During wound regeneration, both cell adhesion and adhesion-inhibitory functions must be controlled in parallel. We developed a membrane with dual surfaces by merging the properties of carboxymethyl cellulose (CMC) and collagen using vitrification.
Carbohydrate Polymers 285 (2022) 119223 Contents lists available at ScienceDirect Carbohydrate Polymers journal homepage: www.elsevier.com/locate/carbpol Bi-layered carboxymethyl cellulose-collagen vitrigel dual-surface adhesion-prevention membrane Yue Wang a, Kei Kanie a, Toshiaki Takezawa b, Miki Horikawa a, b, Kyoshiro Kaneko a, Ayako Sugimoto a, Aika Yamawaki-Ogata c, Yuji Narita c, Ryuji Kato a, d, e, * a Department of Basic Medicinal Sciences, Graduate School of Pharmaceutical Sciences, Nagoya University, Tokai National Higher Education and Research System, Furocho, Chikusa-ku, Nagoya, Aichi 464-8601, Japan Division of Biomaterial Sciences, Institute of Agrobiological Sciences, National Agriculture and Food Research Organization, 1-2 Owashi, Tsukuba, Ibaraki 305-8634, Japan c Department of Cardiac Surgery, Nagoya University Graduate School of Medicine, Tokai National Higher Education and Research System, 65 Tsurumai-cho, Showa-ku, Nagoya, Aichi 466-8550, Japan d Institute of Nano-Life-Systems, Institutes of Innovation for Future Society, Nagoya University, Tokai National Higher Education and Research System, Furocho, Chikusaku, Nagoya, Aichi 464-8601, Japan e Institute of Glyco-core Research (IGCORE), Nagoya University, Tokai National Higher Education and Research System, Furocho, Chikusa-ku, Nagoya, Aichi 464-8601, Japan b A R T I C L E I N F O A B S T R A C T Keywords: Bi-layered carboxymethyl cellulose-collagen vitrigel membrane Cell-adhesion inhibition Wound regeneration Adhesion-inhibitor Collagen hydrogel Anti-adhesion membrane During wound regeneration, both cell adhesion and adhesion-inhibitory functions must be controlled in parallel We developed a membrane with dual surfaces by merging the properties of carboxymethyl cellulose (CMC) and collagen using vitrification A rigid membrane was formed by vitrification of a bi-layered CMC and collagen hydrogel without using cross-linking reagents, thus providing dual functions, strong cell adhesion-inhibition with the CMC layer, and cell adhesion with the collagen layer We referred to this bi-layered CMC-collagen vitrigel membrane as “Bi-C-CVM” and optimized the process and materials The introduction of the CMC layer conferred a “tough but stably wet” property to Bi-C-CVM This enables Bi-C-CVM to cover wet tissue and make the membrane non-detachable while preventing tissue adhesion on the other side The bi-layered vitrification pro cedure can expand the customizability of collagen vitrigel devices for wider medical applications Introduction Recent tissue-engineering research reported that cell-derived medi cal devices have higher therapeutic effectiveness than conventional products with simple materials (Imashiro & Shimizu, 2021) One advantage of cell-derived products is the multiple functions in one product that balances complex in vivo regeneration events, as in some cases, contrary cellular events must be controlled in parallel Wound regeneration is a complex regeneration process that requires delicate control of contrary cellular events to minimize side effects Although cell adhesion enhancement is essential for tissue repair, un wanted tissue adhesion may also occur Tissue adhesion is a critical lifethreatening side effect after various surgeries (Kheilnezhad & Hadjizadeh, 2021; Park et al., 2020) In abdominal surgeries, post operative peritoneal adhesion increases the risk of chronic pain and intestinal obstruction (Hu et al., 2020; Ward & Panitch, 2011) Post cardiac surgeries, postoperative intrapericardial adhesion increases the critical risk of complications (Kikusaki et al., 2021; Wang et al., 2021) Such unwanted adhesion forces patients to undergo a further thor acolaparotomy To prevent such postoperative adhesion, a physical barrier using anti-adhesive polymer membranes has been proposed (Chandel et al., 2021; Hellebrekers et al., 2000; Mayes et al., 2020) To provide anti-adhesive properties, adhesion-preventive membranes are designed to be