Rat tail collagen type 1

ARPE-19 cells (5 × 10⁵ per well) were resuspended in 2 mg/ml Rat tail type I collagen (c8062, Solarbio, Beijing, China) which was dissolved in 500 μl DMEM/F12 with 6 μl 0.1 M NaOH and seeded in a 24 well plate. After incubated at 37 °C for 1 h polymerization, the cell collagen gels were detached from the bottom of the wells and floated in 1 ml DMEM-F12 containing 10% FBS. The medium was changed every two days. After different treatment for 10 days, the surface area of each matrix was observed, recorded and taken picture by a smartphone.

A solution of NaOH (0.1 mol/L) was employed to modify the pH of commercial rat tail type I collagen (Solarbio, China) to neutral. Fibroblasts (1 × 10⁵) were treated with NaCl (20 mmol/L), NaCl plus Pyr3 (10 μmol/L) or without NaCl as a control group for 24, 48 and 72 h. Then, harvested fibroblasts were resuspended in culture medium with a mixed pH neutral rat tail collagen solution containing 1 mg/mL collagen. Each well of 24-well plates was filled with 1 mL of rat tail collagen solution containing fibroblasts, and the plates were kept at room temperature for 20 min to enable coagulation of the solution into a gel. Each group of plates was cultured at 37 °C in a 5% CO₂ incubator after adding appropriate volumes of culture medium and stimulants. Every 12 h, images were taken, and collagen gel areas were measured using ImageJ software (NIH).

Cell counting kit-8 (CCK-8) and polyvinylidene fluoride (PVDF) membrane were purchased from Beyotime Biotechnology (Shanghai, China). BCA protein assay kit, protease inhibitor cocktail and RIPA lysis buffer were purchased from CWBiotech Co. Ltd. (Beijing, China). Rat-tail type I collagen were purchased from Solarbio Science & Technology Co., Ltd. (Beijing, China). Ara-C, DNR and discoidin domain receptor 1 (DDR1)-IN-1 were purchased from MedChemExpress USA (New Jersey, USA), RPMI-1640 media was purchased from Hyclone (GE Healthcare Life Sciences, Logan, UT, USA). Fetal bovine serum (FBS) was purchased from Biological Industries (Beit Haemek, Israel). CD2-PC5 (cat. no. A07745), CD3-ECD (cat. no. A07748), CD4-FITC (cat. no. A07751), CD8-PE (cat. no. A07756), CD34-ECD (cat. no. B49202), (cat. no. B36294), all purchased from Beckman coulter, Inc. (Brea, CA, USA). Stat3 (cat. no. 9139), DDR1 (no. 5583), p-STAT3 (no. 9145), GAPDH (cat. no. 5174s), primary antibodies, goat anti-mouse (cat. no. 7076s) and rabbit HRP-conjugated secondary antibody (cat. no. 14708s) were purchased from Cell Signaling Technology, Inc., (Danvers, MA, USA). PCL scaffolds were purchased from 3D Biotek LLC (New Jersey, USA).
The study was approved by the Ethics Committee of Qingdao University (Qingdao, China).