Toxicological evaluation of ionic liquid in a biological functional tissue construct model based on nano-hydroxyapatite/chitosan/gelatin hybrid scaffolds

Article abstract of DOI:10.1016/j.ijbiomac.2020.04.267

The application of ionic liquid is attracting more attentions as green replacement for volatile organic solvents. However, the toxic effects and risks of ionic liquid in different biological systems for human health and environment are poorly evaluated. Among all ionic liquids, 1-ethyl-3-methylimidazolium diethylphosphate ([Emim]DEP-type) ionic liquid is still at the early phase of development, and its toxicity remains unclear. In this study, we fabricated a 3D biological functional tissue construct model based on nano-hydroxyapatite, chitosan and gelatin hybrid scaffold and evaluated its toxic effects of [Emim]DEP-type ionic liquid. As a control group, the examination of ionic liquid's toxic effects on the pre-osteoblast cell line (MC3T3-E1) was detected in 2D cultures. The MTT assay showed that [Emim]DEP-type ionic liquid inhibited the proliferation of cells on both 2D cultures and 3D tissue constructs. This effect was correlated with culturing time and concentration, while the IC50 on 3D scaffolds (12,566, 9015, 7896 μg/mL, at 24 h, 48 h and 72 h, respectively) was found significantly higher compared to 2D cultures (3959, 2226, 1884 μg/mL). Flow cytometry analysis and scanning electron microscope demonstrated that when [Emim]DEP-type ionic liquid acted on MC3T3-E1 cells for 48 h, the shape of 2D cells shrank, together with decreased surface adhesion.

Full text of DOI:10.1016/j.ijbiomac.2020.04.267

International Journal of Biological Macromolecules 158 (2020) 800–810
Contents lists available at ScienceDirect
International Journal of Biological Macromolecules
journal homepage: http://www.elsevier.com/locate/ijbiomac
Toxicological evaluation of ionic liquid in a biological functional tissue
construct model based on nano-hydroxyapatite/chitosan/gelatin
hybrid scaffolds
Jingjing Zhu a, Qian Kong a, Shuangshuang Zhengb, Yiwei Wangc, Zeren Jiao d, Yi Nie b,e,
,
⁎
Tianqing Liu a, , Kedong Song
⁎
a,⁎⁎
a
State Key Laboratory of Fine Chemicals, Dalian R&D Center for Stem Cell and Tissue Engineering, Dalian University of Technology, Dalian 116024, China
b
c
Zhengzhou Institute of Emerging Industrial Technology, Zhengzhou 450000, China
Burns Research Group, ANZAC Research Institute, University of Sydney, Concord, NSW 2139, Australia
Artie McFerrin Department of Chemical Engineering, Texas A&M University, College Station, TX 77843-3122, USA
Key Laboratory of Green Process and Engineering, Institute of Process Engineering, Chinese Academy of Sciences, Beijing 100190, China
d
e
a r t i c l e i n f o
a b s t r a c t
Article history:
The application of ionic liquid is attracting more attentions as green replacement for volatile organic solvents.
However, the toxic effects and risks of ionic liquid in different biological systems for human health and environ-
ment are poorly evaluated. Among all ionic liquids, 1-ethyl-3-methylimidazolium diethylphosphate ([Emim]
DEP-type) ionic liquid is still at the early phase of development, and its toxicity remains unclear. In this study,
we fabricated a 3D biological functional tissue construct model based on nano-hydroxyapatite, chitosan and gel-
atin hybrid scaffold and evaluated its toxic effects of [Emim]DEP-type ionic liquid. As a control group, the exam-
ination of ionic liquid's toxic effects on the pre-osteoblast cell line (MC3T3-E1) was detected in 2D cultures. The
MTT assay showed that [Emim]DEP-type ionic liquid inhibited the proliferation of cells on both 2D cultures and
3D tissue constructs. This effect was correlated with culturing time and concentration, while the IC50 on 3D scaf-
folds (12,566, 9015, 7896 μg/mL, at 24 h, 48 h and 72 h, respectively) was found significantly higher compared to
2D cultures (3959, 2226, 1884 μg/mL). Flow cytometry analysis and scanning electron microscope demonstrated
that when [Emim]DEP-type ionic liquid acted on MC3T3-E1 cells for 48 h, the shape of 2D cells shrank, together
with decreased surface adhesion.
Received 23 November 2019
Received in revised form 16 April 2020
Accepted 30 April 2020
Available online 05 May 2020
Keywords:
Ionic liquid
Cytotoxicity
Three-dimensional tissue constructs
© 2020 Published by Elsevier B.V.
