Epithelial adherence to polytetrafluoroethylene (PTFE) material
Hans Jacob Grevstad and Knut Norvald Leknes Department of Periodontology, School of Dentistry, University of Bergen, Bergen, Norway
Grevstad HJ, Leicnes KN: Eptthetiat adheretice to potytetraftuoroeliiytene (PTFE) materiai. Scatid J Detn Res 1992; 100: 236-9. The aim of this study was to examine adherence/attachment of gingival cellular elements to polytetrafluoroethylene (PTFE) material with special reference to epithelial cells. Six Gore-Tex membranes used for guided tissue regeneration in patients with severe periodontai disease were studied by transmission electron microscopy. Upon retrieval, after 30 days of healing, the membranes were immediately immersed in formaldehyde-glutaraldehyde, divided in segments (2x4 mm) and embedded in Epon. The majority of segments were devoid of any adhering tissue other than erythrocytes and polymorphonuclear cells. Epithelial cell adherence was mediated by a 2-nm-thick acellular layer containing membraneous profiles with diameter 370 nm or less. Adhering epithelial cells exhibited characteristics of cellular degeneration as evidenced by tonofilamentous and cytoplasmic condensation, vacuolization and loss of structural details. An uneven basement lamina with hemidesmosomal contacts was present at the epithelium-connective tissue interface. It is concluded that epithelial cells adhere to PTFE material without formation of attachment complexes.
Formation of hemidesmosomal complexes and basal lamina is characteristic of epithelial cell attachment to dental hard tissues (1-3). Corresponding attachment mechanisms have been demonstrated at the interface of epithelium and some implant materials like titanium and ceramics (4-7). On other solid materials, e.g. glass and crystal sapphire, the relationship of epithelial cells to surface seems to be a question of cellular adhesion (8, 9). Recent studies by scanning electron and light microscopy have indicated that soft tissue adheres to polytetrafluoroethylene (PTFE) material (10, 11). The aim of the study was to investigate soft tissue adhesion or attachment to PTFE membranes used for guided tissue regeneration with special reference to the ultrastructure of epithelial cells. Material and methods Six polytetrafluoroethylene (PTFE) membranes (Gore-Tex, W. L. Gore and Assoc, Inc., Flagstaff, Arizona, USA) were obtained from six adult patients treated for periodontai disease at the Department of Periodontology, University of Bergen. One intrabony lesion in each patient was scheduled for membrane insertion. The patients received daily
Key words: epithelium; periodontai surgery; polytetrafluoroethylene; transmission electron microscopy Hans Jacob Grevsfad, Department of Periodontology, School of Dentistry, Aarstadveien 17, N-5009 Bergen, Norway Accepted for publication 17 October 1991
650 mg X 2 Femepen (Norges Apotekerforening, Oslo, Norway) and rinsed additionally with 0.2% chlorhexidine digluconate solution during the healing period of 30 days. Following removal, the PTFE membranes were fixed in formaldehyde-glutaraldehyde. Membranes were subsequently divided by a pair of scissors into 18 segments ( 2 x 4 mm) more suitable for preparation of specimens for electron microscopy. The segments were washed in 0.1 M sodium cacodylate buffer containing 0.2 M sucrose and postfixed in 1% phosphate-buffered osmium tetroxide. After dehydration in graded solutions of alcohol, the membrane segments were embedded in Epon and oriented in rubber moulds to obtain sections from three defined areas: 1. Surface facing the bony defect (partially occlusive portion, inner surface). 2. Surface facing flap tissue (partially occlusive portion, outer surface). 3. Surface of open mierostructure portion facing flap tissue. In the following, these parts of membrane segments are more conveniently referred to as apron (1 & 2) and collar (3), respectively. Survey sections at 1 |im were cut with glass knives and stained with toluidine blue. Thin sections were cut with diamond knives, collected on grids and stained
Epithelial adherence of PTFE material with uranyl acetate and lead citrate. The sections were examined in an electron microscope operated at 80 kV. Results
Polymorphonuclear cells, macrophage-like cells erythrocytes and fibrin were found close to membrane on both sides of the apron. Usually a space between tissue and membrane occurred. Even fibrin film had apparently detached from membrane surface, leaving an artifactual split between tissue and membrane in the plane of section. The presence of cells or other constituents within and between the segments was rather inconsistent and several specimens were devoid of any detectable material or tissue. A regular and consistent observation, however, was that membranes (3 of 6) which had been partially exposed during the healing period were heavily coated with plaque mainly located at the membrane collar.
