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Sprayable, antimicrobial and immunoregulation hydrogel loading exosomes based on oxidized sodium alginate for efficient wound healing at skin graft donor sites and health detection
基于氧化海藻酸钠的可喷雾、抗菌和免疫调节水凝胶负载外泌体，用于皮肤移植供体部位的伤口高效愈合和健康检测

Zuyan Shen a a ^(a){ }^{\mathrm{a}}, Lihong Wang b b ^(b){ }^{\mathrm{b}}, Xiaoyun Xie b , ∗ b , ∗ ^(b,**){ }^{\mathrm{b}, *}, Weizhong Yuan a , ∗ a , ∗ ^(a,^(**)){ }^{\mathrm{a},{ }^{*}}
沈祖燕 a a ^(a){ }^{\mathrm{a}} ， 王 b b ^(b){ }^{\mathrm{b}} 立宏 ， 谢 b , ∗ b , ∗ ^(b,**){ }^{\mathrm{b}, *} 晓云 ， 袁 a , ∗ a , ∗ ^(a,^(**)){ }^{\mathrm{a},{ }^{*}} 伟忠 a a  ^("a "){ }^{\text {a }} School of Materials Science and Engineering, Tongii University, Shanghai 201804, PR China
a a  ^("a "){ }^{\text {a }} 同济大学材料科学与工程学院， 上海 201804， 中国 b b ^(b){ }^{\mathrm{b}} Department of Interventional and Vascular Surgery, Shanghai Tenth People's Hospital, School of Medicine, Tongji University, Shanghai 200072, PR China
b b ^(b){ }^{\mathrm{b}} 同济200072大学医学院附属上海市第十人民医院介入血管外科

ARTICLE INFO  文章信息

Keywords:  关键字：

Exosome  外泌体
Antimicrobial  抗菌
Immunoregulation  免疫调节
Sprayable gel  可喷雾凝胶
Sodium alginate oxide  海藻酸钠氧化物

Abstract  抽象

Skin grafting techniques are widely used for large burns, trauma, and various acute and chronic wounds, contributing greatly to the repair of traumatic tissue. However, donor site repair and regeneration are often neglected, resulting in infection and delayed healing. Therefore, it is crucial to reduce the rate of donor site infection and improve the speed and quality of healing. The low-oxidized sodium alginate (OSA) grafting ε ε epsi\varepsilon-polylysine (OSA-g-EPL) prepared through the Schiff base reaction was used to load with mesenchymal stem cell exosomes (Exo), and crosslinked by Ca 2 + Ca 2 + Ca^(2+)\mathrm{Ca}^{2+} to form a gel film (HAE) on the surface of the wound by spraying. EPL provided the hydrogel with good antimicrobial properties, and Exo promoted the polarization of the M2 macrophage, shortened the inflammatory phase of the wound and rapidly transitioned to the proliferation phase, thus accelerating the wound healing process and avoiding the transition to chronic wounds. The excellent electrical conductivity and sensing properties of the hydrogel could be used to monitor the behavioral activities of mice in real time to determine their wound recovery. Therefore, this strategy will provide a promising prospect for efficient and high-quality treatment of donor site wounds.
皮肤移植技术广泛用于大面积烧伤、创伤以及各种急性和慢性伤口，对创伤组织的修复有很大贡献。然而，供体部位的修复和再生往往被忽视，导致感染和延迟愈合。因此，降低供体部位感染率并提高愈合的速度和质量至关重要。通过 Schiff 碱反应制备的低氧化海藻酸钠 （OSA） 接枝 ε ε epsi\varepsilon -聚赖氨酸 （OSA-g-EPL） 用于加载间充质干细胞外泌体 （Exo），并通过喷洒交联 Ca 2 + Ca 2 + Ca^(2+)\mathrm{Ca}^{2+} 在伤口表面形成凝胶膜 （HAE）。EPL 为水凝胶提供了良好的抗菌性能，Exo 促进了 M2 巨噬细胞的极化，缩短了伤口的炎症期并迅速过渡到增殖期，从而加速了伤口愈合过程，避免了向慢性伤口的转变。水凝胶优异的导电性和传感特性可用于实时监测小鼠的行为活动，以确定其伤口恢复情况。因此，该策略将为高效、高质量治疗供体部位伤口提供广阔的前景。

