Self-healing host–guest hydrogels have been developed to treat the myocardial infarction. The self-healing hydrogel, formed through host–guest interactions of adamantine- and β-cyclodextrin-modified HA, was injected into the ischemic myocardium encapsulating endothelial progenitor cells (EPCs) (Gaffey et al., 2015). A rodent model of acute myocardial infarction was employed to confirm that a significant increase in vasculogenesis was noted with the hydrogel encapsulating EPCs, compared to the treatment of EPCs alone or hydrogel alone. Moreover, the hydrogel was designed using adamantane/thiolmodified HA and cyclodextrin/methacrylate-modified HA through host–guest interaction and Michael addition (Rodell et al., 2015a). The reversible host–guest interaction and permanent Michael addition provided shear-thinning injection and high retention, respectively. Epicardial injection of the hydrogel in a rat myocardial infarction model showed significant improvement of the outcome compared to the untreated group and the hydrogel without Michael addition.

Self-healing hydrogels were designed as injectable carriers for growth factors using PEG end-functionalized with four-fold hydrogen-bonding ureidopyrimidinone (UPy) moieties. UPymodified PEG hydrogel incorporated with antifibrotic growth factor was delivered in a pocket introduced under the kidney capsule of rats (Dankers et al., 2012). The kidney capsule was loosened from the kidney to create a small pocket. After injection of growth factor-containing hydrogels, the number of myofibroblasts stayed the same to the contralateral (healthy) kidney, while significantly increased with the injection of saline or hydrogel alone. In another example, growth factors were delivered by UPy-modified PEG hydrogel to repair the infarcted myocardium (Bastings et al., 2014). This pH-switchable hydrogel could be injected through the long and narrow lumen of the catheter mapping system, and rapidly formed a hydrogel in contact with tissue. The growth factor-containing hydrogel reduced scar collagen in a chronic myocardial infarction pig model.

Self-healing hydrogels based on GC-DP was prepared for tissue repairs. In the application of central nervous system (CNS) repair, neurosphere-like progenitors showed better proliferation and differentiation in GC-DP hydrogel, and injection of GC-DP hydrogel combining neurospheres promoted functional recovery in a zebrafish CNS impaired model (Tseng et al., 2015). Moreover, the GC-DP hydrogel combining the optogenetic method was developed as a temporal-spatial approach to treat TABLE 1 | Examples of self-healing hydrogels evaluated by animal models.

neurodegenerative diseases (Hsieh et al., 2018). The hydrogel containing bacteriorhodopsin plasmid and neural stem cells was injected into CNS impaired zebrafish where the neural repair was observed, particularly under green light exposure. Besides, GC-DP hydrogel was also used to induce blood capillary formation. With the incorporation of fibrin gel, a composite hydrogel could form with an interpenetrating polymer network (i.e., double network) of GC-DP and fibrin (Hsieh et al., 2017). The hydrogel induced vascular endothelial cells to form capillarylike structures, and injection of the hydrogel alone promoted angiogenesis in zebrafish and rescued the blood circulation in ischemic hindlimbs of mice.

#### Other Applications

Self-healing hydrogels, based on the host–guest interaction of β-cyclodextrin- and adamantine-modified HA, were used in the 3D printing of high-resolution structures through printing of shearing-thinning ink hydrogel into self-healing support hydrogel (Highley et al., 2015). The multicellular structures could be expediently patterned, such as printing of mesenchymal stem cells within an ink hydrogel into a support hydrogel containing 3T3 fibroblasts. The channel-like structure was achieved by writing the ink hydrogel into the methacrylate-modified support hydrogel, followed by UV irradiation for secondary covalent crosslinks of support hydrogel, followed by removal of the physical (i.e., host–guest) ink hydrogel. Meanwhile, the selfsupporting structure was obtained by covalently crosslinking the ink hydrogel and removing the non-covalent support hydrogel. This system supported the patterning of multiple inks, cells, and channels in 3D space.

A tough self-healing hydrogel was synthesized as cell stimulators and implantable bioelectronics (Han et al., 2017). In the study, graphene oxide was partially converted to conductive graphene through polydopamine reduction, and acrylamide monomers were polymerized in situ to form the hydrogel by interactions between graphene oxide, polydopamine, and polyacrylamide. Meanwhile, the free catechol groups on polydopamine imparted self-healing property and tissue adhesion to the hydrogel via various non-covalent interactions. The hydrogel could be used not only as an adhesive electrode or motion sensor but also as an in vitro cell stimulator and in vivo implantable intramuscular electrode. For example, the hydrogel electrodes were implanted into the rabbit dorsal muscle and connected to a signal detector using the transcutaneous wires. The electrodes could record the electromyographic signal when the rabbit was interfered with external stimulation.

#### CONCLUSIONS

Self-healing hydrogels can be classified as robust and soft hydrogels according to mechanical properties in biomedical applications. Robust self-healing hydrogels are used as soft robots (such as implantable or wearable biosensors) with extended lifetime and mechanical performance due to repairing of the damages or fatigues. Soft self-healing hydrogels with shear-thinning properties are used in cell/drug delivery and 3D printing due to injection through narrow needles and retention at target sites. To facilitate biomedical applications in the future, self-healing hydrogels need to address several major concerns including (1) designing self-healing hydrogels with good biocompatibility and appropriate mechanical properties;

#### REFERENCES

(2) better characterizing the self-healing properties with various assessment tools (such as rheological measurement, mechanical analysis, or other novel tools); (3) developing theories on selfhealing mechanisms and properties (such as chemistry, kinetics, and thermodynamics); and (4) translation by animal experiments and clinical trials. Moreover, because the self-healing properties of hydrogels are mostly determined in non-physiological environments, it would be challenging to verify that the known self-healing properties are well-maintained in physiological conditions such as with electrolytes, under mechanical stress, and in the presence of material–cell interaction. In addition, controllable biodegradability is important in selfhealing hydrogels for tissue engineering and drug delivery. In comparison with permanent crosslinks, reversible crosslinks are broken easily to facilitate biodegradation, while reversible crosslinks recover the macro- and micro-scaled damages to restrain biodegradation. Reversible equilibriums of self-healing hydrogels should be controlled according to the various applications, such as long-term drug release and cell-adaptable materials.

#### AUTHOR CONTRIBUTIONS

All authors listed have made a substantial, direct and intellectual contribution to the work, and approved it for publication.

#### ACKNOWLEDGMENTS

The work was funded by the Higher Education Sprout Project of National Taiwan University (NTU-CC-107L891101).

metal(I)-thiolate/disulfide exchange as biomaterials with tunable mechanical properties. Biomacromolecules 16, 3552–3561. doi: 10.1021/acs.biomac. 5b00980

beta antagonist for treatment of chronic kidney disease. J. Control. Release 206, 131–139. doi: 10.1016/j.jconrel.2015.03.025

**Conflict of Interest Statement:** The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Copyright © 2018 Liu and Hsu. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

# Facile Fabrication of Sandwich Structural Membrane With a Hydrogel Nanofibrous Mat as Inner Layer for Wound Dressing Application

Xueqian Yin1 , Ya Wen1 , Yajing Li 1,2, Pengqing Liu3 , Zhongming Li 3 , Yidong Shi 1 , Jianwu Lan1 , Ronghui Guo1 and Lin Tan1,3 \*

*1 College of Light Industry, Textile and Food Engineering, Sichuan University, Chengdu, China, 2 College of Architecture & Environment, Sichuan University, Chengdu, China, 3 College of Polymer Science and Engineering, Sichuan University, Chengdu, China*

#### Edited by:

*Baolin Guo, Xi'an Jiaotong University, China*

#### Reviewed by:

*Yaobin Wu, Southern Medical University, China Xiaowen Wang, Hong Kong Polytechnic University, Hong Kong*

> \*Correspondence: *Lin Tan<EMAIL_ADDRESS>
#### Specialty section:

*This article was submitted to Polymer Chemistry, a section of the journal Frontiers in Chemistry*

Received: *06 July 2018* Accepted: *26 September 2018* Published: *16 October 2018*

#### Citation:

*Yin X, Wen Y, Li Y, Liu P, Li Z, Shi Y, Lan J, Guo R and Tan L (2018) Facile Fabrication of Sandwich Structural Membrane With a Hydrogel Nanofibrous Mat as Inner Layer for Wound Dressing Application. Front. Chem. 6:490. doi: 10.3389/fchem.2018.00490* A common problem existing in wound dressing is to integrate the properties of against water erosion while maintaining a high water-uptake capacity. To tackle this issue, we imbedded one layer of hydrogel nanofibrous mat into two hydrophobic nanofibrous mats, thereafter, the sandwich structural membrane (SSM) was obtained. Particularly, SSM is composed of three individual nanofibrous layers which were fabricated through sequential electrospinning technology, including two polyurethane/antibacterial agent layers, and one middle gelatin/rutin layer. The obtained SSM is characterized in terms of morphology, component, mechanical, and functional performance. In addition to the satisfactory antibacterial activity against *Staphylococcus aureus* and *Escherichia coli*, and antioxidant property upon scavenging DPPH free radicals, the obtained SSM also shows a desirable thermally regulated water vapor transmission rate. More importantly, such SSM can be mechanically stable and keep its intact morphology without appearance damage while showing a high water-absorption ratio. Therefore, the prepared sandwich structural membrane with hydrogel nanofibrous mat as inner layer can be expected as a novel wound dressing.

Keywords: sandwich structure, hydrogel nanofibrous mat, antibacterial activity, antioxidant activity, wound dressing application

# INTRODUCTION

Hydrogel-based wound dressings are effective materials for the treatment of chronic wound due to the excellent exudates absorption and retaining properties, which allow them to modulate the fluid balance at the site of wound bed and maintain an microenvironment close to that present in native tissue (Fan et al., 2014; Sun et al., 2017; Zhao et al., 2017). Currently, most hydrogels are synthesized in terms of film, powder, particle and bulk 3D constructs. However, some drawbacks existing in those types of hydrogels when applying them as wound dressing materials, such as low gas and liquid permeation, insufficient surface area, poor structural integration (Ravichandran et al., 2014; Chen et al., 2015; Sun et al., 2017). Recently, hydrogel nanofibrous mats which can combine the advantages of both nanofibers and hydrogels have received increasing attention due to their desirable gas/liquid permeability, high surface area, highly porous structure, and excellent water uptake capacity(Brunelle et al., 2017; Sun et al., 2017; Zhao et al., 2017).

Electrospinning is an efficient and versatile technique to produce diversified nanofibers, and hundreds of polymers and their composites have been successfully processed into fibrous structure through electrospinning (Lin et al., 2012; Valizadeh and Mussa, 2014; Ahmed et al., 2015). Recently, several electrospun hydrogel nanofibrous mats, e.g., poly(vinylalcohol) (Bhowmick and Koul, 2016; Oh et al., 2016), Poly-(N-vinyl-2-pyrrolidone) (Kurniawan et al., 2016), poly(acrylic acid)-silica (Wang et al., 2016), poly(N-isopropylacrylamide) (Wen et al., 2015; Yao et al., 2015) have been reported, and the obtained hydrogel nanofibrous mats were mostly proposed to be applied as wound dressing and tissue scaffold. Nevertheless, the maintenance of intact hydrogel nanofibrous structure should be further improved during the functions performing due to the intrinsic poor mechanical stability (Fan et al., 2014; Gonzalez et al., 2014; Wang et al., 2015; Wu et al., 2017), thus additional bottom and top protection barriers for hydrogel nanofibrous layer are necessary (Xia et al., 2015; Kim et al., 2017). Regarding the wound dressing application, susceptible to water erosion and bacterial infection are fatal for wound healing, the membranes possessing two hydrophobic surfaces and one inner hydrophilic layer have greatly promising prospects in wound dressing application accordingly, while each layer can have and perform its own function separately, such as antibacterial property, high water uptake capacity, water resistance against erosion (Fan et al., 2014; Xu et al., 2015; Zhao et al., 2017). In other words, sandwich structure may be an ideal candidate for wound dressing fabrication. However, the conventional electrospun nanofibrous membrane is single layer or double layers. Obviously, the former type has the same wettability for its both surfaces, and the problem of relatively low adhesive force between two individual layers exists in latter type (Tan et al., 2015c; Trinca et al., 2017). Therefore, the fabrication of sandwich structural membrane with hydrogel nanofibrous mat may address the above concerns.

Both electrospun polyurethane and gelatin are popular materials for wound dressings application (Li et al., 2016; Resmi et al., 2016; Sahraro et al., 2016; Nian et al., 2018). Polyurethane (PU) currently is one of the most widely applied synthetic polymers. Compared with conventional polymers, PU possesses comprehensive excellent performances on following aspects, including hydrophobic property, easy preparation, recyclability, and good mechanical features, such as the stretching capability and elasticity (Janik and Marzec, 2015; Kucinska-Lipka et al., 2015); Gelatin (GE) is a natural protein which is derived from the partial and irreversible hydrolysis of collagen, and it also has been extensively employed as a hydrogel polymer (Zhao et al., 2015, 2018), but the poor intrinsic mechanical property of gelatin hinders its application, and the primary way to exert the functions of gelatin is to combine with other polymers (Tan et al., 2015c; Feng et al., 2016; Trinca et al., 2017).

Herein, we put forward the concept of fabricating one novel sandwich structural membrane (SSM) consisting of two hydrophobic electrospun PU layers as bottom and top surfaces, respectively, and one hydrophilic inner layer of gelatin hydrogel nanofibrous mat. Additionally, in order to accelerate the potential wound healing process, both antibacterial activity and free radicals scavenging capacity are introduced into such SSM. We demonstrate that the facilely fabricated SSM shows good performance on antibacterial activity, antioxidant property, water vapor transmission rate, mechanical and water uptake capacity. Therefore, the fabricated SSM in this study can be highly recommended as an ideal cover dressing for promoting wound healing.

