Recently there has been a gradual increase in considering the REPs in prospective clinical experiments for treatment of mature permanent teeth. However, the evidence firmly supporting regeneration/revitalization for apical periodontitis in immature or mature permanent teeth remains of low-quality [12].
In the present trial, REPs were considered for the retreatment of mature teeth even though conventional root canal retreatment is a viable option for the following reasons: 1) REPs are generally less expensive than conventional retreatment because they often require fewer appointments and less complex instrumentation. Conventional retreatment involves removing existing root canal fillings, re-instrumenting the canals, and re-obturating, which can be time-consuming and costly. In contrast, REPs focus on disinfection and revitalization of the pulp-dentin complex, which may reduce the need for additional procedures such as apical surgery or extraction followed by implant placement.2) REPs simplify the process by focusing on disinfection and promoting natural tissue regeneration rather than mechanical debridement and obturation. Additionally, REPs reduce the need for complex instrumentation and minimises the risk of procedural errors [22]. 3) Improve the patient’s outcomes because REPs aim to restore the natural biology of the tooth by promoting the regeneration of pulp-like tissue, which can enhance tooth vitality and function. This contrasts with conventional retreatment, which often results in a non-vital tooth that is more prone to fracture and failure over time. REPs also preserve the natural tooth structure, which is beneficial for long-term oral health [23]. 4) REPs are less invasive and offer the potential for natural tissue regeneration.
However, the use of REPs in the retreatment of mature permanent teeth can be complicated by several factors. The complex root canal anatomy may hinder complete disinfection, increasing the risk of persistent infection and failure of the regenerative procedure. Additionally, cases with insufficient coronal structure or advanced periodontal disease may not be suitable for regenerative endodontics [24]. Nevertheless, ongoing research may expand the indications for REPs in the future.
Although the treatment of mature necrotic permanent teeth with apical periodontitis using REPs is still in its early stages, the limited available studies have shown promising outcomes. This encouraged us to explore the potential benefits of REPs in the retreatment of mature maxillary central incisors with periapical lesions. Therefore, this study hypothesized that there would be no clinical or radiographic difference in periapical lesion healing following the retreatment of mature maxillary central incisors with two REP techniques —revascularization with blood clot (BC) formation versus platelet-rich fibrin (PRF)—over a 24-month period. The other outcome was to assess the pulp responses after thermal and electrical conduction tests.
In the present study, although the difference between the BC and PRF groups was not significant, PRF may offer certain advantages, particularly in inducing bleeding, which was not always possible from the periapical region [25]. One disadvantage of BC is its relatively fragile structure, which may prevent it from completely filling the RC, potentially compromising the coronal seal [26]. Additionally, a systematic review reported deposition of hard tissue in all studies that depended on PRP and PRF scaffolds, while the mineralized tissues were reported in 80% of studies that considered BC in regeneration [27]. However, the need for specialized equipment to collect the venous blood and prepare the PRF was probably the main problem. Moreover, the PRF technique may increase treatment complexity, time and cost, and patient cooperation is necessary.
The impact of age on the regenerative capacity still being controversial. Arslan et al. [13] reported that the age of participants aged 10(-)35 years had no influence on the outcomes of REPs. In contrast, a report of expert consensus on REPs emphasized the significance of age in achieving successful regenerative outcomes [6]. Therefore, this study focused on adolescents to standardize anatomical and physiological factors, such as tooth morphology, root number (only single-rooted maxillary central incisors were included), root length and thickness, and apical foramen size and position. These factors may influence the influx of stem cells and the response to pulp sensibility tests. Additionally, the age limitation aimed to account for the age-related regenerative capacity of stem cells [28].
The biological influence of a patient’s age can be attributed to the reduced potential for revascularization, which is associated with degenerative changes in blood vessels. These changes coincide with continuous dentin apposition and an increased incidence of calcifications [29]. Additionally, the regenerative capacity of multipotent stem cells declines with age [30]. This decline is likely due to the reduced differentiation potential of mesenchymal stem cells (MSCs) over time rather than a decrease in the number of MSCs migrating into the root canal system, as this population remains relatively stable with aging [31].
