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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.

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.

In the rat tibial bone marrow cavity, following colchicine injection, there is a phase of osteogenesis in which bone trabeculae replace the necrotic bone marrow tissues and fill the marrow cavity. The newly formed bone is subsequently resorbed by osteoclasts and normal bone marrow is restored. In this study, we correlated these morphologic events with the pattern of gene expression of bone sialoprotein (BSP), an extracellular matrix protein in mineralized tissues, to elucidate the possible functions of BSP in bone formation and resorption in vivo. The expressions of osteopontin (OPN) and type I collagen were also examined. Northern hybridization of the tibia demonstrated that OPN mRNA was gradually increased and expressed at a maximal level 10 days after colchicine injection (during the bone resorption process), while BSP mRNA expression already reached a maximal level at day 6 (during the initial process of bone formation). Its expression was, thus, quite temporary at the beginning of bone formation and different from that of type I collagen, which was continually elevated from days 6 to 10. In situ hybridization of the newly formed bone induced in the tibia revealed that BSP mRNA was evenly expressed in most osteoblasts and osteocytes, moreover in interconnecting colonies of spindle-shaped cells, possibly preosteoblasts, at day 6. At day 10, however, its expression became restricted to some cells on the bone surfaces, some osteoblasts, and most osteoclasts. These observations suggest that BSP may play an important role mainly in the initiation of bone formation and is also associated with the functions of osteoclast in vivo.

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. Keywords:: tissue engineering, periosteal cell, bone morphogenetic protein-2 (BMP-2), vascular endothelial growth factor (VEGF), gene transfer

Rat osteoblasts were cultured for 4 or 5 days aboard the Space Shuttle and solubilized during spaceflight. Post-flight analyses by quantitative reverse transcriptase-polymerase chain reaction (RT-PCR) determined the relative mRNA levels of matrix proteins, adhesion molecules, and cytoskeletal proteins including osteopontin (OP), osteonectin (ON), CD44, alpha-tubulin, actin, vimentin, fibronectin (FN), and beta1-integrin. The mRNA levels of OP and alpha-tubulin in the flight cultures were decreased by 30% and 50% on day 4 and day 5 of flight, as compared to the ground controls. In contrast, the CD44 mRNA levels in the flight cultures increased by 280% and 570% of the ground controls on day 4 and day 5. The mRNA levels of ON and FN in the flight cultures were slightly increased as compared to ground controls. The mRNA levels of actin, vimentin, or beta1-integrin did not change in spaceflight conditions. The matrix proteins, adhesion molecules, and cytoskeletal proteins may form dynamic network complexity with signaling molecules as an adaptive response to perturbation of mechanical stress under microgravity.

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.

Smad3 is an intracellular signaling molecule that mediates the signal from transforming growth factor-beta (TGF-beta) and activin receptors. In this study, we reveal hypomineralized enamel in mice with the targeted deletion of the Smad3 gene. The Smad3 (-/-) mice had chalky white incisor enamel, while the enamel of the wild-type or Smad3 (+/-) mice was yellow-brown. Histological analysis of the undecalcified sections showed that the enamel thickness of the maxillary incisors in the Smad3 (-/-) mice was similar to that of the wild-type and Smad3 (+/-) mice while that the enamel of the maxillary molars in Smad3 (-/-) mice was disrupted in places. Microcomputed tomography (microCT) analysis revealed that the mineralization of the maxillary incisors and mandibular molars in the Smad3 (-/-) mice showed significant reduction in the degree of mineralization when compared to that of the wild-type and Smad3 (+/-) mice. Scanning electron microscopic (SEM) analysis of the mandibular incisors revealed that the enamel surface of the Smad3 (-/-) mice was irregular and disrupted in places and showed images similar to decalcified mature enamel. The histological analysis of the decalcified sections showed that distinct morphological changes in the ameloblasts at the secretory and maturational stages were not observed between the Smad3 (-/-) and Smad3 (+/-) or wild-type mice, while the enamel matrix was observed in the decalcified sections of the mandibular molars in the Smad3 (-/-) mice. These results suggested that Smad3 was required for enamel biomineralization, and TGF-beta and activin signaling might be critical for its process.

