Expression of dentin matrix protein 1 (DMP1) in nonmineralized tissues.

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.

Aspecto clinicopatológico e inmunohistoquímico en tres casos de ameloblastoma / Clinico-pathologic and immunohistochemical aspects in three ameloblastoma cases

El ameloblastoma es una de las neoplasias odontogénicas más comunes que se presentan en el maxilar inferior con mayor frecuencia, sector posterior, con localización intraósea y periférica (esta última, más rara). Los subtipos histológicos no influencian directamente en el tratamiento ni en el pronóstico porque generalmente se utilizan actos quirúrgicos radicales por la frecuencia de recidiva de los ameloblastomas. Se utilizó en biopsias las técnicas inmunohistoquímicas básicas y Mib-1 (Ki 67 en parafina) para determinar el índice de proliferación celular que indicaría la posible transformación maligna del ameloblastoma o su capacidad de recidiva. En un germen dentario, en estadio de campana, el órgano del esmalte mostró idéntica afinidad inmunohistoquímica con un índice de proliferación celular Mib 1 (Ki 67) de un 1 por ciento (AU)

The immunohistochemical localization of Fas and Fas ligand in jaw bone and tooth germ of human fetuses.

The cellular localization and roles of bone morphogenetic protein (BMP)-2 and apoptosis-associating factors in human orofacial development remain unclear. In this study, BMP-2, osteocalcin, and TGF-beta, which are bone-differentiating markers, apoptosis-associating factors (i.e., Bcl-2, Bax, Fas, and Fas ligand), apoptotic cells detected by the in situ 3'-end labeling method (TUNEL), and proliferating cell nuclear antigen (PCNA) were immunohistochemically examined in the heads (in particular, the jaw bone and tooth germs) of human fetuses of 11-week pregnancy. BMP-2 was positive in osteoblasts and newly formed osteoid of the incisive and palatal bone of the maxilla and the mandible, which indicated that BMP-2 was exclusively involved in intramembranous ossification in the human fetal head. Fas was positive in the cytoplasm of osteocytes and a few osteoblasts. In contrast, Fas ligand was positive in the cytoplasm of osteoblasts and abundant in the stroma of the osteoblastic layer, periosteum, and perichondrium. The Fas ligand in the stroma was recognized as the soluble form, which was possibly produced by osteoblasts. TUNEL-positive apoptotic cells were found in a few osteocytes and a few osteoblastic cells in new bone, and in monocytes of degenerate Meckel's cartilage. The induction of apoptosis observed in monocytes seems to be caused via a Fas-Fas ligand cell death system, because some of these monocytes were Fas-positive, and most of them were Fas ligand-positive. Interestingly, the abundant soluble Fas ligand observed in the periosteum probably protects the bone-formative zone from the invasion of the activated lymphocytes by binding to Fas expressing in these lymphocytes and killing these cells. Fas and Fas ligand were focally positive in the dental lamina and inner enamel epithelium and cusps of the enamel organ, nevertheless, the presence of TUNEL-positive cells was very rare. Bcl-2 was clearly and Bax was weakly positive in the cells throughout the dental lamina and enamel organ. These findings indicated that Fas-mediated apoptosis was inhibited by the Bcl-2 family in the development of teeth.

Loss of deciduous teeth and germs of permanent incisors in a 4-year-old child. An atypic prepubertal periodontitis? A clinical, microbiological, immunological and ultrastructural study.

A 4-year-old child was referred, in April 1988, to Rennes Dental School (France) for deciduous tooth mobility with premature loss of 4 deciduous teeth and germs of 2 permanent incisors. Microbiological examinations by culture revealed the presence of the periodontal pathogen Actinobacillus actinomycetemcomitans. Immunofluorescence of plaque samples revealed the presence of Porphyromonas gingivalis that had not been isolated by culture. Neutrophil functions were within normal ranges. Transmission electron microscopy of gingiva showed a disorganised epithelium. The connective tissue was infiltrated by inflammatory cells. The basement membranes were normal, but the connective tissue-epithelium interface was mainly composed of short rete pegs. Scanning electron microscopy of extracted deciduous teeth revealed lack of cementum, lacunae in the cementum and lack of fibrillar insertion on the middle part of the root. Skin lesions, mainly situated on face, were observed. Treatment was by extraction of mobile deciduous teeth combined with 3-week courses of metronidazole. Clinical and microbiological follow-up was continued over a 7-year period. No periodontal lesions have been detected since eruption of the permanent teeth. The present subgingival and lingual microflora (December 1995) is composed of bacteria associated with periodontal health. However, the future appearance of a hitherto undetected systemic disease is still possible.

Premolar child-to-parent grafting within the framework of orthodontic serial extraction.

