A functional dual responsive CMC/OHA/SA/TOB hydrogel as wound dressing to enhance wound healing | Scientific Reports

Nov 06, 2024

Scientific Reports volume 14, Article number: 26854 (2024) Cite this article

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Within the clinical realm, the complexities of wound healing have consistently presented formidable challenges. Recent advancements, notably in hydrogel technologies, have broadened the therapeutic spectrum. This study focuses on investigating a novel dual responsive composite hydrogel for wound healing. This hydrogel is ingeniously designed to maintain an optimal moist environment, expedite healing, and combat bacterial infection during wound recovery. This study combining carboxymethyl chitosan (CMC), oxidized hyaluronic acid (OHA), and sodium alginate (SA), in addition, tobramycin (TOB) was incorporated to create a CMC/OHA/SA/TOB hydrogel. Hydrogel cross-linking was verified by infrared spectroscopy, and the microstructure was examined with scanning electron microscopy. We explored its swelling and degradation behaviors in different pH environments. The drug release profile and biocompatibility was evaluated via cytotoxicity and hemolysis assays. The antibacterial efficacy of hydrogel was tested in both solid and liquid media. Additionally, the wound models in Sprague–Dawley (SD) rat was employed to investigate the hydrogel’s wound healing capabilities in vivo. Results showed that CMCOHA/SA/TOB hydrogel was effectively cross-linked with a network structure. The hydrogel exhibited pronounced responsiveness in its swelling and degradation characteristics, which was significantly influenced by different levels of pH. In vitro results demonstrated that the CMC/OHA/SA/TOB hydrogel exhibits limited cytotoxicity and hemolysis, coupled with a drug release profile of dual responsive characteristics. Antibacterial activity of the hydrogel against Staphylococcus aureus, Pseudomonas aeruginosa, and Escherichia coli was confirmed. Furthermore, in vivo experiments underscored the hydrogel’s proficiency in promoting wound healing, highlighting its potential for clinical applications. The CMC/OHA/SA/TOB hydrogel not only fosters a moist environment essential for wound healing and enhances structural stability, but it also exhibits functional dual responsive capabilities in swelling and degradation. These distinctive abilities enable the precise release of TOB, thereby optimizing wound healing.

Wound management has always been a complex problem1. With the increased demand for wound management, traditional dressings such as gauze have been gradually replaced by several novel dressings2. Sterile gauze pads are used to absorb exudates from the wound, but traditional dressings have major complications, including the need for frequent dressing changes to prevent infection and the absorption of wound exudates leading to wound adhesion, which makes them less popular at present3. On the other hand, after the wound is formed, the continuity of the skin is destroyed, which is more likely to induce bacterial infection, and the bacteria will produce toxic factors to stimulate inflammation in the wound, which results in affecting the normal healing of the wound4. Nevertheless, traditional dressings are deficient in administering essential therapeutic agents for augmenting wound healing and diminishing the risks of infection5.

As one of advanced dressings, hydrogels have a well-reticulated molecular structure and contain large amounts of water, which makes them as a best candidate to mimic the natural skin microenvironment in wound management6. Maintaining a certain level of moisture in the environment during wound healing can alleviate pain, reduce scarring, stimulate collagen synthesis, promote and facilitate the migration of keratin-forming cells on the wound surface, and support the presence and function of nutrients, growth factors, as well as the delivery of antimicrobial agents within the wound microenvironment7,8. Apart from maintaining a moist environment that is conducive to wound healing, it is crucial to have a deeper understanding of pH and its influence on the biological and biochemical processes involved in wound healing9,10. This understanding is vital for developing targeted treatments that can effectively prevent the transition of acute wounds into chronic infections11.

During the wound-healing process, severe infections can result in a reduction of local pH levels, predominantly attributed to the aerobic respiration and fermentation, resulting in a pH reduction to between 4.5 and 6.5. by bacteria10,12,13. Additionally, pus, dead tissue, and dried serum are signs of a wound with an acidic environment, in which the average pH is around 6.114. Furthermore, the pH of Staphylococcus aureus cultures decreases as bacterial growth occurs due to its facultatively anaerobic nature15. pH sensitive hydrogels are uniquely engineered to detect and adaptively respond to pH variations in the surrounding environment. Available pH sensitive hydrogel species have primarily focused on acid release16, which is cross-linked via coordination bonds17, Schiff base bonds18, intermolecular forces, hydrogen bonding, and π-π stacking19. Thus, pH sensitive hydrogels are engineered to substantially release antimicrobial drugs in response to this decrease in pH, thereby effectively elevating the local drug concentration and precisely targeting the infection site9.

Schiff base bonds, a dynamic chemical bond and a research hotspot in pH-responsive hydrogels, possess significant self-healing capabilities and demonstrate responsiveness to acidic environments20,21. Therefore, we are focusing on developing a pH sensitive hydrogels through the use of carboxymethyl chitosan (CMC) and oxidized hyaluronic acid (OHA), employing dynamic Schiff base bonds. CMC, a chitosan derivative, is extensively utilized in the fabrication of polysaccharide hydrogels22. Hyaluronic acid (HA), a vital component of the extracellular matrix, becomes more conducive to forming hydrogel with other polysaccharides upon oxidation, enabling diverse applications23. These attributes highlight the hydrogels’ significant potential to revolutionize therapeutic strategies for advanced wound care. Tobramycin (TOB) is an aminoglycoside antibiotic with an amino functional group that binds to the aldehyde group of OHA to form a Schiff base bond, which makes pH sensitive tobramycin release to resistant infection24.