1. Introduction
food [12,13], nanomaterials [14–17] and energy field. Moreover, IL are
accepted as “green” solvents for organic/inorganic synthesis, (bio)catal-
Ionic liquid (IL) is a fascinating group of new chemicals with the po-
tential to improve the development in organic chemistry and chemical
technology and stimulate a lot of research fields [1,2]. Over the past
few decades, an increased number of publications regarding ILs as a
novel family of “green solvents” have attracted considerable interests
across different disciplines such as biocatalysis, separation science and
electrochemistry. Due to their unique physiochemical properties, in-
cluding superior solvation properties, negligible vapor pressure, low
combustibility, and high electric conductivity [3,4], they have been
widely investigated in chemical industry [5–10], pharmaceutical [11],
ysis, ion battery, and separation. However, ILs are not always “green” or
“environmentally benign” because they fail to satisfy all the criteria of
green chemistry [3]. IL's toxicological and ecotoxicological are unclear
with minimal available data. Environmental risk of IL was previously re-
ported for its biological toxicity [18], including assessment in molecules,
cells [19–27], tissues, organs to organisms [28–32], plants [33,34], to
populations and ecosystems. By using a large number of different
types of IL, researching the toxicity/toxicology of various life levels and
types to fill the IL toxicity data gap and to lay the foundation for the de-
sign of new ILs which are non-toxic and meet various functional re-
quirements. For cytotoxicity assessment of IL, only 2D in vitro culture
[35] and in vivo animal models (e.g. fish) were reported. Among them,
2D in vitro cultures can hardly simulate the in vivo microenvironment,
frequently leading to inaccurate and unreliable results, while the animal
models fail to monitor and study progression without causing harm or
death of the animals. To bridge the gap between 2D and in vivo models,
3D culture systems on research of cytotoxicity need be developed,
⁎
Corresponding authors.
⁎⁎ Correspondence to: K. Song, State Key Laboratory of Fine Chemicals, Dalian R&D
Center for Stem Cell and Tissue Engineering, Dalian University of Technology, Dalian
116024, China
E-mail addresses: ynie@ipe.ac.cn (Y. Nie), liutq@dlut.edu.cn (T. Liu),
Kedongsong@dlut.edu.cn (K. Song).
https://doi.org/10.1016/j.ijbiomac.2020.04.267
0141-8130/© 2020 Published by Elsevier B.V.

J. Zhu et al. / International Journal of Biological Macromolecules 158 (2020) 800–810
801
which can better mimic in vivo micro-environment for studying cell
behavior.
In the research field of tissue engineering [36], scaffold material is
2.3. Preparation and characterizations of scaffolds
Cs solution was prepared by dissolving Cs powder in 2% (v/v) dilute
acetic solution. After fully dissolved, gelatin particles were added into Cs
solution and stirred for 4 h at 45 °C until both chitosan and gelatin were
fully dissolved (Fig. 1). The nHAp/Cs/Gel scaffolds were prepared by
mixing Cs/Gel in a ratio of 1:0.6 with final concentrations of nHAp at
0%, 20%, 40% and 60%. The nHAp/Cs/Gel liquids were then transferred
to a sizing mold and the mixture liquid was pre-freeze at −20 °C for
12 h, and then freeze dryer over 24 h. The nHAp/Cs/Gel scaffolds were
cross-linked with carbodiimide/N-hydroxy-succinamide/morpholine
ethane sulfonate (EDC/NHS/MES) for 6 h, prior to incubation in
0.1 mol/L Na2HPO4 solution for 2 h at room temperature. The final scaf-
folds were washed in distilled water for three times to remove residual
acetic acid and cross-linking agents.
essential to provide sufficient growth space and functional induction
for inoculated cells [37]. Scaffolds ware also known to have tremen-
dous influence on cell adhesion and proliferation [38]. To date, a
number of materials including both natural and synthetic polymers
have been utilized and validated as 3D cell culture scaffolds. Natu-
rally derived matrix materials, including matrigel, collagen, chitosan,
and fibronectin, have been widely used as extracellular matrix
(ECM) in 3D cell model owing to their good biocompatibility
[39,40]. Chitosan (Cs) is one of the non-toxic natural materials with
superior water absorption, good biocompatibility, high degradabil-
ity, low immunogenicity, antibacterial and anti-inflammatory [39].
Chitosan porous scaffold [40] inoculated mesenchymal stem cells
showed that the cells were capable to adhere to the scaffold in
24 h, well in vivo animal study further confirmed its superior bio-
compatibility with low foreign body reaction and local inflammation.
Gelatin (Gel) is a collagen hydrolysate that is also non-toxic, superior
biocompatibility and absorbable properties [41] with strong cell ad-
hesion and low immunogenicity. Comparing to, gelatin has better
strength, viscoelasticity and degradability. Therefore, in the present
study, chitosan mixed with gelatin and nano hydroxyapatite is hy-
pothesized to enhance the chemical stability and biocompatibility
of the final cast of the 3D scaffolds [42–44].
2.3.1. Mechanical properties
The compression modulus of Cs/Gel scaffolds with 0%, 20%, 40%, and
60% nHAp were examined using SANS static universal material experi-
mental machine. The upper and lower surfaces of the scaffolds were
cut into a cylinder at the size of 5 mm × 5 mm × 5 mm. The displace-
ment control loading speed of universal testing machine was 1 mm/
min. The compression modulus E (%) of each scaffold was calculated
using the following equation:
In the present study, we developed an in vitro MC3T3-E1 cells 3D
models using chitosan/gelatin (Cs/Gel) and nano-hydroxyapatite
(nHAp) scaffolds. Cytotoxicity of [Emim]DEP-type ILs in the 3D models
(e.g. cell distribution, activity, and proliferation inhibition) was exam-
ined and compared to the 2D counterparts.
