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«Pergamon Press.Printed inGt.Britain. Archs or01 Bid. Vol. 13, pp. 271-288, 1968. THE EFFECT OF FORMOCRESOL ON HAMSTER CONNECTIVE TISSUE CELLS, A ...»

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Radioautography; grain counts of the sponge Grain counts were made in at least 100 individual fibroblasts of sponge implants representing each period. Since many cells of the FC-treated sponge were entirely free of overlaying silver grains during the early post-operative period, the average grain counts of labelled cells might not be a valid expression of the total activity of the fibroblast population. Hence, the percentage of unlabelled fibroblasts was studied by counting 100 cells in a randomly selected field at the periphery of the sponge. The results showed that after 5 hr about 40 per cent of the fibroblasts were unlabelled in the experimental animals whereas the percentage of the unlabelled cells of the control was 12 per cent (Figs. 17 and 18). One day after implantation, the experimental hamster showed 16 per cent of the fibroblasts unlabelled and all of the cells in the control showed labelling of varying degrees. By the third day all of the fibroblasts in both groups were labelled (Figs. 19 and 20).

w In view of this difference the quantitative evaluations of silver grains recorded in Figs. 28 and 29 were obtained from counting only the labelled fibroblasts throughout L. H. STRAFFON AND S. S. HAN the experimental period. It might be noted that the patterns of histograms representing 4 hr and 24 hr after proline-Ha are similar to each other, except that generally lower values and less significant differences are shown in the 24-hr post-injection group. The average number of grains/control fibroblast is significant 5 hr after the sponge implantation, appears to reach a peak between 1 and 3 days after the implantation, and is followed by a gradual reduction thereafter. The Fe-treated group shows only a minimal labelling 5 hr after the implantation of the sponge, but the labelling rises rapidly within a day. However, the grain number/experimental fibroblast during the first few days is never as high as that of the control.

35

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FIG. 27. The level of total tritium activity in whole blood of three adult hamsters.

The relative number of extracellularly located grains in samples taken 24 hr after the injection of radioactive precursor shows changes that tend to amplify the results of grain counting (Figs. 21-26). A greater number of extracellular grains may be noted in the control as early as 1 day after implantation of the sponge (Figs. 21 and 22).

The maximum difference was expressed in sponges taken 3 days after the implantation (Figs. 23 and 24). By day 10 and 1 month such difference could not be observed (Figs.

25 and 26).

FORMOCKESOL, CONNECTIVE TISSUE AND PROLINE-H3 RADIOAUTOQRAPHY 277

23 -

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Aside from the generally accepted effects of pulp “mummification”, the mechanism of FC action on the connective tissue cells has not been clarified to a satisfactory extent. It has been shown that one of the components of FC, the formaldehyde, reacts with NH, group of certain ammo acids and creates a methylene bridge linking protein chains together ; for example, lysine to lysine or fromlysine to glutamine (BAKER, 1960; DE ROBERTIS, NOWINSKI and SAEG,1965; DOBBS,1961; LILLIE, 1965; SOLLMANN, 1957; WOLMAN, 1955). FRAENKEL-CONRAT (1954) and STAEHELIN (1958) have reported that the inactivation of the tobacco mosaic virus by formaldehyde is due to its interaction with the amino group of adenine, cytosine and guanine in the ribonucleic acid moiety. They also reported that DNA did not react with formaldehyde under the same conditions. HASELKORN and DOTY (1961) have further elaborated upon this and stated that the reaction of formaldehyde with polynucleotides is first a denaturation step, then the reaction of formaldehyde with freed amino groups occurs.

With respect to the tricresol, which is the other major component of FC, a review of the literature indicates that its specific action at the cellular level has not been determined. The toxicity of cresol is said in standard text books to be less than that of the phenol, although the opposite has been reported (DOBBS,1961; FRANCISand WOOD, 196 1; KRANTZ and CARR, 1954; PAFF, LEHMAN and HALPERIN, 1945 ;

SOLLMANN, 1957). ZANDER and GLASS (1949) stated that phenol placed prior to capping of the exposed dental pulp did not interfere with nor enhance the healing process.

FORMOCRESOL, CONNECTIVE TISSUE AND PROLINE-II’ RADIOAIJTOGRAPHY

The result of the present study strongly indicates that the FC used in l/50 of the normal concentration still causes an effective Gxation of cells in the immediate vicinity of the sponge implant around which the ingrowth of the connective tissue begins.

