Quick and easy sample preparation without resin embedding for the bone quality assessment of fresh calcified bone using fourier transform infrared imaging

Fourier transform infrared (FTIR) imaging is a powerful tool for the assessment of bone quality; however, it requires the preparation of thin bone sections. Conventional poly(methyl methacrylate) (PMMA) embedding for the preparation of sections takes more than two weeks and causes denaturation of the bone. Development of a quick and easy sample preparation technique without denaturation is needed for accurate clinical evaluation of fresh calcified bone using FTIR imaging. Frozen sectioning allows the quick and easy preparation of thin sections without denaturation, but it requires a substrate with good chemical resistance and improved heat shock resistance. Polypropylene (PP) film afforded both good chemical resistance and greater heat shock resistance, and the 4-μm-thick PP film coated with glue was thin enough for the IR beam to pass through it, while the optical anisotropy of infrared bands overlapping with PO43- band was negligible. The bone quality of femoral thin sections prepared by the conventional PMMA embedding and sectioning procedure (RESIN-S) or the newly developed frozen sectioning procedure (FROZEN-S) was evaluated by FTIR imaging. The mineral-to-matrix ratio and crystallinity in the RESIN-S sections were higher than those in the FROZEN-S sections, whereas the carbonate-to-phosphate ratio in the RESIN-S sections was lower than that in the FROZEN-S sections. In RESIN-S, the increased mineral-to-matrix ratio could be caused by dehydration, and the increased crystallinity and decreased carbonate-to-phosphate ratio might be consequence of dissolution of bone mineral during PMMA embedding. Therefore, the combined use of PP film coated with glue and the frozen sectioning procedure without denaturation appears well suited to the assessment of the bone quality of fresh calcified bone using FTIR imaging.

傅里叶变换红外成像技术（FTIR成像）在评估骨质量方面是一项强有力的工具；然而，它需要制备薄骨切片。传统的聚甲基丙烯酸甲酯（PMMA）包埋法用于制备切片耗时超过两周，且会导致骨蛋白变性。因此，迫切需要开发一种快速简便的样本制备技术，以避免变性，从而能够准确地对新鲜钙化骨进行临床评估。冷冻切片技术允许快速且简便地制备薄切片而无需变性，但需要具有良好的化学耐性和改进的热冲击耐性的基底。聚丙烯（PP）薄膜提供了良好的化学耐性和更高的热冲击耐性，4微米厚的PP薄膜涂层粘合剂足以允许红外光束通过，且与磷酸根离子（PO43-）重叠的红外波段的光学各向异性可忽略不计。通过傅里叶变换红外成像技术，对由传统PMMA包埋和切片程序（RESIN-S）或新开发的冷冻切片程序（FROZEN-S）制备的股骨薄切片的骨质量进行了评估。在RESIN-S切片中，矿物质与基质的比率以及结晶度高于FROZEN-S切片，而碳酸盐与磷酸盐的比率在RESIN-S切片中低于FROZEN-S切片。在RESIN-S中，矿物质与基质比率的增加可能是由脱水引起的，而结晶度的增加和碳酸盐与磷酸盐比率的降低可能是由于PMMA包埋过程中骨矿物质溶解的结果。因此，使用涂有粘合剂的PP薄膜和无需变性的冷冻切片程序相结合，似乎非常适合使用FTIR成像技术评估新鲜钙化骨的骨质量。