Temperature Sensitive Simian Virus 40 Large T Antigen Immortalization of Murine Odontoblast Cell Cultures: Establishment of Clonal Odontoblast Cell Line

During tooth formation instructive epithelial-mesenchymal interactions result in the cytodifferentiation of ectomesenchy-mal cells into odontoblasts which produce the dentin extracellular matrix (DECM). The purpose of our study was to establish a stable murine odontoblast cell line by immortalization of odontoblasts using retrovirus transfection. In order to accomplish this goal, we utilized a previously characterized odontoblast monolayer cell culture system supportive of odontoblast cytodifferentiation from dental papilla mesenchyme (DPM), expression and secretion of a DECM and dentin biomineralization. First mandibular molars from E-18 Swiss Webster mice were dissected, the DPM isolated, and pulp cells dissociated. Pulp cells (5 × 10⁵well) were plated as monolayers and grown in α-MEM supplemented with 10% FCS, 100 units/ml penicillin and streptomycin, 50 μg/ml ascorbic acid. Cultures were maintained for 6 days at 37°C in a humidified atmosphere of 95% air and 5% CO₂ with media changes every two days. Immortalization was performed using a recombinant defective retrovirus containing the temperature sensitive SV-40 large T antigen cDNA and the neomycin (G418) resistance gene recovered from CRE packaging cells. Cultures were infected for 24 h with CRE conditioned medium containing 8 μg/ml of polybrene, the media was replaced with selection media containing 300 μg/ml of G418, and the cultures incubated at 33°C for one month with media changes every 3–5 days. Neomycin resistant cells were cloned by serial dilution to single cells in 96-well culture plates and grown in selection medium at 33°C. One established cell line, M06-G3, showed high constitutive expression of dentin phosphoprotein, type I collagen and alkaline phosphatase, characteristic of an odontoblast phenotype. These results demonstrate the establishment of a stable murine odontoblast cell line which retains the tissue-specific expression for a number of DECM proteins.     

A feasibility study for the analysis of reparative dentinogenesis in pOBCol3.6GFPtpz transgenic mice

Frozoni M, Balic A, Sagomonyants K, Zaia AA, Line SRP, Mina M. A feasibility study for the analysis of reparative dentinogenesis in pOBCol3.6GFPtpz transgenic mice. International Endodontic Journal,?45, 907-914, 2012. ABSTRACT: Aim To examine the feasibility of using the pOBCol3.6GFPtpz [3.6-green fluorescent protein (GFP)] transgenic mice as an in vivo model for studying the biological sequence of events during pulp healing and reparative dentinogenesis. Methodology Pulp exposures were created in the first maxillary molar of 12-16-week-old 3.6-GFP transgenic mice with CD1 and C57/Bl6 genetic background. Direct pulp capping on exposed teeth was performed using mineral trioxide aggregate followed by restoration with a light-cured adhesive system (AS) and composite resin. In control teeth, the AS was placed in direct contact with the pulp. Animals were euthanized at various time points after pulp exposure and capping. The maxillary arch was isolated, fixed and processed for histological and epifluorescence analysis to examine reparative dentinogenesis. Results Analysis of teeth immediately after pulp exposure revealed absence of odontoblasts expressing 3.6-GFP at the injury site. Evidence of reparative dentinogenesis was apparent at 4?weeks in 3.6-GFP mice in CD1 background and at 8?weeks in 3.6-GFP mice with C57/Bl6 background. The reparative dentine with both groups contained newly formed atubular-mineralized tissue resembling a dentine bridge and/or osteodentine that was lined by cells expressing 3.6-GFP as well as 3.6-GFP expressing cells embedded within the atubular matrix. Conclusion This study was conducted in a few animals and did not allow statistical analysis. The results revealed that the 3.6-GFP transgenic animals provide a unique model for direct analysis of cellular and molecular mechanisms of pulp repair and tertiary dentinogenesis in vivo. The study also shows the effects of the capping material and the genetic background of the mice in the sequence and timing of reparative dentinogenesis.     

