The GelMA/OSSA/PMB hydrogel was expected to promote diabetic wound healing by the anti-infection and anti-inflammatory. The physicochemical properties of the hydrogel, antibacterial activity, and cell behaviors were investigated in vitro, and the regeneration effect of wound infection was evaluated with the diabetic mice in vivo.
Preparation and Characterization of Hydrogel
Preparation of Hydrogel
The Synthesis process of OSSA and GelMA was presented in Scheme 1. Firstly, SA was sulfated by HClSO3 to get SSA, followed by, SSA being oxidized by sodium periodate to obtain OSSA. In addition, we also prepared the OSA by oxidizing SA. OSSA and PMB were mixed overnight to form polymer OSSA/PMB conjugates, OSSA owned multiple aldehyde and hemiacetal species in the backbone, which could react with amino groups of PMB to form imine linkages (Schiff's base reaction). GelMA and conjugates are mixed and photo-crosslinked to form GelMA/OSSA/PMB hydrogel.
The fourier transform infrared spectroscopy (FT-IR) was conducted to study the OSA and OSSA, as shown in Fig. 1. The hydrophobized sulfated alginate was characterized in SSA by the appearance of a definitive band at 1258 cm− 1. which assigned to the symmetric stretching vibration of S = O stretching of the sulfate group. This successfully demonstrated the introduction of sulfonic acid functional groups into SA [32]. Especially, the peak absorption intensity of OSA and OSSA at 1737cm− 1 was below SA, indicating the carbonyl bond in the aldehyde group. These results designated that the as-obtained products were available for the next application. The FT-IR spectrum of gelation and GelMA displayed the absorption peaks at ∼3310 cm− 1 and ∼1540 cm− 1, which corresponded to the N─H bands stretching vibration amide A and amide B, respectively. As expected, the stretching vibration of C═C─H (3070 cm− 1) was only detected in GelMA (Figure S1a), suggesting that GelMA was successfully synthesized [15]. and the product was available for the next application.
Next, the GelMA/OSSA/PMB hydrogel was further strengthened with the addition of GelMA in the presence of visible blue light and the photoinitiator (LAP), and the clear solution becomes milky white hydrogel (Fig. 1b). Similarly, GelMA, GelMA/OSA, GelMA/OSSA, and GelMA/OSA/PMB were synthesized for the next applications. After light exposure, FT-IR spectrum showed the peak at ∼3070 cm− 1 almost vanished. the peak absorption intensity of GelMA/OSA, GelMA/OSSA, GelMA/OSA/PMB, and GelMA/OSSA/PMB at ∼1737 cm− 1 disappeared, whereas the C = O stretching vibration remarkably enhanced at ∼1650 cm− 1, which indicated the cleavage of the aldehyde group and the formation of the tertiary amide bonds. Maybe A significant peak at ∼1650 cm− 1 (Fig. 1c) might be corresponding to the formation of an imine bond (C = N), ie Schiff's base structure by the reaction of amino groups of PMB and GelMA and aldehyde groups of glutaraldehyde. In addition, the electrostatic interactions between PMB, GelMA, and OSSA can also promote gel formation.
Ability of Water Absorption
A 3D highly porous network structure was observed in representative scanning electron microscopy (SEM) images (GelMA, GelMA/OSA, GelMA/OSSA, GelMA/OSA/PMB, GelMA/OSSA/PMB) (Fig. 1d). The porous structures of hydrogel would promote cell migration when used for wound treatment. The presence of dot-like patterns on the OSSA and PMB-laden GelMA gels in the zoomed-in images of the GelMA/OSSA/PMB was indicative of the Schiff's based conjugates between OSSA with PMB onto the gels (Fig. 1e f). The presence of polymer conjugates was then verified by TEM, Figure S2 showed that particles of about 100nm existed on the surface of the GelMA/OSSA/PMB hydrogel.
