Utilization of snail shells to synthesise hydroxyapatite nanorods for orthopedic applications

Abstract

Snail shells are plentiful in nature and consist of mainly CaCO₃. Here they have been effectively utilized to synthesize hydroxyapatite (HA) nanorods by a facile and rapid microwave irradiation method with the help of ethylenediaminetetraacetic acid (EDTA) as a chelating agent. The conversion of snail shells into HA occurs via the formation of a Ca–EDTA complex which subsequently reacted with phosphate at pH ≈ 10 under microwave irradiation. The obtained product consists of B-type carbonated HA nanorods and can be a potential candidate for biomedical applications.

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Hydroxyapatite (Ca₁₀(PO₄)₆(OH)₂, HA) is the major inorganic component of bone and teeth which has been widely used as a carrier for drug delivery, a bone substitute for filling bone defects, a scaffold matrix for tissue engineering and a coating on metallic implants owing to its excellent biocompatibility and bioactivity.^1–3 However, its usage at high load bearing applications has been restricted because of its poor mechanical performance.^1–3 The recent trend in HA based research is shifting towards nanotechnology, which offers a unique approach to overcome the shortcomings of its conventional forms due to their large surface to volume ratio and unusual chemical/electronic synergistic effects.^3,4 Over the past decades, a number of synthetic techniques such as sol–gel synthesis, solid state reactions, wet precipitation method, hydrothermal method, microemulsion synthesis, microwave irradiation method, mechanochemical synthesis, etc., have been developed to synthesize HA nanocrystals with various sizes and morphologies.^5–10 Among the numerous methods, microwave synthesis is an efficient and facile method for synthesis of HA nanocrystals with different sizes and shapes in a rapid manner.^10–13 Organic modifiers such as cetyltrimethylammonium bromide (CTAB), citric acid, and ethylenediaminetetraacetic acid (EDTA) can be used to control the final shape and size of HA nanocrystals and often used in the microwave irradiation method.^11–13 In particular, EDTA was recognized as an efficient crystal growth modifier to synthesize HA nanorods.¹¹

Recently, natural materials such as corals, fish bone, eggshells, snail shell, etc., have been selected as a source for the synthesis of HA due to their advantage of biological origin as well as recycling of biowaste.^6,14–18 These materials often exhibit a fine-scale microstructure with variable porosity, unusual crystal habits and morphologies, remarkable mechanical and physical properties. Among the several natural materials, snail shells are plentiful in nature and it is a composite material consisting of calcium carbonate and organic matter.^18–20 The use of this waste as a calcium source for the synthesis of HA can reduce the amount of waste to be disposed and reduce the costs from the requirement of using expensive and high purity calcium reagents to synthesize HA. Only few reports were identified on the utilization of snail shells to synthesize HA where high temperature calcination was employed in order to get calcium oxide (CaO) from snail shell for synthesis of HA.^18–20 Consequently, developing a simple method to convert these snail shells into HA is essential from the viewpoint of materials processing. Here, we report a facile and rapid microwave irradiation method to obtain HA nanorods using snail shell as a calcium source with the aid of EDTA as a complexing agent.

The chemicals used were sodium hypochlorite (NaOCl, 99.5%), sodium hydroxide (NaOH, 99.5%), ethylenediamminetetraacetic acid (EDTA) and disodium hydrogen phosphate (Na₂HPO₄, 99%) obtained from Merck. All reagents were used without further purification. Distilled water was employed as the solvent. Snail shells were collected and washed with tap water followed by distilled water to remove the surface contaminants. The cleaned shells were ground into powder and immersed in sodium hypochlorite to remove organic components. Then, they were extensively washed with distilled water and dried in hot air oven for 5 h at 110 °C. Approximately, 1 g of snail shell powder was then mixed with 0.1 M of EDTA solution to form Ca–EDTA complex. Then, 0.06 M of Na₂HPO₄ solution was slowly added to the obtained Ca–EDTA complex and stirred for 30 min. The preliminary pH of the reaction mixture was about 8. The pH of the reaction mixture was adjusted to 10 by addition of NaOH solution. Then, the obtained reaction mixture was put in a microwave oven (2.45 GHz, 900 W, Samsung, India) and irradiated with microwave for 10 min. After cooling to room temperature, the obtained white precipitate was washed with distilled water and dried in hot air oven at 110 °C for 5 h.

