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

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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 b

Warwick Manufacturing Group, University of Warwick, Coventry CV4 7AL, UK School of Chemical Engineering, University of Birmingham, Edgbaston, Birmingham B15 2TT, UK

a r t i c l e

i n f o

Article history: Received 28 May 2013 Received in revised form 26 September 2013 Accepted 19 October 2013 Available online 6 November 2013 Keywords: Bioceramics Substituted hydroxyapatite Aqueous precipitation Simulated body fluid Osteoblasts

a b s t r a c t Hydroxyapatite (HA) substituted with 2 mol% Sr, 10 mol% Mg, and 2 mol% Zn were precipitated under identical alkaline conditions (pH 11) at 20°C from an aqueous solution. As-synthesised materials were confirmed to be phase pure by XRD and samples prepared in air contained surface adsorbed CO2 as observed by FTIR. SEM studies revealed a globular morphology and agglomeration behaviour, typical of precipitated nHA. EDS spectra confirmed nominal compositions and substitution of Sr, Mg and Zn. At the levels investigated cationic doping was not found to radically influence particle morphology. An indication of the potential in-vivo bioactivity of samples was achieved by analysing samples immersed in SBF for up to 28 days by interferometry and complementary SEM micrographs. Furthermore, a live/dead assay was used and confirmed the viability of seeded MC3T3 osteoblast precursor cells on HA and substituted HA substrates up to 7 days of culture. © 2013 Elsevier B.V. All rights reserved.

1. Introduction 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 crystallographically 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 incorporation 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 stoichiometric 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 effects on biomaterial properties, such as the degree of structural order (i.e. crystallinity), solubility in chemical solvents, surface charge and dissolution rate under simulated physiological conditions. This may inturn influence bioactivity, thus attracting much interest in the wider

⁎ Corresponding author. Tel.: +44 2476522324. E-mail address: [email protected] (K.K. Mallick). 0928-4931/$ – see front matter © 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.msec.2013.10.015

research community [1,2,5,6]. In particular, improved degradation of less crystalline HA has been shown to be advantageous due to the ability of these surfaces to provide a dynamic zone for dissolution and reprecipitation [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, thermal 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 decreasing 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 physicochemical properties of Sr, Mg, and Zn substituted HA produced under ‘identical’ experimental conditions has not been previously reported. Notably, it is important that samples be prepared under ‘identical’ conditions to determine the explicit influence of cation doping. Other reaction conditions, such as pH and temperature, are known to affect parameters that are influential in determining the biological performance of HA. Therefore, the authors recognise that it is vital to control

S.C. Cox et al. / Materials Science and Engineering C 35 (2014) 106–114 Table 1 Composition and lattice parameters of bone compared with nominally calculated stoichiometric HA as reported by [2,3]. Composition

Bone [3]

Bone [2]

Stoichiometric HA

Calcium (wt %) Phosphorus (wt %) Carbonate (wt %) Sodium (wt %) Magnesium (wt %) Chloride (wt %) Pyrophosphate (wt %) Potassium (wt %) Fluoride (wt %) Strontium (wt %) Zinc (ppm) Chromium (ppm) Cobalt (ppm) Manganese (ppm) Silicon (ppm) A axis (Å) C axis (Å)

