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Contents lists available at ScienceDirect

Applied Surface Science

journal homepage: www.elsevier.com/locate/apsusc

Full Length Article

Inuence of the modulated two-step synthesis of biogenic

hydroxyapatite on biomimetic products surface
Florin Miculescu a , Aura Catalina Mocanu a,g , George E. Stan b , Marian Miculescu a, ,
Andreea Maidaniuc a,h , Anisoara Cmpean c , Valentina Mitran c , Stefan Ioan Voicu d ,
Teodor Machedon-Pisu e , Lucian Toma Ciocan f
a
University Politehnica of Bucharest, Department of Metallic Materials Science, Physical Metallurgy, 313 Splaiul Independentei, 060042, J Building, District
6, Bucharest, Romania
b
National Institute of Materials Physics, Laboratory of Multifunctional Materials and Structures, Atomistilor Str., No. 405A PO Box MG 7, 077125, Magurele,
Bucharest, Romania
c
University of Bucharest, Department of Biochemistry and Molecular Biology, 91-95 Splaiul Independentei, 050095, District 5, Bucharest, Romania
d
University Politehnica of Bucharest, Advanced Polymer Group, Faculty of Applied Chemistry and Material Science, 1-7 Gh. Polizu Str., Polizu Campus,
011061, L 015 Building, District 1, Bucharest, Romania
e
University Transilvania of Brasov, Department of Materials Engineering and Welding, 1 Universitatii Str., 500177, Room A II 1, Brasov, Romania
f
Carol Davila University of Medicine and Pharmacy, Prosthetics Technology and Dental Materials Department, 37, Dionisie Lupu Street., District 1,
020022, Bucharest, Romania
g
S.C. Nuclear NDT Research & Services S.R.L, Department of Research, Development and Innovation, 104 Berceni Str., Central Laboratory Building, District 4,
Bucharest, Romania
h
S.C. Nuclear NDT Research & Services S.R.L, Destructive and Nondestructive Testing Laboratory, 104 Berceni Str., Central Laboratory Building, District 4,
Bucharest, Romania

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

Article history: Processing calcium-rich natural resources, such as marble and mussel seashells, into biomimetic prod-
Received 7 April 2017 ucts could constitute an environmentally-friendly and economically sustainable alternative given their
Received in revised form 4 July 2017 geographical widespread. Hitherto, their value for biomedicine was demonstrated only for seashells,
Accepted 17 July 2017
with the technological exploitation approaches still facing challenges with respect to the identication
Available online xxx
of generic synthesis parameters capable to allow the reproducible and designed synthesis of calcium
phosphate at an industrial-ready level.
Keywords:
In this study was targeted the optimization of Rathje synthesis method for the fabrication of biogenic
Biogenic hydroxyapatite synthesis
Surface characteristics calcium phosphates, by conveniently adjusting the chemical composition of employed reagents. It was
In vitro assays shown that post-synthesis heat-treatment of compacted powders is the key step for inducing struc-
tural transformations suitable to attain biomimetic products for reconstructive orthopedic applications.
The sintered materials have been multi-parametricallyevaluated from morpho-compositional, structural,
wettability, mechanical and cytocompatibility points of view and the results have been cross-examined
and discussed. Convenient and efcient preparation routes to produce biogenic hydroxyapatite have been
identied. The functional performances of the as-prepared biogenic ceramics endorse their use as a solid
and inexpensive alternative source material for the fabrication of various bone regenerative products
and implant coatings.
2017 Elsevier B.V. All rights reserved.

1. Introduction

Exploitation of marine biogenic resources for preparing bio-

logically relevant calcium phosphates (CaP) attracted considerable
interest since the development, in 1974, of a synthesis method for
coralline hydroxyapatite [1]. Although the method preserved the
Corresponding author.
corals porous architecture, some disadvantages, such as uncon-
E-mail addresses: marian.miculescu@upb.ro, m miculescu@yahoo.com
(M. Miculescu).
trolled resorption or inadequate mechanical properties, led to the

http://dx.doi.org/10.1016/j.apsusc.2017.07.144
0169-4332/ 2017 Elsevier B.V. All rights reserved.

