Exposure to 100% Oxygen Abolishes the Impairment of Fracture Healing after Thoracic Trauma

Abstract

In polytrauma patients a thoracic trauma is one of the most critical injuries and an important trigger of post-traumatic inflammation. About 50% of patients with thoracic trauma are additionally affected by bone fractures. The risk for fracture malunion is considerably increased in such patients, the pathomechanisms being poorly understood. Thoracic trauma causes regional alveolar hypoxia and, subsequently, hypoxemia, which in turn triggers local and systemic inflammation. Therefore, we aimed to unravel the role of oxygen in impaired bone regeneration after thoracic trauma. We hypothesized that short-term breathing of 100% oxygen in the early post-traumatic phase ameliorates inflammation and improves bone regeneration. Mice underwent a femur osteotomy alone or combined with blunt chest trauma 100% oxygen was administered immediately after trauma for two separate 3 hour intervals. Arterial blood gas tensions, microcirculatory perfusion and oxygenation were assessed at 3, 9 and 24 hours after injury. Inflammatory cytokines and markers of oxidative/nitrosative stress were measured in plasma, lung and fracture hematoma. Bone healing was assessed on day 7, 14 and 21. Thoracic trauma induced pulmonary and systemic inflammation and impaired bone healing. Short-term exposure to 100% oxygen in the acute post-traumatic phase significantly attenuated systemic and local inflammatory responses and improved fracture healing without provoking toxic side effects, suggesting that hyperoxia could induce anti-inflammatory and pro-regenerative effects after severe injury. These results suggest that breathing of 100% oxygen in the acute post-traumatic phase might reduce the risk of poorly healing fractures in severely injured patients.

Fig 1. Microscopic and molecular analyses of lungs.

(A) Lung tissue expression of HO-1 3 and 9 hours post-injury. (B) Quantitative analysis of lung histology after 1, 3 and 21 d. (C) Histological scoring of neutrophil stainings in the lungs and (D-F) representative slides 3 hours after injury showing increased numbers of neutrophils after TXT; arrows highlight positively stained neutrophils. (G) Analysis of caspase-3 staining and (H-J) representative slides of each group, 3 hours after injury. Values of outliers: #1 = 7.7x10⁴; #2 = 6.69x10⁴; #3 = 5.7x10⁴; #4 = 2.96x10⁵; #5 = 4.07x10⁵; #6 = 6.16x10⁴. (K-N) Positive nitrotyrosine staining was detected up to 21 days after surgery. O₂ treatment significantly decreased the level of nitrotyrosine. Scale bars: 50 μm; box-plots represent medians and quartiles, whiskers represent the minimum and maximum values. Data represent medians and quartiles. Specimen numbers for each group are depicted. *p<0.05, **p < 0.001.

Fig 2. Biomechanical and μCT analysis of the fracture callus 21 days post-injury.

(A-C) The binding stiffness, moment of inertia and the apparent Young’s modulus of the fracture callus were decreased following TXT, O₂ treatment abolished these effects. Data represent medians and quartiles. Specimen numbers for each group are depicted. *p<0.05, **p < 0.001.

Fig 3. Tissue composition of fracture calli 14 and 21 days after injury.

Callus composition of mice 14 and 21 days post-injury. (A) Mice with TXT displayed significantly more cartilage in comparison to O₂ treated mice after 14 days. (B) Analysis after 21 days did not reveal intergroup differences. (C-E) Representative Safranin-O stained callus sections 14 days after injury. Markedly more cartilage (stained red) was observed in F+TXT mice compared to the other groups. TOT = total osseous tissue, Cg = cartilage, FT = fibrous tissue. Scale bars: 500 μm. Data represent medians and quartiles. Specimen numbers for each group are depicted. *p<0.05.

Fig 4. Immunohistological stainings of fractured femurs for markers of inflammation, nitrosative stress and vascularization.

Left panels are representative slides of mice with isolated fracture, middle panels of animals with additional TXT, and right panels of mice with fracture, TXT and O₂ treatment. Images indicate cortical bone proximal to the fracture gap and periosteal callus, macrophage staining show the marrow cavity. (A-C) Neutrophil staining 3 days post-injury, (D-F) macrophage staining on day 7, (G-L) IL-6 and IL-10 staining on day 7, and (M-R) nitrotyrosine and PECAM-1 staining on day 14. Scale bars: 100 μm.