Immunohistochemical observations on bone morphogenetic protein in normal and abnormal conditions.

Localization of bone morphogenetic protein (BMP) in human tissues and cells is important for investigating the mechanism of bone induction. A stable cell line secreting monoclonal antibody against bovine BMP (bBMP-McAb) was obtained by the hybridoma technique. The result of immunohistochemical staining (ABC method) showed that BMP is distributed along collagen fibers of normal bone, in periosteal cells, and in mesenchymal cells of marrow stroma. Little BMP can be found in bone cells of lamellar bone or in calcified bone matrix. BMP may be abundant in human tooth anlagen such as predentin, cells of the outer and inner enamel epithelium, and cells of dental sac generating bone. BMP is found in the cytoplasm of tumor cells of osteosarcoma and chondrosarcoma. Immunohistochemical staining showed that BMP plays a role in bone fracture healing. The ability of BMP-McAb to detect BMP and to inhibit the generation of new bone also makes it potentially useful in diagnosing, treating, and providing a prognosis for osteosarcoma and other bone diseases.

Evidence for uptake of basement membrane by differentiating ameloblasts in the rat incisor enamel organ.

Demonstration of type-IV collagen and acid phosphatase (ACPase) was carried out in the rat incisor enamel organ after the animals were fixed by perfusion with periodate-lysine-paraformaldehyde. Their incisors were dissected out, demineralized with EDTA, and prepared into 6-microns-thick frozen sections. The sections, which had been treated by means of antibody incubation for type-IV collagen, were washed with a Trismaleate buffer, incubated in Novikoff's medium for acid phosphatase (ACPase), and then incubated in a 3, 3'-diaminobenzidine solution. After osmification, the sections were embedded in epoxy resin for electron microscopy. The plasma membranes of the distal ends of the inner-enamel-epithelial cells were relatively even and were lined with a basement membrane. Type-IV collagen was localized both in the lamina densa and in the filaments attached to the lamina densa. In differentiating ameloblasts, the remarkably undulating distal plasma membranes formed irregular shallow and deep invaginations, and small cytoplasmic processes that penetrated the basement membrane. Coated pits occurred in various parts of these undulating plasma membranes. Positive reaction to type-IV collagen was observed in the invaginations and coated pits. ACPase-positive granules, present in inner-enamel-epithelial cells, increased in number and sometimes appeared close to both shallow and deep invaginations of differentiating ameloblasts. These results indicate that type-IV collagen in the basement membrane of the enamel organ is removed and degraded by differentiating ameloblasts by means of their engulfing system.

Human amelogenins: sequences of "TRAP" molecules.

The extracellular protein matrix of developing enamel includes a major class of proteins, the amelogenins, which are believed to be concerned in regulating enamel biomineralization. Previous studies have shown the amelogenins of the extracellular matrix to be a complex of proline-rich hydrophobic proteins which, it is suggested, arise through posttranslational and postsecretory processing of a primary ameloblast gene product. More recently, it has been shown that the human amelogenin gene is located on both the X and Y chromosomes raising the possibility that polymorphism at the level of the gene may also contribute to the observed complexity of these enamel matrix proteins. To investigate such possible amelogenin polymorphism in developing human dental enamel, individual fractionated by size-exclusion and reversed-phase high pressure liquid chromatography (HPLC). Two tyrosine-rich amelogenin polypeptides (TRAPs) of approximately 5 kDa in size were isolated from an individual human dentition and characterized by automated gas-phase sequencing. These polypeptides were found to be of 42 (TRAP-2) and 44 (TRAP-1) amino acid residues in length; TRAP-2 lacked a carboxy-terminal -Gly-Trp sequence as has previously been described for analogous bovine TRAP molecules. However, residue #25 of the human TRAP-2 sequence was refractory to sequencing, apparently differing from the Trp-25 identified in TRAP-1. These findings suggest (1) two forms of TRAP molecules, differing only by cleavage of a carboxy-terminal dipeptide, are a general feature of human and other mammalian enamel proteins, probably being derived by postsecretory cleavage from the primary extracellular amelogenin; and (2) in human developing enamel four forms of TRAPs may arise either from polymorphism at the level of the gene, or by posttranscriptional alternative splicing of amelogenin mRNAs, coupled with specific post-secretory proteolytic processing.

