Research Article www.acsami.org Tough 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 fulfill the mechanical and cellcompatibility 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 nanofibrils 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 differentiation 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 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 supermolecular 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 fulfill 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.16−18 Among them, chitosan, a natural amino polysaccharide derived from chitin, which is the main © 2016 American Chemical Society component of the exoskeletons of crustaceans (e.g., crabs and shrimp), has received considerable attention because of its excellent biodegradability, biocompatibility, and bioactivity.19−24 The exploitation of chitosan hydrogels for biomaterials is, however, limited by the poor solubility and mechanical integrity, difficulty 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 threedimensional 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 19739 DOI: 10.1021/acsami.6b05302 ACS Appl Mater Interfaces 2016, 8, 19739−19746 Research Article ACS Applied Materials & Interfaces solvent-exchanged with absolute ethanol and then dried from supercritical CO2 to 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 400− 4000 cm−1 were collected in 32 scans at cm−1 resolution 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 Nifiltered 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 multipeak fitting of the XRD profiles Solid-state cross-polarization/magnetic angle spinning (CP/MAS) 13 C nuclear magnetic resonance (NMR) spectra were collected on a Bruker AVANCE-300 Spectrometer (13C frequency = 75.4 MHz) with a standard 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 ultraviolet−visible (UV−vis) spectroscopy (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 −50 to 300 °C The heating rate and frequency were °C min−1 and Hz, respectively The width of the samples was approximately mm Thermogravimetric analysis (TGA) was conducted using a STA 449C (Netzsch, German) from 25 to 600 °C at a heating rate of 10 °C min−1 under nitrogen The hydrogels were subjected to tension tests on a universal tensile tester (CMT 6503, SANS, China) The hydrogel was stretched at a mm min−1 stretch speed at ambient temperature The modulus was calculated from the initial linear regions of the stress−strain curves Mouse bone mesenchymal stem cells (mBMSCs, ATCC, CRL12424) 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 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, 200 μL of the mBMSCs suspension (1 × 104 cells well−1) 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 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 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 After washing again with PBS, the hydrogels were imaged using an Eclipse Ti−U 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 × 104 cells cm−2 After culture for d in the presence of 30 ng mL−1 recombinant human EGF (PeproTech, USA) and 50 ng mL−1 recombinant 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 under 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 nanofibrils 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 differentiation into epithelial cells ■ EXPERIMENTAL SECTION Materials The raw chitin powder was purchased from GoldenShell Biochemical Co Ltd (Zhejiang, China) The raw chitin powder was purified with 0.1 mol L−1 aqueous NaOH at ambient temperature overnight and combined with 0.3% (w/w) aqueous NaClO2 buffered 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 × 104 in 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 −30 °C overnight Subsequently, after thawing at °C, the chitin was dissolved completely and used to form a transparent and viscous 7% (w/w) chitin solution according to our previous method.32 The chitin solution was centrifuged at °C for 15 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 °C for 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 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 He−Ne 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 The fluent was 0.1 M NaAc/HAc buffer (pH = 2.8) with a flow rate of 0.6 mL min−1 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 KH5C8O4 used for calibration The DD value was calculated as follows:35 DD = (V2 − V1) × C × 0.016 0.0994 × W (1) where C is the accurate concentration of aqueous NaOH solution (mol L−1), V1 is the volume of aqueous NaOH solution (mL) at the first titration jump, V2 is 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 mol−1), 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 S4800 instrument The chitin and chitosan physical hydrogels were 19740 DOI: 10.1021/acsami.6b05302 ACS Appl Mater Interfaces 2016, 8, 19739−19746 Research Article ACS Applied Materials & Interfaces Figure (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 (S1−S4) at each heterogeneous deacetylation cycle (c) Macroscopic views of the chitosan physical hydrogels (S4) under stretching, torsional and rolling loading Table Physical Properties of the Chitin Hydrogel and Chitosan Physical Hydrogelsa samples DD, % WH2O, % M × 10−4, g/mol crys % σb, MPa εb, % E, MPa S0 S1 S2 S3 S4 56 80 91 99 84 61 58 51 50 10.7 4.9 4.0 3.9 3.1 50 35 34 36 43 1.7 ± 0.1 3.6 ± 0.5 10.5 ± 0.5 12.1 ± 1.1 12.1 ± 0.7 56 ± 67 ± 106 ± 14 67 ± 57 ± 4.5 7.9 34.5 57.3 92.0 a The DD and WH2O are the degree of deacetylation and the water content, respectively, of the chitin hydrogel (S0) and chitosan physical hydrogels (S1−S4).The viscosity-average molecular weight (Mη) of sample S0 was determined by viscometry, and the weight-average molecular weights (Mw) of samples S1−S4 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 epidermal cell differentiation marker gene K18 and K19 The following primer sequences were used: K18 gene: forward, 5′-AAGGCTGCAGCTGGAGACAGA-3′; reverse, 3′-TGGGCTTCCAGACCTTGGAC-5′; K19 gene: forward, 5′-TGACCTGGAGATGCAGATTGAGA-3′; reverse, 3′- TGGAATCCACCTCCACACTGAC-5′; and GAPDH gene: forward, 5′-TGTGTCCGTCGTGGATCTGA-3′; reverse, 3′-TTGCTGTTGAAGTCGCAGGAG-5′ The relative quantification of the target gene was normalized to GAPDH and determined using the 2−ΔΔCt 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 