Microenvironment-responsive hydrogel for precise sequential repair of acute infectious wounds
微环境响应水凝胶用于急性感染性伤口的精确顺序修复

Zixuan Tang^a,e,1, Qingquan Xia^a,1, Jun Chen^c, Xuhua Wu^a, Jiang Li^a, Jiangyi Liu^a,^d, Xiangchao Meng^a,*, Ke Rong^a,*
Zixuan Tang ^{a, e ,1} , Qingquan Xia ^a,1 , Jun Chen ^c , Xuhua Wu ^a , Jiang Li ^a , Jiangyi Liu ^a , ^d , Xiangchao Meng ^{a *} , Ke Rong ^{a ,*}

^a Department of Orthopedics, Minhang Hospital, Fudan University, Shanghai, China
^a 复旦大学附属闵行医院骨科，上海，中国

^b Department of Sport's Medicine, The Second Affiliated Hospital of Fujian University of Traditional Chinese Medical, Fuzhou, China
^b 福建中医药大学第二附属医院运动医学科，福州，中国

^c Department of Head and Neck Surgery, RenJi hospital, School of medicine, Shanghai JiaoTong University, Shanghai, China
^c 上海交通大学医学院仁济医院头颈外科 ，上海，中国

^d Department of Orthopedics, Ningbo University Affiliated Li Huili Hospital, Ningbo University, Ningbo, China
^d 宁波大学附属李惠丽医院骨科 ，宁波，中国

^e Department of Plastic Surgery and Burn Center, Second Affiliated Hospital, Shantou University Medical College, Shantou, Guangdong 515051, China
汕头大学医学院第二附属医院整形外科暨烧伤中心，广东汕头 ⁵¹⁵⁰⁵¹

¹ These authors contributed equally to this work.
¹ 这些作者对这项工作做出了同等贡献。

* Corresponding authors.
* 通讯作者。

E-mail addresses: mengxiangchao@alumni.sjtu.edu.cn (Xiangchao Meng), rongke1977@163.com (Ke Rong)
邮箱地址： mengyangchao@alumni.sjtu.edu.cn （ 孟向超 ）、 rongke1977@163.com （ 容柯 ）

Abstract: The healing of acute infected wounds is a multi-stage and sequential biological process. Traditional antibacterial dressings are usually a simple superposition of antibacterial properties and active ingredients, lacking effective coupling with the wound microenvironment, and it is difficult to accurately match the continuous process of infected wound healing. In this study, a pH-responsive hydrogel of sodium alginate and carboxymethyl chitosan interpenetrating network was constructed, and tannic acid (TA) and zinc-doped bioglass (BAG) were loaded through hydrogen bonding and hydrophobic interactions. In the acidic environment of infection, the enhancement of intermolecular non-covalent interaction leads to the contraction of hydrogel network and the rapid release of tannic acid. In the alkaline environment of healing, the weakening of intermolecular interaction leads to the expansion of hydrogel network and the continuous release of Zn²⁺ and Ca²⁺. In vitro biological evaluation showed that the hydrogel had an effective antibacterial effects against E.coli and S.aureus, and effectively regulated immune response. In addition, the hydrogel effectively removed excessive ROS and significantly increase the activity of cellular antioxidant enzymes, thereby accelerating the wound healing process in animal experiment. This microenvironment-responsive hydrogel provides a new therapeutic strategy for precise sequential repair of acute infectious wounds.
摘要： 急性感染伤口的愈合是一个多阶段、连续的生物学过程，传统抗菌敷料通常是抗菌特性和活性成分的简单叠加，缺乏与伤口微环境的有效耦合，难以准确匹配感染伤口愈合的连续过程。本研究构建了 pH 响应性的海藻酸钠和羧甲基壳聚糖互穿网络水凝胶，通过氢键和疏水相互作用负载单宁酸(TA)和锌掺杂生物玻璃(BAG)。在酸性感染环境下，分子间非共价相互作用的增强导致水凝胶网络收缩，单宁酸快速释放；在碱性愈合环境下，分子间相互作用的减弱导致水凝胶网络扩张，Zn2 ⁺ 和 Ca2 ⁺ 持续释放 。 体外生物学评价表明，该水凝胶对大肠杆菌和金黄色葡萄球菌具有有效的抗菌作用 ，并有效调节免疫反应。此外，该水凝胶还能有效清除细胞内过量的活性氧（ROS），显著提高细胞抗氧化酶的活性，从而加速动物实验中的伤口愈合过程。这种微环境响应性水凝胶为急性感染性伤口的精准序贯修复提供了一种新的治疗策略。

Keywords: pH-responsive hydrogel, sequential repair, acute infectious wounds, antibacterial,
关键词： pH 响应性水凝胶，顺序修复， 急性感染性伤口，抗菌，

immune regulation.
免疫调节 。

Introduction
介绍

The skin, the largest organ of the human body, plays a crucial role in defending against external physical, chemical, and biological threats, while also maintaining fluid and electrolyte balance. However, as the outermost layer, it is particularly susceptible to damage. Wounds are generally categorized into acute and chronic types, depending on the duration of the healing process[1]. Acute infectious wounds are common in clinical settings and can severely impact patients' health and quality of life, posing significant treatment challenges[2-3]. Traditional wound dressings typically serve as passive barriers, covering the injury site and adhering to the surface of the damaged tissue. However, these dressings have limited ability to manage wound exudates, leading to unstable local humidity and temperature. Over time, their ability to absorb exudates and act as a protective barrier deteriorates, increasing the risk of infection by failing to continuously isolate external pathogens[4]. Moreover, conventional dressings lack inherent antibacterial properties, fail to actively inhibit microbial growth, and do not provide the moist environment essential for cell migration and new tissue formation[5]. Additionally, they lack bioactive components necessary to promote tissue repair, thus hindering the healing process of infected wounds[6-7]. As a result, the development of advanced therapeutic approaches for managing acute infectious wounds is a pressing priority in both clinical medicine and wound care research.
皮肤是人体最大的器官，在抵御外界物理、化学和生物威胁，以及维持体液和电解质平衡方面发挥着至关重要的作用。然而，作为最外层，皮肤特别容易受到损伤。伤口通常根据愈合过程的长短分为急性和慢性伤口 [1] 。 急性感染性伤口在临床中很常见，会严重影响患者的健康和生活质量 ， 给治疗带来巨大的挑战 [2-3] 。传统的伤口敷料通常作为被动屏障，覆盖损伤部位并粘附在受损组织表面。然而，这些敷料对伤口渗出液的控制能力有限，导致局部湿度和温度不稳定。随着时间的推移，它们吸收渗出液和充当保护屏障的能力会下降，无法持续隔离外界病原体，从而增加了感染的风险 [4] 。此外，传统敷料缺乏固有的抗菌特性，不能主动抑制微生物生长，也不能提供细胞迁移和新组织形成所必需的湿润环境 [5] 。此外，它们缺乏促进组织修复所必需的生物活性成分，从而阻碍了感染伤口的愈合过程 [6-7] 。因此，开发用于治疗急性感染性伤口的先进治疗方法是临床医学和伤口护理研究的当务之急。

Acute infectious wounds not only cause localized tissue damage but may also trigger systemic inflammation and facilitate the spread of infection. Traditional wound treatments often provide a single therapeutic function, which limits their ability to address the complex, dynamic needs of wound healing. The wound healing process is a dynamic, multi-phase biological progression comprising inflammatory, proliferative, and remodeling stages, each distinguished by specific physiological and pathological characteristics[8]. Therefore, an ideal wound dressing should dynamically adapt to the distinct phases of wound healing. During the initial antimicrobial phase, the dressing should exhibit effective anti-infection performance by promptly inhibiting pathogenic microorganism growth and preventing further infection progression[9-11]. Once the infection is controlled, the dressing should promptly transition its functionality to support anti-inflammatory activity and tissue repair, fostering cell proliferation, angiogenesis, and tissue remodeling, thereby expediting the wound healing process[12-14]. Hence, developing a multifunctional dressing that can respond to changes in the wound microenvironment and enable a functional transition from anti-infection to pro-healing is essential. By dynamically adjusting the properties of the dressing to deliver tailored therapeutic effects at different healing stages, rapid and complete repair of infectious wounds can be achieved.
急性感染性伤口不仅会造成局部组织损伤，还可能引发全身性炎症，促进感染的扩散。传统的伤口治疗通常提供单一的治疗功能，这限制了它们满足伤口愈合复杂、动态需求的能力。 伤口愈合过程是一个动态的、多阶段的生物学进程，包括炎症、增生和重塑阶段，每个阶段都有其特定的生理和病理特征 [8] 。 因此，理想的伤口敷料应动态地适应伤口愈合的不同阶段。 在初始抗菌阶段，敷料应通过迅速抑制病原微生物生长并防止进一步感染进展而表现出有效的抗感染性能 [9-11] 。 一旦感染得到控制，敷料应迅速转变其功能以支持抗炎活性和组织修复，促进细胞增殖、血管生成和组织重塑，从而加速伤口愈合过程 [12-14] 。 因此，开发一种能够响应伤口微环境变化，实现从抗感染到促进愈合功能的多功能敷料至关重要。 通过动态调节敷料的特性，在不同的愈合阶段提供个性化的治疗效果，可以实现感染性伤口的快速、彻底修复。

Hydrogels are three-dimensional networked materials composed of hydrophilic polymers. With their outstanding biocompatibility and adjustable physicochemical properties, hydrogels have found extensive applications in biomedical engineering, drug delivery, and tissue engineering[15-17]. In skin infections, the local environment is typically acidic and gradually transitions to weakly alkaline as the anti-infection process concludes[18]. Therefore, pH-responsive hydrogels can dynamically adapt to the physiological transition from anti-infection to wound healing by responding to pH variations in the wound microenvironment[19]. As an external stimulus, pH exerts a substantial influence on the sol-gel transition, swelling behavior, and drug release mechanisms of hydrogels[20-21]. Harnessing this pH-responsive property allows for functional modulation of hydrogels across different healing stages, thereby enhancing their application potential in treating infected wounds.
水凝胶是由亲水性聚合物组成的三维网络材料。 由于其出色的生物相容性和可调节的物理化学性质，水凝胶在生物医学工程、药物递送和组织工程中得到了广泛的应用 [15-17] 。 在皮肤感染中，局部环境通常为酸性，随着抗感染过程的结束逐渐转变为弱碱性 [18] 。 因此，pH 响应性水凝胶可以通过响应伤口微环境中的 pH 值变化来动态适应从抗感染到伤口愈合的生理转变 [19] 。 作为外界刺激，pH 值对水凝胶的溶胶-凝胶转变、溶胀行为和药物释放机制具有显著的影响 [20-21] 。 利用这种 pH 响应特性，可以在不同的愈合阶段对水凝胶进行功能性调节，从而增强其在治疗感染伤口中的应用潜力。

Herein, this study introduces a pH-responsive hydrogel formed by the interpenetrating network of sodium alginate (SA) and carboxymethyl chitosan (CMCS), incorporating tannic acid and zinc-doped bioglass through hydrogen bonding and hydrophobic interactions (Fig. 1A). These hydrogels, especially those based on SA and CMCS, offer remarkable potential for wound healing due to their ability to dynamically respond to environmental changes and regulate drug release. Under acidic conditions typical of infection, the carboxyl groups of SA undergo protonation, while enhanced hydrogen bonding between -COOH and -OH groups in CMCS leads to network contraction, promoting the rapid release of tannic acid, which possesses strong antibacterial properties. As the pH shifts to weakly alkaline during the healing phase, the hydrogel expands, absorbing exudates and facilitating the release of growth factors that aid tissue repair. Additionally, the sustained release of zinc and calcium ions fosters angiogenesis and provides anti-inflammatory effects, thereby accelerating wound healing. In summary, this pH-responsive hydrogel demonstrates excellent antibacterial and healing-promoting capabilities, offering a promising solution for managing acute infectious wounds and enhancing tissue regeneration.
本研究介绍了一种 pH 响应性水凝胶，该水凝胶由海藻酸钠 (SA) 和羧甲基壳聚糖 (CMCS) 的互穿网络构成，并通过氢键和疏水相互作用将单宁酸和锌掺杂生物玻璃结合在一起（图 1A）。这些水凝胶，尤其是基于 SA 和 CMCS 的水凝胶，由于其能够动态响应环境变化并调节药物释放，在伤口愈合方面具有显著的潜力。在典型的感染酸性条件下，SA 的羧基会发生质子化，而 CMCS 中 -COOH 和 -OH 基团之间增强的氢键会导致网络收缩，从而促进具有强抗菌特性的单宁酸的快速释放。在愈合阶段，随着 pH 值转变为弱碱性，水凝胶会膨胀，吸收渗出液并促进有助于组织修复的生长因子的释放。此外，锌和钙离子的持续释放可促进血管生成并提供抗炎作用，从而加速伤口愈合。总之，这种 pH 响应水凝胶表现出优异的抗菌和促进愈合的能力，为治疗急性感染性伤口和增强组织再生提供了有希望的解决方案。

2. Materials and Methods
2.材料和方法

2.1. Reagents
2.1. 试剂

Carboxymethyl chitosan (CMCS, MW ~100,000, degree of acetylation ≥75%), sodium alginate (SA, MW ~200,000), tannic acid (TA, MW ~1,701.2), and Bioglass® 45S5 (zinc-modified) were obtained from Sigma-Aldrich (Darmstadt, Germany). Sodium citrate (analytical grade) was sourced from the same supplier and used for solution preparation. Dulbecco’s Modified Eagle’s Medium (DMEM), fetal bovine serum (FBS), penicillin-streptomycin, trypsin-EDTA (0.25%), and phosphate-buffered saline (PBS) were purchased from Gibco (Grand Island, NY, USA). The vascular endothelial growth factor (VEGF), CD86, CD206, inducible nitric oxide synthase (iNOS), Arginase-1 antibodies, and F4/80 antibody were supplied by Proteintech (Wuhan, China) and Santa Cruz Biotechnology (USA). The cell counting kit-8 (CCK-8) was purchased from Biosharp (Hefei, China)., and 2′,7′-Dichlorofluorescin diacetate (DCFH-DA) was obtained from Solarbio (Beijing, China). Other reagents were from China National Pharmaceutical Group Corporation (Shanghai, China), and deionized water was used for all solutions and washing.
羧甲基壳聚糖（CMCS，分子量约 100,000，乙酰化度≥75%）、海藻酸钠（SA，分子量约 200,000）、单宁酸（TA，分子量约 1,701.2）和 Bioglass® 45S5（锌改性）购自 Sigma-Aldrich 公司（德国达姆施塔特）。柠檬酸钠（分析纯）来自同一供应商，用于溶液制备。杜氏改良 Eagle 培养基（DMEM）、胎牛血清（FBS）、青霉素-链霉素、胰蛋白酶-EDTA（0.25%）和磷酸盐缓冲液（PBS）购自 Gibco 公司（美国纽约州大岛）。血管内皮生长因子(VEGF)、CD86、CD206、诱导型一氧化氮合酶(iNOS)、精氨酸酶-1 抗体、F4/80 抗体均由 Proteintech 公司(武汉)和 Santa Cruz Biotechnology 公司(美国)提供。 细胞计数试剂盒 -8 (CCK-8)购自 Biosharp 公司(合肥) ，2′，7′-二氯荧光素二乙酸酯(DCFH-DA)购自 Solarbio 公司(北京)。其他试剂均购自中国医药集团总公司(上海)。所有溶液和洗涤均使用去离子水。

2.2. Construction of hydrogels
2.2. 水凝胶的构建

Sodium alginate (SA)/carboxymethyl chitosan (CMCS) hydrogel was fabricated incorporating tannic acid (TA) and bioglass (BAG) as functional agents (Fig. 1A). Sodium alginate (4% w/v) was dissolved in distilled water, while chitosan (2% w/v) was dissolved in dilute acetic acid (1-2% v/v). Bioglass (10 mg/mL) was added to the chitosan solution and sonicated for homogeneity. The alginate and chitosan solutions were mixed in a 2:1 ratio under continuous stirring. Tannic acid (0.5% w/v) was then incorporated, followed by the addition of sodium citrate (10% w/v) as a crosslinking agent. This process led to the formation of a three-dimensional crosslinked hydrogel network, which was completed at room temperature.
以单宁酸 (TA) 和生物玻璃 (BAG) 作为功能剂，制备海藻酸钠 (SA)/羧甲基壳聚糖 (CMCS) 水凝胶（图 1A）。将海藻酸钠 (4% w/v) 溶解于蒸馏水中，将壳聚糖 (2% w/v) 溶解于稀乙酸 (1-2% v/v) 中。将生物玻璃 (10 mg/mL) 加入壳聚糖溶液中，并进行超声处理使其均匀。将海藻酸钠和壳聚糖溶液以 2:1 的比例混合，并持续搅拌。然后加入单宁酸 (0.5% w/v)，再加入柠檬酸钠 (10% w/v) 作为交联剂。该过程形成三维交联水凝胶网络，并在室温下完成。

