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## Creator

Satsuki Minamisakamoto, [Hiyori Komatsu](https://orcid.org/0000-0002-2525-1362), Shiharu Watanabe, [Shima Ito](https://orcid.org/0000-0002-3233-617X), Hatsune Nishino, [Tetsushi Taguchi](https://orcid.org/0000-0003-2541-2530)

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[The effect of branched structures of alkyl groups on tissue adhesiveness and biocompatibility of alkyl groups-modified Alaska pollock gelatin-based adhesives](https://mdr.nims.go.jp/datasets/ce50541a-2a69-40b2-9a05-057b9e5f9bfe)

## Fulltext

1  The effect of branched structures of alkyl 1 groups on tissue adhesiveness and 2 biocompatibility of alkyl groups-modified 3 Alaska pollock gelatin-based adhesives 4  5 Satsuki Minamisakamoto1,2, Hiyori Komatsu1,2, Shiharu Watanabe2, Shima Ito1,2, 6 Hatsune Nishino2,3, Tetsushi Taguchi1,2* 7 1Graduate School of Science and Technology, Degree Programs in Pure and Applied 8 Sciences, University of Tsukuba, 1-1-1 Tennodai, Tsukuba, Ibaraki 305-8577, Japan 9 2Biomaterials field, Research Center for Macromolecules and Biomaterials, National 10 Institute for Materials Science, 1-1 Namiki, Tsukuba, Ibaraki 305-0044, Japan 11 3College of Engineering Science, School of Science and Engineering, University of 12 Tsukuba, 1-1-1 Tennodai, Tsukuba, Ibaraki 305-8577, Japan 13  14 Correspondence and requests for materials should be addressed to T. Taguchi. (email: 15 TAGUCHI.Tetsushi@nims.go.jp) 16   17 Revised Manuscript File after approval Click here to view linked References 1  2  3  4  5  6  7  8  9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 https://www2.cloud.editorialmanager.com/jmade/viewRCResults.aspx?pdf=1&docID=137412&rev=1&fileID=3215624&msid=83b13ea4-cdd0-4f77-8c1d-3742327aaa04https://www2.cloud.editorialmanager.com/jmade/viewRCResults.aspx?pdf=1&docID=137412&rev=1&fileID=3215624&msid=83b13ea4-cdd0-4f77-8c1d-3742327aaa042  Abstract 18 Tissue adhesives are widely used to prevent air leakage from the lungs and bleeding from 19 vascular anastomoses. However, currently used tissue adhesives still face challenges with 20 either tissue adhesion or biocompatibility. We previously reported tissue adhesives 21 composed of straight alkyl group-modified Alaska pollock gelatin (ApGltn) and 22 pentaerythritol poly (ethylene glycol) ether tetrasuccinimidyl glutarate (4S-PEG). The 23 developed adhesives have sufficient tissue adhesive strength and biocompatibility for 24 biomedical applications; however, the effect of the branched structures of the alkyl groups 25 on these functions has not yet been clarified. In this study, we evaluated the tissue 26 adhesiveness and biocompatibility of three tissue adhesives based on straight/branched 27 alkyl group-modified ApGltns and 4S-PEG. The results showed that branched alkyl-28 group-modified ApGltns-based adhesives had higher tissue adhesion strength than 29 straight alkyl-ApGltn. Furthermore, the burst strength of the branched alkyl group- 30 modified ApGltn-based adhesives 2-fold higher compared to commercial Fibrin. In 31 addition, they were completely biodegraded in rat subcutaneous tissue within 56 days 32 without causing severe inflammation. 33 Keywords: in situ hydrogel; tissue adhesion; Alaska pollock-derived gelatin; 34 hydrophobic interaction. 35   36  1  2  3  4  5  6  7  8  9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 3  1. Introduction 37 Tissue adhesives are widely used in clinical practice to prevent air leakage from the lungs 38 or oozing from vascular anatomic sites. Various tissue adhesives, such as cyanoacrylate, 39 albumin with aldehyde, and fibrin glue, have been developed to close or seal wounds after 40 surgical operations [1]. Cyanoacrylate adhesives exhibit superior adhesive strength and 41 the ability to bond to wet tissues via water-mediated polymerization [2]. Although it has 42 been mainly used for skin adhesion, the release of formaldehyde through the degradation 43 process of cyanoacrylate adhesives is toxic to the body [3]. Other adhesive made from 44 bovine serum albumin and glutaraldehyde as crosslinkers have high adhesive strength 45 owing to Schiff base formation between the primary amine of albumin and glutaraldehyde 46 [4]; however, the release of unreacted glutaraldehyde from cured adhesives also shows 47 toxicity [5]. Therefore, fibrin glue is commonly used post-operatively. It is mainly 48 composed of human blood-derived fibrinogen and thrombin [1, 2] and shows superior 49 biocompatibility; however, its adhesiveness is weak compared to that of cyanoacrylate 50 and albumin-glutaraldehyde adhesives because of its weak interaction with biological 51 tissues. Therefore, commercial tissue adhesives face challenges in terms of strong tissue 52 adhesion and superior biocompatibility. Some functional groups such as N-Hydroxy 53 succinimide (NHS) ester, aldehyde, and catechol have been introduced to the hydrogel 54  1  2  3  4  5  6  7  8  9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 4  for high adhesion to tissue, removing the hydration layer on the tissue surface and 55 building connectivity between the adhesive and tissue through covalent bonds to address 56 these issues [6, 7]. It has also been reported that double network hydrogels, tetra-PEG 