vallet regi calcium phosphate as substitution of bones

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idstchem.2004.07.001

Progress in Solid State Chemistry 32 (2004) 1–31

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Calcium phosphates as substitutionof bone tissues

Marıa Vallet-Regı a,�, Jose Marıa Gonzalez-Calbet b

a Departamento de Quımica Inorganica y Bioinorganica, Facultad de Farmacia, Universidad Complutense

de Madrid, Pza Ramon y Cajal, 28040 Madrid, Spainb Departamento de Quımica Inorganica, Facultad de Ciencias Quımicas, Universidad Complutense de

Madrid, 28040 Madrid, Spain

Received 1 December 2003; received in revised form 1 June 2004; accepted 15 July 2004

Abstract

Calcium phosphates with clinical applications are used in reconstructive surgery. Whendealing with the repairing of a skeletal section, two extremely diverse routes could beinitially considered: to replace the damaged part or to substitute it regenerating the bone.This second option is in fact the role played by calcium phosphates, which can be classifiedamong the bioactive ceramics group. Bioceramics, and therefore, calcium phosphates, exhi-bit very good biocompatibility and bone integration qualities, and constitute the materialsshowing closest similarity to the mineral component of bone; this fact, together with theirbioactivity, make them very good candidates for bone regeneration.This review is focused on calcium phosphate ceramics; therefore, it is important to analyse

firstly the biological calcium phosphates as components of natural hard tissues, that is, boneand teeth, and then to look for synthetic methods able to produce calcium deficient carbon-ate apatites with nanometric size, i.e., showing similar features to the biological apatites.In the present work, we describe the synthesis procedures to obtain in the laboratory cal-

cium deficient carbonate nanoapatite both in bulk and thin film forms, as well as the char-acterization methods applied to these materials, with particular incidence in the electronmicroscopy.# 2004 Elsevier Ltd. All rights reserved.

Contents

1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2

2. Natural hard tissues: bones and teeth . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3

3. Apatites and other phosphates . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5

4. Substituted apatites . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 14

5. Calcium phosphate cements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 19

6. Biphase mixtures of calcium phosphates . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20

7. Fabrications of bulk calcium phosphate pieces . . . . . . . . . . . . . . . . . . . . . . . . . . 21

8. Ceramic coatings . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 23

9. Tissue engineering . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 26

10. Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 27

M. Vallet-Regi, J.M. Gonzalez-Calbet / Progress in Solid State Chemistry 32 (2004) 1–312

1. Introduction

Calcium phosphates with clinical applications constitute an interesting field of

research and development in the production of useful biomaterials for implant fab-

rication and/or fixation. Biomaterials in general, and particularly bioceramics,

allow replacing several parts of our body [1,2].Ceramic materials used in reconstructive surgery can be classified in two large

groups: bioinert and bioactive. Bioinert ceramics have almost no influence in the

surrounding living tissue, and their finest example would be alumina. Bioactive cer-

amics, by contrast, are capable of bonding with living osseous tissues; several cal-

cium phosphates and certain compositions of glasses and ceramic glasses exhibit

such feature. The bioactivity phenomenon is another example of the chemical reac-

tivity of ceramic materials with their environment: namely, an artificial solution

chosen to perform the in vitro assays, or the physiological body fluids during in

vivo assays. The first ceramics to be used in clinical applications, alumina and zir-

conia, are two prototypes of inert ceramics, and this is the main reason why they

were chosen for use in implants. Such ceramics feature extremely slow reaction

kinetics, to such an extent that they can be considered as ‘‘almost inert’’ ceramics.

Obviously, other ceramics have much faster reaction rates, and even very fast

kinetics. As in any other chemical reaction, the product obtained from the reaction

of a substance with its environment could yield an undesirable outcome, such as

the corrosion of a metal, but it could also lead to a favourable reaction product,

through the chemical transformation of the starting material into a sought-after

final product. This is the case with the bioactive ceramics which, when in contact

3M. Vallet-Regi, J.M. Gonzalez-Calbet / Progress in Solid State Chemistry 32 (2004) 1–31

with physiological fluids, react chemically towards the production of newly formedbone. When dealing with the repairing of a skeletal section, two extremely diverseroutes could be initially considered: to replace the damaged part, or to substitute itregenerating the bone. This last option is in fact the role played by the bioactivebioceramics, such as calcium phosphates.From a structural point of view, ceramic materials can be classified as crystalline

solids—ceramics—, amorphous solids—glasses—, or amorphous solids with crys-tallization nuclei—glass-ceramics—, which can in turn be considered as inert, bioac-tive or resorbable. This review is focused on calcium phosphate bioceramics;therefore, it is important to analyse firstly the biological calcium phosphates ascomponents of natural hard tissues, that is, bones and teeth.

2. Natural hard tissues: bones and teeth

The bones and teeth of all vertebrates are natural composite materials, whereone of the components is an inorganic solid, carbonate hydroxyapatite. It amountsto 65% of the total bone mass, with the remaining mass formed by organic matterand water.Most of this organic matter is collagen. Its molecules are bonded forming linear

chains which are in turn arranged in fibres, giving rise to various macroscopicstructures (Fig. 1). In between said molecules there are small interstitial emptycompartments, regularly spaced, where apatite nanocrystals are deposited, in acontrolled biomineralization process involving more than 200 different acid pro-teins. These proteins act as inhibitors, nucleators or templates for the epitaxialgrowth of nanocrystals, anchored to the collagen [3]. The crystallization of thecomplex and hardly soluble apatite structures evolves favourably through the kine-tically controlled formation of metastable intermediate products. Under in vitroconditions, amorphous calcium phosphate is transformed into octacalcium phos-phate (OCP) which, in turn, evolves to carbonate hydroxyapatite; at lower pHvalues, the intermediate phase seems to be dehydrated dicalcium phosphate(DCPD) [4].The bones are characterized by their composition, crystalline structure, mor-

phology, particle size and orientation. The carbonate hydroxyapatite of bones ran-ges between 4% and 8% in carbonate content, which increases with age while thehydrogen phosphate ion decreases. The crystals are nanometer sized, with an aver-age length of 50 nm, 25 nm in width and thicknesses of just 2–5 nm, scattered inthe organic matrix. Their small size is a very important factor related to the solu-bility of biological apatites when compared with mineral apatites. Small dimensionsand low crystallinity are two distinct features of biological apatites which, com-bined with their non-stoichiometric composition, inner crystalline disorder andpresence of carbonate ions in the crystal lattice, allow explaining their specialbehaviour (Fig. 2).The bones, the body supporting scaffold, can exhibit different types of inte-

gration between organic and inorganic materials, leading to significant variations


in their mechanic properties. The ratio of both components reflects the compromisebetween toughness (high inorganic content) and resiliency or fracture strength (lowinorganic content). All attempts to synthesize bone replacement materials for clini-cal applications featuring physiological tolerance, biocompatibility and long termstability have, up to now, had only relative success; it comes to show the superior-ity and complexity of the natural structure where, for instance, human femur canwithstand loads of up to 1650 kg.The bones of vertebrates, as opposed to the shells of molluscs, can be considered

as ‘‘living biominerals’’ since there are cells inside them under permanent activity.The bone formation process starts by the action of osteoblasts, special cells thatsynthesize and release the collagen matrix in the form of a jelly substance, theosteoid, which is subsequently mineralized by controlled deposition of calciumphosphate. The osteoblasts remain trapped inside the mineral phase, evolvingtowards osteocites which continuously maintain the bone formation activity.Meanwhile, another type of cells, the osteoclasts, catabolyse the bone destroying it.This dynamic process of bone formation and destruction accounts for its growthduring the development stages of the body, preserving its shape and consistency,and enabling its regeneration in case of fracture. It also constitutes a storage and

Fig. 1. Cortical or compact bone, and trabecular or spongy bone. Arrangement of carbonate hydro-

xyapatite and collagen in the formation of hard tissues.


hauling mechanism for two essential elements, phosphorus and calcium, which aremainly stored in the bones.The teeth exhibit similar characteristics to the bones, except for their external

surface coating, the enamel. Dental enamel has a much larger inorganic contentthan the bone, up to 90%, and is formed by prismatic crystals, of larger dimensionsand strongly oriented. The differences in crystallinity and carbonate contentbetween bone and dentine (with similar qualities) and enamel are straightforward(Fig. 2). All this explains its different mechanical behaviour. In fact, enamel is con-sidered as the most resistant and tough material in the biological world. However,and in contrast with the bone, dental enamel in an adult body does not containcells, and is therefore unable to regenerate itself; any deterioration that it may suf-fer becomes irreversible. There is no biological process that repairs degraded ordamaged enamel, evidencing the need for enamel-biocompatible materials in therepair of tooth decay.

