Activation of the pattern recognition receptor NOD1 in periodontitis impairs the osteogenic capacity of human periodontal ligament stem cells via p38/MAPK signalling

ObjectivesNucleotide oligomerization domain receptor 1 (NOD1) mediates host recognition of pathogenic bacteria in periodontium. However, the specific role of NOD1 in regulating osteogenesis is unclear. Therefore, this study focused on the activation status of NOD1 in periodontitis and its effect on the osteogenic capacity of human periodontal ligament stem cells (hPDLSCs) as well as the underlying mechanism.MethodsHistological staining and Western blot were utilized to assess NOD1 expression in the periodontium of people with or without periodontitis. HPDLSCs were cultured under NOD1 agonist or antagonist treatment. Q‐PCR and Western blot were employed to assess the expression of osteogenic marker genes and proteins. Alizarin red staining and alkaline phosphatase staining were used to determine the osteogenic capability of hPDLSCs. The activation of downstream signalling was determined and specific inhibitors were utilized to confirm the signalling pathway in NOD1‐regulated osteogenesis.ResultsNOD1 expression is significantly elevated in periodontitis. With NOD1 activated by particular agonist tri‐DAP, the osteogenic potential of hPDLSCs was impaired. NOD1 antagonist co‐incubation partially restored the decreased osteogenesis in hPDLSCs. P38/MAPK was phosphorylated in tri‐DAP‐induced NOD1 activation. The inhibitor of p38 rescued the suppression of osteogenesis induced by tri‐DAP in hPDLSCs.ConclusionsOur study revealed the expression status of NOD1 in periodontitis. Its activation greatly decreased the osteogenic capacity of hPDLSCs which was mediated by the phosphorylation of p38 downstream signalling.

Nucleotide oligomerization domain receptor 1 (NOD1) is substantially expressed in periodontium of individuals with chronic periodontitis. Abnormal activation of NOD1 may result in alveolar bone loss by impairing the osteogenic potential of human periodontal ligament stem cells, which provides a potential target in periodontium regeneration.     

人参皂苷Rg1促进人牙周膜干细胞增殖及骨向分化

人参皂苷Rg1 (GS Rg1) 是人参的主要药理活性成分. GS Rg1有刺激造血干细胞的形成和促进骨髓间充质干细胞增殖和分化的作用.而人牙周膜干细胞（human periodontal ligament stem cells, hPDLSCs）具有自我更新和多向分化的干细胞特性.但目前关于GS Rg1能否促进牙周膜干细胞增殖和分化的研究尚不多见.本研究证明,1×10-5 mol/L GS Rg1作用于hPDLSCs后,能明显促进牙周膜干细胞的增殖与分化.MTT检测细胞增殖显示,培养液中加入了GS Rg1实验组在第2,3,4,5 d增殖情况明显高于对照组,提示GS Rg1成分促进了牙周膜干细胞的增殖. 检测ALP表达量,RUNX2,Collagen I,OPN,OCN表达水平,加药实验组表达水平均高于未加药对照组,它们的表达量的增高提示成骨分化的增强. 牙周膜干细胞与纳米羟基磷灰石支架结合的电镜图片及其支架植入小鼠体内后的免疫组化检测显示,含有GS Rg1成分的细胞支架能促进形成更多的骨样组织.因此,GS Rg1能促进hPDLSCs体内体外的增殖及骨向分化,并在替代传统生长因子应用于牙周组织工程方面有较好前景.     

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.     

Oestrogen retains human periodontal ligament stem cells stemness in long‐term culture

Objectives

During long‐term culture, loss of stemness is observed which greatly restricts the application of human periodontal ligament stem cells (hPDLSCs) in tissue regeneration. Oestrogen (E2) was found to significantly enhance the proliferation and osteogenic differentiation capacity in mesenchymal stem cells. Therefore, in this study, we investigated effects of E2 on hPDLSCs stemness in long‐term culture.

Materials and methods

Effects of E2 on hPDLSCs stemness were systematically evaluated. To characterize underlying the mechanisms, its effects on PI3K/AKT signalling pathway were determined.

