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Bioactive ceramics: From bone grafts to tissue engineering

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RSC Advances

REVIEW

Bioactive ceramics: from bone grafts to tissue

engineering
Cite this: RSC Advances, 2013, 3,
11116
Antonio J. Salinasab and Marı́a Vallet-Regı́*ab

Received 12th January 2013,
Accepted 25th April 2013
DOI: 10.1039/c3ra00166k
www.rsc.org/advances
Bioactive ceramics bond directly with living tissues when implanted. For this reason they have been
profusely investigated as biomaterials. The first synthetic bioactive materials were specific compositions of
glasses and glass ceramics as well as sintered hydroxyapatite. However, all these bioceramics are brittle,
and for this reason their main application for years has been as a grafting material for the filling of small
bone defects and periodontal anomalies. The efforts to expand the applications of bioactive bioceramics
were mainly focused in two areas: (A) the synthesis of organic–inorganic hybrids to apply in tissue
engineering and of ceramic coatings on metallic substrates for applications requiring good mechanical
behavior, and (B) the synthesis of porous materials with very quick bioactive response that can be
upgraded by adding biomolecules or therapeutic inorganic ions to be used in bone tissue engineering. For
these developments, the in vitro studies in solutions mimicking blood plasma played a major role. At the
present, it is universally considered that both bioactive and biodegradable materials are going to play a
central role in the fabrication of porous scaffolds that after being decorated with cells and signals form
constructs: basic elements of tissue engineering. This article reviews the pathway followed by the bioactive
materials from their original applications in bone grafts to the present day where they are widely
investigated as porous scaffolds for bone tissue engineering. After defining the concept of bioactivity,
important bioactive materials will be listed in this article. Then, the specific characteristics of bioactive
materials when used in bulk or coatings as well as the comparison with biodegradable materials will be
presented. Finally, and after describing the in vitro studies for the evaluation of bioactive ceramics, the
main characteristics of template glasses, compared with conventional sol–gel glasses, and the advantages
of using porous bioactive ceramics to obtain scaffolds for bone tissue engineering will be explained.

1. Introduction: what is bioactivity? what
materials are bioactive?
The reactivity of a biomaterial with the living tissues plays an
essential role when used for the implants manufacture.1–3 Due
to the foreign body reaction, inert implants are isolated from
living tissues by a non-adherent fibrous capsule which is
formed after few weeks of implantation.4 This capsule favors
the occurrence of micromovements in the implant-tissue
interface that increase with time leading in many cases to
the prosthesis failure and its subsequent replacement need.
Looking for mechanical stability, two approaches were
investigated: biological fixation and bioactive fixation.5
Biological fixation was searched by designing materials with
rough surfaces and pores over 100 micrometers, to allow tissue
ingrowths and angiogenesis. Some limitations of this fixation
a
Departamento de Quı́mica Inorgánica y Bioinorgánica, Facultad de Farmacia,
Universidad Complutense de Madrid, 28040 Madrid, Spain.
E-mail: vallet@farm.ucm.es; Fax: +34913941786; Tel: +34913941861
b
Networking Research Center on Bioengineering, Biomaterials and Nanomedicine
(CIBER-BBN), Madrid, Spain
include the damage of tissues if interfacial movements
appeared or the prosthesis must be replaced.
On the other hand, bioactive fixation was discovered by
Hench et al. at the beginning of 70 s.6 In bioactive materials an
intimate biomaterial-bone apposition, leading to a mechani-
cally strong bond, is observed. Fig. 1 shows the different
behavior of inert and bioactive materials after implantation.
Bioactive materials have been widely investigated as
biomaterials in the last decades, mainly for hard tissues
substitution. The first bioactive material was a glass obtained
by quenching of a melt of composition (in wt%) 45% SiO2,
24.5% CaO, 24.5% Na2O and 6% P2O5, denoted Bioglass1
45S5.6 In the 70 s and 80 s it was described bioactivity for other
ceramics including new melt glasses,9–11 dense and porous
hydroxyapatite (HA),12–14 and some glass-ceramics.15–17 Later
on, many research efforts were focused in the obtaining of
materials with a quick bioactive response and/or improved
mechanical properties. For instance, glasses with different
composition or obtained by new methods of synthesis such as
sol–gel processing or template techniques were investigated.
Moreover, materials exceeding the mechanical limitations of
glasses, namely brittleness and high elastic module, were

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Fig. 1 Two behaviors of a biomaterial (BM) after implanted 4 weeks in a rabbit
femur. Left: an inert material appears surrounded a fibrous capsule (FC).
Medium and right: bioactive materials exhibit direct apposition with bone
tissues.7,8
synthesized. Thus, glass-ceramics were obtained by partial
devitrification of bioactive glasses. The remaining glassy
matrix was the main bioactivity source whereas the small
crystals formed actuate as reinforcing phase. Furthermore,
new families of bioactive materials were investigated including
organic–inorganic hybrids18–21, star gels,22,23 mesoporous
ordered silica materials,24,25 template glasses,26,27 metallic
alloys coated with bioactive ceramics28,29 or chemically
etched,30,31 and composite materials, frequently based on a
bioactive ceramic and a biocompatible polymer.32,33 In Fig. 2
materials exhibiting a bioactive behavior are depicted. As it
can be observed there are two categories based on ceramics
and metals respectively.
The bioactive behavior of some metals comes from the
ceramic component present on their surfaces. Indeed, the
chemical etch reported to convert an inert metal into bioactive
actually consists in the formation of a ceramic compound in
the metallic surface. In the next section examples of metal-
based bioactive materials will be described.
