Akifumi Akamine

Dentin regeneration by dental pulp stem cell therapy with recombinant human bone morphogenetic protein 2

Regenerative medicine is based on stem cells, signals, and scaffolds. Dental pulp tissue has the potential to regenerate dentin in response to noxious stimuli, such as caries. The progenitor/stem cells are responsible for this regeneration. Thus, stem cell therapy has considerable promise in dentin regeneration. Culture of porcine pulp cells, as a three-dimensional pellet, promoted odontoblast differentiation compared with monolayers. The expression of dentin sialophosphoprotein (Dspp) and enamelysin/matrix metalloproteinase 20 (MMP20) mRNA confirmed the differentiation of pulp cells into odontoblasts and was stimulated by the morphogenetic signal, bone morphogenetic protein 2 (BMP2). Based on the in vitro experiments, an in vivo evaluation of pulp progenitor/stem cells in the dog was performed. The autogenous transplantation of the BMP2-treated pellet culture onto the amputated pulp stimulated reparative dentin formation. In conclusion, BMP2 can direct pulp progenitor/stem cell differentiation into odontoblasts and result in dentin formation.

Expression of growth/differentiation factor 11, a new member of the BMP/TGFβ superfamily during mouse embryogenesis

We have cloned and characterized a new member of the bone morphogenetic protein/transforming growth factor beta (BMP/TGFbeta) superfamily, growth differentiation factor 11 (Gdf11), from rat incisor pulp RNA by reverse transcription-polymerase chain reaction using degenerate primers. The mature carboxyl-terminal domain encoded by Gdf11 is most closely related to Gdf8, being 90% identical to the mouse gene. Northern blot analysis revealed Gdf11 is expressed in adult dental pulp and brain. In situ hybridization of sections and whole-mount embryos demonstrated Gdf11 is first strongly expressed in restricted domains at 8.5 days post coitus (dpc) when it is highest in the tail bud. At 10.5 dpc, it is expressed in the branchial arches, limb bud, tail bud and posterior dorsal neural tube. Later, it is expressed in terminally-differentiated odontoblasts, the nasal epithelium, retina and specific regions of the brain.

Induction of Reparative Dentin Formation by Ultrasound-Mediated Gene Delivery of Growth/Differentiation Factor 11

Bone morphogenetic proteins (BMPs) are morphogens implicated in embryonic and regenerative odontogenic differentiation. Gene therapy has the potential to induce reparative dentin formation for potential pulp capping. We have optimized the gene transfer of Growth/differentiation factor 11 (Gdf11)/Bmp11 plasmid DNA into dental pulp stem cells by sonoporation in vivo. Dental pulp tissue treated with plasmid pEGFP or CMV-LacZ in 5-10% Optison (Molecular Biosystems Inc., San Diego, CA) and stimulated by ultrasound (1 MHz, 0.5 W/cm(2), 30 sec) showed significant efficiency of gene transfer and high level of protein production selectively in the local region, within 500 microm of the amputated site of the pulp tissue. The Gdf11 cDNA plasmid transferred into dental pulp tissue by sonoporation in vitro, induced the expression of dentin sialoprotein (Dsp), a differentiation marker for odontoblasts. The transfection of Gdf11 by sonoporation stimulated a large amount of reparative dentin formation on the amputated dental pulp in canine teeth in vivo. These results suggest the possible use of BMPs using ultrasound-mediated gene therapy for endodontic dental treatment.

Water absorption of poly(methyl methacrylate) containing 4-methacryloxyethyl trimellitic anhydride

The amount of water absorption of poly(methyl methacrylate) (PMMA) containing 0, 1, 3 and 5 wt% of an adhesive monomer, 4-methacryloxyethyl trimellitic anhydride (4-META), was measured at 7 degrees C, 37 degrees C and 60 degrees C. After the water uptake reached equilibrium in specimens, they were desorbed to obtain a constant value and the absorption process was repeated. Mass changes in the second desorption were recorded for the storage temperatures of 37 degrees C and 60 degrees C. Multiple regression analyses were conducted on three independent variables, 4-META concentration, storage temperature and absorption-desorption cycle. A statistically significant relationship was found between the maximum water uptake and 4-META concentration, while there was no relationship between the maximum water uptake and diffusion coefficient obtained using the Ficks law. The negative relationship in the latter did not support the free space theory. The significant and positive relationship between the maximum water uptake and 4-META concentration demonstrates that water molecules diffuse through the formation of a hydrogen bond at polar sites. The maximum water uptake was not influenced by temperature, while the diffusion coefficient increased with the rise in temperature. The activation energy was 41-47 and 50-53 kJ/mol in the first and second absorption tests, respectively.

