Tmem2 Deficiency Leads to Enamel Hypoplasia and Soft Enamel in Mouse.

Teeth consist of 3 mineralized tissues: enamel, dentin, and cementum. Tooth malformation, the most common craniofacial anomaly, arises from complex genetic and environmental factors affecting enamel structure, size, shape, and tooth eruption. Hyaluronic acid (HA), a primary extracellular matrix component, contributes to structural and physiological functions in periodontal tissue. Transmembrane protein 2 (TMEM2), a novel cell surface hyaluronidase, has been shown to play a critical role during embryogenesis. In this study, we demonstrate Tmem2 messenger RNA expression in inner enamel epithelium and presecretory, secretory, and mature ameloblasts. Tmem2 knock-in reporter mice reveal TMEM2 protein localization at the apical and basal ends of secretory ameloblasts. Micro-computed tomography analysis of epithelial-specific Tmem2 conditional knockout (Tmem2-CKO) mice shows a significant reduction in enamel layer thickness and severe enamel deficiency. Enamel matrix protein expression was remarkably downregulated in Tmem2-CKO mice. Scanning electron microscopy of enamel from Tmem2-CKO mice revealed an irregular enamel prism structure, while the microhardness and density of enamel were significantly reduced, indicating impaired ameloblast differentiation and enamel matrix mineralization. Histological evaluation indicated weak adhesion between cells and the basement membrane in Tmem2-CKO mice. The reduced and irregular expressions of vinculin and integrin ß1 suggest that Tmem2 deficiency attenuated focal adhesion formation. In addition, abnormal HA accumulation in the ameloblast layer and weak claudin 1 immunoreactivity in Tmem2-CKO mice indicate impaired tight junction gate function. Irregular actin filament assembly was also observed at the apical and basal ends of secretory ameloblasts. Last, we demonstrated that Tmem2-deficient mHAT9d mouse ameloblasts exhibit defective adhesion to HA-containing substrates in vitro. Collectively, our data highlight the importance of TMEM2 in adhesion to HA-rich extracellular matrix, cell-to-cell adhesion, ameloblast differentiation, and enamel matrix mineralization.

Role of Heparan Sulfate in Vasculogenesis of Dental Pulp Stem Cells.

Dental pulp stem cells (DPSCs) can differentiate into vascular endothelial cells and display sprouting ability. During this process, DPSC responses to the extracellular microenvironment and cell-extracellular matrix interactions are critical in regulating their ultimate cell fate. Heparan sulfate (HS) glycosaminoglycan, a major component of extracellular matrix, plays important roles in various biological cell activities by interacting with growth factors and relative receptors. However, the regulatory function of HS on vasculogenesis of mesenchymal stem cells remains unclear. The objective of this study was to investigate the role of HS in endothelial differentiation and vasculogenesis of DPSCs. Our results show that an HS antagonist suppressed the proliferation and sprouting ability of DPSCs undergoing endothelial differentiation. Furthermore, expression of proangiogenic markers significantly declined with increasing dosages of the HS antagonist; in contrast, expression of stemness marker increased. Silencing of exostosin 1 (EXT1), a crucial glycosyltransferase for HS biosynthesis, in DPSCs using a short hairpin RNA significantly altered their gene expression profile. In addition, EXT1-silenced DPSCs expressed lower levels of endothelial differentiation markers and displayed a reduced vascular formation capacity compared with control DPSCs transduced with scrambled sequences. The sprouting ability of EXT1-silenced DPSCs was rescued by the addition of exogenous HS in vitro. Next, we subcutaneously transplanted biodegradable scaffolds seeded with EXT1-silenced or control DPSCs into immunodeficient mice. Lumen-like structures positive for human CD31 and von Willebrand factor were formed by green fluorescent protein-transduced DPSCs. Numbers of blood-containing vessels were significantly lower in scaffolds loaded with EXT1-silenced DPSCs than specimens implanted with control DPSCs. Collectively, our findings unveil the crucial role of HS on endothelial differentiation and vasculogenesis of DPSCs, opening new perspectives for the application of HS to tissue engineering and dental pulp regeneration.

Fabrication of Vascularized DPSC Constructs for Efficient Pulp Regeneration.

