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Remineralization of human carious dentine in vitro
ArchsoralBiol.Vol. 17, pp. 1005-1008,1972.Pergamon Press.Printed in GreatBritain.
REMINERALIZATION
OF HUMAN IN VITRO R. S.
Department
CARIOUS DENTINE
LEVINE
of Oral Medicine, The Dental School, Bridgeford Street, Manchester Ml5 6FH, England
Summary-Undecalcified sections of carious teeth were divided in two through the lesion so that one half could be stored as a control while the other half was incubated at 37°C in experimental remineralization fluids. A non-toxic phosphate-buffered fluoride solution containing solid dicalcium phosphate dihydrate signs of remineralization under the conditions described.
produced
microradiographic
THEREis evidence that artificially demineralized dentine may be remineralized in vitro (WEI, KAQUELER and MASSLER, 1968). These workers provided radiographic and electron-microscopic evidence that a 10 per cent solution of stannous fluoride would produce rapid precipitation of a mineral, thought to be stannous phosphate (WEI and FORBES, 1968), in naturally carious dentine under in vitro conditions. However, owing to its known toxicity, this agent is unsuitable for use in vivo in deep carious cavities in teeth because of the proximity of the dental pulp. This communication describes a preliminary study to test the ability of a non-toxic agent to remineralize carious dentine in vitro. The remineralization system studied was designed to utilize the ability of the fluoride ion to favour the precipitation of hydroxyapatite from solutions of calcium and phosphate ions (BRUDEVOLDand MCCANN, 1966) and the known epitactic properties of dentine collagen (SOLOMONS and NEUMAN,1960) in addition to the presence of residual hydroxyapatite crystals to encourage remineralization. Freshly extracted carious human teeth were cut on a BOVIS(1968) sectioning machine to produce plano-parallel longitudinal sections approximately 100 pm thick. Sections were then divided in two through the lesion so that one half could be incubated at 37°C in 10 ml of remineralization solution for l-7 days, while the other half was stored for use as the control. After incubation, the experimental half section was washed in distilled water, allowed to dry on a microscope slide and placed next to the similarly dried control half on a Kodak M-R plate. Microradiography was carried out by exposure to Ni filtered Cu radiation excited at 20 kV and 30 mA for 20 min using a cassette described by COOPER(1968). The basic remineralization system used in this preliminary study was a 0 -5 M phosphate buffer (pH 5 -8-7 -0) containing solid brushite (dicalcium phosphate dihydrate), to ensure saturation, which is less insoluble than other calcium phosphate compounds, and concentrations of sodium fluoride over the range O-100 ppm F. The buffer was necessary to counteract the marked fall in pH that occurred in the un1005
1006
R. S.
LEVINE
buffered system on incubation with carious dentine and presumably due to the formation of hydroxyapatite. The results from 10 sections (7 teeth) indicate that a marked increase in radiodensity of carious dentine can be produced by the system described. The typical results shown in Fig. 1 were obtained by incubating the experimental half-section for 7 days in phosphate buffer at pH 6 a0 with a fluoride concentration of 10 ppm, which were found to be the optimum conditions. Fluoride-free solutions and those with less than 5 and more than 50 ppm F produced no detectable remineralization. The solution was neither changed nor agitated. The consistent finding that the increase in radiodensity was not confined to the deeper part of the lesion may reflect not only the wide distribution of residual crystals providing nucleation sites, but also the persistence of intact collagen, even at the surface of the lesion, as suggested by microchemical analysis (LEVINE,1972). Current work on this topic is directed towards the positive identification of the deposited mineral, to be followed by in vivo studies. However, it should be noted that the large surface area in contact with the experimental solution provided by both sides of a tooth section would not be available in vivo. Acknowledgemenrs-The preliminary study was begun in the Department of Oral Pathology of the University of Birmingham and I am indebted to Dr. S. L. ROWLESfor the use of facilities in his laboratory and to Miss J. STEVENSfor technical assistance. R&sum&-Des sections non-decalcifiees de dents car&s ont ttt divisees en deux a travers la lesion, ainsi qu’une moitit puisse etre gard&e comme controle, tandis que I’autre moitit soit incub& a 37°C dans des liquides exp&imentaux de remhteralisation. Une solution de fluorure non-toxique, tamponnee a phosphate, contenant du bihydrate de phosphate bicalcique solide, produisit des signes