Table 1.
Primer sequences.
Fig 1.
In vitro analysis of WT and Col1a1-C5aR1 osteoblasts.
(A) Western blot showing C5aR1 expression in primary osteoblasts of wildtype (WT) and Col1a1-C5aR1 mice after cultivating for 14 d in normal proliferation medium (NM) and osteogenic differentiation medium (ODM). (B) Cell proliferation of WT and Col1a1-C5aR1 osteoblasts determined by BrdU incorporation. (C) Osteogenic differentiation capacity of osteoblasts assessed by alkaline-phosphatase staining after cultivating for 14 d in ODM with or without C5a and (D) by the expression of the osteogenic marker genes Alpl (alkaline phosphatase), Ibsp (bone sialoprotein) and Bglap (bone gamma carboxyglutamate protein/osteocalcin). (E) Western blot showing increased expression of phospho (p)-ERK 1/2 and phospho (p)-p38 after osteoblast stimulation with C5a for 6 h. F) Relative gene expression of interleukin (IL)-6, receptor activator of NF-κB ligand (RANKL) and osteoprotegerin (OPG). *p < 0.05; ***p < 0.001, n = 5–6 per group and treatment.
Table 2.
Bone phenotype of WT and Col1a1-C5aR1 mice aged 12 and 55 weeks.
Fig 2.
Number of inflammatory cells in the fracture callus of WT and Col1a1-C5aR mice.
(A) Quantification of neutrophils (Ly-6G+) at 1 d, (B) representative immunohistological images of neutrophils 1 d after surgery and (C) quantification of neutrophils (Ly-6G+) 3 d after surgery. (D) Quantification of macrophages (F4/80+) 3 d and E) T-lymphocytes (CD8+) 1 d after surgery of wildtype (WT) and Col1a1-C5aR1 mice. Fx: mice with isolated fracture, Fx+TXT: mice with combined fracture and thoracic trauma (TXT). Arrows indicate some of the positively Ly-6G+ stained cells, C: cortex, scale bar 100 μm. *p < 0.05, n = 6 per group.
Table 3.
C5a and IL-6 concentrations in blood, BAL fluid and fracture hematoma 3 h after surgery.
Fig 3.
Fracture healing in WT and Col1a1-C5aR1 mice 14 d after surgery.
(A) Representative histological images of wildtype (WT) and Col1a1-C5aR1 mice with isolated fracture and fracture with additional thoracic trauma (TXT). (B) Callus area, (C) amount of osseous tissue and (D) amount of cartilage. (E) Number of osteoblasts per bone perimeter (N.Ob/B.Pm) and (F) number of osteoclasts per bone perimeter (N.Oc/B.Pm) of WT and Col1a1-C5aR1 mice. (G) Representative tartrate-resistant acid phosphatase (TRAP) staining of fractured calli of WT and Col1a1-C5aR1 mice with isolated fracture. Fx: mice with isolated fracture, Fx+TXT: mice with combined fracture and thoracic trauma. C: cortex, scale bar 100 μm, *p < 0.05, n = 6 per group.
Fig 4.
Representative immunohistological images of bone markers in the fracture callus of WT and Col1a1-C5aR1 mice 14 d after surgery.
(A) Runx2, (B) osteocalcin, (C) receptor activator of nuclear factor-κB ligand (RANKL) and (D) osteoprotegerin (OPG). WT: wildtype, Fx: mice with isolated fracture, Fx+TXT: mice with combined fracture and thoracic trauma (TXT). Arrows indicate some of the positively stained cells, C: cortex, scale bar 100 μm.
Fig 5.
Fracture healing in WT and Col1a1-C5aR1 mice 21 d after surgery.
(A) Representative histological images of wildtype (WT) and Col1a1-C5aR1 mice with isolated fracture and fracture with additional thoracic trauma (TXT). (B) Callus area, (C) amount of osseous tissue and (D) amount of cartilage. (E) Bone mineral density (BMD). (F) Relative flexural rigidity. (G) Number of osteoblasts per bone perimeter (N.Ob/B.Pm) and (H) number of osteoclasts per bone perimeter (N.Oc/B.Pm) of WT and Col1a1-C5aR mice. (I) Representative tartrate-resistant acid phosphatase (TRAP) staining of fractured calli of WT and Col1a1-C5aR1 mice with isolated fracture. Fx: mice with isolated fracture, Fx+TXT: mice with combined fracture and thoracic trauma. C: cortex, scale bar 100 μm, *p < 0.05; **p < 0.005; ***p < 0.001, n = 6–7 per group.
Fig 6.
Fracture healing in WT and Col1a1-C5aR1 mice 25 d after surgery.
(A) Callus area, (B) amount of osseous tissue, (C) amount of cartilage and (D) relative flexural rigidity of wildtype (WT) and Col1a1-C5aR1 mice. Fx: mice with isolated fracture, Fx+TXT: mice with combined fracture and thoracic trauma (TXT). *p < 0.05, n = 6 per group.