Calcium Phosphates and Human Beings

Chemistry for Everyone

Calcium Phosphates and Human Beings Sergey V. Dorozhkin Markell Industrial Group, Kudrinskaja sq. 1 – 155, 123242 Moscow D-242, Russia; [email protected]

There are several classifications of organisms living on our planet, for example, monocellular and multicellular organisms, plants and animals, vertebrates and non-vertebrates, as well as mushrooms, bacteria, viruses, and other types of living organisms. In this article, only animals having hard and dense calcified tissues, such as either a skeleton or mollusk shell, will be considered. The skeletons and shells of animals represent a complex organic–inorganic composite of various substances, with prevalence (in mass) of the inorganic substances (1, 2). We shall not consider organic or biological molecules (there are too many of them in even the simplest living organisms) but will instead focus our attention on the inorganic substances present in the calcified tissues of living organisms. A limited number of inorganic substances are accumulated in significant quantities by living organisms in a process called “biomineralization”. These substances include: calcium carbonate (the basic building material of corals and of the shells of the overwhelming majority of mollusks), calcium oxalate (found in plants and in mammals), silicon dioxide (found in skeletons of algae), sulfates of the alkaline-earth metals (discovered in some plants and jellyfishes), iron oxides (present in bacteria, mollusks, and some plants) and, finally, calcium phosphates, the basic “building material” of bones and teeth of all vertebrates, especially mammals (1, 2). As humans are mammals, the subject of this article is limited to discussion of calcium phosphates and their roles for human beings. Physical and Chemical Properties of Calcium Phosphates Calcium phosphates are chemical compounds of special interest in many interdisciplinary fields of science, including geology, chemistry, biology, and medicine. The first attempts to establish their chemical composition were performed by J. Berzelius in the middle of the 19th century (3). Approximately 70 years later the idea of the existence of different crystal phases of calcium phosphates was introduced (4); mixtures of calcium phosphates had been called “apatites” until then. By definition, all calcium phosphates consist of three chemical elements: calcium (oxidation state +2), phosphorus (oxidation state +5), and oxygen (oxidation state −2). These three chemical elements are present in abundance on the surface of our planet: oxygen is the most widespread chemical element of the earth’s surface with 47 mass percent, calcium occupies the fifth place with 3.3–3.4 mass percent, and phosphorus with 0.08–0.12 mass percent. In addition, the chemical composition of some calcium phosphates includes hydrogen, either as an acidic phosphate anion (for example, HPO42− or H2PO4−), or as incorporated water (for example, CaHPO4⭈2H2O). Diverse combinations of oxides of calcium and phosphorus (both in the presence of water