hydrophilic and water-absorbent Such membranes are wet and slippery, and can therefore detach from the target position after the closure of the surgical site, losing the expected barrier effect If a Abbreviations: CMC, Carboxymethyl cellulose; Bi-C-CVM, Bi-layered CMC-collagen vitrigel membrane; CVM, Collagen vitrigel membrane; EAB, Elongation at breaks * Corresponding author at: Department of Basic Medicinal Sciences, Graduate School of Pharmaceutical Sciences, Nagoya University, Tokai National Higher Education and Research System, Furocho, Chikusa-ku, Nagoya, Aichi 464-8601, Japan E-mail address: kato-r@ps.nagoya-u.ac.jp (R Kato) https://doi.org/10.1016/j.carbpol.2022.119223 Received September 2021; Received in revised form 12 December 2021; Accepted February 2022 Available online February 2022 0144-8617/© 2022 The Authors Published by Elsevier Ltd This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/) Y Wang et al Carbohydrate Polymers 285 (2022) 119223 Fig Procedures for formulating Bi-C-CVM The initial bi-layer image with brighter color indicates layered CMC sol and collagen hydrogel before vitrification: CMC (green), CMC sol; Col (pink), collagen hydrogel The dark-colored bi-layer image represents rehydrated vitrigel The fluorescent image indicates the bi-layer structure of Bi-C-CVM: CMC1 (green), Col (red) The white bar in the image indicates scale bar = 100 μm membrane can control contrary functions by maintaining the antiadhesive function and stably attaching to the wound, it can overcome the drawbacks of the current adhesion-prevention membranes CMC is a biodegradable material used in membranes for post operative adhesion (Ditzel et al., 2012; Kelekci et al., 2004; Park et al., 2011; Lim et al., 2010; Bae et al., 2004) such as Seprafilm® (Helle brekers et al., 2000) However, such CMC membranes are unable to provide cell adhesion for reducing detachment from the wound, and controlling the degradation of stable, mechanically strong membranes remains challenging If a membrane absorbs water and rapidly dis perses, its functionality is hindered Cross-linking is a common strategies ´ et al., 2018; Mohamed et al., used to control CMC degradation (Csapo 2020; Zennifer et al., 2021) However, chemical cross-linking is not favored due to safety concerns For safer cross-linking, enzymatic crosslinking has been reported (Cai et al., 2018) However, simpler mem brane formulating methods are preferred for manufacturing of medical devices Vitrification that concerted the white of boiled egg into a transparent and hard state was first discovered in a process in which bound water was partially removed after free water was completely evaporated by slow drying at ◦ C for more than 10 days (Takushi et al., 1990) Takezawa et al produced collagen vitrigel by gelation of collagen sol, vitrification of collagen gel and rehydration of vitrified collagen gel, and clearly defined vitrigel as “a gel in a stable state produced by rehydra tion after the vitrification of a traditional hydrogel” (Takezawa et al., 2004) The collagen vitrigel membrane (CVM) is an established biomaterial used for cellular scaffolds formulated without any crosslinking reagent, with an excellent balance of both biocompatibility and mechanical properties This high-density collagen fibril-membrane is formulated by rehydration after the vitrification of collagen hydro gel During vitrification, a collagen hydrogel is dried by complete water release (Takezawa et al., 2004) Loss of free water effectively increases collagen intramolecular interactions to form a film composed of highdensity collagen fibrils These intramolecular interactions are retained even after rehydration to form a “wet but rigid” film, which completely differs from the original hydrogel The CVM was first used as a cell culture substratum A nylon membrane-framed CVM was useful for fabricating three-dimensional culture models with paracrine (Takezawa et al., 2007) A plastic cylinder-framed CVM (CVM chamber, commercially available as ad-MED Vitrigel®) facilitated the exposure of chemicals to culture models fabricated on CVM (Oshikata-Miyazaki & Takezawa, 2016; Uzu & Takezawa, 2020; Yamaguchi et al., 2013) Moreover, the attractive properties