|Year : 2011 | Volume
| Issue : 1 | Page : 4-17
Biophysical studies of the gingival epithelium
HS Grover, Himanshu Dadlani
Department of Periodontology and Implantology, Sri Gobind Tricentenary Dental College, Hospital & Research Institute, Gurgaon, Haryana, India
|Date of Web Publication||29-Jul-2013|
Department of Periodontology and Implantology, Sri Gobind Tricentenary Dental College, Hospital & Research Institute, Gurgaon
Source of Support: None, Conflict of Interest: None
| Abstract|| |
Objectives: As the gingival epithelial cells undergo transition between the surface, crevicular and junctional areas, it is reported that their resting potential could also get altered due to change/variations in the membrane transport protein composition. Hence, the purpose of the present study is (i) to study the biophysical properties of the surface, the crevicular, and the junctional epithelial gingival cells and to assess their implications to the cell to cell and the cell to tooth attachment mechanisms; (ii) to study the effect of certain delipidifying agents such as Sodium Deoxycholate (DOC) and Sodium Dodecyl Sulfate (SDS) on the transmembrane electrical activity of the gingival epithelial cells; and, (iii) to study the histological changes due to these treatments and assess their bearings on the biophysical observations. Materials and Methods: The biophysical and histological investigations on the healthy human gingival epithelium were carried out on the fresh biopsy material obtained from a homogeneous group of willing donors between 11-15 years of age, who were advised for extraction of first premolar due to orthodontic reasons. The biophysical measurement involved recording of the transmembrane potentials using glass ultra-microelectrodes. For histologic studies, the hematoxylin and eosin (H&E) staining of the tissue sections was performed. Results: The mean values of the membrane potential in the three types of the gingival epithelium cells vary appreciably. While it was lowest in the junctional cells (2.83 ± 0.98 mV), it was highest in the surface epithelial cells (22.96 ± 5.19 mV). The crevicular cells showed a value greater than the junctional cells but lesser than the surface cells (9.3 ± 1.73 mV). Conclusion: The membrane transport protein density appears to decrease in the following order: surface > crevicular > junctional cells. The crosslinking force of calcium (Ca 2+ ) ions with their increased magnitude from the junctional epithelial cells to the cementum or enamel of the tooth appears to be the most convincing model of the epithelial attachment at the Dentoenamel (DE) junction.
Keywords: Crevicular epithelium, dentoepithelial junction, gingiva, junctional epithelium, surface epithelium, transmembrane potential
|How to cite this article:|
Grover H S, Dadlani H. Biophysical studies of the gingival epithelium. J Int Clin Dent Res Organ 2011;3:4-17
| Introduction|| |
The epithelial cells cover most of the internal and external surfaces of the body.  Like other cells of the body, the epithelial cells also possess specialized cell to cell junctional structures,  and undergo transition from one type to the other, particularly at the junctions with heterogeneous cells. , Thus, the surface, the crevicular and the junctional epithelia of the gingiva have been found to exhibit important differences between them with respect to their junctional structures. , Since these junctional structures play a crucial role in cell to cell transport of chemicals,  transmission of impulses  and holding the cells together or affecting their attachment to the other tissues, considerable amount of histological  and electron microscopic  investigations have been carried out to explain their nature. The human gingival epithelium has been shown to contain the junctional complexes, such as, the tight junctions, the intermediate junctions, the desmosomes and the maculae occludens (or spots of membrane fusion).  These epithelial junctional structures have been shown to exist as gaps of 100-200 Ε between the juxtaposing membranes of adjacent epithelial cells.  The gingival epithelium on the dento - epithelial junction represents a highly specialized part forming tight attachment between the epithelial cells and enamel or cementum. The importance of this junctional tissue is undoubtedly great because in addition to attaching the gingiva to the tooth, it is a barrier to bacterial infiltration, thus requiring a clear understanding of the nature of this junction. 
Although it has been proposed that an area deprived of definitive epithelial cells called sub-lamina lucida of 95 ± 20 Ε is interposed between the lamina densa of the basal lamina and the dental tissues, the mechanism by which it acts as a junctional "glue" is not yet clear.  It has been proposed that Ca 2+ ions might be playing an important cross - linking role in the sub-lamina lucida  which has been thought to contain extra- cellular "epithelial attachment substance",  and fibronectin-like glycoproteins,  (a special mucous secretion).  Moreover, it is also believed that vander Waals electrostatic forces by themselves or in conjunction with the above "biological cements" were involved in binding one surface to another. 
These newer developments on the dento-epithelial junction have by no means given the final picture of the physic-chemical nature of the epithelial attachment, but provide a strong basis for further investigations. In view of the above lacunae in our knowledge about the nature of the dento-epithelial junction which plays a crucial role in the pathophysiology of the periodontal problems, it was considered to investigate the nature of this junction. ,
A novel biophysical approach has been adopted in the present study to investigate the physico-chemical nature of the dento-epithelial junction. The viewpoint behind this approach is that all the living cells, including the epithelial cells exhibit a transmembrane potential difference due to ionic [eg. Sodium (Na + ), Potassium (K + ) and Ca 2+ ] concentration gradients on either sides of the membrane. , The resting membrane potentials reflecting the membrane electro chemical gradient is determined by the membrane spanning transport proteins, particularly the (Na + -K + ) adenosine triphosphatase (ATPase) pump which selectively transports K + inside the cell and Na + outside the cell against their concentration gradients to establish their electrochemical gradients. , Variations in the cell membrane composition particularly with respect to these transport proteins per unit area of the membrane would therefore play a crucial role in determining the magnitude of resting potential which varies from one cell type to the other. , Since, the gingival epithelial cells undergo transition between the surface, crevicular and junctional areas, it is believed that their resting potential could also alter due to change or variations in the membrane transport protein composition.