There are different release mechanisms of entrapped/encapsulated drug in hydrogel such as diffusion controlled, swelling controlled, and chemically controlled mechanisms25. As an anionic polysaccharide, SA is capable of generating pH sensitive hydrogel or nanoparticles in conjunction with cationic polymers, such as CMC, via ionic interactions. Our research includes sodium alginate (SA) to create a hydrogel with the structure of double-network, which results in enhancing the stability of hydrogel with ionic bonds. This ionic bond introduced in hydrogel have pH response ability both in degradation and in swelling, which mitigates the functional loss of hydrogel due to the instability of Schiff base bonds in acidic environments26. The modulation of drug release can be achieved through the alteration in the swelling behavior of the hydrogel, the incorporation of SA endows the hydrogel with dual pH sensitive properties in terms of degradation and swelling.

Therefore, we developed a novel dual responsive CMC/OHA/SA/TOB hydrogel, which is featured with a structure of bilayer network composed of a Schiff base and ionic bonds (Fig. 1). In contrast to the conventional Schiff alkali hydrogel, the innovative hydrogel exhibited dual responsiveness in both its degradation and improved swelling characteristics. This innovative design not only accomplishes exceptional drug release but also actively adjusts the hydrogel’s swelling properties, leading to improve wound healing and provide effective protection against infection.

Synthesis of OHA, schematic diagram of OHA, CMC, SA, and TOB molecules and Schiff base bond and ionic bond ball-and-stick models. Schematic diagram of the principle of CMC/OHA/SA/TOB dual responsive hydrogel.

The successful oxidation of OHA was confirmed by NMR and FTIR. There were three new peaks at 4.9–5.1 ppm in 1HNMR and one new peak at 1732 cm−1 in FTIR, all of which indicated the existence of an aldehyde group in OHA, which proved that HA was successfully oxidized to OHA in this experiment (Fig. 2b,c)27,28. The oxidation rate of OHA was calculated to be approximately 38.5%. Schiff base bonds are dynamic chemical linkages formed between aldehyde and amino groups that impart self-healing properties and make the hydrogel injectable (Fig. 2a). The FTIR of the four groups of hydrogels exhibited distinct peaks at 1630–1640 cm−1, which proved that the hydrogels were successfully crosslinked by Schiff base bonds (Fig. 2c).

(a) COS20 hydrogel used for writing with a syringe and injected through a 24G needle. Self-healing capabilities of COS20 and CO (crystal violet dyed) hydrogels. (b) NMR spectra of HA and OHA, with boxed peaks in OHA indicating successful aldehyde group formation through oxidation. (c) Infrared spectra of OHA and each group of hydrogels, with the peak at 1732 cm−1 indicating OHA’s successful oxidation and peaks between 1628–1632 cm−1 denoting effective hydrogel crosslinking. (d) Scanning Electron Microscopy images depicting the microstructures and pore sizes of each group of hydrogels. Scale bars in each image represent 100 microns.

The three-dimensional network structure of freeze-dried hydrogel was successfully observed by FE-SEM, which further proved the successful preparation of hydrogel. FE-SEM showed that the freeze-dried hydrogel showed porous structure, the addition of SA did not change the surface morphology of the hydrogel significantly. The pore size of hydrogel increases gradually with the gradual increase of SA concentration, average pore size of CO, COS10, COS20, and COS30 group were 56.98 ± 3.094 μm, 60.007 ± 7.832 μm, 75.552 ± 6.243 μm, and 101.843 ± 9.088 μm, respectively. The porosities of hydrogel were 25.224 ± 3.071% (Fig. 2d).

Under neutral (pH = 7.4) and acidic (pH = 5.5) conditions, the degradation experiments were conducted to reveal the varied responses. Initially, we compared the degradation rates of hydrogels across various groups at different pH conditions. Except for the COS30 group, hydrogels in other groups degraded faster in acidic environments than in neutral ones due to the instability of Schiff base bonds under acidic conditions29. In a neutral environment, the degradation rates of the COS10 and COS20 groups were slower than that of the CO group, whereas the COS30 group showed a faster degradation rate than the CO group (Fig. 3a). Under acidic conditions, the degradation rate in ascending order was CO < COS10 < COS20 < COS30 (Fig. 3b,c), indicating that incorporating SA to form ionic bonds decelerated hydrogel degradation30,31. However, excessive SA in the COS30 group led to substantial swelling and instability in a neutral environment, resulting in rapid degradation.

(a) Remaining weights of hydrogels from each group in a neutral (pH 7.4) environment. (b) Remaining weights of hydrogels from each group in a acidic (pH 5.5) environment. (c) Remaining weights of COS20 hydrogels in a neutral (pH 7.4) and acidic (pH 5.5) environment. (d) Swelling ratio of hydrogels in a natural (pH 7.4) environment. (e) Swelling ratio of hydrogels in a acidic (pH 5.5) environment. (f) Swelling ratio of COS20 hydrogels in a neutral (pH 7.4) and acidic (pH 5.5) environment. (g) Release rate curves of TOB from COT and COST hydrogels in neutral and acidic environments. (h) Effects of leachates from each group of hydrogels on the growth of L929 cells. (i) Images depicting the hemolytic effects of hydrogels on red blood cells. (j) Hemolysis rates of hydrogels from each group, there were no significant differences between each pair of groups.

