journal of the mechanical behavior of biomedical materials 62 (2016) 209–221

Available online at www.sciencedirect.com

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Research Paper

Design and characterization of microcapsules-

integrated collagen matrixes as multifunctional
three-dimensional scaffolds for soft
tissue engineering

Loretta L. del Mercatoa,b,nn, Laura Gioia Passionea,b, Daniela Izzoc,

Rosaria Rinaldia,d, Alessandro Sanninoe, Francesca Gervasoe,n
a
Nanoscience Institute-CNR, Euromediterranean Center for Nanomaterial Modelling and Technology (ECMT),
via Arnesano, 73100 Lecce, Italy
b
CNR NANOTEC - Institute of Nanotechnology c/o Campus Ecotekne, Via Monteroni, 73100 Lecce, Italy
c
DHITECH s.c.a.r.l – High Technology Cluster c/o Campus Ecotekne, Via Monteroni s.n., 73100 Lecce, Italy
d
Department of Mathematics and Physics “Ennio De Giorgi” University of Salento, via Arnesano, 73100 Lecce, Italy
e
Department of Engineering for Innovation, University of Salento, Via Monteroni s.n., 73100 Lecce, Italy

art i cle i nfo ab st rac t

Article history: Three-dimensional (3D) porous scaffolds based on collagen are promising candidates for
Received 10 March 2016 soft tissue engineering applications. The addition of stimuli-responsive carriers (nano- and
Received in revised form microparticles) in the current approaches to tissue reconstruction and repair brings about
28 April 2016 novel challenges in the design and conception of carrier-integrated polymer scaffolds. In
Accepted 5 May 2016 this study, a facile method was developed to functionalize 3D collagen porous scaffolds
Available online 12 May 2016 with biodegradable multilayer microcapsules. The effects of the capsule charge as well as

Keywords: the influence of the functionalization methods on the binding efficiency to the scaffolds

Collagen scaffolds were studied. It was found that the binding of cationic microcapsules was higher than that

Multilayer polymer capsules of anionic ones, and application of vacuum during scaffolds functionalization significantly

Tissue engineering hindered the attachment of the microcapsules to the collagen matrix. The physical

Compression test properties of microcapsules-integrated scaffolds were compared to pristine scaffolds.

Confocal microscopy The modified scaffolds showed swelling ratios, weight losses and mechanical properties

Scanning electron microscopy
similar to those of unmodified scaffolds. Finally, in vitro diffusional tests proved that the
collagen scaffolds could stably retain the microcapsules over long incubation time in Tris–
HCl buffer at 37 1C without undergoing morphological changes, thus confirming their
suitability for tissue engineering applications. The obtained results indicate that by tuning
the charge of the microcapsules and by varying the fabrication conditions, collagen

n
Corresponding author at: Department of Engineering for Innovation, University of Salento, Via Monteroni s.n., 73100 Lecce, Italy
nn
Corresponding author at: CNR NANOTEC - Institute of Nanotechnology c/o Campus EcotekneEcotekne, Via Monteroni, 73100 Lecce,
Italy.
E-mail addresses: loretta.delmercato@nanotec.cnr.it (L.L. del Mercato), francesca.gervaso@unisalento.it (F. Gervaso).