An appropriate swelling rate would make wound dressing easier to absorb of wound secretion [22]. The swelling rate indicated that pure GelMA had a good swelling rate of about 800% (Fig. 2a). In the bargain, swelling rate of hydrogel decreased to 600% with the addition of OSA, OSSA, or PMB, mainly due to the increased crosslinking reaction such as Schiff's base reaction and electrostatic interactions resulting in photocurable GelMA network structure (Fig. 2a). An appropriate porosity was observed in all hydrogels, which wound expedites the absorption of wound secretion and gas exchange (Fig. 2b). As proved, 60–90% of porosity was proven to realize nutrient exchange appropriate for cell proliferation and movement in wound healing [33, 34].
Mechanical properties
Generally, protecting the wounds from further injury is required to be strength suitable as an ideal hydrogel dress. Thus, the mechanical characteristics of GelMA/OSSA/PMB hydrogel were so important. To analyze the changelessness, the G′ and G″ were carried out. When the angular frequency turned from 1 to 100 rad·s− 1, the hydrogels’ G′ and G″ showed any modification, indicating the proficient stability (Fig. 2c). Owing to a higher Schiff's base when more − CHO groups existed in the system. Besides, compared with the groups with OSA content, the groups with OSSA content showed a lower G′. This is probably because the higher content of − CHO in OSSA polymer consumed more amine group from PMB or GelMA, leading to the decrease of chemical cross-linking point [35]. The high degree of cross-linking by blue light irradiation of GelMA has a greater effect on modulus than the Schiff base effect present between OSSA and PMB. Generally speaking, the higher OSSA in the GelMA/OSSA/PMB hydrogel led to a lower storage modulus than in GelMA/OSA/PMB hydrogel (Fig. 2c).
Enzyme Responsive Drug Release Properties.
An appropriate degradation rate is essential to an ideal wound dressing under an infected wound microenvironment, which can alleviate PMB release and reduces dressing replacement times. These hydrogels have a slow degradation rate in PBS (Fig. 2d), but the collagenase solution was observably increasing the degradation rate (Fig. 2e f). Collagen concentration in the wound will affect the rate of hydrogel degradation. The relatively long degradation period of hydrogel could meet the wound repair requirement. In addition, the significantly low degradation rate in GelMA/OSA, GelMA/OSSA, GelMA/OSA/PMB, and GelMA/OSSA/PMB than GelMA might be due to the presence of amino groups on the PMB and GelMA (Fig. 2e f), which can react with OSSA/OSA so it improved stability. The skeleton structure of OSSA was relatively shorter than OSA for the sulfated step, which can decrease the hydrogel stability.
pH-Responsive Drug Release Properties.
We evaluate PMB release in PBS at pH 5.5/7.4. During wound healing or in the case of infection, the traumatic inflammatory environment is mostly acidic [36]. The results showed that the behavior of GelMA/OSA/PMB and GelMA/OSSA/PMB hydrogels with slow release PMB lasted about 4–6 days. In the group of GelMA/OSA/PMB, at pH 5.5, the drug release was 7.25% higher than that at pH 7.4 (Fig. 2g). In the group of GelMA/OSSA/PMB and it was also clearly 18.72% higher than that at pH 7.4 when pH is 5.5 (Fig. 2h). This was attributed to the instability and reversibility of Schiff bases (C = N bonds) under acidic conditions. In diabetic wounds, prolonged inflammation usually lead to a lower pH of the wound. This made GelMA/OSSA/PMB hydrogels with higher PMB release properties at low pH advantageous in the treatment of diabetic wound healing.
Antibiotic activity
Whether the releasing PMB remains biologically active must be evaluated before it is put into use for wound healing. MDR-P.a is one of the most problematic pathogenic bacteria and the most common negative bacteria in patients with clinical infections.
We evaluated the antibacterial efficacy of the GelMA/OSA/PMB and GelMA/OSSA/PMB in a broth assay against the model bacteria MDR-P. a. After 12h co-culture of GelMA/OSA/PMB and GelMA/OSSA/PMB hydrogels with MDR-P. a, the antibacterial capability was evaluated by OD600 measure. As shown in Fig. 2i, an equivalent PMB solution can continuously and effectively inhibit the growth of MDR-P. a. Hydrogel containing PMB could weaken the increased rate in absorbance and effectively affect the proliferation of bacteria, which was due to the pH of LB medium dropping to acidic conditions and increased sustained PMB release. These results confirmed the excellent in vitro PMB activity of GelMA/OSA/PMB and GelMA/OSSA/PMB against MDR-P. a.
