Applied Oral Sciences & Community Dental Care, Faculty of Dentistry, The University of Hong Kong, Pokfulam, Hong Kong.
Periodontics and Oral Medicine, School of Dentistry, University of Michigan, Ann Arbor, MI, USA.
J Dent Res. 2022 Sep;101(10):1214-1226. doi: 10.1177/00220345221091528. Epub 2022 Jul 7.
Stem cell-based therapeutics is a promising strategy in dental pulp regeneration. However, low cell viability after transplantation in vivo due to the ischemic microenvironment is still a critical challenge for future clinical application. With the aim of improving postimplantation cell survival and pulp tissue regeneration, stem cells from human exfoliated deciduous teeth (SHED) were preconditioned to a hypoxic condition by hypoxia-inducible factor 1α (HIF-1α) stabilization via knockdown of prolyl hydroxylase domain-containing protein 2 (PHD2) using lentiviral short hairpin RNA. HIF-1α-stabilized SHED were encapsulated in PuraMatrix hydrogel, injected into root canals of human tooth fragments, and implanted in the subcutaneous space of immunodeficient mice. After 28 d, enhanced dental pulp-like tissue formation was observed with a significantly higher level of vascularization, which could be attributed to both endothelial differentiation of SHED and recruitment of host blood vessels. Furthermore, dentin-like tissue formation in vivo and accelerated odontogenic/osteogenic differentiation both in vivo and in vitro were observed. At 7 d postimplantation, significantly less DNA damage and higher Ki67 expression were detected in the HIF-1α-stabilized SHED group compared with the control SHED. Accordingly, cell viability assay and staining for Ki67 and apoptotic cells in vitro showed that HIF-1α stabilization could decrease cell apoptosis and enhance cell survival significantly. We demonstrated that PI3K/AKT pathway activation had resulted in low caspase 3 expression in HIF-1α-stabilized SHED in hypoxic conditions. Furthermore, we found that HIF-1α-induced cell survival could also be attributed to the upregulated expression of PDK1, HK2, and Glut1, which contributes to the maintenance of reactive oxygen species homeostasis and metabolic adaptation in hypoxia. In addition, we identified as 1 of the top 3 upregulated genes through RNA sequencing in HIF-1α-stabilized SHED and demonstrated its essential role in HK2 and Glut1 upregulation. Taken together, HIF-1α stabilization enhances cell survival of SHED through modulating various target genes and potential signaling pathways, as well as odontogenic tissue formation during dental pulp regeneration, which could benefit stem cell-based therapy in general.
基于干细胞的治疗方法是牙髓再生的一种有前途的策略。然而，由于缺血微环境，移植后干细胞的存活率低仍然是未来临床应用的一个关键挑战。为了提高植入后细胞的存活率和牙髓组织的再生能力，研究人员通过慢病毒短发夹 RNA 敲低脯氨酰羟化酶结构域蛋白 2 (PHD2) 稳定缺氧诱导因子 1α (HIF-1α) ，对人乳牙脱落细胞 (SHED) 进行预处理，使其处于缺氧状态。将 HIF-1α 稳定的 SHED 包封在 PuraMatrix 水凝胶中，注入人牙碎片的根管中，并植入免疫缺陷小鼠的皮下空间。28 天后，观察到具有更高血管化水平的增强的牙髓样组织形成，这归因于 SHED 的内皮分化和宿主血管的募集。此外，还观察到体内牙本质样组织的形成以及体内和体外牙源性/成骨分化的加速。在植入后 7 天，与对照 SHED 相比，HIF-1α 稳定的 SHED 组的 DNA 损伤明显减少，Ki67 表达水平更高。相应地，细胞活力测定和体外 Ki67 和凋亡细胞染色表明，HIF-1α 稳定可以显著减少细胞凋亡并增强细胞存活。研究表明，PI3K/AKT 通路的激活导致 HIF-1α 稳定的 SHED 在低氧条件下 caspase 3 表达降低。此外，研究还发现 HIF-1α 诱导的细胞存活也归因于 PDK1、HK2 和 Glut1 的上调表达，这有助于维持低氧条件下的活性氧稳态和代谢适应。此外，通过 RNA 测序，研究人员确定了 HIF-1α 稳定的 SHED 中上调表达的前 3 个基因之一，并证明了其在 HK2 和 Glut1 上调中的重要作用。综上所述，HIF-1α 稳定通过调节各种靶基因和潜在的信号通路以及牙髓再生过程中的牙源性组织形成来增强 SHED 的细胞存活能力，这可能有益于一般的基于干细胞的治疗。
