Additional file 2 of Single-cell profiling reveals periosteal signatures of impaired periosteal cells proliferation in a drill-hole model of type 2 diabetes

Supplementary Material 2: Figure S1. Reduced bone mass in 12-week-old db/db mice. A. Body weight (left) and fasting blood glucose (right) of db/db and db/+ mice at different age. One-way ANOVA, Sidak’s multiple comparison test. Data are shown as mean ± SD. n = 6 (db/+ and db/db, 8- and 12-week-old); n = 3 (db/+ and db/db, 16-week-old). B. Representative stereomicroscopy (left) and 3D μCT images (right) of femurs from db/db and db/+ mice at 12-week-old. Scale bar: 2 mm. C. Femur length of the db/db and db/+ mice at 12- week-old age. Two-tailed Student’s t-test. Data are shown as mean ± SD. n = 6 (db/+); n = 8 (db/db). D. 3D in vivo μCT images of 1 mm cortical bone segments in the mid-diaphysis of the db/db and db/+ femurs at different ages. Scale bar: 2 mm. E. cortical bone area (Ct.ar), total tissue area (Tt.ar) and cortical thickness (Ct.th) of 1 mm cortical bone segments in the mid-diaphysis of the db/db and db/+ femurs at different ages. Two-way ANOVA, Sidak’s multiple comparison test. Data are shown as mean ± SD. n = 3. F. Hematoxylin and eosin staining of the distal femurs from db/db and db/+ mice. Scale bar: 1 mm. *P < 0.05, **P < 0.01, ***P < 0.001. Figure S2. Flow Cytometry and Gene Expression Analysis of Periosteal Cells. A. The flow cytometry gating strategy involved the use of an anti-CD45 antibody to exclude hematopoietic lineage cells. B-I. Vlnplot displaying the expression of key marker genes across different periosteal cell subpopulations identified in the analysis. Subpopulations include proliferating cells, endothelial cells (ECs), Schwann cells, chondrocytes, muscle satellite cells (MuSCs), pericytes, myocytes, IECs and tenocytes. J. UMAP for periosteum from db/+ and db/db. Figure S3. Time-Dependent Changes in the Periosteum Following Drill Hole Surgery. A-F. Panels A to F show the sequential changes in tibias at day 0, 3, 5, 7, 14, and 21 post-drill hole surgery, including: Representative 2D μCT images of horizontal sections of the tibias. Scale bar: 1 mm; Corresponding 3D μCT images of the regions of interest. Scale bar: 500 μm; Hematoxylin and eosin staining; Masson staining; Safranin O staining; Tartrate-resistant acid phosphatase staining. Scale bar: 500 μm and 200 μm G. Quantification of newly forming bone mass at the fracture sites as shown in the 3D μCT images, at 0, 3, 5, 7, 14, and 21 days post-fracture. Statistical analysis was performed using one-way ANOVA with Sidak’s multiple comparison test. Data are presented as mean ± SD, with n = 6. H. Representative 3D μCT images of fractured tibias at different time points post-surgery. Scale bar: 1 mm. Figure S4. Impaired callus remodeling and decreased osteogenic activity in db/db mice during fracture healing. A. Representative micro-CT images of fracture callus at day 5 and day 7 post-fracture in db/+ and db/db mice. Scale bars: 500 µm. B. H&E-stained histological sections of fracture callus at day 14 and day 21 post-fracture. Scale bars: 100 µm. C. Representative dynamic bone formation images using double labeling with alizarin red (red) and calcein (green) in callus regions at day 7 post-fracture. D. Quantification of mineral apposition rate (MAR, µm/day) Data are shown as mean ± SD. ***p < 0.001. E. TRAP-stained sections at day 7 post-fracture showing osteoclasts in callus area. F. Quantification of osteoclast surface per bone surface (Oc.S/BS, %). Data are shown as mean ± SD. ***p < 0.001. Figure S5. Cell Type Composition and Marker Gene Expression in Periosteal Cells. A-G. Vlnplot displaying the expression levels of selected marker genes across various periosteal cell subpopulations. Subpopulations include muscle satellite cells (MuSCs), Pericytes, Schwann cells, Proliferating cells, tenocytes, Endothelial cells (ECs), Chondrocytes. Figure S6. Integrated Single-Cell RNA-Seq Analysis of Periosteal Cells Across Multiple Experimental Conditions. A. UMAP visualizations of unsupervised clustering of periosteal cells from various conditions, including db/+, db/+ injured, db/db, db/db injured, Col2-Cre, Col2-Cre fracture Day 5, Col2-Cre fracture Day 10. Clusters are color-coded and labeled, showing distinct subpopulations across different experimental conditions. B. Heatmap displaying differential gene expression across the identified periosteal cell clusters from the UMAP analysis. Key marker genes associated with specific cell types or functions are highlighted on the right. Figure S7. scRNA-Seq Analysis of Distinct Cellular Dynamics in Bone Repair: Comparing Intramembranous and Endochondral Ossification. A-G. Top: UMAP visualizations of unsupervised clustering of periosteal cells from various conditions, including db/+, db/+ injured, db/db, db/db injured, Col2-Cre, Col2-Cre fracture Day 5, Col2-Cre fracture Day 10, Clusters are color-coded and labeled. H. Dot plot showing the expression levels of key marker genes across different cell types, including FAPs, injury induced fibroblasts (IIFCs), chondrocytes (CHs), PCs, and proliferating cell cluster (Mki67+). I-K. Feature plots showing the expression of representative marker genes (Acan, Postn, and Acta2) across the UMAP space, highlighting distinct gene expression patterns. Figure S8. Effect of FGL2 on Osteoblast Proliferation Under Normal and High Glucose Conditions. A. Osteoblasts were treated with vehicle or varying concentrations of FGL2 (50, 200 ng/mL) under normal (vehicle) and high glucose (4.5 mg/mL) conditions, and relative cell proliferation was assessed after 48 h to compare the effects under these different conditions. Statistical significance was assessed using one-way ANOVA with Sidak’s multiple comparison test. Data are shown as mean ± SD. **P < 0.01, ***P < 0.001. B. RT-qPCR results showing the mRNA levels of Raptor following knockout using CRISPR-Cas9 lentivirus. Statistical significance was determined using a two-tailed Student’s t-test. Data are shown as mean ± SD. **P < 0.01.