Antibiotic loading concentration
The in vitro cytocompatibility of hydrogels was assessed on NIH 3T3 cell lines by live/dead cell staining assays and CCK-8 assays. We used transwell to achieve co-cultured of cells below hydrogels (Fig. 3b). As shown in Fig. 3a, the cells showed excellent viability, growth and proliferation throughout the experiment in the presence of hydrogel at all times. Compared with that of individual GelMA hydrogel, the growth density of cells on co-culture with GelMA/OSSA/PMB hydrogels was affected by 2mg/mL PMB added in the hydrogel. The results of CCK-8 experiments responded that when cells were co-cultured with GelMA/OSSA/PMB (1mg/mL PMB) for 1, 3 or 5 days, there was no statistical difference compared to the control group, cell survival was excellent, and there was no cytotoxic effect of GelMA/OSSA/PMB (1mg/mL PMB) in this group. But free 1mg/mL PMB exhibited remarkable cytotoxic effects to NIH 3T3 (Fig. 3a c). The above results indicate that the same concentration of PMB loaded in the hydrogel caused significantly less damage to the cells than the PMB released suddenly.
Antibacterial and Antibiofilm Properties
In the state of a hyperinflammatory microenvironment, persistent MDR bacterial infections will extend to the surrounding tissues of the wound and even form a biofilm. So we explore the antibacterial activity of group hydrogels against free MDR-P.a and biofilm development.
In sharp contrast, GelMA/OSSA/PMB Hydrogels showed evident MDR-P.a inhibition. Increasing the content of PMB resulted in a significant decrease in colonies in the agar plate. 1mg/mL PMB in GelMA/OSSA/PMB could demonstrate complete anti-bacterial ability (Fig. 4a b). This instant appeared and long-term lasting antibacterial effect evidenced that the GelMA/OSA/PMB and GelMA/OSSA/PMB hydrogels released PMB sustaining released drugs in 4 days, benefit to prevent the infection of wounds for quite a long time. To further confirm the antimicrobial property of GelMA/OSA/PMB and GelMA/OSSA/PMB hydrogels, the two group hydrogels released enough PMB that could completely inhibit free P.a (Fig. 4c) and biofilm (Fig. 4e) compared to the GelMA, GelMA/OSA and GelMA/OSSA groups. In addition, quantitative statistics showed that bacterial kill ratio of MDR-P.a on transwell was greater than 99% (Fig. 4d f). It has been reported that PMB is an FDA-approved cyclic polypeptide antibiotic that could suppress the proliferation of gramnegative bacteria. It has been reported in the literature that the mechanism of the bacterial inhibitory effect of PMB is mainly the binding of PMB to lipopolysaccharide in the outer membrane of bacteria, which eventually leads to bacterial death by increasing cell membrane permeability [17]. Benefiting from this advantage of PMB, hydrogels containing PMB also exhibited the inhibition ability of bacterial proliferation.
Wound healing
The diabetic microenvironment is hyperglycemic and highly inflammatory, and effective treatment of diabetic wounds, a typical chronic trauma, remains a challenge. GelMA/OSSA/PMB hydrogel might be a promising wound dressing according to in vitro studies. We further examined the antibacterial, anti-inflammatory, and wound healing capacity of our hydrogel using a P.a-infected diabetic BALB/c mouse with full-thickness skin defects and chronic wounds (Fig. 5a).
