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The origin of gingival fluid
J. fheor. Biol. (1974) 47, 127-136
The Origin of Gingival Fluid? MICHAEL C. ALFANO$ Oral Science Research Laboratories, Department of Nutrition and Food Science, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139, U.S.A. (Received 26 September 1973, and in revised form 28 January
1974)
Although the existence of gingival crevicular fluid, a fluid which exudes from the crevice between the gingiva and the tooth, has been recognized for decades the origin, function, and composition of this fluid has been the subject of much controversy. In fact, it is unclear whether this fluid results from physiological or pathological processes. This confusion has arisen because by certain parameters (protein concentration) the fluid resembles a physiological transudate, while by others (Na+/K+ Ratio) it appears to be an inflammatory exudate. This report describes a theory which explains the above and other controversies relating to the origin of gingival fluid. It is based on the premise that gingival fluid may arise by two distinct mechanisms: the generation of a standing osmotic gradient, and the initiation of classical inflammation. The osmotic gradient is generated by macromolecular by-products of the bacteria which reside in the subgingival dental plaque. These macromolecules diffuse through the gingival crevicular epithelium to the basement membrane, a structure which restricts further penetration. Consequently, these macromolecules accumulate at the basement membrane resulting in a localized increase in solute concentration, and the establishment of an osmotic gradient. Solvent molecules, drawn across the basement membrane by this gradient, will raise the intercellular hydrostatic pressure and cause the exudation of gingival fluid. The fluid produced by this mechanism may originate from gingival tissues which are clinically and histologically healthy, If the bacterial plaque is not removed, its macromolecular byproducts will eventually penetrate the basement membrane. Depending upon the enzymatic, toxic, and antigenic properties of these molecules, a classical inflammatory exudation may be initiated. Therefore, gingival fluid may progress, at different times or in various areas of the mouth, from an initial osmotically modulated exudate to a secondary inflammatory exudate, with consequent alterations in its composition. Although the concepts developed in this report focus on the origin of gingival fluid, they may be applied to other biological phenomena, such as, the origin of exudate in the lungs during respiratory infection. tThis investigation was supported by USPHS Training Grant No. DE 105-9. Thii is contribution No. 2215 from the Department of Nutrition and Food Science,Massachusetts Institute of Technology. $ Present address: Department of Periodontics and Oral Medicine, Fairleigh Dickinson University, School of Dentistry, Hackensack, New Jersey 07601, U.S.A. 127
128
M.
C.
ALFANO
1. Introduction
Many controversies have arisen in the last decade concerning the origin, composition, and function of the gingival fluid. Perhaps the most fundamental unresolved problem is whether this fluid is the result of physiological or pathological processes. This controversy is more than academic in interest. If the flow of crevicular fluid is a physiological host defense mechanism, efforts might be directed to increase the rate of flow, thereby enhancing the host defenses in the gingival crevice. In contrast, if this flow originates from inflammatory processes, it could provide a useful criterion for diagnosing incipient gingivitis (Lee & Holm-Pedersen, 1965). Most of the previous investigations have indicated that crevicular fluid is present in healthy gingival tissues (Brill & Krasse, 1958; Brill & Bjorn, 1959; Browne, 1962; Egelberg, 1963; Browne, 1964; Bader & Goldhaber, 1966; Weinstein, Mandel, Salkind, Oshrain & Pappas, 1967). These studies have employed a variety of sampling and tracer techniques, and constitute significant evidence indicating that crevicular fluid arises from normal physiological processes. Liie & Holm-Pedersen (1965), however, reported that crevices of normal human gingiva do not exhibit flow of fluid. They attributed this discrepancy to the fact that rigorous criteria to determine gingival health as well as minimally traumatic sampling techniques were employed in their study. In addition, they suggested that the gingival fluid is an inflammatory exudate but, if it is present prior to clinically detectable signs of inflammation, it would appear to be derived from healthy gingival tissue. This interpretation is supported by the observations of Krasse & Egelberg (1962) and Egelberg (1963), that both the ionic and cellular composition of fluid from healthy gingiva are comparable to that found in inflammatory exudates. Based on the investigations discussed above, it is apparent that a reasonable argument could be made that gingival fluid originates either as a physiological transudate or an inflammatory exudate. However, certain facts are inconsistent with both of these explanations. For example, although the gingival fluid has an ionic composition comparable to an inflammatory exudate (Krasse & Egelberg, 1962), its protein composition is considerably lower than one would expect in an inflammatory exudate (Weinstein et al., 1967; Robbins, 1967). Also, Lee & Holm-Pedersen (1965), using a human model of experimental gingivitis, reported that the initial flow of gingival fluid preceded the onset of clinically detectable gingivitis by several days. Considering that in the classical inflammatory response, vascular dilatation and increased blood flow precede the exudation of fluid (Robbins, 1967), it is difficult to explain a delay of several days between the initiation of gingival exudation and the onset of gingivitis. These inconsistencies suggest that a
ORIGIN
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process distinct from either simple filtration or inflammatory exudation may be responsible for the production of crevicular fluid in healthy gingiva. The purpose of this report is to describe a mechanism, based on general concepts of fluid transport across epithelial membranes, which can explain these inconsistencies and may lead to a better understanding of the origin and function of gingival fluid. 2. Transepithelial
Fluid Movement
Several processes have been postulated by which fluid may be transported across epithelial membranes. These processes include hydrostatic filtration, pinocytosis, active transport and classical osmosis. Although each of these mechanisms are possible, they cannot explain certain characteristics peculiar to transporting epithelium. For example, classical osmosis could not account for the production of isomotic secretions such as bile, cerebrospinal fluid, or pancreatic juice; also, the transport of fluid by pinocytosis is inconsistent with the high degree of specificity observed when different compounds are tested for their ability to cross epithelia (Berridge & Oschman, 1972). Thus one might conclude that the secretion of most epithelial membranes must result from complex interactions of the above processes. Recently, Diamond & Tormey (1966qb) have developed a simple model for fluid transport which explains many confusing morphological and physiological characteristics of transporting epithelial surfaces. This model is based upon their observations on the morphological changes which occur during the in vitro transport of water across rabbit gallbladder epithelium (Diamond & Tormey, 1966a). Basically they noted that, under conditions in which water transport was maximal, the intercellular spaces were widely distended; when water transport was either partially or completely inhibited, the spaces either decreased in width or closed down completely. To explain this phenomenon, Diamond & Tormey (19663) postulated that there is a standing osmotic gradient within the intercellular spaces (Fig. l), with the highest concentration of the solute located at the closed apical end of the intercellular channel. This gradient is maintained by the active transport of solute into the closed end of the channel. Therefore, water will be drawn osmotically into the intercellular spaces and out the open end of the channel. As the water proceeds through the channel, the osmolarity is continually reduced so that the emerging solution is isotonic. The intercellular route for fluid transport has been demonstrated on living tissues including isolated renal collecting tubules (Grantham, Canote, Burg & Orloff, 1969), toad bladder (Grantham, Cuppage & Fanestil, 1971), and insect rectum (Wall, Oschman & Schmidt-Nielsen, 1970). Moreover, micro9 T.B.