Please cite this article in press as: F. Miculescu, et al., Inuence of the modulated two-step synthesis of biogenic hydroxyapatite on
biomimetic products surface, Appl. Surf. Sci. (2017), http://dx.doi.org/10.1016/j.apsusc.2017.07.144
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development of alternative synthesis methods which allow the with their mechanical and biological performance, in the search for
preparation of calcium phosphates powders with different com- relevant triggers capable to support a controlled bio-functionality.
positions [2].
Transition to eco-friendly preparation methods, complying
2. Materials and methods
with the current exigent recommendations for environmental
protection, impose adapting traditional synthesis procedures to
2.1. Samples preparation
exploit other various abundant waste-type raw materials. Cur-
rently, besides corals [1,3], waste materials such as marine animal
The raw materials used in this study are dolomitic marble
shells (sea urchins [4] and sea shells [57]) are taken into account
(Ruschita, Romania) and Mediterranean mussels Mytilus gallo-
as CaP precursors in an attempt to satisfy the increasing demand
provincialis (Black Sea coast, Romania). After a mechanical cleaning,
of materials for biomedical applications by using the benets of
the raw materials were calcined in air at 1300 C for 6 h, in an elec-
natural resources in a sustainable manner.
trical furnace. After calcination, the materials were cooled in air and
The frequently investigated synthesis methods use the cal-
stored in Petri dishes.
cium carbonate (CaCO3 ) existing in various natural resources and
The reagents amounts for CaPs synthesis were initially calcu-
a phosphate-based reagent (e.g. phosphoric acid (H3 PO4 )), which
lated based on the stoichiometric reactions described in the Rahtje
are chemically reacted to produce a CaP [7,8]. The reaction takes
method [11,1618]: for 10 g of Ca(OH)2 powder (derived from heat-
place at moderate temperature (150220 C) and requires up to
treated marble/seashells), 200 ml of distilled water, and 5.5 ml of
6 h [9,10]. More promising results have been obtained by applying
H3 PO4 (85%, Sigma-Aldrich) were used. Further, the experiments
an indirect synthesis route, which implies the thermal dissociation
were conducted separately for the marble and seashells-derived
of CaCO3 into calcium oxide (CaO), and its further treatment with
powders, using 1.0, 1.1, 1.2, and 1.3 stoichiometric acid quantity.
H3 PO4 [11]. Although it was initially was introduced for pure syn-
The marble and seashell-derived powders were rstly hydrated in
thetic reagents, the method can be extended for the processing of
distilled water and then treated with H3 PO4 by drop-wise addi-
natural raw materials by adapting the reaction parameters the
tion at a rate of 1 ml/min. The solutions were magnetically stirred
aim of this study.
during the synthesis for 2 h, at 700 rpm. Further, the prepared solu-
The biomaterials prepared by such methods can be thermally
tions were washed, ltered and dried at room temperature (RT)
processed and transformed into biphasic or multiphasic CaPs,
for 168 h, and then in oven at 100 C, for 24 h, until the powders
whose biomedical performance depends on the ratio between the
were obtained. The prepared powders were mixed with normal
insoluble and the resorbable constituent phases. The phase com-
saline solution and pelletized by cold isostatic pressing at 0.1 MPa.
position can be conveniently adjusted, by designed variation of
The pellets were dried at RT for 24 h, and sintered in an electrical
synthesis parameters, such as to induce desired properties (i.e.
furnace at 1200 C, for 10 h.
resorbability, bioactivity, osteoconductivity, or osteoinductivity),
The samples are further referred to by H3 PO4 quantity (e.g.
depending on the local requirements imposed by the implantation
0 = stoichiometric quantity; 3 = 1.3 calculated H3 PO4 quantity)
environment. Besides the biphasic or multiphasic CaPs the mixtures
and by the raw material (M = marble; S = seashell). The as-
of thermally unstable CaPs (e.g. dicalcium phosphate dehydrate
synthesized powders will be referred AS and the thermal-treated
(CaHPO4 2H2 O, DCPD) or brushite, and anhydrous dicalcium phos-
(sintered) samples TT (example: 1S-TT = Thermal Treated pel-
phate (CaHPO4 , DPDA) or monetite) can be used as self-setting
let prepared from Seashells-derived powder treated with 1.1
formulations. In this later case, the setting reactions and the nal
(+10%) stoichiometric H3 PO4 quantity). A bovine hydroxyapatite
product characteristics are governed by each component fraction
(HA) previously prepared and completely characterized [19] was
within the mixture. However, since there has been little discussion
used as reference material in the study. The reference material was
on the stability of such formulations and on the inuence of com-
analyzed as-synthesized (HA-AS) and after sintering (HA-TT) in the
pounds such as calcium oxide or calcium pyrophosphate, which
same conditions as the other samples.
sometimes occur during synthesis reactions [12], further clari-
cations are needed in order to develop competitive high quality
products. 2.2. Bulk and surface physical-chemical characterization
Most of the CaPs synthesis methods based on natural sources of techniques
calcium carbonate are difcult to reproduce because the synthesis
parameters are not completely reported. Also, these studies primar- a) The morphology and composition of the AS powders and TT
ily discuss the rst stages of preparation, although further thermal pellets were analyzed by scanning electron microscopy (SEM)
processing of the obtained powders induces compositional and (Philips Xl 30 ESEM TMP) coupled with energy dispersive spec-
structural changes which inuence considerably the nal products troscopy (EDS) (EDAX Sapphire spectrometer), at an acceleration
performance [1315]. Moreover, the variability of natural sources voltage of 25 kV and working distance of 10 mm. The SEM and
of calcium carbonate (due to their different origin and/or environ- EDS investigations were performed in three randomly chosen
ment) inuences their chemical composition, which in turn affects areas.
the thermal decomposition process into calcium oxide, and the b) The samples structure was evaluated by X-ray diffraction (XRD)
phase composition of the prepared CaPs. (Bruker D8 Advance diffractometer equipped with a LynxEye
This study proposes an adaptation of the conventional indirect detector), in Bragg-Brentano geometry, with Cu K ( = 1.5418 )
synthesis method (known also as Rathje method) [11] for preparing radiation. The scattered intensity was scanned in the 2 range
CaPs using dolomitic marble and Mytilus galloprovincialis seashells 1060 , with a step size of 0.04 and a dwell time of 1 s.
raw source materials. Dolomitic marble represents a new proposal c) The bonding architecture and functional groups presence was
for extending the range of natural calcium carbonate sources used evaluated by Fourier Transform Infrared (FTIR) Spectroscopy
for preparing CaPs. (Perkin Elmer Spectrum BX II spectrometer) in attenuated total
The as-prepared and thermally-treated products have been reectance (ATR) mode (PikeMiracle head). The spectra were
thoroughly characterized, aiming to correlate the composition, recorded in the range 5004000 cm1 , with a resolution of
morphology, structure, and wettability of the biogenic materials 4 cm1 and a total of 32 scans/experiment.
d) The mechanical compression testing of the sintered materials
was performed on cylindrical pellets with a Walter + Bai AG LFV

