Measuring and modulating angiogenesis during fracture healing

Complications in fracture healing often result from insufficient blood supply to the healing bone. In order for bone to form, blood vessels must invade the tissue, bringing proper nutrients and cells to the tissue. The formation of blood vessels, called angiogenesis, is critical to fracture healing. This project examines the growth of blood vessels during fracture healing in a rat femur as well as how the mechanical environment may alter blood vessel growth.
In this project micro-computed tomography (microCT) and laser Doppler and will be used to quantify and visualize angiogenesis at important time points during fracture healing.
Angiogenesis is necessary, but not sufficient, for bone formation during fracture healing. Under certain mechanical environments, bone growth does not occur even with an adequate blood supply. External fixation on a rat femur fracture allows one to alter the mechanical environment during the healing process. The influence of three different loading conditions on fracture healing will be investigated: (1) static fixation, (2) interfragmentary compression, and (3) static tension. Vascular flow, volume, and structure will be assessed for the three loading conditions and compared. After removing the bone from the body, the stiffness properties of the callus, which measure how well it has healed, will be determined.
Understanding the role of angiogenesis in fracture healing is valuable in determining why some fractures do not heal under certain conditions.

Angiogenesis, the growth of blood vessels, is critical for fractures to heal and for bone to grow. There is currently no quantitative way to analyze 3D vasculature during fracture healing. It is also unclear how the local mechanical environment may affect the angiogenic process.

The objectives of this research are to:
(1) Determine how laser Doppler flowmetry and micro-computed tomography (microCT) can be utilized to visualize and quantify angiogenesis during fracture healing in a rat tibia. These techniques will be used to analyze blood flow, vascular volume, three dimensional vascular structure, and location of the angiogenesis in the healing fracture callus.

(2) Identify angiogenic trends during fracture healing. MicroCT and laser Doppler will be used to determine the temporal characteristics of angiogenesis during fracture healing, including when increased vasculature appears and how long it remains.

(3) Explore how the local mechanical environment influences the angiogenic process and ultimately affects fracture healing. By applying different loading conditions with the external fixator, the local mechanical environment of the fracture callus can be modulated. Specifically the external fixator will impose (1) static fixation, (2) interfragmentary compression, or (3) distraction with static tension. Vascular flow will be measured during healing using laser Doppler. After sacrifice the tibias will be scanned in the microCT at high resolution to quantify the vascular volume. Mechanical properties of the explanted tibia will be determined using three-point bending test, and immunohistochemistry will examine cell type and expression of angiogenic and bone proteins in the fracture callus.

This research will help in understanding how angiogenesis develops temporally and spatially over time in fracture healing. This research has clinical implications in the treatment of fractures, especially non-unions, ischemic bone, as well as normal bone formation during growth.