Research Article Cite This: ACS Appl. Mater. Interfaces 2018, 10, 6190−6198
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Tunable Mechanical, Antibacterial, and Cytocompatible Hydrogels Based on a Functionalized Dual Network of Metal Coordination Bonds and Covalent Crosslinking Xin Yi,†,⊥ Jiapeng He,‡,⊥ Xiaolan Wang,∥ Yu Zhang,∥ Guoxin Tan,§ Zhengnan Zhou,§ Junqi Chen,‡,⊥ Dafu Chen,# Renxian Wang,# Wei Tian,# Peng Yu,‡,⊥ Lei Zhou,*,‡,⊥ and Chengyun Ning*,‡,⊥ †
School of Medicine, ‡School of Materials Science and Engineering, and ⊥Guangdong Key Laboratory of Biomedical Sciences and Engineering, South China University of Technology, Guangzhou 510006, China § Institute of Chemical Engineering and Light Industry, Guangdong University of Technology, Guangzhou 510006, China ∥ Hospital of Orthopedics, Guangzhou General Hospital of Guangzhou Military Command of PLA, Guangzhou 510010, China # Laboratory of Bone Tissue Engineering, Beijing Research Institute of Traumatology and Orthopaedics, Beijing Jishuitan Hospital, Beijing 100035, China S Supporting Information *
ABSTRACT: Tissue engineering has become a rapidly developing field of research because of the increased demand from regenerative medicine, and hydrogels are a promising tissue engineering scaffold because of their three-dimensional structures. In this study, we constructed novel hydrogels of gelatin methacrylate (GelMA) hydrogels modified with histidine and Zn2+ (GelMA-His-Zn(II)), which possessed fascinating antibacterial properties and tunable mechanical properties because of the formation of a functionalized dual network of covalent crosslinking and metal coordination bonds. The introduction of metal coordination bonds not only improves the strength of the GelMA hydrogels with covalent crosslinking but also makes their mechanical properties tunable via adjustments to the concentration of Zn2+. The synergistic effect of Zn2+ and the imidazole groups gives the GelMA-His-Zn(II) hydrogels fascinating antibacterial properties (up to 100% inhibition). Counting the colony forming units and compression tests confirmed the fascinating antibacterial abilities and tunable mechanical properties, respectively, of the GelMA-His-Zn(II) hydrogels. In addition, Cell Counting Kit-8 assays, cytoskeletal staining assays, and live/dead assays confirmed the excellent cytocompatibility of the GelMA-His-Zn(II) hydrogels. Therefore, the GelMA-His-Zn(II) hydrogels are promising for applications in tissue engineering. KEYWORDS: dual network, metal coordination, antibacterial hydrogel, cytocompatibility, tunable mechanical properties have become popular in the field of tissue engineering.7 More specifically, GelMA hydrogels contain gelatin as their backbone, which gives them cell-responsive characteristics such as appropriate cell adhesion sites and proteolytic degradability.8−10 GelMA hydrogels are also cytocompatible, inexpensive, and technically simple to prepare.7 Moreover, researchers have shown that methacrylation and photocrosslinking can be used to tune the mechanical and chemical properties of GelMA hydrogels including their applicability in the creation of 3D microarchitectures.9,11,12 However, GelMA hydrogels do not have intrinsic antibacterial properties, which severely restrict their use in tissue engineering. To date, several strategies have been proposed to give GelMA hydrogels antibacterial characteristics. Zhao et al.
1. INTRODUCTION With increased demand for surgeries due to athletic activityrelated injuries, diseases, and aging, the need for regenerative medicine is increasing. Tens of billions of dollars per year are spent to treat approximately 35 million musculoskeletal injuries sustained each year.1−3 Therefore, tissue engineering has attracted increasing attention, and one of the challenges inherent in tissue engineering is replicating the complex three-dimensional (3D) structures of most tissues, such as skeletal muscle, heart, bone, tendon, ligament, artery, and nervous system tissues, as their structures are critical to their function.4 Both natural and synthetic hydrogels are of great interest as engineered tissue materials because of their 3D structures because they can absorb and retain a large amount of water,5 their mass-transfer properties are similar to those of natural tissues,5 and they can be formed into different shapes.6 In particular, gelatin methacrylate (GelMA) hydrogels, which have the advantages of both natural and synthetic biomaterials, © 2018 American Chemical Society
Received: December 11, 2017 Accepted: January 30, 2018 Published: January 30, 2018 6190
DOI: 10.1021/acsami.7b18821 ACS Appl. Mater. Interfaces 2018, 10, 6190−6198
Research Article
ACS Applied Materials & Interfaces
Figure 1. Synthesis of (a) GelMA and (b) N-Acry-L-His. The network architectures of (c) GelMA hydrogels with a single network of covalent bonds and (d) GelMA-His-Zn(II) hydrogels with a dual network of metal coordination and covalent bonds.