ε
σ
10ðL2−L1Þ=S
ðD2−D1Þ=h
E ¼
¼
2.3.2. Water absorption
To determine the water absorption rate of different scaffolds, the dry
weight of each scaffold was first recorded (m0). The scaffolds were
soaked in distilled water at room temperature for 6 h and then placed
on dehydrating filter paper to remove moisture on the surface. The
wet weights (mt) were measured for each scaffold aiming to calculate
the water absorption rate (P (%)) using the following equation:
2. Materials and methods
2.1. Experimental materials
Propidium iodide (PI) and double antibody (penicillin, strepto-
mycin) was obtained from SIGMA (USA). Hoechst 33324, calcein-
AM and MTT kits were purchased from Biyuntian Biological Reagent
Co. LTD. Fetal bovine serum (FBS), pancreas protease was pur-
chased from Gibco (USA). H&E staining kit, 4% paraformaldehyde
solution, chitosan, gelatin was obtained from Beijing Cool Labo
Technology Co., Ltd. Alkaline phosphatase (ALP) detection kit was
obtained from Nanjing JianCheng Biological Reagent Company. An-
hydrous ethanol, glutaraldehyde, glacial acetic acid was purchased
from Tianjin Damao Chemical Reagent Factory. Nano hydroxyapa-
tite, aladddin, 2-(N-morpholinyl)ethanesulfonic acid, N-hydroxyl
succinimide, N-(3-dimethylaminopropyl)-N′-ethylcarbodiimide
hydrochloride was purchased from Beijing Belling Technology Co.,
LTD.
mt−m0
m0
P ð%Þ ¼
ꢀ 100%
2.3.3. Porosity
The porosity of scaffolds was determined by the method of ethanol
substitution. The scaffolds were firstly soaked in absolute ethanol with
a volume of V1 and placed in a vacuum drying oven. After the scaffolds
were completely immersed in absolute ethanol, the total volume of
scaffolds and absolute ethanol were recorded as V2. After removing
the scaffolds out of the ethanol, the remaining volume of ethanol was
defined as V3. The volume of the scaffold was determined as V2 − V1,
while the porosity of the scaffolds was determined as V1 − V3. The ap-
parent volume of scaffold was sum of the volume of the scaffold and
the volume of the scaffold pores, V = (V2 − V1) + (V1 − V3) =
V2 − V3. The porosity A (%) of each scaffold was then calculated using
the following equation:
2.2. Preparation of [Emim] DEP-type ionic liquid
The 1-methylimidazole and triethyl phosphate were mixed in a ratio
of 1:2 and stirred for 8 h at 80 °C, followed by mixing at 150 °C for an-
other 10 h. After cooling down to room temperature, the liquid was
washed with diethyl ether and rotary evaporated under reduced pres-
sure at 75 °C for 4 h to remove all volatile residue, followed by vacuum
dry at 80 °C for 48 h. At the end of the procedure, a pale yellow liquid of
1-ethyl-3-methylimidazolium diethylphosphate ([Emim]DEP) was
obtained.
V1−V3
V2−V3
A ð%Þ ¼
ꢀ 100%
10 mg of [Emim]DEP was dissolved in 10 mL of α-MEM type me-
dium under sterile conditions to prepare a 1 mg/mL stock solution,
which was stored at 4 °C. Dilute to the appropriate concentration with
PBS when ready for use.
2.3.4. Contact angle
The contact angles of the composite scaffolds were measured at a
tableting process of 10 MPa for 1 min by using a contact angle meter.
(OCAH200, Dataphyscics, Filderstadt, Germany).

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J. Zhu et al. / International Journal of Biological Macromolecules 158 (2020) 800–810
Fig. 1. Schematic illustration of the general study design. Preparation of nHAp/Cs/Gel hybrid scaffolds, correlation of nHAp/Cs/Gel hybrid scaffolds, cell extraction and culture process, and
assessment of cytotoxicity of IL on 2D/3D cultures.
2.3.5. Element analysis
at a density of 1 × 107 cells/well. Stock solutions of the ionic liquids
(1 mg/mL) were prepared in culture medium. After 24 h, when cell
growth reached approximately 80% confluence, the medium was re-
placed with fresh medium containing different concentrations of ionic
liquids. The cell-scaffolds were exposed to ionic liquids in the concen-
tration range at 3000 μg/mL, IC50 (12,566, 9015, 7896 μg/mL) and
20,000 μg/mL for 24 h, 48 h, 72 h.
The elements of nHAp/Cs/Gel scaffolds were analyzed for energy dis-
persive spectrometer (EDS) semi-quantitative analysis by using an ele-
mental analyzer. (VarioELIII, Elementar, Langenselbold, Germany).
2.3.6. Degradation rate
The in vitro degradation performance test of the scaffold was com-
pleted by an in vitro immersion test. The scaffolds were weighed (W0)
before immersion. The scaffold samples were soaked in 37 °C PBS solu-
tion for 5, 10, 15, 20, 25, and 30 d, respectively. And three parallel sam-
ples were freeze-dried and subsequently weighed (W1). The
degradation rates D (%) of the scaffolds were calculated using the fol-
lowing equation:
2.5. MTT assay
Cytotoxicity of ionic liquids was assessed using the MTT (3-(4,5-
dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay. To
assay mitochondrial reduction of MTT, test media was removed from
MC3T3-E1 cells in 48 well plates, and then each well was rinsed with
PBS. Following that, each well was incubated with 0.25 mL of
0.3 mg/mL MTT solution in DMEM for 3 h at 37 °C, 5% CO2 incubators,
while protected from light. Supernatant was removed, and blue
formazan dye was dissolved in 0.3 mL of DMSO for 10 min. The absorp-
tion of formazan dye was determined at 540 nm using a microplate
reader (Thermo Fisher Scientific, USA). Cell viability was determined
as the absorption value for test cultures, expressed as a percentage of
absorption for unstimulated control cultures.