1. Introduction  1. 引言

Autografts remain the standard of care for the treatment of burns, large skin defects caused by disease and traumatic injury(Asuku et al., 2021). However, the risk and burden of donor site morbidity are often underestimated, and its healing and regeneration are often neglected (Chalwade et al., 2022). Research has shown that as many as half of donor site wounds showed signs of infection, leading to slow healing and in severe cases, even the formation of chronic wounds that caused more problems for the patient than the original injury (Voineskos et al., 2009). This problem is currently not well addressed.
自体移植物仍然是治疗烧伤、疾病和创伤性损伤引起的大面积皮肤缺陷的标准护理（Asuku 等人，2021 年）。然而，供体部位发病率的风险和负担往往被低估，其愈合和再生往往被忽视（Chalwade et al.， 2022）。研究表明，多达一半的供体部位伤口显示出感染迹象，导致愈合缓慢，在严重的情况下，甚至会形成慢性伤口，给患者带来比原始损伤更多的问题（Voineskos 等人，2009 年）。此问题目前尚未得到很好的解决。
The wound healing process consists of four main phases: hemostasis, inflammation, proliferation and remodeling (Wang et al., 2021), during which the wound may be interrupted or delayed by a variety of deleterious factors that can lead to a change from an acute wound to a chronic one, such as bacterial infections (Valente et al., 2014) and abnormalities of the immune cells (Kwak et al., 2022) that make it difficult to suppress inflammation. The same is true for donor site wounds, which require to be managed by keeping the wound moist, preventing infection
伤口愈合过程包括四个主要阶段：止血、炎症、增殖和重塑（Wang et al.， 2021），在此期间，伤口可能会被各种有害因素中断或延迟，这些有害因素可能导致从急性伤口转变为慢性伤口，例如细菌感染（Valente et al.， 2014）和免疫细胞异常（Kwak et al.， 2022 年），这使得难以抑制炎症。供体部位的伤口也是如此，需要通过保持伤口湿润、防止感染来控制伤口
and accelerating healing through immunoregulation to avoid the formation of chronic wounds. Therefore, targeted wound dressings are needed to address these issues.
并通过免疫调节加速愈合以避免慢性伤口的形成。因此，需要有针对性的伤口敷料来解决这些问题。
Hydrogels are widely used in wound healing applications due to their similarity to the natural extracellular matrix (ECM), ability to provide a moist environment, and excellent biocompatibility (Asadi et al., 2021). However, previously developed hydrogels suffered from poor antimicrobial properties, poor wound shape adaptation and inconvenient handling for use that might limit their ability to support skin tissue regeneration. In addition, most of the traditional therapeutic ideas of hydrogels for wounds referred to the promotion of granulation tissue formation (Mirani et al., 2017), re-epithelialization (Hu et al., 2021), vascular regeneration (Ma et al., 2024) and collagen deposition (He et al., 2021). These studies focused on surface analysis only, and further exploration of the therapeutic mechanisms and wound microenvironmental modulation was lacking (Landén et al., 2016).
水凝胶因其与天然细胞外基质 （ECM） 的相似性、提供潮湿环境的能力以及出色的生物相容性而广泛用于伤口愈合应用（Asadi等人，2021 年）。然而，先前开发的水凝胶具有抗菌性能差、伤口形状适应性差和使用不便等问题，这可能会限制它们支持皮肤组织再生的能力。此外，大多数水凝胶治疗伤口的传统治疗理念是指促进肉芽组织形成（Mirani等人，2017 年）、再上皮化（胡等人，2021 年）、血管再生（马等人，2024 年）和胶原蛋白沉积（He 等人，2021 年）。这些研究仅集中在表面分析上，缺乏对治疗机制和伤口微环境调节的进一步探索（Landén等人，2016 年）。
Compared to prefabricated gels, sprayable gels can be pumped through sprayers to rapidly form a protective gel layer in situ to inhibit bacterial invasion and growth, with the advantages of wound shape
与预制凝胶相比，可喷涂凝胶可以通过喷雾器泵送，在原位快速形成保护凝胶层，以抑制细菌侵袭和生长，并具有伤口形状的优点
Scheme 1. Preparation, mechanism, and application of the HAE hydrogel.
方案 1.HAE 水凝胶的制备、机制和应用。
adaptability and portability. A sprayable hydrogel consisting mainly of gelatin modified with methacrylic anhydride (GelMA) and simulated neutrophil nanoparticles could reduce glucose concentration around diabetic wounds and used to treat diabetic wounds (Liu et al., 2022). However, the sprayable GelMA hydrogel required subsequent crosslinking by blue light to form a gel, reducing ease of use. A sprayable black phosphorus (BP)-based hydrogel with antimicrobial properties promoted angiogenesis during joint wound healing (Ding et al., 2023). However, BP with antimicrobial properties might have limited application due to biotoxicity (Mohamad Latiff et al., 2018). Therefore, designing a sprayable hydrogel system with good biocompatibility, excellent antimicrobial properties, efficient tissue repair properties and ease of use will lead to new therapeutic approaches for wound healing at the donor site.