# MATERIALS AND METHODS

#### Materials

Polyurethane and antibacterial agent N-halamine (N-chloro-2,2,6,6-tetramethyl-4-piperidinyl, Ca) were synthesized according to the previous reports, and the synthesis information of Ca is presented in **Figure S1** (Tan et al., 2015a; Dong et al., 2017). Rutin was purchased from Shanghai Aladdin Biochemical Polytron Technologies Inc.; Gelatin (Type A) from porcine skin was purchased from Sigma Aldrich. N, N-dimethylacetamide (DMAc) and other solvents were purchased from Chengdu Kelong reagent company and used directly without further purification. Dehydrated polycaprolactone diol (PCL, Mn = 4,000 g/mol) was obtained from Perstop U.K. Ltd (United Kindom). 4,4-methylene bis(phenylisocyanate) (MDI), 1,4- Butanediol (BDO) and dibutyltin dilaurate (DBTDL) were purchased from Sigma Aldrich (Unnithan et al., 2015).

# Synthesis of Polyurethane

The applied polyurethane used for electrospinning in this study was synthesized according to our previous method. In particular, the obtained PU composed of PCL, MDI, and chain extender BDO. The reaction to prepare prepolymer was conducted in a 1,000 mL conical flask equipped with a mechanical stirrer. PCL (∼492 g) mixed with MDI (∼24 g) for 2 h at 80◦C, and followed by the chain extension reaction with BDO (∼100 g) for another 2 h under same temperature. The whole isocyanate group content was excessive by 3 mol%, and the hard segment content is around 20%. All the chemicals used in the synthesis process should be dehydrated with 4 Å molecular sieves in advance (Tan et al., 2015a).

#### Preparation of Gelatin-Rutin (GE-R) and Polyurethane-Antibacterial Agent (PU-Ca) Solution

Firstly, 24 wt% gelatin solution was prepared by dissolving gelatin powder into a binary solvent consisting of water and formic acid with the mass ratio of 8:2 accordingly and the homogenous solution was obtained after stirring at room temperature for 3 h; Thereafter, a calculated amount of rutin powder (10 wt% of gelatin) was added to the solution for another 2 h stirring under the same condition. Similarly, PU-Ca dissolved in DMAc was prepared under constant stirring at 80◦C for 4 h, the concentration of PU was fixed at 20 wt%, and the ratio of Ca was 10 wt% of PU content.

# Fabrication of Electrospun Sandwich Structural Membrane (SSM)

SSM was prepared using a manual assembly electrospinning system. The bottom and top layers were fabricated by spinning PU-Ca solution, and the inner hydrogel nanofibrous layer was made from GE-R solution, two voltage gradients of around 1.0 and 1.8 kV/cm were applied for PU-Ca solution and GE-R solution, respectively, and the feeding rate of 1 mL/h was fixed during electrospinning process under an ambient condition (Relative humidity = 50 ± 5%, Working temperature = 22∼26◦C). In the course of spinning process, we controlled the thickness of the electrospun membrane through feeding ratio, spinning time and the width for nanofiber collection, and the thickness was measured with the aid of a micrometer (Mitutoyo, Japan). The PU-Ca/GE-R/PU-Ca sandwich structure was constructed through sequential electrospinning. In our study, two SSMs were prepared by adjusting the thickness ratio among the three layers, particularly including 1:4:1 and 2:2:2, and we named such two SSMs in short as "SSM141" and "SSM222," respectively. In addition, the PU-Ca and GE-R nanofibrous mats were prepared as comparisons. SSM141, SSM222, and GE-R nanofibrous mats were crosslinked by saturated glutaraldehyde vapor for 15 min and followed by placing in a vacuum desiccator to remove residual crosslinker.

#### Characterizations

The surface and cross-section morphologies of nanofibrous mats were characterized by scanning electron microscope (SEM, Hitachi SU3500, Japan), and all specimens were sputter-coated

with gold for 20 s before observation. Nanofiber diameter distribution was determined by measuring the diameters of 100 randomly selected nanofibers in SEM images with the aid of ImageJ software; The chemical structure and conformation of nanofibrous mats were analyzed by attenuated total reflectance model based Fourier transform infrared (ATR-FTIR, Tracer-100, Japan) spectroscopy in the range of 650–4,000 cm−1 at room temperature; Thermal decomposition behavior was investigated by thermogravimetric analysis (DTG-60, Shimadzu, Japan) with a heating scan from 30 to 800◦C, and the flow rate of nitrogen gas was fixed at 50 mL/min; Mechanical properties was investigated using an electronic single yarn strength meter (YM061,China). The width of specimens employed was 3 mm and the distance between two clamps was 20 mm, the stretching speed was 10 mm/min, and each sample was measured at least 10 times, the average values of breaking force (cN) and elongation at break (%) were recorded; Water contact angle (WCA) of the fabricated membranes surface was measured through a contact angle tester (Harke-SPCAX1, China) using the sessile drop method, and at least five individual values were collected and averaged.

## Water-Absorption Ratio and Water Vapor Transmission Rate (WVTR) Investigation

WAR was evaluated by immersing samples in PBS buffer (pH = 7.4) for 12 h, and then weighted them after removing the surface water using wiping paper; WVTR was determined based on evaporation of water vapor through the testing nanofibrous mats under the temperature of 21 and 37◦C with a continuous % RH, including 35, 55, and 75%. WVTR was monitored by measuring the weight loss (mg) of the cups by functions of unit area (cm2 ) and time (h), the cups without samples were applied as comparisons. Tests of both WAR and WVTR were performed in triplicate.

#### Antibacterial Activity Assessment

Antibacterial activity of SSM against Staphylococcus aureus and Escherichia coli was investigated based on agar disk diffusion susceptibility. The samples were cut into discs with the size of 1.5∗ 1.5 cm2 , and sterilized by ultraviolet irradiation 15 min for

both surfaces separately in a laminar flow hood. Gram-negative E. coli and Gram positive S. aureus were selected as representative bacteria and were cultivated overnight in an incubator, and the sterilized samples were placed on the surface of agar plates coated with the E. coli and S. aureus already diluted by PBS buffer. Subsequently, the plates were incubated at 37◦C overnight, and then inhibition zones were observed and photographed.

#### DPPH Radical Scavenging Activity

The antioxidant activity of SSM were determined according to 1,1-diphenyl-2-picrylhydrazyl (DPPH) free radical-scavenging

TABLE 1 | The average tensile strength, thickness, elongation at break of different membranes.

assays. Specifically, a piece of nanofibrous mat with the size of 1.5∗ 1.5 cm2 was immersed in 4 mL DPPH ethanol solution (1.5 mg/mL), scavenging activity assay was carried out by recording the absorbance at 516 nm of DPPH solution at six specific time points, including 2, 4, 8, 16, 32, and 64 min after the mixture reaction, and the lower average absorbance value indicates better antioxidant activity.

# RESULTS AND DISCUSSION

#### Morphological Analysis

SEM images of surface and cross-section of electrospun nanofibrous membranes are presented in **Figure 1**. Firstly, all the as-fabricated nanofibrous mats show desirable fiber morphology and size homogeneity without beads (**Figures 1A–D**), while slow evaporation of DMAc, bending deformation, entanglement of the nanofibers and resulted in the compacted homogenous structure (**Figure 1B**), and the average diameter of GE-R and PU-Ca are 127 and 131 nm, respectively. Secondly, the obvious sandwich structure can be observed, and the distinct boundary exists between the adjacent two layers, while interconnected porous structure due to the entanglement of nanofibers are visible from the cross-sectional sites (**Figures 1E,F**). Unlike the conventional bilayered nanofibrous mats, such kind of sandwich structure can ensure the integration of nanofibrous membrane during the application, in other words, under the protection of bottom and top layers which are hydrophobic and materials-homogenous, such SSM can prevent the mutual stripping, collapse and effusion under the circumstance of water erosion or others. Additionally, the highly porous structure is highly beneficial to the circulation of oxygen gas and water vapor. More importantly, the wound exudates can freely access to the inner hydrogel nanofibrous layer through the porous pathway and stay inside the layer up to the saturated absorption.

# FT-IR Characterization and Thermal Property Investigation

As shown in **Figure 2**, the typical strong peaks of relevant bonds indicate the blending and electrospinning process did

not influence the structure of each component. Particularly, it can be clearly observed that the characteristic peak at ∼1,642 cm−1 represents the C=O stretching vibration of peptide bonds in the backbone of gelatin, and the peak at ∼1,536 cm−1 corresponds to the N-H bending and C-N stretching vibration; Additionally, the sharp peaks at ∼1,722 cm−1 and broad peak at 2,942 cm−1 can be ascribed to C=O stretching vibration of urethane groups and stretching vibration of –NH and – OH groups of PU, respectively (Tan et al., 2015b). After they blended with rutin or antibacterial agent Ca, and even after the formation of SSM, the above typical peaks did not shift, thus, it can be concluded that all the components maintain their own native structure after the physical blending and electrospinning.

The thermal decomposition behavior was also studied, and the decomposition curves are shown in **Figure 3**, **Figures S2A,B**. Regarding the semi-decomposition temperature (SDT) and maximum decomposition temperatures (MDT), 167, 374, 396◦C and 181, 382, 408◦C are recorded for Ca, PU-Ca, and PU, that means the thermal stability of PU decreases over 20◦C after blending with Ca; In contrast, the stability of gelatin was significantly improved after blending with rutin in terms of SDT and MDT, e.g. the MDT of GE-R reach to 326◦C, while only 276◦C was recorded for the MDT of gelatin (**Figures S2C,D**). Additionally, from **Figures 3A,B**, both SSMs shows desirable thermal stability with near 400◦C of SDT and above 400◦C of MDT. In addition, there is no obvious difference between SSM141 and SSM222 in terms of SDT and MDT, indicating that the change of the thickness ratio of each layer has little influence on the thermal stability of the sandwich structural nanofibrous membranes, and such desirable thermal stability can endow the possible thermal sterilization of SSM before use.

#### Mechanical Properties

A certain mechanical strength can cater to the motorial behavior of skin, while weak mechanical strength may cause the collapse of wound dressings and then fail to protect the wound bed. **Figure 4** shows the representative stressstrain curves of different electrospun membranes. In this study, breaking force and elongation were recorded to evaluate the mechanical properties, and the data were summarized in **Table 1**. Obviously, GE-R has the lowest breaking force (∼33 cN) as well as the breaking elongation (∼4.0%), thus gelatin in combination with other polymers, e.g., PU, for wound dressing application is preferable. From the testing results, SSMs show much better mechanical property compared with that of GE-R electrospun membrane, the reason can be mainly attributed to the presence of PU-Ca layers, and higher composition ratio of PU-Ca endows SSM better mechanical property, i.e., SSM222 have desirable breaking force of around 96 cN and elongation of around 99%. Thus, the obtained SSM can withstand the mechanical deformation resulted from the motorial behavior.

#### Surface Wettability

Surface wettability is an important property for wound dressing which can influence not only the biological response but the defense performance against water erosion. **Figure 5** shows the WCA values of two individual layer surfaces which consist the final SSMs, and the layer of PU-Ca is hydrophobic with a WCA value of 108.50 ± 3.99◦ , while the hydrogel nanofibrous layer of GE-R exhibits relative hydrophilic with a WCA value of 58.08 ± 0.76◦ . The WCA values of SSM141 and SSM222 were shown in **Figure S3**, and they have little difference with pristine PU-Ca layer, indicating that the inner layer of the hydrogel nanofiber has no significant effect on the surface wettability of SSM. Consequently, the bottom and top layers of the fabricated SSMs can effectively defense the water or wound fluids erosion and keep the structural integrity of SSMs during the application, and the inner hydrogel nanofibrous layer can absorb the wound fluids which can freely access through the nanofibrous pathway.

#### WAR and WVTR Study

Both WAR and WVTR are critical factors for the wound management, and both of them play their own role separately and synergistically. Particularly, WAR is applied to evaluate the exudates retaining capacity of the wound dressing, and WVTR will influence the wounds dehydration or accumulation of exudates. In virtue of desirable WAR and WVTR, an ideal moisture microenvironment will exist around the site of wound bed, thus promote the wound healing process.

WAR and WVTR are calculated according to following Equation (1) and Equation (2). In Equation (1), W1 represents the weight after the suction, and W0 represents the original weight of the sample.

$$\text{WAR(\%)} = \frac{\text{W}\_1 - \text{W}\_0}{\text{W}\_0} \times 100\% \tag{1}$$

WVTR(mg.cm−2 .h−1 ) = Water weight change (mg) Exposure area (cm2 ) × exposure time(h) (2)

TABLE 2 | WVTR and WAR of different electrospun membranes.

The thickness of the obtained nanofibrous membranes are around 50µm. As shown in **Figure 6**, the WAR values of PU-Ca, SSM222, SSM141, and GE-R are around 56, 119, 282, and 659%, respectively. The WAR of SSM222 and SSM141 increased by 2 and 5 times higher than that of PU-Ca membrane, which indicates the incorporation of hydrogel nanofibrous mat can significantly increase the WAR, and the thicker of GE-R layer incorporated in such sandwich structural membrane renders higher WAR owning to the intrinsic hygroscopicity of gelatin. Therefore, the accumulation of exudates around the wound beds can be avoided and the risk of potential bacteria infection will be reduced. Whatever normal skin or injured skin, body fluid will evaporate through the micropores of skin, while the injured skin normally shows higher WVTR of 1.16– 21.41 mg.cm−2 .h−1 compared to that of normal skin (Saeed et al., 2017). Thus, porous structure with suitable WVTR of wound dressing is essential and required. **Figure 7** and **Figure S4** show the successive recorded WVTR values of different electrospun membranes under different temperature and humidity. It can be clearly seen that the WVTR values for all electrospun membranes reasonably increase with temperature increasing under constant humidity, while WVTR decreases with increasing the ambient humidity. From **Table 2**, all WVTR values of SSMs, lowest value of 3.10 ± 0.02 mg/cm2 .h−1 and the highest value of 19.13 ± 0.04 mg/cm2 .h−1 , are within the body fluids evaporation range of injured skin, which can indicate that the fabricated SSM can meet

FIGURE 9 | Antibacterial activity investigation results. SSM141against *S. aureus* (A) and *E. coli* (D). SSM222 against *S. aureus* (B) and *E. coli* (E), and pristine PU against *S. aureus* (C) and *E. coli* (F) as the comparisons.

the requirement on managing the wound fluids as a qualified wound dressing.

