Facile Fabrication of Biocompatible Gelatin-Based Self-Healing

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Facile Fabrication of Biocompatible Gelatin-Based Self-Healing Hydrogel Jinfeng Lei, Xinying Li, Shen Wang, Lun Yuan, Liming Ge, Defu Li, and Changdao Mu ACS Appl. Polym. Mater., Just Accepted Manuscript • DOI: 10.1021/acsapm.9b00143 • Publication Date (Web): 24 Apr 2019 Downloaded from http://pubs.acs.org on April 24, 2019

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ACS Applied Polymer Materials

Facile Fabrication of Biocompatible Gelatin-Based Self-Healing Hydrogel

Jinfeng Lei,† Xinying Li,‡ Shen Wang,† Lun Yuan,† Liming Ge,† Defu Li,*,† Changdao Mu†

†

Department of Pharmaceutics and Bioengineering, School of Chemical Engineering,

Sichuan University, Chengdu 610065, P. R. China ‡

College of Chemistry and Environment Protection Engineering, Southwest Minzu

University, Chengdu 610041, P. R. China

KEYWORDS: dynamic imine bond, self-healing, self-recovery, gelatin-based hydrogel, cytocompatibility

Corresponding author * E-mail: [email protected] (D.L.)

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ABSTRACT: The self-healing hydrogels are extremely attractive in biological and biomedical fields. Imine bond obtained by Schiff’s base reaction is a commonly used dynamic covalent bond to fabricate self-healing hydrogel. Gelatin is a commonly used natural macromolecule in biomedical field with excellent biocompatibility, biodegradability and non-immunogenicity. However, the gelatin-based hydrogels with self-healing ability are rarely reported based on imine bonds. Herein, we present a facile approach to fabricate a gelatin hydrogel with self-healing ability based on Schiff’s base reaction. The gelatin was firstly reacted with ethylenediamine to increase the content of amino group. Then dialdehyde carboxymethyl cellulose was used to crosslink amino-gelatin to fabricate the self-healing hydrogel. The results showed that the fabricated hydrogel exhibited good self-healing ability as expected owing to the formed dynamic imine bonds between amino-gelatin and dialdehyde carboxymethyl cellulose. The hydrogel also presented good fatigue resistance and self-recovery capacity. Moreover, the self-healing hydrogel possessed ideal hemocompatibility and cytocompatibility. In sum, the fabricated self-healing hydrogel has the application prospects in biomedical fields, such as injectable cell and drug carrier, injectable tissue engineering scaffold.

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1. INTRODUCTION Hydrogel is an attractive soft material possessing 3D network structure and tunable physicochemical properties1. Hydrogel has been widely applied in biomedical fields such as drug delivery system2-3, wound dressing4-5, tissue engineering6-8 and biosensors9-11. Recently, the self-healing hydrogels have drawn much attention because their unique advantages, such as autonomously repairing damage, maintaining structural and performance integrity, stable functionality for long-term application12-14. The fabrication of self-healing hydrogels nowadays mainly focuses on two approaches, based on dynamic covalent bonds 9, 15-18 and noncovalent bonds 10, 19-23.

Dynamic covalent bond has reversibility of non-covalent bond and stability of covalent

bond24. Imine bonds7, 16, acylhydrazone bonds14, 25, phenylboronate esters2, 9, 26, disulfide bonds18, 27, and Diels-Alder reactions17, 25 are the major dynamic covalent bonds which are often employed to fabricate self-healing hydrogels. Many natural macromolecules are used as raw materials to fabricate self-healing hydrogels for biomedical applications owing to the good biocompatibility. For instance, a variety of self-healing hydrogels have been developed using alginate16, hyaluronic acid25 and chitosan28 based on various types of dynamic covalent bonds in the past years. Among them, imine bond obtained by Schiff’s base reaction is a commonly used dynamic covalent bond to fabricate self-healing hydrogel29-30. The Schiff’s base reaction can be carried out simply by blending raw materials without the assistance of catalysts and functional small organic molecules. In addition, the process of Schiff’s base reaction is facile and fast, which can undergo dynamic reaction under neutral conditions. No external stimulus is required during the reaction. Moreover, the vast majority of reported self-healing hydrogels linked by imine bonds are based on chitosan owing to its abundant amino groups4, 31. Gelatin is an another attractive and commonly used natural macromolecule in biomedical fields. Gelatin has many excellent characteristics such as excellent biocompatibility, biodegradability, non-immunogenicity, inducing cell adhesion and proliferation32-33. Many studies have indicated that 3