of CVM prompted its use in tissue engineering and clinical applications including skin regeneration (Aoki et al., 2014), articular cartilage reconstruction (Maruki et al., 2017), and corneal repair (Chae et al., 2015) Although the properties of CVM are ideal for tissue engineering, controlling the cell adhesive-inhibitory property to prevent adhesion is difficult To design an adhesion-prevention membrane to control contrary cellular events in wound regeneration, we hypothesized that “bi-layered vitrification of CMC and collagen hydrogel can create a dual-surface with advantageous properties, thus enabling one membrane to control dual contrary cell-adhesion functions” We developed a reproducible protocol for formulating a bi-layered CMC-collagen vitrigel membrane (Bi-C-CVM) that can provide dual surfaces for contrary functions: cell adhesion-inhibitory function with the stable CMC layer and retention of the cell-adhesion function with the collagen layer The biological and mechanical properties of Bi-C-CVM were evaluated by comparison with CVM to evaluate the novel properties generated by CMC-collagen bilayer vitrification Materials and methods 2.1 Cells and cell-adhesion assay Normal human dermal fibroblasts (NHDF; KF-4109, Kurabo Ltd., Osaka, Japan) were stained with Calcein-AM solution (148504-34-1, Dojindo Laboratories Ltd., Kumamoto, Japan), seeded on the samples (CVM, Bi-C-CVM, polytetrafluoroethylene (PTFE) sheet, or polystyrene (PS) surface of well plates) in serum-free medium, incubated on the sample for h, washed three times with Dulbecco's phosphate-buffered saline (D-PBS; 14249-24, Nacalai Tesque, Inc., Kyoto, Japan), and then the remaining cells were counted by microplate reader (Fluoroskan Ascent; Labsystems Ltd., Helsinki, Finland) The detailed cell culture and cell-adhesion assay protocol is described in the supplementary data Y Wang et al Carbohydrate Polymers 285 (2022) 119223 Table Molecular profiles of examined CMCs (CMC1–CMC5) Type Mw DS Concentration Product no Lot no Provider CMC1 CMC2 CMC3 CMC4 CMC5 250,000 250,000 250,000 90,000 700,000 0.7 0.9 1.2 0.7 0.9 2% w/v* 2% w/v* 2% w/v* 2% w/v* 1% w/v* 419,311 419,303 419,281 419,273 419,338 MKCD5149 MKCB9856 MKBZ8581V MKCG4437 MKCD0622 Sigma-Aldrich Sigma-Aldrich Sigma-Aldrich Sigma-Aldrich Sigma-Aldrich * Concentrations are set to the maximum condition which CMCs can be completely dissolved in D-PBS after autoclaving 2.2 Chemical components 2.3 Formulation of Bi-C-CVM The chemical reagents used in this study are listed in Table S1 with their compound identification numbers The preparation scheme of Bi-C-CVM is illustrated in Fig To form the collagen bottom layer hydrogel, native collagen solution (5 mg mL− 1) in mM HCl at pH 3.0 (IAC-50, Koken Corporation Ltd., Tokyo, Japan) was diluted with D-PBS to a concentration of mg mL− on ice Fig Comparison of CMC types to formulate Bi-C-CVM (a) Representative fluorescent microscopic images of remaining adherent cells after washing The white bar in each image indicates scale bar = mm (b) Bar plot representation of the quantified percentage of remaining adherent cells after washing, with an SD of N = CVM, PTFE sheet, and bare PS surface were compared Y Wang et al Carbohydrate Polymers 285 (2022) 119223 Fig Effect of CMC sol conditions on Bi-C-CVM (a, c, e) Representative fluorescent microscopic images of the remaining adherent cells after washing The white bar in each image indicates scale bar = mm (b, d, f) Bar plot representation of the quantified percentage of remaining adherent cells after washing, with an SD of N = CVM, PTFE sheet, and bare PS surface were compared The pH value of the diluted collagen solution before gelation was 6.78, which was measured using a pH meter (S220, Mettler-Toledo, Colum bus, OH, USA) The dissolved collagen solution was kept at 0.22 mL cm− in containers which size is fit for their further application, and incubated at 37 ◦ C for h until complete gelation CMC (detailed data listed in Table 1) was dissolved in Milli-Q purified water and autoclaved (121 ◦ C, 15 min) to form CMC sol The CMC sol was layered on the collagen hydrogel layer, and the two layers were vitrified together in an air dryer (AA1-AD, As One