a quantitative electron microscopic analysis of the keratinizing epithelium of normal human hard...

Cell Tiss. Res. 158, 177--203 (1975) �9 by Springer-Verlag 1975

A Quantitative Electron Microscopic Analysis of the Keratinizing Epithelium of Normal Human Hard Palate

M a r t i n M e y e r * a n d H u b e r t E . S e h r o e d e r * *

Department of Oral Structural Biology, Dental Institute, University of Zurich, Switzerland

Received November 1, 1974 / in final form January 27, 1975

Summary. The epithelium of normal human hard palate was subjected to stereologic analysis. Ten biopsies were selected from a total of twenty specimens collected from 9 to 16 year old females, and processed for light- and electron microscopy. At two levels of magnification, electron micrographs were sampled from three strata (basale, spinosum, granulosum) in two locations (epithelial ridges and portions over connective tissue papillae). Stereologic point counting procedures were employed to analyse a total 1560 electron micrographs. In general, the thickness of the palate epithelium was 0.12 mm (over papillae) and 0.31 mm (in ridges), the epithelium is distinctly stratified, and homogeneously ortho-keratinized. From basal to granular layers, the composition of strata revealed decreasing densities of nuclei, mitochondria, membrane-bound organelles and aggregates of free ribosomes. Keratohyalin bodies and membrane coating granules increased, and cytoplasmic filaments with a constant diameter of about 85 A increased from 14 to 30% of cytoplasmic unit volume. The cytoplasmic ground substance occupied a stable 50% of the epithelial cytoplasm in all strata. The composition of basal layers in ridges differed from that over connective tissue papillae. The data are discussed in relation to the observations that (1) an increasing gradient of filament density is not the most characteristic feature of ortho-keratinizing oral epithelium and (2) differences in the degree of differentiation in cells of the stratum basale coincided with the comparable frequency distribution pattern of dividing cells.

Key words: Oral epithelium - - Keratinization - - Stereology.

Zusammen/assung. Das Epithel des normalen menschlichen harten Gaumens wurde einer stereologischen Analyse unterworfen. Insgesamt wurden 20 Biopsien aus 9--16 Jahre alten, gesunden M/idchen gewonnen und fiir licht- und elektronenmikroskopische Studien verarbeitet. Aus 10, zufi~llig ausgew~hlten Biopsien wurden Stichproben elektronenmikroskopischer Bilder aus 3 Straten (basale, spinosum, granulosum) und in 2 verschiedenen Bereichen (in epithelialen Leisten und im Epithel fiber Bindegewebspapillen) entnommen. Ein Total yon 1560 elektro- nenmikroskopischen Bildern wurde mit Hills stereologischer Punktziihlverfahren analysiert. Die Dicks des Gaumenepithels sehwankte zwischen durchschnittlich 0,12 (fiber Papillen) und 0,31 mm (in Leistenbereich). Das Epithel ist deutlich geschiehtet und gleiehm~Big ortho- keratinisiert. Vom Stratum basale gegen das Stratum granulosum fiel die volumetrische Dichte fiir Kerne und Mitochondrien, ffir membrangebundene, synthetisierende Organellen und fiir Aggregate freier Ribosomen ab, wiihrend Keratohyalink6rper und membranover-

Send of/print requests to: Dr. H. E. Schroeder, Plattenstrage 11, CIt-8028 Zurich, Switzerland.

* Dr. Meyer is member of the Department of Cariology and Periodontology, Dental Institute, University of Zurich. His work was performed in partial fulfillment of the requirements for a Dr. mad. dent. thesis at the Faculty of Medicine, University of Zurich.

** The authors are thankful to Miss K. Rossinsky for excellent technical assistance, to Mrs. M. Graf-de Beer for competent data computation and to Mrs. S. Mfinzel-Pedrazzoli for help in morphometric analysis. This study was in part supported by Grants Nos. 51 and 106 of the Hartmann Mfiller Foundation and by a Grant from the Foundation of Scientific Research at the University of Zfirieh.

178 M. Meyer and H. E. Schroeder

steifende Granula dichtem~il~ig zunahmen. Der zytoplasmatische Gehalt an Tonofilamenten, die einen konstanten Durchmesser yon 85 A aufwiesen, stieg yon 14 auf 30 % an, w~hrend die strukturlose, zytoplasmatische Grundsubstanz in allen Straten etwa 50% des Zytoplasma- volumens einnahm. Die strukturelle Zusammensetzung des Stratum basale war je nach Lokali- sation (Leisten- und Papillenbereich) verschieden. Die Ergebnisse werden insbesondere im Hinblick auf die Beobachtungen diskutiert, dab 1. ein Ansteigen des Gradienten der Tonofila- mentdichte nicht als das besonders charakteristische Kennzeichen fiir orthokeratinisierendes orales Epithel gelten kann, und 2. die Unterschiede im Differentiationsgrad zwischen Basal- zellen verschiedener Lokalisation mit der vergleichbaren H~ufigkeitsverteilung yon sich teilen- den Zellen gut iibereinstimmte.

Introduction

The epi thel ia of the h u m a n oral cav i ty are f requent ly subjec t to pa thologica l a l te ra t ions which manifes t themselves as changes in the p a t t e r n of epi thel ia l different ia t ion. The most no tab le of these changes is hyperkera t in iza t ion .

The epi the l ium of the h u m a n ha rd pa la te is a most consis tent ly o r thokera t in - izing s t ra t i f ied epi thel ium. This ep i the l ium has been s tud ied only to a minor ex ten t b y l ight microscopy (Meyer and Gerson, 1964; Kryzwick i and Rokicka , 1967), h i s tochemis t ry (F lanagan and Por ter , 1971) and electron microscopy (Haim, 1964, 1965 ; F r i th io f and Wers/~ll, 1965 ; Weins tock and Albr ight , 1966 ; Thi lander , 1968 ; Th i lander and Bloom, 1968; Si lverman, 1971; Si lverman, Barbosa and Kearns , 1971). Most of these inves t iga t ions dea l t wi th some specific fea ture of the epithe- l ium, and only Thi lander (1968) and Si lverman (1971) have a t t e m p t e d to provide an overal l s t ruc tura l descript ion.

I n order to meet the requi rements for baseline d a t a in compara t ive s tudies on epi thel ia l pa thology , a broad, representa t ive , quan t i t a t i ve analysis of the kera- t inizing pa la te ep i the l ium would be desirable, l~ecently, morphomet r i c techniques based on the appl ica t ion of stereologic po in t -count ing procedures (Weibel, 1969) have been tes ted and found to be a powerful and rel iable tool when employed to s tudy oral epi thel ia (Schroeder and Mfinzel-Pedrazzoli , 1970a and b). The present s tudy , therefore, was per formed to provide a quan t i t a t ive , stereologic descr ip t ion of the u l t r a s t ruc tu re of normal ep i the l ium of the h u m a n ha rd pala te .

Materials and Methods

Twenty biopsies of the mucosa of human hard palate were collected from the left side of the pars glandulosa of 9 to 16 year old females, in the area medial to the interdental space between ~-V/5 and ~-6 and approximately halfway between the interdental papilla and the raphe palati. Biopsies were taken only from medically healthy subjects in whom the mucosa was clinically normal.

The biopsies were harvested following nerve block anaesthesia (2 % Lidocain and Epineph- rine 1:80000). A biopsy punch with a 3 mm diameter and a :No. 11 scalpel blade was used. Every attempt was made to avoid injury or compression of the specimen.