While allografting of human teeth holds all the risks and problems of immunological reactions or host-versus-graft reactions encountered in any organ transplantation, root resorption and thus the loss of the grafted tooth may be delayed by several decades if the transfer is between close relatives and minimum histocompatibility is 50%. In 2 cases premolars with incomplete root growth, which had to be extracted because of considerable crowding, were transplanted from child to mother and child to father, respectively. The grafts healed without complications and after a retention time of several weeks the teeth were attached. Three years after transplantation the teeth show only minor signs of root resorption and are fully functional.

Immunohistochemical detection of an enamel protein-related epitope in rat bone at an early stage of osteogenesis.

Monoclonal antibody MI315 was produced against hamster tooth germ homogenate by in vitro immunization. It was found that MI315 reacted with enamel matrix, ameloblasts, and bone matrix at an early stage of osteogenesis. Decalcified tissues of rat femurs and mandibles were examined with MI315 using indirect immunofluorescence. In endochondral ossification of femurs, immunoreactivity was found in bone extracellular matrix (ECM) deposited on the surface of the cartilage core of primary spongiosa, but not in the cartilage core itself. In intramembranous ossification of 0-day-old rat mandibles, intense immunofluorescence was detected in bone ECM and a few young osteocytes, but not in osteoblasts. Immunoreactivity in bone ECM of 2-day-old rats decreased and almost disappeared from bone ECM of 4-day-old rats. Although in nondecalcified sections of 0-day-old rats, negligible immunofluorescence was detected in bone ECM which showed positive staining in decalcified tissues, the immunostaining appeared after decalcification using ethylenediaminetetraacetic acid (EDTA). These results indicate that a substance(s), which had a common epitope with an enamel-derived protein(s), existed in immature bone ECM of both endochondral and intramembranous ossification, and that it might be masked by bone mineral. Monoclonal antibody MI315 is a useful tool to investigate the time- and position-specific changes in osteogenesis and amelogenesis.

Anti-peptide antibodies reactive with epitopic domains of porcine amelogenins at the C-terminus.

This was an immunological investigation of the processing of porcine amelogenins in situ. Rabbit and rat anti-peptide sera reacted specifically with the hydrophilic segment of the intact amelogenins at the C-terminus. The immunogens used were the synthetic peptides: (a) C13 composed of PATDKTKREEVDC and (b) C25 composed of MQSLLPDLPLEAWPATDKTKREEVD. These peptides correspond to the C-terminal 12- and 25-residue segments of porcine amelogenin, respectively. Cystine was introduced at the C-terminus of C12 for KLH-binding (C13). Western blot analysis disclosed that: (i) both rabbit and rat anti-C13 sera reacted selectively with the 25-kDa porcine amelogenin and three other minor components (27, 22 and 18 kDa); (ii) anti-C25 peptide sera, additionally, reacted with the 23-kDa amelogenins (a degradation derivative of the 25-kDa protein, lacking the 12-residue segment at the C-terminus) and as trace components, 20-, 16- and 14-kDa moieties. Importantly, all the proteins reactive with the anti-C13 serum were concentrated in the outer secretory enamel adjacent to the ameloblasts, decreasing significantly in the underlying inner secretory enamel. Immunohistochemical studies applying the anti-peptide sera to the developing tooth germs of a minipig also confirmed the localization of reactivity in the outer secretory region. Neither anti-peptide serum reacted with porcine non-amelogenins, serum proteins nor dentine matrix proteins at the dilutions tested. however, it was found that both the anti-C13 and C25 sera reacted with human keratin.(ABSTRACT TRUNCATED AT 250 WORDS)

Chondroitin sulfates in developing mouse tooth germs. An immunohistochemical study with monoclonal antibodies against chondroitin-4 and chondroitin-6 sulfates.

