258
Periodontal Repair in Dogs: Healing in Experimentally Created Chronic Periodontal Defects UlfM.E. Wikesjö, * Knut A. Selvig, *T Grenith Zimmerman, * and Rolf Nilvéus A
objectively evaluate surgical conditioning and biomaterials in
need exists for well-defined animal models to and a role for biochemical wound
principles possible promoting periodontal regeneration. To test an existing model for its usefulness in quantitative evaluation of periodontal wound healing, large supraalveolar periodontal defects were surgically created around the mandibular premolare (P2, P3, P4) in left or right jaw quadrants in 5 beagle dogs. The defects were exposed to the oral environment for 6 months and were then subjected to reconstructive flap surgery (chronic defects). Healing in these defects was compared to healing following reconstructive surgery in similar contralateral defects which had not been exposed to plaque and calculus (acute defects). The animals were sacrificed after a 4-week healing period and tissue blocks including teeth and surrounding structures were processed for histometric analysis. Mean defect height (± s.d.) for chronic and acute defects amounted to 4.6 ± 0.3 and 4.4 ± 0.4 mm, respectively. Mean connective tissue repair to the root surface in chronic defects amounted to 62% (range 49% to 74%) of the defect height. Mean connective tissue repair in the acute defects exceeded 94% of the defect height in 4 of the dogs, but amounted to only 48% in 1 dog. Regeneration of alveolar bone and cementum was limited under both experimental conditions. Root résorption was frequently encountered, whereas ankylosis was seen in only few teeth. This study indicates that healing may vary not only as a result of controlled experimental variables, but also due to differences in biological response between dogs or to fortuitous traumatic factors. The variability in healing in the chronic defects imposes limitations on the use of this defect model to study periodontal regeneration, especially when a small number of animals is used. J Periodontol 1991;
62:258-263.
Key Words: Models, animals; odontal
wound
diseases/therapy.
healing; surgical flaps;
Despite considerable efforts, predictable and complete periodontal regeneration remains an unsolved problem in periodontal research. Thus, a need exists for continuous development and refinement of suitable animal models. Recent studies in periodontal wound healing have included a
model featuring surgically created and immediately treated large supraalveolar mandibular premolar defects in the dog.1-5 This model heals almost exclusively with connective tissue repair rather than epithelial adhesion to the defect
surface and within 4 weeks may include a modest formation of new cementum and alveolar bone, root résorption, and ankylosis.1 The model may be used to study the nature of the healing root surface-gingival flap interface as well as the effect of various root and wound treatments in enhancing formation of new cementum and alveolar bone and in "Lorna Linda University, Loma Linda, ca. +University of Bergen, Bergen, Norway.
*National Dental Health Service, Kristianstad, Sweden.
bone
regeneration; peri-
preventing healing aberrations such as root résorption and ankylosis. However, this model does not mimic clinical periodontal defects, which generally heal with a long junctional epithelium following flap surgery6 and thus cannot be used to study treatments designed to enhance connective tissue repair to the root surface. In earlier studies, we used small, chronic through-andthrough furcation defects in the mandibular premolare in the dog to study periodontal wound healing.7-12 These defects do not regenerate spontaneously but heal with epithelialization of the fornix of the furcation following flap surgery.7-9 An experimental treatment was considered successful only if the full circumference of the furcation defect healed with connective tissue repair. Further development of this model included more radical removal of tooth-supporting structures to create large circumferential furcation defects, still utilizing complete connective tissue repair in the furcation area as a criterion for
Volume 62 Number 4
WIKESJÖ, SELVIG, ZIMMERMAN, NILVÉUS
EXPERIMENTAL DESIGN DEFECT INDUCTION: CHRONIC DEFECTS
BASELINE
PLAQUE ACCUMULATION RECONSTRUCTIVE SURGERY: CHRONIC DEFECTS DEFECT INDUCTION + RECONSTRUCTIVE SURGERY: ACUTE DEFECTS
6 MONTHS
259
Immediately following reconstructive surgery in the chronic defects, defects of matching size were surgically created in the contralateral quadrant and reconstructive surgery was performed. Average clinical height of these acute defects was 5.3 mm (range 4.3 to 6.3 mm). This examination was part of an institutionally approved protocol in periodontal wound healing.
PLAQUE CONTROL 7 MONTHS
SACRIFICE BLOCK BIOPSIES
Figure 1: Experimental design.
experimental success.13-15 Such analysis is useful in evaluating a biological potential of an experimental procedure but would not be sensitive enough for assessing quantitative differences between experimental conditions. Incomplete connective tissue repair in the furcation will presumably be reflected in adjacent buccal and lingual root
surfaces as well. Therefore, it appears to be of interest to evaluate that model for quantitative evaluation of treatments directed to enhance connective tissue repair to the root surface. Thus, this study was initiated to assess extent and variability of healing on the buccal and lingual surfaces in chronic supraalveolar periodontal defects in the dog following reconstructive flap surgery. Results were compared to healing in surgically created and immediately treated defects. MATERIALS AND METHODS
Animals, Surgical Procedures, Design
and
Experimental
periodontal defects were surgically created in right jaw quadrants in 5 young adult male beagle
Standardized
left and dogs 6 months apart and were then treated with reconstructive flap surgery (Fig. 1). This experimental design allows for intra-animal comparisons of healing in defects exposed to the oral environment for 6 months (chronic defects) and defects without this experience (acute defects). Chronic and acute defects were alternated between left and right jaw quadrants in subsequent dogs. The defects were created around the 2nd, 3rd, and 4th mandibular premolare (P2, P3, and P4) under sodium pentobarbital anesthesia as earlier described.1 Mean height of the chronic defects, measured from the cemento-enamel junction (CEJ) to the reduced alveolar bone with a periodontal probe at time of creation, amounted to 5.2 mm (range 4.2 to 5.9 mm); the defects in P2 always being somewhat smaller than in P3 and P4. The mucoperiosteal flaps were then replaced and sutured immediately above the reduced alveolar bone to expose the surgically denuded root surface to plaque and calculus formation. Six months later the defects were subjected to reconstructive surgery. Clinical height of the chronic defects at that time point averaged 5.1 mm (range 4.1 to 6.2 mm).
