Histologic identification of mast cells in human dental pulp

endodontics Editor: MILTON SISKIN, College of Dentistry

D.D.S.

The University of Tennessee 847 Monroe Avenue Memphis, Tennessee 38 163

Histologic identification of mast cells in human dental pulp Gary Scott Miller, D.D.S., Sam J. Piliero, Ph.D.,*** New York, N. Y.

* Robert N. Sternberg, D.D.S., ** and Paul A. Rosenberg, D.D.S.,*“**

M ast cells

were first discovered in 1877, by Paul Ehrlich, who thought that they were especially abundant in well-fed animals (German, mast = well fed).’ These cells arc found mainly in loose connective tissue, and with few exceptions, their presence in tissues and organs of higher vertebrates can be correlated to the content of connective tissue. It is at the level of fish and amphibians that “true” mast-cell elements appear in larger numbers.2 In man and most other mammals, mast cells vary widely in shape and size. The cells are greatly influenced by surrounding tissue components. In loose or areolar connective tissue they assume plump, rounded shapes, whereas in the dense fibrous tissue of skin or organ capsules they may appear as elongated filiform elements. Cell diameters range from 3.5 to 24 micrometers.2 Mast cells are generally said to contain prominent nuclei which are round or oval in shape and are generally not lobulated. The cytoplasm contains an abundance of spheroidal, basophilic granules which are often so numerous that they may obscure other cytoplasmic structures, especially the nucleus. The presence of sulfated mucopolysaccharides

*Formerly, postgraduate endodontic student, New York University College of Dentistry, New York, N. Y. Presently, in the private practice of endodontics, North Miami Beach, Fla. **Formerly, resident in endodontics, Veterans Administration Hospital, New York, N. Y. Presently, staff endodontist, Veterans Administration Center, Bay Pines, Fla. ***Professor and Chairman, Department of Histology, New York University College of Dentistry, New York, N. Y. ****Clinical Associate Professor and Director of Postgraduate Endodontics, Department of Endodontics, New York University College of Dentistry, New York, N. Y.

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in these granules is responsible for the characteristic metachromatic purple-red appearance of the cell when stained with certain basic aniline dyes. The granules of the mast cells contain a number of important mediators of inflammation. Of prime importance is histamine, an amine whose major effect is realized in the early stages of the inflammatory response. Although histamine has no direct action on capillaries itself, it enhances post-capillary venular permeability by causing separation of endothelial cells along their boundaries, thereby exposing the basement membrane to plasma leakage. Other effects of histamine include peripheral vasodilation of arterioles, stimulation of exocrine glands, and contraction of nonvascular smooth muscle.3 Histamine is bound ionically to another granule constituent, heparin, an acid mucopolysaccharide with striking anticoagulant properties. 4 Heparin inhibits blood coagulation by two different mechanisms, acting as an inhibitor both of prothrombin activation and of the thrombin-fibrinogen reaction.” It is now believed that anticoagulant activity is only one of a number of biologic activities demonstrable with trace amounts of heparin. Heparin inactivates bacterial products and other noxious agents, is involved in the energy metabolism of the affected cells, and has an inhibitory effect on the lymphocyte response.“, 6 Mast cells of rats and mice also contain the amine 5hydroxytryptamine (serotonin), which increases smooth-muscle contraction and vascular permeability. Other primary mediators in mast cells include an eosinophil chemotactic factor of anaphylaxis (ECF-A) which relates to allergic phenomena, a slow-reacting substance of anaphylaxis (SRS-A) which possibly augments and prolongs the action of histamine in inflammatory and hypersensitivity reactions, and a platelet-activating factor (PAF).7 The mast cells are located at strategic points in the vicinity of small blood vessels, and accordingly are in a position to release their products into both the surrounding tissues and the bloodstream. When mast cells are injured or appropriately triggered, degranulation occurs, either by extrusion of granules or by discharge of the granule contents into the surrounding environment. The stimuli that cause degranulation include the following: (1) physical injury-mechanical trauma, heat, irradiation; (2) certain chemical agents“histamine-liberators” ( sueh as (48/80), toxins, snake venoms, bee venom, trypsin, surfactants, dextran, polyvinylpyrrolidone (PVP), and, perhaps more relevant to inflammatory responses, a neutrophil lysosomal cationic protein; (3) immunologic processes-antigenic challenge of homocytotropic antibody IgE sensitized cells, or exposure to anaphylatoxins (C3a and C5a).’ It is now clear that the mast cell is not merely a sac that breaks open when acted upon by drugs and other agents, but a cell capable of secretion of stored materials through a specific process of exocytosis. The release of amines is selective and noncytolytic, as indicated by the fact that histamine is released, whereas other, nongranule constituents, such as lactic dehydrogenase, potassium, and ATP, are retained.x Electron microscopy of mast cells has revealed that secretion of granule material involves fusion of perigranular membranes with the cell surface membrane.8 The granules are, therefore, exposed to the extracellular medium even while they are still within the confines of the cell. Some granules are actually extruded from the cell and stick to the cell surface, whereas others are depleted of amines and remain within the ce11.8Granules that have lost their soluble components are changed in appearance and density. When stimulated with degranulating agents, the granules in the mast cell closest to the surface are