#### Antioxidant Property

Antioxidant property of the fabricated sandwich structural membrane was evaluated in term of the capacity on scavenging free radicals, and the scavenging ratio of DPPH free radicals (SRDPPH) was calculated according to the following Equation (3):

$$\text{SRDPPH(\%)} = \frac{\text{A}\_0 - \text{A}\_t}{\text{A}\_0} \times 100\% \tag{3}$$

Where A0 is the original absorbance of DPPH, At is the absorbance of the DPPH at the specific measuring time. Antioxidant property refers to the ability of scavenging free radicals, and it should be a crucial property for wound dressing which can protect human skin or wound bed from free radicals damage. Antioxidant performance was monitored through measuring the absorbance of DPPH-contained solution at 516 nm, which was also accompanied by a color change from deep purple to yellow. **Figure 8** demonstrates the antioxidant activity of SSM prepared with different contents of rutin, and higher content of rutin renders the higher SRDPPH, that means higher content of GE-R layer can be incorporated to prepare SSM with better antioxidant performance. In addition, the SRDPPH was recorded by the function of time (**Figure 8B**), and the slope of the curve can reflect the scavenging rate of DPPH free radicals. We can clearly observe that the oxidant performance of all the samples include one initial fast SRDPPH stage with a sharp slope and one slower stage. In other words, the electrospun membranes can initially scavenge the DPPH free radicals quickly then reach a plateau gradually.

#### Antibacterial Activity

Antibacterial activity is essential to a qualified wound dressing, that's because a longer healing process will be required once the wound bed is infected by bacteria. **Figure 9** shows the antibacterial activity of two SSMs against E. coli and S. aureus. Compared with the pristine PU membrane, PU-Ca (**Figure S5**), SSM141 and SSM222 have satisfactory antibacterial activity by the evidence of obvious inhibition zones. In addition, we observed that the inhibition zones against E. coli is slightly larger than those against S. aureus, the possible reason can be attributed to the thicker cell wall of the Gram-positive negative bacteria, and thus the encapsulated antibacterial monomer N-halamine (Ca) can more effectively kill the Gram-negative bacteria.

# CONCLUSION

In this study, we have successfully fabricated a novel type of electrospun SSM with a hydrogel nanofibrous mat as the inner layer through lay-by-layer deposition. The obtained SSM has two hydrophobic surfaces (PU-Ca) which can prevent the water erosion, and simultaneously has a high water-uptake capacity because of the hydrophilic hydrogel nanofibrous layer (GE-R), thus the structural integrity can be effectively maintained during the application. Additionally, the SSM also displays favorable antibacterial activity against E. coli and S. aureus, antioxidant activity in terms of scavenging capacity of DPPH free radicals, and a desirable thermally regulated WVTR. More importantly, the functions and properties can be highly adjusted owing to the nanofibrous sandwich structure, and the incorporated functions can be also performed individually, such as the thickness of inner hydrogel nanofibrous layer, wettability of each layer, mechanical strength and antioxidant period. We believe that such type of SSM can work competently as a wound dressing, and more functional SSMs can be prepared based on this concept.

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### AUTHOR CONTRIBUTIONS

XY, YW, and YL completed the experiments. PL and ZL provided some assistance on conducting experiments and testing. YS, JL, and RG gave some suggestions on writing. LT proposed the idea, designed the experiment, gave scientific support, revised, and finalized the manuscript.

### FUNDING

This work was sponsored by the Fundamental Research Funds for the Central Universities (Grant No. YJ201726), Key Research Program of Science & Technology Department of Sichuan Province (Grant No. 2017SZYZF00009), Strategic Project of Lu Zhou Science & Technology Bureau of Sichuan Province (Grant No. 2017CDLZ-S01).

#### SUPPLEMENTARY MATERIAL

The Supplementary Material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/fchem. 2018.00490/full#supplementary-material

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**Conflict of Interest Statement:** The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Copyright © 2018 Yin, Wen, Li, Liu, Li, Shi, Lan, Guo and Tan. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

# Rational Design of Self-Healing Tough Hydrogels: A Mini Review

#### Wenda Wang, Ravin Narain\* and Hongbo Zeng\*

Department of Chemical and Materials Engineering, University of Alberta, Edmonton, AB, Canada

Hydrogels are three-dimensional cross-linked polymer networks which can absorb and retain large amount of water. As representative soft materials with tunable chemical, physical and biological properties, hydrogels with different functions have been developed and utilized in a broad range of applications, from tissue engineering to soft robotics. However, conventional hydrogels usually suffer from weak mechanical properties and they are easily deformed or damaged when they are subjected to mechanical forces. The accumulation of the damage may lead to the permanent structural change and the loss of the functional properties of the hydrogels. Therefore, it is important to develop mechanically robust hydrogels with autonomous self-healing property in order to extend their lifespan for various applications. In this mini review, we focus on the discussion about the appropriate molecular design of the hydrogel network for achieving self-healing and excellent mechanical properties, respectively as well as the corresponding self-healing and toughening mechanisms. We conclude with perspectives on the remaining challenges in the field as well as the recommendations for future development.

Edited by:

Weifeng Zhao, Sichuan University, China

#### Reviewed by:

Baolin Guo, Xi'an Jiaotong University, China Zhipeng Gu, Sun Yat-sen University, China Dongsheng Wang, University of Electronic Science and Technology of China, China

#### \*Correspondence:

Ravin Narain<EMAIL_ADDRESS>Hongbo Zeng<EMAIL_ADDRESS>
#### Specialty section:

This article was submitted to Polymer Chemistry, a section of the journal Frontiers in Chemistry

Received: 20 June 2018 Accepted: 28 September 2018 Published: 18 October 2018

#### Citation:

Wang W, Narain R and Zeng H (2018) Rational Design of Self-Healing Tough Hydrogels: A Mini Review. Front. Chem. 6:497. doi: 10.3389/fchem.2018.00497 Keywords: hydrogels, synthesis, self-healing, tough hydrogels, functional hydrogels

# INTRODUCTION

Hydrogels are three-dimensional networks consisting of cross-linked polymer chains, which form matrices with high water content (Annabi et al., 2014). Over the past few years, significant progress has been achieved in the development of functional hydrogels with tunable chemical, physical and biological properties for various applications, including tissue engineering (Eke et al., 2017; Wang J. et al., 2017), controlled drug delivery systems (Zhang et al., 2011; Chen et al., 2018), sensors and actuators (Lei et al., 2017; Yuk et al., 2017), soft electronics (Lin et al., 2016; Cai et al., 2017), etc. However, conventional hydrogels usually suffer from poor mechanical properties and are easily deformed or damaged when they are subjected to mechanical forces. The propagation and accumulation of the damage would affect the integrity of the hydrogels and cause the loss of the functionalities, which may limit the lifespan of the hydrogels. Therefore, it is desirable to develop mechanically robust hydrogels possessing the self-healing capability in order to not only prolong the life-time of the materials but also increase the durability and the reliability of the hydrogels in certain applications by avoiding accumulation of cracks or damages (**Graphical Abstract**).

Self-healing hydrogels are the hydrogels which can automatically heal damages and restore themselves to normality without the intervention of an external stimuli (Taylor and In Het Panhuis, 2016). Based on different self-healing mechanisms, the self-healing hydrogels can be divided into two categories, chemically and physically self-healing hydrogels. Different design strategies can be employed for fabricating hydrogels with self-healing ability.  For both types of self-healing hydrogels, special functional groups are required to be incorporated into the polymer chains for desirable chemical reactions or physical interactions, which mediate the self-healing process, to occur in the damaged region.

Conventional hydrogels are considered to be mechanically weak (toughness <10 J m−2 ), which limits certain applications such as tissue engineering (cartilages or tendon), since these tissues are natural hydrogels possessing high toughness (1,000 J m−2 ) and high tensile strength (30 MPa) (Taylor and In Het Panhuis, 2016). The brittleness (low toughness) of the conventional hydrogels comes from two reasons. Conventionally, hydrogels are synthesized by free radical polymerization of watersoluble monomers and cross-linkers. Due to the difference in reactivity between the reagents, highly random (heterogeneous) cross-linked network is typically produced. The heterogeneity will lead to uneven stress distribution and stress localization when the hydrogel experiences force loading, which accounts for the brittleness (Naficy et al., 2011). Besides, the conventional hydrogels lack of mechanisms which allow the mechanical energy to be dissipated through the hydrogel upon force loading, which is another reason for the brittleness (Chung et al., 2017). It is generally agreed that the hydrogels with tensile strength of 0.1–1 MPa and fracture energy of 102 -103 J m−2 can be considered as tough hydrogels (Chen et al., 2016). To improve the mechanical properties of the conventional hydrogels, one can either increase the homogeneity of the cross-linked network

[tetra-PEG hydrogels (Ishii et al., 2017; Yang N. et al., 2017), radiation cross-lined hydrogels (Xu et al., 2018)] or design the hydrogels with special structure (double network (DN) hydrogels (Li et al., 2016; Liu and Li, 2016), nanocomposite hydrogels (Jiang et al., 2017; Qin et al., 2017)etc., which allows mechanical energy to be dissipated through the hydrogel.

In this mini-review, we briefly discuss different design strategies for developing self-healing and tough hydrogels, respectively. Examples from recent literature will be illustrated accordingly. Finally, we conclude with a perspective on remaining challenges in the field and the recommendations for future development.

# DESIGN STRATEGIES FOR SELF-HEALING HYDROGELS

#### Chemically Self-Healing Hydrogels

In conventional hydrogels, the polymer networks are usually cross-linked by covalent bonds which are irreversible and too stable for dynamic chemistry to occur for self-healing (Wei et al., 2014). The chemically self-healing hydrogels reform the network through dynamic covalent bonds or reversible chemical reactions, typically including phenylboronic ester bonds, dynamic imine bonds (dynamic Schiff base), disulfide bonds, reversible radical reaction, Diels-Alder reaction, etc. (**Figure 1A**).

#### Phenylboronic Ester Complexation

Phenylboronic ester is the dynamic complex formed based on boronic acid-diol interaction. The dynamic nature (self-healing ability) of the complexation strongly depends on the pKavalue of various phenylboronic acids. Langer et al. reported an injectable self-healing glucose-responsive hydrogel which was constructed by phenylboronic acid-terminated four-armed poly(ethylene glycol) (PEG) and diol-terminated four-armed PEG (Yesilyurt et al., 2016). It was found that the self-healing behavior and the mechanical property of the hydrogels are closely associated with the pKavalue of the terminal phenylboronic acid group. When the hydrogel was formed at pH greater than the pKavalue of

the terminal phenylboronic acid group, the gel was rigid and brittle and the self-healing ability was found to decrease with increasing the pH. However, when the hydrogel was formed around the pKa value of the terminal phenylboronic acid group, the hydrogels showed excellent self-healing ability. When the pH is below the pKa value, there is no gel formation observed. The stability (self-healing ability) of phenylboronic ester complex also depends on the conformation of the diol groups in the

bond, hydrophobic interaction, host-guest interaction and multiple intermolecular interactions.

polymer chains. Schiller et al. determined binding constants between phenylboronic acid and different polysaccharides with different diol conformations (Axthelm et al., 2017). It was found that phenylboronic acid has a higher affinity for the cis-diol groups than that for the trans-diol groups. Therefore, the selfhealing ability of the hydrogel is expected to be enhanced when cis-diol is incorporated into the polymers used to construct hydrogel network since the boron-cis-diol complexation is more thermodynamically stable and more likely to be reformed after being destructed. The self-healing hydrogels which based on phenylboronic ester complexation have been applied to a wide range of biomedical applications. Narain et al. developed a series of self-healing hydrogels for applications such as glucose sensing (Kotsuchibashi et al., 2013), drug delivery (Chen et al., 2018) and 3D cell cultivation (Wang et al., 2016).

#### Dynamic Imine Bond (Schiff Base)

Dynamic imine bonds (dynamic Schiff base) are formed between the aldehyde groups and the primary amine groups. Wei et al. reported a series of self-healing hydrogels based on dynamic Schiff base during the past few years (Zhang et al., 2011; Tseng et al., 2015; Hsieh et al., 2017; Yang X. et al., 2017; Zhang Y. et al., 2017). In most of their work, a benzaldehyde-difunctionalized poly(ethylene glycol) (DF-PEG) was synthesized. Self-healing hydrogels with different properties and applications can be constructed by simply mixing the DF-PEG solution with either natural polymer solutions or synthetic polymer solutions such as glycol chitosan solution (Zhang et al., 2011), amine-modified carboxyethyl cellulose solution (Yang X. et al., 2017), etc. Such self-healing hydrogels can be used in wide biomedical applications such as 3D cell cultivation (Zhang Y. et al., 2017), controlled release of biomolecules (Zhang et al., 2011), blood capillary formation (Hsieh et al., 2017), central nervous system repair (Tseng et al., 2015), etc. Guo et al. also developed self-healing hydrogels which employs dynamic Schiff base for biomedical applications such as cutaneous wound healing (Zhao et al., 2017), cell delivery carrier for cardiac cell therapy (Dong et al., 2016) and drug delivery carrier for hepatocellular carcinoma therapy (Qu et al., 2017). Interestingly, similar to the phenylboronic ester complex, the self-healing ability of the hydrogels based on dynamic Schiff base is also pH dependent. Therefore, appropriate pH condition needs to be met when constructing the hydrogel with desirable self-healing property.