Corporation, Osaka, Japan) at 10 ◦ C in a lowtemperature incubator (IN604, Yamato Scientific Ltd., Tokyo, Japan) for 48 h to produce Bi-C-CVM The Bi-C-CVM was washed three times (10 soaking each time) with D-PBS to rehydrate the sample, wash off the fragile CMC layer surface, and stabilize the surface After washing, the sample was redried in a low-temperature incubator at 10 ◦ C for 24 h and stored until further analysis This form was designated as “redried form.” For density measurements, the weight of Bi-C-CVM was measured by a microbalance (ATX84, Shimadzu Co., Ltd., Kyoto, Japan) For cell-adhesion assays, the samples were vitrified in a 12-well plate, and the redried samples were rehydrated in D-PBS for 10 in the well before the assay D-PBS was removed, and a cell suspension was seeded onto the rehydrated sample For tensile strength measurement, samples were vitrified in an 8-well plate, and the washed membrane was removed from the well using tweezers and redried on a PTFE sheet The membrane was rehydrated with D-PBS for 10 before the measure ment This form was named “rehydrated membrane.” To pick up the membrane by tweezers, a white nylon membrane (HybondTM-N+, RPN1782B, GE Healthcare, Little Chalfont, UK) was kept under the hydrogel for support 2.5 Collagen staining Bi-C-CVM and CVM were stained for 30 using Collagen Stain Kit (K-61, Cosmo Bio Co., Ltd., Tokyo, Japan) and then repeatedly washed with D-PBS until the color of the supernatant disappeared Stained im ages were acquired by phase-contrast microscopy (TS100-F, Nikon Co., Ltd., Tokyo, Japan) equipped with a digital camera (CFI Achromat ADL 10XF, Nikon Co., Ltd) at 10× magnification 2.6 Tensile strength measurements The rehydrated membranes were measured using a tensile test ma chine (AGS-5KNJ, Shimadzu Co., Ltd.) and Trapezium software (Shi madzu Co., Ltd.) The detailed protocol is described in the supplementary data 2.7 Characterization of Bi-C-CVM and CVM The contact angle, swelling ratio, water retention, in vitro degrada tion, tissue adhesion, and handling properties were measured to char acterize Bi-C-CVM and CVM The detailed protocols are described in the supplementary data Results 3.1 Selection of optimum CMC type for Bi-C-CVM To obtain a stable CMC layer on Bi-C-CVM, five types of CMCs with different molecular profiles (Table 1) were examined First, all CMCs were vitrified without the collagen hydrogel but all samples formed fragile flake-like membranes that dispersed immediately in water Therefore, the vitrification of only CMC cannot form a stable membrane However, we found that with the bi-layered vitrification process with collagen hydrogel (Fig 1), all samples formulated visually-similar vit rigel membranes that did not disperse after rehydration From the sectioned image, the clearly formed bi-layer structure and their thick ness of Bi-C-CVM was confirmed In the cell-adhesion assay, CMC1 2.4 Scanning electron microscopy (SEM) characterization Bi-C-CVM (without washing) and CVM (with washing) were lyoph ilized using vacuum deposition equipment (VE-2030, Vacuum Device Ltd., Osaka, Japan), and coated with osmium plasma (OPC-40, Japan laser corporation Ltd., Aichi, Japan) Samples were observed by SEM (JSM-7610F, JEOL Ltd., Tokyo, Japan) at 10,000× magnification Y Wang et al Carbohydrate Polymers 285 (2022) 119223 (a) Bi-C-CVM CMC1 Col CVM Col Bi-C-CVM CMC1 Col CVM Col (b) Fig Surface analysis of CMC1 layer on Bi-C-CVM (a) SEM images of the top surface of Bi-C-CVM (CMC1 layer, without washing) and CVM (b) Phase contrast microscopy images of collagen staining of the top surface of Bi-C-CVM (CMC1 layer) and CVM The white bar in each image indicates scale bar = mm showed the best cell adhesion-inhibitory performance (Fig 2), which was comparable with that of PTFE, one of the most frequently used materials that inhibit cell adhesion Thus, the bi-layered vitrification and optimum CMC type selection are essential for formulating a Bi-CCVM with cell adhesion-inhibitory function CMC1 was selected for further Bi-C-CVM evaluation As the basic property, the average density of the dried state Bi-C-CVM with CMC1 was 1.6 ± 0.2 g/cm3 (N = 3) ´n-Colo ´n et al., affecting the structure and properties of