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affected first. With increasing stimulation, granules toward the cell interior are also involved.8 The mast cell probably responds to stimuli in a graded fashion rather than in an all-or-none manner. When releasing agentsare applied locally to isolated cells, a discrete, localized degranulation develops at one or more sites on the cell surface. Repeated application of the stimulus increasesthe extent of degranulation.8 It hasbeenproposedthat, insteadof regarding mastcells as secretingcells, one should think of them as cells which accumulate material.6, g Heparin, histamine, and related sulfated mucopolysaccharidesare combined with basic protein to form granuleswhich are available for ion exchange. This view of the mast cell allows for the variability in fixation and staining properties of, and variation in biogenic amines present for, mast cells in different speciesand even in different tissueswithin the samespecies.6Padawelgproposed that particles, fluid, and ions are taken into the mastcell by the processesof phagocytosis and pinocytosis. He suggeststhat an analogy could be made between the mast cell and a chromatographiccolumn packed with beads(the specific granules) that has a combination of ion-exchange and immobilized-enzyme properties working in a coordinated way. By identifying histamine as the essentialcation in the chromatographicexchange,he provides for histamine an intracellular physiologic function. This would explain the presenceof large quantities of this amine in one particular cell type (the mast cell) and the fact that histamine is readily synthesized by mast cells. Since mast cells are ubiquitous in connective tissues throughout the body, including the soft tissuesof the oral cavity, it seemspuzzling that so few reports have demonstrated their existence in the pulp. Wislocki, Bunting and Dempsey,‘Owith the use of rat, monkey, and human tissue specimens, found an “authentic mast cell” on only one occasion in the pulp of a monkey’s tooth. Wislocki and Sognnaes,” in studying different mammalian tissues, found that mast cells were numerous in the stroma of the gingival tissue, but entirely lacking in the pulp. Dockrill,‘2 with the use of human biopsy and postmortem material, concluded that, normally, pulp tissue appearsto be devoid of mast cells, but that such cells can be demonstratedin conditions which bring about swelling of the pulp, as in pulp polyps. Anneroth and Brannstrom,13in a study of pulp and gingival specimensfrom eighteen patients, demonstratedthe presenceof mast cells in all of the gingival specimens,but noted an absenceof them in all pulp specimens,except for one tooth examined. Pohto and Antila,14 who used fluorescent microscopy, could not detect the presenceof mast cells in rat, guinea pig, rabbit, cat, or human pulpal tissue, but they did observe a fluorescencesuggestive of histamine in the pulp vasculature of these species. Eda and Langeland i5 used human material demineralized in EDTA and stainedwith toluidine blue and noted mastcells in gingival and periodontal ligament specimensbut not in pulp tissue in the sameblock specimens.Those authorsconcluded that with “all factors taken into consideration, it seemsthat mast cells are a very rare occurrence in the dental pulp although they are frequent in other oral and dental tissues.” Zachrisson and Skogedal16*l7 used human pulp material from clinically caries-free deciduous anterior teeth, caries-free premolars removed for orthodontic reasons, and deciduous molars with carious lesions. Immediately after removal the teeth were divided longitudinally into two halves by forceps and fixed for 24 hours at room temperaturein Newcomer’s fluid or lead acetateformalin. The hard tissue was removed in small pieces, and the pulps were dissectedout and fixed for 24 hours in a fresh volume of the same

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Fig. 1. Photomicrograph of pulp section depicting the presence of mast cells (arrows). inflammatory cells. (Toluidine blue stain. Magnification, X 160.)

