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Biomaterial for Bone and Dental Implants: Synthesis of B-Type Carbonated Hydroxyapatite from Biogenic Source

Written By

Bernard Owusu Asimeng, David Walter Afeke and Elvis Kwason Tiburu

Submitted: 19 February 2020 Reviewed: 25 March 2020 Published: 11 November 2020

DOI: 10.5772/intechopen.92256

From the Edited Volume

Biomaterials

Edited by Petrică Vizureanu and Claudia Manuela Da Cunha Ferreira Botelho

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Abstract

There are several sources from which hydroxyapatite (HAp) can be obtained and may be broadly categorized as synthetic or biogenic. Elevated interest in recent times has pushed for the development of several procedures for extracting HAp from biogenic wastes due to their excellent composition and morphology resemblance to the human calcified tissue (B-type carbonated HAp). Notable biogenic sources reported for HAp extraction span bovine bones, fish scales, corals, eggshells, and snails among other calcium-rich sources. However, most of the synthetic methods are laborious and therefore result in high production costs. In this chapter, we discuss the synthesis of B-type carbonate substituted HAp from an untapped biogenic source, Achatina achatina shells, using a simple precipitation method and a controlled heat-treatment method. This unique treatment method affected the substitution resulting in different crystallographic parameters and revealed a novel material for bone implants and enamel applications.

Keywords

  • biogenic source
  • biomaterial
  • carbonated hydroxyapatite

1. Introduction

Hydroxyapatite (HAp) is a member of the calcium apatite (group of phosphate) family with a high concentration of hydroxyl group [1, 2, 3]. Stoichiometric HAp, Ca10(PO4)6(OH)2 can exhibit either monoclinic or hexagonal crystal structures [4, 5]. The most frequently reported hydroxyapatite crystal structure is the hexagonal system, which consists of unconnected, PO43− tetrahedra with Ca2+ in the interstitial space and a chain of OH ions along the c-axis to balance the unit cell charges [4]. This hexagonal crystal structure allows for replacement (substitution) of ions into the structure. The substitution makes the HAp more reactive and biocompatible [6, 7]. The human calcified tissue (e.g., bone and tooth enamel) consists of mineral components similar to carbonated HAp (CHAp) [8, 9]. As a result, extensive research has been conducted to substitute carbonate into commercial HAp to achieve a suitable material for hard tissue replacement and implants coatings [10, 11].

There are two main types of carbonate (CO3) substitution that occur in hydroxyapatite, namely A-type and B-type. A-type occurs when CO3 replaces OH groups, while B-type substitution occurs when CO3 replaces PO4 in the apatite structure. If these substitutions take place concurrently, an AB-type substitution occurs. Among the types of substitution, literature reports that B-type CHAp is highly similar to the human calcified tissue, which contains 3–5 wt% of carbonate in the lattice of the phosphate [12, 13, 14, 15].

Laboratories have advanced in methods for the synthesis of B-type CHAp but the methods are laborious and expensive. For example, the method most frequently reported in the literature is the one proposed by LeGeros [16]. Herein, the preparation begins by adding PO4/CO3 solution in a stepwise manner into Ca(NO3)2·4H2O solution at a temperature of 90°C. The mixture is stirred for 5 h at a pH of 11 and the precipitates filtered and dried for 10 h at 100°C in an oven. The B-type CHAp is then identified using X-ray diffractometry (XRD) and infra-red (IR) spectrometry. The XRD is a good analytical tool to study the substitution since; CO3 has a smaller planar ion diameter than the tetrahedral PO4 group, and thus the substitution of CO3 with PO4 will result in a decrease of the a-axis and a small increase in the c-axis of the unit cell. On the other hand, in a narrow range, vibrating bonds of functional groups in a material vibrate with a characteristic wavenumber [17], which is why IR spectroscopy is used to study the functional groups.

In this chapter, the authors discussed a simple precipitation and heat treatment method for the preparation of B-type CHAp from Achatina achatina (AA) shells and phosphate-containing solution. Additionally, the XRD and IR results obtained for the substitution are discussed.

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2. Obtaining calcite from the AA shells for HAp preparation

