
==== Front
Bioact Mater
Bioact Mater
Bioactive Materials
2452-199X
KeAi Publishing

S2452-199X(24)00320-7
10.1016/j.bioactmat.2024.08.001
Article
Mussel-inspired sulfated hyaluronan cryogel patch with antioxidant, anti-inflammatory, and drug-loading properties for multifunctional wound adhesives
Song Wonmoon ab1
Choi Young Hwan acd1
Moon Young Gi a
Lee Changyub a
Sundaram M. Nivedhitha a
Hwang Nathaniel S. nshwang@snu.ac.kr
abce⁎
a School of Chemical and Biological Engineering, Institute for Chemical Processes, Seoul National University, Seoul, 08826, Republic of Korea
b Interdisciplinary Program in Bioengineering, Seoul National University, Seoul, 08826, Republic of Korea
c Bio-MAX/N-Bio Institute, Institute of BioEngineering, Seoul National University, Seoul, 08826, Republic of Korea
d Division of Pediatric Cardiac Surgery, Department of Surgery, Johns Hopkins School of Medicine, Baltimore, MD, USA
e Institute of Engineering Research, Seoul National University, Seoul, 08826, Republic of Korea
⁎ Corresponding author. School of Chemical and Biological Engineering, Seoul National University, Seoul, Republic of Korea. nshwang@snu.ac.kr
1 These authors contributed equally to this study.

14 8 2024
10 2024
14 8 2024
40 582596
3 3 2024
30 7 2024
2 8 2024
© 2024 The Authors
2024
https://creativecommons.org/licenses/by-nc-nd/4.0/ This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
Wounds, characterized by the disruption of the continuity of body tissues resulting from external trauma, manifest in diverse types and locations. Although numerous wound dressings are available for various wound scenarios, it remains challenging to find an integrative wound dressing capable of addressing diverse wound situations. We focused on utilizing sulfated hyaluronan (sHA), known for its anti-inflammatory properties and capacity to load cationic drugs. By conjugating catechol groups to sHA (sHA-CA), we achieved several advantages in wound healing: 1) Fabrication of patches through crosslinking with catechol-modified high-molecular-weight hyaluronan (HA(HMW)-CA), 2) Adhesiveness that enabled stable localization, 3) Radical scavenging that could synergize with the immunomodulation of sHA. The sHA-CA patches demonstrated therapeutic efficacy in three distinct murine wound models: diabetic wound, hepatic hemorrhage, and post-surgical adhesion. Collectively, these findings underscore the potential of the sHA-CA patch as a promising candidate for the next-generation wound dressing.

Graphical abstract

Image 1

Highlights

• Adhesive, drug-loadable, and anti-inflammatory dry patch was designed to treat diverse wounds.

• The dry patch healed diabetic wounds via M2 polarization and VEGF accelerated the process.

• The dry patch stopped bleeding by absorbing blood and thrombin could support the hemostasis.

• The dry patch prevented post-surgical adhesion by forming anti-inflammatory physical barrier.

Keywords

Adhesive patch
Anti-inflammation
Sulfated hyaluronic acid
Post-surgical adhesion
Diabetic wound
Hemostasis
==== Body
pmc1 Introduction

“Wound” is defined as injuries or ruptures that disrupt the anatomical and physiological integrity within living tissues [1,2]. There exists a wide spectrum of causes and severities in wound cases. In some cases, only the skin is affected and can regenerate rapidly. However, in the case of diabetes mellitus, dysregulated inflammation and metabolism exacerbate the severity of wounds, resulting in 9.1–26.1 million cases of diabetic ulcer per year worldwide [3]. Conversely, in other scenarios, there are life-threatening situations arising from complex wounds affecting internal organs. For example, liver wounds represent a common lethal injury, primarily due to hemorrhaging [4,5]. Excessive hemorrhage by severe trauma causes over 5.8 million deaths per year [6]. On the other hand, uncontrolled wound healing processes can induce another pathological problem. During laparotomy, peritoneal injury accelerates uncontrolled wound healing such as excessive fibrosis, leading to post-surgical adhesion. In the U.S., over 19 million cases of post-surgical adhesion occur per year [7]. Numerous wound dressings specialized for each wound case have been developed to treat wounds. For instance, wound dressings to repair skin wounds [8], hemostatic gauze to stop bleeding [9], and anti-adhesion barriers to prevent post-surgical adhesion [10]. Furthermore, patches with high performance for addressing wounds have also been actively researched [[11], [12], [13]]. However, there have been few attempts to develop a functional wound dressing that can treat multiple indications [14]. If there existed a multifunctional wound dressing to manage those different types of wounds, it would enhance convenience, reduce cost, and extend its effectiveness to cover wounds with complex symptoms.

Tissue-adhesive hyaluronic acid (HA) hydrogels have been considered as an effective therapeutic option as wound dressing [[14], [15], [16], [17], [18], [19]]. The catechol-conjugated HA (HA-CA) hydrogel can be fabricated by crosslinking between catechol groups in oxidative conditions [20]. This hydrogel easily adhered to wet organ surface and stably located to the target. Furthermore, the catechol-mediated crosslinking enhanced mechanical properties compared to HA, which has been difficult to use in biomedical applications due to its rapid degradation [21]. However, HA-based biomaterials exhibit certain limitations. Unlike other anionic glycosaminoglycans (GAGs) such as heparin, their weak negative charge may not be sufficient for loading cationic drugs through electrostatic interactions [22]. Additionally, there is a risk of inflammation. Low-molecular weight HA and its derivatives can be generated during the degradation process, which can cause inflammation at the implantation site [23]. One strategy to overcome the limitation of HA is to modify it into sulfated HA (sHA) by sulfation of the hydroxyl groups. There are some advantages of sHA compared to HA. First, the stability of sHA is better than HA as the sulfate groups hinder the reaction of hyaluronidase and resist degradation in vivo [24]. Second, due to the enhanced anionic potential of sHA, cationic drugs can be bound effectively and released sustainably [25,26]. Third, the sHA modulates M1 (pro-inflammatory)-M2 (anti-inflammatory) repolarization via suppression of NF-κB nuclear translocation, which can accelerate wound healing due to its anti-inflammatory property [27]. Therefore, the above mentioned multifunctional properties of sHA make it suitable to be used as a wound dressing material.

In this study, we synthesized sHA-CA and fabricated a multifunctional dry adhesive cryogel patch by crosslinking the sHA-CA with HA(HMW)-CA. To crosslink the sHA-CA and HA(HMW)-CA, the patch was prepared using a freeze-thawing method under alkaline conditions. Alkaline conditions help catechol to be oxidized as o-quinone, which can participate in crosslinking reactions with other o-quinones [28]. Freezing accelerates crosslinking between oxidized catechols through inducing ice crystallization and localization of polymers between ice crystals [20,29]. Drying the patch maximized its adhesiveness, allowing it to absorb liquids on the target surface [30]. The dry adhesive cryogel patch exhibited excellent adhesiveness to wet tissue compared to commercial cyanoacrylate glue. Additionally, this patch displayed some unique advantageous characteristics derived from sHA, compared to HA(LMW), which had a similar molecular weight to the sHA we used. sHA-CA overcame the rapid degradation of HA, which has been a representative limitation of HA for biomaterial applications. The anionic charge of sHA enabled the loading of cationic bioactive proteins such as VEGF165 and thrombin, enhancing the utility of sHA for various diseases. Furthermore, due to its intrinsic anti-inflammatory mechanism, the patch demonstrated immunomodulatory function and attenuated inflammation.

To demonstrate the therapeutic potential of the sHA-CA patch as a multifunctional wound dressing, we conducted efficacy tests using three different wound models (Fig. 1A). 1) Diabetic wound model: sHA-CA patches supported diabetic wound healing by building an anti-inflammatory environment through radical scavenging by catechols and M2 polarization of macrophages by sHA. In addition, we loaded cationic VEGF165 into sHA-CA patches, and we demonstrated that sustained release of VEGF165 from the sHA-CA patches accelerated diabetic wound healing. 2) Liver hemorrhage model: hyaluronic acid has the ability to absorb and retain a significant amount of water. Also, catechol has hemostatic activity through interacting with platelets in blood [31]. Harnessing these two aspects, the dry sHA-CA patch exhibited a hemostatic effect by absorbing blood from liver hemorrhages. Additionally, when thrombin was added to the patch, its blood-absorbing capacity synergized with thrombin activity, leading to an extremely rapid hemostatic function. 3) Post-surgical adhesion model: When the sHA-CA patch was applied to the cecal abrasion model, the patch swiftly removed surface moisture and adhered securely to the target area. This physical barrier formation effectively prevented peritoneal adhesion with its intrinsic anti-inflammatory properties. These experiments collectively illustrate the versatility and therapeutic efficacy of the sHA-CA patch as a multifunctional wound dressing in various wound scenarios.Fig. 1 Schematic illustration of the application and fabrication of the multipurpose sHA-CA adhesive dry patch. (A) Applications of the sHA-CA patches in the healing of three types of wounds: diabetic wound healing, liver hemorrhage, and post-surgical adhesion. (B) Schematic illustration of the gelation of the sHA-CA patches through catechol oxidation in an alkaline condition. (C) Schematic illustration of the crosslinking mechanism mediated by catechol oxidation. (D) Representative image of the sHA-CA patch. (E) The roles of sHA-CA in the composition of the sHA-CA patches.

Fig. 1

2 Results and discussion

2.1 Synthesis and characterization of HA-CA and sHA-CA

The sHA was prepared by sulfating the uronic acids in HA (Fig. S1C). We used H2SO4 and DCC as sulfation reagents, and the optimization conditions of sulfation were listed in Table S2 (Sample condition: sHA 1, sHA 2, sHA 3, and sHA 4). The degree of sulfation (DS) of sulfate groups per monosaccharide in HA was measured and calculated by elemental analysis. The sulfur (S) element in sHA increased with the increment of the amount of H2SO4 and DCC (Table S3, Fig. S2B). In contrast, the sulfur element was not detected in the HA sample. The highest DS value (70 ± 3.5 %) was obtained for the sHA at an estimated ratio of 2.8 sulfates per monosaccharide.

After sulfation, the molecular weight (MW) of HA decreased dramatically from 1335.87 ± 29.52 kDa to 89.57 ± 0.12 kDa due to the sulfation reagents in the sample condition ‘sHA 1’ (Table S3 and Fig. S2B). Subsequently, with the increase of the sulfation reagents, the MW of sulfated HA further decreased to 29.07 ± 0.42 kDa. Considering the results of sulfation sample conditions that the DS and MW were saturated after the sample condition ‘sHA 2’, we chose the sample condition ‘sHA 2’ for further experiments (DS = 69 ± 6.5 %, MW = 32.4 ± 2.42 kDa). The chemical compositions of HA and sHA were analyzed by FT-IR to confirm whether the sHA was properly sulfated (Fig. S2A). The FT-IR spectra of sHA revealed a new major peak at 1250 cm−1 and a minor peak at 794 cm−1, as compared to the spectrum of HA. The peak at 1250 cm−1 was attributed to the stretching vibrations of the S=O bond and that at 794 cm−1 to the S–O stretching vibrations, indicating the sulfation of the uronic acids in HA.

