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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.

Previous studies have demonstrated that carbonate apatite (CA) is superior to hydroxyapatite (HA) and β-tricalciumphosphate (β-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, β-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 β-TCP as a bioresorbable bone substitute for tissue engineering.

Immunohistochemical localization of two enamel proteins, amelogenin and enamelin, in comparison with that of keratin, was determined in odontogenic tumors and the allied lesions in order to verify functional differentiation of the tumor cells as ameloblasts. Amelogenin and enamelin were demonstrated in small mineralized foci and in the tumor cells surrounding them in adenomatoid odontogenic tumor (AOT), calcifying epithelial odontogenic tumor (CEOT), and calcifying odontogenic cyst (COC). Hyaline droplets in AOT showed positive staining for both enamel proteins. These mineralized and hyaline materials were not positive for keratin, although tumor cells were positive. On the other hand, no immunoreaction for enamel proteins was obtained in ameloblastoima and odontogenic epithelial cell nests within myxoma and epulis. The results suggest that tumor cells of AOT and CEOT and lining epithelial cells of COC show ameloblastic differentiation in part, but that ameloblastoma cells do not attain functional matauration as secretory phase ameloblasts.

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 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.

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.

Various growth factors are known to play important roles in wound healing, especially in an early inflammatory phase. However, their roles in subsequent scar formation phase are relatively unexplored. The aim of this study is to investigate the mechanisms of regulation in scar formation by these factors. Scar fibroblasts (SF) were obtained from immature scar tissue made at rat hard palate 1 month after excision and normal fibroblasts (NF) were obtained from the palatal mucosa of untreated control animals. SF showed a longer doubling time, and increased level of protein synthesis when compared to NF. Platelet-derived growth factor (PDGF) and epidermal growth factor (EGF) stimulated [ 3 H]-thymidine uptake less effectively in SF than in NF. In both cells, transforming growth factor-β1 (TGF-β1) inhibited EGF-induced stimulation of [ 3 H]-thymidine uptake, but had no effects when it was added alone. TGF-β1 increased collagen synthesis more effectively in SF than in NF. These data indicate that the growth factors may play key roles in regulating proliferation and metabolic activity of fibroblasts during scar formation.

The polymerase chain reaction was used to amplify bovine tooth amelogenin cDNA, resulting in several products which were separated by agarose gel electrophoresis. Sequence determination of one of the products revealed that it encoded an amino acid sequence identical to that of a small leucine-rich amelogenin polypeptide (LRAP) previously characterized by protein sequencing. Comparison of the nucleotide sequence of this cDNA with that determined for the cloned bovine amelogenine gene strongly suggested that the LRAP transcript resulted from alternative splicing of the primary transcript of this gene, thus explaining the origin of the puzzling LRAP sequence. Analysis of the structure of LRAP suggests that the polypeptide might exhibit interesting properties relative to hydroxy apatite crystal formation.

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 (p0.001) and depths (p0.05) 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.

Monoclonal antibody against bovine serum α2HS glycoprotein was raised in mice and the localization of this glycoprotein in bovine and human teeth was examined by immunohistochemical techniques on undemineralized ground sections after surface etching with dilute HCl. Positive reactions were found in both bovine and human peritubular dentin, whereas virtually negative staining was detected in the intertubular dentin. When the ground section was examined without surface etching, no reaction was found in any area of mineralized dentin. These results suggest that α2HS glycoprotein is present mainly in the peritubular dentin associated with the mineral phase. The presence of this component in dentin may not be due simply to nonspecific binding to calcium phosphate crystals.

Chondrocyte differentiation is a fundamental process during endochondral ossification. Retinoic acid (RA) has been shown to regulate this process, however, the mechanisms underlying RA regulation of chondrogenesis are not clearly understood. Chondroprogenitor cells, ATDC5 have been shown to be a useful in vitro model for examining the multiple step differentiation of chondrocytes. The present study investigated the mechanisms underlying RA regulation of chondrogenesis using ATDC5 cell culture. In this study, we show that RA suppresses the cell growth, cartilage nodule formation, accumulation of proteoglycan, alkaline phosphatase (ALPase) activity and mineralization and that RA dose dependently upregulates the levels of type X collagen and matrix metalloproteinase-13 (MMP-13) mRNA which are marker proteins of hypertrophic chondrocytes, in ATDC5 cells. The addition of protein synthesis inhibitor, cycloheximide (CHX), partially inhibits the induction of type X collagen and MMP-13 mRNA by RA. In this system, RA upregulates the mRNA level of Runx2/Cbfa1 (type II), a positive regulator for mineralization, and downregulates the mRNA of Indian hedgehog (Ihh), parathyroid hormone related protein (PTHrP), negative regulators for terminal differentiation. However, RA downregulates ALPase, bone gla protein (BGP) mRNAs and mineralization. These data indicate that RA stimulates cartilage differentiation, however, cell condensation and cartilage nodule formation may be candidates of primary importance in the terminal differentiation of chondrocytes.

