Tibial fracture decreases oxygen levels at the site of injury

Abstract

Objectives: Oxygen is an essential component for many aspects of tissue repair. However, the effect of oxygen levels on differentiation of stem cells into osteoblasts and chondrocytes during fracture healing is unknown, in part because of the difficulty in measuring oxygen during fracture healing. In this study we tested the feasibility of using electron paramagnetic resonance (EPR) oximetry to assess tissue oxygen partial pressure (pO(2)) after tibial fractures in mice.

Methods: Transverse tibia fractures were created by three-point bending in adult mice. Paramagnetic material, lithium phthalocyanine (LiPc), was implanted into the fracture site or adjacent to the periosteum in the contralateral leg immediately after fracture. Tissue pO(2) was assessed by EPR 90-110 minutes after implantation of the crystals. in a second experiment, LiPc was implanted into the fracture site and fracture repair and the bio-compatibility of LiPc were assessed at 14 and 28 days after injury.

Results: At the very early stage after fracture, injury significantly decreased tissue oxygenation at the fracture site. When animals were breathing 21% oxygen, pO(2) at the fracture site ((30.6 +/- 12.7 mmHg, n=7) was lower than that in contralateral legs (45.5 +/- 15.3 mmHg, n=7, p<0.01). breathing 100% inspired oxygen increased the pO(2) in both the fractured (72.8 +/- 28.2 mmHg; n=7) and contralateral legs (148.4 +/- 59.2 mmHg; n=7, p<0.01). in addition, LiPc crystals implanted into fracture sites did not interfere with normal fracture healing at 10 and 28 days post-injury.

Conclusions: EPR oximetry is a valuable tool for monitoring oxygen levels during fracture repair in mice.

Figure 1

Time course of pO₂ change at the fracture site. When animals breathed 100% oxygen, tissue oxygen equilibrium was achieved in 90 minutes in fractures with intact femoral artery. after femoral artery resection, very low tissue pO₂ was detected at fracture sites, which remained low even after 90 minutes of inspiring 100% oxygen.

Figure 2

Fracture and inspired oxygen level significantly affect tissue pO₂. When animals (n=7) were breathing 21% oxygen, the mean pO₂ was 45.5 ± 15.3 mmhg in contralateral limbs and 30.6 ± 12.7 mmhg at fracture sites. When animals were breathing 100% oxygen, the mean pO₂ was 148.4 ± 59.2 mmhg in contralateral legs and 72.8 ± 28.2 mm hg in fractured limbs. ctrl = contralateral limbs.——mean of tissue pO_2· |¯¯¯| p<0.01.

Figure 3

LiPc crystals were located within muscle near the fracture site in the majority of animals. H&E staining. outlined area = fracture ends.

Figure 4

Histological analysis of fracture healing with the presence of LiPc crystals. (A) At 10 days after injury, hypertrophic cartilage and newly formed bone were observed in fracture calluses. (B) High magnification of box B in (A) shows a LiPc crystal is embedded in granulation tissue with minimal fibrosis. (C) High magnification of box C in (A) shows a LiPc crystal is in direct contact with newly formed bone. (D) High magnification of box D in (A) shows a LiPc crystal is in direct contact with cartilage.