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and without it) provide a large variety of calcium phosphates. Calcium phosphates are distinguished by the type of the phosphate anion: ortho, PO43−; meta, PO3−; pyro, P2O74−; and poly, (PO3)nn−. In the case of multi-charged anions (orthophosphate and pyrophosphate), calcium phosphates are also differentiated by the number of hydrogen ions attached to the anion. Examples using the common names include mono-, Ca(H2PO4)2; di-, CaHPO4; tri-, Ca3(PO 4)2; and tetra-, Ca2P2O7, calcium phosphates (5, 6). All chemically pure calcium phosphates are crystals of white color and moderate hardness. However, the natural minerals of calcium phosphates are colored owing to impurities, the most widespread of which are ions of iron and some of the rare-earth elements. The vast majority of calcium phosphates are sparingly soluble in water; however, all of them are easily soluble in acids but not soluble in alkaline solutions. Occurrence in Nature and Industrial Use The most important minerals of calcium phosphates are apatite Ca5(PO4)3X, where X is fluorine, less often chlorine or hydroxyl, and phosphorites (7, 8).1 Large deposits of apatite are found in Russia (on the Kola Peninsula near the border with Finland), Brazil, Finland, and Sweden. Large deposits of phosphorites are found in Morocco, United States (California, Florida, Tennessee), Peru, Kazakhstan, South Africa, Tunisia, Algeria, Egypt, and Israel, as well as in the ocean. Normally, phosphorites occur as irregularly shaped cakes or as black or brown nodules from pellets up to head size. They are assumed to be replacement products of fine-grained lithified carbonates, from mineralization of preexisting organic matter, from precipitates from microorganisms filling cavities within carbonates, or direct precipitates from interstitial waters. Unlike phosphorites, natural apatites have a magmatic origin (8). The majority of natural calcium phosphate ores consist of small crystals with dimensions less than 1 mm (Figure 1). Larger crystals are rare and, in many cases, large and beautiful hexagonal crystals of natural fluorapatite (chemical formula Ca5(PO4)3F) are displayed as examples in mineralogical collections (Figure 2). Most of the natural calcium phosphates are utilized in the production of mineral fertilizers (9). For this purpose, the natural ore is enriched by flotation and then the concentrate is processed by treatment with strong inorganic acids (sulfuric, phosphoric, or nitric) at temperatures of 70–100 ⬚C under intense agitation. Depending on the type of the acid used and the initial mass ratio of the concentrate to the acid, the final products are either ready-to-use fertilizers (for example, single or triple superphosphate) or a wetprocess phosphoric acid that then is neutralized by ammonia, giving rise to ammonium phosphates. The annual manufacture of the phosphorus-containing fertilizers in the world exceeds 25 million tons (as P2O5) (9).

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Another way of processing natural phosphate ores includes sintering a powdered mixture of the ore with silicon dioxide and carbon at temperatures above 1000 ⬚C. Although this method is more expensive, it results in chemically pure products. During the heat-treatment, elemental phosphorus (white phosphorus, chemical formula P4) is produced: 2Ca3(PO4)2 + 6SiO2 + 10C → 6CaSiO3 + P4↑ + 10CO↑ The vapor of sublimed white phosphorus is cooled and collected under a layer of cold water. The solid white phosphorus is dried and combusted in oxygen, producing phosphorus pentoxide, P2O5, which is later reacted with water to form phosphoric acid (called “thermal phosphoric acid”). From the latter all other chemically pure phosphates are easily produced (9). In biological systems, calcified tissues, mainly bones and teeth, of vertebrate animals (fish, amphibians, reptiles, birds, and mammals) are the main location of calcium phosphates. Besides bones and teeth, there are some undesirable (noxious, pathological) calcified tissues, like dental calculi, bladder stones, and atherosclerotic precipitations in blood vessels; all of them either consist of, or contain, calcium phosphates (1, 2, 5). In addition, in some types of ancient mollusks (e.g.,

brachiopod Lingula) the shells consisted not of calcium carbonates but of calcium phosphates (Figure 3). The above-mentioned cases of biologically formed calcium phosphates are defined in the scientific literature as “biological apatite”, which varies widely in structure and chemical composition (1, 2, 5). The main distinction of the biological apatite from the mineral apatite is in their chemical composition. Numerous researches have established that biological apatite is a nonstoichiometric compound in which a part of calcium and phosphate ions is replaced by other ions. For example, ions of calcium are partly replaced with ions of strontium, magnesium, sodium, and potassium; ions of phosphate are partly replaced with ions of a carbonate, and ions of hydroxide, fluoride, chloride, or even carbonate might be present as X ions. The attentive reader will note that some of the above-mentioned replacement ions have charge or dimensions distinct from the “basic” ions of calcium, phosphate, and X. For example, substitutes of doubly charged ions of calcium are single-charged ions of sodium and potassium, while triply charged ions of orthophosphates might be replaced by the doubly charged ions of carbonate or hydrophosphate. Necessary charge compensations in the crystal structure of biological apatite occur by formation of the required quantity of ionic vacancies, which leads to a nonstoichiometric chemical composition for biological apatite. Therefore, an approximate chemical formula of biological apatite can be written, (Ca, Mg, Na)10᎑x(PO4, HPO4, CO3)6(OH, F, CO3)2᎑2x

Figure 1. A polycrystalline sample of natural fluorapatite from Khibiny deposits (Kola peninsula, Russia). Crystals of fluorapatite, scattered through the mineral, have a green–gray color.

