Acta Biomaterialia 8 (2012) 3876–3887

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Acta Biomaterialia
journal homepage: www.elsevier.com/locate/actabiomat

Review

Current views on calcium phosphate osteogenicity and the translation

into effective bone regeneration strategies
Y.C. Chai a,c, A. Carlier b,c, J. Bolander a,c, S.J. Roberts a,c, L. Geris c,d, J. Schrooten c,e, H. Van Oosterwyck b,c,
F.P. Luyten a,c,⇑
a
Laboratory for Skeletal Development and Joint Disorders, KU Leuven, O&N 1, Herestraat 49, PB 813, 3000 Leuven, Belgium
b
Biomechanics Section, KU Leuven, Celestijnenlaan 300 C, PB 2419, 3001 Leuven, Belgium
c
Prometheus, Division of Skeletal Tissue Engineering, KU Leuven O&N 1, Herestraat 49, PB 813, 3000 Leuven, Belgium
d
Biomechanics Research Unit, University of Liege, Chemin des Chevreuils 1 – BAT 52/3, 4000 Liege 1, Belgium
e
Department of Metallurgy and Materials Engineering, KU Leuven, Kasteelpark Arenberg 44, PB 2450, 3001 Leuven, Belgium

a r t i c l e i n f o a b s t r a c t

Article history: Calcium phosphate (CaP) has traditionally been used for the repair of bone defects because of its strong
Received 7 April 2012 resemblance to the inorganic phase of bone matrix. Nowadays, a variety of natural or synthetic CaP-based
Received in revised form 28 June 2012 biomaterials are produced and have been extensively used for dental and orthopaedic applications. This
Accepted 3 July 2012
is justified by their biocompatibility, osteoconductivity and osteoinductivity (i.e. the intrinsic material
Available online 13 July 2012
property that initiates de novo bone formation), which are attributed to the chemical composition, sur-
face topography, macro/microporosity and the dissolution kinetics. However, the exact molecular mech-
Keywords:
anism of action is unknown. This review paper first summarizes the most important aspects of bone
Calcium phosphate
Bone tissue engineering
biology in relation to CaP and the mechanisms of bone matrix mineralization. This is followed by the
Osteoinductivity research findings on the effects of calcium (Ca2+) and phosphate (PO34 ) ions on the migration, prolifera-
Calcium ion tion and differentiation of osteoblasts during in vivo bone formation and in vitro culture conditions. Fur-
Computational modelling ther, the rationale of using CaP for bone regeneration is explained, focusing thereby specifically on the
material’s osteoinductive properties. Examples of different material forms and production techniques
are given, with the emphasis on the state-of-the art in fine-tuning the physicochemical properties of
CaP-based biomaterials for improved bone induction and the use of CaP as a delivery system for bone
morphogenetic proteins. The use of computational models to simulate the CaP-driven osteogenesis is
introduced as part of a bone tissue engineering strategy in order to facilitate the understanding of
cell–material interactions and to gain further insight into the design and optimization of CaP-based bone
reparative units. Finally, limitations and possible solutions related to current experimental and compu-
tational techniques are discussed.
Ó 2012 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.

1. Introduction
Bone is a dynamic, highly vascularized and mineralized tissue
that has self-remodelling and healing capacities under normal
physiological conditions, with bone loss due to disuse and upon in-
jury. It provides structural support to the body for locomotion,
serves as a protective cage for internal organs, and is a site for
haematopoiesis and endocrine regulation. It also maintains the
acid–base balance of blood, and serves as storage for minerals
(mainly calcium (Ca2+) and phosphate (PO34 )) and growth factors
⇑ Corresponding author at: Laboratory for Skeletal Development and Joint
Disorders, Division of Skeletal Tissue Engineering, Katholieke Universiteit Leuven,
O&N 1, Herestraat 49, P.O. Box 7003, 3000 Leuven, Belgium. Tel.: +32 16 342541;
fax: +32 16 342543.
E-mail address: frank.luyten@uz.kuleuven.ac.be (F.P. Luyten).
that are essential for vital physiological events, such as ion homeo-
stasis and other intracellular signalling pathways.
In fact, bone is a biocomposite tissue consisting of an organic
phase (mainly collagen type-1 fibres, 20%) and an inorganic phase
(mainly carbonated hydroxyapatite (Ca10(PO4)6(OH)2), (60%) [1]
(Fig. 1A) that are organized in a lamellar cylindrical osteon system
(i.e. the compact bone) or present as irregular thin trabecular plates
and struts (i.e. the spongy bone) [2]. This special organization of
collagen fibres and mineralized matrix (deposited by the bone-
forming cells, i.e. the osteoblasts) renders the bone tissue with
relatively high elastic modulus and compressive strength, but low
tensile and shear strength [3,4] (Fig. 1B). These mechanical proper-
ties are dependent on the anatomic location [5,6], and are
influenced among others by the porosity and percentage of the
mineral content within a bone tissue to suit a particular functional-
ity (e.g. 80% mineral content within ossicles for sound transduction

1742-7061/$ - see front matter Ó 2012 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.actbio.2012.07.002
 Y.C. Chai et al. / Acta Biomaterialia 8 (2012) 3876–3887 3877

Fig. 1. (A) Composition and (B) mechanical properties of human bone [4,6].

by vibration) [7]. In general, a higher mineral content increases the
stiffness, but decreases the toughness of the bone [8]. Recently,
these stiffness and energy dissipation properties of bone were
found to be attributed to calcium-mediated sacrificial bonds of a
non-fibrillar organic matrix, which act as a ‘‘glue’’ to hold the
mineralized fibrils together (through a hidden length mechanism)
during bone deformation [9,10].
