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Effect of calcium treatment on solubility and calcium uptake of synthetic hydroxyapatite and rat molar enamel
LIKINS— POSNER—STEERE . . . VO LUM E 57, SEPTEMBER 1958 • 335
the housewife, and may in fact explain some o f the differences noted previously between the dental habits o f men and wom en. T hey may also be m ore im portant considerations fo r the wage earner, to w hom , literally, every hour spent in travel, or in waiting and consul tation, costs m oney, than fo r the profes sional worker, w h o often has greater flexibility in working and earning time. A ll these factors can be joined into a whole, from the point o f view o f the patient, by considering the use o f dental services to stem from an over-all assess ment, or definition, o f the situation. T he
prospective patient assesses his dental condition and the seriousness o f the con sequences if he does not seek dental care. This self-diagnosis is weighed or balanced against the factors o f cost, anticipated pain, and inconvenience, to see if going to the dentist “ is worth it.” T h e final result— the use or avoidance o f dental services— is thus a com plex product o f the education involved in self-diagnosis, o f the incom e level involved in weighing costs, o f the dental health and past den tal experience involved in anticipating pain, and o f the social experience in volved in assessing inconvenience.
Effect of calcium treatment on solubility and calcium uptake of synthetic hydroxyapatite and rat molar enamel R . C. Likins,* D .D .S., Bethesda, M d .; A . S. P o s n e r * Ph.D., Washington, D . C., and A . C. Steere,% B.S., Bethesda, M d.
T h e existence o f apatites deficient in d i valent cations is supported by crystallographic evidence . 1 Certain chemicallyprecipitated hydroxyapatites were shown to contain different amounts o f calcium ions missing from structural positions, re sulting in a fam ily o f defect apatites with low calcium -phosphorus ratios. Electrical neutrality was postulated to be obtained by hydrogen bonding between oxygens o f neighboring orthophosphate ions. In this light, the general form ula for apatites may be written: D
M 10-x
2x
(P O ) (O H ) 4 6 2
where D represents the divalent cations (C A ++, Pb++, Sr++, M g ++, and so forth, M
the m onovalent cations (N a +, H +, and so forth ) and x the number o f divalent ca tions missing from structural positions. T h e value o f x has been reported to range from 0 to 2 . 1 W hen x prevails as zero, the stoichiometry o f perfect apatite o b tains. It is quite possible that bone and tooth minéral, which d o not have the ideal calcium -phosphorus ratio o f hydroxyapa-
•National institute of Dental Research, National In stitutes of Health, Public Health Service, U. S. De partment of Health, Education, and Welfare, Bethesda 14, Md. ^Research Associate, American Dental Association Research Division, National Bureau of Standards, Wash ington 25, D. C. I. Posner, A . S., and Perloff, A. Apatites deficient in divalent cations. J . of Research, Natl. Bur. of Stds. 58:279 May 1957.
336 • THE JO U R N A L OF THE A M ER IC A N DENTAL ASSOCIATION
tite, 2 may have calcium defect hydroxyapatite as a m ajor constituent.3’ 4 Syn thetic hydroxyapatites with low calcium phosphorus ratios are thermally unstable when com pared to perfect apatites . 5 It is also possible that the solubility o f apa tites may be related to the calcium con tent, particularly as the content ap proaches the theoretical value. Other experiments have shown that the calcium phosphorus ratios o f synthetic, low -calcium apatites can increase in calcium hydroxide solution . 6 T h e purpose o f the present study was to investigate the effect o f calcium ion treatment on the acid solubility o f synthetic, low -calcium apa tite and rat m olar enamel, and the capac ity o f these materials to rem ove calcium ions from solution. E X P E R IM E N T S