34.8 15.2 7.40 0.90 0.72 0.13 0.07 0.03 0.03 – – – – – – 9.410 6.890

36.6 17.1 4.8 1.0 0.6 0.1 – 0.07 0.1 0.05 39 0.33 b0.025 0.17 500 – –

39.6 18.5 – – – – – – – – – – – – – 9.430 6.891

such reaction conditions to determine the sole influence of the investigated cations. HA containing 2 mol% Sr, 10 mol% Mg, or 2 mol% Zn have been synthesised using an aqueous precipitation method under ‘identical’ experimental conditions. The phase purity, crystallinity, particle morphology, level of ionic substitution, and thermal stability were assessed by X-ray diffraction (XRD), scanning electron microscopy (SEM), energy dispersive spectroscopy (EDS), simultaneous differential thermal analysis thermogravimetry (DTA–TGA), and Fourier transmission infrared spectroscopy (FTIR). Simulated body fluid (SBF) studies up to 28 days were carried out in order to provide an indication of the bioactivity of the substituted samples (in accordance to ISO 23317:2007). Furthermore, a live/dead assay was performed to assess the viability of cultured MC3T3 osteoblast precursor cells seeded on pure and substituted HA substrates. 2. Experimental Identical preparative conditions of alkalinity (pH 11) and temperature (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 by an aqueous precipitation (AP) method. These levels of Sr2+, Mg2+ and Zn2+ cations are intended to replace Ca2+ within the HA lattice [2,24,25]. In order to compare the final products the experimental ratios of Ca/P or (Ca + M)/P (where M = Sr, Mg or Zn) were maintained at 1.67. All reagents were used without further purification and purchased from Sigma Aldrich (UK) unless otherwise stated. 2.1. Aqueous precipitation method to produce pure and substituted HA A stock solution of PO3− was prepared by dissolving 0.03 M of 4 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 adjusted 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%), magnesium nitrate ((Mg(NO3)2 ≥ 99.0% — Johnson Matthey, UK) or zinc nitrate hexahydrate (Zn(NO3)2 · 6H2O ≥ 98.0%) was then dissolved in the solution containing Ca2+. All solutions were stirred until the reagents were fully dissolved. Using a pH metre (pHTestr10, Eutech Instruments, 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− solution was 4 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

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was separated from the supernatant by filtering, washed with DI water, dried and finally ground into a micronised powder. To utilise the CO2 present in the atmosphere and form naturally carbonated HA the procedure outlined above was performed in air. In contrast to other studies [26,27], since the amount of carbonate substituted into the HA structure was not accurately controlled, the products are not deemed as carbonated HA (CHA). 2.2. In-vitro analysis Testing was performed on substrates of substituted HA samples and a pure HA sample, synthesised under the same experimental conditions, was used as a control. 2.2.1. Pellet preparation Each sample was pressed into 10 mm pellets by applying an average uniaxial force of 800 N to 0.25 ± 0.02 g of as-synthesised powder using 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 1°C/min. The powders were characterised by XRD before and after sintering to ensure no phase change occurred. 2.2.2. Simulated body fluid test SBF exhibits ion concentrations nearly equal to those of human blood plasma. It has been suggested that the potential in-vivo bioactivity of a biomaterial may be examined by its ability to form apatite on its surface whilst immersed in SBF. SBF solution was produced in accordance to ISO 23317:2007 by the procedure proposed by Kokubo et al. [28], as outlined in Table 2. Hygroscopic reagents, labelled as 4, 6, 8 and 9 in Table 2, were weighed and quickly added to the solution. During preparation the solution temperature was maintained at 36.5 ± 1.5 °C and stirred continuously at 200 rpm. Four pellets for each sample were immersed in SBF so as to cover the entire sample surface and were placed in an incubator at 37°C. Pellets were removed, dried, and analysed after 1, 7, 14, and 28 days of immersion. 2.2.3. Cell culture The viability of MC3T3 osteoblast precursor cells were analysed by using a Live/Dead® Viability/Cytotoxicity kit after culturing for 1, 3, 5, and 7 days. A final cell density of 2 × 104 cells/well were seeded directly on to the surface of pellets in 24-multiwell plates that were previously coated with 1.5 mL of Sylgard type 184 silicone Elastomer (Dow Corning Corporation, Midland, MI). Calcein-AM and propidium iodide (1 mg/mL; Molecular Probes, Invitrogen, UK) were used to stain live and dead cells, respectively in the dark. 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.

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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 2 3 4 5 6 7

DI water Sodium chloride (NaCl) Sodium bicarbonate (NaHCO3) Potassium chloride (KCl) Potassium phosphate dibasic trihydrate (K2HPO4 · 3H2O) Magnesium chloride hexahydrate (MgCl2 · 6H2O) Hydrochloric acid (HCl)

N/A ≥99.5 99.7–100.3 ≥99.0 ≥99.0 ≥99.0 1M

400 mL 7.996 g 0.350 g 0.224 g 0.228 g 0.305 g 40 mL

N/A Reagents 1–6 including washings, were added individually allowing enough time for each to fully dissolve between each addition

8

Calcium chloride (CaCl2)

≥93.0

0.278 g

9 10 11

Sodium sulfate (Na2SO4) Tris-(hydroxymethyl)aminomethane ((CH2OH)3CNH2) HCl

≥99.0 ≥99.8 1M

0.071 g 6.057 g Appropriate amount for pH adjustment

The size and morphology of particles were investigated by a Zeiss Supra55 FEGSEM. The specimens containing Sr, Mg or Zn were examined by an energy-dispersive spectroscope (X-max 50 mm2, Oxford Instruments, UK) to identify and quantify elemental composition. The chemical absorption characteristics of dried powders were observed 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 magnifications of ×20 was used to visualise cells cultured on the surface of pellets.

