Fluoridated hydroxyapatites synthesized with organic phosphate ester

Fluoridatedhydroqapatites synthesizedwith organicphosphate ester M.Okazaki Depaltment of Dental Technology, Osaka University Faculty of Dentistry. l-8 (Received 2 1 November 1989; accepted 22 November 1989)

Yamadaoka, Suita, Osaka 565, Japan

Fluoridated hydroxyapatites with various fluoride contents were synthesized with organic phosphate ester monomer as a source of P at pfl 9.5 and 80°C. The a-axis dimensions of the fluoridated hydroxyapatites decreased with the increase of the fluoride content in the same way as the fluoridated hydroxyapatites synthesized with inorganic phosphate under the same conditions. However, for the same calculated degree of fluoridation in the feed solution, fluoridated hydroxyapatites synthesized with organic P had higher fluoride contents than those synthesized with inorganic P. The crystallinity of the former decreased initially, then increased with increasing fluoride content; but on the whole it was lower than that of the latter. The apparent solubility at pH 4.0 and 37°C decreased dramatically and monotonically with increasing fluoride content and approached an almost constant value at a fluoride content above that of fluorapatite, at which value calcium fluoride was detected. Keywords: Hydroxyapatites, fluoride, organic phosphate ester, crystallinity

Organic phosphate esters such as ATP, phospholipid and nucleotide have various important roles in the human body’. Being present inside and/or just outside the cells or the matrix vesicles’, it is speculated that they may also be associated directly or indirectly with the formation of calcium phosphates. Some researchers3r4 have reported that organic materials promote crystal growth of hydroxyapatite and that hydroxyapatite was formed epitaxially along the c-axis of collagen. 0thers5-7, however, have reported that organic materials inhibit the diffusion of surrounding ions and finally the crystal growth of hydroxyapatite. Furthermore, these organic monomers and polymers sometimes interact with the crystal interface, may often be adsorbed or chemisorbed directly to the crystal surface and then are apt to inhibit crystal growth. However, the role of organic materials in apatite formation is not fully understood. In our previous study’, using mono(methacryloyloyxethyI) acid phosphate (PM), organic metallic monomer crystals were formed at below pH 10, and at above pH 10 hydroxyapatite was formed. The crystallinity of this hydroxyapatite was inhibited compared with that of hydroxyapatite synthesized with inorganic phosphateg, lo. Fluoride, on the other hand, may be associated with formation of hydroxyapatite as a reaction accelator in its initial stage, and also contribute to prevention of dental caries. Previous studiesg-” have indicated that the crystallinity of fluoridated hydroxyapatites (theoretical formula: CaIo(P04)s Correspondence to Dr M. Okazaki.

(OH), _ 2x FZx) showed unexpected behaviour with the increase of fluoride content, and that the pattern of crystallinity behaviour was sensitive to trace elements. Since the presence of organic materials sometimes inhibits crystal growth, the crystallinity of hydroxyapatite may be affected by phosphate ester. In this study, physicochemical properties of fluoridated hydroxyapatites synthesized with organic phosphate ester were examined.

MATERIALS

Reactions of calcium ions and phosphate ester were examined at 80°C under the various fluoride concentrations. A higher temperature for synthesis than the physiological 37°C was necessary to identify the precipitates as apatites. By a modification of the method used for fluoridated hydroxyapatitesynthesisg, 125 ml of 200 mM Ca(CH3C00)2 Hz0 was fed into 250 ml of mechanically stirred 1.3 M CH,COONH, solution containing 60 mM PM (Kyoeisha Yushi Co. Ltd. Japan) and O-40 mM HF. The calcium solution was supplied at 125 ml/h with a Tokyo Rikakikai microtube pump. The pH was maintained at 9.5 f 0.1 by occasional addition of concentrated NH40H solution. The suspension was stirred at 80 + 1 “C for 2 h, then filtered, and the precipitate was washed with distilled water and dried at 80°C for 2 d. X-ray diffraction was employed to identify precipitates. 0

46

Biomaterials

199 1, Vol 12 January

AND METHODS

1991

Butterworth-Heinemann

Ltd. 0142-9612/91/01DD46-04

FHAp

Measurements

were

made

diffractometer

with

graphite-monochromatized

radiation

on

a

Rigaku

Denki

synthewed

with

organ/c

P. M.

Okarak,

X-ray CuKa

35 kV, 23 mA.

