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Debabrata Palai, Hana Yasue, [Shima Ito](https://orcid.org/0000-0002-3233-617X), [Hiyori Komatsu](https://orcid.org/0000-0002-2525-1362), [Tetsushi Taguchi](https://orcid.org/0000-0003-2541-2530), [Akihiro Nishiguchi](https://orcid.org/0000-0002-3160-6385)

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[Functionalization of viscoelastic gels with decellularized extracellular matrix microparticles enhances tissue adhesion, cell spreading, and tissue regeneration](https://mdr.nims.go.jp/datasets/46e8606e-7a4e-4b80-af3b-2918834ecc26)

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Functionalization of viscoelastic gels with decellularized extracellular matrix microparticles enhances tissue adhesion, cell spreading, and tissue regenerationBiomaterialsSciencePAPERCite this: Biomater. Sci., 2025, 13,3576Received 11th March 2025,Accepted 25th April 2025DOI: 10.1039/d5bm00394frsc.li/biomaterials-scienceFunctionalization of viscoelastic gels withdecellularized extracellular matrix microparticlesenhances tissue adhesion, cell spreading, andtissue regeneration†Debabrata Palai,a Hana Yasue,a,b Shima Ito, a,c Hiyori Komatsu,a,cTetsushi Taguchi *a,c and Akihiro Nishiguchi *a,bThe natural extracellular matrix (ECM) is viscoelastic and fibrous, which are crucial characteristics for con-trolling cellular responses. In contrast, synthetic gels are mostly elastic and less effective at promotingmechanotransduction. Thus, the design of gels that provide mechanical and biochemical cues for tissueregeneration needs to be explored. In this study, we aimed to develop viscoelastic gels functionalizedwith decellularized ECM (dECM) microparticles for tissue regeneration. The incorporation of dECM micro-particles into gels improved not only the tissue adhesive properties of the gels but also their viscoelasti-city. The modulation of the mechanical properties of the gels elicited cell adhesion and spreading.Moreover, the functionalization of viscoelastic gels with dECM microparticles promoted tissue regener-ation in volumetric muscle-loss models. This approach would be a powerful method because functionalscaffolds with sufficient mechanical and biological properties facilitate tissue regeneration.IntroductionThe decellularized extracellular matrix (dECM) has emerged asa primary candidate in the fields of tissue engineering andregenerative medicine to repair and regenerate damagedtissues. The dECM is prepared by removing the cellular com-ponent and associated antigens from native tissue, while con-currently preserving its structural integrity and non-cellularcomponents.1 The dECM comprises a variety of proteins andglycosaminoglycans, providing a fibrous network platform tomediate mechanical and biochemical signaling between cells,which can improve cellular function and promote tissue repairand remodelling.2–6 However, dECM scaffolds, includingsheets and fibers, have drawbacks in their practical usebecause of their lack of injectability and tissue-adhesive pro-perties, which limit their clinical translation. Although solubil-ized dECM can form gels post-injection, enzymatic treatmentimpairs its mechanical and viscoelastic properties.7,8To overcome these challenges, the dECM can be combinedwith hydrogels. Hydrogels are promising biomaterials for tissueregeneration because of their biocompatibility, biodegradability,and tunable mechanical and biological functionalities.9–11 Asidefrom tissue regeneration, hydrogels have been used in variousother biomedical applications, such as tissue scaffoldadhesives12–14 for in vitro vascularization15 and anti-inflammatorydrug carriers.16,17 However, covalently crosslinked synthetichydrogels are mostly elastic and prevent many cellular functionsobserved in the natural ECM.18,19 The natural ECM is viscoelasticand possesses complex structures because of the distinct struc-tural arrangement of various biopolymers. With these features,the ECM regulates cellular functions, such as spreading,migration, proliferation, and differentiation.20,21 Hence, to designtissue regenerative materials, viscoelastic scaffolds with mechani-cal and biochemical properties similar to those of the naturalECM need to be developed.22–24 Recent research has focused ondECM fibers or particles, which are subsequently reconstitutedinto various biomaterial forms, such as hydrogels and electro-spun scaffolds.25,26 Decellularized extracellular matrix (dECM)-based hydrogels have been widely reported as promisingmaterials for tissue regeneration. However, the design and optim-ization of viscoelastic gels modified with dECM microparticlesremain largely unexplored, presenting an opportunity for furtherinvestigation in biomaterial engineering.†Electronic supplementary information (ESI) available: Supplementary Fig. S1and S2. See DOI: https://doi.org/10.1039/d5bm00394faBiomaterials Field, Research Center for Macromolecules and Biomaterials, NationalInstitute for Materials Science, 1-1 Namiki, Tsukuba, Ibaraki 305-0044, Japan.E-mail: nishiguchi.akihiro@nims.go.jp, taguchi.tetsushi@nims.go.jpbDepartment of Materials Science and Technology, Graduate School of AdvancedEngineering, Tokyo University of Science, 6-3-1 Niijuku, Katsushika-ku, Tokyo 125-8585, JapancGraduate School of Pure and Applied Sciences, University of Tsukuba, 1 Chome-1-1Tennodai, Tsukuba, Ibaraki 305-8577, Japan3576 | Biomater. Sci., 2025, 13, 3576–3584 This journal is © The Royal Society of Chemistry 2025Open Access Article. Published on 29 April 2025. Downloaded on 6/25/2025 2:20:25 PM.  