The specimens were immediately placed into a cold fixative containing 5 % glutaraldehyde and 4% paraformaldehyde buffered with 0.02 M sodium cacodylate (pH 7.3, 2010 mOsm; Karnovsky, 1965). This fixative, although highly hypertonic, has been found to provide satis- factory tissue preservation when initial fixation was carried out with comparatively large tissue blocks (Schroeder, 1969). After two hours of fixation at 4 ~ C, the specimens, while immersed in 0.185 M sodium cacodylate buffer (pH 7.4; mOsm), were subdivided into approximately 1 mm thick slices (blocks) cut vertical to the epithelial surface under a dissecting microscope. Thereafter, the blocks were postfixed for two hours at 4 ~ C in 1.33% OsOa, buffered

Stereologic Analysis of Human Hard Palate Epithelium 179

(pH 7.4) in 0.067 M s-collidine (Bennett and Luft, 1959), dehydrated in ethanol, and embedded in Epon (Luft, 1981). The tissue blocks were oriented flat at the bottom of reversed Beem capsules.

From all blocks, approximately 1 to 2 tzm thick cross-sections were prepared, using a Reichert OM-U2 ultramicrotome with glass knives. These sections were examined and photo- graphically recorded in a phase-contrast microscope, in order to study and delineate the site for electron microscopic examination. Semi-thin sections were also stained with PAS and Toluidin blue/Azure I I (Schroeder, 1973).

Measurements of epithelial thickness were performed on one random section from each of five tissue blocks obtained from ten different subjects (i.e. a total of 50 sections). These measurements were made on the projection screen of a Wild sampling microscope M-501 (Wei- bel, 1970) mounted with a lattice test system of regularly spaced vertical and horizontal test lines. The sections were projected at a final magnification of 210 X, and oriented with the epithelial surface parallel to a horizontal test line. Thickness determinations were made along vertical test lines at three different locations: (1) 4 measurements were made where the epithelium was thickest-between the base of epithelial ridges and the epithelial surface; (2) 4 measurements were made where the epithelium was thinnest-between the top of connective tissue papillae and the epithelial surface; (3) 4 measurements were made at random sites indicated by the location of regularly spaced vertical test lines. Average epithelial thickness was then calculated for sites of epithelial ridges, over connective tissue papillae, and at random. The latter value represented an indication of overall general thickness.

From tissue blocks of biopsies selected for morphometric investigation, ultrathin sections were cut, using an LKB-Ultrotome I I with a diamond knife (Dupont). Block faces were trimmed to contain the entire thickness of the epithelium in the selected areas. Section thickness was kept as constant as possible in the range of silver interference colour, suggesting a thickness of 600 to 900/~ (Peachey, 1958). Ribbons of sections were mounted in an oriented fashion (Schroeder, 1967) on R-150 copper grids (VECO) coated with carbon film. The contrast was enhanced by staining with uranyl-magnesium acetate followed by lead citrate (Fraska and Parks, 1985; Reynolds, 1963). Ultrathin sections were examined with a Philips EM-3001. Micrographs were recorded on 35 mm film for morphometrie analysis (Weibcl, Kistler and Scherle, 1966).

Sampling The primary sample consisted of some 100 tissue blocks obtained by subdividing the

20 biopsies. From these, 10 biopsies were randomly selected, each of these 10 was represented by 4 randomly chosen tissue blocks, forming 40 blocks as a secondary sample. Each of these blocks was trimmed to retain one of the two regions of the hard palate epithelium (Fig. 1), either areas of epithelial ridges (R) or areas overlying connective tissue papillae (P). There were two reasons for adopting this procedure: (1) In keratinizing oral epithelium of the hard palate and of the human gingiva, dividing cells occur in clusters and division takes place preferentially at sites of epithelial ridges, when connective tissue ridges and papillae are high and well developed (LSe, Karring and Hara, 1972). (2) The keratinizing epithelium of the dorsum of the human tongue displays a mitotic activity of extremely uneven distribution, most of the dividing ceils occuring at the base of the broad epithelial ridges, while few or none divide in basal layers over secondary papillae of the filiform papillae (Kunze. 1969). Since a striking feature of the human palate is the regular and deep interdigitation of epithelium and connective tissue, a similar distribution of mitotic activities could be expected. Based on this assumption, areas of epithelial ridges and areas over connective tissue papillae were sampled separately in order that any structural differences between frequently dividing ridge ceils and less frequently dividing cells over connective tissue papillae, would be discriminated in the morphometric analysis. Therefore, a total of 20 blocks (2 each of 10 individual biopsies) each of ridge and papilla locations, was subjected to further sampling in these two regions.

The tertiary sample consisted of electron micrographs recorded from one randomly chosen ultrathin section from each of the 2 times 20 tissue blocks of the secondary sample. I t was necessary to further subdivide the tertiary sample in two ways (Fig. 1), firstly into two levels

1 The EM-work was done at the Department of Electron Microscopy at the Insti tute of Anatomy, University of Zurich.


Fig. 1. Representative section of human hard palate epithelium. Note the regular interdigitation of connective tissue papillae (P) with epithelial ridges (R), clear cells (C) and a corpuscle of Meissner (M). The rectangles indicate the stratified sampling at level I in the stratum basale, the upper stratum spinosum and the stratum granulosum of epithelial portions

in ridges and over connective tissue papillae. Magn: • 375

of magnification (levels I and II), and secondly into different strata. Subsampling at two levels of magnification was basically duplicated from a previous study which fully discussed the requirements of sample size, structural recognition and test point density relative to morphometric analysis of stratified epithelia (Schroeder and Miinzel-Pedrazzoli, 1970a). The method of subsampling within and along various epithelial strata has also been previously tested and discussed (Schroeder and Mfinzel-Pedrazzoli, 1970a). However, as the strata subsampling of

Stereologic Analysis of Human Hard Palate Epi thel ium 181

the present s tudy deviates in fundamental ways from tha t discussed previously, a few additional remarks are in order. Oral epithelia are stratified and cells a t different levels in the epithelium differ from one another, the s t ra tum basale being the least differentiated. In addition, the interface between epithelium and connective tissue follows a varying undulated and three-dimensionally complex pa t te rn (Horstmann, 1954) which is not really evident in random histological cross-sections (Karring and LSe, 1970). The density of single connective tissue papillae is very much higher in palatal than in buccal or gingival mucosa (Klein and Schroeder, unpublished observations). Because of this, it is often impossible to determine whether a cell appearing in one section to be located in the s t ra tum spinosum (i.e. in the middle of what appears as an epithelial ridge) is in fact so situated, or whether i t is located adjacent to a connective tissue papilla occuring in adjacent sections, and thus represents a basal cell (LSe, Karr ing and Hara, 1972). These characteristics of epithelial s tructure imply t ha t : (l) the various s t ra ta do not form equidis tant layers vertical to the axis of anisotropy and cell differentiation and (2) all cells in a given random section located between the s t ra tum basale along epithelial ridges and the level beyond the terminat ion of connective tissue papillae are difficult to classify as either s t ra tum basale or s t ra tum spinosum cells. Since the sampling procedure required for morphometric analysis necessitates as much homogeneity and isotropy as possible, the various samples taken from stratified oral epithelia must originate from areas of fine structural homogeneity, i.e. from epithelial s t ra ta which can be distinguished with a certain degree of accuracy (Schroeder and Mfinzel-Pedrazzoli, 1970a). Independent of the plane of cross-section- ing, such strataforming polarized sheets of homogeneous tissue in keratinizing epithelia are the basal layer along the boundaries of epithelial ridges and connective tissue papillae, the s t ra tum granulosum, the s t ra tum corneum and an arbi t rary layer of the s t ra tum spinosum located halfway between the basal layer over papillae and the interface between s t ra tum granulosum and s t ra tum corneum. All of these layers can be reproducibly located. In the present study, therefore, sampling of electron micrographs was performed along and within the basal layer, the arbi trar i ly defined middle layer of spinous cells and the s t ra tum granulosum. Electron micrographs of these three s t ra ta were sampled from all blocks of the ter t iary sample, representing bo th epithelial locations R and P. This produced a standardized sampling procedure which as i l lustrated in Fig. 1.

