Amelogenin as a regenerative endodontic molecule for immature teeth with apical periodontitis. An experimental study

Abstract

Vitality of the dentin-pulp complex depends on cell activity and signalling processes. Amelogenin protein regulates cell signalling pathways during tooth development by activating the Wnt/β-catenin intercellular signalling pathway. This study aimed to regenerate a vascularized pulp using recombinant amelogenin protein, in necrotic root canals by cell homing. Access opening was performed for a total of 120 root canals and were left open to become contaminated with oral microbes for 14 days then cleaned. Canals were divided into 2 groups; in the First group, the canals were filled with amelogenin, while in the 2nd group the canals were left empty. Samples were evaluated histologically and with immunodetection of Sox2, Oct4, Vascular endothelial growth factor (VEGF), Wnt1a, Wnt 3a, Wnt 10b, and Glial Fibrillary Acidic Proteins (GFAP). IC50 was used to determine the cytotoxicity of amelogenin. Regenerated dense cellular tissue was seen in the apical part of amelogenin-treated root canals, and regenerated delicate vascularized tissue was observed in the radicular and pulp chamber. Cells found in the regenerated soft tissue expressed Wnt family members that regulate stem cell pluripotency. Also, Sox2 and Oct4, Pluripotency markers, could be identified in the newly formed apical papilla and dental follicle. Furthermore, VEGF in the regenerated pulps indicated neovascularization. While the GFAP immune reactivity demonstrated that the neuro-sensory organ was being replicated in the regenerated dental pulps. Finally, IC50 test showed that recombinant amelogenin protein has a safe dose at high-level concentrations. Recombinant amelogenin protein induces pulp regeneration most likely from the Sox2 identified stem cells within the apical papilla and can enhance apex formation in non-vital immature teeth.

Graphical abstract

1. Introduction

Dental pulp infections resulting from caries or trauma in immature permanent teeth are the most common challenges to the integrity of a tooth as it matures, both insults can cause loss of vitality of the tooth.¹ Furthermore, tooth fractures are common among traumatized immature permanent teeth due to thin dentin walls and underdeveloped roots.² Consequently, conventional root canal treatment with gutta-percha is contraindicated for infected immature permanent teeth with open root apices and these teeth represent a great clinical challenge. Therefore, studies were directed at regenerating a vascularized pulp in necrotic root canals.³

The investigated regenerative strategies for infected or traumatized immature permanent teeth are either: cell transplantation of ex-vivo cultivated stem/progenitor cells, which are complex procedures of high costs,⁴ or cell homing by molecules that recruit the patient's endogenous cells to achieve tissue repair/regeneration, which is more clinically translatable.^5,6

Homing is the phenomenon whereby cells migrate to the organ of their origin by inducing stem cell mobilization and directing patients' own cells to sites of interest for treating a broad spectrum of diseases or for regeneration. Cell homing in regenerative endodontics has the advantage of the patient's own mesenchymal stem/progenitor cells recruitment into endodontically prepared root canals and inducing them to differentiate along with pulp cell and/or odontoblastic lineages. Recombinant amelogenin protein (RAP) is considered a cell homing molecule tailored as a therapeutic approach for the infected dental pulp of both immature and mature permanent teeth,⁷ Amelogenin expression in vivo is localized mainly around the root, the periodontal ligament, and the alveolar bone.^8,9 It was found that the recruitment of stem cells is facilitated by RAP signalling¹⁰ and the power of stem cells lies in their ability to influence the body's own regenerative processes (Jussila & Thesleff, 2012), as well as, their ability to up-regulate angiogenic and neurogenic differentiation (Ando et al., 2018¹¹; Zhang et al., 2015). Furthermore, amelogenin protein regulates cell signalling pathways during tooth development by activating Wnt/β-catenin signalling pathway.⁸ Wnt signalling pathways are a group of signal transduction pathways that begin with proteins that pass signals into cells through cell surface receptors.¹² There are three signalling pathways; the canonical Wnt pathway, the non-canonical planar cell polarity pathway, and the non-canonical Wnt/calcium pathway.¹³ Amelogenin protein regulates cell signalling pathways during tooth development by activating β-catenin canonical signalling pathways; Wnt signaling molecules and Wnt ligands activate the Wnt/β-catenin signaling pathway, which is one of the several key conserved intercellular signaling pathways in mammals.¹⁴ It plays a fundamental role in proliferation, differentiation, regeneration, and function of cell and tissue types, as well as, multiple and/or complex effects on embryonic stem cell (ES) characteristics. Moreover, it is activated in a dynamic manner during all stages of tooth development and plays multiple roles in these events.¹⁵ The Wnt family of secreted proteins constitutes one such critical pathway: Wnt signalling has multiple and/or complex effects on embryonic stem (ES) cell characteristics. Oct4 is a target of the β-catenin-mediated downstream gene in Wnt-activated cells. SOX proteins also regulate signalling pathways, such as the Wnt pathway by interacting with β-catenin.

