Bovine Tooth Germs Research Articles

Expression and roles of CCN2 in dental mesenchymal cells in primary culture—With findings in a case of odontogenic myxofibroma

Purpose of the researchTooth germ development involves multiple events, including cell proliferation and cell differentiation. Connective tissue growth factor (CTGF/CCN2) is a signaling protein involved in tooth germ development, and we investigated how it is expressed and what roles it may have in primary cultures of mesenchymal cells derived from the developing tooth germ. We also examined the expression of CCN2 in a human odontogenic myxofibroma, a benign tumor of odontogenic mesenchymal origin, and considered the possible roles of CCN2 in the development of myxofibromas. Materials and methodsMesenchymal cells of early bell-stage tooth germs were isolated from Day-90 bovine embryos and placed in primary culture. A resected specimen from a patient with odontogenic myxofibroma was prepared for immunohistochemical studies. Principal resultsThe CCN2 expression level in proliferating odontogenic mesenchymal cells freshly isolated from the early bell stage of developing bovine tooth germs and placed in primary culture was 3 times higher than that in the confluent non-proliferating cells. Recombinant CCN2 significantly increased the proliferation and type I collagen expression in odontogenic mesenchymal cells in primary culture. Immunohistochemical analysis on myxofibroma case revealed that CCN2 was detectable in MIB-1, a cellular marker of proliferation-positive odontogenic mesenchymal cells adjacent to capillary blood vessels and in the endothelial cells of the vessels in the tumor. Major conclusionCCN2 signaling would influence the proliferation of and extracellular matrix production by dental mesenchymal cells. Our results suggest that the same mechanisms of CCN2 action toward dental mesenchymal cells would also be operative in the odontogenic myxofibroma microenvironment.

Unusual amplification in polymerase chain reaction for a plasmid containing an insert derived from bovine genomic DNA

The saliva of various animals contains praline-rich proteins which may play important roles in prevention of mineral precipitation, protection of dietary and digestive proteins from interaction with tannins, and modulation of bacterial colonization on the tooth surface. Previously, we found a segment of Escherichia coli genomic DNA in bovine tooth germ mRNA encoding the proline-rich protein P-B. To examine whether E. coli genomic DNA is present in bovine genomic DNA, we constructed a plasmid library from the bovine DNA. Although results so far have failed to indicate any such presence in the bovine nucleotides examined, experiments using the polymerase chain reaction (PCR) revealed unusual amplification of nucleotides. As an initial step of the study on possible occurrence of E. coli-derived nucleotide sequence in bovine genomic DNA of P-B, we examined the structure of the PCR products generated by unexpected amplification. The determined structure of the PCR products suggested that when the two single strand chains that grow by reading the sequence of the respective template reached a hybridizable short nucleotide structure, they became hybridized and subsequent elongation was continued by reading the sequence of the counter chain that had been elongated by reading the template. It is possible that elongation of the chain was interrupted once before the completion of amplification due to the template’s palindrome region which had formed a double strand structure during the PCR process. Such an unusual amplification made possible under certain conditions in a DNA sequence may be one of the mechanisms for the genetic recombination found in our previous study.

結合組織増殖因子ＣＴＧＦ／ＣＣＮ２が歯胚間葉細胞の象牙芽細胞分化に与える影響

Recent studies using novel bioengineering techniques have demonstrated the regeneration of mammalian dental tissues from tooth-derived cells. In this study, we established a novel in vitro culture system with the use of undifferentiated dental mesenchymal cells from bovine bell-staged tooth germs and promoted differentiation of these cells into functioning odontoblasts. During the. 21-day culture period, the dental mesenchymal cells were exposed to 5 μg/ml ascorbic acid and 1 mM β-glycerophosphate (β-GP). These undifferentiated mesenchymal cells exhibited characteristics of odontoblasts at all stages of differentiation, starting from the immature stage to the terminal mineralization stage. Connective tissue growth factor (CTGF/CCN2) is a potent growth factor that regulates proliferation and differentiation of both chondrocytes and osteoblasts. Since CTGF is also expressed in differentiating preodontoblasts, we examined if CTGF can promote odontoblast differentiation. Recombinant CTGF (rCTGF), in the presence of ascorbic acid and β-GP, stimulated the expressions of the early odontoblast differentiation marker alkaline phosphatase (ALP) and collagen type I mRNA. In addition, the maturation markers osteopontin and dentin sialophosphoprotein (DSPP) mRNA were also expressed during the 9-day culture period. Histological and histochemical analyses confirmed that rCTGF, in the presence of ascorbic acid and β-GP, stimulated high alkaline phosphatase activity, calcium incorporation, and hydroxyapatite deposition. These findings demonstrated that our novel culture system can maintain the osteogenic potential of undifferentiated dental mesenchymal cells and promote their differentiation into functional odontoblasts. Furthermore, rCTGF is capable of promoting the differentiation of tooth germ mesenchymal cells into odontoblasts, suggesting that CTGF may be therapeutically useful in the future for promoting tooth regeneration.

