Design and Fabrication of Injectable Microcarriers Composed of Acellular Cartilage Matrix and Chitosan

Abstract

Cartilage is an avascular tissue with limited self-repair ability. Since the methods for treatment of cartilage defects have not been effective, new therapies based on tissue engineering are considered over the recent years. In this study, human cartilage tissue was decellularized and porous injectable microcarriers (MCs) composed of acellularextracellular matrix (ECM) of cartilage tissue and chitosan(CS), with different ECM weight ratios, were fabricated by electrosprayingtechnique to be used in the treatment of articular cartilage defects. Various properties of ECM/CS MCs such as microstructure, mechanical strength, water uptake behaviour, and biodegradability rate were investigated. MCs with 1% ECM and 2% CS show appropriate characteristics in terms of pore size, density, porosity, and mechanical properties. MTT cytotoxicity assays performed on chondrocyte cells cultured on ECM/CS MCs with various amounts of ECM showed that the sample with 1% ECM content had the greatest cell attachment. Overall, it can be concluded that the prepared MCs with 1% ECM could be a potential candidate to be used in cartilage tissue engineering applications.

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Design and fabrication of injectable microcarriers
composed of acellular cartilage matrix and
chitosan
Farzane Sivandzade & Shohreh Mashayekhan
To cite this article: Farzane Sivandzade & Shohreh Mashayekhan (2018) Design and fabrication
of injectable microcarriers composed of acellular cartilage matrix and chitosan, Journal of
Biomaterials Science, Polymer Edition, 29:6, 683-700, DOI: 10.1080/09205063.2018.1433422
To link to this article: https://doi.org/10.1080/09205063.2018.1433422
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JOURNAL OF BIOMATERIALS SCIENCE, POLYMER EDITION, 2018
VOL. 29, NO. 6, 683700
https://doi.org/10.1080/09205063.2018.1433422
Design and fabrication of injectable microcarriers composed
of acellular cartilage matrix and chitosan
FarzaneSivandzade and ShohrehMashayekhan
Department of Chemical and Petroleum Engineering, Sharif University of Technology, Tehran, Iran
ABSTRACT
Cartilage is an avascular tissue with limited self-repair ability. Since the
methods for treatment of cartilage defects have not been eective,
new therapies based on tissue engineering are considered over the
recent years. In this study, human cartilage tissue was decellularized
and porous injectable microcarriers (MCs) composed of acellular
extracellular matrix (ECM) of cartilage tissue and chitosan (CS), with
dierent ECM weight ratios, were fabricated by electrospraying
technique to be used in the treatment of articular cartilage defects.
Various properties of ECM/CS MCs such as microstructure, mechanical
strength, water uptake behaviour, and biodegradability rate were
investigated. MCs with 1% ECM and 2% CS show appropriate
characteristics in terms of pore size, density, porosity, and mechanical
properties. MTT cytotoxicity assays performed on chondrocyte cells
cultured on ECM/CS MCs with various amounts of ECM showed that
the sample with 1% ECM content had the greatest cell attachment.
Overall, it can be concluded that the prepared MCs with 1% ECM could
be a potential candidate to be used in cartilage tissue engineering
applications.
1. Introduction
e limited self-repair capacity of cartilage has posed signicant clinical challenges to the
elds of orthopedic surgery. Most of cartilage defects require the replacement or regenera-
tion of damaged articular cartilage [1]. Although dierent methods of treatments, such as
mosaicplasty, autologous chondrocyte implantation (ACI), osteochondral allogra trans-
plantation, microfracture, and prosthetic joint replacement, have been devised to repair car-
tilage defects over recent decades, there are still several limitations in producing long-lasting
cartilage with full biological activity [2,3]. Cartilage tissue engineering remains a promising
approach to repair cartilage defects in a functional manner [4]. For such applications, pri-
mary chondrocytes are combined with various scaolds designed with specic architectures
and made of dierent biomaterials, such as natural and synthetic polymers [5–8].
KEYWORDS
Cartilage tissue engineering;
extracellular matrix; chitosan;
microcarriers
ARTICLE HISTORY
Received 27 November 2017
Accepted24 January 2018
© 2018 Informa UK Limited, trading as Taylor & Francis Group
CONTACT Shohreh Mashayekhan mashayekhan@sharif.edu