Visual observation of the wound healing was assessed on corresponding time points (Fig. 5b). Macroscopically, wounds cured by GelMA/OSA/PMB or GelMA/OSSA/PMB showed accelerated wound closure during the wound healing process. More specifically, the wound healing rate of GelMA/OSA/PMB group and GelMA/OSSA/PMB group were significantly faster and the degree of wound healing was much higher than other groups led to 26.6% and 27.8% wound closure on day 7, respectively, which were conspicuously higher than the PBS control group (0.2%), the GelMA/OSA group (4.5%), and the GelMA/OSSA group (5.6%). The GelMA/OSA/PMB and GelMA/OSSA/PMB hydrogels dressed groups displayed effectively controlled drug-resistant bacteria (P.a) infections and a significantly a faster-wound closure rate and dramatic wound area decrease on day 7, which was much higher than that of the other groups, further surpassing them on days 10 and day 14 (Fig. 5cd).
Skin tissue was assessed by tissue homogenate to observe surviving bacteria in vivo on days 0, 3, 7, 10, and 14 (Fig. 5e). The GelMA/OSA/PMB and GelMA/OSSA/PMB hydrogel cured groups displayed an effectively controlled of drug-resistant bacteria (P.a) infections. GelMA/OSSA/PMB hydrogel group showed faster-wound closure rate than GelMA/OSSA/PMB hydrogel group, Both GelMA/OSA/PMB and GelMA/OSSA/PMB hydrogel groups showed the most effective bactericidal action compared to other groups on day 7 (Fig. 5ef). The significant reduction in wound bacterial load likely diminished the stimulation of the inflammatory response, thereby accelerating healing time. This result was consistent with the general observation of wound healing (Fig. 5bc). After a prolonged keep watching, a burst growth of bacteria in other groups were observed on the next days and stayed the same afterward, the surrounding skin gradual ulceration, perhaps due to the increased infection.
In addition, histomorphological analysis of the wound was performed by H&E staining, reflecting the changes in the wound at different phase periods (Fig. 5g). The results were consistent with the wound healing rate (Fig. 5b). H&E histological images showed that PBS, GelMA/OSA, and GelMA/OSSA groups showed obvious defects of the epithelium and dermal layer of the skin tissue, and demonstrated that the number of inflammation cells in the marginal region of wounds was greater when compared with GelMA/OSA/PMB and GelMA/OSSA/PMB groups, after injury, the inflammatory response of GelMA/OSA/PMB and GelMA/OSSA/PMB hydrogels were milder because of the releasing of PMB. Moreover, A new generation of the epithelial layer (yellow dotted line) was observed in GelMA/OSSA/PMB group after 7 days of treatment. Wound re-epithelialization and granulation tissue formation have been identified as typical indicators to evaluate the wound healing process. With the advancement of the repair process, a multilayered, totally connected, and thick epithelium structure (yellow dotted line) that closely resembled the healthy epidermis of the intact skin was formed in the GelMA/OSSA/PMB hydrogel cured group on day 10 (Fig. 6a). The GelMA/OSSA/PMB hydrogel group also exhibited significantly thicker granulation tissue than other groups. No scar tissue was observed on day 14, indicating the successful healing of wounds. New epidermis structures (yellow dotted line) and hair follicle cells (indicated by black arrows) appeared after GelMA/OSA/PMB or GelMA/OSSA/PMB hydrogel treatment, the two groups had already started to show a reconstruction of functional skin (formed a complete epidermis with more regular structure and hair follicles developed on day 14). But the other three groups still had skin defects on day 14. Inflammatory cells almost disappeared compared to the other groups after GelMA/OSSA/PMB hydrogel treatment.
Repairing cascade of wound
Wound healing is a precise process. The process of wound healing with three overlapping but distinct phases: inflammation, proliferation, and remodeling. The following experiments were conducted to evaluate the repairing cascade after different treatments.
In this study, inflammation would have been triggered by the initial wound-creating and MDR-P.a infection to the wound. Persistent inflammation of diabetic infected wounds creates a lower pH environment compared to the normal environment, which is associated with increased metabolic activation [37]. In the inflammation phase, the percolation of proinflammatory cells (such as neutrophils and macrophages) and a high concentration of proinflammatory cytokines are distinctive characteristics. Therefore, the pH responsiveness of the GelMA/OSSA/PMB hydrogel would be appropriately stimulated to trigger PMB during the inflammation. On day 3, infiltration and distribution of pro-inflammatory cells were extremely strong in all groups. On day 7, histological analysis showed that the percolation of proinflammatory cells in the wound area was substantially reduced, especially in the GelMA/OSA/PMB and GelMA/OSSA/PMB hydrogel groups (Fig. 6a). But in the PBS group, inflammatory cells continued to infiltrate the surroundings of the wound at this time. These results demonstrated that a low pH micro-environment during the inflammatory phase favored PMB release, and the wounds cured with GelMA/OSSA/PMB hydrogels transition from the inflammatory to the proliferative phase were accelerated.