The resting potentials can be accurately measured by the glass ultra - microelectrode technique irrespective of the size of the cell,  and it was decided to undertake these measurements in the normal human gingival epithelia. The gingival biopsy material was obtained and the resting potentials measured by the above technique, with or without application of delipidifying agents, Sodium Deoxycholate (DOC) and Sodium Dodecyl Sulfate (SDS). Histological examination of the gingival epithelium was also carried out in the normal samples as well as those treated with the above agents to substantiate, if possible, with the biophysical measurements.
| Aims and Objectives|| |
The aims and objectives of this study were:
- To study the biophysical properties of the surface, the crevicular, and the junctional epithelial cells and to assess their implications to the cell to cell and the cell to tooth attachment mechanisms;
- To study the effect of certain delipidifying agents such as DOC and SDS on the transmembrane electrical activity of the gingival epithelial cells;
- To study the histological changes due to these treatments and assess their bearings on the biophysical observations, if any.
| Materials and Methods|| |
The biophysical and histological investigations on the healthy human gingival epithelium were carried out on the fresh biopsy material obtained from a homogeneous group of willing donors. The biophysical measurement involved recording of the transmembrane potentials using glass ultra-microelectrodes. For histologic studies the H&E staining of the tissue sections were performed. The following are the details of these procedures.
A. Preparations for the tissue biopsy collection:
The material used in this study was obtained from 4 male and 6 female subjects between 11-15 years of age, advised for extraction of first premolar due to orthodontic reasons [Figure 1]. Only those teeth which were having a clinically healthy periodontium were selected. The subjects were oriented to a meticulous oral hygiene maintenance program, and professional brushing was done every alternate day for one week prior to extraction. The samples were collected in the form of biopsies which included the tooth and attached buccal gingiva. Atraumatic extraction techniques were used, preserving the cytological details of the junctional epithelium. Every subject was informed about the type of extraction and a written consent was obtained. Each sample was individually placed in a bakelite screw capped glass vial containing oxygenated Hepes-Tyrode solution. The vial was immediately placed into a thermos-flask containing ice-cubes and transported to the Electrophysiology laboratory of the Central Drug Research Institute, Lucknow, for microdissection, mounting and recording of the cell potentials. Every experiment was thus conducted on a fresh sample.
B. Preparation of the gingival epithelia from biopsy samples and Recording of membrane potentials
Preparation of the gingival epithelial tissue
The tooth with the attached gingiva was placed on the sylgard floor of a dissecting chamber (of perspex), containing oxygenated Hepes-Tyrode solution at room temperature. After separating the junctional epithelium from the tooth with the help of microforceps and stainless steel blade No. 11, the gingiva was pinned to the sylgard floor of the chamber. , The detached gingival tissue was observed under the high power magnification (100x) of the dissecting microscope (Carl Zeiss, West Germany) for the intactness of the epithelial cell layers as evident from the smoothness of the top layers in the crevicular as well as the surface sites. It was noted that the junctional part of the gingiva did not have as smooth a surface as the crevicular part, indicating some rupture of the epithelial layers in this portion. This was also evident from the remaining soft tissue attached to the tooth itself after the mechanical separation of the gingiva. Thus, the chemical separation of gingiva from the tooth was abandoned in favor of mechanical separation as no intact gingival tissue could be retrieved following such a treatment. Thus, the gingival tissue separated mechanically from the tooth by surgical blades as described above, was used in all the experiments. After detaching the gingiva, a small sample containing all the three types of epithelia was dissected out under the dissecting microscope by manipulating the tissue under the microscopic field as required.
Mounting of the preparation for electrophysiological recordings
The dissected preparation was clamped by tungsten pins to the sylgard floor of the lucite electrophysiological chamber, of about 2 ml Capacity, and containing Hepes-Tyrode solution [Table 1]. The epithelial surface to be impaled was kept facing the top. The Hepes-Tyrode solution circulating in the electrophysiological chamber was maintained at 37° ± 0.5 °C, which was preheated in a double jacketted graduated glass container, in the outer jacket of which flowed warm water from a water circulating type 10 V.E.B. thermostat (Ultra thermostat, Prufgerate Werke, DDR). The Hepes-Tyrode solution was oxygenated in the glass container by fine streams of oxygen. The bathing solution was adjusted with the provided regulator to flow into the electrophysiological chamber at 80 ± 5 drops per minute [Figure 2] and [Figure 3]. The preparation was stimulated by suction stimulating electrode which was connected to a Pulse generator (Type-161,Tektronix) and wave form generator (Type-162, Tektronix). The ground electrode for this purpose was placed in the lucite electrophysiological chamber solution, near the other end of the preparation.
Recording of the membrane potentials
The membrane potentials were picked up by 3 M Potassium Chloride filled glass capillary microelectrodes prepared on a CF Palmer H104 microelectrode puller (15 to 30 M Ω resistance and tip potentials < 5mV) and connected to a lucite microelectrode holder containing silver-silver chloride (Ag-AgCl) pellet which in turn was connected to the input stage of a high input impedance microprobe (M701, W.P. Instruments). The microprobe was held on a micromanipulator (Carl Zeiss, Jena). The output of the microprobe was connected to the input of a suitable preamplifier (M701, W. P.1.). An Ag-AgCl pellet electrode placed in the bath and connected to the preamplifier ground, served as the reference (or indifferent) electrode. The output of the preamplifier was connected to one channel of a storage oscilloscope (5113, Tektronix Inc.) to display the cell potentials. The microprobe system and its preamplifier were calibrated using a precision millivolt source (MiMI, W.P.I.). For cellular impalements, the microelectrode tip was placed near the cell surface. The microelectrode tip was then moved using the micromanipulators to impale the cell membrane. The potentials displayed on the oscilloscope screen were recorded on a 35 mm film (Eastman Kodak, USA) by a Kymograph Camera (C4K, Grass Instruments Co). Potentials for all the three areas of gingival epithelium viz, the surface, the crevicular and the junctional epithelium were recorded in the similar fashion. A minimum of 15-20 recordings were done in each experiment. Such 20 experiments including those after DOC and SDS were conducted.