The role of glycosaminoglycans and proteoglycans during ontogenesis is not known. The developing tooth offers a potentially important model for studies of structure-function relationships. In this study, we have analysed the temproal and spatial expression of chondroitins of differing sulfation patterns in embryonic molars and incisors. For this purpose, we have used monoclonal antibodies (Mabs) specific for unsulfated, 4-sulfated, and 6-sulfated forms of chondroitin in conjunction with indirect immunofluorescence or immunoperoxidase labeling. Unsulfated chondroitin was not detected in embryonic teeth. Chondroitin 4- and chondroitin 6-sulfates were present in the stellate reticulum but otherwise they were confined to the dental mesenchyme. The 3B3 and MC21C-epitope, which are markers of 6-sulfated chondroitin, were uniformly distributed in the dental mesenchyme during the bud stage; they disappeared from the dental papilla of the cusps and of the anterior region of the incisor as development proceeded. These epitopes were absent from the basement membrane and from the predentin. In the odontoblastic cell lineage, the 3B3 and MC21C-epitopes were detected only between preodontoblasts at an early stage of differentiation. The monoclonal antibody 2B6 served as a probe to localize chondroitin 4-sulfate. This glycosaminoglycan was detected as early as the dental lamina stage but its expression was restricted to the basement membrane of the teeth until the late bell stage. After the onset of cusp formation, strong staining was also observed over the occlusal region of the dental papilla while the cervical region of the dental papilla remained 2B6-negative. Incisors at the bell stage exhibited a decreasing gradient of immunostaining by 2B6 from their anterior region to their posterior end. The extracellular matrix surrounding preodontoblasts reacted with 2B6 and the predentin, produced by the odontoblasts, was also intensely labeled with this antibody. Comparison between immunostaining with 3B3 and 2B6, on consecutive sections revealed a mutually exclusive pattern of distribution of the corresponding epitopes during odontogenesis. Furthermore, in the continuously growing incisor, a striking positive correlation was found between the immunostaining patterns produced by 3B3 and MC21C and the mitotic indices along the anterior-posterior axis of the tooth. Hence, sulfation of chondroitin seems developmentally regulated. We postulate that changes in the sulfation pattern of chondroitin might play a role in ontogenesis by locally altering the functional properties of the extracellular matrix.

[Production of monoclonal antibodies against tooth germs of hamster or rat by in vitro immunization].

The in vitro immunization procedure for the production of monoclonal antibodies has several advantages over the in vivo procedure; e.g. it requires a much smaller amount of immunogen and only a few days are required for immunization. However, no in vitro immunization procedure for the production of monoclonal antibodies against tooth germs has been reported. By means of in vitro immunization, we tried to produce monoclonal antibodies against the components of tooth germs which would be useful for immunohistochemical investigation of the development of tooth germs. Spleen cells of mice were immunized with a homogenate of molar tooth germs from hamsters or rats. The hybridomas produced were screened by immunohistochemical examination of paraffin sections of tooth germs. We obtained five monoclonal antibodies reacting with the dental tissues; ameloblasts, odontoblasts, enamel, dentin, predentin, and dental sac. One of them reacted specifically with only ameloblasts and one with ameloblasts and enamel, but the others are not specific for individual dental tissues. The results demonstrate that in vitro immunization is a satisfactory method for producing monoclonal antibodies useful for immunohistochemical investigation of tooth germs.

Production of monoclonal antibodies against murine dental papilla.

The mesenchymal component of the embryonic tooth, the dental papilla, shows unique cellular behavior. Only the dental papilla cells are capable of differentiating into odontoblasts. The dental papilla also directs the morphogenesis of the tooth. Our aim is to develop suitable markers for further studies on the molecular mechanisms behind the determination and differentiation of the dental mesenchymal cells. We have produced monoclonal antibodies against this embryonal cell population. Altogether 1114 enzymatically separated mesenchymes from dissected molar tooth germs of 17-day-old mouse embryos were fixed in paraformaldehyde (PFA) and sonicated in saline. A rat was immunized and hybridomas were produced by standard methods. The supernatants were screened by immunohistology, using both frozen sections and sections of PFA-fixed paraffin-embedded mouse molar teeth. Altogether, 19 wells produced antibodies reacting with dental tissues. One monoclonal antibody recognizes a 67,000 dalton intracellular antigen enriched in secretory odontoblasts and ameloblasts. The antigen is also found in osteoblasts and chondrocytes in the developing jaw, and in some cultured murine cells. The method described here appears to be successful for generating and screening monoclonal antibodies against the dental papilla.

Immunoglobulin G in cystic lesions of rat enamel organ following fluoride intoxication.

Fluoride given as a high single dose to young rats with developing molars has earlier been shown to cause subameloblastic cysts with disorganized ameloblasts in the cystic wall and an irregular mineralization pattern of the underlying enamel. In the present study immunohistochemistry has been employed to determine if an increased permeability of the enamel organ occurred at the areas of cell disturbances. For this purpose 5-day-old rats were injected with 60 mg sodium fluoride per kg body weight. They were decapitated after 24 h, the maxilla prepared histologically and paraffin sections incubated for the demonstration of IgG according to the avidin-biotin-peroxidase complex method. Staining for IgG was present in the cystic lumina and in areas of disorganized ameloblasts. No reaction was observed in areas of unaffected ameloblasts. It was suggested that the fluoride-induced cell injury increased the permeability of the ameloblastic cell layer. Diffusion of IgG and most likely other substances as well, through the ameloblastic layer may have contributed to cyst formation and to the irregular mineral deposits that have been found in the ameloblastic layer and at the enamel surface.

Distribution of type 1 and 2 blood group chains in normal and pathological odontogenic epithelium defined by monoclonal antibodies specific for Lea and H type 2.

