rapid fabrication of self-healing, conductive, and injectable ......self-healing hydrogels have...

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Rapid Fabrication of Self-Healing, Conductive, and Injectable Gel as Dressings for Healing Wounds in Stretchable Parts of the Body

Sixiang Li, Le Wang, Wenfu Zheng,* Guang Yang,* and Xingyu Jiang*Q1

Skin wounds on stretchable parts of the body including the elbows, knees, wrists, and nape usually undergo delayed and poor healing due to the interference of their frequent motion. Ordinary dressings that are not flexible enough face difficulty to promote wound healing due to the mismatching between the mechanics of the dressing materials and the wounds. In this study, an injectable, biocompatible, self-healable, and conductive material poly(3,4-ethylenedioxythiophene): poly(styrenesulfonate)/guar slime (PPGS) is developed for healing wounds with various kinds of movements. As a proof-of-principle assay, the healing effect of PPGS is explored on a skin wound model on the nape of rats which often experiences frequent move-ments. PPGS, which can be prepared within 1 min, successfully accelerates the healing of the wounds. The results suggest that PPGS has great potential in the fields of tissue engineering and biomedicine.

DOI: 10.1002/adfm.202002370

Dr. S. Li, Prof. G. YangNational Engineering Research Center for Nano-MedicineDepartment of Biomedical EngineeringCollege of Life Science and TechnologyHuazhong University of Science and TechnologyWuhan 430074, ChinaE-mail: [email protected]. S. Li, Dr. L. Wang, Prof. W. Zheng, Prof. X. JiangBeijing Engineering Research Center for BioNanotechnology and CAS Key Laboratory for Biomedical Effects of Nanomaterials and NanosafetyCAS Center for Excellence in NanoscienceNational Center for NanoScience and TechnologyNo. 11 Zhongguancun Beiyitiao, Beijing 100190, P. R. ChinaE-mail: [email protected]; [email protected]. S. Li, Dr. L. Wang, Prof. W. Zheng, Prof. X. JiangUniversity of Chinese Academy of Sciences19 A Yuquan Road, Shijingshan District, Beijing 100049, P. R. ChinaDr. S. Li, Dr. L. Wang, Prof. X. JiangDepartment of Biomedical EngineeringSouthern University of Science and TechnologyNo. 1088 Xueyuan Rd, Nanshan District, Shenzhen, Guangdong 518055, P. R. China

The ORCID identification number(s) for the author(s) of this article can be found under https://doi.org/10.1002/adfm.202002370.

reconstructing cutaneous function. Con-ventional dressings such as the gauze[2] need to be replaced frequently, which increase the risk for infection and bring secondary damage and pain.[3] The recently developed dressing materials, such as hydrogels, could potentially solve problems associated with wound healing: the secondary damage, the retention of exudate, bacterial infections, dehydration, wound ulcers, and scar formation.[4] How-ever, few studies have paid attention to the wounds occurred in the stretchable parts, for instance, knees, wrists, ankles, and nape (the back of the neck).[5] Due to the frequent activities of these wounds, the application of ordinary dressings without superior compliance and self-healing

capability may cause secondary injury, which inevitably leads to pain, prolonged healing time, and even disabilities.[6] The scar formed during abnormal wound healing will continue to influence the mobility even after healing.[7] Flexible hydrogels with self-healing properties can resolve these problems. Flex-ibility of dressings ensures the integrity of dressings on static wounds or wounds undergoing small movements. In wounds undergoing large movements, having just flexible property is not sufficient: self-healing property make sure spontaneous healing takes place when rupture occurs. Self-healing ability is often accompanied by injectability.[8] Facing an irregular shaped or deep wound, injectable hydrogel can easily fill wound sites.[9] The flexible, self-healing, and injectable materials can reduce the discomfort and pain, extend the life of the dressings, and keep the wounds in a natural way which undoubtedly can pro-mote the healing process.

Self-healing hydrogels have drawn much attention due to their unique properties[10] and self-healing hydrogels real-ized by one or serval types of gelation mechanism including hydrogen bonds,[11] Schiff bases,[5,12] ionic bonds,[13] and boronic ester bonds[14] are widely explored. However, the complicated preparation processes[15] or the introduction of potentially toxic crosslinking agents[16] may limit the large-scale preparation of the self-healing hydrogels.

