Local Concentrations of TGF-ß1 and IGF-1 Appear Determinant in Regulating Bone Regeneration in Human Postextraction Tooth Sockets.

Healing after tooth extraction involves a series of reparative processes affecting both alveolar bone and soft tissues. The aim of the present study was to investigate whether activation of molecular signals during the healing process confers a regenerative advantage to the extraction socket soft tissue (ESsT) at 8 weeks of healing. Compared to subepithelial connective tissue graft (CTG), qRT-PCR analyses revealed a dramatic enrichment of the ESsT in osteogenic differentiation markers. However, ESsT and CTG shared characteristics of nonspecialized soft connective tissue by expressing comparable levels of genes encoding abundant extracellular matrix (ECM) proteins. Genes encoding the transforming growth factor-ß1 (TGF-ß1) and its receptors were strongly enriched in the CTG, whereas the transcript for the insulin-like growth factor-1 (IGF-1) showed significantly high and comparable expression in both tissues. Mechanical stimulation, by the means of cyclic strain or matrix stiffness applied to primary ESsT cells (ESsT-C) and CTG fibroblasts (CTG-F) extracted from the tissue samples, revealed that stress-induced TGF-ß1 not exceeding 2.3 ng/mL, as measured by ELISA, in combination with IGF-1 up to 2.5 ng/mL was able to induce the osteogenic potential of ESsT-Cs. However, stiff matrices (50 kPa), upregulating the TGF-ß1 expression up to 6.6 ng/mL, caused downregulation of osteogenic gene expression in the ESsT-Cs. In CTG-Fs, endogenous or stress-induced TGF-ß1 ≥ 4.6 ng/mL was likely responsible for the complete lack of osteogenesis. Treatment of ESsT-Cs with TGF-ß1 and IGF-1 proved that, at specific concentrations, the two growth factors exhibited either an inductive-synergistic or a suppressive activity, thus determining the osteogenic and mineralization potential of ESsT-Cs. Taken together, our data strongly warrant the clinical exploration of ESsT as a graft in augmentative procedures during dental implant placement surgeries.

The Role of Lactic Acid on Wound Healing, Cell Growth, Cell Cycle Kinetics, and Gene Expression of Cultured Junctional Epithelium Cells in the Pathophysiology of Periodontal Disease.

Lactic acid (LA) is short-chain fatty acid, such as butyric acid and propionic acid, that is produced as a metabolite of lactic acid bacteria, including periodontopathic bacteria. These short-chain fatty acids have positive effects on human health but can also have negative effects, such as the promotion of periodontal disease (PD), which is caused by periodontal pathogens present in the gingival sulcus. PD is characterized by apical migration of junctional epithelium, deepening of pockets, and alveolar bone loss. Thus, the junctional epithelial cells that form the bottom of the gingival sulcus are extremely important in investigating the pathophysiology of PD. The aim of this study was to investigate the effect of LA on wound healing, cell growth, cell cycle kinetics, and gene expression of cultured junctional epithelium cells. The results showed that stimulation with 10 mM LA slowed wound healing of the junctional epithelial cell layer and arrested the cell cycle in the G0/G1 (early cell cycle) phase, thereby inhibiting cell growth. However, cell destruction was not observed. LA also enhanced mRNA expression of integrin α5, interleukin (IL)-6, IL-8, intercellular adhesion molecule-1, and receptor activator of nuclear factor kappa-B ligand. The results of this study suggest that stimulation of junctional epithelial cells with high concentrations of LA could exacerbate PD, similarly to butyric acid and propionic acid.

Structure of junctional epithelium is maintained by cell populations supplied from multiple stem cells.