W0−W1
W0
D ð%Þ ¼
ꢀ 100%
2.3.7. Biocompatibility
The nHAp/Cs/Gel scaffolds were soaked in 75% ethanol and under
UV light for sterilization. The scaffolds were then rinsed with PBS prior
to cell culture. MC3T3-E1 cells were inoculated into the scaffolds at a
density of 1.0 × 107 cells/well and cultured in a 37 °C, 5% CO2 incubators
at static condition for 72 h followed by staining with calcein/Hoechst/
propidium iodide (PI) for 30 min. The PI stained cells seeded on scaf-
folds were examined under fluorescence microscope to assess the cell
distribution and morphology.
2.6. Morphological changes of cell post IL treatment
2.6.1. Fluorescent staining and SEM
Different concentrations (0 μg/mL, 3000 μg/mL, IC50, 20,000 μg/mL)
of IL were added into the cell-scaffold complexes and cultured for 24 h,
48 h or 72 h, respectively. The cell-scaffolds complexes were then
stained with Calcein/Hoechst/PI for 30 min at 37 °C, 5% CO2 incubators
and then scanned under fluorescence microscope (Olympus, Japan).
2.4. Treatment of chemicals
The sterilized scaffolds were prepared as described above. MC3T3-
E1 cells were seeded in 48 well plates on nHAp/Cs/Gel hybrid scaffolds

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803
Another group of cell-scaffold complexes treated with IL were fixed
3. Results and discussions
in 2.5% v/v glutaraldehyde for 3 h. The complexes were dehydrated with
a serial diluted ethanol at 50%, 70%, 90% and 100% prior to dry in a vac-
uum before coating with gold. The cells seeded scaffolds were then
scanned under SEM at voltage of 30 kV.
3.1. MTT assays and Annexin V/PI staining
The Quantitative analysis of IL cytotoxicity on 2D cultured cells
was shown in Fig. 2. Comparing with the blank control group
(Fig. 2a), the OD value of the experimental group was significantly
decreased, indicating that the [Emim]DEP-type IL inhibited the cell
proliferation of MC3T3-E1 cells, and this inhibition was strong cor-
related with the increased concentrations of IL [3]. The inhibition ca-
pability of IL became weaker over time compared to the
proliferation of cells in the concentration range of 0–1500 μg/mL.
The inhibition of IL around 2000 μg/mL was consistent with the ef-
fect of cell proliferation. The inhibition effect of IL above
2500 μg/mL was stronger than that of cell proliferation. The cell in-
hibition rate was calculated from the OD value, and the inhibition
rate-concentration curve was fitted (Fig. 2b) and the IC50 of the IL
for 24 h, 48 h and 72 h was measured at 3959 μg/mL, 2226 μg/mL
and 1884 μg/mL, respectively. The intermediate concentration
values of the ionic liquids setting in the following series of tests
are the IC50 values at the corresponding time.
Annexin V/PI staining was performed to examine the apoptosis of
cells post IL treatment (Fig. 2c). In the control group (0 μg/mL), apopto-
tic cells were accounted for approximately 2.6% of the total initial num-
ber of cells. With increased concentrations of IL, more cells were found
in the apoptotic state, which is approximately 21% for 1000 μg/mL IL,
49.3% for concentration of 3959 μg/mL and 84% for concentration of
5000 μg/mL, respectively. When the IL concentration was 3959 μg/mL,
apoptosis and necrotic cells consist 49.3% of the total number of cells.
This result was supported by flow cytometry data and by MTT assay.
2.6.2. Annexin V-FITC/PI detection of apoptosis after ionic liquid
MC3T3-E1 cells were inoculated at 6-well plate with a density of
1 × 106 cells/well. When cells were attached, IL was added to 6 individ-
ual well plates and cultured for 24 h. Cells were then collected and re-
suspended via adding 100 μL 1 × Binding Buffer solution. Annexin V-
FITC (5 μL) were added into the cell suspension together with 10 μL PI
staining solution reagent. After 15 min reaction in the dark, the cell
numbers were determined using a flow cytometry (BD Biosciences,
USA).
2.6.3. Hematoxylin-eosin staining after ionic liquid
MC3T3-E1 cells were seeded at a density of 1 × 105 cells/well in 96
well plates. After attachment, IL was added to each 96 well plates and
cultured for 24 h in the incubator. Paraformaldehyde solution (4%,
300 μL) was added in the 96 well plate to fix the cells for 10 min and
then rinsed with PBS buffer. All cells were stained with H&E.
2.7. Statistical analysis
Data were presented as mean standard deviations. All data were
statistically analyzed using Origin 9.0. p b 0.05, p b 0.01 and p b 0.001
were considered statistically significant. Three independent trials were
carried out unless otherwise stated.
Fig. 2. Quantitative and semi-quantitative analysis of IL cytotoxicity in 2D culture: (a) OD value of MC3T3-E1 cells, (b) inhibition rate-concentration fitting curve, b1: 24 h, b2: 48 h, b3: 72 h,
(c): Annexin V/PI staining, IL concentration, c1: 0 μg/mL, c2: 1000 μg/mL, c3: 3959 μg/mL, c4: 5000 μg/mL.

