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.

Human dental pulp stem/stromal cells in clinical practice

Dental pulp stem/stromal cells(DPSCs)are fibroblast-like,neural crest-derived,and multipotent cells that can differentiate into several lineages.They are relatively easy to isolate from healthy and inflamed pulps,with little ethical concerns and can be successfully cryopreserved and thawed.The therapeutic effects of DPSCs derived from animal or human sources have been extensively studied through in-vitro and in-vivo animal experiments and the findings indicated that DPSCs are effective not only for dental diseases but also for systemic diseases.Understanding that translational research is a critical step through which the fundamental scientific discoveries could be translated into applicable diagnostics and therapeutics that directly benefit humans,several clinical studies were carried out to generate evidence for the efficacy and safety of autogenous or allogeneic human DPSCs(hDPSCs)as a treatment modality for use in cell-based therapy,regenerative medicine/dentistry and tissue engineering.In clinical medicine,hDPSCs were effective for treating acute ischemic stroke and human exfoliated deciduous teeth-conditioned medium(SHED-CM)repaired vascular damage of the corpus cavernous,which is the main cause of erectile dysfunction.Whereas in clinical dentistry,autologous SHED was able to rege-nerate necrotic dental pulp after implantation into injured teeth,and micrografts enriched with autologous hDPSCs and collagen sponge were considered a treatment option for human intrabony defects.In contrast,hDPSCs did not add a significant regenerative effect when they were used for the treatment of post-extraction sockets.Large-scale clinical studies across diverse populations are still lacking to provide robust evidence on the safety and efficacy of hDPSCs as a new treatment option for various human diseases including dental-related problems.

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.

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.

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.

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.

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.

Application of dental stem cells in three-dimensional tissue regeneration

Dental stem cells can differentiate into different types of cells.Dental pulp stem cells,stem cells from human exfoliated deciduous teeth,periodontal ligament stem cells,stem cells from apical papilla,and dental follicle progenitor cells are five different types of dental stem cells that have been identified during different stages of tooth development.The availability of dental stem cells from discarded or removed teeth makes them promising candidates for tissue engineering.In recent years,three-dimensional(3D)tissue scaffolds have been used to reconstruct and restore different anatomical defects.With rapid advances in 3D tissue engineering,dental stem cells have been used in the regeneration of 3D engineered tissue.This review presents an overview of different types of dental stem cells used in 3D tissue regeneration,which are currently the most common type of stem cells used to treat human tissue conditions.

Effects of low intensity laser irradiation phototherapy on dental pulp constructs

AIM: To investigate low intensity laser irradiation phototherapy(LILIP) on the proliferation, mineralization and degradation of dental pulp constructs.METHODS: Stem cells from human exfoliated deciduous teeth(SHED) were grown to confluence and seeded on collagen scaffolds to create dental pulp constructs. LILIP was delivered to the dental pulp constructs using an 830 nm GaA IAs laser at an output power of 20 m W. The LILIP energy density was 0.4, 0.8, 1.2, and 2.4 J/cm2. After 8 d, the cell proliferation and degradation within the dental pulp constructs were measured using histologic criteria. After 28 d, the effect of LILIP on SHED mineralization was assessed by von Kossa staining.RESULTS: SHED proliferation within the dental pulp constructs varied after exposure to the 0.4, 0.8, 1.2,and 2.4 J/cm2 LILIP energy densities(P < 0.05). The maximum proliferation of SHED in nutrient deficient media was 218% after exposure to a 1.2 J/cm2 LILIP energy density. SHED grown in nutrient deficient media after exposure to a 0.4, 0.8, and 1.2 J/cm2 LILIP energy density, proliferated by 167-218% compared to the untreated(non-LILIP) control group(P < 0.05).SHED exposed to a 0.4, 0.8, and 1.2 J/cm2 LILIP energy density, and grown in optimal nutritional conditions and proliferated by 147%-164% compared to the untreated(non-LILIP) control group(P < 0.05). The exposure of SHED to the highest LILIP energy density(2.4 J/cm2) caused a reduction of the cell proliferation of up to 73% of the untreated(non-LILIP) control(P < 0.05). The amount of mineral produced by SHED increased over time up to 28 d(P < 0.05). The 0.8 and 1.2J/cm2 LILIP energy densities were the most effective at stimulating the increased the mineralization of the SHED from 150%-700% compared to untreated(nonLILIP) control over 28 d(P < 0.05). The degradation of dental pulp constructs was affected by LILIP(P <0.05). The dental pulp constructs grown in optimal nutritional conditions exposed to a 0.8 J/cm2 or 1.2 J/cm2 LILIP energy density had 13% to 16% more degradation than the untreated(non-LILIP) control groups(P < 0.05). The other LILIP energy densities caused a 1%degradation of dental pulp constructs in optimal nutritional conditions(P > 0.05).CONCLUSION: LILIP can enhance or reduce SHED proliferation, degradation and mineralization within dental pulp constructs. LILIP could promote the healing and regeneration of dental tissues.

