Spermidine‐Functionalized Injectable Hydrogel Reduces Inflammation and Enhances Healing of Acute and Diabetic Wounds In Situ

Abstract The inflammatory response is a key factor affecting tissue regeneration. Inspired by the immunomodulatory role of spermidine, an injectable double network hydrogel functionalized with spermidine (DN‐SPD) is developed, where the first and second networks are formed by dynamic imine bonds and non‐dynamic photo‐crosslinked bonds respectively. The single network hydrogel before photo‐crosslinking exhibits excellent injectability and thus can be printed and photo‐crosslinked in situ to form double network hydrogels. DN‐SPD hydrogel has demonstrated desirable mechanical properties and tissue adhesion. More importantly, an “operando” comparison of hydrogels loaded with spermidine or diethylenetriamine (DETA), a sham molecule resembling spermidine, has shown similar physical properties, but quite different biological functions. Specifically, the outcomes of 3 sets of in vivo animal experiments demonstrate that DN‐SPD hydrogel can not only reduce inflammation caused by implanted exogenous biomaterials and reactive oxygen species but also promote the polarization of macrophages toward regenerative M2 phenotype, in comparison with DN‐DETA hydrogel. Moreover, the immunoregulation by spermidine can also translate into faster and more natural healing of both acute wounds and diabetic wounds. Hence, the local administration of spermidine affords a simple but elegant approach to attenuate foreign body reactions induced by exogenous biomaterials to treat chronic refractory wounds.


Fabrication of DN, DN-SPD and DN-DETA hydrogels
The hydrogels were obtained by mixing the precursor solution with 4aPEG-BA solutions at room temperature.Briefly, a 12 wt% GMA solution was prepared by dissolving GMA in phosphate-buffered saline (PBS) containing photoinitiator (LAP, 0.5% w/v) at 60 ℃ and a 3 wt% OCMCS solution was prepared by dissolving OCMCS in PBS at room temperature.Then GMA and OCMCS solutions were mixed at 1:1 ratio to obtain the precursor solution.The 4aPEG-BA solution was prepared by dissolving 0.2 g of the polymer in 1.0 mL PBS.The OCMCS/GMA precursor solution was mixed with 4aPEG-BA solution to form the single network hydrogel (SN) based on Schiff base reaction.Then the SN hydrogel was irradiated with blue light (405 nm, 25 mW/cm 2 ) for 30 s to form the second network by photo-crosslinking GMA.The double network hydrogel without SPD or DETA was named as DN hydrogel.The hydrogels containing different concentrations of SPD or DETA (denoted as DN-SPD or DN-DETA respectively) were prepared by similar protocol except that different volumes of SPD or DETA were introduced into the OCMCS/GMA precursor solution.The SN hydrogels with SPD or DETA before photo-crosslinking were designated as SN-SPD and SN-DETA respectively.

Morphological and chemical characterizations
Scanning electron microscopy (SEM) images were obtained on a XL-30 ESEM FEG FEI COMPANYTM electron microscope (USA).The pore sizes of hydrogels were measured by ImageJ software.Fourier transform infrared (FT-IR) spectra were recorded on a Shimadzu RF-5301PC spectrometer in the transmission mode using KBr pellets of the samples.

Mechanical tests
The mechanical properties of hydrogels were measured by compression tests.
Briefly, hydrogels were prepared as cylinders (10 mm in diameter and 7 mm in height) and compressed at a speed of 0.01 mm• s −1 to a maximum strain of 70% with a universal test machine (Instron 1121, Instron, USA).The compressive moduli were calculated from the slopes of the linear region (0-10%) in stress-strain curves as previously described [1] .

Swelling ratio
For the analysis of swelling property, hydrogels were immersed in PBS (pH = 7.4)     for 24 h (or as otherwise indicated) at 37 ℃, and the weights of swollen hydrogels (Wt) were recorded after removing residual PBS with a filter paper.Subsequently, the swollen hydrogels were lyophilized and weighted to obtain W0.Swelling ratio was calculated according to Eq. (S1): where Wt and W0 represent the weight of swollen hydrogels at each timepoint and the weight of lyophilized hydrogels, respectively.