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.

This article describes the development of an in vitro culture system to enhance the expression of a plasmid DNA for mesenchymal stem cells (MSCs) by a combination of plasmid DNA impregnation into three-dimensional cell scaffolds and culture methods. Gelatin was cationized by introducing spermine to the carboxyl groups for complexation with the plasmid DNA. As the MSC scaffold, poly(glycolic acid) (PGA) fiber fabrics, collagen sponges, and collagen sponges reinforced by incorporation of PGA fibers were used. A complex of cationized gelatin and plasmid DNA encoding bone morphogenetic protein 2 (BMP-2) was impregnated into the scaffolds. Plasmid DNA was released from PGA-reinforced collagen sponge for longer than from the other scaffolds. MCS were seeded into each type of scaffold and cultured by static, stirring, and perfusion methods. When MSCs were cultured in PGA-reinforced sponge, the level of BMP-2 expression was significantly enhanced by perfusion culture compared with the other culture methods, and the time of expression was prolonged. Irrespective of the culture method, the expression level was significantly higher from plasmid DNA impregnated in scaffold than by plasmid DNA in medium. The alkaline phosphatase activity and osteocalcin content of MSCs cultured in PGA-reinforced sponge by the perfusion method were significantly higher compared with those of other methods, and a significantly higher amount of plasmid DNA internalized into MSCs was observed. We conclude that a combination of plasmid DNA-impregnated PGA-reinforced sponge and the perfusion method was promising to promote in vitro gene expression for MSCs.

Many clinical reports suggest that deciduous teeth exhibit a greater susceptibility to resorption than permanent ones. To examine the difference between deciduous and permanent dentine in their susceptibility to osteoclastic resorption, osteoclast-like cells (OCLs) were cultured on deciduous and permanent dentine slices. The number, area, depth, and volume of resorption pits were then measured, using image-analyzing systems. We measured the level of degraded collagen (cross-linked N-telopeptide of type I collagen; NTx) in culture medium using an enzyme-linked immunosorbent assay (ELISA). The levels of cathepsin K, matrix metallo proteinase (MMP)-9, and MMP-13 mRNAs in the cells attached to dentine were also analyzed by real-time reverse transcription polymerase chain reaction (RT-PCR). Deciduous dentine slices exhibited a significant (twofold) increase in resorbed area compared with the permanent slices. Three-dimensional analysis revealed that the volume of pits in deciduous dentine differed significantly (fourfold) compared to that in the permanent dentine. The depth of pits also followed the same trend. However, there was no significant difference in the number of pits or osteoclasts on the dentine slices. The NTx level in deciduous media was significantly more than that in permanent media. The mRNA levels also followed the same trend. These results suggest that deciduous dentine is more susceptible to resorption than permanent dentine and signals from the substrate play an important role in physiological resorption.

Rat osteoblasts were cultured aboard a space shuttle for 4 and 5 days. Cells were treated with 1 nM 1alpha,25-dihydroxyvitamin D(3) (VD) for the last 1 day. The conditioned media were harvested. Cells were solubilized with guanidine solution on board. We examined microgravity effects on the production/expression of osteocalcin, bone sialoprotein (BSP), and VD receptor (VD-R) in osteoblasts. Under VD treatment, the osteocalcin protein level was 243 +/- 117 and 1,718 +/- 534 pg/microg cellular DNA in flight cultures and ground controls, respectively. Without VD treatment, the osteocalcin protein level was not different between flight cultures and ground controls. The osteocalcin mRNA level in the VD-treated flight cultures was as low as 16% of that in ground controls. The VD-R mRNA level in the VD-treated flight cultures was also decreased to 16% of that in ground controls. Microgravity would suppress the VD-inducible production of osteocalcin but not the basal productivity. The BSP mRNA level was increased by microgravity. VD/VD-R binds to the vitamin D-responsive element (VDRE) on the target genes. The rat osteocalcin gene is positively regulated via "enhancer" VDRE, whereas the rat BSP gene is negatively regulated via "repressor " VDRE. Microgravity might modulate osteoblast responsiveness to VD through the suppression of VD-R.

