- Email: [email protected]
Histological Study of Periradicular Tissue Responses to Uninfected and Infected Devitalized Pulps in Dogs
Basic Research—Biology
Histological Study of Periradicular Tissue Responses to Uninfected and Infected Devitalized Pulps in Dogs Louis M. Lin, BDS, DMD, PhD,* Peter M. Di Fiore, DDS, MS,* Jarshen Lin, BDS, DDS,† and Paul A. Rosenberg, DDS* Abstract Uninfected necrotic tissue, such as that which follows a myocardial or cerebral infarct, is capable of inducing an inflammatory reaction. Eventually, the infarct is organized by granulation tissue. Why then, does uninfected devitalized pulp tissue, such as in traumatized teeth, not cause periradicular inflammation and does not become organized by granulation tissue? Four beagle dogs were used in this experiment. A total of 48 teeth, which included 24 maxillary and 24 mandibular incisors, were aseptically devitalized, leaving residual pulp tissues in the root canals, and equally divided into two groups. Group A (24 uninfected): A sterile cotton pellet was placed deep into the canal orifice before the pulp chamber and access opening were closed with a layer of zinc-oxide eugenol cement followed by glass ionomer cement. Group B (24 infected): The teeth were left open to the oral cavity for 7 days and then closed with a cotton pellet and zinc-oxide eugenol and glass ionomer cement. The animals were sacrificed one year after the experiment and prepared for histological examination of periradicular tissue responses to uninfected and infected devitalized pulp tissues. The results indicate that uninfected devitalized pulp tissues did not continuously release inflammatory mediators and cause persistent periradicular inflammation over a period of one year. However, infected devitalized pulp tissues induced various degrees of periradicular inflammation. Only the apical few millimeters of uninfected devitalized pulp tissue in the root canals were organized by granulation tissue from vital periodontal ligament tissue. (J Endod 2006;32: 34–38)
Key Words Devitalization, infected, inflammation, organization, uninfected
*Department of Endodontics, Advanced Education Program in Endodontics, New York University College of Dentistry, New York, New York; and †Department of Restorative and Biomaterials Sciences, Predoctoral Endodontic Program, Harvard School of Dental Medicine, Harvard University, Boston, Massachusetts. Address requests for reprints to Dr. Louis M. Lin, Department of Endodontics, Advanced Education Program in Endodontics, New York University College of Dentistry, New York, NY 10010. 0099-2399/$0 - see front matter Copyright © 2006 by the American Association of Endodontists. doi:10.1016/j.joen.2005.10.010
34
Lin et al.
I
t is well established that colonization of bacteria in the root canal system is the primary cause of periradicular inflammation. The classic study of Kakehashi et al. (1) showed that periradicular inflammation developed in conventional laboratory rats but not in germ-free rats with surgically exposed pulps. In conventional laboratory rats, oral microorganisms entered the pulp cavity and caused pulpal inflammation and necrosis, and subsequent periradicular inflammation 1 to 42 days after surgical pulp exposure. In germ-free rats, even when the root canals were packed with sterile food debris, neither pulpal necrosis nor periradicular inflammation developed. Bergenholtz (2) and Sundqvist (3), in their clinical studies, showed that bacteria could be cultured from the root canals of traumatized necrotic teeth with intact crowns if periradicular lesions were present, but could not be cultured from necrotic teeth if periradicular lesions were absent. Möller et al. (4), in an animal study, demonstrated that uninfected devitalized pulps would not cause periradicular inflammation 6 to 7 months after pulp devitalization, while severe periradicular inflammation developed in devitalized pulps contaminated with indigenous oral microorganisms. Torneck (5) demonstrated that if polyethylene tubes with one end closed were filled with homologous autoclaved muscle and implanted subcutaneously in rats, a mildly inflamed mature granulation tissue was observed at the open ends of the tubes after a period of 60 days. However, if polyethylene tubes were filled with homologous autoclaved muscle contaminated with bacteria and implanted subcutaneously, the reaction ranged from moderate to intense inflammation with abscess formation at the open ends of the tubes. Makkes et al. (6) also reported that perforated polyethylene tubes with both ends closed and filled with homologous, sterile, autolytic muscle tissue were implanted subcutaneously in rabbits, a slight, transient, inflammatory reaction around the tubes was demonstrated after 21 days of the experiment. It is well known that uninfected necrotic tissue, such as myocardium or cerebellum following an infarct, is capable of inducing inflammatory reaction, which is a defense mechanism of living organisms to eliminate necrotic tissue (7). Eventually, the necrotic myocardium is organized by granulation tissue and removed by macrophages before the repair process takes place. The resulting question is why uninfected devitalized pulp tissue, such as in traumatized teeth, does not cause persistent periradicular inflammation and does not become organized by granulation tissue from vital periodontal ligament tissue (2– 4). The purpose of this study was to histologically examine if uninfected devitalized pulp tissue caused persistent periradicular inflammation and could be organized by granulation tissue from vital periodontal ligament tissue in dogs over a period of one year.
Materials and Methods Animal Preparation Four beagle dogs approximately 3 years of age were used in accordance with a research protocol that was approved by the Institutional Animal Care and Use Committee before the experiment. The animals were examined clinically and radiographically to assure their dental health. Anterior teeth were used for the experiment. They were free of caries and periradicular lesions. In addition, they had healthy periodontium and complete root formation. Tooth vitality was confirmed when the pulp chamber was accessed and fresh bleeding was observed. A total of 48 maxillary and mandibular incisors were employed. One canine from each animal served as a control. The animals were sedated with ketamine (10 mg/kg body wt) (Parke-Davis, Morris Plan, NJ) and
JOE — Volume 32, Number 1, January 2006
Basic Research—Biology then anesthetized with intravenous administration of pentobarbital (30 mg/kg body wt) (Abbott Laboratories, North Chicago, IL).
