Review Paper:
Current Views on Calcium Phosphate Osteogenicity and the Translation into Effective
Bone Regeneration Strategies
Yoke Chin Chai1,3, Aurélie Carlier2,3, Johanna Bolander1,3, Scott J. Roberts1,3, Liesbet Geris3,4, Jan
Schrooten3,5, Hans Van Oosterwyck2,3, Frank P. Luyten1,3
Affiliation:
1
Laboratory for Skeletal Development and Joint Disorders, KU Leuven, O&N 1, Herestraat 49, PB
813, 3000 Leuven, Belgium.
2
Biomechanics Section, KU Leuven, Celestijnenlaan 300 C, PB 2419, 3001 Leuven, Belgium.
3
Prometheus, Division of Skeletal Tissue Engineering, KU Leuven O&N 1, Herestraat 49, PB 813,
3000 Leuven, Belgium.
Biomechanics Research Unit, University of Liege, Chemin des Chevreuils 1 – BAT 52/3, 4000 Liege
4
1, Belgium.
5
Department of Metallurgy and Materials Engineering, KU Leuven, Kasteelpark Arenberg 44, PB
2450, 3001 Leuven, Belgium.
Corresponding author:
Prof. Frank P. Luyten
Laboratory for Skeletal Development and Joint Disorders,
Division of Skeletal Tissue Engineering,
Katholieke Universiteit Leuven
O&N 1, Herestraat 49 – Box 7003
3000 Leuven, Belgium.
Tel: +32 16 342541
Fax: +32 16 342543
Email: frank.luyten@uz.kuleuven.ac.be
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Abstract
Calcium phosphate has traditionally been used for the repair of bone defects due to its' strong
resemblance to the inorganic phase of bone matrix. Nowadays, a variety of natural or synthetic CaPbased biomaterials are produced and have been extensively used for dental and orthopedic applications.
This is justified by their biocompatibility, osteoconductivity and osteoinductivity (i.e. the intrinsic
material property that initiates de novo bone formation), which are attributed to the chemical
composition, surface topography, macro-/micro- porosity and the dissolution kinetics. However, the
exact molecular mechanism of action is unknown. This review paper first summarises the most
important aspects of bone biology in relation to CaP and the mechanisms of bone matrix
mineralisation. This is followed by the research findings on the effects of calcium (Ca2+) and
phosphate (PO43-) ions on the migration, proliferation and differentiation of osteoblasts during in vivo
bone formation and in vitro culture conditions. Further, 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 delivery system for bone morphogenetic proteins (BMPs). 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 optimisation of CaP-based bone reparative units. Finally,
limitations and possible solutions related to current experimental and computational techniques are
discussed.
Keywords: calcium phosphate, bone tissue engineering, osteoinductivity, calcium ion, computational
modelling
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(1) Introduction
Bone is a dynamic, highly vascularised and mineralised tissue that has self-remodelling and healing
capacities under normal physiological conditions, with bone loss due to disuse and upon injury. 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 a storage for minerals [mainly calcium (Ca2+) and phosphate (PO43-)] and growth
factors
that are essential for vital physiological events, such as ions homeostasis 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]
(figure 1a) that are organised 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 organisation of
collagen fibres and mineralised 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] (figure 1b). These mechanical properties are dependent on the anatomic
location [5-6], and are among others influenced by the porosity and percentage of the mineral content
within a bone tissue to suit a particular functionality (e.g. 80% mineral content within ossicles for
sound transduction 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 mineralised fibrils together (through a hidden length
mechanism) during bone deformation [9-10].
Indeed, calcium phosphate [11] plays a critical role in the mineralisation of collagen fibres and thus
contributes to physiologically important bone tissue characteristics. Two theories of collagen fibre
biomineralisation are reported: (a) direct nucleation of calcium phosphate crystals onto collagen fibrils
[12], and (b) matrix vesicle (MV) mediated matrix mineralisation [13-14]. Direct nucleation involves
the formation of stable mineral droplets comprised 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 (figure 2a). This amorphous phase is then
transformed into oriented 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 possible mechanisms for the initiation of MV mediated matrix mineralisation have
been suggested [15] (figure 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 intravesicular apatite crystals, which leave the vesicle and
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initiate the mineralisation process; (iii) MV associate directly with collagen and cooperates to initiate
matrix mineralisation.