m olar calcium 4 0 , 4 5 chloride ( 1 m icro curie per milliliter) adjusted to p H 6.5 with N H 4 O H . T h e suspensions were cen trifuged for five minutes, the supernates rem oved by aspiration, and the precipi tates washed three times in 1 0 . 0 ml. p or tions o f distilled water. T h e samples were dried, weighed, dissolved in hydrochloric acid, and analyzed fo r radiocalcium . 2. O n e aliquot from each sample pool was shaken for 25 minutes with 0.001 N lactic acid, and centrifuged fo r five m in utes. T h e supernates were rem oved by aspiration, filtered through sintered glass (nom inal m axim um pore size, 0.9-1.4 m icro n s), and the filtrates analyzed for phosphorus. Studies with R at M ola r T eeth • T h e m o lar crowns o f 16 weanling male SpragueDawley rats were obtained as follow s: A t sacrifice the jaws were excised, freed o f adherent tissue, and the clinical crowns cleaned with a soft brush and distilled water. Using a % inch circular steel saw in a dental handpiece, the teeth were sectioned transversely in situ approxi mately 0 . 1 mm . above the gingival at tachment. A fter sectioning, the first and second molars o f each rat were divided equally into tw o groups. T h e crowns were blotted and sealed, cut surface dow n, on paraffincoated glass coverslips. T hose o f the con -
Studies with Synthetic H ydroxyapatite • A synthetic, low C a /P hydroxyapatite was prepared by chem ical precipitation . 1 Eighteen samples, weighing 50.0 mg. each, were transferred to 1 2 ml. glassstoppered centrifuge tubes. T e n milliliter aliquots o f a 1 M calcium acetate solu tion (p H 7.2) were added to h a lf o f the tubes; 1 0 . 0 ml. o f distilled water was added to each o f the remaining tubes. A ll were shaken for 48 hours at 20° C. and centrifuged. T h e precipitates were washed three times by resuspension and centrifugation in 1 0 . 0 ml. o f distilled 2. Neuman, W . F., and Neuman, M. W . The nature water. A fter drying over calcium chloride, of the mineral phase of bone. Chem. Rev. 53:1 Aug. 1953. the samples were pooled to yield six co m 3. Posner, A. S.; Fabry, C., and Dallemagne, M. J . Defect apatite series in synthetic and natural calcium posites; that is, three control and three phosphates. The concept of pseudoapatites. Biochim. et Biophys. Acta 15:304 Oct. 1954. calcium treated. T hese were analyzed for 4. Posner, A. S. A discussion of a defect apatite calcium 7 and phosphorus . 8 series. Proceedings Int. Union of Pure and Applied Chem., Inorg. Section. Sept. 1954. A ll measurements o f (1 ) radiocalcium 5. Sobel, A. E., and others. Calcification of teeth. uptake and ( 2 ) acid solubility reported II. Comparison of some properties of human and rat teeth. J . D. Res. 28:61 Feb. 1949. here were based on the reaction at 2 0 ° 6. Fabry, C. La fixation de chaux, en milieu aqueux, par les phosphates de calcium synthetiques de structure C. o f 20.0 mg. aliquots o f the six sample apatitique (pseudoapatites). Biochim. et Biophys. Acta 16:377 March 1955. pools with 1 0 . 0 ml. o f the test solution. 7. Tisciall. F. P., and Kramer, B, J . Methods for direct Tw elve milliliter capacity glass-stoppered quantitative determination of sodium, potassium, cal cium and magnesium in urine and stool. J . Biol. Chem. centrifuge tubes served as containers. 48:1 Sept. 1921. 1. O n e aliquot from each sample pool 8. Boltz, D. F., and Mellon, M. G . Determination of phosphorus, germanium, silicon and arsenic by the was shaken for 55 minutes with 8 x 10~ 5 heteropoly blue method. Anal. Chem. 19:873 Nov. 1947.
LI KINS— POSNER—STEERE . . . VO LUM E 57, SEPTEMBER 1958 • 337
Table 1 • Composition, solubility, and radiocalcium uptake of synthetic hydroxyapatite*
Treatment
W a te r Calcium acetate
Caf %
PÎ %
37.1 37.5
18.7 17.8
C a/P weight ratio 1.99 2.11
Total fi g P dissolved in 0.001 N lactic acid§ 168 106
Radio calcium uptake II
1.58 1.47
*Each figure given in this table represents the average of three determinations. fStandard error equals 0.33. ^Standard error equals 0.22. §Standard error equals 7.2. ¡[Figures in this column represent the per cent of total radiocalcium in treating solutions taken up per mg. of enamel. Standard error of each figure equals 0.025.
