Fisher John P. e.a. (ed.) Tissue Engineering

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Calcium Phosphate

Ceramics for Bone

Tissue Engineering

P. Quinten Ruhé

Joop G.C. Wolke

Paul H.M. Spauwen

John A. Jansen

University Medical Center

St. Radboud

9.1 Introduction.............................................. 9-1

9.2 Chemico-Physical Properties of Calcium Phosphate

Ceramics ................................................. 9-2

Crystallinity • Sintering • Stoichiometry • Strength •

Porosity

9.3 Ca-PProducts............................................ 9-5

Natural Ca-P Ceramics • Injectable Ca-P Cements

9.4 In Vivo Interactionsand Osteoinductivity ............ 9-7

9.5 Calcium Phosphate Ceramics for Bone Tissue

Engineering .............................................. 9-8

Ca-P Ceramics and Osteogenic Cells • Ca-P Ceramics and

Osteoinductive Growth Factors

9.6 Conclusion and Future Perspective .................... 9-13

References ....................................................... 9-13

9.1 Introduction

In reconstructive surgery, repair and regeneration of large bone defects is a major challenge. The use of

autologous bone is still the gold standard, although concomitant problems as donor site morbidity and

limited supply have resulted in worldwide endeavors for the development of bone graft substitutes. Each

potential substitute, including tissue engineering approaches with delivery of osteogenic cells or osteoin-

ductive macromolecules, or both is based on an appropriate scaffold biomaterial that is biocompatible,

allows bone ingrowth and shows subsequent degradation of the material. In view of this, a biomaterial

used for tissue replacement shows by preference a resemblance with the inorganic or organic components

of the tissue, or both, to be substituted.

Bone tissue is a living organ composed of an organic and an inorganic component. The organic

substance (approx.

of total dry bone weight) consists of more than 90% of collagen and only for

a small fraction of cells (2%) and noncollagenous proteins (5%). The inorganic bone mineral (approx.

of total dry bone weight) is composed of speciﬁc phases of calcium phosphate (Ca-P), especially

carbonate rich hydroxyapatite (HA). In biologically carbonated HA, PO

is substituted by CO

(so-called

type B carbonation). Biological HA also contains other impurity ions as Cl, Mg, Na, K, and F and

9-1

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9-2 Tissue Engineering

2500

Intensity (counts)

2000

1500

1000

500

bTCP_1200_CAM

HA_1200_CAM

Bone

15 20 25 30 35 40 45 50 55

2u (°)

FIGURE 9.1 X-ray diffraction (XRD) pattern of bone, HA and β-tricalcium phosphate, clearly indicating the

similarity between HA and bone.

trace elements like Sr and Zn [1]. The apatite in bone mineral is composed of small platelet-like crystals

of just 2 to 4 nm in thickness, 25 nm in width, and 50 nm in length [2].

Together with the — organic — collagenous materials, Ca-P materials are among the few biomaterials

that show high similarity to natural tissue. Ca-P biomaterials resemble the mineral phase of bone to a

higher or lesser extent, depending or their stoichiometry and crystallinity (Figure 9.1). In general, Ca-P

biomaterials are crystalline ceramics characterized by a high biocompatibility, the ability of direct bone

bonding and osteoconduction, and a variable resorbability. Since the early seventies, Ca-P ceramics are

clinically used in dentistry, followed by surgical specialties in the eighties. The various available Ca-P

materials differ in their origin (naturally derived or synthetic), chemico-physical composition (calcium

to phosphate ratio, crystallinity, [micro]porosity) and preparation process (prefabricated blocks and

granules, coatings on other biomaterials, self setting cements and composite materials). In this review, the

essential Ca-P characteristics, differences between various Ca-P biomaterials, and proceedings in Ca-P

ceramic bone tissue engineering research will be discussed.