These essential matrix mineralisation events are tightly regulated by several bone-related proteins and
growth factors. For instance, alkaline phosphatase (ALP) is a periplasmic enzyme (membrane of cell
and matrix vesicle) that hydrolyzes pyrophosphate (a mineralisation inhibitor), thereby providing
phosphate ion (PO43-) to promote mineralisation [16-18]. PHOSPHO1 is another phosphatase highly
expressed in bone [19], which is present within matrix vesicles (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 crystal nucleator [22],
whereas osteopontin influences the type of crystal formed [23-24]. On the other hand, mineralisation
inhibitors such as decorin, a member of the small leucine-rich proteoglycans family, negatively
interfere with mineralisation by modulating collagen assembly [25]. Another inhibitor, Matrix Gla
Protein was associated with parathyroid hormone-mediated inhibition of osteoblast mineralisation [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 mineralisation via a Wnt autocrine loop
[27], as well as in the enhancement of PO43- transportation into cells for matrix mineralisation [28].
Fibroblast growth factor-2 (FGF-2), is another growth factor that was found to reduce the expression
of genes associated with matrix mineralisation [29].
(2) The Effect of Calcium (Ca2+) and Phosphate (PO43-) 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 extracellular calcium ion (Ca2+) can be bound to proteins (e.g. albumin).
In this complex form, the ion cannot influence cellular behaviour like differentiation and proliferation
[30-31]. The terms “Ca2+” and “PO43-“ refer, in the remaining part of this paper, to the active, free ions.
During in vivo bone resorption, osteoclasts release Ca2+ and PO43- 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 osteoblasts, 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 that brings together different cell types
required for the initiation of a multicellular process like 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
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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 micro-environments 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 polarisation 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 Ca 2+ 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 calcium/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 (InsP(3)R) [47] present in the osteoblast, which serve to increase
the intracellular 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 were independent of systemic calciotropic factors in a concentration-dependent way [42, 48].
These include osteoblast chemotaxis [36], DNA synthesis [49], proliferation, differentiation [50], and
mineralisation of extracellular matrix [51-52]. Interestingly, Ca2+ also induced the expression of
osteogenic growth factors, such as parathyroid hormone-related peptide (PTHrP) [53], BMP-2 and
BMP-4 [52]. Similar effects were observed when osteoblasts were cultured on Ca2+-functionalised
biomaterials, such as nanocrystalline 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]. Encouragingly,
Ca2+ has been implicated recently as an important messenger involved in the non-canonical β-cateninindependent Wnt/Calcium signalling for bone formation [59]. This pathway relies on an intracellular
release of Ca2+ to activate calcium sensitive enzymes like Ca2+-CaMKII, protein kinase C (PKC) or
calcineurin.
Essentially, the release of PO43- at the resorption site also plays a role in osteoblast proliferation and
differentiation. In fact, PO43- has been identified as an important signalling molecule that regulates cell
5
cycle and proliferation rate, alteration of signal transduction pathways (e.g. Fos-related antigen-1 (Fra1) [60] and extracellular signal-regulated kinase (ERK1/2) [61]), gene expression (e.g. osteopontin)
[62], as well as the secretion of bone-related proteins (e.g. matrix Gla protein (MGP) [63]).
Contradictory, 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 nonphysiological mineral deposition [64]. Nevertheless, Pi is believed to play a critical role in
physiological bone matrix mineralisation [65]. This mineralisation event is mediated by the enzyme
ALP and has been associated to 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]. Unfortunately, the use of Pi-functionalised biomaterials for tissue engineering
applications is rarely described, possibly due to the technical limitations of existing technologies on
handling Pi in an ionic form or the lack of awareness on the potential use of Pi. However, the use of
polymers mixed with phosphate salts as a sustained delivery of Pi to induce in vivo mineralisation of
the carrier has been reported recently [66].
Despite of the vast in vitro research findings, the influences of Ca2+ and PO43- 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 desired in vitro cell behaviour effectively. For instance, our group has recently
reported on the identification of specific Ca2+ and Pi concentrations that could induce higher
proliferation 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
concentration of Ca2+ and Pi) that could optimally initiate higher proliferation, osteogenic
differentiation and bone-like matrix deposition, both in two-dimensional (2D) cultures and threedimensional (3D) constructs. This represents a novel strategy to produce 3D bone reparative units that
may have predictive osteoinductivity for an effective repair of large skeletal defects [69].
