- Email: [email protected]
Microbiological Aspects of Traumatic Injuries
2018 MEETING OF THE WORLD CONGRESS ON DENTAL TRAUMATOLOGY
Ashraf F. Fouad, DDS, MS
Microbiological Aspects of Traumatic Injuries ABSTRACT After traumatic injuries to teeth, microorganisms may invade the compromised pulp tissue and initiate pulp infection and periapical inflammation. In addition to bone resorption that typically accompanies pulp necrosis, root resorption frequently occurs. Root resorption has several variants that may occur shortly after the trauma or at a later stage. The pathological changes seen after traumatic injuries to teeth are invariably linked to the presence of microbial irritants. The presence of bacterial biofilms in the dental pulp space can be treated with regenerative or therapeutic endodontic procedures. However, necrosis of periodontal ligament is usually terminal for the tooth involved. In this review, the sources of bacteria after traumatic injuries are discussed. The types and role of microorganisms involved in the pathogenesis of endodontic pathosis after traumatic injuries are presented, and contemporary approaches for the management of these conditions are reviewed. Contemporary antimicrobial strategies are discussed. The rationale for the use of systemic and topical antimicrobials is presented. Finally, novel approaches to the use of antimicrobial therapies, particularly in regenerative procedures, are reviewed. (J Endod 2019;-:1–10.)
SIGNIFICANCE This articles addresses the likely sources of bacteria in traumatic dental injuries, the microbial ecology within the resultant endodontic infections, and the antimicrobial management of these patients at various presentations after the injuries.
KEY WORDS Antibiotics; endodontics; resorption; traumatic dental injuries
Traumatic injuries to teeth represent a unique form of injury to the dental pulp and periodontal tissues. Instead of following the traditional progression from coronal to radicular and periradicular sites seen with caries and congenital anomalies, the injury with trauma can occur directly at any level of the dental or periradicular tissues. In this regard, the injury can occur directly to the pulp, root apex, cementum, periodontal ligament, or surrounding bone individually or in combination. In most cases, the trauma itself disrupts the pulp and periradicular neurovascular supply and causes injury to the cemental surface. This renders the pulp and periodontal tissues less resistant to bacterial invasion that accompanies the trauma and the root surface less resistant to resorption at the site of cemental injury. In addition, fractures of the crown or root, or avulsion of the tooth, may provide direct pathways of invasion by oral or environmental microorganisms. The capacity of the pulp to resist bacterial invasion after traumatic injuries has been shown to depend on the degree of maturation of the tooth root. Teeth with an immature apex have a much lower probability of developing pulp necrosis after luxation or fracture injuries than teeth with a mature apex1–3. This is thought to be related to the presence of collateral and/or more robust circulation in immature teeth to resist the bacterial invasion that ensues after trauma. Because of the susceptibility of the pulp to degenerate after trauma and the risk of resorption, the urgency of diagnosing the pulp condition of the affected teeth is especially critical. Pulp diagnosis is complicated by the loss of sensibility caused by the traumatic injury and the difficulty of pulp sensibility tests in children3,4. Pulp vitality tests, such as using laser Doppler flowmetry (LDF), were shown to be more accurate than pulp sensibility tests5–7, especially after traumatic injuries. In case reports or case series8–10, LDF showed the onset of vitality signals in luxated teeth much sooner than sensibility testing. However, LDF is not currently widely available or practical. Therefore, a period of uncertainty usually elapses before a definitive diagnosis can be obtained. With the exception of avulsion or intrusion of mature teeth, in which endodontic treatment should be initiated within 1–2 weeks, endodontic interventions after other traumatic injuries are contemplated after a period of observation when the tooth shows signs and symptoms of pulp necrosis, apical periodontitis, and/or infection-related (inflammatory) root resorption. During this period of delay, more bacterial invasion and the establishment of bacterial biofilms inside the pulp space may ensue, and irreparable root resorption may be initiated.
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From the Department of Endodontics, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina Address requests for reprints to Dr Ashraf F. Fouad, Adams School of Dentistry, Department of Endodontics, University of North Carolina at Chapel Hill, Room K5417-J, 385 S. Columbia Street, Chapel Hill, NC 27599-7450. E-mail address: [email protected] 0099-2399/$ - see front matter Copyright © 2019 John Wiley & Sons A/S and American Association of Endodontists. This article is being published concurrently in Dental Traumatology. The articles are identical. Either citation can be used when citing this article. https://doi.org/10.1016/ j.joen.2019.05.011
Microbiological Aspects of Traumatic Injuries
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ORIGIN OF MICROORGANISMS IN TRAUMATIC INJURIES The sources of microorganisms in cases of traumatic injuries, especially if the teeth involved remain intact, have been subject to multiple studies and much debate. The older concept introduced in the 1940s was that circulating bacteria from any cause or source would preferentially diffuse into and grow on degenerated or degenerating tissues, such as the traumatized and ischemic pulp11. This was known as anachoresis. In the decades that followed, there have been several animal studies that supported12,13 or refuted14,15 the concept of anachoresis. One clinical study attempted to localize the bacteria in extracted teeth with pulp necrosis and apical periodontitis and showed that bacterial biofilm in primary infections are present inside the root canal and not at the apical foramen or immediate external surface16. In a classic experiment in the 1960s, Serratia marcescens, a nonoral bacterial species, was inoculated in the oral cavity and gingival crevicular areas of dogs and monkeys17. The teeth were then traumatized by an impact force not strong enough to cause fracture. The results showed that S. marcescens was recovered after various time intervals from the pulp space in about a third of the animals17. Modern studies with sensitive molecular techniques show remarkable
resemblance between the microbiomes of the necrotic pulp and that of the periodontal pocket in general18,19. Moreover, studies on root microflora have failed to identify uniquely nonoral bacteria commonly found in the blood. One example of this concept was a study that failed to identify Streptococcus pneumonia and Chlamydia pneumonia, which are common respiratory pathogens, from the root canals of primary or persistent endodontic pathosis20. Therefore, it is currently thought that after traumatic injuries microbial invasion of the pulp is primarily from the gingival sulcus and/or oral cavity, with rare contributions from hematogenous or other sources. These microorganisms travel locally through the injured gingival sulcus and periodontal attachment and through dentinal tubules, lateral and accessory canals, or injured periodontal tissues to the pulp space, which contains the ischemic and injured pulp. Traumatized teeth may be cracked but not fractured. Cracks are common in anterior traumatized teeth as well as mesiodistally in maxillary first bicuspids because of forced occlusion by mandibular bicuspids in cases of falls on the chin. Bacteria can migrate through cracks, both at the time of crack formation or during normal function21. In general, cracked teeth are thought to occur because of chronic traumatic occlusion, such as with bruxism or unusual masticatory habits, and up to 20% of these cases result in irreversible pulp pathosis
requiring root canal treatment within 6 months from the time of diagnosis according to 1 study22. Traumatic injuries to the dentition are frequently accompanied by soft tissue lacerations, abrasions, and bruises. These soft tissue injuries may also be subject to infections and protracted healing depending on the extent of injury, contamination from oral or nonoral sources, and the presence of embedded foreign material. In cases in which coronal fragments of teeth are fractured, the provider should spend some time documenting the extent of the missing portions of teeth and attempt to locate them. If concomitant deep soft tissue lacerations are present, such as in the labial mucosa, these can be imaged radiographically to ensure that they have not been embedded in these locations. Likewise, the possibility of ingesting or inhaling these tooth fragments should be considered and investigated.
