Materials Science and Engineering C 35 (2014) 106–114

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Materials Science and Engineering C

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Preparation and characterisation of nanophase Sr, Mg, and Zn substituted

hydroxyapatite by aqueous precipitation
Sophie C. Cox a, Parastoo Jamshidi b, Liam M. Grover b, Kajal K. Mallick a,⁎
a
Warwick Manufacturing Group, University of Warwick, Coventry CV4 7AL, UK
b
School of Chemical Engineering, University of Birmingham, Edgbaston, Birmingham B15 2TT, UK

a r t i c l e i n f o a b s t r a c t

Article history: Hydroxyapatite (HA) substituted with 2 mol% Sr, 10 mol% Mg, and 2 mol% Zn were precipitated under identical
Received 28 May 2013 alkaline conditions (pH 11) at 20°C from an aqueous solution. As-synthesised materials were confirmed to be
Received in revised form 26 September 2013 phase pure by XRD and samples prepared in air contained surface adsorbed CO2 as observed by FTIR. SEM studies
Accepted 19 October 2013
revealed a globular morphology and agglomeration behaviour, typical of precipitated nHA. EDS spectra con-
Available online 6 November 2013
firmed nominal compositions and substitution of Sr, Mg and Zn. At the levels investigated cationic doping was
Keywords:
not found to radically influence particle morphology. An indication of the potential in-vivo bioactivity of samples
Bioceramics was achieved by analysing samples immersed in SBF for up to 28 days by interferometry and complementary
Substituted hydroxyapatite SEM micrographs. Furthermore, a live/dead assay was used and confirmed the viability of seeded MC3T3 osteo-
Aqueous precipitation blast precursor cells on HA and substituted HA substrates up to 7 days of culture.
Simulated body fluid © 2013 Elsevier B.V. All rights reserved.
Osteoblasts

1. Introduction research community [1,2,5,6]. In particular, improved degradation of

Calcium phosphates (CaPs) occur in nature and have been
widely used as biomaterials, specifically in the repair or regeneration
of hard tissues. Amongst this group of materials, hydroxyapatite
[(Ca10(PO4)6(OH)2)–(HA)] is the most well-known since it is crystallo-
graphically and chemically similar to the mineralised constituent of
hard tissues [1]. These attributes infer excellent cytocompatibility and
osteoconductivity. It is, however, important to note that native bone
apatite differs from stoichiometric HA in a number of ways, including
non-stoichiometry, nanosized crystal dimensions (nHA), and a relative
crystallinity of 33–37 when stoichiometric HA is assumed to be 100
[2,3].
The non-stoichiometry of biological apatites arises from the incorpo-
ration of foreign ions, either into the crystal lattice or adsorbed onto the
surface. Table 1 presents a comparison of the reported compositions and
unit cell dimensions of bone apatite and nominally calculated stoichio-
metric HA. The variety of cationic and anionic substitutions is justified
by the flexibility of the apatite structure [4]. Reports confirm that
substituting ions present in native hard tissues such as strontium (Sr),
magnesium (Mg), and zinc (Zn) into CaPs can lead to advantageous ef-
fects on biomaterial properties, such as the degree of structural order
(i.e. crystallinity), solubility in chemical solvents, surface charge and dis-
solution rate under simulated physiological conditions. This may in-
turn influence bioactivity, thus attracting much interest in the wider
⁎ Corresponding author. Tel.: +44 2476522324.
E-mail address: k.k.mallick@warwick.ac.uk (K.K. Mallick).
less crystalline HA has been shown to be advantageous due to the ability
of these surfaces to provide a dynamic zone for dissolution and re-
precipitation [7–9].
The doping of naturally occurring ionic species into CaPs, such as HA,
not only alter the space group of the crystal structure, morphology, ther-
mal stability, and mechanical properties but also play an important role
in the biological responses of bone cells [10]. For instance, the depletion
of Mg ions has been linked to the cessation of bone growth, decreased
osteoblastic activity, as well as reduced mechanical properties [11–13].
This makes Mg ions one of the important elements to consider when
substituting into the HA lattice. Equally important is the ability of Sr to
increase the number of osteoblasts in-vitro, whilst at the same time de-
creasing the number and activity of osteoclasts [14–16]. This effect is
particularly beneficial in the treatment of osteoporosis [4,17]. Zn as a
substituting element has been shown to promote osteoblast activity
[18], and deficiency of Zn has been reported to reduce bone density
and ductility resulting in an increased probability of fracture [19]. Of
particular interest to the present study is the potential of these bivalent
ions to enhance the bioactivity of CaPs [2,20–23].
Although there are reported studies in this area, to the best of our
knowledge a systematic characterisation and comparison of the physi-
cochemical properties of Sr, Mg, and Zn substituted HA produced
under ‘identical’ experimental conditions has not been previously re-
ported. Notably, it is important that samples be prepared under ‘identi-
cal’ conditions to determine the explicit influence of cation doping.
Other reaction conditions, such as pH and temperature, are known to af-
fect parameters that are influential in determining the biological perfor-
mance of HA. Therefore, the authors recognise that it is vital to control