3. Apatites and other phosphates

This group includes not only the phosphates (crystalline solid materials), but alsocements and biphasic mixtures with calcium phosphate content.The most used calcium phosphate in implant fabrication is hydroxyapatite,

Ca10(PO4)6(OH)2, since it is the most similar material to the mineral component of

Fig. 2. Crystal structure of carbonate hydroxyapatite. Powder X-ray diffraction patterns and infrared

spectra of enamel, dentine and bone.


bones. Taking into account the hydroxyapatite lattice parameters (a ¼ 0:95 nm andc ¼ 0:68 nm), and its symmetry (hexagonal, S.G. P63/m) (Fig. 3), most likely itsunit cell will be arranged along the c-axis. This would justify a preferred orien-tation that gives rise to an oriented growth along the c-axis and a needle-like mor-phology. It exhibits good properties as biomaterial, such as biocompatibility,bioactivity, osteoconductivity, direct bonding to bone, etc. Among the wide range ofavailable calcium phosphates, or with potential formulation, it is important toknow the close relation between the Ca/P ratio, acidity and solubility. Thus, thelower the Ca/P ratio is, the larger are the acidity and solubility of the mixture. ForCa=P < 1, both acidity and solubility are extremely high, and both parametersdecrease substantially for Ca/P ratios close to 1.67, which is the value of stoichio-metric hydroxyapatite, Ca10(PO4)6(OH)2 [5]. The Ca/P ratio is a very useful para-meter for scientists working in this field. Table 1 shows several calcium phosphatesarranged according to their Ca/P ratio.The lattice parameters of some of these phosphates are quite similar, leading to

overlapping of reflection maxima which makes difficult, in some occasions, theadequate interpretation of the powder X-ray diffraction pattern. Besides, since syn-thesis conditions influence very strongly on crystal size, morphology and structure,a careful characterization by electron microdiffraction and high resolution electrontransmission microscopy (HRTEM) is very useful to investigate small particles andmixtures of calcium phosphates. For instance, hydroxyapatite (OHAp) and b-tri-calcium phosphate (b-TCP), as a function of synthesis conditions, often coexist indistinct proportions. Moreover, when OHAp is heated at 1050

vC it converts par-

tially to b-TCP. When crystal size is small and inhomogeinities appear in the ima-ges obtained by HRTEM, electron microdiffraction is the most powerful tool toascertain the real symmetry. Fig. 4a shows the zero-order Laue zone (ZOLZ)microdiffraction pattern of an OHAp crystallite along [0001]. The sixfold symmetry

Fig. 3. Crystal structure of carbonate hydroxyapatite.


characteristic of the OHAp space group is evident. In addition, the first-order Laue

zone (FOLZ) is also visible although with very weak intensity. Using the formula

H ¼ 2=k ðR=CLÞ2, where H stands for the spacing between ZOLZ and FOLZ, R

stands for the FOLZ radii and CL is the camera constant, the repeat distance

along the c-axis can be approximately calculated [6] giving a value of H ¼ 0:7 nm

which is very close to the OHAp c-axis (c ¼ 0:688 nm). Since crystallites in these

two phosphates usually appear as platelets oriented along [0001], this is a nice pro-

cedure to distinguish between them. Actually, Fig. 4b shows the ZOLZ micro-

diffraction pattern along [0001] corresponding to b-TCP where a threefold

symmetry is observed, in agreement with its space group (R3c). Since the FOLZ is

also visible, the c-axis can be calculated, leading to H ¼ 3:73 nm in agreement with

the b–TCP c-axis (3.79 nm).Calcium phosphate ceramics are unstable under the electron beam after pro-

longed exposure times. Fig. 5 shows the HRTEM image oriented along [0001].

Table 1

Various calcium phosphates with their respective Ca/P atomic ratios

Ca/P N
ame Formula A cronym
2,0 T
etracalcium phosphate Ca4O(PO4)2 T etCP
1,67 H
ydroxyapatite Ca10O(PO4)6(OH)2 O HAp
Amorphous calcium phosphate
Ca10-xH2x(PO4)6(OH)2 A CP
1,50 T
ricalcium phosphate (a,b,c) Ca3(PO4)2 T CP
1,33 O
ctacalcium phosphate Ca8H2(PO4)6�5H2O O CP
1,0 D
icalcium phosphate dihydrate CaHPO4�2H2O D CPD
1,0 D
icalcium phosphate CaHPO4 D CPA
1,0 C
alcium pyrophosphate (a,b,c) Ca2P2O7 C PP
1,0 C
alcium pyrophosphate dihydrate Ca2P2O7�2H2O C PPD
0,7 H
eptacalcium phosphate Ca7(P5O16)2 H CP
0,67 T
etracalcium dihydrogen phosphate Ca4H2P6O20 T DHP
0,5 M
onocalcium phosphate monohydrate Ca(H2PO4)2�H2O M CPM
0,5 C
alcium metaphosphate (a,b,c) Ca(PO3)2 C MP
g. 4. ZOLZ microdiffraction patterns along [0001] of (a) OHAp and (b) b-TC
Fi P.


Irradiation under the electron beam produces small areas (marked with arrows)with fringes different from those of OHAp. Areas showing irregular contrast nearthe edges of the crystal grew with time of exposure and corresponded to densitychanges related to local variations of Ca/P stoichiometry as also observed by Shu-lin et al. [7] on enamel apatites as due to a localized demineralization involving lossof calcium and phosphate ions. Fig. 6 shows the effect of the irradiation damage ofb-TCP. The HRTEM image oriented along [0001], after exposing the crystal for along period, shows regions of patches with fringe spacing different from those ofthe pure b-TCP. The most probable product after the decomposition is CaOformed by the diffusion of Ca2+ and O2� ions.In order to obtain further information on the nature of the observed structural

changes under irradiation, the Moire fringe method has been used. Moire fringepatterns have proven to be not only an excellent tool for the detection of verysmall local crystal spacing variations (undetectable in the image or diffraction pat-tern) but also to enhance the effects of rotations, defects and strain fields [8]. In thecase of OHAp, the study of the evolution of the Moire pattern as a function of theirradiation beam has shown to prove the existence of a secondary phase [9].Finally, although radiation damage in calcium phosphates tends to blur the imagesas a result of progressive amorphization, practical results can be obtained withimage processing by filtering the Fourier transform around amplitude maxima ofdifferent reflections which enhances the image contrast [10].Synthetic apatites aimed at emulating the biological scenario should exhibit

small particle sizes and presence of CO32. In this sense, the wet route is the most

adequate method of synthesis. There are several methods leading to nanometric

. HRTEM image along [0001] corresponding to OHAp. Damaged areas are arro
Fig. 5 wed.


size apatites. Some examples are, for instance:

– A
erosol synthesis technique has been used to produce small particles of differentmaterials [11,12]. Its main advantage is that this technique has the potential tocreate particles of unique composition, for which starting materials are mixed ina solution at atomic level. An ulterior thermal treatment can originate importantmodifications on morphology and texture. In consequence, OHAp preparationby this method was deemed of interest [13]. Hydroxyapatite hollow particleshave been prepared by pyrolysis of an aerosol produced by ultrahigh frequencyof a CaCl2–(NH4)H2PO4 solution. Hollow particles were annealed at differenttemperatures. Thermal treatment at 1050
vC produces the growth of nucleated

crystallites in the particle surface, with remarkable morphology. The raspberry-shaped particles are polycrystalline, the crystallite size being a function of theannealing time (Fig. 7). The mean particle size is 1.2 lm, although there is awide size range of 0.3–2.2 lm. The lattice image obtained by electron microscopyalong the [0001] zone axis clearly shows the sixfold hexagonal symmetry charac-teristic of OHAp and is reflected in the corresponding microdiffraction pattern(left inset). However, different contrast is observed in different areas of the crys-tal marked A–C. Lattice images in parts A and B are different probably due tocrystal tilt. Actually, the microdiffraction pattern in part B of the crystal (rightinset) shows some deviation from the perfect sixfold hexagonal symmetry, which

TEM image along [0001], after irradiation for a long period. Fringes
Fig. 6. b–TCP HR corresponding to
CaO are clearly seen.


is probably related to the crystal tilt of area B with respect to area A. Areas ofwhite contrast (marked C) are possibly due to density variations related to localCa/P stoichiometry.

– M
ethods based on precipitation from aqueous solutions are most suitable for prep-aration of large amounts of apatite, as needed for processing both into ceramicbodies and in association with different matrixes. The difficulty with most of theconventional precipitation methods used is the synthesis of well-defined andreproducible orthophosphates [14,15]. Problems can arise due to the usual lackof precise control on the factors governing the precipitation, pH, temperature,Ca/P ratio of reagents, etc., which can lead to products with slight differences instoichiometry, crystallinity, morphology, etc., that could then contribute to thedifferent ‘‘in vivo/in vitro’’ behaviours described. In this sense, it is important todevelop a methodology able to produce massive and reproducible quantities ofapatite, optimized for any specific application or processing requirements bycontrolling composition, impurities, morphology, and crystal and particle size.
Fig. 7. (a) Schematic representation of the pyrosol equipment used to produce powdered materials. (b)

SEM micrograph showing that polycrystalline OHAp is constituted by small hollow particles. (c) Mag-

nification of a raspberry-shaped particle. (d) Electron diffraction pattern characteristic of polycrystalline

OHAp. (e) Electron micrograph of an OHAp crystallite along [0001]. The microdiffraction pattern at the

right shows some deviation from the perfect hexagonal symmetry observed in the pattern at the left,

probably due to crystal tilt.