Self-healing and injectability assay
The rheological properties of SN-SPD hydrogel were characterized on an Anton Paar MCR302 rheometer in an oscillatory mode.Samples were placed in a plate with a diameter of 25 mm and strain sweep tests were carried out at a fixed frequency of 1 rad• s −1 within a strain range of 1% to 500%.Subsequently, continuous oscillatory stepstrain experiments were conducted at a constant frequency (1 rad• s −1 ) with alternating cycles between 1% and 500% strain to examine the self-healing property of the hydrogel.To further illustrate the self-healing ability of the hydrogel, two star-shaped hydrogels were made in two colors: one is blue and the other is yellow.The two stars were cut into halves and swapped.The halves of different colors were placed next to each other without any external force and incubated at 37 °C.To demonstrate the injectability, the SN-SPD hydrogel was injected through a syringe equipped with a 22G needle to print the letters of "MMB".

Tissue adhesion assays
The tissue adhesion of DN-SPD hydrogel was studied on a pig skin purchased from a local market.First, for qualitative assay, SN-SPD hydrogel was injected onto the pig skin surface and then irradiated with blue light (405 nm, 25 mW/cm 2 ) for 30 s to form DN-SPD hydrogel in situ.The adhesion ability of the DN-SPD hydrogel was evaluated by macroscopic observation of detachment under torsion.Second, for semiquantitative assay, SN-SPD hydrogel was injected onto one end of the pig skin, covered with a glass slide, and then irradiated with blue light (405 nm, 25 mW/cm 2 ) for 30 s through the glass slide to form DN-SPD hydrogel between the pig skin and the glass slide.Different weights were loaded onto the other end of the pig skin until significant detachment occurred, in order to evaluate the tissue adhesion strength.

Chemical antioxidant assays
First, the antioxidant capacity of hydrogels was evaluated by DPPH radical scavenging assay [2] .Briefly, DPPH solution was prepared at a concentration of 100 µM in anhydrous ethanol.Then, 2 mL DPPH solution was incubated with 150 µL hydrogels in dark at 37 ℃.DPPH ethanol solution alone was used as the control.After incubation of different time, the absorbance of DPPH solution was measured by a UV-Vis spectrometer at 517 nm.The scavenging efficiency of DPPH radical was calculated by the following Eq.(S2): where Ab and As represent the absorbances of the DPPH alone and the DPPH solution after incubation with hydrogels, respectively.
Second, the • OH scavenging ability was investigated for free SPD, free DETA and different hydrogels.• OH was generated through Fenton reactions according to a previous report with minor modifications [3] .Briefly, for free SPD and DETA, 100 μL SPD or DETA solutions at different concentrations were mixed with FeSO4 solution (1 mM, 500 μL) and H2O2 solution (100 mM, 500 μL), and then incubated at 37 °C for 1 h.For hydrogels, 150 μL hydrogels were incubated with the mixture of FeSO4 solution (1 mM, 500 μL) and H2O2 solution (100 mM, 500 μL) at 37 °C for 1 h. 100 µL supernatant was collected after cooling to room temperature and mixed with TMB (10 mM, 100 µL).After 10 min, the concentration of • OH was measured by the absorbance at the wavelength of 650 nm using a microplate reader.The scavenging efficiency of • OH was calculated according to Eq. ( S3): where Ab and As represent the absorbances of the blank control and the solution incubated with free SPD, free DETA or different hydrogels, respectively.
Third, the H2O2 scavenging efficiency of hydrogels was characterized as previously described [4] .Briefly, 150 µL hydrogels were incubated with 500 µL H2O2 solution (1 mM) at 37 °C.PBS was added to 500 µL H2O2 solution (1 mM) as the control.After incubation of different time, 20 µL supernatant was collected and mixed with 100 µL Ti(SO4)2 solution (5 mM in 3 M H2SO4).After 30 min, the concentration of H2O2 was determined by the absorbance at the wavelength of 405 nm using a microplate reader.The scavenging efficiency of H2O2 was calculated according to Eq. (S4): where Ab and As represent the absorbances of the PBS control and the solution incubated with different hydrogels, respectively.