In swelling experiments, the rate of swelling in descending order was COS30 > COS20 > COS10 > CO in a neutral environment, indicating that the addition of SA significantly increased the swelling properties of hydrogel, and there was a positive correlation with the amount of SA (Fig. 3d). In an acidic environment, the swelling ability of hydrogels clearly decreased compared with a neutral environment, except those of the CO group, which indicated that the swelling ability of hydrogels had a certain correlation with pH. Also, we found that the addition of SA decrease the the swelling ability of hydrogel with a decrease of pH (Fig. 3e,f)25,31.

TOB is an aminoglycoside antibiotic, its amino groups can form Schiff base bonds with OHA and was released when Schiff base bonds dissociate under acidic conditions. We employed the o-phthalaldehyde method to assess drug release under varying pH conditions. Initially, we established a standard curve for tobramycin using the equation (y = 0.0012x + 0.0436, r2 = 0.9926), where x is the concentration of TOB (mg/mL) and y is the absorbance of the sample at 333 nm, ranging from 0 to 1000 µg/mL. Upon comparing the drug release from COT and COST hydrogels under varying pH conditions, TOB release experiments were carried out. The results revealed that the COST group exhibited a more rapid drug release rate than the COT group during the initial 24 h in a neutral environment. However, in the subsequent 24–48-hour period, the drug release rate of the COT group surpassed that of the COST group. Under acidic conditions, the drug release rate from the COT group’s hydrogel was noticeably faster compared to that of the COST group (Fig. 3g; Table 1).

The results of CCK-8 assay demonstrated that the hydrogels had no significant effect on L929 cells, with COS20 and COS30 even enhancing cell proliferation compared to the control group. In terms of drug toxicity, CCK-8 assays confirmed that both COT and COST hydrogels displayed negligible cytotoxicity (Fig. 3h). The hemolytic results revealed no significant hemolysis in all groups (Fig. 3i). Subsequent optical density testing enabled us to calculate the hemolysis ratio using PBS as a negative control and deionized water as a positive control. The leachate of all tested hydrogels demonstrated that hemolytic activity is below the 5% within safety threshold (Fig. 3j). Consequently, the COS20, COST, and COT groups exhibit commendable biocompatibility, laying a solid foundation for subsequent in vivo experiments.

COST, COT, and CSO20 hydrogels were used to evaluate their antibacterial activity against three common bacterial species in solid and liquid media, respectively. It is evident that both COST and COT showed antibacterial activity against S. aureus, P. aeruginosa, and Escherichia coli compared to the control group COS20 (Fig. 4a). ImageJ analysis indicated a slightly larger inhibition zone for the COST group compared to the COT group, which is potentially due to the rapid release of TOB from COST group within the first 24 h and better water retention (Fig. 4b). After a 24-hour incubation, COST maintained its structure and higher moisture content, whereas COT underwent significant degradation and water loss, adversely affecting the release of TOB. In the liquid LB medium, both COST and COT hydrogels exhibited a decline in OD values from 12 to 48 h post-incubation, compared to their baseline OD values (Fig. 4c–e).

(a) Antibacterial activity exhibited by COST, COT, and CSO20 hydrogels against S. aureus, P. aeruginosa, and E. coli following a 24-hour incubation period in solid medium. (b) Comparison of inhibition zone areas of COST and COT hydrogels against these bacterial types. (c) S. aureus, (d) P. aeruginosa, and (e) E. coli growth curves in COST, COT, COS20, and Blank groups.

To investigate the wound-healing efficacy of hydrogel in vivo, we utilized an SD rat model, which was created an approximately 10 mm circular skin lesions on their backs. The wound was covered with hydrogels immediately after surgery, and the effect of different hydrogels on wound healing was observed. Clearly, the hydrogel dressing has the ability to promote wound healing compared with the Tegaderm group. In the horizontal comparison of different hydrogels, we found that COST could accelerate the wound healing rate than that of COT, indicating that COST hydrogel was a better dressing than COT hydrogel. To the reason, it may be caused by better stability, moisture retention, and excellent biocompatibility. COS20 and COST group rats displayed similar healing trends, indicating that TOB played a limited role in this healing (Fig. 5a,c).

(a) Comparative healing trajectories in rats with different treatments. (b) Histopathological assessments at 12 days post-intervention are depicted, utilizing HE and Masson’s trichrome staining across all groups. Red arrow: the length of granulation tissue, blue: indicates the thickness of granulation tissue, yellow arrow: collagen. (c) Changes in wound area over time for groups of rats with different treatments (n = 5). (d) Comparison of granulation tissue length, (e) thickness, and (f) collagen deposition among different treatment groups.

In our histological analysis using HE staining, we quantified the width and thickness of granulation tissue (Fig. 5b). The granulation tissue in hydrogel-treated groups exhibited a notably reduced in length and increased in thickness compared to that of the control, suggesting an expedited wound-healing process post-hydrogel application (Fig. 5d,e). Notably, the COS20 and COST groups demonstrated more pronounced differences than that of the COT group, underscoring the enhanced healing efficacy imparted by SA in the hydrogel dressings. By Masson staining, we found that collagen deposition was markedly increased in hydrogel-treated samples compared to the Tegaderm group, with no significant variation among hydrogel variants (Fig. 5b,f). This underscores the hydrogel’s potent role in promoting wound healing. In conclusion, our findings indicate that hydrogel dressings facilitate wound healing. The novel COS20 and COST hydrogels, in particular, outperform the traditional COT hydrogel in promoting the healing process.

