In vitro responses of human pulp cells and 3T3 mouse fibroblasts to six contemporary dental restoratives

In vitro responses of human primary pulp cells (HPCs) and 3T3 mouse fibroblasts to six contempo-rary commercial dental restoratives were evaluated using the WST-1 assay. The results show that Fuji II is not cytotoxic to both cells. Fuji II LC is not cyto-toxic to HPCs but cytotoxic to 3T3 cells, indicating that 3T3 cells are more vulnerable to 2-hydroxyethyl methacrylate (HEMA) than HPCs. Vitremer is very cytotoxic probably due to having diphenyliodonium chloride and HEMA in it. Z100 is very cytotoxic probably due to having triethylene glycol dimethacry-late (TEGDMA) in it. P60 is cytotoxic but less cyto-toxic than Z100 probably due to no TEGDMA in it. Durelon is the most cytotoxic among the six materials studied probably due to the high cytotoxicity of zinc ions. Additionally, the cytotoxcity of the tested mate-rials was found to be dose-dependent.

Self-assembly of differentiated dental pulp stem cells facilitates spheroid human dental organoid formation and prevascularization

BACKGROUND The self-assembly of solid organs from stem cells has the potential to greatly expand the applicability of regenerative medicine.Stem cells can self-organise into microsized organ units,partially modelling tissue function and regeneration.Dental pulp organoids have been used to recapitulate the processes of tooth development and related diseases.However,the lack of vasculature limits the utility of dental pulp organoids.AIM To improve survival and aid in recovery after stem cell transplantation,we demonstrated the three-dimensional(3D)self-assembly of adult stem cell-human dental pulp stem cells(hDPSCs)and endothelial cells(ECs)into a novel type of spheroid-shaped dental pulp organoid in vitro under hypoxia and conditioned medium(CM).METHODS During culture,primary hDPSCs were induced to differentiate into ECs by exposing them to a hypoxic environment and CM.The hypoxic pretreated hDPSCs were then mixed with ECs at specific ratios and conditioned in a 3D environment to produce prevascularized dental pulp organoids.The biological characteristics of the organoids were analysed,and the regulatory pathways associated with angiogenesis were studied.RESULTS The combination of these two agents resulted in prevascularized human dental pulp organoids(Vorganoids)that more closely resembled dental pulp tissue in terms of morphology and function.Single-cell RNA sequencing of dental pulp tissue and RNA sequencing of Vorganoids were integrated to analyse key regulatory pathways associated with angiogenesis.The biomarkers forkhead box protein O1 and fibroblast growth factor 2 were identified to be involved in the regulation of Vorganoids.CONCLUSION In this innovative study,we effectively established an in vitro model of Vorganoids and used it to elucidate new mechanisms of angiogenesis during regeneration,facilitating the development of clinical treatment strategies.

Genetic modification of miR-34a enhances efficacy of transplanted human dental pulp stem cells after ischemic stroke

Human dental pulp stem cells(hDPSCs) promote recovery after ischemic stro ke;however,the therapeutic efficacy is limited by the poor survival of transplanted cells.For in vitro expe riments in the present study,we used oxygen-glucose deprivation/reoxygenation in hDPSCs to mimic cell damage induced by ischemia/reperfusion.We found that miRNA-34a-5p(miR-34a) was elevated under oxygen-glucose deprivation/reoxygenation conditions in hDPSCs.Inhibition of miR-34a facilitated the prolife ration and antioxidant capacity and reduced the apoptosis of hDPSCs.Moreove r,dual-luciferase reporter gene assay showed WNT1and SIRT1 as the targets of miR-34a.In miR-34a knockdown cell lines,WNT1 suppression reduced cell prolife ration,and SIRT1 suppression decreased the antioxidant capacity.Togethe r,these results indicated that miR-34a regulates cell prolife ration and antioxidant stress via targeting WNT1 and SIRT1,respectively.For in vivo expe riments,we injected genetically modified hDPSCs(anti34a-hDPSCs) into the brains of mice.We found that anti34a-hDPSCs significantly inhibited apoptosis,reduced cerebral edema and cerebral infarct volume,and improved motor function in mice.This study provides new insights into the molecular mechanism of the cell prolife ration and antioxidant capacity of hDPSCs,and suggests a potential gene that can be targeted to improve the survival rate and efficacy of transplanted hDPSCs in brain after ischemic stroke.

