Colloids and Surfaces B: Biointerfaces 133 (2015) 19–23
Contents lists available at ScienceDirect
Colloids and Surfaces B: Biointerfaces journal homepage: www.elsevier.com/locate/colsurfb
Enhanced biocompatibility and adhesive properties of modified allyl 2-cyanoacrylate-based elastic bio-glues Jin Ik Lim a , Ji Hye Kim b,∗ a Laboratory of Biointerfaces/Tissue Engineering, Department of Chemical Engineering, Institute of Tissue Regeneration Engineering, College of Engineering, Dankook University, Jukjeon-dong, Yongin-si, Gyeonggi-do 448-701, Republic of Korea b Division of Biomedical Polymer Development Center, JISAN Co., Seoul 130-707, Republic of Korea
a r t i c l e
i n f o
Article history: Received 14 November 2014 Received in revised form 2 May 2015 Accepted 5 May 2015 Available online 15 May 2015 Keywords: Ally 2-cyanoacrylate Bio-glue Bond strength Flexibility Biocompatibility Wound healing
a b s t r a c t Despite cyanoacrylate’s numerous advantages such as good cosmetic results and fast application for first aid, drawbacks such as brittleness and local tissue toxicity have limited their applicability. In this study, to improve both the biocompatibility and mechanical properties of cyanoacrylate, allyl 2-cyanoacrylate (AC) was pre-polymerized and mixed with poly(l-lactide-co--caprolactone) (PLCL, 50:50) as biodegradable elastomer. For various properties of pre-polymerized AC (PAC)/PLCL mixtures, bond strength, elasticity of flexure test as bending recovery, cell viability, and in vivo test using rat were conducted and enhanced mechanical properties and biocompatibility were confirmed. Especially, optimal condition for pre-polymerization of AC was determined to 150 ◦ C for 40 min through cytotoxicity test. Bond strength of PAC/PLCL mixture was decreased (over 10 times) with increasing of PLCL. On the other hand, biocompatibility and flexibility were improved than commercial bio-glue. Optimal PAC/PLCL composition (4 g/20 mg) was determined through these tests. Furthermore, harmful side effects and infection were not observed by in vivo wound healing test. These results indicate that PAC/PLCL materials can be used widely as advanced bio-glues in various fields. © 2015 Published by Elsevier B.V.
1. Introduction Tissue adhesives have attracted growing interest as sealants, hemostatic agents, and non-invasive wound-closure materials. The adhesion of biological tissues is challenging in that the adhesive materials must satisfy several conditions, one of these is that suitable adhesion should be an attractive alternative to conventional sutures, rapid adhesion, and close apposition of wound edges for a sufficient period. Furthermore, such adhesive materials must not induce a marked inflammatory response, must be biodegradable, and must have minimal tissue toxicity [1]. Cyanoacrylates have been widely used as structural adhesives for metals, alloys, plastics, rubbers, and ceramics. Due to their biocompatibility and high reactivity under moist conditions, they are also used in medicine as surgical adhesives and coatings [2,3]. The main components of commercial medical cyanoacrylate adhesives for clinical applications are octyl 2-cyanoacrylate and butyl 2-cyanoacrylate, which are longer-chain derivatives [4,5]. However, the application of these polymers has
∗ Corresponding author. Tel.: +82 2 485 5027; fax: +82 2 485 9702. E-mail address: [email protected] (J.H. Kim). http://dx.doi.org/10.1016/j.colsurfb.2015.05.004 0927-7765/© 2015 Published by Elsevier B.V.
been limited because of their unfavorable mechanical properties and the release of cytotoxic chemicals during their degradation [6,7]. Consequently, these materials are mainly used as biological adhesives in emergencies [2,8]. To overcome the limitations associated with these adhesives, researchers have investigated various cyanoacrylate modifications. Petrov reported that combining ethyl 2-cyanoacrylate and poly(methyl methacrylate) or poly(butadiene-co-acrylonitrile) improves the mechanical properties of the adhesive [9], while Tseng modified the cyanoacrylate monomer into ethoxyethyl cyanoacrylate by adding a side chain of relatively low hydrophobicity and high flexibility [10]. Allyl 2-cyanoacrylate (AC), an advanced cyanoacrylate derivative containing a double bond, has been introduced to enhance the mechanical properties of cyanoacrylate [7]. Although the mechanical properties of these polymers have been improved slightly by the abovementioned modifications, their limited biocompatibility for medical applications remains a challenge [11]. We previously reported that partial pre-polymerization of AC results in a longer chain structure, consequently improving the biocompatibility and stability of cyanoacrylate [7]. The application of this product to areas of body movement such as joints, however, remained limited due to the poor flexibility of the product. Thus, there remains a need for similar products with improved
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J.I. Lim, J.H. Kim / Colloids and Surfaces B: Biointerfaces 133 (2015) 19–23
mechanical properties including bond strength and flexibility as well as enhanced biocompatibility of tissue adhesives for medical applications. In this study, the biodegradable elastomer poly(l-lactide-co-caprolactone) (PLCL) was chosen as an additive for elastic pre-polymerized AC (PAC). This copolymer was chosen based on the different elastic recoveries of amorphous polymers and highly crystalline polymers depending on the lactide/caprolactone molar ratio of the material, as previously reported [12]. PLCL is usually used in studies on transplanted biomaterials such as tissue engineered blood vessels, cartilage, and skin [13,14]. The objectives of this study were (1) to select the conditions that yielded the most stable blend of PLCL and PAC for use as elastic bioglue, (2) to determine the PLCL and PAC compositions that yielded the optimal mechanical parameters, and (3) to evaluate the cytotoxicity of the selected bio-glue. This study is thus an in vitro and in vivo assessment of the biocompatibility of the optimum PAC/PLCL mixture for clinical application as a tissue adhesive.
were shaken for 1 h at room temperature and dried in a heatdrying oven for 7 h at 65 ◦ C to completely remove the chloroform solvent. 2.4. Elasticity of flexure test of the PAC/PLCL mixtures Films (size = 10 mm × 50 mm × 0.03 mm) with various PAC/PLCL ratios were prepared by natural drying in 10 wt.% chloroform solutions for 3 weeks. The elasticity of flexure test was performed using a universal testing machine (Instron model 5966, Canton, MAUSA). A 5 N load cell with a crosshead speed of 1 mm/min was used for this experiment. Each film was folded in half at a folding speed of 1 mm/min, immediately after which the stress was released and the recovered angle for 5 min was measured and compared with that of an octyl 2-cyanoacrylate film. The percent elasticity of flexure was calculated as [(recovered angle of film/angle (180◦ ) of unfolded film) × 100]. 2.5. Bond strengths of the PAC/PLCL mixtures
2. Materials and methods 2.1. Preparation and characterization of PACs AC (920; Robinson St. Pottstown, PA, USA) was heated at 150 ◦ C for 0, 5, 10, 20, 40, and 60 min in vacuum vials (10 ml/vial). Samples were then cooled to 0 ◦ C and stored at 4 ◦ C. Change of viscosity according to heating time was measured using viscometer (Brookfield, model LVDV-II+P, Middleboro, MA, USA) and compared with ethyl 2-cyanoacrylate as control.
