In vitro studies demonstrated that mouse bone mesenchymal stem cells mBMSCs cultured on chitosan physical hydrogels had better adhesion and proliferation than those cultured on chitin hy
Trang 1Tough and Cell-Compatible Chitosan Physical Hydrogels for Mouse Bone Mesenchymal Stem Cells in Vitro
Beibei Ding,†,⊥ Huichang Gao,‡,⊥ Jianhui Song,§ Yaya Li,† Lina Zhang,† Xiaodong Cao, *,‡ Min Xu,§ and Jie Cai *,†
†College of Chemistry & Molecular Sciences, Wuhan University, Wuhan 430072, People ’s Republic of China
‡School of Materials Science and Engineering, South China University of Technology, Guangzhou 510641, People ’s Republic of China
§Department of Physics, Shanghai Key Laboratory of Magnetic Resonance, East China Normal University, Shanghai 200062, People ’s Republic of China
*S Supporting Information
ABSTRACT: Most hydrogels involve synthetic polymers and organic
cross-linkers that cannot simultaneously ful fill the mechanical and
cell-compatibility requirements of biomedical applications We prepared a new
type of chitosan physical hydrogel with various degrees of deacetylation
(DDs) via the heterogeneous deacetylation of nanoporous chitin hydrogels
under mild conditions The DD of the chitosan physical hydrogels ranged
from 56 to 99%, and the hydrogels were transparent and mechanically
strong because of the extra intra- and intermolecular hydrogen bonding
interactions between the amino and hydroxyl groups on the nearby chitosan
nano fibrils The tensile strength and Young’s modulus of the chitosan
physical hydrogels were 3.6 and 7.9 MPa, respectively, for a DD of 56% and
increased to 12.1 and 92.0 MPa for a DD of 99% in a swelling equilibrium
state In vitro studies demonstrated that mouse bone mesenchymal stem
cells (mBMSCs) cultured on chitosan physical hydrogels had better
adhesion and proliferation than those cultured on chitin hydrogels In particular, the chitosan physical hydrogels promoted the
di fferentiation of the mBMSCs into epidermal cells in vitro These materials are promising candidates for applications such as stem cell research, cell therapy, and tissue engineering.
KEYWORDS: chitosan, hydrogels, heterogeneous deacetylation, mechanical properties, cell-compatibility
■ INTRODUCTION
Hydrogels, which are three-dimensional polymeric materials
with high water contents and diverse physical properties, have
been extensively used in food, cosmetics, drug-delivery devices,
and other applications.1 The emergence of potential
applications for hydrogels include stem cell and cancer research,
cell therapy, tissue engineering, immunomodulation, and in
vitro diagnostics.2−7 However, the disadvantage of traditional
hydrogels is poor mechanic properties due to high water
content and structural defects at swollen state Therefore,
several new approaches have been introduced to fabricate
mechanically tougher hydrogels,8 including forming
super-molecular interactions,9,10 chemical cross-linking,11,12 and a
double network structure.13−15
Unfortunately, most hydrogels involve synthetic polymers
and organic cross-linkers that cannot simultaneously ful fill the
mechanical and cell-compatibility requirements of biomedical
applications Alternatively, natural polymers, such as alginate,
chitosan, hyaluronidase, and collagen, have been shown to be
promising biomaterials.16−18Among them, chitosan, a natural
amino polysaccharide derived from chitin, which is the main
component of the exoskeletons of crustaceans (e.g., crabs and shrimp), has received considerable attention because of its excellent biodegradability, biocompatibility, and bioactiv-ity.19−24The exploitation of chitosan hydrogels for biomaterials
is, however, limited by the poor solubility and mechanical integrity, di fficulty in fabrication, and requirement for organic cross-linkers.25−31
In our previous works, nanoporous chitin hydrogels prepared
in aqueous NaOH/urea showed remarkable mechanical strength and biocompatibility.32,33 These materials were characterized as having a large interior space with a three-dimensional open network structure, and thus, they may be directly converted into chitosan physical hydrogels via reactions with deacetylation reagents under mild conditions In this work,
we demonstrate the toughness and excellent cell-compatibility
of chitosan physical hydrogels based on the in situ heterogeneous deacetylation of nanoporous chitin hydrogels
Received: May 4, 2016 Accepted: July 13, 2016 Published: July 13, 2016
www.acsami.org
Trang 2under mild conditions and show that these properties can be
attributed to the formation of extra intra- and inter-molecular
hydrogen bonding interactions between the amino and
hydroxyl groups on the nearby chitosan nano fibrils Unlike
the existing chitosan chemical hydrogels described in the
literature, our method allows for the formation of tough and
cell-compatible chitosan physical hydrogels that are suited for
stem cell culture and promote di fferentiation into epithelial
cells.
