Journal of Materials Science

, Volume 49, Issue 4, pp 1461–1475

Extracting hydroxyapatite and its precursors from natural resources

  • Muhammad Akram
  • Rashid Ahmed
  • Imran Shakir
  • Wan Aini Wan Ibrahim
  • Rafaqat Hussain
Review

DOI: 10.1007/s10853-013-7864-x

Cite this article as:
Akram, M., Ahmed, R., Shakir, I. et al. J Mater Sci (2014) 49: 1461. doi:10.1007/s10853-013-7864-x

Abstract

Healing of segmental bone defects remain a difficult problem in orthopedic and trauma surgery. One reason for this difficulty is the limited availability of bone material to fill the defect and promote bone growth. Hydroxyapatite (HA) is a synthetic biomaterial, which is chemically similar to the mineral component of bones and hard tissues in mammals and, therefore, it can be used as a filler to replace damaged bone or as a coating on implants to promote bone in-growth into prosthetic implants when used in orthopedic, dental, and maxillofacial applications. HA is a stoichiometric material with a chemical composition of Ca10(PO4)6(OH)2, while a mineral component of bone is a non-stoichiometric HA with trace amounts of ions such as Na+, Zn2+, Mg2+, K+, Si2+, Ba2+, F, CO32−, etc. This review looks at the progress being made to extract HA and its precursors containing trace amount of beneficial ions from biological resources like animal bones, eggshells, wood, algae, etc. Properties, such as particle size, morphology, stoichiometry, thermal stability, and the presence of trace ions are studied with respect to the starting material and recovery method used. This review also highlights the importance of extracting HA from natural resources and gives future directions to the researcher so that HA extracted from biological resources can be used clinically as a valuable biomaterial.

Introduction

Repair of bone defect due to chronic disease or trauma still remains a challenge for clinicians. Above the critical size, the restoration of bone defect normally requires use of synthetic biomaterial. Due to the restricted supply of autologous bone and threat of possible infection from using allograft, it is necessary to use the synthetic biomaterial or xenograft, a bone segment from different animal species. Benefit of using xenogenous bone is that it is very similar in structure and morphology to human bone. Xenografts such as bovine, sheep, pig, or fish bones contain trace amount of beneficial ions, which are readily available in large supply and require low-cost processing. Xenogenous bone first undergoes a deproteination process followed by calcination at elevated temperatures. Calcination process is carried out to fully remove the organic components and to destroy pathogens; the remaining ash contains the mineral component of the bone. A complete conversion of bovine bone ash to HA can furnish as much as 1 kg of HA from 1.6 kg of compact bone [1]. This extracted HA contains valuable trace ions, which play a crucial role in the bone re-generation process and are known to speed up the bone formation process. Incorporation of one or more trace ions into the synthetic HA is a laborious process and as a result ion-substituted HA is many folds more expensive than simple HA.

HA is an important synthetic biomaterial which is under considerable investigation for many decades due to its similarity with the mammalian hard tissue. Bone and teeth of mammals are composed of Ca (~24 wt%), P (10 wt%), proteins (22 wt%), and trace amount of elements like Na+, Zn2+, Mg2+, K+, Si2+, Ba2+, F, CO32−, etc. These trace elements play a crucial role in the life cycle of hard tissue; therefore, scientists are looking at various ways to incorporate the beneficial ions into the structure of synthetic HA to impart it with better osteoconductive properties. However, a complete match with the mineral components of mammalian hard tissue still remains a challenge. To achieve maximum similarity with the mineral component of bone, biological resources like eggshells, seashells, animal bones, and plants are under extensive investigation to synthesize biological-like HA for various biomedical applications.

Hydroxyapatite and related compounds

Calcium phosphate (CP)-based biomaterials are group of compounds having Ca/P molar ratio in the range of 0.5–2 [2, 3] and are the most sought after biomaterials for the reconstruction of various bone defects especially in the field of dentistry, orthopedic and trauma surgery [46]. A brief list of important CP-based ceramic materials along with their formulas and applications is presented in Table 1.
Table 1

Important calcium phosphate compounds with their Ca/P ratios and Pksa values

No.

Compound

Formula

Ellipsis

Ca/P ratio

−log(Ks)

Application

1

Monocalcium phosphate monohydrate

Ca(H2PO4)2·H2O

MCPM

0.5

1.14

Increase root fluoride uptake [7]

2

Monocalcium phosphate (anhydrous)

Ca(H2PO4)2

MCPA

0.5

1.14

Artificial bone graft

3

Dicalcium phosphate anhydrous

CaHPO4

DCPA

1

6.90

Polishing agent for teeth, source of Ca and P in food supplements

4

Dicalcium phosphate dihydrate

CaHPO4 2H2O

DCPD

1

6.59

Sustained release of highly water-soluble drugs [8]

5

α-Tricalcium phosphate

α-Ca3(PO4)2

α-TCP

1.5

25.5

Biodegradable composite for bone repair [9]

6

β-Tricalcium phosphate

β-Ca3(PO4)2

β-TCP

1.5

28.9

Orthopedic surgery

7

Calcium-deficient hydroxyapatite

Ca10−x(HPO4)x(PO4)6−x(OH)2−x

CDHA

1.5–1.6

85

Bone grafting

8

Hydroxyapatite

Ca10(PO4)6 (OH)2

HA

1.67

116.8

Repairing of hard tissues

9

Fluorapatite

Ca10(PO4)6F2

FAP

1.67

120

Used as source of fluorine in pharmaceutical products

10

Tetracalcium phosphate

Ca4(PO4)2O

TTCP

2

38–44

Applied as cements and coatings on metallic implants

Due to exceptional biocompatibility [10, 11], osteoconductivity [12], and osteointegration [13], CP-based materials have been under intense investigation for over half a century. These CP-based biomaterials have been successfully used to replace and augment damaged, worsened, or degenerated hard tissues of the human body.

