Untreated dental caries, tooth trauma, and dental anatomical variations such as dens invaginatus can result in endodontic infections and inflammatory pulpal reactions, a highly prevalent inflammatory condition worldwide (Bletsa et al., 2006; Agnihotry et al., 2016; Estefan et al., 2016). This kind of inflammation can be reversed with appropriate treatment at an early stage (Bjørndal et al., 2014). However, as inflammation progresses, irreversible pulpitis, which manifests as violently acute pain in the tooth, occurs frequently (Lin et al., 2020). If the irreversible pulpits are not treated promptly, it can induce the dental necrosis, apical periodontitis, abscess, and eventual tooth loss (Bjørndal et al., 2019). Current endodontic treatment concepts for fully developed necrotic permanent teeth include removing inflammatory or necrotic pulp tissue and replacing it with biomaterials (Jung et al., 2019). Nowadays, the most popular therapeutic strategy is root canal therapy (RCT), which includes debridement, instrumentation, and obturation of the pulp canal space (Ma et al., 2016; Manfredi et al., 2016). However, the traditional RCT is not recommended for the immature permanent teeth in children due to their thinner dentinal walls and unclosed apical foramen compared to mature permanent teeth (Mc et al., 2019). The apexogenesis/apexification (Figure 1) is an alternative method to solve this challenge. It entails removing all necrotic pulp tissues and utilizing biomaterials such as mineral trioxide aggregate (MTA) or calcium hydroxide (Ca(OH)2) to create an artificial barrier or stimulate the creation of a mineralized barrier in the root apex after enough debridement (Frank, 1966; Heithersay, 1975; Rafter, 2005). However, apexogenesis does not restore the pulp’s vitality and immunocompetence and cannot promote the root maturation (the closure of the apical foramen as mature permanent teeth and/or the thickening of the root canal walls), which may compromise tooth and root wall stability and eventually increase the likelihood of dental secondary trauma, even tooth loss (Andreasen et al., 2002; Kim et al., 2018; Mc et al., 2019). Likewise, apexification cannot ensure the root maturation completely and it need repeated return visits over a long period to determine the final effect for every patient (Rafter, 2005).

Pulp regeneration (Figure 1) is another approach for immature permanent teeth necrosis. A healthy and alive pulp is required for long-term tooth survival and preservation (Schmalz et al., 2020). In 2001, Iwaya et al. (Iwaya et al., 2001) established a novel treatment technique to address a young permanent tooth which was immature and suffered from apical periodontitis and then named it as “revascularization”. In 2007, “regenerative endodontics”, which is based on a tissue engineering notion, was coined by the American Association of Endodontists, indicating this kind of regeneration included both soft and hard tissues rather than vessels (Murray et al., 2007).

Regenerative endodontics treatment (RET) refers to biologically based procedures to replace damaged tooth tissues with newborn tissues (Schmalz et al., 2020). Following a disinfecting phase, RET induces bleeding by periapical tissue tearing. Blood clot can act as a natural scaffold which provide a platform for stem cells to grow into. Various endodontic studies have shown that RET could improve root maturation and reinforce thin and weak young roots (Kahler and Rossi-Fedele, 2016; Chan et al., 2017). RET also led to comparable survival and success rates compared to apexification (Ribeiro et al., 2020). However, this treatment needs the supplement of innovative biomaterials to improve repeatability and encourage a well-orchestrated regeneration (Keller et al., 2015).

RET includes three crucial parts in regeneration engineering: stem cells, bioactive molecules, and scaffolds. Stem cells can serve as a source to differentiate into different cells and form new regeneration-related tissues. Bioactive molecules can promote the cell migration, growth. The scaffold is considered an essential factor in the RET to coordinate drug delivery of active biomolecules or antibiotics and offers a surface for cells to communicate, migrate, proliferate, and spatially organize (Nakashima and Akamine, 2005; Galler et al., 2011).

With the development of pulp regeneration, the scaffolds become one of the key factors to improve the RET’s success rate. Here we summarized the research and development of nanofibrous scaffolds in RET to highlight the advantages of nanofibrous and discuss the future modification and application of nanofibrous.