Fig. 2 Materials exhibiting bioactivity. Bioactive composite materials formed by
a bioactive ceramic in a polymeric matrix are also studied.32,33
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Bioactive materials are considered osteoconductive because
they provide the suitable surfaces to support the osteoblasts
adhesion and proliferation.34 In addition, a few glasses
exhibiting very quick bioactive response are also osteoinduc-
tive because besides facilitating bone growth they drive the
bone formation.34,35 Osteoinductive ceramics bond to both
hard and soft tissues, important to be considered in clinical
applications like the tympanic ossicles substitution or the
periodontal defects treatment. However, osteoconductive
materials only bond with hard tissues. Osteoconduction was
considered type B bioactivity and attributed to an extracellular
response.36 By contrast osteoinduction, that is, type A
bioactivity, of glasses with the highest bioactivity, was
attributed to an intracellular response provoked by the release
of great amounts of Si(IV) and Ca2+ ions which stimulating the
genes response to produce the formation of bone.37
2. Bioactive materials: bulk versus coatings
Bioactive materials are used in Orthopedics and Dentistry due
to their capacity to promote osteointegration. There, bioactive
calcium phosphates and silica-based glasses exhibit excellent
features as bone grafts in non-bearing applications, where the
bone regeneration kinetics is more important than mechanical
properties.38 However, their brittleness and low resistance to
fatigue is a drawback for repairing large bone defects. This is
especially so in pieces with high porosity percentages and
pores larger than 100 mm. For this reason, they were initially
used in particulate form for small bone or periodontal defects
filling.39,40 Nowadays, they are also used as metallic implants
coatings in load bearing applications like the hip prosthesis. A
very important research line in bioactive materials, that will be
described later, is the designing of scaffolds exhibiting
hierarchical porosity for bone tissue engineering applica-
tions.41
Although clinicians use to prefer autografts, taking bone
from the own patient, allografts, from a bones bank, or
xenografts, of origin animal, synthetic bioactive materials
present numerous advantages.42 For example, they have
neither morbidity problems nor risks of diseases transmis-
sion, they are cheaper and its availability is practically
limitless. For this reason synthetic materials are frequently
used, alone or in mixtures with bone particles, as bioactive
grafts.39
For these applications, an important parameter to control
is the particulate size, which depends on the clinical use.
Thus, HA or hydroxycarbonate apatite (HCA) bioceramics are
commercialized as 0.25–2 mm granules for dental grafts, and
with bigger size, 1–4 mm for orthopedic uses.43 It must be
considered that in these applications, besides favor bone
formation, the particles must remain into the defect con-
tributing to the mechanical stability. Very important is also to
control the particle size in highly bioactive materials whose
extreme superficial reactivity can become in biodegradable to
materials under a certain particle size. For example, it was
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Review
Fig. 3 Ti-6Al-4V coated with a CaO–SiO2–PDMS organic–inorganic hybrid by dip
coating. The coating protects metal from corrosion and brings new properties:
(A) in vitro bioactivity, due to it is coated by HCA after 7 days in SBF,50 and (B)
dumping forces ability, because its mechanical properties: the behavior of
hybrids with different PDMS contents is shown.51
described that bioactive glass particles smaller than 100 mm
are totally degraded into the body.44
Regarding the metallic coatings, a common approach is the
coating of Ti-6Al-4V or commercially pure (c. p.) Ti alloys with
calcium phosphates, such as HA.45 That way, the metal brings
mechanical support and the ceramic bioactivity. In addition,
HA coating protects the metallic substrate from corrosion.
This process is doubly harmful because it leads to the
deterioration of metallic implants and because it leads to the
metallic ions release that can affect biological functions. HA
coatings are usually produced by plasma spray because of the
rapid deposition rate at a relatively low cost. However,
alternative techniques to obtain calcium phosphate coatings
including physical vapor deposition, chemical vapor deposi-
tion, magnetron sputtering, electrophoretic deposition, pulsed
laser deposition and dip coating.46–49
In addition, extra properties can come from the coatings.
For instance, the Ti-6Al-4V coating with a CaO–SiO2–poly-
dimethyl siloxane (PDMS) organic–inorganic hybrid exhibiting
in vitro bioactivity was reported.50 Fig. 3 shows how this
coating confers bioactivity to the metal simultaneously
mitigating the metallic corrosion, but also, due to the elastic
behavior of SiO2–CaO–PDMS, it would be able to dump load
charges, a important property in specific dental and orthope-
dic applications.51
Another approach to obtain bioactive materials exhibiting
good mechanical properties was the synthesis of so called
bioactive metals.52 These metals were obtained by chemical
etch of a metal followed by a heat treatment. In Fig. 4 the
process to become in bioactive c. p. Ti is schematically
depicted. As observed, metal is initially coated by a TiO2
passive layer that confers it environmental stability and
inertness. The chemical reaction of TiO2 with NaOH produces
a sodium hydrogen titanate gel layer. After that, the thermal
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Fig. 4 Amorphous sodium titanate formed by chemical etches with NaOH of a
titanium substrate followed by a heat treatment. Inset: summarizing the process
to induce bioactivity in a metal and the in vitro bioactivity mechanism applied to
c. p. Ti.
treatment produces amorphous sodium titanate able to
exhibit a bioactive response. Although the metal is considered
bioactive, the real responsible of bioactivity was a bioceramic
compound: the sodium titanate.
Analogous treatments to induce bioactivity were reported
for other metals. Inside the rectangle of Fig. 4, the process to
induce bioactivity in a metal (M) coated by a MO2 oxide layer is
summarized. Besides, the in vitro bioactivity mechanism of c.
p. Ti treated is included. As observed in Fig. 4 inset after
soaking the bioactive metal in simulated body fluid (SBF) Na+
release together a Ca2+ uptake from SBF form calcium titanate.
Then, with the PO432 ions uptake amorphous calcium
phosphate is formed that crystallize into nano HCA character-
istic of materials exhibiting in vitro bioactivity.53
3. Biodegradability versus bioactivity
The surface reactions leading to the bond formation of
bioactive materials and living tissues start with a partial
dissolution (leaching) of the material surface.54 In this regard
it was demonstrated that osteoclasts induced degradation of
calcium phosphate ceramics by simultaneous resorption and
phagocytosis mechanisms.55,56 However, in bioactive glasses it
was observed that octeoclasts do not degrade the intact glass
matrix to any significant degree.57,58 Evidently, if small enough
particles of a bioactive ceramic are used, the surface
degradation will finally produce the total degradation of the
particles.59 That is to say, a material generally considered
bioactive could be converted into biodegradable if used in
particulates of very small size. Similarly, it could take place the
inverse situation, i.e., that a degradable material could be
dissolved only at superficial level as happens with bioactive
materials. This could happen when the biodegradable ceramic
was obtained in conditions producing a great particle size.
