The Effect of Mechanical Stimulation on the Keratinization of Sulcular Epithelium*

Raul G. Caffesse,f Carlos J. Nasjleti,! Charles J. Kowalski,§ and Walter A. Castellili

Accepted for publication 2 June 1981

This study was designed to evaluate the effect of mechanical stimulation on the keratinization of the sulcular epithelium in four adult Rhesus monkeys. Each animal received a thorough prophylaxis. One week later, each monkey received one of the following modalities of plaque control: (a) daily intravenous tetracycline and rubber cup prophylaxis, (b) daily rubber cup prophylaxis, (c) daily intravenous tetracycline injections; (d) no treatment, as a control. After sacrifice and tissue processing the histologie sections were evaluated for the presence of sulcular keratinization. The keratin width and length were measured, and an Inflammatory Index determined. It was found that all treatment modalities reduced inflammation signif- icantly, when compared to the control. No differences among the three procedures tested were found. Although all permitted keratinization to develop, sulcular keratinization was signif- icantly increased when daily prophylaxes were performed. It was concluded that mechanical stimulation of the sulcular epithelium, seemingly plays a role in promoting its keratinization.

Previous studies have shown the keratinizing possibil- were used. All animals had full complements of perma- ity of the sulcular epithelium in situ, when different nent teeth with moderate supra- and subgingival regimes of antimicrobial treatments were applied in mon- and moderate One week 2 generalized marginal . keys.1' These treatments have all combined the use of prior to the experiment all teeth were scaled and pol- antimicrobials, such as systemic antibiotics and local ished. applications, with the daily mechanical On day 0, all monkeys were placed on specific plaque removal of by means of a rubber cup control regimes, as follows: Monkey 1 received daily prophylaxis. All combined regimes tested allowed the intravenous (IV) tetracycline hydrochloride (Achromy- sulcular epithelium to keratinize. However, the question cin®) at the maximum safe dosage of 20 mg/kg body remains as to whether the keratinization is mainly due weight and daily rubber cup prophylaxes throughout the to the almost complete elimination of the dental plaque 1 month investigation period. Monkey 2 received daily or to the mechanical stimulation produced by the daily prophylaxes during the whole month investigation pe- rubber cup prophylaxes on the sulcular epithelium. riod. Monkey 3 received daily injections of tetracycline, The purpose of this study was to try to elucidate the using the same route and dosage as that given to Monkey specific effect of the mechanical stimulation produced 1, for 1 month. During this time this monkey received by the prophylaxes on the keratinization of the sulcular no prophylaxes. Monkey 4 served as a control and epithelium. received no additional treatment. the MATERIALS AND METHODS During experimental period, for any treatment, all monkeys were premedicated with ketamine hydrochlo- adult Four male Rhesus monkeys (Macaca mulatta) ride, 25 mg/kg body weight, to insure cooperation. The prophylaxes consisted of a rubber cup with * This polish pro- study was partially supported Dy the Medical Research were Division of the Veterans Administration. phylaxis paste (Nupro®). They performed during the 5 whereas the adminis- t Professor and Chairman, Department of Periodontics, The Uni- weekdays only, tetracycline versity of Michigan School of , Ann Arbor, MI 48109. tration was not interrupted during the weekends. Coordinator, Dental Research Program, Veterans Administration After sacrifice, the animals were decapitated and the Medical Center, Ann Arbor, MI 48105. jaws with the teeth were fixed in 10% neutral buffered Professor of and Associate Scientist in § Dentistry Research the formalin for 4 weeks. Then were divided into small Dental they Research Institute, School of Dentistry, The University of and Michigan. specimens decalcified in 10% formic acid. Following II Professor of Anatomy, The University of Michigan Medical décalcification, the specimens were washed in running School. water overnight, embedded in paraffin and sectioned J. Periodontol. 90 Caffesse, Nasjleti, Kowalski, Castelli February, 1982 buccolingually at 6 µ. The resulting sections were stained RESULTS with either Ehrlich's acid hematoxylin and eosin, Mal- lory connective tissue stain as modified by Ayoub and Each antiplaque regime tested was effective in pro- Shklar,3 or Rhodamine . ducing keratinization of the sulcular epithelium (Fig. 1- Histologie Evaluation. A total of 200 randomly selected 3). As expected, no keratinization was found in the sections per monkey (50 per quadrant) stained with sulcular areas of the control monkey (Fig. 4). Mallory or Rhodamine were evaluated for the presence Individual means for all parameters analyzed, Inflam- or absence of keratin in the sulcular area. When present, matory Index, keratin width and keratin length, were the length and width of the keratin layer was measured determined for the buccal and lingual areas in the ex- using a Filar Micrometer Eyepiece (Bausch and Lomb).2 perimental monkeys. An Inflammatory Index was also In addition, the widths of the keratin layer in the oral determined in the control monkey. Since buccal and gingival epithelium of the same specimens were also lingual means were found to be no different, combined measured. means were obtained and used for further analysis. The corresponding sections stained with hematoxylin Table 1 presents the mean values obtained with the and eosin were used to determine an Inflammatory Index different combinations of treatment as well as in the in the connective tissue underlying the sulcular epithe- control animal. The equality of the mean values of the lium, by counting the number of inflammatory cells variables in each of the relevant groups was tested by within a microscopic field.2 Both buccal and lingual the analysis of variance and significant differences (P < areas were included for all the evaluations. 0.001) were found in each instance. Scheffe's method of The data obtained were statistically analyzed in MI- multiple comparisons was then used to test each of the DAS (Michigan Interactive Data Analysis System) using pairwise differences at the 5% level of significance. For the analysis of variance, Scheffe's method of multiple the Inflammatory Index, the control group was signif- comparisons and the paired / test. icantly different from each of the treatment groups, but

