J. Dent. 1992;
152
20: 152-l
55
Keratinization of palatal mucosa beneath metal-based removable partial dentures* S. Harinasuta* Department
and J. A. Howlett
of Prosthetic
Dentistry,
institute
of Dental Surgery,
University
of London,
UK
ABSTRACT Exfoliative cytology provides a non-invasive method of examining changes in epithelial differentiation beneath dentures. Cell sampling by mechanical scraping may introduce errors due to collection of deeper epithelial cells, therefore an alternative sampling method was evaluated. Cells collected from the hard palates of 24 dentate subjects using either a wooden tongue depressor or transparent, adhesive-coated tape were fixed and stained by Papanicolaou’s technique. The percentage of the total epithelial cells counted that were anucleate (the orthokeratinization index) was 99.95 per cent using the tape technique but 15 per cent lower using the smear technique. It appears that the tape technique gives a more accurate representation of the orthokeratinization of superficial cell layers. The tape technique was used to examine epithelial changes beneath tooth-supported removable partial dentures (RPDs). In 26 subjects, who had worn cobalt chromium based RPDs for at least 6 months, samples taken from beneath the metal denture base had a 50 per cent lower orthokeratinization index than those from uncovered areas of the palate. KEY WORDS:
Dentures,
Epithelium,
152-I
Metals
J. Dent. 1992; 1991)
20:
55 (Received 20 May 1991;
reviewed
Correspondence Dental Surgery,
should be addressed too:Dr J. A. Howlett, Department University of London, Gray’s Inn Road, London WClX
INTRODUCTION The response of the supporting epithelium to dentures is of interest because changes in epithelial structure and functional integrity may be associated with the development of mucosal inflammation and ultimately with underlying bone resorption. Denture-bearing epithelium is comprised predominantly of masticatory mucosa but the peripheral denture flanges often rest upon lining mucosa. The majority of studies on structural changes of denture-bearing tissues have examined mucosa from the palate or maxillary alveolus, lying beneath complete dentures fabricated from acrylic resin. Normal human palatal epithelium is an orthokeratinizing tissue with a well-developed stratum corneum (Chen and Squier, 1984). However, several cytological studies (Al-Ani et al., 1966; McMillan, 1972; Lindholme et al., 1982) and histological studies (Watson and Macdonald, 1982; Sharma and
*Presented in partial fulfilment of the requirements for the degree of MSc in the University of London 1986. Previously presented as a communication at the 1987 Annual BSDR Meeting held at the Institute of Dental Surgery. London. @ 1992 Butterworth-Heinemann 0300-5712/92/030152-04
Ltd.
2 August
1991;
accepted 27 October
of Prosthetic Dentistry, Institute of 8LD. UK.
Mirza, 1986) have found that changes in the degree of keratinization, from orthokeratosis to parakeratosis, and a reduction in the thickness of the stratum corneum occur when the palate is covered by a complete denture base. However, other workers (Kapur and Sklar, 1963; Jani and Bhargava, 1976) have found increased orthokeratinization with denture wear. Cytological techniques have also been used in attempts to correlate changes in the degree of keratinization with the biomechanical features of the dentures. Markov (1969) found that well-designed dentures were conducive to higher levels of orthokeratinization in the underlying tissues, although Stabes et al. (1981) were unable to identify any difference in the behaviour of mucosa beneath dentures as a result of different occlusal schemes. Thus the influences of denture use upon the epithelium remain contentious. The response of epithelium beneath tooth-borne, metal-based, removable partial dentures has not been examined, and may differ from that of the mucosa beneath complete dentures. The former tissues should not be subject to such overt biomechanical stresses, because of the increased retention and stability of the dentures and the reduced soft-tissue loading which they impart.
Harinasuta
Exfoliative cytology has been employed as a simple, non-invasive technique to examine oral epithelia. The methods used in cell collection have differed according to the cell samples required. They include mouth rinsing and cell collection for screening, and mechanical scraping to produce a smear, for examination of selected areas. Previous studies of denture-bearing mucosa have employed the latter method (Sandler, 1964; McMillan, 1972). The disadvantage of such a technique is that the depth of tissue penetrated varies according to the pressure applied. This would seem unsatisfactory for determining the keratinization status of masticatory mucosa, which can be judged only by examination of superficial cell layers. Previous dermatological studies have used adhesivecoated transparent tape to collect corneocytes from skin (Plewig and Marples, 1970). The aims of the present study were twofold, first to assess the use of adhesive tape, as an alternative technique to the smear, in the sampling of oral epithelium, and secondly to use the technique in examining the keratinization of palatal mucosa beneath tooth-supported removable partial dentures.
