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Brain-Derived Neurotrophic Factor Val66Met Polymorphism and Antidepressant Efficacy of Ketamine in Depressed Patients
FULL PAPER Wound Healing
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Bioinspired Multifunctional Hybrid Hydrogel Promotes Wound Healing Guopu Chen, Yunru Yu, Xiuwen Wu, Gefei Wang, Jianan Ren,* and Yuanjin Zhao* hydrogels.[6–11] Among these biomaterials, hydrogels, including chitosan, polyethylene glycol, and their derivatives, are the most attractive candidates because of their functions of absorbing tissue exudates, maintaining a moist wound environment, cooling the wound surface, allowing oxygen to permeate, as well as leading to pain relief for patients.[12–17] In particular, with the application of some injectable hydrogels, wound healing approaches could include the additional functions of in situ drug encapsulation and wound site filling, anti-infection, antioxidation, hemostasis, and adhesiveness.[18–22] Although the use of hydrogel materials has promoted wound healing to a certain degree, biological wound healing occurs in the four unique yet overlapping stages of hemostasis, inflammation, proliferation, and remodeling,[23–26] and the recently developed hydrogels could only contribute their functions in one or two stages. This wound greatly affect the final healing effect of the wound as each stage is highly orchestrated and can directly impact the subsequent events.[27–30] In addition, most of these hydrogels could only repair a single type of wound, which limited their wide applications in the clinic. Thus, the creation of novel hydrogel that can act their functions on the whole healing processes of different wounds is still anticipated. In this paper, inspired by the spontaneous heal-injury process of the organism, which uses coordinated multiple mechanisms for body recovery, we proposed a four-armed benzaldehyde-terminated polyethylene glycol (BAPEG) and dodecyl-modified chitosan (DCS) hybrid hydrogel system with the desired multifunctional features for wound healing, as schemed in Figure 1. The hydrogel was formed and imparted with self-healing capability through the reversible Schiff base between the benzaldehyde and amino groups in the polymer compounds. The existing dodecyl tails can insert themselves into and be anchored onto the lipid bilayer of the cell membrane; thus, the hybrid hydrogel presented outstanding tissue adhesion, blood cell coagulation and hemostasis, and cell recruitment functions. This hybrid hydrogel could also exhibit superior anti-infective properties due to the coordinate effects of the bacterial anchoring ability of the dodecyl, and the bacterial destruction abilities of chitosan and aromatic Schiff base. In
Inspired by the coordinated multiple healing mechanism of the organism, a four-armed benzaldehyde-terminated polyethylene glycol and dodecylmodified chitosan hybrid hydrogel with vascular endothelial growth factor (VEGF) encapsulation are presented for efficient and versatile wound healing. The hybrid hydrogel is formed through the reversible Schiff base and possesses self-healing capability. As the dodecyl tails can insert themselves into and be anchored onto the lipid bilayer of the cell membrane, the hybrid hydrogel has outstanding tissue adhesion, blood cell coagulation and hemostasis, anti-infection, and cell recruitment functions. Moreover, by loading in and controllably releasing VEGF from the hybrid hydrogel, the processes of cell proliferation and tissue remodeling in the wound bed can be significantly improved. Based on an in vivo study of the multifunctional hybrid hydrogel, it is demonstrated that acute tissue injuries such as vessel bleeding and liver bleeding can be repaired immediately because of the outstanding adhesion and hemostasis features of the hydrogel. Moreover, the chronic wound-healing process of an infectious full-thickness skin defect model can also be significantly enhanced by promoting angiogenesis, collagen deposition, macrophage polarization, and granulation tissue formation. Thus, this multifunctional hybrid hydrogel is potentially valuable for clinical applications.
1. Introduction In recent years, wound treatment and its therapeutic impediments have become a fundamental healthcare concern, presenting an significant economically challenging burden worldwide.[1–5] To date, many kinds of biomaterials have been used for rapid wound healing, including electrospun nanofiber, porous foams, biocompatible membranes, and functional Dr. G. P. Chen, Dr. X. W. Wu, Dr. G. F. Wang, Prof. J. A. Ren Department of General Surgery Jinling Hospital Medical School of Nanjing University Nanjing 210002, China E-mail: [email protected] Dr. Y. R. Yu, Prof. Y. J. Zhao State Key Laboratory of Bioelectronics School of Biological Science and Medical Engineering Southeast University Nanjing 210096, China E-mail: [email protected] The ORCID identification number(s) for the author(s) of this article can be found under https://doi.org/10.1002/adfm.201801386.
DOI: 10.1002/adfm.201801386
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Figure 1. a) Schematic illustration of the formation of the hybrid hydrogel by mixing the DCS and BAPEG solutions. b) Photos of the hybrid hydrogel formation process. c) The injecting process of a hybrid hydrogel, which can be casted as a free-standing net. d,e) Self-healing process of the hybrid hydrogel with butterfly shapes: two butterfly-shaped hybrid hydrogels with blue and red colors were cut into two pieces, respectively. Then the total four pieces of alternate colors were combined into two integral butterfly-shaped hydrogels, which could be lifted after 20 min. f) Continuous step-strain measurements were applied to the hybrid hydrogel in step of 1% and 300% oscillatory strain for cycles.
addition, with the loading and controllable release of vascular endothelial growth factor (VEGF) in the hybrid hydrogel, cell proliferation and tissue remodeling in the wound bed could be significantly improved. Based on the proposed multifunctional hybrid hydrogel, we demonstrated through the in vivo study that acute tissue injuries such as vessel bleeding and
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liver bleeding could be repaired immediately by using the outstanding adhesion and hemostasis features of the hydrogel. Moreover, the chronic wound healing process of an infected full-thickness skin defect model could also be significantly enhanced by promoting angiogenesis, collagen deposition, macrophage polarization, and granulation tissue formation.
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These features make the multifunctional hybrid hydrogel ideal for versatile wound healing and other biomedical applications.
2. Results and Discussion In a typical experiment, the DCS was prepared by covalently modifying chitosan with dodecyl aldehyde (Figure S1, Supporting Information), whereas the BAPEG was derived from condensation of amino-terminated PEG with 4-formylbenzoic acid. The hybrid hydrogel system was formed by mixing the DCS and BAPEG solutions at room temperature; the mixture transformed into a transparent hydrogel because of the Schiffbase reaction between the benzaldehyde and amino groups in the polymer compounds (Figure 1a,b). The mechanical strength of the hybrid hydrogel was determined by the molar ratios (R) of CHO and NH2, and various total solid contents (C) of the components, as investigated by rheological analyses (Figure S2, Supporting Information). It was found that the hybrid hydrogel showed a stronger mechanical property when the value of R was 1/2 and that of C was 9% (Figure S2a, Supporting Information), and there was no significant change after being immersed in the phosphate buffered saline (PBS) (Figure S3a, Supporting Information). Under this situation, the gelation kinetics of the hybrid hydrogel were measured by evaluating the storage modulus G′ and the loss modulus G″ versus time (Figure S2b, Supporting Information). As the gelation formed in seconds, it was difficult to detect the cross point of G′ and G″. The gelation hydrogel had the ideal elastic property, and the G′ crossed the G″ curve at the strain around 100% (Figure S2c, Supporting Information). However, when the strain was larger than the cross point, G′ would be lower than G″, indicating the collapse of the hydrogel. Thus, the physical form of the hydrogel changed from solid to fluid. The viscosity of hybrid hydrogel as a function of shear rate decreased progressively with the increasing shear rate, indicating a substantial decrease in the degree of cross-linking (Figure S3b, Supporting Information). To investigate further this feature, the hybrid hydrogel was loaded into a syringe with a 23-gauge needle for injection (Figure 1c). It was found that the linkages of the hydrogel dissociated under pressure when compressed in the syringe, making the hydrogel transform and flow like a liquid to pass through the needle; the linkages then reformed into a solid hydrogel outside the syringe. These properties indicated that the hybrid hydrogel could be applied by injection. As the hybrid hydrogel was formed through aromatic Schiff base, which were much more stable than the aliphatic Schiff base, the hydrogel should have self-healing capacity based on the dynamic equilibria between the Schiff base and the amine and the aldehyde reactants. This should be ascribed to the uncoupling and recoupling of the linkages that occurred dynamically in the hydrogel networks. To investigate this feature, continuous step changes of the oscillatory strain between 1% and 300% at the same frequency (10 Hz) were applied to the hybrid hydrogel (Figure 1f). It was found that the large strain could break the hydrogel network structure and induce gel-to-sol transition, whereas under the low strain, the G′ returned quickly to the initial value, and the hydrogel recovered its normal structure
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again through the sol-to-gel transition. Thus, the broken structure of the hybrid hydrogel could be recovered quickly, and over the cycles of breaking and reforming, the hydrogel nevertheless exhibited its original structure. This self-healing capacity was further evaluated by a macroscopic test based on two pregelled butterfly-shaped hybrid hydrogels with blue (stained with trypan blue) and red colors (stained with rhodamine B) (Figure 1d,e and Figure S4, Supporting Information). It was found that the cut hydrogel pieces could be combined into integral butterflyshaped hydrogels again without any external stimulus, and the self-healed hydrogels were strong enough to be lifted. It was worth mentioning that the self-healing capacity of the hydrogels was influenced slightly after immersed in the PBS (Figure S5, Supporting Information). To demonstrate the potential biomedical value of the hybrid hydrogel, the cytotoxicity of the materials was first investigated. For this purpose, 3T3 mouse fibroblasts were used to assess the biocompatibility of the hybrid hydrogel through the extract solution method (Figure S6, Supporting Information) and cell counting kit-8 (CCK-8) (Figure S7, Supporting Information). The results indicated that the cell viability remained over 95% in different concentrations after incubation for 24 h compared with the control group. This can be attributed to the fact that the basic compositions of the hybrid hydrogel were chitosan and PEG, both of which are well-known biocompatible polymers. Benefiting from the dodecyl tails on the DCS, which could insert themselves into and be anchored onto the lipid bilayer of the cell membrane, the hybrid hydrogel was endowed with outstanding blood cell coagulation and hemostasis function (Figure 2a). To confirm these properties, the blood cell coagulation effect of the hybrid hydrogel was measured using heparinized mouse whole blood, blood cells and plasma. It was found that the mixtures of liquid blood and chitosan or those of liquid blood and CS/BAPEG hydrogel (Figure S8, Supporting Information) remained free-flowing liquids, but the liquid blood was instantly (within a few seconds) transformed into a self-supporting gel when the DCS or DCS/BAPEG hydrogel was added, where the sample held its weight upon tube inversion (Figure 2b). These results indicated that the gelation of blood can be mainly ascribed to the presence of dodecyl on the chitosan chains, which could be embedded within the hydrophobic cores of cell membranes. During this process, Schiff base between the benzaldehyde and amino groups in the cells, as well as the swelling effects of the hydrogel (Figure S9, Supporting Information), were also contributed to the blood gelation; while the reaction between DCS and plasma or inherent viscosity of the materials should have little effects (Figure S10, Supporting Information). Besides the blood cells, the dodecyl tails of the hybrid hydrogel could also host bacteria, which would trigger an inflammation or immune response. As wounds are easily infected in a dirty environment by the gathering of bacteria, thereby impeding the healing process and even causing life-threatening complications, this antibacterial capability was desired for the hybrid hydrogel during its application (Figure 2c). To evaluate the antibacterial efficacy, the hybrid hydrogel was used for separate cultures of gram-positive Staphylococcus aureus and gram-negative Escherichia coli. It was found from LIVE/DEAD bacterial staining that the bacteria were
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Figure 2. a) Schematic illustration of blood cell coagulation and hemostasis function. b) Effects of i) CS, ii) DCS, iii) CS/BAPEG, iv) DCS/BAPEG on heparinized mouse blood. c) Schematic illustration of anti-infective property. d) Live/Dead bacterial viability assay of E. coli and S. aureus before and after in contact with the hybrid hydrogel. e) The kill rate of hybrid hydrogel against S. aureus and E. coli. Scale bars are 10 µm.
