Bulletin of Experimental Biology and Medicine, Vol. 157, No. 3, July, 2014
401
METHODS A Method for Making Preparations from Nondecalcified Articular Cartilage with Sublying Subchondral Bone for Multipurpose Studies T. A. Stupina and M. M. Shchudlo Translated from Byulleten’ Eksperimental’noi Biologii i Meditsiny, Vol. 157, No. 3, pp. 387-389, March, 2014 Original article submitted March 12, 2013 A method for making preparations from nondecalcified articular cartilage and subchondral bone for multipurpose studies is developed. Semithin and ultrathin sections of nondecalcified articular cartilage and subchondral bone prepared by this method are used for light and transmission electron microscopy. The method can also be used for studies of the qualitative and quantitative characteristics of the articular cartilage, scanning electron microscopy, and microanalysis of chemical elements in the articular cartilage and subchondral bone. Key Words: articular cartilage; subchondral bone; light microscopy; electron microscopy; microanalysis Subchondral bone became the focus scientific interest due to new data on osteoarthrosis pathogenesis, when it became clear that this disease manifested by not only articular cartilage loss, but also by changes in the bone tissue [1]. The articular cartilage contact with the sublying subchondral bone was presented by the bone component (mature bone of osteon structure) and the cartilage component (calcified partially resorbing cartilage) [3]. The morphology of the articular cartilage contact with the subchondral bone was studied by microscopy of celloidine, paraffin sections [2,3,7]. However, it was impossible to examine the articular cartilage and subchondral bone in these preparations without preliminary decalcination, involving significant loss of the mineral substances. Moreover, the interface between calcified cartilage and bone could not always be precisely identified under a microscope. Russian G. A. Ilizarov Scientific Center for Restorative Traumatology and Orthopaedics, Ministry of Health of the Russian Federation, Kurgan, Russia. Address for correspondence: StupinaSTA@mail. ru. T. A. Stupina
An important flaw of these histological preparations was their thickness ruling out light microscopy at high magnification. The Holmes effect could be eliminated in these preparations only by using semithin (0.51.0 μ) sections [6]. We developed a method for making preparations of nondecalcified articular cartilage with the sublying subchondral bone for multipurpose studies.
MATERIALS AND METHODS The study was carried out on 14 dogs. Specimens of the articular cartilage with the subchondral bone from the femoral condyles of intact (n=5) and experimental (n=9) animals were studied. The shin bone was elongated by 28-30 mm by the Ilizarov device in these animals (the experiment was carried out by S. A. Erofeev, Doct. Med. Sci.) for 10 days at a rate of 3 mm, in 180 sessions (autodistraction). The specimens were examined by scanning electron microscopy and electron probe microanalysis.
0007-4888/14/15730401 © 2014 Springer Science+Business Media New York
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Bulletin of Experimental Biology and Medicine, Vol. 157, No. 3, July, 2014 METHODS
Fig. 1. Semithin section, methylene blue and basic fuchsine staining. a) Articular cartilage with subchondral bone (ob. 2.5, oc. 12.5); b) deep zone of the cartilage, basophilic line (arrows; ob. 40, oc. 12.5); c) calcified cartilage (CC), osteoclast (OC) resorbing the CC, subchondral bone vessels (arrow; ob. 40, oc. 12.5); d) active osteoblasts on trabecule surface (ob. 40, oc. 12.5).
Fig. 2. Articular cartilage with sublying bone. a) Epoxy block surface (scanning electron microscopy, ×70): 1) noncalcified cartilage; 2) calcified cartilage; 3) bone (arrows: basophilic line); b) Smart Map, reflecting calcium distribution in articular cartilage and subchondral bone.
The animals were kept and sacrificed in accordance with the regulations of the Ministry of Health of the Russian Federation for the Work of Experimental Biological Clinics (Order No. 755 of the Ministry of Health of the USSR, 1977). All manipulations were discussed and approved by the Ethic Committee of the Russian G. A. Ilizarov Scientific Center for Restorative Traumatology and Orthopaedics.