Human pulp tissue dissolution ability of different extracts of Sapindus mukorossi: An in vitro study

Objective:Due to the many negative properties of sodium hypochlorite used in current root canal treatment,interest in biocompatible natural agents is increasing day by day.The aim of this study was to evaluate whether various extract solutions of Sapindus mukorossi have dissolution effects on human pulp tissues.Methods:Primarily powder extracts were obtained by extracting fruit shells of S.mukorossi in different solvents(ethanol,methanol,buthanol and distilled water).The test solutions were prepared and randomly separated into six groups with 10 samples in each group:ethanol extract,methanol extract,butanol extract,distilled water extract of S.mukorossi,sodium hypochlorite(Na OCl)and the control group.Among these,S.mukorossi extracts were separated into two subgroups,depending on their concentration level(50μg/m L and 100μg/m L).The pulp tissues of freshly extracted human molars were used for dissolution test.The weights of the pulpal tissues were measured and recorded for two times after the samples were placed in the solutions.Statistical analysis for all descriptive statistics was performed using SPSS 22(P<0.05).Results:Our results showed that maximum percent yield of preparation was obtained in methanol extract of S.mukorossi.Among all of the groups,the best dissolution capacity was seen in the Na OCl group(positive control group).Among S.mukorossi groups,the best tissue solvent solution was found in SMM group at 50μg/m L and SMB group at 100μg/m L.Conclusion:The different extracts of S.mukorossi had a capacity to dissolve pulp tissue but this capacity was less than Na OCl.Therefore,further studies will enable the creation of a commercial solution for clinical use by increasing the effectiveness of S.mukorossi while combining it with other endodontic irrigation solutions.

TET1 knockdown inhibits the odontogenic differentiation potential of human dental pulp cells

Human dental pulp cells （hDPCs） possess the capacity to differentiate into odontoblast-like cells and generate reparative dentin in response to exogenous stimuli or injury. Ten-eleven translocation 1 （TET1） is a novel DNA methyldioxygenase that plays an important role in the promotion of DNA demethylation and transcriptional regulation in several cell lines. However, the role of TET1 in the biological functions of hDPCs is unknown. To investigate the effect of TET1 on the proliferation and odontogenic differentiation potential of hDPCs, a recombinant shRNA lentiviral vector was used to knock down TET1 expression in hDPCs. Following TET1 knockdown, TET1 was significantly downregulated at both the mRNA and protein levels. Proliferation of the hDPCs was suppressed in the TET1 knockdown groups. Alkaline phosphatase activity, the formation of mineralized nodules, and the expression levels of DSPP and DMP1 were all reduced in the TETl-knockdown hDPCs undergoing odontogenic differentiation. Based on these results, we concluded that TET1 knockdown can prevent the proliferation and odontogenic differentiation of hDPCs, which suggests that TET1 may play an important role in dental pulp repair and regeneration.

Inhibition of matrix metalloproteinases expression in human dental pulp cells by all-trans retinoic acid

All-trans retinoic acid（ATRA） inhibits matrix metalloproteinase（MMP）-2 and MMP-9 in synovial fibroblasts, skin fibroblasts,bronchoalveolar lavage cells and cancer cells, but activates MMP-9 in neuroblast and leukemia cells. Very little is known regarding whether ATRA can activate or inhibit MMPs in human dental pulp cells（HDPCs）. The purpose of this study was to determine the effects of ATRA on the production and secretion of MMP-2 and-9 in HDPCs. The productions and messenger RNA（mRNA） expressions of MMP-2 and-9 were accessed by gelatin zymography and real-time polymerase chain reaction（PCR）, respectively. ATRA was found to decrease MMP-2 level in a dose-dependent manner. Significant reduction in MMP-2 mRNA expression was also observed in HDPCs treated with 25 mmol？L21ATRA. However, HDPCs treated with ATRA had no effect on the pattern of MMP-9 produced or secreted in either cell extracts or conditioned medium fractions. Taken together, ATRA had an inhibitory effect on MMP-2 expression in HDPCs,which suggests that ATRA could be a candidate as a medicament which could control the inflammation of pulp tissue in vital pulp therapy and regenerative endodontics.

