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Aim: A tissue-engineered periodontal ligament (PDL) around implants would represent an important new therapeutic tool to replace lost teeth. The PDL is the key to tooth anchoring; it connects tooth root and alveolar bone, and it sustains bone formation. Materials and Methods: Cells were isolated from PDL and cultured in a bioreactor on titanium pins. After the formation of multiple cellular layers, pins were implanted in enlarged dental alveolae. Main Outcome Measures: Cell-covered implants integrated without adverse effects, and induced bone in their vicinity. Results: A histological examination of a dog model revealed that cells were arranged in a typical ligament-like fashion. In human patients, product safety was ascertained for 6-60 months. Probing and motility assessments suggested that the implants were well integrated with mechanical properties similar to those of teeth. Radiographs demonstrated the regeneration of deficient alveolar bone, the development of a lamina dura adjacent to a mineral-devoid space around the implant and implant migration in an intact bone structure. Conclusions: New tissue consistent with PDL developed on the surface of dental implants after implantation. This proof-of-principal investigation demonstrates the application of ligament-anchored implants, which have potential advantages over osseointegrated oral implants. [ABSTRACT FROM AUTHOR]

Activation of the protein kinase C (PKC)-related signal transduction system has been associated with phenotypic expression in a wide variety of cell types. In in vitro studies, it has often been activated by relatively small increases in the Ca2+ concentration ([Ca22+]) in the medium. The studies reported here explored the hypothesis that localized increases in the extracellular [Ca2+] and activation of the PKC-related pathway may be involved in early dentin and enamel formation. Whole-head, freeze-dried sections through the developing molars of 5-day-old rats were evaluated by methods that localized non-crystalline Ca2+. Immunohistochemical methods were adapted for use with the freeze-dried sections, and two monoclonal antibodies were used to localize PKCα in the formative cells of the developing teeth. Low concentrations of extracellular Ca2+ were observed in the early, unmineralized dentin in the area of ameloblast differentiation. Increased concentrations occurred at the point of initial dentin mineralization, immediately before the beginning of enamel matrix deposition. PKCα was localized in the differentiating odontoblasts, at the beginning of dentin matrix deposition. It was intensely localized in the distal borders of the pre-ameloblasts, and appeared to redistribute in the cells during ameloblast differentiation. These observations suggest that local increases in the extracellular [Ca2+] and the PKC signal transduction pathway may be involved in key inductions in the early stages of dentin and enamel formation. [ABSTRACT FROM AUTHOR]

Living organisms create, copy, and make use of information, the content depending on the level of organization. In cells, a network of signal chain proteins regulates gene expression and other cell functions. Incoming information is encoded through signal reception, processed by the network, and decoded by the synthesis of new gene products and other biological functions. Signaling proteins represent nodes, and signal transmission proceeds via allosteric binding, chemical and structural modifications, synthesis, sequestering, and degradation. The induction of the gene caudal type homeobox 2 (CDX2) in the mammalian preimplantation embryo is outlined as a demonstration of this concept. CDX2 is involved in the decision of cells to enter the trophoblast lineage. Two signal chains are coordinated into an information processing model with the help of logic gates. The model introduces a formal structure that incorporates experimental and morphological data. Above the cell level, information flow relates to tissue formation and functioning, and whole cells play the role of network nodes. This is described for the anatomical patterning of bone with implications for bone formation and homeostasis. The information usage in cells and tissues is set into a context of the nervous system and the interaction of human individuals in societies, both established scenes of information processing., (© 2021 The Authors. Annals of the New York Academy of Sciences published by Wiley Periodicals LLC on behalf of New York Academy of Sciences.)

