- Email: [email protected]
Regenerative endodontics
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ENDODONTICS
Regenerative endodontics A way forward Anibal Diogenes, DDS, MS, PhD; Nikita B. Ruparel, DDS, MS, PhD; Yoav Shiloah, DDS, MBA; Kenneth M. Hargreaves, DDS, PhD
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mmature teeth are at risk of developing pulpal necrosis due to trauma, caries, and anatomic variations such as dens evaginatus and dens invaginatus.1-4 Dental trauma occurs with an incidence that varies from 2.6% to 35% in patients undergoing cranioskeletal development.5-7 Up to one-half of these traumatized teeth may undergo pulpal necrosis, but only 8.5% will exhibit signs and symptoms of disease.8 Dental anomalies also represent a common etiology leading to pulpal necrosis in immature permanent teeth.4 Dens evaginatus and dens invaginatus are the most common anomalies associated with this clinical manifestation.4 Full radicular maturation occurs up to 3 years after the eruption of a tooth in the oral cavity,9 and the loss of pulp vitality during this period arrests further root development. These teeth traditionally have been treated with apexification procedures by using either long-term calcium hydroxide treatment10,11 or immediate placement of a mineral trioxide aggregate (MTA) apical plug.12 Although these treatments often result in the resolution of signs and symptoms of disease, they provide little to no benefit in restoring normal pulpal defenses and nociception and, more importantly, continued root development.13 Thus, immature teeth remain with thin fragile dentinal walls, increasing susceptibility to fractures and lower survival rates.14,15 Tooth loss in patients still undergoing cranioskeletal development has devastating consequences that include altered maxillary and mandibular bone development; Copyright ª 2016 American Dental Association. All rights reserved.
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ABSTRACT Background and Overview. Immature teeth are susceptible to infections due to trauma, anatomic anomalies, and caries. Traditional endodontic therapies for immature teeth, such as apexification procedures, promote resolution of the disease and prevent future infections. However, these procedures fail to promote continued root development, leaving teeth susceptible to fractures. Regenerative endodontic procedures (REPs) have evolved in the past decade, being incorporated into endodontic practice and becoming a viable treatment alternative for immature teeth. The authors have summarized the status of regenerative endodontics on the basis of the available published studies and provide insight into the different levels of clinical outcomes expected from these procedures. Conclusions. Substantial advances in regenerative endodontics are allowing a better understanding of a multitude of factors that govern stem cell–mediated regeneration and repair of the damaged pulp-dentin complex. REPs promote healing of apical periodontitis, continued radiographic root development, and, in certain cases, vitality responses. Despite the clinical success of these procedures, they appear to promote a guided endodontic repair process rather than a true regeneration of physiological-like tissue. Practical Implications. Immature teeth with pulpal necrosis with otherwise poor prognosis can be treated with REPs. These procedures do not preclude the possibility of apexification procedures if attempts are unsuccessful. Therefore, REPs may be considered first treatment options for immature teeth with pulpal necrosis. Key Words. Guided tissue regeneration; revascularization; endodontic therapy; stem cells; outcome assessment; regenerative endodontics. JADA 2016:147(5):372-380 http://dx.doi.org/10.1016/j.adaj.2016.01.009
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interference with pronunciation, breathing, and mastication; and a severe detrimental psychosocial effect.16,17 Because implants are contraindicated in growing young patients because of continued craniofacial development, tooth replacement is not possible until the age of maturity (usually older than 18 years). As an alternative technique, regenerative endodontic procedures (REPs) are intended to promote tooth survival and the function of previously thought hopeless teeth. In this review, we will focus on discussing the historical background and the present and future directions of these clinical procedures. HISTORY
The emergence of regenerative endodontics was catalyzed in the early 2000s with the publication of 2 remarkable case reports.18,19 However, this field has its roots in the seminal work by Dr. Ostby in the early 1960s that aimed to evaluate the role of the apical blood clot in the healing of apical periodontitis and pulp repair.20,21 Regenerative endodontics also relied on contributions from important studies in dental trauma, which provided evidence that the dental pulp in immature teeth often remains vital despite substantial traumatic injuries such as intrusions and avulsions.22-24 This remarkable regenerative potential is highlighted in cases of replantation of avulsed immature teeth with evidence of reestablishment of vitality responses and lack of signs and symptoms of disease.23,25,26 In these cases, clinical success relies heavily on the reestablishment of a blood supply to the ischemic but uninfected dental pulp tissue, followed by reinnervation from sensory axons likely recruited from the apical region. This healing process of a previously necrotic dental pulp in traumatic injuries is crucial for reattaining normal pulpal function after trauma. The term revascularization has emerged from these observations in dental traumatology despite the different application and goals.4 Pulpal revitalization is another commonly used term in the scientific literature. However, for the sake of this review, we simply will address these procedures collectively as REPs. Results from numerous published reports demonstrate that these procedures often lead to resolution of apical periodontitis and signs and symptoms of inflammation, radiographic evidence of continued root development and apical narrowing, and restoration of vitality responses.4,27,28 These published cases establish that REPs address the unmet need of promoting normal physiological development and responses in immature teeth with pulpal necrosis. In most REPs, clinicians rely on creating bleeding from the apical region that passively fills the canal space and forms a blood clot. However, it was not until 2011 that investigators in a clinical study demonstrated that the influx of apical blood into disinfected root canals was accompanied by a clinically significant transfer of mesenchymal stem cells into the root canal system.29
This was an important demonstration in the field of regenerative endodontics because it established that these procedures were, in fact, stem cell–based procedures. The realization that autogenous stem cells can be delivered clinically into root canals without the need for ex vivo stem cell expansion propelled researchers and clinicians to consider principles of tissue engineering to improve treatment protocols and to develop the next generation of procedures. TRANSLATIONAL STUDIES
The balance between adequate disinfection and stem cell survival, proliferation, and differentiation represents an important initial barrier to overcome. The resolution of infection and the disease process remains the primary goal of any endodontic procedure. However, it has become obvious that the philosophy of disinfecting the root canal by using methods typically advocated in traditional root canal therapy had to be modified to attain a biocompatible disinfection strategy. To maintain the physical integrity of the already thin dentinal walls of immature teeth, investigators have advocated chemical debridement as the primary means of disinfection. To this end, investigators in many translational studies have focused efforts on establishing the biological basis for clinical protocols that could achieve both disinfection and optimum regenerative potential.4,27 For example, sodium hypochlorite remains the most used disinfectant in endodontics.30 However, its use at full concentration of 6% denatures crucial growth factors in dentin31 and results in residual detrimental effects greatly affecting stem cell attachment, survival, and differentiation potential.32-35 These deleterious effects largely can be avoided with the use of a 1.5% concentration of sodium hypochlorite followed by 17% ethylenediaminetetraacetic acid.35,36 Furthermore, results from elegant studies have demonstrated that ethylenediaminetetraacetic acid promotes the release of growth factors embedded in dentin, including vascular endothelial growth factor and transforming growth factor beta-1 among others that are known to participate actively in regenerative processes such as angiogenesis and stem cell proliferation, migration, and differentiation, respectively.37-39 Along these lines, it was obvious that the effects of intracanal medicaments be tested on the survival and maintenance of stem cells. A triple antibiotic formulation consisting of ciprofloxacin, metronidazole, and minocycline first was tested in vitro against bacteria isolated from carious lesions and from endodontic infections in primary teeth.40 The investigators found that no bacteria could be recovered after treatment with 100 ABBREVIATION KEY. MTA: Mineral trioxide aggregate. NSRCT: Nonsurgical root canal therapy. REP: Regenerative endodontic procedure.