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Fig. 5 Some biodegradable materials. Non previously defined acronyms: PGA:
polyglicolic acid; PLA: polylactic acid, TCP: tricalcium phosphate; OCP:
octacalcium phosphate; DCPD: dicalcium phosphate dehydrate; DCPA: dical-
cium phosphate anhydrous; TetCP: tetracalcium phosphate.
Examples of how bioactive ceramics can become in biodegrad-
able can be obtained from the literature of Bioglass1 obtained
in particulate form. Thus, Wilson and Noletti found that
particles of 100 mm in diameter of Bioglass were resorbed or
phagocytosed by macrophages in vivo, while larger particles
were bioactive stimulating the bone growth.60 However,
Schepers et al. indicated that the particles under 300 mm in
size were fully resorbed in vivo.61 This controversy supports the
necessity to standardize the evaluation essays.
Due to its close relationship, bioactive and biodegradable
materials are usually studied together. In fact, many authors
considered both as the genuine examples of second generation
biomaterials denoted in this way to distinguish from first
generation, i.e. bioinert, and from third generation ones
actually under investigation as scaffolds for bone tissue
engineering.25,62
Important materials exhibiting biodegradability are
included in Fig. 5. They were divided in three categories: (i)
polymers from both synthetic and natural origins widely used
in sutures and drug delivery systems,63 (ii) ceramics, most of
them calcium salts: phosphates, sulphates or carbonates,64–66
and used as bone grafts and (iii) composites, constituted by a
combination of degradable polymers and/or degradable
ceramics.32,33 Still, a new category of degradable materials is
under investigation in the last years: they are the degradable
metals, mostly belonging to the category of magnesium and its
alloys.67–69 Regarding the degradable polymeric compounds
shown in Fig. 5, most of them are degraded by hydrolytic
cleavage of ester bonds, but some of them suffer an enzymatic
degradation or provoked by bacteria (authentic biodegrada-
tion). Moreover they can experience the degradation processes
by surface or volume mechanisms which clearly influence the
biomedical application of the degradable polymer due to the
most gradual and controllable features of the surface
degradation.70
A challenge in the design of biodegradable materials is to
adapt their degradation kinetics to the living tissue formation,
which is usually slower. Additionally, as resorption takes place,
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a decrease in their mechanical properties, which could be a
drawback depending on the role assigned to the biodegradable
material. It must be considered that these materials are
designed to help in the self-reparation processes of the living
organism for a given period of time and then to be resorbed.
That way, problems associated with long permanence of a
synthetic material into the body are avoided.
Second generation ceramics can display different crystal-
lization degrees, which leads to different properties. Thus,
there are crystalline ceramics such as crystalline hydroxyapa-
tite; amorphous solids, such as glasses; and intermediates,
such as glass-ceramics. In addition, bioactive and biodegrad-
able ceramics have been clinically employed in different
conformations, including powders, porous or dense pieces,
injectable mixtures and coatings depending on the required
application.
Another application of bioactive ceramics are the calcium
phosphate based bone cements.71–74 These materials were
designed to solve the polymeric acrylic bone cements limita-
tions that have been used for years, namely a temperature
close to 70–80 uC reached during the in situ setting that
produces necrosis in the adjacent tissues. These cements that
after setting become in bioactive or biodegradable calcium
phosphates are a great hope of future because they can adapt
to the shape of the bone defect or they even can be injected
without making a surgical incision achieving a dual function
to fix and to regenerate bone.
Finally, very popular bioceramics are biphasic calcium
phosphates containing a mixture of a bioactive ceramic such
as HA and a degradable calcium phosphate, such as
b-Ca3(PO4)2 (b-TCP).75,76 The role of the degradable phosphate
is to release phosphate and calcium ions to favors bone
formation and the generation of an extra porosity as a
consequence of its dissolution which facilitates the biological
fixation of the implant.
4. How to evaluate bioactivity
Along the development of new bioactive materials it was not
ethical, nor, perhaps, economically possible, to start testing
each new material directly in animals of experimentation. In
this sense, it had a great importance the discovering of the
important role on bioactive bond of the bone-like HCA layer
formed on the bioactive surfaces when implanted. The
capacity of a material to be coated by this layer when in
contact with physiological fluids was proposed as the cause of
bioactivity.1 HCA nanocrystals would serve as reinforcement of
collagen fibers during the bone regeneration process. HCA
layer is also formed when bioactive materials are soaked in
fluids simulating human plasma. This was the origin of so
called in vitro bioactivity tests which has played an essential
role in the development of new bioactive materials. In strict
sense, bioactivity refers to the absence of fibrous capsule
under in vivo conditions. Therefore those materials able to be
coated by an apatite layer in simulated body fluids but not
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Review
Table 1 Inorganic ionic composition of the different solutions employed for testing potential implantable materials compared with human blood plasmaa
Na+ K+ Mg2+ Ca2+
Blood plasma 142 5 1.5 2.5
Buffered solution — — — —
SBFc 142 5 1.5 2.5
Corrected SBF 142 5 1.5 2.5
CSIPd 142 5 1.5 2.5
a
Concentrations in nM. b Tris buffer = Tris(hydroxymethyl)aminomethane and hydrochloric acid. c SBF = Simulated Body Fluid.
Carbonated Simulated Inorganic Plasma.
implanted must be considered that exhibit ‘‘in vitro bioactiv-
ity’’.
In this section, different strategies used to determine the
possible bioactivity of a new candidate to implant material are
presented. Evidently a material must be deeply characterized
before be considered as candidate for the manufacture of a
biomedical device. Thus, after a physical-chemical character-
ization similar to materials for other applications, bioactivity
and biocompatibility of the new candidate as biomaterial must
be evaluated. Biocompatibility use to be first evaluated in cell
culture studies, then in experimentation animals, and finally
in clinical assays in humans. However, previously the in vitro
bioactivity studies use to be performed.