Apatite is the general name used for CP class of minerals and have general formula A4B6(MO4)6X2, where A and B are considered as calcium in many living tissues, MO4 is designated as phosphate group, and X indicates the presence of OH group in the apatite structure [14]. HA is an important CP-based material, which resembles mineral component of natural bones and teeth [15, 16]. HA with Ca/P ratio of 1.67 exhibits exceptional biocompatibility [1719] and bioactivity [2022]. It has been used as a bone substitute material [23, 24] and dental implant for over 50 years [25]. HA can promote rapid bone regeneration and direct bonding with regenerated bone without the need of intermediate connective tissues and its synthetic form is mostly applied to reconstruct the hard tissue due to its osteoconductive properties. Due to the growing importance of HA as a biomaterial, continuous attempts are being made to enhance the biological properties of HA. Although HA crystals are frequently used in orthopedic field, its high dissolution rate in the physiological atmosphere limits its applications in the medical field. Research has shown that the dissolution rates of HA can be altered by incorporating biocompatible ions into its ion-friendly crystal structure [26, 27].

Hydroxyapatite can either be synthesized from the inorganic components or from the natural organic based materials. In general, HA synthesized from natural organic sources is non-stoichiometric which may be due to the presence of trace amount of ions found in the natural organic sources. Nevertheless, both types of biomaterials are bioactive and biocompatible in nature and are considered equally well for in vitro applications but the major problem associated with biomaterials synthesized from the inorganic Ca- and P-based sources is the high cost associated with the synthetic process. Most conventional chemical methods involve the synthesis of HA without any trace of beneficial elements such as Na+, Zn2+, Mg2+, K+, Si2+, Ba2+, F, CO32−, etc.; the presence of these ions directly influences various biochemical reactions linked with the bone metabolism. Recently, many research articles have been devoted to the synthesis, characterization, and application of ion-substituted HA. Ions can replace the Ca ions inside the crystal structure or can replace either the OH or the PO43− ions, which is usually referred to as A-type or B-type substitution, respectively. Extraction of HA and its precursors from inexpensive natural biological reservoirs such as mammalian and fish bones [2832], corals [33, 34], eggshells [35, 36], sea shells [37], and plants [38] has opened up attractive and efficient means of preparing ion-doped HA.

Hydroxyapatite prepared from natural bone ash, eggshells, or sea shells can exhibit better biological properties due to the presence of beneficial cations like Na+, K+, Mg2+, Sr2+, Zn2+, and Al3+ or anions like F, Cl, SO42−, and CO32− or the presence of both is proven to be better for different medical applications especially for rapid bone regeneration [39]. The presence of Na+ and Mg2+ ions play a vital role in the development of bone and teeth, while their absence can cause bone loss and fragility.

A simple classification of biological resources for the extraction of HA and its precursors is depicted in Fig. 1.
https://static-content.springer.com/image/art%3A10.1007%2Fs10853-013-7864-x/MediaObjects/10853_2013_7864_Fig1_HTML.gif
Fig. 1

Classification of natural resources for the extraction of HA and its precursors

The aim of this review is to analyze the existing literature and highlight the efforts of scientists to extract HA and its precursors from natural resources. In addition, this review critically evaluates the process parameters and their effect on the thermal stability and phase purity of the extracted HA.

Biological sources for the synthesis of hydroxyapatite

Extraction of hydroxyapatite from mammalian bones

So far HA has been produced using different methods and different synthetic materials, which require strict control over process parameters to produce high-quality stoichiometric HA. Synthetic HA is a stoichiometric material with Ca/P ratio of 1.67, while the HA extracted from mammalian bone is a non-stoichiometric material due to the presence of trace ions and as a result it is considered a potential material for bone graft purposes [40].

Generally, HA crystallites can exist in different forms such as needles, rods, spheres, etc., and are usually synthesized from either natural bone ash or from synthetic chemicals. The properties, efficiency, phase purity, and size distribution of HA extracted from natural resources especially from bones depend upon factors like extraction technique, calcination temperature, and nature of bones. Usually, cortical part of the femoral bones is selected for the preparation of scaffolds for guided bone regeneration purposes. In general, all bones of the animals are thoroughly washed, boiled with distilled water, followed by washing with NaOH or hypochlorite to remove dirt and proteins from the surface of the bones. After this preliminary washing and drying, bones are either cut into small pieces or ball milled for different times which in turn also affects the morphology and particle size of the final product. For calcination process bones are heated in a furnace at various temperatures between 600 and 1400 °C to completely remove the organic matter from the bone and furnish HA. A calcination regime is carefully selected to remove the organic matter and enhance the crystallinity of HA, while avoiding thermal decomposition of the final product, HA. Furthermore, calcination treatment also destroys the pathogens, which are likely to transfer diseases from the cattle to the patient.

HA produced in biological system is naturally grown in an organic matrix of protein molecules with specific mechanism. It is already well known that the organic part of the bone facilitates the regulation of lattice orientation, crystallite size, and size distribution during biomineralization processes and at the same time it helps in controlling the thickness of the apatite crystals. Special group of proteins designated as bone morphogenetic proteins (BMP) are believed to play a vital role as regulator in bone maintenance, repair, and its induction [41, 42]. Apatite crystals produced in the biological system are different in many ways from the crystals synthesized using synthetic precursors. The apatite crystals grown in the living system bear smaller crystallite size thus have large surface area which further allows them to absorb extra amount of ions. In short, biological minerals tend to attain organized structure in a very short time [43].

Extracellular matrix in bovine bone is mainly composed of HA nanocrystals and collagen fibers and, therefore, has been a focus of numerous studies exploring the extraction of natural HA. Extraction of HA from natural bone materials is economically viable and easy to carry out. Bone is a inimitable composite matrix of collagenous fibers (20 wt%), apatite minerals (69 wt%), water (9 wt%), and organic matters such as proteins, lipids, and polysaccharides are present in small quantities. [44].

In order to extract HA from bovine bones, the bovine bone was chemically treated to remove unwanted organic matter followed by overnight calcination 500 °C to furnish poorly crystalline porous carbonated hydroxyapatite with average pore size of 12 nm [45]. Porous HA structures produced at such low calcinations temperatures tend to have poor mechanical properties and, therefore, are only suitable for low load bearing bone grafting or coating purposes.