684 F. SIVANDZADE AND S. MASHAYEKHAN
Recent studies have indicated that decellularized extracellular matrix (ECM) derived
from various tissues can be used to provide bioactive scaolds. Henrique et al. studied a
novel scaold derived from cartilaginous ECM that was used as a growth factor delivery
system to promote chondrogenesis of stem cells in articular cartilage regeneration [9]. In
another study, Cheng et al. investigated the cell growth, matrix accumulation and mechan-
ical properties of neocartilage formed by porcine articular chondrocytes on a porcine car-
tilage-derived matrix for using in cartilage tissue engineering [10]. Kang et al. studied
human cartilage ECM-derived 3D porous acellular scaold using autologous adipose-de-
rived mesenchymal stem cells (MSCs) for cartilage defect repair in in vivo cartilage tissue
engineering [11]. Yang et al. developed a cartilage ECM-derived porous scaold which
showed suitable biocompatibility and 3-D interconnected structure in both in vitro and
in vivo studies. eir results demonstrated appropriate cell repopulation and neocartilage
formation for ECM-derived scaolds [7]. It would be advantageous for a bioactive scaold
to support chondrocyte activity both in vitro and in vivo. is accelerates tissue growth and
improves associated functional biomechanical properties of the engineered construct [12].
Such tissue-derived scaolds may provide a natural microenvironment for cell migration
and dierentiation to promote cartilage tissue formation [13].
Chitosan (CS) is a deacetylated derivative of chitin, the second most abundant polysac-
charide in nature [14,15]. Because of its attractive properties including low immunogenicity,
antibacterial activity, biocompatibility, biodegradability and low cost, CS is recommended
as a suitable material among the applied natural polymers used in diverse biomedical appli-
cations such as tissue engineering, wound healing and drug delivery [14,16–18]. Dierent
blends of synthetic and natural polymer composed of CS have been investigated and showed
promising results both in vitro and in vivo studies in cartilage tissue engineering [19,20].
As for the scaold structure, microcarriers (MCs) have been recently used in several
approaches successfully to expand, redierentiate, and dierentiate chondrocytes because
of their ability to ll irregularly shaped defects by minimally invasive procedures [21].
Schrobback et al. investigated the proliferation and subsequent chondrogenic dierenti-
ation of freshly isolated adult human articular chondrocytes on porous gelatin MCs and
compared them to those expanded using traditional monolayers [22]. Hong et al. fabricated
an injectable scaold by combining collagen-coated polylactide (PLA) MCs and crosslink-
able CS hydrogel and demonstrated the potential application of the composite scaold
in orthopaedics [23]. Georgi et al. tested the use of MSC or chondrocyte laden MCs as
building blocks for cartilage tissue engineering. It led to a compact and ecient macrotis-
sue formation for engineered cartilage tissue [24]. Liao et al. developed a new injectable
three-dimensional alginate hydrogel loaded with biodegradable porous microspheres as a
promising approach for cartilage tissue engineering [21]. In fact, cell expansion on MCs
is a new culture method for chondrocytes leading to the enhancement of proliferation
as well as the conservation of chondrogenic phenotype. Moreover, the cells expanded on
MCs can be used as building units for cartilage tissue engineering constructs [24]. While
recent studies have either focused on non-ECM-derived MCs for engineered cartilage tissue
[23,24]or aimed at fabricating macro-scale cartilage ECM-derived scaolds [14], no study
has been carried out to fabricate and evaluate ECM-derived MCs in the eld of cartilage
tissue engineering. is is despite the fact that the application of such MCs is probably more
promising in cartilage regeneration due to both beneting from various aspects of MCs
potential in tissue engineering reviewed elsewhere [22–25]. Moreover, ECM incorporation

JOURNAL OF BIOMATERIALS SCIENCE, POLYMER EDITION 685
provides informational signals and certain unique composition, which mimick the natural
tissue environment. is, consequently, leads to proper tissue regeneration [7,11].
In our previous study, a hybrid macro-scale CS/ECM scaold, which showed desirable
properties with promissing potentials as a biomimetic matrix for articular cartilage tissue
engineering, was proposed [14]. e aim of this study was to fabricate porous MCs com-
posed of articular cartilage ECM and CS using ionic electrospraying technique. en, their
characteristics were evaluated in terms of physico-mechanical properties, in vitro enzymatic
biodegradation, and biocompatibility.
2. Materials and methods
2.1. Decellularization of articular cartilage for matrix preparation
e decellularization method was performed according to a previous report [14]. In sum-
mary, articular cartilage was obtained from adult human knee cartilage (Baqiyatallah
Hospital, Tehran, Iran). e samples were cut to 2×2mm slices, rinsed with phosphate
buer saline) PBS) (Sigma–Aldrich) immediately, and transferred to a freezer at −20°C
prior to use. Human articular cartilage pieces were rinsed with PBS, pH 7.4. en, they were
lyophilized for 24h at −50°C, 0.6mbar. e samples were then ground into ne powder for
40min with Mixer/Mill device. e cartilage powder was agitated in 0.25% (w/v) trypsin/
EDTA (Gibco) at 37°C for 24h and the solution was changed every 8h. Having rinsed
the samples with PBS for 30min, they were treated with 10mM hypotinic tris(hydroxym-
ethyl)aminomethane hydrochloride (Tris-HCl) (Sigma–Aldrich) solution for 24h followed
by an incubation in Triton X-100 (1% v/v in PBS) (Dae Jung) for another 24h. Finally, the
samples were rinsed in PBS for 24h and the PBS was changed every 8h. e decellularized
cartilage powder was stored at -20°C prior to use. All the solutions contained 1% penicillin/
streptomycin (Sigma–Aldrich) during the decellularization process.
2.2. Preparation of porous ECM/CSMCs
Various hybrid ECM/CS MCs were fabricated with the same diameter (400±50μm) using
CS (medium molecular weight chitosan powder with Mw= 280 kDa (Sigma–Aldrich))
and ECM solution as presented in Table 1. ECM solution was prepared by enzymatic
digestion according to our previous report [14]. In summary ECM powder was briey
digested in pepsin solution (1mg/ml in 0.1M HCl). In order to get a homogeneous solu-
tion, the suspension went under constant stirring for about 60h at room temperature to
complete the digestion process. CS solution (4% w/v acetic acid (Merck)) was prepared
by dissolving CS in 2% (v/v) acetic acid and stirring overnight. en, solubilized ECM
and CS were mixed in certain quantities along with the carbodiimide solution containing
40 mM1-ethyl-3-[3 dimethylaminopropyl] carbodiimide (EDC) (Sigma–Aldrich), 20mM
Table 1.Preparation data of ECM/CS MCs.
Sample C2E0 C2E0.5 C2E1 C2E1.5
CS solution in 2% acetic acid (w/v) 2% 4% 4% 4%
ECM solution in pepsin (w/v) 0% 1% 2% 3%
Final solution for making MCs 2% CS, 0% ECM 2% CS, 0.5% ECM 2% CS, 1% ECM 2% CS, 1.5%ECM