237
mately 2 |im. This layer contained unidentifiable, amorphous material and membraneous profiles, with a diameter of 370 nm or less. Cells abutting this layer appeared as dense masses of tissue with abundant aggregations of tonofilaments and numerous vacuoles suggestive of degenerated epithelial cells. Further from the interface, the signs of cellular degeneration were less conspicuous. Cell membranes as well as cellular contacts were readily traced in these sections (Fig. 3). The epithelial cell layer terminated either with ruptured or apparently intact cells. In the latter situation, alternatively erythrocytes, inflammatory cells and fibroblasts delineated the epithelium. Fig. 4 illustrates an area with fibroblasts and collagen fibrils and infiammatory cells as well. Higher magnification of epithelial cells facing remnants of connective tissue revealed the presence of cytoplasmic condensations suggestive of attachment plaques and of components of basal lamina (Fig. 5). Discussion
Epithelium
,
. \
Light microscopy - Epithelial cells occurred in two segments each originating from two different membranes. The cells were located on the inner aspect of the apron and confined to tiny patches 1 mmor less. Light microscopic examination ofthe tissue close to membrane did not reveal the true nature of these cells (Fig. IA). Cellular elements were absent or barely perceivable in the zone facing the membrane measuring approximately 15 \im. In some areas, this tissue appeared in continuity with epithelial cells primarily identified by their typical intercellular contacts (Fig. IB). The labeled boxes in Fig. IA & B indicate areas which were examined more closely in the electron microscope. Transmission electron microscopy — The moulding effect of the membrane surface on the tissue is apparent in Fig. 2. The layer distinctly outlined by the irregularities of membrane measured approxi-
This study has revealed that PTFE material may be treated with routinely used methods of specimen preparation for light- and transmission electron microscopy. Sectioning with glass and diamond knives usually presented only minor problems. However, ultrathin sections of the material tended to exhibit deformation, i.e. bending and folding when examined in the microscope. Hence, the tissues seemed to have detached from the membrane surface probably during the process of fixation and embedding. These observations indicate that the adhesion of tissue components to PTFE material may be rather weak, consistent with less spreading and adhesion of organic material to low energy sufaces (12, 13). Thus, in many instances, the relationship of surface and tissue had to be evaluated indirectly, i.e. from the appearance of the tissue "moulded" by the membrane surface. Another indirect suggestion involves cellular prolif-
Fig. 1. Histologic sections of membrane-associatedspithelium. A, membrane interface on left. B, remnants of connective tissue and erythrocytes on right. A & B, boxed areas labeled 2, 3 and 4 & 5, respectively, indicate origin of ultrastructural details shown in Figs. 2-5. Toluidine blue. Bars represent 15 |jm. Photomicrographs. x670.
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Grevstad and Leknes
F/g. 2. Membrane-epithelium interface (clear arrow) and serrated cell surface (dense arrow). Acellular layer contains circular and elongated profiles < 370 nm. Cytoplasm of epithelial cells appears homogeneous with abundant vacuoles (v) and densely packed tonofilaments (tO. Cellular spaces are barely perceivable, n; nucleus of degenerating epithelial cell. Bar: 2 nm. Electron micrograph, x 13 330. Fig. 3. Epithelial cells remote from membrane. Outline of individual cells is evident from presence of numerous fingerlike projections and intercellular compartments. Part of inflammatory cells containing abundant dense bodies suggestive of lysosomes are visible in center of illustration, n, nucleus of degenerating epithelial cell. Bar: 2 |im. Electron micrograph, x 8000.