The PAC/PLCL mixtures were applied to bovine skin (area: 10 mm × 10 mm). The specimens were then covered with another section of bovine skin. The volume of sample was 10 l, and the final adhesion thickness was 10 m. After 24 h at room temperature, the bond-strength of the PAC/PLCL mixtures was tested using a universal testing machine (Instron model 4467). The crosshead speed was set to 1 mm/min, and the load at which the specimen deboned from the adherent was recorded. 2.6. In vitro cytotoxicity test of the optimum PAC/PLCL mixture
2.2. Cytotoxicity test of PACs As a direct contact method, the cytotoxicity of PAC was tested using a previously reported method [15]. L929 cells (ATCC, Manassas, VA, USA) were cultured in RPMI 1640D media with 20% fetal bovine serum (FBS), 1% penicillin and 1% streptomycin. The cells were added in 24 well plate (104 cells/well) and maintained in a humidified atmosphere that contained 5% CO2 at 37 ◦ C. After 24 h, the media was aspirated and PACs and Dermabond of 100 l were each added directly to the center of each cultured well. After another 1 h, 2 ml media were slowly added to each well. After 4 h and 24 h, the initial adhesion and proliferation were determined by using a MTT assay. To determine viability, absorbance was measured at a test wavelength of 570 nm using a microplate reader (Molecular Devices, Toronto, Canada). The optical density (O.D.) was calculated as the difference between the reference wavelength and the test wavelength. The percent viability was calculated as [(O.D. of drug-treated sample/O.D. of untreated sample) × 100]. 2.3. Preparation of the PAC/PLCL mixtures PLCL [molar ratio 50:50, number-average molecular weight (Mn): 2.2 × 105 ] was polymerized and purified using a method described previously [10]. The 2% (w/v) PLCL was dissolved in chloroform solution. PLCL solution of various volumes was mixed with a fixed volume of PAC, as presented in Table 1. The mixtures Table 1 Composition of pre-polymerized ally 2-cyanoacrylate (PAC)/poly(l-lactide-co-caprolactone) (PLCL) mixtures. Mixture
PAC (g)
PLCL (mg)
Type-1 Type-2 Type-3 Type-4
4 4 4 4
20 40 80 120
Cytotoxicity testing of the optimum PAC/PLCL mixture was performed using direct contact method (Section 2.2). 2.7. Epidermal growth factor (EGF) release test An EGF (Sigma–Aldrich, St. Louis, MO, USA) solution (500 g/ml) and chitosan (>1000 kDa, 86% DD, Korea Chitosan Co., Ltd.) of 1 wt.% in 1% acetic acid solution was prepared and dropped in liquid nitrogen using a syringe pump (rate: 1 mm/min). And then, the beads were lyophilized for 24 h. The Bradford protein assay was used to measure the EGF released from the chitosan micro-beads, prepared by the EGF included chitosan micro-beads (dry weight 10 mg) and EGF included chitosan micro-beads (dry weight 10 mg)/PAC/PLCL (1 g) mixture, respectively. The samples were suspended in 10 ml PBS and incubated at 37 ◦ C and 30 rpm for 0, 2, 5, 8, 15, 20, 45, and 60 min. After the incubation, the releasate, condensed by lyophilization, was added to a 96-well plate. The Bradford reagent (Bio-Rad) was then added to each well before incubating the plate in the dark at room temperature for 15 min. Absorbance was measured at 595 nm after shaking for 30 s. 2.8. In vivo study Fifteen young male Sprague–Dawley rats (weight, 250–350 g) were used. All animals were anesthetized with inhaled isoflurane (1–5%). After anesthesia, hair from the animal’s back was removed with electric clippers, and a depilatory cream (Nair; Carter Products) was applied for several minutes. The area was washed with wet cotton and dried with a gauze sponge. Povidone-iodine was applied to the surgical site and removed, and then isopropyl alcohol (70%) was applied. Two 1.5 cm long incisions were made on the back of each rat, with at least 1 cm of intact skin between them. The EGF included chitosan micro-beads (20 mg) were applied to
J.I. Lim, J.H. Kim / Colloids and Surfaces B: Biointerfaces 133 (2015) 19–23
the each incision area. And then, the optimum PACA/PLCL mixture and octyl CA were applied as a monolayer over the incision without additional treatment, according to instructions for the commercial tissue adhesive. Rats were sacrificed at 15 days after surgery to examine and compare the tissue reactions between the two groups. Each tissue specimen was fixed in 10% buffered formalin for tissue slicing. Hematoxylin and eosin (H&E) staining was performed to observe inflammatory cell infiltration, and the stained tissues were observed under an optical microscope [16].
