Synthesis of nano-crystalline hydroxyapatite from dead snail shells for biological implantation.
Abstract: Hydroxyapatite [Ca.sub.10][(P[O.sub.4]).sub.6][(OH).sub.2] is an important biomaterial and is the principal replacement inorganic constituent of bones and teeth. It is also used as the replacement of heart valves, hip joints and other implants in the human body. A novel procedure to produce porous hydroxyapatite from dead snail shells is reported. Dead snail shells which are generally discarded as a biological waste are used as the raw material here. The thermal decomposition of the clean and dry snail shells was carried out by DTA/TG analysis. The snail shells were thermally treated and hydroxyapatite was produced through chemical route. The powder was characterized by X-Ray Diffraction, particle size analysis, DTA/TG analysis, scanning electron microscopy and infrared spectroscopy. As a result, the Heap particle exhibited a micrometer-sized spherical shape where Average particle size was found to be 60-80 nm. The crystalinity increased after calcinations and the size of crystallites range from 100-115 nm. Amount of hydroxyapatite obtained from route 1 is less compare to route 2 but purity of route 1 is higher as compare to route 2 in both cases. The snail shells seems to be a promising source of calcium for preparing nanocrystalline hydroxyapatite with excellent properties so essential for hard tissue replacement
Article Type: Report
Subject: Hydroxylapatite (Properties)
Shells (Composition)
Chemical synthesis (Research)
Calcium (Properties)
Authors: Adak, M. Dasgupta
Purohit, K.M.
Pub Date: 07/01/2011
Publication: Name: Trends in Biomaterials and Artificial Organs Publisher: Society for Biomaterials and Artificial Organs Audience: Academic Format: Magazine/Journal Subject: Health Copyright: COPYRIGHT 2011 Society for Biomaterials and Artificial Organs ISSN: 0971-1198
Issue: Date: July, 2011 Source Volume: 25 Source Issue: 3
Topic: Event Code: 310 Science & research
Product: Product Code: 2819821 Calcium NAICS Code: 325188 All Other Basic Inorganic Chemical Manufacturing
Geographic: Geographic Scope: United States Geographic Code: 1USA United States
Accession Number: 304842718
Full Text: Introduction

Materials used for the repair and reconstruction of diseased or damaged parts of the musco-skeletal system is termed as 'Biomaterials such as Hydroxyapatite; Bioglass; Biopolymer; Ca-Phosphates etc (1).

A lot of research has been carried out to find out the substitute to support the bone in the medical field. Presently steel as support to bones are used but they can react with body fluids [T = 37[degrees]C and pH = 7.4] when kept for many years so they need substitute. Next comes the inert materials like bio glass etc but due to Formation of Fibrous Tissue of Variable Thickness they can Leading to Tumor, now ceramics specially the bio ceramics are the better alternatives since they have high corrosion resistance (2) better compressive strength and relatively low density and low weight. Porous bioactive ceramics such as hydroxyapatite and calcium phosphates are attractive for bone regeneration and reconstruction due to their bone bonding ability and good bone in growth property (3). Among them hydroxyapatite is taken as the best alternative as it contains same chemical nature and similar crystallographic structure with bone excellent bio compatibility (4). Bio-activity and osteo-conductivity has increased its use in the clinical fields. It also allows tissue ingrowth which helps for its replacement with bone materials (5,6).

Formation of synthetic HAp is really critical due to the following problems (2,7). 1. Difficult to sinter to achieve full / near-full dense body; 2. Decomposes above 1250[degrees]C; 3. Difficult to control porosity; 4. By the virtue tailoring of microstructure / mechanical properties.

These problems can be solved by synthesis of HAp in nanocrystalline forms, especially by using the nano-sized starting materials which provides (8) very high surface energy, reduction of sintering temperature, control of porosity tailoring of microstructure.

Commercially available HAps are relatively expensive due to the use of high purity of reagents. The HAp derived from natural material has the advantages that they inherit some properties of raw materials such as pore structures, carbonated HAp etc. So for the present purpose natural source of calcium carbonate preferably the dead snail shells (9), a typical biological waste material is used as the raw materials. Snail shells P. notabilis consists of 57.9 mg calcium and 31.5 mg magnesium per 100g of dry shells.