eration. The notion of epithelial cell migration on PTFE material is based on the apron location of the cell, albeit a short distance from the collar. To reach this level, the epithelial cells must have undergone a proliferative phase prior to cellular degeneration. This is consistent with the observation that epithelial cells explanted on PTFE-material do migrate to a limited extent and then degenerate (11). Thus, the clinical significance of epitheli-
um on membranes is limited to the potential possibility that the cells may also fmd their way to the tooth surface during phases of early regeneration. Since the epithelial cells may conceivably, originate from the overlying gingiva, migration had to be preceded by a local and accidental detachment of membrane from the tooth surface. The study was originally designed to correlate cells on membranes with a variety of clinical para-
Epithelial adherence of PTFE material
239
Fig. 4. Epithelium-connective tissue interface runs vertically in center of illustration. Parts of inflammatory cells and fibroblasts (F) surrounded by collagen fibrils on right. Bar: 3 nm. Electron micrograph, x 3600. Fig. 5. Undulating course of basal lamina at epithelium(e)-connective tissue (ct) interface. Bar: 0.5 ycm. Electron micrograph. X 36 000.
meters, e.g. type of bony defect, partial exposure of membrane and assessment of gingival pre- and postoperative conditions. In light of the observations reported here and in view of the limited number of specimens, this approach appeared meaningless and was abandoned, since observation of cells on membranes was also rather undependable in the case of epithelial cells. The peculiar layer facing the membrane (Fig. 2) is apparently not a secretory product of epithelial cells corresponding to what can be seen on other solids (2, 4, 5, 8). Moreover, its width (2 \xm) and content of membraneous profiles with dimensions similar to cytoplasmic organelles indicate the presence of cellular remains rather than a secretory layer. Due to the limitation as to available tissue mentioned above, further investigation may be necessary to establish whether or not epithelial adhesion to PTFE material is merely a question of passive attraction. In conclusion this study indicates that epithelial cell contact with PTFE material is characterized by adhesion rather than attachment. Acknowledgment - This study has been supported in part by grants from A/S Norsk Dental Depot's Fond and Faculty of Dentistry, University of Bergen.
References 1. SELVIG KA. Interfaces between epithelium and soft and hard connective tissues. In: EASTOE J E , PICTON D C A , ALEXANDER AG, eds. The prevention of periodontal disease. London: Kimpton, 1971; 81-8.
2. SCHROEDER H , LISTGARTEN M . Fine structure of developing epithelial attachment of human teeth. In: WOLSKY A, ed. Motiograplts in developtnental biology. Basel: S. Karger, 1971. 3. SMITH C J . Gingival epithelium. In: MELCHER AH, BOWEN
WH, eds. Biology of the periodontiurn. London: Academic Press, 1969; 105-66. 4. GOULD T R L , BRUNETTE DM, WESTBURY L . The attach-
ment mechanism of epithelial cells to titanium in vitro. J Periodont Res 1981: 16: 611-6. 5. GOULD TRL, WESTBURY L, BRUNETTE DM. Ultrastructural
study of the attachment of human gingiva to titanium in vivo. J Prosthet Dent 1984; 52: 418-20. 6. KAVANAGH P, GOULD TRL, BRUNETTE DM, WESTON L: A
rodent model for the investigation of dental implants. / Prosthet Dettt 1985; 54: 252-7. 7. McKiNNEY RV JR., STEFLIK DE, KOTH DL. The epithelium
dental implant interface. J Oral Implantol 1988; 13: 622-A\. 8. FoERSKOv O, THEILADE J, JEPSEN A. Ultrastructure of rat
oral epithelium in long-term cell culture. Scand J Dent Res 1974; 82: 212-28. 9. FARTASH B, ARVIDSON K , ERICSSON J. Histology of tissues
surrounding single crystal sapphire endosseous dental implants. An experimental study in the beagle dog. Clin Oral Impl Res 1990; 1: 13-21. 10. SELVIG KA, NILVEUS RE, FITZMORRIS L , KERSTEN B , K H -
ORSANDi SS. Scanning electron microscopic observations of cell population and bacterial contamination of membranes used for guided tissue regeneration in humans. / Periodontol 1990; 61: 515-20. 11. SALONEN J I , PERSSON R G . Migration of epithelial cells on materials used in guided tissue regeneration. J Periodontol Res 1990; 25: 215-21. 12. TAYLOR AC. Adhesion of cells to surfaces. In: MANLY RS, ed. Adhesion in biological systems. New York: Academic Press, 1970; 51-71. 13. BAIER R E . Surface properties influencing biological adhesion. Iii: MANLY RS, ed. Adhesion in biological systems. New York: Academic Press, 1970; 15-48. - -
Hans Jacob Grevstad and Knut Norvald Leknes Department of Periodontology, School of Dentistry, University of Bergen, Bergen, Norway
