445
Effect of Zinc Upon Cell Attachment and Proliferation on PeriodontallyDiseased Root Surfaces* Kenneth R.
Stookey, Yiming Li,
E.
Brady Hancock,
and
George
Stookey
The purpose of this study was to determine the effect of zinc ions on fibroblast attachment to periodontally-diseased root surfaces in vitro. Extracted periodontally-diseased teeth were treated with 0.5% ZnCl2 by iontophoresis at 0.5 mA for 2 to 6 minutes. Control groups were untreated diseased and untreated healthy teeth. Sections of the root underlying the pocket were cut from the diseased teeth. The specimens were incubated for 18 hours with L929 mouse fibroblasts, then transferred and incubated in cell-free medium for 48 hours. Cell attachment was evaluated by cell counts and scanning electron microscopy (SEM). Root surfaces were sampled with an acid-etch technique and zinc was measured with an atomic absorption spectrophotometer. Two samples from each group were examined for cell attachment with SEM. Data were analyzed using the appropriate statistical methods. The results showed that diseased, untreated root surfaces had significantly fewer cells attached; however, zinc iontophoresis did not significantly improve cell attachment to the diseased root surfaces. Zinc analysis showed that diseased, untreated root surfaces had a higher zinc content than healthy ones. SEM examination showed striking differences in cell attachment to healthy versus diseased root surfaces. The data indicated that zinc iontophoresis did not significantly enhance cell attachment to root surfaces of diseased teeth. / Periodontol 1991; 62:445^451.
Key Words: Fibroblasts; zinc ions; periodontal diseases/therapy; tooth
Interest in the prevention and treatment of periodontal dishas increased among dental researchers during the past decade. One of the goals of periodontal research has been to achieve regeneration of a functionally oriented periodontium at the site of a previously existing pocket.1 A key initial event in this process is the repopulation of the diseased root surface with cells that can participate in the formation of the regenerated periodontium. Measures which facilitate this cellular repopulation could be important in the clinical treatment of periodontal disease. In vitro studies have shown diseased root surfaces to be cytotoxic, permitting little or no cell attachment,2,3 probably due to the presence of lipopolysaccharide (LPS).4'7 Various methodologies to increase cellular attachment have been examined. These include removing the LPS by mechanical means or by extraction using hot phenol,2 or altering the diseased surface or the LPS through chemical treatments.3,8,9 Removal of the LPS by mechanical means is preferable to the clinically impractical use of hot phenol, but other chemical treatments could be easier and as effective. Zinc has been reported to play an important role in cell membrane physiology by inhibiting free radical proease
Indiana
University
School of Dentistry,
Indianapolis,
IN.
root.
duction and lipid peroxidation, thus acting to stabilize the cell membrane.10,11 Snyder and Walker12 found that treatment with ZnCl2 reduced endotoxin-induced lethality in mice. Therefore, adding zinc to the periodontally-diseased root surface might overcome the cytotoxicity of those surfaces by stabilizing the membranes of the repopulating cells. The in vitro studies using cell culture techniques3,6 suggested a possible clinical application for zinc in periodontal treatment, but further in vitro studies are needed to determine whether or not zinc warrants investigation in a clinical setting. The purposes of this study were to examine the effect of zinc iontophoresis on the ability of fibroblasts to attach to periodontally-diseased root surfaces, to determine the presence of any dose-response relationship, and to evaluate the effectiveness of iontophoresis in the incorporation of zinc into the root surface.
MATERIALS AND METHODS Seventy-two extracted human teeth were used in this study. The 60 diseased teeth had periodontal pocket depths of at least 5 mm. The healthy control group consisted of root sections obtained from healthy teeth which were lightly planed to remove rough attachment fibers, giving a smooth healthy root surface. The study design is shown in Table
446
J Periodontol July 1991
CELL ATTACHMENT TO ZINC-TREATED ROOTS
Table 1.
Table 3. Zinc Levels
Study Design
Group
Samples
Status
Iontophoresis
12 12 12 12 12 12
Healthy
NA NA 6 minutes 2 minutes 2x2 minutes 3x2 minutes
Diseased Diseased Diseased Diseased Diseased
Zn++Level Untreated
Group 1: Healthy
2: Diseased 4: Diseased,
(ppm)* Iontophoresed
±
123 1679 551
(9) (12) (12)
3233
4373
±
675
(9)
4685
±
765
5079
±
304
612 1930 4375
± ±
iontophoresed
±
NA NA 1975
(12)
3978
±
2422
(10)
(12) J
3595
±
1774
(12)
(9)
3273
±
1890
(10)
1x2 min.
3:
Diseased,
iontophoresed 1x6 min.
6:
Diseased,
iontophoresed
Table 2. Cell Counts
3x2 min. Number of
Group 1: Healthy
4:
Diseased,
Samples
Average
5:
Number of Cells
( 107mm2)*
10 10
5.0 2.8
±
10
Log
Transformed* 4.6 4.3
±
±
3.5 1.8
±
0.4 0.4
2.2
±
1.3
4.2
±
0.3
1.3
±
0.7
4.1
±
0.3
1.7
±
1.3
4.0
±
0.4
Diseased,
iontophoresed
2x2 min.
*Mean
±
[F>0.05].
SD(n);
values within brackets
are
not
significantly
different
iontophoresed 1x2 min.
3:
Diseased,
iontophoresed 1x6 min.
