The Role of Suicidar A total of 40 flaps were performed, covering observa- tion periods of 1 hour, and 1, 3, 4, 7, 14, 21, 28 and 60 Environment in days. A plastic collar was used as a restraint to prevent Controlling the monkeys from ripping the tissues off during the Keratinization* experiment. Epithelial One hour prior to sacrifice the monkeys received an intraveneous injection of tritiated thymidine|| (1 micro- curie/gm of body weight, specific activity 6.7 Ci/mmole). by The animals were sacrificed by exsanguination. After 10% and R. G. CaFFESSE, D.D.s., M.s., DR. dissection the jaws were fixed in formalin decalcified in a saturated solution of EDTA at pH 7. ODONT.f After décalcification, the specimens were embedded in C. E. Nasjleti, d.d.s4 paraffin, and serially sectioned buccolingually at 6 µ intervals. One out of every four slides was processed for W. A. Castelli, d.d.s., m.s.§ radioautography. The rest were stained with hematoxylin and eosin, Rhodamine and Mallory's trichrome stains. It has been shown that by altering the local environ- Results ment the keratinization potential of epithelium may be Control Sections changed.1'2 In a previous publication from this labora- tory it was shown in monkeys, that when the sulcular Sections from untreated teeth presented a normal epithelium was exposed to the oral environment it had arrangement of the marginal tissues (Fig. 2). The outer the potential for keratinization.3'4 This finding was later surface epithelium showed a definite keratinized epithe- corroborated by other publications.5"7 Consequently al- lium, with areas of both orthokeratinization and para- though the sulcular connective tissue apparently carries keratinization. The sulcular area showed a nonkeratin- the code for epithelial keratinization, it seems that the ized thin epithelium, with shallow rete pegs at the most local environment is influencing the ultimate character coronal area. of the epithelium lining in the sulcular area. The purpose of the present study was to explore the Experimental Specimens role of the sulcular environment in controlling the ker- One Hour and 24 Hour Specimens. Under low mag- atinization of the outer surface gingival epithelium. nification the inverted flap can be seen clearly (Fig. 3). The keratinized is the tooth surface, Materials and Methods gingiva facing keep- ing all the morphological characteristics of the outer Three young adult Rhesus monkeys were used for this surface gingiva and maintaining a close relationship with study. They had a generalized mild , with min- the tooth (Fig. 4a). imal and no significant bone loss. A prophylaxis Under higher magnification, the keratinized surface of was 1 performed week prior to the start of the experi- the epithelium is readily seen using Rhodamine (Fig. ment. The animals were anesthetized using sodium pen- 4b) or Mallory's trichrome (Fig. 4c). The folded epithe- tobarbital intraveneously, and the following surgical pro- lium shows definite thymidine uptake throughout this cedure was performed: Intrasulcular mucoperiosteal period (Fig. 5). flaps were raised on the buccal aspect of individual teeth, without including the approximal papillae. The two ver- Three and Four Day Specimens tical releasing incisions were carried beyond the muco- In 3 and 4 days the epithelium facing the tooth is gingival line. Once the flap was raised, an undermining decreased in thickness, probably due to superficial des- incision through the periosteum was performed mesio- quamation (Fig. 6a). However, no inflammatory infil- at incision of distally the base of the flap. This the trate is seen in the connective tissue and both epithelium periosteum allowed the flap to be pulled coronally. The and connective tissue show increased labeling (Fig. 6b). marginal tissue was folded inwards, in such a way that Rhodamine and Mallory's trichrome specimens the outer surface epithelium came in close contact with showed a discontinuous band of keratin at this stage. the tooth surface (Fig. 1). Atraumatic silk sutures No. 3- and Fourteen 0 were used to maintain the folded flaps in position. Seven Day Specimens The epithelium facing the tooth gets thinner and * Supported in part by the Medical Research Service of the Veterans changes its morphology showing wide and shallow rete Administration. is t Professor of Periodontics, The University of Michigan School of pegs (Fig. 7a). Although superficial desquamation , Ann Arbor, Mich. 48109. evident, no positive stain with Rhodamine or Mallory's Research Veterans % Senior Research Biologist, Dental Section, trichrome is seen (Fig. 7b). Minimal thymidine uptake Administration Hospital. § Professor of Anatomy, The University of Michigan Medical School. II New England Nuclear, Boston, Mass. 1 J. Periodontol. 2 Caffesse, Nasjìeti, Castelli January, 1979

Figure 2. Control section showing a keratinized outer surface gingival epithelium and a nonkeratinized thin sulcular epithelium (H & E, original magnification, x 40).