Phylogenetic distribution of enamel proteins: immunohistochemical localization with monoclonal antibodies indicates the evolutionary appearance of enamelins prior to amelogenins.

The hard covering tissues, enamel or enameloid, of representative vertebrate teeth were immunohistochemically stained using specific monoclonal antibodies against bovine amelogenins and bovine enamelins in order to determine the phylogenetic distribution of enamelin and amelogenin proteins. Immunohistochemically, only enamelin proteins were present in lower vertebrate (shark, bony fish, and larval amphibian) teeth and dermal denticles. Both enamelin and amelogenin proteins were present in higher vertebrate (mammal, reptile, and adult amphibian) teeth. Large hydroxyapatite crystal size and high levels of mineralization, characteristics common to both enamel and enameloid, are probably due to the presence of the common protein enamelin. The evolution of enamel from enameloid in the tetrapods seems to have involved the development of the gene for amelogenin.

Distribution of intermediate-filament proteins in the human enamel organ: unusually complex pattern of coexpression of cytokeratin polypeptides and vimentin.

We applied immunohistochemical techniques and gel electrophoresis to examine the distribution of intermediate filaments in human fetal oral epithelium and the epithelia of the human enamel organ. Both methods demonstrated that human enamel epithelia contain cytokeratins 5, 14, and 17, which are typical of the basal cells of stratified epithelia, as well as smaller quantities of cytokeratins 7, 8, 19, and in trace amounts 18, which are characteristic components of simple epithelial cells. In the external enamel epithelium and stellate-reticulum cells, most of these components appeared to be simultaneously expressed. In contrast, the parental oral epithelium was negative for cytokeratin 7, thus indicating possible "neoexpression" during the course of tooth formation. Immunohistochemical procedures using various monoclonal antibodies against vimentin revealed the transient coexpression of vimentin and cytokeratins in the external enamel epithelium and in stellate-reticulum cells during enamel development. The significance of the coexpression of cytokeratins and vimentin is discussed in relation to previous findings obtained in other normal tissues and in the light of the functional processes characteristic of these epithelia.

Calmodulin immunocytochemistry in rat incisor enamel organ through its life cycle.

The enamel organ of the growing rat incisor was perfusion-fixed with a mixture of formaldehyde and glutaraldehyde and processed for indirect immunogold labeling of calmodulin on post-embedded ultrathin sections. Throughout the zones of presecretion, secretion, and maturation of enamel, specific protein A-immunogold labeling was localized on polyribosomes and those attached to endoplasmic reticulum, mitochondria, nuclear chromatin, phagolysosomes, and cytoplasm adjacent to the plasma membrane, and tonofilaments associated with desmosomes of ameloblasts and cells of outer layer of enamel organ. Golgi membranes, condensing vacuoles, secretion granules, primary lysosomes, and micropinocytotic coated vesicles were hardly labeled. In the presecretion zone, the basal lamina of the preameloblasts and the matrix vesicles and collagen fibrils of the predentin matrix were not immunoreactive. Tomes' process of secretory ameloblast and adjacent enamel crystals were labeled. In addition to the above immunoreactive structures, some phagolysosomes, ferritin granules, and the cytoplasm of the ruffled border zone of maturation ameloblast contained immunogold particles. In control sections incubated with either protein A-gold complex alone, or antiserum preabsorbed with an excess of calmodulin and protein A-gold complex, only a few gold particles were observed to be randomly associated with the tissues. These results indicate that calmodulin is present in the cells of the enamel organ through all stages of amelogenesis. Its wide distribution is consistent with its involvement in various cytoplasmic functions.

Bovine tooth-derived bone morphogenetic protein.