6:
Diseased,
iontophoresed
fes
3x2 min. 5:
Diseased,
10
iontophoresed
2x2 min. 2: Diseased
1.6
±
1.3
4.0
±
0.3
'.
.-
" ·
-
y
-.
*Mean ± SD 'Mean ± SD; values within brackets are not significantly different [P>0.05]
1. After outlining gingival attachment with a dental bur, adherent tissue and calculus were removed by light curettage. Iontophoresis was performed in the manner of Kataoka et al.3 Briefly, the negative electrode of a PM600 Phoresor iontophoresis apparatus1 was applied to a root canal file inserted apically into the root canal. The positive electrode was placed into a 0.5% 02 solution, the tooth was suspended to a demarcation in the zinc solution, and 0.5 mA was applied for the prescribed time (Table 1). Teeth were rinsed immediately after treatment, and between treatment periods in the groups with multiple treatments, with distilled deionized water. After iontophoresis the diseased area of each tooth was removed and divided into treated and untreated sections, the latter consisting of that part of the tooth past the demarcation which was not immersed in the ZnCl2 solution and thus not iontophoresed. All sections were sterilized by autoclaving at 120°C, 15 psi for 20 minutes. Prepared root sections were assigned code numbers and were placed into wells of 24-well plates. A cell suspension (1 105 cell/mL) of NCTC clone 929 mouse fibroblasts,5 in Eagle's minimum essential medium11 supplemented with 10% fetal bovine serum11 and an antibiotic mixture of penmotion Control Inc., Salt Lake City, UT. *Sigma Chemical Co., St. Louis, MO. § American Type Culture Collection, Rockville, MD. «GIBCO Laboratories, Grand Island, NY.
Figure 1. Healthy root surface (original magnification
Um
100).
(100 units/mL) and streptomycin sulfate (100 µ& added to each well. The plates were incubated mL),11 at 37°C in humidified air with 5% C02 for 18 hours. The medium was removed, the root sections were gently washed twice with phosphate-buffered saline (PBS) and then transferred to new 24-well plates. Cell-free medium was added icillin G
was
Volume 62 Number 7
Figure 2. Healthy root surface (original magnification to the
STOOKEY, LI, HANCOCK, STOOKEY
x
1000).
wells, and the plates were incubated as before for 48
hours. The medium was removed and samples were rinsed 3 times with PBS; cells were harvested with trypsinization and counted with a hemocytometer. The root section areas were measured by covering a section with graph paper, trimming the paper to the edges of the section, and measuring the area of the paper. Two randomly selected root sections from each group were not trypsinized and were prepared for scanning electron microscopic (SEM) examination. Specimens selected for SEM were fixed in glutaraldehyde* and in osmium tetroxide,* then were dehydrated with graded ethanol11 and with sublimation dehydrant.* After sputter-coating with gold-palladium (40:60) using a Hummer V sputter-coater,** specimens were examined for cell attachment using a Hitachi S450 SEM,tf and zinc was quantified by an energy dispersive x-ray elemental microanalysis apparatus** connected to an Amray 1830 SEM.§§ An acid-etch technique was used to sample the coded sections for atomic absorption spectrophotometric analysis of zinc content. A window of known area (3.8 mm2) was 'Midwest Grain Products of Illinois, Pékin, IL. 'Ted Pella, Inc., Redding, CA. **Anatech Ltd., Alexandria, VA. "Tracer Northern, Madison, WI. "Hitachi Scientific Instruments, Mountain View, CA. 55Amray, Bedford, MA.
Figure 3.
Diseased
root
surface (original magnification
447
100).
exposed to 0.010 mL of 3N nitric acid1111 for 5 minutes. Etchings were analyzed for zinc content using a Perkin Elmer 2380 atomic absorption spectrophotometer.1111 The etch depth of 10 specimens was measured and averaged; this
used as a constant to estimate the etch volume. The etch volume and the density of dentin13 were then used in the calculation of zinc content. The data were decoded and the means and standard deviations calculated for each group. The homogenicity of the variances was determined at the 0.10 level of significance using Bartlett's test. Because the variances were not homogeneous, log transformation was performed. The variances of the cell attachment data were found to be homogeneous after log transformation and were analyzed using the analysis of variances (ANOVA). Since the zinc content data were found to be non-homogeneous in variance after log transformation, the Welch test was used to replace the ANOVA. Multiple comparisons using Newman-Keul's method were performed to determine the significance of differences among the means.14 was
RESULTS The data representing the number of cells attached to the root samples are summarized in Table 2. Significantly more cell attachment was found on healthy surfaces in comparI «Fisher
Scientific, Fair Lawn, NJ. Corp., Norwalk, CT.
"Perkin Elmer
448
CELL ATTACHMENT TO ZINC-TREATED ROOTS
Figure
4. Diseased root surface
(original magnification
x
J Periodontol July 1991
1000).