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gelatin hydrogel can be used as various scaffolds for tissue engineering34. Gelatin has been employed to fabricate many kinds of hydrogels linked by Schiff’s base for biomedical applications. For example, the modified gelatin was fixed by oxidized dextran to fabricate a fast forming hydrogel linked by Schiff’s base, which can be used to regenerate cartilage in tissue engineering35. Besides, a hydrogel based on gelatin and oxidized alginate has been prepared through Schiff’s base reaction, which was used as injectable scaffold to treat osteoarthritis36. These aforementioned gelatin-based hydrogels present good mechanical properties, bioactive functions, biocompatibility and show application prospect in biomedical fields. However, the self-healing gelatin-based hydrogels based on Schiff’s base reaction are rarely mentioned. This may be due to that the amino content of gelatin is not appropriate for the preparation of self-healing hydrogel compared to chitosan. In this paper, we aim to design a facile approach to fabricate a gelatin-based self-healing hydrogel with good mechanical properties and cytocompatibility based on Schiff’s base reaction. The gelatin was firstly reacted with ethylenediamine to increase the content of amino group. Then dialdehyde carboxymethyl cellulose (DCMC) was used to crosslink the amino-gelatin (Agel) through Schiff’s base to obtain self-healing Agel-DCMC hydrogel. The mechanical properties, self-healing properties, rheological properties, morphology of the Agel-DCMC hydrogel were systematically studied. We further evaluated the cytocompatibility and in vitro hemocompatibility of the Agel-DCMC hydrogel.

2. MATERIALS AND METHODS 2.1. Materials. Phosphate buffered saline, fetal bovine serum, DMEM/high glucose, trypsin solution (0.25%) and penicillin-streptomycin solution are supplied by GE Healthcare Life Sciences (Beijing, China). Calcein-AM is supplied by YEASEN Biotechnology (Shanghai, China). Sulfuric acid and hydrochloric acid (HCl, 36.0-38.0%) are supplied by Kelong Chemical Reagent Company (Chengdu, China). Cell counting kit-8 is supplied by Beyotime Biotechnology (Shanghai, China). 4


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Carboxymethyl

cellulose,

ethylenediamine,

gelatin,

sodium

periodate

and

N-(3-dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride (EDC) are supplied by Aladdin Industrial Corporation (Shanghai, China). 2.2. Preparation and Characterization of Dialdehyde Carboxymethyl Cellulose. DCMC was prepared according to our previous method with an optimized modification37. DCMC was characterized by fourier transform infrared (FTIR) and 1H nuclear magnetic resonance (1H NMR), respectively. FTIR spectra were recorded on a Thermo Scientific Nicolet is10 FTIR spectrometer from 500 to 4000 cm-1 at resolution of 4 cm-1. 1H NMR measurements were carried out on an Agilent 400-MR DD2 1H NMR spectrometer. The DMSO solutions of samples with the concentration of 1.0% (w/v) were used for the measurements. The spectra were recorded with a delay time of 10 s, an acquisition time of 2 s and a pulse angle of 30o. 2.3. Preparation of Amino-gelatin. The gelatin was reacted with ethylenediamine (ED) to increase the content of amino group according to previous method with a slight modification35. The carboxyl groups of gelatin can react with ED with the assistance of EDC. The gelatin (10 g), 100 mL of PBS (pH=5) and 16 mL of ED were mixed together at room temperature. Hydrochloric acid was used to control the pH of mixture at 5.0. EDC (4.6 g) was then added to the mixture. The mixture was reacted at room temperature for 24 h. After that, the resulting products were dialyzed against deionized water for 3 days and then freeze-dried to get the amino-gelatin (Agel). FTIR and 1H

NMR measurements of Agel were performed using the same method as described in section 2.2.

