New Thesis on the Development of Erosion

n vitro decalcification of human enamel was induced with a suspension of dog leukocytes and corn starch in plasma under terile conditions. In controls without leukocytes decalcification did not occur. Enamel was protected against decalcification in the presence of leukocytes under these onditions by addition of sodium fluoride. Englander and Keyes’ demonstrated dental caries and gingival inflammation when a caries-conducive diet and caries-conducive bacteria were introduced to hamsters. When topical application of sodium fluoride was made on control animals under similar experimental conditions, no caries or in- flammation resulted. It is important to emphasize the body’s reactivity to stress- in this instance, the presence of carbohy- drates and caries-conducive bacteria, an approach in d[NextPage]ental research that has not been considered. The presence of leukocytes or corpuscles in saliva has recently received considerable attention. There is a decided fluctuation in the leukocyte count of saliva. The entire mucous membrane is a source of the salivary corpuscles, the highest number of which come from the gingiva. The mechanical stimulation of chewing and brushing and the metabolic activity of microorganisms induce leukocytes to mi- grate from the capillaries to the connective tissues and then to the gingiva.2 According to Florey,3 there is evidence that neutrophils are attracted by many species of bacteria and that starch grains are chemotactic agents. “Chemotaxis exerted by Staphylococcus albus on polymorphonu- This investigation was supported, in part, by Parker Dental Laborato[NextPage]ry, Cleveland, Ohio, USA. Received for publication June 9, 1969. * Present address: Department of Anatomy, School of Medicine, Case Western Reserve University, Cleve- land, Ohio 44106, USA. 1020clear leukocytes can occur in the absence of serum, complement, or glucose and in solutions that have had cheating agents added to remove as much calcium and mag- nesium as possible. The tonicity of solu- tions in which chemotactic experiments are carried out can be varied widely without affecting chemotaxis. The cells can be ac- tive at least ‘for a time in the absence of oxygen and can survive and remain mo- bile in solutions that do not contain a source of energy. It is clear that chemotaxis can occur in tissues even under what would at first sight appear to be extremely adverse conditions.”3 Suc[NextPage]h adverse conditions exist in the mouth. It is known that bacteria are coated with opsonins before phagocytosis occurs; one of these opsonins is lysozyme. Florey4 states that “the influence of the calcium ions in increasing phagocytosis is striking, especially in accelerating it.” Obviously, the complexity of inflamma- tion increases as the zoologic scale is climbed. In lower animals without a vascular system inflammation is represented by phagocytosis.5 a The lysosome, cytoplasmic substructure, been a cellular “killer” or has considered “suicide of its role in inflam- bag” because mation.6 are Although lysosomes probably in most, if not types of cells, present all, are most that are they prominent in cells more in for actively engaged phagocytosis, and example, reticuloendothelial cells p[NextPage]oly- morphonuclear leukocytes. The accumula- of at the tion polymorphs (chemotaxis) site tissue or bacterial invasion of injury results in a concentration of proteolytic enzymes to lyse the offending material. In this sense, one might view the polymorpho- nuclear leukocytes as mobile bags of lysosomes.7In this study the oral conditions tha would be the most conducive to phagocy- tosis were simulated. As has already bee stated, starch grains are chemotactic agents and these were used instead of bacteria t eliminate the latter as the prime cause o proteolysis and decalcification. The opinion that gingival inflammation is the precursor of proteolysis and decalcifi cation of the teeth prompted this study. Materials and Methods Fragments of human enamel were used Dentin was ground away, althoug[NextPage]h a negli gible amount of it remained on the inne surface of the enamel. The fragments wer washed and soaked in distilled water for 48 hours, then sterilized in dry heat at 180 C After sterilization they were again soaked this time in sterile, triple-distilled water fo a period of 45 minutes before the ex periment. Corn starch was sterilized in dry heat a 180 C for 45 minutes and then dissolve in sterile distilled water to give a 5 % solution. Heparinized blood (100 ml) from healthy young mongrel dog supplied th buffy coat, which was separated from th blood by five consecutive centrifugations. No cell count was taken. Scanning of th field proved that there was a high concen tration of leukocytes and a few red bloo cells. Plasma from the same dog, triple-dis tilled water, and 2 % sterile so[NextPage]dium fluorid solution were also used. All the experiments were performed i vitro in standard tissue-culture tubes (Bell co Leighton) under sterile conditions. Solutions were added with Pasteur pipettes. Calcium and phosphate contents were measured by the autoanalyzer. EXPERIMENT 1.