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Universal Testing Machine equipment, using a 300 kN load cell
and a compression rate of 5 mm/min, with an acquisition rate of
0.01 s. The measurements were performed in triplicate.
e) Wettability was evaluated by water contact angle measurements
using a Krss Drop Shape Analyzer DSA100. The experiments
were performed at 19 1 C and 45 5% room humidity. The
images were captured at 1 s and 5 s after water droplet deposi-
tion and the results (average of 3 determinations/sample) were
processed with the ImageJ 1.50 software.
f) Surface energy and roughness parameters were determined
using MountainsMap Technology by Digital Surf Besancon,
France software.
2.3. In vitro cytocompatibility assays
In vitro biological performance of the bioceramic disc samples
was evaluated using murine pre-osteoblastic cell line (MC3T3-E1,
ATCC , CRL-2593TM). The potential of these materials surfaces to
sustain cell survival and growth was assessed by Live/Dead, LDH
and MTT assays, respectively.
The samples were sterilized at 180 C for 1 h, washed four
times for 15 min with Dulbeccos Modied Eagles Medium (DMEM,
Sigma-Aldrich) and then were placed in 24-well plates. MC3T3-
E1 cells were seeded on the samples surface at an initial cell
density of 1.2 104 , allowed to adhere, and incubated in DMEM
supplemented with 10% fetal bovine serum (Gibco) and 1%
(v/v) penicillin/streptomycin (10,000 units mL1 penicillin and
10 mg mL1 streptomycin) (Sigma Aldrich) in a humidied atmo-
sphere of 5% CO2 at 37 C for specic time points. All experiments
have been performed in triplicate.
The Live/Dead viability/cytotoxicity assay kit was used accord-
ing to the Invitrogen Life Technologies manufacturers instructions.
Briey, after 1 day of culture, the analyzed samples were labeled
with a mixture of calcein AM (4 mM) and ethidium homodimer-1
(2 mM) solution for 15 min at RT in dark conditions [20]. The sam-
ples were then washed with DMEM and examined under inversed
microscope (Olympus IX71) to detect the living cells (bright green
uorescence) and dead cells with compromised membrane (red
uorescence). The uorescent images were captured using Cell F
Image acquisition system.
The cytotoxic potential of the analyzed specimens was evalu-
ated by quantication of lactate dehydrogenase (LDH) released in
the culture medium by the MC3T3-E1 cells grown on their surfaces.
This assay was performed after 3 days of culture, by using LDH-
based In Vitro Toxicology Assay Kit (Sigma-Aldrich), as previously
reported [21].
The cell viability/proliferation was investigated using 1 mg/mL
MTT [3-(4, 5-dimethyl thiazol-2-yl) 2,5-diphenyltetrazolium bro-
mide] solution (MTT, Sigma-Aldrich, M 2185) at 3 days after
seeding. This quantitative colorimetric study represents an useful
indicative for cell metabolic activity [22]. The amount of formazan
produced by metabolically active viable cells was recorded at
550 nm using a microplate reader (Thermo Scientic Appliskan).
Statistical analysis of LDH and MTT tests was performed with
GraphPad Prism version 3.03 software using one-way ANOVA with
Bonferronis multiple comparison tests. Triplicate samples were
used to ensure the reproducibility of the results. The data are pre-
sented as mean standard deviation. The results were considered
statistically signicant at p < 0.05.
3. Results and discussion
Indirect synthesis of CaPs started with the thermal decomposi-
tion of CaCO3 into CaO (Eq. (1)) based on the reaction between CaO
and H3 PO4 in aqueous environment. After CaO hydration (Eq. (2)),
Please cite this article in press as: F. Miculescu, et al., Inuence of the modulated two-step synthesis of biogenic hydroxyapatite on
biomimetic products surface, Appl. Surf. Sci. (2017), http://dx.doi.org/10.1016/j.apsusc.2017.07.144