ison to the diseased, untreated group (P < 0.05), indicating a difference between the root surfaces of healthy versus diseased teeth. Although not statistically significant, numerical differences were found in the experimental groups. The 2-minute zinc iontophoresis group (group 4) had the highest cell counts among those iontophoresed, followed by the 6-minute treatment group. Multiple 2-minute treatments decreased the number of attached cells to, or below, that found in the untreated diseased group. The data from the zinc atomic absorption analyses are presented in Table 3. The values are based upon the assumption that the etching volume was the same from sample to sample. The data indicated that before iontophoresis, the sections of diseased roots already had higher levels of surface zinc than the healthy roots (Table 3). Zinc content of root surfaces appeared not to be affected by the iontophoresis process. SEM examination showed that the cells appeared to be tenaciously attached to, and more homogeneous on, healthy surfaces in comparison to diseased or to diseased and treated surfaces. Healthy root sections had a layer of flattened, well-attached cells on their surfaces (Figs. 1 and 2). The untreated diseased surfaces showed very patchy and heterogeneous cell attachment with mostly rounded cells (Figs. 3 and 4). Cell growth on zinc-treated surfaces was also patchy and sparse, with cells forming clusters around a few attached cells (Figs. 5, 6, and 7). Elemental microanalysis of dispersed x-rays indicated heterogeneity in zinc levels
Figure 5. Diseased and iontophoresed (6 minutes) root surface (original magnification xlOO). on diseased root surfaces (Figs. 8, 9.A, and 9.B). An area well-populated by cells (Fig. 8) contained less zinc (429 relative units) when compared to a sparsely-populated area (677 relative zinc units).
DISCUSSION The data from this
study indicate that the iontophoresis of periodontally-diseased root surfaces with zinc ions does not decrease the cytotoxicity of those surfaces. An insignificant increase in cell attachment with zinc iontophoresis was seen. In accord with previous reports,2'3'8 we found periodontally-diseased root surfaces to be cytotoxic, inhibiting cell attachment. In a similar study3 in which zinc iontophoresis was found to significantly increase cell attachment to diseased roots, the significance of differences between groups was determined using the student's i-test. When our data were analyzed using the r-test, our findings were similar to that earlier report:3 cell counts of iontophoresed surfaces (group 4) were not significantly different from counts of healthy surfaces. However, the i-test is not appropriate for comparing multiple groups.14 The SEM examinations showed a large degree of variance in cell attachment, particularly on the diseased samples
Volume 62 Number 7
STOOKEY, LI, HANCOCK, STOOKEY
449
Figure 6. Diseased and iontophoresed (2 minutes) root surface (original magnification xlOO).
Figure 7. Diseased and iontophoresed (2 minutes) root surface (original magnification xlOOO).
(Figs. 1, 3, and 5). The patchy distribution of cells on diseased surfaces, treated or not, indicates much surface heterogeneity which could account for high standard deviations. These local differences in the surface could be due to differences in the oral environment to which the root was exposed or to variations in the disease process, including products of the inflammatory response and the by-products of pathogenic bacteria. Large standard deviations, which tend to decrease the significance of any differences found, were probably due to heterogeneity within and between samples. Since the healthy root surfaces appear to have a more homogeneous distribution of attached cells (Fig. 1), the group standard deviation is most likely due to differences between samples. The ability of the study system to allow differentiation between positive and negative control groups demonstrates its usefulness in examining possible treatments, although any measures which could reduce standard deviation would strengthen conclusions reached.14 Reported zinc values of dentin range from 160 to 199 mg/g,15 but no information on the zinc content of cementum is available. An earlier study,3 which showed a significant increase in cell attachment after zinc iontophoresis, failed to measure zinc surface levels before or after iontophoresis, making a direct correlation between zinc levels and cell attachment impossible. In our study, surface zinc content was measured in order to permit correlation with cell attachment. A significantly greater amount of zinc was found
the diseased surfaces when compared to healthy surfaces. More cells were found to be attached to the surfaces with the lower zinc content, suggesting that increasing the surface zinc level of a root surface would not increase cell attachment by itself. The elemental microanalysis mapping of surface zinc levels (Fig. 8) supports this relationship between zinc and cell attachment. The zinc iontophoresis procedure did not appear to increase the surface zinc content; it can be seen that the diseased root surfaces had numerically higher zinc values before iontophoresis than they did afterwards. This apparent decrease is most likely due to zinc contamination of untreated root portions during iontophoresis, where the zinc chloride solution may have formed an unnoticed film over the unsubmerged (i.e., untreated) root portion. Nevertheless, the untreated diseased group (group 2) provides an estimate of zinc levels of noniontophoresed and diseased surfaces. The mean zinc value for that group is not significantly different from the zinc levels of the iontophoresed surfaces (Table 3). Differences in the volume of root actually etched may have contributed to the high standard deviations in the zinc values, as could differences in the degree of exposure to the oral environment. No trend in surface zinc values after iontophoresis is apparent. Loss of periodontal attachment allows portions of the root to be exposed to saliva, ingested zinc, inflammatory-response products, crevicular fluid, and bacteria, which could on
450
J Periodontol July 1991
CELL ATTACHMENT TO ZINC-TREATED ROOTS
Figure 8. SEM of diseased and iontophoresed (3x2 minutes) root surface containing sparsely-populated area (A) and well-populated area (B) on same sample. Elemental microanalysis of dispersed x-rays found higher levels of zinc in area A (original magnification 920).