适应性和便携性。主要由甲基丙烯酸酐 （GelMA） 改性的明胶和模拟中性粒细胞纳米颗粒组成的可喷涂水凝胶可以降低糖尿病伤口周围的葡萄糖浓度，用于治疗糖尿病伤口（Liu et al.， 2022）。然而，可喷涂的 GelMA 水凝胶需要随后通过蓝光交联才能形成凝胶，从而降低了易用性。一种具有抗菌特性的可喷涂黑磷 （BP） 基水凝胶在关节伤口愈合过程中促进血管生成（Ding et al.， 2023）。然而，由于生物毒性，具有抗菌特性的 BP 的应用可能有限（Mohamad Latiff et al.， 2018）。因此，设计具有良好生物相容性、优异抗菌性能、高效组织修复性能和易用性的可喷涂水凝胶系统将为供体部位伤口愈合带来新的治疗方法。
Bacterial infections cause a persistent inflammatory response at the wound site, further delaying the healing process and, in severe cases, even leading to sepsis (Wang et al., 2023). Antimicrobial materials based on bactericidal substances such as metal derivatives, polyammonium salts and antibiotics have limited their application due to their limited efficiency, toxicity and the substantial increase in drug resistance (Liu et al., 2021). Antimicrobial peptides have shown great potential in the treatment of bacterial infections. ε ε epsi\varepsilon-polylysine (EPL) has the advantages
细菌感染会在伤口部位引起持续的炎症反应，进一步延迟愈合过程，严重时甚至会导致败血症（Wang et al.， 2023）。基于金属衍生物、聚铵盐和抗生素等杀菌物质的抗菌材料由于其效率有限、毒性和耐药性大幅增加而限制了其应用（Liu et al.， 2021）。抗菌肽在治疗细菌感染方面显示出巨大的潜力。 ε ε epsi\varepsilon -聚赖氨酸 （EPL） 具有优点
of high bacteriostatic activity due to its cationic nature, stability, high biodegradability and good safety (Chen, Chen, et al., 2023).
由于其阳离子性质、稳定性、高生物降解性和良好的安全性，具有高抑菌活性（Chen， Chen， et al.， 2023）。
The role of macrophage polarization in wound healing has attracted considerable interesting. Improper function of these immune cells may result in delayed healing of wounds (Sawaya et al., 2020). Exosomes are small extracellular vesicles with a diameter of 30-200 nm (Yang et al., 2022) and the mesenchymal stem cell-derived exosomes (Exo) have received much attention for their immunomodulatory and regenerative functions. Exo can promote macrophage polarization from M1 to M2 phenotype and increase anti-inflammatory cytokines and chemokines, reducing inflammation (Li et al., 2019). Therefore, Exo is expected to shorten the inflammatory phase and accelerate the remodeling phase during the donor site wounds healing process, thus effectively preventing the transformation into chronic wounds and promoting wound healing.
巨噬细胞极化在伤口愈合中的作用引起了相当多的兴趣。这些免疫细胞功能不当可能会导致伤口愈合延迟（Sawaya等人，2020 年）。外泌体是直径为 30-200 nm 的小细胞外囊泡 （Yanget al.， 2022），间充质干细胞衍生的外泌体 （Exo） 因其免疫调节和再生功能而受到广泛关注。外显子细胞可以促进巨噬细胞从 M1 表型极化到 M2 表型，并增加抗炎细胞因子和趋化因子，从而减少炎症 （Li et al.， 2019）。因此，在供体部位伤口愈合过程中，Exo有望缩短炎症期并加速重塑期，从而有效防止其转变为慢性伤口，促进伤口愈合。
Herein, in this work, a sprayable hydrogel was designed and fabricated, which could crosslink in situ at the wound site, adapt to the shape of the donor site wound, function as an antibacterial and antiinflammatory agent, and promote M2 macrophage polarization, thus facilitating tissue repair and skin growth for rapid healing, to provide a simple, rapid, and effective clinical treatment (Scheme 1). Two solutions of low-oxidized sodium alginate (OSA) mixed with MSC exosomes, and
在此，在这项工作中，设计并制备了一种可喷涂的水凝胶，它可以在伤口部位原位交联，适应供体部位伤口的形状，起抗菌和抗炎剂的作用，促进 M2 巨噬细胞极化，从而促进组织修复和皮肤生长快速愈合，提供一种简单、快速、 和有效的临床治疗（方案 1）。两种低氧化海藻酸钠 （OSA） 与 MSC 外泌体混合的溶液，以及
EPL mixed with CaCl 2 CaCl 2 CaCl_(2)\mathrm{CaCl}_{2} were sprayed out at the same time. The aldehyde group in the structure of OSA was crosslinked to the amino group in EPL through Schiff base bond, and the unoxidized carboxyl groups crosslinked with Ca 2 + Ca 2 + Ca^(2+)\mathrm{Ca}^{2+} to form a hydrogel film on the wound, forming a physical barrier to prevent external infections, accelerating wound regeneration and healing through immunoregulation. A full-layer wound model was used to simulate donor site wounds and to assess the wound healing-promoting effect of hydrogel dressings. In addition, the conductive hydrogel was used as a flexible electronic skin sensor that could rapidly sense body movements, achieve message transmission and monitor subtle behavioral activities in different states in mice. Combining the wound healing promotion function with the sensing property allowed real-time monitoring of wound recovery in mice through their behavioral activities during the wound healing process, providing a scientific, efficient and convenient way for clinical performance judgement.
同时喷出混合的 CaCl 2 CaCl 2 CaCl_(2)\mathrm{CaCl}_{2} EPL。OSA 结构中的醛基通过 Schiff 碱键与 EPL 中的氨基交联，未氧化的羧基与之交联 Ca 2 + Ca 2 + Ca^(2+)\mathrm{Ca}^{2+} ，在伤口上形成水凝胶膜，形成物理屏障，防止外部感染，通过免疫调节加速伤口再生和愈合。全层伤口模型用于模拟供体部位伤口并评估水凝胶敷料的伤口愈合促进作用。此外，导电水凝胶被用作柔性电子皮肤传感器，可以快速感应小鼠的身体运动，实现信息传递并监测小鼠在不同状态下的细微行为活动。将伤口愈合促进功能与传感特性相结合，通过小鼠在伤口愈合过程中的行为活动实时监测小鼠的伤口恢复情况，为临床性能判断提供了一种科学、高效、便捷的方法。

