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“The isolectric point of human dental tissues”
“THE
ISOELECTRIC
POINT
H.C. PARREIRA Johnson & Johnson (Peceived
OF HWAH
Research,
Rarch 16th,
DENTAL
TISSUES”
New Brunsnick,
1979; accepted
in fipnl
N-3.
forra October
08903 10th.
(U.S.A.) 1980)
ABSTRACT The isoelectric by the streaming of dental enamel 10’+l KC1 .
point potential nas 3.9
of
human dental tissues was measured technique. The isoelectric point in 10’3H.KCl and 4.0 for dentin in
fNTROBUCTfON The surface electrochemistry of human dental enamel and dentin Is of extreme importance if one is to understand better the comp?ex phenomena involved in plaque formation and other It was concluded by problems of odontolo ical interest. tfeuman and Neuman (1 3 that the electrical potential barrier is the most important variable that affects the rate 0’ exchange betneen calcium and phosphate ions and hydro-yapapteach (2) recognized that the calcium/phospha?e ratio tite. is related to the electric charge of the hydroxyapatite #ore recently, it has been suggested that the zeta surface. potential is an fzepcrtant factor in controlling t%e adhesion It is genl-rally accepted of bacteria to hydrox:rapatite (3.4). that the dental enamer is simulated very well by synthetic very 1 i ttle electrokinetic data hydroxyapati te, Honev?r, are available for human dental tissues. Both the streaming pDtentia1 and microeiectrophoresis have been used in the stud of hydrowyapacitc and other sol id/l iqufd interface*; Hore recently. electrophorcsis (5 1 . (6) and the streaming potential techntque ha-se been used to meas*rre the isoelectric point (I.P.) of the ?.uman dental enamel (7.8). EXPEIIHENTAL The present note describes the values of the I.P. of human obtained by the streaming potential dental enamel and dentin. using the plug method Cnstead of tooth slabs. as technique, in the nork of Kdmbara et al, (7). The plug method uses a bed of coarse particles, and ion pressures are employed to the moderate impeddrive the liquid through the plug. Also, ance of the plug reduces considerably the signal to noise In addition, and el ini na tes cumbersome Faraday cages. ratfo, The latter Ag/AgCl was used instead of platinum electrodes. are known to polarize and to inject undesirable voltages in the system, thus compromising the validity of the calculated zeta potentials, part$cularly when small streaming potentials
are generated (9). The dental enamel was extracted from the dried irouns of human teeth that had been previously stored in nater. The crowns nere separated from the roots with a very fine San. The enamel shells were obtained by drilling out all dentin ni;:h the help of an ordinary dentist’s drill. The Care was taken to eliminate all dentin from the shells. These were dried, dentin was taken from old dental roots. cut lengthwise, and cleaned of cementum and organic matter. Both tissues were crushed separately in a Diamonite mortar and pestle to 20/4D mesh powder. The_&oi;;ctAvi ty of the distilled nater never exceeded 2 x 10 at 25OC KC1 nas added to keep the ionic strength constant and ensure No buffer was used in order to electrode reversibilitv. maintain the system as-simple as possible. The initial and final DH values of each solution differed little since the The liquid’ h,id only a brief contact with the atmosphere. apparatus traces automatically the streaming potential vs pressure line (E vs P) in a few seconds, instead of nearly one hour taken by the manual technique. The device used incorporates a fen improvements over the apparatus tiescribed The streaming potential cell consisted of a previously (10) _ Lucite cylinder fitted nith a pair of perforated AgfAgCI electra des _ These can be adjusted by sliding along inside the cylindric bore of the cell. ‘ihe plug length was ca, 20 mm. The electrodes uere cornected to a Keithley electrometer model 61DC. The pressure of the nitrogen gas used to drive the liquid through the plug was measured with a calibrated Schaevitz pressure trtinsducer PTA-31DD ccrrpled to a Schaevitt linear variable differential transformer (LVDT), series ZAS-025. The outputs of the electrometer and the LVDT were fed to the Y and X axis, respectively, of an Omnigraph KY recorder, series 2000. The liquid flow was controlled by a gradual decrease of the gas pressure, achieved automatically nifh the help of a bleeding needle (10). The cell resistance in the presence of the plug was measured with a General Radio Impedance Bridge, model 165OB. driven by a 1kHz signal provided by a Hewlett-Packard signal generator, model 4D24A and an oscilloscope. The experiments were carried out in a temperature controlled room set at 24 5 loC, RESULTS
AND DISCUSSION
The zeta potent;a;;o;ere This, equation. 5
= 9.43
calculated p reJuces to: K 10
using
the
Smoluchowski
aK/R
where K is the cell constant (contajning the plug), a (mV/cmHg) and R {ohms) are the slope of the E vs P lifle The latter was and the plug resistance, respect!vely, measured while the liquid was flowing through the cell under a pressure of ca 4 cm Hg. Ths E vs P lines were straight, up to 10 cct Hg. and appeared to change very little with the liquid f!on direction. Four to six lines were traced for each value of the zeta potential. The Zeta potez+*al vs pH curves for enamel and dentin. in The curves tend to level at KC’I , are presentet in Figure 1. hi h pH values in clntrart with the results of Kambara et al. (77.
