Baicalein based nano-delivery system restores mitochondrial homeostasis through PPAR signaling pathway to promote wound healing in diabetes | Journal of Nanobiotechnology

Characterization of the nanoparticles and hydrogel

Figure 1A shows the schematic illustration of the synthesis of BA/GOx@ZIF-8 nanoparticles and BGZ@GelMA nanocomposite hydrogels. The morphology of the sample was characterized by SEM (Fig. 1B). The results indicate that the representative SEM of ZIF-8 exhibits a regular dodecahedral structure, consistent with literature reports [36], with a diameter of approximately 150 nm. However, the diameter of ZIF-8 gradually increased to about 180 nm after loading BA and GOx (Fig. 1C). Compared with the homogeneous dodecahedral structure of ZIF-8, the surfaces of BA@ZIF-8 and BA/GOx@ZIF-8 are slightly rougher, and part of the morphology becomes spherical. The characteristic peaks of different nanoparticles were determined by FTIR spectroscopy. The absorption peak at 422 cm^− 1 is attributed to the Zn-N stretching vibrational peak of ZIF-8, which is a bond formed by the coordination of Zn²⁺ and 2- methylimidazole. ZIF-8 has characteristic peaks at 3135, 2926 and 1580 cm^− 1 due to stretching vibrations of aromatic C-H bonds, aliphatic CH bonds and C-N in 2-methylimidazole [37]. The new characteristic peak at 3415 cm^− 1 confirms the successful encapsulation of BA in ZIF-8 due to the presence of a large number of hydroxyl groups in BA. The methyl or methylene groups of GOx exhibit characteristic peaks at 2870 cm^− 1 and 2970 cm^− 1, respectively [38] (Fig. 1D). These results indicated that BA/GOx@ZIF-8 nanoparticles were successfully prepared. The crystal structure of nanocomposites was determined using XRD technology. The XRD showed that ZIF-8, BA@ZIF-8, and BA/GOx@ZIF-8 nanoparticles all had six similar sharp diffraction peaks. The diffraction peaks at 7.26, 10.26, 12.68, 14.6, 16.38 and 17.92° were corresponded to the (101), (102), (103), (006), and (110) planes, respectively [39], indicating that the loading of BA and GOx did not destroy the crystal structure integrity of ZIF-8(Fig. 1E). Due to the negative charge of BA and GOx containing phenolic hydroxyl and carboxyl groups, the positive charge of ZIF-8 is neutralized after drug loading modification, resulting in a significant decrease in its positivity rate (Fig. 1H).

The porous internal structure of hydrogel can simulate the natural ECM, and promote cell adhesion, migration, and ECM deposition, making it an important candidate for accelerating wound healing [40]. In addition, injectable hydrogels are more suitable for wounds with different shapes to meet clinical needs. We have developed injectable photo crosslinked nanocomposite hydrogel. Firstly, we prepared GelMA by reacting gelatin and MA, the chemical structures of gelatin and GelMA were analyzed by ¹H NMR. We found a large number of amino acids and peptides in both gelatin and GelMA from the complex ¹H NMR spectra. It is worth noting that the appearance of methyl (δ = 1.9ppm) and vinyl proton (δ = 5.4/5.7ppm) signals, as well as the decrease in lysine signal intensity (δ = 2.99ppm), demonstrate that gelatin has been successfully modified by MA to synthesize GelMA (Fig. 1F). Compared with sodium alginate and hyaluronic acid, GelMA retains the RGD sequence of gelatin, which can directly promote cell adhesion, proliferation, and differentiation without additional functionalization modifications; it can be specifically degraded by matrix metalloproteinases (MMPs); GelMA is easily composable with nanoparticles, which endows the hydrogel with a variety of functionalities; and its tunability meets the mechanical needs of different tissue repairs.

Subsequently, nanocomposite hydrogels were prepared by doping nanoparticles in GelMA (Z@Gel, BZ@Gel, and BGZ@GelMA). The injectability of the nanocomposite hydrogel was confirmed by extrusion experiments (Fig. 1I). After adding the photoinitiator LAP to the GelMA precursor solution, a jelly-like gel was formed by rapid cross-linking under 405 nm visible light irradiation. The microstructures of lyophilized nanocomposites hydrogels were observed by SEM. The microstructure of pure hydrogel and composite hydrogel doped with nanoparticles are basically the same, and both have 3D porous network structure, which provides the best environment for cell migration, gas exchange and nutrient transport (Fig. 1G).