Microarray analysis of odontoblastic cells treated with sodium fluoride has identified the asporin gene as a fluoride target. Asporin is a member of the small leucine-rich repeat proteoglycan/protein (SLRP) family that is believed to be important in the mineralization process. In this study, asporin expression and distribution were investigated by systematic analysis of dentin and enamel, with and without fluoride treatment. Specific attention was focused on a major difference between the two mineralized tissues: the presence of a collagenous scaffold in dentin, and its absence in enamel. Normal and fluorotic, continually growing incisors from Wistar rats treated with 2.5 to 7.5 mM sodium fluoride (NaF) were studied by immunochemistry, in situ hybridization, Western blotting, and RT-qPCR. Asporin was continuously expressed in odontoblasts throughout dentin formation as expected. Asporin was also found, for the first time, in dental epithelial cells, particularly in maturation-stage ameloblasts. NaF decreased asporin expression in odontoblasts and enhanced it in ameloblasts, both in vivo and in vitro. The inverse response in the two cell types suggests that the effector, fluoride, is a trigger that elicits a cell-type-specific reaction. Confocal and ultrastructural immunohistochemistry evidenced an association between asporin and type 1 collagen in the pericellular nonmineralized compartments of both bone and dentin. In addition, transmission electron microscopy revealed asporin in the microenvironment of all cells observed. Thus, asporin is produced by collagen-matrix-forming and non-collagen-matrix-forming cells but may have different effects on the mineralization process. A model is proposed that predicts impaired mineral formation associated with the deficiency and excess of asporin.

Epithelia in lung, skin, and kidney are often exposed to fluoride, and tissue damage in lung and kidney due to fluoride is well documented. Nevertheless, the biological effects of fluoride on epithelia are poorly investigated. In the present study, we report effects of sodium fluoride (NaF) on the differentiation of a human epithelial cell line, HaCaT. These cells may serve as a keratinocyte model, because they express a wide spectrum of keratins (Ks), and they associate into stratified tissue-like arrangements along with changes in their keratin pattern. NaF was added to the culture medium at concentrations of 0.5 and 5 mM. Cell proliferation remained intact, but cell functions were altered at high dose, and HSP70 was induced. Reverse transcription-polymerase chain reaction and Western blotting revealed that keratin (K) 15 mRNA and protein expression, associated with stratification of epithelia, were inhibited. Also, expression of keratins typical for terminal differentiation, K1 and K10, was decreased and so was the expression of the K1/10 regulating enhancer binding protein c/EBP alpha. Stratification of HaCaT cells was abolished at high fluoride dose, as assessed by electron microscopy. The changes in keratin expression were not reversed by withdrawal of fluoride. Taken together, NaF at high dose blocked terminal differentiation of HaCaT cells, visible by keratin expression and failing stratification. This effect may disturb tissue formation due to altered cell interactions.

Elevated fluoride intake may lead to local tissue disturbances, known as fluorosis. Towards an understanding of this effect, fluoride-induced molecular responses were analyzed in MO6-G3 cultured odontoblasts cells. NaF at 1mM changed expression of genes implicated in tissue formation and growth, without affecting cell proliferation or inducing stress factor RNAs. Up to 1mM NaF, DNA accumulation was not inhibited, whereas at 3mM, cells detached from their support and did not proliferate. Intracellular structures, characterized by EM, were normal up to 1mM, but at 3mM, necrotic features were evident. No sign of apoptotic transformation appeared at any NaF concentration. Fluoride-sensitive genes were identified by microarray analysis; expression levels of selected RNAs were determined by conventional and real-time RT-PCR. At 1mM fluoride, RNAs encoding the extracellular matrix proteins asporin and fibromodulin, and the cell membrane associated proteins periostin and IMT2A were 10-fold reduced. RNA coding for signaling factor TNF-receptor 9 was diminished to one-third, whereas that for the chemokine Scya-5 was enhanced 2.5-fold. These RNAs are present in vivo in tooth forming cells. This was demonstrated by in situ hybridization and RT-PCR on RNA from dissected tissue samples; for the presence and functioning of fibromodulin in dentin matrix, a more comprehensive study has earlier been performed by others [Goldberg, M., Septier, D., Oldberg, A., Young, M.F., Ameye, L.G., 2006. Fibromodulin deficient mice display impaired collagen fibrillogenesis in predentin as well as altered dentin mineralization and enamel formation. J. Histochem. Cytochem. 54, 525-537]. Expression of most other RNA species, in particular of stress factor coding RNAs, was not altered. It was concluded that fluoride could influence the transcription pattern without inducing cell stress or apoptosis. In odontoblasts in vivo, aberrant expression of these fluoride-sensitive genes may impair the formation of the extracellular matrix and influence cell communication, with the possible consequence of fluorotic patterns of normal and deviant dentin.

