Ryuji Yamamoto

Micelle Structure of Amelogenin in Porcine Secretory Enamel

Even during the secretory stage of amelogenesis, enamel crystals thicken as amelogenins (the major protein component) decrease. To explain this phenomenon, we propose a model for amelogenin structure and function based upon the hypothesis that amelogenin forms micelles. Solubility and hydrophobicity analyses suggest that all but the hydrophilic amelogenin C-terminal regions aggregate via hydrophobic bonds to form a micelle core. Amelogenin micelles may form super-assemblies via their C-termini (KTKREEVD), which contain complementary positive (KTKR) and negative (EEVD) elements. Disassembly of the micelles through controlled proteolysis provides space for crystal growth. Initial cleavage (by enamelysin) removes the surface-accessible amelogenin C-terminus, exposing the middle portion to cleavage (by EMSP1). As a result, the 13-kDa amelogenin, a rod-shaped domain based upon ultrafiltration and transmission electron microscopy studies, is released. This model explains how amelogenin is able to ‘space’ and support the ribbon-like crystals and continuously yield space as the crystals thicken, until they are sufficiently mature to support themselves. Abbreviations: dentino-enamel junction (DEJ), sodium dodecyl sulfate (SDS), polyacrylamide gel electro phoresis (PAGE), transmission electron microscope (TEM).

Dentin Sialophosphoprotein–derived Proteins in the Dental Pulp

Porcine dentin sialophosphoprotein (DSPP), the most abundant noncollagenous protein in dentin, is critical for proper mineralization of tooth dentin. DSPP is processed by proteases into 3 major domains: dentin sialoprotein (DSP), dentin glycoprotein (DGP), and dentin phosphoprotein (DPP). There are at least 2 mRNA variants expressed from the Dspp gene: one encodes the full-length DSPP protein (DSP+DGP+DPP); the other encodes only DSP. The shorter transcript is generated through the use of a polyadenylation signal within intron 4, immediately following the DSP coding region (DGP and DPP are encoded by exon 5). We fractionated DSPP-derived proteins from the dental pulp of developing porcine incisors using heparin chromatography. DSP was identified, but little DPP could be detected in any fractions. BMP-1 digestion of DSPP-derived proteins extracted from dental pulp did not generate new DPP bands on sodium dodecyl sulfate–polyacrylamide gel electrophoresis (indicating an absence of intact DSPP), although the results suggested another BMP-1 cleavage site within DSP. We further purified DSPP-derived protein by reversed-phase high-performance liquid chromatography. Its amino acid composition was similar to DSP. Expression of the full-length Dspp mRNA by quantitative real-time polymerase chain reaction analysis was significantly higher in odontoblasts than in pulp, while expression of the DSP-only mRNA was almost equal in odontoblasts and in the body of the pulp. Expression of the full-length Dspp mRNA was also significantly higher than the expression of DSP-only mRNA in odontoblasts. Both the full-length and the DSP-only Dspp mRNA showed only trace expression in the pulp tip. We conclude that use of the 3′ polyadenylation signal in exon 5 predominates in fully differentiated odontoblasts, while both polyadenylation signals are used throughout odontoblast differentiation.

TGF-β1 autocrine signalling and enamel matrix components.

Transforming growth factor-β1 (TGF-β1) is present in porcine enamel extracts and is critical for proper mineralization of tooth enamel. Here, we show that the mRNA of latent TGF-β1 is expressed throughout amelogenesis. Latent TGF-β1 is activated by matrix metalloproteinase 20 (MMP20), coinciding with amelogenin processing by the same proteinase. Activated TGF-β1 binds to the major amelogenin cleavage products, particularly the neutral-soluble P103 amelogenin, to maintain its activity. The P103 amelogenin-TGF-β1 complex binds to TGFBR1 to induce TGF-β1 signalling. The P103 amelogenin-TGF-β1 complex is slowly cleaved by kallikrein 4 (KLK4), which is secreted into the transition- and maturation-stage enamel matrix, thereby reducing TGF-β1 activity. To exert the multiple biological functions of TGF-β1 for amelogenesis, we propose that TGF-β1 is activated or inactivated by MMP20 or KLK4 and that the amelogenin cleavage product is necessary for the in-solution mobility of TGF-β1, which is necessary for binding to its receptor on ameloblasts and retention of its activity.