Figure 2. A single crystal of natural fluorapatite (darker part) embedded into a piece of rock.

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where 0 < x < 2 (5, 6). Owing to its nonstoichiometric nature, it is impossible to specify the exact chemical composition of biological apatite. Moreover, the composition strongly depends on the type of calcified tissue. For example, the chemical composition of biological apatite found in teeth differs from that taken from bones. Nevertheless, it is possible to assert that bones of humans and other mammals, on the average, contain 60–70 mass percent of calcium phosphates, 20–30 mass percent of collagen, and up to 10 mass percent of water (numbers might vary depending on the age, food, presence or absence of diseases, etc.). The average chemical composition and some

Figure 3. The hardened cockleshells of an ancient mollusk brachiopod Lingula embedded into a piece of rock.

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major properties of bones and teeth of humans are summarized in Table 1 (1, 2, 5). The most important details of the structures and chemical properties of the calcified tissues of mammals are discussed below. Bones Bones are the major calcified tissues of humans and other mammals. Two basic functions are carried out by bones in an organism: mechanical support of the body, which also includes the property of creation and maintenance of the body’s shape, and storage source of inorganic ions, mainly ions of calcium and phosphate, that are necessary for many vital functions of living organisms (1, 2, 10). As seen from the data in Table 1, bones of mammals are a composite of both organic (mainly fibers of collagen) and inorganic (mainly calcium phosphates) phases. In this composite, calcium phosphates are responsible for the mechanical durability, hardness, rigidity, and high resistance to compression, while collagen provides some elasticity and viscosity. Contrasting with natural bones, bioceramics consisting of pure calcium phosphates are

brittle and easily collapse on impact or bending. The interior of bones is porous, and the pores are filled with a liquid that acts as a lubricant, thus further improving the elastic properties of bones (Figure 4). In the majority of bones, there is a dense external layer (compact bone) surrounding a less dense and porous internal bone (spongy bone). In the middle of the latter is marrow (Figure 4). The structure of bones is complicated, and several dimensional levels have been outlined (1, 2, 10). In this article we examine only the smallest scale of the hierarchical structure of bones as only this scale includes crystals of biological apatite. The smallest level of the structural organization of bones slightly exceeds dimensions of single molecules. At this scale of measurement, it is possible to differentiate separate threadlike molecules of collagen (which are twirled into spirals) and lamellar nanocrystals of biological apatite (Figure 5). The thickness of these nanocrystals is only 2–4 nm, that is, just several unit cells (Table 1). The flat nanocrystals of biological apatite are stacked parallel to each other and somehow (the details are not clear yet) place themselves in between the

Table 1. Characteristics of Biological Apatite versus Chemically Pure Hydroxyapatite Parameter Calcium

c

Chemical Composition of Apatite (mass percent) Enamela

Dentinea

Bonea

Pureb

36.5

35.1

34.8

39.6

Phosphorusc

17.7

16.9

15.2

18.5

Ca/P (molar ratio)c

1.63

1.61

1.71

1.67

Sodium

c

0.5

0.6

0.9

–

Magnesiumc

0.44

1.23

0.72

–

Potassiumc

0.08

0.05

0.03

–

3.5

5.6

7.4

–

Fluorinec

0.01

0.06

0.03

–

Chlorinec

0.30

0.01

0.13

–

0.022

0.10

0.07

–

Total inorganicd

97

60–70

60–70

100

Total organicd

1.5

20–30

20–30

–

Waterd

1.5

10

10

–

Carbonatesd

Pyrophosphates (as P2O74−)d

Crystal Lattice Parameters (± 0.003 Å) a-axis/Å

9.441

9.421

9.41

9.430

c-axis/Å

6.880

6.887

6.89

6.891

Crystallinity (%)e

70–75

33–37

33–37

100

50,25,4

200–600

Dimensions of the crystals/nm

100f,50,50 35,25,4

Mechanical Properties Elasticity module/GPa

80

15

0.34–13.8

10

Stretching durability/GPa

10

100

150

100

a

Apatitie taken from various calcified tissues of adult humans. bRef 5. Calcined samples. dNon-calcined samples. ePure hydroxyapatite is 100%. fUnit in this dimension is µm.