686 F. SIVANDZADE AND S. MASHAYEKHAN
(N-hydroxysuccinimide) (NHS) (Sigma–Aldrich), and 50mM 2-[N-morpholine] ehanesul-
fonic acid (MES) (Sigma–Aldrich) followed by stirring for 20h to reach a homogenous
mixture. e homogeneous solution was dropped into a crosslinker solution containing 3%
w/v sodium tripolyphosphate) TPP ((Sigma–Aldrich) through 16-gauge needle driven by
a syringe pump under a high voltage electrostatic eld to produce spherical shaped MCs.
e needle was electrically connected to the positive electrode of a high voltage electrostatic
generator, the negative electrode of which was electrically connected to an annular stainless
steel plate xed under the coagulation solution (Figure 1). e optimized potential between
the needle and the stainless steel plate and the pumping rate was obtained between 7 and
11 kV and 300–700 μl/min, respectively. e fabricated MCs were frozen overnight at
−20°C, followed by lyophilisation for 24h. en, the MCs were immersed into 1N NaOH
solution for 30min, rinsed by deionized water, and lyophilized again for 24h.
2.3. Dynamic cell culturing
Human primary chondrocyte cells were derived from the adult human articular cartilage.
Isolation of the cells was carried out as per the instructions by Eslaminejad et al. [26].
Chondrocytes were cultured in dulbecco’s modied eagle’s medium (DMEM) (Sigma–
Aldrich) supplemented with 10% fetal bovine serum (FBS) (Sigma–Aldrich). Cells were
maintained in a humidied CO
2
incubator at 37°C until reaching 90% conuence and were
fed by fresh medium every two days. e prepared porous MCs were sterilized using ethanol
(70%) and rinsed three times with PBS solution for 10min. en, they were incubated with
Figure 1.Schematic illustration of ECM/CS MCs fabrication by electrospraying technique.

JOURNAL OF BIOMATERIALS SCIENCE, POLYMER EDITION 687
DMEM for 24h at 37°C and 5% CO2. A 10mg mass of the sterilized MCs was immersed
in 1ml DMEM and added to a 24-well plate. e cell suspension with a concentration of
2×105 cells/cm2was also added to the plate. e cell-seeded MCs were incubated for 3h at
37°C and 5% CO2 [24]. e plate was then removed and the culture medium was replaced
with a fresh one. e plate was placed on a mini-rocker at 15rpm to facilitate the cell
expansion. Aerwards, it was incubated again at 37°C and 5% CO2 for a specied period
of time. e culture medium was replaced daily with a fresh one.
2.4. Characterization
2.4.1. Evaluation of the decellularization process
Parts of the cartilage samples (native and decellularized) were xed for 24h in 10% neutral
buered formalin solution in PBS (pH 7.4) at room temperature. Subsequently, the sam-
ples were rinsed anddehydrated in graded series of ethanol. en, they were embedded in
paran and sectioned with a thickness of 5μm using microtome. Hematoxylin and eosin
(H&E) staining was performed aer deparanization by xylene and rehydrationto validate
the chondrocyte’s removal from the decellularized cartilage tissue [7,24]. Moreover, total
DNA content was quantied in samples of native and decellularized tissue according tothe
protocol proposed by Rajabi-Zeleti et al. [27]. e amount of DNA was expressed in ng/
mg dry weight of samples.
2.4.2. Fourier transform infrared spectroscopy
e Fourier transform infrared spectroscopy (FTIR) spectra were recorded using a Shimadzu
FTIR spectrometer. All spectra were obtained from 400 to 4000cm−1 at a 4cm−1 resolution
at room temperature.
2.4.3. Morphological characterization
e morphology of porous MCs were investigated by scanning electron microscopy (SEM,
Seron Technology, AIS-2100, Korea). e porous MCs were coated with gold before obser-
vation. Image J analysis soware was used to determine the pore sizes. ree planned
locations were selected for three samples of MCs and a total of 40 measurements were
recorded at each location.
2.4.4. Porosity and density
To obtain the porosity (P) and density (ρs) of MCs, the total volume of MCs (Vs) and pore
volume (Vp) were determined. First, 20 MCs were randomly weighed (W0) and immersed
in ethanol (density ρ). en, the samples were dried in vacuum for 20 min and the excess
ethanol on the surface was removed with blotting paper. en the MCs were immediately
re-weighed (We). Porosity and density were calculated as follows:
(1)
P
=Vp
/
V
s
×
100
(2)
Vp
=
(
We−W0
)/𝜌