Composite resin restoration and postoperative sensitivity: clinical follow-up in an undergraduate program

OBJECTIVES To analyze the relationship between the cavity depth and liners with postoperative sensitivity of resin composite restorations. METHODS A clinical follow-up was conducted on 319 resin composite restorations made in the final year of an undergraduate program over a 3-year period. Along with the analyses of cavity type, cavity depth, type of pulpal protection and the materials used, the postoperative sensitivity was also examined on each restoration. RESULTS Thirty-nine percent of the restorations had no protective layer (Group 1). As the depth of the prepared cavities increased, the restorations received one of the three pulpal protection methods; a calcium hydroxide base (Group 2), glass ionomer cement (Group 3), or protection with a calcium hydroxide base in combination with glass ionomer cement (Group 4). The incidence of postoperative sensitivity showed no significant difference among Groups 1, 2 and 3, but was significantly lower in Group 1 than in Group 4. The restorations made in shallow and medium depth cavities demonstrated significantly less-postoperative sensitivity than those made in deep cavities. The newer generation dentine-bonding agents showed a significantly lower incidence of postoperative sensitivity than the early generation group. CONCLUSIONS Postoperative sensitivity in resin composite restorations was not related to the absence of protective layers but increased with the depth of cavities restored with the resin composite. The type of dentine-bonding agents could also be responsible for postoperative sensitivity.

Investigating a clonal human periodontal ligament progenitor/stem cell line in vitro and in vivo

The lifespan of the tooth is influenced by the periodontal ligament (PDL), a specialized connective tissue that connects the cementum with the tooth socket bone. Generation of a cell line from PDL progenitor/stem cells would allow development of tissue engineering‐based regenerative PDL therapy. However, little is known about the characteristics of PDL progenitor/stem cells because PDL tissue consists of a heterogeneous cell population and there are no pure PDL cell lines. Recently, we succeeded in immortalizing primary human PDL fibroblasts (HPLFs) by transfecting them with SV40 T‐antigen and hTERT (Cell Tissue Res 2006; 324: 117–125). In this study, we isolated three clonal cell lines from these immortalized cells (lines 1–4, 1–11, and 1–24) that express RUNX‐2, Col I, ALP, OPN, OCN, RANKL, OPG, scleraxis, periostin, Col XII, and α‐SMA mRNA. Immunocytochemical analysis demonstrated that CD146 was expressed in cell lines 1–4 and 1–11 and that STRO‐1 was expressed in lines 1–11 and 1–24. Lines 1–4 and 1–11 differentiated into osteoblastic cells and adipocytes when cultured in lineage‐specific differentiation media. Four weeks after transplanting cell line 1–11 into immunodeficient mice with β‐tricalcium phosphate (β‐TCP), the transplant produced cementum/bone‐like tissues around the β‐TCP. Eight weeks after transplantation, the 1–11 cell transplant formed PDL‐like structures on the surface of the β‐TCP. These data suggest that cell line 1–11 was derived from a progenitor/stem cell present in the PDL and should be very useful for studying the biology and regeneration of human periodontium. J. Cell. Physiol. 215: 743–749, 2008.

Construction and characterization of a nonpigmented mutant of Porphyromonas gingivalis: cell surface polysaccharide as an anchorage for gingipains

A nonpigmented mutant of Porphyromonas gingivalis was constructed by using transposon mutagenesis. The mutant possessed the transposon DNA at the novel gene porR. Gene targeted mutagenesis revealed that porR was responsible for pigmentation. The porR gene shared similarities with genes of the degT family, the products of which are now considered to be transaminases involved in biosynthesis of sugar portions of cell-surface polysaccharides and aminoglycosides. The porR mutant showed a pleiotropic phenotype: delayed maturation of fimbrillin, preferential presence of Rgp and Kgp proteinases in culture supernatants, and no haemagglutination. The porR mutant had altered phenol extractable polysaccharide compared to the porR(+) sibling strain. A mAb, 1B5, that reacts with sugar portions of P. gingivalis cell surface polysaccharide and membrane-type Rgp proteinase showed no reaction with the cell lysates of the porR mutant. These results indicate that porR is involved in biosynthesis of cell surface polysaccharide that may function as an anchorage for Rgp, Kgp, haemagglutinins and the haemoglobin receptor protein.