MATERIALS
AND METHODS
The technique The subjects were 12 males and 12 females aged between 17 and 36 years who were fully dentate. All were nonsmokers, in apparent good health and were examined to ensure that they had clinically healthy oral mucosae and that their hard palates showed no signs of inflammation. Two methods were used, in each subject, to sample palatal tissue midway between the gingivae and midline of the palate opposite the premolar and molar teeth. Subjects first rinsed their mouths and then on the right side a wooden tongue depressor was used to scrape the epithelial squames with light pressure. The material was smeared on to a clean glass slide and immediately fixed in 95 per cent ethyl alcohol. On the left side the palatal mucosa was dried, for approximately 5 s, with a stream of air from a three-in-one syringe and a strip of 3M Pressure Sensitive Tape (3M Ltd, Bracknell, UK), 2.0 cm X 1.5 cm, was placed on the mucosa, using sufficient finger pressure in order for it to adhere to the palate. The tape was then peeled off by catching one end with college tweezers and immediately placed in 95 per cent ethyl alcohol for 5 min and then allowed to dry. The tape was attached via its backing to a clean glass slide with DPX mounting medium (BDH Chemicals Ltd, Poole, UK). The smears and tape samples were hydrated and stained by the method of Papanicolaou (1942) using haematoxylin, 0G6 and EA50 (Ortho Diagnostic Systems Ltd, High Wycombe, UK). Smears were then dehydrated using alcohol and xylene and mounted with DPX by standard methods. To avoid dissolution of the adhesive, tape samples were dehydrated in alcohol alone and allowed to dry before mounting with DPX.
and Howlett:
Keratinization
beneath dentures
153
Orthokeratinization This was investigated in 26 partially edentulous subjects using the adhesive tape technique. The subjects, all of whom were patients of the Eastman Dental Hospital, London, were 14 females and 12 males aged between 16 and 78 years. They had worn maxillary, tooth-supported, cobalt chromium-based removable partial dentures for at least 6 months and all the dentures were judged by both clinician and patient to be retentive, stable and comfortable. The subjects did not wear the dentures during the night. All subjects were non-smokers and in apparent good health and were examined to ensure that they had clinically healthy oral mucosa and that the hard palate showed no signs of inflammation.
Tissue sampling Subjects were asked to rinse their mouths after removing their dentures. The adhesive tape technique was then used to sample two areas of the hard palate, which were at least 4.0 mm away from the gingival margin, and which fell entirely beneath the metal base of the denture. For these samples the adhesive tape was cut to the shape dictated by the denture design to produce a sample of approximately 1 cm2. In each of the subjects a control sample was collected from an area of the hard palate which was 4.0 mm from the gingivae but not covered by the denture. The tape samples were fixed and processed as described previously.
Analysis The stained slides were examined in the light microscope at a magnification of X 200. Between 200 and 330 cells were counted from each specimen and classified as orthokeratinized cells or nucleate cells. An index of orthokeratinization was calculated as the percentage of orthokeratinized cells to the total epithelial cell population. This cell sample could be estimated to have a relative standard error of less than 5 per cent according to the method of Aherne and Dunnill (1982). Statistical analysis was performed using the paired and Student’s t tests.
