Periapical lesion-derived decellularized extracellular matrix as a potential solution for regenerative endodontics.

Pulp regeneration remains a crucial target in the preservation of natural dentition. Using decellularized extracellular matrix is an appropriate approach to mimic natural microenvironment and facilitate tissue regeneration. In this study, we attempted to obtain decellularized extracellular matrix from periapical lesion (PL-dECM) and evaluate its bioactive effects. The decellularization process yielded translucent and viscous PL-dECM, meeting the standard requirements for decellularization efficiency. Proteomic sequencing revealed that the PL-dECM retained essential extracellular matrix components and numerous bioactive factors. The PL-dECM conditioned medium could enhance the proliferation and migration ability of periapical lesion-derived stem cells (PLDSCs) in a dose-dependent manner. Culturing PLDSCs on PL-dECM slices improved odontogenic/angiogenic ability compared to the type I collagen group. In vivo, the PL-dECM demonstrated a sustained supportive effect on PLDSCs and promoted odontogenic/angiogenic differentiation. Both in vitro and in vivo studies illustrated that PL-dECM served as an effective scaffold for pulp tissue engineering, providing valuable insights into PLDSCs differentiation. These findings pave avenues for the clinical application of dECM's in situ transplantation for regenerative endodontics.

Wnt7b: Is It an Important Factor in the Bone Formation Process after Calvarial Damage?

OBJECTIVE: Previous studies found that Wnt7b played a unique and indispensable role in the process of osteoblast differentiation and could accelerate the repair of bone loss. However, what is the role of Wnt7B in osteogenesis? Is it possible to increase the expression of Wnt7b to promote the repair of skull defects? This study intends to provide the basic data for the application of Wnt7b in the treatment of craniomaxillofacial bone repair. METHODS: A calvarial defect mouse model that could induce Wnt7b overexpression was established. Three days after the operation, the mice in each group were intraperitoneally injected with tamoxifen (TAM) or oil eight times every other day. There were three groups. The TAMc group (R26Wnt7b/Wnt7b) was injected with tamoxifen. The Oil group (3.2 kb Col1-Cre-ERT2; R26Wnt7b/Wnt7b) was injected with oil. The TAM group (3.2 kb Col1-Cre-ERT2; R26Wnt7b/Wnt7b) was injected with tamoxifen. Four weeks after the surgery, micro-CT scanning was utilized to observe new bone formation and compare the ability to form new bone around the defect area. RESULTS: Four weeks after the operation, bone healing conditions were measured by using micro-CT scanning. The defect area of the TAM group was smaller than that of the other groups. Similarly, the bone volume fraction (BV/TV) significantly increased (p < 0.05), the trabecular number (Tb.N) increased, and the trabecular separation (Tb.Sp) decreased. CONCLUSIONS: Wnt7b participates in the bone formation process after calvarial damage, indicating the important role of Wnt7b in osteogenesis.

Creating a Microenvironment to Give Wings to Dental Pulp Regeneration-Bioactive Scaffolds.

Dental pulp and periapical diseases make patients suffer from acute pain and economic loss. Although root canal therapies, as demonstrated through evidence-based medicine, can relieve symptoms and are commonly employed by dentists, it is still difficult to fully restore a dental pulp's nutrition, sensory, and immune-regulation functions. In recent years, researchers have made significant progress in tissue engineering to regenerate dental pulp in a desired microenvironment. With breakthroughs in regenerative medicine and material science, bioactive scaffolds play a pivotal role in creating a suitable microenvironment for cell survival, proliferation, and differentiation, following dental restoration and regeneration. This article focuses on current challenges and novel perspectives about bioactive scaffolds in creating a microenvironment to promote dental pulp regeneration. We hope our readers will gain a deeper understanding and new inspiration of dental pulp regeneration through our summary.

In vitro evaluation of periapical lesion-derived stem cells for dental pulp tissue engineering.