Dental pulp regeneration is a promising approach to restore the vitality of necrotic teeth. We have previously reported the fabrication of scaffold-free cell constructs containing only dental pulp stem cells (DPSCs) and their ability to form pulp-like tissue in the pulpless tooth. However, the DPSC construct could not build pulp-like tissue with a full root length because it is difficult to induce blood vessels from a small root canal foramen. Therefore, we hypothesized that vascular structure could be preformed in the DPSC construct by employing endothelial differentiation capability of DPSCs, and vascularized constructs might facilitate dental pulp regeneration in the pulpless tooth. In this study, vascularized DPSC constructs were fabricated by inducing endothelial differentiation, and then we investigated the behavior of differentiated DPSCs, the internal structure of cell constructs, and their pulp regenerative ability in vivo. We observed that DPSCs positive for CD31 and von Willebrand factor were localized at the outer layer of constructs and formed a reticulated lumen structure. The cells constituting the outer layer of the construct expressed endothelial differentiation markers at higher levels than cells in the inner part. These results indicated that DPSCs in the outer layer differentiated into endothelial cells and formed vascular-like structures in the cell construct. Next, a vascularized DPSC construct was transplanted into the human pulpless tooth that was implanted into immunodeficient mice in the subcutaneous space. After 6 wk of implantation, the vascularized construct formed pulp-like tissues with higher density of human CD31-positive blood vessels when compared with specimens implanted with a DPSC construct without prevascularization. These results suggest that the vascular structure formed in the DPSC construct facilitated the blood supply and enhanced pulp regeneration. This study demonstrates that a vascularized DPSC construct is a prospective biomaterial as an implant for novel dental pulp regeneration.

VE-Cadherin and Anastomosis of Blood Vessels Formed by Dental Stem Cells.

It is known that dental pulp stem cells (DPSCs) can be induced to differentiate into vasculogenic endothelial (VE) cells. However, the process that results in sprouting and anastomosis of DPSC-derived vessels remains unclear. Here, we performed studies to understand the mechanisms underpinning the anastomosis of the host vasculature with blood vessels generated by DPSCs (a model for mesenchymal stem cells). VE-cadherin-silenced primary human DPSCs seeded in tooth slice/scaffolds and transplanted into the subcutaneous space of immunodeficient mice generated fewer functional blood vessels (i.e., anastomosed with the host vasculature) than control DPSCs transduced with scrambled sequences. Both VE-cadherin-silenced and mitogen-activated protein kinase kinase 1 (MEK1)-silenced cells showed a decrease in the number of capillary sprouts in vitro. Interestingly, DPSC stably transduced with a VE-cadherin reporter demonstrated that vascular endothelial growth factor (VEGF) induces VE-cadherin expression in sprouting DPSCs undergoing anastomosis, but not in quiescent DPSCs. To begin to understand the mechanisms regulating VE-cadherin, we stably silenced MEK1 and observed that VEGF was no longer able to induce VE-cadherin expression and capillary sprout formation. Notably ERG, a transcriptional factor downstream from MEK/ERK, binds to the promoter region of VE-cadherin (chip assay) and is induced by VEGF in DPSCs. Collectively, these data defined a signaling pathway triggered by VEGF that results in phosphorylation of MEK1/ERK and activation of ERG leading to expression of VE-cadherin, which is required for anastomosis of DPSC-derived blood vessels. In conclusion, these results unveiled a signaling pathway that enables the generation of functional blood vessels upon vasculogenic differentiation of DPSCs.

Pulp Regeneration by 3-dimensional Dental Pulp Stem Cell Constructs.

Dental pulp regeneration therapy for the pulpless tooth has attracted recent attention, and clinical trial studies are underway with the tissue engineering approach. However, there remain many concerns, including the extended period for regenerating the dental pulp. In addition, the use of scaffolds increases the risk of inflammation and infection. To establish a basic technology for novel dental pulp regenerative therapy that allows transplant of pulp-like tissue, we attempted to fabricate scaffold-free 3-dimensional (3D) cell constructs composed of dental pulp stem cells (DPSCs). Furthermore, we assessed viability of these 3D DPSC constructs for dental pulp regeneration through in vitro and in vivo studies. For the in vitro study, we obtained 3D DPSC constructs by shaping sheet-like aggregates of DPSCs with a thermoresponsive hydrogel. DPSCs within constructs remained viable even after prolonged culture; furthermore, 3D DPSC constructs possessed a self-organization ability necessary to serve as a transplant tissue. For the in vivo study, we filled the human tooth root canal with DPSC constructs and implanted it subcutaneously into immunodeficient mice. We found that pulp-like tissues with rich blood vessels were formed within the human root canal 6 wk after implantation. Histologic analyses revealed that transplanted DPSCs differentiated into odontoblast-like mineralizing cells at sites in contact with dentin; furthermore, human CD31-positive endothelial cells were found at the center of regenerated tissue. Thus, the self-organizing ability of 3D DPSC constructs was active within the pulpless root canal in vivo. In addition, blood vessel-rich pulp-like tissues can be formed with DPSCs without requiring scaffolds or growth factors. The technology established in this study allows us to prepare DPSC constructs with variable sizes and shapes; therefore, transplantation of DPSC constructs shows promise for regeneration of pulpal tissue in the pulpless tooth.

