Journal of Dental Research http://jdr.sagepub.com/
Modification by Salivary Pellicles of in vitro Enamel Remineralization R.T. Zahradnik J DENT RES 1979 58: 2066 DOI: 10.1177/00220345790580110501 The online version of this article can be found at: http://jdr.sagepub.com/content/58/11/2066
Published by: http://www.sagepublications.com
On behalf of: International and American Associations for Dental Research
Additional services and information for Journal of Dental Research can be found at: Email Alerts: http://jdr.sagepub.com/cgi/alerts Subscriptions: http://jdr.sagepub.com/subscriptions Reprints: http://www.sagepub.com/journalsReprints.nav Permissions: http://www.sagepub.com/journalsPermissions.nav Citations: http://jdr.sagepub.com/content/58/11/2066.refs.html
>> Version of Record - Nov 1, 1979 What is This?
Downloaded from jdr.sagepub.com at Universitats-Landesbibliothek on November 12, 2013 For personal use only. No other uses without permission.
Modification by Salivary Pellicles of in vitro Enamel Remineralization R. T. ZAHRADNIK Forsyth Dental Center, 140 Fen way, Boston, Massachusetts 02115 The effectiveness of inorganic calcifying solutions to remineralize enamel with subsurface demineralization decreased with saliva pre-treatments conducive to the formation of enamel pellicles. Greater reductions in remineralization rates occurred with longer pellicle formation times. It is suggested that enamel pellicles may act to control surface deposition and favor subsurface precipitation.
J Dent Res 58(11):2066-2073, November 1979
Introduction. Partial reconstitution of destroyed enamel tissue has been known to occur both in natural1 ,2 and in experimentally-induced caries in man3 under favorable conditions in the oral environment. Human saliva is capable, therefore, of effecting a regression in carious lesions. Several in vitro studies,4'5 however, have demonstrated that inorganic calcifying solutions are more efficient in depositing mineral in acid-softened enamel than are the salivary secretions; nevertheless, under clinical conditions, practical problems develop with these synthetic solutions (e.g., short shelf life, need for long contact times, and a tendency for surface deposition). For these reasons, a more viable approach to caries reversal may result from the study of the interactions of the inorganic or organic fractions of saliva with tooth enamel; this approach could lead to the development of clinical methods capable of enhancing saliva's natural remineralizing potential. Received for publication January 25, 1979. Accepted for publication April 4, 1979. This investigation was supported by U.S.P.H.S. Grant DE-03187 from the National Institute of Dental Research, National Institutes of Health,
Bethesda, MD. 2066
Several investigators6'7 have attempted to define the conditions which would favor calcium phosphate precipitation by using model systems involving metastable solutions and hydroxyapatite seed materials. However, results of these studies and those found from in vitro enamel tissue remineralization experiments do not correspond completely. The principal problem in dental enamel remineralization is to achieve significant mineral penetration across the length of the subsurface lesion without appreciable surface precipitation. Hard tissue reconstitution is further complicated by the presence of organic macromolecules in saliva. Certain organic salivary components have a significant influence on spontaneous precipitation by highly metastable calcium phosphate-containing solutions.8 9 Furthernjore, several of these salivary components have been shown to have a high affinity for apatite surfaces,1 O,l 1 and separate studies12 have shown that ionic transport is reduced across pellicles of salivary origin formed on apatitic surfaces. Involvement of these active salivary constituents in the formation of natural tooth pellicles could result in a modification of the remineralization rates by saliva and inorganic calcifying solutions. The purpose of this study was to evaluate the effects of salivary pellicles upon in vitro enamel remineralization through the use of a model system incorporating the following features: 1) production of caries-like subsurface demineralization in extracted human teeth; 2) the use of a well-characterized, inorganic calcifying solution; and 3) the quantitative assessment of changes in remineralization rates brought about by saliva pretreatment.
Materials and methods. Preparation of calcifying solutions. -The
Downloaded from jdr.sagepub.com at Universitats-Landesbibliothek on November 12, 2013 For personal use only. No other uses without permission.
