Role of human pulp fibroblasts in angiogenesis

After pulp amputation, complete pulp healing requires not only reparative dentin production but also fibroblast proliferation, nerve fiber growth, and neoangiogenesis. This study was designed to investigate the role of pulp fibroblasts in angiogenesis. Human pulp fibroblasts from third molars co-cultured with human umbilical vein endothelial cells induced the organization of endothelial cells and the formation of tubular structures corresponding to capillaries in vivo. The direct contact between both cells was not necessary to induce angiogenesis, and the observed effect was due to soluble factors. This was confirmed with neutralizing antibodies against FGF-2 and VEGF, which decreased the angiogenic effects of these soluble factors. Immunohistochemistry showed that both FGF-2 and VEGF were expressed in human dental pulp fibroblasts, and this expression increased after injury. These results suggest that the pulp fibroblasts secrete angiogenic factors, which are necessary for complete pulp healing, particularly at the pulp injury site.     

血管内皮细胞对牙周组织细胞迁移的影响

目的: 通过血管内皮细胞与牙周组织细胞的复合培养模拟牙周组织再生环境, 研究血管内皮细胞对牙周组织细胞迁移的影响, 为明确血管内皮细胞在牙周组织再生过程中的作用奠定基础。方法: 应用原代培养的血管内皮细胞, 在Transwell嵌套中实现与人牙周膜成纤维细胞、牙龈成纤维细胞间的复合培养。分别以Transwell嵌套滤膜下腔面24 h的细胞数量检测垂直迁移能力, 以划痕实验后0、8、16和24 h计数划痕区域的细胞数量检测水平迁移能力, 以玻片试验及计算机辅助图像处理软件计算实验第1、4和7天细胞覆盖创面的面积检测覆盖创面能力, 并均与单独培养的2种牙周组织细胞作为对照。采用SPSS13.0软件包对实验结果进行统计学分析。结果: 血管内皮细胞存在时, 2种牙周细胞在垂直和水平方向的迁移量均显著高于单独培养组(P<0.01), 且牙周膜成纤维细胞垂直向的迁移量显著高于牙龈成纤维细胞(P<0.01), 但其水平迁移量在实验24 h时则显著低于牙龈成纤维细胞(P<0.01);在血管内皮细胞共存条件下, 2种牙周细胞创面覆盖能力在实验第7天高于对照组, 且牙周膜成纤维细胞的创面覆盖能力的增加显著高于牙龈成纤维细胞(P<0.01)。结论: 血管内皮细胞对牙周膜、牙龈成纤维细胞的迁移和创面覆盖有明显促进作用, 且血管内皮细胞对牙周膜成纤维细胞的垂直向迁移和创面覆盖能力的促进作用更为明显。     

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、20 μg/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10 μg/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10 μg/mL外泌体浓度促进迁移效果最为明显。     

HPC和L929细胞在修复粘结剂体外毒性评价中灵敏度的比较研究

目的：通过原代培养的人牙髓细胞（HPC）和L929细胞系,对两种树脂改良型玻璃离子粘结剂的体外细胞毒性评价,比较两种细胞的灵敏度,为口腔材料的体外评价建立更接近临床的评价体系。方法：制取Fujiplus和RelyX Luting两种材料浸渍液,实验组为材料的浸出原液、50%原液、25%原液,对照组为DMEM培养基,分别利用原代培养的牙髓细胞和L929细胞系对实验组和对照组进行细胞形态观察、MTT测定细胞相对增值率。结果：树脂改良型玻璃离子粘结剂的体外细胞毒性评价中,和选择的细胞有关,原代培养的牙髓细胞较L929细胞系敏感。结论：原代培养的人牙髓细胞可能较L929细胞系更适用于口腔粘材料的体外毒性评价。     

