# Fileset

[am-2025-14529h.R2_Author Final.pdf](https://mdr.nims.go.jp/filesets/70a71fb0-8394-4f65-9d67-c200353896a8/download)

## Creator

[Yurina Sekine](https://orcid.org/0000-0002-8971-2498), Michiyo Honda, Asako Sugita, [Takuya Nankawa](https://orcid.org/0000-0002-2889-1242), Rimi Sakanaka, Tomoko Ikeda-Fukazawa, [Katsuhiko Ariga](https://orcid.org/0000-0002-2445-2955)

## Rights

This document is the Accepted Manuscript version of a Published Work that appeared in final form in ACS Applied Materials & Interfaces, copyright © 2025 American Chemical Society after peer review and technical editing by the publisher. To access the final edited and published work see https://doi.org/10.1021/acsami.5c14529.[In Copyright](http://rightsstatements.org/vocab/InC/1.0/)

## Other metadata

[Polydopamine-Coating Nanoarchitectonics of Cellulose Hydrogels with Promoted Sustainable and Alkali-Resistant Natures for 3D Cell Culture Application](https://mdr.nims.go.jp/datasets/8c4aa70f-51a5-47d3-b708-bb6305b5f8a3)

## Fulltext

1Polydopamine-Coating Nanoarchitectonics of Cellulose Hydrogels with Promoted Sustainable and Alkali-Resistant Natures for 3D Cell Culture ApplicationYurina Sekine,a,b* Michiyo Honda,c* Asako Sugita,a,b Takuya Nankawa,a,b Rimi Sakanaka,c Tomoko Ikeda-Fukazawa,c Katsuhiko Arigad,ea Laboratory for the Development of Bioresource Technology, Japan Atomic Energy Agency (JAEA), Tokai, Ibaraki 319-1195, Japan. b Promotion office, JAEA, Tokai, Ibaraki 319-1195, Japan. c School of Science and Technology, Meiji University, Kawasaki, Kanagawa 214-8571, Japan. d Research Center for Materials Nanoarchitectonics, National Institute for Materials Science (NIMS), 1-1 Namiki, Tsukuba 305-0044, Japan. e Graduate School of Frontier Sciences, The University of Tokyo, 5-1-5 Kashiwanoha, Kashiwa, Chiba 277-8561, JapanKEYWORDS:  carboxymethyl cellulose nanofibers, hydrogel stability, freeze-drying, heat treatment, polydopamine coating, 3D cell culturePage 1 of 51ACS Paragon Plus EnvironmentACS Applied Materials & Interfaces1234567891011121314151617181920212223242526272829303132333435363738394041424344454647484950515253545556575859602ABSTRACT: Sustainable and chemically resilient hydrogels are critically needed in biointerface engineering, particularly for 3D cell culture systems and surface modification under physiological to mildly alkaline conditions. However, physically crosslinked cellulose nanofiber (CNF) hydrogels—despite their renewable origin and biocompatibility—typically disintegrate at pH > 8, limiting their use in polydopamine (PDA)-based surface functionalization. Here, we present a simple and scalable physical treatment strategy to fabricate alkali-resistant, physically crosslinked hydrogels from carboxymethyl cellulose nanofibers (CMCF). By combining freeze-drying with mild thermal annealing (70 °C), the hydrogel network undergoes partial dehydration and ester bond formation via mild thermal condensation between carboxyl and hydroxyl groups, resulting in enhanced internal densification while preserving porosity and rehydration capacity. The resulting hydrogels remain structurally intact in Tris buffer (pH 9.7) and allow uniform in situ PDA coating at pH 8.5 on a physically crosslinked CMCF scaffold. The PDA-functionalized hydrogels exhibit interconnected porous microstructures, high water content (~97 wt%), and robust mechanical and chemical stability. They support long-term 3D culture of EGFP-expressing human dermal fibroblasts for over 30 days, demonstrating excellent cytocompatibility and deep cell infiltration. This sustainable platform bridges green material design with functional surface engineering under alkaline Page 2 of 51ACS Paragon Plus EnvironmentACS Applied Materials & Interfaces1234567891011121314151617181920212223242526272829303132333435363738394041424344454647484950515253545556575859603conditions, and offers a versatile foundation for applications in regenerative medicine, selective ion adsorption, and environmental remediation.1. INTRODUCTIONThe development of sustainable, biocompatible, and chemically resilient hydrogels is a key challenge in advancing next-generation materials for tissue engineering, biointerfaces, and environmentally friendly systems1-3. As the demand for green and functional soft materials increases, cellulose nanofibers (CNFs) —extracted from renewable biomass—have garnered significant attention due to their biodegradability, high aspect ratio, and tunable surface chemistry4-7. In addition to their favorable biological and mechanical properties, CNFs benefit from global resource abundance and industrial scalability, making them particularly attractive for real-world applications. Particularly, physically crosslinked CNF-based hydrogels formed via hydrogen bonding or ionic interactions exhibit excellent cytocompatibility while avoiding toxic chemical crosslinkers8-11. These features render them ideal for applications requiring environmental sensitivity and biological safety. Such developments align with the broader concept of nanoarchitectonics, which seeks to design functional materials by Page 3 of 51ACS Paragon Plus EnvironmentACS Applied Materials & Interfaces1234567891011121314151617181920212223242526272829303132333435363738394041424344454647484950515253545556575859604organizing nanoscale building blocks into hierarchical architectures bridging molecular and biological interfaces12,13.However, the widespread use of physically crosslinked CNF hydrogels remains limited by their poor structural stability under mildly alkaline conditions (pH>8). In such environments, cellulose networks—regardless of their functional groups—tend to undergo disruption due to hydroxide-induced weakening of hydrogen bonds and, in oxidized CNFs, deprotonation of carboxyl groups (–COOH → –COO⁻), which introduces electrostatic repulsion and further destabilizes the hydrogel matrix4,14,15.This issue is especially critical given the pH environments of many biologically and environmentally relevant systems. For instance, physiological fluids such as blood plasma (pH 7.35–7.45), interstitial fluid, and lymph are slightly alkaline16. Similarly, common biological buffers—including Tris (pH 7.5–8.5), HEPES (pH 7.2–8.2), and bicarbonate-buffered media (pH ~7.4)—are extensively used in 3D cell culture and biointerface experiments. Environmental systems such as natural seawater (pH 8.0–8.3) and alkaline wastewater streams17 further further exemplify the need for hydrogel stability under these conditions.Page 4 of 51ACS Paragon Plus EnvironmentACS Applied Materials & Interfaces1234567891011121314151617181920212223242526272829303132333435363738394041424344454647484950515253545556575859605These stability limitations are particularly detrimental in the context of three-dimensional (3D) cell culture, where hydrogels must serve as both mechanical supports and biologically interactive environments for extended periods. To sustain cell proliferation, migration, and matrix remodeling, such scaffolds must retain high water content, interconnected porosity, and pH stability under near-physiological conditions. Without alkaline-tolerant, cytocompatible, and functionalizable scaffolds, it remains difficult to replicate the dynamic microenvironments required for tissue engineering and organoid culture.A representative use case that highlights this limitation is PDA surface functionalization—a versatile and bioinspired method used to enhance interfacial adhesion, protein immobilization, and cell attachment18-21. Dopamine polymerization is typically carried out at pH ~8.5 in Tris buffer, but physically crosslinked CNF hydrogels generally fail to retain their structure under these conditions, rendering them incompatible with PDA-based coating processes and downstream biofunctionalization strategies.Although some nanocellulose-based systems