Sd rats

One hundred male SD rats (approximately 170 g) were purchased from the Beijing HFK Bioscience Co. Ltd (Beijing, China). The rats were randomly divided into five groups with 20 per group: (1) Negative; (2) STZ; (3) STZ + DMH; (4) STZ + MET; (5) STZ + DMH + MET. The animals were acclimated for 1 week. Subsequently, except those of the Negative group, all rats were provided a high-fat diet. The body weight of the rats was measured once a week. All rats were housed in standard polypropylene cages (4 rats/cage) and maintained under standardized conditions (22 ± 3°C, humidity 50 ± 10%, 12-h light/12-h dark) with free access to food pellets and tap water. All studies were performed with the approval of the animal experimental ethics review committee of Shandong University School of Medicine.

Twenty male SD rats (8–10 weeks) weighing 200–250 g were purchased from Beijing HuaFuKang Biotechnology Co., Ltd. Rats were reared in separate cages under a 12:12-light–dark cycle and were given enough food and water. Fifteen rats were subjected to intraperitoneal injection of MTX 75 mg/kg, and the rest 5 rats were injected with the equal amount of saline. In the seventh day after MTX treatment, 5 MTX-treated rats were injected with PBS, 5 were injected with PBS containing 200 ug of ADSCs­exosomes, and 5 were injected with ADSCs resuspended in PBS (1 × 106). The injections directly into rat brain parenchyma were conducted by a fully automatic brain stereotactic system (RWD, Shenzhen, China). The optimal injection location of rat hippocampus is as follows: lateral to the midline 3 mm, posterior to the bregma 3.8 mm, ventral to the bregma 3.6 mm. Hence, the rats were divided into control group, MTX + PBS group, MTX + ADSCs group, and MTX + ADSC-exo group (5 rats per group). All animal experiments were done in these 20 rats. All animal procedures were approved by the Second Affiliated Hospital of Wenzhou Medical University and conducted according to the institutional guidelines.

To examine the in vivo biocompatibility of DTPU, the printed disc scaffolds with 8 mm diameter were implanted under the skin of SD rats. All the animal experiments were complied with the guidelines of Tianjin Medical Experimental Animal Care, and animal protocols were approved by the Institutional Animal Care and Use Committee of Yi Shengyuan Gene Technology (Tianjin) Co., Ltd. (protocol number: YSY-DWLL-2023252). The printed scaffolds were sterilized using ultraviolet for 2 h and equilibrated in sterile PBS. Subsequently, nine male SD rats (6-week-old, 200 ± 10 g) purchased from Beijing HFK BIOSCIENCE Co., Ltd. (China) were anesthetized with isoflurane using a gas anesthesia machine (ZS-MV-IV). The scaffolds printed with DTPU-0.25-4k or DTPU-0.5-4k were implanted under the skin. At 1-week, 2-week, and 4-week timepoints, the scaffolds along with the surrounding tissues were excised and fixed with 4% paraformaldehyde for 48 h. The local interaction of the implanted scaffolds with the tissues were assessed by histological evaluation based on hematoxylin-eosin (H&E) staining. The sham-control group was set to eliminate the inflammatory response induced by the open wound.

Gingival tissue collection was approved by the Human Ethics Committee of Peking University School and Hospital of Stomatology (Approval number: PKUSSIRB‐202168143). The donors who participated in our study volunteered and signed informed consent forms. The animal surgical procedure was approved by the Institutional Animal Care and Use Committee of the Peking University (Approval number: LA2022394). The SD rats were purchased from Beijing HFK Bioscience Co. Ltd. All efforts were made to minimize the suffering of animals.