Immunolocalization of enamel matrix protein-like proteins in the tooth enameloid of spotted gar, Lepisosteus oculatus, an actinopterygian bony fish.

Enameloid is a well-mineralized tissue covering the tooth surface in fish and it corresponds to the outer-most layer of dentin. It was reported that both dental epithelial cells and odontoblasts are involved in the formation of enameloid. Nevertheless, the localization and timing of secretion of ectodermal enamel matrix proteins in enameloid are unclear. In the present study, the enameloid matrix during the stages of enameloid formation in spotted gar, Lepisosteus oculatus, an actinopterygian, was examined mainly by transmission electron microscopy-based immunohistochemistry using an anti-mammalian amelogenin antibody and antiserum. Positive immunoreactivity with the antibody and antiserum was found in enameloid from the surface to the dentin-enameloid junction just before the formation of crystallites. This immunoreactivity disappeared rapidly before the full appearance of crystallites in the enameloid during the stage of mineralization. Immunolabelling was usually found along the collagen fibrils but was not seen on the electron-dense fibrous structures, which were probably derived from matrix vesicles in the previous stage. In inner dental epithelial cells, the granules in the distal cytoplasm often showed positive immunoreactivity, suggesting that the enamel matrix protein-like proteins originated from inner dental epithelial cells. Enamel matrix protein-like proteins in the enameloid matrix might be common to the enamel matrix protein-like proteins previously reported in the collar enamel of teeth and ganoine of ganoid scales, because they exhibited marked immunoreactivity with the same anti-mammalian amelogenin antibodies. It is likely that enamel matrix protein-like proteins are involved in the formation of crystallites along collagen fibrils in enameloid.

Anteroposterior Patterning of Gene Expression in the Human Infant Sclera: Chondrogenic Potential and Wnt Signaling.

Purpose/Aim: We sought to identify the anteroposterior spatial gene expression hierarchy in the human sclera to develop a hypothesis for axial elongation and deformity of the eyeball. MATERIALS AND METHODS: We analyzed the global gene expression of human scleral cells derived from distinct parts of the human infant sclera obtained from surgically enucleated eyes with retinoblastoma, using Affymetrix GeneChip oligonucleotide arrays, and compared, in particular, gene expression levels between the anterior and posterior parts of the sclera. The ages of three donors were 10M, 4M, and 1Y9M. RESULTS: K-means clustering analysis of gene expression revealed that expression levels of cartilage-associated genes such as COLXIA and ACAN increased from the anterior to the posterior part of the sclera. Microarray analyses and RT-PCR data showed that the expression levels of MGP, COLXIA, BMP4, and RARB were significantly higher in the posterior than in the anterior sclera of two independent infant eyes. Conversely, expression levels of WNT2, DKK2, GREM1, and HOXB2 were significantly higher in the anterior sclera. Among several Wnt-family genes examined, WNT2B was found to be expressed at a significantly higher level in the posterior sclera, and the reverse order was observed for WNT2. The results of luciferase reporter assays suggested that a GSK-3ß inhibitor stimulated Wnt/ß-catenin signaling particularly strongly in the posterior sclera. The expression pattern of RARB, a myopia-related gene, was similar in three independent eyes. CONCLUSIONS: Chondrogenic potential was higher and Wnt/ß-catenin signaling was more potently activated by a GSK-3ß inhibitor in the posterior than in the anterior part of the human infant sclera. Although the differences in the gene expression profiles between the anterior and posterior sclera might be involved only in normal growth processes, this anteroposterior hierarchy in the sclera might contribute to disorders involving abnormal elongation and deformity of the eyeball, including myopia.

Immunohistochemical and Western Blotting Analyses of Ganoine in the Ganoid Scales of Lepisosteus oculatus: an Actinopterygian Fish.

