3.4. In Vitro Assessments

Mouse RAW 264.7 macrophages (RAW 264.7, Chinese Academy of Sciences Cell Bank, Shanghai, China), mouse fibroblasts (L929, Chinese Academy of Sciences Cell Bank, Shanghai, China), and human umbilical vein endothelial cells (HUVEC, Chinese Academy of Sciences Cell Bank, Shanghai, China) were grown in a modified culture medium composed of 90% DMEM, 10% fetal bovine serum, and 1% penicillin–streptomycin. The culture conditions were maintained at 37 °C with a 5% CO₂ atmosphere.

The cytotoxicity evaluation of each hydrogel was carried out using L929 cells and HUVEC cells in a CCK-8 assay. Culture medium containing L929 or HUVEC cells was seeded on 96-well plates at 1 × 10⁴/well. Meanwhile, the extract solution (4 mg/mL) was prepared via immersion of the hydrogel in DMEM for 24 h at 37 °C, and then 100 μL of hydrogel extract was added to each well before incubation for 24 and 48 h. The culture medium without cells added was used as a blank, and the cells without treatment using hydrogel extracts as the control. Finally, CCK-8 was added to the plates, and the optical density of the culture plate solution was measured at 450 nm using a microplate reader. The calculation of the cell viability was as follows:

where O_test, O_blank and O_control were the absorbance of the test, blank, and control, respectively.

For the live/dead assay, L929 or HUVEC cells were evenly spread in 12-well plates at a density of 5 × 10⁴ cells/well and placed in an incubator with 5% CO₂ at 37 °C until the cells adhered completely. The cells in each well were stained according to the instructions for the Calcein-AM/PI Double Stain Kit. The results of the fluorescence staining were obtained under a Nikon DS-Ri2 inverted fluorescence microscope (Tokyo, Japan).

The in vitro blood compatibility of the BSP/BER hydrogel was assessed by directly placing 2% red blood cell suspension in contact with the hydrogel samples at 37 °C. The absorbance of the supernatant at 545 nm was measured spectrophotometrically. For comparison, blood samples were mixed with deionized water and physiological saline as the positive and negative controls, respectively. The calculation of hemolysis rate was as follows:

where OD_n, OD_p, and OD_s were the optical density values of the negative control, positive control, and sample, respectively.

The glucose level in the supernatant of L929 cells was measured to assess the glucose consumption ability of the cells. L929 cells were cultured in a 96-well plate at a density of 0.8 × 10⁴ cells/well and incubated for 48 h with different hydrogel extracts after the cells adhered. Subsequently, the glucose content in each well was detected using a glucose detection kit, and the cell viability was detected using a CCK-8 assay. The culture medium without cells added was used as a blank, and the cells without treatment using hydrogel extracts served as the control. The calculation of glucose consumption was based on the following equation:

where Glu_blank represents the absorbance of the content of glucose in the blank group and Glu_test represents the content of glucose in the hydrogel experimental group.

L929 or HUVEC cells were seeded evenly in 12-well plates at a density of 1 × 10⁵ cells/well and incubated at 37 °C with 5% CO₂ for 24 h until the cells adhered completely. A scratch was created in the plate using a pipette tip, and this was followed by two washes with PBS. Hydrogel extract was added to the plate, and the scratch was observed and photographed at 0 h and 24 h.

The extracellular antioxidant activity of the hydrogel was evaluated using a DPPH scavenging test. Different BSP/BER hydrogel extracts (4 mg/mL) were mixed with a DPPH ethanol solution, with vitamin C as the positive control and deionized water as the negative control. After incubating for 30 min in the dark, the absorbance at 516 nm in the supernatant was measured using a microplate spectrophotometer. The percentage of DPPH free radical clearance was calculated using the following equation:

where A₀ was the absorbance of ethanol with DPPH, A₁ was the absorbance of the sample mixed with ethanol, and A₂ was the absorbance of the sample mixed with DPPH.

In the investigation of the intracellular antioxidant properties of the hydrogel, L929 cells were cultured in 12-well plates at a density of 5 × 10⁵ cells/mL until they adhered. The cells were then pre-stimulated with hydrogel extracts for 12 h. Afterwards, 900 μM H₂O₂ was added to treat the cells for an additional 4 h. The untreated cells served as the control group, while the cells treated only with H₂O₂ served as the model group (MOD). The cells were stained with DCFA–DA, and the fluorescence results were observed using an inverted fluorescence microscope. The staining results were quantitatively analyzed using a Becton Dickinson FAC Scan flow cytometer (Franklin Lakes, NJ, USA).

The RAW264.7 cells at exponential growth were seeded in 6-well plates at a density of 5 × 10⁵ cells/well. The cells were pretreated with culture medium containing hydrogel extracts for 2 h. Afterwards, they were stimulated with lipopolysaccharide (LPS) at a concentration of 1 μg/mL for 24 h. The untreated cells served as the control group, while the cells treated only with LPS served as the model group (MOD). The cell supernatant was collected, and the concentrations of tumor necrosis factor (TNF)-α, monocyte chemotactic protein (MCP)-1, and interleukin (IL)-6 were measured using flow cytometry.

The antibacterial effects of the hydrogel were assessed using liquid medium turbidity assays [48]. A total of 50 mg of hydrogel was added to 5 mL of Luria-Bertani (LB) liquid medium, which contained bacteria (S. aureus, or E. coli) at a concentration of 1 × 10⁶ CFU/mL in the logarithmic growth phase. Untreated bacteria were used as the control group. The mixture was then incubated in a 37 °C thermostat, and the absorbance of the bacterial suspensions at 600 nm was measured using an Epoch 2 microplate reader. Furthermore, 100 μL of the bacterial suspension was evenly coated on a medium plate, and the colonies were observed after incubation at 37 °C for 24 h. The antibacterial activity of the hydrogel was calculated using the following formula:

where OD_con represents the absorbance of the bacterial liquid in the control group and OD_test represents the absorbance of the dressing experimental group.