57 gels, slide-ring gels and nanocomposite hydrogels have a crosslinking style for greater 58 bulk strength [8-11]. We have previously developed hydrophobic-modified Alaska 59 pollock-derived gelatin (ApGltn)-based tissue adhesives [12, 13]. ApGltn has a low sol-60 gel transition temperature for dissolution at room temperature, enabling dissolution in the 61 freeze-dried state without heating. Using ApGltn as a base polymer, we designed tissue 62 adhesives made from straight alkyl group-modified ApGltns and pentaerythritol poly 63 (ethylene glycol) ether tetrasuccinimidyl glutarate (4S-PEG) and demonstrated their 64 excellent tissue adhesiveness and biocompatibility. However, the effects of branched 65 alkyl groups structures on the adhesive strength and biocompatibility of tissue adhesives 66 have not yet been elucidated. It is known that the shorter the main-chain length of an alkyl 67 group, the lower the enthalpy and entropy of melting when the number of carbons is the 68 same [14, 15]. In this study, we synthesized three straight/branched alkyl group-modified 69 ApGltns and evaluated their tissue adhesiveness and biocompatibility of tissue adhesives 70 based on the alkyl group-modified ApGltns and 4S-PEG (Figure 1). It is anticipated that 71 tissue adhesives based on branched alkyl group-modified ApGltns will exhibit superior 72  1  2  3  4  5  6  7  8  9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 5  handling and higher tissue adhesiveness owing to their improved mobility or 73 interpenetration resulting from their lower hydrophobicity and viscosity [16]. 74  75 2. Materials and Methods 76 2.1. Materials 77 ApGltn was purchased from Nitta Gelatin, Inc. (Osaka, Japan). Dulbecco’s Phosphate-78 Buffered Saline (D-PBS), dimethyl sulfoxide (DMSO), triethylamine (TEA), 2,4,6-79 trinitrobenzensulfonic acid (TNBS), 6 N-hydrochloric acid (HCl), boric acid, potassium 80 chloride, sodium hydroxide, phosphoric acid, sodium dihydrogen phosphate, bovine 81 serum albumin (BSA), paraformaldehyde, 4',6-diamidino-2-phenylindole (DAPI) and 82 10% formalin neutral buffer solution were purchased from Wako Pure Chemical 83 Industries, Ltd (Osaka, Japan). n-Octanal and 2-ethylhexanal were purchased from Tokyo 84 Chemical Industry Co., Ltd. (Tokyo, Japan). 2-propyl valeraldehyde was kindly donated 85 by Santa Cruz Biotechnology Inc. (Dallas, TX, USA). 2-Picoline borane was purchased 86 from Junsei Chemical Co. Ltd. (Tokyo, Japan). DMSO-d6, Roswell Park Memorial 87 Institute (RPMI) medium, and fetal bovine serum (FBS)-FITC/Phalloidin were purchased 88 from Sigma-Aldrich Co., LLC (St. Louis, MO, USA). Penicillin/streptomycin was 89 purchased from Thermo Fisher Scientific (Tokyo, Japan). Triton-X was purchased from 90  1  2  3  4  5  6  7  8  9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 6  Cayman Chemical (Ann Arbor, MI, USA). TrypLE Express was purchased from Life 91 Technologies (Grand Island, NY, USA). Collagenase and Water-soluble tetrazolium 92 (WST) -8 were purchased from NACALAI TESQUE, INC. (Kyoto, Japan). The mouse 93 fibroblast-like cell line (L929) was purchased from RIKEN (Saitama, Japan). 99.5% 94 Ethanol was purchased from Kishida Chemical Co., LTD (Osaka, Japan). 4S-PEG was 95 purchased from NOF Co., Ltd. (Tokyo, Japan). Beriplast-P (Fibrin) was purchased from 96 CSL Behring (Tokyo, Japan). Collagen casing was purchased from Nippi, Incorporated 97 (Tokyo, Japan). Saline was purchased from Otsuka Pharmaceutical Co. Ltd. (Tokyo, 98 Japan). Rats (Wistar, male, 6 weeks old) were purchased from Jackson Laboratory Japan 99 (Kanagawa, Japan). 100  101 2.2. Synthesis of C8-ApGltns 102 Three straight/branched alkyl group-modified ApGltns (C8-ApGltns) were synthesized 103 using a previously reported [17, 18]. We employed n-octyl group (straight), 2-ethylhexyl 104 group (branched), and 2-propylpentyl group (branched), which have eight carbons, as the 105 straight/branched alkyl groups. Straight/branched alkyl groups-modified ApGltns were 106 synthesized by reductive amination of the amino groups of the original ApGltn (Org-107 ApGltn) with alkyl aldehydes (Figure 1). Briefly, Org-ApGltn was dissolved in 108  1  2  3  4  5  6  7  8  9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 7  water/EtOH (50/50 (v/v)) mixed solvent to obtain a 15 w/v% solution at 50 °C. Alkyl 109 aldehyde was then added to the solution and stirred for 1 h. After that, 2-picoline borane 110 was added to the reaction solution and the mixture was stirred for 18 h at 50 °C. The 111 resulting C8-ApGltns were re-precipitated in cold EtOH and washed three times with cold 112 EtOH to remove unreacted alkyl aldehyde and 2-Picoline borane. The precipitates were 113 then filtered and dried under reduced pressure for 2 days at room temperature. 114   115  1  2  3  4  5  6  7  8  9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 8  Figure 1. Application of C8-ApGltn adhesives to soft tissue. a) Synthesis of C8-ApGltns 116 through Schiff base formation and reductive amination using straight/branched alkyl 117 aldehydes. b) In situ crosslinking of C8-ApGltn with 4S-PEG. c) Adhesion mechanism 118 of C8-ApGltn adhesives after application onto soft tissue. 