688 F. SIVANDZADE AND S. MASHAYEKHAN
2.4.5. Mechanical properties
Mechanical strength: In order to investigate the mechanical strength of MCs, 100 random
MCs were added to 40mL of water and stirred for 30min (400rpm). en, fragmented
MCs were separated and the residual (unfragmented) MCs were recounted. e mechanical
strength was estimated by noting the percentage of residual MCs [28].
Young’s modulus: e attened samples, which had the same concentrations as prepared
MCs, were generated. In summary, ECM powder was digested in pepsin solution (0.1mg/
100ml in 0.1N HCl) for 60h at 37°C. CS (4% w/v acetic acid) was prepared by dissolving
CS in 2% (v/v) acetic acid and stirring overnight. en, solubilised ECM and CS were
mixed in certain quantities along with carbodiimide solution (40mM EDC, 20mM NHS
and 50mM MES) and stirred for 20h to reach a homogenous mixture. Aerwards, 1ml of
the prepared mixture was poured to each chamber of the 24-well plates. en, they were
frozen overnight at -20°C and lyophilized for 24h. e dried porous scaolds were then
immersed into 1N NaOH solution for 30min, smoothly rinsed by deionized water and
lyophilized again for 24h. To measure the compressive properties, the lyophilized scaolds
were immersed in PBS for 1h at room temperature. en, the mechanical properties of
the scaolds were measured by compressing the hydrated scaolds up to 70% strain using
Hounseld H10KS mechanical tester with a load cell of 1 kN. e loading rate was 0.5mm/
min. e Young’s modulus was determined from the slope of the initial linear section of
stress-strain diagram.
2.4.6. Water Uptake behaviour and in vitro enzymatic biodegradation test
To assess water uptake ratio, 100 MCs of each samples were dried in a vacuum until a con-
stant weight was reached (W0) and then immersed in PBS for 3days at 37°C for pre-dened
time intervals (15, 30min, 1, 2, 4, 6, 8, 24, 48, and 72h). Having removed the excess PBS
on the surface with blotting paper, the MCs were removed from the PBS and weighed (Ww)
immediately. e water uptake ratio (WR) was dened as follows:
e in vitro enzymatic biodegradation test was examined by exposing the MCs in a PBS
solution containing 1.5 μg/ml lysozyme from chicken egg white (Sigma–Aldrich) at 37°C
for various periods up to 4weeks. In summary, the dried MCs (W0) were weighed and
incubated at 37°C in the enzymatic solution. Aerwards, the sample was weighed (Wd)
followed by drying at 60°C for 24h [17]. e extent of in vitro biodegradation rate (BR)
was dened as follows:
(3)
𝜌
s=
W
0
/
V
s
(4)
WR
(%)=
W
w
−W
0
W
0
×
100
(5)
BR
(%)=
W
0
−W
d
W
0
×
100

JOURNAL OF BIOMATERIALS SCIENCE, POLYMER EDITION 689
2.4.7. In vitro cell culture studies
In order to determine the viability and proliferation of the cells cultured on dierent MCs,
3-(4,5-dimethylthiazol-2-yl)-2,5 diphenyltetrazolium bromide) MTT (assay was performed
on the1st, 3rd, 5th, and 7th days of culture. Briey, aer a specied period of incubation of
the cells on MCs, the culture medium in each well were replaced with a 10 μl MTT solution
(1mg/mL) with 400 μl Roswell Park Memorial Institute (RPMI) 1640 medium (Sigma–
Aldrich). Aer a 4h incubation at 37°C with5% of CO2, the medium in all wells were
replaced with 14.95ml isopropyl alcohol and 50 μl of HCl and incubated again for 30min
to ensure solubilizing of crystals. Absorbance was determined using a spectrophotometer
(BioPhotometer, Eppendorf, Germany) at 570nm [17].
2.4.8. Statistical analysis
Each experiment was replicated three times. Values were presented as the means of rep-
licates±standard error of the mean. Mean values were compared by one-way analysis of
variance (ANOVA) using SPSS.18 statistical soware package. P values less than 0.01 were
considered statistically signicant.
3. Results
3.1. Evaluation of decellularization process
Histological ndings from hematoxylin and eosinstaining conrmed that the decellulari-
zation process was successful as there were no visible cell nuclei, as compared to the intact
cartilage tissue (Figure 2(A)). Moreover, DNA quantication showed complete removal of
DNA following mechanical and chemical treatments with signicant dierences in DNA
content observed between native and decellularized tissue (Figure 2(B)).
3.2. FTIR spectroscopy
Figure 3 illustrates the results of the FTIR spectra of ECM, CS, and cross-linked C2E1 MCs.
e FTIR spectrum of CS shows characteristic absorption peaks at 3437, 1811, 1558,1471,
1400, 1205, 1076 and 856cm−1. e broad absorption peak at 3437and 3712cm−1 are
attributed to the amino (–NH2) and hydroxyl (–OH) bond, respectively. e peaks at 1076
and 859cm−1correspond to the saccharide structure of CS [29]. e characteristic peak at
1558cm−1 represents the amide I absorption band, while those at 1400 and 1205cm−1rep-
resent the amide III absorption band, which suggests CS is a partially deacetylated product
[17,30]. e ECM displays absorption peaks at 1680, 1550, and 1290cm−1, which repre-
sent the absorption bands of amide I (C=O bond), amide II (N–H and C–N bonds) and
amide III (C–N bond), respectively. Moreover, the ECM shows the presence of carboxyl
and amine (–NH2) groups at 3420cm−1. e comparison between the graphs of CS and
C2E1 MCs demonstrated signicant reduction in intensity of N-H peaks. is might be
due to the reaction between amino groups in CS and carboxyl group in ECM during the
cross-linking process by EDC/NHS. e intermolecular interaction between CS and ECM
is also conrmed by the peak between 1630 and 1690cm−1that indicates the stretching