alive and maintained their original appearance, i.e., spherical S. aureus and bacilliform E. coli, in the control group of PBS solution. However, after contacting the surface of the hybrid hydrogel, the microbes lost their structure, indicating that the hybrid hydrogel could destroy the cell membranes and cause the nucleic acid inside to outflow and accumulate (Figure 2d). Statistical results indicated that nearly 100% of S. aureus and 99% of E. coli on the surface of the hybrid hydrogel were killed (Figure 2e). The superior anti-infective property of the hybrid hydrogel also related to the bacterial destruction abilities of chitosan and aromatic Schiff-base. Because of the cell membrane anchoring effects of the dodecyl tails in DCS, the hybrid hydrogel was also expected to have cell-recruitment capability after being implanted into the body, which is vital during the wound-healing progress. To investigate this feature, the hybrid hydrogel was subcutaneously injected into the dorsal side of mice (Figure 3). It was found that the amount of hydrogel decreased gradually as a result of the existence of lipase, protease, and hyaluronidase in the mouse body (Figure 3a). During this process, no swelling or redness occurred around the injection site, indicating the good biocompatibility of the hydrogel. To investigate further the microscopic changes of this process, the tissues around the injection sites were stained using hematoxylin–eosin and Masson’s trichrome staining (Figure 3b,c). The results indicated that large amounts of neutrophils were found 12 h after injection, and the number of neutrophils and macrophages increased significantly with the degradation of the hydrogel. In addition, a large number of living fibroblasts were observed in the hydrogel, suggesting that the latter could probably supply a matrix for growth, proliferation, and migration of fibroblasts. These fibroblasts, as well as the disposition of abundant
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collagen, gradually replaced the inflammatory cells over time. These results indicated that the hybrid hydrogel could recruit a large amount of inflammatory and tissue repair cells to effectively promote granulation tissue formation. To demonstrate the practical value of the hybrid hydrogels for clinical applications, they were used for repairing acute tissue injuries, such as vessel bleeding and liver bleeding. In this application, the hydrogels should have strong tissue adhesion ability to anchor themselves on the target wound surface and bear the pressure of the bleeding. To confirm this property, the bursting pressure test was performed when the hydrogel was adhered to the aorta (Figure S11, Supporting Information). The result showed that the bursting pressure of the hybrid hydrogel could reach a value of 160 mmHg, which was clearly higher than the arterial blood pressure of 120 mmHg. Furthermore, the adhesive strength of the hybrid hydrogel on pigskin was observed higher than the CS/BAPEG hydrogel (Figure S12, Supporting Information). These results indicated that the hybrid hydrogel had strong tissue-adhesion capability and was an appropriate candidate for repairing vessel bleeding. This strong tissue adhesion capability could be ascribed to the synergistic effects of the covalent linkage of the Schiff base and the hydrophobic interactions between the hydrogel and the host cell membranes in the tissues. Based on the adhering hydrogel, the repairing process of vessel bleeding in a hemophiliac mouse was performed first. As the jugular vein is an important blood vessel for common injections and catheter insertions, and the femoral vein is a common vessel path for digital subtraction angiography and deep vein catheterization, these two veins were selected for testing by puncturing a hydrogel scrapping-decorated gauge needle inside the veins (Figure 4a). It was found that when the
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Figure 3. a) Gross observation of the degradation assay in vivo during different periods. b) Hematoxylin–eosin staining of the tissues around the injection sites. The host cells recruited around the injection sites (indicated with red arrows). c) Masson’s trichrome staining of the tissues around the injection sites. The deposition of collagen increased with the recruitment of fibroblasts (indicated with red arrows). Scale bars are 100 µm.
needle was removed from the veins and the sustentative alginate hydrogel scrapping, blood poured out from the pinholes in the middle of the scrapping and washed the hydrogel away, which caused a large amount of blood loss (Figure 4b,d, and Movies S1 and S2 in the Supporting Information). However, with the presence of the hybrid hydrogel in the scrapping, the pinholes in the jugular and femoral veins were both repaired because of the strong tissue adhesion and direct blood-gelling capacities of the hydrogel, ignoring the deficiency of the body’s own coagulation mechanism. The pinholes in the scrapping were also filled immediately after needle removal because of the self-healing capacity of the hydrogel. Thus, there was almost no bleeding from the veins, indicating the distinct hemostatic capability of the hybrid hydrogel (Figure 4c,e, and Movies S1 and S2 in the Supporting Information). To demonstrate further the robust hemostatic capability of the hybrid hydrogel, it was used for repairing an acute injury with massive hemorrhage that posed a most significant fatality risk to traumatic patients in critical situations, such as battlefield, natural disasters, and traffic accidents. For this purpose, the liver bleeding model was created by shearing off a part of the liver and
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was used to simulate severe trauma (Figure 4a). It was observed that without any treatment, a large amount of blood was lost from the liver on the hemorrhaging site and the group exhibited a bloodstain with a large area on the filter paper (Figure 4f, and Movie S3 in the Supporting Information). However, when the hybrid hydrogel was placed on the wounds as a physical barrier, the blood loss was quickly stopped and presented excellent hemostatic performance with almost no bloodstain on the filter paper (Figure 4g, and Movie S3 in the Supporting Information). These results indicated that the hybrid hydrogel possessed an excellent in vivo hemostatic capability and was able to overcome the challenges of most bleeding conditions. In addition to hemostasis, the hybrid hydrogel could also be used as a self-healing wound dressing for chronic wound healing. For this purpose, different kinds of actives that can promote angiogenesis, endothelial cell migration, proliferation, and vascular permeability, such as VEGF, were encapsulated into the hybrid hydrogel to improve the effects of the healing process. Benefiting from the chemical and physical properties of the hybrid hydrogel, the encapsulated VEGF was applied with homogeneous distribution and controllable release
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Figure 4. a) Schematic illustration of hybrid hydrogel scrapping-decorated gauge needle, vessel bleeding, and liver bleeding. b, d) Hemostatic capability of the alginate hydrogel scrapping-decorated gauge needle on jugular vein puncture and femoral vein puncture. c, e) Hemostatic capability of the hybrid hydrogel scrapping-decorated gauge needle on jugular vein puncture and femoral vein puncture. Bleeding conditions of liver f) without or g) with the hybrid hydrogel.
(Figure S13, Supporting Information). The hybrid hydrogels with and without VEGF loading were then applied to S. aureusinfected full-thickness skin defect models on the back of a mouse with a diameter of about 1 cm (Figure 5a). The healing capabilities and wound closure processes in these groups were all recorded for a detailed analysis. As wound infection is one of the major causes of death of injured patients, the efficacy of the hydrogels in preventing infection was investigated firstly by immunohistochemistry analysis of the secretion of two typical proinflammatory factors, interleukin-6 (IL-6) and tumor necrosis factor-α (TNF-α), in the wound bed (Figure 5f,g). Large amounts of IL-6 and TNF-α were detected in the control group of PBS solution after one week of healing, which suggested a severe inflammatory response. In contrast, little secretion was observed in the hybrid hydrogel groups, indicating that there were few signs of inflammation or infection formation. This phenomenon can be attributed to the specific chemical groups of the hybrid hydrogels, which have been demonstrated to kill microorganisms and guarantee that the hydrogel has excellent antibacterial capability. Besides preventing infection, the hybrid hydrogels also exhibited an extraordinary wound repair and skin regeneration advantage. As demonstrated above, the abundant dodecyl groups in the hydrogel polymer network exhibited a robust binding affinity to various cytomembranes on the tissue surface when the pregel solution was dripped into the defect. These diverse interfaces promoted the intimate integration of the hybrid hydrogels with the irregular wound bed, thereby providing a biomimetic microenvironment for cell proliferation and migration, and accelerating the growth of new epidermis. In addition, the hybrid hydrogels could also provide structural support for the formation of new granulation tissue and blood
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vessels because of their capability in recruiting host cells for tissue repair and angiogenesis. Thus, the wound closure rate and granulation tissue thickness in the wound bed were both higher in the hybrid hydrogel-treated mouse than in the groups of PBS solution and CS/BAPEG. In particular, with the addition of VEGF, the wound-healing performance was significantly improved (Figure 5b–e). From the microscale observation based on the CD31 staining, it was also found that the blood vessel density in the wound bed was clearly increased in the VEGFloaded hybrid hydrogel-treated mouse (Figure 6a, d). To investigate further the biological mechanism of the repair process, double immunofluorescence staining of collagens and fibroblasts was performed to evaluate collagen deposition in the granulation tissue (Figure 6b). The results showed that the collagens were mainly distributed in and around those fibroblasts, which indicated the collagen fabrication capability of the fibroblasts. These collagens had also shown substantially more deposition and a higher degree of directional alignment in the hybrid hydrogel and the VEGF-loaded hybrid hydrogel groups than in the other groups (Figure 6e). The increased matrix deposition and directional alignment of collagen fibers were essential for improving extracellular matrix construction. Besides the collagens and fibroblasts, macrophages also play an important role in tissue repair and remodeling during the wound repair process, and can be polarized to early M1 macrophages and later M2 macrophages (M1 macrophages participate in pro-inflammatory responses and play a central role in host defense against bacterial and viral infections, while M2 macrophages are associated with anti-inflammatory reactions, tissue remodeling and fibrosis). Hence, immunofluorescence staining was performed to identify the ratio between M2 macrophages (CD68+/CD206+) and M1 macrophages (CD68+/ CD86+) (Figure 6c). The results showed that the ratio of M2
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Figure 5. a) Schematic illustrations of wound healing process: i) hemostasis phase; ii) inflammation phase; iii) proliferation phase; (iv) remodeling phase. b) Representative photos of the skin wounds treated with PBS, CS/BAPEG, DCS/BAPEG, and VEGF-loaded DCS/BAPEG. c) Hematoxylin–eosin staining of wounds after 7 d at low magnification. d) Mean wound area. e) Quantitative analysis of granulation tissue thickness. f,g) Immunostaining of TNF-α and IL-6 in granulation tissues after 7 d. Scale bars in c) are 2 mm, and in (f–g) are 100 µm.