extremely tough and rapidly recoverable double network hydrogels.35 Bu et al. have constructed a double network hydrogels with good biocompatibility.36 Therefore, we want to form a dual network with covalent bonds and metal coordination bonds to improve the mechanical properties of the GelMA hydrogels and to afford GelMA hydrogels with tunable mechanical properties via adjustments to the amount of metal coordination bonds. Most importantly, a promising tissue engineering scaffold should have both excellent antibacterial properties and tunable mechanical properties. We herein report a new family of GelMA-His-Zn(II) hydrogels based on the 3D structures of GelMA hydrogels that possess potent antibacterial properties and tunable mechanical properties. This result was achieved using the functionalized dual network present in the GelMAHis-Zn(II) hydrogels that consist of the covalent bonds of GelMA and the metal coordination bonds between Zn2+ and the imidazole groups. On one hand, the zinc and imidazole groups not only have antibacterial activities but are also beneficial to human health and have low cytotoxicities. On the other hand, the dual network composed of covalent bonds and metal coordination bonds gives the GelMA-His-Zn(II) hydrogels improved and tunable mechanical properties. The antibacterial rate (AR) of the GelMA-His-Zn(II) hydrogels is much higher than that of GelMA hydrogels, and it can be as high as 100%. The compression moduli of the GelMA-HisZn(II) hydrogels can be adjusted from 3.5 to 7.8 kPa, which match with the modulus of some tissues, such as nerve and cardiac tissues.37 The moduli do not appreciably change after five repeated loadings at 80% strain. In addition, the GelMAHis-Zn(II) hydrogels possess brilliant cytocompatibility. A Cell Counting Kit-8 (CCK-8) assay and cytoskeletal staining assay proved that MC3T3-E1 cells can proliferate, adhere, and spread normally after coculture with the GelMA-His-Zn(II) hydrogels, and the average cell spreading area and OD value of cells cocultured with the GelMA-His-Zn(II) hydrogels were noticeably higher than those of cells cultured with GelMA hydrogels. A live/dead assay also showed that the cytotoxicity of the GelMA-His-Zn(II) hydrogels toward MC3T3-E1 cells is acceptable, and the average live cell number of the GelMAHis-Zn(II) hydrogels was higher than that of the GelMA
reported that silver nanoparticles can be loaded onto the surface of the materials to give them antimicrobial activities.13 Moreover, a short cationic antimicrobial peptide known as HHC-36 has been investigated as an antimicrobial component for the surface modification of GelMA hydrogels because of its broad-spectrum antimicrobial activity against common Grampositive and Gram-negative bacteria and enhanced efficacy compared to traditional antibiotics.14−17 However, these surface modification and drug loading methods are generally timeconsuming and require complex, multistep procedures. Imidazole groups and imidazole analogues, antibacterial moieties that exist in various biomolecules such as histidine, histamine, and natural products,18 have generated substantial interest over the years because of their antimicrobial properties. Some imidazole derivatives such as cimetidine, etomidate, ketoconazole, and metronidazole have been used as drug therapies to treat bacterial infections.19,20 In addition, Zn possesses excellent antibacterial abilities,21,22 and it is an important trace element in human biological functions.23,24 Our previous study have proved that Zn2+ can produce reactive oxygen species which can damage the protein and cell wall and kill the bacteria ultimately.25 Tang et al. have reported that the incorporation of Zn into bioglasses and Ca−P and Ca−Si systems can enhance their antibacterial properties.26−28 The mechanical properties of materials are another important consideration for the engineering of tissue scaffolds, and they play crucial roles in regulating the interactions between the cells and extracellular matrices and directing the phenotype and genotype of the cells. However, GelMA hydrogels suffer from poor mechanical properties,29 which need to be improved to make these hydrogels applicable in tissue engineering. On one hand, GelMA hydrogels can be mixed with other polymers, such as hyaluronic acid,30 silk fibroin,31 and gellan gum,32 to improve their mechanical properties. On the other hand, the mechanical properties of GelMA hydrogels can also be improved by introducing nanomaterials, such as graphene oxide33 and carbon nanotubes,34 into the gel. However, with these methods, it is difficult to prepare GelMA hydrogels with tunable mechanical properties. Now, dual network, due to excellent performance, has attracted increasing attention. Yang et al. have fabricated an 6191
DOI: 10.1021/acsami.7b18821 ACS Appl. Mater. Interfaces 2018, 10, 6190−6198
Research Article
ACS Applied Materials & Interfaces
GelMA-His-Zn(II) hydrogels, and Figure 1c shows the synthesis and single network architecture of the GelMA hydrogels.