119   120  1  2  3  4  5  6  7  8  9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 9  2.3. Characterization of C8-ApGltns 121 The degree of modification of the resulting C8-ApGltns were determined based on the 122 residual amino group content using TNBS, method, as reported previously [19, 20]. We 123 prepared 0.1 w/v% each Org and C8-ApGltns solution using the water/DMSO (50/50 124 (v/v)) mixed solvent. 100 μL of each solution was dispensed to well plate (n=9) and added 125 the 100 μL of TEA (0.1 v/v%) and TNBS (0.1w/v%) solution. After reaction at 37 °C for 126 2 h, DS was determined from the ratio of absorbance at 340 nm. Modification of the 127 straight/branched alkyl groups was also confirmed using Fourier transform infrared 128 spectroscopy (FT-IR) (ALPHA II; Bruker Japan K. K., Kanagawa, Japan) and proton 129 nuclear magnetic resonance (1H NMR) (ECZ400S; JEOL, Tokyo, Japan) in DMSO-d6 130 containing 0.03 v/v% of tetramethylsilane (TMS) as a standard substance. 131  132 2.4. Gelation time of C8-ApGltns adhesives 133 C8-ApGltns adhesives were prepared by mixing 4S-PEG (40 mol% of the amino groups 134 of Org-ApGltn in 0.01 M phosphate buffer, pH 4) with Org-ApGltn or straight/branched 135 alkyl groups-modified ApGltns solutions at 1:1 using a double syringe (Bethel Co., Ltd., 136 Ibaraki, Japan) to prepared adhesive hydrogels in situ [21]. The solvent of all ApGltns 137 solutions was borate buffer (0.075 M, pH 9.5). The gelation time of Org and C8-ApGltns 138  1  2  3  4  5  6  7  8  9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 10  adhesives were measured according to a previously reported [22, 23]. Briefly, 500 µL of 139 Org or C8-ApGltns solutions were poured into a 9 mL screw tube and stirred with an 18 140 mm stirrer at 290 rpm and 37 °C. The gelation time was defined as the time until the 141 rotation of the stirring bar was hindered by the gelatin after addition of 500 µL of 4S-PEG 142 solution (n = 3–4). 143  144 2.5. Swelling ratio 145 The swelling ratio of the C8-ApGltn adhesives was measured by immersing Org or C8-146 ApGltns adhesive hydrogels in D-PBS, as previously reported [13, 24]. Briefly, Org-147 ApGltn or C8-ApGltn/4S-PEG mixed solutions were poured into a silicone mold to 148 prepare disks 10 mm in diameter and 1 mm thick. The disks were immersed into 50 mL 149 D-PBS at 37 °C in centrifuge tubes and weighed after immersion for various periods (Ws) 150 [13]. The D-PBS in the centrifuge tube was replaced each time. The swollen adhesives 151 were then immersed in 50 mL of ultrapure water three times every 30 min to remove salt 152 and freeze-dried for 24 h. Finally, the freeze-dried adhesives were weighed (Wd) to 153 determine the swelling ratio using the following equation: 154 𝑆𝑤𝑒𝑙𝑙𝑖𝑛𝑔 𝑟𝑎𝑡𝑖𝑜 =  𝑊𝑠 − 𝑊𝑑𝑊𝑑 155  156  1  2  3  4  5  6  7  8  9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 11  2.6. Rheological measurement 157 Rheological measurements of the Org/stC8/asyC8/syC8-ApGltn hydrogels were 158 performed using a rheometer (MCR301; Anton Paar, Graz, Austria) with a PP10 parallel 159 plate (10 mm) [17]. Adhesive (30 µL) was applied to the stage of the rheometer at 37 °C 160 and sandwiched between plates separated by a 1 mm gap. After removing the excess 161 adhesive from the PP10 plate, the adhesive was left for 10 min to allow sufficient gelation. 162 First, shear strain was measured to determine the linear viscoelastic (LVE) region of the 163 cured adhesive hydrogels. The storage modulus (G’) and loss modulus (G”) were 164 measured at an angular frequency of 10 rad/s with changing shear strains ranging from 165 0.01% to 100%. Then, an angular frequency from 1 to 100 rad/s was measured at strain 166 amplitude within the LVE region at 37 °C. The viscosities of Org and C8-ApGltns 167 solutions at room temperature were measured using a viscometer (VM-10A; SEKONIC, 168 Tokyo, Japan). 169  170 2.7. Tensile strength measurement 171 The tensile strengths of Org and C8-ApGltns adhesive hydrogels were measured 172 according to ASTM D412 [17]. Dumbbell-shaped adhesive hydrogels with a thickness of 173 1 mm were prepared for tensile strength measurements (Figure S3a). The tensile strength 174  1  2  3  4  5  6  7  8  9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 12  was measured using a texture analyzer (TA.XTplusC Texture Analyzer; Stable Micro 175 Systems, Surrey, UK) under fixed conditions (temperature: 25 °C, humidity: 60–70%, 176 strain rate: 10 mm/min). 177  178 2.8. Burst strength measurements 179 The burst strengths of Org and C8-ApGltns adhesives were measured according to ASTM 180 2392-04 (Figure S4). Commercial Fibrin was also used as a control adhesive. The 181 collagen casing was prepared by cutting it into a disk 35 mm in diameter with a hole of 3 182 mm in diameter at the center. Then, a silicone mold with a diameter of 35 mm and a 183 thickness of 1 mm, with a hole 15 mm in diameter at the center, was placed on the surface 184 of the collagen casing. Org and C8-ApGltn adhesives were used to seal the holes. After 185 10 min, a collagen casing with an adhesive was placed on the stage of the test system at 186 37 °C to measure the burst strength. Saline was poured through the bottom of the collagen 187 casing at a flow rate of 2 mL/min. The burst strength for each condition was defined as 188 the maximum burst strength at which the adhesive fractured or peeled off (Figure 4b) 189 [25]. 