Notch-2在大鼠牙髓炎中的时空分布及其意义

目的:研究单纯开髓导致大鼠牙髓炎进程中Notch-2在牙髓损伤修复中的作用.方法:通过开髓建立大鼠牙髓炎模型,用免疫组织化学染色方法研究Notch-2的时空表达变化及其意义.结果:牙髓损伤早期(3 d),牙髓间充质细胞和牙髓成纤维细胞中Notch-2均呈弱阳性表达,成牙本质细胞为阴性表达.牙髓损伤中期(5 d),靠近损伤区的成牙本质细胞深层细胞Notch-2阳性表达达到高峰;同时,新生毛细血管内皮细胞呈强阳性表达.牙髓损伤晚期(7 d),Notch-2在牙髓间充质细胞、血管内皮细胞中表达均减弱,而在成牙本质细胞阳性表达达到高峰.牙髓损伤末期(14 d),Notch-2仅在成牙本质细胞下层细胞中尚有微弱表达,而其余牙髓细胞均为阴性表达.对照组正常牙髓组织Notch-2表达为阴性.结论:Notch-2在牙髓损伤应激情况下在牙髓间充质细胞和成牙本质细胞中上调表达,对于启动牙髓自我修复、诱导牙髓间充质细胞功能性分化以及抑制受损成牙本质细胞凋亡、维系和调动其相对正常的生理功能可能具有重要作用.     

Dentin regeneration in vitro: the pivotal role of supportive cells

The elaboration of dentin-pulp engineering strategies requires the investigation of not only progenitor cell potentials but also their interactions with other non-progenitor "supportive" cells. Under severe caries lesions, progenitor cells may be activated by growth factors released after the acidic dissolution of carious dentin. However, dentin regeneration has also been observed after traumatic injuries without any significant dentin dissolution. This raises questions about the origin of signals involved in progenitor cell activation, migration, and differentiation. Study models such as the entire tooth culture and co-cultures of pulp and endothelial cells highlighted the role of interactions between the different pulp cell types and the pivotal role they play in dentin regeneration. Injured pulp fibroblasts secrete growth factors involved in progenitor cell activation and differentiation as well as neoangiogenesis which may pave the pathways for progenitor cell migration. This appears to be the first paper to focus on this very important field in dental pulp biology.     

正常及炎症状态人牙髓中八聚体结合转录因子4B的表达

目的 研究人正常及炎症牙髓组织中八聚体结合转录因子4B(Oct-4B)的表达特点,检测大肠杆菌脂多糖刺激后人牙髓细胞( HDPCs)中Oct-4B的表达水平,以探讨Oct-4B在牙髓炎症中的可能作用.方法 采用免疫组织化学和反转录聚合酶链反应( RT-PCR)方法检测正常及炎症牙髓组织中Oct-4B的表达情况.RT-PCR检测1 mg/L脂多糖刺激HDPC 24、48、72 h后Oct-4B和热休克蛋白70(HSP70)表达水平的变化.结果 正常牙髓组织中未检测到Oct-4B的表达,炎症牙髓组织病灶处牙髓成纤维细胞和炎症细胞胞质中Oct-4B表达强阳性.炎症牙髓组织中Oct-4B mRNA水平显著高于正常牙髓组织(P<0.05).脂多糖刺激48.72 h后,HDPC中Oct-4B和HSP70 mRNA水平同步上调(P<0.05).结论 Oct-4B在炎症牙髓组织中高表达,且脂多糖刺激可上调牙髓细胞内Oct-4B表达,Oct-4B可能参与牙髓炎症修复过程.     

Toxicity of sonicated extracts of Bacteroides gingivalis on human pulpal cells and L929 cells in vitro.

Human pulpal fibroblasts and L929 cells were treated with sonicated extracts of two strains of Bacteroides gingivalis (W83 and ATCC 33277). The cell reaction was evaluated by monitoring cell growth and DNA synthesis. Light and scanning electron microscopic analysis were used to evaluate morphological changes of the cells. Extracts from both bacterial strains exerted a growth inhibitory effect on the cells. The pulpal cells were more sensitive than L929 cells. The ATCC 33277 strain of B. gingivalis was more cytotoxic than the W83 strain. Pulpal cells appeared to be markedly affected on the microscopic level. The diffusion of these toxic bacterial by-products, through dentin to the pulp, may account for pulpal cell damage that contributes to the initiation of pulpal pathosis.     

Tricalcium Silicate Capping Materials Modulate Pulp Healing and Inflammatory Activity In Vitro

Introduction

On stimulation by lipoteichoic acid or by a physical injury, fibroblasts have been shown to play a major role in the initiation of the pulp inflammatory reaction and healing through secretion of complement proteins and growth factors. The application of direct pulp-capping materials on these cells may interfere with the inflammatory and the healing processes within the pulp's inextensible environment. This work was designed to study in vitro the effects of silicate-based materials on pulp fibroblast modulation of the initial steps of pulp inflammation and healing.