9.2 Chemico-Physical Properties of Calcium Phosphate

Ceramics

Calcium phosphate (Ca-P) compounds exist in several phases. Discern can be made regarding (1). The

crystallinity of various compounds (amorphous vs. various crystalline Ca-P phases), (2). eventual heat

treatment of the materials (sintering), and (3). the calcium to phosphate ratio.

9.2.1 Crystallinity

Amorphous Ca-P (ACP) is the ﬁrst solid phase to appear in solution containing high concentrations

of calcium and phosphate. ACP lacks the orderly internal structure of crystalline Ca-P compounds and

typically has a spherical morphology [3]. Chemically, it has a Ca : P ratio of around 1.5 and is characterized

by the absence of diffraction peaks on x-ray diffraction (XRD) patterns. ACP is unstable in aqueous ﬂuids

and transforms into crystalline phases such as octacalciumphosphate (OCP) and apatite. Heating of ACP

also results in conversion to poorly crystallized apatite (600

◦

C), β-TCP (800

◦

C, dry heat), or HA (800

◦

humid heat) [4]. Therefore, ACP should be considered as a precursor for crystalline Ca-P compounds.

There has been, and still is, considerable debate whether or not ACP is substantially present in skeletal

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Calcium Phosphate Ceramics for Bone Tissue Engineering 9-3

tissue. For example, Transmission Electron Microscope (TEM) analysis has never revealed conclusive

evidence for the presence of ACP in bone mineral [3].

Other phases of Ca-P than ACP reveal a crystalline structure with characteristic peaks on XRD analysis.

There is a broad range in crystal morphology depending on composition and preparation characteristics

such as temperature, pH, impurity, and the presence of macromolecules. Impurities, as commonly occur

in bone mineral, greatly inﬂuence crystallinity (reﬂecting crystal size and crystal strain) but depend on

the type of substitution. For example, type B carbonated apatite (CO

for PO

substitution) has a lower

crystallinity and increased solubility, whereas F substitution (F for OH) give the opposite effects due to a

betterﬁtoftheF

−

ion in the apatite crystal structure.

9.2.2 Sintering

Sintering is the key step in processing of the majority of ceramic materials and conventionally consists of

two separate phases: compacting the initial powder and heating the compacted powder up to temperatures

only a little lower than their melting points. Thereby, atoms and molecules are set in rapid motion, and

the particles coalesce. Fusion of the crystals reduces the porosity and increases the strength and density

of the ﬁnal ceramic product. Currently, several sintering techniques are available, such as continuous

hot pressing, microwave sintering, pressureless sintering, and plasma sintering. Obviously, chemical

composition of initial powders as well as variation of pressure and temperature inﬂuence ﬁnal structure

and composition of the sintered product.

9.2.3 Stoichiometry

The quantitative relationship (stoichiometry) of calcium to phosphate in Ca-P salts is essential for sev-

eral material characteristics as strength, solubility, and crystal structure. Table 9.1 lists the main Ca-P

compounds for biomedical applications. From a crystallographic point of view, the most stable form of

Ca-P has a Ca : P ratio of 1.67 (hydroxyapatite). With a decrease of the Ca to P ratio, other Ca-deﬁcient

phases occur like tricalcium phosphate (TCP, Ca : P ratio 1.50), octacalcium phosphate (OCP, Ca : P ratio

1.33), dicalcium phosphate anhydrous (DCPA or Monetite, Ca : P ratio 1.00), and dicalcium phosphate

dihydrate (DCPD or Brushite, Ca : P ratio 1.00). Most of these calcium deﬁcient compounds are used as

raw material for sintering procedures for ceramics, or as ingredients for example, Ca-P cements. TCP and

HA will be discussed more in detail, since these materials are most used for biomedical applications.