Vascular endothelial growth factor (VEGF) was found to be up-regulated by the Wnt signalling pathway.¹⁶ It is a signalling protein that promotes the growth of new blood vessels, it can induce growth of pre-existing (angiogenesis) or de novo vessel formation (vasculogenesis) and is, therefore, one of the keys to embryonic development and vessel repair. Additionally, VEGF has a broad spectrum of functions like chemotaxis, mitogenesis, and angiogenesis.^17,18

Wnt signalling pathway has a part in the differentiation of new neurons and the development of neuronal circuits.¹³ It was found that the essential elements of neuro-sensory organs persist in the glial networks of the human dental pulp, and cells such as seracytes, telacytes, and alacytes that are analogous to radial glia, astrocytes, and microglia of the central nervous system organs (CNS) were identified in peripheral human dental pulp adjacent to odontoblasts.¹⁹ Glial fibrillary acidic protein (GFAP) is a structural protein that helps in maintaining astrocyte mechanical strength and cell shape by regulating the dynamic properties of the cytoskeleton in the cells. In addition, GFAP is involved in the processes of cellular signalling and modulation of neuron-to-glia interactions, therefore, it plays a key role in the development of reactive astrocytosis, i.e., a typical response of the CNS to injury and is considered the classical marker for astroglia.²⁰ The present manuscript presents a novel approach to dental pulp regeneration, focusing on the regeneration of neural elements inside the dental pulp. We hypothesized that recombinant amelogenin protein might regenerate a neural-vascularized pulp in necrotic root canals by cell homing.

2. Methods

2.1. Preparation of amelogenin

Approximately 1 ml of propylene glycol alginate vehicle (PGA) was mixed with 40 mg of RAP powder M180 (180 amino acid mouse amelogenin), pre-weighed using an aseptic technique, then allowed to rest for 15 min before use.

2.2. Preparation of experimental animals

The approval of the animal studies was given by an Institutional Review Board charged with the safety and protection of vertebrate animals at Pharos University. This study included 12 mongrel dogs 6 months of age. Animals were maintained and observed for health assessment one week before any endodontic procedures were performed. A total of 120 root canals from 60 mandibular and maxillary premolars were used in this study. Animals were anesthetized using sodium pentobarbital intravenous injection (30 mg/kg body weight). Pre-operative periapical x-rays were taken to confirm the presence of open apices in all the premolars included in the study.

The length of each dental canal was determined after obtaining endodontic access, the pulp tissue was removed with K-files and the radiographic apex was identified. Hedstrom files were used to completely remove all pulp tissue remnants. One operator performed all the procedures, aided by a support team, over the course of several days.