A Large Chondroitin Sulfate Proteoglycan, Versican, in Porcine Predentin

Proteoglycans and their constituent glycosaminoglycan (GAG) have been proposed to be involved in the inhibition of mineralization in unmineralized tissue, predentin. Among the proteoglycans secreted by odontoblasts, we focused on the large chondroitin sulfate proteoglycan, versican, for its large binding capacity for calcium ions. The aims of this study were the determination of the full-length sequence and splicing variants of the porcine versican, and the detection of versican in the porcine predentin. The complete coding sequence of the porcine versican mRNA was cloned to be 11,775 nucleotides long and encode 3,924 amino acids, and four splicing variants, V0, V1, V2 and V3, were characterized in the isolated porcine cartilage cells. The number of potential GAG attachment sites was 15 in the V0 variant, 13 in the V1 variant, 2 in the V2 variant and 0 in the V3 variant. They were deposited in DDBJ. The V1 variant was determined by RT-PCR in the odontoblasts, dental papilla cells, dental follicle cells, periodontal ligament cells, dental pulp cells, and gingival cells of pigs, although a small amount of the V0 valiant was found in the dental papilla cells. The predentin was prepared from developing porcine permanent incisor tooth germs and its soluble proteins were extracted in order to be partially characterized by protein and proteinase profiles. The versican V1 cleavage products were detected in the predentin extract by Western blotting analysis. These results suggested that the versican splice variant V1 implicates both the control of the mineralization and the activities of the predentin metalloproteinases, because it has 13 GAG chains that bind a large amount of calcium.

The dynamics of TGF-β in dental pulp, odontoblasts and dentin

Transforming growth factor-beta (TGF-β) is critical for cell proliferation and differentiation in dental pulp. Here, we show the dynamic mechanisms of TGF-β in porcine dental pulp, odontoblasts and dentin. The mRNA of latent TGF-β1 and TGF-β3 is predominantly expressed in odontoblasts, whereas the mRNA expression level of latent TGF-β2 is high in dental pulp. TGF-β1 is a major isoform of TGF-β, and latent TGF-β1, synthesized in dental pulp, is primarily activated by matrix metalloproteinase 11 (MMP11). Activated TGF-β1 enhances the mRNA expression levels of MMP20 and full-length dentin sialophosphoprotein (DSPP) in dental pulp cells, coinciding with the induction of odontoblast differentiation. Latent TGF-β1 synthesized in odontoblasts is primarily activated by MMP2 and MMP20 in both odontoblasts and dentin. The activity level of TGF-β1 was reduced in the dentin of MMP20 null mice, although the amount of latent TGF-β1 expression did not change between wild-type and MMP20 null mice. TGF-β1 activity was reduced with the degradation of DSPP-derived proteins that occurs with ageing. We propose that to exert its multiple biological functions, TGF-β1 is involved in a complicated dynamic interaction with matrix metalloproteinases (MMPs) and/or DSPP-derived proteins present in dental pulp, odontoblasts and dentin.

Effect of Nerve Growth Factor on Osseointegration of Titanium Implants in Type 2 Diabetic Rats.

PURPOSE Compared with the general population, a poorer quality of bone-implant osseointegration occurs and at a higher failure rate in patients with type 2 diabetes mellitus. The aim of this study was to analyze the effects of local injection of nerve growth factor at the bone-implant interface after implantation in type 2 diabetic rats. MATERIALS AND METHODS Goto-Kakizaki (GK) rats (n = 30) were used as a model of type 2 diabetes mellitus, and Wistar rats were used as a control (n = 15). GK rats were assigned randomly into two groups (n = 15/group): the diabetes mellitus group (saline only) and the nerve growth factor group (received nerve growth factor treatment). One titanium implant was placed in each rats left tibia. Immediately postoperatively, nerve growth factor group rats were injected with nerve growth factor (0.4 μg/day) intramuscularly around the implant, daily for 7 days. Diabetes mellitus and control group rats received normal saline in an identical manner. Rats were sacrificed at 2, 4, and 8 weeks following implant surgery. RESULTS Traditional light and confocal laser scanning microscopy were used on nondecalcified sections to investigate fluorochrome labeling changes and histologic features of bone adjoining the implants. Bone-to-implant contact and bone volume percentage in the diabetes mellitus group were significantly less than in the control and nerve growth factor groups, with no statistically significant differences between the control and nerve growth factor groups. Confocal laser scanning microscopy showed a significant increase in marked bone around the nerve growth factor group implant at 4 weeks (P < .01) and 8 weeks (P < .05) compared with the diabetes mellitus group. CONCLUSION This study showed that local injection of nerve growth factor could improve implant-bone osseointegration in diabetic rats and may have important clinical implications.

