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A paraffin-celloidin embedding method for studying soft-hard tissue interfaces
0003-9969/81/090753-0380-2.OC~0 Pergamon Press Ltd
Arch,, orul fl,ol. Vol 26. pp. 753 to 755. 1981 Punted I” Great Entam
SHORT COMMUNICATIONS A PARAFFIN-CELLOIDIN EMBEDDING METHOD FOR STUDYING SOFT-HARD TISSUE INTERFACES J. E. LINDER,’ P. LONGHURST’ and N. W. JOHNSON’ The London Hospital Medical College, London E.l, England ‘Department of Orthodontics & Dentistry for Children, Guy’s Hospital London S.E.l, England
‘Department of Oral Pathology,
Summary-Formaldehyde-fixed tooth-gingiva specimens were embedded whole in low-viscosity nitrocellulose (celloidin) to maintain the soft-hard tissue relationships. The soft tissue-bearing portion was sliced off, demineralized in EDTA and re-infiltrated in paraffin wax. Sections were mounted using a special benzyl alcohol acetone-containing fluid to achieve complete flattening and successfully stained using a variety of stains.
Chronic inflammatory periodontal disease, the major cause of tooth loss in adults, has its foundation in the gingival tissue of the deciduous dentition of young children (McCall, 1933; Longhurst, Johnson and Hopps, 1977; Longhurst, Gillett and Johnson, 1980). The technique described here was developed to enable the earliest stages of human periodontal disease to be studied in more detail than heretofore. The aim was the accurate maintenance of the relationship of gingival and periodontal soft tissue to the hard tissues of the tooth itself. It was also important that the technique should be as simple as possible to perform so that reasonable numbers of specimens could be examined and that the resulting sections should be amenable to a wide variety of stains. Preliminary work using a modification of the method of Brain (1962, 1967) to retain some enamel structure during demineralization gave unreliable results. The method of Schroeder (1969) whilst giving better results, does not support the soft tissue during demineralization and subsequent processing may allow distortions. Further, it relies on plastic (Epon) sections which restricts the range of stains possible. Embedding in agar prior to demineralization was also found to be unsatisfactory. The basis of the technique to be described was the suggestion by Schaffer (1926) that delicate biological specimens could be embedded in celloidin prior to demineralization when maintenance of the orientation of soft and hard tissues was important. Deciduous teeth, extracted because of dental caries, were removed with a piece of gingival tissue approximately 3 x 3 mm attached (Longhurst et al., 1980). Each specimen was fixed for 8-12 h in 4 per cent neutral formaldehyde calcium acetate (Lillie and Fullmer, 1976) at room temperature and dehydrated in the following graded waterethanol mixtures: 70 per cent ethanol, 24 h; 90 per cent ethanol, 24 h; absolute ethanol, 3 x 24 h. The specimen was then transferred to a mixture containing equal parts of absolute ethanol and anaesthetic ether for 24 h and then transferred from the absolute ethanol-ether mixture
through a series of solutions of low-viscosity nitrocellulose (LVN) (Chesterman and Leach, 1949) in absolute ethanolether (5, 10 and 20 per cent LVN), approx. 5 days in each at room temperature. A block of LVN containing the entire tooth-gingiva specimen was then prepared by allowing slow evaporation of the ether which caused the LVN to solidify in the form of a firm gel. This block was hardened by exposure to chloroform vapour, and stored in 70 per cent ethanol to remove volatile chloroform and further harden the nitrocellulose. Once completely hardened, the block was transferred to 30 per cent ethanol, mounted on the chuck of a hard-tissue slitting machine and that portion of the tooth bearing the soft tissue, completely supported by the nitrocellulose, was sliced off. The thickness of the slice was determined by the size of the gingival biopsy and the curvature of the tooth surface and was usually 24mm. The slice was washed in distilled water for several hours and demineralized in a 10 per cent w/v solution of sodium EDTA at pH 7.0. This solution was agitated by slowly bubbling air through it, the specimen being supported in a perforated plastic tube. Completion of demineralization was confirmed by X-ray examination. The specimen was washed carefully in running tapwater to remove the EDTA. To facilitate later orientation, a thin Indian Ink line was drawn on the cut surface of the specimen slice to one side of the soft tissue and parallel to the long axis of the tooth. The specimen was again dehydrated through a graded series of water-ethanol mixtures, 30, 50, 70 and 90 per cent half a day in each grade, taken through two changes of a 50:50 mixture of absolute ethanol