Materials and Methods

Synthesis

Dead snail shells are washed thoroughly and heated in a box furnace at 1000[degrees]C for about 2 hours to decompose organic matters and convert the calcium carbonate to calcium oxide which in turn on exposure to atmosphere forms calcium hydroxide. The product was finely ground in an agate pestle and mortar

According the first route, newly produced calcium hydroxide was weighted and was used to react with prerequisite amount of concentrated HN[O.sub.3] nitric acid to form calcium nitrate. It was then react with ammonia to form ammoniacal suspension and react with diammonium hydrogen phosphate solution corresponding to different stoichiometrioc ratio of Ca/P = ranging (a) 1.17, (b) 1.37, (c) 1.47, (d) 1.57, (e) 1.67 and (f) 1.77 seperately. The entire reaction mixture was allowed to stir constantly for 24 hours maintaining an ice cold condition. The gelatinous product was washed repeatedly with distilled water to remove unwanted ions and dried overnight in an oven at 100[degrees].

According the second route, newly produced calcium hydroxide was weighted and was used to react with pre-prepared acid pre-mix maintaining a pH range 2.1. Then it was added to an aqueous suspension of calcium hydroxide of pH 11. It is allowed to stir continuously for about 24 hours at room temperature. The gelatinous product was washed repeatedly with distilled water to remove unwanted ions and dried overnight in an oven at 100[degrees]C.

Characterization

The particle size analysis of HAp was done by Malvern Particle Size Analyzer (Model--Micro-P, UK).

The weight loss and thermal stability of the samples were also evaluated from the thermogravimetric analysis data.

The X-Ray Powder Diffraction (XRD) analysis of the sample was done by X-ray Diffractometer using Cu-Ka radiation. The X-ray diffraction (XRD) patterns were recorded in the steps of 0.010 interval with 1s counting time at each step.

The functional groups present in newly synthesized products were ascertained by FTIR.

The surface area of the synthesized HAp powder was made by BET surface area analyzer (QUANTACHROME Model: Autosorb1).The specific surface area of each hydroxyapatite was determined by BET analysis which estimates of surface area by nitrogen adsorption at 77K.

The structural characterization of HAp powder synthesized samples were made by Scanning Electron Microscope (SEM).

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Results and Discussion

The TG-DTA thermogram of dead snail shell (Fig 1) shows a weight loss of 42.32% at temperature between 90[degrees]C - 120[degrees]C that is due to the physically adsorbed water. Over a wide range of temperature from 250[degrees]C - 400[degrees]C the weight loss is due to the decomposition of MgC[O.sub.3] combined with the combustion of hydrocarbons. The weight loss along with endothermic peak at 750[degrees]C-850[degrees]C indicates the decomposition of CaC[O.sub.3] following the reaction.

CaC[O.sub.3] [right arrow] CaO + C[O.sub.2]

So it is confirmed from the thermal analysis that snail shell mainly contains CaC[O.sub.3] along with small amount of MgC[O.sub.3] and other organic matters.

The TG-DTA thermograms for the hydroxyapatite powder after drying are illustrated in Fig. 2. The first endothermic region range from 90 to 295[degrees]C with a peak at about 250[degrees]C, which corresponds to the dehydration of the precipitating complex and the loss of physically adsorbed water molecules of the hydroxyapatite powder. The weight loss in this region is 16%. With increasing temperature from 295 to 1200[degrees]C no peak has been observed, except a weight loss of 6% is observed at the TGA curve in the temperature range which is assumed to be resulted gradual dehydroxylation in hydroxyapatite powder. This can be explained by the following reaction (10):

[Ca.sub.10][(P[O.sub.4]).sub.6] [(OH).sub.2] [right arrow] [C.sub.10] [(P[O.sub.4]).sub.6] [(OH).sub.2"2x] [O.sub.x] + x[H.sub.2] O

[FIGURE 3 OMITTED]

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The precipitates obtained from the solution at different temperatures for various lengths of time were identified by XRD and the results are shown in Fig 3. The first and second peaks of pH value were associated with the appearance of dicalcium phosphate anhydrate (CaHP[O.sub.4], DCPA) and fibrous OCP ([Ca.sub.8][H.sub.2][(P[O.sub.4]).sub.6]) respectively, and the last stage corresponded to the phase transition of the precursors into HAp powder.

