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Cytotoxic effects of periodontally involved surfaces of human teeth
ArchsoralBiol.Vol. 16,pp. 465-468,1971.Pergamon Press.P&ted in GreatBritain
CYTOTOXIC EFFECTS OF PERIODONTALLY INVOLVED SURFACES OF HUMAN TEETH C. G.
HATFIELD
and A.
BAUMHAMMFZRS
School of Dental Medicine, University of Pittsburgh, Pittsburgh, Pennsylvania 15213, U.S.A. gingival tissue is separated from a sound tooth and then replaced, uneventful healing with reattachment occurs (RAMFJORD,1951). However, in cases involving teeth effected by periodontal disease of long duration, reattachment to the contaminated root surfaces is unpredictable and difficult despite clinical methods employed (World Workshop in Periodontics, 1966). This study was initiated to clarify the above clinical problem and to examine if there is a cytotoxic effect associated with root surfaces bathed by purulent exudate from a long-standing periodontitis. Root surfaces from impacted third molars not involved with periodontal disease were used for comparison. Following normal extraction techniques, four normal and five periodontally involved teeth were dropped into separate lOm1 test tubes containing a standardized tissue culture medium (Eagles’s medium with Earle’s salts for buffering to a neutral pH). Streptomycin, mycostatin, and penicillin were added to the medium. The crowns were removed by a sterile surgical mallet and chisel to isolate the root samples. The remaining root portion was then scaled and washed in sterile saline and transferred via sterile forceps to separate capped tubes (Leighton tubes) each containing a 11 x 22 mm coverslip on which an epithelial-cell monolayer had been previously established. The cell-line used was a gingival epithelial-like cell isolated by SWLOW and GLICKMAN (1966). The root samples were carefully placed on the monolayers and their positions recorded on the tube. All specimens were incubated at 37°C. Two specimens, one normal and one periodontally involved, were removed at 24 hr intervals at which time respective coverslips were removed and stained with haematoxylin-eosin and then examined microscopically. The remaining involved root surface was wrapped in a cellulose membrane, dampened in saline and allowed to incubate for 96 hr. Two additional tubes containing monolayers only were used as controls. The four monolayers exposed to normal root surfaces displayed identical cellular morphology and mitotic frequency consistent with that of the two controls (Fig. 1). All six samples revealed cells with an open-faced nucleus, distinct nuclear membrane and an average of two nucleoli per cell. The mitotic index was 2-3 figures per field at 450 x . The cytoplasm displayed a frothy appearance with star-like projections. No vacuolizations was noted. All monolayers displayed vital cell lines. All the monolayers exposed to the involved root surfaces reflected irreversible morphologic changes. The 24 hr monolayer exhibited vacuolization, clumping of
IF HEALTHY
465
466
C. G. HATFIELD ANDA. BAUMHAMMERS
chromatin and turgor. No mitotic figures were found. The 48 hr specimen displayed pyknotic, hyperchromatic nuclei, disruption of celluar outline, coagulation of cytoplasm and increased vacuolization (Fig. 2). The 72 hr specimen (Fig. 3) exhibited the same changes with greater severity while the 96 hr sample had complete disruption of all cellular characteristics. The monolayer exposed to the cellulose-enclosed involved root surface showed no pathologic changes and remained morphologically consistent with the controls. The degenerative process induced only by the involved root surfaces suggested that a toxic factor or factors were released by this group. Since the normal root surfaces did not display this effect, the hypothesis is proposed that the toxic factor(s) originate in the purulent contents of the periodontal pocket. These toxic irritants probably then penetrate the root surface and later leach out again. Diffuse penetration of intact enamel and dentine has been demonstrated (WAINWRIGHTand LEMOINE, 1950). It could be speculated that one of the toxic factors is endotoxin released by oral micro-organisms (RIZZO and MERGENHAGEN,1964). This sequence could help in understanding problems in attaining reattachment of periodontal tissues to teeth affected by long-standing periodontal disease. Future studies using this experimental model could clarify the more specific nature of these toxic factors and their mechanism of action. Acknowledgements-We would like to thank Dr. RONALD FLETCHERand Mr. HERMANLANGKEMP,Department of Microbiology, University of Pittsburgh, School of Dental Medicine for preparing the tissue monolayer and their advice. REFERENCES RAMZJORD, S. 1951. Experimental periodontal reattachment in rhesus monkeys. J. Periodont. 22, 67-75. RIZZO, A.
A. and ~~ERGENHAGEN, S. E. 1964. Histopathologic effects of endotoxin injected into rabbit oral mucosa. Archs. oral Biol. 9,659-670. SMULOW, J. B. and GLNXMAN,I. 1966. An epithelial-like cell line in continuous culture from normal adult human gingiva. Proc. Sot. exp. Biol. 121,1294-1296. WAINWRIGHT, Wm. W. and LEMOINE, F. A. 1950. Rapid diffuse penetration of intact enamel and dentin by carbon-14labelled urea. J. Am. Dent. Ass. 41,135-145. World Workshop in Periodontics, p. 324. 1966. University of Michigan Press. Ann Arbor, Michigan.
CYTOTOXIC
EFFECTS
OF PERIODONTALLY
PLATE
INVOLVED
1 OVERLEAF
ROOT
SURFACES
461
468
C. G. HATFIELD ANDA. BAUMHAMMW
FIG. 1. Tissue monolayer exposed to normal root surface for 48 hr. Haematoxylin and eosin X450 FIG. 2. Tissue monolayer exposed to a periodontally involved root surface for 48 hr. Haematoxylin and eosin x450 FIG. 3. Tissue monolayer exposed to a periodontally involved root surface for 72 hr. Haematoxylin and eosin x 450
CYTOTOXIC
EFFECTS
OF PERIODONTALLY
INVOLVED
ROOT
SURFACES
PLA TE 1
A.( 3.0. f.p.
