Metformin facilitates the proliferation,migration, and osteogenic differentiation of periodontal ligament stem cells in vitro

Periodontitis is one of the main causes of tooth loss and has been confirmed as the sixth complication of diabetes. Metformin promotes the osteogenic differentiation of stem cells. Periodontal ligament stem cells (PDLSCs) are the best candidate stem cells for periodontal tissue regeneration. Herein, we aimed to identify the effects of metformin on the proliferation, migration, and osteogenic differentiation of PDLSCs in vitro. PDLSCs were isolated by limiting dilution, and their characteristics were assessed by colony formation assay and flow cytometry. Cell counting and migration assays were used to investigate the effects of metformin on proliferation and migration. The osteogenic differentiation ability of PDLSCs was detected by alkaline phosphatase (ALP) activity and Alizarin Red S staining. Gene and protein levels of osteogenesis‐related markers were determined by quantitative real‐time polymerase chain reaction (qRT‐PCR) and western blot analysis, respectively. Metformin treatment at 10 μM did not affect PDLSC proliferation, while at 50 and 100 μM, metformin time‐dependently enhanced PDLSC proliferation and significantly increased cell numbers after 5 and 7 days of stimulation (P < 0.05). In addition, 50 μM metformin exhibited a maximal effect on migration, ALP activity, and mineral deposition (P < 0.05). Furthermore, 50 μM metformin significantly upregulated the gene expression levels of ALP, BSP, OPN, OCN, and Runx2 and the protein expression of ALP and Runx2 (P < 0.05). In summary, our study confirms that metformin facilitates the proliferation, migration, and osteogenic differentiation of PDLSCs in vitro and could be used as a new strategy for periodontal tissue regeneration.     

p75NTR optimizes the osteogenic potential of human periodontal ligament stem cells by up‐regulating α1 integrin expression

Human periodontal ligament stem cells (hPDLSCs) are a promising source in regenerative medicine. Due to the complexity and heterogeneity of hPDLSCs, it is critical to isolate homogeneous hPDLSCs with high regenerative potential. In this study, p75 neurotrophin receptor (p75NTR) was used to isolate p75NTR⁺ and p75NTR^? hPDLSCs by fluorescence‐activated cell sorting. Differences in osteogenic differentiation among p75NTR⁺, p75NTR^? and unsorted hPDLSCs were observed. Differential gene expression profiles between p75NTR⁺ and p75NTR^? hPDLSCs were analysed by RNA sequencing. α1 Integrin (ITGA1) small interfering RNA and ITGA1‐overexpressing adenovirus were used to transfect p75NTR⁺ and p75NTR^? hPDLSCs. The results showed that p75NTR⁺ hPDLSCs demonstrated superior osteogenic capacity than p75NTR^? and unsorted hPDLSCs. Differentially expressed genes between p75NTR⁺ and p75NTR^? hPDLSCs were highly involved in the extracellular matrix‐receptor interaction signalling pathway, and p75NTR⁺ hPDLSCs expressed higher ITGA1 levels than p75NTR^? hPDLSCs. ITGA1 silencing inhibited the osteogenic differentiation of p75NTR⁺ hPDLSCs, while ITGA1 overexpression enhanced the osteogenic differentiation of p75NTR^? hPDLSCs . These findings indicate that p75NTR optimizes the osteogenic potential of hPDLSCs by up‐regulating ITGA1 expression, suggesting that p75NTR can be used as a novel cell surface marker to identify and purify hPDLSCs to promote their applications in regenerative medicine.     

Spontaneous differentiation of periodontal ligament stem cells into myofibroblast during ex vivo expansion

Periodontitis is characterized by the chronic inflammation and destruction of tooth-supporting tissues. Periodontal ligament stem cell (PDLSC) is the mesenchymal stem cell (MSC) population isolated from periodontal ligament, which is the key tissue for regeneration of periodontal tissues. Although transplantation of PDLSCs is proposed as novel regenerative therapy, limited information is available, regarding the characteristic change of PDLSCs during ex vivo expansion. In this study, we encountered morphological change of PDLSCs during standard cell culture and aimed to investigate the change of PDLSCs in stem cell characteristics and to search for the culture condition to maintain stem cell properties. Characteristics of PDLSCs were examined using in vitro osteoblast and adipocyte differentiation. Myofibroblast differentiation was confirmed using immunohistochemistry and collagen gel contraction assay. Replicative senescence was examined by β-gal staining. PDLSCs changed their morphology from spindle to flat and wide during ex vivo expansion. After the morphological change, PDLSCs showed several features of myofibroblast including extensive stress fiber formation, contraction activity, and myofibroblast marker expression. Upon the morphological change, osteoblastic and adipocyte differentiation capacity were reduced and expression of stem cell-related genes were decreased. β-Gal staining was not always correlated with the morphological change of PDLSCs. Moreover, exogenous addition of bFGF and PDGF-BB served to maintain spindle shape and osteoblastic differentiation potential of PDLSCs. This study demonstrates that spontaneous differentiation of PDLSCs during ex vivo expansion and may provide the important information of cell culture condition of PDLSCs for clinical use.     