Extracorporeal shock wave (ESW) treatment has recently been established as a method to enhance bone repair. Here, we reported that ESW-promoted healing of segmental defect via stimulation of mesenchymal stem cell recruitment and differentiation into bone forming cells. Rats with a segmental femoral defect were exposed to a single ESW treatment (0.16 mJ/mm(2), 1 Hz, 500 impulses). Cell morphology and histological changes in the defect region were assessed 3, 7, 14, and 28 days post-treatment. Presence of mesenchymal stem cell was assayed by immuno-staining for RP59, a recently discovered marker, and also production of TGF-beta 1 and VEGF was monitored. ESW treatment increased total cell density and the proportion of RP59 positive cells in the defect region. High numbers of round- and cuboidal-shaped cells strongly expressing RP59 were initially found. Later, the predominant cell type was spindle-shaped fibroblastic cells, subsequently, aggregates of osteogenic and chondrogenic cells were observed. Histological observation suggested that bone marrow stem cells were progressively differentiated into osteoblasts and chondrocytes. RP59 staining was initially intense and decreased with the appearance of expression depended on the differentiation states of osteogenic and chondrogenic cells during the regeneration phase. Mature chondrocytes and osteoblasts exhibited only slight RP59 immuno-reactivity. Expression of TGF-beta 1 and VEGF-A mRNA in the defect tissues was also significantly increased (P<0.05) after ESW treatment as determined by RT-PCR. Intensive TGF-beta 1 immuno-reactivity was induced immediately, whereas a lag period was observed for VEGF-A. Chondrocytes and osteoblasts at the junction of ossified cartilage clearly exhibited VEGF-A expression. Our findings suggest that recruitment of meseoblasts at the junction of ossified cartilage clearly exhibited mesenchymal stem cells is a critical step in bone reparation that is enhanced by ESW treatment. TGF-beta 1 and VEGF-A are proposed to play a chemotactic and mitogenic role in recruitment and differentiation of mesenchymal stem cells.

Skeletal morphology depends on the local regulation of bone formation, both in a quantitative and qualitative sense. The formative cells, osteoblasts, adjust their synthetic activity in response to signals that influence cell differentiation and matrix production. Here, we review data concerning the morphological patterning during bone ontogenesis and its direct cause: osteoblasts at specific anatomic sites. An overview of the possible origins of osteogenic cells is presented, considering bone growth and homeostasis, and discussing the repair process. A testable model is proposed, in which functional differences between osteoblast populations are explained by homeobox-gene regulation. Newly developed markers for osteoblast recruitment and differentiation provide an experimental system to test the impact of homeobox-gene expression on osteoblast differentiation and bone matrix production.

The interface of bone and aragonite nacre (Margaritifera, fresh water pearl mussel) was studied by in situ hybridization and a tartrate-resistant acid phosphatase (TRAP) histochemical assay. Columnar implants were inserted into rat femora for 4, 7, 14, 28 and 56 days. In medullary region, a burst of transient bone formation was observed, which propagated from the periphery towards the nacre implant. A fused interface of bone and nacre was observed at 14 days. Later, the new medullary bone was resorbed and bone marrow was re-established while a thin layer of bone tissue remained covering the implant surface. Expressions of collagen alpha1(I), osteocalcin, osteopontin mRNAs and TRAP in the surrounding tissue were monitored. Correlated with the histology events, a strong transient induction of collagen alpha1(I) and osteocalcin mRNAs as well as TRAP expression, exhibiting a peak signal intensity on day 7 and subsequent down-regulation after day 14 was observed. Osteopontin mRNA, in contrast, was expressed continuously. The degrading nacre surface appeared in direct contact with macrophages and multinucleated giant cells at both days 14 and 28. These cells expressed osteopontin mRNA intensively and some TRAP enzyme activity occasionally.

We recently described a novel protein in bone marrow of rats, RP59, as a marker for cells with the capacity to differentiate into osteoblasts. In this work, its expression pattern was further investigated to learn about the origin and biological relevance of RP59 expressing marrow cells. As revealed by in situ hybridization and by immunohistochemistry of yolk sac embryos, RP59 was found in the cells of the primitive ectoderm and primitive streak as well as in blood islands and extraembryonal mesoderm. Later, RP59 occurred in fetal liver cells and in circulating blood. From the time around birth, it was found in bone marrow and spleen cells. In addition, in vitro-formed blood vessels contained RP59-positive cells in the lumen. Endothelial cells and the vast majority of cells outside the blood vessels were not labeled. Concerning more mature hematopoietic cell types, RP59 was observed in megakaryocytes and nucleated erythroblasts, but absent from lymphoid cells. In conclusion, RP59 was induced in early mesoderm. It was maintained in the erythroid and megakaryotic lineages and, as earlier described, in young osteoblasts., (Copyright 2002 Wiley-Liss, Inc.)

