Author’s Accepted Manuscript

Biomimetic deposition of carbonate apatite and role

of carbonate substitution on mechanical properties
at nanoscale

Bimal K. Sarma, Pintu Barman, Bikash Sarma,

Apurba Das, Arup R. Pal
[Link]

PII: S0167-577X(16)31493-8
DOI: [Link]
Reference: MLBLUE21466
To appear in: Materials Letters
Received date: 4 May 2016
Revised date: 22 August 2016
Accepted date: 10 September 2016
Cite this article as: Bimal K. Sarma, Pintu Barman, Bikash Sarma, Apurba Das
and Arup R. Pal, Biomimetic deposition of carbonate apatite and role of
carbonate substitution on mechanical properties at nanoscale, Materials Letters,
[Link]
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Biomimetic deposition of carbonate apatite and role of carbonate substitution on
mechanical properties at nanoscale
Bimal K. Sarmaa,*, Pintu Barmana, Bikash Sarmaa, Apurba Dasa, Arup R. Palb

a
Department of Physics, Gauhati University, Guwahati 781014, India
b
Physical Sciences Division, Institute of Advanced Study in Science and Technology, Guwahati 781035,
India
*
sarmabimal@[Link]

ABSTRACT

This work reports on the role of carbonate substitution on the mechanical properties of calcium deficient

carbonate apatite which resembles closely the structure of bone apatite. The carbonate content determined

using vibrational spectroscopy suggests two stage substitutions. The residual stress is evaluated using

non-destructive X-ray diffraction procedure for these polycrystalline aggregates. The crystallinity

increases with deposition time and increased carbonate substitution does not impede the crystal growth

and no phase change is observed. A carbonate content of 6.34 wt. % corresponds to a minimum residual

stress without compromising the crystallinity of the films. The carbonate substitution might be associated

with the calcium vacancy which is considered as the thermodynamically feasible mechanism for B-type

carbonate apatite.

Keywords: biomimetic; hydroxyapatite; carbonate substitution; x-ray diffraction; residual stress

1. Introduction

Calcium phosphate (CaP) is a biomineral and the synthetic form has been used in orthopedic and dental

applications as well as drug delivery systems [1–4]. The most commonly used form is hydroxyapatite

(HAp) which exhibits excellent biocompatibility and bioactivity [1,4]. The bone mineral is calcium

deficient HAp often substituted with carbonate and contains trace ions available in the human blood

plasma. The replacement of PO43– by CO32–, i.e. B-type substitution is thermodynamically favorable if

charge compensation is mediated by the substitution of Na+ for Ca2+ [2,5,6]. Substitution may provoke

changes in the lattice parameters, crystallinity, and morphology of carbonate apatite. The presence of

defects and molecular substitution might influence the mechanical property. In addition, the impact of

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carbonate substitution on the residual stress and lattice distortion is not clear till date. In order to

understand the growth behavior and structure-mechanical property relationship, the biomimetic

depositions of nanostructured carbonate apatite have been carried out and the role of carbonate

substitutions on the observed mechanical properties is interpreted based on x-ray diffraction (XRD)

analyses coupled with Fourier transform infrared (FTIR) spectroscopy studies. The carbonate substitution

is linked to the calcium vacancy and mechanical properties of the apatite.

2. Experimental

A calcifying solution [3] which is a mixture of equal volume calcium and phosphate solutions is used for

the deposition of CaP films at 37 °C. The calcium solution is prepared by adding 0.0277 g CaCl2 in 100

ml de-ionized water and the pH is adjusted at 7.40±0.05 using Tris ((HOCH2)3CNH2). For the phosphate

solution, 0.1512 g NaHCO3 and 0.0950 g Na3PO4.12H2O are added sequentially in 100 ml de-ionized

water and the pH is adjusted at 7.40±0.05 using Hepes (C8H18N2O4S). The depositions are carried out on

polished and ultrasonically cleaned Ti foils (99.2% purity). For the deposition extending to 6 d, the

solution is refreshed after 3 d. FTIR spectra are acquired in the mid IR range with resolution of 4 cm–1.

