Bone Regeneration Molecular and Cellular Interactions

International Journal of Nanomedicine
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Bone regeneration: molecular and cellular

interactions with calcium phosphate ceramics
Florence Barrère, Clemens A van Blitterswijk & Klaas de Groot
To cite this article: Florence Barrère, Clemens A van Blitterswijk & Klaas de Groot (2006)
Bone regeneration: molecular and cellular interactions with calcium phosphate ceramics,
International Journal of Nanomedicine, 1:3, 317-332, DOI: 10.2147/DIJN.1.S627
To link to this article: https://doi.org/10.2147/DIJN.1.S627
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review
Bone regeneration: molecular and cellular

interactions with calcium phosphate ceramics
Florence Barrère Abstract: Calcium phosphate bioceramics are widely used in orthopedic and dental applications
Clemens A van Blitterswijk and porous scaffolds made of them are serious candidates in the field of bone tissue engineering.
Klaas de Groot They have superior properties for the stimulation of bone formation and bone bonding, both
related to the specific interactions of their surface with the extracellular fluids and cells, ie, ionic
Biomedical Technological Institute
(BMTI), University of Twente, exchanges, superficial molecular rearrangement and cellular activity.
Enschede, The Netherlands Keywords: calcium phosphate, biomaterials, tissue engineering, osteoprogenitor cells,
osteoconduction, osteoinduction
Introduction
Calcium phosphate bioceramics have a unique characteristic for bone substitution
compared with other biomaterials. They have such compositional resemblance to bone
mineral that they induce a biological response similar to the one generated during
bone remodeling. Bone remodeling or renewal consists of the resorption of old bone
mineral coupled with the formation of new bone. During resorption, the degradation
products of calcium phosphate bioceramics (calcium and phosphate ions) are naturally
metabolized and they do not induce abnormal calcium or phosphate levels in urine,
serum, or organs (liver, skin, brain, heart, kidney, lung, and intestine) (den Hollander et
al 1991). Calcium phosphate biomaterials are successfully used in cranio-maxillofacial,
dental, and orthopedic surgery. They are of synthetic origin (obtained after aqueous
precipitation or after sintering) or natural origin (freeze-dried or banked bone and
derived coral hydroxyapatite), and they are used as bone fillers in the form of cement
or granules. As they cannot replace as such the load-bearing functions of bone because
of their lower mechanical properties, they are also successfully used as coatings on
metallic hip and dental implants in clinics (Epinette and Manley 2004). Naturally porous
calcium phosphates biomaterials have been selected as relevant scaffold candidates
in bone tissue engineering (Kruyt et al 2004; Arinzeh et al 2005). Technically, several
procedures have been developed to tailor the scaffolds, such as rapid prototyping
(Wilson et al 2004), phase mixing (Li et al 2002), use of porogenic agents (Barralet
et al 2002), or shape replication (Tancred et al 1998). In addition, by selecting and/or
Correspondence: Florence Barrère combining calcium phosphate phases, one can tailor the resorption kinetics, and also
Biomedical Technological Institute their stimulating effect on bone formation (Dhert et al 1991; Dhert et al 1993; Barrere
(BMTI), University of Twente,
Drienerlolaan 5, 7500 AE Enschede, et al 2003a; Rahbek et al 2004; Habibovic et al 2005b; Schopper et al 2005).
The Netherlands In this review, we address the molecular and cellular, interactions that take place
Tel +31 302 295111
Fax +31 302 280255
at the calcium phosphate surfaces and we correlate them with their relevance in bone
Email [email protected] regeneration.
International Journal of Nanomedicine 2006:1(3) 317–332 317

© 2006 Dove Medical Press Limited. All rights reserved
Barrère et al
Bone These values are within the same magnitude as for aluminum
Bone is the component of the skeletal system that is involved or mild steel, but bone is much lighter. The great strength
in body protection, support, and motion. Bone is a protection of bone exists principally along its axis and hence roughly
and production site for specialized tissues such as the parallel both to the collagen fiber axis and to the long axis
blood-forming system, ie, bone marrow. Heart, lungs, and of mineral crystals. Although apparently stiff, bones exhibit
other organs and structures in the chest are protected by the a considerable degree of elasticity, which is important in the
rib cage. The function of these organs involving motion, skeleton’s ability to withstand impact. Estimates of modulus
expansion, and contraction requires flexibility and elasticity of elasticity of bone samples are of the order of 420 to
of the protective rib cage. Bone supports structurally the 700 kg/cm2, values very much less than steel, for example,
indicating much greater elasticity of bone.
mechanical action of soft tissues, like the contraction of
muscles or expansion of lungs. Finally, it is a mineral
reservoir, whereby endocrine systems regulate the level of Bone renewal
calcium and phosphate ions in the circulating body fluids. Bone is a dynamic tissue. In the first year of life, the rate of
turnover of the skeleton approaches 100% per year. This rate
declines to about 10% per year in late childhood, and then
Bone structure
usually continues at approximately this rate or more slowly
Bone macro- and microscopic structures are affected by
throughout life, up to hundred years. After the completion
genetic, metabolic, and mechanical factors. These intrinsic
of skeletal growth, bone turnover results primarily from
factors are the main cause of bone macrostructural diversity.
remodeling: a coordinated cycle of tissue resorption and
For example, broad, flat plates, such as the scapula, anchor
formation over extensive regions of bone and prolonged
large muscle masses, whereas hollow and thick-walled tubes,
periods. Throughout life, physiological remodeling, removal,
such as the femur or radius, support weight. All bone consists
and replacement of bone, at roughly the same location, occur
of a basic double structure, the importance of which varies
without affecting the shape or density of the bone, through
with the function. An external layer, or cortex, covers the
a sequence of events that include (i) osteoclasts activation,
bone; it is smooth, dense, and continuous. In the interior,
(ii) resorption of bone, (iii) osteoblasts activation, and (iv)
cancellous bone is arranged in a network of intersecting plates
formation of new bone at the site of resorption (Buckwalter
and spicules varying in amount and enclosing spaces. These
et al 1996). Owing to these remodeling properties, defects
cavities are filled with blood vessels and marrow, either red, and fractures are easily repaired up to sizes called critical
hematopoietic or yellow, adipose, its character varying with defects, defined as defects of a size that will not heal during
age and site. the lifetime of the animal (Schmitz and Hollinger 1986). For
Microscopically, bone is a highly complex and specialized larger defects, human interventions are necessary in order to
form of connective tissue. It is a mineralized tissue, which help or stimulate the healing.
is composed of an organic matrix strengthened by deposits
of calcium phosphate crystals; in other words bone is a
Bone mineral
natural composite material. The organic matrix is composed
Bone mineral, or enamel (biological apatite) have structure
of collagen type I fibers (approximately 95%) and of
close to a type AB carbonated calcium phosphate apatitic
proteoglycans and numerous non-collagenous proteins (5%).
structure more or less deficient, which can be tentatively
This organic matrix, calcified by calcium phosphate minerals,
represented by the formula (Rey 1990)
embeds bone cells, which participate in the maintenance Ca8.3 1.7(PO4)4.3(CO3)1(HPO4)0.7(OH,CO3)0.3 1.7
and organization of bone, namely osteoprogenitor cells, = vacancy
osteoblasts, osteocytes, and osteoclasts.
However, bone mineral apatite contains non-apatitic
Mechanical properties of bone carbonate and phosphate groups, which are, structurally and
The intimate blend of hard inorganic and resilient organic physically, unstable and very reactive. This high reactivity
components results in excellent mechanical properties. For provides certain physicochemical, biological, functional, and
example, compact bone specimens have been found to have chemical features important in the formation and dissolution
tensile strength in the range of 700 to 1400 kg/cm2, and of the crystals in biological tissues. Furthermore, bone
compressive strength in the range of 1400 to 2100 kg/cm2. contains minor or trace elements, which are not indicated
318 International Journal of Nanomedicine 2006:1(3)