2.3 Material characterization
2.3 材料表征

To investigate the chemical structure of the hydrogel, Fourier-transform infrared spectroscopy (FTIR, Nicolet 6700, Thermo Fisher, USA) was employed. The surface elemental composition and chemical states of the hydrogel were examined using X-ray photoelectron spectroscopy (XPS, AXIS Supra, Kratos Analytical, UK). X-ray diffraction (XRD, Bruker D8 Advance, Germany) was employed to characterize the crystalline structure of the hydrogel. The surface and internal structures of the hydrogel were examined using scanning electron microscopy (SEM, Quanta 250, FEI, USA). Additionally, elemental composition was assessed using energy-dispersive X-ray spectroscopy (EDS).
为了研究水凝胶的化学结构，我们采用了傅里叶变换红外光谱仪 (FTIR, Nicolet 6700, Thermo Fisher, 美国)。 我们利用 X 射线光电子能谱仪 (XPS, AXIS Supra, Kratos Analytical, 英国) 检测了水凝胶的表面元素组成和化学状态。 我们利用 X 射线衍射仪 (XRD, Bruker D8 Advance, 德国) 表征了水凝胶的晶体结构。 我们利用扫描电子显微镜 (SEM, Quanta 250, FEI, 美国) 检测了水凝胶的表面和内部结构。 此外，我们还利用能量色散 X 射线光谱仪 (EDS) 评估了元素组成。

To assess the pH-responsive release behavior of tannic acid (TA), along with zinc (Zn²⁺) and calcium (Ca²⁺) ions from bioglass (BAG) incorporated in the hydrogel, prepared samples were incubated in buffer solutions at pH 5.5 and pH 7.4. These pH conditions were selected to replicate different physiological environments: pH 5.5 simulates the acidic microenvironment commonly associated with infected or inflamed tissues, where bacterial activity and metabolic byproducts lower the pH, while pH 7.4 corresponds to the neutral physiological environment of healthy tissues during later stages of wound healing or in non-infected states. Hydrogel samples, cut into dimensions of 10 mm × 10 mm × 2 mm, were placed in 10 mL of buffer solution within centrifuge tubes. These samples were incubated at 37°C in a thermostatic shaker. At specific time intervals (0, 3, 5, 7, 9, 11, and 14 days), 1 mL of solution was withdrawn and replaced with fresh buffer. The collected solutions were then analyzed using inductively coupled plasma mass spectrometry (ICP-MS) or atomic absorption spectroscopy (AAS) to quantify the concentrations of Zn²⁺ and Ca²⁺ ions. The cumulative ion release at each time point was calculated, and release profiles were generated to compare the hydrogel's ion release characteristics under acidic and neutral pH conditions.
为了评估水凝胶中单宁酸 (TA) 以及生物玻璃 (BAG) 中的锌 (Zn²⁺) 和钙 (Ca²⁺) 离子的 pH 响应释放行为 ， 将制备的样品在 pH 5.5 和 pH 7.4 的缓冲溶液中孵育。选择这些 pH 条件来复制不同的生理环境：pH 5.5 模拟通常与感染或发炎组织相关的酸性微环境，其中细菌活动和代谢副产物会降低 pH 值，而 pH 7.4 对应于健康组织在伤口愈合后期或非感染状态下的中性生理环境。将水凝胶样品切成 10 mm × 10 mm × 2 mm 的尺寸，置于离心管内的 10 mL 缓冲溶液中。将这些样品在恒温摇床中于 37°C 下孵育。在特定时间间隔（0、3、5、7、9、11 和 14 天）抽取 1 mL 溶液并替换为新鲜缓冲液。然后使用电感耦合等离子体质谱法 (ICP-MS) 或原子吸收光谱法 (AAS) 分析收集的溶液，以量化 Zn²⁺ 和 Ca²⁺ 离子的浓度。计算每个时间点的累积离子释放量，并生成释放曲线，以比较水凝胶在酸性和中性 pH 条件下的离子释放特性。

2.4 Cytocompatibility Assessment
2.4 细胞相容性评估

2.4.1 Wound-healing assay
2.4.1 伤口愈合试验

A scratch assay was conducted to investigate the effect of hydrogel extracts on the migration of human umbilical vein endothelial cells (HUVECs). HUVECs were seeded in 6-well plates at a density of 5×10⁴ cells per well and cultured until a confluent monolayer was formed. A vertical scratch was then introduced in the cell layer using a 1000 μL pipette tip. After the culture medium was removed, the wells were rinsed with phosphate-buffered saline (PBS) to eliminate any cell debris. The experimental groups were treated with complete medium containing various hydrogel extracts, while the control group received medium without any hydrogel extracts. Images of the scratched area were captured at both 0 and 24 hours using an inverted microscope. The scratch closure area was measured, and cell migration rates were calculated using ImageJ software. This method provided quantitative data on how hydrogel extracts influenced the migration capacity of HUVECs.
采用划痕实验研究水凝胶提取物对人脐静脉内皮细胞 (HUVEC) 迁移的影响。将 HUVEC 以每孔 5×10⁴ 细胞的密度接种于 6 孔板中，培养至形成汇合单层。然后用 1000 μL 枪头在细胞层上垂直划痕。弃去培养基后，用磷酸盐缓冲液 (PBS) 冲洗孔板以去除细胞碎片。实验组使用含有不同水凝胶提取物的完全培养基，对照组使用不含任何水凝胶提取物的培养基。使用倒置显微镜在 0 小时和 24 小时拍摄划痕区域的图像。测量划痕闭合面积，并使用 ImageJ 软件计算细胞迁移率。该方法提供了水凝胶提取物如何影响 HUVEC 迁移能力的定量数据。

2.4.2 Cytoskeleton Staining
2.4.2 细胞骨架染色

The cytoskeleton and nucleus were stained with fluorescence to evaluate the effect of hydrogel extracts on cell viability and morphology. L929 cells were seeded into 12-well culture plates at a density of 4×10⁵ cells per well. After 8 hours of incubation to allow for cell adhesion, the culture medium was replaced with the hydrogel extract corresponding to the experimental group. The cells were then incubated for an additional 24 hours under standard culture conditions. Following this incubation, the cells were stained according to the manufacturer’s instructions to visualize both the cytoskeleton and nuclei. This approach provided both qualitative and quantitative insights into the effect of hydrogel extracts on cell morphology and viability.
将细胞骨架和细胞核进行荧光染色，以评估水凝胶提取物对细胞活力和形态的影响。 将 L929 细胞以每孔 4×10⁵细胞的密度接种于 12 孔培养板中。孵育 8 小时以使细胞粘附后，用与实验组对应的水凝胶提取物替换培养基。然后将细胞在标准培养条件下再孵育 24 小时。孵育结束后，根据制造商的说明对细胞进行染色，以同时显示细胞骨架和细胞核。这种方法可以定性和定量地了解水凝胶提取物对细胞形态和活力的影响。

2.4.3 Live/dead staining
2.4.3 活/死染色

To evaluate cytotoxicity and cell viability, Calcein-AM/PI dual staining was employed on HUVECs treated with hydrogel extracts. HUVECs were seeded in 24-well plates at a density of 2×10⁴ cells per well and allowed to adhere. After attachment, cells were treated with hydrogel extracts from various experimental groups for 24 hours. Subsequently, A staining solution comprising propidium iodide (PI) and Calcein-AM was then applied, and cells were incubated in the dark at room temperature for 30 minutes. Using a fluorescence microscope, fluorescent pictures of living cells (stained green with Calcein-AM) and dead cells (stained red with PI) were obtained.. The number of live and dead cells was quantified using ImageJ software, and cell viability and cytotoxicity were calculated. This analysis provided a thorough assessment of the biocompatibility of the hydrogel extracts.
为评估细胞毒性和细胞活力，对用水凝胶提取物处理的 HUVEC 进行了 Calcein-AM/PI 双重染色。将 HUVEC 以每孔 2×10⁴ 细胞的密度接种于 24 孔板中并使其粘附。附着后，用来自不同实验组的水凝胶提取物处理细胞 24 小时。 随后，应用包含碘化丙啶 (PI) 和 Calcein-AM 的染色溶液 ，并将细胞在室温下黑暗中孵育 30 分钟。 使用荧光显微镜，获取活细胞（用 Calcein-AM 染成绿色）和死细胞（用 PI 染成红色）的荧光图像。 使用 ImageJ 软件量化活细胞和死细胞的数量，并计算细胞活力和细胞毒性。该分析对水凝胶提取物的生物相容性进行了全面评估。

2.5 In vitro antibacterial performance of hydrogels
2.5 水凝胶的体外抗菌性能

2.5.1 Biofilm 3D imaging
2.5.1 生物膜 3D 成像

To evaluate the inhibitory effects of the hydrogels on bacterial biofilm formation, Escherichia coli (E. coli) and Staphylococcus aureus (S. aureus) were cultured in LB broth at 37°C with shaking at 220 rpm until reaching the logarithmic growth phase. A 1 mL bacterial suspension (10⁷ CFU/mL) was then inoculated onto glass coverslips placed in 24-well plates and incubated at 37°C for 24 hours to allow mature biofilms to form. After 48 hours, the bacterial culture medium was replaced with hydrogel extracts from different experimental groups: PBS (control), SA/CMCS, SA/CMCS/TA, SA/CMCS/BAG, and SA/CMCS/TA/BAG. The samples were then incubated for an additional 12 hours at 37°C.
为了评估水凝胶对细菌生物膜形成的抑制效果， 将大肠杆菌 （E. coli）和金黄色葡萄球菌 （S. aureus）在 37°C 的 LB 肉汤中培养，以 220 rpm 的转速摇动培养至达到对数生长期。然后将 1 mL 细菌悬浮液（10⁷ CFU/mL）接种到置于 24 孔板中的玻璃盖玻片上，并在 37 ° C 下孵育 24 小时，以形成成熟的生物膜。48 小时后，将细菌培养基替换为来自不同实验组的水凝胶提取物：PBS（对照）、SA/CMCS、SA/CMCS/TA、SA/CMCS/BAG 和 SA/CMCS/TA/BAG。 然后将样品在 37°C 下再孵育 12 小时。
To observe biofilm formation and evaluate bacterial viability, a confocal laser scanning microscope (CLSM, Zeiss LSM 880) was used to image the biofilms in three dimensions (3D) after they had been stained with DMAO (green, which indicates live bacteria) and EthD-III (red, which indicates dead bacteria). Biofilm thickness and bacterial viability were analyzed, and the live/dead bacterial ratio was quantified using imaging software. This approach provided detailed insights into the hydrogel’s effectiveness in inhibiting biofilm formation and modulating bacterial viability.
为了观察生物膜的形成并评估细菌活力， 我们使用共聚焦激光扫描显微镜 (CLSM, Zeiss LSM 880) 对生物膜进行三维 (3D) 成像，生物膜先用 DMAO（绿色，表示活菌）和 EthD-III（红色，表示死菌）染色。 分析了生物膜厚度和细菌活力，并使用成像软件量化了活菌/死菌比例。这种方法为水凝胶在抑制生物膜形成和调节细菌活力方面的有效性提供了深入的见解。

2.5.2 Bacterial Live/Dead Staining
2.5.2 细菌活/死染色

To further assess bacterial viability following hydrogel treatment, Escherichia coli and Staphylococcus aureus were cultured in LB broth at 37°C and 220 rpm until they reached the logarithmic phase. The culture medium in each well was then replaced with hydrogel extracts from the respective experimental groups, The samples were subsequently incubated at 37°C for another 12 hours.
为了进一步评估水凝胶处理后的细菌活力， 将大肠杆菌和金黄色葡萄球菌在 LB 肉汤中以 37°C、220 rpm 的转速培养至对数生长期。然后将每个孔中的培养基替换为各实验组的水凝胶提取物。 随后，将样品在 37°C 下继续孵育 12 小时。

After the co-incubation, the bacteria were collected and stained using a bacterial live/dead staining kit according to the manufacturer's protocol. Live bacteria were stained with DMAO, while dead bacteria were labeled with EthD-II dye. The bacterial viability was then observed using a confocal laser scanning microscope (CLSM, Olympus FV1000), and the antimicrobial activity of the hydrogels was quantified based on the live-to-dead bacterial ratio. This analysis allowed for a comparative assessment of the hydrogels' antibacterial efficacy.
共培养后，收集细菌，并根据制造商的说明书使用细菌活/死染色试剂盒进行染色。活细菌用 DMAO 染色，死细菌用 EthD-II 染料标记。然后使用共聚焦激光扫描显微镜（CLSM，奥林巴斯 FV1000）观察细菌活力，并根据活菌与死菌的比例定量水凝胶的抗菌活性。该分析可用于比较评估水凝胶的抗菌效果。

2.5.3 Spread Plate Assay
2.5.3 平板涂布试验

To assess the antibacterial effectiveness of the hydrogel extracts, Escherichia coli and Staphylococcus aureus suspensions treated with hydrogel extracts underwent tenfold serial dilutions. A 100 µL portion from each dilution was then plated onto LB agar plates. The plates were incubated at 37°C for 12 hours, and colony-forming units (CFUs) were subsequently counted. The CFU counts from each group were used to evaluate the antibacterial performance of the hydrogels, enabling a comparative analysis of their inhibitory effects across different formulations.
为了评估水凝胶提取物的抗菌效果， 将经水凝胶提取物处理的大肠杆菌和金黄色葡萄球菌悬浮液进行十倍梯度稀释。 取每份稀释液中的 100 µL 接种到 LB 琼脂平板上。 将平板在 37°C 下孵育 12 小时，然后计数菌落形成单位(CFU)。 每组的 CFU 计数用于评估水凝胶的抗菌性能，以便比较分析不同配方的抑菌效果。

2.6 Anti-inflammatory effect assessment
2.6 抗炎效果评价

2.6.1 Immunofluorescence Analysis
2.6.1 免疫荧光分析

Immunofluorescence staining was employed to evaluate the expression of inflammation-related markers, including CD86, CD206, and iNOS, in cells treated with hydrogel extracts. Cells were seeded onto sterilized glass coverslips in 24-well plates and incubated until they reached 70–80% confluence. After 24 hours of co-incubation with the hydrogel extracts, cells were fixed with 4% paraformaldehyde at room temperature, permeabilized with 0.1% Triton X-100, and blocked with 5% bovine serum albumin (BSA) for 1 hour.
采用免疫荧光染色评估水凝胶提取物处理细胞后炎症相关标志物（包括 CD86、CD206 和 iNOS）的表达。将细胞接种于 24 孔板的无菌玻璃盖玻片上，孵育至细胞汇合度达到 70%-80%。与水凝胶提取物共孵育 24 小时后，室温下用 4%多聚甲醛固定细胞，用 0.1% Triton X-100 进行通透性处理，并用 5%牛血清白蛋白(BSA)封闭 1 小时。

Primary antibodies specific to CD86, CD206, and iNOS (1:200 dilution) were applied and incubated overnight at 4°C. After washing with PBS, cells were incubated with Alexa Fluor 488-labeled secondary antibodies (1:500 dilution) for 1 hour in the dark at room temperature. Nuclei were counterstained with DAPI for 5 minutes. Immunofluorescence images were captured using a confocal laser scanning microscope (CLSM, Zeiss LSM 880), and fluorescence intensities were quantified using ImageJ software. This provided an evaluation of the expression levels of these inflammatory markers and insights into the hydrogel's potential to modulate immune responses.
分别加入针对 CD86、CD206 和 iNOS 的特异性一抗（1:200 稀释），并在 4°C 下孵育过夜。用 PBS 清洗后，将细胞与 Alexa Fluor 488 标记的二抗（1:500 稀释）在室温下避光孵育 1 小时。细胞核用 DAPI 复染 5 分钟。使用共聚焦激光扫描显微镜（CLSM，Zeiss LSM 880）采集免疫荧光图像，并使用 ImageJ 软件定量荧光强度。这可以评估这些炎症标志物的表达水平，并深入了解水凝胶调节免疫反应的潜力。

2.6.2 Western blot analysis
2.6.2 蛋白质印迹分析

Western blotting was performed to quantify the expression of inflammation-related proteins (CD86, CD206, Arg-1, and iNOS) in cell samples treated with hydrogel extracts. After 24 hours of co-incubation, total proteins were extracted using RIPA buffer and subjected to SDS-PAGE. The proteins were then transferred to PVDF membranes, which were incubated overnight at 4°C with primary antibodies specific to CD86, CD206, Arg-1, and iNOS (1:1000 dilution). After that, the membranes were treated for an hour at room temperature with HRP-conjugated secondary antibodies (1:5000 dilution).
采用 Western 印迹法定量分析经水凝胶提取物处理的细胞样本中炎症相关蛋白（CD86、CD206、Arg-1 和 iNOS）的表达。共孵育 24 小时后，用 RIPA 缓冲液提取总蛋白，并进行 SDS-PAGE 电泳。随后，将蛋白转移至 PVDF 膜上，并在 4°C 下与 CD86、CD206、Arg-1 和 iNOS 特异性一抗（1:1000 稀释）孵育过夜。 之后，用 HRP 标记的二抗（1:5000 稀释）在室温下处理膜 1 小时。
A chemiluminescence detection device was used to visualize the protein bands, and enhanced chemiluminescence (ECL) reagents were used for band detection. The band intensities were quantified using ImageJ software. Immunofluorescence staining was performed in parallel to further validate the anti-inflammatory effects of the hydrogels, providing additional confirmation of their potential to modulate inflammatory responses.
使用化学发光检测装置对蛋白质条带进行可视化 ，并使用增强化学发光 (ECL) 试剂进行条带检测。 使用 ImageJ 软件对条带强度进行量化。同时进行免疫荧光染色，以进一步验证水凝胶的抗炎作用，进一步证实其调节炎症反应的潜力。