Vol. S8 No. II
SALI VAR Y PELLICLES AND ENAMEL REMINERALIZA TION
initial study involved a characterization of the inorganic remineralization system, with respect to in vitro enamel reconstitution, so that a reference point could be established for the saliva studies. Inorganic calcifying solutions were prepared from stock solutions of calcium chloride, CaC12, and potassium dihydrogen phosphate, KH2PO4. The water used was distilled, and no attempt was made to keep the system C02-free. The composition of the solutions used in this study and their degrees of supersaturation, DS, at 370C with respect to hydroxyapatite (Ca5OH(PO4)3, HA), octacalcium phosphate (Ca4 H(PO4) 3 *2 1/2 H2 0, and f3-tricalcium OCP), phosphate (3-Ca3(PO4)2, TCP), are given in Table 1. Solutions 5 and 6 were chosen to correspond with Solutions IA and IIA used in a previously-reported study on seeded crystal growth. The procedure used to calculate DS has been outlined in a previous publication ;7 values for D S smaller than unity indicate undersaturation. The calcium-phosphate molar ratio, Ca/P, was always 1.67, and the pH of the solution was adjusted to 7.40 using concentrated potassium hydroxide. It has been suggested13 that the addition of an electrolyte, such as sodium chloride, merely prevents spontaneous precipitation of calcifying solutions and is without effect on the enamel remineralization process. To test this possibility, Solution 1, containing 0.15 M NaCl, was included in this study. All six solutions described in Table 1 were under-saturated with respect to dicalcium phosphate dyhydrate, DCPD, and the anhydrous form of the salt. The solutions were stable to spontaneous precipitation for at least one week when stored at room temperature. Nevertheless, the solutions were prepared fresh every four days throughout a remineralization run. Experimental procedures.-Only clinically-sound, human premolar teeth (extracted for orthodontic reasons) were used in this study; a total of 63 teeth were cleaned, pumiced, and then stored in a moist environment at 40C until use. When selected for use, the teeth were first covered with blue inlay wax except for a 4 mm x 4 mm window on the buccal surface, and then were exposed to a 0.1 M lactate buffer 14 containing calcium and phosphorus at levels of 1.1 x 10-2 M and 6.6 x 10-3 M,
2067
respectively. The final pH of this buffer was 4.23. Three days were adequate for producing caries-like subsurface demineralization patterns extending to over 100 ,m below the enamel surface and constituting about 35-40% total mineral loss. One-half of the demineralized buccal window area was waxed and served as the control; the contralateral half of the window was exposed to one of the calcifying solutions (25 ml/tooth). Thirty-six teeth were divided into six groups, and each group was treated with one of the solutions listed in Table 1. In addition, a total of twelve teeth were used to study the effect of 1 ppm of fluoride upon remineralization, and fifteen teeth were used in the saliva pretreatment study. In this last study, the teeth were introduced into stimulated parotid saliva (5 ml/tooth). Parotid saliva was used since it constitutes a better characterized system than whole saliva and because previous results, relating to salivary pellicle development time and enamel demineralization, 12,5 show that glandular and whole saliva appear to act in a similar fashion. A device comparable to that described by Curby16 was used to collect the saliva from the parotid glands. After discarding the first few ml of saliva, fresh secretion was collected and used immediately. The salivary pellicle was built up as in previous studies,12,17 by exposing the teeth to the saliva for either one day or seven days. In the latter case, there were daily changes of the saliva; continuous stirring was used during this pellicle development process. Three of the teeth with 7-day saliva exposure were used to assess the remineralizing potential of the saliva alone under our experimental conditions, while the remaining teeth were exposed to Solution 2 for 10 days at 370C to assess the effect of saliva pretreatments on the experimental rate of mineral deposition by an inorganic calcifying solution. Quantitative microradiographyl 7 was used to evaluate the enamel modifications (over the length of the artificial lesions) brought about by the previously described remineralizing treatment plans. Teeth were sectioned perpendicular to the window and in a mesialdistal direction so as to include both halves of the exposed window region. The thin sections (100 ,um) were placed against a high resolution photographic film and ex-
Downloaded from jdr.sagepub.com at Universitats-Landesbibliothek on November 12, 2013 For personal use only. No other uses without permission.
2068
ZAHRADNIK
J Dent Res November 19 79
TABLE 1 COMPOSITION OF THE INORGANIC CALCIFYING SOLUTIONS AND THEIR DEGREES OF SUPERSATURATION WITH RESPECT TO HA, TCP, OCP. Solution
1* 2 3 4 5 6
Calcium Mx 103
Phosphorus Mx 103
HA
1.50 1.00 0.90 0.80 0.655 0.443
0.90 0.60 0.54 0.48 0.392 0.265
1.03x 1011 1.05 x 1012 5.41.x 1011 2.52 x 1011 6.68 x 1010 4.81 x 109
Degree of Supersaturation TCP 4.52x 1.40 x 5.18x 1.65 x 2.30 x 73
'
105 104 104 103
OCP 9 66 37 19 6 0.6
* 0.15 M NaCl added to Solution 1.
