Materials Science & Engineering C 118 (2021) 111312

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Materials Science & Engineering C

journal homepage: www.elsevier.com/locate/msec

Chondroitin sulfate modified 3D porous electrospun nanofiber scaffolds T

promote cartilage regeneration
⁎ ⁎
Shuai Chena,1, Weiming Chenb,1, Yini Chenc,1, Xiumei Mod, , Cunyi Fana,
a
Department of Orthopaedics, Shanghai Jiao Tong University Affiliated Sixth People's Hospital, 200233 Shanghai, PR China
b
Shanghai Ninth People's Hospital, Shanghai Jiao Tong University School of Medicine, 200000 Shanghai, PR China
c
Department of Ultrasound in Medicine, Shanghai Jiao Tong University Affiliated Sixth People's Hospital, 200233 Shanghai, PR China
d
College of Chemistry, Chemical Engineering and Biotechnology, Donghua University, 201620 Shanghai, PR China

A R T I C LE I N FO A B S T R A C T

Keywords: 3D electrospun nanofibrous scaffolds have been developed for cartilage regeneration, however, there is no
3D scaffolds consensus on the preferable method for biocompatible scaffolds that enhance regeneration and attenuate in-
Nanofibrous scaffolds flammation. We designed a 3D porous electrospun polylactic acid (PLA) @gelatin-based scaffold by a novel
Cartilage regeneration method. Chondroitin sulfate (CS), commonly used in clinical cartilage treatment, is capable of regulating car-
Anti-inflammation
tilage formation and inhibiting inflammation. Thus we further functionalized the 3D scaffold by crosslinking of
Chondroitin sulfate
CS, assuming that CS-functionalized scaffold (CSS) would promote cartilage regeneration and modulate in-
flammation. We confirmed that CSS exhibits not only appropriate reversible compressibility and mechanical
property, but also appropriate biocompatibility, allowing cell proliferation. In vitro, the potential of CSS for
chondrogenic differentiation was improved compared to control and PLA@gelatin scaffold as chondrogenic
markers Collagen2 and Aggrecan was significantly increased. Meanwhile, significant reduction in two crucial
inflammatory factors (NO and PGE2) in CSS group demonstrated inflammation inhibition. In vivo, rabbit car-
tilage defects were created and CSS effectively promoted cartilage repair. Additionally, superior anti-in-
flammation effect of CSS was demonstrated by reduction in iNOS and PGES, enzymes producing NO and PGE2,
respectively by immunohistology. Our results indicated the preferable property of CSS for cartilage regeneration
and its potential in immunoregulation.

1. Introduction engineering by multimodal electrospinning [13] Hsu et al. [14] and

Damaged cartilage has very limited self-repair capacity and usually
progresses to destruction of the surrounding cartilage. Much effort has
been put into the development of a better therapeutic strategy. Various
electrospun nanofibers have been developed for cartilage regeneration
due to their outstanding performances, including high porosity and
surface-to-volume ratio [1–8]. However, as two-dimensional (2D) bio-
materials, electrospun nanofibers are inappropriate for cartilage re-
generation because of poor mechanical property, limited thickness and
pore size. Three-dimensional (3D) electrospun nanofibers scaffolds
fabricated by a desirable method may provide a favorable micro-
environment for cell adhesion and growth [9–11], and overcome the
above obstacles.
Parker et al. constructed 3D nanofiber structures by rotary jet-
spinning [12]. Traversa et al. fabricated 3D scaffolds for soft tissue
Wang et al. [15] used 3D printing technology to fabricate scaffolds for
cartilage regeneration. Yu et al. [16] generated 3D braid scaffolds for
cartilage regeneration. Although various methods have been reported
to construct 3D electrospun nanofibrous scaffolds [12,13,17–19],
shortcomings still exist [17]. These methods are either time-consuming
or demanding a specific device, complicated technique or strict condi-
tion. Thus, developing a suitable method for 3D nanofibrous scaffold
fabrication, while challenging, is imperative. Recently, a novel, con-
venient strategy to construct 3D electrospun scaffold based on elec-
trospinning and freeze-drying procedures was reported [20–23]. The
resultant 3D scaffold presents desirable reversible compressibility and
mechanics, which are important for tissue regeneration [20–23]. Al-
though this novel method provides superelasticity and multi-
functionality, scaffolds fabricated from inferior biocompatible materials
are inappropriate for tissue regeneration [21,22].

