Effects of delayed stabilization on fracture healing

Abstract

Previous studies have revealed that delayed internal fixation can stimulate fracture callus formation and decrease the rate of nonunion. However, the effect of delayed stabilization on stem cell differentiation is unknown. To address this, we created fractures in mouse tibiae and applied external fixation immediately, at 24, 48, 72, or 96 h after injury. Fracture healing was analyzed at 10 days by histological methods for callus, bone, and cartilage formation, and the mechanical properties of the calluses were assessed at 14 days postinjury by tension testing. The results demonstrate that delaying stabilization for 24-96 h does not significantly affect the volume of the callus tissue (TV) and the new bone (BV) that formed by 10 days, or the mechanical properties of the calluses at 14 days, compared to immediate stabilization. However, delaying stabilization for 24-96 h induces 10-40x more cartilage in the fracture calluses compared with fractures stabilized immediately. These findings suggest that delaying stabilization during the early phase of fracture healing may not significantly stimulate bone repair, but may alter the mode of bone repair by directing formation of more cartilage. Fractures that are not rigidly stabilized form a significantly larger amount of callus tissue and cartilage by 10 days postinjury than fractures stabilized at 24-96 h, indicating that mechanical instability influences chondrocytes beyond the first 96 h of fracture healing.

Fig. 1. Procedures of creating and stabilizing tibia fracture

(A) One 0.25mm pin (arrow) was placed into the marrow cavity. Two 0.25 mm pins (arrowheads) were placed 90° to each other in the proximal and distal segments of tibia. (B) A circular ring oriented perpendicular to the long axis of the tibia was then fixed to the pins in each segment. (C) A closed fracture (arrow) in the tibial diaphysis was created by three-point bending. (D) The tibiae were stabilized immediately, at 24, 48, 72, or 96 hours by connecting the rings with three longitudinal threaded rods (arrows). (This figure is modified from Fig. 2 in “A model for intramembranous ossification during fracture healing. Thompson et al. J Orthop Res. 2002;20(5):1091–8” with permission from John Wiley & Sons, Inc.)

Fig. 2. Comparison of cartilage formation at 10 days post-fracture

(A) A representative histograph of a fracture stabilized immediately after injury, (B) at 24 hours, (C) at 48hours, (D) at 72 hours, (E) at 96 hours, or (F) left non-stabilized (Ctrl). Cartilage was stained red by Safranin O/Fast Green (SO/FG) staining. Scale bar: A–E = 200µm, F = 1mm.bm = bone marrow.

Fig. 3. Histomorphometric analyses of fracture healing at 10 days post-injury

(A) TV (total volume of callus). (B) BV (total volume of new bone). (C) CV (total volume of cartilage). * The non-stabilized control fractures exhibit significantly more callus tissue (TV) and cartilage (CV) compared to the animals with fractures stabilized at 0, 24, 48, 72, or 96 hours (p<0.05).