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Properties of Osteoconductive Biomaterials: Calcium Phosphates

LeGeros, Racquel, Zapanta

Clinical Orthopaedics and Related Research: February 2002 - Volume 395 - Issue - p 81-98
SECTION I SYMPOSIUM: Bioactive Materials in Orthopaedic Surgery
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Bone is formed by a series of complex events involving the mineralization of extracellular matrix proteins rigidly orchestrated by cells with specific functions of maintaining the integrity of the bone. Bone, similar to other calcified tissues, is an intimate composite of the organic (collagen and noncollagenous proteins) and inorganic or mineral phases. The bone mineral idealized as calcium hydroxyapatite, Ca10(PO4)6(OH)2, is a carbonatehydroxyapatite, approximated by the formula: (Ca,X)10(PO4,HPO4,CO3)6(OH,Y)2, where X are cations (magnesium, sodium, strontium ions) that can substitute for the calcium ions, and Y are anions (chloride or fluoride ions) that can substitute for the hydroxyl group. The current author presents a brief review of CaP biomaterials that now are used as grafts for bone repair, augmentation, or substitution. Commercially-available CaP biomaterials differ in origin (natural or synthetic), composition (hydroxyapatite, beta-tricalcium phosphate, and biphasic CaP), or physical forms (particulates, blocks, cements, coatings on metal implants, composites with polymers), and in physicochemical properties. CaP biomaterials have outstanding properties: similarity in composition to bone mineral; bioactivity (ability to form bone apatitelike material or carbonate hydroxyapatite on their surfaces), ability to promote cellular function and expression leading to formation of a uniquely strong boneCaP biomaterial interface; and osteoconductivity (ability to provide the appropriate scaffold or template for bone formation). In addition, CaP biomaterials with appropriate three-dimensional geometry are able to bind and concentrate endogenous bone morphogenetic proteins in circulation, and may become osteoinductive (capable of osteogenesis), and can be effective carriers of bone cell seeds. Therefore, CaP biomaterials potentially are useful in tissue engineering for regeneration of hard tissues.

From the Department of Biomaterials and Biomimetics, New York University College of Dentistry, New York, NY.

The author’s work cited in this paper was supported in part by research grant nos. DE 07223, DE 04123, DE-12388, and S07RR076226 from the National Institute of Dental and Craniofacial Research of the National Institutes of Health and from the Linkow Professorship in Implant Dentistry.

Reprint requests to Racquel Zapanta LeGeros, PhD, Department of Biomaterials, New York University College of Dentistry, 345 East 24th Street, New York, New York 10010.

List of Abbreviations Used: BMP bone morphogenetic protein, ECM extracellular matrix, MSC mesenchymal stem cell

The need to replace missing bones and teeth with other materials was evident even in prehistoric times. The practice of cremation in many societies allowed only limited evidence for identifying substitute materials for replacement of missing teeth. From the Fifth and Fourth centuries BC until the First or Second century AD, archeologic findings exhibited in museums showed that materials used to replace missing human teeth have included ox teeth, shells, coral, ivory (elephant tusk), wood, human teeth from corpses, and metals (gold or silver). 115

In modern times, autografts (bone obtained from another site in the same subject of the same species) are the gold standard for bone repair and substitution. Success with the use of allografts (bone obtained from another subject of the same species (banked freeze-dried bones of human cadavers) also have been reported. However, the use of autografts has some serious disadvantages, such as additional expense and trauma to the patient, possibility of donor site morbidity, and limited availability. In the case of allografts, in addition to limited supply and high costs, other complications such as viral transmission and immunogenicity are of serious concern. 61 Therefore, there was a critical need to develop bone substitute materials approximating the properties of bone but without the drawbacks of autografts or allografts. In recent years, commercial and experimental materials for bone repair, substitution, or augmentation have included metals, polymers, corals, CaP of natural (from corals or bovine bone) or synthetic origin, bioactive glasses (specially formulated silica-based glasses), and polymer and CaP composites. 4,5,22,26,30,34,45,48,51,69,71,84,85,95,98,124 The group of materials, which more closely approximate the properties and composition of the bone mineral is the CaP group 30,51,69,70,85 (Table 1).