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J. Zhu et al. / International Journal of Biological Macromolecules 158 (2020) 800–810
In a previous study, cytotoxicity of imidazole ILs to fish CCO cell lines
time extended to 48 h, most of cells were found broken down (red
arrow) which causes a significantly drop of cell number. After 72 h,
5000 μg/mL IL treatment, only cell debris was appeared. And a large
number of late apoptotic and necrotic cells at the bottom of the well
plate were dyed red during calcein staining (white arrow and dotted
oval) [3]. It is clear that with the increasing of the concentration of IL,
the number of viable cells decreased tremendously, which is in agree-
ment with obtained cytotoxicity data. Correspondingly, apoptotic cell
death caused by 1-ethyl-3-methylimidazolium tetrafluoroborate
[C2MIM][BF4] exposure was detected by AO/EB fluorescence staining
shown in HeLa cell line, where increase in concentration of ionic IL
causes increase in number of apoptotic/necrotic cells. This is probably
due to direct cell membrane destruction [35]. At present, most studies
on IL toxicity are based on different concentrations and ion species of
ILs [22–27]. On this basis, we evaluated the cytotoxicity of ILs exposed
to cells at 24 h, 48 h, and 72 h.
Comparing to the fluorescent staining analysis of MC3T3-E1 cells
that were treated with different cultured times (24 h, 48 h, 72 h) at
the same concentration (1000 μg/mL) the results show that cells
number increased with prolonged culture time. This is because at
low IL concentration, the proliferation of cells is greater than the in-
hibition of ILs. And when the IL concentration reached the corre-
sponding IC50 value (3959, 2226, 1884 μg/mL), the number of cells
showed that treatment with selected imidazolium ILs affected CCO cell
viability on a. anion-dependent manner [20]. However, in the present
study, we found that the toxicity was correlated with the concentration
of ionic liquids and the effects of ILs on MC3T3-E1 cell viability was
found to be dose-dependent, which indirectly showing that not all
“green solvents” are safe for human study [27].
3.2. Cells morphology in 2D culture after ionic liquid
Morphological changes in cell structure were observed by Calcein/
PI/Hoechst 33342 triple staining after the MC3T3-E1 cells were exposed
to different concentrations of ILs, as shown in Fig. 3 for 24 h, 48 h and
72 h, respectively. The Hoechst entered into both viable and nonviable
cells via intercalating into DNA, and Calcein entered in viable cells to
mark the cytoplasm, while PI was excluded from viable cells with intact
cell membrane. Untreated MC3T3-E1 cells showed that nucleus still had
a round and green nuclei, and the MC3T3-E1 cells were able to reach
95% confluency in 72 h and the cells were found well connected and
forming a cell network. However, in all experimental groups, the num-
ber of cells was gradually reduced with increasing concentration of IL.
When the cells were treated with IL at 5000 μg/mL for 24 h, some cells
were observed with shrinking and rupturing. When the treatment
Fig. 3. Evaluation of cytotoxicity of IL at different concentrations to MC3T3-E1 cells in 2D culture. (a–c) The cytotoxicity of IL on cell in 2D culture after 72 h of incubation by a Live/Dead
staining. (40×) Scale: 500 μm. (d) Calcein staining of MC3T3-E1 cells after the treatment of the IL at different concentrations (1000 μg/mL, IC50 (3959, 2226, 1884 μg/mL) and 5000 μg/mL)
(200×) Scale: 250 μm.

J. Zhu et al. / International Journal of Biological Macromolecules 158 (2020) 800–810
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Fig. 4. (a) The cytotoxicity of IL on cell in 2D culture plates after incubation for 48 h by HE staining. (200×) Scale: 250 μm. (b) Scanning electron microscopy (SEM) of MC3T3-E1 cells after
the treatment of [Emim]DEP-type IL for 48 h (3000×). a1–b1: 0 μg/mL, a2-b2: 1000 μg/mL, a3–b3: 2226 μg/mL(IC50), a4–b4: 5000 μg/mL.
in each experimental group was close to half of the corresponding
blank group cell number. When cells treated with 5000 μg/mL IL,
the signals of green fluorescence was significantly reduced, and the
signals of red fluorescence was remarkably increased, which indi-
cates that more cells have been ruptured into fragments resulting
in a decrease in calcein staining.
H&E staining (Fig. 4a) and SEM (Fig. 4b) showed that the cells in
the control groups were well distributed on the surface of well
plate, the morphology of cells remains long fusiform shapes with
good confluency and the cells were well connected via their inter-
cellular matrix. Similar to previous findings in fluorescence micros-
copy, increased dose of IL resulting in significant drop in living cell
number. Moreover, the morphology of cell was found changed.
These results further confirmed that the apoptotic rate of MC3T3-
E1 cells is negatively correlated with the spreading area of single
cells, and positively correlated with the IL concentration, showing
its inhibitory role in cell proliferation as previously reported
study [25].
Fig. 5. Microstructure and element analysis of nHAp/Cs/Gel scaffolds. (a) a1-a4: SEM of hybrid scaffolds (400×) a1: 0% nHAp/Cs/Gel, a2: 20% nHAp/Cs/Gel, a3: 40% nHAp/Cs/Gel, a4: 60%
nHAp/Cs/Gel, a5–a8: SEM of hybrid scaffolds (1600×) Scale: 50 μm. (b) Contact angle of nHAp/Cs/Gel scaffolds, b1: 0% nHAp/Cs/Gel, b2: 20% nHAp/Cs/Gel, b3: 40% nHAp/Cs/Gel, b4: 60%
nHAp/Cs/Gel. (c) Elemental analysis of the nHAp/Cs/Gel scaffold.

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3.3. Physical properties of scaffold
due to the inhomogeneous dispersion of nHAp in the solution (red ar-
rows in Fig. 5a8).
