Bioactive chitosanbased scaffolds with improved properties induced by dextrangrafted nanomaghemite and larginine amino acid

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Bioactive chitosan-based scaffolds with improved properties induced by
dextran-grafted nano-maghemite and L-arginine amino acid
Stefania Scialla ,1 Amilcare Barca,2 Barbara Palazzo,1,3 Ugo D’Amora ,4 Teresa Russo,4
Antonio Gloria,4 Roberto De Santis,4 Tiziano Verri,2 Alessandro Sannino,1 Luigi Ambrosio,4
Francesca Gervaso1
1Department of Engineering for Innovation, University of Salento, Lecce, Italy
2General Physiology Laboratories, Department of Biological and Environmental Sciences and Technologies, University of Salento,
Lecce, Italy
3Ghimas S.p.A., c/o Dhitech Scarl, Campus Ecotekne, Lecce, Italy
4Institute of Polymers, Composites and Biomaterials, National Research Council, Naples, Italy
Received 8 November 2018; revised 14 December 2018; accepted 26 December 2018
Published online 12 February 2019 in Wiley Online Library (wileyonlinelibrary.com). DOI: 10.1002/jbm.a.36633
Abstract: Over the past years, fundamentals of magnetism
opened a wide research area of interest, in the field of tissue engi-
neering and regenerative medicine. The integration of magnetic
nanoarchitectures into synthetic/natural scaffold formulations
allowed obtaining “on demand” responsive structures able to
guide the regeneration process. The aim of this work was the
design and characterization of three-dimensional (3D) chitosan-
based scaffolds containing dextran-graftedmaghemite nanoarchi-
tectures (DM) and functionalized with L-arginine (L-Arg) amino acid
as bioactive agent. A homogeneous pore distribution and a high
degree of interconnection were obtained for all the structures with
DMs, which resulted well distributed inside the polymer matrix.
All the results suggest that the simultaneous presence of DMs and
L-Arg conferred interesting mechano-structural and bioactive
properties toward osteoblast-like and human mesenchymal stem
cells, differentially stimulating their proliferation both in the
absence and in the presence of a time-dependent magnetic field.
© 2019 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 107A: 1244–
1252, 2019.
Key Words: chitosan, magnetic scaffold, magnetic nanoarchi-
tecture, L-arginine
How to cite this article: Scialla S, Barca A, Palazzo B, D’Amora U, Russo T, Gloria A, De Santis R, Verri T, Sannino A,
Ambrosio L, Gervaso F. 2019. Bioactive chitosan-based scaffolds with improved properties induced by dextran-grafted nano-
maghemite and L-arginine amino acid. J Biomed Mater Res Part A 2019:107A:1244–1252.
INTRODUCTION
Scaffolds play a pivotal role in tissue engineering. In particu-
lar, nanocomposite scaffolds with properties that mimic the
tissue-specific microenvironment have revolutionized differ-
ent aspects of health care, due to their capability to enhance
cell adhesion, to induce cytoskeletal re-arrangements, to trig-
ger intracellular pathways, and to drive cell proliferation, dif-
ferentiation, and even tissue growth.1 In this perspective,
scaffolds should not act only as static elements, but they
should be “activated” during cell colonization, adapting their
structure to the different mechanical and anatomical fea-
tures, during different tissue maturation phases.2
Natural polymers such as chitosan, collagen and hyaluro-
nic acid have been widely used as the main component for
scaffold preparation.3–5 Nevertheless, they are not always
ideal materials, if used without any modification; in fact, low
mechanical strength and fast biodegradation rate are key
issues limiting their use in tissue engineering approaches.
Therefore, the need of improving these performances, while
preserving natural polymer good interactions with cells, is
still one of the main goals in biomaterials’ research. Cross-
linking processes and/or composites manufacturing can lead
to natural polymer-based products suitable for tissue engi-
neering and clinics application.3–5
Chitosan is the second most abundant natural biopolymer
currently attracting great attention for tissue engineering
applications due to its intrinsic properties6: excellent bio-
compatibility and controlled biodegradability with safe by-
products and easy processability in multiple ways to produce
a variety of three-dimensional (3D) scaffolds with different
pore structures.3 Chitosan results from alkaline deacetylation
of chitin, a structural element of the exoskeleton of crusta-
ceans, such as shrimps, crabs and lobsters. It is a linear
amino polysaccharide composed of glucosamine and N-acetyl
glucosamine units linked by β-(1-4) glycosidic bonds. As chi-
tin derivative, it is available in many forms different for the
degrees of deacetylation and molecular weights, features
with a strong influence on physico-chemical properties
Additional Supporting Information may be found in the online version of this article.