The condylar cartilage, an important growth site in the mandible, shows characteristic modes of growth and differentiation, unlike the limb bud cartilage. To elucidate the mechanism of chondrogenesis at the condylar cartilage, we analyzed the effects of basic fibroblast growth factor (bFGF) on the growth and development of mouse mandibular condyle using serum-free organ culture and on the expression of genes related to the chondrogenesis. Further, we investigated the localization of bFGF in cultured condyle by immunohistochemistry. The present immunohistochemical observations showed that bFGF is localized in the extracellular matrix of the mesenchymal condylar anlage, the perichondrium and the proliferative cell zone, and that immunostaining was diminished in the metachromatically stained area. In the condyle culture with added recombinant human bFGF (rhbFGF) for 5 days, the area occupied by hypertrophic chondrocytes in the mandibular condylar cartilage was reduced. A reverse transcription-polymerase chain reaction (RT-PCR) assay also showed that the mRNA levels of aggrecan and type X collagen were reduced compared with nontreated tissues. Treatment with rhbFGF for 2 days decreased cell proliferation in the perichondrium, and bFGF downregulated the Indian hedgehog (Ihh), parathyroid hormone-related protein (PTHrP), bone morphogenetic protein 4 (BMP4), and core-binding factor alpha1 (Cbfa1) expression in the RT-PCR assay. These findings suggest that bFGF has the ability for inhibitory regulation of condylar growth, via the inhibition of proliferation and differentiation of chondrocytes, and that this inhibitory regulation is related to the downregulation of growth factors and transcription factors.

Matrix metalloproteinase-9 (MMP-9, or gelatinase B) is an extracellular proteinase that is highly expressed in osteoclasts and has been postulated to play an important role in their resorptive activity. Although MMP-9 has been reported to play a role in bone resorption, the association of this enzyme during deciduous tooth resorption has not yet been clarified. The purpose of the present study was to increase our understanding of the role of MMP-9 during deciduous tooth resorption. Reverse transcription-polymerase chain reaction (RT-PCR) and northern blot analysis of total RNAs extracted from bovine root-resorbing tissues, which lie between the root of a deciduous tooth and its permanent successor, revealed the expression of mRNA for MMP-9 in the tissue. These results indicate that MMP-9 may be involved in the process of deciduous tooth resorption. In addition, in situ hybridization and immunohistochemistry were also performed to identify the cells that produced MMP-9 in bovine root-resorbing tissue. MMP-9 mRNA was highly expressed in odontoclasts that were aligned along the surface of the tissue. Immunohistochemistry confirmed the predominant localization of MMP-9 in odontoclasts. The present data demonstrate that odontoclasts in deciduous root resorption express MMP-9, which may participate in proteolysis during root resorption of deciduous tooth.

Purpose: To investigate the role of mechanical stretch in the regulation of matrix metalloproteinase-2 (MMP-2) in scleral fibroblasts of chick embryos. Methods: Scleral fibroblasts derived from chick embryos were seeded onto flexible bottom culture plates, and subjected to a pulsatile stretch when the cells became subconfluent. After stretching, the cells and the conditioned medium were harvested. One portion of the conditioned medium was activated by reduction, alkylation, and 4-aminophenylmercuric acetate (APMA) treatment. The conditioned medium, with and without the treatment, was subjected to gelatin zymography and quantitative assays. Total cytoplasmic RNA was extracted from the cells, and the expression of MMP-2 and the tissue inhibitor of metalloproteinase-2 (TIMP-2) mRNA was examined by Northern blot analysis. Results: The predominant gelatinolytic enzyme secreted by scleral fibroblasts was MMP-2. The mechanical stretch increased the gelatinolytic activities significantly in the conditioned medium with reduction, alkylation, and APMA treatment. Mechanical stretch also enhanced the expression of MMP-2 and TIMP-2 mRNA in scleral fibroblasts significantly. Conclusions: These results suggest that mechanical stretch may be involved in the regulation of the extracellular matrix in the fibrous sclera of chicks in vivo.

The movement of teeth during orthodontic treatment occasionally induces undesirable root resorption. Although high collagenolytic activity has been detected in resorbing tissue of deciduous teeth, the cellular origin of collagenolytic enzymes in root-resorbing tissue caused by tooth movement has not been identified. Here, rats were subject to 7 days of experimental tooth movement to induce root resorption. In situ hybridization with digoxigenin-labelled RNA probes was performed on sections of the maxillary bone to detect the mRNAs that encode matrix metalloproteinase-1 (MMP-1) and cathepsin K in root-resorbing tissue. MMP-1 mRNA was detected in fibroblastic cells, cementoblasts and osteoblasts, but not in odontoclasts nor osteoclasts. Moreover, MMP-1 mRNA was highly expressed in some cementocytes located near odontoclasts and in many osteocytes. In contrast, cathepsin K mRNA was expressed only in odontoclasts and osteoclasts. These results suggest that MMP-1 and cathepsin K are important in root resorption during tooth movement in a mode similar to bone resorption.