This study describes the distribution of type 1 and type 2 blood group carbohydrate chains in human normal and pathological odontogenic epithelia and in epithelia of human oral mucosa. Odontogenic epithelium was examined from 12 fetal tooth germs, 25 ameloblastomas, 13 odontogenic keratocysts, 13 follicular cysts and 13 radicular cysts. Oral mucosal epithelia was studied from 12 fetuses and 10 adults. Cell surface carbohydrates were detected using antibodies with reactivity for the blood group antigens A, B, type 1 chain Lea and type 2 chain H by an immunofluorescence technique. The expression of Lea and H type 2 chain in fetal palatal epithelium and only H type 2 chain in adult palatal epithelium suggests that a change in synthesis of blood group chains occurs during development. Type 2 blood group chains (antigen H) were found in fetal tooth germs, type 1 (Lea) in ameloblastomas and both type 1 and type 2 in odontogenic cysts. These results indicate that a modulation in synthesis of blood group carbohydrates has occurred in ameloblastomas and odontogenic cysts as compared with the cells from which the lesions presumably are developed. It is suggested that ameloblastomas may be distinguished from odontogenic cysts by the inability of ameloblastomas to synthesize type 2 blood group chains and antigens A and B.

Relation of blood group carbohydrates to differentiation patterns of normal and pathological odontogenic epithelium.

The distribution of epithelial cell surface antigens was studied in normal odontogenic epithelium from 20 fetuses and in odontogenic epithelium from 15 ameloblastomas, 16 odontogenic keratocysts, 15 follicular and 15 radicular cysts. The cell surface carbohydrates were detected using antibodies with reactivity for the blood group antigens A, B, H type 2 (A and B precursor) and N-acetyllactosamine (N-lac, H type 2 precursor) by an immunofluorescence technique. The expression of the blood group carbohydrates differed considerably in normal fetal odontogenic epithelium from that in ameloblastomas and odontogenic cysts. The A, B and H type 2 antigens were demonstrated in odontogenic keratocysts and in follicular and radicular cysts. Expression of the blood group carbohydrates was similar in follicular and radicular cysts but differed from that seen in odontogenic keratocysts by the failure to detect N-lac in the latter. The antigens A, B, H type 2 and N-lac were not expressed in any of the ameloblastomas including types with palisading of basal cells and polarization of basal cell nuclei and types with a plexiform pattern with cuboidal or polyhedral shaped peripheral cells. The findings indicate that epithelium of ameloblastomas can be distinguished from odontogenic cyst epithelium by differences in expression of cell surface carbohydrates with blood group specificity.

Localization of H-2Kk in developing mouse teeth using monoclonal antibody.

The in situ distribution of H-2 antigens during mouse tooth morphogenesis was investigated using monoclonal antibodies to H-2Kk and indirect immunofluorescent techniques. H-2 antigens were detected in the basement membrane region of fetal molars; they were absent from both the epithelial and dental mesenchyme. H-2 antigens were not found in newborn and 4-day-old mouse molars.

Immunofluorescent evidence for the similarity of amelogenins in calf, mouse and pig teeth.

Antiserum was prepared to fetal bovine enamel matrix and was used to localize the amelogenins in developing bovine molars by immunofluorescent microscopy. Amelogenins could be identified to preameloblasts, secretory ameloblasts, stratum intermedium cells, and the newly deposited enamel matrix. Mature enamel matrix did not fluoresce except in a thin line along the DEJ and adjacent to the ameloblasts. Immature enamel matrix of murine and porcine teeth fluoresced when treated with antiserum to bovine enamel matrix. No other portions of tooth buds or other tissues reacted with the specific antiserum.

The nature and nurture of epithelial-mesenchymal interactions during tooth morphogenesis.

Epithelial-mesenchymal interactions during tooth morphogenesis are inductive and instructive developmental processes as well as permissive and regulatory processes. Data is available to support the early influences of enamel organ epithelium upon a responding mesenchyme in the determination of dental morphogenetic fields (Dryburg, 1967; Miller, 1969). Mesenchymal specificity appears to be operant during tooth shape and form and during the induction of secretary amelogenesis (Kollar, 1972). These heterotypic tissue interactions can be observed in vivo and in vitro. The cellular responses to these interactions appear to be transcriptional, translational and post-translational; as a direct consequence of the interactions, new gene products are synthesized and secreted and/or pre-existing gene products are amplified (Hata and Slavkin, 1978). The mechanism(s) by which epithelial-mesenchymal interactions function may best be learned through critical investigations of differentiation alloantigens, receptors, coupling components within the plasma membrane, translating components by which epigenetic external cues become internal chemical information, and the associations between peripheral and integral proteins within the plasma membrane and intracytoplasmic microfilaments and microtubules.