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.

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.

An amelogenin-based peptide hydrogel promoted the odontogenic differentiation of human dental pulp cells

Amelogenin can induce odontogenic differentiation of human dental pulp cells(HDPCs),which has great potential and advantages in dentine-pulp complex regeneration.However,the unstability of amelogenin limits its further application.This study constructed amelogenin self-assembling peptide hydrogels(L-gel or D-gel)by heating-cooling technique,investigated the effects of these hydrogels on the odontogenic differentiation of HDPCs and explored the underneath mechanism.The critical aggregation concentration,conformation,morphology,mechanical property and biological stability of the hydrogels were characterized,respectively.The effects of the hydrogels on the odontogenic differentiation of HDPCs were evaluated via alkaline phosphatase activity measurement,quantitative reverse transcription polymerase chain reaction,western blot,Alizarin red staining and scanning electron microscope.The mechanism was explored via signaling pathway experiments.Results showed that both the L-gel and D-gel stimulated the odontogenic differentiation of HDPCs on both Day 7 and Day 14,while the D-gel showed the highest enhancement effects.Meanwhile,the D-gel promoted calcium accumulation and mineralized matrix deposition on Day 21.The D-gel activated MAPK-ERK1/2 pathways in HDPCs and induced the odontogenic differentiation via ERK1/2 and transforming growth factor/smad pathways.Overall,our study demonstrated that the amelogenin peptide hydrogel stimulated the odontogenic differentiation and enhanced mineralization,which held big potential in the dentine-pulp complex regeneration.

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.

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.

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.

Neural crest derived stem cells from dental pulp and tooth-associated stem cells for peripheral nerve regeneration

The peripheral nerve injuries,representing some of the most common types of traumatic lesions affecting the nervous system,are highly invalidating for the patients besides being a huge social burden.Although peripheral nervous system owns a higher regenerative capacity than does central nervous system,mostly depending on Schwann cells intervention in injury repair,several factors determine the extent of functional outcome after healing.Based on the injury type,different therapeutic approaches have been investigated so far.Nerve grafting and Schwann cell transplantation have represented the gold standard treatment for peripheral nerve injuries,however these approaches own limitations,such as scarce donor nerve availability and donor site morbidity.Cell based therapies might provide a suitable tool for peripheral nerve regeneration,in fact,the ability of different stem cell types to differentiate towards Schwann cells in combination with the use of different scaffolds have been widely investigated in animal models of peripheral nerve injuries in the last decade.Dental pulp is a promising cell source for regenerative medicine,because of the ease of isolation procedures,stem cell proliferation and multipotency abilities,which are due to the embryological origin from neural crest.In this article we review the literature concerning the application of tooth derived stem cell populations combined with different conduits to peripheral nerve injuries animal models,highlighting their regenerative contribution exerted through either glial differentiation and neuroprotective/neurotrophic effects on the host tissue.

Polymeric vs hydroxyapatite-based scaffolds on dental pulp stem cell proliferation and differentiation

AIM: To evaluate adhesion, proliferation and differentiation of human dental pulp stem cells(h DPSCs) on four commercially available scaffold biomaterials. METHODS: hD PSCs were isolated from human dental pulp tissues of extracted wisdom teeth and established in stem cell growth medium. h DPSCs at passage 3-5 were seeded on four commercially available scaffold biomaterials, SureO ss(Allograft), Cerabone(Xenograft), PLLA(Synthetic), and OSTEON Ⅱ Collagen(Composite), for 7 and 14 d in osteogenic medium. Cell adhesion and morphology to the scaffolds were evaluated by scanning electron microscopy(SEM). Cell proliferation and differentiation into osteogenic lineage were evaluated using DNA counting and alkaline phosphatase(ALP) activity assay, respectively. RESULTS: All scaffold biomaterials except Sure Oss(Allograft) supported h DPSC adhesion, proliferation and differentiation. hD PSCs seeded on PLLA(Synthetic) scaffold showed the highest cell proliferation and attachment as indicated with both SEM and DNA counting assay. Evaluating the osteogenic differentiation capability of hD PSCs on different scaffold biomaterials with ALP activity assay showed high level of ALP activity on cells cultured on PLLA(Synthetic) and OSTEON ⅡCollagen(Composite) scaffolds. SEM micrographs also showed that in the presence of Cerabone(Xenograft) and OSTEON Ⅱ Collagen(Composite) scaffolds, the h DPSCs demonstrated the fibroblastic phenotype with several cytoplasmic extension, while the cells on PLLA scaffold showed the osteoblastic-like morphology, round-like shape. CONCLUSION: PLLA scaffold supports adhesion, proliferation and osteogenic differentiation of hD PSCs. Hence, it may be useful in combination with hD PSCs for cell-based reconstructive therapy.

Dental pulp cell bank as a possible future source of individual hepatocytes

Mesenchymal stem cells(MSCs) as a source for regenerative medicine are now the subject of much clinical attention. There are high expectations due to their safety, low tumorigenic risk, and low ethical concerns. MSC therapy has been approved for acute graft-versus host diseases since 2015. Tooth-derived MSCs are known to have a great potential in their proliferation and differentiation capacities, even when compared with bone-marrow-derived MSCs. In particular, stem cells from human exfoliated deciduous teeth(SHEDs) are the best candidates for personal cell banking(dental pulp cell bank), because they can be obtained less invasively in the natural process of individual growth. SHEDs are known to differentiate into hepatocytes. There have been several studies showing the effectiveness of SHEDs on the treatment of liver failure in animal models. They may exert their effects either by repopulation of cells in injured liver or by paracrine mechanisms due to their immuneregulatory functions. Moreover, it may be possible to use each individuals' dental pulp cells as a future source of tailor-made differentiated hepatocytes in the context of a bioartificial liver or liver-on-a-chip to screen for drug toxicity.