To fabricate adhesive patches, we utilized three HA variants: HA(HMW), HA(LMW), and sHA. These were conjugated with catechol to create mussel-inspired adhesives, yielding HA(HMW)-CA, HA(LMW)-CA, and sHA-CA, respectively. Each HA variant was reacted with an excess of EDC, sulfo-NHS, and dopamine, achieving catechol substitution degrees of 31.7 % (HA(HMW)-CA, Fig. S3B), 12.0 % (HA(LMW)-CA, Fig. S3D), and 13.1 % (sHA-CA, Fig. S3F). The HA(HMW)-CA matrix provided mechanical support within patches. To compare the effectiveness of sHA-CA and HA(LMW)-CA, both were incorporated into separate patches with HA(HMW)-CA. Given the similar molecular weights of HA(LMW)-CA and sHA-CA, HA(LMW)-CA was chosen as a positive control to isolate the unique effects of sHA-CA without neglecting the mass contribution in the HA(HMW)-CA matrix. This control design allowed for an effective comparison between patches of HA(HMW)-CA without sHA-CA and HA(HMW)-CA with sHA-CA.

Numerous growth factors, chemokines, and cell adhesion molecules, collectively termed heparin-binding proteins, are known to bind to the highly-negative proteoglycans such as heparin and heparan sulfate [32], primarily through electrostatic interactions [33,34]. To explore the potential for electrostatic interactions between growth factors and sHA-CA, we analyzed the ζ-potential of the synthesized polymer (Fig. S4). The ζ-potential shifted from −25.3 ± 2.02 mV in the free HA(LMW) to −18.53 ± 0.35 mV in the HA(LMW)-CA. This change was attributed to the conjugation of dopamine with the carboxyl groups of HA. Notably, the ζ-potential of sHA-CA was −30.1 ± 2.99 mV, indicating a 1.6-fold increase in negative charge compared to HA(LMW)-CA.

2.2 Fabrication and chemical characterization of the sHA-CA patches

The sHA-CA patches were created by crosslinking a pre-gel mixture of HA(HMW)-CA and sHA-CA, via catechol oxidation. Catechol oxidation is one of the primary strategies for crosslinking in the formation of HA-based hydrogels [35]. We prepared a mixture solution of HA(HMW)-CA and sHA-CA, adjusting its pH to 9 (Fig. 1B). This adjustment promoted the oxidation of catechol to o-quinone, whose radicals subsequently reacted to form crosslinks (Fig. 1C). Additionally, the crosslinking process was enhanced through freeze-thawing. This step concentrated the solution via ice crystal formation, aiding in network formation [29].

After a day of crosslinking, the resultant sHA-CA hydrogel was lyophilized to form a dry patch, which exhibited a grey color due to catechol oxidation (Fig. 1D). This crosslinking method is advantageous compared to other techniques that typically employ strong oxidants such as NaIO4, which induce excessive oxidation. The simplicity and mildness of this gel fabrication process potentially increase its biocompatibility, making it more suitable for biomedical applications.

In the sHA-CA patches, HA(HMW)-CA served as a matrix for mechanical support, while sHA-CA exerted a pivotal role as the functional moiety (Fig. 1E). Catechol moieties enhanced the tissue-adhesiveness of the patch, which helped localization and stabilized action of the sHA-CA patches. Additionally, by scavenging ROS at the wounds, inflammation was attenuated during application. The main role of sHA in the sHA-CA was the immunomodulation of macrophages, promoting their polarization to the M2 phenotype. This anti-inflammation character could be synergized with the ROS-scavenging property of catechol. Furthermore, due to sHA's negative charge, sHA-CA could retain cationic drugs, which could accelerate therapeutic effects and be used for a wide range of applications.

2.3 Physicochemical properties of the HA-CA patch

As previously mentioned, HA(HMW)-CA was employed as the matrix of the patch for physical support. This choice was necessitated by the failure of HA(LMW)-CA or sHA-CA alone to gel, due to their insufficient molecular weight (Data not shown). To optimize the patch formulation, we created patches with varying sHA-CA contents: 0 %, 0.1 %, and 0.2 %. HA(LMW)-CA was added to the 0 % and 0.1 % sHA-CA groups to maintain a constant total content of HA additives (HA(LMW)-CA or sHA). That is, the concentrations of HA(LMW)-CA in the sHA-CA patch (0 %, 0.1 %, and 0.2 % of sHA-CA) were 0.2 %, 0.1 %, and 0 %, respectively (Fig. 2).Fig. 2 Chemical characterization of the sHA-CA patches before and after oxidation. (A) Photographs of the sHA-CA patches before and after oxidation (i), SEM images of the sHA-CA patches before and after oxidation (ii). (B) Absorbance profile of the sHA-CA patches during oxidiation in an alkaline condition at a 280 nm wavelength from the UV/vis absorption spectrum. (C) Quantification of the absorbance of the HA(HMW)-CA matrix at a 280 nm wavelength from the UV/vis absorption spectrum. (D) Schematic illustrations of the status of catechol moieties during oxidation and their XPS peaks. (E) Peak-fitting XPS spectra of the sHA-CA patches before oxidation and after oxidation. (F) Schematic illustration of the crosslinking between sHA-CA and its FT-IR peaks. (G) FT-IR spectra of sHA-CA patches with various concentrations of sHA-CA before and after oxidation. The sHA-CA patches with sHA-CA 0.0 % (Top), 0.1 % (Middle), and 0.2 % (Bottom).

Fig. 2

Auto-oxidation, induced by increasing the pH of the HA(HMW)-CA/sHA-CA pre-gel solution to 9, led to catechol oxidation to quinone and subsequent crosslinking between quinone moieties. This process darkened the color of the sHA-CA patches (Fig. 2A) and resulted in a denser network structure upon oxidation (Fig. 2A; upper). The oxidation altered the absorbance profile at 280 nm over a period of 3 h (Fig. 2B), and significantly increased the overall absorbance of the HA(HMW)-CA/sHA-CA in UV–vis spectroscopic studies (Fig. 2C). There were no specific differences between the sHA-CA groups. The absorbance at 280 nm rose from 0.62 to 1.17 upon oxidation, indicating the presence of various oxidized products derived from crosslinked catechol molecules [36].

To further confirm that catechol oxidation led to sequential crosslinking, X-ray photoelectron spectroscopy (XPS) analysis was employed (Fig. 2D). Before oxidation, the fraction of the C–C/C–H bond the in C 1s peak at 284.7 eV in the sHA-CA 0, 0.1, 0.2 patches made up 35.20 %, 31.98 %, and 33.59 % of the total C 1s species, respectively (Fig. 2E; left). Post-oxidation, these fractions increased to 55.80 %, 45.84 %, and 59.36 %, respectively (Fig. 2E; right), indicating elevated C–C bond levels in sHA-CA patches, thus demonstrating the activation of catechol in the HA-CA patch (Fig. 1C) [[37], [38], [39]]. In contrast, the C=O peak fraction at 287.4 eV dropped in the sHA-CA 0, 0.1, 0.2 patches from 14.86 %, 12.54 %, and 9.68 %–3.28 %, 0.21 %, and 0.12 % after oxidation, respectively, suggesting that the o-quinone groups were reduced and converted to hydroxyl groups after reaction completion. This implies that catechol moieties in HA(HMW)-CA, activated under oxidative alkaline conditions, formed o-quinone groups and subsequently crosslinked.

2.4 Physicochemical properties of the sHA-CA patches

The sHA-CA patches showed decreasing tendencies in FT-IR peaks relative to the pre-gel state (Fig. 2F and G). In HA(HMW)-CA patches, peaks for alcohol C–O bonds (1045 cm−1), aromatic C=C bonds (1634 cm−1), and O–H stretch (3300 cm−1) were diminished after oxidation (Fig. 2G), suggesting quinone-mediated oxidative products and crosslinking. The sulfate group peak (1250 cm−1) arose from the sHA-CA patches. Due to the catechol oxidation, the FT-IR peaks in sHA-CA patches at C–O bonds, aromatic C=C bonds, and O–H stretch were decreased similar to the HA(HMW)-CA patches.

Rheological analysis revealed no significant differences between the sHA-CA groups in storage (G′) and loss modulus (G”) in a frequency sweep test across a frequency range of 1–100 rad/s (Fig. 3A), suggesting stable gel network formation. In the amplitude sweep test, among the strain range from 1 to 100 %, G′ had higher values than G” (Fig. 3B). G′ values in the linear viscoelastic region were 458.98 ± 73.04 Pa, 490.46 ± 10.57 Pa, and 602.66 ± 60.96 Pa for 0 %, 0.1 %, and 0.2 % sHA-CA, respectively (Fig. 3C). Although the highest G’ was observed in the 0.2 % sHA-CA group, the sHA-CA concentration did not dramatically affect the patches' viscoelastic properties, which were primarily influenced by the HA(HMW)-CA matrix.Fig. 3 Physical and mechanical characterization of sHA-CA patches. (A–C) Rheological analysis of sHA-CA patches. Oscillatory frequency sweeps of sHA-CA patches performed at 5 % strain (A), Oscillatory strain sweeps of sHA-CA patches performed at 5 rad/s (B), and storage modulus (G′) of the sHA-CA patches at the linear viscoelastic region during oscillatory strain sweeps (C) (n = 4, mean ± SD, ns: not significant, and *p < 0.05). (D) Degradation profiles of sHA-CA patches after hyaluronidase (10 U/mL) treatment. (E) The swelling ratio of sHA-CA patches after 24 h of PBS treatment (n = 3, mean ± SD, and ns: not significant). (F) Schematic illustration of the standard lap-shear test (ASTM F2255) for measuring the adhesion strength of sHA-CA patches. (G) The adhesion strength curves of sHA-CA patches and cyanoacrylate adhered to wet porcine skin tissues. (H) The shear stress at the maximum during the lap-shear test of sHA-CA patches (n = 3, mean ± SD, ns: not significant, and **p < 0.01). (I) Photographs of sHA-CA patches adhering to diverse types of organs from mice including the liver, kidney, heart, and lung (Scale bar = 10 mm). (J) Photographs of sHA-CA patches adhering to a beating porcine heart (Scale bar = 10 mm).

Fig. 3

The stable gelation behaviors of the sHA-CA patches could be attributed to the freeze-thawing process used during gel fabrication. To assess the impact of this process on enhancing the mechanical properties of the gels, we conducted rheological experiments on sHA-CA patches that had not undergone the freeze-thawing process (Fig. S5). These patches displayed significant differences in the frequency sweep test (Fig. S5A) and amplitude sweep test (Fig. S5B) compared to patches that underwent the freeze-thawing process. Their mechanical properties were considerably lower than the patches with the freeze-thawing process. The G′ values in the linear viscoelastic region were 36.55 ± 2.26, 36.01 ± 1.22, and 35.68 ± 0.66 Pa for 0 %, 0.1 %, and 0.2 % sHA-CA, respectively (Fig. S5C). Similarly, the freeze-thawing process influenced the adhesiveness of sHA-CA patches. In the absence of freeze-thawing process, cohesion failures occurred easily during ASTM F2255 lap-shear tests (Fig. S5D). The sHA-CA patches without freeze-thawing couldn't withstand the shear stress (Fig. S5E) and exhibited significantly lower shear stress values than the patches with freeze-thawing (Fig. S5F). The shear stress values recorded were 0.18 ± 0.06, 0.14 ± 0.02, and 0.09 ± 0.00 kPa for 0 %, 0.1 %, and 0.2 % sHA-CA, respectively. Therefore, the freeze-thaw process significantly enhanced the mechanical properties of the sHA-CA patches, resulting in values that were more than 10-fold higher.