9.2.3.1 TCP (Ca

(PO

)

Tricalcium phosphate (TCP) has a Ca : P ratio of 1.5, similar to the amorphous biologic precursors of bone

[5]. It can be prepared by sintering Ca deﬁcient apatite (Ca : P ratio 1.5). TCP is a polymorph ceramic and

exhibits two phases (α- and β-whitlockite), known as α- and β-TCP. Variation in sintering temperature and

humidity determine, which phase is being formed; α-TCP occurs at dry heat >1300

◦

C and subsequent

quenching in water [4]. Solubility and resorbability of both forms is much higher compared to HA.

However, α-TCP is unstable in water and reacts to produce HA. α-TCP is used mainly as a compound

TABLE 9.1 Ca-P Compounds, Names, Formulas, and Ca : P Ratios

Ca/P Formula Name/mineral Abbreviation

0.5 Ca(H

)

·H

O Monocalcium phosphate monohydrate MCPM

1.0 CaHPO

·2H

O Hydrated calcium phosphate/brushite DCP

1.0 CaHPO

Anhydrous calcium phosphate/Monetite ADCP

1.33 Ca

(PO

)

·5H

O Octacalcium phosphate OCP

1.5 Ca

(PO

)

Tricalcium phosphate/whitlockite TCP

1.67 Ca

(PO

)

(F)

Fluorapatite FA

1.67 Ca

(PO

)

(OH)

Hydroxylapatite HA

2.0 CaO · Ca

(PO

)

Tetracalcium phospate/hilgenstockite TTCP

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9-4 Tissue Engineering

of some Ca-P cements. β-TCP is less soluble and less reactive than α-TCP. It is used pure as bone substitute,

and in combination with HA (biphasic Ca-P ceramic). Due to its solubility, dissolution and reprecipit-

ation occur in vivo. This results in gradual phase transition into carbonated apatite, and resorption by

macrophages, giant cells, and osteoclasts.

9.2.3.2 Hydroxyapatite (Ca

(PO

)

(OH)

)

Hydroxyapatite (HA) is the term commonly used for calcium hydroxyapatite Ca

(PO

)

(OH)

, the most

stable form of Ca-P under physiological pH and body temperature. With ﬂuorapatite and chlorapatite,

hydroxyapatite forms the group of apatite minerals that share a similar hexagonal monoclinic crystal

structure and the general formula A

(PO

)

(OH,F,Cl)

. The A cation can be several metal ions besides

calcium, such as barium, strontium, and magnesium. The phosphate anion can be substituted by a

carbonate anion to a limited extent.

HA has a Ca : P ratio of 1.67 which is similar to bone mineral. It can be prepared by sintering of

precipitated Ca-P salts in a Ca : P ratio of 1.67 at temperatures above 1000

◦

C. Pure HA is hardly soluble

under physiological conditions. Impurities like substitution of Ca

by other metal ions cause variation

in solubility and crystal size due to the differences in ionic radius. With respect to mechanical strength,

pure HA materials are superior to other Ca-P materials. In vivo, however, pure HA hardly shows any

cellular resorption by macrophages, giant cells or osteoclasts unless the particle size is small enough for

phagocytosis. As a consequence, HA should be considered as nonresorbable whereas other compositions

such as β-TCP show substantial dissolution and resorption.

9.2.4 Strength

Biomechanical properties are a considerable concern in the use of Ca-P ceramics. Compressive strength of

cortical and trabecular bone varies, depending on the bone density, from 130 to 180 MPa and 5 to 50 MPa

respectively. For tensile strength these values ﬂuctuate from 60 to 160 MPa and 3 to 15 MPa respect-

ively. Dense sintered Ca-P ceramics may reach compressive strengths much higher than cortical bone

(300–900 MPa), whereas tensile strengths similar and higher than cortical bone (40–300 MPa) have been

reported [6]. Problem, however, is that ceramics do not exhibit substantial elastic or plastic deformation

before fracturing. Due to the lack of ductility, virtually all Ca-P ceramics are brittle. The brittleness can be

explained by the atomic structure of ceramics. Elasticity is manifested as small reversible changes in the

interatomic spacing and stretching of interatomic bonds. Plastic deformation corresponds to breaking of

existing bonds and reforming of bonds with new neighboring atoms. In crystalline materials, this occurs

in planes by means of atomic slip. As a consequence of the electrically charged nature of the ions, both

elastic and plastic deformations in ceramics are limited. The brittleness of ceramics is further enhanced

by very small and omnipresent ﬂaws in the material. These microcracks result in local ampliﬁcation of

applied tensile stresses and cause a relatively low fracture strength.

