Abstract: BACKGROUND: Tissue engineering has brought new hope to the clinical challenge of liver failure, and the preparation of plant-derived decellularized fiber scaffolds holds significant importance in liver tissue engineering.
OBJECTIVE: To prepare apple tissue decellularized scaffold material by using fresh apple slices and a solution of sodium dodecyl sulfate, and assess its biocompatibility. 
METHODS: Fresh apples were subjected to decellularization using phosphate buffer saline and sodium dodecyl sulfate solution, separately. Afterwards, the decellularized apple tissues and apple decellularized scaffold materials were decontaminated with phosphate buffer saline. Subsequently, scanning electron microscopy was used to assess the effectiveness of decellularization of the apple materials. Adipose-derived mesenchymal stem cells were extracted from the inguinal fat BALB/C of mice, and their expression of stem cell-related markers (CD45, CD34, CD73, CD90, and CD105) was identified through flow cytometry. The cells were then divided into a scaffold-free control group and a scaffold group. Equal amounts of adipose-derived mesenchymal stem cells were seeded onto both groups. The biocompatibility of the decellularized scaffold with adipose-derived mesenchymal stem cells was evaluated using CCK-8 assay, hematoxylin-eosin staining, and phalloidine staining. Cell adhesion and growth on the scaffold were observed under light microscopy and scanning electron microscopy. Furthermore, the scaffold was subdivided into the non-induced group and the hepatogenic-induced group. Adipose-derived mesenchymal stem cells were cultured on the decellularized apple scaffold, and they were cultured for 14 days in regular culture medium or hepatogenic induction medium for comparison. Immunofluorescent staining using liver cell markers, including albumin, cytokeratin 18, and CYP1A1, was performed. Enzyme-linked immunosorbent assay was used to detect the secretion of alpha fetoprotein and albumin. Additionally, scanning electron microscopy was employed to observe the morphology of the induced cells on the scaffold, verifying the expression of liver cell-related genes on the decellularized scaffold material. Finally, the cobalt-60 irradiated and sterilized decellularized apple scaffolds were transplanted onto the surface of mouse liver and the degradation of the scaffold was observed by gross observation and hematoxylin-eosin staining after 28 days.
RESULTS AND CONCLUSION: (1) The scanning electron microscopy results revealed that the decellularized apple scaffold material retained a porous structure of approximately 100 μm in size, with no residual cells observed. (2) Through flow cytometry analysis, the cultured cells were identified as adipose-derived mesenchymal stem cells. (3) CCK-8 assay results demonstrated that the prepared decellularized apple tissue scaffold material exhibited no cytotoxicity. Hematoxylin-eosin staining and phalloidine staining showed that adipose-derived mesenchymal stem cells were capable of adhering and proliferating on the decellularized apple tissue scaffold. (4) The results obtained from immunofluorescence staining and enzyme-linked immunosorbent assay revealed that adipose-derived mesenchymal stem cells cultured on the decellularized apple scaffolds exhibited elevated expression of liver-specific proteins, including albumin, alpha-fetoprotein, cytokeratin 18, and CYP1A1. These results suggested that they were induced differentiation into hepatocyte-like cells possessing functional characteristics of liver cells. (5) The decellularized apple scaffold implanted at 7 days has integrated with the liver, with partial degradation of the scaffold observed. By 28 days, the decellularized apple scaffold has completely degraded and has been replaced by newly-formed tissue. (6) The results indicate that the decellularized scaffold material derived from apple tissue demonstrates favorable biocompatibility, promoting the proliferation, adhesion, and hepatic differentiation of adipose-derived mesenchymal stem cells.

Key words: plant tissue, scaffold, decellularization, liver failure, adipose-derived mesenchymal stem cell, tissue engineering, hepatocyte-like cell