Background: Bone allograft preservation may influence graft biology and incorporation. Importantly, preservation strategy also determines tissue-bank workflow, particularly manufacturing lead time, quarantine duration, and release-to-use logistics. However, comparative in vivo evidence remains inconsistent across models and endpoints. Objective: To compare early bone-healing profiles of cryopreserved versus fresh-frozen cortical allografts in a rabbit femoral defect model using radiographic, histological, and immunohistochemical outcomes at 4 and 8 weeks. Methods: A controlled in vivo study was performed in New Zealand white rabbits with a standardized femoral cortical defect. Fresh-frozen grafts underwent a minimum 30-day storage manufacturing period at −80°C prior to implantation. This predefined ≥30-day period was chosen to operationally represent tissue-bank quarantine and release workflows, in which grafts remain in frozen storage until donor eligibility documentation and release criteria are completed. Radiographic union was assessed using mRUST at weeks 4 and 8 by two blinded assessors. Histology quantified osteoblasts and microvascular profiles at the graft–host interface. Immunohistochemistry evaluated VEGF and TGF-β (and additionally BMP and TNF-α) using a predefined semi-quantitative scoring system by two blinded observers, with discrepancies resolved by consensus. Non-parametric tests were used (α=0.05). Results: Both groups showed increasing mRUST from week 4 to week 8 with no between-group differences at week 4 (median 4.5 vs 4.0; p=0.495) or week 8 (median 7.5 vs 6.5; p=0.343). Osteoblast counts were numerically higher in the CRYO group at week 4 (median 85.5 vs 75.5; p=0.070) but not significantly different at week 8 (median 34.0 vs 14.0; p=0.250). Microvascular counts and VEGF/TGF-β scores did not differ significantly at either time point (all p>0.05). BMP and TNF-α showed no apparent between-group differences on semi-quantitative assessment. Conclusion: In this rabbit femoral defect model, cryopreserved and fresh-frozen cortical allografts demonstrated comparable radiographic and tissue-level healing profiles through 8 weeks. Cryopreservation may therefore be considered a feasible alternative preservation option in centers with liquid nitrogen infrastructure, while longer follow-up and functional endpoints are needed to detect potential preservation-related differences in remodeling and mechanical performance.. Graphical abstract A schematic showing (1) graft preparation (fresh-frozen vs cryopreserved), (2) femoral defect creation and graft implantation, and (3) outcomes assessed (mRUST, histology, VEGF/TGF-β/BMP/TNF-α).
Keywords: bone allograft; cryopreservation; fresh-frozen; bone healing; rabbit model
How to cite this article:Widagdo MFA, Edward M, Basuki MH, Mahyudin F., Bone Graft Preservation Methods: Insights from a Rabbit Model Study comparing Fresh-Frozen and Cryopreservation .Int J Drug Deliv Technol. 2026;16(1s): 955-963; DOI: 10.25258/ijddt.16. 955-963