We used nape instead of dorsum (the back of the animal) as the sites for healing wounds in stretchable parts. The self-healing hydrogel for treating stretchable wound such as joint damage has been reported, which selected the dorsum of the rat as the wound site.[5] This part does not move as frequently as the stretchable parts and could not accurately indicate the

1. Introduction

Skin wounds, especially those that have been extensively dam-aged by injuries or illnesses, cannot be repaired immediately by themselves and can cause severe disability or even death.[1] Wound dressings are essential for repairing the skin and

Adv. Funct. Mater. 2020, 2002370

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healing of the wounds at the stretchable sites. The nape is a good site for testing dressings for stretchable wounds since this site experiences various movements including tension, com-pression, and twist. Moreover, this site is large enough for pre-paring wounds with different sizes and shapes and facilitates the fixation of dressings with minimal scratching by the animal itself (as the rats’ paws cannot reach it). However, the reported studies on wounds on the nape have not underscored the suit-ability of the nape to evaluate stretchable wound site.[17] Taking advantage of wounds on the nape for exploring and evaluating novel dressings with excellent mechanical, electrical, and bio-compatible properties is highly desirable.

We used guar gum derivatives based self-healing hydrogel for stretchable wound healing. Guar gum, a water-soluble galacto-mannan derived from soybean gum,[18] has been widely used in the fields of pharmaceuticals,[19] hydraulic fracturing, sensors,[20] absorbents,[21] and skincare.[22] The hydration rate of guar gum or its derivative is very slow,[23,24] which greatly hinds its appli-cations. In this study, we found that the addition of a small amount of acid could greatly promote the hydration of cationic guar gum (CG) and the subsequent neutralization by alkali could change the system to a gel state at physiological pH value, the whole process could be finished within 1 min. The resulting hydrogel was injectable and self-healable, named guar slime (GS). Conductive materials can benefit wound healing.[4c,25] Introduction of conductive polymer utilizing the guar slime system to obtain functional self-healing hydrogel may accel-erate wound healing. We selected commercially available acidic poly(3,4-ethylenedioxythiophene): poly(styrenesulfonate) (PEDOT:PSS, electrically conductive) solution for fabricating conductive PEDOT:PSS/guar slime (PPGS) to improve the wound healing ability. We explored the self-healing and conduc-tive properties of PPGS as dressings in healing skin wounds in the stretchable parts of rats, which showed great improvement in wound closure and tissue reorganization.

2. Results and Discussions

2.1. Preparation of GS and PPGS

The dissolution of different concentrations of cationic guar gum in water is still relatively slow (Figure S1, Supporting Informa-tion), which greatly limits its processing and application. In the present study, we report a facile and green method to fabricate injectable and self-healing gel by the addition of acid and base in sequence (Figure 1). After the introduction of hydrogen ions, the empty electron orbits of hydrogen ions could form hydrogen bonds both with water and the hydroxyl groups in the molecular chain. Hydration of hydroxyl groups (red dots) resulted in the quick dissolution of CG. Unlike pure acidic addition, PEDOT: PSS (black rods) had many negatively charged groups (black dashed circles), which could generate electrostatic interactions (orange dashed circles) with the side chains (green rods) in cati-onic guar gum. PEDOT: PSS would attach to the CG chain. The molecular chain was wrapped with hydronium ions, which hin-dered the intermolecular actions of hydroxyl groups due to the steric hindrance and electrostatic repulsion. After adding alkali to neutralize the excess hydronium ions, the hydrogen bonds

between the hydroxyl groups in molecular chain and the water molecule or hydronium ion were opened, exposing the ipsi-lateral hydroxyl group. Under constant stirring, continuously increasing hydrogen bonding between orthohydroxy groups could form a resultant force, which was strong enough to form a gel (inserted photo) within 1 min. The acid–base method could rapidly fabricate GS and had little effect on the pH of the solution (Figure S2, Supporting Information). The final fabri-cated gel had a physiological pH value.

Ipsilateral hydroxyl groups and branches abound in cati-onic guar gum and can form strong intermolecular hydrogen bonds.[22,26] In the state of cationic guar powder, the molecular chain was crimped and entangled, which was not conducive to rapid hydration, so the dissolution of the gum in water was slow, which limited its processing and application. After the addition of the acid, the large amount of hydronium ions pre-senting in the solution accelerated the hydrogen bonding in the molecular chain, allowing the segments to rapidly hydrate and form a paste. Proper pH could induce guar gum chain to form sufficient stretching state from nonuniform stretching state.[27] The introduction of the basic group could enhance the hydrogen bonding between the molecules and finally forming a hydrogel based on hydrogen bonding.

By this method, we replaced the acid component with acidic PEDOT:PSS solution to obtain the electroactive PPGS. PEDOT:PSS solution could act as acid in accelerating the hydra-tion of CG and provide ionic sources and conjugated compo-nents for conductivity.