Experimental Procedures The teeth and surrounding gingival tissues were first disinfected with 5% tincture of iodine. The teeth were then isolated with rubber dam. The operative field including experimental teeth and surrounding rubber dam were cleansed with 35% hydrogen peroxide and disinfected with 5% tincture of iodine. The teeth were accessed through the lingual surface into the pulp chamber, using sterile carbide round burs. Canal length was determined with a Root ZX electronic apex locator (J Morita, Irvine, CA). The pulp tissues were amputated 3 to 5 mm beyond the electronic apex locator reading at the apex by using Hedström files nos. 15 to 25 ensuring complete devitalization of the pulp tissues. The files were then carefully rotated out of the root canals in a counter-clockwise direction, leaving residual pulp tissue in the root canals according to Möller et al. (4). If the pulp tissue appeared to be completely removed with the files, the tissue was carefully placed back into the root canals. Hemorrhage was controlled with sterile cotton pellets. The coronal 3 mm of the root canals were enlarged with sterile no. 3 Gates Glidden burs after the pulp tissues were compressed deep into the canals, using a small sterile CK condenser (CK Dental specialties, Inc., Orange, CA). All experimental procedures were carefully performed under aseptic condition to avoid contamination. The 48 experimental teeth were equally divided into two groups. Group A (uninfected): 24 teeth, which included three maxillary and three mandibular right incisors from each of four animals. A small sterile cotton pellet was placed deep into the coronal 3 mm of the root canal, and the pulp chamber and access opening were filled with a thick layer of zinc-oxide eugenol cement, followed by a layer of glass ionomer cement. Group B (infected): 24 teeth, which included three maxillary and three mandibular left incisors from each of four animals. The teeth were left open to the oral cavity for 7 days, and then closed with a cotton pellet and zinc-oxide eugenol and glass ionomer cements. The coronal seals of the uninfected devitalized teeth were examined periodically. If the glass ionomer cement in the uninfected teeth was worn away, the teeth were isolated with rubber dam and disinfected with 35% hydrogen peroxide and 5% tincture of iodine. The superficial layer of the glass ionomer cement was removed and newly prepared glass ionomer cement was added. This occurred in three teeth with uninfected devitalized pulp tissues. The animals were sacrificed with an overdose of pentobarbital and perfusion of formaldehyde-glutaraldehyde fixative introduced bilaterally through the external carotid arteries 1 year after the experiment was initiated. Radiographs of all maxillary and mandibular anterior teeth were taken.
Histological Preparation The maxillae and mandibles were removed and carefully block sectioned to include individual teeth and supporting structures. The block specimens were fixed in 10% buffered neutral formalin for 3 days, demineralized in formic acid-sodium citrate solution for 5 wk. and embedded in paraffin. Serial sections of 5 to 6 m were cut from the paraffin blocks and stained with hematoxylin and eosin (H&E), and modified Brown and Brenn stain. The stained sections were examined under a Zeiss light microscope. Sections stained with H&E were used for examination of inflammatory cell infiltration of the pulp and periradicular tissues, as well as root and apical bone resorption. Brown and Brenn stained sections were used for detection of stainable bacteria in the tissue sections. Periradicular tissue responses were histologically classified into four categories, depending on the extent of inflammatory cell infiltrate and apical bone and root resorption: category 1, absence of inflammatory cell infiltrate; category 2, inflammatory cell infiltrate loJOE — Volume 32, Number 1, January 2006
TABLE 1. Periradicular tissue responses to uninfected and infected devitalized pulp tissues at 1-year observation Category of Inflammation
No. of Teeth
Group A (noninfected) B (infected)
24 24
1
2
3
4
21 0
0 0
3* 16
0 8
*These teeth had crown fractures.
calized at the apical foramen with minimal apical bone resorption; category 3, inflammatory cell infiltrate beyond the apical foramen with moderate apical bone and/or root resorption; and category 4, diffuse inflammatory cell infiltrate in the periapical area with a local accumulation of neutrophils (pus), as well as moderate to severe apical bone and root resorption.
Results Three teeth in group A (uninfected) in two animals had crown fractures and developed periradicular lesions (Table 1). Seven teeth in group A still contained vital pulp tissues with intact odontoblast layer along the root canal walls and minimal chronic inflammatory cells in the apical portion of the pulps, which indicated that the pulp tissues were not completely devitalized by the experimental procedure. No periradicular inflammation was present in these seven teeth. The remaining 14 teeth in group A showed complete necrosis of the pulp tissues but no inflammatory cell infiltrate in the periradicular tissues (Fig. 1) (Table 1). The necrotic pulp tissues demonstrated complete loss of cellular structures and became an amorphous denatured mass. Stainable bacteria were not observed along the access cavity walls in 21 teeth without crown fractures in group A. In-growth of connective tissue resembling periodontal ligament tissue into the apical few millimeters of the root canals was present in all completely uninfected devitalized teeth (Fig. 2). Cementoid tissue was observed at the apical foramen in six of 14 completely uninfected devitalized teeth. All 24 teeth in group B (infected) showed moderate to severe periradicular inflammation accompanied by various degrees of apical bone and root resorption (Fig. 3). In some cases, the inflammatory cells were dominated by neutrophilic leukocytes and scattered macrophages. Colonies of stainable bacteria were present in the root canals of devitalized infected teeth. Histologically, control teeth showed normal pulp and periradicular tissues.
Discussion The present study confirms the findings of Möller et al. (4) who demonstrated that bacterial colonization in the root canal is the prerequisite for the development of periradicular inflammation (1–3), and uninfected devitalized pulp would not cause periradicular inflammation. This is in contrast to the finding of Sinai et al. (8) who showed that periradicular tissues of teeth with necrotic pulp stumps, resulting from partial pulp extirpation, invariably broke down in animal and human studies. However, their study did not include a bacteriological examination of the pulp tissues in the root canals to elucidate the possible bacterial cause of periradicular inflammation (9). Clinically, it has also been demonstrated that necrotic pulp tissues in teeth caused by traumatic injuries (2, 3, 10), if uninfected, may remain in the root canals indefinitely without causing periradicular inflammation or clinical symptoms except for occasional crown discoloration. Nevertheless, it is well known that sterile necrotic tissue, such as a myocardium or cerebellum, which has experienced an infarct, is capable of inducing inflammatory reaction by release of inflammatory mediators and cytokines (7). Why then, would uninfected devitalized pulp tissues in the
Periradicular Tissue Responses in Devitalized Pulps
35
Basic Research—Biology
Figure 2. High magnification of the root apex and the periradicular tissues in Fig. 1. Vital connective tissue similar to the periodontal ligament tissue is present in the apical few millimeters of the root canal (arrow). Cementoid tissue is formed at the apical foramen (H&E stain; magnification ⫻200).