trol group were immersed fo r 60 hours in 50.0 ml. o f distilled water, while the crowns o f the experimental group were immersed for the same time in 50.0 ml. o f the 1 M calcium acetate solution. A fter treatment, the teeth were rinsed in eight 1 0 0 ml. portions o f distilled wa ter, rem oved from the coverslips, and re mounted. (I t was necessary to remount the crowns since the rinsing process o c casionally dislodged teeth from the cover slips.) All were immersed fo r one hour in 50.0 ml. o f the radiocalcium solution and rinsed in distilled water as before. T he crowns were p ooled in groups o f four, ground to 60 mesh, and the enamel separated from the dentin . 9 T h e enamel samples were ignited for one hour at 550° C., weighed, dissolved in hydrochloric acid and analyzed for radiocalcium . T o determine the effect o f calcium treatment on solubility, the first and second m olar teeth o f eight weanling, Sprague-Dawley rats were sectioned, di vided into tw o groups, and m ounted on coverslips as before. A fter immersion for 18 hours in 50.0 ml. o f either distilled water or calcium acetate, the teeth were rinsed eight times in distilled water and rem oved from the coverslips. T h e first and second molars were m ounted sep arately in groups o f fou r and each group immersed in 25.0 ml. o f 0.005 N lactic acid at room temperature. Stronger acid was needed to get measurable solubilities in the case o f enamel than in the afore
m entioned synthetic apatite study. After 30 minutes an aliquot o f the solution was rem oved by aspiration, filtered, and the filtrate analyzed for phosphorus. R E S U L T S A N D D IS C U S S IO N
T h e experimental results are summarized in T ables 1, 2, and 3. A standard error o f the reported average is listed for most values in the tables (see footnotes in each table). This standard error was calcu lated by pooling the variability among replicate determinations for all sets of replicate samples in the table-column. A lthough this procedure ignores possible differences in precision for the different treatments, it is useful in assessing real differences in solubility and radiocalcium uptake. (A pparent differences in preci sion am ong the different treatments were not significant.) T h e form ula fo r pure hydroxyapatite, C a io (P 0 4 ) 6 ( O H ) 2, gives 39.8 per cent calcium , 18.6 per cent phosphorus, and a ca lciu m : phosphorus weight ratio o f 2.15. C om paring these values with those o f the samples under test (T a b le 1 ), it is evident that the stoichiometry o f the water-treated apatite specimens differs significantly from the theoretical fo r cal cium, but not for phosphorus. These ana9. Manly, R. S., and Hodge, H. C. Density and re fractive index studies of dental hard tissues. I. Methods for separation and determination of purity. J . D. Res. 18:133 April 1939.
338 • THE JO U R N A L OF THE A M ERICAN DENTAL A SSO CIA TIO N
Table 2 • Solubility and radiocalcium uptake of rat molar enamel
Treat ment
W a te r Calcium acetate
M o lar
1st 2nd 1st 2nd
Total ßg P dissolved in 0.005'N lactic acid*
Radio calcium uptakef
24.9 18.1 15.9 11.6
0.142191 0.14118) 0.044(9) 0.05618)
*Each figure in the column represents the average of four determinations. Standard error of each equals 1.5. {Figures in this column represent the per cent of total radiocalcium in treating solution taken up per mg. enamel. Each figure represents an average of the number of determinations shown in parentheses. Stand ard error of each figure equals 0.006.
lytical data, which yield a ca lciu m : phos phorus m olar ratio o f 9 .2 /6 instead o f the theoretical 1 0 / 6 , indicate that the prep aration is a low -calcium (d efect) hydroxyapatite. Treatm ent o f the apatite with calcium acetate resulted in a change in the cal c iu m : phosphorus weight ratio from 1.99 to 2.11. T his increase in ca lciu m : phos phorus apparently is due primarily to a relative decrease in the phosphorus con tent rather than an increase in the cal cium per cent. T h e reason for the lower phosphorus percentage o f the calcium
acetate-treated synthetic apatite is not clear. Since less than 5 micrograms o f the phosphorus were lost from the solid to the treatment-solutions, it is possible to rule out the surface replacement o f phos phate by acetate. Treatm ent o f the synthetic apatite with calcium acetate resulted in a small but significant (p < 0.05) reduction in the subsequent uptake o f radiocalcium (T a ble 1 ) . M ore striking are the findings with enamel (T able 2 ) , in which calcium treatment decreased (p < 0 .0 1 ) radiocal cium uptake by two thirds. T h e extent o f this decrease appeared to be somewhat greater in the case o f the first molars. These results suggest that some perfec tion o f the calcium content o f the apa tite in the samples tested may have been achieved. Treatm ent with calcium acetate co n siderably reduced the acid solubility (measured as phosphorus in solution) o f both the synthetic apatite (T able 1) and the rat m olar enamel (T a b le 2 ) . In each instance the extent o f this decrease was approxim ately 35 per cent. T h e study o f the acid solubility o f syn thetic apatite was extended to include
Table 3 • Acid solubility of synthetic hydroxyapatite
N o.
