Regenerative endodontic therapy, which replaces damaged tissue instead of restoring it, has been called a "turning point" in treating necrotic teeth with periapical periodontitis. Regenerated dental pulp, unlike endodontically treated teeth, restores immune cells to the root canal's innate immunity, reducing the risk of reinfections, hydrating teeth, and strengthening their mechanical resistance to fractures [5].

According to a recent umbrella review, REPs present a promising substitute for conventional apexification techniques for immature teeth, with paletlet concentrate showing better results in periapical healing than traditional blood clots [25]. Additionally, REPs seem to be a good therapeutic option for adult necrotic teeth with periapical lesions at this stage, according to moderate quality investigation [26]. However, the inconsistent results highlight the necessity of standardized procedures and consistent outcome measures to more accurately assess the effectiveness of various scaffolds in RET.

Therefore, the purpose of this study was to use a randomized clinical trial to treat 51 patients with necrotic pulp and apical periodontitis in mature single-canal teeth. The three different scaffolds used in regenerative endodontic procedures; blood clot, standard PRF, and advanced PRF strategy were evaluated in this study to assess their radiographic and clinical outcomes. Our study results showed that mature teeth with a necrotic pulp and apical periodontitis might gain advantage from treatment with REPs applying any of the three strategies.

To standardize this approach, only single anterior mature tooth was used, as in most clinical studies on adult tooth REP [9,10,11]. Patients' ages were chosen to range from 18 to 35, as physiological alterations occur with ageing as secondary dentin and cementum deposition increases in the apical region of the tooth, resulting in apical foramen constriction, hyper-cementosis, and periodontal ligament stenosis and fibrosis. As a result, these physiological alterations may inhibit the migration of resident stem cells from the surrounding tissue into the root canal. In fact, the ageing process produces physical malfunctions of many cellular and molecular events that result in a loss of immunity and increased inflammation in the aged tissues and surrounding niche, which may hinder the regenerative process and reduce stem cell function [27, 28]. Although, al Arslan et al. observed no effect of age on regeneration outcomes in a regressive investigation of confounding characteristics [9]. Also, Gender was not a confounding factor in this study because it did not affect mesenchymal stem cells' gene expression indicators [29].

Regenerative endodontics can be successful when the root canal system's pathogenic organism and byproducts are eradicated using chemo-mechanical preparation. Like previous studies [9,10,11, 30, 31], the current study used full mechanical instrumentation during the first visit to remove contaminated tissue and make space for irrigants, which decontaminate, detoxify bacterial antigens, eliminate intracanal medicaments, and condition the root surface for scaffolding. The current study used 2.6% sodium hypochlorite since 6% showed cytotoxic effects on dentin, which is essential to healing [32]. The release of embedded growth factors from 17% EDTA boosts stem cell motility, angiogenesis, proliferation, and differentiation into odontoblast-like cells [33, 34]. Double antibiotic paste (DAP) was used for further disinfection, DAP had better tissue development, vascularity, inflammation control, and minimal tooth discolouration than TAP [30]. However, a recent meta-analysis of the regeneration treatment's clinical and radiological success in permanent adult teeth found that all studies had similar success rates, regardless of final irrigants or intracanal medication [35].

Blood columns (BCs) are the most popular scaffold but producing the correct blood clot is not always attainable, which could inhibit stem cells from migrating. BC’s brittle mechanical construction may not be enough to fill the root canal after treatment, causing coronal sealing collapse[34]. The cell-free technique proposes using autologous platelet products like standard or Advanced PRF as scaffolds in the root canal to enclose blood components and platelets as signaling molecules and allow cells to generate vital tissue [13, 36].