Figure 1. Uninfected devitalized tooth. The pulp tissue shows complete necrosis, but the periradicular tissues are free of inflammation (H&E stain; magnification ⫻100).
root canals not cause periradicular inflammation (2– 4)? Tissue that is long dead and coagulated will not induce inflammation. Only dying tissue can cause an intense, transient, acute inflammatory reaction (11). Dying tissue can cause inflammation by a number of mechanisms: (a) release of inflammatory mediators, such as histamine, platelet activating factor, arachidonic acid metabolites, and cytokines by mast cells (12, 13), as well as serotonin, platelet activating factor, clot-promoting factors, thromboxane A2, and complement-cleaving proteases by platelets (14, 15); (b) release of arachidonic acid metabolites and lysosomal enzymes, especially proteases to cleave C3, C4, and C5 by dying cells; (c) activation of the complement system to release C3a, C4a, and C5a, and membrane attack complex C5b6789 (16, 17); (d) activation of kinin system; (e) activation of fibrinolytic system; and (f) activation of clotting cascade. The last four mechanisms are activated by plasma Hageman factor in the blood vessels of the dead tissue, which becomes activated by contact with collagen (11). The necrotic tissue initially releases chemical inflammatory messages to attract inflammatory cells, notably leukocytes and elicit a transient, acute inflammation. However, when the terminal burst of chemical mediators from the necrotic tissue is over, no further inflammatory messages are released. The necrotic tissue will then turn into a mass of coagulated or denatured protein, which no longer acts as an irritant to living tissue and will be removed by macrophages (11). This may in part explain why uninfected devitalized pulps did not cause persistent periradicular inflammation in the present 36
Lin et al.
study. Although over-instrumentation in the present study could cause mechanical injury to the periradicular tissues (18), over-instrumentation alone probably would not perpetuate periradicular inflammation over several weeks or months unless extensive periradicular tissue damage had occurred. This is because of the fact that periradicular tissues are equipped with adequate cellular and humoral defense mechanisms for wound healing. Another possible scenario is that the chemical inflammatory mediators and cytokines, or auto-antigens released by uninfected devitalized pulps through the constricted apical foramen
Figure 3. Infected devitalized tooth. There is local accumulation of inflammatory cells at the apical foramen and a moderate apical bone resorption (H&E, magnification ⫻10).
JOE — Volume 32, Number 1, January 2006
Basic Research—Biology may be limited and quickly neutralized by periradicular tissue defenses. Nevertheless, uninfected devitalized pulps in traumatized immature teeth even with open apexes also would not cause periradicular pathosis (10). Seven teeth in group A (uninfected) were not completely devitalized even though the pulp tissues were amputated 3 to 5 mm beyond the electronic apex locator reading at the apex. This observation is similar to that of Möller et al. (4). The pulp tissues were necrotic in the coronal portion, but remained vital in the apical portion of the root canals, which contained an intact odontoblast layer along the root canal walls and few lymphocytes and plasma cells adjacent to the coronal necrotic pulp tissues. The root canal system appears to be a unique environment in which the inflammatory defense mechanisms of the apical vital pulp tissue is not able to operate effectively to organize the coronal necrotic area because of enclosed dentin walls, constricted apical foramen and a lack of collateral blood circulation (19), which seriously restricts the route of delivery of cellular and humoral defense components. None of the completely devitalized uninfected teeth showed total necrosis of the pulp tissue extending to the apical foramen at 1-yr histological examination. The apical few millimeters of the root canals always contained vital connective tissue, which is similar to periodontal ligament tissue. This finding is in agreement with that of Möller et al. (4). Necrotic tissue in the living body is organized by in-growth of granulation tissue from adjacent vital tissue and slowly removed by macrophages before the repair process takes place (11). Makkes et al. (6) showed that subcutaneous implantation of fragments of sterile, autologous, or heterologous, autolytic connective tissue or muscle tissue in rabbits caused a transient, mild inflammatory reaction, and the implanted tissue fragments were broken down and replaced by vital connective tissue. However, if isologous and homologous, sterile, autolytic muscle tissues were placed in the perforated polyethylene tubes, which were then implanted subcutaneously in rabbits, a slight, transient inflammatory reaction around the tubes was observed. The sterile, autolytic tissues were not broken down or replaced because in-growth of host connective tissue through the holes of the tubes only took place over a very short distance at 21 days of the experiment. Torneck (5) also demonstrated that if polyethylene tubes with one end closed were filled with sterile, autoclaved muscle tissue and implanted subcutaneously in rats, there was some attempt by the granulation tissue to proliferate to a depth of 1 to 2 mm into the tubes at 6 months of the experiment. Presence of vital connective tissue in the apical few millimeters of the root canals in completely devitalized uninfected teeth in the present study indicates organization of that part of the necrotic pulp tissue by in-growth of granulation tissue from vital periodontal ligament tissue through the apical foramen. The vital connective issue in the apical few millimeters of the root canals resembles periodontal ligament tissue, which is not capable of differentiating into odontoblasts (20, 21). This may be resulting the fact that, similar to the polyethylene tubes, the pulp tissue is enclosed within the dentin walls, and the root canal communicates with periodontal ligament tissue only through a constricted apical foramen. Thus, in-growth of granulation tissue from vital periodontal ligament tissue into the root canals is restricted. Therefore, necrotic pulp tissues, if uninfected, can remain in the root canals indefinitely as a mass of nonirritating, denatured protein without being organized by granulation tissue. Nonetheless, in traumatized immature teeth with large open apexes and short roots, organization of completely devitalized, uninfected pulp tissues by vital periodontal ligament tissue has been reported (10). In such cases, in-growth of periodontal ligament tissue into the root canals may cause narrowing of the root canal space, for progenitor cells of the periodontal ligament tissue have the potential to
JOE — Volume 32, Number 1, January 2006
differentiate into cementoblasts (20) and form cementum along the root canal walls (10). Because devitalized pulps were able to remain in the root canals without being contaminated through coronal leakage or exposed root dentinal tubules in the cervical area for 6 months (4), and 1 year in the present study, devitalized pulps could be a suitable model for studying bacteremia and focal infection. Teeth with periradicular lesions did not seem to serve as foci of infection and cause infection of teeth with devitalized pulps on the other side of the jaws in the present study, for none of the teeth in group A (uninfected), other than three teeth with crown fractures, developed periradicular inflammation. The present observation is in agreement with the finding of Möller et al. (4). However, Allard et al. (22) in animal experiment showed that bacteria inoculated in pulpectomized root canals could be transported by the bloodstream into uninfected, pulpectomized root canals in the same animal after 28 to 120 days. They also suggested that establishment of infection in the periradicular area might be necessary for the spread of infection to occur by anachoresis. Nevertheless, Delivanis et al. (23) and Delivanis and Fan (24) were not able to experimentally reproduce similar results in animals if root canals were not over-instrumented. It is possible in the present study that the inflamed periradicular tissues caused by over-instrumentation of the group of teeth containing uninfected devitalized pulps were effectively organized before the group of teeth containing infected devitalized pulps developed periradicular lesions. Accordingly, bacteria from the infected devitalized teeth could not disseminate into the root canals of uninfected devitalized teeth by the bloodstream. Bacteremia occurs only in vascular tissues. In devitalized pulps, the blood circulation is completely cut off. Therefore, it is unlikely that bacteria are able to enter the root canal by way of the bloodstream. Similarly, bacterial myocarditis or cerebral infection usually does not occur in patients suffering from myocardial or cerebral infarct even though transient bacteremia is a common phenomenon (25). However, in diabetic patients, anaerobic cellulitis does occur in ischemic lower extremities (25). Therefore, the predisposing factors to bacteremia, such as bacterial attachment to specific receptors of vascular endothelium and colonization or biofilm formation of bacteria in specific organs and tissues require further investigation. It may be asked that if uninfected devitalized pulp tissues do not cause persistent periradicular inflammation, why should uninfected devitalized pulp tissues be removed and root canal therapy be initiated? The reasons to provide treatment in such cases are: (a) when compared to vital pulp tissues, devitalized pulp tissue is completely devoid of innate and adaptive immune defense mechanisms; (b) the devitalized pulp tissue can be easily colonized and become infected even if a small number of bacteria gain access to the root canal system; and (c) the prognosis of root canal therapy in teeth with periradicular lesions is generally poorer than that without periradicular lesions (26 –28). Therefore, there is an advantage for the patient to have treatment performed in teeth with necrotic pulps before a periradicular lesion develops. Prevention of disease progression or infection is an important treatment strategy.
