Gingiva-derived mesenchymal stromal cells (GMSCs) have been considered as a promising alternative strategy for periodontal regeneration based on their potential for multilineage differentiation in vitro and the ability to form new bone in vivo. In order to investigate the capacity of GMSCs for periodontal regeneration and the fate of GMSCs during periodontal tissue repair, enhanced green fluorescent protein-labeled GMSCs were transplanted into class III furcation defects created in beagle dogs. The results showed that the transplanted GMSCs significantly enhanced the regeneration of the damaged periodontal tissue, including the alveolar bone, cementum and functional periodontal ligament (PDL). Moreover, GMSCs were able to differentiate into osteoblasts, cementoblasts and PDL fibroblasts in vivo. These findings indicate that GMSCs represent a novel cell source for periodontal tissue reconstruction.

Background and Objective Peri‐implantitis and periodontitis are different entities in immune characteristics even though they share similar features in clinical and radiologic signs. Toll‐like receptor 2 (TLR‐2), one of the key pathogen‐recognition receptors in the innate immune system, plays an important role in the progression of periodontitis. However, the role of TLR‐2 in peri‐implantitis remains unclear. The objective of this study was to investigate the role of TLR‐2 in inflammation and alveolar bone loss in a murine model of ligature‐induced peri‐implantitis and to compare it with ligature‐induced periodontitis. Material and Methods Smooth‐surface titanium implants were placed in the alveolar bone of the left maxillary molars of wild‐type (WT) and Tlr2 knockout (Tlr2‐KO) mice 6 weeks after tooth extraction. Silk ligatures were applied to the left implant fixtures and the right maxillary second molars to induce peri‐implantitis and periodontitis 4 weeks after implant placement. Two weeks after ligation, bone loss around the implants and maxillary second molars was analysed by micro‐computed tomography (micro‐CT), and inflammation around the implants and maxillary second molars was assessed at the same time point using histology and TRAP staining, respectively. Expression of mRNA for proinflammatory cytokines (interleukin‐1β [Il1β], tumor necrosis factor‐α [Tnfα]), an anti‐inflammatory cytokine (interleukin‐10 [Il10]) and osteoclastogenesis‐related cytokines (Rankl, osteoprotegerin [Opg]) were evaluated, in gingival tissue, using real‐time quantitative PCR (RT‐qPCR). Results The success rate of implant osseointegration was significantly higher in Tlr2‐KO mice (85.71%) compared with WT mice (53.66%) (P = .0125). Micro‐CT revealed significantly decreased bone loss in Tlr2‐KO mice compared with WT mice (P = .0094) in peri‐implantitis. The levels of mRNA for Il1β (P = .0055), Tnfα (P = .01) and Il10 (P = .0019) in gingiva were significantly elevated in the peri‐implantitis tissues of WT mice, but not in Tlr2‐KO mice, compared with controls. However, the gingival mRNA ratios of Rankl/Opg in peri‐implant tissues were significantly upregulated in both WT (P = .0488) and Tlr2‐KO (P = .0314) mice. Ligature‐induced periodontitis exhibited similar patterns of bone loss and inflammatory cytokine profile in both groups of mice, except that the level of Il10 was elevated (P = .0114) whereas the Rankl/Opg ratio was not elevated (P = .9755) in Tlr2‐KO mice compared with control mice. Histological findings showed increased numbers of TRAP‐positive cells and infiltrated inflammatory cells in ligature‐induced peri‐implantitis in both WT (P < .01) and Tlr2‐KO mice (P < .05), and the numbers of both types of cell were significantly higher in WT mice than in Tlr2‐KO mice (P < .01). Conclusion This study suggests that TLR‐2 mediates bone loss in both peri‐implantitis and periodontitis. However, different molecular features may exist in the pathogenesis of the two diseases.

IL-10-expressing regulatory B cells (B10) play an essential role in immune system balance by suppressing excessive inflammatory responses. In this study, we investigated induction of B 10 cell's IL-10 competency in vitro and its effect on ligature-induced experimental periodontitis in vivo. Spleen B cells were isolated from C57BL/6J mice and cultured for 48 h under the following conditions: control, CD40L, IL-21, anti-Tim1, CD40L +IL-21, CD40L +anti-Tim1, CD40L +IL-21 +anti-Tim1. Silk ligatures were tied around both maxillary second molars of C57BL/6J mice for two weeks. Optimized combination of CD40L, IL-21 and anti-Tim1 and vehicle were injected into contralateral side of palatal gingiva on days 3, 6 and 9. The palatal gingival tissues and maxillary bone were collected on day 14 to determine expressions of IL-10 and periodontal bone resorption respectively. Our results demonstrated that IL-10 expressions of cultured spleen B cells were significantly increased in the presence of CD40L, IL-21 and anti-Tim1 combination when compared with control groups. Gingival IL-10 mRNA and protein expressions were significantly increased after injection of CD40L, IL-21 and anti-Tim1 combination, when compared to the control side. The gingival RANKL expression and periodontal bone loss were significantly decreased on the combination treatment side, as compared to the control side. These results suggest that combination of IL-21, anti-Tim1 and CD40L treatment induced B10 cell's IL-10 competency in vitro and inhibited periodontal bone loss in ligature-induced experimental periodontitis.