690 F. SIVANDZADE AND S. MASHAYEKHAN
band of C=O in amide group. Moreover, the absorption peaks of PO4
−3 in the region of
900–500cm−1 shows the existence of TPP, the ionic cross-linker in the prepared MCs [31].
3.3. Characterization of the prepared MCs in terms of physical and mechanical
properties
In this study, various hybrid ECM/CS MCs were fabricated with the same diameter
(400±50μm) using CS and ECM solution (Table 1). It is noteworthy that the MCs con-
taining more than 2% CS did not show spherical shape. MCs for tissue engineering should
have a highly porous structure to provide enough space for cell attachment and prolifera-
tion [32]. e eect of ECM on the pore parameters of the MCs are shown in Figure 4 and
the results are summarized in Table 2. Figure 4 indicates good porosity and pore-to-pore
interconnectivity within the MCs’ structure. e mean pore size of the MCs decreased
with the increase in ECM content. However, C2E1.5 MCs show the largest pore size and
non-uniform structure. is might be attributed to inecient mixing of the polymers due
to high ECM concentration. Moreover, the mechanical behaviour of the prepared MCs was
investigated in the equilibrium swelling state and the results are summarized in Table 2. By
adding ECM (from 0 to 1.5 wt%), Young’s modulus and mechanical strength decreased. As
shown in Table 2, by an increase in ECM, density of the fabricated MCs decreased, while
porosity increased.
3.4. Water uptake behaviour and in vitro biodegradation test
Water uptake behaviour is one of the most important properties of the scaold that depends
on pore size and interconnectivity between the pores to provide nutrients ow for the cells
[33,34]. e results of the water uptake ratio of the prepared MCs are illustrated in Figure 5.
e weight of all the MCs increased in the rst hours of imersion. Aer 8 h, all the samples
reached equilibrium. C2E1 MCs showed the highest water uptake ratio, compared with
other groups. C2E1.5 MCs showed lower water uptake ratio than other hybrid MCs. is
might be attributed to non-uniform porosity and lost structure.
As the eld of tissue engineering aims to regenerate damaged tissues, the scaold deg-
radation rate should match the new tissue formation rate to allow optimal regeneration.
e results of the in vitro biodegradation rate of the prepared MCs are illustrated in Figure
6. C2E1 MCs showed the highest degradation rate in comparison with the other groups.
ey reached their maximum weight loss of about 55% aer 28days of incubation. As
shown in Figure 6, the biodegradation rate increased with the increase in ECM content.
is might be due to higher porosity, better accessibility of cleavage sites by the enzymes,
or the increased hydrophilicity of the MCs caused by incorporation of higher ECM content.
However, C2E1.5 MCs showed lower biodegradation rate compared with the other hybrid
MCs. is might be attributed to non-uniform porosity and lost structure. Since C2E1.5
MCs demonstrated inappropriate mechanical and structural properties, in the next step,
other groups were examined for the biocompatibility analysis.