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micrograms per milliliter of each antibiotic (300 mg/mL of mixture).40 Next, investigators carried out studies to test the efficacy of triple antibiotic paste in eradicating bacteria from infected dentin. They found that there were no recovered cultivable bacteria at 48 hours, and they found evidence of significant drug penetration into dentin.40 In REPs, the use of the triple antibiotic paste and its modified versions, such as exclusion of minocycline, known as double antibiotic paste, or the addition of cefaclor, were introduced by initial published case reports.19 The drugs were mixed with water, saline, or propylene glycol until a thick creamy mixture was formed. In these case reports, there was no attempt to deliver a specific concentration of the drugs deliberately. Instead, the investigators mixed the drugs until they achieved a certain physical consistency (approximately 1 gram per milliliter) that clinicians deemed suitable. At this concentration, however, the triple antibiotic paste appeared to have long-lasting deleterious effects on stem cell survival through both direct and indirect mechanisms.41,42 This undesirable effect can be avoided greatly by the use calcium hydroxide as an intracanal medicament41,42 or the use of these pastes in lower concentrations (less than 1 milligram/mL); these lower concentrations retain the desirable antibacterial or antibiofilm effect.43-46 Therefore, there have been substantial advances in understanding how to adapt disinfection protocols to the new reality of stem cell–based therapies. In addition to studies on biocompatible disinfection, many other frontiers in regenerative endodontic research are being investigated. These involve tissue engineering strategies that include the evaluation of suitable scaffolds, growth factors, and harvested stem cells to be used in pulp-dentin regeneration.47 Many of the advances from translational research have been evaluated in a clinical setting, including the use of platelet-rich plasma,48,49 platelet fibrin,50 and a gelatin hydrogel51 as scaffolds in patients. In addition, a groundbreaking clinical trial is being conducted in Japan.52 This trial involves harvesting stem cells from a donor site, followed by ex vivo expansion, sorting, and maintenance of these cells in a good manufacturing practice facility, followed by autogenic transplantation into a recipient tooth to promote the regeneration of the once lost functional pulp-dentin complex. These elegant studies highlight the status and sophistication of REPs. CLINICAL OUTCOMES OF REPs
Resolution of infection and the signs and symptoms of inflammation leading to apical periodontitis and restoration of lost function remains the primary goal of any endodontic therapy. Traditional endodontic therapies (nonregenerative procedures) achieve this goal by promoting disinfection followed by a fluid-tight or bacteriatight seal of the root canal system and delivery of a high-quality coronal restoration. The clinical outcome
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of endodontic therapies traditionally has been evaluated on the basis of lack of signs and symptoms of disease, such as pain, swelling, or sinus tracts, and radiographic criteria of healing.10,11,53 REPs, on the other hand, rely on comparably efficient chemical disinfection followed by the stem cell–mediated growth of a reparative tissue that promotes continued root development and reestablishment of pulpal functions such as nociception and immune competency. This paradigm shift in treatment approach also requires a shift in assessment of clinical outcomes.54,55 Last, the success of these procedures must be evaluated by 3 stakeholders: patients and their legal guardians, clinicians, and researchers. Patient-based outcomes. The needs and desires of patients might not always align with the preset criteria of success laid out by clinicians and researchers. There is an ever-increasing need to focus on outcomes that are meaningful to patients because they are the primary stakeholders in their own health and must participate in treatment decisions.56 However, even in the context of long-established therapies such as conventional nonsurgical root canal therapy (NSRCT), there is often a disconnect with patient-centered outcomes. For example, from a patient perspective, the resolution of pain may be the most important criterion for successful NSRCT. However, for most clinicians, success also means healing of a periapical lesion, if present.57 Other patient-centered outcomes may include tooth survival in the mouth and acceptable esthetics. These patient-centered outcomes are also relevant for REPs (Figure 1). Investigators used standardized treatment protocols in a study to compare REPs directly with apexification procedures by using the long-term application of calcium hydroxide or by using MTA as an apical plug.15 REPs and MTA plug apexification procedures were equally effective in resolving signs and symptoms of disease (that is, pain, swelling, or sinus tract) in 100% and 95% of all patients, respectively, whereas apexification procedures with the use of calcium hydroxide were significantly less effective (77%). Investigators in another retrospective study did not include standardization of treatment protocols and found REPs to promote healing in 79% of patients treated, whereas apexification procedures promoted healing in 100% of the patients; however, this difference was not significant.58 In a prospective study, investigators found that both MTA apexification and REPs promoted healing in 100% of patients.51 Further evidence of successful outcomes can be found in case series and retrospective patient cohort studies. Despite significant variations in etiology, inclusion and exclusion criteria, and clinical protocols, results from these studies51,58 demonstrate that both procedures can resolve the signs and symptoms of disease in approximately 90% of patients successfully, without marked difference in resolution of symptoms between REPs and apexification procedures.
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Survival of the tooth is another important patient-centered Scientist-Based outcome. An ideal treatOutcomes ment, from the patient’s Histologic evidence perspective, is one that of complete prolongs the functional regeneration life of an asymptomatic tooth. The survival of Clinician-Based a permanent tooth is • Radiographic healing Outcomes particularly important in • Radiographic root development young patients undergo• Positive vitality responses ing continued cranioskeletal development • Resolution of disease Patient-Based because implants are (absence of swelling, drainage, and pain) Outcomes contraindicated at this • Tooth survival and function 59 • Tooth esthetics stage. Although results from various studies demonstrate the functional survival of mature Figure 1. Trilevel outcome assessment pyramid. Regenerative endodontic procedures must be assessed and evaluated in a systematic manner, acknowledging that there are different levels of possible outcomes that carry teeth treated with conmeanings for patients, clinicians, and scientists. The patient-centered outcomes represent the base of ventional NSRCT,60,61 far different this pyramid, symbolizing the fundamental importance of achieving healing, reestablishment of function, and less is known about the patient satisfaction. effect of either apexification or REPs on tooth survival. REPs promoted 100% survival in a study period Clinician-based outcomes. As mentioned, clinicianof 18 months, which was comparable with the 95% survival based outcomes involve radiographic signs of healing; promoted by MTA apexifications, both being superior however, unique to REPs, clinicians also evaluate for to the 77% survival achieved in calcium hydroxide– continued root development by means of radiographic mediated apexification procedures.15 Another retrospecexamination and responses to pulp sensitivity. Initially, tive study in which the investigators directly compared investigators in case reports and case series relied the survival of REPs and apexification (MTA apical plug) on reporting the nonquantitative assessment of root procedures failed to demonstrate differences between the development, often using a dichotomous dependent treatment alternatives.58 Nonetheless, investigators have measure (that is, yes or no),74 in addition to qualitative shown that REPs arrest the disease process and promote descriptions of radiographic findings.75 In 2009, study retention of teeth with otherwise poor prognosis.4,27 investigators published a new methodology to quanFactors affecting the long-term survival of these teeth titate changes in root width and length13 and further require further investigation by means of randomized refined and revised it in a later study.76 It allows for the digital alignment of nonstandardized radioclinical trials to compare REPs with MTA apexification as the criterion standard during long follow-ups. graphs obtained at different recall visit times in paThere have been esthetic concerns with REPs that result tients undergoing cranioskeletal growth. It allows the unbiased measurement of changes in root development in coronal staining in certain cases.62-64 This coronal staining has been identified as being the result of triple detected by means of conventional 2-dimensional antibiotic paste in which minocycline stains the dentin, periapical radiographs. With use of this methodology, prior intracoronal bleeding, or the coronal placement of teeth treated with REPs showed a significantly greater MTA.63,65-69 This expected staining can be minimized by percentage of increase in root length and width. occluding the dentinal tubules with a dental adhesive if This finding later was confirmed in another retroeither triple antibiotic paste or MTA is used.62 Alternaspective study in which the investigators reported tively, other bioceramic materials, such as Biodentine gains in root length (14.9%) compared with teeth (Septodont), Bioaggregate (Pearson Dental), and Endotreated by means of either MTA apexification (6.1%) Sequence Bioceramic Root Repair Material putty (Brassor calcium hydroxide apexification (0.4%).15 They also reported that the REPs produced a significantly eler), are biocompatible and are osteoinductive when exposed to mesenchymal stem cells70-73 while showing greater percentage of increases in root width (28.2%) minimal to no color change.67 Thus, tooth discoloration as compared with that in teeth treated by means of either an adverse event of REPs largely can be avoided if nonMTA apexification (0.0%) or calcium hydroxide staining medications and restorative materials are used. apexification (1.5%).
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Figure 2. A. A 12-year-old boy sought care for intraoral buccal swelling and sinus tract associated with tooth no. 29. B. Clinical examination revealed a talon cusp (dens evaginatus) on the occlusal aspect of the tooth. Clinical findings led to the diagnosis of pulpal necrosis with chronic apical abscess. C. A preoperative radiograph revealed a large apical radiolucency traced with a gutta-percha point through a sulcular sinus tract, an immature root with a large pulp canal space, and thin dentinal walls. The tooth was treated by means of a regenerative endodontic procedure by using a mixture of ciprofloxacin, metronidazole, and minocycline (triple antibiotic paste) as an intracanal mixture for 40 days. Incision and drainage also was performed at this visit. At the second visit, after removal of the intracanal medicament and evoked bleeding, a 3-millimeter piece of absorbable wound dressing (CollaPlug, Integra LifeSciences) was placed at the level of the cementoenamel junction and restored with a 3-mm layer of mineral trioxide aggregate and light-cured glass ionomer (Fuji II LC, GC America). At the 4-year recall visit, the patient was asymptomatic and demonstrated positive response to electric pulp testing for tooth no. 29. Radiographic examination revealed complete apical closure, as well as clinically significant gain in root length and width with narrowing of the pulp canal space as seen in the periapical radiograph (D) and small-volume cone-beam computed tomography in the coronal (E) and sagittal (F) views. Photographs and radiographs courtesy of Dr. Yoav Shiloah, University of Texas Health Science Center at San Antonio, San Antonio, TX.