#### Disulfide Bond

Disulfide bond is another dynamic covalent bond which is often adopted to make the hydrogel self-healable. The dynamic feature of the disulfide bond is based on the thiol/disulfide exchange reactions which are sensitive to heat, photo-irradiation and mechanical stress. Waymouth et al. developed a self-healing hydrogel which was constructed by an ABA triblock copolymer (Zhang and Waymouth, 2017). The A-block of the polymer contains pendent dithiolane groups which can be cross-linked through reversible ring opening polymerization induced by disulfide exchange reactions between the 1,2-dithiolanes and dithiols. The as-prepared hydrogel underwent reversible sol-gel transition in the presence of external stimuli such as temperature and pH. The hydrogel was able to restore itself after rheological deformation up to a strain value of 800%. Such hydrogel is a promising candidate for stimuli-responsive drug delivery. Wang et al. developed a self-healing hydrogel coating which based on disulfide bond with antibacterial and antifouling properties (Yang et al., 2015). The as-prepared hydrogel coating has great potential as an effective and durable coating in long-term applications for biomaterials.

#### Other Dynamic Chemical Bonds and Reactions

Diels-Alder reaction is one of the "click chemistry" which occurs between a diene and a dienophile (Gregoritza et al., 2017; Guaresti et al., 2018). Yang et al. reported a self-healable cellulose nanocrystal-poly(ethylene glycol) (CNC-PEG) nanocomposite hydrogel via Diels-Alder reaction. The hydrogel possessed both excellent self-healing property and mechanical properties, which can be potentially used for tissue engineering (Shao et al., 2017). Chen et al. reported that the self-healing hydrogel based on Diels-Alder reaction was completely self-healed after 7 h (Wei et al., 2013). For the self-healing hydrogels based on the reversible radical reaction, functional groups such as trithiocarbonate (TTC) units (Amamoto et al., 2011), thiuramdisulfide (TDS) units (Amamoto et al., 2012), etc., need to be integrated into the polymer network. The reversible radical reaction can be triggered by external stimuli such as photo irradiation for the hydrogels to be self-healed (Wei et al., 2014).

### Physically Self-Healing Hydrogels

This section focuses on designing physically self-healing hydrogels based on dynamic non-covalent interactions, including hydrogen bonds, hydrophobic interaction, host-guest interaction and multiple intermolecular interactions (**Figure 1B**).

#### Hydrogen Bond

Hydrogen bonding is one of the most commonly used strategies to construct physically self-healing hydrogels. Zhao et al. prepared a poly(vinyl alcohol) (PVA) hydrogel through facile freezing/thawing method (Zhang H. et al., 2012). It was found that the hydrogel showed excellent self-healing behavior only when the polymer concentration is >35 wt %, which indicates that sufficient number of hydroxyl groups are required to promote the regeneration of enough hydrogen bonds across the interface for the hydrogel to be self-healed. The conformation of hydrogen bond donors and acceptors is another factor which may affect the formation of hydrogen bonds and influence the self-healing performance. Wang et al. designed a selfhealing hydrogel which contains ureido pyrimidinone (UPy) moieties (Zhang G. et al., 2017). The UPy moieties can be dimerized through quadruple hydrogen bonds. The hydrogel showed excellent self-healing performance due to the multiple hydrogen bonds formed in the network. Gu et al. prepared a selfhealing hydrogel which was formed through hydrogen bonding between the cytosine (C) and guanosine (G) modified hyaluronic acid (HA) (Ye et al., 2017). The as-prepared hydrogel could be applied forshort-term injectable drug delivery, tissue engineering and regenerative medicine.

#### Hydrophobic Interaction

Hydrophobic interaction serves as another strategy to design physically self-healing hydrogels. In these hydrogels, hydrophobic monomers are incorporated into the hydrogel network together with the hydrophilic monomers. Hydrophobic associations or hydrophobic domains will be formed within the hydrogel through self-assemble. By carefully controlling the hydrophobe content in the hydrogel, the resulting hydrogel can be self-healed through strong hydrophobic interactions. Okay et al. reported several self-healing hydrogels which were based on hydrophobic interactions and the resulting hydrogels showed excellent self-healing properties (Tuncaboylu et al., 2011; Mihajlovic et al., 2017). Owusu-Nkwantabisah et al. developed a thermoresponsive self-healing hydrogel which incorporated poly(N-isopropylacrylamide) (PNIPAM) nanogels (Owusu-Nkwantabisah et al., 2017). The as-prepared hydrogel showed rapid self-healing behavior through hydrophobic interaction upon raising the temperature. Such hydrogel can be potentially applied for temperature sensors, thermo-switchable windows in biomedical applications.

#### Host-Guest Interaction

Host-guest interaction is one of the supramolecular interactions which is also widely employed to construct physically selfhealing hydrogels. The interaction usually involves two or more molecules forming complex through various dynamic non-covalent interactions such as hydrogen bonds, electrostatic interactions, van der Waals forces, etc., (Wei et al., 2014). Typical examples of the host molecules including, cyclodextrin (CD) (Loebel et al., 2017), cucurbituril (CB) (Xu et al., 2017), crown ether (CE) (Zhang M. et al., 2012), etc., which have been used to make self-healing hydrogels for different applications. The internal cavity of these molecules allows them to accommodate various guest molecules with different binding affinity (Nakahata et al., 2011). Therefore, one should carefully choose the hostguest pair since the self-healing ability strongly depends on the binding affinity between the host and guest molecules. Guo et al. developed several conductive self-healing hydrogels which are based on host-guest interaction. Such hydrogels can be potentially used for a wide range of biomedical applications where electroactivity is in need such as bio sensors (Deng et al., 2018) and carriers for therapeutic agents (Wu et al., 2014).

#### Multiple Intermolecular Interactions

In order to achieve shorter self-healing time and higher self-healing efficiency, multiple intermolecular interactions are employed to endow the hydrogel with self-healing property. Mussel-inspired self-healing hydrogels are typical examples which contain polydopamine (PDA) within their structure. PDA based materials are well known for their remarkable adhesive property through synergestic effect of various interactions such as hydrogen bonds, metal-catechol coordination, cation– π interactions, π-π interactions, etc., (Li et al., 2015b). Zeng et al. reported several mussel-inspired self-healing hydrogels with antifouling and antimicrobial properties (Li et al., 2015c, 2017). The hydrogels showed excellent self-healing performance and such hydrogels have great potential in wound healing application.

#### DESIGN STRATEGIES FOR TOUGH HYDROGELS

#### Homogeneous Tough Hydrogels

Tetra-PEG hydrogel is a typical example of homogeneous hydrogels (**Figure 2A**). Sakai et al. did pioneering work on developing tetra-PEG tough hydrogel (Sakai et al., 2008). In

2008, the first tetra-PEG hydrogel was developed. Two tetra-PEG macro-monomers with the same arm length and different end group were synthesized. The hydrogel was fabricated by simply mixing two tetra-PEG solutions to allow crosslinking reactions between the end groups. The mechanical property of the developed hydrogel was compared with that of agarose gel and acrylamide gel by compression test. It was found that tetra-PEG hydrogel prepared at 1:1 mole ratio of two macromonomers experienced the highest compression strength of ∼2.5 MPa. The enhanced toughness of the hydrogel results from the homogenously cross-linked network, which can not only avoid stress concentration in certain regions but also allows the stress to be distributed evenly (Naficy et al., 2011).

Homogeneously cross-linked hydrogels can also be fabricated through radiation cross-linking method instead of chemical cross-linking method. Wang et al. synthesized a polyacrylic acid (PAA) and polyacrylamide (PAAm) hydrogel throughγradiation-induced polymerization (Wang et al., 2011). It was found that the mechanical property of the PAA-PAAm hydrogel was superior to those produced by the classical chemical polymerization using a crosslinking agent. This may be due to the non-selective initiation effect of the monomers when a high-energy irradiation source is in presence, which could lead to a more homogeneous hydrogel network and improve the mechanical properties of the hydrogel.

# Double-Network Tough Hydrogels (DN Hydrogels)

Double-network hydrogels (DN hydrogels) are considered as one class of tough hydrogels which employ mechanical energy dissipation mechanism (**Figure 2B**). The high strength and toughness of the DN hydrogels are resulted from their unique contrasting network structure and strong network entanglement (Chen et al., 2016). The first network is often designed to be tightly cross-linked and serves as the minor component of the DN hydrogel. The second network is designed to be loosely cross-linked and it is the major component of the DN hydrogel (Gong, 2010). Gong et al. reported the first DN hydrogel in 2003 (Gong et al., 2003), in which the poly(2-acrylamido-2-methylpropanesulfonic acid)/poly(acrylamide) (PAMPS/PAM) DN hydrogel showed fracture stress of 17.2 MPa which is 20 times higher than that of either PAMPS or PAM single network hydrogel. Brown and Tanaka proposed the toughening mechanism of the DN hydrogel (Brown, 2007; Tanaka, 2007). During the fracture process, the tightly cross-linked PAMPS network is firstly fragmented into micro-clusters and they act as the cross-linking points which are able to hold and stabilize the loosely cross-linked PAM network. The brittle PAMPS network serves as the "sacrificial bonds" which allows energy to be dissipated during the formation of the microcracks. However, the largest disadvantage of the DN hydrogel developed by Gong et al. is that the brittle PAMPS network is irreversibly cross-linked and the fracture of the PAMPS network is permanent. Such DN hydrogel will lose its toughness once the first polymer network is ruptured. Later, Suo et al. reported a DN hydrogel which composed of a dynamically cross-linked alginate network based on the ionic interactions between Ca2+ and carboxylic groups (COOH−) on the alginate as the first "sacrificial network" and a chemically cross-linked PAM network as the second network (Sun et al., 2012). The DN hydrogel not only showed superior mechanical property but also recovered most of its mechanical properties after first force loading. Due to the excellent mechanical properties, DN hydrogels attract great interest in biomedical applications. Guo et al. developed biocompatible functional hydrogels with excellent mechanical properties for potential applications of tissue implantation (Li et al., 2015a) and drug delivery (Zhao et al., 2014).

#### Nanocomposite Hydrogels (NC Hydrogels)

Nanocomposite (NC hydrogels) are the hydrogels formed between nano-scaled materials and polymer chains (**Figure 2C**). The concept of NC hydrogel was established by Haraguchi and Takehisa (2002). In this work, they developed a novel NC hydrogel which contains clay and poly(N-isopropyl acrylamide) (PNIPAM) chains. The hydrogels was formed by in-situ free radical polymerization of the NIPAM monomers in well-dispersed clay aqueous solution without using chemical cross-linkers. A mechanism was proposed for the hydrogel formation: First, the initiator could bind to the surface of clay through electrostatic interactions which allows polymer chains to be grafted from the clay nanosheets. In addition, the nucleophilic N(H)CO in PNIPAm chains could coordinate with the Si on the clay. Therefore, the clay nanosheets can act as multifunctional cross-linkers which allow multiple polymer chains to be attached through various interactions. The clay/PNIPAM NC hydrogels possessed excellent mechanical properties with tensile stress of 0.109 MPa and elongation ratio of 1,424%. The outstanding mechanical properties of the NC hydrogel is due to the synergistic effect of the multiple reversible intermolecular interactions between nanomaterials and polymer chains for mechanical energy dissipation (Haraguchi, 2007) and the homogeneity of the well-dispersed nanomaterials within the hydrogel network for even distribution of stress (Chung et al., 2017).

It is worth mentioning that through special design of NC hydrogels, one can fabricate hydrogels with both outstanding mechanical properties and excellent self-healing properties. Lu et al. adopted mussel-inspired surface chemistry to modify the surfaces of the nanomaterials with polydopamine (PDA) and developed a series of tough self-healing NC hydrogels for various applications such as reduced graphene oxide (rGO)/polyacrylamide (PAM) NC hydrogels for implantable bioelectronics (Han et al., 2017b), nanoclay/PAM NC hydrogels for wound healing (Han et al., 2017a), carbon black nanoparticle (CBNP)/PAM NC hydrogels for electricity conduction (Han et al., 2017d), etc. With the modification of PDA, multiple reversible intermolecular interactions from PDA are introduced into the hydrogel system, which not only improve the mechanical properties but also endow the hydrogel with self-healing capability. Similarly, Shao et al. developed a tough self-healing NC hydrogel which based on tannic acid-coated cellulose nanocrystals (TA@CNCs) for wearable strain sensor (Shao et al., 2018). The TA@CNCs act as dynamic bridges in the hydrogel network, which lead to rapid self-healing performance as well as reliable mechanical properties of the as-prepared hydrogel.

# CONCLUDING REMARKS

Mechanically robust hydrogels with self-healing properties present many advantages over conventional hydrogels as they can be utilized in a broad range of areas such as stretchable electronics (Pu et al., 2017; Jing et al., 2018; Hong et al., 2019), sensors and actuators (Francis et al., 2017; Tudor et al., 2017), cartilage repair (Han et al., 2017c; Lolli et al., 2018), with extended life-time and enhanced durability and reliability. Although the field of study shows great promise, there are still challenges to be tackled.

Generally, the self-healing ability of the hydrogels weakens and the time required for the healing process increases with the improvement of the mechanical properties of the hydrogels as summarized by Panhui et al. (Taylor and In Het Panhuis, 2016). Therefore, the development of mechanically robust hydrogels which can be self-healed on the time scale of seconds or minutes should be considered. One possible approach is to introduce multiple chemically or physically self-healing mechanisms into the NC hydrogels/DN hydrogels in order to enhance the self-healing efficiency without sacrificing the mechanical properties.

The fundamental understanding of different self-healing and toughening mechanisms is crucial for designing and optimizing the self-healing tough hydrogels. Both theoretical and experimental studies are needed to understand the self-healing and toughening mechanisms at the atomic-scale or molecularscale. For instance, Zeng et al. used a surface forces apparatus (SFA) to quantitatively study the polymer interactions which account for the self-healing behavior of the hydrogel (Yan et al., 2017). Such non-destructive in situ testing method is highly needed for elucidating the molecular interaction mechanisms of various hydrogel materials.