Calcium absorption

concentrations

were

spectrophotometry.

trations

were

Fluoride

concentration

electrode

determined

(Model

by

were

407,

determined

Total the

method

determined

Orion

by atomic

phosphate

concen-

of

with

20

25

30

35

20

25

3c

35

20

25

30

35

20

25

3c

35

20

25

30

35

20

25

30

35

Eastoe13.

a specific

ion

Co. Ltd. USA).

RESULTS Products

synthesized

monomer

PM as a source

weak

alkaline

formed, pH 9.5,

due

similar

of P varied metallic

alkaline

was to

angle

that

organic

phosphate

with

pH, apatite

formed;

organic of about

of the apatite

and

no

compound

crystals was

4”.

This

formed

X-ray

as

diffraction

found

crystallinity with

and were

of PM. Above

were

synthesized

ester

pH. At acidic

monomer

7. This is due to the hydrolysis

apatite

diffraction than

at higher

in Figure

patterns

the

pH, organic

but

shown

with

at

was

inorganic

lower

Figure

P under

of

To examine

the

physicochemical

hydroxyapatites

(PM-FHAp), chemical Table

synthesized

the

synthesis

compositions

7. With

solutron,

concentrations apatites

similar

synthesized

(FHAp).

with

apatite,

was

to those

1.67.

expected

However, induced

the solution, fluoride

as will

content

2 mmol/g.

In

synthesized

with

equal

degree

fluoride

ratio,

the relatively

Ca/P,

be discussed

was the

case

inorganic

of fluoridation 2

I

L..

the

that

value,

of these

samples,

an ,ncrease

at about

to

hydroxyapat/tes.

half

of

floor/de

IS observed

Degree

content.

WI the peak

due

For to the

28.3”.

than the ratio

3

shows

of

the

(PM-FHAp)

PM-FHAp

rn

are

more organic

as compared

with

confirmed’that Crystallinity

was

1,

apatitic

fluoride

was

a- and

and

patterns

were

detected

with

inhibited

inorganic

inorganic

of F- ions

whole

organic

phosphate

(FHAp).

This indicates

hydroxyapatites,

is needed

phosphate.

as a source

It has already

of a-axis dimension into the OH initially,

then

as shown

with

of P been

is mainly

sites. increased

In Figure

as compared

of

with

fluoridated

phosphate

decreased

on the

dimensions

the same curves.

typical

of fluoridation,

c-axis

synthesized

the contraction

due to substitution the degree

hydroxyapatites content

X,

it, calcium

hydroxyapatites

although

fluorapatite,

fluoride

fluoridation,

fluorapatite.

that

Xr = 1, the

of

i.e. twice

of fluondated equal

Both sets of data gave almost

hydrolysis

Above

fluoridated

in the feed

shows

synthesized

of

than

phosphate,

to the expected

Figure products

higher

later.

patterns

numencally

Above

phosphate

hydroxy-

Fm/POd3-

of

observed. Figure

hydroxy-

was higher

high

is

of CaF2

fluondated

previously

of theoretical

content

diffract/on X,

and 2.15

degree

phosphate

value. This may be due to the incomplete

of PM, which

almost

molar

in

in the feed

and

phosphate

to that

The

shown

of fluoridated

inorganic

also similar

are

calcium

1.45

20:“)

phosphate pH 9.5.

of fluoridation

the

The calcium-phosphate

precipitates

at

X-ray

(1 1 1) reflection

of fluori-

organic

done

precipitates

degree

or below,

were

with

was

of the

a calculated

X,, of 0.5

properties

2

fluoridation,

X =

conditions.

dated

2 Gi”)

the

with

4, and was

that

of

FHAp.

I

I

1

the calculated

solution.

X-ray

diffraction

at a constant

patterns

of

Below

the

pH of 9.5.

0a

.,

.‘“-

5 6

6.92

-

6.87

1 0.5

0

b F/gun? organic at pH

1

Typical X-ray

Pat 7 4 lb)

various

pHs

d/ffraction (al. together

patterns wth

ofprecipitates that

synthesized

I

1

I

X synthesized wth

with

Inorganic

P

F/gure

3

synthesrzed

~-AXIS wth

and

c-ax/s

morgamc

dimensions (FHAp)

(CJ

of

fluoridated

and

organic

hydroxyapatites (PM-FHAp)

(0)

phosphates.