For quantitative reactions in solutions, the reactants must be calcium and phos-phate salts with ions that are unlikely to be incorporated into the apatite lattice.Since it has been claimed that NO�

3 and NHþ4 are not incorporated into crystal-

line apatites, or in the case of NHþ4 present a very limited incorporation [16], the

chosen reaction for this method was 10CaðNO3Þ2 � 4H2Oþ 6ðNH4Þ2HPO4þ8NH4OH ! Ca10ðPO4Þ6ðOHÞ2 þ 20NH4NO3 þ 6H2O.

Thus, apatites with different stoichiometry and morphology have been preparedand the effects of varying synthesis conditions on stoichiometry, crystallinity, andmorphology of the powder are analysed. The effects of varying concentration ofthe reagents, the temperature of the reaction, reaction time, initial pH, aging time,and the atmosphere within the reaction vessel were also studied (Fig. 8). Tempera-tures in the range 25–37

vC are necessary to obtain apatites with crystal sizes in the

range of adult human bone, while 90vC are necessary to obtain apatites with crys-

tal sizes in the range of enamel. Higher reaction times lead to apatites with higherCa/P ratios. Aging of the precipitated powder can lead to the incorporation ofminor quantities of carbonate. It is possible to force the incorporation of carbon-ate ions into the apatitic structure without introducing monovalent cations [17].The main results of the studied variations in the reaction conditions are, in short,that higher concentrations of reagents produce higher amounts of products withminor differences in their characteristics, allowing the production of homogeneoussets of materials.

The application of the liquid mix technique, which is based on the Pechini patent[18]. This patent was originally developed for the preparation of multicomponentoxides, allowing the production of massive and reproducible quantities with a pre-cise homogeneity in both composition and particle size. This method is based onthe preparation of a liquid solution that retains its homogeneity in the solid state.This method not only allows a precise control of the cation concentration, but alsothe diffusion process is enormously favoured by means of the liquid solution, com-pared to other classical methods. The solution of metallic salts in polycarboxylic P

O3�4 acid such as citrates is solidified by the addition of a diol which increases the

solution viscosity due to the formation of ester-type three-dimensional polymers.When the diol reacts with the citric solution a resin is formed, thus avoiding thepartial segregation, which would modify the original homogeneity of the solution[19]. Its application has now extended to the preparation of calcium phosphates.The main difficulty of this synthesis lies on the presence of groups that cannot becomplexed by citric acid, and may cause its segregation and the formation of sepa-rated phosphate phases. The success of this task would suggest the possibility, bymodifying the synthesis conditions, of obtaining large amounts of single phases orbiphasic mixtures with precise proportions of the calcium phosphates. This methodmakes it possible to obtain single phase hydroxyapatite, b-TCP and a-TCP andalso biphasic materials whose content in b-TCP and OHAp can be precisely pre-dicted from the Ca/P ratio in the precursor solutions [20] (Fig. 9).


Obviously, several techniques have been utilized for the preparation of hydro-

xyapatite and other calcium phosphates. The synthetic routes employed can be divided

into solid-state reactions and wet methods, which include precipitation, hydro-

thermal and hydrolysis of other calcium phosphates [17,21–26]. Modifications of

these ‘‘classical’’ methods (precipitation, hydrolysis or precipitation in the pres-

ence of urea, glycine, formamide, hexamethylenetetramine. . .) [27–29] or alterna-

tive techniques have been employed to prepare hydroxyapatite with morphology,

stoichiometry, ion substitution or degree of crystallinity as required for a specific

application. Among them, sol–gel [30–34], microwave irradiation [35,36], freeze-

drying [37], mechanochemical method [38–41], emulsion processing [42–44], spray

pyrolysis [13,45,46], hydrolysis of a-TCP [47], ultrasounds [48,49]. . . can be out-

lined.

matic diagram of a crystallization setup. Micrographs and electron diffracti
Fig. 8. Sche on patterns
(insets) of calcium-deficient apatites obtained from Ca(NO3)2�4H2O and (NH4)2HPO4; all the variable

production parameters were equal for both materials, except the synthesis temperature (as displayed).


Particular attention must be paid at this point to the essays performed on the

absence of gravity. Suvurova and Buffat [50] have compared the results obtained

when calcium phosphate specimens, in particular, OHAp and triclinic octacalcium

phosphate (OCP), are prepared from aqueous solutions under different conditions

of precipitation. When supersaturated solutions of calcium phosphates are pre-

pared by diffusion-controlled mixing in space (EURECA 1992–1993 flight) several

differences are observed in crystal size, morphology and structural features with

respect to those prepared on earth. It is worth stressing that space-grown OCP

crystals possess a maximum growth rate in the [001] direction and a minimum rate

in the [100] one. Space-grown and terrestrial OHAp crystals differ from each other

in size: the former are, at least 1–1.5 orders of magnitude bigger in length. Dif-

fusion-controlled mixing in space seems to provide a lower supersaturation in the

crystallization system comparatively to earth, promoting the crystal growth in the

competition between nucleation and growth. These authors conclude that similar

processes may most probably arise in the human body (under definite internal con-

ditions) during space flying when quite large OHAp crystals start to grow instead

of the small and natural ones. In addition, other modification of OCP crystals with

Fig. 9. Outline of calcium phosphate synthesis by liquid mix technique. Three dimensional phase dia-

gram—Ca/P ratio, annealing temperature, OHAp content—of calcium phosphates.


huge sizes appears. These elements may disturb the Ca dynamical equilibrium inthe body which might lead to possible demineralization of the bone tissue.Biological apatites (mineral component of the bones) are difficult to synthesize at

the laboratory with carbonate contents equivalent to those in the bone. Althoughthe carbonate inclusion in itself is very simple (in fact, when producing stoichio-metric apatites at the laboratory, a strict control of the synthesis conditions is nee-ded to avoid carbonate inclusion), the carbonate content is always different fromthe fraction of carbonates in the natural bone (4–8% wt) [21] and/or are located indifferent lattice positions [51]. At this point, it should be mentioned that this car-bonate content can be slightly different when analysed samples come from othervertebrates [52]. The carbonate easily enters into the apatite structure, but theproblem lies in the amount that should be introduced taking into account the car-bonate content of biological apatites. When the aim is to obtain carbonate hydro-xyapatite and the reaction takes place at high temperatures, the carbonates enterand occupy lattice positions in the OH� sublattice (A type apatites). In contrast,the carbonates in biological apatites always occupy positions in the PO3�

4 sublattice(that is, they are B type apatites) [51]. In order to solve this problem, low tempera-ture synthesis routes have to be followed, allowing obtaining carbonate hydro-xyapatites with carbonates in phosphate positions [21]. But the amount enteredremains to be solved, and it usually is lower than the carbonate content of the min-eral component of the bones.These calcium deficient and carbonated apatites have been obtained in labora-

tory by various techniques; nowadays, it is known that apatites with low crystal-linity, calcium deficiency and carbonate content can be obtained, but withcarbonate contents usually unequal to those of the natural bones [17,53,54]. There-fore, the main problem remains in the control of carbonate content and latticepositioning.

4. Substituted apatites

Hydroxyapatite is clearly a non-stoichiometric compound, with the ability toaccept compositional variations in its three sublattices (Fig. 10). No biologicalhydroxyapatite shows a stoichiometric Ca/P ratio, but they all move towardstissues, which are linked to an increase in crystallinity. These trends have a remark-

ulation of apatite minerals, and potential substitutions in
Fig. 10. Generic form the three sublattices.


able physiological meaning, since the younger, less crystalline tissue can developand grow faster, while storing other elements that the body needs during itsgrowth; this is due to the highly non-stoichiometric quality of OHAp, which catersfor the substitutional inclusion of different amounts of several ions, such as Na+,K+, Mg2+, Sr2+, Cl�, F, HPO2�

4 , etc [1].The more crystalline the OHAp becomes, the more difficult interchanges and

growth are. In this sense, it is worth stressing that the bone is probably a veryimportant detoxicating system for heavy metals due to the ease of substitution inOHAp; heavy metals, in the form of insoluble phosphates, can be retained in thehard tissues without important alterations of their structural properties.But the ability to exchange ions in this structure also allows to design, develop

and characterize new and better calcium phosphates for certain specific applica-tions. It is known that the bone regeneration rate depends on several factors suchas porosity, composition, solubility and presence of certain elements that, releasedduring the resorption of the ceramic material, facilitate the bone regeneration car-ried out by the osteoblasts. Thus, for instance, small amounts of strontium, zinc orsilicates stimulate the action of these osteoblasts and, in consequence, the new boneformation. Carbonate and strontium favour the dissolution, and therefore theresorption of the implant. Silicates increase the mechanical strength, a very impor-tant factor in particular for porous ceramics, and also accelerate the bioactivity ofapatite [55]. The current trend is, therefore, to obtain calcium phosphate bio-ceramics partially substituted by these elements. In fact, bone and enamel are someof the most complex biomineralized structures. The attempts to synthesize bone atthe laboratory are devoted at obtaining biocompatible prosthetic implants, withthe ability to leverage natural bone regeneration when inserted in the human body.Its formation might imply certain temporary structural changes on its components,which demand in turn the presence, at trace levels, of additional ions and mole-cules in order to enable the mineralization process. This is the case, for instance,with bone growth processes, where the localized concentration of silicon-rich mate-rials coincides precisely with areas of active bone growth. The reason is yetunknown, although the evidences are clear; the possible explanation of thisphenomenon would also justify the great activity observed in certain silicon-substi-tuted apatite phases and in some glasses obtained by sol–gel method, regarding cellproliferation and new bone growth.One way to enhance the bioactive behaviour of hydroxyapatite is to obtain sub-

stituted apatites, which resemble the chemical composition and structure of themineral phase in bones [56,57]. These ionic substitutions can modify the surfacestructure and electrical charge of hydroxyapatite, with potential influence on thematerial in biological environments. In this sense, an interesting way to improvethe bioactivity of hydroxyapatite is the addition of silicon to the apatite structure,taking into account the influence of this element on the bioactivity of bioactiveglasses and glass-ceramics [58,59]. In addition, several studies have proposed theremarkable importance of silicon on bone formation and growth [60,61] at in vitroand in vivo conditions.