cells significantly increased in the hybrid hydrogel and the VEGF-loaded hybrid hydrogel groups, indicating a reduction in the inflammatory response and promotion of wound healing (Figure 6f). These results demonstrated that the hybrid hydrogels are ideal scaffolds for remodeling the granulation tissue and repairing different wounds.
3. Conclusion We have developed a BAPEG and DCS hybrid hydrogel for wound healing. Because of the reversible Schiff base between the benzaldehyde and amino groups in the polymer compounds, the hybrid hydrogel was imparted with injectable feature and self-healing capability. As the dodecyl tails could insert themselves into and be anchored onto the lipid bilayer of the cell membrane, the hybrid hydrogel possessed the distinctive functions of strong tissue adhesion, blood cell coagulation and hemostasis, and cell recruitment functions. In addition, with the coordinated effects of the bacterial anchoring ability of the dodecyl, and the bacterial destruction abilities of chitosan and aromatic Schiff-base, the hybrid hydrogel also exhibited superior anti-infective properties. Based on
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the proposed multifunctional hybrid hydrogel, we have demonstrated through the in vivo study that acute tissue injuries such as vessel bleeding and liver bleeding could be repaired immediately by using the outstanding adhesion and hemostasis functions of the hydrogel. Moreover, the chronic wound healing process of an infected full-thickness skin defect model could also be significantly enhanced by using a VEGF-loaded hydrogel to promote angiogenesis, collagen deposition, macrophage polarization, and granulation tissue formation. These features indicate that the multifunctional hybrid hydrogels are efficient and versatile for many kinds of wound healing, and thus we believe that these hybrid hydrogels will be widely used in clinic.
4. Experimental Section Materials, Cell Lines, and Animals: Chitosan (deacetylation degree: 85%, Mw: 4 × 105), dodecyl aldehyde, and sodium cyanoborohydride were purchased from Sigma-Aldrich (USA). Four-armed BAPEG (Mw 20000, 96%) was purchased from Ponsure Biotechnology. 3T3 cell lines were purchased from Nanjing keygen technology development Co., Ltd (China) and cultured in Eagle’s Minimum Essential Medium (Gibco, USA) supplemented with 10% fetal bovine serum (Gibco, USA) under
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Figure 6. a) Double immunofluorescence staining of neovascularization. CD31+ structures (red) were surrounded by α-smooth muscle actin positive cells (green), implying vascular ducts (indicated with white arrows). b) Double immunofluorescence staining of collagen (green) and fibroblast marker vimentin (red). Collagen was found in and around vimentin-expressing fibroblasts. c) Triple immunofluorescence staining of macrophages phenotype markers: CD68+ pan-macrophage (green), CD86+ M1 macrophage (red), and CD206+ M2 macrophage (yellow) colocalized with DAPI stained nuclei (blue). d) Quantification of CD31 labeled structures. e) Quantification of relative collagen expression. f) Statistical analysis of the ratio of M2 and M1. Scale bars in (a)–(c) are 50 µm. the condition of 37 °C, 5% CO2. The 8–12 weeks male Sprague–Dawley and BALBc mice were supplied by Jinling Hospital. Animals were treated in strict accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health, USA. All the animal care and experimental protocols were reviewed and approved by Animal Investigation Ethics Committee of Jinling Hospital. Synthesis of DCS: One gram of chitosan was dissolved in 50 mL of 0.2 m acetic acid. When dissolution was occurred, 40 mL of ethanol was added to allow the dodecyl aldehyde used for the alkylation to be in a solvating medium. The pH was adjusted after complete dissolution to 5.1. The solution of the dodecyl aldehyde in ethanol was added at the adequate ratio prior to an excess of sodium cyanohydroborate NaCNBH4 (3 moles per chitosan mole). The mixture was stirred 18 h at room temperature and the dodecyl-modified chitosan was precipitated with ethanol. The pH was adjusted to 7.0 with a sodium hydroxide solution and the precipitate was washed with ethanol/water mixtures with increasing content from 70% v/v to 100%. Fabrication of the Hybrid Hydrogels: The hybrid hydrogels were obtained by mixing the DCS with BAPEG solutions. Briefly, a 1% (w/w) DCS or CS solution was prepared by dissolving certain amounts of DCS in 0.2 m acetic acid. A 20% (w/w) BAPEG solution was prepared by dissolving 0.2 g of the polymer in 1.0 g of distilled and deionized water. A 10% (w/w) BAPEG solution was prepared by dissolving 0.1 g of the polymer
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in 1.0 g of distilled and deionized water. Hybrid hydrogels with different molar ratios (R) of CHO and NH2, and different total solid contents (C) were made: DCS/BAPEG (R = 1/2, C = 9, 1%DCS/20%BAPEG = 5/4), DCS/BAPEG (R = 1/1, C = 13, 1%DCS/20%BAPEG = 5/8), and DCS/ BAPEG (R = 1/4, C = 5, 1%DCS/10%BAPEG = 5/4). CS/BAPEG hydrogel was made from 1% CS solution and 20% BALEG solution mixed at ratio of 5/4. Rheological Analysis of the Hybrid Hydrogel: Rheological test of the hybrid hydrogel was carried out on an Anton Paar MCR301 rheometer at 25 °C with a 25 mm diameter flat plate attached to a transducer. The storage modulus G′, loss modulus G″ and viscosity were both analyzed for these studies. (1) G′and G″of the 20 min pregelled hybrid hydrogel discs with different weight ratios and total weight contents, 20 min pregelled hybrid hydrogel (R = 1/2, C = 9) immersed in PBS (pH = 7.4) for 4 h, DCS/blood cells mixture, DCS/blood mixture, and CS/ blood mixture were tested under a 1% strain, and the angular frequency was set from 0.1 to 100 Hz. (2) G′ and G″ of the hybrid hydrogel (R = 1/2, C = 9) versus time were tested to obtain the gelation time. (3) The 20 min pregelled hybrid hydrogel (R = 1/2, C = 9) was measured under strain amplitude sweep (γ = 0.1–1000%) at 10 Hz angular frequency. (4) The alternate step strain sweep of 20 min pregelled hybrid hydrogel (R = 1/2, C = 9) was measured at 10 Hz angular frequency, and amplitude oscillatory strains were changed from γ = 1% to 300% with
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150 s for every strain interval. (5) The steady shear viscosity as function of shear rate of CS, DCS, CS/blood mixture, DCS/blood Mixture, and 20 min pregelled hybrid hydrogel (R = 1/2, C = 9) was tested. Swelling Ratio of the Hybrid Hydrogel: The swelling ratio measurements of free-dried hydrogel were carried out gravimetrically. The known weights of free-dried hybrid hydrogels were immersed in the PBS at 37°C. At specific intervals of time, the swollen samples were taken out and weighted. All experiments were carried out in triplicate. The swelling ratio was calculated using the following equation: Swelling ratio (%) = (Ws − Wd)/Wd × 100%, where Ws and Wd were the weights of hydrogels at the swelling state and the dry state. Injectability Test of the Hybrid Hydrogel: A piece of hybrid hydrogel (R = 1/2, C = 9) with 20 min pregelation time was added into 2.5 mL syringe with needle (23-gauge) and injected onto a glass slide to form a net. The hydrogel on the surface of the glass slide formed an integral free-standing net keeping for 20 min at room temperature, and was lifted to test the injectability of the hydrogel. Self-Healing Performance of the Hybrid Hydrogel: Two pieces of butterfly-shaped hybrid hydrogel with 20 min pregelation time stained by rhodamine B and trypan blue were cut into equal two parts, respectively. After 20 min at 25 °C, the total four pieces of alternate colors were combined into two integral blended butterfly-shaped pieces. Self-healing was confirmed by the capacity of the healed butterfly-shaped hydrogel to hold its structure when suspended under gravity. Two disk-shaped hybrid hydrogels were operated as the steps described above, and then immersed in the PBS for 2 h before lift. Cytotoxicity Tests of the Hybrid Hydrogel: To make different extract solutions, hydrogels were added into each well containing 500 µL culture medium to reach the final concentrations of 62.5, 125, 250, and 500 mg mL−1, respectively, and incubated for 24 h, and after which the pH was tested. The 3T3 fibroblasts were plated in 96-well cell culture dishes with 4000 cells per well (100 µL per well) for 24 h to allow attachment before the experiment. Then, the media in the 96-well dish was removed, and the cells were rinsed with PBS. Different 24 h aged extract solutions were added into the wells, and the cells were incubated with the extract solutions (n = 5, for each group) for another 24 h. The cell viability of experimental groups was measured by employing a Cell Counting Kit-8 test and expressed by the percentage of living cells with respect to the control cells. The In Vitro Hemostasis Test of the Hybrid Hydrogel: Heparinized mouse blood, blood cells and blood plasma were used to test the hemostasis property of CS solution, DCS solution, CS/BAPEG hydrogel and DCS/ BAPEG hydrogel. Heparinized mouse blood (300 µL) was mixed with CS or DCS solution (300 µL, 1%, in 0.2 m acetic acid), and CS/BAPEG or DCS/BAPEG hydrogel (150 µL), respectively, in a test tube. Blood cells (300 µL) or plasma (300 µL) was mixed with DCS solution (300 µL, 1%, in 0.2 m acetic acid). In order to test the hemostasis property, the test tube was inverted to observe whether the solution could stand its own weight. Antibacterial Activity of the Hybrid Hydrogel: The Gram-positive bacteria S. aureus and Gram-negative bacteria E. coli were used to investigate the antibacterial activities of the hybrid hydrogels. The precursor solution was sterilized by 200 nm syringe filters. Firstly, 100 µL of the hydrogels were formed in the 96-well plate and incubated for 1 h at 37 °C. PBS buffer (pH 7.4) was added to wash off the unreacted materials for three times. Subsequently, 100 µL of bacteria solution (104 CFU mL−1) was poured onto the surface of the hydrogels, and tissue culture treated polystyrene (TCTP) was used as a control. The bacteria were incubated on the surfaces of hydrogels and TCTP for 24 h at 37 °C after which the suspension was taken out and mixed with 100 µL of LB, and seeded on Nutrient Agar Plate to count the CFU. To visualize the viable microbes on the hybrid hydrogels, bacteria were fluorescently imaged by a live-dead assay with two staining agents, NucView Green Live and propidium lodide. The mixed suspension was further stained with a combination of dye solution (5 µL of NucView Green Live and 5 µL of propidium iodide stock solution dissolved in 10 mL of PBS) in the dark for 15 min at 37 °C. After rinsing with PBS three times, the stained bacteria observed by Opera Phenix