TABLE 1

TABLE 1

Some of the properties of bone (specifically bone mineral), and relevant properties of CaP biomaterials in terms of composition, dissolution or degradation, cell-CaP interaction (ability to facilitate cell function and expression), bioactivity (ability to form bone apatitelike or carbonate hydroxyapatite on their surfaces), osteoconductivity (ability to serve as an interactive template or scaffold for the forming new bone), and the potential role of calcium phosphate biomaterials in hard tissue regeneration using tissue engineering are discussed in the current review.

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PROPERTIES OF BONE

Bone is formed by a series of complex events involving mineralization of ECM proteins rigidly orchestrated by specific cells with specific functions of maintaining the integrity of the bone. 117 Bone is an intimate composite of an organic phase (collagen and noncollagenous proteins) and an inorganic or mineral phase. 60,70,135 The inorganic to organic ratio is approximately 75 to 25 by weight and 65 to 35 by volume.

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The Bone Mineral

The bone mineral consists of irregularly-shaped platelets (of variable lengths with widths 30 to 45 nm and thickness of approximately 5 nm) that are oriented with their c axis parallel to one another and lie along the collagen fibrils. 60,135

Early observations of the similarity in the xray diffraction patterns of the mineral phases of enamel, dentin, and bone when compared with those of mineral apatites (hydroxyapatite, fluorapatite) and the presence of Ca and P ions led to the conclusion that the inorganic or mineral phases of tooth and bone basically were calcium hydroxyapatite idealized as Ca10(PO4)6(OH)2. 10,31,111 McConnell 97 speculated that teeth and bone mineral are more similar to the carbonate-containing mineral apatite, dahllite, but did not have experimental evidence to support his speculations. Others proposed theories on the presence of nonapatitic phases with the apatite phase. 14,15,36,111,118,126 Among such nonapatitic phases assumed to be present with the apatitic phase in the bone mineral were calcium carbonate (CaCO3), dolomite or magnesium calcium carbonate (CaMgCO3), brushite or dicalcium phosphate dihydrate (CaHPO4.2H2O), amorphous CaP, Cax(PO4)y.zH2O and octacalcium phosphate (Ca8H2(PO4)6.5H2O). However, results of combined analyses using xay diffraction, infrared absorption spectroscopy, and transmission electron microscopy showed that only one phase, the apatite phase, is present in normally calcified tissues (enamel, dentin, or bone) 65–67,70 (Figs 1,2). Based on the sharpness of the peaks in the xray diffraction patterns (Fig 1), it can be deduced that the apatite crystals in enamel are much larger than those in dentin or bone. The differences in crystal sizes and morphologic features of these biologic apatites have been confirmed with transmission electron microscopy. In contrast, xray diffraction patterns of pathologically-calcified tissues (dental calculi, urinary stones, vascular calcifications) have shown other CaP phases mixed with the carbonatehydroxyapatite phase. 66,70,76,87,122 These nonapatitic CaP phases included dicalcium phosphate dihydrate, octacalcium phosphate, magnesium-substituted tricalcium phosphate, or amorphous CaP.

Fig 1.

Fig 1.

Fig 2.

Fig 2.

The Ca to P molar ratio of animal bones ranges from below to above 1.67 (the stoichiometric value for pure hydroxyapatite), depending on the species, age, and type of bone. 70 Chemical analyses showed that these biologic apatites (enamel, dentin, and bone) are not pure hydroxyapatite but contain impurity ions including CO3, HPO4, F, Cl, Mg, Na, K ions, and some trace elements (SR, Zn). 65–67,70 Combined results from xray diffraction, infrared absorption, transmission electron microscopy, and chemical analyses of enamel, dentin, and bone mineral, and the results from studies on synthetic carbonate apatites 37,65–67,70,87,92,140 led to the now accepted conclusion that enamel, dentin, and bone mineral consists of only one phase, described as carbonatehydroxyapatite with the approximate formula: (Ca,Mg,Na)10(PO4HPO4CO3)6(OH)2. The mechanism for incorporation of carbonate is principally the coupled substitution of CO3for-PO4 and Na-for-Ca. This type of substitution of carbonate in apatite is described as Type B. 37,65–67,70,87,140 Type A substitution, CO3-for-OH, also has been suggested as being present in biologic apatites. 37,114 This was based on observations made with carbonate apatites prepared under dry conditions at 1000° C. 37 Several investigators have confirmed type B carbonatehydroxyapatite in biologic apatites, such as bone apatite. 65–67,70,87,114,140