Level I . Four electron micrographs as a consecutive field to field sample were recorded from each of the three s t ra ta (see Fig. 1) at a pr imary magnification of 1400 • Thus, 12 fields were obtained from each block, yielding a total ter t iary sample of 2 t imes 240 micrographs representing either R or P locations.

Level I I . Nine electron micrographs as a consecutive field to field sample were recorded from each of the three s t ra ta at a pr imary magnification of 5 200 • In all instances, the level I I sample was contained within the area sampled a t level I. Thus, 27 fields were obtained from each block, yielding a total ter t iary sample of 2 t imes 549 micrographs, representing either R or P locations. The fields recorded in the middle layer as well as in the s t ra tum granulosum were a strictly stratified sample. For reasons discussed previously (Schroeder and Miinzel- Pedrazzoli, 1970a), sampling of the basal layer consisted of al ternat ing fields taken from basal and distal cell portions. In general, sections were oriented within the electron microscope in order to produce an inclination of 20 to 30 ~ of the s t ra tum corneum/stratum granulosum interface to one side of the square field of photographical recording. This procedure served to avoid any parallelism between cytoplasmic structures and lines of the morphometric tes t system.

Before recording micrographs at each level of sampling and at the end of each film, a magnification s tandard was recorded (Weibel, Kistler and Scherle, 1966). All 35 mm films were contact-printed on film. The positive electron micrographs were examined in a table projector uni t (Weibel, 1969), yielding either a 9.6 or an 11.44 • secondary magnification. Thus, the final magnification for morphometric analysis was 13,440 • for level I and 59,325 • for level II .

Two additional series of electron micrographs were taken for the measurement of cytoplasmic fi lament diameter and basal lamina thickness.

The sample for cytoplasmic f i lament diameter s tudy consisted of micrographs at a pr imary magnification of 22,210 • recorded from 1 randomly selected tissue block of ridge location from each of 7 subjects. Four fields were recorded from each block in each of the three strata. These fields were randomly chosen, bu t all fulfilled the criterion t ha t they contained cyto-


Fig. 2. Typical electron micrograph characterizing a sample field at level I of the stratum spinosum in epithelial ridges. The coherent double lattice test system (Weibel, 1969) comprising 100 heavy and 2,500 light volumetric points is superimposed on the micrograph.

Volumetric points falling onto epithelial nuclei are circled. Magn: • 5800

plasmic filaments in cross-section. A total of 84 micrographs was subjected to multiple measurements performed at a final magnification of 253,170 •

The sample for basal lamina thickness determination consisted of electron micrographs at a primary magnification of 9,100 • recorded from 1 randomly selected tissue block each of R and P locations from 5 subjects. Eight randomly chosen fields were sampled along the basal complex from each block. A total sample of 80 micrographs was subjected to multiple measurements at a final magnification of 105,200 •

Stereoloffie Procedures Quantitative analysis of the electron micrographs was performed, using stereologic point-

counting procedures (Weibel, 1969; Weibel and Elias, 1967; Underwood, 1970). A coherent double lattice test system fitted into the viewing screen of the table projector unit was used


for the analysis of micrographs at level I. This test system consisted of a square frame of definite size enclosing a network of heavy and light test lines (Weibel, 1969). The heavy lines crossed to form 100 volumetric points. In applying heavy points of this test system to level I electron mierographs (Fig. 2), the test field comprising a test area of 346 ~zm 2 was randomly placed on each micrograph, one test point representing 3.46 ~zm 2. Test points were differentially counted to estimate the relative volume (Vv) of the following gross tissue components: (1) epithelial cell nuclei (n), (2) epithelial cell cytoplasm (cy), (3) intercellular space (ics), and (4) non- epithelial cells (r). From the above values, the volume fraction occupied by epithelial cells and the nucleus to cytoplasmic ratio were calculated. Intersections of horizontal heavy te~t lines with the nuclear envelope and the cytoplasmic membrane of epithelial ceils were separately counted in order to estimate the surface densities (Sv) and the surface to volume ratios (S/V) of nuclei and epithelial cells. From these values was calculated the ratio of nuclei to cell surfaces. Finally the number of desmosomes was counted and calculated per 100 ~zm 2 test field. The size of the total test sample per stratum examined in level I amounted to 2770 Ezm 2 of R and P locations each per biopsy.

A multipurpose test system consisting of a square frame enclosing 168 test points marked as end points of 84 test lines of length z (Weibel, 1969) was used to analyse level II electron micrographs. In applying this test system (Fig. 3), the test area amounted to 16.4 ptm 2, one test point representing 0.098 tzm 2. Test points were differentially counted in order to estimate the relative volumes (Vv) of the following cytoplasmic components: (1) mitechondria (mi), (2) aggregates of non-membrane bound, free ribosomes (rib), (3) rough endoplasmie reticulum cisternae, including the perinnclear envelope (er), (4) smooth membrane cisternae, including the Golgi apparatus and pinocytotic vesicles (sins), (5) lysosome-like bodies (ly), (6) membrane- coating granules (meg), (7) cytoplasmic filaments organized into tonofilament-like bundles (lib), (8) keratohyalin (kh), and (9) cytoplasmic ground substance or residue (cr). Intersections of test lines with membrane profiles of rough er and sins were counted separately to estimate the surface densities of these structures. The size of the test sample per stratum examined in level II amounted to 295 ~m 2 of R and P locations each per biopsy.

Data Recording and Computation. All single values of points and intersections were recorded on an electronic data collector previously described (Weibel, 1967; Weibel, 1969). To facilitate calculations, the actual pcintcounting procedure at level II was performed by accumulating differential counts up to 100 (Schroeder and Miinzel-Pedrazzoli, 1970a). Calculation of aver- ages was done with a Diehl-Sigmatron calculator. The statistical analysis was performed on a preprogrammed Diehl-Alphatronie calculator. Estimations of volumetric (Vv) and surface densities (Sv) as well as the surface to volume ratio (S/V) of the various structural components were obtained by applying basic stereologic formulas described recently (Weibel, 1969; Weibel, and Bolender, 1973). Significance tests were performed using the classical F-test. Probabilities above 1% were not accepted. For comparative purposes, all data were calculated in relation to 1 cm a volume of epithelial stratum, or to 1 cm 3 of epithelial cytoplasm respectively.

Results

General Description o/ the Ep i the l ium

The epithel ium of the h u m a n hard palate was found to be on average 0.25 • 0.04 m m thick (Table 1). I t showed a very regular pa t t e rn of interdigi ta t ion with the under ly ing connective tissue. Epithel ial ridges had fingershaped outlines and were of ra ther constant width (Fig. 1). At these sites, the epi thel ium was 0.31 • 0.05 m m thick (Table 1). The epithelial ridges were separated by connective tissue papillae of ra ther regular height and width (Fig. 1), Frequent ly , Meissner's cor- puscles were observed at the top of the connective tissue papillae (Fig. 1). The average thickness of the epi thel ium over connective tissue papillae was 0 . 1 2 i 0.02 m m (Table 1). At the base of epithelial ridges, epithelial cells appeared to be small, and nuclei were densely packed (Fig. 1). At the sides of connective tissue papillae, basal cells were ovoid and did no t appear to f la t ten towards the middle


Fig. 3. Typical electron micrograph characterizing a sample field at level II of the stratum spilmsum. The multipurpose test system (Weibel, 1969) comprising 168 volumetric points and 84 test lines is superimposed on the micrograph. Volumetric points falling onto mitochondria

are circled. Magn: ~< 27,600

portion of epithelial ridges (Fig. 1). Over connective tissue papillae, epithelial cells were ovoid and flattened progressively from the upper stratum spinosum to the stratum granulosum. The stratum corneum was of regular width, homogeneously structured and rarely contained pyknotic nuclei (Fig. 1). The basal complex beneath the epithelium was composed of a very distinct lamina lucida and lamina densa, the thickness of which appeared to be identical when measured at epithelial ridges or over connective tissue papillae (503 to 512 A for lamina densa, 414 to 472 A for lamina lucida, Table 2).

