Immunoneurogenic pulp tissue response: a narrative review of the molecular and cellular mechanisms related to pulp tissue regeneration

Abstract

Aim  to evaluate advances in dental pulp regeneration and challenges for regenerative endodontic therapies, particularly nervous tissues and their components.

Methodology  for the analysis of this narrative review, a search for relevant articles was conducted across various scientific databases, including PubMed, Scopus, Web of Science, and Embase, covering the period from 2000 to 2023. The inclusion criteria were set to primarily encompass original research articles, systematic reviews, and meta-analyses that demonstrated relevance to the topics of interest.

Results  neurogenesis occurs during pulp repair but is impaired by ongoing lesions due to reduced vascular supply. The results suggest that while various therapeutic approaches using biomaterials are being explored, efficient neural regeneration though achieved in some studies remains a significant challenge.

Conclusion  Several studies have demonstrated the return of pulp sensitivity and the formation of neural tissue. However, despite growing interest and promising results, challenges remain to be addressed, the variability in individual responses and the complexity of tissue revitalization mechanisms, which are not yet fully understood.

Keywords
Inflammation; Dental pulp; Immune system; Tissue scaffolds

Introduction

The dental pulp is a loose connective tissue located within the root canals, surrounded by dentin walls¹,². It is highly vascularized and contains various cells such as undifferentiated mesenchymal cells, fibroblasts, odontoblasts, vascular and defense cells, blood vessels, and sensory neurons. Among these cells, fibroblasts play a crucial role in pulp regeneration by inducing pluripotent stem cell reprogramming and regulating cellular hypoxia through factors like VEGF and PDGF²,³. Additionally, odontoblasts, located at the dentin interface, are responsible for the formation of primary and secondary dentin, contributing to the structural integrity of the pulp⁴,⁵.

Pulp tissue also has a well-organized sensory system. Most of its nerve fibers are unmyelinated C fibers originating from the trigeminal nerve, while the remaining fibers come from myelinated sympathetic ganglia¹. These neurons release neuropeptides such as substance P (SP), neurokinin A (NKA), and calcitonin gene related peptide (CGRP), which act as neurotransmitters and play roles in the defense system and tissue regeneration³,⁶. Odontogenic pain arises when the physical barriers of the tooth are compromised, leading to the release of inflammatory mediators such as histamine and prostaglandins, causing vasodilation and initial pain, often diagnosed as hyperemia⁷,⁸.

When the infectious process persists, pulp inflammation can progress to pulpitis and eventually pulp necrosis⁹. This occurs when defense cells can no longer control the infection, leading to the destruction of odontoblasts and further stimulation of afferent sensory neurons¹⁰. The resulting release of neuropeptides and activation of receptors like TRP channels contribute to the pain and tissue degeneration associated with pulp disease. These processes highlight the complex mechanisms involved in pulp defense and regeneration¹¹,¹².

A key aspect of the neurogenic pain mechanism is the activation of ion channels such as ASIC and PIEZO channels, which contribute to the perception of touch and temperature¹³,¹⁴. TRP channels, specifically, play a significant role in the acute temperature sensitivity of teeth. Understanding the activation and modulation of these receptors is essential for developing new regenerative therapies aimed at improving sensory function and overall dental tissue health¹,¹².

Then, this review highlights advances in neurogenesis and tissue engineering technologies that are paving the way for innovative methods to regenerate dental tissues¹⁵,¹⁶. Emerging techniques, such as the controlled assembly of cells and biomaterials, show great potential for clinical endodontics¹⁷,¹⁸. The integration of artificial intelligence, tissue engineering, and gene therapy could lead to personalized treatments, revolutionizing dental practices and improving patient quality of life¹⁹.

Methodology

This narrative review included 114 papers using PubMed, Scopus, Web of Science, and Embase databases, covering the period from 2000 to 2023. The search strategy was designed to capture a wide range of studies at the intersection of advanced biomedical engineering and dental science²⁰. Keywords such as “inflammation”, “dental nerve”, “immune system”, and “scaffolds” were used in various combinations to ensure a comprehensive retrieval of articles.