have demonstrated pH-responsive swelling, their practical use under alkaline conditions remains limited. For instance, Zhang et al.22 reported CNC-reinforced hydrogels that swelled at pH 12 but degraded after a single Page 5 of 51ACS Paragon Plus EnvironmentACS Applied Materials & Interfaces1234567891011121314151617181920212223242526272829303132333435363738394041424344454647484950515253545556575859606swelling–deswelling cycle due to loss of intermolecular interactions in ionic environments. This highlights the challenge of achieving sustainable and reversible structural resilience under basic conditions.Chemical crosslinking has been employed to overcome this issue, but these strategies often compromise sustainability, biocompatibility, or porosity due to the use of harsh reagents or UV curing23-25. Furthermore, they introduce permanent covalent bonds that reduce rehydration capacity—undesirable for 3D tissue culture where nutrient diffusion and cell migration are critical. In contrast, a hydrogel that combines alkaline stability, high porosity, and surface modifiability—without chemical additives—would satisfy the demanding requirements of applications such as organoid models, regenerative scaffolds, and microphysiological systems26.Thus, a key challenge remains: how to endow physically crosslinked CNF hydrogels with robust alkaline stability without compromising their sustainability, porosity, or cytocompatibility. In this work, we present a simple, scalable, and additive-free strategy that combines freeze-drying and mild thermal annealing to fabricate structurally robust hydrogels composed of carboxymethyl cellulose nanofibers (CMCF)27.This post-synthetic treatment induces partial dehydration and thermally promoted ester Page 6 of 51ACS Paragon Plus EnvironmentACS Applied Materials & Interfaces1234567891011121314151617181920212223242526272829303132333435363738394041424344454647484950515253545556575859607bond formation between carboxyl and hydroxyl groups, resulting in enhanced internal densification while preserving water uptake and porosity. The resulting CMCF hydrogels remain stable in Tris buffer (pH 9.7) and uniquely enable in situ PDA coating—a previously unachievable feat for physically crosslinked CNF systems (Figure 1(a)). This platform unlocks new possibilities for long-term 3D cell culture, selective adsorption, and environmentally responsive hydrogel materials.Page 7 of 51ACS Paragon Plus EnvironmentACS Applied Materials & Interfaces1234567891011121314151617181920212223242526272829303132333435363738394041424344454647484950515253545556575859608Figure 1. (a) Schematic illustration of the fabrication strategy for alkali-resistant, PDA-compatible CMCF–CA hydrogels and their application in long-term 3D fibroblast culture. The design leverages physical crosslinking, freeze-drying, and mild heating to achieve structural stability under mildly alkaline conditions, enabling in situ surface functionalization and sustained biocompatibility. (b) Molecular structure illustration showing CA-crosslinked CMCF and PDA coating layers. (c) Photograph and SEM image of PDA-CMCF-CA hydrogel.2. EXPERIMENTAL SECTION2.1 MaterialsAn aqueous CMCF sol (2 wt%, TFo-100002) prepared using a water jet system was purchased from Sugino Machine Limited, Japan28. CA, Dulbecco's Modified Eagle Medium (DMEM), fetal bovine serum (FBS), and penicillin–streptomycin (P/S) solution (×100), sodium hydroxide (NaOH), sodium bicarbonate (NaHCO3), and HEPES were purchased from Fujifilm Wako Pure Chemical Co., Ltd., Japan. Tris (Ultra pure, 99.9%) was purchased from MP Biomedicals, LLC, USA. 10× PBS Buffer was purchased from Nippon Gene Co., Ltd., Japan. Hydrochloric acid (HCl) (1 mol L–1) was purchased from Page 8 of 51ACS Paragon Plus EnvironmentACS Applied Materials & Interfaces1234567891011121314151617181920212223242526272829303132333435363738394041424344454647484950515253545556575859609Hayashi Pure Chemical Ind., Ltd., Japan. 3-Hydroxytyamine hydrochloride for PDA coating was purchased from Tokyo Chemical Industry Co., Ltd., Japan. All materials were used as received without further purification. 2.2 Preparation of hydrogel samplesThe hydrogel samples were prepared by freeze-crosslinking using CMCF and CA as a non-toxic crosslinker, following a previously reported procedure with slight modifications27,29. A 2 wt% CMCF aqueous sol was poured into a mold (1.5 mm in height) and frozen at –20°C for 24 hours. The frozen CMCF was subsequently immersed in 1 mol L–1 CA aqueous solution and thawed at 4°C for 24 hours to allow diffusion and crosslinking. The resulting hydrogels were washed repeatedly with Milli-Q water to remove residue for 7 days. The purified hydrogels were punched into disks with a diameter of 8 mm and designated as swollen CMCF-CA Hydrogels. The swollen hydrogels were freeze-dried for 48 hours using a freeze dryer (EYELA, FDU-1100, Japan) to obtain porous sponges, designated as freeze-dried CMCF-CA samples. A subset of these freeze-dried samples was further heated in an oven at 70°C for Page 9 of 51ACS Paragon Plus EnvironmentACS Applied Materials & Interfaces123456789101112131415161718192021222324252627282930313233343536373839404142434445464748495051525354555657585960104, 24, or 48 hours in air to induce partial dehydration and esterification. These were labeled as Heated freeze-dried CMCF-CA-4h, -24h, and -48h, respectively.The water content of the hydrogel was determined using the following equation:Water content (%) = (Wsgel − Wdgel) / Wsgel × 100 (1)where Wsgel (g) and Wdgel (g) are the weight of swollen hydrogel and dried hydrogel, respectively. The dried hydrogel (Wdgel) was obtained by drying the sample at 90 °C for more than 48 hours.2.3 Stability testStability tests were conducted to evaluate the dissolution behavior and long-term stability of the hydrogels under three different pH conditions: Strongly acidic conditions were simulated using HCl (1 mol L–1, pH 0.04) to provide a harsh environment for accelerated degradation. Near-physiological conditions were examined in PBS (0.5 mol L–1, pH 7.6), in which the high ionic strength and buffering capacity ensured a conservative assessment of scaffold durability. Alkaline conditions were tested in Tris buffer (0.01 mol L–1, pH 9.7), corresponding to the standard medium used for dopamine polymerization, which represents the intended downstream application of the material. Page 10 of 51ACS Paragon Plus EnvironmentACS Applied Materials & Interfaces12345678910111213141516171819202122232425262728293031323334353637383940414243444546474849505152535455565758596011For each test, a single hydrogel disk (8 mm in diameter) was immersed in 2 mL of the respective solution and stirred at room temperature using a rotor at 100 rpm to simulate continuous agitation. The dissolution behavior was monitored by observing the time required for complete dissolution in each solution. The hydrogel’s stability was assessed at regular intervals (1 hour, 24 hours, 7 days, and up to 30 days), noting whether the structure remained intact or showed signs of disintegration. Untreated (swollen), freeze-dried, and heat-treated hydrogels were tested under identical conditions to enable direct comparison.2.4 Polydopamine coatingHeated freeze-dried CMCF-CA-48h disks (20 mg) were first immersed in Tris buffer (0.01 mol L–1, 20 mL) for 24 h to allow sufficient rehydration. After this pre-equilibration step, 40 mg of 3-hydroxytyramine hydrochloride was added to the same solution. Following previously reported protocols for PDA deposition under mildly alkaline conditions, the pH was adjusted to 8.5, and the mixture was stirred vigorously for 2 hours at room temperature30. After coating, the samples were rinsed thoroughly with Milli-Q water to remove unreacted chemical. These were labeled PDA-CMCF-CA (Figure 1(b), (c)).2.5 Cell Culture