An essential process of the RET is placing a scaffold to generate a three-dimensional (3D) microenvironment for supporting cells, resulting in better outcomes of restoring tissue function (Gupte and Ma, 2012). A variety of materials can be utilized to construct scaffolds in regeneration engineering (Galler et al., 2011). Synthetic polymers [such as polymers of glycolic acid (PGA), polymers of lactic acid (PLA)], and biological matrices (such as reconstituted collagen and fibrin) are two principal alternatives (Galler et al., 2011; Zein et al., 2019). A suitable scaffold should closely be similar to the natural extracellular matrix (ECM), which is the physiological microenvironment of the cells (Gupte and Ma, 2012). ECM is the cell-environmental nanofibrous protein network in all tissues and consists of various nano-scale proteins, such as fibronectin, vitronectin and collagen (Kwon et al., 2005). This matrix can serve as structural support for cell adhesion and ingrowth and give cells chemical signals to govern their activity and reinforce a specific phenotypic (Galler et al., 2011).

Recent breakthroughs in nanotechnology have tremendously aided in creating innovative ECM-mimicking materials. Nanofibrous scaffolds which are becoming increasingly common in the RET can improve adhesion, allow cells to manifest more normal 3D morphologies when compared with smooth materials, solid films, and tissue culture polymers (Gupte and Ma, 2012; Mc et al., 2019). A nanofibrous scaffold is characterized by the diameter ranging from 1 nm to 100 nm, similar to the size of collagen (Sheikh et al., 2015). And it has a large surface area inversely linked to diameter, facilitating cellular adhesion and migration. Furthermore, the pores of nanofibers are suitable for the cell adhesion and penetration. Cells grown on very porous nanofibrous meshes have been proved to increase cellular adhesion and multiply (Kwon et al., 2005). Thus, nanofibrous scaffolds become an ideal material for RET due to nano-scale fibre, large surface area, and interconnected porosity similar to the ECM.

Biodegradability is also an important character for tissue regeneration. Most nanofibrous scaffolds are biodegradable and can be chemically destroyed in the biological environment by enzymes such as lysozyme (Sheikh et al., 2015). The degradability of the nanofibers is determined by two significant factors: the chemical composition of the fibres and hydrophilicity. Therefore, by adjusting these two factors, the biodegradation speed of scaffolds can be controlled in line with the rate of tissue regeneration for better regeneration. The biocompatibility of scaffolds’ broken down chemical compounds matters as well. To better pulp regeneration, the material of the nanofibrous scaffold should be taken into consideration.

Acting as the reservoirs for stem cells, antimicrobial, anti-inflammatory molecules, and growth factors is a considerable advantage for nanofibrous scaffolds (Albuquerque et al., 2015; Keller et al., 2015). Thus, by providing physical and chemical stimulation, nanofibrous scaffolds have been shown to drive positive cell interactions, stimulate cell growth, preserve cell phenotypics, assist stem cell development, and activate cell-signaling pathways (Sun et al., 2010; Gupte and Ma, 2012; Li et al., 2020).

Currently, several materials processing techniques, including electrospinning, molecular self-assembly, and thermally induced phase separation, have also been developed for the production of nanofibrous scaffolds (Gupte and Ma, 2012; Albuquerque et al., 2014).

Electrospinning is extensively employed to make natural and synthetic polymer fibres because of its ease of usage and compatibility with practically any dissolvable polymer. A strong electric field is applied to control the deposition of polymer fibres with diameters ranging from nanometers to micrometers. Fibre morphology, pore size, and fibre alignment may be altered by adjusting electrospinning parameters as well (Yang et al., 2005; Gupte and Ma, 2012). However, it is frequently unable to make real nanofibers at 100 nm or less using commonly used biodegradable polymers. In addition to low production efficiency, factors such as air humidity, temperature, and air velocity in the spinning environment are difficult to control which affect the physical properties of nanofibrous scaffold can also be its shortcomings to achieve industrial production (Sill and von Recum, 2008).

Molecular self-assembly is a bottom-up technique that has been utilized to create nanofibres as small as 10 nm by using spontaneous molecular organization via weak non-covalent interactions (Dahlin et al., 2011). Molecular self-assembly nanofibers for RET benefit from being built into solutions and producing gels utilized for stem cell encapsulation (Rosa et al., 2013). Besides, the fluid may be administered via a minimally invasive technique, resulting in an in-suit nanofibrous structure. However, this method has limitations in controlling pore size/shape inside the hydrogel scaffold (Gupte and Ma, 2012) and poor mechanical properties in general (Zhang et al., 2012).