2.2. Properties of GS and PPGS

Both GS (Figure 2A) and PPGS (Figure 2B) had a porous struc-ture with a pore size of several hundred micrometers, which may allow the dressing to be gas permeable. In Fourier trans-form infrared spectroscopy (FTIR) spectrum (Figure 2C), the introduction of the inductive components[28] caused blueshift of the characteristic peak, while the hydrogen bonding[29] and conjugation[30] caused the bathochromic-shift. The band around 3342 cm−1 represents the O–H stretching vibration of CG[31] and a redshift occurred in GS (3297 cm−1) might be caused by the formation of hydrogen bond. The introduction of PEDOT:PSS in CG weakened the intermolecular hydrogen bond between CG, which might lead to a blueshift of OH stretching vibration to 3428 cm−1. The hydration effect in GS and PPGS intensified the asymmetric CH stretching vibra-tion at 2920 cm−1. The symmetrical CH stretching vibration obvious in CG (2889 cm−1) and PEDOT:PSS (2848 cm−1) became weak in GS and PPGS. Both PEDOT:PSS and PPGS had the CC in-plane vibrations of thiophene and phenyl rings at 1519 and 1473 cm−1, respectively. The SO vibration at 1166 cm−1 and the OSO signal at 1039 cm−1 belonging to the sulfonic acid for PSS were detected in the PEDOT:PSS.[32] The lack of conju-gate effect between PEDOT and PSS and the increase of induc-tive effect between quaternary ammonium cationic group and sulfonate anion in PPGS might bring a hyperchromatic shift to 1178 and 1079 cm−1. The appearance and changes of these peaks indicated that PEDOT:PSS was successfully doped into CG to form PPGS.


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The cyclic voltammetry curves of GS and PPGS were tested in HCl and NaCl (Figure 2D,E). The PPGS showed an oxida-tion peak at the position of 0.46 V in HCl and a pair of redox peaks at 0.45 and 0.25 V in NaCl. The proton doped PPGS in HCl contributed to a higher current but lower redox property

than that in NaCl.[33] In both solutions PPGS had higher cur-rent and capacity than GS. A circuit was established to show the difference between GS and PPGS. LED had a brighter luminance when connected with PPGS (upper inserted pic-ture in Figure 2D) than that with GS (lower inserted picture in

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Figure 1. Schematic diagram of the fabrication and self-healing of PPGS and GS. GS and PPGS are fabricated from cationic guar gum with two-step methods marked on 1 and 2, respectively. The inserted graphs represent the obtained PPGS and GS. Elements circled with red, green, black, orange, and blue dashed line represent the hydration of hydroxyl, cationic side chain, PEDOT:PSS, electrostatic interactions, and hydrogen bond, respectively. Different background colors in step three indicate the same region before and after self-healing.


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Figure 2D), which was recorded as Movie S1 in the Supporting Information. The PPGS showed an enhanced conductivity of 2.22 mS cm−1 compared with 1.04 mS cm−1 of GS (Table S1, Supporting Information). The combination of ionic and con-jugated structure might result in the enhancement of con-ductivity. We used two block of abdominal muscles of rats to illustrate the transmission of electrical signals between them via PPGS.[34] Electrical stimulations can be transmitted between the two blocks of separate muscles bridged by PPGS and trigger the asynchronous excitation and contraction of the muscles (Movie S2, Supporting Information). The cyclic voltammetry curves, conductivity data, and supporting movies illustrated the conductivity and interfacing properties of PPGS.

Thermogravimetric analysis (TGA, Figure S3, Supporting Information) showed a similar decomposition temperature of PPGS (277.8 °C) and CG (278.8 °C) and a relatively low decomposition temperature of GS (271.8 °C). The ionic inter-actions between the positively charged CG and the negatively charged PEDOT:PSS (Table S2, Supporting Information) was

the possible reason for a higher decomposition temperature of PPGS compared with GS.

2.3. Self-Healing Demonstration and Mechanism

We evaluated the self-healing properties of GS and PPGS. The wounds at the active sites usually experience a high frequency of movement and the gel materials served as dressings may be subjected to external mechanical forces. A gel with self-healing ability can greatly improve the life span of the dressings. In this study, GS and PPGS were injectable to facilitate the fab-rication of gels with arbitrary shapes with appropriate molds (Figure S4A, Supporting Information). In order to evaluate the self-healing properties of GS, we injected the GS gel to irreg-ular shape (Figure S4B, Supporting Information) and remolded them to star shapes (Figure 3A), which proved its excellent deformability. We cut a red and a blue star-shaped gels at the same position and patched two pieces of different colors into

Figure 2. Characterization of GS and PPGS. (A) and (B) shows the SEM images of GS and PPGS, respectively. Scale bar, 200 µm. C) FTIR spectra of CG, GS, PPGS, and PEDOT:PSS. The enlarged graph shows the spectra from 1800 to 700 cm−1. Cyclic voltammetry curve of GS and PPGS in D) 1 m HCl and E) 1 m NaCl. Inserted graphs red and blue dashed box respectively showed the electric performance of GS and PPGS in the circuit.