The toxic effects of ionic liquids on cells in a 2D environment have
been studied. Since the 2D environment only acts on monolayer cells,
the detection results are highly inaccurate and unstable compared
with in vivo experiments. Because of that, a 3D culture systems for cyto-
toxicity research which can better mimic in vivo cell microenvironment
need to be developed to provide more inclusive research for In vitro
studies. There is related research reported that chitosan and gelatin
are used to develop 3D cell culture scaffolds which shows good biocom-
patibility [38]. In the study, it has been found that moderate incorpora-
tion of nHAP can made up the shortcomings of mechanical properties of
natural materials (chitosan, gelatin).
The microstructures of the scaffolds were examined by SEM
(Fig. 5a). SEM analysis showed that the surface of the scaffolds was ir-
regular and had a large specific surface area, which may provide more
initial adhesion sites for the inoculated cells and facilitated the growth
and proliferation of subsequent cells [41]. With the incorporation of
nHAp, the pore size of the nHAp/Cs/Gel scaffolds increased correspond-
ingly. As shown in the SEM images (Fig. 5a), increasing nHAp incorpora-
tion led to a thicker wall and surface of the pores in scaffolds which may
be able to contribute cell capture and adhesion [41]. However, an exces-
sive increase in the pore wall thickness may also have effect on porosity,
which can lead to a reduction in the space of living cells. In addition,
from the SEM partial magnification, we noticed that almost no deposits
were identified on the surface of 20% nHAp scaffold (Fig. 5a6). As the
content of nHAp increased to 40%, a small amount of nHAp particle clus-
ters were deposited on the surface and pores of the scaffolds (yellow ar-
rows in Fig. 5a7). When 60% nHAp was introduced, a large amount of
nHAp particles were deposited on the surface and pores of the scaffolds
The contact angle of the scaffold was used to evaluate its hydrophi-
licity. The contact angle of Cs/Gel scaffolds was the smallest at
54.05 1.42% (Fig. 5b1). The contact angle of 20% nHAp/Cs/Gel scaffold
was 72.15 2.12% (Fig. 5b2). With the increase of nHAp concentration,
the contact angle of the scaffold gradually increased, suggesting that the
hydrophilicity of the scaffold decreased as the content of nHAap in-
creased. It indicated that the addition of hydrophobic nHAp nanoparti-
cles affected the hydrophilicity of the scaffold material which was
consistent with the previous study [45].
In addition, the elemental composition of nHAp/Cs/Gel was analyzed
(Fig. 5c). The results showed that when nHAp was added, the percent-
age of C and H decreased, while the percentage of phosphorus in-
creased. It indicated that nHAp nanoparticles were successfully
introduced into the Cs and Gel.
Results from stress-strain tests demonstrated that the stress of
the scaffolds (Fig. 6a) changes with different nHAp ratio. The elastic
modulus of scaffolds was further calculated based on the stress-
strain curve and the specifications of scaffolds (Fig. 6b). The elastic
modulus of the nHAp/Cs/Gel scaffolds were found significantly
higher compared to the blank Cs/Gel scaffolds (960
2.41%MPa).
The elastic modulus of the 20% nHAp/Cs/Gel scaffolds was measured
at 1002 1.37% MPa, but with additional incorporation of nHAp, the
elastic modulus was gradually decreased. nHAP has unique biologi-
cal activity, which can overcomes low plasticity and high brittleness
of the traditional hydroxyapatite. However, if the incorporation of
nHAp is too much, the brittleness will increase accordingly and the
compression modulus will decrease. The elastic modulus of the 40%
nHAp/Cs/Gel scaffolds was measured at 776
1.29% MPa; 60%
Fig. 6. Physical properties of nHAp/Cs/Gel scaffolds. (a) Stress-strain curves of nHAp/Cs/Gel scaffolds of different proportions. (b) Compressive elastic modulus of nHAp/Cs/Gel hybrid
scaffolds. (c) Porosity and water absorption rate of nHAp/Cs/Gel scaffolds. (d) Degradation rate of nHAp/Cs/Gel scaffolds. Data indicate mean SD. ** p b 0.01.

J. Zhu et al. / International Journal of Biological Macromolecules 158 (2020) 800–810
807
nHAp/CS/Gel scaffolds was measured at 379 2.38% MPa. Which in-
3.4. Biocompatibility of scaffolds
dicates that the 20% nHAp/Cs/Gel scaffolds have the highest elastic
modulus. This is because that nHAp are able to uniformly disperse
in the scaffolds at 20% concentration to improve mechanical proper-
ties of the scaffolds, but the excessive nHAp may induce local densi-
fication and therefore weaken the mechanical properties. Sufficient
space and porosity are known to be the key factors that can affect
cell proliferation in 3D scaffolds.
The porosity and water absorption of scaffolds were also measured
(Fig. 6c). The porosity was measured between approximately 81% and
94% depends on the compositions of scaffolds. Interestingly, 20%
nHAp/Cs/Gel scaffold was found to have the highest porosity of 88.59%
and increased nHAp resulted in decreased porosity, which has the sim-
ilar trend as mechanical property test. This is because incorporating a
certain amount of nHAp into the scaffolds can increase the porosity,
but excessive incorporation will leads to nHAp deposition and local den-
sification which can reduce scaffold porosity. With the increase of nHAp,
the water absorption was found increased at first and then decreased.
