牙周膜干细胞表观遗传学研究进展

牙周膜干细胞(periodontal ligament stem cells,PDLSCs)是牙周膜组织中的间充质干细胞,是牙周组织再生和修复的重要细胞群,牙周微环境的变化会影响牙周膜干细胞的生物学特性.表观遗传(epigenetics)是指不基于DNA序列的变化,而出现稳定可遗传的基因表达水平及功能的改变.环境因素是影响表观遗传的重要因素之一,暴露环境的不同可以引起表观修饰改变,进而影响基因表达.表观遗传是干细胞生物学特性的重要内源性调控机制,在干细胞中的变化稳定可遗传,并具有可逆性.近年来牙周膜干细胞的表观遗传调控已成为研究热点,本文通过对表观遗传调控对牙周膜干细胞生物学特性的影响进行综述,以期使牙周膜干细胞更好地应用于牙周再生修复.     

犬牙周膜干细胞的分化能力研究

目的：体外扩增已鉴定的犬牙周膜干细胞（dog periodontal ligament stem cells,dPDLSCs）,研究其生物学特性。方法：取已鉴定的dPDLSCs细胞株复苏,倒置相差显微镜下观察细胞形态学及数量改变,平板培养并用吉姆萨染色计数克隆形成能力、体外诱导并用Von kossa染色观察体外分化特性,dPDLSCs与陶瓷化骨复合胶原凝胶载体三维培养条件下移植入免疫缺陷（BALB/C）小鼠皮下,分别4周、6周、8周取材,HE染色进行组织学观察。结果：dPDLSCs可在体外培养扩增,克隆形成能力为0.91%,体外矿化液诱导条件下,可向成骨方向分化,形成Von kossa染色阳性的矿化结节。体内实验证实,高密度接种时,dPDLSCs与陶瓷化骨复合胶原凝胶载体三维培养条件下移植入免疫缺陷（BALB/C）小鼠皮下,8周形成牙周膜纤维及牙骨质样复合体结构。结论：dPDLSCs具有很强的增殖能力、分化潜能和自我更新能力,其在适宜培养条件下可向牙周膜和牙骨质分化。     

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.     

人牙周膜干细胞的初步鉴定及体外成骨诱导

目的：体外培养、鉴定人牙周膜干细胞（periodontal ligament stem cells,PDLSCs）并定向诱导分化为成骨细胞,探讨人PDLSCs的多向分化潜能：方法：体外分离、培养人牙周膜细胞,待细胞达一定量后用有限稀释法进行克隆化培养,筛选鉴定牙周膜干细胞（PDLSC）,矿化诱导培养21d后检测钙结节形成情况、ALP活性,免疫细胞化学检测骨涎蛋白（BSP）、Ⅰ型胶原表达,RT—PCR检测ALP、BSP mRNA表达。结果：人PDLSCs体外诱导培养21d后可见钙结节形成,成骨细胞相关蛋白及mRNA均阳性表达。结论：人PDLSCs在体外诱导条件下具有成骨潜能。     

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.     

The Effect of Mechanical Vibration on Osteogenesis of Periodontal Ligament Stem Cells

IntroductionAppropriate occlusal forces can prevent ankylosis after tooth replantation or transplantation. However, the “proper occlusal forces” on periodontal ligament (PDL) healing have not yet been defined due to insufficient in vitro studies and uncertain in vitro models. Herein, we presented a mechanical vibration device as an in vitro model to determine such favorable occlusal forces.MethodsHuman periodontal ligament stem cells (hPDLSCs) were exposed to mechanical vibration force with 4 frequencies (30, 90, 150, and 210 rpm). Cell viability and the expression of osteogenic differentiation-related genes and proteins were tested in vitro. The calvarial transplantation experiment was performed to assess the bone formation ability of 150 rpm mechanical vibration stimulation (MVS).ResultsMVS at 150 and 210 rpm significantly reduced cell viability in the early stages. The 150-rpm MVS decreased osteogenic marker expression at the early time point (3 days) but had no harmful effects at the late time point (14 days). Furthermore, hPDLSC cell sheets treated with 150-rpm MVS had potential to decrease bone formation in rat calvarial defects serendipitously and facilitated functional PDL-like tissue formation.ConclusionsWe found that MVS at a frequency of 150 rpm could provide a strategy for a transient reduction in the osteogenic potential of hPDLSCs and promote PDL-like tissue formation. Thus, 150-rpm MVS could be used as a controllable proper occlusal force to prevent ankylosis and promote PDL healing after tooth replantation or transplantation.     