Several methods for the synthesis of silicon-substituted hydroxyapatites havebeen described. Ruys [62] suggested the use of a sol–gel procedure; however, thesematerials, besides the hydroxyapatite phase, include other crystalline phasesdepending on the substitution degree of silicon. Tanizawa and Suzuki [63] triedhydrothermal methods, obtaining materials with a Ca/(P+Si) ratio higher thanthat of pure calcium hydroxyapatite. Boyer et al. [64] conducted studies on the syn-thesis of silicon-substituted hydroxyapatites by solid-state reaction, but in thesecases the incorporation of a secondary ion, such as La3+ or SO2�

4 , was needed. Inthese examples, no bioactivity studies were performed on the silicon-containingapatites.Gibson et al. [65] synthesized silicon-containing hydroxyapatite by using a wet

method, and its in vitro bioactivity studies gave good results. These authors stud-ied the effects of low substitution levels on the biocompatibility and in vitro bioac-tivity, determining the ability to form the apatite-like layer by soaking thematerials in a simulated body fluid (SBF) [66]. Also, Marques et al. [67] synthe-sized, by wet method, hydroxyapatite with silicon content up to 0.15 wt%, obtain-ing stable materials at 1300

vC and noting that the unit cell volume and the a

parameter length of the hydroxyapatite decreased as the silicon content increased.Hence, the role of silicon substituting part of the phosphorus atoms present in

the hydroxyapatite lattice seems to be an important factor influencing the bioactivebehaviour of the material. However, it is not clearly known whether the siliconpresent in the material substitutes completely the phosphorus in the hydroxyapatitestructure, or whether the replacement is partial, or even if in any of the describedsynthesis the silicon species remain as an independent phase. In all the cited synth-eses, the final product contains silicon, but its chemical nature is not revealed.A similar work focused on the synthesis and bioactivity study of hydroxyapatites

containing orthosilicate anions that isomorphically replace phosphate groups,aimed at improving the bioactivity of the resulting materials as compared with thatof pure calcium hydroxyapatite. To accomplish this purpose, two synthesis proce-dures were used, starting from two different calcium and phosphorus precursorsand the same silicon reagent in both cases. To assess the proposed substitution,surface chemical and structural characterization of the silicon-substituted hydro-xyapatites was performed by means of X-ray diffraction (XRD) and X-ray photo-electron spectroscopy (XPS). The in vitro bioactivity of the so-obtained materialswas determined by soaking the materials in SBF and monitoring the changes of pHand chemical composition of the solution, whereas the modification at the surfacewas followed by means of XPS, XRD, and scanning electron microscopy (SEM).Silicon-containing hydroxyapatites were synthesized by the controlled crystal-lization method. Chemical analysis, N2 adsorption, Hg porosimetry, X-ray diffrac-tion, scanning electron microscopy, energy-dispersive X-ray spectroscopy, and X-ray photoelectron spectroscopy (XPS) were used to characterize the hydroxyapatiteand to monitor the development of a calcium phosphate layer onto the substratesurface immersed in a simulated body fluid, that is, in vitro bioactivity tests. Theinfluence of the silicon content and the nature of the starting calcium and phos-phorus sources on the in vitro bioactivity of the resulting materials were studied. A


sample of silicocarnotite, whose structure is related to that of hydroxyapatite and

contains isolated SiO4�4 anions that isomorphically substitute some PO3�

4 anions,

was prepared and used as reference material for XPS studies. An increase of the

unit cell parameters with the Si content was observed, which indicated that SiO4�4

units are present in lattice positions, replacing some PO3�4 groups. By using XPS it

was possible to assess the presence of monomeric SiO4�4 units in the surface of apa-

tite samples containing 0.8 wt% of silicon, regardless the nature of the starting raw

materials, either CaðNO3Þ2=ðNH4Þ2HPO4=Si ðOCOCH3Þ4 or CaðOHÞ2=H3PO4=Si

ðOCOCH3Þ4. However, an increase of the silicon content up to 1.6 wt% leads to

the polymerization of the silicate species at the surface. This technique shows sili-

con enrichment at the surface of the three samples. The in vitro bioactivity assays

showed that the formation of an apatite-like layer onto the surface of sili-

concontaining substrates is strongly enhanced as compared with pure silicon-free

hydroxyapatite (Fig. 11). The samples containing monomeric silicate species

showed higher in vitro bioactivity than that of silicon-rich sample containing poly-

meric silicate species. The use of calcium and phosphate salts as precursors leads to

materials with higher bioactivity [68].Finally, the results revealed that controlled crystallization is a good procedure to

prepare silicon-substituted hydroxyapatites that can be used as a potential material

for prosthetic applications.The presence of silicon (Si) in HA has shown an important role on the forma-

tion of bone [60]. To study the role of Si, Si-substituted hydroxyapatite (Si-HA)

has been synthesized by several methods [62–65,67,68] but its structural character-

istics and microstructure remain not fully understood. The structural studies car-

ried out until now (mainly by X-ray diffraction) have not demonstrated the Si

incorporation into the apatite structure. In fact, the very similar scattering factor

makes very difficult to determine if Si has replaced some P in the same crystal-

lographic position. Until now, the absence of secondary phases and the different

Fig. 11. SEM images of silicon-substituted apatites, before and after soaking in SBF for 6 weeks.


bioactive behaviour are the best evidences for the Si incorporation. No positive evi-

dence or quantitative study of P substitution by Si has been carried out yet. On the

other hand, the hydroxyls groups sited at the 4e position are one of the most

important sites for the HA reactivity. The movement of H along the c-axis

2contributes to the HA reactivity. However, XRD is not the optimum tool for the

study of light atoms such as H; neutron diffraction (ND) is an excellent alternative

to solve this problem. The Fermi length for Si and P are different enough to be dis-

criminated, whereas neutrons are very sensitive to the H presence. Consequently,

XRD and ND have combined to answer one of the most recent subjects in the

dentistry and orthopaedic surgery fields.In order to explain the higher bioactivity of the silicon-substituted hydro-

xyapatite (SiHA), synthetic ceramic hydroxyapatite (HA) and SiHA have been

structurally studied by neutron scattering. The Rietveld refinements show that the

final compounds are oxy-hydroxyapatites, when obtained by solid-state synthesis

under air atmosphere. By using neutron diffraction, the substitution of P by Si into

oids schematic drawing for hydroxyapatite and silicon-substi
Fig. 12. Thermal ellips tuted hydroxyapatite.


the apatite structure has been corroborated in these compounds. Moreover, thesestudies also allow us to explain the superior bioactive behaviour of SiHA, in termsof higher thermal displacement parameters of the H located at the 4e site [69].The refinements of the structure by the Rietveld method indicate that the ther-

mal treatment produces partial decomposition of the OH groups, leading to oxy-hydroxy apatites in both samples and the higher reactivity of the Si-substituted HAcan be explained in terms of an increasing of the thermal ellipsoid dimension paral-lel to the c-axis for H atoms (Fig. 12).