190  191  1  2  3  4  5  6  7  8  9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 13  2.9. In vitro enzymatic degradation test 192 Disk-shaped adhesive hydrogels (6 mm in diameter, 1 mm in thickness) were immersed 193 in 5 mL of 1.25 U/mL of collagenase in D-PBS To assess the in vitro biodegradability of 194 Org or C8-ApGltns adhesive hydrogels, [12, 26]. After immersion for various periods, the 195 adhesive hydrogels were placed in ultrapure water to remove salts and then freeze-dried 196 (n = 3). The mass of the adhesive hydrogel was measured at each time point. W0 and Wt 197 indicate the mass of the freeze-dried adhesive hydrogels before and after immersion in 198 the collagenase solution, respectively. Biodegradability was calculated using the 199 following equation: 200 𝑊𝑒𝑔ℎ𝑖𝑡 𝑟𝑒𝑚𝑎𝑖𝑛𝑖𝑛𝑔 (%) =  𝑊𝑡𝑊0× 100 201 2.10. Cytocompatibility 202 The cytocompatibility of Org and C8-ApGtlns adhesives was evaluated according to ISO 203 10993-5 and 10993-12 [17]. Adhesive hydrogels with a mass extraction rate of 0.1 g/mL 204 and surface extraction rate of 3 mm2/mL were cut to prepare the extraction medium for 205 each adhesive. Obtained samples were then immersed into RPMI medium for 24 h at 206 37 °C. For the evaluation of cytocompatibility, 1.0 × 104 fibroblasts (L929) were seeded 207 into 20 wells in a 96-well plate and preincubated for 24 h at 37 °C. All media were 208 removed and replaced with the extracted medium (n = 5). The RPMI medium without 209  1  2  3  4  5  6  7  8  9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 14  samples was used as a control. The number of viable cells in each well was measured 210 after incubation for 24 h using the WST-8. Before observing the cell morphology, the cells 211 were fixed with 4% PFA for 15 min at 4 °C and washed twice with D-PBS. BSA (1% in 212 D-PBS) was used for blocking to prevent non-specific binding of antibodies, and Triton-213 X (0.2% in D-PBS) was used for transparent processing. Actin filament was stained with 214 0.1% FITC/phalloidin at D-PBS and nuclear with 0.1% DAPI at D-PBS. The cell 215 morphology was observed using a fluorescence microscope (BZ-X710; KEYENCE, 216 Osaka, Japan). 217  218 2.11. Subcutaneous implantation 219 All animal experiments were approved by the Animal Experiment Committee of the 220 National Institute for Materials Science (approval no. 79-2024-3). Disk-shaped adhesive 221 hydrogels (thickness: 0.5 mm, diameter: 6 mm) were prepared and sterilized using UV 222 irradiation for 15 min. The rats were then anesthetized by isoflurane inhalation, and a 1 223 cm incision was made at the back of each rat after shaving. The tissues around the 224 adhesives were then excised, fixed with a neutral buffered 10% formalin solution, and 225 stained with hematoxylin and eosin (HE) for histological observation. 226  227  1  2  3  4  5  6  7  8  9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 15  2.12. Statistical analysis 228 Statistical analyses were performed using one-way or two-way ANOVA followed by 229 Tukey’s multiple comparison test. Statistical significance was set at p < 0.05. 230  231   232  1  2  3  4  5  6  7  8  9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 16  3. Results and Discussion 233 3.1. Synthesis and characterization 234 C8-ApGltns (stC8/asyC8/syC8-ApGltns) with different alkyl structures and degrees of 235 substitution (DS) were synthesized by reductive amination of alkyl aldehydes to the 236 amino groups of Org-ApGltn. As shown in Table 1, stC8/asyC8/syC8-ApGltns were 237 obtained at high yields (>80%); stC8/asyC8-ApGltns had low (approximately 10 mol%) 238 (L), medium (approximately 30 mol%) (M), and high (approximately 45 mol%) (H) DS; 239 and syC8-ApGltn had only low DS. There were 1.3 alkyl groups per Org-ApGltn 240 molecule in stC8 (L). The FT-IR spectra revealed that the specific peaks corresponding 241 to the N-H stretching vibration of the secondary amine (3,290 cm-1) increased with an 242 increase in DSs (Figure 2a, b). In addition, the specific peaks assigned to the C-H 243 symmetric stretching vibration in CH3 (2,880 cm-1), C-H asymmetric stretching vibration 244 in CH2 (2,940 cm-1), and CH3 (2,960 cm-1) indicated that alkyl groups were successfully 245 introduced to Org-ApGltn. The 1H NMR spectra revealed that the intensity of the stronger 246 specific peaks of CH3 and CH2 at 0.85 and 1.24 ppm, respectively, increased with 247 increasing DSs (Figure 2c). In addition, the peaks of stC8/syC8-ApGltn in the 1H NMR 248 spectra were sharper than those of asyC8-ApGltn because of the diverse proton 249 environment of asyC8-ApGltn. 250  1  2  3  4  5  6  7  8  9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 17  Table 1. Characteristics of C8-ApGltns with straight (stC8), asymmetric (asyC8) and 251 symmetric (syC8) alkyl groups 252 Abbreviation ApGltn (g) Amino groups in ApGltn (μmol/g) Theoretical DS (mol%) DS (mol%) Yield (%) stC8 (L) 5 351 10.3 9.1 87.3 stC8 (L) 30 357 10 8.1 93.2 stC8 (M) 5 351 50 28.6 84.9 stC8 (H) 5 351 154 45.3 86.0 asyC8 (L) 5 351 10 9.4 88.0 asyC8 (L) 30 357 10 8.2 93.4 asyC8 (M) 5 351 40 30.5 85.8 asyC8 (H) 5 351 90 44.5 86.3 syC8 (L) 5 357 10 8.6 82.7  253   254  1  2  3  4  5  6  7  8  9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 18  Figure 2. Characterization of C8-ApGltns. a) FT-IR spectra of stC8-ApGltns. b) FT-IR 255 spectra of branched asyC8/syC8-ApGltns. c) 1H NMR spectra of Org/stC8/asyC8/syC8-256 ApGtlns. 