DEVELOPMENT OF ENDODONTIC BIOFILM AFTER TRAUMATIC INJURIES Bacterial infiltration into the root canal space and dentinal tubules is initially in the form of disparate planktonic cells that can easily be phagocytosed or killed by host defense mechanisms wherever these mechanisms are intact. Wherever the pulp lacks vascularity, the
FIGURE 1 – A diagrammatic representation of the sequence of adherence and colonization of tooth surfaces by bacteria. (A ) Primary colonizing bacteria existing as planktonic cells interact with the conditioning film (eg, acquired pellicle, dentinal tubule fluid, and serum) on the tooth surface using longer-range interactions (eg, pili or shorter-range molecular interactions). (B ) The early colonizers form strong bonds with the surface molecules in the conditioning film or components of the tooth substrate (eg, collagen by a variety of mechanisms and multiple adhesins). In conjunction with adhesion, the bacteria perform other functions such as adapting to the available nutrition, intermicrobial signaling, and production of an extracellular matrix. (C ) Late colonizing bacteria enter the community by coaggregation reactions contributing to sequential binding and colonization of the developing biofilm. In this regard, Fusobacterium has been shown to be an important bridging organism allowing interactions between nonbinding bacteria. Within the biofilm intricate processes and interactions, such as quorum sensing, metabolic communication, genetic exchange, and competitive interactions further shape the membership of the complex community ensuring efficient utilization of nutrients and reduced susceptibility to host defense or therapeutic methods (eg, antimicrobials). (Reprinted with permission23.)
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FIGURE 2 – Microorganisms isolated by different investigators in initial samples from root canals with necrotic pulps. *Gomes et al, 2005 only covers black-pigmented Porphyromonas and Prevotella species. (Reprinted with permission38.) As previously noted, it is frequently difficult to establish the definitive clinical viability of the pulp promptly after a traumatic injury, except in cases of avulsion or intrusion of mature teeth. If the patient is not followed and monitored closely and no symptoms arise, this infection can last for many years or decades before it is diagnosed. Therefore, by the time the clinical diagnosis of pulp necrosis and infection is established, a formidable bacterial biofilm may have developed in the pulp space. This bacterial biofilm is not static because there are changes in bacterial load, community composition, and virulence (Fig. 1) that influence the development of signs and symptoms of disease29–31. This biofilm may expand into lateral canals or periapical tissues causing persistent symptoms32–34. All these changes and expansions of root canal biofilms necessitate
source of cellular and molecular responses is disrupted or eliminated, and the bacterial infiltrate begins to develop a biofilm that is more difficult for host responses to eliminate (Fig. 1A–C)23. Host proteins, such as albumin and fibrinogen, were shown to aid in bacteria growth on dentin surfaces or invasion of dentinal tubules24,25. Most traumatic injuries occur in children and adolescents. It was shown that bacterial invasion of dentinal tubules is significantly deeper and affects more tubules per unit area in young individuals than in older individuals26. It has also been shown that bacterial biofilm on dentin develops within a period of only 3 weeks to a mature biofilm that is difficult to eliminate using traditional endodontic irrigants27. Even intracanal antibiotics may not eliminate this biofilm, especially if low nontoxic concentrations are used28.
substantial antimicrobial strategies and regimens.
MICROBIOLOGY OF TRAUMARELATED ENDODONTIC INFECTIONS
Data from Sundqvist37.
In the late 1960s and early 1970s, the technology of anaerobic microbiology was mastered in endodontic research. Several studies took advantage of this to study the microbiology of intact, traumatically devitalized teeth with pulp necrosis and apical periodontitis35–37. The intent was to determine if the microflora in these cases was not only unique compared with caries but also if it harbored a high content of facultative and strictly anaerobic bacteria. The results of these studies showed a high proportion of strictly anaerobic bacteria (Fig. 2)38. It has always been assumed that the microbiology of endodontic infection after caries, traumatic injuries, congenital anomalies of crowns, or any other etiology shows a similar degree of composition and diversity. Studies have shown that despite uniform distribution of bacterial taxa used to inoculate root canals of monkeys, with time, the composition shifted to favor a prevalence of gram-positive facultative and gramnegative anaerobic bacteria39. Because it is impossible to compare microflora from cases with equivalent durations of the biofilm, it is not clear if this distinction for cases from traumatized teeth is universally consistent. In 1 important human study, patients with
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Microbiological Aspects of Traumatic Injuries
TABLE 1 - Bacteria from Acute Dental Infections of Teeth with Necrotic Pulps after Trauma to the Teeth Bacteria
Patient 1
Patient 2
Patient 3
Fusobacterium spp. F. nucleatum Peptosteptococcus anaerobius Eubacterium spp. E. alactolyticum E. lentum Peptostreptococcus micros Peptococcus spp. Campylobacter sputorum Lactobacillus spp. Bacteroides melaninogenicus ss. intermedius (Prevotella intermedia)
X
X X
X
X
X
X X X X X X
X X X
X X X X X X X X
3
TABLE 2 - Microbiology of Endodontic Infections in Immature Teeth Microorganism
%
Actinomyces naeslundii Porphyromonas endodontalis Parvimonas micra Fusobacterium nucleatum Porphyromonas gingivalis Prevotella intermedia Tannerella forsythia Filifactor alocis Treponema denticola
67 33 33 33 26 26 20 13 13
Data from Nagata et al40.