CVM (Caldero 2012), we examined the vitrification time (Fig 3e–f) Our results showed that the cell adhesion-inhibitory performance of Bi-C-CVM was stable after days of vitrification Moreover, the finalized Bi-C-CVM formation procedure was highly reproducible 3.2 Optimization of CMC1 vitrification conditions for Bi-C-CVM To confirm whether the CMC1 layer existed stably on the collagen layer even after Bi-C-CVM rehydration, we assessed the CMC1 layer surface using SEM (Fig 4a) The CMC1 layer surface was different from that of CVM, although a clear structural pattern was not observed because the sample was never washed to retain the surface To further examine the presence of the CMC1 layer on Bi-C-CVM, collagen was stained (Fig 4b) No collagen with pink staining was detected on the CMC1 layer surface of Bi-C-CVM Collagen staining of all CMC surfaces with different types of CMCs (Fig S1a), concentrations of CMC1 (Fig S1b), and volumes of CMC1 sol (Fig S1c) showed that collagen exposure was found in most samples that lost cell adhesion-inhibitory performance Therefore, although CMC layer of Bi-C-CVM can absorb water and partially disperse, it reproducibly functions as a cell adhesioninhibitory layer under the optimum conditions determined in this study 3.3 Surface analysis of the CMC layer on Bi-C-CVM Vitrification conditions were optimized using CMC1 First, the CMC1 sol concentration was optimized (Fig 3a–b) The desired performance was achieved at a concentration of more than 1.5% wt To make its performance stable and reproducible, we selected 2.0% wt as the opti mum concentration Second, we examined the volume of the CMC layer (Fig 3c–d) and showed that thinner CMC1 layers could not sustain the cell adhesioninhibitory effect, suggesting that the rehydrated CMC1 layer can be lost during washing before cell seeding This result is reasonable because the CMC1 layer is attached to the collagen layer through only simple vitrification without any cross-linking reagents However, the data showed that such defects can be overcome by thickening the CMC1 layer The maximum cell adhesion-inhibitory performance was achieved with 2.6-mm thick CMC1 sol, which was selected as the optimum layer volume Third, since the vitrification condition is an important parameter 3.4 Investigation of layering procedure of Bi-C-CVM After evaluating the conditions and effects of the CMC-layer of Bi-C5 Y Wang et al Carbohydrate Polymers 285 (2022) 119223 Fig Development of a two-step procedure for Bi-C-CVM (a) Schematic illustration of one-step and two-step procedures for forming Bi-C-CVM (b) Bar plot indicating the percentage of remaining adherent cells after washing, with an SD of N = Images above the bar plot indicate the representative fluorescent microscopic images of remaining adherent cells after washing The white bar in each image indicates the scale bar = mm CVM was compared CVM, we investigated the tuning of the collagen layer in Bi-C-CVM As CVM is uniquely tough, we examined whether we can enhance the mechanical property of Bi-C-CVM by controlling the collagen layer Therefore, we modified the bi-layer formulation procedure for Bi-CCVM The one-step procedure established in previous sections was feasible; however, the thickening of the collagen bottom layer was a challenge as the vitrification of bi-layered hydrogels, particularly those containing a CMC layer, take longer to dry By separating the vitrifica tion step of the bottom layer (collagen hydrogel) and the top layer (CMC sol), a two-step procedure was developed (Fig 5a) Based on the cell adhesion-inhibitory performances of Bi-C-CVM formulated with both procedures, the result indicated that the two-step procedure sustained good cell adhesion-inhibitory performance (Fig 5b), enabling the development of a Bi-C-CVM with a thickened collagen layer for subse quent mechanical property measurements 3.5 Tensile strength evaluation of Bi-C-CVM To characterize the mechanical property of Bi-C-CVM, we measured the tensile strength in the rehydrated “wet” state (Fig 6a) Bi-C-CVM (1Col) extended up to ~20%–30% of its total length (20 mm) (Fig 6a upper right) This data also revealed that the mechanical strength of BiC-CVM was nearly equivalent