The water absorption of 20% nHAp incorporated scaffold was measured
The cytocompatibility of the scaffolds was assessed via inoculating
the cells on the scaffolds, following by fluorescence microscopy analysis
(Fig. 7). The images revealed that the cells were well attached and dis-
tributed on the surface of the scaffolds and the pore walls. PI staining
showed that there were no obvious dead cells in the scaffolds of each
group, indicating that the nHAp/Cs/Gel scaffolds had good biocompati-
bility. Indeed, chitosan, as a natural material, has the advantages of
non-toxicity, good biocompatibility, low immunogenicity, which were
good for cell proliferation [35]. When comparing the control group
with the experimental group, 20% nHAp/Cs/Gel scaffolds were found
to have the most cell proliferation and the cells were well stretched
and secreted a large amount of extracellular matrix to form a piece
with other cells. The finding has shown that the addition of nHAP will
improve the mechanical properties of the scaffolds and also increase
the surface roughness of the scaffolds to facilitate cell adhesion [42]. In
this study, by adding different ratios of nHAP to the scaffold, it was
found that only when the incorporation of nHAP was at a suitable
ratio, the biocompatibility of the scaffold was better. Therefore, 20%
nHAp/Cs/Gel hybrid scaffolds were selected for the following
experiments.
the highest at 1300
porosity.
2.67%, which is consistent with the results of
Fig. 6d showed the relationship between the degradation rate
(weight loss rate) of nHAp/Cs/Gel scaffolds and the immersion time.
Since both Cs and Gel components were degradable polymers, the Cs/
Gel scaffold degraded fastest in PBS solution. While nHAp itself hardly
degraded and the introduction of nHAp into Cs/Gel had a stronger inter-
action with Cs/Gel, the degradation rate significantly slowed down.
However, the organic network structure of Cs/Gel failed to support the
high content of nHAp in the early stage of immersion, and part of
nHAp was eluted from the composite scaffold. Therefore, the degrada-
tion rate of 40% and 60% nHAp/Cs/Gel were higher than that of 20%
nHAp/Cs/Gel. Subsequently, with the content of nHAp in the scaffolds
increasing, the degradation rate decreased due to the relatively few de-
gradable components. And the degradation rate curve of 20% nHAp/Cs/
Gel scaffold was similar to that of Cs/Gel scaffold. After 30 days of im-
mersion, the degradation rate of 20% nHAp/Cs/Gel scaffold reached
53.28% 0.39%, which was acceptable for tissue engineering scaffolds.
3.5. MTT assay on 3D scaffold after ionic liquid
Cell biology studies and drug toxicity tests rely heavily on the use of
conventional 2D cell culture systems. But the toxicity tests of drugs
were often found inefficient due to poor correlation between 2D cell
culture models and human pathophysiology. As a result, the OD values
of MC3T3-E1 cells in 3D culture with the treatment of IL were deter-
mined by MTT assay, and the cell inhibition rate-concentration curve
was fitted (Fig. 8a1–a3). When the IL was added to the cell-scaffold con-
structs for over 24 h, 48 h, and 72 h, the IC50 values were measured at
12566 μg/mL, 9015 μg/mL, and 7896 μg/mL, respectively. Compared
with the IC50 value on the 2D level, the IC50 value collected from the
3D model was significantly increased by 3–4 folds (Fig. 8b), suggesting
that the survival rate of cells on the 3D level is significantly higher. In
Fig. 7. Fluorescence staining of MC3T3-E1 cells on nHAp/Cs/Gel hybrid scaffolds. 72 h (40×) (Scale: 500 μm), A1–C1: 0% nHAp/Cs/Gel scaffolds, A2–C2: 20% nHAp/Cs/Gel scaffolds, A3–C3:
40% nHAp/Cs/Gel scaffolds, A4–C4: 60% nHAp/Cs/Gel scaffolds.

808
J. Zhu et al. / International Journal of Biological Macromolecules 158 (2020) 800–810
Fig. 8. (a1–a3) Inhibition rate-concentration fitting curve of [Emim]DEP-type IL acting on cells in 3D culture, a1: 24 h, a2: 48 h, a3: 72 h. (b) Comparison of IC50 value on 2D and 3D.
*p b 0.05, **p b 0.01, ***p b .001 versus the 2D culture.
previous reports, cancer cells cultured in 3D the cell-scaffold construct
was found to exhibit stronger resistance to IL than their 2D counterparts
[26]. This is because the cells in the 3D culture adhered to the surface
and the inside of pores of scaffolds, IL needs to diffuse into the inside
of pores to directly contact the cells, that is, the specific surface area is
increased [41]. In addition, the cells are agglomerated on the hybrid
scaffolds. There may be IL penetration gradients when ILs act on cell
colonies.
Through 2D and 3D comparisons, it is found that the three-
dimensional biomimetic environment is more resistant to ionic liquids,
which indicates that the 3D experimental data is more accurate.
Thereby further filling the [Emim]DEP-type IL cytotoxicity data gap.
Fig. 9. Live/Dead and distribution of MC3T3-E1cells after the treatment of [Emim]DEP-type IL for 24 h, 48 h in 3D scaffolds (100×) Scale: 500 μm. (a) a1–a4: 0 μg/mL, b1–b4: 3000 μg/mL,
c1–c4: 12566 μg/mL, d1–d4: 20000 μg/mL. (b) a1–a4: 0 μg/mL, b1–b4: 3000 μg/mL, c1–c4: 9015 μg/mL, d1–d4: 20000 μg/mL.