The global healthcare system faces significant challenges with skin wounds, particularly as society ages3, which results in a growing prevalence of non-healing skin wounds, imposing substantial social and economic burdens on both patients and healthcare systems32. Over the past few decades, hydrogels have emerged as leading contenders in wound dressing applications, demonstrating a consistent year-over-year increase in their usage16. Current hydrogel applications in wound care often overlook dynamic wound changes, such as pH variations due to bacterial infections and immune responses33,34. Designing pH-responsive hydrogel could enable targeted drug release and more effective wound management by sensing and responding to these changes.

Previous publication has demonstrated that during the healing process, bacteria infected wounds, which generate weak acids, such as lactic acid and acetic acid, through aerobic respiration and fermentation, resulting in a reduction of pH, and the pus within the wound also contributes to an acidic environment10,13. This organism’s metabolism encompasses not only respiratory processes, but also fermentative pathways that convert various carbohydrates-such as glucose-into acidic byproducts14,15. Thus, an acid-sensitive hydrogel that demonstrates on-demand release of antibacterial agents within a specific pathological acidic environment plays a pivotal role in the process of wound healing.

Hydrogels composed of Schiff base bonds can be injected in situ and can integrate as a whole to display their various functions in minimally invasive and precise medical treatments, without the limitation of location or shape21. Among the many polysaccharide hydrogels, the hydrogel with Schiff base bond that formed by CMC and OHA has great prospects for treating wound healing35,36,37. Schiff base bonds cross-link with the amino group of CMC and the aldehyde group of OHA, which dissociate under acidic conditions to enable substantial cargo release in response to changes of pH in the place of wound38. The successful oxidation of OHA and the presence of Schiff base bonds can be found by 1HNMR and FTIR, which proves the successful cross-linking of the hydrogel. Although pH-responsive hydrogels formed by CMC-OHA show considerable potential for applications, but their primary structure, which based on Schiff base bonds, experiences accelerated degradation in acidic environments and shortened their functional lifespan21. Recognized as an anionic polyelectrolyte, SA was incorporated through the interaction of ionic bonds with the positively charged amino groups on CMC39, the ionic bonds form a double network, that could modulate swelling, optimize drug release in a non-acidic environment, and increase water retention.

Therefore, we developed a novel dual responsive hydrogel, which is synthesized through Schiff base bonds and ionic bonding by CMC, OHA and SA to optimize would healing, TOB is encapsulated in with OHA through Schiff base bonds. This novel dual responsive hydrogel not only enables significant drug release to combat infection, but also extends the functional lifespan of hydrogel.

The successful crosslink of Schiff base bonds in hydrogel was confirmed by FTIR, which exhibited distinct peaks at 1630–1640 cm−1. Schiff base bonds are dynamic chemical linkages formed between aldehyde and amino groups that impart self-healing properties and make the hydrogel injectable.

The microstructure of the hydrogel can intuitively reflect its cross-linking network and pore size. The FE-SEM images revealed the three-dimensional network structure of the freeze-dried hydrogel across different groups, confirming successful cross-linking. FE-SEM showed that the freeze-dried hydrogel showed porous structure, the addition of SA did not change the surface morphology of the hydrogel significantly. Noteworthy, the pore sizes within the hydrogel scaffolds varied among different groups, which increase gradually with the gradual increased concentration of SA. The average pore size expanded from 56.98 μm to 75.552 μm with SA addition and further increased to 101.843 μm in the COS30 group. This expansion is likely due to electrostatic repulsion between the negatively charged SA and the similarly charged carboxyl groups on CMC, leading to progressively larger hydrogel pores40. The larger pore size in the hydrogel is more conducive to facilitate cell growth, angiogenesis, and drug release, as well as the absorption of exudates41,42. However, excessively large pores could undermine the hydrogel’s mechanical strength and stability, potentially resulting in a weaker structure and rapid degradation43,44.

The novel dual responsive hydrogel can actively respond to a change in the pH environment, both in degradation and in swelling. Degradation of the Schiff base bond hydrogel was accelerated in the acidic environment due to the Schiff base bond responsive dissociation. Results showed that the average degradation rate of hydrogel in an acidic environment was significantly faster than that in a neutral environment, which was attributed to the cleavage of Schiff bonds under acid condition45. However, we also found that upon entanglement by SA, the degradation rate of hydrogel was inhibited. SA is a kind of polysaccharide, which has been widely used in the biomedical field. Owing to the existence of –COOH in its polymer chains, SA could crosslink with other polymers and form hydrogel under mild conditions or enhance the toughness of hydrogels46,47. Also, SA is capable to conjunction with cationic polymers such as CMC, via ionic interactions26. The formation of enhanced ionic bonds between the positively charged CMC and negatively charged SA helps to counteract the rapid degradation of hydrogel caused by Schiff base bond separation48. Significantly, given that the pKa of the amino group in CMC is around 6.5, and the pKa of the carboxyl group in SA is about 3.5, protonation of the CMC amino group within the pH range of 3.5 to 6.5 is more conducive to forming ionic bonds48,49,50. The formation of enhanced ionic bonds between the positively charged CMC and negatively charged SA helps to counteract the rapid degradation of hydrogels caused by Schiff base bond separation. This might explain why the incorporation of SA enhances the structural stability of the hydrogel and solves the problem that the main hydrogel structure lost in acidic and neutral environment45,50.