Wound Management The root surfaces were instrumented with curets, chisels, and water-cooled flame-shaped finishing burs in an attempt to remove dental plaque and calculus (chronic defects) and the root cementum (chronic and acute defects). The defects were then rinsed with saline and the wounds closed by placing and suturing the flap margins 1 to 2 mm coronal to the cemento-enamel junction as previously described.1
care included a daily administered intramuscular broad spectrum antibiotic§ for 2 weeks, daily topical plaque control with a 2% Chlorhexidine solution11 for the duration of the study, and suture removal 1 week after surgery. The dogs were sacrificed 4 weeks following the reconstructive surgery by an intravenous injection of sodium pentobarbital. Intraoral radiographs were taken of the experimental teeth at the time of sacrifice.
Postoperative
Histological Procedures
Histological processing and evaluation was performed as earlier described.1 Briefly, tissue blocks including teeth and surrounding tissues were removed at sacrifice and fixed in 10% buffered formalin. Following décalcification in 5% formic acid, trimming, dehydration, and embedding in par-
thick, were cut in a buccomesio-distal extension the entire lingual plane throughout of the teeth. Every 14th section, approximately 100 µ apart, was stained with hematoxylin and eosin. The most centrally located stained section of the mesial and the distal root of each tooth was identified by the size of the pulp chamber and the root canal. This section and the 2 step serial sections on either side were used for histometric analysis. Thus, 5 subsequent step serial sections of the mid portion of both the mesial and the distal root were analyzed using a microscope connected to a computer aided manual data collection system.1 The following measurements were made for the buccal and lingual surfaces of each root: Defect Height: the distance between the apical extension of the root planing and the cemento-enamel junction. Junctional Epithelium: the distance from apical to the coronal extension of the junctional epithelium along the root surface. Connective Tissue Repair: the distance between the apaffin, serial sections, 7
µ
§Combiotic, Pfizer Inc, New York, NY. "Hibitane, ICI Ltd., Macclesfield, Great Britain. 'Videoplan, Carl Zeiss Inc., Kontron, Eching bei München, Germany.
260
J Periodontol April 1991
HEALING IN CHRONIC PERIODONTAL DEFECTS
ical extension of the root planing and the apical termination of the junctional epithelium. Cementimi Formation: the distance between the apical extension of the root planing and the coronal extension of a continuous layer of cementum or a cementum-like deposit the root surface. Bone Formation : the distance between the apical extension of the root planing and the coronal extension of newly formed alveolar bone along the root surface. Root Resorption : the combined linear heights of distinct résorption lacunae along the root surface. Ankylosis: the combined linear heights of ankylotic union of newly formed alveolar bone and the root surface. on
Analysis Surface, tooth, and dog Data
for each of the measurethe using 5 selected step serial sections. Differences between dog means for experimental conditions were analyzed using student's i-test for paired observations. Additionally, the frequency of teeth showing root résorption and ankylosis was calculated. Presence of these features in 1 or more of the 10 sections from each tooth resulted in a positive score for the tooth. means
ments were calculated
RESULTS
Clinical Observations
Healing progressed uneventfully leaving all teeth available for analysis. In teeth with chronic defects, gingival recession extended over part of the root surfaces leaving the furcation apertures open to inspection. By contrast, in teeth with acute defects the gingival margins maintained a position slightly coronal to the CEJ, in spite of limited regeneration of alveolar bone revealed by radiographie examination of the surgical sites at sacrifice (Fig. 2). Histometric Observations Histometric evaluation of the chronic and acute defects following 4 weeks of wound healing is shown in Tables 1 and 2 and Figures 3 and 4. Defect height for the chronic defects average 4.6 ± 0.3 mm. Junctional epithelium averaged 30% and connective tissue repair to the root surface averaged 62% (range 49% to 74%) of the defect height. Formation of new cementum and alveolar bone was limited and did not exceed 10% of the defect height. Root résorption was observed in all chronic defects. Ankylosis was observed in 3 specimens. Mean defect height for the acute defects was 4.4 ± 0.4 mm. Junctional epithelium occupied approximately 11% of the root surface. The remainder of the defect, except for 1% of gingival recession, exhibited connective tissue repair. Connective tissue repair to the root surface was significantly smaller in 1 dog (48%) compared to the 4 other dogs (94%, 99%, 100%, and 100%, respectively). Regeneration of alveolar bone and cementum was limited. Root
Figure 2: Representative radiographs of chronic and acute periodontal defects from I of the dogs 4 weeks after reconstructive periodontal surgery. Table 1: Periodontal Repair in Chronic and Acute Supraalveolar = 5 Periodontal Defects (Mean ± s.d. in ram, dogs) Chronic Defect height Junctional epithelium Connective tissue repair Cementum formation Bone formation Root résorption
4.6: 1.4: 2.8: 0.4: 0.2: 0.7:
Ankylosis
0.3 0.3 0.5 0.4
0.3 0.4
0.0: 0.1
Acute
4.4: 0.5: 3.8: 0.1: 0.6: 0.9: 0.1:
0.4 0.9 1.0 0.2 0.2 0.6 0.1
Proportions of Teeth with Root Resorption and Ankylosis Following Reconstructive Surgery in Chronic and Acute Periodontal Defects in 5 Dogs Table 2:
Root
résorption Ankylosis* *Ankylosis was
found in all
Chronic
Acute
15/15 3/15
14/15 5/15
dogs.