http://dx.doi.org/10.1016/j.jmbbm.2016.05.009
1751-6161/& 2016 Elsevier Ltd. All rights reserved.
210 journal of the mechanical behavior of biomedical materials 62 (2016) 209 –221
scaffolds patterned with high or low number of microcapsules can be obtained, and that
the microcapsules-integrated scaffolds fully retain their original physical properties.
1. Introduction
Soft tissue loss and damage associated with trauma and
disease present a significant healthcare burden worldwide
(Rnjak-Kovacina et al., 2015). Among soft tissues there is
cartilage, a predominantly avascular and aneural tissue,
composed by scattered chondrocytes, the only cell type,
embedded within a dense extracellular matrix (ECM). The
regeneration of damaged articular cartilage is limited by poor
ability to self-repair and post-injury cartilage is predomi-
nantly replaced by fibrocartilage through healing from the
subchondral bone. Fibrocartilage lacks the key properties that
characterize hyaline cartilage such as capacity for compres-
sion, hydrodynamic permeability and smoothness of the
articular surface. Traditional methods of repair of chondral
defects include debridement, marrow stimulation, whole
tissue transplantation and chondrocyte implantation techni-
ques (Ye et al., 2013).
In the last years, tissue engineering (TE) approaches have
emerged as potential alternative therapeutic strategies to
treat severely injured patients with minimally invasive tech-
niques (Lee and Shin, 2007). Biomaterials play a unique role in
tissue regeneration and repair: they serve as tunable biophy-
sical and biochemical environments that direct cellular beha-
vior and functions thus promoting the replacement and
regeneration of missing or injured tissues (Rnjak-Kovacina
et al., 2015). Most TE approaches involve the use of 3D porous
scaffolds, constructed from natural or synthetic materials, to
modulate cell proliferation and differentiation (Bitar and
Zakhem, 2014; Boix et al., 2005; Janik and Marzec, 2015; Lee
et al., 2011; Nandakumar et al., 2013; Ribeiro et al., 2014;
Smith and Grande, 2015; Tayalia and Mooney, 2009; Valonen
et al., 2010; Vo et al., 2012). For successful tissue regeneration,
scaffolds must address multiple functions. They must pos-
sess optimal mechanical properties (Zhu et al., 2013), in order
to support new tissue growth over a prolonged period of time;
they must have controllable and high porosity, in order to
promote optimal cell-biomaterial interactions, as well as cell
adhesion and ECM deposition and guarantee adequate trans-
port of nutrients, proteins and gases for cell growth; they
must be biocompatible in order to avoid in vivo inflammation
or toxicity; they must show biocompatibility and bioresorb-
ability at a controllable degradation and resorption
rate accordingly to the rate of tissue regeneration
(Dhandayuthapani et al., 2011; Fuchs et al., 2001; Langer and
Tirrell, 2004). On top of that, sustained release of bioactive
agents (e.g., growth factors and other signaling molecules) in
an appropriate timeframe, is considered a central task to
enhance and guide the regeneration process (Biondi et al.,
2008; O’Brien, 2011; Polo-Corrales et al., 2014). A variety of
growth factors have been used to stimulate cell proliferation,
differentiation and matrix deposition in musculoskeletal
& 2016 Elsevier Ltd. All rights reserved.
tissue engineering. Growth factor choice will depend on
(i) type of employed cells (pluripotent cells or differentiated
cells), (ii) behavior to be induced (differentiation, prolifera-
tion, ECM deposition or blood vessel formation) and (iii)
scaffold phase (cartilage, tendon/ligament, interface or bone)
(Font Tellado et al., 2015). One of the most restricting aspects
of delivering biomolecules is their limited chemical and
physical stability. For the delivery to be successful, however,
the bioactive molecule must remain active in many different
surroundings, e.g. the biological environment of the applica-
tion site or in chemical solvents occasionally used during the
manufacture of the release systems and of course within the
release system that it is incorporated in Tessmar and
Göpferich (2007). One of the possible attempts to control
molecular microenvironment is to use a TE scaffold as a
controlled release platform (Biondi et al., 2008).
Nowadays, various technologies have been exploited to
construct 3D porous scaffolds with different types of materi-
als. Since the major constituent of the ECM is collagen,
scaffolds made up of collagen are considered to be ideal for
TE. Indeed, polymeric scaffolds based on collagen show
unique properties including: biodegradability, high biocom-
patibility, low antigenicity, and appropriate mechanical char-
acteristics. Additionally, collagen scaffolds have been
observed to promote cell and tissue attachment and growth
(de Moraes et al., 2012; Glowacki and Mizuno, 2008; Schoof
et al., 2001), also under inflammatory conditions (Kwon et al.,
2013; O’Brien et al., 2005).
In our previous studies, collagen scaffolds obtained by
freeze-drying technique were tested in vitro and in vivo
(Deponti et al., 2014; Gervaso et al., 2012). The biological tests
proved the ability of the collagen scaffolds to be positively
populated by chondrocytes (Deponti et al., 2014). The cells
produced both aggrecan and type II collagen and showed the
chondrocyte phenotype after three weeks of culture. Notably,
the mechanical analyses performed on TE constructs showed
that their mechanical strength significantly increased after
three weeks of culture (Gervaso et al., 2012). On the other
hand, by studying a novel 3D bicomponent substitute made
of collagen type I and hydroxyapatite for the repair of
osteochondral lesions in a swine model, we recently observed
that the quality of the reparative tissue appeared to be
superior for the lesions treated with the unseeded scaffolds
respect to the scaffolds seeded with autologous chondrocytes
and the untreated group, indicating the promising potential
of this novel biomaterial for use in a one-stage procedure for
osteochondral repair (Sosio et al., 2014). In this scenario, we
want to improve the cell-free collagen scaffold and their
reparative capacity by adding a carrier system (microcap-
sules) for delivery of bioactive molecules. As previously
mentioned, one of the key challenges of research in bioma-
terials science is creating multifunctional and responsive
 journal of the mechanical behavior of biomedical materials 62 (2016) 209 –221
scaffolds that are also able to release biomolecules, such as
growth factors, at established rates, in a predetermined and
optimized spatial distribution. Growth factors play an impor-
tant role during tissue development, homeostasis and repair
(Lam et al., 2015). Given the multiphasic nature of entheses, i.
e. specialized tissue interfaces that integrate soft tissue (e.g.
tendon/ligament) in bone in order to facilitate joint motion,
multiple growth factors as well as growth factor gradients are
necessary to provide the seeded cell populations with differ-
ential proliferation and differentiation stimuli. Therefore, in
an optimal TE approach, multiple growth factors will be
gradually and sequentially released from the scaffold in a
spatiotemporally regulated manner. Numerous approaches
have been proposed for delivering growth factors and achiev-
ing sequential and multifactor delivery (Font Tellado et al.,
2015). The simplest strategy is to incorporate the biomole-
cules within the polymeric matrices directly during the
scaffold fabrication process. However, methods based on this
approach might suffer of certain ineffectiveness. In particu-
lar, the biological integrity of the cargo molecules cannot be
guaranteed if higher processing temperatures are used and if
significant amounts of molecules are already eluted from the
scaffold during processing, especially when water is used to
remove water-soluble porogens as in particulate leaching
processes (Tessmar and Göpferich, 2007; Biondi et al., 2008).
Moreover, the incorporation of a growth factor or a biomole-
cule directly into the scaffold does not allow to accurately
control and modulate the release spatiotemporally because it
strictly depends on the physicochemical properties of the
polymers that are used to fabricate the scaffolds. Biologics
entrapped within bulk matrices often have a large burst