4.1 In vitro bioactivity studies
The origin of in vitro bioactivity tests was the selection of the
optimal biomaterials to continue with their development. In
addition, they became in powerful tools to propose mechan-
isms that explained the bioactive bond formation, conse-
quently influencing the development of new bioactive
materials. Diverse protocols for the in vitro tests were proposed
in which the characterization techniques have also played an
important role. These tests save money and resources since
they are cheaper and quicker than the cell cultures and the in
vivo tests with animals and humans.
4.1.1. In vitro tests with buffered aqueous solution. First
bioactive glasses were initially evaluated in Trishydroxymethyl
aminomethane/hydrochloric acid (Tris-buffer) aqueous solu-
tion buffered at physiological pH of 7.4.77 Because these SiO2–
CaO–Na2O–P2O5 glasses quickly leached amounts of calcium
and phosphate ions to the assay solution, the HCA layer
formation was easily detected with Fourier Transform Infrared
Spectroscopy (FTIR) just looking for the absorption bands of
phosphate not present in the initial glass. When bands of
crystalline phosphate appeared at 603 and 562 cm21 the glass
was considered bioactive. Otherwise was considered not
bioactive.
4.1.2. In vitro test with Simulated Body Fluid. By the end of
the eighties, Kokubo et al. have detected that A–W glass
ceramic bond to bone in vivo, i.e. it was bioactive, but it did not
formed the HCA layer when tested in tris buffer. These
findings inspired them to produce a more elaborated solution
that mimicked human plasma, the so-called Simulated Body
Fluid (SBF).78,79 SBF is an acellular ionic solution with an
inorganic ionic composition almost equal to human plasma
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Cl2 HCO32 HPO422 SO422 pH Buffer
103 27 1 0.5 7.35–7.43 Biological
— — — — 7.4 Trisb
148.8 4.2 1 — 7.4 Tris
147.8 4.2 1 0.5 7.4 Tris
103 24–27 1 0.5 7.4 CO2/HCO32
d
CSIP =
and buffered with tris buffer at physiological pH, as can be
observed in Table 1.
As observed, besides the presence of Tris buffer, the main
difference between SBF and the inorganic part of plasma is the
carbonate concentration: 4.2 mM in SBF and 27 mM in
plasma. The carbonate deficit in SBF is compensated by a
greater concentration of chloride ions, so the electroneutrality
of solution is maintained. On the other hand, the difference
between original SBF and corrected SBF is the presence of
sulphate ions.80 SBF that is a metastable solution because is
supersaturated with respect the apatite. For this reason, it
must be carefully prepared following a strict protocol to avoid
undesired precipitations and it must be used recently
prepared or after being stored refrigerated by one month at
maximum.81
Numerous modifications of both SBF composition and
protocol employed were proposed including the renewal of
solution at time intervals,82 its continuous renewal with a
dynamic system,83 the elimination of tris buffer and the
reaching of a physiological carbonate ions concentration84 or
the enrichment of SBF with proteins of plasma such as the
albumin.85 Many advances in mechanism of reaction were
made by using these protocols. However, the testing of a
material in corrected SBF is generally accepted by the scientific
community as the first step in the evaluation of a new possible
bioactive material. Nevertheless, attempts for the standardiza-
tion of the in vitro bioactivity tests including a round robbing
test for the glasses evaluation in SBF involving 10 laboratories
of 8 countries are in progress.86
Mechanisms bioactivity proposed from studies in SBF have
pushed the development of new bioactive materials. For
instance, it was detected the importance of the presence and
concentration of silanol (Si–OH) groups as well as the textural
properties (surface area and porosity) in the in vitro bioactivity
of glasses. Thus, it was found a direct relationship between
surface area and silanol concentration with the speed of the
bioactive response. This information yield to the synthesis of
porous sol–gel bioactive glasses exhibiting high surface area
and silanol concentration.77 Specific thermal treatments, the
optimum relative CaO to SiO2 proportion, or the role of
different glass components such as P2O5 were revealed.87–89
However, the actual revolution in terms of accelerated
bioactive kinetics came with the development of template
glasses, which will be described later, because of the out-
standing values of surface area and mesoporosity that
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Fig. 6 Different approaches for evaluating the in vitro bioactivity of bioceramics.
promotes a great reaction degree between the glass and
SBF.90,91
4.1.3. Parameters in the in vitro bioactivity tests. During the
in vitro bioactivity tests many parameters can be modified
including the in assay solution composition (see Table 1), the
static or dynamic conditions or the possible shape (powders,
compacts, etc.) of the evaluated materials.
A limitation of SBF comes from the presence of tris buffer
since it is not a blood component and it tends to complex
calcium ions, which could influence on the bioactive process.
For this reason other approaches were attempted. For
instance, our group proposed the substitution of tris buffer
by CO2/HCO32 buffer to produce so-called Carbonated
Simulated Inorganic Plasma (CSIP) with a carbonate concen-
tration of 27 mM.84 In spite of these improvements the system
exhibited higher experimental complexity that made us
recommend it for the study of specific bioactivity mechanisms
more than for the evaluation of new possible bioactive
materials. The use of commercial solutions stable for large
periods of time such as Hanks Balanced Salt Solution (HBBS)
has also be proposed.92
Fig. 6 collects different approaches proposed to perform the
in vitro bioactivity tests. Comparing with classical tests
indicated inside the rectangle, some improvements including
the stirring of solution, the renewal of solution at time
intervals or in a continuous way, the bubbling of gases (to
obtain CSIP) or the addition of plasma proteins, such as, the
albumin are indicated.85
The stirring condition is other parameter that influences the
HCA formation process. A certain level of stirring will ensure
the solution homogenization facilitating the HCA deposi-
tion.86 However, an excessive stirring could hinder the
deposition of the new phase. For this reason to carry out the
tests with moderate orbital stirring (around 100 r.p.m.) is
generally suggested, rather than under more vigorous mag-
netic or mechanical stirring.
