Multifunctional Hydrogel with Good Structure Integrity, Self-Healing

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A Multi-Functional Hydrogel with Good Structure Integrity, Self-healing and Tissue-adhesive Property Formed by Combining Diels-Alder Click Reaction and Acylhydrazone Bond Feng Yu, Xiaodong Cao, Jie Du, Gang Wang, and Xiaofeng Chen ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.5b06896 • Publication Date (Web): 15 Oct 2015 Downloaded from http://pubs.acs.org on October 23, 2015

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ACS Applied Materials & Interfaces

A Multi-Functional Hydrogel with Good Structure Integrity, Self-healing and Tissue-adhesive Property Formed by Combining Diels-Alder Click Reaction and Acylhydrazone Bond Feng Yu,a,b,c Xiaodong Cao,a,c∗ Jie Du,b Gang Wang,a,c Xiaofeng Chen,a,c∗ a. School of Materials Science and Engineering, South China University of Technology, Guangzhou, 510641, PR China.

b. College of Materials and Chemical Engineering, Hainan University, Haikou, 570228, PR China.

c. National Engineering Research Centre for Tissue Restoration and Reconstruction, Guangzhou, 510006, PR China.

KEYWORDS: Hydrogel, self-healing, pH-responsive, dynamic covalent chemistry, Diels-Alder click chemistry, tissue-adhesive

ABSTRACT: Hydrogel, as a good cartilage tissue-engineered scaffold, not only have to possess robust mechanical property but also have to own intrinsic self-healing property to integrate itself or the surrounding host cartilage. In this work a double

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crosslinked network (DN) was designed and prepared by combining Diels-Alder click reaction and acylhydrazone bond. The DA reaction maintained the hydrogel’s structural integrity and mechanical strength in physiological environment, while the dynamic covalent acylhydrazone bond resulted in hydrogel’s self-healing property and controlled the on-off switch of network crosslink density. At the same time, the aldehyde groups contained in hydrogel further promote good integrating of hydrogel to surrounding tissue based on aldehyde-amine Schiff-base reaction. This kind of hydrogel

owned

good

structural

integrity,

autonomous

self-healing

and

tissue-adhesive property simultaneously will have a good application in tissue engineering and tissue repair field.

Introduction It has been known that articular cartilage is a high water content elastomer. The mechanical function of it is to bear and distribute loads and protect the subchondral bone1.

Unfortunately, articular cartilage has a limited capacity to self-heal lesions

due to the lack of blood supply and extracellular matrix secretion2. At present, the best strategy is to develop functional tissue-engineered hydrogel to reconstruct a biomimic microenvironment for cell survival3. It indicated that the cartilage tissue-engineered hydrogel with intrinsic biomimic mechanical properties such as high modulus, good elasticity and repeatedly load bearing properties was urgently demanded4. Diels-Alder (DA) reaction fulfills most of the requirements for the “click” chemistry concept which is based on utilizing rapid, efficient, versatile and selective reactions5-7. At the same time, the DA reaction in aqueous environment results in high 2 ACS Paragon Plus Environment

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yields, produces no byproducts, and occurs under mild reaction conditions without the involvement of any catalysts and coupling reagents5-6, 8-10. Therefore, in recent years, DA click reaction was considered as an efficient crosslinking strategy to synthesize robust biomedical hydrogels which were applied to tissue engineering11-18. In our previous work, a polysaccharide-based hydrogel for cartilage tissue engineering was prepared by DA click reaction successfully19. The hydrogel physicochemical properties were researched particularly and it exhibited outstanding elasticity, shape recovery and anti-fatigue property due to the well-fined network which were similar with the natural cartilage tissue20-21. At the same time, the good cell encapsulation and compatibility of DA crosslinked hydrogel suggested it a potential candidate for cartilage tissue engineering scaffold in vitro. However, as implanted biomaterials, DA crosslinked hydrogel results in poor integration to the surrounding smooth cartilage tissue which always results in the implanted hydrogel looseness or fracture even under gentle daily movement22-24. As we all know, the ability to spontaneously heal injury and recover the body function is the key feature for the organism25-28. We anticipated that if the implanted hydrogels are capable of autonomous healing upon damage and adhesive property to host cartilage tissue, it will be able to make up for the limited self-healing property of natural cartilage. In order to solve this problem, in this work, the dynamic reversible acylhydrazone bond which formed from acylhydrazines and aldehyde was introduced into DA crosslinked network. Acylhydrazone bond is a pH responsive covalent bond which 3 ACS Paragon Plus Environment


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always resulted in the self-healing feature26-27,

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29-33

. It has been reported that the

self-healing process occurs without any outside intervention at ambient temperature34. It's worth exciting that the containing aldehyde groups in the hydrogel can further covalently bind to local cartilage tissue amines based on Schiff-base reaction35-38. Therefore, a double crosslinked hydrogel was designed by integrating DA click reaction and acylhydrazone bond. We anticipate that the DA click chemistry maintains the hydrogel’s structural integrity and mechanical strength in physiological environment, while the dynamic covalent acylhydrazone bond will result in hydrogel’s self-healing property and controlled the on-off switch of network crosslink density. The aldehyde groups contained in hydrogel further promote good integrating of hydrogel to surrounding host cartilage tissue. This kind of smart hydrogel integrated by DA click chemistry and acylhydrazone bond will be a hoping candidate in the field of cartilage tissue engineering. 2. Materials and Methods 2.1 Materials Hyaluronic acid sodium (HA) (Mw = 1×106) was purchased from Shanghai crystal pure

industrial

Co.,

LTD

(Shanghai,

China).