The junctional epithelium (JE) is an epithelial component that attaches directly to the tooth surface and performs the unique function of protecting against bacterial infections; its destruction causes inflammation of the periodontal tissue and loss of alveolar bone. A recent study that used the single-color lineage tracing method reported that JE is maintained by its stem cells. However, the process by which individual stem cells form the entire JE around a whole tooth remains unclear. Using a 4-color lineage tracing method, we performed a detailed examination of the dynamics of individual stem cells that constitute the entire JE. The multicolor lineage tracing method showed that single-color areas, which were derived from each cell color, replaced all the constituent JE cells 168 d after the administration of tamoxifen. The horizontal section of the first molar showed that the single-color areas in the JE expanded widely. We detected putative stem cells at the external basal layer farthest from the enamel. In this study, JE cells that were supplied from different stem cells were visualized as individual monochromatic regions, and the JE around the first molar was maintained by several JE-specific stem cells. These findings indicated that the JE consisted of several cell populations that were supplied from their multiple stem cells and could help to explore the mechanisms involved in periodontal tissue homeostasis.

Aminopyrifen, a novel 2-aminonicotinate fungicide with a unique effect and broad-spectrum activity against plant pathogenic fungi.

Aminopyrifen is a novel 2-aminonicotinate fungicide with unique chemistry and a novel mode of action. The fungicide showed high antifungal activity mainly against Ascomycetes and its related anamorphic fungi under in vitro and pot conditions (EC50 values: 0.0039-0.23 mg/L and 1.2-12 mg/L, respectively). The active ingredient strongly inhibited germ-tube elongation of Botrytis cinerea below 0.1 mg/L and invasion into a plant. The compound exhibited no cross-resistance to commercial fungicides in B. cinerea. The antifungal agent showed high preventive efficacy and translaminar action. In the field, aminopyrifen controlled gray mold and powdery mildew at 150 mg/L. Our findings suggest that aminopyrifen is useful for protecting crops from various plant pathogens.

Effect of Butyric Acid in the Proliferation and Migration of Junctional Epithelium in the Progression of Periodontitis: An In Vitro Study.

PURPOSE: To elucidate the effects of butyric acid (BA), a metabolite of bacteria involved in periodontitis, and a possible enhancer of the junctional epithelial cells. METHODS: A murine junctional epithelial cell line, JE-1, was used to assess the effects of sodium butyrate (NaB) as BA. Cell proliferation, migration and attachment were analyzed. Additionally, gene and promoter expression analysis was performed, i.e., cap analysis of gene expression (CAGE) and gene ontology (GO) term enrichment analysis. RESULTS: NaB affected junctional epithelial cell proliferation, migration and attachment. A high concentration of NaB caused cell death and a low concentration tended to promote migration and adhesion. CAGE analysis revealed 75 upregulated and 96 downregulated genes in the cells after 0.2 mM NaB stimulation for 3 h. Regarding GO term enrichment, the genes upregulated >4-fold participated predominantly in cell migration and proliferation. The results of this study suggest that BA produced from periodontopathic bacteria is involved in periodontal tissue destruction at high concentrations. Furthermore, at low concentrations, BA potentially participates in periodontal disease progression by increasing proliferation, migration and attachment of the junctional epithelium and thereby increasing epithelial down-growth.

Maintaining a protective state for human periodontal tissue.

Periodontitis, caused by infection with periodontal pathogens, is primarily characterized by inflammatory bone resorption and destruction of connective tissue. Simply describing periodontitis as a specific bacterial infection cannot completely explain the various periodontal tissue destruction patterns observed. Periodontal tissue damage is thought to be caused by various factors. In recent years, research goals for periodontal pathogens have shifted from searching for specific pathogens to investigating mechanisms that damage periodontal tissues. Bacteria interact directly with the host in several ways, influencing expression and activity of molecules that evade host defenses, and destroying local tissues and inhibiting their repair. The host's innate and acquired immune systems are important defense mechanisms that protect periodontal tissues from attack and invasion of periodontal pathogens, thus preventing infection. Innate and acquired immunity have evolved to confront the microbial challenge, forming a seamless defense network in periodontal tissues. In the innate immune response, host cells quickly detect, via specialized receptors, macromolecules and nucleic acids present on bacterial cell walls, and this triggers a protective, inflammatory response. The work of this subsystem of host immunity is performed mainly by phagocytes, beta-defensin, and the complement system. In addition, the first line of defense in oral innate immunity is the junctional epithelium, which acts as a physical barrier to the entry of oral bacteria and other nonself substances. In the presence of a normal flora, junctional epithelial cells differentiate actively and proliferate apically, with concomitant increase in chemotactic factor expression recruiting neutrophils. These immune cells play an important role in maintaining homeostasis and the protective state in periodontal tissue because they eliminate unwanted bacteria over time. Previous studies indicate a mechanism for attracting immune cells to periodontal tissue with the purpose of maintaining a protective state; although this mechanism can function without bacteria, it is enhanced by the normal flora. A better understanding of the relationship between the protective state and its disruption in periodontal disease could lead to the development of new treatment strategies for periodontal disease.