Degradation assay in vitro
The initial weights (Wi) of freeze-dried hydrogels were measured.Then the freezedried hydrogels were immersed in PBS and incubated at 37 º C. At different timepoints, the remaining weights of hydrogels (Wt) were recorded.The remaining weight percentages of hydrogels were calculated according to Eq. (S5): where Wt and Wi represent the weight of the remaining hydrogel at each timepoint and the initial weight of the hydrogel, respectively.

Cell viability and proliferation assays
For cell viability assay, L929 and RAW 264.7 cells were seeded in the lower chambers of 24-well transwell plates with 0.4 μm pore size (Corning, USA) at a density of 5×10 4 cells/well and incubated overnight.Subsequently, 150 μL hydrogels were added to the upper chambers of transwell plates.The well without hydrogel was used as the blank control.After incubation at 37 °C for 24 h, the upper chambers were removed.Cells were counted with CCK-8 kits by following manufacturer's protocol and the absorbance at the wavelength of 450 nm was recorded on a microplate reader (MOLECULAR DEVICES, SpectraMax Absorbance Reader, CMax Plus, BK-200 L96C).
The proliferation of L929 cells with hydrogels was examined by two methods.For the method of coculturing in transwell plates, L929 cells were seeded in the lower chambers of 24-well transwell plates with 0.4 μm pore size (Corning, USA) at a density of 5×10 4 cells/well.150 μL hydrogels were added to the upper chambers of transwell plates.The well without the upper chamber was used as the blank control.After incubation at 37 °C for 24 h and 72 h, the upper chambers were removed.Cells were washed with PBS twice and then incubated with Calcein-AM/PI solution at 37 ℃ for 15 min.Cells were observed by a fluorescence microscope (Olympus IX53, Japan).
For the method of coculturing on the surface of hydrogels, the hydrogels (height ~ 1.5 mm) were prepared in 24-well plates under sterile condition and L929 cells were seeded on the surface of hydrogels at a density of 5 × 10 4 cells/well.After incubation of indicated periods, CCK-8 assay was carried out by following manufacturer's protocol and the absorbance at the wavelength of 450 nm was recorded.

Hemolysis assay
The human blood used in the hemolysis assay was collected from the corresponding author, Dr. Zhenning Liu.Hemocompatibility of hydrogels was evaluated by hemolysis assay as previously described [5] .Briefly, red blood cells (RBCs) were obtained by centrifuging fresh human blood at 2000 rpm for 10 min and washed with 0.9% NaCl solution five times.Purified RBCs were resuspended in 0.9% NaCl solution at a rough concentration of 5% (v/v).Then, 200 µL RBC suspension was mixed with 100 µL homogenized hydrogel liquid and 700 µL 0.9% NaCl solution in 1.5 mL tube.After incubation at 37 °C for 1 h, the mixtures were centrifuged at 2000 rpm for 10 min and the absorbance of the supernatant was measured at the wavelength of 540 nm using a microplate reader.Triton X-100 (0.1%) and 0.9% NaCl solution served as the positive and negative controls, respectively.The tests were done in triplicates for each sample.The hemolytic ratio (HR) was calculated according to Eq. (S6): where ODs, ODp, and ODn represent the absorbances of sample, positive control and negative control, respectively.

Scratch healing assay in vitro
The scratch healing assay was performed in 6-well transwell plates.L929 cells were seeded in the lower chambers and allowed to form a confluent monolayer.Cells were scratched with a sterilized 200 L pipette tip to form one linear wound and washed with PBS to remove cell debris.Then fresh culture medium without serum was added, and cells were incubated with or without hydrogels in the upper chamber.The wound sizes at 0, 12, 24 and 48 h were photographed by a microscope (IX53, Olympus, Japan).

