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 cartilage constructs under mechanical stimulation.
METHODS: The chondrocyte-GelMA bioink was prepared and printed by 3D bioprinting technology, and the general observation, the live/dead cell staining, and the cell survival rate at each time point were analyzed; The constructs under compressing stimulation were taken as the experimental group and the constructs without stimulation were taken as the control group. After 2w culture in vitro, the cartilage formation of the two group constructs was observed by histological staining and the relative expression levels of cartilage-related genes was detected by real-time quantitative PCR. The constructs were implanted into nude mice for 5 weeks after 2w culture in vitro with or without mechanical stimulation, and the cartilage formation was observed by gross view and HE staining.
RESULTS AND CONCLUSION:(1) General observation showed stable morphology and clear structure of the chondrocyte-GelMA constructs. The survival rate of cells at 1, 4, and 7 days of culture was above 90%, and the survial rate of cells in 4 and 7d was significantly higher than that in 1d. (2)The qRT-PCR results showed that the chondrogenesis-related genes ELN, COL, A1, COL IIA1, LOX were up-regulated after compressing culture for 2 weaks, and the expressions of COL IIA1 and ELN were significantly up-regulated (P < 0.05). Histology and immunohistochemical staining showed that the cartilage matrix and type I collagen deposition were more obvious in the experimental group than in the control group. (3) The in vivo results showed no obvious difference in appearance between the two groups, and the HE staining results showed that the cartilage formation were more homogeneous in the experimental group. (4) The 3D bioprinted chondrocyte-GelMA construct can maintain a stable three-dimensional structure, has a high cell survival rate, and can form cartilage tissue in vitro and in vivo. Compression stimulation may induce cell death, however, it increases the expression of cartilage specific genes of the survival cells and promotes cartilage formation. 

Key words: three-dimensional bioprinting, mechanical stimulation, chondrocytes, GelMA, cartilage tissue engineering