HCl was added in 5 mL amounts and the solution pH was allowed to stabilise in-between. Washings were also added. CaCl2 was dissolved in 25 mL of DI water and added to solution, including washings Reagent 9 and washings added TRIS was dissolved in 50 mL of DI water and added 5 mL at a time, allowing the pH to stabilise in between up to pH 7.45. HCl 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

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 samples was observed as illustrated in Fig. 2. 3.3. Molecular structure The major functional groups including hydroxyl, phosphate and carbonate 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,

3. Results 3.1. Crystal structure Table 3 summarises the results of the phase purity and average crystallite 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 products 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 increase 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 substituted

Theoretical substitution (wt%)

Main phase(s)

Average crystallite size⁎ (nm)

AP01 AP02 AP03 AP04

N/A (HA) Sr Mg Zn

N/A 2.0 10.0 2.0

HA HA HA HA

15.52 10.54 13.75 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 Table 4 Summary of the average size and morphology of pure and substituted HA particles as observed by SEM. Sample

Average particle size (μm)

Average agglomerate size (μm)

Typical morphology

AP01 AP02 AP03 AP04

2.72 4.89 1.61 3.30

24.61 40.52 19.86 29.53

• Randomly orientated globular particles • Typically rounded particle edges • All samples were largely agglomerated

± ± ± ±

0.51 0.86 0.42 0.43

± ± ± ±

4.27 3.67 4.17 5.08

200–600 and 600–1300°C) that were found to exhibit corresponding thermal reactions (Fig. 3). 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 concentration 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 calculated by Eq. (2): Ra ¼ ðjZ 1 j þ jZ 2 j þ jZ 3 j þ … þ jZ n jÞ=N

ð2Þ

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 apatite for all samples from day 1 onwards. Fig. 5 shows typical micrographs of the grown apatite (red circle) on the surface (blue arrow) of pellets.

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3.7. Cell culture Live (green) and dead (red) assay was used to assess the viability of MC3T3 osteoblast precursor cells, which were seeded onto the surface of pellets formed from powdered samples. Cells were found to be viable on all substrates over the entire culture period (Fig. 6). An increase in the density of osteoblasts between days 1 and 7, as well as a change in the typical morphology from rounded (days 1 and 3) to elongated (days 5 and 7) was visually observed. Very few dead cells were demonstrated and therefore cell death was attributed to the sensitivity of the culture process. 4. Discussion 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 higher degree of broadening and a significantly lower crystallite size, and this may be attributed to the relatively low preparation temperature (20°C) as well as short ageing time (1.5h) [25]. In this study, no discernible 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).

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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 H2O

Soluble CO2 (ν3) and (ν2)

Absorbed H2O (ν2)

CO2− 3 groups (ν3)

PO3− 4 bend (ν3)

PO3− 4 stretch (ν1)

CO2− 3 group

OH structural

PO3− 4 bend (ν4)

Carbonated HA [24,25] AP01 AP02 AP03 AP04

2750–3750 2750–3438 2750–3440 2750–3560 2750–3600

2331–2360 2329–2359 2324–2360 2340–2356 2331–2355

1640 1638 1626 1617 1628

1420, 1455 1422, 1455 1455 1456 1454

1030, 1090 1026, 1090 1029, 1089 1029, 1089 1029, 1089

961 962 962 962 961

873 872 873 871 873

635–632 633 633 634 634

604–600 600 603 604 603

Table 6 Summary of weight loss exhibited by TGA for HA alone (AP01) and HA samples exhibiting substitutions AP02 (2 mol% Sr), AP03 (10 mol% Mg), and AP04 (2 mol% Zn). Sample

AP01 AP02 AP03 AP04

Weight loss (%)