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(PerkinElmer Inc., UK). As a result, live bacteria were fluoresced green and dead bacteria fluoresced red. In Vivo Degradation Behavior and Cell-Recruitment Capability of the Hybrid Hydrogel: Eighteen BALB/c mice were employed to evaluate the in vivo degradation behavior and cell-recruitment capability. 0.4 mL of the hybrid hydrogel was administered by dorsal subcutaneous injection. Three mice from each group were sacrificed at 5 min, 12 h, 1 d, 2 d, 3 d, and 5 d, respectively. The injection sites were opened with a surgical scissors to observe the state of hydrogels. Meanwhile, the tissue around the injected site was carefully removed, fixed in 4% paraformaldehyde, embedded in paraffin, sectioned, and stained with hematoxylin–eosin and Masson’s trichrome for further histopathological examination. Bursting Pressure Test of the Hybrid Hydrogel: The rabbit aorta was used in this experiment. The aorta was linked to a syringe pump and filled with PBS solution. A 1 mm diameter hole was made on the aorta surface after which the hydrogels were formed in situ on the puncture site. The bursting pressure was measured 5 min after the forming of the hydrogels by cardiogram monitor. The pressure at which it began to decrease was considered the bursting pressure. All measurements were repeated for six times. Tissue Adhesive Test of the Hybrid Hydrogel: Adhesion strength testing of hybrid hydrogel and CS/BAPEG hydrogel were performed by a universal testing machine (Testometric Co. Ltd, United Kingdom) using fresh pigskin. The hybrid hydrogel and the CS/BAPEG hydrogel were applied between two pigskin uniformly and placed for 1 h to make the gelation complete. Then, the adhesion strength was determined by the machine equipped with a 20 N load cell at a rate of 10 mm min−1. All the tests were repeated for three times. The Fabrication of the Hybrid Hydrogel Scrapping-Decorated Gauge Needle: The hydrogel scrapping-decorated needle consisted of two layers (alginate hydrogel layer and hybrid hydrogel layer). Alginate hydrogel layer was made by mixing 2% alginate solution and 2% calcium chloride after which the hybrid hydrogel was formed on the surface of the layer in situ to form a double-layer scrapping. Then commercial needle pierced the double-layer scrapping to fabricate the hydrogel scrappingdecorated needle. The alginate hydrogel scrapping only was set as the control group. Evaluating Hemostatic Effects of the Hybrid Hydrogel ScrappingDecorated Gauge Needle: The hemostatic effects of the hybrid hydrogel scrapping-decorated gauge needle were evaluated in vivo using two animal models, mouse femoral vein puncture and jugular vein puncture (Sprague–Dawley, 8–12 weeks old, male, 250–300 g). All animals were chosen at random for the experiments. The mice were anaesthetized by intraperitoneal injection of 10% chloral hydrate. The mouse jugular vein and femoral vein were punctured by alginate or hybrid hydrogel scrapping-decorated gauge needle, respectively, then heparin was injected through the needles to heparinize the blood. The needles were removed from the veins and the alginate or hybrid hydrogel scrapping, and the bleeding conditions were recorded. Mouse Liver Injury Models: A mouse hemorrhaging liver model to investigate the in vivo robust hemostatic ability of the hybrid hydrogels was used. First, the mice were anesthetized as described above. Then the mouse’s liver was exposed and sheared to bleed with a surgical scissors. A filter paper was placed beneath the liver. Immediately, 200 µL of the pregel hybrid hydrogel was injected on the surface of bleeding site as a hemostatic agent. The liver bleeding conditions were observed. Controllable Release of Proteinic Drugs from the Hybrid Hydrogel: 1 mL of the hybrid hydrogel containing 0.1 mg BSA-FITC was injected into 3 mL PBS (pH = 7.4). At each time point (0.5, 1, 3, 6, 12, 24, 48, 72 h), 1 mL PBS was retrieved and the concentration of BSA-FITC were determined by a multi-functional microplate reader (HORIBA, model: FluoroMax-4) at the absorbance of 493 nm. Then, the PBS was poured back. All experiments were performed in triplicates. The concentration of released BSA-FITC from the hybrid hydrogel was calculated according to the following equation: FI = 3.61 × 108 × C + 3.544 × 105 (R2 = 0.928), where the FI referred to the fluorescence intensity and C represented the concentration of released BSA-FITC.
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Chronic Wound Healing Study of the Hybrid Hydrogel: A mouse infected full-thickness cutaneous wound model was used to evaluate the effect of hybrid hydrogel on chronic wound healing. First, a total of 24 healthy male Sprague–Dawley rats (180–250 g, Jinling Hospital, Nanjing, China) were anesthetized and their backs were shaved. A rounded full-thickness cutaneous wound (1 cm × 1 cm) area was created on the back of each rat after which 200 µL S. aureus solution (1 × 108 CFU mL−1) was introduced onto the wounds, and then divided into four groups randomly. The four groups were treated with PBS, CS/ BAPEG hydrogel, DCS/BAPEG hydrogel and VEGF-loaded DCS/BAPEG hydrogel respectively. The precursor solution of hydrogels was sterilized by filtration using 200 nm syringe filters, and the saline was sterilized by autoclaving. Thereafter, the rats were individually housed in cages and allowed to heal for 7d. The wounds were observed on day 0, 3, 5, and 7. The mice were sacrificed after 7d and granulation tissues over the wound bed were excised. Each sample was divided into two pieces. One piece was immersed in neutral formaldehyde for further histology and immunohistochemistry analysis, another was stored in liquid nitrogen for immunofluorescent staining. Histology and Immunohistochemistry: The granulation tissue samples were removed from the neutral formaldehyde, followed by dehydration and embedded in paraffin. Serially sections, 5 µm in thickness, were acquired by a microtome according to standard protocols, and were prepared for hematoxylin-eosin and immunohistochemical staining. Sections for immunohistochemistry were stained with IL-6 and TNF-α. For neovascularization evaluation, sections were reacted with primary antibodies against CD31 (KEYGEN, KGYM0118–7) and α-smooth muscle actin (α-SMA) (KEYGEN, KGYT5053-6). Afterward, sections were washed and incubated with FITC- (KEYGEN, KGAA26) and TRITC(KEYGEN, KGAA98) conjugated secondary antibodies, and were rinsed and mounted with 4, 6-diamidino-2-phenylindole (DAPI) mounting medium to label Nuclei. Immunofluorescence Staining: The frozen samples were fixed in acetone, and then incubated with blocking buffer. To evaluate collagen deposition, sections were incubated with primary antibodies vimentin (Abcam, 200 ab20346) and collagen (Abcam, ab96723) for 2h at room temperature, and then incubated with secondary antibodies Alexa Fluor 555- and 488- conjugated for 1 h. Afterward, the sections were rinsed and mounted with DAPI mounting solution. To evaluate macrophage polarization, sections were incubated with primary antibodies against the pan- macrophage marker CD68 (Abcam, ab955), M1-macrophage marker CD86 (Abcam, ab53004), and the M2-macrophage marker CD206 (Santa Cruz, sc-34577). Afterward, the sections were washed and incubated with the fluorescently conjugated secondary antibodies Alexa Fluor 488-, 555-, and 647-. DAPI was used to label the nuclei. Photos were obtained and analyzed with an Opera Phenix (PerkinElmer Inc., UK).
Supporting Information Supporting Information is available from the Wiley Online Library or from the author.
Acknowledgements G.P.C., Y.R.Y., and X.W.W. contributed equally to this work. This work was supported by the National Science Foundation of China (Grant Nos. 81571881, 21473029, and 51522302), Projects of Jiangsu Social Development (BE2016752 and BE2017722), Innovation Project of Military Medicine (16CXZ007), and the Scientific Research Foundation of Southeast University.
Conflict of Interest The authors declare no conflict of interest.