Results of studies in synthetic systems showed that incorporation of different ions cause changes in morphologic features of crystal (size and shape) and in the dissolution properties of the apatite. 66,70,74,78,81 Magnesium or CO3 the two minor but important constituents in biologic apatites, were shown to cause reduction in crystal size and an increase in the extent of dissolution of synthetic apatites, and that these two ions act synergistically. 70,81,107 The higher concentrations Mg and CO3 in bone or dentin compared with enamel apatite may explain the higher solubility and lower crystallinity (smaller crystal size) of bone or dentin compared with enamel. 66,70,81,91–93

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Bone Porosity

Bone pore sizes (porosity) in normal cortical bone range from 1 to 100 μm whereas trabecular bone has pores ranging from 200 to 400 μm. The size range, extent, and interconnectivity of the pores are critical factors affecting diffusion of nutrients, cell attachment, migration and expression, and tissue ingrowths that are necessary for bone formation and repair or regeneration. 56

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Bone Strength

The high degree of integration and orientation of the mineral and organic components gives the bone its mechanical strength. For the current review, only the properties of the bone mineral are relevant to those of the CaP biomaterials.

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CaP BIOMATERIALS

Historic Development

The first clinical attempt to use a CaP compound (ambiguously described as triple calcium phosphate chemical reagent) was in the successful repair of a bony defect reported by Albee in 1920. 2 A second clinical report by other investigators was published 30 years later. 112 Levitt et al 94 and Monroe et al 99 suggested calcium hydroxyapatite or fluorapatite as a CaP ceramic material for bone and tooth implants. Between 1976 and 1986, serious efforts were made toward development and commercialization of CaP (principally, hydroxyapatite) as biomaterials for bone repair, substitution, and augmentation. 4,5,30,50,51,98

Approximately 60 years after the report by Albee, 2 clinical applications of CaP materials (as calcium hydroxyapatite, tricalcium phosphate) in dentistry were reported. CaP particles described as tricalcium phosphate were used to repair surgicallycreated infrabony defects in dogs. 102 and for alveolar ridge augmentation 103; and dense hydroxyapatite cylinders were used as immediate dental root implants after tooth extraction. 32,33 Denissen’s hydroxyapatite was prepared by precipitation and sintering above 1000° C. 32,33 The tricalcium phosphate used by Nery et al 102,103 was prepared by Hubbard 49 by sintering a chemical reagent labeled as tricalcium phosphate, which similar to most chemical reagents so labeled, actually are Ca-deficient apatites, with a Ca to P molar ratio less than 1.67. 68,70 Xray diffraction analysis of this tricalcium phosphate material showed that the material was not tricalcium phosphate but a mixture of hydroxyapatite and beta-tricalcium phosphate with an hydroxyapatite to betatricalcium phosphate ratio of 80/20. 69 This material and similar materials of varying hydroxyapatite to beta-tricalcium phosphate ratios later were described as biphasic CaP. 12,24,25,77,89,101,137

In the past 2 decades, CaP biomaterials have gained acceptance in dental and orthopaedic applications (Table 1). Some of these applications are repair of bony defects, maintenance or augmentation of the alveolar ridge, immediate tooth root replacements, ear implants, spine fusion, and coatings on orthopaedic and dental implants. 4,19,22,26,33,39,48,69–71,94,95,101–103,124,132,134,137,139

Based on composition, commercial CaP biomaterials may be classified as: (1) hydroxyapatite; (2) β-tricalcium phosphate, Ca3(PO4)2; (3) biphasic calcium phosphate, an intimate mixture of hydroxyapatite and beta-tricalcium phosphate; and (4) unsintered CaP or a calcium-deficient apatite (Ca,Na)10(PO4 HPO4)6(OH)2.

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Hydroxyapatite of Natural Origin

Commercial hydroxyapatite of natural origin is derived from special species of corals (Porites) 48,69–71,124 or from bovine bone. 71,73,132 These hydroxyapatites are not pure but contain some of the minor and trace elements originally present in the coral or in the bone. Coral-derived apatite or coralline hydroxyapatite contain Mg, Sr, CO3, and F as minor elements. 69,85,90 Bovine bone-derived apatite contains Mg, Na, CO3, and other trace elements originally present in the bone. 66,70 These materials have their interconnecting macroporosity conserved from the original materials, coral, and bovine bone (Fig 3).

Fig 3A–C.

Fig 3A–C.

