年龄因素对人牙周膜干细胞培养和性能的影响

目的:探讨年龄因素对人牙周膜干细胞(periodontal ligament stem cells,PDLSCs)体外培养及其生物学特性的影响。方法:收集临床<30岁、>50岁具有完整牙根的第三磨牙各4个,分别刮取根面残留牙周膜进行PDLSCs分离培养,观察其细胞克隆形成能力、MTT方法检测生长曲线、流式细胞仪分析细胞表面分子,并进行体外成骨、成脂诱导,对比分析PDLSCs多向分化能力。结果:两组第三磨牙中均可培养出具有明显间充质干细胞(mesenchymal stem cell,MSC)特性的PDLSCs,但>50岁组获得的PDLSCs克隆形成率显著降低(P<0.05),其增殖、分化能力亦较<30岁组明显减弱(P<0.05)。结论:年龄因素对PDLSCs体外培养及其生物学特性具有明显影响;PDLSCs的基础和临床研究中,应充分考虑到病人年龄因素。     

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.     

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.     

诱导成体人牙周韧带干细胞向软骨细胞分化的实验研究

目的从成体人牙周组织中分离培养牙周韧带干细胞，研究其分化为软骨细胞的可行性。方法选取10～15岁的年轻患者因正畸拔除的健康牙齿，刮取根中1／3的牙周膜，采用组织块培养法得到牙周韧带细胞，待细胞达一定量后用有限元稀释法进行单细胞克隆，筛选牙周韧带干细胞（PDLSC），体外采用离心管聚集体诱导法进行软骨化诱导培养，光镜下观察诱导细胞形态学的改变，甲苯胺蓝染色、免疫组化、RT-PCR等方法检测胶原和糖蛋白的体外表达情况。结果体外离心管聚集体诱导培养3周后，实验组呈白色半透明状，甲苯胺蓝染色显示在外周区域有大量软骨基质成分沉积，组织学显示有较为明显的软骨陷窝。免疫组化及RT-PCR检测到Ⅱ型胶原表达，Ⅱ型胶原染色呈强阳性。对照组细胞团块逐渐收缩、崩解，组织学检测无软骨陷窝结构，Ⅱ型胶原免疫组化结果阴性。结论从成体人牙周组织中可分离培养出干细胞，在体外能有效增殖且保持低分化状态，具有作为软骨组织工程种子细胞来源的可能，也为牙周组织工程的应用奠定了实验基础。     

正畸力调控牙周膜干细胞成骨分化的动物实验研究

目的 研究不同正畸力值对牙周膜干细胞(periodontal ligament stem cells,PDLSCs)分化的作用及机制.方法 分别用50 g、150 g力值建立大鼠牙齿移动模型,加力7d后分离培养PDLSCs,检测其生物学功能和分子信号通路.结果 相比对照组,加力7d后,50 g力值组大鼠第一磨牙近中移动...     

Effects of prostaglandin E2 on clonogenicity,proliferation and expression of pluripotent markers in human periodontal ligament cells

Background and objectiveBased on our earlier work on the response of periodontal ligament (PDL) cells to mechanical stress by induction of cyclooxygenase expression and production of prostaglandin PGE₂ that could regulate mineralization of PDL cells, it was hypothesized that PGE₂ had potential effects on PDL stemness. In this study, we aimed to investigate clonogenicity, proliferation and expression of certain pluripotent markers, considered to be characteristics of PDL stemness, in response to treatment with exogenously-added PGE₂.Material and methodsHuman PDL cells were cultured and treated with various doses of PGE₂, and the aforementioned characteristics of PDL stemness were analyzed.ResultsThe clonogenicity and proliferation were significantly enhanced by PGE₂ at low concentrations (0.01, 0.1 and 1 ng/ml; P < 0.05), but only the proliferation was significantly diminished by PGE₂ at a high concentration (100 ng/ml; P < 0.05). Expression of NANOG and OCT4 mRNA and protein was increased by PGE₂ treatment at 0.1 and 1 ng/ml. Consistently, expression of stage-specific embryonic antigen 4, a putative stem cell marker, was significantly augmented by PGE₂ treatment at 1 ng/ml (P < 0.05).ConclusionOur findings suggest that although a high dose of PGE₂ (100 ng/ml) inhibits proliferation of PDL cells, PGE₂ at low doses appears to play a role in the maintenance of PDL stemness.     

In vitro proliferation of periodontal ligament-like tissue on extracted teeth

ObjectiveTransplantation of autologous teeth is a routine component of orthodontic treatment. The aim of this study was to develop a method for the regeneration of damaged periodontal ligament (PDL) on extracted teeth using a three-dimensional culture system.DesignWe used the maxillary first premolars or third molars extracted from patients for orthodontic treatment. The extracted teeth were stained with toluidine blue to measure the residual PDL area. After confirming damage of the periodontal tissue on the root surface of the extracted teeth, we tried to regenerate the periodontal tissue. Other extracted teeth were inserted into a cell strainer filled with cellulose-based carrier materials to regenerate the periodontal tissue. The strainer was then placed in a 90-mm culture dish filled with culture medium and incubated at 37 °C and 5% CO2 for about 1 month. The cultured teeth were observed under a stereomicroscope and examined by scanning electron microscopy (SEM), and were stained to detect alkaline phosphatase (ALP) activity.ResultToluidine blue staining revealed that the residual periodontal membrane covered an average of 50.4% of the root surface area of each tooth. After culturing extracted teeth with our culture system, globular structures were found on the entire tooth root surface by stereomicroscopy, and PDL-like filamentous tissue was also detected by SEM. The entire tooth root surfaces of the cultured teeth were positive for ALP activity.ConclusionsWe have developed a useful culture method to stimulate the proliferation of cells in PDL-like tissue on the roots of extracted teeth.     