Further evidence of continued root development is available in a prospective randomized clinical trial in which the investigators compared 2 different protocols of REPs with MTA apexification procedures.51 In this study, REPs promoted the gain in root length (12% increase) and width (13% increase), whereas no changes were detected in the MTA apexification group.51 Investigators in other studies with similar methodologies also reported changes in radiographic root structure but failed to detect statistical significance, possibly because of limited sample size or large variability of responses.58,77 Various degrees of radiographic root development have been observed in most published studies. The factors that modulate this varied response are largely unknown because correlation with factors such as tooth type, etiology, sex, or age has not been established.15,58 Thus, although REPs predictably promote healing of apical periodontitis in more than 90% of the cases,4,15,51,58,74 radiographic root development is far less predictable. Figures 2 and 3 provide examples of varied root development. Figure 2 presents a case that shows excellent gain in root length and root width along with complete apical closure, whereas Figure 3 presents a case that shows
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only closure of the apex with no gain in root length or width. Although the lack of radiographic root development in absence of disease does not constitute a treatment failure, future studies with appropriate sample sizes are needed to find prognostic factors for root development. Positive responses to pulpal sensitivity tests are interpreted as evidence favoring the presence of a vital pulp.78 Clinicians routinely use these tests as adjuvants in the diagnosis of pulp status. Investigators have reported positive sensitivity responses to either cold or electric pulp tests in approximately 60% of all published cases.4 Nociception is an important surveillance mechanism that protects tissues against actual or potential damage. The reestablishment of nociception in these teeth must be seen favorably because it suggests the presence of a vascularized tissue with normal physiological responses. Furthermore, primary afferent neurons in the dental pulp are equipped to sense microbial antigens, participating in the inflammatory process evoked by invading microorganisms.79,80 They modulate the immune reaction by the release of neuropeptides with vasoactive properties that increase immune cell recruitment and vascularity, localizing areas of insult to microabcesses
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Figure 3. A 10-year-old boy had trauma of tooth no. 8 with an uncomplicated crown fracture and moderate intrusive luxation. The tooth had pulpal necrosis and symptomatic apical periodontitis approximately 6 months after injury. A. A preoperative periapical radiograph revealed a moderately immature root with an apical radiolucency and inflammatory root resorption. The tooth was treated with a regenerative endodontic procedure by using a mixture of ciprofloxacin and metronidazole (double antibiotic) as an intracanal medicament for 1 month. At the second visit, after medicament removal and evoked bleeding, the access was restored with a 3-millimeter layer of mineral trioxide aggregate placed at the cementoenamel junction over the blood clot, followed by a base layer of glass ionomer and a composite restoration. At the 3-year recall visit, the patient was asymptomatic, and the tooth responded to electric pulp testing with evident resolution of the apical radiolucency, arrestment of the resorptive process, and thickening of the apical one-third with apical closure detected on a periapical radiograph (B). At the 6-year recall visit, the tooth remained asymptomatic with positive responses to electric pulp testing and evidence of apical closure seen on the periapical radiograph (C) and small-volume cone-beam computed tomography in the coronal (D), axial (E), and sagittal (F) views. Radiographs and computed tomographic scans courtesy of Dr. Tyler Lovelace, University of Texas Health Science Center at San Antonio, San Antonio, TX.
within the dental pulp.81 Also, trigeminal neurons increase odontoblastic differentiation and dentinogenesis.82 Lastly, trigeminal neurons harbor an important subpopulation of mesenchymal stem cells along their axonal projections that participate in tooth formation. The role of the perineuronal mesenchymal stem cells in postdevelopment repair or regeneration remains largely
unknown. Mesenchymal stem cells delivered into root canals in REPs may be capable of releasing potent soluble factors that attract nearby neurons to reinnervate the newly formed tissue, possibly participating in the reparative process.83 Clinicians rely on the preestablished criteria of radiographic continued root development and vitality
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responses to evaluate clinical outcomes further. Although these dependent measures add valuable information that can be used to determine whether further interventions are needed, their meaning to patients and their guardians is not entirely clear because their expectation is that the tooth is pain free and functional. The clinician-centered criteria, which often are used to evaluate REPs, are not directly applicable to apexification procedures because root development and vitality responses are not expected. Scientist-based outcomes. Outcomes that are not related directly to the clinical manifestation of the treated tooth but that address a specific question that requires scientific methodology in its evaluation should be categorized as scientist-based outcomes. These are equally important outcomes because they tend to, through science, move the field forward leading to substantial future advances in care. Histologic evaluation of teeth that were treated previously with REPs but later extracted because of recurring trauma and fractures suggests that the newly formed tissue does not resemble the lost dental pulp.84-86 This is the best example of a scientist-based outcome because it has strong scientific merit, but it does not necessarily interfere with consideration of the rate of healing and continued root development seen in cases treated with REPs. There is considerable debate about the use of the term regeneration because existing scientific evidence from histologic studies suggests that the procedures allow for repair instead.87 This is both a semantic and a biological debate; however, it can be argued that true regeneration as defined by the complete recapitulation of the lost tissue with all its constituents, morphology, function, and molecular markers typically is observed only in primitive invertebrates such as hydras and in some amphibians such as salamanders.88 To date, even in sophisticated animal models, the tissues formed closely resemble the native dental pulp, but true odontoblasts are missing; instead, mineralizing cells called odontoblast-like cells are formed. Thus, the definition of regeneration hinges on the methodology used for its assessment and appears to fall short when more advanced molecular techniques are used. Perhaps a more conservative term to be used to describe this procedure is guided endodontic repair (GER). A repaired tissue that promotes the resolution of disease and reestablishment of some or all of the original tissue functions should be a desirable goal. The dental pulp capacity of repair seen in direct and indirect pulp capping procedures is absent once the pulp completely succumbs to infection or trauma. In these cases, clinicians using the knowledge in regenerative endodontics are able to guide endodontic repair, attaining the desirable patient- and clinician-centered outcomes. Clinicians and scientists working in tandem striving to achieve the scientist-based outcome of true regeneration are creating tangible advances in endodontic care, departing from a materials-based to a biologically based approach.
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CONCLUSIONS
Substantial advances in regenerative endodontics are allowing for a better understanding of a multitude of factors that govern stem cell–mediated regeneration and repair of the damaged pulp-dentin complex. REPs are valuable adjuvants to the treatment and retention of immature teeth with pulp necrosis with an otherwise poor prognosis. Despite the clinical success of these procedures, they appear to promote a GER process rather than a true regeneration of physiological-like tissue. For greater regenerative potential to be met, investigators must perform much more research and development. Translational research is crucial in making these procedures more predictable while pushing the boundaries of future procedures that are likely to involve the direct clinical manipulation of scaffolds, growth factors, and stem cells. n Dr. Diogenes is an associate professor, Department of Endodontics, University of Texas Health Science Center at San Antonio, 7703 Floyd Curl Dr., San Antonio, TX 78229, e-mail [email protected]. Address correspondence to Dr. Diogenes. Dr. Ruparel is an assistant professor, Department of Endodontics, University of Texas Health Science Center at San Antonio, San Antonio, TX. Dr. Shiloah is an adjunct professor, Department of Endodontics, University of Texas Health Science Center at San Antonio, San Antonio, TX. Dr. Hargreaves is a professor and the chair, Department of Endodontics, University of Texas Health Science Center at San Antonio, San Antonio, TX. Disclosure. None of the authors reported any disclosures. Endodontics is published in collaboration with the American Association of Endodontists. 1. Cortes MI, Marcenes W, Sheiham A. Prevalence and correlates of traumatic injuries to the permanent teeth of schoolchildren aged 9-14 years in Belo Horizonte, Brazil. Dent Traumatol. 