Cytocompatibility evaluation of the nanocomposite hydrogels

Cytocompatibility is the basis for the in vivo application of nanocomposite hydrogels [11, 12]. We used L929 and HUVECs cells for cytocompatibility evaluation. The functional characteristics of HUVECs, such as surface receptors and signaling pathways, are highly similar to those of adult endothelial cells, and the experimental results have higher clinical reference value. Secondly, HUVECs are commonly used to assess cell migration (simulating wound closure), proliferation (tissue regeneration), and tubular structure formation (in vitro angiogenesis modeling), which can directly reflect the healing potential of the materials, and these characteristics make them ideal for studying the material or drug ideal cells for studying the ability of materials or drugs to promote angiogenesis. We evaluated the effects of hydrogel loaded with different concentrations of ZIF-8, BA@ZIF-8, and BA/GOx@ZIF-8 on the viability of HUVECs. The HUVECs were co-cultured with different concentrations of nanocomposite hydrogels for 24 h. The results showed that there was no effect on cell viability when the concentrations of ZIF-8 and BA@ZIF-8 reached 200 µg/mL, whereas when the concentration of BA/GOx@ZIF-8 reached more than 40 µg/mL caused a significant decrease in cell viability, which provided a concentration reference for our subsequent studies (Fig. S1).

We evaluated the effects of different nanocomposite hydrogels on the proliferation of HUVECs and L929 cells in a high-glucose inflammatory environment using CCK-8 assays. The results (Fig. 2A-B) showed that the viability of the two cells was significantly reduced under the high glucose inflammatory environment. When different nanocomposite hydrogels were added, all the cells showed sustained proliferation with the prolongation of the incubation time, suggesting that these nanocomposite hydrogels might be beneficial to the proliferation of the cells. We further selected L929 and HUVECs cells cultured for 3 days for live-dead staining, and the results showed that under high glucose inflammatory conditions, the number of both types of cells significantly decreased, while the cell viability was good after co-culture with Z@GelMA, BZ@GelMA and BGZ@GelMA hydrogel, and the number of cells in the BGZ@GelMA group significantly increased. The L929 and HUVECs cells are alive (green) in different nanocomposite hydrogels, and there are almost no dead cells (red) (Fig. 2C). Cell flow was further confirmed the apoptosis rate of HUVECs cells increases significantly after being induced with HL, which could be alleviated by treating with Z@GelMA, BZ@GelMA, and BGZ@GelMA hydrogels. In comparison, BGZ@GelMA hydrogels have the most pronounced effect (Fig. S2).

The newly replicated DNA was labeled with EDU to accurately determine cell proliferation. We used EDU staining to evaluate the effect of hydrogel on HUVECs proliferation. The results showed that the BGZ@GelMA group had the highest percentage of EDU-positive cells, which could significantly promote the proliferation of HUVECs (Fig. 2D). However, there were very few newly proliferating cells in the HL groups compared to the control group. The percentage of EDU-labeled cells indicated a consistent trend (Fig. 2E). Furthermore, reduced expression of Ki-67 was observed in HL-induced HUVECs, which was reversed by treatment with the BGZ@GelMA hydrogel (Fig. S3). Collectively, these findings suggested that BGZ@GelMA hydrogel improves the high-glycemic inflammatory microenvironment, provides a suitable environment for cell survival, and exhibits good cytocompatibility.

In addition, based on the results of the hemolysis test, we observed that the different nanocomposite hydrogels did not cause a significant hemolytic reaction, with a hemolysis rate of around 0.7% (Fig. S4).