■ EXPERIMENTAL SECTION
Materials The raw chitin powder was purchased from
Golden-Shell Biochemical Co Ltd (Zhejiang, China) The raw chitin powder
was purified with 0.1 mol Lư1aqueous NaOH at ambient temperature
overnight and combined with 0.3% (w/w) aqueous NaClO2buffered
to pH 4.7 with acetate buffer at 80 °C for 3.5 h Washing with
deionized water was performed after each step to remove any residual
proteins and chemical regents The purification procedures were
repeated twice The purified chitin powder was finally freeze-dried, and
its viscosity-average molecular weight (Mη) was calculated to be 10.7×
104in 5% (w/w) LiCl/N,N-dimethylacetamide (DMAc) at 25± 0.02
°C by viscometry.34
Fabrication of Chitin Hydrogels The purified chitin powder was
dispersed in aqueous 11% NaOH-4% urea (w/w) and then frozen at
ư30 °C overnight Subsequently, after thawing at 5 °C, the chitin was
dissolved completely and used to form a transparent and viscous 7%
(w/w) chitin solution according to our previous method.32The chitin
solution was centrifuged at 5°C for 15 min to prevent gelation and
remove air bubbles The resultant chitin solution was spread on a glass
plate as a 1.0 mm-thick layer and then immersed in ethanol at 5°C for
1 h to produce the chitin gels The gels were then thoroughly washed
with deionized water to create the chitin hydrogels
Heterogeneous Deacetylation of Chitin Hydrogels Typically,
chitin hydrogels were immersed in 35% (w/w) aqueous NaOH at 60
°C for 6 h The deacetylation was stopped by removing the hydrogels
from the aqueous NaOH and then immersing them into 50% (v/v)
aqueous ethanol The degree of deacetylation (DD) of the hydrogels
can be controlled by the number of heterogeneous deacetylation
cycles The deacetylated chitin hydrogels, that is, the chitosan
hydrogels coded as S1, S2, S3, and S4, were obtained by one, two,
three, and four heterogeneous deacetylation cycles, respectively
Characterization The weight-average molecular weight (Mw) of
the chitosan was performed on a size exclusion chromatography
combined with multiangle laser light scattering (SEC-LLS) (DAWN
EOS, Wyatt, USA) equipped with a HeưNe laser (λ = 632.8 nm) A
p100 pump equipped with a TSK GEL G6000 and G4000 PWXL
column (MicroPak, TSK) and an Optilab refractometer (Wyatt, USA)
was combined with the instrument Thefluent was 0.1 M NaAc/HAc
buffer (pH = 2.8) with a flow rate of 0.6 mL minư1
The DDs of the chitin and chitosan physical hydrogels was
calculated by potentiometry The hydrogels were cut into small pieces
and freeze-dried from t-BuOH The quantitative dried gel was
accurately weighed and added to 0.1 M aqueous HCl solution
Subsequently, the mixture was titrated with 0.1 M NaOH, with the
standard substance KH5C8O4used for calibration The DD value was
calculated as follows:35
×
DD V V C
W
0.0994
2 1
(1) where C is the accurate concentration of aqueous NaOH solution
(mol Lư1), V1is the volume of aqueous NaOH solution (mL) at the
first titration jump, V2is the volume of aqueous NaOH solution at the
second titration jump, W is the sample weight (g), 0.016 is the molar
mass weight of NH2(kg molư1), and 0.0994 is the theoretical NH2
percentage in chitosan
Scanning electron microscopy (SEM) images of the cross sections
of the chitin and chitosan hydrogels was carried out on a Hitachi
S-4800 instrument The chitin and chitosan physical hydrogels were
solvent-exchanged with absolute ethanol and then dried from supercritical CO2to give dried gels
Fourier transform infrared spectroscopy (FT-IR) analyses were carried out on a FT-IR spectrometer (Nicolet 5700 FTIR Spectrometer, MA) The powdered samples and KBr were mixed and loaded into the sample holder The spectra in the range of 400ư