257  258  259   260  1  2  3  4  5  6  7  8  9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 19  3.2. Gelation time 261 Gelation of the Org or C8-ApGltns adhesives proceeds by the nucleophilic attack of the 262 amino groups of Org or C8-ApGltns on the carbonyl carbon of the active ester groups in 263 4S-PEG. Figure 3a shows the gelation time of Org and C8-ApGltns adhesives with 264 different DSs. The gelation time increased with an increase in DS. The viscosities of Org 265 and C8-ApGltn solutions were measured using a viscometer to clarify these phenomena. 266 As shown in Figure 3b and Figure S1, the viscosity of the stC8 (H) solution was 6-fold 267 higher than that of the asyC8 (H) solution, indicating that the aggregation property of the 268 stC8 group was higher than that of asyC8. However, the viscosity of the 4S-PEG solution 269 was 6.45 mPa·s. These results indicated a correlation between gelation time and viscosity 270 of C8-ApGltns solutions (Figure 3c), suggesting that reaction kinetics between ApGltn 271 and 4S-PEG was slower by inducing the less opportunity to react when the viscosity of 272 ApGltn solution became high. 273  274 3.3. Swelling ratio 275 The swelling ratios of the adhesive hydrogels were evaluated using 276 Org/stC8(L)/asyC8(L)/syC8(L)-ApGltns cured within 5 s. Swelling ratios of Org- and 277 C8-ApGltns adhesive hydrogels increased up to 5 h and reached equilibrium within 24 h. 278  1  2  3  4  5  6  7  8  9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 20  The swelling ratios of C8-ApGltns adhesive hydrogels were lower than that of the Org-279 ApGltn adhesive hydrogel (Figure 3d, Figure S2). This is because the C8-ApGltns 280 adhesive hydrogels exhibited additional physical crosslinking derived from the 281 straight/branched C8 groups. Among the C8-ApGltns adhesive hydrogels, the swelling 282 ratio of the stC8-ApGltn adhesive hydrogel was lower than that of the asyC8/syC8-283 ApGltns adhesive hydrogel, indicating that the length of the main chain of the C8 group 284 played a key role in decreasing the swelling ratios. The swelling ratio is determined by 285 the osmotic pressure owing to differences in the polymer concentration and elastic 286 pressure of the hydrogel network [27]. Although the polymer concentrations of the Org- 287 and C8-ApGltns adhesive hydrogels were similar, the elastic pressure increased for the 288 formation of physical crosslinking when the hydrophobic groups were introduced, 289 resulting in a lower swelling ratio. In addition, the strength of hydrophobic interactions 290 varies depending on the molecular structure, and the aggregation properties are different 291 [28]. The swelling properties of the C8-ApGltns adhesive hydrogels were determined by 292 the degree of physical crosslinking. 293  1  2  3  4  5  6  7  8  9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 21   294 Figure 3. Physico-chemical properties of C8-ApGltns adhesives (**p < 0.01 using one-295 way ANOVA). a) Gelation time of Org/stC8/asyC8/syC8-ApGtlns adhesives. b) 296 Viscosities of C8-ApGltns solutions with different DS. The red line indicates the viscosity 297 of the 4S-PEG solution. c) Relation of viscosity of C8-ApGltn solution and their gelation 298 time. d) Swelling behavior of Org- and C8-ApGltns adhesive hydrogels. 299   300  1  2  3  4  5  6  7  8  9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 22  3.4. Rheological and mechanical properties of C8-ApGltns adhesive hydrogels 301 The rheological properties were evaluated to clarify the effect of the branched structures 302 of the C8 groups on the modulus of the C8-ApGltns adhesive hydrogels (Figure 4a-c). 303 This rheological measurement was performed to evaluate the viscoelasticity of 304 completely formed hydrogels. We measured the shear strain from 0.01 to 100% to 305 determine the LVE region (Figure 4a). From the results of the shear strain measurements, 306 the strain ranged from 0.01-10%, indicating that these adhesive hydrogels were LVE in 307 that range. Therefore, we measured the angular frequency of the Org- and C8-ApGltns 308 adhesive hydrogels at a strain of 1%. From the angular frequency measurement, the G’ of 309 the Org- and C8-ApGltns adhesive hydrogels showed a plateau in the frequency ranging 310 from 1 to 100 rad/s, indicating that these adhesive hydrogels had no dependence on 311 frequency (Figure 4b). G’ of all C8-ApGltns adhesive hydrogels was larger than that of 312 Org-ApGltn adhesive hydrogel (Figure 4c), meaning that straight/branched C8 groups in 313 C8-ApGltns adhesive hydrogels contributed to the formation of physical crosslinking. 314 Among three C8-ApGltns adhesive hydrogels, syC8(L) had the highest G’, meaning that 315 syC8 adhesive hydrogel was hard and strong against external force by storing the energy 316 in the gel network. In addition, the G” value of stC8(L) was higher than that of syC8(L) 317 and asyC8(L) (Figure 4b). This means that stC8 was weaker against external forces 318  1  2  3  4  5  6  7  8  9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 23  because of its weak hydrogel network, which was prone to not storing energy. 