hydrogels. Finally, the GelMA-His-Zn(II) hydrogels can be used to prepare 3D structures, which is significant for tissue engineering. Therefore, the GelMA-His-Zn(II) hydrogels show promise for use in tissue engineering.
3. RESULTS AND DISCUSSION The microstructural features of hydrogels play a crucial role in the mechanical properties of hydrogels, and we found significant differences in the microstructures of the hydrogels (Figure 2a,b). Notably, GelMA hydrogels were inhomogeneous and composed of a mixture of voids interspersed with a denser material. In contrast, the microstructures of the GelMA-HisZn(II) hydrogels were more homogeneous and showed smaller voids. The average pore sizes of the GelMA and GelMA-HisZn(II) hydrogels were 20 and 10 μm, respectively. The smaller and more uniform distribution of pores could be a key reason for the superior mechanical properties of the GelMA-HisZn(II) hydrogels. Besides, both GelMA and GelMA-His-Zn(II) hydrogels possessed a 3D porous structure, which is useful in tissue engineering. The degree of swelling of the hydrogels is related to the pore size and the amount of metal coordination bonds, which ultimately affects the mechanical strength of the material. As shown in Figure 2c, the degree of swelling of the GelMA hydrogels modified with histidine (GelMA-His) is 30%, which is lower than that of the GelMA hydrogels (36%). This result may be attributed to the introduction of N-Acry-L-His, which decreased the pore size. In addition, when the GelMA-His hydrogels were immersed in the zinc sulfate solution, the degree of swelling of the hydrogels decreased to as low as 24%. This result showed that the formation of a metal coordination bond between Zn2+ and the imidazole group can decrease the degree of swelling, and increasing the number of metal coordination bonds results in a lower degree of swelling. Degradation rate is a key factor in determining the service life of the tissue engineering scaffold, and it is affected by the amount of the crosslinking and metal coordination bonds. Figure 2d shows that the weight loss of the GelMA-His hydrogels is lower than that of the GelMA hydrogels, which can be attributed to the higher cross-linking density in GelMA-His. Moreover, Figure 2d also shows that the presence of the metal coordination bonds can decrease the degradation rate of the GelMA-His-Zn(II) hydrogels relative to that of the GelMA-His hydrogels, and more metal coordination bonds result in a slower degradation rate. The 1H NMR spectra of gelatin and GelMA are shown in Figure 3a. Comparing the spectra of gelatin and GelMA, new signals can be observed at 5.35 and 5.59 ppm, corresponding to the two protons of methacrylate double bond. Therefore, we confirmed that methacrylate has been successfully grafted to the gelatin molecules, and the degree of methacrylation of GelMA was 79.2% by the quantitative 1H NMR analysis. The 1H NMR spectra of histidine and N-Acry-L-His are shown in Figure 3b. Comparing the 1H NMR spectra of histidine and N-Acry-L-His, new signals can be observed at 5.7−6.11 and 6.21 ppm, corresponding to the two protons of acrylamide double bond. Therefore, we confirmed that acryloyl chloride has been successfully grafted to the gelatin molecules. Besides, Figure S1 shows the 13C NMR spectra of N-Acry-L-His. The peaks at 126.8 and 131.1 ppm are attributed to CC, and the peak at 166.8 ppm is attributed to −CCN. Both of it were obtained from the acryloyl chloride molecule. Therefore, the 13C NMR spectra of N-Acry-L-His also proved that acryloyl chloride has been successfully grafted to the gelatin molecules (Figure S1). The Fourier transform infrared (FTIR) spectra of GelMA and