Evidence for inclusion of a segment of Escherichia coli genomic DNA in bovine tooth germ mRNA encoding salivary proline-rich protein P-B

In the course of cloning of bovine cDNA for proline-rich protein (PRP) P-B from bovine tooth germ cDNA, we found that one clone with 662 bp contained a 5'-terminal 393 bp (1-393 bp) sequence essentially identical to that of human P-B cDNA (154-546 in D29833) and bovine P-B cDNA (1-356 bp in AB192573) and a sequence of 233 bp (394-626 bp) highly homologous to the segment of E. coli K12 genomic DNA (365511-365744 in NC000913). Although the latter sequence is contained in the vector pT7Blue, which we used, our results show that this chimeric structure in bovine tooth germ P-B cDNA is not an artifact formed during the cloning process, but intrinsic to the bovine genome since the chimeric structure was detected in bovine tooth germ and bovine genomic DNA.

Progenitor Cells From Dental Follicle Are Able to Form Cementum Matrix In Vivo

To address the molecular mechanisms of cementogenesis, we have isolated dental follicle cells and examined them to see if they contain cementoblast progenitors. Dental follicle tissue was dissected from the root surface of bovine tooth germ and cells were released by digestion with bacterial collagenase. The released cells were maintained as a bovine dental follicle cells (BDFC). To elucidate the differentiation capacity of BDFC, they were transplanted into severe combined immunodeficiency (SCID) mice for 4 weeks. Transplanted BDFC formed cementumlike matrix; in contrast, bovine alveolar osteoblast (BAOB) transplants formed bonelike matrix, and bovine periodontal ligament cells (BPDL) formed a small amount of the cementumlike matrix. Immunohistochemical analysis showed that cementumlike matrix was positive for anti-cementum attachment protein monoclonal antibody, whereas bonelike matrix was negative. These results indicated that the BDFC contained cementoblast progenitors that were able to differentiate to cementoblasts in vivo. They also indicated that the BDFC are phenotypically distinct from BAOB and BPDL, and provide a useful model for investigating molecular mechanisms of cementogenesis.

Development of stratum intermedium and its role as a Sonic hedgehog-signaling structure during odontogenesis.

Stratum intermedium is a transient and subtle epithelial structure closely associated with inner dental epithelium in tooth germs. Little is known about its development and roles. To facilitate analysis, we used bovine tooth germs, predicting that they may contain a more conspicuous stratum intermedium. Indeed, early bell stage bovine tooth germs already displayed an obvious stratum intermedium with a typical multilayered organization and flanking the enamel knot. Strikingly, with further development, the cuspally located stratum intermedium underwent thinning and involution, whereas a multilayered stratum intermedium formed at successive sites along the cusp-to-cervix axis of odontogenesis. In situ hybridization and immunohistochemistry showed that stratum intermedium produces the signaling molecule Sonic hedgehog (Shh). Maximal Shh expression was invariably seen in its thickest multilayered portions. Shh was also produced by inner dental epithelium; expression was not constant but varied with development and cytodifferentiation of ameloblasts along the cusp-to-cervix axis. Interestingly, maximal Shh expression in inner dental epithelium did not coincide with that in stratum intermedium. Both stratum intermedium and inner dental epithelium expressed the Shh receptor Patched2 (Ptch2), an indication of autocrine signaling loops. Shh protein, but not RNA, was present in underlying dental mesenchyme, probably resulting from gradual diffusion from epithelial layers and reflecting paracrine loops of action. To analyze the regulation of Shh expression, epithelial and mesenchymal layers were separated and maintained in organ culture. Shh expression decreased over time, but was maintained in unoperated specimens. Our data show for the first time that stratum intermedium is a highly regulated and Shh-expressing structure. Given its dynamic and apparently interactive properties, stratum intermedium may help orchestrate progression of odontogenesis from cusp to cervix.

