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Journal of Nanostructure in Chemistry

https://doi.org/10.1007/s40097-022-00507-z

ORIGINAL RESEARCH

Chitosan/alginate bionanocomposites adorned with mesoporous

silica nanoparticles for bone tissue engineering
Satar Yousefiasl1 · Hamed Manoochehri2 · Pooyan Makvandi3 · Saeid Afshar2 · Erfan Salahinejad4 ·
Pegah Khosraviyan5 · Massoud Saidijam2 · Sara Soleimani Asl6 · Esmaeel Sharifi2,7 

Received: 22 March 2022 / Accepted: 4 June 2022

© The Author(s), under exclusive licence to Islamic Azad University 2022

Abstract
The regeneration of oral and craniofacial bone defects ranging from minor periodontal and peri-implant defects to large and
critical lesions imposes a substantial global health burden. Conventional therapies are associated with several limitations,
highlighting the development of a unique treatment strategy, such as tissue engineering. A well-designed scaffold for bone
tissue engineering should possess biocompatibility, biodegradability, mechanical strength, and osteoconductivity. For this
purpose, mesoporous silica nanoparticles (MSNs) were synthesized and incorporated at different ratios (10, 20, and 30%)
into alginate/chitosan (Alg/Chit)-based porous composite scaffolds fabricated through the freeze-drying method. The MSN
incorporation significantly improved the mechanical strength of the scaffolds while showing a negligible decreasing effect on
the porosity. All of the samples showed desirable swelling behaviors, which is beneficial for cell attachment and proliferation.
The MSN-containing scaffolds indicated a decreased hydrolytic degradation in an MSN percentage-dependent manner. The
fabricated scaffolds did not depict cytotoxic characteristics. The Alg/Chit/MSN30 scaffolds not only showed noncytotoxic
properties, but also increased the cell viability significantly compared to the control group. The biomineralization proper-
ties of the MSN-containing nanocomposite scaffolds were significantly higher than the Alg/Chit composite, suggesting the
potential of these nanoparticles for bone tissue engineering applications. Taken together, it is concluded that the Alg/Chit/
MSN30 scaffolds are considerable substances for bone tissue regeneration, and MSN has a great tissue engineering potential
in addition to its extensive biomedical applications.

* Esmaeel Sharifi
[email protected]
1
School of Dentistry, Hamadan University of Medical
Sciences, Hamadan 6517838736, Iran
2
Research Center for Molecular Medicine, Hamadan
University of Medical Sciences, Hamadan, Iran
3
Centre for Materials Interface, Istituto Italiano Di
Tecnologia, viale Rinaldo Piaggio 34, 56025 Pontedera, Pisa,
Italy
4
Faculty of Materials Science and Engineering, K. N. Toosi
University of Technology, Tehran, Iran
5
Medical Plants Research Center, Basic Health Sciences
Institute, Shahrekord University of Medical Sciences,
Shahrekord, Iran
6
Department of Anatomy, School of Medicine, Hamadan
University of Medical Sciences, Hamadan, Iran
7
Department of Tissue Engineering and Biomaterials, School
of Advanced Medical Sciences and Technologies, Hamadan
University of Medical Sciences, Hamadan, Iran

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Graphical abstract

Keywords  Bone substitute · Bone tissue regeneration · Maxillofacial rehabilitation · MSN · Nanocomposites

Introduction Scaffolds have an essential role in bone tissue engineer-

ing as a three-dimensional (3D) support for tissue forma-
Bone defects caused by trauma, tumor, and congenital tion by providing a proper microenvironment for cells to
defects are increasing considerably. Accordingly, there is attach, proliferate, and osteogenic differentiation [4, 5].
an increasing demand for bone tissue regeneration. Further- Ideal scaffolds should mimic the structure and function of
more, small periodontal and peri-implant defects to large natural tissue; moreover, they should exhibit biocompat-
and critical lesions caused by trauma, tumor resection, and ibility, controllable biodegradability, non-immunogenicity,
congenital malformations are all instances of oral and crani- and tailorable structural preferences as porosity, intercon-
ofacial bone defects that significantly impact both esthetics nected pore structure, and mechanical strength similar to
and functions in the mouth and face [1–3]. natural bone [6–8]. In this regard, natural polymers play