The remaining weight also confirmed the hypothesis. The remaining weight of hydrogel was significant higher in COS20 group than CO group. Previous publications have proved that when low SA content was added in hydrogel, the hydrogel was still dominated by Schiff base crosslinking, with increase of SA amount in hydrogel, a similar degradation profile was observed at acidic and neutral pH, presumably due to the domination of SA entanglement in the hydrogel51. Similar residual weight of the hydrogels at the end stage in acidic and neutral pH may be attributed to the diminishes of gradient difference between the hydrogel and the surrounding medium. The good swelling performance of the hydrogels also narrowed the gap in the residual weight of the hydrogel52,53.

Beyond its pH responsive degradation characteristics, the newly developed hydrogel demonstrates an improved swelling profile. The excellent swelling ability of hydrogel can enhance its absorptive capacity, efficiently managing wound exudation and preserving a moist wound environment54,55. The swelling behavior of hydrogel are related to many factors, such as such as pH value, ionic strength, cross-linking rate, and ratio of each component. Our results indicated that the swelling ratio of the hydrogel in each group was increased in acidic and neutral environments following the addition of SA. Compared with the CO group, the swelling performance of the hydrogel in each group after the addition of SA was significantly improved in the neutral environment. This was probably due to the electrostatic interaction between the residual of amino groups (-NH2) on CMC and residual SA (-COOH) or CMC (-CH2COOH)56, repel of the carboxyl groups ionize in SA leads to swelling in neutral to alkaline pH environment45. The swelling performance is also related to the pore size of the hydrogel, which lead to enhance the absorption of wound exudate57. Previous publications have confirmed that the pore size of CMC/SA hydrogel increased with increasing proportion of SA at pH 4–7. The porous structures are interconnected and can act as water channels; hence, the hydrogel can take up large quantities of water and leading to a large swelling ratio. In addition, we found that the swelling capacity of COS hydrogel also increased in an acidic environment, but the swelling ratio is significantly less than that in a neutral environment. The reduced swelling in acidic environments can be attributed to the formation of hydrogen bonds between the carboxyl groups of CMC and SA and the electrostatic attraction that reduced the pore size of the hydrogel58. This confirmed the Schott’s law, the lower the pH, the smaller the swelling property45. Based on our results, COS20 and COS30 group exhibit both excellent degradation rate and commendable biological properties, but the excessive swelling performance of the hydrogel in the COS30 group led to the instability of the structure. The improvement of the degradation and swelling performance of the hydrogel in the COS20 group was better than COS10, so the COS20 group was selected for further experiments.

Besides the inherent property alterations in the hydrogel, the variations in their degradation and swelling behaviors significantly influence the drug release of hydrogel24,45. An effective drug release capability can significantly enhance the healing process of skin wounds59. After a severe wound infection, the local pH of the wound decreases9, and Schiff base bond-responsive separation can prompt the release of a large number of drugs to combat the infection. As TOB is an aminoglycoside antibiotic, its amino groups can form Schiff base bonds with OHA24 and was released when Schiff base bonds dissociate under acidic environments. In acidic conditions, due to the instability of the Schiff base bonds at pH levels below 5.520, the COS and COST groups lost the Schiff base bond with the hydrogel and formed free TOB. So the release of TOB in both group is higher at acidic pH than neutral pH. At the same time, the formation of ionic bonds between the CMC and SA helps to counteract the rapid degradation of hydrogels caused by Schiff base bond separation, so the TOB release decreased in COST group compared to COT group at acidic and neutral pH. This dynamic crosslinking does not entirely immobilize TOB, the initial release rate (6 h) of the COST hydrogel in a neutral environment is faster than that of the COT hydrogel, indicates the swelling changes in the hydrogel could also impact the release of TOB26,48. The drug-release properties of COST have substantial clinical implications. The normal skin pH typically ranges from 4 to 6, but acute injuries cause an increase in tissue fluid exudation, gradually elevating the wound’s pH to around 7.4. This pH increase fosters bacterial proliferation60. Traditional Schiff base hydrogels release drugs slowly at pH 7.4, potentially missing the rapid bacterial proliferation phase and hindering infection control. In contrast, COST hydrogel demonstrates a more effective release rate under neutral conditions, aiding early infection management. Under acidic conditions, enhanced stability and reduced swelling ratio ensure sustained drug release, offering prolonged protection to infected wounds16,31,61.

Infection is a critical factor in delayed wound healing, as bacteria and their byproducts disrupt various stages of the wound-healing process62. During the early stages of infection, gram-positive bacteria such as S. aureus are commonly found, predominantly in superficial wound layers. Meanwhile, gram-negative bacteria like E. coli and P. aeruginosa are more frequently encountered in the later stages and the deeper layers of wounds63,64. The COST hydrogel exhibits good antibacterial activity against S. aureus, P. aeruginosa, and E. coli, showcasing its potential broad-spectrum antibacterial abilities. While COT hydrogel exhibited smaller bacteriostasis ring than COST hydrogel in a solid medium. As discussed in the context of drug release, Schiff base bonds exhibit excessively strong binding in neutral environments, resulting in diminished antibacterial efficacy, and the increased swelling of the COST hydrogel facilitates a more effective rate of drug release65. In liquid LB medium, the COST and COT hydrogels showed sustained antibacterial effects against the above three types of bacteria, and their similar OD values might be influenced by ongoing hydrogel degradation. As control group, COS20 hydrogel did not display significant antibacterial activity. In Pseudomonas aeruginosa culture medium, there were uneven areas of reduced bacterial density around the COS20 hydrogel, which was considered to be caused by the swelling of the hydrogel rather than the antibacterial effect of the hydrogel. The sophisticated pH-sensitive hydrogel enables precise and tailored drug delivery, drastically enhancing drug utilization efficiency while alleviating issues like bacterial resistance stemming from the overuse of antibiotics66.