-In experiment 1, 0.4 gm of enamel, 0.5 ml of plasma, 0.5 ml buffy coat in plasma, 0.5 ml 5% corn starch i distilled water, and 0.5 ml saline water were placed in tube 1. Tube 2 was identical to tube 1 except that it lacked enamel. Both tubes were incubated for 75 hours at 37 C and 5% CO2. EXPERIMENT 2.-The same pattern was followed with the addition of two more controls. Tube 3 consisted of 1 ml pure plasma, 0.5 ml distilled water, 0.5 ml 5% starch in distilled water, enamel, and 0.5 mlEROSION AND DENTAL CARIES [NextPage]1021 saline. Tube 4 consisted of only plasma. The experiment was conducted for 86 hours. EXPERIMENT 3.-The same pattern was again followed, but with an additional con- trol and without saline in each tube. Tubes 1 to 3 were the same as in the previous ex- periment. Tube 4 contained the same ma- terial as tube 1, except for the addition of 0.1 ml of 2% sodium fluoride. Tube 5 consisted of pure plasma. The experiment was terminated after 46 hours. Results At the termination of experiment 1, the pH of the supernatant in tubes 1 and 2 was 6.5. The calcium determination of the supernatant material in tube 1 was 27 mg %; tube 2 contained 8 mg % of calcium. At the termination of experiment 2, the pH of the supernatant material in tubes 1 to 3 was 6.5. Determination of calcium and phosphate concen[NextPage]trations in mg % revealed: tube 1, 19.5 and 9.4; tube 2, 2.8 (control) and 1.3; tube 3, 3.5 (control) and 2.0; tube 4, 9.6 and 4.5, respectively. At the termination of the experiment 3, the determination of calcium and phosphate concentrations in mg % revealed: tube 1, 15.8 and 12.3; tube 2, 6.0 (control) and 5.0; tube 3, 4.8 (control) and 3.2; tube 4, 4.9 (control) and 5.3; tube 5, 9.2 and 6.2, respectively. The results show that decalcification oc- curred only in tube 1. Tube 5 represents a calcium content of 9.2 mg % of pure plasma, which was used in all other tubes. Calcium concentration in tubes 1-4 was diluted by 50% to 4.6 mg % with distilled water and solution of starch in distilled water (Illustration). Decalcification oc- curred in tube 1, where enamel, plasma, and leukocytes wer[NextPage]e challenged by starch, which indicates that leukocytes were responsible for decalcification. In the controls decalcifi- cation did not occur. Discussion Regardless of the mechanism of decalcifi- cation that may be finally established, it appears from the present experiments that leukocytes may play an important role in cervical dentin decalcification, erosion, den- tal caries, and possibly in the metabolism of calcium in general. There is a definite correlation between leukocyte activity and caries development. Substances that have an effect on the activ- ity of leukocytes also have an effect on the development of caries. Fluorapatite is known to be less soluble in acid than hydroxyapatite, but only by about 10%. Brudevold and McCann8 re- ported: “It seems unlikely in view of . . this evi[NextPage]dence that reduced solubility as measured in vitro is the main cause of caries reduction. Some additional factors respon- sible for the unique action of fluoride must be found.” Karnovsky (Frei9) has shown that the en- ergy necessary for phagocytosis is provided by the glycolytic system. Considerable de- creases in the ratio adenosine triphosphate to adenosine diphosphate have been ob- served after the addition of fluoride, which inhibits the glycolytic system, whereas carbon monoxide, cyanide, or dinitrophenol have less of an effect.9 “Dental plaque has been reported to con- tain 6 to 180 ppm fluoride in the wet samples compared with 0.1 to 0.2 ppm in the saliva,”‘0 which is a considerable increase of concentration in relation to 1 ppm fluoride in drinking water. These data suggest that b[NextPage]esides transformation, to a certain extent, of hydroxyapatite to fluorapatite, another