Fig 2: In vitro osteogenic differentiation behaviors of MC3T3-E1 cells after different treatments.a Representative ALP staining, ARS staining, and Von Kossa staining assays for MC3T3-E1 cells incubated with different scaffold extracts for 7, 14, and 21 days. Quantitative analysis of b ALP activity, c ARS staining, and d Von Kossa staining in different groups. The insets depict the digital images. Cells cultured without adding scaffold extracts served as a negative control. e The expression of osteogenesis-related genes, including Runx2, Col-1, and OPN, as determined by qRT–PCR assay. Representative immunofluorescent staining images of f Runx2 (green), g Col-1 (green), and h OPN (green) in MC3T3-E1 cells incubated with different extracts for 7 days. F-actin and cell nuclei were labeled with fluorescent red and blue, respectively. Images were captured using confocal microscopy. Scale bar in a: 200 µm, in f–h: 25 µm. Data are expressed as the mean ± SD (n = 3). *P < 0.05 and **P < 0.01 indicate significant differences compared with the control group. #P < 0.05 and ##P < 0.01 indicate significant differences compared with the HS@PDA-LYN/HA group.

Fig 3: Single-cell transcriptomic analysis reveals the transcriptome of myeloid cells in the microenvironment of LUSC. a. tSNE plot of myeloid cells color-coded by their associated clusters. b. tSNE plot of myeloid cells color-coded by their associated sample origins. c. tSNE plot of myeloid cells color-coded by cell subtypes established by marker genes. d. The frequency and proportion of each cell subtype from tumor and normal tissues. e. The boxplot showed the proportion of neutrophils in tumor and normal tissues, respectively, in the TCGA LUSC cohort. (The difference between normal and tumor tissues was calculated by Wilcoxon-test). f. Module scores of genes related to inflammation and N2 signature of neutrophil cluster from tumor and normal tissues, respectively. g. Correlation between neutrophil and expression level of CXCR2(top)/CXCL8(bottom) in TCGA LUSC cohort, respectively (The correlation coefficient and difference were calculated by Pearson-test). h. Heatmap shows the difference in pathway activities scored by GSVA per cell between different monocyte/macrophage groups. i. The boxplot showed the proportion of SPP1+M, SEPP1+M and FABP4+M from tumor and normal tissues, respectively, in the TCGA LUSC cohort. (Significance of difference between tumor and normal tissues was calculated by Wilcoxon-test). j. Association between cell abundance and patient survival from TCGA LUSC cohort (P value was calculated with log-rank test).

Fig 4: RAPA promotes the interaction of ECs and SMCs. a, b Transwell assay was used to study the migration ability of SMCs after co-culturing with NC, TIE2-WT and TIE2-L914F mutant ECs treated with DMSO or rapamycin over a period of 24 h. c, d Different groups of ECs were treated with DMSO or rapamycin (RAPA), and then co-cultured with SMCs. The expression of OPN and a-SMA were measured in SMCs by western blot. Scale bar, 100 µm. The data are means ± SEM (n = 3). ns: no significant, *P < 0.05, **P < 0.01

Fig 5: The osteogenic differentiation stimulated by cyclic mechanical stress in human PDLSCs. (A) The mRNA levels assay of COL1, RUNX2 and OPN were performed using real-time PCR. (B, C) The protein levels assay of COL1, RUNX2 and OPN were performed. (D) The ALP staining was carried out (the scale bar represented 100 and 200 µm). (E) The ALP activity assay was carried out. *P < 0.05 versus 0 h.