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Coralline Hydroxyapatite

Commercially available hydroxyapatite derived from corals or coralline hydroxyapatite (Interpore™ and Pro-Osteon™ manufactured by Interpore International, Inc, Irvine, CA) are prepared by the hydrothermal conversion (260° C, 15,000 psi) of coral (consisting of mostly CaCO3, calcite form) in the presence of ammonium phosphate to hydroxyapatite. 119 Ions such as F, Sr, and CO3 present in the coral become incorporated in the resulting hydroxyapatite whereas other ions (Mg) become incorporated in the minor beta-tricalcium phosphate component that formed after hydrothermal conversion. 69,70

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Bovine-bone Derived Apatites

Commercial and experimental CaP materials derived from bovine bone are of three types, depending on the method of preparation: (1) with the organic matrix, unsintered; (2) without organic matrix, unsintered (Bio-Oss™, from Geistlich Biomaterials, Geistlich, Switzerland); or (3) without the organic matrix and sintered (Osteograf-N™ manufactured by CeraMed Co Denver, CO and Endobon™ manufactured by Merck Co, Darmstadt, Germany). The unsintered bone mineral consists of small crystals of bone apatite (carbonatehydroxyapatite) whereas the sintered bone mineral consists of much larger apatite crystals without CO3 when sintered above 1000° C (Fig 4).

Fig 4.

Fig 4.

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Synthetic CaP Biomaterials

Based on composition, commercially available CaP biomaterials are classified as hydroxyapatite, Ca10(PO4)6(OH)2; unsintered apatite or calcium deficient apatite, Ca(10-x) Nax(HPO4)x(PO4)(6-x)(OH)2; beta-tricalcium phosphate Ca3(PO4)2; and biphasic calcium phosphate, mixture of hydroxyapatite and beta-tricalcium phosphate of hydroxyapatite to beta-tricalcium phosphate ratios. The crystallographic properties (Fig 4), composition, extent of dissolution in acidic buffer (Fig 5), and mechanical properties of these calcium phosphate materials differ from those of cortical or trabecular bone. 51

Fig 5.

Fig 5.

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Hydroxyapatite

Hydroxyapatite is prepared by precipitation under basic conditions and subsequent sintering usually at temperatures above 1000° C. 44,50,51,70,83 Pure hydroxyapatite has the stoichiometric Ca to P ratio of 1.67, lattice parameters: a-axis = 0.9422 nm, and c-axis - 0.688 nm, and the presence of only the OH and PO4 absorption bands in their infrared spectra. Hydroxyapatite can be prepared in dense or macroporous forms, as granules, or blocks. 51,83 Commercial hydroxyapatites include Calcitite™ (Sulzer Calcitek, Carlsbad, CA). In addition to being used as bone substitutes, hydroxyapatite granules also are used as the source material for depositing coatings on commercial dental and orthopaedic prostheses (implants) using the plasma spray technique. 8,30,51,83 Comparative properties of coralline hydroxyapatite and ceramic hydroxyapatite are summarized in Table 2. These properties include differences in crystal size, composition, and dissolution properties 70,80,90 (Fig 6) (Table 2). In terms of mechanical strength, the tensile strength for dense hydroxyapatite is 79 to 106 MPa and porous hydroxyapatite is 42 MPa compared with 69 to 110 MPa for cortical bone. 51

TABLE 2

TABLE 2

Fig 6A–B.

Fig 6A–B.

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Beta-tricalcium Phosphate

Beta-tricalcium phosphate is prepared by sintering precipitated Ca deficient apatite with Ca to P molar ratio of 1.5 or by solid-state reactions at high temperature 70,98 Vitoss™ (Orthovita, Inc, Philadelphia, PA) is one of the commercial beta-tricalcium phosphate.

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Biphasic CaP

Biphasic calcium phosphate of varying hydroxyapatite to beta-tricalcium phosphate weight ratios is obtained by sintering precipitated calcium deficient apatite (Ca/P molar ratio < 1.67) 12,68,77 Commercial biphasic calcium phosphate products include:Triosit™ (Biomatlante Ltd, Nantes, France), MBCP™ (Biomatlante Ltd), and Osteosynt™ (Einco Biomaterial Co Bella Horizonte, Brazil).