Figure 1. Specimen from monkey treated with IV tetracycline and daily Figure 2. Specimen from monkey receiving daily prophylaxes. Observe rubber cup prophylaxes. A definite band of keratin is present lining the the development of sulcular keratinization and minimal inflammation sulcular area (Mallory stain, magnification X 100). within the connective tissue (Mallory stain, magnification X 100). Volume 53 Number 2 Mechanical Stimulation on Keratinization ofEpithelium 91

Figure 3. Specimen from monkey treated with systemic IV tetracycline Figure 4. Specimen from the control monkey. Keratinization stops at the only. Minimal sulcular keratinization is present. Note the almost absence (Mallory stain, magnification X 100). of inflammation within the tissues (Mallory stain, magnification X 100). Table 1 there were no differences between the treat- significant Mean Values, and Their Significance, for Different Modalities ment groups. Keratin length was significantly less in (ANOVA) 1 2 were group 3, but groups and not significantly Keratin Keratin Treatment Inflammatory different. Pairwise comparisons proved similar results for index length width keratin width, with 3 group showing statistically signif- 1. Prophylaxis and 9.48 - icant deviation from group 1 and 2. antibiotics 0.19—j— Table 2 presents the comparison between the keratin 2. Prophylaxis 14.43 2.86"-L- 0.25-L width obtained in the sulcular epithelium and that of the 3. Antibiotics 10.53 0.83 0.06 4. Control 25.06 oral gingival epithelium. Within each of the treatment the t test was used to test the groups paired hypothesis ^Indicates no significant difference (Scheffe's procedure). that the mean difference was equal to zero. This was < in each of the three consid- rejected (P 0.001) groups Additionally, all of these reduced inflammation signif- ered. The largest mean difference was observed in group icantly when compared to the control. The differences 3 and the analysis of variance and Scheffe's method of observed in the mean inflammatory values for the three multiple comparisons applied to the differences showed different treatment regimes were not high enough to that group 3 differed from both 1 and significantly group show among them. but 1 and 2 were not significance group 2, group group significantly all different controlled bacterial different from one another. Although regimes irritation enough to allow the epithelium to complete its full the amount ofkeratin when DISCUSSION differentiation, produced only antibiotic therapy was used was significantly re- As shown in previous studies,1'2 all different combi- duced, compared to the other two groups. This is true nations to control bacterial plaque tested were effective even when the Inflammatory Index obtained for group in producing keratinization of the sulcular epithelium. 3 (antibiotics only) was lower than that obtained in J. Periodontol. 92 Caffesse, Nasjleti, Kowalski, Castelli February, 1982 Table 2 keratin within the sulcus. This is somewhat contrary to Comparison Between Keratin Width in Sulcular and Oral Gingival what has been reported previously when both therapeutic Epithelium (Paired t test) were used in combination, in the sense that Keratin width approaches there seemed to be a correlation between the reduction Treatment Difference Significance -Sulcular Oral of inflammation and the production of keratin.1'2 Con- inflammation needs to be controlled for the 1. Prophylaxis and 0.19 0.33 <0.001 sequently, antibiotics 0.14-p epithelium to exhibit its keratinizing potential, but the 2. Prophylaxis 0.25 0.35 0.10 J- <0.001 magnitude of this response may be well controlled by 3. Antibiotics 0.06 0.28 0.22 <0.001 mechanical stimulation.

^Indicates no significant difference (Scheffe's procedure). CONCLUSIONS 1. antibiotics, and local individ- group 2 (prophylaxes only). As previously reported a Systemic prophylaxes or are effective in inflamma- was observed between the width and the ually combined, reducing relationship within the tissues. of the keratin tion gingival length layer.2 2. The reduction in inflammation obtained either As a consequence, these seem to indicate that by findings allows sulcular keratinization to the addition of the increased the method develop. daily prophylaxes epi- 3. Sulcular keratinization is increased thelial response towards keratinization. The mechanical significantly by effect of the rubber with a performing daily subgingival prophylaxes. daily cupping polishing paste 4. Mechanical stimulation of the sulcular in the gingival area might be responsible for these find- epithelium seems to promote its keratinization. ings. It is worth stressing that the prophylaxes were performed aiming at cleaning the subgingival area as REFERENCES much as possible. These findings agree with the tendency 1. Caffesse, R. G., Kornman, K. S., and Nasjleti, C. E.: The effect towards parakeratinization reported after the evaluation of intensive antibacterial therapy on the sulcular environment in of intracrevicular monkeys. Part II. Inflammation, mitotic activity and keratinization of supervised toothbrushing techniques Periodontol 51: 1980. in humans.4 the sulcular epithelium. J 155, 2. F. L., Caffesse, R. G„ and Nasjleti, C. E.: The effect of is Bye, When the amount of keratinization obtained com- different plaque control modalities on the keratinizing potential of the pared with the width of keratin present in the oral sulcular epithelium in monkeys. J Periodontol 51: 632, 1980. gingival epithelium it is evident that the keratin layer is 3. Ayoub, P., and Shklar, G.: A modification of the Mallory connective tissue stain as a stain for keratin. Oral 16: 580, 1963. always less pronounced regardless of the treatment ap- Surg 4. Fry, H., and App, G.: Histologie evaluation of the effect of this was carried out for plied. However, experiment only intrasulcular tooth brushing on human sulcular epithelium. J Perio- 1 month; longer observations as in previous experiments dontol 49: 163, 1978. might have differed in this respect. From the results it seems safe to conclude that al- Send reprint requests to: Dr. Raul G. Caffesse, Department of though antibiotics control inflammation within the tis- Periodontics, The University of Michigan School of Dentistry, Ann sues, mechanical stimulation is needed to increase the Arbor, MI 48109.