Automated measurement of lamellar thickness in human bone using polarized light microscopy.

Lamellar bone formed in individuals with moderate and severe osteogenesis imperfecta (OI) is often composed of lamellae that are structurally abnormal. Measuring the thickness of these lamellae can be helpful in assessing the effect of specific collagen and collagen-related mutations on OI bone synthesis. Manual measurement of lamellar thicknesses in large quantities is very time consuming. The method for automated measurement described in this article utilizes an image processing script to identify the average thickness of multiple lamellae automatically from histologic images of bone. This allows for faster measurements that are less prone to human error and can account for variability in the thickness of a lamella along its length.•OI and control bone samples are prepared per the glycol methacrylate resin (JB-4 plastic) technique and viewed using polarized light microscopy.•Ideal bone regions for measurement are identified using specific qualitative criteria designed to ensure uniform and accurate thickness measurements.•The method was validated with dataset containing 211 lamellae from control bone and 212 lamellae from OI bone.

Lamellar thickness measurements in control and osteogenesis imperfecta human bone, with development of a method of automated thickness averaging to simplify quantitation.

Lamellar bone that forms in moderate and severe osteogenesis imperfecta (OI) is composed of structurally irregular lamellae compared to those in control bone. OI and control cortical bone fragments were prepared for light microscopy in standardized fashion: decalcified, embedded in plastic, sectioned and stained with toluidine blue. Polarization light microscopy (PLM) was used to demonstrate and quantify bright and dark lamellar thicknesses in cortical bone fragments from 5 patients with moderate to severe OI in whom type I collagen structural/molecular defects were detected and in control bone from 5 patients. Rigid selection criteria identified lamellar regions for quantification. Thicknesses of bright and dark lamellae were measured manually at 20X magnification using a histomorphometric image analysis system. A method of automated thickness averaging was developed to determine lamellar thicknesses from PLM images to make measurement faster. Our study demonstrates, for the first time, that in OI bone from patients with type I collagen structural/molecular defects mean lamellar thickness measurements (along with the bright and dark lamellar thicknesses) were less than those in control bone by statistically highly significant differences. The mean value for bright lamellae was less than that for dark lamellae in both control and OI bone. The ratio of mean values for bright/dark lamellar thicknesses was the same in control and OI bone. The automated method obtained similar results to the manual method. Lamellar bone in moderate and severe OI with type I collagen defects is composed of thinner and less structurally regular lamellae than those in control bone. This finding indicates that lamellar thickness measurements can be helpful in assessing the effect of specific collagen and collagen-related mutations on OI bone synthesis and warrant inclusion in research and clinical histomorphometric assessments.