4-(4,

5-triazin-2-yl)-4-methylmorpholinium chloride (DMTMM),

6-dimethoxy-1,

3,

2-morpholinoethane

sulfonic acid (MES), furylamine, adipic dihydrazide (ADH), sodium periodate were purchased from Sigma-Aldrich (USA). Dimaleimide poly (ethylene glycol) (MAL-PEG-MAL) (Mw = 2×103) was purchased from Shanghai Sunway Pharmaceutical Technology Co., LTD (Shanghai, China). 4 ACS Paragon Plus Environment

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2.2

Methods

2.2.1 Synthesis of HA-furan-ADH （Scheme 1） HA-furan-ADH was prepared by amidation of the carboxyl groups of hyaluronic acid sodium (HA) with the amine groups of furylamine (furan) and adipic dihydrazide (ADH). Briefly, HA (500 mg, 1.25 mmol carboxyl groups) was dissolved in 150 mL of MES buffer (100 mM, pH 5.5), in which DMTMM (0.7 g, 2.5 mmol) was added to active the polysaccharide carboxyl groups for 20 min. Subsequently, furylamine (110 µL, 1.25 mmol) was added dropwise by using a pipette, and then keep stirring at room temperature for 24h. After that, DMTMM (0.7 g, 2.5 mmol) was added to continually active the remaining non-activated polysaccharide carboxyl groups for another 20 min. Then, adipic dihydrazide (0.871 g, 5 mmol) was added and keep stirring at room temperature for another 24 h. After that, it was dialyzed against distilled water for 5 days (Mw cut-off 1.4×104). Finally, HA-furan-ADH derivative was obtained as a white porous sponge by lyophilization. The substitution degree (DS) of furan and ADH function groups on HA was determined by nuclear magnetic resonance (1H-NMR) (BRUKER, Germany). 2.2.2 Synthesis of HA-furan-CHO （Scheme 2） HA-furan was synthesized according to our previous work15,

18-19

.

1.5 g of

HA-furan was dissolved in 150 mL of water, and then 802 mg of sodium periodate were added. After keep stirring for 2 h in dark, 200 µL of ethylene glycol was added to stop the reaction, and then the mixture was dialyzed immediately against water39. The purified product HA-furan-CHO was freeze dried and kept at 4 °C for further 5 ACS Paragon Plus Environment


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using. The aldehyde group was characterized by nuclear magnetic resonance (1H-NMR). The percentage of oxidation of HA was quantified by measuring the number of aldehyde groups using t-butyl carbazate40.

2.2.3 Fabrication of single crosslinked network (SN) and double crosslinked network (DN) (Scheme 3) HA–furan-ADH and HA–furan-CHO were dissolved in deionized water (DI water) separately at a concentration of 20 mg/mL. The SN was formed by mixing of equal volume HA-furan-ADH and HA-furan-CHO solution. The hydrogel was crosslinked by covalent acylhydrazone bond in several minutes. The DN was formed by addition of MAL-PEG-MAL in the mixture solution of equal volume of HA-furan-ADH and HA-furan-CHO. Namely, 20.1 mg MAL-PEG-MAL was added to 1 mL mixture solution. Therefore, the DN hydrogel was crosslinked by both acylhydrazone bond and Diels-Alder click chemistry. In this article, another control group was prepared by mixing HA-furan and MAL-PEG-MAL to get a hydrogel without adhesiveness according to our previous work20. 2.2.4 The self-healing property of SN and DN. SN and DN gels were cut into halves and merged two gels by simply putting halves together in a container for 3h without any outside intervention at ambient temperature. The sol-gel transition process of SN was verified by adding hydrochloric acid and triethylamine successively. 2.2.5 The mechanical property of SN, DN before and after self-healing process 6 ACS Paragon Plus Environment

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In order to obtain the hydrogel’s breaking strength and the elongation at break, the linear ramp force 1N/min up to 18 N was design to test the hydrogel. The compressive modulus was calculated by the slope of stress-strain curve in the linear region. 2.2.6 The swelling and deswelling property of SN and DN To examine swelling and deswelling properties, SN and DN samples were immersed into DI water and acidic solution at 37 oC until equilibrium of swelling had been reached. The swollen hydrogels were weighed with a microbalance quickly after the excess of water on the surfaces was absorbed with a filter paper. The swelling and deswelling ratio (SR) was calculated using the following equation: SR= (Ws-Wd) / Wd Where Ws and Wd are the weights of the hydrogels at equilibrium swelling state and original state, respectively. 2.2.7 The pH responsiveness of DN To examine the pH responsiveness of DN, the sample was alternate immersed into DI water and hydrochloric acid solution (0.1 mol/L) until equilibrium of swelling had been reached. The control group was alternate immersed into DI water and sodium chloride solution (0.1 mol/L). After the swelling equilibrium, all the samples were weighted to calculate the variation of swelling ratio and tested by dynamic mechanical analysis (DMA) (TA Q800, USA) compressive mode to get the change of compressive modulus. 2.2.8 The adhesive property of hydrogel to natural cartilage tissue



