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## Creator

Takumi Ono, Taku Suzuki, Narihito Nagoshi, Yohei Masugi, Kosuke Maeda, Shogo Hashimoto, Shiharu Watanabe, Takuji Iwamoto, [Tetsushi Taguchi](https://orcid.org/0000-0003-2541-2530), Masaya Nakamura

## Rights

This is a pre-copyedited, author-produced version of an article accepted for publication in Spine. The published version of record Spine 49(13):p E200-E207, July 1, 2024. is available online at: https://doi.org/10.1097/BRS.0000000000004985.[In Copyright](http://rightsstatements.org/vocab/InC/1.0/)

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[Alaska Pollock-derived Gelatin Sealant has Higher Sealing Strength than, and Comparable Biocompatibility with, Fibrin Sealant in Porcine and Rat Dural Injury Models](https://mdr.nims.go.jp/datasets/10b2f041-7ebd-470d-afa3-800a370f9c5f)

## Fulltext

Spine (Phila Pa 1976) Alaska pollock-derived gelatin sealant has higher sealing strength than, andcomparable biocompatibility with, fibrin sealant in porcine and rat dural injury models--Manuscript Draft-- Manuscript Number: SPINE 167175R1Full Title: Alaska pollock-derived gelatin sealant has higher sealing strength than, andcomparable biocompatibility with, fibrin sealant in porcine and rat dural injury modelsArticle Type: Basic ScienceKeywords: Alaska pollock-derived gelatin;  Alaska pollock-derived gelatin sealant;  burst strength;cerebrospinal fluid;  cerebrospinal fluid leakage;  dura;  dural injury;  fibrin;  fibrinsealant;  sealant;  sealing strengthCorresponding Author: Taku Suzuki, MD, PhDKeio University School of MedicineShinjuku-ku, Tokyo JAPANCorresponding Author SecondaryInformation:Corresponding Author's Institution: Keio University School of MedicineCorresponding Author's SecondaryInstitution:First Author: Takumi Ono, MDFirst Author Secondary Information:Order of Authors: Takumi Ono, MDTaku Suzuki, MD, PhDNarihito Nagoshi, MDYohei Masugi, MD, PhDKosuke Maeda, MDShogo Hashimoto, MD, PhDShiharu WatanabeTakuji Iwamoto, MD, PhDTaguchi Tetsushi, PhDMasaya Nakamura, MD, PhDOrder of Authors Secondary Information:Additional Information:Question ResponsePlease provide the Word Count of yourmanuscript text. 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The specific changes in the manuscript are highlighted in red. Reviewers' comments: Reviewer #1: This is an animal experimental study using porcine (in vitro) and rat models to evaluate a novel Alaska pollock-derived gelatin sealant for repair of dural injuries. This reviewer has the following comments: 1. The study is generally well conducted and provides new information even if it could be regarded to be "preliminary" findings due to the limited study groups. Thank you for this comment.  2. Abstract: The text here is e.g. "Study Design. Burst strength study in porcine models and functional and histological study in rat models." This sentence must be clarified and include the word "dura", otherwise the reader does not know what the "burst strength" refers to. Thank you for your comment. Based on your advice, the sentence has been revised to “Burst strength study in porcine dural models and functional and histological study in rat dural models.”  3. Abstract: The text here also has this sentence "Summary of Background Data. Disruption of the dura mater occurs during neurosurgery, leading to cerebrospinal fluid leakage." It is NOT ONLY in neurosurgery that disruption of the dura can occur, it of course can happen also in orthopaedic surgery in the spine. This reviewer suggests that "neurosurgery" is changed to "spine surgery". Thank you for your comment. Following your advice, "neurosurgery" has been replaced with "spine surgery".  4. This reviewer suggests that the methods section in the manuscript should specify animal research ethics approval (ID number etc), not only by writing "I agree" in the introductory section before the actual manuscript. Author's Response to Reviewer's (blinded)Thank you for your comment. Following your advice, animal research ethics approval, including the approval number, has been included in lines 27-28 on page 2.  Reviewer #2: Summary: The authors tested a new sealant, Alaska pollock-derived gelatin (ApGltn), for dura mater tears to prevent CSF leakage. Maximum burst strengths of ApGltn was found to be 4.4 x stronger than fibrin using the in vitro porcine dura mater system. In the in vivo dura mater tear rat model, they tested ApGltn, fibrin and no sealant control and found. Histological findings showed that the fibrin sealant was bio absorbed earlier than the ApGltn sealant and that there were no differences in biocompatibility based on the histological scoring system.  General comments: 1. Please include if CSF leakage in the rats was detected after injury and sealant. Thank you for your comment. Both the ApGltn sealant and fibrin sealant groups showed the absence of CSF leakage in all rats at 2, 4, and 8 weeks postoperatively. This information has been documented in lines 143-145 on Page 7.  