It is also necessary to develop self-healing tough hydrogels into multifunctional "smart" materials, which should integrate other properties such as electrical conductivity (Wu et al., 2017),

#### REFERENCES

temperature responsivity (Zubik et al., 2017), photo responsivity (Wang R. et al., 2017), etc., into a single hydrogel system. Such hydrogels can be promisingly applied in different applications such as sensors and actuators. When the hydrogels are endowed with the stimuli responsive properties, one should ensure the chemical and mechanical stability of the hydrogels as the change of environmental conditions may affect the intrinsic properties of the hydrogels. The intermolecular and surface interactions of various components in the hydrogel systems essentially determine the properties and performances of the hydrogel systems, which should be well taken in the rational design of the self-healing tough hydrogels.

#### AUTHOR CONTRIBUTIONS

WW wrote the manuscript. RN and HZ helped to revise the manuscript.

#### ACKNOWLEDGMENTS

We would like to thank the financial support from Natural Sciences and Engineering Research Council of Canada (NSERC).

in Regenerative Medicines," in Hybrid Organic-Inorganic Interfaces, eds M. H. Delville and A. Taubert (Edmonton, AB: Wiley).

**Conflict of Interest Statement:** The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Copyright © 2018 Wang, Narain and Zeng. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

# Functional Hydrogels With Tunable Structures and Properties for Tissue Engineering Applications

Xiaomeng Li 1,2, Qingqing Sun3 , Qian Li 1,2, Naoki Kawazoe4 and Guoping Chen4 \*

*1 School of Mechanics and Engineering Science, Zhengzhou University, Zhengzhou, China, 2 National Center for International Joint Research of Micro-nano Moulding Technology, Zhengzhou University, Zhengzhou, China, 3 Center for Functional Sensor and Actuator, National Institute for Materials Science, Tsukuba, Japan, 4 Research Center for Functional Materials, National Institute for Materials Science, Tsukuba, Japan*

Tissue engineering (TE) has been used as an attractive and efficient process to restore the original tissue structures and functions through the combination of biodegradable scaffolds, seeded cells, and biological factors. As a unique type of scaffolds, hydrogels have been frequently used for TE because of their similar 3D structures to the native extracellular matrix (ECM), as well as their tunable biochemical and biophysical properties to control cell functions such as cell adhesion, migration, proliferation, and differentiation. Various types of hydrogels have been prepared from naturally derived biomaterials, synthetic polymers, or their combination, showing their promise in TE. This review summarizes the very recent progress of hydrogels used for TE applications. The strategies for tuning biophysical and biochemical properties, and structures of hydrogels are first introduced. Their influences on cell functions and promotive effects on tissue regeneration are then highlighted.

Keywords: functional hydrogels, tissue engineering, physical properties, chemical properties, microstructures

Tissue engineering (TE) has emerged as a useful approach to treat tissue damages caused by diseases and trauma, which has shown many advantages as compared to conventional treatment strategies.

#### Edited by:

*Weifeng Zhao, Sichuan University, China*

#### Reviewed by:

*Dongsheng Wang, University of Electronic Science and Technology of China, China Tao Xiang, Southwest Jiaotong University, China*

> \*Correspondence: *Guoping Chen<EMAIL_ADDRESS>
#### Specialty section:

*This article was submitted to Polymer Chemistry, a section of the journal Frontiers in Chemistry*

Received: *31 July 2018* Accepted: *01 October 2018* Published: *22 October 2018*

#### Citation:

*Li X, Sun Q, Li Q, Kawazoe N and Chen G (2018) Functional Hydrogels With Tunable Structures and Properties for Tissue Engineering Applications. Front. Chem. 6:499. doi: 10.3389/fchem.2018.00499*

To afford desirable therapeutic outcome, scaffolds prepared from various kinds of biomaterials have been used for TE to accommodate sufficient amount of cells and to control cell functions (Chen and

Kawazoe, 2016a,b; Chen et al., 2018).

INTRODUCTION

Among of different types of scaffolds, hydrogels have attracted more and more attention in the TE field owing to their similarity to in vivo cellular microenvironment and tunable physiochemical properties (Drury and Mooney, 2003). Hydrogels are generally prepared by translating hydrophilic polymers solution into 3D network structure via physical or chemical crosslinking. During this process, hydrogels can encapsulate cells homogeneously and provide cells a 3D microenvironment similar to the native extracellular matrix (ECM) (Tibbitt and Anseth, 2009). Cell behaviors and functions in vivo are affected by the stimuli that are produced by the surrounding ECM. In a similar way, the structures and physiochemical properties of hydrogels provide critical cues to control the functions of embedded cells and thus guide tissue regeneration.

The structures and physiochemical properties of hydrogels can be designed and controlled through selecting different biomaterials, crosslinking methods and fabrication strategies. This review summarizes the latest developments of functional hydrogels for TE applications. At first, the

**64**

materials and crosslinking methods used for hydrogel preparation are introduced. And then, the approaches to tune the structure and physiochemical properties of hydrogels and their effects on cell functions and tissue regeneration are compared. Finally, the challenges and future perspectives of functional hydrogels are discussed.

### MATERIALS AND CROSSLINKING METHODS FOR FUNCTIONAL HYDROGELS PREPARATION

There are several criterias to prepare hydrogels for TE applications. Firstly, the materials and crosslinking agents should be compatible toward living cells and biological factors (e.g., growth factors). Secondly, the preparation process should be easily occured under mild conditions. Thirdly, the products from hydrogel degradation should be non-toxic to cells and tissues. The materials and crosslinking methods used for hydrogel preparation for TE are summarized in **Table 1**.

#### Hydrogel Construction Materials

Materials used to prepare TE hydrogels can be briefly classified into natural and synthetic polymers. Hydrogels prepared from natural polymers possess intrinsic advantages, such as high biocompatibility, biodegradability and similar microenvironment to that of native tissues. Natural polymers used for hydrogel preparation include protein-based materials (such as gelatin, collagen, fibrin, and silk fibroin) and polysaccharide-based materials (such as hyaluronic acid (HA), chondroitin sulfate (CS), alginate, chitosan and so on). Collagen, as the main ECM component of various tissue, is an attractive material for hydrogels preparation (Heo et al., 2016). It's derivative, gelatin, is also a frequently used protein-based material for hydrogel formation, which has higher solubility and lower cost when compared with collagen (Zhao et al., 2016). Gelatin-based hydrogels are good candidates for various TE. For example, injectable gelatin methacryloyl (GelMA) hydrogels are prepared for cartilage TE (Li et al., 2016). The chondrocytes 3D cultured in these hydrogels have shown excellent viabilities and desirable functions. HA as a glycosaminoglycan is commonly prevalent in body liquid and native ECM (Shendi et al., 2016). Therefore, it has been used to prepare various kinds of hydrogels for cartilage, skin, and many other TE. For example, it has been reported that HA molecular can bond with mesenchymal stem cells (MSCs) through CD44 receptors and promote the chondrogenesis (Chung and Burdick, 2008). CS is a sulfated glycosaminoglycan with a linear structure existing in cartilage tissue ECM (Levett et al., 2014). Chondrocytes cultured in CS hydrogels have round morphology, enhanced gene expression, and secretion of cartilaginous ECM (Levett et al., 2014; Zhu et al., 2014). Other polysaccharide-based materials, such as alginate (obtaining from bacteria and brown seaweed) and chitosan (derived from chitin that is produced from the shells of crabs and shrimps), are also commonly used for hydrogel preparation due to their biocompatibility, degradability and easy modification (Kim et al., 2016; Hunt et al., 2017).

On the other hand, synthetic polymers have also exhibited wide usage due to their controllability, reproducibility, and good mechanical properties (Guan et al., 2017). Representative synthetic polymers used for TE hydrogels include poly(ethylene glycol) (PEG), poly(vinyl alcohol) (PVA),poly (N-isopropylacrylamide) (PNIPAM) (Haq et al., 2017), and polyacrylamide (PAM) (Darnell et al., 2013). PEG and PVA have low toxicity, making them widely used for cell-laden hydrogels and drug carriers (Kim et al., 2016; You et al., 2018). However, due to the lack of biological activity, the biocompatibility of these synthetic polymers is compromised relative to natural materials. Hybridization with natural materials is an efficient and easy-going approach to integrate the advantages of natural and synthetic component. It has been reported that the biocompatibilities of PAM, PVA, PNIPAM, and PEG hydrogels are significantly improved after blend with gelatin (Gao et al., 2015; Kim et al., 2016; Navaei et al., 2016b; Han et al., 2017). The cell spreading, and proliferation in these hybrid hydrogels are enhanced compared with the cells 3D cultured in synthetic hydrogels. Besides, the hybridization of different natural materials can also exhibit novel attractive properties. For example, alginate is interpenetrated and crosslinked into photopolymerized gelatin hydrogel network to generate alginate/gelatin hybrid hydrogel (Pacelli et al., 2018). Such a hydrogel is confirmed to have the function of promoting bone tissue regeneration due to the good biocompatibility and enhanced mechanical property.

#### Crosslinking Methods

There are various methods for crosslinking of hydrophilic polymers chains to form hydrogels, which usually are selected depending on the materials chemistry and expected functions. In general, they can be classified into physical and chemical crosslinking methods.

#### Physical Crosslinking Methods

Physically crosslinked hydrogels can be prepared at very mild conditions without the utilization of crosslinking agents that often cause toxicity to cells or may affect the activity of biological molecules encapsulated in hydrogels (Hennink and Van Nostrum, 2012). There are many methods to prepare physically crosslinked hydrogels for TE applications, such as ionic interaction, guest-host interaction and thermo-gelation.

#### **Ionic crosslinking**

Ionic crosslinking method is frequently used to encapsulate cells and drugs due to the mild crosslinking procedures (Goosen et al., 1985; Gombotz and Wee, 2012). The most representative hydrogels formed by ionic crosslinking is alginate hydrogel. The crosslinking is through the exchange of sodium ions from guluronic acid units with divalent cations such as calcium to form junction zones (Gacesa, 1988). This physically crosslinked alginate hydrogel not only has shown a biocompatible property but also has many other functions, such as self-healing and stress relaxation, due to the reversible dissociation and re-bonding between α-L-guluronic acid in alginate and calcium ions. However, the stability of ionic hydrogel should be considered TABLE 1 | Materials and crosslinking methods for functional hydrogels preparation.

when using under physiological conditions. For instance, calcium crosslinked alginate hydrogel will lose stability in 0.9 wt% sodium chloride solution due to the exchange of calcium ions by sodium ions (Martinsen et al., 1989).

#### **Guest-host chemistry**

The guest-host chemistry holds great potential for hydrogels preparation, because the low cytotoxicity and specific selectivity. In this reaction, host molecules can selectively recognize and physically bind to certain guest molecules to form crosslinked network. The interaction includes hydrophobic association, hydrogen bonding, electrostatic interaction, van der Waals forces, and so on (Steed et al., 2007). Cyclodextrin, as one macrocycle, has hydrophobic interior cavities that have a high affinity for specific hydrophobic guest moieties. Specifically, guest–host pair, adamantane (guest) and βcyclodextrin (host), is widely used to synthesize corresponding macromers. The guest-host bonding can translate mixed solution into hydrogels easily (Rodell et al., 2016). In another study, acrylate β-cyclodextrin can build guest-host binding with the benzene ring of gelatin to form supramolecular hydrogels. These hydrogels have very good bio-adhesive property and sustainable release of hydrophobic drugs, which is promising for TE of bone, cartilage and tendon (Feng et al., 2016).

#### **Thermo-gelation**

Thermo-gelation is to build a physically crosslinked network by altering the temperature. For example, the crystalline nature of PVA has been applied to fabricate physically crosslinked hydrogel by several repeating cycles of freezing and thawing (Hassan and Peppas, 2000). These physically crosslinked PVA hydrogels are reported to have many characteristics, such as high swelling degree and mechanical strength. Gelatin solution becomes hydrogel due to the intermolecular hydrogen bonding formation when the temperature drops below the upper critical solution temperature (UCST). In contrast, some other macromer, such as PNIPAM, becomes hydrogel when temperature increases above the lower critical solution temperature (LCST) due to the balance between hydrogen bonding and hydrophobic effects (Ashraf et al., 2016). These temperature-responsive polymers can transform phases between sol and gel near physiological temperature, which enables the hydrogels injectable. PNIPAMbased injectable hydrogel has been used to encapsulate cardiomyocytes and the encapsulated cells exhibit high viability and mature phenotypes (Navaei et al., 2016b).

#### Chemical Crosslinking Methods

Chemically crosslinked hydrogels have a better performance at stability than physically crosslinked hydrogels due to stronger binding energy and substantially improved flexibility. Hydrogelforming water-soluble polymers have many functional groups, such as OH, COOH, and NH2. 3D network can be established by covalent bonding between these functional groups using glutaraldehyde and EDC/NHS (Balakrishnan et al., 2013; Omobono et al., 2015; Cheaburu Yilmaz et al., 2017). However, the toxicity of crosslinking agents and process limit the TE applications. For example, small molecular crosslinkers like glutaraldehyde and carbodiimides have been reported to be toxic and are not recommended to fabricate cell-laden hydrogels (Balakrishnan, 2016). To meet the requirements for TE, several available strategies for functional hydrogel preparation will be discussed below.

#### **Photopolymerization**

Photopolymerization has been widely used for hydrogel fabrications due to its biocompatibility and spatiotemporal controllability. Usually, macromers are modified with photoreactive moieties, such as methacrylate or acrylate groups. The photoreactive macromer solution with photoinitiator can be crosslinked under UV or visible light. The photoinitiators can generate free radicals that are transferred to the photoreactive carbon double bond groups in the modified macromers to start chain polymerization. However, under high photo exposure, photoinitiator will generate a large number of free radicals which may react with intracellular molecules to induce cell damage. This problem can be addressed by decreasing the light energy and amount of photoinitiator (Bryant et al., 2000; Fedorovich et al., 2009). High density of methacrylate groups has been reported to protect encapsulated cells by quenching free radicals (Bartnikowski et al., 2015). The method can be used for the preparation of hydrogels from various kinds of polymers and a variety of cell types can be encapsulated into the hydrogels for TE applications (Park et al., 2003; Chen et al., 2018). One of the most frequently used macromers is GelMA, which retains cell adhesive peptide sequence (arginine-glycine-aspartic acid, RGD) and matrix metalloproteinase (MMP) degradable peptide (Yue et al., 2015).