Results

Our results showed that E2 was able to enhance the proliferation, modify cell cycle, up‐regulate stemness‐related genes expression, promote osteogenic differentiation and elevate the positive rate of CD146 and STRO‐1 over 10 passages in hPDLSCs. Importantly, PI3K/AKT signing pathway might play a role in these effects.

Conclusions

These findings suggest that E2 retains hPDLSCs stemness in long‐term culture, which might enhance its application in tissue engineering.     

LncRNA SNHG1 modulates adipogenic differentiation of BMSCs by promoting DNMT1 mediated Opg hypermethylation via interacting with PTBP1

Recent evidence indicates that the abnormal differentiation of bone marrow‐derived mesenchymal stem cells (BMSCs) plays a pivotal role in the pathogenesis of osteoporosis. LncRNA SNHG1 has been found to be associated with the differentiation ability of BMSCs. In this study, we aimed to elucidate the role of lncRNA SNHG1 and its associated pathway on the differentiation of BMSCs in osteoporosis. Mice that underwent bilateral ovariectomy (OVX) were used as models of osteoporosis. Induced osteogenic or adipogenic differentiation was performed in mouse BMSCs. Compared to sham animals, lncRNA SNHG1 expression was upregulated in OVX mice. Also, the in vitro expression of SNHG1 was increased in adipogenic BMSCs but decreased in osteogenic BMSCs. Moreover, overexpression of SNHG1 enhanced the adipogenic capacity of BMSCs but inhibited their osteogenic capacity as determined by oil red O, alizarin red, and alkaline phosphatase staining, while silencing of SNHG1 led to the opposite results. LncRNA SNHG1 interacting with the RNA‐binding polypyrimidine tract‐binding protein 1 (PTBP1) promoted osteoprotegerin (Opg) methylation and suppressed Opg expression via mediating DNA methyltransferase (DNMT) 1. Furthermore, Opg was showed to regulate BMSC differentiation. Knockdown of SNHG1 decreased the expressions of adipogenic related genes but increased that of osteogenic related genes. However, the knockdown of Opg partially reversed those effects. In summary, lncRNA SNHG1 upregulated the expression of DNMT1 via interacting with PTBP1, resulting in Opg hypermethylation and decreased Opg expression, which in turn enhanced BMSC adipogenic differentiation and contributed to osteoporosis.     

Nicotine Deteriorates the Osteogenic Differentiation of Periodontal Ligament Stem Cells through α7 Nicotinic Acetylcholine Receptor Regulating wnt Pathway

Aims

Cigarette smoking is one of the high risk factors of adult chronic periodontitis and nicotine is the well established toxic substance in cigarette. However, the mechanism of nicotine induced periodontitis is still unknown. Here we studied whether nicotine impaired the osteogenic differentiation of human periodontal ligament stem cells (hPDLSCs) through activating α7 nicotinic acetylcholine receptor (α7 nAChR).

Methods

hPDLSCs with multi differentiation potential and surface makers for mesenchymal stem cells were harvested by limiting dilution technique. The level of mineralized nodule formation was assessed by alizarin red S staining. Expression level of ostegenic related genes and proteins were detected by real-time PCR and western blot analysis. The expression of α7 nAChR and its downstream signaling pathway were examined by western blot. The role of the receptor and related signaling pathway in nicotine impairing the osteogenic potential of hPDLSCs were also studied in different levels.

Results

Nicotine deteriorated the ostegenic differentiation of hPDLSCs in a dose dependent manner. Activation of α7 nAChR by nicotine treatment activated wnt/β-catenin signaling pathway, leading to osteogenic deficiency of hPDLSCs. Blockage of α7 nAChR and wnt pathway inhibitor treatment rescued nicotine induced osteogenic differentiation deficiency.

Conclusions

These data suggested that nicotine activated α7 nAChR expressed on PDLSCs and further activated wnt signaling downstream, thus deteriorating the osteogenic potential of PDLSCs. The impairment of osteogenic differentiation of PDLSCs by nicotine might lead to cigarette smoking related periodontitis.     