intact, traumatized teeth that developed acute apical abscesses were shown to have several notable facultative and strict anaerobic bacteria (Table 1). In a later analysis of cultivable bacteria from 20 intact traumatically devitalized teeth41, the authors confirmed that the following strict anaerobes had the highest prevalence: Bacteroides gracilis (50%), Propionibacterium
acnes (45%), Fusobacterium nucleatum (30%), Prevotella buccae (30%), and Eubacterium lentum (25%). From the 84 strains that were isolated overall, 81% were strict, and 19% were facultative anaerobes. Furthermore, definitive antibiotic sensitivity was obtained for 66 of these strains. The susceptibility of these strains to amoxicillin and amoxicillin 1 clavulanic acid was much lower than to tetracycline, a finding that was later confirmed in other endodontic antibiotic sensitivity studies42–44. Contemporary molecular techniques, based on 16S ribosomal RNA gene sequencing, are capable of identifying a wide spectrum of endodontic microflora, including many uncultivable taxa45–48. These studies reveal that the abundance of individual species (also known as operational taxonomic units) is low because the depth of coverage shows a high diversity of the bacteria present. Studies examining the microflora in traumatic injuries using these methodologies are underway,49 as this area has not been sufficiently explored. One study
that used a combination of culture and molecular approaches showed that Actinomyces naeslundii was the most prevalent organism in children with immature traumatized teeth with necrotic and infected pulps (Table 2)40. This was an important finding because this microorganism commonly forms periapical microbial colonies that persist despite nonsurgical root canal treatment50–52. In addition, a comprehensive analysis of bacteria in the apical third of infected root canals and their correlation with bone-resorbing inflammatory mediators and matrix metalloproteinases was recently reported53. In this study, it was found that increased matrix metalloproteinase-2 and -9, receptor activator of nuclear factor kappa B, receptor activator of nuclear factor kappa B ligand, and decreased osteoprotegerin were correlated with the phylum Actinobacteria, which contains the genus Actinomyces (53%) and the genus Streptococcus (35%). Fortunately, Actinomyces spp. are very sensitive to commonly used antibiotics54 and
FIGURE 3 – The maxillary left lateral incisor sustained a lateral luxation injury. (A ) Two months later, the tooth continued to have no response to pulp testing and developed external infection-related (inflammatory) root resorption. (B ) Endodontic treatment was initiated, the root canal was debrided, and calcium hydroxide was placed for 4 weeks. (C and D ) The radiographs show stabilization of the resorptive lesion, and the root canal was completed. (E ) Three-month follow-up showing bone deposition and mineralization at the root apex.
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FIGURE 4 – The maxillary right central incisor sustained an extrusive luxation and was repositioned and splinted for several months without sensibility testing or radiographic evaluation. Advanced infection-related (inflammatory) root resorption occurred, rendering the prognosis unfavorable.
other antimicrobials, such as sodium hypochlorite and calcium hydroxide55,56.
ANTIMICROBIAL MANAGEMENT OF PATIENTS WITH TRAUMATIZED TEETH Antibiotics, both systemically and topically, as well as several other topical antimicrobial medicaments have been proposed for use in the treatment of patients who sustain dental traumatic injuries. However, because of the urgent nature of traumatic injuries and the variability of the injuries, randomized clinical trials, which represent the highest level of objective clinical research, are lacking. In many cases, randomized trials would be unethical to conduct, except possibly to compare the
efficacy of 2 therapeutic agents or medicaments. Systemic administration of antibiotics after avulsion or luxation injuries is currently recommended57–59. The evidence for this recommendation is mainly from an older animal study60. In this animal study, teeth were extracted and left to dry for an hour before replantation. The animals were given daily intramuscular injections of 2 mL Streptocillin vet. (Novo, Copenhagen, Denmark), containing dihydrostreptomycin 0.25 g/mL and benzylpenicillin 200,000 IE/mL, on the day of replantation and for 6 days afterward. It was found that this regimen prevented or significantly reduced the development of infection-related (inflammatory) root resorption, similar to early root canal treatment of these teeth60. Another study with a similar
TABLE 3 - Selected Revascularization Cohort or Randomized Studies and the Antimicrobials Used
Study
N
Concentrated NaOCl (%)
Antibiotic
Success/healing (%)
Jeeruphan et al76 Kahler et al77 Nagy et al78 Alobaid et al79 Chan et al80 Lin et al81
20 16 10 19 28 69
2.5 1 2.6 ? 5.25 1.25
TAP TAP DAP 1 doxycycline TAP/DAP/Ca(OH)2 DAP 1 cefaclor DAP 1 clindamycin
80 90 90 78 93 90
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design was reported to evaluate pulp revascularization in immature monkey teeth61. The animals were given 4 mg/kg doxycycline before extraction and 2 mg/kg for 5 days after replantation. This regimen was not found to have any benefit on revascularization. In a more recent human prospective observational study, 140 permanent teeth with intrusion injuries were included62. Many treatment variables were collected, including the administration of antibiotics. Antibiotic treatment had no effect on healing in this study. Older retrospective studies on fractures and replantation also did not show a benefit to using antibiotics63–65. Therefore, the use of systemic antibiotics in patients with traumatic injuries appears to be mainly related to protecting the patient against a possible systemic infection from the site of trauma, and they may reduce infection-related (inflammatory) resorption. However, more studies are needed to confirm that the value of this recommendation outweighs the risks involved with antibiotic prescriptions66. Topical application of antibiotics may be applied on the external root surface before replantation of avulsed teeth or inside the root canal of any traumatized tooth that requires endodontic treatment. The topical application of antibiotics and other antimicrobial medicaments such as calcium hydroxide or Ledermix (Riemser Pharma GmbH, Greifswald, Insel Riems, Germany) paste has been recommended in some traumatic injury cases for decades. As previously noted, the presence of a formidable biofilm in these cases necessitates effective antimicrobial regimens. In addition, it has long been noted that the development of infection-related (inflammatory) root resorption is best treated with calcium hydroxide67 (Fig. 3A–D) or a combination of an antibiotic and a steroid such as Ledermix paste68–71. It is thought that the main action that stops the resorptive lesion (Fig. 4) in these cases is the effective antimicrobial effects of the medicaments72,73, although some have proposed that the alkaline pH (at least in the case of calcium hydroxide) may have a direct effect on the odontoclasts/osteoclasts whose brush border acts in an acidic environment74. Corticosteroids may also stabilize the cell membrane of clastic cells, thus reducing their action. Contemporary regenerative protocols for immature teeth with pulp necrosis involve the use of intracanal antibiotics or calcium hydroxide75. With respect to antibiotics, triple antibiotic paste (comprised of minocycline, ciprofloxacin, and metronidazole) and double antibiotic paste
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5
FIGURE 5 – The size of inhibition zones in millimeters around unit volumes of antibiotic/hydrogel mixture against several endodontic pathogens. Wider zones indicate more effectiveness. Note the enhanced effectiveness of all antibiotics against F. nucleatum and the reduced effectiveness against Enterococcus faecalis. (Data from AlSaeed et al28.)
