Effect of leptin on differentiation of human dental stem cells

Oral Diseases (2011) 17 , 662–669 Objectives: Mesenchymal stem cells (MSCs) were identified in adult human periodontal ligament and dental pulp that are considered as potential stem cell sources for future clinical applications in dentistry. Leptin is known as an important regulator of mesenchymal differentiation. The objective of this study was to elucidate the role of leptin on proliferation and differentiation of dental MSCs. Materials and methods: Enhancement of cemento/odontoblastic differentiation of dental stem cells by leptin was confirmed by alizarin red S staining and alkaline phosphatase activity staining. In contrast, leptin reduced adipogenesis in both dental pulp stem cells (DPSCs) and periodontal ligament stem cells (PDLSCs) confirmed by oil red O staining and RT‐PCR. The expression of adipogenic markers, lipoprotein lipase and proliferator‐activated receptor γ2 (PPARγ2), were suppressed in PDLSCs incubated on media supplemented with leptin for 2 weeks. Results: Leptin had a relatively stronger osteogenesis promoting effect and adipogenesis suppressing effect in PDLSCs than in DPSCs. Conclusions: Collectively, leptin had a relatively stronger promoting effect on cemento/odontoblastic differentiation and a suppressing effect on adipogenesis in PDLSCs than in DPSCs. This study has provided evidence that leptin acts as an important modulator of dental MSCs differentiation.     

不同根吸收阶段乳牙牙周膜干细胞生物学特性研究

目的：比较生理性根吸收不同阶段的乳牙牙周膜干细胞（PDLSCs）的生物学特性,检测其对破骨细胞形成和凋亡相关分子的表达情况,为解释生理性根吸收的调控机制提供实验依据。方法：通过分离、培养处于不同生理性根吸收时期的乳牙PDLSCs（未吸收、中度吸收、重度吸收）与恒牙PDLSCs;采用流式细胞术、MTF实验比较各组PDLSCs的免疫表型、细胞增殖能力;采用成骨、成脂诱导与茜素红染色、油红O染色及Real—timePCR检测各期PDLSCs成骨成脂相关基因表达;采用Westernblot检测各组PDLSCs的RANKL、OPG与FasL表达。结果：所分离获得各组乳牙PDLSCs表达间充质干细胞标志分子,与恒牙PDLSCs相比具有较强的增殖能力。其中,重度吸收组乳牙PDLSCs与其它组相比成骨能力最强、成脂能力最差。不同根吸收时期的PDLSCs差异表达RANKL、OPG与FasL蛋白。结论：不同根吸收时期乳牙PDLSCs生物学特性存在差异,RANKL、OPG与FasL的差异表达可能对生理性根吸收过程中破骨细胞的形成与凋亡起到调控作用。     

Epigenetic regulation of osteogenic differentiation of periodontal ligament stem cells in periodontitis

Periodontitis is an inflammatory disease characterized by alveolar bone loss. Periodontal ligament stem cells (PDLSCs) have osteogenic differentiation potential, which can be influenced by epigenetics regulation in periodontitis. Therefore, this review aimed to shed light on the role of different epigenetic mechanisms in the osteogenic differentiation of PDLSCs and to consider the prospects of their possible therapeutic applications in periodontitis. Databases MEDLINE (through PubMed) and Web of Science were searched for the current knowledge of epigenetics in osteogenic differentiation of PDLSCs using the keywords “periodontal ligament stem cells”, “epigenetic regulation”, “epigenetics”, “osteogenic differentiation”, and “osteogenesis”. All studies introducing epigenetic regulation and PDLSCs were retrieved. This review shows that epigenetic factors like DNMT, KDM6A, HDACi, some miRNAs, and lncRNAs can induce the osteogenic differentiation of PDLSCs in the noninflammatory microenvironment. However, the osteogenic differentiation of PDLSCs is inhibited in the inflammatory microenvironment through the upregulated DNA methylation of osteogenesis-related genes and specific changes in histone modification and noncoding RNA. Epigenetics of osteogenic differentiation of PDLSCs in inflammation exhibits the contrary effect compared with a noninflammatory environment. The application of epigenetic drugs to regulate the abnormal epigenetic status in periodontitis and focus on alveolar bone regeneration is promising.     