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Journal of Nanostructure in Chemistry

a pivotal role for enhancing the biocompatibility [9, 10]. evaluation were carried out to evaluate its capability in bone
For instance, scaffold based on natural polymers have tissue regeneration.
attained great attention for bone tissue engineering owing
to their desirable biocompatibility, biodegradability, and
low immunogenicity. Chitosan is a naturally derived poly-
mer and is deacetylated form of chitin. It shows superior Materials and methods
properties, including availability, biocompatibility, bio-
degradability, and antimicrobial characteristics [11–15]. Materials
Alginate is a polysaccharide with advantageous features,
such as biocompatibility, biodegradability and non-immu- The solvents and materials were purchased from Merck
nogenicity [16, 17]. (Germany), Sigma-Aldrich (USA), and Abcam (USA) unless
Despite the above-mentioned advantages of the natural otherwise noted.
polymers, these polymers show poor mechanical character-
istics, limiting their applications as a bone tissue engineering
Preparation of MSNs
scaffold. Composite scaffolds have been introduced as an
approach to address these limitations. Composite scaffolds
A solution containing 0.400 gr cetyltrimethylammonium
consist of at least two materials with different characteristics
bromide (CTAB)/192 ml DI water/1.4 ml NaOH (2 M) was
to cover their drawbacks and achieve a scaffold with superior
stirred at 50 °C for 30 min at 1000 rpm. Then, 2.8 ml mesi-
properties [18, 19]. Due to covalent interactions between
tylene was added, and the temperature was set at 80 °C. An
the amine groups of chitosan with the carboxyl groups of
aluminum foil was wrapped around the container after the
alginate, it is suggested that chitosan and alginate are com-
mesitylene addition to prevent the undesirable light effect.
bined to create a composite scaffold with superior mechani-
After 4 h, 2 ml tetraethoxysilane (TEOS) was added drop-
cal properties [20].
wise to the solution and continued to be stirred for 2 h.
Furthermore, the addition of nanomaterials to polymeric
Afterward, a filter paper was placed in a Büchner funnel
scaffolds has demonstrated to improve bone regeneration
connected to a Büchner flask with a tube connected to a
capacity, mechanical properties, and swelling behavior
vacuum suction. The prepared solutions from the previous
[21]. For instance, mesoporous silica nanoparticles (MSNs)
steps were poured separately on the filter paper, and the
demonstrated unique merits, including large specific surface
remaining mixture was incubated at 60 °C for 24 h and then
area, adjustable pore volume, controllable size, easy surface
at 120 °C for 24 h to remove the water. Then, the samples
functionalization, and good biocompatibility, explaining
were calcined for 4 h at 550 °C. Finally, the powders were
the increasing biomedical application of MSNs. MSNs are
manually ground using a ball mill to obtain a homogenous
extensively applicated in delivery systems, bioimaging, bio-
MSN powder (Fig. 1A).
sensing, and therapeutic agents. The employment of MSNs
for tissue engineering applications is a relatively emerg-
ing subject that has sparked interest in the scientific commu- Nanoparticle characterization
nity [22, 23].These mentioned properties of MSNs besides
their low cost and availability merit their application in the FTIR
combination of tissue engineering with promising drug,
gene, and protein delivery characteristics. Hence, fabricat- The functional groups of the synthesized MSNs were identi-
ing biodegradable multifunctional scaffolds for promoted fied using Fourier-transform-infrared spectroscopy (FTIR,
bone tissue engineering besides incorporating novel carrier Bruker, Germany). Briefly, 1 mg of each sample was mixed
agents for potential simultaneous delivery applications is the with 300 mg KBr as a standard reference, pelleted, and ana-
novelty of this project. lyzed at 2.60 Hz with a resolution of 4 ­cm−1 in the wave-
In light of this, we fabricated a biocomposite scaffold number range of 4000–400 ­cm−1.
comprising chitosan and alginate possessing promising
physical and biological properties of the composite scaf-
folds for bone tissue engineering applications. Besides, to XRD
improve the physical characteristics and osteogenic differen-
tiation potential, we synthesized and incorporated MSN in The X-ray diffraction (XRD) method was used to identify
the scaffolds. Subsequently, the physicochemical properties different phases present in the synthesized MSNs with
of the synthesized nanoparticles and the host scaffold were a Philips X-ray diffractometer using the Cu-Kα radiation
investigated. Following that, cellular assays, such as cytotox- (λ = 1.54056 Å) with the step size of 0.05°, step time of 2 s,
icity, alkaline phosphatase activity, and calcium deposition and scanning range of 5–70 degrees.