The inclusion criteria were set to primarily encompass original research articles, systematic reviews, and meta-analyses that demonstrated relevance to the topics of interest²⁰. The selected articles were further assessed for quality and relevance, particularly in terms of their findings’ significance for advancing the understanding of topics such as neurogenesis, tissue engineering, and clinical applications in endodontics. Studies from various geographic regions and research contexts were included to provide a global perspective on advancements in these areas. As an exclusion criterion, articles that were not available in English were disregarded, ensuring consistency in data analysis.

Results

A study was conducted to evaluate the interaction between the immune and nervous systems of the dental pulp during pulp pathologies, demonstrating the response of the dentin-pulp complex to microbial, traumatic, or iatrogenic insults. This study revealed that such insults involve complex defense mechanisms and directly affect the nervous tissue (Figure 1). Neurogenesis was observed during pulp repair; however, its occurrence was significantly reduced in cases of continuous lesion progression due to diminished vascular supply, modification of cellular niches, and obliteration of the pulp chamber, which compromises regenerative capacity and leads to pulp necrosis (Figure 2).

Figure 1
Illustrative scheme of pulp tissue after bacterial invasion and detection by odontoblasts and immune cells. Subsequently, inflammatory cytokines, neuropeptides, proteins, and growth factors are released and induce tissue repair or damage. Inflammation, tissue damage and elimination of infectious bacteria are even more events. TLR: toll like receptor; Th: T helper; LPS: lipopolysaccharides; LTA: lipoteichoic acid; NKA: neurokinin A; CGRP: peptide related to the calcitonin gene; NPY: neuropeptide Y; SP: substance P; IL: interleukins; NGF: nerve growth factor; TNF: tumor necrosis factor. Created by BioRender.com.

Figure 2
Tissue drainage and induction of inflammatory cytokines by fibroblasts to the affected region. After the injurious stimulus, pulpal stern cells migrate and act in the nervous tissue repair process. Neurotrophic factor (NTF) has the unique potential to support neuronal survival and enhance neuronal function in the injured and diseased nervous system. Removing the stimulus and action of NFTs, together with immune cells, promotes nerve tissue repair. Created by BioRender.com.

Various studies have explored several therapeutic approaches using cells and natural, biological, and synthetic biomaterials. In regenerative dentistry, scaffolds are developed with biomaterials that enhance the stability and reproducibility of tissue engineering therapies, providing a controlled environment designed for specific applications (Figure 3). Despite advancements in regeneration with the formation of tissues and blood vessels, neural regeneration remains limited and under-documented. Efficient neural regeneration in regenerative treatments continues to be a significant challenge, as evidenced by the reviewed studies (Table 1).

Figure 3
Regenerative potential of stem cells and the use of biomaterials in tissue regeneration. (A) Dental stem cells. (B) Growth factors that regulate cellular events and are important in tissue repair, including transforming growth factor beta (TGF-β), vascular endothelial growth factor (VEGF), platelet-derived growth factor (PDGF), and basic fibroblast growth factor (bFGF). (C) Scaffolds that support cell growth. Biomaterials associated with dental stem cells and growth factors present regenerative potential, enabling tissue formation similar to the original tissue. Created with BioRender.com.

Table 1
Regenerative treatments involving neural response.