⁎
Corresponding authors.
E-mail addresses: mxmdhdx@126.com (X. Mo), cyfan@sjtu.edu.cn (C. Fan).
1
These authors contributed equally to this work.

https://doi.org/10.1016/j.msec.2020.111312
Received 6 January 2020; Received in revised form 6 July 2020; Accepted 14 July 2020
Available online 04 August 2020
0928-4931/ © 2020 Elsevier B.V. All rights reserved.
S. Chen, et al.
The modification of synthetic scaffolds may enhance its capacity to
interact with cells [24]. Various factors and natural processes, such as
cell–extracellular matrix (ECM) interactions, ECM dynamics and cell–-
cell communication and positioning, and mechanical stimulation, pro-
vide a relatively stable microenvironment and direct cell activity [25].
Among these, ECM not only guides the tissue development but also
drives cell fate [25]. Chondroitin sulfate (CS), a member of glycosa-
minoglycan (GAG) family, is a major type of ECM primarily found in
cartilage, bone, and skin [26,27]. CS is capable of regulating cartilage
formation [28]. Elisseeff et al. [29] confirmed the chondrogenic func-
tion of CS-based bioactive hydrogels. Furthermore, CS presents anti-
inflammatory properties, as demonstrated by its clinical value in the
treatment of cartilage degenerative disease [30,31]. Tabata et al. [32]
also reported the anti-inflammatory effects of CS. Therefore, CS is ef-
fective in cartilage regeneration and inflammatory reduction [33,34].
In this study, we fabricated a 3D porous electrospun polylactic acid
(PLA)@gelatin-based scaffold functionalized by the crosslinking of CS
(CS scaffold, CSS). In addition, we fabricated a sole-PLA@gelatin scaf-
fold (SS) to serve as a control. In addition to evaluating the scaffolds in
terms of reversible compressibility and mechanical properties, we as-
sessed their potential for stimulating bone marrow stromal cell (BMSC)
proliferation and chondrogenic differentiation. The anti-inflammatory
effects of scaffolds were also examined. Furthermore, we evaluated the
effects of the scaffold for cartilage regeneration and immunoregulation
in a rabbit model.
2. Materials and methods
2.1. Materials and reagents
CS was purchased from J&K Scientific. Poly(L-lactic acid) (PLLA)
was kindly provided by Medprin Regenerative Medical Technologies
(Guangzhou, China). Type A gelatin was purchased from MP
Biomedicals. Dulbecco's Modified Eagle's Medium (DMEM), fetal bo-
vine serum (Gibco, Carlsbad, CA, USA), and trypsin (Gibco) were pur-
chased from Shanghai Chengpu Biotechnology N-[3-(dimethylamino)
propyl]-N′-ethylcarbodiimide hydrochloride (EDC) and N-hydro-
xysuccinimide (NHS) were purchased from J&K Scientific. The anti-
bodies used for immunohistochemistry were against the following
proteins: Collagen2 (COL2; Boster, Wuhan, China), Aggrecan (ACAN;
Boster), iNOS (BIOSS, Beijing, China), and PGES (Boster).
Chondrogenic medium was purchased from Cyagen Biosciences. All
other chemicals were of analytical grade and were used without further
purification.
2.2. Cell isolation and culture
Rabbit BMSCs were isolated and cultured as previously described
[35]. Rabbits were provided by the Shanghai Animal Experimental
Center, and all procedures were approved by the Animal Research
Committee of the Shanghai Jiao Tong University Affiliated Sixth Peo-
ple's Hospital. All experiments were conducted following the guidelines
of the Animal Research Committee of the Shanghai Jiao Tong Uni-
versity Affiliated Sixth People's Hospital. BMSCs were harvested from
the hind limbs of rabbits. Briefly, primary cells were cultured in DMEM
containing 10% fetal bovine serum and 100 U/mL penicillin. Un-
attached cells were removed after a 24-h culture, and the adherent cells
were further cultured. The medium was replaced every three days.
When reaching 90% confluence, BMSCs were released from the culture
substratum using trypsin/EDTA (0.25% w/v trypsin, 0.02% EDTA),
reseeded into dishes, and cultured at 37 °C in a 5% CO2 atmosphere.
2.3. Preparation of 3D electrospun nanofiber scaffolds
2.3.1. Preparation of coaxial electrospun nanofibers
Nanofiber membranes were fabricated by coaxial electrospinning
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Materials Science & Engineering C 118 (2021) 111312
(Fig. 1). Gelatin and PLA were all dissolved in 1,1,1,3,3,3-hexafluoro-2-
propanol (HFIP). The concentration of gelatin was 12% (w/v), while
that of PLA was 8%. The core solution (PLA) was injected at a flow rate
of 1 mL/h and the shell solution (gelatin) was injected at a rate of 5 mL/
h. The applied voltage was 13 kV. The obtained PLA@gelatin nanofiber
membranes were placed in vacuum for storage.
2.3.2. Preparation of 3D scaffolds