Adv. Funct. Mater. 2018, 1801386
Keywords bioinspired materials, biomaterials, hydrogels, self-healing, wound healing Received: February 22, 2018 Revised: May 9, 2018 Published online:
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Bioinspired Multifunctional Hybrid Hydrogel Promotes Wound Healing Guopu Chen, Yunru Yu, Xiuwen Wu, Gefei Wang, Jianan Ren,* and Yuanjin Zhao* hydrogels.[6–11] Among these biomaterials, hydrogels, including chitosan, polyethylene glycol, and their derivatives, are the most attractive candidates because of their functions of absorbing tissue exudates, maintaining a moist wound environment, cooling the wound surface, allowing oxygen to permeate, as well as leading to pain relief for patients.[12–17] In particular, with the application of some injectable hydrogels, wound healing approaches could include the additional functions of in situ drug encapsulation and wound site filling, anti-infection, antioxidation, hemostasis, and adhesiveness.[18–22] Although the use of hydrogel materials has promoted wound healing to a certain degree, biological wound healing occurs in the four unique yet overlapping stages of hemostasis, inflammation, proliferation, and remodeling,[23–26] and the recently developed hydrogels could only contribute their functions in one or two stages. This wound greatly affect the final healing effect of the wound as each stage is highly orchestrated and can directly impact the subsequent events.[27–30] In addition, most of these hydrogels could only repair a single type of wound, which limited their wide applications in the clinic. Thus, the creation of novel hydrogel that can act their functions on the whole healing processes of different wounds is still anticipated. In this paper, inspired by the spontaneous heal-injury process of the organism, which uses coordinated multiple mechanisms for body recovery, we proposed a four-armed benzaldehyde-terminated polyethylene glycol (BAPEG) and dodecyl-modified chitosan (DCS) hybrid hydrogel system with the desired multifunctional features for wound healing, as schemed in Figure 1. The hydrogel was formed and imparted with self-healing capability through the reversible Schiff base between the benzaldehyde and amino groups in the polymer compounds. The existing dodecyl tails can insert themselves into and be anchored onto the lipid bilayer of the cell membrane; thus, the hybrid hydrogel presented outstanding tissue adhesion, blood cell coagulation and hemostasis, and cell recruitment functions. This hybrid hydrogel could also exhibit superior anti-infective properties due to the coordinate effects of the bacterial anchoring ability of the dodecyl, and the bacterial destruction abilities of chitosan and aromatic Schiff base. In
Inspired by the coordinated multiple healing mechanism of the organism, a four-armed benzaldehyde-terminated polyethylene glycol and dodecylmodified chitosan hybrid hydrogel with vascular endothelial growth factor (VEGF) encapsulation are presented for efficient and versatile wound healing. The hybrid hydrogel is formed through the reversible Schiff base and possesses self-healing capability. As the dodecyl tails can insert themselves into and be anchored onto the lipid bilayer of the cell membrane, the hybrid hydrogel has outstanding tissue adhesion, blood cell coagulation and hemostasis, anti-infection, and cell recruitment functions. Moreover, by loading in and controllably releasing VEGF from the hybrid hydrogel, the processes of cell proliferation and tissue remodeling in the wound bed can be significantly improved. Based on an in vivo study of the multifunctional hybrid hydrogel, it is demonstrated that acute tissue injuries such as vessel bleeding and liver bleeding can be repaired immediately because of the outstanding adhesion and hemostasis features of the hydrogel. Moreover, the chronic wound-healing process of an infectious full-thickness skin defect model can also be significantly enhanced by promoting angiogenesis, collagen deposition, macrophage polarization, and granulation tissue formation. Thus, this multifunctional hybrid hydrogel is potentially valuable for clinical applications.
1. Introduction In recent years, wound treatment and its therapeutic impediments have become a fundamental healthcare concern, presenting an significant economically challenging burden worldwide.[1–5] To date, many kinds of biomaterials have been used for rapid wound healing, including electrospun nanofiber, porous foams, biocompatible membranes, and functional Dr. G. P. Chen, Dr. X. W. Wu, Dr. G. F. Wang, Prof. J. A. Ren Department of General Surgery Jinling Hospital Medical School of Nanjing University Nanjing 210002, China E-mail: [email protected] Dr. Y. R. Yu, Prof. Y. J. Zhao State Key Laboratory of Bioelectronics School of Biological Science and Medical Engineering Southeast University Nanjing 210096, China E-mail: [email protected] The ORCID identification number(s) for the author(s) of this article can be found under https://doi.org/10.1002/adfm.201801386.
DOI: 10.1002/adfm.201801386
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Figure 1. a) Schematic illustration of the formation of the hybrid hydrogel by mixing the DCS and BAPEG solutions. b) Photos of the hybrid hydrogel formation process. c) The injecting process of a hybrid hydrogel, which can be casted as a free-standing net. d,e) Self-healing process of the hybrid hydrogel with butterfly shapes: two butterfly-shaped hybrid hydrogels with blue and red colors were cut into two pieces, respectively. Then the total four pieces of alternate colors were combined into two integral butterfly-shaped hydrogels, which could be lifted after 20 min. f) Continuous step-strain measurements were applied to the hybrid hydrogel in step of 1% and 300% oscillatory strain for cycles.
addition, with the loading and controllable release of vascular endothelial growth factor (VEGF) in the hybrid hydrogel, cell proliferation and tissue remodeling in the wound bed could be significantly improved. Based on the proposed multifunctional hybrid hydrogel, we demonstrated through the in vivo study that acute tissue injuries such as vessel bleeding and
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liver bleeding could be repaired immediately by using the outstanding adhesion and hemostasis features of the hydrogel. Moreover, the chronic wound healing process of an infected full-thickness skin defect model could also be significantly enhanced by promoting angiogenesis, collagen deposition, macrophage polarization, and granulation tissue formation.
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These features make the multifunctional hybrid hydrogel ideal for versatile wound healing and other biomedical applications.
2. Results and Discussion In a typical experiment, the DCS was prepared by covalently modifying chitosan with dodecyl aldehyde (Figure S1, Supporting Information), whereas the BAPEG was derived from condensation of amino-terminated PEG with 4-formylbenzoic acid. The hybrid hydrogel system was formed by mixing the DCS and BAPEG solutions at room temperature; the mixture transformed into a transparent hydrogel because of the Schiffbase reaction between the benzaldehyde and amino groups in the polymer compounds (Figure 1a,b). The mechanical strength of the hybrid hydrogel was determined by the molar ratios (R) of CHO and NH2, and various total solid contents (C) of the components, as investigated by rheological analyses (Figure S2, Supporting Information). It was found that the hybrid hydrogel showed a stronger mechanical property when the value of R was 1/2 and that of C was 9% (Figure S2a, Supporting Information), and there was no significant change after being immersed in the phosphate buffered saline (PBS) (Figure S3a, Supporting Information). Under this situation, the gelation kinetics of the hybrid hydrogel were measured by evaluating the storage modulus G′ and the loss modulus G″ versus time (Figure S2b, Supporting Information). As the gelation formed in seconds, it was difficult to detect the cross point of G′ and G″. The gelation hydrogel had the ideal elastic property, and the G′ crossed the G″ curve at the strain around 100% (Figure S2c, Supporting Information). However, when the strain was larger than the cross point, G′ would be lower than G″, indicating the collapse of the hydrogel. Thus, the physical form of the hydrogel changed from solid to fluid. The viscosity of hybrid hydrogel as a function of shear rate decreased progressively with the increasing shear rate, indicating a substantial decrease in the degree of cross-linking (Figure S3b, Supporting Information). To investigate further this feature, the hybrid hydrogel was loaded into a syringe with a 23-gauge needle for injection (Figure 1c). It was found that the linkages of the hydrogel dissociated under pressure when compressed in the syringe, making the hydrogel transform and flow like a liquid to pass through the needle; the linkages then reformed into a solid hydrogel outside the syringe. These properties indicated that the hybrid hydrogel could be applied by injection. As the hybrid hydrogel was formed through aromatic Schiff base, which were much more stable than the aliphatic Schiff base, the hydrogel should have self-healing capacity based on the dynamic equilibria between the Schiff base and the amine and the aldehyde reactants. This should be ascribed to the uncoupling and recoupling of the linkages that occurred dynamically in the hydrogel networks. To investigate this feature, continuous step changes of the oscillatory strain between 1% and 300% at the same frequency (10 Hz) were applied to the hybrid hydrogel (Figure 1f). It was found that the large strain could break the hydrogel network structure and induce gel-to-sol transition, whereas under the low strain, the G′ returned quickly to the initial value, and the hydrogel recovered its normal structure
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again through the sol-to-gel transition. Thus, the broken structure of the hybrid hydrogel could be recovered quickly, and over the cycles of breaking and reforming, the hydrogel nevertheless exhibited its original structure. This self-healing capacity was further evaluated by a macroscopic test based on two pregelled butterfly-shaped hybrid hydrogels with blue (stained with trypan blue) and red colors (stained with rhodamine B) (Figure 1d,e and Figure S4, Supporting Information). It was found that the cut hydrogel pieces could be combined into integral butterflyshaped hydrogels again without any external stimulus, and the self-healed hydrogels were strong enough to be lifted. It was worth mentioning that the self-healing capacity of the hydrogels was influenced slightly after immersed in the PBS (Figure S5, Supporting Information). To demonstrate the potential biomedical value of the hybrid hydrogel, the cytotoxicity of the materials was first investigated. For this purpose, 3T3 mouse fibroblasts were used to assess the biocompatibility of the hybrid hydrogel through the extract solution method (Figure S6, Supporting Information) and cell counting kit-8 (CCK-8) (Figure S7, Supporting Information). The results indicated that the cell viability remained over 95% in different concentrations after incubation for 24 h compared with the control group. This can be attributed to the fact that the basic compositions of the hybrid hydrogel were chitosan and PEG, both of which are well-known biocompatible polymers. Benefiting from the dodecyl tails on the DCS, which could insert themselves into and be anchored onto the lipid bilayer of the cell membrane, the hybrid hydrogel was endowed with outstanding blood cell coagulation and hemostasis function (Figure 2a). To confirm these properties, the blood cell coagulation effect of the hybrid hydrogel was measured using heparinized mouse whole blood, blood cells and plasma. It was found that the mixtures of liquid blood and chitosan or those of liquid blood and CS/BAPEG hydrogel (Figure S8, Supporting Information) remained free-flowing liquids, but the liquid blood was instantly (within a few seconds) transformed into a self-supporting gel when the DCS or DCS/BAPEG hydrogel was added, where the sample held its weight upon tube inversion (Figure 2b). These results indicated that the gelation of blood can be mainly ascribed to the presence of dodecyl on the chitosan chains, which could be embedded within the hydrophobic cores of cell membranes. During this process, Schiff base between the benzaldehyde and amino groups in the cells, as well as the swelling effects of the hydrogel (Figure S9, Supporting Information), were also contributed to the blood gelation; while the reaction between DCS and plasma or inherent viscosity of the materials should have little effects (Figure S10, Supporting Information). Besides the blood cells, the dodecyl tails of the hybrid hydrogel could also host bacteria, which would trigger an inflammation or immune response. As wounds are easily infected in a dirty environment by the gathering of bacteria, thereby impeding the healing process and even causing life-threatening complications, this antibacterial capability was desired for the hybrid hydrogel during its application (Figure 2c). To evaluate the antibacterial efficacy, the hybrid hydrogel was used for separate cultures of gram-positive Staphylococcus aureus and gram-negative Escherichia coli. It was found from LIVE/DEAD bacterial staining that the bacteria were
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Figure 2. a) Schematic illustration of blood cell coagulation and hemostasis function. b) Effects of i) CS, ii) DCS, iii) CS/BAPEG, iv) DCS/BAPEG on heparinized mouse blood. c) Schematic illustration of anti-infective property. d) Live/Dead bacterial viability assay of E. coli and S. aureus before and after in contact with the hybrid hydrogel. e) The kill rate of hybrid hydrogel against S. aureus and E. coli. Scale bars are 10 µm.