Eight groups of dental tissues were mechanically dissected from the mandibles of one-year-old steers; they were then defatted and decalcified in HCl. The noncollagenous proteins were extracted with various solvents from collections of tissue and bio-assayed for osteo-inductive activity. Collectively, the hard tissue (dentin, enamel, and cementum) noncollagenous proteins were fractionated by molecular sieve chromatography, hydroxyapatite affinity chromatography, and ion exchange chromatography. Osteo-inductive activity of each protein fraction was determined by implantation in the quadriceps muscle pouch of mice. The quantity of bone was measured by computerized image analysis. From 71% to 83% of 41 implants of dental hard tissues induced bone formation. The quantity of bone was greater from unerupted than from erupted teeth. Dental soft tissues that had no osteo-inductive activity were rich in a 14-kDa protein, presumably matrix gamma-carboxyglutamic acid-rich proteins. Proteins with Mr of from 15 to 28 kDa were associated with osteo-inductive activity. Components with Mr greater than 28 kDa had no activity. These observations suggest that bovine teeth have a selection of osteo-inductive proteins that is comparable in range of MW to bovine bone morphogenetic protein.

Patterns of expression of intermediate filaments in ameloblastoma and human fetal tooth germ.

Monoclonal antibodies (Mab) were used to study the expression of cytokeratins and vimentin in various histological types of ameloblastoma and in human fetal tooth germ. The ameloblastoma and the tooth germ epithelia showed characteristics of both simple glandular and stratified squamous epithelial cells. Cytokeratin No. 18 was detected focally in most ameloblastomas studied but not in fetal odontogenic epithelia. Cytokeratins Nos. 8 and 19 were expressed in all epithelial elements of ameloblastomas and tooth germs. Only two tumors showed focally characteristics of keratinizing epithelia also seen in dental lamina but not in the enamel organ. All tumors except the granular cell ameloblastoma showed a variable coexpression of vimentin and cytokeratins in their neoplastic epithelia. A similar coexpression was detected in the stellate reticulum cells of the developing tooth. Ameloblastoma and human tooth germ epithelia share complex pattern of cytokeratin polypeptides together with coexpression of vimentin. The results strongly support the theory that ameloblastomas are of odontogenic origin and not direct derivatives of basal cells of oral epithelium or epidermis.

Micro-PIGE determination of fluorine distribution in developing hamster tooth germs.

A micro-PIGE (Proton-Induced gamma-ray Emission) technique based on the delayed 5/2+----1/2+ nuclear transition of fluorine (E gamma = 197 keV, t1/2 = 87 ns) emitted after 19F(p,p', gamma)19F reaction was used to detect and study the distribution of fluorine in the developing enamel organ during pre-eruptive stages, i.e., the transitional to early maturation stages of enamel formation in neonatal hamsters administered a single IP dose of sodium fluoride (20 mg NaF/kg body weight). The aforementioned nuclear reaction is unique for fluorine, and therefore detection of gamma-rays emanating from this reaction in a biological specimen implies a positive identification of fluorine at that particular site. Calcium and phosphorus X-rays were also recorded and used as parameters for assessment of the relationship between the degree of mineralization and fluoride incorporation into the enamel organ. The highest fluorine concentration in the enamel organ was recorded in the dentin near the dentin-enamel junction (DEJ). In the enamel, the highest concentration of fluorine was found to be associated with the more mature areas of the enamel near the DEJ, but gradually decreased in the direction of the enamel surface. Fluorine was not detected in the control germs. These results suggest that administration of fluoride in high doses during the pre-eruptive stages of enamel formation leads to incorporation of the ion into the forming dentin and enamel mineral, and that the enamel matrix does not seem to bind fluoride avidly.

Cytokeratin expression of the odontogenic epithelia in dental follicles and developmental cysts.

The patterns of cytokeratin expression in the epithelium of 5 dental follicles, 7 dentigerous cysts, 5 odontogenic keratocysts, 3 nasopalatine cysts and an epidermoid cyst have been studies using a panel of monoclonal antibodies. The epithelium of dental follicles and of developmental odontogenic cysts strongly expressed keratins 5 and 19 and showed weaker expression of keratins typical of stratified non-cornified and of simple epithelia. Staining with mAbs against the latter keratins varied with the degree of epithelial differentiation. Nasopalatine cysts strongly expressed simple epithelial keratins and the epidermoid cyst strongly expressed a marker of cornification. Odontogenic cysts thus appear to differ in their pattern of keratin expression from other oral developmental cysts and all derivatives of odontogenic epithelia appear to share similar basic patterns of cytokeratin expression.

