EDTA soluble chemical components and the conditioned medium from mobilized dental pulp stem cells contain an inductive microenvironment, promoting cell proliferation, migration, and odontoblastic differentiation.
EDTA soluble chemical components and the conditioned medium from mobilized dental pulp stem cells contain an inductive microenvironment, promoting cell proliferation, migration, and odontoblastic differentiation.
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DOI：
10.1186/s13287-016-0334-z
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发表时间：
2016-05-25
影响因子：
7.5
通讯作者：
Nakashima M
中科院分区：
文献类型：
--
作者：
Kawamura R;Hayashi Y;Murakami H;Nakashima M
组织工程中的关键挑战是建立干细胞、信号形态发生分子和细胞外基质支架/微环境的最佳组合。在异位牙齿移植生物实验中，牙齿的细胞外基质成分可能被重组为诱导微环境。因此，牙髓/牙本质再生诱导微环境化学成分的分离和鉴定将加速牙组织工程目标的实现。分别用4.0M盐酸胍(GdnHCl)，pH 7.4和0.5M乙二胺四乙酸(EDTA)，pH 7.4依次拔除脱矿牙。将拔除的牙齿与动员的牙髓干细胞(MDPSCs)一起移植到严重联合免疫缺陷(SCID)小鼠的异位部位。未拔除的牙齿作为阳性对照。此外，将诱导微环境的可溶性成分、GdnHCl提取物或EDTA提取物与MDPSC条件培养液(CM)一起或不与MDPSC条件培养液一起系统地重组高压灭菌牙齿，其中化学成分完全失活，仅保留物理微环境。采用组织形态计量学和实时荧光定量RT-PCR方法比较异位牙移植28d后牙髓/牙本质再生能力和血管生成能力。与GdnHCl拔除的牙齿相比，成牙本质细胞标志物釉质溶素和牙髓标志物促甲状腺激素释放激素降解酶(TRH-DE)的表达较低，而牙周细胞标志物抗asporin/牙周膜相关蛋白1(PLAP-1)的表达较高。GdnHCl萃取物或EDTA萃取物重建的高压灭菌牙齿再生能力较弱，新生血管能力较弱，而CM显著增加了这一潜力。EDTA提取物和CM在体内显示了对牙髓/牙本质再生的联合作用，这与它们在体外促进增殖、迁移和成牙本质细胞分化的作用一致。EDTA拔除的牙齿与GdnHCl拔除的牙齿相比，牙髓/牙本质再生潜力显著降低。当EDTA可溶化学成分与高压灭菌牙齿的物理结构重组时，可作为诱导牙髓/牙本质再生的微环境，促进细胞增殖、迁移和成牙本质细胞分化。本文的在线版本(doi：10.1186/s13287-0160334-z)包含补充材料，授权用户可以使用。
The critical challenge in tissue engineering is to establish an optimal combination of stem cells, signaling morphogenetic molecules, and extracellular matrix scaffold/microenvironment. The extracellular matrix components of teeth may be reconstituted as an inductive microenvironment in an ectopic tooth transplantation bioassay. Thus, the isolation and identification of the chemical components of the inductive microenvironment in pulp/dentin regeneration will accelerate progress towards the goal of tissue engineering of the tooth. The teeth demineralized in 0.6 M hydrochloric acid were sequentially extracted by 4.0 M guanidine hydrochloride (GdnHCl), pH 7.4, and 0.5 M ethylenediaminetetraacetic acid (EDTA), pH 7.4. The extracted teeth were transplanted into an ectopic site in severe combined immunodeficiency (SCID) mice with mobilized dental pulp stem cells (MDPSCs). The unextracted tooth served as a positive control. Furthermore, the soluble components for the inductive microenvironment, the GdnHCl extracts, or the EDTA extracts together with or without MDPSC conditioned medium (CM) were reconstituted systematically with autoclaved teeth in which the chemical components were completely inactivated and only the physical microenvironment was preserved. Their pulp/dentin regenerative potential and angiogenic potential were compared 28 days after ectopic tooth transplantation by histomorphometry and real-time RT-PCR analysis. Expression of an odontoblastic marker, enamelysin, and a pulp marker, thyrotropin-releasing hormone degrading enzyme (TRH-DE), was lower, and expression of a periodontal cell marker, anti-asporin/periodontal ligament-associated protein 1 (PLAP-1), was higher in the transplant of the EDTA-extracted teeth compared with the GdnHCl-extracted teeth. The autoclaved teeth reconstituted with the GdnHCl extracts or the EDTA extracts have weak regenerative potential and minimal angiogenic potential, and the CM significantly increased this potential. Combinatorial effects of the EDTA extracts and the CM on pulp/dentin regeneration were demonstrated in vivo, consistent with their in-vitro effects on enhanced proliferation, migration, and odontoblastic differentiation. The EDTA-extracted teeth demonstrated significantly lower pulp/dentin regenerative potential compared with the GdnHCl-extracted teeth. The EDTA soluble chemical components when reconstituted with the physical structure of autoclaved teeth serve as an inductive microenvironment for pulp/dentin regeneration, promoting cell proliferation, migration, and odontoblastic differentiation. The online version of this article (doi:10.1186/s13287-016-0334-z) contains supplementary material, which is available to authorized users.