Excellent biocompatibility, including cytotoxicity and hemolysis, is indeed a crucial prerequisite for the application of hydrogel28. CMC and SA demonstrate outstanding biocompatibility, low toxicity, and minimal immunostimulatory activity67,68. But it was reported that OHA introduces aldehyde groups that could potentially exhibit cytotoxic effects on cells69. However, our results indicated that the hydrogel did not affect the growth of L929 cells. Interestingly, we also observed that the COS20 and COS30 hydrogels, which added SA, could even promote cell proliferation70. This may be due to the reaction between aldehyde and amino groups effectively reduced the free aldehydes, thereby minimizing cellular toxicity and establishing a strong foundation for further in vivo experiments71,72. When loaded with TOB, both COST and COT groups maintained good biocompatibility with hemolysis rates well below the 5% safe threshold and establishing a robust basis for subsequent in vivo experiments.

In vivo experiments have demonstrated that the COST group hydrogel dressing exhibits superior wound healing effects compared to the COT group hydrogel. The study revealed a significant difference in wound healing rates on the 6 day between the COST group and the Tegaderm group, while no notable difference was observed between the COT group and the Tegaderm group throughout the 12-day period. This could be attributed to the enhanced stability and superior absorption and exudation capabilities of the hydrogel following the introduction of SA16.

Histological analysis, including both HE staining of granulation tissue and Masson staining of collagen deposition, indicated that the healing trend with the COST group hydrogel was superior to that of the COT group and significantly distinct from that of the Tegaderm group. While the COT group also facilitated healing, it did not show a significant difference in collagen deposition when compared with the Tegaderm group. Although there was little distinction between COST and COS20 hydrogels, it may be due to the insignificant impact of TOB on wound healing in SPF animals and environments. However, the risk of bacterial infection transforming acute wounds into chronic or non-healing wounds cannot be overlooked considering the potential for bacterial infection during the healing process, therefore effective antibacterial performance remains crucial for hydrogel dressings62. The superior healing effects of the COST and COS20 hydrogels compared to the COT group in vivo may be attributed to several factors.

The addition of SA results in larger pore sizes, facilitating material exchange41. Enhanced swelling ability improves exudate absorption, while increased water retention supports a moist wound-healing environment54. Additionally, the hydrogels’ good biocompatibility and effective simulation of the ECM contribute to their effectiveness in wound healing73.

These findings further demonstrate that hydrogel-modified SA effectively promotes wound healing. Future studies are anticipated to explore the cellular and molecular mechanisms of the COST hydrogel on wound healing and to investigate the incorporation of various drugs for enhanced wound management74.

In this study, a dual-network structure COST hydrogel was created through Schiff base bonding and ionic bonding. This hydrogel exhibits capabilities of pH-responsive drug release and improved properties of swelling. Drug release in COST hydrogel depends on the degradation instead of the of hydrogel disintegration, the ionic bond enhances the stability of COST hydrogel in acidic environment and prolongs its lifespan. The COST hydrogel showed sustained antibacterial effects against bacterial which are commonly found around wounds and exhibit excellent biocompatibility and capability to accelerate wound healing on rat. Overall, the COST hydrogel making it a promising candidate for promoting wound healing and having significant potential for clinical application.

Tobramycin, Phthalaldehyde, Mercaptopropionic acid, Sodium alginate, Hydroxylamine hydrochloride, Carboxymethyl chitosan (substitution degree > 80), Sodium hyaluronate (15–20 kD), Sodium periodate, and Ethylene glycol were purchased from Macklin (China). MD34 dialysis bag MWCO12000-14000 was from Beijing Solarbio Science & Technology (China). CCK-8 was purchased from MCE Corporation (China), Pseudomonas aeruginosa, Staphylococcus aureus, Escherichia coli, LB media, and L929 cells were provided from the Fourth Affiliated Hospital of China Medical University. Specific-Pathogen Free (SPF) SD rats were purchased from Beijing Hfk Bioscience (China), and all animal experiments met the ethical review requirements of China Medical University.

OHA was synthesized following a slightly modified version of a previously established procedure23. Initially, 2.5 g of HA was dissolved in 250 mL of deionized water. Subsequently, 12.5 mL of sodium periodate (0.5 mol/L) was gradually added. The mixture was then covered with aluminum foil and stirred in the dark at room temperature for 5.5 h. To neutralize any remaining sodium periodate, 2.5 mL of ethylene glycol was introduced. After an hour, the solution underwent dialysis using a membrane (molecular weight cut-off: 10,000) for five days, and silver nitrate reaction was used to detect the residual of sodium iodide. The resultant OHA was stored in the dark at 2–4 °C to prevent degradation.

In our experiments, we opted for a CMC: OHA mass ratio of 5:3 after considering factors such as crosslinking time and mechanical properties by referred to the previous publication75. The study divided samples into four groups with varying SA concentrations of 0, 10, 20, and 30 mg/mL (Table 2). CMC, OHA, and SA. Firstly, CMC and SA were dissolved in PBS, OHA solution was prepared at same way, and then the two solutions were homogenized to obtain hydrogel. The gel time of each group is 94 ± 11s (COS10 group), 72 ± 13s (COS20 group), and 40 ± 8s (COS30 group). If the amount of SA was more than 30 mg/mL, the solution would be too viscous and the hydrogel system would be difficult to mix evenly. The hydrogels were produced by homogeneously mixing the polysaccharide solutions of each fraction. For example, 1mL of CO group hydrogel contains 1mL of PBS, 75 mg of CMC, and 45 mg of OHA.