First, heat-treated scaffold was prepared in the following three steps
(Fig. 1): the electrospun nanofiber membranes were cut into pieces. The
pieces were dispersed in tert-butanol using a homogenizer. The revol-
ving speed was 8000–10,000 rpm for 10–30 min. Subsequently, the
dispersed nanofibers were poured into a cylindrical mold and frozen at
−20 °C for 30 min. The materials were freeze-dried for 24 h, followed
by heating at 180 °C for 2 h in air. Different procedures were used to
prepare different scaffolds. For SS, the heat-treated scaffold was im-
mersed in water for 2 h, frozen at −80 °C for 2 h, and freeze-dried for
48 h. For CSS, the heat-treated scaffold was crosslinked with 50 mL
solution containing 1% CS, 30 mM EDC, 8 mM NHS, and 50 mM MES
buffer for 24 h [36], washed with distilled water to remove free CS and
salt, frozen at 80 °C for 2 h, and freeze-dried for 48 h.
2.4. Characterization of scaffolds
2.4.1. Fourier Transform Infrared Spectroscopy (FTIR)
A Nicolet-670 FTIR spectrometer was used for attenuated total re-
flection (ATR)-FTIR of the scaffolds. All spectra were recorded over a
range from 1000 to 4000 cm−1.
2.4.2. Morphology and density of the scaffolds
Scanning electron microscopy (SEM; Hitachi TM-1000, Japan) was
used to observe the morphology of the scaffolds.
The densities were calculated by using the following equation [22]:
ρ = m/v = (4 m)/(d2h) (1)
In this equation, m, v, d, and h represent mass, volume, diameter,
and height of the scaffold, respectively.
2.4.3. Water absorption capacity
Water absorption capacity was tested as previously reported [37].
Dry weights (wd) were determined and recorded. Scaffolds were placed
into distilled water and removed after 2.5, 10, 30, 60, and 120 min. The
size of the specimen was 14 mm in diameter and 10 mm in height. Wet
weights (ww) were determined and recorded after the superficial water
on the scaffolds was removed be gentle blotting with a filter paper. The
water absorption capacity (w) was calculated by using the following
equation:
w = (ww − wd)/wd × 100% (2)
2.4.4. Compressive mechanical property
The Instron 5969 testing system with a 200-N loaded sensor was
used for compressive mechanical property tests of SS and CSS. Five
samples with 13-mm diameter and 14-mm height of each group were
used. The compression strain-stress curves of the scaffolds in wet state
with compression strains (ε = 40% and ε = 60%) were measured at a
strain rate of 10 mm/min.
2.4.5. Micro-computed tomography (CT)
Total porosity of the scaffolds was evaluated on the basis of 3D
micro-CT images generated with a micro-CT scanner (SkyScan 1176;
Bruker, Kontich, Belgium) using Dataviewer and CTAn (Bruker). 3D
reconstruction was conducted using CTvox (Bruker). Five samples of
each group were analyzed in this test. The size of the specimen was
14 mm in diameter and 10 mm in height.
S. Chen, et al.
Fig. 1. Schematic diagram of 3D electrospun scaffold fabrication.
Nanofiber membranes, fabricated by coaxial electrospinning, were cut into pieces and dispersed. The nanofiber dispersions were poured into a cylindrical mold and
frozen and, afterwards, freeze-dried, followed by heating at 180 °C for 2 h. For SS fabrication, the scaffold was subsequently disposed by water. For CSS, scaffold was
crosslinked with CS, EDC, NHS, and MES buffer.
2.5. In vitro study
2.5.1. Cell viability, morphology, and infiltration
Before cell seeding, scaffolds were sterilized by immersion in 75%
ethanol for 3 h. The size of the specimen was 12 mm in diameter and
2 mm in height. Two-hundred microliters of cell suspension containing
2 × 104 cells was seeded on the scaffolds. Cells viability was de-
termined by using a live/dead cell viability assay (KeyGEN BioTECH,
Nanjing, China) on days 3 and 7. Total and live cell numbers were
counted with ImageJ (Version 1.51a; National Institutes of Health,
Bethesda, MD, USA). In addition, live cell percentages were calculated
for comparison. Five samples of each scaffold per time point were used
in this test. Cell morphology was observed by SEM at days 3 and 7.
Scaffolds were fixed with glutaraldehyde for 1 h at 4 °C, followed by
dehydration in an ethanol series. Tert-butanol was used to replace
ethanol in the scaffold. Next, the scaffolds were freeze-dried in a va-
cuum at −60 °C for 24 h. Finally, the samples were gold-coated and
characterized. For evaluation of BMSC infiltration, 2 × 104 cells were
seeded onto scaffolds and grown for 7 days. The scaffolds were fixed in
4% paraformaldehyde for 48 h and then embedded in paraffin. Four-
micrometer-thick sections were cut and stained with hematoxylin-eosin
(HE). Histological observations for infiltration were made under a light
microscope.