3
the exothermic reaction between the calcium hydroxide (Ca(OH)2 )
and H3 PO4 (Eq. (3)) transforms the initial aqueous solution into a
gel whose densication takes place gradually until the mixing of
the gel becomes difcult. In this stage, the gel crystalizes around
the unreacted CaO grains [23,24].
t
CaCO3 CaO + CO2 (1)
CaO + H2 O Ca(OH)2 (2)
10Ca(OH)2 + 6H3 PO4 Ca10 (PO4 )6 (OH)2 + 18H2 O (3)
The controlled release of phosphate anions in water (by drop-
wise addition) and reaction with calcium cations can lead to the
preparation of various biologically relevant CaPs. Hydroxyapatite
[HA, Ca10 (PO4 )6 (OH)2 ], beta-tricalcium phosphate [-TCP, -
Ca3 (PO4 )2 ] and alpha-tricalcium phosphate [-TCP, -Ca3 (PO4 )2 ]
are the most popular biocompatible CaPs used in different
monophasic, biphasic of multiphasic formulations for bone restora-
tion applications [12,25]. Brushite, is a metastable CaP and is known
as a precursor of HA [26]. During indirect synthesis, brushite can
remain stable or can dissolve, reprecipitate and disintegrate [27].
Monetite is the anhydrous form of brushite, and is not present in
the biological environment in neither normal or pathological calci-
cations [25,28].
3.1. EDS analysis
The comparative EDS analysis of the sintered pellets and refer-
ence material (HA-TT) (Fig. 1) aimed to evaluate the samples Ca/P
ratio. Besides the main CaPs elements (i.e., Ca, P, O), the ceram-
ics prepared from dolomitic marble or seashells contain variable
quantities of C and traces of Mg, Na, K and/or Si [29].
The atomic Ca/P ratio calculated from EDS results varied
between 1.18 and 1.63. The Ca/P ratio was found similar for
the samples prepared from marble or seashells under similar
conditions. Therefore, the raw source material type does not sig-
nicantly inuence the CaPs pellets composition. The samples
prepared according to stoichiometric reactions have the closest
Ca/P ratio with respect to the reference material (bovine HA). The
increase of H3 PO4 quantity used in the synthesis process led to
a decrease of Ca/P ratio, and suggested the possible presence of
brushite/monetite/calcium pyrophosphate phases [25,30].
3.2. XRD investigations
The XRD patterns of AS and TT materials are comparatively pre-
sented in Fig. 2. Only the 0S-AS samples derived from seashells
(Fig. 2A), presented a monophasic structure consisting of nano-
sized HA (ICDD: 00-009-0432), as evidenced by the broad and less
dened diffraction maxima [31].
In the case of all the other AS samples, synthesized under either
stoichiometric (i.e. 0M-AS) or non-stoichiometric (i.e. 1M-AS, 1S-
AS, 2M-AS, 2S-AS, 3M-AS, 3S-AS) conditions, a bi- or tri-phasic
composition was revealed, with nano-HA coexisting with brushite
(ICDD: 01-072-1240) and monetite (ICDD: 01-075-1520) crys-
talline phases. The brushite concentration progressively increased
(at the expense of nano-HA) with the quantity of acid used in
the chemical reaction of the natural calcium reagents. This is to
be expected, as in acidic conditions the formation of brushite and
monetite compounds is thermodynamically favored. Although, in
acidic conditions monetite is predicted as the chemical reaction end
product, brushite is usually formed due to its higher precipitation
kinetics [32]. The formation of brushite in the case of the AS samples
derived from marble under stoichiometric conditions can be linked
to the presence of Mg within the dolomitic marble. The inhibitory
effect of Mg on HA growth has been previously documented [33,34],
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Fig. 1. (A) EDS spectra and (B) evolution of Ca/P atomic ratio of the sintered pellets prepared from marble (M) or seashells (S) using different quantities of H3 PO4 .

