Vaterite coatings on electrospun polymeric fibers for

biomedical applications
Maria S. Savelyeva,1 Anatoly A. Abalymov,1 German P. Lyubun,1 Irina V. Vidyasheva,1
Alexey M. Yashchenok,1 Timothy E. L. Douglas,2 Dmitry A. Gorin,1,3 Bogdan V. Parakhonskiy1,2,4
1

Remote Controlled Theranostic Systems Lab, Educational Research Institute of Nanostructures and Biosystem, Saratov State
University, Astrakhanskaya, 83, Saratov, 410026, Russia
2
Department of Molecular Biotechnology, Faculty of Bioscience Engineering, Ghent University,
Coupure Links 653, Ghent 9000, Belgium
3
RASA Center in Tomsk, Tomsk Polytechnic University, 634050, Tomsk, Lenin Avenue, 30, Tomsk 634050, Russia
4
A.V. Shubnikov Institute of Crystallography Russian Academy of Science, Leninskiy prospect 59, Moscow 119333, Russia
Received 31 May 2016; revised 4 August 2016; accepted 17 August 2016
Published online 00 Month 2016 in Wiley Online Library (wileyonlinelibrary.com). DOI: 10.1002/jbm.a.35870
Abstract: The process of porous calcium carbonate (CaCO3)
covering on electrospun poly(e-caprolactone) (PCL) fibers is
described in this study. Uniform CaCO3 coatings, composed
of vaterite microparticles and its aggregates, were formed
on PCL fibers by mineral precipitation from solution under
ultrasonic treatment. The porous structure of CaCO3 in vaterite polymorphic form is useful for loading of various substances (drugs and nanoparticles), and this property makes
vaterite an appropriate material for design of drug delivery
systems. Such mineralization was implemented to attain
therapeutic and/or biological activity of tissue engineering
scaffolds based on electrospun PCL, by means of CaCO3
coatings. Various structures and polymorphs of CaCO3 coatings were obtained by variation of growth conditions (time
of fiber incubation in work solution, ultrasonic treatment of

this system). Coating homogeneity, CaCO3 polymorphic

form, morphology, and CaCO3 mass can be controlled by
number of successive stages of fibrous material treatment.
Cytotoxicity tests showed that PCL fibers mineralized with
CaCO3 did not release substances toxic for cells. SEM
images of PCL/CaCO3 scaffolds cultured with cells demonstrate that scaffolds supported cell adhesion and spreading.
The presented results show the new technique of controlled
PCL scaffold mineralization with vaterite, and an opportunity
C
of using PCL/CaCO3 as scaffolds for tissue engineering. V
2016 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 00A:000000,
2016.

Key Words: calcium carbonate, electrospun scaffolds, composite fibers, functional coating, tissue engineering

How to cite this article: Savelyeva MS, Abalymov AA, Lyubun GP, Vidyasheva IV, Yashchenok AM, Douglas TEL, Gorin DA,
Parakhonskiy BV. 2016. Vaterite coatings on electrospun polymeric fibers for biomedical applications. J Biomed Mater Res Part
A 2016:00A:000000.

INTRODUCTION

Designing of hybrid nanostructured materials consisting of a

polymer and an inorganic component are explored intensively due to their potential for biomedical applications. The
combination of well-known polymer properties (simplicity
of processing and formation, improved mechanical properties) and inorganic moieties properties (biocompatibility,
biodegradability, functional properties), supplemented with
nanostructured morphology, allows benecial applications
in regenerative medicine to be realized.1 For this area, a
number of specic properties (nontoxicity, bioactivity, biodegradability, and so forth) and biomimetic morphology are
critical.2 Moreover, the structure of the polymeric scaffold
has a determinative effect on the growth behavior of the

inorganic material and its resulting morphology.3 In view of

these requirements, nonwoven polymeric materials fabricated by electrospinning (ES) are especially interesting.4
Such materials are structured at nanoscale level, similar to
the natural extracellular matrices of living tissues, and can
be prepared from a wide range of polymers.5 Electrospun
bers can be successfully used as scaffolds for design of tissue engineering materials because of their unique physical
chemical properties due to nanostructured nature of scaffold. To prepare such scaffolds, synthetic [polycaprolactone
(PCL),6,7 and polyurethane8,9], and/or natural [chitosan,10,11
collagen,12] polymers can be used. Such scaffolds have been
used for neural,13 dermal14,15 tissue engineering, vascular
grafts,16 bone,17 and cartilage18 reconstruction.