Antibacterial activities of the nanocomposite hydrogels

The high-sugar environment provides sufficient nutrients for bacterial growth and accelerates bacterial proliferation, so continued bacterial infection is also an important factor in the difficulty of healing diabetic wounds [15]. S. aureus is widely present on the surface of the skin and is one of the most common pathogens causing skin infections. In addition, the proportion of E. coli causing wound infections is gradually increasing. We used a bacterial colony counting test to determine whether the BGZ@GelMA hydrogel could exert an antimicrobial effect. As shown in Fig. 3A, photographs of agar plates show a significant reduction in the number of colonies after S. aureus and E. coli co-culture with different hydrogels. The inhibition of S. aureus and E. coli co-cultured with BGZ@GelMA hydrogel was close to 100% (S. aureus was 95.3% and E. coli was 97.6%), indicating that this hydrogel has a wide range of antimicrobial properties. In contrast, the control group showed almost no antimicrobial effect, whereas Z@GelMA and BZ@GelMA exhibited moderate antimicrobial activity (Fig. 3B-C). We also analyzed the bacterial viability, and the results are shown that the control group exhibited limited inhibitory effects, while the bacterial viability of S. aureus and E. coli in the BGZ@GelMA group decreased significantly (Fig. S5). Similarly, the live/dead bacterial staining results indicated that virtually the majority bacteria remained viable in the control group (in green). The Z@GelMA group exhibited a partial antimicrobial effect due to Zn²⁺ activity. In contrast, the BZ@GelMA group exhibited significantly enhanced antibacterial activity due to the addition of BA. Notably, BGZ@GelMA hydrogel induced extensive bacterial death when co-cultured with S. aureus and E. coli (in red) (Fig. 3E). The same trend was observed for the quantitative ratios corresponding to live and dead bacteria (Fig. S6A-B). Finally, the microstructure of the bacterial membrane after different hydrogel treatments was examined by SEM to explore the antibacterial mechanism of the hydrogel. SEM images showed that S. aureus and E. coli in the control group were regular-shaped spherical and rod-shaped, respectively, with intact and smooth bacterial membrane structures. After co-incubation with BGZ@GelMA hydrogel bacteria, the physiological structure of the S. aureus and E. coli was disrupted, exhibiting varying degrees of contraction and rupture with leakage of contents, indicating a compromised cellular state. We used pseudo-colors to highlight the obviously damaged bacteria (Fig. 3D).

To explore the antibiofilm activity of BGZ@GelMA hydrogel in vitro, crystal violet staining assay and biomass quantitation were performed, and the biofilm in the group treated with BGZ@GelMA hydrogel remained barely viable, validating the significant antibiofilm activity (Fig. S7).

The flow cytometry results showed that after 4 h of intervention with different hydrogels BGZ@GelMA, the positive rates of PI uptake in S. aureus and E. coli were 33.8% and 33.6%, respectively, indicating that Zn²⁺ and BA can damage bacterial cell membranes (Fig. S8). Bacterial plate counting experiment confirm that ZIF-8@BA composite materials have stronger antibacterial ability than ZIF-8 or BA alone, Zn²⁺and BA can synergistically exert antibacterial effects (Fig. S9).

In summary, the antimicrobial mechanism of BGZ@GelMA may be attributed to the following reasons: (1) The sustained release of Zn²⁺ ions disrupts the cell membrane of the bacteria [41]. (2) BA affects bacterial membrane permeability and influences bacterial biosynthesis and metabolism by inhibiting enzyme activity in the bacterium, thus realizing antibacterial effects [42]. (3) Natural polyphenols rich in phenolic hydroxyl groups can chelate metal ions and immobilize them on a substrate, enhancing the antimicrobial efficacy of the material [43]. These results demonstrated the potential of BGZ@GelMA hydrogel to prevent bacterial infection.

