Cacl2

PU was purchased from Sigma-Aldrich (Cat No.81367–5G, America). Fibrin was provided by Thermo Scientific company (Cat No.MA126074, America). Ninety-eight percent formic acid was provided by Aladdin company (Cat No.F112034, China). Dulbecco’s modified eagle’s medium (DMEM), phosphate buffer saline (PBS, Cat No.P1022-500), fetal bovine serum (FBS, Cat No.S9030-200), CaCl₂ (Cat No.G0071) and lipase (Cat No.L8621) were from Solarbio (Beijing, China). Living cell staining kit (Cat No.C2015S), CCK-8 kit (Cat No.C0038), ELISA kit (Cat No.PI328), KCL (Cat No.ST1596) were purchased from Beyotime. Sodium nitroprusside (SNP, Cat No.S817931), adrenaline (AD, Cat No.N814761) and acetylcholine (Ach, Cat No.A886059) were obtained from Macklin (Shanghai, China). Anti-von Willebrand Factor antibody (ab115771), Anti-α-SMA (251411), Mouse Anti-CD68 antibody were obtained from Ruizekang Biotechnology Co., Ltd. (Beijing, China). Safranin O (Cat No.G1031), Verhoeff (Cat No. G1046) and Sirius red (Cat No.G1038) kit were supplied by Servicebio Biochemical Technology Co., Ltd (Wuhan, China). Mesenchymal stromal cells (MSCs) were obtained from Xinxiang Medical University (Henan, China). The use of MSCs has been approved by the Ethics Committee of Xinxiang Medical University.

Astrocytes of the U87 cell line were purchased from the American Type Culture Collection (ATCC, Manassas, USA). U87 cells were cultured in Dulbecco’s modified Eagle’s Medium (Invitrogen, New York, USA) containing 10% fetal bovine serum (GIBCO BRL, Grand Island, NY, USA) and 1% penicillin-streptomycin (Hyclone, South Logan, UT, USA) maintained at 37°C under 5% CO₂. Modified Hanks’ balanced salt solution (MHBSS) was prepared to simulate blood plasma, to which was added albumins (Bovine Serum Albumin Fraction V, BioFRoxx, Einhausen, Germany) and Ca²⁺ (concentrations to 2.4 mmol/L). The electrolyte concentrations were adjusted using CaCl₂, MgCl₂, NaCl and KCl (Solarbio, Beijing, China). The pH and OP were maintained at 7.4 and 300 ± 10 mOsmol/kg. MHBSS components are shown in the Supplementary Materials And MethodsTables S1 and S2.

Following transfection with the pIRVP3IL-18HN recombinant plasmid for 72 h, 2×10⁶ H22 tumor cells were lysed in 300 µl of buffer A, consisting of 10 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid-KOH (pH 7.4), 0.25 M sucrose, 1 mM ethylene glycol-bis(β-aminoethyl ether)-N,N,N',N'-tetraacetic acid, 1 mM EDTA, 1 mM DTT, 10 mM MgCl₂ and 1 mM phenylmethylsulfonyl fluoride (all obtained from Solarbio Science & Technology Co., Ltd.) and homogenized in a Dounce homogenizer (IKA, Guangzhou, China) for 10 min. The supernatant was mixed with TNC buffer, consisting of 10 mM Tris-acetate (pH 8.0) (HD Biosciences Co., Ltd.), 0.5% Nonidet P-40 (HD Biosciences Co., Ltd.) and 5 mM CaCl₂ (Solarbio Science & Technology Co., Ltd.), and the precipitate was dissolved in buffer A to obtain the cytosol, which together with the mitochondria were stored at −80°C for subsequent western blot analysis.

To synthesize the SFF/HAp/MgO (SFFHM) scaffold, MgO (⩽50 nm particle size, Sigma-Aldrich, USA) at different (0, 1, 3, and 5) wt.% and 3 wt.% silk fibers were blended to normal saline solution containing 10 wt.% SF and stirring continuously. After mixing evenly, the SF was cross-linked by ultrasonication for 60 seconds, and the power was 150 watts. The resulting crosslinked mixture was transferred into a 48-well plate and frozen at −80°C for 24 h followed by vacuum freeze-drying for 36 h. The HAp were gradually deposited in the as-prepared scaffolds by stirring evenly in the mineralized solution which consisted of 0.5 M CaCl₂ (Solarbio, English) and 0.2 M Na₂HPO₄ (Solarbio, English) for 6 h at 37ºC. The wet composite scaffolds were frozen at −80°C for 24 h, followed by lyophilization again. Other groups scaffolds were synthesized by following the above procedure without the addition of corresponding ingredients. Finally, the prepared composite scaffolds were sealed with silicone desiccant. (According to the cell activity test results below of the composite scaffolds with different contents of MgO, we selected 1 wt.% MgO in the composite scaffold as the optimal ratio, which was used in all other experiments of this manuscript).