Conclusion The following conclusions are drawn from this animal experiment: 1. Uninfected devitalized pulp tissue does not continuously release inflammatory mediators and cause persistent periradicular inflammation over a period of 1 year. 2. Uninfected devitalized pulp tissue cannot be effectively organized by granulation tissue from vital periodontal ligament tissue.
Periradicular Tissue Responses in Devitalized Pulps
37
Basic Research—Biology 3. Teeth with periradicular lesions do not seem to serve as foci of infection and cause infection of teeth with devitalized pulps on the other side of the jaw in the present study.
References 1. Kakehashi S, Stanley HR, Fitzgerald RJ. The effects of surgical exposures in the dental pulps in germ-free and conventional laboratory rats. Oral Surg Oral Med Oral Pathol Oral Radiol Endod 1965;20:340 –9. 2. Bergenholtz G. Microorganisms from necrotic pulp of traumatized teeth. Odont Revy 1974;25:347–58. 3. Sundqvist G. Bacteriologic studies of necrotic dental pulp. University of Umea Odontology Dissertation no. 7. Umea, Sweden. 1976:1–94. 4. Möller ATR, Fabricius L, Dahlen G, Heyden G. Influence on periapical tissues of indigenous oral bacteria and necrotic pulp tissue in monkeys. Scand J Dent Res 1981;89:475– 84. 5. Torneck CD. Reaction of rat connective tissue to polyethylene tube implants. Part II. Oral Surg Oral Med Oral Pathol Oral Radiol Endod 1967;674 – 83. 6. Makkes PC, Thoden Van Velzen SK, Wesselink PR. Reactions of the living organism to dead and fixed dead tissue. J Endod 1978;4:7–21. 7. Rubin E, Gorstein F, Rubin R, Achwarting R, Strayer D. Rubin’s pathology, 4th ed. Philadelphia: Lippincott Williams & Wilkins, 2004:295-6. 8. Siani I, Seltzer S, Soltanoff W, Goldenberg A, Bender IB. Biologic aspects of endodontics. Part II. Periapical tissue reactions to pulp extirpation. Oral Surg Oral Med Oral Pathol Oral Radiol Endod 1967;23:664 –79. 9. Pitt Ford TR. Vital pulpectomy—an unpredictable procedure. Int Endod J 1982;15:121– 6. 10. Andreasen JO, Andreasen FM. Textbook and color atlas of traumatic injuries to the teeth, 3rd ed. Copenhagen: Munksgaard, 1994 11. Majno G, Joris I. Cells, tissues, and disease, 2nd ed. Oxford: Oxford University Press, 2004:205-8;446-54. 12. Galli SJ. New concepts about the mast cells. N Engl J Med 1993;328:257– 65. 13. Gordon JR, Burd PR, Galli SP. Mast cells as a source of multifunctional cytokines. Immunol Today 1990;11:458 – 64. 14. White JG. Platelet secretory granules and associated proteins. Lab Invest 1993;68:497– 8.
38
Lin et al.
15. Nachman RL, Weksler BB. The platelet as an inflammatory cell. In: WeissmanG, ed. The cell biology of inflammation. Amsterdam: Elsevier/North-Holland Biological Press, 1980:145– 62. 16. Engel AG, Biesecker G. Complement activation in muscle fiber necrosis: demonstration of the membrane attack complex in necrotic fibers. Ann Neurol 1982;12:289 –96. 17. Pinckard RN, Olson MS, Giclas PC, et al. Consumption of classical complement components by heart subcellular membranes in vitro and in patients after acute myocardial infarcts. J Clin Invest 1975;56:740 –50. 18. Seltzer S, Soltanoff W, Sinai I, Goldenberg A, Bender IB. Biologic aspects of endodontics. Part III. Periapical tissue reactions to root canal instrumentation. Oral Surg Oral Med Oral Pathol Oral Radiol Endod1968;26:534 – 46; 694-705. 19. Suda H, Ikeda H. The circulation of the pulp. In: HargreavesKM, GoodisHE, eds. Dental pulp. Chicago: Quintessence Publishing Co., Inc., 2002: 123–50. 20. Melcher AH. On the repair potential of periodontal tissues. J Periodontol 1976;47:256 – 60. 21. D’souza R. Development of the pulpodentin complex. In: Hargreaves KM, Goodis HE, eds. Dental pulp. Chicago: Quintessence Publishing Co., Inc., 2002: 23– 40. 22. Allard U, Nord C-E, Sjöberg L, Strömberg T. Experimental infections with Staphylococcus aureus, Streptococcus sanguis, Pseudomonas aeruginosa, and Bacteroides fragilis in the jaws of dogs. Oral Surg Oral Med Oral Pathol Oral Radiol Endod 1979;48:454 – 62. 23. Delivanis PD, Snowden RB, Doyle RJ. Localization of blood-borne bacteria in instrumented unfilled root canals. Oral Surg Oral Med Oral Pathol Oral Radiol Endod 1981;52:430 –2. 24. Delivanis PD, Fan VSC. The localization of blood-borne bacteria in instrumented unfilled and overinstrumented canals. J Endod 1984;10:521– 4. 25. Mims C, Playfair J, Roitt I, Wakelin D, Williams R. Medical microbiology, 2nd ed. Philadelphia: Mosby 1999:119-26. 26. Strindberg LZ. The dependence of the results of pulp therapy on certain factors. An analytic study based on radiographic and clinical follow-up examination. Acta Odontologia Scand 1956;14(Suppl. 21):1–175. 27. Sjogren U, Hagglund B, Sundqvist G, Wing K. Factors affecting the long-term results of endodontic treatment. J Endod 1990;16:498 –504. 28. Chugal NM, Clive JM, Spangberg LS. A prognostic model for assessment of the outcome of endodontic treatment: effect of biologic and treatment variables. Oral Surg Oral Med Oral Pathol Oral Radiol Endod 2001;91:342–52.
