C.Z. Zhang

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.

Evidence for a local action of growth hormone in embryonic tooth development in the rat

Studies in non-dental embryonic tissues have suggested that an interaction between growth hormone and its receptor may play a role in growth and development before the foetal pituitary gland is competent. This study reports the distribution of growth hormone, its receptor and binding protein in developing rat tooth germs from embryonic day 17 to 21 and postnatal day 0 using antibodies specific for each of these proteins. Four foetal rats were processed at each time point (E17, E18, E20/21 and postnatal day 0). Following routine fixation and paraffin embedding, sections were treated with antisera to rat growth hormone, rat growth hormone binding protein and growth hormone receptor. Localization of antibody/antigen complexes was subsequently visualized by addition of biotinylated IgG and reaction with streptavidin peroxidase and diaminobenzidine. Assessment of the level of staining was qualitative and based on a subjective rankings ranging from equivocal to very strong staining. Overall, growth hormone and its binding protein were located both in the cellular elements and throughout the extracellular matrix, whereas the growth hormone receptor showed an exclusively intra-cellular location. All three proteins were detectable in cells of the dental epithelium and mesenchyme at the primordial bud stage (E17) which occurs prior to expression of pituitary growth hormone. At the cap stage of odontogenesis (E18-19), numerous cells in both the dental epithelium and mesenchyme were intensely immunoreactive for growth hormone, its binding protein and receptor. In the succeeding early bell stage (E20-21), most of the mesenchymal cells in the dental pulp were mildly positive for these proteins, while the dental epithelium and adjacent mesenchyme were more immunoreactive. At the late bell stage (postnatal day 0), all three proteins were localized in dental epithelium, differentiating mesenchymal cells the cuspal surface facing the epithelial-mesenchymal interface, preodontoblasts, and odontoblasts forming dentine. From these observations, immunoreactive growth hormone, its receptor and binding protein appear to be expressed in odontogenic cells undergoing histodifferentiation, morphodifferentiation and dentinogenesis in a cell-type and stage-specific pattern throughout embryonic tooth development. This suggests the possibility that growth hormone, or a growth hormone-like protein, plays a paracrine/autocrine role in tooth development in utero.

Immunocytochemical localization of growth hormone receptor in rat maxillary teeth.

To address the question of what role growth hormone may have in stimulating tooth formation, the distribution of its receptor/binding protein in developing rat incisors and molars was studied immunocytochemically using well-characterized monoclonal antibodies. Ten female 45-day-old Wistar rats were perfused with 4% paraformaldehyde. Five-microns paraffin sections of the growing end of maxillary incisors and molars were cut, deparaffinized and incubated with mouse anti-growth hormone receptor antibodies or control antibodies. A three-layer streptavidin peroxidase technique was used to detect bound antibody. Immunoreaction product was associated primarily with the cytoplasm of cells at certain stages of differentiation. Dividing cells, differentiating preameloblasts and preodontoblasts, secretory ameloblasts and odontoblasts showed immunoreactivity. Undifferentiated dental epithelium cells, stellate reticulum, external dental epithelial cells, mature odontoblasts, and most of cells in the dental papilla were non-reactive. However, at certain stages of tooth development, the stratum intermedium and the external dental epithelium also stained positively. The presence of growth hormone receptor/binding protein in tooth cells at different stages of their development indicates that growth hormone may influence cell proliferation, differentiation and differentiated functions of ameloblasts, odontoblasts and cementoblasts independent of a systemic mediator, and thus may be involved in stimulating odontogenesis directly.

Effect of Growth Hormone on the Distribution of Decorin and Biglycan during Odontogenesis in the Rat Incisor

Previous studies have shown that growth hormone can influence the expression of N-acetylgalactosamine-containing molecules in the extracellular matrix of developing rat incisors. N-acetylgalactosamine is a principal component of proteoglycans containing chondroitin sulfate and dermatan sulfate, as well as of some glycoproteins. Since chondroitin sulfate proteoglycans are identifiable components in enamel, dentin, and cementum, we have tested the hypothesis that growth hormone modulates their expression in developing rat incisors. The distribution of the chondroitin-sulfate-rich proteoglycans, decorin and biglycan, was investigated. We used the Lewis dwarf rat as a model because their circulating growth hormone levels are markedly reduced. Polyclonal antibodies against decorin and biglycan were used to localize these two proteoglycans. Semi-quantitative assessments of the staining patterns and intensities were made for each proteoglycan within compartments of the developing teeth. In normal Lewis rats, decorin and biglycan differentially expressed throughout the enamel organ, dental papilla, and dental follicle. Decorin displayed a wide distribution throughout all three regions and was closely associated with different cellular components. In contrast, biglycan showed little association with cells and was identified in the predentin and osteoid matrices. The expression of both proteoglycans was dramatically decreased in the growth-hormone-deficient animals. Administration of growth hormone to the dwarf rats markedly elevated the expression of both proteoglycans, approximating the distribution and intensity of staining seen in normal animals. These findings confirm that growth hormone status can modulate the expression of decorin and biglycan, and hence matrix deposition, in the rat tooth.

The Influence of Growth Hormone on Cell Proliferation in Odontogenic Epithelia by Bromodeoxyuridine Immunocytochemistry and Morphometry in the Lewis Dwarf Rat

For investigation of how growth hormone affects tooth development, bromodeoxyuridine immunocytochemistry and morphometry were used for the study of cell proliferation in odontogenic epithelial cell layers. The number of cells in the S phase, as revealed by this technique, and in mitosis, were counted in Bouins-perfused and paraffin-embedded undecalcified maxillary incisor enamel organs of normal rats, in growth-hormone-deficient dwarf rats, and in dwarf rats treated with growth hormone (66 μg/100 g body wt) twice daily for six days. Significantly fewer labeled nuclei, unlabeled nuclei, and total nuclei of various odontogenic epithelia were found in dwarf rats, but in dwarf rats treated with growth hormone, numbers of labeled nuclei equivalent to normal were found in the internal enamel epithelium, stratum intermedium, and Hertwig root sheath. Moreover, the mitotic index for pre-ameloblasts was 1.64 in normal rats, 0.92 for dwarf rats, and 1.66 for growth-hormone-treated dwarf rats (SD, 0.10). Other parameters-such as the labeling index and the ratio of positive to negative nuclei-were similarly related to GH status. Thus, growth hormone may play a role in the proliferation of the odontogenic epithelia in the rat.

A Bromodeoxyuridine Immunocytochemical and Morphometric Study of the Influence of Growth-Hormone On Cell-Proliferation in Odontogenic Mesenchyme of the Lewis Dwarf Rat

Cell proliferation was studied in pre-odontoblasts, and in cells of the dental papilla and lingual dental follicle using bromodeoxyuridine immunocytochemistry and morphometry in Bouins perfused and paraffin-embedded, undemineralized maxillary incisors. Cells in DNA synthesis, as shown by this technique, or in mitosis, were counted. Significantly fewer labelled nuclei, unlabelled nuclei and total nuclei were found in the tissues of growth hormone-deficient dwarf rats than in normal tissues. However, in dwarf rats treated for 6 days with bovine growth hormone, their numbers were equivalent to, or in some instances greater than those in normal tissues. The bromodeoxyuridine labelling index, the ratio of positive to negative nuclei and the mitotic index of pre-odontoblasts in dwarf rats were consistently lower than in normal rats, and were reversible by growth hormone. Growth hormone thus plays a part in odontogenic mesenchymal proliferation.