Assessment	Processing technique	Model	Results	Authors
	Hydrogels PVA e CS + CIP + IDR1002	Mice	Formation of pulp-like tissue in both cases	Sousa et al.21 (2022)
	Treated dentin matrix + Matrigel	Mice	Neural formation, tissue formation with blood vessels	Meng et al.22 (2020)
	Atelocollagen	Dogs	Increased angiogenesis and neurites. Neuronal formation	Iohara et al.23 (2020); Murakami et al.24 (2015)
In vivo	BC/ HA:CS/ PC:S	Dogs	Vital vascularized tissue, apical closure, increased thickness and root growth	Palma et al.25 (2017)
	Pulp CD105+ cells/SDF-1/collagen scaffold	Dogs	Regeneration of pulp tissue with vascular and neuronal processes	Nakashima and Iohara26 (2011)
	RAP	Dogs	Distribution pattern similar to authentic pulpal innervation. A and C fibers GFAP specific for astrocytic differentiation, are recognized	Mounir et al.27 (2019)
	BC /PRF	Human	Recovery of sensitivity to different degrees	Abu Zeid et al.28 (2021); Chrepa et al.29 (2020); Ahmed Youssef et al.30 (2021); Nazzal et al.31 (2020); Mittmann et al.32 (2020)
Clinical study	UC-MSCs + PPP	Human	Positive pulp response to cold, hot, and electric tests, both cases	Brizuela et al.18 (2022)
	PRP	Human	Positive response to sensitivity tests, vast majority in electrical test	Bezgin et al.33 (2015)
	PRF/ Collagen / Hydroxyapatite	Human	Both responded positively to cold test but did not respond positively to electrical	Mittal et al.34 (2021)

• BC: Blood Clot; PRF: Platelet Rich Fibrin; UC-MSCs: umbilical cord mesenchymal stem cells; PPP: platelet poor plasma; PRP: platelet rich plasma; CS: chitosan; HA:CS: sodium hyaluronate:chitosan; P:CS:pectin:chitosan scaffolds; RAP: recombinant amelogenin protein.

Discussion

Relationship between the dental pulp immune and nervous systems during dental pulp pathologies

The odontoblasts present at the dentin-pulp interface act as an initial protective barrier through the activation of Pattern Recognition Receptors (PRRs) present on the cell surface, by recognizing Pathogen Associated Molecular Patterns (PAMPs)³⁵. The membrane proteins of the PRRs present in odontoblast cells belong to the Toll-like receptor (TLR) family³⁶. These membrane proteins recognize peptidoglycans and lipoteichoic acid (LTA) from Gram-positive bacteria and lipopolysaccharides (LPS) mainly from Gram-negative bacteria. This recognition leads to the activation of a pro-inflammatory response, including the production of cytokines and chemokines, nitric oxide, and eicosanoids³⁷-³⁹.

After the stimulus is detected by odontoblasts, a repair process begins in the dentin, which promotes the formation of tertiary dentin to block the entry of irritating agents into the pulp tissue. As the intensity of aggression increases, TGF-β1 and TGF-β3 growth factors are released by the odontoblasts, promoting the formation of reparative dentin and, consequently, calcifications of the pulp chamber⁴⁰,⁴¹. The initial confrontation of the invasion of these pathogens also occurs through the production of nitric oxide (NO) and beta-defensins, in addition to the synthesis of pro-inflammatory chemokines such as CCL2, CXCL2, CXCL8, and CXCL10³⁷,⁴².

Bacterial colonization during the progression of pulpal inflammation establishes a biofilm composed mostly of Gram-positive and Gram-negative bacteria, as well as fungi, archaea, and viruses¹²,⁴³. A recent study evaluated different patterns of inflammatory cytokines induced by LPS from E. coli and LPS from P. gingivalis. These bacteria virulence factors significantly increased the expression of pro-inflammatory cytokines after 24h, including interleukins IL-1β, IL-6, tumor necrosis factor (TNF-α), and IL-8. The increase in the production of chemokines, cytokines, and defensins promotes cellular recruitment of leukocytes, neutrophils, macrophages, and dendritic cells, which initiate the innate immune response with the secretion of cytokines (ILs) and influence the adaptive immune response¹²,⁴⁴,⁴⁵.

This process occurs from the presentation of antigens by dendritic cells that activate B lymphocytes and CD4+ T lymphocytes or T helper 0 (Th0) cells, which differentiate into Th1, Th2, and Th17 cells, important regulators in the production of cytokines. Depending on the intensity of the stimulus, these cytokines can act both in the repair and the pathological process of pulp degradation and necrosis, together with matrix metalloproteinases (MMPs), which in uncontrolled production act in synergy with (ILs) in excessive tissue destruction⁹,⁴⁴,⁴⁶.