130
M.
C.
ALFANO
> !t?qM “:
Solute.:,/:.:: pumps .;
..---~-
.
,-
Water
-.
flow
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,’
,, _
I.
)‘:. ,
’
I” __..
‘:
1.; I..
‘/:
i.. ~--I/’
‘..
.-.
‘.
‘.’
..’
FIG. 1. Standing-gradient osmotic flow: a model for fluid transport across epithelia. Solute is pumped into the closed end of a long and narrow channel making it hypertonic and pulling in water osmotically. Osmotic equilibrium is progressively approached as water enters along the length of the channel until the emergent solution is isotonic. (Reproduced by permission of the publisher. Diamond & Tormey. Fed. Pruc., 25: 14.58-1463, 19666.)
puncture samples from the intercellular spaces of insect rectum (Wall et al., 1970) as well as measurement of streaming potentiais in gallbladder epithelium (Machen & Diamond, 1969) confirm that osmotic gradients are developed in the intercellular spaces during fluid transport. It is obvious that the standing osmotic gradient is a useful model for explaining epithelial fluid transport. In addition to providing a mechanism for the secretion of isotonic fluids, it suggests a geometrical function for the narrow, membrane bounded channels often found in transporting epithelium. These channels assist in the maintenance of the osmotic gradient by preventing the solute from rapidly diffusing away. They may be in the form of long intercellular spaces (gallbladder, intestine), basal membrane infoldings (choroid plexus, ciliary body), or intracellular canaliculi (parietal cells of the stomach). As discussed below, the transport model of Diamond St Tormey can be modified to explain the origin of gingival fluid in a manner which would resolve many controversies. 3. Gingival Fluid In adapting the osmotic gradient model of fluid transport to the secretion of gingival fluid, striking similarities are readily apparent. For example, the intercellular spaces in sulcular epithelium form a long, narrow, continuous
ORIGIN
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channel from the basement membrane to the epithelial surface (Gavin, 1968). These intercellular spaces are considerably widened in inflamed gingival tissues which transport fluid maximally (Freedman, Listgarten & Taichman, 1968). Moreover, To10 (1971a) has suggested that the ATPase present in certain segments of epithelial cell membranes (Farquhar & Palade, 1964) indicates the potential for active transport of solute molecules (“solute pump”, Fig. 1) into the intercellular space, with the consequent establishment of an osmotic gradient. This interesting observation is consistent with the originai model of Diamond & Tormey; however, as discussed below, a standing osmotic gradient may be generated in sulcular epithelium without the participation of “solute pumps”. Considering that gingivitis is a reversible process, and that fluid is not present in the crevices of stric:ly healthy gingivae (Lee & Helm-Pedersen, 1965; Oliver, Holm-Pedersen, & Lee, 1969) any model of gingival fluid flow should describe mechanisms by which the flow is both initiated and terminated. Since subgingival bacterial plaque is the primary etiological factor in periodontal disease (Socransky, 1970), and the quantity of plaque increases concurrently with gingival fluid (Lee, Theilade, Jensen & Schiott, 1967), it is reasonable to suspect that subgingival plaque may modulate the flow of gingival fluid. A sequence of events by which subgingival bacterial plaque could establish a standing osmotic gradient, thereby initiating gingival fluid flow, is illustrated diagrammatrically in Fig. 2. In strictly healthy gingiva [Fig. 2(a)] a small amount of subgingival plaque will give rise to limited quantities of free, macromolecular bacterial by-products, including a variety of enzymes, toxins, or other antigenic molecules (Socransky, 1970). These macromolecules could be removed either by adsorbing to the surfaces of sloughing epithelial cells, or through phagocytosis by suprabasal epithelial cells (Lange & Schroeder, 1971). Therefore, only a few of these molecules will reach the basement membrane, and there will be no flow of gingival fluid. When quantities of subgingival plaque increase [Fig. 2(b)], the concentration of macromolecular bacterial by-products will overwhelm the capacity of the epithelium to remove them. Thus, these macromolecules will diffuse intercellularly to the basement membrane (McDougall, 1971). As reviewed by Tolo (1971a), the basement membrane constitutes a barrier to the passage of molecules with a molecular weight greater than 50,000 daltons. Therefore, one would expect the macromolecules derived from the plaque to “back-up” at the basement membrane. This suggestion is supported by the studies of To10 (1971b) and Schwartz, Stinson & Parker (1972) in which the progress of radiolabelled macromolecules was traced autoradiographically through the sulcular epithelium. Both studies reported that the basement membrane
Cmneclwe
tissue
BM
Sulcular
epithellum
GS
P
l-coth
FIG. 2. Diagrammatic representation of the initiation of gingival exudation. (a) There is a small amount of subgingival plaque (P) present in the gingival sulcus (GS). The macromolecular by-products of this plaque (black dots) do not reach the basement membrane (BM), and no exudate is produced. (b) The amount of plaque has increased markedly, and its bacterial by-products accumulate at the basement membrane. This establishes an osmotic gradient which initiates gingival exudation (arrows). The tissue is not inflamed. (c) Considerable quantities of toxic or antigenic molecules have penetrated into the connective tissue and initiated a classical inflammatory exudate.