0928-4931/$ – see front matter © 2013 Elsevier B.V. All rights reserved.
http://dx.doi.org/10.1016/j.msec.2013.10.015
 S.C. Cox et al. / Materials Science and Engineering C 35 (2014) 106–114 107

Table 1 was separated from the supernatant by filtering, washed with DI

Composition and lattice parameters of bone compared with nominally calculated water, dried and finally ground into a micronised powder.
stoichiometric HA as reported by [2,3].
To utilise the CO2 present in the atmosphere and form naturally car-
Composition Bone [3] Bone [2] Stoichiometric HA bonated HA the procedure outlined above was performed in air. In con-
Calcium (wt %) 34.8 36.6 39.6 trast to other studies [26,27], since the amount of carbonate substituted
Phosphorus (wt %) 15.2 17.1 18.5 into the HA structure was not accurately controlled, the products are not
Carbonate (wt %) 7.40 4.8 – deemed as carbonated HA (CHA).
Sodium (wt %) 0.90 1.0 –
Magnesium (wt %) 0.72 0.6 –
Chloride (wt %) 0.13 0.1 – 2.2. In-vitro analysis
Pyrophosphate (wt %) 0.07 – –
Potassium (wt %) 0.03 0.07 – Testing was performed on substrates of substituted HA samples and
Fluoride (wt %) 0.03 0.1 – a pure HA sample, synthesised under the same experimental conditions,
Strontium (wt %) – 0.05 –
was used as a control.
Zinc (ppm) – 39 –
Chromium (ppm) – 0.33 –
Cobalt (ppm) – b0.025 – 2.2.1. Pellet preparation
Manganese (ppm) – 0.17 – Each sample was pressed into 10 mm pellets by applying an average
Silicon (ppm) – 500 –
uniaxial force of 800 N to 0.25 ± 0.02 g of as-synthesised powder using
A axis (Å) 9.410 – 9.430
C axis (Å) 6.890 – 6.891 a Simplemet 2 press (Buehler, UK). To reduce the overall porosity the
pellets were air sintered to 600°C at a constant ramp rate of 1°C/min,
dwelled at this temperature for an hour and then cooled at a rate of
such reaction conditions to determine the sole influence of the investi- 1°C/min. The powders were characterised by XRD before and after
gated cations. sintering to ensure no phase change occurred.
HA containing 2 mol% Sr, 10 mol% Mg, or 2 mol% Zn have been syn-
thesised using an aqueous precipitation method under ‘identical’ experi- 2.2.2. Simulated body fluid test
mental conditions. The phase purity, crystallinity, particle morphology, SBF exhibits ion concentrations nearly equal to those of human
level of ionic substitution, and thermal stability were assessed by X-ray blood plasma. It has been suggested that the potential in-vivo bioactivity
diffraction (XRD), scanning electron microscopy (SEM), energy disper- of a biomaterial may be examined by its ability to form apatite on its sur-
sive spectroscopy (EDS), simultaneous differential thermal analysis face whilst immersed in SBF.
thermogravimetry (DTA–TGA), and Fourier transmission infrared spec- SBF solution was produced in accordance to ISO 23317:2007 by the
troscopy (FTIR). Simulated body fluid (SBF) studies up to 28 days were procedure proposed by Kokubo et al. [28], as outlined in Table 2. Hygro-
carried out in order to provide an indication of the bioactivity of the scopic reagents, labelled as 4, 6, 8 and 9 in Table 2, were weighed
substituted samples (in accordance to ISO 23317:2007). Furthermore, a and quickly added to the solution. During preparation the solution tem-
live/dead assay was performed to assess the viability of cultured MC3T3 perature was maintained at 36.5 ± 1.5 °C and stirred continuously at
osteoblast precursor cells seeded on pure and substituted HA substrates. 200 rpm.
Four pellets for each sample were immersed in SBF so as to cover
2. Experimental the entire sample surface and were placed in an incubator at 37°C. Pel-
lets were removed, dried, and analysed after 1, 7, 14, and 28 days of
Identical preparative conditions of alkalinity (pH 11) and tempera- immersion.
ture (20°C) were used to synthesise a control sample of pure HA as
well as HA substituted with 2 mol% Sr, 10 mol% Mg, and 2 mol% Zn 2.2.3. Cell culture
by an aqueous precipitation (AP) method. These levels of Sr2+, Mg2+ The viability of MC3T3 osteoblast precursor cells were analysed by
and Zn2+ cations are intended to replace Ca2+ within the HA lattice using a Live/Dead® Viability/Cytotoxicity kit after culturing for 1, 3, 5,
[2,24,25]. In order to compare the final products the experimental ratios and 7 days. A final cell density of 2 × 104 cells/well were seeded direct-
of Ca/P or (Ca + M)/P (where M = Sr, Mg or Zn) were maintained at ly on to the surface of pellets in 24-multiwell plates that were previous-
1.67. All reagents were used without further purification and purchased ly coated with 1.5 mL of Sylgard type 184 silicone Elastomer (Dow
from Sigma Aldrich (UK) unless otherwise stated. Corning Corporation, Midland, MI). Calcein-AM and propidium iodide
(1 mg/mL; Molecular Probes, Invitrogen, UK) were used to stain live
2.1. Aqueous precipitation method to produce pure and substituted HA and dead cells, respectively in the dark.