**All solutions adjusted to pH 7.4.
posed to an X-ray source. The resultant microradiographs were scanned with an optical microdensitometer (effective slit of 5 ,um x 100 pm) through each window half; representative microradiographs have been presented previously. 14,17 The demineralization system consistently generated a reasonably parallel demineralization front. Nevertheless, three separate areas in each window half were scanned to evaluate the variability over the length of the exposed area. The recorded scans of optical density vs. distance from the enamel surface were used to generate values for the following four parameters characterizing the mineral content of the enamel section: 17 1) depth of the artificial lesion; 2) percent mineral loss in the body of the lesion; 3) thickness of the apparently "intact" surface layer; and 4) mineral content in this surface layer.
Results. Table 2 contains mineralization results for enamel that had been exposed to Solution 2 for a period of 14 days. The first column in this table lists the mean and standard deviation (control group, N = 6) for each of the four mineralization parameters. Column 2 contains the mean values for the corresponding group of test halves exposed to Solution 2. In the last column the percent differences between the control and the test mean values are given; a negative sign indicates that the value for that particular parameter decreased upon treatment with Solution 2. Trends toward a reduced lesion depth and an increased mineral content in the surface layer are discernible. Moreover, statistically-significant
(P < 0.005) changes are observed in the mineral content of the lesion body and in the thickness of the "intact" surface layer. Based on the remineralization results for the six solutions, the percent change in the level of mineralization in the artificial lesion body was selected to reflect the effectiveness of the different calcifying solutions. A plot of this parameter for the six solutions tested against the total calcium concentration in these solutions is given in Figure 1. Except for Solutions 1 and 6, a direct relationship is suggested. Solution 6 modified the mineral content of the artificial lesion only to an extent that was not significantly different from the control mean value. Solution 1 differed from the other solutions in that it contained 0.15 M NaC 1. Surface precipitation was not detected on any of the teeth exposed to the six calcifying solutions. This was checked by microscopic observation of the surfaces of the thin tooth sections prepared for microradiography. All of the remineralization results reported in this study were obtained from experiments conducted under quiescent conditions (without stirring of the calcifying solutions). This choice was based on a separate study which demonstrated that stirring produced a non-significant (
Modification by Salivary Pellicles of in vitro Enamel Remineralization R.T. Zahradnik J DENT RES 1979 58: 2066 DOI: 10.1177/00220345790580110501 The online version of this article can be found at: http://jdr.sagepub.com/content/58/11/2066
Published by: http://www.sagepublications.com
On behalf of: International and American Associations for Dental Research
Additional services and information for Journal of Dental Research can be found at: Email Alerts: http://jdr.sagepub.com/cgi/alerts Subscriptions: http://jdr.sagepub.com/subscriptions Reprints: http://www.sagepub.com/journalsReprints.nav Permissions: http://www.sagepub.com/journalsPermissions.nav Citations: http://jdr.sagepub.com/content/58/11/2066.refs.html
>> Version of Record - Nov 1, 1979 What is This?
Downloaded from jdr.sagepub.com at Universitats-Landesbibliothek on November 12, 2013 For personal use only. No other uses without permission.
Modification by Salivary Pellicles of in vitro Enamel Remineralization R. T. ZAHRADNIK Forsyth Dental Center, 140 Fen way, Boston, Massachusetts 02115 The effectiveness of inorganic calcifying solutions to remineralize enamel with subsurface demineralization decreased with saliva pre-treatments conducive to the formation of enamel pellicles. Greater reductions in remineralization rates occurred with longer pellicle formation times. It is suggested that enamel pellicles may act to control surface deposition and favor subsurface precipitation.