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appeared to restrict passage of the label. As the macromolecular bacterial by-products accumulate at the basement membrane, a standing osmotic gradient is generated [Fig. 2(b)], and the flow of gingival fluid is initiated. Since few molecules would have penetrated the basement membrane at this time, the gingiva would appear to be clinically and histologically healthy. It is clear that the gingival fluid produced by the process discussed above is not an inflammatory exudate. This fluid could be characterized as an osmotically-modulated, pre-inflammatory exudate, and would be distinct in origin and composition from the exudate produced by frankly inflamed gingival tissues. Therefore, at various times or in different areas of the mouth, gingival fluid may progress from an initial osmotically-modulated to a secondary inflammatory exudate, with consequent alterations in its composition. This progression is reflected in the observation of Weinstein et al. (1967) that the protein/calcium ratio increases markedly in fluid from normal, mildly inflamed, and inflamed gingival tissues. The osmotically-modulated exudate would have a lower protein content than the inflammatory exudate because the intact epithelial basement membrane would restrict the outward passage of serum proteins. The Na+/K+ ratio in both exudates would be similar. The classical inflammatory exudate would have its characteristic high K+ ion concentration as a result of cellular lysis. In the osmotic exudate, a massive influx of Naf ions would cross the basement membrane by solvent drag, thereby overloading the sodium pump of the basal epithelial cells. This would result in the leakage of considerable quantities of K+ ions into the intercellular space creating a Na+/K+ ratio comparable to the inflammatory exudate. In view of the fact that albumin, constituting only a small percentage of the total solute concentration of the plasma, can regulate osmotic pressure in that fluid, it is apparent that a relatively small accumulation of macromolecules at the basement membrane could generate an osmotic gradient. In addition, if these bacterial macromolecular by-products are enzymes such as hyaluronidase (Schultz-Haudt & Scherp, 1955) or collagenase (Gibbons & MacDonald, 1961; Alfano, Morhart, Metcalf & Drummond, 1974), they could fragment both intercellular glycosaminoglycans and the collagen component of the basement membrane (Kefalides, 1971). This would increase the number of solute molecules in the intercellular space thereby maintaining the standing osmotic gradient. Eventually, however, many molecules will penetrate the altered basement membrane, and a classical inflammatory exudate will be initiated [Fig. 2(c)]. The existence of gingival fluid produced by two different mechanisms, inflammation and an osmotic gradient, explains how the flow of gingival fluid could precede the onset of gingival inflammation by several days (Liie
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et al., 1967). It also supports the contention of LBe & Holm-Pedersen (1965) that gingival fluid may derive from clinically healthy gingiva, and is a useful indicator of potential gingival pathology. Based on the very slow rate (BrownGrant & Browne, 1966), as well as the low concentration of immunoglobulins (Weinstein et al., 1967) in the initial gingival exudate, it is doubtful that this fluid contributes significantly to the mechanisms of host defense in the gingival crevice. 4. Conclusion Many controversies on the origin and composition of gingival fluid may be explained by applying the standing osmotic gradient model of transepithelial fluid movement to the sulcular epithelium. Thus, gingival fluid may actually be generated by two mechanisms: pre-inflammatory osmotic exudation, and classical inflammatory exudation. As modified and discussed in this report this model system can account for the observations that : (1) Gingival fluid may be recovered from clinically and histologically healthy tissues (Weinstein et al., 1967). (2) The intercellular spaces in the sulcular epithelium dilate during inflammatory exudation (Freedman et al., 1968). (3) Gingival fluid flow increases several days prior to clinical inflammation (Lee & Holm-Pedersen, 1965). (4) The Na+/K+ ratio of the pre-inflammatory gingival fluid is comparable to an inflammatory exudate (Krasse & Egelberg, 1962; Kaslick et al., 1970), but the protein composition of this fluid is lower than that of an inflammatory exudate (Weinstein et al., 1967). (5) Gingival inflammatory exudation is associated with interruptions in the basement membrane (Dick & Trott, 1971). (6) The protein/calcium ratio increases in the gingival fluid from increasingly inflamed tissues (Weinstein et al., 1967). (7) The flow of gingival fluid increases concurrently with the accumulation of dental plaque (Liie et al., 1967). In addition, the theory presented here is consistent with the findings of Golub, Borden & Kleinberg (1971) that urea concentrations are highest in sulcular fluid from healthy gingiva, and decrease with i::creasing gingival inflammation. If, as suggested by Golub et al. (1971), the sulcular epithelium can concentrate urea, it is probable that a slower transepithelial fluid flow, such as that generated by the pre-inflammatory osmotic gradient, will result in higher concentrations of urea. With the onset of classical inflammatory exudation, and a concomitant increase in transepithelial fluid flow, the concentration of urea in the gingival fluid will be correspondingly lowered.
