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Microradiographic analysis of synthetic salivary calculus
Arch. oral Bid.
Vol.10.
pp.317-318.
1965.
Pergamon
Press
Ltd. Printed inCt.
Britain.
MICRORADIOGRAPHIC ANALYSIS OF SYNTHETIC SALIVARY CALCULUS F. J. DRAUS and F. L. MIKLOS University
of Pittsburgh
School of Dentistry,
Pittsburgh
13, Pa., U.S.A.
IN A recent publication (DRAUS and MIKLOS, 1963) it was suggested that in the formation of synthetic calculus the bovine mucoid serves as a site for nucleation and crystal growth. To further demonstrate this, it became necessary to investigate the calcification pattern over a period of time. If the original hypothesis was correct, the degree of calcification should increase with time of exposure to the calcifying medium. Contact microradiography has been used successfully to demonstrate calcification. WASSERMAN,MANDEL and LEVY (1958) studied calcification of bacterial plaque formed in vitro, with soft X-rays. NYLEN (1960) demonstrated the mineralization of turkey tendon with X-rays in the 2-5 kV range while LITTLE (1960) investigated the calcification patterns of human calculus. From these investigations it appeared that soft X-rays could be used in studying the calcifying process. In this communication we wish to present the results of a microradiographic analysis of synthetic calculus (DRAUSand MIKLOS.1963) on sections of teeth using soft X-rays in the 3 kV range. For the formation of synthetic calculus, sound anterior teeth were cleaned, depulped and suspended in a O-1 % solution of bovine submaxillary gland extract in a calcifying solution prepared according to STRATES(1957). Tbis solution. which essentially contained calcium and phosphate buffered with barbital to pH 7.0, contained 7.6 mg/lOO ml calcium and 6.6 mg/lOO ml phosphorus or a Ca T P product of approximately fifty. The system was closed to minimize evaporation and placed in the dipping apparatus of LEUNG (1957) kept at a constant temperature of 37’C. The teeth were washed and the calcifying solution changed daily. Two teeth were removed each day for a period of 31 days. A Ca x P product of fifty was used in this study in order to eliminate co-precipitaAt this level the Ca x P product would tion as a possible factor in deposit formation. also be in a range of that in whole human saliva. In such a system any deposit having radiopacity could be attributed to an attachment with the matrix and it could be concluded that crystal growth was progressing from these sites. The teeth, on removal from the forming medium, were dehydrated for 24 hr in alcohol solution of 50, 70, 95% and finally in absolute alcohol, then embedded in Bioplastic (Wards Natural Science Est., Rochester, N.Y.). The plastic was allowed to harden at 50°C for 24 hr. Serial sections were cut on a Gillings-Hamco Thin Sectioning Machine (Brownwill Scientific Company, Rochester, N.Y.) with a serrated diamond disk at 50 pm. 317
318
F. J. DRAWSAND F. L.
MIKLOS
The sections were mounted on metal washers and subjected to X-rays with a Phillips CMR-5 Contact Microradiographic Unit (Pitchford Scientific Instrument Company, Pittsburgh, Pa.) for 5 min at 3.3 mA and 3 kV using Kodak Spectroscopic film type 6490. The film was developed with Kodak D-l 1 developer, fixed, mounted on a slide with permount and examined microscopically. Examination of the radiographs of the sections indicated the presence of radiolucent material interspersed with radiopaque areas as early as one day. In Fig. 1 we have illustrated photos of the radiographs from 2, 5, 9 and 17-day sections. The 2-day section (A) shows a small amount of radiopaque material lying in a radiolucent matrix. The amount of radiopaque material increases (B, C, D) with prolonged exposure to the calcifying medium. Almost complete calcification was noted after 17 days. The outer layers at all times are less calcified than the original deposit, indicating that as the new matrix is laid down the calcifying process repeats. With the use of tooth sections it was possible to detect calcified areas as early as 1 day. In order to determine if calcification was taking place before this time it was necessary to modify the original technique since it was impossible to detect any deposit on 50 pm sections before 1 day. Mylar strips were placed in the forming medium and removed at 6-hr intervals, then sectioned at 5 pm with a microtome. By this method is was possible to detect radiopaque areas as early as 6 hr after immersion in the calcifying medium. The results of the examination of the radiographs from the tooth sections and the Mylar strips would indicate that calcification begins immediately after the matrix is deposited. It is impossible to determine at this time if the submaxillary gland extract is premucleated and crystal growth is progressing from these nucleated areas or if the nucleation and crystal growth originate in the calcifying solution. Before this can be determined it will be necessary to exhaustively remove any residual calcium from the gland extract and to subject it to the criteria set up by SOBEL (1963) for the determination of nucleation and crystal growth. Acknowledgement-This investigation was supported by a PHS Research Grant DE-01 588 from the National Institute of Dental Research. Public Health Service. REFERENCES DRAUS, F. J. and MIKLOS, F. L. Res. 42, 1249. LEUNG, S. WAH. 217-221.