#### **Enzyme-enabled crosslinking**

Enzyme-enabled crosslinking is highly selective for a specific enzyme and can be achieved under mild physiological conditions (Ulijn, 2006). Transglutaminase and horse radish peroxidase are two commonly used enzymes. Transglutaminase catalyzes trans-amidation reaction that introduces N ǫ-(γ- glutamyl)lysine cross-links in proteins, converting protein solution into 3D hydrogel network (Chen et al., 2003). Horse radish peroxidase can build networks between polymers by oxidative coupling of hydroxyphenylpropionic acid moieties (Wang et al., 2014). Besides selectivity, this enzyme-mediated crosslinking approach also exhibits rapid gelation and easily tunable mechanical properties by varying the concentration of horse radish peroxidase and H2O2 (Ren et al., 2015).

#### **Click chemistry**

Click chemistry has the characters of high efficiency, high yield, and proceeding with no side products, which make the method wildly studied. TE application requires the reaction to proceed under physiological conditions. The typical click reaction is thiol-ene radical reaction which is hindered by inhibitory capacity of oxygen and shows complicated volume relaxation and stress development compared with classical radical photopolymerization (Hoyle and Bowman, 2010). Other representative chick chemistry includes Diels-Alder reaction, azide-alkyne cycloaddition chemistry (Xu and Bratlie, 2018) and Michal addition. Strain-promoted azide-alkyne cycloaddition (SPAAC) click chemistry has drawn increasing attention recently due to the mild reaction conditions and bioorthogonality (Xu et al., 2014). An injectable and degradable PEG-based hydrogel has been prepared via the bioorthogonal SPAAC click chemistry (Jiang et al., 2015). Michael addition reaction is the nucleophilic addition of a carbanion or a nucleophile to an α, β-unsaturated carbonyl compound. Michael addition-mediated hydrogels can be prepared under physiological conditions, making this kind of hydrogels injectable (Sun et al., 2017). For example, HA with thiol functional groups can form a 3D network with PEG vinysulfone macromers via Michael addition. The gelation time can be controlled by the degree of functionalization and the ratio of the two functional groups (Jin et al., 2010).

#### **Schiff base reaction**

Schiff base reaction has been widely used to form hydrogels via the coupling between aldehyde and amine groups in polymer chains. These hydrogels have been reported to have self-healing capacity due to the dynamic equilibrium of the linkages. For example, glycol chitosan and benzaldehyde functioned PEG have been synthesized and used to form a hydrogel with self-healing property for central nervous regeneration (Tseng et al., 2015). Proliferation rate and differentiation tendency of neurospherelike progenitors are enhanced in the self-healing hydrogel.

The above-described materials and crosslinking reactions have their respective merits and demerits. While, rational combination of natural and synthetic polymers or different crosslinking methods may afford an optimized approach to improve the hydrogel functions. For example, enzymatic crosslinking and Diels-Alder click chemistry can be induced into HA/PEG hydrogels for cartilage TE (Mahadevaiah et al., 2015). Both biocompatibility and mechanical property are improved by blending of HA and PEG polymers. The enzymatic crosslinking provides a suitable gelation speed for injectability, while the click reaction generates second crosslinking that renders an outstanding shape memory and anti-fatigue property. All these characters are required for cartilage TE.

# CONTROL OF PHYSICAL PROPERTIES

Physical properties of hydrogels, such as mechanical strength, stiffness, stress relaxation, self-healing, and degradation, can be controlled at different levels to meet the specific requirements for TE. These physical properties have obvious effects on cell functions, and thus should be investigated and reviewed. The hydrogels prepared with different physical properties, and their effects on cell functions, as well as their applications for TE are summarized in **Table 2**.

# Mechanical Strength and Stiffness

Conventional hydrogels normally possess breakable characters that will decrease their stability and thus cannot be utilized for specific tissue application such as bone, cartilage and tendon. To overcome this issue, two effective strategies have been developed. One is the hybridization of hydrogels with other polymers, nanoparticles or nanofibers. For example, regenerated silk fibroin and chitin nanofiber have been used to improve the mechanical strength of GelMA hydrogels by β-sheet folding and selfassembly, respectively. The hydrogel elastic modulus increases by one-thousand folds and strain-to-failure enhances by around 200% after chitin nanofiber assembly (Hassanzadeh et al., 2016). The hydrogels also demonstrate good cell viability, promotive cell differentiation and stable vasculature formation. Collagen-based hydrogels with a 10-fold increase in stiffness have been realized after mixing with very low amount of chemical functionalized nanoparticles working as crosslinker epicenters to make collagen chains crosslinked on the surface of nanoparticles (Jaiswal et al., 2015). Due to the interaction between nanoparticles and polymer chains, the mechanical properties of hybrid hydrogels can be enhanced. The other strategy is to prepare interpenetrating polymer network (IPN) hydrogels with high resistance to wear and high fracture strength, which has gained a lot of attention recently (Dragan, 2014). Double networks (DN) are introduced in hydrogels to enhance mechanical property for cartilage TE (Gong et al., 2003; Yasuda et al., 2009; Fukui et al., 2014). The feature of DN hydrogels is the formulation of first densely crosslinked hydrogel and second loosely network. The first network serves as sacrificial bonds to disperse the stress, while the second polymer chains work as hidden length that can extend to sustain large deformation (Haque et al., 2012). Similarly, ionic crosslinked chitosan with low molecular weight is used to work as the second crosslinking component to enhance the mechanical strength of the UV initiated PAM hydrogel (Yang et al., 2018).

Macroscopically, the mechanical property is important to maintain the scaffold stability to bear loads and fulfill the defects. Meanwhile, at microscopic level, the mechanical signals play a critical role in affecting cell activities and fates. For example, matrix stiffness has been reported to affect cell spreading, migration, proliferation and differentiation (Wen et al., 2014). Stiffness of hydrogels can be tuned by changing the crosslinking density, crosslinker length and molecular weight of the precursors (Slaughter et al., 2009; Li et al., 2016, 2017b). Differentiation of mesenchymal stem cells (MSCs) cultured on 2D matrix surface depends on substrate stiffness (Engler et al., 2006). MSCs cultured in hydrogels with stiffness of lower (0.1– 1 kPa), intermediate (8–17 kPa) or higher ranges (34 kPa) can differentiate into neural, myogenic or osteogenic phenotypes, respectively. However, the microenvironment provided to the cells in 3D culture differs from that in the traditional 2D system (Baker and Chen, 2012). Osteogenic differentiation of murine MSCs is enhanced in RGD modified alginate, agarose and PEG diacrylate (PEGDA) with intermediate stiffness (11–30 kPa) (Huebsch et al., 2010) (**Figure 1A**). Influence of stiffness on chondrogenic, vascular and neural differentiation has also been studied (Banerjee et al., 2009; Bian et al., 2013; Mahadevaiah et al., 2015). For instance, to investigate the influence of stiffness on the maintaining of chondrocyte phenotype, GelMA hydrogels with different stiffness but the same RGD density are prepared by changing the degree of methacryloyl substitution while using the same GelMA concentration. Bovine articular chondrocytes are encapsulated and cultured in the GelMA hydrogel with low, medium and high stiffness. The chondrocytes encapsulated in

high stiffness hydrogel exhibit round cell morphology and high chondrogenic gene expression, while chondrocytes cultured in low stiffness counterparts show elongated morphology and low chondrogenic gene expression (Li et al., 2016).

(iii). Scale bars are 25µm. Reproduced with the permission from Chaudhuri et al. (2016), Copyright © 2016, Springer Nature.

#### Stress Relaxation

Stress relaxation is another important mechanical property for hydrogels, which also is a common behavior of tissue matrix. It has been reported that ionic and covalent hydrogels both exhibit stress relaxation through uncoupling-reforming of crosslinks and migration of water, respectively (Zhao et al., 2010). Ionic hydrogels have faster stress relaxation and easier modulation than that of covalent hydrogels. Sodium alginate has been commonly used to form hydrogels with tunable stress relaxation. In a previous study, RGD modified alginate has been used to prepare soft but highly stress-relaxing substrate. Cells cultured in this substrate spread similarly to the cell on the surface of stiff elastic substrates (Chaudhuri et al., 2015). The stress relaxation of covalently crosslinked PAM hydrogel, physically crosslinked collagen hydrogel and living tissues like adipose, liver, brain and an initial fracture haematoma are compared by measuring the stress change when fixing the strain at 15% (Chaudhuri et al., 2016). The results indicate that the stress relaxation of collagen hydrogel is faster than that of living tissues and PAM hydrogel, while that of PAM is slowest. RGD coupled alginate with different molecular weights and PEG spacers are used to prepare hydrogels with different stress relaxation properties independent of initial elastic modulus and matrix degradation. Fast stress relaxation enhances cell spreading, proliferation and osteogenic differentiation of stem cell through integrin-RGD binding and clustering (**Figure 1B**). Stress relaxation has also been demonstrated to have the capability to alter chondrocyte phenotype and matrix deposition via modulating cell volume expansion (Lee et al., 2017). Slow relaxing environment restricts cell volume, leading to interleukin-1β secretion increase, which in turn up-regulates the genes related to matrix degradation and cell death. To fully mimic the mechanical and structural cues of native ECM, HA crosslinked with dynamic covalent bonds and fibrillar collagen type I are used to prepare IPN hydrogel with tunable stress relaxation (Lou et al., 2018). Cell spreading, fiber remodeling and focal adhesion formation are enhanced in the faster relaxation hydrogels.

# Self-Healing

Self-healing hydrogel is able to intrinsically and automatically heal the breaks, making itself back to original shape and mechanical property. This property is directly based on the reversible or dynamic crosslinking chemistry (Taylor and in het Panhuis, 2016). One of the representative self-healing hydrogels is prepared by glycol chitosan and benzaldehyde functionalized PEG via Schiff-base reaction (Tseng et al., 2015). The proliferation rate and differentiation tendency of neurosphere-like progenitors cultured in this self-healing hydrogel are enhanced (**Figure 2A**). Thrombin crosslinked fibrinogen can build a 3D network structure, which is used to prepare IPN structure after mixing with the above-mentioned chitosan-PEG hydrogel (Hsieh et al., 2017). This hydrogel encapsulated with vascular endothelial cells shows excellent self-healing and capillary-inducing capacity. Injection of this hydrogel promotes angiogenesis in zebrafish and mice. Dynamic acylhydrazone bond and Diels-Alder click covalent crosslinking are combined to prepare self-healing hydrogels with desirable mechanical property (Lü et al., 2017). These hydrogels increase the viability, reduces apoptosis of MSCs, and enhances bone regeneration in cranial bone defects. Guest-host crosslinking has also been used to prepare self-healing hydrogels. Cyclodextrin conjugated in one polymer backbone can work as the host to engulf hydrophobic moieties of another polymer chain to achieve self-healing. This self-healing property can further be selectively improved when the stimuli-responsive moiety, such as polyacrylamide-ferrocene with a reversible hydrophobic-charge transition at reduced/oxidized state, is included in the above hydrogels (Nakahata et al., 2011).

# Degradation

Covalently crosslinked hydrogels can undergo degradation through ester hydrolysis, enzymatic hydrolysis or photolytic cleavage of the polymer chains (Kharkar et al., 2013). Based on these mechanisms, hydrogels could be designed with good biodegradability and desirable degradation rate, working as temporary supports and being degraded and replaced by the regenerating tissues gradually (Bryant et al., 2004). For example, the photocrosslinkable PVA/PEG hybrid hydrogels with controlled degradation profiles have been used for cartilage TE.

of difunctionalized PEG (DF-PEG) with glycol chitosan (i). The expressions of neuronal-related genes (nestin, β-tubulin, and Map2) of cells after 3 and 7 days of culture in 3D gels, \**p* < 0.05 (ii). Reproduced with the permission from Tseng et al. (2015), Copyright © 2013, John Wiley and Sons. (B) (i) Schematic of sequential crosslinking of MeMaHA using a primary addition and secondary radical polymerization to create -UV and D0 UV hydrogels. (ii) Percentage of hMSCs toward osteogenic or adipogenic differentiation in -UV or D0 UV hydrogels (#*p* < 0:005, *t*-test). Reproduced with the permission from Khetan et al. (2013), Copyright © 2013, Springer Nature.

The degradation time can be tuned from less than 1 to 34 days by altering the ratio of PVA to PEG. The results show that the DNA and GAG contents increase with culture time and the neocartilaginous tissue at 6 weeks is homogeneously distributed in the PVA/PEG hydrogel with the ratio of 1:3, indicating the importance of degradation for TE (Martens et al., 2003). When implanted in vivo, HA-based hydrogel often shows too fast degradation to meet the requirement for cartilage tissue repair. To solve this limitation, sulfate groups are conjugated to HA to decelerate the hyaluronidase degradation rate. Sulfated HA displays slow degradation and enhances protein binding ability, promoting chondrogenesis of hMSCs with reduced hypertrophy (Feng et al., 2017).