In order to compare its characteristics with those of jaw tooth collar enamel, normally developing and experimentally regenerating ganoine from ganoid scales of Lepisosteus oculatus (spotted gar), an actinopterygian fish species, was examined by Western blotting and immunohistochemistry. Amelogenin, a major enamel matrix protein (EMP), is widely found from sarcopterygian fish to mammals. Therefore, we used antimammalian amelogenin antibodies and antisera: an antibody against bovine amelogenin; antiserum against porcine amelogenin; and region-specific antibodies or antiserum against the C-terminus, middle region, or N-terminus of porcine amelogenin in this study. Positive immunoreactivity with the antibody against bovine amelogenin, antiserum against porcine amelogenin, and the middle and C-terminal region-specific antibodies was detected in both normally developing and regenerating ganoine matrix, as well as in granules found within inner ganoine epithelial cells. These immunohistochemical analyses indicated that the Lepisosteus ganoine matrix contains EMP-like proteins with epitopes similar to mammalian amelogenins. In Western blotting analyses of regenerating ganoid scales with the antibovine amelogenin antibody, two protein bands with molecular weights of approximately 78 and 65 kDa were detected, which were similar to those found in Lepisosteus tooth enamel. Our study suggests that in Lepisosteus, EMP-like proteins in the ganoine matrix corresponded to those in tooth enamel. However, it was revealed that the 78 and 65 kDa EMP-like proteins were different from 27 kDa bovine amelogenin.

TGF-ß in dentin matrix extract induces osteoclastogenesis in vitro.

Previously, we have demonstrated that the extracellular matrix from dentin affects osteoclastic activity in co-culture between osteoclast and osteoblast-rich fraction from mouse marrow cells. In the present study, we aimed to investigate the mechanisms of dentin matrix extract-induced osteoclastogenesis in mouse bone marrow macrophages (BMMs). Dentin proteins were extracted from bovine incisor root dentin using 0.6 M HCl. BMMs were cultured in α-MEM containing macrophage colony-stimulating factor/receptor activator of nuclear factor kappa-B ligand in the presence or absence of dentin matrix extract. Tartrate-resistant acid phosphatase (TRAP)-positive cell number, total TRAP activity, and the mRNA levels of osteoclast-related genes, assayed by real-time RT-PCR, were determined as markers of osteoclastogenesis. A neutralizing antibody against transforming growth factor-ß1 (TGF-ß1), SB431542, a TGF-ß receptor inhibitor, and ELISA were used to determine the role of TGF-ß1. We observed increases in TRAP-positive cell number, TRAP activity, and the mRNA levels of osteoclast-related genes of BMMs cultured with dentin extract. The use of a neutralizing antibody against TGF-ß1 or SB431542 inhibited the inductive effect of dentin extract, suggesting TGF-ß1 involvement. The addition of exogenous TGF-ß1, but not bone morphogenic protein-2, also increased osteoclastogenesis, corresponding to the ELISA determination of TGF-ß1 in the dentin extract. In conclusion, our results indicate that proteins from dentin matrix have an inductive effect in osteoclastogenesis, which is mediated, in part, by TGF-ß1.

Characterization of pulp and follicle stem cells from impacted supernumerary maxillary incisors.

PURPOSE: The purpose of this study was to characterize dental pulp and dental follicle stem/progenitor cells (DPSCs and DFSCs) from the same impacted supernumerary maxillary incisors (ISMIs). METHODS: DPSCs and DFSCs were obtained from ISMIs of healthy children (six to 12 years old) by the outgrowth culture method and were compared for proliferation, colony-forming capacity, gene expression, cell surface markers, and trilineage differentiation capacity. RESULTS: The volume of follicle tissue obtained was much larger than that for pulp tissue. DPSCs and DFSCs showed fibroblast-like morphology, expressed the stem cell-associated genes, OCT4 and NANOG, and were positive for CD146, CD90, and CD105 but negative for CD45. The cell proliferation rate and colony-forming capacity of DFSCs were significantly higher than those for DPSCs. Under inductive culture conditions, DPSCs and DFSCs differentiated into osteogenic, adipogenic, and chondrogenic lineage cells. CONCLUSIONS: These results demonstrated that dental pulp and dental follicle stem/progenitor cells have similar mesenchymal stem cell characteristics, but DFSCs are easily accessible for cell culture and have a higher proliferation capacity than DPSCs. It also appears that dental follicle stem/progenitor cells might have some advantages as a stem cell resource for regenerative medicine.

