A mussel-inspired double-crosslinked tissue adhesive intended for internal medical use

Acta Biomaterialia 33 (2016) 51–63

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A mussel-inspired double-crosslinked tissue adhesive intended for internal medical use Changjiang Fan a,b, Jiayin Fu a, Wenzhen Zhu a, Dong-An Wang a,⇑ a b

Division of Bioengineering, School of Chemical and Biomedical Engineering, Nanyang Technological University, Singapore 637457, Singapore Institute for Translational Medicine, College of Medicine, Qingdao University, Qingdao, PR China

a r t i c l e

i n f o

Article history: Received 26 August 2015 Received in revised form 27 January 2016 Accepted 1 February 2016 Available online 2 February 2016 Keywords: Tissue adhesive Dopamine Gelatin Genipin Hydrogel

a b s t r a c t It has been a great challenge to develop aldehyde-free tissue adhesives that can function rapidly and controllably on wet internal tissues with fine adhesion strength, sound biocompatibility and degradability. To this end, we have devised a mussel-inspired easy-to-use double-crosslink tissue adhesive (DCTA) comprising a dopamine-conjugated gelatin macromer, a rapid crosslinker (namely, Fe3+), and a longterm acting crosslinker (namely, genipin). As a mussel-inspired gluing macromer, dopamine is grafted onto gelatin backbone via an one-step reaction, the catechol groups of which are capable of performing strong wet adhesion on tissue surfaces. By addition of genipin and Fe3+, the formation of catechol–Fe3+ complexation and accompanying spontaneous curing of genipin-primed covalent crosslinking of gluing macromers in one pot endows DCTA with the double-crosslink adhesion mechanism. Namely, the reversible catechol–Fe3+ crosslinking executes an controllable and instant adhesive curing; while genipininduced stable covalent crosslinking promises it with long-term effectiveness. This novel DCTA exhibits significantly higher wet tissue adhesion capability than the commercially available fibrin glue when applied on wet porcine skin and cartilage. In addition, this DCTA also demonstrates fine elasticity, sound biodegradability, and biocompatibility when contacting in vitro cultured cells and blood. In vivo biocompatibility and biodegradability are checked and confirmed via trials of subcutaneous implantation in nude mice model. This newly developed DCTA may be a highly promising product as a biological glue for internal medical use including internal tissue adhesion, sealing, and hemostasis. Statement of Significance There is a great demand for ideal tissue adhesives that can be widely used in gluing wet internal tissues. Here, we have devised a mussel-inspired easy-to-use double-crosslink tissue adhesive (DCTA) that meets the conditions as an ideal tissue adhesive. It is composed of gelatin–dopamine conjugates – a gluing macromer, Fe3+ – a rapid crosslinker, and genipin – a long-term acting crosslinker. This DCTA is constructed with a novel complexation-covalent double-crosslinking principle in one pot, in which the catechol–Fe3+ crosslinking executes a controllable and instant adhesive curing, at the same time, genipin-induced covalent crosslinking promises it with long-term effectiveness in physiology conditions. This novel DCTA, with excellent wet tissue adhesion capability, fine elasticity, sound biodegradability, and biocompatibility, is a promising biological glue for internal medical use in surgical operations. Ó 2016 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.

1. Introduction Medical adhesives have facilitated surgical operations for several decades, particularly in the cases when traditional suturing ⇑ Corresponding author at: School of Chemical & Biomedical Engineering, Nanyang Technological University, 70 Nanyang Drive, Blk N1.3-B2-13, Singapore 637457, Singapore. E-mail address: [email protected] (D.-A. Wang). http://dx.doi.org/10.1016/j.actbio.2016.02.003 1742-7061/Ó 2016 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.

poses impractical or ineffective [1,2]. Fibrin glues (such as Tisseel) [3], albumin-glutaraldehyde adhesives (such as BioGlue) [4], and cyanoacrylates (such as Dermabond) [5] are well-known and currently used in many surgical procedures. However, the utilization of fibrin glues involves risks of blood-borne disease transmission and allergic reactions to patients [6]; the high toxicities of aldehyde-containing products severely limit in vivo applications of the revelant adhesive products [7,8]. Currently, there is few commercially available tissue adhesive that can be

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widely used for in vivo applications such as internal tissue or organ adhesion and hemostasis [9]. Mussel adhesive proteins (MAPs) secreted by marine mussels are known capable of building complexes of byssal threads and adhesive plaques to anchor their own bodies firmly onto all surfaces [10]. This finding fascinates the researchers particularly due to their biocompatibility and unique wet adhesion capability [11–14]. Studies have revealed that the excellent affinity of MAPs to wet surfaces can be primarily attributed to the unique catecholic amino acid, L-3,4-dihydroxyphenylalanine (DOPA) [13,15,16]; the pendant catechol group of which is directly responsible for the moisture-resistant adhesion [13,17]. The adhesion mechanism consists of two simultaneous procedures: first, the catechol moieties generate strong non-covalent binding interactions to various surfaces [13,17,23]; second, as a catechol-containing adhesive biopolymer, MAPs are crosslinked by various means, among which the natural way is to convert catechol groups into quinones by polyphenol oxidase enzyme in mussels and then achieve covalent self-crosslinking [13,16,23]. On top of the self-polymerization, strong and reversible coordination interactions between catechol groups and metal ions from ambient environment, such as Fe3+, Cu2+, Ti3+, are widely considered to further strengthen the crosslink to achieve the final hardness [23,45]. Particularly, the catechol– Fe3+ coordination complexation can be rapidly established, which is reversible to serve as a sacrificial load-bearing crosslink to facilitate the extensibility of byssus and play a crucial role in mussels’ adhesion to wet surfaces in turbulent environments [24,26,27,45]. In order to apply these mechanisms to adhesion of biological tissues or organs, various mussel-inspired adhesive (bio)polymers have been developed by incorporating and adopting typical catechol-containing molecule, like dopamine [19,20] or hydrocaffeic acid [21,22], as the functional component for wet-resistant tissue adhesives. Chung et al. have synthesized DOPA-containing adhesive terpolymers that can be cross-linked through the reaction between N-hydroxysuccinimide ester and thiol group without sacrificing DOPA moieties. The adhesion tests of the resultant adhesives indicate the catechol moieties provide a driving force for its adhesion to wet porcine skin [25]. Further studies particularly on crosslinking procedure have shown the catechol group can be oxidized under oxidative or alkaline conditions to produce quinones [9,13]. Several polymeric tissue adhesives are correspondingly developed, and the catechol groups of which are oxidized into quinones by using oxidizing reagents (e.g. sodium periodate, hydrogen peroxide) [9,20,21]. By this means, not only does the quinone trigger intermolecular cross-linking of adhesive polymers but also leads to strong adhesion to tissues through reaction with available nucleophile groups (e.g.
NH2,
SH) on tissues’ surfaces [9,13,20,21]. In addition, the catechol–Fe3+ coordination complexation is also exploited [18,24,26–29]. Previously, it has been adopted to fabricate self-healing hydrogels due to the reversibility [28]. Choi et al. develop a hemostatic hydrogel based on rapidly achieved catechol–Fe3+ coordination crosslinking [29]. The intriguing characteristics of catechol–Fe3+ crosslinking, namely the combination of rapid establishment and reversibility, make it a promising strategy for fabricating tissue adhesives. In this study, we have developed a mussel-inspired synthetic double-crosslink tissue adhesive (DCTA) comprising a dopamineconjugated gelatin macromer, a rapid crosslinker (namely, Fe3+), and a long-term acting crosslinker (namely, genipin). As a mussel-inspired gluing macromer, the dopamine served as interfacial adhesion segment, is grafted onto gelatin backbone via EDC/ NHS coupling chemistry. By addition of Fe3+, rapid formation of catechol–Fe3+ complexation endows the first crosslinking mechanism of gluing. Albeit instant as it is, the reversibility of this complexation reaction makes it unstable afterwards. A second crosslinker, genipin, is applied at the same time. Genipin is a nat-