The inflammatory reaction when subodontoblastic levels are reached promotes a decrease in local blood flow, capable of suppressing the activity of nerve fibers that are located along the pulp tissue, promoting changes such as compression of nerve fibers and consequently induction of pain. These fibers are classified as type A fibers located in the most coronal region of the pulp and which present higher oxygen consumption and lower survival capacity when there are exacerbated inflammatory stimuli. On the other hand, type C fibers that are more resistant to hypoxia are located in the central portion of the pulp tissue²⁷,⁴²,⁴⁷-⁴⁹.

The type A fibers detect early stimuli in cases of reversible pulpitis with localized pain. Upon receiving stimuli, these fibers induce the production of neurotrophins and tissue neuropeptides, such as NGF (neuronal growth factor), CGRP (genetically related peptide to calcitonin), substance P, and neurokinin A, which help in inducing tissue repair, leading to angiogenesis, and production of tertiary dentin by odontoblasts. Type C fibers receive stimulation in the transition from innate to adaptive immune response, mediated by B and T lymphocytes, which becomes an irreversible symptomatic lymphocyte state. It is characterized by severe, intermittent pain; in addition, the ability to recover pulp tissue is lost because the robust stimulus is not removed. Consequently, there is an increase in blood flow permeability and a transient increase in interstitial tissue pressure that compromises pulp vitality. Studies indicate that the production of TNF, responsible for the process of dilation and increased permeability of blood vessels, decreases dramatically as the process of tissue necrosis occurs. This is because infected necrotic pulp tissue is not accessible to both innate and adaptive immune defense mechanisms⁴⁹-⁵¹.

This neural network, together with fibroblasts, acts in tissue protection by recognizing stimuli that impair its functioning, acting in the secretion and recruitment of resident endothelial cells and stem cells involved in repair. Therefore, relatively low stimuli can contain the destructive advance of the inflammatory immune reaction and promote repair, which is possible by directly interfering with the cause of the problem. Chronic levels of infection can induce an irreversible destructive effect on the pulp tissue, due to the exacerbated recruitment of resident cells and the intense action of the immune response²⁷,⁴⁷,⁴⁸.

Cells involved with the neurogenesis process after its destruction

Fibroblasts, which are the main cells of pulp tissue, secrete into the inflamed pulp important factors for the recruitment of stem cells. These cell types recruited during the inflammatory process are directly involved in repair. Various populations of progenitor cells, including dental pulp stem cells (DPSCs), migrate to injured pulp sites and differentiate into odontoblast-like cells during dentinogenesis and reparative neurogenesis. Neurogenesis acts on the formation of new neurons and involves stages of proliferation, migration, and differentiation of neuronal networks. For the neurogenic process to be initiated, the presence of neural stem cells (NSC) in the surrounding microenvironment is important¹⁶,⁵².

The process of neurogenesis occurs during the pulp repair process and is reduced when the injurious progress of pulp tissue is not stopped. This is because the decrease in vascular supply, modification of the niches and obliteration of the pulp chamber alter the regenerative capacity of stem cells. In this way, the ability of pulp tissue to recover if lost, promoting a decrease in local oxygen supply and necrosis of the pulp tissue⁵³,⁵⁴

In the later stage, necrosis extends beyond the apical foramen and into the periapical tissue, resulting in acute/chronic periodontitis. This lesion occurs because of exacerbated pulp inflammation. Consequently, functional interactions occur between the immune and nervous systems that play important roles in the progression of apical periodontitis. Sympathetic nerves inhibit the production of pro-inflammatory bone-resorbing cytokine IL-1α, which can increase the number of osteoclasts by slowing the progression of local bone resorption. In this process, there are changes in the level of innate immune cells and in neural changes which modulate the inflammatory response. As the density of sensory nerves peaks, tissue repair begins to occur⁵⁵-⁵⁸.