Indeed, calcium phosphate [11] plays a critical role in the min-
eralization of collagen fibres and thus contributes to physiologi-
cally important bone tissue characteristics. Two theories of
collagen fibre biomineralization are reported: (a) direct nucleation
of calcium phosphate crystals onto collagen fibrils [12], and (b)
matrix vesicle (MV) mediated matrix mineralization [13,14]. Direct
nucleation involves the formation of stable mineral droplets con-
sisting of calcium phosphate cluster–biopolymer complexes that
bind to a distinct region on the collagen fibres and diffuse through
the interior of the fibril, where they solidify into an amorphous
phase (Fig. 2A). This amorphous phase is then transformed into ori-
ented apatite crystals directed by the collagen fibril arrangement.
The second theory is based on the production of intracellular MV
containing calcium phosphate crystals by osteoblasts. Three possi-
ble mechanisms for the initiation of MV mediated matrix mineral-
ization have been suggested [15] (Fig. 2B): (i) MV only regulates
ion concentrations, leading to the formation of soluble molecular
species which initiate mineral formation in collagen fibrils; (ii)
MV regulates ion compositions, leading to the formation of intra-
vesicular apatite crystals, which leave the vesicle and initiate the
mineralization process; and (iii) MV associate directly with colla-
gen and cooperates to initiate matrix mineralization.
These essential matrix mineralization events are tightly regu-
lated by several bone-related proteins and growth factors. For in-
stance, alkaline phosphatase (ALP) is a periplasmic enzyme

Fig. 2. Theories of biomineralization of collagen fibril. (A) Direct nucleation of CaP crystal: (i) CaP clusters form complexes with the functional biopolymer (e.g. polyaspartic
acid) and (ii) form stable mineral droplets. (iii) The mineral droplets bind to a distinct region on the collagen fibres and enter the fibril and (iv) diffuse through the interior of
the fibril before solidifying into a disordered (amorphous) phase. (v) This amorphous phase is transformed into oriented apatite crystals directed by the collagen orientation
(modified from Colfen [12]). (B) MV-mediated matrix mineralization. Schematic showing the three possible mechanisms for the initiation of MV-mediated matrix
mineralization (adapted from Golub [15]). PPi, pyrophosphate.
3878 Y.C. Chai et al. / Acta Biomaterialia 8 (2012) 3876–3887
(membrane of cell and matrix vesicle) that hydrolyses pyrophos-
phate (a mineralization inhibitor), thereby providing phosphate
ion (PO34 ) to promote mineralization [16–18]. PHOSPHO1 is an-
other phosphatase highly expressed in bone [19], which is present
within MV and plays a role in the initiation of mineral formation
[20]. Osteocalcin, osteonectin, osteopontin and bone sialoprotein
are the four major non-collagenous bone proteins. Osteocalcin
and osteonectin were reported to regulate the size and speed of
crystal formation [21], bone sialoprotein was found to act as a crys-
tal nucleator [22], whereas osteopontin influences the type of crys-
tal formed [23,24]. In contrast, mineralization inhibitors such as
decorin, a member of the small leucine-rich proteoglycans family,
negatively interfere with mineralization by modulating collagen
assembly [25]. Another inhibitor, Matrix Gla Protein, was associ-
ated with parathyroid hormone-mediated inhibition of osteoblast
mineralization [26]. Recently, bone-morphogenetic protein-2
(BMP-2), a potent osteoinductive growth factor, was found to be
involved in the control of ALP expression and osteoblast minerali-
zation via a Wnt autocrine loop [27], as well as in the enhancement
of PO34 transportation into cells for matrix mineralization [28].
Fibroblast growth factor-2 (FGF-2), is another growth factor that
was found to reduce the expression of genes associated with ma-
trix mineralization [29].
2. Effect of calcium (Ca2+) and phosphate (PO34 ) on osteoblastic
cell behaviour and its applications
This section will describe primarily (pre)osteoblasts, since these
cells play a key role during osteogenesis. Notice that the extracel-
lular calcium ion (Ca2+) can be bound to proteins (e.g. albumin). In
this complex form, the ion cannot influence cellular behaviour
such as differentiation and proliferation [30,31]. The terms ‘‘Ca2+’’
and ‘‘PO34 ’’ refer, in the remaining part of this paper, to the active,
free ions.
During in vivo bone resorption, osteoclasts release Ca2+ and
PO34 derived from bone matrix. This causes a local increase in
the ion concentration to supra-physiological levels, which has a
significant impact on the proliferation and differentiation of osteo-
blasts, as well as on the subsequent bone formation process. In fact,
extracellular Ca2+ gradients are present in a number of distinct
microenvironments and represent potent chemical signals for cell
migration (chemotaxis) and directed growth [32]. Additionally,
Ca2+ is an important homing signal, which brings together different
cell types required for the initiation of a multicellular process such
as bone remodelling or wound repair [33]. For instance, high Ca2+
concentrations are shown to stimulate pre-osteoblast chemotaxis
to the site of bone resorption, and their maturation into cells that
produce new bone [34]. This chemotactic response to Ca2+ was also
demonstrated experimentally on different cell types, including
monocytes [35], osteoblasts [36], haematopoietic stem cells [37]
and bone marrow progenitor cells [38]. The latter reported a
dose-dependent relationship, with a maximal effect achieved at
concentrations from 3–10 mM of Ca2+. These findings indicate that
extracellular Ca2+ is a coupling factor between osteoclasts and
osteoblasts [39] (apart from other potential coupling factors, such
as mechanical coupling [40]). Mechanistically, extracellular Ca2+
regulates the migration of osteoblasts via the activation of calcium
sensing receptors (CaSR) and/or by increasing the influx of Ca2+
[41]. In fact, the CaSR is reported to act as a (gradient) sensor, thus
triggering chemotaxis of motile cells to critical microenvironments
and transducing the Ca2+ signal to intracellular signalling pathways
that regulate cell function [33]. Interestingly, studies suggest that
the CaSR in osteoblasts is functionally similar to, but molecularly
distinct from, the CaSR present in the parathyroid and the kidney
[41,42]. Whereas, the influx of Ca2+ elevates intracellular Ca2+ level
and thus the polarization of cell membrane at the leading edge,
which was reported to be critical in determining the persistent
directional cell migration [43,44].