4000 cmư1were collected in 32 scans at 4 cmư1resolution The polymorphisms of the crystals in the chitin and chitosan gels were determined by X-ray diffraction (XRD, D8-Advance, Bruker, USA) over the 2θ range from 5° to 40° with 40 kV and 40 mA Ni-filtered CuKα radiation The powdered samples were used to eliminate the effect of the crystalline orientation The peak position and crystallinity (χc) of the chitin and chitosan gels were estimated from multipeakfitting of the XRD profiles
Solid-state cross-polarization/magnetic angle spinning (CP/MAS)
13C nuclear magnetic resonance (NMR) spectra were collected on a Bruker AVANCE-300 Spectrometer (13C frequency = 75.4 MHz) with
a standard 4 mm rotor at ambient temperature The spinning rate was kept at 5.0 kHz The contact time and relaxation time were 1.0 ms and 4.0 s, respectively Two thousand scans were collected for each sample The light transmittance of the chitin and chitosan physical hydrogels was determined by ultravioletưvisible (UVưvis) spectros-copy (UV-6, Mapada, China) at wavelengths ranging from 400 to 800 nm
Dynamic mechanical analysis (DMA) temperature sweep was performed on a DMA Q800 (TA Instruments, USA) under oscillatory stress in tensile mode from ư50 to 300 °C The heating rate and frequency were 5°C minư1and 1 Hz, respectively The width of the samples was approximately 5 mm
Thermogravimetric analysis (TGA) was conducted using a STA 449C (Netzsch, German) from 25 to 600°C at a heating rate of 10 °C minư1under nitrogen
The hydrogels were subjected to tension tests on a universal tensile tester (CMT 6503, SANS, China) The hydrogel was stretched at a 2
mm minư1 stretch speed at ambient temperature The modulus was calculated from the initial linear regions of the stressưstrain curves Mouse bone mesenchymal stem cells (mBMSCs, ATCC, CRL-12424) were propagated in Dulbecco’s Modified Eagle’s Medium (DMEM, Gibco) supplemented with 10% fetal bovine serum (FBS, Gibco) Chitin and chitosan physical hydrogels were placed in 96-well plates and then sterilized in 75% (v/v) aqueous ethanol for 2 h followed by three rinses with sterilized phosphate-buffered saline (PBS) Subsequently, the hydrogels were prewetted with culture medium for 12 h After removing the culture medium, 200μL of the mBMSCs suspension (1 × 104 cells wellư1) was seeded on the hydrogels and then incubated at 37°C in a humidified incubator at 5%
CO2 The Cell Counting Kit-8 (CCK-8, Dojindo Laboratories, Japan) was used to evaluate the cell proliferation on the hydrogels after 1, 3, 5, and 7 days of culture Briefly, at each time point, the culture medium was removed, and the CCK-8 working solution was added at 37°C for
2 h Subsequently, the supernatant medium was extracted to determine the absorbance at 450 nm using a Thermo 3001 microplate reader (Thermo, USA) (n = 5) The cell viability and morphology on the hydrogels were characterized using a Live/Dead assay kit (Dojindo Laboratories, Japan) The cell-seeded hydrogels were thoroughly washed with PBS Subsequently, the hydrogels were incubated in standard working solution for 30 min After washing again with PBS, the hydrogels were imaged using an Eclipse TiưU fluorescence microscope (Nikon, Japan) SPSS 12.0 software (SPSS, USA) was used to analyze the results with one-way analysis of variance (ANOVA) The data are presented as the mean-standard deviation
To compare the differentiation of the mBMSCs into epidermal cells