reestablishment of a new sulcular epithelium took place by third intention. In this case the folded epithelium (remaining away from the tooth surface) maintained its keratinization while the restored sulcular epithelium did not show any keratinization (Fig. 10). Discussion Figure 1. Diagrams showing theprocedureperformed. See text Sufficient evidence exists today to indicate that the for explanation. sulcular epithelium has the potential for keratinization when moved away from the tooth surface en- is seen (Fig. 7c). The connective tissue underlying the vironment.3"7 "new" sulcular epithelium does not display any inflam- This evidence agrees with findings related to tissue mation. The previous "sulcular epithelium" is now specificity and genetic determination of epithelial differ- embedded in the connective tissue. Total healing is seen entiation.8"10 The results by Karring et al.8 experimenting at the line of fusion (Fig. 7a). with periodontal tissues concur with those reported for and oral tissues.9'10 As mentioned4 and epidermal already Twenty-One, Twenty-Eight Sixty Day Specimens there is no difference in the composition of the connec- The new sulcular epithelium has regained its regular tive tissue underlying the oral and the sulcular epithe- morphology with very few broad rete pegs (Fig. 8a) and lium. As a consequence sulcular epithelium should have displays no keratinization when treated with Rhodamine the potential to keratinize. Such a potential has been and Mallory's trichrome stains (Fig. 8b). The subepi- suggested earlier in the literature.11,12 Furthermore, this thelial connective tissue shows increased inflammation possibility recently has been associated with the eversión (Fig. 8a) and, associated with it, there is an increase in of the sulcular epithelium after orthodontic tooth sepa- the thymidine uptake of the epithelium (Fig. 8c). Similar ration in rats.13 findings are observed in the 60 day specimens (Figs. 9a- If the potential is there, why doesn't the sulcular c). epithelium keratinize? Is it the sulcular environment that In a 28-day-old specimen, where the perfect adapta- is inhibiting epithelial differentiation? tion of the flap to the tooth was lost, the healing and the The findings from the present study seem to corrobo- Volume 50 Number 1 Role of Sulcular Environment 3

Figure 5. Radioautograph showing thymidine uptake in the epithelium of a 24-hour specimen (original magnification, X 125).

Figure 4. Twenty-four hour specimens. A. The outer surface gingiva maintains a close relationship with the tooth surface (H & E, original magnification, X 125). B. A distinct keratinized epithelial surface is seen facing the tooth, with Rhodamine stain (original magnification, X 125). C. Keratin layer stained with Mallory's trichrome (original magnification, X 125). J. Periodontol. 4 Caffesse, Nasjleti, Castelli January, 1979

Figure 6. Three days specimen. A. 77ie epitheliumfacing the tooth is decreased in thickness, probably due to superficial desquamation. The connective tissue is free from inflammatory cells (H & E, original magnification, x 125). B. Radioautograph showing increased labeling both in the epithelium and connective tissue (original magnification, x 125).

Figure 7. Fourteen day specimen. A. The epithelium facing the tooth is thinner, with wide, shallow rete pegs. The former sulcular epithelium is now embedded in connective tissue (H & E, original magnification, x 40). B. Seven day specimen showing negative reaction to the Rhodamine stain (original magnification, x 125). C. Fourteen day specimen showing decreased thymidine uptake (original magnification, x 125).

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m 3

a

Figure 8. Twenty-one day specimens. A. The new sulcular epithelium regains its regular configuration. The underlying connective tissue shows increased inflammation (H & E, original magnification, x 40). B. No keratinization is depicted when stained with Rhodamine (original magnification, x 125). C. Radioautograph showing increased thymidine labeling in the epithelium (original magnification, x 125). Volume 50 Number 1 Role of Sulcular Environment 5

Figure 9. Sixty-day specimen. A. A thin sulcular epithelium is noticed (H & E, original magnification x 64.5). B. No keratinization of the sulcular epithelium is depicted (Rhodamine , original magnification, x 64.5). C. Radioautograph showing labeling of the sulcular epithelium (original magnification, x 64.5).

