biomimetic transformations of calcium phosphates - thermodynamic and kinetic studies d. rabadjieva...
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Biomimetic Transformations of Calcium Phosphates -
Thermodynamic and Kinetic Studies
DD. . RabadjievaRabadjieva11, , SS. . TepavitcharovaTepavitcharova11, , RR. . GergulovaGergulova11, , RR. . TitorenkovaTitorenkova22, , EE. . DyulgerovaDyulgerova33, O. Petrov, O. Petrov22, Chr. Balarew, Chr. Balarew11
September 18-21, 2010, Nessebar, Bulgaria
IGIC-BAS
1Institute of General and Inorganic Chemistry, BAS2Institute of Mineralogy and Crystallography, BAS 3Dental Medicine Faculty, University of Medicine
poly- ((PO3)nn–)
INTRODUCTIONINTRODUCTION CALCIUM PHOSPHATES
September 18-21, 2010, Nessebar, Bulgaria
ortho- (PO43–) meta- (PO3
– )
pyro- (P2O74–)
INTRODUCTIONINTRODUCTIONCALCIUM ORTHOPHOSPHATES (COPh)
Ca/P pH -lgKso
MCPM Ca(H2PO4)2.H2O 0.5 0 - 2 1.14
DCPD Ca(HPO4)2.2H2O 1.0 2 - 6 6.59
OCPt Ca8(PO4)4(HPO4)2.5H2O 1.33 5.5 - 7 96.6
ACP Ca9(PO4)6.nH2O 1.5 5 - 12 25.5
PCA Ca10-xx(PO4)6-x (HPO4)x.((OH)2-xx)
1.33 -1.67 6.5 - 9.5
HA Ca10(PO4)6(OH)2 1.67 9.5 - 12 116.8
COPh precipitated from solutions
Ca/P StabilityMCPA Ca(H2PO4)2 0.5 < 100oC
DCPA Ca(HPO4)2 1.0 < 100oC
TCP -Ca3(PO4)2 1.5 < 800oC
-Ca3(PO4)2 1.5 < 1200oC
HA Ca10(PO4)6(OH)2 1.67 till ~1000oC
ТТCP Ca4(PO4)2О 2.0 < 1500oC
INTRODUCTIONINTRODUCTIONCALCIUM ORTHOPHOSPHATES (COPh)
COPh High-Temperature Forms
Importance for the Biological system
They are the main inorganic component of all body hard tissues
Biological apatite Nano-sizedPoorly crystallized Non-stoichiometric hydroxyapatite Including Na, K, Mg, Cl, F and CO3
INTRODUCTIONINTRODUCTIONCALCIUM ORTHOPHOSPHATES (COPh)
September 18-21, 2010, Nessebar, Bulgaria
INTRODUCTIONINTRODUCTION
MATERIALS FOR BONE REGENERATION
CERAMICS CEMENTSGood
biocompatibility; Low toxicity
HA / -TCP
HA + -TCP
DCPDPCA
Porous, Nano-sized, Adequate biodegradable rates, High strength and elasticity
September 18-21, 2010, Nessebar, Bulgaria
INTRODUCTIONINTRODUCTION BIOMIMETIC APPROACH
BIOMIMETICS (bionics, biognosis and/or biomimicry)
application of the methods and systems found in nature to study, design and construct new engineering systems, materials and modern technologies.
September 18-21, 2010, Nessebar, Bulgaria
INTRODUCTIONINTRODUCTION BIOMIMETIC APPROACH
Biological mineralization (biomineralization) process of in vivo formation of inorganic minerals
September 18-21, 2010, Nessebar, Bulgaria
BLOOD PLASMA It acts as a reservoir
for Ca2+ and PO4
3-
as well asa maturation medium.
Na+ 142.0 142.1 143.5 142.0 142.0 142.0 K+ 5.0 5.3 5.4 5.0 5.0 5.0Ca2+ 2.5 1.26 1.8 2.5 2.5 1.6Mg2+ 1.5 0.9 0.8 1.5 1.5 1.0Cl- 103.0 146.8 123.5 147.8 125.0 103.0HCO3
2- 27.0 4.2 26.2 4.2 27.0 27.0HPO4
2- 1.0 0.78 1.0 1.0 1.0 1.0SO4
2- 0.5 0.41 0.8 0.5 0.5 1.5Ca/P 2.5 1.62 1.8 2.5 2.5 1.6Buffer TRIS TRIS TRISpH 7.4 6.7–6.9 7.2-7.6 7.2-7.4 7.4 7.4
INTRODUCTIONINTRODUCTION BIOMIMETIC SOLUTIONS
Blood HBSS EBSS SBFc SBFr SBFi Plasma
AIMS
To study the biomimetic synthesis and transformations of X-ray ACP and DCPD in conventional, revised and modified with Glycine SBF in order to elucidate some elementary processes of hard tissue mineralization and of the influence of micro-environmental surroundings on synthesis and transformation processes.