Analysis of the cellular potentials
For analysis of the electrical potentials, the original negatives were magnified by a microfilm comparator (Agil, Agfa Gevaert Co.) and the reference zero potential and the membrane potentials were traced on a graph paper. The values of the resting potentials were read out from these tracings and the time scale of the potentials calculated from the sweep speeds of the original records. Only those observations were taken into account in which a single stable impalement in the cell was maintained throughout the experiment. The DOC and SDS were added cumulatively to the stock Hepes-Tyrode solution in the jacketted glass cylinders to attain the desired final concentrations.
C. Preparation for Histological Studies
Paraffin sections for light microscopy were prepared from both the normal gingival tissue serving as a control as well as the DOC and SDS treated gingival preparations. The temperature of wax bath was maintained at 60°C to keep the paraffin at the melting point. Tissue remained in the wax bath for 8 hours. The tissue sections were stained using the Ehrlich's acid haematoxylin and eosin stains.
Photomicrographs showing different areas of gingival epithelium were taken both under low power (6.3x, 16x) and high power (40x), using a 3.2x eyepiece. The photomicrographs were obtained on an ICM-405, Invertoscope (Carl Zeiss) using a 35 mm, 100 ASA Konica Colour Photographic film.
E. Interpretation of the Data
The electrophysiological observations of the membrane potentials recorded from the same epithelial cells during the course of an experiment were measured as described above. The values for the untreated surface, crevicular and junctional epithelial cells were compared with each other. The higher the potentials, the greater the proportion of the membrane transport proteins was assumed to be. The histological findings were expressed in terms of the variations in the epithelial layers and their staining characteristics on the different areas of gingival epithelium. The effects on them of DOC and SDS were also compared.
F. Statistical Analysis
The formulae used for statistical analysis were:
The level of significance or 'p' values were seen from a 't' distribution table.
| Results|| |
The present study comprised of experiments done on 20 freshly obtained human gingival tissue biopsy samples, several cells having been impaled with the microelectrode in each experiment. The mean crevice depth recorded in the 20 samples was 0.88 mm. The results described here represent those obtained from stable impalements in the same cell. Although biopsies were taken from subjects ranging in age from 11-15 years, belonging to either sex, no classification of results was done on this basis. The experiments including those after DOC and SOS treatment, were conducted on all the three areas of gingival epithelium (the surface, the crevicular and the junctional epithelia), as illustrated in [Table 2].
|Table 2: Group wise distribution of the three areas of gingival|
Click here to view
Control Values of the membrane potentials
The membrane potentials recorded were pooled into each of the above group and the mean values and standard deviation (S.D) of control values are shown in [Table 3] and [Figure 4]. In many of the samples, the membrane potentials of 3 or 4 cells in the same group were recorded. The control values in the various groups were as follows:
- Surface epithelium (Group 'A' n = l3): The membrane potential in the surface epithelial cells (or group 'A') ranged from -16 to -36 mV. The mean value being 22.96 ± 5.19 mV.
- Crevicular epithelium (Group 'B' n = 10): The membrane potential in the crevicular epithelial cells (or group 'B') ranged from -7 to -12.5 mV. The mean values being 9.3 ± 1.73 mV.
- Coronal junctional epithelium (Group 'C' n = 6): The membrane potential in the cells of junctional epitheliurn (or group 'C') ranged from -2 to -4 mV. The mean values being 2.83 ± 0.98 mV.
|Figure 4: Control membrane potentials (Em) in the human gingival epithelial cells|
Click here to view
|Table 3: Showing mean, range and S.D. of transmembrane poten tials recorded in clinically healthy surface, crevicular, and coronal junctional epithelia|
Click here to view
|Table 4: Statistical evaluation of membrane potentials among group 'A' (Surface epithelium), 'B' (Crevicular epithelium), and 'C' (Coronal junctional epithelium)|
Click here to view
It is evident from the mean values of the membrane potential that the values in the three types of the gingival epithelium cells vary appreciably. While the membrane potentials were recorded lowest in the junctional cells (2.83 ± 0.98 mV), it was highest in the surface epithelial cells (22.96 ± 5.19 mV), the crevicular cells showing a value greater than the junctional cells but lesser than the surface cells (9.3 ± 1.73 mV). The difference in the mean membrane potential in the surface, the crevicular and the coronal junctional epithelial cells is illustrated in [Table 4]. Comparison of the mean values of the three groups (Group A Vs. Group B; Group B vs. Group C and Group A Vs. Group C) showed a statistically significant difference (p < 0.001) amongst all the three groups.
Effects of Sodium Deoxycholate (DOC)
- Surface epithelium (Group A, n = 3): The effects of the different concentrations of sodium deoxycholate on the membrane potentials in the surface epithelial cells was expressed in terms of percentage change in the membrane potential at particular concentration of the chemical with respect to the control [Table 5]. It is evident from [Figure 5] that a decrease in the membrane potential or depolarization ranging from -8.3% to -25% (at DOC concentration of lx10 -4 to 2.4x10 -3 M), -9.1% to -31.8% (at DOC concentration of 4x10 -4 to 2.8x10 -3 M), and -10% to -50% (at DOC concentration of 1x10 to 2.8x10 -3 M) were brought about by different concentrations of DOC. This clearly shows that this effect of DOC was largely concentration dependant.
- Crevicular epithelium (Group 'B' n = 3): The effects of the different concentrations of DOC on the membrane potentials in the crevicular epithelial cells were expressed in terms of percentage change in the membrane potentials with respect to the control values at the specific concentrations of the chemical agent [Table 6].
|Figure 5: Membrane potentials (Em) recorded in a surface epithelial cell showing the effects of Sodium deoxycholate (DOC)|
Click here to view
|Table 5: Effects of Sodium Deoxycholate (DOC) on the membrane Potential in the surface epithelial cells|
Click here to view
|Table 6: Effects of Sodium Deoxycholate (DOC) on the membrane Potential in the Crevicularcular epithelial cells|
Click here to view
It is evident from [Figure 6] that an increase in membrane potentials or hyperpolarizations ranging from 28.57% to 57.14% (at DOC concentration of 1x10 -4 to 4x10 -4 M), 40% to 83% (at DOC concentration of 4x10 -4 to 4.4x10 -3 M) and 27.27% to 18.18% (at DOC concentration of 1x10 -4 to 4.4x10 -3 M), were observable with respect to the control. Thus, the most prominent effect of the DOC on the crevicular epithelial cells was an increase in the membrane potentials up to a concentration of 2.4 x 10 -3 M (54.54%). The potential started decreasing on further increase of concentration (18.18%) at 4.4 x 10 -3 M. Further increase in the concentration of the chemical agent or 1.01 x 10 -2 M showed a depolarization of -45.45% with respect to the control.