Bone regeneration
in the above formula, and which are difficult to attribute to Table 1 Main biologically relevant calcium phosphate salts
either the mineral phase or the organic matrix (Rey 1990). In Name Formula Abbreviation Ca/P
bone and other mineralized tissues, the mineral crystals are Dicalcium phosphate CaHPO4 DCPA 1.00
formed of thin plates of irregular shapes. Their sizes range anhydrate or monetite
in length from 20 Å for the smallest particles, to 1100 Å for Dicalcium phosphate CaHPO4.2H2O DCPD 1.00
dihydrate or brushite
the largest particles (Moradianoldak et al 1991; Kim et al
1995). These bone crystals expose a very large surface area Octacalcium phosphate Ca8(PO4)4(HPO4)2.5H2O OCP 1.33
to the extracellular fluids, which is critically important for Tricalcium phosphate Ca3(PO4)2 TCP 1.50
the rapid exchange of ions with these fluids. Hydroxyapatite Ca10(PO4)6(OH)2 HA 1.67
Bone mineral starts to nucleate into the holes and
pores present in the collagen fibrils (Glimcher 1987). This
heterogeneous nucleation is catalyzed by the presence phosphate compounds. Calcium phosphate salts vary by their
of phosphated esters groups (Glimcher et al 1984) and composition and their crystal structures, leading to specific
carboxylate groups (Rhee et al 2000) present in the collagen physicochemical properties.
fibrils. Thereafter, the growth, or mineralization, takes place
along the collagen fibrils, eventually interconnecting all of Apatites
the collagen fibrils. Stoichiometric hydroxyapatite (HA) belongs to the general
The nature of the primary mineral phase formed prior and wide apatitic group, represented by the formula:
to mature bone mineral apatite remains controversial. Me10(XO4)6Y2
According to Posner, bone mineral apatite derives from Where Me is a divalent metal (Ca2+, Sr2+, Ba2+, Pb2+ …),
calcium phosphate clusters (Ca9(PO4)6) packing randomly XO4 is a trivalent anion (PO43–, AsO43–, VO43– …), and Y is
with interfacial water to form amorphous calcium phosphate a monovalent anion (F– Cl–, Br–, I–, OH–…). Apatitic crystal
precursor (Posner 1985). This theory is supported by the structure has usually a hexagonal lattice, having a strong ability
presence of several calcium phosphate growth inhibitors such to form solid solutions, and to accept numerous substitutions.
as magnesium that stabilize the amorphous state. However, The structure of biological apatites, namely bone
dicalcium phosphate (DCPD) (Grynpas et al 1984; Roberts mineral, dentine, or tooth enamel, is far different from
et al 1992) and octacalcium phosphate (OCP) phases have stoichiometric HA because of numerous substitutions (i)
been also proposed as bone mineral precursors because by hydrogenophosphate (HPO42–) of XO4 groups and (ii)
of the partial similarities, especially between apatite and by carbonate (CO32–) of Y2 and XO4 groups. In addition,
OCP (Brown et al 1987). DCDP and OCP are kinetically Rey (1990) has shown that bone mineral apatites contains
favored compared with apatitic phases, supporting their non-apatitic carbonate and phosphate groups, which are,
role as precursors in bone apatite formation (Nancollas and structurally and physically, unstable and very reactive (Rey
Wu 2000). However, Kuhn et al (2000) have shown that the 1990). This high reactivity provides certain physicochemical,
apatitic phase seems to be the largely dominant one compared biological, functional, and chemical features important in
with other possible transient phases even at the earliest stages the formation and dissolution of the crystals in biological
of mineralization. tissues. The substitutions affect the apatitic lattice parameters:
the crystal size is decreased, and thereby the surface area
is increased compared with stoichiometric HA (Legeros
Biologically relevant calcium 1991). Biological apatites contain various trace elements
phosphates from intrinsic origins, for example, fluoride is present in
Synthetic calcium phosphates dental apatite and confers to enamel its low dissolution
Calcium phosphates, or more accurately calcium ortho properties to resist acidic attacks; or from extrinsic origins,
phosphates, are salts of orthophosphoric acid (H3PO4), and for example, water pollution is a straightforward intake of
thus can form compounds that contain H2PO4–, HPO32– or trace elements for bone due to its high hosting capacity
PO 43–. The calcium phosphate salts constitute a wide (Cazalbou et al 2004). In addition these trace elements present
group of compounds (Elliot 1994). Table 1 summarizes in extracellular fluids and in bone apatite may have a specific
the chemical name, the formula, the abbreviation, and role on bone quality and health. Table 2 presents the trace
the calcium to phosphorus ratio (Ca/P) of some calcium elements known to have an effect on bone.
International Journal of Nanomedicine 2006:1(3) 319

Barrère et al
Table 2 Selection of some trace elements having an effect on bone as such or in calcium phosphate biomaterials
Trace element Mechanism of action Evidences in combination with calcium
phosphates bioceramics
Copper Cross-linking of collagen and elastin; No
Increasing bone strength
(Hunt 1998; Lowe et al 2002)
Zinc Stimulating osteoblastic activity in vitro; Reducing bone loss at the zinc-containing biomaterial
Inhibiting bone resorption in vivo (Kawamura et al 2003)
(Lowe et al 2002)
Manganese Stimulating alkaline phosphatase activity No

in vitro (Leone et al 1995), and in vivo
(Pabbruwe et al 2004)
Fluoride Stimulating alkaline phosphatase activity Stabilizing bone bonding

Increasing of bone mass (Modrowski et al 1992) Reducing biomaterial’s resorption (Dhert et al 1991, 1993)
Strontium Increasing bone mass: stimulating bone formation No

and reducing bone resorption (Marie 2005)
Lithium Stimulating human mesenchymal stem cells proliferation No