Correspondence to: S. Scialla; e-mail: stefania.scialla@unisalento.it or U. D’Amora; e-mail: ugo.damora@cnr.it
© 2019 WILEY PERIODICALS, INC.1244
including crystallinity, solubility, and degradation. The cat-
ionic nature of this polymer, in a pH range near its pKa, is
particularly important for tissue engineering applications, as
it can form polyelectrolyte complexes with anionic biological
macromolecules. Reactive primary amines and hydroxyl
groups in chitosan remain available for the addition of side
groups, peptides or amino acids, enhancing its properties in
biomedical applications. In addition, chitosan exerts antimi-
crobial effects putatively useful in therapeutic utilization.7
In the last 10 years, physical and biological properties of
polymer-based scaffolds have been improved through different
cross-linking methods.8 Most of these treatments are predomi-
nantly based on physical methods, such as dehydrothermal or
ultraviolet irradiation9; others involve chemical methods, with
potentially cytotoxic effects. Thus, nontoxic and biocompatible
cross-linking agents, such as amino acids, are now being more
considered for cross-linking in scaffold fabrication.10 L-arginine
(L-Arg) is a metabolically versatile amino acid, with different
key physiological roles. Besides being one of the essential
amino acids of proteins, it plays an interesting role in bone
metabolism and growth.11 Indeed, the metabolization of L-Arg
can involve: (1) the use of several nitric oxide synthase iso-
forms, responsible of cytotoxic responses that can lead to cell
killing (e.g., in some macrophage activations)12 or (2) the deg-
radation via arginase isoforms, which are intertwined with
cell proliferation mechanisms (polyamines synthesis depen-
dent), and with collagen deposition and extracellular matrix
dynamics (e.g., via the L-proline synthesis).13 To the best of
our knowledge, most of the few papers reporting the use of
L-Arg as cross-linker agent show the use of this amino acid as
biocross-linker of polymer blends.14
However, mere biochemical stimulation is never enough
in scaffold optimization for tissue engineering; in bone tissue
engineering, for instance, an ideal system should be finely
tuned to dynamically match the physiological needs as well
as the mechano-sensitivity issues; in this respect, mechanical
stimuli and forces have been successfully used to improve
the regenerative skills.15 In this context, scaffold functionali-
zation with magnetic nanoparticles (MNPs) can make it an
ideal mechano-transducer device. MNPs are currently being
combined with biomaterials, as inorganic additives not only
to improve their mechanical properties (e.g., stiffness and
strength) but also the biological responses implied by the
regenerative needs of bone damage, benefiting from the appli-
cation of external magnetic fields.1,2,16 MNPs-incorporating
biomaterials have been shown to have high potential in acti-
vating cells involved in osteogenic processes, being responsive
to external magnetic fields, which affect the cellular microen-
vironment.17 The static or alternating magnetic stimulation
has been highlighted to promote the implant integration and
increase the newly developed bone density, thus promotingfor 3 h. Then, medium was
removed and the formazan crystals produced by cells grown
inside the scaffold structure was brought into solution with
isopropanol/HCl by pipetting. The solutions were finally
transferred into a new 96 multi-well for reading the optical
density (λ = 550 nm) by means of multi-plate reader. For
each type of scaffold used, the MTT procedure was in paral-
lel performed on scaffold samples without cell seeding, to
obtain the blanking values for normalization.
Cytocompatibility in the presence of magnetic stimulation.
