interspersed with fluorosed enamel. Rationalizing why only a small proportion of enamel is abnorm... more interspersed with fluorosed enamel. Rationalizing why only a small proportion of enamel is abnormal is difficult. It appears that different factors (fluoride and non-fluoride) can affect the homeostasis of the cell. The activity of ameloblasts during the maturation stages will greatly influence the pattern of enamel mineralization, which, in turn, may affect fluorosis. Apparently, the ameloblasts at the maturation stage have a complex role which may prove to be significant in the development of normal enamel. There are several changes in maturing ameloblasts, and several agents — including fluoride, vinblastine, and other drugs can disrupt them. A relatively highly-mineralized surface layer overlies a hypomineralized area in fluorotic lesions, but it is not known if the structure of the crystals in the hypomineralized area are affected by fluoride. Epidemiologists can use a fluoride biopsy to help with diagnosis in the case where the defect looks severe (not mild cases). If high levels of fluoride are found, then the opacity is probably fluorosis. There is no doubt that mild fluorosis does occur where low (below 1 ppm in water supplies) levels of fluoride are involved. Individual variation appears to be the explanation. The differences in the prevalence of fluorosis among different socio-economic groups cannot be explained readily. Where such differences occur, the explanation may be a synergistic action of other systemic factors (dietary) on increasing the sensitivity of amelogenesis to low F. Where higher prevalence occurs in higher socioeconomic classes, the explanation may be greater exposure to F supplementation. Russell's differential diagnostic criteria for fluorosis are based solely on appearance and assume a uniqueness for fluorosis. The criteria date back to epidemiological surveys of the 1930's, when an excellent correlation was found between fluoride in the water supply and fluorosis severity. If there are other factors that can cause that type of defect, then fluoride indices are invalid. Every index has a degree of validity, but the underlying issue is what other agents can cause these defects. Thylstrup and Fejerskov's index offers a wider spectrum of classifications than Dean's index, and is particularly useful in differentiating between the mild forms of fluorosis. The Tooth Surface Index of Fluorosis likewise is an improvement on Dean's, and it provides greater sensitivity in identification of the wide variations regularly seen in more severe forms of fluorosis. Dean's surveys in hundreds of communities found an extremely good correlation between fluorosis and fluoride in the water supply, but that doesn't eliminate other factors which may mimic fluorosis. However, more work has to be done to show that other agents can cause diffuse opacities. Several suggestions that calcium availability during mineralization had a role with fluoride during tooth formation did not receive substantial support during the discussion.
Fluoride excess of 0.05-0.07mgF/kgbw/day in water or food additives like salt is the principal ca... more Fluoride excess of 0.05-0.07mgF/kgbw/day in water or food additives like salt is the principal cause of endemic dental fluorosis. How fluoride causes these defects is not clear yet. Recent studies in rodents suggest that development of enamel fluorosis is associated with insufficient neutralization of protons released during the formation of hypermineralized lines. Here we examined whether hypermineralization could also be assessed by MicroCT in developing molar enamel of humans exposed to fluoride. Micro-CT analysis of hypomineralized enamel from human fluorotic molars graded by the Thylstrup-Fejerskov (TF) Index as III-IV showed weak hypermineralized lines and hypermineralized patches not seen in TF-I/II grade enamel. The mesio-distal sides of these molar teeth were significantly smaller (∼18%, p=0.02) than in TF-I/II teeth. The patterns of changes observed in human fluorotic teeth were similar to those in fluorotic rodent incisors. The data are consistent with the hypothesis that...