468
CYTOTOXIC EFFECTS OF PERIODONTALLY INVOLVED SURFACES OF HUMAN TEETH C. G.
HATFIELD
and A.
BAUMHAMMFZRS
School of Dental Medicine, University of Pittsburgh, Pittsburgh, Pennsylvania 15213, U.S.A. gingival tissue is separated from a sound tooth and then replaced, uneventful healing with reattachment occurs (RAMFJORD,1951). However, in cases involving teeth effected by periodontal disease of long duration, reattachment to the contaminated root surfaces is unpredictable and difficult despite clinical methods employed (World Workshop in Periodontics, 1966). This study was initiated to clarify the above clinical problem and to examine if there is a cytotoxic effect associated with root surfaces bathed by purulent exudate from a long-standing periodontitis. Root surfaces from impacted third molars not involved with periodontal disease were used for comparison. Following normal extraction techniques, four normal and five periodontally involved teeth were dropped into separate lOm1 test tubes containing a standardized tissue culture medium (Eagles’s medium with Earle’s salts for buffering to a neutral pH). Streptomycin, mycostatin, and penicillin were added to the medium. The crowns were removed by a sterile surgical mallet and chisel to isolate the root samples. The remaining root portion was then scaled and washed in sterile saline and transferred via sterile forceps to separate capped tubes (Leighton tubes) each containing a 11 x 22 mm coverslip on which an epithelial-cell monolayer had been previously established. The cell-line used was a gingival epithelial-like cell isolated by SWLOW and GLICKMAN (1966). The root samples were carefully placed on the monolayers and their positions recorded on the tube. All specimens were incubated at 37°C. Two specimens, one normal and one periodontally involved, were removed at 24 hr intervals at which time respective coverslips were removed and stained with haematoxylin-eosin and then examined microscopically. The remaining involved root surface was wrapped in a cellulose membrane, dampened in saline and allowed to incubate for 96 hr. Two additional tubes containing monolayers only were used as controls. The four monolayers exposed to normal root surfaces displayed identical cellular morphology and mitotic frequency consistent with that of the two controls (Fig. 1). All six samples revealed cells with an open-faced nucleus, distinct nuclear membrane and an average of two nucleoli per cell. The mitotic index was 2-3 figures per field at 450 x . The cytoplasm displayed a frothy appearance with star-like projections. No vacuolizations was noted. All monolayers displayed vital cell lines. All the monolayers exposed to the involved root surfaces reflected irreversible morphologic changes. The 24 hr monolayer exhibited vacuolization, clumping of
IF HEALTHY
465
466
C. G. HATFIELD ANDA. BAUMHAMMERS
chromatin and turgor. No mitotic figures were found. The 48 hr specimen displayed pyknotic, hyperchromatic nuclei, disruption of celluar outline, coagulation of cytoplasm and increased vacuolization (Fig. 2). The 72 hr specimen (Fig. 3) exhibited the same changes with greater severity while the 96 hr sample had complete disruption of all cellular characteristics. The monolayer exposed to the cellulose-enclosed involved root surface showed no pathologic changes and remained morphologically consistent with the controls. The degenerative process induced only by the involved root surfaces suggested that a toxic factor or factors were released by this group. Since the normal root surfaces did not display this effect, the hypothesis is proposed that the toxic factor(s) originate in the purulent contents of the periodontal pocket. These toxic irritants probably then penetrate the root surface and later leach out again. Diffuse penetration of intact enamel and dentine has been demonstrated (WAINWRIGHTand LEMOINE, 1950). It could be speculated that one of the toxic factors is endotoxin released by oral micro-organisms (RIZZO and MERGENHAGEN,1964). This sequence could help in understanding problems in attaining reattachment of periodontal tissues to teeth affected by long-standing periodontal disease. Future studies using this experimental model could clarify the more specific nature of these toxic factors and their mechanism of action. Acknowledgements-We would like to thank Dr. RONALD FLETCHERand Mr. HERMANLANGKEMP,Department of Microbiology, University of Pittsburgh, School of Dental Medicine for preparing the tissue monolayer and their advice. REFERENCES RAMZJORD, S. 1951. Experimental periodontal reattachment in rhesus monkeys. J. Periodont. 22, 67-75. RIZZO, A.
A. and ~~ERGENHAGEN, S. E. 1964. Histopathologic effects of endotoxin injected into rabbit oral mucosa. Archs. oral Biol. 9,659-670. SMULOW, J. B. and GLNXMAN,I. 1966. An epithelial-like cell line in continuous culture from normal adult human gingiva. Proc. Sot. exp. Biol. 121,1294-1296. WAINWRIGHT, Wm. W. and LEMOINE, F. A. 1950. Rapid diffuse penetration of intact enamel and dentin by carbon-14labelled urea. J. Am. Dent. Ass. 41,135-145. World Workshop in Periodontics, p. 324. 1966. University of Michigan Press. Ann Arbor, Michigan.
CYTOTOXIC
EFFECTS
OF PERIODONTALLY
PLATE
INVOLVED
1 OVERLEAF
ROOT
SURFACES
461
468
C. G. HATFIELD ANDA. BAUMHAMMW
FIG. 1. Tissue monolayer exposed to normal root surface for 48 hr. Haematoxylin and eosin X450 FIG. 2. Tissue monolayer exposed to a periodontally involved root surface for 48 hr. Haematoxylin and eosin x450 FIG. 3. Tissue monolayer exposed to a periodontally involved root surface for 72 hr. Haematoxylin and eosin x 450
CYTOTOXIC
EFFECTS
OF PERIODONTALLY
INVOLVED
ROOT
SURFACES
PLA TE 1
A.( 3.0. f.p.
468