5. Calcium phosphate cements

Cements based on calcium salts, phosphates or sulphates, have attracted muchattention in medicine and dentistry due to their excellent biocompatibility andbone-repair properties [70–73]. Moreover, they have the advantage over the bio-ceramics that they do not need to be delivered in prefabricated forms, becausethese self-setting cements can be handled by the clinician in paste form and injectedinto bone cavities. Depending on the cement formulation, or the presence of additives,different properties, such as setting time, porosity or mechanical behaviour havebeen found in these materials [74–78].On the other hand, in the literature on phosphates focused on calcium phos-

phate cements, the technique employed for obtaining such cements is to mix thedifferent components; one of them is responsible for curing the mixture. Forinstance, in the Constanz cement [79]—the first of its kind to be commercialized—the final product is a carbonateapatite (dahlite) with low crystallinity and a carbon-ate content reaching 4.6%, in substitution of phosphate groups (B type carbonatea-patite) as is the case in bones. Constanz cement is obtained from a dry mixture ofa-tricalcium phosphate, a-Ca3(PO4)2, calcium phosphate monohydrate, Ca(H2-

PO4)�H2O, and calcium carbonate, CaCO3. The Ca/P ratio of the first componentis 1.50, and 0.5 for the second one, both values significantly lower than the Ca/Pratio of 1.67 for hydroxyapatite. A liquid component—a sodium monoacid phos-phate solution—is then added to this solid mixture, which allows the formation ofan easily injectable paste that will cure over time. The paste curing happens after avery reasonable period of time when considering its use in surgery. In fact, after 5min it shows a consistency suitable for injection, and upon 10 min it is solid with-out any exothermal response, exhibiting an initial strength of 10 MPa. Twelvehours later, 90% of its weight has evolved to dahlite, with compression strength of55 and 2.1 MPa when under stress. This cement is then resorbed and graduallyreplaced by newly formed bone.Calcium phosphate cements which can be resorbed and injected are being com-

mercialized by various international corporations [80], with slight differences intheir compositions and/or preparation. Research is still under way in order toimprove the deficiencies still present.These cements cure in field, are very compatible with the bone and seem to

resorb slowly; during this gradual process, the newly formed bone grows and repla-


ces the cement. However, the properties of the calcium phosphate cements are stillinsufficient for their reliable application. There are problems related to their mech-anical toughness, the curing time, the application technique on the osseous defectand the final biological properties. New improvements in the development of thesecements will soon be described, solving at least in part some of these dis-advantages. For instance, the curing time will be shortened, even in contact withblood, and the toughness under compression will also improve.Most of the injectable calcium phosphate cements used evolves to an apatitic cal-

cium phosphate during the setting reaction. One of the main drawbacks of theseapatitic cements is the slow resorption rate of the apatite. On the other hand, cal-cium sulphate dihydrate, gypsum, has been used as bone-void filler during manyyears [70,79–81], although it presents a too fast resorption rate to provide a goodsupport for new bone. The combination of both, calcium sulphate and apatite, canovercome the individual drawbacks, and in the last years studies using this biphasicmaterial have been performed [82–84].Despite the advantages, all these implants can act as foreign bodies and become

potential sources of infections. Then, the in vivo utilization of these materialsrequires a preventive therapy and this may be achieved introducing a drug intothem, which can be locally released ‘‘in situ’’ after implantation. In fact, differentstudies using bioceramics and self-setting materials containing active drugs havebeen performed in the last years [85–89].In this sense, the addition of an antibiotic to calcium sulphate-based cements has

also been studied, in order to determine if the presence of the drug affects the phy-sico-chemical behaviour of the cements and to study the release kinetics of thedrug from the cement. Two system types were chosen: gypsum and apatite/gyp-sum. The antibiotic chosen for this study was cephalexin in crystalline form, i.e.cephalexin monohydrate.The presence of cephalexin into the cements does not alter neither the physico-

chemical behaviour of the cements nor produce structural changes on them. Therelease of the drug is different depending on the composition. For gypsum cements,the cephalexin is quickly released, helped by a dissolution process of the matrix,whereas the drug release is more controlled by the hydroxyapatite presence inhydroxyapatite/gypsum samples. Apatite-containing cements do not only show adifferent drug release process, also the paste viscosity is lower and a faster forma-tion ‘‘in vitro’’ of an apatite-type layer on their surface is observed [90].

6. Biphase mixtures of calcium phosphates

Several attempts have been made to synthesize the mineral component of bonesstarting from biphase mixtures of calcium phosphates [91]. Hence, bone replacingmaterials based on mixtures of hydroxyapatite and b-TCP have been prepared;under physiological conditions, such mixtures evolve to carbonate hydroxyapatite.The chemical reactions are based in equilibrium conditions between the morestable phase, hydroxyapatite, and the phase prone to resorption, b-TCP. As a


consequence, the mixture is gradually dissolved in the human body, acting as astem for newly formed bone and releasing Ca2+ and PO3�

4 to the local environ-ment. This material can be injected, used as coating or in any other form suitablefor application as bulk bone replacement—forming of bulk pieces, filling of bonedefects—[92]. At present, a wide range of biphase mixtures are under preparation,using various calcium phosphates, bioactive glasses, calcium sulphates, etc.[55,84,93,94].Currently, there is an increasing interest on the preparation of mixtures of two

or more calcium phosphates. These materials are commonly prepared with hydro-xyapatite and a more resorbable material such as tricalcium phosphate (a or b) orcalcium carbonate in different proportions depending on the characteristicsrequired for a specific application. Some examples of commercial products basedon these mixtures are: Triosite2, MBCPTM2, Eurocer1,. . .. The synthesis routesemployed commonly in the preparation of these mixtures include the blending ofdifferent calcium phosphates [95,96], and precipitation [97–99]. Other techniquesalso employed are: solid state [100], treatment of natural bone [101], spray pyrol-ysis [102], microwave [103], combustion [104]. . . . Some authors have defended thesuperior properties of the biphasic materials ‘‘directly’’ prepared over thoseobtained by mixing two single phases [105].The promising results obtained with cements and biphase mixtures seem to indi-

cate that it is easier to obtain precursors of synthetic apatites that, when in contactwith the biological environment, can evolve towards similar compositions to thatof the biological apatite, than to obtain apatites in the laboratory with similarcompositional and structural characteristics to those of the biological material, andin adequate quantities, i.e. large, industry-scale amounts with precise compositionand easily repeatable batch after batch, for its use in the production of ceramicbiomaterials.Bioceramics aimed at the replacement or filling of bones could be obtained by

synthesis of apatite precursors through different calcium phosphate mixtures, usinga wet route. If the information gathered from the calcium cements is put to use,it would be necessary to eliminate the solution added to cure the mixture andsearch for compositions and ratios that allow to obtain precursors that, when incontact with the body fluids, evolve chemically towards the formation of carbonatehydroxyapatite crystals, with small particle size and low crystallinity, calciumdeficient and with a carbonate content of approximately 4.5% w/w, located in the

PO3�4 sublattice.

7. Fabrication of bulk calcium phosphate pieces

Traditionally, calcium phosphate ceramics have been processed by high tempera-ture treatments, [5,106]. If the products have previously been synthesized by thewet route at low temperatures, this leads to a very crystalline material, and there-fore not similar to biological apatites. Owing to the thermal decomposition of mostcalcium phosphates at high temperature, this type of process is restricted basically


to obtaining stoichiometric hydroxyapatite and tricalcium phosphate. It can beemployed in the production of both dense and porous pieces. The latter allow thegrowth of natural tissue inside the pores, helping to mechanically stabilize theimplants. Although these treatments are still in use nowadays [107,108], theyreduce significantly the reactivity of the ceramics and the growth kinetics of thebone. Therefore, new forming methods at lower temperatures have been developed[109,110], allowing to obtain pieces without altering the crystallinity of the ceramicstarting material. The combination of the synthesis of calcium deficient, low crys-tallinity carbonate hydroxyapatites with processing methods that preserve theirchemical and microstructural features is an excellent alternative to the productionof bioceramic pieces. Methods such as colloidal processing [107], uniaxial pressingfrom a precipitated powder [108], cold isostatic pressing [111] and starch consoli-dation [112] and combination of gel casting and foams out [113] have yielded excel-lent results. In this sense, porous machinable pieces of OHAp have been preparedfrom polyurethane foams by combination of gelcasting and burn out methods(Fig. 13). The pieces were constituted by polyhedral-like particles with an averagesize of 0.45–1.0 mm that are surrounded by an interconnected network of pores.The porous pieces showed a bimodal distribution of the pores size between 30.8–58.6 and 1.0 lm. The size of the interconnected pores (30.8–58.6 lm) can be con-trolled as a function of the cells in the used foam. The composition of the ceramicsand the volume of the small pores on the particles surface can be modified as afunction of the sintering time. The presence of pores could promote the boneingrowth and also could be used to insert different drugs, which makes theseporous pieces a potential candidate to be used as non-load-bearing bone implantsand as drug delivery systems.If materials in powder form instead of pieces are used for bone filling applica-

tions, the main advantage is their superior adaptability to the profile of eachdefect, while the clinical insertion is not so adequate. Such materials are difficult to

Fig. 13. SEM micrographs of porous pieces sintered at 1300vC for 3 h, at different magnification values.


place and secure in the implant region, and their particles retain the potential tomigrate for weeks or even months. To avoid this drawback, the powder is mixedwith a degradable carrier matrix. At present, the trend is to obtain materials suit-able for injection [114]. Both organic and inorganic matrices can be used. Amongthe organic matrices, non-absorbable polymers such as polymethyl methacrylate(PMMA) [115,116], polyethylene (PE) [117] and polysulphone are being used, butthe main problem is that such matrices reduce the bioactivity of the implant. Bio-degradable polymers are also in use, such as polylactic acid (PLA) [118] and poly-glycolic acid (PGA), as well as natural polymers [119,120] (collagen, cellulose andstarch). A inorganic matrix also in use is calcium sulphate (Hapset) [121].