2.7 Antioxidant Capacity Evaluation
2.7 抗氧化能力评价

Flow cytometry was used to assess the impact of hydrogel extracts on intracellular reactive oxygen species (ROS) levels. Cells were exposed to the hydrogel extracts for a 24-hour period, followed by incubation with the ROS-sensitive fluorescent probe, 2',7'-dichlorofluorescein diacetate (DCFH-DA), at 37°C in the dark for 30 minutes. Following the incubation, the cells were washed with PBS to remove any excess probe, and then collected for analysis. The fluorescence intensity of the oxidized DCFH-DA, which is directly proportional to ROS levels, was quantified using a flow cytometer. The data were analyzed to compare the ROS production between the treated and untreated groups, providing a quantitative assessment of the antioxidant properties of the hydrogels.
采用流式细胞术评估水凝胶提取物对细胞内活性氧 (ROS) 水平的影响。 将细胞暴露于水凝胶提取物中 24 小时，然后与 ROS 敏感的荧光探针 2',7'-二氯荧光素二乙酸酯 (DCFH-DA) 在 37°C 黑暗条件下孵育 30 分钟。孵育结束后，用 PBS 清洗细胞以去除多余的探针，然后收集细胞进行分析。氧化 DCFH-DA 的荧光强度与 ROS 水平成正比，可使用流式细胞仪进行定量分析。分析数据以比较处理组和未处理组之间的 ROS 生成情况，从而定量评估水凝胶的抗氧化性能。

2.8 In Vitro Angiogenesis Assay
2.8 体外血管生成试验

Human umbilical vein endothelial cells (HUVECs) were used in a tube formation experiment to assess the hydrogels' possible pro-angiogenic effects. Matrigel (Corning, USA) was thawed overnight at 4°C, and 50 µL of Matrigel was dispensed into each well of a 96-well plate, which was then incubated at 37°C for 30 minutes to allow solidification. HUVECs were cultivated in a variety of conditions after being seeded onto the solidified Matrigel layer at a density of 2×10⁴ cells/mL, including a control group and experimental groups treated with hydrogel extracts. The cells were incubated in an environment containing 5% CO₂ for six hours at 37°C. During incubation, the cells were stained with Calcein-AM, and the formation of tube-like structures was observed using an inverted fluorescence microscope (Zeiss Axio Observer A1, Zeiss, Germany). Key parameters of tube formation, including the total branch length, number of branch points, and mesh count, were analyzed using the "Angiogenesis Analyzer" plugin in ImageJ software (NIH, USA). Every experiment was carried out in triplicate, and the mean ± standard deviation (SD) was used to express the results. To ascertain the data' significance, statistical analysis was done.
使用人脐静脉内皮细胞 (HUVEC) 进行管形成实验，以评估水凝胶可能产生的促血管生成作用。 将 Matrigel (Corning, USA) 在 4°C 下解冻过夜，将 50 µL Matrigel 分装到 96 孔板的每个孔中，然后在 37°C 下孵育 30 分钟使其凝固。 将 HUVEC 以 2×10⁴ 细胞/mL 的密度接种到凝固的 Matrigel 层上后，在各种条件下进行培养 ，包括对照组和用水凝胶提取物处理的实验组。 将细胞在 37°C 下含有 5% CO₂ 的环境中孵育 6 小时。 在孵育期间，用 Calcein-AM 对细胞进行染色，并使用倒置荧光显微镜 (Zeiss Axio Observer A1, Zeiss, Germany) 观察管状结构的形成。使用 ImageJ 软件（美国国立卫生研究院）中的“血管生成分析器”插件分析了血管形成的关键参数，包括总分支长度、分支点数量和网格数。 每个实验重复三次，结果以平均值±标准差 (SD) 表示。为了确定数据的显著性，进行了统计学分析。

2.9 Hemolysis Assay
2.9 溶血试验

To assess the hemolytic potential of the hydrogels, a red blood cell (RBC) hemolysis assay was conducted. Whole blood was obtained from healthy Sprague-Dawley rats (purchased from the Experimental Animal Center of Shantou University Medical College) and collected in EDTA-coated tubes. The blood was centrifuged at 1500 rpm for 10 minutes at 4°C to isolate RBCs, which were subsequently washed three times with PBS (pH 7.4) and diluted to a 2% (v/v) RBC suspension. A positive control (0.1% Triton X-100 for total hemolysis) and a negative control (PBS) were included in the experimental groups, and groups treated with hydrogel extracts, which were prepared by incubating the hydrogels in PBS at 37°C for 24 hours. 200 µL of each reaction mixture was put into a 96-well plate, incubated for an hour at 37°C, and then centrifuged for five minutes at 4°C at 1500 rpm. The absorbance of the supernatant was measured at 540 nm using a microplate reader (Thermo Fisher Scientific, USA). The hemolysis rate was calculated using the following formula:
为了评估水凝胶的溶血潜力，进行了红细胞 (RBC) 溶血试验。从健康 Sprague-Dawley 大鼠（购自汕头大学医学院实验动物中心）采集全血，并收集于 EDTA 涂层管中。将血液在 4°C 下以 1500 rpm 的速度离心 10 分钟以分离红细胞，然后用 PBS（pH 7.4）洗涤三次，并稀释为 2% (v/v) 红细胞悬浮液。实验组和用水凝胶提取物处理的组均设有阳性对照（0.1% Triton X-100 用于完全溶血）和阴性对照（PBS）。 水凝胶提取物是将水凝胶在 PBS 中在 37°C 下孵育 24 小时后制备的。 将 200 µL 反应液加入 96 孔板中，37 ℃孵育 1 小时，4 ℃以 1500 rpm 离心 5 分钟。 使用酶标仪（美国赛默飞世尔科技公司）在 540 nm 处测定上清液的吸光度。溶血率计算公式如下：

Hemolysis Rate (%) = [(OD_sample − OD_PBS) / (OD_Triton − OD_PBS)] × 100%
溶血率 (%) = [(OD_sample − OD_PBS) / (OD_Triton − OD_PBS)] × 100%

All experiments were performed in triplicate, the mean ± standard deviation (SD) was used to express the results. A hemolysis rate below 5% was considered indicative of non-hemolytic activity.
所有实验均重复进行三次， 结果以平均值±标准差（SD）表示。 溶血率低于 5%视为无溶血活性。

2.10 In vivo full-thickness skin defect rat model
2.10 体内全层皮肤缺损大鼠模型

To assess the therapeutic effects of hydrogels on wound healing, a full-thickness skin defect model was established using male Sprague-Dawley (SD) rats (8 weeks old). The rats were housed in standard conditions and allowed to acclimatize for one week prior to the experiment. Before surgery, sodium pentobarbital (50 mg/kg) was injected intraperitoneally to put the rats to sleep. After shaving and cleaning the dorsal region with iodine solution, a biopsy punch was used to make a full-thickness skin defect that was 10 mm in diameter. Five groups of three animals each were randomly assigned: a blank control group (PBS treatment) and four experimental groups treated with SA/CMCS, SA/CMCS/TA, SA/CMCS/BAG, and SA/CMCS/TA/BAG hydrogels. An equal amount of each hydrogel was evenly applied to the wound site and covered with a breathable dressing.
为评估水凝胶对伤口愈合的治疗作用，使用雄性 Sprague-Dawley (SD) 大鼠（8 周龄）建立全层皮肤缺损模型。将大鼠饲养在标准条件下，并在实验前使其适应一周。术前， 腹腔注射戊巴比妥钠（50 mg/kg）使大鼠睡眠。剃毛并用碘溶液清洁背部区域后，使用活检穿刺器制作直径为 10 mm 的全层皮肤缺损。 随机分为五组，每组三只动物 ：空白对照组（PBS 治疗）和四个实验组，分别用 SA/CMCS、SA/CMCS/TA、SA/CMCS/BAG 和 SA/CMCS/TA/BAG 水凝胶治疗。将等量的每种水凝胶均匀涂抹于伤口部位，并用透气敷料覆盖。

Photographs of the wounds were taken on postoperative days 0, 3, 5, 7, 9, 11, and 14 to monitor changes in wound area. Wound closure rates were calculated using ImageJ software (NIH, USA) according to the formula:
术后第 0、3、5、7、9、11 和 14 天拍摄伤口照片，以监测伤口面积的变化。使用 ImageJ 软件（美国国立卫生研究院）根据以下公式计算伤口闭合率：

Wound Closure Rate (%) = [(Initial Wound Area − Current Wound Area) / Initial Wound Area] × 100%.
伤口闭合率（%）= [（初始伤口面积 − 当前伤口面积）/初始伤口面积] × 100%。

On day 14, the rats were euthanized, and the wound tissues were excised, the wound tissues were removed, preserved in 4% paraformaldehyde, embedded in paraffin, sectioned, and stained with Masson's trichrome and hematoxylin and eosin (H&E) for histological analysis and collagen deposition study. Tissue sections were analyzed using an inverted microscope.
第 14 天，处死大鼠，切除创面组织，取出创面组织，4%多聚甲醛保存，石蜡包埋，切片，Masson 三色染色和苏木精-伊红(H&E)染色，进行组织学分析和胶原沉积研究。倒置显微镜下观察组织切片。

2.11 Statistical Analysis
2.11 统计分析

All experimental data are presented as mean ± standard deviation (SD). Statistical analysis was performed using GraphPad Prism 9.0 (GraphPad Software, USA). Pairwise comparisons were made using an unpaired two-tailed Student’s t-test, while comparisons among multiple groups were analyzed with one-way analysis of variance (ANOVA) followed by Tukey’s post hoc test. A p-value of less than 0.05 (P < 0.05) was considered statistically significant. Graphs were generated to visualize the data, and the statistical tests used are indicated in the figure legends.
所有实验数据均以平均值±标准差（SD）表示。统计学分析使用 GraphPad Prism 9.0（GraphPad Software，美国）进行。成对比较采用非配对双尾学生 t 检验，多组间比较采用单因素方差分析 (ANOVA) 进行，随后进行 Tukey 事后检验。p 值小于 0.05（P < 3C < 0.05）被认为具有统计学意义。我们生成图表以直观呈现数据，所使用的统计学检验方法在图例中注明。

3.Results and discussion
3.结果与讨论

3.1 Fabrication and characterization of hydrogels
3.1 水凝胶的制备与表征

To align with the physiological processes of infection control and wound healing, a pH-responsive hydrogel was developed by exploiting the electrostatic interactions between the amino groups of carboxymethyl chitosan (CMCS) and the carboxyl groups of sodium alginate (SA). The FTIR spectrum (Fig. 2A) of the SA/CMCS/TA/BAG hydrogel revealed several characteristic absorption peaks. The stretching vibrations of the amino (-NH₂) and hydroxyl (-OH) groups were identified as the cause of a large peak at about 3250 cm⁻¹, which reflect interactions between chitosan (CS) and tannic acid (TA)[22]. Furthermore, a prominent peak near 1600 cm⁻¹ corresponds to the C=O stretching vibration of carboxyl (-COOH) and amide I bands, indicating crosslinking between the carboxyl groups of SA and the amino groups of CS, forming a hydrogel network[23]. Peaks in the 1000–1100 cm⁻¹ range correspond to C-O-C stretching vibrations, further confirming the crosslinked polymeric structure of SA and CS [24]. Additionally, a peak around 500 cm⁻¹ was assigned to the bending vibration of Si-O-Si bonds, suggesting the successful incorporation of bioglass (BAG) into the hydrogel matrix. These spectral features confirm the chemical functionality and structural stability of the hydrogel[25].
为了配合感染控制和伤口愈合的生理过程，利用羧甲基壳聚糖 (CMCS) 的氨基和海藻酸钠 (SA) 的羧基之间的静电相互作用，开发了一种 pH 响应性水凝胶。SA/CMCS/TA/BAG 水凝胶的 FTIR 光谱（图 2A）显示出几个特征吸收峰。氨基 (-NH₂) 和羟基 (-OH) 的伸缩振动被确定为在约 3250 cm⁻¹ 处出现大峰的原因 ，这反映了壳聚糖 (CS) 和单宁酸 (TA) 之间的相互作用 [22] 。此外，1600 cm⁻¹ 附近的突出峰对应于羧基 (-COOH) 的 C=O 伸缩振动和酰胺 I 带，表明 SA 的羧基和 CS 的氨基之间发生了交联，形成了水凝胶网络 [23] 。 1000–1100 cm-1 范围内的峰对应于 COC 伸缩振动，进一步证实了 SA 和 CS 的交联聚合物结构 [24] 。此外，500 cm-1 左右的峰归因于 Si-O-Si 键的弯曲振动，表明生物玻璃（BAG）已成功掺入水凝胶基质中。这些光谱特征证实了水凝胶的化学功能和结构稳定性 [25] 。

The chemical composition of the hydrogel was further investigated using XPS to analyze its chemical structure (Fig. 2B). The C 1s signal observed at 284.8 eV was indicative of C–C/C–H bonds, a common feature of all organic components[26]. Peaks at 286.5 eV and 288.2 eV were attributed to C–O and O=C–N bonds, respectively, confirming the presence of tannic acid (TA) and chitosan (CS) [27]. Additionally, peaks for Zn 2p and Ca 2p at 1020–1045 eV and 346–352 eV, respectively, were observed, which are characteristic of zinc ions (Zn²⁺) and calcium ions (Ca²⁺) from the bioglass (BAG), known for their antimicrobial and angiogenic properties[28].
使用 XPS 进一步研究了水凝胶的化学组成，分析了其化学结构（图 2B）。在 284.8 eV 处观察到的 C 1s 信号表明存在 C–C/C–H 键，这是所有有机成分的共同特征 [26] 。286.5 eV 和 288.2 eV 处的峰分别归因于 C–O 和 O=C–N 键，证实了单宁酸 (TA) 和壳聚糖 (CS) 的存在 [27] 。此外，在 1020–1045 eV 和 346–352 eV 处分别观察到 Zn 2p 和 Ca 2p 的峰，这是生物玻璃 (BAG) 中锌离子 (Zn²⁺) 和钙离子 (Ca²⁺) 的特征，生物玻璃以抗菌和血管生成特性而闻名 [28] 。

Further analysis of the XPS N 1s spectra (Fig. 2E) revealed peaks at approximately 399.5 eV and 400.5 eV, which correspond to free amino groups (-NH₂/-NH₃⁺) and amide bonds (-CONH₂), respectively. These peaks demonstrate that the carboxyl groups of SA and the amino groups of CMCS crosslink to produce a stable hydrogel network[29]. Notably, less than 50% of the amino groups in chitosan were protonated, indicating a mixture of protonated and deprotonated chitosan during the binding process. The observed chemical shifts in the N 1s spectra support the presence of hydrogen bonding and electrostatic interactions between TA and CS, as well as possible weak interactions with bioglass[30].
进一步分析 XPS N 1s 谱图（图 2E）发现，峰位于约 399.5 eV 和 400.5 eV 处，分别对应于游离氨基（-NH₂/-NH₃⁺）和酰胺键（-CONH₂）。这些峰表明 SA 的羧基和 CMCS 的氨基交联形成了稳定的水凝胶网络[29]。值得注意的是，壳聚糖中不到 50% 的氨基被质子化，表明在结合过程中存在质子化和去质子化壳聚糖的混合物。N 1s 谱图中观察到的化学位移支持 TA 和 CS 之间存在氢键和静电相互作用，以及与生物玻璃之间可能存在的弱相互作用[30]。

The C 1s spectra (Fig. 2D) showed peaks at 284.8 eV (C–C/C–H) and 286.5 eV (C–N/C–O), suggesting potential interactions, such as hydrogen bonding or electrostatic forces, between CMCS and TA. The O 1s spectra (Fig. 2F) exhibited peaks at 531.0 eV and 532.5 eV, which are associated with carbonyl (C=O) and hydroxyl (-OH) or siloxane (Si–O) bonds, indicating successful incorporation of BAG into the composite[31].
C 1s 谱（图 2D）在 284.8 eV（C-C/C-H）和 286.5 eV（C-N/C-O）处出现峰，表明 CMCS 和 TA 之间存在潜在的相互作用，例如氢键或静电力。O 1s 谱（图 2F）在 531.0 eV 和 532.5 eV 处出现峰，这些峰与羰基（C=O）和羟基（-OH）或硅氧烷（Si-O）键有关，表明 BAG 已成功掺入复合材料中[31]。

XRD analysis of the SA/CMCS/TA/BAG group (Fig. 2C) revealed a broad diffraction peak around 20°–30°, consistent with the amorphous nature of the hydrogel. In contrast, the SA/CMCS/BAG group exhibited weaker diffraction peaks in the 2θ range of 30°–35°, confirming the integration of BAG microcrystalline structures into the hydrogel matrix. The amorphous nature of the hydrogel allows it to retain high flexibility and processability[32].
SA/CMCS/TA/BAG 组的 XRD 分析（图 2C）显示在 20°~30° 左右有一个宽的衍射峰，这与水凝胶的无定形性质相符。相比之下，SA/CMCS/BAG 组在 30°~35° 的 2θ 范围内显示出较弱的衍射峰，证实了 BAG 微晶结构已整合到水凝胶基质中。水凝胶的无定形性质使其保持了较高的柔韧性和可加工性[32]。

SEM images of the composite hydrogels (Fig. 2G) revealed distinct microstructural variations, further supporting the successful incorporation of the individual components into the SA/CMCS/TA/BAG system. The SA/CMCS hydrogel exhibited a porous structure, promoting biocompatibility and facilitating nutrient exchange. The addition of tannic acid (TA) resulted in a denser surface morphology, indicating stronger intermolecular interactions. The presence of bioactive glass (BAG) introduced granular features to the surface, enhancing the hydrogel's rigidity and providing active sites for bioactivity, such as ion release and cellular interactions[33].
复合水凝胶的 SEM 图像（图 2G ） 揭示了明显的微观结构变化，进一步证明了各个组分成功整合到 SA/CMCS/TA/BAG 体系中。SA/CMCS 水凝胶呈现多孔结构，有利于生物相容性并促进营养交换。单宁酸（TA）的添加使表面形貌更致密，表明分子间相互作用更强。生物活性玻璃（BAG）的存在为表面引入了颗粒特征，增强了水凝胶的刚性，并为离子释放和细胞相互作用等生物活性提供了活性位点 [3 3 ] 。