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Table 2 Cell viability and bond strength of pre-polymerized ally 2-cyanoacrylate (PAC) according to heating time (n = 4 for each test, * P < 0.05, # P < 0.05). Heating time (min)
Cell viability (%) After 24 h*
0 5 10 20 40 60 Control
33 47 48 48 61 55 38.4
± ± ± ± ± ± ±
Bond strength (MPa)#
1 9 2 14 11 2 3.3
0.88 0.75 0.79 0.70 0.69 0.58 0.17
± ± ± ± ± ± ±
0.16 0.05 0.03 0.04 0.1 0.1 0.03
n 4 4 4 4 4 4 4
3. Results and discussion 3.1. Characterization of pre-polymerized allyl 2-cyanoacrylates The focus of this study was to achieve low cytotoxicity and good elastic properties for pre-polymerized allyl 2-cyanoacrylate bio-glue. Changes in cytotoxicity following pre-polymerization and altered mechanical properties with varying PLCL content were therefore important parameters to assess. To determine the optimal pre-polymerization conditions, the test materials were assessed in terms of viscosity, bond strength, and cytotoxicity and were compared with octyl 2-cyanoacrylate. The yellowish color and viscosity of the samples were found to increase with heating time, especially in the case of the PAC samples. The observed increase in the viscosity of PAC was attributed to the partial polymerization of another intramolecular double bond instead of the polymerization of a polymerizable double bond by an anion initiator. To confirm this hypothesis, the change in PAC viscosity with increasing heating time was compared to that of ethyl 2-cyanoacrylate (EC) viscosity with heating time. Fig. 1 shows the changes in viscosity and reveals that the viscosity of EC was not affected by heating time, whereas the viscosity of PAC increased with increased heating time. The polymerization reaction of another intramolecular double bond in AC was therefore confirmed. The PAC bond strength was also found to decrease with increasing heating time (Table 1: 0 min, 0.88 ± 0.16 MPa; 5 min, 0.75 ± 0.05 MPa; 10 min, 0.79 ± 0.03 MPa; 20 min, 0.70 ± 0.04 MPa; 40 min, 0.69 ± 0.1 MPa; 60 min, 0.58 ± 0.1 MPa). This trend was attributed to the decreased reactivity of anion initiators because of the high viscosity associated with increased heating time. Decreased PAC bond strength was therefore considered another result of the partial polymerization of the intramolecular double bond; however, the control material octyl 2-cyanoacrylate exhibited the lowest overall bond strength (0.17 ± 0.03 MPa).
Pre-polymerized allyl 2-cyanoacrylate Ethyl 2-cyanoacrylate
350
Viscosity (mPas)
300 250
The cytotoxicity of PAC was determined using a direct-contact culture cell viability test (Table 2). Generally, cytotoxicity decreases with an increase in the length of the intramolecular carbon chains in cyanoacrylates [8] and the cytotoxicity of PAC was therefore expected to decrease with increasing polymerization time. Most samples in this study were found to have a lower cytotoxicity than the control (octyl 2-cyanoacrylate), except the sample that was heat-treated for 0 min (cell viability, 33 ± 1%). The PAC, which was pre-polymerized for 40 min, yielded the highest cell viability (61 ± 11%). Previously reported cell viability data for prepolymerized AC were obtained by indirect methods using NIH3T3 cells [7], while the cytotoxicity testing in this study was conducted according to ISO 10993 (Biological evaluation of medical devices) standards. The optimal heat-treatment time for the production of bio-adhesives with low cytotoxicity was found to be 40 min using these methods. 3.2. Characterization of pre-polymerized allyl 2-cyanoacrylate (PAC)/poly(l-lactide-co--caprolactone) (PLCL) mixtures The elasticity of flexure of the PAC/PLCL films in response to bending stress was compared to that of octyl 2-cyanoacrylate, which was used as the control. The elasticity of flexure of the PAC/PLCL films was found to increase with increasing PLCL concentration (Table 3). Type-3, and -4 films were furthermore found to exhibit the highest elasticity of flexure values and the elasticity of flexure of the PAC/PLCL films was found to be higher than that of PAC and the control. The improved elasticity of flexure is thought to result from the presence of amorphous PLCL (50:50). Fig. 2 shows the bond strengths between porcine skins bound by PAC/PLCL mixtures as a function of PLCL ratio. Bond strengths of the PAC/PLCL mixtures decreased sharply with increasing PLCL content: type-3 and -4 films exhibited notably lower bond strengths than the other samples (PAC, 0.69 ± 0.1 MPa; type-1, 0.354 ± 0.01 MPa; type-2, 0.21 ± 0.03 MPa; type-3, 0.01 ± 0 MPa; type-4, 0.01 ± 0 MPa; and control, 0.17 ± 0.03 MPa). The low bond strength of type-3 and -4 films may be attributable to the increasingly amorphous structure of PAC/PLCL mixtures with increased PLCL concentration. On the other hand, in the case of the type2 film, a bond strength similar to that of the control film was
200 Table 3 Elasticity of flexure (%) of pre-polymerized ally 2-cyanoacrylate (PAC)/poly(llactide-co--caprolactone) (PLCL) mixtures (n = 4 for each test, * P > 0.05, # P > 0.05, *,# P < 0.05).
150 100
Samples (PAC/PLCL ratios)
50
#
0
10
20
30
40
50
60
Heating time (min) Fig. 1. Viscosity of pre-polymerized ally 2-cyanoacrylate (PAC)* and ethyl 2cyanoacrylate# according to heating time (n = 4 for each test, * P < 0.05, # P > 0.05).
PAC (4/0) Type-1 (4/20) Type-2 (4/40) Type-3 (4/80)* Type-4 (4/120)* Control#
Bending recovery (%) 32.4 45.3 48 50 50 31.6
± ± ± ± ± ±
0.7 0.86 0.47 0.27 0.64 0.42
n 4 4 4 4 4 4
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Bond strength (MPa)
0.8
0.6
0.4
* #
0.2
0.0 PAC
Type-1
Type-2
Type-3
Type-4
Control
Adhesives Fig. 2. Bond strength of pre-polymerized ally 2-cyanoacrylate. (PAC)/poly(l-lactideco--caprolactone) (PLCL) mixtures (n = 4 for each test, * P < 0.05, and # P > 0.05 compared with octyl 2-cyanoacrylate as a control)
observed. The type-1 film in particular exhibited increased bond strength compared to the control. Based on these findings, the optimal PAC/PLCL ratio for both enhanced flexibility and high bond strength is 4 g/20 mg. 3.3. Epidermal growth factor (EGF) release from chitosan micro-beads Epidermal growth factor (EGF) has been widely reported to promote wound healing [17–20] and EGF was therefore applied with the bio-glue in this study as a drug delivery system for improved wound healing efficiency. As shown in Fig. 4, the EGF-included chitosan micro-beads exhibited a ∼10-fold higher initial release velocity than the EGF-included chitosan microbead/PAC/PLCL mixture (release time [min]: EGF-included chitosan micro-beads [O.D.]/EGF-included chitosan micro-bead/PAC/PLCL mixture [O.D.], 2: 0.13/0.012). After 5 min, the difference in the amount of EGF released from each sample had increased from ∼10fold to 15-fold or more (release time [min]: EGF-included chitosan micro-beads [O.D.]/EGF-included chitosan micro-bead/PAC/PLCL mixtures [O.D.], 5: 0.44/0.022; 8: 0.57/0.033; 15: 0.65/0.051; 20: 0.75/0.058; 45: 0.98/0.064; 60: 1.1/0.062, Fig. 4). These findings
350
Cell viability (%)
300
TCP Octyl 2-cyanoacrylate Type-1
250 200 150
#
*
100 * 50 0 1 day
2 day
3 day
Culture period Fig. 3. Cell viability (%) test of pre-polymerized ally 2-cyanoacrylate (PAC)/poly(llactide-co--caprolactone) (PLCL) (n = 4 for each test, * P < 0.05, and # P < 0.05 compared with octyl 2-cyanoacrylate of the same period)
Fig. 4. Epidermal Growth Factor (EGF) release test from chitosan porous beads* and bio-glue mixture# in PBS solution at 37 ◦ C (n = 4 for each test, * P < 0.05, and # P > 0.05).