ExperimentsPage 11 of 51ACS Paragon Plus EnvironmentACS Applied Materials & Interfaces12345678910111213141516171819202122232425262728293031323334353637383940414243444546474849505152535455565758596012To evaluate the biocompatibility and 3D cell culture potential, freeze-dried PDA-CMCF-CA hydrogels were sterilized by autoclaving at 121°C for 20 minutes. Sterilized samples were immersed in reconstruction buffer (NaOH (50 mmol L–1), NaHCO3 (260 mmol L–1), and HEPES (200 mmol L–1)) for 24 hours. Following thorough washing with PBS buffer, the samples were transferred to complete culture medium (DMEM supplemented with 10% FBS and 1% P/S) and incubated for 48 hours at 37°C to allow equilibration. Subsequently, normal human dermal fibroblasts (NHDFs) expressing EGFP were seeded onto the hydrogels and cultured for over 30 days under standard conditions (37°C, 5% CO₂, humidified atmosphere). The culture medium was refreshed every 2–3 days to maintain optimal conditions for cell growth. EGFP expression was monitored over time as an indicator of cell proliferation using confocal laser scanning microscopy (CLSM; STELLARISS5, Leica Ltd., Germany).2.6 CharacterizationScanning electron microscopy (SEM) images were obtained using a scanning electron microscope (JSM-6010PLUS/LA, JEOL Ltd., Japan). The Fourier transform infrared (FTIR) spectra were measured at room temperature using a spectral reflectometer (FT/IR-4X JASCO, Japan) with a diffuse reflection unit (ATR PRO 4X, JASCO). The spectra Page 12 of 51ACS Paragon Plus EnvironmentACS Applied Materials & Interfaces12345678910111213141516171819202122232425262728293031323334353637383940414243444546474849505152535455565758596013were analyzed by the IGOR pro software (Wavemetrics, Inc., USA). Powder X-ray diffraction (PXRD) measurements were performed using an X-ray diffractometer (MiniFlex600, Rigaku Co., Ltd., Japan) with a Cu-K radiation ( = 0.15418 nm) at room temperature. The diffraction data were collected in steps of 0.02° at 2 angles between 5° and 50°. Thermo gravimetry analysis (TGA) was carried out using an analyzer (TG-DTA, TG-8120, Rigaku Co., Japan). For compressive tests using a creep meter (EZ-LX, Shimadzu Co., Ltd., Japan), swollen cylindrical gel samples (20 mm in diameter and 10 mm in height) were compressed to a strain of 0.8 with a cylindrical probe (20 mm diameter) at a cross-probe speed of 1 mm s−1. The measurements were repeated more than three times. The pH of samples was measured by a pH meter (SPH70, As ONE Co., Ltd., Japan) at room temperature.3. RESULTS AND DISCUSSION3.1. Dissolution Behavior ComparisonThe dissolution behavior of CMCF-CA hydrogels subjected to different treatments was systematically evaluated in three aqueous environments: HCl (1 mol L–1, pH 0.04), PBS Page 13 of 51ACS Paragon Plus EnvironmentACS Applied Materials & Interfaces12345678910111213141516171819202122232425262728293031323334353637383940414243444546474849505152535455565758596014(0.5 mol L–1, pH 7.6), and Tris buffer (0.01 mol L–1, pH 9.7). Figure 2 and S1 summarizes the morphological stability of the hydrogels under each condition.As shown in Figure 2(a), the swollen CMCF-CA hydrogel exhibited excellent stability in the strongly acidic HCl solution, maintaining its shape for over 30 days. However, in PBS buffer, the gel gradually swelled and disintegrated within 24 hours. In Tris buffer, rapid dissolution was observed, and the hydrogel completely lost its structure within 15 minutes. These results confirm that the physically crosslinked hydrogen-bonded network is highly susceptible to disruption under neutral and alkaline conditions. The freeze-dried CMCF-CA hydrogel (Figure 2(b)) showed moderately improved stability. It remained intact for over 30 days in HCl, for 48 hours in PBS before collapsing, and for approximately 30 minutes in Tris buffer. This suggests that freeze-drying introduces structural densification that partially resists dissolution, particularly in neutral environments. In contrast, the heated freeze-dried CMCF-CA-48h (Figure 2(c)) demonstrated markedly enhanced resistance. The sample retained its shape for over 30 days in both HCl and PBS, and remained visibly intact in Tris buffer for up to 7 days. Complete dissolution occurred only after 18 days, indicating that the combined treatment significantly strengthened the hydrogel’s network, even under alkaline conditions.Page 14 of 51ACS Paragon Plus EnvironmentACS Applied Materials & Interfaces12345678910111213141516171819202122232425262728293031323334353637383940414243444546474849505152535455565758596015The quantitative effect of heating duration on alkaline stability is summarized in Figure 2(d). Hydrogels heated for longer periods (up to 48 hours at 70 °C) exhibited a progressive increase in resistance to dissolution in Tris buffer. The time to complete dissolution increased from less than 1 hour (0 h heating) to nearly 480 hours (48 h heating), demonstrating a time-dependent stabilization effect likely due to gradual dehydration and hydrogen-bonding network rearrangement31. To further assess whether stabilization continue beyond 48 h, additional experiments were performed on 72 h-heated samples under an accelerated alkaline stress condition (0.5 mol L–1, pH 10.7). These samples showed dissolution behavior essentially identical to the 48 h-treated hydrogels, both degrading completely within ~12 hours (Figure S2). The result indicated that the stabilization effect reaches a practical plateau at 48 h, and prolonged heating does not yield further improvement.These results demonstrate that while native swollen hydrogels are highly sensitive to pH changes, particularly in neutral to alkaline conditions, freeze-drying enhances short-term structural retention, and additional mild heating dramatically improves durability by stabilizing the internal network.Page 15 of 51ACS Paragon Plus EnvironmentACS Applied Materials & Interfaces12345678910111213141516171819202122232425262728293031323334353637383940414243444546474849505152535455565758596016Figure 2 Dissolution behavior of CMCF-CA hydrogels subjected to different treatments under acidic, neutral, and alkaline conditions. (a–c) Photographs of swollen (a), freeze-dried (b), and heat-treated freeze-dried (c) hydrogels after immersion in HCl (1 mol L–1, pH 0.04), PBS (0.5 mol L–1, pH 7.6), and Tris buffer (0.01 mol L–1, pH 9.7) at selected time points. (d) Time to complete dissolution of heat-treated hydrogels in Tris buffer (0.01 mol L–1) as a function of heating duration at 70 °C.3.2. Mechanistic Analysis of Dissolution BehaviorThe internal microstructures of the CMCF-CA hydrogels were examined by SEM to elucidate the morphological changes associated with pH-dependent dissolution behavior. Page 16 of 51ACS Paragon Plus EnvironmentACS Applied Materials & Interfaces12345678910111213141516171819202122232425262728293031323334353637383940414243444546474849505152535455565758596017The original CMCF sol exhibited a randomly entangled nanofiber network, with relatively uniform fiber dispersion and no large-scale porous structures32. After freeze-crosslinking with CA, the resulting CMCF-CA hydrogel showed a well-defined porous structure (Figure S3). When this freeze-dried hydrogel was immersed in HCl solution, its porous network remained highly intact, as shown in Figures 3(a) and (b). The structure exhibited interconnected pores ranging from several tens to hundreds of micrometers, indicating that the acidic environment preserved the hydrogen-bonded gel framework without significant degradation. In contrast, the freeze-dried hydrogel immersed in Tris buffer exhibited a markedly altered morphology (Figures 3(c) and (d)). The original porous architecture had collapsed, and the surface appeared fibrous and fragmented, lacking the regular network observed under acidic conditions. This morphological