on the hydrogels, the mBMSCs were seeded onto the chitin and chitosan physical hydrogels at a density of 1.5× 104cells cmư2 After culture for 7 d in the presence of 30 ng mLư1 recombinant human EGF (PeproTech, USA) and 50 ng mLư1recombinant murine IGF-1 (PeproTech, USA), the nuclei were stained by DAPI and the cell morphology were observed by laser scanning confocal microscopy (Leica, Germany) Additionally, reverse transcriptase polymerase chain reaction (RT-PCR) was used to evaluate the expression of the
Trang 3epidermal cell differentiation marker gene K18 and K19 The following
primer sequences were used: K18 gene: forward,
5′-AAGGCTG-CAGCTGGAGACAGA-3′; reverse,
3′-TGGGCTTCCAGACCTTG-GAC-5′; K19 gene: forward,
5′-TGACCTGGAGATGCAGATTGA-GA-3′; reverse, 3′- TGGAATCCACCTCCACACTGAC-5′; and
GAPDH gene: forward, 5′-TGTGTCCGTCGTGGATCTGA-3′;
reverse, 3′-TTGCTGTTGAAGTCGCAGGAG-5′ The relative
quan-tification of the target gene was normalized to GAPDH and
determined using the 2ưΔΔCt method At the end of each PCR, the
melting curve profiles were generated to identify the specific
transcription of the amplification To evaluate the in vitro degradation
of the chitosan hydrogels, certain weights of the chitin hydrogel and
chitosan hydrogels were immersed in 4 mg/mL
three-times-recrystal-lized egg white lysozyme (TSZ, USA) in 0.1 M PBS at pH 7.4 and 37
°C.20After specific time intervals, the hydrogels were removed from
the lysozyme solution, thoroughly washing with double distilled water
and freeze-dried The extent of the in vitro degradation was calculated
from the percentage of the weight of the dried hydrogels before and
after the lysozyme treatment
■ RESULTS AND DISCUSSION
The preparation of the chitosan physical hydrogels from the
nanoporous chitin hydrogel by in situ heterogeneous
deacetylation cycles is described in Figure 1 The chitin
hydrogel is a transparent nanoporous material, which forms via
the hydrogen bonding of the chitin ưNaOHưurea aqueous
solution by a sol ưgel transition in ethanol without an external
cross-linker It has a large interior space that reacts with the
deacetylation reagents and can thus be converted into a
chitosan physical hydrogel under mild conditions The chitin hydrogel was subjected to in situ heterogeneous deacetylation
in 35% (w/w) aqueous NaOH at 60 °C for 6 h Then, the NaOH was almost fully removed using a 50/50 ethanol/water mixture followed by the addition of deionized water, and a chitosan physical hydrogel containing free amine groups formed The ưNHCOCH3 sites were deacetylated to give
ưNH2 groups, leading to the disappearance of hydrophobic interactions between the polymeric chains, which favored physical cross-links corresponding to hydrogen bonding interactions This procedure was repeated four times to obtain chitosan physical hydrogels with 4 di fferent DDs, which, as expected, ranged from 56 to 99% ( Table 1 ) The
weight-Figure 1.(a) Schematic representation of the creation of a chitosan physical hydrogel from a nanoporous chitin hydrogel (b) Photographs of the square chitin hydrogel (S0) and chitosan physical hydrogels (S1ưS4) at each heterogeneous deacetylation cycle (c) Macroscopic views of the chitosan physical hydrogels (S4) under stretching, torsional and rolling loading
Table 1 Physical Properties of the Chitin Hydrogel and Chitosan Physical Hydrogelsa
samples DD, % WH2O, % M × 10 ư4 , g/mol crys % σ b , MPa ε b , % E, MPa S0 8 84 10.7 50 1.7 ± 0.1 56 ± 4 4.5 S1 56 61 4.9 35 3.6 ± 0.5 67 ± 5 7.9 S2 80 58 4.0 34 10.5 ± 0.5 106 ± 14 34.5 S3 91 51 3.9 36 12.1 ± 1.1 67 ± 5 57.3 S4 99 50 3.1 43 12.1 ± 0.7 57 ± 4 92.0
aThe DD and WH2Oare the degree of deacetylation and the water content, respectively, of the chitin hydrogel (S0) and chitosan physical hydrogels (S1ưS4).The viscosity-average molecular weight (Mη) of sample S0 was determined by viscometry, and the weight-average molecular weights (Mw)