2.5.2. Assessment of chondrogenic induction properties of the scaffolds by
quantitative (q)PCR
To study the effects of the scaffolds on chondrogenic differentiation,
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Materials Science & Engineering C 118 (2021) 111312
5 × 105BMSCs were seeded onto the scaffolds and exposed to chon-
drogenic medium (induced condition) or cultured in standard medium
(non-induced condition) for 21 days. BMSCs grown in 6-well plates
with DMEM served as a control group. Total cellular RNA was isolated
using TRIzol (Invitrogen, Carlsbad, CA, USA). The mRNA was reverse-
transcribed into cDNA with the PrimeScript RT Reagent Kit (TaKaRa,
Dalian, China). The cDNA was amplified by Quantitative TaqMan® real-
time PCR. qPCR was carried out using SYBR® Premix Ex Taq™ (TaKaRa)
with the following primers: COL2 A1 (forward 5′-CATCCCA CCCTCTC
ACAGTT-3′ and reverse 5′-GTCTCTGCCT TGACCCAAAG-3′), and ACAN
(forward 5′-TGGTCCACCATTCGGCATA-3′ and reverse 5′-GCCAGAAG
ATTCACCACTACCAC-3′). Data were analyzed by the 2−ΔΔCt method,
with normalization to the Ct of the housekeeping gene glyceraldehyde
3-phosphate dehydrogenase (GAPDH). All experiments were performed
three times.
2.5.3. Anti-inflammatory potential of the scaffolds
To evaluate the anti-inflammatory potential of the scaffolds,
1 × 105 BMSCs were seeded onto the scaffolds and cultured for 24 h
before stimulation. Human recombinant TNF-α (PeproTech) was added
at a concentration of 50 ng/mL for 48 h in all three groups (control, SS,
and CSS) to induce an inflammatory micro-environment, as previously
reported [38]. Cells cultured in normal conditions (without TNF-α sti-
mulation) were evaluated for comparison. Forty-eight hours after sti-
mulation, supernatants were harvested, filtered, and stored at −80 °C.
Nitric oxide (NO) and prostaglandin E2 (PGE2) (two important in-
flammatory factors in cartilage degenerative disease pathology [28])
S. Chen, et al.
levels in culture supernatants were assayed using commercial ELISA
kits from MyBioSource (San Diego, CA, US) and antibodies-online
(Aachen, Germany), respectively, following the manufacturers' in-
structions. All experiments were performed three times.
2.6. In vivo study
2.6.1. Articular joint drilling surgery
The project and experiments were approved by the Animal Research
Committee of the Shanghai Jiao Tong University Affiliated Sixth
People's Hospital. Animals were treated according to the standard
guidelines of our hospital. Rabbits were divided into three treatment
groups; control, SS, and CSS (n = 6 per group). The rabbits were an-
esthetized with pentobarbital sodium (30 mg/kg). Knee joints were
revealed through the lateral approach, followed by patella dislocation
for exposure. Osteochondral drilling defects of 3-mm diameter and 4-
mm depth were created in the trochlear groove of the femur.
Subsequently, two scaffolds were implanted. In the control group, the
defects were untreated. Next, the joint was closed with suture.
Penicillin was given intramuscularly as a prophylactic. The rabbits were
allowed to move freely in cages postoperatively. The sizes of the scaf-
folds used in animal study were all 3 mm in diameter and 4 mm in
height.
2.6.2. Gross examination and histological analysis
Rabbits were euthanized 12 weeks postoperatively. Samples were
observed and photographed for evaluation according to the
International Cartilage Repair Society (ICRS) macroscopic assessment
scale for cartilage repair [39]. Six samples in each group were used for
evaluation. After gross examination, dissected distal femurs were fixed
in 4% paraformaldehyde for 48 h, decalcified with 10% EDTA, and
paraffin-embedded. Four-micrometer-thick sections were cut and
stained with HE or safranin O/fast green to assess GAG distribution.
Histological and immunohistological observations were made under a
light microscope.
2.6.3. Immunohistological analysis
To evaluate cartilage degeneration, the expression of COL2 and
ACAN in defects was analyzed by immunohistological staining.
Deparaffinized sections were hydrated in a graded series of alcohol. The
sections were placed in 10 mM citrate buffer solution (pH 6.0) and
heated in a 95 °C bath to retrieve the antigens. Hydrogen peroxide
(0.3%) and goat serum (1:100 dilution) were used to respectively block
endogenous peroxidase activity and non-specific binding sites. Then,
the sections were incubated overnight at 4 °C with primary antibodies
against the cartilage regeneration markers anti-COL2 and anti-ACAN,
and against iNOS and PGES, which are inducible enzymes that produce
NO and prostaglandins, respectively. Then, the sections were incubated
with secondary antibodies for 1 h at 37 °C and stained in DAB solution