Correspondence to: B. V. Parakhonskiy; e-mail: Bogdan.parakhonskiy@ugent.be

Contract grant sponsor: RFBR Research Project; contract grant number: 1529-01172
Contract grant sponsor: Marie Curie project; contract grant number: PIRSES-GA-2013612673
Contract grant sponsor: Government of the Russian Federation; contract grant number: 14.Z50.31.0004
Contract grant sponsor: Government of the Russian Federation; Russian Governmental Program Nauka; contract grant number: 4002

C 2016 WILEY PERIODICALS, INC.

V

The modication of electrospun bers allows new scaffold

properties to be achieved (biologically active properties,
enhanced mechanical properties, drug delivery capability),
which can be benecial for the design of functional biomedical
materials.4 The widely accepted modication technique is the
incorporation of various llers into the ber structure. The
embedding of mineral particles (calcite nanoparticles,19
hydroxyapatite nanoparticles,20,21 calcium phosphates CaP,22
silicon-doped vaterite23), the embedding of carbon structures
(carbon nanotubes24 and fullerenes25), silver26 or gold11 nanoparticles and the incorporation of nanocontainers with biologically active agents (liposomes27) have been carried out to
obtain scaffolds with improved functionality. However, electrospinning of polymer solution mixed with llers has limitations.
In particular, the ller can be irregularly distributed in bers,
resulting in nonuniform distributions of properties within the
resulting composite material. Another approach is based on the
functionalization of ber surfaces (formation of functional
coatings) by using specic physicalchemical treatment of
bers. Such approaches allow uniform treatment of the material surface to be achieved with uniform properties throughout
the material. Fibers with biomimetic coatings consisting of
osteotropic inorganic materials can be successfully used in
bone tissue repair and substitution. Such composite materials
exhibit properties including interconnected porosity, bioactivity, osteoconductivity, and biodegradability, which are similar
to those of natural bone material, and which all articial substitutes should ideally have. The uniform biomimetic ber coatings of CaP can be obtained by mineralization of brous
scaffold in simulated body uid (SBF).28,29 Similarly, CaP and
CaCO3 can be precipitated on scaffold by its incubation in salt
solutions.3032 In Ref. 33, vaterite particles were deposited on
polymer foam matrix to obtain hydroxyapatite coatings to
design bone tissue engineering scaffold.
The aim of this study was the creation of biocompatible
tissue engineering scaffolds which are suitable for cell culturing and endowed with enhanced functionality. The PCL brous
scaffold was selected as a suitable biomimetic substrate for
CaCO3. In this work, we prepared and studied electrospun
PCL scaffolds modied by functional porous CaCO3 coatings.
The uniform ber shells, consisting of vaterite particle
agglomerates, were synthesized on polymeric bers of PCL by
using a simple, but efcient technique. Vaterite is a metastable
CaCO3 polymorph, which occurs as porous spherical particles,
whose potential for various biomedical applicationsespecially drug deliveryhas been recognized in numerous studies3438; thus, the modication of bers by vaterite can be
benecial for tissue engineering applications. The coating uniformity, CaCO3 morphology, and general CaCO3 coating mass
can be controlled by the mineralization technique presented
in this study. The cytocompatibility test shows the suitability
of PCL/CaCO3 scaffolds for cell culturing. Such scaffolds with
functional coatings have perspectives to be used as drug delivery platforms as well as tissue engineering scaffold.
MATERIALS AND METHODS

Fabrication of electrospun material

Nonwoven nanobrous material was produced by an electrospinning technique. A solution of PCL (Mw-80 kDa,

SAVELYEVA ET AL.