Figure 9. Micrograph of healthy surface (A) and corresponding elemental microanalysis of dispersed x-rays (B) phosphorus, zinc, and visible readouts. Here the zinc is distributed evenly (original magnification x 1000).
explain the high levels of zinc found on the diseased roots. plasma zinc to which the healthy root is normally exposed has been reported to be 0.104 ± 0.014 ppm.16 Salivary zinc levels are higher, but vary greatly, ranging from 0.115 to 8.8 ppm.17 Higher still, but also with much variation, are the amounts of zinc reported to be in dental plaque, ranging from 100 to 1000 ppm.18,19 Because Periodontitis allows the root to be exposed to more zinc than in the
The
clockwise from upper
left: calcium,
healthy situation, our finding that the zinc levels of diseased roots are higher seems reasonable. Differences in dietary zinc, zinc metabolism, and the status of periodontal disease in the patient could explain differences found between the randomly-grouped, diseased sections. The numerical increase in cells attached to zinc-treated diseased root surfaces over untreated diseased surfaces, although not significant, indicates the treated surfaces are not
Volume 62 Number 7
STOOKEY, LI, HANCOCK,
the same as the untreated surfaces. This difference does not appear to be due to surface zinc levels. Zinc iontophoresis for 2 minutes gave a small increase in the number of attached cells. However, the zinc level for this group is the lowest of those iontophoresed. The numerical differences between groups would seem not to be related to any numerical difference in surface zinc levels of iontophoresed groups. The possibility exists that exposure to an electric field by itself could have some effect on the cytotoxicity of diseased root surfaces. However, only conjecture on that point is possible because we did not iontophorese healthy roots.
It has been shown in vitro that the cytotoxic effects of LPS upon cell monolayers can be reduced by addition of zinc chloride to the culture medium.6 In contrast to a previous study,3 we report that zinc iontophoresis of periodontally-diseased root surfaces does not significantly increase the number of cells attached to those surfaces. Although the zinc values remain to be confirmed, no significant increase in surface zinc was seen after zinc iontophoresis of diseased root surfaces. The results of our study indicate that zinc iontophoresis would probably not be useful in the treatment of periodontal disease.
STOOKEY
451
of fibroblasts to periodontally-diseased cementum. J Periodont Res 1987; 22:296-299. 4. Hatfield CG, Baumhammers A. Cytotoxic effects of periodontally involved surfaces of human teeth. Arch Oral Biol 1971; 16:465^168. 5. Aleo JJ, Derenzis FA, Farber PA, Varboncoeur AP. The presence and biologic activity of cementum-bound endotoxin. / Periodontol 1974; 45:672-675. 6. Aleo JJ. Role of zinc++ in endotoxin-challenged fibroblast cultures. Microbios Utters 1976; 3:191-197. 7. Aleo JJ, Derenzis FA. Proliferation of cells in vitro after long-term exposure to endotoxin. / Dent Res 1976; 55:1139. 8. Wirthlin MR, Hancock EB. Biologic preparation of diseased root surfaces. J Periodontol 1980; 51:291-297. 9. Porvaznik M, Cohen ME, Bockowski SW, Mueller EJ II, Wirthlin MR Jr. Enhancement of cell attachment to a substrate coated with oral bacterial endotoxin by plasma fibronectin. J Periodont Res 1982; 17:154-168. 10. Coppen DE, Richardson DE, Cousins RJ. Zinc suppression of free radicals induced in cultures of rat hepatocytes by iron, t-butyl hydroperoxide, and 3-methylindole. Proc Soc Exp Biol Med 1988; 189:100109. 11. Bettger WJ, O'Dell BL. A critical physiological role of zinc in the structure and function of biomembranes. Life Sci 1981; 28:14251438. 12. Snyder SL, Walker RI. Inhibition of lethality in endotoxin-challenged mice treated with zinc chloride. Infect Immun 1976; 13:998-1000. 13. Manly RS, Hodge HC, Ange LE. Density and refractive index studies II. Density distribution curves. / Dent Res of dental hard tissues 1939; 18:203-211. 14. Sokal RR, Rohlf RJ. Biometry. San Francisco: WH Freeman and Company; 1981: 208-262. 15. Frank RM, Sargentini-Maier ML, Turlot JC, Leroy MJF. Zinc and strontium analyses by energy dispersive x-ray fluorescence in human permanent teeth. Arch Oral Biol 1989; 34:593-597. 16. Prasad AS, Halstead JA, Nadimi M. Syndrome of iron deficiency anemia, hepatosphomegaly, hypogonadism, dwarfism, and geophagia. Am J Med 1961; 31:532-546. 17. Freeland-Graves JH, Hendrickson PJ, Ebangit ML, Snowden JY. Salivary zinc as an index of zinc status in women fed a low-zinc diet. Am J Clin Nutr 1981; 34:312-321. 18. Hardwick JL, Martin CJ. A pilot study using mass spectrometry for the estimation of the trace element content of dental tissues. Helv OdontActa 1967; 11:62-70. 19. Schamschula RG, Agus H, Bunzel M, Adkins BL, Barnes DE. The concentrations of selected major and trace minerals in human dental plaque. Archs Oral Biol 1977; 22:321-325. —
Acknowledgments
The authors thank Dr. Barry P. Katz, Indiana University School of Medicine, Department of Medicine, Division of Biostatistics, for the advice and assistance in statistical analyses of the data. We would also like to thank the staff of the Department of Oral Surgery, Indiana University School of Dentistry, for supplying extracted teeth and Dr. Jean D. Schoknecht, of the Electron Microscope Facility, Indiana University School of Dentistry, for the use of and assistance with the scanning electron microscope. REFERENCES 1. Zander HA, Poison AM, Heijl C. Goals of periodontal therapy. / Periodontol 1976; 47:261-266. 2. Aleo JJ, Derenzis FA, Farber PA. In vitro attachment of human gingival fibroblasts to root surfaces. J Periodontol 1975 ; 46:639-645. 3. Kataoka M, Gado , Shimomura Y, Lake F, Sharawy M, Gangarosa LP. The influence of zinc
iontophoresis on in vitro adherence
Send reprint requests to: Dr. Yiming Li, Indiana University School Dentistry, 1121 West Michigan Street, Indianapolis, IN 46202. Accepted for publication February 8, 1991.
of
Effect of Zinc Upon Cell Attachment and Proliferation on PeriodontallyDiseased Root Surfaces* Kenneth R.