JOURNAL OF BIOMATERIALS SCIENCE, POLYMER EDITION 691
3.5. Cell proliferation and cytotoxicity analysis
e biocompatibility of the ECM/CSMCs was conrmed using primary chondrocyte cells
derived from human articular cartilage (Figure 7). e number of attached cells increased
over time during 7days on all MCs due to the high porosities and presence of ECM. e
number of cells on C2E1 MCs increased over time during 7-day culture more than the
other groups. Although the number of attached cells on 2D tissue cell plate (TCP) was
higher than those on all other groups of MCs until day5, it showed a decrease in attached
cells on day7due tothe limited area for cell attachment. In contrast, the MCs provided 3D
porous structure enhancing the overall surface area for cell adhesion and proliferation. By
comparing results of dynamic and static culture (Figure 7), it can be observed that dynamic
culture has a signicant inuence on the growth and proliferation of the attached cells. e
attached cells on CS/ECM MCs aer the 1st day of culture was examined by SEM which
further conrms that the fabricated MCs are nontoxic and biocompatible (data not shown).
4. Discussion
e fundamental concepts guiding the development of tissue-engineered scaolds depend
on the selected biomaterial or the method of scaold production [35]. New trends in tis-
sue engineering have focused on scaold materials mimicking the structure, morphology,
and biological cues of the natural environment [36]. Although various natural and syn-
thetic materials have been explored recently for cartilage tissue engineering, they have not
been considerably capable of mimicking the native ECM environment [37–41]. Herein, we
fabricated porous MCs composed of cartilage ECM and CS designed for cartilage tissue
engineering due to the high potential of biomimeticallyenhanced decellularized cartilage
tissue as a chondroinductive material for cartilage regeneration [7,9,10]. Cartilage ECM is
composed of type II collagen and an interlocking mesh of brous proteins, proteoglycans,
hyaluronic acid, and chondroitin sulfate, which provides cell retention, migration, prolifer-
ation and dierentiation [42]. In fact, the cartilage morphogenetic proteins retained in the
ECM, could serve as a potential source of endogenous biologically active molecules that can
promote cartilage formation [10]. In this study, we demonstrated the complete removal of
cellular contents through a decellularization protocol using SDS, trypsin, and triton-100X
[14,27]. e successful decellularization process was conrmed by H&E staining and DNA
quantication analysis (Figure 2). EDC and NHS were used as cross-linking agents to
Figure 2.(A) H&E staining of cartilage tissue. Cells and empty lacunasin native and decellularized tissue,
respectively are shown by arrows (×20 mag), (B) DNA quantification in native and decellularized tissue.

692 F. SIVANDZADE AND S. MASHAYEKHAN
form amide bonds between the primary amino group of CS and a carboxylic group of
ECM existing in collagen II [43] and glycosaminoglycan (GAG) [44] as recently described
elsewhere [14,45,46]. In order to generate an interconnected structure, hybrid MCs made
of decellularized cartilage matrix and CS were fabricated using an ionic electrospraying
technique. All the prepared MCs have a spherical shape providing a high contact surface for
cell attachment, hence increasing cell growth rate [47]. We have recently reported to apply
the same technique to fabricate porous gelatin-chitosan MCs for MSCs proliferation [48].
e chemical composition of ECM/CS MCs was characterized by FTIR analysis and
compared with its components, which include CS and ECM (Figure 4). e FTIR analysis of
MCs showed a signicant reduction in the free amino group (–NH
2
) with broad absorption
Table 2.Characteristics of the prepared porous MCs.
Sample
The average pore
sizes (μm) Porosity(%) Density (g/cm3)
Mechanical
strength(%)
Young’s modulus
(kPa)
C2E0 85±10 77%±4 1.19±0.01 90%±5 140±10
C2E0.5 50±5 81%±3 1.12±0.02 93%±4 150±10
C2E1 40±5 87%±3 1.06±0.04 84%±3 50±5
C2E1.5 150±15 91%±2 1.02±0.01 81%±3 10±1
Figure 3.FTIR spectra of the samples. (a) ECM, (b) C2E1 MCs, (c) CS.
Figure 4.SEM images of the MCs. (a) C2E0, (b) C2E0.5, (c) C2E1, (d) C2E1.5.

JOURNAL OF BIOMATERIALS SCIENCE, POLYMER EDITION 693
peaks at 3437cm
−1
and 3712cm
−1
, compared with CS. is might be due to the crosslinking
of (–NH
2
) groups of CS with the (–COOH) groups of ECM during the MCs formation. e
intermolecular interaction between CS and ECM is also conrmed by the peak between 1630
and 1690cm−1. is indicates the stretching band of C=O in the amide group. Moreover,
the results of the ECM and cross-linked samples are highly similar. erefore, it can be
concluded that the chemical reaction between the polymers does not disturb the original
ECM structure aer cross-linking.
Mean pore size and its distribution, pore interconnectivity, pore shape, and pore wall
roughness are important parameters that should be considered for designing appropriate
MCs [49]. In other words, the scaold must possess a highly porous structure with an open,
Figure 5.The result of water uptake behaviour test of the MCs.
Figure 6.The result of in vitro biodegradation test of the MCs.