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目的    研究核因子κB受体活化因子配基（RANKL）在人继承恒牙缺失滞留乳牙及乳牙根生理性吸收不同时期的表达。方法    选取2010年6-12月沈阳市口腔医院正畸科及儿童牙病科10～15岁患者因治疗需要拔除的乳牙18颗,按牙根吸收长度不同分为牙根吸收早、中、晚期（早期组、中期组、晚期组）,各6颗。同时选取无恒牙胚滞留乳牙（滞留组）和正畸拔除的正常恒牙（对照组）各6颗。采用免疫组化方法检测RANKL蛋白的表达,并测定累积光密度值。结果    牙髓成纤维细胞：对照组RANKL累积光密度值明显低于其余组（均P < 0.01）;早期组、中期组明显低于晚期组（均P < 0.01）。成牙本质细胞：对照组RANKL累积光密度值亦明显低于其余组（均P < 0.01）;早期组、中期组、晚期组三者间差异均有统计学意义（P < 0.01）。破牙细胞：对照组未见破牙细胞;早期组、中期组与晚期组比较差异有统计学意义（P < 0.01）。结论    RANKL是引起乳牙牙根缓慢吸收的因素之一。     

永生化人成牙本质细胞样细胞系体内矿化特征

目的 研究永生化人成牙本质细胞样细胞系hTERT-hOd-1体内的矿化特征。方法 将细胞和三维支架复合后移植至小鼠背部皮下，采用X线和组织学方法观察复合物生长情况。结果 X线显示复合物阻射像的密度和面积随时间而增加。组织学观察复合物在小鼠体内培养12周可形成矿化组织，部分似牙本质，其中含有牙本质小管样结构。结论 hTERT-hOd-1在体内培养中具矿化能力，可用于牙本质形成和组织工程的研究。     

牙本质矿化过程中的钙离子转运

牙本质矿化过程中的钙离子转运是一种跨细胞主动转运过程，钙离子ATP酶、钠钙离子交换体、L型电压控制钙离子通道、1，4，5—三磷酸肌醇受体多种机制协同作用完成对矿化区钙离子的沉积和成牙本质细胞中钙离子的平衡。     

人成牙本质细胞样细胞的原代培养

目的：培养人原代牙本质细胞。方法：取引产的8月龄死胎，剥离乳磨牙胚牙乳头，组织块法培养。然后采用滤纸片法挑取了3个与成牙本质细胞形态相似的细胞克隆，扩大培养。对培养细胞从形态学、矿化结节、碱性磷酸酶（alkaline phosphatase,ALP）活性、人牙本质基质蛋白-1（human dentin matrix protein-1,hDMP-1）和人牙本质涎磷蛋白（human dentin sialophosphoprotein,hDSPP）在mRNA水平上的表达等方面进行鉴定。结果：有一个克隆来源的细胞呈梭形、有单侧较长细胞突起，未见核极化，同时它们具矿化功能，表达人成牙本质细胞特异性标志hDMP-1、hDSPPmRNA。结论：该培养细胞为人成牙本质细胞样细胞，为进一步的有关研究奠定了基础。     

Effect of antisense oligonucleotide against mouse dentine matrix protein 1 on mineralization ability and calcium ions metabolism in odontoblast-like cell line MDPC-23

AIM: To study the mineralization ability and the dynamic changes of intracellular and extracellular concentrations of calcium ions in the odontoblast-like cell line MDPC-23 affected by antisense oligonucleotide (AS-ODN) against mouse dentine matrix protein 1 (DMP1). METHODOLOGY: The expression of DMP1 in MDPC-23 cells was detected by an immunohistochemical method and its blocking outcome by the Western blot method. The alkaline phosphatase (ALP) activity, size and number of mineralized nodules, and the intracellular free ([Ca2+]if), total ([Ca2+]it) and the extracellular ([Ca2+]e) calcium ion concentrations in MDPC-23 cells in the experimental group affected with AS-ODN were compared with those in the control group (paired-samples t-test). RESULTS: Dentine matrix protein 1 was stably expressed in a stable way in MDPC-23 cells; the expression was only just detectable at 12 h and became negative after 24 h affected by AS-ODN. Compared with the control groups, ALP activity of MDPC-23 cells in the AS-ODN group was decreased (P < 0.05), and both the number and size of mineralized nodules were smaller than those in the control group. [Ca2+]if in the AS-ODN group increased and then decreased after 24 h. [Ca2+]it dropped substantially to the lowest point at 24 h (P < 0.01). [Ca2+]e increased before treatment for 24 h and then dropped, however, it was still higher than that of the control group. CONCLUSIONS: Antisense oligonucleotide against DMP1 could decrease mineralization ability and affect the intracellular and extracellular concentrations of calcium ions in MDPC-23 cells. This would indicate that DMP1 regulates the metabolism and transportation of calcium ions in odontoblasts, and thus boosts dentine mineralization.