2. MATERIALS AND METHODS 2.1. Materials and Reagents. Gelatin, methacrylic anhydride, histidine, acryloyl chloride, and zinc sulfate were purchased from Aladdin Reagent Company (Shanghai, China). Mesenchymal stem cells (MC3T3-E1) were purchased from American Type Culture Collection. Calcein-AM, propidium iodide, 4,6-diamidino-2-phenylindole, F-actin, and a CCK-8 kit were purchased from Biyuntian Biotechnology CO. LTD. All other chemical reagents were of analytical reagent grade and were used without further purification. 2.2. Synthesis of GelMA Monomer. The synthesis of GelMA monomers was reported previously,9 and the synthetic pathway is shown in Figure 1a. First, 0.5 g of gelatin was added to 50 mL of phosphate-buffered saline (PBS) at 60 °C, and the solution was stirred until the gelatin had dissolved. Second, 4 mL of methacrylic anhydride was added to the above solution at 50 °C at a rate of 0.2 mL/min, and the solution was stirred for 3 h until it became milk white. Third, the solution was poured under stirring into PBS to stop the reaction. Finally, the mixed solution was poured into a dialysis bag and placed in deionized water at 50 °C for 1 week to obtain a pure GelMA monomer. Samples were freeze-dried and stored at −20 °C until further use. 2.3. Synthesis of N-Acryloyl-L-histidine Monomer. Figure 1b shows the synthesis of N-acryloyl-L-histidine (N-Acry-L-His). First, 3 g of histidine was added to 7 mL of NaOH solution, and the solution was stirred until the histidine had dissolved. Then, 1.6 mL of acryloyl chloride was added to the above solution while protected from light, and after reacting for 1 h, concentrated HCl was used to adjust the pH of the solution to 3. Finally, acetone ethyl alcohol and deionized water were added successively to wash the mixture three times, and after washing, the product was placed in a refrigerator at −80 °C for 24 h to obtain pure N-Acry-L-His. 2.4. Preparation of GelMA-His-Zn(II) Hydrogels. Figure 1c shows the synthesis of GelMA-His-Zn(II) hydrogels. First, the GelMA monomer (10 w/v %) and N-Acry-L-His (10 w/v %) were dissolved in 50 mL of PBS (pH = 7) at 40 °C. Then, the photoinitiator (0.5 w/v %) was added to the above solution, and the mixture was stirred for 10 min. The mixture was transferred to a 24-well cell culture plate with 300 μL of solution per well, and the plate was placed under UV light for 20 s. Finally, 1 mL of zinc sulfate solutions at certain concentrations was added to each well. The solutions were soaked for 2 h at room temperature, and then each well was washed with PBS three times. By adjusting the concentration of zinc sulfate solution from 12 to 48 mmol/L, different GelMA-His-Zn(II) hydrogels were synthesized, as shown in Table 1. Samples were stored at −4 °C until further use. Figure 1d also shows the dual-network architecture of the GelMA-His-Zn(II) hydrogels with covalent bonds and metal coordination bonds, which gives the GelMA-His-Zn(II) hydrogels excellent antibacterial properties and tunable mechanical properties. In addition, the synthesis of GelMA, GelMA-His, and GelMA-Zn(II) hydrogels followed a procedure similar to what was used for the
Table 1. Concentration of Zn(II) of GelMA-His and GelMA Hydrogels hydrogel name
CZn(II) (mmol/L)
GelMA-His GelMA-Zn(II)-1 GelMA-Zn(II)-2 GelMA-Zn(II)-3 GelMA-His-Zn(II)-1 GelMA-His-Zn(II)-2 GelMA-His-Zn(II)-3
0 12 24 48 12 24 48 6192
DOI: 10.1021/acsami.7b18821 ACS Appl. Mater. Interfaces 2018, 10, 6190−6198
Research Article
ACS Applied Materials & Interfaces
Figure 2. Scanning electron microscopy images of (a) GelMA hydrogel and (b) GelMA-His-Zn(II)-1 hydrogel. (c) Swelling rate and (d) weight loss rate of the GelMA, GelMA-His, and GelMA-His-Zn(II) hydrogels.