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Note surrounding

fixative. Serial sections of 5 to 10 micrometers were stained for mast cells by astra blue and toluidine blue techniques. The results from these studies indicated that pulps from deciduous and young permanent human teeth, without inflammatory cells or with only a few lymphocytes, were devoid of mast cells. However, in pulps with an increased cellular infiltration, significant numbers of mast cells were found adjacent to immature and mature plasma cells and to large lymphocytes. Previous studies which have attempted to identify mast cells in dental pulp have used demineralization or tooth-splitting procedures. Eda and Langeland’j found that mast-cell fluorescence is destroyed by acid-demineralizing agents. Tooth splitting may damage the pulp by crushing it with forceps, or cutting and heating it with burs, stones, or discs. Furthermore, a delay between tooth extraction and pulp extirpation and fixation can result in dehydration of the tissue. The purpose of the present study was to examine inflamed and noninflamed human pulp tissue for mast cells, by means of conventional procedures of endodontic access and extirpation to obtain the pulp tissue. MATERIAL AND METHODS

Pulp tissue was obtained from the Endodontic Clinic and Oral Surgery Clinic at the New York University College of Dentistry. Teeth with deep caries and/or restorations were used to provide specimens of inflamed pulp tissue. In the Endodontic Clinic, after application of the rubber dam, conventional endodontic access was made by means of high-speed rotary instruments with a water spray. Pulps were then removed with barbed broaches. Virgin teeth that were free of periodontal pathosis were obtained from the Oral

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Fig. 2. High-power view of Fig. 1, showing mast cells in area I. (Magnification,

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Surgery Clinic. These teeth, extracted for orthodontic reasons, were used to provide noninflamed pulp tissue which served as the control group. Immediately after extraction, endodontic access openings were made and the pulps were extirpated with barbed broaches. Tissue fragments and severely shredded pulp specimens were excluded from both the experimental and control groups. Teeth with tortuous or narrow canals were not used since they precluded removal of the pulp in-toto. Extirpated pulp tissues were immediately fixed in either 10 per cent neutral buffered formalin or lead acetate formalin for a period of 24 to 48 hours.17-lg Each pulp was stained with one of the following staining solutions: Luna’s stain for mast cells (aldehyde fuchsin solution counterstained with methyl orange solution),20 0.5 per cent toluidine blue at a pH of 1.O, or 1.O per cent toluidine blue at pH of I .0.i6* 17,21 The pulp tissues were embedded in paraffin, and serial sections were made at 6 micrometers. Stained sections were examined by means of bright-field light microscopy for the presence of mast cells. RESULTS

Because of the presence of extensive calcification in many of the inflamed specimens, not all of the pulps could be sectioned properly. Accordingly, results are based on twenty-five inflamed pulps and ten noninflamed pulps that were adequately sectioned. Typical mast cells could not be observed in any of the noninflamed pulps. These pulps generally revealed a few scattered lymphocytes and macrophages in their connective tissue. Numerous mast cells were found in two inflamed-pulp specimens. These pulps were from the group fixed in lead acetate formalin and stained with 1.O per cent toluidine blue.

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Fig. 3. High-power view of Fig. I, showing mast cells in area 2. (Magnification,

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x400.)