Residue (%)

Total

30–200 °C

200–600 °C

600–1300 °C

8.44 9.43 15.28 9.29

4.91 5.17 10.27 5.03

2.38 3.03 3.95 2.98

1.15 1.23 1.06 1.28

91.56 90.57 84.72 90.71

Mg–HA that have shown lattice incorporation up to 10% by NMR [10]. It should be noted, when comparing to other studies it is difficult to observe the key factor influence of substitutions due to the known discernible effect of other reaction parameters (e.g. temperature, pH, ageing) on properties such as crystallinity and crystallite size [33–39]. The average crystallite sizes, calculated using the Scherrer equation suggest that the HA synthesised can be described as nHA (Table 3). Notably, there are inherent inaccuracies in the application of the Scherrer equation, which may arise since a combination of factors contribute to the overall broadening, for example machine broadening and particle coarsening, as well as neglecting the effects of microstrains [40]. Improvement of accuracy has been demonstrated by more advanced crystallographic analysis [41]. In comparison, SEM micrographs show that the crystallites of samples were mostly aggregated (Fig. 2) resulting in the formation of micron-sized particles as well as larger agglomerates (Table 4). A possible explanation of the agglomeration behaviour is

the surface dominated properties associated with nanosized crystallites, which causes them to clump together as observed in Fig. 2. The trend in average particle sizes calculated from SEM micrographs was not consistent with the average crystallite sizes calculated from XRD line broadening analysis. It is suggested that the previously mentioned inaccuracies of the Scherrer equation, the high degree of peak broadening (Fig. 1), and the effect of clumping caused by nanosized particles may explain the inconsistent trends observed between Scherrer analysis and SEM measurements. 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 conditions, such as pH and temperature, influence the resultant phase and particle morphology [42–45]. Since ‘identical’ pH (11) and temperature conditions (20°C) were maintained in the present study this may explain why no discernible change in morphology was observed. EDS spectra confirmed the presence of Sr, Mg or Zn in substituted samples and on average experimental observations were 108, 24, and 36% of the expected theoretical substitutions for samples AP02, AP03, and AP04, respectively. Other studies have reported significantly higher substitutions of 10% Mg, and 20 mol% Zn [10], therefore it cannot be concluded that relatively low levels of Mg (AP03) and Zn (AP04) resulted due to respective substitution limits for these elements in HA. Instead these results are indicative of the fact that the process of Mg and Zn incorporation into HA is difficult to ascertain. The accuracy of Sr content measured for AP02 may be linked to the ability of Sr2+ to replace Ca2+ in the HA structure over the whole range of composition (i.e. 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).

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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 AP02 AP03 AP04

N/A Sr Mg Zn

N/A 2 10 2

N/A 2.17 ± 0.20 2.38 ± 0.35 0.72 ± 0.29

N/A 108 ± 10 24 ± 3.5 36 ± 14

2.17 2.21 1.96 2.20

N/A 2.28 ± 0 2.03 ± 0.10 2.23 ± 0.01

± ± ± ±

0.03 0.01 0.10 0.02

⁎ Where M refers to the appropriate cation substitution.

are 116, 75 and 65% the size, respectively. Since both Mg2+ and Zn2+ doping were found to be significantly more inaccurate than Sr2+ this may indicate that the ability to substitute for Ca2+ in the HA structure is greatly dependent on the difference in ionic size, i.e. the larger the difference in ionic radii the more difficult accurate doping becomes. However, it is recognised that a range of substitution levels would need to be investigated to persuasively draw this conclusion. The inherent inaccuracy of performing EDS analysis on unpolished samples, as well as typical detection limits of approximately 0.1 wt.% [46] should also be noted and may have contributed to measurement inaccuracies. Future work is planned to perform XRF analysis on all samples as characteristic detection limits for this method are within the ppm range, although it has to be borne in mind that the accuracy of XRF is dependent on the molecular weight of the elements to be analysed. The average weight loss recorded by TGA for samples between 30 and 1300 °C was 10.61% (Table 6). In comparison AP03 exhibited a weight loss of 15.28%, which may be attributed to a relatively high content (10.27%) of water contained within the hydrate layer suggested to surround precipitated HA particles [47]. The significantly smaller average particle and agglomerate sizes measured from SEM micrographs for AP03 indicate a potentially larger surface area, which may explain the higher adsorption of surface water in this sample. As expected, corresponding DTA data revealed an endothermic reaction for all samples peaking at approximately 100°C (Fig. 3). On average 3.09% was lost from 200–600°C, which may be associated with the gradual loss of lattice water [48]. Miyaji et al. observed an increase in the amount of adsorbed surface and lattice H2O with increasing Zn substitution but no explanation was offered [49]. In comparison, no significant difference was observed between Zn–HA (AP04) and HA (AP01) in this study. Above 600°C weight loss was attributed to structural dehydroxylation of HA and corresponding endotherms for AP01, AP02 and AP04 were observed at approximately 750°C (Fig. 3). The distinguishable difference in the high temperature behaviour of Mg–HA (AP03) may be associated 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 reaction 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 observed 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 bioactivity of substrates [28]. A surface coating that exhibited spheroidal and needle-like morphology known to be typical of apatite grown in

