Hypoxia preconditioned DPSC-derived exosomes regulate angiogenesis via transferring LOXL2.

Hypoxia was proved to enhance the angiogenesis of stem cells. However, the mechanism of the angiogenic potential in hypoxia-pretreated dental pulp stem cells (DPSCs) is poorly understood. We previously confirmed that hypoxia enhances the angiogenic potential of DPSC-derived exosomes with upregulation of lysyl oxidase-like 2 (LOXL2). Therefore, our study aimed to illuminate whether these exosomes promote angiogenesis via transfer of LOXL2. Exosomes were generated from hypoxia-pretreated DPSCs (Hypo-Exos) stably silencing LOXL2 after lentiviral transfection and characterized with transmission electron microscopy, nanosight and Western blot. The efficiency of silencing was verified using quantitative real-time PCR (qRT-PCR) and Western blot. CCK-8, scratch and transwell assays were conducted to explore the effects of LOXL2 silencing on DPSCs proliferation and migration. Human umbilical vein endothelial cells (HUVECs) were co-incubated with exosomes to assess the migration and angiogenic capacity through transwell and matrigel tube formation assays. The relative expression of angiogenesis-associated genes was characterized by qRT-PCR and Western blot. LOXL2 was successfully silenced in DPSCs and inhibited DPSC proliferation and migration. LOXL2 silencing in Hypo-Exos partially reduced promotion of HUVEC migration and tube formation and inhibited the expression of angiogenesis-associated genes. Thus, LOXL2 is one of various factors mediating the angiogenic effects of Hypo-Exos.

Anti-inflammatory effects of shikonin in human periodontal ligament cells.

CONTEXT: Shikonin (SHI), an active component extracted from Radix Arnebiae, has been reported to possess anti-inflammatory properties in various cells. However, its effect on lipopolysaccharide (LPS)-stimulated human periodontal ligament cells (hPDLCs) is unknown. OBJECTIVE: To investigate the effects of SHI on the expression of inflammatory related cytokines in LPS-stimulated hPDLCs. MATERIALS AND METHODS: The effects of SHI (0.125, 0.25, 0.5, 1, and 2 µg/mL) on hPDLCs proliferation for 1, 3 and 7 days were measured using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. The expression of interleukin-1 (IL-1), IL-6, tumor necrosis factor-α (TNF-α), matrix metalloproteinase-2 (MMP-2), MMP-9 and cyclooxygenase-2 (COX-2) were detected in hPDLCs following SHI treatment (0.25 and 0.5 µg/mL) using Quantitative Reverse Transcriptase Polymerase Chain Reaction (qRT-PCR). The signaling pathways triggered by SHI in hPDLC were evaluated using western blotting. RESULTS: LD50 of SHI is 1.7 µg/mL (day 1) and 1.1 µg/mL (day 3 and 7) in hPDLCs. No morphological changes were observed when hPDLCs were treated with LPS only (1 µg/mL) or LPS with SHI (0.25 and 0.5 µg/mL). Data from qRT-PCR suggests that SHI attenuates LPS-induced increases of IL-1, IL-6, TNF-α, MMP-2, MMP-9 and COX-2 in hPDLCs. Down-regulation of phosphorylated extracellular signal-regulated kinase (ERK) and nuclear factor-κB (NF-κB), and up-regulation of I-κB, were observed in LPS-stimulated hPDLCs after exposed to SHI at 0.25 or 0.5 µg/mL. DISCUSSION AND CONCLUSIONS: SHI possesses anti-inflammatory effects in LPS-stimulated hPDLCs via phospho-ERK and NF-κB/I-κB signaling pathways; this suggests that SHI may hold potential as an anti-inflammatory agent against periodontitis.

Expression of high mobility group box 1 in inflamed dental pulp and its chemotactic effect on dental pulp cells.

High mobility group box 1 protein (HMGB1) is a chromatin protein which can be released extracellularly, eliciting a pro-inflammatory response and promoting tissue repair process. This study aimed to examine the expression and distribution of HMGB1 and its receptor RAGE in inflamed dental pulp tissues, and to assess its effects on proliferation, migration and cytoskeleton of cultured human dental pulp cells (DPCs). Our data demonstrated that cytoplasmic expression of HMGB1 was observed in inflamed pulp tissues, while HMGB1 expression was confined in the nuclei in healthy dental pulp. The mRNA expression of HMGB1 and RAGE were significantly increased in inflamed pulps. In in vitro cultured DPCs, expression of HMGB1 in both protein and mRNA level was up-regulated after treated with lipopolysaccharide (LPS). Exogenous HMGB1 enhanced DPCs migration in a dose-dependent manner and induced the reorganization of f-actin in DPCs. Our results suggests that HMGB1 are not only involved in the process of dental pulp inflammation, but also play an important role in the recruitment of dental pulp stem cells, promoting pulp repair and regeneration.