Expression of cementum-derived attachment protein in bovine tooth germ during cementogenesis

Cementum-derived attachment protein (CAP) is a 56 kDa collagenous protein that promotes attachment of mesenchymal cells. Previous studies have shown that the presence of CAP is restricted to cementum in adult human tissues. In this study, we report generation of a monoclonal antibody against CAP and its use for the investigation of CAP in developing bovine tooth germs. Mice were immunized with CAP purified from bovine cementum, and a monoclonal antibody, 3G9, was produced. Immunohistochemical staining of bovine tooth germ at root forming stage using 3G9 antibody showed that the tissue distribution of CAP expression was limited to cementum matrix and cementoblasts during cementogenesis. Alveolar bone did not stain with the 3G9 antibody, whereas anti-type I collagen stained positively. CAP was purified from bovine tooth germs with immunoaffinity purification using the 3G9 antibody. Examination of the immunoaffinity-purified fraction showed that CAP existed in tooth germ as a 65 kDa protein. The protein was susceptible to bacterial collagenase. To investigate the possible biological function of CAP during cementogenesis, we isolated dental follicle cells from the bovine tooth germ, and showed that they adhered to surfaces containing CAP. These data demonstrate that CAP is expressed by bovine cementoblasts as a 65 kDa protein and that the CAP may have a function in cementogenesis.

Gene Expression of Growth and Differentiation Factors-5, -6, and -7 in Developing Bovine Tooth at the Root Forming Stage

Growth and differentiation factors (GDF)-5, -6, and -7 are members of the bone morphogenetic protein (BMP) family. Previous studies suggest their importance in bone development and in tendon/ligament morphogenesis. The cells of the dental attachment apparatus, cementum, periodontal ligament, and alveolar bone proper are derived from the dental follicle proper. In this study, we investigated the expression of GDF-5, -6, and -7 genes in tissues of the bovine incisor tooth germ at the root forming stage. The results demonstrate distinct expression of GDFs in both the dental follicle and the odontoblast layer. While GDF-5 and -6 mRNAs were expressed in both the dental follicle and the odontoblast layer, GDF-7 mRNA expression was detected only in the dental follicle. These results indicate that GDFs, expressed in the bovine tooth germ including the dental follicle, may be potent regulatory molecules in the development of the dental attachment apparatus.

Expressions of c-jun and jun-B proto-oncogenes in odontoblasts during development of bovine tooth germs.

c-jun and jun-B genes are among the nuclear proto-oncogenes induced by growth factors such as the TGF-beta superfamily and play important roles in cell differentiation. These gene products enhance expressions of proteins including osteocalcin, alkaline phosphatase, and collagens. On the other hand, it is well-known that the TGF-beta superfamily affects odontoblast differentiation, and that differentiated odontoblasts express extracellular and membrane proteins as described above. However, there are few reports of factors that participate in the transcriptional regulation of odontoblasts. Especially, little is known about the expression of c-jun and jun-B genes. In this study, we focused on the examination of expressions of c-jun and jun-B genes in dental papillae of bovine tooth germs. Using in situ hybridization, we found that these genes were expressed only in the odontoblastic lineage, but not in other dental papilla cells. Levels of c-jun and jun-B mRNAs increased along the gradient of differentiation of odontoblasts. These levels of c-jun mRNAs were maintained in both young and mature odontoblasts. However, unlike the c-jun gene, expression of the jun-B gene became sparse in mature odontoblasts compared with young odontoblasts. For further analysis, Northern hybridization of total RNA extracted from differentiated odontoblasts was performed for the examination of levels of jun-B mRNAs, indicating that levels of jun-B mRNAs of mature odontoblasts were clearly less than those of young odontoblasts. These results suggest that c-jun and jun-B genes may participate in the transcriptional regulation of odontoblasts of bovine tooth germs, and may control the odontoblast phenotype. Furthermore, our results suggest that these genes can be markers of odontoblasts during dentinogenesis; especially, high expression of jun-B gene can be a marker of young odontoblasts that start to form the new dentin matrix.