(3) The Rationale of 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 critical). However, delayed healing and non-unions still often develop and will occur
more frequently due the aging of the population. In the United States, approximately 6 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
non-unions [70]. Therefore, the need for bone tissue regeneration is continously increasing and the
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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 at offering 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 micro-environment that promotes local bone repair or regeneration [71-72]. In this
context, CaP bioceramics appear to be interesting candidates for bone tissue engineering applications,
due to their biomimetic properties, supported by the following findings. Firstly, in the event of
endochondral ossification, mineralised cartilaginous matrix is reported to induce osteoprogenitor
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 formation [74]. Secondly, the bone matrix calcification or
mineralisation process is a critical stage of bone formation, either through direct mineralisation of
bone matrix or the pre-formation of cartilaginous tissue template that is mineralised at a later stage.
Thirdly, the dissolution of CaP-based biomaterials, either physicochemically or cell mediated upon
implantation, may also resemble the physiological bone resorption process [75]. Finally, the discussed
data in the previous section show that the effects of Ca2+ and Pi on osteogenic 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 mineralisation of the bone matrix. Due
to chemical and biological similarities, HA derived from natural sources (e.g. bone allograft, autograft,
or coral) or synthetic HA, is widely used as bone filler for treating skeletal defects. However, HA is a
highly stable CaP mineral and has therefore a lower solubility at the physiological pH (7.2 – 7.6) as
compared to other types of CaP that have higher solubility [such as tricalcium phosphate (TCP) and
octacalcium phosphate (OCP)]. Because the dissolution behaviour has been associated to the
osteogenicity as well as the osteoinductivity of CaP [75], the search for an improved CaP-based
biomaterial with higher osteoinductivity, also targets CaP with an appropriately high solubility. This
may be more effective in stimulating osteogenic differentiation of stem cells and initiating bone
formation. However, the exact mechanism of osteoinduction by CaP is currently unknown[67, 71, 76].
In the mid 60’s, 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 (BMPs) as
potent inducers of ectopic bone formation [78], the term “osteoinduction” has generally been used to
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describe an observation of heterotopic bone formation in association with the in vivo ectopic bone
inductivity of a substance, such as growth factors, chemical compounds or biomaterials. In
pathophysiological conditions, ectopic bone formation was induced upon tissue calcification in vivo,
such as in tendons and arteries [79]. This phenomenon was associated to the osteogenic 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 the past decades suggest that:
(i) the presence of a CaP component within a biomaterial (e.g. CaP-based ceramics [82], composite
[83], coating [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 (polyHEMA) sponge [87], and chemical-treated oxidised titanium substrate [88]), are the pre-requisite 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 macrostructures (e.g. dimension, geometry
and porosity), the micro/nano structures [90] (e.g. microporosity [91], grain size, surface topography),
and the chemical composition and characteristics of the biomaterials (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 animal 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 intramuscular than subcutaneous implantation). At
present, the hypothetical mechanisms of CaP-driven osteoinduction from a material perspective can be
classified into four categories:
(a) 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 significant
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 differentiation of stem cells [94], (v) the total surface
area available for protein adsorption (e.g. endogenous BMPs), cell attachment (which activates a
BMP-2 autocrine loop via 2β1 integrin [95] ) and growth, and (vi) ion dissolution and reprecipitation [93] which contributes to the formation of a biological apatite layer that in turn stimulates
the osteogenic differentiation of stem cells.
(b) Indirect effect of the biomaterial
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Osteoinductive proteins such as BMPs and transforming growth factor-beta (TGF-beta) are known to
have a high affinity to CaP [96]. It has been hypothesised that CaP-based biomaterials may act as an
in vivo affinity column/concentrator of the endogenous 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].
(c) Inflammatory response
Upon implantation, CaP-based biomaterials elicit an inflammatory response that attracts the
infiltration of mono- and multinucleated cells, and subsequently activates osteoclastogenesis which
results in CaP degradation and resorption [98]. Subsequently, the released Ca2+ and PO43- stimulate
osteoprogenitor differentiation and bone matrix deposition [99-100].
(d) 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 tendonitis. The
bone tissue formed often lacks functionality and eventually may resolve over the time. This resorption
process may be related to a foreign body reaction or due to the depletion of biochemical cues upon
implantation.
