Abstract: BACKGROUND: Uneven secretion of matrix and insufficient mechanical strength are important factors affecting the formation effect of tissue engineering constructs in vivo. Mechanical stimulation is an effective means to promote the secretion of extracellular matrix.
OBJECTIVE: To explore the biological performance of 3D bioprinted composite scaffolds under mechanical stimulation.
METHODS: The chondrocyte-gelatin methacryloyl composite scaffold was prepared and printed by 3D bioprinting technology. Cell survival was observed by live/dead cell staining. The composite scaffolds were placed in a mechanical pressurized bioreactor for pressurized culture. The composite scaffolds cultured in a 6-well plate without pressurization were used as the control. Live/dead cell staining was used to observe the cell survival. Histological staining was used to observe the in vitro cartilage of the composite scaffolds. The relative expression levels of cartilage-related genes were detected by real-time quantitative PCR. The composite scaffolds were implanted into nude mice for 5 weeks in vitro with or without mechanical stimulation, and the cartilage formation was observed by histological staining.
RESULTS AND CONCLUSION: (1) The appearance of the composite scaffolds showed a clear grid-like structure. When cultured for 1, 4, and 7 days in vitro, the scaffolds had stable morphology and clear structure, and the cell viability was above 90%. (2) After 2 weeks of culture, the cell survival rate in the composite scaffold in the pressurized group was lower than that in the unpressurized group (P < 0.05). Hematoxylin-eosin staining exhibited that composite scaffolds of the two groups had obvious cartilage lacuna structure, and the cells were distributed evenly in the materials. In the pressurized group, more new cartilage tissue was formed in the gap of the composite scaffold. Safranin O staining showed that red cartilage matrix was formed in both groups; the stained pericellular matrix was deeper in the pressurized group. Type I collagen immunohistochemical staining showed that the coloration in the pressurized group was more obvious; the mRNA expression levels of elastin and type II collagen in the pressurized group were higher than those in the unpressurized group (P < 0.05). (3) After 5 weeks of subcutaneous implantation in nude mice, hematoxylin-eosin staining showed that the formation of cartilage tissue in the pressurized group was more uniform; the size of chondrocytes was uniform; the lacuna structure was obvious. (4) The results show that although in vitro stimulation can trigger cell death in the 3D bioprinted chondrocyte-gelatin methacryloyl composite scaffold, it also promotes the ingrowth of surviving cells into the space of the scaffold and increases the expression of their cartilage matrix-related genes, to promote cartilage formation. 

Key words: three-dimensional bioprinting, mechanical stimulation, chondrocyte, gelatin methacryloyl, tissue engineered cartilage, composite scaffold