Human dental pulp stem cells: Applications in future regenerative medicine

Stem cells are pluripotent cells, having a property of differentiating into various types of cells of human body. Several studies have developed mesenchymal stem cells(MSCs) from various human tissues,peripheral blood and body fluids. These cells are then characterized by cellular and molecular markers to understand their specific phenotypes. Dental pulp stem cells(DPSCs) are having a MSCs phenotype and they are differentiated into neuron, cardiomyocytes, chondrocytes, osteoblasts, liver cells and β cells of islet of pancreas. Thus, DPSCs have shown great potentiality to use in regenerative medicine for treatment of various human diseases including dental related problems. These cells can also be developed into induced pluripotent stem cells by incorporation of pluripotency markers and use for regenerative therapies of various diseases. The DPSCs are derived from various dental tissues such as human exfoliated deciduous teeth, apical papilla, periodontal ligament and dental follicle tissue. This review will overview the information about isolation, cellular and molecular characterization and differentiation of DPSCs into various types of human cells and thus these cells have important applications in regenerative therapies for various diseases. This review will be most useful for postgraduate dental students as well as scientists working in the field of oral pathology and oral medicine.

PiggyBac transposon-mediated gene delivery efficiently generates stable transfectants derived from cultured primary human deciduous tooth dental pulp cells(HDDPCs) and HDDPC-derived iPS cells

The ability of human deciduous tooth dental pulp cells（HDDPCs） to differentiate into odontoblasts that generate mineralized tissue holds immense potential for therapeutic use in the field of tooth regenerative medicine. Realization of this potential depends on efficient and optimized protocols for the genetic manipulation of HDDPCs. In this study, we demonstrate the use of a Piggy Bac（PB）-based gene transfer system as a method for introducing nonviral transposon DNA into HDDPCs and HDDPC-derived inducible pluripotent stem cells. The transfection efficiency of the PB-based system was significantly greater than previously reported for electroporation-based transfection of plasmid DNA. Using the neomycin resistance gene as a selection marker, HDDPCs were stably transfected at a rate nearly 40-fold higher than that achieved using conventional methods. Using this system, it was also possible to introduce two constructs simultaneously into a single cell. The resulting stable transfectants, expressing td Tomato and enhanced green fluorescent protein, exhibited both red and green fluorescence. The established cell line did not lose the acquired phenotype over three months of culture. Based on our results, we concluded that PB is superior to currently available methods for introducing plasmid DNA into HDDPCs. There may be significant challenges in the direct clinical application of this method for human dental tissue engineering due to safety risks and ethical concerns. However, the high level of transfection achieved with PB may have significant advantages in basic scientific research for dental tissue engineering applications, such as functional studies of genes and proteins. Furthermore, it is a useful tool for the isolation of genetically engineered HDDPC-derived stem cells for studies in tooth regenerative medicine.

Inductive effect of bovinc bone morphogenetic protein on human dental pulp tissue in vitro

Bone morphogenetic protein (BMP) can promote the proliferation of dental pulp cells and induce reparative dentin formation. In this study, the inductive effect of BMP derived from bovine bone matrix on cultured human dental pulp tissue was observed under light microscope and transmission electron microscope. The results showed that. by the third day of culture, the proliferating star-shaped cells appeared with small cytoplasm and poorly-developed organelles; by the 7th day of the culture, the chondroblast-like cells with rich cytoplasm and well-developed organelles were seen embedded in hyaline matrix. This study suggests that BMP can induce dental pulp cells to differentiate from poorly differentiated state to well-differentiated state.