alive and maintained their original appearance, i.e., spherical S. aureus and bacilliform E. coli, in the control group of PBS solution. However, after contacting the surface of the hybrid hydrogel, the microbes lost their structure, indicating that the hybrid hydrogel could destroy the cell membranes and cause the nucleic acid inside to outflow and accumulate (Figure 2d). Statistical results indicated that nearly 100% of S. aureus and 99% of E. coli on the surface of the hybrid hydrogel were killed (Figure 2e). The superior anti-infective property of the hybrid hydrogel also related to the bacterial destruction abilities of chitosan and aromatic Schiff-base. Because of the cell membrane anchoring effects of the dodecyl tails in DCS, the hybrid hydrogel was also expected to have cell-recruitment capability after being implanted into the body, which is vital during the wound-healing progress. To investigate this feature, the hybrid hydrogel was subcutaneously injected into the dorsal side of mice (Figure 3). It was found that the amount of hydrogel decreased gradually as a result of the existence of lipase, protease, and hyaluronidase in the mouse body (Figure 3a). During this process, no swelling or redness occurred around the injection site, indicating the good biocompatibility of the hydrogel. To investigate further the microscopic changes of this process, the tissues around the injection sites were stained using hematoxylin–eosin and Masson’s trichrome staining (Figure 3b,c). The results indicated that large amounts of neutrophils were found 12 h after injection, and the number of neutrophils and macrophages increased significantly with the degradation of the hydrogel. In addition, a large number of living fibroblasts were observed in the hydrogel, suggesting that the latter could probably supply a matrix for growth, proliferation, and migration of fibroblasts. These fibroblasts, as well as the disposition of abundant
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collagen, gradually replaced the inflammatory cells over time. These results indicated that the hybrid hydrogel could recruit a large amount of inflammatory and tissue repair cells to effectively promote granulation tissue formation. To demonstrate the practical value of the hybrid hydrogels for clinical applications, they were used for repairing acute tissue injuries, such as vessel bleeding and liver bleeding. In this application, the hydrogels should have strong tissue adhesion ability to anchor themselves on the target wound surface and bear the pressure of the bleeding. To confirm this property, the bursting pressure test was performed when the hydrogel was adhered to the aorta (Figure S11, Supporting Information). The result showed that the bursting pressure of the hybrid hydrogel could reach a value of 160 mmHg, which was clearly higher than the arterial blood pressure of 120 mmHg. Furthermore, the adhesive strength of the hybrid hydrogel on pigskin was observed higher than the CS/BAPEG hydrogel (Figure S12, Supporting Information). These results indicated that the hybrid hydrogel had strong tissue-adhesion capability and was an appropriate candidate for repairing vessel bleeding. This strong tissue adhesion capability could be ascribed to the synergistic effects of the covalent linkage of the Schiff base and the hydrophobic interactions between the hydrogel and the host cell membranes in the tissues. Based on the adhering hydrogel, the repairing process of vessel bleeding in a hemophiliac mouse was performed first. As the jugular vein is an important blood vessel for common injections and catheter insertions, and the femoral vein is a common vessel path for digital subtraction angiography and deep vein catheterization, these two veins were selected for testing by puncturing a hydrogel scrapping-decorated gauge needle inside the veins (Figure 4a). It was found that when the
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Figure 3. a) Gross observation of the degradation assay in vivo during different periods. b) Hematoxylin–eosin staining of the tissues around the injection sites. The host cells recruited around the injection sites (indicated with red arrows). c) Masson’s trichrome staining of the tissues around the injection sites. The deposition of collagen increased with the recruitment of fibroblasts (indicated with red arrows). Scale bars are 100 µm.
needle was removed from the veins and the sustentative alginate hydrogel scrapping, blood poured out from the pinholes in the middle of the scrapping and washed the hydrogel away, which caused a large amount of blood loss (Figure 4b,d, and Movies S1 and S2 in the Supporting Information). However, with the presence of the hybrid hydrogel in the scrapping, the pinholes in the jugular and femoral veins were both repaired because of the strong tissue adhesion and direct blood-gelling capacities of the hydrogel, ignoring the deficiency of the body’s own coagulation mechanism. The pinholes in the scrapping were also filled immediately after needle removal because of the self-healing capacity of the hydrogel. Thus, there was almost no bleeding from the veins, indicating the distinct hemostatic capability of the hybrid hydrogel (Figure 4c,e, and Movies S1 and S2 in the Supporting Information). To demonstrate further the robust hemostatic capability of the hybrid hydrogel, it was used for repairing an acute injury with massive hemorrhage that posed a most significant fatality risk to traumatic patients in critical situations, such as battlefield, natural disasters, and traffic accidents. For this purpose, the liver bleeding model was created by shearing off a part of the liver and
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was used to simulate severe trauma (Figure 4a). It was observed that without any treatment, a large amount of blood was lost from the liver on the hemorrhaging site and the group exhibited a bloodstain with a large area on the filter paper (Figure 4f, and Movie S3 in the Supporting Information). However, when the hybrid hydrogel was placed on the wounds as a physical barrier, the blood loss was quickly stopped and presented excellent hemostatic performance with almost no bloodstain on the filter paper (Figure 4g, and Movie S3 in the Supporting Information). These results indicated that the hybrid hydrogel possessed an excellent in vivo hemostatic capability and was able to overcome the challenges of most bleeding conditions. In addition to hemostasis, the hybrid hydrogel could also be used as a self-healing wound dressing for chronic wound healing. For this purpose, different kinds of actives that can promote angiogenesis, endothelial cell migration, proliferation, and vascular permeability, such as VEGF, were encapsulated into the hybrid hydrogel to improve the effects of the healing process. Benefiting from the chemical and physical properties of the hybrid hydrogel, the encapsulated VEGF was applied with homogeneous distribution and controllable release
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Figure 4. a) Schematic illustration of hybrid hydrogel scrapping-decorated gauge needle, vessel bleeding, and liver bleeding. b, d) Hemostatic capability of the alginate hydrogel scrapping-decorated gauge needle on jugular vein puncture and femoral vein puncture. c, e) Hemostatic capability of the hybrid hydrogel scrapping-decorated gauge needle on jugular vein puncture and femoral vein puncture. Bleeding conditions of liver f) without or g) with the hybrid hydrogel.
(Figure S13, Supporting Information). The hybrid hydrogels with and without VEGF loading were then applied to S. aureusinfected full-thickness skin defect models on the back of a mouse with a diameter of about 1 cm (Figure 5a). The healing capabilities and wound closure processes in these groups were all recorded for a detailed analysis. As wound infection is one of the major causes of death of injured patients, the efficacy of the hydrogels in preventing infection was investigated firstly by immunohistochemistry analysis of the secretion of two typical proinflammatory factors, interleukin-6 (IL-6) and tumor necrosis factor-α (TNF-α), in the wound bed (Figure 5f,g). Large amounts of IL-6 and TNF-α were detected in the control group of PBS solution after one week of healing, which suggested a severe inflammatory response. In contrast, little secretion was observed in the hybrid hydrogel groups, indicating that there were few signs of inflammation or infection formation. This phenomenon can be attributed to the specific chemical groups of the hybrid hydrogels, which have been demonstrated to kill microorganisms and guarantee that the hydrogel has excellent antibacterial capability. Besides preventing infection, the hybrid hydrogels also exhibited an extraordinary wound repair and skin regeneration advantage. As demonstrated above, the abundant dodecyl groups in the hydrogel polymer network exhibited a robust binding affinity to various cytomembranes on the tissue surface when the pregel solution was dripped into the defect. These diverse interfaces promoted the intimate integration of the hybrid hydrogels with the irregular wound bed, thereby providing a biomimetic microenvironment for cell proliferation and migration, and accelerating the growth of new epidermis. In addition, the hybrid hydrogels could also provide structural support for the formation of new granulation tissue and blood
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vessels because of their capability in recruiting host cells for tissue repair and angiogenesis. Thus, the wound closure rate and granulation tissue thickness in the wound bed were both higher in the hybrid hydrogel-treated mouse than in the groups of PBS solution and CS/BAPEG. In particular, with the addition of VEGF, the wound-healing performance was significantly improved (Figure 5b–e). From the microscale observation based on the CD31 staining, it was also found that the blood vessel density in the wound bed was clearly increased in the VEGFloaded hybrid hydrogel-treated mouse (Figure 6a, d). To investigate further the biological mechanism of the repair process, double immunofluorescence staining of collagens and fibroblasts was performed to evaluate collagen deposition in the granulation tissue (Figure 6b). The results showed that the collagens were mainly distributed in and around those fibroblasts, which indicated the collagen fabrication capability of the fibroblasts. These collagens had also shown substantially more deposition and a higher degree of directional alignment in the hybrid hydrogel and the VEGF-loaded hybrid hydrogel groups than in the other groups (Figure 6e). The increased matrix deposition and directional alignment of collagen fibers were essential for improving extracellular matrix construction. Besides the collagens and fibroblasts, macrophages also play an important role in tissue repair and remodeling during the wound repair process, and can be polarized to early M1 macrophages and later M2 macrophages (M1 macrophages participate in pro-inflammatory responses and play a central role in host defense against bacterial and viral infections, while M2 macrophages are associated with anti-inflammatory reactions, tissue remodeling and fibrosis). Hence, immunofluorescence staining was performed to identify the ratio between M2 macrophages (CD68+/CD206+) and M1 macrophages (CD68+/ CD86+) (Figure 6c). The results showed that the ratio of M2
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Figure 5. a) Schematic illustrations of wound healing process: i) hemostasis phase; ii) inflammation phase; iii) proliferation phase; (iv) remodeling phase. b) Representative photos of the skin wounds treated with PBS, CS/BAPEG, DCS/BAPEG, and VEGF-loaded DCS/BAPEG. c) Hematoxylin–eosin staining of wounds after 7 d at low magnification. d) Mean wound area. e) Quantitative analysis of granulation tissue thickness. f,g) Immunostaining of TNF-α and IL-6 in granulation tissues after 7 d. Scale bars in c) are 2 mm, and in (f–g) are 100 µm.