Stereologic Analysis of Human Hard Palate Epithelium

Table 1. Thickness of epithelium of hard palate

Location 2 • s Units

Over epithelial ridges 0.310-k 0.050 mm Over connective tissue papillae 0.116 -4- 0.017 mm Random 0.248 • 0.037 mm

Number of biopsies 10

185

Table 2. Size of the lamina densa and lamina lucida in hard palate mucosa at sites of epithelial ridges (R) and over connective tissue papillae (P)

Parameter R P Units 2• 2-ks

Lamina densa a512 ~: 37 503 ~ 47 A Lamina lucida 472 • 35 414 • 57 /~

a Average and standard deviation of data from 5 subjects.

Composition o/Epithelial Tissue The parameters characterizing volumetric and surface densities of epithelial

tissue components of the various strata demonstrated a different structural composition of epithelial portions in ridges (R) and over connective tissue papillae (P).

These two locations will therefore be described separately. Epithelium in Ridges. The average composition of a unit volume of stratum

basale, stratum spinosum and stratum granulosum is illustrated in Fig. 4. In the stratum basale a unit volume of epithelium comprised 58.1% cytoplasm, 32.6% nuclei, 3.3% intercellular space and 5.9% non-epithelial cells (Table 3, Fig. 4). In the stratum spinosum, the volume occupied by cytoplasm rose to 83.7 % and that occupied by nuclei fell to 7.7 %. The intercellular space comprised 7.7 % and non- epithelial cells 0.9%. In the stratum granulosum, 89.5% of the unit volume was cytoplasm, and 5.0 % was nuclei. The intercellular space accounted for 5.4 % and nonepithelial cells occupied 0.06 % (Table 3, Fig. 4).

As is apparent from the above figures, the volume fraction occupied by non- epithelial cells fell sharply and significantly (PF ~ 0.001) f~om stratum basale to stratum spinosum and further still from stratum spinosum to stratum granulosum. This fraction comprised lymphocytes as well as inactive melanocytes, Langerhans- cells and Merkel-cells. The intercellular space was significantly (PF ~ 0.001)lower in the stratum basale as compared to the upper strata.

Epithelial cells occupied 90.8% of the unit volume of stratum basale, 91.4% of the upper stratum spinosum, and increased significantly (PF ~ 0.001) to 94.5 % in the stratum granulosum (Table 3, Figs. 4 and 5). While the volumetric density of epithelial nuclei fell drastically and significantly (P~ ~ 0.001) from the stratum basale to the upper spinous layer (Figs. 4 and 6) and did not change significantly between the upper spinous and the granular layer (Table 3), the volumetric density


Table 3. Stereological parameters of hard palate epithelium over epithelial ridges

Component Parameter Stratum Stratum Stratum basale spinosum granulosum

Units

Epithelial tissue components Epithelial cells

Intercellular space Non-epithelial cells

Epithelial cell components Nuclei

Cytoplasm

Epithelial cytoplasm components Mitochondria Ribosomes Rough endoplasmic reticulum

Smooth membrane system

Lysosomal bodies Membrane coating granules Filament bundles Kerytohyalin granules Cytoplasmic residue

Vve c 907.6 • 913.9 4-19.9 945.3 4-11.5 Sve c 0.554- 0.05 0.564- 0.05 0.914- 0.11 S/Vec 1.164- 0.26 1.224- 0.09 1.934- 0.24 Vvics 33.0 4-15.3 77.4 4-16.8 54.14-11.9 Vvr 59.4 4-42.4 8.7 4- 9.8 0.6 4- 2.2

Vvn 326.3 • 77.1 =t 29.2 50.2 4-17.8 Svn 0.294- 0.03 0.064- 0.02 0.064- 0.02 S/V n 1.814- 0.14 1.744- 0.30 2.794- 1.45 Vvcy 581.3 • 836.8 4-41.1 895.1 4-15.6

Vvm i 45.0 • 5.5 18.7 • 6.2 8.3 4- 4.3 VVrib 102.7 • 80.0 4-18.9 39.4 • 9.0 Vve r 28.4 • 6.9 16.4 • 3.2 12.0 4- 4.5 Sve r 0.80-~: 0.19 0.484- 0.13 0.36• 0.15 S/Ver 29.17• 3.58 29.44• 6.28 31.404- 7.06 Vvsms 11.7 4- 2.9 8.6 4- 2.0 7.0 4- 4.5 Svsms 0.454- 0.07 0.304- 0.06 0.204- 0.12 S/Vsm s 41.714- 8.30 39.194-11.58 29.024- 9.51 Vvly 2.0 4- 1.2 2.3 4- 1.4 2.0 4- 1.2 Vvmcg 0.2 4- 0.5 4.6 -I- 2.5 3.5 4- 1.8 Vvfib 82.2 4- 13.7 273.0 4-23.8 271.3 4-34.8 Vvk h 0.0 4- 0.0 0.7 4- 1.1 37.2 • Vvc r 309.1 4-18.8 432.5 4-49.6 513.5 4-41.2

mma/cm a m2/cm 3 m2/cm 3 mm3/cm a mma/cm a

mma/cm a m2/cm 3 m2/cm 3 mma/cm3

mm3/cm 3 mma/cm 3 mm3/cm 3 m2/cm 3 m2/cm 3 mm3/cm 3 m2/cm 3 m2/cm 3 mm3/cm 3 mm3/em 3 mm3/em 3 mma/cm 3 mm3/cm 3

Volume ratio Nu/Cy Vvn/Vvcy 0.56 • 0.08 0.10• 0.04 0.06 • 0.02 Surface ratio Nu/Cells Svn/Sve c 0.54 4- 0.07 0.12 4- 0.04 0.07 i 0.02 Desmosomes Nd/100[zm2 15.4 =~ 2.2 32.5 4- 4.4 35.7 4- 5.3

Number Number Number

of epithelial cytoplasm increased significantly (PF ~0 .001) both from s t ra tum basale to s t ra tum spinosum and from the latter to the s t ra tum granulosum (Table 3, Fig. 4). This resulted in a sharp drop of the ratio of nuclei/cytoplasmic volume (Table 3).

The decrease in volumetric densi ty of nuclei from basal to spinous layers was paralleled by a similar, significant (PF ~0 .001) decrease in the nuclear surface density (Table 3, Fig. 6). The surface to volume ratio of epithelial nuclei, however, was similar for s t ra tum basale and s t ra tum spinosum and increased towards the s t ra tum granulosum (Table 3, Fig. 6).

The significant increase in volumetric density of epithelial cells f rom s t ra tum basale and s t ra tum spinosum to the s t ra tum granulosum was also paralleled by a corresponding increase (PF ~ 0.001) in surface density (Table 3, Fig. 5). The surface to volume ratio of epithelial cells was similar for s t ra tum basale and s t ra tum spinosum, but increased drastically ( P F ~ 0 . 0 0 1 ) from s t ra tum spinosum to s t ra tum granulosum (Table 3, Fig. 5).

Stereologic Analysis of Human Hard Palate Epithelium

mm 3 VV/cm ~ STRATUM

1000

187

900

800

700

600

500

400

300

200

100

0

R P R

Stratum basale Stratum spinosum Stratum granulosum

Fig. 4. Composition of human hard palate epithelium. Columns representing average volumetric densities per stratum of ridge (R) and papilla (P) epithelium are assembled along an increasing gradient of filament density (left to right). Fractions of intercellular space and

non-epithelial cells were placed below the zero line. For Abbreviations, see Table 3

The ratio of nuclei/epithelial cell surfaces dropped in parallel with the respective volume ratio (Table 3). The average number of desmosomes per 100 ~zm ~ of test field increased significantly (PF ~ 0.001) from 15.4 in the s t ra tum basale to 32.5 in the upper s t ra tum spinosum and remained stable towards the s t ra tum granulosum (35.7; Table 3). The volumetric densities of intercellular space and epithelial nuclei, the surface densi ty of cytoplasmic membranes, and the number of desmo- seines revealed significant (PF ~0 .001) differences between the 10 subjects investigated.