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Thus, while other mechanisms may be postulated to account for the initiation of gingival exudation, it is doubtful that they would explain each of the above observations as simply as does the standing osmotic gradient. Although the standing osmotic gradient has been demonstrated to account for the transport of fluid through several epithelia, the evidence supporting its application to the gingival sulcular epithelium is mostly circumstantial. Therefore, the following studies should be done to confirm the significance of this mechanism in the exudation of gingival fluid. First, the basement membrane should be demonstrated to be the rate-limiting barrier to epithelial penetration by macromolecules. Recent studies in our laboratory have confirmed that the basement membrane does, indeed, limit macromolecular penetration in non-keratinized squamous epithelium. Secondly, osmotic micropuncture measurements should indicate the existence of an intercellular osmotic gradient in sulcular epithelium which is exuding pre-inflammatory gingival fluid. Conversely, in the absence of the production of this fluid, the intercellular osmotic gradient should break down. Finally, if the initial preinflammatory gingival exudate arises strictly by an osmotic mechanism, a macromolecule which is not toxic, enzymatic, or antigenic (i.e. homologous serum albumin) should initiate gingival fluid flow when applied topically to the gingival crevice. REFERENCES ALEANO, M. C., MORHART, R. E., METCALF, G. & DRUMMOND, J. F. (1974). J. dent. Res. 53, 142. BADER, H. I. & GOLDHABER, P. (1966). J. oral Ther. Pharmacol. 2, 324. BERRIDGE, M. J. & OSCHMAN, J. L. (1972). Transporting Epithelium, p.1. New York:
Academic Press. BRILL, N. & KRASSE, B. (1958). Acta odont. Stand. 16, 233. BRILL, N. & BJ~~RN, H. (1959). Acta odont. Stand. 17, Il. BROWNE, R. M. (1962). Proc. R. Sot. Med. 55, 486. BROWNE, R. M. (1964). Dent. Practit. 14, 470.. BROWN-GRANT. K. & BROWNE. R. M. (1966). Arch. oral Biol. 11. 455. DIAMOND, J. G. & TORMEY, J. ‘M. (1966a). Nature, Land. 210, 81;. DIAMOND, J. M. & TORMEY, J. M. (1966b). Fedn Proc. Fedn Am. Sots exp. Biol. 25, 1458. DICK, H. M. & TRO-~~, J. R. (1971). J. Periodont. 42, 796. EGELBERG, J. (1963). Acta oaimt. Stand. 21, 283. FARQUHAR, M. G. & PALADE, G. E. (1964). Proc. natn. Acad. Sci. U.S.A. 51, 569. FREEDMAN, H. L., LISTGARTEN, M. A. & TAICHMAN, N. S. (1968). J. Periodont. Res. 3, 313. GAVIN, J. B. (1968). Am. J. Anat. 123, 283. GOLUB, L. M., BORDEN, S. M. & KLEINBERG, I. (1971). J. Periodont. Res. 6, 243. GIBBONS, R. J. & MACDONALD, J. B. (1961). J. Bact. 81, 614. GRANTHAM, J. J., CUPPAGE, F. E. & FANESTIL, D. (1971). J. Cell Biol. 48, 695. GRANTHAM, J. J., CANOTE, C. E., BURG, M. B. & ORLOFF, J. (1969). J. Cell Biol. 41, 562. KASLICK, R. S., CHASENS, A. I., MANDEL, I. D., WEINSTEIN, D., WALDMAN, R., PLUHAR, T. & LAZZARA, R. (1970). J. Periodont. 25, 93. KEFALIDES, N. A. (1971). Int. Rev. exp. Path. 10, 1.
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KRASSE, B. & EGELBERG, J. (1962). Actu odont. Scund. 20, 143. LANGE, D. & SCHROEDER, H. E. (1971). Helv. odont. Acta 15, 65. ME, H. & HOLM-PEDERSEN, P. (1965). Periodontics 3, 171. LYE, H., THEILADE, E. & JENSEN, S. B. (1965). J. Periodont. 36, 177. Lij~, H., THEILADE, E., JENSEN, S. B. & SCHIOTT, C. R. (1967). J. Periodont. Res. 2,282. MACHEN, T. E. & DIAMOND, J. M. (1969). J. membr. Biol. 1, 194. MCDOUGALL, W. A. (1971). J. periodont. Res. 6, 89. OLIVER, R. C., HOLM-PEDERSEN, P. & LYE, H. (1969). J. Periodont. 40, 201. ROBBINS, S. L. (1967). Pathology. Philadelphia: Saunders, p. 31. SCHULTZ-HAUDT, S. D. & SCHERP, H. W. (1955). J. dent. Res. 34, 924. SCHWARTZ, J., STIN~~N, F. L. & PARKER, R. B. (1972). J. Periodont. 43, 270. S~CRANSKY, S. S. (1970). J. dent. Res. 49, 203. TOLO, K. (1971u). J. Periodont. Res. 6, 237. TOLO. K. (1971b). Arch. oral Bioi. 16. 881. WALL, B. j., O&HMAN, J. L. & SCHM&T-NIELSEN, B. (1970). Science, N. Y. 167, 1497. WEINSTEIN, E., MANDEL, I. D., SALKIND, A., OSHRAIN, H. I. & PAPPAS, G. D. (1967).
Periodontics 5, 161.