release, which can be slightly tuned only by scaffold cross-
linking density and pore size (Lam et al., 2015).
An alternative approach is to load single or multiple
biomolecules within particle-based carriers that allow for
protecting the payload against unwanted degradation while
being able to release it upon different stimuli, such as a pH
decrease, protease degradation, light irradiation, magnetic
field, or ultrasound (Ganta et al., 2008; Iafisco et al., 2013; Lam
et al., 2015; Mura et al., 2013). In these approaches, the
protein-loaded carriers can be easily added onto the prefab-
ricated scaffolds or be added to the polymer phase while
processing the scaffolds (Tessmar and Göpferich, 2007). Poly-
mer capsules fabricated by the layer-by-layer (LbL) method
are considered highly tunable delivery vehicles and easy to
combine with other materials (De Luca et al., 2015; del
Mercato et al., 2016, 2015, 2014; Delcea et al., 2011; Kolbe
et al., 2011; Wohl and Engbersen, 2012; Xiong et al., 2013).
Biodegradable multilayer capsules integrated within 3D scaf-
folds would serve as containers of bioactive molecules that
would allow for controlling the release of the payload and for
protecting it from the surgical procedures (washing, debride-
ment, bleeding, fluid aspiration) applied during the implanta-
tion of the scaffold which challenge the integrity of the
material (De Cock et al., 2012; Facca et al., 2010; She et al.,
2012). The aim of this study was to develop a simple, versatile
and scalable methodology for creating 3D biomimetic scaf-
folds for tissue repair applications. To this aim, advantage
was taken of a simple freeze-drying technique, combined
with the LbL technology, to produce 3D collagen scaffolds
211
stably integrating biodegradable microcapsules sensitive to
proteases action. In order to study how surface interactions
modulate the adsorption of the microcapsules within the
collagen matrix, microcapsules with positive or negative
charges were employed and their attitude to interact with
the matrix was evaluated under several conditions, including
varying the incubation time and the atmospheric pressure.
The applied procedure allowed engineering the scaffolds with
microcapsules while preserving both the scaffold and micro-
capsules structures.
2. Materials and methods
2.1. Materials
Poly-L-arginine hydrochloride (PARG, Mw
70.000 Da), dex-
tran sodium salt from Leuconostoc spp (DEXS, Mw
10.000 Da),
calcium chloride dehydrate (CaCl2, Mw¼147.01 Da), sodium
carbonate (Na2CO3, Mw¼105.99 Da), bovine Serum Albumine
(BSA, Mw
66.000 MW), rhodamine B isothiocyanate (RITC,
Mw¼536.08 Da), fluorescein 5(6)-isothiocyanate (FITC,
Mw¼389.38 Da), dextran (DEX2000, Mw¼2000.000 Da), ethyle-
nediaminetetraacetic acid disodium salt dehydrate (EDTA),
acetic acid (ACS reagent, 99.7%), phosphate buffered saline
(PBS tablets), trizma base, sodium chloride, and sodium azide
were purchased from Sigma-Aldrich. Aminodextran (AM-
dextran, Mw
500.000 Da) was obtained from Invitrogen. All
chemicals were used as received. Ultrapure water with a
resistance greater than 18.2 MΩ cm was used for all
experiments.
2.2. Synthesis of biodegradable microcapsules
Polyelectrolyte multilayer (PEM) microcapsules were pro-
duced via the layer-by-layer (LbL) approach (Sukhorukov
et al., 1998). For LbL, the following degradable polyelectrolyte
pairs were employed: DEXS and PARG. Three types of PEM
microcapsules have been fabricated: (i) cationic microcap-
sules carrying dextran-rhodamine (DEXRITC) into their cav-
ities, (ii) anionic microcapsules carrying dextran-fluorescein
(DEXFITC) into their cavities, and (iii) cationic microcapsules
carrying one layer of bovine serum albumin labeled with
fluorescein (BSAFITC) into the shell. For each capsule system,
calcium carbonate (CaCO3) particles were precipitated from
solutions of calcium chloride (CaCl2) and sodium carbonate
(Na2CO3) under vigorous stirring. Briefly, equal volumes
(2.46 mL) of aqueous CaCl2 and Na2CO3 solutions (0.33 M)
were mixed in the presence of dextran-conjugates, DEXRITC
(150 mL, dye concentration 30 mM) or DEXFITC (84 mL, dye con-
centration 30 mM) and thoroughly agitated on a magnetic
stirrer for 30 s at room temperature. For microcapsules
carrying BSAFITC into the shell, 3 mL (5 mg mL  1) of
2000 kDa dextran (DEX2000) were added during the co-
precipitation reaction. After the agitation, the suspension
was transferred into a centrifuge tube and sonicated for
30 s. Subsequently, the particles have been separated from
the supernatant by centrifugation (1000g, 1 min), then
washed one time with EtOH (8 mL) and two times with pure
water (4 mL) to remove unreacted species. The resulting
212 journal of the mechanical behavior of biomedical materials 62 (2016) 209 –221
dextran-loaded CaCO3 particles have been directed used for
LbL assembly of oppositely charged polyelectrolytes. Alter-
nating layers of negatively charged DEXS (2 mg mL  1 in 0.5 M
NaCl), or BSAFITC (30 mM in 0.5 M NaCl) and positively charged
PARG (2 mg mL  1 in 0.5 M NaCl) were deposited onto the
dextran-loaded CaCO3 cores until two bi-layers of the poly-
electrolytes had been established. For coating with each
layer, the microparticles were suspended in 8 mL of polyelec-
trolyte or protein solution, sonicated for 2 min, shaken gently
for 60 min and washed three times with ultrapure water. The
sacrificial CaCO3 cores were finally removed by complexation
with EDTA buffer (8 mL, 0.2 M, pH 7) for several minutes and
centrifuged for 10 min at 800 rpm. The resulting multilayer
microcapsules were washed three times with ultrapure water
to remove excess EDTA. To obtain cationic microcapsules, an
additional layer of PARG was added following core removal.
The microcapsules were finally stored as suspension in water
at 4 1C. The final architectures of the microcapsules were:
DEXRITC@(DEXS/PARG)2PARG, DEXFITC@(DEXS/PARG)2, and
DEX2000@(BSAFITC/PARG)(DEXS/PARG)PARG, which in the fol-
lowing will be referred to as cationic, anionic and BSA-loaded
cationic microcapsules, respectively.
2.3. Characterization of biodegradable microcapsules
The zeta potential of the microcapsules was measured in
Milli-Q water, after each step of polyelectrolyte adsorption, by
dynamic light scattering (DLS) using a Zetasizer 2000 machine
(Malvern Instruments, UK). The results were averaged from
5 parallel measurements. The microcapsule number per
volume was determined with a hemocytometer under an
optical microscope. A drop of a diluted solution of micro-
capsules was added onto the chamber and the number of
microcapsules in the volume defined by the hemocytometer
was counted by using a 20X objective in phase contrast mode.
2.4. Preparation of collagen scaffolds
Collagen scaffolds have been obtained with a freeze-drying
technique as previously reported (Gervaso et al., 2012). Briefly,
type I equine collagen (Antema s, kindly provided by Opocrin
S.p.A, Italy) were pulverized in a refrigerate mill and the
obtained collagen flakes were suspended in acetic acid solu-
tion (0,5 M) in order to get a collagen concentration of 2 wt%.
The slurry was agitated by a magnetic stirrer for 30 min and
homogenized at a temperature of 4 1C, frozen at 40 1C
(freezing rate of 1 1C/min) then lyophilized for 17 h at 0 1C
under vacuum, using petri dishes as a mold. Then the freeze-
dried collagen membranes were subjected to a dehydrother-
mal crosslinking (DHT) (t¼ 72 h, T ¼121 1C, P¼ 1 bar). Scaf-
folds were cut using a biopsy punch in cylindrical samples of
6 mm in diameter and 2 mm in height for the
subsequent tests.
2.5. Functionalization of collagen scaffolds with
biodegradable microcapsules
Each scaffold has been placed in a plastic tube and exposed to
2 mL of cationic or anionic microcapsule suspension (6.7  107
microcapsules/mL). Two different incubation methods have
been then evaluated to integrate the microcapsules inside
and outside the scaffold's surface. In the first method, herein
abbreviated as method#1, the tubes have been placed on the
plate shaker at 150 oscillations per minute for 2 h and
subsequently washed three times with 2 mL of ultrapure
water (15 min under agitation). In the second method, herein
abbreviated as method#2, the tubes have been placed in a
desiccator under vacuum (max vacuum 20″ Hg) over night,
and subsequently washed three times with 2 mL of ultrapure
water (45 min, under vacuum). Finally, the samples have
