Fig. 2: mSSC mediated skeletal regeneration is estrogen dependent.

A Schematic of fracture creation on sham (sham), ovariectomized (OVX), and ovariectomized with 17-beta estradiol systemic rescue (OVX + E2) mice, and assessment by MST, microCT, FACS analysis, and RNA analysis. B Serum estradiol levels of sham vs OVX vs OVX + E2 demonstrates a significant decrease in serum estradiol levels in OVX vs sham. Elevated levels of estradiol were detected in OVX + E2 mice (n = 5). C Maximal load to fracture (in newtons) of healing femora from sham vs OVX vs OVX + E2 mice (n = 10). D Assessment of bone mass/volume of trabecular bone in healing femora from sham (grey) vs OVX (red) vs OVX + E2 (blue) mice (n = 20) and BV/TV of callus area from sham (grey) vs OVX (red) vs OVX + E2 (blue) mice (n = 4). E Representative photographs and radiographs of sham (left), OVX (center) and OVX + E2 (right) fracture callus at post-fracture day 7 demonstrating a larger callus in OVX + E2 mice and radiological non-union in the OVX mice. F Differences in absolute cell numbers of mSSCs (left) in sham vs ORX vs ORX + E2 for male mice (n = 3) and sham vs OVX vs OVX + E2 for female mice (n = 8), and mBCSPs(right) in sham vs OVX vs OVX + E2 mice (n = 8 per group). G Colony-forming assay of mSSCs (top) and mBCSPs (bottom) isolated at post-fracture day 7 from Sham, OVX, and OVX + E2 mice. There is significant expansion of mSSC and BCSP in systemic estradiol rescue compared to sham mice. The total number of colonies were measured (n = 3). H Heat map showing relative gene expression of sex hormone receptors in mSSCs and mBCSPs from fracture calluses of sham (left), OVX (center left), OVX + E2 (center right), and male (right) mice. I Heat map shows rescue of relative gene expression of mul-tiple skeletogenic factors (Ihh, Fbn2, BMP1, Col14a1, Fgfr2, Osm) in mSSCs and mBCSPs from fracture calluses of sham (left), OVX (center), and OVX + E2 (right) mice. Genes related to (top) Hh signaling; (bottom) skeletal development, growth and repair. Blue, low expression: red, high expression. J Single-cell RNA-se-quencing data demonstrates the percentage of co-expression of estrogen receptors and skeletogenic factors. The data in B,C,D,F,G are expressed as the means ± s.e.m; ordinary one-way ANOVA followed with Tukey’s multiple comparisons test; exact P value are indicated in the figures. For data and statistics, see Source Data File.