Dental pulp tissue engineering is a promising alternative treatment for pulpitis and periapical periodontitis, and dental pulp stem cells (DPSCs) are considered to be the gold standard for dental seed cell research. Periapical lesions harbor mesenchymal stem cells with the capacity for self-renewal and multilineage differentiation. However, it remains unknown whether these periapical lesion-derived stem cells (PLDSCs) are suitable for dental pulp tissue engineering. To investigate this possibility, PLDSCs and DPSCs were isolated using the tissue outgrowth method and cultured under identical conditions. We then performed in vitro experiments to investigate their biological characteristics. Our results indicate that PLDSCs proliferate actively in vitro and exhibit similar morphology, immunophenotype and multilineage differentiation ability as DPSCs. Simultaneously, PLDSCs exhibit stronger migrative ability and express more vascular endothelial growth factor and glial cell line-derived neurotrophic factor than DPSCs, and PLDSC-derived conditioned medium was more effective in tube formation assay. The mRNA expression levels of immunomodulatory genes HLA-G, IDO and ICAM-1 were also higher in PLDSCs. However, regarding osteo/odontogenic differentiation, PLDSCs showed weaker alkaline phosphatase staining and lower calcified nodule formation compared to DPSCs, as well as lower expression of ALP, RUNX2 and DSPP, as confirmed by a quantitative RT-PCR. The osteo/odontogenic protein expression levels of DSPP, RUNX2, DMP1 and SP7 were also higher in DPSCs. The present study demonstrates that PLDSCs demonstrate potential use as seed cells for dental pulp regeneration, especially for achieving enhanced neurovascularization.

Monitoring of canonical BMP and Wnt activities during postnatal stages of mouse first molar root formation.

OBJECTIVE: This study aimed to explore the precise temporospatial distributions of bone morphogenetic protein (BMP) and Wnt signaling pathways during postnatal development of mammalian tooth roots after the termination of crown morphogenesis. METHODOLOGY: A total of two transgenic mouse lines, BRE-LacZ mice and BAT-gal mice, were undertaken. The mice were sacrificed on every postnatal (PN) day from PN 3d up to PN 21d. Then, the first lower molars were extracted, and the dissected mandibles were stained with 5-bromo-4-chloro-3-indolyl-ß-d-galactopyranoside (X-gal) and fixed. Serial sections at 10 µm were prepared after decalcification, dehydration, and embedding in paraffin. RESULTS: We observed BMP/Smads and Wnt/ß-catenin signaling activities in the dental sac, dental pulp, and apical papilla with a certain degree of variation. The position of activation of the BMP/Smad signaling pathway was located more coronally in the early stage, which then gradually expanded as root elongation proceeded and was associated with blood vessels in the pulp and developing complex apical tissues in the later stage. However, Wnt/ß-catenin signaling was highly concentrated in the mesenchyme below the cusps in the early stage, gradually expanded to regions around the root in the transition/root stage, and then disappeared entirely in the later stage. CONCLUSIONS: These results further confirmed the participation of both BMP and Wnt canonical signaling pathways in tooth root development, as well as formed the basis for future studies on how precisely integrated signaling pathways regulate root morphogenesis and regeneration.

Graphene Oxide-Copper Nanocomposites Suppress Cariogenic Streptococcus mutans Biofilm Formation.

INTRODUCTION: Dental caries is a biofilm-dependent disease that largely relies on the ability of Streptococcus mutans to synthesize exopolysaccharide matrix. Graphene oxide-based metal nanomaterials, as the derivatives of graphene, are potent agents against pathogens by their impressive antibacterial and anti-biofilm biofunctions. Previously, we fabricated the novel graphene oxide-copper nanocomposites (GO-Cu), maintaining a long-term release of copper nanoparticles. Here, the biofunctionalization of GO-Cu nanocomposites against cariogenic S. mutans is investigated. METHODS: Growth curve observation and colony forming units counting were applied to detect the antibacterial effect of GO-Cu nanocomposites on S. mutans. Scanning electron microscopy and the crystal violet assay were used to detect nanocomposite effects on biofilm forming ability. The production and distribution of exopolysaccharides within biofilm was analyzed and the expression of genes required for biofilm formation was explored. Moreover, the regulatory landscape of GO-Cu nanocomposites on S. mutans pathogenicity was probed. RESULTS: It has been found that GO-Gu nanocomposites were antibacterial to S. mutans and 10 µg/mL GO-Cu nanocomposites could inhibit the bacteria bioactivity instead of killing them. The biomass of S. mutans biofilm was significantly reduced when treated with 10 µg/mL GO-Cu nanocomposites. Also, 10 µg/mL GO-Cu nanocomposites could alter the biofilm architecture and impair exopolysaccharides production and distribution, and dysregulated the expression of exopolysaccharide-associated genes. CONCLUSION: In all, we found low-dose GO-Cu nanocomposites could disrupt exopolysaccharide matrix assembly and further impair optimal biofilm development with minimal cytotoxicity. Therefore, GO-Cu nanocomposites can open up a new avenue for the development of alternative anti-caries biomaterials.