(excluding minocycline because of staining) have gained the most attention. Other antibiotics, such as Augmentin (GlaxoSmithKline, Research Triangle Park, NC), cefaclor, clindamycin, and doxycycline, have been proposed and used in some clinical studies with good success (Table 3)82. The susceptibility of endodontic pathogens to various antibiotics in hydrogel vehicles or other scaffold materials is currently being explored28 (Fig. 5). Preliminary evidence in this area shows that antibiotic mixtures significantly reduce bacterial presence in infected root canals compared with using sodium hypochlorite alone in an animal study83. In a recent clinical study performed in nontraumatized cases with pulp necrosis and apical periodontitis84, there was a statistically significant (97%) reduction of bacterial DNA with triple antibiotic paste compared with a nonsignificant (39%)
reduction in the calcium hydroxide/ chlorhexidine group. There were also significantly more samples in the antibiotic group with no detectable bacterial DNA than in the calcium hydroxide group84. The outcome of regenerative endodontic procedures was shown to be comparable with both triple antibiotic paste and calcium hydroxide/chlorhexidine in 1 small clinical study85. However, the success of this procedure at the histologic level was significantly improved if bacteria were eliminated compared with cases with persistent bacteria in vivo86. Finally, it appears that the outcome of regenerative endodontic procedures is significantly improved in cases of pulp necrosis caused by dens evaginatus compared with cases with traumatic injuries81. It is not clear whether this difference is consistent (other studies are needed to corroborate this) and if this may be related to the type of
infection present or the damage to the dental tissues in traumatic injuries. Novel approaches to regenerative endodontic procedures involve the use of scaffolds that would have residual antimicrobial properties while the new tissue is developing. This approach allows the antimicrobial effects to persist while the new tissue develops its vascular supply and host resistance mechanisms. Among the approaches explored in this area are antibiotic nanofiber scaffolds that can be printed 3dimensionally and sustain the developing tissues87–91. In the future, specific antibiotics for which the patient’s own microflora may be sensitive can be selected and included in these fabricated scaffold materials.
ACKNOWLEDGMENTS The authors deny any conflicts of interest related to this study.
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Radcliffe CE, Potouridou L, Qureshi R, et al. Antimicrobial activity of varying concentrations of sodium hypochlorite on the endodontic microorganisms Actinomyces israelii, A. naeslundii, Candida albicans and Enterococcus faecalis. Int Endod J 2004;37:438–46.
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Hinckfuss SE, Messer LB. An evidence-based assessment of the clinical guidelines for replanted avulsed teeth. Part II: prescription of systemic antibiotics. Dent Traumatol 2009;25:158–64.
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Andersson L, Andreasen JO, Day P, et al. International Association of Dental Traumatology guidelines for the management of traumatic dental injuries: 2. Avulsion of permanent teeth. Dent Traumatol 2012;28:88–96.
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Andreasen JO, Bakland LK, Andreasen FM. Traumatic intrusion of permanent teeth. Part 3. A clinical study of the effect of treatment variables such as treatment delay, method of repositioning, type of splint, length of splinting and antibiotics on 140 teeth. Dent Traumatol 2006;22:99–111.
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Andreasen JO, Borum MK, Jacobsen HL, Andreasen FM. Replantation of 400 avulsed permanent incisors. 4. Factors related to periodontal ligament healing. Endod Dent Traumatol 1995;11:76–89.
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Andreasen JO, Borum MK, Jacobsen HL, Andreasen FM. Replantation of 400 avulsed permanent incisors. 2. Factors related to pulpal healing. Endod Dent Traumatol 1995;11:59–68.
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Andreasen JO, Andreasen FM, Mejare I, Cvek M. Healing of 400 intra-alveolar root fractures. 2. Effect of treatment factors such as treatment delay, repositioning, splinting type and period and antibiotics. Dent Traumatol 2004;20:203–11.
66.
Fouad A, Cha B, Diogenes A, et al. AAE Position Statement: AAE Guidance on the Use of Systemic Antibiotics in Endodontics. J Endod 2017;43:1409–13.
67.
Cvek M. Treatment of non-vital permanent incisors with calcium hydroxide. II. Effect on external root resorption in luxated teeth compared with effect of root filling with guttapucha. A follow-up. Odontol Revy 1973;24:343–54.
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Pierce A, Lindskog S. The effect of an antibiotic/corticosteroid paste on inflammatory root resorption in vivo. Oral Surg Oral Med Oral Pathol 1987;64:216–20.
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Pierce A, Heithersay G, Lindskog S. Evidence for direct inhibition of dentinoclasts by a corticosteroid/antibiotic endodontic paste. Endod Dent Traumatol 1988;4:44–5.
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Bryson EC, Levin L, Banchs F, et al. Effect of immediate intracanal placement of Ledermix Paste(R) on healing of replanted dog teeth after extended dry times. Dent Traumatol 2002;18:316–21.
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Chen H, Teixeira FB, Ritter AL, et al. The effect of intracanal anti-inflammatory medicaments on external root resorption of replanted dog teeth after extended extra-oral dry time. Dent Traumatol 2008;24:74–8.
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Jiang J, Zuo J, Chen SH, Holliday LS. Calcium hydroxide reduces lipopolysaccharide-stimulated osteoclast formation. Oral Surg Oral Med Oral Pathol Oral Radiol Endod 2003;95:348–54.
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Guo J, Yang D, Okamura H, et al. Calcium hydroxide suppresses Porphyromonas endodontalis lipopolysaccharide-induced bone destruction. J Dent Res 2014;93:508–13.
