An overview on biological effects of trace-element in substituted calcium phosphates
R.M. Guerra Bretaña1, J.R. Guerra-López2, L.A. de Sena3
1
Biomaterials Center, University of Havana, Havana, Cuba
2
Basic Sciences Department, National University of Lujan, Luján, Argentina,
3
Faculty SENAI, Rio de Janeiro, Brazil.
Abstract—Calcium phosphate materials have been widely
used as bone substitute due to its good osteointegration. To im-
prove their osteoinductive and osteoconductive properties sev-
eral element-substitutions in the calcium phosphate matrices
have been explored. In the present work, an overview of biolog-
ical effects of some substitutions in calcium phosphates is done
based on an extensive literature revision. Incorporation of ele-
ments such as magnesium, fluoride, manganese, zinc, silicon
and strontium enhance the bone induction and conduction of
calcium phosphate materials. However, this behavior cannot be
attributed only to the elements release. The analyzed literature
indicated that element substitutions in low concentrations in-
crease the solubility of the calcium phosphate matrices, increas-
ing their bioactivity. Doped materials result osteoinductive and
osteoconductive.
Keywords— calcium phosphates, ionic substitutions, osteoin-
duction, osteoconduction.
I. INTRODUCTION
Calcium hydroxyapatite (CaHap, Ca10(PO4)6(OH)2) and
tricalcium phosphate (TCP) are widely recognized as poten-
tial biomaterial for clinical application due to their chemical
similarity with inorganic component of bone and tooth
mineral. In fact bone mineral phase is a carbonated apatite
that contains trace elements such as magnesium (Mg), so-
dium (Na), fluoride (F), manganese (Mn), zinc (Zn), silicon
(Si) and strontium (Sr) which are considered essential for
bone metabolism [1, 2]. Carbonate, sodium, and magnesium
ions are the major dopants of apatitic bone mineral.
Calcium phosphate (CaP) biomaterials exhibit osteointe-
grative properties, which mean that, after implantation, they
allow deposition of bone cells on their surface followed by
bone grow. However, when a bone defect exceeds a critical
size, it is not enough to repair the defect and osteoinductive
materials, which stimulate the osteogenesis of nearby undif-
ferentiated mesenchymal stem cells, inducing bone for-
mation, and osteoconductive properties, meaning the in-
growth of vascular tissue and mesenchymal stem cells into
the scaffold structure of the graft material are needed [3].
To improve the biofunctionality of synthetic calcium
phosphates several element substitutions have been explored.
These substitutions can be easily obtained by adding the cor-
responding ions into the reactions media. The most common
© Springer Nature Singapore Pte Ltd. 2017
I. Torres et al. (eds.), VII Latin American Congress on Biomedical Engineering CLAIB 2016,
Bucaramanga, Santander, Colombia, October 26th -28th, 2016,
IFMBE Proceedings 60,
DOI: 10.1007/978-981-10-4086-3_20
example is the preparation of carbonate-substituted [4] and
fluoride-substituted CaHap [5]. Another approach is the in-
corporation of divalent cations (Mg(II), Sr(II), or Zn(II)) into
its lattice [1, 5, 6]. TCP also has undergone element substitu-
tions [7]. This paper presents an overview on biological
effects of some substitutions in calcium phosphates.
II. MATERIALSAND METHODS
Selected articles on the topic of interest were analyzed.
The papers were searched on ScienceDirect and Scopus-
Elseiver data bases, using the key words “biological effect”
+ “substituted calcium phosphates”.
III. RESULTS
A. Zn-doped calcium phosphate materials
Among mentioned substituents, Zn(II) deserves some at-
tention because it is one of the essential trace elements in hu-
man bone and plasma [7] and due to their inhibitory activity
to osteoclastic bone resorption and its capacity to induce
apoptosis of mature osteoclasts [8]. Zinc is important in many
biological functions, for instance, this element participates in
the activity of enzymes like alkaline phosphates, which take
part in bone metabolism.
It has been reported that in the concentration range of
10-8–10-5 M (0.65 μg L-1 to 0.65 mg L-1), Zn inhibits osteo-
clast like cell formation in mouse marrow cell cultures and,
in concentrations as low as 10-14 M (0.65 pg L-1) constraints
rats osteoclastic bone resorption in vitro [9]. Moreover, zinc
deficiency causes bone growth retardation and it is a risk fac-
tor of osteoporosis in humans [10]. Because these character-
ristics, it is expected that Zn-containing materials would re-
lease Zn(II), and influence the differentiation of osteogenic
cells promoting the regeneration of adjoining host bone [11].
It was demonstrated that Ca-deficient apatite doped with
Zn could be prepared up to Zn 9.1 wt%, because at higher
concentrations, Zn(II) doping shows inhibitory effect on
apatite formation [12]. Zn-doped CaHap, with Zn content be-
low to 6.5 wt%, has a structure comparable to those ones of
animal bones.
78
An overview on biological effects of trace-element in substituted calcium phosphates
Supplementing zinc in the medium significantly increases
the osteogenic differentiation of both rat and human bone
marrow stromal cells (BMSc) [13]. It is also involved in the
osteoclastic differentiation from macrophages [14].
