Thermo 3001

The cell viability cultured on composite scaffold at day 1 and 5 was evaluated using a calcein-AM/PI double stain kit (Bio Vision, USA). The cell-seeded scaffolds were rinsed with PBS and added with the mixture solution of 1 μM calcein-AM and 3 μM PI. After 30 min incubation, the morphology of stained cells was observed by a confocal laser scanning microscopy (CLSM, Leica SP8, Germany).
The Cell Counting kit-8 (CCK-8, Dojindo, Japan) was used to evaluate the proliferation of mBMSCs on the scaffolds. In brief, the scaffolds were harvested at 1, 4, 7 days and transferred to a new 48-plates, then working solution (1:9 ratio of CCK-8 solution: medium) was add to each well and incubated at 37°C, 5% CO₂ for 1 h in dark. The optical density (OD) value was detected at 450 nm using a micro-plate reader (Thermo 3001, USA). Five samples for each group.

BMSCs (1 × 10⁵ cells per well) were seeded on N‐mGel‐DNA nonporous hydrogel, mGel‐DNA, and N‐mGel‐DNA‐BP macroporous hydrogels in a 24‐well plate. After 7 days and 14 days of culture, the expression of osteogenic genes (Runx‐2, ALP, OCN, and OPN) was detected by quantitative real‐time polymerase chain reaction (qRT‐PCR). The primer sequences are shown in Table S2 (Supporting Information). After 14 days of culture, the formation of mineralized matrix was characterized by Alizarin Red staining. Before staining, BMSCs on the hydrogels were trypsinized and collected by centrifugation. Then, the mineralized matrix of BMSCs was labeled with Alizarin Red solution, and the mineralized matrix was observed using an optical microscope. After that, 100 mm dodecylpyridinium chloride solution was added to each well to quantify the amount of mineralized matrix. Finally, a microplate reader (Thermo 3001, Thermo Scientific, USA) was used to measure the absorbance at 562 nm.

The cell proliferation of each scaffold was analyzed quantitatively by the CCK-8 method. In short, the medium was removed and the cells were washed twice with PBS (pH = 7.2) at specified intervals. CCK-8 solution was added to each well and incubated in an incubator for 2 h. The absorbance was measured at 450 nm wavelength with the microplate reader (Thermo3001, America). Twenty-four hours after the cells were inoculated with the scaffolds, live/dead assay was performed using the calcein-AM/propidium iodide double staining kit (Sigma).

The method previously described by Ekezie et al. [32 (link)] was used with some modifications. The concentrations of both samples were first adjusted to 20 μg/mL using 50 mM carbonate buffer (pH 9.6) and then 100 μL of above samples were placed overnight at 4 °C in the polystyrene 96 well plates (Xiamen YJM Labware Co. Ltd., Xiamen, China). Subsequently, the plates were washed 5 times with 200 μL PBST (0.01 M PBS, pH 7.4, 0.5% Tween-20) and blocked with 5% BSA (150 μL per well) at 37 °C for 2 h. After washing again with PBST, 100 μL/well human sera (1:20 dilution) were added to the plates and incubated at 37 °C for 2 h. After washing, the plates were incubated at 37 °C for 1.5 h with 100 μL/well anti-human IgE (1:5000 dilution in PBS). Then they were washed again, added 100 μL/well TMB was added and they were incubated at 37 °C for 10 min. Finally, 2 M sulfuric acid (50 μL/well) was used to stop the reaction and the OD₄₅₀ was measured by a microplate reader (Thermo3001, Thermo Fisher Scientific Inc., Waltham, MA, USA).

Cell viability was measured by quantitatively cell counting and qualitatively live-dead staining. Cell proliferation was determined using a Cell Counting Kit-8 (CCK-8, Dojindo Laboratories, Japan) following the protocol. Briefly, cells were harvested on days 1, 3, 5, and 7. After the removal of the media, the samples were incubated in H-DMEM medium containing 10% CCK-8 reagent at 37°C for 1 h. Cells cultured without HMBG nanoparticles were used as a control. The absorbance was measured ata wavelength of 450 nm using a micro-plate reader (Thermo 3001, USA). Six specimens for each cultured time point were tested and each test was performed in triplicate.
To further understand the adherence and growth behavior of MG-63, fluorescence microscopy (FM, 40FLAxioskop, Zeiss, Germany) was used after culturing for 1 and 3 days. For the FM assessment, MG-63 cells were stained using a live cell labeling kit (Cell Explorer, AAT Bioquest). The staining process was performed according to the kit instructions. In brief, after culturing for 1 and 3 days, the culture medium was replaced by the staining solution, incubated at 37°C for 1 h, followed by washing with PBS, and then observed by FM.

Alizarin Red S staining and semiquantitative analysis was used to highlight mineralization nodules produced by BMSCs grown for 14 days in Lap (0, 12.5, 50, and 100 μg/mL). The cells were gently rinsed with ddH₂O three times and then were fixed in 4% PFA for 10 min at room temperature. A solution of 1% Alizarin Red S (pH 4.2, Sigma-Aldrich) was added and kept for 10 min to visualize the nodules. Afterward, the cells were washed thoroughly with PBS and images were captured using light microscopy. Quantitative analysis of Alizarin Red S staining was performed by eluting the bound stain with 10% cetylpyridinium chloride for 1 h. The absorbance of the resulting solution at 562 nm was determined using a microplate reader (Thermo3001).