In vitro mineralization in bovine tooth germ cell cultured with sintered hydroxyapatite

Unerupted teeth were extracted from the mandibles of calves, and the enamel surfaces containing ameloblasts and cells of papilla layer were scraped off. Cells migrated from the fragments were transferred into culture flasks containing small bars of sintered hydroxyapatite, and the cultures were incubated long-term. Most of the cells showed a fibroblastic morphology, but some of the cells formed an epithelial cell nest. The fibroblastic cells grew steadily, but the epithelial cells showed poor growth. The fibroblastic cells formed multiple cell layers and were embedded in collagenous matrix containing type I trimer collagen. By 3 months, fibrous bands appeared on the surface of the cell layers and surrounded the bars of sintered hydroxyapatite. Such bands increased in size with incubation time. Mineral deposition was observed in the well-developed bands. The deposited crystals were found by the X-ray powder diffraction method to be hydroxyapatite; the Ca/P molar ratio of 1.49 was determined by the ICP method. These results indicated that cells of papilla layer have the capacity to mineralize in vitro in the presence of sintered hydroxyapatite.

ウシ歯胚の象牙芽細胞層およびエナメル芽細胞層における乳酸脱水素酵素含有基質小胞様小胞に関する研究

ウシ歯胚の象牙芽細胞層およびエナメル芽細胞層における乳酸脱水素酵素含有基質小胞様小胞に関する研究

Presence of Vesicles Containing Lactate Dehydrogenase in the Dentin of Bovine Tooth Germs

The dentin was removed from bovine tooth germs, followed by the separation of the extracellular matrix vesicle fraction after collagenase treatment. Lactate dehydrogenase (LDH)-containing vesicles with a density different from that of matrix vesicles were detected in the matrix vesicle fraction. LDH in these vesicles did not result from cell lysis and vesicle capture during the preparation of the matrix vesicle fraction. The isoenzyme pattern of LDH in LDH-containing vesicles was similar to that of cytosolic LDH of odontoblasts. Other cytosolic enzymes were not detected in LDH-containing vesicles, suggesting the presence of a mechanism for specific uptake of cytosolic LDH during the in vivo formation of the vesicles.

Electrophoretic Demonstration of Glycoproteins, Lipoproteins, and Phosphoproteins in Human and Bovine Enamel

Enamel proteins from fully mineralized human molars and from bovine tooth germs were separated by electrophoresis. The gels were stained for detection of glycoproteins, lipoproteins, and phosphoproteins. Glycoproteins were shown by periodic acid-Schiff staining and lectin blotting. In mature human enamel a number of high molecular weight proteins could be demonstrated after ethylenediaminetetra-acetic acid demineralization and subsequent Triton X-100 extraction. These proteins are suggested to be lipoproteins. Phosphoproteins could only be visualized in enamel matrix from the tooth germs.

Light microscopic and transmission electron microscopic study of initial phases of giant tubule formation in bovine dentin.

Bovine tooth germs dissected from the mandible immediately after bleeding of the less than 1-yr-old calves (Bos taureus) were used to study the initial phases of giant tubule (GT) formation. Three stages could be discerned. Firstly, the initial manifestations were represented by loci about 150 micron apart in the axiomesiodistal plane along the pulpal border of the 100-200-micron-thick incisal dentin. Some of these loci showed only a blunt capillary loop and associated cells, whereas in most others there was a 50-100-micron-wide dense matrix consisting of coarse collagen fibrils and showing a metachromatic staining reaction to toluidine blue. Fibroblasts lined this matrix, and a capillary loop was always present underneath. Both kinds of GT origins were eventually circumvented by the odontoblasts situated incisal to them. The matrices became enclosed in the dentin. In the second stage, the GT grew in length as new dentin was formed. In this stage the vascular loops and the adjoining fibroblasts remained stationary within the GT lumina relative to the origins. In the third stage the GT vessels started to migrate pulpally relative to the origins. This seemed to be the result of a sequestration of the most incisal portions of the vascular loops. In this last stage the formation of collagen fibrils in the GT lumen incisal to the vessel loops started.

A characterization of amelogenin messenger RNA in the bovine tooth germ.