Grevstad HJ, Leicnes KN: Eptthetiat adheretice to potytetraftuoroeliiytene (PTFE) materiai. Scatid J Detn Res 1992; 100: 236-9. The aim of this study was to examine adherence/attachment of gingival cellular elements to polytetrafluoroethylene (PTFE) material with special reference to epithelial cells. Six Gore-Tex membranes used for guided tissue regeneration in patients with severe periodontai disease were studied by transmission electron microscopy. Upon retrieval, after 30 days of healing, the membranes were immediately immersed in formaldehyde-glutaraldehyde, divided in segments (2x4 mm) and embedded in Epon. The majority of segments were devoid of any adhering tissue other than erythrocytes and polymorphonuclear cells. Epithelial cell adherence was mediated by a 2-nm-thick acellular layer containing membraneous profiles with diameter 370 nm or less. Adhering epithelial cells exhibited characteristics of cellular degeneration as evidenced by tonofilamentous and cytoplasmic condensation, vacuolization and loss of structural details. An uneven basement lamina with hemidesmosomal contacts was present at the epithelium-connective tissue interface. It is concluded that epithelial cells adhere to PTFE material without formation of attachment complexes.
Formation of hemidesmosomal complexes and basal lamina is characteristic of epithelial cell attachment to dental hard tissues (1-3). Corresponding attachment mechanisms have been demonstrated at the interface of epithelium and some implant materials like titanium and ceramics (4-7). On other solid materials, e.g. glass and crystal sapphire, the relationship of epithelial cells to surface seems to be a question of cellular adhesion (8, 9). Recent studies by scanning electron and light microscopy have indicated that soft tissue adheres to polytetrafluoroethylene (PTFE) material (10, 11). The aim of the study was to investigate soft tissue adhesion or attachment to PTFE membranes used for guided tissue regeneration with special reference to the ultrastructure of epithelial cells. Material and methods Six polytetrafluoroethylene (PTFE) membranes (Gore-Tex, W. L. Gore and Assoc, Inc., Flagstaff, Arizona, USA) were obtained from six adult patients treated for periodontai disease at the Department of Periodontology, University of Bergen. One intrabony lesion in each patient was scheduled for membrane insertion. The patients received daily
Key words: epithelium; periodontai surgery; polytetrafluoroethylene; transmission electron microscopy Hans Jacob Grevsfad, Department of Periodontology, School of Dentistry, Aarstadveien 17, N-5009 Bergen, Norway Accepted for publication 17 October 1991
650 mg X 2 Femepen (Norges Apotekerforening, Oslo, Norway) and rinsed additionally with 0.2% chlorhexidine digluconate solution during the healing period of 30 days. Following removal, the PTFE membranes were fixed in formaldehyde-glutaraldehyde. Membranes were subsequently divided by a pair of scissors into 18 segments ( 2 x 4 mm) more suitable for preparation of specimens for electron microscopy. The segments were washed in 0.1 M sodium cacodylate buffer containing 0.2 M sucrose and postfixed in 1% phosphate-buffered osmium tetroxide. After dehydration in graded solutions of alcohol, the membrane segments were embedded in Epon and oriented in rubber moulds to obtain sections from three defined areas: 1. Surface facing the bony defect (partially occlusive portion, inner surface). 2. Surface facing flap tissue (partially occlusive portion, outer surface). 3. Surface of open mierostructure portion facing flap tissue. In the following, these parts of membrane segments are more conveniently referred to as apron (1 & 2) and collar (3), respectively. Survey sections at 1 |im were cut with glass knives and stained with toluidine blue. Thin sections were cut with diamond knives, collected on grids and stained
Epithelial adherence of PTFE material with uranyl acetate and lead citrate. The sections were examined in an electron microscope operated at 80 kV. Results
Polymorphonuclear cells, macrophage-like cells erythrocytes and fibrin were found close to membrane on both sides of the apron. Usually a space between tissue and membrane occurred. Even fibrin film had apparently detached from membrane surface, leaving an artifactual split between tissue and membrane in the plane of section. The presence of cells or other constituents within and between the segments was rather inconsistent and several specimens were devoid of any detectable material or tissue. A regular and consistent observation, however, was that membranes (3 of 6) which had been partially exposed during the healing period were heavily coated with plaque mainly located at the membrane collar.