A stock solution of PO3−4 was prepared by dissolving 0.03 M of
diammonium hydrogen phosphate [(NH4)2HPO4 ≥ 98.0%] in deionised
(DI) water. 0.05 M of calcium nitrate tetrahydrate [Ca(NO3)2 · 4H2O ≥
99.0%] was dissolved in DI water to form a Ca2+ solution. To form HA
substituted with Sr, Mg or Zn the amount of calcium nitrate was adjust-
ed to reflect the substitution of 2 mol% Sr, 10 mol% Mg and 2 mol% Zn.
An appropriate amount of strontium nitrate (Sr(NO3)2 ≥ 99.0%), mag-
nesium nitrate ((Mg(NO3)2 ≥ 99.0% — Johnson Matthey, UK) or zinc ni-
trate hexahydrate (Zn(NO3)2 · 6H2O ≥ 98.0%) was then dissolved in
the solution containing Ca2+. All solutions were stirred until the re-
agents were fully dissolved. Using a pH metre (pHTestr10, Eutech In-
struments, USA) the pH of the solutions were measured and adjusted
to 11 ± 0.1 using an appropriate amount of ammonium hydroxide
(NH4OH 28.0–30.0%) solution. Using a burette the PO3− 4 solution was
added dropwise into the solution containing Ca2+ whilst stirring at
400 rpm and maintaining a pH of 11 ± 0.1. The final solution was
stirred for 1.5 h at room temperature (20°C). The formed precipitate
2.3. Material characterisation
A Bruker D5000 X-ray diffractometer in Bragg–Brentano geometry
with a monochromatic CuKα radiation (λ = 1.54056 Å), was used to
analyse the phase purity and degree of crystallinity of as-synthesised
samples. Each run was performed between 2θ values 5 and 70° with a
step size of 0.02° and a count time of 15 s per step using an aluminium
sample holder. The patterns were matched to the Joint Committee on
Powder Diffraction Standards (JCPDS) reference files. An estimation of
the crystallite size was obtained from the line broadening of the (002)
peak of HA using the Scherrer equation (Eq. (1)):
L ¼ K λ=B cos θ ð1Þ
where L = crystallite size, K = Scherrer constant dependent on crystal
habit (0.9), λ = X-ray wavelength, B = peak width at half maximum
intensity (in radians) and θ = Bragg angle.
108 S.C. Cox et al. / Materials Science and Engineering C 35 (2014) 106–114

Table 2
Summary of the procedure used to prepare 1000 mL of SBF solution in accordance to ISO 23317:2007.

Order Reagent Purity (%) Amount Notes

1 DI water N/A 400 mL N/A

2 Sodium chloride (NaCl) ≥99.5 7.996 g Reagents 1–6 including washings, were added individually
3 Sodium bicarbonate (NaHCO3) 99.7–100.3 0.350 g allowing enough time for each to fully dissolve between each
4 Potassium chloride (KCl) ≥99.0 0.224 g addition
5 Potassium phosphate dibasic trihydrate (K2HPO4 · 3H2O) ≥99.0 0.228 g
6 Magnesium chloride hexahydrate (MgCl2 · 6H2O) ≥99.0 0.305 g
7 Hydrochloric acid (HCl) 1M 40 mL HCl was added in 5 mL amounts and the solution pH was
allowed to stabilise in-between. Washings were also added.
8 Calcium chloride (CaCl2) ≥93.0 0.278 g CaCl2 was dissolved in 25 mL of DI water and added to solution,
including washings
9 Sodium sulfate (Na2SO4) ≥99.0 0.071 g Reagent 9 and washings added
10 Tris-(hydroxymethyl)aminomethane ((CH2OH)3CNH2) ≥99.8 6.057 g TRIS was dissolved in 50 mL of DI water and added 5 mL at a
11 HCl 1M Appropriate amount time, allowing the pH to stabilise in between up to pH 7.45. HCl
for pH adjustment was then added to lower the pH to 7.42. TRIS was added up to
pH 7.45 and this was repeated until all of the TRIS was added,
including washings. Final pH was adjusted to 7.25 using HCl