Besides the effect on cell chemotaxis, the release of extracellular
Ca2+ also plays an important role in controlling the proliferation
(via c-fos transcription factor expression) and differentiation (via
dephosphorylation of NFAT transcription factor) of osteoblasts
near the bone resorption site (Howship’s lacunae), through the cal-
cium/calmodulin signalling [41]. These effects are revealed to be
mediated by the calcium-sensing receptor (CaSR) [45], voltage-
gated Ca2+ channels [41,46] or inositol 1,4,5-triphosphate receptors
[47] present in the osteoblast, which serve to increase the intracel-
lular Ca2+ level. In recent years, in vitro studies, based on the
addition of elevated Ca2+ levels into osteoblastic cell cultures
(2–8 mM), demonstrated a profound impact on bone cell fate,
which was independent of systemic calciotropic factors in a con-
centration-dependent way [42,48]. These include osteoblast che-
motaxis [36], DNA synthesis [49], proliferation, differentiation
[50] and mineralization of extracellular matrix [51,52]. Interest-
ingly, Ca2+ also induced the expression of osteogenic growth fac-
tors, such as parathyroid hormone-related peptide [53], BMP-2
and BMP-4 [52]. Similar effects were observed when osteoblasts
were cultured on Ca2+-functionalized biomaterials, such as nano-
crystalline CaP glass [54], Ca2+ implanted titanium substrate [55],
Ca2+-exposed collagen gel [51] and CaP-coated implants [56,57].
In vivo osseointegration and bone formation were also improved
when implants were enriched with calcium ions [58]. Encourag-
ingly, Ca2+ has been implicated recently as an important messenger
involved in the non-canonical b-catenin-independent Wnt/calcium
signalling for bone formation [59]. This pathway relies on an intra-
cellular release of Ca2+ to activate calcium sensitive enzymes such
as Ca2+–CaMKII, protein kinase C or calcineurin.
Essentially, the release of PO34 at the resorption site also plays a
role in osteoblast proliferation and differentiation. In fact, PO34 has
been identified as an important signalling molecule that regulates
cell cycle and proliferation rate, alteration of signal transduction
pathways (e.g. Fos-related antigen-1 (Fra-1) [60] and extracellular
signal-regulated kinase (ERK1/2) [61]), gene expression (e.g. osteo-
pontin) [62], as well as the secretion of bone-related proteins (e.g.
matrix Gla protein (MGP) [63]). In contradiction, several in vitro
studies showed that addition of high concentrations of exogenous
inorganic phosphate (Pi, range from 5 to 7 mM) induced in vitro
osteoblast apoptosis and non-physiological mineral deposition
[64]. Nevertheless, Pi is believed to play a critical role in physiolog-
ical bone matrix mineralization [65]. This mineralization event is
mediated by the enzyme ALP and has been associated with the
bone matrix calcification induced by BMP-2, where BMP-2 was
found to stimulate Pi transport by osteogenic cells primarily via
the sodium-dependent phosphate transporters [28]. Unfortu-
nately, the use of Pi-functionalized biomaterials for tissue engi-
neering applications is rarely described, possibly because of the
technical limitations of existing technologies on handling Pi in an
ionic form or the lack of awareness on the potential use of Pi. How-
ever, the use of polymers mixed with phosphate salts as a sus-
tained delivery of Pi to induce in vivo mineralization of the
carrier has been reported recently [66].
Despite the vast in vitro research findings, the influences of Ca2+
and PO34 differ from cell type to cell type [67]. This implies that
there will be not one optimal Ca2+ and Pi concentration that could
universally drive all cell types toward successful osteogenesis.
Moreover, the optimal concentration may vary according to the
cellular stage including proliferation and differentiation. Therefore,
specific windows of ion concentration need to be determined for a
specific cell type and its cellular stage, in order to initiate the de-
sired in vitro cell behaviour effectively. For instance, the present
authors’ group has recently reported on the identification of
 Y.C. Chai et al. / Acta Biomaterialia 8 (2012) 3876–3887
specific Ca2+ and Pi concentrations that could induce higher prolif-
eration and osteogenic differentiation of a mesenchymal stem cell-
like human osteoprogenitor [68]. These findings were translated
into the formulation of bio-instructive media (containing specific
concentrations of Ca2+ and Pi) that could optimally initiate higher
proliferation, osteogenic differentiation and bone-like matrix
deposition, in both two-dimensional (2D) cultures and three-
dimensional (3D) constructs. This represents a novel strategy to
produce 3D bone reparative units that may have predictive osteo-
inductivity for an effective repair of large skeletal defects [69].
3. Rationale for using calcium phosphate for bone regeneration
Bone is a remarkable organ, as it has impressive self-healing
capacity without scar formation (when the defect size is not criti-
cal). However, delayed healing and non-unions still often develop
and will occur more frequently as a result of the ageing of the pop-
ulation. In the US, approximately six million fractures occur yearly,
of which 5–10% develop into a delayed union or non-union. An
extrapolation of these numbers to the Indian population results
in 240 million fractures a year, of which 12 million are non-unions
[70]. Therefore, the need for bone tissue regeneration is continu-
ously increasing, and the emergence of combined engineering
and life sciences technologies, such as tissue engineering, may lead
to more effective bone-healing therapeutic modalities.
Bone tissue engineering aims to offer a better solution for the
healing of large bone defects and non-unions. This interdisciplinary
research field applies principles of engineering and life sciences to
create an in vivo microenvironment that promotes local bone re-
pair or regeneration [71,72]. In this context, CaP bioceramics ap-
pear to be interesting candidates for bone tissue engineering
applications, owing to their biomimetic properties, supported by
the following findings. First, in the event of endochondral ossifica-
tion, mineralized cartilaginous matrix is reported to induce osteo-
progenitor differentiation and thereafter bone matrix deposition
[73]. This phenomenon has been further investigated in order to
elucidate the necessary physicochemical properties of CaP that
may effectively trigger cellular signalling cascades for bone forma-
tion [74]. Secondly, the bone matrix calcification or mineralization
process is a critical stage of bone formation, either through direct
mineralization of bone matrix or the preformation of cartilaginous
tissue template that is mineralized at a later stage. Thirdly, the dis-
solution of CaP-based biomaterials, either physicochemically or
cell mediated upon implantation, may also resemble the physio-
logical bone resorption process [75]. Finally, the data discussed
in the previous section show that the effects of Ca2+ and Pi on oste-
ogenic cell behaviour are appreciable. Therefore, the rationale of
using CaP-based biomaterials for bone engineering strategies is
clear.