to CVM and suggests that the CMC layer has little an effect on the mechanical strength of Bi-C-CVM, and the collagen layer is the key parameter for controlling strength We next doubled the thickness of the collagen hydrogel layer using our two-step procedure and measured their property (Fig 6a bottom right); the maximum endurable tensile force was increased nearly 7.5fold (up to 1.0–1.5 N) The tensile strain nearly doubled in the Bi-CCVM (2-Col) compared to CVM (2-Col) These data suggest that although the CMC layer did not contribute to the strength, it enhanced Y Wang et al Carbohydrate Polymers 285 (2022) 119223 Fig Tensile strength evaluation of rehydrated form Bi-C-CVM (a) Tensile stretching curves of Bi-C-CVM (1-Col), Bi-C-CVM (2-Col), Bi-C-CVM (10-Col), singlelayered CVM and double-layered CVM (b) Averaged EABs of CVMs and Bi-C-CVMs (c) Averaged tensile stresses of CVMs and Bi-C-CVMs the stretchable property of Bi-C-CVM We further examined the thickening of the collagen layer using a two-step procedure and found that Bi-C-CVM (10-Col) could achieve a tensile force of 4–14 N (Fig 6a) Interestingly, the thickness of Bi-C-CVM (10-Col) in the rehydrated state was 120.4 ± 5.2 μm, whereas that of BiC-CVM (1-Col) was 79.5 ± 9.2 μm These data indicated that during 10 repeated collagen hydrogel layering, the newly layered collagen hydrogel merged with the bottom prior-vitrificated layer during vitri fication to form a denser membrane with small thickness increase To compare the mechanical property with the previously reported anti-adhesive membranes, we converted the stretching profile into two indices: averaged elongation at break (EAB) (Fig 6b) and averaged tensile stresses (Fig 6c) EAB results over 130% were equivalent to those of previously reported crosslinked membranes using CMC, whereas the tensile stress values from Bi-C-CVMs were nearly 10-fold (2-Col) and 40fold (10-Col) higher than those of the cross-linked membranes (Cai et al., 2018) We also confirmed that its cell adhesion-inhibitory function on CMC layer had no negative effect by thickening the collagen layer (Fig S2) These results suggest that our Bi-C-CVM formulated from simple vitrification achieved unique and superior mechanical properties compared to those of other membranes formulated with cross-linking agents From the collagen layer thickening effect, our data also sug gest that the property control is feasible using our two-step procedure (Fig 5a) 3.6 Characterization of Bi-C-CVM for adhesion-prevention membrane application We further characterized the Bi-C-CVM properties to investigate its applicability as an adhesion-prevention membrane (Fig 7) First, the redried form of Bi-C-CVM was evaluated upon physical and visual examination, being semitransparent with a flexible and tough texture (Fig 7a) The Bi-C-CVM was difficult to tear by hand, but easily cut using scissors, and was easy to roll without breaking This handling property was equivalent to that of the CVM; however, it was slightly tougher due to the additional thickness of the CMC layer Second, the moisture-absorbent performance of the redried form of Bi-C-CVM was evaluated The CMC layer side showed higher wettability and rapidly absorbed D-PBS (Fig 7b, Video S1) The contact angle measurements revealed that, after min, the CMC side of Bi-C-CVM quickly absorbed D-PBS and became hydrophilic (Fig 7b) These re sults clearly indicated that the CMC layer added the novel property of moisture-absorbance to Bi-C-CVM Third, the moisture-retaining performance of rehydrated Bi-C-CVM was evaluated The Bi-C-CVM remained wet for h owing to its moisture-retaining property of CMC layer (Fig 7c, Video S2) From the measurements of swelling ratio and water retention ratio, it was found that although both membranes absorbed water within min, Bi-C-CVM has 10-fold higher water absorption capacity than CVM (Fig 7d) Furthermore, when rehydrated form of CVM dried quickly within h, the rehydrated form of Bi-C-CVM retained 80% of absorbed water These Y Wang et al Carbohydrate Polymers 285 (2022) 119223 Fig Characterization of redried form Bi-C-CVM for adhesion-prevention membrane application (a) Image of the CVM and Bi-C-CVM (b) The contact angle of