Cancellous bovine bones from calf femoral condyles were calcined at 800 °C for 6 h to burn off the organic matrix. Dried bone was soaked in diammonium hydrogen phosphate (DAHP) solution at room temperature before calcination at 1300 °C for 1 h to furnish HA. Phase composition of the final product depended on the concentration of DAHP as high concentration of DAHP resulted in transformation of HA into TCP [46]. Structural studies through SEM confirmed that the biphasic structure was well sintered and it retained its porous structure.

Thermally stable phase-pure HA was produced by calcinating bovine femur, sheep femur bone, sheep skull flat bone, and chicken femur bone between 600 and 1100 °C [1]. The calcination process at 800 °C produced phase-pure crystalline HA having crystallite size around 133 nm with trace amounts of Na+, Mg2+, Sr2+, and K+ with Ca/P between 1.46 and 2.01. Increase in calcinations temperature to 1100 °C increased the crystallinity of the HA but this high calcination temperature resulted in the formation of β-TCP.

Hydroxyapatite extracted from bovine cortical bone has been used to fabricate HA/collagen (extracted from bovine tendons) scaffolds for tissue engineering purpose [47]. XRD spectra of bone heat-treated at 1100 °C matched perfectly with the standard XRD pattern of crystalline HA (JCPDS card No. 9-432), while Scherrer equation was used to calculate the average crystallite size (58.4 nm). The presence of secondary phases such as CaO and Ca(OH)2 was attributed to the lack of incubation of bone-derived mass with 1 % phosphoric acid after calcination at elevated temperature. A-type carbonate substitution of HA was confirmed due to the presence of peaks in the FTIR spectra at 1550, 1500, 1462, and 877 cm−1 while B-type substitution was confirmed through the presence of bands at 1455, 1411, and 872 cm−1.

The deprotenized and defatted pork bone pulp has been used to synthesize carbonated HA (cHA) nanoparticles through two-step calcination process at 650 and 950 °C for 3 h [48]. Phase-pure crystalline HA prepared after calcination at 950 °C was mesoporous in nature and contained number of trace elements such as Cr3+, Cu2+, Co2+, Cd2+, Pb2+, and Hg2+ from which Cd2+, Pb2+, Hg2+, and As3+ were within the established standards [ISO standard 13779–1:2000 (E)].

Phase-pure crystalline HA can be extracted from bovine bone using alkaline hydrothermal hydrolysis of organic matrix at 250 °C, subcritical water extraction of collagen at 275 °C, and most commonly used thermal decomposition of collagen and other organic matter at 750 °C [49]. All three extraction methods produced phase-pure HA but the morphology and particle size were directly influenced by the extraction process employed.

Effect of sintering temperature (500–1400 °C) on physical and chemical properties of bovine bone-derived HA revealed that 1000 °C sintering temperature is sufficient to produce HA; however, XRD results confirmed the dehydroxylation of HA during sintering at temperatures above 1000 °C [50]. EDX results confirmed the formation of calcium-rich (Ca/P = 1.85) HA phase, which was attributed to the presence of Na+ and Mg2+ ions observed in the EDX spectra.

Vibro-milling method has been successfully employed to prepare crystalline needle-shaped nano-HA from defatted bovine bone after calcination at 800 °C for 3 h. Vibro-milling time directly influenced the particle shape and size, where the longer vibro-milling time resulted in the breakdown of HA needles into fine powder [51].

Recently a group of researchers have reported the formation of 116-nm-sized crystalline HA nanoparticles of spheroidal and polygonal geometry through thermal method by annealing bovine bone at 800 °C for 2 h [52].

A research group has synthesized natural HA from bovine bone to prepare composites with poly(lactic acid) (PLA) and explored their bioactivity [53]. Results indicated that the resulting powder was crystalline, agglomerated, and irregular in shape. Moreover, it was confirmed from the results that saline coupling made the bovine-derived HA composites thermally more stable.

Formation of HA through transferred arc plasma (TAP) processing at 5 KW in argon plasma for different processing times has been reported [54]. TAP processing for 90 s produced HA with Ca/P ratio of 1.93. Lower TAP processing times of 30 and 45 s resulted in incomplete removal of organic matter whereas higher processing time of 120 s resulted in the decomposition of HA into TCP, tetracalcium phosphate (TTCP), and CaO phases.

Calcination of washed and degreased bovine and porcine bones at 600, 900, and 1200 °C for 18 h furnished porous HA structures [55]. Three main weight losses attributed to the release of water (surface and adsorbed) at temperatures below 200 °C, combustion of organic matter at temperatures between 200 and 600 °C, and release of CO2 due to the carbonate decomposition at temperatures above 700 °C were studied through thermogravimetric analysis. XRD analysis confirmed that the crystallinity of HA increased with calcination temperatures from 600 to 900 °C; however, no increase in crystallinity was observed beyond 900 °C. Chemical analysis of the filtrate from sample washing revealed that the calcination temperature of 1200 °C resulted in the formation of CaO as a secondary phase.

Bovine bone burned in open atmosphere and ground by ball milling was reacted with calcium nitrate at pH 1.3 followed by basification to pH 10 furnished nanosized amorphous HA [56]. HA was heated at 800 and 1100 °C to improve its crystallinity. Bovine HA was further used to prepare composite with PLA (PLLA) and its mechanical and biological properties were studied.

Phase-pure, equiaxed, and polycrystalline HA with uniform porous microstructure was formed by calcinating cortical femoral bovine bone at 900 °C [57]. Authors monitored the weight loss amounting to 37 % associated with water loss and removal of organic matter through TGA analysis. XRD results confirmed the formation of nanosized polycrystalline HA particles (29.5–79.9 nm), while EDX analysis confirmed the presence of Na+, Mg2+, and Al3+ as trace ions.

