https://ijmri.de/index.php/jmsi
volume 4, issue 3, 2025
620
MODIFICATION OF THE METHODOLOGICAL STUDY OF BONE TISSUE
Kurbanova N.K.
Andijan State Medical Institute, Uzbekistan
Abstract:
The article describes the method of obtaining, staining and subsequent morphometry
of non-decalcified bone sections.
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 of volume, remodeling and mineralization of bone tissue.
MАTЕRIАLS АND 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 tissue cells [2].
RЕSULTS АND DISСUSSIОN
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
histomorphometric parameters, which allows us to 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
silver deposition, while the osteoid remains susceptible to contrast staining [3].
Staining technique
1. The sections are placed in a 1% aqueous solution of silver nitrate and illuminated with a strong
light source (sunlight is best) for 2-15 minutes (the duration may vary depending on the intensity
of the light and the freshness of the solution).
2. Rinse in three changes of distilled water.
3. Treat with 2.5% sodium thiosulfate
2-3 minutes.
4. Rinse well in distilled water.
In subsequent stages, cells and osteoid were stained.
https://ijmri.de/index.php/jmsi
volume 4, issue 3, 2025
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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. Rinse in running water.
7. Stain with 0.05% basic fuchsin on 2.5% ethanol for 30 seconds.
8. Rinse in 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,
since in comparison with alcohol, it leads to greater washing out of tetracycline labels.
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 of the American Society of Bone and Mineral Research [4]:
1) parameters of bone formation:
- OV/BV – osteoid volume – the volume of osteoid (%) of spongy bone tissue that has not
undergone calcification;
- Os/Bs – osteoid surface – the surface of osteoid (%) of the total perimeter of spongy bone tissue
that is covered by osteoid;
- O.Th – osteоid thickness – the average thickness (μm) of osteoid layers;
- Ob.s/Bs – osteoblast surface – the surface of osteoblasts – the surface (%) of the total perimeter
of spongy bone tissue covered by active osteoblasts;
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
bone tissue covered 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
millimeter of spongy bone surface.
A similar technique can be used to determine the dynamic parameters of bone remodeling, which
we will do in the future.
RЕFЕRЕNСЕS
1.
Microscopic technology / edited by D. S. Sarkisov, Yu. L. Perov. - M.: Medicine, 2016. -
544 p.
2.
Pikalyuk V. S. Methodological aspects of research on the skeleton of humans and animals
/ V. S. Pikalyuk. - Simferopol, 2017. - 272 p.
3.
Riggs B. L. Osteoporosis / B. L. Riggs, L. J. Melton III. - M.-SPb: BINOM, Nevsky
dialect. - 2210. - 560 p.
4.
Romeis B. Microscopic technology / B. Romeis. - M.: Publishing house of foreign
literature, 2013. - 718 p.