8. Ceramic coatings

At present, for all those clinical applications where load-bearing properties arerequired, most of the implants used are metallic, with subsequent and serious pro-blems due to: (a) the large differences in mechanical properties between the arti-ficial implant and the natural bone, giving rise to ruptures, (b) the presence of ionsthat, released from the artificial implant, could be toxic or harmful and provokepains, and (c) the impossibility to regenerate natural bone. An alternative option,until a more similar material to bone becomes available, is to coat the metallicimplant with ceramics. This technique is being used nowadays, both for dentalimplants and hip joint prosthesis. There is still a long way to follow, but severalmetallic implants with ceramic coatings are commercially available already, and theresearch in problem solving is under way.The ceramic coating process on a metallic substrate is quite complicated, and

several methods are available in this sense. A great deal of the clinical successdepends on this coating, since the quality and durability of the interface attach-ment greatly depend on the purity, particle size, chemical composition of the coat-ing, layer thickness and surface morphology of the substrate. An additionaladvantage when coating a metallic implant with ceramics is the reduction in ionrelease issues from the metal alloy. The ceramic represents a truly effective barrierthat hinders the metallic ion kinetics of release towards the living body. Hydro-xyapatite is being specifically used for this purpose, in order to improve the attachmentof hip joint prostheses, due to its excellent biological properties such as non-tox-icity and lack of inflammatory response and fibrous and/or immunitary reac-tions.Hydroxyapatite (HA), tricalcium phosphate (TCP), and their biphasic combina-

tions are important ceramic materials in the replacement of hard tissues, becausethey can form a strong bond with the bone and favour bone formation. However,the poor mechanical properties of calcium phosphates limit the use of the bulkmaterial to non-load-bearing implants. For this reason, one of most important usesof these calcium phosphates is to coat inert or biotolerable implants with mechan-ical properties adequate for orthopaedic substitutions. In this way, the coatedimplants will not only have the good mechanical properties of the substrate but


also an enhanced osseointegration and bioactivity due to the calcium phosphatelayer. Plasma spray technique is currently the only method commercially availablefor coating metallic substrates [122–124]. The significant deficiencies found in theplasma sprayed HA coatings have promoted the search for new deposition meth-ods, such as ion beam assisted deposition, magnetron sputtering, sol–gel, pulsedlaser deposition. . .[125–128]. Moreover, not only the synthesis method, but thecrystallinity and the calcium phosphate phases present in the film influence thesolubility and biological behaviour of these coatings [129,130].The so-called pyrosol method has been applied for the deposition of a wide var-

iety of ceramic materials with different applications [131–133]. The ability to con-trol the composition and microstructure of the coatings and to obtain uniformfilms onto irregular surfaces, along with the facility for large scale productionmakes this technique potentially very powerful.Thin films of calcium phosphates with different crystallinity and Ca=P ratios

have been prepared by the pyrosol method and dip-coating procedures [134–138]and characterized by XRD, FTIR, EDS and SEM (Fig. 14) [139]. The microstruc-ture, crystallinity and composition of the deposited films can be controlled by mod-ifying the composition of the precursor solution, reactor atmosphere and substratetemperature. By this method, pure hydroxyapatite with varying crystallinity andmicrostructure can be obtained under more oxidant conditions, whereas tricalciumphosphate (a or b) or biphasic hydroxyapatite/tricalcium phosphate mixtures canbe obtained under argon atmosphere. The ability to vary the calcium phosphatephases deposited has significant implications for the use of these films in implantdevices. The results show that the use of a simple and versatile solution methodallows calcium phosphate coatings with different crystallinity and phase compo-

hematic representation of the pyrosol equipment used to produce coatings (left). SE
Fig. 14. Sc M micro-
graphs (above, right) of three hydroxyapatite coatings with different morphologies, obtained by pyrosol.

Powder X-ray diffraction pattern (below, right) of one of these coatings.


sition to be prepared. Dissolution tests show that these calcium phosphate layers

are stable and exhibit different behaviours as a function of their composition.

The ability to modify the phase composition, crystallinity and microstructure of

the coatings should allow designing coatings with tailored in vitro or in vivobehaviour.Although different deposition methods have been applied in the last years [139–

143], sol–gel method offers a good alternative since the synthesis temperatures are

low, and it can be applied to a great number of substrates, including those whichwould oxidize at higher temperatures. Several authors have prepared OHAp via

sol–gel technique using different precursors [144–147], showing that the tempera-

ture required to form OHAp depends on the chemical reactivity of the precursors.

Livage et al. [148] investigated the sol–gel synthesis of phosphates and found thatalkyl phosphates esters or phosphoric acid were unsuitable precursors, either

because the hydrolysis is too slow, or because it reacts so fast that a precipitate is

formed, as in the case of phosphoric acid. In the same way, different works show

the necessity of controlling the ageing effect of the sol, in the synthesis of singlephase OHAp powders by the sol–gel method [149–151]. A similar systematic study

for the optimization of the films deposition has been carried out [152]; single phase

Fig. 15. Immersion stage and thermal treatment followed for the preparation of coatings by dip-coating

technique. The illustration shows a total joint prosthesis partially coated with hydroxyapatite. Atomic

force micrographs of a substrate and two coatings obtained at different immersion velocities.


OHAp coatings were deposited on Ti6Al4V by the sol–gel dipping technique fromaqueous solutions containing triethyl phosphite and calcium nitrate. In order toobtain homogeneous and monophasic hydroxyapatite coatings, the ageing timeand temperature of the sol were, fundamentally, the variables studied. The pHmeasurement was a good tool to evaluate the best conditions of the sols to depositthese coatings. SEM and SFM techniques show that the coatings deposited aredense and homogeneous and with a low roughness (Fig. 15) which depends on thesol viscosity and the film thickness. The higher the sol temperature, the shorter theageing time needed to obtain pure OHAp after coating annealing. When the ageingparameters or the annealing temperature are not adequately controlled, additionalphases or poor surfaces are obtained. The conditions to obtain the best coatingshave been related with the pH decrease on the aqueous sols observed during theageing, according to the polymerization reaction between calcium and phosphor-ous. In order to obtain homogeneous, crack-free coatings, the annealing tempera-ture and thickness of the coatings must be controlled. Films roughness is relatedwith the viscosity of the sol-precursor used to do the deposition, as well as with thenumber of coating layers.

9. Tissue engineering

This field, which started about a decade ago, is now at full research potentialand the first results are currently being developed. The aim is basically to providean artificially made scaffold, e.g. a bioceramic, onto which cells are cultured so thatthe artificial piece becomes colonized. This process can be carried out both underin vitro and in vivo conditions. One of the main objectives is to develop materialsaimed at the functional repair and reconstruction of biological structures. In thissense, much attention is being devoted to the synthesis and surface study of varioussubstrates—such as calcium phosphates, among others—for their application in thedevelopment of three-dimensional scaffolds for tissue engineering. Perhaps themain issue in this subject is the study and tailoring of surface properties of thesesubstrates, in order to control the interaction with biological entities such as mac-rocells and cells.The repair and replacement strategy for damaged parts of the human body is

clearly different from the traditional biomedical implants currently in use where, inorder to perform a bone replacement, the use of donor tissue is the predominanttrend, either from allografts or autografts; there is, however, a certain increase inthe application of artificial materials. Donor tissues exhibit good biocompatibility,but are also handicapped by clear disadvantages: these tissues are costly, scarce,there are risks of disease transmission, etc., and thus it can be predicted that syn-thetic materials will conquer a good share of this market in the long run. The cur-rent global market for these products has an estimated value of more than 1000million euro, with an annual growth rate of 7.7%. With the increasing number ofcomplex revision surgery interventions and, as a consequence, the higher use ofbone filling materials, it seems reasonable to forecast a market for the next decade


in excess of 2000 million euro. There is a general agreement in the fact that this is afield yet to explore [153]. The feasible production of ceramic pieces with tailoredporosity, in order to be used as substrates for tissue engineering, opens up a spec-tacular future for calcium phosphates.

10. Conclusions

In the future, bioceramics could be the ideal biomaterials due to their good bio-compatibility and bone integration, and also to their close similarity to the mineralcomponent of bone. However, the use of bioceramics for load-bearing applicationsis still far from feasible; their inherent rigidity and breakability are the main draw-backs here. Therefore, the applications of bioceramics are currently focused on theproduction of non-load-bearing implants, such as pieces for middle ear surgery,filling of bone defects in oral or orthopaedic surgery, and coating of dentalimplants and metallic prosthesis. Nowadays, the synthesis of organic–inorganichybrids is a strong subject of research, as well as the manufacturing of calciumphosphate-based cements, biphasic mixtures that mimic as closely as possible themineral component of biological apatites, and the production of substrates for cellculture and biochemical factors for tissue engineering. The production of nanos-tructured materials, similar to the complex hierarchical structures of hard tissues—bone and teeth—, is also a very attractive field of research where promising resultsare already being achieved. Finally, the ability to functionalize surfaces with differ-ent molecules of varying nature and dimensions, by means of their attachment tothe substrate, as well as the potential to physical chemically and topographicallynanostructure the surface, will enable in a medium term to act selectively on thebiological species, such as proteins, peptides, . . . .