This article is licensed under a Creative Commons Attribution-NonCommercial 3.0 Unported Licence.View Article OnlineView Journal  | View Issuehttp://rsc.li/biomaterials-sciencehttp://orcid.org/0000-0002-3233-617Xhttp://orcid.org/0000-0003-2541-2530http://orcid.org/0000-0002-3160-6385https://doi.org/10.1039/d5bm00394fhttps://doi.org/10.1039/d5bm00394fhttp://crossmark.crossref.org/dialog/?doi=10.1039/d5bm00394f&domain=pdf&date_stamp=2025-06-19http://creativecommons.org/licenses/by-nc/3.0/http://creativecommons.org/licenses/by-nc/3.0/https://doi.org/10.1039/d5bm00394fhttps://pubs.rsc.org/en/journals/journal/BMhttps://pubs.rsc.org/en/journals/journal/BM?issueid=BM013013In this study, we developed viscoelastic gels functionalizedwith dECM microparticles for tissue regeneration (Fig. 1).Gelatin from two different sources (porcine-skin-derivedgelatin [sG] and porcine tendon-derived gelatin [tG]) waschemically modified with thiol and vinyl sulfone groups andcrosslinked to form elastic and viscoelastic gels, respectively.We addressed the effect of incorporating dECM microparticlesinto gels on mechanical properties, such as stress relaxationand tissue adhesiveness. The effects of these properties on cel-lular behavior and function were thoroughly investigated.Moreover, the treatment of a defect in muscle tissue, volu-metric muscle loss (VML), using dECM microparticle-functio-nalized gels was demonstrated to evaluate the tissue-regenera-tive properties.ExperimentalMaterialsPorcine-skin-derived gelatin (sG, Mw = 180 kDa) and porcine-tendon-derived gelatin (tG, Mw = 344 kDa) were purchasedfrom Nitta Gelatin, Inc. (Osaka, Japan). 2,4,6-Trinitrobenzenesulfonic acid sodium salt dihydrate (TNBS)was purchased from Tokyo Chemical Industry Co. Ltd (Tokyo,Japan). Tris(2-carboxyethyl) phosphine hydrochloride (TCEP)and phosphate-buffered saline (PBS) were purchased fromNacalai Tesque, Inc. (Kyoto, Japan). 5,5′-(2-Nitrobenzoic acid)(DTNB), RPMI 1640 medium, and fetal bovine serum (FBS)were purchased from Sigma-Aldrich (St Louis, MO, USA).Collagen casings were purchased from Nippi (Tokyo, Japan).Penicillin/streptomycin (P/S), trypsin, rhodamine-labelledphalloidin, and 4′,6-diamidino-2-phenyl-indole (DAPI) werepurchased from Thermo Fisher Scientific (Waltham, MA, USA).Amikacin solution was purchased from Meiji Seika Pharma(Tokyo, Japan). Dialysis membranes (molecular weight cut-offvalue: 12 000–14 000) were purchased from Repligen(Waltham, MA, USA). DNase I was purchased from Merck(Darmstadt, Germany). Dimethyl sulfoxide (DMSO), peraceticacid (PA), magnesium sulfate, and proteinase K were pur-chased from Fujifilm (Osaka, Japan). The mouse myoblast cellline (C2C12 cells) was purchased from the EuropeanCollection of Authenticated Cell Cultures (Salisbury, UK).Preparation of dECM microparticlesDecellularization of tissues from the urinary bladder was per-formed as described in a previous report.27 Briefly, a pur-chased porcine urinary bladder (Tokyo Shibaura Zouki, Tokyo,Japan) was cut open, and the mucosal layer was dissectedusing a scalpel. The mucosa was washed with saline and incu-bated in PBS containing 0.1% PA and 4% ethanol for 2 h at25 °C. The tissues were then washed twice with 1 L of salineand 1 L of ultrapure water for 1 h each at 25 °C and freeze-dried. The dried tissues were cut into small pieces using scis-sors and 10 mg of tissue fragments were incubated in 1 mL ofPBS containing DNase I (0.2 mg mL−1, 300 U mL−1; RocheDiagnostics, Indianapolis, IN, USA) and 5 mM magnesiumsulfate at 37 °C for 24 h with stirring. The samples were col-lected by centrifugation at 10 000 rpm for 10 min and washedtwice with 2 mL of PBS and 2 mL of ultrapure water for 10 minat 25 °C, respectively. After repeating the washing and freeze-drying steps, the urinary bladder matrix (UBM)-based dECMwas obtained.dECM microparticle fabrication was performed accordingto a previous report, with slight modifications.28 Freeze-drieddECM sheets were cut into small pieces and ground using agrinder (Wonder Crusher; Osaka Chemical, Osaka, Japan). Theground dECM was further cryo-milled using a cryogenicgrinder machine (6775 FREEZER/MILL; SPEX, USA), loadedinto small grinding vials, precooled for 1 min under liquidnitrogen, and cryo-milled for two cycles at 15 cycles per secondfor 2 min, followed by 2 min of rest for a total of 7 min each.The morphology of the dECM microparticles was observedusing scanning electron microscopy (SEM) (JCM-7000NeoScope; JEOL, Tokyo, Japan). The dECM microparticles wereattached to a carbon tape, and sputtered with gold for 1 min.To obtain the SEM images of the particles, the acceleratingvoltage and working distance were set to 15 kV and 12.7 mm,respectively. The size distribution of the dECM microparticleswas analyzed using ImageJ software (National Institutes ofHealth, Bethesda, MD, USA).Quantification of DNA contentThe DNA content of the tissues before and after the