During the evaluation of highly bioactive glasses, significant
changes on the concentration of the ionic species in solution
were found as a consequence of the leaching processes and the
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HCA formation. In this sense, some authors proposed the
renewal of the solution, which can be made at intervals,82 or in
a continuous way which can be made by using peristaltic
pumps (dynamic method).83 Some differences were found
when a bioactive glass was tested in SBF under both static
(without SBF renewal) and dynamic conditions (continuous
renewal). In dynamic a thicker HCA layer was formed than in
static. Moreover, in static the Ca/P molar ratio of the new layer
was higher, i.e. 1.6 vs. 1.2. The Ca : P molar ratio of 1.2
obtained in dynamic conditions suggested that the precipitate
is formed by a mixture of calcium deficient apatite (CDHA) and
amorphous calcium phosphate (ACP).87 The mechanism to
explain this variation, mainly based on the differences in pH
and the corresponding concentration of phosphate ions in
solution, is depicted in Fig. 7.
Conformation methods allow processing materials in
different shapes, powders, pieces, pellets, etc. Because
bioactivity is a surface process, some authors propose to
increase the surface as much as possible to favor the process,
that is, to use the samples as powders to increase their
reactivity. Consequently, the actual trends propose to evaluate
the materials as powders.86 However, some authors prefer to
use the samples as pieces (pellets or monoliths) so they display
large defined surfaces where it is possible the visualization of
the HCA layer formation by characterization techniques such
as the Scanning Electron Microscopy (SEM).
5. Techniques to verify a bioactive response
In vitro bioactivity is evaluated by analyzing the HCA layer
formed or not on the biomaterial surface. Taking into account
that the size of the crystals of the calcium phosphate layer is
very small at the beginning of the process, diffraction
techniques, such as X-Ray Diffraction (XRD) or Electron
Diffraction (ED) in a Transmission Electron Microscope
(TEM) are scarcely useful, at least in the first stages of the
assays. However, as is observed in Fig. 8, for larger soaking
times, diffuse diffraction bands and rings that can be assigned
to nanocrystalline HCA can be observed by XRD and ED,
respectively.
On the other hand, FTIR spectroscopy has revealed as a
powerful technique to evaluate the in vitro bioactivity, since is
very sensitive to the presence of minimum amounts of
phosphate groups. In addition, FTIR spectroscopy detects
the silanol groups on the unreacted glass surface, which play
an important role in the bioactive process. Moreover, when
HCA is formed, FTIR detects carbonate groups, and also allows
detecting the HCA crystallization because the band at of
amorphous phosphate at 565 cm21 is split in two bands at 563
and 602 cm21 characteristics of phosphate in a crystalline
environment87 (see Fig. 8).
SEM coupled with Energy Dispersive X-ray (EDX) spectro-
scopy are also powerful techniques for visualizing (SEM) the
new layer formed on the bioactive surface and to obtain
information of the layer composition (EDX).
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Fig. 7 Different mechanisms proposed for the in vitro formation of HCA layer on a bioactive glass under static (left) and dynamic (right) conditions.87

The characterization techniques, collected in Fig. 8, allow they bring a great amount of information that can be used to
the use of in vitro tests to assess the potential bioactivity of a improve the material features before be investigated in cell
material. If tests are negative, there is no need to continue with cultures and animal tests. At the bottom of the figure the HCA
cell cultures or in vivo tests. However, if the results are positive,

Fig. 8 Characterization techniques employed to evaluate the bioactive response of bioceramics after be soaked in SBF. Bottom of the figure: schematic representation
of the HCA layer formed on both a sol–gel glass and an ordered mesoporous material.

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Fig. 9 HRTEM micrographs and Fourier transform (FT) of a conventional sol–gel glass, a template glass and a mesoporous silica material. Relevant textural properties
of materials and the required time for exhibiting in vitro bioactivity are also included.27
layer formed on a bioactive sol–gel glass and an ordered
mesoporous material are schematically depicted.
6. Some examples of bioactive materials
In this section, two families of bioactive glasses will be
presented in a comparative way. The in vitro bioactivity of
conventional sol–gel glasses,77 which supposed an advance
with respect to traditional melt glasses,93 will be compared
with mesoporous ordered template glasses26 able to be loaded
with biological active substances and exhibiting an extremely
quick in vitro bioactive response.
6.1. Bioactivity of template glasses vs. conventional sol–gel
glasses
Textural and structural characteristics of mesoporous ordered
bioactive glasses, also called template glasses, explain the
accelerated bioactive response compared with conventional
bioactive sol–gel glasses of analogous composition (Fig. 9). In
fact, some template glasses exhibited a bioactive response
after 1 h of soaking (the fastest bioactive response ever
reported for a bioactive material),94 whereas conventional sol–
gel glasses with analogous composition required 3 days to
display HCA formation. These differences can be explained
considering the higher values of specific surface area and pore
volume in template glasses as well as the higher concentration
of silanol (Si–OH) groups on the template glasses surface.
Fig. 9 also includes High Resolution TEM (HRTEM) micro-
graphs of both types of glasses to visualize the structural
differences at the atomic scale. As is observed, the amorphous
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pore walls of template glass is constituted by an ordered
arrangement of cavities that can be classified in different
crystalline systems in the same way that the atoms are
distributed in a crystalline solid. Relevant textural properties
of the three materials and the required times for exhibiting in
vitro bioactivity are also included in the figure.
For both families of glasses, the major features that
contribute to the in vitro bioactivity are the CaO content, the
textural properties and the concentration of surface silanol
groups. Regarding the calcium oxide concentration, a high
amount of CaO decreases the network connectivity and
consequently improves the glass reactivity and bioactivity.