Undifferentiated human mesenchymal stem cells (hMSCs;
Clonetics, Italy), at the fourth passage, were cultured in
α-MEM (Bio-Whittaker, Belgium) containing 10% v/v FBS
(GibcoTM, Thermo Fisher Scientific), 100 U/mL penicillin
and 0.1 mg/mL streptomycin (HyClone, UK), in a humidified
TABLE I. Summery of synthesized scaffold types
Material
DM
(% w/w)
L-Arg
(M)
Scaffold’s
nomenclature
Chitosan
(1.67% w/w)
– – Cs
5 – Cs/DM5
10 – Cs/DM10
15 – Cs/DM15
– 0.1 Cs-Arg
5 0.1 Cs/DM5-Arg
10 0.1 Cs/DM10-Arg
15 0.1 Cs/DM15-Arg
1246 SCIALLA ET AL. L-ARGININE BIOACTIVATED MAGNETIC CHITOSAN SCAFFOLDS
atmosphere at 37�C and 5% v/v CO2. Scaffolds for cell cul-
tures were prepared. The structures were soaked in 70%
v/v EtOH three times (30 min/cycle), then in phosphate-
buffered saline (PBS, Sigma Aldrich, Italy) with 1% v/v anti-
biotic/antimycotic three times (2 h) and, finally, in cell cul-
ture medium for pre-wetting (2 h). Cells (density 1.0 × 104
cells/sample) suspended in FBS, were statically seeded onto
the scaffolds. After 30 min of incubation, culture medium
was added to each well containing one cell-laden scaffold. At
1 day from cell seeding, some cell-laden scaffolds were
stimulated,18 in a magnetic bioreactor, using a discontinuous
application of a time-dependent magnetic field (70 Hz and
intensity of 25–30 mT), for 6 h per day (20 intervals –
18 min each). Further cell-laden scaffolds, which were not
magnetically stimulated, were used as control and placed in
the same incubator.
Cell metabolic activity was evaluated using the Alamar
Blue Assay (AbD Serotec Ltd, UK). This is based on a redox
reaction that occurs in the mitochondria of the cells; the col-
ored product is transported out of the cell and can be spec-
trophotometrically measured. After 1, 3, and 7 days after cell
seeding, the scaffolds were washed with PBS, and for each
sample, 200 μL of Dulbecco’s modified Eagle’s medium.
(DMEM) without Phenol Red (HyClone, UK) containing 10%
v/v Alamar Blue was added, followed by incubation in 5%
v/v CO2 diluted atmosphere for 4 h at 37�C. One hundred
microliters of the solution was subsequently removed from
the wells and transferred to a 96-well plate. The optical den-
sity was immediately measured with a spectrophotometer
(Sunrise; Tecan, Männedorf, Zurich, Switzerland) at wave-
lengths of 570 and 595 nm. The number of viable cells is
correlated with the magnitude of dye reduction and is
expressed as a percentage of Alamar Blue reduction, accord-
ing to the manufacturer’s protocol.
Statistical analysis
All experiments were performed in quintuplicate, unless oth-
erwise stated. The results of multiple observations are pre-
sented as the mean � standard error of the mean (SEM).
Statistical significance was assessed by the Student’s t test;
values were considered significant at pwith
L-Arg and untreated was measured at a physiological pH, in
PBS and the results are shown in Figure 2A,B, respectively.
Chitosan is known for its high percentage of swelling.30 In
fact, the pure chitosan scaffold showed the highest swelling
ratio, with respect to Cs/DM structures, with a great ten-
dency to de-swell soon, already after 48 h (Fig. 2A). All the
other sample groups reached the maximum swelling value at
1 h since incubation in PBS, remaining stable up to 72 h. It
is worth noting that both untreated magnetic scaffolds and
L-Arg stabilized structures followed this behavior. The lack
of de-swelling in the magnetic composites highlights the effi-
cacy of the nanoarchitectures in stabilizing the scaffold
water retention with respect to the pure samples. Further-
more, an increase of swelling degree proportionally with the
DM content was also observed in the un-crosslinked mag-
netic scaffolds (Fig. 2B). That behavior can be ascribed to
the water uptake capacity of the dextran, which cap the
DMs. Amino acid functionalized magnetic scaffolds (Fig. 2B)
showed lower swelling percentage compared to the
untreated analogues. More specifically, Q was in the range
between 40 and 80, for chitosan27 scaffolds with only DM,
and in the range between 40 and 50, for L-Arg treated mag-
netic scaffolds. Moreover, L-Arg stabilized magnetic scaffolds
showed no significant changes in the swelling behavior as a
function of DMs percentages. While the DMs enhanced the
scaffold ability in retaining water over time, the amino acid
treatment also decreased their water uptake.
This finding confirms the L-Arg capacity of further stabilizing
scaffolds structures if compared to untreated magnetic ones.
