Biomedical subjects

J V Ruch

Publications and source records attributed to J V Ruch.

At least 55 records · Page 3Linked to original sources

PubMed1995

Cultured incisors display major modifications in basal lamina deposition without further effect on odontoblast differentiation.

Matrix-mediated epithelio-mesenchymal interactions play a crucial role in the control of dental cytodifferentiations. Ultrastructural observation of the epithelio-mesenchymal junction in cultured embryonic mouse molars showed discrete zones with duplicated or multilayered basal laminae. The use of synthetic peptides demonstrated that the process was RGD*-independent, did not involve the YIGSR* sequence present on laminin and could occur spontaneously. Cultured incisors showed a similar but much more dramatic multiplication of the basal laminae. Furthermore, the deposition of multilayered basal laminae was specific for the labial aspect of the tooth and could be detected after 6 h of culture. Despite these alterations, preodontoblasts differentiated and gradients of differentiation were maintained, suggesting that among basement membrane constituents, the basal lamina itself does not play a critical role. More important is the inner dental epithelium which may still control odontoblast differentiation by means of diffusible molecules able to reach surface receptors expressed by preodontoblasts or matrix receptors underlying the basal lamina. Gradients of odontoblast differentiation could result from a progressive acquisition of competence by preodontoblasts.

Amino Acid Sequence↗

PubMed1995

Comparison of the effects of growth hormone, insulin-like growth factor-I and fetal calf serum on mouse molar odontogenesis in vitro.

The effects of growth hormone, its mediator insulin-like growth factor-I (IGF-I), and fetal calf serum on odontogenesis were compared to those of serum-free medium. Explanted, 16-day, fetal mouse first molar tooth germs in early bell stage were grown on semisolid, serum-free medium supplemented with ascorbic and retinoic acids. Recombinant human growth hormone at 50 or 100 ng/ml, IGF-I at 100 or 200 ng/ml, or fatal calf serum at 20% concentration were added to the media. Volumetric changes in serial sections of six tooth germs per treatment over 3 days of treatment (4, 5, 6 days in vitro) were compared by digitized morphometry. Mitotic indices were also compared and the cell densities of the dental papillae recorded. Qualitative ratings of differentiation were ascribed to each tooth germ by light microscopy. Differences in volume, mitotic activity and cell densities were found. The growth hormone-treated tooth germs were not larger than the serum-free ones but had increased mitotic indices and higher cell densities in the dental papillae. IGF-I-treated tooth germs had larger volumes than with all other treatments, e.g. germs treated with 200 ng/ml of IGF-I, after 6 days in culture, were significantly larger than with all other treatments (p<0.01-<0.001). Whilst IGF-I-treated germs displayed the greatest extent of differentiation, growth hormone-treated germs also showed advanced differentiation compared to those on serum-free medium. These results suggest that growth hormone and IGF-I are involved in odontogenesis of murine teeth in vitro by affecting mitotic activity, tissue volume and cell differentiation. In conjunction with previous immunohistochemical studies that show expression of growth hormone receptor and IGF-I in developing teeth, these results provide evidence that both growth hormones and its mediator play a part in odontogenesis.

Animals↗

PubMed1995

Induction of odontoblast-like cell differentiation in dog dental pulps after in vivo implantation of dentine matrix components.

The effects of dentine extracellular matrix components on dental mesenchymal cells were studied by light and transmission electron microscopy after their implantation at central sites of mechanically exposed pulps in dog molar teeth. The implants were Millipore filters that had been soaked with solutions containing 30 or 300 micrograms/ml of an EDTA-soluble fraction of rabbit incisor dentine. Control filters were soaked with dog albumin or phosphate buffered saline. Columnar, polarized cells were consistently seen after 8 days in close proximity to the filters coated with both concentrations of dentine matrix components. Characteristic features of these polarized cells included widened cisternae of the rough endoplasmic reticulum, a rich microfilamentous network in the long cytoplasmic extensions invading the filter pores and numerous cytoplasmic bodies. These cells also showed evidence of functional as well as cytological differentiation. Polarized processing of secretory granules could be observed after 8 days' implantation, and also the presence of matrix vesicles and deposition of a fine, collagenous matrix into the filters apically to the distal end of the cytoplasmic processes. After 24 days' implantation, secretion of a tubular matrix could be consistently seen in association with the odontoblast-like cells. No changes in cell organization or matrix synthesis were seen after implantation of control filters. These studies demonstrate that bioactive components present in the EDTA-soluble dentine matrix fraction are able to directly induce cell polarization and apical secretion of tubular matrix when implanted in contact with dental pulp cells at sites remote from the odontoblast layer.

