High resolution micro-computed tomography evaluation of bone morphogenetic protein-4 expression in a mouse model.

Introduction Bone morphogenetic proteins (BMPs) are currently studied to determine their efficacy in inducing bone formation for the treatment of bone pathologies. Adenoviral vectors carrying BMPs effectively produce ectopic bone in a variety of in vivo models. Mice are relevant models in bone research field and ectopic bone formation can be detected and quantified by high resolution micro-computed tomography (micro-CT). We investigated non-invasively by micro-CT the transgene expression of HSVtkgene/ganciclovir system with in mice. Methods Four athymic nude mice, one not treated with viral vector, and three treated by intramuscular injection in left and right hindlimbs with high-titer FG-AdBMP-4TK and FG-Ad-BMP4 (9x1010 viral particles/mouse), were longitudinally monitored with an high resolution micro-CT imaging system (GE Healtcare eXplore Locus, spatial resolution 45?m) at baseline and after 28 days. The acquisition protocol adopted was: x-ray tube voltage: 80 kVp; x ray tube current 450 uA; Number of Views:400; Frames Averaged:2; Exposure Time:100 ms, 45um pixel size; scan time 18 minutes. Images were scaled into Hounsfield units (HU), and post-processed to obtain 3D volume rendering reconstructions. A thresholding technique based on the histogram of the gray-scale values (threshold set 620 HU, 200mg/cc) was used to quantify bone mineral concentration (BMC, mg) and density (BMD, mg/cc) in a volumetric region of interest defined from coxofemoral to tibio-tarsal joints (MicroView, 2.1.1). We compared BMD values by non-parametric analysis. Significance was set at P <0.05. Results Micro-CT of non treated mouse revealed the absence of intramuscular mineralization in quadriceps. In the mice treated with 9 x 1010pv/mouse,an increasing intramuscular ectopic bone formation was observed in both hindlimbs and BMC and BMD values increased at baseline and after 28 days (p=0,0009 and p=0,0043 respectively). Conclusions Micro-CT is a non invasive technique to accurately perform quantitative bone analysis and to monitor over time changes of bone parameters in mice. BMPs expression and the osteogenic process resulted in ectopic bone formation can be accurately demonstrated by qualitative and quantitative micro-CT in the hindlimb muscles of mice. References Sheyn D, et al. Gene Ther. 2008;15(4):257-66. Epub 2007 Nov 22. Lombardo B, et al. Curr Gene Ther. 2013; 13(3): 202-10