Visualization of junctional epithelial cell replacement by oral gingival epithelial cells over a life time and after gingivectomy.

Junctional epithelium (JE), which is derived from odontogenic epithelial cells immediately after eruption, is believed to be gradually replaced by oral gingival epithelium (OGE) over a lifetime. However, the detailed process of replacement remains unclear. The aim of the present study was to clarify the process of JE replacement by OGE cells using a green fluorescent protein (GFP)-positive tooth germ transplantation method. GFP-positive JE was partly replaced by OGE cells and completely replaced on day 200 after transplantation, whereas there was no difference in the expression of integrin ß4 (Itgb4) and laminin 5 (Lama5) between JE before and after replacement by OGE cells. Next, GFP-positive JE was partially resected. On day 14 after resection, the regenerated JE consisted of GFP-negative cells and also expressed both Itgb4 and Lama5. In addition, the gene expression profile of JE derived from odontogenic epithelium before gingivectomy was partly different from that of JE derived from OGE after gingivectomy. These results suggest that JE derived from the odontogenic epithelium is gradually replaced by OGE cells over time and JE derived from the odontogenic epithelium might have specific characteristics different to those of JE derived from OGE.

A novel fungicide aminopyrifen inhibits GWT-1 protein in glycosylphosphatidylinositol-anchor biosynthesis in Neurospora crassa.

Aminopyrifen, 4-phenoxybenzyl 2-amino-6-methylnicotinate, strongly inhibited the mycelial growth of a wild-type Neurospora crassa strain on Vogel's minimal medium containing 1.2% sucrose, with a 0.001 mg/L concentration required for 50% growth inhibition. Similar to micafungin, an inhibitor of beta-1, 3-glucan synthetase, aminopyrifen further inhibited the growth of N. crassa deletion mutants of MAP kinase cascade genes, such as mak-1 and mak-2, than the wild-type strain, suggesting that aminopyrifen perturbs cell wall-related processes. Furthermore, we found that three chitin synthase gene mutants (chs-1, chs-5, and chs-7) were highly sensitive to both chemicals; however, aminopyrifen, but not micafungin, induced a swollen germ tube from the conidia of chs-5 and chs-7 mutants on Vogel's medium containing 1.2% sucrose. To elucidate the target protein of aminopyrifen, we isolated mutants resistant to aminopyrifen after UV treatment of conidia of the wild-type strain or the chs-5 strain. The resistance mutations were localized to the gwt-1 gene that encodes an acyltransferase, GWT-1, which participates in the biosynthesis of the glycosylphosphatidylinositol (GPI) precursor, and were found to result in S180F and V178A alterations in the protein. These results strongly suggest that aminopyrifen works as an inhibitor targeting GWT-1, a protein involved in GPI-anchor biosynthesis.

Cdc42 regulates cranial suture morphogenesis and ossification.