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Fig. 1  Schematic illustration of the (A) synthesizing steps of the MSNs and (B) fabrication process of the Alg/Chit-based nanocomposite scaf-
folds

FESEM and EDS a homogenous mixture. The solution underwent 6 h of

stirring to reach a homogenous solution. Then, the homog-
The size, morphology, and elemental analysis of the nano- enous solutions were pured in 48 well plate, stored at 4 °C
particles were examined by field-emission scanning electron for 2 h, and immediately moved to − 30 °C for 24 h. Before
microscopy (FESEM, MIRA3, Tescan, Brno, Czech Repub- freeze-drying, the samples were moved to − 80 °C for one
lic) equipped with energy dispersive X-ray spectrometry hour and then lyophilized in a freezer dryer (Alpha 1–2
(EDS). LD plus, Christ, Germany) at − 50 °C and 0.040 bar for
48 h. Then, the fabricated scaffolds were sliced in desired
Dynamic light scattering (DLS) thicknesses. The scaffolds were cross-linked by immersing
in a ­CaCl2 solution (1% wt./ml) for 15 min, rinsed with
The synthesized MSNs were well dispersed in DI water DI ­H2O, and moved to − 30 °C after 1 h of incubation at
using sonication. Hydrodynamic particle sizes and size dis- 4 °C. Finally, the scaffolds were lyophilized in the freeze
tributions were acquired using a dynamic light scattering dryer (Alpha 1–2 LD plus, Christ, Germany) at − 50 °C
instrument (DLS, Malvern Panalytical, Worcestershire, UK). and 0.040 bar for 48 h (Fig. 1B).

Preparation of porous chitosan–alginate and MSN

scaffolds Characterization of the fabricated nanocomposite
scaffolds
An alginate solution was made by dissolving alginic acid
sodium salt powder in deionized H ­ 2O to prepare a 5% SEM and EDS
(wt./v) solution. Chitosan (deacetylation degree < 80%,
low MW) was dissolved in acetic acid (1 N) to prepare A thin layer of gold was sputter-coated (SCD 004, Balzers,
5% (wt./v) chitosan solution. Then, 50% alginate and 50% Germany) on the samples before microscopic analyses to
chitosan solutions were mixed under vigorous stirring at ensure an effective conductivity on the surface avoiding
room temperature. For the fabrication of composite scaf- surface charging. Scanning electron microscopy (SEM,
folds, an appropriate amount of the MSNs was added to TESCAN VEGA3 XMU, Tescan, Brno, Czech Repub-
the chitosan-alginate solution to obtain samples contain- lic) equipped with energy dispersive X-ray spectrometry
ing 10–30% MSNs (Alg/Chit/MSN 10%, 20% & 30%). To (EDS) was used to investigate the morphological and
ensure homogenous dispersion and prevent aggregation semi-quantitative elemental properties of the nanocom-
of MSNs in the polymer solution, the ball-milled MSN posite scaffolds at a 20 kV accelerating voltage.
powders were sonicated in deionized water until obtaining

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ATR‑FTIR In vitro degradation assay