Fig. 2. The XRD patterns of the AS and TT samples derived from (A) marble and (B) seashells.

and has been explained by the disturbance of the random aggre-
gation of the (Ca9 (PO4 )6 ) clusters (so-called Posners clusters)
and their interaction with the intercluster aqueous medium, which
hinders the development of the spherical particles, known as the
transient precursors for the formation of HA [13,35]. The advent of
the minor monetite phase in the case of the 2M-AS sample can be a
result of the partial dehydration of brushite during the powder dry-
ing process [15,36]. Traces of unreacted Ca(OH)2 were evidenced
only for the 3M-AS samples (prepared with 1.3 stoichiometric
H3 PO4 quantity) possibly due to a slow kinetic reaction. Given that
the synthesis was not based on pure substances, the inuence of
elemental traces (Mg, Na, K and/or Si) found in the precursors could
also lead to this effect [29,33,34].
The decrease of HA content of the AS samples presented a
monotonous trend in the case of the seashells-derived samples,
and a rapid one for the marble-derived ones (no HA signal being
detected starting with sample 2M-AS).
After the 1200 C thermal-treatment, one can observe a signi-
cant increase in the crystallinity of all samples (Fig. 2). 0M-TT and
0S-TT present a bi-phasic composition, consisting of a HA phase
with increased crystallinity and -TCP (ICDD: 00-055-0898). The
TT samples obtained under non-stoichiometric reaction conditions

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consist of either pure -TCP (i.e. 2M-TT and 1S-TT), or a biphasic -
TCP/Ca2 P2 O7 (calcium pyrophosphate) mixture (i.e. 1M-TT, 2S-TT,
2M-TT, and 3M-TT). These structural modications are supported
also by the Ca/P ratio evolution of the samples (Fig. 1). The HA partial
decomposition into -TCP at high temperatures was anticipated
[37,38]. Furthermore, brushite is known to partially decompose
into monetite above 100 C, and further into Ca2 P2 O7 at 270 C
[36,39]. However, the presence of -TCP phase only for some sam-
ples, which prior to the thermal-treatment contained brushite as
secondary (i.e. 1S-TT) or dominant (i.e. 2M-AS) phase, suggests that
brushite can also serve as a -TCP precursor. The Ca2 P2 O7 (ICDD:
01-073-0440) content of the TT samples gradually increased with
the brushite concentration of AS materials (and thus with the quan-
tity of the acid used during their synthesis).
3.3. FT-IR measurements
The comparative FTIR-ATR spectra of the as-synthesized and
thermal-treated powdered materials are presented in Fig. 3A, B,
together with the envelopes of the reference bovine HA samples.
In all respects the IR absorption data were in good agreement with
the above presented XRD results. The characteristic vibrational
bands of HA were identied: the 4 doubly degenerated bending
(560600 cm1 ), 1 symmetric stretching (961963 cm1 ) and 3
asymmetric triply degenerated stretching (9651100 cm1 ) modes
of orthophosphate functional groups, as well as the vibrational
modes of structural hydroxyl groups (630631 cm1 ) [4042].
Supplementary, the presence of HPO4 2 functional groups have
been reveled by the bands positioned at 871876 cm1 and
11131117 cm1 , in the case of all AS samples [40,41,43]. The
increase in intensity of the HPO4 2 IR bands is directly linked
to the AS samples gradual enrichment in brushite and mon-
etite. The amplication of these bands intensity is accompanied,
in the case of marble derived AS samples prepared under non-
stoichiometric conditions, rich in brushite (i.e. 2M-AS and 3M-AS),
by the advent of the characteristic IR bands of brushite: water
vibration (661 cm1 ), P O H out-of-plane bending vibration
(785 cm1 ), P O(H) 1 stretching (983 cm1 ), P O 3 stretching
(1054 cm1 ), 6 degenerate stretching of HPO4 2 (1132 cm1 ),
and P O H in-plane bending (12071219 cm1 ) [4446]. More-
over, for the above mentioned samples (2M-AS and 3M-AS) a
peak-split was encountered at 1020 cm1 corresponding to the HA
disappearance as a synthesis compound. This was revealed also by
the XRD investigations (Fig. 2A).
After the thermal-treatment at 1200 C these bands disap-
peared, presumably due to the HPO4 2 ions condensation into
P2 O7 4 ions, known to occur above 550 C [40]. This hypothesis
is supported by the emergence of the typical vibrational bands
of P2 O7 4 ions at 755 and 1205 cm1 [47,48], noticed for both
the marble and seashells derived materials (Fig. 3A, B). The bands
positioned at 943, 980981, 11171122, and 11591161 cm1 are
distinctive to -TCP [49].
Regarding the 2500/30003500 cm1 domain, which is spe-
cic for the stretching vibrations of (O H) bonds in absorbed
