31
J. Anat. (1979) 128, 1, pp. 31-51 With 21 figures Printed in Great Britain
Differentiation in normal human buccal mucosa epithelium MERWYN A. LANDAY* AND HUBERT E. SCHROEDERt
Department of Oral Structural Biology, Dental Institute, University of Zurich
(Accepted 19 January 1978) INTRODUCTION
Recently the stratified epithelium of normal human buccal mucosa has been examined stereologically (Landay & Schroeder, 1977). Volumetric density gradients of various cytoplasmic components implied that (1) the most pronounced alterations and the site of major differentiation are in the lower stratum spinosum; (2) shape alterations such as flattening of epithelial cells and their nuclei in the upper stratum spinosum are unrelated to the differentiation process, which at this level is almost complete; (3) a densely developed filament meshwork in the cells of the middle and upper stratum spinosum and the surface stratum may provide the functional matrix for distensibility of this epithelium. These observations prompted a structural re-examination of normal human buccal mucosa epithelium, although its features have already been studied by light microscopy (Watt, 1911; Chambers & Renyi, 1925; Weinmann, 1940; Krzywicki & Rokicka, 1967; Meyer & Gerson, 1964), histochemistry (Falin, 1961; Morgenroth & Themann, 1962; Haim, 1964; Meyer & Gerson, 1964; Billow, 1966; Silverman & Kearns, 1970; Silverman, Barbosa & Kearns, 1971) and electron microscopy (Sognnaes & Albright, 1958; Fasske & Themann, 1959; Zelickson & Hartmann, 1962; Frithiof & Wersall, 1965; Biulow, 1966; Hashimoto, Dibella & Shklar, 1966; Silverman, 1967, 1971; Susi, Belt & Kelly, 1967; Thilander & Bloom, 1968; Llary, Callogny & Deloge, 1969; Susi, 1969; Hutchens, Sagebiel & Clarke, 1971; Epishev, Zufaror & Arzhanykh, 1971; Plackova, Medack, Meyer & Waterhouse, 1971; Whittaker & Adams, 1971; El-Labban & Kramer, 1974). MATERIAL AND METHODS
Twenty biopsies of human buccal mucosa were collected under mandibular block anaesthesia from the left cheek of 10-15 years old females in the area opposite the lower left premolars. Biopsies were taken only when the mucosa was clinically normal, and the subjects medically healthy. The specimen was removed using a biopsy punch with a 3 mm diameter, and a No. 11 scalpel; every attempt was made to minimize injury and compression of the tissue. The specimens were placed in cold fixative containing 2-5 % glutaraldehyde and 2 % paraformaldehyde buffered with 0-02 M sodium cacodylate (pH 7-2 and 900 mOsm; Kamovsky, 1965). This fixative, even though hypertonic, produces minimal change in the size of intercellular spaces in oral epithelium (Squier, Waterhouse & Kraucunas, 1973 a, b). After 2 hours the biopsies were divided into approximately * This investigation was performed while Dr Landay was on leave from the Department of Periodontology, Temple University, School of Dentistry, Philadelphia, USA. t Address for reprints; Plattenstrasse 11, 8028 Zurich, Switzerland.