Stookey, Yiming Li,
E.
Brady Hancock,
and
George
Stookey
The purpose of this study was to determine the effect of zinc ions on fibroblast attachment to periodontally-diseased root surfaces in vitro. Extracted periodontally-diseased teeth were treated with 0.5% ZnCl2 by iontophoresis at 0.5 mA for 2 to 6 minutes. Control groups were untreated diseased and untreated healthy teeth. Sections of the root underlying the pocket were cut from the diseased teeth. The specimens were incubated for 18 hours with L929 mouse fibroblasts, then transferred and incubated in cell-free medium for 48 hours. Cell attachment was evaluated by cell counts and scanning electron microscopy (SEM). Root surfaces were sampled with an acid-etch technique and zinc was measured with an atomic absorption spectrophotometer. Two samples from each group were examined for cell attachment with SEM. Data were analyzed using the appropriate statistical methods. The results showed that diseased, untreated root surfaces had significantly fewer cells attached; however, zinc iontophoresis did not significantly improve cell attachment to the diseased root surfaces. Zinc analysis showed that diseased, untreated root surfaces had a higher zinc content than healthy ones. SEM examination showed striking differences in cell attachment to healthy versus diseased root surfaces. The data indicated that zinc iontophoresis did not significantly enhance cell attachment to root surfaces of diseased teeth. / Periodontol 1991; 62:445^451.
Key Words: Fibroblasts; zinc ions; periodontal diseases/therapy; tooth
Interest in the prevention and treatment of periodontal dishas increased among dental researchers during the past decade. One of the goals of periodontal research has been to achieve regeneration of a functionally oriented periodontium at the site of a previously existing pocket.1 A key initial event in this process is the repopulation of the diseased root surface with cells that can participate in the formation of the regenerated periodontium. Measures which facilitate this cellular repopulation could be important in the clinical treatment of periodontal disease. In vitro studies have shown diseased root surfaces to be cytotoxic, permitting little or no cell attachment,2,3 probably due to the presence of lipopolysaccharide (LPS).4'7 Various methodologies to increase cellular attachment have been examined. These include removing the LPS by mechanical means or by extraction using hot phenol,2 or altering the diseased surface or the LPS through chemical treatments.3,8,9 Removal of the LPS by mechanical means is preferable to the clinically impractical use of hot phenol, but other chemical treatments could be easier and as effective. Zinc has been reported to play an important role in cell membrane physiology by inhibiting free radical proease
Indiana
University
School of Dentistry,
Indianapolis,
IN.
root.
duction and lipid peroxidation, thus acting to stabilize the cell membrane.10,11 Snyder and Walker12 found that treatment with ZnCl2 reduced endotoxin-induced lethality in mice. Therefore, adding zinc to the periodontally-diseased root surface might overcome the cytotoxicity of those surfaces by stabilizing the membranes of the repopulating cells. The in vitro studies using cell culture techniques3,6 suggested a possible clinical application for zinc in periodontal treatment, but further in vitro studies are needed to determine whether or not zinc warrants investigation in a clinical setting. The purposes of this study were to examine the effect of zinc iontophoresis on the ability of fibroblasts to attach to periodontally-diseased root surfaces, to determine the presence of any dose-response relationship, and to evaluate the effectiveness of iontophoresis in the incorporation of zinc into the root surface.
MATERIALS AND METHODS Seventy-two extracted human teeth were used in this study. The 60 diseased teeth had periodontal pocket depths of at least 5 mm. The healthy control group consisted of root sections obtained from healthy teeth which were lightly planed to remove rough attachment fibers, giving a smooth healthy root surface. The study design is shown in Table
446
J Periodontol July 1991
CELL ATTACHMENT TO ZINC-TREATED ROOTS
Table 1.
Table 3. Zinc Levels
Study Design
Group
Samples
Status
Iontophoresis
12 12 12 12 12 12
Healthy
NA NA 6 minutes 2 minutes 2x2 minutes 3x2 minutes
Diseased Diseased Diseased Diseased Diseased
Zn++Level Untreated
Group 1: Healthy
2: Diseased 4: Diseased,
(ppm)* Iontophoresed
±
123 1679 551
(9) (12) (12)
3233
4373
±
675
(9)
4685
±
765
5079
±
304
612 1930 4375
± ±
iontophoresed
±
NA NA 1975
(12)
3978
±
2422
(10)
(12) J
3595
±
1774
(12)
(9)
3273
±
1890
(10)
1x2 min.
3:
Diseased,
iontophoresed 1x6 min.
6:
Diseased,
iontophoresed
Table 2. Cell Counts
3x2 min. Number of
Group 1: Healthy
4:
Diseased,
Samples
Average
5:
Number of Cells
( 107mm2)*
10 10
5.0 2.8
±
10
Log
Transformed* 4.6 4.3
±
±
3.5 1.8
±
0.4 0.4
2.2
±
1.3
4.2
±
0.3
1.3
±
0.7
4.1
±
0.3
1.7
±
1.3
4.0
±
0.4
Diseased,
iontophoresed
2x2 min.
*Mean
±
[F>0.05].
SD(n);
values within brackets
are
not
significantly
different
iontophoresed 1x2 min.
3:
Diseased,
iontophoresed 1x6 min.