cells significantly increased in the hybrid hydrogel and the VEGF-loaded hybrid hydrogel groups, indicating a reduction in the inflammatory response and promotion of wound healing (Figure 6f). These results demonstrated that the hybrid hydrogels are ideal scaffolds for remodeling the granulation tissue and repairing different wounds.
3. Conclusion We have developed a BAPEG and DCS hybrid hydrogel for wound healing. Because of the reversible Schiff base between the benzaldehyde and amino groups in the polymer compounds, the hybrid hydrogel was imparted with injectable feature and self-healing capability. As the dodecyl tails could insert themselves into and be anchored onto the lipid bilayer of the cell membrane, the hybrid hydrogel possessed the distinctive functions of strong tissue adhesion, blood cell coagulation and hemostasis, and cell recruitment functions. In addition, with the coordinated effects of the bacterial anchoring ability of the dodecyl, and the bacterial destruction abilities of chitosan and aromatic Schiff-base, the hybrid hydrogel also exhibited superior anti-infective properties. Based on
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the proposed multifunctional hybrid hydrogel, we have demonstrated through the in vivo study that acute tissue injuries such as vessel bleeding and liver bleeding could be repaired immediately by using the outstanding adhesion and hemostasis functions of the hydrogel. Moreover, the chronic wound healing process of an infected full-thickness skin defect model could also be significantly enhanced by using a VEGF-loaded hydrogel to promote angiogenesis, collagen deposition, macrophage polarization, and granulation tissue formation. These features indicate that the multifunctional hybrid hydrogels are efficient and versatile for many kinds of wound healing, and thus we believe that these hybrid hydrogels will be widely used in clinic.
4. Experimental Section Materials, Cell Lines, and Animals: Chitosan (deacetylation degree: 85%, Mw: 4 × 105), dodecyl aldehyde, and sodium cyanoborohydride were purchased from Sigma-Aldrich (USA). Four-armed BAPEG (Mw 20000, 96%) was purchased from Ponsure Biotechnology. 3T3 cell lines were purchased from Nanjing keygen technology development Co., Ltd (China) and cultured in Eagle’s Minimum Essential Medium (Gibco, USA) supplemented with 10% fetal bovine serum (Gibco, USA) under
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Figure 6. a) Double immunofluorescence staining of neovascularization. CD31+ structures (red) were surrounded by α-smooth muscle actin positive cells (green), implying vascular ducts (indicated with white arrows). b) Double immunofluorescence staining of collagen (green) and fibroblast marker vimentin (red). Collagen was found in and around vimentin-expressing fibroblasts. c) Triple immunofluorescence staining of macrophages phenotype markers: CD68+ pan-macrophage (green), CD86+ M1 macrophage (red), and CD206+ M2 macrophage (yellow) colocalized with DAPI stained nuclei (blue). d) Quantification of CD31 labeled structures. e) Quantification of relative collagen expression. f) Statistical analysis of the ratio of M2 and M1. Scale bars in (a)–(c) are 50 µm. the condition of 37 °C, 5% CO2. The 8–12 weeks male Sprague–Dawley and BALBc mice were supplied by Jinling Hospital. Animals were treated in strict accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health, USA. All the animal care and experimental protocols were reviewed and approved by Animal Investigation Ethics Committee of Jinling Hospital. Synthesis of DCS: One gram of chitosan was dissolved in 50 mL of 0.2 m acetic acid. When dissolution was occurred, 40 mL of ethanol was added to allow the dodecyl aldehyde used for the alkylation to be in a solvating medium. The pH was adjusted after complete dissolution to 5.1. The solution of the dodecyl aldehyde in ethanol was added at the adequate ratio prior to an excess of sodium cyanohydroborate NaCNBH4 (3 moles per chitosan mole). The mixture was stirred 18 h at room temperature and the dodecyl-modified chitosan was precipitated with ethanol. The pH was adjusted to 7.0 with a sodium hydroxide solution and the precipitate was washed with ethanol/water mixtures with increasing content from 70% v/v to 100%. Fabrication of the Hybrid Hydrogels: The hybrid hydrogels were obtained by mixing the DCS with BAPEG solutions. Briefly, a 1% (w/w) DCS or CS solution was prepared by dissolving certain amounts of DCS in 0.2 m acetic acid. A 20% (w/w) BAPEG solution was prepared by dissolving 0.2 g of the polymer in 1.0 g of distilled and deionized water. A 10% (w/w) BAPEG solution was prepared by dissolving 0.1 g of the polymer
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in 1.0 g of distilled and deionized water. Hybrid hydrogels with different molar ratios (R) of CHO and NH2, and different total solid contents (C) were made: DCS/BAPEG (R = 1/2, C = 9, 1%DCS/20%BAPEG = 5/4), DCS/BAPEG (R = 1/1, C = 13, 1%DCS/20%BAPEG = 5/8), and DCS/ BAPEG (R = 1/4, C = 5, 1%DCS/10%BAPEG = 5/4). CS/BAPEG hydrogel was made from 1% CS solution and 20% BALEG solution mixed at ratio of 5/4. Rheological Analysis of the Hybrid Hydrogel: Rheological test of the hybrid hydrogel was carried out on an Anton Paar MCR301 rheometer at 25 °C with a 25 mm diameter flat plate attached to a transducer. The storage modulus G′, loss modulus G″ and viscosity were both analyzed for these studies. (1) G′and G″of the 20 min pregelled hybrid hydrogel discs with different weight ratios and total weight contents, 20 min pregelled hybrid hydrogel (R = 1/2, C = 9) immersed in PBS (pH = 7.4) for 4 h, DCS/blood cells mixture, DCS/blood mixture, and CS/ blood mixture were tested under a 1% strain, and the angular frequency was set from 0.1 to 100 Hz. (2) G′ and G″ of the hybrid hydrogel (R = 1/2, C = 9) versus time were tested to obtain the gelation time. (3) The 20 min pregelled hybrid hydrogel (R = 1/2, C = 9) was measured under strain amplitude sweep (γ = 0.1–1000%) at 10 Hz angular frequency. (4) The alternate step strain sweep of 20 min pregelled hybrid hydrogel (R = 1/2, C = 9) was measured at 10 Hz angular frequency, and amplitude oscillatory strains were changed from γ = 1% to 300% with
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150 s for every strain interval. (5) The steady shear viscosity as function of shear rate of CS, DCS, CS/blood mixture, DCS/blood Mixture, and 20 min pregelled hybrid hydrogel (R = 1/2, C = 9) was tested. Swelling Ratio of the Hybrid Hydrogel: The swelling ratio measurements of free-dried hydrogel were carried out gravimetrically. The known weights of free-dried hybrid hydrogels were immersed in the PBS at 37°C. At specific intervals of time, the swollen samples were taken out and weighted. All experiments were carried out in triplicate. The swelling ratio was calculated using the following equation: Swelling ratio (%) = (Ws − Wd)/Wd × 100%, where Ws and Wd were the weights of hydrogels at the swelling state and the dry state. Injectability Test of the Hybrid Hydrogel: A piece of hybrid hydrogel (R = 1/2, C = 9) with 20 min pregelation time was added into 2.5 mL syringe with needle (23-gauge) and injected onto a glass slide to form a net. The hydrogel on the surface of the glass slide formed an integral free-standing net keeping for 20 min at room temperature, and was lifted to test the injectability of the hydrogel. Self-Healing Performance of the Hybrid Hydrogel: Two pieces of butterfly-shaped hybrid hydrogel with 20 min pregelation time stained by rhodamine B and trypan blue were cut into equal two parts, respectively. After 20 min at 25 °C, the total four pieces of alternate colors were combined into two integral blended butterfly-shaped pieces. Self-healing was confirmed by the capacity of the healed butterfly-shaped hydrogel to hold its structure when suspended under gravity. Two disk-shaped hybrid hydrogels were operated as the steps described above, and then immersed in the PBS for 2 h before lift. Cytotoxicity Tests of the Hybrid Hydrogel: To make different extract solutions, hydrogels were added into each well containing 500 µL culture medium to reach the final concentrations of 62.5, 125, 250, and 500 mg mL−1, respectively, and incubated for 24 h, and after which the pH was tested. The 3T3 fibroblasts were plated in 96-well cell culture dishes with 4000 cells per well (100 µL per well) for 24 h to allow attachment before the experiment. Then, the media in the 96-well dish was removed, and the cells were rinsed with PBS. Different 24 h aged extract solutions were added into the wells, and the cells were incubated with the extract solutions (n = 5, for each group) for another 24 h. The cell viability of experimental groups was measured by employing a Cell Counting Kit-8 test and expressed by the percentage of living cells with respect to the control cells. The In Vitro Hemostasis Test of the Hybrid Hydrogel: Heparinized mouse blood, blood cells and blood plasma were used to test the hemostasis property of CS solution, DCS solution, CS/BAPEG hydrogel and DCS/ BAPEG hydrogel. Heparinized mouse blood (300 µL) was mixed with CS or DCS solution (300 µL, 1%, in 0.2 m acetic acid), and CS/BAPEG or DCS/BAPEG hydrogel (150 µL), respectively, in a test tube. Blood cells (300 µL) or plasma (300 µL) was mixed with DCS solution (300 µL, 1%, in 0.2 m acetic acid). In order to test the hemostasis property, the test tube was inverted to observe whether the solution could stand its own weight. Antibacterial Activity of the Hybrid Hydrogel: The Gram-positive bacteria S. aureus and Gram-negative bacteria E. coli were used to investigate the antibacterial activities of the hybrid hydrogels. The precursor solution was sterilized by 200 nm syringe filters. Firstly, 100 µL of the hydrogels were formed in the 96-well plate and incubated for 1 h at 37 °C. PBS buffer (pH 7.4) was added to wash off the unreacted materials for three times. Subsequently, 100 µL of bacteria solution (104 CFU mL−1) was poured onto the surface of the hydrogels, and tissue culture treated polystyrene (TCTP) was used as a control. The bacteria were incubated on the surfaces of hydrogels and TCTP for 24 h at 37 °C after which the suspension was taken out and mixed with 100 µL of LB, and seeded on Nutrient Agar Plate to count the CFU. To visualize the viable microbes on the hybrid hydrogels, bacteria were fluorescently imaged by a live-dead assay with two staining agents, NucView Green Live and propidium lodide. The mixed suspension was further stained with a combination of dye solution (5 µL of NucView Green Live and 5 µL of propidium iodide stock solution dissolved in 10 mL of PBS) in the dark for 15 min at 37 °C. After rinsing with PBS three times, the stained bacteria observed by Opera Phenix