191
The zeta potential vs pH curves for enamel and dentin shows that both tissues are negati-ely charged above the I.P. and positively charged belor thal: point. The generation of the surface charges on dental tissues can be explained by assuming that the terainal phosphate ions of hydroxyapatite hydrolyze i:fuform a _camplex surface of formula Ca2(HPO4) (OH)2 (11). , the IncorporatIon of fluoride ions rn the enamel or dentin snould not change this mechanism (12). The isoelectric
Fig. KCI.
1. at
Zeta potential 24 c l°C.
vs
pli
for
human
dental
tissues
Sn
points for enam;;ea;dPdentin were found to be 3.9 and 4.0, respectively. found for the enamel is in excellent agreement with 3.8 fluid by Kambara et al, in the presence of Their uork suggests that high concentration of phosphate ions. the phosphate ions were not highly specific for the enamel surface. This is somewhat puzzling because phosphate groups are present fn the hydroxyapaptite lattice. The I .P_ of both dental tissues is close to 4,:: found for the initial I.P. of although aged fluoroapatite gave an I.P.=6.0 fluoroapati te. (12!ie I P found for the enamel is Cn excellent agreement with 3.74 iound by electrophoresis (6). However* the I.P. obtained for dentin is significantly lover than 6.7 reported
192 either the T!ae di f ference suggests th Iat Kumasaki (8). ions are highly specific for denti in or that “K p osphate the age of the extracted dentin used here markedly affec ts the I-P, of this substrate, Rote that fresh dentin conta ins ~.~_ a _ sjanifi_ _ ___ cant amount of collagen. #ore work is necessary to clarify this point. REFERENCES “Chemical Dynamics of %one 1 W.F. Neuman and H.IJ. Reuman, University of Chicago Press. (1958)Mineral”, 2 S.A. Leach. ArchOral Biol. 3. 48-56. (1960). 3 J. Olsson. P. Glantz and B, Krause, Stand, J. Dent. Res. 84, 240-242. (1967)_ 4 W-3. FBrien. P.L. Fan, W.J. Loesche. ?l.C. Hal ker and A. Apostolids, J. Dent. Res. 5f. 910-914, (1978). 5 S-K, 00%~~ J. Dent. Res. SS, 1067-1075,(1976). 6 K. Yoshizaua, Kobyoshi 24. 363-373. (1967). 7 21. Kambara. T. Asai.M. Kumasaki and K. Konishi J, Dent. Res. 57. 366-312, (1978). 0sai:a Odont. Sot. 29. 125-143, (1966). 8 H. Kumasaki , J, 9 H.C. Parreira and J-H, Schulman, AiJv. Chem. Ser. 33, l;;;;;;;,‘““‘) . 20. l-6 (1965). J. Colloid Sci 10 H.C. 11 H.M. ‘A-R, Dietz and FIGFCarpenter. Rootare, J. Colloid Interface Sci. 17. 179-206, (1962). 12 P. Somasundaran, J. Colloid Interface Sci, 27. 659-666. (1968).