The difference is that the used solvent is D2O. 2.4. Preparation of the Self-healing Hydrogel. Agel aqueous solution (10 wt%) and DCMC aqueous solution (10 wt%) were mixed in a tube at the ratio of 1/1 at 37 oC. The resulting mixture was immediately poured into a mold and placed at 37 oC to form the self-healing hydrogel. The hydrogel based on gelatin and DCMC was prepared as a control. The gelation time was determined by the inverted tube method. 2.5. Macroscopic Testing of Self-healing. The hydrogel was made into rod-shaped samples (30 5



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mm ×3 mm). Some hydrogels were stained with azaleine in advance for better visual inspection. The rod-shaped sample was firstly cut into two pieces, and then the new formed cross sections were immediately recombined. The rejoined sample was incubated at 37 oC for a scheduled time. The tensile property of resultant hydrogel was measured by an electronic universal testing machine (MTS CMT6202) according to previous method17. The ratio of tensile strength of healed hydrogel to original hydrogel was used to represent the healing efficiency (HE). 2.6. Rheology Test. The rheology analysis was conducted to qualitatively detect the self-healing properties of samples. Rheology tests were conducted using a TA rheometer (AR2000ex) at 37 oC. Oscillatory frequency sweep measurement was performed at 1% strain amplitude. Oscillatory strain amplitude sweep measurement was performed at 1 rad/s frequency. Step-strain sweep measurements were performed to evaluate the recovery performance of hydrogels on the applied alternate shear force. 2.7. Morphology Observation of the Hydrogel. The hydrogel was firstly sliced using a frozen microtome (EM UC7, Leica, Germany). Then the pieces of hydrogel were fixed on the aluminum stub with carbon tape. The microscopic morphology observation of cross-section was conducted on a Hitachi S4800 SEM. The accelerating voltage was set as 10 kV. The optical microscopy (CKX53, Olympus) was used to detect the juncture of the self-healing hydrogel and corresponding freeze-dried sample too. 2.8. Mechanical Tests. The cylindrical hydrogels were firstly prepared with 10 mm in diameter and 6 mm in height. The uniaxial compressive tests were conducted on the cylindrical samples using an electronic universal testing machine at room temperature. The load cell was set as 200 N while the operating speed of cross-head was set as 50 mm min-1. The fatigue resistance and self-recovery capacity of hydrogel were evaluated by the successive loading-unloading compressive tests according to previous method17. A steel rule (0.7 mm thickness) was used here to detect the shear-resistant property of the cylindrical hydrogel. The nominal stress was defined as the ratio of compressive load to contact area of steel rule and the cylindrical sample. 6


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2.9. Degradability Study. For the in vitro degradation assay, the sample was fully immersed in PBS (pH 7.4) until reaching the equilibrated state. Then the sample was placed in a shaker and incubated at 50 rpm and 37 oC for a scheduled time. The sample was taken out and weighed at regular intervals. The weight remaining was used to reflect the degradability of hydrogel, which was defined as the weight ratio of remaining hydrogel at different time to initial equilibrated hydrogel. The measurement was conducted five times. The average value was calculated and used. 2.10. Swelling Behavior Measurement. The gravimetric method was used to detect the swelling behavior of the hydrogel. The cylindrical sample was immersed in PBS (pH 7.4) for swelling at 37 oC.

The sample was taken out and weighed at regular intervals until reaching the equilibrated state.

The swelling ratio was defined as the weight ratio of swollen hydrogel to initial hydrogel. The measurement was conducted five times. The average value was calculated and used. 2.11. Thermogravimetric Analysis. For the thermal stability analysis, TGA measurement was carried out on a METTLER 1100LF thermal analyzer. To remove the bound water, the gelatin and freeze-dried hydrogels were dried for 24 h at 110 °C before measurement. The measurement was conducted from 50 to 600 °C at 10 °C/min. N2 atmosphere was used as protective gas to avoid thermooxidative reactions. 2.12. Hemolysis Assay. Hemocompatibility is a very important property for biomaterials. In this paper, the hemolysis assays were carried out to evaluate the hemocompatibility of hydrogel using the reported procedure38. The normal saline (0% hemolysis) was used as negative control and deionized water (100% hemolysis) was used as positive control. The hemolysis ratio was detected and calculated by a commonly used method during hemolysis assay38. The measurement was conducted five times. The average value was calculated and used. 2.13. Cytocompatibility Evaluation. The normal hepatocyte (L02) and mouse fibroblast cells (L929)

were

used

to

evaluate

the

cytocompatibility

of

the

Agel-DCMC

hydrogel.

