- Email: [email protected]
Dissolution of hydroxyapatite by calcium complexing agents
Journal of Crystal Growth 110 (1991) 733—738 North-Holland
733
Dissolution of hydroxyapatite by calcium complexing agents Amira Arbel Casali Institute of Applied Chemistry, The Hebrew University of Jerusalem, 91904 Jerusalem, Israel
lony Katz Computer and Research Department, Hadassah Medical Center, The Hebrew University of Jerusalem, 91904 Jerusalem, Israel
and Sara Sang Casali Institute of Applied Chemistry, The Hebrew University of Jerusalem, 91904 Jerusalem, Israel Received 25 July 1990; manuscript received in final form 4 December 1990
Hydroxyapatite (HAP) dissolution effected by calcium complexing ions was studied. The efficiencies of various complexants on the dissolution of HAP at 37°C and pH = 7.4 were compared. This effect was also examined in correlation with parameters such as: the concentration of the complexant solution, the volume of the complexant solution, dissolution times and the perfection of crystals structure. EDTA has been found to be superior complexant to others. The significant dissolution time occurs between 1 and 3 mm and as the volume and concentration of the complexant solution increase, there is a significant increase in the efficiency of dissolution of HAP.
1. Introduction The dissolution of calcium hydroxyapatite (HAP) in water has been extensively studied, particulary by Christoffersen and co-workers. Two consecutive reactions take place when crystals dissolve, a surface process and a transport (diffusion or convective-diffusion) process [1]. In most of the dissolution studies the driving force for dissolution is hydrogen ion catalysis. Hydrogen ions can be transported from the bulk solution to the crystal surface either in the form of free ions or in the form of a weak acid which dissociates at the crystal surface, The rate of dissolution of HAP was measured in the pH range 5—7 using various acids [1—4]. During dissolution, holes are formed in the crystals. A possible explanation of the formation 0022-0248/91/$03.50 © 1991
—
of the holes could be that the rate of dissolution is controlled by a polynuclear mechanism, which causes concave parts of the dissolving surface to grow bigger. Concave parts on a surface could be formed if the crystal contain screw dislocations [5]. Chnstoffersen et al. investigated the effect of various molecules on the kinetics of dissolution of HAP, in order to find inhibitors of the dissolution process [6]. Among the inhibitors, citrate ions appear to reduce the rate of dissolution of HAP. However they increase the rate of dissolution when present in higher concentrations. This effect was attributed to the complex formation of citrate ions with calcium ions [7]. The present study deals with dissolution by complexing agents, at physiological pH. It is very
Elsevier Science Publishers B.V. (North-Holland)
734
A. Arhel et al.
/
Dissolution of hydroxyapatite by calcium complexing agents
probable that it can serve as a theoretical basis for cardiology treatment in which balloon dilatation catheters are used to squeeze atherosclerotic plaques and relieve the obstructions. In this situation it is clear that no acid can be used to dissolve the hard calcified plaques. Complexing agents are used for several purposes; disodium EDTA is used to extract calcium from atherosclerotic aortes [8]. The complex formation causes a decrease in the calcium ion activity, and thereby an increase in the rate of dissolution.
mg of HAP powder (containing 4 mg of calcium) was added to this solution and the time was marked as I 0. After a measured time, the solids in the suspension were separated by a 0.2 ~sm “Schleicher and Schuele” filter. The experiments were repeated with different dissolution times, volumes and concentrations of the attacking reagant (as indicated in table 1). Blank dissolution experiments of buffered solutions without attacking complexants were performed. A series of experiments under constant conditions with HAP of varing degrees of crystallinity was carried out.