The diameter of apical foramen is another factor that may influence the REPs outcome. Nevertheless, the ideal apical foramen size has not yet been established. According to the findings of Fang et al. [32], mature teeth with apical foramen diameters ranging from 0.5 to 1.0 mm had the most favourable outcomes with a clinical success rate of 95.65%. This result is consistent with the findings of the present study. Some reports indicate that REPs have higher clinical and radiographic success rates in younger patients. For example, a study found that nine out of ten maxillary incisors with apical foramen diameters between 0.5 and 1.0 mm in adolescents aged 14–18 showed clinical and radiographic success after one year of follow-up. Additionally, findings from an animal model study suggest that a minimum apical terminus diameter of 0.4 mm is required for successful PEPs [33].
The PAI method was used to track healing status by assessing changes in periapical radiolucency. PAI is a widely used, standardized, and reproducible quantitative method with high inter- and intra-examiner reliability [34, 35]. To enhance reproducibility and diagnostic accuracy, a customized radiographic stent was used for each patient, ensuring consistent angulation and minimizing variations in imaging geometry. Additionally, two calibrated examiners independently scored the radiographic images.
Despite the superiority of three-dimensional imaging techniques such as cone-beam computed tomography (CBCT), this study relied on conventional two-dimensional PAs. CBCT is not recommended for the routine radiographic diagnosis of teeth with periapical periodontitis unless there is a discrepancy between conventional radiographic findings and clinical signs and symptoms [36, 37]. Additionally, ethical concerns regarding CBCT use in children and adolescents must be considered. Growing children are significantly more sensitive to ionizing radiation than adults [38]. Therefore, the potential benefits must be carefully weighed against possible risks, and each case should be evaluated individually to justify the use of CBCT [39].
The current trial outcomes demonstrated the effectiveness of REPs in the retreatment of mature permanent incisors, with no clinically or radiographically significant differences between BC formation and PRF techniques. However, the PAI scores after retreatment with PRF significantly decreased compared to those treated with BC formation at both 3 and 6 months. This suggests that healing occurred more rapidly in teeth retreated with the PRF clot.
This accelerated healing may be attributed to the combination of intentional bleeding induced from the periapical region and the PRF scaffold. This combination likely increases the presence of stem cells and enhances the sustained release of growth factors and cytokines over time. The PRF scaffold, which contains highly concentrated platelets, provides a rich source of bioactive molecules and mesenchymal stem cells (MSCs), promoting greater stem cell proliferation and improving overall healing [40].
Given the limited availability of prior data addressing this topic, a direct comparison of the outcomes of the present trial with previous findings is challenging. Only one randomized trial has investigated the impact of regenerative endodontic procedures (REPs) using blood clot (BC) revascularization on the retreatment of mature permanent incisors with apical periodontitis in adolescents [15]. In that study, involving thirty-three permanent incisors, the clinical success rate at 6 and 12 months was 93.9%, which aligns closely with the results observed in the current study at similar follow-up intervals. However, after 6 months, the PAI median in the previous trial decreased significantly to 1.5, whereas in the present study, the PAI median decreased to 2. This discrepancy may be attributed to differences in baseline PAI medians, which were higher in the present investigation. Additionally, variations in sample size could also contribute to the differences in PAI medians between the two studies.
The findings from the current study confirmed that the difference in clinical success was not significant. This aligns with a recently published study involving 51 mature, permanent, single-rooted teeth with chronic apical periodontitis (PAI > 3). In that study, the teeth were treated using three different approaches: BC formation, standard-PRF, and advanced-PRF, with 17 teeth in each group [41]. The success rates of S-PRF and A-PRF were similar (88.2%), while the BC group had a success rate of 82.4% after eighteen months of follow-up. Another trial, using a similar methodology and age group, reported a clinical success rate of 93.9% for maxillary incisors treated with BC formation, compared to 97% for teeth that underwent conventional retreatment after twelve months of follow-up [15]. Similarly, in cases of immature teeth, most failures were identified within one year after REPs, with the primary cause being persistent periapical infection, accounting for 79% of failures [25].