2001;17(1):22-26. 2. Oehlers FA, Lee KW, Lee EC. Dens evaginatus (evaginated odontome): its structure and responses to external stimuli. Dent Pract Dent Rec. 1967; 17(7):239-244. 3. Levitan ME, Himel VT. Dens evaginatus: literature review, pathophysiology, and comprehensive treatment regimen. J Endod. 2006;32(1):1-9. 4. Diogenes A, Henry MA, Teixeira FB, Hargreaves KM. An update on clinical regenerative endodontics. Endod Topics. 2013;28(1):2-23. 5. Andreasen JO, Ravn JJ. Epidemiology of traumatic dental injuries to primary and permanent teeth in a Danish population sample. Int J Oral Surg. 1972;1(5):235-239. 6. Petti S, Tarsitani G. Traumatic injuries to anterior teeth in Italian schoolchildren: prevalence and risk factors. Endod Dent Traumatol. 1996; 12(6):294-297. 7. Forsberg CM, Tedestam G. Traumatic injuries to teeth in Swedish children living in an urban area. Swed Dent J. 1990;14(3):115-122. 8. Robertson A, Andreasen FM, Bergenholtz G, Andreasen JO, Noren JG. Incidence of pulp necrosis subsequent to pulp canal obliteration from trauma of permanent incisors. J Endod. 1996;22(10):557-560. 9. Moorrees CF, Fanning EA, Hunt EE Jr. Age variation of formation stages for ten permanent teeth. J Dent Res. 1963;42:1490-1502. 10. Strindberg LZ. The dependence of the results of pulp therapy on certain factors: an analytic study based on radiographic and clinical followup examinations. Acta Odontol Scand Suppl. 1956;(14):1-175. 11. Bender IB, Seltzer S, Soltanoff W. Endodontic success: a reappraisal of criteria. II. Oral Surg Oral Med Oral Pathol. 1966;22(6):790-802. 12. Witherspoon DE, Ham K. One-visit apexification: technique for inducing root-end barrier formation in apical closures. Pract Proced Aesthet Dent. 2001;13(6):455-460. 13. Bose R, Nummikoski P, Hargreaves K. A retrospective evaluation of radiographic outcomes in immature teeth with necrotic root canal systems
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treated with regenerative endodontic procedures. J Endod. 2009;35(10): 1343-1349. 14. Cvek M. Prognosis of luxated non-vital maxillary incisors treated with calcium hydroxide and filled with gutta-percha: a retrospective clinical study. Endod Dent Traumatol. 1992;8(2):45-55. 15. 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(10):1330-1336. 16. Judd PL, Casas MJ. Psychosocial perceptions of premature tooth loss in children. Ont Dent. 1995;72(8):16-18, 20, 22-23. 17. Thelen DS, Trovik TA, Bardsen A. Impact of traumatic dental injuries with unmet treatment need on daily life among Albanian adolescents: a case-control study. Dent Traumatol. 2011;27(2):88-94. 18. Banchs F, Trope M. Revascularization of immature permanent teeth with apical periodontitis: new treatment protocol? J Endod. 2004;30(4): 196-200. 19. Iwaya S, Ikawa M, Kubota M. Revascularization of an immature permanent tooth with apical periodontitis and sinus tract. Dent Traumatol. 2001;17(4):185-187. 20. Ostby BN. The role of the blood clot in endodontic therapy: an experimental histologic study. Acta Odontol Scand. 1961;19:324-353. 21. Rule DC, Winter GB. Root growth and apical repair subsequent to pulpal necrosis in children. Br Dent J. 1966;120(12):586-590. 22. Kling M, Cvek M, Mejare I. Rate and predictability of pulp revascularization in therapeutically reimplanted permanent incisors. Endod Dent Traumatol. 1986;2(3):83-89. 23. 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(2):59-68. 24. Andreasen FM, Daugaard-Jensen J. Treatment of traumatic dental injuries in children. Curr Opin Dent. 1991;1(5):535-550. 25. Andreasen JO, Borum MK, Andreasen FM. Replantation of 400 avulsed permanent incisors. 3. Factors related to root growth. Endod Dent Traumatol. 1995;11(2):69-75. 26. 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(2):76-89. 27. Kontakiotis EG, Filippatos CG, Tzanetakis GN, Agrafioti A. Regenerative endodontic therapy: a data analysis of clinical protocols. J Endod. 2015;41(2):146-154. 28. Hargreaves KM, Diogenes A, Teixeira FB. Treatment options: biological basis of regenerative endodontic procedures. Pediatr Dent. 2013; 35(2):129-140. 29. Lovelace TW, Henry MA, Hargreaves KM, Diogenes A. Evaluation of the delivery of mesenchymal stem cells into the root canal space of necrotic immature teeth after clinical regenerative endodontic procedure. J Endod. 2011;37(2):133-138. 30. Harrison JW, Hand RE. The effect of dilution and organic matter on the anti-bacterial property of 5.25% sodium hypochlorite. J Endod. 1981; 7(3):128-132. 31. Zhao S, Sloan AJ, Murray PE, Lumley PJ, Smith AJ. Ultrastructural localisation of TGF-beta exposure in dentine by chemical treatment. Histochem J. 2000;32(8):489-494. 32. Ring KC, Murray PE, Namerow KN, Kuttler S, Garcia-Godoy F. The comparison of the effect of endodontic irrigation on cell adherence to root canal dentin. J Endod. 2008;34(12):1474-1479. 33. Trevino EG, Patwardhan AN, Henry MA, et al. Effect of irrigants on the survival of human stem cells of the apical papilla in a platelet-rich plasma scaffold in human root tips. J Endod. 2011;37(8):1109-1115. 34. Casagrande L, Demarco FF, Zhang Z, et al. Dentin-derived BMP-2 and odontoblast differentiation. J Dent Res. 2010;89(6):603-608. 35. Galler KM, D’Souza RN, Federlin M, et al. Dentin conditioning codetermines cell fate in regenerative endodontics. J Endod. 2011;37(11): 1536-1541. 36. Martin DE, De Almeida JF, Henry MA, et al. Concentrationdependent effect of sodium hypochlorite on stem cells of apical papilla survival and differentiation. J Endod. 2014;40(1):51-55. 37. Galler KM, Buchalla W, Hiller KA, et al. Influence of root canal disinfectants on growth factor release from dentin. J Endod. 2015;41(3):363-368. 38. Galler KM, Widbiller M, Buchalla W, et al. EDTA conditioning of dentine promotes adhesion, migration and differentiation of dental pulp
stem cells [published online ahead of print June 25, 2015]. Int Endod J. http://dx.doi.org/10.1111/iej.12492. 39. Begue-Kirn C, Smith AJ, Ruch JV, et al. Effects of dentin proteins, transforming growth factor beta 1 (TGF beta 1) and bone morphogenetic protein 2 (BMP2) on the differentiation of odontoblast in vitro. Int J Dev Biol. 1992;36(4):491-503. 40. Sato T, Hoshino E, Uematsu H, Noda T. In vitro antimicrobial susceptibility to combinations of drugs on bacteria from carious and endodontic lesions of human deciduous teeth. Oral Microbiol Immunol. 1993;8(3):172-176. 41. Ruparel NB, Teixeira FB, Ferraz CC, Diogenes A. Direct effect of intracanal medicaments on survival of stem cells of the apical papilla. J Endod. 2012;38(10):1372-1375. 42. Althumairy RI, Teixeira FB, Diogenes A. Effect of dentin conditioning with intracanal medicaments on survival of stem cells of apical papilla. J Endod. 2014;40(4):521-525. 43. Sabrah AH, Yassen GH, Liu WC, et al. The effect of diluted triple and double antibiotic pastes on dental pulp stem cells and established Enterococcus faecalis biofilm. Clin Oral Investig. 2015;19(8):2059-2066. 44. Sabrah AH, Yassen GH, Gregory RL. Effectiveness of antibiotic medicaments against biofilm formation of Enterococcus faecalis and Porphyromonas gingivalis. J Endod. 2013;39(11):1385-1389. 45. Sabrah AH, Yassen GH, Spolnik KJ, et al. Evaluation of residual antibacterial effect of human radicular dentin treated with triple and double antibiotic pastes. J Endod. 2015;41(7):1081-1084. 46. Tagelsir A, Yassen GH, Gomez GF, Gregory RL. Effect of antimicrobials used in regenerative endodontic procedures on 3-week-old Enterococcus faecalis biofilm. J Endod. 2016;42(2):258-262. 47. Albuquerque MT, Valera MC, Nakashima M, Nor JE, Bottino MC. Tissue-engineering-based strategies for regenerative endodontics. J Dent Res. 2014;93(12):1222-1231. 48. Sachdeva GS, Sachdeva LT, Goel M, Bala S. Regenerative endodontic treatment of an immature tooth with a necrotic pulp and apical periodontitis using platelet-rich plasma (PRP) and mineral trioxide aggregate (MTA): a case report. Int Endod J. 2015;48(9):902-910. 49. Torabinejad M, Turman M. Revitalization of tooth with necrotic pulp and open apex by using platelet-rich plasma: a case report. J Endod. 2011; 37(2):265-268. 50. Shivashankar VY, Johns DA, Vidyanath S, Kumar MR. Platelet rich fibrin in the revitalization of tooth with necrotic pulp and open apex. J Conserv Dent. 2012;15(4):395-398. 51. 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(2):192-198. 52. Nakashima M, Iohara K. Mobilized dental pulp stem cells for pulp regeneration: initiation of clinical trial. J Endod. 2014;40(4 suppl):S26-S32. 53. Bender IB, Seltzer S, Soltanoff W. Endodontic success: a reappraisal of criteria. 1. Oral Surg Oral Med Oral Pathol. 1966;22(6):780-789. 54. Huang GT. A paradigm shift in endodontic management of immature teeth: conservation of stem cells for regeneration. J Dent. 2008;36(6): 379-386. 55. Murray PE, Garcia-Godoy F, Hargreaves KM. Regenerative endodontics: a review of current status and a call for action. J Endod. 2007; 33(4):377-390. 56. Barry MJ, Edgman-Levitan S. Shared decision making: pinnacle of patient-centered care. N Engl J Med. 2012;366(9):780-781. 57. Reit C. Decision strategies in endodontics: on the design of a recall program. Endod Dent Traumatol. 1987;3(5):233-239. 58. 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(8): 1063-1070. 59. Op Heij DG, Opdebeeck H, van Steenberghe D, Quirynen M. Age as compromising factor for implant insertion. Periodontol 2000. 2003;33: 172-184. 60. Lazarski MP, Walker WA 3rd, Flores CM, Schindler WG, Hargreaves KM. Epidemiological evaluation of the outcomes of nonsurgical root canal treatment in a large cohort of insured dental patients. J Endod. 2001;27(12):791-796. 61. Salehrabi R, Rotstein I. Endodontic treatment outcomes in a large patient population in the USA: an epidemiological study. J Endod. 2004; 30(12):846-850.