change supports the hypothesis that alkaline conditions disrupt intermolecular hydrogen bonding, leading to partial dissolution and loss of hydrogel integrity. These SEM observations reinforce the dissolution results and suggest that the network stability of CMCF-CA hydrogels is strongly influenced by pH, particularly through the preservation or disruption of hydrogen bonding within the structure.Figure 3(e) presents the FTIR spectra of freeze-dried CMCF-CA hydrogels after immersion in aqueous solutions of different pH. To investigate molecular-level changes Page 17 of 51ACS Paragon Plus EnvironmentACS Applied Materials & Interfaces12345678910111213141516171819202122232425262728293031323334353637383940414243444546474849505152535455565758596018that correlate with hydrogel stability, attention was focused on the C=O stretching region (1500–1800 cm–1) and the O–H stretching region (3000–3500 cm–1). Prior to immersion, the freeze-dried CMCF-CA hydrogel exhibited two distinct peaks at 1639.2 cm⁻¹ and 1726.0 cm–1, which are assigned to C=O stretching vibrations of strongly hydrogen-bonded and weakly or non–hydrogen-bonded carboxylic acid groups, respectively33,34. These peaks reflect the presence of different hydrogen bonding environments within the crosslinked network. Upon immersion in PBS (pH 7.6) and Tris buffer (pH 9.7), both peaks shifted toward 1584–1590 cm–1. This shift is indicative of carboxyl group deprotonation and the formation of carboxylate anions (COO–), reflecting a transition from hydrogen-bonded or neutral carboxylic acids to ionized species35,36. The shift was more pronounced in the Tris-buffer-immersed sample, where the major peak appeared around 1584 cm–1, suggesting a greater extent of deprotonation under the more alkaline condition. These spectral changes support the interpretation that neutral and basic environments promote ionization of carboxylic groups, which weakens the hydrogen bonding network and corresponds with the increased dissolution behavior observed under these pH conditions.Furthermore, analysis of the O–H stretching region revealed additional insights. The freeze-dried CMCF-CA hydrogel prior to immersion displayed a broad O–H band, Page 18 of 51ACS Paragon Plus EnvironmentACS Applied Materials & Interfaces12345678910111213141516171819202122232425262728293031323334353637383940414243444546474849505152535455565758596019characteristic of extensive hydrogen bonding15. After immersion in PBS and Tris buffer, the O–H stretching band became broader, indicating increased interactions with water molecules as the network structure loosened. In contrast, the hydrogel immersed in HCl maintained a sharper O–H peak, reflecting a well-maintained hydrogen-bonded structure. These FT-IR results demonstrate that the stability and dissolution behavior of the hydrogels are closely related to the ionization state of the carboxyl groups and the integrity of hydrogen bonding. Neutral and alkaline environments promote carboxylate formation and weaken the hydrogel network, while acidic conditions help preserve hydrogen bonds, contributing to long-term stability.The structural changes of freeze-dried CMCF-CA hydrogels after immersion in different pH environments were investigated using PXRD, and the results are shown in Figure 3(f). The analysis focuses on how immersion in HCl, PBS, and Tris solutions affects the crystalline structure and overall ordering of the hydrogels. The XRD profiles of initial freeze-dried CMCF-CA hydrogel contained three characteristic peaks at around 2 = 12°, 20°, and 22°, which are observed in the profile of cellulose type II37. After immersion in HCl solution (pH 0.04) for 30 days and PBS (pH 7.6) for 48 hours, the PXRD pattern remained largely unchanged, with the cellulose II peaks clearly preserved. This result suggests that the acidic and neutral environment does not disrupt the crystalline structure. Page 19 of 51ACS Paragon Plus EnvironmentACS Applied Materials & Interfaces12345678910111213141516171819202122232425262728293031323334353637383940414243444546474849505152535455565758596020In contrast, hydrogels immersed in Tris buffer (pH 9.7) showed significant changes. The intensity of the diffraction peaks decreased, and the peaks became broader, indicating partial or complete loss of the ordered structure. This reduction in crystallinity is attributed to the swelling and dissolution of the network in alkaline environments, where carboxyl group ionization disrupts hydrogen bonding, as corroborated by FTIR analysis.Page 20 of 51ACS Paragon Plus EnvironmentACS Applied Materials & Interfaces12345678910111213141516171819202122232425262728293031323334353637383940414243444546474849505152535455565758596021Figure 3 (a,b) SEM images of freeze-dried CMCF–CA hydrogel after immersion in 1 mol L⁻¹ HCl for 30 days (low (a) and high (b) magnification) and after immersion in Tris buffer for 30 minutes (low (c) and high (d) magnification). (e) FTIR spectra of freeze-dried CMCF–CA hydrogels before (red) and after immersion in HCl for 30 days (blue), PBS buffer for 48 hours (brown), and Tris buffer for 30 minutes (green). (f) PXRD Page 21 of 51ACS Paragon Plus EnvironmentACS Applied Materials & Interfaces12345678910111213141516171819202122232425262728293031323334353637383940414243444546474849505152535455565758596022patterns of the same samples as in (e). The filled circles represent the diffraction peaks attributed to cellulose II.3.3. Structural Changes Induced by Freeze-Drying and Heat TreatmentThe effects of freeze-drying and subsequent heat treatment on the structural characteristics of CMCF-CA hydrogels were investigated using SEM, FTIR, TGA, water content analysis, internal pH measurements, and mechanical testing. As shown in Figures 4(a), (b) and S3, SEM images revealed that both freeze-dried and heat-treated hydrogels retained a porous, interconnected network structure. No noticeable differences in macroscopic morphology or pore size were observed even after heating at 70 °C, indicating that macroporous morphology was preserved despite molecular rearrangements. PXRD patterns (Figure S4(a)) also showed no shift in peak position or intensity following heat treatment, confirming that the crystallinity of the cellulose framework was preserved. These findings suggest that stabilization occurs primarily at the molecular level rather than through changes in long-range order or bulk morphology.In contrast, FTIR analysis (Figure 4(c)) revealed molecular-level changes in the carbonyl region. The C=O stretching band near 1726 cm–1—typically associated with ester or non-hydrogen-bonded carboxyl groups—exhibited a gradual blue shift with increasing Page 22 of 51ACS Paragon Plus EnvironmentACS Applied Materials & Interfaces12345678910111213141516171819202122232425262728293031323334353637383940414243444546474849505152535455565758596023heating time: from 1726.0 cm–1 (0 and 4 h) to 1726.9 cm–1 (24 h), 1727.9 cm–1 (48 h), and 1729.0 cm–1 (90 °C). Simultaneously, a progressive decrease in band intensity suggests a reduction in hydrogen-bonded carbonyls. Notably, the absorbance at ~1640 cm–1—attributable to strongly hydrogen-bonded carboxylic acids—also diminished upon heating, indicating disruption of hydrogen-bond networks. These spectral changes support a scenario where thermal treatment promotes dehydration and intramolecular condensation between carboxyl and hydroxyl groups, yielding ester linkages. This structural rearrangement, including formation of ester-like carbonyl groups, likely arises from thermal dehydration reactions conducted under aerobic conditions. Such esterification processes lead to carbonyls with higher stretching frequencies, consistent with prior studies38,39. A lower temperature (50–60 °C), by contrast, only minimal spectral shifts were observed (data not shown), indicating insufficient dehydration