The platelet-activating factor (PAF) is a lipid mediator. The G-protein-coupled receptor of PAF (PAF-R) is activated by inflammatory and stressful conditions in numerous cell types. PAF/PAF-R is involved in apoptotic and antiapoptotic processes. We examined microgravity effects on the expression of PAF-R and second messengers in rat osteoblasts. The PAF-R signals are transmitted via arachidonic acid, phospholipase C (PLC), protein kinase C (PKC), and mitogen-activated protein kinase. Rat osteoblasts were cultured for 4 and 5 days aboard a space shuttle and solubilized on board. PAF-R gene expression in flight cultures increased to 2-6-fold higher than in ground controls. Gene expression of the G-protein alpha subunit Galphaq in flight cultures increased to 3-fold and higher than in ground controls. It is known that Galphaq stimulates the effecter PLCbeta, activating PKC. The mRNA levels of PKCdelta and PKCtheta in flight cultures were increased to 2-5-fold higher than in ground controls. The PKCalpha mRNA level in flight cultures was increased to 3-fold higher than in ground controls on the 4th day. Gene expression of catalytic and regulatory subunits of protein kinase A was suppressed in flight cultures. PKCdelta and PKCtheta are novel PKCs that can be target substrates of caspases. The PAF-R gene may act as a mechano-sensitive gene that is involved in the apoptotic and antiapoptotic processes of osteoblasts under microgravity.

Dentin matrix protein 1 (DMP1) is an Arg-Gly-Asp-containing acidic phosphoprotein that was originally identified from a rat incisor cDNA library and was thought to be a dentin-specific protein. DMP1 was later shown to express in a number of hard tissue-forming cells, including osteoblasts, osteocytes, ameloblasts, and cementoblasts, and was considered to play important roles in mineralization. Further, DMP1 gene expression was also detected in fetal bovine brain and in newborn mouse brain. These findings indicate the possibility of DMP1 expression in other soft tissues. In the present study, to clarify the significance of DMP1 expression in nonmineralized tissues, we made a specific antibody to mouse DMP1 peptides and demonstrated that DMP1 protein was localized in mouse brain, pancreas, and kidney by immunohistochemistry. Further DMP1 mRNA was detected in nonmineralized mouse tissues including liver, muscle, brain, pancreas, and kidney by RT-PCR. Based on the evidence that the localization and the expression of DMP1 are not restricted to mineralized tissues, we assume that DMP1 may have functions other than the regulation of mineralization.

MMP-13 appears to be one of the most important MMPs in cartilage remodeling and mineralization, because it exhibits a substrate preference for the cartilage-specific type II collagen. The condylar process is constructed by rapid accumulation of hypertrophic chondrocytes during development, but its mechanism is still unclear. To investigate the role of MMP-13 in developing condylar cartilage, we immunohistochemically examined the localization of MMP-13 in the endochondral ossification of the mandibular condyle and tibiae of newborn mice. In the tibiae, the MMP-13 expression was detected only in the deepest layer of the terminal hypertrophic chondrocytes through every examined stage (day 1 to day 10 after birth). On the other hand, in the condylar cartilage at days 1 and 5, MMP-13 was expressed throughout the proliferating and the hypertrophic chondrocytes, and at day 10, MMP-13 was mainly localized in the deepest edge of the hypertrophic layer. A zymographical study showed that the activity of MMP-13 in the condyle was observed at day 1, earlier than in the tibia, and increased until day 7. The time-dependent and cell-specific expression of MMP-13 and its enzymatic property suggest that in the mandibular condylar cartilage, MMP-13 plays a role in making the space for cell enlargement by degradation of the cartilage matrix and in onset of mineralization during the early stage of development.

The mechanism underlying space flight-induced osteopenia is unknown. In osteoblasts, the inducible nitric oxide (NO) synthase (iNOS) is involved in the early response to mechanical strain and induction of apoptosis. GTP cyclohydrolase I (GTPCH) is a key enzyme that is essential for iNOS activity. The coordinate expression of GTPCH prevents apoptosis that is induced by iNOS/NO. The purpose of this study was to investigate the effects of space flight on the expression of apoptotic/anti-apoptotic molecules iNOS and GTPCH in rat osteoblasts. Rat osteoblasts were cultured aboard a space shuttle and solubilized on the 4th and 5th days of the mission. The mRNA levels for iNOS and GTPCH in the flight cultures were increased to at least 120-fold and threefold higher than the ground (1 × g) controls, respectively. The amount of cellular DNA per flight culture vessel was 53% and 58% of the ground controls on the 4th and 5th days, respectively. However, the increasing rate of the DNA amount from the 4th to the 5th day was not different between the flight cultures and the ground controls. Morphologically, the cells grew in space as well as on the ground. Co-expression of GTPCH and iNOS may indicate a self-protective mode of action in osteoblasts against the harmful stress under microgravity.

