An osteoporotic bone model: developing and validating an ex-vivo bone demineralization protocol

Studies estimate that there are 500 million osteoporotic patients worldwide. Osteoporosis leads to 2.7 million hip fractures annually. These fractures usually require synthetic implants known as total hip replacement. Orthopedic implant development is an ongoing process, which requires numerous tests to evaluate their feasibility, in addition to the International Standards Organization regulatory tests. These tests require human cadaveric bones and animal in vivo experiments, which are associated with high costs, are time-consuming and limited through regulations, which poses a challenge. Following the replacement, reduction and refinement principle in animal testing (3Rs), this study aims to develop and evaluate a protocol where osteoporotic samples are prepared through ex vivo procedure using sheep bones from the food chain, given their availability and size, which is considerably similar to human cadaveric bones. Sheep femurs were demineralized using hydrochloric acid at different time intervals, resulting in changes in the bone architecture, mineral density, and mechanical properties. Demineralized specimens exhibited progressive reductions in mechanical performance and trabecular integrity with increasing demineralization time. Young's modulus decreased from 110.7 ± 28.8 MPa in Control group to 88.7 ± 16.0 MPa and 57.7 ± 6.0 MPa in the 48H and 96H groups, respectively, accompanied by corresponding reductions in failure load. Trabecular microstructure was significantly altered, with trabecular thickness decreasing and trabecular separation and porosity increasing, reaching an approximately 30% higher porosity in the 96H group compared with Control group. Volumetric BMD (vBMD) was significantly reduced by both demineralization treatments (33% relative decrease). Despite the limited sample size, consistent trends indicate meaningful changes with demineralization. The observed structural and mechanical properties fall within reported ranges for human osteoporotic trabecular bone, supporting the use of demineralized sheep bone as a cost-effective ex vivo model for osteoporotic studies and preliminary orthopedic device evaluation.