RESULTS Sampling
techniques
The various types of epithelial cells observed in the samples could be recognized according to their cell size and shape, staining quality and the absence or presence and form of the nucleus, as described in standard cytological texts (Medak et al., 1970). In the smears, cells originating from every layer of the epithelium could be recognized, i.e. basal, spinous, granular and stratum corneum. They were present either singly or frequently in clumps. Isolated cells were easily classified according to
154
J. Dent.
1992;
20:
No. 3
Table 1. Otthokeratinization indices established by the two sampling techniques, from opposite sides of the hard palate, in fully dentate subjects
Orthokeratinization index (mean %) s.e.m. Range (%)
Smear technique
Tape technique
84.95
99.95
1.44 69.12-97.12
0.03 99.38-l 00
type, but when cells clumped the piling of cells prevented identification and quantification. In the tape samples only two types of epithelial cells were observed in all samples, orange cells with and without nuclei-parakeratinized and orthokeratinized squames. The cell density varied across the tape specimens, according to the amount of pressure applied in cell collection and the persistence of their adhesion to the tape during the cytological processing. The adhesive tape which formed the background of the tape samples remained virtually colourless and transparent, and offered no interference to quantification. Cells in the tape samples also occurred either singly or in groups. When they were in groups, they were frequently arranged in such a way that the boundary of one cell contacted those of adjacent cells and did not overlie them, i.e. without piling. Therefore it was possible to distinguish the types of cells and to quantify them with little difficulty. The apparent differences in palatal orthokeratinization identified by the two techniques, for the dentate subjects, are presented in Table I. The tape technique consistently gave a higher orthokeratinization index than the smear technique. Statistical analysis showed that the mean orthokeratinization index was significantly higher than for the smear samples (P < 0.001; paired t test). No significant difference was established been the orthokeratinization indices of male and female subjects by the tape technique, but using the smear technique there was a tendency for females to register lower indices (P > 0.05- < 0.1; Student’s t test).
Orthokeratinization The tape samples from the control areas of palatal tissue uncovered by denture base and the samples taken from beneath the metal denture bases appeared similar to samples from the first study. Fully orthokeratinized and parakeratinized cells were present, no evidence of cells from the deeper epithelial cell layers was found. Isolated cells were present, together with groups of cells with coincident borders, however clumps of piled cells were seen more frequently in the samples from covered tissues. The orthokeratinization indices of the palatal mucosa determined by tape stripping from the 26 patients with the partial dentures are presented in Table ZZ. It was established that the areas covered by the cobalt-chromium showed a lower mean orthokeratinization index than those areas not covered by the dentures.
Table II. Orthokeratinization indices of tissues beneath the metal bases and of uncovered palatal tissues in 26 wearers of partial dentures Uncovered Orthokeratinization index (mean %) s.e.m. Range (%)
sire
98.73 0.53 88.35-l 00
Covered site 49.80 3.1 1 16.07-78.06
The data from this investigation revealed that the differences in keratinization between uncovered and covered tissues in each subject were normally distributed. Therefore a paired r test was performed to evaluate the difference between the two areas. A highly significant difference was found (P < 0.001; paired t test).
DISCUSSION Cytology offers several advantages in studying the response of epithelia to prostheses. The techniques are non-invasive, pain free and provide the opportunity for repeated sampling. However the traditional method of mechanical scraping may collect cells from the deeper epithelial layers by virtue of the pressure applied. This would seem unsatisfactory for examination of variations in the degree of keratinization of oral mucosae. In order to eliminate this variability in sampling an alternative technique was developed. In the present study the use of adhesive tape to collect cells has proved a simple and satisfactory method which warranted only minor modifications to the standard processing regimen. An advantage of the method was that small areas of palate could be sampled and provide sufficient cells for quantitation. Also distribution of cells on the tape facilitated identification and quantitation; generally cells occurred alone or in groups with coincident borders. Piling or clumping of cells was much less