formed metal coordination bonds with the imidazole groups (Figure S2). Figure 4 shows typical compressive stress−strain curves of the GelMA-His-Zn(II) and GelMA-Zn(II) hydrogels. The GelMA-His-Zn(II) hydrogels show significantly higher compression moduli than GelMA-Zn(II) (Figure S3). Briefly, the moduli of the GelMA-His-Zn(II)-1, GelMA-His-Zn(II)-2, and GelMA-His-Zn(II)-3 hydrogels were approximately 3.4-, 3.3-, and 5.8-fold greater than those of the GelMA-Zn(II)-1, GelMAZn(II)-2 and GelMA-Zn(II)-3 hydrogels, respectively. This result is attributable to the formation of metal coordinate bonds between Zn2+ and the imidazole groups, and the dual bonding network provides higher moduli to the hydrogels than a single binding network. In addition, Figure 4b shows (red curve) that the compression moduli of the GelMA-His-Zn(II) hydrogels increase with increasing concentrations of zinc sulfate. This result shows that the compression moduli of the GelMA-HisZn(II) hydrogels can be tuned by adjusting the metal coordination bond density, and the ability to tune the compression modulus of the material means that it can be aligned with the mechanical properties of a greater number of types of tissue. However, the compression modulus of the GelMA-Zn(II) hydrogel did not change noticeably with increasing concentrations of zinc sulfate. A distinctive and advantageous feature of metal coordination bonds is their ability to withstand repeated loading. To quantitatively investigate this ability in the GelMA and GelMAHis-Zn(II) hydrogels, repetitive loading was tested. Stress− strain profiles under the same cyclic compressive strain overlapped with each other, and no appreciable change in the moduli between the loading cycles was observed, indicating elastic recovery at 80% strain (Figure 5a). Moreover, the hysteresis energy (i.e., the energy consumed due to internal bond failure38) was notably higher for the GelMA-His-Zn(II) hydrogels (Figure 5a) than that for the GelMA hydrogels (Figure 5a), indicating energy dissipation due to forceful induction of the metal coordination bond failure. In addition,
Figure 3. 1H NMR spectra of (a) GelMA and gelatin (b) N-Acry-L-His and histidine. (c) FTIR spectrum of GelMA and GelMA-His. (d) EDS of GelMA-His-Zn(II).
GelMA-His are shown in Figure 3c; the characteristic peaks at 3320 and 3314 cm−1 can be ascribed to the stretching vibration of N−H bands. The characteristic peaks at 1660 and 1640 cm−1 can be ascribed to the stretching vibration of CO bands. The characteristic peaks at 1550 and 1243 cm−1 can be ascribed to the stretching vibration of N−H bands. The characteristic peaks at 1086 and 840 cm−1 can be ascribed to the bending vibration and stretching vibration of −CCN bands in imidazole ring. The characteristic peaks at 635 cm−1 can be ascribed to the vibration of imidazole ring. This result suggested that N-Acry-L-His was successfully incorporated into GelMA. Although the GelMAHis-Zn(II) hydrogels were washed several times, free zinc still can be detected in the freeze-dried hydrogels by the energydispersive system (EDS) (Figure 3d). Comparing the EDS of GelMA-His and GelMA-His-Zn(II) confirmed that Zn2+ 6193
DOI: 10.1021/acsami.7b18821 ACS Appl. Mater. Interfaces 2018, 10, 6190−6198
Research Article
ACS Applied Materials & Interfaces
Figure 4. (a) Compressive stress−strain profiles of the GelMA-His-Zn(II) and GelMA-Zn(II) hydrogels with different concentrations of zinc ions. (b) Compression modulus of the hydrogels.
Figure 5. Compressive stress−strain profiles for repeated loadings at 80% strain (five cycles at 0.5 N min−1 as shown, inset) of (a) GelMA-HisZn(II)-1 hydrogels and (b) GelMA hydrogels.