Apical tooth germ cell-conditioned medium enhances the differentiation of periodontal ligament stem cells into cementum/periodontal ligament-like tissues

Background and Objective: Limitations of current periodontal regeneration modalities in both predictability and extent of healing response, especially on new cementum and attachment formation, underscore the importance of restoring or providing a microenvironment that is capable of promoting the differentiatiation of periodontal ligament stem cells (PDLSCs) towards cementoblast‐like cells and the formation of cementum/periodontal ligament‐like tissues. The aim of this study was to investigate the biological effect of conditioned medium from developing apical tooth germ cells (APTG‐CM) on the differentiation and cementogenesis of PDLSCs both in vitro and in vivo. Material and Methods: Using the limiting dilution technique, single‐colony‐derived human PDLSCs were isolated and expanded to obtain homogeneous populations of PDLSCs. Morphological appearance, cell cycle analysis, bromodeoxyuridine incorporation, alkaline phosphatase (ALP) activity, mineralization behavior, gene expression of cementoblast phenotype and in vivo differentiation capacities of PDLSCs co‐cultured with APTG‐CM were evaluated. Results: The induced PDLSCs exhibited several characteristics of cementoblast lineages, as indicated by the morphological changes, increased proliferation, high ALP activity, and the expression of cementum‐related genes and calcified nodule formation in vitro. When transplanted into immunocompromised mice, the induced PDLSCs showed tissue‐regenerative capacity to produce cementum/periodontal ligament‐like structures, characterized by a layer of cementum‐like mineralized tissues and associated periodontal ligament‐like collagen fibers connecting with the newly formed cementum‐like deposits, whereas control, untreated PDLSCs transplants mainly formed connective tissues. Conclusion: Our findings suggest that APTG‐CM is able to provide a cementogenic microenvironment and induce differentiation of PDLSCs along the cementoblastic lineage. This has important implications for periodontal engineering.     

牵张力对牙周膜干细胞内质网线粒体偶联及其介导成骨分化能力的影响研究

目的 探究牙周膜干细胞(periodontal ligament stem cells,PDLSCs)在受到牵张力作用下的成骨分化能力及其内质网线粒体偶联的变化.方法 从牙周膜分离原代PDLSCs并进行传代培养,使用第3代细胞以施加或不施加牵张力处理分别作为实验组和对照组,并在加力后收取细胞进行成骨诱导培养,采用茜素红...     

A Synthetic Oligopeptide Derived From Enamel Matrix Derivative Promotes the Differentiation of Human Periodontal Ligament Stem Cells Into Osteoblast‐Like Cells With Increased Mineralization

Background: In a previous study, the authors obtained a synthetic peptide (SP) for useful periodontal tissue regeneration. Periodontal ligament stem cells (PDLSCs) have multiple potentiality to contribute to tissue regeneration. The aim of this experiment is to investigate the effect of SP on human PDLSCs. Methods: Periodontal ligament cells were obtained from healthy adult human third molars and used to isolate single PDLSC‐derived colonies. The mesenchymal stem cell nature of the PDLSCs was confirmed by immunohistochemical evaluation of STRO‐1 expression. Proliferation and osteoblastic differentiation were investigated by culturing PDLSCs in normal or osteogenic medium with and without SP (100 ng/mL). Osteoblast differentiation was assessed by measuring alkaline phosphatase (ALP) activity, osteocalcin production, mRNA expression of osteonectin, mineralization, and calcium deposition. Results: Isolated PDLSCs were immunohistochemically positive for vimentin and STRO‐1 and negative for cytokeratin. A greater number of calcified nodules were observed in osteogenic medium culture with SP than without. In the early and later stages of PDLSC culture with SP, osteonectin production and osteocalcin production were increased. SP in culture with osteogenic medium significantly enhanced proliferation of PDLSCs, as well as ALP activity, expression of osteonectin, osteocalcin production, formation of calcified nodules, and mineralization. Conclusions: SP enhances the formation of calcified nodules and osteocalcin production in the culture of PDLSCs into osteoblast‐like cells and is a useful material for periodontal tissue regeneration.     