The size and morphology of particles were investigated by a Zeiss
Supra55 FEGSEM. The specimens containing Sr, Mg or Zn were exam-
ined by an energy-dispersive spectroscope (X-max 50 mm2, Oxford In-
struments, UK) to identify and quantify elemental composition.
The chemical absorption characteristics of dried powders were ob-
served by FTIR using a Spectrum One FTIR spectrometer (PerkinElmer,
UK). For each spectrum 8 scans were produced between 4000 and
550 cm−1.
Thermal analysis of powders of substituted HA as well as a control
HA sample were determined by simultaneous DTA–TGA using a TGA/
DSC-1 STARe instrument (Mettler-Toledo, UK) with STARe software.
10 ± 0.05 mg of samples were heated in alumina crucibles from 30–
1300°C at a constant ramp rate of 20 °C/min in flowing air.
The potential growth of apatite on the surface of pellets immersed in
SBF was characterised by SEM and EDS. Furthermore, the average
roughness (Ra) of the pellet surface was profiled quantitatively using a
NT2000 interferometer (Wyco, UK). A fluorescence microscope fitted
with a mercury lamp (Carl Zeiss Ltd, Hertfordshire, UK) at magnifica-
tions of ×20 was used to visualise cells cultured on the surface of pellets.
3. Results
3.1. Crystal structure
3.2. Microstructural development
Table 4 summarises the average particle and agglomerate sizes, as
well as the typical particle morphology observed by SEM. No discernible
difference in the morphology of globular HA and substituted HA sam-
ples was observed as illustrated in Fig. 2.
3.3. Molecular structure
The major functional groups including hydroxyl, phosphate and car-
bonate were identified by FTIR. The observed vibrational frequencies
and corresponding band assignments for carbonated HA as reported
by Panda et al. and Koutsopoulos et al. [29,30] are summarised in
Table 5. As expected, due to sample preparation in air, the formation
of a broad band between 1250 and 1520 cm−1 known to be associated
with adsorption of atmospheric CO2 was observed in all FTIR spectra.
3.4. Thermal behaviour
Simultaneous thermal analysis (DTA–TGA) was performed on all
samples and a summary of the weight loss as well as residue observed
by TGA can be seen in Table 6 for specific temperature ranges (0–200,

Table 3 summarises the results of the phase purity and average crys-
tallite size of all samples analysed by XRD and Scherrer equation
(Eq. (1)), respectively. All samples matched with the standard JCPDS
reference (09–432) file for synthetic HA. No other secondary phases
such as monetite or brushite were identified thus suggesting the prod-
ucts obtained were phase pure. Fig. 1 shows the characteristic peaks of
all samples. Furthermore, XRD analysis of post-sintered pellets prepared
for SBF testing confirmed that no subsequent phase change occurred
due to the sintering protocol employed. An increase in the sharpness
of XRD peaks post-sintering was observed, which is associated to an in-
crease in crystallinity due to the heat treatment.

Table 3
Summary of the XRD characterisation of pure and substituted HA samples produced by AP
method.

Sample Element Theoretical substitution Main Average crystallite

substituted (wt%) phase(s) size⁎ (nm)

AP01 N/A (HA) N/A HA 15.52

AP02 Sr 2.0 HA 10.54
AP03 Mg 10.0 HA 13.75
AP04 Zn 2.0 HA 11.42
⁎ As calculated by Scherrer equation [Eq. (1)] on (002) (25.879°) peak of HA. Fig. 1. Typical XRD patterns of characteristic HA peaks for precipitated samples.
 S.C. Cox et al. / Materials Science and Engineering C 35 (2014) 106–114 109

Table 4 3.7. Cell culture

Summary of the average size and morphology of pure and substituted HA particles as ob-
served by SEM.
Live (green) and dead (red) assay was used to assess the viability of
Sample Average Average Typical morphology MC3T3 osteoblast precursor cells, which were seeded onto the surface
particle agglomerate of pellets formed from powdered samples. Cells were found to be viable
size (μm) size (μm)
on all substrates over the entire culture period (Fig. 6). An increase in
AP01 2.72 ± 0.51 24.61 ± 4.27 • Randomly orientated globular particles the density of osteoblasts between days 1 and 7, as well as a change in
AP02 4.89 ± 0.86 40.52 ± 3.67 • Typically rounded particle edges
the typical morphology from rounded (days 1 and 3) to elongated
AP03 1.61 ± 0.42 19.86 ± 4.17 • All samples were largely agglomerated
AP04 3.30 ± 0.43 29.53 ± 5.08 (days 5 and 7) was visually observed. Very few dead cells were demon-
strated and therefore cell death was attributed to the sensitivity of the
culture process.
200–600 and 600–1300°C) that were found to exhibit corresponding
thermal reactions (Fig. 3). 4. Discussion