RESULTS The articular cartilage with the subchondral bone was cut into fragments 2-3×3-4 mm in size. After aldehyde fixation (without decalcification) the fragments were washed in phosphate buffer, dehydrated in the smooth transition mode from ethanol to acetone in accordance with the method for tissue preparation for transmission electron microscopy [5], but with 1 h prolongation of the exposure at each stage. The next stage (saturation) was carried out in the smooth transition mode from acetone to resin, with 2-fold prolongation of the exposure at each step and additional (before polymerization) saturation with resin at ambient temperature
during up to 10 days. Polymerization was carried out in accordance with the methods for tissue preparation for transmission electron microscopy [5]. The next stage included preparation of blocks for slicing, formation of the pyramid. When preparing the block, the pyramid base area at its height (up to 1 mm) was to be at least 4-fold larger than the section area, in order to prevent the block vibration and formation of mechanical defects on slices during ultratomy. Special attention was paid to the quality and selection of the glass knives. The block for ultratomy was to be oriented so that the section started from the hyaline cartilage, but not from the subchondral bone, this preventing the formation of mechanical defects on the knives and sections because of biomechanical characteristics of the cartilage and bone. Semithin sections of enlarged area, prepared by this method, allowed a significantly higher resolution of the preparations and hence, studies at the organ, tissue, and cell levels of structural organization (Fig. 1) and formation of a representative sample for quantitative studies. The same blocks after slicing could be studied in a scanning electron microscope (Fig. 2, a) and used without additional
T. A. Stupina and M. M. Shchudlo
polishing for microanalysis of the articular cartilage and subchondral bone (Fig. 2, b). Hence, the method [4] is used for preparing semithin and ultrathin sections of nondecalcified articular cartilage and subchondral bone for li and transmission electron microscopy, studies of the qualitative and quantitative characteristics of the articular cartilage and subchondral bone, scanning electron microscopy, microanalysis of chemical elements in the articular cartilage and subchondral bone. This method does not involve different processing of the material for optic, scanning, and transmission electron microscopy, for making sections of different types, and gives objective information on the morphology of the articular cartilage contact with the sublying subchondral bone under different experimental conditions.
403
REFERENCES 1. L. I. Alekseeva and E. M. Zaitseva, Rus. Med. Zh., 12, No. 20, 1133-1136 (2004). 2. P. M. Mazhuga, Morfologiya, 115, No. 1, 43-50 (1999). 3. V. N. Pavlova, G. G. Pavlov, N. A. Shostak, and L. I. Slutskii, The Joint: Morphology, Clinical Picture, Diagnosis, Therapy [in Russian], Moscow (2011). 4. T. A. Stupina and M. M. Shchudlo, Patent RF No. 2466375. A Method for Preparation of Specimens of Nondecalcified Articular Cartilage with Sublying Subchondral Bone for Multipurpose Studies, Bull. No. 31, November 10, 2012. 5. B. Wickley, Electron Microscopy for Beginners [in Russian], Moscow (1975). 6. M. M. Shchudlo, N. A. Shchudlo, T. N. Varsegova, et al., Izv. Chelyabinsk. Nauch. Tsentra, No. 1 (18), 130-134 (2003). 7. D. B. Burr and M. B. Schaffler, Microsc. Res. Tech., 37, No. 4, 343-357 (1997).
401
METHODS A Method for Making Preparations from Nondecalcified Articular Cartilage with Sublying Subchondral Bone for Multipurpose Studies T. A. Stupina and M. M. Shchudlo Translated from Byulleten’ Eksperimental’noi Biologii i Meditsiny, Vol. 157, No. 3, pp. 387-389, March, 2014 Original article submitted March 12, 2013 A method for making preparations from nondecalcified articular cartilage and subchondral bone for multipurpose studies is developed. Semithin and ultrathin sections of nondecalcified articular cartilage and subchondral bone prepared by this method are used for light and transmission electron microscopy. The method can also be used for studies of the qualitative and quantitative characteristics of the articular cartilage, scanning electron microscopy, and microanalysis of chemical elements in the articular cartilage and subchondral bone. Key Words: articular cartilage; subchondral bone; light microscopy; electron microscopy; microanalysis Subchondral bone became the focus scientific interest due to new data on osteoarthrosis pathogenesis, when it became clear that this disease manifested by not only articular cartilage loss, but also by changes in the bone tissue [1]. The articular cartilage contact with the sublying subchondral bone was presented by the bone component (mature bone of osteon structure) and the cartilage component (calcified partially resorbing cartilage) [3]. The morphology of the articular cartilage contact with the subchondral bone was studied by microscopy of celloidine, paraffin sections [2,3,7]. However, it was impossible to examine the articular cartilage and subchondral bone in these preparations without preliminary decalcination, involving significant loss of the mineral substances. Moreover, the interface between calcified cartilage and bone could not always be precisely identified under a microscope. Russian G. A. Ilizarov Scientific Center for Restorative Traumatology and Orthopaedics, Ministry of Health of the Russian Federation, Kurgan, Russia. Address for correspondence: StupinaSTA@mail. ru. T. A. Stupina
An important flaw of these histological preparations was their thickness ruling out light microscopy at high magnification. The Holmes effect could be eliminated in these preparations only by using semithin (0.51.0 μ) sections [6]. We developed a method for making preparations of nondecalcified articular cartilage with the sublying subchondral bone for multipurpose studies.
MATERIALS AND METHODS The study was carried out on 14 dogs. Specimens of the articular cartilage with the subchondral bone from the femoral condyles of intact (n=5) and experimental (n=9) animals were studied. The shin bone was elongated by 28-30 mm by the Ilizarov device in these animals (the experiment was carried out by S. A. Erofeev, Doct. Med. Sci.) for 10 days at a rate of 3 mm, in 180 sessions (autodistraction). The specimens were examined by scanning electron microscopy and electron probe microanalysis.