Micro-CT Evaluation of Different Root Canal Irrigation Protocols on the Removal of Accumulated Hard Tissue Debris: A Systematic Review and Meta-Analysis.

Accumulated hard tissue debris (AHTD) is an inevitable by-product during endodontic treatment and is difficult to remove completely using traditional syringe and needle irrigation (SNI). Adjunctive irrigation is proposed to assist the clean-up of AHTD. This systematic review and meta-analysis aimed to evaluate the AHTD removal efficacy of different root canal irrigation devices using micro-computed tomography (Micro-CT). A literature search was carried out within the main scientific databases until 20 June 2022. All results were screened with detailed eligibility criteria. Eleven studies were included for analysis. SNI, passive ultrasonic irrigation (PUI), negative pressure systems, sonically activated irrigation (SAI), mechanical-activated system and laser-activated irrigation (LAI) were assessed. PUI is superior to SNI for debris removal and LAI has better AHTD removal performance than PUI. The negative pressure system and mechanical-activated system were proved to be less effective. Registration: PROSPERO (CRD42021273892).

Effect of erythropoietin on angiogenic potential of dental pulp cells.

Erythropoietin (EPO) is a 34-kDa glycoprotein that possesses the potential for angiogenesis, as well as anti-inflammatory and anti-apoptotic properties. The present study aimed to examine the effect of EPO on the angiogenesis of dental pulp cells (DPCs) and to explore the underlying mechanisms of these effects. It was demonstrated that EPO not only promoted DPCs proliferation but also induced angiogenesis of DPCs in a paracrine fashion. EPO enhanced the angiogenic capacity by stimulating DPCs to secrete a series of angiogenic cytokines. ELISA confirmed that high concentrations of EPO increased the production of MMP-3 and angiopoietin-1 but decreased the secretion of IL-6. Furthermore, EPO activated the ERK1/2 and p38 signaling pathways in DPCs, while inhibition of these pathways diminished the angiogenesis capacity of DPCs. The present study suggested that EPO may have an important role in the repair and regeneration of dental pulp.

Parthenolide Promotes Differentiation of Osteoblasts Through the Wnt/ß-Catenin Signaling Pathway in Inflammatory Environments.

Periodontitis is a progressive inflammatory disease initiated by bacterial biofilm adhering to the tooth surface. If left untreated, periodontitis may lead to tooth loss and destruction of the alveolar bone. Regaining the lost alveolar bone is a clinical challenge because of the limited differentiation ability of osteoblasts in inflammatory environments. We have previously shown the anti-inflammatory and antiosteoclastogenic activities of parthenolide (PTL) in human periodontal ligament-derived cells by inhibiting nuclear factor kappa B (NF-κB) signaling, indicating its potential for periodontitis treatment. In this study, we further examined whether PTL could stimulate differentiation of osteoblasts from human alveolar bone in inflammatory conditions and investigated the involvement of the Wnt/ß-catenin signaling pathway during this process. The results showed that PTL significantly stimulated alkaline phosphatase activity, mineralization nodule formation, and osteogenesis-related gene/protein expression of osteoblasts under the stimulation of tumor necrosis factor-α (TNF-α). In addition, PTL inhibited the NF-κB/p50 pathway and resisted the inhibition of Wnt/ß-catenin signaling induced by TNF-α. Our results indicate that the stimulatory effect of PTL on the differentiation of osteoblasts in inflammatory environments may involve the activation of the Wnt/ß-catenin signaling pathway, and PTL may be a promising component for bone regeneration in periodontitis treatment.

Anti-inflammatory and antiosteoclastogenic activities of parthenolide on human periodontal ligament cells in vitro.