In-Vitro Cellular Responses of Human Dental Primary Cells to Dental Filling Restoratives

In vitro cytotoxicity of six contemporary commercial dental filling restoratives on human dental primary cells, pulp cells (HPCs) and human gingival fibroblasts (HGFs), were tested using WST-1 assay. Continuous 3T3 mouse fibroblast cell lines were used for comparison. The results show that conventional glass-ionomer cement (GIC) Fuji II is not cytotoxic to all the cells. Resin-modified GIC (RMGIC) Fuji II LC is not cytotoxic to both HPCs and HGFs but cytotoxic to 3T3 cells. RMGIC Vitremer and resin composite Z100 are very cytotoxic to all the cells. Resin composite P60 is cytotoxic but much less cytotoxic than Z100. Polycarboxylate cement Durelon is the most cytotoxic among the six tested materials. It was found that continuous 3T3 cell lines were more vulnerable to leachable cytotoxic components than primary HPCs and HGFs. It was also found that the cytotoxcity of the tested materials was dose-dependent.

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。

研磨处理可注射牙髓细胞外基质促进牙髓再生

背景:可注射细胞外基质材料用于牙髓组织再生时,首先需要经过胃蛋白酶消化,再与其他基质成分如水凝胶结合使用,而这也意味着天然细胞外基质材料所保留的微环境遭到破坏。目的:探讨研磨处理条件下制备的可注射牙髓细胞外基质材料用于牙髓组织再生的效果。方法:(1)将获取的猪牙来源牙髓组织经过脱细胞处理后制备成牙髓脱细胞基质材料,经过液氮冷冻、高速研磨处理后,制备成可注射牙髓细胞外基质材料,比较可注射牙髓细胞外基质材料和天然牙髓细胞外基质在细胞黏附效率和微结构方面的差异,免疫组化分析可注射牙髓细胞外基质中的细胞外基质蛋白质成分;(2)将接种有牙髓干细胞的可注射牙髓细胞外基质材料注入到牙本质基质材料构建的牙髓腔中,植入裸鼠皮下1,3,5周后取材,观察可注射牙髓细胞外基质材料的体内改建速率及其在裸鼠皮下的异位牙髓组织再生能力。结果与结论:(1)与天然牙髓细胞外基质相比,可注射牙髓细胞外基质材料显著提高了细胞黏附率,并保留了与天然牙髓细胞外基质相似的多孔胶原结构;(2)与天然牙髓细胞外基质相比,免疫组化染色显示可注射牙髓细胞外基质材料中层粘连蛋白、Ⅰ型胶原蛋白和整合素β1的表达没有显著变化,而纤连蛋白的表达显著下降;(3)体内实验显示,可注射牙髓细胞外基质材料具有优异的成血管能力,并促进了牙髓样组织的形成。结果表明,研磨处理的可注射牙髓细胞外基质材料可促进牙髓再生。

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。

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。

重组人生长激素促进人牙髓干细胞的成骨分化

背景:既往研究表明人牙髓干细胞具有良好的成骨分化潜能,是骨组织工程中潜在的种子细胞,目前重组人生长激素对人牙髓干细胞的增殖及成骨分化的作用尚不明确。目的:探究重组人生长激素对人牙髓干细胞增殖及成骨分化的影响。方法:采用组织块培养法分离培养人牙髓干细胞,根据药物浓度梯度筛选后,选取10,100,250,500,1000μg/L重组人生长激素干预为实验组,正常培养基培养为对照组。在干预后第1,3,5,7天采用CCK-8法检测人牙髓干细胞的增殖情况。选取含10,100,250,500,1000μg/L重组人生长激素的成骨诱导液干预人牙髓干细胞,在诱导第7天,采用碱性磷酸酶染色及其半定量分析法检测碱性磷酸酶活性,采用荧光定量RT-qPCR检测成骨相关基因Ⅰ型胶原蛋白、骨钙素、Runt相关转录因子2的mRNA表达,在诱导第14天,采用茜素红染色观察成骨矿化情况。结果与结论:①CCK-8检测结果显示,从干预第3天开始,100,250,500,1000μg/L重组人生长激素均能促进人牙髓干细胞增殖,与对照组相比差异有显著性意义(P<0.01);②与对照组比较,100,250,500μg/L重组人生长激素组人牙髓干细胞的碱性磷酸酶活性显著升高(P<0.01);100,250μg/L重组人生长激素组人牙髓干细胞的茜素红染色矿化结节数显著增多(P<0.01);250μg/L重组人生长激素组Ⅰ型胶原蛋白、骨钙素的mRNA表达量升高(P<0.05,P<0.01),100,250μg/L重组人生长激素组Runt相关转录因子2的mRNA表达量升高(P<0.01);③上述结果表明,250μg/L重组人生长激素更适合促进人牙髓干细胞增殖和成骨分化。

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。