microradiographiques de remintralisation sous les conditions d&crites. Zusammenfassung-Nicht entkalkte Teile karioser Zahne wurden in der krankhaften Verlnderung in solcher Weise in zwei Teile geteilt, dass die eine Halfte als Kontrolle aufbewahrt werden konnte, wahrend die andere Halfte bei 37°C in experimenteller Fliissigkeit zur erneuten Mineralisation einer Inkubation unterworfen wurde. Eine nicht toxische, mit Phosphat gepufferte Fluoridlosung mit festem Dikalziumphosphatdihydratgehalt ergab mikrorontgenologische Zeichen erneuter Mineralisation unter den beschriebenen Umstlnden. REFERENCES Bov~s, S. C. 1968. The preparation of plane-parallel sections of calcified tissues. Br. dent. J. 125,502505. BRUDEVOLD, F. and MCCANN, A. G. 1966. The mechanism of caries-inhibiting effect of fluoride. In: Enoironmental Variables in Oral Disease (Ed. Kreshover. S. J. and McClure. F. J.). Am. Ass. Adv. Sci., Washington D.C., pp. 103-128. COOPER, W. E. G. 1968. A microchemical, microradiographic and histological investigation of amelogenesis in the pig. Archs oral Biol. 13,27-48. LEVINE, R. S. 1972.The distribution of hydroxyproline in the dentine of carious teeth. Archs oral Biol. 17, 127-135. SOLOMONS,C. C. and NBUMAN, W. F. 1960. On the mechanisms of calcification: the remineralization of dentine. J. biol. Chem. 235,2502-2506.
REMINERAUZATIONOF CARIOUSDENTINEin Vitro
1007
WEI, S. H., and FORBES,W. C. 1968. X-ray diffraction analysis of carious dentine treated with stannous fluoride. Archs oral Biol. 13,407417. WEI, S. H., KAQUELER, J. C. and MASSLER, M. (1968). Remineralisation of carious dentine. J. dent. Res. 47, 381-391.
PLATE 1 OVERLEAF
1008
R. S. LEVINE
FIGS. la and b. Each of these is a microradiograph of a tooth section which was divided into two. The two parts were then fitted together again for microradiography. The right-hand portions were incubated in the remineralization fluid and the lefthand portions were the controls. The areas of demineralization by caries are more radiopaque in the treated right-hand portions.
REMINERALIZATION
OF CARLOUS
DENTINE
in Vitro
A.O.B.
f.p. 100X
REMINERALIZATION
OF HUMAN IN VITRO R. S.
Department
CARIOUS DENTINE
LEVINE
of Oral Medicine, The Dental School, Bridgeford Street, Manchester Ml5 6FH, England
Summary-Undecalcified sections of carious teeth were divided in two through the lesion so that one half could be stored as a control while the other half was incubated at 37°C in experimental remineralization fluids. A non-toxic phosphate-buffered fluoride solution containing solid dicalcium phosphate dihydrate signs of remineralization under the conditions described.
produced
microradiographic
THEREis evidence that artificially demineralized dentine may be remineralized in vitro (WEI, KAQUELER and MASSLER, 1968). These workers provided radiographic and electron-microscopic evidence that a 10 per cent solution of stannous fluoride would produce rapid precipitation of a mineral, thought to be stannous phosphate (WEI and FORBES, 1968), in naturally carious dentine under in vitro conditions. However, owing to its known toxicity, this agent is unsuitable for use in vivo in deep carious cavities in teeth because of the proximity of the dental pulp. This communication describes a preliminary study to test the ability of a non-toxic agent to remineralize carious dentine in vitro. The remineralization system studied was designed to utilize the ability of the fluoride ion to favour the precipitation of hydroxyapatite from solutions of calcium and phosphate ions (BRUDEVOLDand MCCANN, 1966) and the known epitactic properties of dentine collagen (SOLOMONS and NEUMAN,1960) in addition to the presence of residual hydroxyapatite crystals to encourage remineralization. Freshly extracted carious human teeth were cut on a BOVIS(1968) sectioning machine to produce plano-parallel longitudinal sections approximately 100 pm thick. Sections were then divided in two through the lesion so that one half could be incubated at 37°C in 10 ml of remineralization solution for l-7 days, while the other half was stored for use as the control. After incubation, the experimental half section was washed in distilled water, allowed to dry on a microscope slide and placed next to the similarly dried control half on a Kodak M-R plate. Microradiography was carried out by exposure to Ni filtered Cu radiation excited at 20 kV and 30 mA for 20 min using a cassette described by COOPER(1968). The basic remineralization system used in this preliminary study was a 0 -5 M phosphate buffer (pH 5 -8-7 -0) containing solid brushite (dicalcium phosphate dihydrate), to ensure saturation, which is less insoluble than other calcium phosphate compounds, and concentrations of sodium fluoride over the range O-100 ppm F. The buffer was necessary to counteract the marked fall in pH that occurred in the un1005