2. Please include statistical analysis between histological scores between the 3 groups and between the 3 time points. Thank you for your comment. As you indicated, an analysis of histological scores was performed. Two-way repeated analysis of variance and Tukey’s post hoc comparisons were used to evaluate intergroup differences. Kruskal–Wallis tests were utilized to evaluate intragroup differences across three evaluation periods. As a result, ApGltn showed significantly more vascularization than the control group. There was significant less dural adhesion in the ApGltn than in the fibrin group. No significant differences were observed at the three evaluation time points in each group, except for desmoplasia in the fibrin group between 2 and 8 weeks (p = 0.01). These results may be explained by previous studies about ApGltn sealant. ApGltn sealant promotes cell migration and acts as a scaffold for tissue migration (Mizuno Y. Macromol Biosci. 2019). ApGltn sealant acts as an anti-adhesion barrier on the target surface to prevent adhesion (Mizuta R. Acta Biomater 2021). This has been documented in Statistical analysis (lines 113-116 on page 6), Results (lines 158-164 on page 8), and Discussion (lines 213-218 on page 10).   3. The authors suggest that longer resorption is better for dural mater tears. Please include data indicating how long it takes dura mater tears to heal to prevent CSF leakage. If it is shorter than 2 weeks, than a longer resorption time may not be beneficial. Thank you for your comment. Previous studies have demonstrated that the duration of dural repair in rats is considered to be 3 to 4 weeks or longer. This information has been described in lines 221-222 on page 10 along with references.  4. In the discussion, the authors mentioned ApGltn uses similar crosslinkers to those used in DuraSeal. How similar or different are ApGltn and DuraSeal? Thank you for your comment. The crosslinker (4S-PEG) used is identical to that of DuraSeal and ApGltn. The only distinction between ApGltn sealant and DuraSeal lies in the adhesive components (ApGltn: Dodecyl-group modified ApGltn, DuraSeal: trilysine). This information has been detailed in lines 235-237 on page 11. Alaska pollock-derived gelatin sealant has higher sealing strength than, and comparable biocompatibility with, fibrin sealant in porcine and rat dural injury models  Takumi Ono, MD1*; Taku Suzuki, MD, PhD1*; Narihito Nagoshi, MD, PhD1; Yohei Masugi, MD, PhD2; Kosuke Maeda, MD1, Shogo Hashimoto, MD, PhD1, Shiharu Watanabe3; Takuji Iwamoto, MD, PhD1; Tetsushi Taguchi, PhD3; Masaya Nakamura, MD, PhD1  1. Department of Orthopedic Surgery, Keio University School of Medicine 35 Shinano-machi, Shinjuku, Tokyo 160-8582, Japan  2. Division of Diagnostic Pathology, Keio University School of Medicine 35 Shinano-machi, Shinjuku, Tokyo 160-8582, Japan  3. Biomaterials Field, Research Center for Macromolecules and Biomaterials, National Institute for Materials Science 1-1 Namiki, Tsukuba, Ibaraki 305-0044, Japan  * Takumi Ono and Taku Suzuki contributed equally to this work.  Corresponding authors: Taku Suzuki, MD, PhD Department of Orthopedic Surgery, Keio University School of Medicine  35 Shinanomachi, Shinjuku-ku, Tokyo 160-8582, Japan Email: sutaku49@gmail.com  TEL: +81-3-5363-3812 FAX: +81-3-3353-6597    Title Pagemailto:sutaku49@gmail.comTetsushi Taguchi, PhD Biomaterials Field, Research Center for Macromolecules and Biomaterials, National Institute for Materials Science 1-1 Namiki, Tsukuba, Ibaraki 305-0044, Japan  Email: TAGUCHI.Tetsushi@nims.go.jp TEL: +81-29-860-4498 FAX: +81-29-860-4752  Conflict of Interest Source of Funding: This study was partially supported by the Japan Society for the Promotion of Science (JSPS) KAKENHI (grant no. 22K09364).  Acknowledgments: We are grateful to Prof. M. Matsumoto, Dr. N. Matsumura, and T. Kitagawa in Department of Orthopaedic Surgery, Keio University School of Medicine, and A. Nishiguchi in Polymers and Biomaterials Field, Research Center for Functional Materials, National Institute for Materials Science for their helpful support.   Permission documentation: Any of the illustrations or tables used in this article has not been published previously.  IRB approval: This study was approved by the Institutional Animal Care and Use Committee of our hospital in accordance with institutional guidelines.  ABSTRACT Study Design. Burst strength study in porcine dural models and functional and histological study in rat dural models. Objective. This study aimed to investigate the sealing strength and biocompatibility of Alaska pollock-derived gelatin (ApGltn) and fibrin sealants in disrupted dural injuries. Summary of Background Data. Disruption of the dura mater occurs during spine surgery, leading to cerebrospinal fluid leakage. Fibrin sealant is usually applied to ruptured sites; however, it lacks sealing strength. A novel biocompatible sealant composed of ApGltn was recently demonstrated to have good burst strength and biocompatibility in the porcine aorta. Methods: Ten porcine dura maters with central holes were covered with ApGltn and fibrin sealants (five samples per