Such cytotoxic effect is evidenced by the fact that 40 per cent of the cells in the immediate vicinity are not labelled during the early post-operative period. In this connection it is of a fundamental and clinical importance that the connective tissues around the FC-treated sponge showed far fewer inflammatory cells as compared to the control sponge. It indicates that the cytotoxic effect of FC used at this concentration might have been enough to modify changes in the capillary permeability as well as the leukocytic migration which normally occurs in the immediate vicinity of implanted sponge (HAN and AVERY, 1964). In addition to the few PMN observed, the number of infiltrating mononuclears on day 1 was also smaller in the experimental animal, and the appearance of giant cells was delayed. Thus, the histological appearance of the sponge and the connective tissue in its immediate vicinity indicates that the FC-treated sponge might have effectively reduced the inflammatory response of the region. Of further interest is the fact that by the tenth day both the experimental and control sponges showed a comparable recovery in the pattern of connective tissue ingrowth and in the number of silver grains over fibroblasts. This is further supported by the observation that, even in the femur which was treated with the full concentration of FC, the repair of the bone in the surgical lesion had progressed considerably by the tenth day of the experiment.





The radioautographic studies of fibroblast activity following the administration of proline-HS have been done by numerous authors under different conditions.

HAN, AVERY and BANG(1967), in studying the dental pulp fibroblast following actinomycin D administration, have established a correlation between the regressive changes occurring in the cytoplasmic ultrastructure and the amount of proline-Hs taken up by the fibroblasts as judged by the appearance and the number of silver grains overlaying fibroblasts. It appears, therefore, reasonable to presume that the significant reduction of grain numbers in experimental animals during the first 3 days of study might be taken as evidence for damage incurred in the cytoplasmic structure of the fibroblasts in the region with respect to their protein synthetic capacity.

The increase of the number of grains in the control animals at different times, which appear to be highest somewhere between l-3 days, might reflect the increased function of average fibroblasts in the growing connective tissue and thus support histological observations in which the fibroblasts appeared more robust during this period. The differences between control animals sacrificed 4 hr and 24 hr after the proline-H5 injection could be partially due to differences in the level of available precursors in the blood. It is more likely that the differences are due to the way in which the grain counts were made. Since the cell boundary in fibroblasts was not distinct and often difficult to recognize, only those grains that were clearly inside the cell were counted and the grains that were present along the surface were considered extracellular and not counted.

The cytotoxicity of FC even at the concentration of l/50 of that of the FC in normal use is evidenced by the large percentage of unlabelled fibroblasts in animals A.O.B. 13 No. 3-c VOL.

AND S. S. HAN L. H. STRAFFON sacrificed shortly after the application of the drug. On the other hand, that there was no significant difference between experimental and control groups after 10 days of experiment further strengthens the conclusion that the FC effect is limited to the first 10 days of the experiment.

While histologic examination of the femurs suggests that a satisfactory recovery can be achieved shortly after treatment with FC of full strength, a further study of the relative effects of various FC concentrations on the metabolism of connective tissue cells will be of significance. In the meantime, our observations can be taken as evidence for the fact that FC kills a large number of cells as well as degrading physiological activities of surviving cells in the vicinity even at a fairly low concentration. As the result of such cytotoxic and cytostatic effects, the connective tissue of FC-treated animals could maintain a relatively inflammation-free status which is followed by a normal recovery within the first month. Such a freedom from inflammation might be a beneficial aspect of FC-treatment in tissues such as dental pulp, where an inflammatory response subsequent to pulpotomy could create a serious clinical complication whereas the tooth might be able to withstand the temporary slowdown of the recovery process without manifesting any clinical sign.

Acknowledgement-This study was supported by grants from the Le Gro Fund of the University of Michigan School of Dentistry and U.S.P.H.S. DE-01620, DE-0231 1, DE-0273 1 and HD-03 147.

Resume-La reaction cellulaire conjonctive envers le formocresol a CtC Btudiee au niveau d’eponges implant&es et dam des pertes de substance du femur. a l’aide de radioautographies quantitatives, apres injection de proline-H8. Une eponge est implant& dans le dos de la nuque et une cavite est prepan% chirurgicalement dans le femur droit

de 24 hamsters. Le formocresol est dilue au 1/50eme pour l’etude utilisant des Sponges:

une concentration normale est implantee dans le femur. L’intervalle de temps entre l’application medicamenteuse et le sacrifice est de 5 heures, 1 jour, 3 et 10 jours et un mois. La proline-H3 est inject&e 4 a 24 heures avant le sacrifice. Trois animaux ont Btt utilises pour determiner, au scintillateur, le taux sanguin de tritium de 2,5 min-6 jours.