Physicochemical properties of biological HA derived from deffated, NaOH-treated, and calcined (900 °C) bovine bone have been studied [58]. XRD analysis confirmed the formation of phase-pure HA; however, the defatted bone exhibited the least degree of crystallinity, which was improved upon alkaline treatment, while the calcined bone possessed the highest degree of crystallinity. FTIR and SEM analysis confirmed the presence of organic matter in defatted and alkaline-treated bovine bone samples, hence supporting the fact that these powders were unsuitable for biomedical applications. ICP-OES results confirmed the presence of Mg2+, Na+, Al3+, Ba2+, Cu2+, Fe2+, K+, Mn2+, and Zn2+ as trace ions.

Through thermal decomposition method, HA was synthesized from cow femur bones which was hybridized with silver ions through reduction of silver nitrate using N,N-Dimethylformamide (DMF) in the presence of poly(vinyl acetate) (PVAc) as binding agent [59]. XRD confirmed the formation of phase-pure HA, while peaks due to the (111), (200), (220), and (311) planes of Ag were visible as a separate phase. SEM analysis confirmed the formation of 8–20-nm-sized spherical crystallites, while the TEM image confirmed the attachment of Ag particles to the edge of the HA crystal.

Mammalian bones, especially the bovine bones, are a rich source of ion-doped HA and, therefore, have been exploited by the scientists to extract HA. Table 2 summarizes physicochemical properties of HA extracted from mammalian bones.
Table 2

A brief summary of the properties of HA powder derived from mammalian bones

No

Source

Ca/P ratio (nm/μm)

Particle size (nm)

Shape

Secondary phases

Trace elements

Calcination (T °C)

Reference

1

Bovine bone

1.46–2.01

133

Irregular spheres

Na+, K+, Mg2+, and Sr2+

600–1100

[1]

2

Bovine bone

Co2+, Cr3+, Cu2+, Ni2+, Cd2+, Hg2+, Pb2+ and As3+

[48]

3

Bovine bone

1.56–1.86

300

Nanorod

750

[49]

4

Bovine bone

1.85

500–1400

[50]

5

Bovine bone

1.66

58–62

Needle like

800

[51]

6

Bovine bone (via thermal way and mechonochemical way)

116

Spheroidal and polygonal

800

[52]

7

Bovine bone

1.93

 

TCP

Na+, Mg2+, Al3+, K+, Si2+, S2- and Cl

[54]

8

Human, pig, and porcine

63–104

600–1200

[55]

9

Bovine bone

20

Spherical

800–1100

[56]

10

Bovine bone

29.5–71.1 and 79.9

Equiaxial

900

[57]

11

Bovine bone

>3

 

Porous and interconnected

Al3+, Ba2+, Cu2+, Fe3+, K+, Mn2+, Ni2+ and Zn2+

[58]

12

Bovine bone

1.61

8–20 nm

Spherical

[59]

Marine/river sources

Marine/river captured fisheries provide over more than 50 % of the world’s total fish consumption. Fish and crustaceans consumption results in accumulation of large amount of Ca- and HA-rich waste. Therefore, marine waste has been exploited to prepare various bioactive compounds [60]. Caught fish is typically used to provide fish meat, fish oil, and some low economic value fertilizers. However, recent research studies have identified the presence of several CP salts in these sources; therefore, they are being exploited to prepare bioactive compounds [61]. Fish bones are a rich source of calcium, phosphate, and carbonate which can be used to prepare HA. These bioactive compounds can be synthesized using different simple to complex techniques. Generally, fish bones are used to extract calcium for various dietary products; however, very little attempts have been made to synthesize HA from these natural sources for biomedical application [62]. In order to convert fish bones or related sources into HA, these bones are washed with hot water or steam or different alkaline solutions to remove all types of proteins and other organic impurities. After the removal of protein mass the bones are subjected to high-temperature calcination to furnish HA.

Carbonated HA was extracted from Thunnus obesus (big eye tuna) bone using alkaline hydrolysis with 2 M NaOH and thermal calcination at 900 °C for 5 h [63]. Based on the TGA results (not shown in the manuscript) authors reported that HA prepared from tuna bone was thermally stable up to 1200 °C, higher than the HA prepared from other sources such as bovine or pig bones. XRD analysis of non-treated bone indicated the presence of biologically mineralized poorly crystalline HA embedded in organic matrix. While the XRD patterns of alkaline treated and calcined bone samples revealed that the crystallinity and particle size were increased due to the treatments of the resulting cHA. TEM study revealed that the cHA in alkaline-treated bones existed as nanorods of 17–71 nm length and 5–10 nm width, while the HA from calcined bone existed as microstructured crystals of 0.3–1.0 μm size.

Hydroxyapatite particles having size around 300 nm and spherical in shape were synthesized from the bones of Brazilian river fish such as pentado (Pseudoplatystoma corruscans), jaú (Paulicea lutkeni), and cachara (Pseudoplatystoma fasciatum) [64]. Fish bones were initially calcined at 900 °C for 4–12 h followed by the crushing of bones with high-energy ball mill for 2 and 4 h. SEM analysis indicated that the milling time affected the size of spherical particles. Elemental analysis confirmed the presence of Fe2+, Cr3+, Ni2+, Mn2+, Cu2+, Zn2+, K+, and Na+ as trace elements; however, the presence of first four ions was attributed to the use of stainless steel milling balls.

Highly porous HA showing plate- and rod-like morphology with an average diameter of 200-300 nm were prepared through hydrothermal treatment of cleaned inner bone matrix (lamellae spacing) of aragonite cuttle fish bones with DAHP (Sepia officinalis L Adriatic sea) [65]. XRD pattern confirmed the conversion of aragonite to brushite and HA at hydrothermal temperature of 160 °C and below, while phase-pure HA was formed at 180 °C and above. FTIR analysis confirmed the B-type substitution of carbonate group. SEM analysis showed that the original porous network of the bone was maintained during the hydrothermal treatment, which makes it an interesting candidate for tissue engineering applications.

Bones of Sword (Xiphias gladius) and Tuna (Thunnus thunnus) fish caught from north Atlantic marine have been used to prepare HA [66]. After boiling in water to remove organic waste and flesh, the bones were calcined at 600 and 950 °C in a furnace for 12 h in air. TEM analysis confirmed the isolation of rod-like particles with submicron particle size from both species. FTIR, Raman, and XRD studies confirmed the formation of B-type HA after calcination at 600 °C; however, the formation of β-TCP was confirmed by XRD and Raman spectroscopy after calcination at 950 °C. Deviation of lattice parameters from the standard of HA was attributed to the B-type carbonate substitution.