The degradation profile of the hydrogel is another indicator of crosslinking density. More stable network resists degradation well. The rapid degradation of HA by hyaluronidase has been known as a representative limitation of HA as a biomaterial. However, by hindering the action of hyaluronidase, the sHA can maintain its structure much longer than HA [24]. Therefore, we expected that the sHA-CA patches would have higher stability than HA-CA patches. For the degradation test, sHA-CA patches (sHA-CA 0 %, 0.1 %, and 0.2 %) were treated with hyaluronidase (10 U/mL) and their weight loss was measured at several time points. By 28 d after incubation, only 36.37 ± 9.53 % of the patch was retained in the sHA-CA 0 % group (Fig. 3D). In contrast, the sHA-CA 0.1 % and 0.2 % group maintained 60.52 ± 23.29 % and 68.48 ± 7.63 % of their initial weights, respectively. This indicates that sHA-CA substantially improves retention compared to HA-CA. The enhanced durability of sHA-CA patches suggests a potential for longer-lasting performance, particularly in applications where rapid HA-CA degradation is a concern.

The swelling ratio of the hydrogel represents the crosslinking density of the gel [37]. The mass increase by swelling of sHA-CA patches (sHA-CA 0 %, 0.1 %, and 0.2 %) were 115.52 ± 22.13, 120.17 ± 12.50, and 116.44 ± 14.80, respectively (Fig. 3E). There were no significant difference in the swelling ratio between groups. Because the HA(HMW)-CA matrix was the main component of the mechanical properties of sHA-CA patches, network density would be similar among the groups.

2.5 Adhesiveness of the sHA-CA patches

Adhesive biomaterials have been used with extensive applications in the field of biomedicine, including hemostatic materials [28], drug delivery depots [14], and tissue engineering scaffolds [40]. Their ability to adhere securely to target surfaces is crucial for their proper functioning during application. However, when these adhesive materials are applied to wet tissue surfaces such as mucous tissues or bleeding sites, they tend to lose their adhesive properties. The primary factor contributing to this limitation is the presence of water on the target surface, as water molecules prevent the adhesive moieties of the materials from bonding to the target site. Therefore, achieving adhesion by eliminating water from the target surfaces, a strategy referred to as 'dry adhesion,' has become an intriguing approach for adhering biomaterials to organs [15,41]. To address this challenge, we developed sHA-CA patches with a dry-type adhesive property through a lyophilization process.

To assess the adhesion strength of the sHA-CA patches on wet tissues, we followed the ASTM F2255 guidelines and conducted a standard lap-shear test (Fig. 3F). Each patch was affixed between two wet porcine skins and subjected to stretching using a Universal Testing Machine (UTM). The patches exhibited a significant adhesive strength curve when compared to cyanoacrylate, a representative commercial adhesive (Fig. 3G). The presence of sHA-CA had a statistically insignificant impact on the adhesive strength of the patch. In three different groups of sHA-CA patches containing varying amounts of sHA-CA (0 %, 0.1 %, and 0.2 %), shear stress at failure was observed at approximately 1.75 ± 0.58 kPa, 2.21 ± 0.22 kPa, and 2.63 ± 0.20 kPa, respectively. These values exceeded the adhesion strength of cyanoacrylate (0.99 ± 0.09 kPa), as cyanoacrylate was unable to remove water molecules from the surface of the porcine skin (Fig. 3H). Additionally, we confirmed whether loading cationic drugs (Thrombin and VEGF) could affect the adhesiveness of the patches, because drug loading is one of the main advantages of the sulfated GAG-based biomaterials (Fig. S6). Considering that organs within our body typically maintain a wet condition, the water-absorbing property of the sHA-CA patches is advantageous for biomedical applications as a bioadhesive. From Fig. 3I and J, it was evident that sHA-CA patches could effectively adhere to different wet organs such as the liver, kidney, heart, and lung.

2.6 In vitro biocompatibility and immunomodulatory properties of the sHA-CA patches

Biocompatibility is a crucial prerequisite for therapeutic biomaterials to ensure their safety in biomedical applications [42]. Materials without biocompatibility can induce cytotoxicity, hemolysis, and an inflammatory host response in the human body [43]. To evaluate the biocompatibility of sHA-CA patches, in vitro cytocompatibility was assessed using the Live&Dead assay (Fig. 4A and B). Incubation of NIH-3T3 fibroblasts with sHA-CA patch-conditioned medium for 7 d showed no difference in cell viability among control, sHA-CA 0 %, sHA-CA 0.1 %, and sHA-CA 0.2 % patch groups (Fig. 4A). The percentage of live cells in all groups exceeded 95 %, which fulfilled the requirement of 70 % as an international criteria (Fig. 4B) [44], confirming the cytocompatible nature of sHA-CA patches.Fig. 4 In vitro biocompatibility and in vitro immunomodulation of the sHA-CA patches. (A) Representative Live&Dead assay fluorescence images of NIH-3T3 cells incubated with sHA-CA patches of different sHA concentrations (Scale bar = 100 μm). (B) Quantification of the live NIH-3T3 cells by the Live&Dead assay (n = 4, mean ± SD, ns: not significant). (C–D) Representative H2DCFDA fluorescence images (C), and iNOS/Arg1 immunofluorescence staining images (D) of LPS-treated RAW264.7 cells incubated with sHA-CA patches of different sHA concentrations (Scale bar = 50 μm). (E) Quantification of the H2DCFDA-positive RAW264.7 cells by the H2DCFDA assay (n = 5, mean ± SD, ns: not significant, ***p < 0.001 and **p < 0.01). (F) Radical scavenging effect of sHA-CA patches on DPPH radicals (n = 3, mean ± SD, ns: not significant, ***p < 0.001). (G) Quantification of the iNOS-positive RAW264.7 cells by immunocytochemistry (n = 5, mean ± SD, ns: not significant, ***p < 0.001). (H) Quantification of the Arg1-positive RAW264.7 cells by immunocytochemistry (n = 5, mean ± SD, ns: not significant, ***p < 0.001 and **p < 0.01). (I) Schematic illustration of macrophage morphology change by M1-M2 polarization and definition of the elongation factor. (J) Quantification of elongation factors of LPS-treated RAW264.7 cells (n = 5, mean ± SD, ns: not significant, ***p < 0.001 and **p < 0.01). (K–L) Quantification of TNF-α (K) and NO (L) secreted by LPS-treated RAW264.7 cells (n = 4, mean ± SD, ns: not significant, ***p < 0.001, and *p < 0.05).

Fig. 4

Several reports have demonstrated the immunomodulatory function of sHA, polarizing M1 macrophages to M2 [27,45,46]. To confirm whether sHA-CA in the patch possesses immunomodulatory functions similar to sHA, we analyzed the in vitro immunomodulatory characteristics of the sHA-CA patches. To test the anti-inflammatory characteristics of sHA-CA patches, we first checked for any immunogenic effects in sHA-CA (Fig. S7A). sHA-CA was applied to RAW264.7 macrophages. We quantified the behavior of RAW264.7 macrophages after treating them with HA-CA and sHA-CA with different molecular weights. Furthermore, when macrophages detect pathogens like lipopolysaccharides (LPS) via TLR4, they release pro-inflammatory cytokines explosively, activating immune reactions and recruiting other immune cells [47]. Therefore, we treated sHA-CA to LPS-activated RAW264.7 macrophages and analyzed their behaviors.

HA(LMW)-CA showed a significantly high level of TNF-α expression in the RAW264.7 macrophages (Fig. S7A; left). In contrast, the TNF-α secretion from the sHA-CA-treated group was nearly the same as that of the control group. This contradictory result between HA(LMW)-CA and sHA-CA can be explained by the functions of HA(LMW) and sHA. HA(LMW) is known as a type of damage-associated molecular pattern (DAMPs), which can activate the immunogenicity of macrophages through their CD44-mediated DAMPs recognition mechanism [48,49]. Conversely, sHA has two known mechanisms that mediate anti-inflammatory effects. First, sulfate groups can prohibit DAMPs recognition, hindering the interaction between hyaluronan and CD44 [24,50]. Second, sulfate groups affect the cell signaling pathway of inflammation, attenuating the expression of pro-inflammatory cytokines in macrophages by downregulating NF-kB signaling [27,45,46].

To confirm the anti-inflammatory effect of the sHA in sHA-CA, we treated sHA-CA to RAW264.7 macrophages for 24 h and then LPS (100 ng/mL) was sequentially added and incubated for another 24 h (Fig. S7A; right). LPS treatment caused the RAW264.7 macrophages to increase TNF-α secretion. In this situation, HA(LMW)-CA couldn't reduce TNF-α secretion in the RAW264.7 macrophages. On the contrary, HA(HMA)-CA and sHA-CA lowered TNF-α secretion than control groups. Therefore, compared to HA(LMW)-CA, sHA-CA can reduce immunogenicity by altering the physicochemical behavior of macrophages.

Excessive LPS treatment can affect TNF-α expression due to macrophage apoptosis [51]. To exclude the apoptotic effect that reduces cell numbers, we optimized the LPS dose to 100 ng/mL for treating RAW264.7 cells (Fig. S7B). The addition of HA(LMW)-CA, HA(HMW)-CA, and sHA-CA to LPS-sensitized RAW264.7 cells did not affect the viability of RAW 264.7 cells (Fig. S7C).

Additionally, besides the immunomodulatory function of sHA, we hypothesized that the catechol in sHA-CA would contribute to anti-inflammatory effects through radical scavenging during inflammation. Pathogen-sensitized macrophages produce excessive ROS, accelerating immune reactions [52]. Catechol is known to scavenge harmful radicals from reactive oxygen species in the inflammatory area [28,53]. Thus, a synergy of anti-inflammatory effects based on ROS scavenging by catechol and the action of sHA is possible. The ROS derived from macrophages can be measured by using the fluorescent dye H2DCFDA [54]. LPS-treated RAW264.7 cells exhibited high fluorescence in the H2DCFDA assay (Fig. 4C). However, LPS-treated RAW264.7 cells cultured with sHA-CA patches did not activate H2DCFDA, indicating effective ROS scavenging by the sHA-CA patches. In all macrophage groups treated with sHA-CA patches, over 95 % of the cells were H2DCFDA negative (Fig. 4E). To confirm the ROS scavenging effect of the sHA-CA patches by catechol, we performed a radical scavenging assay using 2,2-Diphenyl-1-picrylhydrazyl (DPPH). DPPH can react with ROS, and the colorimetric absorbance change of DPPH at 517 nm can be measured. All of the patches exhibited significant ROS scavenging ability, 68.3 % of the performance of α-tocopherol, a strong antioxidant reagent used as a positive control (Fig. 4F).