Regulation of pH by ameloblasts during amelogenesis is critical for enamel mineralization. We exa... more Regulation of pH by ameloblasts during amelogenesis is critical for enamel mineralization. We examined the effects of reduced bicarbonate secretion and the presence or absence of amelogenins on ameloblast modulation and enamel mineralization. To that end, the composition of fluorotic and non-fluorotic enamel of several different mouse mutants, including enamel of cystic fibrosis transmembrane conductance regulator-deficient (Cftr null), anion exchanger-2-deficient (Ae2a,b null), and amelogenin-deficient (Amelx null) mice, was determined by quantitative X-ray microanalysis. Correlation analysis was carried out to compare the effects of changes in the levels of sulfated-matrix (S) and chlorine (Cl; for bicarbonate secretion) on mineralization and modulation. The chloride (Cl(-) ) levels in forming enamel determined the ability of ameloblasts to modulate, remove matrix, and mineralize enamel. In general, the lower the Cl(-) content, the stronger the negative effects. In Amelx-null mice, modulation was essentially normal and the calcium content was reduced least. Retention of amelogenins in enamel of kallikrein-4-deficient (Klk4-null) mice resulted in decreased mineralization and reduced the length of the first acid modulation band without changing the total length of all acidic bands. These data suggest that buffering by bicarbonates is critical for modulation, matrix removal and enamel mineralization. Amelogenins also act as a buffer but are not critical for modulation.
Patients with cystic fibrosis (CF) have mild defects in dental enamel. The gene mutated in these ... more Patients with cystic fibrosis (CF) have mild defects in dental enamel. The gene mutated in these patients is CFTR, a Cl(-) channel involved in transepithelial salt and water transport and bicarbonate secretion. We tested the hypothesis that Cftr channels are present and operating in the plasma membranes of mouse ameloblasts. Tissue sections of young mouse jaws and fetal human jaws were immunostained with various anti-Cftr antibodies. Specificity of the antibodies was validated in Cftr-deficient murine and human tissues. Immunostaining for Cftr was obtained in the apical plasma membranes of mouse maturation ameloblasts of both incisor and molar tooth germs. A granular intracellular immunostaining of variable intensity was also noted in bone cells and odontoblasts. In Cftr-deficient mice the incisors were chalky white and eroded much faster than in wild type mice. Histologically, only maturation ameloblasts of incisors were structurally affected in Cftr-deficient mice. Some antibody s...
Hamster first hamster molar tooth germs in early secretory stage of amelogenesis were cultured fo... more Hamster first hamster molar tooth germs in early secretory stage of amelogenesis were cultured for one day in vitro at 6 degrees C, 22 degrees C, 37 degrees C or 45 degrees C in the presence of 3H-proline, 45Ca and 32P-orthophosphate. Other explants were cultured without these labels and after culture examined by histology. The highest temperature tested was lethal to the explants, decreased total dry weight and rapidly increased total uptake of the radiolabelled mineral ions, probably merely due to physicochemical modification of the existing preculture minerals. Optimal synthesis and secretion of amelogenins were measured at physiological temperature (37 degrees C). Effects of exposure to both temperatures below the physiological value were virtually reversible when explants were grown at physiological temperature (37 degrees C) for another day. However, amelogenin secretion during this recovery period did not reach values as high as those found for the first day in explants initi...
The relationship between the distribution of calcium in the cells of the enamel organ and the min... more The relationship between the distribution of calcium in the cells of the enamel organ and the mineralization process in mantle dentin and enamel was investigated at the ultrastructural level in cultured hamster second maxillary molar tooth germs explanted before the onset of mineralization (bell stage). During the early stages of pre-odontoblast and pre-ameloblast differentiation, pyroantimcnate (PA) reaction product for calcium
The developing enamel from three-day-old hamster first maxillary (M1) molar tooth germs exposed t... more The developing enamel from three-day-old hamster first maxillary (M1) molar tooth germs exposed to fluoride (F-) in vitro was analyzed for its mineral content by means of the energy-dispersive x-ray microanalysis technique. The aim of this study was to obtain semi-quantitative data on the F(-)-induced hypermineralization patterns in the enamel and to confirm that the increase in electron density observed in micrographs of F(-)-treated enamel (Lyaruu et. al., 1986, 1987b) is indeed due to an increase in mineral content in the fluorotic enamel. The tooth germs were explanted during the early stages of secretory amelogenesis and initially cultured for 24 hr in the presence of 10 ppm F- in the culture medium. The germs were then cultured for another 24 hr without F-. In order to compare the ultrastructural results directly with the microprobe data, we used the same specimens for both investigations. The net calcium counts (measurement minus background counts) in the analyses were used a...