Gold Nanoparticles Inhibit Matrix Metalloproteases without Cytotoxicity.

Nanoparticles (NPs) are currently the focus of considerable attention for dental applications; however, their biological effects have not been fully elucidated. The long-term, slow release of matrix metalloproteases (MMPs) digests collagen fibrils within resin-dentin bonds. Therefore, MMP inhibitors can prolong the durability of resin-dentin bonds. However, there have been few reports evaluating the combined effect of MMP inhibition and the cytotoxic effects of NPs for dentin bonding. The aim of this study was to evaluate MMP inhibition and cytotoxic responses to gold (AuNPs) and platinum nanoparticles (PtNPs) stabilized by polyvinylpyrrolidone (PVP) in cultured murine macrophages (RAW264) by using MMP inhibition assays, measuring cell viability and inflammatory responses (quantitative reverse transcription polymerase chain reaction [RT-qPCR]), and conducting a micromorphological analysis by fluorescence and transmission electron microscopy. Cultured RAW264 cells were exposed to metal NPs at various concentrations (1, 10, 100, and 400 µg/mL). AuNPs and PtNPs markedly inhibited MMP-8 and MMP-9 activity. Although PtNPs were cytotoxic at high concentrations (100 and 400 µg/mL), no cytotoxic effects were observed for AuNPs at any concentration. Transmission electron microscopy images showed a significant nonrandom intercellular distribution for AuNPs and PtNPs, which were mostly observed to be localized in lysosomes but not in the nucleus. RT-qPCR analysis demonstrated inflammatory responses were not induced in RAW264 cells by AuNPs or PtNPs. The cytotoxicity of nanoparticles might depend on the core metal composition and arise from a "Trojan horse" effect; thus, MMP inhibition could be attributed to the surface charge of PVP, which forms the outer coating of NPs. The negative charge of the surface coating of PVP binds to Zn(2+) from the active center of MMPs by chelate binding and results in MMP inhibition. In summary, AuNPs are attractive NPs that effectively inhibit MMP activity without cytotoxicity or inflammatory responses.

Expression of CD44 splicing isoforms in lung cancers: dominant expression of CD44v8-10 in non-small cell lung carcinomas.

We examined CD44 isoform expression in 138 frozen tissue samples, which included primary lung carcinomas, adjacent non-tumorous lung tissues and benign lung diseases, by both reverse transcriptase polymerase chain reaction (RT-PCR) and immunohistochemical analyses. CD44v8-10 mRNA and protein were dominantly expressed in non-small cell lung carcinomas (NSCLC), while non-tumorous tissues principally expressed CD44s and small cell lung carcinomas (SCLC) expressed either CD44s or no detectable CD44. These results indicate that CD44v8-10 is the dominant splicing isoform in NSCLC and can be practically utilized as a diagnostic marker and therapeutical target in NSCLC.

Sphingosine inhibition of NADPH oxidase activation in a cell-free system.

The effects of sphingoid bases, sphingosine and dihydrosphingosine, which are protein kinase C (PKC) inhibitors, on NADPH oxidase were examined in a cell-free system. The bases inhibited cell-free activation of NADPH oxidase by arachidonic acid at lower concentration than N-acetylsphingosine. Thus, positive charge in the molecules may play a critical role in inhibition of the oxidase. Sphingosine did not change the Km value for NADPH, but shifted the optimum concentration of arachidonic acid for activation of the oxidase. Moreover, sphingosine suppressed the translocation of p47-phox, one of the cytosolic components of the oxidase, to the membrane fraction, suggesting that the base inhibits the assembly of the components.