University of Michigan Department of Surgery, Section of Plastic Surgery, Ann Arbor, MI, USA.
Bone. 2013 May;54(1):28-34. doi: 10.1016/j.bone.2013.01.002. Epub 2013 Jan 11.
Heterotopic ossification (HO), or the abnormal formation of bone in soft tissue, occurs in over 60% of major burn injuries and blast traumas. A significant need exists to improve the current diagnostic modalities for HO which are inadequate to diagnose and intervene on HO at early time-points. Raman spectroscopy has been used in previous studies to report on changes in bone composition during bone development but has not yet been applied to burn induced HO. In this study, we validate transcutaneous, in-vivo Raman spectroscopy as a methodology for early diagnosis of HO in mice following a burn injury.
An Achilles tenotomy model was used to study HO formation. Following tenotomy, mice were divided into burn and sham groups with exposure of 30% surface area on the dorsum to 60° water or 30° water for 18s respectively. In-vivo, transcutaneous Raman spectroscopy was performed at early time points (5 days, 2 and 3 weeks) and a late time point (3 months) on both the tenotomized and non-injured leg. These same samples were then dissected down to the bone and ex-vivo Raman measurements were performed on the excised tissue. Bone formation was verified with Micro CT and histology at corresponding time-points.
Our Raman probe allowed non-invasive, transcutaneous evaluation of heterotopic bone formation. Raman data showed significantly increased bone mineral signaling in the tenotomy compared to control leg at 5 days post injury, with the difference increasing over time whereas Micro CT did not demonstrate heterotopic bone until three weeks. Ex-vivo Raman measurements showed significant differences in the amount of HO in the burn compared to sham groups and also showed differences in the spectra of new, ectopic bone compared to pre-existing cortical bone.
Burn injury increases the likelihood of developing HO when combined with traumatic injury. In our in-vivo mouse model, Raman spectroscopy allowed for detection of HO formation as early as 5 days post injury. Changes in bone mineral and matrix composition of the new bone were also evidenced in the Raman spectra which could facilitate early identification of HO and allow more timely therapy decisions for HO patients.
异位骨化（HO），即在软组织中异常形成骨，在超过 60%的大面积烧伤和爆炸创伤中发生。目前迫切需要改进现有的 HO 诊断方式，因为这些方式不足以在早期诊断和干预 HO。拉曼光谱已在之前的研究中用于报告骨发育过程中骨成分的变化，但尚未应用于烧伤诱导的 HO。在这项研究中，我们验证了经皮、活体拉曼光谱作为一种在烧伤后早期诊断小鼠 HO 的方法。
使用跟腱切开术模型研究 HO 的形成。在切开跟腱后，将小鼠分为烧伤组和假手术组，背部 30%的面积分别暴露于 60°C 水或 30°C 水中 18 秒。在早期（5 天、2 周和 3 周）和晚期（3 个月），对切开和未受伤的腿进行经皮、活体拉曼光谱检测。对相同的样本进行解剖，直至骨骼，并对切除的组织进行离体拉曼测量。在相应的时间点，通过 Micro CT 和组织学验证骨形成。
我们的拉曼探头允许非侵入性、经皮评估异位骨形成。拉曼数据显示，与对照组相比，受伤后 5 天切开的跟腱中骨矿物质信号明显增加，随着时间的推移差异逐渐增加，而 Micro CT 直到 3 周后才显示异位骨。与假手术组相比，烧伤组的离体 Raman 测量显示出 HO 量的显著差异，并且新的异位骨的光谱也与原有的皮质骨不同。
烧伤与创伤结合增加了形成 HO 的可能性。在我们的活体小鼠模型中，拉曼光谱允许在受伤后 5 天内检测到 HO 的形成。新骨的骨矿物质和基质组成的变化也在拉曼光谱中得到证实，这可以促进 HO 的早期识别，并为 HO 患者提供更及时的治疗决策。