been washed three times with ultrapure water (15 min) in
order to remove microcapsules excess. Following functiona-
lization with cationic or anionic microcapsules, all scaffolds
have been lyophilized before SEM and CLSM analyses.
2.6. Fluorescence spectroscopy measurements
To estimate the amount of microcapsules integrated within
the scaffolds, fluorescence spectrophotometry analyses were
performed by means of a Cary Eclipse Fluorescence Spectro-
photometer (Varian Inc., Agilent Technologies, Santa Clara
California) (indirect method). First, the standard curves of the
conjugates DEXRITC and DEXFITC were made. Stock solutions
of 1 mg mL  1 DEXFITC and DEXRITC in ultrapure water were
prepared. All other samples, with DEXRITC and DEXFITC con-
centrations of 0.05 mg mL  1, 0.1 mg mL  1, 0.2 mg mL  1,
0.3 mg mL  1, 0.4 mg mL  1, 0.5 mg mL  1, 0.6 mg mL  1, 0.7 mg
mL  1, 0.8 mg mL  1, 0.9 mg mL  1 were obtained by rigorous
diluting the stock solutions. Second, 1 mL of supernatant
solutions was measured by recording the emission spectra of
DEXFITC from 500–600 nm (λexc ¼490 nm; λemmax ¼518 nm),
and DEXRITC from 545–670 nm (λexc ¼ 520 nm; λemmax ¼ 580
nm). Finally, the concentration of microcapsules detected in
the supernatants had been calculated by comparison to the
standard curves. For BSAFITC-loaded microcapsules, the same
procedure was used. The total amount of microcapsules
adsorbed to the scaffolds was indirectly determined through
comparing the emission spectra of FITC detected in the
supernatants with that of the standard curve obtained from
serial dilution of BSAFITC in ultrapure water (0, 0.1875, 0.375,
0.75, 1.5, 3 μM) (Fig. S3). All measurements were done with a
quartz cuvette and performed in triplicate.
2.7. Morphological analyses of scaffolds
Cross and longitudinal sections of collagen scaffolds, with
and without microcapsules, were observed by Scanning
Electron Microscope (SEM, Zeiss, Jena, Germany) in order to
analyze their microstructure and to obtain information
regarding the features of microcapsules and their mode of
entanglement with the porous supports. The samples were
examined under a voltage of 20.00 KV. To study the distribu-
tion of the microcapsules into the scaffolds, the surface as
well as the longitudinal and cross sectional sections of the
functionalized samples were analyzed by means of confocal
laser scanning microscopy (CLSM, TCS SP5; Leica, Microsys-
tem GmbH, Mannheim, Germany). The scaffolds were
observed with a 20X objective. DEXFITC was excited at
488 nm with an argon laser and its fluorescence was collected
between 500 and 600 nm. DEXRITC was exited at 543 nm using
 journal of the mechanical behavior of biomedical materials 62 (2016) 209 –221
a helium neon laser; the fluorescence was detected with a
long-pass filter 4560 nm. All CLSM images were acquired by
scanning the scaffolds along the x, y and z directions for 3D
reconstruction of the microcapsules-modified scaffolds (scan
speed 200 Hz, pinhole aperture set to 1 Airy). In order to
quantify the spatial distribution of microcapsules present at
different depths, the number of microcapsules along the Z
axis were counted. To this aim, the confocal images were
post-processed using the image processing software Image J
(1.47v). Individual confocal image stacks from microcapsules-
modified scaffolds composed of 250 sections (step size
0.99 μm) were examined and used to create the maximum
intensity projections. Spherical-like structures were identified
and labeled using the automatic segmentation tool and the
segmented elements were automatically counted by the
software.
The quantification of the spatial distribution of both
negative and positive microcapsules incubated within scaf-
fold by methods#1 and method#2 allowed to select the
optimal experimental conditions for successfully integrating
biodegradable charged microcapsules within the 3D scaffolds
(please, see the Section 3). Accordingly, the following char-
acterizations have been performed only on collagen scaffolds
functionalized with cationic biodegradable (DEXS/PARG)2-
PARG microcapsules by using method#1 and on pristine
scaffolds, as comparison.
2.8. Swelling ratio in Phosphate Buffer Solution (PBS)
The collagen scaffolds, with and without microcapsules, were
soaked into PBS, pH 7.4, at room temperature and the
swelling ratio after incubation for 1 h, 2 h, 24 h and 96 h has
been determined. The swelling ratio was calculated as water
uptake per mg of scaffold weight, according to the Eq. (1) (Yan
et al., 2010):
Ww  Wd
Swelling ratio ¼ ð1Þ
Wd
where Ww is the wet scaffold weight and Wd is the dried
scaffold weight. Three specimens were tested for both scaf-
folds, with and without microcapsules, and their average
values were used for data analysis.
2.9. Scaffold's stability in Tris–HCl buffer
The stability of the collagen scaffolds, with and without
microcapsules, was evaluated by measuring the weight loss
of the samples in Tris–HCl buffer 0.05 M, pH 7.4, at 37 1C (Lin
et al., 2007) supplemented with NaCl 0.15 M and Sodium azide
0.01%. After 1, 2, 3 and 4 weeks the samples were removed
from the buffer solution, rinsed with distilled water, frozen at
 40 1C (freezing rate of 1 1C/min), then lyophilized for 17 h at
0 1C under vacuum before characterization. The percentage
weight loss of pristine and microcapsules-embedded collagen
scaffolds was calculated from the weights of the dried
scaffolds before and after incubation in Tris–HCl, according
to Eq. (2) (Liuyun et al., 2009):
m0 md
Weight loss% ¼  100 ð2Þ
m0
where, m0 is the weight of the dried scaffold before
213
incubation times, and md is the weight of the dried scaffold
after 1, 2, 3 and 4 weeks of incubation in Tris–HCl. Three
specimens were tested for each sample and their average
values were used for data analysis.
2.10. Mechanical properties of scaffolds
Uniaxial compression tests in displacement control up to 75%
of strain (rate¼ 0.01 mm/s, load cell¼ 10 N) were performed
on cylindrical samples, immersed in PBS. All samples were
completely hydrated in PBS before testing. In order to verify
the influence of the microcapsule on the mechanical proper-
ties of collagen scaffold, the slope of the linear part of the
stress–strain curve (between 2–10% of strain) was calculated
for pristine and microcapsules-embedded collagen scaffolds.
Besides, the mechanical analysis has been performed on both
groups of samples after the stability test performed for 1 and
4 weeks. All measurements were run in triplicate and their
average values were used for data analysis.
2.11. In vitro BSA diffusion profiles
Diffusion studies were carried out by placing the hybrid
scaffolds in Tris–HCl 0.05 M, NaCl 0.16 M, 0.01% sodium azide
buffer (pH 7.4) at 37 1C. Three scaffolds functionalized with
biodegradable BSA-loaded cationic microcapsules, and three
scaffold without microcapsules, were placed in plastic tubes
with 2 mL of Tris–HCl buffer per scaffold. As additional
control, 2 mL of Tris–HCl buffer were placed in an empty
plastic tube. At each time point (0.5 h, 1.5 h, 18 h, 21 h, 1 day,
5 days, 10 days, 20 days and 25 days), 500 mL of buffer were
removed and stored at 20 1C until analysis. A fresh 500 mL
aliquot of Tris–HCl was added to samples and they were re-
incubated until the following time point. The amount of
BSAFITC released was quantitatively measured by spectro-
photometer analysis by recording the emission spectra of
BSAFITC from 500–600 nm (λexc ¼ 490 nm) in the supernatants.
The BSA diffusion rate was expressed as a percentage of the
total BSA/scaffolds. All samples were run in triplicate and
data points in Fig. 5b are shown as mean7standard devia-
tion. Finally, the concentration of microcapsules detected in
the supernatants has been calculated by comparison to the
standard curve of BSAFITC (Fig. S3b, SI). Scaffolds integrating
BSAFITC-loaded microcapsules were finally imaged via CLSM
on day 25 of exposure to Tris–HCl solution to observe surface
morphology.
2.12. Statistical analysis
Data were analyzed using Origin Pro 8 SR2 software (v.
8.0891). All experiments were performed in triplicate, unless
otherwise stated. The results of multiple observations are
presented as the mean7standard deviation (SD). Statistical
significance was assessed by the Student's t-test; values were
considered significant at po0.05.
214 journal of the mechanical behavior of biomedical materials 62 (2016) 209 –221