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154
J. E.
Linder. P. Longhurst and N. W. Johnson
paraffin wax, ensuring that the Indian Ink line was towards and parallel with the base of the mould. The paraffin block was mounted on the microtome and, as the nitrocellulose portion was approached during preliminary cutting, the Indian Ink line became visible. The block was adjusted so that the microtome knife was parallel with this line. In this way, vertical, bucco-lingually orientated sections were obtained. The difficulties of section cutting produced by the heterogeneity of the block were overcome by using a very sharp knife and slow cutting speed. In addition, rubbing the block face with cotton wool dampened with distilled water prior to each section being cut helped to reduce electrostatic charges, slightly softened the collagen in the tissue and allowed ribbons of serial sections to be achieved. Sections could be cut at specified thicknesses in the range 3-10pm; 6pm was usual. The heterogeneity of both the block and the specimens also meant that flattening and mounting sections in the usual way was impossible.
Therefore,
a special
section-mounting
fluid
was used, prepared as follows: 0.5 ml benzyl alcohol was added to 20ml acetone; 4-5 drops of Mayer’s glycerin-albumin solution (Gatenby and Painter, 1946) was mixed in 30ml distilled water. The albuminized water was added to the acetonebenzyl alcohol solution and ultrasonicated for 5 min to remove any dissolved air. Section mounting and flattening was carried out on a warm plate at about 40°C and the mounted sections allowed to dry at 37’C. If the temperature of the warm plate was too high, large numbers of bubbles formed beneath the sections. The proportions of the constituents of the mounting fluid could be varied; e.g. the quantity of both benzyl alcohol and acetone could be reduced when section flattening was less difficult. However, the amount of benzyl alcohol should not be increased beyond that stated since mounted sections would then be liable to come off during staining. A wide variety of staining methods has been successfully used including haematoxylin and eosin, Hart’s elastin stain, van Gieson, Giemsa, methyl
green-pyronin, periodic acid-Schiff, silver impregnation methods and Gram stain. Typical examples of the results achieved (Figs 14) show that supporting the gingiva against the tooth surface throughout processing has preserved the intimate soft-hard tissue relationship presented by the juxtaposition of teeth and their investing soft tissues and also retained acquired tooth surface deposits. Although the technique presented here was developed to study a soft-hard tissue relationship peculiar to teeth, there seems little doubt that it could have applications elsewhere in biological science. REFERENCES Brain E. H. 1962. A new method for the preparation of decalcified sections of human enamel in situ. Archs. orul Biol. 7, 157-763. Brain E. H. 1967. Rapid demineralization for microscopy of tooth enamel and associated structures. Br. dent. J. 123(4), 177-181. Chesterman W. and Leach E. H. 1949. Low viscosity nitrocellulose for embedding tissues. Q. JI microsc. Sci. 90(4), 43 l--434. Gatenby J. B. and Painter T. S. 1946. The Microtomist’s Vade-Mecum (Belles Lee) Para. 209, p. 114. Churchill, London. Lillie R. D. and Fullmer H. M. 1976. Histopathologic Technic and Practicai Histochemistry, 4th edn, Chap. 3. p, 33. McGraw-Hill, New York. Longhurst P., Gillett R. and Johnson N. W. 1980. Electron microscope quantitation of inflammatory infiltrates in childhood gingivitis. J. periodont. Rex 15, 255-266. Longhurst P., Johnson N. W. and Hopps R. 1977. Differences in lymphocyte and plasma cell densities in inflamed gingiva from adults and young children. J. periodont. 48, 705-710. McCall J. 0. 1933. The Periodonttst looks at Children’s Dentistry. J. Am. Dent. Ass. 20, 1518-1521. Schaffer J. 1926. Enzyklopiidie der Mikroskopischen Technik, Vol. II (Edited bv Krause R.), D. 1150. Urban & Scharzenberg. Berlin. Schroeder H. E. 1969. Ultrastructure of the junctional epithelium of the human gingiva. Heia. odont. Acra 13, 65-83.