319 The bulk strength of the Org/stC8(L)/asyC8(L)/syC8(L)-ApGltns adhesive hydrogel was 320 evaluated using tensile strength measurements. As shown in Figure 4d, the stress-strain 321 curves of each adhesive hydrogel revealed that these adhesive hydrogels were elastic 322 materials. No significant differences in the maximum strain were observed among the 323 Org/stC8(L)/asyC8(L)/syC8-ApGltns adhesive hydrogels (Figure S3c). In contrast, the 324 maximum stress of syC8(L) was significantly higher than Org/ stC8(L)/asyC8(L)-325 ApGltns adhesive hydrogels. In addition, Young’s modulus of the 326 stC8(L)/asyC8(L)/syC8(L)-ApGltns adhesive hydrogels was 2-fold higher than that of 327 the Org-ApGltn adhesive hydrogel (Figure 4e), indicating that the hydrogel network 328 elasticity became stronger owing to the increase in physical crosslinking. As with the 329 rheological measurement, syC8(L) showed the highest young’s modulus. In comparison 330 to straight chains, the gel network is more uniform due to that the shorter the main chain 331 of the branch, the weaker the hydrophobic interaction, which is indicated to result in high 332 storage modulus and Young's modulus. 333  1  2  3  4  5  6  7  8  9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 24   334 Figure 4. Rheological properties and tensile strength of C8-ApGltns adhesives. a) Shear 335 strain dependency of G’ and G”. b) Angular frequency dependence of G’ and G”. c) 336 Storage modulus of Org/stC8/asyC8/syC8-ApGtlns hydrogels at 10 rad/s, 1%. d) Stress-337 strain curve. e) Young’s modulus. (*p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001 338 using one-way ANOVA) 339   340  1  2  3  4  5  6  7  8  9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 25  3.5. Burst strength measurement 341 The burst strengths of the Org/stC8/asyC8-ApGltns adhesives and commercial Fibrin 342 were measured using a collagen casing as the adherend. stC8/asyC8-ApGltns adhesives 343 showed a lower burst strength when their DS increased (Figure 5a). This indicated that 344 stC8/asyC8-ApGltns with low DS were mixed more uniformly with 4S-PEG because of 345 their low viscosity. In contrast, the asyC8/syC8-ApGltn adhesives exhibited higher burst 346 strength than the stC8-ApGltn adhesives at any DS and their burst strength were twice as 347 high as those of Fibrin. We also analyzed the quantitative data of the destruction mode, 348 such as the peeling or fracture of adhesives, after the application of adhesives to collagen 349 casings to discuss these behaviors (Figure 5b). Figure 5c shows the destruction mode of 350 each adhesive after burst strength measurements. The frequency of peeling increased at 351 higher DS of the stC8-ApGltn adhesive, indicating that the interfacial adhesion strength 352 weakened with an increase in DS. In contrast, the asyC8-ApGltn adhesive showed 353 fracture behavior at any DS. This was because asyC8 contributed to hydrophobic 354 interaction/interpenetration with collagen casing rather than self-aggregation because of 355 its higher flexibility compared to stC8, while syC8(L) also showed high burst strength 356 similar to asyC8(L). However, the mode of destruction of syC8(L) was different from that 357 of asyC8(L) (Figure 5c). syC8(L) had a higher storage modulus than asyC8(L) (Figure 358  1  2  3  4  5  6  7  8  9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 26  4a,b), indicating that the bulk strength of syC8(L) was higher than that of asyC8(L). It 359 has been reported that the critical micelle concentration of surfactants with linear alkyl 360 groups is lower than that of surfactants with branched alkyl groups [28], indicating that 361 stC8 induces stronger aggregation than asyC8 or syC8. While, Fibrin showed the lowest 362 burst strength with high peeling ratio, meaning interfacial strength between Fibrin and 363 collagen casings was low. As shown in Figure 6, tissue adhesion of straight/branched 364 alkyl group-modified ApGltns adhesives may be governed by the balance among inner 365 aggregation (bulk strength), interfacial interaction (interfacial adhesive 366 strength/hydrophobicity) and Young’s modulus (elasticity). AsyC8(L)-ApGltn adhesive 367 with middle inner aggregation properties, middle interfacial interaction properties and 368 middle Young’s modulus, results in the high burst strength (Figure 5a). While, syC8(L)-369 ApGltn adhesive may have low interfacial interaction properties, but has high bulk 370 strength and Young’s modulus, results in the high burst strength (Figure 5a).  371  1  2  3  4  5  6  7  8  9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 27  Figure 5. Burst strength of C8-ApGltns adhesives and Fibrin applied on collagen casing. 372 a) Burst strength of Org/stC8/asyC8/syC8-ApGtlns adhesives with different DS and 373 Fibrin. b) Destruction mode during burst strength measurement. c) Ratios of destruction 374 mode of Org/stC8/asyC8/syC8-ApGtlns adhesives and Fibrin. (*p < 0.05, **p < 0.01 375 using one-way ANOVA) 376  377   378  1  2  3  4  5  6  7  8  9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 28   379 Figure 6. Tissue adhesion mechanisms of straight/branched alkyl groups-modified-380 ApGltns adhesives. 