|Figure 6: Membrane potentials (Em) recorded in a crevicular epithelium cell showing the effects of Sodium Deoxycholate (DOC)|
Click here to view
Effects of Sodium Dodecyl Sulfate (SDS)
- Surface epithelium (Group A, n = 3): The effects of the different concentrations of SDS on the membrane potential in the surface epithelial cells were expressed as the percentage change in the membrane potentials with respect to the control values at the specific concentrations of the chemical agent [Table 7]. SDS showed [Figure 7] a mixed response in the surface epithelial cells. On adding the very first dosages of the agent which were 5x10 -3 M, 4x10 -4 M and 1x10 -4 M respectively in the three experiments conducted, it showed an increase in the membrane potentials or hyperpolarizations of 100%, 55% and 20%. However, a decrease in the membrane potentials or depolarization of about 50% at concentrations between 1.2x10 -3 to 2.8x10 -3 M of SDS and about 24% at the same concentration range were observed in the second experiment with SDS. In response to 6.0x10 -3 M SDS, the cells again showed a hyper polarization of about 4% with respect to the control membrane potentials.
- Crevicular epithelium (Group B, n = 2): The effects of the different concentrations of SDS on the membrane potentials in the crevicular epithelial cells were expressed as the percentage change in the membrane potentials with respect to the control at the specific concentrations of the chemical agent [Table 8]. SDS on the crevicular epithelial cells showed a mixed response [Figure 8]. On adding first two dosages of SDS (concentration 1x10 -4 and 4x10 -4 ) there was a decrease in the membrane potentials of -22.22% and -44.44%, with respect to the control showing cellular depolarization. The other three concentrations of 1.2x10 -3 M, 2.8x10 -3 M and 6.0x10 -3 of SDS however, showed an increase in the membrane potentials (or hyperpolarization) of 72.22%, 44.44% and 22.22% in the first experiment and 40%, 30% and 10% respectively, in the second experiment.
|Figure 7: Membrane potentials (Em) recorded in a surface epithelial cell showing the effects of Sodium dodecyl Sulfate (SDS)|
Click here to view
|Figure 8: Membrane potentials (Em) recorded in a crevicular epithelium cell showing the effects of Sodium dodecyl Sulfate (SDS)|
Click here to view
|Table 7: Effects of Sodium Dodycl Sulfate(SDS) on the membrane Potential in the surface epithelial cells|
Click here to view
|Table 8: Effects of Sodium Docecyl Sulfate (SDS) on the membrane potential in the crevicular epithelial Cells|
Click here to view
Histological examination of the clinically normal gingival tissue serving as control as well as the DOC and SDS treated gingival preparations was carried out under light microscope, both at high (128x) and low (28x and 50x) magnifications.
Epithelial structure in control tissue: [Figure 9]
The H and E stained paraffin sections of the control (normal), untreated gingival tissue showed the epithelial structures covering the light pink stained contents of the lamina propria. Two areas of gingival epithelium, clearly identifiable in these samples were the surface and the crevicular epithelia. The surface epithelium shows the presence of rete pegs, whereas the crevicular epithelium is devoid of them. The junctional epithelial cells could not be identified as a specific, separate entity. The nuclei appear purplish whereas the cytoplasmic contents had taken up a pink color in both the types of epithelial cells.
- Surface epithelium: [Figure 10] It appears as a stratified squamous epithelium having different, clearly identifiable layers of distinct groups of cells. The stratum basale, the bottom most layer, is composed of cuboidal or columnar cells. From inside out it is followed by stratum spinosum having polygonal cells, stratum granulosum, consisting of flattened cells and the outermost superficial layer that might be keratinized or parakeratinized. The nuclei having taken haematoxylin stain appear blue. The cytoplasm appears pink with eosin stain. The rete pegs are clearly identifiable in the surface epithelium. The intercellular spaces are also apparent in the stratum spinosum and stratum granulosum.
- Crevicular epithelium: [Figure 11] It appears as a thin non keratinized stratified squamous epithelium without rete pegs. Stratum basale is not seen in the figure but stratum intermedium and stratum superficiale are seen with purplish nuclear components and pink cytoplasmic contents present in the scale like cells. Darker staining membrane areas are also seen in the intercellular sites between the adjacent cells, especially in stratum spinosum.
|Figure 9: Photomicrograph of normal gingiva showing (hematoxylin and eosin stain 20x) S – Surface Epithelium, C – Crevicular epithelium, LP – Lamina Propria|
Click here to view
|Figure 10: Cell layers of the surface epithelium as observed in the photomicrograph of normal gingiva in the study (hematoxylin and eosin stain, 128x), SC – Stratum corneum, SG – Stratum Granulosum, SS – Stratum Spinosum, SB – Stratum Basale|
Click here to view
|Figure 11: Cell layers of crevicular epithelium (hematoxylin and eosin stain, 128x), SI – Stratum Intermedium, SS – Stratum Superfi ciale|
Click here to view
Epithelial structure after treatment with Sodium Deoxycholate (DOC)
The gingival tissue treated with DOC ( 4.4x10 -3 M), showed following structural details in their H and E stained sections:
- Surface epithelium [Figure 12]: In contrast to the control tissue, the cells of stratum corneum in DOC treated prepartions appeared as a homogeneous orange-pink stained layer in which no structural details of the individual cell were discernible. The cells of the stratum granulosum took a dark purplish stain, those of the stratum spinosum and the stratum basale having been stained a light purple. The intercellular spaces took lighter staining compared to those of the control tissue and appear prominently both in the stratum spinosum and stratum granulosum. The nuclear components appear dark purple.