1
2
3 4 5 6
Sample
Calcium acetate treated Calcium acetate treated W a te r treated W a te r treated Residue of N o. 2 Residue of N o. 4
Hours in 0.001 N lactic acid
ßg Ca per 20 ml.*
ßg P per 20 ml.f
l[C a++] [P total])
C a/P
'/2
535
164
1.78 X 10“ 7
3.27
64
521
159
1.67 X 10“ 7
3.28
'A
565
259
2.96 x 10“ 7
2.18
64
561
250
2.82 x 10~7
2.25
Zi
592
272
3.26 x 10“ 7
2.18
'h
604
289
3.51 x 10-7
2.09
*Supernates of two 10 ml. portions pooled for each analysis of calcium. Each value in this column represents an average of two determinations. Standard error for each figure equals 7. •[Figures in this column represent an average of four determinations, with a standard error for each figure shown equal to 4.
LIKIN S— POSNER—STEERE. . .V O LU M E 57, SEPTEMBER 1958 • 339
the calcium release as well as the phos phorus solubility, in order to obtain the (C a ++) ( P total ) product. T h e methods em ployed were those previously described except that all reactions were carried out at room temperature. A 64 hour solubility determination was included fo r com pari son with the 30 minute value. In agree m ent with the findings o f the previous experiment (T a b le 1 ), treatment o f the apatite with calcium acetate resulted in a 35 per cent decrease in the acid release o f phosphorus (T a b le 3 ) . A lthough cal cium release was also diminished, the extent o f this decrease was only 7 per cent. T h e excess o f calcium over phosphorus in the acid filtrates o f the calcium-treated samples most probably can be explained by a release o f adsorbed calcium. It seems unlikely, however, that this surface-held calcium is present as the acetate, since infrared absorption studies o f calcium acetate-treated defect apatites have failed to reveal the presence o f such a phase . 1 0 T h e results o f a second acid leach o f the 64 hour residues revealed no differ ences in solubility between the control
and calcium acetate-treated apatite sam ples (T a b le 3 ) . It appears, therefore, that the decrease in solubility after treatment with calcium acetate is the result of changes in the apatite crystal surface. T h e findings o f the present study, al though not unequivocal, at least provide further basis for postulating the existence o f defect apatites in enamel mineral. T he presence o f such a phase in enamel may be o f im portance in determining suscepti bility to dental caries. Thus, the increased resistance to caries which accompanies advancing age may be due, in part, to a perfection o f defect enamel apatite. SUM M ARY
Treatm ent in calcium acetate solution reduced the acid solubility and radio calcium uptake o f both rat m olar enamel and a synthetic, defect hydroxyapatite. T h e results suggest that the enamel m in eral m ay contain a defect hydroxyapatite as a m ajor phase.
10. Posner, A. S. Unpublished data.
Diseases in Children • T h e um bilical cord o f dependency is not severed at birth. It continues to unite the child to his parents for many years. In the truest sense the sick child is not a sick individual but it belongs to a sick family. W ithin the family the cause o f disease should be sought and treatment should be given to the family. T h e family, however, is not merely a small self-containing aggregate o f organically interrelated individuals; it is the representative and the product o f a particular form o f social organization and o f strata o f this organization, and it is, therefore, also a product o f a particular culture. Different beliefs fashion different atti tudes, and the attitudes produce behavior patterns. T od a y there is abundant evidence to show beyond all doubt that in the social structure and in the culture are forces active which by their actions produce disease. These forces, however, are still to be determined. A sick child is a human being, a part o f an integrated unit, but an individual with fears, aspirations, goals, despairs and compulsions w hich profou ndly affect the state o f his health, and the line that previously had divided his organic processes from his em otional and social life is now seen to be an artifact. F. A. Crewe.