Biological antioxidant assays
Dihydroethidium (DHE) and 2',7'-dichlorofluorescin diacetate (DCFH-DA) were used to evaluate the levels of intracellular superoxide anion and reactive oxygen species (ROS), respectively [6] .Briefly, L929 cells were seeded in the lower chambers of 24well transwell plates at a density of 5 × 10 4 cells/well and incubated at 37 °C for (overnight).The confluent L929 cells were pretreated with hydrogels in upper chambers for 12 h, while the blank control was cultured without the upper chamber.
For mouse tissues, wound tissues were weighed and added to homogenization buffer, followed by mechanical homogenization in ice-water bath to prepare suspensions at a rough concentration of 10% (w/v).The suspensions were centrifuged at 2500 rpm for 10 min at 4 °C to collect the supernatants.The concentrations of TNFα, IL-10, IL-6, interleukin 1β (IL-1β), and transforming growth factor-β (TGF-β) in the supernatants were measured with respective ELISA kits (Invitrogen, USA).
ELISAs were performed according to the manufacturers' protocols.for 60 min at room temperature.After washing with TBST buffer for three times, the PVDF membranes were visualized with a Chemiluminescence Imaging System (ChemiScope 6000, CLINX, China).

Subcutaneous implantation in rats
All animals were housed and treated in accordance with protocols approved by the Jilin GENET-MED Biotechnology (Approval No: IACUC-2022-001.The original approval document in Chinese is available from the authors).Before surgery, male SD rats (200-220 g, 8 weeks, n = 16) were anesthetized by intraperitoneal injection of 2% tribromoethanol (10 mL/kg).Then the dorsa of rats were shaved and sterilized with 75% ethanol.Four linear incisional skin wounds about 10 mm were generated using surgical scissors, and then cylindrical DN, DN-SPD and DN-DETA hydrogels (diameter ≈ 10 mm, height ≈ 3 mm) were embedded inside three wounds on the same rat, whereas the fourth wound without any hydrogel was used as the sham control.Each kind of hydrogels was implanted into twelve animals.After implantation, the incisions were closed by surgical sutures.Three rats were sacrificed on postsurgical Day 3, 7, 14 and     21 (n = 12 = 3 × 4).The remaining hydrogels together with surrounding tissues were photographed and collected for future experiments.In addition, another 3 rats were implanted with 4 hydrogels (diameter ≈ 10mm, height ≈ 3mm) of the same kind to examine the toxicity on various organs.These 3 rats represent DN, DN-SPD, and DN-DETA hydrogels, whereas the last rat that was cut to make four incisions but sutured without implanted hydrogels served as the sham control.These 4 rats were sacrificed on postsurgical Day 21, and their heart, liver, spleen, lung and kidney were harvested and fixed in 4% PFA for hematoxylin and eosin (H&E) staining.

Acute wound healing in normal mice
The acute wound healing experiments were carried out on male C57BL/6 mice (20-25 g, 6-8 weeks) with full-thickness skin defects.Briefly, all mice (n = 27) were anesthetized by intraperitoneal injection of 2% tribromoethanol (0.02 mL/g).Then the dorsa were shaved and sterilized with 75% ethanol.Two full-thickness cutaneous wounds were generated with a circular skin biopsy puncher (8 mm in diameter).
Subsequently, the wounds were treated with commercial product (Duo DERM), DN, DN-SPD 100, DN-SPD 250, or DN-DETA 250 hydrogels, whereas the wound treated with PBS was used as the blank control.Each treatment (including PBS) was done on 9 wounds.Then the wounds were covered with 3M Tegaderm Transparent Film (1624W, USA), which were removed after two days.On postsurgical Day 0, 3, 7 and 12, the wounds were photographed and the wound areas were measured by ImageJ software.Wound closure rates were calculated according to Eq. (S7): where A0 and At represent the wound areas on Day 0 and Day t, respectively.