ural product and nontoxic crosslinker that can spontaneously react with the primary amino groups in polymers (such as gelatin, chitosan, polylysine) to form distinctive blue pigments [30–33]. Genipin has been considered as a promising crosslinker to replace glutaraldehyde for the preparation of biomaterials due to its advantage of biocompatibility [31]. However, the genipin-based crosslinking process needs to cost much time (tens of minutes to hours) that is unacceptable to cure adhesive polymers under clinical conditions [32,33]. Single usage of either Fe3+ or genipin as crosslinker of tissue adhesive cannot meet the demand for the rapid cross-linking and long-term effectiveness in clinical conditions. This novel DCTA, mimicking the double-crosslinking of MAPs in the byssal cuticle [9,23], is fabricated in one pot via a doublecrosslink mechanism. Namely, catechol–Fe3+ complexation executes an instant adhesive curing; while genipin-primed covalent crosslinking promises it with long-term effectiveness under physiological conditions. The formation, adhesion strength, physical properties, degradation profile as well as in vitro cytocompatibility and blood compatibility of DCTA are evaluated in detail. The subcutaneous implantation of DCTA is also carried out to further assess its biocompatibility and degradation under in vivo conditions.

2. Materials and methods 2.1. Synthesis of gelatin–dopamine gluing macromer Gelatin–dopamine conjugate is synthesized as a typical gluing macromer through ethyl-dimethyl-aminopropylcarbodiimide (EDC, Sigma–Aldrich) and N–hydroxy-succinimide (NHS, Sigma–Aldrich) coupling chemistry. Briefly, gelatin (2.0 g, Type A from porcine skin, Sigma–Aldrich) is dissolved in 100 mL of phosphate buffered saline (PBS, pH 7.4) solution at 60 °C. EDC (0.5 g) and NHS (0.3 g) is added into the solution and pH value of the mix solution is adjusted to 5.0. After 30 minutes’ stirring, 1.0 g of dopamine hydrochloride (Sigma–Aldrich) dissolved in 2 mL of deionized (DI) water is added dropwise and pH value of the reaction solution is maintained from 5.0 to 6.0 for 24 h at 37 °C. Subsequently, the reaction solution is dialyzed in DI water for two days and then lyophilized.

2.2. Characterization of gelatin–dopamine gluing macromer To confirm the successful grafting of dopamine onto gelatin, the resultant gelatin–dopamine conjugate is analyzed by proton nuclear resonance spectroscopy (1H NMR). 1.5% (g/mL) of gelatin–dopamine solution in deuterium oxide (D2O) is transferred into a 5 mm NMR tube and recorded on a Bruker Avance-300 NMR spectrometer. Besides, the 1H NMR spectra of parent materials, namely gelatin and dopamine, are also collected. Furthermore, the presence of unoxidzied catechol groups in gelatin–dopamine conjugate is assessed by UV–Vis spectroscopy [9,29]. 10% (g/mL) of gelatin and gelatin–dopamine solutions in DI water are scanned, respectively, at wavelengths from 250 nm to 500 nm using a Nanodrop 2000c spectrophotometer (Thermo Scientific). The content of catechol group is determined with Arnow’s method [34], in which dopamine is used as standard. Typically, 1.0 mL of nitrite-molybdate reagent, namely 10% (g/mL) of sodium nitrite (Sigma–Aldrich) and 10% (g/mL) sodium molybdatein (Sigma–Aldrich) in hydrochloric acid (0.5 M), is added into 1.0 mL of gelatin–dopamine solution (0.15%, g/mL) in DI water. The reaction solution is shaken for five minutes at 100 rpm at room temperature. The pH value and volume of the solution is adjusted to 7.0 and 5 mL, respectively, using sodium hydroxide solution

C. Fan et al. / Acta Biomaterialia 33 (2016) 51–63

(1.0 M) and DI water. The absorbance at 520 nm is measured using the Nanodrop 2000c spectrophotometer (Thermo Scientific). 2.3. Fabrication of tissue adhesive The gelatin–dopamine is dissolved in PBS solution at 37 °C to form 15% (g/mL) gluing macromer solution. To prepare Fe3+-single-crosslink tissue adhesive (Fe3+-SCTA), 40 lL of gluing macromer solution is transferred into a cylindrical mold (diameter 4 mm), and the Fe3+-SCTA gel can be formed within seconds after adding 4 lL of FeCl3 (100 mM) solution. Subsequently, they are mixed with a pipette, forming a homogenous Fe3+-SCTA gel. For the preparation of genipin-single-crosslink tissue adhesive (genipin-SCTA), the genipin solution (10%, g/mL, in 70% ethanol) is added into the gluing macromer solution (15%, g/mL) to make a mixture solution containing 0.5% (g/mL) genipin. The mixture solution (40 lL) is transferred into a cylindrical mold and placed in a humidified incubator at 37 °C for 2 h to form genipin-SCTA. For the fabrication of double-crosslink tissue adhesive (DCTA), the macromer solution (40 lL) containing 0.5% (g/mL) genipin is transferred into a cylindrical mold, followed by adding 4 lL of FeCl3 solution (100 mM). The sticky gel is formed rapidly in the mold, and then which is placed in the humidified incubator for 2 h to complete the covalent crosslink with genipin. 2.4. Stability of tissue adhesives The sticky gel adhesives are prepared via Fe3+-crosslink of 50 lL of gluing macromer solution (15%, g/mL) with or without genipin (0.5%, g/mL) in 1.5 mL centrifuge tubes, respectively; the DCTA is also fabricated, as described above, from 50 lL of gluing macromer solution in a 1.5 mL centrifuge tube. Subsequently, 0.8 mL of simulated body fluid (SBF) supplemented with 0.5% (v/v) fetal bovine serum (FBS, PAA Laboratories) is added into each tube. The changes in appearances for each sample are observed as a function of incubation time at 37 °C.