0007-4888/14/15730401 © 2014 Springer Science+Business Media New York
402
Bulletin of Experimental Biology and Medicine, Vol. 157, No. 3, July, 2014 METHODS
Fig. 1. Semithin section, methylene blue and basic fuchsine staining. a) Articular cartilage with subchondral bone (ob. 2.5, oc. 12.5); b) deep zone of the cartilage, basophilic line (arrows; ob. 40, oc. 12.5); c) calcified cartilage (CC), osteoclast (OC) resorbing the CC, subchondral bone vessels (arrow; ob. 40, oc. 12.5); d) active osteoblasts on trabecule surface (ob. 40, oc. 12.5).
Fig. 2. Articular cartilage with sublying bone. a) Epoxy block surface (scanning electron microscopy, ×70): 1) noncalcified cartilage; 2) calcified cartilage; 3) bone (arrows: basophilic line); b) Smart Map, reflecting calcium distribution in articular cartilage and subchondral bone.
The animals were kept and sacrificed in accordance with the regulations of the Ministry of Health of the Russian Federation for the Work of Experimental Biological Clinics (Order No. 755 of the Ministry of Health of the USSR, 1977). All manipulations were discussed and approved by the Ethic Committee of the Russian G. A. Ilizarov Scientific Center for Restorative Traumatology and Orthopaedics.
RESULTS The articular cartilage with the subchondral bone was cut into fragments 2-3×3-4 mm in size. After aldehyde fixation (without decalcification) the fragments were washed in phosphate buffer, dehydrated in the smooth transition mode from ethanol to acetone in accordance with the method for tissue preparation for transmission electron microscopy [5], but with 1 h prolongation of the exposure at each stage. The next stage (saturation) was carried out in the smooth transition mode from acetone to resin, with 2-fold prolongation of the exposure at each step and additional (before polymerization) saturation with resin at ambient temperature
during up to 10 days. Polymerization was carried out in accordance with the methods for tissue preparation for transmission electron microscopy [5]. The next stage included preparation of blocks for slicing, formation of the pyramid. When preparing the block, the pyramid base area at its height (up to 1 mm) was to be at least 4-fold larger than the section area, in order to prevent the block vibration and formation of mechanical defects on slices during ultratomy. Special attention was paid to the quality and selection of the glass knives. The block for ultratomy was to be oriented so that the section started from the hyaline cartilage, but not from the subchondral bone, this preventing the formation of mechanical defects on the knives and sections because of biomechanical characteristics of the cartilage and bone. Semithin sections of enlarged area, prepared by this method, allowed a significantly higher resolution of the preparations and hence, studies at the organ, tissue, and cell levels of structural organization (Fig. 1) and formation of a representative sample for quantitative studies. The same blocks after slicing could be studied in a scanning electron microscope (Fig. 2, a) and used without additional
T. A. Stupina and M. M. Shchudlo
polishing for microanalysis of the articular cartilage and subchondral bone (Fig. 2, b). Hence, the method [4] is used for preparing semithin and ultrathin sections of nondecalcified articular cartilage and subchondral bone for li and transmission electron microscopy, studies of the qualitative and quantitative characteristics of the articular cartilage and subchondral bone, scanning electron microscopy, microanalysis of chemical elements in the articular cartilage and subchondral bone. This method does not involve different processing of the material for optic, scanning, and transmission electron microscopy, for making sections of different types, and gives objective information on the morphology of the articular cartilage contact with the sublying subchondral bone under different experimental conditions.
403
REFERENCES 1. L. I. Alekseeva and E. M. Zaitseva, Rus. Med. Zh., 12, No. 20, 1133-1136 (2004). 2. P. M. Mazhuga, Morfologiya, 115, No. 1, 43-50 (1999). 3. V. N. Pavlova, G. G. Pavlov, N. A. Shostak, and L. I. Slutskii, The Joint: Morphology, Clinical Picture, Diagnosis, Therapy [in Russian], Moscow (2011). 4. T. A. Stupina and M. M. Shchudlo, Patent RF No. 2466375. A Method for Preparation of Specimens of Nondecalcified Articular Cartilage with Sublying Subchondral Bone for Multipurpose Studies, Bull. No. 31, November 10, 2012. 5. B. Wickley, Electron Microscopy for Beginners [in Russian], Moscow (1975). 6. M. M. Shchudlo, N. A. Shchudlo, T. N. Varsegova, et al., Izv. Chelyabinsk. Nauch. Tsentra, No. 1 (18), 130-134 (2003). 7. D. B. Burr and M. B. Schaffler, Microsc. Res. Tech., 37, No. 4, 343-357 (1997).
