¹BG Klinik Tübingen, Eberhard Karls Universität Tübingen, Tübingen, Deutschland
²Bundeswehrkrankenhaus Ulm, Institute of Pathology and Molecular Pathology, Ulm, Deutschland
³Klinisch-Experimentelle Chirurgie, Universität des Saarlandes, Homburg, Deutschland
⁴BG Trauma Center, Eberhard Karls Universität Tübingen, Siegfried Weller Institute for Trauma Research, Tübingen, Deutschland

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Objectives and questions: Non-unions remain a major complication in trauma and orthopedic surgery. Autologous bone tissue still represents the gold standard in the treatment of large bone defects and non-unions. However, during revision surgery, there may be a significant delay between harvesting of the graft and its transplantation into the bone defect. The interruption of blood supply may lead to an impaired viability of the graft, resulting in healing failure. Therefore, we analyzed the gene expression and viability of autologous bone grafts after different time periods of warm ischemia to determine a critical ischemia time.

Material and methods: During surgeries with autologous bone graft transplantation from the iliac crest, samples were harvested for further analyses (n = 22 patients). The samples of each patient were stored in Ringer solution for 0, 30, 60 and 90 minutes. Gene expression of the grafts was analyzed by arrays analyzing the signaling pathways involving osteogenesis and oxidative stress. Subsequently, the potential signaling pathways were quantified by qRT-PCR analysis. Moreover, immunohistochemistry was used to quantify proliferation, apoptosis, hypoxia and cellular senescence. The data between early (0 and 30 minutes) and late time points (60 and 90 minutes) of ischemia was compared by the wilcoxon-test. Statistical significance was accepted for p < 0.05.

Results: Our results show a significant upregulation of the oxidative stress markers interleukin-8 and dual specificity phosphatase-1 at late time points (p=0.0042 and <0.0001, respectively). Moreover, the expression of matrix genes promoting chondrogenesis, such as cartilage oligomeric matrix protein und collagen 10A1 were significantly increased after late when compared to early time points (p=0.0002 and 0.0013, respectively). On the other hand, genes promoting osteogenesis, such as collagen 1A2, were significantly decreased at late time points (p=0.0021). Furthermore, the immunohistochemical analysis showed a significantly higher number of senescent p16-positive cells at early when compared to late time points of ischemia. The number of proliferating ki67-positive cells, however, decreased at later time points, whereas apoptotic cleaved caspase-3-positive cells significantly increased (p=0.0353).

Discussion and conclusions: These findings indicate the cancellous bone grafts suffer from an early stress response, which results in upregulation of oxidative stress markers and an increase of apoptotic cell death at later time points. This is associated with a decrease in pro-osteogenic gene expression and a reduced cell proliferation. Therefore, an ischemia time of 60 minutes and longer should be avoided to ensure the viability and functionality of cancellous bone grafts after transplantation.