3.5. Compositional analysis
The batch levels of Sr, Mg, and Zn substitution were measured by
EDS and all expected elemental peaks were identified including Sr, Mg
and Zn suggesting that substitution elements were incorporated into
the precipitated HA. Table 7 presents a summary of the average concen-
tration of Sr, Mg or Zn substitution, Ca:P ratio, as well as Ca + M:P ratio
(where M refers to the substituting cation) calculated from three EDS
measurements performed at different sample locations.
3.6. Simulated body fluid test
The effect of immersion time on the average surface roughness (Ra)
measured by interferometry is shown graphically in Fig. 4. Ra denotes
the average roughness calculated over the entire measured array as cal-
culated by Eq. (2):
Ra ¼ ðjZ 1 j þ jZ 2 j þ jZ 3 j þ … þ jZ n jÞ=N
where Z = profile height, and N = number of data points.
Corresponding SEM micrographs and EDS analysis of the surface of
HA pellets illustrated as well as confirmed the growth of carbonated ap-
atite for all samples from day 1 onwards. Fig. 5 shows typical micro-
graphs of the grown apatite (red circle) on the surface (blue arrow) of
pellets.
XRD patterns for as-synthesised samples exhibited broadening
of characteristic peaks ((002) (25.879°), (211) (31.773°), (112)
(32.196°), and (300) (32.902°)), which may be associated to the low
degree of crystallinity known to be typical of precipitated HA [31]. The
degree of broadening was found to exhibit the following trend:
AP03 N AP02 ≈ AP04 N AP01 (Fig. 1). It was expected that an increase
in broadening would relate to a reduction in crystallite size. However,
the crystallite size calculated using the Scherrer equation was observed
to exhibit a different trend: AP02 b AP04 b AP03 b AP01. Specifically,
Mg–HA (AP03) was shown to exhibit the broadest characteristic peaks
despite a larger crystallite size than Sr–HA and Zn–HA. Kanzaki et al.
[32] reported that Mg2+ ions may act as an inhibitor of HA nucleation
and subsequently crystallisation. Likewise the XRD results from this
study infer this assertion. Other studies concerning the synthesis of
Sr–HA have shown the refinement of peaks with increasing Sr doping
[4], which was not demonstrated in the presented XRD patterns but
this may be due to the relatively low levels incorporated. In relation to
other works concerning Zn–HA the XRD pattern for AP04 exhibits a
ð2Þ higher degree of broadening and a significantly lower crystallite size,
and this may be attributed to the relatively low preparation tempera-
ture (20°C) as well as short ageing time (1.5h) [25]. In this study, no dis-
cernible shifts in the characteristic HA peaks were observed for AP02 or
AP04 in relation to AP01, which is consistent with other studies [4,25].
In contrast, a shift to a higher angle for the (002) peak of Mg–HA
(AP03) is shown in Fig. 1, which may indicate lattice incorporation of
the dopant since this would be associated with a reduction in the lattice
parameters. This suggestion is supported by other studies involving

Fig. 2. SEM micrographs illustrating typical particle morphology of pure and substituted HA samples a) AP01, b) AP02, c) AP03, and d) AP04 (scale bars 5 μm).
110 S.C. Cox et al. / Materials Science and Engineering C 35 (2014) 106–114

Table 5
Summary of vibrational frequencies observed by FTIR for carbonated HA as well as synthesised pure and substituted HA samples. Assignments made according to the data reported by
Panda et al. and Koutsopoulos et al. [24,25].

Assignments

Samples Adsorbed Soluble CO2 (ν3) Absorbed CO2−

3 groups PO3−
4 bend PO3−
4 stretch CO2−
3 OH PO3−
4 bend
H2O and (ν2) H2O (ν2) (ν3) (ν3) (ν1) group structural (ν4)