Both SA/CMCS/BAG and SA/CMCS/TA/BAG displayed granular surface morphologies, highlighting BAG's influence on the hydrogel's structure. These granular inclusions likely contribute to the formation of micropores, which increases the surface area and enhances the release of bioactive ions. The synergistic effects of TA and BAG improved the hydrogel's mechanical strength and bioactivity, making it conducive to cellular adhesion, proliferation, and nutrient exchange[34].
SA/CMCS/BAG 和 SA/CMCS/TA/BAG 均呈现出颗粒状表面形貌，凸显了 BAG 对水凝胶结构的影响。这些颗粒状内含物可能有助于微孔的形成，从而增加表面积并增强生物活性离子的释放。TA 和 BAG 的协同作用提高了水凝胶的机械强度和生物活性，使其有利于细胞粘附、增殖和营养交换 [3 4 ] 。

Energy-dispersive X-ray spectroscopy (EDS) (Fig. 2H) further revealed a homogeneous distribution of organic (C, N, O) and inorganic (Ca, Si, Zn) elements within the SA/CMCS/TA/BAG hydrogel. This distribution highlights the successful incorporation of TA and BAG into a stable network, with the SA/CMCS/TA/BAG hydrogel exhibiting the most intricate element distribution. The uniform dispersion of Ca, Si, and Zn throughout the hydrogel matrix underscores the cooperative effects of TA and BAG, significantly contributing to the material’s enhanced mechanical properties and multifunctional bioactivity[35].
能量色散 X 射线光谱(EDS)（图 2 H ）进一步揭示了 SA/CMCS/TA/BAG 水凝胶中有机元素（C、N、O）和无机元素（Ca、Si、Zn）的均匀分布。这种分布表明 TA 和 BAG 已成功整合成稳定的网络结构，其中 SA/CMCS/TA/BAG 水凝胶表现出最为复杂的元素分布。Ca、Si 和 Zn 在整个水凝胶基质中的均匀分散凸显了 TA 和 BAG 的协同作用，显著提高了材料的机械性能和多功能生物活性 [ 35 ] 。

To optimize the hydrogel's response to sequential physiological processes, its drug release behavior was evaluated under different pH conditions. At pH 5.5, simulating an acidic environment in infected wounds, and at pH 7.4, resembling the mildly alkaline environment of healing tissue, the hydrogel demonstrated distinct staged-release profiles (Fig. L, Fig. M).
为了优化水凝胶对连续生理过程的响应，我们评估了其在不同 pH 条件下的药物释放行为。在 pH 5.5（模拟感染伤口的酸性环境）和 pH 7.4（模拟愈合组织中的弱碱性环境）条件下，水凝胶表现出明显的阶段性释放曲线（图 L 和图 M ）。

In the acidic environment (pH 5.5), the hydrogel demonstrated a rapid release of tannic acid (TA), achieving approximately 50% cumulative release within the first two days. By day 8, the release rate slowed but approached 85%, ultimately reaching nearly 100% by day 14. This swift release of TA is attributed to its structural characteristics, as its phenolic hydroxyl groups (-OH) exhibit increased solubility under acidic conditions due to reduced ionization. Furthermore, protonation of the carboxyl groups (-COOH) on the sodium alginate (SA) chains strengthens hydrogen bonding with chitosan (CMCS) via -COOH and -OH interactions, causing the hydrogel network to contract and accelerating the release of TA. This behavior aligns with the early acute inflammation phase in infected wounds, where effective pathogen suppression is critical[36-37].
在酸性环境（pH 5.5）下，水凝胶能快速释放单宁酸 (TA)，在前两天内累计释放量约为 50%。到第 8 天，释放速度减慢但接近 85%，到第 14 天最终达到接近 100%。TA 的快速释放归因于其结构特征，因为其酚羟基 (-OH) 在酸性条件下由于电离降低而表现出更高的溶解度。此外，海藻酸钠 (SA) 链上羧基 (-COOH) 的质子化通过 -COOH 和 -OH 相互作用增强了与壳聚糖 (CMCS) 的氢键结合，导致水凝胶网络收缩并加速 TA 的释放。这种行为与感染伤口的早期急性炎症阶段相一致，此时有效的病原体抑制至关重要 [3 6 -3 7 ] 。

In contrast, at pH 5.5, the release of zinc (Zn²⁺) and calcium (Ca²⁺) ions from bioglass (BAG) was minimal, ensuring that the primary function of the hydrogel during this stage remained antibacterial rather than reparative. This design strategy effectively prioritizes infection control while preserving the bioactive ions for later release during the tissue repair phase at pH 7.4. These stage-specific release profiles emphasize the hydrogel's potential to provide tailored therapeutic responses throughout the wound healing continuum[38].
相比之下，在 pH 5.5 时，生物玻璃（BAG）中锌离子（Zn²⁺）和钙离子（Ca²⁺）的释放量极小，确保了水凝胶在此阶段的主要功能仍然是抗菌而非修复。这种设计策略有效地优先考虑了感染控制，同时保留了生物活性离子，以便在 pH 7.4 的组织修复阶段释放。这些特定阶段的释放曲线强调了水凝胶在整个伤口愈合过程中提供个性化治疗反应的潜力 [ 38 ] 。

There were significant variations in the release patterns of calcium ions (Ca²⁺) and zinc ions (Zn²⁺) from bioglass at pH 5.5 and pH 7.5. (Fig. L, Fig. M). In the acidic environment of pH 5.5, calcium ion release was relatively slow, with concentrations reaching approximately 10 mg/L in the first two days. Over the following 14 days, the concentration gradually increased to around 25 mg/L. In contrast, at pH 7.5, the release of calcium ions accelerated markedly, reaching nearly 30 mg/L within 48 hours and maintaining higher release levels in subsequent days. This indicates that the release of calcium ions is significantly influenced by pH, with a faster release in alkaline conditions supporting endothelial cell activation and angiogenesis during wound healing[39].
在 pH 5.5 和 pH 7.5 下，生物玻璃中钙离子（Ca²⁺）和锌离子（Zn²⁺）的释放模式存在显著差异。 （图 L ，图 M ） 。在 pH 5.5 的酸性环境中，钙离子的释放相对较慢，前两天浓度达到约 10mg/L。在接下来的 14 天里，浓度逐渐增加到 25mg/L 左右。相反，在 pH 7.5 时，钙离子的释放明显加快，在 48 小时内达到近 30mg/L，并在随后的几天保持较高的释放水平。这表明钙离子的释放受 pH 值的显著影响，在碱性条件下释放速度更快，支持伤口愈合过程中的内皮细胞活化和血管生成 [ 39 ] 。

Similarly, the release of zinc ions followed a comparable pH-dependent trend. While the release of zinc ions was relatively low in the acidic environment, reaching approximately 15 mg/L over 14 days, it was significantly enhanced in the alkaline condition, reaching about 20 mg/L within two days and continuing to increase thereafter. This accelerated release aligns with the critical role of zinc ions in promoting fibroblast proliferation and inhibiting microbial growth, thereby creating a favorable microenvironment for tissue repair[40].
同样地，锌离子的释放也呈现出类似的 pH 依赖性趋势。在酸性环境中，锌离子的释放相对较低，在 14 天内达到约 15 毫克/升，但在碱性条件下，锌离子的释放显著增强，在两天内达到约 20 毫克/升，此后持续增加。这种加速释放与锌离子在促进成纤维细胞增殖和抑制微生物生长中的关键作用相一致，从而为组织修复创造了有利的微环境 [ 40 ] 。

Wounds typically experience a progressive pH transition during healing. Acute wounds are initially acidic (pH 5.5–6.5) due to inflammatory responses and tissue damage. As healing progresses, tissue regeneration and repair are often accompanied by an increase in pH toward a neutral or slightly alkaline environment (pH 7.0–7.5). This natural shift in pH facilitates cell proliferation and tissue remodeling[41].
伤口在愈合过程中通常会经历 pH 值的逐渐变化。由于炎症反应和组织损伤，急性伤口最初呈酸性（pH 5.5-6.5）。随着愈合的进展，组织再生和修复通常伴随着 pH 值的升高，逐渐趋向中性或弱碱性环境（pH 7.0-7.5）。pH 值的这种自然变化有助于细胞增殖和组织重塑 [ 41 ] 。

In the early stages, tannic acid (TA) exerts its antibacterial effects by binding to bacterial cell wall proteins, disrupting their structure, and inhibiting microbial growth. Although TA is acidic, its antimicrobial activity does not significantly alter the overall pH of the wound. By reducing bacterial load and associated acidic byproducts, TA indirectly contributes to an increase in wound pH. This pH-responsive release mechanism ensures that TA exerts a potent antibacterial effect during the early acute inflammatory phase, preventing excessive pathogen proliferation without creating an environment overly favorable for bacterial survival[42].
在早期阶段，单宁酸 (TA) 通过与细菌细胞壁蛋白结合、破坏其结构并抑制微生物生长来发挥抗菌作用。尽管 TA 呈酸性，但其抗菌活性不会显著改变伤口的整体 pH 值。通过减少细菌负荷及其相关的酸性副产物，TA 间接促进了伤口 pH 值的升高。这种 pH 响应性释放机制确保 TA 在早期急性炎症期发挥强效抗菌作用，防止病原体过度增殖，同时又不会创造过于有利于细菌生存的环境 [4 2 ] 。

To optimize the bioactivity of bioglass, its composition can be adjusted by reducing SiO₂ content while increasing levels of ZnO and CaO. This modification weakens the silicate network, facilitating faster dissolution and ion release in alkaline environments. The release of Zn²⁺ and Ca²⁺ ions promotes angiogenesis, enhances cell migration, and supports tissue repair, thereby accelerating wound healing while providing sustained reparative support[43].
为了优化生物玻璃的生物活性，可以通过降低 SiO₂含量、增加 ZnO 和 CaO 含量来调整其成分。这种改性会削弱硅酸盐网络，使其在碱性环境中更快地溶解和释放离子。Zn²⁺和 Ca²⁺离子的释放可促进血管生成，增强细胞迁移，并支持组织修复，从而加速伤口愈合，并提供持续的修复支持 [4 3 ] 。

In conclusion, while the release of TA may lead to localized acidification, the pH-responsive hydrogel is designed to maintain its antibacterial efficacy while preventing conditions overly conducive to bacterial growth. This balanced approach effectively addresses both the need for infection control and the promotion of wound healing.
总而言之，虽然 TA 的释放可能导致局部酸化，但 pH 响应性水凝胶的设计旨在保持其抗菌功效，同时防止过度有利于细菌生长的条件。这种平衡的方法有效地满足了控制感染和促进伤口愈合的需求。

3.2 In vitro biocompatibility
3.2 体外生物相容性

To determine the optimal concentrations of tannic acid (TA) and bioglass (BAG) in sodium alginate-chitosan hydrogels, the CCK-8 assay was used to assess the vitality and proliferation of the cells. The findings showed that BAG was most efficient at 10 mg/mL, while the most effective TA concentration was 0.5% (w/v). (Fig. 2I). At this concentration of 0.5% TA, both cell proliferation and viability were significantly enhanced, demonstrating that TA's antioxidative and antibacterial properties are best expressed at this level. It also promotes cell adhesion and proliferation within the hydrogel matrix. In contrast, TA concentrations either below or above 0.5% resulted in diminished cell activity. Low concentrations of TA were insufficient to produce the desired biological effects, while higher concentrations exhibited cytotoxicity, disrupting the cellular microenvironment and hindering normal cell growth. Similarly, BAG displayed the highest bioactivity at a concentration of 10 mg/mL, where the release of bioactive ions was optimal for promoting cell proliferation and differentiation. Concentrations below this level resulted in inadequate ion release, leading to reduced cellular activity, while excessive BAG concentrations overstimulated the cellular environment and compromised the mechanical integrity of the hydrogel. Thus, the combination of 0.5% TA and 10 mg/mL BAG optimized cell viability and proliferation without compromising the hydrogel’s structural or mechanical properties.
为了确定海藻酸钠-壳聚糖水凝胶中单宁酸 (TA) 和生物玻璃 (BAG) 的最佳浓度， 使用 CCK-8 测定法评估细胞的活力和增殖。研究结果表明，BAG 在 10 mg/mL 时最有效，而最有效的 TA 浓度为 0.5% (w/v)。 (图 2I )。在 0.5% TA 浓度下，细胞增殖和活力均显著增强，表明 TA 的抗氧化和抗菌特性在此水平下得到最佳体现。它还能促进细胞在水凝胶基质内的粘附和增殖。相反，低于或高于 0.5% 的 TA 浓度都会导致细胞活性降低。低浓度的 TA 不足以产生所需的生物学效应，而较高浓度则表现出细胞毒性，破坏细胞微环境并阻碍正常细胞生长。类似地，BAG 在浓度为 10 mg/mL 时表现出最高的生物活性，此时生物活性离子的释放最适合促进细胞增殖和分化。低于此浓度会导致离子释放不足，从而降低细胞活性；而过高的 BAG 浓度则会过度刺激细胞环境，并损害水凝胶的机械完整性。因此，0.5% TA 和 10 mg/mL BAG 的组合优化了细胞活力和增殖，同时不损害水凝胶的结构或机械性能。

The biocompatibility of the hydrogel was further assessed using in vitro scratch assays. The SA/CMCS/TA/BAG formulation demonstrated significant scratch closure within 24 hours (Fig. 3A), highlighting the hydrogel’s capacity to enhance HUVEC cell migration and proliferation. As a three-dimensional scaffold, the hydrogel offers essential physical support for cell adhesion and migration, mimicking the structural characteristics of the extracellular matrix (ECM) and creating an environment conducive to cell growth. The incorporation of TA into the hydrogel further supports this microenvironment through its potent antioxidative and anti-inflammatory properties, which reduce the levels of reactive oxygen species (ROS) and inflammatory cytokines. This modulation promotes a favorable environment for cellular proliferation. Additionally, TA may regulate signaling pathways, such as PI3K/Akt, which further enhances cell growth and migration[44].
采用体外划痕试验进一步评估了水凝胶的生物相容性。SA/CMCS/TA/BAG 配方在 24 小时内显示出明显的划痕闭合（图 3A），凸显了水凝胶增强 HUVEC 细胞迁移和增殖的能力。作为三维支架，水凝胶为细胞粘附和迁移提供了必要的物理支撑，模拟了细胞外基质 (ECM) 的结构特征并创造了有利于细胞生长的环境。TA 加入水凝胶中，通过其强大的抗氧化和抗炎特性进一步支持了这种微环境，从而降低了活性氧 (ROS) 和炎性细胞因子的水平。这种调节促进了有利于细胞增殖的环境。此外，TA 还可以调节 PI3K/Akt 等信号通路，从而进一步增强细胞生长和迁移 [4 4 ] 。

BAG also contributed significantly by releasing calcium (Ca²⁺) and zinc (Zn²⁺) ions, which promoted cell proliferation. Calcium ions are crucial for cellular signal transduction, affecting cell cycle progression and division, while zinc ions play a vital role in cellular growth through their antioxidative and anti-inflammatory effects, as well as their involvement in protein synthesis[45]. Additionally, the ions released from BAG support angiogenesis, enhancing nutrient and oxygen delivery to regenerating tissues, thereby accelerating tissue repair.
BAG 还通过释放钙离子（Ca²⁺）和锌离子（Zn²⁺）促进细胞增殖，发挥了重要作用。钙离子对细胞信号转导至关重要，影响细胞周期进程和分裂；而锌离子则通过其抗氧化、抗炎作用以及参与蛋白质合成，在细胞生长中发挥重要作用 [4 5 ] 。此外，BAG 释放的离子支持血管生成，增强营养和氧气向再生组织的输送，从而加速组织修复。

In the cytoskeleton staining experiment (Fig. 3B), cells from the SA/CMCS/TA/BAG group exhibited more organized and complete F-actin structures (FITC staining, green) compared to the control group. The cytoskeletal structures in this group were well-organized and densely aligned, indicating that the hydrogel significantly enhanced cytoskeletal integrity, improving both cellular mechanical properties and migration capacity. Stabilization of the cytoskeleton and improved cell-matrix adhesion are essential for effective cell migration. TA contributed to this by mitigating inflammation and reducing ROS levels, which helped maintain the integrity of the cytoskeleton[46]. Furthermore, calcium ions (Ca²⁺) released from BAG played a pivotal role in intracellular signaling, facilitating cytoskeletal remodeling and enhancing cell migration[47].
在细胞骨架染色实验中（图 3B），与对照组相比，SA/CMCS/TA/BAG 组的细胞表现出更有组织、更完整的 F- 肌动蛋白结构（FITC 染色，绿色）。该组的细胞骨架结构井然有序、排列紧密，表明水凝胶显著增强了细胞骨架的完整性，从而提高了细胞的机械性能和迁移能力。细胞骨架的稳定和细胞-基质粘附的改善对于有效的细胞迁移至关重要。TA 通过减轻炎症和降低 ROS 水平做出了贡献，这有助于维持细胞骨架的完整性 [4 6 ] 。此外，BAG 释放的钙离子 (Ca²⁺) 在细胞内信号传导中起关键作用，促进细胞骨架重塑并增强细胞迁移 [ 47 ] 。