This study assessed the growth pattern, cellular organisation and chemosensitivity of established... more This study assessed the growth pattern, cellular organisation and chemosensitivity of established human tumour cell lines growing as postconfluent cultures in 'V'-bottomed, 96-well microtiter plates. Cross-sections of the colon (HT29, SW620, SW1116), ovarian (A2780) and head and neck (UM-SCC-22B) carcinoma microcultures allowed in situ evaluation of the cellular organisation in the wells. After 5 days of growth, every cell line had reached confluence, but each of them displayed a specific pattern of cell stacking which ranged from two to ten layers. Postconfluent HT29 cells displayed morphologic features suggestive of some degree of enterocytic differentiation. Growth and cytotoxicity could be studied reliably and reproducibly in this system with the sulforhodamine B protein assay. Against HT29 postconfluent cultures, the EC50's (drug concentrations producing absorbance readings 50% lower than those of non-treated wells) of 5-fluorouracil and of the ether lipid, hexadecy...
Patients with cystic fibrosis (CF) have mild defects in dental enamel. The gene mutated in these ... more Patients with cystic fibrosis (CF) have mild defects in dental enamel. The gene mutated in these patients is CFTR, a Cl− channel involved in transepithelial salt and water transport and bicarbonate secretion. We tested the hypothesis that Cftr channels are present and operating in the plasma membranes of mouse ameloblasts.Tissue sections of young mouse jaws and fetal human jaws were
Supra-optimal intake of sodium fluoride (NaF) during early childhood results in formation of irre... more Supra-optimal intake of sodium fluoride (NaF) during early childhood results in formation of irreversible enamel defects. Monofluorophosphate (MFP) was considered as less toxic than NaF but equally cariostatic. We compared the potency of MFP and NaF to induce pre-eruptive sub-ameloblastic cysts and post-eruptive white spots and pits in developing hamster enamel. Hamster pups were injected subcutaneously with either NaF or MFP in equimolar doses of either 9 mg or 18 mg F/kg body weight. At 9 mg F/kg, MFP induced more but smaller sub-ameloblastic cysts with a collective cyst volume twice as large as that induced by NaF. Eight days after F injection, all F-injected groups had formed 4-6 white spots per molar, with an additional 2 pits per molar in the low MFP group. Twenty-eight days after injection, most white spots had turned into pits (5-6 per molar) and only the high MFP group still contained 2 white spots per molar. We conclude that parenterally applied MFP is more potent in inducing enamel defects than NaF. Most white spots formed turn into pits by functional use of the dentition. The higher potency of parenteral MFP may be associated with sustained elevated F levels in the enamel organ by enzymatic hydrolysis of MFP by alkaline phosphatase activity.
The distribution of collagen type V in developing dental and peridental tissues was investigated ... more The distribution of collagen type V in developing dental and peridental tissues was investigated with the indirect immunofluorescence technique using unfixed, frozen sections of jaws from 1-2 day old neonatal rats and hamsters. Immunostaining for type V collagens was found both intracellularly and extracellularly in dental tissues of mesenchymal origin. In non-dental tissues, weak immunostaining was observed in the mesenchymally derived stroma surrounding the developing molar tooth germs but was more pronounced in larger cells, probably young osteoblasts in close vicinity to alveolar bone, and in some cells within developing salivary glands. Decalcification revealed a strong immunostaining in the extracellular bone matrices. In the dental tissues, the mesenchymally derived cells of the papilla exhibited an intracellular staining for type V collagen. In odontoblasts, increased immunostaining over that of other pulpal cells was observed just prior to or coinciding with the onset of predentin secretion and reactivity remained high in fully differentiated odontoblasts. A weak staining was observed in predentin but only after the onset of mineralization. As was the case for bone, after demineralization the dentin matrix stained intensely for type V collagen. The results demonstrate that type V collagen is actively synthesized by mesenchymal cells of developing hard tissues and that this type of collagen is an intrinsic component of hard connective tissue matrices. The data suggest that in developing tooth germs type V collagen is not involved in the differentiation process of either odontoblasts or ameloblasts.
