Agreement Between μCT and Histomorphometrics: Rat Calvaria Defect Model Study

Objectives: The critical-size calvarial defect (CSD) in rats is widely used to investigate the bone regenerative potential of biomaterials and biologics. Historically, histology has been used to evaluate new bone formation, but it is a time consuming, expensive, and destructive method. The aim of this study was to assess agreement among histomorphometric, microcomputed tomography (μCT), and radiographic analyses of new bone formation in the CSD model.
Methods: One hundred and two, 11-13 week-old, male Sprague-Dawley rats were used. Æ8-mm, through-through craniotomy defects were created using standardized procedures. Absorbable collagen sponges soak-loaded with various doses of rhBMP-2 were implanted to support bone formation. Defects were covered with a titanium mesh and wounds were closed using surgical staples. Animals were euthanized at 4 weeks. Samples were radiographed using a digital imaging instrument (Faxitron), scanned using an ex vivo μCT (Skyscan), and then decalcified, sectioned and stained for histological analysis (H&E and picrosirius red). New bone formation inside the defects was measured following established protocols. Volumetric reconstructions were used for uCT analysis, and central histologic sections were used for the histometrics. The region of interest (ROI) was standardized at Æ7.5mm to avoid the margins of the defect, and four different heights were evaluated for μCT and histological analysis: 0.5, 1, 1.5, and 2 mm.
Results: The highest new bone area and volume were obtained when the ROI was Æ7.5x1mm for the uCT and histology (22.2±10.0 and 26.0±9.6, respectively). A moderate agreement was observed between histological percent bone area and μCT percent bone volume (Concordance Correlation Coefficient ≈ 0.70, mean difference <4%), as long as the ROI was ≥1mm in height. Standard digital radiographs significantly overestimated percent bone area fill when compared to histological and μCT measurements.
Conclusions: Under the present circunstances, μCT assessments of bone volume showed moderate agreement with histomorphometric assessments of bone area.