32
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Differentiation of buccal mucosa epithelium
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1 mm thick slices under a dissecting microscope, while immersed in 0-185 M sodium cacodylate buffer (pH 7 4, 350 mOsm), and then replaced into the fixative for a further 1P5 hours. The blocks were post-fixed for 2 hours in 1P33 ° OSO4 buffered (pH 7 4) in 0 067 M s-collidine (Bennett & Luft, 1959), dehydrated in ethanol, and embedded in Epon (Luft, 1961). From all blocks, 1-2 ,tm thick sections were prepared and stained with PAS and methylene blue/azure II (Schroeder, 1973). Thin sections were mounted on carboncoated copper grids and stained with uranyl-magnesium acetate followed by lead citrate (Fraska & Parks, 1965; Reynolds, 1963). The sections were examined in a Philips EM 201. In addition, the surface density of epithelial cells (Scee) residing in an approximately 200 ,um broad surface layer was estimated in nine biopsies by photographical mapping and point counting morphometry. The mapping was performed in one semithin section from each of two blocks of the nine individual biopsies. Photomicrographic reproductions were reconstructed displaying the upper third of the epithelium at x 1900 magnification. On these maps two layers running parallel to the surface contour of the epithelium were arbitrarily defined: a surface layer immediately beneath the uppermost two to four cells, and a subsurface layer parallel to the former. Each of these layers was about 90 Fsm deep. Within each layer a coherent single lattice test system printed into a transparent foil of 11 x 11 cm, and comprising 15 evenly spaced horizontal and vertical lines each, was superimposed on the epithelial structures, the horizontal lines of the test system forming an angle of 30° with the epithelial surface. The intersections of horizontal lines with the intercellular spaces (i.e. two cell borders in juxtaposition) were counted. This procedure was repeated five times along and within each of the surface and subsurface layers. By using the formula _ 2 x EIce
SVe-(L. x N)' where A, is the number of intersections, LT is the total length of horizontal test lines (in ,um) per test field, and N is the number of fields measured, a rough estimate of the surface density of epithelial cells was obtained (Weibel & Bolender, 1973). Averages and standard deviations were calculated per individual biopsy, as well as for groups of biopsies with either wrinkled or flat surface contours. RESULTS
General morphology The buccal epithelium was 0 5-0 6 mm thick. Long and slender connective tissue papillae extended to the upper (i.e. superficial) third of the epithelium (Figs. 1, 2). The papillae frequently branched, bent within the epithelium, and alternated with long epithelial ridges of varying width. Fig. 1. Buccal epithelium with slightly wrinkled surface contour and even or uneven (inset) staining pattern. Only a very slight staining difference between basal/suprabasal and more superficial cells is visible over connective tissue papillae (p). The latter regularly penetrate the basal two thirds of the epithelium, which contains glycogen (g). PAS-methylene blue/azure II. x 130. Inset x 150. Fig. 2. Buccal epithelium with a flat surface contour and uneven staining pattern. Basal and suprabasal cells stain light blue, all other cells dark blue, g, glycogen. Narrow, in part branching connective tissue papillae (p) penetrate the basal two thirds of the epithelium. PAS-methylene blue/azure II. x 130. Inset x 150. 3
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34
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J. Anat. (1979) 128, 1, pp. 31-51 With 21 figures Printed in Great Britain
Differentiation in normal human buccal mucosa epithelium MERWYN A. LANDAY* AND HUBERT E. SCHROEDERt
Department of Oral Structural Biology, Dental Institute, University of Zurich
(Accepted 19 January 1978) INTRODUCTION
Recently the stratified epithelium of normal human buccal mucosa has been examined stereologically (Landay & Schroeder, 1977). Volumetric density gradients of various cytoplasmic components implied that (1) the most pronounced alterations and the site of major differentiation are in the lower stratum spinosum; (2) shape alterations such as flattening of epithelial cells and their nuclei in the upper stratum spinosum are unrelated to the differentiation process, which at this level is almost complete; (3) a densely developed filament meshwork in the cells of the middle and upper stratum spinosum and the surface stratum may provide the functional matrix for distensibility of this epithelium. These observations prompted a structural re-examination of normal human buccal mucosa epithelium, although its features have already been studied by light microscopy (Watt, 1911; Chambers & Renyi, 1925; Weinmann, 1940; Krzywicki & Rokicka, 1967; Meyer & Gerson, 1964), histochemistry (Falin, 1961; Morgenroth & Themann, 1962; Haim, 1964; Meyer & Gerson, 1964; Billow, 1966; Silverman & Kearns, 1970; Silverman, Barbosa & Kearns, 1971) and electron microscopy (Sognnaes & Albright, 1958; Fasske & Themann, 1959; Zelickson & Hartmann, 1962; Frithiof & Wersall, 1965; Biulow, 1966; Hashimoto, Dibella & Shklar, 1966; Silverman, 1967, 1971; Susi, Belt & Kelly, 1967; Thilander & Bloom, 1968; Llary, Callogny & Deloge, 1969; Susi, 1969; Hutchens, Sagebiel & Clarke, 1971; Epishev, Zufaror & Arzhanykh, 1971; Plackova, Medack, Meyer & Waterhouse, 1971; Whittaker & Adams, 1971; El-Labban & Kramer, 1974). MATERIAL AND METHODS
Twenty biopsies of human buccal mucosa were collected under mandibular block anaesthesia from the left cheek of 10-15 years old females in the area opposite the lower left premolars. Biopsies were taken only when the mucosa was clinically normal, and the subjects medically healthy. The specimen was removed using a biopsy punch with a 3 mm diameter, and a No. 11 scalpel; every attempt was made to minimize injury and compression of the tissue. The specimens were placed in cold fixative containing 2-5 % glutaraldehyde and 2 % paraformaldehyde buffered with 0-02 M sodium cacodylate (pH 7-2 and 900 mOsm; Kamovsky, 1965). This fixative, even though hypertonic, produces minimal change in the size of intercellular spaces in oral epithelium (Squier, Waterhouse & Kraucunas, 1973 a, b). After 2 hours the biopsies were divided into approximately * This investigation was performed while Dr Landay was on leave from the Department of Periodontology, Temple University, School of Dentistry, Philadelphia, USA. t Address for reprints; Plattenstrasse 11, 8028 Zurich, Switzerland.