DMEM/high-glucose containing 1% penicillin-streptomycin solution and 10% fetal bovine serum was used as the complete medium. The cytotoxicity of the hydrogel was detected by a conventional 7



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method with some modifications39. The prepared hydrogel was sterilized under UV irradiation for 24 hours. After washing with PBS, hydrogel was immersed in complete medium for 24 hours to gain extract liquid. The extract liquid was sterilized by filtration (S2635 Sterile Syringe Filters, 13 mm). The cells were firstly seeded into 96-well plates containing 100 μL of complete medium. Then the culture was performed at 37 oC for 24 hours in humidified 5% CO2. After that, the extract liquid (100 μL) was employed to replace the complete medium, which was then incubated for another 24 or 72 hours. The cell viability was evaluated using CCK8 assay. All experiments were conducted with five repetitions and averaged. The cells incubated in complete media without hydrogel extracts were used as control group. The cell viability can be calculated by the equation (1): Cell Viability (%) =

OD t est -OD blank 100% ODcontrol -OD blank

(1)

where ODtest, ODcontrol, ODblank are OD values of samples, control, blank groups, respectively. The direct contact measurement was further used to detect the cytotoxicity of hydrogel. The Agel solution and DCMC solution were added into a plate, which was then placed at 37 oC to form hydrogel. The obtained hydrogel was sterilized under UV irradiation for 24 hours. After that the hydrogel was washed by PBS. Then 100 μL of L929 cells or L02 cells suspension and 2 mL of fresh complete medium were placed directly on the surface of hydrogel. After incubation for 24 or 72 hours, the medium was replaced by new medium containing Calcein-AM. Then the culture was conducted for another 25 min. The cell morphology on hydrogel was observed using a fluorescence microscope (CKX53, Olympus). L02 and L929 cells incubated in complete medium were used as controls.

3. RESULTS AND DISCUSSION 3.1. Preparation of Dialdehyde Carboxymethyl Cellulose and Amino-gelatin. FTIR spectra of CMC and DCMC are showed in Figure 1a. It shows that two new peaks at 1732 cm-1 and 886 8


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cm-1 were observed for DCMC, corresponding to C=O stretching vibration of aldehydic carbonyl groups and formed hemiacetal bonds, respectively40. The result indicates that the o-hydroxyl groups of CMC were successfully oxidized to dialdehyde. FTIR spectra of gelatin and amino-gelatin (Agel) are showed in Figure 1b. Gelatin and Agel show the absorption peaks at 2935 and 2880 cm−1, corresponding to C-H asymmetric and symmetrical stretching vibrations of the methylene, respectively. Gelatin and Agel both show the amide I band at 1640 cm-1 and amide II band at 1545 cm-1 37, 41. Specifically, the intensities of amide absorption bands of Agel are obviously higher than that of gelatin, which may attribute to that more amide bands are formed between the gelatin and ED in Agel. Furthermore, Agel shows the higher absorption intensity at ~2935 cm−1. In sum, these results indicate that the carboxyl groups of gelatin have been successfully reacted with ED to increase the content of amino groups.

Figure 1. FTIR spectra of (a) CMC and DCMC, (b) gelatin and amino-gelatin. 1H NMR spectra of (c) CMC and DCMC, (d) gelatin and amino-gelatin. 9



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The chemical structures of DCMC and amino-gelatin were further detected by 1H NMR. Figure 1c

shows the 1H NMR spectra of CMC and DCMC. Clearly, a new peak at 9.27 ppm was observed for DCMC, which corresponds to the H atom signal of aldehyde group. Note that the hemiacetal peaks of DCMC from 3.8 to 5.0 ppm were also observed42. The results further confirm that CMC was successfully oxidized to DCMC. Figure 1d shows the 1H NMR spectra of gelatin and amino-gelatin. The spectrum of gelatin is consistent with the previously reported result in the literature43. Two new characteristic peaks a and b (3.11 and 3.47 ppm) are appeared in the spectrum of amino-gelatin, which are donated as the protons of the ethylenediamine grafted on gelatin. The results further confirm that the amino-gelatin has been successfully prepared.