4. Historical and hypothetical mechanisms of calcium
phosphate osteoinductivity
Carbonated hydroxyapatite (HA) is the prevalent form of CaP
mineral found in the bone. It provides mechanical strength to the
bone and plays a critical role in the mineralization of the bone ma-
trix. Because of chemical and biological similarities, HA derived
from natural sources (e.g. bone allograft, autograft or coral) or syn-
thetic HA, is widely used as bone filler for treating skeletal defects.
However, HA is a highly stable CaP mineral and therefore has lower
solubility at the physiological pH (7.2–7.6) compared with other
types of CaP that have higher solubility (such as tricalcium phos-
phate (TCP) and octacalcium phosphate (OCP)). Because the disso-
lution behaviour has been associated with the osteogenicity as
well as the osteoinductivity of CaP [75], the search for an improved
3879
CaP-based biomaterial with higher osteoinductivity also targets
CaP with an appropriately high solubility. This may be more effec-
tive in stimulating osteogenic differentiation of stem cells and ini-
tiating bone formation. However, the exact mechanism of
osteoinduction by CaP is currently unknown [67,71,76].
In the mid-1960s, osteoinduction was described as ‘‘a process
which supports the mitogenesis of undifferentiated perivascular
mesenchymal cells leading to the formation of osteoprogenitor
cells with the capacity to form new bone’’ [77]. Since the discovery
of bone morphogenetic proteins (BMP) as potent inducers of ecto-
pic bone formation [78], the term ‘‘osteoinduction’’ has generally
been used to describe an observation of heterotopic bone forma-
tion in association with the in vivo ectopic bone inductivity of a
substance, such as growth factors, chemical compounds or bioma-
terials. In pathophysiological conditions, ectopic bone formation
was induced upon tissue calcification in vivo, such as in tendons
and arteries [79]. This phenomenon was associated with the oste-
ogenic differentiation of the calcified tissue [80], including the
expression of BMP-2 by the calcifying cells [81]. In the context of
osteoinduction by synthetic biomaterials, investigations over past
decades suggest that (i) the presence of a CaP component within
a biomaterial (e.g. CaP-based ceramics [82], composite [83], coat-
ing [84], coral-derived ceramic [85] and bioactive glass [86]), or
(ii) the ability of a non-CaP-containing biomaterial to induce
in vivo calcification (e.g. poly-hydroxyethylmethacrylate sponge
[87], and chemical-treated oxidized titanium substrate [88]) are
prerequisites for heterotopic direct bone formation.
Recently, the osteoinductive effect of CaP-based biomaterials
and the in vivo host–biomaterial interactions were reviewed to
gain insight into the physicochemical properties that may govern
osteoinduction [72,89]. These include the effects of the macro-
structures (e.g. dimension, geometry and porosity), the micro/nano
structures [90] (e.g. microporosity [91], grain size, surface topogra-
phy), and the chemical composition and characteristics of the bio-
materials (e.g. active chemical surface for apatite formation, and
dissolution kinetics of CaP biomaterials). Additionally, the in vivo
osteoinductivity by CaP was reported to be dependent on the ani-
mal model used (higher incidence in large animals, possibly due to
higher osteoclastic activity [92]), implantation site [93] and the
duration of implantation [85] (higher incidence and faster in intra-
muscular than subcutaneous implantation). At present, the hypo-
thetical mechanisms of CaP-driven osteoinduction from a
material perspective can be classified into four categories.
4.1. Direct effect of the biomaterial
The physical properties of a CaP-based biomaterial, such as
geometry, macroporosity, microporosity, surface topography and
grain size, have, in combination with its chemical properties, a sig-
nificant impact on: (i) the nutrient, oxygen and waste exchange for
cells within a biomaterial; (ii) the host blood vessel in-growth; (iii)
the total volume of open pores available for cell growth and bone
tissue formation; (iv) the effect on osteochondrogenic differentia-
tion of stem cells [94]; (v) the total surface area available for pro-
tein adsorption (e.g. endogenous BMP), cell attachment (which
activates a BMP-2 autocrine loop via a2b1 integrin [95]) and
growth; and (vi) ion dissolution and re-precipitation [93], which
contributes to the formation of a biological apatite layer which,
in turn, stimulates the osteogenic differentiation of stem cells.
4.2. Indirect effect of the biomaterial
Osteoinductive proteins such as BMP and transforming growth
factor-beta are known to have a high affinity to CaP [96]. It has
been hypothesized that CaP-based biomaterials may act as an
in vivo affinity column/concentrator of the endogenous
3880 Y.C. Chai et al. / Acta Biomaterialia 8 (2012) 3876–3887

osteoinductive molecules upon implantation, which then renders
the biomaterial osteoinductive. This is supported by a study which
showed ectopic bone induction by CaP ceramics implanted
intramuscularly (without cells) in proximity to the fractured fibula,
where osteogenic events were actively ongoing [97].
4.3. Inflammatory response
Upon implantation, CaP-based biomaterials elicit an inflamma-
tory response that attracts the infiltration of mono- and multinu-
cleated cells, and subsequently activates osteoclastogenesis,
which results in CaP degradation and resorption [98]. Subse-
quently, the released Ca2+ and PO34 stimulate osteoprogenitor
differentiation and bone matrix deposition [99,100].
4.4. Pathological ossification/bone formation
Osteoinduction by CaP-based biomaterials may resemble a
pathological condition of heterotopic bone formation due to tissue
calcification in vivo, resembling atherosclerosis or calcified ten-
donitis. The bone tissue formed often lacks functionality and even-
tually may resolve over time. This resorption process may be
related to a foreign body reaction or be due to the depletion of
biochemical cues upon implantation.