group). The maximum burst strength of each sealant was measured, and histological examination was performed after burst testing. Twenty-seven dura maters of male Wistar rats were used for functional and histopathological evaluations. The rats were treated with three surgical interventions: defect + ApGltn sealant; defect + fibrin sealant; defect alone (nine rats per group). Macroscopic confirmation of the sealant, hindlimb motor function analysis, and histopathological examination were performed at 2, 4, and 8 weeks after the procedure. Results: The maximum burst strength of the ApGltn sealant was approximately 4.4 times higher than that of the fibrin sealant (68.1 ± 12.1 vs. 15.6 ± 8.7 mmHg; p < 0.001). Histological examination confirmed that the ApGltn sealant showed tight adhesion to the dural surface, whereas a gap was observed between the fibrin sealant and the dura mater. In the rat model, the ApGltn sealant resulted in spinal function and dural histological findings similar to those of the fibrin sealant. Structured Abstract (300 words)  Biocompatible sealant for dural repair    Conclusions: The ApGltn sealant had a higher sealing strength than, and comparable effect on dura regeneration with, the fibrin sealant. Key words: Alaska pollock-derived gelatin; Alaska pollock-derived gelatin sealant; burst strength; cerebrospinal fluid; cerebrospinal fluid leakage; dura; dural injury; fibrin; fibrin sealant; sealant; sealing strength Key points:  ・The present study investigated the sealing strength and biocompatibility of this sealant in ruptured dura mater in porcine and rat models. ・The burst strength of the ApGltn sealant was 4.4 times higher than that of the fibrin sealant. Histological examination confirmed that the ApGltn sealant adhered tightly to the dural surface compared with the fibrin sealant.  ・Compared with the fibrin sealant, the ApGltn sealant did not prevent spinal function or dura mater regeneration, suggesting the biocompatibility of the ApGltn sealant. ・ApGltn is effective in preventing cerebrospinal fluid leakage and seal disruption of the dura mater. Key Points (3-5 main points of the article)Mini Abstracts This study explores the sealing strength and biocompatibility of Alaska pollock-derived gelatin (ApGltn) and fibrin sealants in porcine and rat dural injury models. ApGltn sealant exhibits 4.4 times greater sealing strength and comparable biocompatibility to fibrin sealant. ApGltn sealant is effective in sealing cerebrospinal fluid leakage of the dura mater. Mini Abstract (50 words)  Biocompatible sealant for dural repair  1  Alaska pollock-derived gelatin sealant has higher sealing strength than, and comparable 1 biocompatibility with, fibrin sealant in porcine and rat dural injury models 2  3 INTRODUCTION 4 Disruption of the dura mater and arachnoid tissue occurs during neurosurgery, which 5 leads to cerebrospinal fluid (CSF) leakage. The reported incidence of CSF leakage ranges from 6 0.8% to 32%.1-3 Primary suturing of the dura is the standard technique for repair, and sealing 7 materials are sometimes used to prevent CSF leakage, with or without suturing. The use of 8 commercially available sealants for dural closure have been reported in the literature.1,2,4 9 Among these, the fibrin sealant is commonly used because of its biocompatibility.1 Many 10 previous studies have used fibrin sealant for CSF leakage and showed the effectiveness of dura 11 mater sealing; however, a disadvantage of fibrin sealant is its lack of sealing strength.1,2,5 Other 12 adhesives such as cyanoacrylate and biopolymers with aldehyde-based crosslinkers are used 13 because of their high bonding strength.6-9 However, the use of these materials is limited owing 14 to their cytotoxicity and low biocompatibility.4, 9 15 Recently, a novel biocompatible sealant composed of Alaska pollock-derived gelatin 16 (ApGltn), partially modified with various alkyl groups and a poly(ethylene glycol)-based 17 crosslinker, was introduced.10,11 The burst strength and biocompatibility of wet tissue have been 18 demonstrated using a porcine aorta or lung model.10-12 The burst strength of the ApGltn sealant 19 was approximately 12 times higher than that of commercial fibrin sealant.10 20 The ApGltn sealant is a promising material, and its use in coating ruptured dura mater 21 during spinal surgery is expected in the future. However, whether this sealant can be applied to 22 ruptured dura mater, where prevention of spinal fluid leakage is needed, is unclear. The purpose 23 Manuscript Text (must include page numbers) 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   Biocompatible sealant for dural repair  2  of this study was to investigate the sealing strength and biocompatibility of this sealant in 24 ruptured dura mater in porcine and rat models. 25 MATERIALS AND METHODS 26 Animal experiments were approved by our institutional animal committee (approval no. 27 A2022-016). 