Abstract
BackgroundWe derived mesenchymal stem cells (MSCs) from rat induced pluripotent stem cells (iPSCs) and transduced them with tumor necrosis factor alpha-stimulated gene-6 (TSG-6), to test whether TSG-6 overexpression would boost the therapeutic effects of iPSC-derived MSCs in experimental periodontitis.MethodsA total of 30 female Sprague-Dawley (SD) rats were randomly divided into four groups: healthy control group (Group-N, n = 5), untreated periodontitis group (Group-P, n = 5), iPS-MSCs-treated and iPSC-MSCs/TSG-6-treated periodontitis groups (Group-P1 and P2, n = 10 per group). Experimental periodontitis was established by ligature and infection with Porphyromonas gingivalis around the maxillae first molar bilaterally. MSC-like cells were generated from rat iPSCs, and transducted with TSG-6. iPSC-MSCs or iPSC-MSCs/TSG-6 were administrated to rats in Group-P1 or P2 intravenously and topically, once a week for three weeks. Blood samples were obtained one week post-injection for the analysis of serum pro-inflammatory cytokines. All animals were killed 3 months post-treatment; maxillae were then dissected for histological analysis, tartrate-resistant acid phosphatase (TRAP) staining, and morphological analysis of alveolar bone loss.ResultsAdministration of iPSC-MSC/TSG-6 significantly decreased serum levels of IL-1β and TNF-α in the Group-P2 rats (65.78 pg/ml and 0.56 pg/ml) compared with those in Group-P (168.31 pg/ml and 1.15 pg/ml respectively) (p<0.05). Both alveolar bone loss and the number of TRAP-positive osteoclasts showed a significant decrease in rats that received iPSC-MSC/TSG-6 treatment compared to untreated rats in Group-P (p<0.05),ConclusionsWe demonstrated that overexpression of TSG-6 in rat iPSC-derived MSCs were capable of decreasing inflammation in experimental periodontitis and inhibiting alveolar bone resorption. This may potentially serve as an alternative stem-cell-based approach in the treatment and regeneration of periodontal tissues.
Highlights
Periodontitis is a chronic infectious disease, leading to periodontal tissue inflammation, attachment loss, alveolar bone resorption, and eventually tooth loss [1]
Materials and Methods induced pluripotent stem cells (iPSCs)-derived mesenchymal stem cells (MSCs) generation Based on our previous study, rat iPSCs were reprogrammed from female rat embryonic fibroblasts by transducing them with Oct4, Sox2, Myc, and Klf4-expressing lentiviral vectors [22]. iPSCs in passage 5 were cultured in a gelatin-coated six-well plate with Minimum Essential Medium (MEM; Gibco, Life Technologies, Grand Island, NY, USA) supplemented with 2% fetal bovine serum (FBS; Fisher Scientific, Pittsburgh, PA, USA), 1% Penicillin/Stremycin (Gibco), 5% knockout serum replacement (Gibco), 1% platelet-derived growth factor, 1% fibroblast growth factor-2, and 0.1% epidermal growth factor
Overexpression of tumor necrosis factor alpha-stimulated gene-6 (TSG-6) in iPSC-MSCs in vitro After lentiviral transfection for 24 hours, iPSC-MSCs were harvested using Accutasefor total mRNA extraction and complimentary DNA (cDNA) synthesis for RT-PCR
Summary
Periodontitis is a chronic infectious disease, leading to periodontal tissue inflammation, attachment loss, alveolar bone resorption, and eventually tooth loss [1]. Certain therapeutic effects of the cells seen in animal models are believed to be the result of MSCs being activated by signals from injured tissues to secrete anti-inflammatory factors [10,11]. Among these factors, the most interesting was tumor necrosis factor alpha-stimulated gene-6 (TSG-6) [12], which has been extensively studied in articular joint diseases [13,14,15]. We derived mesenchymal stem cells (MSCs) from rat induced pluripotent stem cells (iPSCs) and transduced them with tumor necrosis factor alpha-stimulated gene-6 (TSG-6), to test whether TSG-6 overexpression would boost the therapeutic effects of iPSC-derived MSCs in experimental periodontitis
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