5. Translation of calcium phosphate osteogenicity for bone
tissue engineering
In bone tissue engineering, two main forms of CaP materials are
used: (i) bulk (fully dense or open porous) CaP biomaterials (e.g.
bone fillers, carriers, cement) [101], either as pure CaP or as part
of a composite [102], and (ii) CaP coatings to functionalize bioma-
terial surfaces [103]. The use of CaP as growth factor delivery sys-
tem [104] is also discussed, specifically BMP-incorporated CaP
biomaterials for bone formation [105].
5.1. Bulk CaP
The physicochemical properties of bulk CaP are often modified
during the in vitro production process, aiming to find the optimal
construct geometry for CaP scaffolds that could enhance osteoin-
ductivity and hence improve the clinical outcome [106]. This in-
cludes manipulation of the architecture of biomaterials, such as
the geometry, macro- and microporosity and surface topography
[107]. The aim is to adjust the surface area for the entrapment of
endogenous osteoinductive biomolecules and to accommodate
optimum osteogenic cell density, as well as to fine-tune the disso-
lution kinetics that optimally elicit osteogenic differentiation and
bone matrix deposition. For instance, production of cylindrical
HA scaffolds with a hollow centre (2–4 mm) were reported to en-
hance ectopic bone formation [108]. Also, incorporating HA scaf-
folds with cylindrical tunnels of 90–120 lm and 350 lm induced
endochondral and intramembranous ossification [109]. The use
of biphasic CaP (BCP) scaffolds (HA:TCP = 65:35 wt.%) with cubic
pores of 500 lm resulted in the highest bone formation compared
with the scaffold with lower (100 lm) or higher (1000 lm) pore
sizes [110]. Besides that, the surface topography (i.e. surface
roughness) has also been reported to influence the pattern of bone
formation within CaP-based biomaterials [111]. Moreover, the sur-
face topography is beneficial for the formation of bone-like apatite
and provides a surface area that increases the dissolution of Ca2+
and PO34 ions [112]. This surface area was later found to be asso-
ciated with the microporosity within the macropores of CaP, which
affected the material–fluid interface dynamics and triggered oste-
ogenic differentiation [91]. Indeed, CaP discs with larger surface
area induce higher in vitro ALP activity and are capable of concen-
trating more osteoinductive molecules that differentiate cells into
the osteogenic lineage [113]. A similar phenomenon was observed
in an in vivo study in which CaP carriers with lower CaP grain size
and higher microporosity (and thus higher ionic dissolution kinet-
ics) were found to be osteoinductive without using stem cell tech-
nology [75]. The surface area can also be increased by the
production of CaP microparticles, as their osteoinductive proper-
ties are highly correlated to their size. It was shown that micropar-
ticles with size ranges from 80 to 300 lm demonstrated ectopic
bone formation, whereas particle sizes >500 lm were not osteoin-
ductive [114,115]. Indeed, several important in vivo studies have
shown that a CaP-based biomaterial with specific surface area
above a threshold level of 1.0 m2 g 1 was critical for osteoinduc-
tion (Fig. 3). Unfortunately, the availability of a technology to en-
able high controllability and reproducibility of these desired
features is currently lacking.
Also biphasic CaP (BCP) composites, being mixtures of HA with
CaP with higher solubility (such as b-TCP) in a specific ratio, appear
to be an effective alternative to enhance the osteoinductivity of CaP
[116]. The introduction of a more soluble phase of CaP may stimu-
late osteogenic differentiation of osteoprogenitors [117,118] and
meanwhile provide a stable phase of CaP (i.e. the HA), which
may act as a homing site for the implanted cells and promote bone
ingrowth [119]. Indeed, implantation of pure HA or TCP was shown
to be not osteoinductive, due to either too high stability or too high
a dissolution rate [120], whereas implantation of BCP induced bone
formation with a low inflammatory response, with the newly
formed bone being sustainable in vivo during a long-term animal
study [121]. Unfortunately, no optimized HA:TCP ratio has been re-
ported until now [122]. This is mainly due to the large variations in
the bone-forming capacity in studies using BCP with different
HA:TCP ratios, which is also species and material specific [76]. Ow-
ing to the lack of mechanical strength, these biphasic CaP biomate-
rials are more suitable for the treatment of skeletal defects at non-
load-bearing sites, such as bone fillers for cranial defects. Never-
theless, BCP alone was recently reported in the clinic to heal large
bone defects successfully, together with the use of an external fix-
ator [123].
Fig. 3. The influence of specific surface area of CaP-based biomaterials on in vivo
ectopic bone formation. Each data point represents one type of CaP implant with
specific surface area and its in vivo bone forming capacity. Based on these studies, a
threshold level of specific surface area required to induce bone formation was
chosen at 1.0 m2 g 1 (the cut-off point). Data adapted from Habibovic et al. [91], Li
et al. [113] and Yuan et al. [75].
 Y.C. Chai et al. / Acta Biomaterialia 8 (2012) 3876–3887
5.2. CaP coatings on biomaterials
Owing to the brittleness of CaP, it is often combined with a
mechanically superior biomaterial such as titanium. This is neces-
sary, especially in designing an osteoinductive composite for the
repair of skeletal defects that receive high mechanical loading
in situ [124]. To date, various methods have been proposed for
depositing CaP onto the surface of Ti-based biomaterials [125],
including a recent method based on the immobilization of chemi-
cal moieties to induce mineralization on the surface of biomateri-
als both in vitro [69] and in vivo [126]. These methods aim to
produce composites containing mineralized components that
strongly mimic the hierarchies and bioactivity of the mineralized
compositions of the bone, in order to provide an inductive environ-
ment that is capable of triggering bone formation. This again aises
the principle question regarding what type of CaP has to be depos-
ited onto the biomaterial of choice to ensure sufficient and predic-
tive osteoinductivity. Therefore, the controllability and
reproducibility of a technique that deposits CaP with desirable
physicochemical properties, in the context of achieving optimum
osteoinductivity, is highly demanded. A second consideration is
the applicability of a deposition technique to 3D porous structures.