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目的探讨人牙周膜干细胞（periodontal ligament stem cells,PDLSCs）成骨分化过程中miRNA表达谱的变化,以期为揭示PDLSCs成骨分化的分子机制提供基础。方法本研究于2012年1—12月在同济大学口腔医学院口腔生物医学及转化医学实验室、复旦大学公共实验平台完成。原代培养人PDLSCs,经流式细胞仪检测鉴定其干细胞特性（表达干细胞表面标志物STRO-1）;体外诱导其往成骨方向分化,经茜素红S及碱性磷酸酶显色试剂盒染色检测其成骨特性。再采用表达谱基因芯片,分析成骨诱导前后PDLSCs的miRNA表达,筛选出分化前后差异表达的miRNA。结果与未经成骨诱导的细胞相比,PDLSCs在成骨分化14d后,有11个miRNA的表达上调,13个miRNA的表达下调。结论PDLSCs具有成骨潜能,其成骨分化的机制可能与miRNA表达的改变有关。     

Neuropilin Controls Endothelial Differentiation by Mesenchymal Stem Cells From the Periodontal Ligament

Background: Periodontal ligament (PDL) has been reported to be a source of mesenchymal stem cells (MSCs).New vascular networks from undifferentiated cells are essential for repair/regeneration of specialized tissues, including PDL. The current study aims to determine potential of CD105⁺‐enriched cell subsets of periodontal ligament cells (PDLSCs) to differentiate into endothelial cell (EC)‐like cells and to give insights into the mechanism involved. Methods: CD105⁺‐enriched PDLSCs were induced to EC differentiation by endothelial growth medium 2 (EGM‐2) for 3, 7, 14, and 21 days, with mRNA/protein levels and functional activity assessed by: 1) real‐time polymerase chain reaction; 2) Western blotting; 3) fluorescence‐activated cell sorting; 4) immunohistochemistry; 5) immunofluorescence; 6) matrigel; and 7) small interfering RNA assays. Results: Data analyses demonstrated that EGM‐2 treated PDLSCs presented increased expression of EC markers, including: 1) CD105; 2) kinase domain‐containing receptor; and 3) Ulex europaeus agglutinin 1, and were able to form cord/tube‐like structures. Gene and protein expression analysis showed that neuropilin 2 (NRP2), a key factor for vascular development, was significantly downregulated during EC differentiation. NRP2 was constitutively expressed in mouse PDL tissues by immunohistochemistry analysis, and NRP2 knockdown in CD105⁺‐enriched PDLSCs resulted in increased cord/tube‐like structures in a matrigel assay. Conclusion: These findings demonstrated the potential of CD105⁺‐enriched PDLSCs to support angiogenesis, and NRP2 as a pivotal factor regulating this process.     

Cell Responses to Conditioned Media Produced by Patient‐Matched Stem Cells Derived From Healthy and Inflamed Periodontal Ligament Tissues