Degradation of the hydrogel is a chemical process, but it can work as a dynamically physical stimulus to affect cell behaviors and functions for cell-matrix interaction, such as cell spreading, migration, proliferation, and differentiation, to further mimic the native ECM and improve the tissue regeneration. For example, photo-responsive hydrogels can be prepared from PEG derivatives containing nitrobenzyl ether photolabile moieties. When human mesenchymal stem cells (hMSCs) are cultured in the photodegradable hydrogel, cell spreading is enhanced after light exposure (Kloxin et al., 2010). Degradation of hydrogel is always accompanied with decrease of stiffness, making it complicated to discriminate the influences of degradation and stiffness. To decouple the influence from mechanical property of degradable hydrogels, hydrogels are designed to degrade while their mechanical properties remain unchanged. For example, HA functionalized with both maleimide and methacrylate is used to prepare hydrogels by thiol-ene crosslinking via MMP degradable crosslinker and photo-initiated crosslinking of the methacrylate. The MMP-cleavable peptide crosslinker permits cell-mediated degradation. The photo-initiated crosslinking can dynamically control the secondary crosslinking at the very beginning or after a period of cultivation. The stiffness of hydrogels with or without UV exposure is controlled at the same level by changing the molecular weight of HA macromers. When hMSCs are cultured in the tuned hydrogels, they exhibit different osteogenic and adipogenic differentiation. The results indicate that hMSCs differentiation is directed by degradation-mediated cellular traction, independent of the matrix mechanics. (Khetan et al., 2013) (**Figure 2B**).

# CONTROL OF CHEMICAL PROPERTIES

The composition of hydrogels can highly affect the cell behaviors including viability, adhesion, spreading, proliferation, and differentiation (Ruoslahti and Pierschbacher, 1987). Hydrogel composition can be controlled by choosing different precursors and preparation methods. The methods used to tune chemical properties of functional hydrogels are summarized in **Table 3**.

Cell adhesion ligand is a crucial biochemical component in ECM, because many types of cells have to adhere to their microenvironment through cell surface integrin to perform the relative functions and maintain their viability (Huettner et al., 2018). Naturally derived proteins, like collagen and gelatin, retain many cell adhesion ligands. However, polysaccharidebased natural materials and synthetic polymers are lack of these ligands. Hence, bioactive peptide modification has been used to improve the biochemical property of non-adhesive hydrogels. RGD peptide, as one of the potent adhesion ligands, plays an important role on cell adhesion and other functions. To study the impact of dynamic presentation of RGD in matrix on cell functions, a light-responsive PEGDA hydrogel is synthesized, in which a protecting group is used to temporally and spatially control the RGD presentation by transdermal light exposure (**Figure 3A**). PEGDA hydrogels

TABLE 3 | Control of chemical properties of hydrogels for TE applications.

with RGD peptides and light-induced uncaged RGD peptides support high number of adherent cells. On the other hand, PEGDA hydrogels without RGD modifications or with caged RGD support few adherent cells and the cells have round morphology. Besides, it has been reported that RGD peptides can be introduced to polymers, such as HA (Lee et al., 2015), alginate (Sun et al., 2017), chitosan (Kim et al., 2016), and PEG (Long et al., 2017), by chemical binding to fabricated bioactive hydrogels. These functionalized hydrogels show improved biological properties to promote cell adhesion, spreading, proliferation, and differentiation. For instance, RGD peptides promote the survival of MSCs in PEG hydrogels and induce the chondrogenic differentiation (Salinas and Anseth, 2008). RGD ligands in hydrogels increase the chondrogenic gene expression when the hydrogel matrices are loaded with dynamic mechanical force (Steinmetz and Bryant, 2011). Moreover, RGD density affects the redifferentiation of chondrocytes (Schuh et al., 2012).

Hybridization with natural polymers is another facile way to tune the biochemical property of hydrogels, which can change the composition of hydrogels in favor of promotion of cell function and TE. For example, GelMA is used to hybridize with PAM to prepare a photopolymerized hydrogel for cartilage tissue regeneration. Cells cultured in the hybrid hydrogel exhibit higher viability and proliferation rate than those cultured in pure PAM hydrogel (Han et al., 2017). What's more, HA/PEG hybrid hydrogel prepared via the SPAAC crosslinking shows excellent biocompatibility (Fu et al., 2017), which may be due to the fact that HA can bond to some receptors on cell membrane to affect both chondrocyte survival pathway and apoptotic pathway (Knudson and Knudson, 2004).

Besides hybridization with natural polymers or modification with biological moieties to improve the biochemical properties of hydrogels for TE, loading of growth factors and nanoparticles have been frequently adapted to improve hydrogel functions. Growth factors play important roles in cell growth, cell function determination, tissue regeneration, and organ development (Parker et al., 2016; Yan et al., 2018). Therefore, immobilization of growth factors in hydrogels will significantly improve the functionality of hydrogels. There are two strategies for

immobilizing growth factors in hydrogels, namely physical, and chemical immobilization (Nguyen and Alsberg, 2014). Heparin can bind many types of growth factors through the strong electrostatic interactions. The immobilization of fibroblast growth factor-2 and vascular endothelial growth factor in heparin-modified PEG hydrogels has been shown to boost angiogenesis (Zieris et al., 2010). Compared with physical immobilization, chemical immobilization can further improve the stability, prolong the release of growth factors, and reduce the required amount. For example, transforming growth factorbeta 1 (TGF-β1) is covalently tethered to PEG hydrogel through thiol-ene reaction (Sridhar et al., 2014; Mao et al., 2017). Chondrocytes cultured in PEG hydrogel immobilized with TGF-β1 show higher DNA content and chondrogenic matrix production than the cells cultured in PEG hydrogel with soluble TGF-β1 (**Figure 3B**).

A variety of nanoparticles have also been incorporated into different kinds of natural or synthetic polymer networks to prepare nanocomposite hydrogels for TE. The incorporation of nanoparticles can provide not only higher mechanical properties, but also the possibility to tune biochemical characteristics of the 3D network. For examples, nanosilicates are incorporated into GelMA hydrogels to obtain a bioactive nanocomposite (Xavier et al., 2015). Not only the mechanical strength of the hydrogel is enhanced, osteogenic differentiation of preosteoblasts is also promoted (**Figure 4**). In another study, nanosized hydroxyapatite is incorporated into a PEG hydrogel aiming to produce highly tough matrix for bone TE. After incorporation, the morphologies of these hydrogels show highly interconnected porous structures. What's more, the presence of hydroxyapatite nanoparticles can provide osteoblast cells adhesion and bioactive attachment sites compared with pure PEG hydrogels (Gaharwar et al., 2011). For cardiac tissue regeneration, conductive nanoparticles like carbon nanotubes are added into GelMA hydrogels, resulting in improved cell adhesion and maturation (Shin et al., 2013). The percentage of cell retention and viability on carbon nanotubes nanocomposite GelMA hydrogel is higher than those on pristine GelMA. The mature cell/hydrogel sheet with very good electrophysiological and mechanical properties exhibits strong spontaneous and stimulated synchronous beating.

# STRUCTURAL CONTROL OF HYDROGELS

Microstructure is another critical factor to affect cell activities and functions because the optimized architecture of engineered tissue has the function to organize multiple cell types for TE (Stevens et al., 2013). Hydrogels with microarchitectures are briefly classified into microporous, channel-bearing, double-ring, multilayered, and hierarchically structured ones. The structure types of functional hydrogels and their influence on cell functions and tissue regeneration are summarized in **Table 4**.

# Microporous Hydrogels

The structure of bulk hydrogels is dense polymers with absorbed water and nano-sized pores within the network (Hoffman, 2012), in which, the nano-sized pores are too small to promote cell migration, proliferation and ECM diffusion. Thereby,

S staining) (B). Reproduced with the permission from Xavier et al. (2015), Copyright © 2015, American Chemical Society.

TABLE 4 | Structural control of hydrogels and TE applications.

microporous structures have been proposed. The effects of different pore structures and pore size ranges have been studied for cell culture and TE (Zhang et al., 2014; Chen et al., 2016). Various technologies including solvent casting, particle leaching, freeze-drying, and gas foaming can be used to adjust hydrogel porosity (Annabi et al., 2010). Among them, stimuli-responsive porogens, such as gelatin, alginate and HA, are used to create cell-laden hydrogels with tunable porosity. These porogens can be removed by specific stimuli including temperature, chelating, and enzymatic digestion, respectively (Han et al., 2013). Similarly, gelatin microspheres fabricated by water/oil emulsion are used to generate micropores in alginate (Leong et al., 2016) and HA (Fan and Wang, 2015) hydrogels. Plenty of micropores are left after gelatin microbeads are dissolved at 37◦C. Chondrocytes cultured in the porous hydrogels show high proliferation and ECM secretion. Gelatin microparticles have also been used to prepare microporous hydrogels for bone TE (Vo et al., 2016). The pore-forming gelatin microparticles can be used not only for generation of micropores, but also for introduction of living cells in the micropores. Cell-laden gelatin microcubes are prepared by a mesh-cutting method and used to prepare microporous hydrogels with promotive capacity of cell proliferation (Li et al., 2017a). Chondrocytes cultured in the microporous hydrogels show high proliferation and cells prefer to migrate into the microporous cavities (**Figure 5A**).

#### Channel-Bearing Hydrogels

Cells must reside with 100–200µm from adjacent capillary blood vessels to remain viable or else they will undergo necrosis due to insufficient oxygenation and nutrition diffusion (Carmeliet and Jain, 2000). Therefore, hydrogels with micro-channels are highly needed to vascularize artificial tissue or study cell behaviors in a vascular structure, in particular for the regeneration of large and complex tissues and organs. Multiple strategies and techniques have been developed for preparation of channel-bearing hydrogels for TE applications.

PVA-based sacrificial templates are fabricated into branched fluidic architectures by casting (Tocchio et al., 2015). The architecture obtained after washing exhibits promoted regeneration of hierarchically branched endothelium and high cell viability inside bulk hydrogels formed by 2 hydroxyethyl methacrylate, agarose or GelMA. To prepare a complex capillary-like micro-channel vascular structure, solvent-spun PNIPAM microfibers are fabricated and used as temperature-responsive templates (Lee et al., 2016). The PNIPAM microfibers are spun and maintained at a temperature above 32◦C. Cell-suspended gelatin precursor solution is kept at 37◦C prior to embedding the templates. After complete gelation of the gelatin hydrogel, the PNIPAM microfibers are removed by immersion in cell culture medium at room temperature to generate the capillary-like micro-channel vascular structure. Cell viability is higher than 96% during 7 days of cultivation in the perfused micro-channel hydrogel. Carbohydrate-glass microfibers are printed to induce vascular architecture in bulk hydrogels, which can be removed by cell medium perfusion (Miller et al., 2012). The micro-channel hydrogels demonstrate good biocompatibility and enhanced nutrition diffusion. Combination of carbohydrate glass material with 3D printing provides independent control of vascular network

microporous structures. (i). Live/dead staining of chondrocytes in the hydrogels after UV crosslinking (0 week) and after 28 days of *in vitro* culture (4 weeks) (ii). Scale bar: 200 mm. Reproduced with the permission from Li et al. (2017a), Copyright © 2017, Royal Society of Chemistry. (B) Fabrication of hydrogel with channel structure by dissolving 3D printed carbohydrate-glass lattice (i). Representative live/dead image of HUVEC and 10T1/2 co-cultured in the interstitial space of a fibrin gel (ii). Cells survive and spread near open channels (highlighted with white arrow). Scale bar: 200µm. Reproduced with the permission from Miller et al. (2012) Copyright © 2012, Springer Nature.

geometry, endothelialization, and extravascular tissue formation (**Figure 5B**).

### Double-Ring Structural Hydrogels

Osteon-like concentric double-ring structure is prepared with hybrid hydrogels of GelMA and hydroxyapatite via photolithography technology and self-assembly (Zuo et al., 2015). Human umbilical endothelial cells (HUVECs) are encapsulated in the inner ring to mimic blood vessel tubules, while human osteoblast-like cells (MG63s) are located in the outer ring as bone part (**Figure 6A**). Expression of angiogenesisrelated and osteogenesis-related genes is promoted. Osteon-like fibers with HUVECs and MG63s separately encapsulated are constructed with the combination of photolithography and microfluidic chip techniques (Wei et al., 2017). RGD modified alginate precursor with photoreactive functionalization is used for the hydrogel preparation because the modified alginate possesses cell adhesive sites and UV/Ca2+ crosslinkable properties. This not only satisfies cell proliferation but enhances the relative gene expression and protein secretion in the osteon-like architecture.

#### Multilayered Hydrogels

ECM composition and structure of cartilage and bone tissues have an obvious difference. The biochemical and biophysical microenvironments surrounding the cells in cartilage and bone are also different. To regenerate full-thickness articular cartilage defects, bilayered hydrogel architecture is designed.

double-ring structure by photolithograph and self-assembly. Cell viability of MG63s and HUVECs encapsulated in the outer ring and inner ring of the osteon-like module. Reproduced with the permission from Zuo et al. (2015), Copyright © 2015, American Chemical Society. (B) Degradation of the hydrogel through oNB photocleavage (i). Creation of hydrogels with hierarchical vascular structure by programmable photodegradation (ii). Reproduced with the permission from Arakawa et al. (2017), Copyright © 2017, John Wiley and Sons.

For example, bilayered agarose hydrogels are prepared for osteochondral tissue repair (Sheehy et al., 2013). The top hydrogel layer is encapsulated with chondrocytes, while the bottom layer is seeded with MSCs. After 49 days of in vitro cultivation, the top layer promotes production of cartilaginous ECM, while the hypertrophy of MSCs encapsulated in the bottom layer is decreased. The results indicate the bilayered structure has a great potential for spatially separated coculture of chondrocyte and MSCs and osteochondral tissue regeneration. Bilayered structure can also provide tunable biochemical and mechanical microenvironment for differentiation of MSCs. For example, the top hydrogel layer fabricated with low RGD concentration and soft matrix can promote chondrogenesis of MSCs, while, the bottom layer designed to have high RGD amount and stiff mechanical property can promote osteogenesis of MSCs (Steinmetz et al., 2015).