suggest that the high release velocity from the micro-beads was a result of good interconnectivity and a highly porous structure obtained by phase separation (SEM in Fig. 4). The low release velocity of EGF-included chitosan micro-bead/PAC/PLCL mixtures was furthermore likely to be due to low diffusion by the PAC/PLCL mixture coating the chitosan micro-beads. The higher release velocity is moreover indicative of faster diffusion over a large surface area of the 3D porous structure and the presence of exposed protein on the surface, as mentioned in a previous study [21]. To facilitate improved wound healing efficiency, EGF-included chitosan micro-beads were therefore prepared and applied directly to incision areas before the application of bio-glue. 3.4. Assessment of the biocompatibility of the PAC/PLCL mixture by in vitro and in vivo testing Cytotoxicity testing to evaluate short-term in vitro biocompatibility was performed using the optimal PAC/PLCL mixture (type-1) and the control material octyl 2-cyanoacrylate in a direct-contact culture cell viability test for 3 days. As shown in Fig. 3, cell viability in the presence of the PAC/PLCL mixture was significantly higher than that in the presence of octyl 2-cyanoacrylate (1 day, 38.4 ± 3.3%; 2 day, 47.9 ± 13.5%; 3 day, 107.5 ± 8.9%), particularly on the first (68.2 ± 4.2%, *P < 0.05 compared with octyl 2-cyanoacrylate at the same time point) and second (96.7 ± 2.3%, *P < 0.05 compared with octyl 2-cyanoacrylate at the same time point) days. Cytotoxicity therefore seems to decrease as a result of increased polymer chains by pre-polymerization, and the pre-polymerized PAC/PLCL film was shown here to be less cytotoxic than commercial bio-glue. A histological analysis was performed to evaluate long-term in vivo biocompatibility. Fig. 5 shows images of healed skin tissue treated with either the PAC/PLCL mixture or octyl 2cyanoacrylate at 15 days after surgery. Images were obtained after hematoxylin–eosin (H&E) staining, which shows pink cytoplasm and purple nuclei in the tissue. No differences were observed in the images of tissues from wounds closed with the PAC/PLCL film (type1) in comparison to those closed with commercial bio-glue (octyl 2-cyanoacrylate); however, in the case of octyl 2-cyanoacrylate, a weak inflammatory reaction was observed (star, Fig. 5d–f) [16,22]. Although it was difficult to accurately determine the degree of
J.I. Lim, J.H. Kim / Colloids and Surfaces B: Biointerfaces 133 (2015) 19–23
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Fig. 5. H&E stained slice microphotography of closed rat tissue by bioglues [(a–c): Normal, (d–f): Octyl 2-cyanoacrylate, (g–i): pre-polymerized ally 2-cyanoacrylate (PAC)/poly(l-lactide-co--caprolactone) (PLCL)].
inflammation from the small number of specimens in this study, it was inferred that skin tissue closure using the PAC/PLCL mixture caused less inflammation and foreign body reaction for a considerable length of time compared to the octyl 2-cyanoacrylate treatment. 4. Conclusions We prepared and characterized a PAC/PLCL mixture to improve its flexibility and biocompatibility as a tissue adhesive. Prepolymerization of AC by heat treatment at 150 ◦ C for 40 min was the most effective way to reduce cytotoxicity of the short-chain cyanoacrylate derivative. The biocompatible PLCL improved flexibility and cell viability of PAC. Among them, the PAC/PLCL mixture at a ratio of 4 g/20 mg resulted in the highest bond strength, good cell viability, and was an effective skin closure material. Therefore, we expect that this new PAC/PLCL mixture might be useful as an alternative tissue adhesive for skin wound closure along with conventional suture materials and/or commercial tissue adhesives. Further studies are essential to understand the polymerization of cyanoacrylate derivatives to further increase bond strength, decrease cytotoxicity, and make them suitable for applications in various fields including medicine. Acknowledgement The present research was conducted by the research fund of Dankook University in 2014.