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Tronstad L, Andreasen JO, Hasselgren G, et al. pH changes in dental tissues after root canal filling with calcium hydroxide. J Endod 1980;7:17–21.
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Galler KM, Krastl G, Simon S, et al. European Society of Endodontology position statement: revitalization procedures. Int Endod J 2016;49:717–23.
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Jeeruphan T, Jantarat J, Yanpiset K, et al. Mahidol study 1: comparison of radiographic and survival outcomes of immature teeth treated with either regenerative endodontic or apexification methods: a retrospective study. J Endod 2012;38:1330–6.
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Kahler B, Mistry S, Moule A, et al. Revascularization outcomes: a prospective analysis of 16 consecutive cases. J Endod 2014;40:333–8.
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Nagy MM, Tawfik HE, Hashem AA, Abu-Seida AM. Regenerative potential of immature permanent teeth with necrotic pulps after different regenerative protocols. J Endod 2014;40:192–8.
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Alobaid AS, Cortes LM, Lo J, et al. Radiographic and clinical outcomes of the treatment of immature permanent teeth by revascularization or apexification: a pilot retrospective cohort study. J Endod 2014;40:1063–70.
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Chan EK, Desmeules M, Cielecki M, et al. Longitudinal cohort study of regenerative endodontic treatment for immature necrotic permanent teeth. J Endod 2017;43:395–400.
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Lin J, Zeng Q, Wei X, et al. Regenerative endodontics versus apexification in immature permanent teeth with apical periodontitis: a prospective randomized controlled study. J Endod 2017;43:1821–7.
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Fouad AF. Microbial factors and antimicrobial strategies in dental pulp regeneration. J Endod 2017;43:S46–50.
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Windley 3rd W, Teixeira F, Levin L, et al. Disinfection of immature teeth with a triple antibiotic paste. J Endod 2005;31:439–43.
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Arruda ME, Neves MA, Diogenes A, et al. Infection control in tteeth with apical periodontitis using a triple antibiotic solution or calcium hydroxide with chlorhexidine: a randomized clinical trial. J Endod 2018;44:1474–9.
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Nagata JY, Gomes BP, Rocha Lima TF, et al. Traumatized immature teeth treated with 2 protocols of pulp revascularization. J Endod 2014;40:606–12.
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Verma P, Nosrat A, Kim JR, et al. Effect of residual bacteria on the outcome of pulp regeneration in vivo. J Dent Res 2017;96:100–6.
87.
Bottino MC, Yassen GH, Platt JA, et al. A novel three-dimensional scaffold for regenerative endodontics: materials and biological characterizations. J Tissue Eng Regen Med 2013;9:E116–23.
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Albuquerque MT, Ryan SJ, Munchow EA, et al. Antimicrobial effects of novel triple antibiotic paste-mimic scaffolds on Actinomyces naeslundii biofilm. J Endod 2015;41:1337–43.
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Albuquerque MT, Valera MC, Moreira CS, et al. Effects of ciprofloxacin-containing scaffolds on enterococcus faecalis biofilms. J Endod 2015;41:710–4.
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Pankajakshan D, Albuquerque MT, Evans JD, et al. Triple antibiotic polymer nanofibers for intracanal drug delivery: effects on dual species biofilm and cell function. J Endod 2016;42:1490–5.
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Karczewski A, Feitosa SA, Hamer EI, et al. Clindamycin-modified triple antibiotic nanofibers: a stain-free antimicrobial intracanal drug delivery system. J Endod 2018;44:155–62.
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Ashraf F. Fouad, DDS, MS
Microbiological Aspects of Traumatic Injuries ABSTRACT After traumatic injuries to teeth, microorganisms may invade the compromised pulp tissue and initiate pulp infection and periapical inflammation. In addition to bone resorption that typically accompanies pulp necrosis, root resorption frequently occurs. Root resorption has several variants that may occur shortly after the trauma or at a later stage. The pathological changes seen after traumatic injuries to teeth are invariably linked to the presence of microbial irritants. The presence of bacterial biofilms in the dental pulp space can be treated with regenerative or therapeutic endodontic procedures. However, necrosis of periodontal ligament is usually terminal for the tooth involved. In this review, the sources of bacteria after traumatic injuries are discussed. The types and role of microorganisms involved in the pathogenesis of endodontic pathosis after traumatic injuries are presented, and contemporary approaches for the management of these conditions are reviewed. Contemporary antimicrobial strategies are discussed. The rationale for the use of systemic and topical antimicrobials is presented. Finally, novel approaches to the use of antimicrobial therapies, particularly in regenerative procedures, are reviewed. (J Endod 2019;-:1–10.)
SIGNIFICANCE This articles addresses the likely sources of bacteria in traumatic dental injuries, the microbial ecology within the resultant endodontic infections, and the antimicrobial management of these patients at various presentations after the injuries.
KEY WORDS Antibiotics; endodontics; resorption; traumatic dental injuries
Traumatic injuries to teeth represent a unique form of injury to the dental pulp and periodontal tissues. Instead of following the traditional progression from coronal to radicular and periradicular sites seen with caries and congenital anomalies, the injury with trauma can occur directly at any level of the dental or periradicular tissues. In this regard, the injury can occur directly to the pulp, root apex, cementum, periodontal ligament, or surrounding bone individually or in combination. In most cases, the trauma itself disrupts the pulp and periradicular neurovascular supply and causes injury to the cemental surface. This renders the pulp and periodontal tissues less resistant to bacterial invasion that accompanies the trauma and the root surface less resistant to resorption at the site of cemental injury. In addition, fractures of the crown or root, or avulsion of the tooth, may provide direct pathways of invasion by oral or environmental microorganisms. The capacity of the pulp to resist bacterial invasion after traumatic injuries has been shown to depend on the degree of maturation of the tooth root. Teeth with an immature apex have a much lower probability of developing pulp necrosis after luxation or fracture injuries than teeth with a mature apex1–3. This is thought to be related to the presence of collateral and/or more robust circulation in immature teeth to resist the bacterial invasion that ensues after trauma. Because of the susceptibility of the pulp to degenerate after trauma and the risk of resorption, the urgency of diagnosing the pulp condition of the affected teeth is especially critical. Pulp diagnosis is complicated by the loss of sensibility caused by the traumatic injury and the difficulty of pulp sensibility tests in children3,4. Pulp vitality tests, such as using laser Doppler flowmetry (LDF), were shown to be more accurate than pulp sensibility tests5–7, especially after traumatic injuries. In case reports or case series8–10, LDF showed the onset of vitality signals in luxated teeth much sooner than sensibility testing. However, LDF is not currently widely available or practical. Therefore, a period of uncertainty usually elapses before a definitive diagnosis can be obtained. With the exception of avulsion or intrusion of mature teeth, in which endodontic treatment should be initiated within 1–2 weeks, endodontic interventions after other traumatic injuries are contemplated after a period of observation when the tooth shows signs and symptoms of pulp necrosis, apical periodontitis, and/or infection-related (inflammatory) root resorption. During this period of delay, more bacterial invasion and the establishment of bacterial biofilms inside the pulp space may ensue, and irreparable root resorption may be initiated.