The functional groups of the fabricated scaffolds were The in vitro degradation assay was performed according to
identified using Attenuated Total Reflectance-Fourier a previous report [25]. The samples were immersed in PBS
Transform Infrared spectroscopy (ATR-FTIR, Bruker, Ger- for 2 h to attain swelling equilibrium before weighing for the
many). Briefly, the dried scaffold samples were cut into in vitro hydrolytic degradation tests. This record was consid-
10 mm diameter and 1 mm thickness. They were employed ered as the initial weight, and the scaffolds were immersed
for analysis with a resolution of 4 ­cm−1 in the wavenumber in PBS (pH 7.4) and incubated at 37 °C for 21 days. Two-
range of 4000–600 ­cm−1. thirds of the PBS solution was changed with a fresh solu-
tion every three days. At predetermined time points (3, 7,
Porosity and pore size measurement 14, and 21 days), the samples were withdrawn from PBS,
and the weight of each sample was measured. The scaffold
By analyzing electron microscope images using the Image J degradation percentage was determined by the following
software, the porosity and pore distribution of the scaffolds equation: Degradation (%) = (Wt – Wi / Wi) × 100, where Wi
were determined. To determine porosity, the surface area of corresponds to the initial weight of the sample and Wt is
pores was divided by the total image area. This measurement the weight of the sample at various time periods. Data from
was carried out on a series of SEM images, and the mean three scaffolds were obtained and expressed as mean ± SD.
results are expressed. In addition, the average pore size and
pore size distribution were reported after measuring the size Cell toxicity analysis
of the pores randomly in different SEM pictures (at least
100 pores). The biocompatibility of the synthesized MSNs and fabri-
cated scaffolds was assessed by in vitro cytotoxicity tests
Mechanical testing using the 3-(4,5-dimethylthiazol-2-yl)-2, 5-diphenyl tetrazo-
lium bromide (MTT) assay. Rat bone marrow mesenchymal
The compressive mechanical characteristics of the fabri- stem cells (BMSCs) were cultured in the DMEM medium
cated scaffolds were evaluated using monotonic uniaxial supplemented with 10% fetal bovine serum and 1% peni-
unconfined compression, regarding a procedure mentioned cillin/streptomycin antibiotic and incubated at 37 °C with
elsewhere [24]. At least three scaffolds of each composition 5% ­CO2. The MSN powder was well dispersed in the cul-
were prepared in a cylindrical shape with 10 mm height ture media at the concentrations of 250, 500, 750, 1000,
and 10 mm diameter. The samples were immersed in the and 1500 μg/ml, incubated for one day at 37 °C, and steri-
phosphate buffer saline (PBS) for one hour before the test to lized by filtration. BMSCs were seeded on 96-well culture
preserve the physiological conditions of the tissue. The tests plates at a density of 3 × ­103 cells per well. The culture
were performed at a 1 mm/min rate at room temperature medium was replenished with the particle suspensions after
using a universal mechanical tester (SANTAM, STM-20, cellular attachment, and this period was set as the begin-
Iran). The elastic modulus of each sample was determined ning of culture time. The suspensions were refreshed every
from the slope of engineered stress–strain curves. other day. After predetermined periods (1, 3, and 5 days),
the MTT solution (0.5 mg/ml) was added to each well and
Swelling behavior incubated at 37 °C for 4 h. Then, the medium of each well
was removed, and 100 μL Dimethyl sulfoxide (DMSO) was
The swelling behavior of the fabricated scaffolds was meas- added to each well to dissolve insoluble formazan crystals.
ured according to a protocol described elsewhere [25]. The The plates were gently shaken for 15 min in the absence of
initial dry weight of each scaffold with 10 mm diameter light, and formazan product absorbance was measured by a
and 5 mm thickness was first measured, immersed in the microplate spectrophotometer (Tecan's Sunrise, Austria) at
phosphate-buffered saline solution (PBS), and incubated a wavelength of 570 nm.
in vitro at 37 °C. Afterward, the wet weight of the samples at The scaffolds were sectioned in a 1 mm thickness and
predetermined time intervals (5, 10, 20, 30, 40, 50, 60, 120, 6 mm diameter with an approximate weight of 2.4 mg. The
240, 360, and 1440 min) was measured after removing the samples were sterilized using 70% ethanol followed by 2 h of
excess water from the surface of the scaffolds. The scaffold UV radiation and then were washed twice with PBS and cul-
swelling ratio was calculated using the following equation ture media. The prepared scaffolds were placed in a 48-well
(Swelling ratio = Ww—Wd/Wd), where Ww is the weight of plate, and the MSCs were seeded at a density of 5 × ­104
the wet sample, and Wd corresponds to the initial dry weight cells per scaffold on the top of each scaffold. The culture
of the same sample. Data from three samples were obtained media was replaced every other day. After predetermined
and were expressed as mean ± SD. periods (3, 5, and 7 days), the MTT solution (500 μl/ml)