Sigma-Aldrich Chemie, Germany) with 9.4% wt concentration was prepared by dissolving PCL pellets in a solvent
mixture of dichloromethane CH2Cl2 (DM, ComponentReactive, Russia) and N,N-dimethylformamide C3H7NO
(DMF, Component-Reactive, Russia) (weight ratio 77:23) followed by stirring for 3 h at room temperature to obtain a
homogeneous spinning solution. Then, the obtained mixture
was poured into a syringe, which was placed in an experimental electrospinning setup. The formation of nonwoven
material was conducted for 3 h under an applied voltage of
17 kV and at a feeding rate of 0.5 mL/h. The distance
between the needle and the collecting screen was 25 cm.
Treatment of brous material and CaCO3 coating
formation
To carry out the mineralization of PCL bers, a typical process of CaCO3 particles crystallization from a mixture of
CaCl2- and Na2CO3-saturated solutions was applied (solutions were prepared using calcium chloride dihydrate
CaCl2
2H2O, sodium carbonate Na2CO3, all Sigma-Aldrich
Chemie, Germany). Pieces of electrospun PCL mats were
incubated in such a working solution, so that the polymeric
bers acted as substrates for directed growth of CaCO3. To
study the effect of growth conditions on the resulting CaCO3
structures, three experimental methods were used. In all of
them, material samples of 1 3 1 cm and 1 mL of each salt
solution with equal concentrations (1 M or 0.33 M) were
used. Before the mineralization was carried out, all PCL
scaffolds were preliminarily treated with CaCl2 solution in
an ultrasonic bath for 10 min.
In the rst experiment, the scaffold was placed in CaCl2
solution, after which the Na2CO3 solution was added. The
system was settled for 10 min, after which the scaffold was
taken out, washed twice with deionized water, and dried in
a drying oven at 408C for 20 min. The second experiment
was carried out the same way, but the scaffold was incubated in working solution for 30 s, after which it was taken
out, settled for 1 min on a glass slide to complete the crystallization process, washed, and dried the same way as
described above. In the third experiment, the PCL scaffold
and 1 mL of CaCl2 solution were placed in an Eppendorf
tube, which was then placed in an ultrasonic bath (Sapr,
Russia). One milliliter of Na2CO3 solution was added to the
Eppendorf tube upon ultrasonic treatment. The experiment
was performed at working frequency 35 kHz and radiation
intensity 0.64 W/cm2. This system was subjected to ultrasound for 30 s, after which it was removed from the bath
and set aside for completion of the crystallization process.
Finally, the treated scaffold was washed and dried in the
same way.
The formation of CaCO3 uniform ber coatings was carried out by performing repeated successive treatment
stages. The repeated treatment was accomplished by the
third method of CaCO3 mineralization of scaffolds; the material was subjected to one treatment stage, was processed
further in a second stage by the same way, then in a third
stage, and so forth. In such a manner, seven treatment
stages were realized. The material (1 3 1 cm) was

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ORIGINAL ARTICLE

FIGURE 1. SEM images of pure PCL scaffold with fiber size distribution (A) and PCL scaffolds treated by using following methods: incubation in
working solution for 10 min (B); incubation in working solution for 30 s (C); incubation in working solution for 30 s under ultrasonic agitation
(D). Circular curves point to shell-like CaCO3 agglomerates; square curve arrows point to CaCO3 spherulites, similar to those of which the shells
consist.

subjected to procedures; 1 mL of each salt solution (CaCl2

and Na2CO3 with 1 M concentration) was used in each
stage. Mass of the sample was controlled during treatment
stages; measurements of the mass were accomplished with
a Semi-micro analytical balance GR-200 (A&D Company Ltd,
Japan).
Characterization
The structure and morphology of PCL/CaCO3 samples
obtained were investigated by scanning electron microscopy
(SEM) (MIRA II LMU device (Tescan, Czech Republic)) at an
operating voltage of 1530 kV. The observed samples were
sputtered with gold prior to examination.
X-ray diffraction analysis (XRD) of PCL/CaCO3 samples
was performed using a Xcalibur GeminiA device (Oxford Diffraction) equipped with a Cu anode and the operating voltage and current were maintained at 40 kV and 40 mA,
respectively. The database COD (Crystallography Open Database) was used for the diffractogram analysis.
Size distribution of blank PCL bers was performed
using obtained SEM images and Image J software (NIH,