Immunohistochemical and Western blot analyses of collar enamel in the jaw teeth of gars, Lepisosteus oculatus, an actinopterygian fish.

Although most fish have no enamel layer in their teeth, those belonging to Lepisosteus (gars), an extant actinopterygian fish genus, do and so can be used to study amelogenesis. In order to examine the collar enamel matrix in gar teeth, we subjected gar teeth to light and electron microscopic immunohistochemical examinations using an antibody against bovine amelogenin (27 kDa) and antiserum against porcine amelogenin (25 kDa), as well as region-specific antibodies and antiserum against the C-terminus and middle region, and N-terminus of porcine amelogenin, respectively. The enamel matrix exhibited intense immunoreactivity to the anti-bovine amelogenin antibody and the anti-porcine amelogenin antiserum in addition to the C-terminal and middle region-specific antibodies, but not to the N-terminal-specific antiserum. These results suggest that the collar enamel matrix of gar teeth contains amelogenin-like proteins and that these proteins possess domains that closely resemble the C-terminal and middle regions of porcine amelogenin. Western blot analyses of the tooth germs of Lepisosteus were also performed. As a result, protein bands with molecular weights of 78 kDa and 65 kDa were clearly stained by the anti-bovine amelogenin antibody as well as the antiserum against porcine amelogenin and the middle-region-specific antibody. It is likely that the amelogenin-like proteins present in Lepisosteus do not correspond to the amelogenins found in mammals, although they do possess domains that are shared with mammalian amelogenins.

The inhibition effect of non-protein thiols on dentinal matrix metalloproteinase activity and HEMA cytotoxicity.

OBJECTIVES: Phosphoric acid (PA) etching used in etch-and-rinse adhesives is known to activate host-derived dentinal matrix-metalloproteinases (MMPs) and increase dentinal permeability. These two phenomena will result, respectively; in degradation of dentine-adhesive bond and leaching of some monomers especially 2-hydroxyethyl methacrylate (HEMA) into the pulp that would negatively affect the viability of pulpal cells. This study is the first to investigate the inhibitory effect of non-protein thiols (NPSH); namely reduced glutathione (GSH) and N-acetylcysteine (NAC) on dentinal MMPs and compare their effects on HEMA cytotoxicity. METHODS: Dentine powder was prepared from human teeth, demineralized with 1% PA and then treated with 2% GSH, 2% NAC or 2% chlorhexidine (CHX). Zymographic analysis of extracted proteins was performed. To evaluate the effect of GSH, NAC and CHX on HEMA cytotoxicity, solutions of these compounds were prepared with or without HEMA and rat pulpal cells were treated with the tested solutions for (6 and 24h). Cells viability was measured by means of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Cytotoxicity data were analysed by one-way ANOVA and Tukey post hoc tests (p<0.05). RESULTS: The inhibitory effect of GSH and NAC on dentinal MMPs was confirmed. GSH showed similar effectiveness to NAC regarding HEMA cytotoxicity inhibition. CONCLUSION: NPSH were effective to inhibit dentinal MMPs and HEMA cytotoxicity. CLINICAL SIGNIFICANCE: The tested properties of NPSH provide promising clinical use of these agents which would enhance dentine-bond durability and decrease post-operative sensitivity.

The tumor necrosis factor type 2 receptor plays a protective role in tumor necrosis factor-α-induced bone resorption lacunae on mouse calvariae.