(de Boer et al 2004)
Borate Possibly interacting with mineral and vitamin D metabolism No

(Hunt 1998)
Silicate Stimulating extracellular matrix formation and mineralization Stimulating bone remodeling (Porter et al 2004b)
(Carlisle 1988)
HA-based biomaterials have been until now the most Dicalcium phosphate dihydrate
abundant materials used in modern bone substitution DCPD crystals (CaHPO 4.2H2O) are monoclinic. There
(Epinette and Manley 2004). Clinically, apatitic biomaterials are four formulas per unit cell with an asymmetric unit
are widely used in the form sintered macroporous granules, CaHPO4.2H2O. DCPD crystals are one of the most soluble
cement in non-load-bearing applications, and in the form or of the calcium phosphate salts, and are the most stable at
coatings on metallic prostheses. More recently, carbonated pH=5.0. They can be the end product of brushite calcium
apatite biomaterials have been developed as their composition phosphate cement. However, in clinical applications, DCPD
and structure characterics are closer to those of bone mineral. crystals are used as an initial components for bone cements.
But their superiority compared with pure apatite is not yet
proven. Octacalcium phosphate
Octacalcium phosphate (OCP, Ca8H2(PO4)6.5H2O) crystals
Tricalcium phosphate are triclinic and they consist of alternating “apatite layers”
Two major distinct phases of anhydrous tricalcium phosphate (arrangement of calcium and phosphate groups similar to that
(TCP) crystals exist: α-TCP and β-TCP phases. The α-TCP of apatite) and “hydrated layers”. These two layers are linked
crystallizes in the monoclinic space group, and β-TCP to each other by Van der Walls and hydrogen bonds. OCP
crystallizes in the rhombohedral space group. Despite their often occurs as a transient intermediate in the precipitation
similar chemical composition, their different crystallographic of the thermodynamically more stable HA and biological
features confer different resorption features: α-TCP is more apatites. The close relationship between OCP and HA has
soluble than β-TCP, and it is obtained after heating the β-TCP been used to explain the incorporation (via hydrolysis) of
to more than 1170ºC. Clinically, α-TCP is a major reagent in impurities, particularly carbonate, magnesium, and sodium
the composition of cements as they hydrolyze into apatitic ions, and hence the non-stoichiometry of precipitated
structures, but it is also sold under the form of powder, blocks, apatites (Legeros 1991). OCP is biocompatible, resorbable,
or granules, like β-TCP. In pre-clinical studies, TCP coatings and osteoconductive in the form of compacted powder or in
on hip prostheses have been compared with HA coatings for the form of biomimetic coating (Barrere et al 1999, 2003a;
bone formation and bone fixation (Dhert et al 1991, 1993). Sasano et al 1999). In goat muscles, biomimetic OCP-coated

Bone regeneration
porous materials were found to be osteoinductive (Barrere calcium phosphate phases such as deficient apatite, OCP
et al 2003a). However these materials have not been used or DCPD sustain a condensation resulting in the formation
clinically. of pyrophosphates ions (P2O74–) at approximately 300°C
according to the reaction:
Amorphous calcium phosphates 2HPO42– → P2O74– + H2O
Amorphous calcium phosphates vary widely in composition Depending on the substitution mode, the thermal treatment
because of the possible insertion of several secondary ions. of carbonated apatite may lead to the release of CO2 gas with
They are characterized by the broad X-ray diffraction bumps, subsequent rearrangements.
and by infrared mono-component PO4 bands. The basic As a result of a thermal treatment, carbonated and/or
structure unit of amorphous calcium phosphates is a cluster deficient apatite, OCP, and DCPD will transform in a mixture
of ions comprising Ca9(PO4)6 packed with interfacial water of sub-products: pyrophosphate, HA, and TCP.
to form bigger entities. Amorphous calcium phosphates are
the most soluble calcium phosphate salts; they are used as Stability in physiological systems
initial components of some cement. The formation, dissolution, and transformation of calcium
phosphates depend on the nature of the calcium phosphate
Biphasic calcium phosphate body (particle size, crystallographic features, density) and the
Biphasic calcium phosphate (BCP) is a mixture of two nature of the solution (composition, pH, temperature).
different calcium phosphate phases: the sparingly soluble Most of calcium phosphates are sparingly soluble in
HA and highly soluble tricalcium phosphate at different water, and some are very insoluble, but all dissolve in
ratios. BCPs are obtained by sintering of deficient apatites acids. Their solubility, defined as the amount of dissolved
(Ca10-xMx(PO4)6-y(HPO4)y(OH)2, Ca/P<1.67) at or above solute contained in a saturated solution when particles of
700°C according to the following reaction (Legeros et al solute are continually passing into solution (dissolving)
2003): while other particles are returning to the solid solute phase
Ca10-xMx(PO4)6-y(HPO4)y(OH)2 → Ca10(PO4)6(OH)2 + (growth) at exactly the same rate (Wu and Nancollas 1998),
Ca3(PO4)2 decreases with the increase in temperature and in pH (de
The combination these two different calcium phosphate Groot 1983).
phases with different solubility in one biomaterial allows Each calcium phosphate phase possesses its own
its degradation kinetics to be tuned in vitro and in vivo. thermodynamical solubility. For example, at pH=7 and 37ºC,
Clinically, BCPs are used as bone fillers. HA is the most stable phase, followed by TCP, OCP, and
finally DCPD. However, these thermodynamical consider
Stability of calcium phosphates ations are under equilibrium conditions, and therefore they
Thermal stability do not take into account kinetics that dictates the formation
We present here a summary on the thermal transformations of one or the other phase under dynamic conditions.
behavior, extensively developed by Elliot (Elliot 1994). The second important factor in the stability of the calcium
phosphates is the characteristics of the solution in which these
TCP and HA are the only calcium phosphate salts fully salts are formed or placed, namely the solution supersatur
stable up to 1000°C. For TCP, up to 1125ºC, β-TCP is the ation in free calcium and phosphates ions (Tang et al 2001).
most stable phase, whereas above this and up to 1430ºC α- At a given pH and temperature, a free calcium and phosphate
TCP phase is the most stable. For stoichiometric HA, above ion containing solution can be categorized in three different
1000ºC, some hydroxyl groups may condense to produce states: (i) the stable (undersaturated) zone, where crystalliza
water following the reaction: tion is impossible, (ii) the metastable zone (supersaturated),
Ca10(PO4)6(OH)2 ↔ Ca10(PO4)6O + H2O where spontaneous crystallization of calcium phosphate salt
= vacancy is improbable, although the concentrations are higher than
the ones corresponding to the salt solubility. If a crystal seed
DCPD, OCP, carbonates, and/or deficient apatite are were placed in such a metastable solution, growth would
subjected to thermal transformations: occur on the seed; (iii) the unstable or labile (supersaturated)
The water-containing phases such as DCPD or OCP zone, where spontaneous crystallization of calcium
dehydrate at approximately 180°C. The HPO4 containing phosphate is probable, but not inevitable (Mullin 1993).