To investigate the synergistic effect of sHA-CA on the immunomodulation of macrophages, we analyzed activated RAW264.7 cells treated with sHA-CA patches through immunocytochemistry and cytokine quantification. We treated RAW264.7 cells with sHA-CA patches containing different sHA-CA concentrations (0, 0.1, and 0.2 %) for 24 h and then sequancially treated them with LPS for 24 h as a positive control. RAW264.7 cells were then immunostained with antibodies against inducible nitric oxide synthase (iNOS) and arginase1 (Arg1). As shown in Fig. 4D, LPS treatment resulted in RAW264.7 cells expressing an Arg1low/iNOShigh phenotype. In contrast, the patch-treated RAW264.7 cells showed an Arg1high/iNOSlow phenotype, which became more significant with increasing sHA-CA concentration. The sHA-CA patches containing sHA-CA (sHA-CA 0.1 % and 0.2 % patches) reduced iNOS+ cells by nearly 2-fold compared to LPS-treated RAW264.7 cells (Fig. 4G). Additionally, sHA-CA patches increased Arg1+ cells, with over 40 % of RAW264.7 cells being Arg1+ when treated with the sHA-CA 0.2 % patch (Fig. 4H).

Morphological characteristics of LPS-treated RAW264.7 cells were altered by the sHA-CA patches. The morphological difference between the RAW264.7 cells in different groups was quantified as an elongation factor (Fig. 4I). M1 macrophages exhibit a spherical morphology, where the short axis of the cell is similar to the long axis (Elongation factor value is almost 1). In contrast, M2 macrophages have an elongated shape, with an elongation factor value exceeding 1 [55,56].

Higher sHA-CA content in the sHA-CA patches resulted in a higher elongation factor (Fig. 4J). Control, LPS, and sHA-CA 0 % patch groups showed average elongation factor values of 1.20 ± 0.06, 1.48 ± 0.23, and 1.90 ± 0.37, respectively. However, the average elongation factor of RAW264.7 cells in the sHA-CA 0.1 % and 0.2 % patch groups was significantly higher than the other groups, recording values of 4.62 ± 1.55 and 4.53 ± 0.67, respectively. Thus, the sHA-CA patches induced morphological changes in RAW264.7 cells more towards the M2 phenotype than the M1 phenotype.

Interestingly, we found a synergistic relationship for immunomodulation between sHA and catechol. After treating RAW 264.7 cells with the sHA-CA patches, the pro-inflammatory cytokine TNF-α levels and nitric oxide (NO) in the culture supernatant were analyzed (Fig. 4K and L). All sHA-CA patches, including the sHA-CA 0 % group, reduced TNF-α expression (Fig. 4K). The higher the sHA-CA content in the patches, the lower the TNF-α expression. Radical scavenging by the catechol moieties from the patches and the immunomodulatory characteristics of sHA likely contributed to the attenuation of TNF-α expression. All sHA-CA patches significantly lowered NO levels, which could be attributed to the radical scavenging property of the patches (Fig. 4L).

To confirm the immunomodulatory function of the sHA-CA patches, we conducted additional flow cytometry analysis, quantifying the expression of CD80 (M1) and CD206 (M2) in RAW264.7 cells (Fig. S8). LPS-treated RAW264.7 cells exhibited increased CD80 expression, with a mean fluorescence intensity (MFI) of 3186.25 ± 283.92, compared to the control group, which had an MFI of 86.65 ± 11.24. The CD80 MFI values for the sHA-CA 0, 0.1, and 0.2 groups were 1463.75 ± 205.24, 1160.75 ± 174.95, and 1014.25 ± 123.52, respectively. This indicates a 2.2-fold, 2.7-fold, and 3.1-fold decrease in CD80 MFI for the sHA-CA 0, 0.1, and 0.2 groups, respectively. The observed decrease in CD80 expression in the sHA-CA 0 patch group is likely due to the ROS-scavenging activity of the catechol moieties. Furthermore, the addition of sHA-CA appears to increase CD206 expression in RAW264.7 cells, which could be attributed to the anti-inflammatory properties of sHA-CA.

In conclusion, sHA-CA patches demonstrate good biocompatibility and can modulate M1-M2 states of RAW264.7 cells. Interestingly, while only the ROS scavenging activity of catechol groups affected activated RAW264.7 cells, it was not sufficient to attenuate LPS-mediated inflammation. Through synergy with sHA-CA, the sHA-CA patches were able to modulate the immune reactions of RAW264.7 cells.

2.7 Evaluation of the function of catechol moieties in the sHA-CA patch

To investigate the synergistic effects of sHA and catechol groups in the sHA-CA patches, we fabricated sHA patches without catechol groups for comparison. Methacrylation of HA (HA-MA) and sHA (sHA-MA) facilitated gelation through photo-crosslinking mediated by lithium phenyl-2,4,6-trimethylbenzoylphosphinate (LAP). The HA-MA/sHA-MA mixture solutions were crosslinked in a frozen state to form a cryogel structure. We optimized the HA-MA concentration at 1 % and adjusted sHA-MA concentrations at 0 %, 0.1 %, and 0.2 %. These patches exhibited mechanical properties similar to those of the sHA-CA patches in rheological analysis (Fig. S9). In the frequency sweep test, the sHA-MA gels demonstrated stable G′ and G” values from 1 to 100 rad/s (Fig. S9A). The amplitude sweep test (Fig. S9B) showed no significant differences between the sHA-MA groups and similar G′ values to the sHA-CA patches (Fig. S9C). The G’ values in the linear viscoelastic region for sHA-MA 0 %, 0.1 %, and 0.2 % were 1021.40 ± 120.11 Pa, 1146.43 ± 40.44 Pa, and 1118.76 ± 131.19 Pa, respectively. We used sHA-MA patches under these mechanical conditions for further comparisons with sHA-CA patches.

The adhesiveness of the sHA-CA patches primarily relies on the catechol moieties. To evaluate the effect of these moieties, we conducted the ASTM F2255 lap shear test with sHA-MA patches (Fig. S9D). The sHA-MA patches, lacking adhesive moieties, failed to adhere to wet porcine skin, recording adhesion strengths of 0.53 ± 0.45 kPa, 0.56 ± 0.27 kPa, and 0.61 ± 0.46 kPa, respectively (Fig. S9E). These values were approximately 3.87-fold lower than those of the sHA-CA patches. The lack of adhesiveness in the sHA patches makes them less effective for biomedical applications, as it increases the risk of detachment and failure. The inclusion of catechol moieties significantly enhanced the adhesive strength of the sHA-CA patches, rendering them more suitable for biomedical use.

Catechol moieties in the sHA-CA effectively scavenged ROS during oxidative stress, enhancing the patch's utility. In contrast, the sHA-MA patches, devoid of catechol moieties, showed minimal ROS scavenging ability, as evidenced in the DPPH assay, with scavenging abilities of 4.92 ± 3.24 %, 6.90 ± 8.02 %, and 6.40 ± 4.32 % for the 0 %, 0.1 %, and 0.2 % patches, respectively—approximately 9-fold lower than the sHA-CA patches. Since ROS exacerbate inflammation, their scavenging is crucial in supporting the anti-inflammatory properties of the sHA-CA patches. Notably, the sHA-MA patches were ineffective in protecting RAW264.7 cells from oxidative stresses (Fig. S9G).

The absence of catechol moieties also affected blood cell interactions; the sHA-MA patches were unable to retain any RBCs or platelets following blood treatment (Fig. S9H), diminishing their utility. The conjugation of catechol not only conferred adhesiveness to tissues and blood but also enhanced the patches' ROS scavenging capabilities, supporting their anti-inflammatory effects.

2.8 In vivo biocompatibility and in vivo retention of the sHA-CA patch

Before applying the sHA-CA patches to disease models for therapeutic applications, the in vivo biocompatibility of the sHA-CA patches was confirmed. We used luminol as an inflammation detection reagent, which can be activated by myeloperoxidase present in neutrophils and monocytes in inflammation [57]. We subcutaneously implanted 6 groups into the dorsal area of C57BL/6 mice and imaged luminescence signals from the inflammation site after 4 d of implantation by IVIS (Fig. 5A). Polystyrene latex beads were used as a positive control, and sHA-CA patches (sHA-CA 0.2 %) loaded with cationic drugs-VEGF and thrombin-were used as the sample groups. The luminescence was normalized by the basal signal of normal skin. The luminescence caused by inflammation was found to be high at the latex bead implanted site, 51.3-folds higher than normal skin (Fig. 5B). Compared to latex beads, other groups showed weak or no difference in signals from normal skin (Fig. 5C). However, when the sHA-CA patches were compared, except for the latex beads, the sHA-CA patch (sHA-CA 0 %) recorded a 2-fold higher luminescence than normal skin (Fig. 5D). The sHA-CA patches (sHA-CA 0.1 % and 0.2 %) showed basal luminescence, meaning they were not immunogenic in vivo. The luminescence observed for drug-loaded patches was also quite low. From these results, it is clear that sHA-CA patches were safe and could be used as a biomaterial for therapeutic applications.Fig. 5 In vivo biocompatibility and in vivo retention behaviors of the sHA-CA patches. (A) Schematic illustration of the in vivo biocompatibility imaging of the sHA-CA patches with various formulations using luminol assay. (B) Representative IVIS images of bright field (left) and bioluminescence (right) of luminol activity for implanted sHA-CA patches in the mice (Scale bar = 10 mm). (C–D) Quantification of bioluminescence efficiencies of luminol activities for implanted sHA-CA patches in mice. Comparison of the luminescence efficiencies of sHA-CA patches with latex bead as a positive control (C), comparison of the luminescence efficiencies between sHA-CA patches with various formulations (D) (n = 6, mean ± SD, ns: not significant, ***p < 0.001, **p < 0.01 and *p < 0.05). (E) Representative images of subcutaneously implanted sHA-CA patches at various time points (Scale bar = 5 mm). (F) In vivo retention profiles of sHA-CA patches of different sHA concentrations over time (n = 5, mean ± SD). (G) Quantification of in vivo retention time by calculation of the ratio of diameter at 14 d–0 d (n = 5, mean ± SD, ns: not significant, ***p < 0.001 and **p < 0.01, and *p < 0.05).

Fig. 5

Tuning the degradation time of the biomaterials is essential for their therapeutic application [[58], [59], [60]]. Inflammation at the wound site can last at least 2–6 d [[61], [62], [63]], and post-surgical adhesion usually occurs during 3–5 d after surgery [64,65]. Therefore, at least a 7 d of retention time is required for biomaterials. To examine the retention time of the sHA-CA patches in vivo, we subcutaneously implanted the patches (Diameter = 8 mm) into the C57BL/6 mice and harvested the patches at specific time points (4 d, 7 d, 11 d, and 14 d after implantation) (Fig. 5E). The higher the sHA-CA concentration in the sHA-CA patches, the slower their degradation rate (Fig. 5F). After 14 d, the sHA-CA patch (sHA-CA 0 %) was completely degraded. In the sHA-CA 0.1 % group, the patch became smaller and was recorded as 23.69 ± 9.29 % of its initial diameter. The diameter of the residual patch in the sHA-CA 0.2 % group was 41.23 ± 2.40 %. Sulfation of the hyaluronic acid has been known to extend its retention in vivo by inhibiting hyaluronidase activity [24]. Also, we demonstrated the extension of in vitro retention time of the sHA-CA patches by sHA-CA concentration in the patch in Fig. 3D. The diameter of sHA-CA 0.1 % at 7 d after implantation was already 29.63 ± 10.02 % compared to the initial diameter. That is, it was hard to expect that the sHA-CA 0.1 % group would act enough during inflammation. Thus, to maintain their function within 7 d at least, the sHA-CA 0.2 % group would be better to use for wound healing such as diabetic wound healing or peritoneal adhesion. Therefore, we optimized the concentration of the sHA-CA in the sHA-CA patches to 0.2 % and referred to it as the ‘sHA-CA patch’.