CVM and Bi-C-CVM (CMC side and Col side) (N = 3) (c) Water-retaining performance of CVM and Bi-C-CVM (d) Swelling ratio (left) and water retention ratio (right) of CVM and Bi-C-CVM (N = 3) (e) Tissue attachment and re-positioning performance of CVM and Bi-C-CVM The collagen side of Bi-C-CVM is facing the wet tissue (f) Adhesion-prevention performance of CVM and Bi-C-CVM using the wet tissue model (g) Tissue adhesion performance of Bi-C-CVM (CMC side or Col side) under pull-down force (100 G, min) (N = 3) *Student's t-test p < 0.05 (h) Time-course in vitro degradation of Bi-C-CVM (N = 3) Y Wang et al Carbohydrate Polymers 285 (2022) 119223 moisture-retaining performances enabled Bi-C-CVM to achieve the “tough but stably wet” property for more feasible handling Even in highly wet conditions, Bi-C-CVM was tough enough to withstand tweezer manipulation Fourth, the attachment performance of Bi-C-CVM was evaluated The redried form of Bi-C-CVM was flexible enough to attach to the wet tissue with their collagen-side and could smoothly cover the three-dimensional surface with the help of their moisture-absorbent property (Fig 7e, Video S3) Even with such feasible attachment, it was stable enough that considerable effort was required for them to be detached by tweezers However, interestingly, when we tried to re-position the membranes within a short period, Bi-C-CVM could be feasibly re-positioned, whereas CVM shrunk rapidly and became crumpled, hindering repositioning This result further suggest that Bi-C-CVM achieved a “tough but stably wet” property due to the CMC layer, which enabled attachment with flexibility, allowing physicians to reconsider its positioning Fifth, the adhesion-prevention performance of the redried form of BiC-CVM was evaluated with a brief tissue model CVM and Bi-C-CVM were sandwiched between wet tissue samples, and their performances were compared after h (Fig 7f, Video S4) Tissues adhered tightly to both sides of the CVM However, for Bi-C-CVM, the bottom tissue adhered tightly to the collagen layer, whereas the top tissue adjacent to the CMC layer detached easily To quantify the strength of adhesive ability on both sides of Bi-C-CVM, after h of attachment to wet tissue, tissues were pulled down by 100 G for using a centrifuge Even with such severe tissue tearing stress, the tissues on the collagen side of Bi-C-CVM remained 5-fold more than those on the CMC side (Fig 7g) These data strongly suggest that the developed Bi-C-CVM achieved the dual cell-adhesion functions stated in our hypothesis and suggests its potential as a candidate material for an adhesion-preventive barrier with lower risk of detachment Furthermore, to estimate the in vivo degradation potency, the timecourse in vitro degradation was measured (Fig 7h) In the presence of collagenase, Bi-C-CVM degraded within d; however, with only me dium or trypsin, the degradation was slow, and 40% (wt/wt) remained within week preventive devices (Li et al., 2020); thus, our dual-surface membrane BiC-CVM may overcome these limitations In tensile strength evaluation, Bi-C-CVM showed a “J” curve stretching property essential for artificial blood vessel grafts (Sonoda et al., 2003) This “J” curve stretching property is important for avoiding compliance mismatch between the native artery and artificial grafts, which is a critical cause when using small-diameter artificial graft; however, it is difficult to achieve by using hydrogel-based materials (Aussel et al., 2017; Yang et al., 2020) When we compared Bi-C-CVM data with that of porcine blood vessels, our Bi-C-CVM showed a nearly equivalent “J” curve property (Fig S3a) We also found that Bi-C-CVM (10-Col) can maximize the tensile stress up to 2.2 MPa, which is stron ger than that of porcine blood vessels (average 1.7 MPa) (Fig S3b) Therefore, together with the reported hemocompatibility effect of CMC (Basu et al., 2018), our Bi-C-CVM showed a great potential for artificial graft application, and its unique mechanical property with simple des ignability will enable wider medical applications Discussion This study was partially supported by JSPS KAKENHI, grant numbers 23680055 to RK, and 18K14061 and 20K05227 to KK1 (Kanie) This work was also