Dental pulp necrosis in immature permanent teeth, with or without apical periodontitis, presents clinical management challenges characterized by undeveloped roots, thin canal walls, and open apical foramina (Murray, 2023). Achieving an adequate apical seal for an open apical foramen using conventional root canal treatment is difficult. Additionally, thin dentinal walls and incomplete development make the tooth more susceptible to fractures (Xie et al., 2021). In contrast to conventional root canal therapy, Regenerative endodontic procedures (REPs), which aim to physiologically replace damaged tooth structures, including dentin and root structures, as well as cells of the pulp-dentin complex with the concept of tissue engineering (Wei et al., 2022), offer an ideal solution for this condition.

Regenerative endodontics seeks to promote the recovery of normal pulp function in inflamed or necrotic teeth, leading to the restoration of the pulp-dentin complex (Galler, 2016). REPs serve as the alternative to conventional root canal therapy or apexification for treating immature necrotic teeth. For REPs, after chemical decontamination, bleeding is induced, and the ensuing blood clot provides a scaffold for migrated or resident stem cells to attach, proliferate, and differentiate into the essential components within the canal space Continued root development, increased thickness in the dentinal walls, apical closure, and even restoration of pulp vitality was observed following REPs (Chan et al., 2017, Nagy et al., 2014, Nakashima et al., 2017, Peng et al., 2017).

However, since the tissues growing into the canals after REPs mainly consist of fibrous connective tissue and bone-like or cementum-like tissue (Khademi et al., 2014; Martin et al., 2013; Peng et al., 2017), regenerative endodontic treatment at the current stage does not equate to genuine functional dental pulp regeneration. Following the principles of tissue engineering, a cell-based dental pulp regeneration strategy appears to be a reliable approach to achieving functional dental pulp regeneration. A previous study by Xuan et al. (2018) reported that implanting autologous stem cells from human exfoliated deciduous teeth (SHED) led to the regeneration of dental pulp tissue containing blood vessels and sensory nerves, increasing the root's length and thickening the root canal wall. In that same study, the safety of autologous SHED implantation was also confirmed in a preclinical trial with a 24-month follow-up (Xuan et al., 2018). A case report (Meza et al., 2019) showed combination of autologous inflamed dental pulp stem cells and leukocyte platelet-rich fibrin achieved viability of dental pulp in a mature tooth, and concluded it an alternative way to treat irreversible pulpitis in mature permanent teeth. In contrast to allogeneic cell transplantation, autologous cell transplantation is undoubtedly safer in clinical applications due to the potential risk of immune rejection (Li et al., 2018). However, the source of autologous progenitor cells for dental pulp regeneration is currently limited.

It is believed that progenitor cells migrate into the canal space along with the influx of blood when a file is used to stimulate bleeding for clot formation (Hargreaves et al., 2013). While the exact origin of cells that promote root development during REPs remains controversial, it is hypothesized that the cells flowing into the canals have the potential for regenerative endodontic treatment. Nonetheless, there is limited literature on the isolation and culture of cells in canals that migrate along with induced bleeding.

Cell sheets, a type of self-organized cell aggregate rich in extracellular matrix components and devoid of allogenic scaffolds, offer a suitable option for autologous cell transplantation (Li et al., 2019). Cell sheet technology enhances the engraftment and survival of cells on transplant sites and has been applied in regenerative dentistry. Studies (Iwata et al., 2018; Xuan et al., 2018; Yan et al., 2021) have reported the application of cell sheet technology in regenerative dentistry, demonstrating the feasibility of autologous cell sheets in dental pulp tissue engineering.

This study aimed to isolate cells from the blood in root canals (CBRC) induced by over-instrumentation in immature permanent teeth with apical periodontitis, characterize these isolated cells, and preliminarily assess the feasibility of transplanting these cells with cell sheet technology for dental pulp regeneration.