Mechanical characterization
Mechanical strength of scaffolds is a key property usually
assessed via compression testing. Comparing the mechanical
properties of chitosan-based scaffolds among different stud-
ies is difficult because raw material features (molecular
weight, degree of deacetylation, concentration), scaffold fab-
rication process parameters and crosslinking treatment can
significantly differ. However, stress–strain compression tests
results seem to be in agreement with literature.3 In particu-
lar, as reported in Fig. 2C, they highlighted a slight increase
FIGURE 1. (A) Representative SEM micrographs of Cs, Cs/DM10, Cs-Arg, Cs/DM10-Arg cross section (60× of magnification; scale bar 500 μm). (B)
EDS mapping of Fe elemental content into Cs, Cs/DM10, Cs-Arg, Cs/DM10-Arg longitudinal section (100× of magnification; scale bar 200 μm). (C)
Iron content expressed as Fe/C ratio evaluated by EDS analysis for Cs-based scaffolds with incorporated DM nanoarchitectures and stabilized with
L-Arg.
1248 SCIALLA ET AL. L-ARGININE BIOACTIVATED MAGNETIC CHITOSAN SCAFFOLDS
of compressive modulus (Elow = kPa) by increasing the DMs
content up to 10% w/w (Cs = 0.98 � 0.03 kPa; Cs/DM5 =
1.50 � 0.08 kPa; Cs/DM10 = 1.53 � 0.08 kPa) with the
exception of Cs/DM15 (0.87 � 0.04 kPa). In particular, the
compressive modulus of Cs/DM5 and Cs/DM10 resulted sig-
nificantly higher than Cs scaffold modulus (p 0.05); while the compressive mod-
ulus for the Cs/DM15 scaffold was comparable with the Elow
value obtained for the pure chitosan scaffolds. The statistical
analysis in fact revealed no significant difference (p > 0.05)
between the different scaffolds, while a considerable varia-
tion of the compressive modulus occurred between the
Cs/DM5 scaffolds and Cs/DM15, and between Cs/DM10 and
Cs/DM15, with a significance level of p 0.05). The averages are derived from n = 5
tests for independent samples. (C) Compressive modulus trend of the Cs and Cs/DM at 5%–10% to 15% w/w DM and Cs and Cs/DM at 5%–10% to
15% w/w DM, stabilized with 0.1 M L-Arg. The averages are derived from n = 5 tests for independent sample.
JOURNAL OF BIOMEDICAL MATERIALS RESEARCH PART A | JUN 2019 VOL 107A, ISSUE 6 1249
ORIGINAL ARTICLE
the inorganic nanoarchitectures and the clustering effect of
the DMs, which could occur at specific concentration.14 In
particular, the experiments highlighted 15% w/w of DMs as
threshold value, revealing that by further increasing the con-
centration, nanoarchitectures acted as “weak points” instead
of reinforcement for the polymeric matrix.
In the presence of L-Arg, an increase in the Elow value was
observed for most of Cs-based scaffolds with a same DM dose-
dependence trend (Cs-Arg = 2.31 � 0.06 kPa; Cs/DM5-Arg =
3.5 � 0.2 kPa; Cs/DM10-Arg = 3.6 � 0.2 kPa). As already
evidenced by Cs/DM15 scaffolds, Cs/DM15-Arg structures
similarly showed decreased mechanical performances
(2.05 � 0.08 kPa), due to stress concentration at the inter-
face between nanorchitectures and polymer matrix. Anyway,
definitively the increased stiffness of the scaffolds with L-Arg
further confirmed the stabilization effect of the amino acid
as a consequence of weak interactions between the chitosan,
accordingly to swelling data.
In vitro cytocompatibility studies in the absence of
magnetic stimulation
The basic cytocompatibility of the synthesized scaffolds was
assessed by evaluating the metabolic activity of the MG63
human osteoblast-like cells, seeded directly onto the scaffold
FIGURE 3. MTT proliferation assay, in the absence of magnetic stimulation, on MG63 osteoblast-like cells grown for 1, 2, and 3 days on (A) Cs and
Cs/DM scaffolds and (B) Cs-Arg and Cs/DM-Arg scaffolds. For each type of scaffold, the mean values are normalized by subtraction of the mean
values of the blank scaffolds incubated in the absence of cells. Data are expressed as % proliferation (�SEM) with respect to control (Cs = 100%).