water molecules, there were no important modications. This was
expected since the investigated samples were thermally-treated
at 1200 C; at such high temperature we predicted and obtained
completely dehydrated samples, in all the cases.
3.4. SEM analysis
The microstructure evolution of the AS and TT samples is dis-
played in Fig. 4, in comparison to the bovine-HA reference sample.
The reference sample presented particles (with sizes in the range
515 m) having irregular shapes and sharp edges, and submicro-
metric round particle clusters.
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The morphology of AS samples mainly consisted of grains with
sizes between 2 and 15 m. The initial polyhedral shape of the
grains changed with the increase of H3 PO4 quantity to a quasi-
rounded elongated one. At 1.3 stoichiometric H3 PO4 quantity,
the 3M-AS sample is organized in needle-like particles with a
non-uniform size distribution. In this case, the grains of crystal-
lization aggregated as clusters, denoting an incomplete conversion
of Ca(OH)2 , which is consistent with the XRD observations. Sim-
ilarly, seashell-derived samples featured the transformation of
polyhedral grains (with sizes lower than 10 m) into platelets with
narrower quasi-rounded complex shapes (e.g. 3S-AS sample). Com-
pression and sintering of the powders generated, overall, denser
sample morphologies with few residual pores and particle shapes
that are difcult to discriminate. Furthermore, the TT samples
elicited superior compactness which progressively improved with
the increase of the acid quantity used during synthesis. This sug-
gests that the presence of CaPs phases such as monetite or brushite
results in a better sinterability of samples.
3.5. Contact angle measurements
The water contact angles (WCA) have been recorded at 1 s and
5 s after droplet deposition on the TT samples surfaces. The typical
WCA images are comparatively illustrated in Fig. 5A. The WCA val-
ues measured after 1 s varied between 12 and 49 and 1561 for
the marble- and seashells-derived samples, respectively (Fig. 5B).
Except 0M-TT and 0S-TT samples (having a biphasic HA/-TCP
structure (Figs. 2 & 3)), all the other mixed CaPs samples have lower
WCA values with respect to the reference bovine HA specimen. This
suggests that a prominent role upon the surface hydrophilicity of
CaPs samples is played by their phase composition (which progres-
sively turns into full -TCP or -TCP/Ca2 P2 O7 mixes). The surface
morphology (Fig. 4) is the second key inuencing factor for WCA
recorded at 1 s. The lowest WCA values corresponded to samples
with higher surface porosity.
At 5 s after the droplet deposition the WCA decresed sig-
nicantly. All samples, except the 0M-TT and 0S-TT samples
(containing HA as major phase), presented very low WCA values
(<4 ). In some situations, the water droplets were absorbed faster
than image acquisition time, and the WCA value was considered 0 .
Thereby, hydrophilic character (WCA values <90 ) has been
emphasized for all cases, becoming far more pronounced after 5 s.
The WCA values varied function of precursor type and H3 PO4 quan-
tity used for the synthesis of each specimen, and thus of the induced
phase composition (HA, -TCP, or Ca2 P2 O7 , with the latter two
showing a higher solubility in aqueous media). Seashells-derived
pellets presented, overall, higher WCA values with respect to the
marble-derived ones. The increasing H3 PO4 quantity used in syn-
thesis process, and thus the progressive formation of other CaPs
beside HA, led to a decrease of contact angles regardless of natural
calcium precursor type.
The surface hydrophilicity is known to promote harmonious
cell adhesion and protein absorption [50,51], and thus can gov-
ern a given material biofunctionality. Previous studies revealed the
superior hydrophilic character of other CaPs, such as -TCP, with
respect to both pure HA and biphasic calcium phosphates (BCP).
However, it has established that BCPs generally enable better cell
development and protein or other biological moieties adsorption
[12,52,53]. Thus, it suggested that only a certain material surface
hydrophilicity can foster suitable biological interactions.
3.6. Surface energy and roughness investigations
Given that the samples preparation method targeted no prefer-
ential direction, it is important to mention from the beginning the
isotropic character of all the samples. As we indicated above, the
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Fig. 3. Comparative FTIR-ATR spectra of AS and TT samples derived from (A) marble and (B) seashells.