Plate 1. Fig. 1. Gingiva and adjacent tooth; longitudinal bucco-lingual section of upper first deciduous molar; child aged 3 yr. D = dentine; P = bacterial plaque; C = cuticle; E = enamel space; OE = oral epithelium; JE = junctional epithelium; CT = connective tissue. Van Gieson stain. x 42. Fig. 2. Higher Fig. 3. Higher magnification Fig. 4. Higher
magnification
of the sulcus region (S) from the same specimen
x 135
of the same specimen showing the enamel cuticle (C) and adjacent junctional epithelium. x 540 magnification of the same specimen showing the junctional epithelium and subjacent connective tissue (same level as 3). x 540
An embedding method fol soft-hard
Plate 1.
tissue interfaces
755
Arch,, orul fl,ol. Vol 26. pp. 753 to 755. 1981 Punted I” Great Entam
SHORT COMMUNICATIONS A PARAFFIN-CELLOIDIN EMBEDDING METHOD FOR STUDYING SOFT-HARD TISSUE INTERFACES J. E. LINDER,’ P. LONGHURST’ and N. W. JOHNSON’ The London Hospital Medical College, London E.l, England ‘Department of Orthodontics & Dentistry for Children, Guy’s Hospital London S.E.l, England
‘Department of Oral Pathology,
Summary-Formaldehyde-fixed tooth-gingiva specimens were embedded whole in low-viscosity nitrocellulose (celloidin) to maintain the soft-hard tissue relationships. The soft tissue-bearing portion was sliced off, demineralized in EDTA and re-infiltrated in paraffin wax. Sections were mounted using a special benzyl alcohol acetone-containing fluid to achieve complete flattening and successfully stained using a variety of stains.
Chronic inflammatory periodontal disease, the major cause of tooth loss in adults, has its foundation in the gingival tissue of the deciduous dentition of young children (McCall, 1933; Longhurst, Johnson and Hopps, 1977; Longhurst, Gillett and Johnson, 1980). The technique described here was developed to enable the earliest stages of human periodontal disease to be studied in more detail than heretofore. The aim was the accurate maintenance of the relationship of gingival and periodontal soft tissue to the hard tissues of the tooth itself. It was also important that the technique should be as simple as possible to perform so that reasonable numbers of specimens could be examined and that the resulting sections should be amenable to a wide variety of stains. Preliminary work using a modification of the method of Brain (1962, 1967) to retain some enamel structure during demineralization gave unreliable results. The method of Schroeder (1969) whilst giving better results, does not support the soft tissue during demineralization and subsequent processing may allow distortions. Further, it relies on plastic (Epon) sections which restricts the range of stains possible. Embedding in agar prior to demineralization was also found to be unsatisfactory. The basis of the technique to be described was the suggestion by Schaffer (1926) that delicate biological specimens could be embedded in celloidin prior to demineralization when maintenance of the orientation of soft and hard tissues was important. Deciduous teeth, extracted because of dental caries, were removed with a piece of gingival tissue approximately 3 x 3 mm attached (Longhurst et al., 1980). Each specimen was fixed for 8-12 h in 4 per cent neutral formaldehyde calcium acetate (Lillie and Fullmer, 1976) at room temperature and dehydrated in the following graded waterethanol mixtures: 70 per cent ethanol, 24 h; 90 per cent ethanol, 24 h; absolute ethanol, 3 x 24 h. The specimen was then transferred to a mixture containing equal parts of absolute ethanol and anaesthetic ether for 24 h and then transferred from the absolute ethanol-ether mixture
through a series of solutions of low-viscosity nitrocellulose (LVN) (Chesterman and Leach, 1949) in absolute ethanolether (5, 10 and 20 per cent LVN), approx. 5 days in each at room temperature. A block of LVN containing the entire tooth-gingiva specimen was then prepared by allowing slow evaporation of the ether which caused the LVN to solidify in the form of a firm gel. This block was hardened by exposure to chloroform vapour, and stored in 70 per cent ethanol to remove volatile chloroform and further harden the nitrocellulose. Once completely hardened, the block was transferred to 30 per cent ethanol, mounted on the chuck of a hard-tissue slitting machine and that portion of the tooth bearing the soft tissue, completely supported by the nitrocellulose, was sliced off. The thickness of the slice was determined by the size of the gingival biopsy and the curvature of the tooth surface and was usually 24mm. The slice was washed in distilled water for several hours and demineralized in a 10 per cent w/v solution of sodium EDTA at pH 7.0. This solution was agitated by slowly bubbling air through it, the specimen being supported in a perforated plastic tube. Completion of demineralization was confirmed by X-ray examination. The specimen was washed carefully in running tapwater to remove the EDTA. To facilitate later orientation, a thin Indian Ink line was drawn on the cut surface of the specimen slice to one side of the soft tissue and parallel to the long axis of the tooth. The specimen was again dehydrated through a graded series of water-ethanol mixtures, 30, 50, 70 and 90 per cent half a day in each grade, taken through two changes of a 50:50 mixture of absolute ethanol