2.9 Diabetic wound healing effect of the sHA-CA patches

In recent years, engineering efforts have been directed towards developing biomaterials that enable the specific binding of growth factors and their controlled release. A number of growth factors, chemokines, and cell adhesion molecules, collectively referred to as heparin-binding proteins, interact with sulfated GAGs such as heparin. Due to their highly sulfated structures, sulfated GAGs possess a high negative charge, facilitating electrostatic interactions with growth factors such as basic fibroblast growth factor (bFGF), vascular endothelial growth factor (VEGF), bone morphogenetic protein-2 (BMP-2), and bone morphogenetic protein-7 (BMP-7) [66]. Drawing inspiration from these natural interactions, strategies have been devised for efficient growth factor delivery [67,68].

Like heparin, the negative charge of the sHA-CA patches allowed for the retention of cationic drugs, making them suitable for controlled drug release in wound models (Fig. 6A). VEGF165 is a known splicing isoform of VEGF, characterized by its positive electrostatic potential [69] (Fig. 6B). This cationic VEGF165 can bind to the sulfate groups of sHA-CA, resulting in its sustained release from the patch (Fig. 6C). The drug release profile of VEGF165 from the sHA-CA patch was 1.8-fold slower than that from the HA-CA patch. After 7 d of incubation, 11.37 ± 1.09 % of VEGF165 was released from the HA-CA patch, whereas 6.30 ± 1.04 % of VEGF165 was released from the sHA-CA patch. This system, capable of regulating VEGF165 release, contributed to a more stable VEGF165 concentration during the wound healing process.Fig. 6 Accelerated wound healing in diabetic mouse wound model using sHA-CA patches with VEGF loading. (A–B) Schematic illustration of the action of sHA-CA patches on diabetic wound healing. Topical delivery of VEGF to the wound site by sHA-CA patches and modeling diabetic mouse wound healing (A) and the electrostatic potential of the VEGF (B). (C) Release profiles of VEGF from sHA-CA patches (n = 3, mean ± SD). (D) Representative images of wound closure of diabetic wounds treated with VEGF-loaded sHA-CA patches at 0 d, 4 d, 7 d, 11 d, and 14 d after surgery. (E) Profiles of wound closure rate measured up to 14 d after surgery (n = 4, mean ± SD). (F) Representative H&E staining images of diabetic wounds treated with VEGF-loaded sHA-CA patches on 14 d (Scale bar = 500 μm). (G) Representative iNOS/Arg1 immunofluorescence staining images of diabetic wounds on 7 d after surgery (Scale bar = 50 μm). (H) M1/M2 ratio by immunohistochemistry (n = 4, mean ± SD, ns: not significant, **p < 0.01). (I–K) Quantification of pro-inflammatory cytokines TNF-α (I), IL-6 (J), and IL-12p70 (K) from tissue lysates of wound region on 4 d after surgery (n = 4, mean ± SD, ns: not significant, ***p < 0.001, **p < 0.01 and *p < 0.05).

Fig. 6

The pathogenic abnormalities associated with diabetes often impede the wound healing mechanisms in affected patients [70]. To accelerate the abnormal and slow wound healing process, we topically applied the patch to diabetic wound sites. The efficacy of sHA-CA patches, with or without VEGF165, was tested for wound healing in diabetic C57BL/6 mice (Fig. 6D). The negative control group, treated only with PBS, exhibited severely retarded healing, with only 50.12 ± 8.65 % of the wound area closed after 14 d (Fig. 6E). However, when the sHA-CA patch was applied to the wound, wound healing was accelerated, resulting in a wound area of 15.25 ± 13.32 % after 14 d. The anti-inflammatory properties of the sHA-CA patch played a crucial role in this enhanced wound healing. Remarkably, in the group treated with VEGF165-loaded sHA-CA patches, all wounds had completely closed after 14 d. Given that chronic inflammatory conditions in diabetes can reduce angiogenesis [71], the combination of anti-inflammatory action through sHA-CA and angiogenesis support by VEGF165 likely produced a synergistic effect on diabetic wound healing. Thus, this suggests that the action of anti-inflammation through sHA-CA and supporting angiogenesis by VEGF165 can maximize the synergistic effect to diabetic wound healing. Consequently, the anti-inflammatory sHA-CA patch with VEGF165 accelerated diabetic wound healing, achieving nearly 0 % wound area within 14 d of surgery (Fig. 6F). Despite the rapid wound healing, however, there was a possibility that VEGF165 could induce scar formation during wound healing [[72], [73], [74]]. Therefore, sHA-CA patches without VEGF165 would also be a suitable alternative for wound healing, considering scarless wound healing.

To illustrate the effectiveness of the sHA-CA patch as a wound dressing for diabetic wounds, we analyzed the wound tissues treated with sHA-CA patches. Immunohistochemistry of the wound site for iNOS and Arg1 revealed that iNOS+ cells predominated in the control group, while Arg1+ cells were more abundant in the sHA-CA patch and sHA-CA patch/VEGF groups (Fig. 6G). Based on measurements of iNOS+ and Arg1+ cells in the tissue samples, we calculated the M1/M2 ratio (Fig. 6H). In the wound tissues of the control group, M1 macrophages were dominant, resulting in an M1/M2 ratio of 2.24 ± 0.97, reflecting the pro-inflammatory environment of diabetic wounds. However, the groups treated with sHA-CA patches and sHA-CA/VEGF patches exhibited M1/M2 ratios of 0.15 ± 0.11 and 0.14 ± 0.12, respectively. This indicates that the sHA-CA patch strongly promoted M2 polarization of macrophages during application.

To quantify the inflammation level, we measured pro-inflammatory cytokines in the serum of mice at 4 d after surgery. TNF-α (Fig. 6I), IL-6 (Fig. 6J), and IL-12p70 (Fig. 6K) levels in the wound tissue were quantified. The sHA-CA patch and sHA-CA patch/VEGF groups exhibited a significant decrease in the levels of the pro-inflammatory cytokines across all tested cytokines. These results demonstrate that the anti-inflammatory function of sHA-CA, combined with sustained drug release, accelerated wound closure in a diabetic wound model.

2.10 Hemostatic effect of the sHA-CA patch on liver hemorrhage model

It is well-established that catechol possesses hemostatic properties through its interaction with platelets [31]. Given that the sHA-CA patch is a dry-type adhesive patch with the ability to absorb liquids and adhere to target surfaces, we anticipated its potential as a hemostatic agent. The sHA-CA patch could effectively staunch bleeding by absorbing blood and interacting with plasma proteins. Additionally, its adhesive properties at the bleeding site would aid in hemostasis by preventing blood leakage (Fig. 7A).Fig. 7 Hemostasis in mouse hepatic hemorrhage model using sHA-CA patches with thrombin loading. (A–B) Schematic illustration of the action of sHA-CA patches on the bleeding. Blood absorption by sHA-CA patches during hemorrhage (A), and the electrostatic potential of the thrombin (B). (C) Images from the hemolysis test of the sHA-CA patches. (D) Hemolysis ratio of the sHA-CA patches (n = 5, mean ± SD, ns: not significant, ***p < 0.001). (E) Schematic illustrations of an in vitro clotting test by treating recalcified citrated whole blood with thrombin-loaded sHA-CA patches. (F) Representative images of the in vitro blood clotting test. (G) Quantification of clotting time after applying recalcified citrated whole blood to thrombin-loaded sHA-CA patches (n = 4, mean ± SD, ns: not significant, ***p < 0.001). (H) Schematic illustration of modeling mouse hepatic hemorrhage. (I) Representative images of the mouse hepatic hemorrhage model with the application of thrombin-loaded sHA-CA patches on the punctured liver (Scale bar = 10 mm). (J) Quantification of lost blood from the punctured liver treated with thrombin-loaded sHA-CA patches (n = 4, mean ± SD, ns: not significant, ***p < 0.001 and **p < 0.01).

Fig. 7

Moreover, thrombin, a prominent hemostatic protein, features an anionic active site and a cationic exosite (Fig. 7B). Based on this structural characteristic, thrombin is known to bind with heparin, a highly sulfated GAG, which mediates complexation between thrombin and anti-thrombin III, resulting in anticoagulation [75]. However, unlike heparin, sHA doesn't induce anti-coagulation due to the absence of specific 3-O-sulfation of N-acetylglucosamine [76]. The 3-O-sulfation of N-acetylglucosamine is a critical structural element for the stable binding of anti-thrombin III to heparin, essential for anticoagulation [77,78]. Therefore, leveraging the electrostatic interaction between the anionic sHA-CA patch and the cationic exosite of thrombin represents a promising strategy for developing thrombin-based hemostatic materials.

Prior to applying the sHA-CA patch to hemorrhage scenarios, we conducted tests to assess whether sHA-CA could induce any hemotoxic effects on blood cells. Hemocompatibility was evaluated by performing a hemolysis test using the sHA-CA patch, with and without thrombin (Fig. 7C). Compared to Triton X-100 (positive control), both the thrombin and sHA-CA patch, with or without thrombin, exhibited hemocompatibility, with hemolysis ratios below 1 % (Fig. 7D). The data meet the value of 5 % as the international standard ISO 10993-5 [79], confirming the excellent hemocompatibility of sHA-CA patches.

To compare the hemostatic abilities of the materials, we conducted in vitro blood clotting tests. Thrombin, sHA-CA patch, and thrombin-loaded sHA-CA patch were treated with recalcified citrated whole blood for various time intervals (30 s - 10 min) and subsequently washed with PBS. After washing, samples with stable blood clots were imaged (Fig. 7E). Blood required more than 10 min to clot after recalcification, while the sHA-CA patch achieved clot formation within 4–6 min (Fig. 7F). The difference in coagulation time between the blood-alone group and the sHA-CA patch group can be attributed to the blood-absorbing property of the sHA-CA patch. The sHA-CA patch could absorb blood, and the catechol groups could aggregate red blood cells and platelets within the network [80]. The catechols in sHA-CA patches also could exert blood-gel interactions. In SEM images, the sHA-CA patches treated with RBCs (Fig. S10A) and platelets (Figs. S10B and C) retained the cells and induced aggregation. In all groups, significant trends of platelet activation were observed. The platelets aggregated and exhibited blebbed morphologies (Fig. S10C). When thrombin came into contact with blood, it immediately formed a stable clot (Fig. 7G). Commercial thrombin products are in powder form and require solubilization before use [81,82]. The liquid form of thrombin cannot be effectively applied to bleeding sites due to its low viscosity. Therefore, loading thrombin into biomaterials could be a more practical approach to using it as a hemostatic agent. The thrombin-loaded sHA-CA group showed immediate blood coagulation similar to the thrombin group (Fig. 7G). The rapid blood-absorbing property of the sHA-CA patch, combined with the effect of thrombin loaded within the patch, could provide a synergistic effect for the sHA-CA patch to be employed as a hemostatic agent.