低浓度氟化钠对人牙髓细胞的成骨/成牙本质分化的影响

目的探讨低浓度氟化钠对人牙髓细胞(human dental pulp cells,hDPCs)成骨/成牙本质分化的影响。方法本研究已通过单位伦理委员会审查批准。原代培养hDPCs,采用MTT法检测不同浓度氟化钠对hDPCs增殖的影响;选取合适浓度的氟化钠加入成骨/成牙本质分化诱导培养液中,对hDPCs进行体外诱导,通过茜素红染色检测hDPCs成骨/成牙本质分化能力的变化,RT⁃qPCR检测分化相关基因的mRNA表达;同时通过RT⁃qPCR和Western blot检测hDPCs成骨/成牙本质分化过程中内质网应激相关基因的表达。结果低浓度氟化钠(0.1 mmol/L)在体外可刺激hDPCs增殖,高浓度氟化钠(5~10 mmol/L)可抑制hDPCs增殖(P<0.05)。选取0.1 mmol/L氟化钠体外混合成骨/成牙本质分化诱导培养后hDPCs的茜素红染色增加,成骨/成牙本质分化相关基因牙本质涎磷蛋白(dentin sialophosphoprotein,DSPP)、骨涎蛋白(bone sialoprotein,BSP)和骨钙蛋白(osteocalcin,OCN)mRNA表达水平升高(P<0.05)。同时在此过程中RT⁃qPCR检测出mRNA水平hDPCs内质网应激相关基因:剪切x盒结合蛋白1(splicing x⁃box binding protein⁃1,sXBP1)、葡萄糖调节蛋白78(glucose⁃regulated protein 78,GRP78)以及活化转录因子4(activating transcription factor 4,ATF4)表达升高(P<0.05);Western blot检测出氟化钠混合成骨/成牙本质分化培养后细胞磷酸化真核起始因子⁃2α(phosphorylated eukary⁃otic initiation factor⁃2α,p⁃eIF2α)、磷酸化蛋白激酶样内质网激酶(phosphorylated the RNA⁃activated protein kinase⁃like ER⁃resident kinase,p⁃PERK)和ATF4蛋白表达增加(P<0.05)。结论低剂量氟化钠促进人牙髓细胞的成骨/成牙本质分化并伴有内质网应激水平的升高。

现代网络技术支持下的制浆造纸企业管理模式创新研究

随着互联网的普及与发展,现代网络技术正深刻改变着企业生产与管理的方方面面,会有更多的传统行业全面拥抱网络技术,制浆造纸企业也不例外。对此,简述了现代网络技术支持下的制浆造纸企业管理模式与传统的企业管理模式的差异之处,探究了基于现代网络技术进行企业管理模式创新的积极意义,重点从生产管理、财务管理、人力资源管理和营销管理方面提出了网络时代制浆造纸企业管理模式创新举措,以期为现代制浆造纸企业管理模式创新提供参考,为企业的持续稳定发展奠定基础。

甲基丙烯酸酐改性明胶/经处理牙本质基质生物活性支架的制备及性能

背景:牙髓-牙本质再生一直是近几年研究的热点及难点,构建复合生物支架材料为牙髓-牙本质再生提供了新的思路与方法。目的:观察甲基丙烯酸酐改性明胶/经处理牙本质基质支架对人牙髓干细胞增殖、迁移与成骨分化的影响。方法:将不同质量的经处理牙本质基质分散至甲基丙烯酸酐改性明胶溶液中,使甲基丙烯酸酐改性明胶与经处理牙本质基质的质量比分别为2∶1、1∶1、1∶2,采用冷冻干燥法制备甲基丙烯酸酐改性明胶/经处理牙本质基质支架,检测支架的微观形貌、吸水率及机械性能。采用不同质量比的支架浸提液、DMEM培养基(对照组)分别培养人牙髓干细胞,检测细胞增殖与迁移情况。采用不同质量比的支架浸提液+成骨诱导液、DMEM培养基+成骨诱导液(对照组)分别培养人牙髓干细胞,碱性磷酸酶染色分析成骨能力。结果与结论:①扫描电镜下可见3组支架均具有多孔结构,随着支架中经处理牙本质基质质量的增加,支架的孔隙率升高,组间两两比较差异有显著性意义(P<0.05);随着支架中经处理牙本质基质质量的增加,支架的吸水率升高,组间两两比较差异有显著性意义(P<0.05);随着支架中经处理牙本质基质质量的增加,支架的抗压强度、剪切强度增大。②CCK-8检测显示培养3,5,7 d,2∶1、1∶1、1∶2支架组细胞增殖吸光度值均高于对照组(P<0.05),并且随着支架中经处理牙本质基质质量的增加,细胞增殖吸光度值升高(P<0.05);细胞划痕实验显示2∶1、1∶1、1∶2支架组细胞迁移率均大于对照组(P<0.05),并且随着支架中经处理牙本质基质质量的增加,细胞迁移率增大(P<0.05)。③碱性磷酸酶染色显示,2∶1、1∶1、1∶2支架组细胞成骨分化能力均强于对照组,并且随着支架中经处理牙本质基质质量的增加,细胞成骨能力增强。④结果表明,甲基丙烯酸酐改性明胶与经处理牙本质基质质量比为1∶2的支架最适合牙髓干细胞的增殖与分化。