Template glasses show the same trend concerning the initial
formation of an ACP layer upon soaking in SBF. That is, high
amounts of Ca2+ released from x%SiO2–y%CaO–5%P2O5
template glasses with high CaO contents i. e. y = 37% and
20% (denoted Si58m and Si75m by their SiO2 contents of x =
58% and 75%, respectively, and ‘‘m’’ from mesoporous) leads
to the fast formation of a thicker ACP layer compared with the
template glass containing 10% of CaO (Si85m glass).27
However, template glasses show a different trend regarding
the HCA crystallization. In effect, the earliest crystallization
was observed in the template glass with 10% of CaO where the
two bands of phosphate in a crystalline environment were
detected by FTIR after 4 h of soaking. Nevertheless, template
glasses with 20% and 37% of CaO show the first traces of
crystalline phosphate after 24 h and 8 h, respectively. SEM
images show characteristic needle-shaped crystalline aggre-
gates of HCA after 16 h for Si85m and after 3 d in for the other
two template glasses. Because of the in vitro bioactivity of a
material is related with the time required to obtain HCA,
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Review
Fig. 10 SEM micrographs of Si85m after be soaked by different times in SBF.
The corresponding FTIR spectra are also displayed (amorph: amorphous, cryst:
crystalline).
Si85m is the more bioactive of the three glasses compared. The
fast bioactive response of Si85m was attributed to its higher
surface area (427 m2 g21) compared with Si75m (195 m2 g21)
and Si58m (393 m2 g21). In Fig. 10 the SEM micrographs and
the corresponding FTIR spectra of Si85m after different
soaking times in SBF are shown. When the soaking time
increases, the glass surface is coated by a new material and the
band of ACP in the FTIR spectrum is split in two bands of
crystalline phosphate.
On the other hand, the pore structure was proposed as a
possible factor influencing the high bioactive response of
template glasses. In this sense, Si85m shows a 3D-bicontin-
uous cubic structure that could provide superior diffusion
transport mechanism compared with 2D-hexagonal mesopore
structure of Si75m and Si58m. This fact could be responsible
of the quicker bioactive response of Si85m. However, this is
difficult to elucidate because when comparing the in vitro
behavior of template glasses, their composition, textural
properties and structure are varying together, which impede
to identify the governing factor. To clarify that, a glass with
identical composition to Si85m was synthesized in different
experimental conditions to obtain a 2D-hexagonal structure.95
That way in vitro tests in template glasses with identical
composition but different pore structure (3D-bicontinuous
and 2D-hexagonal) were carried out. Both glasses exhibited
identical bioactive behavior, revealing that differences in
mesopore ordering are not decisive for the in vitro response
of template glasses when composition and textural properties
are equivalent.90
6.2. Mechanisms of bioactivity in template glasses
A detailed study by HRTEM has shown notable differences
between the in vitro behavior of Si85m and Si58m glasses
exhibiting different composition, structure and textural
properties. HRTEM study of Si85m, low in CaO (10%) and
with higher textural properties shows the formation of
nanocrystalline calcium deficient hydroxyapatite (CDHA) after
only 1 h in SBF (see Fig. 11). Their 3D-bicontinuous cubic
structure provides not only high surface area and pore volume,
but also facilitates the ionic exchange with the surrounding
medium increasing the mass transport and diffusion pro-
cesses. This material exhibit the fastest apatite kinetic
formation observed for an in vitro biomimetic process.
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Fig. 11 HRTEM micrographs corresponding to the in vitro bioactivity study of
Si85m after 1, 4 and 8 h soaked in SBF. At 1 h the layer formed is composed by
ACP as majority element and CDHA. After 4 h the CDHA proportion increases,
becoming the unique phase at 8 h.
On the other hand, Si58m with higher calcium content (37%
CaO) and 2D hexagonal structure was the first bioceramic that,
after soaking in SBF, exhibits the transition: ACP–OCP
(octacalcium phosphate)–CDHA, similarly to bone biominer-
alization process. All bioceramics investigated up to date
developed CHDA through the direct crystallization of
ACP,2,10,96 without formation of OCP, an intermediate phase
in the natural bone biomineralization.97,98 OCP is a metastable
phase and it precipitate only when the crystallization pH is
below 7.0. Si58m glass exhibits high calcium content and high
surface area (195 m2 g21), both factors providing the requisites
for a very high reactivity. Consequently, a larger ion
interchange between Ca2+ and H3O+ ions takes place leading
to a local pH of 6.7 during the first stages, which leads the OCP
precipitation. On the contrary, this local acid pH does not
occur in the conventional sol–gel glasses surface, where pH is
always .7.4, identical at the surrounding fluid during the full
bioactive process, which explains why OCP was never observed
in this family of glasses.
Biomimetic bone-like in vitro mineralization of Si58m was
investigated by HRTEM (Fig. 12). After 1 h in SBF, the glass is
coated by a large amount of ACP with a Ca/P molar ratio of 1.2.
This event, commonly observed in bioactive sol–gel glasses,
corresponds to the step 4 of the bioactive mechanism
proposed by Hench that will be explained later. After 4 h of
incubation, nanocrystalline oval biphasic nuclei of around 12
nm width and 18 nm length and a Ca/P molar ratio of 1.3,
identified as OCP with a small fraction of CDHA were
observed. The transformation from oval OCP nuclei to
needle-shaped CDHA nanocrystals is evident at 8 h and still
clear at 48 h.
Similarity, the in vivo maturation of calcium phosphates and
the in vitro biomimetism of template glasses are strongly
related with the high Ca2+ content and an open porosity in
glasses. This enforced us to reformulate the bioactivity stages
due to the unique physicochemical characteristics of the
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RSC Advances
Fig. 12 HRTEM images of S58m after being soaked in SBF. After 1 h ACP is
observed. At 4 h in SBF, nanometric oval nuclei constituted by OCP and CDHA
are detected within the ACP matrix. After 8 h needle shaped CDHA, more clear
at 48 h, crystallizes within the ACP matrix.
template glasses surface. Thus, the CDHA formation mechan-
ism on template glasses is similar to that proposed by Hench
for conventional bioactive glasses. However, there are sig-
nificant differences that lead to a biomimetic behavior in
template glasses.99–102 Fig. 13 shows the proposed bioactivity
mechanism for mesoporous template glasses by comparing
with proposed for bioactive glasses.
