Comparison of Bone Healing Using Fresh-Frozen Allografts Preserved by Deep-Freezing Versus Liquid Nitrogen Methods

Abstract

Limb salvage surgery has become the preferred treatment for primary malignant bone tumours, offering functional preservation without compromising oncologic outcomes. Biologic reconstruction using recycled bone allografts processed via deep freezing or liquid nitrogen is increasingly used, particularly in young patients. While both methods aim to maintain structural integrity and osteoconductivity, their effects on bone healing may differ. This study aims to compare the bone healing outcomes of liquid nitrogen-treated and deep-frozen allografts in the context of biologic limb reconstruction. This experimental study employed a post-test control group design involving 16 male New Zealand White rabbits. Animals underwent standardized radial bone defect surgery and were divided into two groups receiving either deep-frozen or liquid nitrogen-preserved bone allografts. Evaluations were conducted histologically, immunohistochemically (VEGF and TGF-β), and radiologically (modified Radiographic Union Scale for Tibial fractures, mRUST) at 4- and 8-weeks post-operation. Comparative analyses demonstrated no significant differences between the two preservation methods regarding osteoblast counts, micro vascularization, VEGF and TGF-β expressions, and mRUST radiological scores. Both methods exhibited similar progression in bone healing with p-values >0,05, respectively. The findings suggest that deep-freezing and liquid nitrogen preservation methods offer equivalent outcomes in supporting bone healing processes. Observed patterns align with known biological phases of bone regeneration, emphasizing comparable efficacy between both methods. Both deep-freezing and liquid nitrogen preservation methods are effective and comparable for bone allograft preservation. Further studies with longer observation periods, biomechanical evaluations, and clinical translational models are recommended to validate these findings