Fig. 4. Representative SEM micrographs for the AS powders and TT samples derived from marble and seashells.

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Fig. 5. (A) Typical contact angle images recorded for the TT samples. (B) Roughness proles of the TT samples obtained with the MountainsMap software.

surfaces hydrophilic character is characterized by a WCA less than
90 (good surface wettability). The mechanical balance of a water
droplet is obtained through the action of all the three interface
stresses and is described by the Youngs equation (Eq. (4)):
LV cos = SV SL
 LV liquid-vapor stress [mN/m],  SV solid-vapor stress [mN/m],
 SL solid-liquid stress [mN/m], contact angle [ ].
By rearranging the above equations terms we obtained the con-
tact angle characteristic equation (Eq. (5)) that offers an overview
on the liquids capacity to wet the surface:
SV SL
cos =
LV
Taking into consideration the reproducible experimental con-
ditions, we assumed a constant solid-vapor tension ( SV = ct.).
The waters surface tension was reported in the literature as
 LV = 72.80 mN/m at 20 C [5459]. Therefore, surface energy
( SV  SL ) values could be determined from Youngs equation.
However, the behavior of solid surfaces in contact with uids can be
better characterized by taking into account the surface roughness
assessment [56,60]. This was conducted in terms of the following
parameters: Ra (the middle arithmetic deection of the prole) for
the surfaces micro-geometry outline and RL0 (the developed length
of the roughness prole) for a better differentiation in case of appro-
priate Ra values, but completely different wettability behavior [59].
Furthermore, given that the roughness is much smaller than the
water droplet dimensions and similar across the analyzed surface
[61], we also used the Wenzel equation, applicable for < 90 , to
determine the ratio between real rough surface and an ideal smooth
surface r which is proportional to the surface area increase
due to its roughness [57]. For a non-rough surface, this parameter
should remain equal to one; otherwise, for hydrophilic surfaces,
its increment is due to the roughness increase. We calculated the
r factor (ISO 4287 standard) [57] by analyzing the 2D roughness
proles (Fig. 5B) obtained from SEM micrographs analysis with the
(4) MountainsMap Technology by Digital Surf Besancon software:
r = 1 + R L0 /100% (6)
All the above-mentioned parameters were determined for the
1 s WCA acquired data (Fig. 5A). Results were comparatively pre-
sented in Fig. 6 and indicated, in terms of precursors type and
(5) chemical composition post thermal treatment, a relative inuence
of the surface energy and roughness factor r on the contact angle
due to the very small dimensional variations of the samples (mea-
sured values were less than 1 m). Compared to the reference
material (HA-TT), surface energy displayed a positive ascending
trend line for all the investigated samples, contrary to the con-
tact angle downtrend [6264]. Given that energy increases during
droplet formation and motion process (surface grooves lling),
these results conrmed the surface wettability ( < 90 ) [63]. Fur-
thermore, the Ra parameter displayed a monotonically ascending
tendency for both precursors, which was consistent with the sur-
face roughness positive effect on the wetting behavior [63].
Overall, the general trend slightly shifted from 2M-TT to 3M-
TT sample, and 2S-TT to 3S-TT sample respectively. Even if the r
factor in case of marble derived samples is an exception, the contact
angle/roughness ratio is similar to all the other investigated data
sets. These results endorsed the initial measuring trend of WCA
and proved that surface roughness had a minor inuence on the
wettability behavior.

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Fig. 6. Surface features of TT samples derived from marble and seashells.

Fig. 7. (A) Relevant uorescence micrographs of green-labeled (viable) and red-labeled (dead) MC3T3-E1 cells, grown in contact with the developed biomaterials and
reference control surfaces, at 1 day post-seeding; (B) Evaluation of LDH activity released into the culture media at 3 days post-seeding; (C) MTT assay assessment of the
viability/proliferation of MC3T3-E1 cells grown on the developed bioceramics.
Data analysis was based on mean SD (n = 3). OD optical density.

3.7. Biocompatibility assays
To explore cell behavior on the surface of the designed bio-
materials, the cellular distribution, morphology, and viability
of MC3T3-E1 cells were evaluated at 24 h post seeding using
Live/Dead assay, which allowed the simultaneous identication of
green-labeled (live) and red-labeled (dead) cells (Fig. 7A). Note-
worthy, the green uorescence also gives information about cell
morphological features. The results showed that the ratio between
the viable and dead cells was signicantly in the favor of the
viable cells on all types of surfaces. Moreover, a higher number
of viable MC3T3-E1 cells was observed on 0M-TT and especially
0S-TT samples, similarly with the control sample represented by
cell culture plates (CCPs). Also, uorescent images showed that
cells could adhere and spread on these surfaces and adopt a typical
spindle-shaped morphology. However, the morphological features
exhibited by MC3T3-E1 cells grown on 0M-TT and 0S-TT sub-
strates, consisting of HA and -TCP, were similar to those recorded
on the reference HA-TT. Slight morphological changes have been
recorded in the case of the other types of sample surfaces. This
dissimilar cell behavior could be ascribed to differences in biomate-
rial chemical composition and surface properties (e.g. morphology
and WCA values). The WCA values, measured at 1 s and 5 s after
liquid-biomaterial surface contact, decreased with the increase
of H3 PO4 quantity, and thereby with the solubility of the sin-
tered end products. This explains the better cell response elicited
by 0S-TT and 0M-TT surfaces which exhibit water contact angle
values close to the CCPs control surface (around 50 60 ). As pre-

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Fig. 8. Stress-strain curves characteristics for TT specimens derived from (A) marble and (B) seashells materials.