Synthesis of phase-pure nanocrystalline cHA from Fish (Tilapia nilotica) scale waste through alkaline heat treatment method was reported recently [67]. Thoroughly washed and dried fish scales were deprotenized and heated with 50 % sodium hydroxide at 100 °C for 1 h to furnish HA. FTIR analysis confirmed the replacement of some of the phosphate groups with the carbonate group (B-type substitution). ICP-OES confirmed that the Ca/P ratio was 1.67, same as the theoretical value.

Porous HA with Ca/P ratio of 1.78 was extracted from washed and crushed tilapia (Oreochromis sp.) fish scales using enzymatic hydrolysis with 1 % protease N followed by hydrolysis with 0.5 % flavourzyme solution [68]. The extracted HA appreciably promoted the cell viability of MG63 type when compared with commercially available HA. This enhanced biological activity was attributed to smaller particle size (719.8 nm).

Thermal extraction of HA from Cod fish bones by annealing the raw bones at temperatures between 900 and 1200 °C was reported recently [69]. XRD images of thermally treated powder confirmed the formation of biphasic mixture containing HA and β-TCP, the biphasic nature was also confirmed by FTIR due to the presence of characteristic peak of β-TCP at 1122 cm−1. Elemental analysis confirmed the formation of calcium-deficient HA with Ca/P ratio of 1.49; the presence of Na+, F, and Cl was also detected in slightly higher concentration than that found in mammalian bones.

Fish bones and scales are also a good source to furnish nano- to micron-sized cHA with good biocompatibility. Table 3 sums up the interesting physicochemical properties of HA extracted from marine life.
Table 3

A brief summary of the properties of HA powder derived from fish sources

No

Source

Ca/P ratio

Particle size (nm)

Shape

Secondary phases

Trace elements

Calcination (°C)

Reference

1

Thunnus obesus bone

1.65–1.76

17–71 and 5-10

Rod like

900

[63]

2

Pseudoplatystoma corruscans, Paulicea lutkeni, and Pseudoplatystoma fasciatum bones

1.64

300

Na+, Fe3+, Cr3+, Cu2+, Mn2+, K+, Mg2+, Ni2+ and Zn2+

900

[64]

3

Sepia officinalis bones

200–300

Rod

Brushite

[65]

4

Tuna and Sword bones

1.87–0.02

50–66

Rod like

β-TCP

K+, Mg2+ and Na+

600

[66]

950

5

Tilapia nilotica scales

1.67

10–25

Hexagonal

[67]

6

Oreochromis sp. scales

1.78

719.8

Irregular spherical

[68]

7

Cod fish bones

1.49

300–500 and 500

Needle like

β-TCP

Na+, Cl and F

900–1200

[69]

Plant sources

During the last few years, significant efforts have been made to synthesize scaffolds to mimic the bone micro and macro structures. Some porous structures with good degree of porosity have already been obtained. Structures with good degree of porosity and interconnected pores are necessary for cell ingrowth and vascularization but the main problem associated with these structures is the lack of proper bio-physical response, which can only be shown by scaffolds endowed with a high degree of hierarchy [70]. Current efforts are insufficient to generate biomimetic and highly organized hierarchal structures. However, researchers are continuously trying to introduce new natural resources to synthesize HA powder and scaffolds. Scientists have now focused their attentions toward natural resources like wood and calcite-rich plants and algae as their hierarchically structures can be exploited to furnish biomedical scaffolds [71]. Wood is composed of highly organized parallel hollow tubes and cellular microstructures that have high strength, stiffness, and toughness at low density; therefore, it can be used to design bone scaffold templates. Some marine species like algae and corals are composed of CaCO3 that are similar in porosity and interconnectivity to human bones and, therefore, can be used to generate HA structures.

Biphasic HA has been made at low pressure and temperature from red Algae [72]. C. Officinals species contain a large amount of calcium carbonate which can be used for the synthesis of HA. Calcium carbonate extracted from red algae after pyrolysis at 650–700 °C for 12 h was used to produce HA by reacting it with ammonium dihydrogen phosphate at 100 °C for 12 h. SEM analysis confirmed the formation of granular-shaped HA with a rough external surface containing open microporous matrix.

Calcite derived from the calcination of red algae (Phymatolithon calcareum) was converted into HA through hydrothermal treatment in the presence of DAHP and magnesium nitrate [73]. SEM study confirmed that red algae retained its original porous nature during the pyrolysis and hydrothermal treatment. Biocompatibility testing using MG63 cells showed good cell attachment to HA, hence confirming its non-cytotoxic nature.

A multistep process has been reported to convert wood to biomimetic HA scaffold for tissue engineering [74]. The five-step process involved removal of raw material to produce carbon template, conversion of carbon template to calcium carbide, formation of calcium oxide from calcium carbide, synthesis of calcium carbonate from calcium oxide, and finally the production of HA through hydrothermal phosphatization. XRD data confirmed the formation of phase-pure crystalline HA, while the FTIR results confirmed the carbonate substitution into the HA structure.

HA has been extracted from the leaves and stalks of C. edulis (Khat), basil, mint, green tea, and trifolium [75]. XRD analysis confirmed that the HA was the main phase in the leaves of C.eduils and basil plants with a minor amount of calcium hydroxide. Authors mentioned the possible presence of other ions in the HA structure but did not provide any qualitative or quantitative data to confirm the presence.

Phycogenic CaCO3 extracted from marine algae (Rodophycophyta division) collected from Brazilian coast was utilized to prepare non-stoichiometric HA and F-substituted HA through hydrothermal synthesis (200 °C for 24–48 h) by reacting with DAHP and ammonium fluoride [76]. The physical–chemical characteristics were studied using techniques like XRD, FTIR, SEM, and EDX. XRD spectra showed that the resulting HA was phase pure in nature. Both A-type and B-type carbonate substitutions were confirmed through FTIR analysis.