The surface morphology and elemental analyses are examined in a field emission scanning electron

microscope (FE-SEM) equipped with energy dispersive x-ray (EDX) spectrometer. XRD patterns (2ϴ:

20–55°; grazing angle: 1°; step size: 0.02°) are recorded in a diffractometer (Cu Kα line: 1.5406 Å;

accelerating voltage: 40 kV; current: 40 mA). The instrumental broadening correction is performed by

recording the XRD pattern of the standard corundum under identical conditions. The (002) peak is chosen

for the residual stress measurements. The shift of the peak is recorded for grazing angles γ = 0.5–5.0°,

thus a range of tilt angle Ψ = ϴ–γ is obtained and the method presented in ref. [7] is adopted for analyses.

3. Results and discussion

FTIR spectra (Fig. 1A) reveal that the CaP films show characteristic peaks of hydroxyapatite with

carbonate substitution. The intense band at 1022 cm–1 is assigned as the ν3 band of PO43–. The detailed

band assignments are summarized in Table S1 of the supplementary data. The strong band at 1432 cm–1 is

assigned as the ν3 band of CO32– characteristic of B-type substitution of apatite [5,6]. The decomposition

of HCO3– ions during deposition results incorporation of CO32– in the CaP films according to the reactions

[8,9]

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NaHCO3  Na   HCO3 (1)

HCO 3  CO 2 OH  (2)

HCO 3 OH   CO 23  H 2 O (3)

An interesting observation is the increase of the intensity of the band at 1432 cm–1 with longer deposition

time while the band at 1022 cm–1 broadens to a noticeable extent indicating increased level of carbonate

with deposition time. A similar behavior was reported by Muller et al. [9] for carbonate apatite grown in

simulated body fluid (SBF) of varying HCO 3– concentrations. The maxima separation of ν4 band of PO43–

at 562 and 602 cm–1 and crystallinity index determined using the method described elsewhere [6] is

shown in Fig. 1B. The maxima separation decreases and crystallinity index increases with deposition time

till 24 h. Beyond 1 d, the maxima separation as well as the crystallinity index reach plateau suggesting no

significant change in the extent of ordering of the lattice with the deposition time. The integrated intensity

ratios of the carbonate (1432 cm–1) to the phosphate (1022 cm–1) bands are determined and the wt. % of

carbonate in the apatite is calculated using the method described in ref. [5]. The wt. % of CO32– increases

with the deposition time (Fig. 1C). The carbonate substitution is rapid till 24 h of deposition time.

However, 1 d onward the carbonate content increases as the deposition time increases but the substitution

takes place at a slower pace as compared to the substitutions till 24 h. Clearly, two stage substitutions of

carbonate are observed and the initial rapid substitution might be associated with the initial higher

deposition rate which in turn is related to the degree of supersaturation in the solution as confirmed by the

initial rapid variation of pH of the calcifying solution with substrate immersion time (Fig. S1 and

subsequent discussion in the supplementary data).

FE-SEM images (Fig. 2) show that the surfaces of carbonate apatite consist of spherical clusters with

diameter ~1 µm. The higher magnification images reveal that these clusters consist of nano-sized plates

arranged in flowery structures. EDX analyses (Fig. 2c and 2f) confirm that the Ca/P atomic percentage

ratio deviates from that of the stoichiometric HAp of 1.67 which further decreases with the deposition

time. Such calcium deficient plate shaped carbonate apatite is a characteristic of bone apatite.

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XRD patterns (Fig. 3A) confirm formation of HAp phase. The HAp is nanocrystalline and the

crystallinity increases with the deposition time. The most intense peak at ~32° might originate from the

collective contributions from (211), (112), and (300) diffraction planes which is obvious from the X-ray

line profile fitting as shown in Fig. 3C. Fig. 2B suggests that the higher deposition time stabilizes (112)

crystal face as compared to (211) face for 3h. The macrostrain changes considerably slowly with tilt angle