decellular-ization process was quantified according to a previousreport.27 Briefly, the dried dECM (10 mg) was dispersed in1 mL of proteinase K (50 μg mL−1) in a mixture of 10 mM Tris-HCl buffer, 10 mM ethylenediaminetetraacetic acid (EDTA),10 mM NaCl, and 0.5% sodium dodecyl sulfate (pH = 8) andincubated at 37 °C for 24 h. The solution was then mixed with500 µL of a phenol/chloroform/isoamyl alcohol (25/24/1) solu-tion and centrifuged at 15 000 rpm at 4 °C for 30 min. Aftercollecting the aqueous layer, 50 μL of acetic acid solution (3 M)was added (final concentration: 300 mM). DNA was precipi-Fig. 1 Schematic of the preparation of dECM microparticle-functiona-lized elastic and viscoelastic gels crosslinked via the thiol–ene reaction.Two types of gelatin from different sources were chemically modifiedwith thiol (TH) and vinyl sulfone (VS) groups: porcine skin-derivedgelatin (sG) with TH (sGTH) and VS (sGVS) and porcine tendon-derivedgelatin (tG) with TH (tGTH) and VS (tGVS). The incorporation of decellu-larized extracellular matrix (dECM) microparticles can alter gel pro-perties, including stress relaxation, tissue adhesion, cell–material inter-action and tissue repair.Biomaterials Science PaperThis journal is © The Royal Society of Chemistry 2025 Biomater. Sci., 2025, 13, 3576–3584 | 3577Open Access Article. Published on 29 April 2025. Downloaded on 6/25/2025 2:20:25 PM.  This article is licensed under a Creative Commons Attribution-NonCommercial 3.0 Unported Licence.View Article Onlinehttp://creativecommons.org/licenses/by-nc/3.0/http://creativecommons.org/licenses/by-nc/3.0/https://doi.org/10.1039/d5bm00394ftated by the addition of a two-fold excess (1 mL) of coldethanol and stored at −20 °C for 1 h. After centrifuging thesolution at 15 000 rpm for 30 min, the supernatant wasremoved, and the precipitate was dried under vacuum for30 min. The samples were resuspended in 1 mL of Tris-HClbuffer (10 mM) with EDTA (1 mM) and diluted 10-fold. Theremaining DNA content was measured using a PicoGreen™dsDNA Assay Kit (Thermo Fisher Scientific) according to themanufacturer’s instructions. Sample fluorescence wasrecorded using a microplate reader (Spark10M; TECAN,Mannedorf, Switzerland). The DNA content was calculatedusing a standard curve.Synthesis of sGTH, tGTH, sGVS, and tGVSTo synthesize gelatin modified with thiol groups, 1 g of sG(amino groups: 292 µmol g−1) and tG (263 μmol g−1) was dis-solved in 13.6 mL and 22 mL of DMSO, respectively, andstirred at 50 °C for 4 h. The number of amino groups in thegelatin samples was determined using the TNBS method.γ-Thiobutyrolactone (60.5 µL, 240 mol% equivalent to theamino groups in sG, and 50 µL, 220 mol% equivalent to theamino groups in tG) was dissolved in 3 mL of DMSO andadded to the solution. The reaction was continued overnight at50 °C with stirring. To reduce the disulfide bonds to thiolgroups, TCEP (1 mM) was added, and the mixture was stirredfor 30 min at room temperature. The resulting solutions wereslowly added to a 20 vol% cold solvent mixture (ethanol andethyl acetate, v/v = 1/1) while stirring, and the precipitates werecollected using a glass filter and washed three times withethanol to remove unreacted reagents. The precipitates col-lected by filtration were dried at room temperature underreduced pressure for 3 days to obtain sGTH and tGTH. Thedegree of substitution of the thiol groups was calculated usingthe Ellman method, as reported elsewhere.12To synthesize gelatin modified with vinyl sulfone (sGVSand tGVS), 1 g of sGTH or tGTH was dissolved in 99 mL ofultrapure water at 50 °C with stirring for 1 h. Divinylsulfone (200 and 120 mol% equivalent to the thiol groupsin sGTH and tGTH, respectively) was dissolved in 1 mL ofultrapure water and added dropwise to the solution. Thereaction was continued for 24 h at 50 °C with continuousstirring. TCEP (1 mM) was added to the solution, and theobtained solution was dialyzed in ultrapure water using adialysis membrane for 3 days. After freeze-drying, sGVS andtGVS were obtained.Evaluation of tissue-adhesive propertiesThe tissue-adhesive properties of the gels were evaluated bymeasuring the burst strength according to the AmericanSociety of Testing and Materials (ASTM) procedure(ASTM-F2392-04R, standard test method for burst strength ofsurgical sealants). Collagen casings (Nippi) were cut into35 mm discs with 3 mm pinholes at the center. A silicone ringmold (outer and inner diameters: 20 and 10 mm, respectively;thickness: 1 mm) was placed on the collagen casings. sGTH,sGVS, tGTH, and tGVS were dissolved in PBS (10 wt%) at 50 °Cwith stirring and the pH was adjusted to 7.4, 6.4, 7.4, and 6.4,respectively, using 1 M NaOH. The solution was maintained at37 °C until further use. Solutions of sGTH (200 µL) and sGVS(200 µL) or tGTH (200 µL) and tGVS (200 µL) were vigorouslymixed using a pipette. The dECM microparticles (40 mg, finalconcentration: 10 wt%) were mixed using a spatula. Threehundred microliters of the mixed solution was placed onto thecollagen casings inside the silicon mold ring. After 60 min ofgelation at 37 °C, the silicone mold was removed, and thesamples were placed in the chamber. Burst strength(maximum pressure until rupture) was measured by runningsaline water using a syringe