In order to investigate the effect of heat treatment on nanocrystallization and phase transformation in hydroxyapatite powder by precipitation method. Fig. 4 presents the XRD spectra at various temperatures for hydroxyapatite calcined powders by precipitation method. Particularly noteworthy, the hydroxyapatite peaks gradually increased in intensity when the sample was heated from 100, 450, 900, up to 1200[degrees]C (1 h holding time at each temperature), indicating further nucleation/growth of the hexagonal-dipyramidal nanocrystals inside the powder particles. At room temperature the stable phase is hexagonal-dipyramidal. This hexagonal-phase-growth XRD data with increasing temperature is reported here for the first time, but the hexagonal- dipyramidal phase was not transformed to the other phases up to 1200[degrees]C. The patterns due to the as-prepared HAp at 100[degrees]C bears with it the characteristic patterns of HAp but not with much resolution and intensity. It contains no other crystalline phase other than HAp. The broad patterns around at (2 1 1) and (0 0 2) indicate that the crystallites are very tiny in nature with much atomic oscillations. The XRD patterns of the heat treatment HAp at 450 and 900 [degrees]C show increase in intensity due to planes around (2 1 1), (0 0 2), (3 0 1), (2 2 2) and (2 1 3). Again it rules out the formation of any new crystalline phase other than HAp. Again at the heat treatment temperature of 1200[degrees]C, new crystalline phases are not seen. This precludes even amorphous phases, as the patterns appear very well resolved. As the increase in intensity is noticed for every pattern with increase in heat treatment temperature. This decomposition into tricalcium phosphate was also not observed in the present study when the HAp was heat treatment at 1200[degrees]C. Reported transformation of HAp into oxyapatite between 1200 and 1400 [degrees]C (14), but at 1200[degrees]C no such transformation is observed in the present study (fig 5).

Infrared characterization was carried out for the sample to study the spectral characteristics indicative of the chemical bonding in the synthesized HAp-e powder (Fig 6). The spectrum can be divided into four regions with peaks having wave numbers around 3500, 1420, 1100 and 600[cm.sup.-1]. The peak observed around 3431.8[cm.sup.-1] is due to the presence of-OH bond (10). This peak is mainly due to O-H stretching vibration in HAp (13). The peak at 1036.2 [cm.sup.-1] is associated with the stretching modes of the P-O bonds of HAp (14,15). The double peak at 603.1 [cm.sup.-1] and 567.4[cm.sup.-1] are due to bending modes of P-O bonds in phosphate group (14). Thus, the presence of P[[O.sub.4].sup.3-] - group in HAp is almost confirm from IR studies. The pH of the medium during synthesis of HAp was maintained using ammonium solution and it was removed from the suspension with repeated washing with distilled water. In spite of all efforts to remove ammonia from the solution, there is a possibility of small amount of it in the HAp powder. The IR analysis shows a small broad peak at 1422.6[cm.sup.-1]; which is characteristics peak of N[H.sub.4.sup.+]-group (16-18). Stretching of P-O at 1040cm-1; Bending of P-O: 567, 602[cm.sup.-1]; Weak band at 2000[cm.sup.-1] (Overtone); C[O.sub.3.sup.2-]: 864, 1417, 1477[cm.sup.-1]; Stretching of O-H: 3447[cm.sup.-1]; O[H.sup.-] band: 633, 3447[cm.sup.-1] (better resolved) 3572[cm.sup.-1] By controlling the temperature during precipitation and ripening time, and with different Ca/P ratio of starting materials, powders with three different values of crystalline degree were produced.

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XRD patterns of the as synthesized powder (HAp - e) showed the presence of an amorphous phase. The sample heated at 750[degrees]C showed broad peaks of an apatite phase. When the temperature was increased, the apatite peaks became sharper, because of crystal growth, (fig 7). Alternatively, calcium carbonate peak at 29.399[degrees] was present together with HAp phase; however, the TCP phase was not detected at any temperature. Also, the CaO peak at 37.469[degrees] and 54.029[degrees] was not detected at any temperature. The presence of hydroxyapatite was confirmed by a strong diffraction peak at 31.773[degrees] (211) plane. The accompanying two peaks at 32.196[degrees] and 32.902[degrees] of equal intensities were also detected. The powder patterns do not indicate any peaks corresponding to CaO. The calcium carbonate peak was minimized and the intensity of hydroxyapatite peaks at 211, 112, 300 plane was increased. The crystallization of the carbonate hydroxyapatite was prepared. From the figure, the secondary impurity phases such as CaO and TCP were not detected at the sintered samples of powder A. Moreover, the O-H stretch band around 3450 [cm.sup.-1] is more accentuated and shifted at lower frequency in powder C characterized by a higher solvation extent. On the other hand, the presence of the C[O.sub.3] group also proved the formation in powder B of some amount of carbonate-apatite (C[O.sub.3]) substituted in both positions for OH and P[O.sub.4] groups.