the housewife, and may in fact explain some o f the differences noted previously between the dental habits o f men and wom en. T hey may also be m ore im portant considerations fo r the wage earner, to w hom , literally, every hour spent in travel, or in waiting and consul tation, costs m oney, than fo r the profes sional worker, w h o often has greater flexibility in working and earning time. A ll these factors can be joined into a whole, from the point o f view o f the patient, by considering the use o f dental services to stem from an over-all assess ment, or definition, o f the situation. T he
prospective patient assesses his dental condition and the seriousness o f the con sequences if he does not seek dental care. This self-diagnosis is weighed or balanced against the factors o f cost, anticipated pain, and inconvenience, to see if going to the dentist “ is worth it.” T h e final result— the use or avoidance o f dental services— is thus a com plex product o f the education involved in self-diagnosis, o f the incom e level involved in weighing costs, o f the dental health and past den tal experience involved in anticipating pain, and o f the social experience in volved in assessing inconvenience.
Effect of calcium treatment on solubility and calcium uptake of synthetic hydroxyapatite and rat molar enamel R . C. Likins,* D .D .S., Bethesda, M d .; A . S. P o s n e r * Ph.D., Washington, D . C., and A . C. Steere,% B.S., Bethesda, M d.
T h e existence o f apatites deficient in d i valent cations is supported by crystallographic evidence . 1 Certain chemicallyprecipitated hydroxyapatites were shown to contain different amounts o f calcium ions missing from structural positions, re sulting in a fam ily o f defect apatites with low calcium -phosphorus ratios. Electrical neutrality was postulated to be obtained by hydrogen bonding between oxygens o f neighboring orthophosphate ions. In this light, the general form ula for apatites may be written: D
M 10-x
2x
(P O ) (O H ) 4 6 2
where D represents the divalent cations (C A ++, Pb++, Sr++, M g ++, and so forth, M
the m onovalent cations (N a +, H +, and so forth ) and x the number o f divalent ca tions missing from structural positions. T h e value o f x has been reported to range from 0 to 2 . 1 W hen x prevails as zero, the stoichiometry o f perfect apatite o b tains. It is quite possible that bone and tooth minéral, which d o not have the ideal calcium -phosphorus ratio o f hydroxyapa-
•National institute of Dental Research, National In stitutes of Health, Public Health Service, U. S. De partment of Health, Education, and Welfare, Bethesda 14, Md. ^Research Associate, American Dental Association Research Division, National Bureau of Standards, Wash ington 25, D. C. I. Posner, A . S., and Perloff, A. Apatites deficient in divalent cations. J . of Research, Natl. Bur. of Stds. 58:279 May 1957.
336 • THE JO U R N A L OF THE A M ER IC A N DENTAL ASSOCIATION
tite, 2 may have calcium defect hydroxyapatite as a m ajor constituent.3’ 4 Syn thetic hydroxyapatites with low calcium phosphorus ratios are thermally unstable when com pared to perfect apatites . 5 It is also possible that the solubility o f apa tites may be related to the calcium con tent, particularly as the content ap proaches the theoretical value. Other experiments have shown that the calcium phosphorus ratios o f synthetic, low -calcium apatites can increase in calcium hydroxide solution . 6 T h e purpose o f the present study was to investigate the effect o f calcium ion treatment on the acid solubility o f synthetic, low -calcium apa tite and rat m olar enamel, and the capac ity o f these materials to rem ove calcium ions from solution. E X P E R IM E N T S