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a new star (Figure 3B). The half parts from which were physi-cally contacted initially can join together and recombine into a new one with heterogeneous colors within 30 min at ambient temperature without any stimuli, showing superior self-healing properties.

We characterized the self-healing capability of PPGS. After introducing PEDOT:PSS as an electroactive component, the resulting PPGS could still heal together with GS bulk (Figure 3C,D).

We tested the mechanical strength of the self-healed gels by stretching a GS strip heterogeneously recombined for 1 h. Over 100% elongation did not influence the stability of the strip (Figure 3E and Movie S3, Supporting Information). We observed the joint position of the gels under microscopy. After connection by patching together two pieces of gels for 1 h, the border between the gels became blurred and the intercon-necting zone became continuous. The two parts of the gel became an integrated one and maintained integrity even under stretching (Figure 3F).

The bonding energies of bidentate hydrogen bonds are gen-erally stronger than those of monodentate hydrogen bonds,[35]

Catechol with ipsilateral hydroxyl groups is used as a triggering material for surface self-healing and the formation of hydrogen bonds between ipsilateral hydroxyl groups was proven to be responsible for the self-healing property.[36] Cationic guar gum has a main chain structure of mannose which contains hydroxyl groups on the same side.[37] These ipsilateral hydroxyl groups resulted in strong intermolecular hydrogen bonds. A large number of hydrogen bonds on molecular chains could work together, forming adequate intermolecular force for gela-tion. Hydrogen bonding has relatively weak molecular interac-tion than covalent bond. External forces such as tension, pres-sure, and even gravity might break parts of the hydrogen bonds (Figure 1). Due to the existence of a large number of ipsilat-eral hydroxyl groups, new stable hydrogen bonds would soon be formed to maintain the stability of the gel (Figure 1, blue dashed lines). This might be the mechanism by which the self-healing and injectable GS and PPGS are achieved.

In order to verify our speculation, we used a method to dis-rupt intermolecular hydrogen bonds of GS to observe the self-healing ability of the gel. Phosphate is capable of disrupting hydrogen bonds.[38] We immersed two blocks of GS into the

Figure 3. Self-healing demonstration and characterization of GS and PPGS. A) The photograph of two star-shaped GS with red and blue staining, respectively. B) A self-healed GS star composed of two halves of stars in different colors. C) Photograph shows two slabs of gels: the white one GS and the black one PPGS. D) Photograph shows a piece of gel formed by the self-healing of GS and PPGS. E) Reactive blue stained and Congo red stained GS were cut into four 1 cm length pieces and recombined color by color. After 1 h of self-healing, the reunited GS could be stretched up to 200% strains and did not break. Scale bars, 1 cm. F) Observation under microscopy with the same operation as (E). Scale bars, 1 mm. Sweep step strain test of G) GS and H) PPGS with amplitude loop between small strain (C = 1%) and large strain (C = 400% or 1200%) with 60 s for each strain interval.


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10 × PBS (red) or water (blue) for 10 s. We brought the red block and blue block into contact with each side of an untreated block of GS for 10 min. The water-treated GS block healed together with the untreated GS block. On the contrary, the PBS-treated GS block was easily pulled away by the tweezers (Figure S5 and Movie S4, Supporting Information). These results dem-onstrated that the formation of dynamic hydrogen bonds is responsible for the self-healing of GS. This mechanism also applies to PPGS due to its similar structure to GS.

2.4. Rheological Behavior of GS and PPGS

In dynamic mechanical analysis, the crosspoint of storage mod-ulus (G′) and loss modulus (G″), namely critical point or gel point, represents a critical state between solid and liquid.[12a] Rheological studies showed that the critical point (500%) of PPGS (Figure S6A, Supporting Information) was higher than that of GS (150%) (Figure S6B, Supporting Information), while the storage modulus (at small deformation) of GS (400 Pa) was higher than that of PPGS (150 Pa). The storage modulus usually represents the energy stored due to elastic deforma-tion. In PPGS, the main storage modulus came from the abundant intermolecular hydrogen bonds in the CG chains. The positively charged quaternary ammonium cationic group in CG chains could interact with the negatively charged sul-fonate anion in PEDOT:PSS molecules to form ionic bond (Figure 1, orange dashed circles). Even though the ionic bond is stronger than the hydrogen bond and may increase the mod-ulus, the steric effect after ionic bonding can greatly influence

and reduce these intermolecular hydrogen bonds nearby. The decrease of hydrogen bond was reflected in FTIR as described earlier (Figure 2C). As a result, the weakened hydrogen bonds made PPGS softer and more flexible, leading to a decrease in G′ and a greater critical tensile elongation for PPGS. We evaluated the self-healing ability of the gels through amplitude oscillation (Figure 3G,H). Small strains were fixed at 1% and large strain were chosen at over twice of the critical points, which were 400% for GS and 1200% for PPGS. For both GS and PPGS, under small strains, the G′ was greater than the G″, meaning that the gels were in gel state. Under large strains, for both GS and PPGS, the G′ became smaller than the G″, meaning that the gels were broken. When returning to small strain again, for both gels, the G′ can recover to the original state, showing good self-healing abilities of GS and PPGS.