interspersed with fluorosed enamel. Rationalizing why only a small proportion of enamel is abnorm... more interspersed with fluorosed enamel. Rationalizing why only a small proportion of enamel is abnormal is difficult. It appears that different factors (fluoride and non-fluoride) can affect the homeostasis of the cell. The activity of ameloblasts during the maturation stages will greatly influence the pattern of enamel mineralization, which, in turn, may affect fluorosis. Apparently, the ameloblasts at the maturation stage have a complex role which may prove to be significant in the development of normal enamel. There are several changes in maturing ameloblasts, and several agents — including fluoride, vinblastine, and other drugs can disrupt them. A relatively highly-mineralized surface layer overlies a hypomineralized area in fluorotic lesions, but it is not known if the structure of the crystals in the hypomineralized area are affected by fluoride. Epidemiologists can use a fluoride biopsy to help with diagnosis in the case where the defect looks severe (not mild cases). If high levels of fluoride are found, then the opacity is probably fluorosis. There is no doubt that mild fluorosis does occur where low (below 1 ppm in water supplies) levels of fluoride are involved. Individual variation appears to be the explanation. The differences in the prevalence of fluorosis among different socio-economic groups cannot be explained readily. Where such differences occur, the explanation may be a synergistic action of other systemic factors (dietary) on increasing the sensitivity of amelogenesis to low F. Where higher prevalence occurs in higher socioeconomic classes, the explanation may be greater exposure to F supplementation. Russell's differential diagnostic criteria for fluorosis are based solely on appearance and assume a uniqueness for fluorosis. The criteria date back to epidemiological surveys of the 1930's, when an excellent correlation was found between fluoride in the water supply and fluorosis severity. If there are other factors that can cause that type of defect, then fluoride indices are invalid. Every index has a degree of validity, but the underlying issue is what other agents can cause these defects. Thylstrup and Fejerskov's index offers a wider spectrum of classifications than Dean's index, and is particularly useful in differentiating between the mild forms of fluorosis. The Tooth Surface Index of Fluorosis likewise is an improvement on Dean's, and it provides greater sensitivity in identification of the wide variations regularly seen in more severe forms of fluorosis. Dean's surveys in hundreds of communities found an extremely good correlation between fluorosis and fluoride in the water supply, but that doesn't eliminate other factors which may mimic fluorosis. However, more work has to be done to show that other agents can cause diffuse opacities. Several suggestions that calcium availability during mineralization had a role with fluoride during tooth formation did not receive substantial support during the discussion.
Fluoride excess of 0.05-0.07mgF/kgbw/day in water or food additives like salt is the principal ca... more Fluoride excess of 0.05-0.07mgF/kgbw/day in water or food additives like salt is the principal cause of endemic dental fluorosis. How fluoride causes these defects is not clear yet. Recent studies in rodents suggest that development of enamel fluorosis is associated with insufficient neutralization of protons released during the formation of hypermineralized lines. Here we examined whether hypermineralization could also be assessed by MicroCT in developing molar enamel of humans exposed to fluoride. Micro-CT analysis of hypomineralized enamel from human fluorotic molars graded by the Thylstrup-Fejerskov (TF) Index as III-IV showed weak hypermineralized lines and hypermineralized patches not seen in TF-I/II grade enamel. The mesio-distal sides of these molar teeth were significantly smaller (∼18%, p=0.02) than in TF-I/II teeth. The patterns of changes observed in human fluorotic teeth were similar to those in fluorotic rodent incisors. The data are consistent with the hypothesis that...