Figure 6. (a,b) Bacterial colonies of Staphylococcus aureus and Escherichia coli after coculture with GelMA, GelMA-His, GelMA-His-Zn(II)-1, GelMA-His-Zn(II)-2, and GelMA-His-Zn(II)-3 hydrogels, respectively. (c,d) AR of S. aureus and E. coli, respectively, n = 4, **p < 0.01, compared to GelMA hydrogels.
with repeated application of 80% strain, Mullins-type softening (i.e., reductions in moduli and strain energy) was not observed
in the GelMA-His-Zn(II) hydrogels. Thus, the GelMA-HisZn(II) hydrogels effectively leverage the primary metal 6194
DOI: 10.1021/acsami.7b18821 ACS Appl. Mater. Interfaces 2018, 10, 6190−6198
Research Article
ACS Applied Materials & Interfaces
Figure 7. Direct culture of MC3T3-E1 cells on surfaces of the hydrogels for up to 2 d; cell density: 2 × 104 cells/mL. Staining of viable (green) and dead (red) cells after coculture with (a) GelMA, (b) GelMA-His, (c) GelMA-His-Zn(II)-1, (d) GelMA-His-Zn(II)-2, and (e) GelMA-His-Zn(II)-3 hydrogels. (f) Quantitative measurements of the number of adhered cells after coculture with the hydrogels; n = 4, *p < 0.05.
Figure 8. MC3T3-E1 cell morphology and cytoskeleton on the (a) GelMA, (b) GelMA-His, (c) GelMA-His-Zn(II)-1, (d) GelMA-His-Zn(II)-2, and (e) GelMA-His-Zn(II)-3 hydrogels; cell density: 1 × 104 cells/mL; F-actin cell staining showing the morphologies after 2 d of culture. (f) Average cell spreading area; n = 4, *p < 0.05.
GelMA-His-Zn(II)-3 hydrogels possess better antibacterial properties than the GelMA-His and GelMA hydrogels and the antibacterial activity of the GelMA-His hydrogel is better than that of the GelMA hydrogels (Figure 6a,b). As shown in Figure 6c,d, the AR of the GelMA-His-Zn(II) hydrogel is significantly different from that of GelMA-His; up to 100% of S. aureus and E. coli were killed by the GelMA-His-Zn(II)-2 and GelMA-His-Zn(II)-3 hydrogels. This result demonstrates that both the imidazole group and Zn2+ possess antibacterial activities, and the synergistic effect of the two can substantially improve the antibacterial activity of the material compared to the presence of either individuals. Because the cytotoxicity of the hydrogels is an important consideration for tissue development, the live/dead assay was conducted to study this parameter. Figure 7 shows MC3T3-E1
coordination bonds to prevent internal rupture of the covalent bonds and maintain their moduli even without lengthy recovery times similar to those needed with other types of crosslinking (e.g., ionic DNs).39,40 Taken together, these results indicate that metal coordination bonds within the GelMA-His-Zn(II) hydrogels immediately repair themselves between the loading cycles and contribute substantially to energy consumption throughout loading, which enables the GelMA-His-Zn(II) hydrogels to withstand repetitive loadings in rapid succession similar to what occurs in biological tissues. An ideal tissue engineering scaffold should possess antibacterial properties. The antibacterial activities of these hydrogels against both E. coli and S. aureus were evaluated using surface antibacterial activity tests. On the basis of 2 h of contact at 37 °C, the GelMA-His-Zn(II)-1, GelMA-His-Zn(II)-2, and 6195
DOI: 10.1021/acsami.7b18821 ACS Appl. Mater. Interfaces 2018, 10, 6190−6198
Research Article
ACS Applied Materials & Interfaces cells spread on the surface of the GelMA, GelMA-His, GelMAHis-Zn(II)-1, GelMA-His-Zn(II)-2, and GelMA-His-Zn(II)-3 hydrogels after being cultured in vitro for 2 d, and the cell densities on the different hydrogels were analyzed, as shown in Figure 7f. The average number of cells on the GelMA-HisZn(II)-1, GelMA-His-Zn(II)-2, and GelMA-His-Zn(II)-3 hydrogels were significantly different from the number of cells on the GelMA and GelMA-His hydrogels (*p < 0.05), and the average number of cells on the GelMA-His hydrogels was higher than that of the GelMA hydrogels. This result can attribute to the presence of histidine and Zn2+, which are important for human health. Additionally, almost all of the adhered cells on the GelMA-His-Zn(II) hydrogels stayed alive after 2 d of culture (cells in green are live cells, and those in red are dead cells), which demonstrated that the GelMA-HisZn(II) hydrogels can provide a habitable 3D environment where the MC3T3-E1 cells can adhere and spread with no apparent toxicity. To understand the interactions