Increased Fibrinolytic Activity in the Inflamed Gingiva of Beagle Dogs OSCAR N. LUCAS
University of Oregon Health Sciences Center, School of Dentistry, Department of Oral Surgery, Portland, Oregon 97201, USA The fibrinolytic activity originated by activation of plasminogen was found to be greater in the inflamed gingiva than in the normal gingiva. The activator of plasminogen present in the normal and inflamed gingiva of the beagle dog was demonstrated histochemically. Plasminogen activator wasfound adjacent to the crenicular epithelium and blood vessels. J Dent Res 56(12): 1533-1538, December 1977.
For the dynamics of healing to occur, a foundation must be provided for the newly forming reparative connective tissue. This foundation or framework for the connective tissue reparative process is regulated in part, and dependent upon, the stability and integrity of the formed fibrin clot. Early dissolution of the clot may result in bleeding and delayed healing. At the same time, retarded disappearance of the clot leads to an exuberant granulation tissue with a hypertrophic scar.' As healing progresses, the fibrin clot must be removed. Kwaan et al.2 indicated that phagocytic and/or nonspecific proteolysis alone is not responsible for the total resolution of the fibrin clot. This is accomplished by the fibrinolytic system following the activation of plasminogen into the active lytic enzyme plasmin. Maintenance and/or dissolution of the fibrin clot which seems to influence connective tissue reparative processes depends upon the dynamic balance of two systems -humoral and cellular.3 The purpose of this study is to demonstrate histochemically the plasminogen activator present in normal and inflamed gingiva of beagle dogs. Received for publication June 4, 1976. Accepted for publication March 7, 1977. The author wishes to thank Drs. W. H. Wright and F. Bremner of the Department of Periodontology for their assistance in determining the gingival condition of the dogs and to D. Fujita for the technical assistance. This study was supported by USPHS Grant DE-03322.
*Parke-Davis, Detroit, Mi.
tFraction I Lot No. Y. 1013 Schwarz/Mann, Orangeburg, NY. tLot No. X 1010 Schwarz/Mann, Orangeburg, NY.
Materials and Methods
Twenty male beagle dogs, ranging from 9 to 13 kgs in body weight, were studied. In all dogs, clinically healthy gingiva was obtained by scaling, polishing, and twice daily brushing for approximately 30 days. Ten dogs used as a control were maintained on a hard diet and a rigorous dental hygiene program throughout the experimental period of time. In the 10 experimental dogs, mild to severe gingivitis was developed by stopping all oral hygiene procedures and feeding a soft diet. Mild gingivitis was usually developed in 21 to 40 days. Gingival biopsies were always obtained from the maxillary right four premolars and mandibular first molar areas. The dogs were allowed to continue on the soft diet until they developed severe gingivitis. This occurred in 60 to 90 days. Gingival biopsies were then obtained from the maxillary left fourth premolar and mandibular first molar areas (Fig 1-B). Normal gingival samples were taken at the same time and from the same areas in the control dogs (Fig 1-A). All gingival biopsies were taken while the dogs were under general anesthesia. The samples were mounted on cryostat chucks with AmesTM O.C.T. compound and quickly frozen. The mounted biopsies were kept at -20 C until ready to be sectioned. To identify the gingival activator, a frozen gingival section was applied over a premade film of fibrin clot on a microscope slide. The fibrin clot film was prepared in the following manner: Ten microliters of a solution containing 20U/ml bovine thrombin* was evenly spread onto a carefully precleaned microscopic slide over an area of 875 mm2 and allowed to dry at room temperature. After this, 60 microliters of a 2% solution of bovine plasminogen-rich fibrinogent were applied over the dried thrombin and evenly distributed with a thin glass rod to facilitate formation of a uniform film of fibrin. The same procedure was repeated with bovine plasminogen-free fibrinogen. + These 1533
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J Dent Res December 1977
Clinical appearance of
normal gingiva (A), and severely in flamed gingiva (B). Arrows indicate areas from which biopsies were taken. Mandibular specimens are depicted in the composite photomicrograph shown in Figure 2.