c

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Figure 4. General structure of a mammalian bone. Taken with permission from http://www.sirinet.net/~jgjohnso/aplongbones.jpg (accessed Feb 2006).

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twirled molecules of collagen, while the latter are united in bunches on the next level of the structural organization of bones (Figure 5). There are still numerous ambiguities in the mechanisms of bone formation and growth. It is generally agreed, that the growth of bones starts with the formation of an initial framework consisting of the spiral-twirled molecules of collagen. The collagen molecules act as nucleators for further formation and growth of the nanocrystals of calcium phosphates. The explanation of why biological apatite forms lamellar crystals that arrange parallel to each other is not yet well established. It is presumed that the size, shape, and relative positioning of the nanocrystals can somehow be correlated to the structural parameters of the collagen framework (for example, it might be defined by the distances between the neighboring molecules of collagen). It is necessary to stress that bones of living organisms are not stable and permanent: the bones are in permanent dynamic balance with the surrounding tissues. The osteoclast cells, by producing an acidic environment, continuously dissolve biological apatite. At the same time, other cells, called osteoblasts, perform continuous crystallization of biological apatite. Such uninterrupted and never-ending processes of dissolution and recrystallization are able to keep the necessary concentrations of calcium

and phosphate ions in the body fluids (blood, saliva, etc.). In addition, the continuous dissolution and recrystallization maintains the bones in good physical condition, because any defective sites that appear due to various reasons are dissolved more rapidly than the non-defective sites and, either simultaneously or immediately afterwards, the healthy bone is crystallized in those places by osteoblasts (1, 2, 10). If one wants to consider the chemistry of bone formation starting from the ions of calcium and phosphate dissolved in blood, many gaps in knowledge will be discovered. It was established a long time ago that simple mixing of aqueous solutions containing ions of calcium (for example, calcium nitrate) and phosphate (for example, ammonium phosphate) in the necessary proportions (Ca兾P molar ratio of 1.67) and under the necessary conditions (solution pH should be above 10) would not directly result in crystallization of hydroxyapatite Ca5(PO4)3OH. Numerous researches proved that crystallization of hydroxyapatite always occurred through formation of one or several intermediate calcium phosphates, called precursor phases (5, 6). Based on those data, the following conclusion was made: if hydroxyapatite were never formed in vitro (i.e., in glass beakers) without the intermediate formation of the precursor phase(s), it would be logical to assume that the biological apatite of bones would also be formed via some intermediate phases. The problem is that, up to now, nobody has ever succeeded in proving either the presence or absence of the intermediate phases during bone formation or bone remodeling. Researchers encounter great experimental difficulties because, although it is easy to perform the crystallization in glass beakers and wait as long as required (periodically taking samples to perform the chemical and structural analysis), such experiments are impossible with living bones. Therefore, scientists are still forced to be content with indirect data only; for example, it was established that both the structure and chemical composition of the biological apatite of young bones, taken from animal cubs, differed from that of the biological apatite found in the bones of old animals (1, 2, 10). Teeth

Figure 5. A schematic image of the smallest level of the structure of mammalian bones.

