1. Biomimetic Transformations of Calcium Phosphates - Thermodynamic and Kinetic Studies
IGIC-BAS
D. Rabadjieva1, S. Tepavitcharova1, R. Gergulova1, R. Titorenkova2, E. Dyulgerova3, O. Petrov2, Chr. Balarew1
1Institute of General and Inorganic Chemistry, BAS
2Institute of Mineralogy and Crystallography, BAS
3Dental Medicine Faculty, University of Medicine
September 18-21, 2010, Nessebar, Bulgaria

2. ortho- (PO43–) meta- (PO3– ) pyro- (P2O74–) poly- ((PO3)nn–)
INTRODUCTION
CALCIUM PHOSPHATES
ortho- (PO43–)
meta- (PO3– )
pyro- (P2O74–)
poly- ((PO3)nn–)
September 18-21, 2010, Nessebar, Bulgaria

3. COPh precipitated from solutions
INTRODUCTION
CALCIUM ORTHOPHOSPHATES (COPh)
COPh precipitated from solutions
Ca/P pH lgKso
MCPM Ca(H2PO4)2.H2O
DCPD Ca(HPO4)2.2H2O
OCPt Ca8(PO4)4(HPO4)2.5H2O
ACP Ca9(PO4)6.nH2O
PCA Ca10-xx(PO4)6-x (HPO4)x.((OH)2-xx)
HA Ca10(PO4)6(OH)

4. INTRODUCTION COPh High-Temperature Forms
CALCIUM ORTHOPHOSPHATES (COPh)
COPh High-Temperature Forms
Ca/P Stability
MCPA Ca(H2PO4) < 100oC
DCPA Ca(HPO4) < 100oC
TCP -Ca3(PO4) < 800oC
-Ca3(PO4) < 1200oC
HA Ca10(PO4)6(OH) till ~1000oC
ТТCP Ca4(PO4)2О < 1500oC

5. INTRODUCTION Importance for the Biological system
CALCIUM ORTHOPHOSPHATES (COPh)
Importance for the Biological system
They are the main inorganic component of all body hard tissues
Biological apatite
Nano-sized
Poorly crystallized
Non-stoichiometric hydroxyapatite
Including Na, K, Mg, Cl, F and CO3
September 18-21, 2010, Nessebar, Bulgaria

6. INTRODUCTION MATERIALS FOR BONE REGENERATION CERAMICS CEMENTS
Good biocompatibility; Low toxicity HA
 / -TCP
HA + -TCP
DCPD
PCA
Porous, Nano-sized, Adequate biodegradable rates,
High strength and elasticity
September 18-21, 2010, Nessebar, Bulgaria

7. INTRODUCTION
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

8. It acts as a reservoir for
INTRODUCTION
BIOMIMETIC APPROACH
Biological mineralization (biomineralization)
process of in vivo formation of inorganic minerals
BLOOD PLASMA
It acts as a reservoir for
Ca2+ and PO43-
as well as
a maturation medium.
September 18-21, 2010, Nessebar, Bulgaria

9. INTRODUCTION BIOMIMETIC SOLUTIONS Blood HBSS EBSS SBFc SBFr SBFi
Plasma Na K Ca Mg Cl
HCO
HPO SO
Ca/P
Buffer TRIS TRIS TRIS
pH –

10. 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

11. Room temperature; Fast mixing
EXPERIMENTS
BIOMIMETIC SYNTHESIS
EXPERIMENTAL CONDITIONS
ACP DCPD
Ca/P = Ca/P = 1
pH (KOH) pH 6
Room temperature; Fast mixing
Filtering
Washing (water : acetone = 1:1)
Freezing -18oC Drying (37oC)
SBFc-Cam
SBFc-Pm
K2HPO4
CaCl2
K2HPO4
September 18-21, 2010, Nessebar, Bulgaria

12. EXPERIMENTS Conditions SBFc, SBFr, SBFg (~SBFc + Glycine)
BIOMIMETIC TRANSFORMATION
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
SBF
September 18-21, 2010, Nessebar, Bulgaria