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(PerkinElmer Inc., UK). As a result, live bacteria were fluoresced green and dead bacteria fluoresced red. In Vivo Degradation Behavior and Cell-Recruitment Capability of the Hybrid Hydrogel: Eighteen BALB/c mice were employed to evaluate the in vivo degradation behavior and cell-recruitment capability. 0.4 mL of the hybrid hydrogel was administered by dorsal subcutaneous injection. Three mice from each group were sacrificed at 5 min, 12 h, 1 d, 2 d, 3 d, and 5 d, respectively. The injection sites were opened with a surgical scissors to observe the state of hydrogels. Meanwhile, the tissue around the injected site was carefully removed, fixed in 4% paraformaldehyde, embedded in paraffin, sectioned, and stained with hematoxylin–eosin and Masson’s trichrome for further histopathological examination. Bursting Pressure Test of the Hybrid Hydrogel: The rabbit aorta was used in this experiment. The aorta was linked to a syringe pump and filled with PBS solution. A 1 mm diameter hole was made on the aorta surface after which the hydrogels were formed in situ on the puncture site. The bursting pressure was measured 5 min after the forming of the hydrogels by cardiogram monitor. The pressure at which it began to decrease was considered the bursting pressure. All measurements were repeated for six times. Tissue Adhesive Test of the Hybrid Hydrogel: Adhesion strength testing of hybrid hydrogel and CS/BAPEG hydrogel were performed by a universal testing machine (Testometric Co. Ltd, United Kingdom) using fresh pigskin. The hybrid hydrogel and the CS/BAPEG hydrogel were applied between two pigskin uniformly and placed for 1 h to make the gelation complete. Then, the adhesion strength was determined by the machine equipped with a 20 N load cell at a rate of 10 mm min−1. All the tests were repeated for three times. The Fabrication of the Hybrid Hydrogel Scrapping-Decorated Gauge Needle: The hydrogel scrapping-decorated needle consisted of two layers (alginate hydrogel layer and hybrid hydrogel layer). Alginate hydrogel layer was made by mixing 2% alginate solution and 2% calcium chloride after which the hybrid hydrogel was formed on the surface of the layer in situ to form a double-layer scrapping. Then commercial needle pierced the double-layer scrapping to fabricate the hydrogel scrappingdecorated needle. The alginate hydrogel scrapping only was set as the control group. Evaluating Hemostatic Effects of the Hybrid Hydrogel ScrappingDecorated Gauge Needle: The hemostatic effects of the hybrid hydrogel scrapping-decorated gauge needle were evaluated in vivo using two animal models, mouse femoral vein puncture and jugular vein puncture (Sprague–Dawley, 8–12 weeks old, male, 250–300 g). All animals were chosen at random for the experiments. The mice were anaesthetized by intraperitoneal injection of 10% chloral hydrate. The mouse jugular vein and femoral vein were punctured by alginate or hybrid hydrogel scrapping-decorated gauge needle, respectively, then heparin was injected through the needles to heparinize the blood. The needles were removed from the veins and the alginate or hybrid hydrogel scrapping, and the bleeding conditions were recorded. Mouse Liver Injury Models: A mouse hemorrhaging liver model to investigate the in vivo robust hemostatic ability of the hybrid hydrogels was used. First, the mice were anesthetized as described above. Then the mouse’s liver was exposed and sheared to bleed with a surgical scissors. A filter paper was placed beneath the liver. Immediately, 200 µL of the pregel hybrid hydrogel was injected on the surface of bleeding site as a hemostatic agent. The liver bleeding conditions were observed. Controllable Release of Proteinic Drugs from the Hybrid Hydrogel: 1 mL of the hybrid hydrogel containing 0.1 mg BSA-FITC was injected into 3 mL PBS (pH = 7.4). At each time point (0.5, 1, 3, 6, 12, 24, 48, 72 h), 1 mL PBS was retrieved and the concentration of BSA-FITC were determined by a multi-functional microplate reader (HORIBA, model: FluoroMax-4) at the absorbance of 493 nm. Then, the PBS was poured back. All experiments were performed in triplicates. The concentration of released BSA-FITC from the hybrid hydrogel was calculated according to the following equation: FI = 3.61 × 108 × C + 3.544 × 105 (R2 = 0.928), where the FI referred to the fluorescence intensity and C represented the concentration of released BSA-FITC.
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Chronic Wound Healing Study of the Hybrid Hydrogel: A mouse infected full-thickness cutaneous wound model was used to evaluate the effect of hybrid hydrogel on chronic wound healing. First, a total of 24 healthy male Sprague–Dawley rats (180–250 g, Jinling Hospital, Nanjing, China) were anesthetized and their backs were shaved. A rounded full-thickness cutaneous wound (1 cm × 1 cm) area was created on the back of each rat after which 200 µL S. aureus solution (1 × 108 CFU mL−1) was introduced onto the wounds, and then divided into four groups randomly. The four groups were treated with PBS, CS/ BAPEG hydrogel, DCS/BAPEG hydrogel and VEGF-loaded DCS/BAPEG hydrogel respectively. The precursor solution of hydrogels was sterilized by filtration using 200 nm syringe filters, and the saline was sterilized by autoclaving. Thereafter, the rats were individually housed in cages and allowed to heal for 7d. The wounds were observed on day 0, 3, 5, and 7. The mice were sacrificed after 7d and granulation tissues over the wound bed were excised. Each sample was divided into two pieces. One piece was immersed in neutral formaldehyde for further histology and immunohistochemistry analysis, another was stored in liquid nitrogen for immunofluorescent staining. Histology and Immunohistochemistry: The granulation tissue samples were removed from the neutral formaldehyde, followed by dehydration and embedded in paraffin. Serially sections, 5 µm in thickness, were acquired by a microtome according to standard protocols, and were prepared for hematoxylin-eosin and immunohistochemical staining. Sections for immunohistochemistry were stained with IL-6 and TNF-α. For neovascularization evaluation, sections were reacted with primary antibodies against CD31 (KEYGEN, KGYM0118–7) and α-smooth muscle actin (α-SMA) (KEYGEN, KGYT5053-6). Afterward, sections were washed and incubated with FITC- (KEYGEN, KGAA26) and TRITC(KEYGEN, KGAA98) conjugated secondary antibodies, and were rinsed and mounted with 4, 6-diamidino-2-phenylindole (DAPI) mounting medium to label Nuclei. Immunofluorescence Staining: The frozen samples were fixed in acetone, and then incubated with blocking buffer. To evaluate collagen deposition, sections were incubated with primary antibodies vimentin (Abcam, 200 ab20346) and collagen (Abcam, ab96723) for 2h at room temperature, and then incubated with secondary antibodies Alexa Fluor 555- and 488- conjugated for 1 h. Afterward, the sections were rinsed and mounted with DAPI mounting solution. To evaluate macrophage polarization, sections were incubated with primary antibodies against the pan- macrophage marker CD68 (Abcam, ab955), M1-macrophage marker CD86 (Abcam, ab53004), and the M2-macrophage marker CD206 (Santa Cruz, sc-34577). Afterward, the sections were washed and incubated with the fluorescently conjugated secondary antibodies Alexa Fluor 488-, 555-, and 647-. DAPI was used to label the nuclei. Photos were obtained and analyzed with an Opera Phenix (PerkinElmer Inc., UK).
Supporting Information Supporting Information is available from the Wiley Online Library or from the author.
Acknowledgements G.P.C., Y.R.Y., and X.W.W. contributed equally to this work. This work was supported by the National Science Foundation of China (Grant Nos. 81571881, 21473029, and 51522302), Projects of Jiangsu Social Development (BE2016752 and BE2017722), Innovation Project of Military Medicine (16CXZ007), and the Scientific Research Foundation of Southeast University.
Conflict of Interest The authors declare no conflict of interest.
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Keywords bioinspired materials, biomaterials, hydrogels, self-healing, wound healing Received: February 22, 2018 Revised: May 9, 2018 Published online:
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