frequent than in the smears. Normal human palatal epithelium is an orthokeratinizing tissue (Chen and Squier, 1984). In cytological smears from hard palate McMillan (1972) found 98 per cent of cells fully keratinized, however Lindholme et al. (1982) reported only 63 per cent of cells were orthokeratinized in healthy subjects. Such a disparity in findings may well reflect the difference in the pressure applied during the smear technique. In the present study the mean percentage of cells that were orthokeratinized when sampled by mechanical scraping was 84.95 per cent. Using the tape technique on the opposite sides. of the palates we identified mean levels of 99.95 per cent, more in keeping with the accepted histology of the tissue. It would seem that the tape technique gave a more accurate representation of the percentage orthokeratinization of the superficial cell layers. In previous studies of smears from the tissues underlying complete dentures made from acrylic resin. the percentage of orthokeratinized cells was between 26 and 50 per cent (Al-Ani ef al., 1966; Markov, 1968; McMillan,
Harinasuta
1972; Lindholme et al., 1982) parts of the mucosa having become parakeratinized as a result of complete denture usage. In the present study, employing the tape technique, the palatal orthokeratinization index also fell in wearers of cobalt-chromium-based tooth-borne removable partial dentures. The mean percentage of cells that were orthokeratinized decreased from 98 per cent for the uncovered mucosa to 50 per cent beneath the metal base of the dentures. Although a direct comparison cannot be made because of the difference in sampling techniques, it appears that, like complete dentures which are entirely mucosa borne, tooth-borne removable partial dentures cause a significant shift in the keratinization pattern of the epithelium beneath the prosthesis from one that is predominantly orthokeratinized to one in which half the surface cells are parakeratinized. Many factors may contribute to changes in epithelial differentiation including tissue loading, the effects of denture plaque, reduced oxygen tension (Riber and Kaaber, 1980) and reduced salivary flow together with thermal and pH changes (Budtz-Jorgenson, 1981). Lindholme et al. (1982) proposed that movement of the denture bases during function may be contributory, while Al-Ani et al. (1966) and Markov (1968) proposed that denture bases may actually ‘protect’ tissues, especially during the process of mastication. Tooth-supported and well-retained partial dentures would not be expected to move nor to load the mucosa to the same extent as complete dentures and consequently not traumatize the tissues as much. They would, however, offer some protection to the palatal tissues against masticatory forces. The present findings show that the use of tooth-supported removable partial dentures is associated with significant changes in epithelial differentiation. Factors such as the role of denture hygiene would also seem to be of considerable importance. Plaque collecting upon the surface of the denture base may initiate a change from orthokeratinization to parakeratinization (Theilade and Budtz-Jorgenson, 1988). Clinical experience indicates that the role of plaque with metal-based partial dentures is as significant as with complete dentures. The present study represents the assessment of a cytological sampling technique which has been shown to be particularly useful in examination of changes in the degree of keratinization beneath oral prostheses. Despite the reduced tissue loading which results from the elements of tooth support and retention which may be incorporated in metal-based partial dentures, the underlying tissues appear likely to undergo structural changes which may affect their functional integrity. The effects of dentures upon the underlying tissues remain poorly understood. In view of the importance of the healthy maintenance of oral mucosa to the long-term success of a denture. further studies of the relationship between denture use and epithelial status are indicated.
and Howlett:
Keratinization
beneath dentures
155
Acknowledgements The authors are indebted to Dr R. Holt of the Department ofchildrens Dentistry and Dr R. Hopps for their help and advice in preparation of this paper.