&omater/als

199 1, Vol

12 Januav

47

FHAp

synthesized

IO

0 T

-6

with

organic

PC M.

Okazakl

Table

I

--IN

I

1

Chemical

compositions

of precipitates

X‘

Ca(mmol/g)

P(mmol/g)

F(mmol/g)

Ca/P

0 0.02 0.04 0.10 0.16 0.20 0.50 1 2

9.57 9.74 9.65 9.74 9.37 9.19 9.06 8.81 8.70

5.73 5.89 5.84 5.80 5.65 5.56 5.19 3.80 3.32

0 0.170 0.280 0.538 0.845 1.07 1.39 2.90 4.30

1.67 1.65 1.65 1.68 1.66 1.65 1.75 2.32 2.62

X,: the calculated degree of fluoridation in feed solution.

Figure organic (300)

4

Ctystallinity P (PM-FHAp)

and

(002)

of shown

fluoridated

hydroxyapatites

as the inverse

synthesized

with

breadths

of the

of the half-value

reflections.

Transmission electron microphotographs supported in part the crystallinity behaviour as shown in Figure 5. The samples with the fluoride content below 2 mmol/g consisted of coagulated needle-like crystals and the crystal size seemed to increase with the increase of fluoride content. Above 2 mmol/g, spherical crystals, which were speculated to be CaF*, were observed in part. This was supported by X-ray diffraction analysis (Figure 2). Figure 6 shows the apparent solubility of PM-FHAp, represented by the dissolved calcium concentration in 0.5 M

acetate buffer at pH 4.0 and 37°C after one month. The solubility decreased dramatically with the increase of fluoride content until near 2 mmol/g, which is equal that of stoichiometrical fluorapatite. Above this content, the decrease was almost negligible, because calcium fluoride was formed. The absolute value of the solubility was higher than those of fluoridated hydroxyapatites synthesized with inorganic phosphate’ ‘.

DISCUSSION Hydrolysis With the acetate buffer used in this study, hydroxyapatite was formed at above pH 9.5, although in non-buffered aqueous system, the boundary pH rose and hydroxyapatite was formed at above pH 10. This tendency was also observed in a previous study’ performed at 60°C. Therefore, it may be said that hydrolysis of PM depends strongly on pH

a

d Figure

5

48

Biomaterials

Odprn

e

Transmission

electron

199 1. Vol

micrographs

12 January

of precipitates.

f Fluoride

content

fmmol/g):

0 (a); 0.170

(b); 0.538

(cl;

1.39

(d); 2.90

(e); 4.30

ff).

FHAp

apatites

PM-FHAp

60

0.5M acetate buffer

50

r

( pH 4.0.37”C

1

the

calcium

imperfection, content

20

conclusion,

1

and

Apparent

solubility

of the

I

4

precipitates

6

as the

calcium

strength.

complex

Below

(PM-Ca)

pH 9.0

the

following

organic

this solubility

behaviour

to the crystallinity crystal

growth

phosphate

was

behaviour.

soluble

by organic apatite

Although

because

of

substances.

crystals

thecrystallinity

in comparison

synthesized

with fluoride

In

ester can serve as a P source

of fluoridated

increased

with

under

highly

is inhibited

with

and

the fluoridated

inorganic

decreases

dramatically

at high fluoride

the strong

stabilizing

action of fluoride.

This

study

was

63570915

was formed: 0

yH3

of

P, the solubility

content

because

of

ACKNOWLEDGEMENTS

dissolved

concentration.

ionic

with

A

5

shown

dramatically

but the concentration

of crystal

alkalineconditions.

6

organic

This may be due to the degree

organic

hydroxyapatites

Ftgure

by the

under the

of crystal

the solubility

calcium

of

than that of FHAp

the apatite

in the formation

IO

content,

was not parallel

the inhibition

3 F(mmol/g)

be shielded

was higher

although

Probably,

2

Okazaki

F- ions. Also, the

of PM decreased

of fluoride

same conditions.

30

I

PC M

of the diffusion

POd3-, OH-,

may

solubility

increase

dissolved

(

by retardation

Ca”,

surface

The apparent

I

orgamc

Solubility

0 0

growth

with

compound.

40

E -

may be inhibited

the main components, crystal

t

synthesized

supported

in part by Research

from the Ministry

of Education

No.

Grant

of Japan.

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