m olar calcium 4 0 , 4 5 chloride ( 1 m icro curie per milliliter) adjusted to p H 6.5 with N H 4 O H . T h e suspensions were cen trifuged for five minutes, the supernates rem oved by aspiration, and the precipi tates washed three times in 1 0 . 0 ml. p or tions o f distilled water. T h e samples were dried, weighed, dissolved in hydrochloric acid, and analyzed fo r radiocalcium . 2. O n e aliquot from each sample pool was shaken for 25 minutes with 0.001 N lactic acid, and centrifuged fo r five m in utes. T h e supernates were rem oved by aspiration, filtered through sintered glass (nom inal m axim um pore size, 0.9-1.4 m icro n s), and the filtrates analyzed for phosphorus. Studies with R at M ola r T eeth • T h e m o lar crowns o f 16 weanling male SpragueDawley rats were obtained as follow s: A t sacrifice the jaws were excised, freed o f adherent tissue, and the clinical crowns cleaned with a soft brush and distilled water. Using a % inch circular steel saw in a dental handpiece, the teeth were sectioned transversely in situ approxi mately 0 . 1 mm . above the gingival at tachment. A fter sectioning, the first and second molars o f each rat were divided equally into tw o groups. T h e crowns were blotted and sealed, cut surface dow n, on paraffincoated glass coverslips. T hose o f the con -
Studies with Synthetic H ydroxyapatite • A synthetic, low C a /P hydroxyapatite was prepared by chem ical precipitation . 1 Eighteen samples, weighing 50.0 mg. each, were transferred to 1 2 ml. glassstoppered centrifuge tubes. T e n milliliter aliquots o f a 1 M calcium acetate solu tion (p H 7.2) were added to h a lf o f the tubes; 1 0 . 0 ml. o f distilled water was added to each o f the remaining tubes. A ll were shaken for 48 hours at 20° C. and centrifuged. T h e precipitates were washed three times by resuspension and centrifugation in 1 0 . 0 ml. o f distilled 2. Neuman, W . F., and Neuman, M. W . The nature water. A fter drying over calcium chloride, of the mineral phase of bone. Chem. Rev. 53:1 Aug. 1953. the samples were pooled to yield six co m 3. Posner, A. S.; Fabry, C., and Dallemagne, M. J . Defect apatite series in synthetic and natural calcium posites; that is, three control and three phosphates. The concept of pseudoapatites. Biochim. et Biophys. Acta 15:304 Oct. 1954. calcium treated. T hese were analyzed for 4. Posner, A. S. A discussion of a defect apatite calcium 7 and phosphorus . 8 series. Proceedings Int. Union of Pure and Applied Chem., Inorg. Section. Sept. 1954. A ll measurements o f (1 ) radiocalcium 5. Sobel, A. E., and others. Calcification of teeth. uptake and ( 2 ) acid solubility reported II. Comparison of some properties of human and rat teeth. J . D. Res. 28:61 Feb. 1949. here were based on the reaction at 2 0 ° 6. Fabry, C. La fixation de chaux, en milieu aqueux, par les phosphates de calcium synthetiques de structure C. o f 20.0 mg. aliquots o f the six sample apatitique (pseudoapatites). Biochim. et Biophys. Acta 16:377 March 1955. pools with 1 0 . 0 ml. o f the test solution. 7. Tisciall. F. P., and Kramer, B, J . Methods for direct Tw elve milliliter capacity glass-stoppered quantitative determination of sodium, potassium, cal cium and magnesium in urine and stool. J . Biol. Chem. centrifuge tubes served as containers. 48:1 Sept. 1921. 1. O n e aliquot from each sample pool 8. Boltz, D. F., and Mellon, M. G . Determination of phosphorus, germanium, silicon and arsenic by the was shaken for 55 minutes with 8 x 10~ 5 heteropoly blue method. Anal. Chem. 19:873 Nov. 1947.
LI KINS— POSNER—STEERE . . . VO LUM E 57, SEPTEMBER 1958 • 337
Table 1 • Composition, solubility, and radiocalcium uptake of synthetic hydroxyapatite*
Treatment
W a te r Calcium acetate
Caf %
PÎ %
37.1 37.5
18.7 17.8
C a/P weight ratio 1.99 2.11
Total fi g P dissolved in 0.001 N lactic acid§ 168 106
Radio calcium uptake II
1.58 1.47
*Each figure given in this table represents the average of three determinations. fStandard error equals 0.33. ^Standard error equals 0.22. §Standard error equals 7.2. ¡[Figures in this column represent the per cent of total radiocalcium in treating solutions taken up per mg. of enamel. Standard error of each figure equals 0.025.