All the canals were irrigated with distilled water and after achieving haemostasis. The teeth were left without a coronal restoration for 14 days to become contaminated with oral microbes. After 14 days, the canals were cleaned to within 1 mm of the radiographic apices using larger files and gentle filing movements, following the protocol recommended by the American Association of Endodontists for simulating regenerative endodontic procedures using 1.5% sodium hypochlorite and irrigated with Ethylenediaminetetraacetic acid (EDTA). Each canal was then dried with sterile paper points, and the teeth were sealed with a temporary filling (Orafil G, Colostol, Fermin, India).

Seven days after closure, the temporary filling was removed, and the canals were divided into the following groups according to the medicament they were receiving:

• 1.Group 1: 100 canals filled using an angulated syringe with amelogenin (completely filling the canal).
• 2.Group 4: 20 teeth that were the negative controls (left without intracanal filling)

Intermediate restorative material (glass ionomer) was then placed, and the access cavity was sealed. Following each operation, the animals received an intravenously delivered painkiller; Voltaren (Novartis Pharma Egypt, under license from Novartis Pharma, Switzerland), and 25 mg/kg. Amoxycillin (Cid Co, Egypt) was administered intramuscularly on the first day and thereafter mixed with food for 7 additional days at a dose of 15 mg/kg. A Soft diet is used postoperatively to avoid traumatizing the operated teeth.

After 1, 3 months postoperatively, the animals were euthanized by intravenous overdose injection of thiopental sodium. The teeth and surrounding bone were removed as a block with a water-cooled diamond disc and samples were evaluated.

2.3. Histology and immunodetection

The treated teeth were recovered at 1 and 3-month time after treatment, then samples were demineralized, and standard histologic procedures were used to prepare tissue sections of 5 μM thickness that was either stained using hematoxylin or eosin or trichrome stain or used for immunodetection.

Immunodetection of several markers was performed. Antibodies for Wnt 1a, Wnt 3a, Wnt 5a, Wnt 10b, Sox2, and Oct4 were obtained from Abcam (Cambridge, UK, ab15251, ab19925, ab 179824, ab 92494, ab 5603 and Anti-Oct4 antibody [EPR2054] respectively), whereas antibody for GFAP was obtained from Spring Bioscience (CA, USA, M3781) and VEGF antibody was obtained from Santa Cruz Biotechnology VEGF Antibody (C-1): sc-7269. All steps were performed following the handling procedures and titters recommended by the supplier. Conjugated secondary antibodies were obtained from Thermo Fisher Scientific (Fremont, CA) and used at a dilution of 1:2000. Sections were imaged using a Nikon Eclipse 80i microscope (Tokyo, Japan) with BX 51 digital camera (Tokyo, Japan) and digital images were recorded.

2.4. Estimation of cytotoxicity

Cytotoxicity of amelogenin was measured on peripheral blood mononuclear cells (PBMCs) to determine the safe dose.

2.4.1. Cell line: PBMCs isolation

The PBMCs were selected for their ease of accessibility and manipulation. Donor PBMCs were prepared by Ficoll density gradient centrifugation of heparinized blood.²¹

2.4.2. Drug exposure

Under aseptic conditions, media was aspirated from all wells in the 96 plates (PBMCs plate was centrifuged first for 10min at 200×g to pellet cells) then 200 μL of growth medium with the corresponding drug concentration loaded into each well. Six wells in the plate received no test compound and were loaded only with the growth medium and the average absorption of these control wells was used for the plate-correction factor.

2.4.3. Estimation of cytotoxicity (neutral red cytotoxicity assay)

A neutral red cytotoxicity assay was used to determine the IC₅₀ on PBMCs.^22,23 A freshly neutral red solution was prepared from a 0.4% aqueous stock solution and shielded from light. A 1:80 dilution of the stock solution was prepared, allowed to precipitate for 24h at room temperature, and centrifuged for 10 min at 1500×g. A clear red solution was used for the assay. PBS (150 μL/well) was used for washing the cells. A neutral red medium (100 μL) was added to each well and incubated for 2 h at 37 °C. The neutral red medium was aspirated then the cells were washed with 150 μL PBS. The plate was tapped gently the washing solution was aspirated. Neutral red de-stain solution (1% acetic acid, 49% de-ionized water, and 50% ethanol) were added as 150 μL/well, and the plates were shaken rapidly on a microtiter plate shaker (Shaker PSU 2T plus, BOECO, Germany) for at least 10 min.