observed in all but 1 tooth. Ankylosis occurred in 5 specimens, 1 in each dog. When comparing experimental conditions, connective tissue repair to the root surface appeared less favorable in the chronic defects. However, statistically significant differences could not be found between acute and chronic defects for this or any other examined parameter. Only when the dog exhibiting reduced connective tissue repair in the acute defects was excluded from statistical testing did
résorption
was
Volume 62 Number 4
WIKESJÖ, SELVIG, ZIMMERMAN, NILVÉUS
261
Previously, a varying extent of connective tissue repair has been reported in acute surgical defects.1_5,16-20 In the model presently described, connective tissue repair surface.
may range from 85% to 100% of the defect height.1^ Indeed, some variation between animals has been observed. A few dogs may show an unusual healing response.1 In the present study, 1 dog significantly deviated from the overall healing pattern in the acute defects. The reason for this is not clear. Failing connective tissue repair may be a reflection of a normal individual variation in wound healing. More likely, however, this may be a result of inadvertent trauma to the wound during early healing, resulting in wound rupture and subsequent formation of a long junctional epithelium. This notion is supported by the fact that healing varied between P2, P3, and P4 and that healing in the chronic defects progressed as expected. Excessive tension on the wound margin is a risk factor particularly in the P2 region due to the insertion of the buccal frenum. It can be argued that the aberrant dog should be exited from the study; however, for the sake of statistical analysis of the chronic model, the complete data were included. Healing in the chronic defects included gingival reces-
sion,
CHRONIC
ACUTE
Figure 3: Histometric results expressed in percent (%) of the defect height in chronic and acute supraalveolar periodontal defects.
P2
P3
CHRONIC DEFECTS
P4
P2
P3
P4
ACUTE DEFECTS
Figure 4: Mean connective tissue repair to the root surface in percent {%) of the defect height for P2, P3, and P4 separately in the 5 dogs. differences in connective tissue repair between chronic and acute defects reach statistical sigificance (P < 0.05). DISCUSSION This study evaluated healing following reconstructive therapy of surgically created supraalveolar periodontal defects with or without long-term exposure to simulated Periodontitis conditions. The striking observation in the acute defects was the almost complete connective tissue repair to the root
a
long junctional epithelium and, consequently,
a
smaller amount of connective tissue repair. All other conditions being equal, one may assume that acquired properties of the root surface due to exposure to periodontitissimulating conditions may have influenced the outcome of healing. Bacterial cells in the dentinal tubules and presence of endotoxins have been demonstrated in roots exposed to periodontal disease.21-25 Presence of residual antigenic material at the root surface at wound closure may possibly trigger an increased recruitment of polymorphonuclear leucocytes and, later, macrophages during the early healing phase with subsequent release of excess proteolytic enzyme, which may impair healing. However, the relative influence of this factor on the outcome of periodontal repair is unclear. Connective tissue repair in this study included on the average 62% of a root surface, which, at least in part, had been exposed to dental plaque. In another recent study, connective tissue repair was demonstrated over large portions of root surfaces which had been intentionally contaminated with saliva including bacterial cells at wound closure.3 However, the impact of root surface properties on periodontal wound healing should not be ruled out. Previous studies have demonstrated failing connective tissue repair to root surfaces treated with stannous fluoride,26 heparin,2'4 and fibronectin,27 whereas almost complete connective tissue repair has been shown repeatedly following root surface treatment with citric acid9-15'18,19'27-30 or
tetracycline.27-31 Variations in
healing response between animals or sites regular experience in experimental research. In chronic periodontal lesions in canines and in non-human primates, healing following regenerative procedures may vary from complete epithelialization to almost complete connective tissue repair to the root surface.32-38 Some difference in is
a
J Periodontol
262
April
HEALING IN CHRONIC PERIODONTAL DEFECTS
healing potential may be ascribed to species and site selecSpecies uniqueness in metabolic rate, site-specific anatomy, and function may play a significant role. Another critical aspect for periodontal would healing is the quality and quantity of flap tissue and the surgical design of the reconstructive procedure. Recession of the gingiva may have reduced the amount of soft tissue available for flap covertion.