Barrère et al
Extracellular fluids that are supersaturated for calcium state of hydration of all tissues; (iii) the transcellular fluid
and phosphate may induce the nucleation and growth of new is a small compartment that represents all those body fluids
calcium phosphate crystals. which are formed from the transport activities of cells; (iv)
bone and dense connective tissues represent 15% of body
Biological milieus water (intracellular:extracellular, 55:45). This water is
In vivo, the interactions between the implant and its mobilized much more slowly than the other components of
“biological surrounding” are highly complex due to the the extracellular fluids. Extracellular fluids are composed of
non-equilibrium conditions and due to the undefined water-soluble molecules: organic (amino acids, sugars, fatty
amount of compounds playing a role in these interactions. acids, coenzymes, vitamins, hormones, neurotransmitters)
Dhert et al (1998) addressed the kinetic of the early events and inorganic (calcium, phosphates, potassium, carbonate,
taking place within the first month at the interface between sulfate, magnesium, chloride, sodium, copper, zinc)
calcium phosphate-coated implants in a press fit model, compounds as well as the waste products from the cellular
ie, implant and defect dimensions are equal, implicating a metabolism.
maximum contact between the hosting bone tissue and the
biomaterial. Regardless of the nature of the biomaterial, its The cellular components
biological surrounding evolves with time. In the first 3 days, As bone formation takes place in an organism during
blood will have invaded all of the empty spaces between the embryonic development, growth remodeling, and fracture
original bone and the implant. Thereafter, at the end of the healing, and after ectopic implantation of osteoinductive
first week of implantation, callus and mesenchymal tissues matrixes, a large reservoir of cells in the body is capable of
will have entirely replaced blood while host bone resorption osteogenesis throughout life (Aubin 2001). Although the
has started. Finally, between the second and fourth week of biomaterials are subjected to numerous cellular interactions
implantation, callus, mesenchymal tissues, and host bone of diverse origin in vivo, we will consider only the cells
will have gradually disappeared in favor of newly formed that are related most directly to bone formation and
bone while bone remodeling takes place (Dhert et al 1998). regeneration.
In a nutshell, components of biological fluids and cells will
interact with the biomaterial. The biomaterials features can The osteoblasts
affect the molecular and cellular interactions at their surface Osteoblasts are derived from pluripotential mesenchymal
and consequently can affect the process of bone formation. stem cells that have the capacity to differentiate into
various lineages. Mature osteoblasts are non-migratory,
The molecular components highly differentiated cells that can differ substantially in
The body can be divided into two major aqueous compart their properties depending on their stage of development.
ments: 2/3 of intracellular fluid within cells, and 1/3 of Their function and phenotype vary and four categories
extracellular fluid. The extracellular fluid is divided into of cells can be described. First, the active osteoblasts
smaller compartments that are distinguished by different are cuboidal in shape, mononuclear and they are rich in
locations and different kinetic characteristics: (i) the alkaline phosphatase activity. They synthesize and secrete
interstitial fluids lie in the interstices of all body tissues collagen type I and glycoproteins (osteopontin, osteocalcin),
that bathes all the cells in the body and is the link between cytokines, and growth factors into a region of unmineralized
the intracellular fluid and the intravascular compartment. matrix (osteoid) between the cell body and the mineralized
Oxygen, nutrients, wastes, and chemical messengers all pass matrix (Kartsogiannis and Ng 2004). Osteoblasts produce
through the interstitial fluid; (ii) the plasma is the only major also calcium phosphate minerals extra- and intracellularly
fluid compartment that exists as a real fluid collection all within vesicles (Annaz et al 2004b). Second, osteocytes are
in one location. It differs from interstitial fluid in its much mature osteoblasts which have become trapped within the
higher protein content and its high bulk flow (transport bone matrix and are responsible for its maintenance. Third,
function). Plasma is the liquid in which red blood cells and bone-lining cells are found along the bone surfaces that are
white blood cells are suspended. The water of the plasma undergoing neither bone formation nor resorption. Finally,
is freely exchangeable with that of body cells and other inactive osteoblasts are elongated cells, undistinguishable
extracellular fluids and is available to maintain the normal morphologically from the bone-lining cells.

Bone regeneration
The osteoclasts bone matrix macromolecules, namely alkaline phosphatase,