During the proliferative phase, fibroblast proliferation, collagen synthesis, and re-epithelialization require neovascularization to supply nutrients and oxygen to the repair site. Thus, the wounds cured with GelMA/OSSA/PMB hydrogel appeared to have improved angiogenesis compared to other control groups as identified via CD31 immunostaining on days 10 and 14. The wound sections from the GelMA/OSA/PMB or GelMA/OSSA/PMB hydrogel groups had more CD31 compared to the other groups on day 10, with GelMA/OSSA/PMB hydrogel group exhibiting the highest level (Fig. 6b). Specifically, the quantitative analysis demonstrated that the relative CD31 coverage area was significantly higher in the GelMA/OSA/PMB and GelMA/OSSA/PMB groups (Figure S5). During the process of wound healing, excessive vascularization also produced side effects such as wound fibrosis and scar formation. Thus, GelMA/OSA/PMB and GelMA/OSSA/PMB could accelerate the remodeling phase entrance and avoid excessive vascularization.
During the remodeling phase, we used Masson’s Trichrome staining to test collagen deposition. Masson staining results showed that the expression of collagen was significantly higher in the GelMA/OSA/PMB hydrogel and GelMA/OSSA/PMB hydrogel. when compared with the PBS, GelMA/OSA, and GelMA/OSSA hydrogels on day 10; however, the expression of collagen in the PBS group was less than in other groups on day 14 (Fig. 6c). A dense and orderly arrangement of collagen fibers was observed in the group of GelMA/OSA/PMB and GelMA/OSSA/PMB treatment on day 10, with a higher collagen index (Figure S6). This demonstrated that GelMA/OSA/PMB and GelMA/OSSA/PMB were capable of promoting collagen deposition (Fig. 6g). Moreover, more skin appendages-like hair follicles were shown in our GelMA/OSSA/PMB hydrogel group, representing the best wound healing effect of GelMA/OSSA/PMB hydrogel among the five groups.
All the above results indicated that GelMA/OSA/PMB and GelMA/OSSA/PMB hydrogels possess great potential for promoting infected diabetic chronic wound healing. By simultaneously eradicating MDR bacteria (P.a), relieving inflammation, promoting angiogenesis and increasing collagen, our GelMA/OSSA/PMB hydrogel showed a significantly accelerated wound closure rate.
3.8. Therapeutic mechanisms of the Hydrogel
A primary challenge in the treatment of diabetic wounds is the prolonged inflammatory phase, which hinders the progression of wound healing. The PBS and GelMA/OSSA/PMB hydrogel treatment groups showed a significant distinction in inflammatory reaction throughout the entire treatment period (Fig. 6g). Results from histological analysis confirmed that the OSSA/PMB-based GelMA/OSSA/PMB hydrogel could accelerate the inflammatory phase to the proliferation phase of diabetic wound healing.