Tumor necrosis factor (TNF)-α exerts its biological function via TNF type 1 and type 2 receptors (TNFR1 and TNFR2). We have previously reported that bone resorption induced by lipopolysaccharide (LPS) in TNFR2-deficient mice is accelerated compared to that in wild-type (WT) mice. Although these results suggested that TNFR2 might have a protective role in bone resorption, we could not exclude the possibility that TNFR2 has no role in bone resorption. To clarify the role of TNFR2, we developed a TNF-α-induced bone resorption model using cholesterol-bearing pullulan nanogel as a TNF-α carrier to minimize the influence of inflammatory cytokines other than TNF-α. Injections of human TNF-α (hTNF), an agonist of mouse TNFR1, stimulated bone resorption lacunae on the calvariae in WT mice, but mouse TNF-α (mTNF), an agonist of both mouse TNFR1 and TNFR2, could not. To eliminate the possibility that the TNFR1 agonistic effects of hTNF were stronger than those of mTNF, we used the same model in TNFR2-deficient mice. Injection of mTNF resulted in clear bone resorption lacunae to the same extent observed after using hTNF in the TNFR2-deficient mice. Histomorphometric analysis of osteoclast number supported the observed changes in bone resorption lacunae. These data suggest that TNFR2 has a protective role in TNF-α-induced bone resorption.

Osteoclast and osteoblast activities on carbonate apatite plates in cell cultures.

Previous studies have demonstrated that carbonate apatite (CA) is superior to hydroxyapatite (HA) and ß-tricalciumphosphate (ß-TCP) with regard to osteoclastic resorption, but evidence on osteoclast and osteoblast response remains controversial. In the present study, the expression of bone related mRNA is examined on CA, HA, ß-TCP, and titanium plates. ICR mouse osteoblast cells are cocultured with ICR mouse bone marrow cells. Crude osteoclast-like cell-rich suspensions are then seeded onto plates and cultured for 48 h. Total RNA is extracted and mRNA expression is examined by real-time RT-PCR. Amounts of vacuolar-type ATPase, cathepsin K, and TRAP mRNA are significantly greater on CA than on the other plates. The amount of osteoprotegerin mRNA is significantly greater on CA than on the other plates. RANKL mRNA expression, which is generally regarded as an osteoblast maker, varies with material, but shows no significant differences between CA and the other plates. The formation and activity of osteoclasts is greater with CA than with the other plates. Thus, CA is superior to ß-TCP as a bioresorbable bone substitute for tissue engineering.

Suppression of NF-kappaB increases bone formation and ameliorates osteopenia in ovariectomized mice.

Bone degenerative diseases, including osteoporosis, impair the fine balance between osteoclast bone resorption and osteoblast bone formation. Single-agent therapy for anabolic and anticatabolic effects is attractive as a drug target to ameliorate such conditions. Inhibition of nuclear factor (NF)-κB reduces the osteoclast bone resorption. The role of NF-κB inhibitors on osteoblasts and bone formation, however, is minimal and not well investigated. Using an established NF-κB inhibitor named S1627, we demonstrated that inhibition of NF-κB increases osteoblast differentiation and bone formation in vitro by up-regulating the mRNAs of osteoblast-specific genes like type I collagen, alkaline phosphatase, and osteopontin. In addition, S1627 was able to increase bone formation and repair bone defect in a murine calvarial defect model. To determine the effect of NF-κB on a model of osteoporosis, we injected two doses of inhibitor (25 and 50 mg/kg·d) twice a day in sham-operated or ovariectomized 12-wk-old mice and killed them after 4 wk. The anabolic effect of S1627 on trabecular bone was determined by micro focal computed tomography and histomorphometry. Bone mineral density of inhibitor-treated ovariectomized animals was significantly increased compared with sham-operated mice. Osteoblast-related indices like osteoblast surface, mineral apposition rate, and bone formation rate were increased in S1627-treated animals in a dose-dependent manner. NF-κB inhibition by S1627 increased the trabecular bone volume in ovariectomized mice. Furthermore, S1627 could inhibit the osteoclast number, and osteoclast surface to bone surface. In vitro osteoclastogenesis and bone resorbing activity were dose-dependently reduced by NF-κB inhibitor S1627. Taken collectively, our results suggest that NF-κB inhibitors are effective in treating bone-related diseases due to their dual anabolic and antiresorptive activities.

