应用背向散射和二次发射扫描电镜观察未脱钙未染色组织块关节软骨形态：一项描述性研究(英文)

Combining back scattered and secondary emission scanning electron microscopy to study articular cartilage morphology on undecalcified unstained samples A descriptive study

背景:在与关节软骨有关的组织工程研究中,应用未脱钙和未染色的样本对于保留关节软骨的三维结构十分有利。当分析大量样本时,应用未脱钙的组织块将减少实验时间和复杂性。目的:检验在未脱钙、未染色的组织块上研究关节软骨形态的可能性,这样可以避免切割并染色薄切片。方法:取新西兰白兔和Sardinian绵羊的股骨髁,在多聚甲醛溶液中固定,乙醇中脱水,包埋于多异丁烯酸甲酯中,切成组织块,并染色。将金钯溅镀于标本上,然后用Jeol JSM 6310电子显微镜进行观察。将背向散射和二次发射扫描电镜对组织块相同部位的观察结果进行合并分析。结果与结论:在兔的关节软骨组织块上,可明显区分未钙化与钙化软骨的传代,但较难辨认位于Ⅱ区和Ⅲ区的小软骨陷窝。在绵羊的软骨组织块中,则能较为容易地区分关节软骨各区和细胞结构。应用背向散射电镜能容易分辨出钙化软骨结构和潮标。合并二次发射扫描电镜和背向散射电镜,可在样本上观察到大的腔道。这些腔道穿过软骨下骨和钙化软骨,终止于未钙化的软骨。结果表明,应用背向散射和二次发射扫描电镜能在未脱钙和未染色的包埋组织块上观察到关节软骨的三维结构。

BACKGROUND： The use of undecalcified and unstained samples for articular cartilage’s study （as Authors suggest） will enable to better preserve its three-dimensional structure. Feasibility of such approach will reduce time and complexity when analyzing a great number of specimens. OBJECTIVE： To test the possibility of studying articular cartilage morphology on the undecalcified inclusion blocks, avoiding cutting and staining thin sections. METHODS： Femoral condyles were obtained from White New Zealand rabbits and from Sardinian sheep, fixed in paraformaldehyde, dehydrated in ethyl alcohol, and embedded into poly-methylmethacrylate. Blocks were cut and ground, sputter-coated with gold-palladium and analyzed by a Jeol JSM 6310 electron microscope, operated between 20 and 25 kV. Data from secondary emission scanning electron microscopy were combined with data from back scattered electron microscopy （BSEM）, performed sequentially over the same area. RESULTS AND CONCLUSION： In the rabbit, it was easy to discern the passage between uncalcified and calcified cartilage but it was difficult to highlight the small chondrocytic lacunae in zones Ⅱ and Ⅲ. The sheep proved to be more suitable for easily discerning all the zones of articular cartilage and its cellularity; BSEM excelled in defining the structure of calcified cartilage and the ＂tidemark＂ front. Large canals could be demarcated, digged through subchondral bone and calcified cartilage, topped by non-calcified cartilage. The results suggested that the possibility of describing articular cartilage morphology on undecalcified and unstained embedding blocks, by avoiding the cutting of thin sections, was illustrated. This provides an obvious advantage in terms of less time needed and less complexity required in comparison with classical histomorphology. It may be an opportunity when a relevant number of samples must be analyzed.