A functional disorder associated with weightlessness is well documented in osteoblasts. The apototic features of this disorder are poorly understood. Harmful stress induces apoptosis in cells via mitochondria and/or Fas. The Bax triggers cytochrome c release from mitochondria, which can be blocked by the Bcl-2. Released cytochrome c then activates the initiator caspase, caspase-9, which can be blocked by the anti-apototic (IAP) family of molecules. The effector caspase, caspase-3, finally exerts DNA fragmentation. We conducted this study to examine the apoptotic effects of vector-averaged gravity on normal human osteoblastic cells. Cell culture flasks were incubated on the clinostat, which generated vector-averaged gravity condition (simulated microgravity) for 12, 24, 48, and 96 hours. Upon termination of clinostat cultures, the cell number and cell viability were assessed. DNA fragmentation was analyzed on the agarose-gel electrophoresis. The mRNA levels for Bax, Bcl-2, XIAP, and caspase-3 genes were analyzed by semi-quantitative RT-PCR. Twenty-four hours after starting clinostat rotation, the ratios of Bax/Bcl-2 mRNA levels (indicator of apoptosis) were significantly increased to 136% of the 1G static controls. However, the XIAP mRNA levels (anti-apoptotic molecule) were increased concomitantly to 138% of the 1G static controls. Thus, cell proliferation or cell viability was not affected by vector-averaged gravity. DNA fragmentation was not observed in clinostat group as well as in control group. Finally, the caspase-3 mRNA levels were not affected by vector-averaged gravity. Simulated microgravity might modulate some apoptotic signals upstream the mitochondrial pathway.

The mechanism of space flight-induced bone mass loss is unknown. We conducted experiments aboard the Space Shuttle using primary cultured osteoblasts. During flight, quadruplicate cultures were treated with 1 nM of 1alpha,25 dihydroxyvitamin D3 for one day, then fixed with guanidine isothiocyanate solution on board. After return to the Earth, the mRNA levels were analyzed by quantitative RT-PCR. Microgravity increased the mRNA levels of JNK, c-Jun N-terminal kinase or stress-activated protein kinase, in rat osteoblasts, as compared to the 1G ground control. Microgravity decreased the mRNA levels of the principal heat shock protein, Hsp 70. JNK is known to play a key role particularly in the stress-activated cell apoptosis. Data suggested apoptotic and anti-apoptotic effects of microgravity on rat osteoblast.

Spaceflight and bed rest induce loss of bone mass. A number of in vivo and in vitro studies have been conducted to clarify the mechanisms, however, the results have been conflicting. The purpose of this study was to investigate the effects of gravity unloading on proliferation, phenotypes, and apoptosis of normal human osteoblastic cells in the presence of 1alpha,25-dihydroxyvitamin D3. We used a vector-averaged gravity condition generated by clinostat rotation to simulate gravity unloading. Clinostat rotation did not affect the cell proliferation. On the first day, the mRNA levels for osteocalcin, ALP, CBFA1, VDR, RANKL, and OPG were reduced by clinostat rotation to 21%, 65%, 62%, 52%, 43%, and 54% of control, respectively. ALP activity was decreased to 75% of control. On the second day, the mRNA levels for osteocalcin and RANKL were reduced to 77% and 61% of control, respectively. The decreased VDR mRNA level might be responsible for the reduction for mRNA levels for osteocalcin, RANKL, and OPG. Clinostat rotation increased the pro-apoptotic index (Bax/Bcl-2 ratio) but did not induce apoptosis due to the simultaneous upregulation of the anti-apoptotic XIAP. Reduction of osteoblast responsiveness to 1alpha,25-dihydroxyvitamin D3 might be involved in osteopenia that is induced by gravity unloading.