References Aherne W. A. and Dunnill M. S. (1987) Preparation of the tissues: sampling. In: Morphometry. London. Edward Arnold, p. 25. Al-Am S., Shklar G. and Yurkstas A. A. (1966) The effect of dentures on the exfoliative cytology of palatal and buccal oral mucosa. .I. Prosthet. Dent. 16, 513-521. Budtz-Jorgenson E. (1981) Oral mucosal lesions associated with the wearing of removable dentures. J. Oral Pathol. 10, 65-80. Chen S. Y. and Squier C. A. (1984) The ultrastructure of the oral epithelium. In: Meyer J., Squier C. A and Gerson S. J. (eds). The Structure and Function of Oral Mucosa. Oxford, Pergamon. pp. 7-30. Jani R. M. and Bhargava K. (1976) A histological comparison of palatal mucosa before and after wearing complete dentures. J. Prosthet. Dent 36, 254-260. Kapur K and Shklar G. (1963) The effect of complete dentures on alveolar mucosa. J. Prosthet. Dent. 13, 1030-1037. Lindholme K., Hakala P. E. and Makila E. (1982) Leukocyte count and keratinization of the palatal denture bearing mucosa. .I. Prosthet. Dent. 41, 440-444. Markov N. J. (1968) Cytologic study of keratinization under complete dentures. J. Prosthet Dent. 20, 8-13. Markov N. J. (1969) Cytologic study of the effect of some biomechanical principles of complete denture construction on keratinization of the mucosa of the edentulous ridge. J. Prosthet. Dent. 21, 132-135. McMillan D. R. (1972) The cytological response of palatal mucosa to dentures. Dent. Pratt. Dent. Rec. 22, 302-304. Medak H.. McGrew E. A., Burlakow B. S. et al. (1970) Atlas of Cytology. Washington, US Department of Health, Education and Welfare, pp. l-6. Papanicolaou G. N. (1942) A new procedure for staining vaginal smears. Science 95, 438-439. Plewig G. and Marples R. R. (1970) Regional differences of cell sizes in human stratum corneum. J. Invest. Dermatol. 54, 13-18. Riber E. and Kaaber S. (1980) A 12-month study on changes in the barrier properties of denture-loaded palatal mucosa in immediate denture wearers. Stand. _J.Dent. Res. 88, 250-256. Sandler H. C. (1964) Oral cytology-a program of cooperative diagnosis. J. Am. Dent. Assoc. 68, 603-606. Sharma A. K. and Mirza F. D. (1986) Palatal mucosa under dentures: a qualitative histologic and histochemical analysis. J. Prosthet. Dent. 56, 574-582. Stabes W. R.. Ellinger C. W., Tucker S. R. et al. (1981) Patient response to variations in denture technique. Part V: microscopic response of the oral mucosa. J. Prosthet. Dent. 45, 11-13. Theilade E. and Budtz-Jorgenson E. (1988) Predominant cultivable microflora of plaque on removable dentures in patients with denture-induced stomatitis. Oral Microbial. Immunol. 3, 8-13. Watson I. B. and MacDonald R. D. (1982) Oral mucosa and complete dentures. J. Prosthet. Dent 47, 133-139.
152
20: 152-l
55
Keratinization of palatal mucosa beneath metal-based removable partial dentures* S. Harinasuta* Department
and J. A. Howlett
of Prosthetic
Dentistry,
institute
of Dental Surgery,
University
of London,
UK
ABSTRACT Exfoliative cytology provides a non-invasive method of examining changes in epithelial differentiation beneath dentures. Cell sampling by mechanical scraping may introduce errors due to collection of deeper epithelial cells, therefore an alternative sampling method was evaluated. Cells collected from the hard palates of 24 dentate subjects using either a wooden tongue depressor or transparent, adhesive-coated tape were fixed and stained by Papanicolaou’s technique. The percentage of the total epithelial cells counted that were anucleate (the orthokeratinization index) was 99.95 per cent using the tape technique but 15 per cent lower using the smear technique. It appears that the tape technique gives a more accurate representation of the orthokeratinization of superficial cell layers. The tape technique was used to examine epithelial changes beneath tooth-supported removable partial dentures (RPDs). In 26 subjects, who had worn cobalt chromium based RPDs for at least 6 months, samples taken from beneath the metal denture base had a 50 per cent lower orthokeratinization index than those from uncovered areas of the palate. KEY WORDS:
Dentures,
Epithelium,
152-I
Metals
J. Dent. 1992; 1991)
20:
55 (Received 20 May 1991;
reviewed
Correspondence Dental Surgery,
should be addressed too:Dr J. A. Howlett, Department University of London, Gray’s Inn Road, London WClX
INTRODUCTION The response of the supporting epithelium to dentures is of interest because changes in epithelial structure and functional integrity may be associated with the development of mucosal inflammation and ultimately with underlying bone resorption. Denture-bearing epithelium is comprised predominantly of masticatory mucosa but the peripheral denture flanges often rest upon lining mucosa. The majority of studies on structural changes of denture-bearing tissues have examined mucosa from the palate or maxillary alveolus, lying beneath complete dentures fabricated from acrylic resin. Normal human palatal epithelium is an orthokeratinizing tissue with a well-developed stratum corneum (Chen and Squier, 1984). However, several cytological studies (Al-Ani et al., 1966; McMillan, 1972; Lindholme et al., 1982) and histological studies (Watson and Macdonald, 1982; Sharma and
*Presented in partial fulfilment of the requirements for the degree of MSc in the University of London 1986. Previously presented as a communication at the 1987 Annual BSDR Meeting held at the Institute of Dental Surgery. London. @ 1992 Butterworth-Heinemann 0300-5712/92/030152-04
Ltd.