agreement with previous data reported from De Geest et al.

3. Results and discussions
3.1. Synthesis and characterization of microcapsules
The binding affinity of the microcapsules to 3D porous
collagen scaffolds obtained by freeze-drying technique
(Deponti et al., 2014) was studied by using microcapsules
with opposite charges and by applying two incubation meth-
ods per each capsule type, namely a plate shaker for 2 h
(method#1) and a vacuum chamber O.N. (method#2). As first
step, biodegradable anionic and cationic microcapsules, car-
rying green fluorescent or red fluorescent dextran-conjugates
into their cavities, respectively, were synthesized (see Section
2 for details). The morphology of the microcapsules was
assessed by means of CLSM analysis. Fig. 1a and b show
two typical images of the employed microcapsules. Both
capsule types are homogenously loaded with fluorescent
dextran-conjugates and show regular spherical shape The
size was determined from CLSM images and ranged between
3.5 and 4.5 μm (N¼100). The alternating adsorption of the
oppositely charged polyelectrolytes was monitored by means
of zeta potential analyses. As shown in Fig. 1c, DEXS and
PARG regularly alternate during the LbL assembly of (DEXS/
PARG)2 multilayer shells. Notably, dissolving the CaCO3 cores
by addition of EDTA resulted in a decrease in zeta potential
from 15.774.8 mV to  29.774.0 mV. This result is in
(2012), who observed that to render the capsule surface
cationic, an additional PARG layer had to be deposited after
the core removal step with EDTA. This phenomenon had
been attributed to a rearrangement of the excess of anionic
compounds released upon dissolution of the CaCO3 tem-
plates and redistributed through the capsule membrane (De
Cock et al., 2011). Accordingly, the deposition of an additional
PARG layer reversed the zeta potential to a cationic value
(10.775.0 mV) (Fig. 1d).
3.2. Scaffold functionalization and morphological
characterization
The as-obtained cationic and anionic biodegradable micro-
capsules were then incubated with the collagen scaffolds and
exposed to two incubation methods (see Section 2 for details).
Fig. 2 shows the representative optical photographs of col-
lagen scaffolds after incubation with anionic and cationic
microcapsules by using method#1 (Fig. 2a) and method#2
(Fig. 2b), and after the washing steps (Fig. 2c and d), respec-
tively. As it can be observed, despite the microcapsules
charge, when the scaffolds are placed on a plate shaker for
2 h (method#1, Fig. 2a), they sediment at the bottom of the
test tubes and acquire a clear yellowish and pinkish color
which qualitatively indicates the binding of the fluorescent
microcapsules. Notably, the color of the scaffolds is