Expanding cells ex vivo is an important step in tissue engineering and the culture medium is usually supplemented with bovine serum. When a patient receives bone marrow stromal cells (BMSCs) grown in a medium containing bovine serum, infection of bovine diseases and/or the patient's unfavorable immune reaction to bovine proteins are of concern. To overcome these problems, we examined whether a patient's autologous serum could support the growth and differentiation of his/her BMSCs.Bone marrow was collected from the iliac by aspiration and cultured in a medium supplemented with 10% autologous serum or 10% fetal bovine serum (FBS). Number and area of the colonies of fibroblast-like cells (colony-forming unit fibroblast, CFU-f) were measured 8 days after the cell inoculation (day 8). Number and area of the alkaline phosphatase (ALP) positive colonies were measured on day 10. On day 40, the cultures were stained with alizarin red S. RNA was prepared from the culture on day 20, and the mRNA expression of osteoblastic genes was examined by reverse transcription polymerase chain reaction (RT-PCR) analysis.BMSCs, which were cultured in the medium supplemented with autologous serum, produced CFU-f, ALP-positive area and mineralized nodules, which is comparable to the BMSCs in the culture supplemented with FBS. The mRNA expressions of osteopontin, parathyroid hormone receptor, osteocalcin, and bone sialoprotein were detected in the culture supplemented with autologous serum.A patient's autologous serum could expand BMSCs without losing their potentiality for osteoblastic differentiation. Patients' autologous serum could be efficient to expand BMSCs and to be utilized safely for bone regeneration therapy.

The primary culture of rat osteoblasts was treated with 1alpha,25 dihydroxyvitamin D(3) and fixed with guanidine isothiocyanate solution during a space shuttle flight. The mRNA levels were analyzed by quantitative reverse transcription-polymerase chain reaction. Microgravity decreased the mRNA levels of insulin-like growth factor-I (IGF-I) and increased reciprocally the IGF-I receptor mRNA levels, as compared to the ground (1 x g) control. Microgravity completely suppressed the mRNA expression of the insulin receptor substrate-1, the postreceptor signaling molecule of IGF-I. Microgravity might deteriorate the action and signaling of IGF-I in rat osteoblasts.

Odontoma-like structures are formed in the jaw bone of osteopetrotic (op/op) mice, which have a congenital deficiency in osteoclastic differentiation due to the absence of functional macrophage colony-stimulating factor (M-CSF).To clarify the histopathogenesis of tooth malformation and odontoma-like structures, a 2-year postnatal process of development of the op/op mandibular incisor was examined radiologically and histologically. At the same time, extracellular matrix (ECM) remodeling around tooth germs was analyzed immunohistochemically.Abnormal forms of op/op tooth germ were noticeable even at 3 days after birth on a radiogram. Histologically, op/op mice were clearly distinguished by the disappearance of dental follicular space at 3 days. With aging, bone trabeculae, which were not remodeled, penetrated into op/op tooth germs and divided them into several daughter germs, which were recognized as odontomas. In mandibular incisor bodies, the immature ECM components, such as heparan sulfate proteoglycan and tenascin, were preserved diffusely in the dental papilla/pulp, which indicates that maturation of the stroma does not take place in op/op mandibular incisors.The observation suggests that the disturbed morphogenesis of op/op tooth germs is functionally explained by the disordered immunolocalization of ECM molecules, and that the dental follicular space is essential for normal tooth development because it prevents bone penetration into the tooth germs.

Young Wistar male rats were exposed to 2G hypergravity by continuous centrifugation for 15 minutes. The nociceptive threshold was measured by using the von Frey type filament on the rat skin surfaces after hypergravity exposure. Following the hypergravity exposure, rats were sacrificed with anesthesia, then perfused and fixed for immunohistochemical examination. The 2G hypergravity elevated the nociceptive threshold up to 2-fold and induced analgesic effects on rats that remained for 2 hours after termination of centrifugation. Expression of Fos-immunoreactive proteins was prominently induced by 2G hypergravity in the arcuate nucleas and the paraventricular nucleus of the hypothalamus. The 15-minute flash exposure to 2G hypergravity induced pain suppression in rats, which might be attributed to change of neuronal activity in rat hypothalamus.

The tooth in the gar-pike, Lepisosteus oculatus, an actinopterygian fish, is characterized by the occurrence of both enamel and enameloid, the former covering the tooth shaft and the latter, the tooth cap. Our previous research demonstrated that the enamel in this species was, as in the lungfish, immunoreactive for amelogenin, indicating its homologous nature with the mammalian tooth enamel, whereas the enameloid was completely immunonegative. The present study demonstrates that, during the early maturation stage of the enameloid formation, the inner enamel epithelial cells (IEECs) synthesize through a well-developed Golgi apparatus a fine-granular substance which is intensely immunoreactive for amelogenin. This substance was accumulated in a large saccule extended in a suprabasal zone of the cell; we were unable to find any morphological sign indicating a connection of the substance with the enameloid matrix. The abortive secretion of the enamel matrix-like substance in the IEEC during an enameloid formation was considered to be an instance of rudimental enamel formation. In the gar-pike, the synthesis of amelogenin in the IEEC has been demonstrated to occur independently from that of the enameloid matrix. The present findings demonstrate a prominent difference between the tooth enamel and enameloid.