TGF-β and Physiological Root Resorption of Deciduous Teeth

The present study was performed to examine how transforming growth factor β (TGF-β) in root-surrounding tissues on deciduous teeth regulates the differentiation induction into odontoclasts during physiological root resorption. We prepared root-surrounding tissues with (R) or without (N) physiological root resorption scraped off at three regions (R1–R3 or N1–N3) from the cervical area to the apical area of the tooth and measured both TGF-β and the tartrate-resistant acid phosphatase (TRAP) activities. The TGF-β activity level was increased in N1–N3, whereas the TRAP activity was increased in R2 and R3. In vitro experiments for the receptor activator of nuclear factor-κB (NF-κB) ligand (RANKL)-mediated osteoclast differentiation revealed that proteins from N1–N3 and R1–R3 enhanced the TRAP activity in RAW264 cells. A genetic study indicated that the mRNA levels of TGF-β1 in N1 and N2 were significantly increased, and corresponded with levels of osteoprotegerin (OPG). In contrast, the expression level of RANKL was increased in R2 and R3. Our findings suggest that TGF-β is closely related to the regulation of OPG induction and RANKL-mediated odontoclast differentiation depending on the timing of RANKL and OPG mRNA expression in the root-surrounding tissues of deciduous teeth during physiological root resorption.

Bone augmentation around a dental implant using demineralized bone sheet containing biologically active substances.

This study was designed to evaluate the volume of alveolar bone augmentation after immediate implant placement using demineralized bone. We examined the collagen matrix of demineralized bone and biologically active substances contained therein. Rat maxillary first molars were extracted, and the animals were divided into five groups as follows: tooth extraction only, implant into the mesial root socket, implant and other root sockets covered with demineralized bone sheet, implant and other root sockets filled with demineralized bone powder under the sheet, and implant and other root sockets covered with demineralized bone sheet from which proteins were extracted. We ascertained whether biologically active substances are contained in extracted proteins. Biologically active substances were detected in extracted proteins. Conditions using demineralized bone sheet with biologically active substances significantly augmented the height of the alveolar bone. Such resorbable membranes containing biologically active substances hold promise as clinical agents for bone augmentation upon implantation.

Effects of Er:YAG and Diode Laser Irradiation on Dental Pulp Cells and Tissues

Vital pulp therapy (VPT) is to preserve the nerve and maintain healthy dental pulp tissue. Laser irradiation (LI) is beneficial for VPT. Understanding how LI affects dental pulp cells and tissues is necessary to elucidate the mechanism of reparative dentin and dentin regeneration. Here, we show how Er:YAG-LI and diode-LI modulated cell proliferation, apoptosis, gene expression, protease activation, and mineralization induction in dental pulp cells and tissues using cell culture, immunohistochemical, genetic, and protein analysis techniques. Both LIs promoted proliferation in porcine dental pulp-derived cell lines (PPU-7), although the cell growth rate between the LIs was different. In addition to proliferation, both LIs also caused apoptosis; however, the apoptotic index for Er:YAG-LI was higher than that for diode-LI. The mRNA level of odontoblastic gene markers—two dentin sialophosphoprotein splicing variants and matrix metalloprotease (MMP)20 were enhanced by diode-LI, whereas MMP2 was increased by Er:YAG-LI. Both LIs enhanced alkaline phosphatase activity, suggesting that they may help induce PPU-7 differentiation into odontoblast-like cells. In terms of mineralization induction, the LIs were not significantly different, although their cell reactivity was likely different. Both LIs activated four MMPs in porcine dental pulp tissues. We helped elucidate how reparative dentin is formed during laser treatments.