2. Experimental
3. Analyses
Materials. Hydroxyapatite (HAP) powder was prepared from stock solutions of CaCl2 and NaH2PO4 [1]. The buffer was prepared from Trizma powder (Sigma) balanced to pH 7.4. Attacking complexants: EDTA, citric acid (AR), ENTMP (ethylene dinitro tetrakis (methylene phosphonic) acid) and Fostex (polyalkylene polyamino polykis (methylene phosphonic) acid, commercial samples. Solutions of the attacking complexants at various concentrations were prepared as indicated in table I and were buffered with Trizma. Procedure. Dissolution experiments were carned out by the following procedure: The attacking solution in an Erlenmayer flask was introduced into a water bath kept at 37°C. The solution was agitated with a magnetic stirrer. A sample of 10 =
=
The HAP powder was subjected to X-ray powder diffractometery (Philips type PW 2233/20 Cu, K~ 1.54). Particles size distribution was measured by the Galai CIS-1 (Computerized Inspection System), a laser based optical analyzer which measures number density and particles size distribution. Dissolution of HAP was determined by measuring calcium concentration in the filtered attacking solutions, using atomic absorbtion. The amount of calcium present in the blank solution, without attacking complexants, but undergoing similar treatment including filtration, was subtracted from each determination. The attacked deposits of the HAP crystals were viewed by scanning electron microscope (SEM). =
4. Results and discussion Table 1 Values of different fundamental parameters of various cornplexants; all experiments were carried Out with 2-3 type HAP (see ta e an ig. ) Material
Concentration
Volume
(M)
(ml)
EDTA EDTA EDTA
0.010 0.025 0.050
100 100 25, 100
4
EDTA
1—60 mm
Citric acid
0.100 0.100
10, 25, 100
5
25
1—5 mm
6 7 8 9
Citric acid ENTMP Fostex Fostex
0.250 0.010 0.025 0.100
25 25 25 25
1—5 mm
1 2
3
Time 1—60 mm 1—15 mm 1 min—24 h
1-60 mm 1—5 mm 1—5 mm
_________________________________________
X-ray diffractograms of four categories of calcium phosphate precipitates are shown in fig. 1. In the prepared HAP one can distinguish between two categories of HAP: the amorphous calcium phosphate (fig. 1 (2) (type 2)) and the calcium phosphate with apatitic character (fig. I (3) (type 3)) [1]. Particles size distribution analysis showed that 78% of the prepared HAP particles were in the range between 0.5 and 2.0 ~sm (fig. 2). Fig. 3 shows the percentage of calcium dissolved from the HAP sample by the various attacking complexants in experiments of I and 5 .
.
.
.
mm duration. It is obvious that EDTA is superior
/
A. Arbel et aL
Dissolution of hydroxyapatite by calcium complexing agents 80
—
—
A
(I
8
—
~
735
60
H!I~ 4
~
FostexO.1M
~ D fl
Fostex Citric CitricO.1M O.25M O.025M
•
EDTA O.05M
40
—
.3
20
12
0
_________________________ I
_V/ C,,
1
=
_________
Time (mm)
o~—~ 5
Fig. 3. Percentage of calcium dissolved from HAP in the
I
25
30
35
28
Fig. 1. X-ray diffraction of calcium phosphate precipitates: (1), (2) amorphous calcium phosphate phases; (3) calcium phosphate with apatitic character; (4) well-defined apatite.
presence of various complexants with dissolution times of I and 5 mm.
______________________________ 40
to the others: compare the effect of 0.05M EDTA to that of five-fold higher concentration (0.25M) of citric acid. The dissolution decreases with concentration as may be seen in the sequences of citric acid and Fostex. Though Fostex, a polyphosphonate, is reported to be an efficient calcium complexant it is not effective as a dissolving agent. Figs. 4 and 5 are kinetic graphs that show the percentage of calcium dissolved from HAP as function of time, measured between 1 and 5 mm.
30 0
~2OI
l0~
50 a ~ 40 0
~ too 60 ~n:O.9pm 40
t
a
a..
I 5
I
3 4 (m in)
6
i
I
I
— —
0
Mean: S.D.:
20 0
2
Fig. 4. A kinetic graph showing an increase in the percentage of calcium dissolved from HAP versus time with 0.1M KCI + 0.1M citric acid.