Regulation of pH by ameloblasts during amelogenesis is critical for enamel mineralization. We exa... more Regulation of pH by ameloblasts during amelogenesis is critical for enamel mineralization. We examined the effects of reduced bicarbonate secretion and the presence or absence of amelogenins on ameloblast modulation and enamel mineralization. To that end, the composition of fluorotic and non-fluorotic enamel of several different mouse mutants, including enamel of cystic fibrosis transmembrane conductance regulator-deficient (Cftr null), anion exchanger-2-deficient (Ae2a,b null), and amelogenin-deficient (Amelx null) mice, was determined by quantitative X-ray microanalysis. Correlation analysis was carried out to compare the effects of changes in the levels of sulfated-matrix (S) and chlorine (Cl; for bicarbonate secretion) on mineralization and modulation. The chloride (Cl(-) ) levels in forming enamel determined the ability of ameloblasts to modulate, remove matrix, and mineralize enamel. In general, the lower the Cl(-) content, the stronger the negative effects. In Amelx-null mice, modulation was essentially normal and the calcium content was reduced least. Retention of amelogenins in enamel of kallikrein-4-deficient (Klk4-null) mice resulted in decreased mineralization and reduced the length of the first acid modulation band without changing the total length of all acidic bands. These data suggest that buffering by bicarbonates is critical for modulation, matrix removal and enamel mineralization. Amelogenins also act as a buffer but are not critical for modulation.
Patients with cystic fibrosis (CF) have mild defects in dental enamel. The gene mutated in these ... more Patients with cystic fibrosis (CF) have mild defects in dental enamel. The gene mutated in these patients is CFTR, a Cl(-) channel involved in transepithelial salt and water transport and bicarbonate secretion. We tested the hypothesis that Cftr channels are present and operating in the plasma membranes of mouse ameloblasts. Tissue sections of young mouse jaws and fetal human jaws were immunostained with various anti-Cftr antibodies. Specificity of the antibodies was validated in Cftr-deficient murine and human tissues. Immunostaining for Cftr was obtained in the apical plasma membranes of mouse maturation ameloblasts of both incisor and molar tooth germs. A granular intracellular immunostaining of variable intensity was also noted in bone cells and odontoblasts. In Cftr-deficient mice the incisors were chalky white and eroded much faster than in wild type mice. Histologically, only maturation ameloblasts of incisors were structurally affected in Cftr-deficient mice. Some antibody s...
Hamster first hamster molar tooth germs in early secretory stage of amelogenesis were cultured fo... more Hamster first hamster molar tooth germs in early secretory stage of amelogenesis were cultured for one day in vitro at 6 degrees C, 22 degrees C, 37 degrees C or 45 degrees C in the presence of 3H-proline, 45Ca and 32P-orthophosphate. Other explants were cultured without these labels and after culture examined by histology. The highest temperature tested was lethal to the explants, decreased total dry weight and rapidly increased total uptake of the radiolabelled mineral ions, probably merely due to physicochemical modification of the existing preculture minerals. Optimal synthesis and secretion of amelogenins were measured at physiological temperature (37 degrees C). Effects of exposure to both temperatures below the physiological value were virtually reversible when explants were grown at physiological temperature (37 degrees C) for another day. However, amelogenin secretion during this recovery period did not reach values as high as those found for the first day in explants initi...
The relationship between the distribution of calcium in the cells of the enamel organ and the min... more The relationship between the distribution of calcium in the cells of the enamel organ and the mineralization process in mantle dentin and enamel was investigated at the ultrastructural level in cultured hamster second maxillary molar tooth germs explanted before the onset of mineralization (bell stage). During the early stages of pre-odontoblast and pre-ameloblast differentiation, pyroantimcnate (PA) reaction product for calcium
The developing enamel from three-day-old hamster first maxillary (M1) molar tooth germs exposed t... more The developing enamel from three-day-old hamster first maxillary (M1) molar tooth germs exposed to fluoride (F-) in vitro was analyzed for its mineral content by means of the energy-dispersive x-ray microanalysis technique. The aim of this study was to obtain semi-quantitative data on the F(-)-induced hypermineralization patterns in the enamel and to confirm that the increase in electron density observed in micrographs of F(-)-treated enamel (Lyaruu et. al., 1986, 1987b) is indeed due to an increase in mineral content in the fluorotic enamel. The tooth germs were explanted during the early stages of secretory amelogenesis and initially cultured for 24 hr in the presence of 10 ppm F- in the culture medium. The germs were then cultured for another 24 hr without F-. In order to compare the ultrastructural results directly with the microprobe data, we used the same specimens for both investigations. The net calcium counts (measurement minus background counts) in the analyses were used a...