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sues, fresh porcine skin and articular cartilage without any purification are chosen as adherends to closely mimic clinical conditions. The porcine skin and cartilage is cut and trimmed into strips with approximately 5  1.5 cm and 2.5  1.2 cm sizes, respectively, and wetted by immersing them into water before use. Four samples for each test are examined. The adhesion tests are carried out under different gluing conditions (described in detail below), in which the adhesion strength is calculated by dividing the maximum load by the corresponding overlapping area [25]. The gluing macromer solution (15%, g/mL) containing genipin (0.5%, g/mL) is applied to the fat layer (inside) of each wet porcine skin, followed by adding drops of FeCl3 solution (100 mM). The solution applied parts, namely working area, are gently pressed together with fingers for 5–10 s. Subsequently, the adhesion tests are performed to get the ‘‘rapid-curing skin-fat gluing” strength of DCTA. The samples, whose fat layers are glued with Fe3+ crosslinking, are placed in a humidified incubator at 37 °C for 2 h to complete the covalent cross-linking with genipin, forming Fe3+-genipin double-crosslink gluing samples, and then the adhesion tests are carried out to get the ‘‘2-hour-curing skin-fat gluing” strength of DCTA. Besides, some samples, achieved by fat-layer double-crosslink gluing, are immersed in SBF solution containing 0.5% (v/v) FBS for 24 h on a rotary shaker (80 rpm), and the adhesion tests are performed to obtain the ‘‘24-hour-curing skin-fat gluing” strength of DCTA. In addition, the gluing macromer solution containing genipin is applied to the fat layer of each wet porcine skin followed by pressing together. They are incubated for 2 h to complete cross-linking, achieving genipin-SCTA gluing samples, and then the adhesion tests are carried out to get the ‘‘genipinSCTA gluing” strength. Besides, the wet collagen layer (outside) of porcine skins and cartilages are double-crosslink glued with DCTA, via the procedure same to 2-hour-curing double-crosslink gluing of the fat layers of porcine skins; the adhesion tests are carried out to get the ‘‘2-h-curing skin-collagen gluing” and ‘‘2-hour-curing cartilage gluing” strength, respectively, which are contrasted to the ‘‘2-hour-curing skin-fat gluing” strength.

2.5. Rheological measurement 2.7. Swelling and degradation Viscoelastic properties are determined by using a rheometer (TA Instruments, Model AR2000ex) equipped with 20 mm diameter stainless steel parallel plate geometry. For Fe3+-SCTA, the data are recorded as soon as the addition of FeCl3 solution into the gluing macromer solution. The samples of DCTA are freshly prepared before rheological tests. The operation temperature is maintained at 37 °C. To ensure the rheological measurements within a linear viscoelastic range, the dynamic strain sweep is conducted prior to the frequency sweep, and the strain is determined to be 5%. Storage modulus (G0 ) and viscous modulus (G00 ) is measured by performing frequency sweeps between 0.01 and 1.0 Hz. The time-sweep experiments are performed at 37 °C to monitor the gelation process of DCTA, in which the frequency and strain is set at 1.0 rad/s and 5%, respectively. The gluing macromer solution containing genipin is loaded, and the data are recorded as soon as the addition of FeCl3 solution. For a comparative study, the timesweep measurements are also carried out to monitor the gelation process of genipin-SCTA. The gluing macromer solution containing genipin is loaded, and the data are recorded without adding FeCl3 solution. 2.6. Tissue adhesion test The adhesion properties are determined by lap shear tests using an Instron mechanical tester (Model 5543) equipped with a 100 N load cell [9,20,25]. The tests are performed at a tensile rate of 1.0 mm/min. Considering the biological similarity to human tis-

DCTA is freshly prepared from 100 lL of gluing macromer solution in cylindrical molds (diameter 6.5 mm) and weighed to find the initial mass (Wi). For determination of swelling kinetics, four samples are immersed in PBS solution. The samples are weighed at pre-determined time intervals (Ws
t) until reaching swelling equilibrium (W1). The swelling percentage of DCTA at time t is determined with the Eq. (1):

Swelling ð%Þ ¼ W s
t =W i  100%

ð1Þ

To measure water content, the samples, reached equilibrium in above swelling experiment, are dried under vacuum to obtain the dry weight (Wdry). The equilibrium water content is calculated using the Eq. (2):

Equilibrium water content ð%Þ ¼ ðW 1
W dry Þ=W 1  100%

ð2Þ

Degradability study of DCTA is performed in enzyme solution according to previous study [48]. The freshly prepared samples are placed into a 50 mL centrifuge tube containing 10 mL of trypsin-EDTA solution (25,200, Life Technologies) that is diluted one fold with high glucose DMEM (Gibco), and incubated at 37 °C on a rotary shaker (150 rpm). At each predetermined time point, three samples are taken out and weighed (Wd
t) after wiping off the solution on the surfaces. The remaining weight fraction of DCTA is determined according to the following Eq. (3):

Remaining weight ð%Þ ¼ W d
t =W i  100%

ð3Þ

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2.8. Cell culture Porcine chondrocytes (PCCs) are isolated from porcine articular cartilage according to previously established protocol [35], and cultured in chondrocyte culture medium (high glucose Dulbecco’s Modified Eagles Medium (DMEM, Gibco) supplemented with 20% (v/v) FBS (‘‘Gold” Standard, PAA Laboratories), 0.4 mM proline (Sigma–Aldrich), 0.1 mM nonessential amino acids (Gibco), 50 mg/L ascorbic acid (Sigma–Aldrich), 10 mM HEPES (Gibco), and 1% penicillin–streptomycin (Invitrogen)) at 37 °C in 5% CO2 [36]. PCCs at passage one are used in this study. Human dermal fibroblasts (HDFs) are purchased from Cambrex (North Brunswick, NJ) and propagated in high glucose DMEM (Gibco) supplemented with 10% (v/v) FBS (‘‘Gold” Standard, PAA Laboratories), 1.5 g/L sodium bicarbonate, 1% (v/v) penicillin–streptomycin (Invitrogen) at 37 °C in an incubator with a 5% CO2. Human mesenchymal stem cells (hMSCs) are purchased from Lonza, cultured in high glucose DMEM (Gibco) supplemented with 15% fetal bovine serum (‘‘Gold” Standard, PAA Laboratories) and 1% penicillin/streptomycin (Invitrogen), and kept in an incubator at 37 °C with 5% CO2. hMSCs at passage eight are used in this study. 2.9. Cytotoxicity studies An indirect contact method, according to ISO10993 standard test, is employed to evaluate the cytotoxicity of DCTA [37], using PCCs and HDFs as model cells. All experiments are carried out in triplicate. Cellular growth and survival is measured by MTT cell viability assay that is based on the reduction of tetrazolium dye (MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide, Sigma–Aldrich) to insoluble purple formazan by viable cells. The formation of formazan can reflect the number of viable cells. The DCTA is prepared as mentioned above in each 15 mL centrifuge tube from 1.0 mL of gluing macromer solution. The bovine serum albumin (BSA)-glutaraldehyde (GA) tissue adhesive is also synthesized according to the reported method [38]. 5 mL of culture medium for PCCs and HDFs culture is filled into the tubes, respectively, and kept in 4 °C for seven days to fully extract the un-reacted materials. These extract solutions are filtered through 0.22 lm filters, respectively. The extract solution of DCTA is diluted with corresponding culture medium (for PCCs or HDFs culture) to make the dilutions containing the initial extract solution of 100%, 50%, and 25% volume percentage, which are named as Extract-100, Extract-50, and Extract-25, respectively. The corresponding extract solution of BSA-GA tissue adhesive is named as ‘‘BioGlue” and served as the positive control, while the corresponding pure culture medium (named as ‘‘Pure medium”) for PCCs and HDFs culture is employed as the negative control, respectively. The PCCs and HDFs are seeded in 96-well plate at a density of 3  103 cells per well respectively, and cultured for 24 h in 200 lL of corresponding culture medium. Thereafter, the culture medium is replaced with corresponding extract solution dilutions or control solutions, which are changed every other day. At predetermined time points, the MTT assay is carried out. Briefly, the culture solution is replaced with a mix solution of 180 lL of fresh DMEM and 20 lL of 5 mg/mL MTT in PBS solution. The plate is incubated at 37 °C for 4 h, the medium is removed, and 200 lL of DMSO is added to dissolve the formazan crystals. The optical density (OD) values are measured at the wavelength of 570 nm using a microplate reader (Thermo Scientific). 2.10. Cell adhesion and proliferation Cell adhesion and proliferation on as-prepared DCTA is studied by the monolayer culture of hMSCs [39]. Briefly, the DCTA is fabri-