References [1] G. Wang, T. Zhang, S. Ahmad, J. Cheng, M. Guo, Int. J. Adhes. Adhes. 41 (2013) 198. [2] M.J. Buckley, E.J. Beckman, Oral Maxillofac. Surg. Clin. N. Am. 22 (2010) 195. [3] P. Losi, S. Burchielli, D. Spiller, V. Finotti, S. Kull, E. Briganti, G. Soldani, J. Surg. Res. 163 (2010) 53. [4] J. Penoff, Plast. Reconstr. Surg. 103 (1999) 730. [5] H.W. Sung, D.M. Huang, W.H. Chang, L.L. Huang, C.C. Tsai, I.L. Liang, J. Biomater. Sci. Polym. Ed. 10 (1999) 751. [6] M.G. Han, S. Kim, S.X. Liu, Polym. Degrad. Stab. 96 (2008) 1243. [7] J.I. Lim, J.H. Kim, H.K. Park, Mater. Lett. 81 (2012) 251. [8] B. Mizrahi, C.F. Stefanescu, C. Yang, M.W. Lawlor, D. Ko, R. Langer, D.S. Kohane, Acta Biomater. 7 (2011) 3150. [9] C. Petrov, B. Serafimov, D.L. Kotzev, Int. J. Adhes. Adhes. 8 (1998) 207. [10] Y.C. Tseng, S.H. Hyon, Y. Ikada, Biomaterials 11 (1990) 73. [11] J.I. Lim, W.K. Lee, Colloids Surf. B: Biointerfaces 122 (2014) 669. [12] J. Fernandez, A. Etxeberria, J.R. Sarasua, J. Mech. Behav. Biomed. Mater. 9 (2012) 100. [13] S.H. Kim, S.H. Kim, Y. Jung, J. Control. Release 20 (2015) 101. [14] T.H. Qazi, D.J. Mooney, M. Pumberger, S. Geißler, G.N. Duda, Biomaterials 53 (2015) 502. ´ Int. J. Oral Maxillofac. Surg. [15] T. Landegren, M. Risling, J.K.E. Persson, A. Sonden, 39 (2010) 705. [16] R.H. Watts, D. Green, G.R. Howells, Stain Technol. 56 (3) (1982) 155. [17] C. Alemdaro˘glu, Z. De˘gim, N. C¸elebi, F. Zor, S. Öztürk, D. Erdo˘gan, Burns 32 (3) (2006) 319. [18] J.K. Choi, J.H. Jang, W.H. Jang, J. Kim, I.H. Bae, J. Bae, Y.H. Park, B.J. Kim, K.M. Lim, J.W. Park, Biomaterials 33 (33) (2008) 8579. [19] A.E. Sakallioglu, A. Yagmurlu, H. Dindar, N. Hasirci, N. Renda, N.S. Deveci, J. Pediatr. Surg. 39 (4) (2004) 591. [20] J. Hardwicke, D. Schmaljohann, D. Boyce, D. Thomas, The Surgeon 6 (3) (2008) 172. [21] S.H. Teng, E.J. Lee, P. Wang, S.H. Jun, C.M. Han, H.E. Kim, J. Biomed. Mater. Res. B: Appl. Biomater. 90 (1) (2009) 275. [22] D. Yoo, J. Vinten-Johansen, L.S. Schmarkey, S.P. Whalen, C.C. Bone, S.L. Katzmark, J. Langberg, Circ. Arrhythm. Electrophysiol. 3 (5) (2010) 505.
Contents lists available at ScienceDirect
Colloids and Surfaces B: Biointerfaces journal homepage: www.elsevier.com/locate/colsurfb
Enhanced biocompatibility and adhesive properties of modified allyl 2-cyanoacrylate-based elastic bio-glues Jin Ik Lim a , Ji Hye Kim b,∗ a Laboratory of Biointerfaces/Tissue Engineering, Department of Chemical Engineering, Institute of Tissue Regeneration Engineering, College of Engineering, Dankook University, Jukjeon-dong, Yongin-si, Gyeonggi-do 448-701, Republic of Korea b Division of Biomedical Polymer Development Center, JISAN Co., Seoul 130-707, Republic of Korea
a r t i c l e
i n f o
Article history: Received 14 November 2014 Received in revised form 2 May 2015 Accepted 5 May 2015 Available online 15 May 2015 Keywords: Ally 2-cyanoacrylate Bio-glue Bond strength Flexibility Biocompatibility Wound healing
a b s t r a c t Despite cyanoacrylate’s numerous advantages such as good cosmetic results and fast application for first aid, drawbacks such as brittleness and local tissue toxicity have limited their applicability. In this study, to improve both the biocompatibility and mechanical properties of cyanoacrylate, allyl 2-cyanoacrylate (AC) was pre-polymerized and mixed with poly(l-lactide-co--caprolactone) (PLCL, 50:50) as biodegradable elastomer. For various properties of pre-polymerized AC (PAC)/PLCL mixtures, bond strength, elasticity of flexure test as bending recovery, cell viability, and in vivo test using rat were conducted and enhanced mechanical properties and biocompatibility were confirmed. Especially, optimal condition for pre-polymerization of AC was determined to 150 ◦ C for 40 min through cytotoxicity test. Bond strength of PAC/PLCL mixture was decreased (over 10 times) with increasing of PLCL. On the other hand, biocompatibility and flexibility were improved than commercial bio-glue. Optimal PAC/PLCL composition (4 g/20 mg) was determined through these tests. Furthermore, harmful side effects and infection were not observed by in vivo wound healing test. These results indicate that PAC/PLCL materials can be used widely as advanced bio-glues in various fields. © 2015 Published by Elsevier B.V.
1. Introduction Tissue adhesives have attracted growing interest as sealants, hemostatic agents, and non-invasive wound-closure materials. The adhesion of biological tissues is challenging in that the adhesive materials must satisfy several conditions, one of these is that suitable adhesion should be an attractive alternative to conventional sutures, rapid adhesion, and close apposition of wound edges for a sufficient period. Furthermore, such adhesive materials must not induce a marked inflammatory response, must be biodegradable, and must have minimal tissue toxicity [1]. Cyanoacrylates have been widely used as structural adhesives for metals, alloys, plastics, rubbers, and ceramics. Due to their biocompatibility and high reactivity under moist conditions, they are also used in medicine as surgical adhesives and coatings [2,3]. The main components of commercial medical cyanoacrylate adhesives for clinical applications are octyl 2-cyanoacrylate and butyl 2-cyanoacrylate, which are longer-chain derivatives [4,5]. However, the application of these polymers has
∗ Corresponding author. Tel.: +82 2 485 5027; fax: +82 2 485 9702. E-mail address: [email protected] (J.H. Kim). http://dx.doi.org/10.1016/j.colsurfb.2015.05.004 0927-7765/© 2015 Published by Elsevier B.V.