The apparent size of hydroxyapatite crystallites obtained from XRD profile analysis by Scherrer method (15-17) is shown in Fig. 7. In this method of broadening contribution due to the crystallites size are taken into account. A gradual increase in crystallite size was observed for hydroxyapatite-e with increasing in heat treatment temperature.

The crystallite sizes were calculated using Scherrer's relationship, d = kl/b cosq,

Where d is the average diameter in [Angstrom], k the shape factor, b stands for full width at half maximum of the peak, and q is the diffraction angle.

The Bragg reflections at (002) planes of HAp were considered to calculate the crystallite size. Taking mean value of b = (0.001373[degrees]A), t = {0.96 x 1.54} / {0.001373 x cos (31.8019 / 2)} = 1120[degrees]A = 112 nm Taking mean value of b =0.00154059 nm t = 998 [degrees]A = 99.8 nm

The expansion does indicate a structural change in the lattice. It should be noted that materials prepared via low temperature wet chemical processes are known to accommodate various ionic species, e.g. [H.sub.3]O+, [(HP[O.sub.4]).sub.2-] etc and they may decompose in a variety of ways leading to structural changes.

The surface areas of the hydroxyapatite powder and calcined HAp are determined using BET plots (fig 8). They are 83 and 12.34 [m.sup.2]/gm respectively (HAp-e, Ca/P ratio = 1.67).

As expected the compaction behaviour of the powders are different, experimentally, higher green density was obtained with HAp-e powder. HAp-e is characterized by larger agglomerates in turn formed by smaller primary particles in respect to HAp-a powder , in which grains are smaller but built by larger particles. Powders are agglomerated during calcinations; but HAp powders have to be calcined to remove volatile impurities like ammonia.

Scanning Electron Micrographs

The morphologies of as synthesized HAp powders are shown in Fig. 9-14. Synthesized HAp powders are almost regular and round in shape; with a little agglomerated structure where as calcined HAp powders are agglomerated. The microstructure as reveals from SEM is in well- agreement with BET surface area analyzer results. HAp -e (Ca/P = 1.667) samples show grains more rounded and with coalescence markedly reduced in respect to other newly produced Hap treated at the same temperature. Calcination treatment (HAp) has a delaying effect on sintering at low temperature: at temperatures in the range 950 to 1000[degrees]C, grains are more rounded and coalscence is reduced, that agrees with values higher in respect to HAp-e and the possibility to identify a preceding mechanism of particle rearrangement; but, at higher temperature, decreasing the probability of grain growth, the densification can be improved.

Conclusion

Homogeneous Hydroxyapatite powders have been synthesized successfully from dead snail shells following two different routes. Amount of hydroxyapatite obtained from route 1 is less compare to route 2 but purity of route 1 is higher as compare to route 2 in both cases. By controlling the temperature during precipitation and ripening time, and with different Ca/P ratio of starting materials, powders with different values of crystalline degree were produced to compare the crystallinity, and sintering behavior. After sintering at 1200[degrees]C, HAp-e samples (Ca/P ratio = 1.67) shows better densification, compatibility and finer particle size with respect to other newly produced HAps,(having lower Ca/P ratio,) treated at the same temperature. The snail shell seems to be a promising source of calcium for preparing nanocrystalline hydroxyapatite with excellent properties so essential for hard tissue replacement. But In order to quantify the biodegradability of these materials, the ISO/FDIS 10993-14:2001 degradation test should be used.

Acknowledgement

We are thankful to the Department of Science and Technology, New Delhi, for financial support under the scheme WOS-A. Our special thanks to Prof Sunil Sarengi, Director, NIT, Rourkela, all the staff and faculty members of Department of Chemistry, NIT, Rourkela for providing laboratory and library facilities.

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M.Dasgupta Adak [1], and K. M. Purohit [2]

[1] Department of Chemistry, [2] Department of Life Sciences, National Institute of Technology, Rourkela, 769008

* Corresponding Author: Dr. M. Dasgupta Adak: m_dasgupta2k6@yahoo.co.in

Received 7 August 2010; Accepted 27 April 2011; Available online 29 May 2011
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