DPSCs have proved to be interesting for dental and non-dental therapeutic applications, at the level of neuronal repair, through the secretion of anti-inflammatory and pro-growth molecular signals, in addition to neurotrophic factors (NTFs). Thus, DPSCs promote a fundamental action in pulp regeneration, since the NTFs released by these cells also regulate the processes of dentinogenesis and repair. This repair process begins with the formation of the vascular network with the local circulatory system and the supply of oxygen and essential nutrients for the elimination of waste, which occurs mainly with the regulatory capacity of DPSCs in inducing platelet-derived growth factor β (PDGFRβ), α-smooth muscle actin (α-SMA), vascular endothelial growth factor (VEGF), platelet-derived growth factor (PDGF), monocyte chemotactic protein-1 (MCP-1), fibroblast growth factor base (bFGF) and endothelin 1 (EDN1)³⁸,⁴⁸.

DPSCs are characterized by the presence of surface markers that determine their properties. Among them are the markers STRO-1, CD29, CD44, CD73, CD90, CD105, CD146, CD166, CD271, CD34, CD117, OCT-3/4 and NANOG. DPSCs can differentiate into neural cells and glial cells, in addition to specific neuronal markers such as neuronal nuclei (NeuNq) that promote neuronal differentiation under neuronal induction conditions. DPSCs grown in neuronal inductive media containing growth factors such as glial cell line-derived neurotrophic factor (GDNF) and brain-derived neurotrophic factor (BDNF) are known to differentiate into neuron-like cells⁵⁹,⁶⁰.

Biotechnological opportunities to regenerate the nervous dental pulp tissue

The regeneration of damaged/lost biological tissues represents a significant challenge for tissue engineering and involves their restoration and return of biological functions. However, for this goal, three objectives must be achieved: elimination of symptoms and evidence of bone healing, increase in root wall thickness and in root length, and a positive response to vitality tests⁶¹-⁶³_. Scientific advances in tissue engineering enable the use of protocols that stimulate tissue regeneration, aiming to imitate the complexity of structures and restore their main functions. The incorporation of synthetic and biological scaffolds with signaling molecules and stem cells are models produced in laboratories that allow in vitro tissue reconstruction. This model, although much studied, still does not allow complete tissue mimicry in line with its specificity. The biggest challenge has been to create an ideal model that enables neural regeneration⁶⁴-⁶⁶.

Given this, more advanced technologies are emerging to reproduce an environment that develops the physiological functions of tissues. This is how bioprinting technologies appear, structures designed to mimic replacement tissues, cell assembly, and biomaterials in a controlled manner⁶⁷-⁶⁸.

Stem cells

Previous studies have provided strong evidence that human dental pulp is an accessible “niche” of stromal stem cells with potential differentiation into adipogenic, osteogenic, chondrogenic, myogenic, and neurogenic lineages without any stimulus for differentiation under basal conditions. It is also important to point out that stem cells from the pulp of permanent teeth represent less than 1% of the cells in the dental pulp. They can produce morphological tissue with functional characteristics like dental pulp, increasing clinical success in conservative endodontic therapies⁶⁹,⁷⁰.

Cells from the pulp tissue remaining from exfoliated primary teeth, SHEDs, were identified as a population of highly proliferative clonogenic cells capable of differentiating into a variety of cell types, including neural cells, adipocytes, and odontoblasts. After in vivo transplantation, SHEDs were found to be able to induce bone formation, generate dentin, and survive in the mouse brain along with the expression of neural markers⁷¹.