Acknowledgements

The authors wish to thank CICYT (Spain) for the financial support throughResearch Project MAT2002-0025. The efforts and publications of the researchgroup on calcium phosphates, Departamento de Quımica Inorganica y Bioinorga-nica, UCM, cited throughout this text, are greatly appreciated. Special thanks to P.Cabanas, F. Conde and J.M. Moreno for their friendship and technical assistance.

References

[1] Vallet-Regı M. Introduction to the world of biomaterials. Ann Quim Int Ed 1997;93:S6–S14.

[2] Black J, Hastings G, editors. Handbook of biomaterials properties. Chapman and Hall; 1998.

[3] Veis A, editor. The chemistry and biology of mineralized connective tissues. Elsevier; 1981, p. 618.

[4] Nancollas GH. Biomineralization. In: Mann S, Webb J, Williams RJP, editors. Chemical and bio-

chemical perspectives. VCH; 1989, p. 157.

[5] Aoki H. Medical applications of hydroxyapatite. Tokyo, St. Louis: Ishikayu Euro America Inc;

1994.


[6] Ayer RJ. Electron Microsc Tech 1989;13:16.

[7] Shulin W, Jingwei F, Airu L, Tingxing L. Proceedings of the XI International Congress on Elec-

tron Microscopy, Kyoto; 1986. p. 3071–2.

[8] Bursill LA, Hutchison JL, Sumida N, Lang AR. Nature 1981;192:518.

[9] Nicolopoulos S, Gonzalez-Calbet JM, Alonso MP, Gutierrez-Rıos MT, de Frutos MI, Vallet-Regı

M. J Solid State Chem 1995;16:265.

[10] Nicolopoulos S, Vallet-Regı M, Gonzalez-Calbet JM. Solid State Ionics 1997;175:101–3.

[11] Cabanas MV, Gonzalez-Calbet JM, Labeau M, Mollard P, Pernet M, Vallet-Regı M. J Solid State

Chem 1992;101:265.

[12] Vallet-Regı M, Ragel V, Roman J, Martınez JL, Labeau M, Gonzalez-Calbet JM. J Mater Res

1993;8(1):138.

[13] Vallet-Regı M, Gutierrez-Rıos MT, Alonso MP, de Frutos MI, Nicolopoulos S. J Solid State

Chem 1994;112:58.

[14] Narasaraju TS, Phebe DE. J Mater Sci 1996;31:1.

[15] Suchanec W, Yoshimura M. J Mater Res 1998;13:98.

[16] Elliott JC. Studies in inorganic chemistry, vol. 18. Elsevier; 1994.

[17] Rodriguez-Lorenzo LM, Vallet-Regı M. Chem Mater 2000;12(8):2460.

[18] Pechini MP. US Patent No. 3330697; 1967.

[19] Vallet-Regı M. In: Rao KJ, editor. Perspectives in solid state chemistry. India: Narosa Pub.

House; 1995, p. 37.

[20] Pena J, Vallet-Regı M. J Euro Ceram Soc 2003;23:1687.

[21] LeGeros RZ. Monographs in oral science, vol. 15. Basel: Karger; 1991.

[22] Elliot JC, editor. Sauramps medical. Montpellier; 1998, p. 25–66.

[23] Suchanek W, Yoshimura MJ. J Mater Res 1998;13(1):94.

[24] Narasaraju TS, Phebe DE. J Mater Sci 1996;31:1.

[25] de Groot K. Ceram Int 1993;19:363.

[26] Bohner M. J Care Injured 2000;31-S-D37–472.

[27] Cuneyt Tas A. Biomaterials 2000;21:1429.

[28] Andres-Verges M, Fernandez-Gonzalez C, Martınez-Gallego M, Solier I, Cachadina JD, Matijevic

EJ. J Mater Res 2000;15(11):2526.

[29] Yasukawa A, Matsuura T, Kakajima M, Kandori K, Ishikawa T. Mater Res Bull 1999;24:589.

[30] Weng W, Baptista JL. Biomaterials 1998;19:125.

[31] Jilavenkatesa A, Condrate RA. J Mater Sci 1998;33:4111.

[32] Chai CS, Gross KA, Ben-Nissan B. Biomaterials 1998;19:2291.

[33] Layrolle P, Ito A, Tateishi T. J Am Ceram Soc 1998;81(6):1421.

[34] Liu DM, Trocynzki T, Tseng W. J Biomater 2001;22:1721.

[35] Sampath Kumar TS, Manjubala I, Gunasekaran. J Biomater 2000;21:1623.

[36] Fang Y, Agrawal DK, Roy DM, Roy RJ. J Mater Res 1992;7(2):490.

[37] Itatani K, Iwafune K, Scott Howellm F, Aizawa M. Mater Res Bull 2000;35:575.

[38] Pena J, del Real RP, Rodriguez-Lorenzo LM, Vallet-Regı M. Bioceramics 1999;12:173.

[39] Kim W, Zang Q, Saito FJ. J Mater Sci 2000;35:5401.

[40] Yeong B, Junmin X, Wang J. J Am Ceram Soc 2001;82:65.

[41] Nakano T, Tokumura A, Umakoshi Y, Imazato S, Ehara A, Ebisu S. J Mater Sci Mater Med

2001;12:703.

[42] Lim GK, Wang J, Ng SC, Chew CH, Gan LM. Biomaterials 1997;18:1433.

[43] Wals D, Mann S. Chem Mater 1996;8:1944.

[44] Furuzono T, Walsh D, Sato K, Sonoda K, Tanaka JJ. J Mater Sci Lett 2001;20:111.

[45] Aizawa M, Hanazawa T, Itatani K, Howell FS, Kishioka A. J Mater Sci 1999;34:2865.

[46] Veilleux D, Barthelemy N, Trombe JC, Verelst M. J Mater Sci 2001;36:2245.

[47] Tenhuisen KS, Brown PW. Biomaterials 1998;19:2209.

[48] Kim W, Satio F. Ultrasonics Sonochem 2001;8:85.

[49] Fang Y, Agrawal DK, Roy DM, Roy R, Brown PW. J Mater Res 1992;7:2294.

[50] Suvurova EI, Buffat PA. Eur Cells Mater 2001;1:27.


[51] Elliot JC, Bond G, Tombe JC. J Appl Crystallogr 1980;13:618.

[52] Tadic D, Epple M. Biomaterials 2004;25:987.

[53] Okazaki M, Matsumoto T, Taira M, Takakashi J, LeGeros RZ. In: Legeros RZ, LeGeros JP, edi-

tors. Bioceramics, vol. 11. New York: World Scientific; 1998, p. 85.

[54] Doi Y, Shibutani T, Moriwaki Y, Kajimoto T, Iwayama YJ. J Biomed Mater Res 1998;39:603.

[55] Vallet-Regı M, Ramila A, Padilla S, Munoz B. J Biomed Mater 2003.

[56] LeGeros RZ. Nature 1965;206:403.

[57] Jha LJJ, Best SMJ, Knowles JC, Rehman I, Santos JD, Bonfield W. J Mater Sci Mater Med

1997;8:185.

[58] Hench LL, Wilson J, Hench LL, Wilson J, editors. An introduction to bioceramics, vol. 20. Boca

Raton (FL): World Scientific; 1992.

[59] Ohura K, Nakamura T, Yamamuro T, Kokubo T, Ebisawa Y, Kotoura Y, et al. J Biomed Mater

Res 1991;25:357.

[60] Carlisle EM. Science 1970;167:179.

[61] Carlisle EMD. Calcif Tissue Int 1981;33:27.

[62] Ruys AJ. J Aust Ceram Soc 1993;29:71.

[63] Tanizawa Y, Suzuki T. J Chem Soc Faraday Trans 1995;91:3499–4350.

[64] Boyer L, Carpena J, Lacout JL. Solid State lonics 1997;95:121.

[65] Gibson IR, Best SM, Bonfield W. J Biomed Mater Res 1999;44:422.

[66] Kokubo T, Kushitani H, Sakka S, Kitsugi T, Yamamuro T. J Biomed Mater Res 1990;24:721.

[67] Marques PAAP, Magalhaes MCF, Correia RN, Vallet-Regı M. Key Eng Mater 2001;247:192–5.

[68] Balas F, Perez-Pariente J, Vallet-Regı MJ. Biomed Mat Res 2003;66A:364.

[69] Arcos D, Rodriguez-Carvajal J, Vallet-Regı M. Physical B 2004;350:607.

[70] Coetzee AS. Arch Otolaryngol 1980;106:405.

[71] Lemaitre J, Mirtchi A, Munting E. Sil Ind Ceram Sci Technol 1987;52:141.

[72] Chow LC. J.Ceram Soc Jpn 1991;99:954.

[73] Sugama T, Allan M. J Am Ceram Soc 1992;75:2076.

[74] Mirtchi AA, Lemaitre J, Munting E. Biomaterials 1991;12:505.

[75] Otsuka M, Matsuda Y, Suwa Y, Fox JL, Higuchi W. J Biomed Mater Res 1995;29:25.

[76] Miyamoto Y, Ishikawa K, Takechi M, Toh T, Yuasa T, Nagayama M, et al. Biomaterials

1998;19:707.

[77] del Real RP, Wolke JCC, Vallet-Regı M, Jansen JA. Biomaterials 2002;23:3673.