http://rsb.info.nih.gov/ij/). One hundred bers per sample

were analyzed.
Cell preparation
Normal human dermal broblasts (NHDF) were provided by
the Department of Cell Engineering, Education and Research
Institute of Nanostructures and Biosystems, Saratov State
University, Russia. Cells were used in passages 26. All cells
were plated separately in tissue culture asks and cultivated in Dulbeccos Modied Eagle Medium (DMEM, SigmaAldrich), containing 10% fetal bovine serum (FBS, Hyclone),
2 mM L-glutamine (Sigma-Aldrich), and 1% penicillinstreptomycin antibiotic antifungal cocktail (Sigma-Aldrich). The
media were replaced every 3 days, and the cells were maintained in a humidied incubator at 378C with 5% CO2. Cell
cultures with 7585% conuence were harvested using
0.25% trypsin (Life technologies) and counted with a
hemocytometer.
All materials were sterilized with 70% ethanol for 30
min, air-dried, and washed three times with PBS. Then cells
were seeded on the sample surfaces in DMEM cell culture

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FIGURE 2. SEM images of the scaffold treated with 0.33 M salt solutions (A); scaffold treated with 1 M salt solutions (B) and scaffold subjected
to two treatment stages with 1 M salt solutions (C).

medium with average density 4 3 105. The samples were

maintained in a CO2-incubator overnight.
Cell viability
NHDF cells were seeded into 24-well cell-culture plates at a
cell density of 10 3 104/well. After 24 h of cultivation, 10
3 10 mm samples were placed into the plate in culture
medium and incubated overnight at 378C under 5% CO2.
Subsequently, cells were incubated (Innova CO-170, New
Brunswick Scientic) at 378C for 4 h, together with added
materials. In the last step, 100 lL of uorescence dye was
added to each well (AlamarBlue, Sigma-Aldrich) and uorescence (540/610 nm) intensity was measured by a spectrophotometer (Synergy H1 Multi-Mode Reader). The cell
viability was studied for up to 3 days using the dye exclusion method. The results of cytotoxicity test were analyzed
using a one-way analysis of variance (ANOVA). Values of
p < 0.05 were considered signicant. The Fishers indexes
were calculated for groups of values (PCL/CaCO3, PCL, control) of each time point separately.
Cell adhesion
To study cell attachment and distribution on the PCL/CaCO3
and PCL scaffolds, the cell cultures were investigated using
scanning electron microscopy. For this, cells on scaffolds
were xed by incubation with 4% formalin for 20 min at
room temperature. Each scaffold was supported individually
with forceps during immersion in xative. After incubation,
scaffolds were transferred immediately to PBS to quench
any unreacted formalin. Finally, the treated scaffolds with
cells were washed with deionized water and dried.
RESULTS

The shell-like vaterite bers coatings formation

The electrospun PCL mesh consisting of thin bers with
average diameter 0.5 6 0.1 lm was used as scaffold for mineralization [Fig. 1(A)]. Figure 1(B,C) represents the SEM
images of scaffolds treated under the following altered
experimental conditions: synthesis time and speed at which
homogenous distribution of reagents in working solution

SAVELYEVA ET AL.