活化α7乙酰胆碱受体促进LPS刺激的人牙髓干细胞牙/骨向分化

目的:探讨活化α7乙酰胆碱受体(alpha 7 nicotinic acetylcholine receptor,α7-nAChR)联合钙离子(calcium ion,Ca^(2+)对LPS刺激的人牙髓干细胞(dental pulp stem cell,DPSC)牙/骨向分化的影响。方法:分离培养DPSC,流式细胞术对DPSC进行表面标志物表达鉴定。CCK-8检测α7-nAChR激动剂PNU-282987和Ca^(2+)对DPSC增殖的影响。通过碱性磷酸酶(alkaline phosphatase,ALP)活性和染色筛选PNU-282987促进DPSC表达ALP活性的最佳浓度。用大肠杆菌脂多糖(lipopolysaccharide,LPS)模拟炎性微环境刺激DPSC。采用免疫印迹分析(Western blot,WB)、实时定量聚合酶链反应(quantitative real-time polymerase chain reaction,RT-qPCR)和茜素红染色等方法检测牙/骨向分化的相关蛋白:Ⅰ型胶原(typeⅠcollagen,COL-I)、牙本质涎磷蛋白(dentin sialoprotein,DSPP)、骨钙素(osteopontin,OPN)、ALP、核心转录因子-2(runt-related transcription factor 2,RUNX2)、成骨细胞特异性转录因子(osterix,OSX),相关基因(COL-I、DSPP、OPN、ALP、RUNX2、OSX)和矿化基质表达情况。Fura-2AM用于检测细胞内Ca^(2+)流动情况。结果:CCK-8实验显示,PNU-282987浓度低于10μmol/L时对细胞增殖无抑制作用,且此浓度处理LPS刺激的DPSC后ALP活性增加最明显;Ca^(2+)浓度低于2 mmol/L对细胞增殖无抑制作用;Western blot和RT-qPCR实验显示,PNU-282987及Ca^(2+)处理后的LPS刺激的DPSC牙/骨向分化相关蛋白(COL-I、DSPP、OPN、ALP、RUNX2、OSX)和相关基因(COL-I、DSPP、OPN、ALP、RUNX2、OSX)的表达及矿化基质形成均明显上调,二者联合后上调最显著(P <0.001)。Fura-2 AM钙离子探针结果显示DPSC细胞内Ca^(2+)浓度增加。结论:10μmol/L PNU-282987联合2 mmol/L Ca^(2+)可以促进LPS刺激的DPSC的牙/骨向分化能力。