753
154
J. E.
Linder. P. Longhurst and N. W. Johnson
paraffin wax, ensuring that the Indian Ink line was towards and parallel with the base of the mould. The paraffin block was mounted on the microtome and, as the nitrocellulose portion was approached during preliminary cutting, the Indian Ink line became visible. The block was adjusted so that the microtome knife was parallel with this line. In this way, vertical, bucco-lingually orientated sections were obtained. The difficulties of section cutting produced by the heterogeneity of the block were overcome by using a very sharp knife and slow cutting speed. In addition, rubbing the block face with cotton wool dampened with distilled water prior to each section being cut helped to reduce electrostatic charges, slightly softened the collagen in the tissue and allowed ribbons of serial sections to be achieved. Sections could be cut at specified thicknesses in the range 3-10pm; 6pm was usual. The heterogeneity of both the block and the specimens also meant that flattening and mounting sections in the usual way was impossible.
Therefore,
a special
section-mounting
fluid
was used, prepared as follows: 0.5 ml benzyl alcohol was added to 20ml acetone; 4-5 drops of Mayer’s glycerin-albumin solution (Gatenby and Painter, 1946) was mixed in 30ml distilled water. The albuminized water was added to the acetonebenzyl alcohol solution and ultrasonicated for 5 min to remove any dissolved air. Section mounting and flattening was carried out on a warm plate at about 40°C and the mounted sections allowed to dry at 37’C. If the temperature of the warm plate was too high, large numbers of bubbles formed beneath the sections. The proportions of the constituents of the mounting fluid could be varied; e.g. the quantity of both benzyl alcohol and acetone could be reduced when section flattening was less difficult. However, the amount of benzyl alcohol should not be increased beyond that stated since mounted sections would then be liable to come off during staining. A wide variety of staining methods has been successfully used including haematoxylin and eosin, Hart’s elastin stain, van Gieson, Giemsa, methyl
green-pyronin, periodic acid-Schiff, silver impregnation methods and Gram stain. Typical examples of the results achieved (Figs 14) show that supporting the gingiva against the tooth surface throughout processing has preserved the intimate soft-hard tissue relationship presented by the juxtaposition of teeth and their investing soft tissues and also retained acquired tooth surface deposits. Although the technique presented here was developed to study a soft-hard tissue relationship peculiar to teeth, there seems little doubt that it could have applications elsewhere in biological science. REFERENCES Brain E. H. 1962. A new method for the preparation of decalcified sections of human enamel in situ. Archs. orul Biol. 7, 157-763. Brain E. H. 1967. Rapid demineralization for microscopy of tooth enamel and associated structures. Br. dent. J. 123(4), 177-181. Chesterman W. and Leach E. H. 1949. Low viscosity nitrocellulose for embedding tissues. Q. JI microsc. Sci. 90(4), 43 l--434. Gatenby J. B. and Painter T. S. 1946. The Microtomist’s Vade-Mecum (Belles Lee) Para. 209, p. 114. Churchill, London. Lillie R. D. and Fullmer H. M. 1976. Histopathologic Technic and Practicai Histochemistry, 4th edn, Chap. 3. p, 33. McGraw-Hill, New York. Longhurst P., Gillett R. and Johnson N. W. 1980. Electron microscope quantitation of inflammatory infiltrates in childhood gingivitis. J. periodont. Rex 15, 255-266. Longhurst P., Johnson N. W. and Hopps R. 1977. Differences in lymphocyte and plasma cell densities in inflamed gingiva from adults and young children. J. periodont. 48, 705-710. McCall J. 0. 1933. The Periodonttst looks at Children’s Dentistry. J. Am. Dent. Ass. 20, 1518-1521. Schaffer J. 1926. Enzyklopiidie der Mikroskopischen Technik, Vol. II (Edited bv Krause R.), D. 1150. Urban & Scharzenberg. Berlin. Schroeder H. E. 1969. Ultrastructure of the junctional epithelium of the human gingiva. Heia. odont. Acra 13, 65-83.
Plate 1. Fig. 1. Gingiva and adjacent tooth; longitudinal bucco-lingual section of upper first deciduous molar; child aged 3 yr. D = dentine; P = bacterial plaque; C = cuticle; E = enamel space; OE = oral epithelium; JE = junctional epithelium; CT = connective tissue. Van Gieson stain. x 42. Fig. 2. Higher Fig. 3. Higher magnification Fig. 4. Higher
magnification
of the sulcus region (S) from the same specimen
x 135
of the same specimen showing the enamel cuticle (C) and adjacent junctional epithelium. x 540 magnification of the same specimen showing the junctional epithelium and subjacent connective tissue (same level as 3). x 540
An embedding method fol soft-hard
Plate 1.
tissue interfaces
755