Epithelium over Connective Tissue Papillae. The average composition of a unit volume of s t ra tum basale, s t ra tum spinosum and s t ra tum granulosum is shown

188 M. Meyer and H. E. Sehroeder

E I0001

~'E 9 0 0 u

E 85O .E

_~ 800

o 1000

.1=

Q .

95O "5

9 0 0

.L-2 850

E

8oo

Svec

y F 2-0 I 1.5

J I [ t.O

0.5

S/Vec / / / t

Basale Spinosum Granulosum

S T R A T A

L0

V2.0

1.5

t.0

0,5

o m

.9

E

o

= 8 o

E

Fig. 5. Volumetric (black dots) and surface (open circles) densities and surface to volume ratios (triangles) for epithelial cells in R- and P-locations. Data (mean and standard deviation)

are expressed in relation to a unit volume of epithelial stratum

in Fig. 4. In the stratum basale, a unit volume of epithelium comprised 74.7% cytoplasm, 16.7 % nuclei, 5.1% intercellular space and 3.5 % non-epithelial cells (Table 4, Fig. 4). In the upper s tratum spinosum, the volume occupied by cytoplasm rose to 86.5% and that of nuclei fell to 7.0%. The intercellular space remained at 5.6% and non-epithelial cells occupied 0.8% (Table 4, Fig. 4). In the s tratum granulosum, 90.1% of the unit volume was cytoplasm and 4.9% was nuclei. The intercellular space was slightly decreased to 4.9% and non-epithelial cells occupied 0.04% (Table 4, Fig. 4). As noticed in ridge epithelium, the volume of non-epithelial cells of papilla-epithelium fell sharply and significantly (PF < 0.001) from stratum basale to stratum spinosum and declined further towards the s t ratum granulosum. The intercellular space, however, was similar in all three strata.

Epithelial cells occupied 91.4% of the unit volume of s tratum basale, 93.6% of the upper s tratum spinosum and 95% of the s tratum granulosum (Table 4, Figs. 4 and 5). While the nuclear volume density fell sharply and significantly (PF < 0.001) from the stratum basale to the s t ratum spinosum and further decre-


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c

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Fig. 6. Volumetric (black dots) and surface (open circles) densities, and surface to volume ratios (triangles) for epithelial nuclei in R- und P-locations. Means and standard deviations

are expressed in relation to a unit volume of epithelial stratum

ased slightly towards the s tratum granulosum (Table 4, Figs. 4 and 6,) the volumetric density of epithelial cytoplasm increased significantly (PF <0.001) from stratum basale to s t ratum spinosum and from the latter to the s tratum granulosum (Table 4, Fig. 4). For this reason, the ratio of nuclei/cytoplasmic volume dropped from stratum basale to s tratum spinosum (Table 4).

The decrease in nuclear volume density was paralleled by a significant (PF < 0.001) drop in nuclear surface density from stratum basale to s t ratum spinosum (Table 4, Fig. 6). The surface to volume ratio of epithelial nuclei, however, increased significantly (PF < 0.001) from stratum basale to s t ratum granulosum but rose gradually and in statistically insignificant steps from basal to spinous to granular layers (Table 4, Fig. 6).

13 Cell Tiss . Res . 158


Table 4. Stereologic parameters of hard palate epithelium over connective tissue papillae

Component Parameter Stratum Stratum Stratum basale spinosum granulosum

~-4-s ~ - - s :F--s

Units

Epithelial tissue components Epithelial cells

Intercellular space Non-epithelial cells

Epithelial cell components Nuclei

Cytoplasm

Epithelial cytoplasm components Mitochondria Ribosomes

Rough endoplasmic reticulum

Smooth membrane system

Lysosomal bodies Membrane coating granules Filament bundles Keratohyalin granules Cytoplasmic residue

Vve c 913.8 • 935.6 --16.8 950.4 --11.2 Sve c 0.53-- 0.05 0.63-- 0.11 0.96:L 0.13 S/Vet 1.17-- 0.12 1.35-- 0.24 2.03• 0.28 Vvics 51.3 Jr 12.8 56.3 -- 10.6 49.2 -- 11.7 VVr 34.9 --29.1 8.1 -- 9.3 0.4 -- 1.2

Vvn 167.2 --33.0 70.3 --21.1 49.0 • Svn 0.14• 0.03 0.07-- 0.02 0.06-1- 0.02 S/V n 1.65-- 0.21 2.00-- 0.32 2.39-- 0.52 Vvcy 746.6 --46.9 865.3 --18.4 901.4 --22.8

Vvm i 48.4 • 7.1 22.2 • 8.3 9.8 • 5.0 Vvrib 83.0 • 58.2 • 23.6 -4- 6.7 Vve r 20.1 • 4.3 18.8 • 5.4 12.6 • 3.3 Sve r 0.60• 0.13 0.53• 0.16 0.31• 0.08 S/Ver 30.42 • 3.62 29.08-- 4.08 26.19~: 5.26 Vvsms 12.6 -- 4.8 9.7 -- 4.2 5.8 -- 3.1 Svsms 0.44• 0.13 0.31-- 0.12 0.13-t- 0.07 S/Vsm s 38.11 -- 10.39 33.40 • 7.41 30.83 -- 14.22 Vvly 1.3 • 0.9 1.5 Jr 1.2 1.1 -- 1.0 Vvmcg 0.2 -- 0.3 5.5 -- 2.4 8.2 -- 2.1 Vvfib 195.9 • 287.0 -4-34.7 272.0 --42.8 Vvk h 0.0 -- 0.0 0.1 -- 0.2 50.3 --28.0 Vvc r 385.1 --29.3 462.3 --37.9 518.0 --28.7

mma/cm a m2/em a m2/cm a mma/cm a mm3/cm a

mm3/cnl 3 m2/cm a m2/cm a mma/cm a

mma/cm 3 mma/cm 3 mma/cm a m2/cm a m2/cm 3 mm3/cm a m2/cm 3 m2/cm a m3/cm mZ/em a m3/em 3 m3/cm a m$/cm a

Volume ratio Nu/Cy Vvn/Vvcy 0.22-- 0.06 0.08 • 0.03 0 05-- 0 02 Surface ratio Nu/Cells Svn/Sve c 0.26 • 0.07 0.11 • 0.03 0.06 ~ 0.02 Desmosomes Nd/1001tim 2 24.5 -- 4.1 29.5 i 3.0 34.8 -- 7.1

Number Number Number

I n con t ras t to the stepwise increase of epi thel ia l cell volume (Table 4, Fig. 5), the surface dens i ty of cy toplasmic membranes was s imilar in s t r a t u m basale and s t r a t um spinosum, bu t increased s ignif icant ly (PF < 0.001) towards the s t r a t um granulosum (Table 4, Fig. 5). The surface to volume ra t io of epi thel ia l cells was similar in s t r a t um basale and s t r a t um spinosum, and increased d ras t i ca l ly towards the s t r a t um granulosum (Table 4, Fig. 5). The ra t io of nuclei to epi thel ia l cell surfaces d ropped in paral le l wi th the respect ive volume ra t io (Table 4). The average number of desmosomes per 100 tzm ~ tes t field was s ignif icant ly h igher ( P < 0 . 0 0 1 ) in the s t r a t um granulosum than the in s t r a t u m basale (34.8 versus 24.5), bu t s imilar in ad jacen t s t r a t a (Table 4).

The volumetr ic densi t ies of in tercel lular space and epi thel ia l nuclei, the surface dens i ty of cy toplasmic membranes , and the number of desmosomes var ied signifi- can t ly (P~, < 0 . 0 1 ) be tween the 10 subjects inves t iga ted .