Bhattacharjee et al. [15] studied the in vivo performance
of Zn-doped porous CaHap scaffolds in critical sized tibial
defects of rabbits. It was observed that Zn-substituted CaHap
showed better osteointegration than undoped material. SEM,
histopathological study, boxytetracycline labeling study and
mechanical push out test further confirmed superior bone–
implant attachment of Zn-doped CaHap.
Other in vivo experiment shows that Zn-TCP induced ec-
topic bone formation in canine muscle related to the concen-
tration of zinc they contained; however, TCP without Zn did
not formed bone in ectopic sites [16].
B. Mg-doped CaP materials
Among substituting cations, magnesium is widely studied,
being the fourth most abundant cation in the human osseous
tissues [17]. Enamel, dentin and bone contain, respectively,
0.44, 1.23, and 0.72 wt% of Mg [18]. It is well known that
Mg is closely associated with the mineralization of calcified
tissues, directly stimulating osteoblast proliferation. Mg
depletion adversely affects all stages of skeletal metabolism,
causing cessation of bone growth, decreased osteoblastic and
osteoclastic activities, osteopenia and bone fragility. Conse-
quently, the incorporation of Mg ions into the CaHap lattice
is of great interest for the developing of artificial bone sub-
stitutes. It has been shown that the presence of Mg(II) within
CaHap lattice sensibly affects apatite crystallization in solu-
tion and its thermal stability, promoting the formation of
β-TCP and thus forming biphasic calcium phosphates (BCP).
Besides, the incorporation of Mg(II) stabilizes the β-TCP
phase, increasing its transition temperature to β-TCP above
1125 ºC. These materials are considered promising substi-
tutes for bone replacement due to their unique biological
features.
Mg 0.55 wt% inhibits the crystal growth of synthetic apa-
tites and it is associated with increased solubility of Mg-Ca-
Hap [8]. Higher cellular activity was observed in vitro with
materilas containing high level of Mg(II) ions in CaHap
lattice [18] in comparison with pure CaHap.
C. Si-substituted CaP materials
In the early 1970s, Carlisle [19] determined that silicon is
an essential trace element for metabolic processes associated
bone and connective tissues development. Schwarz and
Milne [20], who recognized Si as the crosslinking agent in
the connective tissues and its importance to vascular health,
IFMBE Proceedings Vol. 60
79
confirmed these findings. Since that date, one of the most fre-
quently studied substitutions in CaPs is the anionic substitu-
tion of silicate for phosphate in the CaHap structure.
A silicon doped calcium phosphate materials have been
reported as conducive to both osteoblast deposition and oste-
oclast resorption, allowing it to participate in the bone remod-
eling processes [21-25]. Khan et al. [24] presented a compre-
hensive overview about the role of silicon in enhancing the
biological performance and bone forming capabilities of con-
ventional calcium phosphate based bioceramics.
Porter et al. [23] observed that an increase in concentration
of Si in hydroxyapatite (between 0 and 1.5 wt%) increase the
dissolution and the bioactivity of CaHap implants after 6 and
12 weeks in vivo. Their observations confirmed that defects,
in particular those involving grain boundaries, were the start-
ing point of dissolution in vivo.
D. F-substituted CaHap
Fluoride ions added to cell culture media were shown to
have effects on in vitro activities of osteoblasts and osteo-
clasts, influencing on cell proliferation, extracellular matrix
production, and alkaline phosphate activity [26]. Further-
more, NaF not only acts as potent stimulator of ongoing os-
teogenesis from already differentiated osteoblasts, but also it
influences in the initiation of osteogenesis from embryonic
mesenchyme in vitro [27]. However, high F concentrations
may exert a toxic effect on osteoblast function [28]. On this
basis, it has been shown that F ion and F-substituted apatite
promoted osteoblast proliferation and inhibited osteoclast
cell activity. Inoue et al. [29] investigated the in vivo rat tibia
activity on F-substituted unsintered calcium deficient apatite,
with various F concentrations for 1 and 2 weeks. Results
showed that low F concentration (0.48 wt%) induced better
and faster new bone formation in vivo compared to calcium
deficient apatite and high fluoride concentration material
(2.23 wt%). Therefore, they concluded that F had a suitable
effect on bone formation in vivo.
E. Sr-doped CaP materials
Strontium has known influence as promoting bone mass
and bone strength agent, due to a dual mechanism, enhancing
osteoblast differentiation and inhibiting osteoclast differenti-
ation. Even when incorporated into hydroxyapatite Sr(II)
stimulates osteoblast activity and exerts its inhibitory effect
on osteoclast proliferation [30]. Marques et al. [31] studied
the antibiotic release and biocompatibility of strontium and
magnesium co-substituted biphasic hydroxyapatite/β-trical-
cium phosphate powders with composition (Ca+Sr+Mg)/P =
1.62 impregnated with levofloxacin. The Sr-doped granules
exhibited the best drug release profile and the highest proli-
80
feration yields and efficiency in osteoblastic maturation, in-
cluding morphology differentiation and high levels of se-
creted alkaline phosphatase.