Background: Periodontal ligament stem cells (PDLSCs) derived from clinically compromised teeth with periodontitis are considered a readily accessible cell source, but their impaired stem cell functionalities, as observed in various in vitro and in vivo models, necessitate further investigation of these inflamed cells before their translation into therapeutic applications. In this study, the effects of conditioned media (CM) produced by stem cells derived from human healthy periodontal ligament tissues (H‐PDLSCs) or inflamed periodontal ligament tissues (I‐PDLSCs), referred to as H‐CM and I‐CM, respectively, on the biologic properties of H‐PDLSCs and I‐PDLSCs from the same donor are compared to explore the extent to which inflamed cells can be rescued by their extrinsic environment (i.e., by H‐CM). Methods: H‐CM and I‐CM were prepared from in vitro cell cultures, and the cellular responses of H‐PDLSCs and I‐PDLSCs to patient‐matched H‐CM and I‐CM were investigated in terms of colony‐forming ability, cell proliferation, and adipogenic/osteogenic differentiation. Results: In H‐CM and I‐CM, H‐PDLSCs and I‐PDLSCs exhibited similar adipogenic potential. However, when incubated in I‐CM, both cell types demonstrated an increased capacity to proliferate but a decreased capacity to differentiate into osteoblasts. Significantly, the impaired osteogenic differentiation of I‐PDLSCs was partially rescued by incubation in H‐CM under osteo‐inducing conditions. Conclusion: The CM of patient‐matched H‐PDLSCs and I‐PDLSCs differed, and the impaired osteogenic differentiation of inflamed stem cells had the potential to be rescued, at least partially, for therapeutic use via changing the cell culture microenvironment in vitro.     

Periodontal Ligament Stem Cells Possess the Characteristics of Pericytes

Background: Periodontal ligament (PDL) contributes to maintaining homeostasis in periodontal tissues by supplying stem/progenitor cells. It has long been suggested that PDL stem cells/progenitors are located around blood vessels. Recently mesenchymal stem cells (MSCs) have been isolated and cultured from PDL in vitro, although the location of the stem cells in PDL is unclear. The purpose of this study is to test the characteristics of human PDL stem cells (PDLSCs) and examine their similarity to related vascular cell types, such as pericytes and endothelial cells. Methods: PDLSCs were obtained from healthy extracted teeth using the collagenase/dispase enzyme digestion method. MSC and pericyte characteristics of PDLSCs were examined by cell surface marker expression using flow cytometry. The expression of pericyte markers was tested using immunohistochemistry. Pericyte‐like functions of PDLSCs were examined in co‐culture of PDLSCs and umbilical vein endothelial cells on a gel matrix. Results: Cultured PDLSCs were positive for both MSC markers and pericyte markers, including cluster of differentiation 146 (CD146), neural/glial antigen 2 (NG2), and CD140b. When pericyte marker expression was explored in rat periodontal tissue sections, CD146‐ and NG2‐positive signals were observed in the perivascular area of the PDL. Further, when the cells were cultured with human umbilical cord endothelial cells under conditions for forming capillary‐like structures in vitro, PDLSCs localized adjacent to endothelial cells and contributed to the stability of the capillary‐like structure. Conclusions: PDLSCs possess pericyte‐like characteristics and may localize as pericytes in the PDL. These data provide useful information for stem cell biology in periodontal research and stem cell–based periodontal therapy.     

糖原合成酶激酶3β对炎症状态下牙周膜干细胞成骨分化能力的影响

目的：探讨炎症微环境作用下牙周膜干细胞成骨分化能力的变化及糖原合成酶激酶3β（glycogen synthesis kinase,GSK3β）对其成骨分化能力的影响。方法：培养正常及慢性牙周炎组织来源的牙周膜干细胞（H-PDLSCs,P-PDLSCs）,将两种PDLSCs体外成骨诱导7d,实时定量PCR检测细胞Runx2、 ALP、 OCN的基因表达水平, Western Blot检测p-GSK3β, GSK3β的蛋白表达情况。在成骨诱导时使用GSK3β特异性抑制剂LiCl刺激PDLSCs, ALP染色、 RealTime PCR检测PDLSCs成骨分化的情况。结果： P-PDLSCs的成骨化能力显著低于H-PDLSCs。 P-PDLSCs组p-GSK3β表达较H-PDLSCs组增高,成骨诱导后,两种PDLSCs中p-GSK3β表达降低,但GSK3β总蛋白表达水平增高。 LiCl抑制GSK3β后PDLSCs的ALP活性、 Runx2和OCN表达水平显著降低。结论：在慢性炎症微环境中, GSK3β的磷酸化可影响Wnt信号通路,抑制牙周膜干细胞的成骨分化。