and ester bond formation. These results highlight that 70 °C provides the optimal balance, where stabilization occurs effectively while maintaining porosity and rehydration capacity. Furthermore, heating at temperatures above 90 °C is expected to cause undesirable thermal degradation and structural denaturation of cellulose network, which would compromise the material’s mechanical robustness and biocompatibility. Page 23 of 51ACS Paragon Plus EnvironmentACS Applied Materials & Interfaces12345678910111213141516171819202122232425262728293031323334353637383940414243444546474849505152535455565758596024Differential spectra (Figure 4(d)) further support this interpretation, showing increased absorbance near 1729 cm–1 and decreased intensity at ~1688 cm–1 and ~1640 cm–1. These changes suggest a reduction in hydrogen-bonded carboxylic acid groups and the formation of more thermodynamically stable ester carbonyls, which absorb at higher frequencies due to the absence of hydrogen bonding. Importantly, the O–H stretching region (3000–3600 cm–1; Figure S3(b)) remained largely unchanged, indicating that the overall hydroxyl content was preserved and that stabilization arises from local condensation reactions—likely esterification—between hydroxyl and carboxyl groups.Water content measurements (Figure 4(e)) revealed that both freeze-dried and heat-treated hydrogels reached similarly high water contents (~97 wt%) upon rehydration, nearly matching the swollen state (~98 wt%, red diamond). The green and blue curves show that water was readily absorbed by both untreated and heat-treated hydrogels. These findings indicate that heat treatment preserved the hydrogel’s rehydration capacity and water permeability without inducing hydrophobic domains. Thus, the enhanced resistance to alkaline dissolution is not due to limited water uptake, but rather to internal structural reorganization, including densification and ester bond formation. Page 24 of 51ACS Paragon Plus EnvironmentACS Applied Materials & Interfaces12345678910111213141516171819202122232425262728293031323334353637383940414243444546474849505152535455565758596025TGA results (Figure 4(f)) showed that weight loss below 100 °C was primarily due to evaporation of residual water, with negligible degradation observed up to 150 °C. Although the mass loss was minor, it likely facilitated local molecular rearrangements by disrupting hydrogen bonds and enabling condensation reactions such as esterification between carboxyl and hydroxyl groups. This mechanism aligns with the FTIR findings indicating a shift from hydrogen-bonded to non-hydrogen-bonded carbonyl species.Figure 4(g) presents a schematic illustration of the proposed structural changes. Before heating, the hydrogel contains abundant water and extensive hydrogen bonding interactions. Upon heating, partial dehydration reduces hydrogen bonding and promotes the formation of ester linkages between hydroxyl and carboxyl groups, leading to internal crosslinking and stabilization of the hydrogel structure—without compromising high water content.Internal pH measurements further support this interpretation. The pH of rehydrated hydrogels slightly increased from 2.9 (freeze-dried) to 3.0 (70 °C-treated), consistent with partial esterification of acidic carboxyl groups, which reduces the number of proton-releasing moieties. This chemical change contributes to improved pH stability under neutral and alkaline conditions. Mechanical testing demonstrated that heat treatment Page 25 of 51ACS Paragon Plus EnvironmentACS Applied Materials & Interfaces12345678910111213141516171819202122232425262728293031323334353637383940414243444546474849505152535455565758596026enhances mechanical durability. The Young’s modulus increased from 4.1 ± 0.9 kPa (freeze-dried) to 6.4 ± 1.0 kPa (70 °C-treated), indicating increased crosslink density and network rigidity.In summary, freeze-drying combined with mild heat treatment induces molecular-level reorganization—particularly within the carbonyl domain—without altering macroscopic morphology or crystallinity. This simple post-processing strategy promotes partial esterification and improves hydrogel stability, mechanical strength, and resistance to alkaline environments, offering a robust and scalable approach for fabricating cellulose-based hydrogels suitable for biomedical and environmental applications.Page 26 of 51ACS Paragon Plus EnvironmentACS Applied Materials & Interfaces12345678910111213141516171819202122232425262728293031323334353637383940414243444546474849505152535455565758596027Figure 4 (a,b) SEM images of heat-treated freeze-dried CMCF–CA hydrogels at low (a) and high resolution (b). (c) FTIR spectra in the carbonyl region (1535–1835 cm–1) before (red dashed line) and after heat treatment at 70 °C for 4 h (green), 24 h (light blue), 48 h (pink), and at 90 °C for 48 h (dark blue), showing progressive blue shift and intensity Page 27 of 51ACS Paragon Plus EnvironmentACS Applied Materials & Interfaces12345678910111213141516171819202122232425262728293031323334353637383940414243444546474849505152535455565758596028changes. (d) Differential FTIR spectra obtained by subtracting the freeze-dried CMCF–CA spectrum from each heat-treated sample, highlighting loss of hydrogen-bonded carboxylic groups and emergence of ester-type carbonyls. (e) Water content of swollen (red diamond), freeze-dried (green hollow circle), and heat-treated (blue filled circles) hydrogels during rehydration. (f) TGA of freeze-dried CMCF–CA. (g) Schematic illustration of heat-induced molecular changes, including partial dehydration and ester bond formation between –COOH and –OH groups.3.4. PDA Coating and Cell Culture PerformanceTo evaluate whether the thermally stabilized CMCF–CA hydrogel could support chemical functionalization under biologically relevant conditions, we performed in situ PDA coating under mildly alkaline conditions (pH 8.5, Tris buffer). PDA is a widely used surface modifier that improves biointerface properties through catechol and amine functionalities19. As anticipated, only the heat-treated freeze-dried hydrogels retained their structure during the dopamine polymerization process, while unheated or swollen gels disintegrated. This result confirms that thermal stabilization is essential not only for alkaline durability but also for enabling surface modification without chemical crosslinkers.Page 28 of 51ACS Paragon Plus EnvironmentACS Applied Materials & Interfaces12345678910111213141516171819202122232425262728293031323334353637383940414243444546474849505152535455565758596029PDA deposition was visually evident from the uniform browning of the hydrogel (Figure 5(a)). Upon rehydration, the PDA–CMCF–CA hydrogels retained their structural integrity and exhibited high water content (~97 wt%) (Figure 1(c)), even after autoclave sterilization. SEM images (Figure 1(c)) revealed a well-preserved porous microstructure, comparable to the uncoated gels (Figure S3), with no aggregation or roughening, indicating uniform PDA coating throughout the matrix. FTIR spectra (Figure 5(b)) displayed new peaks at 1520 cm–1 and 1578 cm–1, corresponding to C=N and aromatic C=C stretching vibrations of PDA40, confirming successful in situ polymerization.To assess environmental stability, we monitored pH within the hydrogel and in the surrounding medium over 72 hours (Figure S5). Both values remained near pH 8.0 with minimal variation, suggesting that the scaffold does not leach acidic or basic components and can maintain a chemically equilibrated internal environment. The mild buffering capacity of PDA likely contributes to this pH stability.As a representative cell type for dermal tissue models, NHDFs were used to assess whether the scaffold could support long-term viability and infiltration in three-dimensional culture. Notably, no cell attachment was observed