was achieved. Polymorphic and morphological differences of

the resulting CaCO3 structures, obtained by using various
methods, are apparent. The scaffold, treated by incubation
in working solution for 10 min, is shown in Figure 1(B).
Calcium carbonate is present in the form of cubic calcite
crystals, which are attached to and located along bers.
Decreasing the incubation time to 30 s caused the changing
of polymorphic form and resulted in spherical vaterite particle formation [Fig. 1(C)]. Here, vaterite particles were precipitated on the bers. It can be seen that particles are
distributed on the scaffold in an irregular manner. Some of
them are located along bers; most particles are not bound
to the ber surface and are found in gaps between bers. It
can be concluded that the majority of the particles were
synthesized in the bulk of working solution and then precipitated on the bers.
This is supported by the fact that the CaCO3 deposits
are situated on the external surface only, but not on the
scaffold lower layers and in the scaffold interior. The scaffold mineralized by incubation in working solution for 30 s
under ultrasonic treatment is presented in Figure 1(D). We
can see from this image that most of the calcium carbonate
was precipitated in the form of particle aggregates along
bers. Porous morphology and similarity to spherical form
can indicate the presence of the vaterite polymorph. The
shell-like agglomerates consisting of vaterite particles can
be seen on some bers. These shells, having a porous structure, consist of spherical vaterite microparticles.
Figure 2 shows the comparative SEM images of PCL
material mineralized by ultrasonically assisted treatment in
0.33 M salt solution [Fig. 2(A)] and 1 M salt solution [Fig.
2(B)]; and PCL material after two identical treatment stages
in 1 M salt solution [Fig. 2(C)]. These images can be useful
in explanation of the mechanism of formation of CaCO3
coatings and the evolution of CaCO3 structures on bers
during mineralization process, to some extent.
The series of repeated treatments of scaffold with calcium carbonate shells was carried out to attain uniform
coatings throughout the scaffold like sketched in Figure 3
(top panel). Seven treatment stages were realized. The SEM

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ORIGINAL ARTICLE

FIGURE 3. The scheme of fibrous material mineralization under ultrasound treatment and corresponding SEM images of blank PCL fibrous material (A), PCL material on initial mineralization stage (B), and scaffold with uniform CaCO3 coating after second treatment and cross-section of
this scaffold (C).

images of scaffolds at each stage are shown in Figure 4. The

increment of CaCO3 mass was controlled during stages, and
results were reported in Figure 5. Comparing results for the
rst and second stages [Fig. 4(A,B)], we can conclude that
the repeated treatments resulted in an increase of the

number of bers coated with CaCO3. Hence, the scaffold

areas, which were not mineralized by the rst treatment,
were covered with CaCO3 by the second one. The increase
of sample mass after the second treatment is in agreement
with this conclusion (Fig. 5). Further treatment (third stage)

FIGURE 4. SEM images of PCL scaffolds mineralized by one treatment stage (A), two (B), three (C), four (D), five (E), six (F), and seven (G) treatment stages.

JOURNAL OF BIOMEDICAL MATERIALS RESEARCH A | MONTH 2016 VOL 00A, ISSUE 00

FIGURE 7. Results of cytotoxicity tests of PCL/CaCO3 and PCL substrates on NHDF. Error bars show standard deviation. Values labeled
with an asterisk (*) are significantly different from the control based
on the ANOVA test.

FIGURE 5. The increase of the CaCO3 mass on PCL nanofibrous material (1 3 1 cm) depended on number of treatments. Error bars show
standard deviation calculated from 5 measurements for sample in
each treatment stage.

also causes CaCO3 shell enlargement [Fig. 4(C)]. The next

stages are the initiation of the recrystallization process and
the transformation of a part of vaterite to calcite [Fig.
4(D,E)]. After the seventh treatment stage, CaCO3 coatings
consist almost completely of calcite, and shells with a morphology characteristic of calcite can be observed [Fig. 4(G)].
Further treatment of this scaffold is inappropriate, as
mechanical deformation of the sample can be observed
visually.
The X-ray diffractograms obtained from pure PCL nanobrous material and scaffolds mineralized during three treatment stages are shown in Figure 6. The diffractograms
(Fig. 6) exhibit peaks which are characteristic for vaterite
polycrystallites, and one peak which is characteristic for calcite rhombohedra.