To assess the hemostatic property of the sHA-CA patch loaded with thrombin, a liver hemorrhage model was created. The median abdomen of the mice was incised, exposing the liver, which was then punctured using a 19 G needle. The wound area was covered with the sHA-CA patch with or without thrombin loading (Fig. 7H). The amount of blood loss was quantified by weighing the filter paper and the sHA-CA patch before and after the hemorrhage was induced. There was no significant difference in the amount of blood loss between the sHA-CA patch group (50 ± 14.14 mg) and the thrombin group (32.5 ± 29.86 mg). Although thrombin was effective in stopping bleeding, it was challenging to apply it to the bleeding site, and the thrombin solution tended to flow away from the application site (Fig. 7I). In contrast, the sHA-CA patch with thrombin immediately halted bleeding, resulting in blood loss recording 7.5 ± 9.57 mg (Fig. 7J). The blood-absorbing ability of the sHA-CA patch and its stable patch-like structure allowed for the immediate cessation of bleeding, and this clotting process was further accelerated by loading thrombin into the patch.

2.11 The adhesion barrier function of the sHA-CA patch against post-surgical adhesion

Blocking post-surgical adhesion through the formation of a physical barrier on the damaged peritoneum constitutes the fundamental principle of conventional adhesion barriers [65]. Peritoneal adhesions were induced by cecal abrasion on the cecum and abdominal wall of Sprague-Dawley rats (SD rats), and sHA-CA patches were applied to the abraded sites (Fig. 8A). After 7 d post-surgery, the rats were sacrificed, and the level of peritoneal adhesion at the abraded sites was assessed. PBS served as a negative control, and Seprafilm®, a commercial adhesion barrier, was employed as a positive control.Fig. 8 Prevention of peritoneal adhesion in rat cecal abrasion model using sHA-CA patches. (A) Schematic illustrations of the action of sHA-CA patches as adhesion barriers (top) and modeling a rat cecal abrasion (bottom). (B) Representative images of the rat cecal abrasion model on 7 d after surgery (Scale bar = 10 mm). (C) Adhesion formation rates with adhesion scores of sHA-CA patches per rat (n = 5). (D) Quantification of the adhesion scores of sHA-CA patches (n = 5, mean ± SD, **p < 0.01 and *p < 0.05). (E) Representative Masson's trichrome staining images of adhesive tissues on 7 d after surgery (Scale bar = 500 μm). (F) Representative iNOS/Arg1 immunofluorescence staining images of adhesive tissues on 7 d after surgery (Scale bar = 50 μm). (G) M1/M2 ratio quantified by immunohistochemistry (n = 5, mean ± SD, ns: not significant, ***p < 0.001 and *p < 0.05). (H–J) Quantification of pro-inflammatory cytokines IL-1β (H), IL-6 (I), and TNF-α (J) from rat serum on 4 d after surgery (n = 4, mean ± SD, ns: not significant, ***p < 0.001, **p < 0.01 and *p < 0.05).

Fig. 8

In comparison to the control and Seprafilm® treated groups, the sHA-CA patch demonstrated significant effectiveness in preventing post-surgical adhesions (Fig. 8B). The adhesion formation rate in the sHA-CA patch group was only 20 %, indicating that only 1 out of 5 surgeries exhibited adhesions (Fig. 8C). In contrast, the negative control group and the Seprafilm® group showed adhesion formation rates of 100 % and 80 %, respectively. Furthermore, the adhesion score observed for the sHA-CA patch was significantly lower than that of the control and Seprafilm® groups (Fig. 8D). Histological analysis revealed a similar trend. Masson's trichrome staining clearly demonstrated that the control and Seprafilm® groups exhibited severe peritoneal adhesions, resulting in the tight adhesion of the cecum to the abdominal wall. Conversely, a clear separation between the cecum and abdominal wall tissues was observed in the sHA-CA patch-treated samples (Fig. 8E).

The sHA-CA patch proved to be a superior adhesion barrier compared to the commercial product Seprafilm®. This suggests that simply providing a physical barrier between injured tissues is insufficient to prevent adhesions. To gain a deeper understanding of the factors contributing to the improved performance of the sHA-CA patch as an adhesion barrier, we analyzed the inflammation levels in tissue samples collected at 7 d post-surgery. Immunofluorescence staining for iNOS and Arg1 in the cecum and abdominal wall indicated that macrophages recruited at the sites of peritoneal adhesion in the control and Seprafilm® groups were predominantly iNOS+ M1 macrophages (Fig. 8F). In contrast, the sHA-CA patch group exhibited significantly higher levels of Arg1+ M2 macrophages at the surface of the injured abdominal wall and cecum.

The M1/M2 ratio of each group reflected these distinct polarization patterns of peritoneal macrophages. The M1/M2 ratio in the negative control group was 10.51 ± 2.51, indicating that the majority of macrophages exhibited the M1 phenotype (Fig. 8G). Similarly, the M1/M2 ratio in the Seprafilm® group was 3.97 ± 2.19. It is conceivable that Seprafilm® aided in peritoneal wound healing by forming an adhesion barrier and facilitating early termination of inflammation compared to the negative control. The presence of a dominant M1 population, albeit less than in the negative control, might be attributed to the effects of Seprafilm®-supported wound healing. Interestingly, the sHA-CA group reversed this trend, recording M1/M2 ratios of 0.66 ± 0.30 in the cecum and 0.19 ± 0.08 in the abdominal wall. In other words, the sHA-CA group exhibited a highly M2-dominant population. In summary, the sHA-CA patch influenced the M1-M2 macrophage polarization, increasing the presence of M2 macrophages at the injured sites of the cecum and abdominal wall. This supported peritoneal wound healing, and these characteristics contributed to the effective prevention of post-surgical adhesions.

Additionally, the sHA-CA patch significantly reduced pro-inflammatory cytokine levels in the cecum abrasion model (Fig. 8H–J). Blood was collected from the jugular vein of rats 4 d after surgery, and the serum separated from the blood was used for cytokine analysis. Peritoneal lavage fluid was excluded from the cytokine analysis due to the potential risk of rapid clearance of cytokines after surgery [83,84]. Pro-inflammatory cytokines IL-1β (Fig. 8H), IL-6 (Fig. 8I), and TNF-α (Fig. 8J) were significantly decreased in the sHA-CA patch group. Seprafilm® exhibited similar IL-1β and TNF-α expression and elevated IL-6 expression compared to the control. Therefore, the sHA-CA patch effectively prevented peritoneal adhesions by inducing M2 polarization at the injured sites and attenuating pro-inflammatory cytokines.

3 Conclusion

In summary, we have successfully developed multifunctional adhesive dry patches suitable for three different types of wounds. By adjusting the pH to 9 and utilizing freeze-thawing techniques, we were able to manufacture the patches through catechol crosslinking. The sHA-CA patch exhibited improved mechanical properties, including enhanced retention and adhesiveness. Furthermore, the sulfate groups within sHA-CA enabled sustained release of cationic drugs. Additionally, our study revealed that the sHA-CA patch possessed synergic anti-inflammatory potential, with the radical-scavenging capability of its catechol moieties and the ability to downregulate pro-inflammatory cytokine expression through sHA. This anti-inflammatory function was further confirmed in vivo through inflammation level imaging post-implantation of the sHA-CA patch and quantification of cytokine levels. Ultimately, the biocompatible and anti-inflammatory sHA-CA patch exhibited significantly improved therapeutic effects in the treatment of diabetic wounds, liver hemorrhages, and post-surgical wounds. These findings support the notion that the sHA-CA patch could offer a novel and promising therapeutic strategy for a wide range of in vivo applications. However, there are some limitations to the sHA-CA patches. Anti-microbial properties have not been demonstrated, and the patch-type is difficult to use on irregular wounds. Further studies are required to address the limitations, such as loading anti-microbial peptides into the sHA-CA patches.

4 Materials and methods

The detailed description of materials and methods can be found in the Supplementary data file.

Ethics approval and consent to participate

All animal experimental procedures were approved by the Institutional Animal Care and Use Committee (IACUC) of Seoul National University (SNU-200901-7, SNU-230329-1, and SNU-230329-8).

CRediT authorship contribution statement

Wonmoon Song: Writing – review & editing, Writing – original draft, Visualization, Methodology, Investigation, Formal analysis, Conceptualization. Young Hwan Choi: Writing – review & editing, Investigation, Formal analysis, Data curation, Conceptualization. Young Gi Moon: Visualization, Software, Investigation. Changyub Lee: Writing – review & editing, Methodology, Formal analysis. M. Nivedhitha Sundaram: Writing – review & editing. Nathaniel S. Hwang: Writing – review & editing, Supervision, Resources, Project administration, Investigation, Conceptualization.

Declaration of competing interest

All authors declare that they have no financial interests or personal relationships that could be influencing the manuscript entitled.

Appendix A Supplementary data

The following is the Supplementary data to this article:Multimedia component 1

Multimedia component 1

Acknowledgements

This work received financial support from the Ministry of Science and ICT of Korea (NRF-2021R1A2C2008821 and 2022H1D3A2A02093385 ), the Korean Fund for Regenerative Medicine (KFRM) grant funded by the Korean government (21A0301L1-21 ). The Institute of Engineering Research at Seoul National University provided research facilities, and additional support came from the SNU Engineering-Medicine Collaboration grant.

Peer review under responsibility of KeAi Communications Co., Ltd.

Appendix A Supplementary data to this article can be found online at https://doi.org/10.1016/j.bioactmat.2024.08.001.
==== Refs
References