been limited because of their unfavorable mechanical properties and the release of cytotoxic chemicals during their degradation [6,7]. Consequently, these materials are mainly used as biological adhesives in emergencies [2,8]. To overcome the limitations associated with these adhesives, researchers have investigated various cyanoacrylate modifications. Petrov reported that combining ethyl 2-cyanoacrylate and poly(methyl methacrylate) or poly(butadiene-co-acrylonitrile) improves the mechanical properties of the adhesive [9], while Tseng modified the cyanoacrylate monomer into ethoxyethyl cyanoacrylate by adding a side chain of relatively low hydrophobicity and high flexibility [10]. Allyl 2-cyanoacrylate (AC), an advanced cyanoacrylate derivative containing a double bond, has been introduced to enhance the mechanical properties of cyanoacrylate [7]. Although the mechanical properties of these polymers have been improved slightly by the abovementioned modifications, their limited biocompatibility for medical applications remains a challenge [11]. We previously reported that partial pre-polymerization of AC results in a longer chain structure, consequently improving the biocompatibility and stability of cyanoacrylate [7]. The application of this product to areas of body movement such as joints, however, remained limited due to the poor flexibility of the product. Thus, there remains a need for similar products with improved
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J.I. Lim, J.H. Kim / Colloids and Surfaces B: Biointerfaces 133 (2015) 19–23
mechanical properties including bond strength and flexibility as well as enhanced biocompatibility of tissue adhesives for medical applications. In this study, the biodegradable elastomer poly(l-lactide-co-caprolactone) (PLCL) was chosen as an additive for elastic pre-polymerized AC (PAC). This copolymer was chosen based on the different elastic recoveries of amorphous polymers and highly crystalline polymers depending on the lactide/caprolactone molar ratio of the material, as previously reported [12]. PLCL is usually used in studies on transplanted biomaterials such as tissue engineered blood vessels, cartilage, and skin [13,14]. The objectives of this study were (1) to select the conditions that yielded the most stable blend of PLCL and PAC for use as elastic bioglue, (2) to determine the PLCL and PAC compositions that yielded the optimal mechanical parameters, and (3) to evaluate the cytotoxicity of the selected bio-glue. This study is thus an in vitro and in vivo assessment of the biocompatibility of the optimum PAC/PLCL mixture for clinical application as a tissue adhesive.
were shaken for 1 h at room temperature and dried in a heatdrying oven for 7 h at 65 ◦ C to completely remove the chloroform solvent. 2.4. Elasticity of flexure test of the PAC/PLCL mixtures Films (size = 10 mm × 50 mm × 0.03 mm) with various PAC/PLCL ratios were prepared by natural drying in 10 wt.% chloroform solutions for 3 weeks. The elasticity of flexure test was performed using a universal testing machine (Instron model 5966, Canton, MAUSA). A 5 N load cell with a crosshead speed of 1 mm/min was used for this experiment. Each film was folded in half at a folding speed of 1 mm/min, immediately after which the stress was released and the recovered angle for 5 min was measured and compared with that of an octyl 2-cyanoacrylate film. The percent elasticity of flexure was calculated as [(recovered angle of film/angle (180◦ ) of unfolded film) × 100]. 2.5. Bond strengths of the PAC/PLCL mixtures
2. Materials and methods 2.1. Preparation and characterization of PACs AC (920; Robinson St. Pottstown, PA, USA) was heated at 150 ◦ C for 0, 5, 10, 20, 40, and 60 min in vacuum vials (10 ml/vial). Samples were then cooled to 0 ◦ C and stored at 4 ◦ C. Change of viscosity according to heating time was measured using viscometer (Brookfield, model LVDV-II+P, Middleboro, MA, USA) and compared with ethyl 2-cyanoacrylate as control.
The PAC/PLCL mixtures were applied to bovine skin (area: 10 mm × 10 mm). The specimens were then covered with another section of bovine skin. The volume of sample was 10 l, and the final adhesion thickness was 10 m. After 24 h at room temperature, the bond-strength of the PAC/PLCL mixtures was tested using a universal testing machine (Instron model 4467). The crosshead speed was set to 1 mm/min, and the load at which the specimen deboned from the adherent was recorded. 2.6. In vitro cytotoxicity test of the optimum PAC/PLCL mixture
2.2. Cytotoxicity test of PACs As a direct contact method, the cytotoxicity of PAC was tested using a previously reported method [15]. L929 cells (ATCC, Manassas, VA, USA) were cultured in RPMI 1640D media with 20% fetal bovine serum (FBS), 1% penicillin and 1% streptomycin. The cells were added in 24 well plate (104 cells/well) and maintained in a humidified atmosphere that contained 5% CO2 at 37 ◦ C. After 24 h, the media was aspirated and PACs and Dermabond of 100 l were each added directly to the center of each cultured well. After another 1 h, 2 ml media were slowly added to each well. After 4 h and 24 h, the initial adhesion and proliferation were determined by using a MTT assay. To determine viability, absorbance was measured at a test wavelength of 570 nm using a microplate reader (Molecular Devices, Toronto, Canada). The optical density (O.D.) was calculated as the difference between the reference wavelength and the test wavelength. The percent viability was calculated as [(O.D. of drug-treated sample/O.D. of untreated sample) × 100]. 2.3. Preparation of the PAC/PLCL mixtures PLCL [molar ratio 50:50, number-average molecular weight (Mn): 2.2 × 105 ] was polymerized and purified using a method described previously [10]. The 2% (w/v) PLCL was dissolved in chloroform solution. PLCL solution of various volumes was mixed with a fixed volume of PAC, as presented in Table 1. The mixtures Table 1 Composition of pre-polymerized ally 2-cyanoacrylate (PAC)/poly(l-lactide-co-caprolactone) (PLCL) mixtures. Mixture
PAC (g)
PLCL (mg)
Type-1 Type-2 Type-3 Type-4
4 4 4 4
20 40 80 120
Cytotoxicity testing of the optimum PAC/PLCL mixture was performed using direct contact method (Section 2.2). 2.7. Epidermal growth factor (EGF) release test An EGF (Sigma–Aldrich, St. Louis, MO, USA) solution (500 g/ml) and chitosan (>1000 kDa, 86% DD, Korea Chitosan Co., Ltd.) of 1 wt.% in 1% acetic acid solution was prepared and dropped in liquid nitrogen using a syringe pump (rate: 1 mm/min). And then, the beads were lyophilized for 24 h. The Bradford protein assay was used to measure the EGF released from the chitosan micro-beads, prepared by the EGF included chitosan micro-beads (dry weight 10 mg) and EGF included chitosan micro-beads (dry weight 10 mg)/PAC/PLCL (1 g) mixture, respectively. The samples were suspended in 10 ml PBS and incubated at 37 ◦ C and 30 rpm for 0, 2, 5, 8, 15, 20, 45, and 60 min. After the incubation, the releasate, condensed by lyophilization, was added to a 96-well plate. The Bradford reagent (Bio-Rad) was then added to each well before incubating the plate in the dark at room temperature for 15 min. Absorbance was measured at 595 nm after shaking for 30 s. 2.8. In vivo study Fifteen young male Sprague–Dawley rats (weight, 250–350 g) were used. All animals were anesthetized with inhaled isoflurane (1–5%). After anesthesia, hair from the animal’s back was removed with electric clippers, and a depilatory cream (Nair; Carter Products) was applied for several minutes. The area was washed with wet cotton and dried with a gauze sponge. Povidone-iodine was applied to the surgical site and removed, and then isopropyl alcohol (70%) was applied. Two 1.5 cm long incisions were made on the back of each rat, with at least 1 cm of intact skin between them. The EGF included chitosan micro-beads (20 mg) were applied to
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the each incision area. And then, the optimum PACA/PLCL mixture and octyl CA were applied as a monolayer over the incision without additional treatment, according to instructions for the commercial tissue adhesive. Rats were sacrificed at 15 days after surgery to examine and compare the tissue reactions between the two groups. Each tissue specimen was fixed in 10% buffered formalin for tissue slicing. Hematoxylin and eosin (H&E) staining was performed to observe inflammatory cell infiltration, and the stained tissues were observed under an optical microscope [16].