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was added to each well and incubated at 37 °C for 4 h. After Alkaline phosphatase assay
that, the medium of each well was removed, and 150 μL
Dimethyl sulfoxide (DMSO) was added to the each well to ALP enzyme activity was measured through the p-nitro-
dissolve insoluble formazan crystals. The plates were gently phenyl phosphate (pNPP) method using Pars Azmoon kit
shaken for 15 min in the absence of light and then moved to (Iran). To begin, the culture media was removed, and the
a 96-well plate. Finally, the formazan product absorbance scaffolds washed in PBS. The cell-containing scaffolds were
was measured by a microplate spectrophotometer (Tecan's lysed by NP40 buffer. The lysed contents were transferred
Sunrise, Austria) at a wavelength of 570 nm. to a microtube and centrifuged for 10 min at 3000 rpm. The
microtubes supernatant were moved into a 96-well plate,
the ALP substrate was added and reaction was halted after
Osteogenic differentiation
30 min of incubation at 37 °C. Eventually, the plate absorb-
ance was measured at 405 nm and the ALP enzyme activity
The scaffolds were sectioned in a thickness of 1 mm and
was calculated as Unit/L.
diameter of 10 mm and were sterilized using a UV light
for 1 h. Then, each scaffold was placed in a 24-well culture
Statistical analysis
plate. First, 300 μL of complete medium (DMEM contain-
ing 10% FBS and 1% Penicillin–Streptomycin) was dropped
Data are reported as mean ± standard deviation (Mean ± SD)
on top of the scaffolds, and the plate was kept at 37 °C, 5%
and were analyzed using the GraphPad Prisma 9 software
­CO2 incubator for an hour before cell seeding. Afterward,
(San Diego, CA, USA). Statistical data analyses were per-
MSCs were dropped onto the scaffolds with an average den-
formed using one-way analysis of variance (ANOVA) fol-
sity of ­105 cells per well. The plate was placed back in the
lowed by post hoc Tukey's tests. A p-value below 0.05 was
incubator (37 °C, 5% C ­ O2) for 10 min, and the volume of
considered statistically significant for all the tests.
the medium in the wells was raised to 1000 μL with the
complete medium. The complete medium was interchanged
one day after seeding by osteogenic media (BioIdea, Iran).
After that, for the next 21 days, half of the osteogenic media
Results and discussion
in the wells was refreshed every day. Measuring the calcium
Characterization of synthesized MSN
content and alkaline phosphatase level was used to investi-
gate the differentiation process at different time intervals of
The Fourier-transform-infrared (FTIR) spectrum of the
3, 7, 14, and 21 days.
synthesized MSNs is shown in Fig. 2A. The FTIR spec-
troscopy indicates that the MSNs present peaks at 455 and
Alizarin red staining 814 ­cm−1 corresponding to Si–O bonds. The peak observed
at 1088  ­cm−1 also corresponds to the Si–O-Si vibrations
The alizarin red staining assay was used to determine the of silanol groups. The peaks range of 3200–3400 ­cm−1 is
calcium content composed onto the scaffolds during the attributed to the O–H bond. In the XRD pattern of the MSNs
osteogenic differentiation. To summarize, the culture media (Fig. 2B), the broad diffraction peak detected at 2 theta = 23º
was discarded, and the scaffolds were rinsed twice using with a hollow feature confirms an amorphous nature, which
PBS. To fix the scaffolds, they were put in 4% formaldehyde is related to the PDF card standard of silica-cristobalite
for 15 min and then rinsed using PBS. Afterward, 300 μL phase ICDD #00-001-0424 [27]. FESEM images show that
of 1.5% alizarin red (pH 4.2) was added to the scaffolds and the synthesized MSNs have a monodispersed spherical-like
incubated for 4–5 min at room temperature. Then, the scaf- shape with a diameter of approximately 100 nm (Fig. 2C).
folds were rinsed several times with PBS (pH 4.5) to remove The FESEM results also indicate that the sample has a nar-
any remaining dye. Qualitative assessments were performed row particle size distribution. The EDS analysis depicts the
by taking images using an invert microscope (Motic, China). presence of Si and O in the synthesized MSNs, which con-
Subsequently, the scaffolds were incubated with an acetic firms that the synthesized particles are pure MSNs without
acid solution (8%) for 20 min at room temperature under impurities (Fig. 2D). The DLS measurements also showed
gentle shaking to solve deposited dyes. Then, the plates con- an average size of about 270 nm with a narrow particle
tents were moved to a 96-well plate, and the absorbance was size distribution (Fig. 2E). The particle size observed by
measured at a wavelength of 550 nm. The optical absorption FESEM is smaller than the hydrodynamic diameter meas-
of various concentrations of alizarin red was measured at ured by DLS. Variations in DLS and FESEM results are
550 nm wavelength to obtain a standard curve. The calcium commonly indicated in the literature since DLS provides
content was assessed by the fact that an mole of alizarin red a hydrodynamic diameter; thus, the measured particle
interacts with two mole calcium [26]. size is expected to be larger than the exact particle size.