September 18-21, 2010, Nessebar, Bulgaria
September 18-21, 2010, Nessebar, Bulgaria
EXPERIMENTS BIOMIMETIC SYNTHESIS
SBFc-Cam
K2HPO4
SBFc-Pm
CaCl2
K2 H
PO
4
EXPERIMENTAL CONDITIONS
ACP DCPDCa/P = 1.67 Ca/P = 1pH 11.5 (KOH) pH 6
Room temperature; Fast mixing
FilteringWashing (water : acetone = 1:1)
Freezing -18oC Drying (37oC)
September 18-21, 2010, Nessebar, Bulgaria
EXPERIMENTSBIOMIMETIC TRANSFORMATION
SBF
Conditions
SBFc, SBFr, SBFg (~SBFc + Glycine)
37oC, pH 7.3;
Static regime; Solid/liquid ratio 4 g/l ;
Duration – 1, 2, 4, 6, 24 h, 3, 10 days, 1 and 6 months;
SBF solution changes - after 3-rd day
September 18-21, 2010, Nessebar, Bulgaria
EXPERIMENTSTHERMODYNAMIC CALCULATIONS
Precipitation phase Solutions
DATABASE for K, H of the reaction for complex formation and precipitation
pH, toC, Initial solution concentrationsINPUT
CALCULATION DAVIES equations,
OUTPUT
PHREEQCI computer program
Mg Na K Cl Ca/PO4
mmol/g 0.13 0.20 0.45 0.03 1.51 Enamel, Dentin and Bone0.02 - 0.29 0.22 - 0.39 2.10-4 - 0.02 0.03 – 0.1 1.6–1.7
RESULTSRESULTS ACP precipitation
Compositions of the initial precipitated solid phase
0 10 20 30 40 50 60 70 80 902-theta-Scale
XRD powder data and IR spectra
September 18-21, 2010, Nessebar, Bulgaria
4000 3500 3000 2500 2000 1500 1000 500
Wavenumbers, cm-1
P-O
O-P-O
C-O
RESULTSRESULTS ACP precipitation
September 18-21, 2010, Nessebar, Bulgaria
Formation of Posner clusters Ca9(PO4)6 covered with
hydrated shell
CO32- ions from the solution compete with and
partially replace the PO43- ions
RESULT Ca vacancies
Ca vacancies could be occupied by free Na+, K+ and Mg2+ ions from the solution RESULT CawMgxNayKz(PO4)v(CO3)6v
RESULTSRESULTS ACP precipitation
Thermodynamic calculated saturated indices (SI)
SI = lg(IAP/K)where IAP is an ion activity product, and K is a solubility product.
September 18-21, 2010, Nessebar, Bulgaria
0.11 0.78 1.09 2.628.37
26.6334.19
60.18
010203040506070
Mg(
OH)2
Mg3
(PO4)2
:8H2O
MgC
O3:M
g(OH)2
:3H2O
CaCO3
Ca3(PO4)
2(am
)
Ca8H2(P
O4)6:
5H2O
Ca9Mg(
HPO4)(PO4)
6
Ca10(P
O4)6(O
H)2
RESULTSRESULTS ACP precipitation
Ionic substitution Co-precipitation Incorporation of maternal solution
LEAD TO precipitation of calcium deficient, X-ray
amorphous phosphate mineral composition similar to those in the
hard tissues (enamel, dentin, and bone mineral)
September 18-21, 2010, Nessebar, Bulgaria
RESULTSRESULTS ACP transformation
0 10 20 30 40 50 60 70 800.0
0.2
0.4
0.6
0.8
1.0
1.2
1.4
1.6
SBFc SBFr SBFg
PO
43-, m
mo
l/l
time, h
0 10 20 30 40 50 60 70 800.25
0.50
0.75
1.00
1.25
1.50
1.75
2.00
2.25
2.50
2.75
3.00
3.25
3.50
Ca
2+, m
mo
l/l
time,h
SBFc SBFr SBFg
0 10 20 30 40 50 60 70 800.0
0.2
0.4
0.6
0.8
1.0
1.2
1.4
1.6
1.8
time,h
Mg2
+, m
mol
/l
SBFc SBFr SBFg
KINETIC STUDIES
Kinetic profile of PO43-, Ca2+ and Mg2+ contents in liquid
phases after different maturation times
PO43- Ca2+ Mg2+
September 18-21, 2010, Nessebar, Bulgaria
RESULTSRESULTS ACP transformation
KINETIC STUDIES
Kinetic profile of Ca/PO4 and Mg/Ca ratios in solid phases after different maturation times