(Dako, Hamburg, Germany).
2.6.4. Statistical analysis
All quantitative data are presented as means ± standard deviations.
Statistical analysis was carried out using one-way analysis of variance
(ANOVA) followed by the Tukey HSD test for post-hoc comparison
(SPSS, version 22; IBM, Armonk, NY, USA). Differences were con-
sidered significant at p < 0.05.
3. Results
3.1. Characterization of 3D scaffolds
3.1.1. ATR-FTIR
We used ATR-FTIR to evaluate differences in molecular structure
induced by the crosslinking of CS. In Fig. 2A (Pure CS), the character-
istic absorption peaks of pure CS could be observed clearly, the peaks at
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Materials Science & Engineering C 118 (2021) 111312
1056 and 1127 cm−1 result from CeO and S]O stretching vibrations of
hydroxyl groups on CS, respectively. The bands at 1370 and 1412 cm−1
represent C]O and CeO stretching vibrations of ionized carboxyl
groups [40]. For scaffolds without CS (Fig. 2A, SS), a peak at
1240 cm−1, which results from CeN stretching vibration of amide III,
was observed. However, after CS crosslinking (Fig. 2A, CSS), the peak at
1240 cm−1 was decreased due to the influence of S]O stretching vi-
bration of CS. Because CS and gelatin showed many overlapping peaks,
IR spectra indicated limited clear differences.
3.1.2. Compressive mechanical properties
We observed no differences in compressive mechanical properties
induced by CS crosslinking: as shown in Fig. 2B, SS and CSS exhibited
similar compressive strengths, in dry state, the compressive modulus of
SS and CSS were 709 KPa and 657 KPa, respectively. Furthermore, SS
and CSS possessed similar elastic properties in the wet state. Fig. 2C
shows the compressive stress-strain curves of SS and CSS under 40%
and 60% strain, respectively. Highly nonlinear and closed hysteresis
was detected. Thus, the two scaffolds exhibited the same compressive
stress (ε = 40% and ε = 60%).
3.1.3. Water absorption capacity
Both scaffolds presented excellent water absorption capacity
(Fig. 2D), likely owing to the hydrophilic properties of gelatin and the
porous structure. However, CSS exhibited a water absorption behavior
inferior to that of SS, likely because of a decrease in the number of
carboxyl and amino groups after CS crosslinking with EDC/NHS.
3.1.4. Morphology, micro-CT evaluation, and density
Photographs of the scaffolds are shown in Fig. 3A (dry state) and 3B
(wet state). Both scaffolds appeared to be porous. Micro-CT images of
SS and CSS showed porous appearances (Fig. 3C, D). Quantification of
the total porosity (%) indicated no significant difference between SS
and CSS (Fig. 3E), with porosities of 74.67% ± 8.81% and
66.35% ± 4.08%, respectively. The addition of CS may explain the
slightly lower porosity of CSS.
SEM of SS (Fig. 3F, G) and CSS (Fig. 3H, I) demonstrated that the
porous scaffolds are composed of nanofibers. All scaffolds showed an
interconnected and cellular structure that mimicked the natural ECM.
As shown in the images, pores of various sizes and shapes were scat-
tered throughout the scaffolds. However, CSS shows a more compact
structure than SS, which may be the result of CS crosslinking. The
densities of SS and CSS were 94.5 mg/cm3 and 121.7 mg/cm3, re-
spectively. The density of CSS was higher than that of SS, most likely
because of the addition of CS.
3.2. In vitro study
3.2.1. Cell viability, morphology, and infiltration
After BMSCs were seeded on the scaffolds for 3 or 7 days, cell vi-
abilities within SS and CSS were determined by live/dead assay, and the
results are shown in Fig. 4A, B and C, D, respectively. On day 3, large
numbers of live cells were detected on both scaffolds (Fig. 4A, C) in-
dicating appropriate biocompatibility of SS and CSS. The total cell
number in CSS was 1.92-fold change (p < 0.01) in comparison with
that in SS. The percentage of live cells was 1.54-fold change
(p < 0.01). On day 7, the numbers of live cells were increased, and
only few dead cells were detected on both scaffolds (Fig. 4B, D). The
total cell number in CSS was 1.47-fold change (p < 0.01) than that in
SS, while the live-cell percentage was 1.32-fold change (p < 0.01).
These results demonstrated that the scaffolds did not negatively affect
the proliferation of BMSCs. Importantly, BMSCs seeded on CSS pre-
sented significant higher viability than those on SS, both in total cell
number and live cell percentage. BMSC adhesion and proliferation were
evaluated by SEM (Fig. 4E–H). BMSCs adhered and proliferated on the
nanofibers and exhibited normal cell morphology. Cells nearly
S. Chen, et al. Materials Science & Engineering C 118 (2021) 111312

