The regenerative capacity of bone tissue and autograft dust exposed to heat shock induced by high speed surgical cutting

High speed surgical cutting tools induce heat (>47°C) for short durations on bone tissue, leading to a thermally-induced cell death in bone cells. Post-operative bone healing at the cut surface and bone dust are important for ensuring the success of surgery and preventing long recovery time for patients. Autograft bone dust prepared using surgical burs can be re-introduced into patients to enhance bone healing. Recent in vitro and in vivo studies have provided evidence that thermal elevations can in fact trigger osteogenic responses by bone cells, enhancing mineralisation and new bone formation. However, whether bone dust and the cut surface exhibit an osteogenic response to heat shock induced by cutting conditions is unknown. Moreover, the influence of cutting conditions on the osteogenic potential of bone dust is not understood. In particular, the influence of the tissue source, the design of the cutting tool and the use of irrigation have not been investigated. Thus, the global aim of this PhD Thesis is to assess the ex-vivo osteogenic potential of autograft bone dust and the cut surface resected under different cutting conditions, and associated temperature elevations, using surgical burs for cutting and heat induction. The duration of bone surface exposure to elevated temperatures during surgical bone cutting and the extent of heat dissipation and its effect on the bone healing capacity are not well understood. The first study of this PhD thesis sought to address this gap by conducting experimental studies to determine (1) temperature generation and dissipation during surgical bone cutting with no irrigation and (2) the ex-vivo regenerative potential of bone dust and the cut surface. During surgical cutting of bovine bone using burs (Multi-Flute, 2Flute) the relative surface temperatures (using a thermal camera) and dissipated temperatures (measured by embedded thermocouples) were quantified. Ex-vivo culture of bone dust and the cut surface samples were conducted up to 20 days post-cutting. Histological staining and biochemical assays (Alamar Blue, ALP, Alizarin red staining) quantified osteonecrosis and osteogenesis. The results of this study revealed that superficial heat generation induced by high speed surgical burs for short durations triggered an osteogenic response in bone dust, which was associated with apoptotic cell death. The cutting temperature was significantly higher with the Multi-Flute compared to the 2Flute bur, but the % heated tissue volume was lower and faster cooling was observed. Interestingly, the ex-vivo osteogenic capacity of both the bone dust and the cut surface were enhanced when using the Multi-Flute bur, even though increased apoptotic thermal damage was observed. To reduce the risk of excess tissue necrosis during surgical cutting, irrigation is commonly used to minimise temperature elevation. Yet, the influence of irrigation approaches, namely using a surgical assistant to apply irrigation (manual) or using an automated irrigation (continuous) device, have not been investigated and as such there is no consensus regarding a favourable approach to minimise tissue necrosis or osteogenesis. The objective of the second study was to determine how irrigation techniques influence cellular responses to surgical bone cutting. Specifically, this study sought to (1) compare superficial temperature generation and dissipation during high speed surgical bone cutting with either manual or continuous irrigation and (2) determine the influence of irrigation technique on the ex-vivo regenerative potential of bone dust and the cut surface. The results of this study revealed that dissipated temperatures remained below 30°C throughout the cutting for both types of irrigation. Interestingly, the cut surface was preserved when continuous irrigation was used during cutting. In contrast, when using manual irrigation increased apoptotic thermal damage was observed and the ex-vivo osteogenic capacity of bone dust was enhanced. This study can inform surgical irrigation approaches to preserve the osteogenic potential of bone dust and the cut surface. The most common implantation site for such autografts is in the spine, where bone dust is subjected to in vivo biophysical conditions, including compression and torsional forces. However, previous studies of autograft bone dust have been conducted under static conditions and so the mineralisation potential of bone dust in a mechanical environment representative of in vivo conditions is unknown. The objective of the third study was to assess and compare ex-vivo osteogenesis by bone dust using a custom-made compression bioreactor to recreate in vivo mechanical stimulation. The effect of irrigation was assessed under both static and mechanical loading conditions. Interestingly, in this study a 3mm bur was used and the irrigation method (manual or continuous irrigation) did not influence the mineralisation potential of bone dust under either static or mechanically stimulated conditions. Here it is reported for the first time that the osteogenic capacity (in terms of viability and mineralisation responses) was significantly higher for mechanically stimulated bone dust when compared to those cultured under static conditions. These results suggest that the osteogenic potential of bone dust may be enhanced by the mechanical loading conditions that exist in vivo. Together, these studies provide (1) an insight into the osteogenic response of bone tissue resected with different design burs and different irrigation approaches during cutting, and (2) uncovers how the mechanical environment that arises in vivo might influence the osteogenic capacity of autograft bone dust. The information elucidated from this body of work can inform future surgical instrument design to improve post-operative bone tissue regeneration.2023-01-0