products, the epithelium may not develop full differen- tiation due to its increased turnover rate. Such premature desquamation has been suggested as a possible expla- nation for the lack of keratinization in some areas of the oral epithelium.14 The increased thymidine label uptake observed in the sulcular epithelium of the 8 week speci- mens, is undoubtedly associated with increased inflam- mation in the underlying connective tissue. Interestingly enough, in the 28 day specimen showing poor flap adaptation to the tooth (Fig. 9) the folded epithelium maintained its keratinization. This is additional evidence that the contact to the tooth is responsible for the lack of keratinization of the epithelial tissues, probably due to the constant irritation they are subjected to. This report presents further evidence to indicate that the sulcular environment determines the lack of keratin- ization of the Figure 10. third inten- epithelium. Important questions remaining Twenty-eight day specimen. Healing by to be and answered are: can kera- tion occurred due to poorflap adaptation. Thefolded epithelium explored adequately maintained its keratinization (Rhodamine , original magnifica- tinization of the sulcular epithelium be achieved in situ! tion, x 40). If this is possible, what is its clinical significance? If the hypothesis of a premature desquamation of the epithe- rate this previously postulated assumption.4,6 The trans- lium due to constant irritation from bacterial plaque is formation of the outer surface epithelium to a thin true, complete plaque removal should be able to reduce nonkeratinized epithelium, devoid of deep and narrow the turnover rate of the epithelial cells. This in turn, rete pegs, when in contact with the tooth surface supports would allow the sulcular epithelium to fully differentiate. this hypothesis. Since the sulcular epithelium is subjected Contrary to the report by Kopczyk et al.10 who showed to constant irritation from bacterial plaque and its by- a lack of keratinization of sulcular epithelium in dogs J. Periodontol. 6 Caffesse, Nasjleti, Castelli January, 1979 after toothbrushing stimulation for 28 days, Hiatt16 has 2. Banoczy, J.: Keratinization in explanted oral mucosal shown the development of a keratinized sulcular epithe- flaps of rabbits. Arch Oral Biol 16: 1289, 1971. 3. and T.: of lium in after term use of an intracrevicular Caffesse, R., Karring, Keratinizing potential patients long crevicular epithelium. / Dent Res 54: (Special Issue A): L69, brushing technique. The short length of the experiment Abstr. No. L277, 1975. by Kopczyk et al.15 might explain the lack of positive 4. Caffesse, R., Karring, T., and Nasjleti, C: Keratinizing results. Whether a keratinized sulcular epithelium will potential of sulcular epithelium. / Periodontol 48: 140, 1977. act as a better barrier against the penetration of bacterial 5. Bral, M., and Stahl, S.: Keratinization potential of human crevicular J Dent Res 56 Issue is still to be determined. epithelium. (Special A) A96, products Abstr. No. 213, 1977. Summary and Conclusions 6. Bral, M., and Stahl, S.: Keratinizing potential of human crevicular epithelium. / Periodontol 48: 381, 1977. The influence of the sulcular environment on the 7. Stahl, S., Fletcher, P., and Wyman, B.: Crevicular tissues keratinization of the outer surface gingival epithelium as donor tissue for free gingival grafts. J Periodont Res 12: 450, was tested in three young adult Rhesus A total 1977. monkeys. 8. and H.: The role of of 40 were and inverted so Karring, T., Lang, N., Loe, gingival mucoperiosteal flaps raised, connective tissue in differentiation. J as to the outer in contact with determining epithelial bring surface epithelium Periodont Res 10: 1, 1975. and the tooth, were sutured. The experimental time 9. Billingham, R., and Silvers, W.: Studies on the conser- intervals varied from 1 hour to 60 days. The monkeys vation of epidermal specificities of skin and certain mucosas in received H3 thymidine 1 hour to sacrifice. The adult mammals. / Exp Med 125: 429, 1967. prior 10. and H.: Connective tissue in- material was for and radioauto- Stambaugh, R., Gordon, prepared histologie fluence on mucosal keratinization. J Dent Res 52: (Special graphic evaluation. Issue) 147, Abstr. No. 355, 1973. Results indicated that the outer surface epithelium 11. Listgarten M.: Similarity of epithelial relationships in changes its morphology to a nonkeratinized epithelium the gingiva of rat and man. J Periodontol 46: 677, 1975. devoid of deep rete pegs when in close contact with the 12. Takarada, H., Cattoni, M., Sugimoto, ., and Rose, G.: Ultrastructural studies of human / Periodontol 45: 30, in the anatomical characteristics nor- gingiva. tooth, resulting 1974. mally seen in sulcular epithelium. 13. Boisson, M., Gianelli, ., Ruben, M., Randall, F., and It is concluded that the sulcular environment has the Bloom, .: Epithelial changes evident to tooth movement: A capability of controlling the keratinizing potential of the radioautographic analysis. / Periodontol Al: 335, 1976. outer surface gingival epithelium. It is further suggested 14. Hashimoto, K, Dibella, R., and Shklar, G.: Electron- studies of the normal human buccal mucosa. / that the constant irritation of bacterial and its microscopic plaque Invest Dermatol. Al: 512, 1966. products may be responsible for the premature desqua- 15. Kopczyk, R., Lenox, J., and Saxe, S.: Potential for mation of the sulcular epithelium which in turn might keratinization of the sulcular epithelium in the dog. / Dent Res not allow its full differentiation. 53: 137, 1974. 16. Hiatt, W.: An Evaluation of the Present Status of Perio- References dontol Therapy. Continuing Education Course, 59th Annual 1. Innes, P.: The differentiation of cat crevicular epithelium Meeting of the American Academy of . San in diffusion chambers in vivo (Abstr.). Anat Ree 169: 345, 1971. Antonio, Texas, October 27, 1973.

Announcement

UNIVERSITY OF KENTUCKY, COLLEGE OF DENTISTRY This course is designed to focus on the principles of treatment planning for limited tooth movement and to direct attention to the The University of Kentucky, College of Dentistry announces the phenomenon associated with tooth movement. The partici- following continuing education course: biological pants will learn about the techniques of limited tooth movement, along Title: "Tooth Movement"—Orthodontic/Periodontic Interrela- with the physiologic and pathologic responses of the tionships incidental to controlled orthodontic forces. Date: February 22, 1979 Faculty: Orhan C. Tuncay, D.M.D., Assistant Professor, Depart- For further information contact: Ms. Alyce Ferguson, Coordinator, ment of Orthodontics, University of Kentucky. Continuing Education University of Kentucky, College of Dentistry, David C. Vandersall, D.D.S., M.S., Associate Professor, Lexington, Kentucky 40506. Department of Periodontics, University of Kentucky.