Abstract. Identifying a reliable and effective cytokine or growth factor group has been the focus of stem cell osteogenic induction studies. Concentrated growth factors (CGFs) as the novel generation of platelet concentrate products, appear to exhibit a superior clinical and biotechnological application potential, however, there are few studies that have demonstrated this effect. This study investigated the proliferation and differentiation of periodontal ligament stem cells (PDLSCs) co-cultured with CGFs. The rate of proliferation was analyzed by cell counting and an MTT assay. Mineralization nodule counts, alkaline phosphatase activity detection, qPCR, western blot analysis and immunohistochemistry were used to analyze mineralization effects. The results showed that CGF significantly promoted the proliferation of PDLSCs, and exhibited a dose-dependent effect on the activation and differentiation of the stem cells. The application of CGF on PDLSC proliferation and osteoinduction may offer numerous clinical and biotechnological application strategies.
The aim of this study was to compare the osteogenic effects of periodontal ligament stem cells (PDLSCs) versus bone marrow mesenchymal stem cells (BMMSCs) in combination with Bio-Oss scaffolds on subcutaneous and critical-size defects in the immunodeficient rat calvarium. PDLSCs and BMMSCs were obtained from the same canine donor. Twenty-four rats were randomly assigned to one of four experimental groups (n = 6 each): group A (no-graft negative control), group B (Bio-Oss positive control), group C (BMMSC/Bio-Oss test group), and group D (PDLSC/Bio-Oss test group). Eight weeks post-transplantation, ectopic and in situ bone regeneration was evaluated by micro-computed tomography (µ-CT), histology, histomorphometry, and immunohistochemistry. The stem cell/Bio-Oss constructs were significantly superior to the controls in terms of their ability to promote osteogenesis (p < 0.01), while the PDLSC/Bio-Oss construct tended to be superior to the BMMSC/Bio-Oss construct. Thus, engineered stem cell/Bio-Oss complexes can successfully reconstruct critical-size defects in rats, and PDLSCs and BMMSCs are both suitable as seed cells.
Toll-like receptor 4 (TLR4) is a transmembrane receptor responsible for the activation of a number of signal transduction pathways. Despite its involvement in inflammatory processes, the regulation of TLR4 signaling in human periodontal ligament stem cells (hPDLSCs) under inflammatory conditions remains to be fully elucidated. The present study aimed to clarify the regulatory mechanisms of the TLR4 signaling pathway and its role in the differentiation of hPDLSCs under inflammatory conditions. hPDLSCs from the periodontal tissues of healthy subjects and patients with periodontitis were identified by analyzing their cell surface marker molecules, and their osteogenic and adipogenic differentiation abilities. To determine the effect of TLR4 signaling on osteogenic and adipogenic differentiation under inflammatory conditions, cells were challenged with TLR4 agonist and antagonist under pluripotent differentiation conditions. Cell proliferation, apoptosis and migration were then determined using appropriate methods. The alkaline phosphatase (ALP) activity, Alizarin Red staining, Oil red O staining and relative gene and protein levels expression were also determined. The results showed that lipopolysaccharide (LPS)-induced inflammation inhibited cell proliferation and migration, promoted cell apoptosis and affected the cell cycle. Under inflammatory conditions, the activation of TLR4 decreased the activity of ALP and the expression of osteogenic markers, including osteocalcin, Runt-related transcription factor 2 and collagen I, compared with the control group, but increased the expression of adipogenesis-related genes poly (ADP-ribose) polymerase γ and lipoprotein lipase. The activation of TLR4 also induced the expression of proinflammatory cytokines interleukin-1β, tumor necrosis factor-α, nuclear factor-κBP65 and TLR4, compared with that in the control group and the TLR4 antagonist group. The findings showed that LPS-induced upregulation of the TLR4 signaling pathway inhibited osteogenic differentiation and induced adipogenesis of the hPDLSCs under inflammatory conditions. The present study provided a novel understanding of the physiopathology of periodontitis, and a novel avenue for targeted treatments based on stem cell regeneration.
Growing number of studies regarding the role of circRNAs in the development of various diseases have emerged in recent years, but the role of circRNAs in periodontitis pathogenesis remains obscure. Human periodontal ligament stem cells (hPDLSCs) play a critical role in periodontal remodeling, regeneration and repair processes, and their regenerative capacity could be prohibited in local periodontal inflammatory microenvironment. Herein, we sought to uncover the molecular mechanisms of periodontitis pathogenesis by investigating the role of circMAP3K11 (hsa_circ_002284) for regenerative capacity of hPDLSCs under an inflammatory condition. The hPDLSCs isolated from periodontitis patients were used as a cell model of inflammatory microenvironment to study the effect of the circMAP3K11/miR-511-3p/TLR4 axis on the proliferation, apoptosis and migration of hPDLSCs under inflammatory conditions. Compared to the periodontal tissues from normal subjects, those from periodontitis patients exhibited higher expression levels of circMAP3K11 and TLR4, and lower expression level of miR-511-3p. Both the expressions of circMAP3K11 and TLR4 were negatively correlated with the expressions of miR-511-3p in periodontitis. In vitro studies demonstrated that circMAP3K11 is capable of enhancing hPDLSCs proliferation and migration, and reducing the apoptosis of hPDLSCs. We also found that circMAP3K11 could up-regulate the expression of transcription factors that are closely related to periodontal regeneration (Runx2, OSX, ATF4, and BSP). RT-PCR and western blot showed that the inhibitory role of miR-511-3p on TLR4 expression could be reversed by circMAP3K11, which was in line with the results of bioinformatics tools and luciferase reporter assay. Meanwhile, both in vitro and in vivo studies indicated that circMAP3K11 could reverse the effects of miR-511-3p in periodontitis, which further confirmed that circMAP3K11 functioned as a ‘sponge’ of miR-511-3p to positively regulate the expression of TLR4. Taken together, our study preliminarily uncovered a circMAP3K11/miR-511-3p/TLR4 axis that regulates the function of hPDLSCs in periodontitis, providing novel insight and scientific base in the treatment of periodontal tissue regeneration based on stem cells.
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