It has been suggested that microRNAs (miRs) are involved in the immune regulation of periodontitis. However, it is unclear whether and how miRs regulate the function of B cells in the context of periodontitis. This study is to explore the role of miR-146a on the inflammatory cytokine production of B cells challenged by Porphyromonas gingivalis (P. gingivalis) lipopolysaccharide (LPS). Primary B cells were harvested from mouse spleen. Quantitative real-time polymerase chain reaction (qPCR), enzyme-linked immunosorbent assay (ELISA) were used to detect the expression of inflammatory cytokines in B cells in the presence or absence of P. gingivalis LPS and/or miR-146a. Bioinformatics, luciferase reporter assay and overexpression assay were used to explore the binding target of miR-146a. Our results showed that miR-146a level in B cells was elevated by P. gingivalis LPS stimulation, and the mRNA expressions of interleukin (IL)-1β, 6 and 10, and IL-1 receptor associated kinase-1 (IRAK1), but not TNF receptor associated factor 6 (TRAF6), were also upregulated. The expression levels of IL-1β, 6, 10 and IRAK1 were reduced in the presence of miR-146a mimic, but were elevated by the addition of miR-146a inhibitor. MiR-146a could bind with IRAK1 3' untranslated region (UTR) but not TRAF6 3'-UTR. Overexpression of IRAK1 reversed the inhibitory effects of miR-146a on IL-1β, 6 and 10. In summary, miR-146a inhibits inflammatory cytokine production in B cells through directly targeting IRAK1, suggesting a regulatory role of miR-146a in B cell-mediated periodontal inflammation.

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A novel injectable chitosan thermosensitive hydrogel was designed as a target multi-effect scaffold for endogenous repair of the periodontium. The hydrogel complex was designed by embedding chitosan nanoparticles (CSn) loaded with bone morphogenetic protein-2 plasmid DNA (pDNA-BMP2) into a chitosan (CS)-based hydrogel with α,β-glycerophosphate (α,β-GP), termed CS/CSn(pDNA-BMP2)-GP. Characterization, the in vitro release profile for pDNA-BMP2, and cytocompatibility to human periodontal ligament cells (HPDLCs), were then conducted. The average diameter of the CSn(pDNA-BMP2) was 270.1 nm with a polydispersity index (PDI) of 0.486 and zeta potential of +27.0 mv. A DNase I protection assay showed that CSn could protect the pDNA-BMP2 from nuclease degradation. Encapsulation efficiency and loading capacity of CSn(pDNA-BMP2) were more than 80 and 30 %, respectively. The sol-gel transition time was only 3 min when CSn(pDNA-BMP2) was added into the CS/α,β-GP system. Scanning electron microscopy showed that CSn(pDNA-BMP2) was randomly dispersed in a network with regular holes and a porous structure. Weighting method showed the swelling ratio and degradation was faster in medium of pH 4.0 than pH 6.8. An in vitro pDNA-BMP2 release test showed that the cumulative release rate of pDNA-BMP2 was much slower from CS/CSn-GP than from CSn in identical release media. In release media with different pH, pDNA-BMP2 release was much slower at pH 6.8 than at pH 4.0. Three-dimensional culture with HPDLCs showed good cell proliferation and the Cell-Counting Kit-8 assay indicated improved cell growth with the addition of CSn(pDNA-BMP2) to CS/α,β-GP. In summary, the CS/CSn(pDNA-BMP2)-GP complex system exhibited excellent biological properties and cytocompatibility, indicating great potential as a gene delivery carrier and tissue regeneration scaffold for endogenous repair of the periodontium.

Adrenocortical carcinoma (ACC) is a rare type of tumor with a poor prognosis. Ferroptosis is a relatively novel form of programmed cell death driven by iron-dependent lipid peroxidation accumulation. Recent evidence suggests that IFNγ facilitates erastin-induced ferroptosis, which contributed to anticancer therapy in various types of cancer. However, it has remained elusive whether the regulation of IFNγ on ferroptosis has a positive role in the treatment of ACC. Thus, the aim of the present study was to explore the effects of IFNγ on erastin-induced ferroptosis in the ACC cell line NCI-H295R and investigate the underlying mechanisms. Cell viability was assessed using a Cell Counting Kit-8 assay, an ethynyldioxyuridine proliferation assay and Live/Dead staining. The levels of iron, reactive oxygen species, lipid peroxidation and mitochondrial damage were also assessed. Western blot and reverse transcription-quantitative PCR analyses were used to determine the underlying molecular mechanisms involved in the erastin-induced ferroptosis of NCI-H295R cells. The results suggested that IFNγ promoted erastin-induced ferroptotic cell death. Furthermore, IFNγ enhanced erastin-induced ferroptosis, as evidenced by the accumulation of iron, as well as the increase in lipid peroxidation and promotion of mitochondrial damage. Further analysis suggested that IFNγ enhanced ferroptosis by suppressing the expression of solute carrier family 7 member 11, an important negative regulator of ferroptosis, and this was achieved via activation of the JAK/STAT pathway in NCI-H295R cells. The present study provided experimental evidence on the activity and mechanism of ferroptosis enhanced by IFNγ in ACC and may give critical insight into the immunotherapeutic management of ACC.

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