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of preparations permitted differentiation between specific fibrinolytic activity and nonspecific proteolytic activity in the tissue sections.3 The fibrin film clots were allowed to stabilize for one hour in a level moisture chamber at room temperature. Following stabilization of the fibrin clot, three 8M thick tissue sections of each specimen were cut with the cryostat and placed directly on the fibrin clot. This was followed by immersion of the slide in a 50U7 solution of methanol for 30 seconds and rinsing in saline for another 30 seconds. The slides were then immediately returned the moisture chamber and incubated at 37 C for 15, 30, 60, 90, or 120 minutes. After the different incubation periods, the incubated slides were fixed ini a neutral buffered formalin for at least one hour and then were stained with Harris Hematoxylin. The stained slides were examined with a light microscope and the areas of fibrinolytic activity evaluated. Gingival condition was determined according to the criteria repo, ted by two types
Lbe.4 Clinically normal gingiva was pale pink in color, firm in texture, and tightly adapted to the tooth. Mildly inflamed gingiva showed slight edema with minimal change in color and no bleeding on probing. Severely inflamed gingiva showed marked redness, edema, and a tendency to bleed spontaneously. Results
Clinically normal gingiva was obtained in all control dogs after oral prophylaxis followed by twice daily tooth brushing and a hard diet regimen. Figure 1-A is a typical example of clinically normal gingiva. Figure 1 -B is an example of severe gingivitis developed while an experi-
mental dog was on a soft diet for 72 days without receiving oral hygiene care. The gingival tissue activator, plasminogen, was present in both the normal and inflamed gingiva. The lysis of the fibrin film clot due to activation of plasminogen into the specific fi-
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brinolytic enzyme, plasmin, was easily related topographically to the overlaying gingival tissue. The lytic foci were seen microscopically as circular areas of liquefaction when related to blood vessels and a diffuse pattern when related to crevicular epithelium. Figure 2 shows the fibrinolytic areas induced by the normal and severely inflamed gingiva. The degree of lytic activity was related to the time of incubation of the gingival tissue with the fibrin clot and was always greater in the inflamed gingiva. At 15 minutes of incubation, the difference in lytic activity can easily be seen adjacent to the crevi-
cular epithelium and around the blood vessels when compared to the normal and the severely inflamed gingiva (Fig 2-A and 2-D). At 30 minutes of incubation, in the inflamed gingiva, well-defined areas of lysis can be seen with its characteristic circular definition around the blood vessels and in a diffusive pattern along the crevice area (Fig 2-E). It can be seen in this photomicrograph that the lytic areas around the blood vessels are greater in number and larger in size when compared with the normal gingiva (Fig 2-B) incubated for the same period of time. At 60 minutes of incubation, the in-
Ftc; 2. Composite photomicrograph to compare the degree of fibrinolysis indluced by nornmal (A BC, ) and severely inflamed gingiva (D,E,F) at dlifferent periods of incubation. I he backgroutnd (dark color is the stained fibrin film clot and the white areas around the blood vessels and the crevicular epithelium arte specific fibrinolytic activity. It can be seen that the size and number of lytic areas increased with the time of incubation and that the severely inflamed ginigiva showed a greater (legree of activity than the niorm-al
gingiva. The period of incubation shown in Figure A and D was for 15', Figure B and E for 30 and Figure C and F for 60'. Defects observed in the fibrin clot adljacent to the oral epithelium that are cauLsed by separation or shrinkage of the tissue section from the fibrin substrate should not be confused with the true lytic areas evident elsewhere. Arrows outline the lytic areas adjacent to the crevicular epitheliuni and around some blood vessels. Harris Hematoxylin. Original magnificationI 12 x.