The apical papilla is known to be a source of odontoblasts for the pulp tissue, and these odontoblasts are responsible for dentinogenesis, playing an essential role in the root formation process. Studies have shown that apical papilla stem cells (SCAPs) have more significant potential for dentin regeneration than dental pulp stem cells (DPSCs), demonstrating higher levels of essential molecules that act in mediating cell proliferation and differentiation, in addition to expressing a wide variety of neurogenic markers, such as nestin and neurofilament M, after stimulation with a neurogenic medium. For this reason, undifferentiated cells from this tissue have been the focus of several studies in endodontics related to regenerative treatments. Therefore, it was concluded that SCAPs are like DPSCs, but a distinct source of potent dental stem/progenitor cells⁷²,⁷³. Another dental component is the periodontal ligament, which plays a vital role in physically supporting teeth and contributes to the nutrition, homeostasis, and regeneration of adjacent tissues. In addition, it is a niche of stem cells, and, given their specificity, these cells were named periodontal ligament stem cells (PDLSCs). According to the study, which proved the existence of stem cells in the periodontal ligament, they can be obtained by scraping the dental root and then expanded in vitro for future clinical application⁷⁴,⁷⁵.

These cells showed the ability to regenerate rudimentary periodontal ligament and cementum, but subsequent studies revealed that these cells also exhibit osteogenic, adipogenic, chondrogenic, and immunoregulatory/immunosuppressive properties. Given this, several analyses have already demonstrated that stem cells from the periodontal ligament are capable of regenerating periodontal tissues, both the ligament component (connective), as well as bone and cementum, including on implants – as revealed in a study carried out in rats – with possible applicability in humans; and diabetes treatment⁷⁶-⁸².

Thus, stem cells derived from dental tissues have been studied in the treatment of neural injuries as well as neurodegenerative diseases due to their ability to regenerate neural tissue. In 2008, a group of researchers used stem cells from the pulp of human deciduous teeth injected locally into the muscles of puppies with muscular dystrophy (MD). After that, a significant clinical improvement in the dogs was observed; it was suggested, therefore, that this type of cell therapy could delay the progression of the dystrophic process by expressing immunoregulatory paracrine factors⁸²,⁸³.

For degenerative diseases such as Parkinson’s disease, one of the most common neurodegenerative pathologies caused by the loss of dopaminergic neurons, stem cells from the pulp of deciduous teeth, previously induced to neurogenic differentiation in vitro, reduced behavioral deficits after transplantation in Parkinsonian rats. Alzheimer’s disease is another disorder of interest in this area of research. Alzheimer’s is a progressive neurodegenerative disease characterized by a decline in cognitive ability and the formation of myeloid plaques in the brain. Pulp stem cells from deciduous teeth injected intranasally in vivo substantially improved cognitive function in mice with Alzheimer’s. Furthermore, the performance of SHEDs was superior to that of bone marrow stem cells in these animal models¹⁴,⁸³,⁸⁴.

A recent study has compared the neural protection capacity of stem cells from the pulp of deciduous teeth and stem cells from the pulp of permanent teeth with other postnatal stem cells. In adult rats with the spinal cord wholly transected, the cells resulted in the marked recovery of hindlimb locomotor functions. It should also be noted that human nerve pulp stem cells exhibit three central neuro regenerative activities – namely: the cells enhanced apoptosis induced by spinal cord secretion of neurons, astrocytes, and oligodendrocytes, which improved the preservation of neuronal filaments and myelin sheaths; promoted the inhibition of transected axons by directly inhibiting several axon growth inhibitors, including proteoglycans, chondroitin sulfate, and myelin-associated glycoprotein, through paracrine disruption; and replaced the lost cells by differentiating into mature oligodendrocytes under the conditions of spinal cord injury⁸⁵

In this context, the neurogenic potential is already well established, mainly due to the ability of these cells to secrete multiple neurotrophins and which, at least in part, were responsible for stimulating neuroprotection, neurogenesis, and axonogenesis, both in vitro and in vivo for treating optic nerve injuries, and other studies with neural involvement¹³. DPSCs also performed better than bone marrow stem cells restoring neural function after intravitreal transplantation. It is believed, therefore, that these reported facts were due to the origin of these cells being the neural crest and, therefore, presenting neural markers. It is also possible that they are different from other already known types of stem cells, such as those from the bone marrow⁸⁶.