Osteoclasts are derived from hematopoietic stem cells that collagene type I, Cbfa1, bone sialoprotein, osteocalcin, and
differentiate along the monocyte–macrophage lineage. osteopontin (Sodek and Cheifetz 2001).
They are responsible for bone resorption by acidification
of bone mineral leading to its dissolution and by enzymatic Biological events at the calcium
degradation of demineralized extracellular bone matrix.
phosphate substrates
The mature osteoclast is a functionally polarized cell that
attaches via its apical pole to the mineralized bone matrix by
Ionic exchanges at calcium phosphate
forming a tight ring-like zone of adhesion, the sealing zone. surfaces
This attachment involves the specific interaction between the Physico-chemically, calcium phosphate surfaces sustain
cell membrane and some bone matrix proteins via integrins dissolution–reprecipitation cascades as the result of
(transmembrane adhesion proteins mediating cell–substratum exchanges at a solid–liquid interface in supersaturated
and cell–cell interactions). In the resorbing compartment, conditions. In biological systems, this physico-chemical
situated under the cell and delimited by the sealing zone, phenomenon is the result of a multi-component dynamic
osteoclasts generate a milieu acidification resulting in the process involving ions and proteins.
dissolution of bone mineral. This osteoclastic acidification In terms of surface reactivity, ionic transfers occur from
is mediated by the action of carbonate anhydrase that the solid phase to the aqueous liquid via surface hydration of
produces carbon dioxide, water, and protons that are extruded calcium, inorganic phosphate species, and possible impurities
across the cell membrane into the resorbing compartment like carbonate, fluoride, or chloride present in the biomaterial.
(Kartsogiannis and Ng 2004). Under physiological conditions, this dissolution process is
highly dependent on the nature of the calcium phosphate
The mesenchymal stem cells and osteoprogenitor substrate (Okazaki et al 1982; Dhert et al 1993; Ducheyne et
cells al 1993; Radin and Ducheyne 1993; Christoffersen et al 1997;
Adult mesenchymal stem cells can be isolated from bone Doi et al 1999; Barrere et al 2003b), and on the composition
marrow, adipose tissues, or amniotic membrane. Mesenchymal and supersaturation of the environment in vitro (Hyakuna
stem cells are, by definition, of self-renewal capacity and able et al 1990; Raynaud et al 1998; Tang et al 2001), or the
to repopulate all the appropriate differentiation lineages. They implantation site in vivo (Daculsi et al 1990; Barralet et al
are multipotent cells that can differentiate into osteoblastic, 2000). Ionic transfers occur also from the surrounding fluids
myoblastic, adipocytic, chondrocytic, endothelial, and to the calcium phosphate substrate in vitro and in vivo, as
neurogenic lineage through a multi-step differentiation illustrated by the formation of carbonated apatite nanocrystals
sequence as follows: proliferation, commitment, lineage as a result of surface transformation (Heughebaert et al 1988;
progression, maturation, and differentiation. For the Daculsi et al 1989, 1990; Johnsson et al 1991; de Bruijn et
osteogenic lineage, mesenchymal stem cells sustain al 1992a; Radin and Ducheyne 1993; Barrere et al 2003b).
a cascade of differentiation steps as described by the The presence of magnesium and carbonate contributes to the
following sequence: Mesenchymal stem cell → immature formation of a poorly crystallized carbonated apatite that has
osteoprogenitor → mature osteoprogenitor → preosteoblast similar features to the bone mineral phase (Furedi-Milhofer
→ mature osteoblast → osteocyte or lining cells or apoptosis. et al 1976; Legeros 1991; Elliot 1994). In the presence of
The later the differentiation stage, the lower the cell self- proteins, this newly formed mineral phase is also associated
renewal and proliferation capacity (Aubin 2001). In bone with organic compounds (Heughebaert et al 1988; de Bruijn
marrow, osteoprogenitor cells represent a very small et al 1995; Barrere et al 2003b). This phase transformation
percentage (eg <0.005%) of nucleated cell types in healthy occurs for all of the calcium phosphate bioceramics, even
adult bone (Block 2005). Embryonic stem cells are also a the stable apatitic structures, since they have a strong ability
potential source due to their pluripotentiallity and therefore to adapt to their environment by hosting foreign ions and
their ability to differentiate into osteogenic lineage. subsequently to sustain atomic rearrangements (Cazalbou
Osteoprogenitor–stem cell differentiation is controlled by et al 2004). However, crystalline hydroxyapatitic substrates
the “osteogenic mastergene” Cbfa1/Osf2 that intervenes in are often too stable to transform.
skeleton and tooth mineralization (Sodek and Cheifetz 2001). The result of these ionic exchanges favoring either phase
The differentiating osteoprogenitor cells express several transformation or dissolution follows the thermodynamical

Barrère et al
stability order, ie HA>TCP>OCP>DCPD from the least and pattern that can evolve with time (Kawasaki et al 2003).
soluble to the most soluble. This surface reactivity has pivotal These adsorbed proteins can thereafter influence the new
implications in the biological relevance of calcium phosphate formation of calcium phosphate crystals by blocking or
bioceramics and will be discussed further later in this review. not blocking the substrate’s nucleation sites (Koutsopoulos
However, for a given calcium phosphate phase, the crystalline and Dalas 2000; Combes and Rey 2002; Ofir et al 2004),
feature, eg, the presence of defaults in the crystal lattice irrespective of the protein’s isoelectric point (Ofir et al 2004).
or the decrease of crystal size, accelerates the dissolution. These results obtained from in vitro experiments differ
Eventually amorphous biomaterials dissolve faster than strongly from in vivo experiments, as hundreds of proteins
crystalline ones (Legeros 1991; de Bruijn et al 1992b; Barrere are present in biological fluids, and their global effect on
et al 2000). In addition, the presence of some additives of calcium phosphate reactivity is insufficiently understood. It
mineral origins within the calcium phosphate structure can is, however, clear that they play a significant role in the ionic
affect the crystal lattice, and therefore can accelerate the exchanges and their subsequent effect on their biological
dissolution, eg carbonate, silicate, or strontium in HA. On activity, since proteins are detected in close association
the other hand, some additives reduce in vitro and in vivo the with the nanocrystalline carbonated apatite formed on the
dissolution process, eg fluoride in HA, magnesium or zinc surface of calcium phosphate bioceramics in vitro and
in beta-TCP (Okazaki et al 1982; Legeros 1991; Dhert et al in vivo (Heughebaert et al 1988; Johnsson et al 1991; de
1993; Elliot 1994; Christoffersen et al 1997; Ito et al 2002; Bruijn et al 1992b; de Bruijn et al 1994b; Radin et al 1998;
Porter et al 2004a). The incorporation of proteins into calcium Barrere et al 2003b). Consequently, the nature, quantity, and
phosphate can also affect the dissolution kinetics (Liu et al conformation of these proteins at the biomaterial surface
2003). Last but not least, micro- and macroporosity play an will determine the cellular activity (El-Ghannam et al 1999;
important role in the physicochemical dissolution process of Rouahi et al 2006).
calcium phosphates. The larger the exposed surface to the
environment, the faster the biomaterial dissolves, simply Cellular interactions with calcium
because larger quantities of exchanges can take place (Radin phosphate surfaces
and Ducheyne 1994). In general, cell–biomaterial interactions depend on surface
The proteins from the surrounding fluids are also characteristics such as topography, chemistry, and surface
involved in the ionic exchange mechanisms as observed in physics. As mentioned above, surface characteristics
vitro. Their interaction with calcium phosphate substrates determine the ionic exchange dynamics and the protein
depend on the bioceramics characteristics (such as phase, adsorption. They also affect the cellular activity, namely their
crystallinity, composition, texture) (Sharpe et al 1997; attachment, proliferation, and differentiation.
El-Ghannam et al 1999; Rouahi et al 2006) and on the
protein’s features (such as conformation, isoelectric point, Calcium phosphates – osteoblasts and
composition), their concentration, and whether they act in osteoprogenitor cells
solution or on substrates (Johnsson et al 1991; Hunter et al In vitro, cell–biomaterial interactions are assayed by primary
1996; Koutsopoulos and Dalas 2000; Combes and Rey 2002; osteoblasts, osteosarcoma cell lines, and mesenchymal–
Ofir et al 2004). Firstly, in suspension, proteins can inhibit or osteoprogenitor cells issued from bone marrow. More and
support calcium phosphate nucleation and growth (Johnsson more research currently addresses the interaction between
et al 1991; Hunter et al 1996; Boskey and Paschalis 2001). mesenchymal stem–progenitor cells and scaffolds, as these
For phosphorylated proteins, such as collagen, ostepontin, cells participate at the early stage of new bone tissue formation
osteonectin, bone sialoprotein, or ostecalcin, phosphorylated in vivo (Dhert et al 1998; Davies and Hosseini 2001; Devlin
entities have demonstrated their ability to nucleate and grow and Sloan 2002). In contrast with differentiated osteoblasts,
calcium phosphate crystals (Boskey and Paschalis 2001). osteoprogenitor cells are migratory, highly proliferative
However, not all of the phosphorylated proteins induce cells and they have a greater differentiation potential. They
calcium phosphate formation; osteopontin especially is a can migrate on a substrate by generating cycles of weak
strong crystallization inhibitor (Hunter et al 1996) . Secondly, adhesion, traction, movement, and detachment. At the end
when proteins adsorb onto calcium phosphate substrates, their of the migration phase, like osteoblasts, mesenchymal–
charge, their concentration, and the presence of calcium in osteoprogenitor cells adhere to the substrate by developing
the surrounding fluids influence the surface-coverage kinetics strong focal adhesion with substrates in order to start