381   382  1  2  3  4  5  6  7  8  9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 29  3.6. Enzymatic degradation study 383 The enzymatic degradability of the adhesive hydrogels was evaluated by immersion in a 384 collagenase solution. All Org- and C8-ApGltns adhesive hydrogels were enzymatically 385 degraded within 24 h (Figure S5). The weight remaining decreased with increasing 386 immersion time. Among all the adhesive hydrogels examined, the Org-ApGltn adhesive 387 hydrogel degraded rapidly. However, the stC8(L)/asyC8(L)-ApGltns adhesive hydrogels 388 showed the slowest degradation rate among the C8-ApGltns adhesive hydrogels, 389 indicating that their swelling ratio was lower than that of Org/syC8(L)-ApGltns adhesives, 390 limiting the interpenetration of collagenase into the adhesive hydrogel matrix. All 391 adhesive hydrogels can be enzymatically degraded in a similar process in vivo because 392 the matrix metalloproteases (MMP)-2 and -9 selected from the cells during the wound 393 healing process [29] facilitate the decomposition of gelatin from specific amino acid 394 sequences such as Gly Pro-Gln-Gly Ile-Ala-Gly Gln [13]. The hydrolysis of the ester 395 bond in 4S-PEG is believed to play a significant role in the degradation process of 396 adhesive hydrogels. However, in this case, no decrease in mass was observed after 24 397 hours when swelling ratio was evaluated (Figure 3c), suggesting that the gelatin was 398 cleaved by enzymes. 399  1  2  3  4  5  6  7  8  9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 30  3.7. Cytocompatibility 400 The cytocompatibility of each Org or C8-ApGltn adhesive was evaluated according to 401 ISO10993-5 and 10993-12 using an extraction medium. In this experiment, we used 402 adhesive hydrogels from C8-ApGltns with a low DS because of their high burst strength 403 (Figure 5a). Extraction media from all adhesive hydrogels showed over 80% cell viability 404 compared to that of the control group (Figure 7a), indicating that these adhesives had 405 good cytocompatibility. We also observed the cell morphology after staining with DAPI 406 and FITC-phalloidin (Figure 7b). There were no significant differences between the Org 407 and C8-ApGltns adhesive hydrogels and the control groups. These results suggested that 408 straight/branched C8-ApGltns did not affect cell viability or morphology. Therefore, C8-409 ApGltns adhesives with a low DS do not hinder tissue regeneration when applied to 410 wounds. 411 Figure 7. Cytocompatibility test. a) Viability of L929 cultured in extracted medium (ns = 412 not significant using one-way ANOVA). b) Immunofluorescence images. 413  1  2  3  4  5  6  7  8  9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 31  3.8. Subcutaneous implantation 414 The in vivo biodegradation behavior of Org and C8-ApGltns adhesives was evaluated 415 using subcutaneous implantation of disk-shaped adhesive hydrogels on the backs of rats 416 for up to 56 days (Figure 8). After implantation for 7 days, the Org-ApGltn adhesive 417 hydrogel swelled in the subcutaneous tissue compared to the other adhesive hydrogels, 418 similar to the in vitro results (Figure 3d). All adhesive hydrogels became smaller and 419 thinner after implantation for 14 days, indicating that the degradation process had 420 proceeded. Finally, all the adhesive hydrogels almost completely disappeared within 56 421 days. HE staining revealed that the surrounding tissue at the implant site infiltrated the 422 adhesive hydrogel as the duration of the implantation period increased. This suggested 423 that the adhesive hydrogels functioned as scaffolds to facilitate tissue regeneration. 424 Gelatin is a denatured collagen with a cell adhesion peptide sequence of Arg-Gly-Asp 425 [30]. Cells in the surrounding tissues recognized the cell adhesion sequence to infiltrate 426 the adhesive hydrogels. MMPs are expressed in endothelial cells, fibroblasts, neutrophils, 427 and macrophages during wound healing [31-33]. Therefore, specific amino acid 428 sequences in C8-ApGltns adhesive hydrogels are cleaved and enzymatically degraded by 429 MMPs, such as MMP-2 or -9 secreted from these cells [34]. In addition, 4S-PEG 430 decomposes through ester hydrolysis and ether oxidative decomposition [17, 35, 36]. We 431  1  2  3  4  5  6  7  8  9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 32  previously reported that the degradation speed of hydrophobically modified ApGltn 432 adhesive hydrogels in rat subcutaneous tissue was relatively slow compared to that of the 433 Org-ApGltn adhesive hydrogel owing to its additional physical crosslinking by 434 hydrophobic groups [13, 17, 37]. However, we did not observe such differences in this 435 study because of the short alkyl chain (C8) and low DS (~10 mol%). Additionally, we did 436 not observe any foreign body reaction, including the deposition of collagen-rich areas, 437 indicating that the C8-ApGltns adhesive hydrogels are biodegradable and have excellent 438 biocompatibility. 439   440  1  2  3  4  5  6  7  8  9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 33  Figure 8. a) Biodegradability of C8-ApGltn adhesive hydrogels after implantation in rat 441 subcutaneous tissue for various periods. b) Tissues stained by hematoxylin and eosin (HE) 442 extracted for each period. A and T in HE-staining images represent Adhesive and Tissue. 