2 August
1991;
accepted 27 October
of Prosthetic Dentistry, Institute of 8LD. UK.
Mirza, 1986) have found that changes in the degree of keratinization, from orthokeratosis to parakeratosis, and a reduction in the thickness of the stratum corneum occur when the palate is covered by a complete denture base. However, other workers (Kapur and Sklar, 1963; Jani and Bhargava, 1976) have found increased orthokeratinization with denture wear. Cytological techniques have also been used in attempts to correlate changes in the degree of keratinization with the biomechanical features of the dentures. Markov (1969) found that well-designed dentures were conducive to higher levels of orthokeratinization in the underlying tissues, although Stabes et al. (1981) were unable to identify any difference in the behaviour of mucosa beneath dentures as a result of different occlusal schemes. Thus the influences of denture use upon the epithelium remain contentious. The response of epithelium beneath tooth-borne, metal-based, removable partial dentures has not been examined, and may differ from that of the mucosa beneath complete dentures. The former tissues should not be subject to such overt biomechanical stresses, because of the increased retention and stability of the dentures and the reduced soft-tissue loading which they impart.
Harinasuta
Exfoliative cytology has been employed as a simple, non-invasive technique to examine oral epithelia. The methods used in cell collection have differed according to the cell samples required. They include mouth rinsing and cell collection for screening, and mechanical scraping to produce a smear, for examination of selected areas. Previous studies of denture-bearing mucosa have employed the latter method (Sandler, 1964; McMillan, 1972). The disadvantage of such a technique is that the depth of tissue penetrated varies according to the pressure applied. This would seem unsatisfactory for determining the keratinization status of masticatory mucosa, which can be judged only by examination of superficial cell layers. Previous dermatological studies have used adhesivecoated transparent tape to collect corneocytes from skin (Plewig and Marples, 1970). The aims of the present study were twofold, first to assess the use of adhesive tape, as an alternative technique to the smear, in the sampling of oral epithelium, and secondly to use the technique in examining the keratinization of palatal mucosa beneath tooth-supported removable partial dentures.
MATERIALS
AND METHODS
The technique The subjects were 12 males and 12 females aged between 17 and 36 years who were fully dentate. All were nonsmokers, in apparent good health and were examined to ensure that they had clinically healthy oral mucosae and that their hard palates showed no signs of inflammation. Two methods were used, in each subject, to sample palatal tissue midway between the gingivae and midline of the palate opposite the premolar and molar teeth. Subjects first rinsed their mouths and then on the right side a wooden tongue depressor was used to scrape the epithelial squames with light pressure. The material was smeared on to a clean glass slide and immediately fixed in 95 per cent ethyl alcohol. On the left side the palatal mucosa was dried, for approximately 5 s, with a stream of air from a three-in-one syringe and a strip of 3M Pressure Sensitive Tape (3M Ltd, Bracknell, UK), 2.0 cm X 1.5 cm, was placed on the mucosa, using sufficient finger pressure in order for it to adhere to the palate. The tape was then peeled off by catching one end with college tweezers and immediately placed in 95 per cent ethyl alcohol for 5 min and then allowed to dry. The tape was attached via its backing to a clean glass slide with DPX mounting medium (BDH Chemicals Ltd, Poole, UK). The smears and tape samples were hydrated and stained by the method of Papanicolaou (1942) using haematoxylin, 0G6 and EA50 (Ortho Diagnostic Systems Ltd, High Wycombe, UK). Smears were then dehydrated using alcohol and xylene and mounted with DPX by standard methods. To avoid dissolution of the adhesive, tape samples were dehydrated in alcohol alone and allowed to dry before mounting with DPX.
and Howlett:
Keratinization
beneath dentures
153
Orthokeratinization This was investigated in 26 partially edentulous subjects using the adhesive tape technique. The subjects, all of whom were patients of the Eastman Dental Hospital, London, were 14 females and 12 males aged between 16 and 78 years. They had worn maxillary, tooth-supported, cobalt chromium-based removable partial dentures for at least 6 months and all the dentures were judged by both clinician and patient to be retentive, stable and comfortable. The subjects did not wear the dentures during the night. All subjects were non-smokers and in apparent good health and were examined to ensure that they had clinically healthy oral mucosa and that the hard palate showed no signs of inflammation.