Biogenic sources

Every year millions of tons of eggshells are thrown as waste material all around the world. A large amount of eggshells is produced as waste from houses, restaurants, hatcheries, and bakeries. Similarly, a huge amount of sea shells and other calcite materials are also present in the universe. An eggshell contains calcium carbonate (94 %), magnesium carbonate (1 %), CP (1 %), and organic substances (4 %) [77]. Being cheap and easily excessable, eggshells are considered a good source of calcium precursor required for the synthesis of HA.

Traditionally HA is either made dense or porous by sintering it at high temperature where mechanical properties are controlled by controlling the various factors like sintering temperature, duration of sintering, and composition of the ceramic material used. In this regard, sea shells and eggshells are considered the better selection to synthesize good-quality biomaterials as they resemble the human hard tissues. Some Molluscs shells (containing 95–99 % CaCO3) bear better nanoscale regularity, mechanical properties, and compression strength than common mineral crystals [78, 79]. To extract CaO the shells are washed with boiling water or steam to remove the impurities and then the shells are crushed to powder followed by heating at elevated temperature to furnish CaO. This CaO can be reacted with phosphorous precursors to prepare HA according to the following reaction [77].
$$ {\text{CaCO}}_{ 3} \to {\text{CO}}_{ 2} + {\text{CaO}} $$
$$ 3\,{\text{Ca}}_{ 3} \left( {{\text{PO}}_{ 4} } \right)_{ 2} + {\text{CaO}} + {\text{H}}_{ 2} {\text{O}} \to {\text{Ca}}_{ 10} \left( {{\text{PO}}_{ 4} } \right)_{ 6} \left( {\text{OH}} \right)_{ 2}. $$

Carbonated HA nanoparticles constitute the inorganic part of enamel, teeth, and bones and its concentration ranges a few percent in weight [76, 80]. The incorporation of CO32− ions in the apatite structure affects its structure, morphology, and results in better biochemical reactivity of bone minerals [81, 82]. Therefore, CO32−substituted HA nanoparticles are considered to be a good candidate for bone and dental applications [83].

Hydroxyapatite and β-TCP uniform in size were successfully synthesized using recycled eggshells and phosphoric acid [84]. Washed uncrushed eggshells were calcined at 900 °C to remove organic matter. The calcined eggshells were ball milled with phosphoric acid (1:1.1 ratios) for 24 h followed by calcination at 900 °C for 1 h to furnish HA with Ca/P ratio of 1.65. Phase purity of final product depended on the ratio of CaO from eggshells and phosphoric acid used.

Calcium carbonate present in the mollusks shells (Pomacea Lineata) was converted into HA powder through in vitro treatment by soaking in phosphate solution at room temperature [85]. SEM analysis showed the formation of flower-like arrangement of interconnected platelets with sponge-like organization. XRD study indicated the formation of HA from aragonite after 7 days of soaking in phosphate solution, while the FTIR analysis confirmed a complete conversion of aragonite to HA after 30 days.

Hydrothermal and thermal calcination methods were used to synthesize HA from natural coral exoskeleton and xenogeneic bone, respectively [86]. XRD analysis confirmed the formation of phase-pure HA as no peaks for other phases such as TCP, CaO, and CaCO3 were observed. However, peak shifting and deviation of lattice parameters from the standard observed for HA extracted from xenogeneic (bovine) bone was attributed to the presence of carbonate ions in the HA structure.

Coral pre-treated with sodium hypochlorite was subjected to series of hydrothermal treatments to furnish highly porous Si-incorporated HA [87]. XRD analysis confirmed the formation of phase-pure HA, while EDS studies confirmed the presence of Si and Mg in addition to Ca, P, and O. The presence of Mg in the final product was attributed to the high Mg concentration in seawater. The resulting porous structure with 70 % porosity possessed exceptional compressive strength of 5.5 MPa, a value much higher than the cancellous bone and porous HA structures with similar degree of porosity.

In situ HA synthesis was carried out by reacting crushed and washed eggshells with hydrochloric acid and DAHP solution for one week at room temperature [88]. Eggshells were not calcined to remove organic matter such as amino acids, proteins, and glycoproteins prior to their use. Authors concluded that the presence of these molecules helped to control the shape and size of the nanoparticles. However, the presence of these biomolecules in the final product will hinder its biomedical application due to obvious ethical issues.

Calcium oxide from thoroughly washed and calcined hen eggshells was irradiated in microwave at 800 W in the presence of DAHP to furnish nanosized HA (18 nm) [89]. Increased MG-63 osteoblast cells growth after 1 day confirmed the non-cytotoxic nature of the hen eggshell-derived HA.

Polycrystalline nanosized B-type cHA was synthesized through sol–gel reaction between the calcium source from chicken eggshells and potassium dihydrogen phosphate [90]. XRD analysis confirmed the formation of single-phase HA while TEM studies confirmed the formation of polycrystalline material of prolate spheroidal morphology with average particle size of 35–50 nm.

In another research, HA was synthesized by reacting reagent-grade phosphoric acid with CaCO3 extracted from hen eggshells through a precipitation method followed by heating at 900 °C to prepare crystalline powder [91]. XRD and FTIR studies confirmed the formation of HA and the presence of hydroxyl and phosphate groups, respectively, while Ca/P ratio of 1.63 was determined through gravimetric and spectrophotometric analysis.

Microwave irradiation of the reaction mixture containing eggshells and DAHP in the presence of EDTA furnished flower-like nanostructures of HA having crystallite size of 78 nm [92]. The presence of peaks at 1415, 1459, and 875 cm−1 in FTIR spectra confirmed the formation of B-type substituted cHA, while EDX analysis confirmed the presence of Mg as trace ion.

Through precipitation technique nanosized HA was synthesized by employing hen eggshells as calcium source [93]. EDX results showed that the Ca/P ratio proportionally depended on the pH of the reaction mixture. pH of the reaction mixture also influenced the phase composition of HA, where at lower pH value, whitlockite was observed as secondary phase.