J Dent Res December 1977
Composite photomicrograph showing the normal (A) and inflamed gingiva (B) incubated with plasminogen-free film clot for 60'. I'hese are serial sections adjacent to the one shown in Figure 2. Note the lack of lytic activity around blood vessels and crevice area, Harris Hematoxylin. Original magniification 12 x. Fit; 3.
flamed gingiva showed many widely diffused lytic areas that were overlapping each other underneath the connective tissue. Extensive fibrinolytic activity can also be seen in the crevice area that extends on to the connective tissue (Fig 2-F). Conversely, the degree of fibrinolytic activity is considerably less in the normal gingiva (Fig 2-C). Prolongation of the incubation time of the gingival tissue and the fibrin film increased the degree of lytic activity. At 90 minutes of incubation, in the severely inflamed gingiva, the lytic activity was so extensive that in almost all instances the fibrin film underneath the gingival section was completely digested, and tissue folded over or fell off during fixation. In the normal gingiva, extensive dissolution of the fibrin f-ilm was observed at 120 minutes of incubation. Mild gingivitis did produce lysis of the fibrin film clot, following very much the same pattern in intensity and topographical location as the severe gingivitis. There was not any fibrinolytic activity observed when either
the normal or inflamed gingival sections were incubated with the plasminogen-free fibrin film clot. This occurred regardless of the time of incubation (Fig 3). Lytic activity either in the normal or inflamed gingiva was never found associated with the oral epithelium. Separation or shrinkage of the oral epithelium from the fibrin clot may cause some defect in the fibrin film. These artifacts were found in both plasminogen-rich (Fig 2) and plasminogen-free fibrin (Fig 3) clots and, therefore, should not be in any way confused with the true fibrinolytic activity observed around the blood vessels and the crevicular epithelium. I)iscussion The presence of plasminogen activator in the normal and inflamed gingiva of beagle dogs was clearly shown histochemically. Plasminogen activation led to the formation of the spe-
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FIBRINOL YTIC ACTIVITY IN GINGIVA
cific fibrinolytic enzyme, plasmin. Upon its activation, plasmin readily will lyse the fibrinfilm clot underneath the gingival section. The lytic areas were easily seen microscopically and related to blood vessels on almost all occasions as a well-defined circumferential area of liquefaction. Conversely, subjacent to the crevicular epithelium the fibrinolytic activity was always found to be in a diffuse pattern extending away from the sections. The histological localization and the behavior pattern of the activator plasminogen in the gingival tissue have been discussed in detail elsewhere.3 The findings described under results give a clear indication that the fibrinolytic activity induced by the activation of plasminogen increases with the time of incubation and that the degree of lysis at a given incubation time is greater in mild and severe gingivitis than in normal gingiva (Fig 2). This finding is in disagreement with the report by Pandolfi et al,5 indicating that in chronically inflamed gingiva there is a decrease in fibrinolytic activity. Chronically inflamed gingiva has been shown to have increased vascularization.6 Tissue activators have been closely related to blood vessels.7'8 Therefore, it seems reasonable to assume that inflamed gingiva with increased vascularization should produce a greater degree of lytic activity than normal gingiva. Our results substantiate this assumption. Fibrinolytic activity was observed only when the gingival tissue was incubated with a plasminogen-rich fibrin clot. There was no lytic activity induced on the plasminogen-free fibrin clot even when the gingival tissue was incubated with the fibrin clot for 120 minutes. This is a clear indication that the lytic activity observed in this study was due to activation of a specific fibrinolytic enzyme, plasmin, and not to other proteolytic enzymes. If nonspecific proteolytic enzymes were responsible for the lysis of the fibrin substrate observed in this study, they would have been nonselective in lysing the fibrin clot made with plasminogen-rich or plasminogen-free fibrinogen. The size of lytic areas induced by the normal or inflamed gingival tissue was directly related to the time of incubation and the temperature. At 90 minutes of incubation, the fibrin film underneath the inflamed gingival tissue was completely digested, and the tissue section was folded over or fell off the slide during fixation. The same response was found in normal gingiva after 120 minutes of incubation. The activation of plasminogen by tissue activators was postulated to be enzymatic and of first order kinetics. 9