cated from 40 lL of gluing macromer solution in a 96-well plate. Cell suspensions are seeded on top of the DCTA at a density of 3  103 cells/well, and then incubated at 37 °C in 5% CO2 atmosphere. Cell proliferation on samples in triplicate is determined with MTT assay as mentioned above. Besides, the cells are stained with the live/dead kit (Invitrogen) at predetermined time points. Briefly, 200 lL of DMEM supplemented with 0.1 lL of calcein-AM and 0.4 lL ethidium homodimer is added into each well, followed by 30 min of incubation at 37 °C in darkness. The samples are observed under a fluorescence microscope (OLYMPUS-IX71 with BH2-RFL-T3 Fluorescence lamp).

2.11. Hemolysis assay The interactions between blood and freshly prepared DCTA or its extract solution are investigated with hemolysis tests in vitro [40,41]. Approximately 5 ml whole blood is drawn from healthy volunteers (human who are within the ordinary range of clinical and laboratory parameters, not known to suffer any significant hematologic disease, and have given valid consent to this study) into the syringe preloaded with heparin sodium. The DCTA synthesized from 200 lL of gluing macromer solution in cylindrical molds (diameter 6.5 mm) and aforementioned extract solution of DCTA (200 lL) in HDFs culture medium is transferred into 4 mL of 0.9% NaCl solution in 15 mL centrifuge tubes, respectively. 200 lL of whole blood is added to each tube, and they are incubated for 60 min at 37 °C. Positive and negative controls are produced by adding 200 lL of whole blood into 4 mL of DI water and 0.9% NaCl solution, respectively. The samples in triplicate are centrifuged at 1500 rpm for 5 min. The optical density (OD) of the supernatant is measured at the wavelength of 545 nm using a microplate reader (Thermo Scientific), and the hemolysis is calculated calculated using the Eq. (4):

Hemolysis ð%Þ ¼

OD of sample
OD ð
Þ control  100% OD ðþÞ control
OD ð
Þ control

ð4Þ

2.12. In vivo study All animal experiments are performed under guidelines approved by the Institutional Animal Care and Use Committees, SingHealth, Singapore. The in vivo biocompatibility and degradation of DCTA is evaluated by implanting them in the subcutaneous tissues of the back of four-week-old NCr nude mice. Each mouse received four DCTA samples that are freshly prepared from 60 lL of gluing macromer solution in cylindrical molds (diameter 5 mm) [42]. Following euthanasia, the implantation sites are harvested. For degradation assessment, the DCTA explants are carefully dissected from surrounding tissue, dried, and weighed. The average weight of samples on day 4 (W0) is defined as the initial weight and assigned a value of 1.0. The remaining weight (Wt) of samples on day 14 and 28 are normalized to W0, namely, remaining weight (%) = Wt/W0  100%. For histological evaluation, samples are fixed in 4% (g/mL) paraformaldehyde solution, dehydrated through a series of graded alcohol solution, cleared in xylene, and embedded in paraffin overnight. 6 lm sections are mounted on microscope slides and dried for one hour in 50 °C oven. After rehydrating, the sections are stained using hematoxylin and eosin (H&E) for analyzing the degree of tissue response, typically inflammation and fibrosis [43]. The inflammation is characterized based upon the level of neutrophils, giant cells, and lymphocytes, and fibrosis is identified according to collagen deposition [44].

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2.13. Statistical analysis Results were expressed as mean ± standard deviation, with at least three samples. Student’s t-test was performed to analyze the statistical significance between the results of two groups, and a statistically significant difference was defined as p 6 0.05. 3. Results and discussion 3.1. Fabrication of tissue adhesive The gelatin–dopamine conjugate is synthesized by grafting dopamine onto gelatin backbone via EDC/NHS chemistry (Fig. 1a), which serves as the typical gluing macromer for the fabrication of tissue adhesives. Gelatin, derived from collagen, is a biocompatible, bio-absorbable, and inexpensive material; it is also the main constituent of gelatin–resorcinol–formaldehyde/glu taraldehyde (GRF/GRFG) adhesive that have been used clinically for decades in Europe and Japan [17]. As shown in Fig. 1b, compared with gelatin solution, the enhanced absorption of UV light at 280 nm wavelength is observed in the solution of the resultant gelatin–dopamine conjugate, demonstrating the successful incorporation of dopamine (namely, catechol groups) [9]. Moreover, no peaks appear at the wavelength of 395 nm, which indicates the catechol groups in the gelatin–dopamine conjugate are not oxidized to quinones during the synthesis process [16]. The 1H NMR spectra further confirm the presence of dopamine in the gelatin–dopamine conjugate. In Fig. 1c, the peak at d 2.75 ppm can be assigned to protons of methylene group (marked with an asterisk) close to phenyl group in dopamine. It is also observed in the spectrum of gelatin–dopamine conjugate (Fig. 1e), but not appears in the spectrum of gelatin (Fig. 1d). The content of catechol groups in the gelatin–dopamine conjugate is 3.7 lg/mg, determined by Arnow’s method [29,34]. A great deal of catecholcontaining polymers have been developed for preparing tissue adhesives by a certain single cross-linking strategy [17–22].

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Typically, Zhang et al. synthesize a hyperbranched poly (dopamine-co-acrylate) and which is explored to prepare tissue adhesive with different curing agents such as sodium periodate, hydrogen peroxide, FeCl3, fibrinogen, or horseradish peroxidase. Notably, the presence of diacrylate in this kind of adhesive macromer endows it with another capability of rapid photo-crosslinking, however, the limited penetrability of UV light inhibits the internal applications of the tissue adhesive [20]. We are greatly interested in Fe3+ induced crosslinking due to its charming characteristics, namely rapid curing and reversibility. As shown in Fig. 2a, the final gelatin–dopamine gluing macromer product is light yellowish foam that is not adhesive in the solid state. The gluing macromer foam can be dissolved in water to form a viscous solution (Fig. 2b). The Fe3+-SCTA is synthesized herein by adding FeCl3 solution into gluing macromer solution. Considering the fact that the molar ratio of catechol group and Fe3+ affect the structure of catechol–Fe3+ complex [28], the dosage of FeCl3 is evaluated in the preliminary study (data not shown). The results indicate the sticky brown Fe3+-SCTA can be generated even though FeCl3 concentration is elevated to three-fold higher than that currently used. Herein, the gelation of the gluing macromer solution can be achieved rapidly, resulting in sticky Fe3+-SCTA (Fig. 2c). At the same time, the color is deepened from light to dark brown. The sol–gel and color transitions can be attributed to the formation of catechol–Fe3+ complexes, typically tris-catechol–Fe3+ complex (Fig. 2d), which has also been demonstrated in previous study [29]. Because internal human organs/tissues are always wet with body fluid [25], we investigate the stability of the synthetic Fe3+-SCTA in the simulated body fluid (SBF) solution containing 0.5% (v/v) FBS. As seen in Fig. 2e-left, the Fe3+-SCTA, freshly prepared at the bottom of a 1.5 mL centrifuge tube, is brown and the SBF solution is nearly colorless. After 3 hours’ immersion (Fig. 2f-left), the Fe3+-SCTA keeps intact, whereas, whose color become lighter compared to that at the initial stage. Meanwhile, the SBF solution presents a very light brown color. However, interestingly, the Fe3+-SCTA is completely dissolved by 24 hours’