of samples S1ưS4 were determined by SEC-LLS Crys is the degree of crystalline of the dried gels The σb,εb, and E are the tensile strength, elongation at break, and Young’s modulus of the hydrogels, respectively
Figure 2.Top: SEM images of the surfaces of the chitin hydrogel S0 (a) and the chitosan physical hydrogels S1 (b), S2 (c), and S4 (d) Bottom: SEM images of the inner parts of the chitin hydrogel S0 (e) and the chitosan physical hydrogel S1 (f), S2 (g), and S4 (h) (scale bar
=500 nm)
Trang 4average molecular weight (Mw) of the chitosan physical hydrogels decreased from 5.6 × 104to 4.7 × 104g mol−1 At the swelling equilibrium state, the water content of the hydrogels decreased gradually from 84% for the chitin hydrogel (S0) to 50% for the chitosan physical hydrogel (S4) The gross visual appearance of the chitosan physical hydrogels showed that the chitin hydrogel underwent signi ficant volume changes after washing with aqueous ethanol, mainly because of the
di ffusion of ethanol, which disturbed the hydrophobic and hydrogen bonding interactions between the chitosan chains and thereby in fluenced the final density of the physical cross-linking and water content of the hydrogels ( Figure 1 b).19After four heterogeneous deacetylation cycles, the volume of the chitosan physical hydrogel was approximately one-third of that of the pristine chitin hydrogel, generating chitosan physical hydrogels with maximum physical cross-linking density Thus, the chitosan physical hydrogels demonstrated good mechanical integrity under stretching, torsional and rolling loading ( Figure
1 c).
The SEM images of the surface and inner part of the chitin gel ( Figure 2 a and e) show an open nanoporous network structure composed of interconnected chitin nano fibrils The typical diameter of the chitin nano fibrils was approximately 10
nm, which is in good agreement with the Brunauer−Emmett− Teller (BET) surface area of 364 m2 g−1, as determined by nitrogen adsorption and desorption isotherms (see Supporting Information , Figure S1), which corresponds to a fibril width of
7 nm Moreover, the SEM images and the nitrogen adsorption −desorption isotherms of the chitosan physical gels ( Figure 2 b −h, Figure S1 ) show features of a smaller porous structure and surface area after the heterogeneous deacetyla-tion The fibrils that comprise the networks of the chitosan physical hydrogels seem to gradually thicken, and therefore, chitosan likely sticks together to create a close network structure in the fourth deacetylation cycle (S4).
The FT-IR spectrum of the chitin gel ( Figure 3 , S0) shows the characteristic peaks of α-chitin, including broad OH stretching absorption peaks at 3447 and 3268 cm−1, a CH3 stretching absorption peak at 3100 cm−1, and splitting of the
C O stretching absorption peak at 1660 and 1627 cm−1for amide I and 1560 cm−1 for amide II.36−38 After the heterogeneous deacetylation cycles, the CH3 stretching absorption peak of the chitosan physical gel was nearly absent, the amide I and II stretching absorption peaks had gradually weakened, and the new N−H bending absorption peak at 1596
cm−1 was enhanced ( Figure 3 , S1 −S4), indicating the prevalence of NH2 groups and the successful formation of the chitosan physical hydrogels from chitin hydrogel Moreover, the OH stretching absorption peak of the chitosan physical gels shifted from 3447 to 3416 cm−1, indicating a lower-order structure of polymeric chains, which is consistent with the XRD patterns of the chitin and chitosan physical gels.