Regeneration of bone- and tendon/ligament-like tissues induced by gene transfer of bone morphogenetic protein-12 in a rat bone defect.

Members of the bone morphogenetic protein (BMP) family have diverse physiological roles. For instance, BMP-2 stimulates osteogenesis, while BMP-12 induces the formation of tendon/ligament-like tissues. Here, we designed a study to determine whether BMP-12 has bone and/or cartilage regeneration abilities similar to those of BMP-2. We implanted plasmid vectors encoding either BMP-2 or BMP-12 in rats with femur defects, and monitored the bone healing process for 8-weeks. The BMP-12 transgene induced prominent fibrogenesis by 2 weeks, with bone substitution occurring by 8 weeks. BMP-2, however, was associated predominantly with osteogenesis throughout the 8 week period. Thus, we conclude that BMP-12 does not function similarly to BMP-2 during bone healing. Further work is needed to better understand the mechanisms by which it stimulates bony growths to replace the connective tissues formed during the first stages of bone healing.

The pivotal role of the alternative NF-kappaB pathway in maintenance of basal bone homeostasis and osteoclastogenesis.

The alternative NF-kappaB pathway consists predominantly of NF-kappaB-inducing kinase (NIK), IkappaB kinase alpha (IKKalpha), p100/p52, and RelB. The hallmark of the alternative NF-kappaB signaling is the processing of p100 into p52 through NIK, thus allowing the binding of p52 and RelB. The physiologic relevance of alternative NF-kappaB activation in bone biology, however, is not well understood. To elucidate the role of the alternative pathway in bone homeostasis, we first analyzed alymphoplasic (aly/aly) mice, which have a defective NIK and are unable to process p100, resulting in the absence of p52. We observed increased bone mineral density (BMD) and bone volume, indicating an osteopetrotic phenotype. These mice also have a significant defect in RANKL-induced osteoclastogenesis in vitro and in vivo. NF-kappaB DNA-binding assays revealed reduced activity of RelA, RelB, and p50 and no binding activity of p52 in aly/aly osteoclast nuclear extracts after RANKL stimulation. To determine the role of p100 itself without the influence of a concomitant lack of p52, we used p100(-/-) mice, which specifically lack the p100 inhibitor but still express p52. p100(-/-) mice have an osteopenic phenotype owing to the increased osteoclast and decreased osteoblast numbers that was rescued by the deletion of one allele of the relB gene. Deletion of both allele of relB resulted in a significantly increased bone mass owing to decreased osteoclast activity and increased osteoblast numbers compared with wild-type (WT) controls, revealing a hitherto unknown role for RelB in bone formation. Our data suggest a pivotal role of the alternative NF-kappaB pathway, especially of the inhibitory role of p100, in both basal and stimulated osteoclastogenesis and the importance of RelB in both bone formation and resorption.

LPS-induced inhibition of osteogenesis is TNF-alpha dependent in a murine tooth extraction model.