Cytocompatibility testing of PCL/CaCO3

The cytotoxicity tests were conducted with NHDF cells with
the aim of revealing the inuence of PCL and PCL/CaCO3
scaffolds on the cell viability at different time intervals such
as 24, 48, and 72 h. The quantitative assay relating to the
percentage of viable cells for different time intervals is presented in Figure 7. The cell viability on PCL/CaCO3 is comparable with the results for control culture medium at all
time points. At the same time, the control PCL scaffold demonstrates a lower level of cell viability in comparison with
the PCL/CaCO3 scaffold and control culture medium.
Adhesion and spreading of NHDF cells
SEM images of PCL/CaCO3 and control PCL scaffolds cultured with NHDF cells for 24 and 48 h are presented in Figure 8. The cells on both PCL/CaCO3 scaffold [Fig. 8(A,B,E,F)]
and PCL scaffolds [Fig. 8(C,D,G,H)] had a well-spread morphology typical for attached cells, after both 24 h and 48 h
culture. The PCL/CaCO3 scaffold after 48 h culture had
deposits and structural inclusions, which are supposed to
be a product of cell activity. In contrast, no such formations
were observed on the PCL scaffold.
DISCUSSION

FIGURE 6. The X-ray diffraction patterns of blank PCL nanofibrous

material and scaffolds PCL/CaCO3 after various numbers of treatments. Peaks consisting to planes of vaterite (V), calcite (C), and PCL
are indicated.

SAVELYEVA ET AL.

As stated in the introduction, the aim of this study is the

preparation of a biocompatible functional tissue engineering
scaffold based on vaterite-coated PCL bers. The electrospun polymeric matrix consisting of thin bers with average
diameter 0.5 6 0.1 lm [Fig. 1(A)] is similar to natural
extracellular matrices. In living tissues, the collagen bers
have diameters in the range from 50 to 500 nm.39 PCL is a
US Food and Drug Administration (FDA)-approved polymer
for its biocompatibility and biodegradability. Due to these
properties and to the good mechanical properties of PCL,
scaffolds of this polymer have been used in different elds
of tissue engineering.40 Vaterite coatings on bers can provide an enhanced functionality of the scaffold due to benecial properties of its material. The porous structure of
vaterite and mild conditions of its synthesis and decomposition41 allow loading of various bioactive substances (such
as insulin,42 therapeutic enzymes,43 doxorubicin,44 photosesitizers41,45,46) into vaterite structures to be performed.
CaCO3 is biocompatible and able to biodegrade rapidly,41 so

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ORIGINAL ARTICLE

FIGURE 8. SEM images of PCL/CaCO3 scaffolds after culturing NHDF for 24 h (A, B) and 48 h (E, F); and control PCL scaffolds after culturing
NHDF for 24 h (C, D) and 48 h (G, H) with overview (A,C,E,G) and high magnification (B,F,D,H). Red arrows are highlighted the cells.

it can be successfully used as a drug delivery system.35,4547

Vaterite spherical particles have served as vehicles for delivery of photosensitizer for photodynamic therapy in tumor
tissues.41,46 In this way, the vaterite coating on scaffolds can
provide drug delivery functionality due to the possibility of
drug loading into vaterite.
The CaCO3 can be precipitated on PCL bers by using
the classical route of CaCO3 synthesis supplemented with
presence of polymeric bers as CaCO3 growth centers. The
incubation of brous PCL material in CaCl2Na2CO3 working
mixture for 10 min results in calcite crystals [Fig. 1(B)]. Calcite is the most thermodynamically stable CaCO3 polymorph,
and the most abundant form in nature. Here, calcite crystals
have been grown on a brous substrate upon simple mixing
of Ca21 and CO22
ions. Vaterite has the lowest stability in
3
comparison with calcite and the other CaCO3 polymorph,
aragonite. Hence, it usually transforms to calcite in a moist
environment. Decreasing the incubation time to 30 s causes
the interruption of CaCO3 crystallization process due to
removal of the working solution, and therefore the transformation of vaterite to calcite did not have time to occur [Fig.
1(C)].
The vaterite shell-like coatings on bers can be obtained
by incubation in working mixture under US treatment [Fig.
1(D)]. The shell-like formation can be accounted for by the
ultrasonic inuence on the crystallization process and
CaCO3 particle nucleation. To understand the formation of
CaCO3 structures in our system, it is necessary to have an
insight into the classical process of CaCO3 crystallization.
In the study of P. Bots et al. (2012),48 the behavior of

