β tcp

CS was purchased from Shanghai Yuanye Biotechnology Co., Ltd. (Shanghai, China). GM, genipin, β-TCP, and boric acid were purchased from Shanghai Macklin Biochemical Co., Ltd. (Shanghai, China). Gelatin was purchased from Beijing Solarbio Science and Technology Co., Ltd. (Beijing, China). Hematoxylin and eosin were purchased from Sigma Aldrich Trading Co., Ltd. (Shanghai, China). Methyl methacrylate was purchased from Shanghai Zhanyun Chemical Co. Ltd. (Shanghai, China). Paraformaldehyde was purchased from Shanghai Lingfeng Chemical Reagent Co. Ltd. (Shanghai, China). Acetic and hydrochloric acids were purchased from Xilong Scientific Co., Ltd. (Guangdong, China).

The graphene was purchased from Nanjing XFNANO Materials Tech Co., Ltd. (Nanjing, China). Chitosan (CS, degree of deacetylation ≥ 95%,), β-TCP, maleic anhydride, and L-cysteine were obtained from Macklin Biochemical Technology Co., Ltd. (Shanghai, China). The α-minimum essential medium (α-MEM), fetal bovine serum (FBS), penicillin-streptomycin (P/S), and trypsin were purchased from Gibco (ThermoFisher, Shanghai, China). The ascorbic acid, dexamethasone, and β-glycerophosphate were purchased from Sigma Aldrich. All the reagents were used as received without further purification. Malt Sprague–Dawley (SD) rats were purchased from Silaike, Shanghai, China.

Three groups of nano-HA and β-TCP (hereinafter referred to as HCP)/CMC/gelatin scaffolds were made with different HCP contents. Group A (10 wt% HCP), group B (30 wt% HCP), and group C (50 wt% HCP) had 0.17, 1.08, and 2.50 g more HCP, respectively [the ratios of nano-HA (Macklin, Beijing, China) and β-TCP (Macklin) were fixed with 3:2 HCP], in 38 ml of double distilled water. The solution was mixed by ultrasonic shaking for 1 h. An amount of 2 g of gelatin (Macklin) and 0.5 g of CMC (Solarbio, Beijing, China) were dissolved into the mixture at 50°C, and the mixture was mixed at 300 rpm for 12 h to ensure complete dissolution. Freeze drying combined with high-speed stirring was used according to the procedure described by Maji et al. (2018) (link). The solution was stirred using a high-speed blender at 5,000 rpm for 5 min until the foam height was unchanged. The foamed mixture was transferred to 24-well culture plates, frozen at −80°C for 12 h, and then freeze dried for 48 h. The scaffold was soaked in a 0.2 wt% glutaraldehyde solution for 1 h to crosslink. The scaffold was washed with a deionized water solution with sodium borohydride to remove residual glutaraldehyde, washed with deionized water again, and air-dried.