2.4.4. Percentage of cytotoxicity (inhibition)

The optical density (OD) of the neutral red extract was measured at 540 nm in a microtiter plate reader spectrophotometer (Spectrostar^Nano, BMG Labtech), using blanks that contain no cells as a reference. Grubb's test for outliers, also called the ESD method (extreme studentized deviate), to determine whether one of the values in the list was a significant outlier from the rest. The percentage of cytotoxicity (inhibition) was calculated according to the following formula: $\%\text{inhibition}=100-(\frac{\text{O}.\text{D Control}-\text{O}.\text{D Treatment}}{\text{O}.\text{D Control}})$

3. Results

3.1. Histological and immunohistochemical results

Amelogenin did not only regenerate the pulp tissue but also induced the proliferation and regeneration of the apical papilla and dental follicle. However, in the control group, canals showed no regenerated pulp tissues inside the pulp space. Most root canals were still opened and were enclosed by granulation tissue around the thin dentin walls (Fig. 1, Fig. 2A).

Fig. 1.

(A) 1-month post-treatment, control group; canals showed no regenerated pulp tissues inside the pulp space. Most root canals were still opened and were enclosed by granulation tissue around the thin dentin walls. H&E stain, original magnification X40. (B) 1-month post-treatment with amelogenin; canals showed no regenerated pulp tissues inside the pulp space and showed thick dense apical papilla and dental follicle at the periphery of the papilla. H&E stain, original magnification X40. (C) 1-month post-treatment, most root canals were still opened and were enclosed by dense cellular apical papilla and dental follicle cells. Dense cellular tissue representing the regenerated pulp was seen within the newly formed dentin. H&E stain, original magnification X40. (D) 1-month post-treatment showed dense immune reactivity to vascular endothelial growth factor VEGF in the regenerated pulp. VEGF antibody, original magnification X100. (E) 1-month post-treatment control group; showed no immune reactivity to Sox2 proteins in granulation tissue. Pulp space shows no regenerated pulp tissue. Sox2 antibody, original magnification X40. (F) 1-month post-treatment apical root region showed intense immune reactivity to Sox2 proteins in apical papilla, revealed APSC, and dense to moderate immune reactivity in the dental follicle. Pulp space shows no regenerated pulp tissue. Sox2 antibody, original magnification X40. (G) An adjacent tissue section of the boxed area in D showed intense immune reactivity to Oct4 proteins in the apical papilla revealed the APSC that are invading the empty pulp space (black arrow), Oct4 antibody, original magnification X200. (H) 1-month post-treatment showed moderate immune reactivity to Wnt 5a in the soft tissue of regenerating pulp and apical papilla, Wnt 5a antibody, original magnification X40. P pulp, AP apical papilla, DF dental follicle, D dentin, PS pulp space, B bone, PDL periodontal ligament, APSC apical papilla stem cells.

Fig. 2.

