Abstract: BACKGROUND: Liver tissue engineering is an important strategy for treating liver injury. Previous studies have achieved good results in repairing mouse liver injury using adipose derived stem cells combined with plant fiber scaffolds, electrospun silk protein scaffolds, and decellularized liver scaffolds. However, the scaffold materials have poor mechanical properties, irregular microstructures, and cannot simulate the liver microstructure to the greatest extent.
OBJECTIVE: To fabricate microstructured silk fibroin scaffolds using 3D printing technology and evaluate their biocompatibility.
METHODS: (1) The silk fibroin fiber obtained after degummed silkworm cocoon was dissolved in lithium bromide solution, and then glycidyl methacrylate was added for grafting. The mixed solution was dialyzed, lyophilized, and added with distilled water to prepare 3D printing bio-ink. The microstructure and 
compressive elastic modulus of the scaffold were detected. (2) The third generation of mouse adipose derived stem cells were divided into two groups: the control group was cultured in flat plane, and the experimental group was seeded in 3D printed microstructured silk fibroin scaffold. The cytotoxicity of the scaffold was detected by CCK-8 assay. Cell proliferation was detected by live/dead staining. Cell adhesion was observed by scanning electron microscopy. After induction, the secretion of albumin and alpha-fetoprotein in the supernatant of the induced cells was detected by ELISA, and the expression of albumin, cytokeratin 18 and cytochrome P450 mRNA in the induced cells was detected by RT-PCR. (3) Nine BALB/c mice were selected, and the 3D printed microstructured silk fibroin scaffold was sutured and fixed to the surface of the liver after exposing the left lobe of the liver. The degradation of the scaffold and the tissue morphology of the liver-scaffold contact site were observed on days 2, 7, and 28 after surgery.
RESULTS AND CONCLUSION: (1) The 3D printed microstructured silk fibroin scaffold was porous under scanning electron microscopy, with regular and uniform pore structure, pore size of 100 μm, and the compressive elastic modulus of the scaffold was 11.96 kPa. (2) 3D printed microstructured silk fibroin scaffolds were non-cytotoxic, and adipose stem cells could adhere to the scaffolds and proliferate, and were evenly distributed in the scaffolds. After hepatogenic induction culture, compared with the control group, the secretion of albumin and alpha-fetoprotein in the scaffold group increased (P < 0.05), and the expression of albumin, cytokeratin 18, and cytochrome P450 mRNA increased (P < 0.05). (3) With the extension of postoperative time, the scaffold gradually adhered to the liver surface and the scaffold gradually degraded. Hematoxylin-eosin staining showed that on day 2 after surgery, there was a clear junction between the scaffold and the liver surface, and there were more inflammatory cell infiltrations at the junction, and cells began to enter the inside of the scaffold. On day 7 after surgery, the junction between the scaffold and the liver surface began to fuse, the inflammatory cell infiltration at the junction decreased, and more cavities and more cells were formed in the scaffold. On day 28 after surgery, there was no obvious junction between the scaffold and the liver surface, the inflammatory reaction at the junction basically disappeared, and the scaffold was significantly degraded. (4) The results showed that the 3D printed microstructured silk fibroin scaffold had good biocompatibility and could promote the adhesion, proliferation, and hepatocyte-like cell differentiation of adipose-derived stem cells.

Key words: 3D printing, scaffold, tissue engineering, silk fibroin, adipose derived stem cell, liver injury, hepatocyte-like cell, microstructure