2. Materials and methods  2. 材料和方法

2.1. Materials  2.1. 材料

Sodium alginate (SA, M w = 2.0 × 10 5 g / mol M w = 2.0 × 10 5 g / mol M_(w)=2.0 xx10^(5)g//molM_{\mathrm{w}}=2.0 \times 10^{5} \mathrm{~g} / \mathrm{mol}, mannuronate/guluronate ratio = 0.8 = 0.8 =0.8=0.8 ), sodium periodate ( NaIO 4 , 99 % ) NaIO 4 , 99 % (NaIO_(4),99%)\left(\mathrm{NaIO}_{4}, 99 \%\right), Calcium Chloride, ε ε epsi\varepsilon-polylysine (EPL, 99 % 99 % 99%99 \% ), methylene blue ( 95 % 95 % 95%95 \% ), phosphate-buffered saline (PBS, pH = 7.4 pH = 7.4 pH=7.4\mathrm{pH}=7.4 ) and paraformaldehyde solution ( 4 % PFA 4 % PFA 4%PFA4 \% \mathrm{PFA} ) were purchased from Adamas. Escherichia coli (E. coli, ATCC25922) and Staphylococcus aureus (S. aureus, ATCC6358) were obtained from Qingdao Hope Bio-Technology Co., Ltd. Anticoagulated sheep blood was obtained from Nanjing Senbeijia Biotechnology Co. NIH 3 T3 mouse embryonic fibroblast was provided by the National Collection of Authenticated Cell Cultures (Shanghai, China). Mesenchymal stem cell nutristem (MSC NutriStem) was obtained from Sartorius.
海藻酸钠 （SA， M w = 2.0 × 10 5 g / mol M w = 2.0 × 10 5 g / mol M_(w)=2.0 xx10^(5)g//molM_{\mathrm{w}}=2.0 \times 10^{5} \mathrm{~g} / \mathrm{mol} 甘露糖醛酸钠/古尔尿酸比 = 0.8 = 0.8 =0.8=0.8 值 ）、高碘酸 ( NaIO 4 , 99 % ) NaIO 4 , 99 % (NaIO_(4),99%)\left(\mathrm{NaIO}_{4}, 99 \%\right) 钠 ， 氯化钙， ε ε epsi\varepsilon -聚赖氨酸 （EPL， 99 % 99 % 99%99 \% ）、亚甲蓝 （ 95 % 95 % 95%95 \% ）、磷酸盐缓冲盐水 （PBS， pH = 7.4 pH = 7.4 pH=7.4\mathrm{pH}=7.4 ） 和多聚甲醛溶液 （ 4 % PFA 4 % PFA 4%PFA4 \% \mathrm{PFA} ） 购自安道麦斯。大肠埃希菌 （E. coli， ATCC25922） 和金黄色葡萄球菌 （S. aureus， ATCC6358） 购自青岛霍普生物科技有限公司 抗凝绵羊血购自南京森北佳生物科技有限公司 NIH 3 T3 小鼠胚胎成纤维细胞由国家认证细胞培养保藏中心（中国上海）提供。间充质干细胞营养干细胞 （MSC NutriStem） 购自赛多利斯。

