水解鱼胶原诱导大鼠骨髓间充质干细胞成骨分化的作用

目的 探讨水解鱼胶原诱导大鼠骨髓间充质干细胞(rBMSCs)成骨分化的潜能.方法 制备获得水解鱼胶原,对其分子量,氨基酸成分和接触角进行表征,利用MTT 试验和Real-Time PCR 试验分别研究水解鱼胶原对rBMSCs 细胞活力及成骨分化相关基因ALP,OCN 和RUNX2 的影响,利用western blot 技术探讨水解鱼胶原对ERK1/2 信号通路的影响. 结果 水解鱼胶原的分子量为700-1300 Da,接触角约为26 度,主要含甘氨酸,脯氨酸和羟脯氨酸.水解鱼胶原能提高rBMSCs 细胞的活力,促进成骨分化相关基因ALP,OCN 和RUNX2 的表达,Western Blot 结果显示水解鱼胶原可激活ERK1/2 信号通路,进而促进RUNX2 蛋白表达上调. 结论 水解鱼胶原具有诱导大鼠骨髓间充质干细胞成骨分化的潜能.     

细胞外信号调节激酶1/2信号转导通路调控牙周膜细胞成骨分化的研究

目的：探索细胞外信号调节激酶（ERK）1/2信号转导通路是否参与调控牙周膜细胞的成骨分化。方法???取体外培养的第3代牙周膜细胞进行研究。实验分为空白对照组、成骨诱导组和实验组（在成骨诱导培养基中加入10?nmol·L－1?ERK1/2磷酸化的抑制剂PD98059）。培养1周和3周后通过定量聚合酶链反应（qPCR）、碱性磷酸酶（ALP）染色和茜素红染色检测其成骨能力。结果?成骨诱导可促进牙周膜细胞中ERK1/2的磷酸化。培养1周后,抑制ERK1/2的磷酸化可上调成骨标志物Runx2、ALP和骨钙蛋白（OCN）的表达,与成骨诱导组相比较,OCN的表达差异具有统计学意义（P<0.05）,Runx2、ALP的表达差异也具有统计学意义（P<0.01）。培养3周后,实验组牙周膜细胞成骨标志物Runx2、ALP和OCN的表达仍较成骨诱导组高,ALP染色和钙结节形成较成骨诱导组强,其中Runx2、ALP的表达差异具有统计学意义（P<0.05）,OCN的表达差异也具有统计学意义（P<0.01）。结论 ERK?1/2信号转导通路参与了调控体外培养的牙周膜细胞的成骨分化。     

高浓度唑来膦酸对人牙周膜干细胞凋亡及成骨分化影响的实验研究

目的：研究高浓度唑来膦酸对体外分离的健康人牙周膜干细胞增殖、凋亡和成骨分化的影响。方法：体外分离培养健康人牙周膜干细胞,用不同浓度的唑来膦酸（5μmol/L、10 μmol/L）处理,不加药组为空白对照。 以 CCK8 检测细胞增殖,流式细胞技术检测细胞凋亡,ALP、茜素红染色及半定量分析检测细胞体外成骨分化,荧光定量PCR检测成?标志物I型胶原(COL1A1)和骨钙素（OCN）基因的表达,免疫荧光检测 OCN和 COL1A1 蛋白的表达。结果： 唑来膦酸可呈计量依赖性抑制人牙周膜干细胞增殖;唑来膦酸用药组细胞的凋亡率显著?于对照组,且随着药物浓度的增加,凋亡百分比逐渐上升;ALP和茜素红染?结果表示唑来膦酸用药组细胞的体外成骨分化能力较对照组显著下降;荧光定量PCR和免疫荧光结果提示,药物处理后OCN和 COL1A1的基因表达和蛋白表达均降低。结论：高浓度唑来膦酸显著抑制人牙周膜干细胞的增殖及体内外成骨分化,并诱导其凋亡。     

p38信号通路在牙周膜细胞成骨分化中的调控作用

目的 研究p38信号通路在人牙周膜细胞成骨分化中的调控作用。 方法 体外培养人牙周膜细胞,取第三代细胞进行成骨诱导培养,实验组加入p38信号通路抑制剂(SB203580),诱导培养4周后通过定量PCR和茜素红染色检测其成骨能力。 结果 成骨诱导可促进牙周膜细胞中p38的磷酸化（p-p38）。抑制p38的磷酸化可降低成骨标记物Runx相关基因（Runx2）、碱性磷酸酶（ALP）、骨桥蛋白（OPN）和骨钙素（OCN）的表达,减少钙结节形成。 结论 p38信号通路在体外培养的牙周膜细胞成骨分化中具有重要的调控作用。     