To assess the messenger RNA (mRNA) levels in diabetic infected wounds after treatment with GelMA/OSSA/PMB hydrogel, RNA-sequencing (RNA-seq) was performed on the tissue samples collected post-surgery on day 10, which is in the transition phases of inflammation phase to proliferation phase in the wound healing process. Tissue samples collected from the diabetic-infected wounds treated with PBS were taken as control. A significant difference between the transcriptomic profiles of the PBS and GelMA/OSSA/PMB hydrogel groups was observed by the unguided principal component analysis (PCA) (Figure S7a). PBS group and the GelMA/OSSA/PMB group have differentially expressed genes with 1884 upregulated and 1941 downregulated genes, according to the empirical Bayes method (fold change ≥ 2; Padjust < 0.05), as shown in the MA plots (Figure S7b). Up and down-regulation datasets were established to go forward Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis. KEGG pathway enrichment analysis revealed that GelMA/OSSA/PMB hydrogel-treatment enhanced the signal transduction of Wnt signaling pathway and TGF-β signaling pathway (Fig. 7a), which supported migration, proliferation, and production of collagens and matrix metalloproteinases (MMPs). Strong crosstalk of TGF-β and the WNT signaling pathways might facilitate fibrosis development and progression. Therefore, up-regulation of Wnt signaling pathway and TGF-β signaling pathway indicated that GelMA/OSSA/PMB hydrogel promoted wound healing and fibrosis [38]. The KEGG of down-regulation dataset was focused on inflammatory pathways such as NOD-like receptor signaling pathway, TNF signaling pathway, and IL-17 signaling pathway (Fig. 7b). These analysis results suggested that GelMA/OSSA/PMB hydrogel relieved inflammation. For clarity, profound changes in tissue function caused by these gene change, up and down-regulation datasets were further continued GO enrichment analysis. GO analysis revealed that the 1884 up-regulated genes focused on Wnt signaling pathway, regulation of epithelial cell differentiation, and hair cycle process (Fig. 7c). These results suggested that the renewal and regeneration potential of the skin was restored in GelMA/OSSA/PMB hydrogel cured diabetic-infected wounds. GO analysis revealed that the 1941 dawn-regulated genes focused on positive regulation of superoxide metabolic process, T-helper 17 cell differentiation, positive regulation of interleukin-13 production, positive regulation of tyrosine phosphorylation of STAT protein, and regulation of interleukin-1 production (Fig. 7d). Gene expression changes at the level of the single pathway of interest were analyzed by Gene Set Enrichment Analysis (GSEA). GSEA calculated an enrichment score (ES) that reflects the over-representation of a particular gene dataset at the top or bottom of the ranked list of genes found in both expression datasets. Genes are scored and tested for significance by empirical alignment, and then corrected for multiple hypotheses. The Padjust value of wnt signaling pathway was 0.031, clustering of its GSEA results enriched with up regulation with hydrogel treatment (Fig. 7e). These GSEA indicate variants in genes involved in inflammation. The Padjust value of NOD-like receptor signaling pathway was 0, clustering of its GSEA results enriched with gene up (Fig. 7f). NF-κB signaling pathway appeared in both KEGG of down-regulation dataset and KEGG of up-regulation dataset. The GSEA results of NF-κB signaling pathway showed a negative correlation, which meant up regulation related to NF-κB signaling pathway in datasets (Fig. 7g). New dataset would be established for these genes focused on pathways involved in positive and negative regulation. Heatmap showed a difference in gene expression between the PBS and GelMA/OSSA/PMB hydrogel groups partly (Fig. 7h). The expression of wound healing genes (Wnt4, Wnt5a, and Wnt3a) was upregulated after GelMA/OSSA/PMB hydrogel treatment, indicating the cured wound had recovered and had a regenerative potential [39]. The well-known proinflammatory genes-related genes (Tnfrsf11a, Tnfrsf1b, Tnf, Il6, and Tlr4) were also significantly downregulated (Fig. 7i). At same time, we use the dataset to implement protein-protein interaction (PPI) network analysis (Fig. 7j). The up-regulation dataset result not only confirmed the leading role of Wnt4 but also proved the important role played by the neighbouring proteins Wnt3a, Wnt5a, Rspo1 and Fzd5 in promoting epithelialization and regeneration of skin appendages. The down-regulation dataset analysis showed that not only the dominant role of Tnf was confirmed, but also the important role of the accessory proteins IL-1β, IL6 and Vcam1 in reducing inflammation was demonstrated. Down-regulation of these proteins indicated that the GelMA/OSSA/PMB hydrogel reduced inflammation in wounds [40]. Moreover, the downregulation of inflammation-associated Tnf signaling pathways created a directive microenvironment. Collectively, the diabetic infected wounds were effectively treated on day 10 and our treatment system demonstrated continuous delivery of GelMA/OSSA/PMB hydrogel with wound regeneration capability.