Carbonated HA [24,25] 2750–3750 2331–2360 1640 1420, 1455 1030, 1090 961 873 635–632 604–600
AP01 2750–3438 2329–2359 1638 1422, 1455 1026, 1090 962 872 633 600
AP02 2750–3440 2324–2360 1626 1455 1029, 1089 962 873 633 603
AP03 2750–3560 2340–2356 1617 1456 1029, 1089 962 871 634 604
AP04 2750–3600 2331–2355 1628 1454 1029, 1089 961 873 634 603

the surface dominated properties associated with nanosized crystallites,

Table 6 which causes them to clump together as observed in Fig. 2. The trend in
Summary of weight loss exhibited by TGA for HA alone (AP01) and HA samples exhibiting average particle sizes calculated from SEM micrographs was not consis-
substitutions AP02 (2 mol% Sr), AP03 (10 mol% Mg), and AP04 (2 mol% Zn).
tent with the average crystallite sizes calculated from XRD line broaden-
Sample Weight loss (%) Residue (%) ing analysis. It is suggested that the previously mentioned inaccuracies
Total 30–200 °C 200–600 °C 600–1300 °C of the Scherrer equation, the high degree of peak broadening (Fig. 1),
and the effect of clumping caused by nanosized particles may explain
AP01 8.44 4.91 2.38 1.15 91.56
AP02 9.43 5.17 3.03 1.23 90.57 the inconsistent trends observed between Scherrer analysis and SEM
AP03 15.28 10.27 3.95 1.06 84.72 measurements.
AP04 9.29 5.03 2.98 1.28 90.71 The levels of Sr, Mg, and Zn substitutions investigated within this
study were not found to influence particle morphology (Fig. 2). Previous
studies concerning the AP of HA have indicated that experimental con-
Mg–HA that have shown lattice incorporation up to 10% by NMR [10]. It ditions, such as pH and temperature, influence the resultant phase and
should be noted, when comparing to other studies it is difficult to ob- particle morphology [42–45]. Since ‘identical’ pH (11) and temperature
serve the key factor influence of substitutions due to the known discern- conditions (20°C) were maintained in the present study this may ex-
ible effect of other reaction parameters (e.g. temperature, pH, ageing) on plain why no discernible change in morphology was observed.
properties such as crystallinity and crystallite size [33–39]. EDS spectra confirmed the presence of Sr, Mg or Zn in substituted
The average crystallite sizes, calculated using the Scherrer equation samples and on average experimental observations were 108, 24,
suggest that the HA synthesised can be described as nHA (Table 3). No- and 36% of the expected theoretical substitutions for samples AP02,
tably, there are inherent inaccuracies in the application of the Scherrer AP03, and AP04, respectively. Other studies have reported significantly
equation, which may arise since a combination of factors contribute to higher substitutions of 10% Mg, and 20 mol% Zn [10], therefore it cannot
the overall broadening, for example machine broadening and particle be concluded that relatively low levels of Mg (AP03) and Zn (AP04)
coarsening, as well as neglecting the effects of microstrains [40]. Im- resulted due to respective substitution limits for these elements in HA.
provement of accuracy has been demonstrated by more advanced crys- Instead these results are indicative of the fact that the process of Mg
tallographic analysis [41]. In comparison, SEM micrographs show that and Zn incorporation into HA is difficult to ascertain. The accuracy of
the crystallites of samples were mostly aggregated (Fig. 2) resulting in Sr content measured for AP02 may be linked to the ability of Sr2+ to re-
the formation of micron-sized particles as well as larger agglomerates place Ca2+ in the HA structure over the whole range of composition (i.e.
(Table 4). A possible explanation of the agglomeration behaviour is 0–100 wt.%) [2]. Relative to Ca2+ the ionic radii of Sr2+, Mg2+ and Zn2+

Fig. 3. DTA–TGA graphs illustrating thermal behaviour of HA (AP01) and substituted HA samples exhibiting doping of 2 mol% Sr (AP02), 10 mol% Mg (AP03), and 2 mol% Zn (AP04).
 S.C. Cox et al. / Materials Science and Engineering C 35 (2014) 106–114 111

Table 7
Summary of EDS elemental analysis.

Sample Doping element Theoretical substitution (mol%) Experimental substitution (mol%) Theoretical/expected (%) Ca: P ratio Ca + M⁎:P ratio

AP01 N/A N/A N/A N/A 2.17 ± 0.03 N/A

AP02 Sr 2 2.17 ± 0.20 108 ± 10 2.21 ± 0.01 2.28 ± 0
AP03 Mg 10 2.38 ± 0.35 24 ± 3.5 1.96 ± 0.10 2.03 ± 0.10
AP04 Zn 2 0.72 ± 0.29 36 ± 14 2.20 ± 0.02 2.23 ± 0.01
⁎ Where M refers to the appropriate cation substitution.