age. This may limit the extension of wound closure or produce undesired tension on the flap-root surface interface if complete defect coverage is attempted. Placing surgical incisions as distant from the defect as possible may be critical. The limited regeneration generally seen following reconstructive surgery in the interproximal defect, where the incisions usually are placed directly over the lesion, may substantiate this assumption.36-38 On this basis it appears that the observed differences in connective tissue repair in acute and chronic defects arose from properties of the gingival flap rather than the instrumented root surface. A problem inherent in the chronic large supraalveolar defect is the amount of variability in connective tissue repair following a control treatment as replaced flap surgery. In this study, connective tissue repair amounted to 2.8 ± 0.5 mm, which gives a coefficient of variation of 18%. This amount of variability would make it relatively difficult to recognize differences between experimental and control treatments using this model in an experimental design. An alternate way of viewing the problem is to calculate confidence interval estimates for the population. Variability in connective tissue repair at the 95% confidence interval for the standard deviation in chronic defects would be 0.42 to 2.03 mm, which at the upper limit could mean connective tissue repair from close to no repair to total repair in chronic defects rendering flap surgery without additional treatment. To standardize defect height with minimum variability seems essential in this model, since it is not known whether connective tissue repair to the root surface is independent of defect height which appears to be true in the acute defect model.1 Taking these limitations into consideration, the chronic defect model may be used to evaluate biochemical wound conditioning and biomaterials designed to enhance connective tissue repair to the root surface. The potential of these agents to promote cementum formation and regeneration of alveolar bone may also be evaluated. However, it should be noted that it is unlikely that treatments resulting in less than complete connective tissue repair may be tested fairly unless a larger number of animals is used.
Acknowledgments Special thanks are due to Julie Cranfill for secretarial assistance; Norman Medina, MSPH for data management; Elwyn Spaulding and Richard Tinker for illustrations; and
Emily Daniel and Jim Smith for histotechnical preparation.
1991
REFERENCES 1. Wikesjö UME, Nilvéus R. Periodontal repair in dogs: Healing patterns in large circumferential periodontal defects. J Clin Periodontol 1991;18:49-59. 2. Wikesjö UME, Claffey N, Egelberg J. Periodontal repair in dogs: Effect of
heparin
treatment of the root surface. / Clin Periodontol
1991;18:60-64. 3. Wikesjö UME, Hagen , Nielsen DD. Periodontal repair in dogs:
Effect of saliva contamination of the root surface. / Periodontol
1990;61:559-563. Wikesjö UME, Nilvéus R. Periodontal repair in dogs: Effect of wound stabilization on healing. J Periodontol 1990;61:719-724. 5. Claffey N, Motsinger S, Ambruster J, Egelberg J. Placement of a porous membrane underneath the mucoperiosteal flap and its effect on periodontal wound healing in dogs. J Clin Periodontol 1989;16:12-16. 6. Steiner SS, Crigger M, Egelberg J. Connective tissue regeneration to periodontally diseased teeth. II. Histologie observations of cases following replaced flap surgery. J Periodont Res 1981;16:109-116. 7. Johansson O, Nilvéus R, Egelberg J. Experimental bifurcation defects in dogs. J Periodont Res 1978;13:525-531. 8. Nilvéus R, Johansson O, Egelberg J. The effect of autogenous cancellous bone grafts on healing of experimental furcation defects in dogs. J Periodont Res 1978;13:532-537. 9. Crigger M, Bogle G, Nilvéus R, Egelberg J, Selvig ICA. The effect of topical citric acid application on the healing of experimental furcation defects in dogs. / Periodont Res 1978;13:538-549. 10. Nilvéus R, Bogle G, Crigger M, Egelberg J, Selvig KA. The effect of topical citric acid application on the healing of experimental furcation defects in dogs. II. Healing after repeated surgery. / Periodont 4.
Res 1980;15:544-550. 11. Nilvéus R, Egelberg J. The effect of topical citric acid application on the healing of experimental furcation defects in dogs. HI. The relative importance of coagulum support, flap design and systemic antibiotics. J Periodont Res 1980;15:551-560. 12. Selvig KA, Ririe CM, Nilvéus R, Egelberg J. Fine structure of connective tissue attachment following acid treatment of experimental furcation pockets in dogs. J Periodont Res 1981;16:123-129. 13. Klinge , Nilvéus R, Kiger RD, Egelberg J. Effect of flap placement and defect size on healing of experimental furcation defects. / Periodont Res 1981;16:236-248. 14. Klinge B, Nilvéus R, Egelberg J. Effect of crown-attached sutures on healing of experiment furcation defects in dogs. / Clin Periodontol
1985;12:369-373.
15.
Klinge B, Nilvéus R, Bogle G, Badersten A, Egelberg J. Effects of implants on healing of experimental furcation defects in dogs. / Clin
Periodontol 1985;12:321-326. Linghorne WJ, O'Connell DC. Studies in the regeneration and reattachment of supporting structures of teeth. I. Soft tissue reattachment. J Dent Res 1950;29:419^128. 17. Hiatt WH, Stallard RE, Butler ED, Badgett B. Repair following mucoperiosteal flap surgery with full gingival retention. / Periodontol 16.
1968;39:11-16.
18. Poison AM, Proye MP. Fibrin linkage: A precursor for new attachment. J Periodontol 1983;54:141-147. 19. Caton JG, Poison AM, Pini Prato G, Bartolucci EG, Clauser C. Healing after application of tissue-adhesive material to denuded and citric acid-treated root surfaces. / Periodontol 1986;57:385-390. 20. Magnusson I, Stenberg WV, Batich C, Egelberg J. Connective tissue repair in circumferential periodontal defects in dogs following use of a biodegradable membrane. / Clin Periodontol
1990;17:243-248.