21
Table 2 Cell viability and bond strength of pre-polymerized ally 2-cyanoacrylate (PAC) according to heating time (n = 4 for each test, * P < 0.05, # P < 0.05). Heating time (min)
Cell viability (%) After 24 h*
0 5 10 20 40 60 Control
33 47 48 48 61 55 38.4
± ± ± ± ± ± ±
Bond strength (MPa)#
1 9 2 14 11 2 3.3
0.88 0.75 0.79 0.70 0.69 0.58 0.17
± ± ± ± ± ± ±
0.16 0.05 0.03 0.04 0.1 0.1 0.03
n 4 4 4 4 4 4 4
3. Results and discussion 3.1. Characterization of pre-polymerized allyl 2-cyanoacrylates The focus of this study was to achieve low cytotoxicity and good elastic properties for pre-polymerized allyl 2-cyanoacrylate bio-glue. Changes in cytotoxicity following pre-polymerization and altered mechanical properties with varying PLCL content were therefore important parameters to assess. To determine the optimal pre-polymerization conditions, the test materials were assessed in terms of viscosity, bond strength, and cytotoxicity and were compared with octyl 2-cyanoacrylate. The yellowish color and viscosity of the samples were found to increase with heating time, especially in the case of the PAC samples. The observed increase in the viscosity of PAC was attributed to the partial polymerization of another intramolecular double bond instead of the polymerization of a polymerizable double bond by an anion initiator. To confirm this hypothesis, the change in PAC viscosity with increasing heating time was compared to that of ethyl 2-cyanoacrylate (EC) viscosity with heating time. Fig. 1 shows the changes in viscosity and reveals that the viscosity of EC was not affected by heating time, whereas the viscosity of PAC increased with increased heating time. The polymerization reaction of another intramolecular double bond in AC was therefore confirmed. The PAC bond strength was also found to decrease with increasing heating time (Table 1: 0 min, 0.88 ± 0.16 MPa; 5 min, 0.75 ± 0.05 MPa; 10 min, 0.79 ± 0.03 MPa; 20 min, 0.70 ± 0.04 MPa; 40 min, 0.69 ± 0.1 MPa; 60 min, 0.58 ± 0.1 MPa). This trend was attributed to the decreased reactivity of anion initiators because of the high viscosity associated with increased heating time. Decreased PAC bond strength was therefore considered another result of the partial polymerization of the intramolecular double bond; however, the control material octyl 2-cyanoacrylate exhibited the lowest overall bond strength (0.17 ± 0.03 MPa).
Pre-polymerized allyl 2-cyanoacrylate Ethyl 2-cyanoacrylate
350
Viscosity (mPas)
300 250
The cytotoxicity of PAC was determined using a direct-contact culture cell viability test (Table 2). Generally, cytotoxicity decreases with an increase in the length of the intramolecular carbon chains in cyanoacrylates [8] and the cytotoxicity of PAC was therefore expected to decrease with increasing polymerization time. Most samples in this study were found to have a lower cytotoxicity than the control (octyl 2-cyanoacrylate), except the sample that was heat-treated for 0 min (cell viability, 33 ± 1%). The PAC, which was pre-polymerized for 40 min, yielded the highest cell viability (61 ± 11%). Previously reported cell viability data for prepolymerized AC were obtained by indirect methods using NIH3T3 cells [7], while the cytotoxicity testing in this study was conducted according to ISO 10993 (Biological evaluation of medical devices) standards. The optimal heat-treatment time for the production of bio-adhesives with low cytotoxicity was found to be 40 min using these methods. 3.2. Characterization of pre-polymerized allyl 2-cyanoacrylate (PAC)/poly(l-lactide-co--caprolactone) (PLCL) mixtures The elasticity of flexure of the PAC/PLCL films in response to bending stress was compared to that of octyl 2-cyanoacrylate, which was used as the control. The elasticity of flexure of the PAC/PLCL films was found to increase with increasing PLCL concentration (Table 3). Type-3, and -4 films were furthermore found to exhibit the highest elasticity of flexure values and the elasticity of flexure of the PAC/PLCL films was found to be higher than that of PAC and the control. The improved elasticity of flexure is thought to result from the presence of amorphous PLCL (50:50). Fig. 2 shows the bond strengths between porcine skins bound by PAC/PLCL mixtures as a function of PLCL ratio. Bond strengths of the PAC/PLCL mixtures decreased sharply with increasing PLCL content: type-3 and -4 films exhibited notably lower bond strengths than the other samples (PAC, 0.69 ± 0.1 MPa; type-1, 0.354 ± 0.01 MPa; type-2, 0.21 ± 0.03 MPa; type-3, 0.01 ± 0 MPa; type-4, 0.01 ± 0 MPa; and control, 0.17 ± 0.03 MPa). The low bond strength of type-3 and -4 films may be attributable to the increasingly amorphous structure of PAC/PLCL mixtures with increased PLCL concentration. On the other hand, in the case of the type2 film, a bond strength similar to that of the control film was
200 Table 3 Elasticity of flexure (%) of pre-polymerized ally 2-cyanoacrylate (PAC)/poly(llactide-co--caprolactone) (PLCL) mixtures (n = 4 for each test, * P > 0.05, # P > 0.05, *,# P < 0.05).
150 100
Samples (PAC/PLCL ratios)
50
#
0
10
20
30
40
50
60
Heating time (min) Fig. 1. Viscosity of pre-polymerized ally 2-cyanoacrylate (PAC)* and ethyl 2cyanoacrylate# according to heating time (n = 4 for each test, * P < 0.05, # P > 0.05).
