Expression of Toll-like Receptors in Stem Cells of the Apical Papilla and Its Implication for Regenerative Endodontics.

Regenerative therapies to replace cells and tissues damaged due to trauma and dental infections require temporal and spatial controlled recruitment and the differentiation of progenitor/stem cells. However, increasing evidence shows microbial antigens can interfere with this process. Toll-like receptors (TLRs) are crucial in recognizing pathogen-associated molecular patterns. Stem cells of the apical papilla (SCAP) are required for normal dental development and are intimately involved in the reparative and regenerative capacity of developing teeth. We hypothesized that TLRs are expressed in SCAP and that the activation of TLR2/TLR4 or TLR3 by different ligands results in differential cellular fate, impacting their differentiation into a mineralizing phenotype. We found that most TLRs are expressed as detected by PCR except TLR7 and TLR8; exposure to heat-killed E. coli results in upregulating TLR2 and TLR4 and reducing mineralization capacity. In addition, bacterial exposure resulted in the upregulation of 11 genes, of which 9 were chemokines whose proteins were also upregulated and released, promoting in vitro macrophage migration. On the other hand, TLR3 activation resulted in increased proliferation and a dramatic inhibition of osteogenic and odontoblastic differentiation, which was reversed by inhibition or the knockdown of TLR3 expression. The profound effects of TLR activation resulting in different cell fates that are ligand and receptor-specific warrants further evaluation and represents an important therapeutic target to make regenerative approaches more predictable following dental infections.

Effect of 3 Bioceramic Materials on Stem Cells of the Apical Papilla Proliferation and Differentiation Using a Dentin Disk Model.

INTRODUCTION: There is a complex interaction between biomaterials placed as a coronal barrier with stem cells and dentin in regenerative procedures. In this study, the effect of Biodentine (BD; Septodont, Saint-Maurdes-Fossés, France), Endosequence BC Root Repair Material-Putty (ES; Brasseler, Savannah, GA), Endosequence BC Root Repair Material-Putty Fast set (ES-fast, Brasseler), and ProRoot (Dentsply Tulsa Dental Specialties, Johnson City, TN) mineral trioxide aggregate (MTA) on the viability and differentiation of stem cells of the apical papilla (SCAP) was evaluated using an ex vivo dentin disk model. METHODS: Standardized human dentin disks were treated using an established protocol. Disk lumens were filled with BD, ES, ES-fast, or MTA, and SCAP were cultured directly onto the samples. Cell viability was measured at 7 days, whereas differentiation into a mineralizing phenotype was evaluated by real-time reverse-transcription polymerase chain reaction and alizarin red staining at 21 days in culture. Results were analyzed using 1-way analysis of variance with the Bonferroni post hoc test or the Mann-Whitney U test (P ≤ .05). RESULTS: All materials promoted SCAP viability and proliferation with a greater response in the BD and ES groups. Also, a greater expression of alkaline phosphatase messenger RNA and dentin sialophosphoprotein was noted in the BD and ES groups, whereas MTA promoted a greater expression of the osteoblastic marker IBSP. Interestingly, no difference in alizarin red staining was observed with MTA, BD, or ES, which were significantly greater than ES-fast. CONCLUSIONS: These data suggest that BD and ES promoted greater survival and differentiation of SCAP and the increase of the odontoblastic marker DSPP, whereas MTA appeared to promote greater osteoblastic differentiation. Thus, BD and ES can be considered for regenerative and vital pulp therapies.

Evaluation of mesenchymal stem cell modulation of trigeminal neuronal responses to cold.

Tissue engineering protocols, such as regenerative endodontic procedures (REPs), comprise biologically based procedures designed to restore normal physiologic function. For REPs, the goal is reconstitution of the pulp-dentin complex by delivering mesenchymal stem cells (MSCs), including the stem cells of the apical papilla (SCAP) into a root canal system. Many patients regain cold sensitivity after REPs, but the mechanism is not understood. We hypothesized that SCAP modulate nociceptive function through a paracrine mechanism that activates cold-sensitive ion channels in neurons. We established a co-culture system with human SCAP and rat trigeminal (TG) sensory neurons in order to determine the effect of SCAP co-culture on neuronal responses using whole-cell patch-clamp electrophysiology. TG neurons co-cultured with SCAP demonstrated increased TRPA1-mediated (p<0.01) and TRPM8-mediated inward current densities (p<0.01) at 24h in co-culture. Cold stimulation to SCAP significantly increased ATP release (p<0.01), and supernatant collected after cold stimulation to SCAP was able to activate cultured TG neurons. Co-culture with SCAP significantly increased sustained ATP-evoked inward current density (p<0.05). These data suggest that SCAP release trophic factors that act on afferent neurons to enhance cold-sensitive ion channel activity.

Comparative Evaluation of Chemotactic Factor Effect on Migration and Differentiation of Stem Cells of the Apical Papilla.