2.5. Biosafety Evaluation of GS and PPGS

We evaluated the cytotoxicity and proliferation behavior of GS and PPGS using cell counting kit-8 (CCK-8) kit. Compared with the control group, GS and PPGS showed no significant cytotox-icity to Madin–Daby canine kidney (MDCK), 3T3, and human aortic fibroblasts (HAF) cells (Figure 4A–C and Figure S7A,B, Supporting Information) after 24 and 48 h of coculture. We observed the effects of the gels on the cell morphology of MDCK after 24 h of coculture by live/dead staining assay (Figure 4D). Compared with the control group, GS and PPGS-treated groups showed larger cell density and more extended size, indicating the excellent biocompatibility of the gels. We

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Figure 4. Biocompatibility evaluation of GS and PPGS. Evaluation of cytotoxicity and cell proliferation data using CCK-8 assay on A) MDCK, B) 3T3, and C) HAF cells treated by extracts of GS, PPGS, and control. D) Confocal microscope images of live/dead staining on MDCK cells (no dead cell observed) treated by extracts of GS (left), PPGS (middle), and control (cell culture medium, right) for 24 h. Scale bars, 50 µm. The numbers in the top right corner indicate the average percentage of coverage by cells in each group, n = 3. E) Hemolytic tests of GS and PPGS with the blood cells of rats, inserted graphs are the photographs of the samples of water, saline, GS, and PPGS from left to right.


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tested the biocompatibility of GS and PPGS using the blood of rats, which is an important parameter for blood contact mate-rials. GS and PPGS (Figure 4E) had similar hemolysis perfor-mance as the negative control (saline), which meant negligible damage to the erythrocytes.

2.6. In Vivo Wound Healing Performance

We evaluated the performance of dressings based on GS or PPGS on a full-depth skin wound model constructed on the nape of rats. Previous reported studies on wound dressings ignored the needs for applying in the stretchable parts of the body.[12a] The commonly used dorsal area of rats has much less movement compared with joints such as the hip, knee, shoulder, and elbow. These joints on animals are difficult to test dressings due to the small area for creating wounds and the scratching by the animals.

We assumed that the nape is a specific joint position which fre-quently undergo movements but has larger area and is easier to fix the dressings with less animal scratching (Figure 5A). To verify our assumption, we counted and analyzed the movement of nape and back of rats by video recording. We found that the movement of the nape can be divided into three types, namely compression, tension, and twist (Figure 5C), all of which achieved a frequency of more than ten times per minute (Figure 5B). By contrast, the movement of the dorsum occurred only when the body was elongated and the frequency of activities was significantly less than that of the nape. These activities of the nape can cover the behavior of other active parts as the red arrows showing the skin movement (Figure 5A). For instance, knee joints, elbow joints, and knuckles only experience tension or compression, and wrists and ankles may have the twisting activity. The nape is undoubt-edly an appropriate and innovative position for characterizing the wound healing of the stretchable sites.

Figure 5. Movement analysis on rat’s nape area. A) Cartoon pictures of three different behavior patterns of rat on the nape, namely tension, com-pression, and twist from top to bottom. Red arrows indicate the moving direction of skin tissue on the nape. B) Movements counts of different rat’s behavior. ** P < 0.01, **** P < 0.0001, ☆data boundary, □ mean. C) Photocapture of four different movements of rats.


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We created round-shaped full-depth skin wounds on the nape and the dorsum of rats as stretchable wound model and normal wound model, respectively. GS and PPGS were applied to evaluate their wound healing performance compared with gauze in both groups (Figure 6A,B and Figure S8A,B, Sup-porting Information). We measured the wound size and calculated the wound healing rate as the assessment cri-teria. On day 7 postoperation on the nape (Figure 6C), PPGS group achieved ≈71% healing, which was significantly better than those of the GS group (≈57%) (P < 0.05) and the gauze group (≈43%) (P < 0.001). The wound closure rate of the GS group was significantly higher than that of the control group (P < 0.05). On day 14 postoperation, both the GS and PPGS groups almost healed completely, whereas the control group only achieved ≈66% wound closure. The better wound healing effect of PPGS and GS may be ascribed to their novel proper-ties compared with the gauze. During the healing process, GS

and PPGS could not only provide moist condition and avoid adhesion to the wound,[39] but comply with the movements of the nape and maximally reduce the destruction of the wound. It is possible that the conductive components of the PPGS can promote the repair of the tissues[12a,25a,40] especially at the early stage of the wound healing (Figure 6C and Figure S8A, Supporting Information). On day 14 postoperation on the dorsum there was no significant difference among GS (89%), PPGS (87%), and gauze (87%) (Figure 6D). The healing rate of control group on the dorsum (87%) was significantly higher than that on the nape (≈66%), which was consistent with the hypothesis that frequent movement could inhibit wound healing. On the contrary the wounds treated by GS and PPGS healed faster on the nape site than on the dorsal site (Figure 6E).