The Origin of Gingival Fluid? MICHAEL C. ALFANO$ Oral Science Research Laboratories, Department of Nutrition and Food Science, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139, U.S.A. (Received 26 September 1973, and in revised form 28 January
1974)
Although the existence of gingival crevicular fluid, a fluid which exudes from the crevice between the gingiva and the tooth, has been recognized for decades the origin, function, and composition of this fluid has been the subject of much controversy. In fact, it is unclear whether this fluid results from physiological or pathological processes. This confusion has arisen because by certain parameters (protein concentration) the fluid resembles a physiological transudate, while by others (Na+/K+ Ratio) it appears to be an inflammatory exudate. This report describes a theory which explains the above and other controversies relating to the origin of gingival fluid. It is based on the premise that gingival fluid may arise by two distinct mechanisms: the generation of a standing osmotic gradient, and the initiation of classical inflammation. The osmotic gradient is generated by macromolecular by-products of the bacteria which reside in the subgingival dental plaque. These macromolecules diffuse through the gingival crevicular epithelium to the basement membrane, a structure which restricts further penetration. Consequently, these macromolecules accumulate at the basement membrane resulting in a localized increase in solute concentration, and the establishment of an osmotic gradient. Solvent molecules, drawn across the basement membrane by this gradient, will raise the intercellular hydrostatic pressure and cause the exudation of gingival fluid. The fluid produced by this mechanism may originate from gingival tissues which are clinically and histologically healthy, If the bacterial plaque is not removed, its macromolecular byproducts will eventually penetrate the basement membrane. Depending upon the enzymatic, toxic, and antigenic properties of these molecules, a classical inflammatory exudation may be initiated. Therefore, gingival fluid may progress, at different times or in various areas of the mouth, from an initial osmotically modulated exudate to a secondary inflammatory exudate, with consequent alterations in its composition. Although the concepts developed in this report focus on the origin of gingival fluid, they may be applied to other biological phenomena, such as, the origin of exudate in the lungs during respiratory infection. tThis investigation was supported by USPHS Training Grant No. DE 105-9. Thii is contribution No. 2215 from the Department of Nutrition and Food Science,Massachusetts Institute of Technology. $ Present address: Department of Periodontics and Oral Medicine, Fairleigh Dickinson University, School of Dentistry, Hackensack, New Jersey 07601, U.S.A. 127
128
M.
C.
ALFANO
1. Introduction
Many controversies have arisen in the last decade concerning the origin, composition, and function of the gingival fluid. Perhaps the most fundamental unresolved problem is whether this fluid is the result of physiological or pathological processes. This controversy is more than academic in interest. If the flow of crevicular fluid is a physiological host defense mechanism, efforts might be directed to increase the rate of flow, thereby enhancing the host defenses in the gingival crevice. In contrast, if this flow originates from inflammatory processes, it could provide a useful criterion for diagnosing incipient gingivitis (Lee & Holm-Pedersen, 1965). Most of the previous investigations have indicated that crevicular fluid is present in healthy gingival tissues (Brill & Krasse, 1958; Brill & Bjorn, 1959; Browne, 1962; Egelberg, 1963; Browne, 1964; Bader & Goldhaber, 1966; Weinstein, Mandel, Salkind, Oshrain & Pappas, 1967). These studies have employed a variety of sampling and tracer techniques, and constitute significant evidence indicating that crevicular fluid arises from normal physiological processes. Liie & Holm-Pedersen (1965), however, reported that crevices of normal human gingiva do not exhibit flow of fluid. They attributed this discrepancy to the fact that rigorous criteria to determine gingival health as well as minimally traumatic sampling techniques were employed in their study. In addition, they suggested that the gingival fluid is an inflammatory exudate but, if it is present prior to clinically detectable signs of inflammation, it would appear to be derived from healthy gingival tissue. This interpretation is supported by the observations of Krasse & Egelberg (1962) and Egelberg (1963), that both the ionic and cellular composition of fluid from healthy gingiva are comparable to that found in inflammatory exudates. Based on the investigations discussed above, it is apparent that a reasonable argument could be made that gingival fluid originates either as a physiological transudate or an inflammatory exudate. However, certain facts are inconsistent with both of these explanations. For example, although the gingival fluid has an ionic composition comparable to an inflammatory exudate (Krasse & Egelberg, 1962), its protein composition is considerably lower than one would expect in an inflammatory exudate (Weinstein et al., 1967; Robbins, 1967). Also, Lee & Holm-Pedersen (1965), using a human model of experimental gingivitis, reported that the initial flow of gingival fluid preceded the onset of clinically detectable gingivitis by several days. Considering that in the classical inflammatory response, vascular dilatation and increased blood flow precede the exudation of fluid (Robbins, 1967), it is difficult to explain a delay of several days between the initiation of gingival exudation and the onset of gingivitis. These inconsistencies suggest that a
ORIGIN
OF
GINGIVAL
FLUID
129
process distinct from either simple filtration or inflammatory exudation may be responsible for the production of crevicular fluid in healthy gingiva. The purpose of this report is to describe a mechanism, based on general concepts of fluid transport across epithelial membranes, which can explain these inconsistencies and may lead to a better understanding of the origin and function of gingival fluid. 2. Transepithelial
Fluid Movement
Several processes have been postulated by which fluid may be transported across epithelial membranes. These processes include hydrostatic filtration, pinocytosis, active transport and classical osmosis. Although each of these mechanisms are possible, they cannot explain certain characteristics peculiar to transporting epithelium. For example, classical osmosis could not account for the production of isomotic secretions such as bile, cerebrospinal fluid, or pancreatic juice; also, the transport of fluid by pinocytosis is inconsistent with the high degree of specificity observed when different compounds are tested for their ability to cross epithelia (Berridge & Oschman, 1972). Thus one might conclude that the secretion of most epithelial membranes must result from complex interactions of the above processes. Recently, Diamond & Tormey (1966qb) have developed a simple model for fluid transport which explains many confusing morphological and physiological characteristics of transporting epithelial surfaces. This model is based upon their observations on the morphological changes which occur during the in vitro transport of water across rabbit gallbladder epithelium (Diamond & Tormey, 1966a). Basically they noted that, under conditions in which water transport was maximal, the intercellular spaces were widely distended; when water transport was either partially or completely inhibited, the spaces either decreased in width or closed down completely. To explain this phenomenon, Diamond & Tormey (19663) postulated that there is a standing osmotic gradient within the intercellular spaces (Fig. l), with the highest concentration of the solute located at the closed apical end of the intercellular channel. This gradient is maintained by the active transport of solute into the closed end of the channel. Therefore, water will be drawn osmotically into the intercellular spaces and out the open end of the channel. As the water proceeds through the channel, the osmolarity is continually reduced so that the emerging solution is isotonic. The intercellular route for fluid transport has been demonstrated on living tissues including isolated renal collecting tubules (Grantham, Canote, Burg & Orloff, 1969), toad bladder (Grantham, Cuppage & Fanestil, 1971), and insect rectum (Wall, Oschman & Schmidt-Nielsen, 1970). Moreover, micro9 T.B.
