Establishment and Characterization of a Dynamic-Flow Ex Vivo Rat Long-Bone Defect Model for Bone–Biomaterial Interaction Studies

Ex vivo bone culture models preserve aspects of the native three-dimensional bone microenvironment and may provide an intermediate platform for biomaterial screening. However, the application of dynamic-flow whole long-bone defects for time-course evaluation of bone–biomaterial interactions remains insufficiently characterized. We established a dynamic-flow ex vivo rat long-bone defect model and evaluated its capacity to maintain cell viability and to detect biomaterial-associated remodeling responses. Long bones obtained as biological by-products were subjected to standardized metaphyseal defects, with defects either left untreated or filled with injectable bone cement. Explants were maintained under continuous perfusion for 21 days. The presence of viable and membrane-compromised cells at Day 21 was assessed qualitatively by Calcein AM/propidium iodide staining; tissue morphology, mineralized bone and total ossified tissue fractions, and TRAP-positive osteoclast number were assessed on Days 7, 14, and 21. Calcein-positive cells remained detectable in the imaged regions after 21 days of dynamic-flow culture, and mineralized bone was preserved in both groups throughout this period. Cement-treated defects showed a higher mineralized tissue fraction (p < 0.001) and a higher total ossified tissue fraction (p < 0.01) than untreated defects, with no significant effect of culture time on either tissue-fraction endpoint. TRAP-positive osteoclast number was also higher in cement-treated defects (p = 0.004), and the significant Group × Time interaction (p = 0.022) indicates that this difference depended on culture duration. The findings demonstrate the feasibility of using a dynamic-flow whole-bone defect model to study bone–biomaterial interactions ex vivo. The model may serve as an intermediate experimental platform for bone–biomaterial interaction studies before in vivo evaluation.