The long-term outcomes observed in this study are consistent with the findings of a few prior randomized clinical trials that have investigated REPs in the primary root canal treatment of mature teeth with apical periodontitis. For instance, a study by Jha et al. [42], examined 15 mature permanent teeth in children aged 9–15 years treated with BC formation. The study demonstrated a consistent decline in mean PAI values over 18 months period with regular follow-ups. The preoperative mean PAI value of 3.60 decreased to 1.60, 1.40, and 1.10 at subsequent follow-ups, respectively, with an overall success rate of 100%.
Other trials have investigated REPs in older patient groups. One trial reported a clinical and radiographic success rate of 92.3% in patients treated with the BC approach [13]. Another study compared radiographic healing outcomes between two REP techniques—BC and platelet-PRF—in 20 teeth (10 per group). After 12 months, significant healing of periapical lesions was observed, with median PAI scores decreasing from 3.5 and 4 to 2 and 1 in the BC and PRF groups, respectively [5]. Furthermore, a randomized clinical trial assessed the efficacy of MTA and Biodentine in the revascularization of mature permanent incisors with apical lesions. Over an 18-month follow-up, complete healing was observed in 67.7% of treated teeth, while the remaining 32.3% showed progressive healing. These findings highlight the therapeutic potential of REPs in managing mature permanent teeth with periapical lesions [43].
In line with current findings, a study evaluated forty-five mature and immature single-rooted permanent teeth with apical periodontitis using conventional periapical radiographs and CBCT after treatment with three different regenerative endodontic procedures (REPs): blood clot (BC), platelet-rich fibrin (PRF), and platelet-rich plasma (PRP). CBCT analysis revealed that teeth treated with PRF showed the highest median reduction in periapical lesion size (69.57%), followed by BC (44.02%) and PRP (23.08%). However, the differences were not statistically significant [44]. A recent randomized clinical trial used CBCT to evaluate the healing of periapical lesions in eighteen necrotic maxillary permanent incisors. The results showed no significant difference between teeth treated with PRF and those treated with concentrated growth factor (CGF) over a follow-up period of 6 and 12 months [45].
Histological analysis of regenerated tissues following REPs in mature teeth has revealed the formation of mineralized tissue resembling bone (osseous-like) and cementum (cementum-like). These tissues are embedded within dense, highly vascularized, fibrous periodontal-like tissue, but without the induction of reparative dentin due to the absence of odontoblasts [46]. A previous study confirmed the histological similarities between the regenerated connective tissue—containing osseous-like tissue, cementum-like tissue, and vascular components—after REPs in both immature and mature permanent teeth [47].
Regarding the response to the pulp sensibility tests, combining TPT and EPT in the current study was considered. A previous literature review advocated this combination because the outcomes of one test can corroborate the results obtained from the other test, thereby enhancing the reliability and validity of the findings through cross-verification [48].
Previous studies have reported varied pulp sensibility outcomes. A prior investigation on the retreatment of mature incisors with apical periodontitis found positive pulp responses in 54.54% of cases after one year, following the formation of a biological barrier (BC) [15]. This success rate was higher than that observed in the present study, which utilized a revascularization approach. The discrepancy may be attributed to the reliance on EPT alone, whereas the current study employed a combined TPT and EPT methodology.
In the present study, positive pulp responses were observed in 59% of PRF-based retreated mature teeth after one year, increasing to 73% after two years. These results align with previous studies, which reported positive pulp responses in 50% of PRF-treated teeth [5, 13]. Two other studies reported success rates of 60% [10], and over 60% [49] for mature teeth treated with BC formation, which is higher than the 42% observed in the present study after 12 months. This variation may be due to differences in the preoperative status of the included teeth (i.e., presence or absence of periapical radiolucency). However, after 24 months, comparable positive responses were recorded.
Positive responses to pulp sensibility tests in both the blood clot and PRF groups are encouraging, suggesting that both methods may effectively preserve or restore pulp vitality in the short term. However, long-term success depends on ongoing monitoring, the absence of inflammation or infection, and the pulp’s ability to heal and maintain its function over time. Further research, including long-term clinical studies, would be beneficial to compare the effectiveness of these methods in ensuring the lasting success of retreatment procedures.