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From the Department of Endodontics, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina Address requests for reprints to Dr Ashraf F. Fouad, Adams School of Dentistry, Department of Endodontics, University of North Carolina at Chapel Hill, Room K5417-J, 385 S. Columbia Street, Chapel Hill, NC 27599-7450. E-mail address: [email protected] 0099-2399/$ - see front matter Copyright © 2019 John Wiley & Sons A/S and American Association of Endodontists. This article is being published concurrently in Dental Traumatology. The articles are identical. Either citation can be used when citing this article. https://doi.org/10.1016/ j.joen.2019.05.011
Microbiological Aspects of Traumatic Injuries
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ORIGIN OF MICROORGANISMS IN TRAUMATIC INJURIES The sources of microorganisms in cases of traumatic injuries, especially if the teeth involved remain intact, have been subject to multiple studies and much debate. The older concept introduced in the 1940s was that circulating bacteria from any cause or source would preferentially diffuse into and grow on degenerated or degenerating tissues, such as the traumatized and ischemic pulp11. This was known as anachoresis. In the decades that followed, there have been several animal studies that supported12,13 or refuted14,15 the concept of anachoresis. One clinical study attempted to localize the bacteria in extracted teeth with pulp necrosis and apical periodontitis and showed that bacterial biofilm in primary infections are present inside the root canal and not at the apical foramen or immediate external surface16. In a classic experiment in the 1960s, Serratia marcescens, a nonoral bacterial species, was inoculated in the oral cavity and gingival crevicular areas of dogs and monkeys17. The teeth were then traumatized by an impact force not strong enough to cause fracture. The results showed that S. marcescens was recovered after various time intervals from the pulp space in about a third of the animals17. Modern studies with sensitive molecular techniques show remarkable
resemblance between the microbiomes of the necrotic pulp and that of the periodontal pocket in general18,19. Moreover, studies on root microflora have failed to identify uniquely nonoral bacteria commonly found in the blood. One example of this concept was a study that failed to identify Streptococcus pneumonia and Chlamydia pneumonia, which are common respiratory pathogens, from the root canals of primary or persistent endodontic pathosis20. Therefore, it is currently thought that after traumatic injuries microbial invasion of the pulp is primarily from the gingival sulcus and/or oral cavity, with rare contributions from hematogenous or other sources. These microorganisms travel locally through the injured gingival sulcus and periodontal attachment and through dentinal tubules, lateral and accessory canals, or injured periodontal tissues to the pulp space, which contains the ischemic and injured pulp. Traumatized teeth may be cracked but not fractured. Cracks are common in anterior traumatized teeth as well as mesiodistally in maxillary first bicuspids because of forced occlusion by mandibular bicuspids in cases of falls on the chin. Bacteria can migrate through cracks, both at the time of crack formation or during normal function21. In general, cracked teeth are thought to occur because of chronic traumatic occlusion, such as with bruxism or unusual masticatory habits, and up to 20% of these cases result in irreversible pulp pathosis
requiring root canal treatment within 6 months from the time of diagnosis according to 1 study22. Traumatic injuries to the dentition are frequently accompanied by soft tissue lacerations, abrasions, and bruises. These soft tissue injuries may also be subject to infections and protracted healing depending on the extent of injury, contamination from oral or nonoral sources, and the presence of embedded foreign material. In cases in which coronal fragments of teeth are fractured, the provider should spend some time documenting the extent of the missing portions of teeth and attempt to locate them. If concomitant deep soft tissue lacerations are present, such as in the labial mucosa, these can be imaged radiographically to ensure that they have not been embedded in these locations. Likewise, the possibility of ingesting or inhaling these tooth fragments should be considered and investigated.
DEVELOPMENT OF ENDODONTIC BIOFILM AFTER TRAUMATIC INJURIES Bacterial infiltration into the root canal space and dentinal tubules is initially in the form of disparate planktonic cells that can easily be phagocytosed or killed by host defense mechanisms wherever these mechanisms are intact. Wherever the pulp lacks vascularity, the
FIGURE 1 – A diagrammatic representation of the sequence of adherence and colonization of tooth surfaces by bacteria. (A ) Primary colonizing bacteria existing as planktonic cells interact with the conditioning film (eg, acquired pellicle, dentinal tubule fluid, and serum) on the tooth surface using longer-range interactions (eg, pili or shorter-range molecular interactions). (B ) The early colonizers form strong bonds with the surface molecules in the conditioning film or components of the tooth substrate (eg, collagen by a variety of mechanisms and multiple adhesins). In conjunction with adhesion, the bacteria perform other functions such as adapting to the available nutrition, intermicrobial signaling, and production of an extracellular matrix. (C ) Late colonizing bacteria enter the community by coaggregation reactions contributing to sequential binding and colonization of the developing biofilm. In this regard, Fusobacterium has been shown to be an important bridging organism allowing interactions between nonbinding bacteria. Within the biofilm intricate processes and interactions, such as quorum sensing, metabolic communication, genetic exchange, and competitive interactions further shape the membership of the complex community ensuring efficient utilization of nutrients and reduced susceptibility to host defense or therapeutic methods (eg, antimicrobials). (Reprinted with permission23.)