Protecting cell and mitochondrial function, scavenge ROS in vitro

Mitochondria are the primary site of cellular energy production and aerobic respiration, and the main source of intracellular ROS. In a hyperglycemic environment, ROS produced by mitochondria in endothelial cells accumulates and leads to the release of inflammatory factors, excessive oxidative stress causes mitochondrial dysfunction and affects cellular energy metabolism, which makes diabetic wounds difficult to heal [44, 45]. To evaluate the effects of different hydrogel treatments on intracellular ROS levels, we used DCFH-DA and Mito-SOX probes to quantify fluorescence intensity targeting ROS in the cytoplasm and mitochondria, and we used Mito-Tracker to specifically label intracellular mitochondria. After a 24 h incubation period, the fluorescence signal in Fig. 4A shows that the persistent hyperglycemic inflammatory microenvironment (HL group) stimulates the cells to produce excessive ROS. In contrast, the fluorescence intensity of all hydrogel groups decreased significantly, and that in BGZ@GelMA group was the lowest (Fig. 4D). Similarly, as shown in Fig. 4B, the persistent hyperglycemic environment strongly induced the accumulation of mtROS, whereas Z@GelMA and BZ@GelMA hydrogels significantly mitigated, the lowest red fluorescence was observed in the BGZ@GelMA group, suggesting good mtROS elimination, which was supported by the corresponding quantification of fluorescence intensity (Fig. 4E). Accumulation of ROS in mitochondria contributes to the abnormal opening of the mitochondrial permeability transition pore (mPTP), leading to disruption of the electrochemical potential (ΔΨm) across the inner mitochondrial membrane [46]. This disruption leads to mitochondrial matrix osmotic imbalance and disturbances in calcium metabolism. These changes ultimately lead to altered mitochondrial morphology and impaired function, affecting cellular energy metabolism and ultimately apoptosis. Under normal conditions, the mitochondrial membrane potential is high and the JC-1 dye forms aggregates and emits red fluorescence. In contrast, when the membrane potential is abnormally low, the dye forms monomers and emits green fluorescence, indicating a change in mitochondrial membrane permeability. In Fig. 4C, we observed that the red fluorescent signals were significantly weakened or even disappeared in the HL group, indicating that the mitochondrial membrane potential was significantly reduced in the hyperglycemia inflammatory environment. The addition of Z@GelMA and BZ@GelMA slightly enhanced the red fluorescent signals intensity, however, The BGZ@GelMA group showed the most significant increase in fluorescence signal, indicating that BGZ@GelMA hydrogel can reduce the depolarization of mitochondrial membrane potential in hyperglycemia environment, and maintain the polarization of mitochondrial membrane potential at a normal level. The JC-aggregates/monomer fluorescence ratio also confirmed the same results (Fig. 4F). The above research results confirm that even in a sustained hyperglycemic inflammatory microenvironment, BGZ@GelMA nanocomposite hydrogel can also eliminate mtROS, protect mitochondrial function, and maintain cell energy metabolism.

Promoting cell migration and tube formation

Hyperglycemia-induced angiogenic dysfunction is one of the key factors contributing to the difficulty in healing diabetic wounds. Considering the key role of fibroblasts and endothelial cells in wound repair, the effect of BGZ@GelMA hydrogels on cell migration was evaluated. The Transwells assay results showed that the HL environment significantly inhibited the migration of L929 and HUVECs., HUVECs and L929 cells co-cultured with hydrogel extracts revealed a significantly faster migratory rate at 24 h compared with the HL group (Fig. 5A, S10A). Notably, the BGZ@GelMA group showed a superior ability to promote cell migration (Fig. 5D). The scratch assay is another experimental method for evaluating cell migration. As shown in Fig. 5B, S10B, in the HL environment, HUVECs and L929 cells treated with hydrogel extracts migrated rapidly and the scratch area was significantly reduced after 24 h of culture. The migration rate of HUVECs cultured with Z@GelMA, BZ@GelMA and BGZ@GelMA extracts were 36.21%, 42.17%, and 52.19%, respectively, which was higher than 30% of the HL group (Fig. 5E). The results showed that the nanocomposite hydrogels significantly promoted the cell migration of L929 and HUVECs, while Zn²⁺, BA and GOx all acted synergistically.

Endothelial cells are the main cells involved in the process of neovascularization during wound healing. Therefore, we analyzed the tube formation of HUVECs on the BGZ@GelMA hydrogels. The observed photographs in Fig. 5C showed that HUVEC in the BG@GelMA and BGZ@GelMA groups showed an increase in cellular lattices and nodes, and the cells became denser and were able to form good tubular and mesh structures under a hyperglycemic inflammatory environment, which were hardly visible in the HL group. Quantitative analysis showed significant improvement in the number of tube junctions in the BG@GelMA and BGZ@GelMA groups (Fig. 5F).

During wound healing, angiogenesis is closely related to growth factors such as VEGF and CD31 secreted by vascular endothelial cells [47]. It has been shown that Zn²⁺ and BA can promote angiogenesis by upregulating the expression of VEGF [48, 49]. The expression levels of VEGF in HUVECs were evaluated by different methods after treating with different hydrogels for 24 h in a high-glucose inflammatory environment. Immunofluorescence imaging revealed significantly elevated VEGF levels in the BZ@GelMA group and BGZ@GelMA group compared to the HL group (Fig. 6A). Quantitative analysis shows the higher relative fluorescence intensity of the BGZ@GelMA group (Fig. 6D). CD31 immunofluorescence staining and quantitative analysis also showed the same trends (Fig. S11). WB analysis further confirmed that VEGF expression was significantly upregulated in the BGZ@GelMA group (Fig. 6B-C). These results indicate that the BGZ@GelMA hydrogel substantially boosts angiogenic factor expression and fosters neovascularization, potentially aiding in vascular regeneration and endothelial repair in chronic wounds. The above results indicated that BGZ@GelMA hydrogel is expected to accelerate diabetic wound healing by promoting the proliferation and migration of endothelial cells, up-regulating the expression of VEGF and CD31 to promote blood vessel formation.

