Groven Rald V M, Nauta Sylvia P, Gruisen Jane, Claes Britt S R, Greven Johannes, van Griensven Martijn, Poeze Martijn, Heeren Ron M A, Porta Siegel Tiffany, Cillero-Pastor Berta, Blokhuis Taco J
Division of Traumasurgery, Department of Surgery, Maastricht University Medical Center, Maastricht, Netherlands.
Department of Cell Biology-Inspired Tissue Engineering, MERLN Institute for Technology-Inspired Regenerative Medicine, Maastricht University, Maastricht, Netherlands.
Front Chem. 2022 Mar 3;9:780626. doi: 10.3389/fchem.2021.780626. eCollection 2021.
Fracture healing is a complex process, involving cell-cell interactions, various cytokines, and growth factors. Although fracture treatment improved over the last decades, a substantial part of all fractures shows delayed or absent healing. The fracture hematoma (fxh) is known to have a relevant role in this process, while the exact mechanisms by which it influences fracture healing are poorly understood. To improve strategies in fracture treatment, regulatory pathways in fracture healing need to be investigated. Lipids are important molecules in cellular signaling, inflammation, and metabolism, as well as key structural components of the cell. Analysis of the lipid spectrum in fxh may therefore reflect important events during the early healing phase. This study aims to develop a protocol for the determination of lipid signals over time, and the identification of lipids that contribute to these signals, with matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI-MSI) in fxh in healthy fracture healing. Twelve fxh samples (6 porcine; 6 human) were surgically removed, snap frozen, sectioned, washed, and analyzed using MALDI-MSI in positive and negative ion mode at different time points after fracture (porcine: 72 h; human samples: range 1-19 days). A tissue preparation protocol for lipid analysis in fxh has been developed with both porcine and human fxh. Data were analyzed through principal component- and linear discriminant analyses. A protocol for the preparation of fxh sections was developed and optimized. Although hematoma is a heterogeneous tissue, the intra-variability within fxh was smaller than the inter-variability between fxh. Distinctive z values were detected that contributed to the separation of three different fxh age groups: early (1-3 days), middle (6-10 days), and late (12-19 days). Identification of the distinctive z values provided a panel of specific lipids that showed a time dependent expression within fxh. This study shows that MALDI-MSI is a suitable analytical tool for lipid analysis in fxh and that lipid patterns within fxh are time-dependent. These lipid patterns within fxh may serve as a future diagnostic tool. These findings warrant further research into fxh analysis using MALDI-MSI and its possible clinical implications in fracture treatment.
骨折愈合是一个复杂的过程，涉及细胞间相互作用、多种细胞因子和生长因子。尽管在过去几十年中骨折治疗有所改善，但所有骨折中仍有很大一部分显示愈合延迟或未愈合。已知骨折血肿（fxh）在这一过程中发挥着重要作用，但其影响骨折愈合的确切机制仍知之甚少。为了改进骨折治疗策略，需要研究骨折愈合中的调节途径。脂质是细胞信号传导、炎症和代谢中的重要分子，也是细胞的关键结构成分。因此，分析fxh中的脂质谱可能反映早期愈合阶段的重要事件。本研究旨在制定一个方案，用于随时间测定脂质信号，并鉴定对这些信号有贡献的脂质，采用基质辅助激光解吸/电离质谱成像（MALDI-MSI）技术对健康骨折愈合中的fxh进行分析。手术切除12个fxh样本（6个猪样本；6个人样本），速冻、切片、洗涤，并在骨折后的不同时间点使用正离子和负离子模式的MALDI-MSI进行分析（猪样本：72小时；人样本：1-19天）。已开发出一种用于fxh脂质分析的组织制备方案，适用于猪和人的fxh。通过主成分分析和线性判别分析对数据进行分析。制定并优化了fxh切片的制备方案。尽管血肿是一种异质性组织，但fxh内的变异性小于fxh之间的变异性。检测到有助于区分三个不同fxh年龄组的独特z值：早期（1-3天）、中期（6-10天）和晚期（12-19天）。对独特z值的鉴定提供了一组特定脂质，这些脂质在fxh中呈现出时间依赖性表达。本研究表明，MALDI-MSI是一种适用于fxh脂质分析的工具，且fxh内的脂质模式具有时间依赖性。fxh内的这些脂质模式可能成为未来的诊断工具。这些发现值得进一步研究使用MALDI-MSI进行fxh分析及其在骨折治疗中可能的临床意义。