This represents a major drawback for many of the existing meth-
ods, where deposition of CaP onto complex 3D structures is techni-
cally challenging [127]. For instance, in an effort to overcome this
major drawback, the present authors’ research group has devel-
oped perfusion electrodeposition technology to deposit CaP coat-
ing homogeneously onto a complex 3D Ti-scaffold porous
structure, which offers high controllability and reproducibility over
the physicochemical properties of the deposited CaP coatings [127]
and displayed a promising osteoinductive effect [128].
5.3. BMP-incorporated CaP carriers
As an alternative to existing bone grafting techniques, CaP car-
riers have been investigated for enhancing bone formation through
mediated delivery of bone-inducing factors (including biomole-
cules and metallic ions [129]), with some promising results re-
ported to date [130]. An example of such factors are the BMP,
discovered by Urist et al. as potent proteins with the ability to in-
duce heterotopic bone formation [77]. These proteins are members
of the TGF-b family which are secreted by chondrocytes, osteo-
blasts and osteoclasts [131–134], and play pivotal roles throughout
embryonic skeletogenesis and in postnatal bone formation and
endogenous repair mechanisms [130]. Indeed, BMP are attractive
for treatments of critical bone defects such as spinal fusions and
non-unions, and have been successfully combined with substrates
in order to induce bone formation by recapitulating molecular cas-
cades during skeletal development [135–137]. For instance, BMP-7
(also called osteogenic protein-1 or OP-1) and BMP-2, have been
approved for clinical use and are delivered for spinal fusion and
open tibial fractures via bone-derived collagen particles or an
absorbable collagen sponge [138]. Recently, these proteins have
been studied extensively within the field of tissue engineering in
order to mimic natural bone formation from a tissue engineering
point of view. When implanted in animal models, these growth
factors mainly induce bone formation via the endochondral path-
way, through the formation of an intermediate cartilage template
[135,139]. Unfortunately, the currently used delivery system for
BMP causes rapid protein release and diffusion, which besides
inducing bone formation, also causes inflammation and excessive
bone formation. Consequently, the uncontrollable or improper re-
lease kinetics resulted in undesirable side effects such as male ste-
rility, cancer and brain injury [140]. An additional reason for this
could be receptor saturation at higher doses or limited availability
of responding cells, which can lead to stimulation by these BMP at
3881
undesirable locations. Moreover, the carrier composition may af-
fect the regenerative response by BMP, as it may modulate protein
stability and variate release kinetics differently. Nonetheless,
Langer et al. showed as early as 1976 that protein release could
be sustained when BMP were encapsulated in biocompatible bio-
polymers [141]. Therefore, an attractive approach is to investigate
carriers that may favour spatiotemporal physiological protein re-
lease, but yet promote recruitment of endogenous progenitor cells.
These carriers also need to provide a suitable microenvironment
that promotes efficient cell proliferation and differentiation, as this
optimally may lead to bone formation and turnover [109,136]. In
addition to the collagen sponge used in clinical settings today,
studies have shown promising results from the use of osteocon-
ductive and osteoinductive CaP carriers [11,68,142,143]. These car-
riers possess suitable characteristics (e.g. the interconnected
microporosity and the variation in CaP ratio) that are critical for
the release kinetics of BMP, as the electrostatic energy possibly
plays a dominant role in carrier-to-BMP interactions (high binding
affinity of BMP to CaP), as well as in up-scaled constructs where
multiple carriers may be combined [144,145].
Even though CaP carriers in combination with BMP sounds like
a promising approach, an impediment may be variation in acti-
vated bone-forming pathways. The osteoinductive effect of CaP
carriers has been shown to form bone mainly via an intramembra-
nous pathway, whereas BMP induce bone formation mainly via the
endochondral pathway. In 2010, however, Eyckmans et al. showed
a close connection between CaP-induced bone formation and BMP
signalling [71]. In this study, removal of CaP-granules resulted in
loss of bone formation in combination with rescinded BMP signal-
ling as well as abrogated osteoinduction in the construct after inhi-
bition of endogenous BMP [146,147]. This study displayed that CaP
carriers affect BMP signalling in a model similar to physiological
intramembranous bone formation in a BMP- and CaP-dependent
manner.
Another hurdle could possibly be that CaP may inhibit the
osteoinductive effect of BMP, already displayed by Urist et al.
where partially demineralized bone induced inflammation and
inhibited osteogenesis [77]. Additional complications regarding
these issues were shown by Vehof et al., where the osteoinductive
ability in recombinant human BMP-2 (rhBMP-2) coated porous
particles of HA (PPHA) were investigated [148]. Prior to the study,
PPHA had displayed some ability to induce de novo formed bone
through the intramembranous pathway [149,150]. Remarkably,
PPHA in combination with large amounts of rhBMP-2 in this study
displayed an ability to attract a greater number of inflammatory
cells, which increased in time, together with multinucleated cells.
Absence of collagen type II prior to hypertrophic chondrocyte dif-
ferentiation and mineralization of the formed cartilage tissue, to-
gether with lack of bone marrow formation was seen. This
suggests that the de novo formed bone in this construct was not
following the usual BMP-induced endochondral pathway, there-
fore indicating an intermediate pathway that may have been
activated [148].
In conclusion, BMP mainly affect bone formation via an endo-
chondral process, whereas CaP carriers mainly induce an intra-
membranous pathway. Possibly, the combination of appropriate
concentrations of specific BMP and well-defined characteristics in
CaP carriers may function through an intermediate bone forming
pathway.
6. Computer modelling of bone regeneration in CaP scaffolds
Improvements in computer capacity now enable an increased
model realism and complexity (e.g. 3D calculations, complex
geometries, multi-scale and multi-physics) [151]. As a consequence
3882 Y.C. Chai et al. / Acta Biomaterialia 8 (2012) 3876–3887
of this technological revolution, there has been an enormous
increase in the use of mathematical models in biology and medicine.