between the MC3T3-E1 cells and the hydrogels at the interface, we examined cell spreading, which plays key roles in cell survival, growth, and functions (Figure 8). After 48 h of cell incubation, the average cell spreading area on the GelMA-His-Zn(II) hydrogels was significantly higher than those on the GelMA and GelMA-His hydrogels (*p < 0.05); the average cell spreading area on the GelMA-His hydrogels was also higher than that on the GelMA hydrogels. In addition, the cell morphology on the GelMA-HisZn(II) hydrogels was highly elongated in a spindlelike shape, whereas the cell morphology on the GelMA and GelMA-His hydrogels was shown in a polygonal-like shape. The good cell spreading on the GelMA-His-Zn(II) hydrogels was believed to be partially related to the presence of histidine and Zn2+ in the material and proved that MC3T3-E1 cells can positively interact with the interface of the GelMA-His-Zn(II) hydrogels. To evaluate the potential of the GelMA-His-Zn(II) hydrogels for use in long-term clinical applications, we investigated cell proliferation on the hydrogels by CCK-8 assays. As shown in Figure 9, MC3T3-E1 cells grew and proliferated well on all the samples at both 1 and 4 d. Similar viability levels were obtained for each of the hydrogels, and no significant differences were observed. When the culturing time was extended to 7 d, all of the GelMA-His-Zn(II) hydrogels showed even stronger abilities to facilitate the proliferation of MC3T3 cells and with this
culture time, significant differences (*p < 0.05) were observed, which showed that Zn2+ has a positive effect on the proliferation of cells. In addition, there were more cells growing on the GelMA-His hydrogels than on the GelMA hydrogels, which proved that histidine can facilitate the proliferation of cells to some extent.
4. CONCLUSIONS The GelMA-His-Zn(II) hydrogels, which have a dual bonding network and inherit the fascinatingly cytocompatiblity of GelMA hydrogels, possessed both brilliant antibacterial activities and tunable mechanical properties. The synergistic effect of the zinc ions and imidazole groups provides high ARs (up to 100%) to the GelMA hydrogels. The metal coordination bonds between the zinc ions and the imidazole groups not only worked in conjunction with the covalent bond network of the GelMA hydrogels to ensure the proper mechanical properties of the GelMA-His-Zn(II) hydrogels but also gave the GelMAHis-Zn(II) hydrogels tunable mechanical properties via adjustments to the concentration of zinc sulfate. Therefore, these hydrogels may be applicable in the field of tissue engineering.
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ASSOCIATED CONTENT
S Supporting Information *
The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acsami.7b18821. Experimental detail, 13C NMR spectra of N-Acry-L-His, EDS of GelMA-His, and the compress modulus of GelMA and GelMA-His hydrogels immersed into different concentrations of zinc sulfate (PDF)
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AUTHOR INFORMATION
Corresponding Authors
*E-mail:
[email protected] (L.Z.). *E-mail:
[email protected] (C.N.). ORCID
Peng Yu: 0000-0002-2253-7373 Chengyun Ning: 0000-0003-3293-4716 Author Contributions
X.Y. and J.H. contributed equally to this paper. Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS The authors gratefully acknowledge the National Natural Science Foundation of China (grant nos. 51772106, 51702104, 31771080, 51541201, 31430030, and 81501861), the National High Technology Research and Development Program of China (863 Program) (grant no. 2015AA033502), Science and Technology Planning Project of Guangdong Province, China (grant no. 2014A010105048), the Natural Science Foundation of Guangdong Province (grant nos. 2015A030313493, 2016A030308014, and 2015A030312004), Technological Projects of Guangzhou, China (no. 201604020110), the Fundamental Research Funds for the Central Universities (grant no. 2017BQ032), and Beijing Municipal Natural Science Foundation (grant no. 7161001).
Figure 9. Cell proliferation of MC3T3-E1 cells on the GelMA, GelMA-His, GelMA-His-Zn(II)-1, GelMA-His-Zn(II)-2, and GelMAHis-Zn(II)-3 hydrogels after coculture of 1, 4, and 7 d. n = 4, *p < 0.05. 6196
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DOI: 10.1021/acsami.7b18821 ACS Appl. Mater. Interfaces 2018, 10, 6190−6198