Fig. 1. (a) Synthesis scheme of gelatin–dopamine conjugate. (b) UV–vis spectra of gelatin solution (10%, g/mL) and gelatin–dopamine solution (10%, g/mL). 1H NMR spectra of dopamine (c), gelatin (d), and gelatin–dopamine (e); the characteristic proton chemical shift of methylene groups (asterisk-marked) is shown by a vertical dashed-line (c, d, e) and an arrow (e).

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Fig. 2. Property of the gelatin–dopamine conjugate: Dry foam (a) and viscous solution (15%, g/mL) in water (b). The sticky SCTA (c) is achieved within 10 s through forming catechol–Fe3+ complexes, typically tris-catechol–Fe3+ (d). Appearance changes of sticky gel adhesives formed by Fe3+-crosslinking of the gelatin–dopamine solution without (left samples in e, f, and g) or with genipin (right samples in e, f, and g), and DCTA (middle samples in e, f, and g) within SBF solution containing 0.5% FBS after 0 h (e), 3 h (f), and 24 (g) hours’ incubation at 37 °C. In e, f, and g; the upper row is the inverted tubes of the corresponding tubes in the bottom row. (h) Schematic fabrication of DCTA: in one pot, the gelatin–dopamine gluing macromers are first rapidly crosslinked by Fe3+ (first crosslink), at the same time, which are gradually crosslinked with genipin (second crosslink).

incubation, at the same time, the color of the SBF solution become deeper compared with that after 3 hours’ immersion. These phenomena indicate Fe3+ ions are gradually lost from the gel system into the SBF solution containing FBS. However, the similar dissolution of Fe3+-SCTA is not observed in the SBF solution without FBS (data not shown). The dissolution of Fe3+-SCTA in the SBF solution containing FBS might be attributed to Fe3+ binding to serum proteins in the SBF solution. For examples, the serum transferrin existed in body fluids can strongly bind to Fe3+ ions [46]. Therefore, albeit with rapid gelation, the Fe3+-SCTA cannot serve as an ideal tissue adhesive due to lacking long-term effectiveness under in vivo conditions. Actually, the natural MAPs solution, secreted from a mussel, is also solidified through intermolecular covalent crosslinking after oxidizing catechol groups to quinones with the polyphenol oxidase enzyme [16,23]. By learning from mussels, the DCTA is developed by adding a stable covalent crosslinking of gelatin–dopamine chains achieved with genipin. The molar con-

centration of primary amine in gelatin–dopamine should be similar to that in gelatin for two reasons: (1) The dopamine is grafted to the carboxyl groups of gelatin, and (2) the grafted amount of dopamine is relatively low as mentioned above. Therefore, the concentration of genipin (0.5%, g/mL) is selected by referring previous studies, in which genipin is used for crosslinking gelatin hydrogels [31–33]. For the preparation of DCTA, the genipin can be firstly added into the gelatin–dopamine solution since the genipininduced crosslinking needs at least tens of minutes [32,33], and the mixture solution is then subjected to the rapid gelation with Fe3+ ions as the synthesis of Fe3+-SCTA. After about 2 hours’ incubation at 37 °C, the observation of blue pigments indicates the achievement of the genipin-induced covalent crosslinking of gelatin–dopamine chains. As shown in Fig. 2e-middle, freshly prepared DCTA appears dark blue. It keeps intact across the 24 h of incubation in the SBF solution that gradually turns into blue (Fig. 2g-middle). The color

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transition of SBF solution from colorlessness to blue color can be attributed to the diffusion of non-crosslinked gelatin molecules that have reacted with genipin out of the DCTA. These results demonstrate the DCTA is stable in tissue fluids; that is to say, the DCTA will provide long-term effective tissue adhesion under physiological conditions. The stepwise formation of DCTA from Fe3+-SCTA is further evaluated in the presence of SBF solution containing 0.5% FBS. As seen in Fig. 2e-right, the sticky gel adhesive is rapidly obtained via Fe3+-induced gelation of gluing macromer solution (15%, g/mL) containing genipin (0.5%, g/mL) in a 1.5 mL centrifuge tube, and followed by the addition of the SBF solution. Like Fe3+-SCTA, the freshly synthesized gel adhesive containing genipin molecules also exhibits a brown color (Fig. 2e-right). After 3 hours’ incubation (Fig. 2f-right), interestingly, the thin blue pigments are observed on the gel surface contacted with SBF solution, and then the gel fully changed from brown to dark blue after 24 hours’ incubation (Fig. 2g-right). These results indicate the gluing macromers in Fe3+-SCTA can be gradually crosslinked by genipin even though the Fe3+-SCTA is soaked in SBF solution. The one-pot fabrication mechanism of DCTA networks is illustrated in Fig. 2h. The gelatin–dopamine gluing macromers can be rapidly crosslinked through the catechol–Fe3+ complexation at the presence of Fe3+ ions; at the same time, the second crosslink of the gluing macromers is gradually achieved via the reaction between genipin and the primary amino groups of gluing macromers [32,33], forming DCTA. The rheological studies are carried out to characterize the formation and viscoelastic properties of DCTA [29]. Time sweep

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measurements are conducted to investigate the gelation process of DCTA (Fig. 3b). The crossover from the viscous behavior (viscous modulus (G00 ) > storage modulus (G0 )) to an elastic response (G0 > G00 ) could be regarded as the onset of gelation; the time at the crossover point is considered as gelation time. When FeCl3 solution is added as the rapid crosslinker, the gelation time is significantly shortened from about seven minutes (Fig. 3a, genipinSCTA system) to ten seconds (Fig. 3b, DCTA system). In addition, the G0 is increased over time for the DCTA (Fig. 3b), at the same time, the blue pigments appear gradually. All these results indicate the formation of double crosslinks for DCTA. The viscoelastic properties of gelatin–dopamine solution, Fe3+-SCTA, and as-prepared DCTA are evaluated. As shown in Fig. 3c, when FeCl3 solution is added to the gluing macromer solution, its storage modulus (G0 ) and viscous modulus (G00 ) are obviously enhanced compared with the gluing macromer solution. Besides, the G0 and G00 values at 1.0 Hz drastically increase from 0.37 Pa and 1.03 Pa of gluing macromer solution to 47.28 Pa and 31.04 Pa of the resultant Fe3+-SCTA, respectively. The results indicate that the rapid gelation of gluing macromer solution via the formation of catechol–Fe3+ complexes, and which greatly improves the viscoelastic properties [26–29,45]. This instant gelation property for tissue adhesive is highly desirable in practical applications, particularly for controlling bleeding and minimizing surgery time [17]. In addition, as expected, the G0 and G00 values of DCTA are much higher than those of Fe3+-SCTA, especially for G0 , again demonstrating the polymer chains are crosslinked with the irreversible covalent bonds. The superior elasticity and viscosity of DCTA is important to gluing soft