In the live/dead cell assay, minimal cytotoxicity was observed across all experimental groups, with nearly all cells surviving (green fluorescence by Calcein-AM staining), and very few dead cells (red fluorescence by PI staining) detected (Fig. 3B). These results confirm the excellent biocompatibility of the hydrogel with HUVEC cells. Specifically, the SA/CMCS/TA/BAG group demonstrated high cell viability while also promoting cell migration and cytoskeletal remodeling. Overall, the findings suggest that the SA/CMCS/TA/BAG hydrogel exhibits outstanding biocompatibility, as shown by enhanced cell viability and minimal cytotoxicity in all assays, making it highly suitable for biomedical applications, especially in wound healing and tissue regeneration.
在活/死细胞测定中，所有实验组均观察到极小的细胞毒性，几乎所有细胞均存活（Calcein-AM 染色呈绿色荧光），检测到极少的死细胞（PI 染色呈红色荧光）（图 3B ） 。这些结果证实了水凝胶与 HUVEC 细胞具有出色的生物相容性。具体而言，SA/CMCS/TA/BAG 组表现出较高的细胞活力，同时还促进了细胞迁移和细胞骨架重塑。 总体而言，研究结果表明 SA/CMCS/TA/BAG 水凝胶表现出出色的生物相容性，如所有测定中细胞活力增强和细胞毒性极小所示，这使其非常适合生物医学应用，尤其是在伤口愈合和组织再生方面。

3.3 In vitro antibacterial accessment
3.3 体外抗菌评价

To assess whether the SA/CMSC/TA/BAG hydrogels meet antimicrobial requirements and effectively disrupt biofilm formation, we further investigated the killing mechanism of the hydrogels against bacteria.
为了评估 SA/CMSC/TA/BAG 水凝胶是否满足抗菌要求并有效破坏生物膜的形成，我们进一步研究了水凝胶杀灭细菌的机制。

The antibacterial properties of the hydrogels were evaluated through multiple methods, including confocal laser scanning microscopy (Fig. 4A and Fig. 4B), live/dead bacterial staining (Fig. 4C and Fig. 4D), and colony-forming unit (CFU) assays (Fig. 4E and Fig. 4F). In all experiments, the control group consistently displayed robust and intact biofilms, with high bacterial viability, as indicated by strong green fluorescence under microscopy, minimal red fluorescence in live/dead staining, and a large number of bacterial colonies in the CFU assay. The SA/CMSC hydrogel showed limited antibacterial activity, with only a slight reduction in biofilm density, green fluorescence, and CFU counts, suggesting that the hydrogel matrix alone lacks sufficient antibacterial efficacy.
通过多种方法评估水凝胶的抗菌性能，包括共聚焦激光扫描显微镜 （图 4A 和图 4B ） 、活/死细菌染色（图 4C 和图 4D ）和菌落形成单位（CFU）测定（图 4E 和图 4F ） 。在所有实验中，对照组始终显示出坚固完整的生物膜，具有高细菌活力，这表现为显微镜下强的绿色荧光、活/死细菌染色中最小的红色荧光以及 CFU 测定中大量的细菌菌落。SA/CMSC 水凝胶表现出有限的抗菌活性，生物膜密度、绿色荧光和 CFU 计数仅略有降低，这表明单独的水凝胶基质缺乏足够的抗菌功效。

When tannic acid (TA) was incorporated into the hydrogel (SA/CMSC/TA), a notable improvement in antibacterial activity was observed across all testing methods. This included increased red fluorescence in live/dead staining, diminished green fluorescence under microscopy, and a significant reduction in CFU counts. The enhanced antibacterial effect can be attributed to TA’s multifaceted mechanisms, which disrupt bacterial membranes and inhibit bacterial survival. The polyphenolic structure of TA allows it to form hydrogen bonds with bacterial membrane proteins and phospholipids, causing the membrane to become unstable and intracellular materials like proteins and ions to leak out, which eventually results in bacterial cell death[48]. In addition, TA chelates essential metal ions like Fe²⁺ and Zn²⁺, depriving bacteria of key nutrients necessary for their metabolism and growth[49]. TA also induces oxidative stress by generating reactive oxygen species (ROS), which damage bacterial DNA, proteins, and lipids[50]. Furthermore, TA interferes with bacterial adhesion, hindering the establishment and persistence of biofilms[51]. Notably, TA’s antibacterial activity is enhanced in the acidic conditions of biofilms (pH 4.0–6.5), where the proton release from its phenolic groups intensifies its interaction with bacterial membranes and amplifies its bactericidal effects[52]. This pH-dependent behavior explains why TA exhibited stronger antibacterial activity than BAG under the same experimental conditions.
当单宁酸 (TA) 加入水凝胶 (SA/CMSC/TA) 中时，所有测试方法中抗菌活性均显著提高。这包括在活/死染色中红色荧光增加，显微镜下绿色荧光减弱，以及 CFU 计数显著减少。抗菌效果增强可归因于 TA 的多方面机制，它可以破坏细菌膜并抑制细菌存活。TA 的多酚结构使其能够与细菌膜蛋白和磷脂形成氢键， 导致膜变得不稳定，细胞内物质如蛋白质和离子泄漏，最终导致细菌细胞死亡 [ 48 ] 。此外，TA 螯合必需的金属离子如 Fe²⁺ 和 Zn²⁺，使细菌失去代谢和生长所需的关键营养物质 [ 49 ] 。TA 还通过产生活性氧 (ROS) 来诱导氧化应激，从而破坏细菌的 DNA、蛋白质和脂质 [ 50 ] 。此外，TA 会干扰细菌粘附，阻碍生物膜的建立和持久性 [ 51 ] 。值得注意的是，TA 的抗菌活性在生物膜的酸性条件下（pH 4.0-6.5）会增强，此时 TA 的酚基释放的质子会增强其与细菌膜的相互作用，从而增强其杀菌作用 [ 52 ] 。 这种 pH 依赖性行为解释了为什么在相同实验条件下 TA 表现出比 BAG 更强的抗菌活性。

The SA/CMSC/BAG hydrogel also demonstrated significant antibacterial effects, as shown by an increase in bacterial cell death and a reduction in CFU counts compared to the control. BAG exerts its antibacterial action by releasing zinc ions (Zn²⁺), which interfere with bacterial enzymatic processes, compromise membrane integrity, and inhibit biofilm formation[53]. However, in the acidic environment of the biofilm, BAG’s antibacterial performance was slightly less pronounced than that of TA, indicating the important role pH plays in modulating the relative effectiveness of these materials. The ability of TA to release protons in acidic conditions provides it with a distinct advantage, enabling it to outperform BAG under such circumstances.
SA/CMSC/BAG 水凝胶还表现出显著的抗菌效果，与对照组相比，细菌细胞死亡率增加，菌落形成单位 (CFU) 计数减少。BAG 通过释放锌离子 (Zn²⁺) 发挥抗菌作用，锌离子会干扰细菌酶促过程，破坏膜完整性，并抑制生物膜形成 [5 3 ] 。然而，在生物膜的酸性环境中，BAG 的抗菌性能略低于 TA，表明 pH 在调节这些材料的相对有效性方面起着重要作用。TA 在酸性条件下释放质子的能力为其提供了明显的优势，使其在这种情况下的表现优于 BAG。

Among all formulations, the SA/CMSC/TA/BAG hydrogel exhibited the strongest antibacterial performance, with minimal bacterial viability detected in all assays. This outstanding efficacy is mainly attributed to the potent antibacterial mechanisms of TA, which are particularly effective in low-pH environments. These results highlight the crucial role of the biofilm microenvironment in influencing the antibacterial effectiveness of the hydrogels, with TA emerging as the most potent agent in the hydrogel system for combating biofilm-associated infections.
在所有配方中，SA/CMSC/TA/BAG 水凝胶表现出最强的抗菌性能，在所有检测中均检测到极低的细菌活力。这种卓越的功效主要归功于 TA 强大的抗菌机制，尤其是在低 pH 环境下。这些结果凸显了生物膜微环境在影响水凝胶抗菌效果方面的关键作用，其中 TA 已成为水凝胶体系中对抗生物膜相关感染最有效的药物。

3.4 In Vitro Anti-Inflammatory Assessment
3.4 体外抗炎评价

Inflammation is an essential component of the wound healing process, as it helps to clear pathogens, cellular debris, and damaged tissue through the recruitment of immune cells such as neutrophils and macrophages. However, if inflammation persists or becomes excessive, it can impede healing, resulting in delayed cell proliferation, prolonged tissue damage, and hindrance of subsequent healing phases, including proliferation and remodeling. Additionally, the overproduction of reactive oxygen species (ROS) and pro-inflammatory cytokines (such IL-6 and TNF-α) is linked to chronic inflammation[54], which exacerbate tissue damage and prolong the inflammatory phase.
炎症是伤口愈合过程中的一个重要组成部分，它通过募集中性粒细胞和巨噬细胞等免疫细胞来帮助清除病原体、细胞碎片和受损组织。然而，如果炎症持续存在或过度，则会阻碍愈合，导致细胞增殖延迟、组织损伤延长，并阻碍后续的愈合阶段，包括增殖和重塑。 此外，活性氧 (ROS) 和促炎细胞因子（如 IL-6 和 TNF-α）的过量产生与慢性炎症有关 [5 4 ] ，这会加剧组织损伤并延长炎症期。

The inclusion of anti-inflammatory agents, particularly in advanced hydrogel formulations, helps to regulate this response by reducing the excessive production of inflammatory mediators, promoting macrophage polarization toward the pro-repair M2 phenotype, and scavenging ROS. Such controlled modulation of the immune response fosters efficient tissue repair, helps to prevent chronic inflammation, and reduces the risk of complications like scarring and non-healing wounds[55-56].
添加抗炎剂，尤其是在先进的水凝胶配方中，有助于调节这种反应，具体方式包括减少炎症介质的过量产生、促进巨噬细胞向促修复 M2 表型极化以及清除活性氧（ROS）。这种对免疫反应的受控调节有助于促进有效的组织修复，有助于预防慢性炎症，并降低瘢痕形成和伤口不愈合等并发症的风险 [5 5 -5 6 ]。

In our anti-inflammatory experiments, immunofluorescence analysis revealed that the SA/CMCS/TA/BAG hydrogel exhibits substantial anti-inflammatory effects. Specifically, the expression of CD86, a marker associated with pro-inflammatory M1 macrophages, and iNOS (inducible nitric oxide synthase, another M1 marker) was significantly reduced (Fig. 5A and Fig. 5B), indicating effective inhibition of M1 macrophage activation. In contrast, the same hydrogel formulation showed markedly higher expression levels of CD206 and Arginase-1 (Arg-1), both of which are markers indicative of M2 macrophages, suggesting a strong promotion of M2 polarization (Fig. 5C and Fig. 5D). These results were further validated by Western blot analysis (Fig. 5I), which confirmed the significant downregulation of iNOS and CD86, along with an increase in CD206 and Arg-1 expression, highlighting the hydrogel's ability to both suppress M1 macrophage activation and enhance M2 macrophage polarization. These findings are in agreement with the immunofluorescence results.
在我们的抗炎实验中，免疫荧光分析显示 SA/CMCS/TA/BAG 水凝胶表现出显著的抗炎作用。具体而言，促炎性 M1 巨噬细胞相关标志物 CD86 和 iNOS（诱导型一氧化氮合酶，另一种 M1 标志物）的表达显著降低（图 5A 和图 5B），表明有效抑制了 M1 巨噬细胞活化。相反，相同的水凝胶配方显示出明显更高的 CD206 和精氨酸酶-1 (Arg-1) 表达水平，这两者都是 M2 巨噬细胞的标志物，表明其强烈促进了 M2 极化（图 5C 和图 5D）。 Western blot 分析（图 5I）进一步验证了这些结果，结果证实了 iNOS 和 CD86 的显著下调，以及 CD206 和 Arg-1 表达的增加，凸显了该水凝胶能够抑制 M1 巨噬细胞活化并增强 M2 巨噬细胞极化。这些结果与免疫荧光结果一致。

The anti-inflammatory properties of the SA/CMCS/TA/BAG hydrogel are primarily attributed to the action of tannic acid (TA). By scavenging ROS and blocking pro-inflammatory signaling pathways including the NF-κB pathway, which triggers the release of pro-inflammatory cytokines like TNF-α and IL-6, TA functions as a strong antioxidant. This enables TA to reduce oxidative stress and inflammation, while also preventing the activation of M1 macrophages[57]. Additionally, TA promotes a favorable microenvironment for tissue repair by disrupting the oxidative stress cascade and indirectly encouraging M2 macrophage polarization[58]. While the calcium and zinc ions released from the SA/CMSC/BAG hydrogel also contribute to tissue repair and macrophage regulation—where zinc can inhibit the production of pro-inflammatory cytokines and calcium ions can aid macrophage function—the primary anti-inflammatory effects are driven by TA[59].
SA/CMCS/TA/BAG 水凝胶的抗炎特性主要归因于单宁酸 (TA) 的作用。 通过清除 ROS 和阻断促炎信号通路（包括 NF-κB 通路，该通路会触发促炎细胞因子如 TNF-α 和 IL-6 的释放），TA 发挥着强抗氧化剂的作用 。这使得 TA 能够减少氧化应激和炎症，同时还能阻止 M1 巨噬细胞的活化 [57] 。此外，TA 通过破坏氧化应激级联并间接促进 M2 巨噬细胞极化来促进有利于组织修复的微环境 [58] 。虽然 SA/CMSC/BAG 水凝胶释放的钙离子和锌离子也有助于组织修复和巨噬细胞调节（其中锌可以抑制促炎细胞因子的产生，而钙离子可以帮助巨噬细胞发挥功能），但主要的抗炎作用是由 TA 驱动的 [59] 。

In conclusion, the SA/CMCS/TA/BAG hydrogel effectively modulates inflammation through a dual mechanism: it suppresses M1 macrophage activation and promotes M2 macrophage polarization. This dual action makes the hydrogel a promising candidate for accelerating wound healing and enhancing tissue regeneration by providing a balanced anti-inflammatory response.
综上所述，SA/CMCS/TA/BAG 水凝胶通过双重机制有效调节炎症：抑制 M1 巨噬细胞活化，促进 M2 巨噬细胞极化。这种双重作用使该水凝胶成为加速伤口愈合和增强组织再生的有希望的候选材料，因为它能够提供均衡的抗炎反应。

3.5 In vitro antioxidant accessment
3.5 体外抗氧化评价

Excessive reactive oxygen species (ROS) in wounds can lead to oxidative stress, which causes cellular damage, delays healing, impairs angiogenesis, and disrupts collagen synthesis. To counteract this, it is crucial to neutralize ROS in order to mitigate oxidative damage, resolve inflammation, enhance angiogenesis, and promote efficient tissue repair. Antioxidant agents, such as tannic acid (TA), incorporated into wound dressings, help restore the balance of ROS, creating an environment that accelerates wound healing[60].
伤口中过量的活性氧 (ROS) 会导致氧化应激，从而造成细胞损伤、延缓愈合、抑制血管生成并干扰胶原蛋白合成。为了应对这种情况，中和 ROS 至关重要，这样才能减轻氧化损伤、消炎、促进血管生成并促进有效的组织修复。在伤口敷料中添加单宁酸 (TA) 等抗氧化剂有助于恢复 ROS 的平衡，从而创造一个加速伤口愈合的环境 [60] 。

Flow cytometry results demonstrated the varying effects of different hydrogel formulations on ROS levels (Fig. S1). The blank control group showed minimal signal intensity, establishing a baseline, while the positive control group confirmed the proper functioning of the experimental system and provided a reference for maximal ROS scavenging. The untreated control exhibited moderate signal intensity, reflecting an unaltered cellular response. The SA/CMCS hydrogel led to a moderate reduction in signal intensity, indicating limited antioxidant efficacy. In contrast, the SA/CMCS/TA hydrogel significantly reduced the signal, underscoring the potent antioxidant capacity of TA. TA acts as a powerful ROS scavenger due to its polyphenolic structure, which directly neutralizes ROS and interferes with oxidative stress pathways. Additionally, TA inhibits pro-oxidative signaling, such as the NF-κB pathway, which helps reduce oxidative damage and inflammation[61].
流式细胞术结果证明了不同水凝胶配方对 ROS 水平的不同影响（图 S1 ） 。空白对照组显示最小信号强度，建立了基线，而阳性对照组证实了实验系统正常运行，并为最大限度的 ROS 清除提供了参考。未处理的对照组表现出中等信号强度，反映出细胞反应未改变。SA/CMCS 水凝胶导致信号强度适度降低，表明抗氧化功效有限。相反，SA/CMCS/TA 水凝胶显著降低了信号，强调了 TA 强大的抗氧化能力。TA 由于其多酚结构而充当强大的 ROS 清除剂，其可直接中和 ROS 并干扰氧化应激途径。此外，TA 抑制促氧化信号传导，如 NF-κB 通路，这有助于减少氧化损伤和炎症 [ 61 ] 。

Similarly, the SA/CMCS/BAG hydrogel reduced ROS levels, although its effect was less pronounced than that of SA/CMCS/TA. The reduced antioxidant efficacy of the SA/CMCS/BAG hydrogel is likely due to the limited release of bioactive ions from bioglass (BAG) under the acidic conditions typical of the early stages of wound healing. The small amounts of calcium (Ca²⁺) and zinc (Zn²⁺) ions released from BAG play a role in stabilizing mitochondrial function and enhancing the activity of antioxidant enzymes, which helps regulate oxidative stress[62]. Notably, the SA/CMCS/TA/BAG hydrogel exhibited the most significant reduction in ROS signal intensity, highlighting its superior antioxidant performance.
同样地，SA/CMCS/BAG 水凝胶也降低了 ROS 水平，尽管其效果不如 SA/CMCS/TA 明显。SA /CMCS/BAG 水凝胶抗氧化功效降低可能是由于在伤口愈合早期典型的酸性条件下，生物玻璃 (BAG) 中生物活性离子的释放有限。BAG 释放的少量钙 (Ca²⁺) 和锌 (Zn²⁺) 离子在稳定线粒体功能和增强抗氧化酶活性方面发挥作用，这有助于调节氧化应激 [62] 。值得注意的是，SA/CMCS/TA/BAG 水凝胶表现出最显著的 ROS 信号强度降低，突显了其卓越的抗氧化性能。