The mast cells were observed to be rounded or oval and densely granulated. They were intact and well preserved, with none of the cells present in the process of degranulation (Figs. l-3). The presence and distribution of the mast cells could not be correlated to the number and types of other inflammatory cells observed. In fact, although some mast cells were present in certain intlamed areas, other inflamed regions in the same sections were totally devoid of mast cells. In one pulp specimen, mast cells were located in an area in which polymorphonuclear leukocytes predominated, whereas in other regions the picture was one typical of chronic inflammatory cells (lymphocytes and plasma cells). In the second pulp specimen that contained mast cells, chronic inflammatory cell types predominated throughout the area of the mast cells as well as in other regions of the pulp. In general, in both pulps, mast cells were present ahout the small blood vessels. It should be noted that there were a number of cells which were suggestive of mast cells, but they did not exhibit sufficient metachromasia for definitive identification. DISCUSSION

Mast cells are extremely sensitive to any manipulation which may cause rupture of the cell membrane with subsequent degranulation. Once degranulation has occurred, mast cells can no longer be recognized microscopically. I7 Critical steps are involved in obtaining, fixing, and staining pulp tissue. The fragility of mast cells may explain why controversy exists regarding their presence in dental pulp. Furthermore, there is morphologic similarity between mast cells and macrophages with ingested particles. This similarity further complicates identification of mast cells in inflamed, fibrous tissue. Not all mast cells attain levels of metachromasia characteristic of sulfated compounds,

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and less mature cells may bind little or no dye. This may be due to the content of variable amounts of highly sulfated acid mucopolysaccharideswithin the granules. As Zachrisson** stated, mast cells are first recognized by the appearanceof their granules. The granule matrix of the immature mast cells contains characteristic proteases,along with weakly sulfated acid mucopolysaccharides,which are presumably heparin precursors.The sulfate groups of heparin may be presentboth as sulfate estersand sulfamido groups. The heparin precursorsare poor or totally lacking in either or both of thesesulfate groups. This is in contrast to the acid mucopolysaccharidesof the mature mast cells, which probably contain chemically mature heparin. Accordingly, the immature mastcells do not allow for the necessary dye interactions to cause sufficient metachromasianeeded for definitive identification. DelBalso and associates**undertook an investigation to determine possible quantitative changesin porcine pulpal histamine levels subsequentto thermal and electrical insult. Whereaselectrical insult was found to have no significant effect, thermal injury produced a fourfold increase in pulpal histamine levels within 30 minutes, as revealed by quantitative spectrophotofluorometric assays.These findings suggestthat histamine may play a role in the initial stagesof pulpal inflammation. Although mast cells are the only proven repository of histidine decarboxylase(the enzyme which converts histidine to histamine), it has been postulated that there is an inducible form of histidine decarboxylase not contained in mast cells.23This proposal speculatesthat the enzyme is a basic part of the mechanismthrough which the capillaries exert autonomousactions, and there is a correlation between activation of the inducible form of histidine decarboxylaseand the appearance of histamine reproducible events. Accordingly, the level of histamine in the dental pulp may not relate directly to the number of mast cells present. SUMMARY

Previous investigators who attempted to identify mast cells in the dental pulp have useddemineralizing or tooth-splitting proceduresto obtain their tissue samples.However, Eda and Langeland15found that the fluorescence of mast cells is destroyed by acid demineralizing agents. On the other hand, tooth splitting may damagethe pulp by crushing it with forceps, or cutting and heating it with burs, stones, or discs. In the present study, we used the extirpated pulps from teeth in which endodontic accessopenings were made by meansof high-speedrotary instruments with water spray. Metachromatic staining methods failed to demonstrate mast cells in any of the noninflamed pulp specimens.Two of the inflamed pulp specimensrevealed numerous mast cells which appearedintact and well preservedwith no evidence of degranulation. As to the distribution of the mast cells, there was no correlation with the number and types of other inflammatory cells observed. Although severalcells presentin the specimensexamined were suggestiveof mastcells, only thosecells that revealeddefinitive metachromasia were included in this study. We wish to express our appreciation to Mrs. Gloria Turner and Mrs. Alba Velez for their technical assistance in the preparation of the slides for this project. REFERENCES