The averages have been derived from n = 4 independent trials for type of scaffold. Alamar blue proliferation assay on hMSCs cells grown for 1, 3,
and 7 days on Cs and Cs/DM scaffolds with and without L-Arg in (C) absence and (D) presence of magnetic stimulation. Data are expressed as %
proliferation (�SEM) with respect to control (Cs = 100%). The averages have been derived from n = 4.
1250 SCIALLA ET AL. L-ARGININE BIOACTIVATED MAGNETIC CHITOSAN SCAFFOLDS
surface. Figure 3A describes cell proliferation after 1, 2, and
3 days of culture in neat Cs scaffolds and scaffolds contain-
ing DMs synthesized in the absence of L-Arg.
Cs/DM5 showed reduced cell proliferation compared to Cs
at each analyzed time point; interestingly, this result is in agree-
ment with the lower swelling of Cs/DM5 in the culture medium
with respect to Cs, in the first 24 h before cell seeding as well as
throughout the whole 0–3 days interval (Fig. 3A). On the other
hand, both Cs/DM10 and Cs/DM15 showed good proliferation
values after 3 days, with different behaviors at the short- and
mid-time points. In particular, Cs/DM10 reached higher prolifer-
ation than Cs from 2 to 3 days; while Cs/DM15 scaffolds, despite
they allowed cell metabolic activity lower than Cs up to 2 days,
reached Cs control values at 3 days. In both cases, it is worth to
notice that in the 2–3 days interval the observed increase of cell
proliferation can be associated to higherin vivo studies. RSC Adv 2017;7:
25070–25088.
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mechanotransduction. Dev Cell 2006;10(1):11–20.
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Sandri M, Bañobre-López M, Piñeiro-Redondo Y, Uhlarz M,
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netic field stimulation of osteogenesis. Int J Mol Sci 2018;19(2):495.
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Kim EC. Magnetic nanocomposite scaffolds combined with static
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swelling and porosity change on capillarity: DEM coupled with a
pore-unit assembly method. Transport Porous Media 2016;113:
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30. Ren D, Hongfu Y, Wang W, Ma X. The enzymatic degradation and
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N-acetylation. Carbohydr Res 2005;340(15):2403–2410.
31. Petecchia L, Sbrana F, Utzeri R, Vercellino M, Usai C, Visai L,
Vassalli M, Gavazzo P. Electro-magnetic field promotes osteogenic
differentiation of BM-hMSCs through a selective action on Ca2+-related
mechanisms. Sci Rep 2015;5:13856.
32. Rotherham M, Henstock JR, Qutachi O, El Haj AJ. Remote regula-
tion of magnetic particle targeted Wnt signaling for bone tissue
engineering. Nanomedicine 2018;14(1):173–184.
1252 SCIALLA ET AL. L-ARGININE BIOACTIVATED MAGNETIC CHITOSAN SCAFFOLDS(Cs/DM10) or similar
(Cs/DM15) swelling ratios compared to Cs (see Fig. 2A). The
overall parallelism between cell proliferation and swelling
behavior suggest an interesting feature for the different scaffold
compositions that deserves further investigation. When the L-
Arg-treated scaffolds were analyzed, the cell proliferation trend
appeared quite different. At short time (1 day), an evident dose-
dependence related to DM concentration was revealed (Fig. 3B).
Moreover, higher basal proliferation was observed in all the Cs-
Arg scaffolds compared to neat Cs at 1 day after cell seeding,
then decreasing over the culture time (at 2 and 3 days). This
behavior suggests that L-Arg might act as a boosting metabolite
for cell proliferation in the early growth stages (1 day), which
coherently undergoes a time-dependent exhaustion overtime
(Fig. 3B). Furthermore, at 1 day the presence of L-Arg seems to
overcome the previously observed limited proliferation in the
Cs/DM scaffolds without L-Arg. Also, it can be hypothesized that
the positive, dose-dependent trend of DM effect in Cs-Arg scaf-
folds might be elicited based on the structural modification by
the (increasing concentrations of) DM nanoarchitectures and/or
in the presence of L-Arg as a bioactive agent. Overall, the results
from MG63 cells suggest some modular features and apprecia-
ble homogeneity of behavior for Cs/DM-Arg scaffolds compared
to untreated Cs scaffolds. Specifically, the effects of L-Arg at short
time could be further investigated in such applications in which
the early cell–material interactions are critical and thus must be
finely tuned.