- Crevicular epithelium [Figure 13]: No crevicular epithelium could be seen in the section of the DOC treated gingiva. The connective tissue was dark pink in these sections in contrast to the light pink staining in the control tissue. It also showed wide spaces in between the fibers and loosening of the connective tissue elements.
|Figure 12: Photomicrograph of normal gingiva (hematoxylin and eosin stain,128x) treated with 4.4 x 10-3 M Sodium Deoxycholate (DOC) showing the surface epithelium, SC – Stratum corneum, SG – Stratum Granulosum, SS – Stratum Spinosum, SB – Stratum Basale|
Click here to view
|Figure 13: Photomicrograph of normal gingiva (hematoxylin and eosin stain, 128x) treated with 4.4 x 10-3 M Sodium Deoxycholate (DOC) showing the loosening of connective tissue elements|
Click here to view
Epithelial structure after treatment with Sodium Dodecyl Sulfate (SDS)
The gingival tissue treated with SDS (5.0 x 10 -3 M) and processed for H and E staining exhibited the following structural features under optical microscopic examination:
- Surface epithelium [Figure 14]: There is no differentiation between stratum corneum and stratum granulosum in these sections of gingiva. The individual cells in the stratum granulosum are not as clearly identifiable as in the control tissue and are characterized by a hazy pink staining. The intercellular spaces in stratum spinosum appear as lightly stained areas between the individual cells which took up a purplish pink stain. The stratum basale, although not as clearly distinguishable as in the control tissue appears to be composed of light purplish pink stained cells.
- Crevicular epithelium [Figure 15] Distinct crevicular epithelial cellular layers were not seen in the sections of the gingival treated with SDS. The connective tissue or lamina propria could however be identified in these sections as collagen fibers which took a reddish-pink stain.
|Figure 14: Photomicrograph of normal gingiva (128x) treated with 5.0 x 10-3 M Sodium dodecyl Sulfate (SDS) showing the surface epithelium, SC – Stratum corneum, SG – Stratum Granulosum, SS – Stratum Spinosum, SB – Stratum Basale|
Click here to view
|Figure 15: Photomicrograph of normal gingiva (50x) treated with 5.0 x 10-3 M Sodium dodecyl Sulfate (SDS) showing, C – Crevicular epithelium, LP – Lamina Propria|
Click here to view
| Discussion|| |
The present study deals with the biophysical and structural features of the gingival epithelium with an aim to study the nature of the dento-epithelial junction using techniques of measuring their membrane potentials.
Suitability of the biopsy material
The tissues used for recording the membrane potentials and for histological examinations were from normal healthy gingival biopsies from the subjects who were advised extraction of first premolar because of orthodontic reasons. The gingiva was supposed to be healthy in these subjects because the teeth were not being extracted due to any periodontal, periapical or carious involvement which is reported to lead to changes in the gingival epithelium. , In order to bring down subclinical inflammation, if any, professional brushing by Bass brushing technique  was performed every alternate day for one week prior to the extraction. The mean crevice depth recorded in the 20 subjects from whom the biopsy materials were obtained for experimentation were 0.88mm. This is within the range considered to represent the normal values for normal healthy human gingiva. , The biopsy procedure used in study ensured that the cytological details of all the portions of gingival epithelium were well preserved and within physiological limits. , This assumption was strongly supported by the histological and biophysical studies of the epithelia of these samples. Separation of the gingiva from the tooth could be carried out by the mechanical or chemical methods. The relative suitability of these techniques for retrieving the junctional epithelia intact and in good condition as well, was tested initially. The chemical method of separation by treatment with 4 gm% Sodium deoxycholate which had been suggested by Buchanan and Bernard (1978)  as effective chemical agent for the purpose of removing the junctional epithelium cleanly from the tooth, caused rapid and drastic flaking off of the gingival tissue, denuding it of its epithelial cells. At lower concentrations (< 0.5 gm%), DOC failed to isolate the intact gingiva from the tooth at the junction. As such this chemical technique of isolating the gingival junctional epithelium was found to be extremely ineffective for obtaining healthy junctional epithelial cells in this study. Therefore alternatively, mechanical separation by inserting thin steel blades into the gingival crevice as reported by Orban et al. and Weinreb  was used. By this method although the outermost junctional cells forming the dento-epithelial attachment could perhaps not be obtained, the remaining few cell layers especially of the coronal junctional epithelium which came off with the soft tissue could well be taken as representative of the membrane specializations in this area. The histological examination of gingiva obtained from these biopsy samples showed that the epithelium and the subjacent connective tissue exhibited normal staining characteristics and structural features without any sign of inflammation or injury. This finding shows clearly that the gingival epithelial cells investigated for their biophysical properties represented normal healthy cells.
Biophysical properties of the epithelial cells and their modification by delipidifying agents
Resting membrane potential of epithelial cells: Like all living cells, epithelial cells also have an ionic concetration gradient across the cell membrane because of Na + , K + and Ca 2+ concentrations, whose transportation in and out of the cell is the function of transmembrane integral proteins.  In other words the resting membrane potential is a direct indication of the lipid : transport protein ratio in the particular epithelial cell membrane. , The membrane potentials in the epithelial cells could be expressed with the following equation  which is based on the assumption that the epithelial cell membrane contains the transport proteins represented by the ionic pump for eg. Na + , K + - ATPase.
Here, Vm is the measured membrane potential; E K and E Na the Nernst potential for K + and Na + ; R K and R Na , the resistance and I p the pump current. Inhibition or activation of this ionic pump is considered to alter the membrane potential. The resting membrane potential reported for the various inexcitable cells such as the epithelial cells , and erythrocytes  is quite low. The membrane potential recorded in the three types of gingival epithelial cells in the present study was also found to be quite low, ranging between -2 to -36 mV. It may however be emphasised that the values recorded for the three types of epithelial cells exhibited statistically significant difference (p < 0.001); [Table 3] and [Table 4]. The resting membrane potential was lowest in the junctional cells, higher in crevicular cells and highest in the surface epithelial cells. These differences in the membrane potentials of different areas of gingival epithelium may well be related with their biochemical composition and functional specialization. As the membrane potentials are a function of the density of the membrane transport proteins, particularly the ionic pumps,  it may be suggested that the ratio of these proteins to the membrane lipids is reduced in the order surface > crevicular > junctional. In view of the above equation given by Lewis and Wills,  the cell membrane resistances, R K and R Na would consequently be highest for the cells of the junctional epithelium and lowest for the surface epithelium, the values for the crevicular ones lying in between.