Role of PDGF-BB in proliferation,differentiation and maintaining stem cell properties of PDL cells in vitro

ObjectivePlatelet-derived growth factor-BB (PDGF-BB) is one of the most abundant growth factors in platelet derived products and has been shown to stimulate regeneration after tissue injury. There is a population of mesenchymal stem cells (MSC) in human periodontal ligament (PDL) which can contribute to tissue regeneration under appropriate conditions.DesignPDL cells were isolated and characterized using stem cell and differentiation markers via immunofluorescence and flow cytometry and then cultured in vitro and treated with different concentrations of PDGF-BB. The effect of PDGF-BB on cell proliferation, stem cell and differentiation markers expression, soluble collagen production, lysyl oxidase (LOX) activity, alkaline phosphatase (ALP) activity and calcium nodules formation was assessed.ResultsPDGF-BB stimulated the proliferation of cells with the maximum effect at 50 ng/mL. The growth factor increased the expression of stem cell markers and SPARC; Col1a2 expression was decreased, whereas the expression of Col3a1 remain unchanged. Soluble collagen production, ALP activity and calcium nodules formation were also significantly decreased by PDGF-BB; LOX activity was significantly increased.ConclusionsPDGF-BB is a powerful promoter of cell proliferation and increases the expression of stem cell markers; inhibites collagen production and mineraliration but accelerates the maturation of collagen chains through increased LOX activity and SPARC expression.     

Comparison of the properties of human CD146+ and CD146− periodontal ligament cells in response to stimulation with tumour necrosis factor α

ObjectivesPeriodontal ligament stem cells (PDLSCs) can be used in periodontal regeneration. Tumour necrosis factor-alpha (TNF-α) participates in the regulation of cell proliferation, apoptosis, differentiation, and migration. However, whether TNF-α can affect the biological features of PDLSCs is still unclear. The objective of this study was to illustrate the biological effects (proliferation, apoptosis, osteogenesis and migration) of TNF-α on human CD146 positive periodontal ligament cells (CD146+PLDCs) and CD146 negative periodontal ligament cells (CD146?PDLCs).MethodsCD146 ± PDLCs were isolated from human PDLCs and analyzed using a fluorescence-activated cell sorter. The biological effects of TNF-α on CD146 ± PDLCs were evaluated by CCK-8 assay (proliferation), DAPI staining (apoptosis), alizarin red staining and alkaline phosphatase activities assay (osteogenesis), and wounding assay and transwell assay (migration).ResultsCD146+PDLCs, which expressed MSC surface markers CD105, CD90, CD73, CD44, and Stro-1, showed higher proliferative and osteogenic potential than CD146?PDLCs. TNF-α at a dose of 2.5 ng/ml was found to enhance both proliferation and osteogenesis in CD146+PDLCs. At 5 ng/ml, TNF-α promoted proliferation, osteogenesis, and apoptosis in CD146+PDLCs and enhanced osteogenesis in CD146?PDLCs. At 10 ng/ml, TNF-α only aggravated apoptosis in CD146+PDLCs. The migratory ability of both CD146+PDLCs and CD146?PDLCs was not altered by TNF-α.ConclusionsCD146+PDLCs were subpopulation of MSC. It showed greater proliferative and osteogenic potential than CD146?PDLCs. At low concentration, TNF-α was beneficial to CD146+PDLCs on proliferation and osteogenesis, and at high concentration it was detrimental. CD146?PDLCs were found to be less sensitive to TNF-α.     

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.     

Isolation and characterization of multipotent human periodontal ligament stem cells

