Histomorphometric analysis of critical-sized bone defects using Osteomeasure and Aperio systems

Abstract

Most histological evaluations of critical-sized bone defects are limited to the analysis of a few regions of interest at a time. Manual and semi-automated histomorphometric approaches often have intra- and inter-observer subjectivity, as well as variability in image analysis methods. Moreover, the production of large image data sets makes histological assessment and histomorphometric analysis labour intensive and time-consuming. Herein, we tested and compared two image segmentation methods: thresholding (automated) and region-based (manual) modes, for quantifying complete image sets across entire critical-sized bone defects, using the widely used Osteomeasure system and the freely downloadable Aperio Image Scope software. A comparison of bone histomorphometric data showed strong agreement between the automated segmentation mode of the Osteomeasure software with the manual segmentation mode of Aperio Image Scope analysis (bone formation R2 = 0.9615 and fibrous tissue formation R2 = 0.8734). These results indicate that Aperio is capable of handling large histological images, with excellent speed performance in producing highly consistent histomorphometric evaluations compared to the Osteomeasure image analysis system. The statistical evaluation of these two major bone parameters demonstrated that Aperio Image Scope is as capable as Osteomeasure. This study developed a protocol to improve the quality of results and reduce analysis time, while also promoting the standardization of image analysis protocols for the histomorphometric analysis of critical-sized bone defect samples.