MODIFICATION OF THE METHODOLOGICAL STUDY OF BONE TISSUE

Аннотация

The article describes the method of obtaining, staining and subsequent morphometry ofnon-decalcified bonesections. Kеywоrds:technique, histological examination,bone tissue. INTRОDUСTIОN One of the conditions for solving these problems is the ability to distinguish mineralized and non-mineralized (osteoid) bone tissue on histological preparations, which is only possible on non-decalcified sections. It should also be emphasized that only on non-decalcified sections is it possible to conduct a reliable quantitative histomorphometric study of the static and dynamic parameters ofvolume, remodeling and mineralization ofbonetissue. MАTЕRIАLSАND MЕTHОDS In most studies conducted abroad, these problems are solved in several stages. At the first stage, pieces of bone tissue are fixed. The most widely used fixatives are 70% ethanol or 10% formalin (at pH = 7.0). After dehydration, bone samples are filled with monomeric plastics, usually methyl methacrylate, which gives the resulting blocks a strength similar to that of bone. At the next stage, using special microtomes equipped with a diamond knife, sections 1-2 µm thick are prepared. In some laboratories, the plastic is dissolved before staining the sections, while in others this is not done, wishing to preserve the section architecture. Subsequently, the sections are stained. When choosing a staining procedure, it is imperative to use a method that ensures unambiguous identification of osteoid and bone tissuecells [2]. RЕSULTSАNDDISСUSSIОN It should be noted that the above-mentioned methods of filling, obtaining sections and their staining require the presence of special expensive equipment and reagents, and are also characterized by the complexity and duration of the procedure. In this connection, this method is practically not used in Ukraine. We have developed a method for obtaining non-decalcified sections of bone tissue of Wistar rats and their subsequent staining for the study of static histomorphometricparameters, which allows usto avoid these inconveniences. The obtained semi-thin sections are stained using the Koss method with subsequent contrast staining. Unlike staining with Solochrome cyanine R and Goldner trichrome, this method is simple to perform and has readily available reagents. The Koss method is a classic reference method for detecting calcium in tissues. In this case, the bone mineral is stained black due to silverdeposition,while theosteoidremains susceptibleto contrast staining[3]. Stainingtechnique 1.Thesections are placed in a 1%aqueous solution ofsilvernitrate and illuminated witha strong light source (sunlight is best) for 2-15 minutes (the duration may vary depending on the intensity ofthelight and thefreshness ofthe solution). 2.Rinsein three changes ofdistilled water. 3.Treat with2.5%sodium thiosulfate 2-3 minutes. 4.Rinsewell in distilled water. In subsequent stages, cells andosteoid were stained. https://ijmri.de/index.php/jmsi volume 4,issue3,2025 620 5. Stain with 1% methylene blue solution for 30-60 seconds. Method for preparing 1% methylene blue solution: first, prepare a saturated aqueous solution, which is mixed with 90% ethanol in a 1:1 ratio. Then, prepare a 1% aqueous solution of methylene blue from this mother liquor. 6.Rinsein running water. 7.Stainwith 0.05%basic fuchsin on2.5%ethanol for 30seconds. 8.Rinsein running water. Initially, pieces of bone tissue are fixed in 70% ethyl alcohol for 24 hours. Considering the fact that using the same technique it is possible to study dynamic histomorphometric parameters (with preliminary double administration of tetracycline to animals), formalin is less acceptable, sincein comparison with alcohol, it leads togreater washing out oftetracyclinelabels. The sections obtained in this way allow the determination of the following static parameters of bone formation and resorption recommended by the Nomenclature Committee on Histomorphometry oftheAmerican Society ofBone andMineral Research [4]: 1)parameters of boneformation: - OV/BV – osteoid volume – the volume of osteoid (%) of spongy bone tissue that has not undergonecalcification; -Os/Bs –osteoid surface –thesurface ofosteoid (%) ofthetotal perimeter of spongy bonetissue thatis covered byosteoid; -O.Th –osteоid thickness –theaverage thickness (μm)ofosteoid layers; - Ob.s/Bs – osteoblast surface – the surface of osteoblasts – the surface (%) of the total perimeter ofspongy bonetissuecovered byactiveosteoblasts; 2)resorption parameters: - Es/Bs – eroded surface – the part (%) of the surface of spongy bone covered with resorption lacunae. - Oc.s/Bs – osteoclast surface – osteoclast surface – part (%) of the total perimeter of spongy bonetissuecovered with osteoclasts. СОNСLUSIОN Instead of the last indicator, it is possible to determine the number of osteoclasts N.Oc (number of osteoclasts) per square millimeter of bone section or N.Oc/Bs the number of osteoclasts per millimeterofspongy bone surface. Asimilar technique can be used to deter