pump at a flow rate of 2 mL min−1at 37 °C.Rheological analysisRheological measurements were performed using a rheometer(MCR301; Anton Paar GmbH, Graz, Austria). sGTH–sGVS ortGTH–tGVS gels, with or without dECM microparticles (10 wt%),were prepared to evaluate gelation kinetics and viscoelastic pro-perties. The pre-gel solution was placed on the stage of the rhe-ometer (pre-heated at 37 °C) and a jig with a 10 mm diameterwas set up with a gap of 1 mm. After removing the excess gel,measurements were performed at 37 °C at a frequency of10 rad s−1 with 1% strain for 1 h. The stress relaxation propertiesof the gels and dECM-modified gels were measured at 0.2%strain with a deformation rate of 1 mm min−1.Cell encapsulationsGTH, tGTH, sGVS, and tGVS were dissolved in PBS (10 wt%)at 50 °C with stirring and the pH was adjusted to 7.4 and 6.4,respectively, with 1 M NaOH. All solutions were filtered using a0.45 µm syringe filter for sterilization. The solutions werestored at 37 °C until further use. C2C12 cells were cultured inRPMI medium supplemented with 10% FBS and 1% P/S.Cultured cells were treated with trypsin and the cell pellet wascollected after centrifugation at 1200 rpm for 5 min at 4 °C.After removing the supernatant, 200 µL of sGTH–sGVS ortGTH–tGVS mixtures, with or without 10 wt% dECM micropar-ticles, were added to each tube containing the cell pellet (1 ×105 cells) as distinct groups. Twenty microliters of pre-gelsamples containing cells were plated on a chamber cover (10 ×10 mm). The samples were then incubated for 10 min at 37 °Cfor gelation. Four hundred microliters of RPMI medium with10% FBS and 1% P/S was then added to each chamber and thecells were cultured for 1 d at 37 °C in a 5% CO2 incubator.Fluorescence staining, imaging, and analysisCells encapsulated in gels were fixed with 4% paraformalde-hyde for 1 h. After washing with PBS, the cells were permeabi-lized with 0.2% Triton-X for 30 min. After washing the samplewith PBS, the cells were blocked with 1% bovine serumalbumin/PBS for 1 h. For actin staining, the cells were stainedwith rhodamine-labelled phalloidin (1 : 100) overnight at25 °C. After washing with PBS, the cells were stained withDAPI for 1 h at 25 °C. The cell morphology was observed at adepth of 50 µM using confocal laser scanning microscopyPaper Biomaterials Science3578 | Biomater. Sci., 2025, 13, 3576–3584 This journal is © The Royal Society of Chemistry 2025Open Access Article. Published on 29 April 2025. Downloaded on 6/25/2025 2:20:25 PM.  This article is licensed under a Creative Commons Attribution-NonCommercial 3.0 Unported Licence.View Article Onlinehttp://creativecommons.org/licenses/by-nc/3.0/http://creativecommons.org/licenses/by-nc/3.0/https://doi.org/10.1039/d5bm00394f(CLSM; CLSM 900 with Airyscan2; Zeiss, Oberkochen,Germany) and the area of the cells and the average length ofelongated cells were quantified in at least three differentimages using ImageJ. Correlation studies were conductedbetween the average cell length and half relaxation time (t1/2)using Python 3.10.12 run in Google Colaboratory (Colab) toclarify the relationship between the two metrics.Biodegradability testAll animal procedures were performed in accordance with theGuidelines for Care and Use of Laboratory Animals of NationalInstitute for Materials Science and approved by the AnimalEthics Committee of the National Institute for MaterialsScience (no: 76-2023-16). To maintain uniformity in the size ofthe gels, pre-crosslinked gels were used for biodegradabilitytesting. Two milliliters of tGTH-tGVS pre-gel solution with orwithout dECM microparticles (10 wt%) were placed on a1 mm-thick silicone mold, followed by incubation for 1 h at37 °C. The crosslinked gels were then cut into disc shapesusing an 8 mm biopsy punch (KAI Medical, Seki City, Japan).Mice (7 week-old female C57BL/6J mice; Jackson Laboratory,Bar Harbor, ME, USA) were anesthetized via inhalation of 2%isoflurane. The backs of the mice were disinfected with 70%ethanol and the hair was trimmed. Gel discs (1 mm thick)were subcutaneously implanted in each mouse on the left andright sides of the dorsal region. At 3, 7, 14, and 28 days afterimplantation, the mice were euthanized by exsanguination,and tissues were collected. For sham, subcutaneous tissueswithout implantation of gels were collected. The obtainedtissues were fixed in 10% formalin buffer solution for 3 days,embedded in paraffin, sectioned, and stained with hematoxy-lin and eosin (HE). Tissue images were scanned using a digitalslide scanner (NanoZoomer S210; Hamamatsu Photonics,Hamamatsu, Japan).VML modelVolumetric muscle recovery using dECM microparticle-functio-nalized viscoelastic gels was investigated. Mice (7 week-oldfemale C57BL/6J mice, Jackson Laboratory) were anesthetizedvia inhalation of 2% isoflurane. The hair on the hindlimb ofthe mice was trimmed and disinfected using 70% ethanol. Anincision was made to expose the tibialis anterior (TA) muscleand a defect (5 × 2 × 2 mm3) was created. The viscoelastic gelswith dECM microparticles were mixed using a spatula andapproximately 10 µL was transferred carefully to the defectsite. The skin wounds were sutured for closure, and all micewere intraperitoneally administered amikacin (1 mg kg−1).After 