21. Adriaens PA, Edwards CA, DeBoever JA, Loesche WJ. Ultrastructural observations on bacterial invasion in cementum and radicular dentin of periodontally diseased human teeth. / Periodontol
Volume 62 Number 4
WIKESJÖ, SELVIG, ZIMMERMAN, NILVÉUS defects following regenerative surgery in Clin Periodontol 1985;12:837-849.
periodontal
1988;59:493-503.
22. Hatfield CG, Baumhammers A. Cytotoxic effects of periodontally involved surfaces of human teeth. Arch Oral Biol 1971; 16:465468. 23. Aleo JJ, De Renzis FA, Farber PA, Varboncoeur AP. The presence and biologic activity of cementum-bound endotoxin. / Periodontol
1974;45:672-675. 24. Aleo 25.
JJ, De Renzis FA, Farber PA. In
31.
32.
vitro attachment of human
gingival fibroblasts to root surfaces. / Periodontol 1975;46:639-645. Fine DH, Morris ML, Tabak L, Cole JD. Preliminary characterization of material eluted from the roots of periodontally diseased teeth. J
33. 34.
Periodont Res
1980;15:10-19. 26. Wikesjö UME, Claffey N, Nilvéus R, Egelberg J. Periodontal repair in dogs: Effect of root surface treatment with stannous fluoride or citric acid on root résorption. J Periodontol 1991;62:180-184. 27. Wikesjö UME, Claffey N, Christersson LA, et al. Repair of periodontal furcation defects in beagle dogs following reconstructive sur-
including root surface demineralization with tetracycline hydrochloride and topical fibronectin application. / Clin Periodontol gery
1988;15:73-80. Bogle G, Adams D, Crigger M, Klinge , Egelberg J. New attachment after surgical treatment and acid conditioning of roots in naturally occurring periodontal disease in dogs. / Periodont Res 1981;16:130-133. 29. Bogle G, Garrett S, Crigger M, Egelberg J. New connective tissue attachment in beagles with advanced natural Periodontitis. J Periodont Res 1983;18:220-228. 30. Bogle G, Claffey N, Egelberg J. Healing of horizontal circumferential 28.
263
beagle dogs. /
Claffey N, Bogle G, Bjorvatn K, Selvig KA, Egelberg J. Topical application of tetracycline in regenerative periodontal surgery in beagles. Acta Odontol Scand 1987;45:141-146. Linghorne WJ, O'Connell DC. Studies in the reattachment and regeneration of the supporting structures of teeth. III. Regeneration in epithelialized pockets. / Dent Res 1955;34:164-177. Ellegaard B, Karring T, Listgarten M, Löe H. New attachment after treatment of interradicular lesions. J Periodontol 1973;44:209-217. Ellegaard B, Karring T, Davies R, Löe H. New attachment after treatment of intrabony defects in monkeys. / Periodontol 1974;45:368377.
Löe H. Retardation of epithelial migration in new attachment attempts in intrabony defects in monkeys. / Clin Periodontol 1976;3:23-27. 36. Caton J, Zander HA. Osseous repair of an infrabony pocket without new attachment of connective tissue. / Clin Periodontol 1976;3:54-58. 37. Caton J, Ñyman S. Histometric evalution of periodontal surgery. I. The modified Widman flap procedure. J Clin Periodontol 1980;7:212223. 38. Caton J, Nyman S, Zander H. Histometric evaluation of periodontal surgery. II. Connective tissue attachment levels after four regenerative procedures. / Clin Periodontol 1980;7:224-231. 35.
Ellegaard B, Karring T,
Send reprint requests to Dr. Ulf M.E. Wikesjö, School of Loma Linda University, Loma Linda, CA 92350. Accepted for publication October 30, 1990.
Dentistry,
Periodontal Repair in Dogs: Healing in Experimentally Created Chronic Periodontal Defects UlfM.E. Wikesjö, * Knut A. Selvig, *T Grenith Zimmerman, * and Rolf Nilvéus A
objectively evaluate surgical conditioning and biomaterials in
need exists for well-defined animal models to and a role for biochemical wound
principles possible promoting periodontal regeneration. To test an existing model for its usefulness in quantitative evaluation of periodontal wound healing, large supraalveolar periodontal defects were surgically created around the mandibular premolare (P2, P3, P4) in left or right jaw quadrants in 5 beagle dogs. The defects were exposed to the oral environment for 6 months and were then subjected to reconstructive flap surgery (chronic defects). Healing in these defects was compared to healing following reconstructive surgery in similar contralateral defects which had not been exposed to plaque and calculus (acute defects). The animals were sacrificed after a 4-week healing period and tissue blocks including teeth and surrounding structures were processed for histometric analysis. Mean defect height (± s.d.) for chronic and acute defects amounted to 4.6 ± 0.3 and 4.4 ± 0.4 mm, respectively. Mean connective tissue repair to the root surface in chronic defects amounted to 62% (range 49% to 74%) of the defect height. Mean connective tissue repair in the acute defects exceeded 94% of the defect height in 4 of the dogs, but amounted to only 48% in 1 dog. Regeneration of alveolar bone and cementum was limited under both experimental conditions. Root résorption was frequently encountered, whereas ankylosis was seen in only few teeth. This study indicates that healing may vary not only as a result of controlled experimental variables, but also due to differences in biological response between dogs or to fortuitous traumatic factors. The variability in healing in the chronic defects imposes limitations on the use of this defect model to study periodontal regeneration, especially when a small number of animals is used. J Periodontol 1991;
62:258-263.