PDGFD对人牙髓干细胞迁移及成牙本质向分化的作用

目的:探讨人重组血小板来源的生长因子D(platelet-derived growth factor D,PDGFD)对人牙髓干细胞(human dental pulp stem cells,hDPSCs)迁移及成牙本质向分化的作用。方法:利用酶解法分离培养hDPSCs,流式细胞术鉴定所培养的间充质干细胞表面分子标志物的表达,诱导hDPSCs三系分化并使用相应染色鉴定,以表征其多向分化潜能。应用细胞划痕实验检测PDGFD对hDPSCs迁移能力的影响,利用实时荧光定量PCR及蛋白免疫印迹法检测PDGFD对hDPSCs成牙本质相关mRNA及蛋白表达的影响,利用碱性磷酸酶(alkaline phosphataseI,ALP)和茜素红(alizarin red staining,ARS)染色检测PDGFD对hDPSCs矿化的影响。采用SPSS 26.0软件包对数据进行统计学分析。结果:细胞形态学分析、流式细胞术鉴定和三系分化结果显示,所分离得到的细胞符合hDPSCs特征,并且具有多向分化潜能。细胞划痕实验结果表明,12 h时,仅50 ng/mL的PDGFD对hDPSCs的迁移能力有影响;24 h时,10和50 ng/mL的PDGFD对hDPSCs的迁移能力均有影响。PCR结果显示,10与50 ng/mL的PDGFD均对hDPSCs的成牙本质分化有促进作用,50 ng/mL的PDGFD对hDPSCs的成牙本质分化更为显著。蛋白免疫印迹实验、ALP及ARS染色所得结果和PCR结果相同。结论:成功分离并培养了具有典型间充质干细胞表型和多向分化潜能的hDPSCs。10和50 ng/mL浓度的PDGFD对hDPSCs的迁移和成牙本质向分化均有促进作用,其中50 ng/mL的PDGFD的促进作用更为显著。

lncRNA TP53TG1在牙髓中的表达和对牙髓干细胞转录炎症因子的影响

目的探究lncRNA TP53TG1在健康和炎症牙髓中的表达差异,以及对脂多糖(LPS)诱导后人牙髓干细胞(hDPSCs)炎症因子转录水平的影响。方法设计合成lncRNA探针,利用RNA荧光原位杂交技术检测健康和炎症牙髓组织中lncRNA TP53TG1的表达情况。通过酶解组织块法分离培养hDPSCs,对培养的hDPSCs进行多向分化诱导验证,并通过流式细胞术鉴定表型特征。将hDPSCs分为si-NC组、si-NC+LPS组、si-TP53TG1+LPS组,分别转染对照序列和TP53TG1的siRNA,再用LPS处理si-NC+LPS组和si-TP53TG1+LPS组24 h,通过qRT-PCR检测各组炎症因子IL-1β、IL-8以及TNF-α的mRNA表达水平。结果在健康牙髓组织中可观察到lncRNA TP53TG1的表达,主要分布在成牙本质细胞中。炎症状态下,lncRNA TP53TG1的表达量上升,全牙髓可见分布。LPS处理后,hDPSC的TNF-α、IL-1β以及IL-8的mRNA表达水平上升,下调lncRNA TP53TG1导致hDPSCs的TNF-αmRNA表达水平进一步升高。结论lncRNA TP53TG1可能参与调控牙髓炎症过程和成牙本质细胞的免疫调控作用。

负载骨形态发生蛋白2水凝胶诱导牙髓干细胞的成骨分化

背景:前期研究证明,甲基丙烯酸酐改性明胶/经处理牙本质基质复合水凝胶支架可促进人牙髓干细胞的增殖及分化。水凝胶负载骨形态发生蛋白2被认为是骨修复中有前景的材料。目的:观察负载不同质量浓度骨形态发生蛋白2的甲基丙烯酸酐改性明胶/经处理牙本质基质复合水凝胶对人牙髓干细胞成骨向分化的诱导作用。方法:制备含0,50,100,200μg/mL骨形态发生蛋白2的甲基丙烯酸酐改性明胶/经处理牙本质基质复合水凝胶,分别记为GelMA/TDM、BMP-2(50 ng/mL)GelMA/TDM、BMP-2(100 ng/mL)GelMA/TDM、BMP-2(200 ng/mL)GelMA/TDM,检测复合水凝胶对骨形态发生蛋白2的体外缓释性能。采用改良组织块酶消化法提取人牙髓干细胞,分别接种于4种水凝胶表面,采用CCK-8法检测细胞增殖,DAPI染色检测细胞黏附;对各组水凝胶表面的人牙髓干细胞进行成骨诱导,进行碱性磷酸酶染色、碱性磷酸酶活性检测与茜素红染色,采用RT-PCR法检测相关成骨基因(Runx2、骨形态发生蛋白2、骨桥蛋白、骨钙素、Ⅰ型胶原)表达。结果与结论:①甲基丙烯酸酐改性明胶/经处理牙本质基质复合水凝胶可持续释放骨形态发生蛋白2长达21 d,在第3-6天释放较快,第6天之后释放趋于平稳;②4种水凝胶均可促进人牙髓干细胞的增殖,其中以BMP-2(100 ng/mL)-GelMA/TDM复合水凝胶的作用最明显;相较于GelMA/TDM复合水凝胶,BMP-2-GelMA/TDM复合水凝胶可促进人牙髓干细胞的黏附,其中以BMP-2(200 ng/mL)-GelMA/TDM复合水凝胶的作用最明显;③相较于GelMA/TDM复合水凝胶,BMP-2-GelMA/TDM复合水凝胶可提升碱性磷酸酶活性、钙结节含量与相关成骨基因表达,综合分析显示BMP-2(100 ng/mL)-GelMA/TDM复合水凝胶的作用更明显;④结果表明,BMP-2(100 ng/mL)-GelMA/TDM复合水凝胶促进牙髓干细胞成骨向分化的能力更明显。