(A) 3-month post-treatment, control group; most root canals were still opened and showed no regenerated pulp tissues inside the pulp space. Canals were enclosed by dense granulation tissue. H&E stain, original magnification X40. (B) 3-month post-treatment with amelogenin; showed vascular element in dental follicle entering the root canal that was filled with delicate regenerated pulp tissue. Trichrome stain, original magnification X100. (C) 3-month post-treatment showed moderate to mild immune reactivity to Glial Fibrillary Acidic Protein antibody GFAP in regenerated pulp, apical papilla, and dense IR in the pulp periphery. GFAP antibody, original magnification X40. (D) 3-month post-treatment showed intense immune reactivity to Wnt 3a in the regenerated pulp. Wnt 3a antibody, original magnification X40. (E) 3-month post-treatment, control group; showed no immune reactivity to Oct 4 proteins in granulation tissue. Pulp space shows no regenerated pulp tissue. Oct4 antibody, original magnification X40. (F) 3-month post-treatment showed intense immune reactivity to the Wnt10b antibody in the regenerated pulp. Wnt 10b antibody, original magnification X40. (G) Higher magnification of the boxed area in (I) showing dense immune reactivity to Wnt 10b antibody in odontoblast and pulp periphery, while pulp core showed intense-moderate immune reactivity. Wnt 10b antibody, original magnification X400. (H) 3-month post-treatment showed intense to moderate immune reactivity to Wnt1a antibody in odontoblasts, cementoblasts, and regenerated pulp. Original magnification X100. P Pulp, AP Apical papilla, DF Dental follicle, D Dentin, PS Pulp space, Granulation tissue GT, B Bone, PDL Periodontal ligament, BV Blood vessel, APSC apical papilla stem cells, OD odontoblasts, CMBL cementoblasts.

The apical papilla: Showed a thick cellular proliferation surrounding the growing root apices (Fig. 1B, C, F, and G). The cells of the papilla showed intense cellular IR to transcription stem cell regulators Sox2 and Oct4 (Fig. 1F and G), no reaction to Sox2 or Oct4 was seen in the control group (Figs. 1E and 2E, respectively), and intense IR to VEGF (Fig. 1D), but mild IR to GFAP (Fig. 2C). The cells of the papilla also showed intense to moderate IR to Wnt proteins (Figs. 1H, Fig. 2F–H). (Table 1)

Table 1.

Immunodetection of several markers in different areas.

Signalling molecule	Apical papilla	Dental follicle	Regenerated pulp	odontoblasts	Pulp periphery
Wnt 1a IR	++++	++++	++++	++++	++
Wnt 3a IR	++++	+++	++++	++	+
Wnt 5a IR	+++	+++	++	++	+
Wnt 10 b IR	++++	+++	++++	++++	++++
Sox 2 IR	++++	+++	++++	++++	++++
Oct 4 IR	++++	+++	++++	+++	+++
VEGF IR	++++	++++	++++	–	++++
GFAP IR	+	+++	+++	++++	++++

IR immune reactivity.

VEGF vascular endothelial growth factor.

GFAP glial fibrillary acidic protein.

++++ intense immune reactivity.

+++ intense-moderate immune reactivity.

++ moderate immune reactivity.

+ mild immune reactivity.

---- no immune reactivity.

The dental follicle: The regenerated part of the follicle surrounded the apical papilla and growing root apices (Fig. 1C). It showed intense IR to VEGF (Fig. 1D) and to stem cell factors Sox2 and Oct4 (Fig. 1F and G), no reaction to Sox2 or Oct4 was seen in the control group (Figs. 1E and 2E, respectively). It also showed vascular elements migrating to enter the regenerated pulp tissue (Fig. 2B). The dental follicle harbored stem cells that showed intense-moderate IR to Wnt proteins (Figs. 1H, Fig. 2F–H), but mild IR to GFAP (Fig. 2C). (Table 1)

The regenerated pulp tissue: The pulp was loaded with stem cells entering the pulp from apical papilla stem cells (APSC) (Fig. 1F and G). It showed a great cellular proliferation inside the once empty canals (Fig. 2B–D). It showed IR to the Wnt family members (Figs. 1H, Fig. 2F–H), the IR was intense to Wnt 1a, Wnt 3a, and Wnt 10b but moderate to Wnt 5a and Sox2, with increased cellular IR to Wnt 10b at the pulp periphery (Fig. 2G). It also revealed a vascular network with intense IR to VEGF (Fig. 1D) and neuronal elements IR to GFAP especially at the pulp periphery (Fig. 2C). (Table 1)

The odontoblasts in the regenerated pulp: Showed intense IR to Wnt 1a (Fig. 2H) and Wnt 10b (Fig. 2G) but moderate IR to Wnt 3a and Wnt 5a (Fig. 1, Fig. 2H), respectively. It showed intense to moderate IR to GFAP (Fig. 2C) and no IR to VEGF (Fig. 1D). (Table 1)

The periphery of the regenerated pulp tissue: Showed intense IR to Wnt 10b (Fig. 2F and G), Sox2 (Fig. 1F), VEGF (Fig. 1D), and GFAP (Fig. 2C), while it showed mild IR to Wnt 1a.