2.2. Synthesis of oxidized sodium alginate with low oxidation degree
2.2. 低氧化度的氧化海藻酸钠的合成

Oxidized sodium alginate (OSA) was fabricated according to the literature (Wang et al., 2022). 2 g of sodium alginate (SA) was dissolved in 200 mL of deionized water, and 0.2139 g of NaIO 4 NaIO 4 NaIO_(4)\mathrm{NaIO}_{4} was added to SA solution and stirred in the dark for 4 h at room temperature. 5 mL of ethylene glycol was added to terminate oxidation. The product was obtained though dialysis against water for 3 days and subsequently lyophilization.
氧化海藻酸钠 （OSA） 是根据文献制造的 （Wang等人，2022 年）。将 2 g 海藻酸钠 （SA） 溶于 200 mL 去离子水中，向 SA 溶液中加入 0.2139 g NaIO 4 NaIO 4 NaIO_(4)\mathrm{NaIO}_{4} 海藻酸钠，并在室温下避光搅拌 4 h。加入 5 mL 乙二醇终止氧化。该产品是通过用水透析 3 天，然后冻干获得的。

2.3. Extraction procedure of exosomes from MSC nutristem
2.3. 从 MSC 营养干细胞中提取外泌体的程序

The exosomes were extracted from culture supernatants of MSCs by ultracentrifugation. Firstly, the cell supernatant was centrifuged at 2000 g for 20 min to remove large debris and dead cells. Secondly, the supernatant obtained was centrifuged at 10 , 000 g 10 , 000 g 10,000g10,000 \mathrm{~g} for 30 min to remove small cellular debris. Thirdly, the supernatant obtained was filtered with a 0.22 μ m 0.22 μ m 0.22 mum0.22 \mu \mathrm{~m} sterilized filter (Millipore ® ® ^(®){ }^{\circledR} ) and then centrifuged at 100 , 000 g 100 , 000 g 100,000g100,000 \mathrm{~g} at 4 ∘ C 4 ∘ C 4^(@)C4{ }^{\circ} \mathrm{C} for 80 min . Lastly, the supernatant obtained was centrifuged at 100 , 000 g 100 , 000 g 100,000g100,000 \mathrm{~g} at 4 ∘ C 4 ∘ C 4^(@)C4^{\circ} \mathrm{C} for 80 min to remove contaminated proteins. The precipitated exosomes were collected and stored at − 80 ∘ C − 80 ∘ C -80^(@)C-80^{\circ} \mathrm{C}.
通过超速离心从 MSC 的培养上清液中提取外泌体。首先，将细胞上清液以 2000 g 离心 20 min，以去除大碎片和死细胞。其次，将获得的上清液离心 10 , 000 g 10 , 000 g 10,000g10,000 \mathrm{~g} 30 分钟以去除小细胞碎片。然后，将得到的上清液用 0.22 μ m 0.22 μ m 0.22 mum0.22 \mu \mathrm{~m} 灭菌过滤器 （Millipore ® ® ^(®){ }^{\circledR} ） 过滤，然后在 100 , 000 g 100 , 000 g 100,000g100,000 \mathrm{~g} 下 4 ∘ C 4 ∘ C 4^(@)C4{ }^{\circ} \mathrm{C} 离心 80 分钟。最后，将获得的上清液在 AT 4 ∘ C 4 ∘ C 4^(@)C4^{\circ} \mathrm{C} 离心 100 , 000 g 100 , 000 g 100,000g100,000 \mathrm{~g} 80 分钟以去除受污染的蛋白质。收集沉淀的外泌体并储存在 − 80 ∘ C − 80 ∘ C -80^(@)C-80^{\circ} \mathrm{C} 。

2.4. Preparation of OSA hydrogel (H)
2.4. OSA 水凝胶的制备 （H）

3.0 wt % 3.0 wt % 3.0wt%3.0 \mathrm{wt} \% OSA solution and 3.0 wt % CaCl 2 3.0 wt % CaCl 2 3.0wt%CaCl_(2)3.0 \mathrm{wt} \% \mathrm{CaCl}_{2} solution were filled separately into two spray bottles and sprayed simultaneously at a suitable angle. H hydrogels were formed in situ after 2 min .
3.0 wt % 3.0 wt % 3.0wt%3.0 \mathrm{wt} \% 将 OSA 溶液和 3.0 wt % CaCl 2 3.0 wt % CaCl 2 3.0wt%CaCl_(2)3.0 \mathrm{wt} \% \mathrm{CaCl}_{2} 溶液分别填充到两个喷雾瓶中，并以合适的角度同时喷涂。2 分钟后原位形成 H 水凝胶。