IGF-1对人牙周膜干细胞增殖能力的影响

目的：探讨IGF-1是否影响牙周膜干细胞的增殖。方法：分离、培养牙周膜干细胞,细胞增殖检测（MTT、克隆形成能力及细胞周期分析）。结果：实验组细胞克隆形成能力,S＋G2M期明显高于对照组。结论：IGF-1影响牙周膜干细胞在体外的增殖能力。     

模拟微重力培养环境下牙周膜干细胞生长状态的研究

目的探讨模拟微重力培养体系下人牙周膜干细胞（human periodontal ligament stem cells,HPDLSCs）的生长特点。方法在体外用有限稀释法克隆化生长获得HPDLSCs,接种于葡聚糖微载体,观察在旋转微重力细胞培养环境下与普通重力环境下细胞生长状态的差异。结果利用克隆生长法成功获取具备多向分化潜能的HPDLSCs,在微重力环境下的微载体表面细胞多呈半球形,少数铺展为不规则扁平形或长梭形,与普通重力环境相比,细胞生长速度明显加快。结论三维微重力培养环境可以迅速获得大量的HPDLSCs,为构建工程化牙周组织奠定了实验基础。     

人牙周膜干细胞的分离培养及体外诱导分化研究

目的:从成体人牙周组织中分离培养牙周膜干细胞,研究其生物学特性并进行诱导分化,为牙周组织工程提供可靠的种子细胞来源.方法:选取12～20岁的年轻患者因正畸拔除的健康牙齿,采用酶消化组织块培养法得到牙周膜细胞,待细胞达一定量后用有限元稀释法进行单细胞克隆,筛选牙周膜干细胞( PDLSCs).计算细胞克隆形成率(CFU- ...     

Sirtuin 1促进牙周膜干细胞骨向分化的实验研究

目的：研究蛋白酶Sirtuin 1在牙周膜干细胞骨向分化中的作用。方法：筛选人因正畸而拔除的前磨牙,获取牙周膜组织做细胞培养,并行牙周膜干细胞鉴定;实验分为实验组、对照组。实验组1：加入Sirtuin 1激活剂白藜芦醇使其工作浓度为1、5、10mmol/L。实验组2：加入Sirtuin 1抑制剂烟酰胺使其终末工作浓度为1、5、10mmol/L,对照组为空白对照。获取培养细胞后半定量RT-PCR检测Sirtuin 1和各组细胞骨向分化标志物,即碱性磷酸酶,骨桥蛋白,骨钙蛋白和骨涎蛋白。结果：通过检测牙周膜干细胞的蛋白酶Sirtuin 1和骨向分化标志物,即碱性磷酸酶,骨桥蛋白,骨钙蛋白和骨涎蛋白的mRNA含量,显示随着加入白芦藜醇剂量的差异而出现梯度的上调;而随着烟酰胺的浓度的升高而出现梯度的下调。结论：Sirtuin 1可以有效的促进牙周膜干细胞的骨向分化,从而促进牙槽骨再生修复重建,具有良好的临床应用前景。     

USP 12促进循环牵张力作用下人牙周膜干细胞的成骨分化过程

目的:探究泛素特异性蛋白酶12(USP 12)在循环牵张力刺激引起的人牙周膜干细胞(hPDLSCs)成骨分化中的作用.方法:原代培养健康的hPDLSCs,通过成骨、成脂及成软骨诱导分化实验检测其干细胞分化特性.使用循环牵张力刺激hPDLSCs 24 h、48 h、72 h,实时定量PCR、免疫印迹和免疫荧光实验检测USP 12的表达变化,ALP染色检测hPDLSCs的成骨分化,实时定量PCR检测成骨相关因子ALP、OGT、OPN的表达变化.结果:诱导分化试验染色显示本研究使用的原代hPDLSCs具有多向分化潜力.循环牵张力刺激hPDLSCs后,USP 12的表达均呈先升高后降低的趋势.在循环牵张力刺激下hPDLSCs发生成骨分化,ALP、OGT的表达先升高后降低,OPN逐渐升高.结论:USP 12可能促进循环牵张力作用下人牙周膜干细胞的成骨分化.     

USP 12促进循环牵张力作用下人牙周膜干细胞的成骨分化过程

目的:探究泛素特异性蛋白酶12(USP 12)在循环牵张力刺激引起的人牙周膜干细胞(hPDLSCs)成骨分化中的作用.方法:原代培养健康的hPDLSCs,通过成骨、成脂及成软骨诱导分化实验检测其干细胞分化特性.使用循环牵张力刺激hPDLSCs 24 h、48 h、72 h,实时定量PCR、免疫印迹和免疫荧光实验检测USP 12的表达变化,ALP染色检测hPDLSCs的成骨分化,实时定量PCR检测成骨相关因子ALP、OGT、OPN的表达变化.结果:诱导分化试验染色显示本研究使用的原代hPDLSCs具有多向分化潜力.循环牵张力刺激hPDLSCs后,USP 12的表达均呈先升高后降低的趋势.在循环牵张力刺激下hPDLSCs发生成骨分化,ALP、OGT的表达先升高后降低,OPN逐渐升高.结论:USP 12可能促进循环牵张力作用下人牙周膜干细胞的成骨分化.     