Fig. 1 – Characterization of biodegradable anionic and cationic capsules. (a, b) Fluorescence microscopy images of (a) anionic
and (b) cationic capsules (for visualization purposes, FITC-labeled or RITC-labeled dextran conjugates were entrapped into the
cavities). (c, d) Zeta potential values of (c) anionic and (d) cationic microcapsules before and after the core removal step,
respectively. To obtain anionic capsules, the CaCO3 templates are dissolved by EDTA which leads to charge reversal to
negative zeta potential ( 29.774.0 mV). To obtain cationic capsules, an additional layer of PARG is adsorbed onto the
microcapsules after the EDTA treatment to render the zeta potential positive (10.775.0 mV). Scale bars: 10 μm.
 journal of the mechanical behavior of biomedical materials 62 (2016) 209 –221 215

Fig. 2 – Scaffold functionalization with biodegradable cationic and anionic microcapsules. (a, b) Optical photographs of
scaffolds after incubation with anionic (symbol ‘-’) and cationic (symbol ‘þ’) capsules and placed on (a) the plate shaker for 2 h
(method#1) or (b) the vacuum chamber O.N (method#2). (c, d) Optical photographs of scaffolds following incubation with
microcapsules (as described in a, b) and washings steps with ultrapure water. (e) Concentration profiles of red-fluorescent
cationic microcapsules (continuous and dotted red lines) and green-fluorescent anionic microcapsules (continuous and dotted
green lines) detected in the supernatant solutions following incubation and washing steps. Data points represent
mean7standard deviation (n ¼ 3). (For interpretation of the references to color in this figure legend, the reader is referred to the
web version of this article.)

maintained after the washing steps with ultrapure water
(Fig. 2c). Conversely, by placing the scaffolds in a vacuum
chamber for O.N. (method#2, Fig. 2b), the scaffolds stay at the
liquid's meniscus and do not change their color. Additionally,
colored yellowish and pinkish pellets, corresponding to the
microcapsules, can be clearly observed at the bottom of the
test tubes. As expected, following the washing steps these
scaffolds appear white as the pristine scaffolds.
The curves reported in Fig. 2e show the overall concentra-
tion of DEXFITC (continuous and dotted green lines) and
DEXRITC (continuous and dotted red lines) detected in the
feeding solution and in the supernatants after each washing
step (up to three) in ultrapure water. High concentration of
microcapsules detected in the solutions corresponds to a
high number of unbound and free microcapsules. After
incubation, a low concentration of microcapsules is found
in the supernatants recovered by scaffolds exposed to
method#1 (continuous red and green lines), whereas by
applying the method#2 (dotted red and green lines) higher
concentration of microcapsules are recorded. For anionic
microcapsules incubated by method#2 (dotted green line),
the concentration of microcapsules detected in the solution
collected after the incubation appears nearly the same as that
of the feeding solution. This result confirms that the majority
of microcapsules had settled at the bottom of the test tube
during the incubation with the scaffold (Fig. 2b and d).
Extensive washing of the scaffolds with water does not
induce capsule desorption from the scaffolds, indeed a very
low concentration of microcapsules is found after each
washing step.
To obtain a deeper understanding of the influence of the
microcapsules charge on the adsorption efficiency, the pat-
tern distribution of the microcapsules on both the external
and internal surfaces of the modified scaffolds had been
analyzed by scanning electron microscopy (SEM) and confocal
laser scanning microscopy (CLSM) analyses. CLSM and SEM
images of different cross-sections of the modified scaffolds
are shown in Fig. 3 (longitudinal cross-sections) and Fig. S2
(horizontal cross-sections). The images confirm the adsorp-
tion of a higher number of microcapsules in those scaffolds
that had been exposed to method#1 (Fig. 3a, b and e, f). SEM
analyses confirm the data observed by CLSM studies. A lower
concentration of microcapsules is found in scaffolds threated
with method #2 (Fig. 3g and h) and in those scaffold
incubated with anionic microcapsules (Fig. 3f and h). Further-
more, SEM images provide details on the morphology of the
dried multilayer microcapsules, showing intact microcap-
sules, completely enclosed within the porous supports and
retaining their shape fairly well. These data demonstrate that
both the functionalization and freeze-drying procedures do
not affect the microcapsules structure.
The quantitative analysis of the fluorescence images (see
Section 2 for details) confirmed once more that the scaffolds
incubated with cationic microcapsules by method#1 con-
tained the higher number of microcapsules (Fig. 3i). Such
analysis revealed also that the microcapsules preferentially
bind to the inner planes of the scaffolds, suggesting stronger
interactions with inner planes of the supports.
Overall, the obtained results showed a higher concentra-
tion of microcapsules in the scaffolds exposed to cationic
microcapsules by using method#1, likely due to stronger
electrostatic interaction between the positively charged cap-
sules with the negatively charged collagen backbone (Lee
et al., 2007) that resulted to be further promoted under
continuous agitation. It is noteworthy that the experimental
conditions used in method#1 are advantageous for future
scale-up functionalization of the scaffolds as this method
employs only 2 h of incubation and the plate shaker to gentle
mix the test tubes hosting the scaffolds and the capsule
suspensions. Moreover, the data show that the employed
functionalization strategy allows the creation of scaffolds in
which the confinement of the microcapsules occurred in all
216 journal of the mechanical behavior of biomedical materials 62 (2016) 209 –221

Fig. 3 – Characterization of capsules-integrated scaffolds. Morphological observations by CLSM of freeze-dried collagen

scaffolds integrated with (a, c) cationic and (b, d) anionic capsules, following incubation on (a, b) the plate shaker for 4 h or (c,
d) the vacuum chamber O.N. (e, h) Corresponding SEM images of scaffolds (longitudinal sections) as shown in (a, d). Scale bars:
25 lm. (i) Estimation of the amount of capsules enclosed within different sections of the 3D hybrid scaffolds. Columns
represent mean7standard deviation (n ¼3).

