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

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Abstract´╝Ü 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 tess complexity required in comparison with classical histomorphology. It may be an opportunity when a relevant number of samples must be analyzed.

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