In vitro cytocompatibility studies in the presence of
magnetic stimulation
Scaffolds with different DM amounts and/or L-arg were also
seeded by hMSCs undergoing proliferation in the absence or
presence of external magnetic field (see methods for details).
hMSCs are a significant model in the field of bone regenerative
medicine, especially in research approaches aiming at analyz-
ing the time course of proliferation and differentiation.31,32
As summarized in Figure 3C, in the absence of magnetic
stimulation it can be observed that (1) for each DM content
(0%, 5%, 10%, and 15%) viability of hMSCs, grown on Cs/DM-
Arg scaffolds, is equal or higher than cells on Cs/DM, invariably,
at 1, 3, and 7 days post-seeding and (2) the overall proliferation
trend at days 1 and 3 comparatively resembles what described
in MG63 cells, thus it can be hypothesized that both hMSCs and
MG63 cells establish similar proliferation dynamics under com-
parable structural conditions depending on DM content and L-
Arg presence (as previously described). In parallel, hMSCs
were cultured in Cs/DM and Cs/DM-Arg scaffolds in the pres-
ence of 6 h-daily magnetic stimulation (Fig. 3D). At day 1, no
significant effects due to magnetic field were detected on cell
proliferation in each Cs, Cs/DM and Cs/DM-Arg scaffold com-
pared to the respective values in the absence of magnetic stim-
ulation (see Fig. 3C). On the contrary, at day 3 a remarkable
difference was detected for all the Cs/DM-Arg scaffolds on
which cell proliferation under magnetic exposure resulted sig-
nificantly increased compared to all the Cs/DM (Fig. 3D) and to
the respective Cs/DM-Arg in the absence of magnetic field (see
Fig. 3C). Interestingly, the magnetic induction did not enhance
proliferation on L-Arg-deprived scaffolds.
Finally, proliferation of hMSCs under magnetic exposure
was evaluated at a long-term stage post-seeding (7 days).
Under magnetic stimulation (Fig. 3D), Cs/DM scaffolds with-
out L-Arg allowed proliferation rates similar to those
reported in the absence of magnetic field (i.e., it can be
noticed a slight reduction of proliferation compared to neat
Cs regardless of DM content; in contrast, at days 7 prolifera-
tion of cells in the Cs/DM-Arg scaffolds under magnetic stim-
ulation was evidently reduced with respect to Cs (Fig. 3D),
while it was shown to be increased in scaffolds in the
absence of magnetic field (Fig. 3C), regardless of DM content.
Overall, the experimental data from hMCs give hints of a
third level of modulation of cell proliferation in the different
scaffold-nanoarchitectures that is, magnetic stimulation (1),
in addition to the DM modular content (2) and L-Arg dual
activity on structure and cell metabolism (3).
CONCLUSIONS
A method for the design and preparation of chitosan-based
scaffolds with interesting properties due to the presence of
DMs and L-Arg amino acid as a bioactive agent has been pre-
sented in this study. The proposed process has allowed
obtaining scaffolds with a homogeneous pore distribution and
a high degree of porosity interconnection, which are funda-
mental biomaterial requirements to promote cell adhesion
and diffusion of nutrients, and the consequent removal of cel-
lular metabolism products. Furthermore, both components
seem to sinergically act as modulators of the mechano-
structural features, in terms of swelling and mechanical
behavior. Moreover, they confer to the biomaterial specific
requirements toward osteoblast-like and human mesenchymal
stem cells, differentially stimulating their proliferation both in
the absence and in the presence of a time-dependent mag-
netic field. Definitively, both the DM and L-Arg components
seem to imprint versatility to the polymer itself. The optimiza-
tion of these features will represent the starting point for
designing a successful biomimetic structure with “ad hoc”
traits for the regeneration of bone defects.
AKNOWLEDGMENTS
The authors wish to thank Mr. Donato Cannoletta (University
of Salento) for performing SEM and EDS analyses.
JOURNAL OF BIOMEDICAL MATERIALS RESEARCH PART A | JUN 2019 VOL 107A, ISSUE 6 1251
ORIGINAL ARTICLE
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