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In order to investigate the integration of tissue-engineered hydrogel with host cartilage, the porcine articular cartilage was harvested from the knee of edible swine and created as a doughnut-shaped host cartilage explant. The precursor solution of hydrogel was injected into the hole of doughnut-shaped explant36,

41-42

. After the

process of gelation, the hydrogel-cartilage construct was formed. The integration situation was characterized by a scanning electron microscope (SEM) (Quanta 200, Netherlands FEI). Briefly, the construct was freeze-dried to obtain scaffold. Subsequently, the scaffold was immersed into liquid nitrogen and cut off vertically. The section was gold-coated in a sputter coater (Sanyo Denshi, Japan) and observed. The adhesive force between hydrogel and host cartilage was tested by pus-out model. 3. Results and discussion 3.1 Characterization of HA-furan-ADH and HA-furan-CHO. Following

the

synthesis

routes

shown

in

Scheme

1,

HA-furan-ADH

macromolecular was successfully obtained, as determined by the 1H-NMR spectra with evidence of proton peaks from the ADH residue (1, 2 , 3 and 4) and furan residue (5, 6 and 7) (Fig. 1). In Fig. 1, the peak at 1.9 ppm marked by “a” was assigned to the acetamido moiety of the N-acetyl-D-glucosamine residue of HA, and the peaks between 1.4 and 1.8, 2.1 and 2.4 ppm refer to the integration of methylene protons of ADH43, the peaks at 6.26, 6.46 and 7.41 ppm refer to the protons of furan15, 18. The integral ratio on the 1H-NMR spectrum suggested that HA was average modified by 45.19% furan and 29.06% ADH function groups44-46. The calculation of substitution degree was showed below. 8 ACS Paragon Plus Environment

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Areaሺ6.26ppmሻ+Areaሺ6.46ppmሻ+Area（7.41ppm） ×100%=45.19% Area(1.9ppm) Areaሺ1.57ሻ+Areaሺ1.74ሻ+Areaሺ2.21ሻ+Areaሺ2.35ሻ 3 DSሺADHሻ = × ×100%=29.06% Areaሺ1.9ሻ 8 DS(furan) =

Aldehyde groups were introduced to hyaluronic acid by reaction with sodium periodate, which oxidizes the vicinal hydroxyl groups to dialdehydes, thereby opening the sugar ring to form dialdehyde derivatives (Scheme 2). 1H-NMR is a good method to characterize aldehyde groups. The 1H-NMR spectrum was showed in S1. The peak at 8.17 ppm represents the aldehyde protons existing. The percentage oxidation of HA was quantified by measuring the number of aldehydes using t-butyl carbazate and trinitrobenzene-sulfonic acid (TNBS)40,

47

. The actual aldehyde content of

HA-furan-CHO reached an extent of oxidation of 53.1%.

Scheme 1 The synthesis steps of HA-furan-ADH.



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Scheme 2 The synthesis steps of HA-furan-CHO.

Scheme 3 A. The scheme of double crosslinked processes (The dynamic covalent acylhydrazone bond was marked as Red and the Diels-Alder click chemistry was marked as Blue). B. The gelation behavior of double crosslinked hydrogel.


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Figure 1 1H-NMR spectra in D2O (500 MHz) of HA-furan-ADH. Substitution degree of furan was determined by comparing the integrated areas under the proton peaks at 6.26, 6.37, and 7.45 ppm (furan protons), indicated by 5, 6, and 7 to that of the peak at 1.9 ppm indicated by “a” (N-acetyl glucosamine of HA). The peaks labeled by 1, 2, 3, 4 were the protons of ADH. The substitution degree of ADH was calculated as same as that of furan groups.

The mechanism of gelation is attributed to two different crosslinking processes (Scheme 3). The first one is dynamic covalent acylhydrazone bond between acylhydrazine and aldehyde and the second is Diels-Alder click chemistry between furan and maleimide. The gelation time of the hydrogels was determined using a test tube inverting method reported by Jeong et al48. When no fluidity was visually observed upon inverting the vial, the gel state was determined. The solution of 11 ACS Paragon Plus Environment


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HA-furan-ADH at 2 wt/v% and HA-furan-CHO at 2 wt/v% were mixed with same value. No matter SN or DN hydrogel, the gelation time was both kept within several minutes. 3.2 Self-healing property of SN and DN hydrogel. In order to observe the self-healing property of SN and DN gels, the samples were cut into halves and then merged two gels by simply putting halves together in a container for 3h without any outside intervention at ambient temperature as shown in Figure 2B. After 3h healing, the SN gel can stand its own gravity and the DN hydrogel is able to bear pulling force. The healing process can be explained by that the dynamic cleavage and regeneration of the acylhydrazone bond keep occurring in the network to self-heal dissected hydrogel itself automatically without additional stimuli.


