Prolonged ischemia induces oxidative stress, affects extra cellular matrix gene expression and compromises the viability of cancellous bone grafts

Background Despite growing insights into the pathophysiology of non-union formation, failed fracture healing remains a major complication in trauma and orthopedic surgery. The transplantation of cancellous bone grafts represents the gold standard for the treatment of atrophic non-unions and large-scaled bone defects. Depending on the type of procedure and the available personnel, the bone grafts may be exposed to a significant period of intraoperative ischemia before the transplantation to the defect site. This ischemia may have detrimental effects on the quality and functionality of the grafts. Methods Therefore, we analyzed in this study the effects of different periods of ischemia (0, 30, 60 and 90 minutes) on oxidative stress, gene expression and viability of autologous bone grafts, to determine a critical ischemia time window for cancellous bone graft transplantation. Graft samples were harvested from 24 patients undergoing revision surgery due to bone healing failure. The samples were analyzed by mRNA profiler arrays, reverse transcription polymerase chain reaction (RT-PCR) and immunohistochemistry. Results An ischemia of 60 minutes or longer induced the expression of pro-inflammatory and stress-induced genes, such as CXCL8 , JUN and DUSP1 . This was associated with early cell stress within the grafts, as indicated by the presence of hypoxia-inducible factor (HIF)-1α-positive cells and an increased number of senescent p16-positive cells. Additional immunohistochemical analyses revealed a significantly higher number of apoptotic cleaved caspase-3-positive cells at 60 and 90 minutes of ischemia, demonstrating a compromised viability of the grafts. RT-PCR analyses revealed a shift from a pro-osteogenic towards a pro-chondrogenic extracellular matrix (ECM) gene expression profile, along with evidence for potentially compromised angiogenesis and hematoma formation at the later transplantation site. Conclusion Taken together, these findings indicate that periods of ischemia of 60 minutes or longer should be avoided during cancellous bone graft transplantation to preserve graft function and regenerative capacity.