An attempt was made to measure the size of the lytic areas to compare the degree of fibrinolytic activity between the normal gingiva and the gingiva with mild and severe inflammation. This was technically impossible as it is not always feasible to find the same size blood vessels in the gingiva throughout the different periods of incubation. The physiologic role of the fibrinolytic system in maintaining the patency of the blood vessel by its ability to lyse intravascular clots is well known. However, the role played by the gingival tissue activator of fibrinolysis as shown in this study is speculative. It could be postulated that the active fibrinolytic enzyme, plasmin, could continue the breakdown of gingival collagen after the initial hydrolysis by collagenase. Based on this assumption, it was decided that parallel to the evaluation of the activator of plasminogen, the concentration of collagen in the adjacent serial section of the gingiva should be studied. By means of a modified microtechnique,'0 for quantifying hydroxyproline the collagen concentration in the tissue sections was determined. It was found that the increased fibrinolytic activity observed in the inflamed gingiva was correlated with a decrease in collagen. The concentration of hydroxyproline was found to be significantly lower in the inflamed gingiva than in the normal gingiva. I The fibrinolytic system has been associated with many other phenomena occurring in the biologic system. Kwaan'2 reported that an intense fibrinolytic activity was observed in young capillaries in early stages of wound healing that was responsible for the removal of the fibrin clot as soon as its function as a supporting framework was over. Plasmin was also reported to be able to cause cleavage of C '3 to release a fragment that is chemotactically active and an important factor in the mobilization of polymorphonuclear in vivo.'3 Colman'4 indicated that plasmin has a regulatory feedback effect in the kinen forming activity of the plasma. Conclusion
Plasminogen activator in normal and inflamed gingiva of beagle dogs was histochemically identified using a fibrin film-slide technique. Microscopic examination clearly revealed the well-defined areas of liquefaction induced in the fibrin background. The lytic areas were, therefore, topographically related to the overlaying gingival section, thus facilitating the localization of plasminogen activator in the gingival tissue. The degree of fibrinolytic activity at the different times of incubation was
J Dent Res December 1977
greater in inflamed gingiva than in normal gingiva. Mild and severe gingivitis showed very much the same degree of activity. Fibrinolysis was observed only on the plasminogen-rich fibrin clot; there was no activity in the plasminogen-free fibrin clot. This is a clear indication that the lytic areas observed were not due to nonspecific proteolysis but due to specific fibrinolysis. Lytic activity was always associated with blood vessels and the crevicular epithelium. The degree of fibrinolytic activity was related with the time of incubation of the tissue section with the fibrin clot. There was no fibrinolytic activity associated with the oral epithelium.
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Index and The Retention Index System, J Periodontal Res 38:610-161, 1967. PANDOLFI, M.; BJORLIN, G.; and NILSSON, I. M.: Decreased Fibrinolytic Activity in Chronically Inflamed Tissue, Odontol Rev 20:31-35, 1969. ENGELBERG, J.: The Blood Vessel of the Dento-gingival Junction. J Periodontal Res 1:163-179, 1966. TODD, A. S.: The Histological Localization of Fibrinolysis Activator, J Pathol Bacteriol 78:281-283, 1959. WARREN, B. Z.: Fibrinolytic Properties of Vascular Endothelium. BritJ Exp Path 44:356-371, 1963.
9. SHERRY, S.; FLETCHER, A. P.; and ALKJAERSIG,
N.: Fibrinolysis and Fibrinolytic Activity in Man, Physiology Rev 39:343-383, 1959. 10. STANFON, G., and LEVY, M.: Method for the Correlation of Chemical and Histologic Composition,J Dent Res 48:38-42, 1969. 11. LUCAS, 0. N.; FUJITA, D.; and BREMNER, F.: Plasminogen Activator in Normal and Inflamed Dog Gingiva,j Dent Res 53:222, 1974. 12. KWAAN, H. C.: Tissue Fibrinolytic Activity Studied by a Histochemical Method, Federation Proceedings 25:52-56, 1966. 13. WARD, P. A.: A Plasmin-Split Fragment of C'3 as a New Chemotactic Factor, J Exp Med 126:189-206, 1967. 14. COLMAN, R. W.: Formation of Human Plasma Kinin, NEngJMed 291:509-515, 1974.