237
mately 2 |im. This layer contained unidentifiable, amorphous material and membraneous profiles, with a diameter of 370 nm or less. Cells abutting this layer appeared as dense masses of tissue with abundant aggregations of tonofilaments and numerous vacuoles suggestive of degenerated epithelial cells. Further from the interface, the signs of cellular degeneration were less conspicuous. Cell membranes as well as cellular contacts were readily traced in these sections (Fig. 3). The epithelial cell layer terminated either with ruptured or apparently intact cells. In the latter situation, alternatively erythrocytes, inflammatory cells and fibroblasts delineated the epithelium. Fig. 4 illustrates an area with fibroblasts and collagen fibrils and infiammatory cells as well. Higher magnification of epithelial cells facing remnants of connective tissue revealed the presence of cytoplasmic condensations suggestive of attachment plaques and of components of basal lamina (Fig. 5). Discussion
Epithelium
,
. \
Light microscopy - Epithelial cells occurred in two segments each originating from two different membranes. The cells were located on the inner aspect of the apron and confined to tiny patches 1 mmor less. Light microscopic examination ofthe tissue close to membrane did not reveal the true nature of these cells (Fig. IA). Cellular elements were absent or barely perceivable in the zone facing the membrane measuring approximately 15 \im. In some areas, this tissue appeared in continuity with epithelial cells primarily identified by their typical intercellular contacts (Fig. IB). The labeled boxes in Fig. IA & B indicate areas which were examined more closely in the electron microscope. Transmission electron microscopy — The moulding effect of the membrane surface on the tissue is apparent in Fig. 2. The layer distinctly outlined by the irregularities of membrane measured approxi-
This study has revealed that PTFE material may be treated with routinely used methods of specimen preparation for light- and transmission electron microscopy. Sectioning with glass and diamond knives usually presented only minor problems. However, ultrathin sections of the material tended to exhibit deformation, i.e. bending and folding when examined in the microscope. Hence, the tissues seemed to have detached from the membrane surface probably during the process of fixation and embedding. These observations indicate that the adhesion of tissue components to PTFE material may be rather weak, consistent with less spreading and adhesion of organic material to low energy sufaces (12, 13). Thus, in many instances, the relationship of surface and tissue had to be evaluated indirectly, i.e. from the appearance of the tissue "moulded" by the membrane surface. Another indirect suggestion involves cellular prolif-
Fig. 1. Histologic sections of membrane-associatedspithelium. A, membrane interface on left. B, remnants of connective tissue and erythrocytes on right. A & B, boxed areas labeled 2, 3 and 4 & 5, respectively, indicate origin of ultrastructural details shown in Figs. 2-5. Toluidine blue. Bars represent 15 |jm. Photomicrographs. x670.
238
Grevstad and Leknes
F/g. 2. Membrane-epithelium interface (clear arrow) and serrated cell surface (dense arrow). Acellular layer contains circular and elongated profiles < 370 nm. Cytoplasm of epithelial cells appears homogeneous with abundant vacuoles (v) and densely packed tonofilaments (tO. Cellular spaces are barely perceivable, n; nucleus of degenerating epithelial cell. Bar: 2 nm. Electron micrograph, x 13 330. Fig. 3. Epithelial cells remote from membrane. Outline of individual cells is evident from presence of numerous fingerlike projections and intercellular compartments. Part of inflammatory cells containing abundant dense bodies suggestive of lysosomes are visible in center of illustration, n, nucleus of degenerating epithelial cell. Bar: 2 |im. Electron micrograph, x 8000.