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Fig. 2  MSN characterization results: (A) FT-IR spectrum of the syn- and (E) average hydrodynamic size and hydrodynamic size distribu-
thesized MSNs, (B) XRD pattern of the MSNs, (C) representative tion of the synthesized MSNs
FESEM micrographs of the MSNs, (D) EDS analysis of the MSNs,

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Furthermore, the rare presence of large particles besides mapping and profiles also confirm the incorporation and uni-
weak aggregates of the particles can cause the z-average form distribution of the MSNs (the presence of Si element)
particle size to increase [28]. within the scaffolds (Fig. 3).
The ATR-FTIR spectra of the non-crosslinked Alg/Chit,
Characterization of the fabricated composite crosslinked Alg/Chit, Alg/Chit/MSN10, Alg/Chit/MSN20,
scaffolds and Alg/Chit/MSN30 are represented in Fig. 4A. The bands
at 1424 and 1070 ­cm−1 in the Alg/Chit scaffold are attrib-
The SEM micrograph of the scaffolds reveals the porous uted to –COOH and C–O stretching bands, respectively. In
structure of the four types of the scaffolds. The porous and the cross-linked Alg/Chit spectra, amide II bands were con-
rough surface structure of the scaffolds are ideal for stem siderably intensified. The stretching vibrations of the C=O
cell adhesion and shaped cell growth. The EDS elemental groups of the prepared scaffolds are assigned to 1609 ­cm−1,

Fig. 3  (A) SEM micrographs of the fabricated scaffolds (white scale bars indicate 500 µm) and (B) corresponding EDS analyses of the fabri-
cated scaffolds (white scale bars indicate 400 µm)

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Fig. 4  (A) ATR-FTIR spectra of the non-crosslinked Alg/Chit, crosslinked Alg/Chit, Alg/Chit/MSN10, Alg/Chit/MSN20, and Alg/Chit/MSN30
scaffolds, (B) graph and (C) corresponding images of the fabricated scaffolds porosity, and (D) pore size distribution of the scaffolds

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1617 ­cm−1 and 1624 ­cm−1, respectively. The peak observed decreases the mechanical stability of scaffolds [29]. Indeed,
at 1088  ­cm−1 corresponds to the Si–O–Si vibrations of while bulk mechanical parameters for osteogenic scaffolds
silanol groups, confirming the presence of the MSNs in the intended to function as biodegradable temporary structures
Alg/Chit scaffolds. are different from those for load-bearing dense permanent
The porosity of the fabricated Alg/Chit, Alg/Chit/ implants, swelled scaffolds should exhibit sufficient stiffness
MSN10, Alg/Chit/MSN20, and Alg/Chit/MSN30 scaffolds to enable surgical handling and stable graft placement [25].
was measured to be 63.4 (± 4.5) %, 63.4 (± 2.1) %, 62.9 The minimum mechanical strength and elastic module
(± 4.3) %, and 60.3 (± 2.5) %, respectively (Fig. 4B,C). belong to the Alg/Chit scaffold. In our experiments, the
The pore size of the fabricated scaffolds ranges from less mechanical stability is significantly increased with the MSN
than 50 µm to 700 µm. The average pore size of the Alg/ content in the polymer structure. In this regard, it is reported
Chit, Alg/Chit/MSN10, Alg/Chit/MSN20, and Alg/Chit/ that adding MSNs into polymer scaffolds will increase the
MSN30 scaffolds are 221, 160, 124, and 119 µm, respec- strength of composites since MSNs lead to an efficient
tively (Fig. 4B). mechanical stress transfer mechanism. Also, it has been
revealed that the porous nature of MSNs leads to a mechani-
Mechanical testing cal interlocking among the nanoparticles and between the
particles and polymer chains, giving the polymer an oppor-
The mechanical stability of the scaffolds was determined by tunity to form stronger bonds [30].
measuring mechanical strength against vertical dimensional
change and calculating elastic module. We measured the
mechanical stability after soaking the scaffolds in the PBS Swelling behavior
solution to resemble the normal body conditions (Fig. 5A,B).
Mechanical strength is vital for in vivo applications and is Water absorption capacity is critical for tissue engineer-
between 0.3 and 1.3 MPa in our fabricated scaffolds, so they ing scaffolds, as the procedure is dependent on biologi-
are more suitable for engineering cancellous bone rather cal solution absorption, nutrient penetration, and meta-
than cortical bone. However, soaking in PBS significantly bolic wasted  transport  within the biomaterials[31]. The