J Dent Res 58(11):2066-2073, November 1979
Introduction. Partial reconstitution of destroyed enamel tissue has been known to occur both in natural1 ,2 and in experimentally-induced caries in man3 under favorable conditions in the oral environment. Human saliva is capable, therefore, of effecting a regression in carious lesions. Several in vitro studies,4'5 however, have demonstrated that inorganic calcifying solutions are more efficient in depositing mineral in acid-softened enamel than are the salivary secretions; nevertheless, under clinical conditions, practical problems develop with these synthetic solutions (e.g., short shelf life, need for long contact times, and a tendency for surface deposition). For these reasons, a more viable approach to caries reversal may result from the study of the interactions of the inorganic or organic fractions of saliva with tooth enamel; this approach could lead to the development of clinical methods capable of enhancing saliva's natural remineralizing potential. Received for publication January 25, 1979. Accepted for publication April 4, 1979. This investigation was supported by U.S.P.H.S. Grant DE-03187 from the National Institute of Dental Research, National Institutes of Health,
Bethesda, MD. 2066
Several investigators6'7 have attempted to define the conditions which would favor calcium phosphate precipitation by using model systems involving metastable solutions and hydroxyapatite seed materials. However, results of these studies and those found from in vitro enamel tissue remineralization experiments do not correspond completely. The principal problem in dental enamel remineralization is to achieve significant mineral penetration across the length of the subsurface lesion without appreciable surface precipitation. Hard tissue reconstitution is further complicated by the presence of organic macromolecules in saliva. Certain organic salivary components have a significant influence on spontaneous precipitation by highly metastable calcium phosphate-containing solutions.8 9 Furthernjore, several of these salivary components have been shown to have a high affinity for apatite surfaces,1 O,l 1 and separate studies12 have shown that ionic transport is reduced across pellicles of salivary origin formed on apatitic surfaces. Involvement of these active salivary constituents in the formation of natural tooth pellicles could result in a modification of the remineralization rates by saliva and inorganic calcifying solutions. The purpose of this study was to evaluate the effects of salivary pellicles upon in vitro enamel remineralization through the use of a model system incorporating the following features: 1) production of caries-like subsurface demineralization in extracted human teeth; 2) the use of a well-characterized, inorganic calcifying solution; and 3) the quantitative assessment of changes in remineralization rates brought about by saliva pretreatment.
Materials and methods. Preparation of calcifying solutions. -The
Downloaded from jdr.sagepub.com at Universitats-Landesbibliothek on November 12, 2013 For personal use only. No other uses without permission.
Vol. S8 No. II
SALI VAR Y PELLICLES AND ENAMEL REMINERALIZA TION
initial study involved a characterization of the inorganic remineralization system, with respect to in vitro enamel reconstitution, so that a reference point could be established for the saliva studies. Inorganic calcifying solutions were prepared from stock solutions of calcium chloride, CaC12, and potassium dihydrogen phosphate, KH2PO4. The water used was distilled, and no attempt was made to keep the system C02-free. The composition of the solutions used in this study and their degrees of supersaturation, DS, at 370C with respect to hydroxyapatite (Ca5OH(PO4)3, HA), octacalcium phosphate (Ca4 H(PO4) 3 *2 1/2 H2 0, and f3-tricalcium OCP), phosphate (3-Ca3(PO4)2, TCP), are given in Table 1. Solutions 5 and 6 were chosen to correspond with Solutions IA and IIA used in a previously-reported study on seeded crystal growth. The procedure used to calculate DS has been outlined in a previous publication ;7 values for D S smaller than unity indicate undersaturation. The calcium-phosphate molar ratio, Ca/P, was always 1.67, and the pH of the solution was adjusted to 7.40 using concentrated potassium hydroxide. It has been suggested13 that the addition of an electrolyte, such as sodium chloride, merely prevents spontaneous precipitation of calcifying solutions and is without effect on the enamel remineralization process. To test this possibility, Solution 1, containing 0.15 M NaCl, was included in this study. All six solutions described in Table 1 were under-saturated with respect to dicalcium phosphate dyhydrate, DCPD, and the anhydrous form of the salt. The solutions were stable to spontaneous precipitation for at least one week when stored at room temperature. Nevertheless, the solutions were prepared fresh every four days throughout a remineralization run. Experimental procedures.-Only clinically-sound, human premolar teeth (extracted for orthodontic reasons) were used in this study; a total of 63 teeth were cleaned, pumiced, and then stored in a moist environment at 40C until use. When selected for use, the teeth were first covered with blue inlay wax except for a 4 mm x 4 mm window on the buccal surface, and then were exposed to a 0.1 M lactate buffer 14 containing calcium and phosphorus at levels of 1.1 x 10-2 M and 6.6 x 10-3 M,
2067