(5) The 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 (eg. bone fillers, carriers, cement) [101], either as pure CaP or as part of a
composite [102], and (ii) CaP coatings to functionalise biomaterial surfaces [103]. The use of CaP as
growth factor delivery system [104] is also discussed, specifically BMPs-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 at finding the optimal construct geometry for CaP scaffolds that could enhance
osteoinductivity and hence improve the clinical outcome [106]. This includes manipulation of the
architecture of biomaterials, such as the geometry, macro- and micro-porosity and surface topography
[107]. The aim is to adjust the surface area for the entrapment of endogenous osteoinductive
biomolecules and to accomodate optimum osteogenic cell density, as well as to fine-tune the
dissolution kinetics that optimally elicit osteogenic differentiation and bone matrix deposition. For
instance, production of cylindrical hydroxyapatite (HA) scaffolds with a hollow center (2 - 4 mm)
were reported to enhance ectopic bone formation [108]. Also, incorporating HA scaffolds with
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cylindrical tunnels of 90 – 120 μm and 350 μm induced endochondral and intramembranous
ossification [109]. The use of biphasic CaP (BCP) scaffolds (HA:TCP = 65:35 wt%) with cubical
pores of 500 μm resulted in the highest bone formation as compared to the scaffold with lower (100
μm) or higher (1000 μm) 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 surface topography is beneficial for the formation of bone-like apatite and provides a
surface area that increases the dissolution of Ca2+ and PO43- ions [112]. This surface area was later on
found to be associated to the microporosity within the macropores of CaP, which affected the materialfluid interface dynamics and triggered osteogenic differentiation [91]. Indeed, CaP discs with larger
surface area induce higher in vitro ALP activity and are capable to concentrate more osteoinductive
molecules that differentiate cells into the osteogenic lineage [113]. A similar phenomenon was
observed in an in vivo study, where CaP carriers with lower CaP grain size and higher microporosity
(and thus higher ionic dissolution kinetics) were found to be osteoinductive without using stem cell
technology [75]. The surface area can also be increased by the production of CaP microparticles, as
their osteoinductive properties are highly correlated to their size. It was shown that microparticles with
size ranges from 80 – 300 μm demonstrated ectopic bone formation, whereas particle sizes above 500
μm were not osteoinductive [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 was critical for
osteoinduction (figure 3). Unfortunately, the availability of a technology to enable a high
controllability and reproducibility on these desired features is currently lacking.
Also biphasic CaP (BCP) composites, being mixtures of HA with CaP with a higher solubility (such as
β-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 stimulate osteogenic differentiation of
osteoprogenitors [117-118] and meanwhile provide a stable phase of CaP (i.e. the HA) that 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, either due to a too high stability or a too high
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 optimised HA:TCP ratio has been reported until now [122]. This is mainly due to
the large variations in the bone forming capacity by studies using BCP with different HA:TCP ratio,
which is also species and material specific [76]. Due to the lack of mechanical strength, these biphasic
CaP biomaterials are more suitable for the treatment of skeletal defects at non-load bearing sites, such
as bone fillers for cranial defects. Nevertheless, BCP alone was reported recently to successfully heal
large bone defects in the clinic together with the use of an external fixator [123].
5.2 CaP coatings on biomaterials
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Due to the brittleness of CaP, CaP is often combined with a mechanically superior biomaterial like
titanium. This is necessary 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 to deposit CaP onto the surface of Ti-based biomaterials [125], including a recent
method that is based on the immobilisation of chemical moieties to induce mineralisation on the
surface of biomaterials both in vitro [69] and in vivo [126]. These methods aim at producing
composites containing mineralised components that are highly mimicking the hierarchies and
bioactivity of the mineralised compositions of the bone, in order to provide an inductive environment
that is capable of triggering bone formation. This directs us again to the principle question regarding
what type of CaP has to be deposited onto the biomaterial of choice to ensure sufficient and predictive
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 threedimensional porous structures. This represents a major drawback for many of the existing methods,
where deposition of CaP onto complex 3D structures is technically challenging [127]. For instance, as
an effort to overcome this major drawback, our research group has developed the perfusion
electrodeposition (P-ELD) technology to deposit CaP coating homogenously onto a complex 3D Tiscaffold 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-carriers have been investigated to enhance
bone formation through mediated delivery of bone-inducing factors (including biomolecules and
metallic ions [129]), with some promising results reported up to date [130]. An example of such
factors are the bone morphogenetic proteins (BMPs), discovered by Urist et al., 1965 as potent
proteins with the ability to induce heterotopic bone formation [77]. These proteins are members of the
TGF-β family which are secreted by chondrocytes, osteoblasts and osteoclasts [131-134], and play
pivotal roles throughout embryonic skeletogenesis and in postnatal bone formation and endogenous
repair mechanisms [130]. Indeed, BMPs 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 the molecular cascades 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 extensively
studied 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
11
bone formation via the endochondral pathway, through the formation of an intermediate cartilage
template [135, 139]. Unfortunately, the currently used delivery system for BMPs causes rapid protein
release and diffusion, which besides inducing bone formation, also causes inflammation and excessive
bone formation. Consequently, the uncontrollable or improper release kinetics resulted in undesirable
side effects such as male sterility, cancer and brain injury [140]. An additional reason for this could be
receptor saturation at higher doses or limited avaliability of responding cells, which can lead to
stimulation by these BMPs at undesirable locations. Moreover, the carrier composition may affect the
regenerative response by BMPs 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 BMPs were encapsulated in biocompatible biopolymers [141]. Therefore, an attractive approach
is to investigate carriers which may favour spatiotemporal physiological protein release but yet
promote recruitment of endogenous progenitor cells. These carriers also need to provide a suitable
microenvironment which promote 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 by the use of osteoconductive and osteoinductive
CaP-carriers [11, 68, 142-143]. These carriers possess suitable characteristics (eg. the interconnected
microporosity and the variation in CaP ratio) that are critical for the release kinetics of BMPs, as the
electrostatic energy possibly plays a dominant role in carrier-to-BMP interactions (high binding
affinity of BMPs to CaP), as well as in up-scaled constructs where multiple carriers may be combined
[144-145].