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Figure 2 (A) The sol-gel transition property of SN hydrogel. (I) SN hydrogel, (II) 40 µl HCl was added into 1ml hydrogel for 10 min. (III) 20 min later. (IV) 66 µl TEA was added into solution for 25 min. (B) The self-healing property of SN (a-c) and DN hydrogel (d-f).

As we known, acylhydrazone bond is responsive to pH value. When the pH value is below 4, the acylhydrazone bond will cleave which lead the transition from gel to sol. As shown in Figure 2A, the gel to sol transition process of SN could be found in 10 min by adding 40 uL of HCl into 1mL of hydrogel. After 20 min, the gel has been transmitted to solution completely. The sol to gel transition process was stimulated by adding 66 uL of triethylamine(TEA) into the solution. As for DN hydrogel, however,



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the gel structure was kept integrity and the sol-gel transition process couldn’t be found in the same conditions. 3.3 The swelling-deswelling property of SN and DN hydrogel In order to further explore the relationship of network structure and sol-gel transition process, the swelling property of SN and DN hydrogel in DI water and acid solution was tested. Firstly, the SN samples were immersed into DI water. In Figure 3A, the swelling ratio of SN was increasing to 5.5 and the volume of hydrogel became bigger. The network kept absorbing water until the network was broken as shown in Figure 3B. It can be explained by that in DI water the swollen network enlarged the reaction distance of acylhydrazine and aldehyde groups and lead to less chance for forming dynamic acylhydrazone bond. When the SN samples were immersed into acid solution, the hydrogel sample degraded and collapsed rapidly in 3h due to the reverse reaction of acylhydrazone bond (Figure 3C, D). As a conclusion, no matter in DI water or acid solution, it will result in irreversible structural failure for SN hydrogel.


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Figure 3 The swelling property of SN in DI water and collapse of SN in acid solution (pH value is 1), respectively. (A) The relation curve of swelling ratio and swelling time in DI water. (B) The images of hydrogel’s volume change in DI water at different swelling time. (C) The relation of remained hydrogel mass and soak time in acid solution. (D) The images of hydrogel’s volume change in acid solution at different swelling time.

As for DN hydrogel, the swelling property in DI water and acid solution was obviously different from that of SN. As shown in Figure 4A, the swelling ratio of DN in DI water increased to the peak value 1.0 within 2h and kept a plateau with increasing time further. The volume of hydrogel samples expanse but keep structure



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integrity as shown in Figure 4B. When the DN hydrogel was immersed in acid solution, the hydrogel had a dehydrated stage until the equilibrium was reached (in Figure 4C, D). No matter in DI water or acid solution, therefore, all the DN samples could keep the structural integrity just along with reversible expansion and shrinkage of hydrogel volume due to the stable DA network.

Figure 4 The swelling and deswelling property of DN hydrogel in DI water and acid solution (pH value is 1), respectively. (A) The relation curve of swelling ratio and swelling time in DI water. (B) The images of hydrogel’s volume change in DI water. (C) The relation curve of deswelling ratio and soak time in acid solution. (D) The images of hydrogel’s volume change in acid solution.

3.4 The pH responsiveness of DN hydrogel


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Generally, the swelling and deswelling process is the common property for a hydrogel due to the osmotic driving force of the network towards isoconcentration tendency with the surrounding solution49. Manifold cycles of swelling and deswelling processed were tested for DN hydrogel and the results were shown in Figure 5A. In the control group, the samples were alternately soaking in DI water and NaCl solution. The swelling process was tested in DI water, while the deswelling process was tested in 0.1 mol/L NaCl solution. After four swelling-deswelling cycles, it can be found that the finally swelling ratio was about 1.4 g/g and the deswelling ratio was at 0.6 g/g. In the experiment group, the samples were alternate soaking in DI water and 0.1 mol/L HCl solution. The swelling and the deswelling processed can be also observed. In the first swelling course, the swelling ratio curve of control group and experiment group has no significant difference. But after the hydrogel reaching the first deswelling equilibrium in NaCl and HCl solution respectively, it had a significant lower deswelling ratio (0.4 g/g) in HCl compared with in NaCl solution. It indicated that in HCl solution the acylhydrazone bonds were cleaved and a looser network was obtained compared with control group which promoted the desweling process. After then, the subsequent swelling-deswelling curve of experiment groups was significant lower than the control group showed. In Figure 5B, the enlarge area of one swelling process, the swelling ratio linearly increased for control group in first 3h and reached a peak value (1.4 g/g) after 12h. But for experiment group, the swelling ratio had no significant change in first 3h and increased slowly to a peak (0.9 g/g) after 12h swelling. It can be explained that the cleaved bonds regenerated again in DI water and 17 ACS Paragon Plus Environment


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restricted the water absorbing process. As a result, the experiment group had a lower swelling ratio and deswelling ratio compared with control group. It’s very interesting that the change tendency of compressive modulus at swelling and deswelling equilibrium state was consistent with its swelling-deswelling property. In Figure 5C, the compressive modulus was periodic changed between 20-30 kPa in control group. But in experiment group, the modulus was periodic changed between 15-22 kPa. In order to further explore the reason of responsiveness of DN, the DA crosslinked hydrogel was prepared as control group. The swelling property and compressive modulus of DA and DN was studied and the results were showed in Figure S2-S3.We found that the compressive modulus of DA hydrogel has no significant difference along with the change of pH value. But in DN hydrogel, the compressive modulus has an obvious responsiveness phenomenon at pH < 4 vs. pH > 4 situation. From the result, we got the conclusion that DA is used here to develop the “backbone” of the hydrogel while the acylhydrazone bond provides the hydrogel pH responsiveness.