Cdc42 (cell division cycle 42) is ubiquitously expressed small GTPases belonging to the Rho family of proteins. Previously, we generated limb bud mesenchyme-specific Cdc42 inactivated mice (Cdc42 conditional knockout mice; Cdc42 fl/fl; Prx1-Cre), which showed short limbs and cranial bone deformities, though the mechanism related to the cranium phenotype was unclear. In the present study, we investigated the role of Cdc42 in cranial bone development. Our results showed that loss of Cdc42 caused a defect of intramembranous ossification in cranial bone tissues which is related to decreased expressions of cranial suture morphogenesis genes, including Indian hedgehog (Ihh) and bone morphogenetic proteins (BMPs). These findings demonstrate that Cdc42 plays a crucial role in cranial osteogenesis, and is controlled by Ihh- and BMP-mediated signaling during cranium development.

Establishment of mouse gingival junctional epithelial cell line using a bioengineered tooth system.

Junctional epithelium (JE), one of the constituents of periodontal tissue, has several unique features to prevent bacterial infection. However, the molecular mechanisms of these cells remain to be completely elucidated because there has been no JE cell line to date. We have succeeded in isolating JE cells expressing green fluorescent protein (GFP) by using a bioengineered tooth technique in mice. The gene expressions of GFP-positive JE cells, isolated from around the erupted bioengineered teeth using flow cytometry, were analyzed by RNA sequencing. GFP-positive cells derived from the bioengineered tooth germs showed similar gene expression patterns to primary JE cells. The isolated GFP-positive JE cells were immortalized by transducing the simian virus 40 large T antigen using lentiviral vectors. The established GFP-positive JE cells maintained proliferative activity for more than 20 passages, and did not show cellular senescence as demonstrated by ß-galactosidase assay. These cells also expressed similar gene expression patterns to primary JE cells. The established cell lines may prove useful for future investigation of JE characteristics in vitro.

Expression of nephronectin is enhanced by 1α,25-dihydroxyvitamin D3.

The extracellular matrix protein nephronectin (Npnt), also called POEM, is considered to play critical roles as an adhesion molecule in development and functions of various tissues, such as the kidneys, liver, and bone. In the present study, we examined the molecular mechanism of Npnt gene expression and found that vitamin D3 (1α,25-dihydroxyvitamin D3,VD 3) strongly enhanced Npnt mRNA expression in MC3T3-E1 cells from a mouse osteoblastic cell line. The VD 3-induced increase in Npnt expression is both time- and dose-dependent and is mediated by the vitamin D receptor (VDR).

Rho GTPase protein Cdc42 is critical for postnatal cartilage development.

Cdc42, a small Rho GTPase family member, has been shown to regulate multiple cellular functions in vitro, including actin cytoskeletal reorganization, cell migration, proliferation, and gene expression. However, its tissue-specific roles in vivo remain largely unknown, especially in postnatal cartilage development, as cartilage-specific Cdc42 inactivated mice die within a few days after birth. In this study, we investigated the physiological functions of Cdc42 during cartilage development after birth using tamoxifen-induced cartilage-specific inactivated Cdc42 conditional knockout (Cdc42 (fl/fl); Col2-CreERT) mice, which were generated by crossing Cdc42 flox mice (Cdc42 (fl/fl)) with tamoxifen-induced type II collagen (Col2) Cre transgenic mice using a Cre/loxP system. The gross morphology of the Cdc42 cKO mice was shorter limbs and body, as well as reduced body weight as compared with the controls. In addition, severe defects were found in growth plate chondrocytes of the long bones, characterized by a shorter proliferating zone (PZ), wider hypertrophic zone (HZ), and loss of columnar organization of proliferating chondrocytes, resulting in delayed endochondral bone formation associated with abnormal bone growth. Our findings demonstrate the importance of Cdc42 for cartilage development during both embryonic and postnatal stages.

Cdc42 is crucial for facial and palatal formation during craniofacial development.