28 days, the mice were euthanized by exsanguination andmuscle tissues were collected. The obtained tissues wereweighed and fixed in 10% formalin buffer for 3 days,embedded in paraffin, sectioned, and stained with HE andMasson’s trichrome (MT). The tissue images were scannedusing a digital slide scanner. Muscle area was quantified fromcross-sectional HE-stained images using ImageJ and the areaof the control sample was set as 100%.Statistical analysisThe results are expressed as the mean ± standard deviation.One-way analysis of variance (ANOVA) followed by Tukey’s mul-tiple comparisons post-hoc test was used to assess differencesamong groups. Experiments were repeated multiple times onindependent occasions. The data shown in each figure arecomplete datasets from representative independent experi-ments. None of the samples were excluded from the analysis.Statistical significance is indicated as *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001. Statistical analyses were per-formed using GraphPad Prism software (version 8.0; GraphPadSoftware, San Diego, CA, USA). Pearson’s correlation analysiswas performed using Google Colab and Python version3.10.12.Results and discussionStress relaxation property in gels with dECM microparticlesLiving tissues and organisms microscopically appear to besolid or elastic, but they are not exactly so.29 They exhibit time-dependent mechanical responses or dissipate partially theenergy applied to deform them. This is known as viscoelasticbehaviour.30 The viscoelasticity of natural tissues is regulatedby the temporal and spatial arrangement of the surroundingECM, which regulates cell behaviors in favor of tissueregeneration.24,29 Therefore, artificial scaffolds designed fortissue regeneration need to possess appropriate mechanicalcharacteristics, especially viscoelastic behavior, together withthe necessary biochemical properties. To develop tissue-regen-erative scaffolds, the dECM was incorporated into gels. ThedECM was prepared by the decellularization and cryo-millingof UBM samples (Fig. 2a). UBM, consisting of the laminapropria and basal lamina, has been suggested to provide a pro-regenerative microenvironment in injured tissues because ofits inherent immunomodulatory properties, such as therecruitment of immune cells and macrophage polarization.31After the decellularization process of the urinary bladder usingPA and DNase treatment, the DNA content decreased from2126 ng mg−1 in the native tissue to 20 ng mg−1 in the dECM(Fig. 2b). This result satisfied the minimal criterion of aremaining DNA level of <50 ng mg−1.36 SEM observationsshowed that the cryo-milled dECM consisted of micrometer-sized fragments with different diameters, which we termeddECM microparticles (Fig. 2c). The results of particle size ana-lysis revealed that the average particle diameter was 9 µm(Fig. 2d).To prepare the gels, two types of gelatin derived fromdifferent sources, skin and tendon (sG and tG), were used asthe main polymers. sG and tG gelatins were chemically modi-fied with thiol and vinyl sulfone groups to obtain sGTH, tGTH,sGVS, and tGVS, respectively. The introduction of the thiol andvinyl sulfone groups was confirmed using the TNBS method(Table 1). The sGTH, sGVS, and tGTH, tGVS solutions weremixed (v/v = 1 : 1) at 37 °C to prepare sGTH–sGVS or tGTH–tGVS gels via a Michael addition reaction. To prepare dECM-Biomaterials Science PaperThis journal is © The Royal Society of Chemistry 2025 Biomater. Sci., 2025, 13, 3576–3584 | 3579Open Access Article. Published on 29 April 2025. Downloaded on 6/25/2025 2:20:25 PM.  This article is licensed under a Creative Commons Attribution-NonCommercial 3.0 Unported Licence.View Article Onlinehttp://creativecommons.org/licenses/by-nc/3.0/http://creativecommons.org/licenses/by-nc/3.0/https://doi.org/10.1039/d5bm00394fmodified gels, dECM microparticles (10 wt%) were mixed withthe gels at 37 °C using a spatula. Rheological measurementsrevealed that the tGTH–tGVS gels possessed viscoelastic pro-perties with rapid stress relaxation, whereas the sGTH-sGVSgels were elastic with minimal stress relaxation (Fig. 2e).Previously, we reported that tG possessed a higher sol–gel tran-sition temperature and stronger non-covalent intermolecularinteractions (e.g., hydrogen bonding) compared to sG, partlybecause sG and tG differ in their molecular structures, includ-ing their molecular weight (191 kDa for sG and 344 kDa fortG) and amino acid compositions.30 Since it has been reportedthat hydrogels made of high-molecular-weight polymers showfaster stress relaxation compared to those made from low-molecular-weight polymers,32 gels composed of higher-mole-cular-weight tG may display rearrangement of their networkstructures under force, showing viscoelastic behaviors. Basedon these results, we refer to sGTH–sGVS gels as “elastic gels”and tGTH–tGVS gels as “viscoelastic gels”.Next, we investigated the effect of incorporating dECMmicroparticles into the gels on their rheological properties.Time-dependent rheological evaluation revealed that theelastic modulus of each gel reached a plateau within 1 h, indi-cating the completion of gel formation (ESI Fig. S1†). Theincorporation of the dECM microparticles also increased gela-tion