respectively. The final pH of this buffer was 4.23. Three days were adequate for producing caries-like subsurface demineralization patterns extending to over 100 ,m below the enamel surface and constituting about 35-40% total mineral loss. One-half of the demineralized buccal window area was waxed and served as the control; the contralateral half of the window was exposed to one of the calcifying solutions (25 ml/tooth). Thirty-six teeth were divided into six groups, and each group was treated with one of the solutions listed in Table 1. In addition, a total of twelve teeth were used to study the effect of 1 ppm of fluoride upon remineralization, and fifteen teeth were used in the saliva pretreatment study. In this last study, the teeth were introduced into stimulated parotid saliva (5 ml/tooth). Parotid saliva was used since it constitutes a better characterized system than whole saliva and because previous results, relating to salivary pellicle development time and enamel demineralization, 12,5 show that glandular and whole saliva appear to act in a similar fashion. A device comparable to that described by Curby16 was used to collect the saliva from the parotid glands. After discarding the first few ml of saliva, fresh secretion was collected and used immediately. The salivary pellicle was built up as in previous studies,12,17 by exposing the teeth to the saliva for either one day or seven days. In the latter case, there were daily changes of the saliva; continuous stirring was used during this pellicle development process. Three of the teeth with 7-day saliva exposure were used to assess the remineralizing potential of the saliva alone under our experimental conditions, while the remaining teeth were exposed to Solution 2 for 10 days at 370C to assess the effect of saliva pretreatments on the experimental rate of mineral deposition by an inorganic calcifying solution. Quantitative microradiographyl 7 was used to evaluate the enamel modifications (over the length of the artificial lesions) brought about by the previously described remineralizing treatment plans. Teeth were sectioned perpendicular to the window and in a mesialdistal direction so as to include both halves of the exposed window region. The thin sections (100 ,um) were placed against a high resolution photographic film and ex-
Downloaded from jdr.sagepub.com at Universitats-Landesbibliothek on November 12, 2013 For personal use only. No other uses without permission.
2068
ZAHRADNIK
J Dent Res November 19 79
TABLE 1 COMPOSITION OF THE INORGANIC CALCIFYING SOLUTIONS AND THEIR DEGREES OF SUPERSATURATION WITH RESPECT TO HA, TCP, OCP. Solution
1* 2 3 4 5 6
Calcium Mx 103
Phosphorus Mx 103
HA
1.50 1.00 0.90 0.80 0.655 0.443
0.90 0.60 0.54 0.48 0.392 0.265
1.03x 1011 1.05 x 1012 5.41.x 1011 2.52 x 1011 6.68 x 1010 4.81 x 109
Degree of Supersaturation TCP 4.52x 1.40 x 5.18x 1.65 x 2.30 x 73
'
105 104 104 103
OCP 9 66 37 19 6 0.6
* 0.15 M NaCl added to Solution 1.
**All solutions adjusted to pH 7.4.
posed to an X-ray source. The resultant microradiographs were scanned with an optical microdensitometer (effective slit of 5 ,um x 100 pm) through each window half; representative microradiographs have been presented previously. 14,17 The demineralization system consistently generated a reasonably parallel demineralization front. Nevertheless, three separate areas in each window half were scanned to evaluate the variability over the length of the exposed area. The recorded scans of optical density vs. distance from the enamel surface were used to generate values for the following four parameters characterizing the mineral content of the enamel section: 17 1) depth of the artificial lesion; 2) percent mineral loss in the body of the lesion; 3) thickness of the apparently "intact" surface layer; and 4) mineral content in this surface layer.
Results. Table 2 contains mineralization results for enamel that had been exposed to Solution 2 for a period of 14 days. The first column in this table lists the mean and standard deviation (control group, N = 6) for each of the four mineralization parameters. Column 2 contains the mean values for the corresponding group of test halves exposed to Solution 2. In the last column the percent differences between the control and the test mean values are given; a negative sign indicates that the value for that particular parameter decreased upon treatment with Solution 2. Trends toward a reduced lesion depth and an increased mineral content in the surface layer are discernible. Moreover, statistically-significant
(P < 0.005) changes are observed in the mineral content of the lesion body and in the thickness of the "intact" surface layer. Based on the remineralization results for the six solutions, the percent change in the level of mineralization in the artificial lesion body was selected to reflect the effectiveness of the different calcifying solutions. A plot of this parameter for the six solutions tested against the total calcium concentration in these solutions is given in Figure 1. Except for Solutions 1 and 6, a direct relationship is suggested. Solution 6 modified the mineral content of the artificial lesion only to an extent that was not significantly different from the control mean value. Solution 1 differed from the other solutions in that it contained 0.15 M NaC 1. Surface precipitation was not detected on any of the teeth exposed to the six calcifying solutions. This was checked by microscopic observation of the surfaces of the thin tooth sections prepared for microradiography. All of the remineralization results reported in this study were obtained from experiments conducted under quiescent conditions (without stirring of the calcifying solutions). This choice was based on a separate study which demonstrated that stirring produced a non-significant (