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Unsintered Calcium Deficient Apatite

Calcium deficient apatite is prepared either by precipitation at temperatures 25° to 100° C (depending on the desired crystallinity) or by hydrolysis of amorphous calcium phosphate, dicalcium phosphate dihydrate, alpha-tricalcium phosphate, or octacalcium phosphate. 71 An example of commercial unsintered apatite product is Osteogen™ (Impladent Co, Long Island, NY) advertised as resorbable hydroxapatite. 73,134

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Other Potential CaP Biomaterials

Noncommercial CaP materials also recommended for bone grafts include octacalcium phosphate, 52 whitlockite, or Mg-substituted tricalcium phosphates, 40 Znsubstituted tricalcium phosphate, 53 carbonate-substituted apatites, 108 and fluoride-substituted apatites. 38,40,55,99 Substitution in the betatricalcium phosphate or apatite structure affects the crystal and dissolution properties of the CaP. For example, carbonate substitution causes the formation of smaller and more soluble apatite, whereas fluoride incorporation has the opposite effects. 66,70,88,91–93,100,140 Magnesium incorporation in apatite is limited but causes reduction in crystallinity (smaller crystal size) and increases its extent of dissolution. 66,70,78,81,91 Strontium causes an increase in solubility. 70 Magnesium or Zn substitution in the beta-tricalcium phosphate structure also affects the properties. 70,74,107

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CaP COATINGS ON DENTAL AND ORTHOPAEDIC IMPLANTS

The rationale for the development of hydroxyapatite-coated orthopaedic and dental implants is to combine the demonstrative bioactive properties of hydroxyapatite and the strength of the metal, usually Ti or Ti alloy. 8,51,63,70,82,83 Plasma-spray technique is the method used for depositing the coating. Although the source material used is pure hydroxyapatite, the plasma-sprayed coatings are not pure hydroxyapatite but a mixture of crystalline (principally hydroxyapatite) and the noncrystalline component, or amorphous calcium phosphate. 63,82,86 Other minor crystalline components that may be present include alpha- or beta-tricalcium phosphate, tetracalcium phosphate, and sometimes CaO. The hydroxyapatite component can make up approximately 95% of the crystalline component; however, the hydroxyapatite to amorphous calcium phosphate ratio of the coating can vary from 30 to 70 to 70 to 30 and this ratio is higher in the layer closest to the metal compared with the outermost layer. 63,70,86 Accelerated skeletal attachment and a stronger bone-implant interface have been reported for hydroxyapatite-coated implants compared with the uncoated ones implants. 20,42 Bloebaum et al 11 reported implant failure attributed to the hydroxyapatite coating that resorbs or delaminates before bone can attach to it. It is speculated that the hydroxyapatite to amorphous calcium phosphate ratio is critical to the stability of the coating and also may affect the stability of the implant. 82 Coating with a low hyroxyapatite to amorphous calcium phosphate ratio is expected to be more soluble because the amorphous calcium phosphate component preferentially will dissolve. 70,72,82 An alternative to the plasma-spray method of depositing CaP coating is electrochemical deposition. This allows deposition of the desired coating of homogeneous composition. Dicalcium phosphate dihydrate, octacalcium phosphate, or apatite coatings have been obtained using this method. 64,123,133

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CaP CEMENTS

The concept of developing apatitic cement or CaP cements first was introduced by LeGeros et al 75 and Chow et al. 17,18 CaP cements consist of solid and liquid components. A combination of CaP with or without other calcium compounds make up the solid components and the liquid component may be inorganic (sulfuric acid, phosphoric acid) or organic acids (lactic acid, tartaric acid) or NaP solutions. 17–19,35,62,75 Calcium phosphate compounds that have been used include: amorphous calcium phosphate, dicalcium phosphate dihydrate, dicalcium phosphate anhydrous, monocalcium phosphate monohydrate, alpha-tricalcium phosphate, beta-tricalcium phosphate, calcium deficient apatite, and tetracalcium phosphate. The products obtained after setting depends on the solid and liquid reactants. Setting products include: calcium deficient apatite, carbonate apatite, dicalcium phosphate dihydrate. Commercial CaP cements presently available are αBSM™ (Etex Corporation, Boston, MA) consisting of amorphous calcium phosphate and dicalcium phosphate dihydrate mixed with saline solution;62 Bonesource™ (Orthofix Inc, McKinney, TX) consisting of tetracalcium phosphate and anhydrous dicalcium phosphate mixed with phosphate solution or water, 17,18 and monocalcium phosphate monohydrate and CaCO3 mixed with water. 35 Some of these cements are injectable. 41,96

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PROPERTIES OF CaP MATERIALS

The many desirable properties of CaP materials have been documented, 6,9,16,26,30,51,69,79,80,85,101–106,109,110 including similarity in composition to bone mineral (Table 2); bioactivity (ability to form bone apatitelike or carbonate hydroxyapatite on their surfaces); ability to promote cellular function and expression, ability to form a direct uniquely strong interface with bone; and osteoconductivity (ability to provide a scaffold or template for the formation of new bone).