28  29 Characteristics and preparation of sealants 30 ApGltn sealant 31 The preparation and characterization of dodecyl group–modified ApGltn, a component 32 of the ApGltn sealant, was performed according to previously reported procedures.10,11 The 33 resulting dodecyl group–modified ApGltn was combined with a biocompatible poly(ethylene 34 glycol)-based four-arm crosslinker, pentaerythritol poly(ethylene glycol) ether 35 tetrasuccinimidyl glutarate (4S-PEG). The pH of the modified ApGltn–0.1 M borate buffer 36 solution was adjusted to 8, and a solvent of 4S-PEG was prepared with 0.01 M phosphate 37 solution (pH 4). The solutions were mixed in equal volumes using a dual-injection device. 38 ApGltn hardened within 60 s of injection. 39  40 Fibrin glue 41 The fibrin glue (Bolheal®; KM Biologics, Kumamoto, Japan) used in this study was 42 refrigerated (4°C) before use. Fibrinogen powder (40 mg) and coagulation factor XIII (37.5 IU) 43 were reconstituted in an aprotinin solution (500 KIE/0.5 mL). Thrombin concentrate powder 44  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   Biocompatible sealant for dural repair  3  (125 IU) was dissolved in a calcium chloride solution (2.95 g/0.5 mL). Fibrinogen and thrombin 45 solutions were cured by mixing each component in equal volumes. 46  47 Burst strength and histological evaluation using a porcine dural defect model 48 The primary outcome was the maximum burst strength of the ApGltn and fibrin sealants 49 using a fresh porcine dural defect, according to American Society of Testing and Materials 50 (ASTM) F2392-04. Ten samples were tested to determine the burst strength of each sealant (n 51 = 5 each) (Figure 1). Tissue samples (diameter, 30 mm) were prepared. After opening a 3-mm 52 pinhole in the center of each sample, the ApGltn and fibrin sealants (15-mm diameter and 1-53 mm thickness) were applied using a silicone ring (Figure 2). The maximum burst strength was 54 determined by running a saline solution through the system at a flow rate of 2 mL/min at 37C 55 and measuring the maximum value (Figure 3). Then, the samples were fixed with 10% formalin 56 neutral buffer solution (Wako Pure Chemical Industries, Ltd, Japan) and stained with 57 hematoxylin and eosin (HE). Cross sections of the stained samples (slice thickness, 4 mm) were 58 visualized using a BX51 light microscope (Olympus, Tokyo, Japan). The burst style with a gap 59 between the dura and the sealant was examined. 60  61 Functional and histological evaluation using a rat dural defect model 62  The secondary outcome was the functional and histological recovery of the repaired 63 dura mater in a rat model. Male Wistar rats (Sankyo Labo, Tokyo, Japan; age, 8 weeks, mean 64 body weight at the time of surgery, 198 g; range, 190–212 g) were used for the dural rupture 65 model. 66  67  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   Biocompatible sealant for dural repair  4  Surgical treatments of dura defect with the sealant 68 All surgical procedures and surgical treatment assignments were performed by a single 69 orthopedic surgeon. All rats were deeply anesthetized with intraperitoneal injections of 70 midazolam (0.39 mL/kg; Sandoz, Tokyo, Japan), medetomidine (0.37 mL/kg; Zenoaq, 71 Fukushima, Japan), butorphanol (0.49 mL/kg; Meiji Seika, Tokyo, Japan), and saline (3.74 72 mL/kg; Otsuka, Tokyo, Japan). A dorsal longitudinal skin incision was made, and the lamina 73 was explored by splitting the paravertebral muscle. Laminectomy was performed at the T10 74 level using a bone rongeur, and the dura was exposed. A 3-mm defect in the dura was made 75 using a 30-G needle to avoid damage to the spinal cord, and CSF leakage was observed. The 76 defects were treated randomly using three surgical interventions: defect + ApGltn sealant, 77 defect + fibrin sealant, defect without sealant (n = 9 rats per group). In the surgical interventions 78 involving defect + ApGltn sealant and defect + fibrin sealant, approximately 0.5 mL of the 79 ApGltn or fibrin sealant was placed around the dura rupture site. One suture was placed in the 80 surrounding muscle at the same level as the site of the dural injury. Then, the fascia and skin 81 were closed with interrupted sutures, and the rats were allowed unrestricted motion. No rats 82 were excluded due to death. 83 To evaluate the regeneration of the dura and spinal function, hind-limb motor function 84 analysis was conducted at 2, 4, and 8 weeks after the initial procedure. The rats were killed at 85 2 (n = 3 per group), 4 (n = 3 per group), and 8 weeks (n = 3 per group) after surgery for 86 macroscopic and histological examinations. 87  88 Macroscopic examination 89  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   Biocompatible sealant for dural repair  5  The macroscopic appearance of the tissue surrounding the dural injury site was evaluated 90 weekly. The resorption of the ApGltn and fibrin sealants was also examined. 91  92 Hind-limb motor function analysis 93 Hind-limb motor function was tested using the open-field Basso, Beattie, and Bresnahan 94 (BBB) locomotor scale.13 BBB scores were measured 2, 4, and 8 weeks after the initial 95 procedure, with 0 denoting total loss of hind-limb movement and 21 denoting normal function.  