3.6 in vitro angiogenesis assessment
3.6 体外血管生成评估

Angiogenesis is a pivotal process in wound healing, as it ensures an adequate supply of oxygen and nutrients, supporting fibroblast activity, collagen synthesis, and epithelialization. Angiogenesis also facilitates the recruitment of immune cells and the transport of growth factors such as VEGF and FGF, which promote the proliferation of endothelial cells and fibroblasts, stimulate granulation tissue formation, and aid in wound remodeling. Additionally, angiogenesis helps transition the wound from the inflammatory phase to the proliferative phase by resolving inflammation and supporting the polarization of macrophages toward the M2 phenotype. Impaired angiogenesis can lead to chronic wounds, hypoxia, and delayed healing, underscoring its critical role in effective tissue repair[63].
血管生成是伤口愈合的关键过程，因为它能确保充足的氧气和营养供应，支持成纤维细胞活性、胶原合成和上皮形成。血管生成还促进免疫细胞的募集和生长因子（例如血管内皮生长因子 (VEGF) 和成纤维细胞生长因子 (FGF)）的运输，这些因子促进内皮细胞和成纤维细胞的增殖，刺激肉芽组织形成，并有助于伤口重塑。此外，血管生成还能通过消退炎症和支持巨噬细胞向 M2 表型极化，帮助伤口从炎症期过渡到增殖期。血管生成受损可导致慢性伤口、缺氧和愈合延迟，凸显了其在有效组织修复中的关键作用 [63] 。

The results of the angiogenesis experiment demonstrated that the SA/CMCS/TA/BAG hydrogel significantly promoted the formation of capillary-like structures within 6 hours, indicating its strong angiogenic potential (Fig. S2A and Fig. S2B). In comparison, the SA/CMCS/BAG hydrogel also exhibited some angiogenic activity, though it was slightly less pronounced than that of the SA/CMCS/TA/BAG group. The SA/CMCS/TA hydrogel showed moderate angiogenic effects, while the SA/CMCS group and the control group exhibited minimal to no capillary formation. This enhanced angiogenesis can be attributed to the synergistic actions of TA and BAG at different stages of wound healing.
血管生成实验结果表明，SA/CMCS/TA/BAG 水凝胶在 6 小时内显著促进了毛细血管样结构的形成，表明其具有强大的血管生成潜力（图 S2 A 和图 S2 B ）。相比之下，SA/CMCS/BAG 水凝胶也表现出一定的血管生成活性，但其作用略弱于 SA/CMCS/TA/BAG 组。SA/CMCS/TA 水凝胶表现出中等程度的血管生成效果，而 SA/CMCS 组和对照组的毛细血管形成极少甚至没有。这种增强的血管生成作用可归因于 TA 和 BAG 在伤口愈合不同阶段的协同作用。

During the early inflammatory phase, TA plays a crucial role by utilizing its antioxidant and anti-inflammatory properties. TA scavenges excessive ROS and inhibits pro-inflammatory pathways such as NF-κB, reducing oxidative stress and protecting endothelial cells from damage. These effects create a favorable microenvironment for angiogenesis[64]. In the subsequent proliferative phase, BAG further supports angiogenesis by releasing bioactive ions, such as silicon (Si⁴⁺) and calcium (Ca²⁺). Silicon ions enhance the formation of the extracellular matrix (ECM), providing a stable and functional scaffold for capillary network growth. Calcium ions stimulate endothelial cell proliferation and migration by activating angiogenesis-related signaling pathways, such as PI3K/Akt, thereby accelerating the formation of capillary-like structures[65].
在炎症早期，TA 利用其抗氧化和抗炎特性发挥着至关重要的作用。TA 清除过量的 ROS 并抑制 NF-κB 等促炎通路，从而减少氧化应激并保护内皮细胞免受损伤。这些作用为血管生成创造了有利的微环境 [64] 。在随后的增殖阶段，BAG 通过释放生物活性离子（如硅 (Si⁴⁺) 和钙 (Ca²⁺)）进一步支持血管生成。硅离子可增强细胞外基质 (ECM) 的形成，为毛细血管网络生长提供稳定且功能性的支架。钙离子通过激活血管生成相关信号通路（如 PI3K/Akt）刺激内皮细胞增殖和迁移，从而加速毛细血管样结构的形成 [65] 。

Crucially, the pH-responsive release properties of BAG ensure a sustained and stage-specific delivery of bioactive ions. In the acidic environment of the early wound healing phase, BAG releases small amounts of silicon and calcium ions, which gradually increase as the pH shifts toward neutral or alkaline conditions. This dynamic ion release supports angiogenesis and tissue regeneration through multiple stages of healing[66]. Therefore, the superior angiogenic capability of the SA/CMCS/TA/BAG hydrogel results from the antioxidant and anti-inflammatory effects of TA in the inflammatory phase and the direct angiogenic effects of BAG during the proliferative phase.
至关重要的是，BAG 的 pH 响应释放特性确保了生物活性离子的持续和阶段特异性递送。在伤口愈合早期的酸性环境中，BAG 会释放少量的硅离子和钙离子，随着 pH 值向中性或碱性转变，这些离子的释放量逐渐增加。这种动态离子释放支持愈合过程中多个阶段的血管生成和组织再生 [66] 。因此，SA/CMCS/TA/BAG 水凝胶卓越的血管生成能力源于 TA 在炎症期的抗氧化和抗炎作用，以及 BAG 在增殖期的直接血管生成作用。

3.7 in vitro hemolysis assay
3.7 体外溶血试验

Hemolysis, which refers to the rupture of red blood cells (RBCs) and the subsequent release of hemoglobin, is a key parameter for evaluating the biocompatibility of wound healing materials. Excessive hemolysis can damage RBCs, impair oxygen transport, and disrupt the wound microenvironment, ultimately hindering the healing process. Therefore, wound healing hydrogels must exhibit minimal hemolytic activity to avoid exacerbating tissue damage or delaying recovery.
溶血是指红细胞 (RBC) 破裂并随后释放血红蛋白，是评估伤口愈合材料生物相容性的关键参数。过度溶血会损害红细胞，阻碍氧气运输，破坏伤口微环境，最终阻碍愈合过程。因此，伤口愈合水凝胶必须具有最低的溶血活性，以避免加剧组织损伤或延缓愈合。

The hemolysis assay results demonstrated that the SA/CMCS/TA/BAG hydrogel exhibited a very low hemolysis rate of less than 5%, suggesting excellent hemocompatibility. In comparison, the SA/CMCS/BAG hydrogel showed a slightly higher hemolysis rate of approximately 7%, while the SA/CMCS/TA hydrogel also exhibited a low hemolysis rate (below 6%). The SA/CMCS hydrogel, acting as the base matrix, demonstrated negligible hemolysis, similar to the negative control group (Fig. S3).
溶血试验结果表明，SA/CMCS/TA/BAG 水凝胶的溶血率极低，低于 5%，表明其具有优异的血液相容性。相比之下，SA/CMCS/BAG 水凝胶的溶血率略高，约为 7%，而 SA/CMCS/TA 水凝胶的溶血率也较低（低于 6%）。作为基质的 SA/CMCS 水凝胶的溶血率可忽略不计， 与阴性对照组相似 （图 S3 ） 。

The minimal hemolysis observed with the SA/CMCS/TA/BAG hydrogel can be attributed to the antioxidative and membrane-stabilizing properties of tannic acid (TA), which helps protect RBCs from oxidative stress-induced damage. The phenolic hydroxyl groups in TA neutralize reactive oxygen species (ROS), reducing oxidative damage and preventing lipid peroxidation on RBC membranes.
SA/CMCS/TA/BAG 水凝胶中观察到的最低溶血率可归因于单宁酸 (TA) 的抗氧化和膜稳定特性，它有助于保护红细胞免受氧化应激引起的损伤。TA 中的酚羟基可以中和活性氧 (ROS)，从而减少氧化损伤并防止红细胞膜上的脂质过氧化 。

Additionally, bioglass (BAG) contributes to the hydrogel's low hemolytic activity by exhibiting minimal disruption to RBC membranes, due to its controlled ion release profile. BAG releases bioactive ions such as calcium (Ca²⁺) and silicon (Si⁴⁺) in a sustained manner, ensuring gradual ion release and minimizing potential damage to RBCs.
此外，生物玻璃 (BAG) 凭借其可控的离子释放特性，最大程度地降低了对红细胞膜的破坏，从而有助于水凝胶降低溶血活性。BAG 能够持续释放生物活性离子，例如钙离子 (Ca²⁺) 和硅离子 (Si⁴⁺)，确保离子缓慢释放，最大限度地减少对红细胞的潜在损害。

However, under acidic conditions, BAG's hemolytic potential may slightly increase, which could explain the higher hemolysis rate observed in the SA/CMCS/BAG hydrogel compared to the SA/CMCS/TA/BAG hydrogel[79].
然而，在酸性条件下，BAG 的溶血潜力可能会略有增加，这可以解释与 SA/CMCS/TA/BAG 水凝胶相比，SA/CMCS/BAG 水凝胶中观察到的溶血率更高 [79] 。

A rapid release of calcium and silicon ions from BAG could alter the osmotic balance around RBCs, leading to some degree of hemolysis. Nevertheless, the controlled-release system in the hydrogel mitigates these effects by regulating ion release, ensuring that ion concentrations remain within safe limits for RBC integrity. This controlled release not only reduces the risk of RBC damage but also allows the therapeutic benefits of BAG to be delivered effectively without compromising hemocompatibility.
BAG 中钙离子和硅离子的快速释放可能会改变红细胞周围的渗透平衡，导致一定程度的溶血。然而，水凝胶中的控释系统通过调节离子释放来减轻这些影响，确保离子浓度保持在维护红细胞完整性的安全范围内。这种控释系统不仅降低了红细胞损伤的风险，还能在不损害血液相容性的情况下有效发挥 BAG 的治疗功效。

The negligible hemolysis seen with the SA/CMCS matrix alone further supports the non-hemolytic nature of the hydrogel network. When combined with TA and BAG, the hydrogel maintains both protective and therapeutic benefits, ensuring it remains hemocompatible while promoting wound healing.
单独使用 SA/CMCS 基质时观察到的溶血几乎可以忽略不计，这进一步证明了水凝胶网络的非溶血特性。当与 TA 和 BAG 结合时，水凝胶兼具保护和治疗功效，确保其在促进伤口愈合的同时保持血液相容性。

In summary, the SA/CMCS/TA/BAG hydrogel achieves an optimal balance between therapeutic functionality and hemocompatibility. Its minimal hemolytic activity suggests that it will not negatively affect the wound microenvironment, supporting its potential for use as a biocompatible material in wound-healing applications.
综上所述，SA/CMCS/TA/BAG 水凝胶在治疗功能和血液相容性之间实现了最佳平衡。其极低的溶血活性表明它不会对伤口微环境产生负面影响，从而有望作为生物相容性材料应用于伤口愈合领域。

3.8 In vivo wound healing and histological analysis
3.8 体内伤口愈合和组织学分析

To evaluate the effectiveness of the hydrogels in promoting wound healing, an acute skin infection wound model in rats was used. The results showed that the SA/CMCS/TA/BAG composite hydrogel significantly accelerated wound healing. Compared to the control group, the SA/CMCS/TA/BAG hydrogel exhibited a consistently higher wound contraction rate at all time points, with near-complete healing observed by day 14(Fig. 6D,E,F). This accelerated healing process indicates the hydrogel's ability to improve both the speed and quality of wound repair. The heatmaps further confirmed these findings(Fig. 6F), with the SA/CMCS/TA/BAG group showing the most pronounced reduction in wound area, indicative of effective tissue regeneration and repair.
为了评估水凝胶促进伤口愈合的有效性，我们采用了大鼠急性皮肤感染伤口模型。结果表明， SA/CMCS/TA/BAG 复合水凝胶明显加速了伤口愈合。与对照组相比，SA/CMCS/TA/BAG 水凝胶在所有时间点均表现出更高的伤口收缩率，到第 14 天时伤口已接近完全愈合 （图 6D 、E 、F ） 。这种加速的愈合过程表明水凝胶能够提高伤口修复的速度和质量。热图进一步证实了这些发现 （ 图 6F ） ，其中 SA/CMCS/TA/BAG 组伤口面积减少最为明显，表明组织再生和修复有效。

The superior healing performance of the SA/CMCS/TA/BAG hydrogel can be attributed to the synergistic effects of its components. Tannic acid (TA) contributes anti-inflammatory and antioxidant properties, establishing a favorable microenvironment for tissue regeneration. Additionally, bioactive ions such as calcium (Ca²⁺) and zinc (Zn²⁺) released from bioglass (BAG) enhance angiogenesis and promote cellular proliferation, further accelerating the healing process. Histological analysis using H&E staining (Fig.7A and Fig.7C) revealed that by day 14, skin tissue in the SA/CMCS/TA/BAG group closely resembled normal tissue, with an intact epidermal layer and well-organized dermal fibers, indicating near-complete wound healing. In contrast, other groups, especially the control group, exhibited significant tissue defects and disorganized fiber arrangements.
SA/CMCS/TA/BAG 水凝胶优异的愈合性能可归因于其成分的协同作用。单宁酸 (TA) 具有抗炎和抗氧化特性，为组织再生建立了良好的微环境。此外，生物玻璃 (BAG) 释放的钙 (Ca²⁺) 和锌 (Zn²⁺) 等生物活性离子可增强血管生成并促进细胞增殖，进一步加速愈合过程。使用 H&E 染色进行的组织学分析（ 图 7A 和图 7C ）显示，到第 14 天，SA/CMCS/TA/BAG 组的皮肤组织与正常组织非常相似，具有完整的表皮层和组织良好的真皮纤维，表明伤口接近完全愈合。相反，其他组，尤其是对照组，表现出严重的组织缺损和混乱的纤维排列。

Masson’s trichrome staining further revealed that the SA/CMCS/TA/BAG group exhibited the highest density of collagen fibers, indicating efficient collagen deposition and enhanced tissue repair. Conversely, the SA/CMCS and control groups showed lower collagen generation with irregular and incomplete collagen structures, suggesting suboptimal healing. Immunofluorescence staining results (Fig.7B and Fig.7D) corroborated these findings. The expression of iNOS, a marker of pro-inflammatory M1 macrophages, was significantly reduced in the SA/CMCS/TA/BAG group, indicating effective suppression of local inflammation. At the same time, the expression of CD206, a marker of anti-inflammatory M2 macrophages, was markedly elevated, demonstrating the hydrogel's ability to promote M2 macrophage polarization, thereby enhancing wound healing and tissue reconstruction.
Masson 三色染色进一步显示 SA/CMCS/TA/BAG 组胶原纤维密度最高，提示胶原沉积有效，组织修复增强。而 SA/CMCS 组和对照组胶原生成较低，胶原结构不规则、不完整，提示愈合不良。免疫荧光染色结果（ 图 7B 和图 7D ） 证实了上述结果。SA/CMCS/TA/BAG 组促炎性 M1 型巨噬细胞标志物 iNOS 表达显著降低，提示局部炎症得到有效抑制。同时，抗炎性 M2 型巨噬细胞标志物 CD206 表达显著升高，表明水凝胶能够促进 M2 型巨噬细胞极化，从而促进伤口愈合和组织重建。

In conclusion, the SA/CMCS/TA/BAG composite hydrogel effectively accelerates the healing of acute infected wounds through multiple mechanisms, including anti-inflammatory and antioxidant effects, improved angiogenesis, and enhanced collagen deposition. These results support the potential of this hydrogel as a biocompatible material for promoting wound healing in clinical applications.
综上所述，SA/CMCS/TA/BAG 复合水凝胶通过多种机制有效加速急性感染伤口的愈合，包括抗炎和抗氧化作用、促进血管生成和增强胶原沉积。这些结果支持了该水凝胶作为生物相容性材料在临床应用中促进伤口愈合的潜力。

4. Conclusion
4. 结论

In conclusion, the pH-responsive SA/CMCS-based hydrogel developed in this study offers a highly effective and adaptable therapeutic strategy for the precision-driven, stepwise treatment of acute infected wounds. By dynamically tuning its structural properties in response to local pH changes, the hydrogel ensures well-timed release of antibacterial tannic acid and tissue-repairing ions, effectively mitigating bacterial load, modulating inflammatory responses, and attenuating oxidative stress. This dual-phase mechanism fosters an optimal wound microenvironment, accelerates tissue regeneration, and enhances overall healing outcomes. The encouraging in vitro and in vivo evidence underscores the clinical potential of this microenvironment-responsive hydrogel as a next-generation wound dressing for achieving targeted, sequential repair of complex infectious wounds.
综上所述，本研究开发的 pH 响应型 SA/CMCS 基水凝胶，为精准分步治疗急性感染伤口提供了一种高效且适应性强的治疗策略。该水凝胶通过响应局部 pH 值变化动态调整其结构特性，确保适时释放抗菌单宁酸和组织修复离子，有效减轻细菌负荷，调节炎症反应，并减弱氧化应激。这种双相机制可促进最佳伤口微环境，加速组织再生，并增强整体愈合效果。令人鼓舞的体外和体内证据凸显了这种微环境响应型水凝胶作为下一代伤口敷料的临床潜力，可用于实现复杂感染性伤口的靶向、序贯修复。

Declaration of competing interest
利益竞争声明

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.
作者声明，他们没有任何已知的竞争性经济利益或个人关系可能会影响本文所报告的工作。

Acknowledgements
致谢

This work was supported in part by the Youth Program of Minhang Hospital(2023MHPY01), Zhejiang Provincial Medicine and Health Technology Project
本研究得到闵行医院青年项目（2023MHPY01）、浙江省医药卫生科技项目等资助。

(2025710477).