INTRODUCTION: Cell homing strategies could potentially be used in regenerative endodontic procedures (REPs) to promote the progressive coronal migration of stem cells, including stem cells of the apical papilla (SCAPs), along with formation of a new vascular network without the need for intentional apical trauma and intracanal bleeding. Although many chemotactic factors have been investigated for different mesenchymal stem cells, their effect on SCAP migration and differentiation is not fully understood. This study aimed to comparatively evaluate the effect of stromal cell-derived factor 1 (SDF-1), transforming growth factor beta 1 (TGF-ß1), platelet-derived growth factor, granulocyte colony-stimulating factor (G-CSF), or fibroblast growth factor 2 (FGF-2) on the migration and differentiation of SCAPs. METHODS: A characterized SCAP cell line was fluorescently labeled with Vybrant DiO dye (Life Technologies, Grand Island, NY) and used in transwell migration assays. Cells were subjected to 1, 10, or 100 ng/mL of each factor or a combination of factors followed by detection in a fluorescent plate reader. Lastly, SCAP differentiation into a mineralizing phenotype was evaluated in the presence or absence of the tested factors by quantitative alizarin red staining and alkaline phosphatase activity. Data were analyzed with 1-way analysis of variance with the Tukey post hoc test. RESULTS: Maximum migration was observed with G-CSF or FGF-2, which was significantly greater than the effects observed by the other tested factors. A combination of G-CSF with TGF-ß1 significantly augmented both migration and differentiation into a mineralizing phenotype. CONCLUSIONS: G-CSF appears to be well suited to be further investigated as a key chemotactic factor in cell homing-based regenerative endodontic procedures.

Effect of Bacterial Biofilm on the Osteogenic Differentiation of Stem Cells of Apical Papilla.

INTRODUCTION: Although clinical success in regenerative endodontics is substantially high, histological success is limited to finding bone/cementum-like tissue instead of dentin within the canal space. The aims of this study were to investigate (1) the effect of bacterial biofilm on osteogenic gene expression in stem cells of the apical papilla (SCAP) and (2) the effect of bacterial antigens on the functional differentiation of SCAP into a mineralizing phenotype. METHODS: Using an ex vivo organotypic root canal model and an American Association of Endontists-recommended regenerative endodontic procedures, we evaluated SCAP differentiation in the presence and absence of an Enterococcus faecalis biofilm. Gene expression analysis for dentinogenic and osteoblastic markers was performed with real-time polymerase chain reaction. The effect of E. faecalis antigens on SCAP differentiation into mineralizing cells in vitro was evaluated with 2 functional assays: Alizarin Red and alkaline phosphatase activity assays. RESULTS: After regenerative endodontic procedures, residual bacteria continued to sustain within the root canal system. SCAP in the presence of E. faecalis biofilm significantly downregulated dentinogenic genes such as dentin sialophosphoprotein and upregulated osteoblastic genes such as bone sialoprotein, osteocalcin, distal-less homeobox 5, and runt-related transcription factor 2. E. faecalis antigens significantly inhibited SCAP differentiation into a mineralizing phenotype when alizarin red staining and alkaline phosphatase assays were used in vitro. CONCLUSIONS: Current disinfection protocols were ineffective in eliminating bacteria from root tips and the levels of the residual bacterial biofilm, and its byproducts, were able to significantly alter osteogenic-differentiation of SCAP.

Cathepsin K Inhibitor Regulates Inflammation and Bone Destruction in Experimentally Induced Rat Periapical Lesions.

INTRODUCTION: Cathepsin K is highly expressed in osteoclasts and plays an essential role in bone resorption. NC-2300 is an artificially designed cathepsin K inhibitor, and its application to experimentally induced arthritis induces down-regulation of bone destruction. In this study, we evaluated the effects of NC-2300 on inflammation and bone destruction in experimentally induced rat periapical lesions. METHODS: The dental pulps of lower first molars in rats were extirpated, and the pulp chambers were left open to the oral environment. NC-2300 and phosphate-buffered saline were administered orally twice a day in the experimental and control groups, respectively. Animals were sacrificed on day 21, and the mandibles were extracted. The left hemimandibles were used for micro-computed tomographic and histologic examination. For the right hemimandibles, RNA was extracted from the periapical tissues surrounding the root apices, and inflammatory mediator expression was examined by real-time polymerase chain reaction using complementary DNA converted from extracted RNA. RESULTS: The size of the periapical lesion, number of tartrate-resistant acid phosphatase-positive osteoclasts and major histocompatibility complex class II molecule-expressing macrophages in the experimental group decreased significantly when compared with the control group. The expression of proinflammatory cytokines in the experimental group was significantly suppressed when compared with the control group. CONCLUSIONS: These results suggest that the cathepsin K inhibitor may inhibit not only cathepsin K activity in osteoclasts but also inflammatory mediator synthesis relating to osteoclastogenesis, and these synergistic effects may be involved in the suppression of periapical lesion expansion.

Down-regulation of inflammatory mediator synthesis and infiltration of inflammatory cells by MMP-3 in experimentally induced rat pulpitis.