1963. Calcifying potential of bovine submaxillary
mucoid. J. rienr.
1957. A new method for the production of synthetic calculus. J. Periodont.
28,
LITTLE, M. F., ROWLEY, J., BARBER, J. and COSCIANI. C. 1960. Calcification patterns and inorganic composition on natural dental calculus. J. denr. Res. 39, 710.
NYLEN, M., SCOTT. D. and MOSLEY, V. 1960. Mineralization biological systems, p. 129, AAAS No. 64. SOBEL, A. E.
1963.
Differentiating
of turkey leg tendon.
between nucleation and crystal growth.
Calcification in
J. den!.
Res.
(Abs.
Supp.) 42,45. STRATES, B. S., NEUMAN, W. F. and LEVINSKAS, G. J. J. ph.vs. Chem. 61, 279-282. WASSERMAN, B. H., MANDEL, I. D. and LEVY, B. M. J. Periodont. 29, 144-147.
1957. The solubility
of bone mineral--II.
1958. In vitro calcification of dental calculus.
MICKDHADIOGKAPHIC
days.
, 318
FIG. I. Contact x 450.
ANALYSIS
IIIlCrOradiOgmphs.
OF SYNTHLllC
A, 2 days;
SALIV,\Kl
CL(‘UI
B, 5 days;
US
C, 9 dir+:
I>. I7 PLA? t
F. J. DRAUS AND
PLATE.
2
F. L.
MIKLOS
Vol.10.
pp.317-318.
1965.
Pergamon
Press
Ltd. Printed inCt.
Britain.
MICRORADIOGRAPHIC ANALYSIS OF SYNTHETIC SALIVARY CALCULUS F. J. DRAUS and F. L. MIKLOS University
of Pittsburgh
School of Dentistry,
Pittsburgh
13, Pa., U.S.A.
IN A recent publication (DRAUS and MIKLOS, 1963) it was suggested that in the formation of synthetic calculus the bovine mucoid serves as a site for nucleation and crystal growth. To further demonstrate this, it became necessary to investigate the calcification pattern over a period of time. If the original hypothesis was correct, the degree of calcification should increase with time of exposure to the calcifying medium. Contact microradiography has been used successfully to demonstrate calcification. WASSERMAN,MANDEL and LEVY (1958) studied calcification of bacterial plaque formed in vitro, with soft X-rays. NYLEN (1960) demonstrated the mineralization of turkey tendon with X-rays in the 2-5 kV range while LITTLE (1960) investigated the calcification patterns of human calculus. From these investigations it appeared that soft X-rays could be used in studying the calcifying process. In this communication we wish to present the results of a microradiographic analysis of synthetic calculus (DRAUSand MIKLOS.1963) on sections of teeth using soft X-rays in the 3 kV range. For the formation of synthetic calculus, sound anterior teeth were cleaned, depulped and suspended in a O-1 % solution of bovine submaxillary gland extract in a calcifying solution prepared according to STRATES(1957). Tbis solution. which essentially contained calcium and phosphate buffered with barbital to pH 7.0, contained 7.6 mg/lOO ml calcium and 6.6 mg/lOO ml phosphorus or a Ca T P product of approximately fifty. The system was closed to minimize evaporation and placed in the dipping apparatus of LEUNG (1957) kept at a constant temperature of 37’C. The teeth were washed and the calcifying solution changed daily. Two teeth were removed each day for a period of 31 days. A Ca x P product of fifty was used in this study in order to eliminate co-precipitaAt this level the Ca x P product would tion as a possible factor in deposit formation. also be in a range of that in whole human saliva. In such a system any deposit having radiopacity could be attributed to an attachment with the matrix and it could be concluded that crystal growth was progressing from these sites. The teeth, on removal from the forming medium, were dehydrated for 24 hr in alcohol solution of 50, 70, 95% and finally in absolute alcohol, then embedded in Bioplastic (Wards Natural Science Est., Rochester, N.Y.). The plastic was allowed to harden at 50°C for 24 hr. Serial sections were cut on a Gillings-Hamco Thin Sectioning Machine (Brownwill Scientific Company, Rochester, N.Y.) with a serrated diamond disk at 50 pm. 317