3.2. Synthesis of the Self-healing Hydrogel. In this paper, DCMC was prepared from CMC. The gelatin was reacted with ED to increase the number of amino groups. Hence, the Agel-DCMC hydrogel could be fabricated through the Schiff’s base reaction between amino groups of Agel and aldehyde groups of DCMC. The schematic diagram of the fabrication route of self-healing Agel-DCMC hydrogel is showed in Scheme 1. Figure 2a shows the photographs of the mixed solution of Agel and DCMC and the formation of hydrogel. After homogeneously mixing Agel and DCMC, the mixture was incubated under 37 oC and the gelation time was about 2 hours. Note that the mixture of gelatin and DCMC did not form hydrogel under the same condition. The number of amino groups of Agel is more than that of gelatin, so more Schiff’s base cross-linkages would be formed between Agel and DCMC to promote the formation of hydrogel. Figure 2b shows that Agel-DCMC hydrogel was flexible and soft under pressing and bending, which may be attributed to the dynamic imine bonds between Agel and DCMC.

10


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Scheme 1. Schematic diagram of the fabrication route of self-healing Agel-DCMC hydrogel

Figure 2. (a) Photographs of the mixed solution of amino-gelatin and DCMC and the formation of hydrogel. (b) The bending and pressing shapes of the self-healing hydrogel.

3.3. Self-healing Properties. The mechanical performance testing and visual inspection were combined to detect self-healing capability of Agel-DCMC hydrogel. Figure 3a shows the cutting and rejoining of the self-healing hydrogels. The rod-shaped samples with or without staining with azaleine were cut into two pieces and rejoined, and then the rejoined samples were incubated at 37 11



oC

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for a certain time. Figure 3b shows the manual stretching of the original hydrogel and healed hydrogel

for 30 min. It shows that after healing for 30 min, the hydrogel can withstand considerable tension. The

tensile properties and healing efficiency were investigated to further quantitatively examine the self-healing behavior of Agel-DCMC hydrogel. Figure 3d shows that healing efficiency is increasing along with the healing time. The healing efficiency reaches up to 50% when healing for 40 min. When healing for 60 min, the healed hydrogel would not fracture from the juncture (Figure 3c). The healing efficiency can reach 90% (Figure 3d). The results indicate that the hydrogel could almost completely heal and regain the tensile properties after healing for one hour. It was reported that the healing efficiency of the nanocomposite hydrogel based on cellulose nanocrystal and PEG reached up to 80% when curing for 24 hours17. Thus, the Agel-DCMC hydrogel possesses excellent self-healing capacity thanks to the reversible imine linkage in the hydrogel networks.

Figure 3. (a) Cutting and rejoining of the self-healing hydrogels. (b) Manual stretching of the original hydrogel and healed hydrogel for 30 min. (c) Stretching of the healed hydrogel by machine. (d) Stress-strain curves of the hydrogels healing for different times. One hydrogel was stained with azaleine for better

visual inspection. 12


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3.4. Rheology Analysis. Figure 4a shows the storage modulus (G′) and loss modulus (G″) of the Agel-DCMC hydrogel at different frequency. It shows that the G′ value is significantly larger than G″ over the whole range of frequency, demonstrating the formation of hydrogel Figure 4b shows that the curves of G′ and G″ intersect at 20% strain with fixed frequency. The result indicates that the hydrogel was at critical point of liquid and solid. The G′ value is dramatically decreased and smaller than G″ value when the strain is larger than 20%. It indicates the loose of the network structure of hydrogel. Figure 4c shows the G′ and G″ values of the Agel-DCMC hydrogel at alternate strain between 1.0% and large strain (80%, 300%, 500%) with fixed frequency (1.0 rad/s). The results are used to evaluate the recovery capacity of the Agel-DCMC hydrogel. Figure 4c shows that G″ value is smaller than G′ at the low strain (1.0%, kept for 300 s), illustrating that the hydrogel maintains its mechanical stability. When the strain is increased (80%, kept for 100 s), G″ displays higher value than G′. It indicates that the network of the hydrogel is loosed, and the hydrogel loses its mechanical stability (“damage”). Note that G″ and G′ immediately recover their original values when the strain returns to initial low strain (1.0%). The result indicates that the network structure of hydrogel recovers to original state (“healing”). In addition, the G″ and G′ of hydrogel can quickly restore initial values when larger (300% and 500%) and small strains (1.0%) are alternatively applied. These repeated damage-healing experiments indicate the self-recovery capacity of the Agel-DCMC hydrogel.