Fig. 3. (a) The effect of genipin-primed covalent crosslink on the gelation of genipin-SCTA. (b) The combined effect of Fe3+ and genipin on the gelation of DCTA. (c) Storage modulus (G0 ) and viscous modulus (G00 ) of DCTA (double-crosslinked tissue adhesive), Fe3+-SCTA (gelatin–dopamine + Fe3+), and gluing macromer solution (gelatin–dopamine solution) as a function of frequency (Hz) at 37 °C.

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tissues, which can better bear load and dissipate elastic energy by viscous deformation and keep in harmony with flexible soft tissues [9]. The usage and probable working principle of DCTA is shown in Fig. 4. The mixture solution of gluing macromer (15%, g/mL) and genipin (0.5%, g/mL) is applied on the surfaces of porcine skin that is acted as a typical tissue (Fig. 4a). Because of the surface tension, the solution is readily filmed on the surfaces of tissues even in the upright position. At the same time, the catechol groups of gluing macromers can be interacted with tissue surfaces by strong noncovalent interactions [17,25]. The gluing macromers are rapidly cross-linked through catechol–Fe3+ coordination complexation on each tissue as soon as the addition of FeCl3 solution, forming sticky brown hydrogel (Fig. 4b). Both tissues are glued together instantaneously after simple overlapping and gently pressing for 5–10 s (Fig. 4c and e), which is attributed to the reestablishment of stiffness and cohesiveness (self-healing property) of the sticky gel based on catechol–Fe3+ crosslinking [28]. This manipulation procedure shows the characteristics of DCTA, namely rapid and controllable gluing for substrates. After then, the covalent crosslinking with genipin is gradually achieved within 2 h (Fig. 4d and f). In physiological environments, the Fe3+ ions in DCTA may gradually lose (Fig. 4g) owing to the complexation by proteins (such as transferrin) existed in body fluids [46], however, the tissues can be stably glued together due to the presence of covalent genipin crosslinking and the strong wet-resistant interactions of catechol groups with tissues [51]. Taken together, the Fe3+-SCTA possesses the virtue of rapid and controllable gelation but lacks stability under in vivo conditions. However, significantly, the Fe3+-genipin DCTA not only bears the advantages of Fe3+-SCTA but also achieves superior elasticity and long-term effectiveness, namely structural stability, in physiological environments. The curing of DCTA mimics the formation of byssal cuticles in mussels that is achieved by both selfcrosslinking and catechol–metal interactions of MAPs [9,23]. 3.2. Swelling and degradation The swelling behaviors and degradability are important structural parameters for tissue adhesives. The equilibrium water

content of the DCTA is 88.2 ± 1.0%, this high water content is beneficial to increasing its biocompatibility. Fig. 5a shows the swelling kinetics profile of as-prepared DCTA in PBS solution. The swelling kinetics curve is plotted as the mean value of four samples, and the equilibrium swelling of DCTA is achieved within 5 hours’ immersion. Approximately 15% of maximum expansion is observed compared with the freshly prepared DCTA, which is dramatically lower than that of the PEG-based tissue adhesive that swells up to about 400% of its original size [1]. The high swelling ratio of tissue adhesive may build up extreme pressure on the surrounding tissues, especially when used in closed cavities [1]. The light swelling of DCTA will not produce the pressure effect on the surrounding tissues, and at the same time, which can offer great advantage for stopping the leakage of fluid [47]. As shown in Fig. 5b, the DCTA exhibits a degradable property in enzyme solution, which is similar to previous reported gelatin hydrogels [48]. The biodegradability of DCTA can avoid the secondary surgery for its removal in practical applications, particularly for large-area in vivo use. Besides, the degradation process will aid the healing or regeneration of tissues [49,50]. 3.3. Tissue adhesion The adhesion properties of DCTA are investigated in detail by lap-shear tests (Fig. 6a) using fresh porcine skin and cartilage as typical adherends. The objective of this work is to develop tissue adhesives that can be extensively used in vivo, and therefore, we evaluate the adhesive capability of DCTA for the inside (fat layer) and outside (collagen layer) of porcine skin as well as cartilage tissue. These are all the potential target gluing substrates in surgical operations. Besides, the gluing properties of DCTA are studied as a function of curing time with the fat layer of porcine skin as a model substrate. The samples for adhesion tests are typically prepared as illustrated in Fig. 4. In order to evaluate the wet adhesion property, the substrates are firstly wetted with water, and then the gluing macromer solution containing genipin is applied on the surfaces of tissues. The tissues are glued together after adding FeCl3 solution on each tissue with overlapping, and then the DCTA will spontaneously form at 37 °C without other manipulations. The

Fig. 4. Schematic illustration of the usage and probable working principle of DCTA.

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Fig. 5. (a) Swelling kinetics of DCTA in PBS solution. (b) Degradation of DCTA in trypsin-EDTA solution.

Fig. 6. (a) Representative image of lap shear test. Representative stress–strain curves of DCTA gluing on skin fat layer (b), after rapid-curing skin-fat gluing (1), 2-hour-curing skin-fat gluing (2), and 24-hour-curing skin-fat gluing (3) respectively, skin collagen layer (c), cartilage tissue (d); and, representative stress–strain curve of genipin-SCTA gluing on skin fat layer (e).

operation is much simpler and shorter in operation time than previous reported catechol-based tissue adhesives that require at least 10 minutes’ compression [9,25]. The controllable, rapid curing, and successively spontaneous formation of DCTA great facilitates its use in surgical procedures, especially for internal tissues’ adhesion. As shown in Fig. 6b, c and Table 1, the ‘‘2-hour-curing skin-fat gluing” and ‘‘2-hour-curing skin-collagen gluing” strength is 24.7 ± 3.3 and 20.4 ± 4.0 kPa, respectively, which are obviously higher than that of commercially available fibrin glue (15.38 ± 2.82 kPa, [9]) obtained from similar shear tests. No statistically significant difference is observed in the adhesion strength of DCTA for the adhesion of fat and collagen layers of porcine skin, which can be attributed to the universal non-covalent bonding of catechol groups to various substrates [17,25]. This endow DCTA

Table 1 Adhesion strength of DCTA under different conditions. Adhesion pattern

Adhesion strength (kPa)

Rapid-curing skin-fat gluing 2-hour-curing skin-fat gluing 24-hour-curing skin-fat gluing 2-hour-curing skin-collagen gluing 2-hour-curing cartilage gluing