J. Zhu et al. / International Journal of Biological Macromolecules 158 (2020) 800–810
809
Fig. 10. SEM of MC3T3-E1 cell-scaffold construct acted by the [Emim]DEP-type IL for 48 h, a1–a3: 0 μg/mL, b1–b3: 3000 μg/mL, c1–c3: 9015 μg/mL, d1–d3: 20000 μg/mL, (a1–d1) × 200,
(a2–d2) × 800, (a3–d3) × 1600.
3.6. Cell morphologies in 3D hybrid scaffolds after ionic liquid
apparent interconnection between the cells, and the number of cells
was significantly reduced in Fig. 8b3. When the concentration of IL
reached 20,000 μg/mL, no intact cells were apparent on the surface of
the scaffolds, but only cell debris (red arrows in Fig. 10d4). In summary,
these findings suggest that the IL inhibited cell proliferation of the cell-
scaffold microstructure was successfully constructed, and the higher the
concentration of the IL, the stronger the inhibition of cell proliferation.
Previous studies have shown that ionic liquids are less toxic to mono-
layer cultured skin cells than to three-dimensionally reconstructed
skin models. A 3D reconstituted human full skin model is composed of
well-differentiated keratinocytes and fibroblasts, and it has a certain
barrier function that is absent in 2D monolayer cell culture. Therefore,
this model is expected to exhibit more in vivo-like morphological and
growth characteristics, in addition to providing much more accurate
data for the evaluation of dermal toxicity of ionic liquids.
When the [Emim]DEP-type IL acted on the MC3T3-E1 cell-
scaffold constructs for 24 h and 48 h, the cell-scaffold constructed
was stained with Calcein/PI/Hoechst fluorescent dye (Fig. 9). The
cells adhered to scaffold and grew well when there was no IL treat-
ment. The cells were well connected to each other and formed a
cell network. However, it has been founded that the numbers of
cells in the experimental groups were continually reduced with the
increase of the concentration of IL. Cells were found to have uneven
distribution which is because that dead cells after IL treatment can
be easily washed out during the sample preparation. When the
cells were treated with 20,000 μg/mL IL for 72 h, 90% of the cells
showed cell apoptotic and could not adhere to the scaffold. When
the same concentration of IL was used but with different action
time, the results further confirmed that the cells inhibition was pos-
itively correlated with the action time of IL. When the concentration
of IL was at 3000 μg/mL, the number of cells cultured for 48 h in-
creased compared to cells cultured for 24 h, because at a lower IL
concentration, the cell proliferation ability was stronger than the in-
hibition of IL; when 20,000 μg/mL IL acted on the cells, N90% cells are
apoptotic. And the number of cells cultured for 48 h is less in compar-
ison with culture for 24 h, which indicated that the cell proliferation
ability was stronger than the inhibition of IL when the concentration
of IL was at 20,000 μg/mL. This proved that when the ionic liquid con-
centration increased, the inhibition effect on cell proliferation was
enhanced.
4. Conclusion
In this experiment, the toxicity of [Emim]DEP-type IL was examined
using both 2D and 3D cell-scaffold models in vitro. By characterizing and
analyzing the mechanical properties and biocompatibility of hybrid
scaffolds, 20% nHAp/Cs/Gel scaffold was selected as the 3D stereo cell
models in vitro. Both 2D and 3D models experiments found that the IL
inhibited the cell proliferation and the inhibitory effects were positively
correlated with increased concentration and action time of IL. If the IL
reached a half lethal dose in both 2D cells culture and 3D cells-scaffold
culture, the IL concentration required for 3D was 3.17, 4.05, and 4.19
times, respectively that of 2D, which is related to the increased specific
surface area of the cells on the scaffold and the depth of penetration of
IL. In this study, we reported that a 3D scaffolds prepared with nHAp/
Cs/Gel can be utilized as a 3D model of biological environment to assess
the toxicity of IL In vitro. Moreover, the 3D culture systems are easy to be
reproduced, which can forms a fundamental base between 2D and 3D
in vitro models in studying IL toxicology.
To further assess the cytotoxicity of IL on the cell-scaffolds, the mi-
crostructure of the cell-scaffold construct cultured for 48 h was scanned
using SEM at 200×, 800× and 1600× magnification (Fig. 10). SEM anal-
ysis indicated that MC3T3-E1 cells without the treatment of IL adhered
to the wall of scaffolds (yellow arrow) and embed with other cells in the
pores of scaffolds (blue arrow). When 3000 μg/mL of IL was used to treat
cells, the number of cells dropped and the morphology of cells shrank
into sphere from the stretched state (Fig. 10b1–b3). There was no

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CRediT authorship contribution statement
Jingjing Zhu:Data curation, Writing - original draft, Writing - review
& editing.Qian Kong:Software, Writing - original draft.Shuangshuang
Zheng:Investigation, Validation.Yiwei Wang:Writing - review &
editing.Zeren Jiao:Writing - review & editing.Yi Nie:Supervision.
Tianqing Liu:Conceptualization, Methodology.Kedong Song:Supervi-
sion, Project administration, Conceptualization, Methodology.
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Declaration of competing interest
The authors have no conflicts of interest.
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This work was supported by the National Natural Science Founda-
tion of China (31670978/21676041), the Fok Ying Tung Education
Foundation (132027), the State Key Laboratory of Fine Chemicals
(KF1111), and the Natural Science Foundation of Liaoning
(20180510028) and the Fundamental Research Funds for the Central
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