levels (Movie S1, SI). The capability to decorate the 3D
scaffolds with delivery systems homogenously patterned
within the support, is particularly relevant for tissue repair
applications, where there is the need to strictly confine the
release of growth factors in the proximity of the defect site
reducing potential side effects and supporting tissue regen-
eration which normally occurs in long time frames (Tessmar
and Göpferich, 2007).
3.3. Swelling, stability and mechanical studies
After selecting the experimental conditions for successfully
integrating biodegradable charged microcapsules within the
3D scaffolds, we investigated the swelling capacity, the
stability and the mechanical properties of the hybrid scaf-
folds. For all these studies, collagen scaffolds functionalized
with cationic biodegradable (DEXS/PARG)2PARG microcap-
sules by using method#1 were produced and compared to
pristine scaffolds. Both pristine and microcapsules-
embedded collagen scaffolds have a high porous and inter-
connected structure, as shown in Figs. S1 and S2, and so, to
determine the possible influence of microcapsules content on
the water uptake ability, the water absorption of collagen
scaffolds, with and without microcapsules, was measured. As
shown in Fig. 4a, in both groups of analyzed samples, the
maximum swelling was observed after two hours in PBS
buffer (36.9070.36 for pristine scaffold and 35.2977.77 for
microcapsules-embedded scaffolds) and it remained more or
less constant up to 96 h indicating that in two hours both
scaffolds, with and without microcapsules, are completely
hydrated without any statistically significant difference
between the two groups (p40.05). The water binding capacity
of the scaffold plays an important role in tissue regeneration;
besides, the swelling ratio of the scaffold strongly depends on
the hydrophilic nature and microstructure of the scaffold
(Yan et al., 2010). Therefore, scaffold porosity and swelling
capacity are critical factors in predicting their performances
in vivo: an adequate swelling ratio and an interconnected
 journal of the mechanical behavior of biomedical materials 62 (2016) 209 –221 217

Fig. 4 – (a) Swelling ratio calculated as water uptake per mg of pristine scaffolds (light gray curve) and capsules-embedded
scaffolds (dark grey curve), after various soaking time in PBS at RT. (b) Medium weight loss of pristine scaffolds (light grey
squares) and capsules-embedded scaffolds (dark grey spheres) after incubation in Tris–HCl at 37 1C at different time points (1,
2, 3, and 4 weeks). (c) Average elastic moduli obtained by compression tests performed on pristine collagen scaffolds (light
grey histograms) and embedded-capsules scaffolds (dark grey histograms) before degradation (time 0) and after 1 and 4 weeks
of degradation in Tris–HCl at 37 1C. All data points and columns represent mean7standard deviation (n ¼3).

porosity allow a good infiltration of cells of a specific tissue
and the required exchange of gases and nutrients, needed to
support the cell growth in the early stage of tissue regenera-
tion, as well as degradation rates that match the kinetics of
de novo tissue formation (Rnjak-Kovacina et al., 2015). The
swelling measurements results provided strong evidences
that neither the presence of microcapsules nor the functio-
nalization procedure influenced the overall swelling proper-
ties of the collagen scaffold.
Moreover, soft tissue biomaterials need to be pliable to
match the contours of the tissue defect, while offering ade-
quate mechanical support to prevent the collapse of the defect
(Rnjak-Kovacina et al., 2015). Therefore, in order to predict the
structural behavior of the hybrid scaffolds in vitro, stability test
and mechanical compression were performed; first of all, we
determined the weight loss percentage of pristine and
microcapsules-embedded collagen scaffolds, after incubation
in Tris–HCl solution at 37 1C, and the results are represented in
Fig. 4b (weight loss percentage vs different incubation times).
As it can be observed in the graph, both scaffold typologies lost
weight in the first week in buffer solution (11.5373.88% and
13.5974.31%, for pristine scaffolds and microcapsules-
embedded scaffolds, respectively), without any significant
weight loss in the next weeks and without significant differ-
ences between two scaffolds typologies (p40.05).
Next, we compared the mechanical properties of pristine
scaffolds and microcapsules-embedded scaffolds, before and
after incubation in Tris–HCl buffer and the corresponding
elastic moduli were measured (Fig. 4c). The average elastic
modulus (E) at time 0 resulted equal to 1.6470.31 kPa for
pristine scaffold and to 1.5970.10 kPa for microcapsules-
embedded, indicating that the microcapsules did not signifi-
cantly affect the overall collagen robustness (p40.05).
Furthermore, the elastic moduli of scaffolds maintained in
Tris–HCl buffer for 1 and 4 weeks, respectively, did not
decrease with respect to time 0, both in absence (1w:
2.2570.31 kPa, 4w: 2.4170.23 kPa) and in presence of micro-
capsules (1w: 2.87 7 1.11 kPa, 4w: 2.23 7 0.96 kPa), without
significant differences between the two scaffolds typologies
(p40.05). This behavior has been observed despite the sam-
ples weight loss after 1 and 4 weeks of permanence in Tris–
HCl was about 15% (Fig. 4b). However, from the scaffold
diameter measurements we observed that the dimension of
the scaffold decreased accordingly to the weight loss (data
not shown). The observed reduced dimensions of the scaffold
did not influence their mechanical compressive properties,
suggesting that a scaffold surface erosion occurred during the
first week of incubation in Tris–HCl, probably due to the soft
thermal collagen cross-linking. From the stability and
mechanical studies we can assume that, after one week of
incubation in Tris–HCl buffer, scaffolds are stable in size,
218 journal of the mechanical behavior of biomedical materials 62 (2016) 209 –221