supported by the Toyoaki Scholarship Foundation to KK1, and Tatematsu Foundation to KK Conclusions We developed a biocompatible membrane with dual surfaces for controlling contrary cell-adhesion functions with one membrane, named as Bi-C-CVM By simple bi-layered vitrification of CMC and collagen hydrogels, the difficulty of controlling CMC degradation for membrane formation was overcome, resulting in a rigid membrane without using cross-linking agents and supporting our hypothesis Our Bi-C-CVM exhibited novel properties exerted by the CMC layer while retaining the advantages of CVM Therefore, it may be applicable for controlling more complex regenerative events in vivo such as postoperative adhesion prevention Furthermore, by taking advantage of its unique mechanical properties and its dual-surface functions, Bi-C-CVM might be considered as a basal material to formulate a tube-like structure for developing novel-type artificial blood vessels Supplementary data to this article can be found online at https://doi org/10.1016/j.carbpol.2022.119223 Funding We developed a dual-functional membrane device for controlling contrary cellular events in wound regeneration by combining the ad vantages of CMC and CVM Using a simple but robust bi-layer vitrifi cation procedure, we developed Bi-C-CVM that retains the advantageous properties of both CMC and collagen without using any cross-linking agents Our combinatorial application of CMC and collagen in one bilayered membrane will accelerate not only the use of CVM devices but also other medical applications of CMC-based membranes (Rahman et al., 2021) and collagen-based membranes (Sayani & Raines, 2014) Uncontrolled detachment of adhesion-prevention membranes after the closure of a surgical site is a practical and serious risk Most animal models of postoperative adhesion are designed using small animals by creating abdominal defects between small spaces with flat surfaces (e.g., abdominal walls, or organ surfaces); therefore, the membrane detach ment risk in humans (larger space between more structurally complex surfaces) is difficult to determine However, even in small animals, Hellebrekers et al (2000) showed that 11 of 20 PolyActive™ were de tached from the wound, suggesting the potential for such risk Our data indicated the novel potential of the “dual-surface adhesion-prevention membrane,” which can attach and cover the wound on one side, while preventing adhesion on the other Cauterizing the surface negatively affects the opposite intact peritoneum surface within h, damaging protective mesothelial cells (Suzuki et al., 2015) Therefore, not only providing barrier effect but also rapid ensealing of post-operative tissue surface is important for improving adhesion prevention There are still few reports that propose such wound protection effect for adhesion CRediT authorship contribution statement Yue Wang: Data curation, Investigation, Methodology, Validation, Visualization, Writing – original draft, Writing – review & editing Kei Kanie: Conceptualization, Methodology, Funding acquisition, Project administration, Supervision, Visualization, Writing – review & editing Toshiaki Takezawa: Conceptualization, Methodology Miki Hori kawa: Conceptualization, Data curation, Validation, Methodology Kyoshiro Kaneko: Conceptualization, Data curation, Validation, Methodology Ayako Sugimoto: Data curation, Methodology Aika Yamawaki-Ogata: Methodology Yuji Narita: Conceptualization, Methodology Ryuji Kato: Conceptualization, Funding acquisition, Methodology, Project administration, Supervision, Writing – review & editing Declaration of competing interest 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 Y Wang et al Carbohydrate Polymers 285 (2022) 119223 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a dual-surface. .. properties, thus enabling one membrane to control dual contrary cell-adhesion functions” We developed a reproducible protocol for formulating a bi-layered CMC-collagen vitrigel membrane (Bi-C-CVM) that... Characterization of Bi-C-CVM for adhesion-prevention membrane application We further characterized the Bi-C-CVM properties to investigate its applicability as an adhesion-prevention membrane (Fig 7) First,