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After bones, teeth occupy the second most important place among all calcified tissues of humans and mammals. From the chemical point of view, the structure of the teeth of humans and mammals appears to be even more complicated than the structure of bones. A tooth consists of the external (very hard) part named enamel and internal (relatively soft) part named dentine (Figure 6). As shown in Table 1, both the chemical composition and mechanical properties of dentine and bone are rather close and therefore, almost everything previously mentioned about bone is valid for dentine as well. Dental enamel, however, strongly differs from bone (1, 2, 5, 11). As shown in Table 1, the chemical composition of dental enamel is very close to that of pure hydroxyapatite. The main difference of enamel from both dentine and bones is that enamel contains almost no organic phase. For this reason, dental enamel is the hardest part of the body of humans and other mammals. In addition, the hardness of dental enamel is increased by the presence of fluoride ions. Here it is necessary to stress that the presence of fluorides results in

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Figure 6. The structure of a mammalian tooth.

formation of fluorapatite, Ca5(PO4)3F, which is the hardest, densest, and least soluble calcium phosphate (5, 6). For this reason, fluoride-containing toothpastes are extremely important since, upon contact with dental enamel, the ions of fluoride interact with enamel, giving rise to the formation of fluorapatite on the enamel surface. The latter raises both the mechanical hardness of enamel and its resistance to dissolution in the acidic environment produced by bacteria living in the mouth. From a chemical point of view, dental caries is the dissolution of biological apatite by weak organic acids (5, 11). It is thought that the mechanism of formation of dental enamel is similar to that of bone (e.g., mineralization of an organic matrix, probable presence of phases-precursors, etc.). The essential differences of dental enamel from dentine and bone are: (i) the absence of almost any organic compounds in enamel, (ii) great distinctions in the organic phase (which does not contain fibers of collagen), and (iii) dimensions of biological apatite crystals (the crystals of enamel are up to 100 µm in length, Figure 7). Interestingly, it is presumed that at the initial stages of formation, dental enamel contains only about 50% of biological apatite, but during aging the quantity of inorganic phase increases up to 98–99% (Table 1). In addition, unlike bone, the damaged dental enamel is not restored by cells similar to osteoclasts and osteoblasts (1, 2, 11). Hence, it is possible to consider enamel as a “dead” calcified tissue of living organisms (contrary to living bone). To conclude this section, it is necessary to mention one more phase from teeth called enameloid, that exists at the interface between enamel and dentine (Figure 6). It is established that enameloid consists of the crystals of biological apatite similar to those found in dental enamel, but these crystals are embedded into the organic matrix of collagen, as in dentine and bones. However, the structure and other properties of this interfacial phase are still insufficiently investigated (1, 11). www.JCE.DivCHED.org

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Biomaterials and Bioceramics As the inorganic component of bones and teeth of humans and mammals consists of calcium phosphates of a biological origin, it is obvious that, from the point of view of biocompatibility (i.e., the ability of live organisms to accept unknown substances without any inflammation or tearing away), artificial bone substitutes made of calcium phosphates should have the optimum chemical and physiological properties. However, owing to poor mechanical properties (a high brittleness) and poor workability (i.e., it is difficult to adjust the shape of calcium phosphate ceramics to the desired profile) artificial implants made of pure calcium phosphates2 are seldom used in surgery (12, 13). Here it is necessary to remember that bones and dentine have a porous structure and contain up to 30% of the organic phase, which improves their mechanical properties (10). Hence, the ideal bone substitute should also contain an organic phase to provide the necessary toughness and be porous to allow soft tissues of living organisms to grow into the artificial implants. Several

Figure 7. Needlelike crystals of biological apatite of dental enamel (1).

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Figure 8. Artificial hip joints made of titanium. One of the joints is partly covered with a layer of calcium phosphates (white color) for better biocompatibility.

Figure 9. A tooth-holder made of titanium to be implanted into gums for fastening of artificial teeth. The surface of the holder is covered with a biocompatible layer of calcium phosphates.

Figure 10. A cylindrical item made of porous calcium phosphates.

Figure 11. Some commercial samples of ready-to-use artificial bone substitutes made of calcium phosphates.