Enterococcus Faecalis activates NLRP3 inflammasomes leading to increased interleukin-1 beta secretion and pyroptosis of THP-1 macrophages.

OBJECTIVES: Enterococcus faecalis is the bacterial species closely related to persistent infection in root canals. Interleukin-1 beta (IL-1ß) is the most commonly detected proinflammatory cytokine in periapical granulation tissue and plays a critical role in host defenses against microbial infection. The synthesis and secretion of IL-1ß are mediated mainly by Toll-like receptors and inflammasome activation. The previous study found that the nucleotide-binding oligomerization domain-like receptor protein 3 (NLRP3) and the absent in Melanoma 2 (AIM2) inflammasomes are positively expressed in periapical granulation tissue. The aim of this study was to investigate the pathogenicity of E. faecalis and the molecular mechanisms of IL-1ß secretion by THP-1 macrophages infected with E. faecalis. METHODS: The IL-1ß and lactate dehydrogenase (LDH) levels induced by E. faecalis were investigated with enzyme-linked immunosorbent assay (ELISA) kit and cytotoxicity assay kit, caspase-1 and inflammasome expression levels were investigated using real time PCR and Western blot analysis. Then the effect of caspase-1, NLRP3, adenosine triphosphate (ATP), and extracellular K+ on IL-1ß and LDH secretion, Gasdermin-D (GSDMD) cleavage induced by E. faecalis were analyzed. RESULTS: E. faecalis significantly increased IL-1ß and LDH release, caspase-1 and GSDMD cleavage, and NLRP3 inflammasome activation. It also showed that IL-1ß and LDH release, GSDMD cleavage required caspase-1 and NLRP3 activation. Furthermore, the expression and activation of caspase-1 and NLRP3 were blocked by oxidized ATP and extracellular K+. CONCLUSION: E. faecalis infection activated caspase-1 and the NLRP3 inflammasome to induce IL-1ß secretion and inflammatory cell death (pyroptosis). Furthermore, the activation and expression of NLRP3 induced by E. faecalis required P2X7R and K+ efflux. This study furthers our understanding of the inflammatory response mechanism induced by E. faecalis indicates that NLRP3 may be a potential target for treatment and prevention of persistent periodontitis caused by E. faecalis.

Analysis of the expression of NLRP3 and AIM2 in periapical lesions with apical periodontitis and microbial analysis outside the apical segment of teeth.

OBJECTIVE: To detect the distribution and expression levels of the nucleotide-binding oligomerization domain-like receptor protein 3 (NLRP3) and the absent in Melanoma 2 (AIM2) inflammasomes in periapical lesions and to analyse the possible microbial stimuli outside of teeth. DESIGN: The distribution of NLRP3 and AIM2 inflammasomes in sixteen periapical lesions was investigated by immunohistochemistry. Meanwhile, the relative gene expression levels of NLRP3 and AIM2 in sixteen periapical lesions and three health periodontal tissue were quantified by real-time polymerase chain reaction (PCR). Moreover, forty-seven teeth without sinus tracts were obtained in the clinic and included in bacterial analysis using PCR. Then, the mRNA levels of apoptosis-associated speck-like protein (ASC), caspase-1, interleukin-1ß (IL-1ß), NLRP3 and AIM2 in THP-1-derived macrophages treated with lipopolysaccharides (LPS) of Porphyromonas were also quantified by real-time PCR, and the IL-1ß secretion level was investigated using enzyme-linked immunosorbent assay (ELISA). RESULTS: NLRP3 and AIM2 were positively expressed in periapical lesions and were mainly distributed in inflammatory cells. Most of the samples that demonstrated up-regulation of NLRP3 mRNA also demonstrated up-regulation of caspase-1 mRNA. Microbial analysis revealed that Porphyromonas endodontalis was the most commonly detected species and was detected in 27 of 47 cases (57.4%), followed by Fusobacterium nucleatum (20/47, 42.6%), Porphyromonas gingivalis (19/47, 40.4%), Tannerella forsythia (19/47, 40.4%), Actinomyces sp. (17/47, 36.17%), Treponema denticola (10/47,21.28%), Actinomyces israelii (9/47,19.15%), Prevotella intermedia (6/47, 12.77%), Enterococcus faecalis (1/47,2.13%) and Enterococcus faecium (0/47,0). Furthermore, we found that LPS of P. gingivalis induced THP-1 cells to produce IL-1ß and to activate NLRP3 and AIM2 inflammasomes. CONCLUSIONS: Our results suggest that the NLRP3 and AIM2 proteins play a part in the pathogenesis of periapical periodontitis. Anaerobes, such as P. endodontalis, P. gingivalis, F. nucleatum and T. forsythia, were the main detected microbial stimuli that might activate inflammasomes in periapical tissues.