Morphological cell changes due to chemical toxicity of a dental material: an electron microscopic study on human periodontal ligament fibroblasts and L929 cells

New endodontic materials with polymer bases may be more difficult to evaluate in cell cultures in vitro than conventional zinc oxide-eugenol cements. In order to study the morphological changes taking place in cells exposed to such materials, L929 cells and human periodontal fibroblasts were observed using scanning electron microscopic and transmission electron microscopic techniques. The morphological changes of the cells were correlated to the quantitative results observed simultaneously in cytotoxicity studies using the radiochromium release method. Results showed there was a relationship between the chromium release and the degree of individual cell damage. The periodontal ligament fibroblasts were more resistant to this kind of chemical injury than the L929 cells. Consequently, it may be proper to use periodontally derived cells for the study of cytotoxic mechanisms of polymer endodontic filling materials.     

3种义齿粘附剂的体外细胞毒性研究

目的:体外研究3种商品化义齿粘附剂的细胞毒性。方法:采用MTT法检测不同浓度义齿粘附剂浸提液对人原代口腔黏膜角质细胞、成纤维细胞和小鼠成纤维细胞系L929的细胞毒性。结果:3种义齿粘附剂对人原代口腔黏膜角质细胞有1~2级细胞毒性,对人原代口腔黏膜成纤维细胞有1级细胞毒性,对L929细胞却无毒性。高浓度义齿粘附剂浸提液一般比低浓度浸提液具有更强的细胞生长抑制作用。结论:义齿粘附剂对人原代口腔黏膜角质细胞和成纤维细胞有细胞毒性,对L929细胞却无细胞毒性,提示在义齿粘附剂的细胞毒性测试方面,人原代口腔黏膜细胞可能会提供更有价值的信息。     

复合树指单体对人牙髓细胞毒性的研究

目的：评价复合树脂单体对人牙髓的毒性作用,探讨不同细胞系对检测敏感性的影响。方法：选用人牙髓细胞（ＬＳＣ）为实验细胞,以Ｌ－９２９小鼠成纤维细胞作为对照细胞系,采用ＭＴＴ比色分析法,对两种牙科用复合树脂单体（ＴＥＧＤＭＡ和ＵＤＭＡ）进行体外细胞毒性研究。结果：在低于ＩＣ５０的各浓度组中,ＬＳＣ细胞的生存率高于Ｌ－９２９细胞;ＭＴＴ比色法作为一种能定量检测牙科材料细胞毒性的有效方法,实验时应注重考虑     

体外人牙髓细胞的矿化特性

为探讨体外人牙髓细胞的生物学特性，采用体外细胞连续培养、钙质染色、Ｘ射线能谱分析与透射电镜观察等方法，对体外人恒牙牙髓细胞的矿化特性进行了研究并与人牙龈成纤维细胞（ｈｕｍａｎｇｉｎｇｉｖａｆｉｂｒｏｂｌａｓｔｓ，ＨＧＦｓ）进行了比较。结果表明，人牙髓细胞在体外可以复层生长并形成细胞结节，ＨＧＦｓ则无此能力，牙髓细胞的碱性磷酸酶活性亦较ＨＧＦｓ者高；细胞结节钙质染色呈阳性，结节内钙磷含量明显增高；人牙髓细胞有许多与成牙本质细胞相似的超微结构特征，胞间基质中可见致密晶状小体。结果提示，体外连续培养的人牙髓细胞有向成牙本质细胞分化的可能，这可为研究人牙髓细胞的分化和矿化提供有价值的思路。     