Animals↗

PubMed1995

Tooth crown morphogenesis and cytodifferentiations: candid questions and critical comments.

Teeth are probably meristic units and crown morphogenesis leads to tooth specific distribution of functional cells. Since heterodonty is derived from homodonty, one way to understand tooth morphogenesis would be to unravel the involved phenomena in homodont species and then to characterize the "put up job" of evolution leading to species specific dentitions with particular functional abilities. Interaction of paleontologists and developmental biologists should be initiated. My naive "developmental" point of view will illustrate only one facet of mouse tooth morphogenesis and cytodifferentiation. The main concern will be to try to discriminate between known facts and speculations, between hypotheses and anticipated or deduced certitudes, to call attention to conflicting data, to suggest some further investigations and to advocate the point of view that molecular interpretations should be founded on indisputable morphological data.

Animals↗

PubMed1995

Influence of substrate nature and immobilization of implanted dentin matrix components during induction of reparative dentinogenesis.

The biological effects of isolated soluble dentin extracellular matrix components on the induction of reparative dentinogenesis in exposed cavities in ferret canine teeth have been shown to be blocked by immobilizing the extracellular matrix components on nitrocellulose or Millipore membranes during implantation. This contrasts with the picture of induction of odontoblast-like cell differentiation and reparative dentin deposition on existing insoluble dentin matrix of the exposure walls when the extracellular matrix components are implanted in lyophilized form. These data indicate the importance of an existing insoluble dentin matrix in providing a substrate to potentiate the growth factor-like activity of soluble isolated dentin extracellular matrix components in the induction of reparative dentinogenesis.

Animals↗

PubMed1995

Comparison of expression of the msx-1, msx-2, BMP-2 and BMP-4 genes in the mouse upper diastemal and molar tooth primordia.

The existence of transient putative tooth anlagen in the prospective mouse upper diastema region has been documented previously in morphological studies. By in situ hybridization we investigated the expression patterns of the msx-1, msx-2, BMP-2 and BMP-4 genes, supposed to regulate early tooth development, in day 10-14 mouse embryonic upper diastema and molar regions, using 49 series of frontal sections. On the basis of comparison of the temporo-spatial expression patterns in both diastemal and molar tooth primordia we conclude that each of the four genes was expressed at least for some period simultaneously and at a comparable developmental stage in the transient and persisting dental primordia. BMP-2 and BMP-4 expression was downregulated in the diastemal dental primordia during their regression starting at day 13. The temporo-spatial pattern of BMPs expression may be associated with the disappearance of diastemal rudiments. Contrary to the molar anlage, we did not detect msx-2 gene expression in the diastemal dental rudiments after the stage of epithelial thickening. The deficiency of the msx-2 gene products may play a role in the growth retardation of diastemal dental primordia resulting in their subsequent involution.

Animals↗

PubMed1995

Contribution of 3-D computer-assisted reconstructions to the study of the initial steps of mouse odontogenesis.

The specific arrangement of mouse dentition in each dental quadrant (1-0-0-3) is supposed to result from the initiation of two independent dental laminae--one for the incisor and one for the three molars. In order to verify whether the adult mouse dental pattern really corresponds to the initial patterning, an analysis of development of the mouse antemolar part of the upper dental quadrant was performed in 10-13 day embryos using histological sections and computer-assisted 3-D reconstructions. Six primary dental laminae contributed to the formation of the upper incisor anlage, which is, therefore, a structure of multiple origin. In contrast to the lower diastema, where only a low epithelial band extended mesially from the first lower molar in 12-13 day embryos, in the upper diastema a dental lamina existed interconnecting transiently the incisor and molar anlagen and giving rise to 3 distinct epithelial rudiments. The rudiments exhibited growth retardation and regressed after reaching a maximum at the bud stage. Our results showed a discrepancy between the embryonic and adult dental patterns in the mouse upper jaw. The specific arrangement of the mouse dentition implied a reduction of the embryonic dental anlagen, which was achieved either by their integration into the one incisor primordium or regression in the prospective diastema. Odontogenesis in the mouse upper jaw provides a model of hypodontia of evolutionary origin, which can be employed in molecular studies of the control mechanisms of initiation, spatial organization and specific morphogenesis of teeth.

