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

R A Mukhamadiyarov; L A Bogdanov; S V Mishinov; A G Kutikhin

doi:10.17691/stm2020.12.4.02

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

Sovrem Tekhnologii Med. 2021;12(4):13-20. doi: 10.17691/stm2020.12.4.02. Epub 2020 Aug 27.

Authors

R A Mukhamadiyarov¹, L A Bogdanov², S V Mishinov³, A G Kutikhin⁴

Affiliations

¹ Senior Researcher, Laboratory for Fundamental Aspects of Atherosclerosis, Department of Experimental and Clinical Cardiology; Research Institute for Complex Issues of Cardiovascular Diseases, 6 Sosnovy Blvd, Kemerovo, 650002, Russia.
² Junior Researcher, Laboratory for Fundamental Aspects of Atherosclerosis, Department of Experimental and Clinical Cardiology; Research Institute for Complex Issues of Cardiovascular Diseases, 6 Sosnovy Blvd, Kemerovo, 650002, Russia.
³ Senior Researcher, Neurosurgeon, Neurosurgery Unit; Novosibirsk Scientific Research Institute of Traumatology and Orthopedics named after Ya.L. Tsivyan of the Ministry of Health of the Russian Federation, 17 Frunze St., Novosibirsk, 630091, Russia.
⁴ Head of the Laboratory for Fundamental Aspects of Atherosclerosis, Department of Experimental and Clinical Cardiology Research Institute for Complex Issues of Cardiovascular Diseases, 6 Sosnovy Blvd, Kemerovo, 650002, Russia.

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.

Keywords: biocompatibility.; implantation; metal implants; scanning electron microscopy; stents; vascular calcification; vascular mineralization.

Publication types

Research Support, Non-U.S. Gov't

MeSH terms

Alloys
Metals*
Microscopy, Electron, Scanning
Osmium Tetroxide*
Staining and Labeling

Substances

Alloys
Metals
Osmium Tetroxide

Grants and funding

Study funding. This study was supported by the Comprehensive Program of Basic Research of the Siberian Branch of RAS within the framework of the fundamental theme of Research Institute for Complex Issues of Cardiovascular Diseases No.0546-2015-0011 “Pathogenetic basis for the development of cardiovascular implants from biocompatible materials using patient-oriented approach, mathematical modeling, tissue engineering, and genomic predictors”.