Diabetic wound healing in situ
The in situ diabetic wound healing experiments were carried out on male db/db mice (35-40 g, 6-8 weeks) with full-thickness skin defects.Briefly, all mice (n = 24) were anesthetized by intraperitoneal injection of 2% tribromoethanol (0.02 mL/g).Then the dorsa were shaved and sterilized with 75% ethanol.Two full-thickness cutaneous wounds were generated with a circular skin biopsy puncher (8 mm in diameter).
Subsequently, SN, SN-SPD or SN-DETA hydrogels were injected onto the wounds and irradiated with blue light (405 nm, 25 mW/cm 2 ) to form in situ DN, DN-SPD or DN-DETA hydrogels, respectively.The wound treated with PBS and irradiated with blue light was used as the blank control.Each treatment (including PBS) was done on 12 wounds.On postsurgical Day 0, 3, 7, 10, 14, 18 and 21, the wounds were photographed and the wound areas were measured by ImageJ software.Wound closure rates were also calculated according to Eq. (S7).Six mice were sacrificed on postsurgical Day 3, 7, 14, and 21 (n = 24 = 6 × 4), which contained 12 wounds for 4 treatments in triplicates.
For the animal experiments in this work, including the subcutaneous implantation in rats, acute wound healing in normal mice and diabetic wound healing in db/db mice, the tissues collected around the implanted hydrogels or the wounds were divided for various assays.One portion of a harvested tissue was fixed in 4% PFA for 24 h, dehydrated and embedded in paraffin.The paraffin-embedded samples were sliced into 5-μm thick sections for histological analyses, including H&E staining, Masson's trichrome staining, immunohistochemistry (IHC) and immunofluorescence (IF).The remaining portion of the tissue was immediately frozen in liquid nitrogen after collection, and then stored at -80 ℃ for ELISA and RT-qPCR experiments.
H&E and Masson's trichrome stainings were performed in accordance with standard or published protocols [7] .Images were collected with a microscope (Eclipse C1, Nikon, Japan).

Immunohistochemistry (IHC) and immunofluorescence (IF)
Antigen recovery and endogenous peroxidase elimination were performed on paraffin-embedded tissues before immunostaining.For IHC, samples were blocked with 3% BSA for 30 min, and then incubated with primary antibodies overnight at 4 °C .
After washing with PBS three times, samples were incubated with HRP-conjugated goat anti-rabbit IgG antibody (1:400, 5220-0336, Seracare, USA) at room temperature for 50 min.After washing with PBS three times, freshly prepared diamindbenzidine (DAB) solution was added for color development.Finally, samples were incubated with hematoxylin for 3 min to counterstain nuclei.IHC images were collected by a microscope (E100, Nikon, Japan) and analyzed by ImageJ software.The primary antibodies used in IHC were listed in Table S3.

Supplementary figures
three times, followed by incubation with DHE(5 μM) or DCFH-DA (10 μM) for 30 min.Subsequently, cells were washed with PBS three times and counterstained with DAPI for 10 min in the dark to label nuclei.The images were collected by a fluorescence microscope (Eclipse C1, Nikon, Japan) and the mean fluorescence intensity (M.F.I.) was quantified with ImageJ software.1.15Macrophage polarization in vitroRaw 264.7 cells were seeded at a density of 5 × 10 4 cells/well in the lower chambers of 24-well transwell plates and cultured overnight.Cells were stimulated with 1 μg/mL LPS and treated with different hydrogels in the upper chambers for 48 h.Cells treated with 1 μg/mL LPS and 40 ng/mL IL-4 were used as the negative control and positive control, respectively.The blank control was without any treatment.1.16Immunofluorescence (IF)RAW 264.7 cells were cocultured with different hydrogels as described above and fixed on slides with 4% paraformaldehyde (PFA).Then, the cells were blocked with 3% bovine serum albumin (BSA) for 30 min at room temperature, washed with PBS for three times, and then incubated with rabbit anti-CD86 antibody (1:200, 13395-1-AP, Proteintech, USA) overnight at 4 ℃.After washing with PBS, cells were incubated with Alexa Fluor 488-conjugated goat anti-rabbit IgG antibody(1:200, ab150077,     Abcam, UK) for 50 minutes at room temperature.The cells were washed with PBS three times and incubated with DAPI for 10 min at room temperature to label nuclei.IF images were collected by a fluorescence microscope (Eclipse C1, Nikon, Japan) and analyzed by ImageJ software.For CD206 marker, cells were stained with rabbit anti-CD206 antibody (1:1000, ab64693, Abcam, UK) and Cy3-conjugated goat anti-rabbit