#### Hierarchically Structured Hydrogels

Hierarchical architecture exists in living tissues. For example, blood vessels possess many scales of size to adapt to tissue requirements. They include large arteries, smaller arterioles and smallest capillaries, which have the functions to permit large blood volume flow and support mass transport via diffusion (Michiels, 2003). The above mentioned methodologies for channel structures have limitations to introduce such hierarchical structures in cell-laden hydrogel constructs, especially for the size of diameter less than 150 µm (Lee et al., 2014). Multiphoton lithography-assisted photoscission, as a cytocompatible fabrication strategy, is used to create hierarchical vessels spanning nearly all the size ranges in the human body (Arakawa et al., 2017) (**Figure 6B**). The hydrogel network is crosslinked by azide-alkyne cycloaddition between PEG tetrabicyclononyne and diazide-functionalized peptide. The photo degradation is based on the orthonitrobenzyl ester (oNB) moiety that undergoes photolysis upon exposure to pulsed near-infrared light (DeForest and Tirrell, 2015). Human bone marrow-derived hS5 stromal cells are encapsulated inside the hydrogel, followed by preparation of photodegradable vessels. The hS5 cells are viable during the whole culture period. This complex and hierarchical structures can maintain cell viability and functions, which is essential for regeneration of heterogeneous tissues. Furthermore, the hierarchical vascular structure is promising for investigation of endothelialization and blood-capillary interaction in the various environments.

3D stamping technique can also be used to prepare hydrogels with hierarchical structures. AngioChip with hierarchical vascular and porous architecture is prepared by this method

#### REFERENCES

Abbadessa, A., Blokzijl, M. M., Mouser, V., Marica, P., Malda, J., Hennink, W., et al. (2016). A thermo-responsive and photo-polymerizable chondroitin sulfatebased hydrogel for 3D printing applications. Carbohydr. Polym. 149, 163–174. doi: 10.1016/j.carbpol.2016.04.080

(Zhang et al., 2016). Nanopores and micro-holes are fabricated into the vascular walls to promote molecular exchange and cell migration. The permeability for large molecular (70 kDa TRITC-dextran) of AngioChip with micro-holes is more than 4 times higher than that of the counterparts without micro-holes. A confluent endothelium is exhibited on the microchannel surface and endothelial sprouts are observed through the micro-holes. Human embryonic stem cell-derived hepatocytes and hMSCs are encapsulated in a collagen matrix and seeded into the parenchymal space to engineer the AngioChip. Urea secretion per cell from this hierarchical construct is much higher than that of collagen sandwich control. Furthermore, this complex hierarchical structural AngioChip is also used for in vitro regeneration of cardiac tissue models and implantation for direct surgical anastomosis. Due to the complex and hierarchical architectures, cell viabilities and relative functions are enhanced.

# CONCLUSION AND OUTLOOKS

Control of the structures and physiochemical properties of hydrogels is important for their utilization in TE. Cells first sense the signals from surrounding hydrogel matrix, which makes the physiochemical properties of hydrogel critical for cell functions. Various structures of hydrogels can further alter the microenvironments and provide the possibility of flow stimulus by micro-channel structure or co-culture microenvironment by spatially layered structures. A variety of preparation methods and functional hydrogels have been developed for TE of various tissues and organs. Despite of these progresses, hydrogels with further improved biochemical and biophysical properties, as well as biomimetic structures are deserved to be further studied. The explored hydrogels until now have some similar but not completely matched functions to the in vivo cell-surrounding microenvironments. Dynamically responsive biophysical and biochemical stimuli and spatiotemporally controlled architecture may become the main direction of design and preparation of functional hydrogels to realize the complete functionality for TE applications.

# AUTHOR CONTRIBUTIONS

All authors listed have made a substantial, direct and intellectual contribution to the work, and approved it for publication.

# ACKNOWLEDGMENTS

This work was supported by JSPS KAKENHI Grant Number 15H03027 and 18K19947.

Annabi, N., Nichol, J. W., Zhong, X., Ji, C., Koshy, S., Khademhosseini, A., et al. (2010). Controlling the porosity and microarchitecture of hydrogels for tissue engineering. Tissue Eng. B Rev. 16, 371–383. doi: 10.1089/ten.teb.2009.0639

Arakawa, C. K., Badeau, B. A., Zheng, Y., and DeForest, C. A. (2017). Multicellular vascularized engineered tissues through user-programmable biomaterial photodegradation. Adv. Mat. 29:1703156. doi: 10.1002/adma.201703156

for perfusable engineered three-dimensional tissues. Nat. Mater. 11:768. doi: 10.1038/nmat3357

articular cartilage matrix. J. Mech. Behav. Biomed. Mater. 36, 32–46. doi: 10.1016/j.jmbbm.2014.04.003

**Conflict of Interest Statement:** The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Copyright © 2018 Li, Sun, Li, Kawazoe and Chen. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

# Preparation and Characterization of Chitosan/β-Glycerophosphate Thermal-Sensitive Hydrogel Reinforced by Graphene Oxide

#### Han Qin, Jian Wang, Tong Wang, Xiaomeng Gao, Qianbing Wan\* and Xibo Pei\*

State Key Laboratory of Oral Diseases, Department of Prosthodontics, National Clinical Research Center for Oral Diseases, West China Hospital of Stomatology, Sichuan University, Chengdu, China

#### Edited by:

Baolin Guo, Xi'an Jiaotong University, China

#### Reviewed by:

Yaobin Wu, Southern Medical University, China Artur J. M. Valente, University of Coimbra, Portugal

#### \*Correspondence:

Qianbing Wan<EMAIL_ADDRESS>Xibo Pei<EMAIL_ADDRESS>
#### Specialty section:

This article was submitted to Polymer Chemistry, a section of the journal Frontiers in Chemistry

Received: 29 August 2018 Accepted: 31 October 2018 Published: 22 November 2018

#### Citation:

Qin H, Wang J, Wang T, Gao X, Wan Q and Pei X (2018) Preparation and Characterization of Chitosan/β-Glycerophosphate Thermal-Sensitive Hydrogel Reinforced by Graphene Oxide. Front. Chem. 6:565. doi: 10.3389/fchem.2018.00565 Thermal-sensitive hydrogel based on chitosan (CS) and β-glycerophosphate (GP) has shown good biocompatibility and biodegradability. But the application of such hydrogel is limited due to its poor mechanical property. Recently, graphene oxide(GO) is widely used as a reinforcement agent to prepare nanocomposites with different polymers for improving the properties of the materials. In this study, CS/GP-based hydrogels with different weight ratio of GO/CS (0.5, 1, 2%) were fabricated. The gelation time of the hydrogels at body temperature was evaluated by tube inverting method. The gelation process during heating was monitored by rheological measurement. The morphology, porosities, chemical structure, swelling properties of the lyophilized hydrogels were investigated by scanning electron microscopy, liquid displacement method, Fourier transform infrared spectroscopy and gravimetric method. Mechanical property of the hydrogels was analyzed by rheological measurement and unconfined compression test. MC3T3-E1 mouse pre-osteoblast cell line was used to assess the biological properties of the hydrogels. The results obtained from those assessments revealed that the addition of GO into CS/GP improved the properties of the prepared hydrogels without changing the high porous and interconnected microstructure and swelling ability of the hydrogels. The gelation time at body temperature was significantly reduced by nearly 20% with the addition of small amount of GO (0.5% weight ratio of CS). The mechanical properties of the hydrogels containing GO were improved significantly over that of CS/GP. The storage (G′ )/loss (G′′) moduli of the hydrogels with GO were 1.12 to 1.69 times that of CS/GP at the gelling temperature. The Young's modulus of 0.5%GO/CS/GP hydrogel is 1.76 times that of CS/GP. Moreover, the 0.5%GO/CS/GP hydrogel revealed remarkable biological affinity such as cellular attachment, viability and proliferation. All of these results suggest that 0.5%GO/CS/GP hydrogel has great potential for practical application in biomedical field.

Keywords: thermal-sensitive, hydrogel, chitosan, β-glycerophosphate, graphene oxide

# INTRODUCTION

Hydrogels have some unique physical properties, which make them widely used in biomedical fields such as tissue engineering, wound dressing and drug delivery systems (Bhattarai et al., 2010). A good candidate to form hydrogel is chitosan (CS), a copolymer of β[1,4]-linked 2-acetamido-2-deoxy-Dglucopyranose and 2-amino-2-deoxy-D-glucopyranosea. This biopolymer with polycationic structure is generally obtained by alkaline deacetylation from chitin which is the second most abundant polysaccharide in nature after celluse (Rinaudo, 2006). Because of its non-toxicity, biocompatibility, biodegradability, antibacterial ability, and low immunogenicity, chitosan has attracted large interest in biological application such as tissue engineering, drug delivery and wound healing (Riva et al., 2011; Zhao et al., 2015, 2018; Dong et al., 2016). These admirable characteristics of chitosan is ascribed to its unique chemical structure. For example, the polysaccharide unit of chitosan resembles the structure of glycosaminoglycans (GAGs), which is the major component of the extracellular matrix (ECM) of bone and cartilage (Khor and Lim, 2003). The cationic character of chitosan enables it to form complexes with anionic molecules, including proteoglycans, GAGs, growth factors, receptors and adhesion proteins that help to regulate cellular activity (Lehr et al., 1992; Dutta et al., 2011). In addition, chitosan can create networks with highly porous and interconnected pore structure which is helpful for cell adhesion, proliferation, and differentiation as it enhances the diffusion of nutrients and provide room for neovascularization (Dutta et al., 2011). Hence, chitosan can be used to form hydrogels as scaffolds or substrate for tissue engineering.

Generally, chitosan hydrogels can be prepared by either covalently or ionically crosslinking methods with the addition of different crosslinkers. Covalently cross-linked hydrogels possess a stable and permanent network structure as irreversible chemical bonding are formed. However, covalent cross-linkers such as glutaraldehyde are usually toxic to organism. And the chemical crosslinking makes hydrogels hard to degrade. On the other hand, ionically cross-linked hydrogels are formed by reversible links and generally considered biocompatible, exhibiting a higher swelling sensitivity to pH changes compared with covalently cross-linked chitosan hydrogels (Berger et al., 2004). Because chitosan is a polycationic polymer, one of the anionic molecules—β-glycerophosphate (GP) can be used as an ionic crosslinking agent. It is an organic compound naturally found in the body and has been used as an osteogenic supplement for culturing human bone marrow stem cells (Zhou et al., 2015). Chitosan/β-Glycerophosphate (CS/GP) is an in situ thermal-sensitive gelling system, which maintains fluidity at low temperature (room temperature or below) and becomes gel at higher temperature (body temperature) (Berger et al., 2004; Zhou et al., 2015). With this characteristics, CS/GP hydrogel has attracted great attention as it does not require an surgical operation when implanted (Zheng et al., 2010). However, the usage of the material is usually limited due to its poor mechanical properties. Hence, many studies aimed at improving its mechanical functionality by blending CS/GP system with other nanomaterials, such as nano-attapulgite (Wang and Chen, 2016), nano-hydroxyapatite (Huang et al., 2011; Chen et al., 2016), nanosilver (Tsai et al., 2011), carbon nanotubes(CNTs) (Gholizadeh et al., 2017) and etc.

In addition to those nanofillers, a recently attractive material has yet not been studied as a reinforcing agent in CS/GP system graphene. Graphene is expected for application in various fields because of its exceptional physico-chemical properties (Zhu et al., 2010). It is considered as a promising nanofiller as it possesses high aspect ratio which is a very useful parameter for improving the properties of materials, such as thermal, electrical and mechanical properties (Stankovich et al., 2006; Cote et al., 2009; Salavagione et al., 2009; Vickery et al., 2009). Compared with its allotrope CNTs, graphene offers enhanced mechanical properties to the composite, due to the planar structure and the aspect ratio, which allows better stress transfer within the host medium during the loading process (Mittal et al., 2015). Moreover, graphenebased materials promote stem cell attachment and growth, and enhance osteogenic differentiation, indicating its application as an alternative material for bone regeneration research (Dubey et al., 2015). A widely used form of graphene is graphene oxide (GO), which is amphiphilic due to the multiple oxygencontaining groups such as hydroxyls, epoxides, and carboxyls on its surface (Li et al., 2008; Marcano et al., 2010). These functional groups (oxygen-containing) allow GO to disperse at an individual sheet and highly negatively charged in aqueous solution, which makes it possible to form special interaction between GO and protonated polymers such as CS. Meanwhile, these functional groups (oxygen-containing) as well as the large aromatic (πconfiguration) interface endow GO with the ability to interact with proteins, peptides, or DNA via chemical bonding or physical adsorption (Shin et al., 2016). Adding GO into CS to form films or scaffolds have been reported with improved properties, including mechanical strength in both wet and dry state, storage modulus, and thermal stability (Fan et al., 2010; Yang et al., 2010; Depan et al., 2011; Han et al., 2011; Dinescu et al., 2014). Because of the advantages of graphene and its derivatives and previous studies of CS and GO mentioned above, we considered adding graphene oxide into CS/GP system to improve the properties of CS/GP hydrogel.

In the present work, GO/CS/GP hydrogels with different GO/CS weight ratios were prepared as the amount of GO in nanocomposites plays an important role to the properties of materials. Meanwhile, the physical, chemical and biological properties of each hydrogel were evaluated. We expect to develop a composite hydrogel of GO/CS/GP with suitable mechanical properties while maintaining biological functionality for biomedical applications.

#### MATERIALS AND METHODS

#### Materials

β-Glycerophosphate disodium salt was obtained from Sigma-Aldrich Co.(US). Medium molecular weight chitosan(molecular weight≈21,0000 Da, deacetylation degree≥95%) was purchased from Tokyo Chemical Industry Co., Ltd. Acetic acid (chemical reagents of analytical grade) was provided by Baoxin Bio-Technology Co., Ltd. (Chengdu, China). Single-layered graphene oxide, purity>99%, diameter of 0.5–5µm, thickness of 0.8–1.2 nm was purchased from Nanjing/Jiangsu XFNANO Materials Tech Co., Ltd. (Nanjing, China).

#### Preparation of the Hydrogels