Accelerated fracture healing in mice lacking the 5-lipoxygenase gene

Figures & data

Figure 1. Radiographic examination of fracture healing in 5-LO^KO mice and control C57BL/6 mice. Shown are serial dorsal-ventral radiographs made from a C57BL/6 mouse (column A) and a 5-LO^KO mouse (column B) of identical age and genetic background at 7, 10, 14, 21, and 28 days after fracture, as indicated.

Figure 2. Fractures heal by endochondral ossification in 5-LO^KO mice. Shown are fracture callus specimens from control C57BL/6 mice (A), and 5-LO^KO mice (B) 7 days after fracture. Specimens were embedded in polymethylmethacrylate, sectioned, and stained with Stevenel's blue and van Gieson's picrofuchsin to identify mineralized tissue (Bn; red) and proteoglycan-rich cartilage (Ca; deep blue). The fracture site (Fx) is in the lower, left quadrant of each image. Digital images were collected at the same magnification and the white bar in panel A corresponds to 500 μm. In the 5-LO^KO specimen, a portion of the femoral cortical bone was lost during preparation (M).

Figure 3. Endochondral ossification is accelerated in 5-LO^KO mice. Callus area (A), percent cartilage (B), and percent mineralized tissue (C) were measured for femoral fracture specimens at 7, 10, 14, and 21 days after fracture. Group size was 5 at each time point for the C57BL/6 strain (blue) and 5-LO^KO strain (green). Each rectangle represents the 25th and 75th percentiles and the median value is indicated with a line. Differences between the C57BL/6 and 5-LO^KO samples are indicated with the corresponding p-values above the x-axis.

Figure 4. Fracture calluses from 5-LO^KO mice have enhanced mechanical properties. Fractured and contralateral femurs after 28 days (blue) and 84 days (green) of healing from control C57BL/6 mice (n = 11 at 28 and 84 days) and 5-LO^KO mice (n = 11 and 8 at 28 and 84 days, respectively) were mechanically tested to failure in torsion. Peak torque (panel A), maximum rigidity (panel B), maximum shear stress (panel C), and shear modulus (panel D) were calculated from the torque to angular displacement curves and callus dimensions. Fractured femur values were normalized to values from the contralateral femur of that mouse as a percentage. The limits of each rectangle represent the 25th and 75th percentile of the normalized values. The median value is indicated in each rectangle. The 5th and 95th percentile values are shown below and above each rectangle for those with group sizes of 11. Normalized values were compared between genotypes at each time point and p-values are indicated in each graph above the x-axis.

Contralateral mechanical values for unfractured femurs

A	B	C	D	E	F	G	H	I	J
C57BL/6	14–16	11	14.67 (0.26)	1.73 (0.07)	1.20 (0.05)	31.9 (5.0)	819 (200)	144 (30)	4.2 (1.0)
5-LO^KO	14–16	11	14.30 (0.21)	1.64 (0.08)	1.16 (0.05)	24.8 (4.9)	688 (168)	126 (28)	4.2 (1.0)
p-value	—	—	0.002	0.011	0.077	0.003	0.111	0.176	0.997
C57BL/6	22–24	11	15.26 (0.21)	1.77 (0.10)	1.23 (0.04)	37.2 (8.4)	784 (208)	156 (37)	3.8 (1.2)
5-LO^KO	22–24	8	15.11 (0.39)	1.53 (0.07)	1.17 (0.03)	23.8 (5.7)	645 (157)	130 (29)	4.5 (0.8)
p-value	—	—	0.293	< 0.001	0.002	0.001	0.131	0.117	0.130

A Genotype

B Age (weeks)

C Sample size

D Length (mm)

E Max. diameter (mm)

F Min. diameter (mm)

G Peak torque (Nmm)

H Max. rigidity (Nmm²/rad)

I Max. shear stress (MPa)

J Shear modulus (Ga)

Figure 5. Loss of COX-1, COX-2, or 5-LO function alters callus eicosanoid levels. Rectangles represent the 25th and 75th percentiles with median values shown for fracture callus PGE₂ levels (A), PGF_2α levels (B), and LTB₄ levels (C) measured 4 days after fracture in control B6;129P2 mice (n = 6), COX-2^HET mice (n = 6), COX-1^KO mice (n = 4), COX-1^KO mice treated with 30 mg/kg of rofecoxib (n = 3), COX-2^KO mice (n = 6), COX-2^KO mice treated with 30 mg/kg of SC-560 (n = 5), control C57BL/6 mice (n = 6), and 5-LO^KO mice (n = 6). Significant differences are indicated as a, p < 0.001 vs. COX-2^HET; b, p < 0.001 vs. COX-1^KO; c, p < 0.001 vs. COX-2^KO; d, p = 0.04 vs. COX-1^KO; e, p = 0.02 vs COX-2^HET; and f, p = 0.002 vs. C57BL/6. Red bars indicate values from mice with the mixed B6;129P2 background and yellow bars indicate values from mice with the C57BL/6 background.