Figure 4. Storage modulus (G′) and loss modulus (G″) of the Agel-DCMC hydrogel at different frequency (a), in a strain range of 0.1% to 100% with fixed frequency (1.0 rad/s) (b), and at alternate strain 13



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between 1.0% and large strain (80%, 300%, 500%) with fixed frequency (1.0 rad/s) (c).

3.5. Study of the Morphology. Figure 5a shows the photograph of the cylinder-shaped Agel-DCMC hydrogel after healing for 60 min. Figure 5b shows the optical microscopy image of the juncture of the healed Agel-DCMC hydrogel. It is clear that no crack was observed at the juncture of the healed hydrogel. Figure 5c shows the optical microscopy image of the freeze-dried healed hydrogel. Note that there is no discernable difference between the uncut area and juncture. The cross-section morphology of juncture of freeze-dried healed hydrogel was detected by SEM. The result is presented in Figure 5d. It shows that the healed hydrogel exhibits a porous structure with a diameter of ~100 μm. The porous structure of the juncture is almost the same as that of the undamaged area. The above results indicate that Agel-DCMC hydrogel possesses good self-healing ability, which can rebuild its microstructure after being damaged.

Figure 5. (a) Photograph of the cylinder-shaped Agel-DCMC hydrogel after healing for 60 min. (b) Optical microscopy photographs of the juncture of the healed hydrogel and (c) the freeze-dried healed sample. (d) SEM image of the cross-section of juncture of the freeze-dried healed hydrogel.

3.6. Mechanical Properties. The compressive tests were conducted to examine the strength and compressibility of Agel-DCMC hydrogel. Figure 6a shows the typical compressive strain-stress curve of hydrogel. It shows that the Agel-DCMC hydrogel exhibits a maximum compressive strength of about 200 kPa, without fracturing until 80% of deformation (E = 28.92 ± 0.68 kPa). It indicates that the Agel-DCMC hydrogel is soft and ductile. Then the cyclic loading-unloading compressive tests were implemented to further study the self-recovery and fatigue resistance 14


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capabilities of the Agel-DCMC hydrogel. Figure 6d shows the photographs of the process of loading-unloading compression tests. The shape of the Agel-DCMC hydrogel could recover immediately after removing the external force. As shown in Figure 6b, the hydrogel could continuously recover its original strength during the 10 cyclic compressive loading-unloading tests. The results indicate that the Agel-DCMC hydrogel possesses good fatigue resistance and self-recovery capacity. Here a steel rule was used to shear the hydrogel to check the shear-resistant property. The results are showed in Figure 6c and e. They show that the hydrogel could withstand the shearing with the steel rule without any damage even at a strain of 70% and recover immediately after removing the external force. In sum, the above results indicate that the Agel-DCMC hydrogel possesses excellent mechanical properties, including fast self-recovery from compressive deformation and shear deformation as well as fatigue resistance properties.

Figure 6. (a) Compression strain-stress curve. (b) Loading-unloading continuous compression tests at 70% strain for 10 cycles. (c) The shear strain-stress curve. (d) Digital photograph of the process of loading-unloading compression tests. (e) Digital photograph of the process to shear the hydrogel with a steel rule. 15



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3.7. Swelling Behavior, Degradability and Thermostability. The swelling behavior of hydrogel was measured to demonstrate the stability in aqueous environment. Figure 7a shows the swelling kinetics of the hydrogel in PBS (pH 7.4). The hydrogel swells slightly until equilibrium state after 12 h. The equilibrium swelling ratio is about 1.28. The inset of Figure 7a exhibits that the hydrogel maintained the initial shape intact after swelling for 48 h. It is known that gelatin will completely dissolve in water. The results indicate that the Agel-DCMC hydrogel is stable in aqueous solution thanks to the crosslinking effect of DCMC. Figure 7b shows the degradation profile of Agel-DCMC hydrogel in PBS in vitro. It shows that the hydrogel exhibits a fast degradation in first

12 days. After that, the remaining weight decreases slowly and stays around 20%. The result indicates the degradability of the Agel-DCMC hydrogel.

Figure 7. Swelling kinetics (a) and degradation profile (b) of the Agel-DCMC hydrogel in PBS (pH 7.4). TGA (c) and DTG (d) curves of gelatin, amino-gelatin and Agel-DCMC hydrogel. Inset of 7a is the photographs of initial hydrogel and swollen hydrogel after 48 h. Inset of 7b is the photographs of equilibrium 16


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swollen hydrogel and hydrogel after degradation for 60 days.