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Figure 5 (A) The pH sensitive property of DN hydrogel after 4 cycles. (B) The enlarged area of DN hydrogel swelling ratio. (C) The compressive modulus of DN hydrogel changing with pH value. The crosslinking mechanism of SN and DN network was shown in Figure 6. For SN hydrogel, the network was crosslinked by dynamic acylhydrazone bond and the network would cleave in acid solution (Figure 6A). But for DN hydrogel, the two-step crosslinking lead to an interpenetrating polymer networks in which only dynamic acylhydrazone bond was switched on-off by acid solution or DI water. While, the DA network was kept stable and complete no matter in acid solution or DI water as shown in Figure 6B.



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Figure 6 The pH sensitive principle diagram of hydrogels. A. SN hydrogel. B. DN hydrogel.

3.5 The mechanical property of SN and DN hydrogel The interpenetrating network not only provided hydrogel the intelligent self-healing and pH-responsiveness but also had a big progress in hydrogel mechanical property. Typical compressive stress-strain curves of original SN, DN and heal SN, DN hydrogels were shown in Figure 7A. It could be seen that the stress-strain curve of healed SN and DN hydrogel had no significant difference with their original hydrogel. It demonstrated that the self-healing process could promote the fully healing of mechanical property. At the same time, it’s obvious that the DN hydrogel had a higher static compressive modulus judging from the slope of the stress-strain curve linear region. In Figure 7B, the dynamic compressive modulus was tested by DMA. The 20 ACS Paragon Plus Environment

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results were consistent with the stress-strain test. The storage modulus of heal hydrogel had no significant difference with original hydrogel. And the storage modulus of DN was about 18 kPa, which was significantly higher than that of SN (2.5 kPa).

Figure 7 (A) Typical compressive stress-strain curves for original SN, DN and heal SN, DN hydrogels. (B) The compressive modulus of the hydrogels.

3.6 The adhesive property of DN hydrogel Due to the existing of aldehyde groups in the hydrogel system, the Schiff-base reaction between aldehyde and local cartilage tissue amines occurred as showed in Figure 8A. In Figure 8B, the schematic of the experiment examining integration of tissue engineered hydrogel to native cartilage was showed and the push-out test was utilized to determine the adhesive strength. In the experiment, we regarded DA crosslinked hydrogel without adhesiveness as the control group. As showed in Figure 9, the adhesive strength of DN hydrogel was 10.3±0.7 kPa which was significant higher than the control group (1.2±0.5 kPa). It demonstrated that the covalent bond between hydrogel and cartilage played a key role in progressing the adhesive strength. 21 ACS Paragon Plus Environment


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Figure 8 (A) Schematic of hydrogel’s adhesive property based on Schiff-base reaction. (B)Schematic of the experiment examining integration of tissue engineered hydrogel to native cartilage and the push-out test was utilized to determine the adhesive force.


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Figure 9 The adhesive strength of control hydrogel and DN hydrogel with significant difference. The control group was DA crosslinked non-adhesive hydrogel.

In order to further validate the interface integration of hydrogel and cartilage, the microstructure of construct was observed by SEM pictures. As showed in Figure 10 A and B, an obviously gap was found between hydrogel and hydrogel after freeze-dry process. It exhibited that the control group hydrogel couldn’t bond itself with the host cartilage tissue due to the absence of covalent bonds. In the contrary, in experiment groups Figure 10 C and D, the construct was integrated together without any cracks and the boundary between hydrogel and cartilage was impartible. From the microstructure images of construct, it demonstrated that the aldehyde-amine Schiff-base reaction created the integration of materials and cartilage tissue.

Figure 10 The SEM observation of integration of hydrogel and cartilage. (A, B) DA crosslinked non-adhesive hydrogel was regarded as a control group. Figure B was the 23 ACS Paragon Plus Environment


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enlarged area showed in figure A. (C, D) The DN hydrogel with adhesive property. Figure D was the enlarged area showed in figure C.

4. Conclusion As a result, the integration of DA click chemistry and acylhydrazone bond is an efficient way to form a multi-functional hydrogel. It not only maintains the perfect mechanical properties which is due to the DA click crosslinking, but also has the intelligent self-healing property due to the reversible acylhydrazone bond and good tissue-adhesive property due to the aldehyde-amine Schiff-base reaction. The hydrogels after healing process owned the same compressive modulus with the original hydrogel. The results demonstrate that DN hydrogel has more stable structure and higher compressive modulus compared with SN hydrogel no matter in acid solution or DI water. At the same time, the DN hydrogel owns switch on-off network structure stimulated by pH value which resulted in good self-healing and pH responsive behavior. This method enlarged the application of the DA reaction in the area of self-healing hydrogels. CORRESPONDING AUTHOR INFORMATION 1.

