The results of this study indicate that EPR oximetry will be a valuable tool for monitoring oxygen levels during fracture repair in mice. EPR oximetry detected significantly lowered tissue oxygen levels at fracture sites after injury and it also successfully detected the expected changes of tissue oxygen levels after switching the breathing oxygen concentration from 21% to 100%. These data suggest that the sensitivity of EPR oximetry is adequate to assess tissue pO2 during fracture healing. In addition, we confirmed that the paramagnetic probe used in current study, LiPc, is biocompatible. LiPc crystals did not induce obvious inflammatory response and interfere with normal fracture healing. Further, LiPc crystals gave us the ability to make repeated measurements in the same location and to assess tissue formation at each site, providing a unique opportunity to examine the effect that oxygen has on stem cell differentiation and function. For instance, this technique will allow us to assess pO2 at a particular site and then at a later time point we can examine what type of tissue has formed in this region using histologic methods. Developing and employing these technologies for studying fracture repair in mice is of great importance, because compared to other rodent models, a murine model offers the advantage of using a multitude of genetically engineered strains, and there is an abundance of reagents for molecular and cellular analyses available.
The level of tissue oxygenation may affect fracture repair. Decreasing tissue oxygenation by inducing chronic systemic hypoxia11
or by ligating femoral artery20
delays fracture healing. Increasing tissue oxygenation by providing hyperbaric oxygen, on the contrary, can accelerate bone repair.25
Understanding the mechanisms underlying these effects is of great important. There is evidence suggesting that oxygen levels may have direct effects on stem cells. For example, the function and differentiation of chondrocytes and osteoblasts is affected by oxygen levels in vitro.26,27,28
Under experimental conditions that induce chronic hypoxia, fractured femurs did not exhibit signs of bone formation. Rather the calluses were comprised of abundant cartilage11
which indicates that chondrocyte differentiation can occur in a more hypoxic environment than osteoblast differentiation. In addition to the effect of hypoxia on stem cells, tissue oxygen levels may play other important roles during bone repair. Studies on wound healing have demonstrated that oxygen is involved in multiple processes, including cell survival, collagen and extracellular matrix deposition,4,5
and production of the oxidative burst used for intracellular bacterial killing.9,10
When tissue pO2 falls below 40 mmHg, angiogenesis, extracellular matrix formation, and resistance to infection are all impaired.29
In current study, we detected that tissue oxygen level at fracture sites dropped below this level after injury, especially after femoral artery ligation, warranting further studies on the effects of oxygen on cell death, angiogenesis, and infection during fracture healing.
In summary, these data illustrate that EPR oximetry is a promising technique to assess tissue oxygenation in murine fracture models. We have used EPR to examine the effect that tibial fracture has on pO2
during fracture repair. We have demonstrated that these fractures generate vascular damage that significantly reduces tissue oxygen levels to levels that could impair healing. However, mice exhibit exceptional regenerative capacity. Normally no difficulties in skeletal healing are detected without the inclusion of a major traumatic insult such as cautery of the periosteum30
or resection of the femoral artery.20
Even in the latter situation the mice salvage their limbs, and eventually the skeletal injury heals.20
These results suggest that tissue oxygenation returns to normoxic levels rapidly after injury. Thus, understanding this time course and evaluating the role that oxygen plays during fracture repair are important parameters for elucidating and exploiting the mechanisms that underlie the regenerative potential during fracture repair in mice.