Purpose:  Biomechanical, densitometric, and histologic analyses have been the mainstay for reproducible outcome measures for investigation new bone formation and osseous healing. Here we report the addition of radiomorphometric vascular analysis as a quantitative measure of vascularity and vasculogenesis in the murine mandible. To our knowledge this is the first description of this technique in the mandible and will lay down the groundwork for using micro-CT to quantitatively evaluate the temporal and spatial pattern of angiogenesis during distraction osteogenesis (DO) as well as the forms of bone regeneration and repair in the craniofacial skeleton. 

Methods:  Sprague-Dawley rats were euthanized and the left ventricle was catheterized and flushed with normal saline containing heparin (1000u/ml). After pressure fixation, the vasculature is injected with 15ml of a radiopague silicone rubber compound containing lead chromate.  The mandible was then treated with Cal-EX II solution to decalcify the bone and facilitate image thresholding of the mandibular vasculature from the surrounding tissues.  The left hemimandibles were imaged using high-resolution micro-CT imaging system.  The scanner is set to a voltage of 80kV and a current of 80цA.  The volume of interest (VOI) is defined as the volume counting from the end of third molar every 15slices for a total of 10 times (Figure 1). Six separate radiomorphometric vascular metrics including vessel volume (VV), connectivity, number, thickness, and separation as well as vessel volume fraction (VVF) were calculated.     

Results:  Radiomorphometric values were analyzed using 3 different thresholds at 1000, 1200, and 1400 on micro-CT.  Alteration of the threshold value had an observable effect on the radiomorphometric parameters of the vasculature (Figure 2).  As threshold was increased, imaging of smaller vasculature was minimized and that of lager vasculature was maximized. To capture the maximal vascular content of the bone it was evident from the imaging that 1000 was the optimal threshold for analysis. Data from 6 hemimandibles were compared.  One of the hemimandibles was excluded from the study due to less optimal perfusion technique.  Descriptive statistics were used to compare variance among all radiomorphometric parameters.  Minimal variance in quantitative measurement resulted in reproducible metrics for each of the radiomorphometric parameters. Vascular metrics variances of the native murine mandible were as follows:  Vessel Volume (0.034), Vessel Volume Fraction (0.000), Vessel Number (0.004), Vessel Thickness (0.000), Vessel Separation (0.079), and Connectivity (0.159).   

Conclusions:  For the first time, to our knowledge, we have demonstrated that micro-CT imaging combined with the use of perfused contrast agents and bone decalcification provides a robust methodology for evaluation of rat mandibular vascular networks.  Specifically, micro-CT is an advantageous quantitative method to calculate vascularization compared with x-ray microangiography, laser Doppler flow, micro sphere or histology because it provides high-resolution, quantitative, 3D, and objective data analysis.  The data presented here will serve as baseline reproducible measurements that can be used to compare and assess the role of angiogenesis during mechanically induced bone formation in DO as well as other forms of bone regeneration and repair in the craniofacial skeleton.