1006
R. S.
LEVINE
buffered system on incubation with carious dentine and presumably due to the formation of hydroxyapatite. The results from 10 sections (7 teeth) indicate that a marked increase in radiodensity of carious dentine can be produced by the system described. The typical results shown in Fig. 1 were obtained by incubating the experimental half-section for 7 days in phosphate buffer at pH 6 a0 with a fluoride concentration of 10 ppm, which were found to be the optimum conditions. Fluoride-free solutions and those with less than 5 and more than 50 ppm F produced no detectable remineralization. The solution was neither changed nor agitated. The consistent finding that the increase in radiodensity was not confined to the deeper part of the lesion may reflect not only the wide distribution of residual crystals providing nucleation sites, but also the persistence of intact collagen, even at the surface of the lesion, as suggested by microchemical analysis (LEVINE,1972). Current work on this topic is directed towards the positive identification of the deposited mineral, to be followed by in vivo studies. However, it should be noted that the large surface area in contact with the experimental solution provided by both sides of a tooth section would not be available in vivo. Acknowledgemenrs-The preliminary study was begun in the Department of Oral Pathology of the University of Birmingham and I am indebted to Dr. S. L. ROWLESfor the use of facilities in his laboratory and to Miss J. STEVENSfor technical assistance. R&sum&-Des sections non-decalcifiees de dents car&s ont ttt divisees en deux a travers la lesion, ainsi qu’une moitit puisse etre gard&e comme controle, tandis que I’autre moitit soit incub& a 37°C dans des liquides exp&imentaux de remhteralisation. Une solution de fluorure non-toxique, tamponnee a phosphate, contenant du bihydrate de phosphate bicalcique solide, produisit des signes microradiographiques de remintralisation sous les conditions d&crites. Zusammenfassung-Nicht entkalkte Teile karioser Zahne wurden in der krankhaften Verlnderung in solcher Weise in zwei Teile geteilt, dass die eine Halfte als Kontrolle aufbewahrt werden konnte, wahrend die andere Halfte bei 37°C in experimenteller Fliissigkeit zur erneuten Mineralisation einer Inkubation unterworfen wurde. Eine nicht toxische, mit Phosphat gepufferte Fluoridlosung mit festem Dikalziumphosphatdihydratgehalt ergab mikrorontgenologische Zeichen erneuter Mineralisation unter den beschriebenen Umstlnden. REFERENCES Bov~s, S. C. 1968. The preparation of plane-parallel sections of calcified tissues. Br. dent. J. 125,502505. BRUDEVOLD, F. and MCCANN, A. G. 1966. The mechanism of caries-inhibiting effect of fluoride. In: Enoironmental Variables in Oral Disease (Ed. Kreshover. S. J. and McClure. F. J.). Am. Ass. Adv. Sci., Washington D.C., pp. 103-128. COOPER, W. E. G. 1968. A microchemical, microradiographic and histological investigation of amelogenesis in the pig. Archs oral Biol. 13,27-48. LEVINE, R. S. 1972.The distribution of hydroxyproline in the dentine of carious teeth. Archs oral Biol. 17, 127-135. SOLOMONS,C. C. and NBUMAN, W. F. 1960. On the mechanisms of calcification: the remineralization of dentine. J. biol. Chem. 235,2502-2506.
REMINERAUZATIONOF CARIOUSDENTINEin Vitro
1007
WEI, S. H., and FORBES,W. C. 1968. X-ray diffraction analysis of carious dentine treated with stannous fluoride. Archs oral Biol. 13,407417. WEI, S. H., KAQUELER, J. C. and MASSLER, M. (1968). Remineralisation of carious dentine. J. dent. Res. 47, 381-391.
PLATE 1 OVERLEAF
1008
R. S. LEVINE
FIGS. la and b. Each of these is a microradiograph of a tooth section which was divided into two. The two parts were then fitted together again for microradiography. The right-hand portions were incubated in the remineralization fluid and the lefthand portions were the controls. The areas of demineralization by caries are more radiopaque in the treated right-hand portions.
REMINERALIZATION
OF CARLOUS
DENTINE
in Vitro
A.O.B.
f.p. 100X