In the XRD patterns, the chitin gel ( Figure 4 , S0) shows characteristic peaks at 9.4 °, 12.8°, 19.3°, 20.8°, 23.4°, and 26.4°, corresponding to the (020), (021), (110), (120), (130), and (013) re flections, respectively, of an α-chitin crystal.37 , 39 , 40
The XRD patterns of the chitosan physical gels ( Figure 4 , S1 −S4) show near-systematic superposition with those of pure α-chitin and chitosan (DD of 100%), indicating a homogeneous distribution of the two components in the structure resulting from the in situ heterogeneous deacetylation cycles Moreover, the intensities of the (020), (021), (130), and (013) re flections
of the chitosan physical gels decreased gradually, and the
Figure 3 FT-IR spectra of the chitin hydrogel (S0) and chitosan
physical hydrogels (S1−S4)
Figure 4 XRD patterns of the chitin hydrogel (S0) and chitosan
physical hydrogels (S1−S4)
Figure 5 Solid-state CP/MAS 13C NMR spectra of the chitin
hydrogel (S0) and chitosan physical hydrogels (S1−S4)
Trang 5di ffraction angle of the (110) reflection increased, suggesting
that the crystallinity and crystallite size of the chitosan physical
gels decreased as the number of heterogeneous deacetylation
cycles increased The crystallinities estimated from multipeak fitting were 50% for the chitin gel and between 34 and 43% for the chitosan physical gels ( Table 1 ) Additionally, the crystallite sizes evaluated based on the (110) re flection using the Scherrer equation were 4.4 nm for the chitin gel (S0) and 3.7 nm for the chitosan physical gel (S4) These results are also consistent with the higher optical transmittance of the chitosan physical hydrogels relative to the chitin hydrogel (94% vs 81% at 800 nm) (see Supporting Information , Figure S2).
The structure change of the chitin hydrogel caused by the heterogeneous deacetylation was con firmed by the solid-state CP/MAS 13C NMR analysis ( Figure 5 ) The corresponding chemical shifts are listed in Table 2 The spectrum of the chitin gel shows the characteristic eight resonances of α-chitin: C1 (104.5 ppm), C2 (55.9 ppm), C3 (74.2 ppm), C4 (83.6 ppm), C5 (76.1 ppm), C6 (61.8 ppm), CH3(23.3 ppm), and C O (174.1 ppm).41−44Compared with the spectrum of the chitin gel, the C1 and C4 resonances of the chitosan physical hydrogels became weak and broad and shifted slightly, indicating a loosely packed structure and altered internal torsion angles of the polymeric chains.45,46 Moreover, the signals of C3 and C5 merged into a single resonance centered
at 75.9 ppm, and the signal intensities of the methyl and carbonyl carbons of the chitosan physical gels decreased gradually, disappearing after the final deacetylation cycle Simultaneously, all of the resonances of the deacetylated C2, which are primarily involved in hydrogen bonding with glucosamine units, of the chitosan physical gels shifted down field These spectral and morphological characteristics indicate that nanoscale heterogeneous deacetylation was achieved in the nanoporous chitin hydrogel; that is, the N-acetylglucosamine units on the surface of the interconnected chitin nano fibrils were removed, and the resultant amino groups interacted with the hydroxyl groups on the nearby nano fibrils to form extra intra- and inter-molecular hydrogen bonds.