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62. Reynolds K, Johnson JD, Cohenca N. Pulp revascularization of necrotic bilateral bicuspids using a modified novel technique to eliminate potential coronal discolouration: a case report. Int Endod J. 2009;42(1):84-92. 63. Kim JH, Kim Y, Shin SJ, Park JW, Jung IY. Tooth discoloration of immature permanent incisor associated with triple antibiotic therapy: a case report. J Endod. 2010;36(6):1086-1091. 64. Petrino JA, Boda KK, Shambarger S, Bowles WR, McClanahan SB. Challenges in regenerative endodontics: a case series. J Endod. 2010;36(3): 536-541. 65. Ioannidis K, Mistakidis I, Beltes P, Karagiannis V. Spectrophotometric analysis of coronal discolouration induced by grey and white MTA. Int Endod J. 2013;46(2):137-144. 66. Camilleri J. Staining potential of Neo MTA Plus, MTA Plus, and Biodentine used for pulpotomy procedures. J Endod. 2015;41(7):1139-1145. 67. Kohli MR, Yamaguchi M, Setzer FC, Karabucak B. Spectrophotometric analysis of coronal tooth discoloration induced by various bioceramic cements and other endodontic materials. J Endod. 2015;41(11): 1862-1866. 68. Krastl G, Allgayer N, Lenherr P, et al. Tooth discoloration induced by endodontic materials: a literature review. Dent Traumatol. 2013;29(1):2-7. 69. Lenherr P, Allgayer N, Weiger R, et al. Tooth discoloration induced by endodontic materials: a laboratory study. Int Endod J. 2012;45(10): 942-949. 70. Chen I, Karabucak B, Wang C, et al. Healing after root-end microsurgery by using mineral trioxide aggregate and a new calcium silicatebased bioceramic material as root-end filling materials in dogs. J Endod. 2015;41(3):389-399. 71. Luo Z, Kohli MR, Yu Q, et al. Biodentine induces human dental pulp stem cell differentiation through mitogen-activated protein kinase and calcium-/calmodulin-dependent protein kinase II pathways. J Endod. 2014; 40(7):937-942. 72. Luo Z, Li D, Kohli MR, et al. Effect of Biodentine on the proliferation, migration and adhesion of human dental pulp stem cells. J Dent. 2014; 42(4):490-497. 73. Zanini M, Sautier JM, Berdal A, Simon S. Biodentine induces immortalized murine pulp cell differentiation into odontoblast-like cells and stimulates biomineralization. J Endod. 2012;38(9):1220-1226. 74. Chen MY, Chen KL, Chen CA, et al. Responses of immature permanent teeth with infected necrotic pulp tissue and apical periodontitis/ abscess to revascularization procedures. Int Endod J. 2012;45(3):294-305.
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75. Chueh LH, Ho YC, Kuo TC, et al. Regenerative endodontic treatment for necrotic immature permanent teeth. J Endod. 2009;35(2):160-164. 76. Flake NM, Gibbs JL, Diogenes A, Hargreaves KM, Khan AA. A standardized novel method to measure radiographic root changes after endodontic therapy in immature teeth. J Endod. 2014;40(1):46-50. 77. Kahler B, Mistry S, Moule A, et al. Revascularization outcomes: a prospective analysis of 16 consecutive cases. J Endod. 2014;40(3):333-338. 78. Weisleder R, Yamauchi S, Caplan DJ, Trope M, Teixeira FB. The validity of pulp testing: a clinical study. JADA. 2009;140(8):1013-1017. 79. Diogenes A, Ferraz CC, Akopian AN, Henry MA, Hargreaves KM. LPS sensitizes TRPV1 via activation of TLR4 in trigeminal sensory neurons. J Dent Res. 2011;90(6):759-764. 80. Ferraz CC, Henry MA, Hargreaves KM, Diogenes A. Lipopolysaccharide from Porphyromonas gingivalis sensitizes capsaicin-sensitive nociceptors. J Endod. 2011;37(1):45-48. 81. Byers MR, Taylor PE. Effect of sensory denervation on the response of rat molar pulp to exposure injury. J Dent Res. 1993;72(3):613-618. 82. Kubota K, Yonaga T, Hosaka K, et al. Experimental morphological studies on the functional role of the pulpal nerves in dentinogenesis. Anat Anz. 1985;158(4):323-336. 83. de Almeida JF, Chen P, Henry MA, Diogenes A. Stem cells of the apical papilla regulate trigeminal neurite outgrowth and targeting through a BDNF-dependent mechanism. Tissue Eng Part A. 2014;20(23-24):30893100. 84. Becerra P, Ricucci D, Loghin S, Gibbs JL, Lin LM. Histologic study of a human immature permanent premolar with chronic apical abscess after revascularization/revitalization. J Endod. 2014;40(1):133-139. 85. Shimizu E, Ricucci D, Albert J, et al. Clinical, radiographic, and histological observation of a human immature permanent tooth with chronic apical abscess after revitalization treatment. J Endod. 2013;39(8): 1078-1083. 86. Martin G, Ricucci D, Gibbs JL, Lin LM. Histological findings of revascularized/revitalized immature permanent molar with apical periodontitis using platelet-rich plasma. J Endod. 2013;39(1):138-144. 87. Simon SR, Tomson PL, Berdal A. Regenerative endodontics: regeneration or repair? J Endod. 2014;40(4 suppl):S70-S75. 88. Yun MH, Gates PB, Brockes JP. Sustained ERK activation underlies reprogramming in regeneration-competent salamander cells and distinguishes them from their mammalian counterparts. Stem Cell Reports. 2014; 3(1):15-23.
ENDODONTICS
Regenerative endodontics A way forward Anibal Diogenes, DDS, MS, PhD; Nikita B. Ruparel, DDS, MS, PhD; Yoav Shiloah, DDS, MBA; Kenneth M. Hargreaves, DDS, PhD
I
mmature teeth are at risk of developing pulpal necrosis due to trauma, caries, and anatomic variations such as dens evaginatus and dens invaginatus.1-4 Dental trauma occurs with an incidence that varies from 2.6% to 35% in patients undergoing cranioskeletal development.5-7 Up to one-half of these traumatized teeth may undergo pulpal necrosis, but only 8.5% will exhibit signs and symptoms of disease.8 Dental anomalies also represent a common etiology leading to pulpal necrosis in immature permanent teeth.4 Dens evaginatus and dens invaginatus are the most common anomalies associated with this clinical manifestation.4 Full radicular maturation occurs up to 3 years after the eruption of a tooth in the oral cavity,9 and the loss of pulp vitality during this period arrests further root development. These teeth traditionally have been treated with apexification procedures by using either long-term calcium hydroxide treatment10,11 or immediate placement of a mineral trioxide aggregate (MTA) apical plug.12 Although these treatments often result in the resolution of signs and symptoms of disease, they provide little to no benefit in restoring normal pulpal defenses and nociception and, more importantly, continued root development.13 Thus, immature teeth remain with thin fragile dentinal walls, increasing susceptibility to fractures and lower survival rates.14,15 Tooth loss in patients still undergoing cranioskeletal development has devastating consequences that include altered maxillary and mandibular bone development; Copyright ª 2016 American Dental Association. All rights reserved.
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ABSTRACT Background and Overview. Immature teeth are susceptible to infections due to trauma, anatomic anomalies, and caries. Traditional endodontic therapies for immature teeth, such as apexification procedures, promote resolution of the disease and prevent future infections. However, these procedures fail to promote continued root development, leaving teeth susceptible to fractures. Regenerative endodontic procedures (REPs) have evolved in the past decade, being incorporated into endodontic practice and becoming a viable treatment alternative for immature teeth. The authors have summarized the status of regenerative endodontics on the basis of the available published studies and provide insight into the different levels of clinical outcomes expected from these procedures. Conclusions. Substantial advances in regenerative endodontics are allowing a better understanding of a multitude of factors that govern stem cell–mediated regeneration and repair of the damaged pulp-dentin complex. REPs promote healing of apical periodontitis, continued radiographic root development, and, in certain cases, vitality responses. Despite the clinical success of these procedures, they appear to promote a guided endodontic repair process rather than a true regeneration of physiological-like tissue. Practical Implications. Immature teeth with pulpal necrosis with otherwise poor prognosis can be treated with REPs. These procedures do not preclude the possibility of apexification procedures if attempts are unsuccessful. Therefore, REPs may be considered first treatment options for immature teeth with pulpal necrosis. Key Words. Guided tissue regeneration; revascularization; endodontic therapy; stem cells; outcome assessment; regenerative endodontics. JADA 2016:147(5):372-380 http://dx.doi.org/10.1016/j.adaj.2016.01.009
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interference with pronunciation, breathing, and mastication; and a severe detrimental psychosocial effect.16,17 Because implants are contraindicated in growing young patients because of continued craniofacial development, tooth replacement is not possible until the age of maturity (usually older than 18 years). As an alternative technique, regenerative endodontic procedures (REPs) are intended to promote tooth survival and the function of previously thought hopeless teeth. In this review, we will focus on discussing the historical background and the present and future directions of these clinical procedures. HISTORY
The emergence of regenerative endodontics was catalyzed in the early 2000s with the publication of 2 remarkable case reports.18,19 However, this field has its roots in the seminal work by Dr. Ostby in the early 1960s that aimed to evaluate the role of the apical blood clot in the healing of apical periodontitis and pulp repair.20,21 Regenerative endodontics also relied on contributions from important studies in dental trauma, which provided evidence that the dental pulp in immature teeth often remains vital despite substantial traumatic injuries such as intrusions and avulsions.22-24 This remarkable regenerative potential is highlighted in cases of replantation of avulsed immature teeth with evidence of reestablishment of vitality responses and lack of signs and symptoms of disease.23,25,26 In these cases, clinical success relies heavily on the reestablishment of a blood supply to the ischemic but uninfected dental pulp tissue, followed by reinnervation from sensory axons likely recruited from the apical region. This healing process of a previously necrotic dental pulp in traumatic injuries is crucial for reattaining normal pulpal function after trauma. The term revascularization has emerged from these observations in dental traumatology despite the different application and goals.4 Pulpal revitalization is another commonly used term in the scientific literature. However, for the sake of this review, we simply will address these procedures collectively as REPs. Results from numerous published reports demonstrate that these procedures often lead to resolution of apical periodontitis and signs and symptoms of inflammation, radiographic evidence of continued root development and apical narrowing, and restoration of vitality responses.4,27,28 These published cases establish that REPs address the unmet need of promoting normal physiological development and responses in immature teeth with pulpal necrosis. In most REPs, clinicians rely on creating bleeding from the apical region that passively fills the canal space and forms a blood clot. However, it was not until 2011 that investigators in a clinical study demonstrated that the influx of apical blood into disinfected root canals was accompanied by a clinically significant transfer of mesenchymal stem cells into the root canal system.29
This was an important demonstration in the field of regenerative endodontics because it established that these procedures were, in fact, stem cell–based procedures. The realization that autogenous stem cells can be delivered clinically into root canals without the need for ex vivo stem cell expansion propelled researchers and clinicians to consider principles of tissue engineering to improve treatment protocols and to develop the next generation of procedures. TRANSLATIONAL STUDIES
The balance between adequate disinfection and stem cell survival, proliferation, and differentiation represents an important initial barrier to overcome. The resolution of infection and the disease process remains the primary goal of any endodontic procedure. However, it has become obvious that the philosophy of disinfecting the root canal by using methods typically advocated in traditional root canal therapy had to be modified to attain a biocompatible disinfection strategy. To maintain the physical integrity of the already thin dentinal walls of immature teeth, investigators have advocated chemical debridement as the primary means of disinfection. To this end, investigators in many translational studies have focused efforts on establishing the biological basis for clinical protocols that could achieve both disinfection and optimum regenerative potential.4,27 For example, sodium hypochlorite remains the most used disinfectant in endodontics.30 However, its use at full concentration of 6% denatures crucial growth factors in dentin31 and results in residual detrimental effects greatly affecting stem cell attachment, survival, and differentiation potential.32-35 These deleterious effects largely can be avoided with the use of a 1.5% concentration of sodium hypochlorite followed by 17% ethylenediaminetetraacetic acid.35,36 Furthermore, results from elegant studies have demonstrated that ethylenediaminetetraacetic acid promotes the release of growth factors embedded in dentin, including vascular endothelial growth factor and transforming growth factor beta-1 among others that are known to participate actively in regenerative processes such as angiogenesis and stem cell proliferation, migration, and differentiation, respectively.37-39 Along these lines, it was obvious that the effects of intracanal medicaments be tested on the survival and maintenance of stem cells. A triple antibiotic formulation consisting of ciprofloxacin, metronidazole, and minocycline first was tested in vitro against bacteria isolated from carious lesions and from endodontic infections in primary teeth.40 The investigators found that no bacteria could be recovered after treatment with 100 ABBREVIATION KEY. MTA: Mineral trioxide aggregate. NSRCT: Nonsurgical root canal therapy. REP: Regenerative endodontic procedure.