To study osteoblast recruitment from bone marrow cells, a rat femur cDNA library was screened by in situ hybridization for novel mRNA sequences that are frequently expressed in both marrow cells and osteoblasts. One isolated clone, called RP59, is described here. Northern blots indicated two bands of 2.6 and 2.8 kb in femur and spleen, tissues containing high amounts of immature mesenchymal cells, and no or little expression in other tissues. The cDNA sequence revealed a reading frame for a repetitive protein composed of arrays of 14-mers and phased phosphorylation sites. Antisera versus RP59 detected a single band of 90 kDa by Western blotting of femur extract. Immunohistochemistry indicated strong RP59 presence in the cytoplasm of bone marrow cells and weaker presence in nuclei of osteoblasts. Intermediate stages were found between strongly labeled, round, free bone marrow cells and weaker labeled, fibroblast-like young osteoblasts associated with bone matrix. These data indicated that marrow cells with high RP59 content were recruited into growing bone tissue. RP59 may help to study the transition of bone marrow cell to osteoblast in more detail., (Copyright 2001 Academic Press.)

The pacemaker of endochondral bone growth is cell division and hypertrophy of chondrocytes. The developmental stages of chondrocytes, characterized by the expression of collagen types II and X, are arranged in arrays across the growth zone. Mutations in collagen II and X genes as well as the absence of their gene products lead to different, altered patterns of chondrocyte stages which remain aligned across the growth plate (GP). Here we analyze GP of rats bearing the mutation toothless (tl) which, apart from bone defects, develop a progressive, severe chondrodystrophy during postnatal weeks 3 to 6. Mutant GP exhibited disorganized, non-aligned chondrocytes and mineralized metaphyseal bone but without cartilage mineralization or cartilaginous extensions into the metaphysis. Expression of mRNA coding for collagen types II (Col II) and X (Col X) was examined in the tibial GP by in situ hybridization. Mutant rats at 2 weeks exhibited Col II RNA expression and some hypertrophied chondrocytes (HC) but no Col X RNA was detected. By 3rd week, HC had largely disappeared from the central part of the mutant GP and Col II RNA expression was present but weak and in 2 separate bands. Peripherally the GP contained HC but without Col X RNA expression. This abnormal pattern was exacerbated by the fourth week. Bone mineralized but cartilage in the GP did not. These data suggest that the tl mutation involves a regulatory function for chondrocyte maturation, including Col X RNA synthesis and mineralization, and that the GP abnormalities are related to the Col X deficiency. The differences in patterning in the tl rat GP compared to direct Col X mutations may be explained by compensatory effects.

The effect of the addition of titanium ion (Ti) on osteoblast function and the mineralization of osteoid nodules in rat calvaria cultures was characterized. Concentrations of 10 ppm of Ti or more inhibited cell proliferation; 5 ppm or less either had no effect or stimulated proliferation. The number of nodules formed was not influenced by 5 ppm of Ti, but mineral deposition in nodules was suppressed, as revealed by von Kossa staining. Likewise, 5 ppm of Ti inhibited the incorporation of [(45)Ca] in cultures during nodule formation even if the Ti was withdrawn from the medium when mineralization was initiated. In order to test whether the synthesis of osteoid components was affected, the expression of osteonectin (OSN), osteopontin (OPN), osteocalcin (OSC), and alkaline phosphatase (ALP) mRNAs as well as ALP enzyme activity was analyzed. The expression of OSN and OPN mRNAs was reduced dramatically, but OSC mRNA was little affected by 5 ppm of Ti. Ti delayed the development of ALP mRNA expression and enzyme activity relative to the controls. Thus Ti treatment changed the proportional composition of cellular mRNA contributing the osteoblast phenotype., (Copyright 1999 John Wiley & Sons, Inc.)

