Dio green

The transendothelial migration assay of cancer cells crossing the BBB was performed as previously described with minor modification48 (link). Prior to assay, the astrocytes and BMECs were stained with Hoechst 33258 and DiI live staining dyes, respectively. The different cancer cells were digested and resuspended to the density of 5 × 10⁴ cells/mL, loaded into the medium channel, and adhered to the BBB by turning the device on its side for 5 min, as done to originally establish the BBB, and then the device was incubated in a humidified incubator at 37 °C. Tumor cells were allowed to invade for 72 h, and images were collected every 24 h by a fluorescence microscope equipped with a CCD digital camera. For the invasion assay of endogenous U87 brain tumor cells, the cells were digested and resuspended to the density of 1 × 10⁶ cells/mL and pre-dyed with DiO (green, Beyotime) prior to use. Before establishing the BBB, the U87 cells were mixed together with collagen solution and loaded into the collagen channels. After gelling, the U87 cells were embedded in the collagen gel in a 3D microenvironment to mimic the brain tumor in vivo. The 3D high throughput BBB system was subsequently established, and images were obtained every 24 h beginning 48 h after the BBB was formed. All of the invasion assays were performed under flow conditions.

The Rhodamine B solution was added to the RSF solution to obtain a final mixture with a Rhodamine B concentration of approximately 5 ppm. Degradation experiments were conducted under accelerated degradation conditions using rats. At specified time points (three and four months), three disks were removed from the subcutaneous tissues and fixed with a 4 wt% polyformaldehyde solution containing 30 wt% sucrose for 48 h. Frozen sections were made and stained with DAPI, and the fluorescence area of Rhodamine was measured and analyzed. Chondrocytes and BMSCs were transfected with DIL (red) and DIO (green) (Beyotime, Shanghai, China) fluorescent probes, respectively. The cell labeling rate was detected by flow cytometry, and the cell scaffold complex was removed from the subcutaneous tissue on the back of nude mice after seven days, and then fixed in 4% paraformaldehyde containing 30% sucrose for 48 h. Frozen Sects. (5–8 μm) were made and DAPI staining was performed. The fluorescence areas of DIL and DIO were analyzed.
The detailed experimental method is shown in the Supplementary Information of the in vivo osteochondral repair and evaluation, including histological evaluation of cartilage repair, RNA extraction, and mRNA-seq.