We characterized the detail of the wound healing by his-tochemical staining. Hematoxylin-eosin (H&E) and Masson

Figure 6. Wound healing experiment of GS and PPGS-treated wounds on the nape and dorsum area of rats. A) Photographs of wounds show the gauze (control), GS, or PPGS treated wounds on the nape and dorsum area of rats on day 0 and day 14 postoperation. B) The enlarged images of nape and dorsum areas with ruler corresponding to (A) on day 14. (C), (D), and (E) are statistics of wound healing rates at different time points on nape or dorsum (* P < 0.05, *** P < 0.001).


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staining (Figure 7), and all the groups showed the distribution of neovascularization (green arrow) and fibroblasts (yellow arrow). In each group, there was a process of fibrous cell migration and angiogenesis (Figure S9, Supporting Infor-mation) during wound healing. Quantified data (Figure 7A) showed that the granulation tissue (Figure 7E, indicated with red double headed arrows and black dotted area) of PPGS was significantly thicker than those of GS and the control groups on days 14 (Figure 7C,D). PPGS group showed larger area of blue colored collagen fiber contents (Figure 7H) than those in other groups (Figure 7F,G). PPGS group had significantly higher collagen volume fraction (76%) (Figure 7B) than GS group (56%) and control group (20%). The deposition of col-lagen and formation of granulation are positive correlation with wound healing.[4i] Thus, thicker granulation tissue and

higher collagen contents indicated a better healing effect in PPGS group than other groups.

On day 14, hair follicles can be seen in the PPGS group (Figure 7H,K,N, black arrow). On the contrary, at the same time point, there were large number of fibroblasts in the GS group (Figure 7G,J,M, yellow arrow). Generally, excessive fibroblasts will result in the formation of dense scar tissues, reducing the mobility of new skin and hindering the formation of hair fol-licles.[41] The PPGS group showed better wound healing results and neotissue maturation. In the gauze group, infiltration of inflammatory cells was still visible on day 14 (Figure 7F,I,L, blue arrow), indicating its poor wound healing. Higher wound healing rate, thicker granulation tissue, higher collagen fiber content, and hair follicle regeneration showed a better wound healing effect of PPGS in stretchable parts.

Figure 7. Images and analysis of Masson and hematoxylin-eosin staining of wound tissues on day 14 postoperation. A) Statistical data of the granu-lation tissue thickness of GS, PPGS, and the control groups on day 14 indicated by H&E staining. B) Calculation of collagen volume fraction using imageJ software. (*** P < 0.001, **** P < 0.0001). Images of hematoxylin-eosin stained nape wound tissues of C) gauze, D) GS, and E) PPGS on day 14 postoperation. Red double-headed arrows represent the range of granulation tissue. Black dotted lines represent the boundary of granulation tissue. Yellow dotted lines represent the boundary between wound and normal tissue. (F), (G), and (H) are the wide field observation of the Masson stained tissues of the control group, GS, and PPGS, respectively. (I), (J), and (K) show the enlarged part of the black dotted zones from (F), (G), and (H), respectively. (L), (M), and (N) show the H&E stained tissues at the same sites of (I), (J), and (K). Red, green, yellow, black, and blue arrows represent vessels, collagen fibers, fibroblast, hair follicle, and inflammatory cells, respectively.


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Conducting dressings showed better skin wound healing than nonconducting dressings.[25b] PEDOT:PSS improved the conductivity of PPGS to a level similar to human dermis (2.2 mS cm−1).[42] The similarity of conductivity to dermis would potentially benefit PPGS to transfer the bio-electrical signals around the skin and accelerate the wound healing.[25a,43] It is also possible that high fixed charge content could absorb the exudate, maintain the balance of skin hydra-tion,[44] and benefit wound healing.[45] On the other hand, external electric field has been proven to promote both wound healing and growth of hair follicle.[46] The movement induced generation of electricity in conductive hydrogel had also been reported.[47] The synergistic effects of movements in animal and conductive self-healing gel might lead to an induced elec-tric field in PPGS, promoting wound healing in the stretch-able site,[48] thereby making the wounds on the nape heal faster than those on the dorsum, although treated by the same gels. In conclusion, the GS could promote wound healing in stretchable parts and the addition of PEDOT: PSS showed accelerated wound healing, epithelialization, and hair follicle regeneration.