As is observed, the main differences in the bioactive process
steps are: (i) the exchange of Ca2+ in glass with H+ in the
solution is quicker and more intense for template glasses due
Fig. 13 Bioactive mechanisms for conventional sol–gel glasses1 compared to template glasses.103 Approximated scales of time are included.
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to their higher and accessible porosity and surface area, which
speeds up the surface reactivity. Thus, a higher H+ incorpora-
tion is detected on template glasses, producing (ii) a higher
density of silanol groups on their surface. (iii) The polycon-
densation of Si–OH groups to form hydrated silica gel is
somewhat different in both cases. For template glasses, a
highly protonated silica gel is formed leading to an acid local
pH in the surface of 6.7. However, the conventional sol–gel
glasses surface exhibit always along the assays a basic pH ¢
7.4, identical to the in vitro fluid. (iv) Bigger amounts of ACP
are precipitated on template glasses. (v) The crystallization of
ACP by incorporation of Ca2+ and HPO422 ions to form OCP
takes place only in template glasses. The acidic surface of
template glasses allows the formation of OCP in the first
hours. (vi) For template glasses, the OCP maturation into
CDHA takes place through a dehydration process, which
occurs only within small OCP crystallites; the hydrolysis
reaction of the PO432 and HPO422 is the critical chemical
transformation of OCP to CDHA. For conventional bioactive
glasses a direct crystallization of ACP occurs by incorporation
of OH2 and CO322 ions. The time period for the CDHA
formation is very different in both types of glasses. For
conventional sol–gel glasses, CDHA formation takes place after
approximately 3 days in SBF, whereas for template glasses it
requires only 8 h, or even lower times.
6.3. Some mechanisms of bioactivity proposed for bioactive
glasses
Fig. 14 shows the sequence of reactions on the surface of a
conventional bioactive glass when in contact to physiological
fluids leading to the new bone formation.1 The first 5 stages
can be reproduced in vitro. In highly bioactive glasses 1–3
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Review
Fig. 14 Sequence of 11 stages taking place on the bioactive glass surface when in contact with physiological fluids leading to the new bone formation.
stages overlap with 4 and 5, and thus HCA is formed in few
days. For template glasses, the HCA formation, via an OCP
intermediate phase, is still quicker, and can be detected after
just a few hours.103
In Fig. 15 different mechanisms of formation of the HCA
layer are compared, including those of Hench,1 Salinas et al.87
(Fig. 7) and Izquierdo et al.103 (Fig. 13). In addition, other two
mechanisms proposed by Kokubo et al.104 and Vallet-Regi
et al.88 are included. In 1992, Kokubo et al. stated that the
most important event in the in vitro formation of HCA on CaO–
P2O5–SiO2 glasses was the leaching of Ca2+ ions, which
promoted the formation of extra Si–OH and increase the
saturation of solution, both factors favoring the HCA forma-
tion. Once the new layer is formed, collagen fibers that reach
the implant trying to encapsulate it find the HCA layer forming
new bone. On the other hand, in 2005 Vallet-Regi et al.
described the strong effect of variations in the composition of
conventional sol–gel glasses on the new layer formation
mechanism. Thus, for a phosphorous-free SiO2–CaO sol–gel
glass the ACP was formed just in a few hours, but the HCA
formation required around 7 days. On the other hand, when
5% of P2O5 was included in the glass composition, 2 days were
required for the ACP formation, but nanocrystalline HCA was
detected after only 4 days.89 Therefore the presence of
phosphorous accelerated the bioactive response. When analyz-
ing these different behaviors by HRTEM it was found that
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phosphorous binds to calcium forming Si doped b-TCP
nanocrystals. Thus, in P-containing glass, the Ca2+ leaching
is hindered impeding the formation of new silanol groups and
the increasing the solution supersaturation. Consequently,
longer time was required to form ACP, but once formed, b-TCP
nanocrystals act as crystallization nuclei accelerating the HCA
crystallization.
7. Positive aspects of bioactive materials for
the scaffolds manufacture
Tissue engineering is based on three basic pillars: scaffolds,
cells and signals.105,106 In this regard, second generation
bioceramics, i.e. bioactive or biodegradable, were profusely
investigated in the last few years for the production of
scaffolds for bone tissue engineering. The use of bioactive
materials for the scaffold manufacture has several advantages
including the mechanical stability provided by the bioactive
bond, and cell attachment and proliferation favored by their
osteconductive capability.
Besides bioactivity, scaffolds must comprise a hierarchical
and interconnected porosity close to 90% analogous to
bone.107–111 Fig. 16 shows a bioceramic scaffold functionalized
to reach specific interactions with biomolecules and drugs.
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RSC Advances Review

Fig. 15 Different mechanisms proposed for the formation of the new layer of HCA on the surface of bioactive ceramics.

The relative sizes of elements involved in osteogenesis are also First, the scaffold must contain giant pores, visible with the
included. As observed, a scaffold must include three different human eye, with sizes ranging from 150 to 450 mm or even
levels of porosity to satisfy specific physiological functions. greater than that. These pores are necessary for the cells

Fig. 16 Construct designed for bone tissue engineering composed by a bioceramic scaffold interacting with signals and cells. The relative sizes of species involved in
bone formation and important pore size regions that must include a scaffold for fulfilling specific physiological functions are also included.

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Review
colonization and vascularization, that is, formation of blood
vessels, for the nutrients supply to the new formed tissues.
Besides, macropores under 20 mm are needed for capillary
internal growth and interactions between the cells and the
matrix. Finally, the presence of mesopores ranging from 2 to
50 nm allows decorating the scaffolds with molecules with
biological activity that present sizes in this region. Thus,
osteogenic substances such as growth factors, peptides and
hormones can be included in the scaffolds, but also they can
be loaded with drugs for the treatment of pathologies such as
bone infection or cancer.