speed. Importantly, the incorporation of dECM micropar-ticles into the gels modulated the viscoelastic properties of thegels, resulting in faster stress relaxation under a fixed strain of20% for both gels. The half relaxation time (t1/2) of the visco-elastic gels decreased from 5.3 min to 2.6 min and the relax-ation time for the elastic gels changed from undefined to17 min, indicating that the incorporation of the dECM signifi-cantly accelerated the rate of stress relaxation in the gels(Fig. 2f). Previous studies have reported that incorporating col-lagen into gels can improve the stress relaxation rate.10 Theincorporated dECM microparticles may function as fillers inthe gels and dissipate energy under stress by rearranging theirstructures. The incorporation of dECM microparticles into theelastic and viscoelastic gels affected their elastic moduli differ-ently, but the dECM microparticle-incorporated elastic andviscoelastic gels possessed almost the same elastic modulus(Fig. 2g).Enhanced tissue adhesiveness by dECM microparticlesThe tissue adhesion of gels to defects is essential for closingwounds and avoiding the leakage of body fluids or blood. ItFig. 2 (a) Schematic illustration of the decellularization process of sub-mucosal tissues obtained from the porcine urinary bladder to preparedECM microparticles. (b) DNA content of native tissue and dECM (n = 3).(c) Scanning electron microscopy images of dECM microparticles. (d)Distribution of dECM microparticles with different diameter sizes. (e)Rheological measurements of the stress relaxation of gels and dECM-modified gels at 20% strain. The sGTH–sGVS gel and tGTH–tGVS gelwere referred to as the elastic gel and viscoelastic gel, respectively. (f )Quantification of the timescale at which the stress is relaxed to half ofits original value, t1/2, from the stress relaxation tests in (e) (n = 3). (g)Measurements of the elastic modulus of the gels (n = 3). Data are pre-sented as mean ± standard deviation (SD). *P < 0.05, **P < 0.01, and****P < 0.0001, analyzed using a two-tailed Student’s t-test and one-way analysis of variance (ANOVA), followed by Tukey’s multiple compari-sons post hoc test. n.d. and n.s. denote not determined and notsignificant.Table 1 Synthesis of thiolated and vinyl sulfonated gelatin with different degrees of substitutionγ-Thiobutyrolactone(equivalent to amino group in gelatin)Divinyl sulfone(mol% to thiol)Amount of TH(µmol g−1)Amount of VS(µmol g−1) DS (%) Yield (%)sGTH 240 — 232 — 40 88sGVS — 200 1 231 79 85tGTH 220 — 130 — 50 90tGVS — 120 9 121 36 91TH, thiol; VS, vinyl sulfone; DS, degree of substitution; sG, skin-derived gelatin; and tG, tendon-derived gelatin.Paper Biomaterials Science3580 | Biomater. Sci., 2025, 13, 3576–3584 This journal is © The Royal Society of Chemistry 2025Open Access Article. Published on 29 April 2025. Downloaded on 6/25/2025 2:20:25 PM.  This article is licensed under a Creative Commons Attribution-NonCommercial 3.0 Unported Licence.View Article Onlinehttp://creativecommons.org/licenses/by-nc/3.0/http://creativecommons.org/licenses/by-nc/3.0/https://doi.org/10.1039/d5bm00394fcontributes to the promotion of tissue regeneration and theprevention of postoperative complications. The burst strengthsof the gels were evaluated using an adhesion test setup with acollagen casing (Fig. 3a). The dECM microparticle-functiona-lized gels remained intact in the defect of the collagen casingfor a long period of time and revealed a much higher burstpressure than the gels without the dECM (Fig. 3b and c).dECM paste without gels often shows low mechanical strength,poor stability, and rapid degradation, which was also evidentin this study, where the dECM possessed a burst strength of0.4 kPa. In contrast, the incorporation of dECM microparticlesinto gels significantly improved the bulk strength of the gelsand the burst strength because of the combination of chemicalbonding between the thiol–ene reaction and the physical inter-action between collagen fibers present in the dECM micropar-ticles. Furthermore, chemical anchoring with functionalgroups in collagen casings may support interfacial adhesion toimprove the adhesion strength to tissues.31,32 The burststrength increased 2.4-fold in an elastic gel and 2-fold in aviscoelastic gel after incorporating the dECM microparticles byincreasing the cohesive strength of gels through physical inter-actions between the polymer and microparticles. When dECMpowder that was not cryo-milled was incorporated with gels,the burst strength reduced by almost half compared to thatwith dECM microparticles, indicating that the size of thedECM has a great impact on the cohesive strength of gels (ESIFig. S2†). The elastic gel exhibited more improvement instrength because of the presence of a large number of thioland vinyl sulfone groups compared to the viscoelastic gels.Viscoelastic hydrogels may have lower burst strength comparedto purely elastic gels, due to their ability to dissipate energythrough viscous flow rather than maintaining structural integ-rity under sudden stress. Such differences in strength are alsoreported in previous studies.33,34Effect of stress relaxation on cell spreadingFor tissue remodeling and regeneration, it is crucial to designgel scaffolds with