1−/− mice and examined the role of T1R1 and mGluRs in taste detection. •  The T1R1−/− mice exhibited seriously diminished synergistic responses to glutamate and inosine monophosphate but not to glutamate alone and significantly smaller responses to sweeteners. •  Addition of mGluR antagonists significantly inhibited responses to glutamate in both T1R1−/− and heterozygous T1R1+/− mice. •  Taken together, these results suggest that T1R1 mainly contributes to umami taste synergism and partly to sweet sensitivity, while mGluRs are involved in the detection of umami compounds.

Bone-like nodules formed in vitro by rat periodontal ligament cells

The periodontal ligament has been shown to possess the ability to regenerate both new cementum and alveolar bone as well as a self-regenerative capacity; however, the source of cementoblasts and osteoblasts is not still clear. We investigated the development of bone-like tissue in vitro by periodontal ligament cells, in order to determine whether the periodontal ligament contains osteoprogenitor cells. Periodontal ligament cells were obtained from periodontal ligament tissue attached to the maxillary incisors of 6-week-old WKA rats by means of the explant technique. Cells at passage #3 were cultured for long term in α-minimum essential medium containing 10% fetal bovine serum, antibiotics, and 50 μg/ml ascorbic acid, and were then examined using phase-contrast microscopy, histochemistry, transmission electron microscopy, X-ray microanalysis, and electron diffraction. Nodules were formed in the cultures, and when 10 mM Na-β-glycerophosphate was added, these nodules became mineralized. The mineralized nodules were identified as bone-like elements in view of the presence of osteoblast-like and osteocyte-like cells, collagenous matrix, a mineral composed of hydroxyapatite, and intense alkaline phosphatase activity. The results show that the periodontal ligament contains osteoprogenitor cells, which differentiate into osteoblasts and produce bone-like tissue.

Specific immunocytochemical localization of cathepsin E at the ruffled border membrane of active osteoclasts

The immunocytochemical localization of cathepsin E, a non-lysosomal aspartic proteinase, was investigated in rat osteoclasts using the monospecific antibody to this protein. At the light-microscopic level, the preferential immunoreactivity for cathepsin E was found at high levels in active osteoclasts in the physiological bone modeling process. Neighboring osteoblastic cells were devoid of its immunoreactivity. At the electron-microscopic level, cathepsin E was exclusively confined to the apical plasma membrane at the ruffled border of active osteoclasts and the eroded bone surface. Cathepsin E was also concentrated in some endocytotic vacuoles of various sizes in the vicinity of the ruffled border membrane, some of which appeared to be secondary lysosomes containing the phagocytosed materials. These results strongly suggest that this enzyme is involved both in the extracellular degradation of the bone organic matrix and in the intracellular breakdown of the ingested substances in osteoclasts.

Differentiation-Inducing Factor-1 Alters Canonical Wnt Signaling and Suppresses Alkaline Phosphatase Expression in Osteoblast-Like Cell Lines

Because DIF‐1 has been shown to affect Wnt/β‐catenin signaling pathway, the effects of DIF‐1 on osteoblast‐like cell lines, SaOS‐2 and MC3T3‐E1, were examined. We found that DIF‐1 inhibited this pathway, resulting in the suppression of ALP promoter activity through the TCF/LEF binding site.

Histologic evaluation of tetracalcium phosphate-based cement as a direct pulp-capping agent

Histologic healing processes were observed at 1, 3, 7, and 10 days after application with either tetracalcium phosphate cement or calcium hydroxide cement to the exposed pulp of the rat maxillary incisors. In teeth applied with calcium hydroxide cement, necrotic tissue was present beneath the cement before new hard tissue formed. In contrast, tetracalcium phosphate cement elicited a dentine bridge formation with no evidence of either intervening tissue necrosis or marked inflammation. Furthermore on ultrastructural examination the newly formed hard tissue was in direct contact with the material. This study suggests that 4CP cement possesses a biocompatible property, which indicates its potential for use as a direct pulp-capping agent.

An analogue of lipid A and LPS from Rhodobacter sphaeroides inhibits neutrophil responses to LPS by blocking receptor recognition of LPS and by depleting LPS-binding protein in plasma.

When incubated with lipopolysaccharide (LPS) in the presence of plasma, neutrophils become primed for enhanced release of superoxide in response to triggering by formyl‐Met‐Leu‐Phe (fMLP). The effect of LPS on phagocytes is inhibited by a synthetic lipid A precursor, LA‐14‐PP (lipid IVa) or by LPS from Rhodobacter sphaeroides (Rs). We studied the mechanisms by which LA‐14‐PP or Rs‐LPS inhibited LPS‐induced responses. When neutrophils were exposed to LA‐14‐PP or Rs‐LPS for 3 min and then to Escherichia coli‐LPS, the antagonists inhibited priming for superoxide release, and also blocked up‐regulation of CD11b and adherence. This inhibition was dependent on plasma, was not overcome by higher amounts of E. coli‐LPS or plasma, and was not observed at 0°C, suggesting that E. coli‐LPS was not able to interact with its receptor or other cellular recognition molecule in neutrophils that had been exposed to the antagonists. The alternative possibility that LA‐14‐PP or Rs‐LPS depleted a plasma cofactor, resulting in inhibition of priming, was investigated by using LPS from Porphyromonas gingivalis (Pg) and Bordetella pertussis (Bp). These LPS primed neutrophils in a plasma‐dependent and CD14‐dependent manner, but were not blocked by LA‐14‐PP or Rs‐LPS. When sub‐optimal concentrations of plasma were exposed to LA‐14‐PP or Rs‐LPS, and then mixed with Pg‐IPS or Bp‐LPS, followed by incubation with neutrophils, priming and up‐regulation of GD11b were inhibited, and this inhibition was overcome by increasing the concentration of plasma. Binding of LPS‐binding protein (LBP) in plasma to immobilized E. coli‐LPS was inhibited by pre‐incubation of plasma with LA‐14‐PP or Rs‐LPS. Together with the result that treatment of plasma with anti‐LBP antibody abolished the cofactor activity of plasma, these results indicated that LA‐14‐PP and Rs‐LPS depleted LBP from plasma, resulting in inability of LPS to act on neutrophils. Thus LA‐14‐PP and Rs‐LPS inhibited the action of LPS on neutrophils by at least two mechanisms, blocking of LPS receptor recognition and depletion of the cofactor LBP.