3. Conclusion

To overcome the shortcoming of present dressings in dealing with the skin wounds at the stretchable parts of the body, we developed a facile strategy to prepare injectable self-healable and conductive slime based on cationic guar gum within 1 min. The injectable and self-healing ability endows the hydrogel with the capability to comply with the attached objec-tives and reorganize its shape following the varying of environ-mental topology. The conductivity might benefit a better wound healing and regeneration. Thus, the self-healable and conduc-tive slime can serve as excellent wound dressings in healing wounds on the nape of rats, which experiences complex and frequent movements. Yet the mechanism of conductive mate-rials accelerated wound healing remains to be explored. The combination of other functional molecules and the self-healing hydrogel remains a great potential in wounds healing and other applications.

4. Experimental SectionFabrication of GS and PPGS: 0.03 g cationic guar gum (guar gum

hydroxypropyl trimethyl ammonium chloride, CG) (Yuanye Biotechnology co., LTD) was suspended into 1 mL distilled water and then 20 µL 1% phytic acid (Sinopharm Group) was added to the suspension which became homogenous after stirring for 10 s. 8 µL 0.2 m NaOH solution was introduced into the solution which formed the guar gum slime within 1 min of stirring. The preparation steps of PPGS were similar to that of GS, and the only difference was that 50 µL 1.5% w/v PEDOT:PSS (Macklin) was used as the acidic part followed by the addition of 10 µL NaOH.

FTIR Spectra: The FTIR spectra of CG, GS, PPGS, and PEDOT:PSS were recorded in the range of 4000–500 cm−1 by employing a Spectrum One FTIR spectrometer (Perkin Elmer Instruments).

Morphology Characterization: Scanning electron microscopy (SEM U8220, Japan) was used to observe the surface morphology of GS and PPGS. The samples were lyophilized and gold sputtered before observation.

TGA: The thermogravimetric performance of CG, GS, and PPGS with nitrogen atmosphere at a heating rate of 10 °C min−1 between 25 and 500 °C by TG 209 F3 Tarsus (NETZSCH, German) was measured.

Conductivity Tests: DDS-307A conductivity meter was used to test the conductivity of GS and PPGS. Multi Auto lab/M204 was used to test the cyclic voltammetry through a three-electrode system. GS or PPGS covered conducting ITO glasses with size of 2 mm × 10 mm × 20 mm as working electrode, the platinum electrode as the counter electrode, and Ag/AgCl as the reference electrode was used. A scan ranging from 0 to 0.8 V at 50 mV s−1 rate was applied for tests in both 1 m HCl and 1 m NaCl solution. All samples were tested three times.

Rheological Tests: The DMA of GS and PPGS were conducted by a Malvern Panalytical Kinexus Pro+ rotational rheometer with 40 mm parallel plates and 500 µm gap, fixing angular frequency at 6.28 rad s−1 and temperature at 25 °C. The slime was injected into the plates and the extra materials were removed before the tests. Two methods were used to characterize the rheological performance of GS and PPGS. A strain amplitude sweep test (C = 1–1000%) was performed to find the gel point. Then a sweep step strain test was activated with amplitude loop between small strain (C = 1%) and large strain (C = 400% or 1200%) with 60 s as the interval for each strain.

Self-Healing Assays: 0.1% reactive blue 19 (Macklin) or Congo red (Macklin) was added to the GS solution to obtain a blue or red GS gel. The gel was loaded into a syringe and injected into water by a 17 G needle to show the injectability. The gel block was chopped and reshaped into a star using a mold. Two stars in different colors were cut into halves at the same positions and the two pieces in different colors were framed together and left at room temperature for 30 min. The gel pieces can self-heal to form a complete star. The above processes were recorded as photographs.

The blue and red GS were reshaped into cylinder with cross-section diameter at 5 mm and were cut into 1 cm bulks. They were put together color by color. After 1 h healing, the reunited GS was stretched. The above process was recorded by Cannon 60D as photographs and movie as well as microscopy images.

GS and PPGS were cut into 1 cm cubes and were put together to heal for 30 min. The resulting slime was picked up with a pair of tweezers to show the self-healing ability. The hydrogel blocks could combine into one piece when they were attached together. No extra force was needed during the self-healing process.