Animals↗

PubMed1995

Reactionary dentinogenesis.

Reactionary dentinogenesis is the secretion of a tertiary dentine matrix by surviving odontoblast cells in response to an appropriate stimulus. Whilst this stimulus may be exogenous in nature, it may also be from endogenous tissue components released from the matrix during pathological processes. Implantation of isolated dentine extracellular matrix components in unexposed cavities of ferret teeth led to stimulation of underlying odontoblasts and a response of reactionary dentinogenesis. Affinity chromatography of the active components prior to implantation and assay for growth factors indicated that this material contained significant amounts of TGF-beta 1, a growth factor previously shown to influence odontoblast differentiation and secretory behavior. Reactionary dentinogenesis during dental caries probably results from solubilization of growth factors, TGF-beta in particular, from the dentine matrix which then are responsible for initiating the stimulatory effect on the odontoblasts. Compositional differences in tertiary dentine matrices beneath carious lesions in human teeth have also been shown indicating modulation of odontoblast secretion during reactionary and reparative dentinogenesis.

Animals↗

PubMed1995

Odontoblast differentiation.

Odontoblasts are post-mitotic, neural crest-derived, cells which overtly differentiate according to tooth specific temporo-spatial patterns and secrete predentin-dentin components. Neither the timing nor the molecular mechanisms of their specification are known and the problem of their patterning in the developing jaws is far from being solved. On the other hand, some significative strides were made concerning the control of their terminal differentiation. Fibronectin interacting with a 165 kDa, non integrin, membrane protein intervenes in the cytoskeletal reorganization involved in odontoblast polarization and their terminal differentiation can be triggered in vitro by immobilized members of the TGF beta family. Histological aspects and the transcriptional phenotypes (transcripts of TGF beta s, BMPs, msxs, IGF1, fibronectin, osteonectin, bone sialoprotein genes) are very similar in vivo and in vitro. In vivo members of the TGF beta super family secreted by preameloblasts, trapped and activated by basement membrane associated components, might initiate odontoblast terminal differentiation.

Animals↗

PubMed1995

Polyamine depletion-mediated effects on murine odontogenesis are dependent on tooth developmental stage and culture conditions.

Polyamines are known to play a central role in processes such as growth and development. Virtually nothing is known about their importance in tooth development, an attractive and frequently used experimental model for studies of developmental processes. A polyamine-depleted state was created in tooth cells in an organ culture system. First lower molar germs from 16-and 17.5-day old mouse fetuses were used. alpha-difluoromethylornithine (DFMO) and methylglyoxal bis-(guanylhydrazone) (MGBG) were used to deplete the cells from their polyamine content. Polyamine interconversion and catabolism were prevented by aminoguanidine sulfate (AG). In day-16 germs cultured in serum-containing medium, DFMO reduced the frequency of cycling cells as shown by [3H]thymidine incorporation, and induced a delay of odontoblast differentiation of about 24 h. Under the same conditions, MGBG induced an arrest of histo-morphogenesis, correlated to a significant decrease in the rate of cell proliferation. Addition of polyamines prevented DFMO- and MGBG-induced delay of tooth differentiation. Interestingly, MGBG did not delay the terminal differentiation of odontoblasts and ameloblasts in cultured day-17.5 molars; in these, cells at the tip of the cusps are only a few hours before their withdrawal from the cell cycle. In serum-deprived medium, dental cytodifferentiations did not occur. Addition of putrescine or spermidine to serum-free media, however, allowed for tooth morphogenesis and cytodifferentiation. Tooth explants in a serum-deprived medium reacted to DFMO in a cytocidal fashion, whereas MGBG showed only a mild toxicity in some cell types. Addition of putrescine to DFMO-containing medium prevented its cytotoxic effect. Addition of spermidine to MGBG-containing medium not only prevented its mild toxicity but also allowed for predentin secretion by differentiated odontoblasts. The results are discussed with regard to the well-established developmental events of tooth germs cultured in vitro and with respect to present knowledge of polyamine metabolism and their involvement in cellular processes.

Animals↗

PubMed1994

In vitro effects of retinoic acid on mouse incisor development.