tunable viscoelasticity and profoundmechanical strength.35,36 Cell encapsulation tests were per-formed to address the influence of stress relaxation and bio-chemical cues in gels on cellular behavior. Mouse myoblasts(C2C12 cells) were encapsulated and cultured in elastic andviscoelastic gels, with and without dECM microparticles. TheCLSM observations revealed that cell adhesion and spreadingwere suppressed in the elastic gel, which required a longertime to relax the applied stress (Fig. 4a). Although the dECMmicroparticle functionalization of elastic gels improved thetimescale for stress relaxation of elastic gels (t1/2 ∼18 min), themorphology of most of the cells remained round. In contrast,the cell adhesion area and length of the adhered cells signifi-cantly increased in viscoelastic gels with dECM microparticleswith faster stress relaxation (t1/2–3 min) compared to gelswithout the dECM (Fig. 4b and c). Cell proliferation was con-firmed over time in a viscoelastic gel containing the dECM(ESI Fig. S3†). This indicated that C2C12 cell adhesion andspreading were influenced by the viscoelasticity of the matrix.Notably, the incorporation of dECM microparticles into visco-elastic gels significantly improved cell spreading compared tothat of viscoelastic gels alone, indicating that both mechanical(enhanced stress relaxation) and biological (cell-adhesiveligands on the dECM) cues may be associated with cell behav-ior. Although bioactive molecules in dECM-incorporated gelsinfluenced cell adhesion, the length of the elongated cell hada moderate negative correlation (correlation co-efficient: −0.31)with t1/2, suggesting that the faster stress relaxation of thematrix likely supports cell adhesion and spreading (Fig. 4d).Many previous studies have highlighted the importance of gelmatrix remodeling on cell functions.10,29,35 Cells initiallyemploy strain on the gel matrix, and depending on the elasticmodulus, the matrix resists strain and prevents deformation.In this study, the elastic gel showed no relaxation of theapplied forces or a prolonged time. When the matrix structureis chemically crosslinked, cells are unable to spread throughmatrix remodeling, which results in the suppression of cellularactivity.37 Conversely, in viscoelastic gels composed of tGTHand tGVS, the forces can relax over time through physical inter-actions between tG. In addition, the inclusion of dECM micro-particles introduced weak physical interactions between theFig. 3 Tissue-adhesive properties of gels. (a) Setup for the measure-ment of burst strength. (b) Macroscopic images of burst pressuremeasurements. (c) Burst strength of the dECM and elastic gels andviscoelastic gels with dECM microparticles (n = 3). Data are presented asthe mean ± SD. *P < 0.05, ***P < 0.001, and ****P < 0.0001, analyzedusing one-way ANOVA, followed by Tukey’s multiple comparisons posthoc test.Biomaterials Science PaperThis journal is © The Royal Society of Chemistry 2025 Biomater. Sci., 2025, 13, 3576–3584 | 3581Open Access Article. Published on 29 April 2025. Downloaded on 6/25/2025 2:20:25 PM.  This article is licensed under a Creative Commons Attribution-NonCommercial 3.0 Unported Licence.View Article Onlinehttp://creativecommons.org/licenses/by-nc/3.0/http://creativecommons.org/licenses/by-nc/3.0/https://doi.org/10.1039/d5bm00394fcollagen fibers. This contributed to faster stress relaxation andimproved cell adhesion and spreading (Fig. 4e).Host tissue response to viscoelastic gels with dECMmicroparticlesFrom the in vitro analysis, it was determined that the cellslargely adhered to the dECM microparticle-functionalizedviscoelastic gels by exerting strain and remodeling the gelmatrix. To evaluate the host tissue response and cell–matrixinteraction in vivo, a biodegradability test of viscoelastic gelswith and without dECM microparticles was conducted by sub-cutaneous implantation into the dorsal region of mice(Fig. 5a). Irritation and local reaction were not observed at theimplant sites in any group during the course of the study. Forimplantable biomaterial applications, scaffolds must haverobust mechanical properties and degrade at a rate thatmatches the rate of new tissue ingrowth. Macroscopic imagesshowed that the viscoelastic gels swelled on day three anddegraded completely on day seven after implantation (Fig. 5b).In contrast, the viscoelastic gels with dECM microparticlesshowed longer structural stability and were almost completelydegraded after 28 days. Histological observations of the HE-stained tissues revealed that immune cells infiltrated the visco-elastic gels within 3 days, and the gel structures degraded onday 7 (Fig. 5c). In viscoelastic gels containing dECM micropar-ticles, immune cells accumulated on the gel surface and infil-trated the gel over time. The dECM-functionalized gels havesuitable biocompatibility and rates of cell infiltration andmight be useful as scaffolds for regenerative applications, suchas muscle tissue regeneration.Tissue regeneration in VML modelsFinally, we evaluated the therapeutic efficacy of the engineeredgels against VML. VML is a traumatic or surgical injury to theskeletal muscles. Irrecoverable muscle tissue loss often resultsin chronic functional deficits and long-term disability.Although the dECM is a potent therapeutic biomaterial againstVML for structurally and functionally reconstructing muscletissues,35 the suspension of the dECM may detach fromimplantation sites due to poor structural integrity and tissueFig. 