Data availability
数据可用性

Data will be made available on request.
数据将根据要求提供。

Reference
参考

[1] X. Y. Zhang, Y. Wu, H. Gong, Y. Xiong, Y. Chen, L. Li, B. Zhi, S. Q. Lv, T. Peng, H. Zhang, SMALL 2024, 20.
[1] 张学友, 吴勇, 龚浩, 熊勇, 陈勇, 李丽, 支本, 吕世强, 彭涛, 张浩, SMALL 2024, 20.

[2] Q. Q. Wu, R. J. Yang, W. X. Fan, L. Wang, J. Zhan, T. T. Cao, Q. M. Liu, X. S. Piao, Y. H. Zhong, W. X. Zhao, S. H. Zhang, J. A. Yu, S. Liang, T. M. Roberts, B. D. Wang, Z. N. Liu, ADVANCED SCIENCE 2024, 11.
[2] 吴 QQ, 杨荣杰, 范文新, 王丽, 詹俊, 曹涛, 刘庆明, 朴晓生, 钟云辉, 赵维新, 张世华, 于嘉, 梁世强, TM Roberts, 王 BD, 刘正宁, ADVANCED SCIENCE 2024, 11.

[3] J. C. Xu, L. L. Chang, Y. H. Xiong, Q. Peng, ADVANCED HEALTHCARE MATERIALS 2024, DOI: 10.1002/adhm.202401490.
[3] JC Xu, LL Chang, YH Xiong, Q. Peng，ADVANCED HEALTHCARE MATERIALS 2024，DOI：10.1002/adhm.202401490。

[4] B. Xu, A. Li, R. Wang, J. Zhang, Y. L. Ding, D. Pan, Z. J. Shen, ADVANCED FUNCTIONAL MATERIALS 2021, 31.
[4] B. Xu, A. Li, R. Wang, J. Zhang, YL Ding, D. Pan, ZJ Shen, ADVANCED FUNCTIONAL MATERIALS 2021, 31.

[5] M. Cai, Z. Liu, X. Sun, Y. Qi, X. L. Mei, S. Liu, C. L. Zhang, X. Zhang, Z. G. Zong, P. P. Ma, T. Wang, W. G. Xu, T. Zhang, CHEMICAL ENGINEERING JOURNAL 2024, 498.
[5] 蔡明，刘志，孙晓玲，齐勇，梅晓玲，刘胜，张春林，张晓玲，宗志刚，马平平，王婷，徐文伟，张婷，化学工程学报，2024，498。

[6] C. Fan, Q. Xu, R. Q. Hao, C. Wang, Y. M. Que, Y. X. Chen, C. Yang, J. Chang, BIOMATERIALS 2022, 287.
[6] 范春、徐清、郝荣强、王春、阙明明、陈一新、杨春、张建，生物材料 2022, 287.

[7] F. H. Afruzi, M. Abdouss, E. N. Zare, E. R. Ghomi, S. Mahmoudi, R. E. Neisiany, COORDINATION CHEMISTRY REVIEWS 2025, 524.
[7] FH Afruzi、M. Abdouss、EN Zare、ER Ghomi、S. Mahmoudi、RE Neisiany，配位化学评论 2025 年，524。

[8] J. R. Chen, M. Z. Luo, Y. Chen, Z. W. Xing, C. Peng, D. Li, CHEMICAL ENGINEERING JOURNAL 2024, 500.
[8] 陈 JR，罗明哲，陈勇，幸志伟，彭晨，李德军，化学工程学报，2024，500。

[9] C. Huang, L. L. Dong, B. H. Zhao, Y. F. Lu, S. R. Huang, Z. Q. Yuan, G. X. Luo, Y. Xu, W. Qian, CLINICAL AND TRANSLATIONAL MEDICINE 2022, 12.
[9] 黄成，董丽华，赵宝华，陆一峰，黄升华，袁 ZQ，罗国兴，徐勇，钱伟，临床与转化医学 2022，12。

[10] L. P. Zhou, W. Pi, S. Y. Cheng, Z. Gu, K. X. Zhang, T. T. Min, W. M. Zhang, H. W. Du, P. X. Zhang, Y. Q. Wen, ADVANCED FUNCTIONAL MATERIALS 2021, 31.
[10] LP Zhou, W. Pi, SY Cheng, Z. Gu, KX Zhang, TT Min, WM Zhang, HW Du, PX Zhang, YQ Wen, ADVANCED FUNCTIONAL MATERIALS 2021, 31.

[11] J. S. Chin, L. Madden, S. Y. Chew, D. L. Becker, Advanced Drug Delivery Reviews 2019, 149-150, 2.
[11] JS Chin, L. Madden, SY Chew, DL Becker, 高级药物输送评论 2019, 149-150, 2.

[12] Y. P. Liang, J. H. He, B. L. Guo, ACS NANO 2021, 15, 12687.
[12] YP Liang, JH He, BL Guo, ACS NANO 2021, 15, 12687。

[13] X. Zhou, X. Z. Yu, T. T. You, B. H. Zhao, L. L. Dong, C. Huang, X. Q. Zhou, M. L. Xing, W. Qian, G. X. Luo, ADVANCED SCIENCE 2024, DOI: 10.1002/advs.202404580.
[13] X. Zhou, XZ Yu, TT You, BH Zhao, LL Dong, C. Huang, XQ Zhou, ML Xing, W. Qian, GX Luo, ADVANCED SCIENCE 2024，DOI：10.1002 / advs.202404580。

[14] T. Zivari-Ghader, M. R. Rashidi, M. Mehrali, INTERNATIONAL JOURNAL OF BIOLOGICAL MACROMOLECULES 2024, 279.
[14] T. Zivari-Ghader，MR Rashidi，M. Mehrali，国际生物大分子杂志 2024，279。

[15] W.-C. Huang, R. Ying, W. Wang, Y. Guo, Y. He, X. Mo, C. Xue, X. Mao, Advanced Functional Materials 2020, 30, 2000644.
[15] W.-C. Huang, R. Ying, W. Wang, Y. Guo, Y. He, X. Mo, C. Xue, X. Mao, Advanced Functional Materials 2020, 30, 2000644.

[16] H. E. Emam, T. I. Shaheen, Carbohydrate Polymers 2022, 278, 118925.
[16] HE Emam，TI Shaheen，碳水化合物聚合物 2022，278，118925。

[17] I. Singh, S. Kumar, S. Singh, M. Y. Wani, International Journal of Biological Macromolecules 2024, 278, 135022.
[17] I.Singh，S.Kumar，S.Singh，MYWani，国际生物大分子杂志 2024，278，135022。

[18] K. Liu, Y. Kang, X. Dong, Q. Li, Y. Wang, X. Wu, X. Yang, Z. Chen, H. Dai, Chemical Engineering Journal 2023, 470, 143987.
[18] 刘坤，康勇，董晓，李强，王勇，吴晓，杨晓，陈正，戴红，化学工程，2023, 470, 143987.

[19] X. Wu, Y. Wang, X. Liu, Q. Ding, S. Zhang, Y. Wang, G. Chai, Y. Tang, J. Yang, T. Yu, W. Liu, C. Ding, Carbohydrate Polymers 2025, 349, 122960.
[19] 吴 X.，Y.王，X.刘，Q.丁，S.张，Y.王，G.柴，Y.唐，J.杨，T.Yu，W.刘，C.丁，碳水化合物聚合物 2025, 349, 122960.

[20] D. Ghosal, N. Majumder, P. Das, S. Chaudhary, S. Dey, P. Banerjee, P. Tiwari, P. Das, P. Basak, S. K. Nandi, S. Ghosh, S. Kumar, Advanced Healthcare Materials 2024, n/a, 2402024.
[20] D. Ghosal、N. Majumder、P. Das、S. Chaudhary、S. Dey、P. Banerjee、P. Tiwari、P. Das、P. Basak、SK Nandi、S. Ghosh、S. Kumar，先进医疗材料 2024，n/a，2402024。

[21] Z. Yang, C. Wang, Z. Zhang, F. Yu, Y. Wang, J. Ding, Z. Zhao, Y. Liu, International Journal of Biological Macromolecules 2024, 264, 130741.
[21] Z. Yang, C. Wang, Z. Zhuang, F. Yu, Y. Wang, J. Ding, Z. Zhao, Y. Liu, International Journal of Biological Macromolecules 2024, 264, 130741.

[22] Z. Han, M. Wang, Z. Hu, Y. Wang, J. Tong, X. Zhao, W. Yue, G. Nie, Communications Materials 2024, 5, 137.
[2 2 ] Z. Han, M. Wang, Z. Hu, Y. Wang, J. Tong, X. Zhao, W. Yue, G. Nie, Communications Materials 2024, 5, 137。

[23] F. Azadikhah, A. R. Karimi, G. H. Yousefi, M. Hadizadeh, International Journal of Biological Macromolecules 2021, 188, 114.
[2 3 ] F. Azadikhah、AR Karimi、GH Yousefi、M. Hadizadeh，国际生物大分子杂志 2021, 188, 114。

[24] T. Li, X. Hu, Q. Zhang, Y. Zhao, P. Wang, X. Wang, B. Qin, W. Lu, Polymers for Advanced Technologies 2020, 31, 1648.
[2 4 ] T. Li, X. Hu, Q. Zhang, Y. Zhao, P. Wang, X. Wang, B. Qin, W. Lu, Polymers for Advanced Technologies 2020, 31, 1648。

[25] X. Zhou, Q. Zhou, Q. Chen, Y. Ma, Z. Wang, L. Luo, Q. Ding, H. Li, S. Tang, ACS Biomaterials Science & Engineering 2023, 9, 437.
[2 5 ] X. Zhou, Q. Zhou, Q. Chen, Y. Ma, Z. Wang, L. Luo, Q. Ding, H. Li, S. Tang, ACS 生物材料科学与工程 2023, 9, 437。

[26] T. Lin, J. Su, Chemosphere 2022, 302, 134892.
[2 6 ] T. Lin，J. Su，Chemosphere 2022，302，134892。

[27] L. Zhang, B. Shen, C. Zheng, Y. Huang, Y. Liang, P. Fei, J. Chen, W. Lai, Food Hydrocolloids 2024, 156, 110368.
[ 27 ] 张丽, 沉本, 郑成, 黄勇, 梁勇, 费鹏, 陈建, 赖伟, 食品亲水胶体 2024, 156, 110368.

[18] Z. Song, W. Yao, X. Zhang, Y. Dong, Z. Zhang, Y. Huang, W. Jing, L. Sun, Y. Han, F. Hu, Z. Yuan, B. Zhao, P. Wei, X. Zhang, Nano Today 2024, 54, 102060.
[ 18 ] Z. Song, W. Yao, X. Zhang, Y. Dong, Z. Zhang, Y. Huang, W. Jing, L. Sun, Y. Han, F. Hu, Z. Yuan, B. Zhao, P. Wei, X. Zhang, Nano Today 2024, 54, 102060。

[29] R. Jia, Y. He, J. Liang, L. Duan, C. Ma, T. Lu, W. Liu, S. Li, H. Wu, H. Cao, T. Li, Y. He, iScience 2024, 27, 109197.
[ 29 ] R. Jia, Y. He, J. Liang, L. Duan, C. Ma, T. Lu, W. Liu, S. Li, H. Wu, H. Cao, T. Li, Y. He, iScience 2024, 27, 109197。

[20] N. Zhao, W. Yuan, Composites Part B: Engineering 2022, 242, 110095.
[ 20 ] N. Zhao, W. Yuan, 复合材料 B 部分:工程 2022, 242, 110095。

[31] R. Azmi, F. Lindgren, K. Stokes-Rodriguez, M. Buga, C. Ungureanu, T. Gouveia, I. Christensen, S. Pal, A. Vlad, A. Ladam, K. Edström, M. Hahlin, ACS Applied Materials & Interfaces 2024, 16, 34266.
[ 31 ] R. Azmi、F. Lindgren、K. Stokes-Rodriguez、M. Buga、C. Ungureanu、T. Gouveia、I. Christensen、S. Pal、A. Vlad、A. Ladam、K. Edström、M. Hahlin，ACS 应用材料与界面 2024, 16, 34266。

[32] Q. Wu, Y. Xu, S. Han, A. Chen, J. Zhang, Y. Chen, X. Yang, L. Guan, ACS Nano 2024, 18, 31148.
[ 32 ] 吴强，徐勇，韩胜，陈阿，张建，陈勇，杨旭，管丽，ACS Nano 2024, 18, 31148.

[33] H. Chang, P. Tian, L. Hao, C. Hu, B. Liu, F. Meng, X. Yi, X. Pan, X. Hu, H. Wang, X. Zhai, X. Cui, J. Pui Yin Cheung, X. Liu, H. Pan, S. Bian, X. Zhao, Chemical Engineering Journal 2024, 481, 148768.
[3 3 ] H. Chang, P. Tian, L. Hao, C. Hu, B. Liu, F. Meng, X. Yi, X. Pan, X. Hu, H. Wang, X. Zhai, X. Cui, J. Pui Yin Cheung, X. Liu, H. Pan, S. Bian, X. Zhao, Chemical Engineering Journal 2024, 481, 148768.

[34] S. Chen, M. Li, M. Michálek, H. Kaňková, L. Zhao, A. R. Boccaccini, D. Galusek, K. Zheng, Materialia 2024, 36, 102165.
[3 4 ] S. Chen，M. Li，M. Michálek，H. Kaňková，L. Zhu，AR Boccaccini，D. Galusek，K. Cheng，Materialia 2024, 36, 102165。

[35] L. Krishnan, P. Chakrabarty, K. Govarthanan, S. Rao, T. S. Santra, International Journal of Biological Macromolecules 2024, 277, 133073.
[3 5 ] L. Krishnan，P. Chakrabarty，K. Govarthanan，S. Rao，TS Santra，国际生物大分子杂志 2024，277，133073。

[36] S. Chen, J. Bao, Z. Hu, X. Liu, S. Cheng, W. Zhao, C. Zhao, ACS Applied Materials & Interfaces 2024, 16, 23855.
[3 6 ] S. Chen, J. Bao, Z. Hu, X. Liu, S. Cheng, W. Zhao, C. Zhao, ACS Applied Materials & Interfaces 2024, 16, 23855。

[37] G. Mitchell, R. R. Isberg, Cell Host & Microbe 2017, 22, 166.
[ 37 ] G. Mitchell、RR Isberg，Cell Host & Microbe 2017，22，166。

[38] N. Ullah, D. Khan, N. Ahmed, A. Zafar, K. U. Shah, A. ur Rehman, Journal of Drug Delivery Science and Technology 2023, 80, 104110.
[ 38 ] N. Ullah，D. Khan，N. Ahmed，A. Zafar，KU Shah，A. ur Rehman，《药物输送科学与技术杂志》2023，80，104110。

[39] P. Zhuang, Y. Yao, X. Su, Y. Zhao, K. Liu, X. Wu, H. Dai, Composites Part B: Engineering 2022, 242, 110030.
[ 39 ] 庄鹏，姚耀，苏旭，赵勇，刘坤，吴旭，戴红，复合材料 B 部分：工程 2022, 242, 110030。

[40] F. Yang, Y. Xue, F. Wang, D. Guo, Y. He, X. Zhao, F. Yan, Y. Xu, D. Xia, Y. Liu, Bioactive Materials 2023, 26, 88.
[ 40 ] F. Yang, Y. Xue, F. Wang, D. Guo, Y. He, X. Zhao, F. Yan, Y. Xu, D. Xia, Y. Liu, Bioactive Materials 2023, 26, 88.

[41] I. J. Das, T. Bal, International Journal of Biological Macromolecules 2024, 279, 135118.
[4 1 ] IJ Das，T. Bal，国际生物大分子杂志 2024，279，135118。

[42] M. Xiong, Y. Chen, H.-J. Hu, H. Cheng, W.-X. Li, S. Tang, X. Hu, L.-M. Lan, H. Zhang, G.-B. Jiang, Carbohydrate Polymers 2024, 341, 122348.
[4 2 ] M. Xiong, Y. Chen, H.-J. Hu, H. Cheng, W.-X. Li, S. Tang, X. Hu, L.-M. Lan, H. Zhang, G.-B. Jiang, Carbohydrate Polymers 2024, 341, 122-348。

[43] J. Feng, F. Wang, X. Pan, Y. Shao, A. Jin, L. Lei, X. Lin, APL Materials 2024, 12, 101124.
[4 3 ] J. Feng, F. Wang, X. Pan, Y. Shao, A. Jin, L. Lei, X. Lin, APL Materials 2024, 12, 101124。

[44] Y. Zhang, S. Chen, X. Qin, A. Guo, K. Li, L. Chen, W. Yi, Z. Deng, F. R. Tay, W. Geng, L. Miao, Y. Jiao, B. Tao, Advanced Healthcare Materials 2024, 13, 2400318.
[4 4 ] Y. Zhang, S. Chen, X. Qin, A. Guo, K. Li, L. Chen, W. Yi, Z. Deng, FR Tay, W. Geng, L. Miao, Y. Jiao, B. Tao, Advanced Healthcare Materials 2024, 13, 2400318.

[45] J. Chen, X. Jia, S. Wang, X. Wang, L. Li, G. Han, Y. Dou, J. Li, Chemical Engineering Journal 2024, 497, 154521.
[4 5 ] 陈建, 贾晓, 王胜, 王晓, 李立, 韩刚, 窦勇, 李建, 化学工程, 2024, 497, 154521.