Ball-milled slurry of crushed oyster shells (Crassostrea gigas) and calcium pyrophosphate in 4:3 ratio was dried in oven at 150 °C for 12 h, followed by heat treatment at temperatures between 900 and 1100 °C furnished HA with β-TCP and calcite as secondary phases [94]. However, in case of reaction between oyster shell powder and DCPD followed by milling for 5 h and heat treatment at 1000 °C, no secondary phase was observed. At heating temperature of 900 °C the product existed in multiple phases mentioned above; however, increase in the heating temperature resulted in the conversion of calcite into β-TCP due to their structural similarities and β-TCP was transformed to HA due to the higher thermodynamic stability of HA than β-TCP when calcination was carried out at 1100 °C.

Ground waste eggshells were hydrothermally treated with DAHP at 80–160 °C for 2 days to furnish multi-phase CPs [95]. Phases like monetite (CaHPO4), whitlockite Ca3(PO4)2, and HA were formed at low heating temperature of 80 °C; however, the increase in heating temperature to 160 °C resulted in the predominant formation of HA. This increase in heating temperature also influenced the particle morphology, which changed from small particles with the laminar-plate structure at 80 °C to scattered flower-like agglomerates and small prisms with smooth surfaces at 140 °C followed by hashed micro-sheets at 160 °C.

Formation of HA nanoparticles from eggshell wastes and fruit waste extracts was reported recently [96]. Biomolecules present in the fruit waste were used to provide nucleation site for HA nuclei; furthermore the biomolecules along with hydrothermal treatment substantially influenced the particle size and morphology. XRD analysis confirmed the formation of poorly crystalline monophase of HA, this low degree of crystallinity was attributed to the presence of A-type and B-type carbonate group in the HA structure. ICP-AES confirmed the presence of Na (0.410 wt%), Mg (0.390 wt%), and Sr (0.289 wt%) in addition to Ca (37.2 wt%) and P (17 wt%), however, it was not clear whether the presence of trace elements was due to the eggshells used or the extract from the pomelo peel.

Solid-state reaction between CaO produced from eggshell waste with dicalcium phosphate dehydrate (DCPD) resulted in the formation of HA [97]. The phase purity was dependant on the heating temperature and duration, where higher heating temperature of 1100 °C for 5 h furnished pure HA. High Ca/P ratio of 2.20 was attributed to the presence of B-type carbonate group, which had substituted the phosphate group.

A pyrolysis wet-slurry precipitation method was used to synthesize HA using CaO derived from mussel shells (Perna canaliculus), which are abundantly available in New Zealand [98]. XRD of the as-synthesized HA confirmed the formation of nanosized, crystalline HA with trace amount of calcite, which was removed through calcination at 800 °C. The presence of carbonate peaks in FTIR spectra was attributed to the adsorbed CO2 from the atmosphere. EDX spectra of the heat-treated HA confirmed the Ca/P ratio to be 1.66, while the presence of potassium ions was attributed to the inadequate washing of the samples.

Thoroughly washed, cleaned eggshells were reacted with nitric acid to produce calcium nitrate, which was later reacted with phosphoric acid to furnish cHA [99]. Affinity of HA for cations was exploited and the synthesized HA was used for the removal of Pb2+ ions from the aqueous solution under acidic conditions. Good affinity of HA toward Pb2+ ions was attributed to the adsorption of Pb2+ ions on the surface of HA and an ion-exchange process between Ca2+ of HA and Pb2+ ions in aqueous solution.

Hydrothermal treatment of calcined eggshells and DAHP in mixed solvent system comprising DMF and hydrogen peroxide under acidic conditions at 120 °C for 24 h resulted in the formation of flower-like cHA structures [100]. XRD results showed well-crystallized 002 plane, while the broadness of other peaks width indicated partial crystallization of the material. Authors reported that the morphology of particles was strictly dependent on the temperature of hydrothermal treatment and the ratio of the mixed solvent systems.

Biogenic calcite extracted from spines collected from various species of Paracentrotus lividus sea urchins was converted to HA through hydrothermal treatment in the presence of sodium phosphate solution at 180 °C for 24 h [101]. During this pseudomorphic transformation the calcite crystals of the original spine were replaced by the nanosized elongated apatite nanocrystals, which resulted in the change of density and porosity in the final product. Furthermore the product not only inherited the external morphological features of the sea urchin spines but the complex internal porous structure was also retained.

Micrometer-sized whiskers of HA were successfully synthesized using CaHPO4 and CaO obtained from eggshells through a hydrothermal method at 170 °C for 48 h [102]. Surface area (50 m2/g) and particle size (0.06 μm) were calculated using BET results and XRD of the resulting calcined powder (700 °C) confirmed the formation of HA and CaHPO4 in 3:1 ratio.

Phase-pure HA was synthesized through a self-assisted chemical method using eggshells [103]. Results indicate that the grain size and crystallinity of the material is dependent upon the soaking time of CaO derived from eggshell in the K2HPO4 solution, where smaller sized particles were formed over longer soaking time resulting in slight peak broadening of XRD pattern. TEM and SEM analysis confirmed the formation of nanocrystalline circular particles.

The latest research has shown that the biogenic resources are a good source of Ca precursors for HA synthesis and as a result they have been widely used to prepare HA with different physical and chemical properties (Table 4).
Table 4

A brief summary of the properties of HA powder derived from biogenic sources

No

Source

Ca/P ratio

Particle size (nm/μm)

Morphology

Secondary phases

Trace elements

Calcination (T °C)

References

1

Eggshells

1.65

β-TCP

900–1200

[84]

2

Coral shells

Mg2+ and Si2+

[87]

3

Eggshells

=1.67

50

Rectangular

[88]

4

Eggshells

>1.67

18

Spherulite

800–1200

[89]

5

Eggshells

35–50

Prolate spheroidal

700

[90]

6

Eggshells

1.63

900

[91]

7

Eggshells

 

78

Flower like

 

Mg2+

 

[92]

8

Eggshells

=1.67

35

Like globules

900

[93]

9

Oyster shells

  

Rod like

 

1000

[94]

10

Eggshells

  

Flower like

[95]

11

Eggshells

1.57–1.77

12–49

Needle and rod like

Na+, Mg2+ and Sr2+

[96]