Fig. 2. Mechanical properties of the scaffolds.

(A) ATR-FTIR spectra of SS, CSS, and pure CS. (B) Compressive stress−strain curves of SS and CSS. (C) Compressive stress−strain curves of the SS and CSS under
compressing and releasing cycles (ε = 40% or 60%). (D) Water absorption rate of the SS and CSS.

contacted each other and formed a cell sheet. These findings demon- infiltration was examined by HE, which demonstrated better pro-
strated an appropriate biocompatibility of both scaffolds. In addition, in liferation on CSS as indicated by the large number of cells (violet blue)
comparison with those on SS (Fig. 4E, F), cells seeded on CSS (Fig. 4G, observed throughout the scaffold (Fig. 4I–L).
H) presented a better morphology, exhibiting a spindle shape. Cell

Fig. 3. Appearance, micro-CT, total porosity, and SEM of 3D scaffolds.

(A, B) The appearances of scaffolds in dry (A) and wet (B) state. (C, D) Micro-CT images of SS (C) and CSS (D). (E) Total porosity of the scaffolds. Results are
mean ± S.D. n = 5 per group. NS, no significant difference. (F–I) SEM images of SS (F, G) and CSS (H, I). Magnification: 200 × (F, H), 1000 × (G, I).

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S. Chen, et al. Materials Science & Engineering C 118 (2021) 111312

Fig. 4. CSS exhibits favorable cell viability, morphology, and infiltration in comparison with SS.
(A–D) Live/dead assay of BMSCs seeded on SS (A, B) and CSS (C, D). Magnification, 100×. (E–H) SEM images of BMSCs seeded on SS (E, F) and CSS (G, H).
Magnification, 500×. (I–L) The infiltration of BMSCs cultured on SS (I, J) and CSS (K, L) by HE staining. BMSCs in scaffolds were stained with violet blue.
Magnification: 40 × (I, K), 100 × (J, L). (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

3.2.2. Chondrogenic differentiation
mRNA expression of two chondrogenic genes, COL2 and ACAN, was
assessed by qPCR to evaluate the chondrogenic inductive potential of
the scaffolds after 21 days (Fig. 5). BMSCs cultured in plates served as a
control group. In standard medium (non-induced condition), the ex-
pression of COL2 in the CSS group was higher than that in the control
(8.00-fold, p < 0.05) and SS (8.30-fold, p < 0.05) groups. Further-
more, in chondrogenic medium (induced condition), COL2 expression
was significantly higher in the CSS than in the control (4.56-fold,
p < 0.01) and SS (2.29-fold, p < 0.05) groups. In addition, COL2
expression in the SS group was significantly higher than that in the
control group (1.99-fold, p < 0.05) (Fig. 5A). Similarly, ACAN mRNA
expression was significantly higher in the CSS group than in the control
(12.97-fold, p < 0.05) and SS (13.65-fold, p < 0.05) groups in
standard medium. In chondrogenic medium, ACAN expression was
significantly higher in the CSS than in the control (3.02-fold, p < 0.01)
and SS (1.60-fold, p < 0.05) groups. In addition, ACAN expression was
significantly higher in the SS than in the control group (1.89-fold,
p < 0.05) (Fig. 5B). These findings suggested that CSS is effective in
inducing chondrogenic differentiation of BMSCs in both standard and
chondrogenic medium.
3.2.3. Immunosuppressive potential of scaffolds in vitro
To test the immunosuppressive potential of the scaffolds in vitro, we
examined the expression of NO and PGE2 in BMSCs seeded onto the
different scaffolds, with and without TNF-α stimulation (Fig. 5C, D).
Under normal condition (without stimulation), no significant difference
was found among three groups with regard to NO production (Fig. 5C).
After stimulation, the NO level increased in all groups. CSS produced
the lowest level of NO, with significant differences as compared to the
control (0.82-fold, p < 0.05) and SS (0.74-fold, p < 0.01) groups
(Fig. 5C). These findings indicated that CSS may have preferable im-
munosuppressive potential than SS under TNF-α stimulation, which
imitated the inflammatory environment in cartilage damage.
Similarly, under normal condition, no significant difference in PGE2
level was found among the three groups, while after stimulation, PGE2
production in all groups increased (Fig. 5D). PGE2 release after TNF-α
treatment was the highest in the SS group. The level of PGE2 in the CSS
group was significantly lower than that in the control (0.87-fold,
p < 0.05) and SS (0.83-fold, p < 0.01) groups (Fig. 5D). PGE2 pro-
duction was 1.04-fold higher in the SS than in the control group, but the
difference was not significant. These results demonstrated that CSS may
have superior immunosuppressive effect in comparison with SS. Taken
together, although the anti-inflammatory effect of SS was uncertain, all
findings demonstrated the potential immunosuppressive property of
CSS, which indicated that crosslinking of CS may be effective for anti-
inflammation.
3.3. In vivo study using a rabbit model
3.3.1. Gross appearance and ICRS score
Twelve weeks after transplantation, an obvious defect and little
white neo-tissue was observed in the boundary area in the control
group (Fig. 6A). In the SS and CSS groups, neo-tissue appeared to be
integrated with the surrounding tissue (Fig. 6B, C). In the SS group, the
defect was filled with white tissue with small fissures (Fig. 6B). The
defect in the CSS group was filled with cartilage-like tissue that was
similar to the surrounding cartilage. The defect was well integrated
with the surrounding tissue, with a smooth and flat surface (Fig. 6C).
ICRS scores obtained from macroscopic evaluation were 1.7 ± 0.6,
7.3 ± 2.5, and 9.7 ± 2.5 in the control, SS, and CSS groups, re-
spectively. The ICRS scores showed substantial improvement in the CSS

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S. Chen, et al. Materials Science & Engineering C 118 (2021) 111312

Fig. 5. CSS exhibits superior chondrogenic inductive and immunosuppressive potential in vitro.
(A, B) mRNA expression levels of COL2 (A) and ACAN (B). Gene expression levels were expressed as fold change compared to GAPDH expression. n = 5; *p < 0.05,
**p < 0.01. (C, D) NO (C) and PGE2 (D) concentrations in the supernatants of BMSCs cultured on scaffolds in normal and TNF-α stimulation condition. n = 5;
*p < 0.05, **p < 0.01.