This study assessed the growth pattern, cellular organisation and chemosensitivity of established... more This study assessed the growth pattern, cellular organisation and chemosensitivity of established human tumour cell lines growing as postconfluent cultures in 'V'-bottomed, 96-well microtiter plates. Cross-sections of the colon (HT29, SW620, SW1116), ovarian (A2780) and head and neck (UM-SCC-22B) carcinoma microcultures allowed in situ evaluation of the cellular organisation in the wells. After 5 days of growth, every cell line had reached confluence, but each of them displayed a specific pattern of cell stacking which ranged from two to ten layers. Postconfluent HT29 cells displayed morphologic features suggestive of some degree of enterocytic differentiation. Growth and cytotoxicity could be studied reliably and reproducibly in this system with the sulforhodamine B protein assay. Against HT29 postconfluent cultures, the EC50's (drug concentrations producing absorbance readings 50% lower than those of non-treated wells) of 5-fluorouracil and of the ether lipid, hexadecy...
Patients with cystic fibrosis (CF) have mild defects in dental enamel. The gene mutated in these ... more Patients with cystic fibrosis (CF) have mild defects in dental enamel. The gene mutated in these patients is CFTR, a Cl− channel involved in transepithelial salt and water transport and bicarbonate secretion. We tested the hypothesis that Cftr channels are present and operating in the plasma membranes of mouse ameloblasts.Tissue sections of young mouse jaws and fetal human jaws were
Supra-optimal intake of sodium fluoride (NaF) during early childhood results in formation of irre... more Supra-optimal intake of sodium fluoride (NaF) during early childhood results in formation of irreversible enamel defects. Monofluorophosphate (MFP) was considered as less toxic than NaF but equally cariostatic. We compared the potency of MFP and NaF to induce pre-eruptive sub-ameloblastic cysts and post-eruptive white spots and pits in developing hamster enamel. Hamster pups were injected subcutaneously with either NaF or MFP in equimolar doses of either 9 mg or 18 mg F/kg body weight. At 9 mg F/kg, MFP induced more but smaller sub-ameloblastic cysts with a collective cyst volume twice as large as that induced by NaF. Eight days after F injection, all F-injected groups had formed 4-6 white spots per molar, with an additional 2 pits per molar in the low MFP group. Twenty-eight days after injection, most white spots had turned into pits (5-6 per molar) and only the high MFP group still contained 2 white spots per molar. We conclude that parenterally applied MFP is more potent in inducing enamel defects than NaF. Most white spots formed turn into pits by functional use of the dentition. The higher potency of parenteral MFP may be associated with sustained elevated F levels in the enamel organ by enzymatic hydrolysis of MFP by alkaline phosphatase activity.
The distribution of collagen type V in developing dental and peridental tissues was investigated ... more The distribution of collagen type V in developing dental and peridental tissues was investigated with the indirect immunofluorescence technique using unfixed, frozen sections of jaws from 1-2 day old neonatal rats and hamsters. Immunostaining for type V collagens was found both intracellularly and extracellularly in dental tissues of mesenchymal origin. In non-dental tissues, weak immunostaining was observed in the mesenchymally derived stroma surrounding the developing molar tooth germs but was more pronounced in larger cells, probably young osteoblasts in close vicinity to alveolar bone, and in some cells within developing salivary glands. Decalcification revealed a strong immunostaining in the extracellular bone matrices. In the dental tissues, the mesenchymally derived cells of the papilla exhibited an intracellular staining for type V collagen. In odontoblasts, increased immunostaining over that of other pulpal cells was observed just prior to or coinciding with the onset of predentin secretion and reactivity remained high in fully differentiated odontoblasts. A weak staining was observed in predentin but only after the onset of mineralization. As was the case for bone, after demineralization the dentin matrix stained intensely for type V collagen. The results demonstrate that type V collagen is actively synthesized by mesenchymal cells of developing hard tissues and that this type of collagen is an intrinsic component of hard connective tissue matrices. The data suggest that in developing tooth germs type V collagen is not involved in the differentiation process of either odontoblasts or ameloblasts.
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Papers by D.M. Lyaruu