ISOELECTRIC
POINT
H.C. PARREIRA Johnson & Johnson (Peceived
OF HWAH
Research,
Rarch 16th,
DENTAL
TISSUES”
New Brunsnick,
1979; accepted
in fipnl
N-3.
forra October
08903 10th.
(U.S.A.) 1980)
ABSTRACT The isoelectric by the streaming of dental enamel 10’+l KC1 .
point potential nas 3.9
of
human dental tissues was measured technique. The isoelectric point in 10’3H.KCl and 4.0 for dentin in
fNTROBUCTfON The surface electrochemistry of human dental enamel and dentin Is of extreme importance if one is to understand better the comp?ex phenomena involved in plaque formation and other It was concluded by problems of odontolo ical interest. tfeuman and Neuman (1 3 that the electrical potential barrier is the most important variable that affects the rate 0’ exchange betneen calcium and phosphate ions and hydro-yapapteach (2) recognized that the calcium/phospha?e ratio tite. is related to the electric charge of the hydroxyapatite #ore recently, it has been suggested that the zeta surface. potential is an fzepcrtant factor in controlling t%e adhesion It is genl-rally accepted of bacteria to hydrox:rapatite (3.4). that the dental enamer is simulated very well by synthetic very 1 i ttle electrokinetic data hydroxyapati te, Honev?r, are available for human dental tissues. Both the streaming pDtentia1 and microeiectrophoresis have been used in the stud of hydrowyapacitc and other sol id/l iqufd interface*; Hore recently. electrophorcsis (5 1 . (6) and the streaming potential techntque ha-se been used to meas*rre the isoelectric point (I.P.) of the ?.uman dental enamel (7.8). EXPEIIHENTAL The present note describes the values of the I.P. of human obtained by the streaming potential dental enamel and dentin. using the plug method Cnstead of tooth slabs. as technique, in the nork of Kdmbara et al, (7). The plug method uses a bed of coarse particles, and ion pressures are employed to the moderate impeddrive the liquid through the plug. Also, ance of the plug reduces considerably the signal to noise In addition, and el ini na tes cumbersome Faraday cages. ratfo, The latter Ag/AgCl was used instead of platinum electrodes. are known to polarize and to inject undesirable voltages in the system, thus compromising the validity of the calculated zeta potentials, part$cularly when small streaming potentials
are generated (9). The dental enamel was extracted from the dried irouns of human teeth that had been previously stored in nater. The crowns nere separated from the roots with a very fine San. The enamel shells were obtained by drilling out all dentin ni;:h the help of an ordinary dentist’s drill. The Care was taken to eliminate all dentin from the shells. These were dried, dentin was taken from old dental roots. cut lengthwise, and cleaned of cementum and organic matter. Both tissues were crushed separately in a Diamonite mortar and pestle to 20/4D mesh powder. The_&oi;;ctAvi ty of the distilled nater never exceeded 2 x 10 at 25OC KC1 nas added to keep the ionic strength constant and ensure No buffer was used in order to electrode reversibilitv. maintain the system as-simple as possible. The initial and final DH values of each solution differed little since the The liquid’ h,id only a brief contact with the atmosphere. apparatus traces automatically the streaming potential vs pressure line (E vs P) in a few seconds, instead of nearly one hour taken by the manual technique. The device used incorporates a fen improvements over the apparatus tiescribed The streaming potential cell consisted of a previously (10) _ Lucite cylinder fitted nith a pair of perforated AgfAgCI electra des _ These can be adjusted by sliding along inside the cylindric bore of the cell. ‘ihe plug length was ca, 20 mm. The electrodes uere cornected to a Keithley electrometer model 61DC. The pressure of the nitrogen gas used to drive the liquid through the plug was measured with a calibrated Schaevitz pressure trtinsducer PTA-31DD ccrrpled to a Schaevitt linear variable differential transformer (LVDT), series ZAS-025. The outputs of the electrometer and the LVDT were fed to the Y and X axis, respectively, of an Omnigraph KY recorder, series 2000. The liquid flow was controlled by a gradual decrease of the gas pressure, achieved automatically nifh the help of a bleeding needle (10). The cell resistance in the presence of the plug was measured with a General Radio Impedance Bridge, model 165OB. driven by a 1kHz signal provided by a Hewlett-Packard signal generator, model 4D24A and an oscilloscope. The experiments were carried out in a temperature controlled room set at 24 5 loC, RESULTS
AND DISCUSSION
The zeta potent;a;;o;ere This, equation. 5
= 9.43
calculated p reJuces to: K 10
using
the
Smoluchowski
aK/R
where K is the cell constant (contajning the plug), a (mV/cmHg) and R {ohms) are the slope of the E vs P lifle The latter was and the plug resistance, respect!vely, measured while the liquid was flowing through the cell under a pressure of ca 4 cm Hg. Ths E vs P lines were straight, up to 10 cct Hg. and appeared to change very little with the liquid f!on direction. Four to six lines were traced for each value of the zeta potential. The Zeta potez+*al vs pH curves for enamel and dentin. in The curves tend to level at KC’I , are presentet in Figure 1. hi h pH values in clntrart with the results of Kambara et al. (77.