1 Kujath P. Michelsen A. Wounds - from physiology to wound dressing Dtsch. Arztebl. Int. 105 13 2008 239 248 10.3238/arztebl.2008.0239 19629204
2 Nagle S.M. Stevens K.A. Wilbraham S.C. Wound Assessment 2023 StatPearls Treasure Island (FL)
3 Oliver T.I. Mutluoglu M. Diabetic Foot Ulcer 2023 StatPearls Treasure Island (FL)
4 Tang W.R. Wu C.H. Yang T.H. Yen Y.T. Hung K.S. Wang C.J. Shan Y.S. Impact of trauma teams on high grade liver injury care: a two-decade propensity score approach study in Taiwan Sci. Rep. 13 1 2023 5429 10.1038/s41598-023-32760-9 37012308
5 Coccolini F. Coimbra R. Ordonez C. Kluger Y. Vega F. Moore E.E. Biffl W. Peitzman A. Horer T. Abu-Zidan F.M. Liver trauma: WSES 2020 guidelines World J. Emerg. Surg. 15 1 2020 24 10.1186/s13017-020-00302-7 32228707
6 Li M. Zhang Z. Liang Y. He J. Guo B. Multifunctional tissue-adhesive cryogel wound dressing for rapid nonpressing surface hemorrhage and wound repair ACS Appl. Mater. Interfaces 12 32 2020 35856 35872 10.1021/acsami.0c08285 32805786
7 Stapleton L.M. Lucian H.J. Grosskopf A.K. Smith A.A.A. Totherow K.P. Woo Y.J. Appel E.A. Dynamic hydrogels for prevention of post-operative peritoneal adhesions Adv. Ther. 4 3 2021 10.1002/adtp.202000242
8 Norahan M.H. Pedroza-González S.C. Sánchez-Salazar M.G. Álvarez M.M. Trujillo de Santiago G. Structural and biological engineering of 3D hydrogels for wound healing Bioact. Mater. 24 2023 197 235 10.1016/j.bioactmat.2022.11.019 36606250
9 Montazerian H. Davoodi E. Baidya A. Baghdasarian S. Sarikhani E. Meyer C.E. Haghniaz R. Badv M. Annabi N. Khademhosseini A. Weiss P.S. Engineered hemostatic biomaterials for sealing wounds Chem. Rev. 122 15 2022 12864 12903 10.1021/acs.chemrev.1c01015 35731958
10 He Y. Li Q. Chen P. Duan Q. Zhan J. Cai X. Wang L. Hou H. Qiu X. A smart adhesive Janus hydrogel for non-invasive cardiac repair and tissue adhesion prevention Nat. Commun. 13 1 2022 7666 10.1038/s41467-022-35437-5 36509756
11 Li Y. Zhao W. Chen S. Zhai H. Wu S. Bioactive electrospun nanoyarn-constructed textile dressing patches delivering Chinese herbal compound for accelerated diabetic wound healing Mater. Des. 237 2024 112623 10.1016/j.matdes.2023.112623
12 Zhang C. Yang X. Yu L. Chen X. Zhang J. Zhang S. Wu S. Electrospun polyasparthydrazide nanofibrous hydrogel loading with in-situ synthesized silver nanoparticles for full-thickness skin wound healing application Mater. Des. 239 2024 112818 10.1016/j.matdes.2024.112818
13 Zhou F. Sun S. Cui C. Li X. Wu S. Ma J. Chen S. Li C.M. Zinc ions and ciprofloxacin-encapsulated chitosan/poly(ɛ-caprolactone) composite nanofibers promote wound healing via enhanced antibacterial and immunomodulatory Int. J. Biol. Macromol. 253 2023 127086 10.1016/j.ijbiomac.2023.127086
14 Shin J. Choi S. Kim J.H. Cho J.H. Jin Y. Kim S. Min S. Kim S.K. Choi D. Cho S.W. Tissue tapes-phenolic hyaluronic acid hydrogel patches for off-the-shelf therapy Adv. Funct. Mater. 29 49 2019 10.1002/adfm.201903863
15 Xu X. Xia X. Zhang K. Rai A. Li Z. Zhao P. Wei K. Zou L. Yang B. Wong W.K. Bioadhesive hydrogels demonstrating pH-independent and ultrafast gelation promote gastric ulcer healing in pigs Sci. Transl. Med. 12 558 2020 10.1126/scitranslmed.aba8014
16 Shin J. Kang E.H. Choi S. Jeon E.J. Cho J.H. Kang D. Lee H. Yun I.S. Cho S.W. Tissue-adhesive chondroitin sulfate hydrogel for cartilage reconstruction ACS Biomater. Sci. Eng. 7 9 2021 4230 4243 10.1021/acsbiomaterials.0c01414 33538598
17 Ryu J.H. Kim H. Kim K. Yoon G. Wang Y. Choi G.S. Lee H. Park J.S. Multipurpose intraperitoneal adhesive patches Adv. Funct. Mater. 29 29 2019 10.1002/adfm.201900495
18 Shin J. Lee J.S. Lee C. Park H.J. Yang K. Jin Y. Ryu J.H. Hong K.S. Moon S.H. Chung H.M. Tissue adhesive catechol-modified hyaluronic acid hydrogel for effective, minimally invasive cell therapy Adv. Funct. Mater. 25 25 2015 3814 3824 10.1002/adfm.201500006
19 Park H.J. Jin Y. Shin J. Yang K. Lee C. Yang H.S. Cho S.W. Catechol-functionalized hyaluronic acid hydrogels enhance angiogenesis and osteogenesis of human adipose-derived stem cells in critical tissue defects Biomacromolecules 17 6 2016 1939 1948 10.1021/acs.biomac.5b01670 27112904
20 Pinnataip R. Lee B.P. Oxidation chemistry of catechol utilized in designing stimuli-responsive adhesives and antipathogenic biomaterials ACS Omega 6 8 2021 5113 5118 10.1021/acsomega.1c00006 33681552
21 Lee J.S. Cho J.H. An S. Shin J. Choi S. Jeon E.J. Cho S.W. In situ self-cross-linkable, long-term stable hyaluronic acid filler by gallol autoxidation for tissue augmentation and wrinkle correction Chem. Mater. 31 23 2019 9614 9624 10.1021/acs.chemmater.9b02802
22 Koehler L. Ruiz-Gómez G. Balamurugan K. Rother S. Freyse J. Möller S. Schnabelrauch M. Köhling S. Djordjevic S. Scharnweber D. Dual action of sulfated hyaluronan on angiogenic processes in relation to vascular endothelial growth factor-A Sci. Rep. 9 1 2019 18143 10.1038/s41598-019-54211-0
23 Funt D.K. Treatment of delayed-onset inflammatory reactions to hyaluronic acid filler: an algorithmic approach Plast. Reconstr. Surg. Glob. Open 10 6 2022 e4362 10.1097/gox.0000000000004362
24 Feng Q. Lin S. Zhang K. Dong C. Wu T. Huang H. Yan X. Zhang L. Li G. Bian L. Sulfated hyaluronic acid hydrogels with retarded degradation and enhanced growth factor retention promote hMSC chondrogenesis and articular cartilage integrity with reduced hypertrophy Acta Biomater. 53 2017 329 342 10.1016/j.actbio.2017.02.015 28193542
25 Koehler L. Samsonov S. Rother S. Vogel S. Kohling S. Moeller S. Schnabelrauch M. Rademann J. Hempel U. Pisabarro M.T. Sulfated hyaluronan derivatives modulate TGF-beta1:receptor complex formation: possible consequences for TGF-beta1 signaling Sci. Rep. 7 1 2017 1210 10.1038/s41598-017-01264-8 28446792
26 Rother S. Samsonov S.A. Moeller S. Schnabelrauch M. Rademann J. Blaszkiewicz J. Kohling S. Waltenberger J. Pisabarro M.T. Scharnweber D. Sulfated hyaluronan alters endothelial cell activation in vitro by controlling the biological activity of the angiogenic factors vascular endothelial growth factor-A and tissue inhibitor of metalloproteinase-3 ACS Appl. Mater. Interfaces 9 11 2017 9539 9550 10.1021/acsami.7b01300 28248081
27 Hauck S. Zager P. Halfter N. Wandel E. Torregrossa M. Kakpenova A. Rother S. Ordieres M. Rathel S. Berg A. Collagen/hyaluronan based hydrogels releasing sulfated hyaluronan improve dermal wound healing in diabetic mice via reducing inflammatory macrophage activity Bioact. Mater. 6 12 2021 4342 4359 10.1016/j.bioactmat.2021.04.026 33997511
28 Kim S.H. Kim K. Kim B.S. An Y.H. Lee U.J. Lee S.H. Kim S.L. Kim B.G. Hwang N.S. Fabrication of polyphenol-incorporated anti-inflammatory hydrogel via high-affinity enzymatic crosslinking for wet tissue adhesion Biomaterials 242 2020 119905 10.1016/j.biomaterials.2020.119905
29 Park E. Ryu J.H. Lee D. Lee H. Freeze-thawing-induced macroporous catechol hydrogels with shape recovery and sponge-like properties ACS Biomater. Sci. Eng. 7 9 2021 4318 4329 10.1021/acsbiomaterials.0c01767 33821606
30 Yuk H. Varela C.E. Nabzdyk C.S. Mao X. Padera R.F. Roche E.T. Zhao X. Dry double-sided tape for adhesion of wet tissues and devices Nature 575 7781 2019 169 174 10.1038/s41586-019-1710-5 31666696
31 Kim K. Ryu J.H. Koh M.Y. Yun S.P. Kim S. Park J.P. Jung C.W. Lee M.S. Seo H.I. Kim J.H. Coagulopathy-independent, bioinspired hemostatic materials: a full research story from preclinical models to a human clinical trial Sci. Adv. 7 13 2021 10.1126/sciadv.abc9992
32 Chen B.L. Arakawa T. Hsu E. Narhi L.O. Tressel T.J. Chien S.L. Strategies to suppress aggregation of recombinant keratinocyte growth-factor during liquid formulation development J. Pharmaceut. Sci. 83 12 1994 1657 1661 10.1002/jps.2600831204
33 Freeman I. Kedem A. Cohen S. The effect of sulfation of alginate hydrogels on the specific binding and controlled release of heparin-binding proteins Biomaterials 29 22 2008 3260 3268 10.1016/j.biomaterials.2008.04.025 18462788
34 Felder S. Masasa H. Orenbuch A. Levaot N. Goldenberg M.S. Cohen S. Reconstruction of the ovary microenvironment utilizing macroporous scaffold with affinity-bound growth factors Biomaterials 205 2019 11 22 10.1016/j.biomaterials.2019.03.013 30901634
35 Kim S.H. Lee S.H. Lee J.E. Park S.J. Kim K. Kim I.S. Lee Y.S. Hwang N.S. Kim B.G. Tissue adhesive, rapid forming, and sprayable ECM hydrogel via recombinant tyrosinase crosslinking Biomaterials 178 2018 401 412 10.1016/j.biomaterials.2018.04.057 29752077
36 Cheynier V. Moutounet M. Oxidative reactions of caffeic acid in model systems containing polyphenol oxidase J. Agric. Food Chem. 40 11 1992 2038 2044 10.1021/jf00023a002
37 Lee H. Dellatore S.M. Miller W.M. Messersmith P.B. Mussel-inspired surface chemistry for multifunctional coatings Science 318 5849 2007 426 430 10.1126/science.1147241 17947576
38 Lee B.P. Messersmith P.B. Israelachvili J.N. Waite J.H. Mussel-inspired adhesives and coatings Annu. Rev. Mater. Res. 41 2011 99 132 10.1146/annurev-matsci-062910-100429 22058660
39 Matos-Perez C.R. White J.D. Wilker J.J. Polymer composition and substrate influences on the adhesive bonding of a biomimetic, cross-linking polymer J. Am. Chem. Soc. 134 22 2012 9498 9505 10.1021/ja303369p 22582754
40 Kim S.H. An Y.H. Kim H.D. Kim K. Lee S.H. Yim H.G. Kim B.G. Hwang N.S. Enzyme-mediated tissue adhesive hydrogels for meniscus repair Int. J. Biol. Macromol. 110 2018 479 487 10.1016/j.ijbiomac.2017.12.053 29229249
