RU2466375C1 - Method for preparing samples of non-decalcified articular cartilage with subjacent subchondral bone for multipurpose study - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: method for preparing samples of non-decalcified articular cartilage with subjacent subchondral bone for multipurpose study involves tissue preparation for transmission electronic microscopy. Tissue samples 3-4 mm are taken and dehydrated in the mode of smooth alcohol to acetone transition with time increase at the stages from 70% to 96% of alcohol per 1 h. Then they are dehydrated in 2 changes 70% of alcohol for 1 h, in 2 changes 80% of alcohol for 1 h, in 2 changes 90% of alcohol for 1 h, in 2 changes 96% of alcohol for 1 h, in 2 changes 100% of alcohol for 30 min, in 2 changes 100% of acetone for 30 min. It is followed by impregnation in the mode of smooth alcohol to acetone transition: resin-acetone composition 100% 3:1 for 2 h, resin-acetone composition 100% 1:1 for 2 h, resin-acetone composition 100% 1:3 for 2 h. Before polymerisation, the sampled are impregnated by resin at room temperature to 10 days.

EFFECT: provided qualitative and quantitative study with the use of microscopy techniques.

3 cl, 6 dwg

Description

The invention relates to experimental biology and medicine and can be used to study the influence of various factors on the condition of articular cartilage and subchondral bone, the study of biopsy material in order to diagnose articular pathology.

Interest in the study of subchondral bone arose due to the emergence of new information about the pathogenesis of osteoarthritis, when it became clear that this disease is manifested not only by loss of articular cartilage, but also by changes in bone tissue. The contact of the articular cartilage with the subchondral bone underlying it is represented by the bone component — the mature bone of the osteon structure and the cartilage component — calcified partially resorbable cartilage (Joint: Morphology, clinic, diagnosis, treatment / Edited by V.N. Pavlova, G.G. Pavlova, N.A.Shostak, L.I. Slutsky. M .: LLC “Publishing house“ Medical Information Agency ”, 2011. 552 p .; Alekseeva L.I., Zaitseva E.M. Subchondral bone in osteoarthritis: new opportunities Therapy // Breast Cancer. 2004. Volume 12. No. 20. P.1133-1136).

A known method of preparing samples of biological tissues for research in a scanning electron microscope, including fixing, washing and dehydrating the samples, the samples after dehydration, without drying, transferred to a mixture of dehydrant and camphene, then sequentially impregnated in two portions of molten camphene at a temperature of 51-60 ° C (Patent 2397472 RU, publ. 08/20/2010, No. 23, Method for preparing samples of biological tissues for research in a scanning electron microscope).

However, the known invention is not intended for microanalysis of articular cartilage and subchondral bone.

Known methods for studying the morphological features of the contact of the articular cartilage with the underlying subchondral bone using light microscopy on celloidin, paraffin sections (Mazhuga P.M. Sources of trophic and structural self-renewal of articular cartilage // Morphology. 1999. No. 1. S. 43-50; Polyakov V.Yu., Kudryavtseva IP, Antipov AV On the issue of replacing defects in articular surfaces with bone-cartilage allografts (experimental study) // Traumatology and Orthopedics of Russia. 2000. No. 1. P. 37-40; Burr DB, Schaffler MB The involvement of subchondral mineraliz ed tissues in osteoarthrosis: guantitative microscopic evidence // Microsc Res Tech. 1997. No. 37 (4). P.343-357).

However, the study of articular cartilage and subchondral bone using such preparations without a preliminary decalcification procedure, in which a significant loss of mineral substances occurs, is impossible. In addition, light microscopy does not always make it possible to accurately determine the boundary between calcified cartilage and bone. A significant drawback of such histological preparations is their significant thickness, which does not allow conducting light-optical studies at high magnifications. For articular cartilage (chondrocyte diameter 10–20 μm), sections 0.5–1 μm thick are optimal; It is impossible to produce high-quality paraffin sections of undecalcified articular cartilage with a underlying bone of such thickness; a denser filling medium, for example, epoxy resins, is required.

A known method of preparing tissues for transmission electron microscopy, followed by the manufacture of semi-thin and ultra-thin sections (Wickley B. Electron microscopy for beginners. M: Mir, 1975. 324 S.).

However, the known technique does not allow the use of semi-thin epoxy sections, because standard technology for their manufacture involves filling pieces of tissue with sizes of 0.5 - 1 mm, which makes it difficult to obtain representative samples of images for stereological studies.

The objective of the present invention is to develop a methodology for preparing samples of undecalcified articular cartilage with the underlying subchondral bone for conducting qualitative and quantitative studies using light, scanning, transmission electron microscopy and electron probe microanalysis.

This problem is achieved by the fact that in the method of preparing samples of undecalcified articular cartilage with the underlying subchondral bone for multi-purpose studies, including a method of preparing tissues for transmission electron microscopy, for which tissue samples are taken 3-4 mm and dehydrated in the smooth transition from alcohol to acetone with an increase in time at stages from 70% to 96% alcohol to 1 hour, dehydrated - in 2 shifts of 70% alcohol for 1 hour; in 2 shifts of 80% alcohol for 1 hour; in 2 shifts of 90% alcohol for 1 hour; in 2 shifts, 96% alcohol for 1 hour; in 2 shifts of 100% alcohol for 30 minutes; in 2 shifts of 100% acetone for 30 minutes, then impregnation is carried out in a smooth transition from acetone to resin: a mixture of resins - acetone 100% 3: 1 - 2 hours; a mixture of resins - acetone 100% 1: 1 - 2 hours; a mixture of resins - acetone 100% 1: 3 - 2 hours and before polymerization is impregnated in the resin at room temperature for up to 10 days. The resulting block is ground in the form of a pyramid, while the area of its base at a height of 1.0 mm should be 4 times the area of the cut. The slice of the block begin to produce with hyaline cartilage.