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“roundabout” ways are possible to overcome the poor mechanical properties of ceramics made of pure calcium phosphates. It is possible to fabricate artificial bone substitutes from any strong and inert material (titanium, stainless steel, etc.) and, for better biocompatibility, to cover the entire outer surface with a layer of calcium phosphates by either plasma-spray deposition or precipitation from supersaturated solutions. In this case, the metallic core provides mechanical strength, while a superficial layer of calcium phosphates is responsible for good biocompatibility with the surrounding soft tissues. In this way artificial hip joints (Figure 8) and special holders for artificial teeth (usually, the artificial teeth are made of ceramics; see Figure 9) are fabricated (11–13). It is possible to mimic nature and prepare organic–inorganic composites consisting of calcium phosphates and any biologically compatible (highly desirable) or inert (worse) polymer. The simplest way to produce such composites is to add powdered calcium phosphate either to a polymer-containing solution or a pure melted polymer and to mix thoroughly with the subsequent formation of the desired bone substitutes. Such composites (e.g., HAPEX) have been developed and are used for manufacturing small artificial bones (14). For filling the small defects in large bones (e.g., cracks or artificially removed small fragments), it is possible to use viscous suspensions of calcium phosphates in aqueous solutions of a water-soluble and biologically compatible polymer (for example, starch or cellulose ethers). Such suspensions can be placed into a syringe and injected directly into cavities in the bone (15). Osteoclasts and osteoblasts will use this suspension as a building material to construct new bone. Self-hardening calcium phosphate cements represent another option. The cements consist of powders of two different calcium phosphates, usually an acidic calcium phosphate (for example, CaHPO4) and a basic calcium phosphate [for example, Ca4(PO4)2O] and powders of calcium hydroxide or calcium carbonate. The powders are mixed in the necessary proportions and later either water or a dilute aqueous solution of phosphoric acid is added. Owing to chemical reactions between the components, the cement is hardened, forming apatite. An ability to fill bone defects having awkward geometrical forms is the great advantage of the self-setting calcium phosphate cements (16). It is possible to prepare porous bone substitutes from calcium phosphates (Figure 10) and then to toughen them with a surface layer of a biocompatible polymer. A structure similar to triplex, the material used in automobile windshields, is created by this technique. Owing to the polymeric film on its surface, triplex does not disintegrate into pieces during a collision. The polymer-toughened calcium phosphates have similar mechanical properties. The advantages of such materials covered with a polymeric layer are obvious: a surgeon can easily cut off a piece of the desired shape and dimensions from the initial large piece of porous ceramic. In addition, the polymeric coating might be enriched by the addition of drugs (e.g., antibiotics) or bioactive compounds (e.g., hormones, growth factors, etc.). This type of artificial implant could also be used as a slow release drug-delivery system.

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Finally, pieces cut from calcined natural (e.g., bovine) bones might also be used as artificial bone substitutes. As calcination occurs at very high temperatures (about 1000 ⬚C) in oxidative conditions (airflow), all carbon-containing organic and biological compounds are burned out completely and the solid residue consists of porous ceramics of calcium phosphates containing a small quantities of substituting ions (see Table 1). In principle, after cooling, such substitutes are ready-touse in surgery because calcining acts as sterilization as well. Unfortunately, implants cut from the calcined bovine bones possess poor mechanical properties, much lower than that of the artificially prepared porous bone substitutes. In addition, it is difficult to prepare standard samples (i.e., having the desired chemical composition, porosity, shape, and dimensions) in large quantities. This difficulty causes problems in getting the approval of the U.S. Food and Drug Administration and other similar organizations to allow use of calcined natural bones as artificial implants in surgery; administrative rules strongly reduce application of calcined natural bones for implants. Some commercial examples of the artificial bone substitutes made of calcium phosphates are shown in Figure 11. Conclusion The inorganic part of mammalian hard tissues (bones and teeth) consists of calcium phosphates mainly of apatitic structure. Similarly, most undesired calcifications (i.e., those appearing because of various diseases) of mammals also contain calcium phosphates. For example, atherosclerosis results in blood vessel blockage caused by a solid composite of cholesterol with calcium phosphate. Dental caries results in a replacement of less soluble and hard apatite by more soluble and softer calcium hydrogenphosphates. Osteoporosis is a demineralization of bone. Therefore, from a chemical point of view, processes of normal (bone and teeth formation and growth) and pathological (atherosclerosis and dental calculus) calcifications are just an in vivo crystallization of calcium phosphate. Similarly, dental caries and osteoporosis can be considered as in vivo dissolution of calcium phosphates. Owing to the chemical similarity with biological calcified tissues, all calcium phosphates are remarkably biocompatible. This property is widely used in surgery to prepare bone substitutes that are either entirely made of, or only coated with, calcium phosphates. For example, self-setting bone cements made of calcium phosphates are helpful in bone repairing and titanium substitutes covered by a surface layer of calcium phosphates are used for hip joint endoprostheses and tooth substitutes. There is a great biological and medical significance of calcium phosphates and, in this article, a brief overview on the current knowledge in this subject is given.