2.5. Preparation of OSA-EPL hydrogel (HA)
2.5. OSA-EPL 水凝胶 （HA） 的制备

EPL was mixed with CaCl 2 CaCl 2 CaCl_(2)\mathrm{CaCl}_{2} solution ( 3.0 wt % 3.0 wt % 3.0wt%3.0 \mathrm{wt} \% ) at a concentration of 1.0 wt % 1.0 wt % 1.0wt%1.0 \mathrm{wt} \%. The above solution and 3.0 wt % 3.0 wt % 3.0wt%3.0 \mathrm{wt} \% OSA solution were filled separately into two spray bottles and sprayed simultaneously. HA
将 EPL 与 CaCl 2 CaCl 2 CaCl_(2)\mathrm{CaCl}_{2} 浓度为 1.0 wt % 1.0 wt % 1.0wt%1.0 \mathrm{wt} \% 的溶液 （ 3.0 wt % 3.0 wt % 3.0wt%3.0 \mathrm{wt} \% ） 混合。将上述溶液和 3.0 wt % 3.0 wt % 3.0wt%3.0 \mathrm{wt} \% OSA 溶液分别灌装到两个喷雾瓶中，同时喷涂。医 管 局
hydrogels were formed in situ after 2 min.
2 分钟后原位形成水凝胶。

2.6. Preparation of OSA-EPL-Exo hydrogel (HAE)
2.6. OSA-EPL-Exo 水凝胶 （HAE） 的制备

An exosome@PBS solution with a concentration of 2 × 10 12 mL − 1 2 × 10 12 mL − 1 2xx10^(12)mL^(-1)2 \times 10^{12} \mathrm{~mL}^{-1} was evenly mixed with OSA solution ( 3.0 wt % 3.0 wt % 3.0wt%3.0 \mathrm{wt} \% ) at a volume ratio of 1 : 10. EPL was mixed with calcium chloride solution (3.0 wt%) at a concentration of 1.0 wt % 1.0 wt % 1.0wt%1.0 \mathrm{wt} \%. The above two solutions were filled into two separate spray bottles and sprayed simultaneously. HAE hydrogels were obtained in situ after 2 min .
将浓度为 的 2 × 10 12 mL − 1 2 × 10 12 mL − 1 2xx10^(12)mL^(-1)2 \times 10^{12} \mathrm{~mL}^{-1} exosome@PBS 溶液与 OSA 溶液 （ 3.0 wt % 3.0 wt % 3.0wt%3.0 \mathrm{wt} \% ） 以 1 ： 10 的体积比均匀混合。将 EPL 与氯化钙溶液 （3.0 wt%） 混合，浓度为 1.0 wt % 1.0 wt % 1.0wt%1.0 \mathrm{wt} \% 。将上述两种溶液灌装到两个单独的喷雾瓶中，同时喷涂。2 分钟后原位获得 HAE 水凝胶。

2.7. Characterization  2.7. 表征

Fourier transform infrared (FT-IR) instrument (INVENIO) spectroscopy was used to analyze the FT-IR spectra of SA, OSA, EPL and OSAEPL. Nuclear magnetic resonance spectroscopy ( 1 H 1 H ^(1)H{ }^{1} \mathrm{H} NMR) spectra of SA and OSA were recorded using nuclear magnetic resonance spectrometer (Bruker 400 M ) with the samples dissolved in D 2 O D 2 O D_(2)O\mathrm{D}_{2} \mathrm{O}. X-ray powder polycrystalline diffractometer (XRD, DX-2700BH) was used to analyze the structure of SA and OSA. The diffraction angle (20) was scanned over a region from 5 ∘ 5 ∘ 5^(@)5^{\circ} to 45 ∘ 45 ∘ 45^(@)45^{\circ} at a scan rate of 2 ∘ / min 2 ∘ / min 2^(@)//min2^{\circ} / \mathrm{min}. The UV-vis absorption spectra of H and HA were measured by Lambda 35 (PerkinElmer) from 200 to 800 nm , and the scanning rate was 1 nm s − 1 1 nm s − 1 1nms^(-1)1 \mathrm{~nm} \mathrm{~s}{ }^{-1}. SEM images of lyophilized hydrogels were obtained using scanning electron microscopy (SEM) (ZEISS GeminiSEM 300). High-resolution transmission electron microscopy (HR-TEM, JEM-2100F) was used to obtain exosome morphology. Laser confocal microscopy (Leica, stellaris5) images were examined for the distribution of Dio-labelled exosomes in hydrogels.
傅里叶变换红外 （FT-IR） 仪器 （INVENIO） 光谱用于分析 SA、OSA、EPL 和 OSAEPL 的 FT-IR 光谱。使用核磁共振波谱仪 （Bruker 400 M） 记录 SA 和 OSA 的核磁共振波谱 （ 1 H 1 H ^(1)H{ }^{1} \mathrm{H} NMR） 波谱，样品溶解在 D 2 O D 2 O D_(2)O\mathrm{D}_{2} \mathrm{O} 中。采用 X 射线粉末多晶衍射仪 （XRD， DX-2700BH） 分析 SA 和 OSA 的结构。衍射角 （20） 以 的扫描速率 2 ∘ / min 2 ∘ / min 2^(@)//min2^{\circ} / \mathrm{min} 从 到 5 ∘ 5 ∘ 5^(@)5^{\circ} 45 ∘ 45 ∘ 45^(@)45^{\circ} 的区域进行扫描。用 Lambda 35 （PerkinElmer） 在 200 至 800 nm 范围内测量 H 和 HA 的紫外-可见吸收光谱，扫描速率为 1 nm s − 1 1 nm s − 1 1nms^(-1)1 \mathrm{~nm} \mathrm{~s}{ }^{-1} 。使用扫描电子显微镜 （SEM） （ZEISS GeminiSEM 300） 获得冻干水凝胶的 SEM 图像。高分辨率透射电子显微镜 （HR-TEM， JEM-2100F） 用于获得外泌体形态。检查激光共聚焦显微镜 （Leica， stellaris5） 图像中 Dio 标记的外泌体在水凝胶中的分布。