These mathematical models can propose and test possible biological
mechanisms, contributing to the unravelling of the complex nature
of biological systems, such as bone regeneration processes inside
CaP structures. Moreover, they can be used to design and test
possible experimental strategies in silico before they are tested
in vitro or in vivo [152,153]. Eventually, all this knowledge can be
used to develop clinically relevant CaP-based bone reparative units
(Fig. 4).
Currently, there are many computational models of bone forma-
tion and regeneration in general [154] or even in scaffolds specifi-
cally [11]. Bohner et al. propose a theoretical approach to
determine the effect of geometrical factors on the resorption rate
of CaP scaffolds [155]. Their theoretical model was based on five
assumptions: (i) the sphericity of the pores; (ii) face-centred cubic
packing of the pores; (iii) surface-controlled resorption; (iv)
resorption requires the presence of blood vessels (50 lm in diam-
eter); and (v) the resorption time is proportional to the net amount
of material [156]. The model calculations show that, based on these
assumptions, the resorption time of a macroporous block depends
on the pore radius, which is determined by the size of the bone
substitute and interpore distance [156]. Subsequently, the model
was used to optimize the pore size of CaP scaffolds and was
validated with experimental data.
The theoretical model mentioned above, however, looks exclu-
sively at geometrical scaffold properties and does not include bio-
logical variables such as cell or matrix densities. Byrne et al.
developed a 3D mechanoregulatory model of bone regeneration
in a regular scaffold to investigate the effect of porosity, Young’s
modulus and dissolution rate on bone regeneration in different
loading conditions [157]. They modelled the scaffold as a poroelas-
tic material, which resorbs in a linear, load-independent fashion,
i.e. the porosity will be increased by 0%, 0.5%, 1% per iteration for
low, intermediate and high dissolution rates, respectively [158].
Consequently, the size of all scaffold elements decreases uniformly,
Fig. 4. The concept of integrating manufacturing process, stem cell technology and computational modelling as a novel strategy for designing and optimizing a CaP-based
bone reparative unit for effective bone regeneration that can also assist the translation to personalized bone regeneration.
resulting in an overall volumetric reduction, while the scaffold
geometry remains unaltered. Their calculations show that, as scaf-
fold degradation progresses, the regenerating tissue must take over
the mechanical function of the bone–scaffold system, which would
otherwise collapse due to a lack of mechanical strength. Moreover,
all three variables (i.e. porosity, Young’s modulus and dissolution
rate) appear to influence the amount of bone formation in a non-
intuitive way, demonstrating the need to optimize scaffolds for
site-specific loading requirements. This model was improved by
including blood vessel growth, thereby establishing a framework
to investigate the effect of vascularization on bone formation [159].
Other studies have modelled the bone regeneration process in-
side biodegradable polymer-based scaffolds. Stops et al. further
investigated the influence of mechanical strain and perfusive fluid
flow on cell differentiation and proliferation within a collagen–
glycosaminoglycan scaffold [160]. Sanz-Herrera et al. presented a
multi-scale model of bone regeneration inside a porous scaffold
[161]. The degradation mechanism of the biodegradable polymer
scaffold was modelled as a hydrolysis process, i.e. the water
content in the polymer chemically reacts and breaks down the
polymer molecules, resulting in biomaterial bulk erosion. The
mechanical properties of the polymer were assumed to relate lin-
early to its molecular weight. The model was used to predict the
evolution of the bone formation process in a scaffold implanted
in the femoral condyle of a rabbit. They found good qualitative
agreement between the obtained computational and experimental
results. Although further validation is necessary, the proposed
multi-scale model is a useful tool for investigating the complex
phenomena that occur at different length and time scales, i.e. the
bone formation and scaffold resorption at the microscopic scale
and the change in mechanical properties at the macroscopic scale.
Lacroix et al. reviewed the current techniques used for scaffold
development: from scaffold optimization of scaffolds by mathe-
matical models (e.g. FEM) to scaffold design using computer-aided
design and scaffold characterization by computed tomography (CT)
[162].
 Y.C. Chai et al. / Acta Biomaterialia 8 (2012) 3876–3887
Although the above models can be used to optimize some
(mechanical) properties of scaffolds, e.g. the porosity, the microar-
chitecture, Young’s modulus and dissolution rate, they neglect the
influence of growth factors and other biochemical signals on the
bone formation process. Moreover, the dissolution process is only
crudely modelled, neglecting the influence of the degradation
products (e.g. Ca2+ and Pi) on the cellular activities and bone forma-
tion processes. Carlier et al. developed and implemented an exper-
imentally informed bioregulatory model of the effect of calcium
ions released from CaP-based biomaterials on the activity of oste-
ogenic cells and mesenchymal stem cell driven ectopic bone for-
mation [163]. The dissolution kinetics of the CaP scaffold were
modelled by a general empirical equation [164], assuming that
the dissolution rate is proportional to the driving force (i.e. the dif-
ference between the current and the saturated calcium concentra-
tion) and that the rate constant is time independent. The amount of
bone formation predicted by the model of Carlier et al. corre-
sponded to the amount measured experimentally under similar
conditions. Moreover, experimentally impaired bone formation
due to conditions such as insufficient cell seeding and scaffold
decalcification, was also retrieved in silico. Subsequently, this mod-
el was used to optimize CaP scaffold selection to make their use in
combination with cells more clinically relevant.
Although computational models can contribute to the general
knowledge on bone formation inside CaP structures and are useful
for customizing the CaP carriers to patient-specific needs as well as
the particular bone application, experimental research is necessary
to establish and validate the mathematical models. The most
important parameters and their respective parameter values
should be experimentally quantified. Consequently, the multidisci-
plinary problem of optimizing scaffold architecture and seeding
protocols for bone tissue engineering strategies requires an integra-
tive approach, which was nicely summarized by Bohner et al. [165]:
a combination of mathematical modelling to explain a mechanism
of biomaterial–cell interactions, with experimental research to pro-
vide data for the determination of model parameters as well as the
validation of the mathematical model. This integrative approach re-
quires careful design and extensive characterization of the scaffold.