The invention is illustrated by an example of a qualitative and quantitative study of articular cartilage with the underlying subchondral bone by light, scanning electron microscopy and electron probe microanalysis and illustrations, which depict:

Figure 1 (a, b, c, d) - semi-thin section, staining with methylene blue-basic fuchsin:

a - a general view of the articular cartilage with subchondral bone, about 2.5; OK. 12.5;

b - deep cartilage zone, basophilic line (arrows), volume 40; approx. 12.5;

c - calcified cartilage (CH), osteoclast resorbing calcified cartilage (OK), vessels of the subchondral bone (arrow), volume 40, approx. 12.5;

g - active osteoblasts on the surface of the trabecula, vol. 40, approx. 12.5.

Figure 2 (a, b) - articular cartilage with the underlying bone:

a - surface of the epoxy block, SEM, magnification 70, 1 - non-calcified cartilage, 2 - calcified cartilage, 3 - bone, arrows - basophilic line;

b - element map (Smart Map), reflecting the distribution of calcium in the articular cartilage and subchondral bone.

The method is as follows.

The articular cartilage with the underlying subchondral bone was cut into pieces 2-3 to 3-4 mm in size, which, after aldehyde fixation, without the stage of decalcification, were washed in phosphate buffer.

The next stage of fixation in a 1% solution of osmium tetrooxide was excluded with the aim of conducting a subsequent electron probe microanalysis. Then the material was dehydrated in a smooth transition from alcohol to acetone, in accordance with the methods [Weekley B., 1975], but with an increase in time from 70% to 96% alcohol for up to 1 hour.

Dehydration scheme: in 2 shifts of 70% alcohol for 1 hour; in 2 shifts of 80% alcohol for 1 hour; in 2 shifts of 90% alcohol for 1 hour; in 2 shifts, 96% alcohol for 1 hour; in 2 shifts of 100% alcohol for 30 minutes; in 2 shifts of 100% acetone for 30 minutes.

The next stage — impregnation was carried out in a smooth transition from acetone to resin with a 2-fold increase in time at each stage.

Impregnation scheme: resin-acetone mixture 100% 3: 1 - 2 hours; resin-acetone mixture 100% 1: 1 - 2 hours (can be left overnight); resin-acetone mixture 100% 1: 3 - 2 hours.

And additional, before polymerization, impregnation in resin at room temperature for up to 10 days.

The polymerization stage was carried out according to the methods of preparing tissues for transmission electron microscopy [Wickley B., 1975].

The next stage is the preparation of blocks for cutting, the formation of a pyramid. To obtain a sufficient area of semi-thin sections of undecalcified articular cartilage with the underlying subchondral bone, the following techniques were used.

When sharpening a block, the area of the base of the pyramid, with its height up to 1.0 mm, should be at least 4 times larger than the cut area, which avoids vibration of the block and the formation of mechanical defects on the slices during ultratomization. In the manufacture of slices, careful quality control and selection of glass knives are important. During ultra-tomography, the block should be oriented so that the section begins with hyaline cartilage, and not with the subchondral bone, which avoids the formation of mechanical defects on the knife and sections due to various biomechanical properties of the cartilage and bone. Obtained in this way, semi-thin sections of an increased area significantly increase the resolution of drugs, allow studies at the organ (Fig. 1 a), tissue (Fig. 1 a, b) and cellular (Fig. 1 b, c, d) levels of structural organization, representative sample for quantitative research. The same blocks after the manufacture of sections can be studied in a scanning electron microscope (Fig.2 a) and used without additional grinding for microanalysis of the articular cartilage and subchondral bone (Fig.2 b).

The proposed method used in the FSI "RRC" WTO "them. Acad. GA Ilizarova »Ministry of Health and Social Development, does not require the use of various methods of processing the material for light, scanning and transmission electron microscopy, does not require the manufacture of various types of sections and allows without additional technical and material costs to obtain objective information about the morphological features of the contact of the articular cartilage with the underlying subchondral bone in various experimental conditions.

This method can be recommended for processing biopsy material in order to diagnose articular pathology and study the influence of various factors on the condition of articular cartilage and subchondral bone.

Claims (3)

1. A method of preparing samples of undecalcified articular cartilage with the underlying subchondral bone for multi-purpose studies, including a method of preparing tissues for transmission electron microscopy, characterized in that they take 3-4 mm tissue samples and dehydrate in a smooth transition from alcohol to acetone with an increase in time by stages from 70% to 96% alcohol for up to 1 hour, dehydrated - in 2 shifts of 70% alcohol for 1 hour, in 2 shifts of 80% alcohol for 1 hour, in 2 shifts of 90% alcohol for 1 hour, in 2 shifts of 96% alcohol 1 h, in 2 shifts of 100% alcohol for 30 minutes, in 2 shifts of 100% ac tones of 30 minutes each, then impregnation is carried out in a smooth transition from acetone to resin: resin-acetone mixture 100% 3: 1 - 2 hours, resin-acetone mixture 100% 1: 1 - 2 hours, resin-acetone mixture 100% 1 : 3 - 2 hours and before polymerization, impregnated in resin at room temperature for up to 10 days. 2. The method according to claim 1, characterized in that the resulting block is sharpened in the form of a pyramid, while the area of its base at a height of 1.0 mm should be 4 times the area of the cut. 3. The method according to claim 1, characterized in that the section of the block begin to produce with hyaline cartilage.

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