target or action of fluoride is the glycolytic system of neutro- phil granulocytes. The present experiments seem to confirm this. No decalcification wasdetected in controls where fluoride was added. Another inhibitor of glycolysis is iodo- acetate, which blocks phagocytosis com- pletely.” Jodoacetic acid reduced caries when added to the drinking water of rats.’2″3 Again, these data suggest that the formation of fluorapatite is of secondary importance in the prevention of caries. The data also suggest that fluoride and iodo- acetate function mainly in the prevention of caries as glycolysis inhibitors, which block phagocytosis and the subsequent re- lease of enzymes. Englander and Keyes’4 found that the th[NextPage]ird molars of every hamster that received 10 ppm of fluoride in drinking water before and throughout the posteruptive period re- mained caries free, whereas all the third- molar crowns were destroyed in each hamster that received the fluoride in drink- ing water only during a preeruptive period, by intubation or injection. Minimum dental caries activity occurred in the third molars of all hamsters that received 10 or 50 ppm of fluoride in drinking water for only 14 days posteruptively. Apparently, the caries- free condition of the posteruptively treated third molars is associated with the inhibi- tion of the glycolytic system and phagocy- tosis by neutrophil granulocytes. When hamsters were treated during the preerup- tive period only, there was no such inhibi- tion in the posteruptive per[NextPage]iod and caries resulted.Burger and Leonhardt (Gaillard’5) foun that in human neutrophil granulocytes, estra diol (, 5y/ml) stimulated the ingestion o carmine or trypan blue stains. Moreover, with this hormone the known inhibiting ef fect of NaF could be neutralized. The ad ministration of estradiol to control an gonadectomized rats resulted in an increase incidence of dental caries in all rats.’6 Th last two experiments demonstrate the cor relation between the activity of leukocyte and the development of caries, in this in stance under the stimulus of estradiol. Injury to cells resulting from the libera tion of lysosomal enzymes has been ob served. The hemolysins of streptococci 0 and S disrupt lysosomes in vivo and i vitro.17,18 The earliest morphologic conse quence of streptolysin action[NextPage] is the disrup tion of leukocyte lysosomes.’9 With thes facts the etiologic role of streptococci i dental caries can be understood more fully Possibly, the process starts with the hemo- lysins of streptococci disrupting leukocyte lysosomes, followed by release of enzymes, and finally proteolysis and decalcification. When there is stress (release of cortisone into the bloodstream) the situation is more complicated. Cortisone is an agent that stabilizes lysosomal membranes and, there- fore, we would expect less caries. Prisoners of war and people confined in concentration camps were subjected to severe mental stress and, in many instances, to an increase in disease and to severe undernutrition or starvation, a fundamental biologic stress and injury.20 A number of reports indicate a considera[NextPage]ble decrease in the incidence of caries in civilian populations during the war. Caries incidence decreased among prisoners of war.20 Moreover, when corti- sone is given over a four-day period to human subjects in a dose of 75 mg every six hours, there is a considerable increase of neutrophils21 (potential danger: increase of lysosomes). Lattes, Martin, and Ragan,22 found that bacterial infection would inhibit the effect of cortisone on inflammation. On the basis of this evidence, a contradictory conclusion may be drawn: cortisone may under certain circumstances increase the incidence of caries in experimental animals. The incidence of carious lesion formation in rats given 1.0 mg of hydrocortisoneacetate daily was significantly higher than that of rats given 0.5 mg daily.23 It seems that a[NextPage] moderate increase in systemic cortisone leads to a decrease in caries, whereas a sharp increase leads to an increase in caries. These opposite effects arise through the mediation of neutrophils and their lysosomes. Some authors have reported that the ammonia content of saliva of caries- resistant persons was consistently higher than that of caries-susceptible patients.24 Ammonia may inactivate the fourth com- ponent of complement and thus interfere with chemotactic mechanisms or opsonic activity that are dependent on comple- ment.’1 An