The tensile strength ( σb), elongation at break ( εb), and Young ’s modulus (E) of the chitin hydrogel ( Figure 6 , S0;
Table 2 Chemical Shifts of the Chitin Hydrogel and Chitosan Physical Hydrogel Determined by CP/MAS13C NMR
chemical shift/ppm samples C7 (C O) C1 C4 C5 C3 C6 C2 C8 (CH3) S0 174.1 104.5 83.6 76.1 74.2 61.8 55.9 23.3 S1 174.3 104.5 83.2 75.9 75.9 61.1 58.2 23.5 S2 174.3 105.5 82.9 75.9 75.9 61.2 58.0 23.0 S3 105.3 82.9 75.7 75.7 61.7 58.5 24.0 S4 105.7 82.7 75.9 75.9 61.5 58.3
Figure 6.Typical stress−strain curves of the chitin hydrogel (S0) and
chitosan physical hydrogels (S1−S4) The inset is the stress−strain
curve of the chitosanfilm dried from sample S3
Figure 7.Proliferation of the mBMSCs cultured on the surfaces of the
chitin hydrogel (S0) and chitosan physical hydrogels (S1−S4)
Figure 8.Viability and morphology of the mBMSCs cultured on the
chitin hydrogel (S0) and chitosan physical hydrogels (S1−S4) for 48
h The green bright spots represent the living mBMSCs stained by
Calcein-AM The red spots indicate the dead mBMSCs stained by PI
(scale bar =100μm)
Figure 9.Morphology of the mBMSCs (a−e) and the nuclei staining
by DAPI (f−j) on the chitin hydrogel (S0) and chitosan physical hydrogels (S1−S4) after a 7-day differentiation period (scale bar =100 μm)
Trang 6Table 1 ) were 1.7 MPa, 56% and 4.5 MPa, respectively In
contrast, the tensile behavior of the chitosan physical hydrogels
( Figure 6 , S1 −S4; Table 1 ) showed remarkable strengthening
and toughening e ffects after the heterogeneous deacetylation.
The σb, εb, and E values of the chitosan physical hydrogels were
3.6 MPa, 67% and 7.9 MPa, respectively, for sample S1 (DD of
56%) and increased to 12.1 MPa, 57% and 92.0 MPa for sample
S4 (DD of 99%) at the swelling equilibrium state These values
were much higher than those of chitosan hydrogels obtained by
neutralization or chemical cross-linking of their acidic solutions
( σb, from 0.1 to 2.9 MPa; E, from 31 kPa to 4 MPa).22,24,47−50
Interestingly, the chitosan physical hydrogel (sample S2) had a
moderate σb of 10.5 MPa and, remarkably, an εb of 106%,
probably because of the lower degree of crystallinity and
physical cross-linking density in the chitosan hydrogel at a DD
of 80% Moreover, upon drying, the σb and E of the
heterogeneous-deacetylated chitosan film were 107.1 and
3053 MPa, respectively, con firming that the good mechanical
properties of the chitosan physical hydrogels are attributable to
hydrogen bonding interactions between the amino and
hydroxyl groups of the chitosan chains The DMA of the
chitosan film (see Supporting Information , Figure S3) revealed
typical behavior of a semicrystalline polymer The tensile
storage modulus (E ′) of the chitosan film was reduced from 58
to 24 MPa at temperatures from −50 to 200 °C, demonstrating
signi ficant mechanical stability Moreover, in the TGA of the
chitosan physical gels, decomposition was observed between
230 and 400 °C, regardless of the DD value (see Supporting
Information , Figure S4) As evidenced by the tensile, DMA, and
TGA results, the chitosan physical hydrogels demonstrated
strong mechanical properties and sufficient thermal adaptivity
for applications in biomaterials after heterogeneous
deacetyla-tion.
In this study, we aimed to exploit chitosan physical hydrogels
in tissue engineering repair, especially as adaptive substrates for
stem cells For this application, we chose mBMSCs as model
cells to assess the biological performance of our chitin and
chitosan physical hydrogels The proliferation and viability of
the mBMSCs cultured on the hydrogels were studied in vitro.
