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. 2013 Jan;52(1):220-9.
doi: 10.1016/j.bone.2012.09.037. Epub 2012 Oct 12.

The role of oxygen during fracture healing

Chuanyong Lu  1 , Neema Saless, Xiaodong Wang, Arjun Sinha, Sebastian Decker, Galateia Kazakia, Huagang Hou, Benjamin Williams, Harold M Swartz, Thomas K Hunt, Theodore Miclau, Ralph S Marcucio
Affiliations

The role of oxygen during fracture healing

Chuanyong Lu et al. Bone. 2013 Jan.

Abstract

Oxygen affects the activity of multiple skeletogenic cells and is involved in many processes that are important for fracture healing. However, the role of oxygen in fracture healing has not been fully studied. Here we systematically examine the effects of oxygen tension on fracture healing and test the ability of hyperoxia to rescue healing defects in a mouse model of ischemic fracture healing. Mice with tibia fracture were housed in custom-built gas chambers and groups breathed a constant atmosphere of 13% oxygen (hypoxia), 21% oxygen (normoxia), or 50% oxygen (hyperoxia). The influx of inflammatory cells to the fracture site, stem cell differentiation, tissue vascularization, and fracture healing were analyzed. In addition, the efficacy of hyperoxia (50% oxygen) as a treatment regimen for fracture nonunion was tested. Hypoxic animals had decreased tissue vascularity, decreased bone formation, and delayed callus remodeling. Hyperoxia increased tissue vascularization, altered fracture healing in un-complicated fractures, and improved bone repair in ischemia-induced delayed fracture union. However, neither hypoxia nor hyperoxia significantly altered chondrogenesis or osteogenesis during early stages of fracture healing, and infiltration of macrophages and neutrophils was not affected by environmental oxygen after bone injury. In conclusion, our results indicate that environmental oxygen levels affect tissue vascularization and fracture healing, and that providing oxygen when fractures are accompanied by ischemia may be beneficial.

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Figures

Fig. 1
Fig. 1
Effects of environmental oxygen on fracture healing in a mouse tibia fracture model. Histomorphometric analyses of the volume of callus, new bone, and cartilage in the callus were performed on HBQ stained tissue sections under a stereology microscope (n=4–6/group/time point). (A) Hypoxia-treated fractures. (B) Hyperoxia-treated fractures. * p<0.05, ** p<0.01.
Fig. 2
Fig. 2
Effects of environmental oxygen on tissue vascularization at 3 days after fracture (n=5–6/group). Lactate and VEGF levels were measured in tissue lysates of fracture calluses. Length density and surface density of blood vessels were determined using stereology. * p<0.05, ** p<0.01.
Fig. 3
Fig. 3
Environmental oxygen does not affect chondrocyte differentiation at 5 days after fracture. (A–D) Non-stabilized tibia fracture treated with normoxia. (E–H) Non-stabilized tibia fracture treated with hypoxia. (I–L) Non-stabilized tibia fracture treated with hyperoxia. (A,E,I) HBQ staining shows fracture callus and cartilage was stained blue. (B,F,J) Transcripts of collagen type II (Col 2), (C,G,K) collagen type X (Col 10), and (D,H,L) vascular endothelial growth factor (VEGF) were detected by in situ hybridization. Col 2 is a marker for chondrocytes while Col 10 and VEGF are markers for hypertrophic chondrocytes. No significant difference in the expression of these markers was detected between three groups. (M) Histomorphometric analysis shows similar ratio of the volume of cartilage and the volume of new bone in fracture calluses of three groups at 5 days after injury. Scale bar: A,E,I = 400μm, B–D,F–G,J–L = 200μm.
Fig. 4
Fig. 4
Hypoxia does not induce more cartilage formation in stabilized fractures. (A) Tibia fractures were rigidly stabilized with an external fixator. Under normoxic conditions, a small amount of bone and cartilage (Cart) is present at the fracture site. (B) Under hypoxic conditions, fracture healing is inhibited and no obvious bone and cartilage formation was observed in the callus. Scale bar = 1mm.
Fig. 5
Fig. 5
Longitudinal measurements of tissue O2 levels during fracture healing. (A) Longitudinal oxygen measurements made by EPR during the first 10 days of healing. (B–E) Histologic sections at day 10 illustrate that LiPc crystals are located in cartilage (B, C), in muscle (D), and in the outer region of the callus (E). (F) O2 levels differ depending on crystal location. In general areas of cartilage and bone formation have lower O2 levels than the outer area of the callus and the muscle during early fracture healing, especially at day 1 (p < 0.05). Scale bar=100μm.
Fig. 6
Fig. 6
Environmental hyperoxia improves the repair of ischemic fractures. (A–E) Histomorphometry. ** p<0.01. Length density and surface density of blood vessels were analyzed at 5 days after fracture. (F) Ischemic fracture treated with normoxia did not heal completely at 28 days after fracture. HBQ staining and the blue color shows cartilage remnant in the callus. Outlined area is fibrous tissue. (G) Ischemic fracture treated with hyperoxia healed at 28 days after fracture and no cartilage remnant was detected. Scale bars = 2mm.
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