F. Doped calcium phosphate nanoparticles and coatings
Devanand et al. [32] developed a drug delivery system
with zinc-doped CaHap nanoparticles loaded with Cipro-
floxacin for use in bone infections. They found that the anti-
microbial activity against Pseudomonas aeruginosa and
Staphylococcus aureus increases with increase in the drug
concentration and the amount of Zn and that the drug release
from zinc-doped hydroxyapatite is higher than from pure hy-
droxyapatite. The implantation of these nanoparticles is
expected to induce cell growth and antimicrobial activity.
Zinc substituted CaHap nanoparticles (1.6 wt% Zn) have
been found to enhance bioactivity to human adipose-derived
mesenchymal stem cells, along with the increase in the bone
cell differentiation markers [33]. Zn-CaHap nanoparticles
also exhibit antimicrobial capability against Gram-negative
and Gram-positive bacterial strains [34, 35]
Due to good biocompatibility calcium phosphate coatings
on metallic biomedical implants are also widely employed in
orthopedic and dental applications, with the aim of improv-
ing osteointegration between bones and metallic implants
[35]. Chung and Long [36] attempt to enhance the osseointe-
gration of titanium implants by fabricating Sr-CaHap
coatings. Obtained results indicate Sr-CaHap coating had
higher osteoblast compatibility than raw titanium metal and
the CaHap coating. Moreover, Sr(II) in low concentrations
stimulates osteoblast adhesion and proliferation. However,
high Sr concentrations (38.9 at.% Sr) significantly inhibits
osteoclast differentiation.
Composite coatings, containing silicon substituted CaHap
and poly-(ε-caprolactone) on titanium substrate, have im-
proved bonding strength [37].After immersion in simulated
body fluid for 8 days, the composite coatings have the ability
to induce the bone-like apatite formation.
Mg-substituted and Sr-substituted octacalcium phosphate
(OCP) was also studied as thin films on titanium substrates
[38]. Human osteoblast like MG-63 cells were cultured on
the studied biomaterials up to 14 days. MgOCP and SrOCP
coatings promote osteoblast proliferation and differentiation
with respect to OCP. The level of differentiation of the cells
grown on the different coatings increased in the order
SrOCP>MgOCP>OCP.
IV. DISCUSSION
Studied ion-substitutions enhance the osteoinductive and
osteoconductive properties of calcium phosphate materials.
IFMBE Proceedings Vol. 60
R.M. Guerra Bretaña, J.R. Guerra-López and L.A. de Sena
The in vitro cellular results and release behavior of the minor
elements studied correlate with the in vivo responses. These
effects are synergistically combined with the osteointegrative
properties of the calcium phosphate materials, due to their
ability for protein adsorption based on surface features,
chemical composition and hydrophobic properties.
Despite that, the biological mechanisms of the interactions
of bioceramics and proteins are still not completely under-
stood [39]; the widely accepted theory for bone bonding to
calcium phosphate based bioceramics is based on the disso-
lution–reprecipitation mechanism of apatite nucleation [40-
42]. As it is explained in this theory, dissolution of calcium
and phosphate ions from the bioceramic surface increases
their concentration locally, leading to the precipitation of bi-
ological apatite heterogeneously on the surface of proteins
present close to the bioceramic implant or coating or directly
on its surface. Next, more proteins and osteoblasts are ab-
sorbed on the modified bioceramic surface promoting cell ad-
hesion and bone formation.
Even though, Porter [43] determined that dissolution is not
a prerequisite for osteointegration, he also confirmed that it
accelerates nucleation of biological apatite. Thus, the gener-
ation of defect centers with elemental substitution in CaP im-
plants or coatings enhances the in vitro dissolution of the ma-
terials with a corresponding increase in their osteoinductive
capacity.
Although many studies of HA substitutions have already
been made, and many papers have been published, there
search results are in many cases so far from commercial
implementation. Studies relating co-doped CaHap materials
have also been conducted [44]. Much interdisciplinary work
remains to be done to transfer interesting research findings
into valuable materials for use in medical applications.
V. CONCLUSIONS
Doping of CaP materials, with elements important to bone
metabolism such as Zn, Mg, Sr, Si, F, brings an increment in
bone induction and conduction. This behavior cannot be at-
tributed only to the elements release. The analyzed literature
indicated that element substitutions in low concentrations in-
crease the solubility of the calcium phosphate matrices, and
play an important role in their bioactivity. This review con-
tribute to deeper understanding that addition of mentioned el-
ements is a useful strategy to convert calcium phosphates in
really osteoinductive and osteconductive biomaterials.
CONFLICT OF INTEREST
The authors declare that they have no conflict of interest.
An overview on biological effects of trace-element in substituted calcium phosphates 81

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Author: Rosa Mayelin Guerra Bretaña
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Institute: Biomaterials Center, University of Havana
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Street: Ave Universidad
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City: Havana
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Country: Cuba
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Email: mayelin@biomat.uh.cu
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