(Fig. 2H), Wnt 3a (Fig. 2D), Wnt 5a (Fig. 1H), and intense to moderate IR to Oct4 (Fig. 1G). (Table 1)

Cytotoxicity result: Neutral red cytochrome assay IC50 for Drug = 0.486 mg/m. The amelogenin treatment with has a safe dose at a high-level concentration according to the IC50 (Table 2).

Table 2.

Cytotoxicity assay.

Sample	Concentration (mg/ml)	Viability %	Inhibition %
	5	18	82
	2.5	26	74
	1.25	36	64
	0.625	54	46
	0.3125	59	41
	0.1562	71	29
	0.0781	92	8

IC50 for Drug = 0.486 mg/ml.

4. Discussion

The success of regenerative endodontic therapy depends majorly on pulp regeneration. The ultimate goal of regenerative endodontics in mature teeth with opened apex and pulp necrosis is the resolution of clinical signs and symptoms with maturation and completion of the roots, restoring the lost attachment apparatus, the regeneration of the pulp, and return of vasculogenesis and neurogenesis to restore the tooth vitality.¹¹ A unique and complex neural control system is triggered by chewing, this system is established to protect teeth’ structure. The ability of a tooth to endure the rigors of mastication depends on having a complex neural control system to maintain the tooth's integrity. The cornerstone of this neural control system is the intact dental pulp.²⁴

Several biological advantages for pulp-dentin regeneration are maintained. For example, tooth homeostasis and improved immune defence system. On top is promoting root development which represents a clinical advantage.²⁵ The regeneration and preservation of vital dental pulp with vasculature and nerve components remain one of the significant challenges in modern therapies.⁷

Various experimental conditions showed regeneration of pulp-like tissue (Orti et al., 2018). The field of regenerative endodontics relies on stem cell activation and in vivo cell mobilization and homing inducing endogenous reparative cells. The regenerative powers of RAP may be due to the utilization and activation of the canonical Wnt/β-catenin signalling. Which in turn increases nuclear and cytoplasmic β-catenin. DNA replication is initiated by the increased β-catenin and eventually cell growth and proliferation of stem cells. Wnt/β–catenin signalling is activated in a dynamic fashion in cranial neural crest cells.¹⁰ All regenerated tissues that are seen post amelogenin therapy revealed the expression of Wnt family members. Wnt 1a was expressed in apical papilla, dental follicle, regenerated pulp tissue, odontoblasts, and to a lesser extent in regenerated pulp periphery. Wnt 3a was expressed mainly in the regenerated pulps, while Wnt 5a was expressed in the regenerated pulps, apical papilla, and periapical tissues, and Wnt 10b expression in regenerated pulps, especially pulp peripheries and odontoblasts.

The common feature seen in this study is the replacement of the necrotic, destructed, and inflamed periapical tissues with regenerated tissues following amelogenin therapy; the previously bare infected periapical area is now filled with a thick, dense cellular cushion representing the regenerated apical papilla that showed the presence of abundant stem/progenitor cells, apical papilla stem cells that were immune reactive to Sox2 and Oct4 transcription factors It lies quite near to it are the dental follicle cells.