Previous studies have demonstrated the morphological similarity of the enamel-like layer found in the teeth of the coelacanth, lungfish and gar-pike to the enamel of tetrapods. In order to clarify the phylogenetic continuity between both structures, tooth germs of the gar-pike were immunocytochemically studied using an anti-bovine amelogenin polyclonal antibody. Intense immunoreaction was shown over the enamel-like matrix layer. Certain cell organelles associated with the secretory pathway of the ameloblasts were recognized as immunoreactive. These results indicate that the enamel-like layer of the gar-pike is a tissue homologous with the mammalian enamel because both possess a common, amelogenin-like substance.

A number of studies have indicated that microgravity induces osteopenia and modulates functions of mammalian cells. However, the molecular mechanisms underlying these effects of microgravity are still unknown. Rat osteoblasts were cultured for 4 and 5 days during Shuttle-Spacelab flight, and fixed by guanidine isothiocyanate solution on board after treatment with 1α, 25 (OH)2 vitamin D3. The mRNA levels for platelet-derived growth factor (PDGF)-β receptor, epidermal growth factor (EGF) receptor, the growth factor receptor adaptor protein Shc, and c-fos were determined using the method of quantitative reverse transcription-polymerase chain reaction. The mRNA levels for EGF receptor were not altered by microgravity. However, the mRNA levels for PDGF-β receptor, Shc, and c-fos were decreased to 62, 55 and 25% on the 4th day of flight, and 47, 40, and 43% on the 5th day, respectively, as compared to the corresponding ground controls. Expression of the growth factor receptor and the receptor adaptor protein was modulated in rat osteoblasts during spaceflight. Data suggest that signal transduction via growth factor receptors in rat osteoblasts is impaired by microgravity. Dysfunction of osteoblasts might be involved in spaceflight-induced osteopenia.

Expression of the TGF-β and type I collagen genes during healing of extracted tooth sockets was studied using in situ hybridization and immunohistochemistry.1. Histological findingsA clot was formed in the extracted tooth socket 1 day after extraction. By 2days after extraction, fibrogenesis was evident in the clot. Bone trabeculae began to form from the basal wall of the tooth socket from 3days, and from the lateral walls from 3 to 5 days after extraction. The tooth sockets were filled by newly formed bone within 14 days after extraction.2. Gene expression of TGF-β 1At 1 day after extraction, the gene for TGF-β 1 was expressed in leukocytes near the clot, and in fibroblasts and osteoblasts in the remaining periodontal ligament after tooth extraction. The signals of osteoblasts associated with newly formed bone were increased from 3 days after extraction. Within 5 to 7 days after extraction, the most intense signals were observed in osteoblasts along the newly formed bone trabeculae and in fibroblasts. The signals in the cellular component at 7 days after extraction were stronger than those at 14 days after extraction.3. Gene expression of type I collagenThe gene for type I collagen was expressed more strongly than that of TGF-β 1. At 1 day, no signals were found in the cells of the clot, but fibroblasts and osteoblasts showed intense expression. After 2 days the distribution of signals was essentially the same as that for TGF-β 1.4. Immunohistochemistry of TGF-β 1At 1 day after extraction, immunolocalization of TGF-β 1 was observed in the clot, leukocytes, and in fibroblasts and osteoblasts in the remaining periodontal ligament after tooth extraction. Then, immunoreactions were observed in osteoblasts and fibroblasts. These results suggest that expression of the TGF-β gene regulates the synthesis of type I collagen by fibroblasts and osteoblasts, and plays a central role in cell proliferation and differentiation during the healing of extracted tooth sockets.

The concentration and distribution of alpha 2HS glycoprotein in teeth from patients with heritable dentin disorders were examined utilizing immunological techniques. ELISA for the quantitative determination showed lower concentrations of alpha 2HS glycoprotein in dentins of patients with heritable disorders than in the control; some cases contained very low and others intermediate concentrations. Immunohistochemical findings indicated that the concentration of this protein was correlated to the regularity of the dentinal tubules. This result suggests that alpha 2HS glycoprotein is incorporated into dentin in concomitance with the development of the dentinal tubule, especially with the deposition of peritubular dentin. Incorporation of this protein into dentin is thought to involve the function of odontoblasts.