PAC (4/0) Type-1 (4/20) Type-2 (4/40) Type-3 (4/80)* Type-4 (4/120)* Control#
Bending recovery (%) 32.4 45.3 48 50 50 31.6
± ± ± ± ± ±
0.7 0.86 0.47 0.27 0.64 0.42
n 4 4 4 4 4 4
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Bond strength (MPa)
0.8
0.6
0.4
* #
0.2
0.0 PAC
Type-1
Type-2
Type-3
Type-4
Control
Adhesives Fig. 2. Bond strength of pre-polymerized ally 2-cyanoacrylate. (PAC)/poly(l-lactideco--caprolactone) (PLCL) mixtures (n = 4 for each test, * P < 0.05, and # P > 0.05 compared with octyl 2-cyanoacrylate as a control)
observed. The type-1 film in particular exhibited increased bond strength compared to the control. Based on these findings, the optimal PAC/PLCL ratio for both enhanced flexibility and high bond strength is 4 g/20 mg. 3.3. Epidermal growth factor (EGF) release from chitosan micro-beads Epidermal growth factor (EGF) has been widely reported to promote wound healing [17–20] and EGF was therefore applied with the bio-glue in this study as a drug delivery system for improved wound healing efficiency. As shown in Fig. 4, the EGF-included chitosan micro-beads exhibited a ∼10-fold higher initial release velocity than the EGF-included chitosan microbead/PAC/PLCL mixture (release time [min]: EGF-included chitosan micro-beads [O.D.]/EGF-included chitosan micro-bead/PAC/PLCL mixture [O.D.], 2: 0.13/0.012). After 5 min, the difference in the amount of EGF released from each sample had increased from ∼10fold to 15-fold or more (release time [min]: EGF-included chitosan micro-beads [O.D.]/EGF-included chitosan micro-bead/PAC/PLCL mixtures [O.D.], 5: 0.44/0.022; 8: 0.57/0.033; 15: 0.65/0.051; 20: 0.75/0.058; 45: 0.98/0.064; 60: 1.1/0.062, Fig. 4). These findings
350
Cell viability (%)
300
TCP Octyl 2-cyanoacrylate Type-1
250 200 150
#
*
100 * 50 0 1 day
2 day
3 day
Culture period Fig. 3. Cell viability (%) test of pre-polymerized ally 2-cyanoacrylate (PAC)/poly(llactide-co--caprolactone) (PLCL) (n = 4 for each test, * P < 0.05, and # P < 0.05 compared with octyl 2-cyanoacrylate of the same period)
Fig. 4. Epidermal Growth Factor (EGF) release test from chitosan porous beads* and bio-glue mixture# in PBS solution at 37 ◦ C (n = 4 for each test, * P < 0.05, and # P > 0.05).
suggest that the high release velocity from the micro-beads was a result of good interconnectivity and a highly porous structure obtained by phase separation (SEM in Fig. 4). The low release velocity of EGF-included chitosan micro-bead/PAC/PLCL mixtures was furthermore likely to be due to low diffusion by the PAC/PLCL mixture coating the chitosan micro-beads. The higher release velocity is moreover indicative of faster diffusion over a large surface area of the 3D porous structure and the presence of exposed protein on the surface, as mentioned in a previous study [21]. To facilitate improved wound healing efficiency, EGF-included chitosan micro-beads were therefore prepared and applied directly to incision areas before the application of bio-glue. 3.4. Assessment of the biocompatibility of the PAC/PLCL mixture by in vitro and in vivo testing Cytotoxicity testing to evaluate short-term in vitro biocompatibility was performed using the optimal PAC/PLCL mixture (type-1) and the control material octyl 2-cyanoacrylate in a direct-contact culture cell viability test for 3 days. As shown in Fig. 3, cell viability in the presence of the PAC/PLCL mixture was significantly higher than that in the presence of octyl 2-cyanoacrylate (1 day, 38.4 ± 3.3%; 2 day, 47.9 ± 13.5%; 3 day, 107.5 ± 8.9%), particularly on the first (68.2 ± 4.2%, *P < 0.05 compared with octyl 2-cyanoacrylate at the same time point) and second (96.7 ± 2.3%, *P < 0.05 compared with octyl 2-cyanoacrylate at the same time point) days. Cytotoxicity therefore seems to decrease as a result of increased polymer chains by pre-polymerization, and the pre-polymerized PAC/PLCL film was shown here to be less cytotoxic than commercial bio-glue. A histological analysis was performed to evaluate long-term in vivo biocompatibility. Fig. 5 shows images of healed skin tissue treated with either the PAC/PLCL mixture or octyl 2cyanoacrylate at 15 days after surgery. Images were obtained after hematoxylin–eosin (H&E) staining, which shows pink cytoplasm and purple nuclei in the tissue. No differences were observed in the images of tissues from wounds closed with the PAC/PLCL film (type1) in comparison to those closed with commercial bio-glue (octyl 2-cyanoacrylate); however, in the case of octyl 2-cyanoacrylate, a weak inflammatory reaction was observed (star, Fig. 5d–f) [16,22]. Although it was difficult to accurately determine the degree of
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Fig. 5. H&E stained slice microphotography of closed rat tissue by bioglues [(a–c): Normal, (d–f): Octyl 2-cyanoacrylate, (g–i): pre-polymerized ally 2-cyanoacrylate (PAC)/poly(l-lactide-co--caprolactone) (PLCL)].
inflammation from the small number of specimens in this study, it was inferred that skin tissue closure using the PAC/PLCL mixture caused less inflammation and foreign body reaction for a considerable length of time compared to the octyl 2-cyanoacrylate treatment. 4. Conclusions We prepared and characterized a PAC/PLCL mixture to improve its flexibility and biocompatibility as a tissue adhesive. Prepolymerization of AC by heat treatment at 150 ◦ C for 40 min was the most effective way to reduce cytotoxicity of the short-chain cyanoacrylate derivative. The biocompatible PLCL improved flexibility and cell viability of PAC. Among them, the PAC/PLCL mixture at a ratio of 4 g/20 mg resulted in the highest bond strength, good cell viability, and was an effective skin closure material. Therefore, we expect that this new PAC/PLCL mixture might be useful as an alternative tissue adhesive for skin wound closure along with conventional suture materials and/or commercial tissue adhesives. Further studies are essential to understand the polymerization of cyanoacrylate derivatives to further increase bond strength, decrease cytotoxicity, and make them suitable for applications in various fields including medicine. Acknowledgement The present research was conducted by the research fund of Dankook University in 2014.
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