mg/mL three-times-recrystallized egg white lysozyme (TSZ, USA) in 0.1 M PBS at pH 7.4 and 37 °C.20 After 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 Figure 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) 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 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 −NHCOCH3 sites were deacetylated to give −NH2 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 different DDs, which, as expected, ranged from 56 to 99% (Table 1) The weight- ■ 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 The chitin hydrogel is a transparent nanoporous material, which forms via the hydrogen bonding of the chitin−NaOH−urea aqueous solution by a sol−gel 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 19741 DOI: 10.1021/acsami.6b05302 ACS Appl Mater Interfaces 2016, 8, 19739−19746 Research Article ACS Applied Materials & Interfaces average molecular weight (Mw) of the chitosan physical hydrogels decreased from 5.6 × 104 to 4.7 × 104 g 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 significant volume changes after washing with aqueous ethanol, mainly because of the diffusion of ethanol, which disturbed the hydrophobic and hydrogen bonding interactions between the chitosan chains and thereby influenced the final density of the physical cross-linking and water content of the hydrogels (Figure 1b).19 After 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 1c) The SEM images of the surface and inner part of the chitin gel (Figure 2a and e) show an open nanoporous network structure composed of interconnected chitin nanofibrils The typical diameter of the chitin nanofibrils 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 nm Moreover, the SEM images and the nitrogen adsorption−desorption isotherms of the chitosan physical gels (Figure 2b−h, Figure S1) show features of a smaller porous structure and surface area after the heterogeneous deacetylation 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 CO stretching absorption peak at 1660 and 1627 cm−1 for amide I and 1560 cm−1 for amide II.36−38 After the heterogeneous deacetylation cycles, the CH 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) reflections, 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) reflections of the chitosan physical gels decreased gradually, and the Figure FT-IR spectra of the chitin hydrogel (S0) and chitosan physical hydrogels (S1−S4) Figure XRD patterns of the chitin hydrogel (S0) and chitosan physical hydrogels (S1−S4) Figure Solid-state CP/MAS 13C NMR spectra of the chitin hydrogel (S0) and chitosan physical hydrogels (S1−S4) 19742 DOI: 10.1021/acsami.6b05302 ACS Appl Mater Interfaces 2016, 8, 19739−19746 Research Article ACS Applied Materials & Interfaces Table Chemical Shifts of the Chitin Hydrogel and Chitosan Physical Hydrogel Determined by CP/MAS 13C NMR chemical shift/ppm samples C7 (CO) C1 C4 C5 C3 C6 C2 C8 (CH3) S0 S1 S2 S3 S4 174.1 174.3 174.3 104.5 104.5 105.5 105.3 105.7 83.6 83.2 82.9 82.9 82.7 76.1 75.9 75.9 75.7 75.9 74.2 75.9 75.9 75.7 75.9 61.8 61.1 61.2 61.7 61.5 55.9 58.2 58.0 58.5 58.3 23.3 23.5 23.0 24.0 Figure 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) Figure Typical stress−strain curves of the chitin hydrogel (S0) and chitosan physical hydrogels (S1−S4) The inset is the stress−strain curve of the chitosan film dried from sample S3 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) reflection 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 confirmed by the solid-state CP/MAS 13C NMR analysis (Figure 5) The corresponding chemical shifts are listed in Table 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 CO (174.1 ppm).41−44 Compared 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 downfield These spectral and morphological characteristics indicate that nanoscale heterogeneous deacetylation was achieved in the nanoporous chitin hydrogel; that is, the Nacetylglucosamine units on the surface of the interconnected chitin nanofibrils were removed, and the resultant amino groups interacted with the hydroxyl groups on the nearby nanofibrils 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; Figure Proliferation of the mBMSCs cultured on the surfaces of the chitin hydrogel (S0) and chitosan physical hydrogels (S1−S4) Figure 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 =100 μm) diffraction 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 19743 DOI: 10.1021/acsami.6b05302 ACS Appl Mater Interfaces 2016, 8, 19739−19746 Research Article ACS Applied Materials & Interfaces Figure 10 Relative gene expression of the K18 (a) and K19 (b) as epidermal cell differentiation markers by RT-PCR 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 hydrogels51,52 and chitosan chemical hydrogels,53 the 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.20,54,55 Furthermore, after culturing the cells for 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.56,57 RT-PCR analysis of the epidermal cell differentiation marker genes K18 and K19 were performed to verify the epidermal cell differentiation 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 differentiation of the mBMSCs into epidermal cells in cooperation with EGF and IGF-1 in vitro Thus, the chitosan physical hydrogels constructed via the heterogeneous deacetylation of nanoporous chitin hydrogel have excellent mechanical properties and good cell-compatibility with potential applications in stem cell research and tissue engineering Table 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 effects 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 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, confirming 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 significant 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 deacetylation 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 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 Significant differences for all of the samples were observed after 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 ■ 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 offers a facile approach to synthesize chitosan physical hydrogels with excellent mechanical properties and cell compatibility In vitro studies showed that mBMSCs cultured 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