60 ~80
//2
-
a
11111
0.5
I
I 2
iilliii
5
0
I 20
4.5pm 7.6jjm
________________
30~______________________ 2 3 4
5
6
111111
50
100 ISO
Size (in microns) Log Scale Fig. 2. Probability number distribution graph of HAP crystals, used in this study.
( minI Fig. 5. A kinetic graph showing an increase in the percentage of calcium dissolved from HAP versus time with 0.1M KCI + 0.05M EDTA.
736
A. Arhel et al. I
70
-
65
-
60
-
/
I
Dissolution of hydroxyapatite by calcium complexing agents I
Table2
I
.
ent types of calcium phosphates 0
_____________________________________________ Calcium phosphate type % Ca, I mm % Ca, 5 mm The percentage of calcium dissolved from HAP of three differ-
55-.
1 2
Well-defined 2—3 Type
26.75 48.00
71.50 77.50
3
Amorphous
47.25
71.25
. S
50-
• I
0
I
.02
I
.04 .06 Con. (M
.08
.10
Fig. 6. The increase in the percentage of calcium dissolved from HAP in correlation with the concentration.
The attacking complexant solutions were 25 ml of 0.05M EDTA and 0.IM citric acid respectively. In both of the kinetic graphs the significant dissolution time occurs between 1 and 3 mm. Other dissolution experiments examined dissolution periods between 5 mm and 24 h. However, they did not show significant increase in the percentage of dissolved calcium. Fig. 6 shows the percentage of calcium dissolved from the HAP as a function of concentration. The attacking solution was a 100 ml sample of EDTA. Experiments with 0.O1M, 0.025M, 0.05M and 0.1M concentrations were performed. The dissolution is enhanced significantly with increasing the concentration of the attacking agent. The percentage of calcium dissolved from the HAP as a function of volume is shown in fig. 7, The attacking complexant solution was 0.05M EDTA. Experiments with 10, 25 and 100 ml were performed.
(1) well-defined HAP (Sigma); (2) HAP of crystallinity between types 2 and 3; (3) amorphous calcium phosphate. The type characteristics were determined by X-ray diffractograms. The dissolution experiments were carried out with 25 ml 0.05M EDTA attacking solution. Dissolution times were 1 mm and 5 mm. Comparison of the percentage of calcium dissolved from the three samples of HAP in I mm experiments shows that well defined HAP (Sigma) dissolved significantly less than the 2—3 HAP type and amorphous calcium phosphate; the last two had similar dissolution values. The experiments of 5 mm show similar dissolution values for all three types of HAP. Fig. 8 shows the HAP crystal surfaces before and after being attacked by the complexant solutions, as viewed using the scanning electron microscope. Fig. 8a shows a HAP crystal surface before being attacked, whereas fig. 8b shows a HAP crystal surface after being attacked by 0.1M EDTA, during 10 mm of dissolution time. Cornparison of the two photomicrographs indicates that the HAP crystal surface before being attacked, though not perfectly smooth, is intact but after being attacked by 0.1M EDTA solution con-
_______________________
I’ll
Table 2 indicates the percentage of calcium dissolved from HAP of three different types:
I’
75 •
.
a
55
-
45
.
• 0
I
20
I
I
I
40 60 80 Volume (ml)
I
cave holes are created on the crystal surface. Christoffersen et al.’s explanation was that concave parts on a surface could be formed if the tion concerning the mechanism of dissolution is crystal contained a screw dislocation. His assumpthat the centre has a higher rate of dissolution, so that the cores of the screws open up [5].
00
Fig. 7. The increase in the percentage of calcium dissolved from HAP in correlation with the volume,
The EDTA has 6 bonding functional groups (4 of carboxylic acid and 2 of nitrogen) which are 2~) on the able to bond one calcium cation (Ca
.4. .4rhel et a!.