The craniofacial skeleton develops from a base in which coordinated growth at sutures and growth centres assures the development of normal form. In this report we describe features of retarded postnatal craniofacial development in the osteopetrotic mutation, toothless (tl), in the rat in which bone growth in both the nasal area and the cranial base is reduced, suggesting that the mutation affects bone formation in sutures and growth plates. We began a systematic search for potential mechanisms by analysing the expression in time and intensity of RNA coding for collagens type I (Col I) and type III (Col III) analysed by in situ hybridisation of cells in the premaxillary-maxillary suture (PMMS). In the centre of the PMMS of tl rats, cells expressing Col I and Col III appeared later than in normal littermates and exhibited lower signal. During osteoblast recruitment from the suture centre into the bone domains, Col III RNA expression is switched off. Osteoblasts expressing Col I in abundance, but no Col III, appeared in the flanking bone regions of tl rats later than in normal littermates. It is proposed that the tl mutation restricts the number of available osteoblast progenitor cells, and that the shortage of these cells affects bone growth in the PMMS and in the cranial base. Additional analyses are needed to test this hypothesis and to understand the developmental dynamics in the cranial base.

The toothless (osteopetrotic) mutation in the rat is characterized by retarded development of the anterior facial skeleton. Growth of the anterior face in rats occurs at the premaxillary-maxillary suture (PMMS). To identify potential mechanisms for stunted facial growth in this mutation we compared the temporospatial expression of collagen I (Col I) and collagen III (Col III) RNA around this suture in toothless (tl) rats and normal littermates by in situ hybridization of specific riboprobes in sagittal sections of the head. In normal rats, the suture is S shaped at birth and becomes highly convoluted by 10 days with cells in the center (fibroblasts and osteoblast progenitors) expressing Col III RNA and those at the periphery (osteoblasts) expressing no Col III RNA but high amounts of Col I RNA throughout the growth phase (the first 2 postnatal weeks). In the mutant PMMS, cells were reduced in number, less differentiated, and fewer osteoblasts were encountered. Expression of Col I RNA was at normal levels, but centrosutural cells expressed Col III RNA only after day 6 and then only weakly. A highly convoluted sutural shape was never achieved in mutants during the first 2 postnatal weeks. Treatment of tl rats with the cytokine CSF-1 improved facial growth and restored cellular diversity and Col III RNA expression in the PMMS to normal levels. Taken together, these data suggest that normal facial growth in rats is related to expression of Col III RNAby osteoblast precursors in the PMMS, that these cells are deficient in the tl mutation and are rescued following treatment with CSF-1.

Collagen alpha1(I) mRNA is composed of two variants of 5 and 6 kb, differing in the length of the 3' untranslated region. In this work, the nucleotide sequences of the two rat mRNA variants were compared, and their expression pattern in cells forming bone, dentin, and cementum were analyzed. The sequences were determined from cDNA inserts of tooth and bone libraries plus directly from PCR fragments, obtained from bone. A total of 5721 bases of the rat collagen alpha1(I) sequence from cDNA of tooth and bone was determined. All sequences of the short variant were represented in the long variant. Only the alternatively poly-A additions gave rise to the variants in hard tissue. Two oligonucleotides were chosen as probes, one of which recognized, on Northern blots, the two bands of 5 and 6 kb, and the other the 6-kb variant only. The oligonucleotides were used in in situ hybridization experiments, for study of the distribution of the variants in different extracellular matrix-forming cells. Osteoblasts, odontoblasts, and cementum-associated cells were closely examined in sections from rat maxillae from 2 to 25 days of age. A similar or identical pattern of mRNA expression was observed with both oligonucleotides, indicating that the two mRNA variants were co-expressed in all cases.

Bone formation of the maxilla and premaxilla of rats was studied by in situ hybridization, using probes for fibrillar collagen mRNAs. Chondroblasts, osteoblasts, fibroblasts and peripheral bone cells differed in their expression patterns. Prospective nasal chondroblasts expressed collagen alpha1(II) and alpha1(XI) RNA from day 15 post coitum. Bone formation in the adjacent maxilla and premaxilla started around day 17: groups of osteoblasts, representing ossification centers, expressed collagen alpha1(I) RNA strongly, and alpha1(V), alpha2(V) and alpha1(XI) RNA weakly, but they were deficient in collagen alpha1(III) RNA. As the centers expanded, osteoblasts in the resulting bone domains expressed collagen alpha1(I) RNA in abundance, whereas collagen alpha1(III) RNA was absent. Bone domains were surrounded by fibroblasts containing collagens alpha1(I), alpha1(III) and alpha2(V) RNA. Widely separated fibroblasts underwent condensation into densely packed periosteum and sutural soft tissues. Cells at the periphery of fast-growing bone domains also displayed, apart from collagen alpha1(I) RNA, collagens alpha2(V) and alpha1(XI) RNA. Given the continuous recruitment of cells from the periosteum, peripheral bone cells represent differentiating osteoblasts synthetizing collagens alpha2(V) and alpha1 (XI) RNA transiently. Thus, gene expression during osteoblast differentiation reflects synthesis of fiber components during bone growth, since collagen V is located in the center of fibers consisting primarily of collagen I.