Interferon-gamma (IFN-gamma), a multifunctional cytokine, has been noted as a potential therapeutic agent for various fibrotic disorders, including excessive scar tissue formation. We previously reported that transforming growth factor-beta1 (TGF-beta1) induced the myofibroblastic phenotype in palatal fibroblasts derived from palatal mucosa, and that such effects might have a close link to palatal scar formation. In the present study, we examined the effects of IFN-gamma on TGF-beta1-pretreated palatal fibroblasts for the purpose of clarifying the suppressive potency against myofibroblastic phenotype expression in vitro. IFN-gamma significantly altered the spindle morphology of TGF-beta1-pretreated palatal fibroblasts into the polygonal one that was similar to the non-treated palatal fibroblasts. This change was parallel with a decrease in the expression of alpha-smooth muscle actin protein, a marker for myofibroblast, as determined by immunoblot analysis. Northern blot analysis showed that IFN-gamma inhibited proalpha2(I) collagen mRNA expression that was stimulated by TGF-beta1 pretreatment for 24 h. Furthermore, IFN-gamma decreased the cell contractility enhanced by TGF-beta1 pretreatment for 24 h in a three-dimensional collagen gel culture system. These results suggest that IFN-gamma may have negative effects with regard to controlling the myofibroblastic phenotype induced by TGF-beta1 in palatal fibroblasts.

Rat osteoblasts were cultured for 4 or 5 days during a Space Shuttle mission. After 20-h treatment with 1α,25-dihydroxyvitamin D3, conditioned media were harvested and cellular DNA and/or RNA were fixed on board. The insulin-like growth factor binding protein (IGF BP)-3 levels in the media were three- and tenfold higher than in ground controls on the fourth and fifth flight days, as quantitated by Western ligand blotting and radioimmunoassay, respectively. The increased IGF BP-3 protein levels correlated with two- to threefold elevation of IGF BP-3 mRNA levels, obtained by reverse transcription-polymerase chain reaction. The IGF BP-5 mRNA levels in flight cultures were 33–69% lower than in ground controls. The IGF BP-4 mRNA levels in flight cultures were 75% lower than in ground controls on the fifth day but were not different on the fourth day. The glucocorticoid receptor mRNA levels in flight cultures were increased by three- to eightfold on the fourth and fifth days compared with levels in ground controls. These data suggest potential mechanisms underlying spaceflight-induced osteopenia.

Amelogenesis imperfecta is a hereditary disease of the enamel that is unassociated with generalized defects. Cases of the condition are clinically classified into three groups: hypoplastic, hypomaturation, and hypocalcified. In this study, soluble protein fractions of the enamel from three patients with hypocalcified amelogenesis imperfecta were examined through the use of immunochemical and biochemical techniques. In immunochemical analyses done with a polyclonal anti-amelogenin antibody, all samples from enamel in which there was amelogenesis imperfecta were found to contain considerable amounts of amelogenin peptides. When an enamel sample from one patient was examined by Western-blot transfer and immunobinding analysis, the amelogenin fraction was found to consist of a 26-kDa molecule thought to be normally present in the outer layer of secretory-stage enamel. This enamel was also found to contain albumin as one of the major constituents of the protein fraction. These results suggest that hypocalcified amelogenesis imperfecta may in part be caused by a disturbance in matrix protein degradation during the maturation phase.

The effect of basic fibroblast growth factor (bFGF) on apoptosis in normal rat palatal fibroblasts and rat palatal scar fibroblasts was examined by the TUNEL method in order to clarify the mechanism of apoptosis induction in myofibroblasts during the scar formation process. A percentage of scar fibroblasts undergoing apoptosis was significantly higher than that of palatal fibroblasts when they were treated with bFGF succeeding to serum starvation. Palatal fibroblasts, phenotypically modulated into myofibroblasts by the pretreatment with transforming growth factor-beta 1 (TGF-beta 1), similarly showed a higher level of apoptosis induction by bFGF-treatment. TGF-beta 1 elevated protein and mRNA level of FGF receptor (FGFR) in palatal fibroblasts. Tyrosine autophosphorylation of FGFR upon stimulation by bFGF was significantly higher in scar fibroblasts than in normal palatal fibroblasts. These findings suggested that bFGF may be a potential stimulator of apoptosis in myofibroblasts during palatal scar formation and that FGFR may be responsible for this process.