/
Dissolution of hvdroxvapatite hr ealaum compleving agents
—
737
~
~
—
4 4
~
‘~
.~
44,
.4
Fig. 8. Photomicrographs of the surface of HAP crystals’. (a) x4100. before being attacked by the complexant: (h) ~4l00. after being attacked by 0.lM EDTA; Ic) v.8200. after being attacked by 0.25M citric acid: (d) Y4100. after being attacked h~0.OIM ENTMP.
crystal surface. At pH = 7.4. EDTA is a very strong complexant with a chelate structure. The EDTA complexant adsorbs on the crystal surface and forms, as it is able. 6 coordinative bonds surrounding the calcium cation from all sides and
this is evidently able to leech the calcium out of the crystal surface. Using a molecular model of EDTA. one may show that this chelating molecule is able to enwrap the calcium ion perfectly, forming a symmetric octahedral complex [9]. This
738
A. Arbel et aL
/ Dissolution
of hydroxyapatite by calcium complexing agents
leech out of the cation weakens the crystal’s electrostatic stabilizing forces and permits phosphate groups to leave. The same phenomenon occurs when the attacking complexant is 0.25M citric acid at 5 mm dissolution time but the holes created are much smaller (fig. 8c). The citric acid has only four bonding functional groups to react with the calcium cation. Its leeching-out ability is weaker than that of the EDTA. Fig. 8d shows that when the attacking solution is 0.O1M of ENTMP, the crystal surface is similar to that of HAP before being attacked by a solution containing the cornplexant. ENTMP can adsorb on the crystal surface but presumably cannot leech the calcium cation out of the crystal. The ENTMP structure is similiar to that of EDTA but instead of having carboxylic groups it has phosphonate groups. This modification renders the molecule heavy and unflexible, not allowing it to fold freely and form an octahedral complex. Most probably ENTMP serves only as a tridentade complexing agent, granting a stability constant lower than that of the EDTA complex [10]. The result of the scanning electron microscope (SEM) and those of the dissolution ability of the attacking complexants support each other. The results as presented in fig. 3 also indicate the superior effect of EDTA as a dissolving agent. Previous dissolution experiments were carried out with various acids by Christoffersen et al. They assumed that the dissolution process of HAP depends on the pH, because the electrostatic bonds between calcium ions and phosphate ions were weakened by the reaction between hydrogen ion
and phosphate ion. Christoffersen et al. investigated also the effect of inhibitors on the dissolution of HAP. The inhibitor polyisoprene phosphonic acid gave, contrary to the usual trend, an increase of the dissolution rate up to 10 s. Citrate ions at pH 7.2 at a high concentration also induced an increase in the dissolution rate [7]. Christoffersen et al. explained it as due to cornplex formation between the inhibitors and the calcium ions, but no experiments investigating the increase of HAP dissolution in correlation with the complexant agents were performed in his group. The dissolution of HAP by organic complexants may be of importance in biological systerns. =
References [1] J. Christoffersen, M.R. Christoffersen and N. Kjaergaard,
J. Crystal Growth 43 (1978) 501. [2j J. Christoffersen and M.R. Chnstoffersen, J. Crystal Growth 47 (1979) 671. [31J. Christoffersen and M.R. Christoffersen, J. Crystal Growth 57 (1982) 21. [4] J. Christoffersen, Calcif. Tissue Intern. 33 (1981) 557. [5] J. Christoffersen, J. Crystal Growth 49 (1980) 29. [6] J. Christoffersen, M.R. Christoffersen, S.B. Christensen and G.H. Nancollas, J. Crystal Growth 62 (1983) 254. [7] J. Christoffersen and M.R. Christoffersen, Croatica Chem. Acta 56 (1983) 769. [8] W. Hollander, Exptl. Mol. Pathol. 25 (1976) 106. [9] Vogel’s Text book of Quantitative Inorganic Analysis Including Elementary Instrumental Analysis, 4th ed. (Longman, London, 1979). [10] E. Martell and R.M. Smith, Critical Stability Constants, Vols. 2, 3 and 5 (Plenum, New York, 1975, 1977, 1982).