6:
Diseased,
iontophoresed
fes
3x2 min. 5:
Diseased,
10
iontophoresed
2x2 min. 2: Diseased
1.6
±
1.3
4.0
±
0.3
'.
.-
" ·
-
y
-.
*Mean ± SD 'Mean ± SD; values within brackets are not significantly different [P>0.05]
1. After outlining gingival attachment with a dental bur, adherent tissue and calculus were removed by light curettage. Iontophoresis was performed in the manner of Kataoka et al.3 Briefly, the negative electrode of a PM600 Phoresor iontophoresis apparatus1 was applied to a root canal file inserted apically into the root canal. The positive electrode was placed into a 0.5% 02 solution, the tooth was suspended to a demarcation in the zinc solution, and 0.5 mA was applied for the prescribed time (Table 1). Teeth were rinsed immediately after treatment, and between treatment periods in the groups with multiple treatments, with distilled deionized water. After iontophoresis the diseased area of each tooth was removed and divided into treated and untreated sections, the latter consisting of that part of the tooth past the demarcation which was not immersed in the ZnCl2 solution and thus not iontophoresed. All sections were sterilized by autoclaving at 120°C, 15 psi for 20 minutes. Prepared root sections were assigned code numbers and were placed into wells of 24-well plates. A cell suspension (1 105 cell/mL) of NCTC clone 929 mouse fibroblasts,5 in Eagle's minimum essential medium11 supplemented with 10% fetal bovine serum11 and an antibiotic mixture of penmotion Control Inc., Salt Lake City, UT. *Sigma Chemical Co., St. Louis, MO. § American Type Culture Collection, Rockville, MD. «GIBCO Laboratories, Grand Island, NY.
Figure 1. Healthy root surface (original magnification
Um
100).
(100 units/mL) and streptomycin sulfate (100 µ& added to each well. The plates were incubated mL),11 at 37°C in humidified air with 5% C02 for 18 hours. The medium was removed, the root sections were gently washed twice with phosphate-buffered saline (PBS) and then transferred to new 24-well plates. Cell-free medium was added icillin G
was
Volume 62 Number 7
Figure 2. Healthy root surface (original magnification to the
STOOKEY, LI, HANCOCK, STOOKEY
x
1000).
wells, and the plates were incubated as before for 48
hours. The medium was removed and samples were rinsed 3 times with PBS; cells were harvested with trypsinization and counted with a hemocytometer. The root section areas were measured by covering a section with graph paper, trimming the paper to the edges of the section, and measuring the area of the paper. Two randomly selected root sections from each group were not trypsinized and were prepared for scanning electron microscopic (SEM) examination. Specimens selected for SEM were fixed in glutaraldehyde* and in osmium tetroxide,* then were dehydrated with graded ethanol11 and with sublimation dehydrant.* After sputter-coating with gold-palladium (40:60) using a Hummer V sputter-coater,** specimens were examined for cell attachment using a Hitachi S450 SEM,tf and zinc was quantified by an energy dispersive x-ray elemental microanalysis apparatus** connected to an Amray 1830 SEM.§§ An acid-etch technique was used to sample the coded sections for atomic absorption spectrophotometric analysis of zinc content. A window of known area (3.8 mm2) was 'Midwest Grain Products of Illinois, Pékin, IL. 'Ted Pella, Inc., Redding, CA. **Anatech Ltd., Alexandria, VA. "Tracer Northern, Madison, WI. "Hitachi Scientific Instruments, Mountain View, CA. 55Amray, Bedford, MA.
Figure 3.
Diseased
root
surface (original magnification
447
100).
exposed to 0.010 mL of 3N nitric acid1111 for 5 minutes. Etchings were analyzed for zinc content using a Perkin Elmer 2380 atomic absorption spectrophotometer.1111 The etch depth of 10 specimens was measured and averaged; this
used as a constant to estimate the etch volume. The etch volume and the density of dentin13 were then used in the calculation of zinc content. The data were decoded and the means and standard deviations calculated for each group. The homogenicity of the variances was determined at the 0.10 level of significance using Bartlett's test. Because the variances were not homogeneous, log transformation was performed. The variances of the cell attachment data were found to be homogeneous after log transformation and were analyzed using the analysis of variances (ANOVA). Since the zinc content data were found to be non-homogeneous in variance after log transformation, the Welch test was used to replace the ANOVA. Multiple comparisons using Newman-Keul's method were performed to determine the significance of differences among the means.14 was
RESULTS The data representing the number of cells attached to the root samples are summarized in Table 2. Significantly more cell attachment was found on healthy surfaces in comparI «Fisher
Scientific, Fair Lawn, NJ. Corp., Norwalk, CT.