Cell to cell junction in gingival epithelia
Inspite of a great importance of the cell to cell junctions in epithelial structures in general,  not much information is was available on the physio-chemical aspects of these junctions in gingival epithelial tissue. An attempt was made in the present study to stimulate the epithelial cells at one point and study the electronic spread of the wave of excitation. Since these cells are inexcitable in nature, no action potential could be recorded* and therefore the spread of the wave of excitation could also not be measured. Although, it is likely that must have happened due to relative lack of gap - junctions in the gingival epithelial cells. A short circuit current measurement will have to be carried out to obtain conclusive information in this aspect. 
[*Note: Since no action potentials were experimentally observable following electrical stimulation of the gingival epithelial cells this portion has not been included in the results.]
Effects of Sodium Deoxycholate (DOC):
DOC, an anionic detergent, when used in lower concentrations alters the membrane permeability to certain ions.  Since, it also acts on the Ca 2+ channels in excitable cells and since Ca 2+ has been implicated in the epithelio-epithelial junctions , and the dentoepithelial junction of the gingiva,  it was tested for its effects on the membrane potentials of these cells. The results indicated that DOC caused a decrease in the membrane potential or depolarization of the surface epithelial cells but had reverse effects on the crevicular ones, where it caused increase in membrane potentials or hyperpolarization. It is likely that these effects are due to changes in the membrane permeability to Na + , K + or even Ca 2+ ions. The decrease in the membrane potential of the surface epithelial cells could be due to a leakage of K + from inside or due to inhibition of the Na + K + pump resulting into an increase in R K and R Na of the epithelial cell membranes (equation mentioned above). Increase in the membrane potential of the crevicular epithelium in the same concentration as those employed in the surface epithelium is indicative of increase in the K + permeability and decrease in the R K in these cells. The most likely site of action of DOC for these effects is again on the Na + , K + - ATPase. It may however be pointed out that the maximal hyperpolarization of these cells does not go beyond the normal membrane potential of the surface epithelium cells as evident from [Table 5] and [Table 6]. Moreover, the higher concentration of DOC (l.01x10 -2 M) also caused a depolarization of the crevicular cells which had been previously hyperpolarized by its lower concentrations. This shows that the membrane permeability of the crevicular epithelial cells was increased by DOC to reach the values closer (but not equal) to the resting values of the surface epithelial cell, thereafter bringing about similar changes in the two types of epithelia. The effects of DOC on the junctional epithelial cells were not studied because they showed very low membrane potentials. Such low potentials are strong indications that the values of R K and R Na are tremendously high in these cells. This could be due to low density of the transport proteins in the membranes of these cells or due to extreme rigidity of the cell membrane in these regions.
Effects of Sodium Dodecyl Sulfate (SDS)
SDS is a detergent used commonly in the dentifrices and was studied to see its effect on the gingival epithelial cells. It caused a biphasic change in the membrane potentials of both the surface and crevicular epithelial cells, depending upon its concentration and duration of application. Unlike, DOC it caused an increase in the membrane potentials on surface epithelial cells following initial application indicating cellular hyperpolarization. The higher concentrations applied subsequently however caused depolarization [Table 7] in the same cells as evident from reduction in the membrane potential. Since, a change in the membrane potentials is a function of alterations in the membrane permeability to ions, it is likely that SDS increases the pump activity reducing the value of R K and causing an increase in the membrane potential.
In the crevicular cells however SDS caused a reduction in the membrane potential subsequent to its initial application followed by an increase in these values as evident from cellular depolarization and hyperpolarization respectively [Table 8]. On the basis of the above points of view it may again be suggested that SDS brought about these changes by altering the membrane permeability for the K + ions. In this case the change in the membrane potentials was in the opposite direction as compared to the surface epithelium. It may be pointed out that inspite of somewhat complex effects produced by SDS on the membrane potential of the different types of gingival epithelial cells', the values never crossed the maximal limits of transmembrane potentials observed in epithelial cells. Such change in membrane potentials appear to be certainly due to the limited number of transport proteins in these cell membranes. The above findings on the effects of SDS on membrane potentials when considered together with those of DOG show definitely that the gingival epithelial cells have by nature low membrane potentials which cannot be altered beyond certain values even by treatment with these delipidifying agents. This could be best explained by the limiting effect of sparsely distributed ionic pump proteins in the epithelial cell membranes. , It may further be added that SDS does not cause any deleterious effects on the physiological properties of the gingival epithelial cells and could safely be used.
Histological evidences for the differential structure of the gingival epithelium
It is evident from the histological studies that the gingival epithelium of the biopsy samples retained its structural integrity although the junctional epithelium could not be identified as a specific, separate entity at the magnifications at which the photomicrographs were taken in the present study. The multilayered epithelium is widest on the surface side; narrower in crevicular portion and tapers down to very thin layers in the junctional portion, retaining their staining properties to haematoxylin and eosin.
