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. 2024 Jul 18;27(8):110537. doi: 10.1016/j.isci.2024.110537

Figure 4.

Figure 4

BMP signaling is increased in stable, hyaline-like cartilage upon ectopic transplantation

(A) hBMSCs/SSCs were incubated with HyA-FMBs for 2 h and transplanted ectopically into NSG mice, followed by digestion of transplanted tissue 8 weeks post-transplant and scRNA-seq analysis shown in (B).

(B) Dot plot demonstrating global gene expression of integrated in vitro HyA-FMB datasets (from days 0, 1, 3, 5, and 10 of chondrogenic differentiation) and in vivo transplant of hBMSCs/SSCs attached to HyA-FMBs analyzed 8 weeks post-transplant. Genes associated with BMP signaling (purple) and chondro-osteogenesis (green) are shown. Asterisks indicate differentially expressed genes (logfc>0.35) in transplanted hBMSCs/SSCs attached to HyA-FMBs, compared with all in vitro HyA-FMB datasets.

(C and D) Toluidine blue (C) and H&E (D) staining of ectopic transplant of hBMSCs/SSCs attached to HyA-FMBs at 8 weeks post-transplant with high-magnification insets. Scale bars, 500 μm. Asterisks (∗) indicate HyA-FMBs.

(E) Immunofluorescence analysis of pSMAD5 (red) at 8 weeks post-transplant with high-magnification insets. Nuclei counterstained with DAPI (blue). Scale bar, 100 μm. Asterisks (∗) indicate HyA-FMBs.

(F) hBMSCs/SSCs were incubated with HyA-FMBs for 2 h and transplanted into a 2-mm defect at the femoral trochlear groove in SRG rats, followed by histology analyses in (G).

(G) Toluidine blue staining of defect areas (dashed lines) from chondral transplants at 2 and 4 months post-transplant in SRG rats. Scale bars, 500 μm. High-magnification images shown below each image.

(H) hBMSCs/SSCs were cultured with HyA-FMBs in chondrogenic medium for 10 days and transplanted into a 2-mm defect at the femoral trochlear groove in SRG rats, followed by histology analyses in (I).

(I) Toluidine blue staining of defect areas (dashed lines) from organoid chondral transplants at 2 and 4 months post-transplant in SRG rats. Scale bars, 500 μm. High-magnification images shown below each image (see also Figure S3; Table S1).