Fig. 5  Mechanical behavior of the fabricated scaffolds based on: (A) stress–strain curve and (B) elastic modulus. (C) Swelling ratio of the
freeze-dried scaffolds and (D) In vitro hydrolytic degradation of the fabricated scaffolds

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above-mentioned characteristics can be maintained using a concentration show significantly decreased viability. How-
high swelling ratio in the fabricated scaffolds. ever, all of the MSN concentrations do not induce significant
In terms of water uptake by the dry scaffolds, all the sam- cytotoxicity and have a similar cellular viability compared
ples demonstrate a high swelling capacity (Fig. 5C). After with the control group on 3rd and 5th days. Particle size and
soaking for the extended periods, the increased swelling concentration play a key role in MSN cytotoxicity. It has
ratio indicates an increase in the water absorption amounts; been reported that MSNs ranging from 30 to 300 nm have no
however, the increasing water absorption rate is decreased, significant cytotoxicity toward HeLa cells [36]. Still, there
and the scaffold reached a swelling equilibrium. The Alg/ is controversy regarding the results of MSN cytotoxicity,
Chit scaffold shows the highest swelling rate compared to which can be related to variations in the MSN synthesis
the nanocomposite scaffolds. Even though the incorporation method and cytotoxicity measurement assays [37, 38].
of the MSNs does not alter the swelling kinetics (all the It is essential for composites to remain inert in the
samples are swelled after 1 h), the swelling ratio is decreased human body and be compatible with bone formation when
with increasing the reinforcement levels. The decrease in the implanted. In addition, scaffolds should be biocompatible in
swelling ratio with increasing the MSN concentration can addition to their physicochemical characteristics. The results
be attributed to the porosity changes of the scaffolds and the indicated that all of the scaffolds show no toxicity compared
relative reduction of the polymer concentration in the scaf- with the two-dimensional cultured cells as the control group
folds. Because the biopolymer swelling capacity is generally after 3, 5, and 7 days (Fig. 6B). Furthermore, the Alg/Chit/
related to the interaction of polymer components with water MSN30 shows significantly higher cell viability compared
molecules. Furthermore, these results are in accordance with with the control group after 3 and 5 days. The composition
the presented results of recent studies on the swelling ratio of the scaffolds largely influences biocompatibility charac-
of Alg/Chit composite and nanocomposite scaffolds [32, 33]. teristics. FDA has approved the use of chitosan and alginate
as biomaterials; In fact, the biocompatibility of these constit-
In vitro degradation assay uent polymers is reflected in the cytotoxicity findings [39].

The degradation behavior of scaffolds is critical for tissue Osteogenic differentiation

regeneration and is regarded as one of the most desired char-
acteristics for a scaffold developed for bone tissue regenera- The differentiation efficiency of the MSCs into mature
tion. Scaffold degradation should occur at a nearly identical osteocytes on the different scaffolds was examined using
rate to that of tissue repair to support regenerative tissue the alizarin red and alkaline phosphatase assays. The min-
ingrowth. The analysis of the scaffold stability under hydro- eralization extent is higher on Alg/Chit/MSN20 than the
lytic conditions for 21 days reveals that approximately 50% other scaffolds, followed by Alg/Chit/MSN30 at 7, 14, and
of the scaffolds were degraded after 21 days, and the Alg/ 21 days (Fig. 7A). Also, the content of calcium deposited
Chit and Alg/Chit/MSN30 scaffolds showed the highest and on the Alg/Chit/MSN20 scaffolds is significantly higher
lowest degradation percentages, respectively (Fig. 5D). The than the Alg/Chit/MSN10 and Alg/Chit/MSN30 scaffolds
results also indicated that the combination of the MSNs with at 7 and 14 days. However, there is no significant difference
the Alg/Chit scaffolds leads to a decrease in biodegradabil- between the calcium deposition of the Alg/Chit/MSN20 and
ity, so that this decline was commensurate with the increase Alg/Chit/MSN30 scaffolds at 21 days. Based on previous
in the MSN percentage. This can be related to the low degra- studies, silica nanoparticles improve the osteogenic nature
dation rate of the MSNs [34]. Still, the in vitro recapitulation of scaffolds [40, 41] mainly through releasing biologically
of the surrounding environment following the implantation active ions, such as Si [42], inducing collagen production,
of the scaffold is exceedingly complex due to the existence enhancing ALP activity, and upregulation of bone-related
of multiple types of proteases that regulate extracellular genes, such as OPN RUNX2 and OCN [43]. Released Si
matrix remodeling. Hence, the addition of MSNs could be ions can stimulate osteogenesis pathways, including TGF-
beneficial for bone tissue engineering applications since it β1[43], Wnt [44], ERK, and SHH signaling pathways [45].
can prevent its rapid degradation of implants [35]. Yang et al. [46] showed that silica nanoparticles significantly
enhance the ALP activity and mineralization rate of human
Cell proliferation and viability (MTT) MSCs. Also, Ha et al. [47] reported that silica nanoparti-
cles could stimulate the osteogenic differentiation of mouse
The clinical translation of MSNs is conceivable only if MC3T3-E1 cells by increasing ALP activity, enhancing nod-
they do not exhibit cytotoxic activity toward normal cell ules formation, and upregulation of osteocyte-related genes.
lines. The viability of cultured BMSCs on the synthe- In the present study, the higher osteogenic efficiency of the
sized MSNs was assessed by the MTT assay after 1, 3, and Alg/Chit/MSN20 scaffold than Alg/Chit/MSN30 in 7 and
5 days (Fig. 6A). On the 1st day, the MSNs with 1.5 mg/ml 14 days can originate from a slight reduction of the cell