Due to the inferior biomechanical properties, Ca-P ceramics are less suitable for clinical application

under weight-bearing conditions compared to, for example, metallic or polymeric biomaterials. Obvi-

ously, mechanical properties of porous ceramics deteriorate even further with an increasing porosity.

Nevertheless, compressive strengths similar to trabecular bone have been reported [7].

9.2.5 Porosity

Cortical bone has pores ranging from 1 to 100 µm (volumetric porosity 5 to 10%), whereas trabecular

bone has pores of 200 to 400 µm (volumetric porosity 70 to 90%). Porosity in bone provides space for

nutrients supply in cortical bone and marrow cavity in trabecular bone. As mentioned in the previous

paragraph, porosity is devastating for mechanical properties of Ca-P ceramics. On the other hand, it

is known that porosity enhances degradation of Ca-P ceramics and determines the nature of ingrow-

ing tissue. Consequently, this delicate equilibrium depends on more factors than mechanical material

properties alone, that is, application site (vascularization, weight loading, defect dimensions), biological

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Calcium Phosphate Ceramics for Bone Tissue Engineering 9-5

material properties (biocompatibility, osteoconductivity, and degradation) and other aspects as pore

geometry and the use of tissue engineering strategies (addition of osteogenic cells or bioactive factors).

Therefore, no general optimal pore size and architecture for all applications and biomaterials can be

given.

9.2.5.1 Microporosity

Microporosity covers pores sizes smaller than — an arbitrary — 5 µm: too small for penetration an

ingrowth of cells, but sufﬁcient for penetration of ﬂuids. Crystalline Ca-P materials intrinsically exhibit,

depending on crystal size and structure, a nano- or microporous structure. However, microporosity of

ceramics is highly decreased or virtually absent due to high pressure compaction and (partial) fusion of

crystals after sintering. Microporous Ca-P structures have an increased surface area, which inﬂuences

the biological behavior and the Ca-P dissolution/precipitation characteristics. It has even been reported

that microstructure plays a crucial role in osteoinductive properties of ceramics. Yuan et al. [8] observed

heterotopic bone formation in macro- and microporous ceramics implanted in the dorsal muscle in dogs,

whereas similar implants without microporous structure did not reveal bone formation. They suggested

that the increased surface area, resultant of the microporous structure, could have caused accumulation

of adsorbed proteins (e.g., BMPs) which could have triggered osteogenesis.

9.2.5.2 Macroporosity

Pores larger than 10 µm can be considered as macropores. Various methods can be used to induce

macroporosity in Ca-P material. For example, porous ceramics can be obtained by merging the Ca-P slurry

with a polymer sponge-like mold or polymer beads before sintering. During the sintering, the polymer

is completely burnt out, which results in a porous ceramic structure. In this way, architecture of porous

ceramics can be controlled and a completely interconnected structure with any desirable pore size and

pore geometry is feasible. Obviously, interconnection of pores is essential for tissue growth throughout

the scaffold. Tsuruga and Kuboki have investigated the inﬂuences of pore size and geometry in induced

bone formation in ceramics. They reported that for porous sintered HA blocks with pore sizes ranging

from 106–212 µm to 500–600 µm, the highest amount of bone was produced in implants with pore sizes

of 300 to 400 µm [9]. In another study, Kuboki et al. [10] observed that an optimal vascular ingrowth was

achieved in pores with a diameter of 350 µm, and claimed that this was the key factor for effective bone

formation. Macroporous dimensions are also reported to play a role in osteoinductive behavior of Ca-P

ceramics. In a series of experiments in primates, Ripamonti et al. [11,12] reported that implant geometry

is of critical importance for cell shape, cell locomotion and cell differentiation. They hypothesized that

concavities in sintered HA mediate intrinsic osteoinduction.