2.8. Rheological behavior, swelling and mechanical properties
2.8. 流变行为、溶胀和机械性能

Rheological behaviors were conducted on Thermo Fisher HAKKE MARS rheometer with a 20 mm diameter plate and gap set at 1 mm at 37 ∘ C 37 ∘ C 37^(@)C37{ }^{\circ} \mathrm{C}. The storage modulus G ′ G ′ G^(')\mathrm{G}^{\prime} and loss modulus G " of hydrogels with different CaCl 2 CaCl 2 CaCl_(2)\mathrm{CaCl}_{2} concentrations (3.0, 6.0, 9.0 wt % 9.0 wt % 9.0wt%9.0 \mathrm{wt} \% ) were measured at a constant deformation of 1 % 1 % 1%1 \% strain (angular frequency, 0.1 − 10 Hz 0.1 − 10 Hz 0.1-10Hz0.1-10 \mathrm{~Hz} ). The hydrogel with CaCl 2 CaCl 2 CaCl_(2)\mathrm{CaCl}_{2} concentrations of 3.0 wt % 3.0 wt % 3.0wt%3.0 \mathrm{wt} \% was measured under strain amplitude sweep ( γ = 1 % − 10 % γ = 1 % − 10 % gamma=1%-10%\gamma=1 \%-10 \% ) at a fixed angular frequency (10 rad s − 1 s − 1 s^(-1)\mathrm{s}^{-1} ). The swelling properties of the hydrogels were tested using the following method. The initial weights of the sample were recorded as W 0 W 0 W_(0)\mathrm{W}_{0}. Two kinds of hydrogels were immersed in PBS solution ( pH = 7.4 pH = 7.4 pH=7.4\mathrm{pH}=7.4 ) at 37 ∘ C 37 ∘ C 37^(@)C37{ }^{\circ} \mathrm{C} for a period of time and then removed. The sample was weighed again at regular intervals. The weight of the hydrogel at different time was recorded as W t W t W_(t)W_{\mathrm{t}}. Swelling ratio can be calculated by eq. 1 .
流变行为是在 Thermo Fisher HAKKE MARS 流变仪上进行的，该流变仪的板直径为 20 mm，间隙设置为 1 mm。 37 ∘ C 37 ∘ C 37^(@)C37{ }^{\circ} \mathrm{C} 在 1 % 1 % 1%1 \% 应变恒定变形 （角频率， 0.1 − 10 Hz 0.1 − 10 Hz 0.1-10Hz0.1-10 \mathrm{~Hz} ） 下测量不同 CaCl 2 CaCl 2 CaCl_(2)\mathrm{CaCl}_{2} 浓度 （3.0， 6.0， 9.0 wt % 9.0 wt % 9.0wt%9.0 \mathrm{wt} \% ） 的水凝胶的储能模量 G ′ G ′ G^(')\mathrm{G}^{\prime} 和损耗模量 G ”。在应变幅扫描 （ ） 下以固定角频率 （10 rad） 测量浓度为 3.0 wt % 3.0 wt % 3.0wt%3.0 \mathrm{wt} \% 的水凝胶 CaCl 2 CaCl 2 CaCl_(2)\mathrm{CaCl}_{2} s − 1 s − 1 s^(-1)\mathrm{s}^{-1} 。 γ = 1 % − 10 % γ = 1 % − 10 % gamma=1%-10%\gamma=1 \%-10 \% 使用以下方法测试水凝胶的溶胀性能。样品的初始重量记录为 W 0 W 0 W_(0)\mathrm{W}_{0} 。将两种水凝胶在 PBS 溶液 （ pH = 7.4 pH = 7.4 pH=7.4\mathrm{pH}=7.4 ） 37 ∘ C 37 ∘ C 37^(@)C37{ }^{\circ} \mathrm{C} 中浸泡一段时间，然后去除。定期再次称量样品。水凝胶在不同时间的重量记录为 W t W t W_(t)W_{\mathrm{t}} 。膨胀率可以通过方程 1 计算。
Swelling (%) = W t W 0 × 100 % = W t W 0 × 100 % =(W_(t))/(W_(0))xx100%=\frac{W_{t}}{W_{0}} \times 100 \%  肿胀 （%） = W t W 0 × 100 % = W t W 0 × 100 % =(W_(t))/(W_(0))xx100%=\frac{W_{t}}{W_{0}} \times 100 \%
The H and HA hydrogel were made into elongated strips of specimens. The mechanical properties of the hydrogels were investigated by a digital tensile machine (Instron 5565). The initial distance between the two fixtures was 3 mm , the specimen width was 5 mm , and the thickness was 2 mm . The extension rate was set at 100 mm min − 1 100 mm min − 1 100mmmin^(-1)100 \mathrm{~mm} \mathrm{~min}^{-1}. The compression experiments were performed at a constant compression rate of 5 mm min − 1 5 mm min − 1 5mmmin^(-1)5 \mathrm{~mm} \mathrm{~min}{ }^{-1}. The cylindrical specimens were prepared with a diameter of 20 mm and a height of 1 mm .
将 H 和 HA 水凝胶制成细长的标本条。通过数字拉伸机 （Instron 5565） 研究水凝胶的机械性能。两个夹具之间的初始距离为 3 mm，试样宽度为 5 mm，厚度为 2 mm。扩展速率设置为 100 mm min − 1 100 mm min − 1 100mmmin^(-1)100 \mathrm{~mm} \mathrm{~min}^{-1} 。压缩实验以恒定的压缩速率 进行 5 mm min − 1 5 mm min − 1 5mmmin^(-1)5 \mathrm{~mm} \mathrm{~min}{ }^{-1} 。制备直径为 20 mm、高度为 1 mm 的圆柱形试样。