Figure 7 shows that the heterogeneous deacetylation of the
chitin hydrogels enhanced the adhesion and proliferation of the
mBMSCs on the surface of the resultant chitosan physical
hydrogels As the culture time increased, the mBMSCs
gradually proliferated in the chitin and chitosan physical
hydrogels Signi ficant differences for all of the samples were
observed after 5 days The proliferation rate of mBMSCs on the
surface of the chitosan physical hydrogels was superior to that
on the chitin hydrogel and was independent of the DD value of
the chitosan physical hydrogels.
Cell viability was also examined by a Live/Dead assay kit As shown in Figure 8 , high cell viability (green) was achieved for all samples, but more cells were visible on the chitosan physical hydrogels, indicating increased cell proliferation, consistent with the cell proliferation results Compared with the synthetic polymer hydrogels51,52and chitosan chemical hydrogels,53the chitosan physical hydrogels showed a greater cell proliferation rate, improved mechanical properties, and less cytotoxicity Moreover, the chitosan physical hydrogels showed a lower degradation rate than the chitin hydrogel (see Supporting Information , Figure S5) These results are, in part, attributable
to the nanoporous structure and saturated positively charged amino acids of the chitosan physical hydrogels resulting from the heterogeneous deacetylation, which allow for stronger electrostatic interactions with glycosaminoglycan In addition, the surface properties of the chitosan physical hydrogels promote cell growth and proliferation.20,54,55Furthermore, after culturing the cells for 7 d in the presence of EGF and IGF-1, the mBMSCs cultured on the chitin hydrogel and chitosan physical hydrogels took on a cobblestone morphology under the light microscope with the cell nucleolus in the middle of each cell ( Figure 9 ), which is characteristic of epidermal cells.56,57RT-PCR analysis of the epidermal cell di fferentiation marker genes K18 and K19 were performed to verify the epidermal cell di fferentiation of the BMSCs cultured on the hydrogels ( Figure 10 ) The results demonstrated that the chitin hydrogel (S0) and chitosan physical hydrogels (S3 and S4) induced the di fferentiation of the mBMSCs into epidermal cells
in cooperation with EGF and IGF-1 in vitro Thus, the chitosan physical hydrogels constructed via the heterogeneous deacety-lation of nanoporous chitin hydrogel have excellent mechanical properties and good cell-compatibility with potential applica-tions in stem cell research and tissue engineering.
■ CONCLUSIONS
In summary, we developed a novel chitosan physical hydrogels cross-linked by hydrogen bonding with considerable technical and commercial importance The heterogeneous deacetylation
of the nanoporous chitin hydrogel o ffers a facile approach to synthesize chitosan physical hydrogels with excellent mechan-ical properties and cell compatibility In vitro studies showed that mBMSCs cultured on these chitosan physical hydrogels exhibited good adhesion and proliferation Furthermore, the chitosan physical hydrogels also induced the di fferentiation of mBMSCs into epidermal cells in cooperation with EGF and IGF-1 in vitro The simplicity of the process and the widely tunable properties of the chitosan physical hydrogels make them promising candidates for potential biomedical applica-tions in stem cell culture and di fferentiation.
Figure 10.Relative gene expression of the K18 (a) and K19 (b) as epidermal cell differentiation markers by RT-PCR
Trang 7■ ASSOCIATED CONTENT
*S Supporting Information
The Supporting Information is available free of charge on the
ACS Publications website at DOI: 10.1021/acsami.6b05302
Nitrogen adsorption and desorption, UV −visible spectra,
DMA, and TGA ( PDF )
■ AUTHOR INFORMATION
Corresponding Authors
*E-mail: caijie@whu.edu.cn ; Telephone: +86-27-6878-9321.
*E-mail: caoxd@scut.edu.cn
Author Contributions
⊥B.D and H.G contributed equally to this work.
Notes
The authors declare no competing financial interest.
■ ACKNOWLEDGMENTS
This work was supported by the National Natural Science
Foundation of China (21422405, 51373125, 21574045) and
the Major Program of National Natural Science Foundation of
China (21334005) The authors thank the facility support of
the Natural Science Foundation of Hubei Province and the
Fundamental Research Funds for the Central Universities.
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