Lipopolysaccharide can modify differentiation and immunomodulatory potential of periodontal ligament stem cells via ERK1,2 signaling

Lipopolysaccharide (LPS) is a pertinent deleterious factor in oral microenvironment for cells which are carriers of regenerative processes. The aim of this study was to investigate the emerging in vitro effects of LPS (Escherichia coli) on human periodontal ligament stem cell (PDLSC) functions and associated signaling pathways. We demonstrated that LPS did not affect immunophenotype, proliferation, viability, and cell cycle of PDLSCs. However, LPS modified lineage commitment of PDLSCs inhibiting osteogenesis by downregulating Runx2, ALP, and Ocn mRNA expression, while stimulating chondrogenesis and adipogenesis by upregulating Sox9 and PPARγ mRNA expression. LPS promoted myofibroblast‐like phenotype of PDLSCs, since it significantly enhanced PDLSC contractility, as well as protein and/or gene expression of TGF‐β, fibronectin (FN), α‐SMA, and NG2. LPS also increased protein and gene expression levels of anti‐inflammatory COX‐2 and pro‐inflammatory IL‐6 molecules in PDLSCs. Inhibition of peripheral blood mononuclear cells (MNCs) transendothelial migration in presence of LPS‐treated PDLSCs was accompanied by the reduction of CD29 expression within MNCs. However, LPS treatment did not change the inhibitory effect of PDLSCs on mitogen‐stimulated proliferation of CD4⁺ and the ratio of CD4⁺CD25^high/CD4⁺CD25^low lymphocytes. LPS‐treated PDLSCs did not change the frequency of CD34⁺ and CD45⁺ cells, but decreased the frequency of CD33⁺ and CD14⁺ myeloid cells within MNCs. Moreover, LPS treatment attenuated the stimulatory effect of PDLSCs on CFC activity of MNCs, predominantly the CFU‐GM number. The results indicated that LPS‐activated ERK1,2 was at least partly involved in the observed effects on PDLSC differentiation capacity, acquisition of myofibroblastic attributes, and changes of their immunomodulatory features.     

Odontogenesis and neuronal differentiation characteristics of periodontal ligament stem cells from beagle dog

Periodontal ligament stem cells (PDLSCs) from beagle dogs had the characteristics of multi‐directional differentiation and had great application potential in tissue engineering and cell regenerative medicine. In this study, we analysed the odontogenesis and neuronal differentiation characteristics of PDLSCs in vitro. Results showed that the calcined tooth powder (CTP) and silver nanoparticles (AgNPs) additives could induce the PDLSCs into odontogenesis differentiation; besides, the immunofluorescence staining identified that the high dosage calcined tooth powder (400 μg/mL) significantly facilitated the odontogenesis associated with BMP4 expression. While the nutritional factor (L‐glutamine, NGF (nerve growth factor), bFGF (basic fibroblast growth factor), IGF‐1 (insulin‐like growth factor‐1) and EGF (epidermal growth factor)) additives were prior to induce the PDLSCs into neuronal differentiation. Simultaneously, PDLSCs had high proliferation ability with the different supplemented additives. Importantly, the Western blot results also proved the BMP4 and SMAD1 proteins were highly expressed in the induced odontoblast, while the SOX1, NCAM1, GFAP and VEGFA proteins were all obviously expressed in the induced neurons. Hence, PDLSCs had characteristics of both odontogenesis and neuronal differentiation.     