JOE — Volume 32, Number 1, January 2006
Histological Study of Periradicular Tissue Responses to Uninfected and Infected Devitalized Pulps in Dogs Louis M. Lin, BDS, DMD, PhD,* Peter M. Di Fiore, DDS, MS,* Jarshen Lin, BDS, DDS,† and Paul A. Rosenberg, DDS* Abstract Uninfected necrotic tissue, such as that which follows a myocardial or cerebral infarct, is capable of inducing an inflammatory reaction. Eventually, the infarct is organized by granulation tissue. Why then, does uninfected devitalized pulp tissue, such as in traumatized teeth, not cause periradicular inflammation and does not become organized by granulation tissue? Four beagle dogs were used in this experiment. A total of 48 teeth, which included 24 maxillary and 24 mandibular incisors, were aseptically devitalized, leaving residual pulp tissues in the root canals, and equally divided into two groups. Group A (24 uninfected): A sterile cotton pellet was placed deep into the canal orifice before the pulp chamber and access opening were closed with a layer of zinc-oxide eugenol cement followed by glass ionomer cement. Group B (24 infected): The teeth were left open to the oral cavity for 7 days and then closed with a cotton pellet and zinc-oxide eugenol and glass ionomer cement. The animals were sacrificed one year after the experiment and prepared for histological examination of periradicular tissue responses to uninfected and infected devitalized pulp tissues. The results indicate that uninfected devitalized pulp tissues did not continuously release inflammatory mediators and cause persistent periradicular inflammation over a period of one year. However, infected devitalized pulp tissues induced various degrees of periradicular inflammation. Only the apical few millimeters of uninfected devitalized pulp tissue in the root canals were organized by granulation tissue from vital periodontal ligament tissue. (J Endod 2006;32: 34–38)
Key Words Devitalization, infected, inflammation, organization, uninfected
*Department of Endodontics, Advanced Education Program in Endodontics, New York University College of Dentistry, New York, New York; and †Department of Restorative and Biomaterials Sciences, Predoctoral Endodontic Program, Harvard School of Dental Medicine, Harvard University, Boston, Massachusetts. Address requests for reprints to Dr. Louis M. Lin, Department of Endodontics, Advanced Education Program in Endodontics, New York University College of Dentistry, New York, NY 10010. 0099-2399/$0 - see front matter Copyright © 2006 by the American Association of Endodontists. doi:10.1016/j.joen.2005.10.010
34
Lin et al.
I
t is well established that colonization of bacteria in the root canal system is the primary cause of periradicular inflammation. The classic study of Kakehashi et al. (1) showed that periradicular inflammation developed in conventional laboratory rats but not in germ-free rats with surgically exposed pulps. In conventional laboratory rats, oral microorganisms entered the pulp cavity and caused pulpal inflammation and necrosis, and subsequent periradicular inflammation 1 to 42 days after surgical pulp exposure. In germ-free rats, even when the root canals were packed with sterile food debris, neither pulpal necrosis nor periradicular inflammation developed. Bergenholtz (2) and Sundqvist (3), in their clinical studies, showed that bacteria could be cultured from the root canals of traumatized necrotic teeth with intact crowns if periradicular lesions were present, but could not be cultured from necrotic teeth if periradicular lesions were absent. Möller et al. (4), in an animal study, demonstrated that uninfected devitalized pulps would not cause periradicular inflammation 6 to 7 months after pulp devitalization, while severe periradicular inflammation developed in devitalized pulps contaminated with indigenous oral microorganisms. Torneck (5) demonstrated that if polyethylene tubes with one end closed were filled with homologous autoclaved muscle and implanted subcutaneously in rats, a mildly inflamed mature granulation tissue was observed at the open ends of the tubes after a period of 60 days. However, if polyethylene tubes were filled with homologous autoclaved muscle contaminated with bacteria and implanted subcutaneously, the reaction ranged from moderate to intense inflammation with abscess formation at the open ends of the tubes. Makkes et al. (6) also reported that perforated polyethylene tubes with both ends closed and filled with homologous, sterile, autolytic muscle tissue were implanted subcutaneously in rabbits, a slight, transient, inflammatory reaction around the tubes was demonstrated after 21 days of the experiment. It is well known that uninfected necrotic tissue, such as myocardium or cerebellum following an infarct, is capable of inducing inflammatory reaction, which is a defense mechanism of living organisms to eliminate necrotic tissue (7). Eventually, the necrotic myocardium is organized by granulation tissue and removed by macrophages before the repair process takes place. The resulting question is why uninfected devitalized pulp tissue, such as in traumatized teeth, does not cause persistent periradicular inflammation and does not become organized by granulation tissue from vital periodontal ligament tissue (2– 4). The purpose of this study was to histologically examine if uninfected devitalized pulp tissue caused persistent periradicular inflammation and could be organized by granulation tissue from vital periodontal ligament tissue in dogs over a period of one year.
Materials and Methods Animal Preparation Four beagle dogs approximately 3 years of age were used in accordance with a research protocol that was approved by the Institutional Animal Care and Use Committee before the experiment. The animals were examined clinically and radiographically to assure their dental health. Anterior teeth were used for the experiment. They were free of caries and periradicular lesions. In addition, they had healthy periodontium and complete root formation. Tooth vitality was confirmed when the pulp chamber was accessed and fresh bleeding was observed. A total of 48 maxillary and mandibular incisors were employed. One canine from each animal served as a control. The animals were sedated with ketamine (10 mg/kg body wt) (Parke-Davis, Morris Plan, NJ) and
JOE — Volume 32, Number 1, January 2006
Basic Research—Biology then anesthetized with intravenous administration of pentobarbital (30 mg/kg body wt) (Abbott Laboratories, North Chicago, IL).