733
Dissolution of hydroxyapatite by calcium complexing agents Amira Arbel Casali Institute of Applied Chemistry, The Hebrew University of Jerusalem, 91904 Jerusalem, Israel
lony Katz Computer and Research Department, Hadassah Medical Center, The Hebrew University of Jerusalem, 91904 Jerusalem, Israel
and Sara Sang Casali Institute of Applied Chemistry, The Hebrew University of Jerusalem, 91904 Jerusalem, Israel Received 25 July 1990; manuscript received in final form 4 December 1990
Hydroxyapatite (HAP) dissolution effected by calcium complexing ions was studied. The efficiencies of various complexants on the dissolution of HAP at 37°C and pH = 7.4 were compared. This effect was also examined in correlation with parameters such as: the concentration of the complexant solution, the volume of the complexant solution, dissolution times and the perfection of crystals structure. EDTA has been found to be superior complexant to others. The significant dissolution time occurs between 1 and 3 mm and as the volume and concentration of the complexant solution increase, there is a significant increase in the efficiency of dissolution of HAP.
1. Introduction The dissolution of calcium hydroxyapatite (HAP) in water has been extensively studied, particulary by Christoffersen and co-workers. Two consecutive reactions take place when crystals dissolve, a surface process and a transport (diffusion or convective-diffusion) process [1]. In most of the dissolution studies the driving force for dissolution is hydrogen ion catalysis. Hydrogen ions can be transported from the bulk solution to the crystal surface either in the form of free ions or in the form of a weak acid which dissociates at the crystal surface, The rate of dissolution of HAP was measured in the pH range 5—7 using various acids [1—4]. During dissolution, holes are formed in the crystals. A possible explanation of the formation 0022-0248/91/$03.50 © 1991
—
of the holes could be that the rate of dissolution is controlled by a polynuclear mechanism, which causes concave parts of the dissolving surface to grow bigger. Concave parts on a surface could be formed if the crystal contain screw dislocations [5]. Chnstoffersen et al. investigated the effect of various molecules on the kinetics of dissolution of HAP, in order to find inhibitors of the dissolution process [6]. Among the inhibitors, citrate ions appear to reduce the rate of dissolution of HAP. However they increase the rate of dissolution when present in higher concentrations. This effect was attributed to the complex formation of citrate ions with calcium ions [7]. The present study deals with dissolution by complexing agents, at physiological pH. It is very
Elsevier Science Publishers B.V. (North-Holland)
734
A. Arhel et al.
/
Dissolution of hydroxyapatite by calcium complexing agents
probable that it can serve as a theoretical basis for cardiology treatment in which balloon dilatation catheters are used to squeeze atherosclerotic plaques and relieve the obstructions. In this situation it is clear that no acid can be used to dissolve the hard calcified plaques. Complexing agents are used for several purposes; disodium EDTA is used to extract calcium from atherosclerotic aortes [8]. The complex formation causes a decrease in the calcium ion activity, and thereby an increase in the rate of dissolution.
mg of HAP powder (containing 4 mg of calcium) was added to this solution and the time was marked as I 0. After a measured time, the solids in the suspension were separated by a 0.2 ~sm “Schleicher and Schuele” filter. The experiments were repeated with different dissolution times, volumes and concentrations of the attacking reagant (as indicated in table 1). Blank dissolution experiments of buffered solutions without attacking complexants were performed. A series of experiments under constant conditions with HAP of varing degrees of crystallinity was carried out.
