D000521

Animal studies were approved by the Laboratory Animal Welfare and Ethics Committee of the First Affiliated Hospital of Nanjing Medical University. About 1 × 10⁶ cells were suspended in 100 µl PBS and then injected subcutaneously into BALB/c nude mice (5 weeks old, Gempharmatech, D000521). Every 4–5 days, tumor volume was measured and calculated using the following equation: (longer axes×shorter axes^²)/2. Tumor weight was calculated after executed and then fixed in 4% formalin for IHC analysis. All animal experiments were approved by the Committee on the Ethics of Animal Experiments of the Nanjing Medical University (IACUC-2,310,034).

Briefly, 1 × 10⁷ tumor cells of Mia‐Paca2 were inoculated subcutaneously in the flank of 5‐week‐old female BALB/c mice (GemPharmatech, D000521). After the xenograft tumors reached ≈100 mm³, the mice were randomly divided into five groups to receive either vehicle (sterilized deionized water) or various doses of WBC100(0.1, 0.2, 0.4 mg kg^–1) via oral administration twice a day for 28 consecutive days. The positive drug gemcitabine was injected intraperitoneally with a dose of 100 mg kg^–1 twice a week (BIW) for 4 weeks. Tumor volume and mouse body weight were measured at different time points. At the end of the experiment, all mice were euthanized for analysis of body weight, tumor weight, and survival rate. TGI % was calculated using the following equation: (1 − tumor volume of treated day x/tumor volume of control day x) × 100.

A murine dorsal subcutaneous pocket model was chosen to estimate bone formation on various hydrogels encapsulated BMSCs. After co-culturing with BMSCs in vitro for one week, BMSCs-laden hydrogels were implanted subcutaneously into nude mice (n = 6, D000521, GemPharmatech Co., Ltd, China). Each mouse received dorsal subcutaneous implants (laden 2 × 10⁶ cells) at both sides of the back, and implants were allowed to develop in vivo for 4 weeks. The mouse was euthanized at week 4 post-surgery, and the samples were excised and fixed overnight in 4% paraformaldehyde. The total bone volume formed in hydrogels was visualized and quantified using micro-CT. The degree of bone ingrowth was further determined by the AV/TV (Apatite volume/total volume) ratio. Afterwards, samples were embedded in paraffin wax and sectioned for H&E, VEGF (ab32152, Abcam, 1:150), CD31 (MA1-26196, Invitrogen, 1:100), and Runx2 (Invitrogen, PA5-87299, 1:100) staining. Goat anti-mouse second antibody (Abcam, ab150113, 1:400) was used as the second antibody. Microstructure and mineralized components were characterized by SEM and EDS, while the mechanical property of post-implants was evaluated by DMA.