We utilized FTIR to verify the presence of aldehyde groups in OHA and assess hydrogel cross-linking. The measurements were conducted at room temperature using a blank potassium bromide (KBr) film as the background. The analyzed samples included OHA powder and freeze-dried hydrogel products. A 0.5 mg sample was finely ground, mixed with KBr powder, and compressed into a transparent pellet for analysis. The FTIR spectra were recorded in the wavenumber range of 3000–400 cm−1, with results compiled from 20 scans.

The structure of OHA was validated using NMR spectroscopy with a BRUKER AV-500 instrument. The oxidation rate (OR) was calculated using the formula76:

For FE-SEM analysis, hydrogels from each group were freeze-dried and mounted on a scaffold as described71, and their surface morphology was observed under the Sigma 300(Zeiss, German) FE-SEM at 15 kV.

In vitro degradation study was performed by immersing the hydrogel samples in neutral PBS buffer solution (pH = 7.4) and acidic solution (pH = 5.5) environments at room temperature77. Total of 3 samples per groups were analyzed. The initial weight of the hydrogel was measured (Wi). At 12-hour intervals, the soaking solution was carefully removed, and the surface water of the hydrogel was dried before the hydrogels were weighed (Wt). Degradation was determined using the following equation.

The hydrogels were firstly freeze-dried to obtain lyophilized products, after which measured their dry weight (Wd). Subsequently, they were immersed in both neutral PBS buffer (pH 7.4) and acidic (pH 5.5) solutions until achieving swelling equilibrium. Upon achieving equilibrium, the swollen hydrogel were blotted with filter paper to remove excess surface water and then immediately weighed (Ws). The equilibrium swelling ratio was calculated using the specified equation78. Total of 3 samples per groups were analyzed.

After considering factors such as crosslinking time and mechanical properties, we opted the COS20 group loaded with 1 mg/mL TOB as the experimental group, the CO group loaded with an equal amount of TOB serve as control (Table 3).

In this study, we investigated the release profile of Tobramycin (TOB) from COST and COT hydrogels by the o-phthalaldehyde (OPA) method. First, 1.5 mL hydrogel were measured for each group, then 1.5 mL of phosphate-buffered saline (PBS) were added to immerse the hydrogel in a 24 well plate, TOB release were measured under two pH conditions: acidic (pH 5.5) and neutral (pH 7.4). To emulate a sustained release environment, the medium was methodically replaced every 12 h. This involved extracting 1.5 mL of the solution for subsequent analysis and replacing it with an equal volume of fresh PBS at the same pH. The OPA test solution was prepared by dissolving 100 mg of o-phthalaldehyde in a borate buffer solution (pH 10.5), followed by the addition of 1 ml of mercaptoethanol, and adjusting the final volume to 100 ml to ensure a consistent pH of 10.5. For the analysis, 1.5 mL of the OPA solution was mixed with 4.5 ml of isopropanol and 1.5 ml of the release medium from the hydrogels. This mixture was then incubated in a water bath at 60 °C for 15 min. After cooling to room temperature, the TOB concentration was determined by measuring the absorbance at 333 nm with a microplate reader.

The cytotoxicity of CO, COS10, COS20, COS30, COT, and COST group hydrogels was evaluated in vitro using the Cell Counting Kit-8 (CCK-8)71, each group have 3 samples and the measurement are performed 3 times. Leachates from each hydrogel group were prepared at 20 mg/mL by incubation in Dulbecco’s Modified Eagle Medium (DMEM) for 48 h. L929 cells were seeded in 96-well plates at a density of 1 × 104 cells/well and incubated for 24 h. Subsequently, hydrogel leachates were added to the respective wells, and the cells were co-incubated with these leachates at 37 °C in a 5% CO2 atmosphere for another 24 h. After this period, CCK-8 solution was introduced into each well and incubated as per the manufacturer’s protocol for 2 h. Absorbance was measured at 450 nm using a microplate reader. Cells treated with DMEM served as the control, with their viability set at 100%.

As: absorbance value of the sample (hydrogel group). Ad: absorbance value of the DMEM group Ab: absorbance value of the blank.

In the blood compatibility assay, red blood cells (RBCs) were isolated from rat blood via centrifugation for 10 min. The isolated RBCs were thrice washed in PBS. Subsequently, 20 µL of these RBCs were combined with 1000 µL of hydrogel leachate (20 mg/mL) in a microcentrifuge tube. This mixture was then agitated at 37 °C and 100 rpm for four hours. Post incubation, the contents of each tube were centrifuged for 10 min to sediment any intact RBCs. Carefully, 100 µL of the supernatant was transferred to a clear 96-well plate. The absorbance at 540 nm was measured to assess hemolysis. Deionized water and PBS were employed as positive and negative controls, respectively. The hemolysis percentage was calculated using the following formula76. The experiments are performed 3 times.

Ah: absorbance value of the hydrogel group supernatant. At: absorbance value of deionized water group Ap: absorbance value of PBS group.