The developing dentition is known to express the complete set of retinoic acid (RA) nuclear receptors and cytoplasmic RA-binding proteins (CRABPI and II), and RA is required for in vitro mouse molar morphogenesis, so the role of RA during in vitro mouse incisor development was investigated. Histological procedures, immunocytochemical detection of proliferating cells, immunofluorescence detection of laminin, and in situ hybridization with RNA probes for CRABPI and II were done on the tooth-germ cultures either in the presence or in the absence of RA. RA appeared to control initial morphogenesis, particularly the asymmetrical growth of the cervical loop, and to regulate required differential mitotic activity. RA seemed also to be involved in asymmetrical laminin deposition. The distribution of the CRABP gene transcripts was similar during in vivo and in vitro incisor development. However, CRABPI gene transcript distribution in the labial part of the epithelial loop was detected in vitro only in the presence of RA. A direct role of the CRABPs during tooth development is, however, unlikely because Ch55, a synthetic RA analogue that does not bind to CRABP, had the same effects as RA on in vitro incisor development.

Animals↗

PubMed1994

Odontoblast stimulation in ferrets by dentine matrix components.

The possible effects of isolated dentine matrix components on odontoblast secretory activity were investigated in vivo by implantation of lyophilized fractions of these components into cavities prepared in ferret canine teeth. After implantations as short as 14 days there was significant deposition of reactionary dentine by the odontoblasts beneath the cavity and this response increased in a non-linear manner with time of implantation. In contrast, control cavities lacking the dentine matrix components showed no evidence of reactionary dentine deposition. Examination of teeth at early periods of implantation (2 and 5 days) indicated that odontoblast death had not occurred as a result of the operative procedures and that the response was one of stimulation of existing odontoblasts rather than that of induction of a new generation of odontoblast-like cells. The mechanisms of odontoblast stimulation by the dentine matrix components remain to be elucidated, but could be mediated by growth factors within the dentine matrix preparations.

Animals↗

PubMed1994

Immunocytochemical localization of choline-phospholipids in postnatal mouse molars.

This localization was studied using anti-choline phospholipid monoclonal IgM antibody (MC22-33F). By immunofluorescence, the apical portions of enamel-secreting ameloblasts and dentine-secreting odontoblasts were positively stained with MC22-33F. By immunoelectron microscopy, lysosomes in preodontoblasts, odontoblasts, preameloblasts and ameloblasts as well as matrix vesicles in dentine were strongly stained with MC22-33F. On the other hand, plasma membrane of these cells did not stain with MC22-33F. The frequency of positively reacting lysosomes in ameloblasts and odontoblasts and of matrix vesicles increased with cell maturation and mineral deposition.

Ameloblasts↗

PubMed1994

Effects of suramin, a polyanionic drug inducing lysosomal storage disorders on tooth germs in vitro.

Suramin, a potent inhibitor of lysosomal enzymes, is commonly employed as a tool for inducing experimental mucopolysaccharidosis and lipidosis. The effects of the drug on embryonic mouse molars were analysed. Presecretory ameloblasts and odontoblasts were loaded with lysosome-like vacuoles. Staining with MC22-33F, an antibody to choline phospholipids and sphingomyelin, was completely reversed in the suramin-treated germs, in that it stained only presecretory ameloblasts (versus odontoblasts and some pulpal cells in the control group), according to a developmentally regulated pattern. The suramin-induced cytoplasmic changes were reminiscent of the features of mucopolysaccharidoses and lipidoses. The basement membrane, separating the enamel organ from the dental papilla, displayed suramin-induced patches, and in predentin collagen fibrillogenesis was found to be disturbed. Furthermore, autoradiography was employed to reveal uptake and distribution of [3H] suramin in the cells and predentin. Finally, a suramin-induced disturbance of the metabolism of sulphated macromolecules was found. The results imply that suramin effects in vitro on tooth germs can be used as a useful experimental model with to study both the action of the drug as well as cell and extracellular matrix perturbations in a mucopolysaccharidosis-like condition.

Animals↗

PubMed1994

Expression of nuclear retinoic acid receptors during mouse odontogenesis.