Fig. 5 – Scaffold integrated with BSA-loaded polymer microcapsules. (a) CLSM image of scaffolds integrated with positive
BSAFITC-loaded capsules. The overlay of green and bright field channels is displayed (maximum intensity projection of 178
planes, longitudinal section). Scale bar¼100 μm. (b) Maximum emission peaks of BSAFITC detected over time in the
supernatant solutions of hybrid scaffolds incubated at 37 1C. Data points represent mean7standard deviation (n ¼ 3). Insets:
optical photograph of test tubes containing scaffolds suspended in Tris HCl at 37 1C. (For interpretation of the references to
color in this figure legend, the reader is referred to the web version of this article.)

weight and mechanical stiffness, indicating they could be
suitable for tissue engineering application. However, since
the initial scaffold weight loss is accompanied with a size
decrease, this dimension reduction should to be taken into
account during the scaffold design.
3.4. In vitro diffusional tests
The tight entrapment of particle-based carriers into porous
3D scaffolds represents a key issue. It must avoid passive
diffusion of the embedded cargo, as they would not be
suitable for controlled release of molecules over long period
of times. To evaluate this matter, we fabricated 3D collagen
scaffolds integrated with biodegradable microcapsules carry-
ing the model protein Bovine Serum Albumine labelled with
the FITC dye (BSAFITC) into the multilayer walls. For this
purpose, BSAFITC was electrostatically entrapped within the
multilayer shells by adsorbing it as first layer, followed by
additional adsorption of four layers of biodegradable poly-
electrolytes [(BSAFITC/PARG)(DEXS/PARG)PARG] (Fig. S3a). The
loading of BSAFITC as inner layer of the multilayer shell is
expected to protect the payload from the surrounding envir-
onment, as well as to facilitate the prolonged release of the
protein over a long time period. This is particularly relevant,
as most clinical applications require hundreds of nanograms
of bioactive factors per day to achieve an effective dose
(Gavenis et al., 2010; Srouji et al., 2011). According to the data
described in Section 3.2, to integrate the microcapsules
within the 3D collagen scaffolds we adopted the experimental
conditions used in method#1. The binding efficiency of
BSAFITC-loaded microcapsules to the scaffolds was estimated
by spectrofluorimetric analysis of the supernatant solutions
(Fig. S3c). Notably, the concentration profile of free BSAFITC-
loaded microcapsules (Fig. S3c) resulted similar to that
obtained for scaffolds functionalized with cationic DEXRITC-
loaded microcapsules (Fig. 2e, red continuous line). These
data demonstrated that the loading of BSAFITC (or other
bioactive molecules) within the inner layer of the shells did
not affect the overall efficiency of binding of the microcap-
sules to the collagen matrixes, another confirmation that the
interactions between microcapsules and scaffolds were pri-
marily governed by the electrostatic interactions between the
outermost polyelectrolyte layer of the microcapsules, PARG,
and the collagen backbone. Fig. 5a shows the maximum
intensity projection of a 3D confocal microscopy scan of the
scaffolds integrated with positive BSAFITC-loaded capsules, in
which the microcapsules appear homogenously distributed
and fully integrated within the collagen matrix as optimized
for cationic capsules incubated by using method#1.
Then, the diffusion of BSAFITC from the scaffolds was
monitored over 25 days in vitro and is summarized in the
percentage cumulative release profile shown in Fig. 5b. Small
traces of BSAFITC were detected in the supernatants collected
from the hybrid scaffolds kept at 37 1C in buffer solution for
30 min, likely due to desorption of a some microcapsules
weakly bound at the scaffold surface. However, the initial
burst effect was low (
5.2% by 30 min) and the maximum
percentage of the BSAFITC release appeared negligible (
6.5%)
after 25 days, suggesting a stable absorption of the micro-
capsules to the scaffolds. These data demonstrated that the
loading of proteins inside the multilayer biodegradable
microcapsules and their subsequent electrostatic absorption
into the scaffolds was highly efficient. Indeed, only a low
percentage of BSAFITC was lost over 25 days. CLSM analyses
performed onto the microcapsules-embedded scaffolds kept
for 25 days in buffer solution at 37 1C further confirmed these
observations. The images reported in Fig. S4, show
microcapsules-embedded scaffolds that are fully integrated
with fluorescent microcapsules.
4. Conclusions
Porous 3D collagen scaffolds integrating biodegradable micro-
capsules have been successfully prepared by a simple method
based on freeze drying and layer-by-layer techniques. The stable
 journal of the mechanical behavior of biomedical materials 62 (2016) 209 –221
integration of the microcapsules has been confirmed by CLSM
and SEM analyses. It turned out that both the charge of the
microcapsules and the incubation method influence the binding
efficiency of the microcapsules and their distribution pattern
over the entire surface of the 3D porous scaffolds. Additionally, it
was observed that, the structure, the porosity and other physical
features (i.e., swelling capacity, stability and mechanical proper-
ties) of the composite microcapsules-embedded scaffolds were
almost identical to that of pristine collagen scaffolds. Also the
capsule's structure remained unaltered following integration into
the scaffolds and showed negligible diffusion profiles under
in vitro conditions. These findings suggest that the combined
use of scaffolds and biodegradable protein-loaded microcapsules
is a promising strategy for the design of multifunctional
microcapsules-integrated scaffolds for controlled delivery of
bioactive molecules. Further studies are being devoted to assess
the biocompatibility of the produced composite scaffolds as well
as their in vivo performances. It is noteworthy to mention that
the developed method is facile and highly tunable, as there is no
requirement for expensive or customized equipment, which
indicate that such methodology may be easily and cheaply
implemented.
Associated content
Supporting Information. The SEM and CLSM images of var-
ious cross-sections of the pristine and microcapsules-
integrated collagen scaffolds (Figs. S1, S2, S4); the synthesis
of BSAFITC-loaded biodegradable microcapsules and integra-
tion within collagen scaffolds (Figure S3); a 3D reconstruction
of a Z-stack (movie S1; size-depth: 251 μm). The following files
are available free of charge.
Acknowledgements
The authors acknowledge the National Operational Pro-
gramme for Research and Competitiveness (PONREC) ‘RINO-
VATIS’ (PON02_00563_3448479).
Appendix A. Supplementary material
Supplementary data associated with this article can be found
in the online version at http://dx.doi.org/10.1016/j.jmbbm.
2016.05.009.
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