The X-ray diffraction (XRD) patterns were recorded using Rigaku MiniFlex II powder X-ray diffractometer in the range between 20° ≤ 2θ ≤ 60° with Cu Kα monochromatic radiation (1.5406 Å). Crystallographic identification of the phases of the samples was accomplished by comparing the experimental XRD pattern with JCPDS data. FT-IR spectrum was recorded in the 4000–400 cm⁻¹ region with 4 cm⁻¹ resolution using a Perkin Elmer RX1 FT-IR spectrometer by KBr pellet technique. The surface of snail shell was examined using JEOL-6390 scanning electron microscope. The morphological feature was examined using JEOL JEM-2100 high resolution transmission electron microscope (HRTEM) and the elemental analysis were done using the Oxford INCA energy dispersive X-ray fluorescence (EDX) microanalysis. Thermal stability of prepared sample was analyzed using thermogravimetry (TG) analyzer (Make: TA Instruments, Model: Q600).

Fig. 1 shows the XRD, SEM and EDX profile of snail shell. The XRD pattern (Fig. 1(b)) of snail shell was compared with JCPDS data for aragonite (JCPDS file no. 05-0453) which matched well with standard data. This suggested that the crystalline phase present in the as-collected snail shell was aragonite having orthorhombic crystal system. The lattice constants and unit cell volume for aragonite crystal were calculated as a = 4.9553 Å, b = 7.9470 Å, c = 5.7337 Å and V = 225.7977 ų, respectively. EDX spectrum (Fig. 1(b)) indicates the presence of Ca, P, C, and O in snail shell. SEM images (Fig. 1(c)–(e)) shows the morphological feature of surface of snail shell with different magnifications. It is found that snail shell consist of oriented arrays of aragonite crystals at nanoscale level. Moreover, two neighbour arrays are oriented in opposite directions as indicated by arrows in Fig. 1(d) and (e). This fine-scale architecture with unusual crystal habits and morphology is due to organic–inorganic hybrid nature of the snail shell.

Fig. 1 (a) XRD pattern, (b) EDX spectra and (c–e) SEM images of snail shell.

Fig. 2 shows the XRD pattern and FT-IR spectrum of obtained HA powder by using snail shell as a calcium source. The X-ray diffraction peaks of synthesized sample (Fig. 2(a)) matched well with JCPDS data for HA (JCPDS file no. 09-0432), indicating that it is composed of phase pure crystalline HA having hexagonal crystal system. The lattice constants and unit cell volume for the obtained HA were calculated as a = b = 9.4106 ± 0.0049 Å, c = 6.8514 ± 0.0509 Å and V = 525.47 ± 4.14 ų, respectively.

Fig. 2 (a) XRD pattern and (b) FT-IR spectrum of obtained HA powder.