Standardized and calibrated 2-dimensional radiographic examination was done for REPs evaluation [37]. Further,the apical lesions were assessed using periapical index scoring(PAI), since PAI scoring offers a reliable and precise measuring method [38]. According to the current investigation, the radiographic healing of the periapical lesion appears encouraging. At 12 months, it showed radiographic periapical healing success (PAI < 3) (35.3% in the blood group, 64.7% in the S-PRF group, and 41.2% in the A-PRF group). At 18 months, it showed success in radiographic periapical healing (PAI < 3) (58.8% in the blood group, 94.1% in the S-PRF group, and 76.5% in the A-PRF group). There was no significant difference between the three groups in terms of the occurrence of healing scores at 6, 12, and 18 months (p = 0.442, p = 0.204, and p = 0.173), respectively.

Unfortunately, these methods have not been directly compared for periapical healing in adult mature tooth revascularization. However, for immature teeth, a meta-analysis conducted by Murray et al.,2018 evaluated the clinical efficacy of the revascularization in immature teeth after one year and concluded that the periapical lesion healing response is 88.9% for blood clot and 100% for PRF (p > 0.05) [39]. Although, Sabeti et al., 2023 concluded that PRF exhibit the greatest success (75%) in periapical lesion resolution outcome within 12 months postoperatively compared to the blood clot technique (57%) [40].

To our knowledge for mature teeth studies, just one study comparing blood clot and standard PRF found that both groups' PAI scores of periapical lesions decreased significantly in the first 6 and 12 months after a REP. At 6 and 12 months, S-PRF showed a better periapical lesion healing rate than BC [11].

The repair of periapical lesions may have been affected by antimicrobial elimination and the early phase of the physiologic wound healing process, which is activated by tissue bleeding [41]. Additionally, blood contains immunoglobulins and cytokines from the innate and adaptive immune systems, which help locate and opsonize bacteria for phagocytosis [42].

All published research has shown that biological effects of autologous platelet concentrate depend on platelet concentration and number and type of leukocyte trapped in the fibrin membrane. S-PRF, a second-generation platelet concentrate, releases growth factors such as PDGF, TGF-β, b-FGF, and VEGF for up to ten days when used as a scaffold in REPs [43,44,45]. These growth factors control angiogenesis, promote stem cell migration, proliferation, and differentiation, and are essential for the self-regulation of infectious and inflammatory processes [17, 18]. Furthermore, S-PRF also forms a fibrin network that binds infection-fighting immune cells and cytokines and protects growth factors from proteolysis [46, 47].

A-PRF was treated like previous platelet concentrates, but with more evenly distributed platelets and leukocytes across the membrane, except for the acellular zone. Naturally, growth factors particularly IGF-1, EGF, and fibrin matrix protein impact A-PRF success. These factors have been found at higher concentrations than those found in blood and significantly enhance the process of tissue regeneration [48].

However, platelet rejuvenation is crucial because platelets can signal cell migration and start tissue healing. Masuki et al., [49] found that A-PRF didn't give good results regarding platelet renewal, which may explain its lower success rate (41.2%, 76.5%) compared to S-PRF (64.7%, 94.1%) at 12 and 18 months.

Most literature studies had limited follow-ups, which may underestimate failure rates. The current study has 18 months of follow-up, but long-term follow-up is needed to ensure long-term results. Even though 86.2% of teeth showed clinical success with the REPs, clinical failure after 18 months was 11.8% for both S-PRF and A-PRF and 17.6% for blood column.

According to the AAE, the ultimate objective of REPs should be a positive pulp vitality test which is commonly measured by electric or thermal test. Inaccurate results and considerable stimulus attenuation occur when cold testing is done because bioactive cements prevent cold stimuli from reaching living tissue [21]. It is critical to note positive pulp responses do not indicate canal space pulp tissue regeneration, and negative pulp responses do not indicate failure [10, 50]. Using regenerative endodontic therapy to restore pulp tissue's neuronal architecture is controversial and requires more research. Revitalization studies show increased sensitivity. In this regard, Arslan et al., [9] found that 50% of the teeth responded positively, agreeing with the findings of Nageh et al., [31] which found that 60% of the teeth responded, along with El-Kateb et al., [10] which also demonstrated the increased sensitivity of the treated teeth (77%).