Biomaterials and biomolecules

The ideal materials for dentine-pulp complex regeneration should be biocompatible and have clinical applicability. They should therefore allow adhesion to the cell surface and promote migration, differentiation, and cell function¹⁹,⁶¹,⁸⁷,⁸⁸. The number of nerve fibers in the regenerated pulp determines the degree of sensory function. Given this, neural regeneration is indispensable for maintaining the structure and function of the pulp tissue.

Recent animal studies have reported pulpal regeneration with vascularization and neural innervation; however, there was no significant response to clinical sensitivity test. Similar results were verified for some human patients, as only a few patients with post-treatment pulp sensitivity were reported. Another study, evaluating two dental elements submitted to a revascularization treatment, observed the presence of peripheral afferent neurons in the space of the necrotic canal, suggesting a solid indication of regeneration/repair. Furthermore, these elements responded positively to the sensitivity test, which indicates a possible regenerative success¹⁸,⁸⁹-⁹².

Currently, the biggest challenge has been to find compatible biomaterials associated with stem cells and growth factors that promote regenerative success based on the formation of nerve fibers, complete root formation, absence of periapical lesions, and asymptomatic dental elements after treatment. Endodontics has evolved considerably, with a wide range of concepts and treatment options in this practice. The homing cell technique, for example, has been applied more frequently, followed by methods with natural scaffolds of hydrogels/collagen, PRF, and PRP. These scaffolds, in addition to the characteristics already mentioned, must be degradable after implantation and replaced by newly formed tissues. Degradation of the scaffold too quickly can compromise its cellular support function, while a degradation rate that is too slow can prevent the formation of new tissues⁹³-⁹⁷.

Natural materials such as fibrin, collagen, gelatin, and decellularized matrix offer a favorable environment for differentiation and biodegradability control. In in vitro studies, the results prove that these scaffolds allow significantly greater cell viability, proliferation, and migration. Recent studies have demonstrated that human pulp tissue can regenerate itself using decellularization methods, making use of the dental pulp as a scaffold. It is possible to identify a variety of cells present in the tissue, including stem cells that have the potential to induce differentiation into several different cell classes, including osteoblasts, odontoblasts, neural cells, and adipocytes⁹³,⁹⁸,⁹⁹.

New tissue regeneration strategies are being combined with the aforementioned scaffolds, because there are still major challenges regarding the risk of infection and non-vascular formation. In the search for possibilities for neural inheritance, nanometric materials present physical-chemical and biological properties, because their size favors the ability to interact with cells that promote characteristics of adhesion, migration, and differentiation of fibroblasts, neural and vascular cells. These nanomaterials can be applied alone or accompanied by medications that favor antibacterial activity, or they can also be associated with stem cells and growth factors along with scaffolds, increasing the efficiency of their functionality.

Regenerative endodontic therapy must be associated with a previous decontamination protocol before inducing apical bleeding. The most used protocol includes the use of sodium hypochlorite (NaOCl) due to its antimicrobial potential and ability to dissolve organic matter, associated with ethylenediaminetetraacetic acid (EDTA) due to its ability to accommodate the dentin¹⁰⁰. Recent studies have evaluated the influence of EDTA irrigation on regenerative endodontic processes, and it is possible to observe improved results when compared with groups using only NaOCl¹⁰⁰,¹⁰¹.

In addition, recent studies have evaluated the influence of irrigation with EDTA on regenerative endodontic processes, and better results are observed when compared with groups that used only sodium hypochlorite (NaOCl)¹⁰². These studies reported total closure of the apex while the groups with only NaOCl adopted partial closure, in addition to the release of important cytokines in the endodontic regenerative process, such as IL-1, TGF-β and IGF, favoring cell recruitment and differentiation¹⁰³-¹⁰⁵. Although it has a high capacity to induce cell proliferation and migration, other studies have observed that the use of EDTA after irrigation with NaOCl and without association with saline solution to remove residues can compromise cell differentiation. Another study proved that the activation of ultrasound irrigation can also positively favor cell recruitment. These characteristics may induce the neurogenic potential of DPSCs recruited in the root canal¹⁰⁶,¹⁰⁷.