TNF-alpha is a major etiologic factor of inflammatory bone diseases such as periodontitis and rheumatoid arthritis. In addition, patients with metabolic diseases such as chronic heart disease and diabetes have significantly increased plasma levels of TNF-alpha. Several lines of evidence show inhibition of osteoblastogenesis by TNF-alpha in vitro. Therefore, bone formation and osteogenesis in these patients might be inhibited because of TNF-alpha. However, little is known about the inhibitory role of TNF-alpha in bone formation/osteogenesis in vivo. The purpose of this study was to investigate the role of TNF-alpha in osteogenesis using a murine tooth extraction model. Lipopolysaccharide (LPS) was injected subcutaneously into the calvariae of either wildtype (WT) or TNF-alpha-deficient (KO) mice. The left incisor was extracted 4 days after LPS injection. The measuring area was established as the tooth socket under the mesial root of the first molar. A significant increase in serum TNF-alpha levels after LPS injection was observed in WT mice. The BMD of the tooth socket was significantly decreased by LPS injection 21 days after extraction in WT but not in KO mice. Histomorphometric analysis showed a significant decrease in the mineral apposition rate after LPS injection, which appeared at an early stage in WT but not in KO mice. Injection of a peptide that blocked the TNF-alpha signaling pathway by preventing transmission of the NF-kappaB signal recovered the inhibition of osteogenesis observed after LPS injection. In conclusion, TNF-alpha might play a major role in LPS-induced inhibition of osteogenesis under inflammatory conditions.

Bovine dentine organic matrix down-regulates osteoclast activity.

Physiological root resorption is a phenomenon that normally takes place in deciduous teeth; root resorption of permanent teeth occurs only under pathological conditions. The molecular mechanisms underlying these processes are still unclear. Our previous study showed that osteoclasts cultured on deciduous dentine exhibited a higher degree of resorption and higher levels of cathepsin K and MMP-9 mRNA than osteoclasts cultured on permanent dentine. These results could be because of different susceptibilities to acid and the different organic matrices between deciduous and permanent dentine. Thus, the purpose of this study was to investigate the effect of dentine extracts from bovine deciduous and permanent dentine on osteoclast activity. Osteoclasts, obtained from mouse bone marrow cells co-cultured with an osteoblast-rich fraction in the presence of 1,25-(OH)(2)-vitamin D3 and PGE2, were incubated with or without 0.6 M HCl extracts from bovine deciduous or permanent dentine for 48 h. TRAP positive cell number, TRAP activity, the areas of resorption pits, and mRNA levels of TRAP, v-ATPase, calcitonin receptor, cathepsin K, and MMP-9 were examined. The results illustrated that TRAP activity, the resorbed area, and the mRNA levels of osteoclast marker genes seemed to be suppressed by both deciduous and permanent dentine extracts. These findings indicate that some factors that suppress osteoclast activity are contained in both deciduous and permanent dentine extracts. Although there was no significant difference in osteoclast activity between deciduous and permanent dentine extracts, osteoclasts incubated with permanent dentine extracts tend to exhibit less resorption activity than those incubated with deciduous dentine extracts. However, we could not clearly explain the causes of this.

Inhibition of the classical NF-kappaB pathway prevents osteoclast bone-resorbing activity.

The classical NF-kappaB pathway plays an important role in osteoclast formation and differentiation; however, the role of NF-kappaB in osteoclast bone-resorbing activity is not well understood. To elucidate whether NF-kappaB is important for osteoclast bone-resorbing activity, we used a selective peptide inhibitor of the classical NF-kappaB pathway named the NBD peptide. Osteoclasts were generated using bone marrow macrophages in the presence of M-CSF and RANKL. The NBD peptide dose-dependently blocked the bone-resorbing activity of osteoclasts by reducing area, volume (p < 0.001) and depths (p < 0.05) of pits. The reduced resorption by the peptide was due to reduced osteoclast bone-resorbing activity, but not reduced differentiation as the number of osteoclasts was similar in all groups. The peptide inhibited bone resorption by reducing TRAP activity, disrupting actin rings and preventing osteoclast migration. Gene expressions of a panel of bone resorption markers were significantly reduced. The NBD peptide dose-dependently reduced the RANKL-induced c-Src kinase activity, which is important for actin ring formation and osteoclast bone resorption. Therefore, these data suggest that the classical NF-kappaB pathway plays a pivotal role in osteoclast bone-resorbing activity.