Craniofacial deformities with multifactorial etiologies, such as cleft palate and facial dysmorphism, represent some of the most frequent congenital birth defects seen in humans. Their pathogeneses are often related to cranial neural crest (CNC) cells. During CNC cell migration, changes in cell shape and formation, as well as maintenance of subcellular structures, such as filopodia and lamellipodia, are dependent on the complex functions of Rho family small GTPases, which are regulators of actin cytoskeletal organization. Cdc42, a member of the Rho family of small GTPases, is known to play critical roles in organogenesis of various tissues. To investigate the physiological functions of Cdc42 during craniofacial development, we generated CNC-derived cell-specific inactivated Cdc42 mutant mice (Cdc42fl/fl ;P0-cre). Most of the Cdc42fl/fl ;P0-cre neonates were viable at birth, though they appeared weaker and no milk was found in their stomachs, and all died within a few days. They had a short face and intracranial bleeding, and abnormal calcification of the cranium. Cdc42fl/fl ;P0-cre neonates also demonstrated a cleft palate and there was no fusion of the secondary palate because of failure of palatal shelf elongation for the process of palate closure. Cdc42 is crucial for facial and palatal formation during craniofacial development.

Cdc42 is critical for cartilage development during endochondral ossification.

Cdc42 is a widely expressed protein that belongs to the family of Rho GTPases and controls a broad variety of signal transduction pathways in a variety of cell types. To investigate the physiological functions of Cdc42 during cartilage development, we generated chondrocyte-specific inactivated Cdc42 mutant mice (Cdc42(fl/fl); Col2-Cre). The gross morphology of mutant neonates showed shorter limbs and body as compared with the control mice (Cdc42(fl/fl)). Skeletal preparations stained with alcian blue and alizarin red also revealed that the body and the long bone length of the mutants were shorter than those of the control mice. Furthermore, severe defects were found in growth plate chondrocytes in the femur sections of mutant mice, characterized by a reduced proliferating zone height, wider hypertrophic zone, and loss of columnar organization in proliferating chondrocytes. The expression levels of chondrocyte marker genes, such as Col2, Col10, and Mmp13, in mutant mice were decreased as compared with the control mice. Mineralization of trabecular bones in the femur sections was also decreased in the mutants as compared with control mice, whereas osteoid volume was increased. Together these results suggested that chondrocyte proliferation and differentiation in growth plates in the present mutant mice were not normally organized, which contributed to abnormal bone formation. We concluded that Cdc42 is essential for cartilage development during endochondral bone formation.

Procedure-dependent construction of two isomers of trimeric self-assembled boronic esters.

A procedure-dependent selective construction of two isomers of trimeric boronic esters was realized. A capsule-like trimeric boronic ester was constructed by the direct connection of two kinds of self-assembled boronic esters via [3+2] cycloaddition, while the tricyclic trimeric boronic ester was obtained by the self-assembly of pre-connected components.

BMP-2 Induced Expression of Alx3 That Is a Positive Regulator of Osteoblast Differentiation.

Bone morphogenetic proteins (BMPs) regulate many aspects of skeletal development, including osteoblast and chondrocyte differentiation, cartilage and bone formation, and cranial and limb development. Among them, BMP-2, one of the most potent osteogenic signaling molecules, stimulates osteoblast differentiation, while it inhibits myogenic differentiation in C2C12 cells. To evaluate genes involved in BMP-2-induced osteoblast differentiation, we performed cDNA microarray analyses to compare BMP-2-treated and -untreated C2C12 cells. We focused on Alx3 (aristaless-like homeobox 3) which was clearly induced during osteoblast differentiation. Alx3, a homeobox gene related to the Drosophilaaristaless gene, has been linked to developmental functions in craniofacial structures and limb development. However, little is known about its direct relationship with bone formation. In the present study, we focused on the mechanisms of Alx3 gene expression and function during osteoblast differentiation induced by BMP-2. In C2C12 cells, BMP-2 induced increase of Alx3 gene expression in both time- and dose-dependent manners through the BMP receptors-mediated SMAD signaling pathway. In addition, silencing of Alx3 by siRNA inhibited osteoblast differentiation induced by BMP-2, as showed by the expressions of alkaline phosphatase (Alp), Osteocalcin, and Osterix, while over-expression of Alx3 enhanced osteoblast differentiation induced by BMP-2. These results indicate that Alx3 expression is enhanced by BMP-2 via the BMP receptors mediated-Smad signaling and that Alx3 is a positive regulator of osteoblast differentiation induced by BMP-2.