Hemolytic Activity Test: The hemolytic activity of GS and PPGS was measured according to the reported method[49] with fresh blood of rats. In brief, the blood with anticoagulant tube through the carotid arteries of Sprague-Dwaley (SD) rats was collected. the 4% w/v erythrocyte suspension was prepared by centrifuging the blood (1500 rpm, 15 min) and resuspending the sediment with saline solution. For the experimental group, GS and PPGS were molded into round shaped hydrogel at a size of 1 mm × 0.32 cm2 and were put into a 96-well plate with 100 µL saline solution in each well. The same volume of deionized water and saline as positive control and negative control respectively were set. 100 µL of the erythrocyte suspension was added to each well to reach a final test concentration of 2% w/v. After incubation for 4 h at 37 °C, it was centrifuged at 1500 rpm for 15 min. 100 µL of solution was taken in each sample and the hemoglobin release was evaluated at 540 nm by measuring UV–vis absorbance. The data were presented as average value with standard deviation (n = 3).

In Vitro Cytocompatibility Evaluation: The cytocompatibility of the hydrogels (GS and PPGS) was evaluated by culturing MDCK, mouse fibroblasts (3T3), and HAF cells in the extracts of the slimes according to reported literature.[25a,50] Briefly, sterilized GS and PPGS were immersed in cell culture medium (Dulbecco’s modified Eagle’s medium with 10% fetal bovine serum (Invitrogen) and 1% penicillin-streptomycin solution (MP Biomedicals)) at a concentration of 0.1 g mL−1 and incubated for 24 h at 37 °C. Then the supernatant was collected and filtered with a filter (0.22 µm, Millipore) as GS or PPGS extracts. Cells were seeded at a density of 5 × 103 per well on 96-well plate and a density of 3 × 105 mL−1 on 20 mm confocal dish (Corning, American). After incubating for 24 h, the cells were rinsed with PBS twice and GS or PPGS extracts

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were added as the experimental groups. Cells were seeded at a density of 1 × 104 per well on 96-well plate. After incubating for 4 h, the cells were rinsed with PBS twice and GS or PPGS hydrogel was added as the experimental groups for contact evaluation. The cells cultured in fresh culture medium were set as control. The incubation was carried out for 24–48 h at 37 °C, 5% CO2. The cell viability was evaluated at 24 and 48 h with CCK-8 kit (Dojindo, Japan). A confocal scanning microscopy (Zeiss LCSM 710) was used to observe the cells after dual fluorescence calcein AM/PI (Dojindo, Japan) staining.

In Vivo Wound Healing in a Full-Thickness Skin Defect Model: The materials for preparing GS and PPGS were sterilized before use. In brief, cationic guar gum was sterilized at 121 °C for 20 min, and phytic acid, sodium hydroxide, and PEDOT:PSS solution were filtered through a 0.22 µm filter. GS and PPGS were then prepared in clean bench.

In vivo animal study was approved by the Institutional Animal Care and Use Committee of the National Center for Nanoscience and Technology, China. In this study, 6–8-week aged female SD rats weighing 200–250 g were used. Rats were divided randomly into three groups with six rats in each group (n = 6) and raised for 2 d to get used to the environment before surgery. A dose of 5 mL kg−1 10% chloral hydrate was used in anesthesia through intraperitoneal injection. The nape and dorsum area of rats were shaved and alcohol sterilized, then full-thickness skin damage with a diameter of 1.8 cm in each area was created using scissors and forceps. GS and PPGS were retained in the wound sites followed by fixation of gauze and medical tape. The wounds covered by gauze were set as a control group. After dressing, the rats were housed individually in cages at ambient temperature.

The wound area was measured and the closure rate was calculated on day 3, 7, and 14, respectively. At each time point, photographs were taken with a ruler beside the wound and a rat in each group was sacrificed for histochemical analysis. The skin wound tissues were excised and stained with H&E or Masson trichrome after fixation and embedment and for morphological observations and collagen production evaluation, respectively.

Statistical Analysis: ImageJ was used to analyze the photographs of wound area, one-way ANOVA in SPSS statics 25 was used for the P value, and Origin 2016 was used for data plotting. Wound closure rate was calculated by the following equation

)( = − ×% 1000

0R

S SSn

n

(1)

where Rn and Sn are the closure rate and wound area at day n, respectively. S0 is the initial wound area. n = 3, 7, or 14.

Supporting InformationSupporting Information is available from the Wiley Online Library or from the author.

AcknowledgementsS.L. and L.W. contributed equally to this work. The authors thank the National Natural Science Foundation of China (21574050, 51603079, 21535001, 81730051, 21761142006, and 81673039), the National Key R&D Program of China (2018YFA0902600 and 2017YFA0205901), the Chinese Academy of Sciences (QYZDJ-SSW-SLH039, 121D11KYSB20170026, was XDA16020902), and Tencent Foundation through the XPLORER PRIZE for financial support.

Conflict of InterestThe authors declare no conflict of interest.

Keywordsconductive hydrogels, guar gum, self-healing, stretchable wounds, wound healing

Received: March 13, 2020Revised: April 9, 2020

Published online:

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