Key Words: Models, animals; odontal
wound
diseases/therapy.
healing; surgical flaps;
Despite considerable efforts, predictable and complete periodontal regeneration remains an unsolved problem in periodontal research. Thus, a need exists for continuous development and refinement of suitable animal models. Recent studies in periodontal wound healing have included a
model featuring surgically created and immediately treated large supraalveolar mandibular premolar defects in the dog.1-5 This model heals almost exclusively with connective tissue repair rather than epithelial adhesion to the defect
surface and within 4 weeks may include a modest formation of new cementum and alveolar bone, root résorption, and ankylosis.1 The model may be used to study the nature of the healing root surface-gingival flap interface as well as the effect of various root and wound treatments in enhancing formation of new cementum and alveolar bone and in "Lorna Linda University, Loma Linda, ca. +University of Bergen, Bergen, Norway.
*National Dental Health Service, Kristianstad, Sweden.
bone
regeneration; peri-
preventing healing aberrations such as root résorption and ankylosis. However, this model does not mimic clinical periodontal defects, which generally heal with a long junctional epithelium following flap surgery6 and thus cannot be used to study treatments designed to enhance connective tissue repair to the root surface. In earlier studies, we used small, chronic through-andthrough furcation defects in the mandibular premolare in the dog to study periodontal wound healing.7-12 These defects do not regenerate spontaneously but heal with epithelialization of the fornix of the furcation following flap surgery.7-9 An experimental treatment was considered successful only if the full circumference of the furcation defect healed with connective tissue repair. Further development of this model included more radical removal of tooth-supporting structures to create large circumferential furcation defects, still utilizing complete connective tissue repair in the furcation area as a criterion for
Volume 62 Number 4
WIKESJÖ, SELVIG, ZIMMERMAN, NILVÉUS
EXPERIMENTAL DESIGN DEFECT INDUCTION: CHRONIC DEFECTS
BASELINE
PLAQUE ACCUMULATION RECONSTRUCTIVE SURGERY: CHRONIC DEFECTS DEFECT INDUCTION + RECONSTRUCTIVE SURGERY: ACUTE DEFECTS
6 MONTHS
259
Immediately following reconstructive surgery in the chronic defects, defects of matching size were surgically created in the contralateral quadrant and reconstructive surgery was performed. Average clinical height of these acute defects was 5.3 mm (range 4.3 to 6.3 mm). This examination was part of an institutionally approved protocol in periodontal wound healing.
PLAQUE CONTROL 7 MONTHS
SACRIFICE BLOCK BIOPSIES
Figure 1: Experimental design.
experimental success.13-15 Such analysis is useful in evaluating a biological potential of an experimental procedure but would not be sensitive enough for assessing quantitative differences between experimental conditions. Incomplete connective tissue repair in the furcation will presumably be reflected in adjacent buccal and lingual root
surfaces as well. Therefore, it appears to be of interest to evaluate that model for quantitative evaluation of treatments directed to enhance connective tissue repair to the root surface. Thus, this study was initiated to assess extent and variability of healing on the buccal and lingual surfaces in chronic supraalveolar periodontal defects in the dog following reconstructive flap surgery. Results were compared to healing in surgically created and immediately treated defects. MATERIALS AND METHODS
Animals, Surgical Procedures, Design
and
Experimental
periodontal defects were surgically created in right jaw quadrants in 5 young adult male beagle
Standardized
left and dogs 6 months apart and were then treated with reconstructive flap surgery (Fig. 1). This experimental design allows for intra-animal comparisons of healing in defects exposed to the oral environment for 6 months (chronic defects) and defects without this experience (acute defects). Chronic and acute defects were alternated between left and right jaw quadrants in subsequent dogs. The defects were created around the 2nd, 3rd, and 4th mandibular premolare (P2, P3, and P4) under sodium pentobarbital anesthesia as earlier described.1 Mean height of the chronic defects, measured from the cemento-enamel junction (CEJ) to the reduced alveolar bone with a periodontal probe at time of creation, amounted to 5.2 mm (range 4.2 to 5.9 mm); the defects in P2 always being somewhat smaller than in P3 and P4. The mucoperiosteal flaps were then replaced and sutured immediately above the reduced alveolar bone to expose the surgically denuded root surface to plaque and calculus formation. Six months later the defects were subjected to reconstructive surgery. Clinical height of the chronic defects at that time point averaged 5.1 mm (range 4.1 to 6.2 mm).
Wound Management The root surfaces were instrumented with curets, chisels, and water-cooled flame-shaped finishing burs in an attempt to remove dental plaque and calculus (chronic defects) and the root cementum (chronic and acute defects). The defects were then rinsed with saline and the wounds closed by placing and suturing the flap margins 1 to 2 mm coronal to the cemento-enamel junction as previously described.1
care included a daily administered intramuscular broad spectrum antibiotic§ for 2 weeks, daily topical plaque control with a 2% Chlorhexidine solution11 for the duration of the study, and suture removal 1 week after surgery. The dogs were sacrificed 4 weeks following the reconstructive surgery by an intravenous injection of sodium pentobarbital. Intraoral radiographs were taken of the experimental teeth at the time of sacrifice.