山羊骨髓基质干细胞向成骨细胞诱导后成骨分化相关基因表达的变化

目的:研究山羊骨髓基质干细胞向成骨细胞诱导后成骨分化相关基因ALP、OCN、COL I mRNA表达水平的变化.方法:采用全骨髓培养法培养骨髓基质干细胞,使用成骨条件培养液体外诱导成骨细胞,通过细胞形态学观察、免疫组化染色、钙结节等检测手段进行成骨细胞鉴定,采用RT-PCR和实时定量PCR检测诱导2周后骨髓基质干细胞ALP、OCN、COL Ⅰ的表达水平,未诱导的骨髓基质干细胞作为对照组.结果:通过成骨细胞鉴定,骨髓基质干细胞成功诱导分化为成骨细胞:OCN和COL Ⅰ基因存细胞诱导组表达上调,ALP基因表达在诱导组和未诱导组间无显著差异.结论:成功诱导山羊BMSCs向成骨细胞分化,成骨分化相关基因在诱导后为适应成骨细胞的功能发生变化.     

人牙周膜干细胞成骨相诱导后相关基因的表达变化

目的:对人牙周膜干细胞进行筛选纯化,骨向诱导后观察其成骨能力,并对成骨相分化过程中相关基因的表达变化进行检测。方法:用组织块联合磁珠分选法分离纯化人牙周膜干细胞,初步探讨其成骨性能,碱性磷酸酶(ALP)和茜素红染色观察其成骨情况,实时定量PCR检测其相关成骨基因的表达变化。利用SPSS12.0软件包对数据进行统计学分析。结果:组织块联合磁珠分选法能成功培养出高纯度的人牙周膜干细胞。体外培养中,PDLSCs具备成脂和成骨的双相分化能力。在成骨诱导过程中,FOXO1和RUNX2的表达先升高再降低,ALP和OCN的基因表达水平持续增高。结论:成骨相关基因 ALP、RUNX2、FOXO1和OCN均参与其向成骨样组织分化的过程,其表达变化具有时间规律性,与成骨过程类似。     

犬不同部位BMSCs对牙周膜细胞增殖和成骨分化的影响

目的:研究犬髂骨骨髓基质细胞(iliac bone marrow stromal cells,I-BMSCs)和犬颌骨骨髓基质细胞(maxilla bone marrow stromal cells,M-BMSCs)对犬牙周膜细胞(periodontal ligament cells,PDLCs)增殖和成骨分化的影响,了解不同部位来源的骨髓基质细胞对牙周膜细胞调控作用的差异性。方法:原代培养犬髂骨骨髓基质细胞、颌骨骨髓基质细胞和牙周膜细胞。分别建立犬髂骨和颌骨来源的骨髓基质细胞与牙周膜细胞的Transwell共培养体系;制备骨髓基质细胞条件培养液培养牙周膜细胞,MTT法检测牙周膜细胞生长曲线;利用实时荧光定量PCR法检测牙周膜细胞成骨相关基因核心结合因子2(Runx2)、碱性磷酸酶(ALP)、骨钙素(OCN)的变化;Westernblot法检测牙周膜细胞Runx2和OCN蛋白的表达变化。结果:髂骨骨髓基质细胞条件培养液能促进牙周膜细胞的增殖;颌骨骨髓基质细胞条件培养液对牙周膜细胞增殖有抑制作用;QPCR检测到共培养组牙周膜细胞Runx2、OCN、ALP基因表达高于对照组,Westernblot检测到共培养组牙周膜细胞的Runx2和OCN的蛋白表达高于对照组,且颌骨骨髓基质细胞诱导牙周膜细胞成骨分化效果更显著。结论:犬颌骨骨髓基质细胞条件培养液可能会抑制牙周膜细胞的增殖,相较于髂骨骨髓基质细胞,颌骨骨髓基质细胞促进牙周膜细胞成骨分化效果更显著,颌骨骨髓基质细胞和牙周膜细胞共同作为种子细胞可能更有利于牙周组织再生。     