Tissue sampling Subjects were asked to rinse their mouths after removing their dentures. The adhesive tape technique was then used to sample two areas of the hard palate, which were at least 4.0 mm away from the gingival margin, and which fell entirely beneath the metal base of the denture. For these samples the adhesive tape was cut to the shape dictated by the denture design to produce a sample of approximately 1 cm2. In each of the subjects a control sample was collected from an area of the hard palate which was 4.0 mm from the gingivae but not covered by the denture. The tape samples were fixed and processed as described previously.
Analysis The stained slides were examined in the light microscope at a magnification of X 200. Between 200 and 330 cells were counted from each specimen and classified as orthokeratinized cells or nucleate cells. An index of orthokeratinization was calculated as the percentage of orthokeratinized cells to the total epithelial cell population. This cell sample could be estimated to have a relative standard error of less than 5 per cent according to the method of Aherne and Dunnill (1982). Statistical analysis was performed using the paired and Student’s t tests.
RESULTS Sampling
techniques
The various types of epithelial cells observed in the samples could be recognized according to their cell size and shape, staining quality and the absence or presence and form of the nucleus, as described in standard cytological texts (Medak et al., 1970). In the smears, cells originating from every layer of the epithelium could be recognized, i.e. basal, spinous, granular and stratum corneum. They were present either singly or frequently in clumps. Isolated cells were easily classified according to
154
J. Dent.
1992;
20:
No. 3
Table 1. Otthokeratinization indices established by the two sampling techniques, from opposite sides of the hard palate, in fully dentate subjects
Orthokeratinization index (mean %) s.e.m. Range (%)
Smear technique
Tape technique
84.95
99.95
1.44 69.12-97.12
0.03 99.38-l 00
type, but when cells clumped the piling of cells prevented identification and quantification. In the tape samples only two types of epithelial cells were observed in all samples, orange cells with and without nuclei-parakeratinized and orthokeratinized squames. The cell density varied across the tape specimens, according to the amount of pressure applied in cell collection and the persistence of their adhesion to the tape during the cytological processing. The adhesive tape which formed the background of the tape samples remained virtually colourless and transparent, and offered no interference to quantification. Cells in the tape samples also occurred either singly or in groups. When they were in groups, they were frequently arranged in such a way that the boundary of one cell contacted those of adjacent cells and did not overlie them, i.e. without piling. Therefore it was possible to distinguish the types of cells and to quantify them with little difficulty. The apparent differences in palatal orthokeratinization identified by the two techniques, for the dentate subjects, are presented in Table I. The tape technique consistently gave a higher orthokeratinization index than the smear technique. Statistical analysis showed that the mean orthokeratinization index was significantly higher than for the smear samples (P < 0.001; paired t test). No significant difference was established been the orthokeratinization indices of male and female subjects by the tape technique, but using the smear technique there was a tendency for females to register lower indices (P > 0.05- < 0.1; Student’s t test).
Orthokeratinization The tape samples from the control areas of palatal tissue uncovered by denture base and the samples taken from beneath the metal denture bases appeared similar to samples from the first study. Fully orthokeratinized and parakeratinized cells were present, no evidence of cells from the deeper epithelial cell layers was found. Isolated cells were present, together with groups of cells with coincident borders, however clumps of piled cells were seen more frequently in the samples from covered tissues. The orthokeratinization indices of the palatal mucosa determined by tape stripping from the 26 patients with the partial dentures are presented in Table ZZ. It was established that the areas covered by the cobalt-chromium showed a lower mean orthokeratinization index than those areas not covered by the dentures.
Table II. Orthokeratinization indices of tissues beneath the metal bases and of uncovered palatal tissues in 26 wearers of partial dentures Uncovered Orthokeratinization index (mean %) s.e.m. Range (%)
sire
98.73 0.53 88.35-l 00
Covered site 49.80 3.1 1 16.07-78.06
The data from this investigation revealed that the differences in keratinization between uncovered and covered tissues in each subject were normally distributed. Therefore a paired r test was performed to evaluate the difference between the two areas. A highly significant difference was found (P < 0.001; paired t test).