96  97 Histological examination  98 Three of the nine rats from each group killed at 2, 4, and 8 weeks (27 rats in total) for 99 histological examination. The spinal cord was resected along with the dura mater and fixed in 100 10% formalin buffer solution for at least 3 days. Tissue specimens were cut stepwise at 2-mm 101 intervals and embedded in paraffin. Paraffin-embedded tissues were cut into 4-mm-thick slices 102 and stained with HE. A pathologist, blinded to clinical information, selected one representative 103 section that contained the border region between the spinal cord and the dura mater for each rat 104 and performed a semiquantitative evaluation of pathological changes, including inflammation, 105 myelitis, neuronal damage, edema, desmoplasia, vascularization, necrosis, foreign body 106 reaction, and adhesion between the dura and the surrounding tissues. These findings were 107 classified into four categories according to a modified previous method (0, none; 1, mild; 2, 108 moderate; 3, severe) (Table 1) .9,14,15 The average score for each of the three rats was calculated.  109  110 Statistical analysis 111  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   Biocompatible sealant for dural repair  6  Student’s t test was used to evaluate the burst strength of the ApGltn and fibrin groups. 112 The sample size for burst testing was determined according to previous reports.10,11,16 An 113 analysis of histological scores involved the use of two-way repeated analysis of variance and 114 Tukey’s post hoc comparisons to assess intergroup differences. Kruskal–Wallis tests were 115 utilized to evaluate intragroup differences across three evaluation periods. A post-hoc power 116 analysis was conducted to confirm whether the sample size was adequate to detect a significant 117 difference ( = 0.05). The effect sizes are expressed as mean  standard deviations. The power 118 analysis demonstrated a statistical power of 100% for burst testing. Statistical significance was 119 set at p < 0.05.  120  121 RESULTS 122 Burst strength using a fresh porcine dural defect model 123 Burst strength 124 The maximum burst strengths of the ApGltn and fibrin sealants (68.1 ± 12.1 and 15.6 ± 125 8.7 mmHg, respectively) significantly differed (p < 0.001). Macroscopic examination of the 126 burst behavior showed that the ApGltn sealant ruptured and saline leaked from the ruptured site 127 in all five samples. In the fibrin group, the sealant detached from the dura surface, and saline 128 leaked from the interface between the fibrin sealant and the dura mater in all five samples. 129  130 Histological findings after the burst testing 131 Histological images of the tissues stained with HE after burst testing are shown in Figure 132 4. The ApGltn sealant ruptured and adhered tightly to both the defect and the surrounding dura 133 in all five samples (Figure 4, left). A gap was observed between the fibrin sealant and the 134  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   Biocompatible sealant for dural repair  7  surface of the dura, both in the defect and in the surrounding dura, in all five samples (Figure 135 4, right). These findings are consistent with the macroscopic examination of the burst behavior. 136  137 Functional evaluation using a rat dural defect model 138 Macroscopic examination 139 Scar tissue was observed around the dural injury site in all three groups at each 140 examination. ApGltn remained on the dura mater in three rats at 2 weeks and in two rats at 4 141 weeks and was resorbed in three rats at 8 weeks. The fibrin sealant remained on the dura mater 142 in one rat at 2 weeks and was resorbed in three rats at 4 and 8 weeks (Figure 5). Both the ApGltn 143 sealant and fibrin sealant groups demonstrated the absence of CSF leakage in all rats at 2, 4, 144 and 8 weeks postoperatively. 145  146 Hind-limb motor function analysis 147 All rats in the three procedures showed a BBB score of 21 points at 2, 4, and 8 weeks 148 after initial repair, indicating no loss of hind-limb motor function. 149  150 Histological examinations 151 The results of the semiquantitative analyses of the histological findings are summarized 152 in Table 2. Mild, moderate, and severe inflammatory cell infiltration and myelitis were 153 observed in all three groups at 2 weeks. At 4 and 8 weeks, the inflammation gradually became 154 mild or disappeared in the ApGltn group, whereas it only lightly decreased or was unchanged 155 in the control and fibrin groups. Mild or no neuronal damage or edema of the spinal cord was 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   Biocompatible sealant for dural repair  8  observed in all three groups every week. All three groups had mild to severe desmoplasia and 157 vascularization at 2 weeks, but these findings were moderate or absent at 8 weeks. ApGltn 158 showed significantly more vascularization than the control group (p = 0.03). No soft tissue 159 necrosis was observed in the three groups. Dural adhesion between the dura and connective 160 tissues was mild or absent in the ApGltn group and mild or moderate in the fibrin group at 2, 4, 161 and 8 weeks with significant differences (p = 0.02). No significant differences were found at 162 the three evaluation time points in each group, except desmoplasia in fibrin group between 2 163 and 8 weeks (p = 0.01). Representative histological images of HE staining at 2, 4, and 8 weeks 164 are shown in Figure 6. 