Bone regeneration
their differentiation phase. The migration and adhesion of et al 1992a, 1994b). As explained earlier the calcium and
bone cells in general are mediated via integrins which are phosphate concentrations of the environment may increase
transmembrane proteins (Anselme 2000). Among the integrin or decrease when calcium phosphate ceramics are immersed.
superfamily, fibronectin and vitronectin are proteins involved This level of modification induced by immersing calcium
in osteoblasts adhesion on biomaterials in vitro (El-Ghannam phosphate ceramics can even induce cell death (Hyakuna
et al 1999; Anselme 2000). Regarding their interaction with et al 1989). Changes of calcium or phosphate contents in
calcium phosphates, osteoblasts are intimately in contact culture medium also affect directly the osteoblastic activities
with calcium phosphate surfaces thanks to the production (Bellows et al 1992; Meleti et al 2000; Adams et al
of extracellular collagen firmly attached perpendicular or 2001; Dvorak et al 2004). As a consequence of surface
parallel to the substrate (Annaz et al 2004b). transformation the cellular activity might be further affected
The osteogenic differentiation phase comprises three as the substrate has significantly changed (Anselme et al
principal biological periods: cellular proliferation, cellular 1997);
maturation, and matrix mineralization. Through these (ii) Composition. A change in calcium to phosphorus ratio
periods, the osteoblasts are known to synthesize and secrete in the bioceramics means a change in phase composition
subsequently type I collagen, alkaline phosphatase, and and consequently a direct effect on the ionic exchanges
other non-collagenous extracellular bone matrix proteins mechanisms, as discussed earlier. The incorporation of
such as osteocalcin, osteopontin, osteonectin, and bone mineral ions such as zinc (to a certain extent) or silicate
sialoprotein. Type I collagen is expressed during the initial in calcium phosphate bioceramics showed an increase of
period of proliferation and extracellular matrix biosynthesis, osteoblasts attachment and proliferation (Ishikaw et al
whereas alkaline phosphatase is expressed during the post- 2002; Thian et al 2005). While the presence of carbonate
proliferative period of extracellular matrix maturation, and in the apatitic network of the biomaterial had contradictory
the expression of osteopontin, osteocalcin, and bone effects on osteoblastic proliferation and alkaline phosphatase
sialoprotein occurs later during the third period of production compared with the HA substrate (Anselme et al
extracellular-matrix mineralization (Sodek and Cheifetz 1997; Redey et al 2000; Midy et al 2001);
2001). Consequently, because there is no specific single (iii) Topography. On one hand, grooved calcium phos
marker for osteoblasts, the cellular expression of a range phate surfaces influence the osteoblastic guidance, and the
of non-collagenous and collagenous proteins as well as groove profile independently of nature of the substrate (Lu
alkaline phosphatase and Cbfa1 has to be investigated, when and Leng 2003). On the other hand, in contact with micro-
examining osteoblastic differentiation. Finally, osteoblasts and macroporous calcium phosphate ceramic osteoblasts
participate actively to the biomaterial mineralization (Radin sense the surface microporosity and they can bridge even
et al 1998) by producing calcium phosphate containing large pores many times larger than fully flattened osteoblasts
vesicles (Annaz et al 2004b). In practice, the mineralization (Annaz et al 2004a). For osteoblasts differentiation, Chou et
of osteoblasts cannot be assayed on calcium phosphates al have demonstrated in vitro that MC3T3 cells were sensitive
biomaterials because of the similarities in composition to the crystal shape: large apatite crystals induced more bone
between the substrate and the extracellular mineral. sialoprotein and osteocalcin expression after 3 weeks of
Several calcium phosphate parameters can affect the culture than small apatite crystals (Chou et al 2005b).
cellular activity: (iv) Surface energy. The study by Redey et al (2000)
(i) Dissolution. Depending on the calcium phosphate showed that the surface energy strongly affected the initial
reactivity (ie, dissolution–precipitation behavior) osteoblast osteoblastic activity for proliferation and function, while at a
function can be affected, in their proliferation, their later time the less favorable surface exhibited a comparable
differentiation, and their maturation phenotypes (de Bruijn osteoblastic activity to the most favorable surface.
et al 1992a, 1994b; Midy et al 2001; Knabe et al 2004;
Siebers et al 2004; Wang et al 2004; Arinzeh et al 2005; Calcium-phosphates – osteoclasts
Berube et al 2005). De Bruijn et al demonstrated a clear link Heymann et al (1999) have extensively addressed the
between dissolution rate and early bone formation in vivo cellular degradation of calcium phosphate ceramics in
and the osteogenic differentiation in vitro of osteoprogenitor vitro and in vivo. After colonization of the substrate by
cells, suggesting therefore the influence of free calcium monocytes–macrophages that are recruited during the
and inorganic phosphates on bone formation (de Bruijn inflammatory reaction following surgery (Basle et al 1993;