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micrograms per milliliter of each antibiotic (300 mg/mL of mixture).40 Next, investigators carried out studies to test the efficacy of triple antibiotic paste in eradicating bacteria from infected dentin. They found that there were no recovered cultivable bacteria at 48 hours, and they found evidence of significant drug penetration into dentin.40 In REPs, the use of the triple antibiotic paste and its modified versions, such as exclusion of minocycline, known as double antibiotic paste, or the addition of cefaclor, were introduced by initial published case reports.19 The drugs were mixed with water, saline, or propylene glycol until a thick creamy mixture was formed. In these case reports, there was no attempt to deliver a specific concentration of the drugs deliberately. Instead, the investigators mixed the drugs until they achieved a certain physical consistency (approximately 1 gram per milliliter) that clinicians deemed suitable. At this concentration, however, the triple antibiotic paste appeared to have long-lasting deleterious effects on stem cell survival through both direct and indirect mechanisms.41,42 This undesirable effect can be avoided greatly by the use calcium hydroxide as an intracanal medicament41,42 or the use of these pastes in lower concentrations (less than 1 milligram/mL); these lower concentrations retain the desirable antibacterial or antibiofilm effect.43-46 Therefore, there have been substantial advances in understanding how to adapt disinfection protocols to the new reality of stem cell–based therapies. In addition to studies on biocompatible disinfection, many other frontiers in regenerative endodontic research are being investigated. These involve tissue engineering strategies that include the evaluation of suitable scaffolds, growth factors, and harvested stem cells to be used in pulp-dentin regeneration.47 Many of the advances from translational research have been evaluated in a clinical setting, including the use of platelet-rich plasma,48,49 platelet fibrin,50 and a gelatin hydrogel51 as scaffolds in patients. In addition, a groundbreaking clinical trial is being conducted in Japan.52 This trial involves harvesting stem cells from a donor site, followed by ex vivo expansion, sorting, and maintenance of these cells in a good manufacturing practice facility, followed by autogenic transplantation into a recipient tooth to promote the regeneration of the once lost functional pulp-dentin complex. These elegant studies highlight the status and sophistication of REPs. CLINICAL OUTCOMES OF REPs
Resolution of infection and the signs and symptoms of inflammation leading to apical periodontitis and restoration of lost function remains the primary goal of any endodontic therapy. Traditional endodontic therapies (nonregenerative procedures) achieve this goal by promoting disinfection followed by a fluid-tight or bacteriatight seal of the root canal system and delivery of a high-quality coronal restoration. The clinical outcome
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of endodontic therapies traditionally has been evaluated on the basis of lack of signs and symptoms of disease, such as pain, swelling, or sinus tracts, and radiographic criteria of healing.10,11,53 REPs, on the other hand, rely on comparably efficient chemical disinfection followed by the stem cell–mediated growth of a reparative tissue that promotes continued root development and reestablishment of pulpal functions such as nociception and immune competency. This paradigm shift in treatment approach also requires a shift in assessment of clinical outcomes.54,55 Last, the success of these procedures must be evaluated by 3 stakeholders: patients and their legal guardians, clinicians, and researchers. Patient-based outcomes. The needs and desires of patients might not always align with the preset criteria of success laid out by clinicians and researchers. There is an ever-increasing need to focus on outcomes that are meaningful to patients because they are the primary stakeholders in their own health and must participate in treatment decisions.56 However, even in the context of long-established therapies such as conventional nonsurgical root canal therapy (NSRCT), there is often a disconnect with patient-centered outcomes. For example, from a patient perspective, the resolution of pain may be the most important criterion for successful NSRCT. However, for most clinicians, success also means healing of a periapical lesion, if present.57 Other patient-centered outcomes may include tooth survival in the mouth and acceptable esthetics. These patient-centered outcomes are also relevant for REPs (Figure 1). Investigators used standardized treatment protocols in a study to compare REPs directly with apexification procedures by using the long-term application of calcium hydroxide or by using MTA as an apical plug.15 REPs and MTA plug apexification procedures were equally effective in resolving signs and symptoms of disease (that is, pain, swelling, or sinus tract) in 100% and 95% of all patients, respectively, whereas apexification procedures with the use of calcium hydroxide were significantly less effective (77%). Investigators in another retrospective study did not include standardization of treatment protocols and found REPs to promote healing in 79% of patients treated, whereas apexification procedures promoted healing in 100% of the patients; however, this difference was not significant.58 In a prospective study, investigators found that both MTA apexification and REPs promoted healing in 100% of patients.51 Further evidence of successful outcomes can be found in case series and retrospective patient cohort studies. Despite significant variations in etiology, inclusion and exclusion criteria, and clinical protocols, results from these studies51,58 demonstrate that both procedures can resolve the signs and symptoms of disease in approximately 90% of patients successfully, without marked difference in resolution of symptoms between REPs and apexification procedures.
SPECIALTY UPDATE
Survival of the tooth is another important patient-centered Scientist-Based outcome. An ideal treatOutcomes ment, from the patient’s Histologic evidence perspective, is one that of complete prolongs the functional regeneration life of an asymptomatic tooth. The survival of Clinician-Based a permanent tooth is • Radiographic healing Outcomes particularly important in • Radiographic root development young patients undergo• Positive vitality responses ing continued cranioskeletal development • Resolution of disease Patient-Based because implants are (absence of swelling, drainage, and pain) Outcomes contraindicated at this • Tooth survival and function 59 • Tooth esthetics stage. Although results from various studies demonstrate the functional survival of mature Figure 1. Trilevel outcome assessment pyramid. Regenerative endodontic procedures must be assessed and evaluated in a systematic manner, acknowledging that there are different levels of possible outcomes that carry teeth treated with conmeanings for patients, clinicians, and scientists. The patient-centered outcomes represent the base of ventional NSRCT,60,61 far different this pyramid, symbolizing the fundamental importance of achieving healing, reestablishment of function, and less is known about the patient satisfaction. effect of either apexification or REPs on tooth survival. REPs promoted 100% survival in a study period Clinician-based outcomes. As mentioned, clinicianof 18 months, which was comparable with the 95% survival based outcomes involve radiographic signs of healing; promoted by MTA apexifications, both being superior however, unique to REPs, clinicians also evaluate for to the 77% survival achieved in calcium hydroxide– continued root development by means of radiographic mediated apexification procedures.15 Another retrospecexamination and responses to pulp sensitivity. Initially, tive study in which the investigators directly compared investigators in case reports and case series relied the survival of REPs and apexification (MTA apical plug) on reporting the nonquantitative assessment of root procedures failed to demonstrate differences between the development, often using a dichotomous dependent treatment alternatives.58 Nonetheless, investigators have measure (that is, yes or no),74 in addition to qualitative shown that REPs arrest the disease process and promote descriptions of radiographic findings.75 In 2009, study retention of teeth with otherwise poor prognosis.4,27 investigators published a new methodology to quanFactors affecting the long-term survival of these teeth titate changes in root width and length13 and further require further investigation by means of randomized refined and revised it in a later study.76 It allows for the digital alignment of nonstandardized radioclinical trials to compare REPs with MTA apexification as the criterion standard during long follow-ups. graphs obtained at different recall visit times in paThere have been esthetic concerns with REPs that result tients undergoing cranioskeletal growth. It allows the unbiased measurement of changes in root development in coronal staining in certain cases.62-64 This coronal staining has been identified as being the result of triple detected by means of conventional 2-dimensional antibiotic paste in which minocycline stains the dentin, periapical radiographs. With use of this methodology, prior intracoronal bleeding, or the coronal placement of teeth treated with REPs showed a significantly greater MTA.63,65-69 This expected staining can be minimized by percentage of increase in root length and width. occluding the dentinal tubules with a dental adhesive if This finding later was confirmed in another retroeither triple antibiotic paste or MTA is used.62 Alternaspective study in which the investigators reported tively, other bioceramic materials, such as Biodentine gains in root length (14.9%) compared with teeth (Septodont), Bioaggregate (Pearson Dental), and Endotreated by means of either MTA apexification (6.1%) Sequence Bioceramic Root Repair Material putty (Brassor calcium hydroxide apexification (0.4%).15 They also reported that the REPs produced a significantly eler), are biocompatible and are osteoinductive when exposed to mesenchymal stem cells70-73 while showing greater percentage of increases in root width (28.2%) minimal to no color change.67 Thus, tooth discoloration as compared with that in teeth treated by means of either an adverse event of REPs largely can be avoided if nonMTA apexification (0.0%) or calcium hydroxide staining medications and restorative materials are used. apexification (1.5%).