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FIGURE 2 – Microorganisms isolated by different investigators in initial samples from root canals with necrotic pulps. *Gomes et al, 2005 only covers black-pigmented Porphyromonas and Prevotella species. (Reprinted with permission38.) As previously noted, it is frequently difficult to establish the definitive clinical viability of the pulp promptly after a traumatic injury, except in cases of avulsion or intrusion of mature teeth. If the patient is not followed and monitored closely and no symptoms arise, this infection can last for many years or decades before it is diagnosed. Therefore, by the time the clinical diagnosis of pulp necrosis and infection is established, a formidable bacterial biofilm may have developed in the pulp space. This bacterial biofilm is not static because there are changes in bacterial load, community composition, and virulence (Fig. 1) that influence the development of signs and symptoms of disease29–31. This biofilm may expand into lateral canals or periapical tissues causing persistent symptoms32–34. All these changes and expansions of root canal biofilms necessitate
source of cellular and molecular responses is disrupted or eliminated, and the bacterial infiltrate begins to develop a biofilm that is more difficult for host responses to eliminate (Fig. 1A–C)23. Host proteins, such as albumin and fibrinogen, were shown to aid in bacteria growth on dentin surfaces or invasion of dentinal tubules24,25. Most traumatic injuries occur in children and adolescents. It was shown that bacterial invasion of dentinal tubules is significantly deeper and affects more tubules per unit area in young individuals than in older individuals26. It has also been shown that bacterial biofilm on dentin develops within a period of only 3 weeks to a mature biofilm that is difficult to eliminate using traditional endodontic irrigants27. Even intracanal antibiotics may not eliminate this biofilm, especially if low nontoxic concentrations are used28.
substantial antimicrobial strategies and regimens.
MICROBIOLOGY OF TRAUMARELATED ENDODONTIC INFECTIONS
Data from Sundqvist37.
In the late 1960s and early 1970s, the technology of anaerobic microbiology was mastered in endodontic research. Several studies took advantage of this to study the microbiology of intact, traumatically devitalized teeth with pulp necrosis and apical periodontitis35–37. The intent was to determine if the microflora in these cases was not only unique compared with caries but also if it harbored a high content of facultative and strictly anaerobic bacteria. The results of these studies showed a high proportion of strictly anaerobic bacteria (Fig. 2)38. It has always been assumed that the microbiology of endodontic infection after caries, traumatic injuries, congenital anomalies of crowns, or any other etiology shows a similar degree of composition and diversity. Studies have shown that despite uniform distribution of bacterial taxa used to inoculate root canals of monkeys, with time, the composition shifted to favor a prevalence of gram-positive facultative and gramnegative anaerobic bacteria39. Because it is impossible to compare microflora from cases with equivalent durations of the biofilm, it is not clear if this distinction for cases from traumatized teeth is universally consistent. In 1 important human study, patients with
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Microbiological Aspects of Traumatic Injuries
TABLE 1 - Bacteria from Acute Dental Infections of Teeth with Necrotic Pulps after Trauma to the Teeth Bacteria
Patient 1
Patient 2
Patient 3
Fusobacterium spp. F. nucleatum Peptosteptococcus anaerobius Eubacterium spp. E. alactolyticum E. lentum Peptostreptococcus micros Peptococcus spp. Campylobacter sputorum Lactobacillus spp. Bacteroides melaninogenicus ss. intermedius (Prevotella intermedia)
X
X X
X
X
X
X X X X X X
X X X
X X X X X X X X
3
TABLE 2 - Microbiology of Endodontic Infections in Immature Teeth Microorganism
%
Actinomyces naeslundii Porphyromonas endodontalis Parvimonas micra Fusobacterium nucleatum Porphyromonas gingivalis Prevotella intermedia Tannerella forsythia Filifactor alocis Treponema denticola
67 33 33 33 26 26 20 13 13
Data from Nagata et al40.
intact, traumatized teeth that developed acute apical abscesses were shown to have several notable facultative and strict anaerobic bacteria (Table 1). In a later analysis of cultivable bacteria from 20 intact traumatically devitalized teeth41, the authors confirmed that the following strict anaerobes had the highest prevalence: Bacteroides gracilis (50%), Propionibacterium
acnes (45%), Fusobacterium nucleatum (30%), Prevotella buccae (30%), and Eubacterium lentum (25%). From the 84 strains that were isolated overall, 81% were strict, and 19% were facultative anaerobes. Furthermore, definitive antibiotic sensitivity was obtained for 66 of these strains. The susceptibility of these strains to amoxicillin and amoxicillin 1 clavulanic acid was much lower than to tetracycline, a finding that was later confirmed in other endodontic antibiotic sensitivity studies42–44. Contemporary molecular techniques, based on 16S ribosomal RNA gene sequencing, are capable of identifying a wide spectrum of endodontic microflora, including many uncultivable taxa45–48. These studies reveal that the abundance of individual species (also known as operational taxonomic units) is low because the depth of coverage shows a high diversity of the bacteria present. Studies examining the microflora in traumatic injuries using these methodologies are underway,49 as this area has not been sufficiently explored. One study
that used a combination of culture and molecular approaches showed that Actinomyces naeslundii was the most prevalent organism in children with immature traumatized teeth with necrotic and infected pulps (Table 2)40. This was an important finding because this microorganism commonly forms periapical microbial colonies that persist despite nonsurgical root canal treatment50–52. In addition, a comprehensive analysis of bacteria in the apical third of infected root canals and their correlation with bone-resorbing inflammatory mediators and matrix metalloproteinases was recently reported53. In this study, it was found that increased matrix metalloproteinase-2 and -9, receptor activator of nuclear factor kappa B, receptor activator of nuclear factor kappa B ligand, and decreased osteoprotegerin were correlated with the phylum Actinobacteria, which contains the genus Actinomyces (53%) and the genus Streptococcus (35%). Fortunately, Actinomyces spp. are very sensitive to commonly used antibiotics54 and
FIGURE 3 – The maxillary left lateral incisor sustained a lateral luxation injury. (A ) Two months later, the tooth continued to have no response to pulp testing and developed external infection-related (inflammatory) root resorption. (B ) Endodontic treatment was initiated, the root canal was debrided, and calcium hydroxide was placed for 4 weeks. (C and D ) The radiographs show stabilization of the resorptive lesion, and the root canal was completed. (E ) Three-month follow-up showing bone deposition and mineralization at the root apex.
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FIGURE 4 – The maxillary right central incisor sustained an extrusive luxation and was repositioned and splinted for several months without sensibility testing or radiographic evaluation. Advanced infection-related (inflammatory) root resorption occurred, rendering the prognosis unfavorable.
other antimicrobials, such as sodium hypochlorite and calcium hydroxide55,56.