Gene expression and localization of amelogenin were studied in the developing rat incisor by the methods of in situ hybridization and immunohistochemistry. ISH revealed the first expression of amelogenin mRNA in the inner enamel epithelium of the cervical loop. The signals were clearly observed in pre-ameloblasts in the region bordering on predentin formation and became more intense toward the cells on the initial enamel matrix secretion. The maximal signals were found in the cytoplasm of secretory ameloblasts. From the terminal secretion zone, the signals then became gradually weaker toward the incisal edge but were still evident in the cytoplasm of shortening, transitional ameloblasts and those at the early maturation stage. No signals were found in the cells of the stratum intermedium and stellate reticulum throughout amelogenesis. Immunohistochemistry by means of an antibody against amelogenin C-telopeptide consisting of 12 amino acids revealed immunoreaction in the secretory ameloblasts reacting to the ISH. When a polyclonal antibody against amelogenin was used, immunoreaction was found in the distal ends of ruffle-ended ameloblasts (RA) in the maturation zone. Those results indicated that amelogenin is synthesized by ameloblastic cells from the inner enamel epithelium to the early maturation stage and is then resorbed by the RA.

A genomic DNA clone (14.5 kbp) for amelogenin, a tooth enamel specific protein, has been isolated from a mouse genomic library. The nucleotide structure of this DNA was determined by the polymerase chain reaction and sequence analysis. The result indicated that the cloned DNA contained all the exons in addition to approximately 5.5 kbp of upstream sequence.

PURPOSE To determine factors that control ocular enlargement in experimental form-deprivation myopia and to clarify the mechanism of form-deprivation myopia. METHODS After the left eyes of 20 chicks were monocularly occluded for 2 weeks, protein, basic fibroblast growth factor (bFGF) and transforming growth factor (TGF)-beta 2 contents in samples of constant area (circular button, diameter = 8.5 mm) in the retina-retinal pigment epithelium (RPE)-choroid and the sclera in the posterior region of control and myopic eyes were determined by enzyme-linked immunosorbent assay. RESULTS The bFGF content (per circular button) and bFGF concentration (per mg protein) were significantly lower in the sclera in the posterior region of the myopic eyes than in control eyes. The bFGF content and concentration were similar in the retina-RPE-choroid in myopic and control eyes. The TGF-beta 2 content and concentration were significantly higher in myopic eyes in both the retina-RPE-choroid and the sclera (P < 0.05). CONCLUSIONS These results are consistent with the possibility that bFGF and TGF-beta 2 regulate ocular enlargement or respond to myopiagenic mechanisms in form-deprivation myopia.

Screening of a bovine odontoblast cDNA library from developing incisor with murine Msx-1 and Msx-2 cDNA probes led to the isolation of three positive clones. All of them encoded for a sequence of a protein containing 297 amino acids. The responsible gene was designated as bovine Msx-1 (bMsx-1) due to the high homology with the human MSX-1 and mouse Msx-1 sequences.

The odontoclast, which is morphologically similar to osteoclast, is thought to play a major role in root resorption of deciduous teeth. High collagenolytic activity has been detected in the root resorbing tissue. In order to identify collagenase-producing cells and the role of collagenase in root resorption of deciduous tooth, in situ hybridization of collagenase mRNA in bovine root-resorbing tissue sections was performed using a digoxigenin-labeled, nonradioactive RNA probe. Collagenase mRNA expression was clearly observed in odontoclasts in addition to the macrophages, fibroblasts, odontoclasts, and cementoblasts. Multinuclear odontoclasts showed intense tartrate-resistant acid phosphatase (TRAP) activity. We also examined interleukin-1 (IL-1) mRNA expression in the root-resorbing tissue by in sity hybridization. IL-1 transcripts were found to be expressed in odontoclasts, fibroblasts, and macrophages suggesting that IL-1 might be an important factor for promoting root resorption. These results suggest that the collagenase produced by odontoclasts may play a key role in dentin collagen degradation in root resorption.