Analyses of key mRNAs and lncRNAs for different osteo-differentiation potentials of periodontal ligament stem cell and gingival mesenchymal stem cell

Both human periodontal ligament stem cells (hPDLSCs) and human gingival mesenchymal stem cells (hGMSCs) are candidate seed cells for bone tissue engineering, but the osteo-differentiation ability of the latter is weaker than the former, and the mechanisms are unknown. To explore the potential regulation of mRNAs and long non-coding RNAs (lncRNAs), this study obtained the gene expression profiles of hPDLSCs and hGMSCs in both undifferentiated and osteo-differentiated conditions by microarray assay and then analysed the common and specific differentially expressed mRNAs and lncRNAs in hPDLSCs and hGMSCs through bioinformatics method. The results showed that 275 mRNAs and 126 lncRNAs displayed similar changing patterns in hPDLSCs and hGMSCs after osteogenic induction, which may regulate the osteo-differentiation in both types of cells. In addition, the expression of 223 mRNAs and 238 lncRNAs altered only in hPDLSCs after osteogenic induction, and 177 mRNAs and 170 lncRNAs changed only in hGMSCs. These cell-specific differentially expressed mRNAs and lncRNAs could underlie the different osteo-differentiation potentials of hPDLSCs and hGMSCs. Finally, dickkopf Wnt signalling pathway inhibitor 1 (DKK1) was proved to be one regulator for the weaker osteo-differentiation ability of hGMSCs through validation experiments. We hope these results help to reveal new mRNAs-lncRNAs-based molecular mechanism for osteo-differentiation of hPDLSCs and hGMSCs and provide clues on strategies for improving stem cell–mediated bone regeneration.     

HOTAIRM1 as a potential biomarker for diagnosis of colorectal cancer functions the role in the tumour suppressor

Recent studies have revealed many different long noncoding RNAs (lncRNA), however, the investigation for their function and clinical value as tumour biomarkers has scarcely begun. Here, we found that expression of HOTAIRM1 was reduced in colorectal cancer (CRC) tissues compared with matched normal tissues, and plasma HOTAIRM1 levels in CRC patients were less than in controls. The cut‐off point was chosen as 0.003 with a sensitivity of 64.00% and a specificity of 76.50% in the validation set. The performance of HOTAIRM1 was highly comparable to carcinoembryonic antigen (CEA), and better than CA19‐9 and CA125. The combined assay of HOTAIRM1 and CEA raised the sensitivity and specificity to 84.00%. HOTAIRM1 knockdown resulted in obvious changes in expression of the cell proliferation related to genes and promoted cell proliferation. HOTAIRM1 plays a role of tumour suppressor in CRC; Down‐regulation of HOTAIRM1 can serve as a biomarker for CRC, and combined HOTAIRM1 and CEA assay might provide a promising diagnosis for CRC.     

Localization of STRO-1, BMP-2/-3/-7, BMP receptors and phosphorylated Smad-1 during the formation of mouse periodontium

Bone morphogenetic proteins (BMPs) and BMP receptors (BMPRs) are known to regulate the development of calcified tissues by directing mesenchymal precursor cells differentiation. However, their role in the formation of tooth-supporting tissues remains unclear. We investigated the distribution pattern of STRO-1, a marker of mesenchymal progenitor cells and several members of the BMP pathway during the development of mouse molar periodontium, from the post-natal days 6 to 23 (D6 to D23). STRO-1 was mainly localized in the dental follicle (DF) at D6 and 13 then in the periodontal ligament (PDL) at D23. BMP-2 and -7 were detected in Hertwig's epithelial root sheath (HERS) and in DF, then later in differentiated periodontal cells. BMP-3 was detected after D13 of the periodontal development. BMPRs-Ib, -II, the activin receptor-1 (ActR-1) and the phosphorylated Smad1 were detected in DF and HERS at D6 and later more diffusely in the periodontium. BMPR-Ia detection was restricted to alveolar bone. These findings were in agreement with others data obtained with mouse immortalized DF cells. These results suggest that STRO-1 positive DF cells may be target of BMPs secreted by HERS. BMP-3 might be involved in the arrest of this process by inhibiting the signaling provided by cementogenic and osteogenic BMPs.     