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。

明胶-羟基磷灰石-磷酸三钙支架对hDPSCs的成骨刺激作用

目的:探讨明胶-羟基磷灰石-磷酸三钙支架(明胶-HA-TCP支架)对人牙髓干细胞(human dental pulp stem cells,hDPSCs)的成骨刺激作用。方法:通过溶剂浇铸/粒子沥滤法制备明胶支架及明胶-HA-TCP支架,利用场发射扫描电镜观察支架微观特征,立式电子万能试验机检测支架应力强度(stress intensity,SS)及弹性模量(E),制备成骨培养基,根据介质分为对照组(单纯成骨培养基)、实验1组(明胶支架提取物+成骨培养基)和实验2组(明胶-HA-TCP支架+成骨培养基),采用CCK8法评估支架对hDPSCs增殖的影响,测定碱性磷酸酶(alkaline phosphatase,ALP)活性并进行ALP染色,通过茜素红染色和反转录-聚合酶链反(RT-PCR)应检测hDPSCs中成骨因子的表达。采用SPSS 20.0软件包对数据进行统计学分析。结果:与明胶支架相比,明胶-HA-TCP支架SS显著提高,E显著降低(P<0.05);CD146和CD105在hDPSCs呈阳性表达,CD34和CD45在hDPSCs呈阴性表达;实验1组3、5和7 d细胞数量显著多于对照组,实验2组1、3、5和7 d细胞数量显著多于对照组(P<0.05);实验2组4、7和12 d ALP活性显著大于对照组及实验1组(P<0.05);与对照组相比,实验1组有少量红色视野,钙化沉淀量较少且分布不均匀,实验2组大部分视野呈红色,钙化沉淀量较多且分布均匀;实验1组与实验2组第4天Runt相关转录因子2(runt related transcription factor 2,Runx2)mRNA表达水平显著高于对照组(P<0.05);实验2组第7天成骨相关转录因子(osterix,OSX)mRNA表达水平显著高于对照组(P<0.05);实验2组第4天骨涎蛋白(bone sialoprotein,BSP)mRNA表达水平显著高于对照组和实验1组(P<0.05)。结论:明胶-HA-TCP支架的微观表征与天然骨结构相似,具有良好的抗压性,不易变形,对hDPSCs增殖和分化具有促进作用,并具有显著的骨诱导性,可作为骨组织工程新型复合材料。

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。

hUCMSCs促进VEGF分泌对LPS刺激下牙髓细胞增殖和凋亡及成牙本质细胞分化能力的影响

目的探讨人脐带间充质干细胞促进血管内皮生长因子(vascular endothelial growth factor,VEGF)分泌对脂多糖(lipopolysaccharide,LPS)刺激下牙髓细胞增殖、凋亡及成牙本质细胞分化能力的影响。方法体外培养牙髓细胞,将其分为牙髓细胞组(A组,常规培养)、牙髓细胞+LPS组(B组,牙髓细胞+10 mg/L LPS)、过表达VEGF牙髓细胞+LPS组(C组,牙髓细胞+转染过表达VEGF质粒+10 mg/L LPS)、hUCMSCs+牙髓细胞+LPS组(D组,牙髓细胞和hUCMSCs1∶1混合+10 mg/L LPS)及过表达VEGF牙髓细胞+hUCMSCs+LPS组(E组,转染过表达VEGF质粒的牙髓细胞和hUCMSCs1∶1混合+10 mg/L LPS);采用Real-time PCR法检测VEGF mRNA表达,细胞计数试剂盒8(CCK-8)法检测细胞增殖,末端标记法(TUNEL)检测细胞凋亡,茜素红染色检测成牙本质细胞向分化,免疫印迹法检测碱性磷酸酶(alkaline phosphatase,ALP)、Ⅰ型胶原(collagen typeⅠ,COLⅠ)及牙本质涎磷蛋白(dentin sialophosphoprotein,DSPP)蛋白表达。结果与A组比较,B组、C组、D组、E组细胞VEGF mRNA表达、凋亡率均显著升高,细胞增殖率、矿化结节、ALP、COLⅠ及DSPP蛋白表达均显著降低(P<0.05);与B组比较,C组、D组、E组细胞VEGF mRNA表达、增殖率、矿化结节、ALP、COLⅠ及DSPP蛋白表达明显升高,细胞凋亡率明显降低,差异有统计学意义(P<0.05)。结论人脐带间充质干细胞对LPS刺激下牙髓细胞活性具有显著改善作用,且可显著促进牙髓细胞成牙本质细胞向分化,其机制可能与促VEGF分泌有关。

脐静脉内皮细胞外泌体和内皮祖细胞外泌体对hDPSCs增殖及迁移能力的比较研究

目的将人脐静脉内皮细胞外泌体(human umbilical vein endothelial cells-derived exosome,HUVECs-exo)和内皮祖细胞外泌体(endothelial progenitor cells-derived exosome,EPCs-exo)对人牙髓干细胞(human dental pulp stem cells,hDPSCs)增殖和迁移能力的影响进行比较研究,以期为外泌体在牙髓再生中的应用积累经验。方法采用超速离心法分离提取外泌体,透射电镜观察外泌体形态,纳米粒子跟踪分析技术(nanoparticle tracking analysis,NTA)检测外泌体粒径大小,Western blot蛋白印迹检测外泌体标志蛋白CD9、CD63、TSG101的表达。将两种外泌体按照5、10、20μg/mL浓度梯度分别作用于hDPSCs,通过CCK-8实验检测hDPSCs增殖能力,细胞划痕实验和Transwell实验检测hDPSCs迁移能力。结果透射电镜下观察两种外泌体均呈圆盘状,粒径集中在30~150 nm,阳性表达CD9、CD63、TSG101三种标志蛋白。与对照组(Control组)相比,两种外泌体在不同浓度下均对hDPSCs增殖能力无显著促进作用,差异无统计学意义(P>0.05)。细胞划痕实验和Transwell实验表明,与对照组相比,两种外泌体均可促进hDPSCs迁移,差异有统计学意义(P<0.05),其中10μg/mL外泌体浓度作用效果最明显(P<0.01)。相同浓度的两种外泌体组间作用效果差异无统计学意义(P>0.05)。结论本实验成功分离提取到两种细胞来源的外泌体并进行鉴定,将不同浓度的两种细胞来源的外泌体作用于hDPSCs,发现对其增殖能力无明显促进作用,但可以促进其迁移,尤以10μg/mL外泌体浓度促进迁移效果最为明显。