Comparison o/ R- and P-Epithelium. The composi t ion of epi thel ial t issue in ridges differed from t h a t of epi the l ium over connect ive t issue papi l lae wi th regard


to nuclear and cytoplasmic densities in the stratum basale. Epithelium in ridges revealed significantly (PF~0.001) higher volumetric and surface densities of epithelial nuclei (326.3 versus 167.2 mma/cm 3, and 0.29 versus 0.14 m2/cm 8) when compared to epithelium over connective tissue papillae. On the other hand, the density of epithelial cytoplasm in ridges was significantly (PF ~ 0.001) lower than in papillae-epithelium (581.3 versus 746.6 mma/cmS). These differences can be seen in Fig. 1. In addition, the density of intercellular space tended to be lower in the stratum basale of R- than P-epithelium, and higher in the stratum spinosum of R- than P-epithelium. Desmosomes in the stratum basale appeared to be less numerous in R- than in P-epithelium.

Composition o/ Epithelial Cytoplasm The average composition of epithelial cytoplasm calculated in relation to a

unit volume of epithelial stratum in R- and P-epithelium is given in Tables 3 and 4, and illustrated in Fig. 4. Since density parameters of epithelial nuclei and non- epithelial cells differed drastically between strata and locations, the presentation of cytoplasmic composition is preferentially based on parameters calculated in relation to a unit volume of epithelial cytoplasm. Furthermore, the composition of epithelial cytoplasm and changes from stratum to stratum can be described more meaningfully by considering data grouped for functionally related parameters and presenting these separately for R- and P-epithelium.

Epithelial Cytoplasm in Ridges. In all 3 strata, specific organelles occupied around 40 to 50 % while the structureless residue of cytoplasmic ground substance accounted for the remaining 50 to 60 % of the cytoplasm.

Mitochondria. The volumetric density of mitochondria (in mma/cm a cytoplasm) fell significantly (PF ~ 0.001) from 77.4 ~: 9.4 in the stratum basale to 22.4 ~ 7.4 in the upper stratum spinosum. I t decreased further (PF ~ 0.001) to 9.3 :[: 4.8 in the stratum granulosum (Fig. 7).

ER and SMS. Both, volumetric and surface density data of the rough endoplasmic reticulum dropped significantly (PF ~ 0.001) and in almost parallel fashion from stratum basale to stratum spinosum (48.84- 11.9 to 19.6~3.8 mma/cm a cytoplasm, and 1.37 ~: 0.32 to 0.57 4- 0.15 m2/cm a cytoplasm respectively; Fig. 8). The further slight decrease of both parameters towards the stratum granulosum (13.44-5.0 mma/cm a, and 0.40:~0.17 m2/cm a) was not statistically significant, Fig. 8). This parallel decrease of surface and volume parameters resulted in a stable surface to volume ratio through all three strata (Table 3, Fig. 8).

The volumetric and surface density data of the smooth membrane system revealed a pattern somewhat different from that of er. Both parameters fell significantly (PF~0.001) but not in parallel, from the stratum basale to the stratum spinosum (20.1 =~5.0 to 10.3 =~2.4 mma/cm 3 cytoplasm, and 0.77 4-0.12 to 0.36 • 0.07 m2/cm 3 cytoplasm respectively, Fig. 9). They fell further, again in non-parallel fashion, towards the stratum granulosum (8.84-5.0 mmS/cm a, and 0.224-0.13 m2/cma; Fig. 9). This latter drop was not statistically significant. Accordingly, the surface to volume ratio decreased slightly but insignificantly from stratum to stratum (Table 3, Fig. 9).

13"

192 M. Meyer and H. E. Sehroeder

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S T R A T A

Fig. 7. Volumetric densities (mean -F standard deviation) of mitochondria per unit volume of epithelial cytoplasm in the three strata of ridge (R) and papilla (P) epithelium

The er-parameters but not the sins-data differed significantly (Pv <0.001) between the 10 subjects investigated.

Free Ribosomes, Filament Bundles and Cytoplasmic Residue. The volumetric density of aggregates of non-membrane bound, free ribosomes decreased significantly (PF < 0.001) from stratum to s t ratum (Fig. 10). I t amounted to 176.7 • 36.6 in the s tratum basale, 95.6 =E 22.6 in the upper s tratum spinosum, and 44.0 • 10.0 mma/cm a cytoplasm of the s tratum granulosum.

Cytoplasmic filaments which always occurred organized into bundles (Figs. 2, 3 and 12) were the most prominent albeit not most voluminous cytoplasmic component. Bundled filaments occupied 141.4 • 23.5 mma/cm a of the cytoplasm in stratum basale and increased significantly (PF <0.001) towards the upper s t ratum spinosum (326.6 =E 28.5 mma/cm a, Fig. 10). There was no further increase in bundled filament density from stratum spinosum to stratum granulosum (Fig. 10, 303.1 • 38.9 mma/cma).

Individual filaments in the bundles of s tratum basale, s t ra tum spinosum and s t ratum granulosum were found to have an average diameter of 83.4, 84.5 and 84.9/~ respectively (Table 5). The most voluminous part of the epithelial cyto-


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Fig. 8. Volumetric (black dots) and surface (open circles) densities, and surface to volume ratios (triangles) for rough endoplasmic reticulum in the 3 strata of ridge (R) and papilla (P) epithelium. Means and standard deviations are expressed per unit volume of epithelial cyto-

plasm

Table 5. Diameter of cytoplasmic filaments in epithelium of hard palate

Location ~ • s Units

Stratum basale a83.4 fl: 11 .8

Stratum spinosum 84.5 ~_ 10.3 A Stratum granulosum 84.9 ~: 14.2 A

Mean 84.3 ~: 12.1 /~

a Average of multiple measurements in sections of ridge epithelium from 7 subjects.

plasm was occupied by the structureless residue of cytoplasmic ground substance (Fig. 10). Its volumetric density remained essentially constant through all three strata and occupied 531.7 • 32.3 in the stratum basale, 516 ~-59.3 in the stratum spinosum, and 573.7 • 46.0 mma/cm a cytoplasm in the stratum granulosum.


E 5 0 - ( I )

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m ~ ~ e s o -

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Fig. 9. Volumetric (black dots) and surface (open circles) densities, and surface to volume ratios (triangles) for the smooth membrane system in 3 strata of ridge (R) and papilla (P) epithelium. Means and standard deviations are expressed per unit volume of epithelial

cytoplasm

The volumetric densities of ribosomes (PF<0.01) , filament bundles ( P F < 0.001) and of the cytoplasmic residue (P~ < 0.001) differed significantly between the 10 subjects investigated.

Lysosomal Bodies, Membrane Coating Granules and Keratohyalin. Lysosomal bodies and membrane coating granules occupied very small fractions of the epithelial cell cytoplasm (Fig. 11). The volumetric density of lysosomal bodies remained constant throughout the three strata and occupied around 3 mm3/cm a cytoplasm (Fig. 11). The volumetric density of membrane coating granules was practically zero in the s t ratum basale, rose significantly (PF < 0.001)towards the upper s t ratum spinosum (5.5• 3.1 mm3/em a cytoplasm), and fell slightly towards the s tratum granulosum (3.9 • mma/cma; Fig. 11).


700 - A ~, 6 0 0

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,[,.t / / / ~ .[/______.~ Filament bundles

Basale Spinosum Granulosum S T R A T A

Fig. 10. Volumetric densities (mean • standard deviation) of aggregates of free ribosomes (black dots), filament bundles (triangles), and the cytoplasmic ground substance residue (circles) in 3 strata of epithelial ridges (solid lines) and epithelium over connective tissue

papillae (broken lines). Data are expressed per unit volume of epithelial cytoplasm

Keratohyalin bodies were practically absent in the stratum basale, very rare in the stratum spinosum (0.8• 1.3 mma/cm a cytoplasm) and increased dramatically (PF < 0.001) towards the stratum granulosum (41.5 • 14.8 mm3/cma; Fig. 11). None of these parameters revealed significant differences between subjects.