Experimental Procedures The teeth and surrounding gingival tissues were first disinfected with 5% tincture of iodine. The teeth were then isolated with rubber dam. The operative field including experimental teeth and surrounding rubber dam were cleansed with 35% hydrogen peroxide and disinfected with 5% tincture of iodine. The teeth were accessed through the lingual surface into the pulp chamber, using sterile carbide round burs. Canal length was determined with a Root ZX electronic apex locator (J Morita, Irvine, CA). The pulp tissues were amputated 3 to 5 mm beyond the electronic apex locator reading at the apex by using Hedström files nos. 15 to 25 ensuring complete devitalization of the pulp tissues. The files were then carefully rotated out of the root canals in a counter-clockwise direction, leaving residual pulp tissue in the root canals according to Möller et al. (4). If the pulp tissue appeared to be completely removed with the files, the tissue was carefully placed back into the root canals. Hemorrhage was controlled with sterile cotton pellets. The coronal 3 mm of the root canals were enlarged with sterile no. 3 Gates Glidden burs after the pulp tissues were compressed deep into the canals, using a small sterile CK condenser (CK Dental specialties, Inc., Orange, CA). All experimental procedures were carefully performed under aseptic condition to avoid contamination. The 48 experimental teeth were equally divided into two groups. Group A (uninfected): 24 teeth, which included three maxillary and three mandibular right incisors from each of four animals. A small sterile cotton pellet was placed deep into the coronal 3 mm of the root canal, and the pulp chamber and access opening were filled with a thick layer of zinc-oxide eugenol cement, followed by a layer of glass ionomer cement. Group B (infected): 24 teeth, which included three maxillary and three mandibular left incisors from each of four animals. The teeth were left open to the oral cavity for 7 days, and then closed with a cotton pellet and zinc-oxide eugenol and glass ionomer cements. The coronal seals of the uninfected devitalized teeth were examined periodically. If the glass ionomer cement in the uninfected teeth was worn away, the teeth were isolated with rubber dam and disinfected with 35% hydrogen peroxide and 5% tincture of iodine. The superficial layer of the glass ionomer cement was removed and newly prepared glass ionomer cement was added. This occurred in three teeth with uninfected devitalized pulp tissues. The animals were sacrificed with an overdose of pentobarbital and perfusion of formaldehyde-glutaraldehyde fixative introduced bilaterally through the external carotid arteries 1 year after the experiment was initiated. Radiographs of all maxillary and mandibular anterior teeth were taken.
Histological Preparation The maxillae and mandibles were removed and carefully block sectioned to include individual teeth and supporting structures. The block specimens were fixed in 10% buffered neutral formalin for 3 days, demineralized in formic acid-sodium citrate solution for 5 wk. and embedded in paraffin. Serial sections of 5 to 6 m were cut from the paraffin blocks and stained with hematoxylin and eosin (H&E), and modified Brown and Brenn stain. The stained sections were examined under a Zeiss light microscope. Sections stained with H&E were used for examination of inflammatory cell infiltration of the pulp and periradicular tissues, as well as root and apical bone resorption. Brown and Brenn stained sections were used for detection of stainable bacteria in the tissue sections. Periradicular tissue responses were histologically classified into four categories, depending on the extent of inflammatory cell infiltrate and apical bone and root resorption: category 1, absence of inflammatory cell infiltrate; category 2, inflammatory cell infiltrate loJOE — Volume 32, Number 1, January 2006
TABLE 1. Periradicular tissue responses to uninfected and infected devitalized pulp tissues at 1-year observation Category of Inflammation
No. of Teeth
Group A (noninfected) B (infected)
24 24
1
2
3
4
21 0
0 0
3* 16
0 8
*These teeth had crown fractures.
calized at the apical foramen with minimal apical bone resorption; category 3, inflammatory cell infiltrate beyond the apical foramen with moderate apical bone and/or root resorption; and category 4, diffuse inflammatory cell infiltrate in the periapical area with a local accumulation of neutrophils (pus), as well as moderate to severe apical bone and root resorption.
Results Three teeth in group A (uninfected) in two animals had crown fractures and developed periradicular lesions (Table 1). Seven teeth in group A still contained vital pulp tissues with intact odontoblast layer along the root canal walls and minimal chronic inflammatory cells in the apical portion of the pulps, which indicated that the pulp tissues were not completely devitalized by the experimental procedure. No periradicular inflammation was present in these seven teeth. The remaining 14 teeth in group A showed complete necrosis of the pulp tissues but no inflammatory cell infiltrate in the periradicular tissues (Fig. 1) (Table 1). The necrotic pulp tissues demonstrated complete loss of cellular structures and became an amorphous denatured mass. Stainable bacteria were not observed along the access cavity walls in 21 teeth without crown fractures in group A. In-growth of connective tissue resembling periodontal ligament tissue into the apical few millimeters of the root canals was present in all completely uninfected devitalized teeth (Fig. 2). Cementoid tissue was observed at the apical foramen in six of 14 completely uninfected devitalized teeth. All 24 teeth in group B (infected) showed moderate to severe periradicular inflammation accompanied by various degrees of apical bone and root resorption (Fig. 3). In some cases, the inflammatory cells were dominated by neutrophilic leukocytes and scattered macrophages. Colonies of stainable bacteria were present in the root canals of devitalized infected teeth. Histologically, control teeth showed normal pulp and periradicular tissues.
Discussion The present study confirms the findings of Möller et al. (4) who demonstrated that bacterial colonization in the root canal is the prerequisite for the development of periradicular inflammation (1–3), and uninfected devitalized pulp would not cause periradicular inflammation. This is in contrast to the finding of Sinai et al. (8) who showed that periradicular tissues of teeth with necrotic pulp stumps, resulting from partial pulp extirpation, invariably broke down in animal and human studies. However, their study did not include a bacteriological examination of the pulp tissues in the root canals to elucidate the possible bacterial cause of periradicular inflammation (9). Clinically, it has also been demonstrated that necrotic pulp tissues in teeth caused by traumatic injuries (2, 3, 10), if uninfected, may remain in the root canals indefinitely without causing periradicular inflammation or clinical symptoms except for occasional crown discoloration. Nevertheless, it is well known that sterile necrotic tissue, such as a myocardium or cerebellum, which has experienced an infarct, is capable of inducing inflammatory reaction by release of inflammatory mediators and cytokines (7). Why then, would uninfected devitalized pulp tissues in the
Periradicular Tissue Responses in Devitalized Pulps
35
Basic Research—Biology
Figure 2. High magnification of the root apex and the periradicular tissues in Fig. 1. Vital connective tissue similar to the periodontal ligament tissue is present in the apical few millimeters of the root canal (arrow). Cementoid tissue is formed at the apical foramen (H&E stain; magnification ⫻200).