"Perkin Elmer
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CELL ATTACHMENT TO ZINC-TREATED ROOTS
Figure
4. Diseased root surface
(original magnification
x
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1000).
ison to the diseased, untreated group (P < 0.05), indicating a difference between the root surfaces of healthy versus diseased teeth. Although not statistically significant, numerical differences were found in the experimental groups. The 2-minute zinc iontophoresis group (group 4) had the highest cell counts among those iontophoresed, followed by the 6-minute treatment group. Multiple 2-minute treatments decreased the number of attached cells to, or below, that found in the untreated diseased group. The data from the zinc atomic absorption analyses are presented in Table 3. The values are based upon the assumption that the etching volume was the same from sample to sample. The data indicated that before iontophoresis, the sections of diseased roots already had higher levels of surface zinc than the healthy roots (Table 3). Zinc content of root surfaces appeared not to be affected by the iontophoresis process. SEM examination showed that the cells appeared to be tenaciously attached to, and more homogeneous on, healthy surfaces in comparison to diseased or to diseased and treated surfaces. Healthy root sections had a layer of flattened, well-attached cells on their surfaces (Figs. 1 and 2). The untreated diseased surfaces showed very patchy and heterogeneous cell attachment with mostly rounded cells (Figs. 3 and 4). Cell growth on zinc-treated surfaces was also patchy and sparse, with cells forming clusters around a few attached cells (Figs. 5, 6, and 7). Elemental microanalysis of dispersed x-rays indicated heterogeneity in zinc levels
Figure 5. Diseased and iontophoresed (6 minutes) root surface (original magnification xlOO). on diseased root surfaces (Figs. 8, 9.A, and 9.B). An area well-populated by cells (Fig. 8) contained less zinc (429 relative units) when compared to a sparsely-populated area (677 relative zinc units).
DISCUSSION The data from this
study indicate that the iontophoresis of periodontally-diseased root surfaces with zinc ions does not decrease the cytotoxicity of those surfaces. An insignificant increase in cell attachment with zinc iontophoresis was seen. In accord with previous reports,2'3'8 we found periodontally-diseased root surfaces to be cytotoxic, inhibiting cell attachment. In a similar study3 in which zinc iontophoresis was found to significantly increase cell attachment to diseased roots, the significance of differences between groups was determined using the student's i-test. When our data were analyzed using the r-test, our findings were similar to that earlier report:3 cell counts of iontophoresed surfaces (group 4) were not significantly different from counts of healthy surfaces. However, the i-test is not appropriate for comparing multiple groups.14 The SEM examinations showed a large degree of variance in cell attachment, particularly on the diseased samples
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Figure 6. Diseased and iontophoresed (2 minutes) root surface (original magnification xlOO).
Figure 7. Diseased and iontophoresed (2 minutes) root surface (original magnification xlOOO).
(Figs. 1, 3, and 5). The patchy distribution of cells on diseased surfaces, treated or not, indicates much surface heterogeneity which could account for high standard deviations. These local differences in the surface could be due to differences in the oral environment to which the root was exposed or to variations in the disease process, including products of the inflammatory response and the by-products of pathogenic bacteria. Large standard deviations, which tend to decrease the significance of any differences found, were probably due to heterogeneity within and between samples. Since the healthy root surfaces appear to have a more homogeneous distribution of attached cells (Fig. 1), the group standard deviation is most likely due to differences between samples. The ability of the study system to allow differentiation between positive and negative control groups demonstrates its usefulness in examining possible treatments, although any measures which could reduce standard deviation would strengthen conclusions reached.14 Reported zinc values of dentin range from 160 to 199 mg/g,15 but no information on the zinc content of cementum is available. An earlier study,3 which showed a significant increase in cell attachment after zinc iontophoresis, failed to measure zinc surface levels before or after iontophoresis, making a direct correlation between zinc levels and cell attachment impossible. In our study, surface zinc content was measured in order to permit correlation with cell attachment. A significantly greater amount of zinc was found
the diseased surfaces when compared to healthy surfaces. More cells were found to be attached to the surfaces with the lower zinc content, suggesting that increasing the surface zinc level of a root surface would not increase cell attachment by itself. The elemental microanalysis mapping of surface zinc levels (Fig. 8) supports this relationship between zinc and cell attachment. The zinc iontophoresis procedure did not appear to increase the surface zinc content; it can be seen that the diseased root surfaces had numerically higher zinc values before iontophoresis than they did afterwards. This apparent decrease is most likely due to zinc contamination of untreated root portions during iontophoresis, where the zinc chloride solution may have formed an unnoticed film over the unsubmerged (i.e., untreated) root portion. Nevertheless, the untreated diseased group (group 2) provides an estimate of zinc levels of noniontophoresed and diseased surfaces. The mean zinc value for that group is not significantly different from the zinc levels of the iontophoresed surfaces (Table 3). Differences in the volume of root actually etched may have contributed to the high standard deviations in the zinc values, as could differences in the degree of exposure to the oral environment. No trend in surface zinc values after iontophoresis is apparent. Loss of periodontal attachment allows portions of the root to be exposed to saliva, ingested zinc, inflammatory-response products, crevicular fluid, and bacteria, which could on
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CELL ATTACHMENT TO ZINC-TREATED ROOTS
Figure 8. SEM of diseased and iontophoresed (3x2 minutes) root surface containing sparsely-populated area (A) and well-populated area (B) on same sample. Elemental microanalysis of dispersed x-rays found higher levels of zinc in area A (original magnification 920).