32
M. A. LANDAY AND H. E. SCHROEDER
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;.
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Differentiation of buccal mucosa epithelium
33
1 mm thick slices under a dissecting microscope, while immersed in 0-185 M sodium cacodylate buffer (pH 7 4, 350 mOsm), and then replaced into the fixative for a further 1P5 hours. The blocks were post-fixed for 2 hours in 1P33 ° OSO4 buffered (pH 7 4) in 0 067 M s-collidine (Bennett & Luft, 1959), dehydrated in ethanol, and embedded in Epon (Luft, 1961). From all blocks, 1-2 ,tm thick sections were prepared and stained with PAS and methylene blue/azure II (Schroeder, 1973). Thin sections were mounted on carboncoated copper grids and stained with uranyl-magnesium acetate followed by lead citrate (Fraska & Parks, 1965; Reynolds, 1963). The sections were examined in a Philips EM 201. In addition, the surface density of epithelial cells (Scee) residing in an approximately 200 ,um broad surface layer was estimated in nine biopsies by photographical mapping and point counting morphometry. The mapping was performed in one semithin section from each of two blocks of the nine individual biopsies. Photomicrographic reproductions were reconstructed displaying the upper third of the epithelium at x 1900 magnification. On these maps two layers running parallel to the surface contour of the epithelium were arbitrarily defined: a surface layer immediately beneath the uppermost two to four cells, and a subsurface layer parallel to the former. Each of these layers was about 90 Fsm deep. Within each layer a coherent single lattice test system printed into a transparent foil of 11 x 11 cm, and comprising 15 evenly spaced horizontal and vertical lines each, was superimposed on the epithelial structures, the horizontal lines of the test system forming an angle of 30° with the epithelial surface. The intersections of horizontal lines with the intercellular spaces (i.e. two cell borders in juxtaposition) were counted. This procedure was repeated five times along and within each of the surface and subsurface layers. By using the formula _ 2 x EIce
SVe-(L. x N)' where A, is the number of intersections, LT is the total length of horizontal test lines (in ,um) per test field, and N is the number of fields measured, a rough estimate of the surface density of epithelial cells was obtained (Weibel & Bolender, 1973). Averages and standard deviations were calculated per individual biopsy, as well as for groups of biopsies with either wrinkled or flat surface contours. RESULTS
General morphology The buccal epithelium was 0 5-0 6 mm thick. Long and slender connective tissue papillae extended to the upper (i.e. superficial) third of the epithelium (Figs. 1, 2). The papillae frequently branched, bent within the epithelium, and alternated with long epithelial ridges of varying width. Fig. 1. Buccal epithelium with slightly wrinkled surface contour and even or uneven (inset) staining pattern. Only a very slight staining difference between basal/suprabasal and more superficial cells is visible over connective tissue papillae (p). The latter regularly penetrate the basal two thirds of the epithelium, which contains glycogen (g). PAS-methylene blue/azure II. x 130. Inset x 150. Fig. 2. Buccal epithelium with a flat surface contour and uneven staining pattern. Basal and suprabasal cells stain light blue, all other cells dark blue, g, glycogen. Narrow, in part branching connective tissue papillae (p) penetrate the basal two thirds of the epithelium. PAS-methylene blue/azure II. x 130. Inset x 150. 3
ANA I28
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34
M. A. LANDAY AND H. E. SCHROEDER
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