130
M.
C.
ALFANO
> !t?qM “:
Solute.:,/:.:: pumps .;
..---~-
.
,-
Water
-.
flow
.i . ...’
,’
,, _
I.
)‘:. ,
’
I” __..
‘:
1.; I..
‘/:
i.. ~--I/’
‘..
.-.
‘.
‘.’
..’
FIG. 1. Standing-gradient osmotic flow: a model for fluid transport across epithelia. Solute is pumped into the closed end of a long and narrow channel making it hypertonic and pulling in water osmotically. Osmotic equilibrium is progressively approached as water enters along the length of the channel until the emergent solution is isotonic. (Reproduced by permission of the publisher. Diamond & Tormey. Fed. Pruc., 25: 14.58-1463, 19666.)
puncture samples from the intercellular spaces of insect rectum (Wall et al., 1970) as well as measurement of streaming potentiais in gallbladder epithelium (Machen & Diamond, 1969) confirm that osmotic gradients are developed in the intercellular spaces during fluid transport. It is obvious that the standing osmotic gradient is a useful model for explaining epithelial fluid transport. In addition to providing a mechanism for the secretion of isotonic fluids, it suggests a geometrical function for the narrow, membrane bounded channels often found in transporting epithelium. These channels assist in the maintenance of the osmotic gradient by preventing the solute from rapidly diffusing away. They may be in the form of long intercellular spaces (gallbladder, intestine), basal membrane infoldings (choroid plexus, ciliary body), or intracellular canaliculi (parietal cells of the stomach). As discussed below, the transport model of Diamond St Tormey can be modified to explain the origin of gingival fluid in a manner which would resolve many controversies. 3. Gingival Fluid In adapting the osmotic gradient model of fluid transport to the secretion of gingival fluid, striking similarities are readily apparent. For example, the intercellular spaces in sulcular epithelium form a long, narrow, continuous
ORIGIN
OF
GINGIVAL
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channel from the basement membrane to the epithelial surface (Gavin, 1968). These intercellular spaces are considerably widened in inflamed gingival tissues which transport fluid maximally (Freedman, Listgarten & Taichman, 1968). Moreover, To10 (1971a) has suggested that the ATPase present in certain segments of epithelial cell membranes (Farquhar & Palade, 1964) indicates the potential for active transport of solute molecules (“solute pump”, Fig. 1) into the intercellular space, with the consequent establishment of an osmotic gradient. This interesting observation is consistent with the originai model of Diamond & Tormey; however, as discussed below, a standing osmotic gradient may be generated in sulcular epithelium without the participation of “solute pumps”. Considering that gingivitis is a reversible process, and that fluid is not present in the crevices of stric:ly healthy gingivae (Lee & Helm-Pedersen, 1965; Oliver, Holm-Pedersen, & Lee, 1969) any model of gingival fluid flow should describe mechanisms by which the flow is both initiated and terminated. Since subgingival bacterial plaque is the primary etiological factor in periodontal disease (Socransky, 1970), and the quantity of plaque increases concurrently with gingival fluid (Lee, Theilade, Jensen & Schiott, 1967), it is reasonable to suspect that subgingival plaque may modulate the flow of gingival fluid. A sequence of events by which subgingival bacterial plaque could establish a standing osmotic gradient, thereby initiating gingival fluid flow, is illustrated diagrammatrically in Fig. 2. In strictly healthy gingiva [Fig. 2(a)] a small amount of subgingival plaque will give rise to limited quantities of free, macromolecular bacterial by-products, including a variety of enzymes, toxins, or other antigenic molecules (Socransky, 1970). These macromolecules could be removed either by adsorbing to the surfaces of sloughing epithelial cells, or through phagocytosis by suprabasal epithelial cells (Lange & Schroeder, 1971). Therefore, only a few of these molecules will reach the basement membrane, and there will be no flow of gingival fluid. When quantities of subgingival plaque increase [Fig. 2(b)], the concentration of macromolecular bacterial by-products will overwhelm the capacity of the epithelium to remove them. Thus, these macromolecules will diffuse intercellularly to the basement membrane (McDougall, 1971). As reviewed by Tolo (1971a), the basement membrane constitutes a barrier to the passage of molecules with a molecular weight greater than 50,000 daltons. Therefore, one would expect the macromolecules derived from the plaque to “back-up” at the basement membrane. This suggestion is supported by the studies of To10 (1971b) and Schwartz, Stinson & Parker (1972) in which the progress of radiolabelled macromolecules was traced autoradiographically through the sulcular epithelium. Both studies reported that the basement membrane
Cmneclwe
tissue
BM
Sulcular
epithellum
GS
P
l-coth
FIG. 2. Diagrammatic representation of the initiation of gingival exudation. (a) There is a small amount of subgingival plaque (P) present in the gingival sulcus (GS). The macromolecular by-products of this plaque (black dots) do not reach the basement membrane (BM), and no exudate is produced. (b) The amount of plaque has increased markedly, and its bacterial by-products accumulate at the basement membrane. This establishes an osmotic gradient which initiates gingival exudation (arrows). The tissue is not inflamed. (c) Considerable quantities of toxic or antigenic molecules have penetrated into the connective tissue and initiated a classical inflammatory exudate.