Barrère et al
Heymann et al 1999), osteoclasts are responsible for bone activity known to promote the osteoclastic acidic secretion
mineral degradation, ie, bone resorption. It has been observed in vitro (Doi et al 1999; Leeuwenburgh et al 2001). On
that osteoclasts degrade calcium phosphate ceramics in a the other hand, zinc and fluoride included in calcium
similar way to bone mineral: osteoclasts attach firmly to the phosphate biomaterials have shown also an inhibiting effect
substrate sealing zone. In the center of this sealing zone, they on osteoclastic resorption in vitro (Sakae et al 2000; Ito
secrete H+ leading to a local pH=4–5. In vivo, osteoclasts et al 2002) and in vivo (Kawamura et al 2003; Sakae et al
participate partially in the degradation of calcium phosphate 2003) for specific concentration in zinc-containing calcium
ceramics (Gauthier et al 1999; Lu et al 2002; Ooms et al phosphates;
2002; Wenisch et al 2003; Zerbo et al 2005). In contact with (iii) Surface energy of the calcium phosphate biomaterial:
osteoclasts, implanted bioceramics exhibited resorption pits the polar component of the surface energy was found to
associated with newly formed bone (Gauthier et al 1999; modulate the osteoclastic adhesion in vitro. However, the
Ooms et al 2002; Wenisch et al 2003). However, the in vivo further spreading and resorption was not influenced by
degradation of calcium phosphate materials is also associated surface energy considerations (Redey et al 1999);
with a dissolution phenomenon (Gauthier et al 1999; Lu et (iv) Surface roughness is known in general to influence
al 2002). The osteoclastic activity versus dissolution process the cell attachment in vitro and it was shown also in vitro
of a calcium phosphate ceramic depends on the nature of the for osteoclast attachment. Rough apatitic surfaces appear to
calcium phosphate (such as, cement, bulk ceramic, particles, enhance osteoclastic attachment compared with smooth ones
highly soluble TCP, sparingly soluble HA) (Gauthier et al (Gomi et al 1993).
1999; Lu et al 2002). In the the degradation of highly soluble
TCP ceramics in vivo, Zerbo et al (2005) have shown that Future directions in cell–calcium phosphate
physicochemical dissolution took place to a larger extent than interactions
osteoclastic resorption. Osteoclasts can also degrade calcium Cell sources At present, the reactivity of calcium phosphate
phosphates by phagocytic processes, ie, incorporation in the substrates is evaluated from primary osteoblasts, osteosarcoma
cytoplasm of the cell and thereafter dissolved by acid attack or cell lines, pre-osteoblasts, and osteoprogenitor cells. Although
enzymatic process when biomaterials particles are generated human osteoprogenitor cells seem to be the most adapted for
as the result of either local dissolution at grain boundaries or bone tissue engineering, very few reports deal with the culture
by mechanical stress generating debris (Heymann et al 1999). of human osteoprogenitor cells. These cells are donor- and
The osteoclastic activity is usually determined by a specific culture-dependent (Mendes et al 2002) and their pluripotency
osteoclastic enzyme (tartrate-resistant acid phosphatase) decreases with passage numbers. Unless many donors’ cells
depends on intrinsic properties of the calcium phosphate are used to screen calcium phosphate biomaterials, human
ceramics that can vary from study to stud. However, some osteoprogenitor cells are not a handy tool for understanding
general trends can be outlined: their interactions with calcium phosphate, as another donor’s
(i) Physicochemical dissolution kinetics of the bio cell may act very differently. To our knowledge, no extensive
material: calcium phosphate ceramics do not all interact studies on different human osteoprogenitor cells have been
in the same way with osteoclasts. The release of calcium performed in general on biomaterials. On the other hand, cell
ions from the biomaterial seems to play a critical role in lines giving more reproducible results are “manipulated” and
the osteoclastic activity; above a critical range of calcium may not represent the real situation, as different cell lines have
ions levels, osteoclastic resorption is inhibited (de Bruijn demonstrated a variation in responsiveness when cultured on
et al 1994a; Yamada et al 1997; Doi et al 1999). Together a similar calcium phosphate materials (Rochet et al 2003).
with the dissolution behavior, the structure of the calcium Embryonic stem cells are also a potential source in tissue
phosphate ceramic and the crystallinity influence the engineering and they are investigated on calcium phosphate
osteoclastic activity (de Bruijn et al 1994a; Yamada et al biomaterials (Both et al 2005; Melville et al 2006).
1997; Leeuwenburgh et al 2001). The question remains open of which cell type to focus on
(ii) Carbonates and other mineral ions: carbonated apatitic to understand the cell–calcium phosphate interactions for a
salts, eg dentin, bone mineral, synthetic carbonated apatite, tissue engineering approach.
and calcium carbonate structures (ie aragonite, calcite), Co-culture systems, ie, culturing two types of cells,
are resorbed by osteoclasts. It has been proposed that the are the new center of interest in the domain of bone tissue
carbonate content may stimulate the carbonic anhydrase engineering. For example, bone tissue-engineered constructs

Bone regeneration
lack viability due to insuff icient vascularization. As 2005b). The quantification of alkaline phosphatase activity
angiogenesis in the bone marrow is closely associated with is the most common and straightforward biological assay
osteogenesis in developing and mature bone, co-cultures performed on osteogenic differentiation, but the presence
of osteoprogenitor cells with endothelial cells are initiated of inorganic phosphate may inhibit the production by the
in order to create simultaneously vascularization and bone cells of alkaline phosphatase activity without inhibiting the
formation. However, to our knowledge there are no reports further mineralization process by osteoblasts (Bellows et
including the influence of a calcium phosphate biomaterial al 1992). So measuring alkaline phosphatase activity as a
(or any other biomaterial) with osteoprogenitor–endothelial unique differentiation assay on reactive calcium phosphate
cell co-cultures. This should be taken into account in future, bioceramics may not represent a valid quantification of
as it was shown that a calcium phosphate substrate, in osteogenic differentiation.
contact with a hematopoietic–osteoblasts co-culture system,
induced a entirely specific differentiation pathway than tissue Relevance of calcium phosphates
culture-treated plastic (Rochet et al 2002). Could the prior
for bone tissue regeneration
osteoclastic resorption of the calcium phosphate biomaterial
influence the osteoblastic activity as observed in natural bone
Bone-bonding ability
remodeling? Ionic exchange phenomena occurring with calcium
Finally, live observation of transgenic luminescent cell phosphate bioceramics are associated with reactivity towards
cultures offers great opportunities to evaluate long-term bone bonding, ie, the formation of an interfacial mineralized
activities on the same sample in vivo and in vitro (Cao et layer between bioceramics and bone tissue that insures their
al 2005; Kotobuki et al 2005; de Boer et al 2006). More cohesion. Structurally, this layer is comparable to the films
specifically for bone, de Boer et al has recently addressed the grown in vitro by dissolution–precipitation mechanisms, ie,
application of transgenic luminescent cell cultures coupled nanocrystals of carbonated apatite in simulated body fluids
with osteogenic reporters (de Boer et al 2006). that mimic the mineral composition of blood plasma. When
formed in the presence of osteogenic cells experiments,
Limitations of in vitro tests Calcium phosphates are this mineralized layer is comparable with the cement lines
reactive and their reactivity depend on their characteristics synthesized in vivo (de Bruijn et al 1995; Davies 1996). In
(such as composition, dissolution, sintering temperature, vivo (osseous and non-osseous environment), physico
microstructure). As a consequence, the calcium and phos chemical, and crystallographic continuity are observed
phate levels in the cell culture medium can vary substantially between the calcium phosphate implant and the newly formed
without being regulated and this can affect cellular function mineralized layer (Daculsi et al 1989; de Bruijn et al 1992b;
(Bellows et al 1992; Meleti et al 2000; Adams et al 2001; Neo et al 1993). Its occurrence and thickness are related
Midy et al 2001; Dvorak et al 2004; Wang et al 2004; to the reactivity (dissolution–precipitation) of the calcium
Arinzeh et al 2005; Berube et al 2005); while in vivo, these phosphate substrate (Neo et al 1993), the so-called bioactivity
variations are automatically and rapidly regulated. Therefore (Hench and Wilson 1984). This mineralized interface insures
the in vitro predictability of the biomaterial as such is often a physicochemical and mechanical cohesion between the
inconclusive (Habibovic et al 2005a). In the context of bone implant and the host bone. It is particularly relevant for
tissue engineering, scaffolds and cells are in contact in vitro load-bearing applications, ie, hip metallic prostheses coated
for several days before being implanted, hence testing the in with calcium phosphate which undoubtedly improves the
vitro cellular activity remains pertinent. However, Kruyt et mechanical stability of the implant by augmenting and
al (2004) did not find any differences in vivo between the accelerating the bone apposition (Geesink et al 1987; Dhert
bone tissue engineering constructs cultured in stimulating et al 1993; Rahbek et al 2004).
or non-stimulating osteogenic differentiation medium for 6
days, while they measured a significant difference in alkaline Osteoinduction by calcium phosphates
phosphatase activity in vitro between these two conditions. Osteoinductive materials are biomaterials that have intrinsic
On the other hand, the number of biological assays increases properties to induce bone formation in a non-osseous
with time, but they are still in the developmental stage. environment. Recently, it has been demonstrated in goats, that
It is sometimes difficult to understand the regulations of osteoinductive macroporous calcium phosphates stimulate
some proteins as reported by Chou et al (Chou et al 2005a, more bone formation as tissue-engineered constructs