9.3 Ca-P Products

Numerous Ca-P products are currently marketed for bone regenerative purposes. Most frequently used

Ca-P products in dentistry and surgery are Ca-P coatings applied to prostheses. Several techniques, such

as plasma spraying and RF magnetron sputtering can provide a thin bio-active Ca-P coating on the inert

metal prosthesis surface [13]. Consequently, the superior mechanical metal properties can be combined

with favorable bone bonding Ca-P properties. Coatings applied usually consist of HA to prevent resorption

of the coating in time.

Other Ca-P products are marketed as bone ﬁller and are available in various forms as granules, blocks,

and injectable cements. Both granules and blocks are available with a dense or porous structure and

several resorption rates. Most synthetic products are composed of β-TCP, HA or a combination of both

(so called bi-phasic ceramics [Figure 9.2]). Besides synthetic ceramics, several naturally derived ceramics

are commercially available. These and injectable Ca-P cements are being discussed in detail in the next

paragraphs.

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9-6 Tissue Engineering

FIGURE 9.2 µCT representation of a highly porous biphasic ceramic implant material (Camceram®, Cam Implants,

Leiden, The Netherlands) with a HA : TCP ratio of 60 : 40, porosity of 90%, and macropores ranging from 300 to

500 µm. Bar represents 1 mm.

9.3.1 Natural Ca-P Ceramics

Natural Ca-P biomaterials can be prepared from the inorganic bone mineral matrix of bone. Chemical,

or high temperature heat treatment, or both, eliminate organic substances responsible for immunologic

response and potential disease transmission (e.g., prions). Chemically treated materials derived from

human spongiosa (Puros Tutoplast®, Zimmer Dental, Carslbad, CA, USA) or bovine spongiosa (Ortoss®

and Bio-oss®, Geistlich Biomaterials, Geistlich, Switzerland) maintain the macroporous structure of

spongious bone as well as the natural small carbonatehydroxyapatite crystal structure. Sintered bovine

spongious bone products, such as Osteograf®-N (Dentsply Ceramed, Denver Co, USA) and Endobon®

(Biomet Merck, Darmstadt, Germany), also maintain its macroporous structure, but due to the high

sintering temperatures (>1100

◦

C) the natural carbonate HA crystals convert into larger HA crystals

without CO

, resembling synthetic HA.

Besides bovine origin, natural Ca-P biomaterials can also be derived from special species of coral

(genus Porites and Goniopora). Hydrothermal “replamiform” treatment (260

◦

C; 15.000PSI) of the

calcium carbonate (calcite) exoskeletal microstructure of these corals results in conversion into hydroxy-

apatite [14]. Like natural bone, this hydroxyapatite contains minor elements of Mg, Sr, F, and CO

The porous coralline hydroxyapatite is completely interconnected and available in various porosities

(Interpore

, Pro Osteon

, Interpore Cross International Inc, Irvine, CA, USA). Different hydro-

thermal treatment of coral used for Pro Osteon-R results in only partial conversion of the calcium

carbonate to HA [5,15]. As a result, the composite of HA/CaCO

is being resorbed faster than pure

HA. Another coral derived bone substitute, which has not been converted into HA at all and therefore

cannot be considered as a Ca-P ceramic, shows even faster degradation. This CaCO

material (Biocoral®,

Inoteb, St. Gonnery, France) currently does not have FDA approval, but is available for clinical use in

Europe.