(Fig. 4A) for carbonate apatite deposited for 3 d and 6 d suggesting lesser distortion of HAp unit cell. The

residual stress are determined from the gradient of the plots using the estimated values of the Young’s

modulus (138.4 GPa) and the Poisson ratio (0.31) of carbonate apatite along [001] direction and applying

the method described elsewhere [7,8]. The detailed calculations for the Young’s modulus, Poisson ratio

and residual stress are shown in the Table S2 of the supplementary data. The residual stress is quite high

for 3 h and 12 h and is the lowest for the deposition time of 3 d. The effect of carbonate substitution on

the residual stress and unit cell parameters are shown in Fig. 4B. The residual stress decreases as the

carbonate content increases and the residual stress attains the lowest value corresponding to the

deposition time of 3 d. The residual stress is significantly high when carbonate substitution is rapid even

if the carbonate content is low. The CO32– structure is pyramidal and possesses lower rotational symmetry

compared to the tetrahedral PO43– [8]. Accordingly, HAp lattice with B-type substitution where the

carbonate content is higher must experience lower residual stress. However, beyond a certain limit, the

residual stress marginally increases with the carbonate content which could be attributed to the higher

calcium vacancies associated with the charge compensation due to substitution of PO 43– by CO32–. This

observation fairly agrees with FTIR and EDX studies. The unit cell parameters approaches standard HAp

results for carbonate apatite deposited for 6 d and deviations in a, c, and V for shorter deposition time

cannot be solely attributed to the carbonate content although the role of carbonate substitution is directly

reflected in the residual stress and calcium vacancy.

4. Conclusion

Nanostructured HAp films are deposited at physiological temperature and pH of human blood plasma.

FTIR, FE-SEM, and XRD studies confirm formation of calcium deficient carbonate apatite with flowery

structure characteristic of bone apatite. The carbonate content increases rapidly till 1 d of deposition and

then increase slowly. The extent of carbonate substitution does not inhibit the HAp crystal growth.

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However, rapid substitution results higher residual stress at low carbonate level. The calcium deficiency

is related to the carbonate level and the higher the carbonate content the greater is the calcium deficiency.

Acknowledgements

BKS acknowledges the University Grants Commission for the financial support under the Grant No. F.

30–20/2014 (BSR).

References

[1] D.F. Williams, Biomaterials 29 (2008) 2941.

[2] L. Wang, G.H. Nancollas, Chem. Rev. 108 (2008) 4628.

[3] A. Bigi, E. Boanini, B. Bracci, A. Facchini, S. Panzavolta, F. Segatti, L. Sturba, Biomaterials 26

(2005) 4085.

[4] E. Altinoglu, J.H. Adair, Future Oncol. 5 (2009) 279.

[5] A. Grunenwald, C. Keyser, A.M. Sautereau, E. Crubezy, B. Ludes, C. Drouet, J. Archaeol. Sci. 49
(2014) 134.

[6] A. Shemesh, Geochim. Cosmochim. Acta 54 (1990) 2433.

[7] A.J. Perry, J.A. Sue, P.J. Martin, Surf. Coat. Technol. 81 (1996) 17.

[8] B.K. Sarma, A. Das, P. Barman, A.R. Pal, J. Phys. D: Appl. Phys. 49 (2016) 145304.

[9] L. Muller, F.A. Muller, Acta Biomater. 2 (2006) 181.

Fig 1: A. FTIR spectra of carbonate apatite for deposition time (a) 3 h, (b) 12 h, (c) 1 d, (d) 3 d, and (e) 6

d, B. variations of maxima separation of ν4 of PO43– and crystallinity index with deposition time, C. plot

of wt % of CO32– in apatite with deposition time.

Fig. 2: FE-SEM images and EDX spectra of carbonate apatite deposited for (a)–(c) 3 d, (d)–(f) 6 d.

Fig. 3: A. XRD patterns of carbonate apatite for deposition time (a) 3 h, (b) 12 h, (c) 3 d, (d) 6 d. The

standard data for HAp is plotted as vertical lines. The crystallite size is shown in the inset. Peaks marked

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by asterisk are the substrate contributions, B. XRD patterns showing the possible change of orientation of

crystals, C. X-ray line profile fitting of carbonate apatite deposited for 6 d.

Fig. 4: A. Sin2Ψ plots for carbonate apatite for different deposition time, B. Carbonate substitution

dependence of (a) residual stress, (b) lattice parameter c, (c) lattice parameter a, (d) unit cell volume. The

standard data a0, c0, and V0 are shown as dotted lines.

Highlights

 Biomimetic deposition of calcium deficient carbonate apatite with flowery structure

 Two stage carbonate substitution is predicted by vibrational spectroscopy

 Residual stress is evaluated using non-destructive X-ray diffraction procedure

 Role of carbonate substitution is reflected in the residual stress and calcium vacancy

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