group as compared to the non-treated group at 12 weeks (p < 0.01). indicated by the intense red staining on the defect surfaces. The neo-
The ICRS score for the SS group was significantly higher that for the tissues were integrated with the native tissue and showed a similar
non-treated group (p < 0.05). In addition, the CSS group presented a color and organized architecture, demonstrating favorable cartilage
higher score than the SS group (Fig. 6D). These findings demonstrated regeneration (Fig. 7I, L).
that CSS presents superior outcomes for cartilage regeneration in rab-
bits.
3.3.3. Immunohistochemistry
The expression levels of COL2 and ACAN in vivo were examined by
immunohistochemistry (Fig. 8A). In the non-treated group, limited
3.3.2. HE and safranin O/fast green staining
COL2 and ACAN expression was observed, and only at the bottom of the
Six weeks after implantation, the defects still existed in the non-
defects. Compared with the non-treated group, the SS group presented
treated group, with thin fibrous-like tissue appearing on the surface as
an increased expression level of both markers. However, they were even
shown by HE staining (Fig. 6E, H). However, in the SS group, loose neo-
more strongly expressed in the CSS group, indicating superior cartilage
tissues were found in the defect (Fig. 6F, I). In the CSS group, dense
regeneration. Immunohistochemistry largely matched the findings of
fibrous-like tissue was found in defects infiltrated by large numbers of
HE and safranin O/fast green staining, and corroborated that CSS ex-
cells (Fig. 6G, J). Moreover, safranin O/fast green showed few light-red-
hibited the best properties allowing cartilage regeneration.
stained neo-tissues, indicating that almost no GAG was deposited in the
To evaluate the anti-inflammatory effect of the scaffolds, we ex-
non-treated group (Fig. 6K, N). In contrast, GAG was deposited in the
amined the expression of iNOS and PGES in vivo (Fig. 8B). Compared to
defects of SS and CSS groups, with the CSS group showing the highest
the non-treated group, the expression levels of iNOS and PGES were
deposition (Fig. 6L, M, O, P).
decreased in the SS group as shown by immunohistochemistry. In the
At 12 weeks after implantation, the defects in the non-treated group
CSS group, both markers were significantly decreased as compared to
were not healed, and we observed little thin connective tissue on the
the non-treated and SS group. These results indicated that CSS possesses
surface (Fig. 7A, D). In the SS group, HE staining showed that the de-
anti-inflammatory properties in vivo.
fects were filled up with dense neo-tissues, which was likely connective
tissue with a disordered structure. The cells were mostly fibroblast-like
cells (Fig. 7B, E). In contrast, the defects in the CSS group were almost 4. Discussion
entirely filled up with thick, more cartilage-like tissue. The cells were
mostly chondrocyte-like cells (Fig. 7C, F). In addition, safranin O/fast In this study, we successfully fabricated 3D electrospun scaffolds
green staining demonstrated poor GAG deposition in the non-treated possessing distinctive mechanical properties and evaluated their po-
group (Fig. 7G, J). Limited red staining was detected on the defect tential for cartilage regeneration and immunoregulation. The results
surfaces, indicating inferior cartilage regeneration (Fig. 7H, K). How- demonstrated that BMSCs seeded on CSS showed better chondrogenic
ever, in the CSS group, GAG formation was clearly detectable, as differentiation than cells seeded on SS and the control scaffold. In

7
S. Chen, et al. Materials Science & Engineering C 118 (2021) 111312

(caption on next page)

8
S. Chen, et al. Materials Science & Engineering C 118 (2021) 111312

Fig. 6. Gross observation and ICRS score at 12 weeks after implantation, and preferable cartilage repair in CSS as compared SS and non-treated scaffold by
histological evaluation at 6 weeks.
(A–C) Representative gross observations of cartilage defects. The black dotted lines show the defects. (D) ICRS evaluation of cartilage repair. n = 6; *p < 0.05,
**p < 0.01. (E–J) HE of cartilage regeneration in defects. (K–P) Safranin O/fast green staining of cartilage regeneration. The edges of defects are indicated by black
arrows. Blue dotted lines indicate the implantation areas. (For interpretation of the references to color in this figure legend, the reader is referred to the web version
of this article.)

addition, CSS showed better anti-inflammatory properties than control
and SS in vitro. Furthermore, in a rabbit model, CSS enhanced cartilage
regeneration as compared to control and SS. Finally, the anti-in-
flammatory effect of CSS is also superior.
Scaffolds play a vital role in controlling cell growth and differ-
entiation for tissue regeneration [41–43]. Karbasi et al. [4–8] used
multi-walled carbon nanotubes to fabricate electrospun scaffolds which
showed appropriate potential for cartilage regeneration. However,
these materials are relatively two dimensional which may limit its
porosity to some degree in comparison with 3D structure. Previous
studies reported cartilage regeneration by 3D printed or braid scaffolds
that were fabricated with custom-made machinery [14–16]. The
printed scaffolds were efficient in chondrogenic regeneration [14,15],
and the braid scaffold displayed mechanical properties of cartilage re-
pair in vitro [16]. These findings indicated the potential of 3D scaffolds
in cartilage repair. The complicated production process and relatively
low porosity of materials remain challenge for scaffolds fabrication.
However, our 3D scaffolds were fabricated by a more convenient and
economic method. Additionally, our previous study, in which the same
method was applied, showed the feasibility of 3D scaffolds in cartilage

Fig. 7. The favorable cartilage regeneration in CSS by histological evaluation at 12 weeks after implantation.
(A–F) HE of cartilage regeneration in defects. (G–L) Safranin O/fast green staining of cartilage regeneration. The edges of defects are indicated by black arrows. (For
interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