Figure 1. Uninfected devitalized tooth. The pulp tissue shows complete necrosis, but the periradicular tissues are free of inflammation (H&E stain; magnification ⫻100).
root canals not cause periradicular inflammation (2– 4)? Tissue that is long dead and coagulated will not induce inflammation. Only dying tissue can cause an intense, transient, acute inflammatory reaction (11). Dying tissue can cause inflammation by a number of mechanisms: (a) release of inflammatory mediators, such as histamine, platelet activating factor, arachidonic acid metabolites, and cytokines by mast cells (12, 13), as well as serotonin, platelet activating factor, clot-promoting factors, thromboxane A2, and complement-cleaving proteases by platelets (14, 15); (b) release of arachidonic acid metabolites and lysosomal enzymes, especially proteases to cleave C3, C4, and C5 by dying cells; (c) activation of the complement system to release C3a, C4a, and C5a, and membrane attack complex C5b6789 (16, 17); (d) activation of kinin system; (e) activation of fibrinolytic system; and (f) activation of clotting cascade. The last four mechanisms are activated by plasma Hageman factor in the blood vessels of the dead tissue, which becomes activated by contact with collagen (11). The necrotic tissue initially releases chemical inflammatory messages to attract inflammatory cells, notably leukocytes and elicit a transient, acute inflammation. However, when the terminal burst of chemical mediators from the necrotic tissue is over, no further inflammatory messages are released. The necrotic tissue will then turn into a mass of coagulated or denatured protein, which no longer acts as an irritant to living tissue and will be removed by macrophages (11). This may in part explain why uninfected devitalized pulps did not cause persistent periradicular inflammation in the present 36
Lin et al.
study. Although over-instrumentation in the present study could cause mechanical injury to the periradicular tissues (18), over-instrumentation alone probably would not perpetuate periradicular inflammation over several weeks or months unless extensive periradicular tissue damage had occurred. This is because of the fact that periradicular tissues are equipped with adequate cellular and humoral defense mechanisms for wound healing. Another possible scenario is that the chemical inflammatory mediators and cytokines, or auto-antigens released by uninfected devitalized pulps through the constricted apical foramen
Figure 3. Infected devitalized tooth. There is local accumulation of inflammatory cells at the apical foramen and a moderate apical bone resorption (H&E, magnification ⫻10).
JOE — Volume 32, Number 1, January 2006
Basic Research—Biology may be limited and quickly neutralized by periradicular tissue defenses. Nevertheless, uninfected devitalized pulps in traumatized immature teeth even with open apexes also would not cause periradicular pathosis (10). Seven teeth in group A (uninfected) were not completely devitalized even though the pulp tissues were amputated 3 to 5 mm beyond the electronic apex locator reading at the apex. This observation is similar to that of Möller et al. (4). The pulp tissues were necrotic in the coronal portion, but remained vital in the apical portion of the root canals, which contained an intact odontoblast layer along the root canal walls and few lymphocytes and plasma cells adjacent to the coronal necrotic pulp tissues. The root canal system appears to be a unique environment in which the inflammatory defense mechanisms of the apical vital pulp tissue is not able to operate effectively to organize the coronal necrotic area because of enclosed dentin walls, constricted apical foramen and a lack of collateral blood circulation (19), which seriously restricts the route of delivery of cellular and humoral defense components. None of the completely devitalized uninfected teeth showed total necrosis of the pulp tissue extending to the apical foramen at 1-yr histological examination. The apical few millimeters of the root canals always contained vital connective tissue, which is similar to periodontal ligament tissue. This finding is in agreement with that of Möller et al. (4). Necrotic tissue in the living body is organized by in-growth of granulation tissue from adjacent vital tissue and slowly removed by macrophages before the repair process takes place (11). Makkes et al. (6) showed that subcutaneous implantation of fragments of sterile, autologous, or heterologous, autolytic connective tissue or muscle tissue in rabbits caused a transient, mild inflammatory reaction, and the implanted tissue fragments were broken down and replaced by vital connective tissue. However, if isologous and homologous, sterile, autolytic muscle tissues were placed in the perforated polyethylene tubes, which were then implanted subcutaneously in rabbits, a slight, transient inflammatory reaction around the tubes was observed. The sterile, autolytic tissues were not broken down or replaced because in-growth of host connective tissue through the holes of the tubes only took place over a very short distance at 21 days of the experiment. Torneck (5) also demonstrated that if polyethylene tubes with one end closed were filled with sterile, autoclaved muscle tissue and implanted subcutaneously in rats, there was some attempt by the granulation tissue to proliferate to a depth of 1 to 2 mm into the tubes at 6 months of the experiment. Presence of vital connective tissue in the apical few millimeters of the root canals in completely devitalized uninfected teeth in the present study indicates organization of that part of the necrotic pulp tissue by in-growth of granulation tissue from vital periodontal ligament tissue through the apical foramen. The vital connective issue in the apical few millimeters of the root canals resembles periodontal ligament tissue, which is not capable of differentiating into odontoblasts (20, 21). This may be resulting the fact that, similar to the polyethylene tubes, the pulp tissue is enclosed within the dentin walls, and the root canal communicates with periodontal ligament tissue only through a constricted apical foramen. Thus, in-growth of granulation tissue from vital periodontal ligament tissue into the root canals is restricted. Therefore, necrotic pulp tissues, if uninfected, can remain in the root canals indefinitely as a mass of nonirritating, denatured protein without being organized by granulation tissue. Nonetheless, in traumatized immature teeth with large open apexes and short roots, organization of completely devitalized, uninfected pulp tissues by vital periodontal ligament tissue has been reported (10). In such cases, in-growth of periodontal ligament tissue into the root canals may cause narrowing of the root canal space, for progenitor cells of the periodontal ligament tissue have the potential to
JOE — Volume 32, Number 1, January 2006
differentiate into cementoblasts (20) and form cementum along the root canal walls (10). Because devitalized pulps were able to remain in the root canals without being contaminated through coronal leakage or exposed root dentinal tubules in the cervical area for 6 months (4), and 1 year in the present study, devitalized pulps could be a suitable model for studying bacteremia and focal infection. Teeth with periradicular lesions did not seem to serve as foci of infection and cause infection of teeth with devitalized pulps on the other side of the jaws in the present study, for none of the teeth in group A (uninfected), other than three teeth with crown fractures, developed periradicular inflammation. The present observation is in agreement with the finding of Möller et al. (4). However, Allard et al. (22) in animal experiment showed that bacteria inoculated in pulpectomized root canals could be transported by the bloodstream into uninfected, pulpectomized root canals in the same animal after 28 to 120 days. They also suggested that establishment of infection in the periradicular area might be necessary for the spread of infection to occur by anachoresis. Nevertheless, Delivanis et al. (23) and Delivanis and Fan (24) were not able to experimentally reproduce similar results in animals if root canals were not over-instrumented. It is possible in the present study that the inflamed periradicular tissues caused by over-instrumentation of the group of teeth containing uninfected devitalized pulps were effectively organized before the group of teeth containing infected devitalized pulps developed periradicular lesions. Accordingly, bacteria from the infected devitalized teeth could not disseminate into the root canals of uninfected devitalized teeth by the bloodstream. Bacteremia occurs only in vascular tissues. In devitalized pulps, the blood circulation is completely cut off. Therefore, it is unlikely that bacteria are able to enter the root canal by way of the bloodstream. Similarly, bacterial myocarditis or cerebral infection usually does not occur in patients suffering from myocardial or cerebral infarct even though transient bacteremia is a common phenomenon (25). However, in diabetic patients, anaerobic cellulitis does occur in ischemic lower extremities (25). Therefore, the predisposing factors to bacteremia, such as bacterial attachment to specific receptors of vascular endothelium and colonization or biofilm formation of bacteria in specific organs and tissues require further investigation. It may be asked that if uninfected devitalized pulp tissues do not cause persistent periradicular inflammation, why should uninfected devitalized pulp tissues be removed and root canal therapy be initiated? The reasons to provide treatment in such cases are: (a) when compared to vital pulp tissues, devitalized pulp tissue is completely devoid of innate and adaptive immune defense mechanisms; (b) the devitalized pulp tissue can be easily colonized and become infected even if a small number of bacteria gain access to the root canal system; and (c) the prognosis of root canal therapy in teeth with periradicular lesions is generally poorer than that without periradicular lesions (26 –28). Therefore, there is an advantage for the patient to have treatment performed in teeth with necrotic pulps before a periradicular lesion develops. Prevention of disease progression or infection is an important treatment strategy.