AGEs are also the key factors leading to delayed healing in diabetes. The AGEs activate downstream signaling pathways (e.g., NF-κB) by binding to cell-surface receptors (RAGEs), resulting in increased secretion of the cytokines IL-6, IL-1β, and TNF-α secretion to increase, triggering inflammatory responses and oxidative stress [50]. In addition, AGEs inhibit endothelial cell proliferation and migration, leading to delayed diabetic wound healing [51]. Therefore, anti-glycosylation may help to improve the diabetic wound environment. In this study, we further used AGEs to interfere with HUVECs, to detect the expression of inflammatory factors, cell proliferation and migration and to observe the anti-AGEs effect of different hydrogels. The experimental results indicate that the expression of inflammatory factors was significantly increased under AGEs stimulation, and cell migration was inhibited, the Z@GelMA, BZ@GelMA and BGZ@GelMA hydrogel extract can reverse this phenomenon, and BGZ@GelMA hydrogel can significantly reduce the expression of inflammatory factors (Fig. S12A-C) and promote cell migration (Fig. S12D-G).

Promoting wound healing mechanism in vitro

To further explore the regulatory mechanism of the BGZ@GelMA hydrogel regulating HUVECs. We determined the mRNA profile of HUVECs treated with BGZ@GelMA using RNA-Seq analysis. As shown in Fig. 7A, principal component analysis (PCA) shows that the samples in each group are independently clustered. The volcano map in Fig. 7B shows that there are 235 differentially expressed genes (DEGs), including 70 upregulated genes and 165 downregulated genes between the two groups. We conducted Gene Ontology (GO) enrichment analysis, including categories of biological processes (BP), molecular functions (MF), and cellular components (CC) to analyze these DEGs. It focuses on epidermal tissue development, keratinocyte differentiation, endothelial cell proliferation, immune defense response, and metabolism-related enzymes, which are closely related to the whole process of wound healing (Fig. 7C). The Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis was then applied to analyze potential signaling pathways, and furthermore showed changes in the expression of specific genes in a complex clustering diagram (Fig. 7D). The KEGG enrichment analysis shows BGZ@GelMA regulated the peroxisome proliferator-activated receptor (PPAR) signaling pathway and some metabolic pathways in endothelial cells. The expression of specific genes was shown in the clustered string plot, such as COL7A1 and COL4A4, involved in collagen formation, while ACADL, BCKDHA, and HMGCS1 were involved in mitochondrial metabolism. According to reports, the PPAR family and signaling pathways are involved in multiple stages of skin repair after injury [52]. PPAR consists of three subtypes, PPAR -αmainly participates in the oxidative metabolism of fatty acids, provides sufficient energy for endothelial cells, and participates in early inflammatory repair of the skin [53]. PPARβ/δ is primarily involved in cell growth and differentiation and regulates mitochondrial function and metabolism and promotes proliferation and adhesion of keratinocytes [54]. PPARγ is widely involved in lipid metabolism and plays an important role in inflammatory response, immune regulation, cell proliferation, and survival [35]. PPARγ is an upstream signaling molecule of NF-κB. Activated PPARγ can inhibit the activation of NF-κB transcription factors, thus suppressing the inflammatory response [55, 56]. These results suggest that BGZ@Gel hydrogel may be involved in the healing process of diabetic wounds by promoting keratinized fiber formation, endothelial cell proliferation, mitochondrial metabolism, activation of the PPAR pathway, and immunomodulation. BA may affect metabolism and inflammatory response by directly or indirectly regulating the expression and activity of PPARs (PPARα, PPARγ). Some studies have shown that baicalein is able to inhibit mitochondrial function and kinetic changes, exert mitochondrial protective and antioxidant effects, and inhibit apoptosis through the upregulation of PPARγ coactivator 1α, Nrf2, and related redox signaling pathways [57]. Baicalein exerts beneficial effects on macrophage lipid accumulation and inflammatory responses through activation of the PPARγ/liver X receptor α (LXRα) signaling pathway [58].