2. Experimental
3. Analyses
Materials. Hydroxyapatite (HAP) powder was prepared from stock solutions of CaCl2 and NaH2PO4 [1]. The buffer was prepared from Trizma powder (Sigma) balanced to pH 7.4. Attacking complexants: EDTA, citric acid (AR), ENTMP (ethylene dinitro tetrakis (methylene phosphonic) acid) and Fostex (polyalkylene polyamino polykis (methylene phosphonic) acid, commercial samples. Solutions of the attacking complexants at various concentrations were prepared as indicated in table I and were buffered with Trizma. Procedure. Dissolution experiments were carned out by the following procedure: The attacking solution in an Erlenmayer flask was introduced into a water bath kept at 37°C. The solution was agitated with a magnetic stirrer. A sample of 10 =
=
The HAP powder was subjected to X-ray powder diffractometery (Philips type PW 2233/20 Cu, K~ 1.54). Particles size distribution was measured by the Galai CIS-1 (Computerized Inspection System), a laser based optical analyzer which measures number density and particles size distribution. Dissolution of HAP was determined by measuring calcium concentration in the filtered attacking solutions, using atomic absorbtion. The amount of calcium present in the blank solution, without attacking complexants, but undergoing similar treatment including filtration, was subtracted from each determination. The attacked deposits of the HAP crystals were viewed by scanning electron microscope (SEM). =
4. Results and discussion Table 1 Values of different fundamental parameters of various cornplexants; all experiments were carried Out with 2-3 type HAP (see ta e an ig. ) Material
Concentration
Volume
(M)
(ml)
EDTA EDTA EDTA
0.010 0.025 0.050
100 100 25, 100
4
EDTA
1—60 mm
Citric acid
0.100 0.100
10, 25, 100
5
25
1—5 mm
6 7 8 9
Citric acid ENTMP Fostex Fostex
0.250 0.010 0.025 0.100
25 25 25 25
1—5 mm
1 2
3
Time 1—60 mm 1—15 mm 1 min—24 h
1-60 mm 1—5 mm 1—5 mm
_________________________________________
X-ray diffractograms of four categories of calcium phosphate precipitates are shown in fig. 1. In the prepared HAP one can distinguish between two categories of HAP: the amorphous calcium phosphate (fig. 1 (2) (type 2)) and the calcium phosphate with apatitic character (fig. I (3) (type 3)) [1]. Particles size distribution analysis showed that 78% of the prepared HAP particles were in the range between 0.5 and 2.0 ~sm (fig. 2). Fig. 3 shows the percentage of calcium dissolved from the HAP sample by the various attacking complexants in experiments of I and 5 .
.
.
.
mm duration. It is obvious that EDTA is superior
/
A. Arbel et aL
Dissolution of hydroxyapatite by calcium complexing agents 80
—
—
A
(I
8
—
~
735
60
H!I~ 4
~
FostexO.1M
~ D fl
Fostex Citric CitricO.1M O.25M O.025M
•
EDTA O.05M
40
—
.3
20
12
0
_________________________ I
_V/ C,,
1
=
_________
Time (mm)
o~—~ 5
Fig. 3. Percentage of calcium dissolved from HAP in the
I
25
30
35
28
Fig. 1. X-ray diffraction of calcium phosphate precipitates: (1), (2) amorphous calcium phosphate phases; (3) calcium phosphate with apatitic character; (4) well-defined apatite.
presence of various complexants with dissolution times of I and 5 mm.
______________________________ 40
to the others: compare the effect of 0.05M EDTA to that of five-fold higher concentration (0.25M) of citric acid. The dissolution decreases with concentration as may be seen in the sequences of citric acid and Fostex. Though Fostex, a polyphosphonate, is reported to be an efficient calcium complexant it is not effective as a dissolving agent. Figs. 4 and 5 are kinetic graphs that show the percentage of calcium dissolved from HAP as function of time, measured between 1 and 5 mm.
30 0
~2OI
l0~
50 a ~ 40 0
~ too 60 ~n:O.9pm 40
t
a
a..
I 5
I
3 4 (m in)
6
i
I
I
— —
0
Mean: S.D.:
20 0
2
Fig. 4. A kinetic graph showing an increase in the percentage of calcium dissolved from HAP versus time with 0.1M KCI + 0.1M citric acid.