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Figure 2. A. A 12-year-old boy sought care for intraoral buccal swelling and sinus tract associated with tooth no. 29. B. Clinical examination revealed a talon cusp (dens evaginatus) on the occlusal aspect of the tooth. Clinical findings led to the diagnosis of pulpal necrosis with chronic apical abscess. C. A preoperative radiograph revealed a large apical radiolucency traced with a gutta-percha point through a sulcular sinus tract, an immature root with a large pulp canal space, and thin dentinal walls. The tooth was treated by means of a regenerative endodontic procedure by using a mixture of ciprofloxacin, metronidazole, and minocycline (triple antibiotic paste) as an intracanal mixture for 40 days. Incision and drainage also was performed at this visit. At the second visit, after removal of the intracanal medicament and evoked bleeding, a 3-millimeter piece of absorbable wound dressing (CollaPlug, Integra LifeSciences) was placed at the level of the cementoenamel junction and restored with a 3-mm layer of mineral trioxide aggregate and light-cured glass ionomer (Fuji II LC, GC America). At the 4-year recall visit, the patient was asymptomatic and demonstrated positive response to electric pulp testing for tooth no. 29. Radiographic examination revealed complete apical closure, as well as clinically significant gain in root length and width with narrowing of the pulp canal space as seen in the periapical radiograph (D) and small-volume cone-beam computed tomography in the coronal (E) and sagittal (F) views. Photographs and radiographs courtesy of Dr. Yoav Shiloah, University of Texas Health Science Center at San Antonio, San Antonio, TX.
Further evidence of continued root development is available in a prospective randomized clinical trial in which the investigators compared 2 different protocols of REPs with MTA apexification procedures.51 In this study, REPs promoted the gain in root length (12% increase) and width (13% increase), whereas no changes were detected in the MTA apexification group.51 Investigators in other studies with similar methodologies also reported changes in radiographic root structure but failed to detect statistical significance, possibly because of limited sample size or large variability of responses.58,77 Various degrees of radiographic root development have been observed in most published studies. The factors that modulate this varied response are largely unknown because correlation with factors such as tooth type, etiology, sex, or age has not been established.15,58 Thus, although REPs predictably promote healing of apical periodontitis in more than 90% of the cases,4,15,51,58,74 radiographic root development is far less predictable. Figures 2 and 3 provide examples of varied root development. Figure 2 presents a case that shows excellent gain in root length and root width along with complete apical closure, whereas Figure 3 presents a case that shows
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only closure of the apex with no gain in root length or width. Although the lack of radiographic root development in absence of disease does not constitute a treatment failure, future studies with appropriate sample sizes are needed to find prognostic factors for root development. Positive responses to pulpal sensitivity tests are interpreted as evidence favoring the presence of a vital pulp.78 Clinicians routinely use these tests as adjuvants in the diagnosis of pulp status. Investigators have reported positive sensitivity responses to either cold or electric pulp tests in approximately 60% of all published cases.4 Nociception is an important surveillance mechanism that protects tissues against actual or potential damage. The reestablishment of nociception in these teeth must be seen favorably because it suggests the presence of a vascularized tissue with normal physiological responses. Furthermore, primary afferent neurons in the dental pulp are equipped to sense microbial antigens, participating in the inflammatory process evoked by invading microorganisms.79,80 They modulate the immune reaction by the release of neuropeptides with vasoactive properties that increase immune cell recruitment and vascularity, localizing areas of insult to microabcesses
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Figure 3. A 10-year-old boy had trauma of tooth no. 8 with an uncomplicated crown fracture and moderate intrusive luxation. The tooth had pulpal necrosis and symptomatic apical periodontitis approximately 6 months after injury. A. A preoperative periapical radiograph revealed a moderately immature root with an apical radiolucency and inflammatory root resorption. The tooth was treated with a regenerative endodontic procedure by using a mixture of ciprofloxacin and metronidazole (double antibiotic) as an intracanal medicament for 1 month. At the second visit, after medicament removal and evoked bleeding, the access was restored with a 3-millimeter layer of mineral trioxide aggregate placed at the cementoenamel junction over the blood clot, followed by a base layer of glass ionomer and a composite restoration. At the 3-year recall visit, the patient was asymptomatic, and the tooth responded to electric pulp testing with evident resolution of the apical radiolucency, arrestment of the resorptive process, and thickening of the apical one-third with apical closure detected on a periapical radiograph (B). At the 6-year recall visit, the tooth remained asymptomatic with positive responses to electric pulp testing and evidence of apical closure seen on the periapical radiograph (C) and small-volume cone-beam computed tomography in the coronal (D), axial (E), and sagittal (F) views. Radiographs and computed tomographic scans courtesy of Dr. Tyler Lovelace, University of Texas Health Science Center at San Antonio, San Antonio, TX.
within the dental pulp.81 Also, trigeminal neurons increase odontoblastic differentiation and dentinogenesis.82 Lastly, trigeminal neurons harbor an important subpopulation of mesenchymal stem cells along their axonal projections that participate in tooth formation. The role of the perineuronal mesenchymal stem cells in postdevelopment repair or regeneration remains largely
unknown. Mesenchymal stem cells delivered into root canals in REPs may be capable of releasing potent soluble factors that attract nearby neurons to reinnervate the newly formed tissue, possibly participating in the reparative process.83 Clinicians rely on the preestablished criteria of radiographic continued root development and vitality
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responses to evaluate clinical outcomes further. Although these dependent measures add valuable information that can be used to determine whether further interventions are needed, their meaning to patients and their guardians is not entirely clear because their expectation is that the tooth is pain free and functional. The clinician-centered criteria, which often are used to evaluate REPs, are not directly applicable to apexification procedures because root development and vitality responses are not expected. Scientist-based outcomes. Outcomes that are not related directly to the clinical manifestation of the treated tooth but that address a specific question that requires scientific methodology in its evaluation should be categorized as scientist-based outcomes. These are equally important outcomes because they tend to, through science, move the field forward leading to substantial future advances in care. Histologic evaluation of teeth that were treated previously with REPs but later extracted because of recurring trauma and fractures suggests that the newly formed tissue does not resemble the lost dental pulp.84-86 This is the best example of a scientist-based outcome because it has strong scientific merit, but it does not necessarily interfere with consideration of the rate of healing and continued root development seen in cases treated with REPs. There is considerable debate about the use of the term regeneration because existing scientific evidence from histologic studies suggests that the procedures allow for repair instead.87 This is both a semantic and a biological debate; however, it can be argued that true regeneration as defined by the complete recapitulation of the lost tissue with all its constituents, morphology, function, and molecular markers typically is observed only in primitive invertebrates such as hydras and in some amphibians such as salamanders.88 To date, even in sophisticated animal models, the tissues formed closely resemble the native dental pulp, but true odontoblasts are missing; instead, mineralizing cells called odontoblast-like cells are formed. Thus, the definition of regeneration hinges on the methodology used for its assessment and appears to fall short when more advanced molecular techniques are used. Perhaps a more conservative term to be used to describe this procedure is guided endodontic repair (GER). A repaired tissue that promotes the resolution of disease and reestablishment of some or all of the original tissue functions should be a desirable goal. The dental pulp capacity of repair seen in direct and indirect pulp capping procedures is absent once the pulp completely succumbs to infection or trauma. In these cases, clinicians using the knowledge in regenerative endodontics are able to guide endodontic repair, attaining the desirable patient- and clinician-centered outcomes. Clinicians and scientists working in tandem striving to achieve the scientist-based outcome of true regeneration are creating tangible advances in endodontic care, departing from a materials-based to a biologically based approach.