Even though CaP carriers in combination with BMPs sounds like a promising approach, an
impediment may be variation in activated bone forming pathways. The osteoinductive effect of CaPcarriers have displayed to form bone mainly via an intramembranous pathway, whereas BMPs mainly
induce bone formation via the endochondral pathway. However, Eyckmans et al., showed in 2010 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 BMPsignalling as well as abrogated osteoinduction in the construct after inhibition of endogenous BMPs
[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 BMPs, already
displayed by Urist et al., 1965, where partially demineralised bone induced inflammation and inhibited
osteogenesis [77]. Additional complications regarding these issues were shown by Wehof et al., 2002
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
12
combination with large amounts of rhBMP-2 in this study displayed an ability to attract a greater
amount of inflammatory cells, which increased in time, together with multinucleated cells. Absence of
collagen type II prior to hypertrophic chondrocyte differentiation and mineralization of the formed
cartilage tissue, together 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,
therefore indicating an intermediate pathway that may have been activated [148].
In conclusion, BMPs mainly affect bone formation via an endochondral process whereas CaP-carriers
mainly induce an intramembranous pathway. Possibly, the combination of appropriate concentrations
of specific BMPs 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 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, like 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 (figure 4).
Currently, many computational models of bone formation and regeneration in general [154], or even in
scaffolds specifically [11] exist. 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) a face-centered cubic packing of the pores, (iii)
surface-controlled resorption, (iv) the resorption requires the presence of blood vessels (50 µm in
diameter) 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
validated with experimental data.
The theoretical model mentioned above looks, however, exclusively at geometrical scaffold properties
and does not include biological 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
13
[157]. They modelled the scaffold as a poroelastic material which resorbs in a linear, load-independent
fashion, i.e. the porosity will be increased by a 0%, 0.5%, 1% per iteration for low, intermediate and
high dissolution rates respectively [158]. Consequently, the size of all scaffold elements decreases
uniformly resulting in an overall volumetric reduction while the scaffold geometry remains unaltered.
Their calculations show that as scaffold 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 modeled the bone regeneration process inside 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]. SanzHerrera 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
linearly 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 a good qualitative
agreement between the obtained computational and experimental results. Although further validation
is necessary, the proposed multi-scale model is a useful tool to investigate 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 of mechanical properties at the macroscopic scale. Lacroix et al.
reviewed the current techniques used for scaffold development: from scaffold optimization of
scaffolds by mathematical models (e.g. FEM) to scaffold design using computer aided design (CAD)
and scaffold characterization by computed tomography (CT) [162].
Although the above models can be used to optimize some (mechanical) properties of scaffolds, e.g. the
porosity, the micro-architecture, the 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 modeled, neglecting the influence of the degradation products (e.g.