1. Greep, R. 0.) and Weiss, L., editors: Histology, ed. 3, New York, 1973,McGraw-Hill Book Co., Inc., pp. 149-151.

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2. Zweifach, B. W., Grant, L., and McCluskey, R. T., editors: The Inflammatory Process, New York, 1965, Academic Press Inc., pp. 355-388. 3. Rosenberg, P. A.: Lecture: Mediators of Inflammation, New York University College of Dentistry, Feb. I& 1977. 4. Weissman, G., editor: Mediators of Inflammation, New York, 1974, Plenum Press, pp. 26-30, 141-161. 5. Zachrisson, B. U., and Schultz-Haudt, S. D.: Biologically Active Substances of the Mast Cell, J. Periodontol. Res. 2: 21-35, 1967. 6. Jaques, L. B.: Review-The Mast Cells in the Light of New Knowledge of Heparin and Sulfated Mucopolysaccharides, Gen. Pharmac. 6: 235-245, 1975. 7. Ryan, G. B., and Majno, G.: Inflammation, Kalamazoo, Michigan, 1977, The Upjohn Company, pp. 48-50. 8. Goth, A., and Johnson, A. R.: Current Concepts on the Secretory Function of Mast Cells, Life Sci. 16: 1201-1213, 1975. 9. Padawer, J.: Editorial-The Ins and Outs of Mast Cell Function, Am. J. Anat. 141: 299-302, 1974. IO. Wislocki, G. B., Bunting, H., and Dempsey, E. W.: Metachromasia in Mammalian Tissues and Its Relationship to Mucopolysaccharides, Am. J. Anat. 81: l-38, 1947. I I. Wislocki, G. B., and Sognnaes, R. F.: Histochemical Reactions of Normal Teeth, Am. J. Anat. 87: 239-276, 1950. 12. Do&ill, T. E.: Tissue Mast Cells in the Oral Cavity, Aust. Dent. J. 6: 210-214, 1961. 13. Anneroth, G., and Brannstrom, M.: Autofluorescent Granular Cells and Mast Cells in the Human Gingiva, Odontol. Revy. 15: 10-14, 1964. 14. Pohto, P., and Antila, R.: Assay of Histamine in Dental Pulps, Acta. Odontol. Stand. 28: 691-699, 1970. 15. Eda, S., and Langeland, K.: The Alteration of Mast Cell Staining Due to Various Fixatives and Demineralizing Agents, Stand. J. Dent. Res. 78: 217-231, 1970. 16. Zachrisson, B. U.: Mast Cells in Human Dental Pulp, Arch. Oral Biol. 16: 555-556, 1971. 17. Zachrisson, B. U., and Skogedal, 0.: Mast Cells in Inflamed Human Dental Pulp, Stand. J. Dent. Res. 79: 488-492, 1971. 18. Haust, D. M., and Landing, B. H.: Histochemical Studies in Hurler’s Disease: A New Method for Localization of Acid Mucopolysaccharides, and An Analysis of Lead Acetate “Fixation,” J. Histochem. Cytochem. 9: 69-86, 1961. 19. Mathiesen, A.: Preservation and Demonstration of Mast Cells in Human Apical Granulomas and Radicular Cysts, Stand. J. Dent. Res. 81: 218-229, 1973. 20. Luna, L. G.: Manual of Histologic Staining Methods of the Armed Forces Institute of Pathology, ed. 3, New York, 1968, McGraw-Hill Book Co., Inc., pp. 114-I 15. 21, Zachrisson, B. U.: Mast Cells of the Human Gingiva. III. Histochemical Demonstration of Immature Mast Cells in Chronically Inflamed Tissue, J. Periodontol. Res. 3: I36 145, 1968. 22. DelBalso, A. M., Nishimura, R. S., and Setterstrom, J. A.: The Effects of Thermal and Electrical Injury on Pulpal Histamine Levels, ORAL SURG. 41: I IO-1 13, 1976. 23. Schayer, R. W.: Histidine Decarboxylase in Mast Cells, Ann. N. Y. Acad. Sci. 103: 164-178, 1963. Reprint requests to: Dr. Paul A. Rosenberg 133 East 58th St., Suite 306 New York. N. Y. 10022