The sequence of rat osteonectin mRNA was determined. Comparison with osteonectin (ON) mRNA sequences of other vertebrates showed a great similarity, but a stretch of codons deviated with respect to another rat strain, suggesting the possibility of two ON variants in rats. Northern blots exhibited one band of ON mRNA only. The expression of ON and collagen alpha1(I) RNA in tissues of the developing rat maxilla was studied by in situ hybridization. Both ON and collagen alpha1(I) RNA were observed concomitantly in osteoblasts, starting from the onset of bone formation at day 17 post coitum through the oldest age examined, day 20 after birth. A strict co-expression of the two sequences was also observed in odontoblasts as well as in fibroblasts of the periodontal ligament. In ameloblasts, neither ON nor collagen alpha1(I) RNA was detected under stringent hybridization conditions, but lower stringency led to an ON signal. Considering that ON is a secretory protein and the high stability of the ON mRNA, the co-expression of collagen alpha1(I) and ON RNA sequences in matrix-forming cells provided evidence that ON is a substantial component of collagen matrices.

In the active Balbiani ring (BR) genes of the dipteran Chironomus tentans, the assembly of a specific pre-mRNP particle can be analyzed in situ, and the incorporation of hnRNP proteins into the nascent pre-mRNP can be directly visualized by immunoelectron microscopy. In the present study we have shown that hrp36, one of the major hnRNP proteins in Chironomus tentans, is continuously added to the nascent BR pre-mRNP particle throughout transcription and is localized along the entire BR RNP fiber. Interestingly, hrp36 becomes concealed during the structural transition that occurs during the formation of the mature BR RNP particle. This conclusion is based on the observation that hrp36 can be revealed by a monoclonal antibody during the initial assembly of the BR RNP fiber but becomes almost undetectable in the final packaging stage. The hrp36 protein, however, is not removed from the BR RNP particle since the ability of the monoclonal antibody to reveal hrp36 is restored by artificial relaxation of mature BR RNP particles. Another major hnRNP protein, hrp45, is also incorporated in a continuous manner into the nascent pre-mRNP fiber but remains accessible in mature BR RNP particles. Our results provide immunocytochemical evidence for drastic structural changes occurring in the final stage of BR pre-mRNP packaging, and suggest that different hnRNP proteins might be differently involved in the pre-mRNP assembly process.

Two variants of an mRNA sequence are identified that are expressed at high levels in rat ameloblasts during the formation of the enamel matrix. The sequences contain open reading frames for 407 and 324 amino acid residues, respectively. The encoded proteins, which we call amelins, are rich in proline, glycine, leucine, and alanine residues and contain the peptide domain DGEA, an integrin recognition sequence. The sequences coding for the C-terminal 305 amino acid residues, the 3' nontranslated part, and a microsatellite repeat at the nontranslated 5' region are identical in both mRNA variants. The remaining 5' regions contain 338 nucleotides unique to the long variant, 54 common nucleotides, and 46 nucleotides present only in the short variant. Eleven nucleotides have the potential to code for 5 amino acids of both proteins in different reading frames. The reading frame of the longer variant includes codons for a typical N-terminal signal peptide. The amelins are likely to be constituents of the enamel matrix and the only proteins that have so far been implicated in binding interactions between the ameloblast surface and its extracellular matrix.

In the larval salivary glands of C. tentans, it is possible to visualize by electron microscopy how Balbiani ring (BR) pre-mRNA associates with proteins to form pre-mRNP particles, how these particles move to and through the nuclear pore, and how the BR RNA is engaged in the formation of giant polysomes in the cytoplasm. Here, we study C. tentans hrp36, an abundant protein in the BR particles, and establish that it is similar to the mammalian hnRNP A1. By immuno-electron microscopy it is demonstrated that hrp36 is added to BR RNA concomitant with transcription, remains in nucleoplasmic BR particles, and is translocated through the nuclear pore still associated with BR RNA. It appears in the giant BR RNA-containing polysomes, where it remains as an abundant protein in spite of ongoing translation.