60 ~80
//2
-
a
11111
0.5
I
I 2
iilliii
5
0
I 20
4.5pm 7.6jjm
________________
30~______________________ 2 3 4
5
6
111111
50
100 ISO
Size (in microns) Log Scale Fig. 2. Probability number distribution graph of HAP crystals, used in this study.
( minI Fig. 5. A kinetic graph showing an increase in the percentage of calcium dissolved from HAP versus time with 0.1M KCI + 0.05M EDTA.
736
A. Arhel et al. I
70
-
65
-
60
-
/
I
Dissolution of hydroxyapatite by calcium complexing agents I
Table2
I
.
ent types of calcium phosphates 0
_____________________________________________ Calcium phosphate type % Ca, I mm % Ca, 5 mm The percentage of calcium dissolved from HAP of three differ-
55-.
1 2
Well-defined 2—3 Type
26.75 48.00
71.50 77.50
3
Amorphous
47.25
71.25
. S
50-
• I
0
I
.02
I
.04 .06 Con. (M
.08
.10
Fig. 6. The increase in the percentage of calcium dissolved from HAP in correlation with the concentration.
The attacking complexant solutions were 25 ml of 0.05M EDTA and 0.IM citric acid respectively. In both of the kinetic graphs the significant dissolution time occurs between 1 and 3 mm. Other dissolution experiments examined dissolution periods between 5 mm and 24 h. However, they did not show significant increase in the percentage of dissolved calcium. Fig. 6 shows the percentage of calcium dissolved from the HAP as a function of concentration. The attacking solution was a 100 ml sample of EDTA. Experiments with 0.O1M, 0.025M, 0.05M and 0.1M concentrations were performed. The dissolution is enhanced significantly with increasing the concentration of the attacking agent. The percentage of calcium dissolved from the HAP as a function of volume is shown in fig. 7, The attacking complexant solution was 0.05M EDTA. Experiments with 10, 25 and 100 ml were performed.
(1) well-defined HAP (Sigma); (2) HAP of crystallinity between types 2 and 3; (3) amorphous calcium phosphate. The type characteristics were determined by X-ray diffractograms. The dissolution experiments were carried out with 25 ml 0.05M EDTA attacking solution. Dissolution times were 1 mm and 5 mm. Comparison of the percentage of calcium dissolved from the three samples of HAP in I mm experiments shows that well defined HAP (Sigma) dissolved significantly less than the 2—3 HAP type and amorphous calcium phosphate; the last two had similar dissolution values. The experiments of 5 mm show similar dissolution values for all three types of HAP. Fig. 8 shows the HAP crystal surfaces before and after being attacked by the complexant solutions, as viewed using the scanning electron microscope. Fig. 8a shows a HAP crystal surface before being attacked, whereas fig. 8b shows a HAP crystal surface after being attacked by 0.1M EDTA, during 10 mm of dissolution time. Cornparison of the two photomicrographs indicates that the HAP crystal surface before being attacked, though not perfectly smooth, is intact but after being attacked by 0.1M EDTA solution con-
_______________________
I’ll
Table 2 indicates the percentage of calcium dissolved from HAP of three different types:
I’
75 •
.
a
55
-
45
.
• 0
I
20
I
I
I
40 60 80 Volume (ml)
I
cave holes are created on the crystal surface. Christoffersen et al.’s explanation was that concave parts on a surface could be formed if the tion concerning the mechanism of dissolution is crystal contained a screw dislocation. His assumpthat the centre has a higher rate of dissolution, so that the cores of the screws open up [5].
00
Fig. 7. The increase in the percentage of calcium dissolved from HAP in correlation with the volume,
The EDTA has 6 bonding functional groups (4 of carboxylic acid and 2 of nitrogen) which are 2~) on the able to bond one calcium cation (Ca
.4. .4rhel et a!.
