A Novel Technique for Preparation, Staining, and Visualization of Tissue with Metal Implants and Extraskeletal Calcification Areas.

R.A Mukhamadiyarov,S.V Mishinov,A.G Kutikhin,L.A Bogdanov

doi:10.17691/stm2020.12.4.02

Abstract

The aim of the study was to evaluate the efficacy of a novel technique for preparation, staining, and visualization of tissues containing extra-skeletal mineralization areas, all-metal implants or their prototypes for their subsequent examination using scanning electron microscopy in the backscattered electron mode.Materials and Methods.After fixation in 10% formalin (24 h), the biomaterial (a titanium nickelide plate with the surrounding tissues after subcutaneous implantation, patented titanium alloy plates with the surrounding tissues after cranioplasty, primary and secondary calcified atherosclerotic plaques) were fixed with 1% osmium tetroxide (12 h) and then stained with 2% aqueous solution of osmium tetroxide (48 h). The samples were further stained with 2% alcoholic uranyl acetate (5 h), dehydrated with isopropanol (5 h) and acetone (1 h), impregnated with a mixture of acetone and epoxy resin Epon (1:1, 6 h) and then embedded into a fresh portion of epoxy resin (24 h), which was followed by polymerization at 60°C. After grinding and polishing, epoxy blocks were counterstained with lead citrate (7 min) and sputter-coated with carbon, then the samples were visualized by scanning electron microscopy in the backscattered electron mode. The elemental composition was studied using X-ray microanalysis.Results.The developed technique allows obtaining high-quality images at five thousand-fold magnifications, provides the possibility to identify the shape and structure of intact metal and mineral inclusions, and to type the surrounding cells, distinguishing mesenchymal and immunocompetent cells by shape and cytoplasmic content. Apart from connective tissue capsule thickness and leukocyte infiltration, this technique makes it possible to estimate the number and area of newly formed small-caliber vessels representing a surrogate marker of inflammation.Conclusion.The proposed technique provides the possibility to investigate adequately the structure of samples when their sectioning is impossible or significantly complicated, with image quality remarkably higher than that obtained by light microscopy.

Highlights

Despite the rapid evolvement of histological and immunohistochemical methods, improvement of imaging techniques, development of new models of light, fluorescence, and electron microscopes [1,2,3], to date there are no effective methods for histological analysis of tissues containing all-metal implants and ectopic calcifications [4].Stents [5, 6] and metal plates [7, 8] are the most commonly used metal implants
The aim of the study was to evaluate the efficacy of a novel technique for preparation, staining, and visualization of tissues containing extra-skeletal mineralization areas, all-metal implants or their prototypes for their subsequent examination using scanning electron microscopy in the backscattered electron mode
Epoxy blocks were counterstained with lead citrate (7 min) and sputter-coated with carbon, the samples were visualized by scanning electron microscopy in the backscattered electron mode

Summary

Introduction

Despite the rapid evolvement of histological and immunohistochemical methods, improvement of imaging techniques, development of new models of light, fluorescence, and electron microscopes [1,2,3], to date there are no effective methods for histological analysis of tissues containing all-metal implants and ectopic calcifications [4].Stents [5, 6] and metal plates [7, 8] are the most commonly used metal implants. Routine histological or immunohistochemical staining requires visualization by light microscopy, the limited resolution of which does not allow obtaining high-quality images at more than 400fold magnification. It is unlikely to solve this problem even with confocal microscopy that, firstly, allows no adequate assessment of tissue structure and, secondly, is usually limited by 630-fold magnification. With this range of magnifications, it is rather difficult to carry out adequate assessment of neovascularization, which is a surrogate marker of inflammation during the implantation of medical device prototypes. In practice, newly formed small-caliber vessels are rarely stained with antibodies to classical endothelial markers

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Conclusion