大气压常温等离子体对人牙周膜干细胞成骨分化的影响

目的:探究大气压常温等离子体(atmospheric room temperature plasma, ARTP)对人牙周膜干细胞(human periodontal ligament stem cells, hPDLSCs)成骨分化的影响。方法:原代培养人牙周膜细胞,采用免疫磁珠法从中分选出hPDLSCs,通过成骨和成脂诱导培养检测其分化潜能;利用不同时长ARTP处理hPDLSCs,行成骨诱导培养,测定细胞碱性磷酸酶(alkaline phosphatase, ALP)活性,茜素红染色(alizarin red staining, ARS)及半定量分析法观察细胞矿化结节形成状况,RT-qPCR测定细胞成骨相关基因的表达状况,测定细胞内活性氧粒子(reactive oxygen species, ROS)含量变化。结果:采用免疫磁珠法分选出的hPDLSCs呈长梭形、多角形,表现出良好的成骨和成脂分化潜能;ARTP处理时长1 min可提高hPDLSCs的ALP活性,增加细胞内矿化结节形成量;能够提升hPDLSCs成骨相关基因ALP、COL-I、RUNX2的表达水平;ARTP提高了hPDL...     

仿生鱼胶原复合膜引导牙周/骨组织再生的体外研究

目的 研制一种综合性能优异的仿生鱼胶原复合膜,探讨其作为引导牙周/骨组织再生（GTR/GBR）膜的生物学潜能。方法 制备一种新型仿生静电纺鱼胶原/生物玻璃/壳聚糖（Col/BG/CS）复合纳米纤维膜,分别研究复合膜的结构、性能、抗菌性及其诱导人牙周膜韧带细胞（HPDLCs）的生物学效应。结果 复合膜具有仿生结构和一定的力学强度。相比单纯的鱼胶原膜,复合膜对变形链球菌显示出一定的抑菌效果。复合膜被证明能明显促进HPDLCs的粘附、增殖成骨分化以及RUNX-2,OPN蛋白的表达。结论 本研究首次开发一种具有主动诱导牙周组织再生能力并兼有一定抗菌性的多功能仿生鱼Col/BG/CS复合纳米纤维膜,为其作为GTR/GBR膜的应用提供可能。     

Asiaticoside induces osteogenic differentiation of human periodontal ligament cells through the Wnt pathway

1 Background

Asiaticoside is a compound isolated from Herb Centella asiatica, which has been shown to promote osteogenic differentiation of human periodontal ligament (hPDL) cells. This study investigated the molecular mechanism underlying the asiaticoside‐induced osteogenic differentiation of hPDL cells.

2 Methods

hPDL cells were incubated with various concentrations of asiaticoside to test cell viability by MTT assay. The mRNA expression levels were analyzed by using quantitative real‐time polymerase chain reaction (PCR). Osteogenic differentiation was determined by alkaline phosphatase activity assay and alizarin red staining. The subcellular localization of β‐catenin was analyzed by both immunofluorescence and western blot.

3 Results

The results showed that asiaticoside had no effect on the cell viability at any of the tested concentrations. Real‐time PCR revealed that osterix (OSX) and dentin matrix protein1 (DMP1) mRNA were significantly enhanced by asiaticoside treatment. Alkaline phosphatase activity and in vitro mineralization were also significantly induced. Interestingly, asiaticoside dose‐dependently increased WNT3A mRNA expression, but not WNT5A and WNT10B. The activation of Wnt signaling was shown to result in nuclear accumulation of β‐catenin as evaluated by immunofluorescence staining and western blot analysis. Pre‐treatment with recombinant human Dickkopf1 (rhDKK1) inhibited asiaticoside‐induced β‐catenin nuclear translocation and osteoblast marker gene expression. Moreover, rhDKK1 attenuated asiaticoside‐induced DMP1 protein expression.