Lipopolysaccharide-induced bone resorption is increased in TNF type 2 receptor-deficient mice in vivo.

The release of tumor necrosis factor (TNF)-alpha from macrophages upon stimulation of lipopolysaccharide (LPS) is a major etiological factor of inflammatory bone disease and elicits the effects through TNF receptors type 1 and 2. Given the importance of TNF-alpha action on osteoclastic bone resorption, the role of TNF type 2 receptor (TNFR2) on bone resorption has not been elucidated well so far. The purpose of this study is to investigate the role of TNFR2 on LPS-induced inflammatory bone resorption in vivo. LPS at 10 mg/kg (Re 595) was injected subcutaneously on calvariae of wild-type (WT), TNF type 1 receptor (TNFR1)-deficient (KO), and TNFR2 KO mice, killed on day 5 after the LPS injection. The calvarial bone mineral density (BMD) was significantly decreased by LPS compared to the vehicle-injected control in WT mice, but not in TNFR1 KO mice. Interestingly, the decrease of calvarial BMD and the increase of the osteoclast number by LPS in TNFR2 KO mice seemed to be more than those in WT mice. Furthermore, the significant decrease by LPS on the BMD of tibiae, femurs, and lumber vertebrae were observed only in TNFR2 KO mice. Histomorphometric analysis of tibiae showed the significant increases of osteoclast number and surface in the LPS-injected TNFR2 KO mice, and the levels of urinary deoxypyridinoline reflected these increases of bone resorption parameters. The present data indicate that TNFR1 is critical for bone resorption at the site of LPS injection and that TNFR2 might have a protective role on the LPS-induced inflammatory bone resorption process.

Bone morphogenetic protein-2 (BMP-2) and vascular endothelial growth factor (VEGF) transfection to human periosteal cells enhances osteoblast differentiation and bone formation.

Periosteum has been demonstrated to contain mesenchymal progenitor cells differentiating to osteoblasts, and both bone morphogenetic protein-2 (BMP-2) and vascular endothelial growth factor (VEGF) may play important roles in cell-based approaches to bone regeneration. The purpose of this study was to evaluate the feasibility and efficacy of BMP-2 and/or VEGF on periosteal cell differentiation to osteoblasts in vitro and ectopic bone formation in vivo. Human periosteum-derived cells were transfected with BMP-2, VEGF, BMP-2 + VEGF, or vehicle as a control by non-viral gene transfer and then cultured and implanted to nude mice intramuscularly. Real-time polymerase chain reaction analysis of the culture revealed that transgenes for BMP-2 and BMP-2 + VEGF induced more mRNA expression of alkaline phosphatase, collagen type I, and osteocalcin than VEGF and vehicle treatments; additionally, alizarin red S staining, alkaline phosphatase staining, and alkaline phosphatase activity were significantly higher in the BMP-2 + VEGF transgene than in the other versions. After implantation, ectopic bone was observed at 4 weeks and greatly increased at 8 weeks in all groups. In particular, the combination of BMP-2 and VEGF formed significantly more bone at 4 weeks, and VEGF transfection resulted in more blood vessels relative to the conditions without VEGF. Thus, VEGF might enhance BMP2-induced bone formation through modulation of angiogenesis.

Small GTPase Ras and Rho expression in rat osteoblasts during spaceflight.