vaterite microparticles formation was studied. When solutions containing Ca21 and CO22
3 ions are mixing, amorphous
calcium carbonate (ACC) nanoparticles form initially. These
nanoparticles have sizes in the range from a few to tens of
nanometers. In agglomerates of nanoparticles, the growth
centers appear and there vaterite particles start to form.
Decomposition of ACC around growth centers provides
delivery of Ca21 and CO22
ions to growing vaterite par3
ticles. ACC transformation to vaterite is described by the
mechanism of spherulitic growth of polycrystalline particles,
where new particles grow via the continual nucleation of
misaligned structurally equal crystallites on surfaces of
growing spherulites.49 This process results in formation of
spherical polycrystalline particles consisting of crystallites
of approximately equal sizes. The further evolution of the
spherical CaCO3 particles is described by means of the classical Ostwald ripening process, which accounts for the
growth of larger particles at the expense of dissolution of
smaller particles.48,50 In this case, the increase of vaterite
crystallites size occurs via a dissolutionreprecipitation
mechanism, and this process nally leads to calcite crystallization. To prevent the fast recrystallization of metastable
vaterite to calcite and inhibit it to an extent, particles
should be stored in an environment with sufciently low
humidity. In some studies, for example, in Ref. 51, the vaterite formation was considered as an aggregation of primary
ACC nanoparticles to spherical microparticles. However,
nonuniformity of size of the resulting particles can hardly
be explained by nanoaggregation. On the contrary, it can be
described by the supposed spherulitic crystal growth.52

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In a great number of previous articles, the effects of various experimental conditions on nal CaCO3 morphology
and polymorph have been studied. The use of supersaturated salt solutions promotes the increase of the number of
growth centers. As the amount of reactant in the system is
nite, the nal particles sizes will decrease as a result.48,52
In the study of L. Dupont et al.,53 it was shown that vigorous agitation of working solution during CaCO3 particle formation, which aids rapid homogenous distribution of
reagents in the volume, leads to the occurrence of a larger
number of growth centers. The cavitation effects in solution,
occurring from ultrasonic impact, sustain the intensive stirring of the solution and have the same inuence.54 The
ultrasound prevents formation of big particles and their
aggregates in the bulk solution and stimulates their dissolution, and at the same time sustains a free circulation of
Ca21 and CO22
ions in the system. In addition, the ultra3
sonic treatment provides the penetration of solutions into
pores and interior of the brous material, which facilitates
the homogenous mineralization of the scaffold.
In our case, the bers serve as the growth centers for
CaCO3 crystallization, and ACC or/and forming nuclei are
precipitating on bers and are organizing growth centers
for vaterite particle formation. The vaterite particles, some
of which formed on the bers and some of which formed in
the bulk solution and then precipitated on the bers, also
can serve as sites for agglomeration with other particles.
Figure 2 shows the bers with CaCO3 structures which indicate the mechanism of calcium carbonate coating formation.
The bers treated with 0.33 M salt solutions [Fig. 2(A)]
exhibit the CaCO3 spherical particles with sizes <1 lm,
located on bers and tending to agglomeration on the ber
surface. The increasing of solution concentrations to 1 M
causes an increase of the amount of CaCO3 produced, and
therefore it causes an increase in the number of forming
vaterite particles [Fig. 2(B)]. In addition, an increase in the
size of some particles occurred. The particles are organized
in agglomerates on the ber surface, which cover the bers,
and thus form the shell-like structure. The bers on Figure
2(C) were treated twice by the same way as the previous
ones. They have a CaCO3 coating with slightly different morphology. In this case, it is difcult to distinguish the outlines
of spherical particles in some CaCO3 formations. They
seemed to consist of crystallites which are similar to those
of which vaterite particles are composed. These formations
can be composed of associations of incompletely formed
spherulites and their aggregations with formed particles. In
such a manner, the shell-like structures, located on bers,
can be formed by growth and organization of vaterite particles and crystallites on bers. The nal CaCO3 structures,
resulting from complicated associations of agglomerates
consisting of microparticles and crystallites, make up the
uniform ber calcium carbonate shells.
The scheme in Figure 3 illustrates the described mechanism of ber mineralization. In this way, the uniform CaCO3
shell-like ber coatings over the whole material can be
obtained by using ultrasound treatment in CaCO3 synthesis
[Fig. 3(C)]. It should be noted that the CaCO3 was