Figure 9. Micrograph of healthy surface (A) and corresponding elemental microanalysis of dispersed x-rays (B) phosphorus, zinc, and visible readouts. Here the zinc is distributed evenly (original magnification x 1000).
explain the high levels of zinc found on the diseased roots. plasma zinc to which the healthy root is normally exposed has been reported to be 0.104 ± 0.014 ppm.16 Salivary zinc levels are higher, but vary greatly, ranging from 0.115 to 8.8 ppm.17 Higher still, but also with much variation, are the amounts of zinc reported to be in dental plaque, ranging from 100 to 1000 ppm.18,19 Because Periodontitis allows the root to be exposed to more zinc than in the
The
clockwise from upper
left: calcium,
healthy situation, our finding that the zinc levels of diseased roots are higher seems reasonable. Differences in dietary zinc, zinc metabolism, and the status of periodontal disease in the patient could explain differences found between the randomly-grouped, diseased sections. The numerical increase in cells attached to zinc-treated diseased root surfaces over untreated diseased surfaces, although not significant, indicates the treated surfaces are not
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the same as the untreated surfaces. This difference does not appear to be due to surface zinc levels. Zinc iontophoresis for 2 minutes gave a small increase in the number of attached cells. However, the zinc level for this group is the lowest of those iontophoresed. The numerical differences between groups would seem not to be related to any numerical difference in surface zinc levels of iontophoresed groups. The possibility exists that exposure to an electric field by itself could have some effect on the cytotoxicity of diseased root surfaces. However, only conjecture on that point is possible because we did not iontophorese healthy roots.
It has been shown in vitro that the cytotoxic effects of LPS upon cell monolayers can be reduced by addition of zinc chloride to the culture medium.6 In contrast to a previous study,3 we report that zinc iontophoresis of periodontally-diseased root surfaces does not significantly increase the number of cells attached to those surfaces. Although the zinc values remain to be confirmed, no significant increase in surface zinc was seen after zinc iontophoresis of diseased root surfaces. The results of our study indicate that zinc iontophoresis would probably not be useful in the treatment of periodontal disease.
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of fibroblasts to periodontally-diseased cementum. J Periodont Res 1987; 22:296-299. 4. Hatfield CG, Baumhammers A. Cytotoxic effects of periodontally involved surfaces of human teeth. Arch Oral Biol 1971; 16:465^168. 5. Aleo JJ, Derenzis FA, Farber PA, Varboncoeur AP. The presence and biologic activity of cementum-bound endotoxin. / Periodontol 1974; 45:672-675. 6. Aleo JJ. Role of zinc++ in endotoxin-challenged fibroblast cultures. Microbios Utters 1976; 3:191-197. 7. Aleo JJ, Derenzis FA. Proliferation of cells in vitro after long-term exposure to endotoxin. / Dent Res 1976; 55:1139. 8. Wirthlin MR, Hancock EB. Biologic preparation of diseased root surfaces. J Periodontol 1980; 51:291-297. 9. Porvaznik M, Cohen ME, Bockowski SW, Mueller EJ II, Wirthlin MR Jr. Enhancement of cell attachment to a substrate coated with oral bacterial endotoxin by plasma fibronectin. J Periodont Res 1982; 17:154-168. 10. Coppen DE, Richardson DE, Cousins RJ. Zinc suppression of free radicals induced in cultures of rat hepatocytes by iron, t-butyl hydroperoxide, and 3-methylindole. Proc Soc Exp Biol Med 1988; 189:100109. 11. Bettger WJ, O'Dell BL. A critical physiological role of zinc in the structure and function of biomembranes. Life Sci 1981; 28:14251438. 12. Snyder SL, Walker RI. Inhibition of lethality in endotoxin-challenged mice treated with zinc chloride. Infect Immun 1976; 13:998-1000. 13. Manly RS, Hodge HC, Ange LE. Density and refractive index studies II. Density distribution curves. / Dent Res of dental hard tissues 1939; 18:203-211. 14. Sokal RR, Rohlf RJ. Biometry. San Francisco: WH Freeman and Company; 1981: 208-262. 15. Frank RM, Sargentini-Maier ML, Turlot JC, Leroy MJF. Zinc and strontium analyses by energy dispersive x-ray fluorescence in human permanent teeth. Arch Oral Biol 1989; 34:593-597. 16. Prasad AS, Halstead JA, Nadimi M. Syndrome of iron deficiency anemia, hepatosphomegaly, hypogonadism, dwarfism, and geophagia. Am J Med 1961; 31:532-546. 17. Freeland-Graves JH, Hendrickson PJ, Ebangit ML, Snowden JY. Salivary zinc as an index of zinc status in women fed a low-zinc diet. Am J Clin Nutr 1981; 34:312-321. 18. Hardwick JL, Martin CJ. A pilot study using mass spectrometry for the estimation of the trace element content of dental tissues. Helv OdontActa 1967; 11:62-70. 19. Schamschula RG, Agus H, Bunzel M, Adkins BL, Barnes DE. The concentrations of selected major and trace minerals in human dental plaque. Archs Oral Biol 1977; 22:321-325. —
Acknowledgments
The authors thank Dr. Barry P. Katz, Indiana University School of Medicine, Department of Medicine, Division of Biostatistics, for the advice and assistance in statistical analyses of the data. We would also like to thank the staff of the Department of Oral Surgery, Indiana University School of Dentistry, for supplying extracted teeth and Dr. Jean D. Schoknecht, of the Electron Microscope Facility, Indiana University School of Dentistry, for the use of and assistance with the scanning electron microscope. REFERENCES 1. Zander HA, Poison AM, Heijl C. Goals of periodontal therapy. / Periodontol 1976; 47:261-266. 2. Aleo JJ, Derenzis FA, Farber PA. In vitro attachment of human gingival fibroblasts to root surfaces. J Periodontol 1975 ; 46:639-645. 3. Kataoka M, Gado , Shimomura Y, Lake F, Sharawy M, Gangarosa LP. The influence of zinc
iontophoresis on in vitro adherence
Send reprint requests to: Dr. Yiming Li, Indiana University School Dentistry, 1121 West Michigan Street, Indianapolis, IN 46202. Accepted for publication February 8, 1991.
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