Isolation, Processing and Analysis of Murine Gingival Cells

Mizraji, Gabriel; Segev, Hadas; Wilensky, Asaf; Hovav, Avi‐Hai

doi:10.3791/50388

Cited by 34 publications

(36 citation statements)

References 9 publications

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“…In our study, we collected gingiva from mouse maxilla following previous research . According to the previous position paper, defining MSCs needed to meet the following three minimal criteria: Under the standard culture conditions, MSCs should be adherent to plastic. Surface markers should have specific expression: CD105, CD73, CD90 ≥ 90% and CD45, CD34, CD14 or CD11b, CD79α or CD19 and HLA‐DR ≤ 2%. MSCs should have potential of differentiating into osteoblasts adipocytes and chondroblasts. …”

Section: Discussionmentioning

confidence: 99%

“…In our study, we collected gingiva from mouse maxilla following previous research. 31 According to the previous position paper, de-fining MSCs needed to meet the following three minimal criteria 32 : the expression and regulation of miR-146a and miR-155 are more significant in gingiva than in other tissues. 17 According to this study, we checked the expression of two gingiva-dependent miRNAs:…”

Section: Discussionmentioning

confidence: 99%

See 1 more Smart Citation

Comparison of gingiva‐derived and bone marrow mesenchymal stem cells for osteogenesis

Sun

Nakata

Yamamoto

et al. 2019

J Cellular Molecular Medi

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Presently, bone marrow is considered as a prime source of mesenchymal stem cells; however, there are some drawbacks and limitations. Compared with other mesenchymal stem cell (MSC) sources, gingiva‐derived mesenchymal stem cells (GMSCs) are abundant and easy to obtain through minimally invasive cell isolation techniques. In this study, MSCs derived from gingiva and bone marrow were isolated and cultured from mice. GMSCs were characterized by osteogenic, adipogenic and chondrogenic differentiation, and flow cytometry. Compared with bone marrow MSCs (BMSCs), the proliferation capacity was judged by CCK‐8 proliferation assay. Osteogenic differentiation was assessed by ALP staining, ALP assay and Alizarin red staining. RT‐qPCR was performed for ALP, OCN, OSX and Runx2. The results indicated that GMSCs showed higher proliferative capacity than BMSCs. GMSCs turned more positive for ALP and formed a more number of mineralized nodules than BMSCs after osteogenic induction. RT‐qPCR revealed that the expression of ALP, OCN, OSX and Runx2 was significantly increased in the GMSCs compared with that in BMSCs. Moreover, it was found that the number of CD90‐positive cells in GMSCs elevated more than that of BMSCs during osteogenic induction. Taking these results together, it was indicated that GMSCs might be a promising source in the future bone tissue engineering.

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Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

Comparison of gingiva‐derived and bone marrow mesenchymal stem cells for osteogenesis

Sun

Nakata

Yamamoto

et al. 2019

J Cellular Molecular Medi

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“…The isolation, processing and analysis of murine maxilla gingival cells collected from uninfected or F. nucleatum -infected mice was as described by Mizraji et al., 2013 [51] . Briefly, after the protocol for oral infection, mice were euthanized and both upper and lower mandibles were collected.…”

Section: Methodsmentioning

confidence: 99%

Oral infection of mice with Fusobacterium nucleatum results in macrophage recruitment to the dental pulp and bone resorption