*Xiaodong Cao. School of Materials Science and Engendering, South China University of Technology, Guangzhou, 510641, PR China. Fax: +86-20-87111752; Tel: +86-20-22236066; E-mail: [email protected]


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2.

*Xiaofeng Chen. School of Materials Science and Engendering, South China University of Technology, Guangzhou, 510641, PR China. Fax: +86-20-22236083; Tel: +86-20-22236283; E-mail: [email protected]

Author Contributions The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript.

ACKNOWLEDGMENT

This work was financially supported by National Basic Research Program of China (973 Program) (No. 2012CB619100), National Natural Science Foundation of China (No. 51372085, 21404028), Guangdong-Hong Kong common technology bidding project (No.2013B010136003) ， Fundamental Research Funds for the Central Universities and the Science and Technology Program of Guangdong Province (Grant No. 2012A061100002).

Supporting Information. The characterization of HA-furan-CHO, the mechanical property, swelling ratio and pH responsiveness of DA vs DN hydrogel was supplied as Supporting Information.

REFERENCES 1. Bhosale, A. M.; Richardson, J. B., Articular Cartilage: Structure, Injuries and Review of Management. Br. Biomed. Bull. 2008, 87 (1), 77-95. 2. Tatari, H., The Structure, Physiology, and Biomechanics of Articular Cartilage: Injury and Repair. Acta Orthop Traumatol Turc 2007, 2, 1-5. 3. Solchaga, L. A.; Goldberg, V. M.; Caplan, A. I., Cartilage Regeneration Using Principles of Tissue Engineering. Clin Orthop Relat Res 2001, 391 (70), 161-170. 25 ACS Paragon Plus Environment


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 26 of 30

4. Schindler, O. S., Current Concepts of Articular Cartilage Repair. Acta Orthop Belg 2011, 77 (6), 709-726. 5. Tasdelen, M. A., Diels–Alder “click” Reactions: Recent Applications in Polymer and Material Science. Polym. Chem. 2011, 2 (10), 2133-2145. 7. Kolb, H. C.; Finn, M. G.; Sharpless, K. B., Click Chemistry: Diverse Chemical Function from a Few Good Reactions. Angew. Chem., Int. Ed. Engl. 2001, 40 (11), 2004-2021. 8. Goiti, E.; Huglin, M. B.; Rego, J. M., Some Properties of Networks Produced by the Diels–Alder Reaction Between Poly(styrene-co-furfuryl methacrylate) and Bismaleimide. Eur. Polym. J. 2004, 40 (2), 219-226. 9. Hong Liang, W.; Jun, Y.; Hui Juan, C.; Zhe, Y.; Cun Cai, M.; Kai, Y., Diels-Alder Reaction in Water for the Straightforward Preparation of Thermoresponsive Hydrogels. J Appl Polym Sci 2011, 120 (2), 974-980. 10. Imbesi, P. M.; Fidge, C.; Raymond, J. E.; Cauët, S. I.; Wooley, K. L., Model Diels–Alder Studies for the Creation of Amphiphilic Cross-Linked Networks as Healable, Antibiofouling Coatings. ACS Macro Lett. 2012, 1 (4), 473-477. 11. Liu, Y. L.; Hsieh, C. Y.; Chen, Y. W., Thermally Reversible Cross-linked Polyamides and Thermo-responsive Gels by Means of Diels–Alder Reaction. Polymer 2006, 47 (8), 2581-2586. 12. Wei, H. L.; Yang, Z.; Zheng, L.M.; Shen, Y.M., Thermosensitive Hydrogels Synthesized by Fast Diels-Alder Reaction in Water. Polymer 2009, 50 (13), 2836-2840. 13. Wei, H.L.; Yang, Z.; Chen, Y.; Chu, H.J.; Zhu, J.; Li, Z.C., Characterisation of N-vinyl-2-pyrrolidone-based Hydrogels Prepared by a Diels-Alder Click Reaction in Water. Eur. Polym. J. 2010, 46 (5), 1032-1039. 14. Wei, H. L.; Yang, Z.; Chu, H. J.; Zhu, J.; Li, Z. C.; Cui, J. S., Facile Preparation of Poly(N-isopropylacrylamide)-Based Hydrogels via Aqueous Diels–Alder Click Reaction. Polymer 2010, 51 (8), 1694-1702. 15. Nimmo, C. M.; Owen, S. C.; Shoichet, M. S., Diels-Alder Click Cross-Linked Hyaluronic Acid Hydrogels for Tissue Engineering. Biomacromolecules 2011, 12 (3), 824-830. 16. Polaske, N. W.; McGrath, D. V.; McElhanon, J. R., Thermally Reversible Dendronized Linear AB Step-Polymers via “Click” Chemistry. Macromolecules 2011, 44 (9), 3203-3210. 17. Sheridan, R. J.; Adzima, B. J.; Bowman, C. N., Temperature Dependent Stress Relaxation in a Model Diels-Alder Network. Aust J Chem 2011, 64 (8), 1094-1099. 18. Tan, H.; Rubin, J. P.; Marra, K. G., Direct Synthesis of Biodegradable Polysaccharide Derivative Hydrogels Through Aqueous Diels-Alder Chemistry. Macromol. Rapid Commun. 2011, 32 (12), 905-911. 19. Yu, F.; Cao, X.; Zeng, L.; Zhang, Q.; Chen, X., An Interpenetrating HA/G/CS biomimic Hydrogel via Diels-Alder Click Chemistry for Cartilage Tissue Engineering. Carbohydr Polym 2013, 97 (1), 188-195. 20. Yu, F.; Cao, X.; Li, Y.; Zeng, L.; Zhu, J.; Wang, G.; Chen, X., Diels-Alder Crosslinked HA/PEG Hydrogels with High Elasticity and Fatigue Resistance for Cell 26 ACS Paragon Plus Environment