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appeared to restrict passage of the label. As the macromolecular bacterial by-products accumulate at the basement membrane, a standing osmotic gradient is generated [Fig. 2(b)], and the flow of gingival fluid is initiated. Since few molecules would have penetrated the basement membrane at this time, the gingiva would appear to be clinically and histologically healthy. It is clear that the gingival fluid produced by the process discussed above is not an inflammatory exudate. This fluid could be characterized as an osmotically-modulated, pre-inflammatory exudate, and would be distinct in origin and composition from the exudate produced by frankly inflamed gingival tissues. Therefore, at various times or in different areas of the mouth, gingival fluid may progress from an initial osmotically-modulated to a secondary inflammatory exudate, with consequent alterations in its composition. This progression is reflected in the observation of Weinstein et al. (1967) that the protein/calcium ratio increases markedly in fluid from normal, mildly inflamed, and inflamed gingival tissues. The osmotically-modulated exudate would have a lower protein content than the inflammatory exudate because the intact epithelial basement membrane would restrict the outward passage of serum proteins. The Na+/K+ ratio in both exudates would be similar. The classical inflammatory exudate would have its characteristic high K+ ion concentration as a result of cellular lysis. In the osmotic exudate, a massive influx of Naf ions would cross the basement membrane by solvent drag, thereby overloading the sodium pump of the basal epithelial cells. This would result in the leakage of considerable quantities of K+ ions into the intercellular space creating a Na+/K+ ratio comparable to the inflammatory exudate. In view of the fact that albumin, constituting only a small percentage of the total solute concentration of the plasma, can regulate osmotic pressure in that fluid, it is apparent that a relatively small accumulation of macromolecules at the basement membrane could generate an osmotic gradient. In addition, if these bacterial macromolecular by-products are enzymes such as hyaluronidase (Schultz-Haudt & Scherp, 1955) or collagenase (Gibbons & MacDonald, 1961; Alfano, Morhart, Metcalf & Drummond, 1974), they could fragment both intercellular glycosaminoglycans and the collagen component of the basement membrane (Kefalides, 1971). This would increase the number of solute molecules in the intercellular space thereby maintaining the standing osmotic gradient. Eventually, however, many molecules will penetrate the altered basement membrane, and a classical inflammatory exudate will be initiated [Fig. 2(c)]. The existence of gingival fluid produced by two different mechanisms, inflammation and an osmotic gradient, explains how the flow of gingival fluid could precede the onset of gingival inflammation by several days (Liie
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et al., 1967). It also supports the contention of LBe & Holm-Pedersen (1965) that gingival fluid may derive from clinically healthy gingiva, and is a useful indicator of potential gingival pathology. Based on the very slow rate (BrownGrant & Browne, 1966), as well as the low concentration of immunoglobulins (Weinstein et al., 1967) in the initial gingival exudate, it is doubtful that this fluid contributes significantly to the mechanisms of host defense in the gingival crevice. 4. Conclusion Many controversies on the origin and composition of gingival fluid may be explained by applying the standing osmotic gradient model of transepithelial fluid movement to the sulcular epithelium. Thus, gingival fluid may actually be generated by two mechanisms: pre-inflammatory osmotic exudation, and classical inflammatory exudation. As modified and discussed in this report this model system can account for the observations that : (1) Gingival fluid may be recovered from clinically and histologically healthy tissues (Weinstein et al., 1967). (2) The intercellular spaces in the sulcular epithelium dilate during inflammatory exudation (Freedman et al., 1968). (3) Gingival fluid flow increases several days prior to clinical inflammation (Lee & Holm-Pedersen, 1965). (4) The Na+/K+ ratio of the pre-inflammatory gingival fluid is comparable to an inflammatory exudate (Krasse & Egelberg, 1962; Kaslick et al., 1970), but the protein composition of this fluid is lower than that of an inflammatory exudate (Weinstein et al., 1967). (5) Gingival inflammatory exudation is associated with interruptions in the basement membrane (Dick & Trott, 1971). (6) The protein/calcium ratio increases in the gingival fluid from increasingly inflamed tissues (Weinstein et al., 1967). (7) The flow of gingival fluid increases concurrently with the accumulation of dental plaque (Liie et al., 1967). In addition, the theory presented here is consistent with the findings of Golub, Borden & Kleinberg (1971) that urea concentrations are highest in sulcular fluid from healthy gingiva, and decrease with i::creasing gingival inflammation. If, as suggested by Golub et al. (1971), the sulcular epithelium can concentrate urea, it is probable that a slower transepithelial fluid flow, such as that generated by the pre-inflammatory osmotic gradient, will result in higher concentrations of urea. With the onset of classical inflammatory exudation, and a concomitant increase in transepithelial fluid flow, the concentration of urea in the gingival fluid will be correspondingly lowered.