Moreover, this process is intrinsically iterative: new experimental
results can be fed into the model, and new research hypotheses
can result from thorough model analysis.
7. Limitations, future perspectives and conclusion
As discussed above, it is clear that bone induction by CaP bio-
materials is influenced by the physicochemical properties of the
material and thus the subsequent cellular events of osteogenesis.
However, the exact key determinant(s) of CaP osteoinduction,
meaning the molecular mechanisms involved and the stem
cell–material–host interactions upon implantation is/are still
underdetermined. Therefore, further study to decipher the molec-
ular signalling at the cellular level (such as receptor binding of the
released Ca2+ and PO34 and the activation of critical intracellular
osteogenesis pathway), and to understand the critical biological
parameters that are essential for implanted cells to communicate
and integrate effectively with the host system are required [166].
Unfortunately, the translation of the complex in vivo osteogenesis
environment into an in vitro system is far from trivial, and the pre-
dictiveness of in vitro observations often does not correlate well
with the in vivo bone formation capability [89]. This is due to some
critical issues such as the use of correct cell types for a specific type
of CaP-based biomaterial and the selection of the correct culture
conditions. Therefore, there is a need for customization of the
CaP-based biomaterial to overcome these limitations. Moreover,
other parameters present in vivo, including the compositions and
3883
dynamics of body fluids and blood vessel in-growth, are technically
challenging to be translated into a simplified in vitro setting. Re-
cently, the reactivity of CaP-based biomaterials in culture medium
was revealed to have significant influence on Ca2+ and PO34 disso-
lution kinetics and the subsequent cellular behaviour, which was
not correlated to the ion dissolution behaviour evaluated in simu-
lated body fluid (SBF) or phosphate-buffered saline. This must be
carefully considered when evaluating the osteoinductive potential
of the material [167]. The present authors propose the standardiza-
tion of in vitro dissolution tests to evaluate the ion release kinetics
of a CaP-based biomaterial, where the in vitro dissolution experi-
ment should resemble and be customized to the in vivo environ-
ment as close as possible [156]. First, the CaP scaffolds should be
thoroughly characterized (porosity, pore size distribution, grain
size, surface topography and surface area, and composition) before
and after the dissolution test. Here, the highlight is on the use of
non-invasive methods, including Raman spectroscopy [158] and
micro- or nano-focus X-ray CT to obtain quantitative characteriza-
tion of the material properties [168]. Secondly, a dual solution sys-
tem is proposed for in vitro dissolution testing using SBF [169] and
culture medium (with and without the presence of cells) [170].
This will allow for the characterization of the intrinsic ionic disso-
lution behaviour of the biomaterial in a solution with similar ionic
composition to the bodily fluid, followed by understanding of the
interactions or the influences of the cells and proteins on the ionic
dissolution kinetics. In fact, more and more studies have shown the
reduction in Ca2+ in the culture medium, indicating the formation
of new CaP crystals onto the existing CaP substrate [128]. Thirdly,
the experimental setting should also account for the local in vivo
hydrodynamic of the bodily fluid: for instance, at the implantation
site. This can be achieved by performing the dissolution test under
dynamic conditions (e.g. using perfusion system) at a physiologi-
cally relevant flow rate [118,127].
Moreover, the in vitro and in vivo experiments should be de-
signed in such a way that they minimize variability and enable
quantification, thus providing the essential data for the determina-
tion of model parameters as well as for the model validation. It is of
note that, as mathematical models predict the dynamics at differ-
ent scales (e.g. molecular, cellular and tissue) as a function of time
and space, temporal and spatial quantitative data are crucial to the
success of mathematical models. Clearly, computational modelling
plays an essential role to further unravel the complex mechanism
of CaP osteoinduction in vivo. Most of the current models look
either at mechanoregulatory or bioregulatory stimuli, depending
on the specific research question that is being answered. In the fu-
ture, however, these models could be combined to further improve
the predictive capabilities of the model.
The limitations mentioned above underline the importance of
an interdisciplinary strategy for the optimization of CaP scaffolds
for bone tissue engineering applications [165]. An essential charac-
teristic of these integrative research efforts is that their iterativity:
model analysis can lead to new research hypotheses, and new
experimental findings can be used to improve the predictive capac-
ities of the model.
In conclusion, this review has discussed the importance of CaP
for physiological bone homeostasis as well as its potential for bone
tissue engineering. Although numerous studies have been devoted
to unravelling the mechanisms of osteoinductivity of CaP scaffolds,
many questions still remain unresolved. To overcome this bottle-
neck, it is therefore essential that experimental and computational
research are combined so that the complex in vivo biological pro-
cess of bone regeneration inside CaP constructs can be deciphered
in an effective and systematic manner. This includes computa-
tional modelling and correlation analysis between the physico-
chemical properties of CaP constructs and the influences on
in vitro and in vivo biological outcomes, thus identifying the
3884 Y.C. Chai et al. / Acta Biomaterialia 8 (2012) 3876–3887
critical parameters and obtaining a thorough understanding of the
underlying biology that governs osteoinductivity of CaP constructs.
Only in this way will it be possible to make CaP a clinically relevant
and predictive tissue engineering construct for effective bone
defect repair.
Acknowledgements
This work is funded by the KU Leuven IDO project 05/009-
QuEST, Stem Cell Institute of Leuven—KU Leuven, ENDEAVOUR
project G.0982.11N. Aurélie Carlier and Johanna Bolander are
Ph.D. fellows of the Research Foundation Flanders (FWO Vlaander-
en). The work is part of Prometheus, the Leuven Research and
Development Division of Skeletal Tissue Engineering of KU Leuven:
http://www.kuleuven.be/Prometheus. The authors declare that
they have no potential conflict of interest, and they had and will
have no financial relationships with companies whose products
are relevant to the subject of this study.
A. Figures with essential colour discrimination
Certain figures in this article, particularly Figs. 1 and 2, are dif-
ficult to interpret in black and white. The full color images can be
found in the on-line version, at http://dx.doi.org/10.1016/
j.actbio.2012.07.002.
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