In this study, three bacterial strains were selected as test subjects: Staphylococcus aureus (Gram-positive), Pseudomonas aeruginosa (Gram-negative), and Escherichia coli (Gram-negative). The antibacterial efficacy of COT and COST hydrogels against these pathogens was evaluated using the agar diffusion method, with COS20 hydrogel serving as a blank control. Each group has 3 samples and the measurements are performed 3 times. Briefly, Lysogeny Broth (LB) solid medium plates were prepared, each uniformly inoculated with 100 µL of a bacterial suspension (OD600 = 0.6) of Staphylococcus aureus (S. aureus), Pseudomonas aeruginosa (P. aeruginosa), and Escherichia coli (E. coli), respectively. Before testing, the hydrogel groups were sterilized using bacterial filters and 500 µL injected into the molds. Subsequently, these hydrogels were placed onto the agar plates and incubated at 37 °C24. The antibacterial activity was assessed by measuring the diameter of inhibition zones formed around the hydrogels.

In this series of experiments conducted in liquid culture medium, the antibacterial properties of the hydrogels were quantified using S. aureus, P. aeruginosa, and E. coli. The bacterial strains were cultured in liquid beef extract medium and incubated at 37 °C until they reached an optical density (OD) of 0.4 at 600 nm. For the antibacterial assays, 500 µL of each hydrogel group (COT, COST, and COS20) was combined with 500 µL of the bacterial suspension in a 6-well plate. These mixtures were then incubated at 37 °C for time intervals of 12, 24, 36, and 48 h. Following each incubation period, the OD600 was measured to determine bacterial growth, and inhibition curves were plotted to quantify the antibacterial activity of the hydrogels, Each group have 3 samples and the measurement are performed 3 times24.

The animal experiment was approved by the China Medical University Animal Care and Use Committee in Sep. 2023 (CMU20231083) and we have ensured adherence to the ARRIVE guidelines in reporting our animal experiments. After sample size calculation, twenty SD male rats, each weighing approximately 260 ± 10 g, were randomly selected and anesthetized using a suitable dose of isoflurane. These rats were then randomly assigned to four experimental groups. Following hair removal and disinfection, circular full-thickness skin defects with a diameter of 10 mm were surgically created on the dorsal median area of each rat. The wounds were left open for two hours before treatment. Subsequently, wounds in the COS20, COT, and COST groups were treated with 300 µL of the respective hydrogels. The Tegaderm group covered transparent film dressing frame style (3 M Tegaderm) on their wounds. To prevent hydrogel displacement, a 12-mm silicone rubber washer was placed around each wound for 24 h, followed by covering with a 3 M Tegaderm transparent dressing. The analysis included those rats that survived 12 days after the operation, excluding any that died accidentally, and the experimental subjects were adjusted accordingly. Wound healing was monitored over time, with photographic documentation and observations for any abnormalities such as exudation, abscess formation, or non-healing, recorded on days 1, 3, 6, 9, and 12 post-surgery and the experiment was terminated at day 12, and rats were euthanized by CO2.

After 12 days, three rats from each group were selected for histological analyses. Wound tissues were harvested and immediately fixed in 4% paraformaldehyde for 4 days. Following fixation, the tissues were processed and sectioned into 7-µm-thick slices. These sections were prepared to facilitate the evaluation of scar formation and skin regeneration associated with different hydrogel treatments. For detailed histopathological examination, the sagittal sections were stained using hematoxylin and eosin (HE) and Masson’s trichrome stains. These staining methods obtained the length and thickness of granulation tissue and the collagen content of granulation tissue to assess scar tissue development and the regeneration of skin appendages, measurements are replicate 3 times for each sample.

All experimental data were expressed as mean ± standard deviation (SD). Statistical analyses of the data inside the group were compared by one-way analysis of variance (ANOVA) and pairwise comparison was conducted by LSD-t test. Differences were considered significant at P < 0.05. (*P < 0.05, **P < 0.001, ***P < 0.001).

The datasets used or analyzed during the current study are available from the corresponding author on reasonable request.

Carboxymethyl chitosan

Chitosan

Hyaluronic acid

Oxidized hyaluronic acid

Sodium alginate

Nuclear magnetic resonance

Fourier transform infrared spectroscopy

Tobramycin

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The authors thank Dr. Xiaochong Guo, Department of laboratory animal science, China Medical University, for the assistance with the animal experiments.

The work was supported by Foundation of Liaoning Province Education Administration (LJKZ0772).

Department of Stomatology, The 4th Affiliated Hospital of China Medical University, No.4 Chongshan Dong Road, Shenyang, 110032, China

Jiajun Xiao, Yanming Liang, Ting Sun & Xiaoning He

Department of Epidemiology and Biostatistics, The 4th Affiliated Hospital of China Medical University, No.4 Chongshan Dong Road, Shenyang, 110032, Liaoning, China

Ming Liu

Department of Periodontology, Jinzhou Stomatological Hospital, Jinzhou, Liaoning, China

Ting Sun

Department of Endodontics, Shanghai Fengxian Dental Institute, Shanghai, China

Jiajun Xiao

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XH ML JX YL TS conceived and designed the experiments, JX YL TS XH performed the experiments, ML XH JX analyzed the data, XH ML JX wrote the paper.

Correspondence to Ming Liu or Xiaoning He.

The authors declare no competing interests. Funding The work was supported by Foundation of Liaoning Province Education Administration(LJKZ0772)

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Xiao, J., Liang, Y., Sun, T. et al. A functional dual responsive CMC/OHA/SA/TOB hydrogel as wound dressing to enhance wound healing. Sci Rep 14, 26854 (2024). https://doi.org/10.1038/s41598-024-78044-8

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DOI: https://doi.org/10.1038/s41598-024-78044-8

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