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Notes 1. Sometimes the chemical formula of apatite is written as 3Ca3(PO4)2⭈CaX2. The phosphorites might be described as calcium orthophosphate, Ca3(PO4)2. 2. Stoichiometric hydroxyapatite, nonstoichiometric hydroxyapatite, tri-calcium orthophosphate, and multiphase mixtures of these compounds are some examples of pure calcium phosphates.

Literature Cited 1. Lowenstam, H. A.; Weiner, S. On Biomineralization; Oxford University Press: New York, 1989. 2. Biomimetic Materials Chemistry; Mann, S., Ed.: VCH Publishers, Inc.: New York, 1996. 3. Berzelius, J. Ann. Chem. Pharmac. 1845, 53, 286–288. 4. Hausen, H. Acta Acad. Abo. Ser. B: Mat. Phys. Mat. Natur. Teknik. 1929, 5, 62–65. 5. LeGeros, R. Z. Calcium Phosphates in Oral Biology and Medicine. In Monographs in Oral Science; Myers, H. M., Ed.; S. Karger AG: Basel, Switzerland, 1991; Vol. 15. 6. Elliott, J. C. Structure and Chemistry of the Apatites and Other Calcium Orthophosphates. In Studies in Inorganic Chemistry; Elsevier: Amsterdam, 1994; Vol. 18. 7. McConnell, D. Apatite: Its Crystal Chemistry, Mineralogy, Utilization and Biologic Occurrences; Springer-Verlag: New York, 1973. 8. Phosphate Deposits of the World; Notholt, A. J. G., Sheldon, R. P., Davidson, D. F., Eds.; Cambridge University Press: Cambridge, 1989; Vol. 2. 9. Becker, P. Phosphates and Phosphoric Acid: Raw Materials Technology and Economics of the Wet Process, 2nd ed.; In Fertilizer Science and Technology Series; Marcel Dekker: New York, 1989; Vol. 6. 10. Currey, J. D. Bones: Structure and Mechanics; Princeton University Press: Princeton, NJ, 2002. 11. Dental Caries: The Disease and Its Clinical Management; Kidd, E. A. M., Fejerskov, O., Eds; Iowa State University Press: Ames, IA, 2003. 12. Ravaglioli, A.; Krajewski, A. Bioceramics: Materials, Properties, Applications; Chapman & Hall: London, 1991. 13. Biomaterials: Principles and Applications; Park, J. B., Bronzino, J. D., Eds.; CRC Press: Boca Raton, FL, 2002. 14. Deb, S.; Wang, M.; Tanner, K. E.; Bonfield, W. J. Mat. Sci.: Mat. Med. 1996, 7, 191–193. 15. Iooss, P.; Le Ray, A. M.; Grimandi, G.; Daculsi, G.; Merle, C. Biomaterials 2001, 22, 2785–2794. 16. Bohner, M. Eur. Spine J. 2001, 10, S114–S121.

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