Long noncoding RNA ATB participates in the development of renal cell carcinoma by downregulating p53 via binding to DNMT1

Long noncoding RNA (lncRNA) exerts an essential role in the pathological processes of many diseases. Our previous study found that lncRNA ATB was highly expressed in renal cell carcinoma (RCC). Cell Counting Kit-8 (CCK-8), 5-ethynyl-2′-deoxyuridine (EdU), and migration-related assays were conducted to access the regulatory effects of lncRNA ATB on proliferative and migratory capacities of RCC cells. Flow cytometry was carried out to determine cell cycle and apoptosis influenced by lncRNA ATB. The interaction among lncRNA ATB, DNMT1, and p53 was evaluated through RNA immunoprecipitation (RIP), chromatin immunoprecipitation (ChIP), and western blot analyses. The results showed that lncRNA ATB knockdown in RCC cell line ACHN inhibited proliferative and migratory capacities and promoted apoptosis. Meanwhile, overexpression of lncRNA ATB in RCC cell line A-498 promoted proliferative and migratory capacities but inhibited apoptosis. RIP and ChIP assays confirmed that lncRNA ATB can bind to DNMT1 and stabilize its expression; meanwhile, it can promote the binding of DNMT1 to p53. Overexpression of p53 partially reversed the proliferative and migratory changes caused by lncRNA ATB. To sum up, our study revealed that high expression of lncRNA ATB could accelerate the proliferative and migratory rates of RCC cells and inhibit cell apoptosis through downregulating p53 via binding to DNMT1.     

Mitochondria transfer reverses the inhibitory effects of low stiffness on osteogenic differentiation of human mesenchymal stem cells

Microenvironment biophysical factors such as matrix stiffness can noticeably affect the differentiation of mesenchymal stem cells (MSCs). In this mechanobiology transduction process, mitochondria are shown to be an active participant. The present study aims to systematically elucidate the phenotypic and functional changes of mitochondria during the stiffness-mediated osteogenic differentiation. Additionally, the effect of mitochondria transfer on the osteogenesis of impaired MSCs caused by stiffness was investigated. Human periodontal ligament stem cells (PDLSCs) were used as model cells in the current study. Low stiffness restrained the cell spreading and significantly inhibited the proliferation and osteogenic differentiation of PDLSCs. Mitochondria of PDLSCs cultured on low stiffness exhibited shorter length, rounded shape, fusion/fission imbalance, ROS and mitophagy level increase, and ATP production reduction. The inhibited mitochondria function and osteogenic differentiation capacity were recovered to near-normal levels after transferring the mitochondria of PDLSCs cultured on the high stiffness. This study indicated that low matrix stiffness altered the mitochondrial morphology and induced systematical mitochondrial dysfunction during the osteogenic differentiation of MSCs. Mitochondria transfer was proved to be a feasible technique for maintaining MSCs function in vitro by reversing the osteogenesis ability.     

A feasibility study of an in vitro differentiation potential toward insulin-producing cells by dental tissue-derived mesenchymal stem cells

Dental tissue-derived mesenchymal stem cells have been proposed as an alternative source for mesenchymal stem cells. Here, we investigated the differentiation ability toward insulin producing cells (IPCs) of human dental pulp stem cells (hDPSCs) and human periodontal ligament stem cells (hPDLSCs). These cells expressed mesenchymal stem cell surface markers and were able to differentiate toward osteogenic and adipogenic lineages. Upon 3 step-IPCs induction, hDPSCs exhibited more colony number than hPDLSCs. The mRNA upregulation of pancreatic endoderm/islet markers was noted. However, the significant increase was noted only for PDX-1, NGN-3, and INSULIN mRNA expression of hDPSCs. The hDPSCs-derived IPCs expressed PRO-INSULIN and released C-PEPTIDE upon glucose stimulation in dose-dependent manner. After IPCs induction, the Notch target, HES-1 and HEY-1, mRNA expression was markedly noted. Notch inhibition during the last induction step or throughout the protocol disturbed the ability of C-PEPTIDE release upon glucose stimulation. The results suggested that hDPSCs had better differentiation potential toward IPCs than hPDLSCs. In addition, the Notch signalling might involve in the differentiation regulation of hDPSCs into IPCs.