In order to study the nature of amelogenin mRNA, we isolated ameloblast-rich tissue from the unerupted permanent incisor tooth germs of 18-month-old steers and subjected it to guanidine HC1 solubilization for extraction of mRNA. When poly A+ ameloblast RNA was incubated with radioactive deoxynucleotides and reverse transcriptase, four major transcripts were detected with sizes of 1.9, 1.4, 0.7, and 0.4 kb in length. One of the transcripts (0.7 kb) corresponded precisely in length to that predicted from the size of the major in vitro translated amelogenin proteins (27,000 daltons). To determine whether the transcripts did indeed encode amelogenin mRNA, we constructed a λgt11 cDNA library and isolated several amelogenin cDNA's by screening with amelogenin antibody. Four clones were amplified and insert sizes determined by acrylamide gel electrophoresis. Two of the clones had insert sizes of ~ 0.7 kb (λAm 16, XAm 7), and two had insert sizes of ~ 0.4 kb (λAm 11, λAm 4). When the amelogenin cDNA was radiolabeled and used for northern analysis, two species of amelogenin message (0.75 and 0.45 kb) were evident, both of which showed extensive hybridization to λAm 16 (large) and λAm 11 (small) cDNA. These data indicate that: (1) Amelogenin mRNA is heterogeneous in the bovine tooth germ, having two major species 800 and 400 bases long; and (2) the major species of amelogenin share extensive sequence homology. Based on these data, we suggest that at least part of the heterogeneity of amelogenin matrix protein may arise from the production of heterogeneous amelogenin mRNA's that share some common nucleotide sequences.

Heterogeneity of amelogenin mRNA in the bovine tooth germ.

The amelogenins are a complex mixture of hydrophobic proteins that are the major organic component of developing enamel. To study the molecular mechanisms underlying the heterogeneity of the amelogenins we isolated cDNA clones encoding these proteins. The clones were definitively identified by hybrid-selected translation experiments and by comparison of the DNA sequence with the protein-derived amino acid sequence. Southern hybridization of bovine genomic DNA indicated that amelogenin is a single copy gene. However, Northern hybridization experiments distinctly showed two major species of mRNA, each of which were sufficiently large enough to encode the highest known molecular weight species of amelogenin proteins. Furthermore, immunoprecipitation of hybrid-selected translation products using isolated amelogenin cDNA showed multiple, translated protein products. These data are supportive of a differential mRNA processing mechanism involved in generating a heterogeneous family of amelogenin matrix proteins from a single gene.

Cell Culture of Dental Sac from Bovine Tooth Germ

Cell Culture of Dental Sac from Bovine Tooth Germ

Histochemical demonstration of activity of acid phosphatase and beta-glucuronidase in bovine incisor tooth germs.

Activity of acid phosphatase and beta-glucuronidase was shown in bovine preodontoblasts and preameloblasts prior to the onset of secretion. In the preameloblasts the rather weak reaction consisted of small discrete granules dispersed in the cytoplasm apical, lateral, and proximal to the nucleus. After initiation of enamel formation, a change in localization and intensity of the colored reaction product was observed in the ameloblasts. The activity appeared stronger and was restricted to a narrow zone just apical to the nucleus. It is proposed that the acid hydrolases in the tooth forming cells are located to the Golgi complex. The differences in activity of acid hydrolases between bone and tooth forming cells are expounded.

The separation and culture of cell populations from bovine molar tooth germs

Separated components of the maxillary molar tooth germs were cultured. The typical epithelial characteristics of enamel organ cells were retained. The cultured cells of the reduced enamel epithelium, outer enamel epithelium and stratum intermedium became polygonal with numerous microvilli, in contrast with the lack of microvilli and elongated profile of stellate reticulum and dental papilla cells. A plexiform organization of cytoskeletal microfila-ments was noted in cells which became polygonal, whereas in elongated cells, microfilaments were arranged in parallel bundles beneath the cell surface. In the cultured epithelial cells, but not dental papilla cells, cytoskeletal tonofilaments as well as cell surface specializations at contact sites between cells were common.

Proteolytic Activity in Developing Bovine Enamel

Partially mineralized enamel matrix removed from bovine incisor tooth germs contains proteolytic activity capable of completely degrading enamel matrix proteins at neutral and slightly alkaline pH. The protease activity also degrades common protease substrates such as casein and azocoll with optimal activity at pH 8-9. Inhibitor studies revealed that the enzyme involved is a serine protease of the chymotrypsin family.