trol group were immersed fo r 60 hours in 50.0 ml. o f distilled water, while the crowns o f the experimental group were immersed for the same time in 50.0 ml. o f the 1 M calcium acetate solution. A fter treatment, the teeth were rinsed in eight 1 0 0 ml. portions o f distilled wa ter, rem oved from the coverslips, and re mounted. (I t was necessary to remount the crowns since the rinsing process o c casionally dislodged teeth from the cover slips.) All were immersed fo r one hour in 50.0 ml. o f the radiocalcium solution and rinsed in distilled water as before. T he crowns were p ooled in groups o f four, ground to 60 mesh, and the enamel separated from the dentin . 9 T h e enamel samples were ignited for one hour at 550° C., weighed, dissolved in hydrochloric acid and analyzed for radiocalcium . T o determine the effect o f calcium treatment on solubility, the first and second m olar teeth o f eight weanling, Sprague-Dawley rats were sectioned, di vided into tw o groups, and m ounted on coverslips as before. A fter immersion for 18 hours in 50.0 ml. o f either distilled water or calcium acetate, the teeth were rinsed eight times in distilled water and rem oved from the coverslips. T h e first and second molars were m ounted sep arately in groups o f fou r and each group immersed in 25.0 ml. o f 0.005 N lactic acid at room temperature. Stronger acid was needed to get measurable solubilities in the case o f enamel than in the afore
m entioned synthetic apatite study. After 30 minutes an aliquot o f the solution was rem oved by aspiration, filtered, and the filtrate analyzed for phosphorus. R E S U L T S A N D D IS C U S S IO N
T h e experimental results are summarized in T ables 1, 2, and 3. A standard error o f the reported average is listed for most values in the tables (see footnotes in each table). This standard error was calcu lated by pooling the variability among replicate determinations for all sets of replicate samples in the table-column. A lthough this procedure ignores possible differences in precision for the different treatments, it is useful in assessing real differences in solubility and radiocalcium uptake. (A pparent differences in preci sion am ong the different treatments were not significant.) T h e form ula fo r pure hydroxyapatite, C a io (P 0 4 ) 6 ( O H ) 2, gives 39.8 per cent calcium , 18.6 per cent phosphorus, and a ca lciu m : phosphorus weight ratio o f 2.15. C om paring these values with those o f the samples under test (T a b le 1 ), it is evident that the stoichiometry o f the water-treated apatite specimens differs significantly from the theoretical fo r cal cium, but not for phosphorus. These ana9. Manly, R. S., and Hodge, H. C. Density and re fractive index studies of dental hard tissues. I. Methods for separation and determination of purity. J . D. Res. 18:133 April 1939.
338 • THE JO U R N A L OF THE A M ERICAN DENTAL A SSO CIA TIO N
Table 2 • Solubility and radiocalcium uptake of rat molar enamel
Treat ment
W a te r Calcium acetate
M o lar
1st 2nd 1st 2nd
Total ßg P dissolved in 0.005'N lactic acid*
Radio calcium uptakef
24.9 18.1 15.9 11.6
0.142191 0.14118) 0.044(9) 0.05618)
*Each figure in the column represents the average of four determinations. Standard error of each equals 1.5. {Figures in this column represent the per cent of total radiocalcium in treating solution taken up per mg. enamel. Each figure represents an average of the number of determinations shown in parentheses. Stand ard error of each figure equals 0.006.
lytical data, which yield a ca lciu m : phos phorus m olar ratio o f 9 .2 /6 instead o f the theoretical 1 0 / 6 , indicate that the prep aration is a low -calcium (d efect) hydroxyapatite. Treatm ent o f the apatite with calcium acetate resulted in a change in the cal c iu m : phosphorus weight ratio from 1.99 to 2.11. T his increase in ca lciu m : phos phorus apparently is due primarily to a relative decrease in the phosphorus con tent rather than an increase in the cal cium per cent. T h e reason for the lower phosphorus percentage o f the calcium
acetate-treated synthetic apatite is not clear. Since less than 5 micrograms o f the phosphorus were lost from the solid to the treatment-solutions, it is possible to rule out the surface replacement o f phos phate by acetate. Treatm ent o f the synthetic apatite with calcium acetate resulted in a small but significant (p < 0.05) reduction in the subsequent uptake o f radiocalcium (T a ble 1 ) . M ore striking are the findings with enamel (T able 2 ) , in which calcium treatment decreased (p < 0 .0 1 ) radiocal cium uptake by two thirds. T h e extent o f this decrease appeared to be somewhat greater in the case o f the first molars. These results suggest that some perfec tion o f the calcium content o f the apa tite in the samples tested may have been achieved. Treatm ent with calcium acetate co n siderably reduced the acid solubility (measured as phosphorus in solution) o f both the synthetic apatite (T able 1) and the rat m olar enamel (T a b le 2 ) . In each instance the extent o f this decrease was approxim ately 35 per cent. T h e study o f the acid solubility o f syn thetic apatite was extended to include
Table 3 • Acid solubility of synthetic hydroxyapatite
N o.