Although the difference in pulp sensibility records between the BC and PRF techniques was not significant, the faster healing and the higher number of teeth responding positively to pulp sensibility tests in the first year after retreatment with PRF could be due to the abundant and sustained release of growth factors—such as TGF-β, PDGF, insulin-like growth factor 1, and bFGF—for 7–28 days, which mediate new vascular and nerve fiber formation [10, 50]. Additionally, the presence of leukocytes, cytokines, and lymphocytes in PRF creates a balanced and self-regulating system that effectively manages inflammation and infection while enhancing the healing potential of the PRF clot [10].
The findings of the present study confirm the success of REPs even in the presence of infection and inflammation in the pulp and periapical tissues. This might be due to the presence of residual vital pulp tissue around the root apex [6]. Generally, the mature teeth possess a lesser number of stem cells than those available in the immature teeth [51]. Upon evoking apical bleeding, periodontal ligament stem cells (PDLSCs), bone marrow mesenchymal stem cells (BMSCs), and residual dental pulp stem cells (DPSCs) around the root apex of mature necrotic teeth migrate into the canal space, organize on the three-dimensional matrix, and contribute to the repair process [51, 52].
Despite the relatively long follow-up period in the current study, this trial may offer a new perspective on the retreatment of mature teeth with apical periodontitis, rather than relying exclusively on conventional root canal retreatment. However, certain limitations should be acknowledged, such as the reliance on two-dimensional radiographs to assess periapical healing, which is less sensitive than three-dimensional imaging [53]. Nevertheless, some reports have demonstrated consistency between two-dimensional and three-dimensional imaging techniques, such as CBCT [7, 54]. Further clinical trials incorporating CBCT for a subset of cases could provide a more detailed assessment of healing, particularly in complex cases.
To expand the benefits, studies should include other age groups, varying apical foramen sizes, and different tooth types. Another limitation is the relatively small number of treated teeth, which were confined solely to maxillary mature permanent teeth. This may restrict the generalizability of the findings. Therefore, larger cohort randomized clinical trials with at least five years of follow-up are needed to assess the durability of outcomes, the potential for late failures, and to provide more robust evidence. Additionally, histological studies are essential to determine the nature of newly regenerated tissues after the retreatment of mature teeth and to validate the regenerative potential of REPs in mature teeth.
Finally, relying solely on sensibility tests presents challenges, as a positive response does not necessarily indicate true pulp regeneration. Alternative methods, such as laser Doppler flowmetry or pulse oximetry, may offer more accurate and reliable data regarding the actual pulp status of retreated mature permanent teeth.
While REPs have shown promise in treating immature teeth with necrotic pulps, their application in mature teeth presents several significant challenges. One of the primary limitations is the difficulty of achieving adequate disinfection in mature teeth with complex root canal systems. These teeth often have narrower root canals, denser dentin, and intricate anatomical variations, such as lateral canals and isthmuses, which can harbour biofilm and hinder effective disinfection [1]. Another major challenge is the potential for incomplete regeneration of the pulp-dentin complex. Unlike immature teeth, mature teeth have a limited blood supply and a less favourable microenvironment for stem cell recruitment and differentiation [23].
The regenerative potential of REPs in mature teeth is further limited by the reduced number of resident stem cells and the diminished capacity for angiogenesis and neurogenesis. Consequently, the outcomes of REPs in mature teeth are often less predictable, with a higher likelihood of fibrous or osseous tissue formation rather than true pulp regeneration. Additionally, clinical protocols for REPs in mature teeth are not yet standardized, leading to variability in treatment outcomes. Factors such as the choice of irrigants, intracanal medicaments, and scaffold materials can significantly influence the procedure’s success [55].
Although conventional root canal retreatment remains the gold standard for retreating mature permanent teeth, the promising outcomes of REPs in previous clinical trials encouraged us to explore and compare alternative REP techniques (BC vs. PRF). This paradigm shift in treatment approach also necessitates a shift in the assessment of clinical outcomes. Therefore, this trial aimed to compare two corresponding REP approaches. Further research comparing different REP techniques with conventional root canal retreatment is needed to optimize treatment protocols and develop strategies to enhance the regenerative potential of REPs in mature teeth.
Additionally, multicenter studies with larger sample sizes and a follow-up period of at least five years, utilizing advanced imaging techniques and histological investigations to validate regenerative outcomes, are mandatory.