Thermally-induced phase separation (TIPS) involves drastically quenching a single-phase homogenous polymer solution, inducing separation into a solvent-rich phase and a polymer-rich phase and with fiber diameters between 50 and 500 nm. It is a technique that has been utilized for a long time to make membranes and scaffolds. This method is common to create nanofibrous scaffolds. TIPS can also be used with other approaches to create macro/micropore/channel networks within 3D nanofibrous scaffolds (Gupte and Ma, 2012).

The application of nanofiber scaffolds in RET can be devived into two parts in immature teeth based on tissue-engineering-techniques (Gupte and Ma, 2012): 1) bioactive nanofibrous scaffold with antibiotics for root canal disinfection. 2) nanofibrous scaffolds with stem cells and/or biomolecules for dental regeneration in a broad sense, which contains four parts: pulp connective-tissue formation; dentin formation; revascularization; reinnervation (Figure 2, Figure 3).

Pulp regeneration processes can be quite complex (Schmalz et al., 2020), and bacteria in the root canal further complicates the RET (Kim et al., 2018). Over 600 bacterial species have been identified as potentially involved with pulp infections (Sampaio-Maia et al., 2016). The microbial ecology in the pulp canals of infected young permanent teeth was comparable to that of mature permanen teeth (Nagata et al., 2014). Enterococcus faecalis (E. faecalis) is commonly isolated from early-root canal infections. Prophyromonas, Treponema, Bacteroides, Fusobacterium, Prevotella, Peptostreptococcos, Eubacterium, and Campylobacter species are the most commonly found endodontic bacteria in primary endodontic infections (Neelakantan et al., 2017). Bacteria can not only enter the dentinal canal tubules of infected teeth but form biofilms on the radicular canal walls (Lin et al., 2014). Previously, the most commonly used drug in clinical practice for successful disinfection is triple antibiotic paste (TAP), which is made up of equivalent doses of metronidazole (MET), ciprofloxacin (CIP) and minocycline (MINO) (Arruda et al., 2018). The antibacterial effects of this drug are widely recognized in the therapeutic setting, but it has garnered numerous complaints (Kim et al., 2018). One of the most crucial issues reported at TAP was dentin discolouration induced by the presence of MINO (Porter et al., 2016). Therefore, the double antibiotic paste (DAP) combining MET and CIP or the incorporation of ampicillin (AMP) into a new TAP formulation were used (Arslan et al., 2015). However, antibiotic pastes at therapeutically recommended doses will impair the viability of human apical papilla stem cells (SCAPs) (Ruparel et al., 2012). In view of the above disadvantages, Ca (OH)₂ is commonly used in RET currently. However, it also has several disadvantages. For example, it can form a calcified barrier in the root canal, hindering the growth of pulp tissue (Song et al., 2017; Almutairi et al., 2022). And because of its high pH, it may destroy cells from the apical papilla and periapical tissue as well (Banchs and Trope, 2004; Jadhav et al., 2012).

Thus, it is crucial to employ biomaterials that reduce germs in the root canal while remaining non-cytotoxic and encouraging the creation of new tissues (Murray, 2018). New nano-scaffolds which are capable of serving as drug delivery vehicles in a regulated manner depending on therapeutic use are developed (Sundararaj et al., 2014). Compared to propylene glycol, which is the most often utilized carrier for delivering antimicrobial pastes to the root canal, nanofibers have the potential benefit of increasing the surface area and, presumably, allowing for controllable drug release over a more extended period (Bottino et al., 2013; Palasuk et al., 2014). Figure 2 and Table 1 summarized the use of nanofibrous scaffolds in root canal disinfection.

In 2013, Bottino et al. (Bottino et al., 2013) mixed polydioxanone monofilament solution material (PDS-Ⅱ) with MET or CIP in different concentrations (5% or 25%), and typical electrospinning procedures were applied. Almost all scaffolds demonstrated an early burst followed by a general linear continuous drug release. Within the first 48 h, all nanofibrous scaffolds only release a portion of the medication from 22.4% to 51.4% indicated the sustained drug release from the scaffolds. All CIP-containing scaffolds strongly reduced the growth of Porphyromonas gingivalis (P.gingivalis) and E. faecalis. However, MET-containing’s have no E. faecalis resistance. Although they all have good antibacterial properties, only the 25% wt CIP-containing extract had a substantial adverse effect on Human dental pulp stem cells (hDPSCs).

