Glucocorticoid-induced delayed fracture healing and impaired bone biomechanical properties in mice

Figures & data

Figure 1 X-ray imaging of the fracture healing process in mice with prednisone exposure.

Notes: GC-L: CD-1 mice exposed to prednisone (6 mg/kg/day); GC-M: CD-1 mice exposed to prednisone (9 mg/kg/day); GC-H: CD-1 mice exposed to prednisone (12 mg/kg/day). Control: wild-type CD-1 mice.

Figure 2 X-ray imaging analysis of fracture callus tissue post-fracture.

Notes: GC-H: CD-1 mice exposed to prednisone (12 mg/kg/day); Control: wild-type CD-1 mice. (A) Representative radiographs of the fracture healing process of mice in the GC-H and control groups. (B) Radiographs of fractured callus area in mice quantified using ImageJ software.

Table 1 Fracture callus quantified by histologic analysis at 2 weeks post-closed femur fracture

Variables	Control	GC-H
Chondrocyte area (mm^2)	0.29±0.23	1.65±0.87*
Total callus area (mm^2)	8.60±2.26	8.01±2.44
Bone area (mm^2)	3.49±0.73	1.49±0.42*
Bone/total callus (%)	40.86±7.48	18.93±3.26*
Chondrocyte/total callus (%)	3.47±2.01	20.08±4.67*

Notes:

* P<0.05, GC-H: CD-1 fracture mice exposed to prednisone 12 mg/kg/d; Control: healthy CD-1 fracture mice. Chondrocyte area, total callus area and bone area in the callus were quantified with Image Pro Plus 6.0 based on histologic staining.

Table 2 Fracture callus quantified by micro-CT analysis at 2 weeks post-closed femur fracture

Parameter	Control	GC-H
BV/TV (%)	4.9±1.1	3.7±1.3*
BS/TV (1/mm)	5.0±1.2	3.7±1.2*
Conn.D (1/mm^3)	442.1±86.8	381.4±132.9
BMC (mg)	6.0±1.6	3.6±1.4*

Notes:

* P<0.05, GC-H: CD-1 fracture mice exposed to prednisone 12 mg/kg/day; Control: healthy CD-1 fracture mice.

Abbreviations: BV/TV, bone volume to tissue volume; BS/TV, callus bone surface to volume; Conn.D, connectivity density; BMC, bone mineral content.

Figure 3 Representative histological images of fracture callus at 2 weeks post-closed femur fracture in mice.

Notes: GC-H: CD-1 mice exposed to prednisone (12 mg/kg/day); Control: wild-type CD-1 mice. Longitudinal sections of the calluses were subjected to Safranin O and H&E staining. Red color indicates the chondrocyte area in the callus. Chondrocyte area, total callus area and bone area in the callus were quantified with Image-Pro Plus 6.0 based on histological staining (Table 1).

Abbreviation: H&E, hematoxylin and eosin.

Figure 4 Micro-CT images of fractured femoral callus from representative specimens at 2 weeks post-fracture.

Notes: GC-H: CD-1 fracture mice exposed to prednisone 12 mg/kg/day. Control: healthy CD-1 fracture mice. Fracture callus at 2 weeks post-closed femur fracture was quantified by micro-CT (Table 2).

Figure 5 Micro-CT analysis of fractured femoral callus from representative specimens at 9 weeks post-fracture.

Notes: (A) Representative micro-CT images of fracture callus at 9 weeks post-closed femur fracture; (B) BMD and callus BV/TV were quantified by micro-CT analysis. *P<0.05; GC-H: CD-1 fracture mice exposed to prednisone 12 mg/kg/day; Control: healthy CD-1 fracture mice.
Abbreviations: BMD, bone mineral density; BV/TV, bone volume to tissue volume.

Table 3 Analysis of the biomechanical properties of the healed femur at 9 weeks post-femur fracture

Variables	Control	GC-H
Stiffness (N/mm)	198.2±45.3	130.2±33.5*
Ultimate stress (N/mm^2)	195.6±33.1	93.1±32.2*
Yield stress (N/mm^2)	160.9±37.7	82.1±30.5*
Flexural modulus (N/mm^2)	1,823.2±548.5	694.3±297.1*
Toughness (N-mm)	3.27±1.87	2.48±1.34
Resiliance (N-mm)	1.76±0.82	1.51±0.92

Notes:

* P<0.05, GC-H: CD-1 fracture mice exposed to prednisone 12 mg/kg/d. Control: healthy CD-1 fracture mice.