To further verify the transcriptional results, we collected wounds in all groups at 3 rd, 7 rd, 10 rd, and 14 rd days throughout progress to detect a key proteins of Tnf Detection of key protein. The ELISA kit to analyze the proinflammatory cytokines expression of TNF-α, IL-1β, and IL-6 was performed. Moderate inflammation can facilitate wound healing, but excessive inflammatory cell infiltration can damage normal tissue structures. As shown in Fig. 7. the PBS group, GelMA/OSA group, and GelMA/OSSA group showed severe inflammatory cell infiltration due to delayed healing during treatment in the whole process, but the wound of the GelMA/OSA/PMB and GelMA/OSSA/PMB groups contained few inflammatory cells and few bacterial on day10 (Fig. 6a). Inflammation during wound healing was assessed by ELISA kit in the different groups to analyze the expression of TNF-α (Fig. 7k)、IL-1β (Fig. 7l), and IL-6 (Fig. 7m) on days 3, 7, 10, and 14. All groups exhibited apparent acute inflammation on days 3 and 7, which was mainly caused by bacterial infection and the migration of inflammatory cells. The level of IL-1β, IL-6, and TNF-α expressed in GelMA/OSA/PMB and GelMA/OSSA/PMB hydrogel groups were lower than that of other groups. Therefore, the introduction of PMB could clear bacterial infection and reduce the expression of pro-inflammatory factors IL-1β, IL-6, and TNF-α. The study results were consistent with the available results that OSSA can reduce inflammatory cell infiltration and accelerate wound healing by downregulating the expression of IL-1β, IL-6, and TNF-α. The results showed that GelMA/OSSA/PMB exhibited anti-inflammatory properties which accelerated transition from inflammatory phase to proliferation phase. All the above results revealed that the GelMA/OSA/PMB or GelMA/OSSA/PMB demonstrated excellent performance in promoting wound healing. In diabetic wound, lipid peroxidation was remarkably increased [41]. Malondialdehyde (MDA) is a final product in lipid peroxidation, which establishes a relationship with lipid peroxidation. MDA increased in the inflammatory phase, which indicates tissue damage and wound repair delay. Compared to the PBS and other groups, MDA level was significantly decreased after the GelMA/OSSA/PMB or GelMA/OSA/PMB hydrogel was cured (Fig. 7n). In summary, the GelMA/OSSA/PMB or GelMA/OSA/PMB hydrogel could preclude oxidative stress in vivo, which was consistent with better wound healing. The contrast of inflammatory transformation on the 10rd day was very obvious. These results proved that the wounds cured with GelMA/OSSA/PMB hydrogels showed a positive effect on the outcome of inflammation.
Biosafety in vitro and in vivo
After 1, 3, and 5 days of co-culture with GelMA, GelMA/OSA, GelMA/OSSA, GelMA/OSA/PMB, and GelMA/OSSA/PMB (1mg/mL PMB) hydrogels, it was unmistakable that almost all NIH 3T3 cells were alive in transwell which indicated that the formulated hydrogels could promote cell proliferation. NIH 3T3 proliferation observed no apparent cytotoxicity. The GelMA/OSA/PMB, and GelMA/OSSA/PMB hydrogel samples exhibited similar cell viability compared to hydrogels without PMB. hydrogels exhibited good biocompatibility and non-cytotoxicity (Fig. 8a b). Moreover, hydrogels revealed a low hemolysis (less than 7%) ratio without any apparent hemolysis, indicating its excellent hemocompatibility (Fig. 8c).
In vivo safety evaluation was overseen by inspecting the main organs (heart, liver, spleen, lung, kidney) after each group hydrogel medicament. As shown in Fig. 8d, H&E exhibited no visible visceral injury, indicating that the hydrogels had no obvious
conceivable hostile reactions during utilization. Hence, the addition of PMB in GelMA/OSSA/PMB hydrogel was secure and scalable.