Epithelial Cytoplasm over Connective Tissue Papillae. As seen in the epithelium of ridges, around 40 to 50 % of the cytoplasm in all three strata of the epithelium over papillae was occupied by specific organelles, while the remaining 50 to 60 % belonged to the structureless cytoplasmic ground substance.

Mitochondria. The volumetric fraction occupied by mitochondria was 64.9• 9.5 mma/cm 3 cytoplasm in the stratum basale (Fig. 7). I t fell drastically to the upper stratum spinosum (25.6 ~: 9.6 mm3/cm 3) and continued to decrease towards the stratum granulosum (10.9 • 5.6 mma/cma). At both steps, the differences were statistically significant (P~, < 0.001 ; Fig. 7).

E R and SMS. Both volumetric and surface density parameters for the rough endoplasmic reticulum (er), fell slightly, in parallel and almost linear fashion, from stratum basale to stratum granulosum (Fig. 8). There were no statistically


E 88

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~ 8 4 Z ' $ $

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STRATA

Vvmcg

Fig. 11. Volumetric densities (mcan:L standard deviation) of lysosomal bodies (black dots), membrane coating granules (circles), and keratohyalin bodies (triangles) in 3 strata of epithelial ridges (solid lines) and epithelium over connective tissue papillae (broken lines). Data

are expressed per unit volume of epithelial cytoplasm

significant differences between stratum basale (26.9=k 5.8 mma/cm 3, 0.80=[=0.17 m2/em 8 cytoplasm) and stratum spinosum (21.8 =t= 6.2 mma/em a, 0.61 =t= 0.18 m2/ cma). However, the volumetric and surface densities of er in the stratum granulosum (13.9 • 3.7 mma/cm 3, 0.34 -4- 0.09 m2/cm 3) were significantly (PF < 0.001) smaller than that of the lower strata (Fig. 8). The parallelism in declining volume and surface densities resulted in a practically stable surface to volume ratio (Table 4, Fig. 8).

Volumetric and surface density parameters of s ins decreased steadily, but in non-parallel fashion, from the stratum basale (16.9 • 6.4 mma/cm 3, 0.59 i 0.17 m2/ cm a cytoplasm) to the upper stratum spinosum (11.2 • 4.8 mmS/cm 8, 0.36 • 0.16 m2/cm 3) and to the stratum granulosum (6.4 • 3.4 mma/cm s, 0.14 i 0.08 m*/cm s ; Fig. 9). There was no statistically significant difference between the basal and spinous layer, but parameters of both were significantly ( P F < 0.001) larger than those of the stratum granulosum. The surface to volume ratio, therefore, fell slightly from basal to granular layers. The er-data but not the s i n s - p a r a m e t e r s

varied significantly between the 10 subjects investigated.


Free Ribosomes, Filament Bundles and Cytoplasmic Residue. The volume fraction occupied by aggregates of non-membrane bound, free ribosomes amounted to 111.2 • 35.6 mma/cm 8 cytoplasm in the stratum basale (Fig. 10). This fraction fell almost linearly to 67.3:E20.0mma/cm s in the stratum spinosum, and to 26.7 J: 7.4 mma/cm a in the stratum granulosum. In both instances, the differences were statistically significant (PF H0.001).

The volumetric density of cytoplasmic filament bundles was already high in the stratum basale (262.4• mmS/cm a cytoplasm; Fig. 10). I t increased significantly (P ~ 0.001) towards the upper stratum spinosum (331.7:{: 40.1 mma/ cm a) and decreased slightly towards the stratum granulosum (301.7 :L 47.5 mmS/ cma). The difference at the latter step was not statistically significant.

The most voluminous part of the cytoplasm in epithelium over papillae was again the cytoplasmic ground substance. Its volumetric density remained rather stable from the stratum basale to the upper stratum spinosum (515.8~39.2 versus 534.3:t:43.8 mma/cm 3 cytoplasm) and increased slightly towards the stratum granulosum (547.7• 31.8 mm3/cma; Fig. 10).

The volumetric density of all three parameters differed significantly (VVrib PF H 0.01; Vvfib and Vvcr--'PF <: 0.001) between the 10 subjects investigated.

Lysosomal Bodies, Membrane Coating Granules and Keratohyalin. Lysosomal bodies regularly occupied a very small fraction of the cytoplasm in all three strata, ranging between 1.2 and 1.8 mma/cm a and maintaining a rather constant level (Fig. 11). The volumetric density of membrane coating granules was practically zero in the stratum basale, rose significantly (PFH 0.001) to the stratum spinosum (6.4• mma/cm a cytoplasm) and further on increased slightly towards the stratum granulosum (9.1 ~ 2.4 mma/cm a ; Fig. 11).

Keratohyalin bodies were practically absent in both stratum basale and stratum spinosum, but dramatically rose to 55.7~:31.2 mm3/cm 8 cytoplasm in the stratum granulosum (Fig. 11). Neither lysosomal bodies, membrane coating granules nor keratohyalin bodies displayed significant differences between subjects.

Cytoplasmic Di//erences in R- and P-Epithelium. The cytoplasmic composition differed most remarkably between basal cells located at the base of epithelial ridges and those situated at the top of connective tissue papillae. These differences concerned the volumetric densities of rough endoplasmic reticulum (Fig. 8), aggregates of free ribosomes (Fig. 10), and filament bundles (Fig. 10), as well as the surface density of rough endoplasmic reticulum (Fig. 8). These were all highly statistically significant (PF H0.001). The cytoplasm of basal cells in epithelial ridges was more densely filled with er and aggregates of free ribosomes than that of basal cells over connective tissue papillae. On the other hand, basal cells over connective tissue papillae were more densely packed with filament bundles than those of epithelial ridges (Fig. 12). Less obvious, but still statistically significant differences were observed in the cytoplasmic composition of the stratum granulosum cells residing in R- or P-locations. While er and sins parameters were almost identical in both locations, the volumetric density of aggregates of free ribosomes was significantly higher (PFH 0.001) in the stratum granulosum cells of R- than of P-epithelium (Fig. 10). Furthermore, the volumetric density of membrane coating granules was significantly (PFH0.01) lower in stratum granulosum cells of R- than of P-epithelium (Fig. 11).


Fig. 12


Discussion The present study, by providing a detailed quantitative characterization of

structural constituents in the normal orthokeratinizing epithelium of hard palate, furnished base line data for comparison and better understanding of the various normal and pathologically altered stratified epithelia which occur in the human oral cavity. The essential findings can be summarized as follows.

The human hard palate, epithelium is relatively thin (0.25 mm) and is charac- terized by regularly spaced interdigitations with the underlying connective tissue, a distinct stratification into four strata (basale, spinosum, granulosum, corneum), and a homogeneous, orthokeratinized stratum corneum of even thickness. I t contains a variable number of non-epithelial cells, such as inactive melanocytes, Langerhans cells, Merkel cells, and small lymphocytes, most of which are confined to the basal and suprabasal layers and are more numerous at the base of epithelial ridges than over connective tissue papillae. The pattern of differentiation is similar in ridge and papilla epithelium. Mitochondria, rough endoplasmic reticulum, free ribosomes, and smooth membrane systems (including the Golgi apparatus) decrease in volumetric and surface density from stratum basale to stratum granulosum. Constituents which in part are considered key structures for the process of keratinization, such as bundled cytoplasmic filaments, keratohyalin granules, and membrane coating granules (keratinosomes) increase in volumetric density between either stratum basale and stratum spinosum (Vv~ib, Vvmcg) or between stratum spinosum and stratum granulosum (Vvkh). The cytoplasmic ground substance, however displays a rather stable density throughout all strata. On the other hand, there are distinct differences in organelle density between basal cells residing at epithelial ridges and those over connective tissue papillae. In the stratum basale of ridges, the epithelial nuclei are volumetrically denser and the epithelial cytoplasm less dense than in the stratum basale over connective tissue papillae. In addition, basal cells in ridges possess more rough endoplasmic reticulum and free ribosomes and less bundled filaments than basal cells over connective tissue papillae. The ribosome density differences persist even in the stratum granulosum.