The apical papilla and dental follicle not only represent derivatives of the neural crest but were also loaded with stem cells showing immune reactivity to Sox2 and Oct4 pluripotent factors. As early as one month, all surrounding stem cells invade the empty canal. Some vascular elements from the dental follicle may migrate into the opened canal too creating pulp vasculature. The regenerated pulp tissue showed immune reactivity to vascular endothelial growth factor VEGF indicating growth of new blood vessels, chemotaxis, mitogenesis, and angiogenesis and consequently healing and regeneration. Wnt/β–catenin signalling pathway was found to strongly up-regulate VEGF⁹ and is associated with embryonic stem cell markers (Sox2 and Oct) expression¹⁷; Liu et al., 2011.¹⁶;

Pluripotency Oct4 and Sox2 factors have higher expressions in the apical papilla and cells that invade the empty canals representing the regenerated pulps. Oct4 pluripotent marker accentuates Sox2 signalling which in turn regulates Wnt/β–catenin signalling pathway by interacting with β-catenin. Oct4 enhances the function of Sox2 by forming a trimeric complex on DNA that controls the expression of several genes involved in directing it to a key pluripotent transcription factor and a neural factor forming a continuous link of enhancing pluripotency and neural differentiation. After Wnt/β–catenin pathway is activated, it is self-sustaining.²⁶ This may explain the unique regeneration ability of the amelogenin protein (Bernard & Harley, 2010; Liu et al., 2011; Zhang et al., 2019). The immunoreactivity to Sox2 at an early stage of dental pulp regeneration suggests neural initiation and differentiation. It is highly expressed in proliferating neural progenitor cells in the apical papilla and in the cells that invade the empty canals and is also expressed in post-mitotic neuronal and glial cells. This transcription factor may be partly responsible for regulating the pluripotency and self-renewal of stem cell apical papilla surrounding the regenerated root apices, and their adoption of a neural fate (Liapatas et al., 2003; Nusse et al., 2008).

In previous studies using amelogenin therapy, the apical papilla was present in around 100% of roots at 1 month and persisted in 57.5% of roots till the third month, in addition, peripherin neuronal intermediate filaments and Calcitonin gene-related peptide were released from sensory nerves were seen in the regenerated pulps indicating the presence of nociceptive nerves fibres similar to authentic pulp innervation¹¹; Miki et al., 2011; Zhang et al., 2014). In the present study immunoreactivity to GFAP was seen in-between and underneath odontoblast cells, around vascular elements, and at the periphery of the regenerated pulps mainly, and to a lesser extent, dispersed in the core of the regenerated pulp. This arrangement is similar to GFAP immune reactivity in the authentic pulp. The development and differentiation of new neurons and maintenance of the stem cell pool are related to the Wnt signalling pathway. It also has a part in the development of neuronal circuits.¹³ The intense immune reactivity at the regenerated pulp periphery may be interpreted by the ability of GFAP for active regeneration following nerve injury by increasing the number of astroglial cells i.e., reactive astrocytosis to regenerate neurons in the empty canals and guide the regeneration process.¹⁹ The regenerated pulp periphery showed cellular condensation immune reactive to Wnt 10 b. This may be indicative that the cumulative effect of these factors leads the pulp regeneration towards the crown to fill the whole pulp canal and chamber, which can be useful in clinical endodontics as it can easily induce pulp regeneration in addition to the regeneration of the lost periapical tissues due to periodontitis in one or two visits only.

5. Conclusions

We conclude that recombinant amelogenin protein has a safe dose at a high-level concentration and induces pulp regeneration most likely from the Sox2 identified stem cells within the apical papilla and can enhance apex formation in non-vital immature teeth. It can be used clinically as a therapy in regenerative endodontic procedures and for the treatment of periapical pathology.

Authorship confirmation statement

MM conceived the idea, designed the study, drafted the manuscript, and supervised the experimental part. FR contributed to the experimental part, collected and analyzed the data, and reviewed the drafts. SB gave suggestions and significantly revised and refined the manuscript. All authors read and approved the final manuscript.

Author disclosure statement

The authors have no conflicting interests.

Funding statement

This work was supported in part by Misr El Kheir Foundation, Egypt with Grant Number: 3993.