biomimetic transformations of calcium phosphates - thermodynamic and kinetic studies d. rabadjieva...

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

[email protected]

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


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


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



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


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.


INTRODUCTIONINTRODUCTION BIOMIMETIC APPROACH

Biological mineralization (biomineralization) process of in vivo formation of inorganic minerals


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.



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)


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


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


4000 3500 3000 2500 2000 1500 1000 500

Wavenumbers, cm-1

P-O

O-P-O

C-O



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


Thermodynamic calculated saturated indices (SI)

SI = lg(IAP/K)where IAP is an ion activity product, and K is a solubility product.


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


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)


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+



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



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



IR STUDIES

IR spectra of solid phases after different maturation times


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


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



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)


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


RESULTSRESULTS DCPD precipitation

Thermodynamic simulations of the precipitation process in the studied system (pH 4 - 6)


-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


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


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



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



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


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


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.


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.


biomimetic transformations of calcium phosphates - thermodynamic and kinetic studies d. rabadjieva...

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