The FT-IR spectrum of obtained HA powder is shown in Fig. 2(b). A typical structure of HA is characterized by different vibrational modes of the phosphate PO₄³⁻ and hydroxyl OH⁻ groups. The peaks at 478, 563, 604, 961, 1042 and 1106 cm⁻¹ are attributed to vibrations of tetrahedral phosphate group in HA.^21–23 The peak at 3569 cm⁻¹ is due to stretching vibration of OH group of HA.^21–23 The broad band extending from 2500 to 3600 cm⁻¹ is attributed to the ν₃ and ν₁ stretching vibrations of water molecules.^21–23 The peak at 1640 cm⁻¹ is attributed to the ν₂ bending mode of the water molecules.^21–23 Besides, it showed additional peaks at 874 cm⁻¹, 1418 cm⁻¹ and 1460 cm⁻¹ which are due to vibrations of CO₃²⁻ group.^22–27 It is generally agreed that the carbonate ions can substitute at two sites in HA structure, namely the hydroxyl and the phosphate ion positions, giving A and B-type CHA, respectively. Normally A-type CHA showed vibrations of carbonate group at 1550 cm⁻¹, 1457 cm⁻¹ and 880 cm⁻¹ whereas B-type CHA showed at 1462 cm⁻¹, 1418 cm⁻¹ and 876 cm⁻¹.^22–27 The CO₃²⁻ vibration modes observed in FT-IR spectrum (Fig. 2(b)) suggests that the obtained product is a B-type carbonated HA, i.e. part of the phosphate (B-type) groups in the HA structure is replaced by carbonate groups.

Fig. 3(a) and (b) shows HRTEM image at different magnifications which indicates the morphological feature of prepared sample. It showed rod-like morphology of size 500–700 nm long and 40–60 nm wide. Moreover HRTEM image at high magnification displays the resolved lattice fringes. The EDX (Fig. 3(c)) taken from individual nanorod confirms that the nanorods are composed of calcium (26.35 wt%), phosphorous (15.44 wt%), oxygen (50.60 wt%) and carbon (7.61 wt%).

Fig. 3 HRTEM images of synthesized HA (a) lower magnification, (b) higher magnification and (c) EDX profile of HA.

TG analysis of prepared samples is shown Fig. 4. A significant weight loss (around 10%) was observed between room temperature and 250 °C which is due to desorption of adsorbed water and elimination of lattice water from the sample.^10,22 With increasing temperature, there is no significant weight loss which indicates EDTA residue not present in the sample. This result was also supported by FT-IR analysis. The FT-IR spectrum of sample shows absorption peaks only due to B-type carbonated HA and water molecules not of EDTA molecules. Hence there is no possibility for the presence of EDTA residue in the prepared sample.

Fig. 4 TG analysis of prepared HA.

Schematic illustration for utilization of snail shells to synthesize of HA nanorods is shown in Fig. 5. EDTA, a member of the polyamino carboxylic acid family, is a complex reagent. With Ca²⁺, it forms Ca–EDTA complexes.^11,28 When snail shell powder was added with the EDTA solution, it dissolves readily and CO₂ gas was evolved. Progressively, the complete snail shell powder was dissolved and a stable Ca–EDTA complex was formed according to the following reaction

CaCO₃ + EDTA⁴⁻ → Ca–EDTA²⁻ + CO₃²⁻

Fig. 5 Schematic representation for utilization of snail shells to synthesis of HA nanorods.

Further the obtained Ca–EDTA solution was added with phosphate solution, a clear solution was formed which is due to the stability of the Ca–EDTA complex at pH ≈ 10. EDTA can form stable complex with calcium ions especially under basic condition. In the complex, EDTA acts as a hexadentate unit by packaging itself around Ca²⁺ with four atoms of oxygen and two atoms of nitrogen and forms several chelate rings.¹¹ However microwave irradiation is an efficient heating of matter by microwave dielectric heating. The energy of a microwave photon at a frequency of 2.45 GHz is only 10⁻⁵ eV or about 1 J mol⁻¹.²⁹ This energy is transferred to the material by interaction of the electromagnetic field at the molecular level. The dielectric properties determine the effect of the electromagnetic field on the material. The interaction of microwave radiation with a dielectric material results in translational motions of free or bound charges and rotation of the dipoles. The resistance of these induced motions due to inertial, elastic, and frictional forces causes losses resulting in rapid volumetric heating.^29,30 Under this rapid microwave heating, Ca ions can be easily released from Ca–EDTA complexes and react with phosphate ions forming a three dimensional cluster of critical size which act as nucleus and develop into HA crystallite under further microwave irradiation. However further growth of HA crystallites will be controlled by two important key factors which determine the final shape of HA nanocrystals. The first one is an intrinsic factor, that is, the crystallographic phase of the nucleated seeds. In general, for the materials having hexagonal structure, the anisotropic growth along the c-axis is preferable which form the one dimensional nanostructure. The second key factor is external factors including pH value and stability of Ca–EDTA complex, which may render the anisotropic growth of HA nanocrystals along possible crystallographically reactive directions.^11,28 At pH ≈ 10, the crystal growth habit is mainly affected by the intrinsic factor rather than the external condition since Ca ions can be easily released from Ca–EDTA complexes under microwave radiation. The free Ca ions will incorporate into the active site of the obtained HA nucleus and will grow anisotropically along the c-axis. Consequently, HA nanorods were formed at pH ≈ 10.

