Fig. 1

Elevated active TGF-β levels in human HO. a H&E staining of normal muscle (left) and HO in early osteogenesis stage (middle) and late osteogenesis stage (right) after elbow fracture (EF) and central nervous system trauma (CNST). Scale bar, 100 μm. b Safranin O and Fast Green (SOFG) staining of normal muscle (left) and HO. Proteoglycan (red) and heterotopic bone (green). Scale bar, 100 μm. c TRAP⁺ cells (red) and d quantitative analysis of the number of TRAP⁺ osteoclast surface (OCS) per bone surface (BS). Scale bar, 100 μm. Immunohistochemical staining of e pSmad2/3⁺ cells (brown) 
and g PDGF-BB⁺ cells (brown) and quantitative analysis of the number of f pSmad2/3⁺ cells or h PDGF-BB⁺ cells per bone marrow area (mm²). i, j Quantitative analysis of i active TGF-β and j PDGF-BB in serum in HO patients at early osteogenesis stage and late mature stage by ELISA. Dotted line indicates the average concentration of healthy people with active TGF-β of 1.23 ng/ml and PDGF-BB of 1.68 ng/ml. k Immunofluorescent staining and l quantitative analysis of CD73⁺ (green) and CD90⁺ cells (red) in bone marrow of ectopic bone marrow in HO patients. Blue color indicates DAPI staining of nuclei. Scale bar, 20 μm. C cartilage, B bone, BM bone marrow, M muscle, NM normal muscle, EF elbow fracture, CNST central nervous system trauma, OR osteogenesis MR maturation. All data are shown as the mean ± s.d. n = 9 per group for d, f, h, l histomorphometry analysis. *p < 0.05 as determined by unpaired, two-tailed Student’s t-test. n = 8 per group for i, j ELISA analysis. *p< 0.05 as determined by paired, two-tailed Student’s t-test