9.3 ± 4.9 24.7 ± 3.3 12.9 ± 0.5 20.4 ± 4.0 194.4 ± 20.7

with the general gluing for all tissues, expanding its in vivo applications. Interestingly, the adhesion strength of genipin-SCTA (43.8 ± 8.3 kPa) is significantly higher than ‘‘2-hour-curing skinfat gluing” strength (Fig. 6b and e). This result might be attributed

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to the formation of polymer networks within the skins. The gluing macromer solution containing genipin gradually penetrates into the skins during the period of incubation before gelation and then gelates to form interpenetrating networks between genipin-SCTA and skin tissues, which can obviously enhance the adhesion strength. Nonetheless, since we must pursue rapid gelation, as well, by Fe3+ induced crosslinking for practical applications, this adhesion-strengthening but time-consuming effect has to be sacrificed as the Fe3+ induced rapid gelation in the very first minutes will severely compromise the penetration of DCTA macromer solution into the skin (fatty) substrate. The gluing capability of DCTA is further estimated using more strong and rigid cartilage tissue as the substrate. Significantly, the ‘‘2-hour-curing cartilage gluing” strength is dramatically increased to 194.4 ± 20.7 kPa (Fig. 6d and Table 1) that is 24-fold higher than that of commercial fibrin glue (about 8.0 kPa, [42]) obtained from similar shear tests. This higher adhesion strength is important for cartilage transplantation due to its load-bearing property in vivo. Compared to adhesion of porcine skins, the distinctly higher adhesion strength on cartilage gluing can be attributed to their different physicochemical properties. The cartilage tissue is much more hydrophilic than skin tissue that contains lipids secreted by sebaceous glands; therefore, the gluing macromers easily interact with the collagen of cartilage surfaces. Subsequently, they are intermolecularly crosslinked by genipin, which can greatly increase adhesion strength of DCTA. Aiming to fully evaluate the adhesion properties of DCTA, we also investigate its adhesion capability after ‘‘rapid-curing skin-fat gluing” and ‘‘24-hour-curing skin-fat gluing” (Fig. 6b and Table 1). Albeit lower than the ‘‘2-hour-curing skin-fat gluing” strength, they both are still comparable to the reported tissue adhesive based on catechol moieties’ non-covalent interaction to tissue surfaces [25]. The increase of adhesion strength from ‘‘rapid-curing skin-fat gluing” (after 5–10 seconds’ catechol–Fe3+ crosslinking) to ‘‘2-hourcuring skin-fat gluing” (after Fe3+-genipin double-crosslinking), probably stems from (1) the increase in mechanical strength from Fe3+-SCTA to DCTA due to the added covalent crosslinking, and (2) the more sufficient bonding of catechol groups to substrate. The adhesion strength decreases from ‘‘2-hour-curing skin-fat gluing” to ‘‘24-hour-curing skin-fat gluing” (Fig. 6b and Table 1). In addition to the strong interaction of catechol groups with substrate, (other) physical interactions (e.g. van der Waals forces) also contribute to the adhesion between DCTA and substrate, however, which can be weakened by the displacement with water during soaking and agitating within SBF solution [25]. Besides, the slight swelling of DCTA (Fig. 5a) also can reduce its adhesion capability [1]. These results demonstrate that the DCTA not only controllably rapidly glues wet tissues regardless of tissue types, but also exhibits long-term adhesion effects. This double-crosslink strategy may be widely applied to prepare other DCTAs using other gluing macromers containing both amino and catechol groups. For instance, the chitosan that possesses amino groups has been conjugated with catechol and then successfully used as the macromer to prepare nano-particles and hydrogels for mucoadhesive drug vehicles [51–53]. Among them, Xu et al. fabricate the hydrogels by crosslinking the chitosan-catechol macromers with genipin, and which exhibit good mucoadhesion capability due to the presence of catechol groups [51].

liferation from day 1 to 3 and 5. The HDFs cultured in Extract-100 and pure medium exhibit significantly higher cell viabilities than those cultured in BioGlue at any time points. This result demonstrates that the DCTA is more cytocompatible than aldehydebased tissue adhesives. Interestingly, cell viability of HDFs cultured in Extract-100 is always much higher than those cultured in pure medium; at the same time, the cell viability of HDFs shows a decreasing trend from those cultured in Extract-100 to Extract50 and Extract-25 on day 1 and 3. In other words, the increase of volume percentage of extract solution in culture medium can enhance the growth of HDFs. Considering the fact that gelatin coating on substrates or scaffolds is a common method to enhance cell attachment and proliferation, the higher cell viability of HDFs cultured in Extract-100 compared with those cultured in pure medium or other extract solutions (Extract-50 and Extract-25) should be attributed to (more) coating of gelatin–dopamine conjugate that is diffused out from DCTA. In addition to HDFs, PCCs are also employed as model cell to examine the cytocompatibility of DCTA. As shown in Fig. 7b, the cell viabilities of PCCs cultured in Extract100, -50, and -25 show increasing trend from day 1 to 3, and which are significantly increased from day 3 to 5, respectively. However, cell viabilities of PCCs cultured in BioGlue drastically decrease from day 3 to 5, after increasing from day 1 to 3. The PCCs cultured in the medium containing extract solutions (Extract-100, Extract-50 and Extract-25) always show much higher cell viabilities than those in BioGlue at each time point, in particular, the cell viability of PCCs cultured in Extract-100 is 9-fold higher than those cultured in BioGlue after 5 days of culture. Similar to the observation in HDFs culture, the PCCs cultured in Extract-100, Extract-50, and Extract-25 also show higher cellular growths than those cultured in the pure PCCs culture medium (Pure medium) after 5 days of culture. Compared to HDFs, interestingly, the PCCs are more susceptible to the BioGlue, which further highlights the importance to develop aldehyde-free tissue adhesives. These results of PCCs and HDFs culture demonstrate the excellent cytocompatibility and non-toxic nature of the DCTA. To further assess the cytocompatibility of DCTA, cellular adhesion and proliferation on freshly prepared DCTA is typically studied by the monolayer culture of hMSCs. As shown in Fig. 7c, hMSCs show excellent attachment and spreading on freshly prepared DCTA, and cell density increases along with culture time from day 1 to 3 and 5. The proliferation of hMSCs cultured on DCTA is quantitatively determined via MTT assay. There is no statistical difference in cell viability from day 1 to 3, and it exhibits statistically significant increase after 5 days’ culture compared with that on day 1 and 3 (Fig. 7d). These results suggest the DCTA can well support hMSCs adhesion and proliferation. Hemolysis is a great problem associated with bioincompatibility, and hemolysis test is the widely accepted and frequently used method to determine hemo-compatibility of biomaterials. As shown in Table 2, the hemolysis of freshly prepared DCTA and its extract solutions is 0.498 ± 0.273% and 0.677 ± 0.292%, respectively, which is well within the permissible limit of 5% for biomaterials [41]. All above results demonstrate that the DCTA is cytocompatible and hemocompatible as well as suitable for cell adhesion and proliferation. 3.5. In vivo degradation and tissue response

3.4. In vitro cytocompatibility and hemocompatibility The cytocompatibility of DCTA is first evaluated by determining cellular growth and survival of HDFs and PCCs cultured within the extract solutions of DCTA using the MTT cell viability assay. As shown from the OD value in Fig. 7a, all the HDFs cultured in extract solution medium (Extract-100, Extract-50, and Extract-25) and control medium (BioGlue and pure medium) shows significant pro-

In order to preliminarily evaluate the in vivo degradation and biocompatibility of DCTA, freshly prepared DCTA samples in cylindrical molds are subcutaneously implanted in the back of nude mice. Their degradation and the degree of the tissue responses to the foreign DCTA implants is assessed over implantation time. As shown in Fig. 8a, the dark blue DCTA maintains the cylindrical shape after 4 days’ implantation. It becomes slightly flattened on

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Fig. 7. Proliferation of HDFs (a) and PCCs (b) in Extract-100, Extract-50, Extract-25, BioGlue (positive control), and Pure medium (negative control). Fluorescence micrographs (c) and proliferation (d) of hMSCs seeded on freshly prepared DCTA as a function of culture time. Statistical significance is indicated with *(p 6 0.05) and **(p 6 0.01). Scale bar is 100 lm.