巨噬细胞游走抑制因子在人牙髓中的表达及对牙髓细胞增殖的影响

目的 研究正常及炎症牙髓组织中巨噬细胞游走抑制因子(macrophage migration-inhibitory factors,MIF)的表达及其对人牙髓细胞(human dental pulp cells,HDPC)增殖的影响,以期探讨MIF在牙髓炎症中的可能作用.方法采用免疫组织化学和实时荧光定量聚合酶链反应(PCR)法检测健康及炎症牙髓组织中MIF的表达情况,并以不同质量浓度[0(对照组)、0.1、1.0、10.0 mg/L]脂多糖刺激HDPC 24 h,ELISA法检测HDPC培养上清液中MIF含量的变化;不同质量浓度(10、30、60 μg/L)的重组人MIF分别作用于HDPC 24和48 h,细胞计数试剂盒法检测细胞增殖率.结果 健康牙髓组织中MIF主要分布于成牙本质细胞层,炎症牙髓组织中MIF分布于成牙本质细胞、炎症细胞、牙髓成纤维细胞和血管内皮细胞中;MIF mRNA在正常牙髓组织和炎症牙髓组织中的表达差异无统计学意义(P>0.05);不同质量浓度脂多糖刺激HDPC后对照组MIF质量浓度为(1048.53±161.81) ng/L,0.1和1.0 mg/L组MIF分泌量[分别为(1772.58±495.05)、(1692.58±337.45) ng/L]与对照组相比均显著升高(P<0.05),约为对照组的1.5倍;10、30、60 μg /L 重组人MIF均可促进HDPC的增殖(P<0.05).结论 MIF在人牙髓组织中有表达且一定浓度脂多糖可促进HDPC MIF的分泌,重组人MIF可促进HDPC增殖,MIF可能在牙髓炎症发展过程中发挥一定作用.     

正常及炎症牙髓组织中白细胞介素1的活性检测

用细菌内毒素建立豚鼠的牙髓炎模型，借助体外培养的Ｌ９２９细胞，在培养液中加入正常和炎症的牙髓组织上清液，采用成纤维细胞增殖法测定正常和炎症牙髓组织中白细胞介素１（ＩＬ－１）的活性。结果发现正常牙髓组织无ＩＬ－１的活性，炎症牙髓组织产生明显的ＩＬ－１活性，并随机内毒素诱导时间的延长而增强，ＩＬ－１为牙髓炎的主要介质之一，介导了牙髓炎的发生和发展。     

Adenovirus-mediated recombinant human bone morphogenetic protein-7 expression promotes differentiation of human dental pulp cells

Recombinant human bone morphogenetic protein-7 (BMP-7) has been shown to stimulate new reparative dentin formation in animal models. However, little is known about whether BMP-7 could promote the odontoblast-like differentiation and the formation of mineralized nodules in human dental pulp cells. Here, we reported that the infection with adenovirus-BMP-7 (Ad-BMP-7), a BMP-7-expressing adenoviral vector, induced the expression of BMP-7 in primarily cultured human dental pulp cells in the long term with little effect on their proliferation and viability. Importantly, BMP-7 expression significantly increased alkaline phosphatase activity and induced the dentin sialophosphoprotein expression in a dose- and time-dependent manner, suggesting that BMP-7 promoted the odontoblast differentiation. Furthermore, BMP-7 expression stimulated the formation of many mineralized dentin-like calcified nodules. Our data suggest that Ad-BMP-7-mediated BMP-7 expression can promote the differentiation of human pulp cells into odontoblast-like cells and mineralization in vitro, which may provide insight for the design of new gene therapy for the pulp capping in the clinic.     

Bisphosphonates affect migration ability and cell viability of HUVEC,fibroblasts and osteoblasts in vitro

Oral Diseases (2011) 17 , 194–199 Objectives: Bisphosphonate‐associated osteonecrosis of the jaw (BP‐ONJ) is a side effect in patients being treated with bisphosphonates. The bisphosphonates most often associated with BP‐ONJ are the highly potent nitrogen‐containing bisphosphonates, e.g. pamidronate or zoledronate. In terms of BP‐ONJ aetiology, several theories are being discussed: inhibition of bone remodelling, effect on soft tissues, and antiangiogenic effect of bisphosphonates. The aim of this in vitro study was to investigate the effect of different potent bisphosphonates on osteoblasts, fibroblasts and human umbilicord vein endothelial cells (HUVEC). Materials and methods: Three nitrogen‐containing bisphosphonates (ibandronate, pamidronate and zoledronate) and one non‐nitrogen‐containing bisphosphonate (clodronate) were compared concerning their potency on apoptosis induction (tunel), cell viability (calcein assay) and migration potency (boyden chamber) on osteoblasts, fibroblasts and HUVEC. Results: The nitrogen‐containing bisphosphonates, particularly pamidronate and zoledronate, affect cell viability, cell migration and the induction of apoptosis of osteoblasts, fibroblasts and HUVEC. Conclusions: These results support the theory that BP‐ONJ is a multifactorially caused disease because several cell lines of the oral cavity which are responsible for integrity and wound healing are negatively affected by nitrogen‐containing bisphosphonates. Perioperative interruption of bisphosphonate application during dental surgical procedures – if possible – might be feasible to promote better wound healing.