165  166 DISCUSSION 167 In this study, the sealing strength and histological recovery of dural defects were 168 compared between ApGltn and fibrin sealants. The burst strength of the ApGltn sealant was 169 approximately 4.4 times higher than that of the fibrin sealant. Histological examination 170 confirmed that the ApGltn sealant adhered tightly to the dural surface compared with the fibrin 171 sealant. These results indicated that ApGltn sealant showed cohesion failure when applied to 172 the dura surface, which means that the interfacial strength between the cured ApGltn sealant 173 and the dura tissue was higher than that of the fibrin sealant. Compared with the fibrin sealant, 174 the ApGltn sealant did not prevent spinal function or dura mater regeneration, suggesting the 175 biocompatibility of the ApGltn sealant. 176 Taguchi et al. developed the first hydrophobically modified ApGltn-based biocompatible 177 sealant and reported sufficient burst strength for its clinical application in burst porcine aortas 178 and in an air-leak model of a rat lung.10 The burst strength of the ApGltn sealant was 11.6 times 179  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   Biocompatible sealant for dural repair  9  higher than that of a commercial fibrin sealant (341 vs. 29 mmHg). Yamaoka et al. validated 180 the burst strength of ApGltn and fibrin sealants using an air-leak porcine lung.12 The burst 181 pressure of ApGltn sealant was significantly higher than that of fibrin sealant (52 vs. 38 cm 182 H2O). Our results are consistent with those of previous studies in that the breaking strength of 183 the ApGltn sealant was higher than that of the fibrin sealant. Doormaal et al. evaluated burst 184 strength using a fresh porcine dura model to compare the sealing effect of nine different sealants 185 for dural closure using the same methods as our study (ASTM F2392-04; 30 mm in diameter 186 with a 3-mm hole of the dura and sealant with a diameter of 15 mm and thickness of 1 mm).2 187 Adherus® (polyethylene glycol-based hydrogel) had the highest burst pressure (87 ± 47 mmHg), 188 followed by Tachosil® (hemostatic collagen; 71 ± 16 mmHg); Tisseel® (fibrin sealant) showed 189 a significantly lower burst pressure (12 ± 9 mmHg) than these two sealants. Although the type 190 of fibrin sealant used in our study was different, its breaking strength of the fibrin sealant was 191 low when applied to porcine dural defects. Normal adult CSF pressure is approximately 5–15 192 mmHg.17,18 The use of a sealant with greater breaking strength than intracranial pressure is 193 desirable to prevent CSF leakage. 194 Histologically, the ApGltn sealant tightly adhered to the dural surface, and the bond was 195 ruptured only by the destruction of the bulk ApGltn sealant. In contrast, a gap was observed 196 between the fibrin sealant and the surface of the dura, suggesting weak interfacial strength 197 between the sealant and the dura. This burst style is consistent with previous histological studies 198 that showed tight adhesion of the ApGltn sealant in porcine aorta or lung burst model.11,12 These 199 results were due to increased burst strength and hydrophobic interactions between the dodecyl 200 group of the ApGltn and extracellular matrix proteins.11 Under wet conditions, tight adhesion 201 of the ApGltn sealant contributes to the prevention of CSF leakage. 202  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   Biocompatible sealant for dural repair  10  Previous studies that used fibrin sealants in animal models have demonstrated that fibrin 203 sealants do not prevent the regeneration of the dura and lead to axonal damage.1,14 In the present 204 study, the ApGltn sealant demonstrated the same functional and histological findings of the 205 dura mater as those of the fibrin sealant. Previous studies have demonstrated that severe 206 inflammatory reactions are not observed at the bonding site of ApGltn,11,16,19 which is consistent 207 with the results of our study. This may be because the hexanoyl group induces weak 208 inflammation in the tissue, resulting in the secretion of inflammatory cytokines or growth 209 factors.20 Masuda et al. demonstrated that rat sciatic nerves repaired with ApGltn sealant 210 showed similar recovery of axons as those repaired with sutures and fibrin sealant.21 Our data 211 suggest that the ApGltn sealant can be applied not only to the peripheral nerve but also to the 212 central nerve field. Mizuno et al. showed that ApGltn sealant promotes cell migration and acts 213 as a scaffold for tissue migration, which might lead to vascularization around the dura.22 214 Because it is composed of gelatin, this sealant does not prevent tissue regeneration. Furthermore, 215 Mizuta et al. showed that ApGltn sealant acts as an anti-adhesion barrier on the target surface 216 to prevent adhesion.23 This may have contributed to the prevention of dural adhesions in the 217 current study. 