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Porosity

The study of Klawitter 56 indicated that the size of pores at the surface, the extent, and the interconnectivity of the pores of the implant should allow bony ingrowth and allow blood vessels and canaliculi to form in the porosities. He recommended pore sizes of at least 50 μm for the ingrowth of blood vessels and 200 μm for osteonal ingrowths. These pore size requirements are achieved in coral-derived and bovine-bone derived hydroxyapatite, which have approximately maintained the porosity characteristics of the original material (coral and bovine bone, respectively) during the processing (Fig 3). Coralline hydroxyapatite has macroporosity with a mean diameter of 230 μm and interconnecting pores with a mean diameter of 190 μm. For synthetic hydroxyapatite, beta-tricalcium phosphate or biphasic calcium phosphate, porosity (microporosity and macroporosity) may be introduced by mixing the CaP with a volatile material (H2O2 or naphthalene), which leave pores on sintering. 49 However, porosity by this method is not easy to control and interconnecting porosity has been difficult to achieve. Commercial CaP materials give the pore size range of 100 to 400 μm. 51 Macroporous biphasic CaP (Biomatlante, Ltd, Nantes, France) has interconnecting porosity comparable to those of coralline hydroxyapatite or bone (Fig 3). However, from a review of the literature and results from his own work, Liu 96 reported that no direct evidence indicates that bone ingrowths or formation will be inhibited by the degree of interconnectivity porosity (12% to 80%) and pore size (range, < 1 μm to 1500 μm).

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Dissolution and Degradation

Cell-mediated biodegradation occurs under acidic conditions. 7,28,72 Therefore, in vitro dissolution studies of CaP biomaterials may be predictive of their in vivo dissolution or biodegradation. 72 In vitro dissolution of CaP materials depend on composition (hydroxyapatite versus beta-tricalcium phosphate), and for materials of similar composition (hydroxyapatite from natural or synthetic origin), the extent of dissolution will depend on particle size, porosity (microporosity and macroporosity), specific surface area, and crystallinity (reflecting crystal size and perfection). 57,70–73,109 The extent of dissolution of the bovine-derived apatite will depend on its method of preparation. The extent of dissolution in acidic buffer was observed to be the greatest for the unsintered bone without organic matrix followed by unsintered bone with organic matrix, and the least for sintered bone. The crystal size of sintered bone apatite is of much greater magnitude than that of the unsintered bones. The lower extent of dissolution observed for bone with organic matrix compared with that without may be caused by the protective effect of the organic matrix. 91 For synthetic CaP, the extent of dissolution in acidic buffers is the greatest for amorphous calcium phosphate, followed by tetracalcium phosphate, alpha-tricalcium phosphate, beta-tricalcium phosphate, and is the least for hydroxyapatite. 70,72,73 The extent of dissolution of unsintered bone apatite is close to that of beta-tricalcium phosphate. The stability of the plasma-sprayed hydroxyapatite coating in orthopaedic or dental implants may be affected by the composition of the coating, principally the amorphous calcium phosphate to hydroxypatite ratio: the higher the ratio, the more soluble the coating 82,86 because of the higher solubility of amorphous calcium phosphate (Fig 5). The preferential dissolution of amorphous calcium phosphate component of a coating with a high amorphous calcium phosphate to hydroxyapatite ratio may lead to premature resorption and delamination of the coating. 82,86

The extent of dissolution of coralline hydroxyapatite is much higher than that of ceramic hydroxyapatite. 73,90 This difference can be attributed to the difference in crystal size and composition (Table 2): coralline apatite crystals are much smaller (Fig 6) and contain magnesium and carbonate ions that cause an increase in the dissolution of apatite. 70,81,93