Effects of delipidifying agents
Deoxycholate when applied in higher concentrations of 4.4 x 10 -3 M caused no structural change in the surface epithelium although the crevicular epithelium was completely denuded. This is due to the strong detergent properties of this agent which in the above concentration range is reported to cause severe loss of cellular components in the soft tissue.  Since, the surface epithelium was still intact under these conditions inspite of some changes in the staining characteristics of their cellular components, it appears that the whole of the epithelium undergoes gradual change in the physico-chemical properties from the surface to the junctional portion. One possibility in this respect is that the surface epithelium is covered with stratum corneum which might be preventing rapid diffusion of DOG in the deeper cells. ,, It is further substantiated by the fact that the surface epithelial cells treated with these concentrations of DOG were still showing membrane potentials in the physiological limits. The question of how membrane potentials could be recorded from crevicular epithelial cells in presence of DOC which denuded them can be satisfied by assuming that the epithelial cell being studied remain attached to the microelectrode tip even after the neighboring cells softened and flaked off. In contrast to the tissue treated with DOC, SDS application did not appear to cause any structural damage in the epithelial cells. The staining characteristics of the surface as well as crevicular epithelium were however altered marginally following application of higher concentrations of SDS. This provides additional evidence to support that SDS does not have deleterious effects on the gingival cells.
Biophysical nature of the Dento-epithelial (DEJ) junction
Several hypothesis have been put forward to explain the precise nature of the interaction between the junctional epithelial cells with the cementum or enamel of the tooth. ,, None of them have however, provided a final answer due to a lack of understanding about the physico-chemical characteristics of the epithelial cells and the tooth components immediately involved in such junction. It is generally believed now that there is a thin layer of relatively electron opaque components in osmium tetraoxide stained electron micrographs of the DEJ which plays a crucial role in the formation of this junction.  According to them, this layer represents the "interplay of electrostatic forces of repulsion and a force of attraction arising from the van der Waals dispersion force", multiple hemidesmosomes playing an important role in these adhesions.  According to this hypothesis, the hemidesmosomes act as pegs to which the sheet of junctional epithelial cells is anchored, leaving an acellular space called sub-lamina lucida (95 ± 20 Ε) immediately at the junction. This hypothesis does not however exclude the possibility of a 'cross linking' role of Ca 2+ ions between the adhering molecules at this junction (Culp, 1974) 
It also does not dispense with the possibility that a fluid filled mucous secretion  or serum proteins  could be present as important junctional components on the dento-epithelial junction. The question of whether some microexudates  from the basal layers of junctional epithelial cells are crucial in providing the necessary force of attraction between the epithelium and the hard dental tissues presents an interesting possibility requiring further investigation. The findings of the present investigation clearly demonstrate that the junctional epithelial cells have extremely low membrane potentials due to sparse membrane transport proteins. Whether such cells could be able to secrete mucous or protein substances suggested by some studies , appears to be a remote possibility because most of the secretory epithelial cells in the body have higher membrane potentials  indicating greater metabolic activity. 
The next point of whether the negative charge on the isolated human gingival cells studied by electrophoretic mobility technique  could represent the physicochemical properties of the junctional epithelial cells requires further elucidation. Present findings have clearly demonstrated that trans-membrane potential and therefore the membrane transporting protein densities per unit area of the membrane are substantially different between the surface, crevicular and junctional epithelium. The very low values obtained for the junctional epithelium indicate that they have undergone drastic reduction in the "fluid-mosaic" nature of cell membrane.  The membrane rigidity thus attained by the junctional cells could be an indication of formation of stronger bonds between the elements of the membrane of these cells and the adjoining cementum or enamel.
The occasional junctional epithelial cells recovered from the gingival sulcus where they are shed, , are those coming out from the layer of the junctional epithelium immediately in contact with the hard dental structures by a lateral movement along the tooth. A detailed study of the physico-chemical characteristics of these shed junctional epithelial cells could provide valuable information on this hypothesis. In addition to these aspects an investigation of the transepithelial potential in different regions of the gingival epithelium will throw valuable information about the physic-chemical and functional characteristics of these cells as has been done for the other epithelial cells of the body. 
| Conclusions|| |
The transmembrane potentials of epithelial cells in different regions of the gingiva exhibit extremely low potentials ranging between -2 to -36 mV. The magnitude of mean potential difference follows a descending order at surface epithelium (22.9 mV), crevicular epithelium (9.3 mV), and junctional epithelium (2.8 mV). The membrane transport protein density also appears to decrease in the similar order surface > crevicular > junctional cells. But the membrane rigidity and resistance to transport of K + and Na + ions increased in the reverse order; junctional > crevicular > surface. In all the experiments carried out the gingival epithelial cells were inexcitable in nature as they did not elicit any action potential. DOC could depolarize the surface epithelial cells and hyperpolarize of the crevicular ones. SDS showed a biphasic effect of depolarizing and hyperpolarizing the surface and crevicular epithelial cells. DOC brought about removal of the epithelial cell layers from crevicular epithelium and has no effect on the surface epithelium. DOC did not show have any deleterious effect on any of the epithelial cells. The crosslinking force of Ca 2+ ions with their increased magnitude from the junctional epithelial cells to the cementum or enamel of the tooth appears to be the most convincing model of the epithelial attachment at the DE junction.