218 In addition to its burst strength, the ApGltn sealant has other advantages over fibrin glue. 219 The fibrin sealant was resorbed in approximately 2 weeks, whereas the ApGltn sealant was 220 resorbed in approximately 4–8 weeks, suggesting a longer-lasting adhesive capacity.10,12,21 The 221 duration of dural repair in rats is considered to be 3 to 4 weeks or longer.24,25 Sealants with 222 long-lasting adhesive capacity are desirable for repairing the dura mater to prevent CSF leakage. 223 The fibrin sealant is composed of fibrinogen (lyophilized pooled human concentrate) and 224 thrombin; therefore, viral infections may occur. The ApGltn sealant is composed of 225  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   Biocompatible sealant for dural repair  11  biocompatible ApGltn purified from Alaska pollock skin by alkali treatment; thus, the risk of 226 viral infection may be reduced. Furthermore, ApGltn is derived from the waste products of 227 Alaska pollock skin; therefore, the cost of these materials is quite low. In clinical practice, the 228 cost of ApGltn sealants is lower than that of fibrin sealants. Based on these observations and 229 the results of this study, we believe that ApGltn could be used as a substitute for fibrin glue in 230 the future. 231 ApGltn is prepared from Alaska pollock skin via demineralization and alkaline treatment; 232 therefore, it is used in the same manner as previously approved bovine- and porcine-derived 233 gelatin. The chemical structure and molecular weight of the crosslinker (4S-PEG) are the same 234 as those of FDA/Japanese FDA-approved sealant (DuraSeal®) used in brain surgery. The 235 difference between ApGltn sealant and DuraSeal lies in the adhesive components (ApGltn: 236 Dodecyl-group modified ApGltn, DuraSeal: trilysine). We believe that ApGltn sealant could 237 overcome the regulatory challenges of its use in humans. Clinical trials on humans will be 238 needed in the future.  239 The ApGltn sealant has greater sealing strength than fibrin glue. The functional and 240 histological findings in the spinal cord and dura mater are similar to those of the fibrin glue. To 241 prevent CSF leakage, ApGltn is a promising material for disrupting the dura mater in clinical 242 settings. 243  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 REFERENCES 1. Esposito F, Angileri FF, Kruse P, et al. Fibrin Sealants in Dura Sealing: A Systematic Literature Review. PLoS One. 2016;11:e0151533. 2. van Doormaal T, Kinaci A, van Thoor S, et al. Usefulness of Sealants for Dural Closure: Evaluation in an In Vitro Model. Oper Neurosurg (Hagerstown). 2018;15:425-32. 3. Kizmazoglu C, Ozyoruk S, Husemoglu RB, et al. Comparison of dural closure alternatives: an experimental study. Br J Neurosurg. 2019;33:655-8. 4. Epstein NE. Dural repair with four spinal sealants: focused review of the manufacturers’ inserts and the current literature. Spine J. 2010;10:1065-8. 5. Chauvet D, Tran V, Mutlu G, George B, et al. Study of dural suture watertightness: an in vitro comparison of different sealants. Acta Neurochir (Wien). 2011;153:2465-72. 6. Cohen-Gadol AA, Bellew MP, Akard W, et al. The application of n-butyl 2-cyanoacrylate to repair CSF fistulas for 221 patients who underwent transsphenoidal surgery. Minim Invasive Neurosurg. 2010;53:207-9. 7. Asan Z, Kilitci A. Use of cyanoacrylate to prevent cerebrospinal fluid fistulas after cranial surgery. Br J Neurosurg. 2018;32:544-7. 8. Kawai H, Nakagawa I, Nishimura F, et al. Usefulness of a new gelatin glue sealant system for dural closure in a rat durotomy model. Neurol Med Chir (Tokyo). 2014;54:640-6. 9. Kalsi P, Thom M, Choi D. Histological effects of fibrin glue and synthetic tissue glues on the spinal cord: are they safe to use? Br J Neurosurg. 2017;31:695-700. 10. Taguchi T, Ryo M, Ito T, et al. Robust Sealing of Blood Vessels with Cholesteryl Group-Modified, Alaska Pollock-Derived Gelatin-Based Biodegradable Sealant Under Wet Conditions. J Biomed Nanotechnol. 2016;12:128-34. References (cited in order of appearance)  Biocompatible sealant for dural repair    11. Mizuno Y, Mizuta R, Hashizume M, et al. Enhanced sealing strength of a hydrophobically-modified Alaska pollock gelatin-based sealant. Biomater Sci. 2017;5:982-9. 12. Yamaoka M, Maki N, Wijesinghe A, et al. Novel Alaska Pollock Gelatin Sealant Shows High Adhesive Quality and Conformability. Ann Thorac Surg. 2019;107:1656-62. 13. Basso DM, Beattie MS, Bresnahan JC. A sensitive and reliable locomotor rating scale for open field testing in rats. J Neurotrauma. 1995;12:1-21. 14. de Vries J, Menovsky T, van Gulik S, et al. Histological effects of fibrin glue on nervous tissue: a safety study in rats. Surg Neurol. 2002;57:415-22. 15. Bakar B, Kose EA, Balci M, et al. Evaluation of the neurotoxicity of the polyethylene glycol hydrogel dural sealant. Turk Neurosurg. 