The developmental expression of retinoic acid (RA) nuclear receptors RAR(alpha, beta, gamma) and RXR(alpha, beta, gamma) was analysed during mouse odontogenesis by in situ hybridization on frozen sections and compared with the expression patterns of the cellular retinoic acid binding proteins CRABPI and II. The transcripts distribution of each RAR and RXR was basically similar in developing molars and incisors. RAR alpha and RXR alpha were preferentially expressed in dental epithelia, whereas RAR gamma and RXR gamma were transcribed in the dental mesenchyme. RAR beta, RAR gamma and RXR beta displayed both epithelial and mesenchymal expression. RAR beta expression was initiated during bell stage. RXR gamma transcripts were observed only at day 19.5 post coitum in the mitogenic mesenchyme facing the epithelial loops. Odontoblasts expressed RAR beta and RAR gamma, RXR alpha and RXR beta. Preameloblasts expressed RXR alpha and RXR beta and ameloblasts RXR gamma, RXR alpha and RXR beta. RAR alpha transcription in the incisor preameloblasts and ameloblasts was not observed in the first molar. The coexpression between RARs and RXRs might be important to form RAR/RXR heterodimers which are necessary to activate the transcriptions of target genes. CRABPI and CRABPII demonstrated graded variation of expression during odontogenesis in the mesenchyme and in the inner dental epithelium respectively. The pattern of CRABPI transcripts overlapped at least partially with expressions of all the studied nuclear receptors whereas CRABPII epithelial expression was superimposed with the transcription of RAR alpha, RXR alpha and RXR beta. These cytoplasmic proteins might participate in the storage and/or metabolism of RA and then distribute RA to colocalized nuclear receptors.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1994

Comparative analysis of TGF beta s, BMPs, IGF1, msxs, fibronectin, osteonectin and bone sialoprotein gene expression during normal and in vitro-induced odontoblast differentiation.

Immobilized TGF beta 1 and BMP2 are able to promote the differentiation of odontoblast-like cells in isolated mouse dental papillae cultured in vitro. These cells polarize and accumulate predentin-like matrix at their apical pole. Immobilized IGF1 mainly promoted polarization with disturbed matrix accumulation. In situ hybridization demonstrated that TGF beta 1 combined with heparin mirrored the physiological processes of odontoblast differentiation. Normal odontoblast and in vitro induced odontoblast-like cells expressed transcripts encoding for TGF beta 1 and 3, BMP2 and 4, bone sialoprotein and osteonectin whereas either ubiquitous expression or no expression could be detected for TGF beta 2, IGF1 or fibronectin mRNAs. Odontoblast-like cells obtained in the presence of IGF-1 combined with heparin did not express TGF beta 1 transcripts and expressed weakly TGF beta 3 transcripts. Our results suggest that in vivo an epithelial-derived member of the TGF beta family trapped by basement membrane-associated components interacts with competent preodontoblasts and promotes the polarization by triggering the transcription of growth factor gene(s) like TGF beta itself and/or selector gene(s) like msx2.

Animals↗

PubMed1993

Absence of interaction between the 165-kDa fibronectin-binding protein involved in mouse odontoblast differentiation and vinculin.

Previous data suggested that matrix could control the organization of microfilaments in differentiating odontoblasts and that this process involved a complex of fibronectin-165-kDa membrane protein-vinculin. The use of two different gel systems and microsequence analysis demonstrated that two distinct 165-kDa proteins interact, one with fibronectin and the other with vinculin.

Actin Cytoskeleton↗

PubMed1993

Interaction of vinculin with the clathrin heavy chain.

To further document the interaction of vinculin with the clathrin heavy chain (CHC) which was observed by using gel overlay, co-sedimentation experiments were performed and attempts were made to localize the domains involved on both molecules. The binding properties of proteolytic fragments of vinculin were investigated after cleavage with V8 protease. Neither the isolated globular domain, nor the C-terminal rod domain were able to interact with the CHC. Either the interaction involved the portion of vinculin which links these two domains, or the region of vinculin mediating the interaction was present on one of the two major fragments, but the cleavage itself resulted in conformational changes which abolished the binding. The first hypothesis could be ruled out using alpha-chymotrypsin generated fragments of vinculin, suggesting that the native conformation of vinculin might play an important role. Proteolytic cleavage of CHC with trypsin demonstrated that the interaction with vinculin is mediated by the proximal or distal segment of the CHC. Presence of clathrin light chain (CLC) associated with the CHC did not affect its interaction with vinculin. Vinculin did not interact with the CLC.

Animals↗