Page 27 of 30

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Encapsulation and Articular Cartilage Tissue Repair. Polym. Chem. 2014, 5 (17), 5116-5123. 21. Yu, F.; Cao, X.; Li, Y.; Zeng, L.; Yuan, B.; Chen, X., An Injectable Hyaluronic acid/PEG Hydrogel for Cartilage Tissue Engineering Formed by Integrating Enzymatic Crosslinking and Diels-Alder "Click Chemistry". Polym. Chem. 2014, 5 (3), 1082-1090. 22. Khan, I. M.; Gilbert, S. J.; Singhrao, S. K.; Duance, V. C.; Archer, C. W., Cartilage Integration: Evaluation of the Reasons for Failure of Integration during Cartilage Repair. A review. Eur Cell Mater 2008, 16, 26-39. 23. Huey, D. J.; Hu, J. C.; Athanasiou, K. A., Unlike Bone, Cartilage Regeneration Remains Elusive. Science 2012, 338 (6109), 917-921. 24. Ramaswamy, S.; Wang, D. A.; Fishbein, K. W.; Elisseeff, J. H.; Spencer, R. G., An Analysis of the Integration Between Articular Cartilage and Nondegradable Hydrogel Using Magnetic Resonance Imaging. J Biomed Mater Res B Appl Biomater 2006, 77 (1), 144-148. 25. Gao, Y.; Luo, Q.; Qiao, S.; Wang, L.; Dong, Z.; Xu, J.; Liu, J., Enzymetically Regulating the Self-Healing of Protein Hydrogels with High Healing Efficiency. Angew Chem Int Edit 2014, 53 (35), 9343-9346. 26. Meng, H.; Xiao, P.; Gu, J.; Wen, X.; Xu, J.; Zhao, C.; Zhang, J.; Chen, T., Self-healable Macro-/microscopic Shape Memory Hydrogels Based on Supramolecular Interactions. Chem Commun (Camb) 2014, 50 (82), 12277-12280. 27. Lu, X. K.; Chan, C. Y.; Lee, K. I.; Ng, P. F.; Fei, B.; Xin, J. H.; Fu, J., Super-tough and Thermo-healable Hydrogel - promising for Shape-memory Absorbent Fiber. J. Mater. Chem. B 2014, 2 (43), 7631-7638. 28. Harada, A.; Takashima, Y.; Nakahata, M., Supramolecular Polymeric Materials via Cyclodextrin-Guest Interactions. Acc. Chem. Res. 2014, 47 (7), 2128-2140. 29. Wei, Z.; Yang, J. H.; Zhou, J.; Xu, F.; Zrinyi, M.; Dussault, P. H.; Osada, Y.; Chen, Y. M., Self-healing Gels Based on Constitutional Dynamic Chemistry and Their Potential Applications. Chem. Soc. Rev. 2014, 43 (23), 8114-8131. 30. Sichao, L.; Peng, H.; Huaping, X., Self-Healing Polymeric Materials. Prog. Chem. 2012, 24 (7), 1346-1352. 31. Meng, H.; Zheng, J.; Wen, X. F.; Cai, Z. Q.; Zhang, J. W.; Chen, T., pH- and Sugar-Induced Shape Memory Hydrogel Based on Reversible Phenylboronic Acid-Diol Ester Bonds. Macromol. Rapid Commun. 2015, 36 (6), 533-537. 32. Guo, W. W.; Lu, C. H.; Orbach, R.; Wang, F. A.; Qi, X. J.; Cecconello, A.; Seliktar, D.; Willner, I., pH-Stimulated DNA Hydrogels Exhibiting Shape-Memory Properties. Adv. Mater. 2015, 27 (1), 73-78. 33. Nejadnik, M. R.; Yang, X.; Bongio, M.; Alghamdi, H. S.; Van den Beucken, J. J. J. P.; Huysmans, M. C.; Jansen, J. A.; Hilborn, J.; Ossipov, D.; Leeuwenburgh, S. C. G., Self-healing Hybrid Nanocomposites Consisting of Bisphosphonated Hyaluronan and Calcium Phosphate Nanoparticles. Biomaterials 2014, 35 (25), 6918-6929. 34. Deng, G.; Li, F.; Yu, H.; Liu, F.; Liu, C.; Sun, W.; Jiang, H.; Chen, Y., Dynamic Hydrogels with an Environmental Adaptive Self-healing Ability and Dual Responsive Sol–Gel Transitions. ACS Macro Lett. 2012, 1 (2), 275-279. 27 ACS Paragon Plus Environment