191
The zeta potential vs pH curves for enamel and dentin shows that both tissues are negati-ely charged above the I.P. and positively charged belor thal: point. The generation of the surface charges on dental tissues can be explained by assuming that the terainal phosphate ions of hydroxyapatite hydrolyze i:fuform a _camplex surface of formula Ca2(HPO4) (OH)2 (11). , the IncorporatIon of fluoride ions rn the enamel or dentin snould not change this mechanism (12). The isoelectric
Fig. KCI.
1. at
Zeta potential 24 c l°C.
vs
pli
for
human
dental
tissues
Sn
points for enam;;ea;dPdentin were found to be 3.9 and 4.0, respectively. found for the enamel is in excellent agreement with 3.8 fluid by Kambara et al, in the presence of Their uork suggests that high concentration of phosphate ions. the phosphate ions were not highly specific for the enamel surface. This is somewhat puzzling because phosphate groups are present fn the hydroxyapaptite lattice. The I .P_ of both dental tissues is close to 4,:: found for the initial I.P. of although aged fluoroapatite gave an I.P.=6.0 fluoroapati te. (12!ie I P found for the enamel is Cn excellent agreement with 3.74 iound by electrophoresis (6). However* the I.P. obtained for dentin is significantly lover than 6.7 reported
192 either the T!ae di f ference suggests th Iat Kumasaki (8). ions are highly specific for denti in or that “K p osphate the age of the extracted dentin used here markedly affec ts the I-P, of this substrate, Rote that fresh dentin conta ins ~.~_ a _ sjanifi_ _ ___ cant amount of collagen. #ore work is necessary to clarify this point. REFERENCES “Chemical Dynamics of %one 1 W.F. Neuman and H.IJ. Reuman, University of Chicago Press. (1958)Mineral”, 2 S.A. Leach. ArchOral Biol. 3. 48-56. (1960). 3 J. Olsson. P. Glantz and B, Krause, Stand, J. Dent. Res. 84, 240-242. (1967)_ 4 W-3. FBrien. P.L. Fan, W.J. Loesche. ?l.C. Hal ker and A. Apostolids, J. Dent. Res. 5f. 910-914, (1978). 5 S-K, 00%~~ J. Dent. Res. SS, 1067-1075,(1976). 6 K. Yoshizaua, Kobyoshi 24. 363-373. (1967). 7 21. Kambara. T. Asai.M. Kumasaki and K. Konishi J, Dent. Res. 57. 366-312, (1978). 0sai:a Odont. Sot. 29. 125-143, (1966). 8 H. Kumasaki , J, 9 H.C. Parreira and J-H, Schulman, AiJv. Chem. Ser. 33, l;;;;;;;,‘““‘) . 20. l-6 (1965). J. Colloid Sci 10 H.C. 11 H.M. ‘A-R, Dietz and FIGFCarpenter. Rootare, J. Colloid Interface Sci. 17. 179-206, (1962). 12 P. Somasundaran, J. Colloid Interface Sci, 27. 659-666. (1968).