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CONCLUSIONS
Substantial advances in regenerative endodontics are allowing for a better understanding of a multitude of factors that govern stem cell–mediated regeneration and repair of the damaged pulp-dentin complex. REPs are valuable adjuvants to the treatment and retention of immature teeth with pulp necrosis with an otherwise poor prognosis. Despite the clinical success of these procedures, they appear to promote a GER process rather than a true regeneration of physiological-like tissue. For greater regenerative potential to be met, investigators must perform much more research and development. Translational research is crucial in making these procedures more predictable while pushing the boundaries of future procedures that are likely to involve the direct clinical manipulation of scaffolds, growth factors, and stem cells. n Dr. Diogenes is an associate professor, Department of Endodontics, University of Texas Health Science Center at San Antonio, 7703 Floyd Curl Dr., San Antonio, TX 78229, e-mail [email protected]. Address correspondence to Dr. Diogenes. Dr. Ruparel is an assistant professor, Department of Endodontics, University of Texas Health Science Center at San Antonio, San Antonio, TX. Dr. Shiloah is an adjunct professor, Department of Endodontics, University of Texas Health Science Center at San Antonio, San Antonio, TX. Dr. Hargreaves is a professor and the chair, Department of Endodontics, University of Texas Health Science Center at San Antonio, San Antonio, TX. Disclosure. None of the authors reported any disclosures. Endodontics is published in collaboration with the American Association of Endodontists. 1. Cortes MI, Marcenes W, Sheiham A. Prevalence and correlates of traumatic injuries to the permanent teeth of schoolchildren aged 9-14 years in Belo Horizonte, Brazil. Dent Traumatol. 2001;17(1):22-26. 2. Oehlers FA, Lee KW, Lee EC. Dens evaginatus (evaginated odontome): its structure and responses to external stimuli. Dent Pract Dent Rec. 1967; 17(7):239-244. 3. Levitan ME, Himel VT. Dens evaginatus: literature review, pathophysiology, and comprehensive treatment regimen. J Endod. 2006;32(1):1-9. 4. Diogenes A, Henry MA, Teixeira FB, Hargreaves KM. An update on clinical regenerative endodontics. Endod Topics. 2013;28(1):2-23. 5. Andreasen JO, Ravn JJ. Epidemiology of traumatic dental injuries to primary and permanent teeth in a Danish population sample. Int J Oral Surg. 1972;1(5):235-239. 6. Petti S, Tarsitani G. Traumatic injuries to anterior teeth in Italian schoolchildren: prevalence and risk factors. Endod Dent Traumatol. 1996; 12(6):294-297. 7. Forsberg CM, Tedestam G. Traumatic injuries to teeth in Swedish children living in an urban area. Swed Dent J. 1990;14(3):115-122. 8. Robertson A, Andreasen FM, Bergenholtz G, Andreasen JO, Noren JG. Incidence of pulp necrosis subsequent to pulp canal obliteration from trauma of permanent incisors. J Endod. 1996;22(10):557-560. 9. Moorrees CF, Fanning EA, Hunt EE Jr. Age variation of formation stages for ten permanent teeth. J Dent Res. 1963;42:1490-1502. 10. Strindberg LZ. The dependence of the results of pulp therapy on certain factors: an analytic study based on radiographic and clinical followup examinations. Acta Odontol Scand Suppl. 1956;(14):1-175. 11. Bender IB, Seltzer S, Soltanoff W. Endodontic success: a reappraisal of criteria. II. Oral Surg Oral Med Oral Pathol. 1966;22(6):790-802. 12. Witherspoon DE, Ham K. One-visit apexification: technique for inducing root-end barrier formation in apical closures. Pract Proced Aesthet Dent. 2001;13(6):455-460. 13. Bose R, Nummikoski P, Hargreaves K. A retrospective evaluation of radiographic outcomes in immature teeth with necrotic root canal systems
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62. Reynolds K, Johnson JD, Cohenca N. Pulp revascularization of necrotic bilateral bicuspids using a modified novel technique to eliminate potential coronal discolouration: a case report. Int Endod J. 2009;42(1):84-92. 63. Kim JH, Kim Y, Shin SJ, Park JW, Jung IY. Tooth discoloration of immature permanent incisor associated with triple antibiotic therapy: a case report. J Endod. 2010;36(6):1086-1091. 64. Petrino JA, Boda KK, Shambarger S, Bowles WR, McClanahan SB. Challenges in regenerative endodontics: a case series. J Endod. 2010;36(3): 536-541. 65. Ioannidis K, Mistakidis I, Beltes P, Karagiannis V. Spectrophotometric analysis of coronal discolouration induced by grey and white MTA. Int Endod J. 2013;46(2):137-144. 66. Camilleri J. Staining potential of Neo MTA Plus, MTA Plus, and Biodentine used for pulpotomy procedures. J Endod. 2015;41(7):1139-1145. 67. Kohli MR, Yamaguchi M, Setzer FC, Karabucak B. Spectrophotometric analysis of coronal tooth discoloration induced by various bioceramic cements and other endodontic materials. J Endod. 2015;41(11): 1862-1866. 68. Krastl G, Allgayer N, Lenherr P, et al. Tooth discoloration induced by endodontic materials: a literature review. Dent Traumatol. 2013;29(1):2-7. 69. Lenherr P, Allgayer N, Weiger R, et al. Tooth discoloration induced by endodontic materials: a laboratory study. Int Endod J. 2012;45(10): 942-949. 70. Chen I, Karabucak B, Wang C, et al. Healing after root-end microsurgery by using mineral trioxide aggregate and a new calcium silicatebased bioceramic material as root-end filling materials in dogs. J Endod. 2015;41(3):389-399. 71. Luo Z, Kohli MR, Yu Q, et al. Biodentine induces human dental pulp stem cell differentiation through mitogen-activated protein kinase and calcium-/calmodulin-dependent protein kinase II pathways. J Endod. 2014; 40(7):937-942. 72. Luo Z, Li D, Kohli MR, et al. Effect of Biodentine on the proliferation, migration and adhesion of human dental pulp stem cells. J Dent. 2014; 42(4):490-497. 73. Zanini M, Sautier JM, Berdal A, Simon S. Biodentine induces immortalized murine pulp cell differentiation into odontoblast-like cells and stimulates biomineralization. J Endod. 2012;38(9):1220-1226. 74. Chen MY, Chen KL, Chen CA, et al. Responses of immature permanent teeth with infected necrotic pulp tissue and apical periodontitis/ abscess to revascularization procedures. Int Endod J. 2012;45(3):294-305.
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75. Chueh LH, Ho YC, Kuo TC, et al. Regenerative endodontic treatment for necrotic immature permanent teeth. J Endod. 2009;35(2):160-164. 76. Flake NM, Gibbs JL, Diogenes A, Hargreaves KM, Khan AA. A standardized novel method to measure radiographic root changes after endodontic therapy in immature teeth. J Endod. 2014;40(1):46-50. 77. Kahler B, Mistry S, Moule A, et al. Revascularization outcomes: a prospective analysis of 16 consecutive cases. J Endod. 2014;40(3):333-338. 78. Weisleder R, Yamauchi S, Caplan DJ, Trope M, Teixeira FB. The validity of pulp testing: a clinical study. JADA. 2009;140(8):1013-1017. 79. Diogenes A, Ferraz CC, Akopian AN, Henry MA, Hargreaves KM. LPS sensitizes TRPV1 via activation of TLR4 in trigeminal sensory neurons. J Dent Res. 2011;90(6):759-764. 80. Ferraz CC, Henry MA, Hargreaves KM, Diogenes A. Lipopolysaccharide from Porphyromonas gingivalis sensitizes capsaicin-sensitive nociceptors. J Endod. 2011;37(1):45-48. 81. Byers MR, Taylor PE. Effect of sensory denervation on the response of rat molar pulp to exposure injury. J Dent Res. 1993;72(3):613-618. 82. Kubota K, Yonaga T, Hosaka K, et al. Experimental morphological studies on the functional role of the pulpal nerves in dentinogenesis. Anat Anz. 1985;158(4):323-336. 83. de Almeida JF, Chen P, Henry MA, Diogenes A. Stem cells of the apical papilla regulate trigeminal neurite outgrowth and targeting through a BDNF-dependent mechanism. Tissue Eng Part A. 2014;20(23-24):30893100. 84. Becerra P, Ricucci D, Loghin S, Gibbs JL, Lin LM. Histologic study of a human immature permanent premolar with chronic apical abscess after revascularization/revitalization. J Endod. 2014;40(1):133-139. 85. Shimizu E, Ricucci D, Albert J, et al. Clinical, radiographic, and histological observation of a human immature permanent tooth with chronic apical abscess after revitalization treatment. J Endod. 2013;39(8): 1078-1083. 86. Martin G, Ricucci D, Gibbs JL, Lin LM. Histological findings of revascularized/revitalized immature permanent molar with apical periodontitis using platelet-rich plasma. J Endod. 2013;39(1):138-144. 87. Simon SR, Tomson PL, Berdal A. Regenerative endodontics: regeneration or repair? J Endod. 2014;40(4 suppl):S70-S75. 88. Yun MH, Gates PB, Brockes JP. Sustained ERK activation underlies reprogramming in regeneration-competent salamander cells and distinguishes them from their mammalian counterparts. Stem Cell Reports. 2014; 3(1):15-23.