[78] Nilsson M, Fernandez E, Sarda S, Lidgren L, Planell JA. J Biomed Mater Res 2002;61:600–7.

[79] Constanz BR, Ison IC, Fulmer MT, Fulmer RD, Poser RD, Smith ST, et al. Science

1995;267:1796.

[80] Takagi S, Chow LC, Ishikawa K. Biomaterials 1998;9:1593.

[81] Pietrzak WS, Ronk R. J Craniofac Surg 2001;11:327.

[82] Rawlings III CE, Wilkins RH, Hanker JS, Georgiade NG, Harrelson JM. J Neurosurg

1988;69:269.

[83] Sato S, Koshino T, Saito T. Biomaterials 1998;19:1895.

[84] Cabanas MV, Rodrıguez-Lorenzo LM, Vallet-Regı M. Chem Mater 2002;14:3550.

[85] Yu D, Wong J, Matsuda Y, Fox JL, Higuchi WI, Otsuka M. J Pharm Sci 1992;81:529.

[86] Hamanishi C, Kitamoto K, Tanaka S, Osuka M, Doi Y, Kitahashi T. J Biomed Mater Res Appl

Biomater 1996;33:139.

[87] Mousset B, Benoit MA, Delloye C, Bouillet R, Guillard. Int Orthop 1997;21:403.

[88] Meseguer L, Ros MJ, Clavel M, Vicente V, Alcaraz M, Lax A, et al. J Biomed Mater Res

2002;61:458.

[89] Ratier A, Gibsn IR, Best SM, Freche M, Lacout JL, Rodrıguez F. Biomaterials 2001;22:897.

[90] Doadrio JC, Arcos D, Cabanas MV, Vallet-Regı M. Biomaterials 2004;25:2629.

[91] Daculsi G. Biomaterials 1998;19:1473.

[92] Drimandi G, Weiss P, Millot F, Daculsi G. J Biomed Mater Res 1998;39:660.

[93] Ragel CV, Vallet-Regı M, Rodriguez-Lorenzo LM. Biomaterials 2002;23:1865.

[94] Ramila A, Padilla S, Munoz B, Vallet-Regı M. Chem Mater 2002;14:2439.


[95] Tancred DC, McCormack BAO, Carr AJ. Biomaterials 1998;19:2303.

[96] Bouler JM, Trecant M, Delecrin J, Royer J, Passuti N, Daculsi GJ. J Biomed Mater Res

1996;32:603.

[97] Slosarczyk A, Piekarcyk J. Ceram Int 1999;25:561.

[98] Kivrak N, Cuneyt Tas A. J Am Ceram Soc 1998;82:2245.

[99] Petrov OE, Dyulgerova E, Petrov L, Ropova R. Mater Lett 2001;48:162.

[100] Yang X, Wang Z. J Mater Chem 1998;8:2233.

[101] Lin FH, Liao CJ, Chen KS, Sun JS, Lin CY. J Biomed Mater Res 2000;51:157.

[102] Itatani K, Nishioka T, Seike S, Howell FS, Kishiota A, Kinoshita M. J Am Ceram Soc

1994;77:801.

[103] Manjubala I, Sivakimar M. Mater Chem Phys 2001;71:272.

[104] Cunneyt Tas A. J Eur Ceram Soc 2000;20:2389.

[105] Gauthier O, Bouler JM, Aguado E, LeGeros RZ, Pilet P, Daculsi G. J Mater Sci Mater Med

1999;10:199.

[106] Aoki H, Kato K, Ogiso M, Tabata T. J Dent Outlook 1997;49:567.

[107] Rodrıguez-Lorenzo LM, Vallet-Regı M, Ferreira JMF. Biomaterials 2001;22:583.

[108] Rodrıguez-Lorenzo LM, Vallet-Regı M, Ferreira JMF. Biomaterials 2001;22:1847.

[109] Wan ACA, Khor G, Hasting GW. J Biomed Mater Res 1998;41:541.

[110] Du C, Cui FZ, Feng QL, Zhu XD, de Groot K. J Biomed Mater Res 1999;44:407.

[111] Tadic D, Beckmann F, Schwarz K, Epple M. Biomaterials 2004;25:3335.

[112] Rodrıguez-Lorenzo LM, Vallet-Regı M, Ferreira JMF. J Biomed Mater Res 2002;60:232.

[113] Padilla S, Roman J, Vallet-Regı M. J Mater Sci: Mater Med 2002;13:1193.

[114] Dupraz A, Nguyen TP, Richard M, Daculsi G, Passuti N. Biomaterials 1999;20:663.

[115] Pena J, Vallet-Regı M, San Roman J. J Biomed Mat Res 1997;35:129.

[116] Rodriguez-Lorenzo LM, Salinas AJ, Vallet-Regı M, San Roman J. J Biomed Mat Res

1996;30:515.

[117] Wang M, Kokubo T, Bonfield W. In Kokubo T, Nakamura T, Miyaji F, editors. Bioceramics,

vol. 9. Japan; 1996. p. 387.

[118] Kikuchi M, Sato K, Suetsugu Y, Tanaka J. In: LeGeros RZ, LeGeros JP, editors. Bioceramics,

vol. 11. NY: World Scientific; 1998, p. 153.

[119] Bakos D, Soldan M, Hernandez-Fuentes I. Biomaterials 1999;20:191.

[120] Cherng A, Takagi S, Chow LC. J Biomed Mater 1997;35:273.

[121] Constantino PD, Friedman CD. Clin North Am 1996;27:1037.

[122] de Groot K, Gessink R, Klein CPAT, Serekain P. J Biomed Mater Res 1987;21:1375.

[123] Park E, Condrate RA, Hoelzer DT, Fischman GS. J Mat Sci Mater Med 1998;9:643.

[124] MacDonal DE, Betts F, Stranick M, Doty S, Boskey AL. J Biomed Mater Res 2001;54:480.

[125] Van Dijk K, Schaehen HG, Wolke JGC, Jansen JA. Biomaterials 1996;17:405.

[126] Zeng HT, Lacefield WR. Biomaterials 2000;21:23.

[127] Cui FZ, Lou ZS, Feng QL. J Mat Sci Mater Med 1997;8:403.

[128] Spoto G, Ciliberto E, Allen GC. J Mater Chem 1994;4:1849.

[129] Cao Y, Weng J, Chen J, Fen J, Yang Z, Zhang X. Biomaterials 1996;17:419.

[130] Chou L, Marek B, Wagner WR. Biomaterials 1999;20:977.

[131] Labeau M, Gautheron B, Cellier F, Vallet-Regı M, Garcıa E, Gonzalez-Calbet JM. J Solid State

Chem 1993;102:434.

[132] Martınez A, Pena J, Labeau M, Gonzalez-Calbet JM, Vallet-Regı M. J Mater Res 1995;10:1307.

[133] Patil PS. Mater Chem Phys 1999;59:185.

[134] Barrere F, Layrolle P, Van Blitterswijk CA, de Groot K, Laydolle P. J Am Ceram Soc

2002;85:517.

[135] Choi J, Bogdanski D, Koller M, Esenwein SA, Muller D, Muhr G, et al. Biomaterials

2003;24:3689.

[136] Ozawa N, Ideta Y, Yao T, Kokubo T. Bioceramics 2003;15:71.

[137] Vallet-Regı M, Izquierdo-Barba I, Gil FJ. J Biomed Mater Res 2003;67:674.

[138] Hijon N, Cabanas MV, Izquierdo-Barba I, Vallet-Regı M. Chem Mater 2004;16:1451.


[139] Cabanas MV, Vallet-Regı MJ. Mater Chem 2003;13:1104.

[140] Lin S, LeGeros RZ, LeGeros JP. J Biomed Mater Res 2003;66:819.

[141] Cotell CM. Appl Surface Sci 1993;69:140.

[142] Suchanek W, Yoshimur MJ. J Mater Res 1998;13:94.

[143] Tkalcec E, Sauer M, Nonninger R, Schmidt H. J Mater Sci 2001;36:5253.

[144] Layrolle P, Lebugle A. Chem Mater 1994;6:1996.

[145] Jillavenkatesa A, Condrate Sr RA. J Mater Sci 1998;33:4111.

[146] Ben-Nissan B, Green DD, Kannangara GSK, Chai CS, Mile A. J Sol–Gel Sci Tech 2001;21:27.

[147] Bezzi A, Celotti G, Landi E, La Torretta TMG, Sopyan I, Tampieri A. Mater Chem Phys

2003;78:816.

[148] Livage J, Barboux P, Vandenborre MT, Schmutz C, Taulelle F. J Non Cryst Solids 1992;147–

148:18.

[149] Chai CS, Gross KA, Ben-Nissan B. Biomaterials 1998;19:2291.

[150] Hsieh M, Perng L, Chin T, Perng H. Biomaterials 2001;22:2601.

[151] Liu D, Troczynski T, Tseng WJ. Biomaterials 2002;23:1227.

[152] Hijon N, Cabanas MV, Izquierdo-Barba I, Vallet-Regı M. Key Eng Mater 2004;254–256:363.

[153] Vallet-Regı M. Perspective article. J Chem Soc Dalton Trans 2001;2:97.

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