2013;23:16-24. 16. Ichimaru H, Mizuno Y, Chen X, et al. Prevention of pulmonary air leaks using a biodegradable tissue-adhesive fiber sheet based on Alaska pollock gelatin modified with decanyl groups. Biomater Sci. 2021;9:861-73. 17. Ren R, Zhang X, Wang N, et al. Cerebrospinal fluid pressure in ocular hypertension. Acta Ophthalmol. 2011;89:e142-8. 18. Fleischman D, Berdahl JP, Zaydlarova J, et al. Cerebrospinal fluid pressure decreases with older age. PLoS One. 2012;7:e52664. 19. Mizuta R, Ito T, Taguchi T. Effect of alkyl chain length on the interfacial strength of surgical sealants composed of hydrophobically-modified Alaska-pollock-derived gelatins and poly(ethylene)glycol-based four-armed crosslinker. Colloids Surf B Biointerfaces. 2016;146:212-20. 20. Yoshizawa K, Mizuta R, Taguchi T. Enhanced angiogenesis of growth factor-free porous biodegradable adhesive made with hexanoyl group-modified gelatin. Biomaterials. 2015;63:14-23.   Biocompatible sealant for dural repair    21. Masuda S, Suzuki T, Shibata S, et al. A Novel Alaska Pollock Gelatin Sealant Shows Higher Bonding Strength and Nerve Regeneration Comparable to That of Fibrin Sealant in a Cadaveric Model and a Rat Model. Plast Reconstr Surg. 2021;148:742e-52e. 22. Mizuno Y, Taguchi T. Promotion of Cell Migration into a Hydrophobically modified Alaska Pollock Gelatin-Based Hydrogel. Macromol Biosci. 2019;19:e1900083. 23. Mizuta R, Mizuno Y, Chen X, et al. Evaluation of an octyl group-modified Alaska pollock gelatin-based surgical sealant for prevention of postoperative adhesion. Acta Biomater. 2021;121:328-38.   24. Nurata H, Cemil B, Kurt G, et al. The role of fibroblast growth factor-2 in healing the dura mater after inducing cerebrospinal fluid leakage in rats. J Clin Neurosci. 2009;16:542-4.   25. Kawai  H, Nakagawa I, Nishimura F, et al. Usefulness of a new gelatin glue sealant system for dural closure in a rat durotomy model. Neurol Med Chir (Tokyo). 2014;54:640-6. Table 1. Grading system used for quantifying histological findings   0 1 2 3 Inflammation No cell/few inflammatory cells Mild  inflammatory cells Moderate inflammatory cells Severe  inflammatory cells Myelitis None Few fibroblasts Moderate fibroblasts Severe fibroblasts Neuronal damage None Mild neuronal disintegration Moderate neuronal disintegration Severe neuronal disintegration Edema None Mild rarification of the intercellular tissue  Moderate rarification of the intercellular tissue Severe rarification of the intercellular tissue Desmoplasia None Thin layer Moderate thickness Thick layer Vascularization None Few new capillaries Moderate capillaries Dense capillaries Necrosis None Mild Moderate Severe Foreign body reaction None Mild giant cells Moderate giant cells Severe giant cells Dural adhesion None Mild Moderate Severe Table1Table 2. Semiquantitative analyses of the histological findings  2 Weeks (n = 3 per group) 4 Weeks (n = 3 per group) 8 Weeks (n = 3 per group) Control ApGltn Fibrin Control ApGltn Fibrin Control ApGltn Fibrin Inflammation 1.7  0 2.3  0.5 2.3  0.5 1.0  0.8 1.3  0.5 1.7  0.5 1.7  0.5 1.0  0 2.0  0 Myelitis 1.0  1.4 1.3  1.3 1.0  0 1.0  0.8 0.7  0.5 1.0  0 1.7  0.5 0.3  0.5 1.3  0.5 Neuronal damage 0.7  0.9 1.0  0.8 1.0  0 0.7  0.9 0.3  0.5 1.3  0.5 1.7  0.5 0  0 1.0  0 Edema 0  0 0.7  0.5 1.0  0 0.3  0.5 1.0  0 0.3  0.5 0.3  0.5 0.3  0.5 0.7  0.5 Desmoplasia 2.0  0.8 1.7  0.5 3.0  0 1.0  0.8 2.0  0 2.0  0 1.3  0.5 1.0  0 1.0  0 Vascularization 1.7  0.5 1.7  0.5 2.0  0 0.3  0.5 2.0  0.8 1.3  0.5 0.3  0.5 1.7  0.5 1.0  0.8 Necrosis 0  0 0  0 0  0 0  0 0  0 0  0 0  0 0  0 0  0 Foreign body reaction 0.3  0.5 1.3  0.9 0.7  0.5 1.0  1.4 0.3  0.5 0.3  0.5 1.0  0.8 2.0  0.8 1.7  0.5 Dural adhesion 0.7  0.9 0.7  0.5 1.3  0.5 0.7  0.5 0.3  0.5 1.7  0.5 2.3  1.2 1.0  0 2.0  0 Values are presented as mean  standard deviation. Table 2FIGURE LEGENDS Figure 1. Burst strength testing and functional testing using the disruption of the dura mater in porcine and rat models. ApGltn, Alaska pollock-derived gelatin.  Figure 2. Fresh porcine dura was used in this study. The sealants were applied with a diameter of 15 mm and thickness of 1 mm. (left) ApGltn sealant. (right) Fibrin sealant.  Figure 3. (left) Measurement equipment of the burst strength of sealants according to American Society of Testing and Materials F2392-04. (right) System for evaluating burst strength.   Figure 4. Histological images of the tissue after burst testing. (left) ApGltn; The ApGltn adhered tightly to the dura (arrowhead). (right) Fibrin: A gap was observed between the fibrin and the dura (arrowhead).  Figure 5. Macroscopic examination of the dura after each procedure at 2 weeks. The ApGltn (arrowhead) remained on the dura mater in all three rats, and the fibrin sealant (arrowhead) remained in one of three rats.   Figure 6. Histological images of the rat dura mater (arrowhead) and spinal cord after burst testing at 2, 4, and 8 weeks. ApGltn, Alaska pollock-derived gelatin; SC, spinal cord.  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