ANTIMICROBIAL MANAGEMENT OF PATIENTS WITH TRAUMATIZED TEETH Antibiotics, both systemically and topically, as well as several other topical antimicrobial medicaments have been proposed for use in the treatment of patients who sustain dental traumatic injuries. However, because of the urgent nature of traumatic injuries and the variability of the injuries, randomized clinical trials, which represent the highest level of objective clinical research, are lacking. In many cases, randomized trials would be unethical to conduct, except possibly to compare the
efficacy of 2 therapeutic agents or medicaments. Systemic administration of antibiotics after avulsion or luxation injuries is currently recommended57–59. The evidence for this recommendation is mainly from an older animal study60. In this animal study, teeth were extracted and left to dry for an hour before replantation. The animals were given daily intramuscular injections of 2 mL Streptocillin vet. (Novo, Copenhagen, Denmark), containing dihydrostreptomycin 0.25 g/mL and benzylpenicillin 200,000 IE/mL, on the day of replantation and for 6 days afterward. It was found that this regimen prevented or significantly reduced the development of infection-related (inflammatory) root resorption, similar to early root canal treatment of these teeth60. Another study with a similar
TABLE 3 - Selected Revascularization Cohort or Randomized Studies and the Antimicrobials Used
Study
N
Concentrated NaOCl (%)
Antibiotic
Success/healing (%)
Jeeruphan et al76 Kahler et al77 Nagy et al78 Alobaid et al79 Chan et al80 Lin et al81
20 16 10 19 28 69
2.5 1 2.6 ? 5.25 1.25
TAP TAP DAP 1 doxycycline TAP/DAP/Ca(OH)2 DAP 1 cefaclor DAP 1 clindamycin
80 90 90 78 93 90
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design was reported to evaluate pulp revascularization in immature monkey teeth61. The animals were given 4 mg/kg doxycycline before extraction and 2 mg/kg for 5 days after replantation. This regimen was not found to have any benefit on revascularization. In a more recent human prospective observational study, 140 permanent teeth with intrusion injuries were included62. Many treatment variables were collected, including the administration of antibiotics. Antibiotic treatment had no effect on healing in this study. Older retrospective studies on fractures and replantation also did not show a benefit to using antibiotics63–65. Therefore, the use of systemic antibiotics in patients with traumatic injuries appears to be mainly related to protecting the patient against a possible systemic infection from the site of trauma, and they may reduce infection-related (inflammatory) resorption. However, more studies are needed to confirm that the value of this recommendation outweighs the risks involved with antibiotic prescriptions66. Topical application of antibiotics may be applied on the external root surface before replantation of avulsed teeth or inside the root canal of any traumatized tooth that requires endodontic treatment. The topical application of antibiotics and other antimicrobial medicaments such as calcium hydroxide or Ledermix (Riemser Pharma GmbH, Greifswald, Insel Riems, Germany) paste has been recommended in some traumatic injury cases for decades. As previously noted, the presence of a formidable biofilm in these cases necessitates effective antimicrobial regimens. In addition, it has long been noted that the development of infection-related (inflammatory) root resorption is best treated with calcium hydroxide67 (Fig. 3A–D) or a combination of an antibiotic and a steroid such as Ledermix paste68–71. It is thought that the main action that stops the resorptive lesion (Fig. 4) in these cases is the effective antimicrobial effects of the medicaments72,73, although some have proposed that the alkaline pH (at least in the case of calcium hydroxide) may have a direct effect on the odontoclasts/osteoclasts whose brush border acts in an acidic environment74. Corticosteroids may also stabilize the cell membrane of clastic cells, thus reducing their action. Contemporary regenerative protocols for immature teeth with pulp necrosis involve the use of intracanal antibiotics or calcium hydroxide75. With respect to antibiotics, triple antibiotic paste (comprised of minocycline, ciprofloxacin, and metronidazole) and double antibiotic paste
Microbiological Aspects of Traumatic Injuries
5
FIGURE 5 – The size of inhibition zones in millimeters around unit volumes of antibiotic/hydrogel mixture against several endodontic pathogens. Wider zones indicate more effectiveness. Note the enhanced effectiveness of all antibiotics against F. nucleatum and the reduced effectiveness against Enterococcus faecalis. (Data from AlSaeed et al28.)
(excluding minocycline because of staining) have gained the most attention. Other antibiotics, such as Augmentin (GlaxoSmithKline, Research Triangle Park, NC), cefaclor, clindamycin, and doxycycline, have been proposed and used in some clinical studies with good success (Table 3)82. The susceptibility of endodontic pathogens to various antibiotics in hydrogel vehicles or other scaffold materials is currently being explored28 (Fig. 5). Preliminary evidence in this area shows that antibiotic mixtures significantly reduce bacterial presence in infected root canals compared with using sodium hypochlorite alone in an animal study83. In a recent clinical study performed in nontraumatized cases with pulp necrosis and apical periodontitis84, there was a statistically significant (97%) reduction of bacterial DNA with triple antibiotic paste compared with a nonsignificant (39%)
reduction in the calcium hydroxide/ chlorhexidine group. There were also significantly more samples in the antibiotic group with no detectable bacterial DNA than in the calcium hydroxide group84. The outcome of regenerative endodontic procedures was shown to be comparable with both triple antibiotic paste and calcium hydroxide/chlorhexidine in 1 small clinical study85. However, the success of this procedure at the histologic level was significantly improved if bacteria were eliminated compared with cases with persistent bacteria in vivo86. Finally, it appears that the outcome of regenerative endodontic procedures is significantly improved in cases of pulp necrosis caused by dens evaginatus compared with cases with traumatic injuries81. It is not clear whether this difference is consistent (other studies are needed to corroborate this) and if this may be related to the type of
infection present or the damage to the dental tissues in traumatic injuries. Novel approaches to regenerative endodontic procedures involve the use of scaffolds that would have residual antimicrobial properties while the new tissue is developing. This approach allows the antimicrobial effects to persist while the new tissue develops its vascular supply and host resistance mechanisms. Among the approaches explored in this area are antibiotic nanofiber scaffolds that can be printed 3dimensionally and sustain the developing tissues87–91. In the future, specific antibiotics for which the patient’s own microflora may be sensitive can be selected and included in these fabricated scaffold materials.
ACKNOWLEDGMENTS The authors deny any conflicts of interest related to this study.
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