13. pH, toC, Initial solution concentrations
EXPERIMENTS
THERMODYNAMIC CALCULATIONS
PHREEQCI computer program
INPUT
pH, toC, Initial solution concentrations
DATABASE for K, H of the reaction for complex formation and precipitation
CALCULATION
DAVIES equations,
OUTPUT
Precipitation phase
Solutions
September 18-21, 2010, Nessebar, Bulgaria

14. RESULTS ACP precipitation
Compositions of the initial precipitated solid phase
Mg Na K Cl Ca/PO4
mmol/g
Enamel, Dentin and Bone
– –1.7
XRD powder data and IR spectra
September 18-21, 2010, Nessebar, Bulgaria

15. RESULTS ACP precipitation
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
September 18-21, 2010, Nessebar, Bulgaria

16. where IAP is an ion activity product, and K is a solubility product.
RESULTS
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

17. Incorporation of maternal solution LEAD TO
RESULTS
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

18. RESULTS ACP transformation KINETIC STUDIES
PO Ca2+ Mg2+
Kinetic profile of PO43-, Ca2+ and Mg2+ contents in liquid phases after different maturation times
September 18-21, 2010, Nessebar, Bulgaria

19. RESULTS ACP transformation KINETIC STUDIES
Ca/PO Mg/Ca
Kinetic profile of Ca/PO4 and Mg/Ca ratios in solid phases after different maturation times
September 18-21, 2010, Nessebar, Bulgaria

20. XRD powder data of solid phases after different maturation times
RESULTS
ACP transformation
XRD STUDIES
XRD powder data of solid phases after different maturation times
September 18-21, 2010, Nessebar, Bulgaria

21. IR STUDIES RESULTS ACP transformation
IR spectra of solid phases after different maturation times
September 18-21, 2010, Nessebar, Bulgaria

22. IR STUDIES RESULTS ACP transformation
Changes in the carbonate content of the samples treated in SBFc and SBFr
September 18-21, 2010, Nessebar, Bulgaria

23. RESULTS ACP transformation THERMODYNAMIC STUDIES
Calculated saturated indices (SI) Pr
Mg(OH)
Mg3(PO4)2.8H2O +
MgCO3.Mg(OH)2.3H2O +
CaCO
Ca3(PO4)2(am) +
Ca8H2(PO4)6.5H2O +
Ca9Mg(HPO4)(PO4)6 +
Ca10(PO4)6(OH)2 +
M(m) -
M(eq) -
September 18-21, 2010, Nessebar, Bulgaria

24. RESULTS DCPD precipitation Chemical composition of the precipitate
Ca/PO4 Mg Na K Cl
mmol/g
XRD and IR spectra of the precipitate
September 18-21, 2010, Nessebar, Bulgaria

25. RESULTS DCPD precipitation
Thermodynamic simulations of the precipitation process in the studied system (pH 4 - 6)
September 18-21, 2010, Nessebar, Bulgaria

26. RESULTS DCPD transformation PO43- Ca2+ Mg2+ KINETIC STUDIES
Kinetic profile of PO43-, Ca2+ and Mg2+ contents in liquid phases after different maturation times
September 18-21, 2010, Nessebar, Bulgaria

27. RESULTS DCPD transformation KINETIC STUDIES
Kinetic profile of Ca/PO4 ratios in solid phases after different maturation times
September 18-21, 2010, Nessebar, Bulgaria

28. XRD powder data of matured solid phases in different SBFs
RESULTS
DCPD transformation
XRD
STUDIES
XRD powder data of matured solid phases in different SBFs
___ DCPD; ____ DCPD+OCP; _____ OCP; _____ PCA
September 18-21, 2010, Nessebar, Bulgaria

29. RESULTS DCPD transformation IR studies
IR spectra of matured solid phases in different SBFs
September 18-21, 2010, Nessebar, Bulgaria

30. RESULTS DCPD transformation THERMODYNAMIC STUDIES
Calculated (____) and experimental (■ ;; ▲)
pH and Ca values of liquid phase
September 18-21, 2010, Nessebar, Bulgaria

31. 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

32. 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

33. Thank you for your attention
September 18-21, 2010, Nessebar, Bulgaria