explanation for caries resistance in this instance would be the block or slowdown of influx of neutrophils and there- fore the absence of lysosomal enzymes. In this study, the emphasis has been on the role of leukocytes in decalcification, without intention[NextPage] to neglect the role of bacteria or other factors in the development of dental caries. The functions of leukocytes and their response to stress depend on gen- etic, metabolic, and immunologic param- eters. These factors, together with are environment, responsible either for the to or resistance the development of dental caries. Conclusions Decalcification of enamel occurred with- out bacteria. Sodium fluoride prevented decalcification under sterile conditions. Cervical erosion of the teeth may develop through the action of salivary leukocytes. The development of caries may be initiated by the release of enzymes by neutrophils. References 1. ENGLANDER, H.R., and KEYES, P.H.: The Prevention of Dental Caries in the Syrian Hamster after Repeated Topical Applica- tion of Sodium Fluoride Gels, J[NextPage]ADA 73: 1342-1347, 1966. 2. NOLTE, W.A.: Oral Microbiology, St Louis: C.V. Mosby Co., 1968, p 50. 3. FLOREY, H.: General Pathology, 3rd ed, Philadelphia: W.B. Saunders Co., 1962, pp 98-126. 4. FLOREY, H.: Ibid, p 109. 5. TAMAYO, R.P.: Mechanisms of Disease, Philadelphia: W.B. Saunders Co., 1961, p 54. 6. THOMAS, L.: Papain, Vitamin A, LysQ-somes, and Endotoxin, Arch Int Med 110: 785, 1962. 7. BLACK, M.M., and WAGNER, B.M.: Dy- namic Pathology, St Louis: C.V. Mosby Co., 1964, pp 30-31. 8. BRUDEVOLD, F., and MCCANN, H.G.: Fluo- ride and Caries Control-Mechanism of Ac- tion, in NIZEL, A.E. (ed): The Science of Nutrition and Its Application in Clinical Dentistry, 2d ed, Philadelphia: W.B. Saun- ders Co., 1967, p 344. 9. FREI, J.: Energy Levels in the Human Cir- culating Leucocyte, in WOLSTENHO[NextPage]LME, G.E.W., and O’CONNOR, M. (ed): Biolog- ical Activity of the Leucocyte, Ciba Foun- dation Study Group No. 10, Boston: Little, Brown and Co., 1961, pp 87, 90, 91. 10. ZIPKIN, I.: The Metabolism and Safety of Fluorides, in NIZEL, A.E. (ed): The Sci- ence of Nutrition and Its Application in Clinical Dentistry, 2d ed, Philadelphia: W.B. Saunders Co., 1967, p 121. 11. HIRSCH, J.G.: Neutrophil and Eosinophil Leucocytes, in ZWEIFACH, B.W.; GRANT, L.; and MCCLUSKEY, R.T. (ed): The Inflam- matory Process, New York: Academic Press, 1965, pp 251, 265. 12. HARRIS, R.S.: Iron and Micro Elements, in NIZEL, A.E. (ed): The Science of Nutri- tion and Its Application in Clinical Den- tistry, 2d ed, Philadelphia: W.B. Saunders Co., 1967, p 105. 13. MCCLURE, F.J.; FOLK, J.E.; and RUST, J.D.: Smooth Surfac[NextPage]e Caries in White Rats: Ef- fects of Fluoride, Jodoacetate, Penicillin, Crisco, Butterfat and a Salt Mixture, in MCCLURE (ed): Fluoride Drinking Waters, Washington, DC: USPHS Publication No. 825, 1962, p 487. 14. ENGLANDER, H.R., and KEYES, P.H.: Pre- and Post-Eruptive Effect of Sodium Fluo-ride on Dental Caries in the Syrian Ham- ster, J Dent Res 45:1149-1153, 1966. 15. GAILLARD, P.T.: The Effect of Sex Hor- mones on the Phagocytotic Activity of Sur- viving Human Neutrophil Granulocytes, Exp Cell Res 8(Suppl):210, 1961. 16. SHAFER, W.G., and MUHLER, J.G.: Experi- mental Dental Caries, I Dent Res 33:842- 848, 1954. 17. WEISSMANN, G.; KEISER, H.; and BERN- HEIMER, A.W.: Studies on Lysosomes: III. The Effects of Streptolysins 0 and S on the Release of Acid Hydrolases from a Gran- ular Fracti[NextPage]on of Rabbit Liver, J Exp Med 118:205, 1963. 1 8. HIRSCH, J.G.; BERNHEIMER, A.W.; and WEISSMANN, G.: A Motion Picture Study of the Toxic Action of Streptolysins on Leucocytes, J Exp Med 118:223, 1963. 19. WEISSMANN, G.: Lysosomes, Blood 24:600, 1964. 20. SUTTON, P.R.N.: Stress in Dental Caries, in STAPLE, P.H. (ed): Advances in Oral Biology, Vol 2, New York: Academic Press, 1966, pp 116-119. 21. STURGIS, C.C.: 2d Hematology, ed, Spring- field, Ill: Charles C Thomas, 1955, 708. 22. LATTES, R.; MARTIN, J.R.; and RAGAN, C.: Suppression of Cortisone Effect on Repair in the Presence of Local Bacterial Infec- tion, Amer J Path 30:901, 1954. 23. Liu, F.T.Y., and LIN, H.S.: Effect of Hy- drocortisone Acetate on Dental Caries and Salivary Glands in Adrenalectomized Fe- male Rats, J Dent Res 47:158,[NextPage] 1968. 24. FINN, S.B.; CHERASKIN, E.; VOLKER, J.F.; HITCHCOCK, H.P.; LAZANSKY, J.P.; PARFITT, G.J.; THOMAS, A.E.; and SHARRY, J.H.: Clinical Pedodontics, Philadelphia: W.B. Saunders Co., 1957, p 623.