Mineral Trioxide Aggregate Induces Bone Morphogenetic Protein-2 Expression and Calcification in Human Periodontal Ligament Cells

INTRODUCTION Mineral trioxide aggregate (MTA) is a therapeutic, endodontic repair material that is reported to exhibit calcified tissue-conductive activity although the mechanisms remain unclear. We hypothesize that the dissolution of calcium from MTA into the surrounding environment may play an important role in the osteoblastic/cementoblastic differentiation of human periodontal ligament cells (HPLCs). METHODS Two populations of HPLCs were obtained from two patients, respectively, and were cultured in the presence or absence of MTA discs and/or CaCl(2) in order to investigate calcium release, calcification activity, calcium-sensing receptor (CaSR) gene expression and bone morphogenetic protein-2 (BMP-2), and BMP-2 receptor protein and gene expression. RESULTS MTA released a substantial accumulation of calcium (4 mmol/L) within 14 days into culture media. After 4 weeks, the two populations of HPLCs independently exhibited calcification as well as BMP-2 distribution in the vicinity of MTA. HPLCs inherently expressed genes encoding for the CaSR and BMP-2 receptors. Exogenous CaCl(2) media supplementation induced CaSR gene expression in HPLCs and calcification and BMP-2 synthesis throughout the entire HPLC cultures, whereas MgCl(2) had no effect. Both MTA and CaCl(2) stimulated BMP-2 gene expression above that of baseline levels. CONCLUSION Here we show the first report showing that HPLCs cocultured directly with MTA up-regulated BMP2 expression and calcification. These results may be through CaSR interactions that were potentially activated by the release of calcium from MTA into the culture environment.

Visualization of Irrigant Flow and Cavitation Induced by Er:YAG Laser within a Root Canal Model

INTRODUCTION Laser-activated irrigation (LAI) has recently been introduced as an innovative method for root canal irrigation. However, there is limited information about the cleaning mechanism of an Er:YAG laser. In this study, we visualized the action of laser-induced bubbles and fluid flow in vitro to better understand the physical mechanisms underlying LAI. METHODS An Er:YAG laser was equipped with a novel cone-shaped tip with a lateral emission rate of approximately 80%. Laser light was emitted at a pulse energy of 30, 50, or 70 mJ (output energy: 11, 18, or 26 mJ) and a repetition rate of 1 or 20 pulses per second, without air or water spray. Fluid flow dynamics in a root canal model were observed by using glass-bead tracers under a high-speed camera. Moreover, laser-induced bubble patterns were visualized in both free water and the root canal model. RESULTS Tracers revealed high-speed motion of the fluid. A full cycle of expansion and implosion of vapor and secondary cavitation bubbles were clearly observed. In free water, the vapor bubble expanded for 220 microseconds, and its shape resembled that of an apple. In the root canal model, the vapor bubble expanded in a vertical direction along the canal wall, and bubble expansion continued for ≥700 microseconds. Furthermore, cavitation bubbles were created much more frequently in the canal model than in free water. CONCLUSIONS These results suggest that the cleaning mechanism of an Er:YAG laser within the root canal might depend on rapid fluid motion caused by expansion and implosion of laser-induced bubbles.

Effects of TGF-β1 on the proliferation and differentiation of human periodontal ligament cells and a human periodontal ligament stem/progenitor cell line

Periodontal ligament (PDL) is a specialized connective tissue that influences the lifespan of the tooth. Transforming growth factor-β1 (TGF-β1) is a multifunctional cytokine, but little is known about the effects of TGF-β1 on PDL cells. Our aim has been to demonstrate the expression of TGF-β1 in rat PDL tissues and to evaluate its effects on the proliferation and gene expression in human PDL cells (HPLCs) and a human PDL stem/progenitor cell line, line 1-11, that we have recently developed. The expression of TGF-β1 in the entire PDL tissue was confirmed immunohistochemically, and both HPLCs and cell line 1-11 expressed mRNA from the TGF-β1, TGF-β type I receptor, and TGF-β type II receptor genes. Although exogenous TGF-β1 stimulated the proliferation of HPLCs, it did not upregulate the expression of alpha-smooth muscle actin (α-SMA), type I collagen (Col I), or fibrillin-1 (FBN1) mRNA or of α-SMA protein in HPLCs, whereas expression for these genes was attenuated by an anti-TGF-β1 neutralizing antibody. In contrast, exogenous TGF-β1 reduced the proliferation of cell line 1-11, although it upregulated the expression of α-SMA, Col I, and FBN1 mRNA and of α-SMA protein in this cell line. In addition, interleukin-1 beta stimulation significantly reduced the expression of TGF-β1 mRNA and protein in HPLCs. Thus, TGF-β1 seems to play an important role in inducing fibroblastic differentiation of PDL stem/progenitor cells and in maintaining the PDL apparatus under physiological conditions.