Only few reports were identified on the synthesis of HA using snail shell where calcium oxide (CaO) was obtained by calcination of snail shells at high temperature and then it was either converted into calcium nitrate [Ca(NO₃)₂] by treating with required amount of nitric acid or converted into calcium hydroxide [Ca(OH)₂] by exposing it in atmosphere. The complexity with these methods is need of high temperature calcinations (1000 °C) of snail shell to obtain CaO for deriving Ca precursor.^18–20 However we have developed facile and rapid microwave irradiation method for synthesis of HA nanorods using snail shell as a calcium source. Moreover, synthesis of materials more similar to bone mineral remains one of the most interesting objectives of biomaterials research. Bone minerals are nanostructured non-stoichiometric carbonated HA with variety of ionic (cationic and anionic) substitutions, such as Mg, Zn, Na, F, Sr, etc. The amount of ionic substitutions in the HA of bone varies from the wt% level (e.g. 3–8 wt% CO₃) to ppm–ppb level (e.g. Mg, Sr, etc.).³¹ It is rational to synthesize non-stoichiometric carbonated HA, mimicking bone apatite, to achieve improved properties in comparison with pure HA ceramics. Incorporation of carbonate into HA has enhance the biological activity than pure HA because carbonate substitution in HA caused a change in crystal morphology, a decrease in crystallinity, an increase in solubility and increases the local concentration of calcium and phosphate ions that are necessary for new bone formation.^{23–27,31–33} A-CHA and B-CHA are abundant in bone of the old and the young, respectively. The B-type CHA is preferred because the high reactivity of young bone could be related to the greater presence of B-CHA compared to the old bone. B-CHA showed enhanced solubility, collagen deposition in vitro and resorption in vivo compared with HA or A-CHA.^{23–27,31–33} The obtained product is B-CHA and it can be used in various forms including as powder/particulate for drug delivery, filling bone defects, coating of metallic implants, as scaffolds for bone tissue engineering and drug delivery depending on clinical needs.

In conclusion, HA nanorods have been successfully synthesized by utilization of snail shells as a calcium source via facile microwave irradiation method in rapid manner using EDTA as a chelating agent. The obtained product was identified to be B-type carbonated HA with hexagonal crystal system having a = b = 9.4106 ± 0.0049 Å, c = 6.8514 ± 0.0509 Å and V = 525.47 ± 4.14 ų. The formation of nanorods is due to the anisotropic growth habit of HA crystallites under microwave irradiation. Consequently it can provide new prospects in the development of biomaterials for orthopedic applications.

Acknowledgements

The authors (G. S. K. and L. S.) express their sincere thanks to Dr V. Radhakrishnan, Principal, K. S. Rangasamy College of Arts and Science (Autonomous), Tiruchengode, India, for his constant encouragement to carry out this work. The authors would like to express their special thanks to Prof. B. Viswanathan, Head, National Centre for Catalysis Research, Department of Chemistry, Indian Institute of Technology-Madras, India for providing HRTEM facility.

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Footnote

† Electronic supplementary information (ESI) available. See DOI: 10.1039/c5ra04402b

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