Heterogeneity of the periosteum at steady state

A. Experimental design. Nuclei were extracted from the periosteum of uninjured tibia and processed for single-nuclei RNAseq. B. Sorting strategy of nuclei stained with Sytox-7AAD for snRNAseq. Sorted nuclei are delimited by a red box. C. UMAP projection of color-coded clustering of the uninjured periosteum dataset. Six populations are identified and delimited by black dashed lines. D. Violin plots of key marker genes of the different cell populations. E. UMAP projection of color-coded clustering of the subset of SSPC/fibroblasts. F. Feature plots of Prrx1 and Pdgfra in the subset of SSPC/fibroblasts. G. Feature plots of key marker genes of the different cell populations. H. Dot plot of the stemness markers Pi16, Ly6a (Sca1), Cd34, and Dpp4. I. Violin and feature plots of Cytotrace scoring in the subset of SSPC/fibroblasts, showing that Sca1+ SSPCs (cluster 0) are the less differentiated cells in the dataset.

Periosteal response to fracture at single-nuclei resolution

A. Experimental design. Nuclei were extracted from the periosteum of uninjured tibia of wild-type mice and from the periosteum and hematoma at days 3, 5 and 7 post-tibial fracture and processed for single-nuclei RNAseq. B. UMAP projection of color-coded clustering of the integration of uninjured, day 3, day 5 and day 7 datasets. Eleven populations are identified and delimited by black dashed lines. C. Violin plots of key marker genes of the different cell populations. D. UMAP projection of the combined dataset separated by time-point. E. Percentage of cells in SSPC, injury-induced fibrogenic cell, osteoblast, chondrocyte, and immune cell clusters in uninjured, day 3, day 5 and day 7 datasets.

Cellular organization of the fracture callus

A. Safranin’O staining of longitudinal callus sections at day 3-post tibial fracture. B. Immunofluorescence on adjacent sections show absence of SSPCs (SCA1+), and presence of IIFCs (POSTN+) and immune cells (CD45+) in the activated periosteum and hematoma at day 3-post fracture. Chondrocytes (SOX9+, arrowhead), osteoblasts (OSX+, arrowhead) and endothelial cells (PECAM1+) are detected in activation periosteum (n=3 per group). C. Safranin’O staining of longitudinal callus sections at day 5-post tibial fracture. D. Immunofluorescence on adjacent sections show IIFCs (POSTN+), chondrocytes (SOX9+, arrowhead), osteoblasts (OSX+, arrowhead), immune cells (CD45+) and endothelial cells (PECAM1+) in the fibrosis, chondrocytes (SOX9+) in cartilage and osteoblasts (OSX+), immune cells (CD45+, arrowhead) and endothelial cells (PECAM1+) in new bone. (n=3 per group). E. Safranin’O staining of longitudinal callus sections at day 7-post tibial fracture. F. Immunofluorescence on adjacent sections show IIFCs (POSTN+), chondrocytes (SOX9+, arrowhead), osteoblasts (OSX+, arrowhead), immune cells (CD45+) and endothelial cells (PECAM1+) in the fibrosis, chondrocytes (SOX9+) in cartilage and osteoblasts (OSX+), immune cells (CD45+, arrowhead) and endothelial cells (PECAM1+) in new bone (n=3 per group). Scale bars: A-B-E: 1mm, B-D-F: 100µm.

Periosteal SSPCs activate through a common fibrogenic state prior to undergoing osteogenesis or chondrogenesis

A. SSPCs, injury-induced fibrogenic cells (IIFCs), chondrocytes and osteoblasts from integrated uninjured, day 3, day 5 and day 7 post-fracture samples were extracted for a subset analysis. B. UMAP projection of color-coded clustering (left), color-coded sampling (middle) and monocle pseudotime trajectory (right) of the subset dataset. The four populations are delimited by black dashed lines. C. (top) Feature plots of the stem/progenitor, fibrogenic chondrogenic and osteogenic lineage scores (middle) Scatter plot of the lineage scores along pseudotime. (bottom) Violon plot of the lineage score per time point. D. Schematic representation of the activation trajectory of pSSPCs after fracture.