BMP2 differentially regulates the expression of Gremlin1 and Gremlin2, the negative regulators of BMP function, during osteoblast differentiation.

Bone morphogenetic proteins (BMPs) control the expressions of many genes involved in bone formation. On the basis of our hypothesis that BMP2 stimulation-regulated gene expression plays a critical role in osteoblast differentiation, we performed genome-wide screening of messenger RNA from BMP2-treated and -untreated C2C12 cells using a DNA microarray technique. We found that the expressions of Gremlin1 and Gremlin2, which are known BMP antagonists, were bidirectionally regulated by BMP2. Gremlin1 was down-regulated by BMP2, while Gremlin2 was up-regulated in both time- and dose-dependent manners. Ablation of Gremlin1 or Gremlin2 enhanced osteoblast differentiation induced by BMP2. On the other hand, treatment with recombinant Gremlin1 inhibited BMP2-induced osteoblast differentiation. Furthermore, treatment with Smad4 siRNA and the p38 MAPK inhibitor SB203580 suppressed BMP2-induced Gremlin2 gene expression. The differential regulation of Gremlin1 and Gremlin2 gene expressions by BMP2 may explain the critical function of these genes during osteoblast differentiation.

Cdc42 is required for chondrogenesis and interdigital programmed cell death during limb development.

Cdc42, a member of the Rho subfamily of small GTPases, is known to be a regulator of multiple cellular functions, including cytoskeletal organization, cell migration, proliferation, and apoptosis. However, its tissue-specific roles, especially in mammalian limb development, remain unclear. To investigate the physiological function of Cdc42 during limb development, we generated limb bud mesenchyme-specific inactivated Cdc42 (Cdc42(fl/fl); Prx1-Cre) mice. Cdc42(fl/fl); Prx1-Cre mice demonstrated short limbs and body, abnormal calcification of the cranium, cleft palate, disruption of the xiphoid process, and syndactyly. Severe defects were also found in long bone growth plate cartilage, characterized by loss of columnar organization of chondrocytes, and thickening and massive accumulation of hypertrophic chondrocytes, resulting in delayed endochondral bone formation associated with reduced bone growth. In situ hybridization analysis revealed that expressions of Col10 and Mmp13 were reduced in non-resorbed hypertrophic cartilage, indicating that deletion of Cdc42 inhibited their terminal differentiation. Syndactyly in Cdc42(fl/fl); Prx1-Cre mice was caused by fusion of metacarpals and a failure of interdigital programmed cell death (ID-PCD). Whole mount in situ hybridization analysis of limb buds showed that the expression patterns of Sox9 were ectopic, while those of Bmp2, Msx1, and Msx2, known to promote apoptosis in the interdigital mesenchyme, were down-regulated. These results demonstrate that Cdc42 is essential for chondrogenesis and ID-PCD during limb development.

Expression of POEM, a positive regulator of osteoblast differentiation, is suppressed by TNF-α.

POEM, also known as nephronectin, is an extracellular matrix protein considered to be a positive regulator of osteoblast differentiation. In the present study, we found that tumor necrosis factor-α (TNF-α), a key regulator of bone matrix properties and composition that also inhibits terminal osteoblast differentiation, strongly inhibited POEM expression in the mouse osteoblastic cell line MC3T3-E1. TNF-α-induced down-regulation of POEM gene expression occurred in both time- and dose-dependent manners through the nuclear factor kappa B (NF-κB) pathway. In addition, expressions of marker genes in differentiated osteoblasts were down-regulated by TNF-α in a manner consistent with our findings for POEM, while over-expression of POEM recovered TNF-α-induced inhibition of osteoblast differentiation. These results suggest that TNF-α inhibits POEM expression through the NF-κB signaling pathway and down-regulation of POEM influences the inhibition of osteoblast differentiation by TNF-α.