0 10 20 30 40 50 60 70 80 90 700 750 800
0.010
0.015
0.020
0.025
0.030
0.035
0.040
0.045
0.050
0.055 SBFc SBFr SBFg
time, hm
ola
r ra
tio M
g/C
a0 10 20 30 40 50 60 70 80 90 700 750 800
1.40
1.42
1.44
1.46
1.48
1.50
1.52
1.54
1.56
1.58
1.60
1.62
1.64
1.66
1.68
mol
ar r
atio
Ca/
PO
4
time, h
SBFc SBFr SBFg
Ca/PO4 Mg/Ca
September 18-21, 2010, Nessebar, Bulgaria
RESULTSRESULTS ACP transformation
XRD STUDIES
XRD powder data of solid phases after different maturation times
SBFg
002 300
2-theta-Scale
initial
SBFc SBFr
10 20 30 40 50 60
10 20 30 40 50 60
10 20 30 40 50 60
1h
2h
4h
720h
002 300
002 300
September 18-21, 2010, Nessebar, Bulgaria
RESULTSRESULTS ACP transformation
IR STUDIES
IR spectra of solid phases after different maturation times
September 18-21, 2010, Nessebar, Bulgaria
3300 1500 1000 500
72 h
1 h
initial
initial
1 h
2 h
72 h
Wavenumbers, cm-1
SBFc
3300 1500 1000 500
C-O
P-OSBFg
SBFr
O-P-O
C-OH-O-H
3300 1500 1000 500
2 h
RESULTSRESULTS ACP transformation
IR STUDIES
0 1 2 3 4 5 6 7 8 9 70 72 74
0.04
0.06
0.08
0.10
0.12
0.14
0.16
Inte
grat
ed a
rea
ratio
CO
3/P
O4
stre
tchi
ng r
egio
ns
time, h
matured in SBFc matured in SBFr
Changes in the carbonate content of the samples treated in SBFc and SBFr
September 18-21, 2010, Nessebar, Bulgaria
RESULTSRESULTS ACP transformation
THERMODYNAMIC STUDIES
PrMg(OH)2 +Mg3(PO4)2.8H2O +MgCO3.Mg(OH)2.3H2O +CaCO3 +Ca3(PO4)2(am) +Ca8H2(PO4)6.5H2O +Ca9Mg(HPO4)(PO4)6 +Ca10(PO4)6(OH)2 +
M(m)---00
M(eq)-------0
Calculated saturated indices (SI)Calculated moles of the crystallized salts
0.00E+002.00E-034.00E-036.00E-038.00E-031.00E-021.20E-021.40E-021.60E-021.80E-02
mo
les
of
cry
sta
lliz
ed
sa
lts
CaCO3Ca3(PO4)2(am)
September 18-21, 2010, Nessebar, Bulgaria
RESULTSRESULTS DCPD precipitation
10 20 30 40 50 60 70 80
0
500
1000
1500
2000
2500
3000
2-theta-Scale
inte
nsity
4000 3500 3000 2500 2000 1500 1000 500
(HPO4)
(PO4)
(PO4)
H2O
(OH)
(P-OH)
(PO4)
Chemical composition of the precipitate
XRD and IR spectra of the precipitate
Ca/PO4 Mg Na K Cl mmol/g0.95 0.001 0.025 0.001 0.003
September 18-21, 2010, Nessebar, Bulgaria
RESULTSRESULTS DCPD precipitation
Thermodynamic simulations of the precipitation process in the studied system (pH 4 - 6)
September 18-21, 2010, Nessebar, Bulgaria
-15.00
-10.00
-5.00
0.00
5.00
10.00
15.00
20.00
25.00
30.00
35.00
CaHPO4:2H
2O
Ca3(PO4)
2(am
)
Ca8H2(P
O4)6:
5H2O
Ca9Mg(
HPO4)(PO4)
6
Ca10(P
O4)6(O
H)2
pH 6
pH 5
pH 4
RESULTSRESULTS DCPD transformation
KINETIC STUDIES
PO43- Ca2+ Mg2+
Kinetic profile of PO43-, Ca2+ and Mg2+ contents in liquid
phases after different maturation times
September 18-21, 2010, Nessebar, Bulgaria
0 10 20 30 40 50 60 70 800.0
0.5
1.0
1.5
2.0
2.5
3.0
3.5
4.0
4.5
5.0
5.5
6.0
PO
43-, m
mo
l/l
time, h
SBFc SBFr SBFg
0 10 20 30 40 50 60 70 800.0
0.5
1.0
1.5
2.0
2.5
3.0
3.5
Ca
2+, m
mol
/l
time, h
SBFc SBFr SBFg
0 10 20 30 40 50 60 70 800.0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
0.9
1.0
1.1
1.2
1.3
1.4
1.5
1.6
Mg
2+, m
mol
/l
time, h
SBFc SBFr SBFg