DISCUSSION Cytology offers several advantages in studying the response of epithelia to prostheses. The techniques are non-invasive, pain free and provide the opportunity for repeated sampling. However the traditional method of mechanical scraping may collect cells from the deeper epithelial layers by virtue of the pressure applied. This would seem unsatisfactory for examination of variations in the degree of keratinization of oral mucosae. In order to eliminate this variability in sampling an alternative technique was developed. In the present study the use of adhesive tape to collect cells has proved a simple and satisfactory method which warranted only minor modifications to the standard processing regimen. An advantage of the method was that small areas of palate could be sampled and provide sufficient cells for quantitation. Also distribution of cells on the tape facilitated identification and quantitation; generally cells occurred alone or in groups with coincident borders. Piling or clumping of cells was much less frequent than in the smears. Normal human palatal epithelium is an orthokeratinizing tissue (Chen and Squier, 1984). In cytological smears from hard palate McMillan (1972) found 98 per cent of cells fully keratinized, however Lindholme et al. (1982) reported only 63 per cent of cells were orthokeratinized in healthy subjects. Such a disparity in findings may well reflect the difference in the pressure applied during the smear technique. In the present study the mean percentage of cells that were orthokeratinized when sampled by mechanical scraping was 84.95 per cent. Using the tape technique on the opposite sides. of the palates we identified mean levels of 99.95 per cent, more in keeping with the accepted histology of the tissue. It would seem that the tape technique gave a more accurate representation of the percentage orthokeratinization of the superficial cell layers. In previous studies of smears from the tissues underlying complete dentures made from acrylic resin. the percentage of orthokeratinized cells was between 26 and 50 per cent (Al-Ani ef al., 1966; Markov, 1968; McMillan,
Harinasuta
1972; Lindholme et al., 1982) parts of the mucosa having become parakeratinized as a result of complete denture usage. In the present study, employing the tape technique, the palatal orthokeratinization index also fell in wearers of cobalt-chromium-based tooth-borne removable partial dentures. The mean percentage of cells that were orthokeratinized decreased from 98 per cent for the uncovered mucosa to 50 per cent beneath the metal base of the dentures. Although a direct comparison cannot be made because of the difference in sampling techniques, it appears that, like complete dentures which are entirely mucosa borne, tooth-borne removable partial dentures cause a significant shift in the keratinization pattern of the epithelium beneath the prosthesis from one that is predominantly orthokeratinized to one in which half the surface cells are parakeratinized. Many factors may contribute to changes in epithelial differentiation including tissue loading, the effects of denture plaque, reduced oxygen tension (Riber and Kaaber, 1980) and reduced salivary flow together with thermal and pH changes (Budtz-Jorgenson, 1981). Lindholme et al. (1982) proposed that movement of the denture bases during function may be contributory, while Al-Ani et al. (1966) and Markov (1968) proposed that denture bases may actually ‘protect’ tissues, especially during the process of mastication. Tooth-supported and well-retained partial dentures would not be expected to move nor to load the mucosa to the same extent as complete dentures and consequently not traumatize the tissues as much. They would, however, offer some protection to the palatal tissues against masticatory forces. The present findings show that the use of tooth-supported removable partial dentures is associated with significant changes in epithelial differentiation. Factors such as the role of denture hygiene would also seem to be of considerable importance. Plaque collecting upon the surface of the denture base may initiate a change from orthokeratinization to parakeratinization (Theilade and Budtz-Jorgenson, 1988). Clinical experience indicates that the role of plaque with metal-based partial dentures is as significant as with complete dentures. The present study represents the assessment of a cytological sampling technique which has been shown to be particularly useful in examination of changes in the degree of keratinization beneath oral prostheses. Despite the reduced tissue loading which results from the elements of tooth support and retention which may be incorporated in metal-based partial dentures, the underlying tissues appear likely to undergo structural changes which may affect their functional integrity. The effects of dentures upon the underlying tissues remain poorly understood. In view of the importance of the healthy maintenance of oral mucosa to the long-term success of a denture. further studies of the relationship between denture use and epithelial status are indicated.
and Howlett:
Keratinization
beneath dentures
155
Acknowledgements The authors are indebted to Dr R. Holt of the Department ofchildrens Dentistry and Dr R. Hopps for their help and advice in preparation of this paper.
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