A nanofibrous scaffold by combining MET and CIP in different ratios and using PDS as a drug carrier is created in another study (Palasuk et al., 2014). All scaffolds containing antibiotics suppressed the growth of P. gingivalis, E. faecalis, and Fusobacterium nucleatum (F. nucleatum), except for the MET-containing scaffold. When compared to scaffolds integrated with individual antimicrobials, scaffolds with two antibiotics did not increase their antimicrobial abilities. Moreover, antibiotic-containing aliquots slightly reduced cell viability to hDPSC.

To further determine the drug release abilities of the new dual-mix antibacterial scaffolds and their effect on the proliferation and activity of hDPSCs. Kamocki et al. (Kamocki et al., 2015) uses electrospinning to create PDS-based nanofibrous scaffolds containing MET and CIP. Pure PDS scaffolds are utilized as the negative (non-toxic) controls, while saturated CIP/MET solution scaffolds are treated as the positive (toxic) groups. The results show no differences in cell proliferation in the pure PDS or MET scaffold groups. For pure CIP-containing scaffolds, increasing drug concentration led to decreased cell viability. However, no cells could be observed after exposing to the saturated CIP/MET solution after 3 days, indicating that antibiotic-containing nanofibrous scaffolds had considerably reduced impacts on hDPSC proliferation. Pankajakshan et al. (Pankajakshan et al., 2016) discovered a similar experimental phenomenon: antibiotic–containing (MINO, CIP, MET) polymer nanofibers resulted in numerous bacterial killing but had no effect on hDPSC adhesion and growth on dentin when compared to a saturated TAP solution. These studies suggest that nanofibrous scaffolds are more biologically friendly to cells when compared with antibiotic paste.

Karczewski et al. (Karczewski et al., 2018) changed the formula of triple antibiotics, including MET, CIP, and clindamycin (CLIN). Hydrated triple antibiotic-containing nanofibrous scaffolds showed pronounced antimicrobial activity against Actinomyces naeslundii (A. naeslundii), Aggregatibacter actinomycetemcomitans (A. actinomycetemcomitans), and E. faecalis, but with no cytotoxic effects on hDPSC indicating that this kind of triple antibiotic-containing scaffolds might be a feasible option in the RET. Bottino et al. (Mc et al., 2019) optimized electrospinning parameters and created a 3D tubular triple antibiotic-eluting construct to increase the antibacterial activity and the ability to remove bacterial biofilm. The 3D nanofibrous scaffold created an optimal environment with apical foramen closure and a thin layer of osteodentin-like tissue within the root canal.

In general, compared with antibiotic pastes, utilizing nanofibrous scaffolds as drug-carrying carriers is more conducive to drug release and increase the capacity of pathogen clearance in the first stage of pulp regeneration. Moreover, it has no toxic effects on stem cells during regeneration. However, there are also some limitations in these researches. The main problem is the lack of in vivo studies including antibacterial, degradability and compatibility. Only one article established a canine model of periapical disease. The rest of the studies were still limited to in vitro experiments which lack of enough reliability. At the same time, as drug-loaded scaffolds, they also lack the exploration of the best speed for drug release as well as the best degradation rate of the scaffolds which determine the long-term bactericidal ability.

Currently, there are two significant challenges in the pulp regeneration, including the disinfection of the pulp and the regeneration of tooth-related issues. Regarding pulp disinfection, antibiotic pastes at therapeutically recommended doses impaired the viability of stem cells. And pastes cannot be released at a steady and lasting pace as well. As for the regeneration of tooth-related issues, RET has to be supplemented with scaffolds to improve repeatability and encourage a well-orchestrated regeneration. To address the challenges, nanofibrous scaffolds mimicking extra-cellular matrix (ECM) have been developed. They can act as antibacterial molecule reservoirs and provide growth factors, stem cells to guide the various cells’ migration, growth, and differentiation. However, most of the application of nanofibrous scaffolds is still at the laboratory research stage and lack of the in vivo and clinical investigation and evaluation. Meanwhile, we still have a lot of details to work out, including: the optimal degradation rate and drug release rate, the best hardness, suitable physical properties etc. Moreover, no single material can perfectly combine the antibacterial characteristics and stimulate regeneration of connective-tissue, dentin, nerves, and blood vessels simultaneously. New materials are still needed to construct the composite nanofibrous scaffolds for all needs in RET. Furthermore, the in vivo and clinical evaluation are also critical for the development of new nanofibrous scaffolds.