1
2
3 4 5 6
Sample
Calcium acetate treated Calcium acetate treated W a te r treated W a te r treated Residue of N o. 2 Residue of N o. 4
Hours in 0.001 N lactic acid
ßg Ca per 20 ml.*
ßg P per 20 ml.f
l[C a++] [P total])
C a/P
'/2
535
164
1.78 X 10“ 7
3.27
64
521
159
1.67 X 10“ 7
3.28
'A
565
259
2.96 x 10“ 7
2.18
64
561
250
2.82 x 10~7
2.25
Zi
592
272
3.26 x 10“ 7
2.18
'h
604
289
3.51 x 10-7
2.09
*Supernates of two 10 ml. portions pooled for each analysis of calcium. Each value in this column represents an average of two determinations. Standard error for each figure equals 7. •[Figures in this column represent an average of four determinations, with a standard error for each figure shown equal to 4.
LIKIN S— POSNER—STEERE. . .V O LU M E 57, SEPTEMBER 1958 • 339
the calcium release as well as the phos phorus solubility, in order to obtain the (C a ++) ( P total ) product. T h e methods em ployed were those previously described except that all reactions were carried out at room temperature. A 64 hour solubility determination was included fo r com pari son with the 30 minute value. In agree m ent with the findings o f the previous experiment (T a b le 1 ), treatment o f the apatite with calcium acetate resulted in a 35 per cent decrease in the acid release o f phosphorus (T a b le 3 ) . A lthough cal cium release was also diminished, the extent o f this decrease was only 7 per cent. T h e excess o f calcium over phosphorus in the acid filtrates o f the calcium-treated samples most probably can be explained by a release o f adsorbed calcium. It seems unlikely, however, that this surface-held calcium is present as the acetate, since infrared absorption studies o f calcium acetate-treated defect apatites have failed to reveal the presence o f such a phase . 1 0 T h e results o f a second acid leach o f the 64 hour residues revealed no differ ences in solubility between the control
and calcium acetate-treated apatite sam ples (T a b le 3 ) . It appears, therefore, that the decrease in solubility after treatment with calcium acetate is the result of changes in the apatite crystal surface. T h e findings o f the present study, al though not unequivocal, at least provide further basis for postulating the existence o f defect apatites in enamel mineral. T he presence o f such a phase in enamel may be o f im portance in determining suscepti bility to dental caries. Thus, the increased resistance to caries which accompanies advancing age may be due, in part, to a perfection o f defect enamel apatite. SUM M ARY
Treatm ent in calcium acetate solution reduced the acid solubility and radio calcium uptake o f both rat m olar enamel and a synthetic, defect hydroxyapatite. T h e results suggest that the enamel m in eral m ay contain a defect hydroxyapatite as a m ajor phase.
10. Posner, A. S. Unpublished data.
Diseases in Children • T h e um bilical cord o f dependency is not severed at birth. It continues to unite the child to his parents for many years. In the truest sense the sick child is not a sick individual but it belongs to a sick family. W ithin the family the cause o f disease should be sought and treatment should be given to the family. T h e family, however, is not merely a small self-containing aggregate o f organically interrelated individuals; it is the representative and the product o f a particular form o f social organization and o f strata o f this organization, and it is, therefore, also a product o f a particular culture. Different beliefs fashion different atti tudes, and the attitudes produce behavior patterns. T od a y there is abundant evidence to show beyond all doubt that in the social structure and in the culture are forces active which by their actions produce disease. These forces, however, are still to be determined. A sick child is a human being, a part o f an integrated unit, but an individual with fears, aspirations, goals, despairs and compulsions w hich profou ndly affect the state o f his health, and the line that previously had divided his organic processes from his em otional and social life is now seen to be an artifact. F. A. Crewe.