9.3.2 Injectable Ca-P Cements

Difﬁculties with the clinical applicability of preformed ceramic blocks and granules have led to the

development of injectable ceramic bone graft substitutes. In the early eighties, Brown and Chow were

mikos: “9026_c009” — 2007/4/9 — 15:51 — page7—#7

Calcium Phosphate Ceramics for Bone Tissue Engineering 9-7

the ﬁrst to describe the principles of a self setting Ca-P paste [16,17]. Currently, several different

Ca-P cements are commercially available like BoneSource® (Stryker Leibinger, Kalamazoo, MI, USA),

Norian® (Synthes, Oberdorf, Switzerland), α-BSM/Biobon® (Etex Corporation, Cambridge, MA, USA),

and Calcibon® (Biomet Merck, Darmstadt, Germany). Each cement has a (slightly) different chemical

composition, which results in differences in handling properties, setting time, strength and resorbabil-

ity [18]. The major advantage of Ca-P cements is the injectability of the cement paste. Obviously, this

results in favorable moldability of the bone graft, but also it results in immediate, seamless contact of

cement to the surrounding bone [19]. Moreover, Ca-P cements have been found to exhibit excellent bone

biocompatibility [20,21].

Roughly, the different Ca-P cements can be divided into four distinct groups [17]:

1. Cements composed of a solid Ca-P powder compound with an aqueous liquid component with or

without Ca or P ions. After mixing, hardening of the cement is the result of the formation of one

or more Ca-P compounds.

2. Cements with a powder component similar to those in (1), but an organic acid as liquid component.

Hardening of the cement is the result of complex formation of calcium and the organic acid.

3. Cements similar to those in (2), except that the liquid component is a polymer solution.

Cement hardening can be as described in (1) or complex formation of calcium and the polymer

solution or both.

4. Cement composites on a polymer basis. Hardening of the cement is based on polymerization of

the monomers. The Ca-P compounds present in these materials act as ﬁllers and do not play

a signiﬁcant role in the mechanism of cement hardening.

In this review, we will focus only on the ﬁrst group, as these cements currently play the most important

role in research and clinical applications. The chemico-physical reaction that occurs during and after

mixing of the powder and the liquid components is complex. After mixing, soluble compounds dis-

solve in the aqueous environment and subsequently precipitate, which results in formation and growth

of different forms of Ca-P crystals. This crystallization ﬁnally results in entanglement of the insoluble

Ca-P compounds and hardening of the cement. The setting time and phase transformation depend on

the solubility of the various solid compounds, as well as of external factors as pH and temperature

[22]. The setting reaction can be accelerated when the liquid phase contains phosphate ions. There-

fore, setting time is different for each Ca-P cement formulation and may vary from minutes to hours.

After setting, Ca-P cements may have an amorphous appearance under low magniﬁcation, but high

magniﬁcation (100,000×) reveals extremely small crystals. Crystal size and interparticle micropores

were observed to grow in time [17]. The ﬁnal result is a microporous structure of nanometer sized

crystals composed of more or less apatitic compounds, resembling the small crystal size of biological

apatite.

After setting of the cement, gradual phase transformation of the Ca-P compounds occurs into more

apatitic phases. Due to the nonsintered nature and the higher surface area of Ca-P cements, dissolution

of calcium and phosphate ions in physiological conditions occurs to a higher extent than in sintered

ceramics. However, the relatively large surface area in combination with supersaturated body ﬂuids also

results in high Ca-P precipitation. Ishikawa et al. [23] reported that the total precipitation of (B-type)

carbonate HA at the surface of Ca-P cement increased the total surface dissolution in simulated blood

plasma in vitro for a period of 20 weeks. In vivo, formation of carbonated HA on cement surface has

been reported to occur within 12 h of implantation [24]. This phenomenon could be one of the essential

aspects of the exceptionally positive osteoconductive behavior of Ca-P cements.

9.4 In Vivo Interactions and Osteoinductivity

In general, Ca-P compounds are found to be biocompatible and favorable for the ﬁnal bone response

[4]. The ability of Ca-P ceramics to bond to bone is a rather unique property. For example, it has been