| References|| |
|1.||Cormack DH. Ham's Histology. 9 th ed. Philadelphia: JB Lippincott Co; 1987. |
|2.||Raleigh DR, Boe DM, Yu D, Weber CR, Marchiando AM, Bradford EM, et al. Occludin S408 phosphorylation regulates tight junction protein interactions and barrier function. J Cell Biol 2011;193:565-82. |
|3.||Schild L. The epithelial sodium channel and the control of sodium balance. Biochim Biophys Acta 2010;1802:1159-65. |
|4.||Karpushev AV, Levchenko V, Ilatovskaya DV, Pavlov TS, Staruschenko A. Novel role of Rac1/WAVE signaling mechanism in regulation of the epithelial Na+ channel. Hypertension 2011;57:996-1002. |
|5.||Schroeder HE, Theilade J. Electron microscopy of normal human gingival epithelium. J Periodont Res 1966;1:95-119. |
|6.||Thilander H, Bloom GD. Cell contacts in oral epithelia. J Periodont Res 1968;3:96-110. |
|7.||Dewey MM, Barr L. Intercellular connection between smooth muscle cells: The Nexus. Science 1962;137:670-2. |
|8.||Farquhar MC, Palade GE. Junctional complexes in various epithelia. J Cell Biol 1963;17:375-412. |
|9.||Geisenheimer J, Han SS. A quantitative electron microscopic study of desmosomes and hemidesmosomes in human crevicular epithelium. J Periodontol 1971;42:395-405. |
|10.||Buchanan W, Bernard GW. A technique for the removal of junctional epithelium from the epithelial attachment. J Dent Res 1978;57:895-8. |
|11.||Kobayashi K, Rose GG, Mahan CJ. Ultrastructure of the dentoepithelial junction. J Periodont Res 1976;11:313-30. |
|12.||McKinney RV Jr, Steflik DE, Koth DL. Evidence for a junctional epithelial attachment to ceramic dental implants. A transmission electron microscopic study. J Periodontol 1985;56:579-91. |
|13.||Dorfman JD. Surface energy effects of implant bio-materials on the implant tissue interface: Implications for the rate, character and quality of post-surgical healing. J Oral Implant 1986;12:661-72. |
|14.||Marikova Z. Ultrastructure of normal and newly formed dento-epithelial junction in rats. J Periodont Res 1983;18:459-68. |
|15.||Katz B. Nerve, muscle and synapse. New York City, U.S: McGraw Hill Inc; 1966. |
|16.||Lewis SA, Wills NK. An electrogenic sodium pump in a mammalian tight epithelium. In current topics in membranes and transport, Vol. 16, Waltham, Massachusetts: Academic Press; 1982. |
|17.||Skou JC. The (Na+K+) activated enzyme system and the relationship to transport of sodium and potassium. Q Rev Biophys 1975;7:401-34. |
|18.||Sperelakis SN. Origin of the cardiac resting potential. In: Berne RM, Sperelakis N, Geiger SR, editors. The handbook of physiology Section 2, The cardiovascular system Section 1. Minnesota, Springer: American Physiological Society; 1979. p. 187-267. |
|19.||Bhatnagar A, Tripathi ON. A comparative study of the (Na+ K+) ATPase activity in rabbit SA node and atrial tissue. Med J Biochem Biophys 1988;40;806-8. |
|20.||Purves RD. Microelectrode methods for intracellular recording and ionophoresis. Waltham, Massachusetts: Academic Press; 1981. |
|21.||Box HK. Treatment of the periodontal pocket. Toronto: The University of Toronto Press; 1928. |
|22.||Orban B, Kohler J. The physiologic gingival sulcus. Z Stomatol 1924;22:353. |
|23.||Listgarten MA. Electron microscopic study of the gingiva-dental junction of man. Am J Anat 1966;119:147-78. |
|24.||Schroeder HE, Listgarten MA. Fine structure of the developing epithelial attachment of human teeth. In: Monographs in Developmental Biology. Wolsky A, editor. Vol. 2. S. Basel: Karger; 1977. p. 23-36. |
|25.||Gibson JA, Wade AB. Plaque removal by the Bass and Roll brushing techniques. J Periodontol 1977;48:456-9. |
|26.||Orban B, Bhatia H, Kollar JA, Wentz FM. The epithelial attachment (The attached epithelial cuff). J Periodontol 1956;27:167-80. |
|27.||Weinreb HM. The epithelial attachment. J Periodontol 1960;31:186-96. |
|28.||Lindemann B. Impalement artifacts in microelectrode recordings of epithelial membrane potentials. J Biophys 1975;5:1161. |
|29.||Baker GF, Shukla H. A method for studying the electrophysiology of isolated epithelial cells. Physiol 1985;6:362-6. |
|30.||Hoffman F, Laris PC. Determination of membrane potentials in human and amphiuma red blood cells by means of a fluorescent probe. J Physiol 1974;239:519-52. |
|31.||Neilsen R. A couple electrogenic Na+K+ pump for mediating transepithelial sodium transport in frog skin. In current topics in membranes and transport. Vol. 16. Waltham, Massachusetts: Academic Press; 1982. |
|32.||Tripathi ON, Ray M, Bajpai VK, Bhatnagar A, Kumar R, Singh YK, et al. Effects of Sodium deoxycholate on mechanical and electrical activities and ultra- structure of guinea pig atria. Biomedica Biochem Acta; 1988;47:901-14. |
|33.||Culp LA. Substrate attached Glycoproteins mediating adhesion of Normal and virus transformed mouse fibroblasts. J Cell Biol 1974;63:71-83. |
|34.||Curtis AS. Cell contact and adhesion. Biolog Rev 1962;37:82-129. |
|35.||Dewey MM, Barr L. A study of the structure and distribution of the nexuses. J Cell Biol 1964;23:553-86. |
|36.||Curtis AS. The mechanism of adhesion of cells to glass - A study by interference reflection microscopy. J Cell Biol 1964;20:199-215. |
|37.||Flaxman BA, Lutzner MA, Van Scott EJ. Ultra-structure of cell attachment to substratum in vitro. J Cell Biol 1968;36:406-10. |
|38.||Slayman CL. Current topics in membranes and transport. Vol. 16. Waltham, Massachusetts: Academic Press; 1982. |
|39.||Lorento de No R. Membrane potential of frog nerve. Vol. 131. New York Stud Rockefeller Inst Med Res Repr.; 1947. p. 1-113. |
|40.||Neiders ME, Weiss L. The electric charge at the surfaces of isolated human epithelial gingival cells. J Periodontol 1971;42:761-5. |
|41.||Singer SJ, Nicolson GL. The fluid mosaic model of the structure of cell membranes. Science 1972;175;720-31. |
|42.||Beagrie GS, Skougaard MR. Observations on the life cycle of the gingival epithelial cells of mice as revealed by autoradiography. Acta Odontol Scand 1962;20:15. |
|43.||McHugh WD, Zander HA. Cell division in the periodontium of developing and erupted teeth. Dent Pract 1965;15:451. |
[Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5], [Figure 6], [Figure 7], [Figure 8], [Figure 9], [Figure 10], [Figure 11], [Figure 12], [Figure 13], [Figure 14], [Figure 15]
[Table 1], [Table 2], [Table 3], [Table 4], [Table 5], [Table 6], [Table 7], [Table 8]