Localization of lead and fluoride in cultured tooth germs by laser microprobe mass analysis.

Trace elements can influence dental health, possibly by altering tooth resistance during preeruptive development. Therefore, it was investigated whether lead and fluoride would be incorporated into the calcifying matrices or the cellular parts of tooth germs in vitro. Using laser microprobe mass analysis, the localization of lead and fluoride was studied in the different layers or tooth germs that had been cultured in a medium to which PbCl2 of NaF had been added in different concentrations. Both elements could only be detected in the dentine layer. Hence, the enamel organ in the secretory stage of tooth development excludes lead and fluoride from the enamel, even when enamel formation by the ameloblasts is visibly disturbed. Furthermore, there seemed to be a process of saturation in the accumulation of lead and fluoride in the dentine.

Histochemical localization of calcium in the enamel organ of rat incisors in early-stage amelogenesis.

The localization of calcium in the enamel organ of rapidly-frozen, freeze-substituted rat incisors in early-stage amelogenesis was examined by a histochemical calcium-staining method. In secretory ameloblasts, glyoxal bis(2-hydroxyanil) (GBHA) staining revealed intense red reactions in mitochondria and tubulovesicular structures located throughout the cytoplasm, while no reaction was seen in the nucleus and cytosol, nor along the plasma membranes of the respective cells. No significant GBHA reaction was observed in the intercellular compartment and other cells of the enamel organ. Some granular reactions were localized in the cells of the adjacent connective tissue. Control tests confirmed the specificity of GBHA reactions for calcium. Thus, the present observations provide histochemical evidence indicating an exclusive localization of calcium in mitochondria and tubulovesicular structures of the secretory ameloblast, and support their contributions to the translocation of calcium from the proximal to the distal pole of the cytoplasm.

Ultrastructural localization of dentine phosphoprotein in rat tooth germs by immunogold staining.

Dentine phosphoprotein (DPP) was localized on thin frozen sections of fixed rat tooth germs by indirect immunogold staining. Antisera were directed against DPP and against glutaraldehyde-treated DPP and were characterized by immuno-electroblotting. In odontoblasts, DPP was found to be localized in the cisternae of the rough endoplasmic reticulum (RER) and the Golgi apparatus and in Golgi-associated vesicles. Odontoblastic processes were moderately positive for DPP and dentine was intensely labeled on frozen sections of unfixed tissue. Predentine showed a slight immunoreactivity. These results indicate the synthesis of DPP in the RER, its accumulation in the Golgi apparatus and its vesicular transport and secretion via the odontoblastic processes into dentine. The close association of the gold particles with the dentinal collagen fibres makes a role of DPP in linking mineral to collagen conceivable. Matrix vesicles were negative for DPP, suggesting that the protein is not present at the sites of matrix vesicle-associated nucleation.

A histochemical demonstration of calcium in the maturation stage enamel organ of rat incisors.

The location of calcium in a rapid-frozen and freeze-substituted maturation stage enamel organ of the rat incisors was demonstrated by means of the glyoxal bis(2-hydroxyanil) (GBHA) staining method, which formed insoluble red precipitates of calcium-GBHA complex. In the ameloblast layer, highly GBHA-reactive tubulo-vesicular structures corresponding to mitochondria and some other membrane-bound structures were localized in both ruffle-ended and smooth-ended ameloblasts, although no significant GBHA reaction was localized in the nucleus, Golgi region, nor along the plasma membrane of these cells. In addition, numerous granular GBHA reactions appeared exclusively in association with the ruffled border of ruffle-ended ameloblasts. GBHA reactions were positive, but were considerably weaker in papillary cells than in the ameloblast. These observations provide a first published histochemical mapping of calcium in the maturation stage enamel organ, and suggest the active participation of mitochondria in maturation stage ameloblasts in calcium regulation.