0 10 20 30 40 50 60 70 80
6.5
7.0
7.5
8.0
8.5
9.0
pH
time, h
SBFc SBFr SBFg
RESULTSRESULTS DCPD transformation
KINETIC STUDIES
0 10 20 30 40 50 60 70 800.90
0.95
1.00
1.05
1.10
1.15
1.20
Ca/
PO
4
time, h
SBFc SBFr SBFg
Kinetic profile of Ca/PO4 ratios in solid phases after different maturation times
September 18-21, 2010, Nessebar, Bulgaria
RESULTSRESULTS DCPD transformation
XRD STUDIES
XRD powder data of matured solid phases in different SBFs ___ DCPD; ____ DCPD+OCP; _____ OCP; _____ PCA
OC
P
OC
P OC
P
10 20 30 40 50 60 70
2-theta-Scale2-theta-Scale2-theta-Scale
10 20 30 40 50 60 70 10 20 30 40 50 60 70
SBFrSBFgSBFc
initial
3 days
10 days
1 month
6 months
September 18-21, 2010, Nessebar, Bulgaria
RESULTSRESULTS DCPD transformation
H-O-H
P-OC-O
P-O
Wavenumber/cm-1
C-O
P-O
P-O
H-O-H H-O-H
P-O
P-O
C-O
C-O
H-O-H
P-O
C-O
O-H H-O-H
O-H P-O-HP-O(H)
C-O
4000 3500 3000 2500 2000 1500 1000 500
P-O
P-O(H)
P-O-HP-O
H-O-H
O-HOH
4000 3500 3000 2500 2000 1500 1000 500
H-O-HOH
P-OP-O
P-O-HO-H
4000 3500 3000 2500 2000 1500 1000 500
SBFc SBFg SBFr
initial
72h
240h
720h
6 monts
IR studies
IR spectra of matured solid phases in different SBFsSeptember 18-21, 2010, Nessebar, Bulgaria
RESULTSRESULTS DCPD transformation
THERMODYNAMIC STUDIES
Initi
al
Dis
solu
tion
of D
CPD
Tran
sfor
mat
ion
of D
CPD
to O
CP
Dis
solu
tion
of O
CP
Tran
sfor
mat
ion
of O
CP
to H
A
5.5
6.0
6.5
7.0
7.5
8.0
8.5
Calculated data SBFc SBFcg SBFr
Experimental data SBFc SBFcg SBFr
pH
1hmaturation
72hmaturation
0.0
0.5
1.0
1.5
2.0
2.5
3.0
3.5
4.0
4.5
72hmaturation
1hmaturation
Calculated data SBFc SBFcg SBFr
Experimental data SBFc SBFcg SBFr
Ca
, m
mo
l/l
Calculated (____) and experimental (■ ;; ▲) pH and Ca values of liquid phase
September 18-21, 2010, Nessebar, Bulgaria
CONCLUSSIONS
Kinetic reasons determine the biomimetic precipitation of XRD amorphous calcium phosphate (ACP) and dicalcium phosphate dihydrate (DCPD) that are a less thermodynamic stable phases in comparison with calcium hydroxyapatite;
The precipitated salts always contain impurities due to the parallel co-precipitation, ion substitution and maternal liquor incorporation. Their content depends on the nature and crystallinity of the precipitants;
Both ACP and DCPD transform into poorly crystalline apatite in the studied SBFs microenvironments. An intermediate phase of octacalcium phosphate (OCP) was registered for DCPD only.
September 18-21, 2010, Nessebar, Bulgaria
CONCLUSSIONS
The SBF composition influences the polymorphous phase transformation and its rate - HCO3
- ions accelerate the transformation rates both of ACP and DCPD while the Glycine increases the transformation rate of ACP only.
The phase transformations of ACP and DCPD leaded to changes in the chemical compositions of solid and liquid phases. Thermodynamic simulations reveal that these phenomena could be explained by the processes of dissolution/crystallization/co-crystallization/ion-exchange.
September 18-21, 2010, Nessebar, Bulgaria
September 18-21, 2010, Nessebar, Bulgaria