Pulp regeneration and new perspectives

All recent results are important for tissue regeneration, although they do not present enough clinical data for the clinical applicability of many studied models. These difficulties in translating to human models are due to experiences regarding the use of correct protocols for instrumentation and interference. Human studies, in addition to clinical case studies, should be further explored to obtain accurate results. There are still few studies where there was complete pulp revitalization, although some studies are related to the recovery of partial sensitivity, mainly in electrical tests. In human studies, few recovered sensitivities after regeneration; however, they developed pulp tissue similar to living tissue, increased length/thickness of the root, and remission of the inflammatory/infectious process, with no symptoms. These findings indicate that although neural formation is an indicator of expected success, the results obtained so far indicate a great clinical advance for regenerative endodontic therapy⁹⁷,⁹⁸.

New approaches are being tested in order to enhance the neurogenic potential in regenerative therapies and improve clinical outcomes. Synthetic biology has contributed to the construction of genetic circuits that allow the predictable programming of new cellular functions. These synthetic gene schedules can control the location, timing, and dosage of therapeutic activities in response to disease-specific biomarkers. The construction of culture models and 3D and 4D scaffolds presents conditions for improving the techniques already performed today¹⁰⁸-¹¹².

Synthetic biology has also developed clustered regulatory interspaced short palindromic experiments technology (CRISPR) – Cas9, for example, allows a new genetic configuration in most of the cells present in the organism. DNA is fragmented by genetic processing that stimulates cellular DNA repair change and results in changes to the original DNA sequence¹¹³,¹¹⁴. Tests carried out in vitro and in vivo influence neural influence and the influence of other important tissues. This is mainly due to the genetic ability of this technique to reprogram a type of cell that is present in greater quantity in the body, by another type of reduced cell to restore compromised tissue areas. This strategy may possibly facilitate neural recovery, which remains a research challenge¹¹⁵,¹¹⁶.

A recently conducted study obtained strong results in promoting neural cells, neurite outgrowth in vivo, axons and remyelination by inducing transdifferentiation. The study developed a baculovirus (BV) hybrid vector to target the CRISPRa system in activating BDNF, GDNF and NGDF: these neurotrophic factors were activated for 21 days and promoted a positive result for the sciatic nerve immunity of mice¹¹⁷. These techniques use a viral vector to target the CRISPRa system to the desired location, while techniques with non-viral vectors offer reduced efficiency as they use microparticle or nanoparticle delivery methods. In addition to CRISPRa, genetically modified spheroids also showed controlled regenerative potential in preclinical trials¹¹⁸.

New perspectives are addressed with the aim of reproducing an environment that has all the ideal characteristics and regulation of natural biological processes. The construction of a scaffold/synthetic cell that self-regenerates and self-replicates and that reproduces a natural biological environment has evolved, and despite seeming a distant goal, it represents a great advance for technical engineering.

Despite the advances in dental pulp tissue engineering, challenges remain, such as variability in individual responses and the lack of standardized protocols. The future of this field points to more precise and personalized approaches, using technologies like gene therapy and artificial intelligence to optimize the creation of biomaterials tailored to each patient’s specific needs. Interdisciplinary collaboration and the development of new technologies will be essential for ensuring more effective and predictable regenerative treatments.

Acknowledgments

This study was supported by Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) (305242/2022-9), Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) (200085891), Fundação de Apoio à Pesquisa do Distrito Federal (FAPDF) (00193– 00000782/2021-63 and 00193-00001118/2021-31).

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• Data Availability:
  Datasets related to this article will be available upon request to the corresponding author.

Edited by

• Editor:
  Dr. Altair A. Del Bel Cury

Data availability

Datasets related to this article will be available upon request to the corresponding author.

Publication Dates

• Publication in this collection
  24 July 2026
• Date of issue
  2026

History

• Received
  02 Feb 2024
• Accepted
  30 Jan 2026