318
F. J. DRAWSAND F. L.
MIKLOS
The sections were mounted on metal washers and subjected to X-rays with a Phillips CMR-5 Contact Microradiographic Unit (Pitchford Scientific Instrument Company, Pittsburgh, Pa.) for 5 min at 3.3 mA and 3 kV using Kodak Spectroscopic film type 6490. The film was developed with Kodak D-l 1 developer, fixed, mounted on a slide with permount and examined microscopically. Examination of the radiographs of the sections indicated the presence of radiolucent material interspersed with radiopaque areas as early as one day. In Fig. 1 we have illustrated photos of the radiographs from 2, 5, 9 and 17-day sections. The 2-day section (A) shows a small amount of radiopaque material lying in a radiolucent matrix. The amount of radiopaque material increases (B, C, D) with prolonged exposure to the calcifying medium. Almost complete calcification was noted after 17 days. The outer layers at all times are less calcified than the original deposit, indicating that as the new matrix is laid down the calcifying process repeats. With the use of tooth sections it was possible to detect calcified areas as early as 1 day. In order to determine if calcification was taking place before this time it was necessary to modify the original technique since it was impossible to detect any deposit on 50 pm sections before 1 day. Mylar strips were placed in the forming medium and removed at 6-hr intervals, then sectioned at 5 pm with a microtome. By this method is was possible to detect radiopaque areas as early as 6 hr after immersion in the calcifying medium. The results of the examination of the radiographs from the tooth sections and the Mylar strips would indicate that calcification begins immediately after the matrix is deposited. It is impossible to determine at this time if the submaxillary gland extract is premucleated and crystal growth is progressing from these nucleated areas or if the nucleation and crystal growth originate in the calcifying solution. Before this can be determined it will be necessary to exhaustively remove any residual calcium from the gland extract and to subject it to the criteria set up by SOBEL (1963) for the determination of nucleation and crystal growth. Acknowledgement-This investigation was supported by a PHS Research Grant DE-01 588 from the National Institute of Dental Research. Public Health Service. REFERENCES DRAUS, F. J. and MIKLOS, F. L. Res. 42, 1249. LEUNG, S. WAH. 217-221.
1963. Calcifying potential of bovine submaxillary
mucoid. J. rienr.
1957. A new method for the production of synthetic calculus. J. Periodont.
28,
LITTLE, M. F., ROWLEY, J., BARBER, J. and COSCIANI. C. 1960. Calcification patterns and inorganic composition on natural dental calculus. J. denr. Res. 39, 710.
NYLEN, M., SCOTT. D. and MOSLEY, V. 1960. Mineralization biological systems, p. 129, AAAS No. 64. SOBEL, A. E.
1963.
Differentiating
of turkey leg tendon.
between nucleation and crystal growth.
Calcification in
J. den!.
Res.
(Abs.
Supp.) 42,45. STRATES, B. S., NEUMAN, W. F. and LEVINSKAS, G. J. J. ph.vs. Chem. 61, 279-282. WASSERMAN, B. H., MANDEL, I. D. and LEVY, B. M. J. Periodont. 29, 144-147.
1957. The solubility
of bone mineral--II.
1958. In vitro calcification of dental calculus.
MICKDHADIOGKAPHIC
days.
, 318
FIG. I. Contact x 450.
ANALYSIS
IIIlCrOradiOgmphs.
OF SYNTHLllC
A, 2 days;
SALIV,\Kl
CL(‘UI
B, 5 days;
US
C, 9 dir+:
I>. I7 PLA? t
F. J. DRAUS AND
PLATE.
2
F. L.
MIKLOS