This is the first detailed report about the collar enamel of the teeth of Polypterus senegalus. We have examined the fine structure of the collar enamel and enamel organ of Polypterus during amelogenesis by light and transmission electron microscopy. An immunohistochemical analysis with an antibody against bovine amelogenin, an antiserum against porcine amelogenin and region-specific antibodies or antiserum against the C-terminus, middle region and N-terminus of porcine amelogenin has also been performed to examine the collar enamel matrix present in these teeth. Their ameloblasts contain fully developed Golgi apparatus, rough endoplasmic reticulum and secretory granules. During collar enamel formation, an amorphous fine enamel matrix containing no collagen fibrils is found between the dentin and ameloblast layers. In non-demineralized sections, the collar enamel (500 nm to 1 μm thick) is distinguishable from dentin, because of its higher density and differences in the arrangement of its crystals. The fine structural features of collar enamel in Polypterus are similar to those of tooth enamel in Lepisosteus (gars), coelacanths, lungfish and amphibians. The enamel matrix shows intense immunoreactivity to the antibody and antiserum against mammalian amelogenins and to the middleregion- and C-terminal-specific anti-amelogenin antibodies. These findings suggest that the proteins in the enamel of Polypterus contain domains that closely resemble those of bovine and porcine amelogenins. The enamel matrix, which exhibits positive immunoreactivity to mammalian amelogenins, extends to the cap enameloid surface, implying that amelogenin-like proteins are secreted by ameloblasts as a thin matrix layer that covers the cap enameloid after enameloid maturation.

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.

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.

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.

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 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.

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.

Direct determination of the stable keto-imine forms of [3H]NaBH4-reducible precursors of dihydroxylysinonorleucine [(OH)2Lys-norLeu] and hydroxylysinonorleucine [(OH)Lys-norLeu] in bone and dentin collagen are described. Acidic thermal conditions were used to break labile imminium bonds and acetylate the resulting amino groups to prevent reformation of the original cross-links under the conditions used for NaBH4-reduction.

Rabbit polyclonal antibodies against bovine amelogenins and enamelins which did not show any cross-reaction were raised, and ultrathin sections of rat incisors were examined by the protein A-gold and ABC methods. The immunoreactivity of amelogenins was found in dense granules in the intercellular spaces between preameloblasts, and later over the fine- and coarse-textured material. The immunoreactivity was present over the cell organelles associated with the secretory pathway, as well as pale and dark lysosomes of the presecretory and secretory ameloblasts. Here the enamel was immunolabeled in the intercrystal spaces. The immunoreactivity in multivesicular bodies was stronger in preameloblasts than in secretory ameloblasts. In the region of second ruffle-ended ameloblasts at the maturation stage, the immunolabeling was intense in the ruffled-border, but in the rough endoplasmic reticulum and Golgi apparatus, the immunolabeling was much weaker than at the secretory stage. The immunolabeling for enamelins showed essentially the same intracellular topographical pattern as that for amelogenins by the secretory stage, but was weaker. The immunoreactivity was found mainly attached to the enamel crystals. Double immunostaining of amelogenins and enamelins revealed that both immunoreactivities were present over the same cell organelles associated with secretion and lysosomal systems. It is suggested that the presecretory and secretory ameloblasts are actively involved in the secretion, degradation and resorption of enamel proteins, and that multivesicular bodies and lysosomes in the cells take part in these processes. Ameloblasts are considered to be related to the synthesis of enamelins.

A peptide containing 59 amino acid residues with a stoichiometric amount of dihydroxylysinonorleucine (0.7 mole) and hydroxylysinonorleucine (0.2 mole) was isolated from reduced 3H labelled bovine bone collagen sequentially cleaved with CNBr and trypsin. Further cleavage of the isolated crosslinked peptide with periodate yielded a radioactive peptide of 45 residues and a non-radioactive peptide of 16 residues. From the characteristic amino acid composition of these peptides it was deduced that the peptide was derived from an intermolecularly crosslinked region between lysyl or hydroxylysl residues in the carboxy-terminal extension of alpha 1-CB6 (17C residue) and alpha 1-CB5 (87th residue). This finding supports the observation that the alpha 1-CB6 peak was prominent on carboxymethyl cellulose chromatography of the CNBr digest of bone collagen only after limited pepsin digestion, and is consistent with the results obtained from a smaller crosslinked peptide previously isolated from calf bone collagen.

A specific enzyme-linked immuno-adsorbent assay for enamel protein of the developing tooth was devised, using antibody against antigen prepared from immature bovine enamel. Samples containing 5–500 ng of amelogenin protein in 50 μl gave consistent results. Bovine enamelin cross-reacted with the antibody but was less reactive than amelogenin. Proteins in other tissues or fluids did not react to the antibody.