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 28 of 30

35. Wiltsey, C.; Kubinski, P.; Christiani, T.; Toomer, K.; Sheehan, J.; Branda, A.; Kadlowec, J.; Iftode, C.; Vernengo, J., Characterization of Injectable Hydrogels Based on Poly(N-isopropylacrylamide)-g-chondroitin Sulfate with Adhesive Properties for Nucleus Pulposus Tissue Engineering. J. Mater. Sci.-Mater. Med. 2013, 24 (4), 837-847. 36. Theodoropoulos, J. S.; De Croos, J. N.; Park, S. S.; Pilliar, R.; Kandel, R. A., Integration of Tissue-engineered Cartilage with Host Cartilage: an in Vitro Model. Clin Orthop Relat Res 2011, 469 (10), 2785-2795. 37. Artzi, N.; Shazly, T.; Baker, A. B.; Bon, A.; Edelman, E. R., Aldehyde-Amine Chemistry Enables Modulated Biosealants with Tissue-Specific Adhesion. Adv. Mater. 2009, 21 (32-33), 3399-3403. 38. Wang, D.-A.; Varghese, S.; Sharma, B.; Strehin, I.; Fermanian, S.; Gorham, J.; Fairbrother, D. H.; Cascio, B.; Elisseeff, J. H., Multifunctional Chondroitin Sulphate for Cartilage Tissue–Biomaterial Integration. Nat. Mater. 2007, 6 (5), 385-392. 39. Ito, T.; Yeo, Y.; Highley, C. B.; Bellas, E.; Benitez, C. A.; Kohane, D. S., The Prevention of Peritoneal Adhesions by in Situ Cross-linking Hydrogels of Hyaluronic Acid and Cellulose Derivatives. Biomaterials 2007, 28 (6), 975-983. 40. Kamal H. Bouhadir; Debra S. Hausman; Mooney, D. J., Synthesis of Cross-linked Poly(aldehyde guluronate) Hydrogels. Polymer 1999, 40, 3575-3584. 41. Athens, A. A.; Makris, E. A.; Hu, J. C., Induced Collagen Cross-Links Enhance Cartilage Integration. PLoS ONE 2013, 8 (4), e60719. 42. Strehin, I.; Nahas, Z.; Arora, K.; Nguyen, T.; Elisseeff, J., A Versatile pH Sensitive Chondroitin Sulfate-PEG Tissue Adhesive and Hydrogel. Biomaterials 2010, 31 (10), 2788-97. 43. Pouyani, T.; Prestwich, G. D., Functionalized Derivatives of Hyaluronic Acid Oligosaccharides: Drug Carriers and Novel Biomaterials. Bioconjugate Chem. 1994, 5 (4), 339-347. 44. Prestwich, G. D.; Marecak, D. M.; Marecek, J. F.; Vercruysse, K. P.; Ziebell, M. R., Controlled Chemical Modification of Hyaluronic Acid: Synthesis, Applications, and Biodegradation of Hydrazide Derivatives. J. Controlled Release 1998, 53 (1–3), 93-103. 45. Bulpitt, P.; Aeschlimann, D., New Strategy for Chemical Modification of Hyaluronic Acid: Preparation of Functionalized Derivatives and Their Use in the Formation of Novel Biocompatible Hydrogels. J Biomed Mater Res 1999, 47 (2), 152-169. 46. Park, K. M.; Yang, J. A.; Jung, H.; Yeom, J.; Park, J. S.; Park, K. H.; Hoffman, A. S.; Hahn, S. K.; Kim, K., In Situ Supramolecular Assembly and Modular Modification of Hyaluronic Acid Hydrogels for 3D Cellular Engineering. ACS Nano 2012, 6 (4), 2960-2968. 47. Yeo, Y.; Highley, C. B.; Bellas, E.; Ito, T.; Marini, R.; Langer, R.; Kohane, D. S., In Situ Cross-linkable Hyaluronic acid Hydrogels Prevent Post-operative Abdominal Adhesions in a Rabbit Model. Biomaterials 2006, 27 (27), 4698-4705.


Page 29 of 30

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


48. Jeong, B.; Bae, Y. H.; Kim, S. W., Thermoreversible Gelation of PEG-PLGA-PEG Triblock Copolymer Aqueous Solutions. Macromolecules 1999, 32 (21), 7064-7069. 49. Hoffman, A. S., Hydrogels for Biomedical Applications. Adv. Drug Delivery Rev. 2012, 64, 18-23.



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Table of Content Graphic


Multifunctional Hydrogel with Good Structure Integrity, Self-Healing

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