are 116, 75 and 65% the size, respectively. Since both Mg2+ and Zn2+ SBF was demonstrated on all samples from days 1–28 of immersion
doping were found to be significantly more inaccurate than Sr2+ this (Fig. 5) [52]. In an attempt to quantitatively distinguish the amount of
may indicate that the ability to substitute for Ca2+ in the HA structure apatite growth on samples the average surface roughness (Ra) was cal-
is greatly dependent on the difference in ionic size, i.e. the larger the dif- culated from white-light interferometry measurements (Fig. 4). Assum-
ference in ionic radii the more difficult accurate doping becomes. How- ing that an increase in Ra corresponds to an increase in surface apatite,
ever, it is recognised that a range of substitution levels would need to be the interferometry study suggests that in comparison to pure HA all
investigated to persuasively draw this conclusion. The inherent inaccu- substituted samples exhibited more apatite growth at day 1. In compar-
racy of performing EDS analysis on unpolished samples, as well as typ- ison, Ra values calculated at days 14 and 28 revealed that Sr (AP02) and
ical detection limits of approximately 0.1 wt.% [46] should also be noted Mg (AP03) HA samples exhibited more apatite compared with pure
and may have contributed to measurement inaccuracies. Future work is (AP01) and Zn–HA (AP04) substrates (Fig. 4). However, SEM micro-
planned to perform XRF analysis on all samples as characteristic detec- graphs do not qualitatively corroborate these assertions. It is important
tion limits for this method are within the ppm range, although it has to to note that the validity of the SBF test is debated in the literature.
be borne in mind that the accuracy of XRF is dependent on the molecu- Bohner and Lemaitre [53] reported the possibility of false positive and
lar weight of the elements to be analysed. false negative results from this testing procedure and this calls into
The average weight loss recorded by TGA for samples between 30 question the reproducibility of the presented interferometry study. Nu-
and 1300 °C was 10.61% (Table 6). In comparison AP03 exhibited a merous studies have reported the bioactivity of HA in-vivo [6,54,55],
weight loss of 15.28%, which may be attributed to a relatively high con- which strongly suggests that the growth of apatite shown on the surface
tent (10.27%) of water contained within the hydrate layer suggested to of samples immersed in SBF is a true positive result (Fig. 5). However, a
surround precipitated HA particles [47]. The significantly smaller aver- combination of other factors: 1) variation in initial surface roughness
age particle and agglomerate sizes measured from SEM micrographs of pellets, as indicated by the significant difference in Ra values at
for AP03 indicate a potentially larger surface area, which may explain day 0 (Fig. 4), 2) dissolution of substrates during the SBF study, and
the higher adsorption of surface water in this sample. As expected, cor- 3) variability in SBF solution and resultant apatite formation, may
responding DTA data revealed an endothermic reaction for all samples have influenced the quantitative surface roughness values obtained by
peaking at approximately 100°C (Fig. 3). On average 3.09% was lost interferometry. Therefore it may be concluded, that despite observing
from 200–600°C, which may be associated with the gradual loss of lat- significant differences in the value of Ra between AP01 and substituted
tice water [48]. Miyaji et al. observed an increase in the amount of samples, this assessment does not directly correlate to bioactivity
adsorbed surface and lattice H2O with increasing Zn substitution but and therefore this test may only be used as a positive/negative indicator.
no explanation was offered [49]. In comparison, no significant differ- However, it should be noted that if factors 1) and 2) can be minimised it
ence was observed between Zn–HA (AP04) and HA (AP01) in this is expected that this technique may be used quantifiably, as highlighted
study. Above 600°C weight loss was attributed to structural dehydroxyl-
ation of HA and corresponding endotherms for AP01, AP02 and AP04
were observed at approximately 750°C (Fig. 3). The distinguishable dif-
ference in the high temperature behaviour of Mg–HA (AP03) may be as-
sociated with the reported decomposition of nonstoichiometric Mg–HA
(Table 7) to whitlockite above 600°C [24]. To confirm this assertion
phase analysis (XRD or FTIR) of heat treated AP03 is required. Notably,
AP01 and AP02 exhibited an endothermic reaction at approximately
1180°C, and this may indicate a partial transformation of HA to β-TCP
since this reaction is known to occur at approximately 1125°C [50].
AP04 was also observed to exhibit a high temperature endothermic re-
action but at a comparatively lower temperature range (1000–1100°C)
to other samples.
The FTIR results and summarised assignments (Table 5) show that
the control HA and substituted HA samples synthesised contain CO2− 3
species. According to Rehman and Bonfield, the peaks assigned in the
range of 1300–1600 cm−1 are attributed to surface carbonate ions as
opposed to carbonate ions bound in the crystal structure [51]. Therefore
the demonstrated carbonate composition within the samples is likely to
be reduced after air sintering [29]. The presence of broad bands ob-
served at 1645 and 2750–3750 cm−1 indicate the presence of adsorbed
hydroxyl ions, which corroborates weight losses recorded below 200°C
by TGA (Table. 6).
Kokubo et al. suggest that the growth of carbonated apatite on the
surface of samples immersed in SBF indicates the potential in-vivo bio-
activity of substrates [28]. A surface coating that exhibited spheroidal Fig. 4. Graphical illustration of the change in average surface roughness (Ra) for pure and
and needle-like morphology known to be typical of apatite grown in substituted HA pellets immersed in SBF solution for up to 28 days.
112 S.C. Cox et al. / Materials Science and Engineering C 35 (2014) 106–114