The expression of amelogenin mRNA in growing rat molars was studied. Northern blotting and the analysis of cDNA isolates revealed two predoninant variants. One group of cDNA inserts contained sequences of a long mRNA version and the other group contained mRNA sequences of the shorter leucin-rich amelogenin polypeptide (LRAP). The LRAP group was deficient in an internal stretch which coded for a peptide with a high potential for beta turns. Northern blot experiments showed that most amelogenin RNA in rat teeth was represented by two bands of 1.1 and 0.8 kb. Two oligonucleotide probes were designed that were specific for the long version and for the LRAP variant. The probes were used for in situ hybridization experiments on sections of developing maxillar teeth of rats between day 2 and day 15 after birth. Both RNA species were accumulated concomitantly and exclusively in cells of the inner enamel epithelium. Expression was first observed at the mesial cusp sides and finally involved the whole ameloblast layer except for the cells adjacent to the enamel-free region at the tip of the cusps. The early amelogenin RNA expression occurred adjacent to the initial deposition of the dentin matrix. Low amounts of amelogenin RNA persisted after the differentiation of ameloblasts into the maturative stage. The sequence of events was similar in all three molars.

In the developing tooth, the bisphosphonate HEBP causes hypoplasias and hypomineralization of the enamel and dentine as well as inhibition of acellular cementum formation. Here, we describe a novel effect, associated with dentine mineralization. HEBP was administrated to young rats, and the maxillary molars were analyzed histologically. Localized dentinal nodules, protruding towards the pulp, were found in the developing crown of the molars. They occurred in regions, where the mantle dentine was about to mineralize at the time of the injection, and were more frequent at the mesial cusp side. The nodules accumulated mineral, as evidenced by the fluorescence after calcein and tetracyclin labelling. Histologically, the nodules were separated from the enamel by a layer of mantle dentine and were progressively surrounded by predentine and dentine. The nodules were interpreted to contain transport or metabolism intermediates, which were locally accumulated due to the interruption of the mineralization process by HEBP.

Activation of the protein kinase C (PKC)-related signal transduction system has been associated with phenotypic expression in a wide variety of cell types. In in vitro studies, it has often been activated by relatively small increases in the Ca2+ concentration ([Ca2+]) in the medium. The studies reported here explored the hypothesis that localized increases in the extracellular [Ca2+] and activation of the PKC-related pathway may be involved in early dentin and enamel formation. Whole-head, freeze-dried sections through the developing molars of 5-day-old rats were evaluated by methods that localized non-crystalline Ca2+. Immunohistochemical methods were adapted for use with the freeze-dried sections, and two monoclonal antibodies were used to localize PKC alpha in the formative cells of the developing teeth. Low concentrations of extracellular Ca2+ were observed in the early, unmineralized dentin in the area of ameloblast differentiation. Increased concentrations occurred at the point of initial dentin mineralization, immediately before the beginning of enamel matrix deposition. PKC alpha was localized in the differentiating odontoblasts, at the beginning of dentin matrix deposition. It was intensely localized in the distal borders of the pre-ameloblasts, and appeared to redistribute in the cells during ameloblast differentiation. These observations suggest that local increases in the extracellular [Ca2+] and the PKC signal transduction pathway may be involved in key inductions in the early stages of dentin and enamel formation.

Balbiani ring premessenger ribonucleoprotein (RNP) particles are built from a 7-nm RNP fiber which is tightly folded into a ring-shaped RNP ribbon. Isolated particles are known to disintegrate in all-or-none fashion upon RNase A treatment. In the present study we investigated whether this mode of disintegration is dependent on an intact particle structure or is inherent in the 7-nm fiber. When treated at low ionic strength, the Balbiani ring (BR) particles lost their higher order structure and the 7-nm fiber was unpacked, as evidenced by sucrose gradient sedimentation and electron microscopy. When treated with RNase A, unfolded as well as intact particles disintegrated in the all-or-none fashion, with similar kinetics and without apparent intermediates. Proteinase K treatment, however, obliterated this pattern: the protein-free particle RNA degraded progressively. As the typical disintegration pattern of the particles was not altered by unfolding, but was lost by deproteinization, the all-or-none mode of disintegration is likely to be a property of the 7-nm RNP fiber.