Aspiration and characterization of predentin fluid in developing rat teeth by means of a micropuncture and micro-analytical technique.

A fluid phase was aspirated in vivo and in vitro from predentin or pulp of developing rat teeth by means of a micropuncture technique. Pooled aspirates (approx. 2 nL) were analyzed for P, Na, K, Ca, Mg, and S by electron probe microtechniques (Lechene and Warner, 1979). Compared with pulp fluid, currently and previously studied cartilage fluids, as well as serum, predentin fluid showed elevated K, depressed Na, Cl, and Ca, as well as increased P. Statistical analysis was possible for only a few groups of comparisons among the elemental profiles. Ultrastructural examination of the aspiration site and of the aspirates showed no evidence of contamination with cell organelles or other formed elements. The micropuncture technique used was a critically precise and laborious procedure; possible contamination with intracellular fluid could not be avoided. The consistently low Mg concentration found in the aspirates, however, supports our view that the samples were primarily extracellular.

Ultrastructural localization of osteocalcin in rat tooth germs by immunogold staining.

Osteocalcin was localized by indirect immunogold staining of thin frozen sections of rat tooth germs which had been fixed by different methods. Acrolein fixation proved to be satisfactory considering the preservation of fine structure and antigenicity. In odontoblasts, osteocalcin was found to be localized in the cisternae of the rough endoplasmic reticulum and Golgi apparatus. Few positive transport vesicles were found. Staining for osteocalcin in odontoblastic processes was only observed after strong fixation and was intense in odontoblasts engaged in early dentine formation. Predentine was slightly positive in the neighbourhood of positive processes. Matrix vesicles were negative and strong osteocalcin labeling of dentine seemed to appear after the onset of mineralization.

Proteins in the enamel fluid of immature porcine teeth.

The fluid was separated from the immature soft enamel of porcine permanent teeth in the secretory stage according to procedures reported previously (Aoba and Moreno, 1987). The protein content of the fluid was about 2.8% w/v; its amino-acid composition was characterized by high contents of Pro, Glx, Leu, and His, showing composition similar to that of the 20 kilo-dalton (kd) amelogenin or its C-terminal segments. The two major protein species in the fluid had apparent molecular weights of 13 kd and 11 kd, as determined by SDS electrophoresis; the N-terminal residue of the former was Leu, while that of the latter was Ala. The C-terminal sequence of both of them was -Met-Phe-Ser. By comparison with the published sequence of 20-kd porcine amelogenin, it is concluded that the main fluid constituents were derived by cleavages of N-terminal segments from the 20-kd amelogenin.

Immunocytochemical localization of osteocalcin in developing rat teeth.

Osteocalcin was purified by gel chromatography from a crude extract obtained after decalcification of rat incisors. The apparent molecular weight, as determined by 5-15% SDS-polyacrylamide gel electrophoresis, was 18,000, and amino acid analysis revealed 60 gamma-carboxyglutamic acid residues per 1000. Antisera against osteocalcin, raised in rabbits, reacted specifically with osteocalcin when investigated by immuno-electroblotting of dentin crude extract. 4-micron cryosections of formaldehyde-fixed tooth germs showed positive immunocytochemical staining for osteocalcin in dentin and odontoblasts. The staining of the mantle dentin at the coronal sides of the tooth germs was more intense than that of the adjacent circumpulpal dentin, while the odontoblasts involved in the formation of mantle dentin showed stronger immunoreactivity than did odontoblasts involved in circumpulpal dentin formation. This marked difference was not observed on the root sides of the tooth germs. In 1-micron cryosections, osteocalcin immunoreactivity was found evenly distributed throughout the entire cell body, with the exception of the Golgi region, which was less intensely stained, while the nucleus and the cell process were negative. The positive staining reaction with anti-osteocalcin antiserum was found in dentin from the very onset of its formation in the fetus. In conclusion, our results demonstrate the presence of osteocalcin in odontoblasts and dentin. Its immunocytochemical localization may be compatible with a distinct role in early dentinogenesis.