In the current study, incidence of the regaining sensitivity after 12 months was 29.4% with A-PRF group and 41.2% within the S-PRF group, 17.6% with BC group. This is in line with the findings of Youssef et al., who observed that 20% of the BC group and 50% of the S-PRF group had recovered their pulp sensitivity [11]. The overall rate of regaining sensitivity with an 18-month follow-up was 29.4%, which is comparable to the findings of Lu et al., where 35% of teeth regained sensitivity after 4.3 years follow up [12].

Numerous reasonable avenues for regaining sensitivity exist, even if the specific procedure responsible for that after REPs is yet unidentified. An increase in the percentage of patients who regained tooth sensibility in the PRF group, compared to the BC group, may be attributed to firstly the different growth factors available in PRF, which is important for the neurogenesis process. Transforming growth factor beta regulates mitogenic effects of other growth factors (nerve growth factor and brain-derived neurotrophic factor), stimulates neurite outgrowth, and regulates differentiation of Schwann cells (SCs) [51]. Platelet derived growth factor (PDGF) induces SC proliferation, differentiation, and myelin formation [52]. Vascular endothelial growth factor promotes blood vessel growth, neurogenesis, and neuroprotection [53]. Insulin growth factor 1 supports the forward extension of the nerve fibers and suppresses apoptosis in motor, sensory, and sympathetic neurons [54]. Basic fibroblast growth factor facilitates neuroprotection and SC regeneration [55].

The ability of some dental pulp stem cells to survive a peri-radicular lesion and proliferate into neural cells, which initiates axon guidance, is the basis for the second theory [56]. According to the third mechanism, stem cells have the capacity to differentiate into neurogenic lineages and may originate from the periodontal ligament, which expresses markers of undifferentiated neural crest cells [57]. The bone marrow mesenchymal stem cells, which can develop into neurons and astrocytes under the right circumstances, are an integral component of the fourth potential mechanism [58]. The fifth potential technique involves the use of 17% EDTA irrigation, which stimulates neurogenesis by releasing neurotrophic factors from the dentin matrix locally. These elements are crucial for maintaining neurons because they encourage neuronal development and axonal regeneration [59]. The sixth proposed mechanism is that nearby ipsilateral nerves may have sprouted or ingrown, resulting in a collateral reinnervation of the pulp canal. Still, more research is necessary to understand the underlying mechanism of its activity [60].

Although this study's results seem encouraging, there are several constraints. The study's limitations include the inability to generalize findings across tooth kinds, a small number of participants, and a short follow-up period. However, future therapeutic directions will include extended monitoring and follow-up. It was difficult to induce blood in teeth with closed apices was another drawback. Another drawback was performance bias, which is unavoidable given the nature of the intervention. Finally, because the histological assessment of regenerated tissues is intrusive and necessitates tooth extraction, the findings cannot be correlated with them as well restoring pulp tissue after regenerative endodontic treatment is uncertain and requires clear data and objective testing.

Regeneration therapy is an attractive new alternative to traditional endodontic treatments. However, there are no established guidelines regarding REPs in mature teeth also, the prognosis of it is still vague, so a greater number of strong clinical investigations with prolonged follow up periods are needed. The higher success rates observed for PRF scaffolds could suggest a preference for PRF-based treatments in similar cases, which could be beneficial in guiding clinical decision-making.

Taking into consideration from the clinical point of view, before undergoing REPs, the patient must be well informed of the treatment possibilities and potential complications. In addition, the therapy might require longer follow-ups and stronger patient motivation. Furthermore, some endodontists may lack the training and experience to provide regenerative endodontic procedures [61]. Moreover, for future directions in REPs, it must be accompanied with respect to the following: new scaffolds, antimicrobial medicaments, the determination of pulpal blood flow might be a much better objective way to assess pulp vitality, also determentaion of potential markers like IL-8 or matrix metalloproteases MMP.