694 F. SIVANDZADE AND S. MASHAYEKHAN
fully interconnected geometry which provides a large surface area allowing cell in growth
and uniform cell distribution. It should also facilitate the neovascularization of the con-
struct [50]. Moreover, the porous architecture can protect cells grown inside the pore from
physical damage exerted by the shear stress that occurs during cell culture or injection [51].
Hence, the pore size can play an important role in the biological function of MCs [10,52].
Moreover, it was discovered that water content, evaporation rate, and evaporation time of
the solvent considerably aect the pore parameters of the MCs [33,53,54]. Hence, all the
prepared MCs were kept in PBS solution for 48h to reach the equilibrium swelling state,
then freeze-dried at the same evaporation rate and time. e only variable is this process
was the amount of ECM. As Figure 4 shows, the SEM images of the fabricated MCs indi-
cated desirable porosity and pore-to-pore interconnectivity. e mean pore size of the MCs
decreased with the increase in ECM content. is can be due to an increase in the amide
band between (–NH2) groups of CS and (–COOH) groups of ECM existing in collagen II
[43] and glycosaminoglycan (GAG) [44]. Similarly, Vrana et al. indicated that amide band
between (–NH2) groups of CS and (–COOH) groups of collagen causes a reduction in the
pore size of collagen-based scaolds [55].
In this study, the eect of ECM content on MCs density and porosity was investigated.
Corresponding results are presented in Table 2. e density and porosity of the prepared
MCs decreased and increased with the increase in the ECM content, respectively. is can
be due to the stronger intermolecular interactions between molecules in hybrid MCs [55,56].
Scaolds with high porosity provide a high surface area for cell-matrix interactions and act
as a temporary template for the new tissue’s growth and reorganization. is, consequently,
increases cell adhesion, sucient space for ECM regeneration, and uniform and ecient
cell seeding [48]. e ideal porosity varies for dierent cells and tissues for a more ecient
cell/tissue in growth and regeneration [57]. In previous studies, the porosity percentage
was reported 70–92% in cartilage tissue engineering [49,58]. As illustrated in Table.2, the
porosity values of all fabricated MCs are in the range of 77–91%, which highly comply with
Figure 7.The results of the MTT assay for the cells cultured on the prepared MCs in dynamic culture(*
p<0.01 statistically significant).

JOURNAL OF BIOMATERIALS SCIENCE, POLYMER EDITION 695
the reported range. Moreover, it was postulated that in dynamic cell culturing, the MCs
density is an important factor. It is due to the fact that a buoyant density of between 1.03
and 1.10g/cm3 allows MCs suspension with slow agitation [25]. At a density lower than
1g/cm3, the MCs would be oating in the culture medium and the cell attachment would
not be uniform.
Mechanical stability of the MCs are of the most crucial properties for successful gra
uptake by the host tissues. Besides, the MCs must be compatible with and harmless to the
surrounding tissues. ey should be also capable of resisting structural collapse during in
vitro culture and in vivo implantation, which may cause necrosis and inammation [59]. e
eect of ECM content on the MCs mechanical properties was investigated since the main
purpose of the prepared MCs is to provide mechanical and structural support for attach-
ment and proliferation of the cells in a 3D environment which mimicks the native cartilage.
All the prepared MCs showed the ability to reabsorb water and retain their original shape
aer removing the mechanical load. is is an essential feature for engineered tissue since
it makes it capable of tolerating the pressures during physiological high repetitive loading
[60]. Both the mechanical strength and Young’s moduli of the prepared MCs decreased with
the increase in ECM content (Table 2). As anticipated, CS has a tightening eect. In fact,
as CS to ECM ratio increases, the mechanical strength and Young’s moduli of the prepared
MCs improves. Another study by Correia et al. showed a similar eect of hyaluronic acid
on the mechanical properties of the fabricated scaolds which decreased with increasing
hyaluronic acid content [61]. It should be noted that the biomechanical properties of MCs
would increase dramatically following in vivo implantation. It is due to cellular microen-
vironment changes and ECM secretion by chondrocytes in tissue-engineered device [37].
Overall, C2E0.5 and C2E1 MCs show appropriate characteristics in terms of pore size,
density, porosity, and mechanical properties.
Fluid absorption and exchange of nutrients and waste are critical features of scaolds
in tissue engineering applications [48,62]. e water uptake ratio depends on both the
scaold’s hydrophilic nature and microstructure [63]. As Figure 5 shows, the ratio of CS to
ECM considerably aected the MCs’ water uptake ratio andC2E1 MCs showed the highest
water uptake ratio. is might be due to an increase in hydrophilicity of the MCs caused
by incorporation of higher ECM content. In a similar study, Correia et al. showed the
eect of the collagen and hyaluronic acid presence in scaolds. e eects were similar to
those of ECM presence, leading to a signicant increase in absorption of water due to high
hydrophilicity of collagen and hyaluronic acid [61]. Although C2E1.5 contained a higher
amount of ECM in its component, it showed less PBS absorption, compared to C2E1 and
C2E0.5. In homogeneity of the polymeric solution caused poor mechanical properties and a
collapsed pore network which prevented retention of PBS in the MCs. erefore, it showed
a lower water uptake ratio in comparison with the other hybrid samples.
Scaolds need to provide an appropriate condition for the cells to attach, proliferate,
and synthesize their own matrix molecules. Upon forming new ECM by the cells during in
vivo tissue regeneration the scaold should degrade or be absorbed by the body gradually.
erefore, to have an optimal regeneration, the rate of degradation should keep up with
the speed of new tissue formation [64]. e rst four weeks following graing is a critical
period for cartilage regeneration [10]. erefore, the in vitro biodegradation of the prepared
MCs in PBS/lysozyme solution during four weeks was evaluated and the corresponding
results are presented in Figure 6. It can be observed that C2E1 MCs showed the highest