12

Eggshell

2.20

Spheroidal

β-TCP

Na+ and Mg2+

1100

[97]

13

Mussel shells

1.61

[98]

14

Eggshells

600

[99]

15

Eggshells

35–15

Flower like (microspheres)

[100]

16

Sea urchin spines

Rod like

Mg2+ and Na+

900

[101]

17

Eggshells

0.06

Whiskers

CaHPO4

700

[102]

18

Eggshells

41

Circular

[103]

Summary

Due to the growing importance of HA in different medical fields, a large number of review papers have been written on the synthesis of HA nanoparticles with reference to its usage and synthetic techniques. However, so far no critical analyses have been carried out of the work done on the synthesis of HA from the natural waste materials or other natural materials. Owing to its increasing demand in day-to-day applications it is essential to carry out a detailed study regarding its preparation, phase purity, and thermal stability in case of its synthesis from natural sources and natural waste materials. A comparison among the HA powder synthesized from these different natural resources is given in Table 5.
Table 5

Comparison among HA derived from various natural sources and natural bone

Parameters

Bovine bone

Fish bones

Plants

Egg and sea shells

Natural bone

Ca/P ratio

>1.67

= or <1.67

= or <1.67

1.67

Particle size

Micro to nanometer range

In nanometer range

In nanometer range

<50 nm

Nano composite

Morphology

Needle, rod, plate, spherical, etc.

Needle, rod, spherical, etc.

Rod or granular like

Rod or needle

Rod or needle

Phase composition

Phase pure and TCP

Phase pure

Phase pure

Phase pure and TCP

Composite

TCP

Thermal stability

800–1000 °C

600–800 °C

600–850 °C

<800 °C

>1200 °C

Trace elements

Na+, Mg2+, K+, Ti2+, etc.

Fe3+, Cr3+, Cu2+, K+, Mg2+, etc.

CO32− major element

Na+, K+, Mg2+, Zn2+, CO32− etc.

Hydroxyapatite can be synthesized from different resources like eggshells, bone of different animals, seashells, and plants. Research carried out so far suggests that these natural resources can be a good source of biological HA or the promising alternative to Ca and P precursor for the production of phase-pure and thermally stable HA. Furthermore, HA synthesized from the natural raw materials or natural waste can be more beneficial as it often contains useful ions, which are also found in the biological HA.

Hydroxyapatite derived from bovine bone resembles more closely to the HA present in the mammalian bones but the presence of trace elements is seldomly reported. There is always a danger of HA derived from biological sources being contaminated with toxic ions, especially HA recovered from sources around the industrial area. It is, therefore, imperative that researchers report the presence of trace ions when reporting the synthesis of HA from biological resources or waste products. Furthermore, certain ethical issues will always be attached to the HA recovered from animal sources; these ethical issues will no doubt will limit the use of such HA.

This review has highlighted that fish and bovine bones are a valuable candidate for the production of phase-pure HA, whereas use of biogenic and plant sources tend to furnish thermally unstable HA, which could be due to the incomplete conversion of precursors to HA or due to the presence of carbonate ions in the natural materials. This carbonate may be due to incomplete calcination of calcite or due to absorption of CO2 from air or water. Processing parameters such as milling time, calcination time, and temperature also affect the phase purity of the final product (Table 6).
Table 6

Effect of source, method, calcination temperature on phase stability, and phase decomposition of HA synthesized from natural sources

Source

Method

Calcination (T °C)

Phases

References

Bovine bone

Calcination

650–950

HA

[48]

Bovine bone

Thermal

750

HA

[49]

Bovine bone

Thermal

500–1400

HA

[50]

Bovine bone

Vibro milling/thermal

800

HA

[51]

Bovine bone

Arc plasma (30–90 s)

HA

[54]

(120 s)

HA, CaO and β-TCP

Bovine bone

Thermal

850

HA

[59]

Fish bone

Alkaline hydrolysis

HA

[63]

Thermal

900

HA

Fish bone

Thermal/milling

900

HA

[64]

Fish bones

Hydrothermal

HA and brushite

[65]

Fish bones

Thermal

600

HA

[66]

900

HA and β-TCP

Cod fish bones

Thermal

900–1200

HA and β-TCP

[69]

Red algae

Hydrothermal

650

HA, CaCO3 and β-TCP

[72]

700

HA

Plant leaves and stalk

Thermal

600–800

HA

[75]

Eggshells

Thermal

900

HA, CaO and β-TCP

[84]

Eggshells

Thermal

800–1200

HA

[89]

1250

HA and CaO

Eggshells

Microwave

β-TCP

[92]

Eggshells

Precipitation

400

HA (amorphous)

[93]

700

HA (crystalline)

900

HA and whitlockite

Oyster shells

Ball milling/thermal

900

HA, β-TCP, calcite & Ca2P2O7

[94]

1000

HA, β-TCP

1100

HA and β-TCP

Eggshells

Solid state

800

HA, DCPD, CaO and β-TCP

[97]

900

HA β-TCP

1000

HA and β-TCP

1100

HA and β-TCP

1150

HA

Mussel shells

Precipitation

800

HA

[98]

Acknowledgements

Authors would like to acknowledge the financial support from UTM through IDF UTM. J. 10.01/13. 14/1/128(201105M10019).

Copyright information

© Springer Science+Business Media New York 2013

Authors and Affiliations

  • Muhammad Akram
    • 1
  • Rashid Ahmed
    • 2
  • Imran Shakir
    • 3
  • Wan Aini Wan Ibrahim
    • 1
  • Rafaqat Hussain
    • 4
  1. 1.Department of Chemistry, Faculty of ScienceUniversiti Teknologi MalaysiaUTM Johor BahruMalaysia
  2. 2.Department of Physics, Faculty of ScienceUniversiti Teknologi MalaysiaUTM Johor BahruMalaysia
  3. 3.Department of Sustainable EnergyKing Saud UniversityRiyadhSaudi Arabia
  4. 4.Ibnu Sina Institute for Fundamental Science StudiesUniversiti Teknologi MalaysiaUTM Johor BahruMalaysia