Postoperative
Histological Procedures
Histological processing and evaluation was performed as earlier described.1 Briefly, tissue blocks including teeth and surrounding tissues were removed at sacrifice and fixed in 10% buffered formalin. Following décalcification in 5% formic acid, trimming, dehydration, and embedding in par-
thick, were cut in a buccomesio-distal extension the entire lingual plane throughout of the teeth. Every 14th section, approximately 100 µ apart, was stained with hematoxylin and eosin. The most centrally located stained section of the mesial and the distal root of each tooth was identified by the size of the pulp chamber and the root canal. This section and the 2 step serial sections on either side were used for histometric analysis. Thus, 5 subsequent step serial sections of the mid portion of both the mesial and the distal root were analyzed using a microscope connected to a computer aided manual data collection system.1 The following measurements were made for the buccal and lingual surfaces of each root: Defect Height: the distance between the apical extension of the root planing and the cemento-enamel junction. Junctional Epithelium: the distance from apical to the coronal extension of the junctional epithelium along the root surface. Connective Tissue Repair: the distance between the apaffin, serial sections, 7
µ
§Combiotic, Pfizer Inc, New York, NY. "Hibitane, ICI Ltd., Macclesfield, Great Britain. 'Videoplan, Carl Zeiss Inc., Kontron, Eching bei München, Germany.
260
J Periodontol April 1991
HEALING IN CHRONIC PERIODONTAL DEFECTS
ical extension of the root planing and the apical termination of the junctional epithelium. Cementimi Formation: the distance between the apical extension of the root planing and the coronal extension of a continuous layer of cementum or a cementum-like deposit the root surface. Bone Formation : the distance between the apical extension of the root planing and the coronal extension of newly formed alveolar bone along the root surface. Root Resorption : the combined linear heights of distinct résorption lacunae along the root surface. Ankylosis: the combined linear heights of ankylotic union of newly formed alveolar bone and the root surface. on
Analysis Surface, tooth, and dog Data
for each of the measurethe using 5 selected step serial sections. Differences between dog means for experimental conditions were analyzed using student's i-test for paired observations. Additionally, the frequency of teeth showing root résorption and ankylosis was calculated. Presence of these features in 1 or more of the 10 sections from each tooth resulted in a positive score for the tooth. means
ments were calculated
RESULTS
Clinical Observations
Healing progressed uneventfully leaving all teeth available for analysis. In teeth with chronic defects, gingival recession extended over part of the root surfaces leaving the furcation apertures open to inspection. By contrast, in teeth with acute defects the gingival margins maintained a position slightly coronal to the CEJ, in spite of limited regeneration of alveolar bone revealed by radiographie examination of the surgical sites at sacrifice (Fig. 2). Histometric Observations Histometric evaluation of the chronic and acute defects following 4 weeks of wound healing is shown in Tables 1 and 2 and Figures 3 and 4. Defect height for the chronic defects average 4.6 ± 0.3 mm. Junctional epithelium averaged 30% and connective tissue repair to the root surface averaged 62% (range 49% to 74%) of the defect height. Formation of new cementum and alveolar bone was limited and did not exceed 10% of the defect height. Root résorption was observed in all chronic defects. Ankylosis was observed in 3 specimens. Mean defect height for the acute defects was 4.4 ± 0.4 mm. Junctional epithelium occupied approximately 11% of the root surface. The remainder of the defect, except for 1% of gingival recession, exhibited connective tissue repair. Connective tissue repair to the root surface was significantly smaller in 1 dog (48%) compared to the 4 other dogs (94%, 99%, 100%, and 100%, respectively). Regeneration of alveolar bone and cementum was limited. Root
Figure 2: Representative radiographs of chronic and acute periodontal defects from I of the dogs 4 weeks after reconstructive periodontal surgery. Table 1: Periodontal Repair in Chronic and Acute Supraalveolar = 5 Periodontal Defects (Mean ± s.d. in ram, dogs) Chronic Defect height Junctional epithelium Connective tissue repair Cementum formation Bone formation Root résorption
4.6: 1.4: 2.8: 0.4: 0.2: 0.7:
Ankylosis
0.3 0.3 0.5 0.4
0.3 0.4
0.0: 0.1
Acute
4.4: 0.5: 3.8: 0.1: 0.6: 0.9: 0.1:
0.4 0.9 1.0 0.2 0.2 0.6 0.1
Proportions of Teeth with Root Resorption and Ankylosis Following Reconstructive Surgery in Chronic and Acute Periodontal Defects in 5 Dogs Table 2:
Root
résorption Ankylosis* *Ankylosis was
found in all
Chronic
Acute
15/15 3/15
14/15 5/15
dogs.
observed in all but 1 tooth. Ankylosis occurred in 5 specimens, 1 in each dog. When comparing experimental conditions, connective tissue repair to the root surface appeared less favorable in the chronic defects. However, statistically significant differences could not be found between acute and chronic defects for this or any other examined parameter. Only when the dog exhibiting reduced connective tissue repair in the acute defects was excluded from statistical testing did
résorption
was
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WIKESJÖ, SELVIG, ZIMMERMAN, NILVÉUS
261
Previously, a varying extent of connective tissue repair has been reported in acute surgical defects.1_5,16-20 In the model presently described, connective tissue repair surface.
may range from 85% to 100% of the defect height.1^ Indeed, some variation between animals has been observed. A few dogs may show an unusual healing response.1 In the present study, 1 dog significantly deviated from the overall healing pattern in the acute defects. The reason for this is not clear. Failing connective tissue repair may be a reflection of a normal individual variation in wound healing. More likely, however, this may be a result of inadvertent trauma to the wound during early healing, resulting in wound rupture and subsequent formation of a long junctional epithelium. This notion is supported by the fact that healing varied between P2, P3, and P4 and that healing in the chronic defects progressed as expected. Excessive tension on the wound margin is a risk factor particularly in the P2 region due to the insertion of the buccal frenum. It can be argued that the aberrant dog should be exited from the study; however, for the sake of statistical analysis of the chronic model, the complete data were included. Healing in the chronic defects included gingival reces-
sion,
CHRONIC
ACUTE
Figure 3: Histometric results expressed in percent (%) of the defect height in chronic and acute supraalveolar periodontal defects.