on unmodified CMCF–CA hydrogels, highlighting the intrinsic lack of cell-adhesive functionality in the native Page 29 of 51ACS Paragon Plus EnvironmentACS Applied Materials & Interfaces12345678910111213141516171819202122232425262728293031323334353637383940414243444546474849505152535455565758596030material (Figure S5). In contrast, cells adhered and spread across the PDA-coated scaffold by day 14 (Figures 5(c) and (d)), and continued to proliferate over 30 days, with uniform distribution in the 3D matrix (Figure S6). Fibroblasts maintained a normal spindle-shaped morphology throughout the culture period, with no signs of cytotoxicity, aggregation, or detachment. Importantly, the scaffold maintained its structural integrity throughout the entire culture period. These results demonstrate that PDA–CMCF–CA scaffolds provide a supportive microenvironment for long-term dermal cell culture and may serve as a promising platform for skin tissue engineering or wound healing models.To benchmark the performance of the PDA–CMCF–CA hydrogel, we systematically compared its material origin, structural characteristics, and functional attributes with three widely used scaffolds—collagen41,42, GelMA43,44, and PEGDA45,46—as summarized in Table 1. In contrast to animal-derived or synthetic scaffolds, the PDA–CMCF–CA hydrogel is fully plant-based and fabricated via an additive-free route involving CA–mediated physical crosslinking. This green approach yields highly porous and rehydratable architectures without the need for toxic reagents or initiators. The versatility of cellulose-based materials in maintaining structural integrity, porosity, and biocompatibility has also been demonstrated in bacterial cellulose composites, such as BC/Hap/GO scaffolds developed for bone tissue engineering41, and further supported by Page 30 of 51ACS Paragon Plus EnvironmentACS Applied Materials & Interfaces12345678910111213141516171819202122232425262728293031323334353637383940414243444546474849505152535455565758596031recent reviews highlighting sustainable nanocellulose-based biomaterials for tissue regeneration47,48. These examples reinforce the potential of plant-derived scaffolds as green, high-performance alternatives to traditional animal- or synthetic-based systems.A key distinguishing feature is the incorporation of a mild thermal treatment step, which induces partial dehydration and the formation of ester-like carbonyl groups with reduced hydrogen-bonding. This structural densification enhances alkaline resistance (stable in Tris buffer at pH 9.7) while maintaining high water uptake (~97 wt%). This modification is critical for enabling in situ PDA coating, which is typically unachievable in physically crosslinked CNF-based gels due to their instability in mildly basic media. In contrast, collagen and GelMA scaffolds are pH-sensitive and require chemical or photo-initiated crosslinking, while PEGDA, though chemically robust, suffers from low porosity and poor sustainability. Collectively, these comparisons underscore a key limitation in conventional scaffolds: none concurrently achieve sustainability, alkali resistance, and long-term cytocompatibility.The PDA–CMCF–CA hydrogel, in contrast, supports 3D culture of fibroblasts for over 30 days without structural degradation, demonstrating superior cytocompatibility and deep cellular infiltration throughout the matrix. These results highlight its potential as a Page 31 of 51ACS Paragon Plus EnvironmentACS Applied Materials & Interfaces12345678910111213141516171819202122232425262728293031323334353637383940414243444546474849505152535455565758596032multifunctional scaffold that bridges sustainable design, chemical stability, and interfacial bioactivity—an integration rarely achieved in a single hydrogel system.Page 32 of 51ACS Paragon Plus EnvironmentACS Applied Materials & Interfaces12345678910111213141516171819202122232425262728293031323334353637383940414243444546474849505152535455565758596033Figure 5 (a) Photographs of freeze-dried PDA–CMCF–CA hydrogel. (b) FTIR spectra of freeze-dried PDA–CMCF–CA (brown) and freeze-dried CMCF–CA (green). (c) Fluorescence (left) and merged (right) images of EGFP-expressing fibroblasts cultured on the hydrogel for 14 days. (d) Confocal 3D reconstruction showing cellular infiltration and distribution throughout the hydrogel matrix. The Z-axis images as pseudocolors show three-dimensional distribution of EGFP.Page 33 of 51ACS Paragon Plus EnvironmentACS Applied Materials & Interfaces12345678910111213141516171819202122232425262728293031323334353637383940414243444546474849505152535455565758596034Table 1. Comparative summary of material origin, structural properties, and functional performance of PDA–CMCF–CA hydrogels and representative scaffold materials. "Alkaline stability" and "PDA compatibility" are based on experimental evaluations in this work. Other data are derived from literature benchmarks41-46.To provide a comprehensive visual comparison, we constructed a radar chart (Figure 6) evaluating six critical criteria: sustainability, alkali stability, PDA compatibility, porosity, cytocompatibility, and long-term 3D culture support. Each attribute was rated on a 5-point scale based on experimental results (e.g., immersion tests, PDA coating success, SEM porosity, and fibroblast culture longevity) and literature benchmarks.Page 34 of 51ACS Paragon Plus EnvironmentACS Applied Materials & Interfaces12345678910111213141516171819202122232425262728293031323334353637383940414243444546474849505152535455565758596035PDA–CMCF–CA exhibits consistently high scores across all categories (5/5), reflecting its rare combination of environmental, chemical, and biological functionality. By contrast, collagen excels in cytocompatibility but lacks alkali stability and sustainability; GelMA offers moderate PDA compatibility but relies on UV crosslinking and degrades over time; PEGDA demonstrates chemical robustness but suffers from low porosity and lacks biodegradability due to its synthetic origin.The superior performance profile of PDA–CMCF–CA stems from its plant-based, additive-free fabrication and mild post-treatment strategy, yielding a porous yet chemically resilient scaffold capable of supporting PDA modification and long-term 3D culture. As such, Figure 6 highlights its unique position among current hydrogel technologies and supports its potential as a next-generation bioactive scaffold platform.Page 35 of 51ACS Paragon Plus EnvironmentACS Applied Materials & Interfaces12345678910111213141516171819202122232425262728293031323334353637383940414243444546474849505152535455565758596036Figure 6 Comparative radar chart illustrating key performance metrics of PDA–CMCF–CA hydrogel and representative hydrogel materials (Collagen, GelMA, PEGDA). The evaluation criteria include sustainability, alkali stability, PDA compatibility, porosity, cytocompatibility, and 3D culture support. Scores are based on literature evidence and experimental results detailed in Table 1.4. CONCLUSIONSIn this study, we developed a sustainable and alkali-resistant cellulose hydrogel by combining freeze-drying and mild thermal treatment of CMCF. The resulting physically crosslinked scaffold exhibited high porosity, mechanical integrity, and excellent water retention even under mildly alkaline conditions. Spectroscopic and thermal analyses revealed that the thermal treatment promoted internal densification via partial dehydration and ester bond formation between hydroxyl and carboxyl groups, enhancing chemical stability.Unlike conventional CNF-based hydrogels that readily dissolve under such conditions, our thermally stabilized system enabled in situ PDA coating without requiring chemical crosslinkers or photoinitiators. The PDA–CMCF–CA hydrogels supported long-term 3D Page 36 of 51ACS Paragon Plus EnvironmentACS Applied Materials & Interfaces12345678910111213141516171819202122232425262728293031323334353637383940414243444546474849505152535455565758596037culture of human dermal fibroblasts for over 30 days, highlighting their cytocompatibility, structural robustness, and biofunctionality.Beyond 3D cell culture, this hydrogel platform provides a generalizable strategy to develop multifunctional and environmentally responsive biomaterials—all without compromising sustainability. Its chemical stability under alkaline conditions opens pathways for downstream surface functionalization, metal ion adsorption, and catalytic reactions in aqueous media—processes often incompatible with conventional hydrogels. Furthermore, the mild, additive-free fabrication process supports sterilization and scalability, making the material attractive not only for regenerative medicine and tissue engineering, but also for smart filtration, wearable sensors, and radionuclide capture. By bridging the gap between sustainable material design and functional performance, this work lays the foundation for next-generation cellulose-based materials that are both biointeractive and industrially relevant.ASSOCIATED CONTENTSupporting Information. The following files are available free of charge.Additional experimental data are provided in the Supporting Information. Figure S1 Page 37 of 51ACS Paragon Plus EnvironmentACS Applied Materials & Interfaces12345678910111213141516171819202122232425262728293031323334353637383940414243444546474849505152535455565758596038presents time-resolved visual assessments of hydrogel stability under different pH conditions. Figure S2 shows the stability of hydrogels treated at 70 °C for 48 h and 72 h in 0.5 mol L⁻¹ Tris buffer. Figures S3–S4 provide detailed SEM, PXRD, and FTIR analyses of freeze-dried and heat-treated CMCF–CA hydrogels. Figure S5 shows the time-dependent pH stability of PDA–CMCF–CA hydrogels. Figures S6 and S7 include fluorescence and phase-contrast images of EGFP-expressing fibroblasts cultured on unmodified and PDA-coated hydrogels over time. These data further support the morphological, chemical, and biological characterizations described in the main text. AUTHOR INFORMATIONCorresponding Author*Yurina Sekine - Laboratory for the Development of Bioresource Technology, Japan Atomic Energy Agency Tokai, Ibaraki 319-1195, Japan., Promotion office, JAEA, Tokai, Ibaraki 319-1195, Japan. E-mail: sekine.yurina@jaea.go.jp, *Michiyo Honda - School of Science and Technology, Meiji University, Kawasaki, Kanagawa 214-8571, Japan. E-mail: michiyoh@meiji.ac.jpPage 38 of 51ACS Paragon Plus EnvironmentACS Applied Materials & Interfaces123456789101112131415161718192021222324252627282930313233343536373839404142434445464748495051525354555657585960mailto:sekine.yurina@jaea.go.jpmailto:michiyoh@meiji.ac.jp39AuthorsAsako Sugita - Laboratory for the Development of Bioresource Technology, Japan Atomic Energy Agency Tokai, Ibaraki 319-1195, Japan.Takuya Nankawa - Laboratory for the Development of Bioresource Technology, Japan Atomic Energy Agency Tokai, Ibaraki 319-1195, Japan.Rimi Sakanaka - School of Science and Technology, Meiji University, Kawasaki, Kanagawa 214-8571, Japan.Tomoko Ikeda-Fukazawa - School of Science and Technology, Meiji University, Kawasaki, Kanagawa 214-8571, Japan.Katsuhiko Ariga - Research Center for Materials Nanoarchitectonics, National Institute for Materials Science (NIMS), 1-1 Namiki, Tsukuba 305-0044, Japan., Graduate School of Frontier Sciences, The University of Tokyo, 5-1-5 Kashiwanoha, Kashiwa, Chiba 277-8561, Japan.Author Contributions.Yurina Sekine and Michiyo Honda contributed equally to this work. All authors read and commented on the manuscript.Page 39 of 51ACS Paragon Plus EnvironmentACS Applied Materials & Interfaces12345678910111213141516171819202122232425262728293031323334353637383940414243444546474849505152535455565758596040Funding Sources. This work was supported in part by JSPS KAKENHI (Grants 24K01409 (T.N., Y.S), 24K10550 (Y.S.,T.N.), and 23K20875 (T.F.,Y.S.,T.N.)).ABBREVIATIONS3D, three-dimensional; CNF, cellulose nanofiber; CMCF, carboxymethylcellulose nanofiber; PDA, polydopamine; Normal Human Dermal Fibroblasts, NHDF; CA, citric acid; DMEM, Dulbecco's Modified Eagle Medium; FBS, fetal bovine serum; HCl, Hydrochloric acid; CMCF-CA hydrogel, Freeze-crosslinked CMCF-CA hydrogel; CLSM, confocal laser scanning microscopy; SEM, scanning electron microscopy; FT–IR, Fourier transform-infrared; PXRD, powder X-ray diffraction; TGA, Thermo gravimetry analysis.References(1) Zhang, Y. S.; Khademhosseini, A. Advances in engineering hydrogels. Science 2017, 356 (6337). DOI: 10.1126/science.aaf3627(2) Wang, Z.; Wei, H.; Huang, Y.; Wei, Y.; Chen, J. Naturally sourced hydrogels: emerging fundamental materials for next-generation healthcare sensing. Chem. So.c Rev. 2023, 52 (9), 2992-3034. DOI: 10.1039/d2cs00813k(3) Bao, Z.; Xian, C.; Yuan, Q.; Liu, G.; Wu, J. Natural Polymer-Based Hydrogels with Enhanced Mechanical Performances: Preparation, Structure, and Property. Adv. Healthc. Mater. 2019, 8 (17), e1900670. DOI: 10.1002/adhm.201900670(4) Isogai, A.; Saito, T.; Fukuzumi, H. TEMPO-oxidized cellulose nanofibers. Nanoscale 2011, 3 (1), 71-85. DOI: 10.1039/c0nr00583e(5) Eichhorn, S. J.; Dufresne, A.; Aranguren, M.; Marcovich, N. E.; Capadona, J. R.; Rowan, S. J.; Weder, C.; Thielemans, W.; Roman, M.; Renneckar, S.; et al. Review: Page 40 of 51ACS Paragon Plus EnvironmentACS Applied Materials & Interfaces12345678910111213141516171819202122232425262728293031323334353637383940414243444546474849505152535455565758596041current international research into cellulose nanofibres and nanocomposites. J. Mater. Sci. 2010, 45 (1), 1-33. DOI: 10.1007/s10853-009-3874-0.(6) Thomas, B.; Raj, M. C.; B, A. K.; H, R. M.; Joy, J.; Moores, A.; Drisko, G. L.; Sanchez, C. Nanocellulose, a Versatile Green Platform: From Biosources to Materials and Their Applications. Chem. Rev. 2018, 118 (24), 11575-11625. DOI: 10.1021/acs.chemrev.7b00627(7) Klemm, D.; Kramer, F.; Moritz, S.; Lindstrom, T.; Ankerfors, M.; Gray, D.; Dorris, A. Nanocelluloses: a new family of nature-based materials. Angew. Chem. Int. Ed. Engl. 2011, 50 (24), 5438-5466. DOI: 10.1002/anie.201001273(8) Du, H.; Liu, W.; Zhang, M.; Si, C.; Zhang, X.; Li, B. Cellulose nanocrystals and cellulose nanofibrils based hydrogels for biomedical applications. Carbohydr. Polym. 2019, 209, 130-144. DOI: 10.1016/j.carbpol.2019.01.020(9) Hickey, R. J.; Pelling, A. E. Cellulose Biomaterials for Tissue Engineering. Front. Bioeng. Biotechnol. 2019, 7, 45. DOI: 10.3389/fbioe.2019.00045(10) Chang, C.; Zhang, L. Cellulose-based hydrogels: Present status and application prospects. Carbohydr. Polym. 2011, 84 (1), 40-53. DOI: 10.1016/j.carbpol.2010.12.023.(11) Kundu, R.; Mahada, P.; Chhirang, B.; Das, B. Cellulose hydrogels: Green and sustainable soft biomaterials. Curr. Res. Green Sustain. Chem. 2022, 5. DOI: 10.1016/j.crgsc.2021.100252.(12) Song, J.; Lyu, W.; Kawakami, K.; Ariga, K. Bio-gel nanoarchitectonics in tissue engineering. Nanoscale 2024, 16 (28), 13230-13246. DOI: 10.1039/d4nr00609g(13) Song, J.; Jancik-Prochazkova, A.; Kawakami, K.; Ariga, K. Lateral nanoarchitectonics from nano to life: ongoing challenges in interfacial chemical science. Chem. Sci. 2024, 15 (45), 18715-18750. DOI: 10.1039/d4sc05575f(14) Saito, T.; Nishiyama, Y.; Putaux, J. L.; Vignon, M.; Isogai, A. Homogeneous suspensions of individualized microfibrils from TEMPO-catalyzed oxidation of native cellulose. Biomacromolecules 2006, 7 (6), 1687-1691. DOI: 10.1021/bm060154s(15) Sekine, Y.; Nankawa, T.; Hiroi, K.; Oba, Y.; Nagakawa, Y.; Sugita, T.; Shibayama, Y.; Ikeda-Fukazawa, T. Nanocellulose hydrogels formed via crystalline transformation from cellulose I to II and subsequent freeze cross-linking reaction. Carbohydr. Polym. 