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.

The revolution in genetics has rapidly increased our knowledge of human and mouse genes that are critical for the formation of dental enamel and helps us understand how enamel evolved. In this graphical review we focus on the roles of 41 genes that are essential for the secretory stage of amelogenesis when characteristic enamel mineral ribbons initiate on dentin and elongate to expand the enamel layer to the future surface of the tooth. Based upon ultrastructural analyses of genetically modified mice, we propose a molecular model explaining how a cell attachment apparatus including collagen 17, α6ß4 and αvß6 integrins, laminin 332, and secreted enamel proteins could attach to individual enamel mineral ribbons and mold their cross-sectional dimensions as they simultaneously elongate and orient them in the direction of the retrograde movement of the ameloblast membrane.

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.

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.

Recent studies have shown that native phosphorylated full-length porcine amelogenin (P173) and its predominant cleavage product (P148) can inhibit spontaneous calcium phosphate formation in vitro by stabilizing an amorphous calcium phosphate (ACP) precursor phase. Since full-length amelogenin undergoes proteolysis by matrix metalloproteinase 20 (MMP20, enamelysin) soon after secretion, the present study was conducted to assess the effect of amelogenin proteolysis on calcium phosphate formation. Calcium and phosphate were sequentially added to protein solutions without and with added MMP20 (ratio = 200:1) under physiological-like conditions of ionic strength (163 mM) in 50 mM Tris-HCl (pH 7.4) at 37 °C. Protein degradation with time was assessed by gel-electrophoresis, and mineral products formed were characterized by transmission electron microscopy (TEM). MMP20 was found to cleave P173 to primarily generate P148, along with P162, P46-148, and P63/64-148. In sharp contrast, MMP20 did not cleave P148. In addition, the formation of well-aligned bundles of enamel-like hydroxyapatite (HA) crystals was promoted in the presence of P173 with added MMP20, while only ACP particles were seen in the absence of MMP20. Although P148 was found to have a somewhat lower capacity to stabilize ACP and prevent HA formation compared with P173 in the absence of MMP20, essentially no HA formation was observed in the presence of somewhat higher concentrations of P148 regardless of MMP20 addition, due to the lack of observed protein proteolysis. Present findings suggest that ACP transformation to ordered arrays of enamel crystals may be regulated in part by the proteolysis of full-length native amelogenin, while the predominant amelogenin degradation product in developing enamel (e.g., P148) primarily serves to prevent uncontrolled mineral formation during the secretory stage of amelogenesis.

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.