BACKGROUND: Periodontal ligament (PDL) repair is thought to involve mesenchymal progenitor cells capable of forming fibroblasts, osteoblasts and cementoblasts. However, full characterization of PDL stem cell (SC) populations has not been achieved. OBJECTIVE: To isolate and characterize PDLSC and assess their capability to differentiate into bone, cartilage and adipose tissue. METHODS: Human PDL cells were stained for STRO-1, FACS sorted and expanded in culture. Human bone marrow SC (BMSC) served as a positive control. PDLSC and BMSC were cultured using standard conditions conducive for osteogenic, chondrogenic and adipogenic differentiation. Osteogenic induction was assayed using alizarine red S staining and expression of alkaline phosphatase (ALP) and bone sialoprotein (BSP). Adipogenic induction was assayed using Oil Red O staining and the expression of PPAR gamma 2 (early) and LPL (late) adipogenic markers. Chondrogenic induction was assayed by collagen type II expression and toluidine blue staining. RESULTS: Human PDL tissue contains about 27% STRO-1 positive cells with 3% strongly positive. In osteogenic cultures ALP was observed by day-7 in BMSC and day-14 in PDLSC. BSP expression was detectable by day-7; with more intense staining in PDLSC cultures. In adipogenic cultures both cell populations showed positive Oil Red O staining by day-25 with PPAR gamma 2 and LPL expression. By day-21, both BMSC and PDLSC chondrogenic induced cultures expressed collagen type II and glycosaminoglycans. CONCLUSIONS: The PDL contains SC that have the potential to differentiate into osteoblasts, chondrocytes and adipocytes, comparable with previously characterized BMSC. This adult PDLSC population can be utilized for potential therapeutic procedures related to PDL regeneration.     

几种胚胎干细胞标志在牙周膜细胞中的表达及意义

目的检测部分胚胎干细胞标志在牙周膜细胞中的表达。方法通过酶解组织块法分离牙周膜细胞,利用多向分化实验和流式细胞术分析表面标志对获得细胞进行鉴定。利用免疫荧光法和RT-PCR法检测牙周膜细胞中Oct4、Nanog、Sox2、SSEA-4、TRA-1-60、TRA-1-81这些胚胎干细胞标志的表达。结果牙周膜细胞为成纤维细胞样,可分化为成骨细胞和成软骨细胞,可均一表达间充质细胞标志CD44、CD90、CD105,异质性表达间充质标志CD146、Stro-1。细胞免疫荧光实验显示牙周膜细胞表达部分胚胎干细胞标志:Oct4、Nanog、SSEA-4、TRA-1-60阳性,Sox2、TRA-1-81阴性。半定量RTPCR结果显示P3和P8代牙周膜细胞表达Oct4和Nanog,表达量差异不大,Sox2则为阴性。结论牙周膜细胞除了表达常用间充质干细胞标志外,也可表达部分胚胎干细胞标志,这对牙周膜细胞的鉴定和进一步分选纯化有一定应用价值。     

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- ...     

Comparative proteomic profiling of human dental pulp stem cells and periodontal ligament stem cells under in vitro osteogenic induction

ObjectiveThis study aimed to compare the proteomic profiling of human dental pulp stem cells (DPSCs) and periodontal ligament stem cells (PDLSCs) under in vitro osteogenic induction, which imitates the microenvironment during osteo-/odontogenesis of DPSCs and PDLSCs.DesignThe proteomic profiles of osteoinduced DPSCs and PDLSCs from a single donor were compared using the isobaric tag for relative and absolute quantitation (iTRAQ) technique and subsequent bioinformatics analysis.ResultsA total of 159 differentially expressed proteins in PDLSCs and DPSCs were identified, 82 of which had a higher expression level in PDLSCs, while 77 were more highly expressed in DPSCs. Among these enriched proteins, certain members from the collagen, heat shock protein and protein S100 families may distinguish osteoinduced PDLSCs and DPSCs. Gene ontology (GO) classification revealed that a large number of the enriched terms distinguishing PDLSCs and DPSCs are involved in catalytic activity, protein binding, regulation of protein metabolic processes and response to stimulus. Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis indicated several involved pathways, including the fatty acid biosynthesis pathway, pantothenate and CoA biosynthesis pathway, arachidonic acid metabolism pathway and PPAR signaling pathway. Further verification showed that the mineralization and migration capacities of PDLSCs were greater than those of DPSCs, in which heat shock protein beta-1, Protein S100-A10 and S100-A11 may play a part.ConclusionsLess than 5% of the differentially expressed proteins make up the comparative proteomic profile between osteoinduced PDLSCs and DPSCs. This study helps to characterize the differences between osteoinduced PDLSCs and DPSCs in vitro.     

High-plasticity mesenchymal stem cells isolated from adult-retained primary teeth and autogenous adult tooth pulp—A potential source for regenerative therapies?

PurposeThe objective of this study was to compare the growth rate, morphology, immunohistology and plasticity of autogenous adult-retained SHEDs (arSHEDs) and adult dental pulp stem cells (DPSCs) obtained from the same donor.MethodsExpression of the mesenchymal stem cell markers CD44, CD90, CD105, caspase-3 and GAPDH were assessed using RT-PCR. Caspase-3 and CD44 were also evaluated at the protein level by western blotting of cell lysates. Plasticity of DPSCs and arSHEDs were tested by culture in adipogenic, chondrogenic, osteogenic and Schwann cells induction media.ResultsDPSCs and arSHEDs were isolated by explant culturing and were similarly positive for growth rate and all tested markers. Furthermore, DPSCs and arSHEDs could be driven to adipocyte, chondrocyte, osteocyte and Schwann cells lineages thus indicating similar plasticity as precursor cells.ConclusionThis study demonstrates the similarities between DPSCs and arSHEDs in a unique situation, where both stem cells (SC) types were obtained from a single patient and thus represent an alternative source of SC’s for tissue engineering and regeneration.