4 Conclusion

The data demonstrate that asiaticoside induces osteogenic differentiation of hPDL cells by activating the Wnt/β‐catenin signaling pathway. The findings suggest that asiaticoside could be used as a novel therapeutic drug for periodontal tissue regeneration.     

Effects of relaxin on collagen type I released by stretched human periodontal ligament cells

Authors – Takano M, Yamaguchi M, Nakajima R, Fujita S, Kojima T, Kasai K Objectives – Relapse of teeth that have moved during orthodontic treatment is a major clinical issue with respect to the goals of successful treatment. Such relapse is a physiologic response of the supporting tissues to application of force, and is mainly attributed to occlusal instability and increased mechanical tension exerted by the periodontal ligament (PDL). Relaxin, a member of the insulin/relaxin family of structurally related hormones, has an influence on many physiologic processes, such as collagen turnover, angiogenesis, and antifibrosis. Therefore, relaxin may also affect orthodontic tooth movement through alterations of the PDL, though little is known regarding the relationship between relaxin and stretched human PDL (hPDL) cells. In the present study, we investigated the effects of relaxin on the expression of collagen type I (Col‐I) and matrix metalloproteinase 1 (MMP‐1) in stretched hPDL cells in vitro. Materials and Methods – The release and gene expression of Col‐I, as well as those of MMP‐1 in stretched hPDL cells treated with relaxin were investigated using enzyme‐linked immunosorbent assay and real‐time PCR methods. Results – Relaxin decreased the release and gene expression of Col‐I, and increased those of MMP‐1 by stretched hPDL cells in a magnitude‐dependent manner. Conclusion – Our results indicate that relaxin modulates collagen metabolism in stretched hPDL cells via the release and expression of Col‐I and MMP‐1. This hormone may be useful to prevent orthodontic relapse following orthodontic treatment.     

What is the function of osteocalcin?

BackgroundOsteocalcin is the most abundant non-collagenous protein in bone and is specifically expressed in osteoblasts. Previous studies using osteocalcin-deficient (Ocn^–/–) mice demonstrated that osteocalcin inhibits bone formation, and serum uncarboxylated osteocalcin functions as a hormone that improves glucose metabolism, induces testosterone synthesis in the testes, and maintains muscle mass. Furthermore, the relationship between serum osteocalcin and glucose metabolism or cardiovascular risk in humans has been reported. However, new Ocn^–/– mice exhibited different phenotypes.HighlightBone volume, formation, and resorption were normal in the new Ocn^–/– mice. The orientation of collagen fibers was parallel to the bone longitudinal direction and the size of apatite crystals was normal, but the c-axis of apatite crystals was random and bone strength was reduced in new Ocn^–/– mice. Glucose metabolism, testosterone synthesis, and muscle mass were normal in new Ocn^–/– mice. Exercise improved glucose metabolism and increased bone formation, leading to an increase in the serum osteocalcin level, which is a marker for bone formation.ConclusionContrary to previous findings, new Ocn^–/– mice revealed that osteocalcin has no function in the regulation of bone quantity, but instead, functions to direct the parallel alignment of the c-axis of apatite crystals with collagen fibrils. Moreover, it has no physiological function as a hormone that regulates glucose metabolism, testosterone synthesis, or muscle mass. These controversial phenotypes require further investigation. The relationship of serum osteocalcin with glucose metabolism or cardiovascular risk suggests the importance of exercise for their improvement.     

Dental pulp stem cells from traumatically exposed pulps exhibited an enhanced osteogenic potential and weakened odontogenic capacity

Objectives

Traumatic pulp exposure can bring about some permanent damages to tooth tissues including dental pulps. This study was designed to evaluate the effects of traumatic pulp exposure on the osteo/odontogenic capacity of dental pulp stem cells (DPSCs).

Methods

Rat incisors were artificially fractured and dental pulps were exposed to the oral environment for 48 h. Then, multi-colony-derived DPSCs from the injured pulps (iDPSCs) were isolated. Their osteo/odontogenic differentiation and the involvement of NF-κB pathway were subsequently investigated.