Table 2 Hemolysis of blood by DCTA and its extract solution. Sample

OD value

Hemolysis (%)

0.9% NaCl Water DCTA Extract solution

0.136 ± 0.002 1.341 ± 0.011 0.142 ± 0.003 0.144 ± 0.003


ve control +ve control 0.498 ± 0.273 0.677 ± 0.292

day 14, and only a little DCTA is left over on day 28 after implantation. This degradation process is determined gravimetrically by weighing method and shown in Fig. 8b. These results indicate DCTA bears an appropriate degradation in vivo, which is beneficial to tissues’ healing and regeneration [9].

To assess the effect of DCTA and its degradation products on tissue response, the histological sections of the tissues adjacent to DCTA are stained with H&E and assessed blindly. As can be seen from Fig. 8c, on day 4 post-operation, lots of inflammatory cells, mainly neutrophils, are emigrated from the blood vessels and accumulated in the surrounding tissues, indicating the moderate acute inflammatory tissue response. This phenomenon is in agreement with previous implantation studies of gelatin hydrogel [54]. Actually, the surgical wounding is sufficient to induce neutrophils’ accumulation [43]. This result, that the use of acidic FeCl3 solution does not induce further tissue response, might be resulted from the buffering effect of gluing macromer (PBS) solution. On day 14, the inflammatory cells decrease obviously (Fig. 8d), suggesting

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Fig. 8. (a) Gross view of the DCTA implants (with murine skins) extracted on day 4, 14, and 28, respectively, after subcutaneous implantation in mice. (b) Degradation of DCTA over time after implantation. H&E staining of the tissues surrounding DCTA after 4 (c), 14 (d), and 28 (e) days’ implantation; the DCTA is marked with an asterisk and scale bar is 100 lm.

the decline in degree of inflammatory reaction over the degradation of DCTA [54]. Significantly, these inflammatory cells are not observed on day 28 after implantation, which demonstrates the disappearance of the inflammatory reaction (Fig. 8e). Furthermore, no giant cells reaction and fibrosis is observed across the whole 28 days’ implantation. These observations on tissue responses demonstrate the biodegradability and biocompatibility of the DCTA in vivo. Collectively, the novel DCTA described herein is designed and developed by constituting gelatin–dopamine conjugates – as a typical gluing macromer, Fe3+ – as a rapid crosslinker, and genipin – as a long-acting crosslinker. It meets the conditions as an ideal tissue adhesive. The typical gluing macromer, gelatin–dopamine, is synthesized in a convenient one-step grafting reaction of dopamine onto gelatin backbone by EDC/NHS chemistry, where no special conditions (e.g. high temperature, high vacuum, high pressure, and specific catalyst) and equipments are required. Genipin is more biocompatible than aldehyde-containing crosslinkers, and Fe3+ ions are always found in human body. The DCTA is convenient to prepare and utilize in clinical environments. The flowability of the gluing macromer solution facilitate to film it on the surfaces of tissues, the adhesion of tissues is accomplished in a rapid and controllable fashion by the simple overlapping after the addition of FeCl3 solution on each tissue, and subsequently the stable covalent crosslinking with genipin can be achieved spontaneously. The DCTA displays much higher wet adhesion strength than the fibrin glue that has been reported to achieve a certain level of success for some in vivo applications. Besides, it also possesses biodegradability and excellent cyto/tissue-compatibility both in vitro and in vivo. The current results support that the DCTA will be an attractive candidate as a tissue adhesive for internal medical use.

4. Conclusion In this study, we have designed and developed novel DCTA from gelatin–dopamine conjugate by a Fe3+-genipin double-crosslink methodology for internal medical use. The DCTA combines the

qualities of catechol–Fe3+ coordination complexation (rapid and controllable gelation) and genipin-induced covalent crosslinking (structural stability), which facilitates its utilization in clinics. The results presented above demonstrate the promising potential of DCTA for internal tissue adhesion and sealing, and hemostasis. The double-crosslink strategy may be envisaged to be extensively employed to fabricate tissue adhesives with the polymers containing both amino and catechol groups, such as catechol functionalized chitosan, polyamino acids, and proteins. The efficacy of this DCTA for in vivo applications, such as seroma prevention and transplantation of tissue-engineered cartilage, will be evaluated on basis of our current studies. Acknowledgements The work is financially supported by AcRF Tier 1 RG 30/15 and AcRF Tier 2 ARC 1/13, Singapore. References [1] J.C. Wheat, J.S. Wolf Jr, Advances in bioadhesives, tissue sealants, and hemostatic agents, Urol. Clin. North Am. 36 (2009) 265–275. [2] W.D. Spotnitz, S. Burks, Hemostats, sealants, and adhesives: components of the surgical toolbox, Transfusion 48 (2008) 1502–1516. [3] M. Radosevich, H.I. Goubran, T. Burnouf, Fibrin sealant: scientific rationale, production methods, properties, and current clinical use, Vox Sanguinis 72 (1997) 133–143. [4] G. Hidas, A. Kastin, M. Mullerad, J. Shental, B. Moskovitz, O. Nativ, Sutureless nephron-sparing surgery: use of albumin glutaraldehyde tissue adhesive (BioGlue), Urology 67 (2006) 697–700. [5] J.V. Quinn, Tissue adhesives in clinical medicine, in: J.V. Quinn (Ed.), Tissue Adhesives in Clinical Medicine, second ed., BC Decker Inc, Hamilton, 2005, pp. 27–76. [6] C. Joch, The safety of fibrin sealants, Cardiovasc. Surg. 11 (2003) 23–28. [7] W. Fürst, A. Banerjee, Release of glutaraldehyde from an albuminglutaraldehyde tissue adhesive causes significant in vitro and in vivo toxicity, Ann. Thoracic Surg. 79 (2005) 1522–1528. [8] J. Ennker, I.C. Ennker, D. Schoon, H.A. Schoon, S. Dorge, M. Meissler, M. Rimpler, R. Hetzer, The impact of gelatin–resorcinol glue on aortic tissue: a histomorphologic evaluation, J. Vasc. Surg. 20 (1994) 34–43. [9] M. Mehdizadeh, H. Weng, D. Gyawali, L. Tang, J. Yang, Injectable citrate-based mussel-inspired tissue bioadhesives with high wet strength for sutureless wound closure, Biomaterials 33 (2012) 7972–7983.

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