41 Mao X. Yuk H. Zhao X. Hydration and swelling of dry polymers for wet adhesion J. Mech. Phys. Solid. 137 2020 10.1016/j.jmps.2020.103863
42 Huzum B. Puha B. Necoara R.M. Gheorghevici S. Puha G. Filip A. Sirbu P.D. Alexa O. Biocompatibility assessment of biomaterials used in orthopedic devices: an overview (Review) Exp. Ther. Med. 22 5 2021 1315 10.3892/etm.2021.10750 34630669
43 Bernard M. Jubeli E. Pungente M.D. Yagoubi N. Biocompatibility of polymer-based biomaterials and medical devices - regulations, in vitro screening and risk-management Biomater. Sci. 6 8 2018 2025 2053 10.1039/c8bm00518d 29968869
44 Schafer S. Al-Qaddo H. Gosau M. Smeets R. Hartjen P. Friedrich R.E. Nada O.A. Vollkommer T. Rashad A. Cytocompatibility of bone substitute materials and membranes In Vivo 35 4 2021 2035 2040 10.21873/invivo.12472 34182478
45 Franz S. Allenstein F. Kajahn J. Forstreuter I. Hintze V. Moller S. Simon J.C. Artificial extracellular matrices composed of collagen I and high-sulfated hyaluronan promote phenotypic and functional modulation of human pro-inflammatory M1 macrophages Acta Biomater. 9 3 2013 5621 5629 10.1016/j.actbio.2012.11.016 23168224
46 Hempel U. Matthaus C. Preissler C. Moller S. Hintze V. Dieter P. Artificial matrices with high-sulfated glycosaminoglycans and collagen are anti-inflammatory and pro-osteogenic for human mesenchymal stromal cells J. Cell. Biochem. 115 9 2014 1561 1571 10.1002/jcb.24814 24706396
47 Lu Y.C. Yeh W.C. Ohashi P.S. LPS/TLR4 signal transduction pathway Cytokine 42 2 2008 145 151 10.1016/j.cyto.2008.01.006 18304834
48 Yang C. Cao M. Liu H. He Y. Xu J. Du Y. Liu Y. Wang W. Cui L. Hu J. The high and low molecular weight forms of hyaluronan have distinct effects on CD44 clustering J. Biol. Chem. 287 51 2012 43094 43107 10.1074/jbc.M112.349209 23118219
49 Ruppert S.M. Hawn T.R. Arrigoni A. Wight T.N. Bollyky P.L. Tissue integrity signals communicated by high-molecular weight hyaluronan and the resolution of inflammation Immunol. Res. 58 2–3 2014 186 192 10.1007/s12026-014-8495-2 24614953
50 Bhattacharya D.S. Svechkarev D. Bapat A. Patil P. Hollingsworth M.A. Mohs A.M. Sulfation modulates the targeting properties of hyaluronic acid to P-selectin and CD44 ACS Biomater. Sci. Eng. 6 6 2020 3585 3598 10.1021/acsbiomaterials.0c00115 32617404
51 Xaus J. Comalada M. Valledor A.F. Lloberas J. Lopez-Soriano F. Argiles J.M. Bogdan C. Celada A. LPS induces apoptosis in macrophages mostly through the autocrine production of TNF-alpha Blood 95 12 2000 3823 3831 10.1182/blood.V95.12.3823 10845916
52 Hsu H.Y. Wen M.H. Lipopolysaccharide-mediated reactive oxygen species and signal transduction in the regulation of interleukin-1 gene expression J. Biol. Chem. 277 25 2002 22131 22139 10.1074/jbc.M111883200 11940570
53 Ju K.Y. Lee Y. Lee S. Park S.B. Lee J.K. Bioinspired polymerization of dopamine to generate melanin-like nanoparticles having an excellent free-radical-scavenging property Biomacromolecules 12 3 2011 625 632 10.1021/bm101281b 21319809
54 Lyublinskaya O.G. Ivanova J.S. Pugovkina N.A. Kozhukharova I.V. Kovaleva Z.V. Shatrova A.N. Aksenov N.D. Zenin V.V. Kaulin Y.A. Gamaley I.A. Redox environment in stem and differentiated cells: a quantitative approach Redox Biol. 12 2017 758 769 10.1016/j.redox.2017.04.016 28426982
55 Tu Z.L. Chen M. Wang M. Shao Z.X. Jiang X.Q. Wang K.Y. Yao Z. Yang S.W. Zhang X.X. Gao W.Y. Engineering bioactive M2 macrophage-polarized anti-inflammatory, antioxidant, and antibacterial scaffolds for rapid angiogenesis and diabetic wound repair Adv. Funct. Mater. 2021 10.1002/adfm.202100924
56 Hu J.J. Wang M. Lei X.X. Jiang Y.L. Yuan L. Pan Z.J. Lu D. Luo F. Li J.H. Tan H. Scarless healing of injured vocal folds using an injectable hyaluronic acid-waterborne polyurethane hybrid hydrogel to tune inflammation and collagen deposition ACS Appl. Mater. Interfaces 14 38 2022 42827 42840 10.1021/acsami.2c07225 36121932
57 Tseng J.C. Kung A.L. In vivo imaging of inflammatory phagocytes Chem. Biol. 19 9 2012 1199 1209 10.1016/j.chembiol.2012.08.007 22999887
58 Cao Y. Wang B. Biodegradation of silk biomaterials Int. J. Mol. Sci. 10 4 2009 1514 1524 10.3390/ijms10041514 19468322
59 Deshayes S. Kasko A.M. Polymeric biomaterials with engineered degradation J. Polym. Sci., Polym. Chem. Ed. 51 17 2013 3531 3566 10.1002/pola.26765
60 Hofmann D. Entrialgo-Castano M. Kratz K. Lendlein A. Knowledge-based approach towards hydrolytic degradation of polymer-based biomaterials Adv. Mater. 21 32–33 2009 3237 3245 10.1002/adma.200802213 20882494
61 Mele E. Electrospinning of natural polymers for advanced wound care: towards responsive and adaptive dressings J. Mater. Chem. B 4 28 2016 4801 4812 10.1039/c6tb00804f 32263137
62 Mautner K. Malanga G. Colberg R. Optimization of ingredients, procedures and rehabilitation for platelet-rich plasma injections for chronic tendinopathy Pain Manag. 1 6 2011 523 532 10.2217/pmt.11.56 24645763
63 Manchon E. Hirt N. Bouaziz J.D. Jabrane-Ferrat N. Al-Daccak R. Stem cells-derived extracellular vesicles: potential therapeutics for wound healing in chronic inflammatory skin diseases Int. J. Mol. Sci. 22 6 2021 10.3390/ijms22063130
64 Ergul E. Korukluoglu B. Peritoneal adhesions: facing the enemy Int. J. Surg. 6 3 2008 253 260 10.1016/j.ijsu.2007.05.010 17617231
65 Schnuriger B. Barmparas G. Branco B.C. Lustenberger T. Inaba K. Demetriades D. Prevention of postoperative peritoneal adhesions: a review of the literature Am. J. Surg. 201 1 2011 111 121 10.1016/j.amjsurg.2010.02.008 20817145
66 Rabenstein D.L. Heparin and heparan sulfate: structure and function Nat. Prod. Rep. 19 3 2002 312 331 10.1039/b100916h 12137280
67 Kim I. Lee S.S. Bae S. Lee H. Hwang N.S. Heparin functionalized injectable cryogel with rapid shape-recovery property for neovascularization Biomacromolecules 19 6 2018 2257 2269 10.1021/acs.biomac.8b00331 29689163
68 Choi Y.H. Ahn H.J. Park M.R. Han M.J. Lee J.H. Kwon S.K. Dual growth factor-immobilized bioactive injection material for enhanced treatment of glottal insufficiency Acta Biomater. 86 2019 269 279 10.1016/j.actbio.2018.12.047 30599245
69 Kazemi M. Carrer A. Moimas S. Zandona L. Bussani R. Casagranda B. Palmisano S. Prelazzi P. Giacca M. Zentilin L. VEGF(121) and VEGF(165) differentially promote vessel maturation and tumor growth in mice and humans Cancer Gene Ther. 23 5 2016 125 132 10.1038/cgt.2016.12 27033458
70 Matoori S. Veves A. Mooney D.J. Advanced bandages for diabetic wound healing Sci. Transl. Med. 13 585 2021 10.1126/scitranslmed.abe4839
71 Baltzis D. Eleftheriadou I. Veves A. Pathogenesis and treatment of impaired wound healing in diabetes mellitus: new insights Adv. Ther. 31 8 2014 817 836 10.1007/s12325-014-0140-x 25069580
72 Wilgus T.A. Vascular endothelial growth factor and cutaneous scarring Adv. Wound Care 8 12 2019 671 678 10.1089/wound.2018.0796
73 Wilgus T.A. Ferreira A.M. Oberyszyn T.M. Bergdall V.K. Dipietro L.A. Regulation of scar formation by vascular endothelial growth factor Lab. Invest. 88 6 2008 579 590 10.1038/labinvest.2008.36 18427552
74 Korntner S. Lehner C. Gehwolf R. Wagner A. Grutz M. Kunkel N. Tempfer H. Traweger A. Limiting angiogenesis to modulate scar formation Adv. Drug Deliv. Rev. 146 2019 170 189 10.1016/j.addr.2018.02.010 29501628
75 Fu X. Ning J.P. Synthesis and biocompatibility of an argatroban-modified polysulfone membrane that directly inhibits thrombosis J. Mater. Sci. Mater. Med. 29 5 2018 66 10.1007/s10856-018-6054-4 29744595
76 Miura T. Kawano M. Takahashi K. Yuasa N. Habu M. Kimura F. Imamura T. Nakayama F. High-sulfated hyaluronic acid ameliorates radiation-induced intestinal damage without blood anticoagulation Adv. Radiat. Oncol. 7 3 2022 100900 10.1016/j.adro.2022.100900
77 Miura T. Yuasa N. Ota H. Habu M. Kawano M. Nakayama F. Nishihara S. Highly sulfated hyaluronic acid maintains human induced pluripotent stem cells under feeder-free and bFGF-free conditions Biochem. Biophys. Res. Commun. 518 3 2019 506 512 10.1016/j.bbrc.2019.08.082 31439376
78 Kusche M. Backstrom G. Riesenfeld J. Petitou M. Choay J. Lindahl U. Biosynthesis of heparin. O-sulfation of the antithrombin-binding region J. Biol. Chem. 263 30 1988 15474 15484 10.1016/S0021-9258(19)37613-6 3139669
79 I.O.f. Standardization ISO 10993-5: 2009 Biological Evaluation of Medical Devices - Part 5: Tests for in Vitro Cytotoxicity 2009
80 He H. Sun C. Weng Y. Huang H. Ni P. Fang Y. Xu R. Wang Z. Liu H. Catechol modification of non-woven chitosan gauze for enhanced hemostatic efficacy Carbohydr. Polym. 286 2022 119319 10.1016/j.carbpol.2022.119319
81 Lew W.K. Weaver F.A. Clinical use of topical thrombin as a surgical hemostat Biologics 2 4 2008 593 599 10.2147/btt.s2435v 19707440
82 Weaver F.A. Lew W. Granke K. Yonehiro L. Delange B. Alexander W.A. Study I. A comparison of recombinant thrombin to bovine thrombin as a hemostatic ancillary in patients undergoing peripheral arterial bypass and arteriovenous graft procedures J. Vasc. Surg. 47 6 2008 1266 1273 10.1016/j.jvs.2008.01.034 18440754
83 Hendriks T. Bleichrodt R.P. Lomme R.M. De Man B.M. van Goor H. Buyne O.R. Peritoneal cytokines predict mortality after surgical treatment of secondary peritonitis in the rat J. Am. Coll. Surg. 211 2 2010 263 270 10.1016/j.jamcollsurg.2010.03.038 20670866
84 Chung D.R. Chitnis T. Panzo R.J. Kasper D.L. Sayegh M.H. Tzianabos A.O. CD4+ T cells regulate surgical and postinfectious adhesion formation J. Exp. Med. 195 11 2002 1471 1478 10.1084/jem.20020028 12045245