Barrère et al
ectopically (Kruyt et al 2004) and as such in critical-sized Load-bearing applications

orthopedic defect models (Habibovic et al 2005b). Although Calcium phosphate applied as coatings on metallic prostheses
the mechanism of osteoinduction remains unclear, the ionic have a highly successful clinical record for hip arthroplasty
exchanges properties of the calcium phosphate scaffolds (Epinette and Manley 2004). These coatings significantly
with the surrounding milieu have been pointed out as a accelerate bone growth onto the metallic implant, improve
relevant parameter, among others (Yuan et al 2001). By fixation of the implants, and extend the prostheses’ longevity.
implanting intramuscularly in goats two macroporous Extensive animal studies have been conducted taking into
calcium phosphate scaffolds identical in composition, account bone formation rate versus resorption rate and
crystallinity, and porosity but with different microporosities, mechanical stability. By changing the coating’s parameters
Habibovic et al (2005a) have demonstrated that an elevated (technique, temperature, composition) one can change the
microporosity was responsible for ectopic bone formation. coatings’ degradation characteristics. For hip prostheses, the
This high microporosity is directly correlated with the calcium phosphate coatings’ resorption kinetic has a certain
exposed surface, and therefore an elevated dissolution in the paradigm. On the one hand, soluble coatings enhance bone
pores where the level of stable critical level of free calcium formation at the early implantation stage, inducing a fast
ions and possibly free orthophosphate ions might trigger cell early fixation, but soluble coatings may lead to a second
differentiation into osteogenic lineage. In addition, through stage mechanical instability between the metallic implant and
a dissolution–precipitation process, the development of a the surrounding bone. On the other hand, insoluble coatings
bone-like mineral layer might initiate bone formation either delay bone fixation, ie, the first stage mechanical stability,
but insoluble coatings stabilize the long-term fixation of the
by mimicry with the bone mineral structure or by the presence
implant with the surrounding tissues (Dhert et al 1993, 1998;
of osteogenic compounds (for example bone morphogenetic
de Bruijn et al 1994b; Rahbek et al 2004).
proteins, BMPs) contained naturally in body fluids that
might have concentrated at the newly formed mineral layer
Non-load bearing applications
(Ripamonti 1996).
In non-loading situations, bare calcium phosphates are
used as granules or cement. Their composition can have
Tailoring the resorption kinetics of virtually limitless variations with regard to their structure,
calcium phosphates the incorporation of additives, and the mixture of phases.
In large defects that cannot be healed naturally by bone, The current scaffolds either degrade too fast (and therefore
adjusting the degradation kinetics of the calcium phosphate do not allow sufficient stability for new bone formation), or
bone filler with the kinetics of bone formation rate remains scaffolds do not resorb fully (and remain encapsulated by
a great challenge in bone tissue regeneration. While the newly formed bone, avoiding the entire restoration of
contributing to bone formation, the scaffold should degrade in natural bone mechanical strength). A common way to tune
a controlled fashion to leave gradually more space for newly the degradation properties of calcium phosphate scaffolds is
formed bone until full tissue regeneration. Mixing at various to combine a highly soluble phase (TCP) with a non-soluble
ratios a low soluble phase (HA) with a highly soluble phase phase (HA), to create so called BCP ceramics (Legeros et
(amorphous TCP) resulting in biphasic calcium phosphates al 2003). Depending on the HA/TCP ratio, bone formation
ceramics (BCP), including additives (magnesium, carbonate, versus biomaterial resorption can be significantly improved
fluoride) in a given crystalline phase, or selecting different or decreased (Schopper et al 2005).
calcium phosphate phases (amorphous, DCPD, OCP, HA,
TCP), are the options to tailor the degradation kinetics of Functionalization of calcium phosphates
calcium ceramics from a few weeks to a few years. In theory for triggering bone regeneration
and in practice, one can change the resorption kinetics In view of the dissolution properties of calcium phosphate,
of calcium phosphate ceramics; however, no universal several groups have used calcium phosphate ceramics as
degradable scaffolds have been developed yet, and their delivery systems by (i) adsorption on powder followed by
degradation properties should be designed depending on compaction, (ii) co-precipitation, or (iii) adding the cement
their application. Below, two types of degradation patterns paste applied in bone regeneration and related fields of
are described, one for load-bearing and non-load bearing gene therapy (Shen et al 2004), or local drug delivery (Urist
applications. et al 1987; Lebugle et al 2002; Stigter et al 2004; Kroese-

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Bone Regeneration Molecular and Cellular Interactions

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International Journal of Nanomedicine

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Bone regeneration: molecular and cellular

Florence Barrère, Clemens A van Blitterswijk & Klaas de Groot

To link to this article: https://doi.org/10.2147/DIJN.1.S627

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Bone regeneration: molecular and cellular

International Journal of Nanomedicine 2006:1(3) 317–332 317

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Manganese Stimulating alkaline phosphatase activity No

Fluoride Stimulating alkaline phosphatase activity Stabilizing bone bonding

Strontium Increasing bone mass: stimulating bone formation No

Lithium Stimulating human mesenchymal stem cells proliferation No

Borate Possibly interacting with mineral and vitamin D metabolism No

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322 International Journal of Nanomedicine 2006:1(3)

The osteoclasts bone matrix macromolecules, namely alkaline phosphatase,

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ectopically (Kruyt et al 2004) and as such in critical-sized Load-bearing applications

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