牙龈卟啉单胞菌感染通过Wnt通路调节牙周膜干细胞的成骨分化

目的 观察牙龈卟啉单胞菌(P.gingivalis)感染通过Wnt通路调节牙周膜干细胞(PDLSCs)成骨分化的作用。 方法 培养原代PDLSCs,分为常规处理的对照组、P.gingivalis感染的P.gingivalis组和P.gingivalis感染并用Wnt3a处理的P.gingivalis+Wnt3a组,成骨诱导后茜素红染色并检测A405值,Western blot检测Wnt通路分子的蛋白表达量,碱性磷酸酶(ALP)试剂盒检测ALP活力,PCR检测成骨标志基因Runt相关转录因子2(Runx2)、骨钙素(OCN)的mRNA表达量。 结果 与对照组比较,P.gingivalis组Wnt3a、βcatenin、pGSK3β的蛋白表达水平(0.33±0.07)、(0.27±0.08)、(0.44±0.09)以及成骨诱导后A405值(0.55±0.08)、ALP活力(20.14±6.54)U/mL和Runx2、OCN的mRNA表达量(0.45±0.09)、(0.51±0.07)均明显减少;与P.gingivalis组比较,P.gingivalis+Wnt3a组成骨诱导后A405值(0.89±0.15)、ALP活力(29.44±5.26)U/mL及Runx2、OCN的mRNA表达量(0.89±0.17)、(0.81±0.18)均明显增加。 结论 P.gingivalis感染能够抑制PDLSCs的成骨分化,抑制Wnt通路是可能的分子机制。     

The inhibition of periodontal ligament stem cells osteogenic differentiation by IL-17 is mediated via MAPKs

Periodontal disease (PD), a degenerative bacterially induced disease of periodontium, can lead to bone resorption and teeth loss. Development of PD includes a strong inflammatory reaction, which involves multiple immune cells and their secreting factors including interleukin-17 (IL-17), which is not only an important modulator of immune and hematopoietic responses but also affects bone metabolism. In the present study we aimed to determine whether IL-17 affects the regenerative potential of periodontal ligament mesenchymal stem cells (PDLSCs) by investigating its ability to modulate osteogenic differentiation of these cells in vitro along with associated signaling pathways. Our results revealed that IL-17 inhibited both the proliferation and migration of PDLSCs and decreased their osteogenic differentiation by activating ERK1,2 and JNK mitogen-activated protein kinases. Obtained data suggested that IL-17 might contribute to alveolar bone loss in PD.     

Novel insights into the pharmacological modulation of human periodontal ligament stem cells by the amino-bisphosphonate Alendronate

Alendronate (ALN) is a second-generation bisphosphonate widely used for osteoporosis and cancer-induced bone lesions. Many studies have confirmed a strong relationship between osteonecrosis of the jaws (ONJ) development and oral bisphosphonates, especially ALN, although the molecular mechanisms underlying this pathology have not yet been elucidated. The reduction in bone turnover and vascularization usually observed in ONJ are the result of ALN action on different cell types harboured in oral microenvironment, such as osteoclasts, endothelial cells, and periodontal ligament stem cells (PDLSCs). In this perspective, the present study aims to investigate the effects of different ALN concentrations (2 μM, 5 μM, 10 μM, 25 μM, 50 μM) on the phenotype and functional properties of human PDLSCs (hPDLSCs). hPDLSCs showed a decrease in cell viability (MTT assay) only when treated with ALN concentration of 10 μM or larger for 48 h and 72 h. Cell cycle analysis revealed a moderate increase in proportion of S-phase cells after exposure to low ALN concentration (2–5 μM), an effect that was reverted after exposure to 10–50 μM ALN. Conversely, cell death was evidenced via Annexin V/PI assay at very high concentration of ALN (50 μM) after 4 days of treatment. In addition, we explored whether the effects of ALN on hPDLSCs growth and survival can be mediated by its ability to modulate oxidative stress. To this, we quantified the intracellular ROS amount and lipid peroxidation by using DCF probe and Bodipy staining, respectively. Flow cytometry analysis showed that ALN induced a dose-dependent reduction of intracellular oxidative stress and lipid peroxidation upon treatment with low concentrations at both 48 h and 72 h. Increased levels of oxidative stress was reported at 50 μM ALN and was also confirmed via TEM analysis. Despite the stability of the cellular immunophenotype, hPDLSCs showed impaired mobility after ALN exposure. Chronic exposure (7–14 days) to ALN in the range of 2–10 μM significantly decreased the expression of the differentiation-related factors ALP, RUNX2, COLI, and OPN as well as the osteogenic ability of hPDLSCs compared with untreated cells. Conversely, higher doses were found to be neutral. Our findings indicated that the effects of ALN on hPDLSCs behavior are dose-dependent and suggest a role for oxidative stress in ALN-induced cell death that may lead to novel therapeutic approaches for ONJ.