For substituted apatites, the type and extent of substitution affects the solubility: carbonate substitution contributes to increasing the solubility and F substitution to decreasing the solubility of apatite. 71,86,101 Comparing unsubstituted apatite with carbonate- or Fsubstituted apatite, carbonate-substituted apatite is most soluble, and F-substituted apatite, the least souble. With biphasic calcium phosphates, the dissolution property depends on the hydroxyapatite to beta-tricalcium phosphate ratio: the higher the ratio, the lower the extent of dissolution. 77

In vivo data confirm the in vitro observations on comparative degradation of CaP biomaterials. The factors that influence dissolution properties in vitro (composition, surface area, surface topography, microporosity and macroporosity) also were operative in vivo. Therefore, it is possible to use an appropriate CaP material with the biodegradation desired for particular clinical applications. With biphasic calcium phosphates, control of biodegradation (and bioactivity) by adjusting the hydroxyapatite to beta-tricalcium phosphate ratio was suggested. 77

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INTERACTION BETWEEN CELLS AND CaP BIOMATERIALS

In vitro studies on bone cell culture models are used routinely to determine biocompatibility and to investigate bone and biomaterial interactions. Osteoblastlike cells (responsible for bone formation) are used most frequently 1,6,27,28,38,43,54,105,139 and osteoclasts (cells responsible for bone resorption) are used less frequently. 29,121,136,138 Interaction with periodontal ligament cells also has been reported. 3,21,125 Cell proliferation, attachment, and phenotypic expression seem to be influenced by composition, surface roughness, or topography of the CaP biomaterial. 21,38,72,109,120,125,138,139 In vitro, no difference in cell proliferation was observed between coralline hydroxyaptite and synthetic (ceramic) hydroxyapatite. 80 Fluoride-treated bovine bone seemed to stimulate cell activity including proliferation and collagen production (Fig 7) compared with the nontreated bone. 38 Fluoride also seems to inhibit osteoclastic resorption of F-treated bone 136 or F-containing apatite. 38,55,121 In vivo, implanted F-substituted apatite showed higher levels of osteoblastic activity at the bone-biomaterial interface compared with F-free apatite. 55

Fig 7.

Fig 7.

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BIOACTIVITY AND OSTEOCONDUCTIVITY: BONE-CaP MATERIAL INTERFACE

Bioactivity

Bioactivity is defined as the property of the material to develop a direct, adherent, and strong bonding with the bone tissue. 45,46,110 This property was observed first by Hench et al 45,46 with silica-based glasses with special formulation referred to as bioactive glass and associated with a CaP-rich layer that forms between the bioactive glass and the bone tissue. In vitro, bioactivity has been attributed to materials that have the ability to form carbonate hydroxyapatite on its surface when exposed to simulated body fluid. 45,59,77 Therefore, Ti, formerly described as biotolerant and not bioactive, 110 can be treated chemically (with NaOH) to form a TiOH layer that allows a formation of a bioactive carbonatehydroxyapatite layer when exposed to simulated or natural body fluid, imparts a bioactive property to the Ti implant. 59

In vivo, microcrystals were observed to be associated with ceramic CaP biomaterials (hydroxyapatite or biphasic calcium phosphate) implanted in osseous 9,39,77,79,80,129 or nonosseous sites. 47 These microcrystals were identified as carbonatehydroxyapatite intimately associated with an organic matrix. 47,77,79,80 In vivo, the formation of carbonatehydroxyapatite on surfaces of CaP biomaterials at the bone-biomaterial interface (Fig 8) is thought to be a cellmediated dissolution and precipitation processes. 13,77,79,80 Cellular activity induces partial dissolution of the CaP biomaterials and liberation of Ca and P ions onto the microenvironment. In addition to the Ca and P ions, other ions (Mg, CO3) from the biologic fluid become incorporated in the carbonatehydroxyapatite microcrystals that form in intimate association with an organic component on the surfaces of the much larger crystals of the CaP biomaterial (Fig 8). This initial action may trigger a mineralization of the ECM leading to bone formation. The enrichment of Ca and P ions in the microenvironment seem to promote bone mineralization and enhance bone formation. 16,77,80

Fig 8.

Fig 8.