Diabetic wounds healing in vivo

Combined with BGZ@GelMA nanocomposite hydrogel has good antibacterial, pro-angiogenic, and mitochondrial homeostasis restoration effects in vitro. We further verified the in vivo therapeutic effect through a diabetic rat wound model. Diabetic SD rats were equally divided into three groups, blank control group without any treatment, GelMA hydrogel dressing, and BGZ@GelMA hydrogel dressing. The schematic illustration is shown in Fig. 8A. In all groups, the BGZ@GelMA hydrogel represents a faster wound healing process, in which, on the 21st day, the BGZ@GelMA group observed good wound healing ability and a wound healing rate of 98.5%, which was superior to other groups (Fig. 8B-D). After that, we performed a comprehensive histological analysis to assess the change in wound length, wound healing rate, and skin morphology. H&E staining analysis showed that in the BGZ@GelMA hydrogel group, the inflammatory cell infiltration level was significantly reduced on the 7th day, and after 21 days of BGZ@GelMA hydrogel treatment, epithelial metaplasia has typical skin layer structure and morphological feature (Fig. 8E). In addition, we have found that after 7 days of BGZ@GelMA hydrogel treatment, the length of the wound gape was significantly reduced compared to the other groups (Fig. 8F). Subsequently, Masson staining was used to evaluate the collagen deposition in each group. After 21 days of BGZ@GelMA hydrogel treatment, a large amount of well-structured collagen and skin appendages were observed in the subepidermal tissue of the wound compared to the GelMA hydrogel group and the control group. (Figures 8G and 9A). We found that the hydrogel-only group also had some collagen fiber deposition, which can be attributed to the fact that GelMA hydrogels contain the common Arg-Gly-Asp (RGD) fraction, a tripeptide that promotes cell proliferation, differentiation, and adhesion [59]. The results indicated that BGZ@GelMA hydrogel significantly promoted the formation and maturation of granulation tissue and accelerated collagen deposition in diabetic wounds.

Due to the complex physiological process of wound healing, it can be roughly divided into three stages: inflammation, proliferation, and recovery. These three stages appear to be independent yet interconnected. To explore the therapeutic effects of BGZ@GelMA hydrogel in vivo in the inflammatory stage, we evaluated the expression of pro-inflammatory cytokines at the wound site after 7 days of treatment using immunohistochemistry, as shown in Fig. 9B, the expression level of IL-6 and TNF-α was highest in the DM group on day 7, indicating a severe inflammatory response. Compared with the DM group and GelMA group, the levels of TNF-αand IL-6 in the BGZ@GelMA hydrogel group were significantly reduced (Fig. 9C-D). This indicates that the BGZ@GelMA sustained release of BA alleviates local inflammatory reactions and reduces the expression of pro-inflammatory cytokines. These results suggest that, the BGZ@GelMA hydrogel treatment can significantly alleviate the inflammatory reaction of chronic diabetes wounds.

Newly formed blood vessels actively contribute to the wound repair process by supplying nutrients and oxygen to growing tissues. VEGF can accelerate wound healing by stimulating angiogenesis, and vascular markers CD31 was performed to assess neovascularization. however, local VEGF production is often limited in diabetic wounds, resulting in delayed wound healing. Therefore, we evaluated angiogenesis in each group of wounds during the proliferation stage. On the 14st day, we performed immunohistochemical staining on VEGF and CD31 in the treated wound tissue, as shown in the Fig. 9E, the levels of VEGF and CD31 in the BGZ@GelMA hydrogel group were significantly higher than those in the blank control group and GelMA group, and a large number of new blood vessels can be seen at the wound. Quantitative analysis shows the same trend (Fig. 9F-G). This may be due to the adhesive of hydrogel and the synergistic effect of Zn²⁺ and BA to promote the proliferation and migration of endothelial cells, create a microenvironment conducive to angiogenesis, and thus induce angiogenesis. H&E staining showed no obvious damage to the heart, liver, spleen, lung, kidney and other major organs (Fig. S13). These results confirmed that BGZ@GelMA hydrogel has good biological safety and can accelerate the growth of diabetes wounds.

So far, despite our success, our research still has some limitations. First of all, in terms of the synthesis of nanomaterials, we lacked in-depth research on different material concentrations, and did not systematically compare the injectable hydrogel concentration, crosslinking time and light intensity; Secondly, we designed to use GOx to reduce local blood glucose without considering its impact on systemic blood glucose. These provide direction and ideas for our future research, and are also worth further exploration in the future.