Rat osteoblasts were cultured for 4 and 5 days aboard a space shuttle and solubilized after a 24-h treatment with 1alpha,25 dihydroxyvitamin D(3). The quantitative RT-PCR determined the mRNA levels of signaling molecules upstream and downstream Ras. The small GTPase is activated by guanine nucleotide exchange protein (GEF) and deactivated by GTPase-activating protein (GAP). When external stimuli are transduced into intracellular signals, various pathways are recruited: focal adhesion kinase (FAK) is associated with integrin-beta, and directs tyrosine phosphorylation of downstream substrates, including phospholipase C-gamma (PLC-gamma) and son of sevenless (SOS, a Ras GEF). The mRNA levels of FAK and PLC-gamma1 and -gamma2 in the flight cultures were increased 150% and 250% of the ground controls. The SOS mRNA levels in the flight cultures were increased 520% and 320% of the ground controls. Signals via G protein-coupled receptors are transmitted through PLC-beta and Ras GRF (another Ras GEF). Activated Ras then stimulates Raf, mitogen-activated protein kinase (MAPK) cascades. The mRNA levels of Raf, extracellular signal-regulated protein kinase of MAPK family (ERK-1 and -2), and PLC-beta were increased during spaceflight. Rho GAP expression in the flight cultures was increased twofold of the ground controls. Since Rho GAP deactivates Rho, microgravity may suppress Rho signals, regulating actin filament rearrangement. Microgravity signals may involve two pathways (G protein-coupled receptor-mediated pathway and tyrosine phosphorylation-mediated pathway) that activate Ras, Raf, and MAPK cascades in rat osteoblasts.

Expression of MT1-MMP during deciduous tooth resorption in odontoclasts.

Membrane type 1-matrix metalloproteinase (MT1-MMP) is a membrane-bound matrix metalloproteinase capable of mediating pericellular proteolysis of extracellular matrix components. In osteoclasts, the localization of MT1-MMP has been reported at the tips of specialized membrane protrusions (podosomes and lamellipodia) so that osteoclasts might use MT1-MMP to perform focal proteolysis and move through the extracellular matrix to the bone surface. The objectives of this study were to investigate an association of MT1-MMP in physiological root resorption of the deciduous tooth by reverse transcriptase-polymerase chain reaction (RT-PCR) and Northern blot analysis, and to identify MT1-MMP-producing cell during deciduous tooth resorption by in situ hybridization and immunohistochemistry. RT-PCR and Northern blot analysis revealed the exclusively high expression of MT1-MMP mRNA in bovine root-resorbing tissue, which lies between the root of the deciduous tooth and its permanent successor. Expression of MT1-MMP mRNA was seen in odontoclasts aligning in the surface layer of the root-resorbing tissue at sites of root resorption. Furthermore, immmunohistochemistry also confirmed the localization of MT1-MMP protein to the odontoclasts. The present identification of MT1-MMP in odontoclasts during deciduous tooth resorption might be relevant to the migration activity that these cells have to gain access to the root surface.

Combination of 3D tissue engineered scaffold and non-viral gene carrier enhance in vitro DNA expression of mesenchymal stem cells.

The objective of this study is to enhance the expression of a plasmid DNA for mesenchymal stem cells (MSC) by combination of 3-dimensional (3D) tissue engineered scaffolds and non-viral gene carrier. As a carrier of plasmid DNA, dextran-spermine cationic polysaccharide was prepared by means of reductive-amination between oxidized dextran and the natural oligoamine, spermine. As the MSC scaffold, collagen sponges reinforced by incorporation of poly(glycolic acid) (PGA) fibers were used. A complex of the cationized dextran and plasmid DNA of BMP-2 was impregnated into the scaffolds. MCS were seeded into each scaffold and cultured by a 3D culture method. When MSC were cultured in the PGA-reinforced sponge, the level of BMP-2 expression was significantly enhanced by the cationized dextran-plasmid DNA complex impregnated into the scaffold than by the cationized dextran-plasmid DNA complex in 2-dimensional (2D) (tissue culture plate) culture method. The alkaline phosphatase activity and osteocalcin content of transfected MSC cultured in the PGA-reinforced sponge were significantly higher compared with 2D culture method. We conclude that combination of cationized dextran plasmid DNA complex and 3D tissue engineered scaffold was promising to promote the in vitro gene expression for MSC.