Fig. 9. Comparative (A) compressive strength and (B) breaking strain evaluation for the materials derived from derived from marble and seashells.

viously mentioned, it is well known that the surface wettability
plays an important role in determining the dynamics of protein-
surface interaction and it can be considered one of the main factors
reecting the extent of cell adhesion onto a given surface. Gener-
ally, the anchorage dependent mammalian cells prefer moderately
hydrophilic surfaces (water contact angle around 60 ) showing
good spreading, proliferation and differentiation [65,66].
Low cytotoxicity is an important feature of any biomaterial that
is suitable for biomedical applications. To determine the possible
detrimental effects exerted on cell viability by the analyzed bioma-
terials, the release of LDH into the culture media was quantied as
an index of cell death [31,67]. The LDH activity prole revealed no
signicant differences between all studied biomaterial surfaces at
3 days after cells seeding (Fig. 7B). Furthermore, these biomaterials
proved to display low LDH release in culture medium suggesting
that none of them exerted cytotoxic effects.
Also, the in vitro studies investigated the MC3T3-E1 cells via-
bility/proliferation on the surface of the analyzed specimens using
the MTT assay, which provides information on the cell growth and
metabolic activity. Fig. 7C presents the levels of MTT conversion
after 3 days of cell culturing. It was showed that pre-osteoblast
cells maintained their metabolic activity during this incubation
period. No signicant differences between the analyzed samples
were observed. However, it can be seen a slight increase in cell via-
bility/proliferation for samples 0S-TT versus 0M-TT; 1S-TT versus
1M-TT, and 2S-TT versus 2M-TT, suggesting that seashell-derived
bioceramics are slightly more cytocompatible than the marble-
derived ones.
Taken together, the biological evaluation of the developed bio-
ceramics, in terms of pre-osteoblast survival, morphology and
viability/proliferation, demonstrated their potential to be used as
bone graft substitutes.
3.8. Compressive strength evaluation
The samples mechanical performance was evaluated by stress-
strain curve measurements (Fig. 8) and compressive and tensile
strength assays (Fig. 9). All the analyzed specimens resemble the
typical fragile mechanical characteristics of ceramic materials,
slightly accentuated in the case of the seashell-derived ones.
Youngs modulus (elastic modulus) of marble-derived TT sam-
ples decreased with the increase of H3 PO4 quantity used during
synthesis and with respect to the bovine HA-TT reference sample
(e.g. 0M-TT decreased with 61%) (Fig. 8A). A similar variation could
be noticed for seashells-derived TT samples (e.g. 0S-TT decreased
with 32% compared to HA-TT sample) (Fig. 8B), only that the mini-
mum value in this specic case was recorded for the 2S-TT material
(corresponding to 1.2 stoichiometric H3 PO4 quantity).
The compressive strength values (Fig. 9A) varied inversely pro-
portional with the H3 PO4 quantity used for synthesis, similarly to
elastic modulus evolution. However, suitable results, compared to
those of reference HA-TT specimen, were identied only for sam-
ples synthesized under stoichiometric regime (i.e., 0M-TT, 0S-TT
with biphasic HA/-TCP composition). Their compressive strength
values fall within the 210 MPa range, corresponding to the human
cortical bone [41]. All analyzed samples revealed higher break-
ing strain values (Fig. 9B) with respect to the reference HA-TT
specimen. The seashells-derived materials withstood the highest
deformations. The high recorded standard deviations suggest the
presence of non-homogeneity induced by porosity.
4. Conclusions
The proposed hydroxyapatite synthesis method focused on
the optimization of Rathje technique due to limited information
reported on the subject and the involved biogenic precursors,

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dolomitic marble and Mytilus galloprovincialis mussel shell. Of the
key parameters governing this route performance (calcination tem-
perature, pH, magnetic stirring), the H3 PO4 amount modulation
allowed for a wide range of calcium phosphates synthesis before
and after the thermal treatment: from 100% HA (stoichiometric
conditions) and bi-phasic HA/DCPA compounds (after acid mod-
ulation) to bi-phasic HA/-TCP, pure -TCP or -TCP/Ca2 P2 O7
compounds. This chemical dynamism improved the samples sin-
terability, generating denser morphologies with great effect on
the contact angle measurements performed on thermally treated
samples: the lowest WCA values corresponded to samples with
increased H3 PO4 amount and higher surface porosity. A particular
inuence of phosphoric acid increment was outlined in terms of
surface roughness and energy, which displayed a positive ascend-
ing trend line for both precursors, from stoichiometric to maximum
acid amount, and was consistent to the contact angle downtrend.
Further, the surface wettability was emphasized in all cases, by
means of good cell spreading, proliferation and differentiation, with
no cytotoxic effects.
Taken together, these results conrm the possibility for HA
preparation from sustainable and cost-efcient raw materials and
a viable precursors range expansion using dolomitic marble. This
alternative synthesis method allows for highly performing bio-
ceramics fabrication, destined for medical applications. Further
investigations are required to thoroughly establish the optimum
HA/DCPA ratio for an improved performance of the bi-phasic CaPs.
Acknowledgements
This work was supported by a grant of the Romanian National
Authority for Scientic Research and Innovation, CNCS UEFISCDI,
project number PN-III-P2-2.1-PED-2016-0892. G.E.S. expresses
thanks to Core Programme PN 16 48-3/2016.
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