[46] C. Zhou, L. Zhang, Z. Xu, T. Sun, M. Gong, Y. Liu, D. Zhang, Small 2023, 19, 2206408.
[4 6 ] C. Zhou, L. Zhang, Z. Xu, T. Sun, M. Gong, Y. Liu, D. Zhang, Small 2023, 19, 2206408.

[47] G. Bonsignore, S. Martinotti, E. Ranzato, CELLS 2024, 13.
[ 47 ] G. Bonsignore、S. Martinotti、E. Ranzato，细胞 2024，13。

[48] Z. Zhou, J. Xiao, S. Guan, Z. Geng, R. Zhao, B. Gao, Carbohydrate Polymers 2022, 285, 119235.
[ 48 ] Z. Zhou, J. Xiao, S. Guan, Z. Geng, R. Zhao, B. Gao, Carbohydrate Polymers 2022, 285, 119-235.

[49] K. Yang, X. Zhou, Z. Li, Z. Wang, Y. Luo, L. Deng, D. He, ACS Applied Materials & Interfaces 2022, 14, 43010.
[ 49 ] 杨凯、周晓、李子、王子、罗勇、邓丽、何德, ACS 应用材料与界面 2022, 14, 43010.

[50] C. Pucci, C. Martinelli, D. De Pasquale, M. Battaglini, N. di Leo, A. Degl’Innocenti, M. Belenli Gümüş, F. Drago, G. Ciofani, ACS Applied Materials & Interfaces 2022, 14, 15927.
[5 0 ] C. Pucci、C. Martinelli、D. De Pasquale、M. Battaglini、N. di Leo、A. Degl'Innocenti、M. Belenli Gümüş、F. Drago、G. Ciofani，ACS 应用材料与界面 2022, 14, 15927。

[51] K.-B. Bu, M. Kim, J.-S. Sung, A. A. Kadam, ACS Applied Nano Materials 2024, 7, 313.
[5 1 ] K.-B. Bu，M. Kim，J.-S. Sung，AA Kadam，ACS Applied Nano Materials 2024，7，313。

[52] D. Li, J. Li, S. Wang, Q. Wang, W. Teng, Advanced Healthcare Materials 2023, 12, 2203063.
[5 2 ] D. Li, J. Li, S. Wang, Q. Wang, W. Teng, Advanced Healthcare Materials 2023, 12, 2203063。

[53] B. Lu, J. Zhang, G. Zhu, T. Liu, J. Chen, X. Liang, Journal of Nanobiotechnology 2023, 21, 491.
[5 3 ] 陆博，张建，朱桂，刘涛，陈建，梁晓，纳米生物技术杂志 2023, 21, 491.

[54] Z. H. Wang, M. Y. Li, J. Chen, S. M. Zhang, B. Wang, J. L. Wang, ADVANCED HEALTHCARE MATERIALS 2024, DOI: 10.1002/adhm.202402080.
[5 4 ] ZH Wang, MY Li, J. Chen, SM Zhang, B. Wang, JL Wang，ADVANCED HEALTHCARE MATERIALS 2024，DOI：10.1002/adhm.202402080。

[55] X. Wei, C. Liu, Z. Li, Z. Gu, J. Yang, K. Luo, Carbohydrate Polymers 2024, 331, 121873.
[5 5 ] X. Wei, C. Liu, Z. Li, Z. Gu, J. Yang, K. Luo, 碳水化合物聚合物 2024, 331, 121873.

[56] Y. Zhu, Z. Ma, L. Kong, Y. He, H. F. Chan, H. Li, Biomaterials 2020, 256, 120216.
[5 6 ] Y. Zhu, Z. Ma, L. Kong, Y. He, HF Chan, H. Li, Biomaterials 2020, 256, 120216。

[57] N. Xu, Y. Gao, Z. Li, Y. Chen, M. Liu, J. Jia, R. Zeng, G. Luo, J. Li, Y. Yu, Chemical Engineering Journal 2023, 466, 143173.
[ 57 ] 徐娜，高勇，李子，陈勇，刘明，贾建，曾荣，罗国强，李建，余勇，化学工程杂志 2023, 466, 143173.

[58] Y. Ding, G. Liu, S. Liu, X. Li, K. Xu, P. Liu, K. Cai, Advanced Healthcare Materials 2023, 12, 2300722.
[ 58 ] Y. Ding, G. Liu, S. Liu, X. Li, K. Xu, P. Liu, K. Cai, Advanced Healthcare Materials 2023, 12, 2300722。

[59] N. Ninan, A. Forget, V. P. Shastri, N. H. Voelcker, A. Blencowe, ACS Applied Materials & Interfaces 2016, 8, 28511.
[ 59 ] N. Ninan，A. Forget，VP Shastri，NH Voelcker，A. Blencowe，ACS Applied Materials & Interfaces 2016，8，28511。

[60] a) L.-L. Zhao, J.-J. Luo, J. Cui, X. Li, R.-N. Hu, X.-Y. Xie, Y.-J. Zhang, W. Ding, L.-J. Ning, J.-C. Luo, T.-W. Qin, ACS Applied Materials & Interfaces 2024, 16, 15879.
[6 0 ] a) L.-L.赵杰杰罗建军，崔晓玲，李 R.-N.胡，X.-Y.谢永杰张伟，丁 L.-J.宁，J.-C.罗，T.-W. Qin，ACS 应用材料与界面 2024，16，15879。

[61] a) Y. Wu, L. M. Zhong, Z. R. Yu, J. H. Qi, DRUG DEVELOPMENT RESEARCH 2019, 80, 262; b) S. Nag, K. Das Saha, ACS Omega 2021, 6, 28752.
[6 1 ] a) Y. Wu，LM Zhong，ZR Yu，JH Qi，药物开发研究 2019，80，262； b) S. Nag、K. Das Saha，ACS Omega 2021, 6, 28752。

[62] H. Liu, H. Yang, Y. Fang, K. Li, L. Tian, X. Liu, W. Zhang, Y. Tan, W. Lai, L. Bian, B. Lin, Z. Xi, Science of The Total Environment 2020, 705, 135809.
[6 2 ] 刘红、杨红、方勇、李凯、田丽、刘晓、张文、谭勇、赖伟、卞丽、林本、奚，总体环境科学 2020, 705, 135809。

[63] a) H. Yan, L. Wang, H. Wu, Y. An, Y. Qin, Z. Xiang, H. Wan, Y. Tan, L. Yang, F. Zhang, Q. Jiang, R. Luo, Y. Wang, Chemical Engineering Journal 2024, 490, 151893.
[6 3 ] a) 严华、王丽、吴华、安亚、秦亚、向祥、万华、谭亚、杨丽、张凤、蒋庆、罗瑞、王亚,《化学工程》2024, 490, 151893 .

[64] R. Q. Wang, Y. C. Ma, S. H. Zhan, G. B. Zhang, L. Cao, X. G. Zhang, T. G. Shi, W. C. Chen, CELL DEATH & DISEASE 2020, 11.
[6 4 ] RQ Wang, YC Ma, SH Zhan, GB Zhang, L. Cao, XG Zhang, TG Shi, WC Chen, CELL DEATH & DISEASE 2020, 11.

[65] F. Shan, N. Zhang, X. Yao, Y. Li, Z. Wang, C. Zhang, Y. Wang, Cell Communication and Signaling 2024, 22, 93.
[6 5 ] F. Shan, N. Zhang, X. Yao, Y. Li, Z. Wang, C. Zhang, Y. Wang, 细胞通讯和信号传导 2024, 22, 93。

[66] S. Shang, K. Zhuang, J. Chen, M. Zhang, S. Jiang, W. Li, Bioactive Materials 2024, 34, 298.
[6 6 ] S. Shang, K. Zhuang, J. Chen, M. Zhang, S. Jiang, W. Li，生物活性材料 2024, 34, 298。

Figure:
数字：

Fig. 1. Schematic representation of the pH-responsive multifunctional hydrogel designed for the healing of infected wounds. (A) Zn @ TA and SA / CMCS composites were synthesized by chemical and physical interactions to construct pH-responsive hydrogels with interpenetrating networks. (B) Under acidic conditions, the hydrogel contracts, releasing tannic acid (TA), while in alkaline conditions, it swells, promoting the release of zinc ions. (C) TA exerts antibacterial effects and scavenges reactive oxygen species (ROS), whereas Zn@TA supports macrophage polarization, angiogenesis, and tissue regeneration.
图 1. 用于感染伤口愈合的 pH 响应多功能水凝胶示意图。 （A） 通过化学和物理相互作用合成 Zn @ TA 和 SA / CMCS 复合材料，以构建具有互穿网络的 pH 响应水凝胶 。（ B ）在酸性条件下，水凝胶收缩，释放单宁酸 (TA)，而在碱性条件下，它会膨胀，促进锌离子的释放。（ C ）TA 发挥抗菌作用并清除活性氧 (ROS)，而 Zn@TA 支持巨噬细胞极化、血管生成和组织再生。

Fig.2. Characterization of SA/CMCS-based hydrogels incorporating Tannic Acid (TA) and Bioactive Glass (BAG). (A) FTIR spectra of different hydrogel formulations: SA/CMCS, SA/CMCS/TA, SA/CMCS/BAG, and SA/CMCS/TA/BAG. (B)XPS spectra showing elemental composition and chemical states of SA/CMCS and its variants. (C) XRD patterns of the hydrogels. SA/CMCS and SA/CMCS/TA exhibit broad amorphous peaks around 20°–30°, consistent with the amorphous structure of polysaccharide-based hydrogels. (D) SEM micrographs of freeze-dried hydrogels illustrating their surface morphology. (E) EDX elemental mapping of carbon (C), calcium (Ca), nitrogen (N), sodium (Na), oxygen (O), silicon (Si), and zinc (Zn) for the hydrogels.
图 2 . 添加单宁酸 (TA) 和生物活性玻璃 (BAG) 的 SA/ CMCS 基水凝胶的表征。 (A) 不同水凝胶配方的 FTIR 光谱：SA/ CMCS 、 SA/ CMCS /TA、SA/ CMCS /BAG 和 SA/ CMCS /TA/BAG。 (B) XPS 光谱显示 SA/ CMCS 及其变体的元素组成和化学状态。 (C) 水凝胶的 XRD 图案。SA/CMCS 和 SA/CMCS/TA 在 20°–30° 左右表现出宽的无定形峰，与多糖基水凝胶的无定形结构一致。(D) 冻干水凝胶的 SEM 显微照片，显示其表面形貌。(E) 水凝胶中碳 (C)、钙 (Ca)、氮 (N)、钠 (Na)、氧 (O)、硅 (Si) 和锌 (Zn) 的 EDX 元素映射。

Fig.3. In vitro biocompatibility of SA/CMCS/TA/BAG hydrogel.(A) Wound healing scratch assay images at 0 hours (0H) and 24 hours (24H) for control, SA/CMCS, SA/CMCS/TA, SA/CMCS/BAG, and SA/CMCS/TA/BAG groups. Red dashed lines indicate the wound edges. (B) Immunofluorescence staining of FITC (green) and DAPI (blue) to evaluate cellular responses for different hydrogel formulations (control, SA/CMCS, SA/CMCS/TA, SA/CMCS/BAG, and SA/CMCS/TA/BAG). (C) Quantification of wound healing rates for each group. (D) Live/Dead assay results showing live cells (green) and dead cells (red) in different hydrogel formulations after treatment, with merged images indicating overall cell viability. (*p <**p < 0.01, and ***p < 0.001, respectively, in comparison to the control group).
图 3 . SA/ CMCS /TA/BAG 水凝胶的体外生物相容性 。 ( A) 对照组、SA/CMCS 组、SA/CMCS/TA 组、SA/CMCS/BAG 组和 SA/CMCS/TA/BAG 组在 0 小时 (0H) 和 24 小时 (24H) 的伤口愈合划痕试验图像 。 红色虚线表示伤口边缘 。 ( B ) FITC（绿色）和 DAPI（蓝色）免疫荧光染色以评估不同水凝胶配方（对照组、SA/ CMCS 组 、SA/ CMCS /TA 组、SA/ CMCS /BAG 组和 SA/ CMCS /TA/BAG 组）的细胞反应。 (C) 量化每组的伤口愈合率。 (D) 活/死试验结果显示治疗后不同水凝胶配方中的活细胞（绿色）和死细胞（红色），合并图像显示整体细胞活力。 （ 与对照组相比，分别为 *p <**p < 0.01 和 ***p < 0.001 ）。

Fig.4. In vitro antibacterial evaluation of SA/CMCS/TA/BAG hydrogels.(A) Confocal laser scanning microscopy pictures of S. aureus and E. coli biofilms following hydrogel treatment, showing live (green) and dead (red) bacteria. (B)Quantitative fluorescence intensity analysis of DMAO (live) and EthD-III (dead) staining of E. coli and S. aureus biofilms. (C) Live/Dead staining images of bacterial cells treated with different hydrogels,demonstrating bacteria viability.(D)Fluorescence intensity statistics for bacterial live/dead staining. (E) Typical pictures of CFU tests for hydrogel-treated S. aureus and E. coli. (F)Quantitative analysis of bacterial colony numbers after hydrogel treatment. (*p < 0.05, **p < 0.01, and ***p < 0.001, compared with the control group).
图 4 . SA/CMCS/TA/BAG 水凝胶的体外抗菌评价 。 ( A) 水凝胶处理后金黄色葡萄球菌和大肠杆菌生物膜的共聚焦激光扫描显微镜照片 ，显示活菌（绿色）和死菌（红色）。(B)大肠杆菌和金黄色葡萄球菌生物膜的 DMAO（活菌）和 EthD-III（死菌）染色的定量荧光强度分析。(C)用不同水凝胶处理的细菌细胞的活/死菌染色图像，显示细菌活力。(D)细菌活/死菌染色的荧光强度统计。(E) 水凝胶处理的金黄色葡萄球菌和大肠杆菌的 CFU 检测典型图片。 (F)水凝胶处理后细菌菌落数量的定量分析。 （与对照组相比，*p < 0.05、**p < 0.01 和 ***p < 0.001）。

Fig.5. In vitro evaluation of the immunomodulatory properties of SA/CMCS/TA/BAG hydrogels. (A–D) Representative fluorescence images showing the expression of CD86, iNOS (M1 macrophage markers), CD206, and Arginase-1 (M2 macrophage markers) in RAW264.7 cells treated with different hydrogels after LPS-induced inflammatory stimulation. (E–H) Quantitative analysis of the relative fluorescence intensity of CD86, iNOS, CD206, and Arginase-1. (I) Western blotting results showing protein expression levels of CD86, iNOS, CD206, and Arginase-1 in RAW264.7 cells. (J–K) Statistical analysis of western blot band intensities to quantify the relative expression levels of M1 and M2 markers. (*p < 0.05, **p < 0.01, and ***p < 0.001, compared with the control group).
图 5. SA/CMCS/TA/BAG 水凝胶免疫调节特性的体外评估 。 （A–D）代表性荧光图像显示在 LPS 诱导的炎症刺激后，用不同水凝胶处理的 RAW264.7 细胞中 CD86、iNOS（M1 巨噬细胞标志物）、CD206 和精氨酸酶-1（M2 巨噬细胞标志物）的表达。（E–H）定量分析 CD86、iNOS、CD206 和精氨酸酶-1 的相对荧光强度。（I）蛋白质印迹结果显示 RAW264.7 细胞中 CD86、iNOS、CD206 和精氨酸酶-1 的蛋白质表达水平。（J–K）对蛋白质印迹条带强度进行统计分析，以量化 M1 和 M2 标志物的相对表达水平。 （与对照组相比，*p < 0.05、**p < 0.01 和 ***p < 0.001）。

Fig.6. In vivo experiment results on wound healing.(A) Diagram of acute wound in rats.(B) Comparison of wound healing percentage among treatment groups at various time points.(C) Macroscopic observation of wound healing at different time points (0 day, 3 days, 5 days, 7 days, 9 days, 11 days, 14 days) for various treatment groups (Control, SA/CMSC, SA/CMSC/TA, SA/CMSC/BAG, SA/CMSC/TA/BAG).(D) Statistical analysis of the wound areas on Days 0, 3,5,7,9,11and 14 days.
图 6 . 体内实验伤口愈合结果。 (A) 大鼠急性伤口示意图 。 (B) 不同时间点各治疗组伤口愈合率比较 。 (C) 各治疗组 (Control、SA/CMSC、SA/CMSC/TA、SA/CMSC/BAG、SA/CMSC/TA/BAG) 在不同时间点 (0 天、3 天、5 天、7 天、9 天、11 天、14 天) 伤口愈合情况宏观观察。 ( D ) 0、3、5、7、9、11 和 14 天伤口面积统计分析 。

Fig.7. Histological and immunofluorescence analysis of tissue sections.(A) Hematoxylin - Eosin (HE) staining of tissue sections for different treatment groups (Control, SA/CMSC, SA/CMSC/TA, SA/CMSC/BAG, SA/CMSC/TA/BAG). (B) Masson's trichrome staining of tissue sections for different treatment groups. (C) Immunofluorescence staining of tissue sections for different treatment groups, targeting specific markers (DAPI,CD206 and INOS).
图 7 . 组织切片的组织学和免疫荧光分析 。 ( A) 不同治疗组（对照组、SA/CMSC、SA/CMSC/TA、SA/CMSC/BAG、SA/CMSC/TA/BAG）组织切片的苏木精-伊红 (HE) 染色。(B) 不同治疗组组织切片的 Masson 三色染色。(C) 不同治疗组组织切片的免疫荧光染色，针对特定标志物（ DAPI、 CD206 和 INOS）。