P2
P3
CHRONIC DEFECTS
P4
P2
P3
P4
ACUTE DEFECTS
Figure 4: Mean connective tissue repair to the root surface in percent {%) of the defect height for P2, P3, and P4 separately in the 5 dogs. differences in connective tissue repair between chronic and acute defects reach statistical sigificance (P < 0.05). DISCUSSION This study evaluated healing following reconstructive therapy of surgically created supraalveolar periodontal defects with or without long-term exposure to simulated Periodontitis conditions. The striking observation in the acute defects was the almost complete connective tissue repair to the root
a
long junctional epithelium and, consequently,
a
smaller amount of connective tissue repair. All other conditions being equal, one may assume that acquired properties of the root surface due to exposure to periodontitissimulating conditions may have influenced the outcome of healing. Bacterial cells in the dentinal tubules and presence of endotoxins have been demonstrated in roots exposed to periodontal disease.21-25 Presence of residual antigenic material at the root surface at wound closure may possibly trigger an increased recruitment of polymorphonuclear leucocytes and, later, macrophages during the early healing phase with subsequent release of excess proteolytic enzyme, which may impair healing. However, the relative influence of this factor on the outcome of periodontal repair is unclear. Connective tissue repair in this study included on the average 62% of a root surface, which, at least in part, had been exposed to dental plaque. In another recent study, connective tissue repair was demonstrated over large portions of root surfaces which had been intentionally contaminated with saliva including bacterial cells at wound closure.3 However, the impact of root surface properties on periodontal wound healing should not be ruled out. Previous studies have demonstrated failing connective tissue repair to root surfaces treated with stannous fluoride,26 heparin,2'4 and fibronectin,27 whereas almost complete connective tissue repair has been shown repeatedly following root surface treatment with citric acid9-15'18,19'27-30 or
tetracycline.27-31 Variations in
healing response between animals or sites regular experience in experimental research. In chronic periodontal lesions in canines and in non-human primates, healing following regenerative procedures may vary from complete epithelialization to almost complete connective tissue repair to the root surface.32-38 Some difference in is
a
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April
HEALING IN CHRONIC PERIODONTAL DEFECTS
healing potential may be ascribed to species and site selecSpecies uniqueness in metabolic rate, site-specific anatomy, and function may play a significant role. Another critical aspect for periodontal would healing is the quality and quantity of flap tissue and the surgical design of the reconstructive procedure. Recession of the gingiva may have reduced the amount of soft tissue available for flap covertion.
age. This may limit the extension of wound closure or produce undesired tension on the flap-root surface interface if complete defect coverage is attempted. Placing surgical incisions as distant from the defect as possible may be critical. The limited regeneration generally seen following reconstructive surgery in the interproximal defect, where the incisions usually are placed directly over the lesion, may substantiate this assumption.36-38 On this basis it appears that the observed differences in connective tissue repair in acute and chronic defects arose from properties of the gingival flap rather than the instrumented root surface. A problem inherent in the chronic large supraalveolar defect is the amount of variability in connective tissue repair following a control treatment as replaced flap surgery. In this study, connective tissue repair amounted to 2.8 ± 0.5 mm, which gives a coefficient of variation of 18%. This amount of variability would make it relatively difficult to recognize differences between experimental and control treatments using this model in an experimental design. An alternate way of viewing the problem is to calculate confidence interval estimates for the population. Variability in connective tissue repair at the 95% confidence interval for the standard deviation in chronic defects would be 0.42 to 2.03 mm, which at the upper limit could mean connective tissue repair from close to no repair to total repair in chronic defects rendering flap surgery without additional treatment. To standardize defect height with minimum variability seems essential in this model, since it is not known whether connective tissue repair to the root surface is independent of defect height which appears to be true in the acute defect model.1 Taking these limitations into consideration, the chronic defect model may be used to evaluate biochemical wound conditioning and biomaterials designed to enhance connective tissue repair to the root surface. The potential of these agents to promote cementum formation and regeneration of alveolar bone may also be evaluated. However, it should be noted that it is unlikely that treatments resulting in less than complete connective tissue repair may be tested fairly unless a larger number of animals is used.
Acknowledgments Special thanks are due to Julie Cranfill for secretarial assistance; Norman Medina, MSPH for data management; Elwyn Spaulding and Richard Tinker for illustrations; and
Emily Daniel and Jim Smith for histotechnical preparation.
1991
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Ellegaard B, Karring T,
Send reprint requests to Dr. Ulf M.E. Wikesjö, School of Loma Linda University, Loma Linda, CA 92350. Accepted for publication October 30, 1990.
Dentistry,
