Live dead staining kit

Meloxicam (>99% purity) was purchased from TCI chemicals Co., Ltd. (Shanghai, China). Distearoyl phosphaethanolamine-polyethylene glycol 2000 (DSPE-PEG2000), hydrogenated soybean phosphatidylcholine (HSPC), and cholesterol were purchased from Nippon Fine Chemical Co., Ltd. (Osaka, Japan). Divalent metal salts (analytical grade) used in this study, including calcium acetate, zinc sulfate, magnesium sulfate, and manganese sulfate, and Dowex resin were purchased from Merck KGaA (Darmstadt, Germany). Live-Dead Staining Kit was purchased from Beyotime Biotech Co., Ltd. (Shanghai, China). Cell Counting Kit-8 was purchased from Dojindo Laboratories (Kyushu, Japan). Apoptosis Detection Kit was purchased from KGI Biotechnology Development Co., Ltd. (Shanghai, China). Purified water (18.2 ​MΩ ​cm resistivity at 25 ​°C) from Milli-Q® Direct System (Merck, Darmstadt, Germany) was employed for preparation of all the solutions.

The electrospun nanofibrous membranes underwent sterilization with 75% ethanol for 30 min, followed by an overnight immersion in α-MEM. Cell viability was determined using a live/dead staining kit (Beyotime, Haimen, China). Cells were seeded in 24-well plates at a density of 1 × 10⁵ cells per well. After 1, 3, and 5 days of culture, the cells were stained with the working solution of the live/dead cells staining for 30 min at 37°C. The stained cells were observed and captured using an automated fluorescence microscope (Zeiss).

Gelatin (porcine skin, type A), dopamine hydrochloride, 2-(dimethylamino)ethyl methacrylate (DMAEMA), polyethylene glycol diacrylate (PEGDA), visible light initiator lithium phenyl(2,4,6-trimethylbenzoyl) phosphinate (LAP), DCFH-DA, Hoechst 33342, β-sodium glycerophosphate, l-ascorbic acid and dexamethasone were purchased from Sigma Aldrich (St. Louis, MO, USA). Heparin sodium salt (Mn = 1.25 kDa, >189 U/mg, Sinopharm Chemical Reagent Co. Shanghai, China), 2, 2-diphenyl-1-picrylhydrazyl (DPPH) (Mackline, China), Recombinant Human/Murine/Rat BMP-2 (E.coli derived, PeproTech, USA), Human/Murine/Rat BMP-2 Standard TMB ELISA Development Kit (PeproTech, USA), Live/Dead Staining Kit (Beyotime, China), CCK8 assay (ApexBio, USA), fetal bovine serum (FBS; HyClone, USA), α-MEM (HyClone, USA), 1% penicillin-streptomycin solution (HyClone, USA), trypsin (Gibco, USA), type II collagenase (BioFroxx, Germany), DAPI (Abcam, USA), Alizarin Red S (ARS) Staining Kit (Beyotime, China), Alp staining (Beyotime, China), Hematoxylin and Eosin (H&E) Staining Kit (Solarbio, China) and Masson's Trichrome Stain Kit (Solarbio, China) were used for in vitro and in vivo study. Unless otherwise stated, all other regents and solvents were used as received without further purification or modification.

The proliferation of L929 cells in four different extracts was investigated by cell counting kit (CCK-8, Beyotime, Shanghai, China). In other words, the cell suspension (10⁴ cells/mL) was separately cultured in 100 μL of extract for 24, 48 and 72 h, respectively. The culture medium and CCK-8 were put in a 96-well plate and maintained in 37 °C incubator for 4 h. After transferring the above mixed solution to a new well plate, the absorbance was read at 450 nm using a micro-plate reader (Bio-Rad 680, Hercules, CA, USA). According to the instruction manual, the cells cultured for 24, 48 and 72 h were qualitatively detected by a live/dead staining kit (Beyotime, Shanghai, China). Cell morphology and distribution were observed by fluorescence microscopy (Zeiss Axioskop 40, Jena, Germany).

FeCl₃·6H₂O, polyvinylpyrrolidone, and urea were bought from Macklin Reagent (Shanghai, China). 2‐Methylimidazole (2‐MI) was purchased from Bide Pharmatech_. Ltd (Shanghai, China). Methanol and dimethyl sulfoxide (DMSO) were obtained from Sinopharm Chemical Reagent (Shanghai, China). 5, 5′‐dithiobis (2‐nitrobenzoic acid) (DTNB), 3,3′,5,5′‐tetramethyl‐benzidine (TMB), glutathione (GSH), and glacial acetic acid were purchased from Aladdin Bio‐Chem Technology Co (Shanghai). Agar powder and tryptone soya broth (TSB) were purchased from Solarbio (Beijing, China). Live/Dead staining kit and ROS staining test kit were commercially obtained from Beyotime Biotechnology (Shanghai, China). All chemical reagents were obtained from commercial companies and used as applicable without further purification.

The biocompatibility of the piGEL was measured by live/dead cell staining. VK2 cells were seeded on piGEL in 48-well culture plates. After 24 h of incubation, the viability of VK2 cells was measured using a live/dead staining kit (Beyotime Technology, China) according to the manufacturer’s instructions. The images were observed under a fluorescence microscope (Leica DM6B, Germany).