Periodontitis is an inflammatory disease that causes osteolysis and tooth loss. It is known that the nuclear factor kappa B (NF-κB) signalling pathway plays a key role in the progression of inflammation and osteoclastogenesis in periodontitis. Parthenolide (PTL), a sesquiterpene lactone extracted from the shoots of Tanacetum parthenium, has been shown to possess anti-inflammatory properties in various diseases. In the study reported herein, we investigated the effects of PTL on the inflammatory and osteoclastogenic response of human periodontal ligament-derived cells (hPDLCs) and revealed the signalling pathways in this process. Our results showed that PTL decreased NF-κB activation, I-κB degradation, and ERK activation in hPDLCs. PTL significantly reduced the expression of inflammatory (IL-1ß, IL-6, and TNF-α) and osteoclastogenic (RANKL, OPG, and M-CSF) genes in LPS-stimulated hPDLCs. In addition, PTL attenuated hPDLC-induced osteoclastogenic differentiation of macrophages (RAW264.7 cells), as well as reducing gene expression of osteoclast-related markers in RAW264.7 cells in an hPDLC-macrophage coculture model. Taken together, these results demonstrate the anti-inflammatory and antiosteoclastogenic activities of PTL in hPDLCs in vitro. These data offer fundamental evidence supporting the potential use of PTL in periodontitis treatment.

The ionic products from bredigite bioceramics induced cementogenic differentiation of periodontal ligament cells via activation of the Wnt/ß-catenin signalling pathway.

Periodontitis results from the destructive inflammatory reaction of the host elicited by a bacterial biofilm adhering to the tooth surface and if left untreated, may lead to the loss of the teeth and the surrounding tissues, including the alveolar bone. Cementum is a specialized calcified tissue covering the tooth root and an essential part of the periodontium which enables the attachment of the periodontal ligament to the root and the surrounding alveolar bone. Periodontal ligament cells (PDLCs) represent a promising cell source for periodontal tissue engineering. Since cementogenesis is the critical event for the regeneration of periodontal tissues, this study examined whether inorganic stimuli derived from bioactive bredigite (Ca7MgSi4O16) bioceramics could stimulate the proliferation and cementogenic differentiation of PDLCs, and further investigated the involvement of the Wnt/ß-catenin signalling pathway during this process via analysing gene/protein expression of PDLCs which interacted with bredigite extracts. Our results showed that the ionic products from bredigite powder extracts led to significantly enhanced proliferation and cementogenic differentiation, including mineralization-nodule formation, ALP activity and a series of bone/cementum-related gene/protein expression (ALP, OPN, OCN, BSP, CAP and CEMP1) of PDLCs in a concentration dependent manner. Furthermore, the addition of cardamonin, a Wnt/ß-catenin signalling inhibitor, reduced the pro-cementogenesis effect of the bredigite extracts, indicating the involvement of the Wnt/ß-catenin signalling pathway in the cementogenesis of PDLCs induced by bredigite extracts. The present study suggests that an entirely inorganic stimulus with a specific composition of bredigite bioceramics possesses the capacity to trigger the activation of the Wnt/ß-catenin signalling pathway, leading to stimulated differentiation of PDLCs toward a cementogenic lineage. The results indicate the therapeutic potential of bredigite ceramics in periodontal tissue engineering application.

[Macrophage migration-inhibitory factors expression and its effects on proliferation in human dental pulps].

OBJECTIVE: To investigate the expression of macrophage migration-inhibitory factors (MIF) in clinically healthy and inflamed human pulp tissues and the effects of rhMIF on the proliferation of human dental pulp cells (HDPC). METHODS: Immunohistochemistry was used to detect the localization of MIF expression in clinically healthy pulp and inflamed pulp tissues. Quantitative real-time polymerase chain reaction (PCR) was performed to evaluate the mRNA levels of MIF in pulp specimens. In addition, the culture supernatants of HDPC were collected after HDPC was stimulated by lipopolysaccharide (LPS) for 24 h, and then the MIF levels were assayed by quantitative sandwich enzyme-linked immunosorbent assay. Meanwhile, the effects of rhMIF on the proliferation of HDPC at different concentrations for 24 and 48 h were observed by cell counting kit-8 (CCK-8). RESULTS: MIF was mainly distributed in odontoblasts of healthy pulp tissue, however, in inflamed pulp tissue, it was widely detected in fibroblasts, inflammatory infiltrates and endothelial cells as well as odontoblasts. Quantitative real-time PCR showed that there was no significant difference in MIF mRNA levels between inflamed pulps and healthy pulps (P > 0.05). Additionally, the secretion of MIF was significantly increased by stimulation with LPS at the concentration of 0.1 and 1.0 mg/L [(1772.58 ± 495.05), (1692.58 ± 337.45) ng/L] (P < 0.05), and the concentration was (1048.53 ± 161.81) ng/L in control group. rhMIF stimulated the HDPC's proliferation at the concentration of 10, 30, 60 µg/L for 24 and 48 h. CONCLUSIONS: MIF was expressed in pulp tissue and its expression was increased after stimulation by LPS. rhMIF increased the proliferation of HDPC. These results suggest that MIF may be involved in the process of pulpal inflammation.