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Fig. 6  (A) Cell viability of BMSCs cultured at 0.25, 0.5, 0.75, 1, and dimensional cultured cells (control), Alg/Chit, Alg/Chit/MSN10, Alg/
1.5  mg/ml MSNs for 1, 3, and 5  days with the corresponding heat- Chit/MSN20, and Alg/Chit/MSN30 scaffolds for 3, 5, 7 days with the
map of the cell viability (*p < 0.05) and (B) cell viability of the two- corresponding heatmap of the cell viability (*p < 0.05, **p < 0.01)

proliferation rate on the Alg/Chit/MSN30 scaffold compared concentrations of the MSNs (10, 20, and 30%). All of the
to the Alg/Chit/MSN20 scaffold in a time-dependent man- fabricated scaffolds showed a proper swelling profile. The
ner. However, the ALP activity in the Alg/Chit/MSN30 scaf- MSN-reinforced systems exhibited significantly increased
folds is relatively higher than the others at all times. Since mechanical properties with a slight decrease in porosity.
the ALP activity was normalized by the cell number, this Besides, the MSN incorporation into the polymeric struc-
test was not affected by the cell proliferation rate. A reduc- ture prolonged the hydrolytic degradation that can depict
tion is observed in the ALP activity of all the samples on the positive effect of these nanoparticles in the inhibition
21 days. In this regard, previous studies reported that ALP of rapid degradation upon implantation. The fabricated
activity is increased in osteoblasts and declined in mature scaffolds showed no toxicity toward BMSCs and a slight
osteocytes [48]. decrease in the cell viability compared with two-dimensional
cultured cells. In addition, Alg/Chit/MSN30 demonstrated
significantly higher cell viability compared to the control
Conclusions group on 3rd and 5th day. Mineral deposition and alkaline
phosphatase expression measurements indicated the posi-
In the present study, we successfully designed a nanocom- tive effect of the MSN incorporation on osteogenic differ-
posite scaffold via integrating MSNs into a Alg/Chit-based entiation. Overall, the porous nanocomposite Alg/Chit/MSN
polymeric network. MSNs were synthesized and then scaffolds developed with desired characteristics could be a
employed to fabricate the nanocomposites with the varying proper implant for craniofacial bone defects regeneration.

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Fig. 7  Osteogenic potential of BMSCs cultured on the fabri- (C) ALP activity secreted by BMSCs after 3, 7, 14, and 21  days of
cated scaffolds: (A) Alizarin red staining images and (B) relative cal- culture. (*p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001)
cium content quantification after 3, 7, 14, and 21 days of cell culture.

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Acknowledgements  The study was funded by Vice-chancellor for and mechanical properties. Polym. Technol. Mater. 58, 2031–2040
Research and Technology, Hamadan University of Medical Sciences (2019)
(Grant Number: 9804042537). 15. Zafari, M., Mansouri, M., Omidghaemi, S., Yazdani, A., Pour-
motabed, S., Hasanpour Dehkordi, A., Nosrati, H., Validi, M.,
Sharifi, E.: Physical and biological properties of blend-electrospun
polycaprolactone/chitosan-based wound dressings loaded with
N-decyl-N, N -dimethyl-1-decanaminium chloride: An in vitro
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