SBF was demonstrated on all samples from days 1–28 of immersion (Fig. 5) [52]. In an attempt to quantitatively distinguish the amount of apatite growth on samples the average surface roughness (Ra) was calculated from white-light interferometry measurements (Fig. 4). Assuming that an increase in Ra corresponds to an increase in surface apatite, the interferometry study suggests that in comparison to pure HA all substituted samples exhibited more apatite growth at day 1. In comparison, Ra values calculated at days 14 and 28 revealed that Sr (AP02) and Mg (AP03) HA samples exhibited more apatite compared with pure (AP01) and Zn–HA (AP04) substrates (Fig. 4). However, SEM micrographs do not qualitatively corroborate these assertions. It is important to note that the validity of the SBF test is debated in the literature. Bohner and Lemaitre [53] reported the possibility of false positive and false negative results from this testing procedure and this calls into question the reproducibility of the presented interferometry study. Numerous studies have reported the bioactivity of HA in-vivo [6,54,55], which strongly suggests that the growth of apatite shown on the surface of samples immersed in SBF is a true positive result (Fig. 5). However, a combination of other factors: 1) variation in initial surface roughness of pellets, as indicated by the significant difference in Ra values at day 0 (Fig. 4), 2) dissolution of substrates during the SBF study, and 3) variability in SBF solution and resultant apatite formation, may have influenced the quantitative surface roughness values obtained by interferometry. Therefore it may be concluded, that despite observing significant differences in the value of Ra between AP01 and substituted samples, this assessment does not directly correlate to bioactivity and therefore this test may only be used as a positive/negative indicator. However, it should be noted that if factors 1) and 2) can be minimised it is expected that this technique may be used quantifiably, as highlighted

Fig. 4. Graphical illustration of the change in average surface roughness (Ra) for pure and substituted HA pellets immersed in SBF solution for up to 28 days.

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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 [56]. Fluorescence micrographs confirmed the viability of MC3T3 cells on all substrates up to 7 days of culture (Fig. 6). An increase in the density

of seeded cells was revealed between days 1 and 7, and the change in morphology from rounded to elongated over this period is indicative of adhesion, spreading, and proliferation of seeded cells [57]. Since 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.

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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 invitro 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 estimate the size of crystallites, which were confirmed by EDS to contain the investigated cations. Crystallite sizes were not found to be consistent 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 agglomerate sizes exhibited by Mg–HA (AP03) was suggested to explain the higher surface water content calculated from TGA analysis. No discernible 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 substrates, the interferometry results presented in this study may not directly 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 precipitated 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 important to recognise the influence of reaction conditions (e.g. pH, temperature, reagent concentration, ageing time) on material properties, in particular crystallinity, crystallite size, and particle morphology, that ultimately 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 prepared under ‘identical’ experimental conditions. Acknowledgements The authors gratefully acknowledge the financial support of the Warwick Chancellor's scholarship. References [1] P.N. Kumta, et al., Nanostructured calcium phosphates for biomedical applications: novel synthesis and characterization, Acta Biomater. 1 (1) (2005) 65–83. [2] E. Boanini, M. Gazzano, A. Bigi, Ionic substitutions in calcium phosphates synthesized at low temperature, Acta Biomater. 6 (6) (2010) 1882–1894.

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