443   444  1  2  3  4  5  6  7  8  9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 34  4. Conclusion 445 The adhesion properties and biocompatibility of the tissue adhesives composed of 446 straight/branched C8-ApGltns and 4S-PEG were evaluated. Branched alkyl groups 447 (asyC8/syC8) contributed to a higher burst strength than straight alkyl groups (stC8). The 448 high burst strength of asyC8-ApGltn adhesive came from interfacial strength, achieved 449 by having adequate hydrophobicity to interact with tissues and mobility of the alkyl 450 groups from their self-aggregation property. In contrast, the syC8-ApGltn adhesive 451 hydrogel had the highest bulk strength, as revealed by rheological measurements. 452 Additionally, the stC8/asyC8/syC8-ApGltns adhesive exhibited excellent 453 cytocompatibility and completely degraded subcutaneously within 56 days without 454 severe inflammation. The structural comparison of alkyl groups in this study will provide 455 new insight into the role of hydrophobic modification on tissue adhesion. 456  457   458  1  2  3  4  5  6  7  8  9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 35  Data Availability 459 The authors declare that all data supporting the findings of this study are available in the 460 paper and the associated Supporting Information. 461 Conflicts of Interest 462 The authors have no competing interests to declare. 463 Acknowledgements 464 This work was financially supported in part by the Japan Society for the Promotion of 465 Science (JSPS KAKENHI) Grant-in-Aid for JSPS Fellows (grant nos. 24KJ0500 and 466 22KJ0418) and JSPS KAKENHI (grant nos. 23K25216, 23K26411, 24K21677, and 467 24K22399). 468 Appendix a. 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B Biointerfaces. 146 (2016) 581 212–220. https://doi.org/10.1016/j.colsurfb.2016.06.017. 582  583   584  1  2  3  4  5  6  7  8  9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 https://doi.org/10.1002/adma.202405805https://doi.org/10.1002/anie.201602610https://doi.org/10.1021/la500332shttps://doi.org/10.1016/j.jcma.2017.11.002https://doi.org/10.1021/acs.chemrev.7b00522https://doi.org/10.1016/j.ejmech.2020.112260https://doi.org/10.1007/978-3-319-28140-7_31https://doi.org/10.1007/s00726-010-0689-xhttps://doi.org/10.1016/S0021-9258(17)30519-7https://doi.org/10.1002/jbm.a.37339https://doi.org/10.1002/jbm.a.35096https://doi.org/10.1016/j.colsurfb.2016.06.01739  The effect of branched structures of alkyl 585 groups on tissue adhesiveness and 586 biocompatibility of alkyl groups-modified 587 Alaska pollock gelatin-based adhesives 588  589 Satsuki Minamisakamoto1,2, Hiyori Komatsu1,2, Shiharu Watanabe2, Shima Ito1,2, 590 Hatsune Nishino2,3, Tetsushi Taguchi1,2* 591 1Graduate School of Science and Technology, Degree Programs in Pure and Applied 592 Sciences, University of Tsukuba, 1-1-1 Tennodai, Tsukuba, Ibaraki 305-8577, Japan 593 2Biomaterials field, Research Center for Macromolecules and Biomaterials, National 594 Institute for Materials Science, 1-1 Namiki, Tsukuba, Ibaraki 305-0044, Japan 595 3College of Engineering Science, School of Science and Engineering, University of 596 Tsukuba, 1-1-1 Tennodai, Tsukuba, Ibaraki 305-8577, Japan 597  598 Correspondence and requests for materials should be addressed to T. Taguchi. (email: 599 TAGUCHI.Tetsushi@nims.go.jp) 600  601  602  1  2  3  4  5  6  7  8  9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 40  Figure S1. Appearance of Org/stC8/asyC8-ApGltn solution (15% (w/v)). DS of stC8 or 603 asyC8-ApGltn were 45.3(H) and 44.5(H) mol%, respectively. 604   605  1  2  3  4  5  6  7  8  9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 41   606 Figure S2. Appearance of Org/stC8/asyC8-ApGltn adhesive hydrogels before and after 607 immersed in D-PBS. DS of stC8/asyC8-ApGltns were 9.1(L) and 9.4(L) mol%, 608 respectively. 609   610  1  2  3  4  5  6  7  8  9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 42   611 Figure S3. Tensile strength of cured Org/stC8(L)/asyC8(L)syC8(L)-ApGltn adhesives. 612 a) Dumbbell-shape adhesive hydrogels for tensile strength measurement. b) Maximum 613 stress. c) Maximum strain. (*p < 0.05, **p < 0.01, ***p < 0.001, ns = not significant 614 using one-way ANOVA)  615  1  2  3  4  5  6  7  8  9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 43   616 Figure S4. Measurement of burst strength using ASTM 2392-04. 617  618   619  1  2  3  4  5  6  7  8  9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 44   620 Figure S5. Enzymatic degradability of Org/stC8/asyC8/syC8-ApGltn adhesive hydrogels 621 in a collagenase solution. (*p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, ns = no 622 significant using two-way ANOVA) 623  624 0 1 5 9 24050100150Time (h)Weight remaining (%)OrgstC8 (L)asyC8 (L)syC8 (L)nsns✱✱✱✱✱✱✱✱ ✱✱✱✱✱✱✱✱ 1  2  3  4  5  6  7  8  9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 Declaration of interests   ☒ The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.   ☐ The authors declare the following financial interests/personal relationships which may be considered as potential competing interests:          Declaration of Interest Statement