In vivo validation of pSSPC activation trajectory

A. (Top) Representative Safranin’O staining on longitudinal sections of the hematoma/callus at day 5 post-fracture. The callus is composed of fibrosis, cartilage (red dashed line) and bone (green dashed line). (Middle, box 1) Immunofluorescence on adjacent section shows decreased expression of POSTN (green) and increased expression of SOX9 (red) in the fibrosis-to-cartilage transition zone. (Bottom, box 2) Immunofluorescence on adjacent section shows decreased expression of POSTN (green) and increased expression of OSX (red) in the fibrosis-to-bone transition zone (n=3 per group). B. Experimental design: GFP+ Sca1+ SSPCs were isolated from uninjured tibia of Prx1Cre; R26mTmG mice and grafted at the fracture site of wild type mice. Safranin’O staining of callus sections at day 5 post-fracture and high magnification of POSTN immunofluorescence of adjacent section showing that GFP+ cells contribute to the callus and that grafted SSPCs differentiate into POSTN+ IIFCS (white arrowheads) (n=4 per group). C. Experimental design: GFP+ IIFCs from day 3 post-fracture tibia were isolated from Prx1Cre; R26mTmG mice and grafted at the fracture site of wild type mice. Safranin’O of callus sections at day 14 post-fracture and high magnification of OSX and SOX9 immunofluorescence of adjacent sections showing that GFP+ cells contribute to the callus and that grafted IIFCs differentiate into OSX+ osteoblasts (box 3, white arrowheads) and SOX9+ chondrocytes (box 4, white arrowheads) (n=4 per group). Scale bars: Low magnification: A: 500µm, B-C: 1mm. High magnification: 100µm.

Characterization of injury-activated fibrogenic cells

A. Gene ontology analyses of upregulated genes in IIFCs (clusters 2 to 6 of UMAP clustering from Fig. 4). B. Dot plot of ECM genes in UMAP clustering from Fig. 4. C. Feature plot per cluster and scatter plot along pseudotime of the mean expression of ECM genes. D. Gene regulatory network (GRN)-based tSNE clustering of the subset of SSPCs, IIFCs, chondrocytes and osteoblasts. E. Activation of Mta3, Six1, Sox9 and Sp7 regulons in SSPCs, IIFCs, chondrocytes and osteoblasts. Blue dots mark cells with active regulon. F. Number of regulons activated per cell in the SSPC, IIFC, osteoblast (Ob) and chondrocyte (Ch) populations. Statistical differences were calculated using one-way ANOVA. ***: p-value < 0.001. G. Heatmap of activated regulons in SSPC, IIFC, osteoblast and chondrocyte populations. H. Scatter plot of the activity of the combined fibrogenic regulons along monocle pseudotime from Fig. 4. I. Reactome pathway analyses of the fibrogenic regulons shows that the 3 most significant terms are related to Notch signaling (blue). J. Feature plot in Seurat clustering and scatter plot along monocle pseudotime of the Notch signaling score.

Gene regulatory network analyses identifies gene cores driving fibrogenic to chondrogenic and osteogenic transition.

A. Activation of Maf, Arntl, and Nfatc2 regulons in SSPCs, IIFCs, chondrocytes and osteoblasts. B. STRING interaction network of the chondro-core 1 and 2 transcription factors (blue and orange respectively). C. Feature plot of chondro-core 1 (top) and chondro-core 2 (bottom) activities in SSPCs, IIFCs, chondrocytes and osteoblasts in Seurat UMAP from Fig. 4. D. Scatter plot of chondro-core 1 (top) and chondro-core 2 (bottom) activities along monocle pseudotime and Acan expressing. E. Activation of Tcf7, Bclb11b and Tbx2 regulons in SSPCs, IIFCs, chondrocytes and osteoblasts. F. STRING interaction network of the osteo-core transcription factors (green) and their related genes shows that most of osteo-core related genes are involved in Wnt pathway (purple). G. Feature plot of the osteo-core activity in SSPCs, IIFCs, chondrocytes and osteoblasts in Seurat UMAP from Fig. 4. H. Scatter plot of osteo-core activity along monocle pseudotime and Ibsp expressing.

IIFCs are the main source of paracrine factors after fracture.

A. Outgoing interaction strengths of the different cell populations of the fracture environment determined using CellChat package. B. Comparison of incoming and outgoing interaction strengths across SSPC, IIFC, chondrogenic and osteogenic populations. C. Outgoing and incoming signaling from and to SSPCs, IIFCs, chondrocytes and osteoblasts. D. Cell-cell interactions identified between SSPCs, IIFCs, chondrocytes and osteoblasts. E. Feature plots of the score of BMP, TGFβ, PDGF, POSTN, PTN and ANGPTL signaling per timepoint. F. Scatter plot along pseudotime and feature plot per time point of the mean expression of the ligand and receptors involved in signaling from IIFCs. G. Circle plot of the interactions between SSPCs, IIFCs, chondrocytes and osteoblasts, showing that most signals received by SSPCs are coming for IIFCs.