Les tissus sont fixes dans la solution de Bouin, inclus puis coupes. Une emulsion Kodak NTB-3 est utilisee nom l’autoradioarauhie. Le formocresol a 1150eme nrovoaue une dfgenerescence cellulaire au voisinageimmediat de l’implant--+ponge. Cependant, dans tous les animaux trait&s par le formocresol, une reduction marquee des cellules inflammatoires est observee. Au lObme jour, les Sponges experimentales et controles montrent des stades similaires. Ce fait est egalement valable pour la cicatrisation observee au niveau du femur. 11semble que le formocresol n’emp&he pas la cicatrisation conjonctive, tout en supprimant significativement la reponse inflammatoire initiale.

Zusammenfassung-Mit Hilfe quantitativer Autoradiographie nach Prolin-H3-Itrjektion wurde der EinfluB von Formokresol auf Bindegewebszellen in Schwammimplantaten und Femurwunden untersucht. Bei 24 Hamstern wurde im Nackenrticken ein Schwamm implantiert und der rechte Femur chirurgisch eriiffnet. Fur den Versuch mit Schwammimplantaten wurde Formokresol auf 1/SO der normalen Konzentration verdtinnt, wahrend am Femur die normale Konzentration getestet wurde. Die Zeitintervalle von der Anwendung des Pharmakons bis zur Tiitung betrugen 5 Stunden, 1,3 und 10 Tage sowie 1 Monat. H3-Prolin wurde 4 oder 24 Stunden vor der Totung injiziert. 3 Tiere wurden dazu verwendet, den Tritiumspiegel im Blut von 2,5 min bis 6.zu Tagen mit Hilfe der Scintiallationszlhluna zu bestimmen. Die Gewebe wurden in Bouin’s-Lijsune fixiert, und in tiblicher Weiseeingebettet und geschnitten. Die Schnitte wurden rn;

FORMOCRESOL, CONNECTIVE TISSUEAND PROLINE-H’RADIOAUTOGRAPHY 281 Kodak-NTB-3 Filmen bedeckt und ausreichend lange exponiert. Formokresol in l/50 Konzentration verursachte in der unmittelbaren Umgebung des Schwammimplantates Zelldegeneration, wie histologisch und durch autoradiographische Kornzahlungen nachweisbar war. Bei allen mit Formokresol behandelten Tieren wurde jedoch eine deutliche Reduktion der Anzahl infiltrierender Entztindungszellen beobachtet. Urn den zehnten Tag herum zeigten sowohl die Versuchs- als such die Kontrollschwlmme einen vergleichbaren Stand des einwachsenden Bindegewebes. Dies traf such fur die Heilung der Femurwunde zu. Daraus wurde geschlossen, daD Formokresol die verziigerte Wiederherstellung des Bindegewebes nicht stiirt und zugleich initiale Entziindungsreaktionen signifikant herabzudrticken vermag.

REFERENCES

ANDERSON, A. 1965. The protein matrices of the teeth and periodontium in hamsters-a A. tritiated proline study. M. SC. Thesis, Univ. Michigan, p. 84.

BAKER,J. R. 1960. Cytological Technique (4th ed.), p. 150. Wiley, New York.

BARTELS, A. 1941. A bacteriologic appraisal of some material used in root-canal therapy. J. Am.

H.

dent. AS. 28, 1108-1112.

BEAVER,H. A., KOPEL, H. M. and SABES,W. R. 1966. The effect of zinc oxide-eugenol cement on a formocresolized pulp. J. dent. Child. 33, 381-396.

BERGER, E. 1963. An evaluation of the effects of formocresol on the pulps of human primary molars J.

following pulpotomies. M. SC. Thesis, Univ. Michigan, p. 92.

BLACK, G. V. 1915. Special Dental Pathology, p. 489. Medico-Dental, Chicago.

BONNECKER, and Par~z, H. (translator). 1899. Modem methods of treatment of diseased pulps.

Ohio dent. J., Toledo. 19, 183-184.

BONSACK,C. 1930. Technique of mummification with the trio paste of Prof. Gysi. Dent. Items 52, 681-685.

BUCKLEY,J. P. 1904. A rational treatment for putrescent pulps. Dent. Rev. 18, 1193-1197.

COOLIDGE, D. 1932. Reaction of dog tissue to drugs used in root canal treatment. J. Am. dent. Ass.

E.

19,747-759.

DE ROBERTIS,E. D. P., NOWINSKI, W. W. and SAEG, F. A. 1965. Cell Biology (4th ed.), p. 446.

Saunders, Philadelphia.

DIETZ, D. R. 1961. A histologic study of the effects of formocresol on normal primary pulpal tissue.

M. SC. Thesis, Univ. Washington, p. 55.

DOBBS,E. C. 1961. Pharmacology and Oral Therapeutics (12th ed.), p. 578. Mosby, St. Louis.

DOYLE, W. A. 1961. A comparison of the formocresol pulpotomy technique with the calcium hydroxide pulpotomy technique. M. SC. Thesis, Indiana Univ., p. 68.



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