SAVELYEVA ET AL.

precipitated on inner bers as well [Fig. 3(C), the crosssection of PCL scaffold after second treatment].
The realization of successive treatment stages can allow
uniform vaterite coatings on bers to be achieved and can
allow the observation of the changes of CaCO3 coating morphology to be carried out. The number of vaterite-coated
bers increases during the second and third mineralization
stages [Fig. 4(B,C)]; after the second stage, the vaterite covers almost the whole surface of bers.
The increase of sample mass (Fig. 5) conrms the addition of CaCO3 to PCL material. Thus, two mineralization
stages are sufcient to produce uniform coatings. The third
stage causes an enlargement and densication of preexisting shells, which can be explained by the precipitation
of CaCO3 on the existing CaCO3 structures which are serving
as sites for crystal growth, which was also conrmed by the
corresponding increase of sample mass (Fig. 5). The vateritecalcite transformation in CaCO3 initiated by the fourth
treatment can be explained by the Ostwald ripening process,
which is occurring during each repeated treatment stage in
parallel with the CaCO3 precipitation. When the size of
vaterite crystallites becomes critical, the process of recrystallization from vaterite to calcite takes place.48 The
increases of scaffold mass by CaCO3 coating mass increments (Fig. 5) are steadily occurring during the fourth to
the sixth treatment stages. This fact also conrms the
replacement of vaterite by calcite, as vaterite has a lower
density than calcite due to its porosity. Thus, uniform CaCO3
ber coatings in the electrospun scaffold can be obtained by
using two stages of treatment.
The cytotoxicity test conducted with NHDF (Fig. 7) show
that PCL/CaCO3 scaffolds do not have an inuence on the
cytotoxicity. Moreover, based on ANOVA test studies, the
NHDF cell viability on the scaffold is comparable with the
cell viability in control culture medium at all time points,
thus it can be concluded that cells have the sufcient activity and ability to ssion in the PCL/CaCO3 scaffold, along
with control culture medium. SEM images of cultured scaffolds (Fig. 8) show cells with a morphology typical for
spreading broblasts. Fibroblasts are attached with their
pseudopodia to scaffold bers and exhibit sufcient cell
adhesion to scaffold and homogeneous distribution. The
scaffold cultured for 48 h [Fig. 8(E,F)] contains cell aggregates and substances, which are cross-linked bers of material, indicating the activity of living broblasts in the
scaffold. In the case of control PCL scaffolds, there is lower
cell viability in comparison with mineralized scaffolds and
control culture medium, though pure PCL bers also demonstrate well-spread and attached broblasts. It can be supposed that cells are better able to adhere to mineralized
bers.
CONCLUSIONS

In this study, we demonstrated the formation of functional

porous vaterite coverings on PCL ber surfaces. The uniform vaterite coatings cover the whole surface of the electrospun material, as well as its interior, and can be obtained

VATERITE COATINGS ON ELECTROSPUN POLYMERIC FIBERS

ORIGINAL ARTICLE

by using the ultrasound technique described. The variation

of experimental conditions (reagent concentration, time)
and number of treatment stages allow control over the
mass of CaCO3 deposited, the polymorph (vaterite vs. calcite), and the coating uniformity. The normal human dermal
broblasts have been successfully cultivated on PCL/CaCO3
scaffolds. The mineralized scaffolds demonstrate no inuence on cell viability and nice adhesion over different time
intervals up to 72 h. As the result, the presented PCL/
CaCO3 materials are suitable for cell culturing and further
development of tissue engineering materials.
ACKNOWLEDGMENTS

The authors thank Andrey Zakharevich for providing scanning

electron microscopy measurements and Aleksander Scaptsov
for providing X-ray diffraction measurements. This study was
partially supported by the Government of the Russian Federation to support scientic research projects implemented under
the supervision of leading scientists at Russian institutions
and Russian institutions of higher education. T.E.L.D. and B.P.
thank the Research Foundation Flanders (FWO) for postdoctoral research fellowships. This work was in part supported
by Russian Governmental Program Nauka, No 4002).
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