miR-152 induces human dental pulp stem cell senescence by inhibiting SIRT7 expression.

Human dental pulp stem cells (HDPSCs) have potential applications in regenerative medicine. The molecular mechanisms underlying HDPSC senescence are not completely understood. Here, we investigated the significance of miR-152 in HDPSC senescence. We show that miR-152 is upregulated during HDPSC senescence and its overexpression in early passaged HDPSCs induced senescence. Sirtuin 7 (SIRT7) was identified as a target of miR-152. SIRT7 was downregulated in senescent HDPSCs, whereas miR-152 inhibition upregulated SIRT7 and suppressed the senescent phenotype and SIRT7 overexpression rescued miR-152-induced senescence. Our results demonstrate that the miR-152/SIRT7 axis plays a key role in the regulation of HDPSC senescence and provide a candidate target to improve the functional and therapeutic potential of HDPSCs.

Dentin tubule invasion by Enterococcus faecalis under stress conditions ex vivo.

Enterococcus faecalis is the species most frequently isolated from failed endodontic treatments because it can survive under stress conditions imposed by root canal treatment. The objective of this study was to determine the ability of E. faecalis to invade dentine tubules under alkaline and energy-starvation stress and to explore the potential mechanisms. Roots from single-rooted human teeth were infected with E. faecalis under alkaline and energy-starvation stress conditions. After 4 wk of culture, samples were processed to establish the tubule-penetration distance. In addition, the hydrophobicity of E. faecalis cells under these conditions was analysed and the expression of genes involved in adhesion was quantified by real-time quantitative PCR. Culture of E. faecalis under alkaline and energy-starvation stress conditions resulted in a marked reduction of tubule-penetration distance, a significant increase in hydrophobicity of the bacterial surface, and marked down-regulation of most adhesin genes compared with E. faecalis cultured in tryptic soy broth. The results indicate that the dentine tubule invasion ability of E. faecalis was markedly decreased under alkaline and glucose-starvation stress conditions, possibly because of the increase of hydrophobicity and down-regulation of some adhesion genes.

Human Dental Pulp Stem Cells via the NF-κB Pathway.

BACKGROUND/AIMS: Odontogenic differentiation of human dental pulp stem cells (HDPSCs) is regulated by multiple factors and signaling molecules. However, their regulatory mechanisms are not completely understood. In this study, we investigated the role of Zinc finger and BTB domain-containing 20 (ZBTB20) in odontoblastic differentiation of HDPSCs. METHODS: HDPSCs were obtained from human third molars and ZBTB20 expression was examined by qRT-PCR and western blot. Their osteo/odontogenic differentiation and the involvement of NF-κB pathway were subsequently investigated. RESULTS: The expression of ZBTB20 is upregulated in a time-dependent manner during odontogenic differentiation of hDPSCs. Inhibition of ZBTB20 reduced osteogenic medium (OM)-induced odontogenic differentiation, reflected in decreased alkaline phosphatase (ALP) activity, mineralized nodule formation and mRNA expression of odonto/osteogenic marker genes. In contrast, overexpression of ZBTB20 enhanced ALP activity, mineralization and the expression of differentiation marker genes. Furthermore, the expression of IκBα was increased by ZBTB20 silencing in HDPSCs, whereas ZBTB20 overexpression decreased IκBα and enhanced nuclear NF-κB p65. Inhibition of the NF-κB pathway significantly suppressed the odontogenic differentiation of HDPSCs induced by ZBTB20. CONCLUSION: This study shows for the first time that ZBTB20 plays an important role during odontoblastic differentiation of HDPSCs and may have clinical implications for regenerative endodontics.