This porosity implies a certain sacrifice of their mechanical
properties which limit the clinical uses of the constructs
formed the bioceramic scaffold decorated with signals and
cells. Several authors proposed the addition of certain
polymers such as poly(caprolactone) to obtain composite
scaffolds with sponge-like mechanical properties.112
Furthermore, a stimuli-responsive behavior of the construct,
so that it can modify their properties in response to external
stimuli is also pursued. For instance, the construct included,
as an extra feature, the capacity of release an antibiotic when
an infection is detected.
Of all the employed materials for the fabrication of 3D
scaffolds for bone injures treatment, bioactive ceramics are
excellent candidates due to their similarities with the
inorganic component of bone. As it was previously mentioned
some template glasses compositions are the synthetic materi-
als with faster in vitro bioactive response. Furthermore, these
glasses exhibit high surface area and pore volume with a very
narrow pore size distribution, which make possible a great
capacity and a homogenous behavior of material in terms of
biomolecules adsorption and release. Therefore, processing
these materials by methods that allow obtaining macroporos-
ity without destroying their intrinsic mesoporosity is an
interesting subject in bone tissue engineering research.113
Many conformation methods of scaffolds can be used
including porogens that melt, like wax, or dissolve like sugar
or salt,114 as well as, by electrospinning, supercritical proces-
sing, and freeze-drying from suspensions using replicas of
porous sponges.115 However, to obtain porous scaffolds that
maintain the small particle size of the bioactive and
biodegradable ceramics is necessary to use methods that do
not need high temperatures. This approach has an extra
advantage because allows the inclusion of thermal sensitive
biomolecules in the same processing step. In this regards it
was proposed the synthesis of scaffolds by rapid prototyping
(RP) 3D printing.116 This method allows the preparation of 3D
scaffolds with complex geometries and fine structures together
with porosity control. In addition, this approach can start from
the tomography information of the bone tissue to be repaired.
Then, and by using computed assisted design (CAD) the
appropriate scaffold for each clinical application can be
obtained. A paste including the bioactive template glass is
prepared to load in the injector to mould a scaffold with
previously designed porosity.117 Thus, a combination of
methods such as sol–gel, for the synthesis of template glasses,
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polymer templating, to obtain the injectable paste, and RP to
obtain the 3D piece lead to the bioactive scaffold with the
three levels of required porosity. (Fig. 16).
Results in this field were reached in pure silica (SiO2)
mesoporous materials with SBA-15 structure. SBA-15 is a
scarcely bioactive material, but after being loaded with
osteostatin, produced excellent in vitro results in MC3T3-E1
osteoblast cell line,118 and in rabbit femur cavity defect in both
healthy8 and osteoporotic rabbits.119 These in vitro and in vivo
results show the very promising future of osteostatin loaded
silica mesoporous materials for bone repair and regeneration
technologies. In addition, they are a good example of how a
bioceramic can be noticeably upgraded with the addition of a
signaling biomolecule.
On the other hand, results in binary SiO2–P2O5 mesoporous
scaffolds were obtained producing better biocompatible
response and less cellular damage than pure silica materi-
als.101 The highly bioactive mesoporous glasses in the ternary
SiO2–CaO–P2O5 system are the first and more widely studied
template glasses.102,120–125 Significant features of this family of
compounds has been explained in the section 6.
In addition, scaffolds made with quaternary glasses, where
the fourth element respect the ternary system is added with
therapeutic aims, were fabricated. Thus, quaternary glasses
based in 80%SiO2–15%CaO–5%P2O5 glass to which amounts
(from 0.2% to 7%) of a fourth oxide were added.126 Ce3+ Ga3+
or Zn2+ were selected as therapeutic ions because their
beneficial biological properties. Thus, Cerium favors the
osteoblast growth, reduces the enamel demineralization and
is neuroprotective; Gallium inhibits the osteoclasts activity,
increases the bone calcium content improving the bone
mechanical properties, and is antimicrobial and Zinc is a
potent inhibitor of osteoclasts resorption, stimulates the bone
formation and is also antimicrobial.127,128 In general, for
moderate substitutions, the scaffolds exhibited in vitro
bioactivity, mesoporous order and textural properties some-
what smaller than in the unsubstituted parent glass, but
useful to be clinically applied. These scaffolds based on
template glasses are good candidates for bone tissue engineer-
ing because they include as extra property the added value of
substituents.
8. Future perspectives
Clinicians were always more confident in autografts, which
they consider the gold standard, allografts or xenografts than
in synthetic bioactive materials. In fact, they were indicating to
us basic investigators the pathway to follow. These biomater-
ials of biological origin have a porous structure optimized
throughout millennia of evolution and they contain signals
and cells, basic pillars of which today we know as tissue
engineering. However, these materials of biological origin
present problems of morbidity, disease transmission and high
costs that totally justify the research efforts into new synthetic
bioactive materials.
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Synthetic materials used alone are not enough to drive
processes of living tissues regeneration. The living tissues have
a great self-repairing capacity that must be used. Today the
investigating activity in biomaterials goes to which some
authors denoted as third generation biomaterials what
actually suppose the application of the tissue engineering
and regenerative medicine principles. Here bioactive and
biodegradable ceramics are called to play an essential and
exclusive role. Thus, many efforts are done in the obtaining of
3D constructs for bone tissue engineering. Scaffolds must
promote bone tissue growth in interconnected pore architec-
ture. Important features come from required porosity that
should be hierarchical and interconnected to allow the
development of the physiological functions and the composi-
tion should promote natural tissue growing. Bioactive cera-
mics containing different levels of porosity and the
development of processing methods allow to obtain at the
surgeon demand 3D bioactive scaffolds for bone tissue
engineering. Such scaffolds can still be upgraded including
osteogenic or antimicrobial agents and including therapeutic
inorganic ions in the bioceramic composition.
Acknowledgements
Financial support thought Comisión Interministerial de
Ciencia y Tecnologı́a (CICYT, Spain) (MAT2008-736),
Comunidad Autónoma de Madrid (S2009/MAT-1472) and the
Network of Excellence of Spanish MICINN (CSO2010-11384-E)
is acknowledged.
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