Achatina achatina (AA) is the giant African snail ubiquitously scattered throughout the African continent particularly in West Africa, Ghana. The land strata of the soil are made up of various ions, and the eating habit of the snails enables the ingestion of these ions into their shells in trace amounts. The AA shells, however, serve as a rich source of calcium carbonate (CaCO3)—a precursor for HAp preparation compared to other biogenic sources like bovine bones, fish scales, corals, eggshells, and other landmark snail shells. CaCO3 mainly exists in two crystal forms: aragonite and calcite. The aragonite structure as indicated by the XRD pattern in Figure 1(a) exists in ‘raw ground’ AA shells after the shells are washed with running water to remove sludge and dried in the open air for 6 h. The structure contains impurities like sulphate (SO42−) and is indicated in the IR spectra in Figure 2(a). These impurities consequently rule out raw AA powders as carbonate precursors for the HAp formation. Thus, the raw AA powder is calcined at high temperatures to burn the impurities and convert the aragonite structure into calcite. The calcination temperature typically used is between 600 and 800°C [18] [see Figure 1(b)(d)]. The IR spectra in Figure 2(b) and (c) support XRD patterns, which show that the temperatures (600 and 700°C) facilitate the conversion of the aragonite structure to calcite. The calcite functional groups occur at wavenumbers 712, 856, and 1418 cm−1. The calcite structure is formed at the two temperatures, however, the ideal calcination temperature for HAp synthesis is between 800 and 850°C. These temperatures bring an additional phase to the calcite to initiate the HAp preparation. The evidence is shown in Figure 2(d) where a dual-phase of calcite and calcium hydroxide [Ca(OH)2] is observed. The calcite absorbed moisture from the atmosphere during the open-air drying; so, under high temperature (800°C) the CaCO3 decomposes to form Ca(OH)2. The reaction pathway is given by Eqs. (1) and (2):

Figure 1.

XRD patterns of (a) raw uncalcined AA powder and (b) calcined AA powder at 600°C (c) 700°C (d) 800°C.

Figure 2.

IR spectra of (a) raw uncalcined AA powder and (b) calcined AA powder at 600°C (c) 700°C and (d) 800°C.

CaCO3+H2Oheat800°CCaO.H2O+CO2E1
CaO.H2OCaOH2E2

The reaction is confirmed with IR results in Figure 2(d) showing the hydroxyl group (OH) of calcium hydroxide at wavenumber 3642 cm−1, and calcium oxide (Ca▬O bonding) stretches from wavenumbers 400 to 700 cm−1 in calcite.

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3. HAp preparation

HAp is prepared from 0.3 M solution of diammonium hydrogen phosphate (DHP, pH of 8.12) and 5.7 g of calcite. The calcite is dissolved in distilled water of volume 0.15 cm3 and the same volume of calcite is measured for DHP. The DHP is then added stepwise while stirring at 40°C for 1 h. The stirring time controls the particle size. The longer the stirring time the smaller the particle size [14]. The calcite mostly preferred for HAp preparation has Ca(OH)2 phase; so, in aqueous solution, the Ca(OH)2 quickly dissociates into 2Ca2+ and 2OH, whereas (NH4)2HPO4 produces (NH4)2OH and a monophosphoric acid (HPO4). The monophosphoric acid reacts with OH groups released by the calcium hydroxide to form a phosphate ester through a condensation reaction. The phosphate ester reacts with Ca to form HAp. The reaction is allowed to age for 24 h to enable HAp crystals growth [14]. The ammonium hydroxide is filtered together with water to obtain the HAp crystals. The reaction pathway is given in Eq. (3):

10CaOH2+6NH42.HPO4+2H2OCa10PO46OH2+12NH4OHl+8H2OE3
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4. B-type carbonated HAp preparation

The calcite (CaCO3) in the mixed-phase of the HAp precursor decomposes into calcium (Ca2+) and carbonate (CO32) during stirring. The calcium is used as part of the HAp formation and the CO32 lies behind the lattice of phosphate (PO43). The ionic diameter of PO43 is larger than that of CO32, thus the first masked the latter and is not detected using XRD. The IR spectra in Figure 3(a) show major functional groups of HAp and minor functional groups of carbonate. The PO43groups of HAp occur at wavenumbers 1028, 963, 600, and 563 cm−1 whereas CO32groups are found at 1435 and 874 cm−1. The HAp is subjected to a temperature of 850°C for 12 h for annealing and normalizing. The heat treatment initiates the substitution of CO32 for PO43as indicated in Eq. (4). Figure 3(b) shows functional groups of the heat-treated HAp. The CO32 groups at 1435 cm−1 are now prominent and the OH groups, which are absent in HAp without heat treatment, are also intense, which serves as evidence of B-type carbonate HAp.

Ca10x2PO46xOH2+CO3xheattreatmentCa9.5PO45CO3OH2E4

Figure 3.

IR spectra of (a) HAp without heat treatment and (b) HAp with heat treatment at 850°C. The figure is obtained from Asimeng et al.

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5. Discussion

Natural sources for hydroxyapatite synthesis have generated a lot of interest in recent times due to the various advantages it presents over synthetic sources in terms of similarity to the biological apatite (bone and dental). Some of these sources include but are not limited to plants, bovine bone, eggshells, and snail shells. All these biogenic sources have calcium carbonate as a precursor for HAp synthesis. Plant and algae, however, do not have a lot of calcium carbonate as compared to the other sources like bovine, eggshells, and snail shells [19]. The extraction of HAp, therefore, becomes somewhat laborious as there is the need for the addition of more calcium and/or phosphate precursors. This complicated process and the usage of additional calcium or phosphate source increases the cost of production. Clearly, this relegates plant and algae as a great source of HAp for biomedical applications. Biogenic sources such as seashells are rich in CaCO3; however, due to their aquatic nature, they require additional processes/treatment to transform them into a pure phase of HAp [20]. The employment of a combination of extraction methods to achieve the transformation affects the cost of production greatly.