Porphyromonas gingivalis lipopolysaccharide activates canonical Wnt/ß-catenin and p38 MAPK signalling in stem cells from the apical papilla.

As dental precursor cells, stem cells from the apical papilla (SCAP) are capable of forming roots and undergoing apexogenesis, which are impaired upon exposure to bacterial infection. Porphyromonas gingivalis is a common Gram-negative bacterium that is involved in pulpal and periapical infection. The purpose of this study was to investigate the effects of P. gingivalis lipopolysaccharide (LPS) on the Wnt/ß-catenin and p38 mitogen-activated protein kinase (MAPK) signalling pathways in SCAP. As indicated by the IL-1ß and TNF-α mRNA levels, P. gingivalis LPS induced the expression of pro-inflammatory cytokines in a dose-dependent manner. In addition, activation of the p38 MAPK and Wnt/ß-catenin pathways was confirmed by the augmentation of phospho-p38 and ß-catenin protein expression and increased expression of c-myc and cyclin D1 mRNA. Despite no significant increase in ß-catenin mRNA expression, increased phosphorylation of glycogen synthase kinase (GSK)-3ß suggested that GSK-3ß was responsible for the accumulation of ß-catenin in the cytoplasm and translocation to the nucleus. Previous studies have shown that GSK-3ß plays a critical role in crosstalk between the Wnt/ß-catenin and p38 MAPK pathways. In the present study, we showed that the level of p38 phosphorylation decreased upon pretreatment with a p38 MAPK inhibitor for 1 h before stimulating SCAP with 10 µg/ml P. gingivalis LPS. However, the levels of GSK-3ß and ß-catenin phosphorylation in the cytoplasm and nucleus were not significantly altered. Our results suggest that the p38 MAPK and canonical Wnt/ß-catenin signalling pathways are activated by P. gingivalis LPS in SCAP, but we have no evidence that p38 MAPK is upstream of GSK-3ß in the Wnt/ß-catenin signalling pathway.

Histone acetylation regulates osteodifferentiation of human dental pulp stem cells via DSPP

Dental pulp stem cells (DPSCs) are a unique population of precursor cells isolated from postnatal human dental pulp, with the ability to regenerate a reparative dentin-like complex. We examined the regulation of odontoblast-like differentiation of DPSCs by histone acetylation. Western blot analysis showed that histone H3 acetylation was strongly induced in osteodifferentiation medium. Inhibition of histone acetyltransferase by garcinol reversed osteodifferentiation and mineral formation. Real-time polymerase chain reaction assay indicated that the dentin sialophosphoprotein (DSPP) gene, which is mainly expressed in odontoblasts and preameloblasts in teeth and plays an important role in tooth function, was also down-regulated in garcinol-treated cells. Moreover, lentivirus-mediated knockdown of DSPP in human DPSCs was associated with significant inhibition of mineral formation, but not osteoblast differentiation. In conclusion, the results of this study suggest that DSPP positively affects mineral formation, and that odontoblast-like differentiation and maturation of DPSCs can be regulated by histone acetylation of the DSPP gene.

[Quantitive study of interleukin 1beta in periapical exudates of chronic periapical periodontitis in the course of root canal therapy].

PURPOSE: To evaluate the relationship between IL-1beta and clinical findings of chronic apical periodontitis and to explore the function of IL-1beta during the endodontic interappointment flare-ups. METHODS: Periapical exudates samples were obtained from 19 teeth suffering from endodontic flare-ups after root canal preparation and 20 teeth without any symptoms and signs at the second visit after root canal preparation. The levels of IL-1beta were determined by ELISA and the data was analyzed by SAS6.12 software package. RESULTS: Significantly higher levels of IL-1beta were found in periapical exudates from teeth suffering from endodontic flare-ups than that before canal preparation(P<0.001). The mean IL-1beta levels significantly decreased following the endodontic therapy if there were no symptom at the second visit (P<0.001). CONCLUSION: The level of IL-1beta in the exudates of root canals were related with the exist of infection which might take an active part in the occurrence of endodontic interappointment flare-up.