Figure
Figure S1.a) Photographs of the preparation of SN hydrogel with dynamic single network.b) Chemical structures of SPD and DETA.c) FT-IR spectra of OCMCS, GMA, and two hydrogels (DN and DN-SPD 100).

Figure S2 .
Figure S2.Stress-strain curves of compression tests for hydrogels.

Figure S4 .
Figure S4.Photographs of SN-SPD 100 hydrogels formed on pig skin subjected to various forces.

Figure S5 .
Figure S5.Antioxidant properties of hydrogels in chemical experiments.a) ⋅OH scavenging efficiencies for free SPD and DETA.b) ⋅OH scavenging efficiencies for various hydrogels.c) H2O2 scavenging efficiencies for various hydrogels treated with H2O2 for different times.d) UV-Vis spectra of DPPH after 6 h incubation with various hydrogels.e) DPPH scavenging efficiencies for various hydrogels at different timepoints.f) Photograph of DPPH solution after 6 h incubation with various hydrogels.Data are presented as mean ± SD (n = 3) in (a-c) and (e).

Figure
Figure S6.a) H&E and Masson's stainings of hydrogels on Day 14. b) Quantified collagen density within 100 microns from the implanted hydrogels.Data are presented as mean ± SD (n = 3) in (b).Statistical significance was determined by using one-way

Figure S11 .
Figure S11.H&E staining of major organs (heart, liver, spleen, lung and kidney) from SD rats implanted with different hydrogels for 21 days.

Figure
Figure S12.a) H&E staining of wounds on Day 3. b) H&E and Masson's trichrome stainings of wounds on Day 7.

Figure S14. a )
Figure S14.a) Immunohistochemical stainings of collagen I and collagen III in wound tissues on Day 7 and 12. b, c) Quantified mean optical densities (M.O.D.) of collagen I (b) and collagen III (c).Data are presented as mean ± SD (n = 4) in (b) and (c).Statistical significance was determined by using one-way ANOVA with Tukey's

Figure
Figure S18.a) H&E staining of the wounds in db/db mice on Day 7. b) H&E and Masson's trichrome stainings of the wounds in db/db mice on Day 14.

Figure S19 .
Figure S19.Sirius red staining of the wounds in db/db mice on Day 21.

Figure S20 .
Figure S20.Immunofluorescent staining of ROS in the wounds of db/db mice on Day 14.

Figure
Figure S21.a, b) Immunohistochemical staining of HIF-1α (a) and VEGF (b) in the wounds of db/db mice on Day 21.

Figure S22 .
Figure S22.Immunofluorescent staining of CD31 in the wounds of db/db mice on Day 21.

1.18 Real-time quantitative polymerase chain reaction (RT-qPCR)
.5 mL centrifuge tubes.Cells were lysed on ice for 30 minutes and centrifuged at 12000 rpm for 10 min at 4 ℃.The supernatants were collected and the total protein concentrations were quantified with BCA protein assay kit (MD913053, 1.19 ImmunoblottingRAW 264.7 cells were washed with PBS for three times and then lysed with RIPA lysis buffer (G2002, Servicebio, China).The cells were collected with a cell scraper and transferred into 1conjugated goat anti-rabbit IgG antibody (MD6553, Medical Discovery Leader, China)