These data were obtained by stereologic point counting procedures as described previously (Schroeder and Miinzel-Pedrazzoli, 1970a). The sampling design used had to cope with some difficulties which were overcome by various measures. First, for reasons mentioned above, sampling of electron microscopic fields did not include the middle portion of epithelial ridges. However, as this portion comprised only a small fraction of the total epithelial volume (see Fig. 1), and because the cells of the stratum spinosum have been shown to constitute a rather well defined population which undergoes major differentiation changes at the level of the termination of connective tissue papillae (Thilander, 1968; Sil- verman, 1971), the restriction of sampling to the stratum basale and the upper

Fig. 12a--d. Typical electron micrographs illustrating the cytoplasmic organelle distribution pattern in basal cells of R-epithelium (a), basal cells of P-epithelium (b), and spinous cells of R- (c) and P- (d) epithelium. Bundles of filaments (lib), aggregates of free ribosomes (rib), rough endoplasmie reticulum cisternae (er) and portions of the smooth membrane system

(sins) are indicated. Magn: X 36,500


stratum spinosum would still appear to have detected the major steps in cellular differentiation. Secondly, the sample size per biopsy, which was similar to that used for a methodological study on human buccal gingival epithelium (Schroeder and Miinzel-Pedrazzoli, 1970a) might have been below the optimal level for quantitative characterization of rarely occuring organelles especially in the upper strata. However, a rather large group of subjects was included in the present study and rare organelles such as lysosomal bodies, keratohyalin granules, and membrane coating granules did not reveal significant differences between subjects. Therefore, the average data reported in this study originated from a sample large enough overall to provide valid and representative estimates for comparative purposes (Schroeder and Miinzel-Pedrazzoli, 1970a). The underestimation of rare organelle density caused by a slightly inadequate sample size was estimated vo lie between 10 and 20%, an order of magnitude not affecting the significance of differences found in the present study or of differences found between the palate and other oral stratified epithelia (Schroeder and Miinzel-Pedrazzoli, 1970a and b). The present data partly confirm and are partly incompatible with previous observations. For example, the thickness reported for palate epithelium in this study confirms measurements taken by Krzywicki and Rokicka (1967) who found ridge portions to range between 0.21 and 0.32 ram, and papillae portions between 0.11 and 0.18 mm in thickness. Meyer and Gerson (1964) reported an overall thickness of 0.27 • mm. Good agreement was also found between our data on the thickness of the basal lamina complex and the data of Thilander (1968). In addition, some of the morphometric data confirmed earlier morphological descriptions. Thilander (1968) and Silverman (1971) reported that tonofilament bundles were sparse in basal cells and increased in density towards the stratum spinosum and stratum granulosum. We have confirmed this increase quantitatively for cells moving from stratum basale to the upper stratum spinosum. Also, there has been general agreement in previous studies on the absence of glycogen in all strata, on the occurence of membrane coating granules in the upper spinous and granular layer, on the numerical increase of desmosomes from stratum basale to stratum spinosum, and on the homogeneously orthokeratinized character of the stratum corneum (Thilander, 1968; Silverman, 1971). On the other hand, there is complete disagreement in relation to the average diameter of single cytoplasmic filaments. Thilander (1968) reported an increase in filament diameter from about 50 A in the stratum basale to about 100 A in the stratum granulosum. Similar data were given by Brody (1960) for filaments in normal human epidermis. In the present study, however, filament diameter was found to be similar in all strata, with an average of 84 • 12 A. This size is almost identical to the filament diameter found for human cheek epithelium (Landay and Sehroeder, 1975). Therefore, the increase in filament bundle density from basal to higher strata was not related to increases in the size of individual filaments. The filaments of palate epithelial cells are similar to the 80 A filaments of a variety of other keratinizing epithelia that show an s-diffraction pattern (Parakkal and Alexander, 1972; Montagna and Parakkal, 1974).

The present stereologic data reported for the human palate epithelium, can also he compared to similar data for the epithelium of human buccal gingiva (Schroeder and Miinzel-Pedrazzoli, 1970b) and of the epithelium of human cheek


mucosa (Landay and Schroeder, 1975). Gingival epithelium which is mainly a parakeratinizing integument ranging between 0.15 and 0.30 mm in thickness (Schroeder and Theilade, 1966), differs in some aspects from the orthokeratinizing hard palate epithelium. Nuclei and cytoplasm show a similar density in basal ridge portions of both epithelia. However, the volumetric and surface density (per unit cytoplasmic volume) of rough endoplasmic reticulum and the smooth membrane system is about 50 % smaller, the filament bundle density 25 % larger, and the volumetric density of mitochondria 15 % larger in basal cells of gingival epithelium than in those of hard palate epithelium (Schroeder and Miinzel- Pedrazzoli, 1970b). These remarkable differences persist in higher strata. In the stratum granulosum, rough endoplasmic reticulum and the smooth membrane system were still noticeably less dense in gingival than in palate epithelium, while the filament bundle content comprises 50% of the cytoplasmic volume in gingival epithelium compared to 30% in the palate (Schroeder and Miinzel- Pedrazzoli, 1970b). In both epithelia there was a statistically significant difference in filament density between subjects. These differences become more meaning- ful when compared to the respective organelle parameters in human cheek epithelium. In the latter, the stratum basale is remarkably sirni]ar to that of the palate epithelium, except that filament bundles occupy 22 % of the cytoplasm. In higher strata, the density of mitochondria, and rough endoplasmic reticulum is similar to those of palate epithelium. The smooth membrane system is less dense than in palate epithelium but denser than in gingival epithelium, while the cytoplasmic filaments, occuring in a meshwork of individual filaments, rise to occupy around 70% of the cytoplasm in the cheek epithelium (Landay and Schroeder, 1975). Silverman (1971) stated that "increasingly dense packing of tonofilaments is one of the main events of cell differentiation in keratinizing epithelium ". This view is generally accepted in keratinization research (Mat- oltsy, 1969; Parakkal and Alexander, 1972; Fraser et al., 1972; Montagna and Parakkal, 1974). Stereologic data, however, reveal that in a regularly orthokeratinizing epithelium (hard palate) filament density ranges from 14 to 30% (basal to granular layers) in para-keratinizing epithelium (buccal gingiva) from 20 to 50% and in "non-keratinizing" normal cheek epithelium from 20 to 70% (basal to subsurface layers).

The present data, furthermore, revealed significant differences in the structure of basal cells situated in epithelial ridges compared to those lying over connective tissue papillae. These differences imply that the gradient of organelle distribution from basal to granular layers is steeper in epithelial ridges than over connective tissue papillae. This suggests, that basal cells in ridges are less differentiated towards keratinization than those over papillae. At the latter location, the organelle pattern of basal cells was more similar to stratum spinosum cells than to basal cells in ridges. In fact, when epithelial components per stratum in ridges and over papillae were arranged along a gradient of decreasing volumetric density for nuclei and aggregates of free ribosomes, and along increasing density for cytoplasmic filaments (Fig. 4), a general trend of differentiation became apparent. While nuclear volume gradually decreased, the density of energy producing and synthesizing organelles also decreased, while filament and keratohyalin density increased. The assumption that basal cells over connective tissue papillae are


already somewhat more differentiated t han basal cells in ridges, appears to be supported by data concerning the frequency dis t r ibut ion of dividing cells in oral kerat inizing epithelia other t h a n the palate (Kunze, 1969; L6e, Kar r ing and Hara, 1972).

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a quantitative electron microscopic analysis of the keratinizing epithelium of normal human hard...

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