Ca2+ and Pi) on the cellular activities and bone formation processes. Carlier et al. developed and
implemented an experimentally informed bioregulatory model of the effect of calcium ions released
from CaP-based biomaterials on the activity of osteogenic cells and mesenchymal stem cell driven
ectopic bone formation [163]. The dissolution kinetics of the CaP scaffold were modeled by a general
14
empirical equation [164], assuming that the dissolution rate is proportional to the driving force (i.e. the
difference between the current and the saturated calcium concentration) and that the rate constant is
time-independent. The amount of bone formation predicted by the model of Carlier et al. corresponded
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 model was used to optimise 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 useful to customise the CaP carriers to the 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 multidisciplinary problem of optimizing scaffold
architecture and seeding protocols for bone tissue engineering strategies requires an integrative
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
provide data for the determination of model parameters as well as the validation of the mathematical
model. This integrative approach requires a careful design and an extensive characterisation of the
scaffold. Moreover, this process is intrinsically iterative: new experimental results can be fed to 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 the bone induction by CaP biomaterials 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 molecular signalling at the cellular level (such as receptor
binding of the released Ca2+ and PO43- 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 predictiveness 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 customisation of the CaP-based biomaterial to overcome these limitations.
Moreover, other parameters present in vivo, including the compositions and dynamics of body fluids
and blood vessel in-growth, are technically challenging to be translated into a simplified in vitro
15
setting. Recently, the reactivity of CaP-based biomaterials in culture medium was revealed to have
significant influences on Ca2+ and PO43- dissolution kinetics and the subsequent cellular behaviour,
which was not correlated to the ion dissolution behaviour evaluated in simulated body fluid (SBF) or
phosphate-buffered saline. This must be carefully considered when evaluating the osteoinductive
potential of the material [167]. We propose the standardisation of in vitro dissolution tests to evaluate
the ions release kinetics of a CaP-based biomaterial, where the in vitro dissolution experiment should
resemble and be customised to the in vivo environment as close as possible [156]. Firstly, the CaP
scaffolds should be thoroughly characterised (porosity, pore size distribution, grain size, surface
topography and surface area, composition and etc.) before and after the dissolution test. Here, we
highlight on the use of non-invasive methods including Raman spectroscopy [158] and micro- or
nano-focus X-ray computed tomography
to obtain quantitative characterisation of the material
properties [168]. Secondly, we propose a dual solution system for the in vitro dissolution testing using
simulated body fluid [169] and culture medium (with and without the presence of cells) [170]. This
will allow for the characterisation of the intrinsic ionic dissolution behaviour of the biomaterial in a
solution having similar ionic composition to the bodily fluid, followed by the understanding on 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 of 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 condition (e.g. using perfusion
system) at physiologically relevant flow rate [118, 127].
Moreover, the in vitro and in vivo experiments should be designed in such a way that they minimise
variability and enable quantification, thus providing the essential data for the determination of model
parameters as well as for the model validation. It is of note that as mathematical models predict the
dynamics at different 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 future, 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
optimisation of CaP scaffolds for bone tissue engineering applications [165]. An essential
characteristic of these integrative research efforts is their iterativity: model analysis can lead to new
research hypotheses and new experimental findings can be used to improve the predictive capacities of
the model.
16
In conclusion, this review 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 unravel
the mechanisms of osteoinductivity of CaP scaffolds, many questions still remain unresolved. To
overcome this bottleneck, it is therefore essential that experimental and computational research are
combined so that the complex in vivo biological process of bone regeneration inside CaP constructs
can be deciphered in an effective and systematic manners. This includes computational modelling and
correlation analysis between the physicochemical properties of CaP constructs and the influences on in
vitro and in vivo biological outcomes, thus identifying the critical parameters and thorough
understanding of the underlying biological that govern osteoinductivity of CaP constructs. Only in
this way, it will 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 PhD
fellows of the Research Foundation Flanders (FWO Vlaanderen). The work is part of Prometheus, the
Leuven Research and Development Division of Skeletal Tissue Engineering of KU Leuven:
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.
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Figure Legends:
Figure 1: (a) Composition and (b) mechanical properties of human bone [Athanasiou KA et al. 2000;
Keaveny TM et al. 2001].
Figure 2: Theories of biomineralisation 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 H. Nat Mater. 2010]. (b) Matrix vesicle (MV)
mediated matrix mineralisation. Schematic showing the three possible mechanisms for the initiation of
MV-mediated matrix mineralisation [adapted from Golub E.E. Biochimical et Biophysica Acta. 2009].
PPi, pyrophosphate.
Figure 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 around 1.0 m2/g (the cut-off point). [Data are adopted from
Habibovic et al. 2005; Li et al. 2008; Yuan et al. 2010].
Figure 4: The concept of integrating manufacturing process, stem cell technology and computational
modelling as a novel strategy to design and optimise CaP-based bone reparative unit for effective bone
regeneration that can also assist the translation to personalised bone regeneration.
25