Formation of carbonatehydroxyapatite on surfaces of calcium phosphate biomaterials in vitro and in vivo seems to be related to the extent of dissolution of the calcium phosphate biomaterials. When exposed to serum, carbonatehydroxyapatite formed after a shorter time on the coralline hydroxyapatite surfaces compared with that on ceramic hydroxyapatite. 80,90 Biphasic calcium phosphates with lower hydroxyapatite to beta-tricalcium phosphate ratios (more soluble) showed greater abundance of the carbonatehydroxyapatite microcrystals. 24,77

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Osteoconductivity

Urist 130 and Urist et al 131 described osteoconductivity as the property of a material to support tissue ingrowth, osteoprogenitor cell growth, and development for bone formation to occur. Important features of this property are the appropriate chemical composition and appropriate architectural geometry. The osteoconductive CaP biomaterials (hyroxyapatite, beta-tricalcium phosphate) allows attachment, proliferation, migration, and phenotypic expression of bone cells leading to formation of new bone in direct apposition to the CaP biomaterial 13,40,109 (Fig 9).

Fig 9.

Fig 9.

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Bone and CaP Biomaterial Interface

Bioactive materials allow attachment of cells and their differentiation directly on the surface to result in intimate bonding with the newly-formed bone creating a uniquely strong interface. 46,110 Cohesive failure of the material, bone tissue, or both, occurred with bioactive materials (CaP materials, bioactive glasses, hydroxyapatite-coated implants) and not at the bone-material interface, whereas failure occurred at the bone-material interface with nonbioactive materials (metals, polymers). Failure observed at the bone-Ti interface in the case of uncoated Ti implant, and at the coating-Ti interface in the case of hydroxyapatite-coated implant, is attributable to the stronger bond between the bone and the bioactive coating compared with that between the Ti and the coating. 23 The bone-CaP biomaterial interface is a dynamic one with the CaP biomaterial interacting with and becoming part of the newly forming bone.

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CaP BIOMATERIALS IN TISSUE ENGINEERING

CaP biomaterials have a high affinity for proteins, making them ideal carriers for bioactive peptides, bone growth factors, or MSCs. 106,113,127 It generally is accepted that CaP biomaterials are osteoconductive but not osteoinductive (property to form bone when implanted in nonbony sites). However, there are reports of its possible osteoinductive properties. 58,116,128 This osteoinductive property was found to be associated with specific geometry of the CaP biomaterial. 113,116 Reddi 113 attributed this phenomenon to the binding of an optimum amount of endogenous BMP in circulation to the CaP biomaterial allowing the biomaterial to become osteoinductive. Kuboki et al 58 concluded that the geometry of the BMP carrier is one of the important factors controlling the efficacy of the phenotype induction. These investigators determined that the optimal pore size of porous blocks of hyroxyapatite as BMP carriers to induce direct bone formation was 300 to 400 μm.

Ohgushi and Caplan, 106 Toquet et al, 127 and Kawai et al 54 reported that MSCs from bone marrow can be cultured in porous CaP biomaterials (ceramic hydroxyapatite, coralline hydroxyapatite, biphasic CaP) in vitro and implanted as a tissue engineered material for bone regeneration. Takata et al 125 described periodontal tissue regeneration on the surface of synthetic hydroxyapatite implanted into root surface.

Commercial CaP biomaterials used as bone substitutes in several orthopaedic and dental applications generally differ from bone in mechanical strength and physicochemical properties. 30,51,69,85 They cannot be used in load bearing applications because of their low fracture strength. 30,51 Used as coatings on orthopaedic and dental metal (Ti or Ti alloy) implants, CaP biomaterials impart bioactivity to the metal substrate enhancing and accelerating skeletal fixation. 8,30,42,51 The bioactive and osteoconductive properties of CaP biomaterial allow direct and intimate apposition of the newly formed bone to their surfaces resulting in formation of a uniquely strong interface. With the appropriate geometry, CaP biomaterials can serve as carriers of bone growth factors (BMPs), bioactive peptides, or MSCs that allow regeneration of hard tissues using tissue engineering. 58,106,113,116

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Acknowledgments

The author thanks Drs R. Craig, G. Daculsi, C. Frondoza, A. Gatti, M. Heughebaert, R. Kijkowska, J.P. LeGeros, D. Mijares, S. Lin, E. Nery, I. Orly, R. Rohanizadeh, T. Sakae, and M. Okazaki for professional collaboration and Dr. M. Retino, Ms. Bleiwas, and Mr. J. Wong for technical collaboration in the author’s work cited in this review.

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Thomas W. Bauer, MD, PhD; and George F. Muschler, MD—Guest Editors

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