eration. The notion of epithelial cell migration on PTFE material is based on the apron location of the cell, albeit a short distance from the collar. To reach this level, the epithelial cells must have undergone a proliferative phase prior to cellular degeneration. This is consistent with the observation that epithelial cells explanted on PTFE-material do migrate to a limited extent and then degenerate (11). Thus, the clinical significance of epitheli-
um on membranes is limited to the potential possibility that the cells may also fmd their way to the tooth surface during phases of early regeneration. Since the epithelial cells may conceivably, originate from the overlying gingiva, migration had to be preceded by a local and accidental detachment of membrane from the tooth surface. The study was originally designed to correlate cells on membranes with a variety of clinical para-
Epithelial adherence of PTFE material
239
Fig. 4. Epithelium-connective tissue interface runs vertically in center of illustration. Parts of inflammatory cells and fibroblasts (F) surrounded by collagen fibrils on right. Bar: 3 nm. Electron micrograph, x 3600. Fig. 5. Undulating course of basal lamina at epithelium(e)-connective tissue (ct) interface. Bar: 0.5 ycm. Electron micrograph. X 36 000.
meters, e.g. type of bony defect, partial exposure of membrane and assessment of gingival pre- and postoperative conditions. In light of the observations reported here and in view of the limited number of specimens, this approach appeared meaningless and was abandoned, since observation of cells on membranes was also rather undependable in the case of epithelial cells. The peculiar layer facing the membrane (Fig. 2) is apparently not a secretory product of epithelial cells corresponding to what can be seen on other solids (2, 4, 5, 8). Moreover, its width (2 \xm) and content of membraneous profiles with dimensions similar to cytoplasmic organelles indicate the presence of cellular remains rather than a secretory layer. Due to the limitation as to available tissue mentioned above, further investigation may be necessary to establish whether or not epithelial adhesion to PTFE material is merely a question of passive attraction. In conclusion this study indicates that epithelial cell contact with PTFE material is characterized by adhesion rather than attachment. Acknowledgment - This study has been supported in part by grants from A/S Norsk Dental Depot's Fond and Faculty of Dentistry, University of Bergen.
References 1. SELVIG KA. Interfaces between epithelium and soft and hard connective tissues. In: EASTOE J E , PICTON D C A , ALEXANDER AG, eds. The prevention of periodontal disease. London: Kimpton, 1971; 81-8.
2. SCHROEDER H , LISTGARTEN M . Fine structure of developing epithelial attachment of human teeth. In: WOLSKY A, ed. Motiograplts in developtnental biology. Basel: S. Karger, 1971. 3. SMITH C J . Gingival epithelium. In: MELCHER AH, BOWEN
WH, eds. Biology of the periodontiurn. London: Academic Press, 1969; 105-66. 4. GOULD T R L , BRUNETTE DM, WESTBURY L . The attach-
ment mechanism of epithelial cells to titanium in vitro. J Periodont Res 1981: 16: 611-6. 5. GOULD TRL, WESTBURY L, BRUNETTE DM. Ultrastructural
study of the attachment of human gingiva to titanium in vivo. J Prosthet Dent 1984; 52: 418-20. 6. KAVANAGH P, GOULD TRL, BRUNETTE DM, WESTON L: A
rodent model for the investigation of dental implants. / Prosthet Dettt 1985; 54: 252-7. 7. McKiNNEY RV JR., STEFLIK DE, KOTH DL. The epithelium
dental implant interface. J Oral Implantol 1988; 13: 622-A\. 8. FoERSKOv O, THEILADE J, JEPSEN A. Ultrastructure of rat
oral epithelium in long-term cell culture. Scand J Dent Res 1974; 82: 212-28. 9. FARTASH B, ARVIDSON K , ERICSSON J. Histology of tissues
surrounding single crystal sapphire endosseous dental implants. An experimental study in the beagle dog. Clin Oral Impl Res 1990; 1: 13-21. 10. SELVIG KA, NILVEUS RE, FITZMORRIS L , KERSTEN B , K H -
ORSANDi SS. Scanning electron microscopic observations of cell population and bacterial contamination of membranes used for guided tissue regeneration in humans. / Periodontol 1990; 61: 515-20. 11. SALONEN J I , PERSSON R G . Migration of epithelial cells on materials used in guided tissue regeneration. J Periodontol Res 1990; 25: 215-21. 12. TAYLOR AC. Adhesion of cells to surfaces. In: MANLY RS, ed. Adhesion in biological systems. New York: Academic Press, 1970; 51-71. 13. BAIER R E . Surface properties influencing biological adhesion. Iii: MANLY RS, ed. Adhesion in biological systems. New York: Academic Press, 1970; 15-48. - -