Epi-1对脂多糖诱导人牙髓干细胞炎症反应的影响

目的研究生物活性肽Epi-1在脂多糖(LPS)诱导下的炎症微环境中对人牙髓干细胞(hDPSC)表达炎症因子的影响和可能机制。方法通过组织块酶消化法分离、培养hDPSC,并通过流式细胞术鉴定。利用CCK-8试剂检测Epi-1对hDPSC细胞活性的影响,筛选出适宜的浓度。实验分为4组,分别为空白对照组(不含LPS和Epi-1)、LPS组(1.0μg/mL LPS)、2.5μg/mL Epi-1组(1.0μg/mL LPS和2.5μg/mL Epi-1)和5.0μg/mL Epi-1组(1.0μg/mL LPS和5.0μg/mL Epi-1),通过实时荧光定量聚合酶链式反应检测Epi-1对hDPSC白介素(IL)-6、IL-1β、IL-8基因表达的影响,进一步通过蛋白质免疫印迹法检测核因子κB信号通路关键蛋白p65、p-p65的表达情况,使用荧光探针检测活性氧生成情况。采用SPSS 22.0分析数据。结果在2.5、5.0μg/mL的质量浓度下,Epi-1无明显细胞毒性;Epi-1可显著降低LPS诱导后hDPSC中IL-6、IL-1β、IL-8的mRNA表达(P<0.01),减少p-p65的表达(P<0.05)和活性氧的生成(P<0.001)。结论Epi-1可能通过抑制核因子κB信号通路的激活和减轻氧化应激反应,从而降低LPS诱导的hDPSC的炎症反应。

MTA和三种改良盖髓剂对人牙髓干细胞增殖及分化为成牙本质细胞的促进作用观察

目的观察矿物三氧化物凝聚体(Mineral Trioxide Aggregate,MTA)和三种改良盖髓剂(nRoot、Vitapex、iRoot BP Plus)对人牙髓干细胞(hDPSCs)增殖、分化为成牙本质细胞的促进作用。方法①不同浓度四种盖髓剂对hDPSCs增殖的促进作用观察。取第5代hDPSCs细胞分为MTA组、iRoot BP Plus组、nRoot组、Vitapex组及空白组,MTA组、iRoot BP Plus组、nRoot组、Vitapex组分别加入不同浓度(0.02、0.2、1、2 mg/mL)的MTA、iRoot BP Plus、nRoot和Vitapex培养液,空白组加入含10%FBS的DMEM/F12培养液。分别于培养24、48 h时,采用CCK-8法测算各组细胞增殖活性。②四种盖髓剂对hDPSCs分化为成牙本质细胞的促进作用观察。通过ALP活性检测筛选出四种材料对hDPSCs最适诱导浓度。取第4代hDPSCs分为五组:空白组、MTA组、iRoot BP Plus组、nRoot组和Vitapex组换为0.2 mg/mL的MTA、iRoot BP Plus、nRoot和Vitapex成骨培养基,空白组为正常成骨培养基。培养第21天时采用茜素红染色和半定量分析法观察各组细胞矿化结节形成情况,培养第7天时采用Western Blotting法检测细胞相关蛋白类核转录因子(Runt-related transcription factor 2,RUNX-2)、骨钙素(Osteocalcin,OCN)、牙本质涎磷蛋白(Dentin sialophosphoprotein,DSPP)以及牙本质基质蛋白1(Dentin matrix protein 1,DMP-1)。结果与空白组相比,培养第2天时0.02、0.2、1 mg/mL的MTA组、iRoot BP Plus在和nRoot组细胞增殖活性高(P均<0.05);与培养第1天时相比,培养第2天时2 mg/mL的MTA组和Vitapex组细胞增殖活性低(P均<0.05)。筛选0.2 mg/mL为后续受试浓度。与nRoot组和Vitapex组相比,iRoot BP Plus组和MTA组细胞钙沉积量高(P均<0.05)。与空白组相比,MTA组、iRoot BP Plus组、nRoot组和Vitapex组细胞OCN、RUNX-2、DSPP、DMP-1相对表达量均升高(P均<0.05);与nRoot组、Vitapex组相比,iRoot BP Plus组细胞OCN、RUNX-2、DSPP、DMP-1相对表达量均高(P均<0.05)。结论相比于MTA、Vitapex,nRoot和iRoot BP Plus更能促进hDPSCs的细胞增殖、诱导细胞分化为成牙本质细胞。

