Prolonged Ischemia Induces Cellular Stress, Stimulates Extracellular Matrix Remodeling and Compromises the Viability of Human Cancellous Bone Grafts

Abstract

Fracture healing failure remains a major complication in trauma and orthopedic surgery. The transplantation of autologous cancellous bone grafts represents the gold standard for the treatment of atrophic non-unions. However, during revision surgery the grafts can be exposed to a significant period of intraoperative ischemia, which may have detrimental effects on their quality and functionality. Therefore, we analyzed the effects of different periods of ischemia (0, 30, 60 and 90 min) on cellular stress, gene expression and viability of the bone grafts, to determine a critical ischemia time window for transplantation. Graft samples were harvested from 24 patients undergoing revision surgery due to bone healing failure. Analyses included mRNA profiler arrays, reverse transcription polymerase chain reaction (RT-PCR) and immunohistochemistry. Ischemia lasting 60 min or longer induced the expression of stress-induced genes, such as JUN and DUSP1. This was associated with early cellular 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 at early time points of ischemia. Additional analyses revealed a significantly higher number of apoptotic cleaved caspase-3-positive cells at 60 and 90 min of ischemia, demonstrating a compromised viability of the grafts. Moreover, RT-PCR analyses revealed a shift from a pro-osteogenic towards a pro-chondrogenic extracellular matrix (ECM) gene expression profile. Taken together, periods of ischemia of 60 min or longer after tissue harvesting should be avoided during cancellous bone graft transplantation to preserve graft viability.