Balb c nu nu

Recombinants (n = 15) were implanted under the renal capsule of an immunocompromised PN 6-week male mouse (BALB/C nu/nu purchased from Nara Biotech Co.) for 12 weeks. Since immunocompromised mice do not have a thymus, they are commonly used in xenotransplantation experiments and tissue calcification. A total of five mice were randomly selected for the recombinant transplantation experiment for calcification. Mice that died due to transplantation were excluded; however, there was no exclusion in this experiment. The mice were incised on the dorsal side, and the kidney was collected. Using the tungsten needle, a small hole at the renal capsule covering the kidney was made to insert recombinants. Once this procedure was completed, the kidney was transferred back inside the abdominal cavity, and the incision was sutured. Furthermore, 16 weeks after the surgery, the mice were euthanized in a CO₂ chamber to collect the kidney. The collected kidney was fixed in 4% paraformaldehyde and scanned using a micro-CT scanner. Samples were decalcified with 10% EDTA at 50°C, and histological analysis was performed after reviewing.

Chitosan (CS; CAS No. 9012-76-4, low molecular weight −50,000–190,000 Da; 75–85% deacetylated chitin, poly (D-glucosamine), polyethylene glycol (PEG 6000), L-lysine α-oxidase (LO) from Trichoderma viride, ethidium bromide (EB), acridine orange (AO), hydrochloric acid, 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC: HCl), rhodamine 123 (Rh123), N-hydroxysuccinimide (NHS), propodium iodine (PI), dichlorofluorescein diacetate (DCFH-DA) were purchased from Sigma-Aldrich (St. Louis, MO, USA). Antibody anti-HER2 (Herceptin, trastuzumab; A1046-100 Lot. 4E31A10460) was obtained from BioVision Inc., Milpitas, CA, USA. The cell viability assay kit CELLO MAXTM was procured from MediFab, Republic of Korea. Cell culture media (Roswell Park Memorial Institute Medium; RPMI), fetal bovine serum (FBS), SnakeSkin™ Dialysis Tubing (10 K MWCO, 35 mm), penicillin and streptomycin (P&S), and apoptosis kit (Annexin V FITC and PI) for flow cytometry were acquired from ThermoFisher Scientific (Waltham, MA, USA). Human breast cancer cell line (BT474) was purchased from Korean Cell Line Bank, (KCLB), Seoul, Korea. Seven-week-old BALB/c/nu/nu female mice weighing 20–22 g were purchased from Nara Biotech, Seoul, Korea.

All animal experiments were performed according to the guidelines of the Yonsei University Health System, Intramural Animal Care and Use Committee (YUHS‐IACUC). YUHS‐IACUC complies with the Guide for the care and use of laboratory animals (National Research Council). The animal study plan was reviewed and approved by this committee (2020‐1043). Immunocompromised PN 6 weeks male mice, with bodyweight around 20 g (BALB/C nu/nu purchased from Nara Biotech Co.), were orthotopically implanted with five hydrogel embedded‐spheroids, into the tooth socket after maxillary first molar extraction. Then, to avoid spheroid detach from the tooth socket, the OssGuide (Bioland) were covered. During the tooth extraction and AM‐1 spheroid transplantation procedure, mice were anaesthetised. After 1 week of monitorization, the Verapamil intraperitoneal injection was performed every 48 h for 3 weeks (n = 7), and as a mock group (n = 7), the PBS was injected. A total of 30 days after inoculation, the mice were sacrificed, and the micro‐CT was performed after sacrifice. The whole maxilla was harvested and fixed in 4% paraformaldehyde (Sangon Biotech Co. Ltd.) at 4°C for 24 h and after dehydration, it was embedded into the paraffin for the histological study.