促红细胞生成素干预牙髓损伤后修复性牙本质形成及骨形态发生蛋白2的表达

背景:促红细胞生成素具有抗炎、抗凋亡、促进骨缺损修复等作用,目前关于促红细胞生成素对牙髓损伤后修复性牙本质形成及其相关分子机制的研究较少。目的:探究促红细胞生成素对牙髓损伤后修复性牙本质形成的影响。方法:(1)动物实验:采用随机数字表法将32只大鼠随机分为对照组(n=16)与实验组(n=16),实验组牙髓损伤处使用含促红细胞生成素的胶原蛋白海绵直接盖髓,对照组露牙髓损伤处使用含PBS的胶原蛋白海绵直接盖髓,用玻璃离子粘固剂封闭窝洞;治疗2,4周后取上颌骨,采用免疫组化染色检测巢蛋白在牙本质中的表达,苏木精-伊红染色观察修复性牙本质生成。取4只SD大鼠上颌骨,免疫组化染色检测磨牙与切牙中促红细胞生成素表达。(2)细胞实验:从人牙髓组织、牙周韧带组织及牙龈组织中分别分离获取人牙髓细胞、人牙周韧带细胞和人牙龈成纤维细胞,采用RT-PCR检测促红细胞生成素mRNA表达。在成牙本质细胞诱导或未诱导分化条件下,采用RT-PCR检测人牙髓细胞中促红细胞生成素、牙本质涎磷蛋白、牙本质基质蛋白1、巢蛋白m RNA表达。在成牙本质细胞诱导或未诱导分化条件下,下调促红细胞生成素表达或外源性给予促红细胞生成素干预后,采用RT-PCR检测人牙髓细胞牙本质涎磷蛋白、牙本质基质蛋白1 mRNA相对表达,茜素红S染色检测矿化结节形成,RT-PCR与Western blotting检测骨形态发生蛋白2 mRNA与蛋白表达。结果与结论:(1)动物实验:与对照组相比,实验组治疗2,4周后的修复性牙本质生成量更大,牙本质中巢蛋白表达更高。促红细胞生成素在大鼠上颌第一磨牙牙髓、成牙本质细胞层和牙周膜中呈弱阳性表达,在成牙本质细胞中呈强阳性表达。(2)细胞实验:与其他两种细胞相比,人牙髓细胞中的促红细胞生成素m RNA表达更高。与未诱导分化相比,成牙本质细胞诱导分化下人牙髓细胞中牙本质涎磷蛋白、牙本质基质蛋白1、巢蛋白、促红细胞生成素与骨形态发生蛋白2 mRNA表达升高,矿化结节生成增加。在成牙本质细胞诱导分化下,下调促红细胞生成素表达后人牙髓细胞中牙本质涎磷蛋白、牙本质基质蛋白1、巢蛋白、骨形态发生蛋白2 mRNA表达与矿化结节生成均降低,骨形态发生蛋白2蛋白表达降低;外源性给予促红细胞生成素干预后,人牙髓细胞中上述指标表达均升高。(3)结果表明:促红细胞生成素对牙本质形成具有一定的促进作用。