2024, 327, 121538. DOI: 10.1016/j.carbpol.2023.121538(16) Guyton, A. C. a. H., John E. Textbook of Medical Physiology; Elsevier Saunders, 2011.(17) Von Sperling, M. Wastewater Characteristics, Treatment and Disposal; IWA Publ.: 2007; Biological wastewater treatment series. DOI: 10.2166/9781780402086.Page 41 of 51ACS Paragon Plus EnvironmentACS Applied Materials & Interfaces12345678910111213141516171819202122232425262728293031323334353637383940414243444546474849505152535455565758596042(18) Lee, H.; Dellatore, S. M.; Miller, W. M.; Messersmith, P. B. Mussel-inspired surface chemistry for multifunctional coatings. Science 2007, 318 (5849), 426-430. DOI: 10.1126/science.1147241(19) Ryu, J. H.; Messersmith, P. B.; Lee, H. Polydopamine Surface Chemistry: A Decade of Discovery. ACS Appl. Mater. Interfaces 2018, 10 (9), 7523-7540. DOI: 10.1021/acsami.7b19865(20) Park, S. E.; Georgescu, A.; Oh, J. M.; Kwon, K. W.; Huh, D. Polydopamine-Based Interfacial Engineering of Extracellular Matrix Hydrogels for the Construction and Long-Term Maintenance of Living Three-Dimensional Tissues. ACS Appl. Mater. Interfaces 2019, 11 (27), 23919-23925. DOI: 10.1021/acsami.9b07912(21) Liu, X.; Wu, Y.; Lin, Q.; Cheng, J.; Lin, F.; Tang, L.; Huang, B.; Lu, B. Polydopamine-coated cellulose nanocrystal as functional filler to fabricate nanocomposite hydrogel with controllable performance in response to near-infrared light. Cellulose 2021, 28 (4), 2255-2271. DOI: 10.1007/s10570-020-03630-8(22) Zhang, H.; Pechstedt, J.; Xiong, G.; Plohl, O.; Chen, Q.; Armand, M.; Sun, J.-Y.; Karakurt, I.; Aili, D. CNC-reinforced zwitterionic hydrogels with bioinspired mechanical adaptability. Nat. Commun. 2023, 14 (1), 6483. DOI: 10.1038/s41467-023-41874-7(23) Hoffman, A. S. Hydrogels for biomedical applications. Adv. Drug Deliv. Rev. 2012, 64, 18-23. DOI: 10.1016/j.addr.2012.09.010(24) Caló, E.; Khutoryanskiy, V. V. Biomedical applications of hydrogels: A review of patents and commercial products. Eur. Polym. J. 2015, 65, 252-267. DOI: 10.1016/j.eurpolymj.2014.11.024(25) Li, J.; Mooney, D. J. Designing hydrogels for controlled drug delivery. Nat. Rev. Mater. 2016, 1 (12). DOI: 10.1038/natrevmats.2016.71(26) Tibbitt, M. W.; Anseth, K. S. Hydrogels as extracellular matrix mimics for 3D cell culture. Biotechnol. Bioeng. 2009, 103 (4), 655-663. DOI: 10.1002/bit.22361(27) Sekine, Y.; Nankawa, T.; Yunoki, S.; Sugita, T.; Nakagawa, H.; Yamada, T. Eco-friendly Carboxymethyl Cellulose Nanofiber Hydrogels Prepared via Freeze Cross-Linking and Their Applications. ACS Appl. Polym. Mater. 2020, 2 (12), 5482-5491. DOI: 10.1021/acsapm.0c00831(28) Watanabe, Y.; Kitamura, S.; Kawasaki, K.; Kato, T.; Uegaki, K.; Ogura, K.; Ishikawa, K. Application of a water jet system to the pretreatment of cellulose. Biopolymers 2011, 95 (12), 833-839. DOI: 10.1002/bip.21686Page 42 of 51ACS Paragon Plus EnvironmentACS Applied Materials & Interfaces12345678910111213141516171819202122232425262728293031323334353637383940414243444546474849505152535455565758596043(29) Sekine, Y.; Nankawa, T. Freeze-Concentrated Layers as a Unique Field for the Formation of Hydrogels. Bull. Chem. Soc. Jpn. 2023, 96 (10), 1150-1155. DOI: 10.1246/bcsj.20230146(30) Qian, Y.; Yuan, Y.; Wang, H.; Liu, H.; Zhang, J.; Shi, S.; Guo, Z.; Wang, N. Highly efficient uranium adsorption by salicylaldoxime/polydopamine graphene oxide nanocomposites. J. Mater. Chem. A 2018, 6 (48), 24676-24685. DOI: 10.1039/c8ta09486a(31) Zhang, X. N.; Wang, Y. J.; Sun, S.; Hou, L.; Wu, P.; Wu, Z. L.; Zheng, Q. A Tough and Stiff Hydrogel with Tunable Water Content and Mechanical Properties Based on the Synergistic Effect of Hydrogen Bonding and Hydrophobic Interaction. Macromolecules 2018, 51 (20), 8136-8146. DOI: 10.1021/acs.macromol.8b01496(32) Miura, D.; Sekine, Y.; Nankawa, T.; Sugita, T.; Oba, Y.; Hiroi, K.; Ohzawa, T. Microscopic structural changes during the freeze cross-linking reaction in carboxymethyl cellulose nanofiber hydrogels. Carbohydr. Polym. Technol. Appl. 2022, 4. DOI: 10.1016/j.carpta.2022.100251(33) Katsumoto, Y.; Tanaka, T.; Ozaki, Y. Molecular interpretation for the solvation of poly(acrylamide)s. I. Solvent-dependent changes in the C=O stretching band region of poly(N,N-dialkylacrylamide)s. J. Phys. Chem. B 2005, 109 (44), 20690-20696. DOI: 10.1021/jp052263r(34) Sekine, Y.; Ikeda-Fukazawa, T. Structural changes of water in a hydrogel during dehydration. J. Chem. Phys. 2009, 130 (3), 034501. DOI: 10.1063/1.3058616(35) Max, J.-J.; Chapados, C. Infrared Spectroscopy of Aqueous Carboxylic Acids:  Comparison between Different Acids and Their Salts. J. Phys. Chem. A 2004, 108 (16), 3324-3337. DOI: 10.1021/jp036401t(36) Cuba-Chiem, L. T.; Huynh, L.; Ralston, J.; Beattie, D. A. In situ particle film ATR FTIR spectroscopy of carboxymethyl cellulose adsorption on talc: binding mechanism, pH effects, and adsorption kinetics. Langmuir 2008, 24 (15), 8036-8044. DOI: 10.1021/la800490t(37) French, A. D. Idealized powder diffraction patterns for cellulose polymorphs. Cellulose 2013, 21 (2), 885-896. DOI: 10.1007/s10570-013-0030-4(38) Hou, L.; Wu, P. Two-dimensional correlation infrared spectroscopy of heat-induced esterification of cellulose with 1,2,3,4-butanetetracarboxylic acid in the presence of sodium hypophosphite. Cellulose 2019, 26 (4), 2759-2769. DOI: 10.1007/s10570-019-02255-wPage 43 of 51ACS Paragon Plus EnvironmentACS Applied Materials & Interfaces12345678910111213141516171819202122232425262728293031323334353637383940414243444546474849505152535455565758596044(39) Suenaga, S.; Osada, M. Hydrothermal Gelation of Pure Cellulose Nanofiber Dispersions. ACS Appl. Polym. Mater. 2019, 1 (5), 1045-1053. DOI: 10.1021/acsapm.9b00076(40) Zangmeister, R. A.; Morris, T. A.; Tarlov, M. J. Characterization of polydopamine thin films deposited at short times by autoxidation of dopamine. Langmuir 2013, 29 (27), 8619-8628. DOI: 10.1021/la400587j(41) Challa, A. A.; Saha, N.; Zhivkova, T.; Alexandrova, R.; Saha, P. Bacterial Cellulose/Graphene Oxide/Hydroxyapatite Biocomposite: A Scaffold from Sustainable Sources for Bone Tissue Engineering. ACS Appl. Mater. Interfaces 2025, 17 (1), 572-582. DOI: 10.1021/acsami.4c17306(42) Ferreira, A. M.; Gentile, P.; Chiono, V.; Ciardelli, G. Collagen for bone tissue regeneration. Acta Biomater. 2012, 8 (9), 3191-3200. DOI: 10.1016/j.actbio.2012.06.014(43) Nichol, J. W.; Koshy, S. T.; Bae, H.; Hwang, C. M.; Yamanlar, S.; Khademhosseini, A. Cell-laden microengineered gelatin methacrylate hydrogels. Biomaterials 2010, 31 (21), 5536-5544. DOI: 10.1016/j.biomaterials.2010.03.064(44) Yue, K.; Trujillo-de Santiago, G.; Alvarez, M. M.; Tamayol, A.; Annabi, N.; Khademhosseini, A. Synthesis, properties, and biomedical applications of gelatin methacryloyl (GelMA) hydrogels. Biomaterials 2015, 73, 254-271. DOI: 10.1016/j.biomaterials.2015.08.045(45) Nemir, S.; Hayenga, H. N.; West, J. L. PEGDA hydrogels with patterned elasticity: Novel tools for the study of cell response to substrate rigidity. Biotechnol. Bioeng. 2010, 105 (3), 636-644. DOI: 10.1002/bit.22574(46) Zhu, J. Bioactive modification of poly(ethylene glycol) hydrogels for tissue engineering. Biomaterials 2010, 31 (17), 4639-4656. DOI: 10.1016/j.biomaterials.2010.02.044(47) Parthasarathy, V.; Kumar, P. S.; Aureen Albert, A.; Krishnasamy, S.; Chandrasekar, M. Recent progress in nanocellulose-based biocomposites for bone tissue engineering and wound healing applications. Carbohydr. Polym. 2025, 357, 123455. DOI: 10.1016/j.carbpol.2025.123455(48) Joseph, A.; Umamaheswari, S.; Vassou, M. C. Bacterial cellulose: A versatile biomaterial for biomedical application. Carbohydr. Res. 2025, 552, 109350. DOI: 10.1016/j.carres.2024.109350 Page 44 of 51ACS Paragon Plus EnvironmentACS Applied Materials & Interfaces123456789101112131415161718192021222324252627282930313233343536373839404142434445464748495051525354555657585960