Fig. 5. Typical SEM micrographs illustrating growth of apatite on the surface of substituted HA pellets immersed in SBF a) AP02 day 1, b) AP02 day 7, c) AP04 day 14, and d) AP03 day 28.
Red circles indicate grown carbonated apatite and blue arrows highlight the surface of pellets.

by an interferometry study of stainless steel substrates immersed in SBF of seeded cells was revealed between days 1 and 7, and the change in
[56]. morphology from rounded to elongated over this period is indicative
Fluorescence micrographs confirmed the viability of MC3T3 cells on of adhesion, spreading, and proliferation of seeded cells [57]. Since
all substrates up to 7 days of culture (Fig. 6). An increase in the density only a few dead cells were observed on substrates this was associated

Fig. 6. Typical fluorescence micrographs illustrating the viability of MC3T3 osteoblast precursor cells after 1, 3, 5, and 7 days of culture on a) AP01, b) AP02, c) AP03, and d) AP04. Cell
staining: dead = red and live = green.
 S.C. Cox et al. / Materials Science and Engineering C 35 (2014) 106–114
to the sensitivity of the culture process and not to any cytotoxicity. The
in-vitro results presented here advocates that HA and substituted HA
substrates support the growth of MC3T3 cells as might be expected.
Since it is difficult to distinguish between the proliferative rates of
cells visualised by fluorescence micrographs further quantitative in-
vitro testing is planned to assess the degree of proliferation. It will be
useful to compare these results with those presented in this study to
more accurately evaluate whether the bioactivity of different samples
immersed in SBF can be differentiated and if interferometry analysis
may be used as a quantitative tool.
5. Conclusions
The Scherrer equation was applied to the (002) XRD peak to esti-
mate the size of crystallites, which were confirmed by EDS to contain
the investigated cations. Crystallite sizes were not found to be consis-
tent with the broadening exhibited of in XRD patterns (Fig. 1) and this
difference was associated with the reported inhibitory effect of Mg2+
ions on HA nucleation and crystallisation. Furthermore, a higher angle
shift was observed for Mg–HA (AP03), which suggests this cation was
incorporated into the apatite lattice.
SEM micrographs confirmed bulk agglomeration within all samples
resulting in the formation of micron sized particles (1.6–4.9 μm) and
larger agglomerates (20–30 μm). The smaller average particle and ag-
glomerate sizes exhibited by Mg–HA (AP03) was suggested to explain
the higher surface water content calculated from TGA analysis. No dis-
cernible change in particle morphology was observed via SEM, which
was attributed to ‘identical’ preparation conditions.
The presence of apatite growth on the surface of all substituted and
pure HA samples immersed in SBF for up to 28 days is suggested to be a
positive result that indicates bioactivity. It was concluded that due to a
combination of factors that may influence the surface roughness of sub-
strates, the interferometry results presented in this study may not di-
rectly be correlated to increased apatite growth and therefore cannot
reliably be used as a quantitative tool. However, it is suggested that
comparison with results of a quantitative cellular in-vitro assay would
provide a better assessment of reliability and accuracy. A live/dead
assay was used to confirm the viability of MC3T3 osteoblast precursor
cells on all substrates as visualised by fluorescence microscopy. Cellular
in-vitro results advocate that all substrates support the adhesion,
spreading, and proliferation of seeded cells.
In summary, non-toxic biologically relevant phase pure and HA
substituted with 2 mol% Sr, 10 mol% Mg, and 2 mol% Zn were precipi-
tated under ‘identical’ conditions. The results from this study compared
with others concerning substituted HA highlight the importance and
value of performing a key factor assessment of the influence of doping.
When comparing results of studies regarding substituted HA it is impor-
tant to recognise the influence of reaction conditions (e.g. pH, tempera-
ture, reagent concentration, ageing time) on material properties, in
particular crystallinity, crystallite size, and particle morphology, that ul-
timately define biological performance. It is suggested that due to the
variation of reported precipitation procedures it is more accurate to
compare samples substituted with different cations that have been pre-
pared under ‘identical’ experimental conditions.
Acknowledgements
The authors gratefully acknowledge the financial support of the
Warwick Chancellor's scholarship.
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