Osteonectin, gamma-carboxyglutamic acid-containing (Gla) protein, and dentin phosphoprotein were covalently attached to sepharose beads and inoculated in solutions at two different degrees of supersaturation with respect to both octacalcium phosphate (OCP) and hydroxyapatite. In both solutions, the inhibitory activity towards de novo formation of calcium phosphate that these proteins display when freely dissolved in solution was completely eliminated when they were immobilized on the sepharose at concentrations of up to 5 micrograms/mg wet beads. In the solution that was more highly supersaturated with respect to OCP, the immobilized dentin phosphoprotein, moreover, was found to induce de novo formation of OCP in proportion to the concentration of the protein immobilized. For example, at 10 micrograms/ml of the immobilized dentin phosphoprotein, the induction period was reduced more than 50%. However, in the solution considerably less supersaturated with respect to OCP, none of the immobilized proteins were capable of inducing OCP or apatite deposition. These findings suggest that the immobilized dentin phosphoprotein could work as a nucleating substrate for the OCP phase in solutions where calcium and phosphate concentrations are sufficiently higher than equilibrium saturation levels for the OCP phase.

The present studies were undertaken to investigate the presence of common epitopes of mammalian amelogenins at the C-terminus and the possible functional importance of the conserved C-terminal domain in enamel mineralization during mammalian amelogenesis. Enamel proteins, including the intact amelogenins and their degraded polypeptides, were isolated from the secretory enamel of pig, cow, rat, and rabbit incisors. Rabbit and rat antipeptide sera, as well as rat anti-25 kD and 20 kD pig amelogenin sera, were used to identify the amelogenins among the isolated matrix proteins of each of the animal species. The antipeptide sera were developed previously (Aoba et al. [19]) using as immunogens the two synthetic peptides, C13 and C25, which correspond to the last 12 (plus Cys for KLH-conjugation) and 25 amino acid residues of pig intact amelogenin, respectively. Reactivity of the enamel proteins with each antiserum was examined by Western blot analysis. The results of immunoblotting showed that a few enamel matrix proteins in each of the mammalian species were recognized by the anti-C13 serum, specifically, pig amelogenin at 25 kD (and trace components at 27, 22, and 18 kD), cow amelogenin at 28 kD (trace components at 26, 22, 19, and 14 kD), rat amelogenins at 28 and 26 kD (and a trace component at 20 kD), and rabbit amelogenins at 24 and 21 kD (and a trace at 13 kD). The anti-C25 serum reacted additionally with pig amelogenin at 23 kD, cow amelogenin at 27 kD (a major matrix constituent), and rabbit protein at 19 kD. The anti-pig 20 kD amelogenin (lacking the last 25 amino acid residues at the C-terminus) serum reacted with a large number of pig, cow, and rat amelogenins but, interestingly, with none of the rabbit enamel proteins. Probing of rat enamel proteins with Maclura pomifera lectin showed the heterogeneity of glycosylation of rat amelogenins, particularly between the 28 and 26 kD intact amelogenins. In parallel adsorption studies, part of the enamel protein samples isolated from each of the species was used as adsorbates to investigate the selective adsorption of amelogenins onto hydroxyapatite. Immunoblot analysis of the proteins adsorbed onto the crystals revealed that the mammalian amelogenins having the common epitopes at the C-terminus, in general, adsorb preferentially onto hydroxyapatite. The adsorption affinity of the degraded amelogenins decreased significantly with the loss of reactivity toward the anti-C13 serum. The overall results support the contention that the intact mammalian amelogenins, including rat and rabbit amelogenins, share common epitopes at the C-terminus and that the conserved C-terminal domain plays an important role in setting the molecular structures of the intact amelogenins so as to facilitate the protein-enamel mineral interaction.

Dentin phosphoprotein (DPP) and dephosphorylated DPP (deP-DPP) were isolated from developing bovine incisor and purified by DEAE-Sephacel and Sepharose CL-6B chromatography. Intact DPP showed partial resistance to proteolytic cleavage; while deP-DPP was completely susceptible to protease digestion. This result suggested that phosphate residues within DPP molecule may protect its structural integrity. DPP was also very sensitive to heat. When DPP was heated at 100°C in the presence of sodium dodecyl sulfate, increasing concentrations of SDS resulted in a protective effect from degradation. An analysis of DPP biosynthesis was carried out by isolation of the odontoblast tissue/cells attached to forming dentin or scraped from dentin surface followed by incubation with radioactive amino acids and phosphate. DPPs produced in vitro were larger in size than those DPPs extracted from mature dentin matrix. To determine the size of the unprocessed form of DPP, Xenopus leavis oocytes were incubated in [ 32 p] -phosphate-containing medium after microinjection of poly(A + mRNA extracted from the bovine odontoblasts. Radioactive DPP analyzed by two-dimensional polyacrylamide gel electrophoresis and immunoprecipitation with an anti-DPP monoclonal antibody was shown to have an apparent M r of 150-160 k. These data imply that changes in molecular size of DPP may occur by post-translational cleavage or other modification such as degradation within dentin matrix in vivo .