2.9. Cytocompatibility and skin toxicity tests of hydrogels
2.9. 水凝胶的细胞相容性和皮肤毒性试验

NIH/3 T3 cells were inoculated at a density of 5 × 10 3 5 × 10 3 5xx10^(3)5 \times 10^{3} cells/well on 96-well plates. After soaking 2 mL of UV-irradiated sterilized hydrogel in 40 mL of complete DMEM medium for 24 h , different concentrations of hydrogel extracts ( 0 % , 25.0 % , 50.0 % , 75.0 % , 100.0 % 0 % , 25.0 % , 50.0 % , 75.0 % , 100.0 % 0%,25.0%,50.0%,75.0%,100.0%0 \%, 25.0 \%, 50.0 \%, 75.0 \%, 100.0 \% ) were prepared using complete DMEM medium. After replacing the original medium with different hydrogel extracts, incubation was continued for 24 h . The relative growth rate (RGR) of cells was determined by CCK-8 assay. The
在 96 孔板上以细胞 5 × 10 3 5 × 10 3 5xx10^(3)5 \times 10^{3} 密度/孔接种 NIH/3 T3 细胞。将 2 mL 紫外线照射灭菌水凝胶浸泡在 40 mL 完全 DMEM 培养基中 24 小时后，使用完全 DMEM 培养基制备不同浓度的水凝胶提取物 （ 0 % , 25.0 % , 50.0 % , 75.0 % , 100.0 % 0 % , 25.0 % , 50.0 % , 75.0 % , 100.0 % 0%,25.0%,50.0%,75.0%,100.0%0 \%, 25.0 \%, 50.0 \%, 75.0 \%, 100.0 \% ）。用不同的水凝胶提取物替换原始培养基后，继续孵育 24 小时。CCK-8 法测定细胞的相对生长速率 （RGR）。这
    • Corresponding authors.  通讯作者。
    E-mail addresses: xiaoyunxietj@126.com (X. Xie), yuanwz@tongji.edu.cn (W. Yuan).
    电子邮件地址：xiaoyunxietj@126.com （X. Xie）， yuanwz@tongji.edu.cn （W. Yuan）。