内皮素-1对牙周膜干细胞肿瘤坏死因子表达的影响

目的:探讨内皮素-1(endothelin-1,ET-1)对牙周膜干细胞(periodontal ligament stem cells,PDLSCs)肿瘤坏死因子(Tumor Necrosis Factor,TNF-α)表达的影响。方法:体外培养鉴定牙周膜干细胞,加入不同浓度(1,10,100 nmol/L)的ET-1培养12、24、72 h,以未作任何处理的牙周膜干细胞为对照,采用ELISA及Western blot检测ET-1作用下牙周膜干细胞TNF-α分泌及蛋白表达的改变。结果:与对照组相比,ET-1对牙周膜干细胞TNF-α分泌及蛋白表达具有显著促进作用,且促进作用具有剂量依赖性及时间依赖性。结论:内皮素-1可以诱导牙周膜干细胞分泌TNF-α,而且这种促进作用呈浓度依赖性及时间依赖性。     

Response of Human Periodontal Ligament Cells to Baicalin

Background: Periodontitis is the most common cause of tooth loss in adults. Periodontal ligament cell (PLC)–based therapy is considered one of the most promising methods in periodontal tissue regeneration. The traditional Chinese medicine baicalin has been shown to possess antimicrobial and anti‐inflammatory activities and enhance cell proliferation and alkaline phosphatase activity. The aim of this study is to investigate the response of human PLCs (HPLCs) to baicalin. Methods: The effect of baicalin on cultured HPLC proliferation was measured with a 3‐(4,5‐dimethylthiazol‐2‐yl)‐2,5‐diphenyltetrazolium bromide assay. The effect of baicalin on the expression of osteoprotegerin (OPG), receptor activator of nuclear factor‐κB ligand (RANKL), core binding factor α1 (Cbfα1), and osteocalcin (OC) was determined by quantitative real‐time polymerase chain reaction and immunodetection. Results: Baicalin at a concentration of 0.01 μg/mL promoted HPLC proliferation, upregulated OPG messenger RNA (mRNA) and protein expression, and downregulated RANKL mRNA and protein expression. In addition to reducing the RANKL/OPG expression ratio significantly, it also increased Cbfα1 and OC mRNA and protein expression. Conclusion: Baicalin showed multifaceted regulation of genes with important roles in tissue growth and differentiation, and thus it has the potential to be a promising candidate for HPLC‐based periodontal regeneration therapy.     

High Glucose Concentrations Suppress the Proliferation of Human Periodontal Ligament Stem Cells and Their Differentiation Into Osteoblasts

Background: Diabetes mellitus (DM) is a major risk factor for periodontal disease and affects various cellular functions. Periodontal ligament stem cells (PDLSCs) play an important role in periodontal tissue regeneration; however, the effect of hyperglycemia on PDLSCs is unclear. The aim of this study is to investigate whether hyperglycemia affects periodontal tissue regeneration, using human PDLSCs and high‐glucose medium as a model of DM. Methods: PDLSCs were obtained from healthy adult human mandibular third molars. Cell proliferation, osteoblastic differentiation, and proinflammatory cytokine expression were investigated by culturing PDLSCs in media supplemented with four different glucose concentrations representative of control patients (5.5 mM), patients with postprandial or controlled DM (8.0 mM), and patients with uncontrolled DM (12.0 and 24.0 mM). The molecular effects of hyperglycemia on PDLSC physiology were examined with a focus on the nuclear factor (NF)‐(κB signaling pathway. The involvement of NF‐κB was investigated with a specific NF‐κB inhibitor in PDLSCs under hyperglycemic conditions. Results: High glucose levels inhibited PDLSC proliferation and differentiation into osteoblasts but induced NF‐κB activation and subsequent interleukin (IL)‐6 and IL‐8 expression. Treatment with an NF‐κB inhibitor rescued the defects in cell proliferation and osteoblastic differentiation and inhibited the IL‐6 expression caused by the high‐glucose environment. Conclusion: The results of this study demonstrate that hyperglycemia inhibits human PDLSC proliferation and osteoblastic differentiation.     

Potential of Periodontal Ligament Cells to Regenerate Alveolar Bone

Regeneration of periodontal tissues, lost as a result of periodontal disease, is a key objective of periodontal treatment. Although several periodontal regeneration therapies have been devised, the origin of the undifferentiated cells that regenerate periodontal tissues remains unknown. Therefore, in the present study, to clarify the existence of osteoblast progenitor cells in the periodontal ligament, as well as to investigate the mechanism of alveolar bone regeneration without any effects from the original bone, we evaluated osteoblast differentiation induced by transplantation of GFP-transgenic rat molars into the subcutaneous tissues of wild-type rats. Ten days after transplantation, initial alveolar bone was formed apart from the cementum in the bifurcation region. After 20 days, this bone tissue had expanded to almost all of the bifurcation. GFP localization showed that the osteoblasts were derived from the transplant. Alpha-SMA- and BMP4-positive cells were observed near the root surface at 5 days after transplantation. With the progress of alveolar bone regeneration, osteoblasts expressing Runx2 and Osterix appeared in the bone-forming region. These results indicate that periodontal ligament tissue remaining on the root surface after a tooth extraction contains undifferentiated cells that have the ability to regenerate alveolar bone. The process of osteoblast differentiation in this model might be similar to that for normal alveolar bone formation. Thus, periodontal ligament cells might be useful for the regeneration of alveolar bone in tissue engineering applications.