The increasing consumption of snails (Achatina sp.) as a delicacy across all of West Africa has caused a significant increase in snail shell waste production. The recovery of these shells allows for HAp synthesis and reduction in solid wastes. It is recorded in the literature that eggshells and snail shells have a lot of calcium carbonate but the percentage composition in snail shells is more than in eggshells [21]. Snail shells are reported to have about 95–99% calcium carbonate [21]. Bovine bones, comparable to snail shells, present excellent properties to extract HAp but using bovine bones would require a processing technique (hydrothermal process), which is very expensive.

Several extraction methods have been documented for the synthesis of HAp. These fabrication methods include dry methods (solid-state and mechanochemical), wet methods (chemical precipitation, hydrolysis, sol-gel, hydrothermal, emulsion, and sonochemical), and high-temperature processes (combustion and pyrolysis) [22]. It is mostly reported in the literature that wet chemical precipitation tops the chart when it comes to cost-effectiveness considering the materials used and their availability. Also, the use of the wet chemical precipitation method typically results in a low crystalline material compared to a hydrothermally extracted HAp. According to ISO 23317, calcium apatite similar to the bone or enamel apatite should be Ca-deficient and made up of impurities such as CO32− and Na+ and have low crystallinity. It should be noted that for biomedical applications, one desirable property is nano-sized particles. The extraction of nano-sized HAp has advantages in terms of high surface activity and ultrafine structures [23, 24, 25], higher bioactivity, and better resorbability than micron-sized particles. With this in mind, the author has successfully extracted HAp from AA shells with particle size in the nanosize range in his previous works. Also, it should be noted that results of an earlier work conducted by Asimeng et al. on the extraction of HAp from the AA shells highlight particle size, lattice parameters, physiochemical properties, and morphological characteristics typical of the human enamel apatite. HAp obtained from the AA shells has further been subjected to a heat treatment to obtain a B-type carbonated HAp and this has significantly improved the physicochemical properties for applications in dentistry and orthopedics. The unit cell parameters of HAp with and without heat treatment are reported by Asimeng et al. The crystallographic information of the B-type carbonated HAp supports the theory that the unit cell a-axis decreases whereas c-axis increases as compared to the HAp without heat treatment.

Table 1 provides a comparative summary of properties of HAp extracted from the different sources and AA shell.

PropertiesMammalian (bovine bone, porcine bone, horse bone, camel bone)Aquatic/marine (fish scales, fish bone)Other shells (cockle shell, clam, sea shell, eggshell)Plant/algaeAA shell
MorphologyIrregular, rod-like, flakelike, needle-like, plate-likeFlat-plate, hexagonal, rod-like, irregular, nearly spherical, agglomerate, varySpherical, needle-like, rod like, globules, agglomerate polygonalFlakes, cluster, rectangular, elongateNeedle-like, rod like, spheroids
Particle size20 nm–500 μm5 nm–1.0 μm5 nm–10.4 μm50–500 nm12–17 nm
CrystallinityHigh (calcination)
Low (chemical treatment)
High (calcination)
Low (chemical treatment)
High (combination method)
Low (chemical treatment)
High (calcination)
Low (chemical treatment)
78.2–83.32%
Crystalline PhasesHAp, β-TCP, CaOHAp, β-TCP, TCPHAp, calcite, βTCPHAp þ whitlockite, CaCO3, βTCP, SiO2HAp, calcite
References[26, 27, 28, 29, 30][31, 32, 33, 34, 35, 36][37, 38, 39, 40, 41][42, 43, 44, 45, 46]Asimeng et al.

Table 1.

A summary of the properties of HAp from different biogenic sources.

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6. Conclusion

This book chapter discussed the synthesis of heat-treated B-type carbonated hydroxyapatite (CHAp) from Achatina achatina snail shells and diammonium hydrogen phosphate (DHP) for human calcified tissue replacement and coatings. The synthesis process is in three steps: [1] conversion of the shell crystal structure from aragonite to calcite, [2] precipitation of calcite and DHP to form hydroxyapatite (HAp), and [3] annealing and normalizing the HAp to form B-type CHAp. The synthesis reveals that calcination temperature plays a key role in step [1]. It was noticed that temperatures from 800 to 850°C provided an additional phase (calcium hydroxide) that is required to produce hydroxyl groups for the phosphoric ester formation needed for the HAp synthesis. Also, it is recorded herein that heat treatment facilitated the substitution of CO32−.

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Acknowledgments

The authors acknowledge Mr. Osman Wahidu and Mr. Richard Asiamah for the support provided during material synthesis.

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Written By

Bernard Owusu Asimeng, David Walter Afeke and Elvis Kwason Tiburu

Submitted: 19 February 2020 Reviewed: 25 March 2020 Published: 11 November 2020