Conclusion The following conclusions are drawn from this animal experiment: 1. Uninfected devitalized pulp tissue does not continuously release inflammatory mediators and cause persistent periradicular inflammation over a period of 1 year. 2. Uninfected devitalized pulp tissue cannot be effectively organized by granulation tissue from vital periodontal ligament tissue.
Periradicular Tissue Responses in Devitalized Pulps
37
Basic Research—Biology 3. Teeth with periradicular lesions do not seem to serve as foci of infection and cause infection of teeth with devitalized pulps on the other side of the jaw in the present study.
References 1. Kakehashi S, Stanley HR, Fitzgerald RJ. The effects of surgical exposures in the dental pulps in germ-free and conventional laboratory rats. Oral Surg Oral Med Oral Pathol Oral Radiol Endod 1965;20:340 –9. 2. Bergenholtz G. Microorganisms from necrotic pulp of traumatized teeth. Odont Revy 1974;25:347–58. 3. Sundqvist G. Bacteriologic studies of necrotic dental pulp. University of Umea Odontology Dissertation no. 7. Umea, Sweden. 1976:1–94. 4. Möller ATR, Fabricius L, Dahlen G, Heyden G. Influence on periapical tissues of indigenous oral bacteria and necrotic pulp tissue in monkeys. Scand J Dent Res 1981;89:475– 84. 5. Torneck CD. Reaction of rat connective tissue to polyethylene tube implants. Part II. Oral Surg Oral Med Oral Pathol Oral Radiol Endod 1967;674 – 83. 6. Makkes PC, Thoden Van Velzen SK, Wesselink PR. Reactions of the living organism to dead and fixed dead tissue. J Endod 1978;4:7–21. 7. Rubin E, Gorstein F, Rubin R, Achwarting R, Strayer D. Rubin’s pathology, 4th ed. Philadelphia: Lippincott Williams & Wilkins, 2004:295-6. 8. Siani I, Seltzer S, Soltanoff W, Goldenberg A, Bender IB. Biologic aspects of endodontics. Part II. Periapical tissue reactions to pulp extirpation. Oral Surg Oral Med Oral Pathol Oral Radiol Endod 1967;23:664 –79. 9. Pitt Ford TR. Vital pulpectomy—an unpredictable procedure. Int Endod J 1982;15:121– 6. 10. Andreasen JO, Andreasen FM. Textbook and color atlas of traumatic injuries to the teeth, 3rd ed. Copenhagen: Munksgaard, 1994 11. Majno G, Joris I. Cells, tissues, and disease, 2nd ed. Oxford: Oxford University Press, 2004:205-8;446-54. 12. Galli SJ. New concepts about the mast cells. N Engl J Med 1993;328:257– 65. 13. Gordon JR, Burd PR, Galli SP. Mast cells as a source of multifunctional cytokines. Immunol Today 1990;11:458 – 64. 14. White JG. Platelet secretory granules and associated proteins. Lab Invest 1993;68:497– 8.
38
Lin et al.
15. Nachman RL, Weksler BB. The platelet as an inflammatory cell. In: WeissmanG, ed. The cell biology of inflammation. Amsterdam: Elsevier/North-Holland Biological Press, 1980:145– 62. 16. Engel AG, Biesecker G. Complement activation in muscle fiber necrosis: demonstration of the membrane attack complex in necrotic fibers. Ann Neurol 1982;12:289 –96. 17. Pinckard RN, Olson MS, Giclas PC, et al. Consumption of classical complement components by heart subcellular membranes in vitro and in patients after acute myocardial infarcts. J Clin Invest 1975;56:740 –50. 18. Seltzer S, Soltanoff W, Sinai I, Goldenberg A, Bender IB. Biologic aspects of endodontics. Part III. Periapical tissue reactions to root canal instrumentation. Oral Surg Oral Med Oral Pathol Oral Radiol Endod1968;26:534 – 46; 694-705. 19. Suda H, Ikeda H. The circulation of the pulp. In: HargreavesKM, GoodisHE, eds. Dental pulp. Chicago: Quintessence Publishing Co., Inc., 2002: 123–50. 20. Melcher AH. On the repair potential of periodontal tissues. J Periodontol 1976;47:256 – 60. 21. D’souza R. Development of the pulpodentin complex. In: Hargreaves KM, Goodis HE, eds. Dental pulp. Chicago: Quintessence Publishing Co., Inc., 2002: 23– 40. 22. Allard U, Nord C-E, Sjöberg L, Strömberg T. Experimental infections with Staphylococcus aureus, Streptococcus sanguis, Pseudomonas aeruginosa, and Bacteroides fragilis in the jaws of dogs. Oral Surg Oral Med Oral Pathol Oral Radiol Endod 1979;48:454 – 62. 23. Delivanis PD, Snowden RB, Doyle RJ. Localization of blood-borne bacteria in instrumented unfilled root canals. Oral Surg Oral Med Oral Pathol Oral Radiol Endod 1981;52:430 –2. 24. Delivanis PD, Fan VSC. The localization of blood-borne bacteria in instrumented unfilled and overinstrumented canals. J Endod 1984;10:521– 4. 25. Mims C, Playfair J, Roitt I, Wakelin D, Williams R. Medical microbiology, 2nd ed. Philadelphia: Mosby 1999:119-26. 26. Strindberg LZ. The dependence of the results of pulp therapy on certain factors. An analytic study based on radiographic and clinical follow-up examination. Acta Odontologia Scand 1956;14(Suppl. 21):1–175. 27. Sjogren U, Hagglund B, Sundqvist G, Wing K. Factors affecting the long-term results of endodontic treatment. J Endod 1990;16:498 –504. 28. Chugal NM, Clive JM, Spangberg LS. A prognostic model for assessment of the outcome of endodontic treatment: effect of biologic and treatment variables. Oral Surg Oral Med Oral Pathol Oral Radiol Endod 2001;91:342–52.
JOE — Volume 32, Number 1, January 2006