INTRODUCTION: Matrix metalloproteinase (MMP)-3 is a member of the MMP family that degrades the extracellular matrix. Application of MMP-3 to injured pulp tissue induces angiogenesis and wound healing, but its anti-inflammatory effects are still unclear. Here, we evaluated the anti-inflammatory functions of MMP-3 in vitro and in vivo. METHODS: Nitric oxide and inflammatory mediator synthesis in macrophages activated by lipopolysaccharide (LPS) was measured in the presence or absence of MMP-3. The mouse Mmp3 (mMmp3) expression vector containing full length cDNA sequence of mMmp3 or cDNA sequence of mMmp3 missing the signal peptide and pro-peptide regions was transfected to RAW264, a mouse macrophage cell line, and NO synthesis and inflammatory mediator expression were evaluated. Pulpal inflammation was histologically and immunohistochemically evaluated in a rat model of incisor pulpitis induced by the application of LPS for 9 hours in the presence or absence of MMP-3. RESULTS: NO and pro-inflammatory mediator synthesis promoted by LPS was significantly down-regulated by MMP-3 in vitro. The full length of mMmp3 down-regulated the LPS-induced NO synthesis and chemical mediator mRNA expression, however the mMmp3 missing the signal peptide failed to block the NO synthesis induced by LPS. The numbers of major histocompatibility complex class II+ and CD68+ cells, which infiltrated into the rat incisor pulp tissues in response to the topical application of LPS, were significantly decreased by the application of MMP-3 in vivo. CONCLUSIONS: These results indicate that MMP-3 possesses anti-inflammatory functions, suggesting its potential utility as an anti-inflammatory agent for pulpal inflammation.

Three-dimensional spheroid culture promotes odonto/osteoblastic differentiation of dental pulp cells.

OBJECTIVE: Three-dimensional (3D) spheroid culture is a method for creating 3D aggregations of cells and their extracellular matrix without a scaffold mimicking the actual tissues. The aim of this study was to evaluate the effects of 3D spheroid culture on the phenotype of immortalized mouse dental papilla cells (MDPs) that have the ability to differentiate into odontoblasts. METHODS: We cultured MDPs for 1, 3, 7, and 14 days in 96-well low-attachment culture plates for 3D spheroid culture or flat-bottomed plates for two-dimensional (2D) monolayer culture. Cell proliferation and apoptosis were detected by immunohistochemical staining of Ki67 and cleaved caspase-3, respectively. Hypoxia was measured by the hypoxia probe LOX-1. Odonto/osteoblastic differentiation marker gene expression was evaluated by quantitative PCR. We also determined mineralized nodule formation, alkaline phosphatase (ALP) activity, and dentine matrix protein-1 (DMP1) expression. Vinculin and integrin signalling-related proteins were detected immunohistochemically. RESULTS: Odonto/osteoblastic marker gene expression and mineralized nodule formation were significantly up-regulated in 3D spheroid-cultured MDPs compared with those in 2D monolayer-cultured MDPs (p<0.05). Histologically, 3D spheroid colonies consisted of two compartments: a cell-dense peripheral zone and cell-sparse core zone. Proliferating cells with high ALP activity and DMP1 expression were found mainly in the peripheral zone that also showed strong expression of vinculin and integrin signalling-related proteins. In contrast, apoptotic and hypoxic cells were detected in the core zone. CONCLUSION: 3D spheroid culture promotes odonto/osteoblastic differentiation of MDPs, which may be mediated by integrin signalling.

Wnt11 expression in rat dental pulp and promotional effects of Wnt signaling on odontoblast differentiation.

Congenital anomalies of wingless-type mouse mammary tumor virus (MMTV) integration site family (Wnt) are frequently accompanied with tooth and dentin abnormality. The aim of this study was to investigate the effects of Wnt signaling on odontoblast differentiation of mouse dental papilla cells (MDPs). Mouse dental papilla cells were cultured in α-modified minimum essential medium containing 10% fetal bovine serum and antibiotics. Odontoblast differentiation was induced by bone morphogenic protein 2 (BMP2), and the expression of odontoblast-specific markers and Wnt-related signaling molecules was analyzed by real-time reverse transcription-polymerase chain reaction and immunohistochemistry. Odontoblast differentiation was evaluated by dentin sialophosphoprotein (Dspp) and dentin matrix protein (DMP) 1 expression. Localization of ß-catenin in MDPs was detected by immunocytochemistry using an anti-ß-catenin antibody. Dspp expression in MDPs was upregulated in the presence of BMP2. Wnt5a, Wnt11, Lef1 and Tcf4 expression was upregulated in BMP2-treated MDPs. Wnt11 expression was detected in rat dental pulp in vivo, and particularly strong expression of Wnt11 was detected in odontoblasts. Enhanced Dspp and DMP1 expression and alkaline phosphatase activity induced by BMP2 were completely negated by the Wnt antagonist: IWR-1-endo treatment. Nuclear translocation of ß-catenin observed in BMP2-treated MDPs was also negated by IWR-1-endo treatment. These results indicate that Wnt signaling upregulates odontoblast marker expression in MDPs, suggesting a promoting effect of Wnt signaling on odontoblast differentiation.