Exploiting Dentine Matrix Proteins in Cell-Free Approaches for Periradicular Tissue Engineering

Abstract

The recent discovery of mesenchymal stem cells within periapical lesions (PL-MSC) has presented novel opportunities for managing periradicular diseases in adult teeth by way of enhancing tissue regeneration. This discovery coincides with the current paradigm shift toward biologically driven treatment strategies in endodontics, which have typically been reserved for non-vital immature permanent teeth. One such approach that shows promise is utilizing local endogenous non-collagenous dentine extracellular matrix components (dECM) to recruit and upregulate the intrinsic regenerative capacity of PL-MSCs in situ. At picogram levels, these morphogens have demonstrated tremendous ability to enhance the cellular activities in in vitro and in vivo animal studies that would otherwise be necessary for periradicular regeneration. Briefly, these include proliferation, viability, migration, differentiation, and mineralization. Therefore, topical application of dECMs during ortho- or retrograde root canal treatment could potentially enhance and sustain the regenerative mechanisms within diseased periapical tissues that are responsible for attaining favorable clinical and radiographic outcomes. This would provide many advantages when compared with conventional antimicrobial-only therapies for apical periodontitis (AP), which do not directly stimulate healing and have had stagnant success rates over the past five decades despite significant advances in operative techniques. The aim of this narrative review was to present the novel concept of exploiting endogenous dECMs as clinical tools for treating AP in mature permanent teeth. A large scope of literature was summarized to discuss the issues associated with conventional treatment modalities; current knowledge surrounding PL-MSCs; composition of the dECM; inductive potentials of dECM morphogens in other odontogenic stem cell niches; how treatment protocols can be adapted to take advantage of dECMs and PL-MSCs; and finally, the challenges currently impeding successful clinical translation alongside directions for future research. Impact statement Apical periodontitis (AP) is an inflammatory condition that is associated with a great degree of morbidity and ultimately leads to tooth loss. The purpose of this review was to summarize the current evidence pertaining to stem cell therapy in endodontics and present a novel clinical methodology through which they may be utilized to address AP. A comprehensive overview of the basic science, clinical translation, and potential challenges are presented in this review.

Keywords: dentine extracellular matrix components; endodontics; regenerative medicine; stem cells; tissue regeneration; wound healing.

Conflict of interest statement

All authors declare no potential conflicts of interest with respect to the authorship and/or publication of this article.

Figures

FIG. 1.

(A–C) A schematic illustration of osteogenic, adipocytic, and neuronal differentiation pathways in mesenchymal stem cells. Akt, protein kinase B; BDNF, brain-derived neurotrophic factor; BMP, bone matrix protein; Ca2+, calcium ions; CEBP, enhancer binding protein; CREB, cAMP response element-binding protein; EGF, epithelial growth factor; ERK, extracellular signal-regulated kinases; FGF, fibroblast growth factor; GDNF, glial cell line-derived neurotrophic factor; GR, glucocorticoid receptor; HES, hairy and enhancer of split-1; IGF, insulin-like growth factor; Jnk, c-Jun N-terminal kinases; MAPK, mitogen-activated protein kinase; PI3K, phosphoinositide 3-kinase; PPAR, peroxisome proliferator-activated receptor; Runx2, runt-related transcription factor 2; TGFβ, transforming growth factor beta; TNF-α, tumour necrosis factor-alpha; Wnt, wingless/integrated. Color images are available online.

FIG. 2.

Multipotent potential of primary PL-MSCs. (A) PL-MSCs were isolated from the apical granuloma of extracted teeth via a collagenase type 1 enzyme digestion technique. Cells were cultured in a T25 flask with 20% fetal bovine serum supplemented α-MEM media, which was changed every 2 days. (B, C) Phase-contrast microscopy at 10 × magnification of PL-MSC cultures at day 1 (B) and day 7 (C). (D–G) Osteogenic differentiation after 21 days of culture with control or osteogenic induction media (α-MEM, 20% FBS, 1% penicillin/streptomycin, 2 mM glutamine, 0.2 mM ascorbic acid, 100 nm dexamethasone, 10 mM β-glycerophosphate). Staining with Alizarin Red S confirmed absence in control wells (D) and presence in test wells of mineral deposits (E, F). Staining was solubilised with 10% acetic acid, and subsequent intensity was quantified by using a microplate reader with an excitation wavelength set at 405 nm (G). (H–K) Adipogenic differentiation after 21 days of culture with control or adipogenic induction media (α-MEM, 20% FBS, 1% penicillin/streptomycin, 2 mM glutamine, 0.5 mM IBMX, 200 μM indomethacin, 10 μM insulin, 1 μM dexamethasone). Staining with Oil Red O confirmed absence in control wells (H) and presence in test wells of lipid droplets (I, J). Staining was solubilized with isopropanol, and subsequent intensity was quantified by using a microplate reader with an excitation wavelength set at 540 nm (K). All experiments were conducted up to passage 2 by using three biological replicates. Scale bars represent 1000 μm. Color images are available online.

FIG. 3.

Human growth factor anti-body array of lyophilized dECM components extracted from dentine powder using 10% EDTA. A semi-quantitative autoradiographic image analysis technique was used to determine the relative radiographic intensity for a total of 41 different cytokines. A representative autoradiographic image is displayed in the top left corner. AR, amphiregulin; EGFR, epidermal growth factor receptor; GCSF, granulocyte colony-stimulating factor; GM-CSF, granulocyte macrophage colony-stimulating factor; HB-EGF, heparin-binding epidermal growth factor; HGF, hepatocyte growth factor; IGFBP, insulin-like growth factor binding protein; M-CSF, macrophage colony-stimulating factor; M-CSFR, macrophage colony-stimulating factor receptor; NGF, nerve growth factor; NT, neurotrophin; PDGF, platelet-derived growth factor; PDGFR, platelet-derived growth factor receptor; PlGF, placental growth factor; SCF, stem cell factor; SCFR, stem cell factor receptor; VEGF, vascular endothelial growth factor; VEGFR, vascular endothelial growth factor receptor. Color images are available online.

FIG. 4.

Data obtained from our research group demonstrating the deleterious effects of sodium hypochlorite on the solubilization of dentine extracellular matrix components when delivered into prepared root canals of extracted mature permanent human teeth (n = 10) via conventional needle irrigation. A sandwich ELISA technique was used to detect TGF-β1 concentration (pg/mL) from 100 μL of EDTA after irrigation with or without 2% sodium hypochlorite. Error bars represent standard deviation. Color images are available online.

FIG. 5.

A schematic illustration of the proposed protocol for enhancing periradicular tissue regeneration in mature permanent teeth by using endogenous dECM components. (A) Single-rooted mature permanent tooth diagnosed with apical periodontitis; (B) accessing pulp chamber and conservative pre-enlargement of apical foramen; (C) chemomechanical preparation of root canal using a chelating agent; (D) passive ultrasonic activation of irrigant to stimulate release of dECMs into the root canal; (E) manual dynamic activation to encourage periapical bioavailability of dECMs; (F) interappointment calcium hydroxide medicament; (G) irrigation and passive ultrasonic activation to release dECMs; (H) manual dynamic activation to encourage periapical bioavailability of dECMs; (I) obturation; (J) annual clinical and radiographic review. Color images are available online.