9
 S. Chen, et al.

10
Fig. 8. The potential immunosuppressive property of CSS at 12 weeks after implantation.
Immunohistochemistry images of cartilage regeneration (A) and anti-inflammation (B). The edges of the defect are indicated by black arrows.
Materials Science & Engineering C 118 (2021) 111312
S. Chen, et al.
repair [44]. In this study, CSS showed appropriate compressive me-
chanical properties and water absorption capacity. In addition, micro-
CT and SEM images indicated a favorable porous structure. Further, SS
exhibited appropriate properties for cell proliferation and infiltration in
vitro.
Native ECM is an attractive choice for scaffold modification owing
to the inherent cues that regulate specific cell proliferation and differ-
entiation [45–47]. Among native ECMs, CS plays an important role in
cell communication, which regulates crucial physiological processes
such as induction of differentiation [33,48]. Our findings showed that
after the crosslinking of CS, COL2 and ACAN mRNA expression in
BMSCs seeded in CSS was significantly higher than that in the SS and
control groups in vitro, which suggested a better chondrogenic effect of
CSS. Further, CSS was superior to SS in terms of cartilage regeneration
with higher COL2 and ACAN expression in our rabbit model, as shown
by immunohistology. These findings are in accordance with previous
studies showing that CS regulates cartilage regeneration by promoting
collagen and proteoglycan synthesis [27,49]. Moreover, the functio-
nalization of CS did not significantly influence the physical properties
of the scaffold, as shown by the compressive mechanical properties and
water absorption capacity, as well as micro-CT image and SEM ana-
lyses.
CS not only is a major component of the ECM, it also plays a more
important role in inflammation and immunity than other components,
owing to its intrinsic stability and low immunogenicity [50,51]. Our
results showed that CSS reduce the generation of pro-inflammatory
cytokines, PGE2 and NO, in vitro. NO is an important factor in the pa-
thology of cartilage damage because it can induce inflammatory com-
ponents and may lead to tissue injury [28]. Meanwhile, the crucial role
played by PGE2 in cartilage damage has been demonstrated in animal
models [52–54]. PGE2 increases the generation of catabolic factors,
which in turn induce further deterioration of the cartilage [28]. Our
results are consistent with those of previous studies showing that CS
efficiently suppresses the production of pro-inflammatory cytokines
[55,56]. We further examined the immunomodulatory properties of
CSS in a rabbit model, which demonstrated reduced expression of PGES
and iNOS, inducible enzymes producing PGE2 and NO, respectively
[57], in the CSS group. Taken together, these findings indicate that
functionalization by CS potentially improve the immunomodulatory
properties of the scaffold. As chronic inflammation in cartilage de-
struction may provide an inferior environment for regeneration [58],
the regulation of inflammation could therefore create favorable con-
ditions for cartilage repair [59]. Thus, the immunomodulatory effect of
CSS may have positive impacts on cartilage regeneration. For cartilage
degenerative diseases that are accompanied by persistent and intense
inflammation, such as osteoarthritis, CSS holds promise for cartilage
repair.
In this study, we fabricate 3D porous electrospun nanofiber scaffolds
by a convenient method, and functionalize it by CS to promote cartilage
regeneration. However, there are limitations to this study. Further
studies are required to compare our method with conventional elec-
trospinning. In addition, the feasibility of CSS for cartilage tissue re-
generation needs to be tested. The mechanisms underlying the chon-
drogenic and anti-inflammatory effects of CSS were not studied, and
signaling pathways related to the pharmacological functions of CS were
not evaluated. Future studies will be conducted focusing on the ma-
nipulation of CS dosage to achieve optimal cartilage repair. In addition,
evaluation of CSS in big animal models, such as dogs, is needed because
these are more suitable for the evaluation of cartilage repair than
rabbits.
5. Conclusion
In this study, we designed a 3D porous electrospun scaffold by a
novel method and further functionalized it by the crosslink of CS to
promote cartilage regeneration and modulate inflammation. We
11
Materials Science & Engineering C 118 (2021) 111312
confirmed that CSS exhibits not only appropriate mechanical property,
but also appropriate biocompatibility. In vitro, CSS exhibited superior
chondrogenic differentiation potential and better anti-inflammation
effect than the control and SS. In vivo, CSS effectively promoted carti-
lage repair in rabbit cartilage defects. Additionally, superior anti-in-
flammation effect of CSS was found in animal study. Our results in-
dicated the preferable property of CSS for cartilage regeneration and its
potential in immunoregulation.
Author statement
Shuai Chen: Conceptualization, Methodology, Investigation,
Software, Data curation, Writing-Original draft preparation. Weiming
Chen: Methodology, Visualization, Investigation, Validation. Yini Chen:
Software, Validation. Xiumei Mo: Supervision. Cunyi Fan: Supervision,
Writing- Reviewing and Editing,
Declaration of competing interest
The authors declare that they have no known competing financial
interests or personal relationships that could have appeared to influ-
ence the work reported in this paper.
Acknowledgements
This work was supported by the Youth Program of National Natural
Science Foundation of China (81802157), National Natural Science
Foundation of China (81830076, 81672146), and clinical project of
Shanghai Jiao Tong University Affiliated Sixth People's Hospital
(ynlc201821).
Appendix A. Supplementary data
Supplementary data to this article can be found online at https://
doi.org/10.1016/j.msec.2020.111312.
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