TGA was employed to evaluate the thermal stability of Agel-DCMC hydrogel. TGA and DTG results of gelatin, amino-gelatin, Agel-DCMC hydrogel are presented in Figure 7c and d. For all the samples, the initial weight losses (below 150 oC) correspond to the release of residual adsorbed water, which were not removed by beforehand drying in the oven. The temperature at the maximum rate of weight loss is denoted as Tmax. Figure 7d shows that the Tmax decreases from 318.9 oC to 308.7 oC when gelatin is converted to amino-gelatin, indicating the decrease of thermal stability. The increased amino groups of gelatin maybe prevent the interaction between protein molecules, resulting in decrease of thermal stability. Note that the Tmax of Agel-DCMC hydrogel increases to 326.6 oC thanks to the crosslinking effect of DCMC44. It indicates that Agel-DCMC is more thermal stable than that of gelatin and amino-gelatin.

3.8. Blood Compatibility and Cytocompatibility. Cytocompatibility and blood compatibility are important properties for biomaterials. We tested the degree of hemolysis of Agel-DCMC hydrogel to evaluate the blood compatibility. Figure 8a shows that the hemolytic ratio of blood contracting with hydrogel for 1 h is about 2.70%, which is below the international permissible level of 5%45. The result indicates that the hydrogel is nonhemolytic. The L929 cells and L02 cells were employed here to evaluate the cytocompatibility of Agel-DCMC hydrogel. To assess the toxicity of the hydrogel, the cell viabilities of L02 and L929 cells were tested when incubating in the extract liquid of hydrogel. Figure 8b shows the cell viabilities of L02 and L929 cells incubated with the extract liquid of Agel-DCMC hydrogel. It shows that the viabilities of L02 and L929 cells are all higher than

90% after 24 and 72 hours incubation, indicating non cytotoxicity of hydrogel towards L02 and L929 cells. Then, the direct contact tests were employed to investigate the cytotoxicity of Agel-DCMC hydrogel. The L929 cells and L02 cells suspensions were placed directly on the 17



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surface of hydrogel and incubated for 24 and 72 hours. After that, the Calcein-AM was used to stain the live cells (green-fluorescent) to observe the cell morphology under a fluorescence microscope. Figure 8c and d show the images of live L929 and L02 cells after culture on the surface of Agel-DCMC hydrogel for 24 and 72 hours along with controls. Clearly, most of the L929 cells and L02 cells adhered to the hydrogel and exhibited a green-fluorescent, spindle-shaped morphology, which is consistent with normal cell morphology as controls. The results further reveal that Agel-DCMC hydrogel exerts no cytotoxicity on L929 cells and L02 cells.

Figure 8. (a) Hemolytic extents of the Agel-DCMC hydrogel at 37 oC for 60 mins. (b) Cell viabilities of L02 and L929 cells incubated with the extract liquid of Agel-DCMC hydrogel measured by CCK-8 assay. Images of the live (c) L929 and (d) L02 cells after culturing on the surface of Agel-DCMC hydrogel for 24 and 72 hours along with controls. 18


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4. CONCLUSION Dialdehyde carboxymethyl cellulose and amino modified gelatin were employed to synthesize a self-healing hydrogel without additional crosslinking agent. Dialdehyde carboxymethyl cellulose and amino modified gelatin mixture could form hydrogel based on Schiff’s base reaction, which endowed the hydrogel with self-healing properties as expected. The fabricated hydrogel also exhibited good mechanical properties such as good fatigue resistance and self-recovery capacity. Moreover, the hydrogel possessed a porous structure with a diameter of ~100 μm. Furthermore, the hydrogel had good blood compatibility and cytocompatibility. Our findings suggest that the fabricated self-healing hydrogel has the application prospects in biomedical fields, such as injectable cell and drug carrier, injectable tissue engineering scaffold.

AUTHOR INFORMATION Corresponding Author E-mail: [email protected] (D.L.). ORCID Defu Li: 0000-0001-6466-4947 Notes The authors declare no competing financial interest.

ACKNOWLEDGMENTS This work was financially supported by the Key Research and Development Project of Sichuan Province (SCST18ZDYF1426), Sichuan Science and Technology Program (2019YFH0110).

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Facile Fabrication of Biocompatible Gelatin-Based Self-Healing

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