et al. 2018

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BackgroundFusobacterium nucleatum is a Gram-negative anaerobic bacterium associated with periodontal disease. Some oral bacteria, like Porphyromonas gingivalis, evade the host immune response by inhibiting inflammation. On the other hand, F. nucleatum triggers inflammasome activation and release of danger-associated molecular patterns (DAMPs) in infected gingival epithelial cells.MethodsIn this study, we characterized the pro-inflammatory response to F. nucleatum oral infection in BALB/c mice. Western blots and ELISA were used to measure cytokine and DAMP (HMGB1) levels in the oral cavity after infection. Histology and flow cytometry were used to observe recruitment of immune cells to infected tissue and pathology.ResultsOur results show increased expression and production of pro-inflammatory cytokines during infection. Furthermore, we observe that F. nucleatum infection leads to recruitment of macrophages in different tissues of the oral cavity. Infection also contributes to osteoclast recruitment, which could be involved in the observed bone resorption.ConclusionsOverall, our findings suggest that F. nucleatum infection rapidly induces inflammation, release of DAMPs, and macrophage infiltration in gingival tissues and suggest that osteoclasts may drive bone resorption at early stages of the inflammatory process.

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“…Periodontal tissue was harvested from CD18 +/and CD18 -/and single-cell suspensions were prepared as previously described. 46 The cells were stained with LIVE/DEAD TM Fixable Aqua Dead Cell Stain Kit (Invitrogen) to gate out dead cells and then with anti-CD45 (Clone 30-F11; Biolegend) and anti-Ly6G (Clone 1A8, Biolegend) Abs to detect neutrophils. 47 Flow cytometry data were acquired on a NovoCyte flow cytometer (ACEA Biosciences) and analyzed with NovoExpress software (ACEA Biosciences).…”

Section: Neutrophil Detection By Flow Cytometrymentioning

confidence: 99%

Frontline Science: Activation of metabolic nuclear receptors restores periodontal tissue homeostasis in mice with leukocyte adhesion deficiency-1

Kajikawa

Wang

et al. 2020

Journal of Leukocyte Biology

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2 Integrins mediate neutrophil-endothelial adhesion and recruitment of neutrophils to sites of inflammation. The diminished expression of 2 integrins in patients with mutations in the ITGB2 (CD18) gene (leukocyte adhesion deficiency-Type 1; LAD1) results in few or no neutrophils in peripheral tissues. In the periodontium, neutrophil paucity is associated with up-regulation of IL-23 and IL-17, which drive inflammatory bone loss. Using a relevant mouse model, we investigated whether diminished efferocytosis (owing to neutrophil scarcity) is associated with LAD1 periodontitis pathogenesis and aimed to develop approaches to restore the missing efferocytosis signals. We first showed that CD18 −/− mice phenocopied human LAD1 in terms of IL-23/IL-17driven inflammatory bone loss. Ab-mediated blockade of c-Mer tyrosine kinase (Mer), a major efferocytic receptor, mimicked LAD1-associated up-regulation of gingival IL-23 and IL-17 mRNA expression in wild-type (WT) mice. Consistently, soluble Mer-Fc reversed the inhibitory effect of efferocytosis on IL-23 expression in LPS-activated M s. Adoptive transfer of WT neutrophils to CD18 −/− mice down-regulated IL-23 and IL-17 expression to normal levels, but not when CD18 −/− mice were treated with blocking anti-Mer Ab. Synthetic agonist-induced activation of liver X receptors (LXR) and peroxisome proliferator-activated receptors (PPAR), which link efferocytosis to generation of homeostatic signals, inhibited the expression of IL-23 and IL-17 and favorably affected the bone levels of CD18 −/− mice. Therefore, our data link diminished efferocytosisassociated signaling due to impaired neutrophil recruitment to dysregulation of the IL-23-IL-17 axis and, moreover, suggest LXR and PPAR as potential therapeutic targets for treating LAD1 periodontitis.

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Isolation, Processing and Analysis of Murine Gingival Cells

Cited by 34 publications

References 9 publications

Comparison of gingiva‐derived and bone marrow mesenchymal stem cells for osteogenesis

Comparison of gingiva‐derived and bone marrow mesenchymal stem cells for osteogenesis

Oral infection of mice with Fusobacterium nucleatum results in macrophage recruitment to the dental pulp and bone resorption

Frontline Science: Activation of metabolic nuclear receptors restores periodontal tissue homeostasis in mice with leukocyte adhesion deficiency-1

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