Results

iDPSCs presented a lower proliferative capacity than normal DPSCs (nDPSCs), as indicated by MTT and FCM assay. ALP levels in iDPSCs were significantly higher (P < 0.01) than those in nDPSCs. Alizarin red staining revealed that iDPSCs exhibited an increased capacity of calcium deposition. Moreover, iDPSCs expressed stronger osteogenic markers (Runx2/RUNX2 and Ocn/OCN) and less odontogenic gene/protein (Dspp/DSP) than nDPSCs in vitro. In vivo transplantation showed that nDPSCs implants generated the typical dentine-pulp complex while all iDPSCs pellets formed the osteodentin-like tissues which were immunopositive for OCN. Mechanistically, iDPSCs expressed the higher levels of cytoplasmic phosphorylated IκBα/P65 and nuclear P65 than nDPSCs, indicating an active cellular NF-κB pathway in iDPSCs. After the inhibition of NF-κB pathway, the osteogenic potential in iDPSCs was significantly down-regulated while odontogenic differentiation was up-regulated, as indicated by the decreased Alp/Runx2/Ocn and uprised Dspp expression.

Conclusions

Pulp exposure for 48 h decreased the odontogenic capacity and enhanced the osteogenic potential of DPSCs via the NF-κB signalling pathway.     

Fibronectin-loaded Collagen/Gelatin Hydrogel Is a Potent Signaling Biomaterial for Dental Pulp Regeneration

IntroductionGuided tissue regeneration has been considered a promising biological strategy to replace conventional endodontic therapies of teeth with incomplete root formation. Therefore, in the present study, a collagen/gelatin hydrogel either containing dosages of fibronectin (FN), or not, was developed and assessed concerning their bioactive and chemotactic potential on human apical papilla cells (hAPCs).MethodsHydrogels were prepared by varying the ratio of collagen and gelatin (Col/Gel; v/v), and used to establish the following groups: Collagen (positive control); Col/Gel 4:6; Col/Gel 6:4; Col/Gel 8:2. The viability, adhesion, and spreading of cells seeded on the hydrogels were evaluated. Different concentrations of FN (0, 5, or 10 μg/mL) were incorporated into the best formulation of the collagen/gelatin hydrogel selected. Then, the hAPCs seeded on the biomaterials were assessed concerning the cell migration, viability, adhesion and spreading, and gene expression of ITGA5, ITGAV, COL1A1, and COL3A1.ResultsThe Col/Gel 8:2 group exhibited better cell viability, adhesion and spreading in comparison with Control. Higher values of hAPC migration, viability, adhesion, spreading and gene expression of pulp regeneration markers were found, the higher the concentration was of FN incorporated into the collagen/gelatin hydrogel.ConclusionCollagen/gelatin hydrogel with 10 μg/mL of FN had potent bioactive and chemotactic effects on cultured hAPCs.     

Characterization of a bioactive Jagged1-coated polycaprolactone-based membrane for guided tissue regeneration

ObjectiveThe aim of the present study was to develop a Jagged1-coated polycaprolactone (PCL) membrane and to evaluate the response of human periodontal ligament cells (hPDL) on this membrane in vitro.MethodsMembranes were prepared from PCL and PCL-incorporated hydroxyapatite (PCL/HA). The membranes’ surface roughness, surface wettability, and mechanical properties were examined. An indirect affinity immobilization technique was used to coat the membranes with Jagged1. Membrane cytotoxicity was evaluated using LIVE/DEAD and MTT assays. The morphology of the cells on the membranes was observed using scanning electron microscopy. hPDL alkaline phosphatase (ALP) enzymatic activity and mineral deposition were examined using an ALP assay and Alizarin Red S staining, respectively. Notch target gene mRNA expression was determined using real-time polymerase chain reaction.ResultsThe PCL/HA membranes exhibited a significantly reduced surface contact angle, decreased maximum tensile strain, and ultimate tensile stress. However, the surface roughness parameters were significantly increased. The PCL and PCL/HA membranes were not cytotoxic to hPDL in vitro. hPDLs attached and spread on both membrane types. Further, indirect affinity immobilized Jagged1 on the membranes upregulated hPDL Notch target gene expression. After culturing in osteogenic medium, Jagged1-immobilized PCL/HA membranes significantly enhanced hPDL ALP enzymatic activity.ConclusionIndirect immobilized Jagged1 PCL/HA membranes could be further developed as an alternative guided tissue regeneration membrane to promote osteogenic differentiation in periodontal defects.