/
Dissolution of hvdroxvapatite hr ealaum compleving agents
—
737
~
~
—
4 4
~
‘~
.~
44,
.4
Fig. 8. Photomicrographs of the surface of HAP crystals’. (a) x4100. before being attacked by the complexant: (h) ~4l00. after being attacked by 0.lM EDTA; Ic) v.8200. after being attacked by 0.25M citric acid: (d) Y4100. after being attacked h~0.OIM ENTMP.
crystal surface. At pH = 7.4. EDTA is a very strong complexant with a chelate structure. The EDTA complexant adsorbs on the crystal surface and forms, as it is able. 6 coordinative bonds surrounding the calcium cation from all sides and
this is evidently able to leech the calcium out of the crystal surface. Using a molecular model of EDTA. one may show that this chelating molecule is able to enwrap the calcium ion perfectly, forming a symmetric octahedral complex [9]. This
738
A. Arbel et aL
/ Dissolution
of hydroxyapatite by calcium complexing agents
leech out of the cation weakens the crystal’s electrostatic stabilizing forces and permits phosphate groups to leave. The same phenomenon occurs when the attacking complexant is 0.25M citric acid at 5 mm dissolution time but the holes created are much smaller (fig. 8c). The citric acid has only four bonding functional groups to react with the calcium cation. Its leeching-out ability is weaker than that of the EDTA. Fig. 8d shows that when the attacking solution is 0.O1M of ENTMP, the crystal surface is similar to that of HAP before being attacked by a solution containing the cornplexant. ENTMP can adsorb on the crystal surface but presumably cannot leech the calcium cation out of the crystal. The ENTMP structure is similiar to that of EDTA but instead of having carboxylic groups it has phosphonate groups. This modification renders the molecule heavy and unflexible, not allowing it to fold freely and form an octahedral complex. Most probably ENTMP serves only as a tridentade complexing agent, granting a stability constant lower than that of the EDTA complex [10]. The result of the scanning electron microscope (SEM) and those of the dissolution ability of the attacking complexants support each other. The results as presented in fig. 3 also indicate the superior effect of EDTA as a dissolving agent. Previous dissolution experiments were carried out with various acids by Christoffersen et al. They assumed that the dissolution process of HAP depends on the pH, because the electrostatic bonds between calcium ions and phosphate ions were weakened by the reaction between hydrogen ion
and phosphate ion. Christoffersen et al. investigated also the effect of inhibitors on the dissolution of HAP. The inhibitor polyisoprene phosphonic acid gave, contrary to the usual trend, an increase of the dissolution rate up to 10 s. Citrate ions at pH 7.2 at a high concentration also induced an increase in the dissolution rate [7]. Christoffersen et al. explained it as due to cornplex formation between the inhibitors and the calcium ions, but no experiments investigating the increase of HAP dissolution in correlation with the complexant agents were performed in his group. The dissolution of HAP by organic complexants may be of importance in biological systerns. =
References [1] J. Christoffersen, M.R. Christoffersen and N. Kjaergaard,
J. Crystal Growth 43 (1978) 501. [2j J. Christoffersen and M.R. Chnstoffersen, J. Crystal Growth 47 (1979) 671. [31J. Christoffersen and M.R. Christoffersen, J. Crystal Growth 57 (1982) 21. [4] J. Christoffersen, Calcif. Tissue Intern. 33 (1981) 557. [5] J. Christoffersen, J. Crystal Growth 49 (1980) 29. [6] J. Christoffersen, M.R. Christoffersen, S.B. Christensen and G.H. Nancollas, J. Crystal Growth 62 (1983) 254. [7] J. Christoffersen and M.R. Christoffersen, Croatica Chem. Acta 56 (1983) 769. [8] W. Hollander, Exptl. Mol. Pathol. 25 (1976) 106. [9] Vogel’s Text book of Quantitative Inorganic Analysis Including Elementary Instrumental Analysis, 4th ed. (Longman, London, 1979). [10] E. Martell and R.M. Smith, Critical Stability Constants, Vols. 2, 3 and 5 (Plenum, New York, 1975, 1977, 1982).