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Thus, while other mechanisms may be postulated to account for the initiation of gingival exudation, it is doubtful that they would explain each of the above observations as simply as does the standing osmotic gradient. Although the standing osmotic gradient has been demonstrated to account for the transport of fluid through several epithelia, the evidence supporting its application to the gingival sulcular epithelium is mostly circumstantial. Therefore, the following studies should be done to confirm the significance of this mechanism in the exudation of gingival fluid. First, the basement membrane should be demonstrated to be the rate-limiting barrier to epithelial penetration by macromolecules. Recent studies in our laboratory have confirmed that the basement membrane does, indeed, limit macromolecular penetration in non-keratinized squamous epithelium. Secondly, osmotic micropuncture measurements should indicate the existence of an intercellular osmotic gradient in sulcular epithelium which is exuding pre-inflammatory gingival fluid. Conversely, in the absence of the production of this fluid, the intercellular osmotic gradient should break down. Finally, if the initial preinflammatory gingival exudate arises strictly by an osmotic mechanism, a macromolecule which is not toxic, enzymatic, or antigenic (i.e. homologous serum albumin) should initiate gingival fluid flow when applied topically to the gingival crevice. REFERENCES ALEANO, M. C., MORHART, R. E., METCALF, G. & DRUMMOND, J. F. (1974). J. dent. Res. 53, 142. BADER, H. I. & GOLDHABER, P. (1966). J. oral Ther. Pharmacol. 2, 324. BERRIDGE, M. J. & OSCHMAN, J. L. (1972). Transporting Epithelium, p.1. New York:
Academic Press. BRILL, N. & KRASSE, B. (1958). Acta odont. Stand. 16, 233. BRILL, N. & BJ~~RN, H. (1959). Acta odont. Stand. 17, Il. BROWNE, R. M. (1962). Proc. R. Sot. Med. 55, 486. BROWNE, R. M. (1964). Dent. Practit. 14, 470.. BROWN-GRANT. K. & BROWNE. R. M. (1966). Arch. oral Biol. 11. 455. DIAMOND, J. G. & TORMEY, J. ‘M. (1966a). Nature, Land. 210, 81;. DIAMOND, J. M. & TORMEY, J. M. (1966b). Fedn Proc. Fedn Am. Sots exp. Biol. 25, 1458. DICK, H. M. & TRO-~~, J. R. (1971). J. Periodont. 42, 796. EGELBERG, J. (1963). Acta oaimt. Stand. 21, 283. FARQUHAR, M. G. & PALADE, G. E. (1964). Proc. natn. Acad. Sci. U.S.A. 51, 569. FREEDMAN, H. L., LISTGARTEN, M. A. & TAICHMAN, N. S. (1968). J. Periodont. Res. 3, 313. GAVIN, J. B. (1968). Am. J. Anat. 123, 283. GOLUB, L. M., BORDEN, S. M. & KLEINBERG, I. (1971). J. Periodont. Res. 6, 243. GIBBONS, R. J. & MACDONALD, J. B. (1961). J. Bact. 81, 614. GRANTHAM, J. J., CUPPAGE, F. E. & FANESTIL, D. (1971). J. Cell Biol. 48, 695. GRANTHAM, J. J., CANOTE, C. E., BURG, M. B. & ORLOFF, J. (1969). J. Cell Biol. 41, 562. KASLICK, R. S., CHASENS, A. I., MANDEL, I. D., WEINSTEIN, D., WALDMAN, R., PLUHAR, T. & LAZZARA, R. (1970). J. Periodont. 25, 93. KEFALIDES, N. A. (1971). Int. Rev. exp. Path. 10, 1.
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KRASSE, B. & EGELBERG, J. (1962). Actu odont. Scund. 20, 143. LANGE, D. & SCHROEDER, H. E. (1971). Helv. odont. Acta 15, 65. ME, H. & HOLM-PEDERSEN, P. (1965). Periodontics 3, 171. LYE, H., THEILADE, E. & JENSEN, S. B. (1965). J. Periodont. 36, 177. Lij~, H., THEILADE, E., JENSEN, S. B. & SCHIOTT, C. R. (1967). J. Periodont. Res. 2,282. MACHEN, T. E. & DIAMOND, J. M. (1969). J. membr. Biol. 1, 194. MCDOUGALL, W. A. (1971). J. periodont. Res. 6, 89. OLIVER, R. C., HOLM-PEDERSEN, P. & LYE, H. (1969). J. Periodont. 40, 201. ROBBINS, S. L. (1967). Pathology. Philadelphia: Saunders, p. 31. SCHULTZ-HAUDT, S. D. & SCHERP, H. W. (1955). J. dent. Res. 34, 924. SCHWARTZ, J., STIN~~N, F. L. & PARKER, R. B. (1972). J. Periodont. 43, 270. S~CRANSKY, S. S. (1970). J. dent. Res. 49, 203. TOLO, K. (1971u). J. Periodont. Res. 6, 237. TOLO. K. (1971b). Arch. oral Bioi. 16. 881. WALL, B. j., O&HMAN, J. L. & SCHM&T-NIELSEN, B. (1970). Science, N. Y. 167, 1497. WEINSTEIN, E., MANDEL, I. D., SALKIND, A., OSHRAIN, H. I. & PAPPAS, G. D. (1967).
Periodontics 5, 161.