4 (a) Confocal laser-scanning microscopy images of cells encap-sulated in elastic and viscoelastic gels with dECM microparticles for24 h. Representative immunofluorescence staining for actin (red) andnuclei (blue). (b) Area of adherent cells in gels (n = 3). (c) Average lengthof cells in the respective gel groups (n = 5). (d) Pearson’s correlationanalysis between the length of the cells and the t1/2 of each samplegroup. (e) Illustrations depicting the possible mechanism whereby theelasticity and stress relaxation of the matrix regulated cellular behaviors.Data are presented as the mean ± SD. *P < 0.05 and **** P < 0.0001 ana-lyzed using one-way ANOVA, followed by Tukey’s multiple comparisonpost hoc test. n.s. denotes not significant.Fig. 5 Evaluation of the biodegradability of the gels with dECM micro-particles after subcutaneous implantation in mice. (a) Schematic depict-ing the implantation of viscoelastic gels, with and without dECM, intothe subcutaneous space of mice. (b) Images of the tissues and visco-elastic gels, with and without the dECM embedded, in tissues at days 3,7, 14, and 28. (c) Histological observation of hematoxylin and eosin–stained images of viscoelastic gels with and without dECM microparti-cles. The asterisk represents remaining gels or their fragments. Scalebars represent 250 µm (top) and 50 µm (bottom).Paper Biomaterials Science3582 | Biomater. Sci., 2025, 13, 3576–3584 This journal is © The Royal Society of Chemistry 2025Open Access Article. Published on 29 April 2025. Downloaded on 6/25/2025 2:20:25 PM.  This article is licensed under a Creative Commons Attribution-NonCommercial 3.0 Unported Licence.View Article Onlinehttp://creativecommons.org/licenses/by-nc/3.0/http://creativecommons.org/licenses/by-nc/3.0/https://doi.org/10.1039/d5bm00394fadhesive properties under physiological conditions. Thus,tissue- and cell-adhesive gels have the potential to improvetherapeutic efficacy against VML. VML defects (5 mm × 2 mm× 2 mm) were created in the TA muscle of the mice, and thegels were implanted for tissue regeneration (Fig. 6a). Twenty-eight days after VML treatment and gel implantation, muscleweight recovered in tissues treated using viscoelastic gels withdECM microparticles compared to tissues treated with shamand viscoelastic gels without the dECM (Fig. 6b). Histologicalobservations of HE-stained tissue sections revealed that visco-elastic gels with dECM microparticles increased the area ofmuscle tissues compared to viscoelastic gels alone (Fig. 6c andd). Moreover, Masson’s trichrome staining showed that thefibrotic area did not increase in muscle tissues treated withviscoelastic gels with dECM microparticles compared to thecontrol. These results indicated that engineered gels withviscoelastic, tissue-adhesive, and cell-adhesive properties canpromote muscle tissue regeneration in VML models. Inaddition to the mechanical features (viscoelasticity and tissueadhesiveness) provided by dECM microparticles, the biochemi-cal features of the dECM, including the presentation of cell-adhesive ligands, the secretion of biological signals, and theinteraction with immune cells, may affect therapeutic efficacy.ConclusionsIn conclusion, this study presents the engineering of a visco-elastic, biocompatible, and tissue-adhesive dECM-based gelscaffold. Thiol–ene crosslinking improved mechanical strengthand tissue adhesion, whereas dECM microparticles providedthe necessary biological cues for cell adhesion and tissue mod-eling. The gelation speed and mechanical properties of theviscoelastic gel were also improved by incorporating dECMmicroparticles. The dECM-based gels had a faster rate of stressrelaxation than the gels themselves, which led to enhancedcell adhesion and spreading. The dECM-modified gels showedhigh biocompatibility when implanted subcutaneously inmice. Thus, dECM-incorporated viscoelastic gels can providetissue regenerative properties through biological interactionswith tissues. This dECM-based viscoelastic gel can be used asa biofunctional and tissue-adhesive scaffold to promote tissueremodeling and regeneration.Data availabilityData generated during the study are available from the corres-ponding author upon request.Conflicts of interestThere are no conflicts to declare.AcknowledgementsWe acknowledge the financial support from the Japan Societyfor the Promotion of Science (JSPS) KAKENHI (grant no.22H03962, 23H01718, 23K26411, and 24K21677) and theUehara Memorial Foundation.References1 M. T. Wolf, Adv. Drug Delivery Rev., 2015, 84, 208–221.2 M. P. Lutolf and J. A. Hubbell, Nat. Biotechnol., 2005, 23,47–55.3 D. O. Freytes, J. Martin, S. S. Velankar, A. S. Lee andS. F. Badylak, Biomaterials, 2008, 29, 1630–1637.4 E. Vorotnikova, Matrix Biol., 2010, 29, 690–700.5 B. N. Brown, J. E. Valentin, A. M. Stewart-Akers,G. P. McCabe and S. F. Badylak, Biomaterials, 2009, 30,1482–1491.6 M. T. Wolf, K. A. Daly, E. P. Brennan-Pierce, S. A. Johnson,C. A. Carruthers, A. 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