Paraffin Fixed Human Trabecular Bone Specimens for Study of Osteoporosis

Abstract

Examining and quantifying specific measures of trabecular bone to investigate the progression of osteoporosis presents a challenge. Specifically, the development of analytical and computational strategies to interpret computed tomography (CT) generated data sets requires gross anatomical specimens that closely approximate the intraosseous matrix of the marrow while maintaining the structural integrity of the trabecular network. Moreover, these specimens must be durable, easily transported, and stored at room temperature (e.g., do not require freeze/thaw cycles). Here we investigated if paraffin impregnated bone specimens maintained the trabecular network and bone density of prepared fresh‐frozen specimens used in imaging studies for osteoporosis. Standardized fresh‐frozen human distal tibia specimens (n = 12), from the institution’s Deeded Body Program, were scanned using multi‐row detector computed tomography (MDCT) and micro computed tomography (micro‐CT) imaging at different times in the preparation process to evaluate and quantify the preservation of microstructure and continuity of cortical and trabecular bone constituents. Once scanned, the specimens were placed in a series of acetone baths to remove water and marrow. With the de‐marrowing procedure complete, four specimens were rescanned using MDCT and micro‐CT. De‐marrowed specimens were then impregnated, under vacuum controlled conditions, with either a silicone polymer, gelatin matrix solution, or paraffin wax. After impregnation and curing, the specimens were re‐scanned. Analysis demonstrated a high correlation in the region of interest (ROI) for trabecular bone network area density from micro‐CT data sets of the preprocessed and de‐marrowed states (r = 0.99). Quantitatively, wax impregnated specimens demonstrated congruence in MDCT bone volume fraction (r = 0.92) and trabecular network area (r = 0.94) measures when the preprocessed and impregnated states were correlated. Results were more robust in the micro‐CT data in the bone volume fraction (r = 0.97) and the trabecular network area (r = 0.99). MDCT analysis at the (ROI) corresponding to 14‐16% length measured from the distal aspect of specimens demonstrated an increased estimate of bone mineral density (BMD) relative to scans acquired with the unmodified tissue (polymer +160 mg/cc; gelatin +58 mg/cc), whereas the paraffin wax condition slightly underestimated BMD (‐21 mg/cc). Of the impregnating procedures, paraffin wax provided the best qualitative specimens for handling, storage, and processing time. Together these data suggest that the trabecular structure is preserved through the de‐marrowing procedure and demonstrates that specimens can be transferred away from cold storage. Impregnating bone specimens with paraffin wax effectively provides a space occupying matrix that protects the delicate trabecular network without altering the ability to quantify important biomechanical measures in clinical CT as well as micro‐CT imaging for assessing bone integrity for the study of osteoporosis. This procedure creates specimens that are easily stored and transported, can be scanned across various imaging modalities, and effectively retain the trabecular micro‐structure.