696 F. SIVANDZADE AND S. MASHAYEKHAN
rate of degradation. is might be due to increased hydrophilicity caused by the incorpo-
ration of greater ECM content, and therefore caused substantial degradation of the MCs
as it enhances the interaction of the biomaterial with the enzymatic solution. e increase
in the biodegradation rate of chitosan by adding hydrophilic material, such as gelatin [37]
and hyaluronic acid [61], was shown in previous studies. Correia et al. reported the same
eect for adding hyaluronic acid to CS on biodegradability as that of adding ECM to CS.
ey also indicated that the scaolds containing more hyaluronic acid show a higher rate
of biodegradation compared to other samples due to their higher porosity, accessibility
of cleavage sites by the enzymes, and the biodegradable nature of hyaluronic acid [61].
Although the ECM content of C2E1.5 was higher than other hybrid MCs, the percentage
of weight loss was not higher due to reduced uid uptake and lost structure leading to less
accessibility for enzymatic solution, hence less biodegradation.
Previous studies regarding primary chondrocyte cultures performed in monolayer and
pellet culture may have limitations in their applications while applying these studies to func-
tional tissue engineering in a human clinical setting [65,66]. Despite the numerous studies
on primary chondrocytes seeded on 3D scaolds [10,14,38], they may be less applicable
compared to MCs due to the fact that they require a surgery process to be transplanted in a
human body. us in the present study, the possibility of using MCs for primary chondrocyte
cultivation derived from human articular cartilage was investigated [10]. Despite the limited
access to primary chondrocytes and diculties of the isolation process, they are applicable
in tissue engineering and regenerative medicine due to their potential to avoid allogenic
rejection following transplantation [67] Herein, the ability of porous MCs to support the
cell growth without inducing cytotoxicity was evaluated using the MTT proliferation assay
during 7days of culture and the results are presented in Figure 7. As illustrated, all the MCs
showed a rising trend in all time intervals, meaning that MCs supported the attachment
and proliferation of chondrocytes. A signicant increase in the number of attached cells
during culturing on the MCs, compared to TCP, conrmed the unique properties of MCs,
such as higher porosity. e greatest attached cell number was that of C2E1 MCs during the
7-day dynamic culture. Similarly, Chang et al. observed that an increase in the trend of cell
attachment for a 3D porous scaold composed of polycaprolactone improved by collagen
for cartilage tissue engineering. is is due to the increase in the amount of collagen as a
biological material providing appropriate cell binding site and environmental cues to the
cells [39]. O’Brien et al. showed that pore size has a signicant eect on mechanical and
structural properties, and cell adhesion in collagen-GAG scaolds since pore size should
be large enough to help cell migration and small enough to provide sucient surface [68].
e collapsed oversized pore structure of C2E1.5 led to smaller adhesion area and, hence,
less cell viability, compared to other hybrid MCs. erefore, it seems that the biocompatible
properties of the ECM not only play a pivotal role in mimicking the native cartilage envi-
ronment for primary chondrocytes attachment and proliferation, but also in the incorpo-
ration of higher ECM content leading to favourable biochemical and structural properties
enhancing the biocompatible properties of MCs [33,48,69,70]. Hence, the proposed C2E1
MCs provide the most desirable biocompatible properties and support cell attachment and
proliferation in vitro.
While in our previous study, 2D macro-scale CS/ECM scaolds were fabricated, in this
study, porous MCs composed of the same materials were designed and fabricated in order
to provide injectable MCs for 3D cell cultivation in cartilage tissue engineering [14]. Overall,

JOURNAL OF BIOMATERIALS SCIENCE, POLYMER EDITION 697
the ndings of this study reveal that MCs composed of 1% ECM and 2% CS (C2E1 MCs)
can be considered as a potential candidate to be used in cartilage tissue engineering due
to their appropriate biocompatibility, biodegradability, and structural properties. Ongoing
studies focus on using fabricated porous, cell-laden MCs as building blocks for cartilage
tissue engineering.
5. Conclusion
In this study, we successfully designed and fabricated porous MCs composed of acellular
cartilage-derived ECM and CS due to their potential application in the regeneration of a
damaged cartilage following injury. MCs for cartilage regeneration must have key charac-
teristics, including appropriate mechanical strength, porous microstructure, and pore size
as well as controlled biodegradability and biocompatibility. Here, the eect of CS and ECM
content on various characteristics of the prepared MCs was investigated. Overall, based on
our observations, C2E1 MCs can be considered as a potential candidate to be used in car-
tilage tissue engineering applications due to appropriate biodegradation, suitable structural
properties, and favourable biocompatibility.
Acknowledgment
e authors are grateful to Dr Hasanzadeh, the head of Persian Orthopedic Trauma Association,
for providingthe required articular cartilage specimens during total knee operations. We are also
thankful to Mr Fallahfor isolating MSCs from the human articular cartilage specimens and Mr Liles
for help with the nal editing.
Disclosure statement
No potential conict of interest was reported by the authors.
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